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Sample records for bovine blastocyst development

  1. Melatonin improves the quality of in vitro produced (IVP bovine embryos: implications for blastocyst development, cryotolerance, and modifications of relevant gene expression.

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    Feng Wang

    Full Text Available To evaluate the potential effects of melatonin on the kinetics of embryo development and quality of blastocyst during the process of in vitro bovine embryo culture. Bovine cumulus-oocyte complexes (COCs were fertilized after in vitro maturation. The presumed zygotes were cultured in in vitro culture medium supplemented with or without 10(-7 M melatonin. The cleavage rate, 8-cell rate and blastocyst rate were examined to identify the kinetics of embryo development. The hatched blastocyst rate, mortality rate after thawing and the relevant transcript abundance were measured to evaluate the quality of blastocyst. The results showed that melatonin significantly promoted the cleavage rate and 8-cell embryo yield of in vitro produced bovine embryo. In addition, significantly more blastocysts were observed by Day 7 of embryo culture at the presence of melatonin. These results indicated that melatonin accelerated the development of in vitro produced bovine embryos. Following vitrification at Day 7 of embryo culture, melatonin (10(-7 M significantly increased the hatched blastocyst rate from 24 h to 72 h and decreased the mortality rate from 48 h to 72 h after thawing. The presence of melatonin during the embryo culture resulted in a significant increase in the gene expressions of DNMT3A, OCC, CDH1 and decrease in that of AQP3 after thawing. In conclusion, melatonin not only promoted blastocyst yield and accelerated in vitro bovine embryo development, but also improved the quality of blastocysts which was indexed by an elevated cryotolerance and the up-regulated expressions of developmentally important genes.

  2. Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro.

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    Dessie Salilew-Wondim

    Full Text Available Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY, 4-cell (4C or 16-cell (16C were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP. Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic

  3. Native plants ( and extracts act as antioxidants to support developmental competence of bovine blastocysts

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    Geon-Yeop Do

    2017-09-01

    Full Text Available Objective Phellodendron amurense (P. amurense and Humulus japonicus (H. japonicus are closely involved in anti-oxidative response and increasing antioxidant enzymes activities. However, the effects of their extracts on development of preimplantation bovine embryos have not been investigated. Therefore, we investigated the effects of P. amurense and H. japonicus extracts on developmental competence and quality of preimplantation bovine embryos. Methods After in vitro fertilization, bovine embryos were cultured for 7 days in Charles Rosenkrans amino acid medium supplemented with P. amurense (0.01 μg/mL and H. japonicus (0.01 μg/mL. The effect of this supplementation during in vitro culture on development competence and antioxidant was investigated. Results We observed that the blastocysts rate was significantly increased (p<0.05 in P. amurense (28.9%±2.9%, H. japonicus (30.9%±1.5%, and a mixture of P. amurense and H. japonicus (34.8%± 2.1% treated groups compared with the control group (25.4%±1.6%. We next confirmed that the intracellular levels of reactive oxygen species (ROS were significantly decreased (p<0.01 in P. amurense and/or H. japonicus extract treated groups when compared with the control group. Our results also showed that expression of cleaved caspase-3 and apoptotic cells of blastocysts were significantly decreased (p<0.05 in bovine blastocysts derived from both P. amurense and H. japonicus extract treated embryos. Conclusion These results suggest that proper treatment with P. amurense and H. japonicus extracts in the development of preimplantation bovine embryos improves the quality of blastocysts, which may be related to the reduction of ROS level and apoptosis.

  4. Identification and expression analysis of genes associated with bovine blastocyst formation

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    Van Zeveren Alex

    2007-06-01

    Full Text Available Abstract Background Normal preimplantation embryo development encompasses a series of events including first cleavage division, activation of the embryonic genome, compaction and blastocyst formation. First lineage differentiation starts at the blastocyst stage with the formation of the trophectoderm and the inner cell mass. The main objective of this study was the detection, identification and expression analysis of genes associated with blastocyst formation in order to help us better understand this process. This information could lead to improvements of in vitro embryo production procedures. Results A subtractive cDNA library was constructed enriched for transcripts preferentially expressed at the blastocyst stage compared to the 2-cell and 8-cell stage. Sequence information was obtained for 65 randomly selected clones. The RNA expression levels of 12 candidate genes were determined throughout 3 stages of preimplantation embryo development (2-cell, 8-cell and blastocyst and compared with the RNA expression levels of in vivo "golden standard" embryos using real-time PCR. The RNA expression profiles of 9 (75% transcripts (KRT18, FN1, MYL6, ATP1B3, FTH1, HINT1, SLC25A5, ATP6V0B, RPL10 were in agreement with the subtractive cDNA cloning approach, whereas for the remaining 3 (25% (ACTN1, COPE, EEF1A1 the RNA expression level was equal or even higher at the earlier developmental stages compared to the blastocyst stage. Moreover, significant differences in RNA expression levels were observed between in vitro and in vivo produced embryos. By immunofluorescent labelling, the protein expression of KRT18, FN1 and MYL6 was determined throughout bovine preimplantation embryo development and showed the same pattern as the RNA expression analyses. Conclusion By subtractive cDNA cloning, candidate genes involved in blastocyst formation were identified. For several candidate genes, important differences in gene expression were observed between in vivo and in

  5. 309 proteomic analysis of the blastocoel fluid and remaining cells of bovine blastocysts

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    Jensen, P L; Groendahl, M L; Beck, Helle

    2012-01-01

    Human embryonic stem cells (hESC) are derived from the human blastocyst and possess the potential to differentiate into any cell type present in the adult human body. Human ESC are considered to have great potential in regenerative medicine for the future treatment of severe diseases and conditions...... such as Parkinson's disease, diabetes, and spinal cord injury. One of today's challenges in regenerative medicine is to define proper culture conditions for hESC. The natural milieu in the blastocyst may provide clues on how to improve culture conditions, and the aim of the present study was to determine...... the proteome of the blastocoel fluid and the remaining cells of bovine blastocysts. Bovine blastocysts were produced by in vitro fertilization of oocytes retrieved from slaughterhouse ovaries. The blastocoel from 195 blastocysts (1-8nL per blastocyst) were isolated by micromanipulation and analysed by nano...

  6. Vitamin C supplementation enhances compact morulae formation but reduces the hatching blastocyst rate of bovine somatic cell nuclear transfer embryos.

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    Li, Qian; Wang, Yong-Sheng; Wang, Li-Jun; Zhang, Hui; Li, Rui-Zhe; Cui, Chen-Chen; Li, Wen-Zhe; Zhang, Yong; Jin, Ya-Ping

    2014-08-01

    Vitamin C, an antioxidant that reduces reactive oxygen species (ROS) in cells, is capable of significantly improving the developmental competence of porcine and mouse somatic cell nuclear transfer (SCNT) embryos, both in vitro and in vivo. In the present study, the effects of vitamin C on the developmental competence of bovine SCNT embryos were investigated. The results indicated that vitamin C (40 μg/mL) positively affected the scavenging of intracellular ROS, cleavage rate at 24 h (76.67 vs. 68.26%, pvitamin C supplementation did not significantly affect the blastocyst formation rate and proportion of inner cell mass over total cells per blastocyst on day 7. Moreover, vitamin C supplementation obviously impaired the total cell numbers per blastocyst (97.20 ± 11.35 vs. 88.57 ± 10.43, pVitamin C supplementation preferentially improved the viability of bovine SCNT embryos prior to the blastocyst stage, but did not enhance the formation and quality of blastocysts in vitro. In conclusion, the effect of vitamin C on the development of bovine SCNT embryos is complex, and vitamin C is not a suitable antioxidant chemical for the in vitro culture of bovine SCNT embryos.

  7. Insulin during in vitro oocyte maturation has an impact on development, mitochondria, and cytoskeleton in bovine day 8 blastocysts.

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    Laskowski, Denise; Båge, Renée; Humblot, Patrice; Andersson, Göran; Sirard, Marc-André; Sjunnesson, Ylva

    2017-10-01

    Insulin is a key metabolic hormone that controls energy homeostasis in the body, including playing a specific role in regulating reproductive functions. Conditions associated with hyperinsulinemia can lower developmental rates in bovine in vitro embryo production and are linked to decreased fertility in humans, as in cases of obesity or type 2 diabetes. Embryo quality is important for fertility outcome and it can be assessed by choosing scoring standards for various characteristics, such as developmental stage, quality grade, cell number, mitochondrial pattern or actin cytoskeleton structure. Changes in the embryo's gene expression can reflect environmental impacts during maturation and may explain morphological differences. Together with morphological evaluation, this could enable better assessment and possibly prediction of the developmental potential of the embryo. The aim of this study was to use a bovine model to identify potential gene signatures of insulin-induced changes in the embryo by combining gene expression data and confocal microscopy evaluation. Bovine embryos were derived from oocytes matured in two different insulin concentrations (10 µg mL - 1 and 0.1 µg mL - 1 ), then stained to distinguish f-Actin, DNA and active mitochondria. The total cell number of the embryo, quality of the actin cytoskeleton and mitochondrial distribution were assessed and compared to an insulin-free control group. A microarray-based transcriptome analysis was used to investigate key genes involved in cell structure, mitochondrial function and cell division. Our results indicate that insulin supplementation during oocyte maturation leads to lower blastocyst rates and a different phenotype, characterised by an increased cell number and different actin and mitochondrial distribution patterns. These changes were reflected by an up-regulation of genes involved in cell division (MAP2K2; DHCR7), cell structure (LMNA; VIM; TUBB2B; TUBB3; TUBB4B) and mitochondrial activation

  8. Effect of Culture System on Developmental Competence, Cryosurvival and DNA-Fragmentation of In Vitro Bovine Blastocysts

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    Mahdi Hajian

    2011-01-01

    Full Text Available Background: This study investigated the effect of two in vitro embryo culture systems (co-culturesystem versus cell-free sequential-media on developmental competence, cryosurvival and DNAfragmentationof in vitro developed bovine blastocysts.Materials and Methods: Bovine presumptive zygotes were cultured in Ménézo's B2 (B2 plusvero-cells or sequential synthetic oviductal fluid (SOF for eight days. Subsequently, half of theexpanded blastocysts developed in both groups were vitrified, warmed within 30 minutes and postwarmingembryos along with their corresponding non-vitrified embryos were cultured for twoadditional days in the same medium used before vitrification. Embryo development, cryosurvivaland apoptosis were compared between the groups.Results: For non-vitrified embryos, culture in SOF significantly promoted the potency of embryosto develop into blastocysts compared with the co-culture system. The difference in post vitrificationsurvival rate of SOF blastocysts (83.3% was insignificant compared with co-culture (84.3%.However, while total cell number of warmed blastocysts in the co-culture system was significantlyhigher in the co-culture versus the sequential system (215.4 vs. 170.4, the quality of survived embryosin terms of hatching ability and apoptosis was adversely affected by co-culture compared with SOF(65.0% vs. 74.3%, and 13.5% vs. 10.0%, respectively; p<0.05.Conclusion: Although co-culture system may increase the viability of embryos followingcryopreservation, the potency and dynamics of blastocyst formation significantly increased withsequential media compared to the co-culture system which can compensate for the lower efficiency ofsequential media for vitrification/warming purposes.

  9. Ploidy of Bovine Nuclear Transfer Blastocysts Blastomere Donors

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    Booth, P J; VIUFF, D; THOMSEN, P D

    2000-01-01

    The higher rate of embryonic loss in nuclear transfer compared to in vitro produced embryos may be due to chromosome abnormalities that occur during preimplantation in vitro devel- opment. Because little is known about ploidy errors in nuclear transfer embryos, this was ex- amined using embryos...... cultured until day 7 at which time blastocyst nuclei were extracted and chromosome abnormalities were evaluated by fluorescent in situ hybridization using two probes that bind to the subcentromeric regions on chromosomes 6 and 7. In 16 nuclear transfer blastocysts generated from 5 donor embryos, 53.8 6 20...... comprised mainly triploid (8.2 6 10.3 [0–26.3]: SD [range]) and tetraploid (10.6 6 19.9 [0–54.9]) nuclei with other ploidy com- binations accounting for only 0.9 6 2.1 [0–2.1]% of deviant nuclei. The proportion of com- pletely normal nuclear transfer embryos was no less than those produced by in vitro...

  10. Vitrification of bovine matured oocytes and blastocysts in a paper container.

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    Paul, Ashit Kumar; Liang, Yuanyuan; Srirattana, Kanokwan; Nagai, Takashi; Parnpai, Rangsun

    2018-02-01

    In the present study, we aimed to determine the applicability of a paper container for the vitrification of in vitro matured (IVM) bovine oocytes. In experiment 1, IVM oocytes were exposed to vitrification solution (20% dimethylsulfoxide (DMSO), 20% ethylene glycol (EG), and 5 mol/L sucrose), using a two-step method, for 30 s; loaded onto either a paper container or Cryotop; and stored in liquid nitrogen. No significant difference (P container and Cryotop. In experiment 2, IVM oocytes were exposed to either a two- or three-step vitrification solution. The three-step vitrification solution was not significantly different from the two-step solution in terms of oocyte survival, cleavage and blastocyst rates. In experiment 3, in vitro produced blastocysts were graded according to the manual of the International Embryo Transfer Society (grades 1 and 2) and vitrified using the two- and three-step methods. For grade 2 blastocysts, the three-step method showed significantly higher (P < 0.05) survival and hatched blastocyst rates than the two-step method, whereas for grade 1 blastocysts, no significant difference was observed. In conclusion, the paper device and three-step technique are suitable for oocytes and embryo vitrification. © 2017 Japanese Society of Animal Science.

  11. Susceptibility of in vitro produced hatched bovine blastocysts to infection with bluetongue virus serotype 8

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    Vandaele Leen

    2011-01-01

    Full Text Available Abstract Bluetongue virus serotype 8 (BTV-8, which caused an epidemic in ruminants in central Western Europe in 2006 and 2007, seems to differ from other bluetongue serotypes in that it can spread transplacentally and has been associated with an increased incidence of abortion and other reproductive problems. For these reasons, and also because BTV-8 is threatening to spread to other parts of the world, there is a need for more information on the consequences of infection during pregnancy. The aim of the present study was to investigate whether hatched (i.e. zona pellucida-free in vitro produced bovine blastocysts at 8-9 days post insemination are susceptible to BTV-8 and whether such infection induces cell death as indicated by apoptosis. Exposure of hatched in vitro produced bovine blastocysts for 1 h to a medium containing 103.8 or 104.9 TCID50 of the virus resulted in active viral replication in between 25 and 100% of the cells at 72 h post exposure. The infected blastocysts also showed growth arrest as evidenced by lower total cell numbers and a significant level of cellular apoptosis. We conclude from this in vitro study that some of the reproductive problems that are reported when cattle herds are infected with BTV-8 may be attributed to direct infection of blastocysts and other early-stage embryos in utero.

  12. RNA-Seq analysis uncovers transcriptomic variations between morphologically similar in vivo- and in vitro-derived bovine blastocysts

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    Driver Ashley M

    2012-03-01

    Full Text Available Abstract Background A valuable tool for both research and industry, in vitro fertilization (IVF has applications range from gamete selection and preservation of traits to cloning. Although IVF has achieved worldwide use, with approximately 339,685 bovine embryos transferred in 2010 alone, there are still continuing difficulties with efficiency. It is rare to have more than 40% of fertilized in vitro cattle oocytes reach blastocyst stage by day 8 of culture, and pregnancy rates are reported as less than 45% for in vitro produced embryos. To investigate potential influences in-vitro fertilization (IVF has on embryonic development, this study compares in vivo- and in vitro-derived bovine blastocysts at a similar stage and quality grade (expanded, excellent quality to determine the degree of transcriptomic variation beyond morphology using RNA-Seq. Results A total of 26,906,451 and 38,184,547 fragments were sequenced for in vitro and in vivo embryo pools, respectively. We detected expression for a total of 17,634 genes, with 793 genes showing differential expression between the two embryo populations with false discovery rate (FDR Conclusions Thus, our results support that IVF may influence at the transcriptomic level and that morphology is limited in full characterization of bovine preimplantation embryos.

  13. A Method Based on Artificial Intelligence To Fully Automatize The Evaluation of Bovine Blastocyst Images.

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    Rocha, José Celso; Passalia, Felipe José; Matos, Felipe Delestro; Takahashi, Maria Beatriz; Ciniciato, Diego de Souza; Maserati, Marc Peter; Alves, Mayra Fernanda; de Almeida, Tamie Guibu; Cardoso, Bruna Lopes; Basso, Andrea Cristina; Nogueira, Marcelo Fábio Gouveia

    2017-08-09

    Morphological analysis is the standard method of assessing embryo quality; however, its inherent subjectivity tends to generate discrepancies among evaluators. Using genetic algorithms and artificial neural networks (ANNs), we developed a new method for embryo analysis that is more robust and reliable than standard methods. Bovine blastocysts produced in vitro were classified as grade 1 (excellent or good), 2 (fair), or 3 (poor) by three experienced embryologists according to the International Embryo Technology Society (IETS) standard. The images (n = 482) were subjected to automatic feature extraction, and the results were used as input for a supervised learning process. One part of the dataset (15%) was used for a blind test posterior to the fitting, for which the system had an accuracy of 76.4%. Interestingly, when the same embryologists evaluated a sub-sample (10%) of the dataset, there was only 54.0% agreement with the standard (mode for grades). However, when using the ANN to assess this sub-sample, there was 87.5% agreement with the modal values obtained by the evaluators. The presented methodology is covered by National Institute of Industrial Property (INPI) and World Intellectual Property Organization (WIPO) patents and is currently undergoing a commercial evaluation of its feasibility.

  14. Embryonic stem-like cells derived from in vitro produced bovine blastocysts

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    Erika Regina Leal de Freitas

    2011-06-01

    Full Text Available The aim of this work was to study the derivation of bovine embryonic stem-like (ES-like cells from the inner cell mass (ICM of in vitro produced blastocysts. The ICMs were mechanically isolated and six out of seventeen (35% ICMs could attach to a monolayer of murine embryonic fibroblasts (MEF. Ten days after, primary outgrowths were mechanically dissected into several small clumps and transferred to a new MEF layer. Cells were further propagated and passaged by physical dissociation over a 60 days period. The pluripotency of the bovine ES-like cells was confirmed by RT-PCR of Oct-4 and STAT-3 gene markers. The colonies were weakly stained for alkaline phosphatase and the mesoderm and endoderm differentiation gene markers such as GATA-4 and Flk-1, respectively, were not expressed. Embryoid bodies were spontaneously formed at the seventh passage. Results showed that bovine ES-like cells could be obtained and passaged by mechanical procedures from the fresh in vitro produced blastocysts.

  15. Proteomic analysis of bovine blastocoel fluid and blastocyst cells

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    Jensen, Pernille Linnert; Grøndahl, Marie Louise; Beck, Hans Christian

    2014-01-01

    by micromanipulation. From two independent replicates, 23 proteins were identified in the blastocoel fluid while 803 proteins were identified in the remaining cell material. The proteins were grouped into categories according to their gene ontology (GO) terms by which proteins involved in cell differentiation, cell...... proliferation, development, and reproduction could be derived. Proteins classified in these categories could be candidates for further functional studies to understand pluripotency and early mammalian development....

  16. Effects of High Hydrostatic Pressure on Expression Profiles of In Vitro Produced Vitrified Bovine Blastocysts

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    Jiang, Zongliang; Harrington, Patrick; Zhang, Ming; Marjani, Sadie L.; Park, Joonghoon; Kuo, Lynn; Pribenszky, Csaba; Tian, Xiuchun (Cindy)

    2016-01-01

    High hydrostatic pressure (HHP) has been used to pre-condition embryos before essential, yet potentially detrimental procedures such as cryopreservation. However, the mechanisms for HHP are poorly understood. We treated bovine blastocysts with three different HHP (40, 60 and 80 MPa) in combination with three recovery periods (0, 1 h, 2 h post HHP). Re-expansion rates were significantly higher at 40 and 60 but lower at 80 MPa after vitrification-warming in the treated groups than controls. Microarray analysis revealed 399 differentially expressed transcripts, representing 254 unique genes, among different groups. Gene ontology analysis indicated that HHP at 40 and 60 MPa promoted embryo competence through down-regulation of genes in cell death and apoptosis, and up-regulation of genes in RNA processing, cellular growth and proliferation. In contrast, 80 MPa up-regulated genes in apoptosis, and down-regulated protein folding and cell cycle-related genes. Moreover, gene expression was also influenced by the length of the recovery time after HHP. The significantly over-represented categories were apoptosis and cell death in the 1 h group, and protein folding, response to unfolded protein and cell cycle in the 2 h group compared to 0 h. Taken together, HHP promotes competence of vitrified bovine blastocysts through modest transcriptional changes. PMID:26883277

  17. In-straw cryoprotectant dilution of IVP bovine blastocysts vitrified in hand-pulled glass micropipettes.

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    Vieira, A D; Forell, F; Feltrin, C; Rodrigues, J L

    2007-06-01

    The aim of this study was to determine the influence of two ethylene glycol-based vitrification solutions on in vitro and in vivo survival after in-straw cryoprotectant dilution of vitrified in vitro-produced bovine embryos. Day-7 expanded blastocysts were selected according to diameter (> or = 180 microm) and osmotic characteristics and randomly assigned to one of three groups (i) VSa: vitrification in 40% EG+17.1% SUC+0.1% PVA; (ii) VSb: vitrification in 20% EG+20% DMSO; (iii) control: non-vitrified embryos. Vitrification was performed in hand-pulled glass micropipettes (GMP) and cryoprotectant dilution in 0.25 ml straws after warming in a plastic tube. Embryo viability was assessed by re-expansion and hatching rates after 72 h of IVC and by pregnancy rates after direct transfer of vitrified embryos. No differences in re-expansion rates were observed between vitrified groups after 24 h in culture (VSa=84.5%; VSb=94.8%). However, fewer VSa embryos (55.2%, Pstraw cryoprotectant dilution and direct embryo transfer.

  18. Effect of exogenous glutathione in medium fertilization to improve blastocyst rates of bovine embryos

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    E Triwulaninngsih

    2002-06-01

    Full Text Available Glutathione (C10H17N3O6S is a tripeptide (γ-Glu-Cys-Gly widespread in living organism. Glutathione (GSH at the 5 mM concentration increased the motility and fertility of frozen-thawed sperm. Intracellulair glutathione improved the cleavage rate and embryo development to the blastocyst rate. Research on in vitro embryos production through the implementation of GSH during IVF (in vitro fertilization on embryo development has been conducted at the Laboratorium Reproductive of Physiology, Research Institute for Animal Production. Ovaries of beef cattle as source of oocytes were collected from the slaughterhouse in a thermo flask with 350C PBS as medium and transported to the laboratory. The oocytes were fertilized in vitro with selected motile sperm using Percoll gradient (90 and 45%. Ten COCs (cumulus oocytes complexes were transfered to 44 μl of fertilization medium (mTALP was performed with either 0; 0.25; 0.50; 0.75 and 1.00 mM of glutathione as treatment A, B, C, D and E respectively, and inseminated with 2 μl of capacitated sperm and added 2 μl of heparin and 2 μl of PHE (consisting of 20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine. Incubation between sperm and oocytes in the 5% CO2 incubator and RH 90% in fertilization media (TALP for 20 hours. All zygotes were cultured in modification of CR1aa medium up to blastocyst and were fed serum 5 μl/50μl medium on day 6. Results of this experiment showed that the effect of concentration of glutathione (0, 0.25; 0.50; 0.75 and 1.00 mM on fertilization medium to the percentage of cleavage rates were 69.35 + 29.40; 79.07 + 13.17; 67.88 + 10.65; 98.10 + 3.30 and 82.62 + 24.19% not significant different (P>0.05 among treatments A, B, C, D dan E respectively.The precentages of morula and blastocyst for treatment D were 50.45 + 42.64% and 31.28 + 24.28%; while 36.55 + 24.13% and 17.74 + 17.86% for treatment E respectively.

  19. Increased cleavage and blastocyst rate in ewes treated with bovine somatotropin 5 days before the end of progestin-based estrous synchronization.

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    Montero-Pardo, A; Hernández-Cerón, J; Rojas-Maya, S; Valencia, J; Rodríguez-Cortez, A; Gutiérrez, C G

    2011-05-01

    Treatment with bovine somatotropin (bST) during estrous synchronization increased fertility and prolificacy in sheep. In the present study, a single dose of bST 5 days before the end of progestin treatment improved cleavage and embryo development. Stage of estrous cycle was synchronized in ewes (n=32) with progestin and superovulation was induced by use of FSH. Five days before the end of progestin treatment, ewes were randomly assigned to two groups: bST group (n=16) received a depot injection of 125 mg of bST sc (Lactotropina, Elanco, México) and the control group (n=16) received saline solution. Estrous was detected with rams fitted with an apron every 2 h and estrous sheep were mated every 8 h whilst in estrous. Embryos were recovered on Day 7 post mating, assessed microscopically and fixed in 4% paraformaldehyde. Cell number in blastocysts was counted after Hoechst 33342 staining. Plasma concentrations of IGF-I, insulin and progesterone were determined in eight sheep per group from the day of bST treatment to the day of embryo recovery. Cleavage rate, percentage of transferable embryos (transferable embryos/cleaved) and percentage of embryos reaching the blastocyst stage (blastocyst/cleaved) were compared between groups by logistic regression. IGF-I, insulin and progesterone plasma concentrations were analyzed by ANOVA for repeated measurements and cell number by ANOVA. Cleavage rate was greater (Psheep. Plasma concentrations of IGF-I and insulin were greater (Pprogesterone concentrations (P=0.5). It is concluded that bST injection 5 days before progestin removal increases cleavage rate and the proportion of embryos that reach the blastocyst stage. These effects are associated with an increase in IGF-I and insulin concentrations. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Effect of Hyaluronan on Developmental Competence and Quality of Oocytes and Obtained Blastocysts from In Vitro Maturation of Bovine Oocytes

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    Jolanta Opiela

    2014-01-01

    Full Text Available The objective of the present study was to evaluate the effect of hyaluronan (HA during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC, and obtained blastocysts. COCs were matured in vitro in control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P<0.001 was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P<0.01. Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higher Bax mRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure.

  1. Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization

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    Jin, Muzi; Wu, Asga; Dorzhin, Sergei; Yue, Qunhua; Ma, Yuzhen; Liu, Dongjun

    2012-01-01

    Although isolation and characterization of embryonic stem cells have been successful in cattle, maintenance of bovine embryonic stem cells in culture remains difficult. In this study, we compared different methods of cell passaging, feeder cell layers and medium conditions for bovine embryonic stem cell-like cells. We found that a murine embryonic fibroblast feeder layer is more suitable for embryonic stem cell-like cells than bovine embryonic fibroblasts. When murine embryonic fibroblasts we...

  2. Coasting, embryo development and outcomes of blastocyst transfer: a case-control study.

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    Talebi Chahvar, Solmas; Zosmer, Ariel; Caragia, Alina; Balestrini, Simona; Sabatini, Luca; Tranquilli, Andrea Luigi; Al-Shawaf, Talha

    2014-08-01

    This study compared the effect on blastocyst development and clinical outcome of coasting in women at increased risk of moderate-severe ovarian hyperstimulation syndrome (OHSS; n=389) with a control group matched for age and basal FSH that did not undergo coasting (n=386) in IVF/intracytoplasmic sperm injection (ICSI) cycles. The main outcome measures were rate of blastocyst development and live birth. More cycles progressed to the blastocyst stage in the coasted group (n=169) compared with the control group (n=83; 43.4% versus 21.5%; P<0.001). The biochemical pregnancy, clinical pregnancy and live birth rates were similar (46.5% versus 42.0%; 40.6% versus 37.8%; 31.6% versus 30.1%). The duration of coasting up to 4 days did not affect progression to blastocyst stage. The multivariate model showed that coasting (OR 1.73, P=0.004) and the number of oocytes retrieved (OR 1.17, P=0.001) were positively correlated with blastocyst formation. Coasting, a measure to reduce the risk of OHSS, does not impair blastocyst development or clinical outcome. Coasting should remain an effective measure to prevent OHSS. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  3. Numerical Chromosome Errors in Day 7 Somatic Nuclear Transfer Bovine Blastocysts

    DEFF Research Database (Denmark)

    Booth, Paul J.; VIUFF, Dorte; Tan, Shijian

    2002-01-01

    Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona-free manipulat......Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona...

  4. Cryotop vitrification for in vitro produced bovine and buffalo (Bubalus bubalis embryos at different stages of development

    Directory of Open Access Journals (Sweden)

    B. Gasparrini

    2010-02-01

    Full Text Available The aim of this study was to evaluate the possibility to vitrify in vitro produced (IVP buffalo and bovine embryos at different stages of development by an advanced version of the “minimal volume approaches”: the Cryotop method. In both experiments, the embryos were vitrified at the tight morula (TM, early blastocyst (eBl, blastocyst (Bl, expanded blastocyst (xBl and, only for buffalo, at the hatched blastocyst (hBl stage. After warming, the embryos were cultured in vitro for 24 hours. Stage of development affected the freezability of IVP embryos of both species with the highest embryo survival rates at advanced stages (xBl=76% and hBl=75% for buffalos and xBl=75% for bovine. These results suggest that Cryotop vitrification is an efficient method for buffalo and bovine IVP embryo cryopreservation.

  5. Blastocyst Development in a Single Medium Compared to Sequential Media: A Prospective Study With Sibling Oocytes.

    Science.gov (United States)

    Sfontouris, Ioannis A; Kolibianakis, Efstratios M; Lainas, George T; Petsas, George K; Tarlatzis, Basil C; Lainas, Trifon G

    2017-09-01

    The aim of the present study was to compare blastocyst formation rates after embryo culture in a single medium (Global) as compared to sequential media (ISM1/BlastAssist). In this prospective trial with sibling oocytes, 542 metaphase II (ΜΙΙ) oocytes from 31 women were randomly and equally divided to be fertilized and cultured to the blastocyst stage in either sequential media (ISM1/BlastAssist; n = 271 MII oocytes) or a single medium (Global; n = 271 MII oocytes). In both groups, embryos were cultured in an interrupted fashion with media changes on day 3. Embryo transfer was performed on day 5. Blastocyst formation rates on day 5 (61.7% ± 19.9% vs 37.0% ± 25.5%, P ISM1/BlastAssist, respectively. Fertilization rates, cleavage rates, and percentage of good quality embryos on day 3 were similar between Global and ISM1/BlastAssist, respectively. The percentages of good quality blastocysts (63.0% ± 24.8% vs 32.1% ± 37.2%, P ISM1/BlastAssist, respectively. In conclusion, culture in Global was associated with higher blastocyst formation rates compared to ISM1/BlastAssist, suggesting that the single medium may provide better support to the developing embryo.

  6. Infertility diagnosis has a significant impact on the transcriptome of developing blastocysts.

    Science.gov (United States)

    McCallie, Blair R; Parks, Jason C; Griffin, Darren K; Schoolcraft, William B; Katz-Jaffe, Mandy G

    2017-08-01

    Is the human blastocyst transcriptome associated with infertility diagnosis, specifically: polycystic ovaries (PCO), male factor (MF) and unexplained (UE)? The global blastocyst transcriptome was significantly altered in association with a PCO, MF and UE infertility diagnosis. Infertility diagnosis has an impact on the probability for a successful outcome following an IVF cycle. Limited information is known regarding the relationship between a specific infertility diagnosis and blastocyst transcription during preimplantation development. Blastocysts created during infertility treatment from patients with specific infertility diagnoses (PCO, MF and UE) were analyzed for global transcriptome compared to fertile donor oocyte blastocysts (control). Surplus cryopreserved blastocysts were donated with patient consent and institutional review board approval. Female patients were infertility diagnosis: PCO (n = 50), MF (n = 50), UE (n = 50) and fertile donor oocyte controls (n = 50). Pooled blastocysts were lysed for RNA isolation followed by microarray analysis using the SurePrint G3 Human Gene Expression Microarray. Validation was performed on significant genes of interest using real-time quantitative PCR (RT-qPCR). Transcription alterations were observed for all infertility etiologies compared to controls, resulting in differentially expressed genes: PCO = 869, MF = 348 and UE = 473 (P 2-fold). Functional annotation of biological and molecular processes revealed both similarities, as well as differences, across the infertility groups. All infertility etiologies displayed transcriptome alterations in signal transducer activity, receptor binding, reproduction, cell adhesion and response to stimulus. Blastocysts from PCO patients were also enriched for apoptotic genes while MF blastocysts displayed enrichment for genes involved in cancer processes. Blastocysts from couples with unexplained infertility displayed transcription alterations related to various disease states

  7. Effects of retinol on the in vitro development of Bos indicus embryos to blastocysts in two different culture systems.

    Science.gov (United States)

    Lima, P F; Oliveira, M A L; Gonçalves, P B D; Montagner, M M; Reichenbach, H-D; Weppert, M; Neto, C C C; Pina, V M R; Santos, M H B

    2004-10-01

    The objective of this study was to evaluate the effect of retinol on the in vitro development of early embryos of cultured Bos indicus (Expt 1) to the blastocyst stage in medium simplex of optimization (KSOM) or sintetic fluid of oviduct (SOF) or co-cultured (Expt 2) with an oviduct cell monolayer (OCM) in KSOM or SOF. A total of 3149 cumulus-oocyte complexes obtained by aspirating follicles (2-5 mm diameter) from ovaries of slaughtered animals were selected for IVM and incubated in TCM 199 supplemented with 25 mM HEPES at 39 degrees C in air with 5% CO(2) and maximum humidity for 24 h. In vitro fertilization (IVF) was performed in modified defined medium (mDM) medium. Eighteen hours after IVF, cumulus cells were removed and presumptive zygotes were randomly allocated to the experimental groups. Zygotes cultured (Expt 1) in KSOM + retinol, KSOM, SOF + retinol and SOF were incubated in maximum humidity at 39 degrees C, 5% CO(2), 5% O(2) and 90% N(2). Zygotes co-cultured (Expt 2) in KSOM + retinol + OCM, KSOM + OCM, SOF + retinol + OCM and SOF + OCM were incubated at 39 degrees C, 5% CO(2). In both experiments media were partially changed 48 h after IVF and unfertilized ova were removed. Afterwards embryos were kept in culture or co-culture for further 9 days. In Expt 1, blastocyst rates (day 7) were 14.6% (KSOM + retinol), 15.8% (KSOM), 16.4% (SOF + retinol) and 15.9% (SOF). In Expt 2, the blastocyst rates (day 7) were 25.4% (KSOM + retinol + OCM) 14.2% (KSOM + OCM), 24.3% (SOF + retinol + OCM) and 15.9% (SOF + OCM). The same influence profile of retinol was observed in the formation of the expanded (day 9) and hatched (day 11) blastocysts. The results obtained in Expt 2 demonstrated that the addition of 0.28 microg/ml retinol to the embryo culture media used in this study had a significant (p < 0.05) positive effect on bovine early embryonic development, under the conditions tested, and can be used to enhance in vitro embryo production.

  8. N, N-Dimethylglycine decreases oxidative stress and improves in vitro development of bovine embryos.

    Science.gov (United States)

    Takahashi, Toshikiyo; Sasaki, Kouya; Somfai, Tamas; Nagai, Takashi; Manabe, Noboru; Edashige, Keisuke

    2016-04-22

    The antioxidant effect of N, N-dimethylglycine (DMG) on in vitro-produced (IVP) bovine embryos was examined. After in vitro fertilization, presumptive zygotes were cultured with or without 0.1 μM DMG under different oxygen tensions. The percentage of embryos developing to the blastocyst stage was lowest under a 20% oxygen concentration without DMG, and it was significantly increased (P DMG significantly improved blastocyst development, which was nearly equal to that achieved under 5% oxygen without DMG. Furthermore, a tendentious increase (P = 0.06) in blastocyst cell numbers was observed when DMG was applied. In the second experiment, addition of H2O2 (0.5 mM) to the culture medium significantly (P DMG supplementation prevented this reduction. In conclusion, DMG enhanced the invitro development of IVP bovine embryos by acting as an antioxidant.

  9. In Vitro Maturation and Embryo Development to blastocyst Mouse Germinal Vesicle Oocytes after Vitrification

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    M Nikseresht

    2013-05-01

    Full Text Available Abstract Background & aim: Vitrification is a simple and ultra rapid technique for the conservation of fertility. Improving pregnancy rate associate with the use of cryopreserved oocytes would be an important advanced in human assisted reproductive technology (ART. The purpose of this study was to evaluate survival, oocytes maturation and embryo development to the blastocyst stage after vitrification of oocytes germinal vesicle-stage and multi stage Methods: In the present experimental study, germinal vesicle oocytes with or without cumulus cells were transferred to vitrification solution containing 30% (v/v ethylene glycol, 18% (w/v Ficoll-70, and 0.3 M sucrose, either by single step or in a step-wise way. After vitrification and storage in liquid nitrogen, the oocytes were thawed and washed twice in culture medium TCM119, and then subjected to in vitro maturation, fertilization, and culture. Data analysis was performed by using One-way variance and Tukey tests. Results: Oocytes survival, metaphase 2 stage oocyte maturation, fertilization and embryo formed blastocyst in vitrification methods multistage were significantly higher than the single step procedure (P<0/05 Conclusion: The Germinal vesicle stage oocytes vitrified with cumulus cells and stepwise procedure had positive effect on the survival, maturation and developmental rate on blastocyst compared to oocytes without cumulus cell and single step procedure. Key words: Germinal Vesicle Oocyte, Blastocyst, Vitrification, Ethylene glycol

  10. Bivariate analysis of basal serum anti-Mullerian hormone measurements and human blastocyst development after IVF

    LENUS (Irish Health Repository)

    Sills, E Scott

    2011-12-02

    Abstract Background To report on relationships among baseline serum anti-Müllerian hormone (AMH) measurements, blastocyst development and other selected embryology parameters observed in non-donor oocyte IVF cycles. Methods Pre-treatment AMH was measured in patients undergoing IVF (n = 79) and retrospectively correlated to in vitro embryo development noted during culture. Results Mean (+\\/- SD) age for study patients in this study group was 36.3 ± 4.0 (range = 28-45) yrs, and mean (+\\/- SD) terminal serum estradiol during IVF was 5929 +\\/- 4056 pmol\\/l. A moderate positive correlation (0.49; 95% CI 0.31 to 0.65) was noted between basal serum AMH and number of MII oocytes retrieved. Similarly, a moderate positive correlation (0.44) was observed between serum AMH and number of early cleavage-stage embryos (95% CI 0.24 to 0.61), suggesting a relationship between serum AMH and embryo development in IVF. Of note, serum AMH levels at baseline were significantly different for patients who did and did not undergo blastocyst transfer (15.6 vs. 10.9 pmol\\/l; p = 0.029). Conclusions While serum AMH has found increasing application as a predictor of ovarian reserve for patients prior to IVF, its roles to estimate in vitro embryo morphology and potential to advance to blastocyst stage have not been extensively investigated. These data suggest that baseline serum AMH determinations can help forecast blastocyst developmental during IVF. Serum AMH measured before treatment may assist patients, clinicians and embryologists as scheduling of embryo transfer is outlined. Additional studies are needed to confirm these correlations and to better define the role of baseline serum AMH level in the prediction of blastocyst formation.

  11. The 'GO' system--a novel method of microculture for in vitro development of mouse zygotes to the blastocyst stage.

    Science.gov (United States)

    Thouas, G A; Jones, G M; Trounson, A O

    2003-08-01

    A novel system of in vitro culture termed the 'glass oviduct' or 'GO' culture system is described. Mouse zygotes were cultured in pairs to the blastocyst stage in open-ended 1 microl glass capillaries. 'GO' culture supported the development of significantly more hatching or hatched blastocysts than did a standard microdroplet (10 zygotes per 20 microl) control culture (48.3 versus 3.3%, respectively). 'GO' bslastocysts contained significantly larger populations of cells (92+/-3 versus 75+/-3), and inner cell mass (25+/-1 versus 21+/-1) and trophectoderm (68+/-2 versus 53+/-3) subpopulations, compared with microdroplet-derived blastocysts. Before blastulation, 'GO'-derived morulae were found to contain significantly more cells than microdroplet-derived morulae (27+/-0.7 versus 14+/-0.5). After implantation, 'GO' blastocysts formed fetuses at a similar rate to microdroplet-derived blastocysts (55 versus 62%), but at a lower rate than blastocysts derived in vivo (80%). 'GO'- and microdroplet-derived fetuses were similar in wet weight to each other (0.412 and 0.415 g, respectively) but were heavier than fetuses derived from flushed blastocysts (0.390 g). An additional experiment investigated whether the beneficial effect of 'GO' culture was due to the significantly increased embryo density. Proportions of hatching or hatched blastocysts after 'GO' culture (50%) were higher than after standard microdroplet culture (7.6%), but were not different from culture in high embryo density microdroplets (20 zygotes per 10 microl; 42%). 'GO' blastocysts contained more cells (79.6+/-2.1) than did standard microdroplet-derived blastocysts (68.7+/-2.0), but were similar to high density microdroplet-derived blastocysts (85.8+/-2.7). Similarly, 'GO' blastocysts contained more trophectoderm cells (62.2+/-2.0) than did standard microdroplet-derived blastocysts (52.7+/-1.7), but were similar to the high density microdroplet blastocysts (68.8+/-2.5). Numbers of inner cell mass cells ('GO

  12. Development of buffalo (Bubalus bubalis embryonic stem cell lines from somatic cell nuclear transferred blastocysts

    Directory of Open Access Journals (Sweden)

    Syed Mohmad Shah

    2015-11-01

    Full Text Available We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

  13. Cytotoxic Effects of Dillapiole on Embryonic Development of Mouse Blastocysts in Vitro and in Vivo

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    Wen-Hsiung Chan

    2014-06-01

    Full Text Available We examined the cytotoxic effects of dillapiole, a phenylpropanoid with antileishmanial, anti-inflammatory, antifungal, and acaricidal activities, on the blastocyst stage of mouse embryos, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation via embryo transfer. Blastocysts treated with 2.5–10 μM dillapiole exhibited a significant increase in apoptosis and corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with dillapiole were lower than those of their control counterparts. Moreover, in vitro treatment with 2.5–10 μM dillapiole was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that dillapiole induces apoptosis and retards early post-implantation development, both in vitro and in vivo. However, the extent to which this organic compound exerts teratogenic effects on early human development is not known at present. Further studies are required to establish effective protection strategies against the cytotoxic effects of dillapiole.

  14. The effect of prefreezing the diluent portion of the straw in a step-wise vitrification process using ethylene glycol and polyvinylpyrrolidone to preserve bovine blastocysts.

    Science.gov (United States)

    Mtango, N R; Varisanga, M D; Dong, Y J; Otoi, T; Suzuki, T

    2001-03-01

    A total of 678 bovine blastocysts, which had been produced by in vitro maturation, fertilization, and culture, were placed into plastic straws and were vitrified in various solutions of ethylene glycol (EG) + polyvinylpyrrolidone (PVP). Part of the straw was loaded with TCM199 medium + 0.3 M trehalose as a diluent; the diluent portions of the straw were prefrozen to either -30 or -196 degrees C. Then, the embryos suspended in the vitrification solution were pipetted into the balance of the straw and vitrified by direct immersion into liquid nitrogen. For thawing, the straws were warmed for 3 s in air and 20 s in a water bath at 39 degrees C and then agitated to mix the diluent and cryoprotectant solution for 5 min followed by culture in TCM199 + 10% FCS + 5 + microg/ml insulin + 50 microg/ml gentamycin sulfate for 72 h. Variables that were examined were the time of exposure to EG prior to vitrification, the PVP concentration, and the temperature of exposure to EG + PVP prior to vitrification. Survival and hatching rates of the blastocysts exposed to 40% EG in four steps at 4 degrees C were higher than those of embryos exposed in two steps (81.3 +/- 4.3% and 80.2 +/- 3.4% vs 67.6 +/- 4.5% and 71.5 +/- 4.7%, respectively; P straws do favor developmental competence of in vitro produced embryos.

  15. Bovine conceptus of Bos indicus produced by somatic cell nuclear transfer and parthenogenesis present morphological variations since the blastocyst stage

    Directory of Open Access Journals (Sweden)

    F.D. Oliveira

    2015-12-01

    Full Text Available In cattle, embryo development is characterized by the appearance of two distinct cell layers, the trophectoderm and the inner cell mass. The latter will undergo differentiation to form the embryonic disc consisting of the epiblast and hypoblast. The aim of this study was to ultrastructurally characterize the bovine embryo from different in vitro production techniques, with emphasis on trophectoderm and inner cell mass cells. Bovine embryos on day 7 (conception = D1 of pregnancy, derived via in vitro production techniques, were fixed for light and transmission electron microscopy processing. Results suggested that embryos produced by nuclear transfer of somatic cells and parthenogenesis showed significant changes in macroscopic and microscopic structure. Size was reduced, and the inner cell mass had no defined shape. Furthermore, organelles responsible for the absorption processes, communication, growth, and cellular metabolism were fewer and had changes in shape, when compared to results in embryos produced by in vitrofertilization. We concluded that embryos produced by parthenogenesis and SCNT exhibit morphological differences when compared with IVF embryos, such as undeveloped blastocoel, poorly defined distribution of ICM, and morphological differences in organelles.

  16. Melatonin promotes the in vitro development of pronuclear embryos and increases the efficiency of blastocyst implantation in murine.

    Science.gov (United States)

    Wang, Feng; Tian, XiuZhi; Zhang, Lu; Tan, DunXian; Reiter, Russel J; Liu, GuoShi

    2013-10-01

    When a defect occurs in the in vitro development of a pronuclear embryo, the interruption of the subsequent implantation limits the success of assisted conception. This common problem remains to be solved. In this study, we observed that melatonin at its physiological concentration (10(-7)  m) significantly promoted the in vitro development of murine pronuclear embryos. This was indicated by the increased blastocyst rate, hatching blastocyst rate, and blastocyst cell number with melatonin treatment. In addition, when these blastocysts were implanted into female recipient mice, the pregnancy rates (95.0% versus control 67.8%), litter sizes (4.1 pups/litter versus control 2.7 pups/litter), and postnatal survival rates of offspring (96.84% versus control 81.24%) were significantly improved compared with their non-melatonin-treated counterparts. Mechanistic studies revealed that melatonin treatment upregulates gene expression of the antioxidant enzyme, superoxide dismutase (SOD), and the anti-apoptotic factor bcl-2 while downregulating the expression of pro-apoptotic genes p53 and caspase-3. Due to these changes, melatonin treatment reduces ROS production and cellular apoptosis during in vitro embryo development and improves the quality of blastocysts. The implantation of blastocysts with higher quality leads to more healthy offspring and increased pup survival. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems.

    Science.gov (United States)

    Somfai, Tamás; Inaba, Yasushi; Aikawa, Yoshio; Ohtake, Masaki; Kobayashi, Shuji; Konishi, Kazuyuki; Nagai, Takashi; Imai, Kei

    2010-12-01

    The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O2 compared to 5% O2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O2 tension, whereas IVD101 supported blastocyst formation only under low O2 levels but enhanced the proliferation of ICM cells.

  18. Rhein Induces Oxidative Stress and Apoptosis in Mouse Blastocysts and Has Immunotoxic Effects during Embryonic Development

    Directory of Open Access Journals (Sweden)

    Chien-Hsun Huang

    2017-09-01

    Full Text Available Rhein, a glucoside chemical compound found in a traditional Chinese medicine derived from the roots of rhubarb, induces cell apoptosis and is considered to have high potential as an antitumor drug. Several previous studies showed that rhein can inhibit cell proliferation and trigger mitochondria-related or endoplasmic reticulum (ER stress-dependent apoptotic processes. However, the side effects of rhein on pre- and post-implantation embryonic development remain unclear. Here, we show that rhein has cytotoxic effects on blastocyst-stage mouse embryos and induces oxidative stress and immunotoxicity in mouse fetuses. Blastocysts incubated with 5–20 μM rhein showed significant cell apoptosis, as well as decreases in their inner cell mass cell numbers and total cell numbers. An in vitro development assay showed that rhein affected the developmental potentials of both pre- and post-implantation embryos. Incubation of blastocysts with 5–20 μM rhein was associated with increased resorption of post-implantation embryos and decreased fetal weight in an embryo transfer assay. Importantly, in an in vivo model, intravenous injection of dams with rhein (1, 3, and 5 mg/kg body weight/day for four days resulted in apoptosis of blastocyst-stage embryos, early embryonic developmental injury, and decreased fetal weight. Intravenous injection of dams with 5 mg/kg body weight/day rhein significantly increased the total reactive oxygen species (ROS content of fetuses and the transcription levels of antioxidant proteins in fetal livers. Additional work showed that rhein induced apoptosis through ROS generation, and that prevention of apoptotic processes effectively rescued the rhein-induced injury effects on embryonic development. Finally, the transcription levels of the innate-immunity related genes, CXCL1, IL-1 β and IL-8, were down-regulated in the fetuses of dams that received intravenous injections of rhein. These results collectively show that rhein has

  19. Blastomere nucleation: Predictive factors and influence of blastomere with no apparent nuclei on blastocyst development and implantation.

    Science.gov (United States)

    Setti, Amanda Souza; Figueira, Rita Cássia Sávio; Braga, Daniela Paes de Almeida Ferreira; Iaconelli, Assumpto; Borges, Edson

    2018-06-01

    To investigate whether embryos presenting blastomere(s) with no apparent nucleus (BNAN) on days 2 and 3 are more likely to fail to develop into blastocysts, hatch and implant. A total of 5705 zygotes obtained from 743 intracytoplasmic sperm injection (ICSI) cycles were analyzed. The presence and incidence of BNAN on days 2 and 3 of embryo development were recorded and then associated with ICSI outcomes. The occurrence of BNAN on day 2 of embryo development was determinant to the decreased odds of blastocyst formation (OR: 0.57, CI: 0.50-0.65), quality (OR: 0.56, CI: 0.43-0.73) and hatching status (OR: 0.66, CI: 0.50-0.87). The presence of BNAN on day 3 of embryo development was determinant to the decreased odds of blastocyst formation (OR: 0.67, CI: 0.58-0.78) and hatching status (OR: 0.61, CI: 0.45-0.83). The occurrence of BNAN on day 2 of embryo development was determinant to the decreased odds of blastocyst implantation (OR: 0.50, CI: 0.27-0.94). The presence of BNAN on day 2 or day 3 reduces development to blastocyst stage, hatching and implantation. Careful nuclear observation, taking into account the absence of blastomere nucleus, should be part of the strategies used for embryo selection.

  20. Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media.

    Science.gov (United States)

    Hennings, Justin M; Zimmer, Randall L; Nabli, Henda; Davis, J Wade; Sutovsky, Peter; Sutovsky, Miriam; Sharpe-Timms, Kathy L

    2016-03-01

    Validate single versus sequential culture media for murine embryo development. Prospective laboratory experiment. Assisted Reproduction Laboratory. Murine embryos. Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization. On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4',6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality. Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = culture. Human embryo studies are needed. © The Author(s) 2015.

  1. Effects of embryo-derived exosomes on the development of bovine cloned embryos.

    Directory of Open Access Journals (Sweden)

    Pengxiang Qu

    Full Text Available The developmental competence of in vitro cultured (IVC embryos is markedly lower than that of their in vivo counterparts, suggesting the need for optimization of IVC protocols. Embryo culture medium is routinely replaced three days after initial culture in bovine, however, whether this protocol is superior to continuous nonrenewal culture method under current conditions remains unclear. Using bovine somatic cell nuclear transfer (SCNT embryos as the model, our results showed that compared with routine renewal treatment, nonrenewal culture system significantly improved blastocyst formation, blastocyst quality (increased total cell number, decreased stress and apoptosis, enhanced Oct-4 expression and ratio of ICM/TE, as well as following development to term. Existence and function of SCNT embryo-derived exosomes were then investigated to reveal the cause of impaired development induced by culture medium replacement. Exosomes were successfully isolated through differential centrifugation and identified by both electron microscopy and immunostaining against exosomal membrane marker CD9. Supplementation of extracted exosomes into freshly renewed medium significantly rescued not only blastocyst formation and quality (in vitro development, but also following growth to term (in vivo development. Notably, ratio of ICM/TE and calving rate were enhanced to a similar level as that in nonrenewal group. In conclusion, our results for the first time indicate that 1: bovine SCNT embryos can secrete exosomes into chemically defined culture medium during IVC; 2: secreted exosomes are essential for SCNT blastocyst formation, blastocyst quality, and following development to term; 3: removal of exosomes induced by culture medium replacement impairs SCNT embryo development, which can be avoided by nonrenewal culture procedure or markedly recovered by exosome supplementation.

  2. Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination.

    Science.gov (United States)

    Jeong, Young-Hee; Kim, Yeong Ji; Kim, Eun Young; Kim, Se Eun; Kim, Jiwoo; Park, Min Jee; Lee, Hong-Gu; Park, Se Pill; Kang, Man-Jong

    2016-06-01

    Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine β-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the β-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine β-casein gene.

  3. Effects of N, N-dimethylglycine on the development of in vitro produced bovine embryos.

    Science.gov (United States)

    Takahashi, Toshikiyo; Itoh, Ryu; Nagai, Takashi

    2009-06-01

    This study investigated the effects of N, N-Dimethylglycine (DMG) on the development of in vitro produced (IVP) bovine embryos. IVP embryos were obtained by in vitro fertilization of in vitro matured oocytes for 6 h. In Experiment 1, IVP embryos were cultured in mSOFaa supplemented with bovine serum albumin but without glucose (SOF1) for 4 days, transferred to mSOFaa (with 5% fetal bovine serum and 1.5 mM glucose; SOF2) supplemented with 0 (control), 0.1,1 or 10 microM DMG and cultured for an additional 7 days (11 days in total) to assess their development in vitro. When cultured in the medium with 0.1 microM DMG, a significantly higher number of IVP embryos developed to the blastocyst and hatched blastocyst stages (40.3 and 40.8%, respectively) compared with the other groups (18.7-31.0% and 15.0-28.7%, respectively; PDMG for 4 days, transferred to SOF2 with or without 0.1 microM DMG and further cultured as in Experiment 1; DMG was added to either SOF1 or SOF2 and to both of them to assess its exposure effects on embryo development. When cultured continuously with DMG for 11 days, significantly higher rates of IVP embryos developed into blastocyst and hatched blastocyst stages (39.0 and 47.7%, respectively) compared with the other groups (31.0-32.2% and 29.5-31.0%, respectively; PDMG to IVC medium after 7 days of IVC. When DMG was added to IVC medium, the ratio of embryos developed to advanced developmental stages (No. of embryos developed to the blastocyst and expanded blastocyst stages/No. of embryos developed to the morula stage) was 28.7% (86/3) and 7 times higher than that of those cultured without DMG, 4.0% (52/13). These results suggest that addition of 0.1 microM DMG to mSOFaa during IVC of IVP bovine embryos has a promoting effect on their development.

  4. Bivariate analysis of basal serum anti-Müllerian hormone measurements and human blastocyst development after IVF

    Directory of Open Access Journals (Sweden)

    Sills E Scott

    2011-12-01

    Full Text Available Abstract Background To report on relationships among baseline serum anti-Müllerian hormone (AMH measurements, blastocyst development and other selected embryology parameters observed in non-donor oocyte IVF cycles. Methods Pre-treatment AMH was measured in patients undergoing IVF (n = 79 and retrospectively correlated to in vitro embryo development noted during culture. Results Mean (+/- SD age for study patients in this study group was 36.3 ± 4.0 (range = 28-45 yrs, and mean (+/- SD terminal serum estradiol during IVF was 5929 +/- 4056 pmol/l. A moderate positive correlation (0.49; 95% CI 0.31 to 0.65 was noted between basal serum AMH and number of MII oocytes retrieved. Similarly, a moderate positive correlation (0.44 was observed between serum AMH and number of early cleavage-stage embryos (95% CI 0.24 to 0.61, suggesting a relationship between serum AMH and embryo development in IVF. Of note, serum AMH levels at baseline were significantly different for patients who did and did not undergo blastocyst transfer (15.6 vs. 10.9 pmol/l; p = 0.029. Conclusions While serum AMH has found increasing application as a predictor of ovarian reserve for patients prior to IVF, its roles to estimate in vitro embryo morphology and potential to advance to blastocyst stage have not been extensively investigated. These data suggest that baseline serum AMH determinations can help forecast blastocyst developmental during IVF. Serum AMH measured before treatment may assist patients, clinicians and embryologists as scheduling of embryo transfer is outlined. Additional studies are needed to confirm these correlations and to better define the role of baseline serum AMH level in the prediction of blastocyst formation.

  5. Function of JARID2 in bovines during early embryonic development

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    Yao Fu

    2017-12-01

    Full Text Available Histone lysine modifications are important epigenetic modifications in early embryonic development. JARID2, which is a member of the jumonji demethylase protein family, is a regulator of early embryonic development and can regulate mouse development and embryonic stem cell (ESC differentiation by modifying histone lysines. JARID2 can affect early embryonic development by regulating the methylation level of H3K27me3, which is closely related to normal early embryonic development. To investigate the expression pattern of JARID2 and the effect of JARID2-induced H3K27 methylation in bovine oocytes and early embryonic stages, JARID2 mRNA expression and localization were detected in bovine oocytes and early embryos via qRT-PCR and immunofluorescence in the present study. The results showed that JARID2 is highly expressed in the germinal vesicle (GV, MII, 2-cell, 4-cell, 8-cell, 16-cell and blastocyst stages, but the relative expression level of JARID2 in bovine GV oocytes is significantly lower than that at other oocyte/embryonic stages (p < 0.05, and JARID2 is expressed primarily in the nucleus. We next detected the mRNA expression levels of embryonic development-related genes (OCT4, SOX2 and c-myc after JARID2 knockdown through JARID2-2830-siRNA microinjection to investigate the molecularpathwayunderlying the regulation of H3K27me3 by JARID2 during early embryonic development. The results showed that the relative expression levels of these genes in 2-cell embryos weresignificantly higher than those in the blastocyst stage, and expression levels were significantly increased after JARID2 knockdown. In summary, the present study identified the expression pattern of JARID2 in bovine oocytes and at each early embryonic stage, and the results suggest that JARID2 plays a key role in early embryonic development by regulating the expression of OCT4, SOX2 and c-myc via modification of H3K27me3 expression. This work provides new data for improvements in the

  6. Fusion of blastomeres in mouse embryos under the action of femtosecond laser radiation. Efficiency of blastocyst formation and embryo development

    Energy Technology Data Exchange (ETDEWEB)

    Osychenko, A A; Zalesskii, A D; Krivokharchenko, A S; Zhakhbazyan, A K; Nadtochenko, V A [N N Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow (Russian Federation); Ryabova, A V [A M Prokhorov General Physics Institute, Russian Academy of Sciences, Moscow (Russian Federation)

    2015-05-31

    Using the method of femtosecond laser surgery we study the fusion of two-cell mouse embryos under the action of tightly focused femtosecond laser radiation with the fusion efficiency reaching 60%. The detailed statistical analysis of the efficiency of blastomere fusion and development of the embryo up to the blastocyst stage after exposure of the embryos from different mice to a femtosecond pulse is presented. It is shown that the efficiency of blastocyst formation essentially depends on the biological characteristics of the embryo, namely, the strain and age of the donor mouse. The possibility of obtaining hexaploid embryonal cells using the methods of femtosecond laser surgery is demonstrated. (extreme light fields and their applications)

  7. Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos

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    Daniel R. Arnold

    2015-07-01

    Full Text Available Abstract In vitro production (IVP of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS. Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst. Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05. Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05, 16-cell (P<0.05 and morula (P<0.05 stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01. Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation.

  8. mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

    Science.gov (United States)

    Kropp, Jenna; Khatib, Hasan

    2015-01-01

    In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

  9. The effect of IVM and IVC media on in vitro development of bovine embryos

    Directory of Open Access Journals (Sweden)

    E.T Mergawati

    2000-12-01

    Full Text Available The purpose of this study was to examine the effect of medium combination of IVM and IVC on the in vitro development of bovine embryos. The study involved 4 groups in a 2 (IVM medium x 2 (IVC medium factorial in a randomized block design. Each group was replicated for 5 times. The treatments were as follows: TCM-199/CR1aa (T1; TCM-199/SOF (T2; B- 199/CR1aa (T3 and B-199/SOF (T4. Oocytes were aspirated from ovaries collected at local abattoirs using aspiration medium of PBS supplemented with 3% FCS and 0.1% Penicillin and Streptomycin. The oocytes were matured in medium of TCM-199 or B-199 supplemented with 10% FCS, hormones: 10μg/ml FSH+ 10μg/ml hCG+ 1μg/ml Estradiol. Maturation was maintained at 37oC for 22 hours in 5% CO2 incubator with high humidity. A method of BRACKETT & Oliphant (BO was used to fertilize the matured oocytes. The fertilization was incubated for 7 hours in the 5% CO2 incubator. Two culture media of CR1aa or SOF/AA/BSA were used to develop the fertilized oocytes undergo to morula and blastocyst embryos. The findings showed that the proportion of oocytes cleaved and formation of blastocysts were affected significantly by a combination of IVM and IVC media (P<0.05. A combination of B-199/SOF (T4 resulted in a higher blastocyst rate (32% than others (T3= 29%; T2=T1= 23%. This study suggests that either SOF/AA/BSA or CR1aa has similar competence in development of bovine embryos in vitro.

  10. Effect of embryo density on in vitro development and gene expression in bovine in vitro-fertilized embryos cultured in a microwell system.

    Science.gov (United States)

    Sugimura, Satoshi; Akai, Tomonori; Hashiyada, Yutaka; Aikawa, Yoshio; Ohtake, Masaki; Matsuda, Hideo; Kobayashi, Shuji; Kobayashi, Eiji; Konishi, Kazuyuki; Imai, Kei

    2013-01-01

    To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 μl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density.

  11. Kinetics of early in vitro development of bovine in vivo- and in vitro-derived zygotes produced and/or cultured in chemically defined or serum-containing media

    DEFF Research Database (Denmark)

    Holm, P; Booth, P J; Callesen, H

    2002-01-01

    The kinetics of the in vitro development of early embryos from bovine zygotes derived in vitro and in vitro were compared, investigating the effect of serum during in vitro maturation and fertilization (IVM-IVF) and in culture. Zygotes were collected from superovulated heifers or produced in vitro...... to the compact morula or blastocyst stages (87% versus 47-54 respectively; P

  12. Quality of porcine blastocysts produced in vitro in the presence of absence of GH

    NARCIS (Netherlands)

    Kidson, A.; Rubio-Pomar, F.J.; Knegsel, van A.; Tol, van H.T.A.; Hazeleger, W.; Ducro-Steverink, D.W.B.; Colenbrander, B.; Dieleman, S.J.; Bevers, M.M.

    2004-01-01

    GH receptor (GHR) mRNA is expressed in bovine in vitro produced embryos up to the blastocyst stage and GH improves the quality of bovine embryos by increasing blastocyst cell numbers and reducing the incidence of apoptosis as evaluated by DNA strand-break labelling. Porcine in vitro produced

  13. Gene expression of bovine embryos developing at the air-liquid interface on oviductal epithelial cells (ALI-BOEC).

    Science.gov (United States)

    van der Weijden, Vera A; Chen, Shuai; Bauersachs, Stefan; Ulbrich, Susanne E; Schoen, Jennifer

    2017-11-25

    We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array™ on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.

  14. Characterization of the onset of embryonic control and early development in the bovine embryo

    International Nuclear Information System (INIS)

    Barnes, F.L.

    1988-01-01

    Bovine embryos were used to determine if morphological and molecular features of early development are similar to in vivo recovered bovine embryos and to determine at what level early bovine development is regulated. Radiolabeling of IVP embryos and in vivo recovered embryos with 35 S-methionine for SDS-polyacrylamide gel electrophoresis reveals that these embryos are equivalent. Few differences in protein profiles are observed between 1- and early 4-cell embryos. A change in protein profiles begins at the mid 4-cell stage and continues into the 8-cell stage. Few differences in protein profiles are observed between 1- and early 4-cell embryos. A change in protein profiles begins at the mid 4-cell stage and continues into the 8-cell stage. Few differences in protein profiles are observed between late 8-cells and morulae. This transition is α-amanitin sensitive therefore due to de novo embryonic transcription. Embryonic transcription is partially responsible for terminating the post-transcriptionally regulated period of early bovine development. Argentophillic nucleolar organizing regions (Ag-NORs) indicate onset of nucleolar activation. Ag-NORs were absent in 2- and 4-cell IVP embryos and rarely occurred in 8-cell IVP embryos cultured in vitro. IVP 1- and 2-cell embryos cultured to blastocysts in sheep oviducts demonstrated Ag-NORs. Thus the lack of nucleolar activation of IVP embryos cultured in vitro is culture induced between the 2- and 8-cell stage

  15. In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.

    Science.gov (United States)

    Gerger, R P C; Ribeiro, E S; Forell, F; Bertolini, L R; Rodrigues, J L; Ambrósio, C E; Miglino, M A; Mezzalira, A; Bertolini, M

    2010-02-18

    The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

  16. Effect of the association of IGF-I, IGF-II, bFGF, TGF-beta1, GM-CSF, and LIF on the development of bovine embryos produced in vitro.

    Science.gov (United States)

    Neira, J A; Tainturier, D; Peña, M A; Martal, J

    2010-03-15

    This study examined the influence of the following growth factors and cytokines on early embryonic development: insulin-like growth factors I and II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-beta), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Synthetic oviduct fluid (SOF) was used as the culture medium. We studied the development of bovine embryos produced in vitro and cultured until Day 9 after fertilization. TGF-beta1, bFGF, GM-CSF, and LIF used on their own significantly improved the yield of hatched blastocysts. IGF-I, bFGF, TGF-beta1, GM-CSF, and LIF significantly accelerated embryonic development, especially the change from the expanded blastocyst to hatched blastocyst stages. Use of a combination of these growth factors and cytokines (GF-CYK) in SOF medium produced higher percentages of blastocysts and hatched blastocysts than did use of SOF alone (45% and 22% vs. 24% and 12%; PGM-CSF, produces similar results to 10% fetal calf serum for the development of in vitro-produced bovine embryos. This entirely synthetic method of embryo culture has undeniable advantages for the biosecurity of embryo transfer. Copyright 2010 Elsevier Inc. All rights reserved.

  17. Bovine rotavirus pentavalent vaccine development in India.

    Science.gov (United States)

    Zade, Jagdish K; Kulkarni, Prasad S; Desai, Sajjad A; Sabale, Rajendra N; Naik, Sameer P; Dhere, Rajeev M

    2014-08-11

    A bovine rotavirus pentavalent vaccine (BRV-PV) containing rotavirus human-bovine (UK) reassortant strains of serotype G1, G2, G3, G4 and G9 has been developed by the Serum Institute of India Ltd, in collaboration with the National Institute of Allergy and Infectious Diseases (NIAID), USA. The vaccine underwent animal toxicity studies and Phase I and II studies in adults, toddlers and infants. It has been found safe and immunogenic and will undergo a large Phase III study to assess efficacy against severe rotavirus gastroenteritis. Copyright © 2014. Published by Elsevier Ltd.

  18. Transfer of spontaneously hatching or hatched blastocyst yields better pregnancy rates than expanded blastocyst transfer

    Directory of Open Access Journals (Sweden)

    Natachandra M Chimote

    2013-01-01

    Full Text Available Context: Blastocyst stage embryo transfer (ET has become routine practice in recent years. However, probably due to limitations of assisted hatching techniques, expanded blastocyst transfer (EBT is still the preferred mode. Inexplicably, not much consideration has been given to spontaneously hatching/hatched blastocyst transfer (SHBT. Aim: This study aimed to investigate developmental potential of spontaneously hatching/hatched blastocyst against EBT in in vitro fertilization (IVF cycles. Settings and Design: Prospective study of 146 women undergoing their first IVF- ET cycle. SUBJECTS AND Methods: On the basis of blastocyst status, women were classified into SHBT and EBT groups. Intracytoplasmic sperm injection cycles were excluded to remove male factor bias. Implantation rate (IR, clinical pregnancy rate, and live birth rate were the main outcome measures. Statistical Analysis: Graph-pad Prism 5 statistical package. Results: SHBT group showed significantly higher blastocyst formation rate (53.3 ± 17.5 vs. 43.1 ± 14.5%, P = 0.0098, top-quality blastocysts (71.8 vs. 53.7%, P = 0.0436, IR (43.6 vs. 27.9%, P = 0.0408, pregnancy rate (59.4 vs. 45.1%, P = 0.0173, and live birth rate (36.8 vs. 22.8%, P = 0.003 compared to EBT group. Multiple pregnancy rates remained comparable between the two groups. Implantation correlated strongly with top-quality blastocysts (Pearson, r = 0.4441 in SHBT group, while the correlation was nonsignificant in EBT group. Conclusion: Extending culture of expanded blastocysts by a few hours to allow transfer of spontaneously hatching/hatched blastocysts gives higher implantation and pregnancy rates with no added risk of multiple gestations. Spontaneously hatching/hatched blastocysts have a better potential to implant and develop into a positive pregnancy.

  19. Tumor necrosis factor alpha inhibits in vitro bovine embryo development through a prostaglandin mediated mechanism

    Directory of Open Access Journals (Sweden)

    Jackson Lauren R

    2012-03-01

    Full Text Available Abstract Mastitis or other infectious diseases have been related to reduced fertility in cattle. Inflammatory cytokines such as tumor necrosis factor α (TNFα are released in response to infection and may have negative effects on embryo development. In the current study the effect of exposure to TNFα on the development of in vitro fertilized bovine embryos was examined. Indomethacin, a prostaglandin synthesis inhibitor, was used to determine if blockade of prostaglandin synthesis would alter the effects of TNFα. Ovaries were obtained from a local abattoir and immature COC were isolated from 2-10 mm follicles, in vitro matured and fertilized. After fertilization, groups of presumptive zygotes were randomly placed into either control development medium, medium containing 25 ng/mL TNFα or medium containing 25 ng/mL TNFα plus 1 μg/mL indomethacin. The proportion of blastocysts formed was assessed at day 7 of culture. Fewer embryos exposed to TNFα alone reached the blastocyst stage (17.5 ± 2.4%, P

  20. mRNA Fragments in In-Vitro Culture Media are Associated with Bovine Preimplantation Embryonic Development

    Directory of Open Access Journals (Sweden)

    Jenna eKropp

    2015-08-01

    Full Text Available In vitro production (IVP systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerated conditioned media. Differential expression was confirmed by quantitative real-time PCR for

  1. The effects of melatonin on bovine uniparental embryos development in vitro and the hormone secretion of COCs

    Directory of Open Access Journals (Sweden)

    Shujuan Wang

    2017-07-01

    Full Text Available Melatonin is a unique multifunctional molecule that mediates reproductive functions in animals. In this study, we investigated the effects of melatonin on bovine parthenogenetic and androgenetic embryonic development, oocyte maturation, the reactive oxygen species (ROS levels in parthenogenetic and androgenetic embryos and cumulus—oocyte complexes (COCs hormone secretion with melatonin supplementation at four concentrations (0, 10, 20, and 30 pmol/mL, respectively. The results showed that melatonin significantly promoted the rates of bovine parthenogenetic and androgenetic embryonic cleavage and morula and blastocysts development (P < 0.05. The rate of cleavage was higher in the androgenetic embryo than that in the parthenogenetic embryo. Compared with the parthenogenetic embryos, the androgenetic embryos had a poor developmental competence from morula to blastocyst stage. Moreover, the levels of ROS were significantly lower in the parthenogenetic and androgenetic embryoes with melatonin-treated group than that of the control group (P < 0.05. Melatonin supplemented significantly increased the maturation rate of oocyte in vitro (P < 0.05. More importantly, melatonin significantly promoted the secretion of progesterone and estradiol by COCs (P < 0.05. To reveal the regulatory mechanism of melatonin on steroids synthesis, we found that steroidogenic genes (CYP11A1, CYP19A1 and StAR were upregulated, suggesting that melatonin regulated estradiol and progesterone secretion through mediating the expression of steroidogenic genes (CYP11A1, CYP19A1 and StAR. In addition, MT1 and MT2 were identified in bovine early parthenogenetic and androgenetic embryos using western blot. It could be concluded that melatonin had beneficial effects on bovine oocyte in vitro maturation, COC hormone secretion, early development of subsequent parthenogenetic and androgenetic embryos. It is inferred that melatonin could be used to enhance the efficiency of in

  2. Protective effects of resveratrol on ethanol-induced apoptosis in embryonic stem cells and disruption of embryonic development in mouse blastocysts

    International Nuclear Information System (INIS)

    Huang, L.-H.; Shiao, N.-H.; Hsuuw, Y.-D.; Chan, W.-H.

    2007-01-01

    Previous studies have established that ethanol induces apoptosis, but the precise molecular mechanisms are currently unclear. Here, we show that 0.3-1.0% (w/v) ethanol induces apoptosis in mouse blastocysts and that resveratrol, a grape-derived phytoalexin with known antioxidant and anti-inflammatory properties, prevents ethanol-induced apoptosis and inhibition of cell proliferation. Moreover, ethanol-treated blastocysts show normal levels of implantation on culture dishes in vitro but a reduced ability to reach the later stages of embryonic development. Pretreatment with resveratrol prevented ethanol-induced disruption of embryonic development in vitro and in vivo. In an in vitro cell-based assay, we further found that ethanol increases the production of reactive oxygen species in ESC-B5 embryonic stem cells, leading to an increase in the intracellular concentrations of cytoplasmic free Ca 2+ and NO, loss of mitochondrial membrane potential, mitochondrial release of cytochrome c, activation of caspase-9 and -3, and apoptosis. These changes were blocked by pretreatment with resveratrol. Based on these results, we propose a model for the protective effect of resveratrol on ethanol-induced cell injury in blastocysts and ESC-B5 cells

  3. Retinol improves bovine embryonic development in vitro

    Directory of Open Access Journals (Sweden)

    Edwards J Lannett

    2004-12-01

    Full Text Available Abstract Retinoids are recognized as important regulators of vertebrate development, cell differentiation, and tissue function. Previous studies, performed both in vivo and in vitro, indicate that retinoids influence several reproductive events, including follicular development, oocyte maturation and early embryonic development. The present study evaluated in vitro effects of retinol addition to media containing maturing bovine oocytes and developing embryos in both a low oxygen atmosphere (7% and under atmospheric oxygen conditions (20%. In the first experiment, abbatoir collected bovine oocytes were matured in the presence or absence of varying concentrations of retinol. After a 22–24 hour maturation period the oocytes were fertilized, denuded 18 hours later and cultured in a modified synthetic oviductal fluid (mSOF in a humidified atmosphere at 38.5 degrees C, 5% CO2, 7% O2 and 88% N2. Cleavage rates did not differ among control and retinol-treated oocytes in all three experiments. Addition of 5 micromolar retinol to the maturation medium (IVM tended (p

  4. Proteomic Analysis of Human Blastocoel Fluid and Blastocyst Cells

    DEFF Research Database (Denmark)

    Linnert Jensen, Pernille; Beck, Hans Christian; Petersen, Jørgen

    The human blastocyst consists of 100-200 cells that are organized in an outer layer of differentiated trophectoderm (TE) cells lining the blastocyst cavity into which the undifferentiated inner cell mass (ICM) protrudes. The cavity of the blastocyst is filled with blastocoel fluid to which all...... the cells of the blastocyst are exposed. The ICM is the starting point for the development of undifferentiated human embryonic stem cells (hESCs), which posses the potential to develop into any cell type present in the adult human body [1,2]. This ability makes hESCs a potential source of cells...

  5. Time-lapse cinematography-compatible polystyrene-based microwell culture system: a novel tool for tracking the development of individual bovine embryos.

    Science.gov (United States)

    Sugimura, Satoshi; Akai, Tomonori; Somfai, Tamás; Hirayama, Muneyuki; Aikawa, Yoshio; Ohtake, Masaki; Hattori, Hideshi; Kobayashi, Shuji; Hashiyada, Yutaka; Konishi, Kazuyuki; Imai, Kei

    2010-12-01

    We have developed a polystyrene-based well-of-the-well (WOW) system using injection molding to track individual embryos throughout culture using time-lapse cinematography (TLC). WOW culture of bovine embryos following in vitro fertilization was compared with conventional droplet culture (control). No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality and inner cell mass (ICM) and trophectoderm (TE) cell numbers were not different between control- and WOW-derived blastocysts; however, apoptosis in both the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at Days 30 and 60 after embryo transfer (P < 0.05). TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pickup; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of two blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers.

  6. Blastocyst development in single medium with or without renewal on day 3: a prospective cohort study on sibling donor oocytes in a time-lapse incubator.

    Science.gov (United States)

    Costa-Borges, Nuno; Bellés, Marta; Meseguer, Marcos; Galliano, Daniela; Ballesteros, Agustin; Calderón, Gloria

    2016-03-01

    To evaluate the efficiency of using a continuous (one-step) protocol with a single medium for the culture of human embryos in a time-lapse incubator (TLI). Prospective cohort study on sibling donor oocytes. University-affiliated in vitro fertilization (IVF) center. Embryos from 59 patients. Culture in a TLI in a single medium with or without renewal of the medium on day-3. Embryo morphology and morphokinetic parameters, clinical pregnancy, take-home baby rate, and perinatal outcomes. The blastocyst rates (68.3 vs. 66.8%) and the proportion of good-quality blastocysts (transferred plus frozen) obtained with the two-step (80.0%) protocol were statistically significantly similar to those obtained in the one-step protocol (72.2%). Similarly, morphokinetic events from early cleavage until late blastocyst stages were statistically significantly equivalent between both groups. No differences were found either in clinical pregnancy rates when comparing pure transfers performed with embryos selected from the two-step (75.0%), one-step (70.0%, respectively), and mixed (57.1%) groups. A total of 55 out of 91 embryos transferred implanted successfully (60.4%), resulting in a total of 37 newborns with a comparable birth weight mean among groups. Our findings support the idea that in a TLI with a controlled air purification system, human embryos can be successfully cultured continuously from day 0 onward in single medium with no need to renew it on day-3. This strategy does not affect embryo morphokinetics or development to term and offers more stable culture conditions for embryos as well as practical advantages and reduced costs for the IVF laboratory. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  7. The Effect of Lysophosphatidic Acid during In Vitro Maturation of Bovine Oocytes: Embryonic Development and mRNA Abundances of Genes Involved in Apoptosis and Oocyte Competence

    Directory of Open Access Journals (Sweden)

    Dorota Boruszewska

    2014-01-01

    Full Text Available In the present study we examined whether LPA can be synthesized and act during in vitro maturation of bovine cumulus oocyte complexes (COCs. We found transcription of genes coding for enzymes of LPA synthesis pathway (ATX and PLA2 and of LPA receptors (LPAR 1–4 in bovine oocytes and cumulus cells, following in vitro maturation. COCs were matured in vitro in presence or absence of LPA (10−5 M for 24 h. Supplementation of maturation medium with LPA increased mRNA abundance of FST and GDF9 in oocytes and decreased mRNA abundance of CTSs in cumulus cells. Additionally, oocytes stimulated with LPA had higher transcription levels of BCL2 and lower transcription levels of BAX resulting in the significantly lower BAX/BCL2 ratio. Blastocyst rates on day 7 were similar in the control and the LPA-stimulated COCs. Our study demonstrates for the first time that bovine COCs are a potential source and target of LPA action. We postulate that LPA exerts an autocrine and/or paracrine signaling, through several LPARs, between the oocyte and cumulus cells. LPA supplementation of maturation medium improves COC quality, and although this was not translated into an enhanced in vitro development until the blastocyst stage, improved oocyte competence may be relevant for subsequent in vivo survival.

  8. Post-hatching development of the porcine and bovine embryo-defining criteria for expected development in vivo and in vitro

    DEFF Research Database (Denmark)

    Vejlsted, Morten; Du, Yutao; Vajta, Gábor

    2006-01-01

    ) Somite stage(s) where paraxial mesoderm gradually condensates to form somites. Post-hatching development of bovine embryos in vitro is compromised and although hatching occurs and elongation can be physically provoked by culture in agarose tunnels, the embryonic disk characterizing the pre-streak stage 1......Particular attention has been paid to the pre-hatching period of embryonic development although blastocyst development is a poor indicator of embryo viability. Post-hatching embryonic dev elopment in vitro would allow for establishment of more accurate tools for evaluating developmental potential...... without the need for transfer to recipient animals. Such a system would require (1) definition of milestones of expected post-hatching embryonic development in vivo; and (2) development of adequate culture systems. We propose a stereomicroscopical staging system for post-hatching embryos defining...

  9. Physiology and culture of the human blastocyst.

    Science.gov (United States)

    Gardner, David K; Lane, Michelle; Schoolcraft, William B

    2002-01-01

    The human embryo undergoes many changes in physiology during the first 4 days of life as it develops and differentiates from a fertilized oocyte to the blastocyst stage. Concomitantly, the embryo is exposed to gradients of nutrients within the female reproductive tract and exhibits changes in its own nutrient requirements and utilization. Determining the nature of such nutrient gradients in the female tract and the changing requirements of the embryo has facilitated the formulation of stage-specific culture media designed to support embryo development throughout the preimplantation period. Resultant implantation rates attained with the culture and transfer of human blastocysts are higher than those associated with the transfer of cleavage stage embryos to the uterus. Such increases in implantation rates have facilitated the establishment of high pregnancy rates while reducing the number of embryos transferred. With the introduction of new scoring systems for the blastocyst and the non-invasive assessment of metabolic activity of individual embryos, it should be possible to move to single blastocyst transfer for the majority of patients.

  10. Bovine preimplantation embryos with silenced nucleophosmin mRNA are able to develop until the blastocyst stage

    Czech Academy of Sciences Publication Activity Database

    Toralová, Tereza; Benešová, Veronika; Vodičková Kepková, Kateřina; Vodička, Petr; Šušor, Andrej; Kaňka, Jiří

    2012-01-01

    Roč. 144, č. 3 (2012), 349-359 ISSN 1470-1626 R&D Projects: GA ČR GA523/09/1035; GA ČR(CZ) GD204/09/H084 Institutional support: RVO:67985904 Keywords : double stranded DNA * in vitro * gene expression Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.555, year: 2012

  11. Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development.

    Science.gov (United States)

    Morató, Roser; Izquierdo, Dolors; Paramio, Maria Teresa; Mogas, Teresa

    2008-10-01

    Two experiments were designed to assess the effectiveness of cryopreserving bovine MII oocytes using cryotops as the carrier system for vitrification. In the first experiment, we examined the developmental competence of oocytes after: (i) vitrification in open-pulled straws (OPS method); or (ii) vitrification in plastic handle (Cryotop method). In the second experiment, warmed oocytes that had been vitrified in OPS or cryotops were fixed to analyze spindle and chromosome configuration. In all experiments both cow and calf oocytes were used. Significantly different fertilization rates were observed between the vitrification groups: 31.5% and 20.2% for the cow and calf oocytes vitrified in OPS, respectively, versus 46.1% and 46.4% for the oocytes vitrified using cryotops. After in vitro fertilization, 3.8% of the calf oocytes and 5.3% of the cow oocytes developed to the blastocyst stage. All blastocysts from vitrified oocytes resulted from the Cryotop method. A significantly lower percentage of the OPS-vitrified calf oocytes showed a normal spindle configuration (37.8%) compared to control fresh oocytes (69.9%), while normal spindle and chromosome configurations were observed in a significantly higher proportion of the cryotop-vitrified calf oocytes (60.2%). For the cow oocytes, 60.6% in the OPS group and 60.3% in the Cryotop group exhibited a normal morphology after warming. These findings suggest the cryotop system is a more efficient carrier for vitrification than OPS for the cryopreservation of bovine oocytes.

  12. Effect of the time interval between fusion and activation on epigenetic reprogramming and development of bovine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Liu, Jun; Wang, Yongsheng; Su, Jianmin; Wang, Lijun; Li, Ruizhe; Li, Qian; Wu, Yongyan; Hua, Song; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2013-04-01

    Previous studies have shown that the time interval between fusion and activation (FA interval) play an important role in nuclear remodeling and in vitro development of somatic cell nuclear transfer (SCNT) embryos. However, the effects of FA interval on the epigenetic reprogramming and in vivo developmental competence of SCNT embryos remain unknown. In the present study, the effects of different FA intervals (0 h, 2 h, and 4 h) on the epigenetic reprogramming and developmental competence of bovine SCNT embryos were assessed. The results demonstrated that H3 lysine 9 (H3K9ac) levels decreased rapidly after fusion in all three groups. H3K9ac was practically undetectable 2 h after fusion in the 2-h and 4-h FA interval groups. However, H3K9ac was still evidently detectable in the 0-h FA interval group. The H3K9ac levels increased 10 h after fusion in all three groups, but were higher in the 2-h and 4-h FA interval groups than that in the 0-h FA interval group. The methylation levels of the satellite I region in day-7 blastocysts derived from the 2-h or 4-h FA interval groups was similar to that of in vitro fertilization blastocysts and is significantly lower than that of the 0-h FA interval group. SCNT embryos derived from 2-h FA interval group showed higher developmental competence than those from the 0-h and 4-h FA interval groups in terms of cleavage rate, blastocyst formation rate, apoptosis index, and pregnancy and calving rates. Hence, the FA interval is an important factor influencing the epigenetic reprogramming and developmental competence of bovine SCNT embryos.

  13. The HIST1 Locus Escapes Reprogramming in Cloned Bovine Embryos

    Directory of Open Access Journals (Sweden)

    Byungkuk Min

    2016-05-01

    Full Text Available Epigenetic reprogramming is necessary in somatic cell nuclear transfer (SCNT embryos in order to erase the differentiation-associated epigenetic marks of donor cells. However, such epigenetic memories often persist throughout the course of clonal development, thus decreasing cloning efficiency. Here, we explored reprogramming-refractory regions in bovine SCNT blastocyst transcriptomes. We observed that histone genes residing in the 1.5 Mb spanning the cow HIST1 cluster were coordinately downregulated in SCNT blastocysts. In contrast, both the nonhistone genes of this cluster, and histone genes elsewhere remained unaffected. This indicated that the downregulation was specific to HIST1 histone genes. We found that, after trichostatin A treatment, HIST1 histone genes were derepressed, and DNA methylation at their promoters was decreased to the level of in vitro fertilization embryos. Therefore, our results indicate that the reduced expression of HIST1 histone genes is a consequence of poor epigenetic reprogramming in SCNT blastocysts.

  14. Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos

    Science.gov (United States)

    Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

    2011-01-01

    In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers. ?? Copyright Cambridge University Press 2010.

  15. Proteomic analysis of human blastocoel fluid and blastocyst cells

    DEFF Research Database (Denmark)

    Jensen, Pernille; Beck, Hans Christian; Petersen, Jørgen

    2013-01-01

    Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the blastocyst and can differentiate into any cell type in the human body. These cells hold a great potential for regenerative medicine, but to obtain enough cells needed for medical treatment, culture is required......, the blastocoel fluid, which is in contact with all the cells in the blastocyst, including hESCs. Fifty-three surplus human blastocysts were donated after informed consent, and blastocoel fluid was isolated by micromanipulation. Using highly sensitive nano-high-pressure liquid chromatography-tandem mass...... from the ICM of the human blastocyst are exposed to via the blastocoel fluid. These results can be an inspiration for the development of improved culture conditions for hESCs....

  16. Development of a Vaccine for Eradicating Contagious Bovine ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Since eventual success depends on the vaccine's acceptability by livestock keepers, the project involves them in field-testing and assesses their willingness to pay for the vaccine. The project also ... Outputs. Studies. Developing vaccines for animals, the case of Contagious Bovine Pleuropneumonia (CBPP) in Africa. 53232.

  17. Cell and genetic predictors of human blastocyst hatching success in assisted reproduction.

    Science.gov (United States)

    Syrkasheva, Anastasiya G; Dolgushina, Nataliya V; Romanov, Andrey Yu; Burmenskaya, Olga V; Makarova, Nataliya P; Ibragimova, Espet O; Kalinina, Elena A; Sukhikh, Gennady T

    2017-10-01

    The aim was to identify cell and genetic predictors of human blastocyst hatching success in assisted reproduction programmes via a prospective case-control study. Blastocysts, donated by couples in assisted reproduction programmes were used. Hatching success assessment was performed after 144-146 h post-fertilization. The mRNA expression levels of cathepsin V (CTSV), GATA-binding protein 3 (GATA3) and human chorionic gonadotropin beta subunit 3, 5, 7 and 8 (CGB) genes were detected by quantitative real-time polymerase chain reaction. The odds ratio (OR) of hatching due to zona pellucida (ZP) thickness, oocyte and sperm quality, embryo quality and mRNA expression of CTSV, GATA3 and CGB genes in blastocysts was determined. From 62 blastocysts included in the study, 47 (75.8%) were unable to hatch spontaneously. The ZP thickening, and oocyte and sperm quality did not affect human blastocyst ability to hatch, except the combination of cytoplasmic and extracytoplasmic oocyte dysmorphisms (OR = 1.25; 95% confidence interval = 1.08, 1.45). Hatching-capable blastocysts had higher Gardner scale grade and mRNA expression of CTSV, GATA3 and CGB genes than hatching-incapable blastocysts. The human blastocyst hatching success depends on the blastocyst Gardner grade, but not on ZP and gamete quality. Blastocyst development was regulated by CTSV, GATA3 and CGB gene expression.

  18. Transcriptomic profiling of bovine IVF embryos revealed candidate genes and pathways involved in early embryonic development

    Directory of Open Access Journals (Sweden)

    Yandell Brian S

    2010-01-01

    Full Text Available Abstract Background Early embryonic loss is a large contributor to infertility in cattle. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. The objective of this study was to identify genes differentially expressed between blastocysts and degenerative embryos at early stages of development. Results Using microarrays, genome-wide RNA expression was profiled and compared for in vitro fertilization (IVF - derived blastocysts and embryos undergoing degenerative development up to the same time point. Surprisingly similar transcriptomic profiles were found in degenerative embryos and blastocysts. Nonetheless, we identified 67 transcripts that significantly differed between these two groups of embryos at a 15% false discovery rate, including 33 transcripts showing at least a two-fold difference. Several signaling and metabolic pathways were found to be associated with the developmental status of embryos, among which were previously known important steroid biosynthesis and cell communication pathways in early embryonic development. Conclusions This study presents the first direct and comprehensive comparison of transcriptomes between IVF blastocysts and degenerative embryos, providing important information for potential genes and pathways associated with early embryonic development.

  19. Leucemia inhibitory factor; investigating the time-dependent effect on viability of vitrified bovine embryos.

    Science.gov (United States)

    Kocyigit, A; Cevik, M

    2017-12-01

    Leucemia inhibitory factor (LIF) is involved in various reproductive processes, including sperm development, regulation of ovulation, as well as blastocyst formation, hatching and implantation in embryos. Moreover, LIF has also been shown significantly to enhance the blastocyst formation rates of bovine embryos, a finding that remains controversial. Our purpose was to investigate time-dependent effect of LIF on bovine embryo culture, especially in terms of addition timing. Presumptive zygotes were cultured in five different groups. In this study, 100 ng/ml LIF was added to the culture medium were as follows; control: SOF alone, group A: at day 0 (fertilization day), group B: at day 4 post-insemination (p.i.), group C: at day 4 to 7 (p.i. before vitrification) and group D: at day 8 (p.i. after thawing). Addition of LIF to the culture medium at day 4 significantly increased the percentage of blastocyst rate when compared day 0, day 4 at 6/7 and control group (41.8% versus 24.3%, 19.7%, 34.6%). In conclusion, the addition of LIF only on day 4 (p.i.) to the culture medium was found to be beneficial for bovine embryonic development based on several measures, including blastocysts rate, re-expansion rate and cellular cryotolerance after vitrification. © 2017 Blackwell Verlag GmbH.

  20. Culture medium composition affects the gene expression pattern and in vitro development potential of bovine somatic cell nuclear transfer (SCNT embryos

    Directory of Open Access Journals (Sweden)

    María E Arias

    2013-01-01

    Full Text Available Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively. No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01 in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28% compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively. Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA. Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.

  1. Non-invasive analysis of bovine embryo metabolites during in vitro embryo culture using nuclear magnetic resonance

    Directory of Open Access Journals (Sweden)

    Marcello Rubessa

    2016-12-01

    Full Text Available The ability to identify embryos that have the highest developmental potential from a cohort would significantly increase the chances of achieving pregnancy. Metabolic analysis is a well-established analytical approach in biological systems. Starting from this idea, we chose to use high-resolution nuclear magnetic resonance (1H-NMR spectroscopy. The aim of this study was to determine if it is possible to select viable embryos after 48 h of culture using metabolic activity as the parameter. We evaluated embryo metabolism after the first 48 h of culture and compared the activity of cleaved embryos that became blastocysts to cleaved embryos that did not develop to blastocysts, and in vitro fertilized (IVF blastocysts and parthenogenetic-activated (PA blastocysts. Our results show that citrate, pyruvate, myo-inositol and lysine have great impact on predicting embryo development. When we compared IVF and PA blastocysts, we found that acetate and phenylalanine concentrations are excellent parameters for evaluating blastocyst quality. Combining all these results, we were able to create a formula that predicts zygote development after 2 days of culture. In conclusion, we found that it is possible predict the future development of in vitro produced bovine embryos after only 2 days of culture using 1H-NMR.

  2. Culture medium composition affects the gene expression pattern and in vitro development potential of bovine somatic cell nuclear transfer (SCNT) embryos.

    Science.gov (United States)

    Arias, María E; Ross, Pablo J; Felmer, Ricardo N

    2013-01-01

    Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (Pculture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.

  3. Accumulation of RNA in blastocysts during embryonic diapause and the periimplantation period in the western spotted skunk

    International Nuclear Information System (INIS)

    Mead, R.A.; Rourke, A.W.

    1985-01-01

    The in vivo incorporation of 3 H-uridine into RNA was studied in delayed implanting and activated blastocysts obtained from 33 western spotted skunks. 3 H-uridine was incorporated into RNA by all blastocysts; however, significantly more label was incorporated as blastocyst diameter increased. Activated blastocysts with diameters of 1.6 mm or greater on average incorporated 65 times more 3 H-precursor in 5 hr than diapausing blastocysts with diameters of 1.1 mm or less. Polyadenylated RNA was likewise synthesized by delayed implanting and activated skunk blastocysts; however, the proportion of polyadenylated RNA synthesized by the former was greater than in the latter. The data suggest that the transition from embryonic diapause to fully activated blastocysts first occurs gradually for several days before entering a 1-2-day period of rapid development characterized by an abrupt increase in RNA accumulation

  4. Blastocyst Morphology Holds Clues Concerning The Chromosomal Status of The Embryo

    Directory of Open Access Journals (Sweden)

    Rita de Cassia Savio Figueira

    2015-07-01

    Full Text Available Background: Embryo morphology has been proposed as an alternative marker of chromosomal status. The objective of this retrospective cohort study was to investigate the association between the chromosomal status on day 3 of embryo development and blastocyst morphology. Materials and Methods: A total of 596 embryos obtained from 106 cycles of intracytoplasmic sperm injection (ICSI followed by preimplantation genetic aneuploidy screening (PGS were included in this retrospective study. We evaluated the relationship between blastocyst morphological features and embryonic chromosomal alteration. Results: Of the 564 embryos with fluorescent in situ hybridization (FISH results, 200 reached the blastocyst stage on day 5 of development. There was a significantly higher proportion of euploid embryos in those that achieved the blastocyst stage (59.0% compared to embryos that did not develop to blastocysts (41.2% on day 5 (P<0.001. Regarding blastocyst morphology, we observed that all embryos that had an abnormal inner cell mass (ICM were aneuploid. Embryos with morphologically normal ICM had a significantly higher euploidy rate (62.1%, P<0.001. As regards to the trophectoderm (TE morphology, an increased rate of euploidy was observed in embryos that had normal TE (65.8% compared to embryos with abnormal TE (37.5%, P<0.001. Finally, we observed a two-fold increase in the euploidy rate in high-quality blastocysts with both high-quality ICM and TE (70.4% compared to that found in low-quality blastocysts (31.0%, P<0.001. Conclusion: Chromosomal abnormalities do not impair embryo development as aneuploidy is frequently observed in embryos that reach the blastocyst stage. A high-quality blastocyst does not represent euploidy of chromosomes 13, 14, 15, 16, 18, 21, 22, X and Y. However, aneuploidy is associated with abnormalities in the ICM morphology. Further studies are necessary to confirm whether or not the transfer of blastocysts with low-quality ICM should be

  5. Effect of early addition of bone morphogenetic protein 5 (BMP5) to embryo culture medium on in vitro development and expression of developmentally important genes in bovine preimplantation embryos.

    Science.gov (United States)

    García, Elina V; Miceli, Dora C; Rizo, Gabriela; Valdecantos, Pablo A; Barrera, Antonio D

    2015-09-01

    Previous studies have reported that bone morphogenetic protein 5 (BMP5) is differentially expressed in the isthmus of bovine oviducts and it is present in the oviductal fluid. However, the specific action of this factor is unknown. To evaluate whether BMP5 exerts some effect during early bovine embryo development, gene expression of BMP5, BMP receptors, and the effect of exogenous BMP5 on in vitro development and expression of developmentally important genes were assessed. In experiment 1, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from in vitro fertilization, were collected for analysis of BMP5 and BMP receptors (BMPR1A, BMPR1B, and BMPR2) messenger RNA (mRNA) expression. On the basis of previous results, in experiment 2, presumptive zygotes were cultured for the first 48 hours after insemination in CR1aa medium assaying three different treatments: (1) control (CR1aa); (2) vehicle control (CR1aa + 0.04 mM HCl), and (3) BMP5 treatment (CR1aa + 100 ng/mL of BMP5). The cleavage rate was evaluated 48 hours after insemination (Day 2), and then, embryos were transferred to CR1aa + 10% fetal bovine serum. The blastocyst rate was determined on Day 7. In experiment 3, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from control and BMP5-treated groups, were collected for analysis of ID2 (BMP target gene), OCT4, NANOG, and SOX2 (pluripotency genes) mRNA expression. BMP5 transcripts were not detectable in any of the embryonic stages examined, whereas the relative mRNA abundance of the three BMP receptors analyzed was greater in early embryo development stages before maternal-embryonic transition, raising the possibility of a direct effect of exogenous BMPs on the embryo during the first developmental period. Although early addition of 100 ng/mL of BMP5 to the embryo culture medium had no effect on the cleavage rate, a significantly higher proportion of cleaved embryos developed to the

  6. Numerical chromosome errors in day 7 somatic nuclear blastocysts

    DEFF Research Database (Denmark)

    Booth, Paul J; Viuff, Dorthe; Tan, Shijian J

    2003-01-01

    Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona-free manipulat......Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona...... families, consisting of 112 blastocysts reconstructed from five different primary granulosa cell cultures, were examined. Overall, the mean chromosome complement within embryos was 86.9 +/- 3.7% (mean +/- SEM) diploid, 2.6 +/- 0.5% triploid, 10.0 +/- 3.1% tetraploid, and 0.5 +/- 0.2% pentaploid or greater......; the vast majority (>75%) of the abnormal nuclei were tetraploid. Completely diploid and mixoploid embryos represented 22.1 +/- 4.5% and 73.7 +/- 5.5%, respectively, of all clones. Six totally polyploid blastocysts, containing or=5N chromosome complements, respectively) between two clone families were...

  7. Development of a Radioimmunoassay for Bovine Chymosin B

    Directory of Open Access Journals (Sweden)

    Borceux, JP.

    2007-01-01

    Full Text Available The present study was conducted to develop and validate a specific radioimmunoassay system for measurement of bovine chymosin B (bChyB concentrations in plasma samples. Bovine ChyB was used for immunization of rabbits and as standard and tracer. Chymosin B concentrations were measured in plasma samples from two groups of calves (Group 1: calves sampled from birth to 24 hours; Group 2: calves sampled from Day 1 to 21 after birth and from one cow during the peri-partum period. Detection limit of the assay was 9.0 ng/ml. Recovery was higher than 89.3%. Repeatability and reproducibility ranged from 1.52% to 5.23% and from 1.52% to 12.57% respectively. No cross-reaction was found with pepsinogen A from bovine, porcine or human origins. In Group 1, bChyB concentrations increased from 47.3 ± 45.1 ng/ml (5 min after birth to 325.5 ± 161.2 ng/ml (12 hours after birth, then no significant change was observed till 24 hours after birth (293.0 ± 161.5 ng/ml. In Group 2, concentrations decreased from Day 1 (455.3 ± 191.1 ng/ml to Day 21 (117.9 ± 85.1 ng/ml. In adult cow, mean concentration was 136.0 ± 32.3 ng/ml. In conclusion, bChyB is able to cross the stomach basal membrane and to reach the blood circulation at detectable levels in both young calves and adult cows.

  8. Apicomplexan profilins in vaccine development applied to bovine neosporosis.

    Science.gov (United States)

    Mansilla, Florencia C; Capozzo, Alejandra V

    2017-12-01

    Neospora caninum, an intracellular protozoan parasite from the phylum Apicomplexa, is the etiologic agent of neosporosis, a disease considered as a major cause of reproductive loss in cattle and neuromuscular disease in dogs. Bovine neosporosis has a great economic impact in both meat and dairy industries, related to abortion, premature culling and reduced milk yields. Although many efforts have been made to restrain bovine neosporosis, there are still no efficacious control methods. Many vaccine-development studies focused in the apicomplexan proteins involved in the adhesion and invasion of the host cell. Among these proteins, profilins have recently emerged as potential vaccine antigens or even adjuvant candidates for several diseases caused by apicomplexan parasites. Profilins bind Toll-like receptors 11 and 12 initiating MyD88 signaling, that triggers IL-12 and IFN-γ production, which may promote protection against infection. Here we summarized the state-of-the-art of novel vaccine development based on apicomplexan profilins applied as antigens or adjuvants, and delved into recent advances on N. caninum vaccines using profilin in the mouse model and in cattle. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Eimeria tenella: in vitro development in irradiated bovine kidney cells

    Energy Technology Data Exchange (ETDEWEB)

    Crane, M.St.J.; Schmatz, D.M.; Stevens, S.; Habbersett, M.C.; Murray, P.K. (Merck Sharp and Dohme Research Labs., Rahway, NJ (USA))

    1984-06-01

    The initial infection and first-generation development of Eimeria tenella was quantified using a cloned MDBK (Madin-Darby Bovine Kidney) cell line, irradiated with gamma radiation prior to infection, as the host cell. Irradiated cell cultures were found to be more susceptible to infection and had a greater capacity to support parasite development than non-irradiated cultures. It was suggested that the larger proportion of cells in the G/sub 2/ phase of the cell cycle, the larger individual cell size and the inhibition of cell division in the irradiated cultures were all factors contributing to the increased susceptibility to infection and capacity to support parasite growth and development. The application of this technique (host cell irradiation) to the cultivation of other intracellular, protozoan parasites is discussed.

  10. Eimeria tenella: in vitro development in irradiated bovine kidney cells

    International Nuclear Information System (INIS)

    Crane, M. St.J.; Schmatz, D.M.; Stevens, S.; Habbersett, M.C.; Murray, P.K.

    1984-01-01

    The initial infection and first-generation development of Eimeria tenella was quantified using a cloned MDBK (Madin-Darby Bovine Kidney) cell line, irradiated with gamma radiation prior to infection, as the host cell. Irradiated cell cultures were found to be more susceptible to infection and had a greater capacity to support parasite development than non-irradiated cultures. It was suggested that the larger proportion of cells in the G 2 phase of the cell cycle, the larger individual cell size and the inhibition of cell division in the irradiated cultures were all factors contributing to the increased susceptibility to infection and capacity to support parasite growth and development. The application of this technique (host cell irradiation) to the cultivation of other intracellular, protozoan parasites is discussed. (author)

  11. Effect of B-mercaptoethanol on the viability of IVM/IVF/IVC bovine embryos during long-distance transportation in plastic straws.

    Science.gov (United States)

    Takahashi, H; Kuwayama, M; Hamano, S; Takahashi, M; Okano, A; Kadokawa, H; Kariya, T; Nagai, T

    1996-10-15

    Experiments were conducted to assess the effect of beta-mercaptoethanol (beta-ME) on the quality and viability of bovine blastocysts derived from in-vitro culture (IVC) of in-vitro matured and fertilized (TVM-IVF) oocytes during their transport between 2 distant places. Follicular oocytes were collected from ovaries obtained at a slaughterhouse and were cultured for 20 to 21 h in modified TCM-199. The IVM oocytes were fertilized in vitro with frozen-thawed spermatozoa. Fertilized oocytes were cultured for 7 d, and embryos that developed to the blastocyst stage were used for the experiments. The blastocysts, packed in straws with transportation medium that consisted of modified TCM-199 with HEPES equilibrated in air and supplemented with 20 % calf serum and 0, 10, 50, 100 or 150 microM beta-ME, were transported at 37 degrees C from Tokyo to Sapporo by air (18.3 h). The quality of blastocysts was assessed and ranked as excellent (A), good (B), fair (C) or poor (D) after transportation. The percentages of blastocysts ranked as A or B were significantly higher (P plastic straws for several hours without control of CO2 and that the concentration of beta-ME used in this experiment is not detrimental to the blastocysts.

  12. Bovine lymphosarcoma: development of a radioimmunologic techniques for detection of the etiologic agent

    International Nuclear Information System (INIS)

    Devare, S.G.; Stephenson, J.R.; Sarma, P.S.; Aaronson, S.A.

    1976-01-01

    A highly sensitive and specific radioimmunoassay has been developed for the major structural protein of an oncornavirus etiologically associated with bovine lymphosarcoma. This test can be used to identify cattle which have been exposed to the bovine leukemia virus and may thus develop or transmit the disease. Analysis of randomly obtained serums indicates that infection with this virus is widespread among cattle

  13. Improved parthenogenetic development of vitrified-warmed bovine oocytes activated with 9% ethanol plus 6-DMAP

    DEFF Research Database (Denmark)

    Hou, Y -p; Liu, Ying; Dai, Y -p

    2009-01-01

    The objective was to compare various activation protocols on developmental potential of vitrified bovine oocytes. Bovine oocytes matured in vitro for 23 h were vitrified with EDFSF30 in open pulled straws. After warming, they were cultured in vitro for 1 h, followed by parthenogenetic activation....... Vitrified-warmed oocytes had a morphologically normal rate similar to that of controls (nonvitrified oocytes cultured in vitro for 24 h; 98.6% vs. 100%, P > 0.05). When vitrified-warmed oocytes were first activated with 7% ethanol for 5 min and then incubated in 6-dimethylaminopurin (6-DMAP) for 4 h...... (for 5 min) or in combination with 6-DMAP (4 h) was used to activate vitrified-warmed oocytes, cleavage rates ranged from 22.3% to 61.1% and blastocyst rates ranged from 1.1% to 30.6%. These rates were optimized when oocytes were treated with 9% ethanol plus 6-DMAP; this was verified in experiments...

  14. In vivo and in vitro development of Tibetan antelope (Pantholops hodgsonii interspecific cloned embryos

    Directory of Open Access Journals (Sweden)

    Guanghua SU,Lei CHENG,Yu GAO,Kun LIU,Zhuying WEI,Chunling BAI,Fengxia YIN,Li GAO,Guangpeng LI,Shorgan BOU

    2014-02-01

    Full Text Available The Tibetan antelope is endemic to the Tibetan Plateau, China, and is now considered an endangered species. As a possible rescue strategy, the development of embryos constructed by interspecies somatic cell nuclear transfer (iSCNT was examined. Tibetan antelope fibroblast cells were transferred into enucleated bovine, ovine and caprine oocytes. These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals. Less than 0.5% of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage. However, when the cloned antelope-bovine embryos were transferred to caprine oviducts, about 1.6% of the embryos developed to the blastocyst stage. In contrast, only 0.7% of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts. The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine. When the antelope-bovine embryos, constructed from oocytes treated with roscovitine or trichostatin A, were cultured in rabbit oviducts 2.3% and 14.3% developed to blastocysts, respectively. It is concluded that although some success was achieved with the protocols used, interspecies cloning of Tibetan antelope presents difficulties still to be overcome. The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming.

  15. Advances in development and evaluation of bovine herpesvirus 1 vaccines

    NARCIS (Netherlands)

    Oirschot, van J.T.; Kaashoek, M.J.; Rijsewijk, F.A.M.

    1996-01-01

    This review deals with conventional and modern bovine herpesvirus 1 (BHV1) vaccines. Conventional vaccines are widely used to prevent clinical signs of infectious bovine rhinotracheitis. The use of conventional vaccines, however, does not appear to have resulted in reduction of the prevalence of

  16. Expression of the CTCF gene in bovine oocytes and preimplantation embryos

    Directory of Open Access Journals (Sweden)

    Álvaro F.L. Rios

    2007-01-01

    Full Text Available The CCCTC - binding factor (CTCF is a protein involved in repression, activation, hormone-inducible gene silencing, functional reading of imprinted genes and X-chromosome inactivation. We analyzed CTCF gene expression in bovine peripheral blood, oocytes and in different cellular stages (2-4 cells, 8-16 cells, 16-32 cells, morulae, and blastocysts of in vitro fertilized embryos. This is the first report of CTCF expression in oocytes and preimplantation bovine embryos and has implications for the production of embryonic stem cells and the development of novel medical technologies for humans.

  17. Culture of bovine embryos on a polydimethylsiloxane (PDMS) microwell plate.

    Science.gov (United States)

    Akagi, Satoshi; Hosoe, Misa; Matsukawa, Kazutsugu; Ichikawa, Akihiko; Tanikawa, Tamio; Takahashi, Seiya

    2010-08-01

    We fabricated a polydimethylsiloxane (PDMS)-based microwell plate (PDMS-MP) containing 100 microwells with a rounded bottom and examined whether it can be used for culture of individual in vitro fertilized (IVF) embryos or parthenogenetically activated zona-free embryos in cattle. In Experiment 1, we examined the in vitro developmental ability of IVF embryos cultured individually on PDMS-MP. After IVF, 20 embryos were transferred into 100 microl drops on PDMS-MP and cultured individually in each well of PDMS-MP (PDMS group). After 7 days of culture, the embryos in the PDMS group developed to the blastocyst stage at the same rate of those in the control group cultured in a group of 20 embryos without PDMS-MP. There were no differences in total number of cells and the ratio of inner cell mass to total cells between the PDMS and control groups. In Experiment 2, we examined the in vitro developmental ability of parthenogenetically activated zona-free bovine embryos cultured individually on PDMS-MP. The zona-free embryos were cultured individually in each well of a PDMS-MP or in each well produced by pressing a darning needle onto the bottom of a culture dish (WOW group). After 7 days of culture, the blastocyst formation rate and cell number of blastocysts in the PDMS group did not differ from those of the zona-intact embryos in the control group. Also, there were no differences in the blastocyst formation rate and cell number of blastocysts between the WOW and PDMS groups. These results suggest that the culture system using PDMS-MP is useful for individual embryos or zona-free embryos in cattle.

  18. Single blastocyst transfer: The key to reduce multiple pregnancy rates without compromising the live birth rate

    Directory of Open Access Journals (Sweden)

    Uma M Sundhararaj

    2017-01-01

    Full Text Available Background: Historically, to achieve higher pregnancy rates, multiple embryos were transferred after an in-vitro fertilisation (IVF. However, this practice is being reassessed, because it leads to multiple pregnancies that is known to cause adverse maternal and fetal outcomes. Aim: To compare the pregnancy outcomes in fresh IVF or intracytoplasmic sperm injection (ICSI cycles among women undergoing elective single blastocyst transfer (eSBT vs. those undergoing double blastocyst transfer (DBT. Settings and Design: It is a retrospective data analysis of 582 patients undergoing fresh IVF/ICSI cycles performed from January 2012 to June 2015. Materials and Methods: Patients, who underwent IVF/ICSI and developed more than one blastocyst, were included in the study. Donor cycles were excluded from the study. All the embryos were cultured to blastocyst stage in sequential media followed by transfer of two blastocysts (DBT or eSBT and cryopreservation of the remaining. Statistical Analysis: Statistical analysis was performed using chi square test. Results: Out of 582 patients, in 149 patients one blastocyst was transferred and in 433 patients two blastocysts were transferred. There was no statistical difference in the biochemical pregnancy rate, clinical pregnancy rate and live birth rate in both the groups. Statistics demonstrated a significant drop in miscarriage rate in eSBT group. There was no incidence of twins in eSBT group, whereas twin birth rate per clinical pregnancy was 29.02% in DBT group. Conclusion: Single blastocyst transfer is an effective method to reduce the risk of multiple births without compromising the pregnancy outcomes. Given the promising potential of vitrification; the remaining blastocyst can be cryopreserved.

  19. Dual effect of fetal bovine serum on early development depends on stage-specific reactive oxygen species demands in pigs.

    Directory of Open Access Journals (Sweden)

    Seong-Eun Mun

    Full Text Available Despite the application of numerous supplements to improve in vitro culture (IVC conditions of mammalian cells, studies regarding the effect of fetal bovine serum (FBS on mammalian early embryogenesis, particularly in relation to redox homeostasis, are lacking. Herein, we demonstrated that early development of in vitro-produced (IVP porcine embryos highly depends on the combination of FBS supplementation timing and embryonic reactive oxygen species (ROS requirements. Interestingly, FBS significantly reduced intracellular ROS levels in parthenogenetically activated (PA embryos regardless of the developmental stage. However, the beneficial effect of FBS on early embryogenesis was found only during the late phase (IVC 4-6 days treatment group. In particular, developmental competence parameters, such as blastocyst formation rate, cellular survival, total cell number and trophectoderm proportion, were markedly increased by FBS supplementation during the late IVC phase. In addition, treatment with FBS elevated antioxidant transcript levels during the late IVC phase. In contrast, supplementation with FBS during the entire period (1-6 days or during the early IVC phase (1-2 days greatly impaired the developmental parameters. Consistent with the results from PA embryos, the developmental competence of in vitro fertilization (IVF or somatic cell nuclear transfer (SCNT embryos were markedly improved by treatment with FBS during the late IVC phase. Moreover, the embryonic stage-specific effects of FBS were reversed by the addition of an oxidant and were mimicked by treatment with an antioxidant. These findings may increase our understanding of redox-dependent early embryogenesis and contribute to the large-scale production of high-quality IVP embryos.

  20. Live imaging of primitive endoderm precursors in the mouse blastocyst.

    Science.gov (United States)

    Grabarek, Joanna B; Plusa, Berenika

    2012-01-01

    The separation of two populations of cells-primitive endoderm and epiblast-within the inner cell mass (ICM) of the mammalian blastocyst is a crucial event during preimplantation development. However, many aspects of this process are still not very well understood. Recently, the identification of platelet derived growth factor receptor alpha (Pdgfrα) as an early-expressed protein that is also a marker of the later primitive endoderm lineage, together with the availability of the Pdgfra(H2B-GFP) mouse strain (Hamilton et al. Mol Cell Biol 23:4013-4025, 2003), has made in vivo imaging of primitive endoderm formation possible. In this chapter we present two different approaches that can be used to follow the behavior of primitive endoderm cells within the mouse blastocyst in real time.

  1. The efficacy of the well of the well (WOW) culture system on development of bovine embryos in a small group and the effect of number of adjacent embryos on their development.

    Science.gov (United States)

    Kang, Sung-Sik; Ofuji, Sosuke; Imai, Kei; Huang, Weiping; Koyama, Keisuke; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Nagano, Masashi

    2015-06-01

    The aim of the present study was to clarify the efficacy of the well of the well (WOW) culture system for a small number of embryos and the effect of number of adjacent embryos in a WOW dish on blastocyst development. In conventional droplet culture, embryos in the small-number group (5-6 embryos/droplet) showed low blastocyst development compared with a control group (25-26 embryos/droplet). However, small and large numbers of embryos (5-6 and 25 embryos, respectively) in a WOW dish showed no significant differences in cleavage, blastocyst rates, and mean cell number in blastocysts compared with the control group (25-30 embryos/droplet). In addition, the number of adjacent embryos in a WOW dish did not affect the development to blastocysts and cell number in blastocysts. In conclusion, a WOW dish can provide high and stable blastocyst development in small group culture wherever embryos are placed in microwells of the WOW dish.

  2. The effects of ovalbumin as a protein source during the in vitro production of bovine embryos

    Directory of Open Access Journals (Sweden)

    Tatiane Almeida Drummond Tetzner

    2011-10-01

    Full Text Available Embryo quality is influenced by the culture conditions that affect in vitro maturation (IVM, fertilization (IVF and culture (IVC rates. The present study investigated the feasibility of producing bovine embryos after the replacement of fetal calf serum (FCS and bovine serum albumin (BSA by ovalbumin (OVA. The IVM and IVC medium were supplemented with 10% FCS, 4 mg/mL BSA, or 4 mg/mL OVA. The IVF medium was supplemented with 6 mg/mL BSA or OVA. For IVM, supplementation with FCS, BSA, and OVA did not affect nuclear maturation or cortical granule migration. Higher rates of formation of two pronuclei were obtained when FCS was employed for IVM (79.97%, regardless of the supplement used for IVF, and when BSA was used for IVF (59.4%, regardless of the supplement used for IVM. Supplementation with OVA for IVM+IVC (20.40% and for IVF (22.15% was inferior to supplementation with FCS for IVM+IVC (30.47% and with BSA for IVF (28.91% for blastocyst development. Hatching rates were lower using OVA for IVM+IVC (23.02% and for IVF (28.93% compared with FCS and BSA under the same conditions (40.78 and 34.82%, respectively and BSA for IVF (36.82%. Supplementation with OVA for IVM+IVC and IVF resulted in reduced inner cell mass, trophectoderm cells and total blastocyst cell numbers (17.29, 37.88, and 55.17, respectively. In conclusion, OVA is a protein source for bovine in vitro embryo production, although the quantity and quality of bovine blastocysts using only ovalbumin in the entire in vitro production process are lower than those obtained in the presence of FCS and BSA, when used as supplements in any step of bovine in vitro embryo production.

  3. Changes in Sperm Motility and Capacitation Induce Chromosomal Aberration of the Bovine Embryo following Intracytoplasmic Sperm Injection.

    Directory of Open Access Journals (Sweden)

    Yoku Kato

    Full Text Available Intracytoplasmic sperm injection (ICSI has become the method of choice to treat human male infertility. One of the outstanding problems associated with this technique is our current lack of knowledge concerning the effect of sperm capacitation and motility upon the subsequent development of oocytes following ICSI. In the present study, we first examined the capacitation state of sperm exhibiting normal motility, along with sperm that had been activated, and examined the effect of reactive oxygen species (ROS produced by these sperm types upon embryogenesis following bovine in vitro fertilization (IVF and ICSI. Data showed that activated sperm reduced the chromosomal integrity of IVF/ICSI embryos at the blastocyst stage, while capacitated sperm produced ROS in capacitation media. Secondly, we treated sperm with carbonyl cyanide m-chlorophenyl hydrazine (CCCP, a chemical known to uncouple cell respiration within the mitochondria, and investigated the effect of this treatment upon blastocyst formation and chromosomal integrity at the blastocyst stage. Activated sperm in which the mitochondria had been treated with CCCP reduced levels of chromosomal aberration at the blastocyst stage following ICSI, by reducing mitochondrial activity in activated sperm. In conclusion, these findings suggest that capacitated sperm exhibiting activated motility induced chromosomal aberration during development to the blastocyst stage following ICSI. The injection of sperm exhibiting normal motility, or activated sperm in which mitochondrial activity had been reduced, improved the quality of ICSI-derived embryos. Therefore, the selection of sperm exhibiting progressive motility may not always be better for early embryo development and fetal growth following human ICSI, and that the use of a bovine model may contribute to a deeper understanding of sperm selection for human ICSI embryo development.

  4. Finding biomarkers in non-model species: literature mining of transcription factors involved in bovine embryo development

    Directory of Open Access Journals (Sweden)

    Turenne Nicolas

    2012-08-01

    approach based on literature-mining and score arrangement of data from model organisms. This approach was applied to identify novel transcription factors during bovine blastocyst elongation, a process that is not observed in rodents and primates. As a result, searching through human and mouse corpuses, we identified numerous bovine homologs, among which 11 to 14% of transcription factors including the gold standard TF as well as novel TF potentially important to gene regulation in ruminant embryo development. The scripts of the workflow are written in Perl and available on demand. They require data input coming from all various databases for any kind of biological issue once the data has been prepared according to keywords for the studied topic and species; we can provide data sample to illustrate the use and functionality of the workflow. Results To do so, we created a workflow that allowed the pipeline processing of literature data and biological data, extracted from Web of Science (WoS or PubMed but also from Gene Expression Omnibus (GEO, Gene Ontology (GO, Uniprot, HomoloGene, TcoF-DB and TFe (TF encyclopedia. First, the human and mouse homologs of the bovine proteins were selected, filtered by text corpora and arranged by score functions. The score functions were based on the gene name frequencies in corpora. Then, transcription factors were identified using TcoF-DB and double-checked using TFe to characterise TF groups and families. Thus, among a search space of 18,670 bovine homologs, 489 were identified as transcription factors. Among them, 243 were absent from the high-throughput data available at the time of the study. They thus stand so far for putative TF acting during bovine embryo elongation, but might be retrieved from a recent RNA sequencing dataset (Mamo et al. , 2012. Beyond the 246 TF that appeared expressed in bovine elongating tissues, we restricted our interpretation to those occurring within a list of 50 top-ranked genes. Among the transcription

  5. Modification of mitochondrial function, cytoplasmic lipid content and cryosensitivity of bovine embryos by resveratrol.

    Science.gov (United States)

    Abe, Takahito; Kawahara-Miki, Ryouka; Hara, Tomotaka; Noguchi, Tatsuo; Hayashi, Takeshi; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

    2017-10-18

    Resveratrol is a potent activator of NAD-dependent deacetyltransferase sirtuin-1 (SIRT1) and affects lipid metabolism and ATP generation in somatic cells. In the present study, the effects of supplementing culture medium with resveratrol on lipid metabolism, ATP generation, and cryosensitivity of bovine in vitro produced embryos were investigated. Bovine early cleaved-stage embryos were cultured in medium containing 0 or 0.5 µM resveratrol for 1 or 5 days. Resveratrol treatment for both 1 day and 5 days increased the expression levels of SIRT1 and phosphorylated AMP-activated protein kinase (pAMPK) in the embryos. Furthermore, resveratrol treatment was effective to increase ATP generation and reduce lipid content of the embryos. The effects of resveratrol treatment were diminished by the SIRT1 inhibitor "EX527", and the reduced lipid content was reversed by treatment with etomoxir (a potent inhibitor of beta-oxidation). Blastocysts developed after resveratrol treatment showed low levels reactive oxygen species and increased cryotolerance. These results demonstrate that resveratrol improves in vitro development of bovine embryos, while reducing cytoplasmic lipid content through activation of beta-oxidation, thereby effective for production of bovine blastocysts with enhanced cryotolerance.

  6. Efeito de diferentes meios de cultivo no desenvolvimento e proporção do sexo de embriões bovinos produzidos in vitro Effect of different culture media on development and sex ratio of bovine embryos fertilized in vitro

    Directory of Open Access Journals (Sweden)

    S.G.T. Gilardi

    2004-10-01

    Full Text Available Avaliou-se o efeito da suplementação de meios de cultivo sobre o desenvolvimento e proporção do sexo de embriões bovinos fertilizados in vitro. Complexos cumulus-oócitos obtidos de ovários de matadouro foram maturados e fertilizados in vitro. Os zigotos (n= 484 foram distribuídos aleatoriamente em meio CR2aa, contendo soro fetal bovino (SFB (T1, albumina sérica bovina (BSA (T2 ou BSA mais insulina:transferrina:selênio e vitaminas (BSA+ (T3, no cultivo embrionário in vitro, a uma atmosfera de 5% CO2 a 38,8ºC em ar. A taxa de clivagem foi observada 72-76 horas pós-fertilização (PF e a taxa de blastocistos com sete e oito dias PF. Os blastocistos (n= 63 foram sexados pela técnica de reação em cadeia de polimerase. A taxa de clivagem em T2 foi maior (P0,05 entre T2 e T3, porém menor (P0,05 entre os tratamentos. O T1 influenciou o desenvolvimento de blastocistos, mas não teve efeito sobre a proporção do sexo.The effect of culture media on the development and on the sex ratio of bovine embryos fertilized in vitro was studied. Cumulus oocyte-complexes from slaughterhouse ovaries were matured and fertilized in vitro. Zygotes (n= 484 were randomly allotted to different culture media and cultured with their cumulus cells in CR2aa medium and an atmosphere of 5% CO2 in air at 38.8ºC. The fetal calf serum (FCS, bovine seric albumin (BSA or BSA plus insulin:transferrin:selenium and vitamins (BSA+ supplementation effect on embryo culture was evaluated. Cleavage rate was assessed at 72-76h post-fertilization (PF and blastocyst rate on days 7 and 8 PF. The blastocysts (n= 63 were also sexed using polymerase chain reaction. Cleavage rate for BSA medium supplemented was higher (P0.05, but lower (P<0.01 than FCS. Culture medium FCS supplemented affected blastocyst development but not the sex ratio.

  7. Effects of green tea polyphenols, insulin-like growth factor I and glucose on developmental competence of bovine oocytes

    Directory of Open Access Journals (Sweden)

    Zhengguang Wang

    2012-12-01

    Full Text Available The present study examined the effects of green tea polyphenols (GTP, insulin-like growth factor-I (IGF-I and glucose on oocyte in vitro maturation, subsequent embryo development and blastocyst quality in bovine. Cumulus-oocyte complexes (COC were aspirated from the ovaries and cultured in synthetic oviduct fluid supplemented with MEM amino acids (SOFaa media supplemented with one of the following supplements: GTP (0, 10, 15 and 20 µM, IGF-I (0, 50, 100 and 150 ng/mL or glucose (0, 1.5, 5.6 and 20 mM for 24 h. The results showed that oocytes cultured in media supplemented with 15 µM GTP, 100 ng/mL IGF-I and 5.6 mM glucose, in separate experiments, have higher cleavage and blastocyst rates compared with oocytes cultured in media without or with other concentration of GTP, IGF-I and glucose. Then these three substances with the concentration above were added together into SOFaa media and constituted a modified medium (Modified SOFaa. The COC were cultured in control SOFaa media and modified SOFaa media, respectively. The results showed that modified SOFaa media increased the intracellular glutathione concentration of matured oocytes, blastocyst rates and total cell numbers and cell numbers of inner cell mass per blastocyst compared with the control. Supplementing of GTP, IGF-I and glucose synchronously to maturation media can increase the intracellular GSH concentration of oocytes after in vitro maturation, and improve the embryo development and blastocyst quality in bovine.

  8. Active caspase-3 and ultrastructural evidence of apoptosis in spontaneous and induced cell death in bovine in vitro produced pre-implantation embryos

    DEFF Research Database (Denmark)

    Gjørret, Jakob O.; Fabian, Dusan; Avery, Birthe

    2007-01-01

    In this study we investigated chronological onset and involvement of active caspase-3, apoptotic nuclear morphology, and TUNEL-labeling, as well as ultrastructural evidence of apoptosis, in both spontaneous and induced cell death during pre-implantation development of bovine in vitro produced...... microscopy in both treated and untreated blastocysts. Activation of caspase-3 is likely involved in both spontaneous and induced apoptosis in bovine pre-implantation embryos, and immunohistochemical staining of active caspase-3 may be used in combination with other markers to identify apoptosis in pre...... embryos. Pre-implantation embryos (2-cell to Day 8 blastocysts) were cultured with either no supplementation (untreated) or with 10 µM staurosporine for 24 hr (treated). Embryos were subjected to immunohistochemical staining of active caspase-3, TUNEL-reaction for detection of DNA degradation and DAPI...

  9. Effect of triiodothyronine on developmental competence of bovine oocytes.

    Science.gov (United States)

    Costa, N N; Cordeiro, M S; Silva, T V G; Sastre, D; Santana, P P B; Sá, A L A; Sampaio, R V; Santos, S S D; Adona, P R; Miranda, M S; Ohashi, O M

    2013-09-01

    Developmental competence of in vitro-matured bovine oocytes is a limiting factor in production of embryos in vitro. Several studies have suggested a potential positive effect of thyroid hormones on cultured oocytes and/or their supporting cells. Therefore, the aim of the present study was to ascertain whether medium supplementation with triiodothyronine (T3) improved subsequent developmental competence of in vitro-matured bovine oocytes. For this purpose, we first documented (using reverse transcription PCR) that whereas bovine cumulus cells expressed both thyroid hormone receptor (TR)-α and TRβ, immature bovine oocytes expressed TRα only. Thereafter, to test the effects of TH on developmental competence, abattoir-derived oocytes were matured in vitro in a medium containing 0, 25, 50, or 100 nM T3 and subjected to in vitro fertilization. Embryo quality was evaluated by assessing cleavage and blastocyst rates, morphological quality, development kinetics, and total cell number on Day 8 of culture. Notably, addition of 50 or 100 nM T3 to the in vitro maturation medium increased (P 0.05) on gene expression. We concluded that supplementation of bovine oocyte in vitro maturation medium with T3 may have a beneficial effect on the kinetics of embryo development. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Comparative evaluation of various solid phases for the development of coated tube assays for the estimation of progesterone in human serum, bovine serum and bovine milk

    International Nuclear Information System (INIS)

    Karir, Tarveen; Samuel, Grace; Sivaprasad, N.; Venkatesh, Meera

    2009-01-01

    Immobilization of progesterone antibody using three polystyrene surfaces and two progesterone radiotracers for use in the development of a coated tube assay for the evaluation of progesterone levels in human serum, bovine serum and bovine milk was studied. The selection of the solid phase and the tracers were based on the maximum binding, non-specific binding, sensitivity and percentage recovery. Amongst the polystyrene tubes studied, streptavidin coated tubes showed the acceptable assay features such as low non-specific binding (0.5-1.0%), adequate sensitivity (0.13-0.16 ng/ml) and recovery (85-115%) for all the three sample matrices, human serum, bovine serum and bovine milk.

  11. Superoxide dismutase and taurine supplementation improves in vitro blastocyst yield from poor-quality feline oocytes.

    Science.gov (United States)

    Ochota, Małgorzata; Pasieka, Anna; Niżański, Wojciech

    2016-03-15

    Blastocyst production in vitro seems to be crucial part of assisted reproduction techniques in feline species. However, the results of cats' oocyte maturation and embryo development are still lower than those in other species. The aim of this study was to evaluate whether the supplementation with superoxide dismutase (SOD) and taurine during maturation or culture would improve the blastocyst yield obtained from lower grades of oocytes, that are usually discarded, as not suitable for further in vitro purposes. To investigate the effect of antioxidants' addition, the good- and poor-quality oocytes, were cultured with the addition of 10-mmol taurine and 600 UI/mL SOD. The nuclear maturity, embryo development, and blastocyst quality were subsequently assessed. In control group, without antioxidant supplementation, significantly less poor-quality oocytes matured (42% vs. 62%) and more degenerated (35% vs. 20%), comparing to the experimental group supplemented with SOD and taurine. The amount of obtained blastocyst was much higher, when poor quality oocytes were supplemented with SOD and taurine (supplementation to IVM-4%; supplementation to IVC-5.5%; supplementation to IVM and IVC-5.9% of blastocyst), comparing to not supplemented control group (1.3%). The best blastocysts were obtained when poor oocytes had antioxidants added only during embryo culture (185 ± 13.4 blastomeres vs. 100 ± 1.5 in control). In the present study, we reported that the lower grades of oocytes can better mature and form significantly more blastocysts with better quality, when cultured with addition of SOD and taurine. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Developments in diagnosis and control of bovine paratuberculosis

    DEFF Research Database (Denmark)

    Nielsen, Søren Saxmose

    2014-01-01

    the exposure of susceptible animals to the milk and faeces of infected animals. However, cost-effectiveness may depend on labour costs, and strategic use of diagnostics may have certain appeals through the information provided. Current bulk tank milk tests are not deemed to have a role in MAP control, whereas......Bovine paratuberculosis can be costly to farmers who, as a consequence, may be interested in control of the causative agent, Mycobacterium avium subsp. paratuberculosis (MAP). Between-herd spread is primarily due to movement of MAP-infected livestock, and within-herd transmission most often occurs...

  13. Development of a new live rough vaccine against bovine brucellosis

    International Nuclear Information System (INIS)

    Comerci, D.J.; Ugalde, J.E.; Ugalde, R.A.

    2005-01-01

    Brucella abortus S19 is the most commonly used attenuated live vaccine to prevent bovine brucellosis. In spite of its advantages, S19 has several drawbacks: it is abortive for pregnant cattle, is virulent for humans, and re-vaccination is not advised due to the persistence of anti-lipopolysaccharide (LPS) antibodies that hamper the immunoscreening procedures. For these reasons, there is a continuous search for new bovine vaccine candidates. We have previously characterized the phenotype of the phosphoglucomutase (pgm) gene disruption in Brucella abortus S2308, as well as the possible role for the smooth LPS in virulence and intracellular multiplication. Here we evaluate the vaccine properties of an unmarked deletion mutant of pgm. Western blot analysis of purified lipopolysaccharide and whole-cell extract from Δpgm indicate that it synthesizes O-antigen but is incapable of assembling a complete LPS. In consequence Δpgm has a rough phenotype. Experimental infections of mice indicate that Δpgm is avirulent. Vaccination with Δpgm induces protection levels comparable to those induced by S19, and generates a splenocyte proliferative response and cytokines profile typical of a Th-1 response. The ability of the mutant to generate a strong cellular Th-1 response without eliciting specific O-antigen antibodies highlights the potential use of this mutant as a new live vaccine for cattle. (author)

  14. Production of bovine hand-made cloned embryos by zygote-oocyte cytoplasmic hemi-complementation.

    Science.gov (United States)

    Mezzalira, Joana Claudia; Ohlweiler, Lain Uriel; da Costa Gerger, Renato Pereira; Casali, Renata; Vieira, Fabiano Koerich; Ambrósio, Carlos Eduardo; Miglino, Maria Angélica; Rodrigues, José Luiz; Mezzalira, Alceu; Bertolini, Marcelo

    2011-02-01

    The aim of this study was to evaluate the effect of the cytoplast type and activation process on development of cloned embryos. Bovine oocytes (MII) or zygotes at the one-cell stage (IVF) were manually bisected and segregated in MII or IVF hemi-cytoplasts or hemi-karyoplasts. Adult skin cells from a bovine female were used as nucleus donors (SC). Experimental groups were composed of IVF embryos; parthenogenetic embryos; hand-made cloned (HMC) embryos; and reconstructed HMC embryos using IVF hemi-cytoplast + MII hemi-cytoplast + SC (G-I); IVF hemi-cytoplast + IVF hemi-cytoplast + SC (G-II); MII hemi-cytoplast + IVF hemi-karyoplast (G-III); and IVF hemi-cytoplast + IVF hemi-karyoplast (G-IV). Embryos from G-I to G-IV were allocated to subgroups as sperm-activated (SA) or were further chemically activated (SA + CA). Embryos from all groups and subgroups were in vitro cultured in the WOW system. Blastocyst development in subgroup G-I SA (28.2%) was similar to IVF (27.0%) and HMC (31.4%) controls, perhaps due to a to a more suitable activation process and/or better complementation of cytoplasmic reprogramming factors, with the other groups and subgroups having lower levels of development. No blastocyst development was observed when using IVF hemi-karyoplasts (G-III and G-IV), possibly due to the manipulation process during a sensitive biological period. In summary, the presence of cytoplasmic factors from MII hemi-oocytes and the sperm activation process from hemi-zygotes appear to be necessary for adequate in vitro development, as only the zygote-oocyte hemi-complementation was as efficient as controls for the generation of bovine cloned blastocysts.

  15. Effect of supplementation of green tea polyphenols on the developmental competence of bovine oocytes in vitro

    Directory of Open Access Journals (Sweden)

    Z.G. Wang

    2007-08-01

    Full Text Available The objective of the present study was to examine the effect of green tea polyphenols (GTPs supplementation during in vitro maturation, in vitro fertilization, and in vitro culture on the developmental competence of bovine oocytes. Cumulus-oocyte complexes aspirated from the ovaries were matured in vitro (38.5ºC for 24 h and fertilized (38.5ºC for 15-18 h and embryos were cultured (38.5ºC for 192 h in a defined conditioned medium with or without GTPs supplementation. The GTPs used in the present study contained 99% catechin derivatives, with the major components being 50% (--epigallocatechin gallate, 22% (--epicatechin gallate, 18% (--epigallocatechin, and 10% (--epicatechin. Four replicate trials were done for each type of experiment. GTPs supplementation (15 µM of the maturation medium led to a significant increase in the rate of blastocyst formation (34.0 vs 21.4%, P < 0.05. However, the rate of blastocyst formation was not improved when higher GTPs concentrations (20 or 25 µM were added to the in vitro maturation medium. During in vitro fertilization, supplementation with higher GTPs concentrations (20 or 25 µM significantly reduced the rate of blastocyst formation (P < 0.05. Supplementation of the culture medium with 15 µM GTPs improved the rate of blastocyst formation, while higher GTPs concentrations (25 µM significantly reduced embryo development (P < 0.05. In conclusion, these results demonstrate that supplementation with GTPs at low concentration (15 µM during in vitro maturation and in vitro culture improved the developmental competence of bovine oocytes.

  16. Expression of microRNAs in bovine and human pre-implantation embryo culture media

    Science.gov (United States)

    Kropp, Jenna; Salih, Sana M.; Khatib, Hasan

    2014-01-01

    MicroRNAs (miRNA) are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, miR-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy. PMID:24795753

  17. An Epigenetic Modifier Results in Improved In Vitro Blastocyst production after Somatic Cell Nuclear Transfer

    DEFF Research Database (Denmark)

    Zhang, Yunhai; Li, Juan; Villemoes, Klaus

    2007-01-01

    The present study was designed to examine the effect of trichostatin A (TSA), an inhibitor of histone deacetylase, on development of porcine cloned embryos. Our results showed that treatment of cloned embryos derived from sow oocytes with 50 nM TSA for up to 24 h after the onset of activation cou...... were tested, and for all cell lines an enhancement in blastocyst development compared to their corresponding control was observed. Our data demonstrate that TSA treatment after somatic cell nuclear transfer in the pig can significantly improve the in vitro blastocyst production...

  18. Development to term of sheep embryos reconstructed after inner cell mass/trophoblast exchange.

    Science.gov (United States)

    Loi, Pasqualino; Galli, Cesare; Lazzari, Giovanna; Matsukawa, Kazutsugu; Fulka, Josef; Goeritz, Frank; Hildebrandt, Thomas B

    2018-04-13

    Here we report in vitro and term development of sheep embryos after the inner cell mass (ICM) from one set of sheep blastocysts were injected into the trophoblast vesicles of another set. We also observed successful in vitro development of chimeric blastocysts made from sheep trophoblast vesicles injected with bovine ICM. First, we dissected ICMs from 35 sheep blastocysts using a stainless steel microblade and injected them into 29 re-expanded sheep trophoblastic vesicles. Of the 25 successfully micromanipulated trophoblastic vesicles, 15 (51.7%) re-expanded normally and showed proper ICM integration. The seven most well reconstructed embryos were transferred for development to term. Three ewes receiving manipulated blastocysts were pregnant at day 45 (42.8%), and all delivered normal offspring (singletons, two females and one male, average weight: 3.54 ± 0.358 kg). Next, we monitored in vitro development of sheep trophoblasts injected with bovine ICMs. Of 17 injected trophoblastic vesicles, 10 (58.8%) re-expanded after 4 h in culture, and four (40%) exhibited integrated bovine ICM. Our results indicate that ICM/trophoblast exchange is feasible, allowing full term development with satisfactory lambing rate. Therefore, ICM exchange is a promising approach for endangered species conservation.

  19. The presence of acylated ghrelin during in vitro maturation of bovine oocytes induces cumulus cell DNA damage and apoptosis, and impairs early embryo development.

    Science.gov (United States)

    Sirini, Matias A; Anchordoquy, Juan Mateo; Anchordoquy, Juan Patricio; Pascua, Ana M; Nikoloff, Noelia; Carranza, Ana; Relling, Alejandro E; Furnus, Cecilia C

    2017-10-01

    The aim of this study was to investigate the effects of acylated ghrelin supplementation during in vitro maturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus-oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. The in vitro effects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

  20. Effect of adiponectin on bovine granulosa cell steroidogenesis, oocyte maturation and embryo development

    Directory of Open Access Journals (Sweden)

    Coyral-Castel Stéphanie

    2010-03-01

    Full Text Available Abstract Background Adiponectin is an adipokine, mainly produced by adipose tissue. It regulates several reproductive processes. The protein expression of the adiponectin system (adiponectin, its receptors, AdipoR1 and AdipoR2 and the APPL1 adaptor in bovine ovary and its role on ovarian cells and embryo, remain however to be determined. Methods Here, we identified the adiponectin system in bovine ovarian cells and embryo using RT-PCR, immunoblotting and immunohistochemistry. Furthermore, we investigated in vitro the effects of recombinant human adiponectin (10 micro g/mL on proliferation of granulosa cells (GC measured by [3H] thymidine incorporation, progesterone and estradiol secretions measured by radioimmunoassay in the culture medium of GC, nuclear oocyte maturation and early embryo development. Results We show that the mRNAs and proteins for the adiponectin system are present in bovine ovary (small and large follicles and corpus luteum and embryo. Adiponectin, AdipoR1 and AdipoR2 were more precisely localized in oocyte, GC and theca cells. Adiponectin increased IGF-1 10(-8 M-induced GC proliferation (P Conclusions In bovine species, adiponectin decreased insulin-induced steroidogenesis and increased IGF-1-induced proliferation of cultured GC through a potential involvement of ERK1/2 MAPK pathway, whereas it did not modify oocyte maturation and embryo development in vitro.

  1. Vaccines for bovine neosporosis: current status and key aspects for development.

    Science.gov (United States)

    Horcajo, P; Regidor-Cerrillo, J; Aguado-Martínez, A; Hemphill, A; Ortega-Mora, L M

    2016-12-01

    Bovine neosporosis is a worldwide concern due to its global distribution and great economic impact. Reproductive failure in cattle due to abortion leads to major economic losses associated with the disease. Currently, there is no treatment or vaccine available against abortion or transmission caused by Neospora caninum infection in cattle. However, vaccination is considered the best measure of control against bovine neosporosis. Several host and parasite factors can influence the dynamics of the infection in bovines. Moreover, the availability of well-defined infection models is a key factor for the evaluation of vaccine candidates. However, working with cattle is not easy due to difficult handling, facilities and costs, and therefore, 'more affordable' models could be used for screening of promising vaccines to establish proof of concept. So far, live-attenuated vaccines have shown good efficacy against exogenous transplacental transmission; however, they have relevant disadvantages and associated risks, which render inactivated or subunit vaccines the best way forward. The identification of novel potential targets and vaccines, and the application of innovative vaccine technologies in harmonized experimental animal models, will accelerate the development of an effective vaccine against bovine neosporosis. © 2016 John Wiley & Sons Ltd.

  2. Radiofrequency Ablation (RFA): Development of a Flow Model for Bovine Livers for Extensive Bench Testing

    International Nuclear Information System (INIS)

    Lubienski, Andreas; Bitsch, Rudi G.; Lubienski, Katrin; Kauffmann, Guenter; Duex, Markus

    2006-01-01

    Purpose. To develop a flow model for bovine livers for extensive bench testing of technical improvements or procedure-related developments of radiofrequency ablation excluding animal experiments. Methods. The perfusion of bovine livers directly from the slaughterhouse was simulated in a liver perfusion tank developed for the experimental work. The liver perfusion medium used was a Tyrode solution prepared in accordance with physiologic criteria (as for liver transplants) which was oxygenated by an oxygenator and heated to 36.5 deg. C. Portal vein circulation was regulated via a flow- and pressure-controlled pump and arterial circulation using a dialysis machine. Flow rate and pressure were adjusted as for the physiology of a human liver converted to bovine liver conditions. The fluid discharged from the liver was returned into the perfusion system through the vena cava. Extendable precision swivel arms with the radiofrequency probe attached were mounted on the liver perfusion tank. RFA was conducted with the RF3000 generator and a 2 cm LeVeen needle (Boston Scientific, Ratingen, Germany) in a three-dimensional grid for precise localization of the generated thermolesions. Results. Four bovine livers weighing 8.4 ± 0.4 kg each were prepared, connected to the perfusion system, and consecutively perfused for the experiments. Mean arterial flow was 569 ± 43 ml/min, arterial pressure 120 mmHg, portovenous flow 1440 ± 305 ml/min, and portal pressure 10 mmHg. Macroscopic evaluation after the experiments revealed no thrombi within the hepatic vessels. A total of 136 RF thermolesions were generated with an average number of 34 per liver. Mean RF duration was 2:59 ± 2:01 min:sec with an average baseline impedance of 28.2 ± 3.4 ohms. The mean diameter of the thermolesions along the puncture channel was 22.98 ± 4.34 mm and perpendicular to the channel was 23.27 ± 4.82 mm. Conclusion. Extracorporeal perfusion of bovine livers with consecutive standardized RF ablation was

  3. Development of an antibody to bovine IL-2 reveals multifunctional CD4 T(EM) cells in cattle naturally infected with bovine tuberculosis.

    Science.gov (United States)

    Whelan, Adam O; Villarreal-Ramos, Bernardo; Vordermeier, H Martin; Hogarth, Philip J

    2011-01-01

    Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2). Using recombinant phage display technology, we have identified an antibody that recognises biologically active bIL-2. Using this antibody, we have developed a polychromatic flow cytometric staining panel that has allowed the investigation of multifunctional CD4 T-cells responses in cattle naturally infected with M. bovis. Assessment of the frequency of antigen specific CD4 T cell subsets reveals a dominant IFN-γ(+)IL-2(+)TNF-α(+) and IFN-γ(+) TNF-α(+) response in naturally infected cattle. These multifunctional CD4 T cells express a CD44(hi)CD45RO(+)CD62L(lo) T-effector memory (T(EM)) phenotype and display higher cytokine median fluorescence intensities than single cytokine producers, consistent with an enhanced 'quality of response' as reported for multifunctional cells in human and murine systems. Through our development of these novel immunological bovine tools, we provide the first description of multifunctional T(EM) cells in cattle. Application of these tools will improve our understanding of protective immunity in bovine TB and allow more direct comparisons of the complex T cell mediated immune responses between murine models, human clinical studies and bovine TB models in the future. © 2011 Whelan et al.

  4. Development of an antibody to bovine IL-2 reveals multifunctional CD4 T(EM cells in cattle naturally infected with bovine tuberculosis.

    Directory of Open Access Journals (Sweden)

    Adam O Whelan

    Full Text Available Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2. Using recombinant phage display technology, we have identified an antibody that recognises biologically active bIL-2. Using this antibody, we have developed a polychromatic flow cytometric staining panel that has allowed the investigation of multifunctional CD4 T-cells responses in cattle naturally infected with M. bovis. Assessment of the frequency of antigen specific CD4 T cell subsets reveals a dominant IFN-γ(+IL-2(+TNF-α(+ and IFN-γ(+ TNF-α(+ response in naturally infected cattle. These multifunctional CD4 T cells express a CD44(hiCD45RO(+CD62L(lo T-effector memory (T(EM phenotype and display higher cytokine median fluorescence intensities than single cytokine producers, consistent with an enhanced 'quality of response' as reported for multifunctional cells in human and murine systems. Through our development of these novel immunological bovine tools, we provide the first description of multifunctional T(EM cells in cattle. Application of these tools will improve our understanding of protective immunity in bovine TB and allow more direct comparisons of the complex T cell mediated immune responses between murine models, human clinical studies and bovine TB models in the future.

  5. Expression of HSG is essential for mouse blastocyst formation

    International Nuclear Information System (INIS)

    Jiang Guangjian; Pan Lei; Huang Xiuying; Han Mei; Wen Jinkun; Sun Fangzhen

    2005-01-01

    It has been shown recently that hyperplasia suppressor gene (HSG) is a powerful regulator for cell proliferation and has a critical role in mitochondrial fusion in many cells. However, little is known about its expression, localization, and function during oocyte maturation and early embryogenesis. In this study, with indirect immunofluorescent staining and Western blotting, we found that HSG was expressed in mouse oocytes and preimplantation embryos which primarily exhibited a submembrane distribution pattern in the cytoplasm. Moreover, HSG mainly associated with β-tubulin during oocyte maturation and early embryonic development. When mouse zygotes were injected with HSG antisense plasmid and cultured in vitro, their capacity to form blastocysts was severely impaired. Our results indicate that HSG plays an essential role in mouse preimplantation development

  6. Development of LC-MS/MS confirmatory method for the determination of testosterone in bovine serum

    Directory of Open Access Journals (Sweden)

    Woźniak Barbara

    2017-03-01

    Full Text Available Introduction: In the European Union the use of steroid growth promoters is prohibited under Council Directive 96/22/EC. For effective control of illegal use of natural steroids, highly sensitive analytical methods are required, because sex hormones can be present in very low concentrations in biological samples. The aim of the study was to develop a confirmatory method for the detection of testosterone in bovine serum at ppt level.

  7. PRODUCTION OF EMBBRYONIC STEM CELLS FROM INNER CELL MASS OF BLASTOCYST ISOLATED BY ENZYMATIC AND IMMUNOSURGERY METHODS

    Directory of Open Access Journals (Sweden)

    Thomas Mata Hine

    2008-03-01

    Full Text Available The objective of the research is determining the ICM isolation method to produce ESC. Blastocyst stage of DDy mice embryos were used in this study. Zona pellucida of blastocysts were removed by 0.25% pronase, the ICM isolation were done by enzimatic or immunosurgery method, and then they were cultured in DMEM-high glucose supplemented with mercaptoethanol, gentamycin, fetal bovine serum, and cumulus cells as feeder layer. The result of the research indicated that immunosurgery method yielding attachment rate and number ESC colony 93.85% and 43.08%, respectively, higher (P<0.05 than enzimatic method that weree 79.63% and 18.52%, respectively, but the viability of ICM cells were equal (P >0.05 that are 93.59% in enzymatic method and 98.56% in immunosurgery method. This research concluded that immunosurgery more effective method for isolation of ICM and ESC production than enzymatic method.

  8. Ouabain stimulates a Na+/K+-ATPase-mediated SFK-activated signalling pathway that regulates tight junction function in the mouse blastocyst.

    Directory of Open Access Journals (Sweden)

    Holly Giannatselis

    Full Text Available The Na(+/K(+-ATPase plays a pivotal role during preimplantation development; it establishes a trans-epithelial ionic gradient that facilitates the formation of the fluid-filled blastocyst cavity, crucial for implantation and successful pregnancy. The Na(+/K(+-ATPase is also implicated in regulating tight junctions and cardiotonic steroid (CTS-induced signal transduction via SRC. We investigated the expression of SRC family kinase (SFK members, Src and Yes, during preimplantation development and determined whether SFK activity is required for blastocyst formation. Embryos were collected following super-ovulation of CD1 or MF1 female mice. RT-PCR was used to detect SFK mRNAs encoding Src and Yes throughout preimplantation development. SRC and YES protein were localized throughout preimplantation development. Treatment of mouse morulae with the SFK inhibitors PP2 and SU6656 for 18 hours resulted in a reversible blockade of progression to the blastocyst stage. Blastocysts treated with 10(-3 M ouabain for 2 or 10 minutes and immediately immunostained for phosphorylation at SRC tyr418 displayed reduced phosphorylation while in contrast blastocysts treated with 10(-4 M displayed increased tyr418 fluorescence. SFK inhibition increased and SFK activation reduced trophectoderm tight junction permeability in blastocysts. The results demonstrate that SFKs are expressed during preimplantation development and that SFK activity is required for blastocyst formation and is an important mediator of trophectoderm tight junction permeability.

  9. MicroRNA-212 post-transcriptionally regulates oocyte-specific basic-helix-loop-helix transcription factor, factor in the germline alpha (FIGLA, during bovine early embryogenesis.

    Directory of Open Access Journals (Sweden)

    Swamy K Tripurani

    Full Text Available Factor in the germline alpha (FIGLA is an oocyte-specific basic helix-loop-helix transcription factor essential for primordial follicle formation and expression of many genes required for folliculogenesis, fertilization and early embryonic survival. Here we report the characterization of bovine FIGLA gene and its regulation during early embryogenesis. Bovine FIGLA mRNA expression is restricted to gonads and is detected in fetal ovaries harvested as early as 90 days of gestation. FIGLA mRNA and protein are abundant in germinal vesicle and metaphase II stage oocytes, as well as in embryos from pronuclear to eight-cell stage but barely detectable at morula and blastocyst stages, suggesting that FIGLA might be a maternal effect gene. Recent studies in zebrafish and mice have highlighted the importance of non-coding small RNAs (microRNAs as key regulatory molecules targeting maternal mRNAs for degradation during embryonic development. We hypothesized that FIGLA, as a maternal transcript, is regulated by microRNAs during early embryogenesis. Computational predictions identified a potential microRNA recognition element (MRE for miR-212 in the 3' UTR of the bovine FIGLA mRNA. Bovine miR-212 is expressed in oocytes and tends to increase in four-cell and eight-cell stage embryos followed by a decline at morula and blastocyst stages. Transient transfection and reporter assays revealed that miR-212 represses the expression of FIGLA in a MRE dependent manner. In addition, ectopic expression of miR-212 mimic in bovine early embryos dramatically reduced the expression of FIGLA protein. Collectively, our results demonstrate that FIGLA is temporally regulated during bovine early embryogenesis and miR-212 is an important negative regulator of FIGLA during the maternal to zygotic transition in bovine embryos.

  10. Characterization of SCF-Complex during Bovine Preimplantation Development

    Czech Academy of Sciences Publication Activity Database

    Benešová, Veronika; Kinterová, Veronika; Kaňka, Jiří; Toralová, Tereza

    2016-01-01

    Roč. 11, č. 1 (2016), e0147096-e0147096 E-ISSN 1932-6203 R&D Projects: GA ČR GP13-24730P Institutional support: RVO:67985904 Keywords : F-box protein * early development Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.806, year: 2016

  11. Development of a Subunit Vaccine for Contagious Bovine ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Their work has set the stage for commercial development of a sub-unit vaccine. ... The sub-unit vaccine will be cost-effective, easy to produce, and safe. How it will make a ... IDRC invites applications for the IDRC Doctoral Research Awards.

  12. Post-hatching development of the porcine and bovine embryo-defining criteria for expected development in vivo and in vitro

    DEFF Research Database (Denmark)

    Vejlsted, Morten; Du, Yutao; Vajta, Gábor

    2006-01-01

    without the need for transfer to recipient animals. Such a system would require (1) definition of milestones of expected post-hatching embryonic development in vivo; and (2) development of adequate culture systems. We propose a stereomicroscopical staging system for post-hatching embryos defining......Particular attention has been paid to the pre-hatching period of embryonic development although blastocyst development is a poor indicator of embryo viability. Post-hatching embryonic dev elopment in vitro would allow for establishment of more accurate tools for evaluating developmental potential...

  13. [Investigation of neural stem cell-derived donor contribution in the inner ear following blastocyst injection].

    Science.gov (United States)

    Volkenstein, S; Brors, D; Hansen, S; Mlynski, R; Dinger, T C; Müller, A M; Dazert, S

    2008-03-01

    Utilising the enormous proliferation and multi-lineage differentiation potentials of somatic stem cells represents a possible therapeutical strategy for diseases of non-regenerative tissues like the inner ear. In the current study, the possibility of murine neural stem cells to contribute to the developing inner ear following blastocyst injection was investigated. Fetal brain-derived neural stem cells from the embryonic day 14 cortex of male mice were isolated and expanded for four weeks in neurobasal media supplemented with bFGF and EGF. Neural stem cells of male animals were harvested, injected into blastocysts and the blastocysts were transferred into pseudo-pregnant foster animals. Each blastocyst was injected with 5-15 microspheres growing from single cell suspension from neurospheres dissociated the day before. The resulting mice were investigated six months POST PARTUM for the presence of donor cells. Brainstem evoked response audiometry (BERA) was performed in six animals. To visualize donor cells Lac-Z staining was performed on sliced cochleas of two animals. In addition, the cochleas of four female animals were isolated and genomic DNA of the entire cochlea was analyzed for donor contribution by Y-chromosome-specific PCR. All animals had normal thresholds in brainstem evoked response audiometry. The male-specific PCR product indicating the presence of male donor cells were detected in the cochleas of three of the four female animals investigated. In two animals, male donor cells were detected unilateral, in one animal bilateral. The results suggest that descendants of neural stem cells are detectable in the inner ear after injection into blastocysts and possess the ability to integrate into the developing inner ear without obvious loss in hearing function.

  14. Rho-associated kinase activity is required for proper morphogenesis of the inner cell mass in the mouse blastocyst.

    Science.gov (United States)

    Laeno, Arlene May A; Tamashiro, Dana Ann A; Alarcon, Vernadeth B

    2013-11-01

    The blastocyst consists of the outer layer of trophectoderm and pluripotent inner cell mass (ICM), the precursor of the placenta and fetus, respectively. During blastocyst expansion, the ICM adopts a compact, ovoidal shape, whose proper morphology is crucial for normal embryogenesis. Rho-associated kinase (ROCK), an effector of small GTPase RHO signaling, mediates the diverse cellular processes of morphogenesis, but its role in ICM morphogenesis is unclear. Here, we demonstrate that ROCK is required for cohesion of ICM cells and formation of segregated tissues called primitive endoderm (PrE) and epiblast (Epi) in the ICM of the mouse blastocyst. Blastocyst treatment with ROCK inhibitors Y-27632 and Fasudil caused widening or spreading of the ICM, and intermingling of PrE and Epi. Widening of ICM was independent of trophectoderm because isolated ICMs as well as colonies of mouse embryonic stem cells (mESC) also spread upon Y-27632 treatment. PrE, Epi, and trophectoderm cell numbers were similar between control and treated blastocysts, suggesting that ROCK inhibition affected ICM morphology but not lineage differentiation. Rock1 and Rock2 knockdown via RNA interference in mESC also induced spreading, supporting the conclusion that morphological defects caused by the pharmacological inhibitors were due to ROCK inactivation. When blastocysts were transferred into surrogates, implantation efficiencies were unaffected by ROCK inhibition, but treated blastocysts yielded greater fetal loss. These results show that proper ICM morphology is dependent on ROCK activity and is crucial for fetal development. Our studies have wider implication for improving efficiencies of human assisted reproductive technologies that diminish pregnancy loss and promote successful births.

  15. Effect of two activation treatments and age of blastomere karyoplasts on in vitro development of bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Booth, Paul J; Holm, Peter; Vajta, Gabor

    2001-01-01

    The yield and quality of (a) parthenogenetic blastocysts produced by two activation treatments (cycloheximide [CHX] or 6-dimethylaminopurine [DMAP]) and (b) nuclear transfer blastocysts generated using these two activation treatments and three different ages of karyoplast derived from day 3, 4......, or 5 in vitro produced donor embryos, were examined in order to define an optimal nuclear transfer protocol. The two activation protocols comprised calcium ionophore followed by either CHX or DMAP. Parthenogenetic blastocyst yields were greater (P ....7 +/- 5.1 vs. 31.4 +/- 4.5 [mean +/- SEM]). In contrast, nuclear transfer blastocyst rates per fused embryo were lower (P

  16. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

    Directory of Open Access Journals (Sweden)

    Page Grier P

    2009-04-01

    Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT. Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively. However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

  17. Decreased Blastocyst Production in Mice Exposed to Increased Rack Noise

    Science.gov (United States)

    Zamora, Bernadette M; Jiang, Meisheng; Wang, Ying; Chai, Minghua; Lawson, P Timothy; Lawson, Gregory W

    2009-01-01

    This study was conducted to investigate the possible effect of rack type on the blastocyst yield of mouse embryo donors. The first phase of the study consisted of housing some mice (group A) in a ventilated rack and others (group B) in a static rack in the same room for 3 d, followed by euthanasia for blastocyst collection and corticosterone assay. Parametric tests were used to compare groups. The number of blastocysts per donor was lower in group A (5.0 ± 1.4 blastocysts) than group B (13.1 ± 3.7 blastocysts). Mean noise was higher in the ventilated rack (80.4 dBC) than in the static rack (69.2 dBC). Serum corticosterone concentrations did not differ between groups. For the second phase of the study, a third group of mice (group C) was housed in a static rack without a ventilated rack in the same room. The noise level for group C was even lower (45.18 ± 2.91 dBC), and the blastocyst count per donor (16.4 ± 2.4) was higher than that of group B. The mean noise levels of empty ventilated and static racks differed significantly between groups for 10 different sound frequencies. Plotting mean blastocyst production against mean rack noise revealed a negative linear relationship with good strength of correlation. These results support the earlier observation that decreased blastocyst count occurs following housing of bred C57BL/6 donor mice in ventilated cages. PMID:19807968

  18. Gene expression studies of developing bovine longissimus muscle from two different beef cattle breeds

    Directory of Open Access Journals (Sweden)

    Byrne Keren A

    2007-08-01

    Full Text Available Abstract Background The muscle fiber number and fiber composition of muscle is largely determined during prenatal development. In order to discover genes that are involved in determining adult muscle phenotypes, we studied the gene expression profile of developing fetal bovine longissimus muscle from animals with two different genetic backgrounds using a bovine cDNA microarray. Fetal longissimus muscle was sampled at 4 stages of myogenesis and muscle maturation: primary myogenesis (d 60, secondary myogenesis (d 135, as well as beginning (d 195 and final stages (birth of functional differentiation of muscle fibers. All fetuses and newborns (total n = 24 were from Hereford dams and crossed with either Wagyu (high intramuscular fat or Piedmontese (GDF8 mutant sires, genotypes that vary markedly in muscle and compositional characteristics later in postnatal life. Results We obtained expression profiles of three individuals for each time point and genotype to allow comparisons across time and between sire breeds. Quantitative reverse transcription-PCR analysis of RNA from developing longissimus muscle was able to validate the differential expression patterns observed for a selection of differentially expressed genes, with one exception. We detected large-scale changes in temporal gene expression between the four developmental stages in genes coding for extracellular matrix and for muscle fiber structural and metabolic proteins. FSTL1 and IGFBP5 were two genes implicated in growth and differentiation that showed developmentally regulated expression levels in fetal muscle. An abundantly expressed gene with no functional annotation was found to be developmentally regulated in the same manner as muscle structural proteins. We also observed differences in gene expression profiles between the two different sire breeds. Wagyu-sired calves showed higher expression of fatty acid binding protein 5 (FABP5 RNA at birth. The developing longissimus muscle of

  19. Beneficial effect of two culture systems with small groups of embryos on the development and quality of in vitro-produced bovine embryos.

    Science.gov (United States)

    Cebrian-Serrano, A; Salvador, I; Silvestre, M A

    2014-02-01

    Currently, in vitro-produced embryos derived by ovum pick up (OPU) and in vitro fertilization (IVF) technologies represent approximately one-third of the embryos worldwide in cattle. Nevertheless, the culture of small groups of embryos from an individual egg donor is an issue that OPU-IVF laboratories have to face. In this work, we tested whether the development and quality of the preimplantation embryos in vitro cultured in low numbers (five embryos) could be improved by the addition of epidermal growth factor, insulin, transferrin and selenium (EGF-ITS) or by the WOW system. With this aim, immature oocytes recovered from slaughtered heifers were in vitro matured and in vitro fertilized. Presumptive zygotes were then randomly cultured in four culture conditions: one large group (LG) (50 embryos/500 μl medium) and three smaller groups [five embryos/50 μl medium without (control) or with EGF-ITS (EGF-ITS) and five embryos per microwell in the WOW system (WOW)]. Embryos cultured in LG showed a greater ability to develop to blastocyst stage than embryos cultured in smaller groups, while the blastocyst rate of WOW group was significantly higher than in control. The number of cells/blastocyst in LG was higher than control or WOW, whereas the apoptosis rate per blastocyst was lower. On the other hand, the addition of EGF-ITS significantly improved both parameters compared to the control and resulted in similar embryo quality to LG. In conclusion, the WOW system improved embryo development, while the addition of EGF-ITS improved the embryo quality when smaller groups of embryos were cultured. © 2013 Blackwell Verlag GmbH.

  20. Proteomic analysis of the early bovine yolk sac fluid and cells from the day 13 ovoid and elongated preimplatation embryos

    DEFF Research Database (Denmark)

    Jensen, Pernille L.; Beck, Hans Christian; Petersen, Tonny S.

    2014-01-01

    differentiate into the hypoblast and epiblast, which remain surrounded by the trophectoderm. The formation of the hypoblast epithelium is also accompanied by a change in the fluid within the embryo, i.e., the blastocoel fluid gradually alters to become the primitive yolk sac (YS) fluid. Our previous research......The bovine blastocyst hatches 8 to 9 days after fertilization, and this is followed by several days of preimplantation development during which the embryo transforms from a spherical over an ovoid to an elongated shape. As the spherical embryo enlarges, the cells of the inner cell mass...... describes the protein composition of human and bovine blastocoel fluid, which is surrounded by the trophectoderm and undifferentiated cells of the inner cell mass. In this study, we further examine the changes in the protein composition in both the primitive YS fluid and the embryonic cells during early...

  1. Vitamin K2 improves developmental competency and cryo-tolerance of in vitro derived ovine blastocyst.

    Science.gov (United States)

    Sefid, Fatemeh; Ostadhosseini, S; Hosseini, S M; Ghazvini Zadegan, F; Pezhman, M; Nasr Esfahani, Mohammad Hossein

    2017-08-01

    Vitamin K2 (VK2), acts as an electron carrier in mitochondria and thereby effects reactive oxygen species (ROS) and ATP production. This study evaluates role of VK2 on in vitro developmental competency and cryo-survival of pre-implantation ovine embryos. Initially the optimal and beneficial concentration of VK2 on compaction and blastocyst formation rates was defined (0.1 μM). Subsequently, it was shown that 0.1 μM VK2, at blastocyst stage, reduces H2O2 production, increase the expression of mitochondrial related gene and improved embryos quality. We further assessed presence VK2 supplementation before and/or after vitrification of in vitro derived blastocysts. Our results reveal that presence of VK2 before and after vitrification improves rates of blastocysts re-expansion (88.19± 3.37% vs 73.68± 1.86%, P < 0.05) and hatching (49.55± 4.37% vs 32.7± 3.32%) compared to control group. These observation were consistent with reduction in H2O2 production and improved in expression of mitochondrial related genes. However, VK2 before or after vitrification, not only had no positive effect on these two parameters, but also significantly reduced these parameters. Therefore, in concordance with pervious report in bovine, we show that VK2 supplementation post genomic activation (Day 3-7) improved developmental competency of ovine in vitro derived embryos. We also showed that presence of VK2 after vitrification improves the cryo-survival of ovine embryos. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Engineered Bovine Antibodies in the Development of Novel Therapeutics, Immunomodulators and Vaccines

    Directory of Open Access Journals (Sweden)

    Madhuri Koti

    2014-05-01

    Full Text Available Some bovine antibodies across all classes are unique, such as the CDR3 of the variable heavy-domain (VH CDR3, which is exceptionally long (up to 66 amino acids, unlike most conventional antibodies where the VH CDR3 loops range from 10 to 25 amino acids. The exceptionally long VH CDR3 is encoded by unusually long germline IGHD genes together with insertion of novel “a” nucleotide rich conserved short nucleotide sequence (CSNS specifically at the IGH V-D junction. Such an exceptionally long VH CDR3 confers unique “knob and stalk” structural architecture where the knob, formed by intra-VH CDR3 disulfide bridges, is separated by 20 Å solvent exposed stalk composed of anti-parallel beta strands. The substitution of the knob with cytokines, such as, erythropoietin and granulocyte colony stimulating factor 3 (granulocyte colony stimulating factor, results in expression of functional fusion proteins with enhanced pharmacokinetics. The beta stranded stalk can be substituted with other rigid structures, for example, repeat alpha helices to form coiled-coil that mimics the beta-stranded stalk and, thus, opens opportunities for insertion of this structure in the CDRs of antibodies across species. Given the versatility of such a structural platform in bovine antibody VH CDR3, it provides the opportunity for the development of new generation of diagnostics, therapeutics, vaccines and immunomodulating drugs.

  3. Prepubertal goat oocytes from large follicles result in similar blastocyst production and embryo ploidy than those from adult goats.

    Science.gov (United States)

    Romaguera, R; Moll, X; Morató, R; Roura, M; Palomo, M J; Catalá, M G; Jiménez-Macedo, A R; Hammami, S; Izquierdo, D; Mogas, T; Paramio, M T

    2011-07-01

    Developmental competence of oocytes from prepubertal females is lower than those from adult females. Oocyte development competence is positively related to follicular diameter. Most of the follicles of prepubertal goat ovaries are smaller than 3 mm. The aim of this study was to compare oocytes of two follicle sizes (goats with oocytes from adult goats in relation to their in vitro production and quality of blastocysts. Oocytes from prepubertal goats were obtained from slaughterhouse ovaries and selected according to the follicle diameter whereas oocytes from adult goats were recovered in vivo by LOPU technique without prior selection of follicle size. COCs were IVM for 27 h, IVF at the conventional conditions with fresh semen and presumptive zygotes were cultured in SOF medium for 8 days. Blastocysts obtained were vitrified and after warming their blastocoele re-expansion and the ploidy by FISH technique were assessed. We found significant differences between blastocysts yield of oocytes recovered from follicles smaller than 3 mm of prepubertal goats compared to those from adult goats (5.45% vs 20. 83%, respectively) however, these differences disappear if oocytes were recovered form large follicles (18.07%). A total of 28 blastocysts were analysed and 96.43% showed mixoploidy. Age did not affect the number of embryos with abnormal ploidy or blastocyst re-expansion after warming. Furthermore, the percentage of diploid blastomeres per embryo was similar in the 3 groups studied, adult, prepubertal from follicles ≥ 3 mm and goats 45 days old were not different to the blastocysts produced from adult goats, both in terms of quantity and quality. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Targeted organ generation using Mixl1-inducible mouse pluripotent stem cells in blastocyst complementation.

    Science.gov (United States)

    Kobayashi, Toshihiro; Kato-Itoh, Megumi; Nakauchi, Hiromitsu

    2015-01-15

    Generation of functional organs from patients' own cells is one of the ultimate goals of regenerative medicine. As a novel approach to creation of organs from pluripotent stem cells (PSCs), we employed blastocyst complementation in organogenesis-disabled animals and successfully generated PSC-derived pancreas and kidneys. Blastocyst complementation, which exploits the capacity of PSCs to participate in forming chimeras, does not, however, exclude contribution of PSCs to the development of tissues-including neural cells or germ cells-other than those specifically targeted by disabling of organogenesis. This fact provokes ethical controversy if human PSCs are to be used. In this study, we demonstrated that forced expression of Mix-like protein 1 (encoded by Mixl1) can be used to guide contribution of mouse embryonic stem cells to endodermal organs after blastocyst injection. We then succeeded in applying this method to generate functional pancreas in pancreatogenesis-disabled Pdx1 knockout mice using a newly developed tetraploid-based organ-complementation method. These findings hold promise for targeted organ generation from patients' own PSCs in livestock animals.

  5. Bovine ovarian follicular growth and development correlate with lysophosphatidic acid expression.

    Science.gov (United States)

    Sinderewicz, Emilia; Grycmacher, Katarzyna; Boruszewska, Dorota; Kowalczyk-Zięba, Ilona; Staszkiewicz, Joanna; Ślężak, Tomasz; Woclawek-Potocka, Izabela

    2018-01-15

    The basis of successful reproduction is proper ovarian follicular growth and development. In addition to prostaglandins and vascular endothelial growth factor, a number of novel factors are suggested as important regulators of follicular growth and development: PGES, TFG, CD36, RABGAP1, DBI and BTC. This study focuses on examining the expression of these factors in granulosa and thecal cells that originate from different ovarian follicle types and their link with the expression of lysophosphatidic acid (LPA), known local regulator of reproductive functions in the cow. Ovarian follicles were divided into healthy, transitional, and atretic categories. The mRNA expression levels for PGES, TFG, CD36, RABGAP1, DBI and BTC in granulosa and thecal cells in different follicle types were measured by real-time PCR. The correlations among expression of enzymes synthesizing LPA (autotaxin, phospholipase A2), receptors for LPA and examined factors were measured. Immunolocalization of PGES, TFG, CD36, RABGAP1, DBI and BTC was examined by immunohistochemistry. We investigated follicle-type dependent mRNA expression of factors potentially involved in ovarian follicular growth and development, both in granulosa and thecal cells of bovine ovarian follicles. Strong correlations among receptors for LPA, enzymes synthesizing LPA, and the examined factors in healthy and transitional follicles were observed, with its strongest interconnection with TFG, DBI and RABGAP1 in granulosa cells, and TFG in thecal cells; whereas no correlations in atretic follicles were detected. A greater number of correlations were found in thecal cells than in granulosa cells as well as in healthy follicles than in transitional follicles. These data indicate the role of LPA in the growth, development and physiology of the bovine ovarian follicle. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Conditional deletion of Msx homeobox genes in the uterus inhibits blastocyst implantation by altering uterine receptivity.

    Science.gov (United States)

    Daikoku, Takiko; Cha, Jeeyeon; Sun, Xiaofei; Tranguch, Susanne; Xie, Huirong; Fujita, Tomoko; Hirota, Yasushi; Lydon, John; DeMayo, Francesco; Maxson, Robert; Dey, Sudhansu K

    2011-12-13

    An effective bidirectional communication between an implantation-competent blastocyst and the receptive uterus is a prerequisite for mammalian reproduction. The blastocyst will implant only when this molecular cross-talk is established. Here we show that the muscle segment homeobox gene (Msh) family members Msx1 and Msx2, which are two highly conserved genes critical for epithelial-mesenchymal interactions during development, also play crucial roles in embryo implantation. Loss of Msx1/Msx2 expression correlates with altered uterine luminal epithelial cell polarity and affects E-cadherin/β-catenin complex formation through the control of Wnt5a expression. Application of Wnt5a in vitro compromised blastocyst invasion and trophoblast outgrowth on cultured uterine epithelial cells. The finding that Msx1/Msx2 genes are critical for conferring uterine receptivity and readiness to implantation could have clinical significance, because compromised uterine receptivity is a major cause of pregnancy failure in IVF programs. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. Rabbit blastocysts accumulate [3H]prostaglandins in vitro

    International Nuclear Information System (INIS)

    Jones, M.A.; Harper, M.J.

    1984-01-01

    Rabbit blastocysts obtained on days 5, 6, and 6.8 of pregnancy were incubated in vitro in Tyrode's buffer with 3 H-labeled prostaglandins (PGs). Accumulation of PGs was studied, using Whatman GF/F filters to separate bound and free ligands. The uptake and efflux of [ 3 H]PGs were studied as a function of PG type, incubation time, temperature, and effect of metabolic inhibitors as well as age and number of blastocysts. Blastocysts of the same age accumulated approximately the same amount of [ 3 H]PGE2 and [ 3 H]PGF2 alpha from their environment; however, there was no apparent saturation over a PG concentration range of 1-1000 nM. Both the uptake and efflux of PG were age dependent, with older blastocysts accumulating more PGs. Approximately 90% of the [ 3 H]PGs appear to be transported into the blastocoelic fluid, with little PG remaining in the blastomeres. PG accumulation was relatively insensitive to azide, ouabain, cyanide, or bromcresol green, but was affected by incubation at 0 C or the addition of indomethacin (10 micrograms/ml). No catabolism of the accumulated PGs was observed. The release of PGE2 in general did not differ from that of PGF2 alpha, except on day 6.8 of pregnancy when PGE2 was released more rapidly than on day 6. The authors conclude that rabbit blastocysts can accumulate PGs from their environment, which may imply a storage potential in the blastocyst and release before implantation

  8. H2O2-induced mild stress in relation with in vitro ovine oocyte developmental competence: implications for blastocyst apoptosis and related genes expression.

    Science.gov (United States)

    Nikdel, K; Aminafshar, M; Mohammadi-Sangcheshmeh, A; EmamJomeh-Kashan, N; Seyedjafari, E

    2017-05-20

    In this study, in vitro maturation was performed in presence of various concentrations (0, 10, 100, or 1000 µM) of H2O2. The intracellular glutathione (GSH) level, fertilization, cleavage, and blastocyst rates, total cell number, and apoptotic cell number and expression of Bax, Bcl-2, and p53 genes in blastocyst-stage embryos were studied. At 10 μM H2O2 concentration, a higher GSH level was detected in comparison to the other groups while oocytes exposed to 1000 μM H2O2 had the lowest GSH level. Treatment of oocytes with 1000 μM H2O2 decreased the rate of two pronuclei formation as compared with other groups. A higher rate of blastocyst formation was seen in 100 μM H2O2 group as compared with the control group. However, exogenous H2O2 in maturation medium did not affect total cell numbers and apoptotic cell ratio at the blastocyst stage. Moreover, mRNA transcript abundance of Bax, Bcl-2, and p53 genes was similar between blastocysts derived from H2O2-induced oocytes and control blastocysts. Treatment of oocytes with H2O2 at mild level during in vitro maturation had a positive effect on GSH level and this, in turn, may lead to improvement in preimplantation embryonic development.

  9. Application of preimplantation genetic diagnosis in equine blastocysts

    Directory of Open Access Journals (Sweden)

    Grady ST

    2016-08-01

    Full Text Available Pre-implantation genetic diagnosis (PGD is a procedure used to screen in vitroproduced embryos or embryos recovered after uterine flush to determine genetic traits by DNA testing prior to transfer into the uterus. Biopsy methods to obtain a sample of cells for genetic analysis before implantation have been successful in small embryos (morulae and blastocysts 300 µm diameter. The successful biopsy of expanded equine blastocysts via micromanipulation, with subsequent normal pregnancy rates, was first reported in 2010. Direct PCR may be performed when evaluating only one gene, such as for embryo sexing, while whole genome amplification is effective for subsequent multiplex PCR of multiple genes.

  10. Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications.

    Directory of Open Access Journals (Sweden)

    Jens Popken

    Full Text Available The present study demonstrates a major remodeling of the nuclear envelope and its underlying lamina during bovine preimplantation development. Up to the onset of major embryonic genome activation (MGA at the 8-cell stage nuclei showed a non-uniform distribution of nuclear pore complexes (NPCs. NPCs were exclusively present at sites where DNA contacted the nuclear lamina. Extended regions of the lamina, which were not contacted by DNA, lacked NPCs. In post-MGA nuclei the whole lamina was contacted rather uniformly by DNA. Accordingly, NPCs became uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions which control the integration of NPCs into the nuclear envelope. The switch from maternal to embryonic production of mRNAs was accompanied by multiple invaginations covered with NPCs, which may serve the increased demands of mRNA export and protein import. Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Since the abundance of these invaginations and vesicles increased in concert with a massive nuclear volume reduction, we suggest that they reflect a mechanism for fitting the nuclear envelope and its lamina to a shrinking nuclear size during bovine preimplantation development. In addition, a deposit of extranuclear clusters of NUP153 (a marker for NPCs without associated lamin B was frequently observed from the zygote stage up to MGA. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided NUP153 mRNA and little unspliced, newly synthesized RNA prior to MGA, which increased strongly at the initiation of embryonic expression of NUP153 at MGA.

  11. Efeito do número da passagem e do gênero das células doadoras de núcleo no desenvolvimento de bovinos produzidos por transferência nuclear Effect of culture time and gender of nuclei donor cells on bovine development produced by nuclear transfer

    Directory of Open Access Journals (Sweden)

    Giovana Krempel Fonseca Merighe

    2010-10-01

    Full Text Available Objetivou-se com este trabalho avaliar o efeito do número da passagem e do sexo das células doadoras de núcleo no desenvolvimento embrionário e fetal após transferência nuclear. Para isso, oócitos bovinos foram maturados, enucleados e reconstruídos com células somáticas de animal adulto. Após a fusão e ativação química, os zigotos reconstituídos foram cultivados em Charles Rosenkranz 2 (CR2 com monocamada de células da granulosa a 38,8ºC em atmosfera umidificada a 5% de CO2 em ar, durante sete dias, e transferidos para receptoras sincronizadas. As taxas de clivagem e desenvolvimento a blastocisto de embriões reconstruídos com células cultivadas por tempo maior foram inferiores às obtidas com os demais tempos de cultivo. Além disso, os blastocistos produzidos não resultaram no desenvolvimento de uma gestação a termo. Embora a taxa de clivagem em embriões fêmeas tenha sido maior, o número de embriões que atingiram o estádio de blastocisto foi maior nos embriões machos. No período gestacional, fêmeas apresentaram maior taxa de aborto entre 90 e 120 dias de gestação. Esses resultados indicam que células doadoras de núcleos cultivados por longos períodos dificultam a produção de blastocistos e aumentam as chances de perdas durante a gestação. Embriões clonados machos têm maior competência para se desenvolver a blastocisto e resultam em menor taxa de perda gestacional.The objective of this study was to evaluate the effects of culture time and sex of nuclei donor cells on embryo and fetal development after nuclear transfer. Thus, bovine oocytes were matured, enucleated and reconstructed with somatic cells from an adult animal. After fusion and chemical activation, the reconstituted zygotes were cultured in Charles Rosenkranz 2 (CR2 on a granular monolayer cell at 38.8ºC in a humidified atmosphere 5% CO2 in air for seven days, and transferred to synchronized receptors. Cleavage rates and development to

  12. Effect of glycine and alanine supplementation on development of cattle embryos cultured in CR1aa medium with or without cumulus cells

    Directory of Open Access Journals (Sweden)

    Kr. BREDBACKA

    2008-12-01

    Full Text Available The effect of alanine (1 mM and glycine (10 mM supplementation on bovine embryo development in vitro was investigated. Presumptive bovine zygotes, produced by in vitro maturation and insemination of oocytes, were cultured for 144 h in CR1aa medium in the absence (Experiments 1 and 2 or presence of cumulus cells (Experiment 3. In Experiment 1, the proportion of morulae and blastocysts of cleaved embryos in glycine-supplemented medium was not different from that of the control medium (34% in both mediaglycine-enriched medium (69.5 vs. 53.3, P = 0.016. In Experiment 2, addition of alanine did not improve the formation of morulae and blastocysts (13% vs. 21% in control medium, and the mean cell numbers in morulae and blastocysts were lower than those in the control group (34.3 vs. 68.7, P = 0.007. In the presence of cumulus cells, the combined supplementation of glycine and alanine increased the proportion of morulae and blastocysts over that in the control medium (31% vs. 14%, P = 0.003.;

  13. Blastocyst morphology, actin cytoskeleton quality and chromosome content are correlated with embryo quality in the pig

    NARCIS (Netherlands)

    Zijlstra, C.; Kidson, A.; Schoevers, E.J.; Daemen, A.J.J.M.; Tharasanit, T.; Kuijk, E.W.; Hazeleger, W.; Ducro-Steverink, D.W.B.; Colenbrander, B.; Roelen, B.A.J.

    2008-01-01

    Embryo survival rates obtained after transfer of in vitro produced porcine blastocysts are very poor. This is probably related to poor quality of the embryos. The aim of the present study was to determine markers for good quality blastocysts. Therefore, we tried to link blastocyst morphology to

  14. Maturation, fertilisation and culture of bovine oocytes and embryos in an individually identifiable manner: a tool for studying oocyte developmental competence.

    Science.gov (United States)

    Matoba, Satoko; Fair, Trudee; Lonergan, Patrick

    2010-01-01

    The ability to successfully culture oocytes and embryos individually would facilitate the study of the relationship between follicle parameters and oocyte developmental competence, in order to identify markers of competent oocytes, as well as the ability to use small numbers of oocytes from an individual donor such as when ovum pick-up is carried out. Using a total of 3118 oocytes, the aim of the present study was to develop a system capable of supporting the development of immature bovine oocytes to the blastocyst stage in an individually identifiable manner. Initially, post-fertilisation embryo culture in the Well-of-the-Well (WOW) system, on the cell adhesive Cell-Tak or in polyester mesh was tested and shown to result in similar development to embryos cultured in standard group culture. The results demonstrate that it is possible to culture bovine oocytes to the blastocyst stage in an individually identifiable manner in all three culture systems with comparable success rates. This permits the localisation and identification of individual embryos throughout preimplantation development in vitro while retaining the developmental benefits of group culture. In terms of ease of preparation and use, culture in isolation within the strands of a polyester mesh is preferable.

  15. Blastocyst utilization rates after continuous culture in two commercial single-step media: a prospective randomized study with sibling oocytes.

    Science.gov (United States)

    Sfontouris, Ioannis A; Kolibianakis, Efstratios M; Lainas, George T; Venetis, Christos A; Petsas, George K; Tarlatzis, Basil C; Lainas, Tryfon G

    2017-10-01

    The aim of this study is to determine whether blastocyst utilization rates are different after continuous culture in two different commercial single-step media. This is a paired randomized controlled trial with sibling oocytes conducted in infertility patients, aged ≤40 years with ≥10 oocytes retrieved assigned to blastocyst culture and transfer. Retrieved oocytes were randomly allocated to continuous culture in either Sage one-step medium (Origio) or Continuous Single Culture (CSC) medium (Irvine Scientific) without medium renewal up to day 5 post oocyte retrieval. Main outcome measure was the proportion of embryos suitable for clinical use (utilization rate). A total of 502 oocytes from 33 women were randomly allocated to continuous culture in either Sage one-step medium (n = 250) or CSC medium (n = 252). Fertilization was performed by either in vitro fertilization or intracytoplasmic sperm injection, and embryo transfers were performed on day 5. Two patients had all blastocysts frozen due to the occurrence of severe ovarian hyperstimulation syndrome. Fertilization and cleavage rates, as well as embryo quality on day 3, were similar in the two media. Blastocyst utilization rates (%, 95% CI) [55.4% (46.4-64.1) vs 54.7% (44.9-64.6), p = 0.717], blastocyst formation rates [53.6% (44.6-62.5) vs 51.9 (42.2-61.6), p = 0.755], and proportion of good quality blastocysts [36.8% (28.1-45.4) vs 36.1% (27.2-45.0), p = 0.850] were similar in Sage one-step and CSC media, respectively. Continuous culture of embryos in Sage one-step and CSC media is associated with similar blastocyst development and utilization rates. Both single-step media appear to provide adequate support during in vitro preimplantation embryo development. Whether these observations are also valid for other continuous single medium protocols remains to be determined. NCT02302638.

  16. Analysis of embryo morphokinetics, multinucleation and cleavage anomalies using continuous time-lapse monitoring in blastocyst transfer cycles.

    Science.gov (United States)

    Desai, Nina; Ploskonka, Stephanie; Goodman, Linnea R; Austin, Cynthia; Goldberg, Jeffrey; Falcone, Tommaso

    2014-06-20

    Time-lapse imaging combined with embryo morphokinetics may offer a non-invasive means for improving embryo selection. Data from clinics worldwide are necessary to compare and ultimately develop embryo classifications models using kinetic data. The primary objective of this study was to determine if there were kinetic differences between embryos with limited potential and those more often associated with in vitro blastocyst formation and/or implantation. We also wanted to compare putative kinetic markers for embryo selection as proposed by other laboratories to what we were observing in our own laboratory setting. Kinetic data and cycle outcomes were retrospectively analyzed in patients age 39 and younger with 7 or more zygotes cultured in the Embryoscope. Timing of specific events from the point of insemination were determined using time-lapse (TL) imaging. The following kinetic markers were assessed: time to syngamy (tPNf), t2, time to two cells (c), 3c (t3), 4c ( t4), 5c (t5), 8c (t8), morula (tMor), start of blastulation (tSB); tBL, blastocyst (tBL); expanded blastocyst (tEBL). Durations of the second (cc2) and third (cc3) cell cycles, the t5-t2 interval as well as time to complete synchronous divisions s1, s2 and s3 were calculated. Incidence and impact on development of nuclear and cleavage anomalies were also assessed. A total of 648 embryos transferred on day 5 were analyzed. The clinical pregnancy and implantation rate were 72% and 50%, respectively. Morphokinetic data showed that tPNf, t2,t4, t8, s1, s2,s3 and cc2 were significantly different in embryos forming blastocysts (ET or frozen) versus those with limited potential either failing to blastulate or else forming poor quality blastocysts ,ultimately discarded. Comparison of embryo kinetics in cycles with all embryos implanting (KID+) versus no implantation (KID-) suggested that markers of embryo competence to implant may be different from ability to form a blastocyst. The incidence of multinucleation

  17. Lower blastocyst quality after conventional vs. Piezo ICSI in the horse reflects delayed sperm component remodeling and oocyte activation.

    Science.gov (United States)

    Salgado, R M; Brom-de-Luna, J G; Resende, H L; Canesin, H S; Hinrichs, Katrin

    2018-04-10

    The aim of this study was to evaluate the differential effects of conventional and Piezo-driven ICSI on blastocyst development, and on sperm component remodeling and oocyte activation, in an equine model. In vitro-matured equine oocytes underwent conventional (Conv) or Piezo ICSI, the latter utilizing fluorocarbon ballast. Blastocyst development was compared between treatments to validate the model. Then, oocytes were fixed at 0, 6, or 18 h after injection, and stained for the sperm tail, acrosome, oocyte cortical granules, and chromatin. These parameters were compared between injection techniques and between sham-injected and sperm-injected oocytes among time periods. Blastocyst rates were 39 and 40%. The nucleus number was lower, and the nuclear fragmentation rate was higher, in blastocysts produced by Conv. Cortical granule loss started at 0H after both sperm and sham injection. The acrosome was present at 0H in both ICSI treatments, and persisted to 18H in significantly more Conv than Piezo oocytes (72 vs. 21%). Sperm head area was unchanged at 6H in Conv but significantly increased at this time in Piezo; correspondingly, at 6H significantly more Conv than Piezo oocytes remained at MII (80 vs. 9.5%). Sham injection did not induce significant meiotic resumption. These data show that Piezo ICSI is associated with more rapid sperm component remodeling and oocyte meiotic resumption after sperm injection than is conventional ICSI, and with higher embryo quality at the blastocyst stage. This suggests that there is value in exploring the Piezo technique, utilized with a non-toxic fluorocarbon ballast, for use in clinical human ICSI.

  18. Comparison of the efficacy of conventional slow freezing and rapid cryopreservation methods for bovine embryos

    NARCIS (Netherlands)

    Wagtendonk-de Leeuw, van A.M.; Daas, den J.H.; Kruip, T.A.; Rail, W.F.

    1995-01-01

    Day 7 bovine morulae and early blastocysts were randomly assigned to one of four cryopreservation methods: (i) a modified conventional controlled slow freezing and stepwise dilution after thawing; and three methods which enable direct transfer of the embryo into the recipient upon thawing: (ii)

  19. Development of magnetic nanoparticle based calorimetric assay for the detection of bovine mastitis in cow milk.

    Science.gov (United States)

    Chinnappan, Raja; Al Attas, Sana; Kaman, Wendy E; Bikker, Floris J; Zourob, Mohammed

    2017-04-15

    Mastitis in dairy cattle is an inflammatory reaction of the udder tissue. Mastitis increases plasmin levels, leading to an increased proteolysis of milk proteins such as casein, resulting in a significant decrease in milk quality and related dairy products. Due to its key-role in mastitis, we used plasmin proteolytic activity as a biomarker for the detection of mastitis in bovine mastitic milk. Inspired by earlier studies on protease activity using mastitic milk samples, we developed a simple colorimetric assay to distinguish mastitic milk from milk derived from healthy animals. The plasmin substrate coupled to magnetic nanoparticles form a black self-assembled monolayer on a gold sensor surface. In the presence of increased levels of plasmin, the substrate is cleaved and the peptide fragment attached to the magnetic beads, will be attracted by the magnet which is present under the sensor strips revealing the golden surface. We found the area of the golden color surface proportional to plasmin activity. The sensitivity of this method was determined to be 1 ng/ml of plasmin in vitro. Next, we tested the biosensor using mastitis positive milk of which infection is confirmed by bacterial cultures. This newly developed colorimetric biosensor has high potential in applications for the diagnosis of mastitis with potential spin offs to health, food and environmental sectors. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Over-expression of CXCR4, a stemness enhancer, in human blastocysts by low level laser irradiation

    Directory of Open Access Journals (Sweden)

    Mohammad Hossein Tahmasbi

    2013-09-01

    Full Text Available The key role of chemokine receptor CXCR4 in the maintenance of stemness property of stem cells has been shown recently. The low level laser irradiation (LLLI is being used currently in a wide variety of clinical cases as a therapeutic tool for wound healing, relieving pain and destroying tumor cells. The aim of this study was to evaluate the effect of LLLI mimicking low level laser therapy (LLLT on the expression level of CXCR4 gene a few hours after irradiation on human blastocysts. After the development of human embryos to the first grade blastocyst stage, they were irradiated with a low power Ga-Al-As laser at a continuous wavelength of 650 nm and a power output of 30 mW. The total RNA of the irradiated blastocysts and control groups were isolated in groups of 1x2 J/cm2, 2x2 J/cm2, 1x4 J/cm2 and 2x4 J/cm2 LLLI. Specific Real-Time PCR primers were designed to amplify all the two CXCR4 isoforms yet identified. RNA amplifications were done for all the groups. We showed for the first time that LLLI makes the human blastocysts to increase the expression level of CXCR4 a few hours after irradiation. Moreover, it was shown that two irradiation doses with one day interval can cause a significant increase in CXCR4 expression level in human blastocysts. This study revealed that LLLI could be a proliferation motivator for embryonic cell divisions through enhanced over-expression of CXCR4 level.

  1. Two-cell embryos are more sensitive than blastocysts to AMPK-dependent suppression of anabolism and stemness by commonly used fertility drugs, a diet supplement, and stress.

    Science.gov (United States)

    Bolnick, Alan; Abdulhasan, Mohammed; Kilburn, Brian; Xie, Yufen; Howard, Mindie; Andresen, Paul; Shamir, Alexandra M; Dai, Jing; Puscheck, Elizabeth E; Secor, Eric; Rappolee, Daniel A

    2017-12-01

    This study tests whether metformin or diet supplement BR-DIM-induced AMP-activated protein kinase (AMPK) mediated effects on development are more pronounced in blastocysts or 2-cell mouse embryos. Culture mouse zygotes to two-cell embryos and test effects after 0.5-1 h AMPK agonists' (e.g., Met, BR-DIM) exposure on AMPK-dependent ACCser79P phosphorylation and/or Oct4 by immunofluorescence. Culture morulae to blastocysts and test for increased ACCser79P, decreased Oct4 and for AMPK dependence by coculture with AMPK inhibitor compound C (CC). Test whether Met or BR-DIM decrease growth rates of morulae cultured to blastocyst by counting cells. Aspirin, metformin, and hyperosmotic sorbitol increased pACC ser79P ~ 20-fold, and BR-DIM caused a ~ 30-fold increase over two-cell embryos cultured for 1 h in KSOMaa but only 3- to 6-fold increase in blastocysts. We previously showed that these stimuli decreased Oct4 40-85% in two-cell embryos that was ~ 60-90% reversible by coculture with AMPK inhibitor CC. However, Oct4 decreased only 30-50% in blastocysts, although reversibility of loss by CC was similar at both embryo stages. Met and BR-DIM previously caused a near-complete cell proliferation arrest in two-cell embryos and here Met caused lower CC-reversible growth decrease and AMPK-independent BR-DIM-induced blastocyst growth decrease. Inducing drug or diet supplements decreased anabolism, growth, and stemness have a greater impact on AMPK-dependent processes in two-cell embryos compared to blastocysts.

  2. Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (20 C)

    Science.gov (United States)

    Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

    2009-01-01

    Summary Usually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 C/min in a low-temperature (80 C) freezer (LTF) before storage in liquid nitrogen (LN2); however, a LTF is not always available. The purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen in a LTF or conventional freezer (CF; 20 C) and their subsequent ability for development to blastocyst stage after fusion with enucleated bovine oocytes. Percentages of live cells frozen in LTF (49.5%) and CF (50.6%) were similar, but significantly less than non-frozen control (88%). In both CF and LTF, percentages of live apoptotic cells exposed to LN2 after freezing were lower (4% and 5%, respectively) as compared with unexposed cells (10% and 18%, respectively). Cells frozen in a CF had fewer cell doublings/24 h (0.45) and required more days (9.1) to reach 100% confluence at the first passage (P) after thawing and plating as compared with cells frozen in a LTF (0.96 and 4.0 days, respectively). Hypoploidy at P12 was higher than at P4 in cells frozen in either a CF (37.5% vs. 19.2%) or in a LTF (30.0% vs. 15.4%). A second-generation cryo-solution reduced the incidence of necrosis (29.4%) at 0 h after thawing as compared with that of a first generation cryo-solution (DMEM + DMSO, 60.2%). The percentage of apoptosis in live cells was affected by cooling rate (CF = 1.9% vs. LFT = 0.7%). Development of bovine cloned embryos to the blastocyst stage was not affected by cooling rate or freezer type. ?? 2009 Cambridge University Press.

  3. Oogenesis, follicular development and reproductive performance in the prenatally irradiated bovine

    International Nuclear Information System (INIS)

    Erickson, B.H.; Reynolds, R.A.

    1978-01-01

    To determine the radioresponse of the various developmental stages of the prenatal bovine germ cell, cows bearing foetuses varying in age from 40+-5 (point of gonadal sex differentiation) to 270+-10 (approximately 13 days before parturition) days of gestation were irradiated with 300 R of 60 Co gamma radiation at 25-50 R/min, a level just below that likely to cause maternal deaths and gross foetal abnormalities at some developmental stages. Dose to the foetal gonad was approximately 100 rad. When the prenatally irradiated heifers were approximately 10 months old their ovaries were recovered at slaughter, serially sectioned, and prepared for microscopic analysis. A complete quantitative analysis of oocytes in primordial, growing and vesicular follicles was effected. Follicular development (reflected by counts of growing and vesicular follicles) was apparently unaffected at all ages tested (P>0.25), whereas oogenesis (reflected by counts of oocytes in primordial follicles) was significantly impeded (64% of control) only between 70 and 90 days of gestation (P<0.05). The germ-cell population at this period differs from later developmental stages principally by the presence of a high proportion of mitotically active oogonia, hence it appears that the oogonium is the most vulnerable cell type. Neither the viability nor reproductive performance of 60 cows that were prenatally irradiated (100 rad) at either 80+-10 or 130+-20 days of gestation was affected through a minimum of 5 years of postnatal life and three pregnancies. (author)

  4. Increase of essential amino acids in the bovine uterine lumen during preimplantation development.

    Science.gov (United States)

    Groebner, Anna E; Rubio-Aliaga, Isabel; Schulke, Katy; Reichenbach, Horst D; Daniel, Hannelore; Wolf, Eckhard; Meyer, Heinrich H D; Ulbrich, Susanne E

    2011-05-01

    Amino acids (AAs) are crucial for the developing conceptus prior to implantation. To provide insights into the requirements of the bovine embryo, we determined the AA composition of the uterine fluid. At days 12, 15, and 18 post-estrus, the uteri of synchronized pregnant and non-pregnant Simmental heifers were flushed for the analysis of 41 AAs and their derivatives by liquid chromatography-tandem mass spectrometry. The ipsilateral endometrium was sampled for quantitative PCR. In addition to a pregnancy-dependent increase of the essential AAs (P<0.01), we detected elevated concentrations for most non-essential proteinogenic AAs. Histidine (His) and the expression of the His/peptide transporter solute carrier 15A3 (SLC15A3) were significantly increased at day 18 of pregnancy in vivo. In addition, SLC15A3 was predominantly stimulated by trophoblast-derived interferon-τ in stroma cells of an in vitro co-culture model of endometrial cells. Our results show an increased concentration of AAs most likely to optimally provide the elongating pre-attachment conceptus with nutrients.

  5. Naturally occurring mastitis disrupts developmental competence of bovine oocytes.

    Science.gov (United States)

    Roth, Z; Dvir, A; Kalo, D; Lavon, Y; Krifucks, O; Wolfenson, D; Leitner, G

    2013-10-01

    We examined the effects of naturally occurring mastitis on bovine oocyte developmental competence in vitro. Specifically, we investigated the effects of intramammary infection on the ovarian pool of oocytes (i.e., follicle-enclosed oocytes) and their ability to undergo in vitro maturation, fertilization, and further development to the blastocyst stage. Culled Holstein cows (n=50) from 9 commercial dairy farms in Israel were allotted to 3 groups according to somatic cell count (SCC) records of the last 3 monthly milk tests as well as of quarter samples collected before slaughter: (1) low SCC (n=7), (2) medium SCC (n=16), or (3) high SCC (n=27). Means of SCC values differed among low-, medium-, and high-SCC groups: 148,000, 311,000 and 1,813,000 cell/mL milk, respectively. Milk yield and days in milk did not differ among the 3 groups. Bacterial isolates included coagulase-negative staphylococci, Escherichia coli, Streptococcus dysgalactiae, or no bacteria found. Ovaries were collected at the abattoir and brought to the laboratory. Cumulus oocyte complexes were recovered separately from each cow and subjected individually to in vitro maturation and fertilization, followed by 8d in culture. The number of aspirated oocytes did not differ among groups, with a range of 17 to 21 oocytes per cow. The proportion of oocytes that cleaved into 2- to 4-cell-stage embryos (86.1 ± 3.4%) did not differ among groups. In contrast, mean percentages of embryos developed to the blastocyst stage on d 7 and 8 after fertilization were less in both medium- and-high SCC groups than in the low-SCC group (5.6 ± 2.3 and 4.1 ± 1.8 vs. 18.1 ± 4.6%, respectively). Additional analysis indicated that cleavage and blastocyst-formation rates did not differ among the bacterial types in the low-, medium-, and high-SCC groups. These are the first results to demonstrate that naturally occurring mastitis disrupts the developmental competence of the ovarian pool of oocytes, (i.e., oocytes at the

  6. Feeder Cell Type Affects the Growth of In Vitro Cultured Bovine Trophoblast Cells

    Directory of Open Access Journals (Sweden)

    Islam M. Saadeldin

    2017-01-01

    Full Text Available Trophectoderm cells are the foremost embryonic cells to differentiate with prospective stem-cell properties. In the current study, we aimed at improving the current approach for trophoblast culture by using granulosa cells as feeders. Porcine granulosa cells (PGCs compared to the conventional mouse embryonic fibroblasts (MEFs were used to grow trophectoderm cells from hatched bovine blastocysts. Isolated trophectoderm cells were monitored and displayed characteristic epithelial/cuboidal morphology. The isolated trophectoderm cells expressed mRNA of homeobox protein (CDX2, cytokeratin-8 (KRT8, and interferon tau (IFNT. The expression level was higher on PGCs compared to MEFs throughout the study. In addition, primary trophectoderm cell colonies grew faster on PGCs, with a doubling time of approximately 48 hrs, compared to MEFs. PGCs feeders produced a fair amount of 17β-estradiol and progesterone. We speculated that the supplementation of sex steroids and still-unknown factors during the trophoblasts coculture on PGCs have helped to have better trophectoderm cell’s growth than on MEFs. This is the first time to use PGCs as feeders to culture trophectoderm cells and it proved superior to MEFs. We propose PGCs as alternative feeders for long-term culture of bovine trophectoderm cells. This model will potentially benefit studies on the early trophoblast and embryonic development in bovines.

  7. Four simple rules that are sufficient to generate the mammalian blastocyst

    DEFF Research Database (Denmark)

    Nissen, Silas Boye; Perera Pérez, Marta; Martin Gonzalez, Javier

    2017-01-01

    requiring any initial transcriptional variation. It also suggests that a fixed time point for the cells’ competence of fibroblast growth factor (FGF)/extracellular signal—regulated kinase (ERK) sets an embryonic clock that enables certain scaling phenomena, a concept that we evaluate quantitatively......Early mammalian development is both highly regulative and self-organizing. It involves the interplay of cell position, predetermined gene regulatory networks, and environmental interactions to generate the physical arrangement of the blastocyst with precise timing. However, this process occurs...

  8. Aseptic minimum volume vitrification technique for porcine parthenogenetically activated blastocyst.

    Science.gov (United States)

    Lin, Lin; Yu, Yutao; Zhang, Xiuqing; Yang, Huanming; Bolund, Lars; Callesen, Henrik; Vajta, Gábor

    2011-01-01

    Minimum volume vitrification may provide extremely high cooling and warming rates if the sample and the surrounding medium contacts directly with the respective liquid nitrogen and warming medium. However, this direct contact may result in microbial contamination. In this work, an earlier aseptic technique was applied for minimum volume vitrification. After equilibration, samples were loaded on a plastic film, immersed rapidly into factory derived, filter-sterilized liquid nitrogen, and sealed into sterile, pre-cooled straws. At warming, the straw was cut, the filmstrip was immersed into a 39 degree C warming medium, and the sample was stepwise rehydrated. Cryosurvival rates of porcine blastocysts produced by parthenogenetical activation did not differ from control, vitrified blastocysts with Cryotop. This approach can be used for minimum volume vitrification methods and may be suitable to overcome the biological dangers and legal restrictions that hamper the application of open vitrification techniques.

  9. Bovine oocytes and early embryos express mRNA encoding glycerol kinase but addition of glycerol to the culture media interferes with oocyte maturation.

    Science.gov (United States)

    Okawara, Sumika; Hamano, Seizo; Tetsuka, Masafumi

    2009-04-01

    Glycerol plays multi-functional roles in cellular physiology. Other than forming the backbone molecule for glycerophospholipid and triglyceride (TG), glycerol acts as an energy substrate for glycolysis. Spermatozoa are known to utilize glycerol for energy production, but there are no reports of this in oocytes. In this study, the value of glycerol as an energy substrate for bovine oocyte maturation (Exp. 1) and the gene expression of glycerol kinase (GK), an enzyme crucial for cellular glycerol utilization, in bovine oocytes and early embryos (Exp. 2) were examined. In Exp. 1, in vitro maturation (IVM) was conducted using synthetic oviduct fluid supplemented with/without glucose (1.5 mM) and/or glycerol (1.0 mM), and maturation rate, degree of cumulus expansion, glucose consumption and lactate production by cumulus-oocyte complexes (COC) were examined. In Exp. 2, to examine the developmental expression of GK mRNA, cumulus cells, oocytes and embryos at the 2-, 8- and 16-cell, morula, expanded blastocyst and hatched blastocyst stages were obtained in separate experiments, and the expression of GK mRNA was quantified using a real-time PCR. Glycerol did not support oocyte maturation or cumulus expansion. Addition of glycerol to glucose-supplemented media significantly decreased the maturation rate. Expression of GK mRNA was very low in cumulus cells, whereas an appreciable level of the transcript was observed in the oocytes. GK mRNA was detected in embryos at all the stages examined, and its expression significantly increased at the morula stage. These results indicate that glycerol, at least at the present concentration, is not beneficial as a constituent of the medium for bovine oocyte maturation. However, the appreciable levels of GK mRNA found in the oocyte and embryo imply a physiological role for glycerol in bovine oocyte maturation and embryo development.

  10. Oocyte activation and preimplantation development of bovine embryos obtained by specific inhibition of cyclin-dependent kinases Ativação oocitária e desenvolvimento pré-implantação de embriões bovinos obtidos com o uso de inibidores específicos das quinases dependentes de ciclina

    Directory of Open Access Journals (Sweden)

    F. Perecin

    2007-04-01

    Full Text Available The efficiency of bohemine and roscovitine in combination with ionomycin on parthenogenetic activation and initial embryonic development of bovine oocytes was studied. Two experiments were performed: in the first, different concentrations (0, 50, 75 or 100µM and different exposure periods (2, 4 or 6 hours to bohemine or roscovitine were tested for activation rates of in vitro matured (IVM bovine oocytes, which were pre-exposed to ionomycin. The best treatments, 75µM bohemine and 50µM roscovitine, both for 6h, were used in the second experiment, in which IVM bovine oocytes were exposed to ionomycin, followed or not by bohemine or roscovitine treatment, and evaluated for nuclear status, activation rate and blastocyst development were assessed. The combined treatments (ionomycin + cyclin-dependent kinases inhibitors - CDKIs showed better results for activation rates (77.3% and initial embryonic development (35.2% than the single ionomycin treatment (69.4% for activation and 21.9% for development; and also lead to a more uniform activation (nearly 90% single pronucleus development. The results showed that CDKIs improve the effects of ionomycin on parthenogenetic activation and blastocyst development in bovine oocytes and could help to achieve more efficient activation protocols, increasing the developmental competence of embryos obtained by reproductive biotechniques.Realizaram-se dois experimentos para avaliar a eficiência da bohemina e roscovitina associadas à ionomicina para ativação partenogenética e desenvolvimento embrionário inicial de bovinos. No primeiro, foram testadas diferentes concentrações (0, 50, 75 ou 100µM e diferentes tempos de exposição (2, 4 ou 6 horas à bohemina ou à roscovitina na ativação de oócitos bovinos maturados in vitro (MIV pré-expostos à ionomicina. Os melhores tratamentos, bohemina 75µM e roscovitina 50µM, ambos por seis horas, foram utilizados no segundo experimento, no qual oócitos bovinos

  11. Sperm DNA fragmentation index does not correlate with blastocyst aneuploidy or morphological grading.

    Directory of Open Access Journals (Sweden)

    Itai Gat

    Full Text Available High DNA fragmentation index (DFI may be associated with poor outcome after IVF. Our aim was to determine whether DFI impacts blastocyst quality or clinical outcome. This retrospective study included 134 couples who underwent 177 IVF-ICSI and pre-implantation genetic screening (PGS cycles during January 1st, 2014-March 31st, 2016 and had documented previous DFI. Group 1 (DFI>30% encompassed 25 couples who underwent 36 cycles; Group 2 (DFI 15-30% included 45 couples and 57 cycles; group 3 (DFI<15% included 64 couples and 83 cycles. Male partners within group 1 were older (45.1 compared to 40.6 and 38.3 years, respectively, p<0.05, had higher BMI (32.4 compared to 26.6 and 25.8 respectively, p<0.05 and lower sperm count and motility (46*106/ml and 35.5%, respectively compared to groups 2 (61.8*106/ml and 46.6%, respectively and 3 (75.8*106/ml and 55.1%, respectively, p<0.05. Female parameters including ovarian reserve and response and embryo development were similar. Total numbers of biopsied blastocysts were 116, 175 and 259 in groups 1, 2 and 3, respectively. PGS for 24 chromosomes revealed comparable euploidy rate of 46-50.4%, with a similar morphological classification. No significant differences were found regarding pregnancy rates or pregnancy loss. It seems that DFI doesn't correlate with blastocyst aneuploidy or morphological grading.

  12. Development of Fibroblast Cell Lines From the Cow Used to Sequence the Bovine Genome

    Science.gov (United States)

    Two cell lines, designated MARC.BGCF.2 and MARC.BGCF.1-3, were initiated from skin biopsies obtained from the Hereford cow whose DNA was used in sequencing the bovine genome. These cell lines were submitted to American Type Culture Collection (ATCC, Manassas, VA, USA) and will be made publicly avai...

  13. Knowledge Gaps Impacting the Development of Bovine Viral Diarrhea Virus Control Programs in the United States

    Science.gov (United States)

    This paper identifies knowledge gaps that impact on the design of programs to control and or eradicate bovine viral diarrhea viruses (BVDV) in the United States. Currently there are several voluntary regional BVDV control programs in place. These control programs are aimed at the removal of animals ...

  14. Gene expression and apoptosis in bovine embryos during in vitro culture and in vivo development

    NARCIS (Netherlands)

    Knijn, H.W.

    2004-01-01

    The first attempts to fertilise in vitro bovine oocytes were done in the late sixties but only in 1982 the first calf was born after transplantation of a complete in vitro produced embryo. Since then the in vitro production system improved a lot but it is still impossible to mimic the in vivo

  15. Influence of antral follicle size on oocyte characteristics and embryo development in the bovine

    DEFF Research Database (Denmark)

    Lequarre, Anne Sophie; Vigneron, Céline; Ribaucour, Fabrice

    2005-01-01

    The developmental competence of bovine oocytes isolated from antral follicles of different sizes was assessed in three European laboratories (Belgium, UCL; Denmark, DIAS; France, INRA). Using the same protocol for in vitro production of embryos, the oocytes isolated from follicles with a diameter...

  16. Centrosome Clustering in the Development of Bovine Binucleate Trophoblast Giant Cells.

    Science.gov (United States)

    Klisch, Karl; Schraner, Elisabeth M; Boos, Alois

    2017-01-01

    Binucleate trophoblast giant cells (BNC) are the characteristic feature of the ruminant placenta. During their development, BNC pass through 2 acytokinetic mitoses and become binucleate with 2 tetraploid nuclei. In this study, we investigate the number and location of centrosomes in bovine BNC. Centrosomes typically consist of 2 centrioles surrounded by electron-dense pericentriolar material. Duplication of centrosomes is tightly linked to the cell cycle, which ensures that the number of centrosomes remains constant in proliferating diploid cells. Alterations of the cell cycle, which affect the number of chromosome sets, also affect the number of centrosomes. In this study, we use placentomal tissue from pregnant cows (gestational days 80-230) for immunohistochemical staining of γ-tubulin (n = 3) and transmission electron microscopy (n = 3). We show that mature BNC have 4 centrosomes with 8 centrioles, clustered in the angle between the 2 cell nuclei. During the second acytokinetic mitosis, the centrosomes must be clustered to form the poles of a bipolar spindle. In rare cases, centrosome clustering fails and tripolar mitosis leads to the formation of trinucleate "BNC". Generally, centrosome clustering occurs in polyploid tumor cells, which have an increased number of centrioles, but it is absent in proliferating diploid cells. Thus, inhibition of centrosome clustering in tumor cells is a novel promising strategy for cancer treatment. BNC are a cell population in which centrosome clustering occurs as part of the normal life history. Thus, they might be a good model for the study of the molecular mechanisms of centrosome clustering. © 2016 S. Karger AG, Basel.

  17. Development of an Indirect ELISA Based on a Recombinant Chimeric Protein for the Detection of Antibodies against Bovine Babesiosis.

    Science.gov (United States)

    Jaramillo Ortiz, José Manuel; Montenegro, Valeria Noely; de la Fournière, Sofía Ana María; Sarmiento, Néstor Fabián; Farber, Marisa Diana; Wilkowsky, Silvina Elizabeth

    2018-01-23

    The current method for Babesia spp. serodiagnosis based on a crude merozoite antigen is a complex and time-consuming procedure. An indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant multi-antigen of Babesia bovis (rMABbO) was developed for detection of antibodies in bovines suspected of infection with this parasite. The multi-antigen comprises gene fragments of three previously characterized B. bovis antigens: MSA-2c, RAP-1 and the Heat Shock protein 20 that are well-conserved among geographically distant strains. The cutoff value for the new rMABbo-iELISA was determined using 75 known-positive and 300 known-negative bovine sera previously tested for antibodies to B. bovis by the gold-standard ELISA which uses a merozoite lysate. A cutoff value of ≥35% was determined in these samples by receiver operator characteristic (ROC) curve analysis, showing a sensitivity of 95.9% and a specificity of 94.3%. The rMABbo-iELISA was further tested in a blind trial using an additional set of 263 field bovine sera from enzootic and tick-free regions of Argentina. Results showed a good agreement with the gold standard test with a Cohen's kappa value of 0.76. Finally, the prevalence of bovine babesiosis in different tick enzootic regions of Argentina was analyzed where seropositivity values among 68-80% were obtained. A certain level of cross reaction was observed when samples from B. bigemina infected cattle were analyzed with the new test, which can be attributed to shared epitopes between 2 of the 3 antigens. This new rMABbo-iELISA could be considered a simpler alternative to detect anti Babesia spp. antibodies and appears to be well suited to perform epidemiological surveys at the herd level in regions where ticks are present.

  18. Oil-free culture system for in vitro bovine embryo production

    Directory of Open Access Journals (Sweden)

    Paulo B.D. Gonçalves

    2010-04-01

    Full Text Available The use of oil to avoid water evaporation from cell culture has several disadvantages, amongst which there is the migration of compounds from media to oil and from oil to media. The aim of this study was to evaluate the osmolality of a culture system using four-well plates with water in the central hole as an alternative to in vitro bovine embryo production (IVP. In addition, the osmolality changes of the oocyte washing medium were assessed in 35mm dishes with or without 2 mL of silicon oil overlay. Osmolality of oocyte washing medium changed a great deal over time after 60 minutes on a 39°C heated plate (291 mOsm kg-1, which was not detected when the medium was overlaid with silicon oil (280 mOsm kg-1; P0.05. Blastocyst rates were higher when embryos were cultured in presence of water or oil (29.7 and 29.9% for water and 33% in oil conventional microdrop system, except in the group that oocytes were washed in hyperosmotic washing medium (15.1%; P<0.05. Groups cultured in absence of water in the central hole had lower blastocyst rates (P<0.05 independently of exposure (15.5% or not (16.2 and 16.8% to hyperosmotic washing medium. In conclusion, four-well plates with water in the central hole can be an alternative to replace oil overlay for bovine IVP, maintaining stable osmolality and embryo development rates.

  19. Oocyte transport: Developmental competence of bovine oocytes arrested at germinal vesicle stage by cycloheximide under air.

    Science.gov (United States)

    Hashimoto, Shu; Kimura, Kouji; Iwata, Hisataka; Takakura, Ryo

    2003-02-01

    The effects of the medium (TCM 199 or SOFaa) and temperature (20 or 39 C) during meiotic arrest by cycloheximide (CHX) under air on the developmental competence of bovine oocytes after in vitro maturation (IVM) and fertilization (IVF) were investigated. Oocytes were maintained in meiotic arrest by 10 microg/ml CHX in a 50-microl droplet of 25-mM HEPES-buffered TCM 199 (H199) at 39 C or synthetic oviduct fluid (HSOFaa) at 20 or 39 C in air for 24 h. After release from the arrest, the oocytes was matured and fertilized in vitro and their developmental competence was examined. The developmental rate of oocytes arrested in HSOFaa at 20 C to the blastocyst stage was similar to that of non-arrested oocytes but was significantly higher (Ptransport conditions, we also investigated the meiotic arrest of oocytes maintained in a 0.25-ml straw by CHX individually with 10 microl HSOFaa or as a group (40-50 oocytes) with 170-200 microl HSOFaa at 20 C in air for 24 h. After release from meiotic arrest, the developmental competence of these oocytes was assessed similarly. The developmental rate of oocytes treated with CHX individually was similar to that of those treated with CHX in 50-microl droplet of HSOFaa at 20 C. However, the developmental rate of oocytes treated with CHX as a group was lower than that of oocytes treated with CHX in a 50-microl droplet. Five blastocysts developed from oocytes maintained in meiotic arrest in a plastic straw were transferred to five recipient heifers. Consequently, three recipients became pregnant and 2 calves were delivered. The results of the present study indicate that bovine oocytes treated with CHX in HSOFaa at 20 C under air retain the same developmental competence as non-arrested oocytes.

  20. Effect of roscovitine treated donor cells and different activation methods on development of handmade cloned goat (Capra hircus) embryos.

    Science.gov (United States)

    Akshey, Y S; Malakar, D; De, A Kumar; Jena, M Kumar; Pawar, S Kumar; Dutta, R; Sahu, S

    2011-05-01

    The aim of the present investigation was to find out the effects of roscovitine treatment of donor cells and different activation methods on development of HMC goat embryos. Goat fetal fibroblast cells were cultured and divided into three treatment groups-contact inhibition group, roscovitine treatment group and serum starvation group. There was a significant decrease in blastocyst yield in serum starvation group (6.82%) compared to roscovitine treatment group (19.31%) and contact inhibition group (18.52%), however, no significant difference was found between roscovitine treatment group and contact inhibition group. To see the effect of different methods of activation, the reconstructed embryos were randomly divided into two groups and activated by two methods-one half by 2 μM Ca ionophore and another half by 2.31 kV/cm for 15 μSec electrical pulse. Subsequently, cloned embryos were cultured in TCM-199 based embryo development medium supplemented with 10 mg/mL bovine serum albumin in WOW culture system. There was a significant increase in the rate of cleavage and blastocyst production in electric pulse activation of 78.57% and 21.43% than Ca ionophore activation of 62.63% and 10.61% respectively. In conclusion, treatment of donor cells with roscovitine yields a significantly increased blastocyst than serum starved donor cells but equivalent blastocyst to contact inhibition group and electrical pulse activation (EPA) improves the production of HMC goat embryos. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Influence of maternal nutrition and heat stress on bovine oocyte and embryo development

    Directory of Open Access Journals (Sweden)

    Alzahraa M. Abdelatty

    Full Text Available The global population is expected to increase from 7.6 to 9.6 billion people from 2017 to 2050. Increased demand for livestock production and rising global temperatures have made heat stress (HS a major challenge for the dairy industry. HS been shown to have negative effects on production parameters such as dry matter intake, milk yield, and feed efficiency. In addition to affecting production parameters, HS has also been shown to have negative effects on the reproductive functions of dairy cows. Mitigation of HS effects on dairy cow productivity and fertility necessitate the strategic planning of nutrition, and environmental conditions. The current review will discuss the potential nutriepigenomic strategies to mitigate the effect of HS on bovine embryo. Keywords: Bovine embryo, Dairy cow, Fertility, Heat stress, Maternal nutrition, Oocyst

  2. Dysfunction in gap junction intercellular communication induces aberrant behavior of the inner cell mass and frequent collapses of expanded blastocysts in mouse embryos.

    Science.gov (United States)

    Togashi, Kazue; Kumagai, Jin; Sato, Emiko; Shirasawa, Hiromitsu; Shimoda, Yuki; Makino, Kenichi; Sato, Wataru; Kumazawa, Yukiyo; Omori, Yasufumi; Terada, Yukihiro

    2015-06-01

    We investigated the role of gap junctions (GJs) in embryological differentiation, and observed the morphological behavior of the inner cell mass (ICM) by time-lapse movie observation (TLM) with gap junction inhibitors (GJis). ICR mouse embryos were exposed to two types of GJis in CZB medium: oleamide (0 to 50 μM) and 1-heptanol (0 to 10 mM). We compared the rate of blastocyst formation at embryonic day 4.5 (E4.5) with E5.5. We also observed and evaluated the times from the second cleavage to each embryonic developing stage by TLM. We investigated embryonic distribution of DNA, Nanog protein, and Connexin 43 protein with immunofluorescent staining. In the comparison of E4.5 with E5.5, inhibition of gap junction intercellular communication (GJIC) delayed embryonic blastocyst formation. The times from the second cleavage to blastocyst formation were significantly extended in the GJi-treated embryos (control vs with oleamide, 2224 ± 179 min vs 2354 ± 278 min, p = 0.013). Morphological differences were traced in control versus GJi-treated embryos until the hatching stage. Oleamide induced frequent severe collapses of expanded blastocysts (77.4 % versus 26.3 %, p = 0.0001) and aberrant ICM divisions connected to sticky strands (74.3 % versus 5.3 %, p = 0.0001). Immunofluorescent staining indicated Nanog-positive cells were distributed in each divided ICM. GJIC plays an important role in blastocyst formation, collapses of expanded blastocysts, and the ICM construction in mouse embryos.

  3. Lipid content and cryotolerance of in vitro-produced bovine embryos treated with forskolin before vitrification

    Directory of Open Access Journals (Sweden)

    Melissa Meneghel

    Full Text Available ABSTRACT: The aim of the present study was to evaluate the intracytoplasmic lipid content, development and cryotolerance of in vitro-produced bovine embryos treated with different concentrations of forskolin before vitrification. Embryos were produced from abattoir-derived ovaries and allocated into four groups. In the treatment groups, forskolin was added to the in vitro culture medium on Day 6 and incubated for 24 hours in one of the following concentrations: 2.5μM (Forsk 2.5 group, 5.0μM (Forsk 5.0 group or 10.0μM (Forsk 10.0 group. Embryos from the control group were cultured without forskolin. On Day 7 of culture, the expanded blastocysts were stained with the lipophilic dye Sudan Black B for determination of the intracytoplasmic lipid content or were cryopreserved via the Vitri-Ingá® procedure. Although there were no significant differences (P>0.05 in the blastocyst rates between the Control group (44.9% and the other treatments, the embryo production was lower (P0.05 to that found in Forsk 2.5 (0.92±0.03 and Forsk 10.0 groups (1.06±0.03 groups; however the lipid accumulation in blastocysts from Forsk 5.0 group (0.82±0.04 was lower than in the Control group (P<0.05. Based on these results, Forsk 5.0 treatment was tested for cryotolerance and it was observed that the blastocoel re-expansion rate evaluated 24 hours after warming was greater (P<0.05 in Forsk 5.0 group (72.2% compared to the Control group (46.2%. In conclusion, pre-treatment with forskolin at a concentration of 5.0 μM for 24 hours before vitrification is effective in reducing the intracytoplasmic lipid content and, consequently, improves cryotolerance of IVP bovine embryos.

  4. The impact of food intake and social habits on embryo quality and the likelihood of blastocyst formation.

    Science.gov (United States)

    Braga, Daniela Paes Almeida Ferreira; Halpern, Gabriela; Setti, Amanda S; Figueira, Rita Cássia S; Iaconelli, Assumpto; Borges, Edson

    2015-07-01

    The aim of this study was to evaluate the influence of patients' lifestyle factors and eating habits on embryo development. A total of 2659 embryos recovered from 269 patients undergoing intracytoplasmic sperm injection cycles were included. The frequency of intake of food items and social habits were registered and its influences on embryo development evaluated. The consumption of cereals, vegetables and fruits positively influenced the embryo quality at the cleavage stage. The quality of the embryo at the cleavage stage was also negatively correlated with the consumption of alcoholic drinks and smoking habits. The consumption of fruits influenced the likelihood of blastocyst formation, which was also positively affected by the consumption of fish. Being on a weight-loss diet and consumption of red meat had a negative influence on the likelihood of blastocyst formation. The likelihood of blastocyst formation was also negatively influenced by the consumption of alcoholic drinks and by smoking habits. The consumption of red meat and body mass index had a negative effect on the implantation rate and the likelihood of pregnancy. In addition, being on a weight-loss diet had a negative influence on implantation rate. Our evidence suggests a possible relationship between environmental factors and ovary biology. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  5. Differential Effect of Medium on the Ratio of ICM/TE of Bovine Embryos in a Co-culture System

    Directory of Open Access Journals (Sweden)

    Mohsen Forouzanfar

    2010-01-01

    Full Text Available Background: This study was undertaken to investigate the efficiency of two differentembryo somatic cell co-culture conditions, tissue culture medium 199 (TCM199–vero cellsand Menezo B2 (B2-vero cells, for the in vitro developmental quantity and quality of bovineembryos.Materials and Methods: Bovine oocytes were allowed to mature and subsequently undergofertilization in vitro. Their presumptive zygotes were cultured in either TCM199 or B2 culturemedia in conjunction with vero cells for up to nine days. The culture media were refreshedevery two days and the proportion of embryos that cleaved and further developed to themorula and blastocyst (early, expand and hatched stages were recorded. Hatched blastocystsunderwent differential staining in order to determine the numbers of inner cell mass (ICMand tropho ectoderm (TE and total cell number (TCN.Results: Of the two groups, no significant difference was seen between the proportions ofthe presumptive zygotes cleaved, those which developed to 8-16 cells, morula and reacheddays 7or 8 blastocyst stage or hatched. However, the values for TCN and TE of the TCM199-vero embryos were significantly greater than those of B2-vero embryos. The values for ICM/TCN and ICM/TE were significantly greater in the B2-vero group versus the TCM199-verogroup.Conclusion: Both TCM199 and B2 culture media in conjunction with vero cells were ofthe same efficiency when used for in vitro development of bovine presumptive zygotes.However, TCM199 was superior in providing embryos with more embryo cell numbers,whereas B2 medium was superior in providing embryos with greater ICM/TE and ICM/TCN ratios.

  6. Distinct differences in global gene expression profiles in non-implanted blastocysts and blastocysts resulting in live birth

    DEFF Research Database (Denmark)

    Kirkegaard, Kirstine Kjær; Fredsted, Palle Villesen; Jensen, Jacob Malte

    2015-01-01

    Results from animal models points towards the existence of a gene expression profile that is distinguishably different in viable embryos compared with non-viable embryos. Knowledge of human embryo transcripts is however limited, in particular with regard to how gene expression is related...... to clinical outcome. The purpose of the present study was therefore to determine the global gene expression profiles of human blastocysts. Next Generation Sequencing was used to identify genes that were differentially expressed in non-implanted embryos and embryos resulting in live birth. Three trophectoderm...

  7. Human Endometrial CD98 Is Essential for Blastocyst Adhesion

    Science.gov (United States)

    Domínguez, Francisco; Simón, Carlos; Quiñonero, Alicia; Ramírez, Miguel Ángel; González-Muñoz, Elena; Burghardt, Hans; Cervero, Ana; Martínez, Sebastián; Pellicer, Antonio; Palacín, Manuel; Sánchez-Madrid, Francisco; Yáñez-Mó, María

    2010-01-01

    Background Understanding the molecular basis of embryonic implantation is of great clinical and biological relevance. Little is currently known about the adhesion receptors that determine endometrial receptivity for embryonic implantation in humans. Methods and Principal Findings Using two human endometrial cell lines characterized by low and high receptivity, we identified the membrane receptor CD98 as a novel molecule selectively and significantly associated with the receptive phenotype. In human endometrial samples, CD98 was the only molecule studied whose expression was restricted to the implantation window in human endometrial tissue. CD98 expression was restricted to the apical surface and included in tetraspanin-enriched microdomains of primary endometrial epithelial cells, as demonstrated by the biochemical association between CD98 and tetraspanin CD9. CD98 expression was induced in vitro by treatment of primary endometrial epithelial cells with human chorionic gonadotropin, 17-β-estradiol, LIF or EGF. Endometrial overexpression of CD98 or tetraspanin CD9 greatly enhanced mouse blastocyst adhesion, while their siRNA-mediated depletion reduced the blastocyst adhesion rate. Conclusions These results indicate that CD98, a component of tetraspanin-enriched microdomains, appears to be an important determinant of human endometrial receptivity during the implantation window. PMID:20976164

  8. In vitro culture of bovine embryos in murine ES cell conditioned media negatively affects expression of pluripotency-related markers OCT4, SOX2 and SSEA1.

    Science.gov (United States)

    Oliveira, C S; de Souza, M M; Saraiva, N Z; Tetzner, T A D; Lima, M R; Lopes, F L; Garcia, J M

    2012-06-01

    Despite extensive efforts, establishment of bovine embryonic stem (ES) cell lines has not been successful. We hypothesized that culture conditions for in vitro-produced (IVP) embryos, the most used source of inner cell mass (ICM) to obtain ES cells, might affect their undifferentiated state. Therefore, the aim of this work was to improve pluripotency of IVP blastocysts to produce suitable ICM for further culturing. We tested KSR and foetal calf serum (FCS) supplements in SOF medium and ES cell conditioned medium (CM) on IVC (groups: KSR, KSR CM, FCS and FCS CM). Cleavage and blastocyst rates were similar between all groups. Also, embryonic quality, assessed by apoptosis rates (TUNEL assay), total cell number and ICM percentage did not differ between experimental groups. However, expression of pluripotency-related markers was affected. We detected down-regulation of OCT3/4, SOX2 and SSEA1 in ICM of FCS CM blastocysts (p < 0.05). SOX2 gene expression revealed lower levels (p < 0.05) on KSR CM blastocysts and a remarkable variation in SOX2 mRNA levels on FCS-supplemented blastocysts. In conclusion, pluripotency-related markers tend to decrease after supplementation with ES cell CM, suggesting different mechanisms regulating mouse and bovine pluripotency. KSR supplementation did not differ from FCS, but FCS replacement by KSR may produce blastocysts with stable SOX2 gene expression levels. © 2011 Blackwell Verlag GmbH.

  9. Live birth potential of good morphology and vitrified blastocysts presenting abnormal cell divisions

    DEFF Research Database (Denmark)

    Azzarello, Antonino; Høst, Thomas; Hay-Schmidt, Anders

    2017-01-01

    a lower live birth rate (17.0%) than blastocyst with solely regular cell divisions (29.3%). ACDs could occur at more than one cell division in the same good morphology blastocyst. Reported as independent events, we observed ACDs occurring more frequently at the later cell cycles (1st: 1.3%; 2nd: 8.0%; 3rd...

  10. Effect of antifreeze glycoprotein 8 supplementation during vitrification on the developmental competence of bovine oocytes.

    Science.gov (United States)

    Liang, Shuang; Yuan, Bao; Kwon, Jeong-Woo; Ahn, Mija; Cui, Xiang-Shun; Bang, Jeong Kyu; Kim, Nam-Hyung

    2016-07-15

    The purpose of this study was to investigate the effect of antifreeze glycoprotein 8 (AFGP8) supplementation during vitrification on the survival, fertilization, and embryonic development of bovine oocytes and the underlying molecular mechanism(s). Survival, fertilization, early embryonic development, apoptosis, DNA double-strand breaks, reactive oxygen species levels, meiotic cytoskeleton assembly, chromosome alignment, and energy status of mitochondria were measured in the present experiments. Compared with that in the nonsupplemented group; survival, monospermy, blastocyst formation rates, and blastomere counts were significantly higher in the AFGP8-supplemented animals. Oocytes of the latter group also presented fewer double-strand breaks and lower cathepsin B and caspase activities. Rates of normal spindle organization and chromosome alignment, actin filament impairment, and mitochondrial distribution were significantly higher in the AFGP8-supplemented group. In addition, intracellular reactive oxygen species levels significantly decreased in the AFGP8-supplemented groups, maintaining a higher ΔΨm than that in the nonsupplemented group. Taken together, these results indicated that supplementation with AFGP8 during vitrification has a protective effect on bovine oocytes against chilling injury. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. DNA methylation at a bovine alpha satellite I repeat CpG site during development following fertilization and somatic cell nuclear transfer.

    Directory of Open Access Journals (Sweden)

    Christine Couldrey

    Full Text Available Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT. Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5 during development of cattle generated either by artificial insemination (AI or in vitro fertilization (IVF and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic

  12. Algorithms for automatic segmentation of bovine embryos produced in vitro

    International Nuclear Information System (INIS)

    Melo, D H; Oliveira, D L; Nascimento, M Z; Neves, L A; Annes, K

    2014-01-01

    In vitro production has been employed in bovine embryos and quantification of lipids is fundamental to understand the metabolism of these embryos. This paper presents a unsupervised segmentation method for histological images of bovine embryos. In this method, the anisotropic filter was used in the differents RGB components. After pre-processing step, the thresholding technique based on maximum entropy was applied to separate lipid droplets in the histological slides in different stages: early cleavage, morula and blastocyst. In the postprocessing step, false positives are removed using the connected components technique that identify regions with excess of dye near pellucid zone. The proposed segmentation method was applied in 30 histological images of bovine embryos. Experiments were performed with the images and statistical measures of sensitivity, specificity and accuracy were calculated based on reference images (gold standard). The value of accuracy of the proposed method was 96% with standard deviation of 3%

  13. Developmental kinetics of the first cell cycles of bovine in vitro produced embryos in relation to their in vitro viability and sex

    DEFF Research Database (Denmark)

    Holm, Peter; Shukri, Naseer Mahmoud; Vajta, Gabor

    1998-01-01

    The development of bovine IVP-embryos was observed in a time-lapse culture system to determine cell cycle lengths of 1) embryos that developed into compact morulae (CM) or blastocysts (BL) within 174 h after insemination (viable), 2) embryos that arrested during earlier stages (nonviable) and 3......) male and female embryos. In 4 replicates, inseminated oocytes were cultured on a microscope stage in 3 to 4 groups on a granulosa cell monolayer in supplemented TCM 199. Images were sequentially recorded and stored at 30-min intervals. All embryos that could be identified throughout the culture period...... were included (n=392), and the times of cleavage events noted. After culture, 100 CM or BL were randomly selected for sexing by PCR. BL developed equally well in the time-lapse and control culture systems (36 vs 38. The respective lengths of the first 4 cell cycles of viable embryos were 32.0 + 3.9, g...

  14. The use of embryonic stem cell derived bioactive material as a new protein supplement for the in vitro culture of bovine embryos.

    Science.gov (United States)

    Kim, Eun Young; Lee, Jun Beom; Park, Hyo Young; Jeong, Chang Jin; Riu, Key Zung; Park, Se Pill

    2011-06-01

    Embryonic stem (ES) cells are expanded versions of the inner cell mass cells that compose the early mammalian blastocyst. Components derived from ES cells may contain various bioactive materials (BM) helpful for early preimplantation embryo growth. In this study, we examined the effect of human ES cell derived BM (hES-BM) on in vitro culture of bovine embryos. When bovine parthenogenetic day 2 embryos were cultured in 10% hES-BM, a significantly higher embryo development rate (44.3%) and increased cell numbers were observed relative to control medium containing 3 mg/ml BSA (19.5%; Pculture environment to support the growth of bovine embryos in vitro (P<0.05). Little difference was observed between 10% hES-BM and 10% FBS treatment in the examined parthenogenetic or in vitro fertilized embryos, although the hES-BM group developed at a slightly better rate. However, the ICM cell numbers were significantly higher in the hES-BM group in irrespective of embryo origin (P<0.05). In addition, the relative levels of pluripotency (Oct4, × 1.8 fold; Nanog. × 3.3 fold), embryogenesis (Stat3, × 2.8 fold; FGF4, × 18.8 fold; E-cad, × 2.0 fold) and growth (Glut5, × 2.6 fold) genes were significantly higher in the 10% hES-BM group than in the 10% FBS group (P<0.05), while the levels of other genes (Bax, Bcl2, MnSOD and Connexin43) were not different. This is the first report examining the positive effects of hES-BM on bovine embryo development in vitro. Based on our results, we conclude that hES-BM can be used as a new protein supplement for bovine preimplantation embryo development.

  15. Expression profile of genes as indicators of developmental competence and quality of in vitro fertilization and somatic cell nuclear transfer bovine embryos.

    Directory of Open Access Journals (Sweden)

    Maria Jesús Cánepa

    Full Text Available Reproductive biotechnologies such as in vitro fertilization (IVF and somatic cell nuclear transfer (SCNT enable improved reproductive efficiency of animals. However, the birth rate of in vitro-derived embryos still lags behind that of their in vivo counterparts. Thus, it is critical to develop an accurate evaluation and prediction system of embryo competence, both for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA of diverse transcripts and thus compromise embryo quality. The aim of this work was to analyze the RA of a set of genes involved in cellular stress (heat shock protein 70-kDa, HSP70, endoplasmic reticulum (ER stress (immunoglobulin heavy chain binding protein, Bip; proteasome subunit β5, PSMB5 and apoptosis (BCL-2 associated X protein, Bax; cysteine aspartate protease-3, Caspase-3 in bovine blastocysts produced by IVF or SCNT and compare it with that of their in vivo counterparts. Poly (A + mRNA was isolated from three pools of 10 blastocysts per treatment and analyzed by real-time RT-PCR. The RA of three of the stress indicators analyzed (Bax, PSMB5 and Bip was significantly increased in SCNT embryos as compared with that of in vivo-derived blastocysts. No significant differences were found in the RA of HSP70 and Caspase-3 gene transcripts. This study could potentially complement morphological analyses in the development of an effective and accurate technique for the diagnosis of embryo quality, ultimately aiding to improve the efficiency of assisted reproductive techniques (ART.

  16. Effect of the cryopreservation method used, the embryonic stage and the use of conjugated linoleic acid isomers on the cryotolerance of in vitro-produced bovine embryos

    Directory of Open Access Journals (Sweden)

    Luciana Simões Rafagnin Marinho

    2015-12-01

    Full Text Available Conjugated linoleic acid (CLA might be able to improve the cryotolerance of in vitro-produced (IVP embryos. The effect of two CLA isomers on the cryotolerance of bovine IVP embryos, as well as that of the stage of embryonic development and the method used for cryopreservation was evaluated by three experiments. In Experiment 1, oocytes (n = 3,917 were fertilized in vitro and cultured with 0, 50, 100, or 200 ?M trans-10, cis-12 (t10, c12 CLA. In Experiment 2, fertilized oocytes (n = 2,131 were cultured with 100 ?M t10, c12 or cis-9, trans-11 (c9, t11 CLA, or a combination of both isomers. The embryos were vitrified at the blastocyst (BL or the expanded blastocyst (EB stage. In Experiment 3, oocytes (n = 1,720 were fertilized and cultured with or without 100 ?M t10, c12 CLA, and the blastocysts were vitrified or frozen. Blastocyst development rate as well as the rates of re-expansion and hatching after thawing was recorded. Moreover, the mean cell number and mRNA expression of acetyl-CoA carboxylase (ACC1 and stearoyl-CoA desaturase (SCD1 as well as fatty acid synthase (FASN multienzyme complex were determined. In Experiment 1, the highest concentration of t10, c12 CLA that did not reduce blastocyst development rate was 100 ?M. In Experiment 2, the rates of re-expansion and hatching among the EBs obtained through IVP after supplementation with t10, c12 CLA (73.1% and 57.7%, with c9, t11 CLA (80.0% and 68.6%, with the combination (78.3% and 52.2%, and with the control group (85.4% and 58.3% were similar. At the BL stage, the rates of re-expansion and hatching were lower than those at the EB stage, and CLA combination allowed a hatching rate (8.0% lower than that observed in the control group (40.0%. In Experiment 3, the hatching rates for vitrified EBs (vitrified control; 67.4% and vitrified CLA EBs (65.8% were higher than those obtained for frozen EBs, exposed (13.3% or not exposed (28.6% to CLA. In addition, in Experiment 3, the hatching rate was

  17. TallyHO obese female mice experience poor reproductive outcomes and abnormal blastocyst metabolism that is reversed by metformin.

    Science.gov (United States)

    Louden, Erica D; Luzzo, Kerri M; Jimenez, Patricia T; Chi, Tiffany; Chi, Maggie; Moley, Kelle H

    2014-12-01

    Obese women experience worse reproductive outcomes than normal weight women, specifically infertility, pregnancy loss, fetal malformations and developmental delay of offspring. The aim of the present study was to use a genetic mouse model of obesity to recapitulate the human reproductive phenotype and further examine potential mechanisms and therapies. New inbred, polygenic Type 2 diabetic TallyHO mice and age-matched control C57BL/6 mice were superovulated to obtain morula or blastocyst stage embryos that were cultured in human tubal fluid (HTF) medium. Deoxyglucose uptake was determined for individual insulin-stimulated blastocysts. Apoptosis was detected by confocal microscopy using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay and Topro-3 nuclear dye. Embryos were scored for TUNEL-positive as a percentage of total nuclei. AMP-activated protein kinase (AMPK) activation, tumour necrosis factor (TNF)-α expression and adiponectin expression were analysed by western immunoblot and confocal immunofluorescent microscopy. Lipid accumulation was assayed by BODIPY. Comparisons were made between TallyHO morulae cultured to blastocyst embryos in either HTF medium or HTF medium with 25 μg mL(-1) metformin. TallyHO mice developed whole body abnormal insulin tolerance, had decreased litter sizes and increased non-esterified fatty acid levels. Blastocysts from TallyHO mice exhibited increased apoptosis, decreased insulin sensitivity and decreased AMPK. A possible cause for the insulin resistance and abnormal AMPK phosphorylation was the increased TNF-α expression and lipid accumulation, as detected by BODIPY, in TallyHO blastocysts and decreased adiponectin. Culturing TallyHO morulae with the AMPK activator metformin led to a reversal of all the abnormal findings, including increased AMPK phosphorylation, improved insulin-stimulated glucose uptake and normalisation of lipid accumulation. Women with obesity and

  18. 76 FR 38602 - Bovine Tuberculosis and Brucellosis; Program Framework

    Science.gov (United States)

    2011-07-01

    ...] Bovine Tuberculosis and Brucellosis; Program Framework AGENCY: Animal and Plant Health Inspection Service... framework being developed for the bovine tuberculosis and brucellosis programs in the United States. This... proposed revisions to its programs regarding bovine tuberculosis (TB) and bovine brucellosis in the United...

  19. Development of a solid-phase radioimmunoassay for antibody to antigens of Babesia bovis infected bovine erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kahl, L.P.; Anders, R.F.; Mitchell, G.F. (Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia)); Callow, L.L.; Rodwell, B.J. (Animal Research Inst. Wacol (Australia). Tick Fever Research Centre)

    1982-06-01

    A quantitative solid-phase radioimmunoassay (RIA) has been developed for the purpose of ranking sera from exposed animals according to their titre of anti-B. bovis antibody. The antigen was a sonicate of lysed infected blood cells, and antibody binding was detected with /sup 125/I-labelled anti-bovine IgG. The assay was tested using sera from experimentally infected splenectomized and intact cattle and from animals resident in an endemic area. A high specificity for B. bovis was demonstrated. There was good agreement in identifying exposed cattle when the test was compared with an indirect fluorescent antibody (IFA) test although no correlation was seen between titres obtained in the two tests. Analysis of the effects of challenge infection with B. bovis on IFA and RIA titres in previously exposed animals illustrated that the RIA was a more sensitive test for detecting changes in antibody titre.

  20. Peptidomic Approach to Developing ELISAs for the Determination of Bovine and Porcine Processed Animal Proteins in Feed for Farmed Animals.

    Science.gov (United States)

    Huet, Anne-Catherine; Charlier, Caroline; Deckers, Elise; Marbaix, Hélène; Raes, Martine; Mauro, Sergio; Delahaut, Philippe; Gillard, Nathalie

    2016-11-30

    The European Commission (EC) wants to reintroduce nonruminant processed animal proteins (PAPs) safely into the feed chain. This would involve replacing the current ban in feed with a species-to-species ban which, in the case of nonruminants, would only prohibit feeding them with proteins from the same species. To enforce such a provision, there is an urgent need for species-specific methods for detecting PAPs from several species in animal feed and in PAPs from other species. Currently, optical microscopy and the polymerase chain reaction are the officially accepted methods, but they have limitations, and alternative methods are needed. We have developed immunoassays using antibodies raised against targets which are not influenced by high temperature and pressure. These targets were identified in a previous study based on an experimental approach. One optimized competitive ELISA detects bovine PAPs at 2% in plant-derived feed. The detection capability demonstrated on blind samples shows a good correlation with mass spectrometry results.

  1. Phytohemagglutinin facilitates the aggregation of blastomere pairs from Day 5 donor embryos with Day 4 host embryos for chimeric bovine embryo multiplication.

    Science.gov (United States)

    Simmet, Kilian; Reichenbach, Myriam; Reichenbach, Horst-Dieter; Wolf, Eckhard

    2015-12-01

    Multiplication of bovine embryos by the production of aggregation chimeras is based on the concept that few blastomeres of a donor embryo form the inner cell mass (ICM) and thus the embryo proper, whereas cells of a host embryo preferentially contribute to the trophectoderm (TE), the progenitor cells of the embryonic part of the placenta. We aggregated two fluorescent blastomeres from enhanced green fluorescent protein (eGFP) transgenic Day 5 morulae with two Day 4 embryos that did not complete their first cleavage until 27 hours after IVF and tested the effect of phytohemagglutinin-L (PHA) on chimeric embryo formation. The resulting blastocysts were characterized by differential staining of cell lineages using the TE-specific factor CDX2 and confocal laser scanning microscopy to facilitate the precise localization of eGFP-positive cells. The proportions of blastocyst development of sandwich aggregates with (n = 99) and without PHA (n = 46) were 85.9% and 54.3% (P chimeric blastocysts analyzed by confocal laser scanning microscopy, nine had eGFP-positive cells (three of them in the ICM, three in the TE, and three in both lineages). When integration in the ICM occurred, the number of eGFP-positive cells in this compartment was 8.3 ± 2.3 (mean ± standard error of the mean). We conclude that PHA is advantageous for the formation of aggregation chimeras, but the approach tested in the present study with only two donor blastomeres and two host embryos did not result in multiplication of genetically valuable donor embryos. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Development of a recombinant poxvirus expressing bovine herpesvirus-1 glycoprotein D

    International Nuclear Information System (INIS)

    Ruiz Saenz, Julian; Osorio, Jorge E; Vera, Victor J.

    2012-01-01

    Bovine herpesvirus-1 is a DNA virus belonging to the family herpesviridae, which affects cattle, causing a wide spectrum of clinical manifestations and economic losses. The main immunogenic component is its envelope glycoprotein d (GD), which has been characterized and used as immunogen in different expression systems. The aim of this work was to generate a recombinant poxvirus (raccoonpox [RCN]) expressing a truncated version of BHV-1 GD to be used as a vaccine. to do this, it was amplified the gene for a truncated version of GD which subsequently was cloned in transfer plasmid PTK/IRES/TPA which has homology to sites of poxvirus thymidine kinase, an internal site of ribosome entry (IRES) and a secretory signal (TPA), generating the construct PTK/GD/IRES/TPA. to generate the recombinant RCN, we took BSC-1 cells and we infected with a wild type RCN (CDC/v71-i-85a) at a multiplicity of infection of 0.05, then cells were transfected with the construct PTK/GD/IRES/TPA, generating different viral populations with and without the gene of interest. To select recombinant viruses expressing the gene of interest, we performed a selection of recombinant thymidine kinase negative and positive for GD by three rounds of plaque purification on rat-2 cells monolayers which are thymidine kinase null and using bromodeoxyuridine. Recombinant viruses were recovered and confirmed by PCR and nucleotide sequencing and so called RCN-GD.

  3. Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

    Science.gov (United States)

    Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe

    2016-09-01

    The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes

  4. Vitrified/warmed single blastocyst transfer in preimplantation genetic diagnosis/preimplantation genetic screening cycles.

    Science.gov (United States)

    Huang, Jin; Li, Rong; Lian, Ying; Chen, Lixue; Shi, Xiaodan; Qiao, Jie; Liu, Ping

    2015-01-01

    To investigate the single blastocyst transfer in preimplantation genetic diagnosis (PGD)/preimplantation genetic screening (PGS) cycles. 80 PGD/PGS cycles undergoing blastocyst biopsy were studied. There were 88 warming cycles during the study period. Only one warmed blastocyst was transferred per cycle. The outcomes were followed up to the infants were born. The embryo implantation rate was 54.55% (48/88). The clinical pregnancy rate was 54.55% (48/88) per transfer cycle and 60% (48/80) per initial PGD/PGS cycle. There was no multi-pregnant in this study. The live birth rate was 42.05% (37/88) per transfer cycle and 46.25% (37/80) per initial PGD/PGS cycle. In PGD/PGS cycles, single blastocyst transfer reduces the multiple pregnancy rate without affecting the clinical outcomes.

  5. Model systems to analyze the role of miRNAs and commensal microflora in bovine mucosal immune system development.

    Science.gov (United States)

    Liang, Guanxiang; Malmuthuge, Nilusha; Guan, Le Luo; Griebel, Philip

    2015-07-01

    Information is rapidly accumulating regarding the role of miRNAs as key regulators of immune system development and function. It is also increasingly evident that miRNAs play an important role in host-pathogen interactions through regulation of both innate and acquired immune responses. Little is known, however, about the specific role of miRNAs in regulating normal development of the mucosal immune system, especially during the neonatal period. Furthermore, there is limited knowledge regarding the possible role the commensal microbiome may play in regulating mucosal miRNAs expression, although evidence is emerging that a variety of enteric pathogens influence miRNA expression. The current review focuses on recent information that miRNAs play an important role in regulating early development of the bovine mucosal immune system. A possible role for the commensal microbiome in regulating mucosal development by altering miRNA expression is also discussed. Finally, we explore the potential advantages of using the newborn calf as a model to determine how interactions between developmental programming, maternal factors in colostrum, and colonization of the gastrointestinal tract by commensal bacteria may alter mucosal miRNA expression and immune development. Identifying the key factors that regulate mucosal miRNA expression is critical for understanding how the balance between protective immunity and inflammation is maintained to ensure optimal gastrointestinal tract function and health of the whole organism. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Development and Characterization of A Multiplexed RT-PCR Species Specific Assay for Bovine and one for Porcine Foot-and-Mouth Disease Virus Rule-Out

    Energy Technology Data Exchange (ETDEWEB)

    Smith, S M; Danganan, L; Tammero, L; Vitalis, B; Lenhoff, R; Naraghi-arani, P; Hindson, B

    2007-08-06

    Lawrence Livermore National Laboratory (LLNL), in collaboration with the Department of Homeland Security (DHS) and the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Services (APHIS) has developed candidate multiplexed assays that may potentially be used within the National Animal Health Laboratory Network (NAHLN), the National Veterinary Services Laboratory (Ames, Iowa) and the Plum Island Animal Disease Center (PIADC). This effort has the ability to improve our nation's capability to discriminate between foreign animal diseases and those that are endemic using a single assay, thereby increasing our ability to protect food and agricultural resources with a diagnostic test which could enhance the nation's capabilities for early detection of a foreign animal disease. In FY2005 with funding from the DHS, LLNL developed the first version (Version 1.0) of a multiplexed (MUX) nucleic-acid-based RT-PCR assay that included signatures for foot-and-mouth disease virus (FMDV) detection with rule-out tests for two other foreign animal diseases (FADs) of swine, Vesicular Exanthema of Swine (VESV) and Swine Vesicular Disease Virus (SVDV), and four other domestic viral diseases Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus 1 (BHV-1), Bluetongue virus (BTV) and Parapox virus complex (which includes Bovine Papular Stomatitis Virus [BPSV], Orf of sheep, and Pseudocowpox). In FY06, LLNL has developed Bovine and Porcine species-specific panel which included existing signatures from Version 1.0 panel as well as new signatures. The MUX RT-PCR porcine assay for detection of FMDV includes the FADs, VESV and SVD in addition to vesicular stomatitis virus (VSV) and porcine reproductive and respiratory syndrome (PRRS). LLNL has also developed a MUX RT-PCR bovine assay for detection of FMDV with rule out tests for the two bovine FADs malignant catarrhal fever (MCF), rinderpest virus (RPV) and the domestic diseases vesicular stomatitis

  7. Quantitation and histochemical localization of galectin-1 and galectin-1-reactive glycoconjugates in fetal development of bovine organs.

    Science.gov (United States)

    Kaltner, H; Lips, K S; Reuter, G; Lippert, S; Sinowatz, F; Gabius, H J

    1997-10-01

    The display of cellular oligosaccharide chains is known to undergo marked developmental changes, as monitored histochemically with plant lectins. In conjunction with endogenous lectins respective ligand structures may have a functional role during fetal development. The assumption of a recognitive, functionally productive interplay prompts the study of the expression of a tissue lectin and of lectin-reactive glycoconjugates concomitantly. Focusing on common beta-galactosides as constituents of oligosaccharide chains and the predominant member of the family of galectins in mammals, namely galectin-1, the question therefore is addressed as to whether expression of lectin and lectin-reactive glycoconjugates exhibits alterations, assessed in three morphologically defined fetal stages and in adult bovine organs. Using a sandwich ELISA, the level of the rather ubiquitous galectin-1 is mostly increased in adult organs relative to respective fetal stages, except for the case of kidney. This developmental course is seen rather seldom, when the amounts of lectin-reactive glycoproteins or glycolipids are quantitated in solid-phase assays after tissue homogenization. Western blotting, combined with probing by labeled galectin-1, discloses primarily quantitative changes in the reactivity of individual glycoproteins. Performing the same assays on extract aliquots with a plant agglutinin, namely the galactoside-binding mistletoe lectin, whose fine specificity is different from galectin-1, its reduced extent of binding in solid-phase assays and the disparate profile of lectin-reactive glycoproteins reveal a non-uniform developmental alteration within the group of structural variants of beta-galactosides. Although sample preparation can affect ligand preservation and/or presentation and thus restricts the comparability of biochemical and histochemical results, especially for soluble reactants, the histochemical studies on frozen and paraffin-embedded sections of bovine heart

  8. Expression of intracellular interferon-alpha confers antiviral properties in transfected bovine fetal fibroblasts and does not affect the full development of SCNT embryos.

    Directory of Open Access Journals (Sweden)

    Dawei Yu

    Full Text Available Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α (without secretory signal sequence gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT. Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9% became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR and the 2'-5'-oligoadenylate synthetase gene (2'-5' OAS, which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2'-5' OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.

  9. Development of a loop-mediated isothermal amplification assay for the detection of Streptococcus agalactiae in bovine milk.

    Science.gov (United States)

    Bosward, Katrina L; House, John K; Deveridge, Amber; Mathews, Karen; Sheehy, Paul A

    2016-03-01

    Streptococcus agalactiae is a well-characterized bovine mastitis pathogen that is known to be highly contagious and capable of spreading rapidly in affected dairy herds. Loop-mediated isothermal amplification (LAMP) is a novel molecular diagnostic method that has the capability to provide rapid, cost-effective screening for pathogens to support on-farm disease control and eradication programs. In the current study, a LAMP test was developed to detect S. agalactiae in milk. The assay was validated on a bank of existing clinical mastitis milk samples that had previously been identified as S. agalactiae positive via traditional microbiological culture techniques and PCR. The LAMP assay was conducted on bacterial colonies and DNA extracted from milk in tube- and plate-based formats using multiple detection platforms. The 1-h assay conducted at 64 °C exhibited repeatability (coefficient of variation) of 2.07% (tube) and 8.3% (plate), sensitivity to ~20 pg of extracted DNA/reaction, and specificity against a panel of known bacterial mastitis pathogens. Of the 109 known S. agalactiae isolates assessed by LAMP directly from bacterial cells in culture, 108 were identified as positive, in accordance with PCR analysis. The LAMP analysis from the corresponding milk samples indicated that 104 of these milks exhibited a positive amplification curve. Although exhibiting some limitations, this assay provides an opportunity for rapid screening of milk samples to facilitate on-farm management of this pathogen. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  10. F1F0-ATP synthase from bovine heart mitochondria: development of the purification of a monodisperse oligomycin-sensitive ATPase.

    OpenAIRE

    Lutter, R; Saraste, M; van Walraven, H S; Runswick, M J; Finel, M; Deatherage, J F; Walker, J E

    1993-01-01

    A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20...

  11. 77 FR 29914 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Science.gov (United States)

    2012-05-21

    ... Spongiform Encephalopathy; Importation of Bovines and Bovine Products AGENCY: Animal and Plant Health... derived from bovines with regard to bovine spongiform encephalopathy. This action will allow interested... importation of live bovines and products derived from bovines with regard to bovine spongiform encephalopathy...

  12. NanoSMGT: transgene transmission into bovine embryos using halloysite clay nanotubes or nanopolymer to improve transfection efficiency.

    Science.gov (United States)

    Campos, Vinicius Farias; de Leon, Priscila Marques Moura; Komninou, Eliza Rossi; Dellagostin, Odir Antônio; Deschamps, João Carlos; Seixas, Fabiana Kömmling; Collares, Tiago

    2011-11-01

    The objectives were to investigate whether: 1) nanotransfectants are more effective than other common transfection methods for SMGT; 2) NanoSMGT is able to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay nanotubes (HCNs) can be used as a transfection reagent to improve transgene transmission. Four transfection systems were used: naked DNA (without transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid uptake by sperm and its transfer to embryos were quantified by conventional and real-time PCR, as well as EGFP expression by fluorescence microscopy. Furthermore, sperm motility and viability, and embryo development were investigated. Mean number of plasmids taken up was affected (P < 0.05) by transfection procedure, with the nanopolymer being the most effective transfectant (∼ 153 plasmids per spermatozoon). None of the treatments affected sperm motility or viability. The mean number of plasmids transmitted to four-cell stage embryos was higher (P < 0.05) in nanopolymer and HCNs than liposomes and naked DNA groups. The number of embryos carrying the transgene increased from 8-10% using naked DNA or liposomes to 40-45% using nanopolymer or HCN as transfectants (P < 0.05). There were no significant differences among transfection procedures regarding blastocyst formation rate of resulting embryos. However, no EGFP-expressing embryo was identified in any treatment. Therefore, nanotransfectants improved transgene transmission in bovine embryos without deleterious effects on embryo development. To our knowledge, this was the first time that bovine embryos carrying a transgene were produced by NanoSMGT. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. A Newly Developed Perfused Umbrella Electrode for Radiofrequency Ablation: An Ex Vivo Evaluation Study in Bovine Liver

    International Nuclear Information System (INIS)

    Bruners, Philipp; Pfeffer, Jochen; Kazim, Rana M.; Guenther, Rolf W.; Schmitz-Rode, Thomas; Mahnken, Andreas H.

    2007-01-01

    The purpose of this study was to evaluate the effectiveness of a newly developed perfused monopolar radiofrequency (RF) probe with an umbrella-shaped array. A perfused umbrella-shaped monopolar RF probe based on a LeVeen electrode (Boston Scientific Corp., Natick, MA, USA) with a 3-cm array diameter was developed. Five different configurations of this electrode were tested: (a) perfusion channel/endhole, (b) perfusion channel/endhole + sideholes, (c) 1 cm insulation removed at the tip, (d) 1 cm insulation removed at the tip + perfusion channel/endhole, and (e) 1 cm insulation removed at the tip + perfusion channel/endhole + sideholes. An unmodified LeVeen electrode served as a reference standard. RF ablations were performed in freshly excised bovine liver using a commercial monopolar RF system with a 200-W generator (RF 3000; Boston Scientific Corp.). Each electrode was tested 10 times applying the vendor's recommended ablation protocol combined with the preinjection of 2 ml 0.9% saline. Volumes and shapes of the lesions were compared. Lesions generated with the original LeVeen electrode showed a mean volume of 12.74 ± 0.52 cm 3 . Removing parts of the insulation led to larger coagulation volumes (22.65 ± 2.12 cm 3 ). Depending on the configuration, saline preinjection resulted in a further increase in coagulation volume (25.22 ± 3.37 to 31.28 ± 2.32 cm 3 ). Besides lesion volume, the shape of the ablation zone was influenced by the configuration of the electrode used. We conclude that saline preinjection in combination with increasing the active tip length of the umbrella-shaped LeVeen RF probe allows the reliable ablation of larger volumes in comparison to the originally configured electrode

  14. Development of intramammary delivery systems containing lasalocid for the treatment of bovine mastitis: impact of solubility improvement on safety, efficacy, and milk distribution in dairy cattle

    Science.gov (United States)

    Wang, Wen; Song, Yunmei; Petrovski, Kiro; Eats, Patricia; Trott, Darren J; Wong, Hui San; Page, Stephen W; Perry, Jeanette; Garg, Sanjay

    2015-01-01

    Background Mastitis is a major disease of dairy cattle. Given the recent emergence of methicillin-resistant Staphylococcus aureus as a cause of bovine mastitis, new intramammary (IMA) treatments are urgently required. Lasalocid, a member of the polyether ionophore class of antimicrobial agents, has not been previously administered to cows by the IMA route and has favorable characteristics for development as a mastitis treatment. This study aimed to develop an IMA drug delivery system (IMDS) of lasalocid for the treatment of bovine mastitis. Methods Minimum inhibitory concentrations (MICs) were determined applying the procedures recommended by the Clinical and Laboratory Standards Institute. Solid dispersions (SDs) of lasalocid were prepared and characterized using differential scanning calorimetry and Fourier transform infrared spectroscopy. IMDSs containing lasalocid of micronized, nano-sized, or as SD form were tested for their IMA safety in cows. Therapeutic efficacy of lasalocid IMDSs was tested in a bovine model involving experimental IMA challenge with the mastitis pathogen Streptococcus uberis. Results Lasalocid demonstrated antimicrobial activity against the major Gram-positive mastitis pathogens including S. aureus (MIC range 0.5–8 μg/mL). The solubility test confirmed limited, ion-strength-dependent water solubility of lasalocid. A kinetic solubility study showed that SDs effectively enhanced water solubility of lasalocid (21–35-fold). Polyvinylpyrrolidone (PVP)-lasalocid SD caused minimum mammary irritation in treated cows and exhibited faster distribution in milk than either nano or microsized lasalocid. IMDSs with PVP-lasalocid SD provided effective treatment with a higher mastitis clinical and microbiological cure rate (66.7%) compared to cloxacillin (62.5%). Conclusion Lasalocid SD IMDS provided high cure rates and effectiveness in treating bovine mastitis with acceptable safety in treated cows. PMID:25653501

  15. Development of intramammary delivery systems containing lasalocid for the treatment of bovine mastitis: impact of solubility improvement on safety, efficacy, and milk distribution in dairy cattle.

    Science.gov (United States)

    Wang, Wen; Song, Yunmei; Petrovski, Kiro; Eats, Patricia; Trott, Darren J; Wong, Hui San; Page, Stephen W; Perry, Jeanette; Garg, Sanjay

    2015-01-01

    Mastitis is a major disease of dairy cattle. Given the recent emergence of methicillin-resistant Staphylococcus aureus as a cause of bovine mastitis, new intramammary (IMA) treatments are urgently required. Lasalocid, a member of the polyether ionophore class of antimicrobial agents, has not been previously administered to cows by the IMA route and has favorable characteristics for development as a mastitis treatment. This study aimed to develop an IMA drug delivery system (IMDS) of lasalocid for the treatment of bovine mastitis. Minimum inhibitory concentrations (MICs) were determined applying the procedures recommended by the Clinical and Laboratory Standards Institute. Solid dispersions (SDs) of lasalocid were prepared and characterized using differential scanning calorimetry and Fourier transform infrared spectroscopy. IMDSs containing lasalocid of micronized, nano-sized, or as SD form were tested for their IMA safety in cows. Therapeutic efficacy of lasalocid IMDSs was tested in a bovine model involving experimental IMA challenge with the mastitis pathogen Streptococcus uberis. Lasalocid demonstrated antimicrobial activity against the major Gram-positive mastitis pathogens including S. aureus (MIC range 0.5-8 μg/mL). The solubility test confirmed limited, ion-strength-dependent water solubility of lasalocid. A kinetic solubility study showed that SDs effectively enhanced water solubility of lasalocid (21-35-fold). Polyvinylpyrrolidone (PVP)-lasalocid SD caused minimum mammary irritation in treated cows and exhibited faster distribution in milk than either nano or microsized lasalocid. IMDSs with PVP-lasalocid SD provided effective treatment with a higher mastitis clinical and microbiological cure rate (66.7%) compared to cloxacillin (62.5%). Lasalocid SD IMDS provided high cure rates and effectiveness in treating bovine mastitis with acceptable safety in treated cows.

  16. Clinical outcome of fresh and vitrified-warmed blastocyst and cleavage-stage embryo transfers in ethnic Chinese ART patients

    Directory of Open Access Journals (Sweden)

    Tong Guo

    2012-10-01

    Full Text Available Abstract Objectives This study sought to evaluate the outcome of fresh and vitrified-warmed cleavage-stage and blastocyst-stage embryo transfers in patients undergoing ART treatment within an ethnic Chinese population. Study design We compared the clinical results of embryo transfer on the 3rd (cleavage stage or 5th (blastocyst stage day after oocyte retrieval, including clinical pregnancy rates, implantation rates and multiple pregnancy rates. Results Our data showed that blastocyst transfer on day 5 did not significantly increase clinical pregnancy rate (41.07% vs 47.08%, p>0.05 and implantation rate (31.8% vs 31.2%, p>0.05 in patients under 35 years of age, in comparison with day 3 cleavage stage embryo transfer. In patients older than 35 years of age, the clinical pregnancy rate after blastocyst transfer was slightly decreased compared with cleavage stage embryo transfer (33.33% vs 42.31%, p>0.05. Unexpectedly, It was found that vitrified-warmed blastocyst transfer resulted in significantly higher clinical pregnancy rate (56.8% and implantation rate (47% compared with fresh blastocyst transfer in controlled stimulation cycles (41.07% and 31.8%, respectively. For patients under 35 years of age, the cumulative clinical pregnancy rate combining fresh and vitrified-warmed blastocyst transfer cycles were significantly higher compared to just cleavage-stage embryo transfer (70.1% versus 51.8%, p Conclusions In an ethnic Chinese patient population, fresh blastocyst transfer does not significantly increase clinical pregnancy rate. However, subsequent vitrified-warmed blastocyst transfer in a non-controlled ovarian hyperstimulation cycle dramatically improves clinical outcomes. Therefore, blastocyst culture in tandem with vitrified-warmed blastocyst transfer is recommended as a favourable and promising protocol in human ART treatment, particularly for ethnic Chinese patients.

  17. Clinical outcome of fresh and vitrified-warmed blastocyst and cleavage-stage embryo transfers in ethnic Chinese ART patients.

    Science.gov (United States)

    Tong, Guo Qing; Cao, Shan Ren; Wu, Xun; Zhang, Jun Qiang; Cui, Ji; Heng, Boon Chin; Ling, Xiu Feng

    2012-10-05

    This study sought to evaluate the outcome of fresh and vitrified-warmed cleavage-stage and blastocyst-stage embryo transfers in patients undergoing ART treatment within an ethnic Chinese population. We compared the clinical results of embryo transfer on the 3rd (cleavage stage) or 5th (blastocyst stage) day after oocyte retrieval, including clinical pregnancy rates, implantation rates and multiple pregnancy rates. Our data showed that blastocyst transfer on day 5 did not significantly increase clinical pregnancy rate (41.07% vs 47.08%, p>0.05) and implantation rate (31.8% vs 31.2%, p>0.05) in patients under 35 years of age, in comparison with day 3 cleavage stage embryo transfer. In patients older than 35 years of age, the clinical pregnancy rate after blastocyst transfer was slightly decreased compared with cleavage stage embryo transfer (33.33% vs 42.31%, p>0.05). Unexpectedly, It was found that vitrified-warmed blastocyst transfer resulted in significantly higher clinical pregnancy rate (56.8%) and implantation rate (47%) compared with fresh blastocyst transfer in controlled stimulation cycles (41.07% and 31.8%, respectively). For patients under 35 years of age, the cumulative clinical pregnancy rate combining fresh and vitrified-warmed blastocyst transfer cycles were significantly higher compared to just cleavage-stage embryo transfer (70.1% versus 51.8%, p<0.05). However, the cumulative multiple pregnancy rates showed no significant difference between the two groups. In an ethnic Chinese patient population, fresh blastocyst transfer does not significantly increase clinical pregnancy rate. However, subsequent vitrified-warmed blastocyst transfer in a non-controlled ovarian hyperstimulation cycle dramatically improves clinical outcomes. Therefore, blastocyst culture in tandem with vitrified-warmed blastocyst transfer is recommended as a favourable and promising protocol in human ART treatment, particularly for ethnic Chinese patients.

  18. Next generation sequencing for preimplantation genetic testing of blastocysts aneuploidies in women of different ages

    Directory of Open Access Journals (Sweden)

    Krzysztof Lukaszuk

    2015-12-01

    Full Text Available Most of the current preimplantation genetic screening of aneuploidies tests are based on the low quality and low density comparative genomic hybridization arrays. The results are based on fewer than 2,700 probes. Our main outcome was the association of aneuploidy rates and the women’s age. Between August–December 2013, 198 blastocysts from women (mean age 36.3+-4.6 undergoing in vitro fertilization underwent routine trophectoderm biopsy. NGS was performed on Ion Torrent PGM (Life Technologies. The results were analyzed in five age groups ( 40. 85 blastocysts were normal according to NGS results. The results in the investigated groups were (% of normal blastocyst in each group: 40 (38.5%. Our study suggests that NGS PGD is applicable for routine preimplantation genetic testing. It allows also for easy customization of the procedure for each individual patient making personalized diagnostics a reality.

  19. Factors Analysis of Spontaneous Abortion after Thawed-vitrified Blastocysts Transfer

    Institute of Scientific and Technical Information of China (English)

    Dong YANG; Zheng-yi SUN; Cheng-yan DENG; Qi YU; Fang-fang HE

    2008-01-01

    Objective To investigate the factors resulting in spontaneous abortion after transferring frozen-thawing blastocysts. Methods A total of 108 cases transferring vitrified blastocysts were divided into two groups: abortion group (n =20) and ongoing group (n=88). Cytogenetic analysis of apoblemas was performed in 12 cases of the abortion.Results The overall spontaneous abortion rate was 18.50%(20/108) and the early spontaneous rate was 16.67%(18/108). ,4 significant difference in maternal age was observed (abortion group: 33.3±4.0 years, ongoing group: 31.0±3.6 years, P=0.02). No difference in other parameters was found. Cytogenetic analysis of apoblemas was obtained for 12 cases, and 2 specimens were contaminated. Seven of ten patients had abnormal karyotypes. Conclusion The underlying cause of spontaneous abortion after transferring frozen thawing blastocysts appears to be abnormal karyotypes.Advancing maternal age seems to increase the risk of spontaneous abortion.

  20. Synthesis and Blastocyst Implantation Inhibition Potential of Lupeol Derivatives in Female Mice

    Directory of Open Access Journals (Sweden)

    Anita Mahapatra

    2015-06-01

    Full Text Available Blastocyst implantation which is analogous to pro-inflammatory response, mediated by different inflammatory mediators and ovarian hormones found to be an effective target for the development of emergency contraceptives. In the present study, a series of derivatives an anti-inflammatory natural scaffold, lupeol were synthesized under mild reaction conditions and good yield. All the compounds were evaluated for acute anti-inflammation. The three active compounds with 62-92% edema protection were screened for chronic anti-inflammation. The analogue 3-(p-chlorocinnamoyl lupeol ( 2 with potent anti-inflammatory activity (85% protection was evaluated for t he anti-implantation activity by studying changes in superoxide dismutase (SOD and lipid peroxidation (LPO levels, visualization of implantation site and anti-estrogenic activity. As expected, a sharp decrease in superoxide anion radical and increase in SOD activity was seen in the endometrium of treated animals. Also no implantation sites were observed in the uterus of treated animals. The active compound also exhibited anti-estrogenic activity.

  1. Stimulation of mitochondria during maturation influences development and gene expression in bovine embryos derived from oocytes with differenet meiotic competence.

    Czech Academy of Sciences Publication Activity Database

    Hulínská, P.; Hanzalová, K.; Knitlová, D.; Němcová, Lucie; Kaňka, Jiří; Jeseta, M.; Machatková, M.

    2016-01-01

    Roč. 49, č. 4 (2016), s. 174-174 ISSN 1335-3683. [International Scientific Conference "Animal Biotechnology " /4./. 08.12.2016-08.12.2016, Nitra] R&D Projects: GA MZe(CZ) QJ1510138 Institutional support: RVO:67985904 Keywords : bovine embryos Subject RIV: EB - Genetics ; Molecular Biology

  2. Preservation of bovine hide using less salt with low concentration of antiseptic, part I: effectiveness of developed formulations

    Science.gov (United States)

    Bovine raw hides are commercially cured either with high salt concentration of about half the weight of actual hide or 95% saturated brine solution. This conventional technique is very popular due to the availability of common salt (sodium chloride) and its cost effective procedure but it generates ...

  3. Development and validation of an ultra high performance liquid chromatography tandem mass spectrometry method for simultaneous determination of sulfonamides, quinolones and benzimidazoles in bovine milk.

    Science.gov (United States)

    Hou, Xiao-Lin; Chen, Guo; Zhu, Li; Yang, Ting; Zhao, Jian; Wang, Lei; Wu, Yin-Liang

    2014-07-01

    A simple, sensitive and reliable analytical method was developed for the simultaneous determination of 38 veterinary drugs (18 sulfonamides, 11 quinolones and 9 benzimidazoles) and 8 metabolites of benzimidazoles in bovine milk by ultra high performance liquid chromatography-positive electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS). Samples were extracted with acidified acetonitrile, cleaned up with Oasis(®) MCX cartridges, and analyzed by LC-MS/MS on an Acquity UPLC(®) BEH C18 column with gradient elution. The method allows such multi-analyte measurements within a 13min runtime while the specificity is ensured through the MRM acquisition mode. The method was validated according to the European Commission Decision 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), recovery, precision, linearity and stability. For compounds which have MRLs in bovine milk, the CCα values fall into a range from 11 to 115μg/kg, and the CCβ values fall within a range of 12-125μg/kg. For compounds which have not MRLs in bovine milk, the CCα values fall into a range from 0.01 to 0.08μg/kg, and the CCβ values fall within a range of 0.02-0.11μg/kg. The mean recoveries of the 46 analytes were between 87 and 119%. The calculated RSD values of repeatability and within-laboratory reproducibility experiments were below 11% and 15% for the 46 compounds, respectively. The method was demonstrated to be suitable for the simultaneous determination of sulfonamides, quinolones and benzimidazoles in bovine milk. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. The p66(Shc adaptor protein controls oxidative stress response in early bovine embryos.

    Directory of Open Access Journals (Sweden)

    Dean H Betts

    Full Text Available The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2-4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2-4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos.

  5. The antiprogesterone Org 31710 inhibits human blastocyst-endometrial interacttions in vitro

    DEFF Research Database (Denmark)

    Petersen, A; tin-Ley, U; Ravn, V

    2005-01-01

    OBJECTIVE: To investigate the effect of the anti-P Org 31710 on human blastocyst attachment to cultured endometrial epithelial cells. DESIGN: Experimental in vitro study. SETTING: University hospital. PATIENT(S): Eleven fertile endometrial donors. INTERVENTION(S): Timed endometrial biopsy for cell...... statistical significance. The presence of swollen microvilli, precursors of endometrial pinopodes, was significantly reduced on cultures with Org 31710 (P=.03). CONCLUSION(S): The study presents a model for human blastocyst-endometrial interactions responding to an anti-P drug. The exact mechanism...

  6. The antiprogesterone Org 31710 inhibits human blastocyst-endometrial interactions in vitro

    DEFF Research Database (Denmark)

    Petersen, Astrid; Bentin-Ley, Ursula; Ravn, Vibeke

    2005-01-01

    OBJECTIVE: To investigate the effect of the anti-P Org 31710 on human blastocyst attachment to cultured endometrial epithelial cells. DESIGN: Experimental in vitro study. SETTING: University hospital. PATIENT(S): Eleven fertile endometrial donors. INTERVENTION(S): Timed endometrial biopsy for cell...... statistical significance. The presence of swollen microvilli, precursors of endometrial pinopodes, was significantly reduced on cultures with Org 31710 (P=.03). CONCLUSION(S): The study presents a model for human blastocyst-endometrial interactions responding to an anti-P drug. The exact mechanism...

  7. Clinical outcomes after IVF or ICSI using human blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum.

    Science.gov (United States)

    Itoi, Fumiaki; Asano, Yukiko; Shimizu, Masashi; Nagai, Rika; Saitou, Kanako; Honnma, Hiroyuki; Murata, Yasutaka

    2017-04-01

    In this study the clinical and neo-natal outcomes after transfer of blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum (SER) were compared between IVF and intracytoplasmic sperm injection (ICSI) cycles. Clinical and neo-natal outcomes of blastocysts in cycles with at least one SER metaphase II oocyte (SER + MII; SER + cycles) did not significantly differ between the two insemination methods. When SER + MII were cultured to day 5/6, fertilization, embryo cleavage and blastocyst rates were not significantly different between IVF and ICSI cycles. In vitrified-warmed blastocyst transfer cycles, the clinical pregnancy rates from SER + MII in IVF and ICSI did not significantly differ. In this study, 52 blastocysts (27 IVF and 25 ICSI) derived from SER + MII were transferred, yielding 15 newborns (5 IVF and 10 ICSI) and no malformations. Moreover, 300 blastocysts (175 IVF and 125 ICSI) derived from SER-MII were transferred, yielding 55 newborns (24 IVF and 31 ICSI cycles). Thus, blastocysts derived from SER + cycles exhibited an acceptable ongoing pregnancy rate after IVF (n = 125) or ICSI (n = 117) cycles. In conclusion, blastocysts from SER + MII in both IVF and ICSI cycles yield adequate ongoing pregnancy rates with neo-natal outcomes that do not differ from SER-MII. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  8. Development of an ELISA for evaluation of swab recovery efficiencies of bovine serum albumin.

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    Nadja Sparding

    Full Text Available After a potential biological incident the sampling strategy and sample analysis are crucial for the outcome of the investigation and identification. In this study, we have developed a simple sandwich ELISA based on commercial components to quantify BSA (used as a surrogate for ricin with a detection range of 1.32-80 ng/mL. We used the ELISA to evaluate different protein swabbing procedures (swabbing techniques and after-swabbing treatments for two swab types: a cotton gauze swab and a flocked nylon swab. The optimal swabbing procedure for each swab type was used to obtain recovery efficiencies from different surface materials. The surface recoveries using the optimal swabbing procedure ranged from 0-60% and were significantly higher from nonporous surfaces compared to porous surfaces. In conclusion, this study presents a swabbing procedure evaluation and a simple BSA ELISA based on commercial components, which are easy to perform in a laboratory with basic facilities. The data indicate that different swabbing procedures were optimal for each of the tested swab types, and the particular swab preference depends on the surface material to be swabbed.

  9. Successful application of the strategy of blastocyst biopsy, vitrification, whole genome amplification, and thawed embryo transfer for preimplantation genetic diagnosis of neurofibromatosis type 1

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    Yi-Lin Chen

    2011-03-01

    Conclusion: We first demonstrate successful application of blastocyst biopsy, vitrification, WGA, and thawed embryo transfer for PGD of a monogenic disease. Vitrification of blastocysts after biopsy permits sufficient time for shipment of samples and operation of molecular diagnosis.

  10. Extended Culture of Encapsulated Human Blastocysts in Alginate Hydrogel Containing Decidualized Endometrial Stromal Cells in the Presence of Melatonin.

    Science.gov (United States)

    Arjmand, Fatemeh; Khanmohammadi, Manijeh; Arasteh, Shaghayegh; Mohammadzadeh, Afsaneh; Kazemnejad, Somaieh; Akhondi, Mohammad-Mehdi

    2016-10-01

    Extended in vitro culture of human embryos beyond blastocyst stage could serve as a tool to explore the molecular and physiological mechanisms underlying embryo development and to identify factors regulating pregnancy outcomes. This study presents the first report on the maintenance of human embryo in vitro by alginate co-encapsulation of human blastocyst and decidualized endometrial stromal cells (EnSCs) under melatonin-fortified culture conditions. The effectiveness of the 3D culture system was studied through monitoring of embryo development in terms of survival time, viability, morphological changes, and production of the two hormones of 17b-oestradiol and human chorionic gonadotropin. The embryo structural integrity was preserved during alginate encapsulation; however, only 23 % of the encapsulated embryos could retain in the hydrogels over time and survived until day 4 post-encapsulation. The culture medium fortification with melatonin significantly elevated the maintenance rate of expanded embryos in alginate beads by 65 % and prolonged survival time of human embryos to day 5. Furthermore, embryo co-culture with EnSCs using melatonin-fortified medium increased the survival time of encapsulated embryos to 44 %. The levels of two measured hormones significantly rose at day 4 in comparison with day 2 post-encapsulation especially in the group co-encapsulated with EnSCs and cultivated in melatonin-fortified culture medium. These data are the first evidence representing in vitro development of human embryos until day 10 post-fertilization. This achievement can facilitate the investigation of the mechanisms regulating human embryo development.

  11. Long-term culture of undifferentiated spermatogonia isolated from immature and adult bovine testes.

    Science.gov (United States)

    Suyatno; Kitamura, Yuka; Ikeda, Shuntaro; Minami, Naojiro; Yamada, Masayasu; Imai, Hiroshi

    2018-03-01

    Undifferentiated spermatogonia eventually differentiate in the testis to produce haploid sperm. Within this cell population, there is a small number of spermatogonial stem cells (SSCs). SSCs are rare cells in the testis, and their cellular characteristics are poorly understood. Establishment of undifferentiated cell line would provide an indispensable tool for studying their biological nature and spermiogenesis/spermatogenesis in vitro. However, there have been few reports on the long-term culture of undifferentiated spermatogonia in species other than rodents. Here, we report the derivation and long-term in vitro culture of undifferentiated spermatogonia cell lines from immature and adult bovine testes. Cell lines from immature testes were maintained in serum-free culture conditions in the presence of glial-cell-line-derived neurotropic factor (GDNF) and bovine leukemia inhibitory factor (bLIF). These cell lines have embryonic stem (ES)-like cell morphology, express pluripotent-stem-cell-specific and germ-cell-specific markers at the protein and mRNA levels, and contributed to the inner cell mass (ICM) of embryos in the blastocyst stage. Meanwhile, cell lines established from adult testes were maintained in low-serum media in the presence of 6-bromoindirubin-3'-oxime (BIO). These cell lines have characteristics resembling those of previously reported male mouse germ cell lines as confirmed by their botryoidally aggregated morphology, as well as the expression of germ-cell-specific markers and pluripotent stem cell markers. These findings could be useful for the development of long-term culture of undifferentiated spermatogonia, which could aid in conservation of species and improvement of livestock production through genome editing technology. © 2018 Wiley Periodicals, Inc.

  12. Chromosomal analysis of blastocyst derived from monopronucleated ICSI zygotes: approach by double trophectoderm biopsy.

    Science.gov (United States)

    Mateo, Silvia; Vidal, Francesca; Coll, Lluc; Veiga, Anna; Boada, Montserrat

    2017-09-01

    This study aims to increase the knowledge about monopronucleated ICSI-derived blastocysts, analyzing trophectoderm biopsies by aCGH and FISH to evaluate their chromosome constitution. Fifteen monopronucleated ICSI-derived blastocysts were studied. Double trophectoderm biopsy was performed and analyzed by FISH and aCGH. The blastocysts were classified according to chromosome constitution. Disagreements between the two techniques were assessed. Results obtained after FISH and aCGH analyses showed the following: 20% (3/15) and 60% (9/15) diploid females, respectively; 26.7% (4/15) and 26.7% (4/15) diploid males, respectively; and 53.3% (8/15) and 13.3% (2/15) mosaics, respectively. No mosaic male embryos were found using FISH or aCGH. There were disagreements in 40% (6/15) of the cases due to the higher detection of mosaicism by FISH compared to aCGH. The combination of FISH and aCGH has been shown to be a suitable approach to increase the knowledge about monopronucleated ICSI-derived embryos. FISH analysis of blastocysts derived from monopronucleated ICSI zygotes enabled us to conclude that aCGH underestimates haploidy. Some diploid embryos diagnosed by aCGH are in fact mosaic. In cases where these embryos would be used for reproductive purposes, extra analysis of parental genome origin is recommended.

  13. Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes.

    Science.gov (United States)

    Cremades, N; Sousa, M; Silva, J; Viana, P; Sousa, S; Oliveira, C; Teixeira da Silva, J; Barros, A

    2004-02-01

    Vitrification of human blastocysts has been successfully applied using grids, straws and cryoloops. We assessed the survival rate of human compacted morulae and early blastocysts vitrified in pipette tips with a smaller inner diameter and solution volume than the previously described open pulled straw (OPS) method. Excess day 5 human embryos (n = 63) were experimentally vitrified in vessels. Embryos were incubated at 37 degrees C with sperm preparation medium (SPM) for 1 min, SPM + 7.5% ethylene glycol (EG)/dimethylsulphoxide (DMSO) for 3 min, and SPM + 16.5% EG + 16.5% DMSO + 0.67 mol/l sucrose for 25 s. They were then aspirated (0.5 microl) into a plastic micropipette tip (0.36 mm inner diameter), exposed to liquid nitrogen (LN(2)) vapour for 2 min before being placed into a pre-cooled cryotube, which was then closed and plunged into LN(2). Embryos were warmed and diluted using 0.33 mol/l and 0.2 mol/l sucrose. The survival rate for compacted morulae was 73% (22/30) and 82% (27/33) for early blastocysts. The survival rates of human compacted morulae and early blastocysts after vitrification with this simple technique are similar to those reported in the literature achieved by slow cooling and other vitrification protocols.

  14. Monoclonal antibodies against trophectoderm-specific markers during mouse blastocyst formation.

    Science.gov (United States)

    Brûlet, P; Babinet, C; Kemler, R; Jacob, F

    1980-01-01

    Two-dimensional gel electrophoresis has allowed the detection of proteins characteristic of inner cell mass and trophectoderm in mouse blastocyst. Certain of the proteins characterizing trophectoderm copurify with intermediate filaments from trophectoderm and a trophoblastoma cell line. A monoclonal antibody prepared against proteins of these intermediate filaments labels a filament network in trophectoderm but not in inner cell mass cells. Images PMID:6933460

  15. The effect of anandamide on uterine nitric oxide synthase activity depends on the presence of the blastocyst.

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    Micaela S Sordelli

    2011-04-01

    Full Text Available Nitric oxide production, catalyzed by nitric oxide synthase (NOS, should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot(-1 h(-1 compared to days 4 (0.34±0.03 and 5 (0.35±0.02 of pregnancy and to day 6 implantation sites (0.33±0.01. This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA, an endocannabinoid, binds to cannabinoid receptors type 1 (CB1 and type 2 (CB2, and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04 and URB-597 (1.08±0.09 vs 0.83±0.06 inhibited NOS activity in the absence of a blastocyst (pseudopregnancy through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05. While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02, a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01. Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These

  16. Inner cell mass incarceration in 8-shaped blastocysts does not increase monozygotic twinning in preimplantation genetic diagnosis and screening patients

    Science.gov (United States)

    Zhou, Qin-Wei; Zhang, Shuo-Ping; Lu, Chang-Fu; Gong, Fei; Tan, Yue-Qiu; Lu, Guang-Xiu; Lin, Ge

    2018-01-01

    Background The use of assisted reproductive technology (ART) has been reported to increase the incidence of monozygotic twinning (MZT) compared with the incidence following natural conception. It has been hypothesized that splitting of the inner cell mass (ICM) through a small zona hole may result in MZT. In this study, using a cohort of patients undergoing preimplantation genetic diagnosis/screening (PGD/PGS), we compared the clinical and neonatal outcomes of human 8-shaped blastocysts hatching with ICM incarceration with partially or fully hatched blastocysts, and attempted to verify whether this phenomenon increases the incidence of MZT pregnancy or negatively impact newborns. Methods This retrospective study included 2059 patients undergoing PGD/PGS between March 1, 2013, and December 31, 2015. Clinical and neonatal outcomes were only collected from patients who received a single blastocyst transfer after PGD/PGS (n = 992). A 25- to 30-μm hole was made in the zona of day 3 embryos by laser. The blastocysts were biopsied and vitrified on day 6. The biopsied trophectoderm (TE) cells were analyzed using different genetic methods. One tested blastocyst was thawed and transferred to each patient in the subsequent frozen embryo transfer cycle. All the biopsied blastocysts were divided into three types: 8-shaped with ICM incarceration (type I), partially hatched without ICM incarceration (type II), and fully hatched (type III). ICM/TE grading, clinical and neonatal outcomes were compared between the groups. Results The percentage of grade A ICMs in type I blastocysts (22.2%) was comparable to that in type III blastocysts (20.1%) but higher than that in type II blastocysts (4.5%). The percentage of grade A TEs in type I blastocysts (4.2%) was comparable to that in type II (3.6%) but lower than that in type III (13.5%). There were no significant differences in clinical pregnancy, MZT pregnancy, miscarriage, live birth, MZT births, and neonatal outcomes between the

  17. Are there ethnic differences in pregnancy rates in African-American versus white women undergoing frozen blastocyst transfers?

    Science.gov (United States)

    Csokmay, John M; Hill, Micah J; Maguire, Marcy; Payson, Mark D; Fujimoto, Victor Y; Armstrong, Alicia Y

    2011-01-01

    To determine whether frozen-thawed blastocyst transfer pregnancy rates (PR) are lower in African-American compared with white women. Retrospective review of frozen blastocyst cycles. University-based assisted reproductive technology (ART) program. All patients who underwent a frozen blastocyst transfer between 2003 and 2008. None. Live birth rate. One hundred sixty-nine patients underwent transfer of a frozen-thawed blastocyst. African-American women had a higher incidence of leiomyoma (40% vs. 10%) and tubal and uterine factor infertility. There was no difference in the live birth rate for African-American patients (28.0%) compared with white patients (30.2%). Of the patients who underwent a frozen-thawed blastocyst transfer, 58% (n=98) had their fresh, autologous IVF cycle, which produced the cryopreserved blastocyst, at Walter Reed Medical Center. A higher peak serum E2 level was noted in African-American patients (5,355 pg/mL) compared with white patients (4,541 pg/mL). During the fresh cycle, the live birth rates between African-American and white patients were significantly different at 16.7% versus 39.7%, respectively. Live birth rates after frozen blastocyst transfer are not different between African-American and white women despite a fourfold higher incidence of leiomyomas in African-American women. Copyright © 2011. Published by Elsevier Inc.

  18. Avances en el desarrollo de vacunas contra la neosporosis bovina Advances in the development of vaccines for bovine neosporosis

    Directory of Open Access Journals (Sweden)

    Yanina P. Hecker

    2012-09-01

    Full Text Available La neosporosis es una enfermedad que ocasiona abortos en bovinos y está causada por un protozoo intracelular obligado denominado Neospora caninum. Las graves pérdidas económicas que provoca en los sistemas de producción de bovinos justifica la necesidad de avanzar en el desarrollo de vacunas. La resistencia a parásitos Apicomplexa está asociada a una respuesta inmune T helper 1 mediada por linfocitos T CD4 citotóxicos y a la producción de interferón-gamma, interleuquina-12, factor de necrosis tumoral e inmunoglobulina G2. La disminución de la transmisión vertical en las sucesivas preñeces y el bajo nivel de repetición de abortos en animales infectados sugieren la existencia de mecanismos inmunitarios de protección. Hasta el momento se conoce que la inoculación pre-servicio con taquizoítos vivos protege contra la infección y el aborto. Los antecedentes de desarrollo de vacunas vivas contra otros protozoos estimulan a los investigadores a continuar en la búsqueda de una vacuna de este tipo contra N. caninum de buena eficacia. Por otra parte, una vacuna inactivada, aun con una baja eficacia, es útil en la prevención del aborto en aquellos establecimientos donde la enfermedad es epizoótica. Una vacuna contra la neosporosis debería evitar el aborto, la transmisión transplacental y la persistencia de la infección. Este trabajo menciona los diversos tipos de vacunas que han sido evaluados hasta el momento, incluyendo inmunógenos inactivos, taquizoítos vivos, antígenos recombinantes y vacunas en vectores.Neosporosis, a disease caused by the obligate intracellular protozoan Neospora caninum, produces abortions in cattle. The severe economic losses in cattle industry justify the need to develop control measures for preventing bovine abortion. Apicomplexan parasitic resistance is associated with T helper 1 immune response mediated by CD4 cytotoxic T lymphocytes, the production of interferon-gamma, interleukin-12, tumor necrosis

  19. X-linked gene transcription patterns in female and male in vivo, in vitro and cloned porcine individual blastocysts.

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    Chi-Hun Park

    Full Text Available To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF, and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process

  20. Completely ES cell-derived mice produced by tetraploid complementation using inner cell mass (ICM deficient blastocysts.

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    Duancheng Wen

    Full Text Available Tetraploid complementation is often used to produce mice from embryonic stem cells (ESCs by injection of diploid (2n ESCs into tetraploid (4n blastocysts (ESC-derived mice. This method has also been adapted to mouse cloning and the derivation of mice from induced pluripotent stem (iPS cells. However, the underlying mechanism(s of the tetraploid complementation remains largely unclear. Whether this approach can give rise to completely ES cell-derived mice is an open question, and has not yet been unambiguously proven. Here, we show that mouse tetraploid blastocysts can be classified into two groups, according to the presence or absence of an inner cell mass (ICM. We designate these as type a (presence of ICM at blastocyst stage or type b (absence of ICM. ESC lines were readily derived from type a blastocysts, suggesting that these embryos retain a pluripotent epiblast compartment; whereas the type b blastocysts possessed very low potential to give rise to ESC lines, suggesting that they had lost the pluripotent epiblast. When the type a blastocysts were used for tetraploid complementation, some of the resulting mice were found to be 2n/4n chimeric; whereas when type b blastocysts were used as hosts, the resulting mice are all completely ES cell-derived, with the newborn pups displaying a high frequency of abdominal hernias. Our results demonstrate that completely ES cell-derived mice can be produced using ICM-deficient 4n blastocysts, and provide evidence that the exclusion of tetraploid cells from the fetus in 2n/4n chimeras can largely be attributed to the formation of ICM-deficient blastocysts.

  1. Preimplantation genetic diagnosis for chromosomal rearrangements with the use of array comparative genomic hybridization at the blastocyst stage.

    Science.gov (United States)

    Christodoulou, Christodoulos; Dheedene, Annelies; Heindryckx, Björn; van Nieuwerburgh, Filip; Deforce, Dieter; De Sutter, Petra; Menten, Björn; Van den Abbeel, Etienne

    2017-01-01

    To establish the value of array comparative genomic hybridization (CGH) for preimplantation genetic diagnosis (PGD) in embryos of translocation carriers in combination with vitrification and frozen embryo transfer in nonstimulated cycles. Retrospective data analysis study. Academic centers for reproductive medicine and genetics. Thirty-four couples undergoing PGD for chromosomal rearrangements from October 2013 to December 2015. Trophectoderm biopsy at day 5 or day 6 of embryo development and subsequently whole genome amplification and array CGH were performed. This approach revealed a high occurrence of aneuploidies and structural rearrangements unrelated to the parental rearrangement. Nevertheless, we observed a benefit in pregnancy rates of these couples. We detected chromosomal abnormalities in 133/207 embryos (64.2% of successfully amplified), and 74 showed a normal microarray profile (35.7%). In 48 of the 133 abnormal embryos (36.1%), an unbalanced rearrangement originating from the parental translocation was identified. Interestingly, 34.6% of the abnormal embryos (46/133) harbored chromosome rearrangements that were not directly linked to the parental translocation in question. We also detected a combination of unbalanced parental-derived rearrangements and aneuploidies in 27 of the 133 abnormal embryos (20.3%). The use of trophectoderm biopsy at the blastocyst stage is less detrimental to the survival of the embryo and leads to a more reliable estimate of the genomic content of the embryo than cleavage-stage biopsy. In this small cohort PGD study, we describe the successful implementation of array CGH analysis of blastocysts in patients with a chromosomal rearrangement to identify euploid embryos for transfer. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  2. Development of intramammary delivery systems containing lasalocid for the treatment of bovine mastitis: impact of solubility improvement on safety, efficacy, and milk distribution in dairy cattle

    Directory of Open Access Journals (Sweden)

    Wang W

    2015-01-01

    Full Text Available Wen Wang,1 Yunmei Song,1 Kiro Petrovski,2 Patricia Eats,2 Darren J Trott,2 Hui San Wong,2 Stephen W Page,3 Jeanette Perry,2 Sanjay Garg11School of Pharmacy and Medical Science, University of South Australia, Adelaide, SA, Australia; 2School of Animal and Veterinary Sciences, University of Adelaide, Adelaide, SA, Australia; 3Luoda Pharma Pty Ltd, Caringbah, NSW, AustraliaBackground: Mastitis is a major disease of dairy cattle. Given the recent emergence of methicillin-resistant Staphylococcus aureus as a cause of bovine mastitis, new intramammary (IMA treatments are urgently required. Lasalocid, a member of the polyether ionophore class of antimicrobial agents, has not been previously administered to cows by the IMA route and has favorable characteristics for development as a mastitis treatment. This study aimed to develop an IMA drug delivery system (IMDS of lasalocid for the treatment of bovine mastitis.Methods: Minimum inhibitory concentrations (MICs were determined applying the procedures recommended by the Clinical and Laboratory Standards Institute. Solid dispersions (SDs of lasalocid were prepared and characterized using differential scanning calorimetry and Fourier transform infrared spectroscopy. IMDSs containing lasalocid of micronized, nano-sized, or as SD form were tested for their IMA safety in cows. Therapeutic efficacy of lasalocid IMDSs was tested in a bovine model involving experimental IMA challenge with the mastitis pathogen Streptococcus uberis.Results: Lasalocid demonstrated antimicrobial activity against the major Gram-positive mastitis pathogens including S. aureus (MIC range 0.5–8 µg/mL. The solubility test confirmed limited, ion-strength-dependent water solubility of lasalocid. A kinetic solubility study showed that SDs effectively enhanced water solubility of lasalocid (21–35-fold. Polyvinylpyrrolidone (PVP-lasalocid SD caused minimum mammary irritation in treated cows and exhibited faster distribution in milk than

  3. Development and Characterization of a Multiplexed RT-PCR Species Specific Assay for Bovine and one for Porcine Foot-and-Mouth Disease Virus Rule-Out Supplemental Materials

    Energy Technology Data Exchange (ETDEWEB)

    Smith, S; Danganan, L; Tammero, L; Lenhoff, R; Naraghi-arani, P; Hindson, B

    2007-08-06

    Lawrence Livermore National Laboratory (LLNL), in collaboration with the Department of Homeland Security (DHS) and the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Services (APHIS) has developed advanced rapid diagnostics that may be used within the National Animal Health Laboratory Network (NAHLN), the National Veterinary Services Laboratory (Ames, Iowa) and the Plum Island Animal Disease Center (PIADC). This effort has the potential to improve our nation's ability to discriminate between foreign animal diseases and those that are endemic using a single assay, thereby increasing our ability to protect animal populations of high economic importance in the United States. Under 2005 DHS funding we have developed multiplexed (MUX) nucleic-acid-based PCR assays that combine foot-and-mouth disease virus (FMDV) detection with rule-out tests for two other foreign animal diseases Vesicular Exanthema of Swine (VESV) and Swine Vesicular Disease (SVD) and four other domestic viral diseases Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus 1 (BHV-1 or Infectious Bovine Rhinotracheitus IBR), Bluetongue virus (BTV) and Parapox virus complex (which includes Bovine Papular Stomatitis Virus BPSV, Orf of sheep, and Pseudocowpox). Under 2006 funding we have developed a Multiplexed PCR [MUX] porcine assay for detection of FMDV with rule out tests for VESV and SVD foreign animal diseases in addition to one other domestic vesicular animal disease vesicular stomatitis virus (VSV) and one domestic animal disease of swine porcine reproductive and respiratory syndrome (PRRS). We have also developed a MUX bovine assay for detection of FMDV with rule out tests for the two bovine foreign animal diseases malignant catarrhal fever (MCF), rinderpest virus (RPV) and the domestic diseases vesicular stomatitis virus (VSV), bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitus virus (BHV-1), bluetongue virus (BTV), and the Parapox

  4. Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells

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    Andrea Pecora

    2015-03-01

    Full Text Available Bovine viral diarrhea virus (BVDV is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2 was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 µg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle.

  5. Development of anti-bovine IgA single chain variable fragment and its application in diagnosis of foot-and-mouth disease

    Science.gov (United States)

    Sridevi, N. V.; Shukra, A. M.; Neelakantam, B.; Anilkumar, J.; Madhanmohan, M.; Rajan, S.; Dev Chandran

    2014-01-01

    Recombinant antibody fragments like single chain variable fragments (scFvs) represent an attractive yet powerful alternative to immunoglobulins and hold great potential in the development of clinical diagnostic/therapeutic reagents. Structurally, scFvs are the smallest antibody fragments capable of retaining the antigen-binding capacity of whole antibodies and are composed of an immunoglobulin (Ig) variable light (VL) and variable heavy (VH) chain joined by a flexible polypeptide linker. In the present study, we constructed a scFv against bovine IgA from a hybridoma cell line IL-A71 that secretes a monoclonal antibody against bovine IgA using recombinant DNA technology. The scFv was expressed in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The binding activity and specificity of the scFv was established by its non-reactivity toward other classes of immunoglobulins as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Kinetic measurement of the scFv indicated that the recombinant antibody fragment had an affinity in picomolar range toward purified IgA. Furthermore, the scFv was used to develop a sensitive ELISA for the detection of foot and mouth disease virus (FMDV) carrier animals. PMID:24678404

  6. Development of an in vitro test battery for assessing chemical effects on bovine germ cells under the ReProTect umbrella

    International Nuclear Information System (INIS)

    Lazzari, Giovanna; Tessaro, Irene; Crotti, Gabriella; Galli, Cesare; Hoffmann, Sebastian; Bremer, Susanne; Pellizzer, Cristian

    2008-01-01

    Current European legislation for the registration and authorisation of chemicals (REACH) will require a dramatic increase in the use of animals for reproductive toxicity testing. Since one objective of REACH is to use vertebrates only as last resort, the development and validation of alternative methods is urgently needed. For this purpose ReProTect, an integrated research project funded by the European Union, joining together 33 partners with complementary expertise in reproductive toxicology, was designed. The study presented here describes a battery of two tests developed within ReProTect. The objective of these tests is the detection of chemical effects during the processes of oocyte maturation and fertilisation in a bovine model. The corresponding toxicological endpoints are the reaching of metaphase II and the formation of the pronuclei respectively. Fifteen chemicals have been tested (Benzo[a]pyrene, Busulfan, Butylparaben, Cadmium Chloride, Carbendazim, Cycloheximide, Diethylstilbestrol, Genistein, Ionomycin, Ketoconazole, Lindane, Methylacetoacetate, Mifepristone, Nocodazole and DMSO as solvent) demonstrating high intra-laboratory reproducibility of the tests. Furthermore, the responses obtained in both tests, for several substances, had a good correlation with the available in vivo and in vitro data. These tests therefore, could predictably become part of an integrated testing strategy that combines the bovine models with additional in vitro tests, in order to predict chemical hazards on mammalian fertility

  7. Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells.

    Science.gov (United States)

    Pecora, Andrea; Malacari, Darío A; Pérez Aguirreburualde, María S; Bellido, Demian; Escribano, José M; Dus Santos, María J; Wigdorovitz, Andrés

    2015-01-01

    Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 μg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  8. Male gender explains increased birthweight in children born after transfer of blastocysts.

    Science.gov (United States)

    Kaartinen, N M; Kananen, K M; Rodriguez-Wallberg, K A; Tomás, C M; Huhtala, H Sa; Tinkanen, H I

    2015-10-01

    Does extended embryo culture have a different effect on the birthweight of girls and boys? The mean birthweight of boys born after fresh and frozen-thawed blastocyst transfer was increased compared with those born after cleavage stage embryo transfer. This effect was not detected among girls. Previous studies indicate that newborns from frozen-thawed cleavage stage embryos may present with a higher weight than newborns from fresh embryo transfers. With regard to fresh embryos, newborns after a blastocyst transfer have been reported as having higher birthweights than newborns from cleavage stage embryos. Retrospective multicentre case-control cohort study. All IVF/ICSI treatments were performed in the time-period from January 2008 to March 2014. Birthweight of singletons born at full-term (≥37 weeks), after fresh or frozen blastocyst embryo transfers (n = 277), were compared with weights of children born after fresh or frozen cleavage stage embryo transfers (Day 2-3) (n = 277). The cases and controls were matched by delivery week, and by gender. Data of IVF/ICSI treatments, and the treatments' outcomes were collected and analysed. The birthweight after a fresh blastocyst transfer was significantly higher (mean 3530.6 g) than that after a transfer of cleavage stage embryos (mean 3418.8 g; weight difference 111.8 g, P = 0.047). The weights of newborns after frozen-thawed blastocyst transfers (mean 3647.5 g) and the frozen-thawed cleavage stage embryo transfers (mean 3650.9 g), were similar (weight difference 3.4 g, P = 0.95). The boys born after transfer of frozen-thawed blastocysts had a significantly higher birthweight (mean 3767.9 g) than girls (3525.2 g; weight difference 242.7 g, P = 0.002), whereas the difference of birthweights between genders was only 13.5 g in cleavage stage (P = 0.863). The same effect was seen after fresh blastocyst transfers (weight difference 211.5 g, P = 0.011), but not after fresh Day 2-3 embryo transfers (weight difference 53.6 g, P

  9. Duration of blastulation may be associated with ongoing pregnancy rate in single euploid blastocyst transfer cycles.

    Science.gov (United States)

    Mumusoglu, Sezcan; Ozbek, Irem Y; Sokmensuer, Lale K; Polat, Mehtap; Bozdag, Gurkan; Papanikolaou, Evangelos; Yarali, Hakan

    2017-12-01

    Not all euploid embryos implant, necessitating additional tools to select viable blastocysts in preimplantation genetic screening cycles. In this retrospective cohort study, 129 consecutive patients who underwent 129 single euploid blastocyst transfers in cryopreserved embryo transfer cycles were included. All embryos were individually cultured in a time-lapse incubator from intracytoplasmic sperm injection up to trophoectoderm biopsy. Twenty-three time-lapse morphokinetic variables were tested among patients with (n = 68) or without (n = 61) ongoing pregnancy. All 23 time-lapse morphokinetic variables, apart from duration of blastulation (tB-tSB), were comparable between patients with or without ongoing pregnancy. Duration of blastulation was significantly shorter in patients with ongoing pregnancy (8.1 ± 3.2 versus 9.5 ± 3.4 h; P = 0.014); shorter duration of blastulation remained an independent predictor for ongoing pregnancy, when tested by logistic regression analysis (OR 0.81; 95% CI 0.70 to 0.93). One important limitation of this study, and a reason for caution, is the use of multiple comparisons, which can lead to differences at the 0.05 level simply by chance or random variation. Nonetheless, the study suggests that when more than one euploid blastocyst is available, priority might be given to those with a shorter duration of blastulation. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  10. Heat stress effects on the cumulus cells surrounding the bovine oocyte during maturation: altered matrix metallopeptidase 9 and progesterone production.

    Science.gov (United States)

    Rispoli, L A; Payton, R R; Gondro, C; Saxton, A M; Nagle, K A; Jenkins, B W; Schrick, F N; Edwards, J L

    2013-08-01

    When the effects of heat stress are detrimental during maturation, cumulus cells are intimately associated with the oocyte. To determine the extent to which heat stress affects these cells, in this study, transcriptome profiles of the cumulus that surrounded control and heat-stressed oocytes (41 °C during the first 12 h only and then shifted back to 38.5 °C) during in vitro maturation (IVM) were compared using Affymetrix bovine microarrays. The comparison of cumulus-derived profiles revealed a number of transcripts whose levels were increased (n=11) or decreased (n=13) ≥ twofold after heat stress exposure (P1.7-fold decrease in the protein levels of latent matrix metallopeptidase 9 (proMMP9). Heat-induced reductions in transcript levels were noted at 6 h IVM with reductions in proMMP9 protein levels at 18 h IVM (P=0.0002). Independent of temperature, proMMP9 levels at 24 h IVM were positively correlated with the development rate of blastocysts (R²=0.36; P=0.002). The production of progesterone increased during maturation; heat-induced increases were evident by 12 h IVM (P=0.002). Both MMP9 and progesterone are associated with the developmental competence of the oocyte; thus, it seems plausible for some of the negative consequences of heat stress on the cumulus-oocyte complex to be mediated through heat-induced perturbations occurring in the surrounding cumulus.

  11. Comparative efficacy of enrofloxacin to that of tulathromycin for the control of bovine respiratory disease and prevalence of antimicrobial resistance in Mannheimia haemolytica in calves at high risk of developing bovine respiratory disease.

    Science.gov (United States)

    Crosby, S; Credille, B; Giguère, S; Berghaus, R

    2018-04-14

    Bovine respiratory disease (BRD) is the most common cause of morbidity and mortality in North American beef cattle. Mannheimia haemolytica is the bacterial pathogen most often isolated from cattle with BRD, and the prevalence of antimicrobial resistance (AMR) in this organism has increased in recent years. Antimicrobials are commonly used to prevent BRD in cattle at high risk of developing BRD; however, recent work would suggest that this practice might be one factor contributing to the increased prevalence of AMR in M. haemolytica. We hypothesized that the administration of the short-acting fluoroquinolone, enrofloxacin, would be just as effective as the long-acting triamilide, tulathromycin, in preventing BRD but would be less likely to select for AMR M. haemolytica in stocker calves at high risk of developing BRD. Three hundred forty-one stocker calves were enrolled in the study with 172 calves in 4 pens being randomly assigned to treatment with enrofloxacin and 169 calves in 4 pens randomly assigned to treatment with tulathromycin. Calves within each treatment group were allocated to one of 4 replicate pens based on the week of enrollment. Of calves receiving enrofloxacin, 33.7% required treatment for BRD at least once within 45 d after arrival, compared with 18.3% of calves receiving tulathromycin (P = 0.040). The percentages of calves that required more than one treatment for BRD within 45 d after arrival did not differ statistically for those receiving enrofloxacin compared with those receiving tulathromycin (10.5% and 4.7%, respectively; P = 0.107) Likewise, the percentages of calves that died during the 45-d follow-up period did not differ for those receiving enrofloxacin compared with those receiving tulathromycin (12.2% and 10.1%, respectively; P = 0.592). Mannheimia haemolytica was cultured from 11% of calves sampled at arrival and from 50% of calves sampled at revaccination 14 to 17 d later. There was a significanst effect of sampling time on the

  12. Development, characterization and biocompatibility of chondroitin sulfate/poly(vinyl alcohol)/bovine bone powder porous biocomposite.

    Science.gov (United States)

    da Silva, Gabriela T; Voss, Guilherme T; Kaplum, Vanessa; Nakamura, Celso V; Wilhelm, Ethel A; Luchese, Cristiane; Fajardo, André R

    2017-03-01

    Chondroitin sulfate (ChS), a sulfated glycosaminoglycan, poly(vinyl alcohol) (PVA) and bovine bone powder (BBP) were blended to form a novel eco-friendly biocomposite through cyclic freeze-thawing under mild conditions. The systematic investigation reveals that the content of BBP has a remarkable effect on the pore size, porosity, mechanical and liquid uptake properties and biodegradability. At 10wt.% BBP the biocomposite exhibited enhanced mechanical properties and biodegradability rate as compared to the pristine sample. Further, different properties of the biocomposite can be tailored according to the content of BBP. In vitro assays showed that ChS/PVA-BBP does not exert cytotoxicity against healthy cells. In vivo and ex vivo experiments revealed that ChS/PVA-BBP biocomposites are biocompatibility materials without exert pro-inflammatory responses. The biocomposite was completely biodegraded and bioresorbed after 15days of treatment. Taken together, BBP is a low-cost source of hydroxyapatite and collagen, which are insurance. All these results suggest that the biocomposite designed in this study is a promising biomaterial for potential skin tissue engineering. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Bovine Herpesvirus 4 infections and bovine mastitis

    NARCIS (Netherlands)

    Wellenberg, Gerardus Johannus

    2002-01-01

    Mastitis is an often occurring disease in dairy cattle with an enormous economic impact for milk producers worldwide. Despite intensive research, which is historically based on the detection of bacterial udder pathogens, still around 20-35% of clinical cases of bovine mastitis have an unknown

  14. Successful vitrification of bovine immature oocyte using liquid helium instead of liquid nitrogen as cryogenic liquid.

    Science.gov (United States)

    Yu, Xue-Li; Xu, Ya-Kun; Wu, Hua; Guo, Xian-Fei; Li, Xiao-Xia; Han, Wen-Xia; Li, Ying-Hua

    2016-04-01

    The objectives of this study were to compare the effectiveness of liquid helium (LHe) and liquid nitrogen (LN2) as cryogenic liquid for vitrification of bovine immature oocytes with open-pulled straw (OPS) system and determine the optimal cryoprotectant concentration of LHe vitrification. Cumulus oocyte complexes were divided into three groups, namely, untreated group (control), LN2 vitrified with OPS group, and LHe vitrified with OPS group. Oocyte survival was assessed by morphology, nuclear maturation, and developmental capability. Results indicated that the rates of normal morphology, maturation, cleavage, and blastocyst (89.3%, 52.8%, 42.7%, and 10.1%, respectively) in the LHe-vitrified group were all higher than those (79.3%, 43.4%, 34.1%, and 4.7%) in the LN2-vitrified group (P 0.05). The maturation rate of the EDS35 group (65.0%) was higher than those of the EDS30 (51.3%), EDS40 (50.1%), EDS45 (52.1%), and EDS50 groups (36.9%; P liquid for vitrification of bovine immature oocytes, and it is more efficient than LN2-vitrified oocytes in terms of blastocyst production. EDS35 was the optimal cryoprotectant agent combination for LHe vitrification in this study. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Effect of Collection Technique on Yield of Bovine Oocytes and the Development Potential of Oocytes from Different Grades of Oocytes

    Directory of Open Access Journals (Sweden)

    R.G Sianturi

    2002-10-01

    Full Text Available Oocyte collection technique is important to obtain a maximum number of oocytes to be employed on in vitro production of embryos. In this study, immature bovine oocytes were collected from slaughterhouse ovaries by two techniques: aspiration of 2- to 6-mm follicles and slicing. Following collection, oocyte qualities were classified into four categories (A, B, C, and D on the basis of cumulus attachment. Oocytes of each category were matured in vitro in CO2 incubator for 22-24 hours and cumulus expansion and maturation rates were observed. The total number of oocytes (group A+B+C+D and yield of good quality oocytes (only group A and B recovered per ovary by aspiration were 12.02 and 8.21, and by slicing were 29.38 and 19.65 (P<0.01, respectively. The total cumulus cells expansion rates of A, B, C and D oocytes were 97.1%, 88.3%, 6.0% and 20.6% respectively. Maturation rates for A, B and C categories of oocytes were 91.4%, 82.3% and 35.0% respectively while no matured oocyte was observed for group D oocytes. Maturation rates were significantly different between group A and C and also between B and C but not between A and B (P<0.05. In conclusion, slicing technique recovered more oocytes per ovary (2.4 times than that of aspiration and the best maturation rate was observed from category A oocytes which surrounded by more than 3 layers of cumulus cells. However oocytes of category A and B can be considered as good quality oocytes.

  16. Frozen and fresh ovarian tissue require different culture media to promote in vitro development of bovine preantral follicles.

    Science.gov (United States)

    Castro, Simone Vieira; Carvalho, Adeline Andrade; Silva, Cleidson Manoel Gomes; Santos, Francielli Weber; Campello, Cláudio Cabral; de Figueiredo, José Ricardo; Rodrigues, Ana Paula Ribeiro

    2014-10-01

    The aim of this study was to evaluate the efficiency of different media in the in vitro culture of bovine preantral follicles that were used either fresh or following slow freezing treatment. Frozen and fresh noncultured or cultured ovarian fragments were processed for histological, viability, and cell proliferation analyses. For cryopreservation, a solution containing 1.5 M ethylene glycol was frozen in a programmable biological freezer. After thawing, a portion of the samples was destined for frozen controls. The remainder were cultured in vitro for 5 days in three media: α-MEM, McCoy, or M199. Samples from these culture media were collected on days 1 and 5 for quantification of reactive oxygen species (ROS) and for hormonal assays. In fresh-cultured tissues, the percentage of morphologically normal follicles was significantly higher when cultured in M199 compared to that in the other media. In frozen-cultured tissues, McCoy medium was significantly superior to the other media, and was the only treatment that helped in maintaining the viability similar to fresh and frozen controls. Upon quantification of the nucleolus organizer region, we observed greater proliferation of granulosa cells in the frozen-cultured tissues with McCoy medium, and lesser proliferation in fresh-cultured tissues only with α-MEM. In frozen-cultured tissues, ROS levels were highest at day 1 and progressively reduced during culture, independent of the media used. In conclusion, under the conditions used in this study, the M199 and McCoy media are recommended for the culture of follicles derived from fresh and frozen ovarian tissues, respectively.

  17. Successful pregnancy following single blastocyst transfer in a renal transplant recipient.

    Science.gov (United States)

    Muthuvel, V Arun; Ravindran, Manipriya; Chander, Aravind; Veluswamy, Chandralekha

    2016-01-01

    Numerous spontaneous pregnancies have been reported in renal transplant recipients; however, only a few pregnancies after the use of assisted reproductive techniques. The authors report a case of renal transplant recipient with secondary infertility who delivered a healthy baby without any complications. The report highlights the importance of minimal stimulation protocol during ovarian stimulation, single embryo transfer, and the need for multispecialty care for these patients. To the best of the authors' knowledge, the present report is the first such case from India and also the second in the world to report a blastocyst transfer among renal transplant recipients.

  18. Live Birth from Previously Vitrified Oocytes, after Trophectoderm Biopsy, Revitrification, and Transfer of a Euploid Blastocyst

    Directory of Open Access Journals (Sweden)

    Jamie A. Grifo

    2013-01-01

    Full Text Available Our objective is to describe a successful live birth from oocyte vitrification followed by thaw, fertilization, blastocyst culture, trophectoderm biopsy, vitrification, and subsequent thaw. Fifteen mature oocytes were frozen from a patient with uterine factor infertility. Thirteen oocytes survived the thaw, and five underwent trophectoderm biopsy and were refrozen. Three euploid embryos were obtained. A single euploid embryo was transferred in the second thaw cycle to a known recipient leading to the delivery of a normal male infant. This case report is proof of the concept that preimplantation screening and diagnosis is an option for fertility preservation patients.

  19. Bovine cysticercosis situation in Brazil

    Directory of Open Access Journals (Sweden)

    Gabriel Augusto Marques Rossi

    2014-02-01

    Full Text Available The taeniasis-cysticercosis complex is a long known zoonotic parasitosis characteristic of underdeveloped countries. In addition to its public health significance, this parasitosis is cause of economic losses to the beef production chain, and synonymous of technical inadequacy in relation to the adoption of Good Agricultural Practices. The occurrences of both human teniasis and bovine cysticercosis could and should be controlled with basic sanitary measures. However, there is much variation in the occurrence of the disease in cattle, characterizing a low rate of technical development as well as problems related to the adoption of basic sanitation measures. This review describes, in details, the causative agent and its epidemiological chain, besides raising current information about the occurrence of bovine cysticercosis in different regions of Brazil, aiming at the adoption of prophylactic measures by different segments responsible.

  20. Additional mitochondrial DNA influences the interactions between the nuclear and mitochondrial genomes in a bovine embryo model of nuclear transfer.

    Science.gov (United States)

    Srirattana, Kanokwan; St John, Justin C

    2018-05-08

    We generated cattle embryos using mitochondrial supplementation and somatic cell nuclear transfer (SCNT), named miNT, to determine how additional mitochondrial DNA (mtDNA) modulates the nuclear genome. To eliminate any confounding effects from somatic cell mtDNA in intraspecies SCNT, donor cell mtDNA was depleted prior to embryo production. Additional oocyte mtDNA did not affect embryo development rates but increased mtDNA copy number in blastocyst stage embryos. Moreover, miNT-derived blastocysts had different gene expression profiles when compared with SCNT-derived blastocysts. Additional mtDNA increased expression levels of genes involved in oxidative phosphorylation, cell cycle and DNA repair. Supplementing the embryo culture media with a histone deacetylase inhibitor, Trichostatin A (TSA), had no beneficial effects on the development of miNT-derived embryos, unlike SCNT-derived embryos. When compared with SCNT-derived blastocysts cultured in the presence of TSA, additional mtDNA alone had beneficial effects as the activity of glycolysis may increase and embryonic cell death may decrease. However, these beneficial effects were not found with additional mtDNA and TSA together, suggesting that additional mtDNA alone enhances reprogramming. In conclusion, additional mtDNA increased mtDNA copy number and expression levels of genes involved in energy production and embryo development in blastocyst stage embryos emphasising the importance of nuclear-mitochondrial interactions.

  1. Extension of the culture period for the in vitro growth of bovine oocytes in the presence of bone morphogenetic protein-4 increases oocyte diameter, but impairs subsequent developmental competence.

    Science.gov (United States)

    Yang, Yinghua; Kanno, Chihiro; Sakaguchi, Kenichiro; Yanagawa, Yojiro; Katagiri, Seiji; Nagano, Masashi

    2017-11-01

    Bone morphogenetic protein-4 (BMP-4) inhibits luteinization of granulosa cells during in vitro growth (IVG) culture of bovine oocytes; however, oocytes derived from a 12 day IVG were less competent for development than in vivo-grown oocytes. We herein investigated whether an extended IVG culture with BMP-4 improves oocyte growth and development to blastocysts after in vitro fertilization. Oocyte-granulosa cell complexes (OGCs) were cultured for 14 or 16 days with BMP-4 (10 ng/mL), while a 12 day culture with BMP-4 served as the in vitro control. OGC viability was maintained for the 16 day culture with BMP-4 (83.2%), but was significantly lower without BMP-4 (58.9%) than the control (83.0%). Prolong-cultured oocytes at 16 days had statistically greater diameter (114.6 μm) than the control (111.7 μm). IVG oocytes with BMP-4 for the 16 day culture had a similar nuclear maturation rate to the control (approximately 67%); however, blastocyst rates in BMP-4 treated oocytes of 14 (1.8%) and 16 day (0%) IVG were statistically lower than that of 12 day IVG (9.0%). In conclusion, BMP-4 maintained OGC viability and promoted oocyte growth in a prolonged culture, but impaired the developmental competence of oocytes. Prolonged culture may not be an appropriate strategy for enhancing the developmental competence of IVG oocytes. © 2017 Japanese Society of Animal Science.

  2. PREVALENCE OF BOVINE (1)

    African Journals Online (AJOL)

    408 heads of cattle to determine the prevalence of bovine tuberculosis and assess its public health implications. A comparative ..... (78.6%) of the respondents consume raw and poorly heat ... compromises related to certain stress factors.

  3. Extracellular Vesicles from BOEC in In Vitro Embryo Development and Quality.

    Directory of Open Access Journals (Sweden)

    Ricaurte Lopera-Vásquez

    Full Text Available To evaluate the effect of conditioned media (CM and Extracellular Vesicles (EVs derived from bovine oviduct epithelial cell (BOEC lines on the developmental capacity of bovine zygotes and the quality of embryos produced in vitro, presumptive zygotes were cultured under specific conditions. In experiment 1, zygotes were cultured either on monolayers from BOEC extended culture (E, together with fresh BOEC suspension cells, or with BOEC-CM from fresh or E-monolayers. In experiment 2, EVs were isolated from BOEC-CM and characterized (150-200 nm by Nanosight® and electron microscopy. Zygotes were cultured in the presence of 3x10(5 EVs/mL, 1.5x10(5 EVs/mL or 7.5x10(4 EVs/mL of fresh or frozen BOEC-EVs. In experiment 3, zygotes were cultured in absence of FCS but with EVs from BOEC-E that had been cultured in different culture media. In experiment 4, zygotes were cultured in SOF+5% normal-FCS, or EV-depleted-FCS. In all cases, cleavage rate (Day 2 and blastocyst development (Day 7-9 was assessed. Blastocysts on Days 7/8 were used for quality evaluation through differential cell count, cryotolerance and gene expression patterns. No differences were found among all FCS-containing groups in cleavage rate or blastocyst yield. However, embryos derived from BOEC-CM had more trophectoderm cells, while embryos derived from BOEC-EVs, both fresh and frozen, has more trophectoderm and total cells. More embryos survived vitrification in the BOEC-CM and BOEC-EV groups. In contrast, more embryos survived in the EV-depleted-FCS than in normal-FCS group. Gene expression patterns were modified for PAG1 for embryos cultured with EVs in the presence of FCS and for IFN-T, PLAC8, PAG1, CX43, and GAPDH in the absence of FCS. In conclusion, EVs from FCS have a deleterious effect on embryo quality. BOEC-CM and EVs during in vitro culture had a positive effect on the quality of in vitro produced bovine embryos, suggesting that EVs have functional communication between the

  4. Cryopreservation of human oocytes, zygotes, embryos and blastocysts: A comparison study between slow freezing and ultra rapid (vitrification methods

    Directory of Open Access Journals (Sweden)

    Tahani Al-Azawi

    2013-12-01

    Full Text Available Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans. It can also be done by preservation of ovarian tissue or entire ovary for transplantation, followed by oocyte harvesting or natural fertilization. Two basic cryopreservation techniques rule the field, slow-rate freezing, the first to be developed and vitrification which in recent years, has gained a foothold. The slow-rate freezing method previously reported had low survival and pregnancy rates, along with the high cost of cryopreservation. Although there are some recent data indicating better survival rates, cryopreservation by the slow freezing method has started to discontinue. Vitrification of human embryos, especially at early stages, became a more popular alternative to the slow rate freezing method due to reported comparable clinical and laboratory outcomes. In addition, vitrification is relatively simple, requires no expensive programmable freezing equipment, and uses a small amount of liquid nitrogen for freezing. Moreover, oocyte cryopreservation using vitrification has been proposed as a solution to maintain women’s fertility by serving and freezing their oocytes at the optimal time. The aim of this research is to compare slow freezing and vitrification in cryopreservation of oocytes, zygotes, embryos and blastocysts during the last twelve years. Therefore, due to a lot of controversies in this regard, we tried to achieve an exact idea about the subject and the best technique used.

  5. The effect of air bubble position after blastocyst transfer on pregnancy rates in IVF cycles.

    Science.gov (United States)

    Friedman, Brooke E; Lathi, Ruth B; Henne, Melinda B; Fisher, Stephanie L; Milki, Amin A

    2011-03-01

    To investigate the relationship between air bubble position after blastocyst transfer (BT) and pregnancy rates (PRs). Retrospective cohort study. University-based infertility center. Three hundred fifteen consecutive nondonor BTs by a single provider. Catheters were loaded with 25 μL of culture media, 20 μL of air, 25 μL of media containing the blastocysts, 20 μL of air, and a small amount of additional media. The distance from the air bubble to the fundus, as seen on abdominal ultrasound examination, was measured at the time of transfer. Air bubble location was categorized as 20 mm from the fundus. Clinical pregnancy rate. After controlling for age, parity, FSH and frozen transfers, and accounting for repeated cycles per patient, the PRs for both the >20-mm (38.3%) and the 10-20-mm (42.0%) from the fundus group were significantly reduced compared with the group in which the bubble was Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  6. Modified natural cycle for embryo transfer using frozen-thawed blastocysts: A satisfactory option.

    Science.gov (United States)

    Le, Quoc V; Abhari, Sina; Abuzeid, Omar M; DeAnna, Jennifer; Satti, Mohamed A; Abozaid, Tarek; Khan, Iqbal; Abuzeid, Mostafa I

    2017-06-01

    To describe pregnancy outcomes of frozen-thawed blastocysts cycles using modified natural cycle frozen embryo transfers (NC-FET) and down-regulated hormonally controlled frozen embryo transfers (HC-FET) protocols. This retrospective cohort study included all patients undergoing either modified NC-FET or down-regulated HC-FET using frozen-thawed day 5 embryos. Cycles with donor blastocysts were excluded. Four hundred twenty eight patients underwent a total of 493 FET cycles. Patients with regular menses and evidence of ovulation underwent modified NC-FET. These patients were given hCG 10,000 IU IM on the day of LH-surge. Vaginal progesterone (P4) was started two days later and blastocyst transfer was planned seven days after detecting the LH surge. Anovulatory patients and some ovulatory patients underwent down-regulated HC-FET. These patients were placed on medroxy-progesterone acetate (10mg) for 10days to bring on menses and were also given a half-dose of GnRH-agonist (GnRH-a) on the third day of medroxy-progesterone acetate. Exogenous estradiol was initiated on the third day of menses. Once serum E2 levels reached >500pg/mL and endometrial lining reached >8mm, intramuscular (IM) P4 in oil was administered. Blastocyst FET was planned 6days after initiating P4. The primary outcomes included clinical pregnancy and delivery rates. There were 197 patients in the modified NC-FET protocol and 181 in the down-regulated HC-FET protocol. Mean age (years), day-3 FSH levels (mIU/mL) and percentage of patients with male factor infertility were significantly higher and mean BMI (kg/m 2 ) was significantly lower in modified NC-FET compared to HC-FET, respectively. Analysis of the first cycle pregnancy outcomes revealed no significant differences in clinical pregnancy rate (54.3% vs. 52.5%) and delivery rate (47.2% vs. 43.6%) between modified NC-FET and HC-FET. Logistic regression analysis showed age (OR=0.939, 95% CI 0.894-0.989, p=0.011), number of blastocysts transferred (OR

  7. The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation.

    Directory of Open Access Journals (Sweden)

    Kátia R L Schwarz

    Full Text Available This study aimed to determine the influence of cyclic guanosine 3'5'-monophosphate (cGMP and cGMP-dependent kinase (PKG during in vitro maturation (IVM on lipolysis-related parameters in bovine cumulus-oocyte complexes (COCs, and on embryo development and cryosurvival. COCs were matured with cGMP/PKG modulators and assessed for metaphase II rates (MII, cGMP levels, lipid content in oocytes (OO, transcript abundance for genes involved in lipolysis (ATGL and lipid droplets (PLIN2 in cumulus cells (CC and OO, and presence of phosphorylated (active hormone sensitive lipase (HSLser563 in OO. Embryo development, lipid contents and survival to vitrification were also assessed. Phosphodiesterase 5 inhibition (PDE5; cGMP-hydrolyzing enzyme with 10-5M sildenafil (SDF during 24 h IVM increased cGMP in COCs (56.9 vs 9.5 fMol/COC in untreated controls, p<0.05 and did not affect on maturation rate (84.3±6.4% MII. Fetal calf serum (FCS in IVM medium decreased cGMP in COCs compared to bovine serum albumin (BSA + SDF (19.6 vs 66.5 fMol/COC, respectively, p<0.05. FCS increased lipid content in OO (40.1 FI, p<0.05 compared to BSA (34.6 FI, while SDF decreased (29.8 and 29.6 FI, with BSA or FCS, respectively p<0.05. PKG inhibitor (KT5823 reversed this effect (38.9 FI, p<0.05. ATGL and PLIN2 transcripts were detected in CC and OO, but were affected by cGMP and PKG only in CC. HSLser563 was detected in OO matured with or without modulators. Reduced lipid content in embryos were observed only when SDF was added during IVM and IVC (27.6 FI compared to its use in either or none of the culture periods (34.2 FI, p<0.05. Survival to vitrification was unaffected by SDF. In conclusion, cGMP and PKG are involved in lipolysis in OO and possibly in CC and embryos; serum negatively affects this pathway, contributing to lipid accumulation, and cGMP modulation may reduce lipid contents in oocytes and embryos, but without improving embryo cryotolerance.

  8. Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts

    DEFF Research Database (Denmark)

    Jakobsen, Jannik E; Li, Juan; Moldt, Brian

    2011-01-01

    We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon-based do......We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon...

  9. Detection of lipomannan in cattle infected with bovine tuberculosis

    Science.gov (United States)

    Early and rapid detection of bovine tuberculosis (bTB) is critical to controlling the spread of this disease in cattle and other animals. In this study, we demonstrate the development of an immunoassay for the direct detection of the bovine bTB biomarker, lipomannan (LM) in serum using a waveguide-...

  10. Sequential Analysis of Global Gene Expression Profiles in Immature and In vitro Matured Bovine Oocytes: Potential Molecular Markers of Oocyte Maturation

    LENUS (Irish Health Repository)

    Mamo, Solomon

    2011-03-16

    Abstract Background Without intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis. Results 8489 transcripts were detected across the two oocyte groups, of which ~25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of α-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation. Conclusion Using sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource

  11. Development of a confirmatory method for detecting recombinant bovine somatotropin in plasma by immunomagnetic precipitation followed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry.

    Science.gov (United States)

    Robert, Christelle; Huet, Anne-Catherine; Suárez-Pantaleón, Célia; Brasseur, Amaury; Delahaut, Philippe; Gillard, Nathalie

    2017-11-01

    Recombinant bovine somatotropin (rbST), a synthetic growth hormone, is used to stimulate growth and enhance milk production in dairy cows. Both its use and the sale of dairy products from treated animals are prohibited in the European Union, as well as in Australia, Canada, Japan, and New Zealand, but authorised in several countries (e.g. Brazil, USA). Screening methods involve detecting anti-rbST antibodies (biomarkers) in treated cows. Confirmatory methods are required to prove rbST abuse. The major challenges in determining rbST are its potentially low levels, its high similarity to native bST, and matrix interferences. To overcome these obstacles, we have developed a method involving immunomagnetic precipitation followed by UHPLC-MS/MS for rbST detection. Briefly, protein G magnetic beads pre-coated with an in-house produced monoclonal antibody were added to plasma. Incubation at room temperature allowed rbST present in the sample to bind to the magnetic beads. After that, magnetic beads were isolated by centrifugation and thoroughly washed (PBS, PBS + 0.2% Tween 20). Finally, rbST was released by alkalinisation and the samples were trypsin digested prior to UHPLC-MS/MS analysis in the MRM mode. Validation was done in accordance with European Commission Decision 2002/657/CE. Matrix-matched calibration with internal standards was used. The decision limit (CCα) reached with this approach was 0.11 µg l -1 .

  12. Radioimmunoassay of bovine heart protein kinase

    International Nuclear Information System (INIS)

    Fleischer, N.; Rosen, O.M.; Reichlin, M.

    1976-01-01

    Immunization of guinea pigs with bovine cardiac cAMP-dependent protein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) resulted in the development of precipitating antibodies to the cAMP-binding subunit of the enzyme. Both the phosphorylated and nonphosphorylated cAMP-binding protein of the protein kinase reacted with the antiserum. A radioimmunoassay was developed that detects 10 ng of holoenzyme and permits measurement of enzyme concentrations in bovine cardiac muscle. Bovine liver, kidney, brain, and skeletal muscle contain protein kinases which are immunologically identical to those found in bovine cardiac muscle. However, the proportion of immunoreactive enzyme activity differed for each tissue. All of the immunologically nonreactive enzyme in skeletal muscle and heart was separable from immunoreactive enzyme by chromatography on DEAE-cellulose. Rat tissues and pig heart contained protein kinase activity that cross reacted immunologically in a nonparallel fashion with bovine cardiac enzyme. These results indicate that cAMP-dependent protein kinases within and between species are immunologically heterogeneous

  13. Selection of Suitable Internal Control Genes for Accurate Normalization of Real-Time Quantitative PCR Data of Buffalo (Bubalus bubalis) Blastocysts Produced by SCNT and IVF.

    Science.gov (United States)

    Sood, Tanushri Jerath; Lagah, Swati Viviyan; Sharma, Ankita; Singla, Suresh Kumar; Mukesh, Manishi; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

    2017-10-01

    We evaluated the suitability of 10 candidate internal control genes (ICGs), belonging to different functional classes, namely ACTB, EEF1A1, GAPDH, HPRT1, HMBS, RPS15, RPS18, RPS23, SDHA, and UBC for normalizing the real-time quantitative polymerase chain reaction (qPCR) data of blastocyst-stage buffalo embryos produced by hand-made cloning and in vitro fertilization (IVF). Total RNA was isolated from three pools, each of cloned and IVF blastocysts (n = 50/pool) for cDNA synthesis. Two different statistical algorithms geNorm and NormFinder were used for evaluating the stability of these genes. Based on gene stability measure (M value) and pairwise variation (V value), calculated by geNorm analysis, the most stable ICGs were RPS15, HPRT1, and ACTB for cloned blastocysts, HMBS, UBC, and HPRT1 for IVF blastocysts and RPS15, GAPDH, and HPRT1 for both the embryo types analyzed together. RPS18 was the least stable gene for both cloned and IVF blastocysts. Following NormFinder analysis, the order of stability was RPS15 = HPRT1>GAPDH for cloned blastocysts, HMBS = UBC>RPS23 for IVF blastocysts, and HPRT1>GAPDH>RPS15 for cloned and IVF blastocysts together. These results suggest that despite overlapping of the three most stable ICGs between cloned and IVF blastocysts, the panel of ICGs selected for normalization of qPCR data of cloned and IVF blastocyst-stage embryos should be different.

  14. Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk

    NARCIS (Netherlands)

    Wellenberg, G.J.; Verstraten, E.; Belak, S.; Verschuren, S.B.E.; Rijsewijk, F.A.M.; Peshev, R.; Oirschot, van J.T.

    2001-01-01

    A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were

  15. Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses.

    Science.gov (United States)

    Furusawa, Tadashi; Ohkoshi, Katsuhiro; Kimura, Koji; Matsuyama, Shuichi; Akagi, Satoshi; Kaneda, Masahiro; Ikeda, Mitsumi; Hosoe, Misa; Kizaki, Keiichiro; Tokunaga, Tomoyuki

    2013-08-01

    Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages.

  16. Xenogeneic chimera-Generated by blastocyst complementation-As a potential unlimited source of recipient-tailored organs.

    Science.gov (United States)

    Oldani, Graziano; Peloso, Andrea; Lacotte, Stéphanie; Meier, Raphael; Toso, Christian

    2017-07-01

    Blastocyst complementation refers to the injection of cells into a blastocyst. The technology allows for the creation of chimeric animals, which have the potential to be used as an unlimited source of organ donors. Pluripotent stem cells could be generated from a patient in need of a transplantation and injected into a large animal blastocyst (potentially of a pig), leading to the creation of organ(s) allowing immunosuppression-free transplantation. Various chimera combinations have already been generated, but one of the most recent steps leads to the creation of human-pig chimeras, which could be studied at an embryo stage. Although still far from clinical reality, the potential application is almost unlimited. The present review illustrates the historical steps of intra- and interspecific blastocyst complementation in rodents and large animals, specifically looking at its potential for generation of organ grafts. We also speculate on how it could change transplant indications, on its economic impact, and on the linked ethical concerns. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. A feasible strategy of preimplantation genetic diagnosis for carriers with chromosomal translocation: Using blastocyst biopsy and array comparative genomic hybridization

    Directory of Open Access Journals (Sweden)

    Chu-Chun Huang

    2013-09-01

    Conclusion: Our study demonstrates an effective PGD strategy with promising outcomes. Blastocyst biopsy can retrieve more genetic material and may provide more reliable results, and aCGH offers not only detection of chromosomal translocation but also more comprehensive analysis of 24 chromosomes than traditional FISH. More cases are needed to verify our results and this strategy might be considered in general clinical practice.

  18. Na--dependent transport of basic, zwitterionic, and bicyclic amino acids by a broad-scope system in mouse blastocysts

    International Nuclear Information System (INIS)

    Van Winkle, L.J.; Christensen, H.N.; Campione, A.L.

    1985-01-01

    Mouse blastocysts which had been activated from diapause in utero appeared to take up amino acids via a Na - -dependent transport system with novel characteristics. In contrast to other cell types, uptake of 3-aminoendobicyclo [3,2,1]octane-3-carboxylic acid (BCO) by blastocysts was largely Na - dependent. Moreover, L-alanine and BCO met standard criteria for mutual competitive inhibition of the Na - -dependent transport of each other. The Ki for each of these amino acids as an inhibitor of transport of the other had a value similar to the value of its Km for transport. In addition, both 2-aminoendobicyclo [2,2,1]heptane-2-carboxylic acid and L-valine appeared to inhibit Na - -dependent transport of alanine and BCO competitively. Finally, alanine and L-lysine appeared to compete for the same Na+-dependent transport sites in blastocysts. For these reasons, the authors conclude that lysine, alanine, and BCO are transported by a common Na+-dependent system in blastocysts. In addition, the apparent interaction of the system with other basic amino acids, such as 1-dimethylpiperidine-4-amino-4-carboxylic acid, which has a nondissociable positive charge on its side chain, and L-arginine and L-homoarginine, whose cationic forms are highly predominant at neutral pH, suggests that the cationic forms of basic amino acids are transported by the wide-scope system

  19. Blastocysts production and collection in albino Syrian hamster using superovulation and intrauterine artificial insemination in non-breeding season

    Directory of Open Access Journals (Sweden)

    A. Amiri Divani

    2017-06-01

    Full Text Available In vivo blastocyst production and collection using superovulation and intrauterine insemination was established in albino Syrian hamsters. Twenty female albino hamsters were injected pregnant mare serum gonadotropin (PMSG, 25 IU in non-breeding season and 48 h or 56 h later, 25 IU of human chorionic gonadotropin (hCG were injected. Both groups were divided into two subgroups of natural mating and artificial insemination. The former group was mated with a fertile male (1 male for 2 fe-males after hCG injection and in the next morning, the hamsters with vaginal plug were regarded as pregnant. In the artificial insemination group, intrauterine artificial insemination of 1×108 sperms was done 12 h after hCG injection. Blastocysts were counted at 3.5 days after mating or insemination. However, 48 h and 56 h hCG and natural mating and 48 h hCG and artificial insemination were without blastocyst; however the method of 56 h hCG and artificial insemination produced of 15±5 (mean and standard deviation blastocysts in each albino hamster in the winter.

  20. Generation of human embryonic stem cells from abnormal blastocyst diagnosed with albinism.

    Science.gov (United States)

    Sun, Yi; Zhou, Xiaoying; Chen, Jing; Du, Juan; Lu, Guangxiu; Lin, Ge; Ouyang, Qi

    2016-11-01

    Human embryonic stem cell (hESC) line chHES-478 was derived from abnormal blastocyst diagnosed with albinism after preimplantation genetic diagnosis (PGD) treatment. DNA sequencing analysis confirmed that chHES-478 cell line carried a compound heterozygous mutation, c.896G>A(p.Arg299His) and c.929_930insC(p.Pro310Glnfs*9), of TYR gene. Characteristic tests proved that the chHES-478 cell line presented typical markers of pluripotency and had the capability to form the three germ layers both in vitro and in vivo. Copyright © 2016 Michael Boutros, German Cancer Research Center, Heidelberg, Germany. Published by Elsevier B.V. All rights reserved.

  1. Recent Progress in Cryopreservation of Bovine Oocytes

    Directory of Open Access Journals (Sweden)

    In-Sul Hwang

    2014-01-01

    Full Text Available Principle of oocyte cryoinjury is first overviewed and then research history of cryopreservation using bovine oocytes is summarized for the last two decades with a few special references to recent progresses. Various types of cryodevices have been developed to accelerate the cooling rate and applied to the oocytes from large domestic species enriched with cytoplasmic lipid droplets. Two recent approaches include the qualitative improvement of IVM oocytes prior to the vitrification and the short-term recovery culture of vitrified-warmed oocytes prior to the subsequent IVF. Supplementation of L-carnitine to IVM medium of bovine oocytes has been reported to reduce the amount of cytoplasmic lipid droplets and improve the cryotolerance of the oocytes, but it is still controversial whether the positive effect of L-carnitine is reproducible. Incidence of multiple aster formation, a possible cause for low developmental potential of vitrified-warmed bovine oocytes, was inhibited by a short-term culture of the postwarm oocytes in the presence of Rho-associated coiled-coil kinase (ROCK inhibitor. Use of an antioxidant α-tocopherol, instead of the ROCK inhibitor, also supported the revivability of the postwarm bovine oocytes. Further improvements of the vitrification procedure, combined with pre- and postvitrification chemical treatment, would overcome the high sensitivity of bovine oocytes to cryopreservation.

  2. Seroprevalence of bovine theileriosis in northern China

    Directory of Open Access Journals (Sweden)

    Yaqiong Li

    2016-11-01

    Full Text Available Abstract Background Bovine theileriosis is a common disease transmitted by ticks, and can cause loss of beef and dairy cattle worldwide. Here, an indirect enzyme-linked immunosorbent assay (iELISA based on Theileria luwenshuni surface protein (TlSP was developed and used to carry out a seroepidemiological survey of bovine theileriosis in northern China. Methods We used the BugBuster Ni-NTA His•Bind Purification Kit to purify recombinant TlSP (rTlSP, which was subsequently analyzed by Western Blotting to evaluate cross-reactivity with other pathogen-positive sera. The iELISA method based on rTlSP was successfully developed. Sera from 2005 blood samples were tested with the rTlSP-iELISA method, and blood smears from these samples were observed by microscopy. Results The specificity of iELISA was 98.9%, the sensitivity was 98.5%, and the cut-off was selected as 24.6%. Western Blot analysis of rTlSP confirmed that there were cross-reactions with Theileria luwenshuni, Theileria uilenbergi, Theileria ovis, Theileria annulata, Theileria orientalis and Theileria sinensis. The epidemiological survey showed that the highest positive rate of bovine theileriosis was 98.3%, the lowest rate was 84.1%, and the average positive rate was 95.4% by iELISA. With microscopy, the highest positive rate was 38.9%, the lowest rate was 5.1%, and the relative average positive rate was 13.7%. Conclusions An rTlSP-iELISA was developed to detect circulating antibodies against bovine Theileria in northern China. This is the first report on the seroprevalence of bovine theileriosis in northern China, and it also provides seroepidemiological data on bovine theileriosis in China.

  3. Development and evaluation of a real-time fluorescent polymerase chain reaction assay for the detection of bovine contaminates in cattle feed.

    Science.gov (United States)

    Rensen, Gabriel; Smith, Wayne; Ruzante, Juliana; Sawyer, Mary; Osburn, Bennie; Cullor, James

    2005-01-01

    A real-time fluorescent polymerase chain reaction assay for detecting prohibited ruminant materials such as bovine meat and bone meal (BMBM) in cattle feed using primers and FRET probes targeting the ruminant specific mitochondrial cytochrome b gene was developed and evaluated on two different types of cattle feed. Common problems involved with PCR based testing of cattle feed include the presence of high levels of PCR inhibitors and the need for certain pre-sample processing techniques in order to perform DNA extractions. We have developed a pre-sample processing technique for extracting DNA from cattle feed which does not require the feed sample to be ground to a fine powder and utilizes materials that are disposed of between samples, thus, reducing the potential of cross contamination. The DNA extraction method utilizes Whatman FTA card technology, is adaptable to high sample throughput analysis and allows for room temperature storage with established archiving of samples of up to 14 years. The Whatman FTA cards are subsequently treated with RNAse and undergo a Chelex-100 extraction (BioRad, Hercules, CA), thus removing potential PCR inhibitors and eluting the DNA from the FTA card for downstream PCR analysis. The detection limit was evaluated over a period of 30 trials on calf starter mix and heifer starter ration feed samples spiked with known concentrations of BMBM. The PCR detection assay detected 0.05% wt/wt BMBM contamination with 100% sensitivity, 100% specificity, and 100% confidence. Concentrations of 0.005% and 0.001% wt/wt BMBM contamination were also detected in both feed types but with varying levels of confidence.

  4. The role of RNA polymerase I transcription and embryonic genome activation in nucleolar development in bovine preimplantation embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, F.; Petrovicova, I.

    2008-01-01

    The aim of the present study was to investigate the role of RNA polymerase I (RPI) transcription in nucleolar development during major transcriptional activation (MTA) in cattle. Late eight-cell embryos were cultured in the absence (control group) or presence of actinomycin D (AD) (RPI inhibition...

  5. Identification of the bovine Arachnomelia mutation by massively parallel sequencing implicates sulfite oxidase (SUOX in bone development.

    Directory of Open Access Journals (Sweden)

    Cord Drögemüller

    2010-08-01

    Full Text Available Arachnomelia is a monogenic recessive defect of skeletal development in cattle. The causative mutation was previously mapped to a ∼7 Mb interval on chromosome 5. Here we show that array-based sequence capture and massively parallel sequencing technology, combined with the typical family structure in livestock populations, facilitates the identification of the causative mutation. We re-sequenced the entire critical interval in a healthy partially inbred cow carrying one copy of the critical chromosome segment in its ancestral state and one copy of the same segment with the arachnomelia mutation, and we detected a single heterozygous position. The genetic makeup of several partially inbred cattle provides extremely strong support for the causality of this mutation. The mutation represents a single base insertion leading to a premature stop codon in the coding sequence of the SUOX gene and is perfectly associated with the arachnomelia phenotype. Our findings suggest an important role for sulfite oxidase in bone development.

  6. Identification of the Bovine Arachnomelia Mutation by Massively Parallel Sequencing Implicates Sulfite Oxidase (SUOX) in Bone Development

    Science.gov (United States)

    Drögemüller, Cord; Tetens, Jens; Sigurdsson, Snaevar; Gentile, Arcangelo; Testoni, Stefania; Lindblad-Toh, Kerstin; Leeb, Tosso

    2010-01-01

    Arachnomelia is a monogenic recessive defect of skeletal development in cattle. The causative mutation was previously mapped to a ∼7 Mb interval on chromosome 5. Here we show that array-based sequence capture and massively parallel sequencing technology, combined with the typical family structure in livestock populations, facilitates the identification of the causative mutation. We re-sequenced the entire critical interval in a healthy partially inbred cow carrying one copy of the critical chromosome segment in its ancestral state and one copy of the same segment with the arachnomelia mutation, and we detected a single heterozygous position. The genetic makeup of several partially inbred cattle provides extremely strong support for the causality of this mutation. The mutation represents a single base insertion leading to a premature stop codon in the coding sequence of the SUOX gene and is perfectly associated with the arachnomelia phenotype. Our findings suggest an important role for sulfite oxidase in bone development. PMID:20865119

  7. Y-27632 enhances differentiation of blastocyst like cystic human embryoid bodies to endocrinologically active trophoblast cells on a biomimetic platform

    Directory of Open Access Journals (Sweden)

    Totey Satish M

    2009-09-01

    Full Text Available Abstract Trophoblast differentiation and formation of the placenta are important events linked to post-implantation embryonic development. Models mimicking the biology of trophoblast differentiation in a post-implantation maternal microenvironment are needed for understanding disorders like placental-ischemia or for applications in drug-screening, and would help in overcoming the ethical impasse on using human embryos for such research. Here we attempt to create such a model by using embryoid bodies (EBs and a biomimetic platform composed of a bilayer of fibronectin and gelatin on top of low-melting agarose. Using this model we test the hypothesis that cystic-EBs (day 30 that resemble blastocysts morphologically, are better sources as compared to noncytic EBs (day 10, for functional trophoblast differentiation; and that the Rho kinases inhibitor Y27632 can enhance this differentiation. Non/cytic EBs with/out Y27632 were grown on this platform for 28 days, and screened from secretion and expression of trophoblast and other lineage markers using ECLIA, RT-PCR, and Immunofluorescence. All EBs attached on this surface and rapidly proliferated into hCG and progesterone (P2 secreting functional trophoblast cells. However, the cells derived from cytic-EBs and cytic-EBs+ Y27632 showed the maximum secretion of these hormones and expressed IGF2, supporting our hypothesis. Also Y27632 reduced extraembryonic endoderm and trophoblast lineage differentiation from early noncystic-EBs, whereas, it specifically enhanced the induction of trophoblast and multinucleated syncitiotrophoblast differentiation from late cystic-EBs. In vivo trophoblast differentiation can be replicated in fibronectin based biomaterials, using cytic-EBs and by maneuvering the Rho-ROCK pathways. Response of EBs to a compound may vary temporally, and determination of their right stage is crucial for applications in directed-differentiation or drug-screening.

  8. Characterization of bovine gamma delta T cells phenotype during post-natal development and following Mycobacterium bovis vaccination or virulent infection

    Science.gov (United States)

    Bovine tuberculosis caused by Mycobacterium bovis is a globally significant veterinary health problem. Gamma delta T cells are known to participate in the immune control of mycobacterial infections. Data in human and non-human primates suggest that mycobacterial infection regulates memory/effector p...

  9. Development of risk-based trading farm scoring system to assist with the control of bovine tuberculosis in cattle in England and Wales.

    Science.gov (United States)

    Adkin, A; Brouwer, A; Simons, R R L; Smith, R P; Arnold, M E; Broughan, J; Kosmider, R; Downs, S H

    2016-01-01

    Identifying and ranking cattle herds with a higher risk of being or becoming infected on known risk factors can help target farm biosecurity, surveillance schemes and reduce spread through animal trading. This paper describes a quantitative approach to develop risk scores, based on the probability of infection in a herd with bovine tuberculosis (bTB), to be used in a risk-based trading (RBT) scheme in England and Wales. To produce a practical scoring system the risk factors included need to be simple and quick to understand, sufficiently informative and derived from centralised national databases to enable verification and assess compliance. A logistic regression identified herd history of bTB, local bTB prevalence, herd size and movements of animals onto farms in batches from high risk areas as being significantly associated with the probability of bTB infection on farm. Risk factors were assigned points using the estimated odds ratios to weight them. The farm risk score was defined as the sum of these individual points yielding a range from 1 to 5 and was calculated for each cattle farm that was trading animals in England and Wales at the start of a year. Within 12 months, of those farms tested, 30.3% of score 5 farms had a breakdown (sensitivity). Of farms scoring 1-4 only 5.4% incurred a breakdown (1-specificity). The use of this risk scoring system within RBT has the potential to reduce infected cattle movements; however, there are cost implications in ensuring that the information underpinning any system is accurate and up to date. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

  10. Oral Serum-Derived Bovine Immunoglobulin/Protein Isolate Has Immunomodulatory Effects on the Colon of Mice that Spontaneously Develop Colitis.

    Directory of Open Access Journals (Sweden)

    Anna Pérez-Bosque

    Full Text Available Dietary immunoglobulin concentrates prepared from animal plasma can modulate the immune response of gut-associated lymphoid tissue (GALT. Previous studies have revealed that supplementation with serum-derived bovine immunoglobulin/protein isolate (SBI ameliorates colonic barrier alterations in the mdr1a-/- genetic mouse model of IBD. Here, we examine the effects of SBI on mucosal inflammation in mdr1a-/- mice that spontaneously develop colitis. Wild type (WT mice and mice lacking the mdr1a gene (KO were fed diets supplemented with either SBI (2% w/w or milk proteins (Control diet, from day 21 (weaning until day 56. Leucocytes in mesenteric lymph nodes (MLN and in lamina propria were determined, as was mucosal cytokine production. Neutrophil recruitment and activation in MLN and lamina propria of KO mice were increased, but were significantly reduced in both by SBI supplementation (p < 0.05. The increased neutrophil recruitment and activation observed in KO mice correlated with increased colon oxidative stress (p < 0.05 and SBI supplementation reduced this variable (p < 0.05. The Tact/Treg lymphocyte ratios in MLN and lamina propria were also increased in KO animals, but SBI prevented these changes (both p < 0.05. In the colon of KO mice, there was an increased production of mucosal pro-inflammatory cytokines such as IL-2 (2-fold, IL-6 (26-fold and IL-17 (19-fold, and of chemokines MIP-1β (4.5-fold and MCP-1 (7.2-fold. These effects were significantly prevented by SBI (p < 0.05. SBI also significantly increased TGF-β secretion in the colon mucosa, suggesting a role of this anti-inflammatory cytokine in the modulation of GALT and the reduction of the severity of the inflammatory response during the onset of colitis.

  11. Effects of recipient oocyte age and interval from fusion to activation on development of buffalo (Bubalus bubalis) nuclear transfer embryos derived from fetal fibroblasts.

    Science.gov (United States)

    Lu, F; Jiang, J; Li, N; Zhang, S; Sun, H; Luo, C; Wei, Y; Shi, D

    2011-09-15

    The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P fusion (P fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Vaccination of cattle against bovine viral diarrhoea

    NARCIS (Netherlands)

    Oirschot, van J.T.; Bruschke, C.J.M.; Rijn, van P.A.

    1999-01-01

    This brief review describes types and quality (efficacy and safety) of bovine viral diarrhoea virus (BVDV) vaccines that are in the market or under development. Both conventional live and killed vaccines are available. The primary aim of vaccination is to prevent congenital infection, but the few

  13. NUTRIENTS AND EPIGENETICS IN BOVINE CELLS

    Science.gov (United States)

    This is a chapter for a book titled “Livestock Epigenetics” edited by Dr. Hasan Khatib and published by Wiley-Blackwell. This chapter is focused on the research development in our laboratory in the area of interaction of nutrients and genomic phonotype in bovine cells. Briefly, the Research on nutri...

  14. The functional role of insulin in fertility and embryonic development-What can we learn from the bovine model?

    Science.gov (United States)

    Laskowski, D; Sjunnesson, Y; Humblot, P; Andersson, G; Gustafsson, H; Båge, R

    2016-07-01

    Insulin is a key metabolic hormone that plays a crucial role in regulating energy homeostasis in the body. In addition, insulin-dependent signaling has important functions in reproduction and early embryo development. As metabolism and reproduction are closely linked, metabolic challenges may be the source of reproductive disorders and decreased fertility. This is known for the dairy cow and for other species including the human. Although metabolic disorders in the dairy cow often derive from a failure to adapt to a high milk production, the situation in the human is often linked to emerging conditions and associated diseases in our modern society such as obesity and diabetes, where an excess energy intake causes decreased fertility in women. Both energy excess and energy deficit are associated with a deviation of insulin concentrations in serum and follicular fluid from normal levels. Although many studies have shown that extreme variation in energy supply can negatively influence early embryo development by inducing changes in circulating concentrations of several metabolites or hormones like insulin, several in vitro culture media are still supplemented with insulin in high concentrations. In this review, direct and indirect effects of insulin on fertility will be described. Differences between the in vivo and in vitro situations will also be discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Heterogeneity of Bovine Peripheral Blood Monocytes

    Directory of Open Access Journals (Sweden)

    Jamal Hussen

    2017-12-01

    Full Text Available Peripheral blood monocytes of several species can be divided into different subpopulations with distinct phenotypic and functional properties. Herein, we aim at reviewing published work regarding the heterogeneity of the recently characterized bovine monocyte subsets. As the heterogeneity of human blood monocytes was widely studied and reviewed, this work focuses on comparing bovine monocyte subsets with their human counterparts regarding their phenotype, adhesion and migration properties, inflammatory and antimicrobial functions, and their ability to interact with neutrophilic granulocytes. In addition, the differentiation of monocyte subsets into functionally polarized macrophages is discussed. Regarding phenotype and distribution in blood, bovine monocyte subsets share similarities with their human counterparts. However, many functional differences exist between monocyte subsets from the two species. In contrast to their pro-inflammatory functions in human, bovine non-classical monocytes show the lowest phagocytosis and reactive oxygen species generation capacity, an absent ability to produce the pro-inflammatory cytokine IL-1β after inflammasome activation, and do not have a role in the early recruitment of neutrophils into inflamed tissues. Classical and intermediate monocytes of both species also differ in their response toward major monocyte-attracting chemokines (CCL2 and CCL5 and neutrophil degranulation products (DGP in vitro. Such differences between homologous monocyte subsets also extend to the development of monocyte-derived macrophages under the influence of chemokines like CCL5 and neutrophil DGP. Whereas the latter induce the differentiation of M1-polarized macrophages in human, bovine monocyte-derived macrophages develop a mixed M1/M2 macrophage phenotype. Although only a few bovine clinical trials analyzed the correlation between changes in monocyte composition and disease, they suggest that functional differences between

  16. Field trial of a synthetic tsetse-repellent technology developed for the control of bovine trypanosomosis in Kenya.

    Science.gov (United States)

    Bett, B; Randolph, T F; Irungu, P; Nyamwaro, S O; Kitala, P; Gathuma, J; Grace, D; Vale, G; Hargrove, J; McDermott, J

    2010-12-01

    We conducted a field trial among Maasai cattle-keepers in Nkuruman and Nkineji areas of Kenya to evaluate the effectiveness of a synthetic tsetse-repellent technology developed for the control of trypanosomosis in cattle. The technology was a repellent (2-methoxy 4-methylphenol) emitted from dispensers attached to collars worn by cattle. Treatment was allocated at the herd level to ensure adequate protection of all the animals in a herd, with measurements of effectiveness conducted at the individual-animal level. The trial began in April 2005 and ran for 16 months including a baseline phase of 4 months. We recruited 12 herds in each area using a restricted random-sampling technique and distributed them equally into intervention (repellent) and control groups. Sample size was determined using a formal power calculation. Effectiveness or minimal worthwhile difference was defined as a 50% reduction in the incidence of trypanosome infection in the treated versus control group (effectiveness below which the technology was considered by experts as not viable compared to existing control techniques). All the animals in the recruited herds were screened monthly (buffy-coat technique) for trypanosome infections. The analysis followed the principle of intention-to-treat by which subjects are analysed according to their initial treatment assignment, regardless of the mechanical performance of the device. Crude and adjusted effects of the technology were 23% (ptechnology estimated in this study did not achieve the threshold of 50% reduction in the trypanosome infection incidence set a priori to indicate effectiveness (ptechnology package was not sufficiently effective in reducing trypanosome infection incidence under natural tsetse challenge to merit commercial development. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. Control of Maillard-type off-flavor development in ultrahigh-temperature-processed bovine milk by phenolic chemistry.

    Science.gov (United States)

    Kokkinidou, Smaro; Peterson, Devin G

    2014-08-13

    The application of phenolic compounds to suppress Maillard chemistry and off-flavor development in ultrahigh-termperature (UHT)-processed milk during processing and storage was investigated. Five phenolic compounds were examined for structure-reactivity relationships (catechin, genistein, daidzein, 1,2,3-trihydroxybenzene, and 1,3,5-trihydroxybenzene). The levels of key transient Maillard reaction (MR) intermediates (reactive carbonyl species) and select off-flavor markers (methional, 2-acetyl-2-thiazoline, 2-acetyl-1-pyrroline) were quantified by LC-MS/MS and GC-MS/ToF, respectively. The addition of phenolic compounds prior to UHT processing significantly reduced the concentration of MR intermediates and related off-flavor compounds compared to a control sample (p markers and reactive carbonyl species. Sensory studies were in agreement with the analytical data. The cooked flavor intensity was rated lower for the recombination model samples of the catechin-treated UHT milk compared to the control UHT milk. Additionally, consumer acceptability studies showed catechin-treated UHT milk to have significantly higher liking scores when compared the control sample (Fisher's LSD = 0.728).

  18. The diagnosis of bovine basesiosis (babesia bovis) by means of the test of ELISA

    International Nuclear Information System (INIS)

    Leon Arenas, Edgar; Guillen, Ana Teresa; Silva, Maglene

    1997-01-01

    Between 1994 and 1996 a kit ELISA, developed by the FAO - IAEA for the diagnose of bovine babesiosis produced by Babesia bovis, was validated. There were processed a total of 547 blood serums from bovine between 9 and 18 months old, coming from high and low risk to illness areas. The point obtained for the test was 0.178 (DO) and the resulting percentages inside the population studied was 48% animal positive and 52% bovine negative. These results confirm that bovine population in Venezuela is in enzootic uncertainty areas for bovine babesiosis [es

  19. Bovine somatic cell nuclear transfer.

    Science.gov (United States)

    Ross, Pablo J; Cibelli, Jose B

    2010-01-01

    Somatic cell nuclear transfer (SCNT) is a technique by which the nucleus of a differentiated cell is introduced into an oocyte from which its genetic material has been removed by a process called enucleation. In mammals, the reconstructed embryo is artificially induced to initiate embryonic development (activation). The oocyte turns the somatic cell nucleus into an embryonic nucleus. This process is called nuclear reprogramming and involves an important change of cell fate, by which the somatic cell nucleus becomes capable of generating all the cell types required for the formation of a new individual, including extraembryonic tissues. Therefore, after transfer of a cloned embryo to a surrogate mother, an offspring genetically identical to the animal from which the somatic cells where isolated, is born. Cloning by nuclear transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. Cattle were the second mammalian species to be cloned after Dolly the sheep, and it is probably the most widely used species for SCNT experiments. This is, in part due to the high availability of bovine oocytes and the relatively higher efficiency levels usually obtained in cattle. Given the wide utilization of this species for cloning, several alternatives to this basic protocol can be found in the literature. Here we describe a basic protocol for bovine SCNT currently being used in our laboratory, which is amenable for the use of the nuclear transplantation technique for research or commercial purposes.

  20. Camel and bovine chymosin

    DEFF Research Database (Denmark)

    Jensen, Jesper Langholm; Mølgaard, Anne; Poulsen, Jens-Christian Navarro

    2013-01-01

    chymosin. Both enzymes possess local positively charged patches on their surface that can play a role in interactions with the overall negatively charged C-terminus of κ-casein. Camel chymosin contains two additional positive patches that favour interaction with the substrate. The improved electrostatic......Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105-Met106 bond of the milk protein κ-casein, releasing its predominantly negatively charged C-terminus, which leads to the separation of the milk into curds and whey. Despite...... interactions arising from variation in the surface charges and the greater malleability both in domain movements and substrate binding contribute to the better milk-clotting activity of camel chymosin towards bovine milk....

  1. Aortic reconstruction with bovine pericardial grafts

    Directory of Open Access Journals (Sweden)

    Silveira Lindemberg Mota

    2003-01-01

    Full Text Available INTRODUCTION: Glutaraldehyde-treated crimped bovine pericardial grafts are currently used in aortic graft surgery. These conduits have become good options for these operations, available in different sizes and shapes and at a low cost. OBJECTIVE:To evaluate the results obtained with bovine pericardial grafts for aortic reconstruction, specially concerning late complications. METHOD: Between January 1995 and January 2002, 57 patients underwent different types of aortic reconstruction operations using bovine pericardial grafts. A total of 29 (50.8% were operated on an urgent basis (mostly acute Stanford A dissection and 28 electively. Thoracotomy was performed in three patients for descending aortic replacement (two patients and aortoplasty with a patch in one. All remaining 54 underwent sternotomy, cardiopulmonary bypass and aortic resection. Deep hypothermia and total circulatory arrest was used in acute dissections and arch operations. RESULTS: Hospital mortality was 17.5%. Follow-up was 24.09 months (18.5 to 29.8 months confidence interval and complication-free actuarial survival curve was 92.3% (standard deviation ± 10.6. Two patients lately developed thoracoabdominal aneurysms following previous DeBakey II dissection and one died from endocarditis. One "patch" aortoplasty patient developed local descending aortic pseudoaneurysm 42 months after surgery. All other patients are asymptomatic and currently clinically evaluated with echocardiography and CT scans, showing no complications. CONCLUSION: Use of bovine pericardial grafts in aortic reconstruction surgery is adequate and safe, with few complications related to the conduits.

  2. Functional and gene network analyses of transcriptional signatures characterizing pre-weaned bovine mammary parenchyma or fat pad uncovered novel inter-tissue signaling networks during development

    Directory of Open Access Journals (Sweden)

    Lewin Harris A

    2010-05-01

    Full Text Available Abstract Background The neonatal bovine mammary fat pad (MFP surrounding the mammary parenchyma (PAR is thought to exert proliferative effects on the PAR through secretion of local modulators of growth induced by systemic hormones. We used bioinformatics to characterize transcriptomics differences between PAR and MFP from ~65 d old Holstein heifers. Data were mined to uncover potential crosstalk through the analyses of signaling molecules preferentially expressed in one tissue relative to the other. Results Over 9,000 differentially expressed genes (DEG; False discovery rate ≤ 0.05 were found of which 1,478 had a ≥1.5-fold difference between PAR and MFP. Within the DEG highly-expressed in PAR vs. MFP (n = 736 we noted significant enrichment of functions related to cell cycle, structural organization, signaling, and DNA/RNA metabolism. Only actin cytoskeletal signaling was significant among canonical pathways. DEG more highly-expressed in MFP vs. PAR (n = 742 belong to lipid metabolism, signaling, cell movement, and immune-related functions. Canonical pathways associated with metabolism and signaling, particularly immune- and metabolism-related were significantly-enriched. Network analysis uncovered a central role of MYC, TP53, and CTNNB1 in controlling expression of DEG highly-expressed in PAR vs. MFP. Similar analysis suggested a central role for PPARG, KLF2, EGR2, and EPAS1 in regulating expression of more highly-expressed DEG in MFP vs. PAR. Gene network analyses revealed putative inter-tissue crosstalk between cytokines and growth factors preferentially expressed in one tissue (e.g., ANGPTL1, SPP1, IL1B in PAR vs. MFP; ADIPOQ, IL13, FGF2, LEP in MFP vs. PAR with DEG preferentially expressed in the other tissue, particularly transcription factors or pathways (e.g., MYC, TP53, and actin cytoskeletal signaling in PAR vs. MFP; PPARG and LXR/RXR Signaling in MFP vs. PAR. Conclusions Functional analyses underscored a reciprocal influence in

  3. BOVINE TUBERCULOSIS (BTB) AS A RISK FACTOR FOR DEVELOPING TUBERCULOSIS IN HUMANS IN THE RURAL COMMUNITY OF ETHIOPIA: A CASE-CONTROL STUDY.

    Science.gov (United States)

    Mengistu, Araya; Enquselassi, Fikre; Aseffa, Abraham; Beyen, Demissew

    2015-01-01

    The current study aimed at assessing BTB as a possible risk factor for human TB in the rural community of North Eastern and Western parts of Ethiopia. A case-control design was conducted among cattle owning households with TB and without TB. Comparative cervical intradermal test using purified protein derivatives were used to test cattle. Reading of the reaction was done 72 ± 4hrs after antigen injection. Based on the skin test reaction measurement, cattle categorized as negative, doubtful and positive. Questionnaires were used to collect the required factors. Thirty-five with TB and 105 households without TB participated in this study of which 49.3% and 61.4% had the habit of drinking raw milk and eating uncooked meat, respectively. About 70.7% knew about the chance of disease transmission from animals to humans. Among the TB households 31.43% shared their house with their cattle. Of the attendants, approximately 38% shared utensil. Based on > 2mms as a cutoff value 23.6% an overall apparent bovine tuberculosis (BTB) and 48.6% apparent BTB in households with TB were recorded. The odds for households having bovine TB in their cattle to get tuberculosis was more than 8 times (95% CI; 2.82-24.60) higher than those owned by households without TB. Bovine TB has been seen as an exposure to human pulmonary TB occurrence. A separate house for cattle should be constructed to minimize the fear of cross infections and further study regarding the possible infection of cattle with M. tuberculosis is suggested. Key wordsi bovine tuberculosis, households, human TB, M. tuberculosis, risk.

  4. Development and evaluation of a replicon particle vaccine expressing the E2 glycoprotein of bovine viral diarrhea virus (BVDV in cattle

    Directory of Open Access Journals (Sweden)

    Loy John Dustin

    2013-01-01

    Full Text Available Abstract Background Bovine viral diarrhea virus is one of the most significant and costly viral pathogens of cattle worldwide. Alphavirus-derived replicon particles have been shown to be safe and highly effective vaccine vectors against a variety of human and veterinary pathogens. Replicon particles are non-propagating, DIVA compatible, and can induce both humoral and cell mediated immune responses. This is the first experiment to demonstrate that Alphavirus-based replicon particles can be utilized in a standard prime/boost vaccination strategy in calves against a commercially significant bovine pathogen. Findings Replicon particles that express bovine viral diarrhea virus sub-genotype 1b E2 glycoprotein were generated and expression was confirmed in vitro using polyclonal and monoclonal antibodies specific to E2. Vaccine made from particles was generated in Vero cells and administered to BVDV free calves in a prime/boost regimen at two dosage levels. Vaccination resulted in neutralizing antibody titers that cross-neutralized both type 1 and type 2 BVD genotypes following booster vaccination. Additionally, high dose vaccine administration demonstrated some protection from clinical disease and significantly reduced the degree of leukopenia caused by viral infection. Conclusions Replicon particle vaccines administered in a prime/boost regimen expressing BVDV E2 glycoprotein can induce cross-neutralizing titers, reduce leukopenia post challenge, and mitigate clinical disease in calves. This strategy holds promise for a safe and effective vaccine to BVDV.

  5. Epidemiology of Bovine Mastitis in Cows of Dharwad District

    OpenAIRE

    Kurjogi, Mahantesh M.; Kaliwal, Basappa B.

    2014-01-01

    Bovine mastitis is very common in cows of both developed and developing countries. The prevalence of clinical and subclinical mastitis (SCM) varies from region to region. Hence, the present study was carried out to determine the prevalence of mastitis using three diagnostic tests by considering different risk factors like age, lactation, breed, season, quarters, and herd. The results showed that surf field mastitis test (SFMT) is the most sensitive test for diagnosis of bovine mastitis, the o...

  6. Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation

    International Nuclear Information System (INIS)

    Sheth, Bhavwanti; Nowak, Rachael L.; Anderson, Rebecca; Kwong, Wing Yee; Papenbrock, Thomas; Fleming, Tom P.

    2008-01-01

    Apicolateral tight junctions (TJs) between epithelial cells are multiprotein complexes regulating membrane polarity and paracellular transport and also contribute to signalling pathways affecting cell proliferation and gene expression. ZO-2 and other ZO family members form a sub-membranous scaffold for binding TJ constituents. We investigated ZO-2 contribution to TJ biogenesis and function during trophectoderm epithelium differentiation in mouse preimplantation embryos. Our data indicate that ZO-2 is expressed from maternal and embryonic genomes with maternal ZO-2 protein associated with nuclei in zygotes and particularly early cleavage stages. Embryonic ZO-2 assembled at outer blastomere apicolateral junctional sites from the late 16-cell stage. Junctional ZO-2 first co-localised with E-cadherin in a transient complex comprising adherens junction and TJ constituents before segregating to TJs after their separation from the blastocyst stage (32-cell onwards). ZO-2 siRNA microinjection into zygotes or 2-cell embryos resulted in specific knockdown of ZO-2 mRNA and protein within blastocysts. Embryos lacking ZO-2 protein at trophectoderm TJs exhibited delayed blastocoel cavity formation but underwent normal cell proliferation and outgrowth morphogenesis. Quantitative analysis of trophectoderm TJs in ZO-2-deficient embryos revealed increased assembly of ZO-1 but not occludin, indicating ZO protein redundancy as a compensatory mechanism contributing to the mild phenotype observed. In contrast, ZO-1 knockdown, or combined ZO-1 and ZO-2 knockdown, generated a more severe inhibition of blastocoel formation indicating distinct roles for ZO proteins in blastocyst morphogenesis

  7. LukMF′ is the major secreted leukocidin of bovine Staphylococcus aureus and is produced in vivo during bovine mastitis

    Science.gov (United States)

    Vrieling, Manouk; Boerhout, Eveline M.; van Wigcheren, Glenn F.; Koymans, Kirsten J.; Mols-Vorstermans, Tanja G.; de Haas, Carla J. C.; Aerts, Piet C.; Daemen, Ineke J. J. M.; van Kessel, Kok P. M.; Koets, Ad P.; Rutten, Victor P. M. G.; Nuijten, Piet J.M.; van Strijp, Jos A. G.; Benedictus, Lindert

    2016-01-01

    Staphylococcus aureus is a major human and animal pathogen and a common cause of mastitis in cattle. S. aureus secretes several leukocidins that target bovine neutrophils, crucial effector cells in the defence against bacterial pathogens. In this study, we investigated the role of staphylococcal leukocidins in the pathogenesis of bovine S. aureus disease. We show that LukAB, in contrast to the γ-hemolysins, LukED, and LukMF′, was unable to kill bovine neutrophils, and identified CXCR2 as a bovine receptor for HlgAB and LukED. Furthermore, we assessed functional leukocidin secretion by bovine mastitis isolates and observed that, although leukocidin production was strain dependent, LukMF′ was most abundantly secreted and the major toxin killing bovine neutrophils. To determine the role of LukMF′ in bovine mastitis, cattle were challenged with high (S1444) or intermediate (S1449, S1463) LukMF′-producing isolates. Only animals infected with S1444 developed severe clinical symptoms. Importantly, LukM was produced in vivo during the course of infection and levels in milk were associated with the severity of mastitis. Altogether, these findings underline the importance of LukMF′ as a virulence factor and support the development of therapeutic approaches targeting LukMF′ to control S. aureus mastitis in cattle. PMID:27886237

  8. The bovine QTL viewer: a web accessible database of bovine Quantitative Trait Loci

    Directory of Open Access Journals (Sweden)

    Xavier Suresh R

    2006-06-01

    Full Text Available Abstract Background Many important agricultural traits such as weight gain, milk fat content and intramuscular fat (marbling in cattle are quantitative traits. Most of the information on these traits has not previously been integrated into a genomic context. Without such integration application of these data to agricultural enterprises will remain slow and inefficient. Our goal was to populate a genomic database with data mined from the bovine quantitative trait literature and to make these data available in a genomic context to researchers via a user friendly query interface. Description The QTL (Quantitative Trait Locus data and related information for bovine QTL are gathered from published work and from existing databases. An integrated database schema was designed and the database (MySQL populated with the gathered data. The bovine QTL Viewer was developed for the integration of QTL data available for cattle. The tool consists of an integrated database of bovine QTL and the QTL viewer to display QTL and their chromosomal position. Conclusion We present a web accessible, integrated database of bovine (dairy and beef cattle QTL for use by animal geneticists. The viewer and database are of general applicability to any livestock species for which there are public QTL data. The viewer can be accessed at http://bovineqtl.tamu.edu.

  9. PREVALENCE OF BOVINE HERPESVIRUS-1,PARAINFLUENZA-3,BOVINE ROTAVIRUS, BOVINE VIRAL DIARRHEA, BOVINE ADENOVIRUS-7,BOVINE LEUKEMIA VIRUS AND BLUETONGUE VIRUS ANTIBODIES IN CATTLE IN MEXICO

    OpenAIRE

    SUZAN, Victor M.; ONUMA, Misao; AGUILAR, Romero E.; MURAKAMI, Yosuke

    1983-01-01

    Sera were collected from dairy and beef cattle in 19 different states of Mexico. These sera were tested for bovine herpesvirus-1 (BHV-1), parainfluenza-3 virus (PIV-3), bovine rotavirus (BRV), bovine leukemia virus (BLV), bovine adenovirus-7 (BAV-7), bluetongue virus (BTV) and bovine viral diarrhea virus (BVDV). Seropositive rates for each virus for dairy cattle tested were 158/277(57.0%) for BHV-1,217/286(75.0%) for PIV-3,541/1498(36.1%) for BLV, 134/144(93.1%) for BRV, 39/90(43.3%) for BTV,...

  10. Cysteamine supplementation during in vitro maturation of slaughterhouse- and opu-derived bovine oocytes improves embryonic development without affecting cryotolerance, pregnancy rate, and calf characteristics.

    Science.gov (United States)

    Merton, J S; Knijn, H M; Flapper, H; Dotinga, F; Roelen, B A J; Vos, P L A M; Mullaart, E

    2013-09-01

    Optimization of ovum pick up (OPU) followed by in vitro embryo production (IVP) is strongly driven by the needs of both beef and dairy cattle breeders to enhance genetic improvement. The rapidly growing use of genomic selection in cattle has increased the interest in using OPU-IVP technology to increase the number of embryos and offspring per donor, thus allowing enhanced selection intensity for the next generation. The aim of this study was to optimize embryo production through supplementation of cysteamine during in vitro maturation (IVM) and in vitro culture (IVC) of both slaughterhouse- and OPU-derived oocytes. The effects on embryo production and on embryo cryotolerance, post-transfer embryo survival, and calf characteristics, including gestation length, birth weight, perinatal mortality, and sex ratio were studied. In study 1, immature slaughterhouse-derived cumulus-oocyte complexes (COCs) were matured in IVM medium supplemented with or without 0.1 mM cysteamine, fertilized and cultured for 7 days in 0.5 ml SOFaaBSA. In study 2, cysteamine was present during both IVM (0.1 mM) and IVC (0.01, 0.05, 0.1 mM) from Days 1 to 4. In study 3, OPU-derived COCs were matured in medium supplemented with or without 0.1 mM cysteamine in a 2 × 2 factorial design (OPU week and cysteamine treatment). Embryos were evaluated for stage and grade on Day 7 and, depending on the number of transferable embryos and recipients available, the embryos were transferred either fresh or frozen-thawed at a later date. The presence of cysteamine during IVM significantly increased the embryo production rate with slaughterhouse-derived COCs (24.0% vs. 19.4%). The higher number of embryos at Day 7 was due to an increased number of blastocysts, whereas the distribution of embryos among different quality grades and cryotolerance was not affected. Embryo production rate was negatively affected when cysteamine was present during both the processes of IVM and IVC during Days 1 to 4 of culture (13

  11. Effect of vitrification on number of inner cell mass in mouse blastocysts in conventional straw, closed pulled straw, open pulled straw and cryoloop carriers

    International Nuclear Information System (INIS)

    Ghasem, S.; Negar, K.

    2013-01-01

    Objective: To compare the effect of using open and closed carriers on count of inner cell mass in vitrified mouse blastocyst after warming. Methods: The experimental study was conducted at Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran, from April to September 2010. Forty female NMRI (Naval Medical Research Institute, USA) mice were injected with pregnant mares serum gonadotropin and human chorionic gonadotropin in order to induce super ovulation. Following the latter injection, two or three females were caged with the same-breed male mice. The presence of vaginal plug was examined the following morning. To collect blastocyst embryos, the pregnant females were sacrificed by cervical dislocation at 88-90 hours after the injection and dissected. Blastocysts were collected in phosphate-buffered saline and allocated to four groups: vitrification in conventional straw, closed pulled straw, open pulled straw and cryoloop. The vitrification solution was ethylene glycol, Ficol and sucrose (EFS) 20% and 40%. After storage for 1 month in liquid nitrogen, the blastocysts were thawed in 0.5 M sucrose then cultured in M16 medium. After 6 hours of culture, the number of expanded blastocysts was recorded and stained by double-dye technique. After staining, the number of total cell and inner cell mass was calculated. Results: The re-expansion rate of blastocysts in the cryoloop group (n=90; 78.26%) was significantly higher (p<0.05) than open pulled straw (n=83; 69.16%), closed pulled straw (n=68; 54.83%) and conventional straws (n=63; 51.21%) groups. Significant differences (p<0.05) in the number of inner cell mass in blastocysts vitrified in open pulled straws, closed straws and cryoloop with blastocysts cryopreserved in conventional straws. Conclusion: The re-expansion rate and total cell number of mouse blastocysts vitrified using open system had a better result compared with the closed system. The value of cryoloop and open pulled straws as carriers in

  12. Bovine Virus Diarrhea (BVD)

    OpenAIRE

    Hoar, Bruce R.

    2004-01-01

    Bovine virus diarrhea (BVD) is a complicated disease to discuss as it can result in a wide variety of disease problems from very mild to very severe. BVD can be one of the most devastating diseases cattle encounter and one of the hardest to get rid of when it attacks a herd. The viruses that cause BVD have been grouped into two genotypes, Type I and Type II. The disease syndrome caused by the two genotypes is basically the same, however disease caused by Type II infection is often more severe...

  13. Characterization Of Bovine Adipose-Derived Stem Cells

    OpenAIRE

    Daniel Cebo

    2017-01-01

    Bovine adipose-derived stem cells were obtained from the subcutaneous abdominal adipose tissue. The cells were cultured by the modified tissue-explants method developed in our laboratory and then analyzed using optical microscopy and flow cytometry. These cells were able to replicate in our cell culture conditions. cell Flow cytometry showed that bovine adipose-derived stem cells expressed mesenchymal stem cell markers CD73 and CD90. Meanwhile haematopoietic markers CD45 and CD34 are absent f...

  14. Proteomic Analysis of Bovine Nucleolus

    Institute of Scientific and Technical Information of China (English)

    Amrutlal K.Patel; Doug Olson; Suresh K. Tikoo

    2010-01-01

    Nucleolus is the most prominent subnuclear structure, which performs a wide variety of functions in the eu-karyotic cellular processes. In order to understand the structural and functional role of the nucleoli in bovine cells,we analyzed the proteomie composition of the bovine nueleoli. The nucleoli were isolated from Madin Darby bo-vine kidney cells and subjected to proteomie analysis by LC-MS/MS after fractionation by SDS-PAGE and strongcation exchange chromatography. Analysis of the data using the Mascot database search and the GPM databasesearch identified 311 proteins in the bovine nucleoli, which contained 22 proteins previously not identified in theproteomic analysis of human nucleoli. Analysis of the identified proteins using the GoMiner software suggestedthat the bovine nueleoli contained proteins involved in ribosomal biogenesis, cell cycle control, transcriptional,translational and post-translational regulation, transport, and structural organization.

  15. Effect of the microenvironment and embryo density on developmental characteristics and gene expression profile of bovine preimplantative embryos cultured in vitro.

    Science.gov (United States)

    Hoelker, Michael; Rings, Franka; Lund, Qamaruddin; Ghanem, Nasser; Phatsara, Chirawath; Griese, Josef; Schellander, Karl; Tesfaye, Dawit

    2009-03-01

    The Well of the Well (WOW) system has been developed to culture embryos in small groups or to track the development of single embryos. In the present study, we aimed to examine the effects of the microenvironment provided by the WOW system and embryo density on developmental rates, embryo quality and preimplantative gene expression profile of the resulting embryos. Embryos cultured in a group of 16 reached the blastocyst stage at a significantly lower level than zygotes cultured in a group of 50 (22.2 vs 30.3%), whereas zygotes cultured in WOW were able to compensate against low embryo densities, reaching a blastocyst rate as high as embryos cultured in a group of 50 (31.3 vs 30.3%). Moreover, embryos derived from WOW culture did not differ in terms of differential cell counts and apoptotic cell index compared with controls. The gene expression analysis revealed 62 transcripts to be upregulated and 33 transcripts to be downregulated by WOW culture. Comparing the in vivo derived blastocysts with the blastocysts derived from WOW culture, and group culture, expression of ATP5A1, PLAC8 and KRT8 was more similar to the embryos derived from WOW culture, whereas expression of S100A10 and ZP3 genes was more similar to blastocysts cultured in a group. In conclusion, microenvironment as well as embryo density significantly affected developmental rates. While subsequent blastocysts did not differ in terms of differential cell counts and apoptotic cell index, significant differences were observed in terms of the relative abundance of transcripts in the resulting embryos.

  16. In vitro development rate of preimplantation rabbit embryos cultured with different levels of melatonin.

    Science.gov (United States)

    Mehaisen, Gamal Mohamed Kamel; Saeed, Ayman Moustafa

    2015-02-01

    This study aimed to investigate the effect of melatonin supplementation at different levels in culture medium on embryo development in rabbits. Embryos of 2-4 cells, 8-16 cells and morula stages were recovered from nulliparous Red Baladi rabbit does by laparotomy technique 24, 48 and 72 h post-insemination, respectively. Normal embryos from each stage were cultured to hatched blastocyst stages in either control culture medium (TCM-199 + 20% fetal bovine serum) or control supplemented with melatonin at 10(-3) M, 10(-6) M or 10(-9) M. No effect of melatonin was found on development of embryos recovered at 24 h post-insemination. The high level of melatonin at 10(-3) M adversely affected the in vitro development rates of embryos recovered at 48 h post-insemination (52 versus 86, 87 and 80% blastocyst rate; 28 versus 66, 78 and 59% hatchability rate for 10(-3) M versus 10(-9) M, 10(-6) M and control, respectively, P< 0.05). At the morula stage, melatonin at 10-3 M significantly increased the in vitro development of embryos (92% for 10(-3) M versus 76% for control, P < 0.05), while the hatchability rate of these embryos was not improved by melatonin (16-30% versus 52% for melatonin groups versus control, P < 0.05). Results show that a moderate level of melatonin (10(-6) M) may improve the development and hatchability rates of preimplantation rabbit embryos. The addition of melatonin at a 10-3 M concentration enhances the development of rabbit morulae but may negatively affect the development of earlier embryos. More studies are needed to optimize the use of melatonin in in vitro embryo culture in rabbits.

  17. Formation of nucleoli in interspecies nuclear transfer embryos derived from bovine, porcine, and rabbit oocytes and nuclear donor cells of various species.

    Science.gov (United States)

    Lagutina, Irina; Zakhartchenko, Valeri; Fulka, Helena; Colleoni, Silvia; Wolf, Eckhard; Fulka, Josef; Lazzari, Giovanna; Galli, Cesare

    2011-04-01

    The most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genes de novo in the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages.

  18. Viral infections and bovine mastitis: a review

    NARCIS (Netherlands)

    Wellenberg, G.J.; Poel, van der W.H.M.; Oirschot, van J.T.

    2002-01-01

    This review deals with the role of viruses in the aetiology of bovine mastitis. Bovine herpesvirus 1, bovine herpesvirus 4, foot-and-mouth disease virus, and parainfluenza 3 virus have been isolated from milk from cows with clinical mastitis. Intramammary inoculations of bovine herpesvirus 1 or

  19. Radioimmunoassay of bovine leukosis virus antibodies

    International Nuclear Information System (INIS)

    Franz, J.; Hampl, J.; Svoboda, I.; Granatova, M.; Hofirek, B.; Skrobak, F.

    1986-01-01

    A RIA method was developed for identifying the presence of serum antibodies to the bovine leukosis virus. The chosen procedure uses the ability of the virus antigen to bind to the solid phase of a polystyrene carrier. The method was compared with the ELISA method and with the pseudoneutralization and immunodiffusion tests. A high level of agreement was achieved between the RIA and the ELISA methods (95%). By its accuracy the RIA method proves superior to the immunodiffusion test. (author)

  20. Radioimmunoassay of bovine leukosis virus antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Franz, J; Hampl, J; Svoboda, I; Granatova, M; Hofirek, B; Skrobak, F

    1986-08-01

    A RIA method was developed for identifying the presence of serum antibodies to the bovine leukosis virus. The chosen procedure uses the ability of the virus antigen to bind to the solid phase of a polystyrene carrier. The method was compared with the ELISA method and with the pseudoneutralization and immunodiffusion tests. A high level of agreement was achieved between the RIA and the ELISA methods (95%). By its accuracy the RIA method proves superior to the immunodiffusion test.

  1. Relationship between growth hormone concentrations in bovine oocytes and follicular fluid and oocyte developmental competence

    Directory of Open Access Journals (Sweden)

    S Modina

    2009-08-01

    Full Text Available In the last few years, several works suggest that Growth Hormone (GH is involved in follicular development and oocyte maturation. These actions may reflect endocrine roles of pituitary GH and also account for local autocrine or paracrine activities of GH produced in reproductive tissue. This study was aimed to verify whether the developmental competence of bovine female gametes might be related to ovarian GH.We evaluated the localisation and distribution of GH in the cumulus oocytes complexes (COCs and the concentration of GH in the oocytes and in the follicular fluids (FF from ovaries classified on the basis of the follicles number. Oocytes retrieved from ovaries with more than 10 follicles of 2 to 5 mm in diameter (High ovaries, Hi show higher rate of maturation and blastocyst formation than those retrieved from ovaries with less than 10 follicles (Low ovaries, Lo. At the same time we measured Estrogen (E2 and Progesterone (P4 concentrations in FF, to relate oocytes quality, GH concentration and follicle health. GH localization in COCs and oocytes was performed by indirect immunofluorescence and its concentration within the ooplasm was evaluated by microspectrophotometer analysis. GH, E2 and P4 concentrations in FF were measured by an Enzyme Linked ImmunoSorbent assay (ELISA.We observed a positive, diffuse signal at cytoplasmic level in most of the cumulus cells, with no differences between COCs collected from Hi and Lo ovaries. On the contrary, GH level was significantly higher in the oocytes collected from Lo ovaries than in those recovered from Hi ovaries. Finally we found that also GH level in the FF was inversely related to the oocytes developmental capability. We suggest that the increase of GH in the oocytes and in the FF derived from Lo ovaries might be interpreted as attempt of the follicular environment to improve ovarian activity and in turn oocytes developmental competence in a autocrineparacrine manner. Moreover, E2, and P4 levels

  2. Embryonic stem cells derived from in vivo or in vitro-generated murine blastocysts display similar transcriptome and differentiation potential.

    Directory of Open Access Journals (Sweden)

    Rhodel K Simbulan

    Full Text Available The use of assisted reproductive technologies (ART such as in vitro fertilization (IVF has resulted in the birth of more than 5 million children. While children conceived by these technologies are generally healthy, there is conflicting evidence suggesting an increase in adult-onset complications like glucose intolerance and high blood pressure in IVF children. Animal models indicate similar potential risks. It remains unclear what molecular mechanisms may be operating during in vitro culture to predispose the embryo to these diseases. One of the limitations faced by investigators is the paucity of the material in the preimplantation embryo to test for molecular analysis. To address this problem, we generated mouse embryonic stem cells (mESC from blastocysts conceived after natural mating (mESCFB or after IVF, using optimal (KSOM + 5% O2; mESCKAA and suboptimal (Whitten's Medium, + 20% O2, mESCWM conditions. All three groups of embryos showed similar behavior during both derivation and differentiation into their respective mESC lines. Unsupervised hierarchical clustering of microarray data showed that blastocyst culture does not affect the transcriptome of derived mESCs. Transcriptomic changes previously observed in the inner cell mass (ICM of embryos derived in the same conditions were not present in mESCs, regardless of method of conception or culture medium, suggesting that mESC do not fully maintain a memory of the events occurring prior to their derivation. We conclude that the fertilization method or culture media used to generate blastocysts does not affect differentiation potential, morphology and transcriptome of mESCs.

  3. Transferência de blastocisto após descongelamento de embriões em mórula resultando em gestação gemelar: relato de caso Twin pregnancy after thawing of morula embryos and blastocyst transfer: case report

    Directory of Open Access Journals (Sweden)

    Luiz Eduardo T. Albuquerque

    1999-12-01

    Full Text Available A criopreservação de embriões em estágios mais tardios do desenvolvimento parece apresentar resultados satisfatórios. Com o objetivo de melhor testar a sobrevivência e o desenvolvimento de embriões, os mesmos foram criopreservados e descongelados em estadio de mórula ou blastocisto e deixados em cultura para que pudesse ser avaliada sua evolução natural. Dos 2 blastocistos e 5 mórulas congelados, 4 mórulas sobreviveram ao descongelamento, tendo sido transferidas em estadio de blastocisto, 24 horas depois. A transferência, realizada em paciente jovem, segundo casamento de homem vasectomizado há dez anos, resultou em gestação gemelar. O descongelamento de embriões em estadio de mórula e a observação in vitro da retomada de seu desenvolvimento até o estadio de blastocisto fornecem um parâmetro adicional na avaliação da qualidade do embrião e, provavelmente, melhore as taxas de gravidez.The cryopreservation of embryos in late developing stages seems to present satisfactory results. With the purpose of better testing the embryos' survival, they were cryopreserved in the morula or blastocyst stage, thawed and left in culture for 24 hours so that their natural evolution could be observed. Amongst the frozen 2 blastocysts and 5 morulas, 4 morulas survived the thawing process, being transferred as blastocysts 24 hours later. The transfer was performed in a young patient, second marriage of a ten-year vasectomized man and resulted in twin pregnancy. Thawing morula embryos and the in vitro observation of their development resumption until the blastocyst stage give us an additional parameter in the quality evaluation of the embryo and probably an improvement in pregnancy rates.

  4. Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos Melatonina no meio de maturação não melhorou as taxas de maturação dos ovócitos, de desenvolvimento embrionário e a fragmentação do DNA dos embriões bovinos

    Directory of Open Access Journals (Sweden)

    Luciana Takada

    2010-08-01

    Full Text Available Melatonin (MEL acts as a powerful scavenger of free radicals and direct gonadal responses to melatonin have been reported in the literature. Few studies, however, have evaluated the effect of MEL during in vitro maturation (IVM on bovine embryos. This study tested the addition of MEL to maturation medium (MM with no gonadotropins on nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos. Cumulus-oocyte complexes were aspirated from abattoir ovaries and cultured in MM (TCM-199 medium supplemented with 10% fetal calf serum - FCS at 39ºC and 5% CO2 in air. After 24 hours of culture in MM with 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH; 10-9 M MEL or 10-9 M MEL, 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH, the oocytes were stained with Hoechst 33342 to evaluate nuclear maturation rate. After in vitro fertilization and embryo culture, development rates were evaluated and the blastocysts were assessed for DNA damage by Comet assay. There was no effect of melatonin added to the MM, alone or in combination with gonadotropins, on nuclear maturation, cleavage and blastocyst rates. These rates ranged between 88% to 90%, 85% to 88% and 42% to 46%, respectively. The extent of DNA damage in embryos was also not affected by MEL supplementation during IVM. The addition of 10-9 M MEL to the MM failed to improve nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos, but was able to properly substitute for gonadotropins during IVM.Melatonin (MEL atua como um potente redutor de radicais livres. Efeito direto da MEL na função gonadal também foi observado. Existem poucos estudos relacionados ao efeito da MEL durante a maturação no desenvolvimento embrionário in vitro. Avaliou-se a adição de MEL no meio de maturação (sem gonadotrofinas nas taxas de maturação nuclear e de desenvolvimento embrionário e na incidência de fragmentação do DNA nos embriões produzidos in vitro

  5. Effects of heat stress on bovine preimplantation embryos produced in vitro.

    Science.gov (United States)

    Sakatani, Miki

    2017-08-19

    Summer heat stress decreases the pregnancy rate in cattle and has been thought to be associated with the early embryonic death caused by the elevation of maternal body temperature. In vitro cultures have been widely used for the evaluation of effects of heat stress on oocytes, fertilization, preimplantation, and embryonic development. Susceptibility to heat stress is present in developmental stages from oocytes to cleavage-stage (before embryonic gene activation, EGA) embryos, leading to a consequent decrease in developmental competence. On the other hand, advanced-stage embryos such as morula or blastocysts have acquired thermotolerance. The mechanism for the developmental stage-dependent change in thermotolerance is considered to be the accumulation of antioxidants in embryos in response to heat-inducible production of reactive oxygen species. The supplementation of antioxidants to the culture media has been known to neutralize the detrimental effects of heat stress. Besides, EGA could be involved in acquisition of thermotolerance in later stages of embryos. Morulae or blastocysts can repair heat-induced unfolded proteins or prevent DNA damage occurring in processes such as apoptosis. Therefore, embryo transfer (ET) that can bypass the heat-sensitive stage could be a good solution to improve the pregnancy rate under heat stress. However, frozen-thawed ET could not improve the pregnancy rate as expected. Frozen-thawed blastocysts were more sensitive to heat stress and showed less proliferation upon heat exposure, compared to fresh blastocysts. Therefore, further research is required to improve the reduction in pregnancy rates due to summer heat stress.

  6. Expression of proposed implantation marker genes CDX2 and HOXB7 in the blastocyst does not distinguish viable from non-viable human embryos

    DEFF Research Database (Denmark)

    Kirkegaard, Kirstine; Hindkjær, Johnny Juhl; Ingerslev, Hans Jakob

    2012-01-01

    expression differs between viable and non-viable embryos in both human and non-humans, suggesting transcriptome analysis of trophectoderm (TE) as a novel method of improving embryo selection. Potential candidate marker genes have been identified with array studies on animal blastocysts. The aim of this study...... was to investigate the expression of selected genes in human blastocysts in relation to the outcome of implantation. Materials and methods: Embryos from 10 oatients undergoing in vitro fertilization treatment were included in the project. A single blastocyst was chosen for biopsy on the morning of day 5 after oocyte...... of 15 key genes associated with developmental competence in animals were evaluated in high quality human embryos with monogenic or chromosomal disorders from a pre-implantation genetic disorder program. Triplicate cDNA amplifications for quantitative (q) RT-PCR were performed using pre-designed gene...

  7. Strategical control of cattle tick in the Milk Bovine: A Revision

    OpenAIRE

    Furlong, Jonh; EMBRAPA - Gado de Leite; Sales, Ronaldo de Oliveira; Universidade Federal do Ceará

    2013-01-01

    In this bibliographical revision the different types of controls used in the eradication of the bovine cattle tick are presented that to develop itself, it needs to pass a phase of its life in the animals. It is important to know that the carrapato of the bovines is different of the cattle tick of the equines. In this text the common cattle tick of the bovines will be argued only (Boophilus microplus), mainly of the milk bovines, whose way of combat is different of that it is made for cut bov...

  8. The role of the PI3K-Akt signaling pathway in the developmental competence of bovine oocytes.

    Directory of Open Access Journals (Sweden)

    Gabriella Mamede Andrade

    Full Text Available The ovarian follicle encloses oocytes in a microenvironment throughout their growth and acquisition of competence. Evidence suggests a dynamic interplay among follicular cells and oocytes, since they are constantly exchanging "messages". We dissected bovine ovarian follicles and recovered follicular cells (FCs-granulosa and cumulus cells and cumulus-oocyte complexes (COCs to investigate whether the PI3K-Akt signaling pathway impacted oocyte quality. Following follicle rupture, COCs were individually selected for in vitro cultures to track the follicular cells based on oocyte competence to reach the blastocyst stage after parthenogenetic activation. Levels of PI3K-Akt signaling pathway components in FCs correlated with oocyte competence. This pathway is upregulated in FCs from follicles with high-quality oocytes that are able to reach the blastocyst stage, as indicated by decreased levels of PTEN and increased levels of the PTEN regulators bta-miR-494 and bta-miR-20a. Using PI3K-Akt responsive genes, we showed decreased FOXO3a levels and BAX levels in lower quality groups, indicating changes in cell cycle progression, oxidative response and apoptosis. Based on these results, the measurement of levels of PI3K-Akt pathway components in FCs from ovarian follicles carrying oocytes with distinct developmental competences is a useful tool to identify putative molecular pathways involved in the acquisition of oocyte competence.

  9. Effect of Intracytoplasmic Sperm Injection (ICSI on Mouse Embryos Preimplantational Development

    Directory of Open Access Journals (Sweden)

    Claudia Cârstea

    2012-05-01

    Full Text Available It is known that the in vitro culture (IVC of preimplantation embryos is associated with changes in gene expression. It is however, not known if the method of fertilization affects the global pattern of gene expression. We compared the development of mouse blastocysts produced by intracytoplasmic sperm injection (ICSI versus blastocysts fertilized in vivo and cultured in vitro from the zygote stage (IVC. At the end of cultivation (96 hrs for blastocyst stage embryos, expanded blastocysts of each group were randomly selected, and ICM and total cells number were differentially stained. The total cell number of blastocysts was estimated by counting the total number of nuclei using DAPI staining. Cell number for inner cell mass (ICM was estimated by counting the OCT4 (POU5FL positive cells. Digitally recombined, composite images were analyzed using the Zeiss Axion Vision software and Zeiss Apotome. All 5–10 optical sections were divided using a standard grid over each layer to count all. Comparing the total cells and the ICM cells number, it appears that each method of fertilization has a unique pattern development. The developmental rate and the total cell number of the blastocyst were significantly lower in ICSI versus in vivo fertilized embryos which affect the embryonic developmental rate and the total cell number of blastocysts.

  10. Bovine Tuberculosis, A Zoonotic Disease

    Directory of Open Access Journals (Sweden)

    Tarmudji

    2008-12-01

    Full Text Available Bovine tuberculosis is caused by the infection of Mycobacterium tuberculosis var. bovis (M. bovis. This species is one of Mycobacterium tuberculosis complex, can infect wide range of hosts: cattle and other domesticated animals, wild mammals and humans (zoonotic. M. bovis bacterium from infected hosts can be transmitted to other susceptible animals and humans through respiratory excretes and secretion materials. Humans can be infected with M. bovis by ingested M. bovis contaminated animal products, unpasteurised milk from tuberculosis cows or through respiratory route of contaminated aerosol. Bovine tuberculosis at the first stage does not show any clinical sign but as the disease progress in the next stage which may take several months or years, clinical signs may arise, suh as: fluctuative body temperature, anorexia, lost body weight, coughing, oedema of lymph nodes, increased respiratory frequencies. Pathological lesion of bovine tuberculosis is characterised by the formation of granulomas (tubercles, in which bacterial cells have been localised, most in lymph nodes and pulmonum, but can occur in other organs. The granulomas usually arise in small nodules or tubercles appear yellowish either caseus, caseo-calcareus or calcified. In Indonesia, bovine tuberculosis occurred in dairy cattle since 1905 through the imported dairy cows from Holland and Australian. It was unfortunate that until recently, there were not many research and surveilances of bovine tuberculosis conducted in this country, so the distribution of bovine tuberculosis is unknown. Early serological diagnosis can be done on live cattle by means of tuberculin tests under field conditions. Confirmation can be done by isolation and identification of excreted and secreted samples from the slaughter house. Antibiotic treatment and vaccination were uneffective, therefore the effective control of bovine tuberculosis is suggested by tuberculin tests and by slaughtering the selected

  11. Efeito do transporte no desenvolvimento de embriões bovinos cultivados in vitro a fresco ou reaquecidos após vitrificação Effect of transportation on development of fresh or vitrified-warmed bovine embryos

    Directory of Open Access Journals (Sweden)

    Alessandra de Almeida Ramos

    2006-12-01

    bovine embryos, fresh or warmed, after submitted to different periods of transportation (6h-12h. Oocytes obtained from ovaries collected from slaughterhouse were matured, fertilized and cultured in vitro. After seven days, grades I and II blastocysts (according to IETS manual were selected and vitrified after exposition to PBS solution with 5% fetal calf serum (HM, added with 10% ethylene glycol (EG and 10% of dymetil sulfoxide (DMSO, for one minute, followed by HM solution with 20% EG and 20% DMSO, for 20 seconds. Embryos were loaded into open pulled straws (OPS and plunged into liquid nitrogen. Warming was performed at 39ºC by embryo exposure to decreasing concentration of sucrose (0.25 and 0.15M, for five minutes in each step. The warmed embryos were distributed in three groups: V0: in vitro cultured after warmed; V6: embryos loaded into straws and kept for 6 hours at 35ºC, before in vitro culture; and V12: embryos loaded into straws and kept for 12 hours at 35ºC, before in vitro culture. Each group was evaluated by control groups of fresh embryos (C0, C6 and C12, respectively. The embryos were co-cultured with cumulus cells in TCM-199 micro droplets added with SFB. Re-expansion and hatching rates after 48 hours in culture were evaluated and results were compared by the Chi-square test. Re-expanded rates among groups V0, V6 and V12 as well as hatching rates among vitrified groups and among control groups did not differ. However, hatching rates were different between vitrified groups and their respective controls. The satisfactory rates of hatching suggest that it is possible to transport warmed and fresh in vitro produced embryos for periods up to 12 hours.

  12. Late effects of 60Co γ radiation on the bovine oocyte as reflected by oocyte survival, follicular development, and reproductive performance

    International Nuclear Information System (INIS)

    Erickson, B.H.; Reynolds, R.A.; Murphree, R.L.

    1976-01-01

    Long-term effects of ionizing radiation on the oocyte of a long-lived mammal have not been determined. Therefore, 280 15- to 18-month-old grade Herefords were partitioned among five groups and irradiated with either 0, 200, 300, 400, or 300 + 300 (55 days between fractions) R of 60 Co γ radiation at exposure rates of 0.69 or 0.78 R/min. Dose to the ovaries was 37 +- 3 percent of the dose measured in air. Germ cell effects were assessed in the survivors through (1) counts of germ cells and follicles in serially sectioned ovaries, (2) incidence of ovarian abnormalities, and (3) reproductive performance. Neither germ cell nor follicular counts were significantly affected by irradiation (P greater than 0.05). Incidence of ovarian abnormalities was not altered nor was reproductive performance or quality of offspring. It was concluded, therefore, that a near lethal postpuberally applied dose of γ radiation would not affect the reproductive capacity of the bovine female

  13. SELECTIVITY PROFILE OF SOME RECENT MUSCARINIC ANTAGONISTS IN BOVINE AND GUINEA-PIG TRACHEA AND HEART

    NARCIS (Netherlands)

    ROFFEL, AF; HAMSTRA, JJ; ELZINGA, CRS; ZAAGSMA, J

    1994-01-01

    The functional affinities of some recently developed subtype-selective muscarinic antagonists towards bovine tracheal smooth muscle muscarinic M(3) receptors were established and compared to binding affinities for bovine cardiac M(2) and functional affinities for guinea-pig tracheal smooth muscle

  14. Production and manipulation of bovine embryos: techniques and terminology.

    Science.gov (United States)

    Machaty, Z; Peippo, J; Peter, A

    2012-09-15

    There are numerous publications regarding bovine embryos, ranging from descriptions of their appearance and development to emerging techniques in the field of assisted reproductive technology. Concurrently, several specialized terms have been developed to describe the bovine embryo. The purpose of the current review is two-fold; it is primarily to describe techniques involved in the in vivo and in vitro production of bovine embryos and their manipulation, and secondarily to summarize specialized terms used in these processes. The intention is not to review these techniques in detail, but instead to provide salient points and current knowledge regarding these techniques, with a focus on terminology. The first review dealt with classical and contemporary terminology used to describe morphologic aspects of ovarian dynamics in cattle. Subsequently, the terms and current understanding of processes involved in preattachment bovine embryos were described in the second review. As the third article in a series, this mini-review is focused on defining the production, manipulation, and transfer of bovine preattachment embryos. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Epidemiology of Bovine Mastitis in Cows of Dharwad District

    Science.gov (United States)

    Kurjogi, Mahantesh M.; Kaliwal, Basappa B.

    2014-01-01

    Bovine mastitis is very common in cows of both developed and developing countries. The prevalence of clinical and subclinical mastitis (SCM) varies from region to region. Hence, the present study was carried out to determine the prevalence of mastitis using three diagnostic tests by considering different risk factors like age, lactation, breed, season, quarters, and herd. The results showed that surf field mastitis test (SFMT) is the most sensitive test for diagnosis of bovine mastitis, the older age and cows with later part of lactation period were more prone to bovine mastitis, and exotic breeds like Holstein freshen (HF) were more susceptible to bovine mastitis. The highest incidence of mastitis was recorded in monsoon season. The prevalence of subclinical and clinical mastitis was more in single and two quarters, respectively, and the rate of bovine mastitis was more in unorganized herds. The study concluded that SCM is directly associated with age, lactation period, and environmental factors of the cow and clinical mastitis is more associated with breed of the cow and environmental conditions. PMID:27382623

  16. Bovine Mastitis Resistance: Novel Quantitative Trait Loci and the Role of Bovine Mammary Epithelial Cells

    OpenAIRE

    Kurz, Jacqueline P.

    2018-01-01

    Bovine mastitis, or inflammation of the mammary gland, has substantial economic and animal welfare implications. A genetic basis for mastitis resistance traits is recognized and can be used to guide selective breeding programs. The discovery of regions of the genome associated with mastitis resistance, and knowledge of the underlying molecular mechanisms responsible, can facilitate development of efficient mastitis control and therapeutic strategies. The objectives of this dissertation resear...

  17. Cryostorage duration does not affect pregnancy and neonatal outcomes: a retrospective single-centre cohort study of vitrified-warmed blastocysts.

    Science.gov (United States)

    Ueno, Satoshi; Uchiyama, Kazuo; Kuroda, Tomoko; Yabuuchi, Akiko; Ezoe, Kenji; Okimura, Tadashi; Okuno, Takashi; Kobayashi, Tamotsu; Kato, Keiichi

    2018-06-01

    A retrospective cohort study of 8736 autologous single vitrified-warmed blastocyst transfer cycles was conducted in a single centre to investigate the effect of cryostorage on clinical and neonatal outcomes. Cryostorage duration was classified into three groups: (A) 0-2 months (n = 4702); (B) 2-13 months (n = 2853) and (C) 13-97 months (n = 1181). Blastocysts were vitrified using the Cryotop method. No significant differences were observed in live birth rates: (A) 37.3%; (B) 34.9%; (C) (35.2%). Gestational period was significantly shorter in group C: (A) 38.7 ± 1.8; (B) 38.6 ± 1.6; (C) 38.1 ± 1.7; P limitation of this study was that maximum storage duration was 8 years; most blastocysts were in cryostorage for much shorter periods. Long-term storage of blastocysts that are vitrified using an open device vitrification system has no negative effect on pregnancy and neonatal outcomes. Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  18. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study

    Directory of Open Access Journals (Sweden)

    Yang Zhihong

    2012-05-01

    Full Text Available Abstract Background Single embryo transfer (SET remains underutilized as a strategy to reduce multiple gestation risk in IVF, and its overall lower pregnancy rate underscores the need for improved techniques to select one embryo for fresh transfer. This study explored use of comprehensive chromosomal screening by array CGH (aCGH to provide this advantage and improve pregnancy rate from SET. Methods First-time IVF patients with a good prognosis (age Results For patients in Group A (n = 55, 425 blastocysts were biopsied and analyzed via aCGH (7.7 blastocysts/patient. Aneuploidy was detected in 191/425 (44.9% of blastocysts in this group. For patients in Group B (n = 48, 389 blastocysts were microscopically examined (8.1 blastocysts/patient. Clinical pregnancy rate was significantly higher in the morphology + aCGH group compared to the morphology-only group (70.9 and 45.8%, respectively; p = 0.017; ongoing pregnancy rate for Groups A and B were 69.1 vs. 41.7%, respectively (p = 0.009. There were no twin pregnancies. Conclusion Although aCGH followed by frozen embryo transfer has been used to screen at risk embryos (e.g., known parental chromosomal translocation or history of recurrent pregnancy loss, this is the first description of aCGH fully integrated with a clinical IVF program to select single blastocysts for fresh SET in good prognosis patients. The observed aneuploidy rate (44.9% among biopsied blastocysts highlights the inherent imprecision of SET when conventional morphology is used alone. Embryos randomized to the aCGH group implanted with greater efficiency, resulted in clinical pregnancy more often, and yielded a lower miscarriage rate than those selected without aCGH. Additional studies are needed to verify our pilot data and confirm a role for on-site, rapid aCGH for IVF patients contemplating fresh SET.

  19. Time-lapse cinematography of dynamic changes occurring during in vitro development of human embryos.

    Science.gov (United States)

    Mio, Yasuyuki; Maeda, Kazuo

    2008-12-01

    The purpose of this study was to clarify developmental changes of early human embryos by using time-lapse cinematography (TLC). For human ova, fertilization and cleavage, development of the blastocyst, and hatching, as well as consequent changes were repeatedly photographed at intervals of 5-6 days by using an inverse microscope under stabilized temperature and pH. Photographs were taken at 30 frames per second and the movies were studied. Cinematography has increased our understanding of the morphologic mechanisms of fertilization, development, and behavior of early human embryos, and has identified the increased risk of monozygotic twin pregnancy based on prolonged incubation in vitro to the blastocyst stage. Using TLC, we observed the fertilization of an ovum by a single spermatozoon, followed by early cleavages, formation of the morula, blastocyst hatching, changes in the embryonic plates, and the development of monozygotic twins from the incubated blastocysts.

  20. A monoclonal antibody to pestviruses in bovine and ovine sera

    International Nuclear Information System (INIS)

    Mweene, A.S.

    1991-01-01

    An enzyme-linked immunoabsorbent assay (ELISA) has been developed to defeat antibodies to pestviruses in bovine and ovine sera. Single sera from 211 cattle and 22 sheep from 7 different farms were tested using ELISA and Serum Neutralisation Test (SNT). 17 Monoclonal antibodies (Mabs) directed against P80, gp48 and gp53 were tested for ability to coat ELISA plates and capture the bovine viral diarrhea antigen. 5 mabs(WB 103, WB, 105, WB 112 against P80 kDa protein, WB 210 and WB 214 directed against gp48 and gp 53 kDa protein. Specific antibody to BVDV was detected by rabbit anti-bovine and anti-ovine IgG antisera. The quantitative correlation between two tests was good

  1. Control of Bovine Mastitis: Old and Recent Therapeutic Approaches.

    Science.gov (United States)

    Gomes, Fernanda; Henriques, Mariana

    2016-04-01

    Mastitis is defined as the inflammatory response resulting of the infection of the udder tissue and it is reported in numerous species, namely in domestic dairy animals. This pathology is the most frequent disease of dairy cattle and can be potentially fatal. Mastitis is an economically important pathology associated with reduced milk production, changes in milk composition and quality, being considered one of the most costly to dairy industry. Therefore, the majority of research in the field has focused on control of bovine mastitis and many efforts are being made for the development of new and effective anti-mastitis drugs. Antibiotic treatment is an established component of mastitis control programs; however, the continuous search for new therapeutic alternatives, effective in the control and treatment of bovine mastitis, is urgent. This review will provide an overview of some conventional and emerging approaches in the management of bovine mastitis' infections.

  2. Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells.

    Directory of Open Access Journals (Sweden)

    Hongmei Wang

    Full Text Available BACKGROUND: Although it is known that RNA interference (RNAi targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV, it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge. PRINCIPAL FINDING: Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2, VP3 (RNAi-VP3, or VP4 (RNAi-VP4 of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID(50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h. CONCLUSION: RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.

  3. Diprosopia em bovino Bovine diprosopus

    Directory of Open Access Journals (Sweden)

    I.T. Rotta

    2008-04-01

    Full Text Available This work describes a malformation in one newborn female bovine, with two faces and two skull fused, showing one single head. Duplications of the nasal and oral structures, tetraofthalmy, two brains, one single cerebellum, and pons were observed. The right thyroid was hypertrophic and the other organs had normal morphology. Every change observed in this case was compatibles with diprosopus.

  4. Diprosopia em bovino Bovine diprosopus

    OpenAIRE

    I.T. Rotta; M.B.A.M. Torres; R.G. Motta

    2008-01-01

    This work describes a malformation in one newborn female bovine, with two faces and two skull fused, showing one single head. Duplications of the nasal and oral structures, tetraofthalmy, two brains, one single cerebellum, and pons were observed. The right thyroid was hypertrophic and the other organs had normal morphology. Every change observed in this case was compatibles with diprosopus.

  5. The value of blastocyst culture on preimplantation genetic diagnosis%囊胚培养在植入前遗传学诊断中的价值

    Institute of Scientific and Technical Information of China (English)

    偶健; 王玮; 马燕琳; 周知; 丁洁; 王馥新; 段程颖; 李林江; 郑爱燕

    2015-01-01

    Objective To estimate the value of blastocyst culture for preimplantation genetic diagnosis (PGD).Methods Day 3 embryos were biopsied and analyzed with fluorescence in situ hybridization (FISH) technique.Embryos with normal FISH results were cultured into blastocysts,and the ones with better morphology scores were transferred.Fourteen embryos with abnormal FISH results were cultured into blastocysts.Part of the cells taken from the blastocysts were amplified by whole genomic amplification (WGA) and assessed by array-based comparative genomic hybridization (array-CGH) analysis.Results Six blastocysts with normal FISH results were transferred in 5 cycles.Four healthy babies of 3 cycles were delivered.Another one was a singleton pregnancy but with embryo growth arrest,whose villus karyotype was normal.Fourteen embryos with abnormal FISH results were cultured into blastocysts and analyzed by array-CGH.Six blastocysts were normal by array-CGH.Conclusion FISH combined with blastocyst culture may further ensure the accuracy of PGD result.Detection at the blastocyst stage can avoid false positive results and mosaic interferences on Day 3 stage and are therefore more authentic.%目的 探讨囊胚培养在植入前遗传学诊断(preimplantation genetic diagnosis,PGD)中的应用价值.方法 受精后第3天(Day 3)行胚胎活检,进行荧光原位杂交(fluorescent in situ hybridization,FISH).对于诊断为正常的胚胎,培养到囊胚阶段后选择形态评分优良的囊胚进行移植;对于诊断为异常的胚胎,有14个培养到囊胚阶段,各取其一部分细胞用于全基因组扩增(whole genomic amplification,WGA),将扩增后的DNA用微阵列比较基因组杂交(array-based comparative genomic hybridization,arrayCGH)进行再次检测.结果 FISH诊断为正常的6个囊胚进行了5个周期的胚胎移植,3个周期成功生育了4个健康婴儿,1个周期单胎妊娠见胚囊后流产,绒毛染色体检测为正常核型.FISH诊断为异常的14

  6. Development of a HS-SPME-GC-MS/MS Method for the Quantitation of Thymol and Carvacrol in Bovine Matrices and To Determine Residue Depletion in Milk and Tissues.

    Science.gov (United States)

    Armorini, Sara; Yeatts, James E; Mullen, Keena A E; Mason, Sharon E; Mehmeti, Elmira; Anderson, Kevin L; Washburn, Steve P; Baynes, Ronald E

    2016-10-11

    Thymol and carvacrol may be present in several phytoceutical products but there are no well-defined methods to measure these compounds in meat and milk from treated animals. U.S. regulatory authorities deem their presence as an adulteration of food. A rapid and sensitive HS-SPME-GC-MS/MS method was developed for the detection of thymol and carvacrol in bovine milk, plasma, liver, kidney, and fat. Inter- and intraday precision values were all less than 15.7 and 20.2% for thymol and carvacrol, respectively. The accuracy was in ranges of 69.9-111.8% for thymol and 74.0-119.2% for carvacrol. With the exception of fat tissue, stability studies showed that both compounds are stable over a 2 month period. A pilot pharmacokinetic study was conducted to evaluate the developed analytical method and to provide initial estimates of thymol and carvacrol depletion in plasma, milk, and several tissues. Treatment of lactating dairy cattle with phytoceutical products containing these substances resulted in low but measurable residue levels at 96 h for liver and 36 h for milk with very short apparent plasma and milk half-lives (<3.0 h).

  7. Human exposure to bovine polyomavirus: a zoonosis

    Energy Technology Data Exchange (ETDEWEB)

    Parry, J V; Gardner, S D

    1986-01-01

    A competitive-type solid phase radioimmunoassay (RIA) was developed for the detection of antibody to bovine polyomavirus. Comparison of RIA and counter-immunoelectrophoresis (CIE) results on 273 cattle sera indicated that both techniques were detecting antibody of like specificity. Human sera from 256 blood donors, 219 people recently vaccinated against polio, rubella or rabies, 50 immunosuppressed patients and 472 people with various occupational exposure to cattle were tested for antibody to bovine polyomavirus, the foetal rhesus monkey kidney strain, (anti-FRKV) by RIA. Apart from one blood donor and one of 108 rabies vaccinees only those in close contact with cattle possessed anti-FRKV. Compared with 62 per cent seropositive in the natural hosts, cattle, 71 per cent of veterinary surgeons, 50 per cent of cattle farmers, 40 per cent of abattoir workers, 16 per cent of veterinary institute technical staff and 10 per cent of veterinary students were anti-FRKV positive. Our findings indicate that the theoretical hazard of FRKV infection from undetected contamination of current tissue culture derived vaccines may, in practice, be remote. Proposed wider use of primate kidney cells as substrates for new vaccines may increase this risk.

  8. Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the oosight imaging system.

    Science.gov (United States)

    Kim, Eun Young; Park, Min Jee; Park, Hyo Young; Noh, Eun Ji; Noh, Eun Hyung; Park, Kyoung Sik; Lee, Jun Beom; Jeong, Chang Jin; Riu, Key Zung; Park, Se Pill

    2012-08-01

    In somatic cell nuclear transfer (SCNT) procedures, exquisite enucleation of the recipient oocyte is critical to cloning efficiency. The purpose of this study was to compare the effects of two enucleation systems, Hoechst staining and UV irradiation (hereafter, irradiation group) and Oosight imaging (hereafter, Oosight group), on the in vitro production of bovine SCNT embryos. In the Oosight group, the apoptotic index (2.8 ± 0.5 vs. 7.3 ± 1.2) was lower, and the fusion rate (75.6% vs. 62.9%), cleavage rate (78.0% vs. 63.7%), blastocyst rate (40.2% vs. 29.2%), and total cell number (128.3±4.8 vs. 112.2 ± 7.6) were higher than those in the irradiation group (all p<0.05). The overall efficiency after SCNT was twice as high in the Oosight group as that in the irradiation group (p<0.05). The relative mRNA expression levels of Oct4, Nanog, Interferon-tau, and Dnmt3A were higher and those of Caspase-3 and Hsp70 were lower in the Oosight group compared with the irradiation group (p<0.05). This is the first report to show the positive effect of the Oosight imaging system on molecular gene expression in the SCNT embryo. The Oosight imaging system may become the preferred choice for enucleation because it is less detrimental to the developmental potential of bovine SCNT embryos.

  9. Assessment of swim-up and discontinuous density gradient in sperm sex preselection for bovine embryo production

    Directory of Open Access Journals (Sweden)

    A.C Lucio

    2012-06-01

    Full Text Available The purpose of this work was to associate the modified swim-up method with centrifugation in density gradient for the separation of X-bearing spermatozoa. Sperm viability and integrity were evaluated through the Trypan Blue/Giemsa staining method. Quality control of centrifuged spermatozoa was performed in in vitro produced embryos. The results were validated by the sex ratio of in vitro produced embryos using PCR by Y- specific sequences present in bovine male genomic DNA. After determining genetic sex of in vitro produced embryos, the results showed difference (P<0.05 in deviation of sex ratio when comparing the control group (45.2% females with the other spermatozoa selection procedures (60.6% females (P<0.05. The sperm selection methods are capable of selecting X-bearing spermatozoa without compromising the spermatozoa fertility (cleavage and blastocyst rates, 70% and 26%, respectively and were considered relevant methods to be introduced in bovine in vitro produced embryo programs.

  10. Comparative validation using quantitative real-time PCR (qPCR and conventional PCR of bovine semen centrifuged in continuous density gradient

    Directory of Open Access Journals (Sweden)

    M.V. Resende

    2011-06-01

    Full Text Available The objective of the present study was to determine the sperm enrichment with X-bearing spermatozoa, after one centrifugation in a Percoll or OptiPrep continuous density gradient, using quantitative real-time polymerase chain reaction (qPCR of sperm DNA and resultant in vitro-produced bovine embryos by PCR. Frozen/thawed sperm was layered on density gradients and the tubes were centrifuged. Supernatants were gently aspirated and the sperm recovered from the bottom of the tubes. Cleavage and blastocyst rates were determined through in vitro production of embryos and PCR was performed to identify the embryos' genetic sex. A difference in blastocyst rate was found in the Percoll treatment compared to OptiPrep (P<0.05. The percentage of female embryos in the Percoll and OptiPrep groups was 62.0% and 47.1%, respectively. These results were confirmed by qPCR of spermatozoa DNA and underestimation was seen only in the Percoll group. It was possible to sexing sperm using simple approach.

  11. Influence of irradiation (Co60) in pre-implant rabbits embryos: effect on blastocyst diameters and embryos smaller than 2 mm

    International Nuclear Information System (INIS)

    Approbato, Mario S.; Oliveira Moura, Katia K.V. de; Souza Florencio, Rodopiano de; Cunha Junior, Carlos; Garcia, Ricardo; Faria, Renato S.; Benedetti, Leonardo N.; Goulart, Flamarion B.

    1995-01-01

    We studied the effect of ionizing irradiation on 12 New Zealand rabbits (65 embryos), in three different times: at match time (zero hour), two days after and four days after, with two different irradiation doses, 5 c Gy and 10 c Gy. Six rabbits (36 blastocysts) were used as controls. The matching instant was the zero hour. Exactly six days after (± 60 minutes) the embryos of each rabbit was picked up by flushing the uterus with culture media. The embryos were fixed in methanol for 48 hours, and colored with acid Mayer hematoxylin. The following embryos parameters were studied: diameter growth; percentage of embryos smaller than 2 mm. We observed that only the irradiation time influenced the blastocysts diameter (no irradiation dose). There was no relation between percentage of embryos smaller than 2 mm and the irradiation. (author)

  12. Monozygotic twinning after in vitro fertilization/intracytoplasmic sperm injection treatment is not related to advanced maternal age, intracytoplasmic sperm injection, assisted hatching, or blastocyst transfer

    Directory of Open Access Journals (Sweden)

    Dennis Wu

    2014-09-01

    Conclusion: Until definite conclusions are drawn from larger trials, patients receiving IVF should not be overly concerned about the increase in MZT risk when proceeding to various assisted reproductive procedures (i.e., ICSI, AH, and blastocyst transfer. However, there is some evidence that the incidence of monochorionic–monoamniotic twins may be significantly increased after IVF/ICSI cycles. Patients should be informed about the possible obstetric complications regarding this rare type of MZT.

  13. Comparison of male chimeric mice generated from microinjection of JM8.N4 embryonic stem cells into C57BL/6J and C57BL/6NTac blastocysts.

    Science.gov (United States)

    Fielder, Thomas J; Yi, Charles S; Masumi, Juliet; Waymire, Katrina G; Chen, Hsiao-Wen; Wang, Shuling; Shi, Kai-Xuan; Wallace, Douglas C; MacGregor, Grant R

    2012-12-01

    To identify ways to improve the efficiency of generating chimeric mice via microinjection of blastocysts with ES cells, we compared production and performance of ES-cell derived chimeric mice using blastocysts from two closely related and commonly used sub-strains of C57BL/6. Chimeras were produced by injection of the same JM8.N4 (C57BL/6NTac) derived ES cell line into blastocysts of mixed sex from either C57BL/6J (B6J) or C57BL/6NTac (B6NTac) mice. Similar efficiency of production and sex-conversion of chimeric animals was observed with each strain of blastocyst. However, B6J chimeric males had fewer developmental abnormalities involving urogenital and reproductive tissues (1/12, 8%) compared with B6NTac chimeric males (7/9, 78%). The low sample size did not permit determination of statistical significance for many parameters. However, in each category analyzed the B6J-derived chimeric males performed as well, or better, than their B6NTac counterparts. Twelve of 14 (86%) B6J male chimeras were fertile compared with 6 of 11 (55%) B6NTac male chimeras. Ten of 12 (83%) B6J chimeric males sired more than 1 litter compared with only 3 of 6 (50%) B6NTac chimeras. B6J male chimeras produced more litters per productive mating (3.42 ± 1.73, n = 12) compared to B6NTac chimeras (2.17 ± 1.33, n = 6). Finally, a greater ratio of germline transmitting chimeric males was obtained using B6J blastocysts (9/14; 64%) compared with chimeras produced using B6NTac blastocysts (4/11; 36%). Use of B6J host blastocysts for microinjection of ES cells may offer improvements over blastocysts from B6NTac and possibly other sub-strains of C57BL/6 mice.

  14. Female age, serum antimüllerian hormone level, and number of oocytes affect the rate and number of euploid blastocysts in in vitro fertilization/intracytoplasmic sperm injection cycles.

    Science.gov (United States)

    La Marca, Antonio; Minasi, Maria Giulia; Sighinolfi, Giovanna; Greco, Pierfrancesco; Argento, Cindy; Grisendi, Valentina; Fiorentino, Francesco; Greco, Ermanno

    2017-11-01

    To study the relative role of female age and ovarian reserve, measured through serum antimüllerian hormone (AMH) in determining the rate and number of euploid blastocysts in in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles. Retrospective analysis of cycles performed in 2014-2015. Tertiary referral IVF center. A total of 578 infertile couples undergoing IVF/ICSI and preimplantation genetic screening (PGS) analysis. All embryos were cultured and biopsied at the blastocyst stage. The method involved whole-genome amplification followed by array comparative genome hybridization. Serum AMH was measured by means of the modified Beckman Coulter AMH Gen II assay. The rate and number of euploid blastocysts and their correlation with ovarian reserve and response to stimulation. The mean (±SD) age of patients was 37.6 ± 4.1 years, and the mean number of blastocysts per patient was 3.1 ± 2. The total number of blastocysts available to the analysis was 1,814, and 36% of them were euploid after PGS. Age and serum AMH were significantly and independently related to the rate of euploid blastocysts available for patients. As an effect of the cohort size, the number of mature oocytes positively affected the total number of euploid blastocysts per patient. A strong positive age-independent relationship between AMH level and the rate of euploid blastocysts was found. This confirms that the measurement of ovarian reserve by means of AMH has high relevance when counseling infertile patients. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  15. Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration

    DEFF Research Database (Denmark)

    Vajta, G.; Holm, P.; Greve, T.

    1996-01-01

    %) and no hatching of these embryos was observed. In the second experiment, Day 7 expanded blastocysts were vitrified using PBS, PBS + albumin, TCM199 and TCM 199 + calf serum as holding media. No differences in re-expansion and hatching rates were seen. However, when incubation with the concentrated cryoprotectant......The aim of this work was to investigate the possibilities of simplification, and to outline the limits of application, of a vitrification method for cow embryos. Morulae and blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen...... and developmental stage (Day 5 compacted morulae, Day 6 early blastocysts, Days 6 and 7 blastocysts, Day 7 expanded blastocysts and Day 8 hatched blastocysts) as well as Days 7 and 5 blastocysts previously subjected to partial zona dissection were vitrified. After thawing, the re-expansion rates of blastocysts...

  16. In vitro fertilization outcomes after fresh and frozen blastocyst transfer in South Asian compared with Caucasian women.

    Science.gov (United States)

    Shah, Meera Sridhar; Caballes, Marissa; Lathi, Ruth Bunker; Baker, Valerie Lynn; Westphal, Lynn Marie; Milki, Amin A

    2016-06-01

    To study pregnancy outcomes between South Asian and Caucasian women undergoing frozen blastocyst transfer cycles. Retrospective cohort study. Not applicable. Caucasian and South Asian patients undergoing frozen blastocyst transfer between January 2011 and December 2014. Not applicable. Live birth rate. A total of 196 Caucasian and 117 South Asian women were included in our study. Indians were on average 2.2 years younger than Caucasian women (34.9 vs. 37.1 years), and were more likely to be nulliparous (59% vs. 43%). All other baseline characteristics were similar. In women undergoing their first frozen ET cycle, implantation rate (49% vs. 47%), clinical pregnancy rate (PR; 54% vs. 49%), and live birth rate (43% vs. 43%) were similar between South Asians and Caucasians, respectively. In patients who underwent a prior fresh blastocyst transfer, the live birth rate was significantly lower in South Asian versus Caucasian women (21% vs. 37%). Our data demonstrate that IVF outcomes are better in frozen versus fresh cycles among South Asian women. The IVF clinics may wish to consider these findings when counseling South Asian patients about the timing of ET. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  17. Hepes na produção de embriões bovinos in vitro Hepes on in vitro production of bovine embryos

    Directory of Open Access Journals (Sweden)

    Marcelo Marcos Montagner

    2000-06-01

    of pH changes in maturation and embryo development media, buffered with different HEPES concentrations. Initially, the effect of different concentrations of HEPES (0, 12.5 and 25.0mM on the variation of pH in the maturation (modified TCM-199 and embryonic development (modified KSOM media was evaluated at room temperature (25ºC and in an atmosphere of 5% CO2 in air at 39ºC. In another experiment, the effect of HEPES on in vitro oocyte maturation was determined. Oocytes were maturated in TCM-199 modified either with 25.0mM of HEPES (HEPES group; n = 137 or without HEPES (control group; n = 142, performing 7 replicates and evaluating the rate of blastocyst. In this study, the medium used for fertilization was Fert-TALP while for embryo development was KSOM with 10% of fetal bovine serum with monolayer of oviduct epithelial cells. A third experiment was designed to determine the effect of HEPES on embryo development. The zygotes were divided in two groups and co-incubated with oviduct epithelial cells in modified KSOM with 10% of fetal bovine serum without HEPES (n = 95 or with 25.0mM of HEPES (n = 92. For this experiment, it was used embryos with two or more cells and the embryo development was considered from cleavage to expanded blastocyst (Bx, 7 and 9 days after insemination. The oocytes and embryos were incubated at temperature of 39ºC, an atmosphere containing 5% CO2 in air and saturated humidity. The media with 25.0mM of HEPES were more efficient in minimizing the range of pH than those with 12.5mM or without HEPES. To determine the effect of HEPES during in vitro oocyte maturation, the percentage of Bl considered either the total number of oocytes or the total number of cleavages was higher in the HEPES group (21.9% or 42.9%, respectively than those obtained in the control group (10.56% or 16.67%, respectively. When HEPES was added to embryo culture medium, the percentage of Bx (45.65% was higher than that obtained in medium without HEPES (11.58%; p<0.01. The

  18. Proteomic analyses of host and pathogen responses during bovine mastitis.

    Science.gov (United States)

    Boehmer, Jamie L

    2011-12-01

    The pursuit of biomarkers for use as clinical screening tools, measures for early detection, disease monitoring, and as a means for assessing therapeutic responses has steadily evolved in human and veterinary medicine over the past two decades. Concurrently, advances in mass spectrometry have markedly expanded proteomic capabilities for biomarker discovery. While initial mass spectrometric biomarker discovery endeavors focused primarily on the detection of modulated proteins in human tissues and fluids, recent efforts have shifted to include proteomic analyses of biological samples from food animal species. Mastitis continues to garner attention in veterinary research due mainly to affiliated financial losses and food safety concerns over antimicrobial use, but also because there are only a limited number of efficacious mastitis treatment options. Accordingly, comparative proteomic analyses of bovine milk have emerged in recent years. Efforts to prevent agricultural-related food-borne illness have likewise fueled an interest in the proteomic evaluation of several prominent strains of bacteria, including common mastitis pathogens. The interest in establishing biomarkers of the host and pathogen responses during bovine mastitis stems largely from the need to better characterize mechanisms of the disease, to identify reliable biomarkers for use as measures of early detection and drug efficacy, and to uncover potentially novel targets for the development of alternative therapeutics. The following review focuses primarily on comparative proteomic analyses conducted on healthy versus mastitic bovine milk. However, a comparison of the host defense proteome of human and bovine milk and the proteomic analysis of common veterinary pathogens are likewise introduced.

  19. A dynamic model to calculate cadmium concentrations in bovine tissues from basic soil characteristics

    International Nuclear Information System (INIS)

    Waegeneers, Nadia; Ruttens, Ann; De Temmerman, Ludwig

    2011-01-01

    A chain model was developed to calculate the flow of cadmium from soil, drinking water and feed towards bovine tissues. The data used for model development were tissue Cd concentrations of 57 bovines and Cd concentrations in soil, feed and drinking water, sampled at the farms were the bovines were reared. Validation of the model occurred with a second set of measured tissue Cd concentrations of 93 bovines of which age and farm location were known. The exposure part of the chain model consists of two parts: (1) a soil-plant transfer model, deriving cadmium concentrations in feed from basic soil characteristics (pH and organic matter content) and soil Cd concentrations, and (2) bovine intake calculations, based on typical feed and water consumption patterns for cattle and Cd concentrations in feed and drinking water. The output of the exposure model is an animal-specific average daily Cd intake, which is then taken forward to a kinetic uptake model in which time-dependent Cd concentrations in bovine tissues are calculated. The chain model was able to account for 65%, 42% and 32% of the variation in observed kidney, liver and meat Cd concentrations in the validation study. - Research highlights: → Cadmium transfer from soil, drinking water and feed to bovine tissues was modeled. → The model was based on 57 bovines and corresponding feed and soil Cd concentrations. → The model was validated with an independent data set of 93 bovines. → The model explained 65% of variation in kidney Cd in the validation study.

  20. Impairments of mecA gene detection in bovine Staphylococcus spp.

    Directory of Open Access Journals (Sweden)

    Dayanne Araújo de Melo

    2014-09-01

    Full Text Available Staphylococcus aureus antimicrobial resistance, especially to beta-lactams, favors treatment failures and its persistence in herd environment. This work aimed to develop a more specific primer for mecA gene detection based on the comparison of the conserved regions from distinct host origins and also investigated the presence of homologue mecA LGA251 in bovine strains. A total of 43 Staphylococcus spp. were included in this study, comprising 38 bovine S. aureus, two human and three equine coagulase-negative staphylococci (CNS. Phenotypical methicillin-resistance detection was performed through oxacillin agar-screening and cefoxitin disk-diffusion test. None isolate tested positive for mecA LGA251 gene. For mecA gene PCR, new primers were designed based on the sequences of human S. aureus (HE681097 and bovine S. sciuri (AY820253 mecA. The new primers based on the S. aureus mecA sequence amplified fragments of human and equine CNS and the ones based on S. sciuri mecA sequence only yielded fragments for S. aureus bovine strains. Multiples alignments of mecA gene sequences from bovine, human and equine revealed punctual but significant differences in bovine strains that can lead to the mecA gene detection impairment. The observed divergences of mecA gene sequences are not a matter of animal or human origin, it is a specificity of bovine samples.

  1. Bovine β-casein

    NARCIS (Netherlands)

    Atamer, Zeynep; Post, Antonie E.; Schubert, Thomas; Holder, Aline; Boom, Remko Marcel; Hinrichs, Jörg

    2017-01-01

    In recent years there has been an increasing interest in pure casein fractions, particularly β-casein due to its physiochemical properties as well as its bio- and techno-functional properties. A range of methods has been developed for the fractionation of casein into its individual proteins. The

  2. No cytotoxic effects from application of pentoxifylline to spermatozoa on subsequent pre-implantation embryo development in mice

    Directory of Open Access Journals (Sweden)

    Mohammad Ali Khalili

    2017-06-01

    Full Text Available The aim was to assess the effect of spermatozoa exposed to PTX on the rates of fertilization and embryo development and apoptotic cells within blastocysts in an animal model. Mice Oocytes were inseminated with spermatozoa exposed to 3.6 mmol PTX for 30 min, or with neat spermatozoa. Then fertilization and embryo development rate, blastocyst formation and quality, as well as total cell number of blastocyst, and DNA fragmentation index (DFI in blastocysts were surveyed in both groups. Fertilization and embryo development rate were similar between the groups. The rates of blastocyst formation did not differ significantly between control and PTX groups (52.4% vs. 51.8%. The average of total cell count in blastocysts and DFI in control and PTX groups were also insignificant (31.08 ± 1.5 vs. 34.14 ± 1.5 and 9.76 ± 5.0 vs. 11.77 ± 5.4. Application of PTX for enhancing sperm motility does not cause a cytotoxic effect on subsequent embryo development and embryo genome integrity.

  3. Immunochromatographic Lateral-flow test strip for the rapid detection of added bovine rennet whey in milk and milk powder

    NARCIS (Netherlands)

    Martin-Hernandez, C.; Munoz, M.; Daury, C.; Weymuth, H.; Kemmers-Voncken, A.; Corbation, V.; Toribo, T.; Bremer, M.G.E.G.

    2009-01-01

    An immunochromatographic lateral-flow test dipstick test was developed for the fast detection of bovine rennet whey in liquid milk and milk powder. The test is based on the binding of casein glycomacropeptide (cGMP) by two specific anti-bovine ¿-casein monoclonal antibodies and has a visual

  4. Blastocyst culture using single versus sequential media in clinical IVF: a systematic review and meta-analysis of randomized controlled trials.

    Science.gov (United States)

    Sfontouris, Ioannis A; Martins, Wellington P; Nastri, Carolina O; Viana, Iara G R; Navarro, Paula A; Raine-Fenning, Nick; van der Poel, Sheryl; Rienzi, Laura; Racowsky, Catherine

    2016-10-01

    The purpose of this study was to undertake a review of the available evidence comparing the use of a single medium versus sequential media for embryo culture to the blastocyst stage in clinical IVF. We searched the Cochrane Central, PubMed, Scopus, ClinicalTrials.gov, Current Controlled Trials and WHO International Clinical Trials Registry Platform to identify randomized controlled trials comparing single versus sequential media for blastocyst culture and ongoing pregnancy rate. Included studies randomized either oocytes/zygotes or women. Eligible oocyte/zygote studies were analyzed to assess the risk difference (RD) and 95 % confidence intervals (CI) between the two media systems; eligible woman-based studies were analyzed to assess the risk ratio (RR) and 95 % CI for clinical pregnancy rate. No differences were observed between single and sequential media for either ongoing pregnancy per randomized woman (relative risk (RR) = 0.9, 95 % CI = 0.7 to 1.3, two studies including 246 women, I 2  = 0 %) or clinical pregnancy per randomized woman (RR = 1.0, 95 % CI = 0.7 to 1.4, one study including 100 women); or miscarriage per clinical pregnancy: RR = 1.3, 95 % CI = 0.4 to 4.3, two studies including 246 participants, I 2  = 0 %). Single media use was associated with an increase blastocyst formation per randomized oocyte/zygote (relative distribution (RD) = +0.06, 95 % CI = +0.01 to +0.12, ten studies including 7455 oocytes/zygotes, I 2  = 83 %) but not top/high blastocyst formation (RD = +0.05, 95 % CI = -0.01 to +0.11, five studies including 3879 oocytes/zygotes, I 2  = 93 %). The overall quality of the evidence was very low for all these four outcomes. Although using a single medium for extended culture has some practical advantages and blastocyst formation rates appear to be higher, there is insufficient evidence to recommend either sequential or single-step media as being superior for the culture of

  5. Simplification of Bovine Somatic Cell Nuclear Transfer by Application of a Zona-Free Manipulation Technique

    DEFF Research Database (Denmark)

    Booth, Paul J; Tan, Shijian; Reipurth, Rikke

    2001-01-01

    Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods. The techni......Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods....... The technique comprises the bisection of zona-free oocytes and the reconstruction of embryos comprising two half cytoplasts and a somatic cell by adherence using phytohaemagglutinin-P (PHA) followed by an electropulse and subsequent culture in microwells (termed WOWs--well of the well). The development......-intact zygotes were not different in either blastocyst yield (44.6 +/- 2.4% versus 51.8 +/- 13.5% [mean +/- SEM]) or quality (126.3 +/- 48.4 versus 119.9 +/- 32.6 total cells), and exposure of zygotes to PHA-P did not reduce blastocyst yields compared to vehicle control (40.8 +/- 11.6% versus 47.1 +/- 20...

  6. PCR assay with host specific internal control forStaphylococcus aureus from bovine milk samples

    Directory of Open Access Journals (Sweden)

    Zafer Cantekin

    2015-03-01

    Full Text Available Staphylococcus aureus is considered as one of the most important and common pathogens of bovine mastitis. Polymerase Chain Reaction is frequently proposed in the diagnosis of S. aureus directly from milk samples instead of classical culture. However, false-negative results may occur in the polymerase chain reaction analysis performed directly from clinical material. For the purpose of disclosing the false negative results, the use of internal amplification controls can be beneficial. Therefore, in this study a new polymerase chain reaction technique with host specific internal amplification control was developed by optimizing S. aureus-specific primers in combination with bovine specific primers. The effectiveness of the developed technique in this study was attempted in milk samples from bovine subclinical mastitis. This technique has the potential to detect S. aureus from bovine milk samples or dairy products.

  7. Biomimetic acellular detoxified glutaraldehyde cross-linked bovine pericardium for tissue engineering

    International Nuclear Information System (INIS)

    Mathapati, Santosh; Bishi, Dillip Kumar; Guhathakurta, Soma; Cherian, Kotturathu Mammen; Venugopal, Jayarama Reddy; Ramakrishna, Seeram; Verma, Rama Shanker

    2013-01-01

    Glutaraldehyde (GLUT) processing, cellular antigens, calcium ions in circulation, and phospholipids present in the native tissue are predominantly responsible for calcification, degeneration, and lack of natural microenvironment for host progenitor cell migration in tissue implants. The study presents an improved methodology for adhesion and proliferation of endothelial progenitor cells (EPCs) without significant changes in biomechanical and biodegradation properties of the processed acellular bovine pericardium. The anti-calcification potential of the processed tissue was enhanced by detoxification of GLUT-cross-linked bovine pericardium by decellularization, pretreating it with ethanol or removing the free aldehydes by citric acid treatment and lyophilization. The treated tissues were assessed for biomechanical properties, GLUT ligand quantification, adhesion, proliferation of EPCs, and biodegradability. The results indicate that there was no significant change in biomechanical properties and biodegradability when enzymatic hydrolysis (p > 0.05) is employed in detoxified acellular GLUT cross-linked tissue (DBP–G–CA–ET), compared with the native detoxified GLUT cross-linked bovine pericardium (NBP–G–CA–ET). DBP–G–CA–ET exhibited a significant (p > 0.05) increase in the viability of EPCs and cell adhesion as compared to acellular GLUT cross-linked bovine pericardium (p < 0.05). Lyophilized acellular detoxified GLUT cross-linked bovine pericardium, employed in our study as an alternative to conventional GLUT cross-linked bovine pericardium, might provide longer durability and better biocompatibility, and reduce calcification. The developed bovine pericardium patches could be used in cardiac reconstruction and repair, arteriotomy, soft tissue repair, and general surgical procedures with tissue regeneration dimensions. - Highlights: ► We improved the quality of patch biomaterial for cardiovascular surgical procedures. ► Bovine pericardium was

  8. Biomimetic acellular detoxified glutaraldehyde cross-linked bovine pericardium for tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Mathapati, Santosh; Bishi, Dillip Kumar [Stem Cell and Molecular Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Madras, Chennai (India); Frontier Lifeline Pvt Ltd. and Dr. K. M. Cherian Heart Foundation, Mogappair, Chennai (India); Healthcare and Energy Materials Laboratory, NUSNNI, Faculty of Engineering, National University of Singapore (Singapore); Guhathakurta, Soma [Departmet of Engineering Design, Indian Institute of Technology Madras, Chennai (India); Cherian, Kotturathu Mammen [Frontier Lifeline Pvt Ltd. and Dr. K. M. Cherian Heart Foundation, Mogappair, Chennai (India); Venugopal, Jayarama Reddy; Ramakrishna, Seeram [Healthcare and Energy Materials Laboratory, NUSNNI, Faculty of Engineering, National University of Singapore (Singapore); Verma, Rama Shanker, E-mail: vermars@iitm.ac.in [Stem Cell and Molecular Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Madras, Chennai (India)

    2013-04-01

    Glutaraldehyde (GLUT) processing, cellular antigens, calcium ions in circulation, and phospholipids present in the native tissue are predominantly responsible for calcification, degeneration, and lack of natural microenvironment for host progenitor cell migration in tissue implants. The study presents an improved methodology for adhesion and proliferation of endothelial progenitor cells (EPCs) without significant changes in biomechanical and biodegradation properties of the processed acellular bovine pericardium. The anti-calcification potential of the processed tissue was enhanced by detoxification of GLUT-cross-linked bovine pericardium by decellularization, pretreating it with ethanol or removing the free aldehydes by citric acid treatment and lyophilization. The treated tissues were assessed for biomechanical properties, GLUT ligand quantification, adhesion, proliferation of EPCs, and biodegradability. The results indicate that there was no significant change in biomechanical properties and biodegradability when enzymatic hydrolysis (p > 0.05) is employed in detoxified acellular GLUT cross-linked tissue (DBP–G–CA–ET), compared with the native detoxified GLUT cross-linked bovine pericardium (NBP–G–CA–ET). DBP–G–CA–ET exhibited a significant (p > 0.05) increase in the viability of EPCs and cell adhesion as compared to acellular GLUT cross-linked bovine pericardium (p < 0.05). Lyophilized acellular detoxified GLUT cross-linked bovine pericardium, employed in our study as an alternative to conventional GLUT cross-linked bovine pericardium, might provide longer durability and better biocompatibility, and reduce calcification. The developed bovine pericardium patches could be used in cardiac reconstruction and repair, arteriotomy, soft tissue repair, and general surgical procedures with tissue regeneration dimensions. - Highlights: ► We improved the quality of patch biomaterial for cardiovascular surgical procedures. ► Bovine pericardium was

  9. Radioimmunoassay for progesterone in bovine milk

    International Nuclear Information System (INIS)

    Ruiz, Miriam; Figueredo, Nancy; Castillo, Sonia; Pizarro

    2002-01-01

    A system for the measurement of progesterone in bovine milk by radioimmunoassay has been developed and validated. This assay includes an iodine tracer purified by HPLC, the standard prepared in fat-free milk and an antibody anti-progesterone combined with second antibody. The detection limit of the assay is at 0.2 nmol/L calculated from the maximum binding menus two standard deviations and the precision is satisfactory. In the recovery assay was used 4 milk different samples and the result was 98% of recuperation. The progesterone was determinate in milk samples from post-partum animals taking samples three times per week for 40 days. The assay is simple, rapid and possibility the progesterone measurement without sample dilution, distinguish the cyclic changes of this hormone that reflect the ovarian activity in the animals. (author)

  10. Effect of mating between the donor cow and bull (Holstein versus Gyr on the in vitro production of bovine embryos

    Directory of Open Access Journals (Sweden)

    Ana Paula Toledo Barbosa da Silva

    2015-03-01

    Full Text Available The objective of this study was to evaluate the effect of the breed of the oocyte donor cow and bull (Holstein versus Gyr on in vitro production (IVP parameters of bovine embryos comparing the mean number of recovered oocytes and oocytes suitable for culture, the rate of suitable oocytes, and cleavage and blastocyst rates. Data from 1,000 follicular aspiration sessions (OPU, including 500 in donor cows of the Holstein breed and 500 of the Gyr breed, were collected. The results were analyzed by the unpaired Student t-test and chi-square test, adopting a level of significance of 5%. The mean number and standard deviation of recovered oocytes and oocytes suitable for culture were 15.1±13.0 and 8.7±7.6 for the Holstein breed and 15.5±11.9 and 9.1±7.9 for the Gyr breed. The rates of suitable oocytes were 57.7% and 58.5% for Holstein and Gyr breeds, respectively. A significant difference between breeds was observed for the number of oocytes suitable for culture (P<0.05, but not for the number of recovered oocytes or rates of suitable oocytes (P>0.05. Similarly, the breed of the oocyte donor cow and bull influenced cleavage and blastocyst rates (P<0.05. The cleavage rates were 65.7, 60.3, 59.6 and 56.5% for the combinations (donor breed x bull breed Holstein x Holstein (G1, Holstein x Gyr (G2, Gyr x Holstein (G3 and Gyr x Gyr (G4, respectively, with G1>G2, G1>G3, G1>G4, G2=G3, G2>G4, and G3>G4. The blastocyst rates were 28.1, 33.3, 26.8 and 31.0%, respectively, with G1>G2, G1=G3, G1

  11. Desenvolvimento de um sistema computacional para dimensionamento e evolução de rebanhos bovinos Development of software for dimensioning and evolution of bovine herds

    Directory of Open Access Journals (Sweden)

    Marcos Aurélio Lopes

    2000-10-01

    Full Text Available Os objetivos deste estudo foram desenvolver um sistema computacional que efetue o dimensionamento e a evolução de rebanhos bovinos e criar uma ferramenta que possibilite ao usuário efetuar simulações em um sistema de produção de carne e/ou leite. Foi utilizada a linguagem CA Clipper. As rotinas foram desenvolvidas de forma conversacional, com acesso aos diversos programas, por meio de menus auto-explicativos. O sistema desenvolvido pode auxiliar o técnico e o pecuarista no dimensionamento e na evolução de um rebanho bovino com precisão e considerável rapidez; possibilita ao usuário efetuar inúmeras simulações; e constitui -se em importante ferramenta no auxílio da tomada de decisões.The objectives of this study were to develop a software that accomplishes both the dimensioning and evolution of cattle herds and develop a tool which makes it possible for the user to perform simulations with production systems of milk and or beef. The language employed was CA Clipper. The routines have been developed in a conversational form, with an access to the several programs by means of self-explicative menus. The developed system can aid both the technician and the raiser in dimensioning and evolution of a cattle herd with precision and outstanding rapidity; it allows to the user to perform a number of simulations; and it is considered an important tool in the assistance to decision-making.

  12. DNA Methylation at a Bovine Alpha Satellite I Repeat CpG Site during Development following Fertilization and Somatic Cell Nuclear Transfer

    OpenAIRE

    Couldrey, Christine; Wells, David N.

    2013-01-01

    Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT bla...

  13. Insulin-like growth factor I (IGF-I) and long R(3) IGF-I differently affect development and messenger ribonucleic acid abundance for IGF-binding proteins and type IIGF receptors in in vitro produced bovine embryos

    Czech Academy of Sciences Publication Activity Database

    Motlík, Jan; Prelle, K.; Stojkovic, M.; Ewald, D.; Arnold, G. J.; Welf, E.

    2001-01-01

    Roč. 142, - (2001), s. 1309-1316 ISSN 0013-7227 R&D Projects: GA ČR GV524/96/K162; GA AV ČR KSK5052113 Keywords : Insulin Like Growth Factor * bovine embryos Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.971, year: 2001

  14. Bovine cysticercosis in the European Union

    DEFF Research Database (Denmark)

    Blagojevic, Bojan; Robertson, Lucy J.; Vieira-Pinto, Madalena

    2017-01-01

    -only inspection of slaughtered cattle in order to reduce the potential for cross-contamination with bacteria that are of greatest public health risk, is expected in the European Union in the near future. With this system, the detection sensitivity for bovine cysticercosis that is already low with the current meat...... of bovine cysticercosis in the European Union....

  15. Bovine Necrotic Vulvovaginitis Associated with Porphyromonas levii

    Science.gov (United States)

    Friedgut, Orly; Alpert, Nir; Stram, Yehuda; Lahav, Dan; Tiomkin, Doron; Avramson, Miriam; Grinberg, Kalia; Bernstein, Michael

    2004-01-01

    An outbreak of bovine necrotic vulvovaginitis associated with Porphyromonas levii, an emerging animal and human pathogen, affected 32 cows on a dairy farm in the northeast of Israel. Five animals had to be culled. This report appears to be the first that associates P. levii with bovine necrotic vulvovagnitis. PMID:15109423

  16. Comparison of nine DNA extraction methods for the diagnosis of bovine tuberculosis by real time PCR

    OpenAIRE

    Moura, André; Hodon, Mikael Arrais; Soares Filho, Paulo Martins; Issa, Marina de Azevedo; Oliveira, Ana Paula Ferreira de; Fonseca Júnior, Antônio Augusto

    2016-01-01

    ABSTRACT: Bovine tuberculosis is an infectious disease with a high impact on the cattle industry, particularly in developing countries. PCR is a very sensitive method for detection of infectious agents, but the sensitivity of molecular diagnosis is largely dependent on the efficiency of the DNA extraction methods. The objective of this study was to evaluate DNA extraction methods for direct detection of Mycobacterium bovis in bovine tissue. Nine commercial kits for DNA extraction were evalua...

  17. Stromal fibroblasts derived from mammary gland of bovine with mastitis display inflammation-specific changes

    OpenAIRE

    Chen, Qing; He, Guiliang; Zhang, Wenyao; Xu, Tong; Qi, Hongliang; Li, Jing; Zhang, Yong; Gao, Ming-Qing

    2016-01-01

    Fibroblasts are predominant components of mammary stromal cells and play crucial roles in the development and involution of bovine mammary gland; however, whether these cells contribute to mastitis has not been demonstrated. Thus, we have undertaken biological and molecular characterization of inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis and normal fibroblasts (NFs) from slaughtered dairy cows because of fractured legs during lactation...

  18. Clinical applications of bovine colostrum therapy

    DEFF Research Database (Denmark)

    Rathe, Mathias; Müller, Klaus; Sangild, Per Torp

    2014-01-01

    Bovine colostrum, the first milk that cows produce after parturition, contains high levels of growth factors and immunomodulatory components. Some healthy and diseased individuals may gain health benefits by consuming bovine colostrum as a food supplement. This review provides a systematic...... to populations, outcomes, and methodological quality, as judged by the Jadad assessment tool. Many studies used surrogate markers to study the effects of bovine colostrum. Studies suggesting clinical benefits of colostrum supplementation were generally of poor methodological quality, and results could...... not be confirmed by other investigators. Bovine colostrum may provide gastrointestinal and immunological benefits, but further studies are required before recommendations can be made for clinical application. Animal models may help researchers to better understand the mechanisms of bovine colostrum supplementation...

  19. A close look at the mammalian blastocyst: epiblast and primitive endoderm formation.

    Science.gov (United States)

    Artus, Jérôme; Chazaud, Claire

    2014-09-01

    During early development, the mammalian embryo undergoes a series of profound changes that lead to the formation of two extraembryonic tissues--the trophectoderm and the primitive endoderm. These tissues encapsulate the pluripotent epiblast at the time of implantation. The current model proposes that the formation of these lineages results from two consecutive binary cell fate decisions. The first controls the formation of the trophectoderm and the inner cell mass, and the second controls the formation of the primitive endoderm and the epiblast within the inner cell mass. While early mammalian embryos develop with extensive plasticity, the embryonic pattern prior to implantation is remarkably reproducible. Here, we review the molecular mechanisms driving the cell fate decision between primitive endoderm and epiblast in the mouse embryo and integrate data from recent studies into the current model of the molecular network regulating the segregation between these lineages and their subsequent differentiation.

  20. Development and application of radioimmunological methods to determine an anabolic compound (trenboloneacetate sup(R)) and its major metabolite (trenbolone) in various tissues and in bovine plasma

    International Nuclear Information System (INIS)

    Oettel, G.M.

    1976-01-01

    For quantitation of the anabolic compound trenboloneacetate sup(R)(TBA) and its major metabolite trenbolone (TBOH) a radioimmunoassay (RIA) was established after raising antibodies in rabbits immunized with TBA-3-(o-carboxi-methyl)-oxime-BSA and TBOH-17β-hemisuccinate-BSA. In order to apply this assay for residue determinations in muscle, liver kidney and fat as well as in plasma of treated animals specific extraction procedures were developed. Specifity, sensitivity, accuracy and reproductibility were tested and the developed methods were shown to be highly reliable. Besides free also conjugated TBOH could be determined after enzyme hydrolysis. The lower limit of sensitivity was around 45 pg; at present 100 tissue samples can be analyzed by one person during one week. With the methods described tissue and plasma samples of 41 heifer calves, 10 bull calves and two steers were analyzed for residues of TBA and TBOH. The animals were treated with various dosages of TBA, alone or in combination with oestradiol-17β. In treated animals the highest residue levels were found in liver, with conjugated TBOH being the major residue fraction (76,4%). Second ranking after liver were fat and kidney tissue with similarly high total residue concentrations, however, free TBOH being the major residue fraction (84,1%) in fat, while each, free and conjugated TBOH, accounted for about 50% in kidney. Lowest residue concentrations with free TBOH being the major residue fraction (91,5%) were found in general in muscular tissue. TBA could only be quantitated in fat (14.2% of total residues). (orig.) [de

  1. Modeling the relationship between scanned rump and 12th-rib fat in young temperate and tropical bovines: model development and evaluation.

    Science.gov (United States)

    Walmsley, B J; Wolcott, M L; McPhee, M J

    2010-05-01

    A decision support tool for predicting subcutaneous fat depths called BeefSpecs, based on the Davis growth model (DGM), has been developed by the Cooperative Research Centre for Beef Genetic Technologies. Currently, the DGM predicts 12th-rib fat thickness (RFT, mm). To allow predictions of fat thickness at the P8 rump (P8FT, mm) site, the standard carcass fat measurement in the Australian beef industry, a relationship was developed between ultrasound RFT and P8FT in steers and heifers from temperate (Angus, Hereford, Shorthorn, and Murray Grey) and tropical (Brahman, Belmont Red, and Santa Gertrudis) breed types. Model development involved fitting various combinations of sex, breed type (BrT), BW, age, and RFT to produce 6 models. The models were challenged with data from 3 independent data sets: 1) Angus steers from 2.4 generations of divergent selection for and against residual feed intake; 2) 2 tropically adapted genotypes [Brahman and tropically adapted composites (combinations of Belmont Red, Charbray, Santa Gertrudis, Senepol, and Brahman breeds)]; and 3) a study using sires from Charolais, Limousin, Belgian Blue, and Black and Red Wagyu breeds and 3 genetic lines of Angus to create divergence in progeny in terms of genetic potential for intramuscular fat percent and retail beef yield. When challenged with data from Angus cattle, the mean biases (MB, mm) for models A to F were -1.23, -0.56, -0.56, -0.02, 0.14, and 0.04, and the root mean square errors of predictions (mm) were 1.53, 0.97, 0.97, 0.92, 0.93, and 0.91, respectively. When challenged with data from Brahman cattle, MB were 0.04, -0.22, -0.14, 0.05, -0.11, and 0.02 and root mean square errors of predictions were 1.30, 1.29, 1.27, 1.23, 1.37, and 1.29, respectively. Generally, model accuracy indicated by MB tended to be less for model E, which contained age rather than BW as a covariate. Models B and C were generally robust when challenged with data from Angus, Brahman, and Tropical Composite cattle

  2. In vitro susceptibility of bovine mastitis pathogens to a combination of penicillin and framycetin: development of interpretive criteria for testing by broth microdilution and disk diffusion.

    Science.gov (United States)

    Pillar, C M; Stoneburner, A; Shinabarger, D L; Abbeloos, E; Goby, L; Bradley, Andrew J

    2014-10-01

    Dry cow therapy is an important part of mastitis control. This therapy typically consists of an antibiotic or antibiotics administered at a single dose by intramammary infusion at dry off to treat or prevent infection by prevalent mastitis pathogens. A combination dry cow therapy consisting of the active components penicillin and framycetin is currently used in several countries. Despite its use, standardized methods for the susceptibility testing of this combination against mastitis pathogens have not been established. In this study, which used Clinical and Laboratory Standards Institute methodology, preliminary interpretive criteria for the broth microdilution minimum inhibitory concentration (MIC) testing of mastitis pathogens to penicillin combined with framycetin (2:1 wt/wt) were established based on the amount of drug achieved and maintained postadministration in the udder. Based on resulting MIC distributions of recent veterinary field isolates and a subset of isolates preselected for resistance to β-lactams or aminoglycosides and concentrations achieved postadministration, criteria for broth microdilution testing of the combination (susceptible, intermediate, resistant in micrograms per milliliter) were set as follows: Escherichia coli ≤8/4, 16/8, ≥32/16; Staphylococcus spp. ≤2/1, 4/2-8/4, >16/8; Streptococcus uberis and Streptococcus dysgalactiae 4/2. A disk diffusion test using disks containing 100 μg of framycetin and 10 IU of penicillin was also developed, and preliminary interpretive criteria (susceptible, intermediate, resistant in millimeters) were set based on correlation to broth MIC values and the minimization of interpretive errors between isolates tested concurrently by broth microdilution and disk diffusion as follows: E. coli ≥18, 16-17, ≤15; Staphylococcus spp. ≥21, 18-20, ≤17; Strep. uberis and Strep. dysgalactiae ≥21, 19-20, ≤18. In addition, ranges for the quality control of the testing of this combination by both broth

  3. Immunohistochemical study on the ontogenetic development of the regional distribution of peptide YY, pancreatic polypeptide, and glucagon-like peptide 1 endocrine cells in bovine gastrointestinal tract.

    Science.gov (United States)

    Pyarokhil, Asadullah Hamid; Ishihara, Miyuki; Sasaki, Motoki; Kitamura, Nobuo

    2012-04-10

    The regional distribution and relative frequency of peptide YY (PYY)-, pancreatic polypeptide (PP)-, and glucagon-like peptide 1 (GLP-1)-immunoreactive (IR) cells were determined immunohistochemically in the gastrointestinal tract at seven ontogenetic stages in pre- and postnatal cattle. Different frequencies of PYY-, PP-, and GLP-1-IR cells were found in the intestines at all stages; they were not found in the esophagus and stomach. The frequencies varied depending on the intestinal segment and the developmental stage. The frequencies of PYY- and PP-IR cells were lower in the small intestine and increased from ileum to rectum, whereas GLP-1-IR cells were more numerous in duodenum and jejunum, decreased in ileum and cecum, and increased again in colon and rectum. The frequencies also varied according to pre- and postnatal stages. All three cell types were most numerous in fetus, and decreased in calf and adult groups, indicating that the frequencies of these three types of endocrine cells decrease with postnatal development. The results suggest that these changes vary depending on feeding habits and adaptation of growth, secretion, and motility of intestine at different ontogenetic stages of cattle. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Development of Fourier transform mid-infrared calibrations to predict acetone, β-hydroxybutyrate, and citrate contents in bovine milk through a European dairy network.

    Science.gov (United States)

    Grelet, C; Bastin, C; Gelé, M; Davière, J-B; Johan, M; Werner, A; Reding, R; Fernandez Pierna, J A; Colinet, F G; Dardenne, P; Gengler, N; Soyeurt, H; Dehareng, F

    2016-06-01

    To manage negative energy balance and ketosis in dairy farms, rapid and cost-effective detection is needed. Among the milk biomarkers that could be useful for this purpose, acetone and β-hydroxybutyrate (BHB) have been proved as molecules of interest regarding ketosis and citrate was recently identified as an early indicator of negative energy balance. Because Fourier transform mid-infrared spectrometry can provide rapid and cost-effective predictions of milk composition, the objective of this study was to evaluate the ability of this technology to predict these biomarkers in milk. Milk samples were collected in commercial and experimental farms in Luxembourg, France, and Germany. Acetone, BHB, and citrate contents were determined by flow injection analysis. Milk mid-infrared spectra were recorded and standardized for all samples. After edits, a total of 548 samples were used in the calibration and validation data sets for acetone, 558 for BHB, and 506 for citrate. Acetone content ranged from 0.020 to 3.355mmol/L with an average of 0.103mmol/L; BHB content ranged from 0.045 to 1.596mmol/L with an average of 0.215mmol/L; and citrate content ranged from 3.88 to 16.12mmol/L with an average of 9.04mmol/L. Acetone and BHB contents were log-transformed and a part of the samples with low values was randomly excluded to approach a normal distribution. The 3 edited data sets were then randomly divided into a calibration data set (3/4 of the samples) and a validation data set (1/4 of the samples). Prediction equations were developed using partial least square regression. The coefficient of determination (R(2)) of cross-validation was 0.73 for acetone, 0.71 for BHB, and 0.90 for citrate with root mean square error of 0.248, 0.109, and 0.70mmol/L, respectively. Finally, the external validation was performed and R(2) obtained were 0.67 for acetone, 0.63 for BHB, and 0.86 for citrate, with respective root mean square error of validation of 0.196, 0.083, and 0.76mmol/L. Although

  5. Nonenzymatic glycosylation of bovine myelin basic protein

    International Nuclear Information System (INIS)

    Hitz, J.B.

    1987-01-01

    In the CNS myelin sheath the nonenzymatic glycosylation reaction (at the early stage of the Amadori product) occurs only with the myelin basic protein and not with the other myelin proteins. This was observed in isolated bovine myelin by in vitro incubation with [ 14 C]-galactose and [ 14 C]-glucose. The respective in-vitro incorporation rates for purified bovine myelin basic protein with D-galactose, D-glucose and D-mannose were 7.2, 2.4 and 2.4 mmoles/mole myelin basic protein per day at 37 0 C. A more rapid, HPLC method was devised and characterized to specifically analyze for the Amadori product. The HPLC method was correlated to the [ 14 C]-sugar incorporation method for myelin basic protein under a set of standard reaction conditions using [ 14 C]-glucose and [ 14 C]-mannose with HPLC values at 1/6 and 1/5 of the [ 14 C]-sugar incorporation method. A novel myelin basic protein purification step has been developed that yields a relativity proteolytic free preparation that is easy to work with, being totally soluble at a neutral pH. Nine new spots appear for a trypsinized glycosylated MBP in the paper peptide map of which eight correspond to positions of the [ 3 H]-labeled Amadori product in affinity isolated peptides. These studies provide a general characterization of and a structural basis for investigations on nonenzymatically glycosylated MBP as well as identifying MBP as the only nonenzymatically glycosylated protein in the CNS myelin sheath which may accumulate during aging, diabetes, and demyelinating diseases in general

  6. Bovine colostrum to children with short bowel syndrome

    DEFF Research Database (Denmark)

    Aunsholt, Lise; Jeppesen, Palle Bekker; Lund, Pernille

    2014-01-01

    Background: Management of short bowel syndrome (SBS) aims to achieve intestinal autonomy to prevent fluid, electrolyte, and nutrient deficiencies and maintain adequate development. Remnant intestinal adaptation is required to obtain autonomy. In the newborn pig, colostrum has been shown to support...... intestinal development and hence adaptive processes. Aim: The efficacy of bovine colostrum to improve intestinal function in children with SBS was evaluated by metabolic balance studies. Materials and Methods: Nine children with SBS were included in a randomized, double-blind, crossover study. Twenty percent...... of enteral fluid intake was replaced with bovine colostrum or a mixed milk diet for 4 weeks, separated by a 4-week washout period. Intestinal absorption of energy and wet weight was used to assess intestinal function and the efficacy of colostrum. Results: Colostrum did not improve energy or wet weight...

  7. Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos

    Directory of Open Access Journals (Sweden)

    Van Zeveren Alex

    2005-12-01

    Full Text Available Abstract Background Real-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the succesful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published GAPD, ACTB or 18S rRNA have been used as a single reference gene without prior verification of their expression stability. Normalization of the data using unstable controls can result in erroneous conclusions, especially when only one reference gene is used. Results In this study the transcription levels of 8 commonly used reference genes (ACTB, GAPD, Histone H2A, TBP, HPRT1, SDHA, YWHAZ and 18S rRNA were determined at different preimplantation stages (2-cell, 8-cell, blastocyst and hatched blastocyst in order to select the most stable genes to normalize quantitative data within different preimplantation embryo stages. Conclusion Using the geNorm application YWHAZ, GAPD and SDHA were found to be the most stable genes across the examined embryonic stages, while the commonly used ACTB was shown to be highly regulated. We recommend the use of the geometric mean of those 3 reference genes as an accurate normalization factor, which allows small expression differences to be reliably measured.

  8. Simulating the mammalian blastocyst--molecular and mechanical interactions pattern the embryo.

    Directory of Open Access Journals (Sweden)

    Pawel Krupinski

    2011-05-01

    Full Text Available Mammalian embryogenesis is a dynamic process involving gene expression and mechanical forces between proliferating cells. The exact nature of these interactions, which determine the lineage patterning of the trophectoderm and endoderm tissues occurring in a highly regulated manner at precise periods during the embryonic development, is an area of debate. We have developed a computational modeling framework for studying this process, by which the combined effects of mechanical and genetic interactions are analyzed within the context of proliferating cells. At a purely mechanical level, we demonstrate that the perpendicular alignment of the animal-vegetal (a-v and embryonic-abembryonic (eb-ab axes is a result of minimizing the total elastic conformational energy of the entire collection of cells, which are constrained by the zona pellucida. The coupling of gene expression with the mechanics of cell movement is important for formation of both the trophectoderm and the endoderm. In studying the formation of the trophectoderm, we contrast and compare quantitatively two hypotheses: (1 The position determines gene expression, and (2 the gene expression determines the position. Our model, which couples gene expression with mechanics, suggests that differential adhesion between different cell types is a critical determinant in the robust endoderm formation. In addition to differential adhesion, two different testable hypotheses emerge when considering endoderm formation: (1 A directional force acts on certain cells and moves them into forming the endoderm layer, which separates the blastocoel and the cells of the inner cell mass (ICM. In this case the blastocoel simply acts as a static boundary. (2 The blastocoel dynamically applies pressure upon the cells in contact with it, such that cell segregation in the presence of differential adhesion leads to the endoderm formation. To our knowledge, this is the first attempt to combine cell-based spatial

  9. Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples.

    Science.gov (United States)

    Pulford, D J; Gias, E; Bueno, I M; McFadden, Amj

    2016-01-01

    To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis. Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay. The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R(2) = 0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with T. orientalis, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with T. orientalis ranged between 5.6 × 10(4) and 3.3 × 10(6) genomes per

  10. 21 CFR 184.1034 - Catalase (bovine liver).

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Catalase (bovine liver). 184.1034 Section 184.1034... Listing of Specific Substances Affirmed as GRAS § 184.1034 Catalase (bovine liver). (a) Catalase (bovine liver) (CAS Reg. No. 81457-95-6) is an enzyme preparation obtained from extracts of bovine liver. It is...

  11. Antibacterial performance of bovine lactoferrin-fish gelatine electrospun nanocomposites

    OpenAIRE

    Padrão, Jorge; Machado, Raul; Casal, Margarida; Rodrigues, L. R.; Dourado, Fernando; Lanceros-Méndez, S.; Sencadas, V.

    2014-01-01

    Antibacterial performance of bovine lactoferrin-fish gelatine electrospun nanocomposites The alarming increase of antibiotic resistant microorganisms urged the development and synthesis of novel antimicrobial biomaterials, to be employed in a broad range of applications, ranging from food casings to medical devices [1 – 3]. This work describes the processing and characterization of an innovative fully biobased eletrctrospun nanocomposite material displaying antibacterial properties. Its c...

  12. The copper transporter (SLC31A1/CTR1) is expressed in bovine spermatozoa and oocytes: Copper in IVF medium improves sperm quality.

    Science.gov (United States)

    Anchordoquy, J P; Anchordoquy, J M; Pascua, A M; Nikoloff, N; Peral-García, P; Furnus, C C

    2017-07-15

    Adequate dietary intake of copper (Cu) is required for normal reproductive performance in cattle. The objective of this study was to investigate the pregnancy rates from cattle with deficient, marginal and adequate Cu plasma concentration at the beginning of artificial insemination protocol. Moreover, we determined Cu concentrations present in bovine oviductal fluid (OF), and the effects of Cu on fertilizing ability of bovine spermatozoa. Also, the presence of Cu transporter, SLC31A1 (also known as CTR1), in spermatozoa and in vitro matured oocyte were investigated. We found no differences in pregnancy rates among animals with adequate, marginal, and deficient Cu concentrations measured in plasma at the beginning of fixed-time artificial insemination (FTAI) protocol. Copper concentrations in OF were 38.3 ± 2.17 μg/dL (mean ± SEM) regardless of cupremia levels. The addition of 40 μg/dL Cu to IVF medium enhanced total and progressive motility, sperm viability, functional sperm membrane integrity (HOST), sperm-zona binding, and pronuclear formation. On the other hand, the presence of Cu in IVF medium did not modify acrosome integrity and cleavage rates after IVF, but impaired blastocyst rates. Cu transporter SLC31A1 was detected in bovine spermatozoa in the apical segment of acrosome, and in the oocyte matured in vitro. In conclusion, the results obtained in the present study determined that cupremia levels at the beginning of FTAI protocol did not influence the pregnancy rates at 60 d after insemination. The presence of CTR1 in bovine mature oocyte and spermatozoa, as well as the beneficial effect of Cu on sperm quality would suggest an important role of this mineral during the fertilization process. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Molecular cytogenetic analysis of human blastocysts andcytotrophoblasts by multi-color FISH and Spectra Imaging analyses

    Energy Technology Data Exchange (ETDEWEB)

    Weier, Jingly F.; Ferlatte, Christy; Baumgartner, Adolf; Jung,Christine J.; Nguyen, Ha-Nam; Chu, Lisa W.; Pedersen, Roger A.; Fisher,Susan J.; Weier, Heinz-Ulrich G.

    2006-02-08

    Numerical chromosome aberrations in gametes typically lead to failed fertilization, spontaneous abortion or a chromosomally abnormal fetus. By means of preimplantation genetic diagnosis (PGD), we now can screen human embryos in vitro for aneuploidy before transferring the embryos to the uterus. PGD allows us to select unaffected embryos for transfer and increases the implantation rate in in vitro fertilization programs. Molecular cytogenetic analyses using multi-color fluorescence in situ hybridization (FISH) of blastomeres have become the major tool for preimplantation genetic screening of aneuploidy. However, current FISH technology can test for only a small number of chromosome abnormalities and hitherto failed to increase the pregnancy rates as expected. We are in the process of developing technologies to score all 24 chromosomes in single cells within a 3 day time limit, which we believe is vital to the clinical setting. Also, human placental cytotrophoblasts (CTBs) at the fetal-maternal interface acquire aneuploidies as they differentiate to an invasive phenotype. About 20-50% of invasive CTB cells from uncomplicated pregnancies were found aneuploidy, suggesting that the acquisition of aneuploidy is an important component of normal placentation, perhaps limiting the proliferative and invasive potential of CTBs. Since most invasive CTBs are interphase cells and possess extreme heterogeneity, we applied multi-color FISH and repeated hybridizations to investigate individual CTBs. In summary, this study demonstrates the strength of Spectral Imaging analysis and repeated hybridizations, which provides a basis for full karyotype analysis of single interphase cells.

  14. bovine

    African Journals Online (AJOL)

    'n Radio-irnmunologiese bepalingsmetode vir luteihiserende hormoon (LH) in bloed van die bees is ontwikkel duer die gebruik van buisies bestryk met teeliggame. .... proportion (%) of labelled LH bound by unadsorb- ed antisera in a double ... the location of the "protein" (elution volume 10-20 rnI) and "free iodine" (elution ...

  15. Developing a vaccine for eradicating contagious bovine ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    sub-Saharan Africa. CBPP kills up to 50% of infected animals, when newly introduced into a population, and many ... animal and human diseases. While vaccines for CBPP are available, they have several ... project involves them in field testing and assesses their willingness to pay for the vaccine. The project also examines.

  16. Bovine bone for white ceramic

    International Nuclear Information System (INIS)

    Souza, J.L. de; Harima, E.; Leite, J.I.P.; Monteiro, F.M.; Bezerra, M.T.T.

    2011-01-01

    The porcelain is composed of feldspar, kaolin and about 50% for bovine bone ashes. This work aims to analyze the properties acquired by the substitution of kaolin by its waste. For characterization of raw materials chemical analyzes were made by X-Ray Fluorescence (XRF) and mineralogical analysis by X-Ray Diffraction (XRD). Four formulations were produced varying the percentage of waste materials of kaolin and bone ashes of 25 and 55% by weight. The samples were sintered at temperatures of 1150, 1200 and 1250 deg C. The technological tests realized were: water absorption (WA), apparent porosity (AP), apparent density (AD) and linear retraction (LR). Improvement in the physical-mechanical properties of the samples with increasing temperature were observed, and 1250 deg C obtained 0.69% of WA, 1.22% AP, 2.26 g / cm3 AD, and 0.52% LR

  17. 两种助孕方式下D5、D6囊胚行冻融移植后妊娠结局的比较%Comparison of pregnancy outcome after D5 and D6 blastocysts under freeze-thawing in two kinds of ART

    Institute of Scientific and Technical Information of China (English)

    冷芹; 周平; 陈蓓丽; 章志国; 陈大蔚; 曹云霞; 魏兆莲

    2017-01-01

    目的 比较体外受精-胚胎移植(IVF-ET)与胚胎植入前遗传学诊断或胚胎植入前遗传学筛查(PGD/PGS)周期中D5、D6囊胚形成情况及妊娠结局,同时探究D5、D6囊胚的发育潜能及两种助孕方式的优劣.方法 回顾性分析行IVF-ET助孕的138个周期及PGD/PGS筛查助孕的148个周期,按移植胚胎的发育天数分为IVF-D5组、IVF-D6组、PGD/PGS-D5组和PGD/PGS-D6组,分析比较各组的一般情况、囊胚形成情况及冻融移植后的种植率、临床妊娠率、流产率等.结果 IVF-D5组与IVF-D6组相比、PGD/PGS-D5组与PGD/PGS-D6组相比,D5组囊胚形成率、优质胚胎率、种植率、临床妊娠率均高于D6组(P<0.05);PGD/PGS周期中D5组整倍体囊胚检出率高于D6组(p<0.05);IVF-D5组与PGD/PGS-D5组相比、IVF-D6组与PGD/PGS-D6组相比,两组间的囊胚形成率、优质胚胎率、种植率及临床妊娠率均差异无统计学意义.结论 两种助孕方式下冻融移植D5囊胚比D6囊胚可获得较好的临床妊娠结局;与IVF相比,PGD/PGS并未明显改善患者的妊娠结局.%Objective To compare the embryogenesis and the outcome of pregnancy about D5 and D6 blastocyst of in vitro fertilization-embryo transfer(IVF-ET) and preimplantation genetic diagnosis/preimplantation genetic screening(PGD/PGS) cycles,meanwhile explore the development potential of D5 and D6 blastocysts and the advantages and disadvantages of the two methods.Methods 138 cycles of in vitro fertilization-embryo transfer and 148 cycles of preimplantation genetic diagnosis/preimplantation genetic screening were analysed retrospectively.According to the development of transplanted embryos,the patients were divided into four groups:IVF-D5 group,IVF-D6 group,PGD/PGS-D5 group and PGD/PGS-D6 group.The general situation of each group,the formation of blastocyst and the implantation rate after freeze-thawing,clinical pregnancy rate,abortion rate were analysed.Results Compared with IVF-D6 group

  18. Bovine Mastitis: Frontiers in Immunogenetics

    Science.gov (United States)

    Thompson-Crispi, Kathleen; Atalla, Heba; Miglior, Filippo; Mallard, Bonnie A.

    2014-01-01

    Mastitis is one of the most prevalent and costly diseases in the dairy industry with losses attributable to reduced milk production, discarded milk, early culling, veterinary services, and labor costs. Typically, mastitis is an inflammation of the mammary gland most often, but not limited to, bacterial infection, and is characterized by the movement of leukocytes and serum proteins from the blood to the site of infection. It contributes to compromised milk quality and the potential spread of antimicrobial resistance if antibiotic treatment is not astutely applied. Despite the implementation of management practises and genetic selection approaches, bovine mastitis control continues to be inadequate. However, some novel genetic strategies have recently been demonstrated to reduce mastitis incidence by taking advantage of a cow’s natural ability to make appropriate immune responses against invading pathogens. Specifically, dairy cattle with enhanced and balanced immune responses have a lower occurrence of disease, including mastitis, and they can be identified and selected for using the high immune response (HIR) technology. Enhanced immune responsiveness is also associated with improved response to vaccination, increased milk, and colostrum quality. Since immunity is an important fitness trait, beneficial associations with longevity and reproduction are also often noted. This review highlights the genetic regulation of the bovine immune system and its vital contributions to disease resistance. Genetic selection approaches currently used in the dairy industry to reduce the incidence of disease are reviewed, including the HIR technology, genomics to improve disease resistance or immune response, as well as the Immunity+™ sire line. Improving the overall immune responsiveness of cattle is expected to provide superior disease resistance, increasing animal welfare and food quality while maintaining favorable production levels to feed a growing population. PMID

  19. Bovine Mastitis: Frontiers in Immunogenetics

    Directory of Open Access Journals (Sweden)

    Kathleen eThompson-Crispi

    2014-10-01

    Full Text Available Mastitis is one of the most prevalent and costly diseases in the dairy industry with losses attributable to reduced milk production, discarded milk, early culling, veterinary services, and labor costs. Typically, mastitis is an inflammation of the mammary gland most often, but not limited to, bacterial infection, and is characterized by the movement of leukocytes and serum proteins from the blood to the site of infection. It contributes to compromised milk quality and the potential spread of antimicrobial resistance if antibiotic treatment is not astutely applied. Despite the implementation of management practises and genetic selection approaches, bovine mastitis control continues to be inadequate. However, some novel genetic strategies have recently been demonstrated to reduce mastitis incidence by taking advantage of a cow’s natural ability to make appropriate immune responses against invading pathogens. Specifically, dairy cattle with enhanced and balanced immune responses have a lower occurrence of disease, including mastitis, and they can be identified and selected for using the High Immune Response (HIR technology. Enhanced immune responsiveness is also associated with improved response to vaccination, increased milk and colostrum quality. Since immunity is an important fitness trait, beneficial associations with longevity and reproduction are also often noted. This review highlights the genetic regulation of the bovine immune system and its vital contributions to disease resistance. Genetic selection approaches currently used in the dairy industry to reduce the incidence of disease are reviewed, including the HIR technology, genomics to improve disease resistance or immune response, as well as the Immunity+TM sire line. Improving the overall immune responsiveness of cattle is expected to provide superior disease resistance, increasing animal welfare and food quality while maintaining favourable production levels to feed a growing

  20. Melatonin Promotes the In Vitro Development of Microinjected Pronuclear Mouse Embryos via Its Anti-Oxidative and Anti-Apoptotic Effects.

    Science.gov (United States)

    Tian, Xiuzhi; Wang, Feng; Zhang, Lu; Ji, Pengyun; Wang, Jing; Lv, Dongying; Li, Guangdong; Chai, Menglong; Lian, Zhengxing; Liu, Guoshi

    2017-05-05

    CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats) combined with pronuclear microinjection has become the most effective method for producing transgenic animals. However, the relatively low embryo developmental rate limits its application. In the current study, it was observed that 10 -7 M melatonin is considered an optimum concentration and significantly promoted the in vitro development of murine microinjected pronuclear embryos, as indicated by the increased blastocyst rate, hatching blastocyst rate and blastocyst cell number. When these blastocysts were implanted into recipient mice, the pregnancy rate and birth rate were significantly higher than those of the microinjected control, respectively. Mechanistic studies revealed that melatonin treatment reduced reactive oxygen species (ROS) production and cellular apoptosis during in vitro embryo development and improved the quality of the blastocysts. The implantation of quality-improved blastocysts led to elevated pregnancy and birth rates. In conclusion, the results revealed that the anti-oxidative and anti-apoptotic activities of melatonin improved the quality of microinjected pronuclear embryos and subsequently increased both the efficiency of embryo implantation and the birth rate of the pups. Therefore, the melatonin supplementation may provide a novel alternative method for generating large numbers of transgenic mice and this method can probably be used in human-assisted reproduction and genome editing.

  1. Protective effect of bovine milk against HCl and ethanol-induced gastric ulcer in mice.

    Science.gov (United States)

    Yoo, Jeong-Hyun; Lee, Jeong-Sang; Lee, You-Suk; Ku, SaeKwang; Lee, Hae-Jeung

    2018-05-01

    The purpose of this study was to investigate the gastroprotective effects of bovine milk on an acidified ethanol (HCl-ethanol) mixture that induced gastric ulcers in a mouse model. Mice received different doses of commercial fresh bovine milk (5, 10, and 20 mL/kg of body weight) by oral gavage once a day for 14 d. One hour after the last oral administration of bovine milk, the HCl-ethanol mixture was orally intubated to provoke severe gastric damage. Our results showed that pretreatment with bovine milk significantly suppressed the formation of gastric mucosa lesions. Pretreatment lowered gastric myeloperoxidase and increased gastric mucus contents and antioxidant enzymes catalase and superoxide dismutase. Administration of bovine milk increased nitrate/nitrite levels and decreased the malondialdehyde levels and the expression of proinflammatory genes, including transcription factor nuclear factor-κB, cyclooxygenase-2, and inducible nitric oxide synthase in the stomach of mice. These results suggest that bovine milk can prevent the development of gastric ulcer caused by acid and alcohol in mice. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  2. The influence of zygote pronuclear morphology on in vitro human embryo development

    Directory of Open Access Journals (Sweden)

    Lidija Križančić-Bombek

    2007-09-01

    Full Text Available Background: The selection of embryos with largest implantation potential is an important part in assisted reproduction. Besides the embryo or blastocyst morphology, selection criteria such as position and orientation of pronuclei (PN in relation to polar body positioning and the number, size and distribution of nucleolar precursor bodies (NPB have been proposed. In our study, a correlation between PN and NBP morphology with the development of early embryos (day 2 of cultivation and blastocysts (day 5 was investigated.Methods: 653 zygotes from 113 IVF (in vitro fertilization and ICSI (intracytoplasmic sperm injection patients, younger than 40 years, were assessed 18–20 hours post-insemination. Optimal zygotes (Z1 had thouching centrally located PN with equall numbers of alligned NPB. Other zygote types differred from Z1 in having scattered NPB in both PN (Z2 or alligned NPB in one PN (Z3 or in PN beeing distant from one another (Z4. For each zygote type a percentage of normal early embryos and blastocysts was calculated.Results: Among 653 assessed zygotes 21.8 % were Z1; 29.1 % Z2, 34.6 % Z3 and 14.5 % Z4. The percentage of normal early embryos decreased from Z1 to Z4 zygote type (70.4 % vs. 55.3 % vs. 59.7 % vs.45.3 %; p < 0.05 as well as the percentage of developed blastocysts (63.4 % vs. 55.3 % vs. 58.8 % vs. 43.2 %. However, the percentages of optimal blastocysts in the four groups did not differ (11.3 % vs. 11.1 % vs. 8.4 % vs. 6.3 %.Conclusions: Best grade zygotes result in batter early embryo and blastocyst development suggesting that zygote morphology can be used in combination with embryo and/or blastocyst evaluation as a method for embryo selection prior to embryo transfer.

  3. Spectroscopic study of gamma irradiated bovine hemoglobin

    International Nuclear Information System (INIS)

    Maghraby, Ahmed Mohamed; Ali, Maha Anwar

    2007-01-01

    In the present study, the effects of ionizing radiation of Cs-137 and Co-60 from 4.95 to 743.14 Gy and from 40 Gy to 300 kGy, respectively, on some bovine hemoglobin characteristics were studied. Such an effect was evaluated using electron paramagnetic resonance (EPR) spectroscopy, and infra-red (IR) spectroscopy. Bovine hemoglobin EPR spectra were recorded and analyzed before and after irradiation and changes were explained in detail. IR spectra of unirradiated and irradiated Bovine hemoglobin were recorded and analyzed also. It was found that ionizing radiation may lead to the increase of free radicals production, the decrease in α-helices contents, which reflects the degradation of hemoglobin molecular structure, or at least its incomplete performance. Results also show that the combined application of EPR and FTIR spectroscopy is a powerful tool for determining structural modification of bovine hemoglobin samples exposed to gamma irradiation

  4. PREVALENCE OF BOVINE FASCIOLOSIS AT THE IBADAN ...

    African Journals Online (AJOL)

    Ismail

    Bovine fasciolosis is a parasitic disease of cattle caused by trematodes usually. Fasciola gigantic ... and impaired fertility [6,7]. The value of the .... especially males with good body condition, are transported to these cities with higher population ...

  5. Closed system for bovine oocyte vitrification

    Directory of Open Access Journals (Sweden)

    Helena Ševelová

    2012-01-01

    Full Text Available The aim of our study was to develop a vitrification carrier for bovine oocyte cryopreservation. The carrier was to be cheap enough, elementary in its construction and meet contemporary requirements for a safe closed system. In a closed system, a cell is prevented from direct exposure to liquid nitrogen, thus minimizing the risk of cross-contamination. Furthermore, two questions regarding the proper vitrification technique were resolved: if it is necessary to partially denude the oocytes before the vitrification process or whether intact cumulus oocyte complexes should be frozen; and if it is more advantageous to preheat the vitrification solutions to female body temperature (39 °C or to keep them at room temperature. Our results show that it is better to partially denude the oocytes prior to vitrification because cryopreserved intact cumulus oocyte complexes often proved dark, non-homogeneous or fragmented cytoplasm after warming, with many of them having visibly widened perivitelline spaces or fractured zonae pellucidae as a result of extensive damage during vitrification. Consequently, intact cumulus oocyte complexes showed significantly lower numbers of cleavage stage embryos on Day 3 compared to partially denuded oocytes (7.4% and 26%, respectively. On the other hand, the survival rate and following development of fertilized oocytes in preheated vitrification solution were equal to results reached at room temperature conditions. In conclusion, results achieved with the newly developed carrier were comparable to previously published studies and therefore they could be recommended for common use.

  6. Human and bovine viruses in the Milwaukee River Watershed: hydrologically relevant representation and relations with environmental variables

    Science.gov (United States)

    Corsi, Steven R.; Borchardt, M. A.; Spencer, S. K.; Hughes, Peter E.; Baldwin, Austin K.

    2014-01-01

    To examine the occurrence, hydrologic variability, and seasonal variability of human and bovine viruses in surface water, three stream locations were monitored in the Milwaukee River watershed in Wisconsin, USA, from February 2007 through June 2008. Monitoring sites included an urban subwatershed, a rural subwatershed, and the Milwaukee River at the mouth. To collect samples that characterize variability throughout changing hydrologic periods, a process control system was developed for unattended, large-volume (56–2800 L) filtration over extended durations. This system provided flow-weighted mean concentrations during runoff and extended (24-h) low-flow periods. Human viruses and bovine viruses were detected by real-time qPCR in 49% and 41% of samples (n = 63), respectively. All human viruses analyzed were detected at least once including adenovirus (40% of samples), GI norovirus (10%), enterovirus (8%), rotavirus (6%), GII norovirus (1.6%) and hepatitis A virus (1.6%). Three of seven bovine viruses analyzed were detected including bovine polyomavirus (32%), bovine rotavirus (19%), and bovine viral diarrhea virus type 1 (5%). Human viruses were present in 63% of runoff samples resulting from precipitation and snowmelt, and 20% of low-flow samples. Maximum human virus concentrations exceeded 300 genomic copies/L. Bovine viruses were present in 46% of runoff samples resulting from precipitation and snowmelt and 14% of low-flow samples. The maximum bovine virus concentration was 11 genomic copies/L. Statistical modeling indicated that stream flow, precipitation, and season explained the variability of human viruses in the watershed, and hydrologic condition (runoff event or low-flow) and season explained the variability of the sum of human and bovine viruses; however, no model was identified that could explain the variability of bovine viruses alone. Understanding the factors that affect virus fate and transport in rivers will aid watershed management for minimizing

  7. Activation of bovine neutrophils by Brucella spp.

    Science.gov (United States)

    Keleher, Lauren L; Skyberg, Jerod A

    2016-09-01

    Brucellosis is a globally important zoonotic infectious disease caused by gram negative bacteria of the genus Brucella. While many species of Brucella exist, Brucella melitensis, Brucella abortus, and Brucella suis are the most common pathogens of humans and livestock. The virulence of Brucella is largely influenced by its ability to evade host factors, including phagocytic killing mechanisms, which are critical for the host response to infection. The aim of this study was to characterize the bovine neutrophil response to virulent Brucella spp. Here, we found that virulent strains of smooth B. abortus, B. melitensis, B. suis, and virulent, rough, strains of Brucella canis possess similar abilities to resist killing by resting, or IFN-γ-activated, bovine neutrophils. Bovine neutrophils responded to infection with a time-dependent oxidative burst that varied little between Brucella spp. Inhibition of TAK1, or SYK kinase blunted the oxidative burst of neutrophils in response to Brucella infection. Interestingly, Brucella spp. did not induce robust death of bovine neutrophils. These results indicate that bovine neutrophils respond similarly to virulent Brucella spp. In addition, virulent Brucella spp., including naturally rough strains of B. canis, have a conserved ability to resist killing by bovine neutrophils. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Bovine TLR2 and TLR4 mediate Cryptosporidium parvum recognition in bovine intestinal epithelial cells.

    Science.gov (United States)

    Yang, Zhengtao; Fu, Yunhe; Gong, Pengtao; Zheng, Jingtong; Liu, Li; Yu, Yuqiang; Li, Jianhua; Li, He; Yang, Ju; Zhang, Xichen

    2015-08-01

    Cryptosporidium parvum (C. parvum) is an intestinal parasite that causes diarrhea in neonatal calves. It results in significant morbidity of neonatal calves and economic losses for producers worldwide. Innate resistance against C. parvum is thought to depend on engagement of pattern recognition receptors. However, the role of innate responses to C. parvum has not been elucidated in bovine. The aim of this study was to evaluate the role of TLRs in host-cell responses during C. parvum infection of cultured bovine intestinal epithelial cells. The expressions of TLRs in bovine intestinal epithelial cells were detected by qRT-PCR. To determine which, if any, TLRs may play a role in the response of bovine intestinal epithelial cells to C. parvum, the cells were stimulated with C. parvum and the expression of TLRs were tested by qRT-PCR. The expression of NF-κB was detected by western blotting. Further analyses were carried out in bovine TLRs transfected HEK293 cells and by TLRs-DN transfected bovine intestinal epithelial cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs. The expression of TLR2 and TLR4 were up-regulated when bovine intestinal epithelial cells were treated with C. parvum. Meanwhile, C. parvum induced IL-8 production in TLR2 or TLR4/MD-2 transfected HEK293 cells. Moreover, C. parvum induced NF-κB activation and cytokine expression in bovine intestinal epithelial cells. The induction of NF-κB activation and cytokine expression by C. parvum were reduced in TLR2-DN and TLR4-DN transfected cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs, and bovine intestinal epithelial cells recognized and responded to C. parvum via TLR2 and TLR4. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Embryonic stem-like cells from rabbit blastocysts cultured with melatonin could differentiate into three germ layers in vitro and in vivo.

    Science.gov (United States)

    Wei, Ruxue; Zhao, Xueming; Hao, Haisheng; Du, Weihua; Zhu, Huabin

    2016-11-01

    The rabbit is considered an important model animal from which to obtain embryonic stem cells because of the utility of this animal in physiology and reproductive research. Here, we derived rabbit ES-like (rES-like) cells from blastocysts of superovulated Japanese white rabbits using culture medium containing 10 -7  M melatonin, 10 ng/mL basic fibroblast growth factor, and 1,000 IU/mL human leukemia inhibitory factor. This concentration of melatonin had the most significant positive effects on the proliferation inner cell mass-derived cells (improving rates from 19.97% to 34.57%) and the longevity of passaging rES-like cells. Melatonin also enhanced the expression of pluripotent genes-including alkaline phosphatase, Pou5f1, Sox2, Klf4, c-Myc, Nanog, Line28a, and surface marker proteins-in fifth-passage rES-like cells. In vitro, these rES-like cells could spontaneously differentiate into some somatic cells, such as beating cardiomyocytes; formed embryoid bodies; expressed markers of the three germ layers after differentiation; and formed teratomas after injection into non-obese diabetic-severe combined immune deficient (NOD-SCID) mice. Thus, melatonin helped coax ES-like cells from rabbit blastocysts, which raises intriguing questions about the relationship between pluripotency and proliferation in rabbit embryonic stem cells. Mol. Reprod. Dev. 83: 1003-1014, 2016 © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. Blastocyst transfer does not improve cycle outcome as compared to D3 transfer in antagonist cycles with an elevated progesterone level on the day of hCG.

    Science.gov (United States)

    Demirel, Cem; Aydoğdu, Serkan; Özdemir, Arzu İlknur; Keskin, Gülşah; Baştu, Ercan; Buyru, Faruk

    2017-09-01

    To evaluate the association between progesterone elevation on the day of human chorionic gonadotropin (hCG) administration and clinical pregnancy rates of gonadotropin-releasing hormone (GnRH) antagonist in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles with the transfer of embryos at different developmental stages (day-3 versus day-5 ETs). This is a retrospective analysis of fresh IVF/ICSI; 194 cycles out of 2676 conducted in a single center. A total of 2676 cycles were analyzed, of which 386 had no progesterone measurements available. Two hundred eighteen cycles had progesterone elevation (p>1.5 ng/mL) giving an overall incidence of 9.5%. Twenty-four cycles were excluded from further analysis. Of the remaining 194 cycles, 151 had day-3 transfers and 43 had blastocyst transfers. There was no statistically significant difference in pregnancy and clinical pregnancy rates per transfer between the D3-ET and D5-ET groups (46% vs. 49%, and 39% vs. 35%, respectively). The results of this study suggest that blastocyst transfer does not improve cycle outcomes compared with D3 transfer in GnRH antagonist cycles with an elevated progesterone level on the day of hCG.

  11. Three-step in vitro maturation culture of bovine oocytes imitating temporal changes of estradiol-17β and progesterone concentrations in preovulatory follicular fluid

    Directory of Open Access Journals (Sweden)

    M. Matsuo

    2017-10-01

    Full Text Available The objective of the article is to evaluate the effect of three-step in vitro maturation (IVM culture system imitating estradiol-17β (E2 and progesterone (P4 concentrations in preovulatory follicles on in vitro bovine embryo production. The cumulus–oocyte complexes (COCs were collected from follicles (2 to 8 mm in diameter of bovine ovaries obtained from a local slaughterhouse. For IVM, the COCs were cultured for 22 h in a three-step system: (1 culture in medium 199, containing 700 ng mL−1 E2 and 50 ng mL−1 P4, for 5 h, followed by the medium containing 150 ng mL−1 E2 and 150 ng mL−1 P4 for 11 h, and then the medium containing 20 ng mL−1 E2 and 300 ng mL−1 P4 for 6 h (EP group; (2 culture in the medium containing 700 ng mL−1 E2 for 5 h, followed by the medium containing 150 ng mL−1 E2 for 11 h, and then the medium containing 20 ng mL−1 E2 for 6 h (E group; or (3 culture in the medium containing 50 ng mL−1 P4 for 5 h, followed by the medium containing 150 ng mL−1 P4 for 11 h, and then the medium containing 300 ng mL−1 P4 for 6 h (P group. The COCs were cultured in the medium containing 1000 ng mL−1 E2 for 22 h (control group. After IVM, the COCs were co-incubated with sperm and further cultured. At 48 h after insemination, the cleavage rate of embryos was not different among the groups. At 192 h after insemination, the blastocyst formation rate of EP group was significantly higher than that of the other groups. The total cell number of blastocysts did not differ among the groups. In conclusion, these results demonstrate that the three-step IVM culture system of bovine oocytes imitating temporal changes of E2 and P4 concentrations in preovulatory follicular fluid improves the developmental potential of embryos in vitro.

  12. The loss of imprinted DNA methylation in mouse blastocysts is inflicted to a similar extent by in vitro follicle culture and ovulation induction.

    Science.gov (United States)

    Saenz-de-Juano, M D; Billooye, K; Smitz, J; Anckaert, E

    2016-06-01

    Does in vitro follicle culture (IFC) have an effect on maintenance of imprinted DNA methylation in preimplantation mouse embryos? We report similar alterations in the methylation pattern of H19 imprinted maternally expressed transcript (H19), small nuclear ribonucleoprotein polypeptide N (Snrpn) and mesoderm specific transcript (Mest) imprinted genes in mouse blastocysts obtained after ovulation induction and IFC. Furthermore, we observed no differences in the gene expression of maternal effect proteins related with imprinting maintenance between superovulated in vivo grown or IFC oocytes. Assisted reproductive technology is associated with adverse post-natal outcomes such as increased risk of premature birth, altered birthweight, congenital anomalies and genomic imprinting syndromes in human and in animal models. Previous studies have shown that ovulation induction allowed normal imprinting establishment in mouse oocytes, but interfered with imprinting maintenance during preimplantation . Normal imprinting establishment was also observed in mouse oocytes derived from a standardized IFC from the early pre-antral follicle stage. The methylation profiles of differentially methylated regions (DMRs) of three key imprinted genes (H19, Snrpn and Mest) were compared at hatched blastocyst stage between embryos obtained from IFC or superovulated oocytes, each subjected to IVF and preimplantation in vitro culture (IVC); in non-manipulated in vivo produced late blastocyst (control) and in in vivo produced 2-cell embryos that were in vitro cultured until the hatched blastocyst stage (to assess the effect of IVC). Two different mice strains (Mus musculus C57BL/6J X CBA/Ca and Mus musculus B6 (CAST7)) were used to discriminate between maternal and paternal alleles of imprinted genes. Additionally, a limiting-dilution bisulfite-sequencing technique was carried out on individual embryos in order to avoid amplification bias. To assess whether IFC and ovulation induction

  13. Effect of culture medium volume and embryo density on early mouse embryonic development: tracking the development of the individual embryo.

    Science.gov (United States)

    Dai, Shan-Jun; Xu, Chang-Long; Wang, Jeffrey; Sun, Ying-Pu; Chian, Ri-Cheng

    2012-07-01

    To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly. (1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining. The blastocyst development was significantly higher (P WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P WOW system than without. Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 μl of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced

  14. Bioelectrical impedance analysis of bovine milk fat

    Science.gov (United States)

    Veiga, E. A.; Bertemes-Filho, P.

    2012-12-01

    Three samples of 250ml at home temperature of 20°C were obtained from whole, low fat and fat free bovine UHT milk. They were analysed by measuring both impedance spectra and dc conductivity in order to establish the relationship between samples related to fat content. An impedance measuring system was developed, which is based on digital oscilloscope, a current source and a FPGA. Data was measured by the oscilloscope in the frequency 1 kHz to 100 kHz. It was showed that there is approximately 7.9% difference in the conductivity between whole and low fat milk whereas 15.9% between low fat and free fat one. The change of fatness in the milk can be significantly sensed by both impedance spectra measurements and dc conductivity. This result might be useful for detecting fat content of milk in a very simple way and also may help the development of sensors for measuring milk quality, as for example the detection of mastitis.

  15. Bioelectrical impedance analysis of bovine milk fat

    Energy Technology Data Exchange (ETDEWEB)

    Veiga, E A; Bertemes-Filho, P [Department of Electrical Eng., State University of Santa Catarina, Joinville (Brazil)

    2012-12-20

    Three samples of 250ml at home temperature of 20 Degree-Sign C were obtained from whole, low fat and fat free bovine UHT milk. They were analysed by measuring both impedance spectra and dc conductivity in order to establish the relationship between samples related to fat content. An impedance measuring system was developed, which is based on digital oscilloscope, a current source and a FPGA. Data was measured by the oscilloscope in the frequency 1 kHz to 100 kHz. It was showed that there is approximately 7.9% difference in the conductivity between whole and low fat milk whereas 15.9% between low fat and free fat one. The change of fatness in the milk can be significantly sensed by both impedance spectra measurements and dc conductivity. This result might be useful for detecting fat content of milk in a very simple way and also may help the development of sensors for measuring milk quality, as for example the detection of mastitis.

  16. Bioelectrical impedance analysis of bovine milk fat

    International Nuclear Information System (INIS)

    Veiga, E A; Bertemes-Filho, P

    2012-01-01

    Three samples of 250ml at home temperature of 20°C were obtained from whole, low fat and fat free bovine UHT milk. They were analysed by measuring both impedance spectra and dc conductivity in order to establish the relationship between samples related to fat content. An impedance measuring system was developed, which is based on digital oscilloscope, a current source and a FPGA. Data was measured by the oscilloscope in the frequency 1 kHz to 100 kHz. It was showed that there is approximately 7.9% difference in the conductivity between whole and low fat milk whereas 15.9% between low fat and free fat one. The change of fatness in the milk can be significantly sensed by both impedance spectra measurements and dc conductivity. This result might be useful for detecting fat content of milk in a very simple way and also may help the development of sensors for measuring milk quality, as for example the detection of mastitis.

  17. Anti-Bovine Programmed Death-1 Rat–Bovine Chimeric Antibody for Immunotherapy of Bovine Leukemia Virus Infection in Cattle

    Directory of Open Access Journals (Sweden)

    Tomohiro Okagawa

    2017-06-01

    Full Text Available Blockade of immunoinhibitory molecules, such as programmed death-1 (PD-1/PD-ligand 1 (PD-L1, is a promising strategy for reinvigorating exhausted T cells and preventing disease progression in a variety of chronic infections. Application of this therapeutic strategy to cattle requires bovinized chimeric antibody targeting immunoinhibitory molecules. In this study, anti-bovine PD-1 rat–bovine chimeric monoclonal antibody 5D2 (Boch5D2 was constructed with mammalian expression systems, and its biochemical function and antiviral effect were characterized in vitro and in vivo using cattle infected with bovine leukemia virus (BLV. Purified Boch5D2 was capable of detecting bovine PD-1 molecules expressed on cell membranes in flow cytometric analysis. In particular, Biacore analysis determined that the binding affinity of Boch5D2 to bovine PD-1 protein was similar to that of the original anti-bovine PD-1 rat monoclonal antibody 5D2. Boch5D2 was also capable of blocking PD-1/PD-L1 binding at the same level as 5D2. The immunomodulatory and therapeutic effects of Boch5D2 were evaluated by in vivo administration of the antibody to a BLV-infected calf. Inoculated Boch5D2 was sustained in the serum for a longer period. Boch5D2 inoculation resulted in activation of the proliferation of BLV-specific CD4+ T cells and decrease in the proviral load of BLV in the peripheral blood. This study demonstrates that Boch5D2 retains an equivalent biochemical function to that of the original antibody 5D2 and is a candidate therapeutic agent for regulating antiviral immune response in vivo. Clinical efficacy of PD-1/PD-L1 blockade awaits further experimentation with a large number of animals.

  18. Transporte de oócitos bovinos em meio de maturação sem controle de atmosfera gasosa Transport of bovine oocytes in maturation medium without a controlled gaseous atmosphere

    Directory of Open Access Journals (Sweden)

    Fábio Gallas Leivas

    2004-02-01

    aspirated from 2 to 8mm follicles obtained from bovine slaughterhouse ovaries (11 replications were randomly distributed in four treatments. Oocytes were matured for 24h with modified TCM-199 Earle salts, plus 25mM bicarbonate, 25 mM HEPES, rFSH-h, Estrus Cow Serum (ECS, and piruvate at 39ºC, in incubator with 5% CO2 and saturated humidity (Control Group, n=296 or exposed to a simulated transport for 6 (T6, n=286, 12 (T12, n=294 or 18h (T18, n=301 in maturation medium containing TCM + HEPES, in a 39ºC water bath, with the same components used in the Control Group, but with 1mM bicarbonate. At the conclusion of each transport period, oocytes were transferred to dishes with maturation medium to reach 24h in incubator, under the same conditions described for the Control group. Fertilization was accomplished during 18h, with the same temperature and gaseous atmosphere, in FERT-TALP plus heparin. The insemination dose was 1x106 spermatozoa/mL, sorted by swim-up. Presumptive zygotes were cultured in SOF medium + 5% ECS for 8 days, in incubator at 39ºC using gasified bags with 5% CO2, 5% O2 and 90% N2. Cleavage rates did not differ between treatments. Embryonic development rates at D7 were similar for Control (20.9%, T6 (19.2% and T12 (21.4% groups, with a reduction (P0.05 in hatched blastocyst rate. The average number of cells of hatched blastocysts was similar (P>0.05 in Control (136, T6 (125.5 and T12 (126.8 groups. These results indicate the possibility of transporting bovine oocytes in maturation medium containing TCM + HEPES, without controlled gaseous atmosphere environment, at 39ºC, for up to 12 hours. This technique offers a practical and efficient alternative for the transport of bovine oocytes for in vitro production of bovine embryos (IVP.

  19. Antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni

    Science.gov (United States)

    Bovine NK-lysins, which are functionally and structurally similar to human granulysin and porcine NK-lysin, are predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Although antimicrobial activity of bovine NK-lysin has been assessed for several bacterial pathogens, not all t...

  20. DESENVOLVIMENTO DE FOLÍCULOS PRÉ-ANTRAIS BOVINOS IN VITRO EM MONOCAMADA DE CÉLULAS OVARIANAS IN VITRO DEVELOPMENT OF BOVINE PREANTRAL FOLLICLES IN MONOLAYER OF OVARIAN CELLS

    Directory of Open Access Journals (Sweden)

    Luís Fabiano Santos da Costa

    2001-04-01

    Full Text Available O presente trabalho teve como objetivos determinar a influência de células ovarianas no desenvolvimento in vitro de folículos pré-antrais, avaliar a viabilidade das células ovarianas em monocamada e a influência do soro na manutenção de folículos pré-antrais in vitro. Folículos pré-antrais (FPs e células ovarianas foram isolados de ovários de fetos bovinos, com idade entre 6 e 8 meses de gestação, oriundos de matadouro. Células ovarianas em monocamada foram cultivadas em meio TCM-199, e a viabilidade celular, após o cultivo na presença ou ausência de FSH, foi determinada com o corante vital azul de tripan. FPs foram distribuidos em quatro tratamentos e cultivados em TCM-199 modificado, contendo soro de novilho castrado (SNC, SNC em monocamada de células ovarianas (MCO, MCO com FSH ou meio definido com álcool polivinílico (PVA como macromolécula. A viabilidade celular não foi afetada em conseqüência da presença ou ausência de FSH. No entanto, houve um incremento significativo no tamanho dos FPs cultivados na presença de SNC, MCO e FSH (PThe aim of the present work was to determine the influence of ovarian cells in the in vitro development of preantral follicles (PF. The viability of monolayer ovarian cells and the effect of the serum in the survive of in vitro PF was also investigated. Ovarian cells and PF were isolated from ovaries of bovine fetus between 6 and 8 months of pregnancy, obtained in a slaughterhouse. Monolayer of ovarian cells were cultured in a modified TCM-199 in the presence and absence of FSH and its viability after incubation was determined with Trypan Blue. PFs were divided in four different treatments, cultured in modified TCM-199, containing serum of castrated steer (SCS, SCS in monolayer of ovarian cells (MOC, MOC with FSH or a defined medium with polyvinyl alcohol (PVA as macromolecule. The cellular viability was not affected by the presence or absence of FSH. However, PFs had a significant

  1. Bovine reproduction in tropical environment

    International Nuclear Information System (INIS)

    Jimenez Lopez, J.

    2001-01-01

    In this document it has met relating data to the reproduction of bovine and their handling for the man that it can serve as norms to judge reproductive efficiency but always view in the aspect of the nutritious, climatic circumstances and of handling under which met. Under the previous description one can say that the fertility is the resultant of the interaction among the inheritance, the means and the handling, they vary in particular for each region and property. The fertility can be good, regulate or bad in the measure in that the factors that intervene. The environmental effect on the reproductive processes of the cow represents 80 percent of the variation factors and they include climate, effect of the light, effect of the temperature, effect of the nutritious contribution, effect of psychological factors: the loss of the tendency to the seasonal reproduction is in fact an answer from the animals to its association with the man. The influence of the environment and the feeding of the animals are more intense in the females than in the males, being evidenced that the reproduction control is under the influence hormonal joint with the nutrition. An appropriate nutrition is prerequisite for the beginning of the sexual maturation with an appropriate weight and corporal condition. It is also described the effect and the relationship of the energy contribution about the fertility, the restart of the ovarian activity, its cause of the continuation of the interval childbirth-conception, silent ovulation, organic ancestry and interval among childbirths

  2. Bovine petechial fever (Ondiri disease).

    Science.gov (United States)

    Davies, G

    1993-02-01

    Bovine petechial fever is a Rickettsial disease of cattle, which has been diagnosed, only in Kenya, East Africa. Other countries in the region share some of the biotopes in which the disease occurs, and may well have the infection. The disease is characterised by widespread petechial and ecchymotic haemorrhages on the mucosal surfaces, and throughout the serosal and subserosal surfaces of the body organs and cavities. It may be fatal in up to 50% of untreated cases. The causal organism may be demonstrated most readily in the cytoplasm of polymorphonuclear granulocytes of the peripheral blood, as well as other leucocytes, and has been classified as Cytoecetes ondirii, a member of the tribe Ehrlichiae. Circumstantial and other evidence suggests that the disease is transmitted by an arthropod vector, which has yet to be identified. The blood of a naturally infected wild ruminant, the bushbuck, Tragelaphus scriptus has been shown to remain infective for at least 2 years, and other species such as the African buffalo, Syncercus caffer for at least 5 weeks. These and possibly other species, may serve as the amplifying and reservoir hosts.

  3. DEVELOPMENT OF A MICROCONTROLLED TEMPERATURE MONITORING SYSTEM AND EVALUATION OF THE SENSOR ELEMENT IMPLANT IN BOVINES DESENVOLVIMENTO DE UM SISTEMA MICROCONTROLADO DE MONITORAÇÃO DA TEMPERATURA E AVALIAÇÃO DO IMPLANTE DO ELEMENTO SENSOR DIGITAL EM BOVINOS

    Directory of Open Access Journals (Sweden)

    Ernane José Xavier Costa

    2007-09-01

    Full Text Available

    this paper presents a complete system for tempe-rature monitoring. the system was developed to speed up bovine behavior studies under temperature exposure. the equipment uses digital technology with custom setup ca-pability by means of computer program and the sensor can be implanted in to animal. results obtained show that the developed system is able to monitor bovine temperatures with a sample rate of five minutes during 30 days with accuracy of 0.0625 oc.

    KEY-WORDS: Heat stress sensors, optical, temperature, transceptor.

    um sistema completo de instrumentação para monitoração de temperatura é apresentado neste artigo. o sistema foi desenvolvido para auxiliar estudo de estresse térmico em bovinos. neste equipamento, o elemento sensor pode ser implantado no animal. o equipamento consiste de tecnologia digital e óptica com capacidade de configuração através de programa de computador. os resultados obtidos mostram que o sistema desenvolvido é capaz de monitorar a temperatura de bovinos a cada cinco minutos durante trinta dias com resolução de 0.0625 oc.

    PALAVRAS-CHAVE: estresse térmico sensores, óptico, temperatura, transceptor.  

  4. Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro

    OpenAIRE

    Cong, Shan; Cao, Guifang; Liu, Dongjun

    2014-01-01

    To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic ...

  5. Insights into the bovine rumen plasmidome

    Science.gov (United States)

    Kav, Aya Brown; Sasson, Goor; Jami, Elie; Doron-Faigenboim, Adi; Benhar, Itai; Mizrahi, Itzhak

    2012-01-01

    Plasmids are self-replicating genetic elements capable of mobilization between different hosts. Plasmids often serve as mediators of lateral gene transfer, a process considered to be a strong and sculpting evolutionary force in microbial environments. Our aim was to characterize the overall plasmid population in the environment of the bovine rumen, which houses a complex and dense microbiota that holds enormous significance for humans. We developed a procedure for the isolation of total rumen plasmid DNA, termed rumen plasmidome, and subjected it to deep sequencing using the Illumina paired-end protocol and analysis using public and custom-made bioinformatics tools. A large number of plasmidome contigs aligned with plasmids of rumen bacteria isolated from different locations and at various time points, suggesting that not only the bacterial taxa, but also their plasmids, are defined by the ecological niche. The bacterial phylum distribution of the plasmidome was different from that of the rumen bacterial taxa. Nevertheless, both shared a dominance of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. Evidently, the rumen plasmidome is of a highly mosaic nature that can cross phyla. Interestingly, when we compared the functional profile of the rumen plasmidome to two plasmid databases and two recently published rumen metagenomes, it became apparent that the rumen plasmidome codes for functions, which are enriched in the rumen ecological niche and could confer advantages to their hosts, suggesting that the functional profiles of mobile genetic elements are associated with their environment, as has been previously implied for viruses. PMID:22431592

  6. Membrane associated phospholipase C from bovine brain

    International Nuclear Information System (INIS)

    Lee, K.; Ryu, S.H.; Suh, P.; Choi, W.C.; Rhee, S.G.

    1987-01-01

    Cytosolic fractions of bovine brain contain 2 immunologically distinct phosphoinositide-specific phospholipase (PLC), PLC-I and PLC-II, whose MW are 150,000 and 145,000 respectively, under a denaturing condition. Monoclonal antibodies were derived against each form and specific radioimmunoassays were developed. Distribution of PLC-I and PLC-II in cytosolic and particulate fractions was measured using the radioimmunoassay. More than 90% of PLC-II was found in the cytosolic fraction, while the anti-PLC-I antibody cross-reacting protein was distributed nearly equally between the soluble fraction and the 2 M KCl extract of particulate fraction. The PLC enzyme in the particulate fraction was purified to homogeneity, yielding 2 proteins of 140 KDa and 150 KDa when analyzed on SDS-PAGE. Neither of the 2 enzymes cross-reacted with anti-PLC-II antibodies, but both could be immunoblotted by all 4 different anti-PLC-I antibodies. This suggests that the 140 KDa PLC was derived from the 150 KDa form. The 150 Kda form from particulate fraction was indistinguishable from the cytosolic PLC-I when their mixture was analyzed on SDS-PAGE. In addition, the elution profile of tryptic peptides derived from the 150 KDa particulate form was identical to that of cytosolic PLC-I. This result indicates that PLC-I is reversibly associated to membranes

  7. Advances in lactoferrin research concerning bovine mastitis.

    Science.gov (United States)

    Shimazaki, Kei-Ichi; Kawai, Kazuhiro

    2017-02-01

    Lactoferrin is a multifunctional, iron-binding glycoprotein found in milk and other exocrine secretions. Lactoferrin in milk plays vital roles in the healthy development of newborn mammals, and is also an innate resistance factor involved in the prevention of mammary gland infection by microorganisms. Inflammation of the udder because of bacterial infection is referred to as mastitis. There have been many investigations into the relationships between lactoferrin and mastitis, which fall into several categories. The main categories are fluctuations in the lactoferrin concentration of milk, lactoferrin activity against mastitis pathogens, elucidation of the processes underlying the onset of mastitis, participation of lactoferrin in the immune system, and utilization of lactoferrin in mastitis treatment and prevention. This minireview describes lactoferrin research concerning bovine mastitis. In the 1970s, many researchers reported that the lactoferrin concentration fluctuates in milk from cows with mastitis. From the late 1980s, many studies clarified the infection-defense mechanism in the udder and the contribution of lactoferrin to the immune system. After the year 2000, the processes underlying the onset of mastitis were elucidated in vivo and in vitro, and lactoferrin was applied for the treatment and prevention of mastitis.

  8. Origin and implications of bovine spongiform encephalopathy.

    Science.gov (United States)

    Narang, H

    1996-04-01

    All spongiform encephalopathies in animals, including humans, are slow developing infectious diseases. The current working theory links the origin of bovine spongiform encephalopathy (BSE) to the feeding of cattle with meat and bone meal prepared from scrapie-infected sheep remains. Recycling of cattle meat and bones (MBM) essentially resulted in the selection of a single strain from the "wild type", a mixture of 20 strains. The BSE agent is easily transmitted through ingestion, with some evidence of vertical transmission. Paradoxically, cattle have selected a major new strain which appears to be more virulent than an unselected strain found in scrapie sheep. The same strain of BSE agent is implicated in the occurrence of spongiform encephalopathy in domestic cats, tiger, and some exotic species of ruminants in zoos. The properties of BSE and its spread into cattle are still disputed. Since our understanding of the disease and its transmissibility in humans must await observations that will be made over some years to come, it is important to keep a reasonable perspective and ensure that any speculative comment is consistent with fact. In risk assessment in such circumstances, it is tempting give too much credence to persuasive parallels when direct relevant information is not available. On the other hand, it would also not be wise to assume that the disease will die by itself and will have no effect on humans.

  9. Assessing Tn5 and Sleeping Beauty for transpositional transgenesis by cytoplasmic injection into bovine and ovine zygotes

    Science.gov (United States)

    Bevacqua, R. J.; Fernandez-Martin, R.; Canel, N. G.; Gibbons, A.; Texeira, D.; Lange, F.; Vans Landschoot, G.; Savy, V.; Briski, O.; Hiriart, M. I.; Grueso, E.; Ivics, Z.; Taboga, O.; Kues, W. A.; Ferraris, S.

    2017-01-01

    Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes. In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected. In the case of SB, the supercoiled plasmids encoding a transposon and the SB transposase were co-injected. In vitro produced bovine zygotes were used to establish the cytoplasmic injection conditions. The in vitro cultured blastocysts were evaluated for reporter gene expression and genotyped. Subsequently, both transposon systems were injected in seasonally available ovine zygotes, employing transposons carrying the recombinant human factor IX driven by the beta-lactoglobulin promoter. The Tn5 approach did not result in transgenic lambs. In contrast, the Sleeping Beauty injection resulted in 2 lambs (29%) carrying the transgene. Both animals exhibited cellular mosaicism of the transgene. The extraembryonic tissues (placenta or umbilical cord) of three additional animals were also transgenic. These results show that transpositional transgenesis by cytoplasmic injection of SB transposon components can be applied for the production of transgenic lambs of pharmaceutical interest. PMID:28301581

  10. Hydrometer test for estimation of immunoglobulin concentration in bovine colostrum.

    Science.gov (United States)

    Fleenor, W A; Stott, G H

    1980-06-01

    A practical field method for measuring immunoglobulin concentration in bovine colostrum has been developed from the linear relationship between colostral specific gravity and immunoglobulin concentration. Fourteen colostrums were collected within 24 h postpartum from nursed and unnursed cows and were assayed for specific gravity and major colostral constituents. Additionally, 15 colostrums were collected immediately postpartum prior to suckling and assayed for specific gravity and immunoglobulin concentration. Regression analysis provided an equation to estimate colostral immunoglobulin concentration from the specific gravity of fresh whole colostrum. From this, a colostrometer was developed for practical field use.

  11. Dual effects of fluoxetine on mouse early embryonic development

    International Nuclear Information System (INIS)

    Kim, Chang-Woon; Choe, Changyong; Kim, Eun-Jin; Lee, Jae-Ik; Yoon, Sook-Young; Cho, Young-Woo; Han, Sunkyu; Tak, Hyun-Min; Han, Jaehee; Kang, Dawon

    2012-01-01

    Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50 μM) for different durations. When late 2-cells were incubated with 5 μM fluoxetine for 6 h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetine (5 μM) over 24 h showed a reduction in blastocyst formation. The addition of fluoxetine (5 μM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K + channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ∼ 30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating. Highlights: ► Short-term exposure of 2-cells to fluoxetine enhances mouse blastocyst formation. ► The enhancive effect of fluoxetine is resulted from CaMKII activation

  12. Dual effects of fluoxetine on mouse early embryonic development

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chang-Woon [Department of Physiology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751 (Korea, Republic of); Department of Obstetrics and Gynecology, Samsung Changwon Hospital, Sungkyunkwan University, Changwon 630-723 (Korea, Republic of); Choe, Changyong [National Institute of Animal Science, RDA, Cheonan 330-801 (Korea, Republic of); Kim, Eun-Jin [Department of Physiology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751 (Korea, Republic of); Lee, Jae-Ik [Department of Obstetrics and Gynecology, Gyeongsang National University Hospital, Jinju 660-702 (Korea, Republic of); Yoon, Sook-Young [Fertility Center of CHA Gangnam Medical Center, CHA University, Seoul 135-081 (Korea, Republic of); Cho, Young-Woo; Han, Sunkyu; Tak, Hyun-Min; Han, Jaehee [Department of Physiology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751 (Korea, Republic of); Kang, Dawon, E-mail: dawon@gnu.ac.kr [Department of Physiology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751 (Korea, Republic of)

    2012-11-15

    Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50 μM) for different durations. When late 2-cells were incubated with 5 μM fluoxetine for 6 h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetine (5 μM) over 24 h showed a reduction in blastocyst formation. The addition of fluoxetine (5 μM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K{sup +} channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ∼ 30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating. Highlights: ► Short-term exposure of 2-cells to fluoxetine enhances mouse blastocyst formation. ► The enhancive effect of fluoxetine is resulted from Ca

  13. Raman-microscopy investigation of vitrification-induced structural damages in mature bovine oocytes.

    Directory of Open Access Journals (Sweden)

    Giulia Rusciano

    Full Text Available Although oocyte cryopreservation has great potentials in the field of reproductive technologies, it still is an open challenge in the majority of domestic animals and little is known on the biochemical transformation induced by this process in the different cellular compartments. Raman micro-spectroscopy allows the non-invasive evaluation of the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. The aim of this work was to assess the biochemical modifications of both the zona pellucida and cytoplasm of vitrified/warmed in vitro matured bovine oocytes at different post-warming times. By taking advantage of Principal Component Analysis, we were able to shed light on the biochemical transformation induced by the cryogenic treatment, also pointing out the specific role of cryoprotective agents (CPs. Our results suggest that vitrification induces a transformation of the protein secondary structure from the α-helices to the β-sheet form, while lipids tend to assume a more packed configuration in the zona pellucida. Both modifications result in a mechanical hardening of this cellular compartment, which could account for the reduced fertility rates of vitrified oocytes. Furthermore, biochemical modifications were observed at the cytoplasmic level in the protein secondary structure, with α-helices loss, suggesting cold protein denaturation. In addition, a decrease of lipid unsaturation was found in vitrified oocytes, suggesting oxidative damages. Interestingly, most modifications were not observed in oocytes exposed to CPs, suggesting that they do not severely affect the biochemical architecture of the oocyte. Nevertheless, in oocytes exposed to CPs decreased developmental competence and increased reactive oxygen species production were observed compared to the control. A more severe reduction of cleavage and blastocyst rates after in vitro fertilization was obtained from vitrified oocytes. Our

  14. Expression of infectious bovine rhinotracheitis virus glycoprotein D ...

    African Journals Online (AJOL)

    USER

    2010-06-14

    Jun 14, 2010 ... Bovine Herpesvirus 1 (BHV-1) belongs to the genus of ... through vaccination with recombinant vaccines of thymidine kinase, manufacturing and applying ..... Resistance to bovine respiratory syncytial virus (BRSV) induced in.

  15. The Well-of-the-Well system: an efficient approach to improve embryo development.

    Science.gov (United States)

    Vajta, Gábor; Korösi, Tamás; Du, Yutao; Nakata, Kumiko; Ieda, Shoko; Kuwayama, Masashige; Nagy, Zsolt Peter

    2008-07-01

    Transfer of human embryos at the blastocyst stage may offer considerable benefits including an increased implantation rate and a decreased risk of multiple pregnancies; however, blastocyst culture requires an efficient and reliable in-vitro embryo culture system. In this study, the effect of the Well-of-the-Well (WOW) system consisting of microwells formed on the bottom of the culture dish was tested in three mammalian species, including humans. The WOW system resulted in significant improvement when comparing the drops for culture of in-vitro-matured and parthenogenetically activated porcine oocytes, and in-vivo-derived mouse zygotes. In human embryos, using a sibling oocyte design, embryos cultured in WOW developed to the blastocyst stage in a significantly higher proportion than did embryos cultured traditionally (55% in WOW and 37% in conventional culture; P WOW system or in microdrops. Transferable quality blastocyst development (48.9% of cultured zygotes) was observed in the WOW system. Ninety-four blastocysts transferred to 45 patients resulted in clinical pregnancy rates of 48.9%, including nine twin pregnancies, seven single pregnancies, five miscarriages and one ectopic pregnancy. The results indicate that the WOW system provides a promising alternative for microdrop culture of mammalian embryos, including human embryos.

  16. Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro.

    Science.gov (United States)

    Cong, Shan; Cao, Guifang; Liu, Dongjun

    2014-12-01

    To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1-5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

  17. Bovine besnoitiosis emerging in Central-Eastern Europe, Hungary

    OpenAIRE

    Hornok, Sándor; Fedák, András; Baska, Ferenc; Hofmann-Lehmann, Regina; Basso, Walter

    2014-01-01

    BACKGROUND: Besnoitia besnoiti, the cause of bovine besnoitiosis, is a cyst-forming coccidian parasite that has recently been shown to be spreading in several Western and Southern European countries. FINDINGS: Clinical cases of bovine besnoitiosis were confirmed for the first time in Hungary, by histological, serological and PCR analyses. CONCLUSIONS: This is the first report of autochthonous bovine besnoitiosis in Central-Eastern Europe. The emergence of bovine besnoitiosis in this region re...

  18. Importance of bovine mastitis in Africa.

    Science.gov (United States)

    Motaung, Thabiso E; Petrovski, Kiro R; Petzer, Inge-Marie; Thekisoe, Oriel; Tsilo, Toi J

    2017-06-01

    Bovine mastitis is an important animal production disease that affects the dairy industry globally. Studies have estimated the prevalence of this disease in approximately 30% of African countries, with the highest prevalence found in Ethiopia. This is despite the wide cattle distribution in Africa, and the largest number of dairy farms and herds in countries such as South Africa, Kenya and Uganda. Furthermore, the estimated financial losses due to direct and indirect impacts of bovine mastitis are lacking in this continent. Therefore, intensive research efforts will help determine the continent-wide economic impacts and advance careful monitoring of disease prevalence and epidemiology. Here, published cases supporting the occurrence and importance of bovine mastitis in certain regions of Africa are outlined.

  19. Bovine herpes virus infections in cattle.

    Science.gov (United States)

    Nandi, S; Kumar, Manoj; Manohar, M; Chauhan, R S

    2009-06-01

    Bovine herpes virus 1 (BHV-1) is primarily associated with clinical syndromes such as rhinotracheitis, pustular vulvovaginitis and balanoposthitis, abortion, infertility, conjunctivitis and encephalitis in bovine species. The main sources of infection are the nasal exudates and the respiratory droplets, genital secretions, semen, fetal fluids and tissues. The BHV-1 virus can become latent following a primary infection with a field isolate or vaccination with an attenuated strain. The viral genomic DNA has been demonstrated in the sensory ganglia of the trigeminal nerve in infectious bovine rhinotracheitis (IBR) and in sacral spinal ganglia in pustular vulvovaginitis and balanoposthitis cases. BHV-1 infections can be diagnosed by detection of virus or virus components and antibody by serological tests or by detection of genomic DNA by polymerase chain reaction (PCR), nucleic acid hybridization and sequencing. Inactivated vaccines and modified live virus vaccines are used for prevention of BHV-1 infections in cattle; subunit vaccines and marker vaccines are under investigation.

  20. Increased blastocyst formation of cloned porcine embryos produced with donor cells pre-treated with Xenopus egg extract and/or digitonin

    DEFF Research Database (Denmark)

    Liu, Ying; Østrup, Olga; Li, Juan

    2012-01-01

    from Xenopus laevis eggs. In Experiment 1, fetal fibroblasts were permeabilized by digitonin, incubated in egg extract and, after re-sealing of cell membranes, cultured for 3 or 5 days before use as donor cells in handmade cloning (HMC). Controls were produced by HMC with non-treated donor cells....... The blastocyst rate for reconstructed embryos increased significantly when digitonin-permeabilized, extract-treated cells were used after 5 days of culture after re-sealing. In Experiment 2, fetal and adult fibroblasts were treated with digitonin alone before re-sealing the cell membranes, then cultured for 3...... cells after pre-treatment with permeabilization/re-sealing and Xenopus egg extract. Interestingly, we observe a similar increase in cloning efficiency by permeabilization/re-sealing of donor cells without extract treatment that seems to depend on choice of donor cell type. Thus, pre-treatment of donor...

  1. Cloning and sequencing of the bovine gastrin gene

    DEFF Research Database (Denmark)

    Lund, T; Rehfeld, J F; Olsen, Jørgen

    1989-01-01

    In order to deduce the primary structure of bovine preprogastrin we therefore sequenced a gastrin DNA clone isolated from a bovine liver cosmid library. Bovine preprogastrin comprises 104 amino acids and consists of a signal peptide, a 37 amino acid spacer-sequence, the gastrin-34 sequence followed...

  2. Effect of melatonin on in vitro maturation of bovine oocytes ...

    African Journals Online (AJOL)

    ... Vs 17.67, 15.68, 16.53). In conclusion in this experiment, melatonin cannot improve cumulus cell expansion and nuclear maturation of bovine oocytes. When concentrations is high, melatonin may affect bovine oocytes meiotic maturation at metaphase-1 stage, but it is improbable melatonin be toxic for bovine oocytes.

  3. Sexing bovine pre-implantation embryos using the polymerase ...

    African Journals Online (AJOL)

    The paper aims to present a bovine model for human embryo sexing. Cows were super-ovulated, artificially inseminated and embryos were recovered 7 days later. Embryo biopsy was performed; DNA was extracted from blastomeres and amplified using bovine-specific and bovine-Y-chromosomespecific primers, followed ...

  4. 76 FR 26239 - Bovine Tuberculosis and Brucellosis; Public Meetings

    Science.gov (United States)

    2011-05-06

    ... Inspection Service [Docket No. APHIS-2011-0044] Bovine Tuberculosis and Brucellosis; Public Meetings AGENCY... bovine tuberculosis and brucellosis programs in the United States. The meetings are being organized by... tuberculosis (TB) and bovine brucellosis in the United States. In keeping with its commitment to partnering...

  5. Study of the immune response by antibodies against the Bothrops asper venom for the elaboration of a antiophidic vaccine for bovines

    International Nuclear Information System (INIS)

    Gonzalez Rojas, Katherine

    2014-01-01

    Active immunization has determined against Bothrops asper snake venom (tested in murine and bovine models) a induced response by antibody able to prevent in immunized animals. A coagulopathy or death is developed after of be administered with adequate doses of poison. The amount of B. asper venom has defined the poisoning induced in bovine and murine models. The plasmatic concentration of equine antibodies against B. asper venom is specified to prevent coagulopathy and lethality induced by this venom in murine and bovine models. Murine and bovine models have verified the active immunization reached in a concentration of antibodies against B. asper venom equal or greater to the maximum concentration achieved by intravenous administration of antivenoms from equine origin. The concentration of antibodies induced by the active immunization is evaluated against B. asper venom to prevent the development of coagulopathy and lethality induced by the venom in murine and bovine models [es

  6. Radiography of the bovine cranioventral abdomen

    International Nuclear Information System (INIS)

    Partington, B.P.; Biller, D.S.

    1991-01-01

    This study consists of a review of 115 consecutively accrued bovine cranial abdomial radiographs. The sensitivity and specificity of radiography in detecting traumatic reticuloperitonitis or pericarditis was 83% and 90%, respectively. Increased reticular size was associated with vagal indigestion. Increased reticulo-diaphragmatic separation did not correlate with a specific disease process, 25/35 (71%) cattle presented for radiographic examination with a 1 cm or longer metallic reticular foreign body unattached to a magnet had traumatic reticuloperitonitis. Standing lateral abdominal radiographs were determined to be a valuable tool in the diagnosis of cranial abdominal disorders in the bovine

  7. Mapping and polymorphism of bovine ghreline gene

    OpenAIRE

    Colinet, Frédéric; Eggen, André; Halleux, Caroline; Arnould, Valérie; Portetelle, Daniel; Renaville, Robert

    2006-01-01

    Bovine ghrelin, a 27-amino-acid peptide has been identified in bovine oxyntic glands of the abomasum. It is an endogenous growth hormone secretagogue. Total mRNA was extracted from abomasum and complete ghrelin mRNA was sequenced by rapid amplification of cDNA ends. The gene contains five exons and four introns with a short noncoding first exon of 17 bp similar to mouse and human ghrelin gene. Using a radiation hybrid panel, the gene was mapped to chromosome 22 near microsat...

  8. Effect of gamma irradiation on in vitro bovine lens proteins

    International Nuclear Information System (INIS)

    Bernardes, D.M.L.; Mastro, N.L. del.

    1988-07-01

    The radiosensitivity of the ocular lens manifested by cataract formation has been of considerable interest in the study on the biological efects of radiations. Cataract can ben produced by different causes and also for the normal process of ageing. The aim of this work was to develop an in vitro system similar to in vivo cataract formation. It was used an aqueous solution of bovine lenses. The lenses after surgical removal mechanical and ultrasonic disrupted. The suspension was centrifuged and the supernatant was dialyzed and irradiated with different doses of 60 Co radiation. The opacification extent was measured in an spectrophotometer. (author) [pt

  9. Effect of chloramphenicol on sister chromatid exchange in bovine fibroblasts.

    Science.gov (United States)

    Arruga, M V; Catalan, J; Moreno, C

    1992-03-01

    The genotoxic potential of different chloramphenicol concentrations (5, 20, 40 and 60 micrograms ml-1) was investigated in bovine fibroblast primary lines by sister chromatid exchange assay. Chloramphenicol acted for long enough to ensure similar effects to persistent storage in the kidney. In this experiment 10 micrograms ml-1 of 5-bromodeoxyuridine was added for 60 hours for all doses of chloramphenicol and to the control. When the tissue culture cells were exposed to increasing doses, increased numbers of sister chromatid exchanges developed. Differences were significantly different to the control.

  10. Non-specific esterases in partly mineralized bovine enamel

    DEFF Research Database (Denmark)

    Moe, D; Kirkeby, S

    1990-01-01

    Activity for non-specific esterase was demonstrated in the matrix of developing bovine enamel with alpha-naphthyl acetate and 5-bromoindoxyl acetate as the esterase substrates. By use of high-performance liquid chromatography gel filtration, ion-exchange chromatography, and electrophoresis three...... esterases were shown to be present in the enamel matrix. The enzymes showed highest activity at pH 6.5-7.5. In sections a strong reaction was observed in the secretory ameloblasts. The esterases may be proteolytic enzymes that participate in the degradation of the matrix proteins....

  11. Pathogenesis and Treatment of Bovine Foot Rot.

    Science.gov (United States)

    Van Metre, David C

    2017-07-01

    Bovine foot rot (BFR) is an infectious disease of the interdigital skin and subcutaneous tissues of beef and dairy cattle that occurs under a variety of management and environmental settings. The anaerobic, gram-negative bacteria Fusobacterium necrophorum, Porphyromonas levii, and Prevotella intermedia are commonly isolated from lesions. A multitude of host, agent, and environmental factors contribute to the development of BFR. Initiation of systemic antimicrobial therapy early in the course of disease commonly leads to resolution. Delays in treatment may result in extension of infection into deeper bone, synovial structures, or ligamentous structures, and the prognosis for recovery is reduced. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Pre- and Peri-/Post-Compaction Follistatin Treatment Increases In Vitro Production of Cattle Embryos.

    Directory of Open Access Journals (Sweden)

    Guo Zhenhua

    Full Text Available Our previous studies demonstrated that maternal (oocyte derived follistatin (FST expression is positively associated with bovine oocyte competence and exogenous follistatin treatment during the pre-compaction period of development (d 1-3 post insemination is stimulatory to bovine early embryogenesis in vitro [blastocyst rates and cell numbers/allocation to trophectoderm (TE]. In the present study, bovine embryos were treated with exogenous follistatin during d 1-3, d 4-7 and d 1-7 post insemination to test the hypothesis that embryotropic effects of exogenous follistatin are specific to the pre-compaction period (d 1-3 of early embryogenesis. Follistatin treatment during d 4-7 (peri-/post-compaction period of embryo culture increased proportion of embryos reaching blastocyst and expanded blastocyst stage and total cell numbers compared to controls, but blastocyst rates and total cell numbers were lower than observed following d 1-3 (pre-compaction follistatin treatment. Follistatin supplementation during d 1-7 of embryo culture increased development to blastocyst and expanded blastocyst stages and blastocyst total cell numbers compared to d 1-3 and d 4-7 follistatin treatment and untreated controls. A similar increase in blastocyst CDX2 mRNA and protein (TE cell marker was observed in response to d 1-3, d 4-7 and d 1-7 follistatin treatment. However, an elevation in blastocyst BMP4 protein (TE cell regulator was observed in response to d 1-3 and d 1-7, but not d 4-7 (peri-/post-compaction follistatin treatment. In summary, our study revealed the potential utility of follistatin treatment for increasing the success rate of in vitro embryo production in cattle. Such results also expand our understanding of the embryotropic actions of follistatin and demonstrate that follistatin actions on blastocyst development and cell allocation to the TE layer are not specific to the pre-compaction period.

  13. Extraction and characterization of highly purified collagen from bovine pericardium for potential bioengineering applications

    International Nuclear Information System (INIS)

    Santos, Maria Helena; Silva, Rafael M.; Dumont, Vitor C.; Neves, Juliana S.; Mansur, Herman S.; Heneine, Luiz Guilherme D.

    2013-01-01

    Bovine pericardium is widely used as a raw material in bioengineering as a source of collagen, a fundamental structural molecule. The physical, chemical, and biocompatibility characteristics of these natural fibers enable their broad use in several areas of the health sciences. For these applications, it is important to obtain collagen of the highest possible purity. The lack of a method to produce these pure biocompatible materials using simple and economically feasible techniques presents a major challenge to their production on an industrial scale. This study aimed to extract, purify, and characterize the type I collagen protein originating from bovine pericardium, considered to be an abundant tissue resource. The pericardium tissue was collected from male animals at slaughter age. Pieces of bovine pericardium were enzymatically digested, followed by a novel protocol developed for protein purification using ion-exchange chromatography. The material was extensively characterized by electrophoresis, scanning electron microscopy, energy dispersive X-ray spectroscopy, and infrared spectroscopy. The results showed a purified material with morphological properties and chemical functionalities compatible with type I collagen and similar to a highly purified commercial collagen. Thus, an innovative and relatively simple processing method was developed to extract and purify type I collagen from bovine tissue with potential applications as a biomaterial for regenerative tissue engineering. - Highlights: ► Type I collagen was obtained from bovine pericardium, an abundant tissue resource. ► A simple and feasible processing technique was developed to purify bovine collagen. ► The appropriate process may be performed on industrial scale. ► The pure collagen presented appropriate morphological and molecular characteristics. ► The purify collagen has shown potential use as a biomaterial in tissue engineering.

  14. Extraction and characterization of highly purified collagen from bovine pericardium for potential bioengineering applications

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Maria Helena, E-mail: mariahelena.santos@gmail.com [Department of Dentistry, Federal University of Vales do Jequitinhonha e Mucuri-UFVJM, Diamantina/MG 39100-000 (Brazil); Center for Assessment and Development of Biomaterials-BioMat, Federal University of Vales do Jequitinhonha e Mucuri-UFVJM, Diamantina/MG 39100-000 (Brazil); Silva, Rafael M.; Dumont, Vitor C. [Department of Dentistry, Federal University of Vales do Jequitinhonha e Mucuri-UFVJM, Diamantina/MG 39100-000 (Brazil); Center for Assessment and Development of Biomaterials-BioMat, Federal University of Vales do Jequitinhonha e Mucuri-UFVJM, Diamantina/MG 39100-000 (Brazil); Neves, Juliana S. [Center for Assessment and Development of Biomaterials-BioMat, Federal University of Vales do Jequitinhonha e Mucuri-UFVJM, Diamantina/MG 39100-000 (Brazil); Mansur, Herman S. [Department of Metallurgical and Materials Engineering, Federal University of Minas Gerais-UFMG, Belo Horizonte/MG 31270-901 (Brazil); Heneine, Luiz Guilherme D. [Department of Health Science, Ezequiel Dias Foundation-FUNED, Belo Horizonte/MG 30510-010 (Brazil)

    2013-03-01

    Bovine pericardium is widely used as a raw material in bioengineering as a source of collagen, a fundamental structural molecule. The physical, chemical, and biocompatibility characteristics of these natural fibers enable their broad use in several areas of the health sciences. For these applications, it is important to obtain collagen of the highest possible purity. The lack of a method to produce these pure biocompatible materials using simple and economically feasible techniques presents a major challenge to their production on an industrial scale. This study aimed to extract, purify, and characterize the type I collagen protein originating from bovine pericardium, considered to be an abundant tissue resource. The pericardium tissue was collected from male animals at slaughter age. Pieces of bovine pericardium were enzymatically digested, followed by a novel protocol developed for protein purification using ion-exchange chromatography. The material was extensively characterized by electrophoresis, scanning electron microscopy, energy dispersive X-ray spectroscopy, and infrared spectroscopy. The results showed a purified material with morphological properties and chemical functionalities compatible with type I collagen and similar to a highly purified commercial collagen. Thus, an innovative and relatively simple processing method was developed to extract and purify type I collagen from bovine tissue with potential applications as a biomaterial for regenerative tissue engineering. - Highlights: Black-Right-Pointing-Pointer Type I collagen was obtained from bovine pericardium, an abundant tissue resource. Black-Right-Pointing-Pointer A simple and feasible processing technique was developed to purify bovine collagen. Black-Right-Pointing-Pointer The appropriate process may be performed on industrial scale. Black-Right-Pointing-Pointer The pure collagen presented appropriate morphological and molecular characteristics. Black-Right-Pointing-Pointer The purify

  15. Selection of possible signature peptides for the detection of bovine lactoferrin in infant formulas by LC-MS/MS.

    Directory of Open Access Journals (Sweden)

    Mingmei Yuan

    Full Text Available An LC-MS/MS assay based on a signature peptide was developed and fully validated for the quantitation of bovine lactoferrin in infant formulas. Three unreported signature peptides were derived and identified from the tryptic peptides of bovine lactoferrin. The peptide ETTVFENLPEK was used for quantification based on assay performance. The blank matrix camel milk powder and bovine lactoferrin protein standards were mixed and spiked with stable isotope-labeled internal standard to establish a calibration curve. The established method was extensively validated by determining the linearity (R2 > 0.999, sensitivity (limit of quantitation, 0.16 mg/100 g, recovery (83.1-91.6%, precision (RSD < 5.4% and repeatability (RSD < 7.7%. To validate the applicability of the method, four different brands of infant formulas in China were analysed. The acquired contents of bovine lactoferrin were 52.60-150.56 mg/100 g.

  16. Gamma irradiation attenuation of Anaplasma Marginale and its possible use as vaccine against bovine anaplasmosis

    International Nuclear Information System (INIS)

    Castro Z, J.; Pupiales T, N.

    1986-01-01

    Bovine Anaplasmosis is one of the limiting in the cattle development at Colombia and others tropical countries. Bovine Anaplasmosis is an infectious disease caused by a microorganism called Anaplasma Marginale, it with invades the red globules producing erythrofagocitosis. In this job, were irradiated with different gamma beam intensity; microorganisms of Anaplasma Marginale, those which were used as innocuous. Animals of the groups (I,II,III) presented good protection against the disease; animals of the Group IV presented mild symptoms with subsequent recovery and strong symptoms

  17. Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos

    International Nuclear Information System (INIS)

    Souza, D.K. de; Salles, L.P.; Rosa e Silva, A.A.M.

    2015-01-01

    Although the metabolism of early bovine embryos has not been fully elucidated, several publications have addressed this important issue to improve culture conditions for cattle reproductive biotechnologies, with the ultimate goal of producing in vitro embryos similar in quality to those developing in vivo. Here, we review general aspects of bovine embryo metabolism in vitro and in vivo, and discuss the use of metabolic analysis of embryos produced in vitro to assess viability and predict a viable pregnancy after transference to the female tract

  18. Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos

    Energy Technology Data Exchange (ETDEWEB)

    Souza, D.K. de [Laboratório de Biotecnologia da Saúde, Faculdade de Medicina, Universidade de Brasília, Brasília, DF (Brazil); Faculdade da Ceilândia, Universidade de Brasília, Brasília, DF (Brazil); Salles, L.P. [Laboratório de Biotecnologia da Saúde, Faculdade de Medicina, Universidade de Brasília, Brasília, DF (Brazil); Departamento de Biologia Molecular, Instituto de Biologia, Universidade de Brasília, Brasília, DF (Brazil); Rosa e Silva, A.A.M. [Laboratório de Biotecnologia da Saúde, Faculdade de Medicina, Universidade de Brasília, Brasília, DF (Brazil)

    2015-01-23

    Although the metabolism of early bovine embryos has not been fully elucidated, several publications have addressed this important issue to improve culture conditions for cattle reproductive biotechnologies, with the ultimate goal of producing in vitro embryos similar in quality to those developing in vivo. Here, we review general aspects of bovine embryo metabolism in vitro and in vivo, and discuss the use of metabolic analysis of embryos produced in vitro to assess viability and predict a viable pregnancy after transference to the female tract.

  19. Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos

    Directory of Open Access Journals (Sweden)

    D.K. de Souza

    2015-03-01

    Full Text Available Although the metabolism of early bovine embryos has not been fully elucidated, several publications have addressed this important issue to improve culture conditions for cattle reproductive biotechnologies, with the ultimate goal of producing in vitro embryos similar in quality to those developing in vivo. Here, we review general aspects of bovine embryo metabolism in vitro and in vivo, and discuss the use of metabolic analysis of embryos produced in vitro to assess viability and predict a viable pregnancy after transference to the female tract.

  20. Control of bovine hepatic fatty acid oxidation

    International Nuclear Information System (INIS)

    Jesse, B.W.; Emery, R.S.; Thomas, J.W.

    1986-01-01

    Fatty acid oxidation by bovine liver slices and mitochondria was examined to determine potential regulatory sites of fatty acid oxidation. Conversion of 1-[ 14 C]palmitate to 14 CO 2 and total [ 14 C]acid-soluble metabolites was used to measure fatty acid oxidation. Oxidation of palmitate (1 mM) was linear in both liver slice weight and incubation time. Carnitine stimulated palmitate oxidation; 2 mM dl-carnitine produced maximal stimulation of palmitate oxidation to both CO 2 and acid-soluble metabolites. Propionate (10 mM) inhibited palmitate oxidation by bovine liver slices. Propionate (.5 to 10 mM) had no effect on palmitate oxidation by mitochondria, but malonyl Coenzyme A, the first committed intermediate of fatty acid synthesis, inhibited mitochondrial palmitate oxidation (inhibition constant = .3 μM). Liver mitochonndrial carnitine palmitoyltransferase exhibited Michaelis constants for palmitoyl Coenzyme A and l-carnitine of 11.5 μM and .59 mM, respectively. Long-chain fatty acid oxidation in bovine liver is regulated by mechanisms similar to those in rats but adapted to the unique digestive physiology of the bovine

  1. Pathogenesis of bovine spongiform encephalopathy in sheep

    NARCIS (Netherlands)

    Keulen, van L.J.M.; Vromans, M.E.W.; Dolstra, C.H.; Bossers, A.; Zijderveld, van F.G.

    2008-01-01

    The pathogenesis of bovine spongiform encephalopathy (BSE) in sheep was studied by immunohistochemical detection of scrapie-associated prion protein (PrPSc) in the gastrointestinal, lymphoid and neural tissues following oral inoculation with BSE brain homogenate. First accumulation of PrPSc was

  2. diagnosis of bovine cysticercosis in Kenyan cattle

    African Journals Online (AJOL)

    A total of 55 cattle divided into two groups of experimentally (n =30) and naturally ... sensitive than meat inspection in the diagnosis of bovine cysticercosis, detecting .... The second group of 15 calves was ... ined for the presence of C. bovis. .... variable and pour ' ..... appropriate intermediate host is dependent on:- the state.

  3. Aggregation and fibrillation of bovine serum albumin

    DEFF Research Database (Denmark)

    Holm, NK; Jespersen, SK; Thomassen, LV

    2007-01-01

    The all-alpha helix multi-domain protein bovine serum albumin (BSA) aggregates at elevated temperatures. Here we show that these thermal aggregates have amyloid properties. They bind the fibril-specific dyes Thioflavin T and Congo Red, show elongated although somewhat worm-like morphology...

  4. Chemotherapy of experimental bovine petechial fever.

    Science.gov (United States)

    Snodgrass, D R

    1976-01-01

    A dithiosemicarbazone was compared with two tetracycline formulations in the treatment of bovine petechial fever (BPF) in experimentally infected sheep, and was then used to treat the disease in experimental cattle. The dithiosemicarbazone was found to be more efficacious than either of the other two drugs in treating ovine BPF, and also to be effective against BPF in cattle.

  5. Concomitant infection of Neospora caninum and Bovine Herpesvirus type 5 in spontaneous bovine abortions

    Directory of Open Access Journals (Sweden)

    Maia S. Marin

    2013-11-01

    Full Text Available Bovine Herpesvirus type 5 (BoHV-5 has not been conclusively demonstrated to cause bovine abortion. Brain lesions produced by Neospora caninum and Bovine Herpesvirus type 1 (BoHV-1 exhibit common features. Therefore, careful microscopic evaluation and additional diagnostic procedures are required to achieve an accurate final etiological diagnosis. The aim of the present work was to investigate the occurrence of infections due to BoHV-1, BoHV-5 and N. caninum in 68 cases of spontaneous bovine abortions which showed microscopic lesions in the fetal central nervous system. This study allowed the identification of 4 (5.9% fetuses with dual infection by BoHV-5 and N. caninum and 33 (48.5% cases in which N. caninum was the sole pathogen identified. All cases were negative to BoHV-1. The results of this study provide evidence that dual infection by BoHV-5 and N. caninum occur during pregnancy in cattle; however, the role of BoHV-5 as a primary cause of bovine abortion needs further research. Molecular diagnosis of BoHV-5 and N. caninum confirmed the importance of applying complementary assays to improve the sensitivity of diagnosing bovine abortion.

  6. Detection and identification of the atypical bovine pestiviruses in commercial foetal bovine serum batches.

    Directory of Open Access Journals (Sweden)

    Hongyan Xia

    Full Text Available The recently emerging atypical bovine pestiviruses have been detected in commercial foetal bovine serum (FBS of mainly South American origin so far. It is unclear how widely the viruses are presented in commercial FBS of different geographic origins. To further investigate the possible pestivirus contamination of commercially available FBS batches, 33 batches of FBS were obtained from ten suppliers and analysed in this study for the presence of both the recognised and the atypical bovine pestiviruses. All 33 batches of FBS were positive by real-time RT-PCR assays for at least one species of bovine pestiviruses. According to the certificate of analysis that the suppliers claimed for each batch of FBS, BVDV-1 was detected in all 11 countries and BVDV-2 was detected exclusively in the America Continent. The atypical pestiviruses were detected in 13 batches claimed to originate from five countries. Analysis of partial 5'UTR sequences showed a high similarity among these atypical bovine pestiviruses. This study has demonstrated, for the first time that commercial FBS batches of different geographic origins are contaminated not only with the recognised species BVDV-1 and BVDV-2, but also with the emerging atypical bovine pestiviruses.

  7. Endometrial thickness as a predictor of the reproductive outcomes in fresh and frozen embryo transfer cycles: A retrospective cohort study of 1512 IVF cycles with morphologically good-quality blastocyst.

    Science.gov (United States)

    Zhang, Tao; Li, Zhou; Ren, Xinling; Huang, Bo; Zhu, Guijin; Yang, Wei; Jin, Lei

    2018-01-01

    To evaluate the relationship between endometrial thickness during fresh in vitro fertilization (IVF) cycles and the clinical outcomes of subsequent frozen embryo transfer (FET) cycles.FET cycles using at least one morphological good-quality blastocyst conducted between 2012 and 2013 at a university-based reproductive center were reviewed retrospectively. Endometrial ultrasonographic characteristics were recorded both on the oocyte retrieval day and on the day of progesterone supplementation in FET cycles. Clinical pregnancy rate, spontaneous abortion rate, and live birth rate were analyzed.One thousand five hundred twelve FET cycles was included. The results showed that significant difference in endometrial thickness on day of oocyte retrieval (P = .03) was observed between the live birth group (n = 844) and no live birth group (n = 668), while no significant difference in FET endometrial thickness was found (P = .261) between the live birth group and no live birth group. For endometrial thickness on oocyte retrieval day, clinical pregnancy rate ranged from 50.0% among patients with an endometrial thickness of ≤6 mm to 84.2% among patients with an endometrial thickness of >16 mm, with live birth rate from 33.3% to 63.2%. Multiple logistic regression analysis of factors related to live birth indicated endometrial thickness on oocyte retrieval day was associated with improved live birth rate (OR was 1.069, 95% CI: 1.011-1.130, P = .019), while FET endometrial thickness did not contribute significantly to pregnancy outcomes following FET cycles. The ROC curves revealed the cut-off points of endometrial thickness on oocyte retrieval day was 8.75 mm for live birth.Endometrial thickness during fresh IVF cycles was a better predictor of endometrial receptivity in subsequent FET cycles than FET cycle endometrial thickness. For those females with thin endometrium in fresh cycles, additional estradiol stimulation might be helpful for adequate

  8. Novel Polymerase Spiral Reaction (PSR) for rapid visual detection of Bovine Herpesvirus 1 genomic DNA from aborted bovine fetus and semen.

    Science.gov (United States)

    Malla, Javed Ahmed; Chakravarti, Soumendu; Gupta, Vikas; Chander, Vishal; Sharma, Gaurav Kumar; Qureshi, Salauddin; Mishra, Adhiraj; Gupta, Vivek Kumar; Nandi, Sukdeb

    2018-02-20

    Bovine herpesvirus-1 (BHV-1) is a major viral pathogen affecting bovines leading to various clinical manifestations and causes significant economic impediment in modern livestock production system. Rapid, accurate and sensitive detection of BHV-1 infection at frozen semen stations or at dairy herds remains a priority for control of BHV-1 spread to susceptible population. Polymerase Spiral Reaction (PSR), a novel addition in the gamut of isothermal techniques, has been successfully implemented in initial optimization for detection of BHV-1 genomic DNA and further validated in clinical samples. The developed PSR assay has been validated for detection of BHV-1 from bovine semen (n=99), a major source of transmission of BHV-1 from breeding bulls to susceptible dams in artificial insemination programs. The technique has also been used for screening of BHV-1 DNA from suspected aborted fetal tissues (n=25). The developed PSR technique is 100 fold more sensitive than conventional PCR and comparable to real-time PCR. The PSR technique has been successful in detecting 13 samples positive for BHV-1 DNA in bovine semen, 4 samples more than conventional PCR. The aborted fetal tissues were negative for presence of BHV-1 DNA. The presence of BHV-1 in bovine semen samples raises a pertinent concern for extensively screening of semen from breeding bulls before been used for artificial insemination process. PSR has all the attributes for becoming a method of choice for rapid, accurate and sensitive detection of BHV-1 DNA at frozen semen stations or at dairy herds in resource constrained settings. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Evaluation of ethanol vortex ELISA for detection of bovine tuberculosis in cattle and deer

    Science.gov (United States)

    Background The use of serological assays for diagnosis of bovine tuberculosis (TB) has been intensively studied and use of specific antigens have aided in improving the diagnostic accuracy of the assays. In the present study, we report an in-house enzyme linked immunosorbent assay (ELISA), developed...

  10. Virulence and genotype of a bovine herpesvirus 1 isolate from semen of a subclinically infected bull

    NARCIS (Netherlands)

    Oirschot, van J.T.; Rijsewijk, F.A.M.; Straver, P.J.; Ruuls, R.C.; Quak, J.; Davidse, A.; Westenbrink, E.; Gielkens, A.L.J.; Dijk, van J.E.; Moerman, A.

    1995-01-01

    A bovine herpesvirus 1 (BHV-1) isolate from the semen of a subclinically infected bull was administered to cattle by various routes to assess its virulence. Cattle that were artificially inseminated or inoculated intrapreputially did not develop clinical signs, but did transmit the virus to contact

  11. Measuring bovine gamma delta T cell function at the site of Mycobacterium bovis infection

    Science.gov (United States)

    Bovine gamma delta T cells are amongst the first cells to accumulate at the site of Mycobacterium bovis infection; however, their role in the developing lesion remains unclear. We utilized transcriptomics analysis, in situ hybridization, and a macrophage/gamma delta T cell co-culture system to eluc...

  12. Sensitive detection of Myobacterium avium subsp paratuberculosis in bovine semen by real-time PCR

    NARCIS (Netherlands)

    Herthnek, D.; Englund, S.; Willemsen, P.T.J.; Bolske, G.

    2006-01-01

    Aims: To develop a fast and sensitive protocol for detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine semen and to make a critical evaluation of the analytical sensitivity. Methods and Results: Processed semen was spiked with known amounts of MAP. Semen from different bulls as

  13. Effect of oxygen concentration on human embryo development evaluated by time-lapse monitoring

    DEFF Research Database (Denmark)

    Ingerslev, Hans Jakob; Hindkjær, Johnny Juhl; Kirkegaard, Kirstine

    2012-01-01

    -points for each cell division and blastocyst stages were registered until 120 hours after oocyte retrieval. Only 2PN embryos completing the first cleavage were evaluated. The groups were compared using one-way ANOVA or Kruskall-Wallis test. Estimates are reported as medians with 95% confidence intervals. Time......Introduction: Data from a number of studies indicate -but not unequivocally- that culture of embryos in 5% O2 compared to 20% O2 improves blastocyst formation in humans and various animal species and may yield better pregnancy rates in IVF. The detrimental effects of atmospheric oxygen were...... was to evaluate the influence of oxygen tension on human pre-implantation development using time-lapse monitoring. Materials and methods: Human embryos were cultured to the blastocyst stage in a time-lapse incubator (EmbryoScope™) in 20% O2 (group 1), 20% O2 for 24 hours followed by culture in 5% O2 (group 2...

  14. Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

    Science.gov (United States)

    Lee, Jae Eun; Lee, Jae Young; Kim, Hong Rye; Shin, Hyun Young; Lin, Tao; Jin, Dong Il

    2015-01-01

    Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum. PMID:25925056

  15. Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

    Directory of Open Access