WorldWideScience

Sample records for bovine embryos cultured

  1. 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.

  2. Effect of sericin on preimplantation development of bovine embryos cultured individually.

    Science.gov (United States)

    Isobe, T; Ikebata, Y; Onitsuka, T; Wittayarat, M; Sato, Y; Taniguchi, M; Otoi, T

    2012-09-01

    The silk protein sericin has been identified as a potent antioxidant in mammalian cells. This study was conducted to examine the effects of sericin on preimplantation development and quality of bovine embryos cultured individually. When two-cell-stage embryos were cultured individually for 7 days in CR1aa medium supplemented with 0, 0.1, 0.5, or 1% sericin, rates of total blastocyst formation and development to expanded blastocysts from embryos cultured with 0.5% sericin were higher (P sericin. When embryos were cultured individually for 7 days in the CR1aa medium supplemented with 0 or 0.5% sericin under two oxidative stress conditions (50 or 100 μm H(2)O(2)), the addition of sericin significantly improved the blastocyst formation rate of embryos exposed to 100 μm H(2)O(2). However, the protective effect of sericin was not observed in development of embryos exposed to 50 μm H(2)O(2). When embryos were exposed to 100 μm H(2)O(2) during culture, the DNA fragmentation index of total blastocysts from embryos cultured with 0.5% sericin was lower than blastocysts derived from embryos cultured without sericin (4.4 vs. 6.8%; P sericin to in vitro culture medium improved preimplantation development and quality of bovine embryos cultured individually by preventing oxidative stress. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Supplementation of bovine embryo culture medium with L-arginine improves embryo quality via nitric oxide production.

    Science.gov (United States)

    Santana, Priscila Di Paula Bessa; Silva, Thiago Velasco Guimarães; da Costa, Nathália Nogueira; da Silva, Bruno Barauna; Carter, Timothy Frederick; Cordeiro, Marcela da Silva; da Silva, Bruno José Martins; Santos, Simone do Socorro Damasceno; Herculano, Anderson Manoel; Adona, Paulo Roberto; Ohashi, Otávio Mitio; Miranda, Moysés dos Santos

    2014-10-01

    Nitric oxide (NO) is a cell-signaling molecule that regulates a variety of molecular pathways. We investigated the role of NO during preimplantation embryonic development by blocking its production with an inhibitor or supplementing in vitro bovine embryo cultures with its natural precursor, L-arginine, over different periods. Endpoints evaluated included blastocyst rates, development kinetics, and embryo quality. Supplementation with the NO synthase inhibitor N-Nitro-L-arginine-methyl ester (L-NAME) from Days 1 to 8 of culture decreased blastocyst (P L-arginine decreased blastocyst rates (P L-arginine improved embryo hatching rates (P supplementing the embryo culture medium with L-arginine favors preimplantation development of bovine embryos. © 2014 Wiley Periodicals, Inc.

  4. 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

  5. Influence of "Solcoseryl" during culture on the sex-dependent repair of bovine demi-embryos.

    Science.gov (United States)

    Tominaga, K; Yoneda, K; Utsumi, K

    1996-03-01

    The purpose of this experiment was to determine the effect of culture conditions on the development of split embryos after bisection and on the sex ratio of resultant bovine demi-embryos. Embryos that had developed into blastocysts on days 6 1/2 to 7 or on days 7 1/2 to 8 from oocytes matured and fertilized in vitro were bisected in BMOC-3 medium supplemented with 33% calf serum. The medium also contained 0%, 0.1% or 1.0% Solcoseryl, a deproteinized hemodialysate product from calf blood. The demi-embryos were first cultured for 4 hours in the same medium in which they had been bisected and then co-cultured with cumulus cells in TCM199 supplemented with 1% calf serum for an additional 20 hr. The rate of production of good to excellent quality demi-embryos obtained from days 6 1/2 to 7 blastocysts was higher than from those on days 7 1/2 to 8. The rate was also significantly improved when blastocysts were bisected in medium containing 0.1% or 1.0% Solcoseryl, compared to the medium without Solcoseryl. Male embryos seemed to recover more rapidly than female embryos, as assessed by morphological quality at 4 hr, although the quality of female embryos had improved by 24 hr. The percentage of males after culture was higher in the medium without Solcoseryl than in its presence. Thus, addition of Solcoseryl at either 0.1% or 1.0% to BMOC-3 medium seemed to improve the production efficiency of good quality demi-embryos, but did not influence the sex ratio. It appears as if female demi-embryos required more time than male embryos to be repaired after bisection.

  6. 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.

  7. 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.

  8. Inclusion of bovine lipoproteins and the vitamin E analogue, Trolox, during in vitro culture of bovine embryos changes both embryo and fetal development.

    Science.gov (United States)

    Rooke, J A; Watt, R G; Ashworth, C J; McEvoy, T G

    2012-01-01

    This experiment investigated effects of lipoproteins and Trolox (vitamin E analogue) on bovine embryo and fetal development. The treatments were: in vitro culture (IVC) in synthetic oviducal fluid alone (SOF); with bovine lipoproteins (2% v/v; SOFLP); with Trolox (100μM; SOFT); and with lipoproteins and Trolox (SOFLPT). In vitro culture with lipoproteins increased fatty acid content of blastocysts (Peffect (P>0.05). Whereas lipoproteins reduced zygote development to blastocysts (P=0.03), Trolox facilitated increased development (P0.05) had no effect on blastocyst morphological grade. Pregnancy rates resulting from synchronous transfer of IVP embryos were not affected by IVC treatment. At Day 70 of pregnancy, compared with SOF, fetal weight was lower in SOFLP but not SOFLPT (interaction, Pembryo grades were also associated with increased numbers of placentomes (P=0.024). In conclusion, the interactive effects of lipoprotein and Trolox inclusion on in vitro embryo development were also evident in fetal development at Day 70.

  9. Effect of supplementation of different growth factors in embryo culture medium with a small number of bovine embryos on in vitro embryo development and quality.

    Science.gov (United States)

    Ahumada, C J; Salvador, I; Cebrian-Serrano, A; Lopera, R; Silvestre, M A

    2013-03-01

    When embryos are cultured individually or in small groups, blastocyst yield efficiency and quality are usually reduced. The aim of this work was to investigate the effect of supplementation of the embryo culture medium (CM) with several growth factors (GFs) on embryo development and apoptosis rate when a reduced number of embryos were in vitro cultured. Two experimental studies (ES) were carried out. In ES 1, five treatments were tested to study the effect of GF on embryo development: Control (∼30 to 50 embryos cultured in 500 μl of CM); Control 5 (Five embryos cultured in 50 μl microdrops of CM), without addition of GF in either of the two control groups; epidermal GF (EGF); IGF-I; and transforming GF-α (TGF-α) (Five embryos were cultured in 50 μl microdrops of CM with 10 ng/ml EGF, 10 ng/ml IGF-I or 10 ng/ml TGF-α, respectively). In ES 2, following the results obtained in ES 1, four different treatments were tested to study their effect on embryo development and quality (number of cells per blastocyst and apoptotic rate): Control; Control 5; EGF, all three similar to ES 1; EGF + IGF-I group (five embryos cultured in 50 μl microdrops of CM with 10 ng/ml EGF and 10 ng/ml IGF-I). In both ESs, it was observed that a higher proportion of embryos cultured in larger groups achieved blastocyst stage than embryos cultured in reduced groups (22.6% v. 14.0%, 12.6% and 5.3% for Control v. Control 5, IGF-I, TGF-α groups in ES 1, and 24.9% v. 17.1% and 19.0% for Control v. Control 5 and EGF in ES 2, respectively; P embryos cultured in medium supplemented with EGF (18.5%) or with EGF + IGF-I (23.5%), in ES 1 and ES 2, respectively. With regard to blastocyst quality, embryos cultured in reduced groups and supplemented with EGF, alone or combined with IGF-I, presented lower apoptosis rates than embryos cultured in reduced groups without GF supplementation (11.6% and 10.5% v. 21.9% for EGF, EGF + IGF-I and Control 5 groups, respectively; P embryos, increasing blastocyst

  10. In vitro development of OPU-derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen-thawed embryos after transfer.

    Science.gov (United States)

    Isobe, Tomohiro; Ikebata, Yoshihisa; Do, Lanh Thi Kim; Tanihara, Fuminori; Taniguchi, Masayasu; Otoi, Takeshige

    2015-07-01

    The optimization of single-embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick-up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen-thawed embryos after a direct transfer. When two-cell-stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen-thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen-thawed embryos after transfer to recipients. © 2014 Japanese Society of Animal Science.

  11. Effect of supplemented sericin on the development, cell number, cryosurvival and number of lipid droplets in cultured bovine embryos.

    Science.gov (United States)

    Hosoe, Misa; Inaba, Yasushi; Hashiyada, Yutaka; Imai, Kei; Kajitani, Kenji; Hasegawa, Yuichi; Irie, Mamoru; Teramoto, Hidetoshi; Takahashi, Toru; Niimura, Sueo

    2017-02-01

    Sericin was investigated as an alternative to fetal bovine serum (FBS) for bovine embryo culture. In vitro matured oocytes were developed using 0.05%, 0.1% or 0.15% sericin. The developmental rate, cryosurvival rate and blastulation time of these embryos were compared with those of embryos developed using 5% FBS. The number of lipid droplets was compared among the blastocysts developed using 5% FBS, using 0.05% sericin and in vivo. The rate of cleavage and blastocyst formation was similar among all groups. Blastulation occurred significantly earlier in the embryos developed using 5% FBS than in those developed using sericin at any concentration (P sericin were significantly higher compared with those developed using 0.1% and 0.15% sericin (P sericin and in vivo produced a significantly fewer number of medium and large lipid droplets than those developed using 5% FBS. These results suggest that the blastocysts developed using 0.05% sericin show characteristics similar to those of the blastocysts developed in vivo and that the use of sericin as an alternative to FBS is feasible. © 2016 Japanese Society of Animal Science.

  12. Plating Efficiency for Primary Hamster Embryo Cells as an Index of Efficacy of Fetal Bovine Serum for Cell Culture

    Science.gov (United States)

    Goodheart, C. R.; Casto, B. C.; Zwiers, A.; Regnier, P. R.

    1973-01-01

    Attachment and growth of mammalian cells plated at low cell density require optimum conditions for the cells to form colonies. Reliability, reproducibility, and validity of the plating efficiency test for evaluating cell culture sera were determined by measuring the plating efficiency of 37 lots of fetal bovine serum obtained from 8 suppliers (5 lots from each of 7, 2 lots from 1 supplier), by using hamster embryo fibroblasts plated at low cell density. The test revealed considerable variation between lots of serum and between suppliers. The five lots from some suppliers had consistently high plating efficiencies, whereas one or more lots from other suppliers had quite low efficiencies. The results were reproducible in repeated tests, and control experiments indicated that the test measured the efficiency of the test serum independently of the efficiency of the serum used for the primary outgrowth of the hamster embryo cells. PMID:4584591

  13. Lipofection of siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well culture system.

    Science.gov (United States)

    Ikeda, Shuntaro; Sugimoto, Miki; Kume, Shinichi

    2018-04-13

    Bovine preimplantation embryos exhibit dramatic biological changes between before and after the 8-16-cell stage. Here we report a simple lipofection method to transfect siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well (WOW) culture system. Bovine one-cell embryos produced in vitro were freed from the zona pellucida and cultured up to the 8-16-cell stage in WOW dishes. The 8-16-cell embryos were lipofected with siRNA and the transfection efficiency was assessed at 48 h of transfection. Lipofection with a red fluorescent non-targeting siRNA revealed the importance of zona removal for transfection of siRNA into embryos. Using this method, we knocked down the methionine adenosyltransferase 2A (MAT2A) gene, achieving a significant reduction in MAT2A expression (P < 0.05) concomitant with the marked inhibition of blastocyst development. Our proposed method, tentatively named 'Octo-lipofection', may be useful to analyze gene functions in bovine preimplantation embryos without expensive equipment and skill-intensive techniques.

  14. The influence of in vitro fertilization and embryo culture on the embryo epigenetic constituents and the possible consequences in the bovine model.

    Science.gov (United States)

    Sirard, M-A

    2017-08-01

    Medically assisted reproductive technologies, such as in vitro embryo production, are increasingly being used to palliate infertility. Eggs are produced following a hormonal regimen that stimulates the ovaries to produce a large number of oocytes. Collected oocytes are then fertilized in vitro and allowed to develop in vitro until they are either frozen or transferred to mothers. There are controversial reports on the adverse impacts of these technologies on early embryos and their potential long-term effects. Using newly developed technological platforms that enable global gene expression and global DNA methylation profiling, we evaluated gene perturbations caused by such artificial procedures. We know that cells in the early embryo produce all cells in the body and are able to respond to their in vitro environment. However, it is not known whether gene perturbations are part of a normal response to the environment or are due to distress and will have long-term impacts. While the mouse is an established genetic model used for quality control of culture media in clinics, the bovine is a large mono-ovulating mammal with similar embryonic kinetics as humans during the studied developmental window. These model systems are critical to understand the effects of assisted reproduction without the confounding impact of infertility and without the limitations imposed by the scarcity of donated human samples and ethical issues. The data presented in this review come mostly from our own experimentation, publications, and collaborations. Together they demonstrate that the in vitro environment has a significant impact on embryos at the transcriptomic level and at the DNA methylation level.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Sexing bovine pre-implantation embryos using the polymerase ...

    African Journals Online (AJOL)

    Yomi

    2012-03-06

    Mar 6, 2012 ... 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-chromosome-.

  1. 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 ...

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

    Science.gov (United States)

    Qu, Pengxiang; Qing, Suzhu; Liu, Ruiqi; Qin, Hongyu; Wang, Weiwei; Qiao, Fang; Ge, Hui; Liu, Jun; Zhang, Yong; Cui, Wei; Wang, Yongsheng

    2017-01-01

    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.

  3. Rabbit whole embryo culture.

    Science.gov (United States)

    Marshall, Valerie A; Carney, Edward W

    2012-01-01

    Although the rabbit is used extensively in developmental toxicity testing, relatively little is known about the fundamental developmental biology of this species let alone mechanisms underlying developmental toxicity. This paucity of information about the rabbit is partly due to the historic lack of whole embryo culture (WEC) methods for the rabbit, which have only been made available fairly recently. In rabbit WEC, early somite stage embryos (gestation day 9) enclosed within an intact amnion and attached to the visceral yolk sac are dissected from maternal tissues and placed in culture for up to 48 h at approximately 37°C and are continuously exposed to an humidified gas atmosphere mixture in a rotating culture system. During this 48 h culture period, major phases of organogenesis can be studied including cardiac looping and segmentation, neural tube closure, and development of anlagen of the otic system, eyes and craniofacial structures, somites and early phases of limb development (up to bud stage), as well as expansion and closure of the visceral yolk sac around the embryo. Following completion of the culture period, embryos are evaluated based on several growth and development parameters and also are assessed for morphological abnormalities. The ability to sustain embryo development independent of the maternal system allows for exposure at precise development stages providing the opportunity study the direct action of a teratogen or one of its metabolites on the developing embryo. Rabbit WEC is perhaps most useful when used in conjunction with rodent WEC methods to investigate species-specific mechanisms of developmental toxicity.

  4. 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

  5. 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.

    OpenAIRE

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

    2014-01-01

    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 n...

  6. 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.

  7. 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

  8. 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.

  9. Bovine in vitro embryo production : An overview

    Directory of Open Access Journals (Sweden)

    V. S. Suthar

    Full Text Available Dairy industry perfected the application of the first reproductive biotechnology, i.e. artificial insemination (AI - a great success story and also remains the user of embryo transfer technology (ETT. In addition, recently the researchers taking interest to embraced the field of Transvaginal OocyteRecovery (TVOR and in vitro production (IVEP of embryos. IVF provides the starting point for the generation of reproductive material for a number of advanced reproduction techniques including sperm microinjection and nuclear transfer (cloning. In several countries commercial IVF facilities are already being employed by cattle ET operators. Various research groups have reported on modification of TVOR technique to give greater efficiency. Much research is still needed in domestic animal (Especially Indian species on mechanisms controlling embryo development and on development of totally in vitro system for embryo culture. [Vet World 2009; 2(12.000: 478-479`

  10. Bovine in-vitro embryo production and its contribution towards ...

    African Journals Online (AJOL)

    Bovine in-vitro embryo production and its contribution towards improved food security in Kenya. ... African Journal of Food, Agriculture, Nutrition and Development ... be low valued, it can be raised to higher reproductive potential using current reproductive technologies such as in-vitro embryo production and embryo transfer.

  11. Microorganisms in cryopreserved semen and culture media used in the in vitro production (IVP) of bovine embryos identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS).

    Science.gov (United States)

    Zampieri, Dávila; Santos, Vanessa G; Braga, Patrícia A C; Ferreira, Christina R; Ballottin, Daniela; Tasic, Ljubica; Basso, Andréa C; Sanches, Bruno V; Pontes, José H F; da Silva, Bárbara Pereira; Garboggini, Fabiana Fantinatti; Eberlin, Marcos N; Tata, Alessandra

    2013-09-01

    Commercial cattle breeders produce their own herd offspring for the dairy and beef market using artificial insemination. The procedure involves sanitary risks associated with the collection and commercialization of the germplasm, and the in vitro production and transfer of the bovine embryos must be monitored by strict health surveillance. To avoid the spreading of infectious diseases, one must rely on using controlled and monitored germplasm, media, and reagents that are guaranteed free of pathogens. In this article, we investigated the use of a new mass spectrometric approach for fast and accurate identification of bacteria and fungi in bovine semen and in culture media employed in the embryo in vitro production process. The microorganisms isolated from samples obtained in a commercial bovine embryo IVP setting were identified in a few minutes by their conserved peptide/protein profile, obtained applying matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), matched against a commercial database. The successful microorganisms MS identification has been confirmed by DNA amplification and sequencing. Therefore, the MS technique seems to offer a powerful tool for rapid and accurate microorganism identification in semen and culture media samples. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. 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.

  13. Preimplantation bovine embryos: Pathobiology of Haemophilus somnus exposure and resistance mechanisms to vesicular stomatitis virus

    International Nuclear Information System (INIS)

    Thomson, M.S.

    1988-01-01

    Preimplantation bovine embryos were exposed in vitro to H. somnus to determine if the bacteria would adhere to zona pellucida-intact (ZP-I) embryos or adhere to or infect ZP-free embryos. The effect of H. somnus on embryonic development in vitro was also investigated. Electrophoretic comparisons of outer membrane proteins of H. somnus revealed 2 major protein bands common to 10 H. somnus isolates. A monoclonal antibody produced against the outer membrane proteins reacted to one of the major protein bands. The sensitivity of a nucleic acid probe for detection of vesicular stomatitis virus (VSV) was validated in cells in culture and used to determine if the synthetic double-stranded complex of polyriboinosinic and polyribocytidylic acids (poly I:C) would induce viral resistance in cultured bovine embryos. Two 32 P-nick translated probes of high specific activity prepared from plasmids containing nucleic acid sequences of VSV virus were employed for viral mRNA detection in the tissue culture cells using a DNA-hybridization dot-blot technique. Using one of the probes, the technique was applied to detect differences in viral replication between four groups of bovine embryos (nonexposed, exposed to VSV virus, poly I:C-treated, and poly I:C-treated and exposed to VSV). The nucleic acid probe was sufficiently sensitive to detect differences in quantities of VSV mRNA among embryo treatment groups, resulting in the demonstration that resistance to viral infection was induced in day 9 bovine embryos

  14. In vitro production of bovine embryos

    DEFF Research Database (Denmark)

    Stroebech, L.; Mazzoni, Gianluca; Pedersen, Hanne Skovsgaard

    2015-01-01

    In vitro production (IVP) of bovine embryos has become a widespread technology implemented in cattle breeding and production. The implementation of genomic selection and systems biology adds great dimensions to the impact of bovine IVP. The physical procedures included in the IVP process can stil...

  15. Analysis of the expression of putatively imprinted genes in bovine peri-implantation embryos

    DEFF Research Database (Denmark)

    Tveden-Nyborg, Pernille Yde; Alexopoulos, N.I.; Cooney, M.A.

    2008-01-01

    The application of assisted reproductive technologies (ART) has been shown to induce changes in the methylation of the embryonic genome, leading to aberrant gene expression, including that of imprinted genes. Aberrant methylation and gene expression has been linked to the large offspring syndrome...... procedures, either by in vitro maturation, fertilization or culture. In conclusion, effects of genomic imprinting and of in vitro procedures for embryo production may influence the success of bovine embryo implantation....... (LOS) in bovine embryos resulting in increased embryonic morbidity and mortality. In the bovine, limited numbers of imprinted genes have been studied and studies have primarily been restricted to pre-implantation stages. This study reports original data on the expression pattern of 8 putatively...... imprinted genes (Ata3, Dlk1, Gnas, Grb10, Magel2, Mest-1, Ndn and Sgce) in bovine peri-implantation embryos. Two embryonic developmental stages were examined, Day 14 and Day 21. The gene expression pattern of single embryos was recorded for in vivo, in vitro produced (IVP) and parthenogenetic embryos...

  16. Nucleolar ultrastructure in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Kaňka, Jiří; Smith, Steven Dale; Soloy, Eva

    1999-01-01

    all three cell cycles. In the eight-cell stage embryo, a primary vacuole appeared as an electron lucid area originating in the centre of the nucleolar precursor body. In nuclear transfer embryos reconstructed from nonactivated cytoplasts, the nuclear envelope was fragmented or completely broken down...... vacuoles. A nucleolar precursor body typical for the two-cell stage control embryos was never observed. None of the reconstructed embryos of this group reached the eight-cell stage. Nuclear transfer embryos reconstructed from activated cytoplasts, in contrast, exhibited a complete nuclear envelope at all...... time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear...

  17. Studies of In Vitro Embryo Culture of Guppy (Poecilia reticulata).

    Science.gov (United States)

    Liu, LiLi; Lee, Ki-Young

    2014-09-01

    Different with other fishes, the guppies (Poecilia reticulata) is ovoviviparity, which retain their fertilized eggs within the follicle throughout gestation. The synchronously growing diplotene oocytes store nutrients in droplets and yolk, before their maturation and fertilization. The lecithotrophic strategy of development entails the provisioning of embryos with resources from the maternal yolk deposit rather than from a placenta, it allows the extracorporeal culture of guppy embryo. Studies on their early development of live bearers like the guppy including lineage tracing and genetic manipulations, have been limited. Therefore, to optimize conditions of embryo in vitro culture, explanted embryos from pregnant females were incubated in embryo medium (L-15 medium, supplemented with 5, 10, 15, 20% fetal bovine serum, respectively). We investigated whether the contents of FBS in vitro culture medium impact the development of embryos, and whether they would hatch in vitro. Our study found that in 5% of FBS of the medium, although embryos developed significantly slower in vitro than in the ovary, it was impossible to exactly quantify the developmental delay in culture, due to the obvious spread in developmental stage within each batch of eggs, and embryos can only be maintained until the early-eyed. And although in culture with 20% FBS the embryos can sustain rapid development of early stage, but cannot be cultured for the entire period of their embryonic development and ultimately died. In the medium with 10% and 15% FBS, the embryos seems well developed, even some can continue to grow after follicle ruptures until it can be fed. We also observed that embryonic in these two culture conditions were significantly different in development speed, in 15% it is faster than 10%. But 10% FBS appears to be more optimizing condition than 15% one on development process of embryos and survival rate to larvae stage.

  18. Transcriptome analysis of primary bovine extra-embryonic cultured cells

    Directory of Open Access Journals (Sweden)

    Séverine A. Degrelle

    2015-12-01

    Full Text Available The dataset described in this article pertains to the article by Hue et al. (2015 entitled “Primary bovine extra-embryonic cultured cells: A new resource for the study of in vivo peri-implanting phenotypes and mesoderm formation” [1]. In mammals, extra-embryonic tissues are essential to support not only embryo patterning but also embryo survival, especially in late implanting species. These tissues are composed of three cell types: trophoblast (bTCs, endoderm (bXECs and mesoderm (bXMCs. Until now, it is unclear how these cells interact. In this study, we have established primary cell cultures of extra-embryonic tissues from bovine embryos collected at day-18 after artificial insemination. We used our homemade bovine 10K array (GPL7417 to analyze the gene expression profiles of these primary extra-embryonic cultured cells compared to the corresponding cells from in vivo micro-dissected embryos. Here, we described the experimental design, the isolation of bovine extra-embryonic cell types as well as the microarray expression analysis. The dataset has been deposited in Gene Expression Omnibus (GEO (accession number GSE52967. Finally, these primary cell cultures were a powerful tool to start studying their cellular properties, and will further allow in vitro studies on cellular interactions among extra-embryonic tissues, and potentially between extra-embryonic vs embryonic tissues.

  19. The recovery of ureaplasmas from bovine embryos following in vitro exposure and ten washes.

    Science.gov (United States)

    Britton, A P; Miller, R B; Ruhnke, H L; Johnson, W H

    1988-01-01

    Twenty-six unhatched embryos and ova were exposed to Ureaplasma diversum strain 2312 in vitro for 16 h and subsequently washed ten times. Fifteen of the embryos and their wash fluids were cultured for ureaplasmas. Of the remaining 11 embryos, six were incubated with rabbit anti-Ureaplasma immunoglobulin (RAI) and five were incubated with serum from naive rabbits(NRS), after which all were incubated with protein A gold and prepared for electron microscopy. On ultrastructural examination, ureaplasmas were observed on the outer surface of the zona pellucida of all 11 embryos. The ureaplasmas on the six embryos incubated with RAI were labeled with gold particles, while those on the five embryos incubated with NRS were not labeled. Ureaplasmas were recovered from all 15 of the cultured embryos and all of the first and second wash fluids as well as intermittently from the third, fourth, sixth, seventh, eighth and ninth wash but not fom the fifth or tenth wash. It was concluded that viable ureaplasmas adhered to the zona pellucida during in vitro exposure of bovine embryos and were not removed by ten washes.

  20. Pyridoxine supplementation during oocyte maturation improves the development and quality of bovine preimplantation embryos.

    Science.gov (United States)

    Aboelenain, Mansour; Balboula, Ahmed Zaky; Kawahara, Manabu; El-Monem Montaser, Abd; Zaabel, Samy Moawad; Kim, Sung-Woo; Nagano, Masashi; Takahashi, Masashi

    2017-03-15

    Recently, inhibition of cathepsin B (CTSB) activity during in vitro maturation (IVM) and culture (IVC) improved the developmental competence and quality of bovine oocytes and embryos. E-64 is a widely used inhibitor to inhibit CTSB activity, however, E-64 inhibits not only CTSB activity but also the activities of other proteases including cathepsin L (CTSL), papain, calpain, and trypsin. Pyridoxine, the catalytically active form of vitamin B6, plays a crucial role in several cellular processes and has the ability to inhibit CTSB activity. However, whether pyridoxine has an improving effect during IVM of bovine oocytes is still unknown. In this study, we investigated the effect of pyridoxine supplementation during IVM on the developmental competence of bovine oocytes and the quality of the produced blastocysts. Supplementation of pyridoxine to the maturation medium significantly decreased the activity of CTSB in both bovine cumulus cells and oocytes. Moreover, pyridoxine improved both the blastocyst and hatched blastocyst rates. In addition, the presence of pyridoxine during IVM also significantly improved the quality of the produced embryos by increasing the total cell number as well as decreasing the CTSB mRNA expression and apoptotic rate. These results indicate that pyridoxine is a promising tool to improve the developmental competence of bovine oocytes and subsequent embryo quality. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Improvement of bovine in vitro embryo production by vitamin K₂ supplementation.

    Science.gov (United States)

    Baldoceda-Baldeon, Luis Manuel; Gagné, Dominic; Vigneault, Christian; Blondin, Patrick; Robert, Claude

    2014-11-01

    Mitochondria play an important role during early development in mammalian embryos. It has been shown that properly controlled follicular preparation increases the likelihood of in-vitro-produced bovine embryos reaching the blastocyst stage and that competent embryos exhibit heightened expression of genes associated with mitochondrial function. We hypothesized that apparently incompetent embryos could be rescued by restoring mitochondrial function. It has been shown that vitamin K2 (a membrane-bound electron carrier similar to ubiquinone) can restore mitochondrial dysfunction in eukaryotic cells. The aim of this study was therefore to investigate the effects of vitamin K2 on bovine embryonic development in vitro. The vitamin was found most effective when added 72 h after fertilization. It produced a significant (Pembryos of better morphological quality. It improved the mitochondrial activity significantly and had a measurable impact on gene expression. This is the first demonstration that current standard conditions of in vitro production of bovine embryos may be inadequate due to the lack of support for mitochondrial function and may be improved significantly by supplementing the culture medium with vitamin K2. © 2014 Society for Reproduction and Fertility.

  2. 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.

  3. In vitro culture of Arabidopsis embryos.

    Science.gov (United States)

    Sauer, Michael; Friml, Jirí

    2008-01-01

    Embryogenesis of Arabidopsis thaliana follows a nearly invariant cell division pattern and provides an ideal system for studies of early plant development. However, experimental manipulation with embryogenesis is difficult, as the embryo develops deeply inside maternal tissues. Here, we present a method to culture zygotic Arabidopsis embryos in vitro. It enables culturing for prolonged periods of time from the first developmental stages on. The technique omits excision of the embryo by culturing the entire ovule, which facilitates the manual procedure. It allows pharmacological manipulation of embryo development and does not interfere with standard techniques for localizing gene expression and protein localization in the cultivated embryos.

  4. Identification of abnormal gene expression in bovine transgenic somatic cell nuclear transfer embryos

    OpenAIRE

    Cho, Jongki; Kang, Sungkeun; Lee, Byeong Chun

    2014-01-01

    This study was conducted to investigate the expression of three genes related to early embryonic development in bovine transgenic cloned embryos. To accomplish this, development of bovine transgenic somatic cell nuclear transfer (SCNT) embryos was compared with non-transgenic embryos. Next, mRNA transcription of three specific genes (DNMT1, Hsp 70.1, and Mash2) related to early embryo development in transgenic SCNT embryos was compared between transgenic and non-transgenic SCNTs, parthenogene...

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

    OpenAIRE

    Meneghel, Melissa; Dall’Acqua, Priscila Chediek; Ambrogi, Marcela; Leão, Beatriz C.S.; Rocha-Frigoni, Nathália A.S.; Mingoti, Gisele Z.

    2017-01-01

    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 ...

  6. Effects of Insulin-like Growth Factor-1 on Development of Somatic Cell Cloned Bovine Embryos.

    Science.gov (United States)

    Qu, Pengxiang; Li, Yanyan; Deng, Tengfei; Jia, Dan; Qing, Suzhu; Su, Jianmin; Zhang, Yong; Wang, Yongsheng

    2016-06-01

    The aim of this study was to assess the effect of insulin-like growth factor-1 (IGF-1) on the developmental competence of somatic cell nuclear transfer (SCNT) bovine embryos. First, the expression levels of IGF-1 receptor (IGF-1R) and IGF-1 in the oocytes and embryos of different developmental stages were examined. Then the effects of exogenous IGF-1 on the development of SCNT embryos were evaluated both in vitro and in vivo. The results showed that IGF-1 was not expressed in both IVF and SCNT embryos, whereas IGF-1R could be detected throughout the preimplantation stages in both protein and mRNA levels. Also, exogenous IGF-1 had no obvious impact on the developmental competence of IVF embryos. However, it could improve the developmental competence of SCNT embryos in terms of blastocyst developmental rate (31.3% vs. 43.2%, p embryo transfer, there was an upward tendency in both examined terms in the IGF-1-supplemented group when compared with the control group. In conclusion, the present study showed that supplementing exogenous IGF-1 to the culture medium has an obvious positive effect on the development competence of SCNT embryos.

  7. 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.

  8. 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.

  9. Nucleolar development and allocation of key nucleolar proteins require de novo transcription in bovine embryos

    DEFF Research Database (Denmark)

    Svarcova, Olga; Laurincik, Jozef; Avery, Birthe

    2007-01-01

    The goal of the present study was to investigate whether key nucleolar proteins involved in ribosomal RNA (rRNA) transcription and processing are transcribed de novo or from maternally inherited messenger RNAs (mRNA) in bovine embryos, and to which extent de novo transcription of these proteins m......RNA is required for the development of functional nucleoli during the major activation of the embryonic genome. Immunofluorescence for localization of key nucleolar proteins, autoradiography for detection of transcriptional activity, and transmission electron microscopy were applied to in vitro produc ed bovine...... embryos cultured from the 2-cell stage with or without (control groups) a-amanitin, which blocks the RNA plymerases II and III transcription and, thus the synthesis of mRNA. In the control groups, weak autoradiographic labelling was initially observed in the periphery of few nuclei at the 4-cell...

  10. Effect of chilling on the development of in vitro produced bovine embryos at various cleavage stages

    OpenAIRE

    Balasubramanian, S.; Rho, Gyu-Jin

    2006-01-01

    Purpose: Bovine embryos and zygotes are known to be sensitive to “temperature shock” when cooled to temperatures near 0°C. The effect of chilling on in vitro derived embryos at various cleavage stages was investigated.

  11. Experimental risk assessment of bovine viral diarrhea virus transmission via in vitro embryo production using somatic cell nucleus transfer.

    Science.gov (United States)

    Gregg, K; Chen, S H; Sadeghieh, S; Guerra, T; Xiang, T; Meredith, J; Polejaeva, I

    2009-07-01

    The objective of this study was to perform a comprehensive risk assessment on infectious disease transmission in the system of in vitro embryo production via somatic cell nucleus transfer (SCNT) technology using bovine viral diarrhea virus (BVDV) as a model. The risks of BVDV transmission in each step of the SCNT embryo production procedure, from donor cells to preimplantation SCNT embryo culture, were carefully examined using a sensitive real-time polymerase chain reaction assay. The identified primary source of BVDV transmission in SCNT embryo production was donor cell infection, most likely caused by contaminated fetal bovine serum in the culture medium. The risk of disease transmission through contaminated oocytes from an abattoir was relatively low, and it can be greatly minimized by cumulus cell removal and adequate oocyte washing procedures. Of the 200 cumulus-oocyte complexes (COCs) and more than 1500 cumulus cell-free oocyte (CFO) samples collected from multiple sources over a course of 7 months, only 2.5% of the COCs were BVDV positive, and all of the CFOs (100%) were BVDV negative. To evaluate the risk of BVDV introduction during in vitro SCNT embryo culture, 324 SCNT embryos were produced from 18 different cell lines using oocytes from 26 different batches collected over a course of 9 months. The embryos were cultured in vitro for 7 days and then tested for BVDV. All of the 324 SCNT embryos (100%) were negative, indicating that the embryo culture system is virtually risk-free for BVDV transmission. Based on these results, a standard operational protocol (SOP) for SCNT embryo production was proposed to greatly minimize the risk of BVDV transmission through the SCNT embryo production system. This SOP could be a starting point to produce a SCNT system that is virtually risk-free for disease transmission in general.

  12. Recombinant human growth differentiation factor-9 improves oocyte reprogramming competence and subsequent development of bovine cloned embryos.

    Science.gov (United States)

    Su, Jianmin; Hu, Guangdong; Wang, Yongsheng; Liang, Dong; Gao, Mingqing; Sun, Hongzheng; Zhang, Yong

    2014-08-01

    Previously, we found that oocyte-secreted factors (OSFs) secreted by denuded oocytes during in vitro maturation (IVM) enhance subsequent development of bovine somatic cell nuclear transfer (SCNT) embryos. This treatment requires many oocytes during IVM. Hence, the aim of this study was to investigate whether supplementing with recombinant growth differentiation factor-9 (GDF9), one of crucial OFSs, in oocyte maturation medium could improve developmental competence of bovine oocytes and subsequent development of cloned embryos. Cumulus-oocyte complexes (COCs) from antral follicles of bovine ovaries collected from an abattoir were cultured with (SCNT+GDF9 group) or without (SCNT group) 200 ng/mL recombinant human GDF9 in oocyte maturation medium. After 22 h, metaphase II (MII) oocytes were used for SCNT. The presence of 200 ng/mL GDF9 significantly increased oocyte maturation rates, the cleavage rate, and blastocyst formation rates of bovine cloned embryos. The blastocyst total, inner cell mass (ICM) cell numbers, and ratio of ICM:TE were higher, whereas the rate of apoptosis in bovine cloned blastocysts was lower in the SCNT+GDF9 group than in the SCNT group. The histone modifications at various sites were also different between each group. These results suggest that COCs cultured with recombinant GDF9 in oocyte maturation medium improve oocyte developmental competence and subsequent developmental competence of cloned embryo in cattle.

  13. Homologous recombination and non-homologous end-joining repair pathways in bovine embryos with different developmental competence

    Energy Technology Data Exchange (ETDEWEB)

    Henrique Barreta, Marcos [Universidade Federal de Santa Catarina, Campus Universitario de Curitibanos, Curitibanos, SC (Brazil); Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Garziera Gasperin, Bernardo; Braga Rissi, Vitor; Cesaro, Matheus Pedrotti de [Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Ferreira, Rogerio [Centro de Educacao Superior do Oeste-Universidade do Estado de Santa Catarina, Chapeco, SC (Brazil); Oliveira, Joao Francisco de; Goncalves, Paulo Bayard Dias [Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Bordignon, Vilceu, E-mail: vilceu.bordignon@mcgill.ca [Department of Animal Science, McGill University, Ste-Anne-De-Bellevue, QC (Canada)

    2012-10-01

    This study investigated the expression of genes controlling homologous recombination (HR), and non-homologous end-joining (NHEJ) DNA-repair pathways in bovine embryos of different developmental potential. It also evaluated whether bovine embryos can respond to DNA double-strand breaks (DSBs) induced with ultraviolet irradiation by regulating expression of genes involved in HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro insemination and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation. All studied genes were expressed before, during and after the genome activation period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before genome activation in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-insemination in response to DSBs induced by ultraviolet irradiation. In conclusion, important genes controlling HR and NHEJ DNA-repair pathways are expressed in bovine embryos, however genes participating in these pathways are only regulated after the period of embryo genome activation in response to ultraviolet-induced DSBs.

  14. Homologous recombination and non-homologous end-joining repair pathways in bovine embryos with different developmental competence

    International Nuclear Information System (INIS)

    Henrique Barreta, Marcos; Garziera Gasperin, Bernardo; Braga Rissi, Vitor; Cesaro, Matheus Pedrotti de; Ferreira, Rogério; Oliveira, João Francisco de; Gonçalves, Paulo Bayard Dias; Bordignon, Vilceu

    2012-01-01

    This study investigated the expression of genes controlling homologous recombination (HR), and non-homologous end-joining (NHEJ) DNA-repair pathways in bovine embryos of different developmental potential. It also evaluated whether bovine embryos can respond to DNA double-strand breaks (DSBs) induced with ultraviolet irradiation by regulating expression of genes involved in HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro insemination and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation. All studied genes were expressed before, during and after the genome activation period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before genome activation in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-insemination in response to DSBs induced by ultraviolet irradiation. In conclusion, important genes controlling HR and NHEJ DNA-repair pathways are expressed in bovine embryos, however genes participating in these pathways are only regulated after the period of embryo genome activation in response to ultraviolet-induced DSBs.

  15. In vitro bovine embryo production in a synthetic medium: embryo development, cryosurvival, and establishment of pregnancy.

    Science.gov (United States)

    Moreno, D; Neira, A; Dubreil, L; Liegeois, L; Destrumelle, S; Michaud, S; Thorin, C; Briand-Amirat, L; Bencharif, D; Tainturier, D

    2015-10-15

    The aim of this study was to develop an in vitro embryo culture medium without either fetal calf serum or BSA, using various growth factors and cytokines (GFs-CYKs; IGF-I, IGF-II, bFGF, LIF, GM-CSF, TGF-β1, and PDGF-BB), and other molecules with surfactant and embryotrophic properties, such as recombinant albumin (RA) and hyaluronan (HA). The first part of the study was dedicated to define the best combination of GFs-CYKs + RA + HA for optimal embryonic development. Next, we compared development rates and embryo quality (inner cell mass [ICM]-to-total cell number [TCN] ratio), and postthaw survival and hatching rates using this synthetic medium (T1) and a control medium: synthetic oviduct fluid + BSA + ITS (insulin, transferrin, and selenium). The blastocyst rates were significantly higher with T1 than those with the control at 7 and 8 days after fertilization. There was no significant difference in TCN or the ICM/TCN ratio between the two treatments. Survival and hatching rates 48 hours after thawing were similar for both treatments. Finally, nine embryo transfers were conducted using fresh and previously frozen Day-7 blastocysts to evaluate the in vivo viability of embryos produced in this synthetic medium; four gestations were obtained from six fresh embryos and one gestation from three frozen embryos. In conclusion, the fetal calf serum and BSA-free medium, supplemented with GFs-CYKs + RA + HA, improved embryo development and gave comparable ICM/TCN ratios and postthaw survival rates to the control with BSA. Fresh and frozen embryos produced in this medium are viable for embryo transfer. This fully synthetic method of embryo culture is a useful means of reducing the risk of disease transmission via embryo transfer. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Genome stability of bovine in vivo-conceived cleavage-stage embryos is higher compared to in vitro-produced embryos.

    Science.gov (United States)

    Tšuiko, Olga; Catteeuw, Maaike; Zamani Esteki, Masoud; Destouni, Aspasia; Bogado Pascottini, Osvaldo; Besenfelder, Urban; Havlicek, Vitezslav; Smits, Katrien; Kurg, Ants; Salumets, Andres; D'Hooghe, Thomas; Voet, Thierry; Van Soom, Ann; Robert Vermeesch, Joris

    2017-11-01

    Is the rate and nature of chromosome instability (CIN) similar between bovine in vivo-derived and in vitro-cultured cleavage-stage embryos? There is a major difference regarding chromosome stability of in vivo-derived and in vitro-cultured embryos, as CIN is significantly lower in in vivo-derived cleavage-stage embryos compared to in vitro-cultured embryos. CIN is common during in vitro embryogenesis and is associated with early embryonic loss in humans, but the stability of in vivo-conceived cleavage-stage embryos remains largely unknown. Because human in vivo preimplantation embryos are not accessible, bovine (Bos taurus) embryos were used to study CIN in vivo. Five young, healthy, cycling Holstein Friesian heifers were used to analyze single blastomeres of in vivo embryos, in vitro embryos produced by ovum pick up with ovarian stimulation (OPU-IVF), and in vitro embryos produced from in vitro matured oocytes retrieved without ovarian stimulation (IVM-IVF). Single blastomeres were isolated from embryos, whole-genome amplified and hybridized on Illumina BovineHD BeadChip arrays together with the bulk DNA from the donor cows (mothers) and the bull (father). DNA was also obtained from the parents of the bull and from the parents of the cows (paternal and maternal grandparents, respectively). Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the genomic architecture of 171 single bovine blastomeres of 16 in vivo, 13 OPU-IVF and 13 IVM-IVF embryos. The genomic stability of single blastomeres in both of the in vitro-cultured embryo cohorts was severely compromised (P vivo. Only 18.8% of in vivo-derived embryos contained at least one blastomere with chromosomal anomalies, compared to 69.2% of OPU-IVF embryos (P vivo-developed embryos, in vitro procedures exacerbate chromosomal abnormalities during early embryo development. Hence, the present study highlights that IVF treatment compromises embryo viability and should be applied

  17. Embryo forming cells in carrot suspension cultures

    NARCIS (Netherlands)

    Toonen, M.A.J.

    1997-01-01


    Somatic cells of many plant species can be cultured in vitro and induced to form embryos that are able to develop into mature plants. This process, termed somatic embryogenesis, was originally described in carrot (Daucus carota L.). Somatic embryos develop through the same characteristic

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

    Science.gov (United States)

    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.

  19. 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, N. M.; Vajta, Gábor

    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...... 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...

  20. Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos

    DEFF Research Database (Denmark)

    Smith, S. D.; Soloy, E.; Kanka, J.

    1996-01-01

    Nucleus transfer for the production of multiple embryos derived from a donor embryo relies upon the reprogramming of the donor nucleus so that it behaves similar to a zygotic nucleus. One indication of nucleus reprogramming is the RNA synthetic activity. In normal bovine embryogenesis, the embryo...

  1. Comparison of transcriptomic landscapes of bovine embryos using RNA-Seq

    Directory of Open Access Journals (Sweden)

    Khatib Hasan

    2010-12-01

    Full Text Available Abstract Background Advances in sequencing technologies have opened a new era of high throughput investigations. Although RNA-seq has been demonstrated in many organisms, no study has provided a comprehensive investigation of the bovine transcriptome using RNA-seq. Results In this study, we provide a deep survey of the bovine embryonic transcriptomes, the first application of RNA-seq in cattle. Embryos cultured in vitro were used as models to study early embryonic development in cattle. RNA amplified from limited amounts of starting total RNA were sequenced and mapped to the reference genome to obtain digital gene expression at single base resolution. In particular, gene expression estimates from more than 1.6 million unannotated bases in 1785 novel transcribed units were obtained. We compared the transcriptomes of embryos showing distinct developmental statuses and found genes that showed differential overall expression as well as alternative splicing. Conclusion Our study demonstrates the power of RNA-seq and provides further understanding of bovine preimplantation embryonic development at a fine scale.

  2. Large scale in vivo risk assessment of bovine viral diarrhea virus (BVDV) transmission through transfer of bovine embryos produced via somatic cell nuclear transfer (SCNT).

    Science.gov (United States)

    Gregg, K; Gosch, G; Guerra, T; Chen, S H; Xiang, T; Broek, D; Bruner, B; Polejaeva, I

    2010-10-15

    The objective was to use the bovine viral diarrhea virus (BVDV) as a model to assess the risk of infectious disease transmission in the system of in vitro embryo production and transfer via somatic cell nuclear transfer (SCNT) technology. The risks of BVDV transmission in the SCNT embryo production were previously evaluated. In that in vitro study, following standard operating procedures (SOP), including pre-nuclear transfer donor cell testing, oocyte decontamination and virus-free cell and embryo culture conditions, SCNT embryos produced were free of detectable viral RNA. The current study focused on the evaluation of the potential risk of disease transmission from SCNT embryos to recipients, and the risk of producing persistently infected animals via SCNT embryo transfer. Blood samples were collected from 553 recipients of SCNT embryos and 438 cloned calves and tested for the presence of BVDV viral RNA via a sensitive real time PCR method. All samples tested were negative. These results, in conjunction with the previous in vitro study, confirmed that the established SCNT embryo production and transfer system is safe and presents no detectable risk of disease transmission. Copyright © 2010 Elsevier Inc. All rights reserved.

  3. Proteome analysis of early lineage specification in bovine embryos.

    Science.gov (United States)

    Demant, Myriam; Deutsch, Daniela R; Fröhlich, Thomas; Wolf, Eckhard; Arnold, Georg J

    2015-02-01

    During mammalian embryo development, the zygote undergoes embryonic cleavage in the oviduct and reaches the uterus at the morula stage, when compaction and early lineage specification take place. To increase knowledge about the associated changes of the embryonic protein repertoire, we performed a comprehensive proteomic analysis of in vitro produced bovine morulae and blastocysts (six biological replicates), using an iTRAQ-based approach. A total of 560 proteins were identified of which 502 were quantified. The abundance of 140 proteins was significantly different between morulae and blastocysts, among them nucleophosmin (NPM1), eukaryotic translation initiation factor 5A-1 (EIF5A), receptor of activated protein kinase C 1 (GNB2L1/RACK1), and annexin A6 (ANXA6) with increased, and glutathione S-transferase mu 3 (GSTM3), peroxiredoxin 2 (PRDX2), and aldo-keto reductase family 1 member B1 (AKR1B1) with decreased abundance in blastocysts. Seventy-three percent of abundance altered proteins increased, reflecting an increase of translation activity in this period. This is further supported by an increase in the abundance of proteins involved in the translation machinery and the synthesis of ATP. Additionally, a complementary 2D saturation DIGE analysis led to the detection of protein isoforms, e.g. of GSTM3 and PRDX2, relevant for this period of mammalian development, and exemplarily verified the results of the iTRAQ approach. In summary, our systematic differential proteome analysis of bovine morulae and blastocysts revealed new molecular correlates of early lineage specification and differentiation events during bovine embryogenesis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Effect of embryo density on in vitro developmental characteristics of bovine preimplantative embryos with respect to micro and macroenvironments.

    Science.gov (United States)

    Hoelker, M; Rings, F; Lund, Q; Phatsara, C; Schellander, K; Tesfaye, D

    2010-10-01

    To overcome developmental problems as a consequence of single embryo culture, the Well of the Well (WOW) culture system has been developed. In this study, we aimed to examine the effect of embryo densities with respect to both microenvironment and macroenvironment on developmental rates and embryo quality to get a deeper insight into developmentally important mechanisms. WOW diameter and depth significantly affected developmental rates (p WOWs with diameter of 500 μm reached significantly higher blastocyst rates (32.5 vs 21.1% vs 20.3%) compared to embryos cultured in WOWs of 300 μm diameter or plain cultured controls. Embryos cultured in WOWs with 700 μm depth reached significant higher developmental rates compared with embryos cultured in WOWs of 300 μm depth and control embryos (30.6 vs 22.6% vs 20.3%). Correlation of the embryo per WOW volume with developmental rates was higher (r(2) = 0.92, p = 0.0004) than correlation of WOW diameter or WOW depth with developmental rates. However, the embryo per WOW volume did not affect differential cell counts. An embryo per culture dish volume of 1 : 30 μl was identified to be optimal when the embryo per WOW volume was 1 : 0.27 μl increasing developmental rates up to the level of mass embryo production. Giving the opportunity to track each embryo over the complete culture period while keeping high developmental rates with normal mitotic dynamics, the results of this work will provide benefit for the single culture of embryos in human assisted reproduction, mammalian embryos with high economic interest as well as for scientific purpose. © 2009 Blackwell Verlag GmbH.

  5. Reprogrammed transcriptome in rhesus-bovine interspecies somatic cell nuclear transfer embryos.

    Directory of Open Access Journals (Sweden)

    Kai Wang

    Full Text Available Global activation of the embryonic genome (EGA, one of the most critical steps in early mammalian embryo development, is recognized as the time when interspecies somatic cell nuclear transfer (iSCNT embryos fail to thrive.In this study, we analyzed the EGA-related transcriptome of rhesus-bovine iSCNT 8- to 16-cell embryos and dissected the reprogramming process in terms of embryonic gene activation, somatic gene silencing, and maternal RNA degradation. Compared with fibroblast donor cells, two thousand and seven genes were activated in iSCNT embryos, one quarter of them reaching expression levels comparable to those found in in vitro fertilized (IVF rhesus embryos. This suggested that EGA in iSCNT embryos had partially recapitulated rhesus embryonic development. Eight hundred and sixty somatic genes were not silenced properly and continued to be expressed in iSCNT embryos, which indicated incomplete nuclear reprogramming. We compared maternal RNA degradation in bovine oocytes between bovine-bovine SCNT and iSCNT embryos. While maternal RNA degradation occurred in both SCNT and iSCNT embryos, we saw more limited overall degradation of maternal RNA in iSCNT embryos than in SCNT embryos. Several important maternal RNAs, like GPF9, were not properly processed in SCNT embryos.Our data suggested that iSCNT embryos are capable of triggering EGA, while a portion of somatic cell-associated genes maintain their expression. Maternal RNA degradation seems to be impaired in iSCNT embryos. Further understanding of the biological roles of these genes, networks, and pathways revealed by iSCNT may expand our knowledge about cell reprogramming, pluripotency, and differentiation.

  6. Effects of downregulating TEAD4 transcripts by RNA interference on early development of bovine embryos

    OpenAIRE

    SAKURAI, Nobuyuki; TAKAHASHI, Kazuki; EMURA, Natsuko; HASHIZUME, Tsutomu; SAWAI, Ken

    2016-01-01

    Transcription factor TEA domain family transcription factor 4 (Tead4) is one of the key factors involved in the differentiation of the trophectoderm (TE) in murine embryos. However, knowledge on the roles of TEAD4 in preimplantation development during bovine embryos is currently limited. This study examined the transcript and protein expression patterns of TEAD4 and attempted to elucidate the functions of TEAD4 during bovine preimplantation development using RNA interference. TEAD4 mRNA was f...

  7. 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

  8. Mouse embryos cultured in amniotic fluid | Oettle | South African ...

    African Journals Online (AJOL)

    The embryos were cultured for 72 hours at 37°C in 5% carbon dioxide in air. Osmolarity, pH, partial arterial carbon dioxide pressure and HCO; were checked before culture and again at the end of culture. Embryos were assessed according to the stage of development, namely degenerate embryos, morulae and blastocysts.

  9. Mouse embryos cultured In amnIotIc fluid

    African Journals Online (AJOL)

    Mouse embryos are used as a monitor of human in vicro fertilisation (IVF) culture conditions. Embryo culture media are often complicated solutions requiring careful monitoring to ensure uniformity for successful embryo culture.1-3 Much of the quality control is outside the scope of the IVF laboratory, for example the source of ...

  10. Cryopreservation for bovine embryos in serum-free freezing medium containing silk protein sericin.

    Science.gov (United States)

    Isobe, Tomohiro; Ikebata, Yoshihisa; Onitsuka, Takeshi; Do, Lanh Thi Kim; Sato, Yoko; Taniguchi, Masayasu; Otoi, Takeshige

    2013-10-01

    Because the use of serum in the embryo cryopreservation increases the probability of animal health problems such as bovine spongiform encephalopathy (BSE) and viral infections, this study was conducted to examine the effects of sericin supplementation for serum-free freezing medium on the survival and development of bovine embryos after freezing-thawing and direct transfer to recipients. When in vitro-produced bovine embryos were frozen conventionally in the freezing medium supplemented with various concentrations (0.1%, 0.5%, and 1.0%) of sericin, the percentages of damaged zona pellucida, survival, and development of frozen-thawed embryos were similar to those of embryos frozen in freezing medium supplemented with 0.4% bovine serum albumin (BSA) and 20% fetal bovine serum (FBS) (0.4BSA/20F; control). When in vivo-derived embryos were frozen with 0.4BSA/20F (control), 0.5% sericin +20% FBS (0.5S/20F) or 0.5% sericin (0.5S) and were subsequently transferred directly to recipients, the percentages of recipients with pregnancy and normal calving in the 0.5S/20F group were higher than those in the control group (47.3% vs. 40.1% and 94.6% vs. 87.3%, respectively). Moreover, the percentages of recipients with pregnancy and normal calving (42.2% and 92.4%, respectively) in the 0.5S group were similar with those of other groups. In conclusion, these results indicate that serum-free freezing medium supplemented with sericin is available for the cryopreservation of bovine embryos and that it is beneficial for the elimination of a potential source of biological contamination by serum or BSA. Copyright © 2013. Published by Elsevier Inc.

  11. Regulation of heat-inducible HSPA1A gene expression during maternal-to-embryo transition and in response to heat in in vitro-produced bovine embryos.

    Science.gov (United States)

    Lelièvre, Jean-Marc; Peynot, Nathalie; Ruffini, Sylvie; Laffont, Ludivine; Le Bourhis, Daniel; Girard, Pierre-Marie; Duranthon, Véronique

    2017-09-01

    In in vitro-produced (IVP) bovine embryos, a burst in transcriptional activation of the embryonic genome (EGA) occurs at the 8-16-cell stage. To examine transcriptional regulation prior to EGA, notably in response to heat stress, we asked (1) whether the spontaneous expression of a luciferase transgene that is driven by the minimal mouse heat-shock protein 1b (hspa1b) gene promoter paralleled that of HSPA1A during EGA in IVP bovine embryo and (2) whether expression of the endogenous heat-inducible iHSPA group member HSPA1A gene and the hspa1b/luciferase transgene were induced by heat stress (HS) prior to EGA. Using two culture systems, we showed that luciferase activity levels rose during the 40-h long EGA-associated cell cycle. In contrast, iHSPA proteins were abundant in matured oocytes and in blastomeres from the two-cell to the 16-cell stages. However, normalised results detected a rise in the level of HSPA1A and luciferase mRNA during EGA, when transcription was required for their protein expression. Prior to EGA, HS-induced premature luciferase activity and transgene expression were clearly inhibited. We could not, however, establish whether this was also true for HSPA1A expression because of the decay of the abundant maternal transcripts prior to EGA. In bovine embryos, heat-induced expression of hspa1b/luciferase, and most likely of HSPA1A, was therefore strictly dependent on EGA. The level of the heat-shock transcription factor 1 molecules that were found in cell nuclei during embryonic development correlated better with the embryo's capacity for heat-shock response than with EGA-associated gene expression.

  12. Full-term development of gaur-bovine interspecies somatic cell nuclear transfer embryos: effect of trichostatin A treatment.

    Science.gov (United States)

    Srirattana, Kanokwan; Imsoonthornruksa, Sumeth; Laowtammathron, Chuti; Sangmalee, Anawat; Tunwattana, Wanchai; Thongprapai, Thamnoon; Chaimongkol, Chockchai; Ketudat-Cairns, Mariena; Parnpai, Rangsun

    2012-06-01

    Trichostatin A (TSA) has previously been used in somatic cell nuclear transfer (SCNT) to improve the cloning efficiency in several species, which led our team to investigate the effects of TSA on the full-term development of bovine SCNT and gaur-bovine interspecies SCNT (gaur iSCNT; gaur somatic cells as donors and bovine oocytes as recipients) embryos. Treatment with 50 nM TSA for 10 h after fusion had no positive effects on the rates of fusion, cleavage, or the development to eight-cell or morula stages in both bovine SCNT and gaur iSCNT embryos. However, TSA treatment significantly enhanced the blastocyst formation rate in bovine SCNT embryos (44 vs. 32-34% in the TSA-treated and TSA-untreated groups, respectively), but had no effects on gaur iSCNT embryos. The fresh blastocysts derived from bovine SCNT and gaur iSCNT embryos (fresh groups), as well as vitrified bovine SCNT blastocysts (vitrified group), were transferred to bovine recipients. We found that TSA treatment increased the pregnancy rates only in recipients receiving fresh bovine SCNT embryos. In recipients receiving TSA-treated bovine SCNT embryos, three cloned calves from the fresh group and twin cloned calves from the vitrified group were delivered; however, no calf was born from the TSA-untreated bovine SCNT embryos. In contrast, one gaur iSCNT calf was born from a recipient receiving blastocysts from the TSA-untreated group. In summary, TSA improved the preimplantation development and pregnancy rates of bovine SCNT embryos, but did not have any beneficial effect on gaur iSCNT embryos. However, one gaur iSCNT calf reached full-term development.

  13. The effect of Ureaplasma diversum activated mononuclear leukocytes on the development and interferon-tau production by bovine IVF-derived embryos.

    Science.gov (United States)

    Chełmońska-Soyta, A; Katska, L; Kurpisz, M; Stefaniak, T; Zimecki, M

    2001-08-01

    Ureaplasma diversum is an opportunistic pathogen of the bovine genital tract causing herd outbreaks of granular vulvitis, abortion and infertility. Early embryonic death probably contributes to reduction of the reproductive performance in cows, however, pathogenesis of the disease remains obscure. The aim of the study was to examine whether activation of mononuclear leukocytes by U. diversum may affect embryo development and IFN-tau production. Bovine peripheral blood mononuclear leukocytes were cultured with U.diversum antigen for 24 h. The levels of IL-1, TNF-alpha, NO and GM-CSF in the cell culture supernatants were measured. IVF-derived embryos were cultured in the presence of supernatants from activated leukocytes. The development of embryos until day 6 postinsemination and the rate of morulae/blastocysts were determined. IFN-tau production in supernatants of cultured embryos was examined by inhibition of a virally-induced cytopathic effect. The results showed that U. diversum stimulated mononuclear leukocyte production of IL-1, TNF-alpha and NO. Supernatants from U. diversum-activated cells did not impair the rates of the embryo development and blastocyst formation. The products of activated leukocytes increased the IFN-tau production by cultured blastocysts. This suggest that U. diversum infection provides leukocyte-mediated signals for developing embryos for generation of additional production of cytokine - an important component of innate immunity.

  14. [Association of human chorionic gonadotropin level in embryo culture media with early embryo development].

    Science.gov (United States)

    Wang, Haiying; Zhang, Renli; Han, Dong; Liu, Caixia; Cai, Jiajie; Bi, Yanling; Wen, Anmin; Quan, Song

    2014-06-01

    To investigate the association of human chorionic gonadotropin (HCG) level on day 3 of embryo culture with embryo development. Spent culture media were collected from individually cultured embryos on day 3 of in vitro fertilization and embryo transfer (IVF-ET) cycles. HCG concentration in the culture media was measured using an ELISA kit and its association with embryo development was assessed. In the 163 samples of embryo culture media from 60 patients, HCG was positive in 153 sample (93.8%) with a mean level of 0.85 ± 0.43 mIU/ml. The concentration of hCG in the culture media increased gradually as the number of blastomeres increased (F=2.273, P=0.03), and decreased as the morphological grade of the embryo was lowered (F=3.900, P=0.02). ELISA is capable of detecting HCG levels in spent culture media of embryos on day 3 of in vitro culture. The concentration of HCG in spent culture media is positively correlated with the status of early embryo development and implantation rate and thus serves as a useful marker for embryo selection in IVF-ET procedure.

  15. Molecular Characterization of the First Bovine Herpesvirus 4 (BoHV-4 Strains Isolated from In Vitro Bovine Embryos production in Argentina.

    Directory of Open Access Journals (Sweden)

    Erika González Altamiranda

    Full Text Available Bovine herpesvirus 4 (BoHV-4 is increasingly considered as responsible for various problems of the reproductive tract. The virus infects mainly blood mononuclear cells and displays specific tropism for vascular endothelia, reproductive and fetal tissues. Epidemiological studies suggest its impact on reproductive performance, and its presence in various sites in the reproductive tract highlights its potential transmission in transfer-stage embryos. This work describes the biological and genetic characterization of BoHV-4 strains isolated from an in vitro bovine embryo production system. BoHV-4 strains were isolated in 2011 and 2013 from granulosa cells and bovine oocytes from ovary batches collected at a local abattoir, used as "starting material" for in vitro production of bovine embryos. Compatible BoHV-4-CPE was observed in the co-culture of granulosa cells and oocytes with MDBK cells. The identity of the isolates was confirmed by PCR assays targeting three ORFs of the viral genome. The phylogenetic analyses of the strains suggest that they were evolutionary unlinked. Therefore it is possible that BoHV-4 ovary infections occurred regularly along the evolution of the virus, at least in Argentina, which can have implications in the systems of in vitro embryo production. Thus, although BoHV-4 does not appear to be a frequent risk factor for in vitro embryo production, data are still limited. This study reveals the potential of BoHV-4 transmission via embryo transfer. Moreover, the high variability among the BoHV-4 strains isolated from aborted cows in Argentina highlights the importance of further research on the role of this virus as an agent with the potential to cause reproductive disease in cattle. The genetic characterization of the isolated strains provides data to better understand the pathogenesis of BoHV-4 infections. Furthermore, it will lead to fundamental insights into the molecular aspects of the virus and the means by which these

  16. Numerical calculations for diffusion effects in the well-of-the-well culture system for mammalian embryos.

    Science.gov (United States)

    Matsuura, Koji

    2014-06-01

    Recent studies suggest that the microenvironment and embryo density used during embryo culture considerably affect development to the blastocyst stage. High embryo density allows for autocrine secretions to diffuse to neighbouring embryos during group culture, with a positive effect on further development. A variation of group culture is the well-of-the-well (WOW) culture system, allowing for individual identification of embryos cultured in small holes in a microdroplet. Bovine blastocyst development is higher in the WOW culture system than in conventional group culture. To compare the concentration of chemical factors between conventional and WOW culture, a model was constructed to calculate the concentration of secreted factors based on Fick's second law of diffusion using spreadsheet software. Furthermore, model was used to determine the concentration of growth factors and waste materials adjacent to the embryo periphery. The results of these calculations suggest that the highest difference in the concentration of secreted small molecules and macromolecules was at the most two- to threefold, with the concentrations reduced more and diffusion kinetics facilitated to a greater extent in the WOW culture system. The average ratio of the concentration of secreted macromolecules (10nm diameter) around the embryos was also compared between systems with well widths of 0.1 and 0.3mm. The concentration of secreted materials surrounding embryos increased in a narrow tapered well. The findings suggest that the WOW culture system is better than conventional group culture because of the increased final concentration of autocrine factors and higher diffusion kinetics of waste materials.

  17. New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system

    DEFF Research Database (Denmark)

    Vajta, G; Peura, T T; Holm, P

    2000-01-01

    (WOW) system. Small wells (WOWs) were formed in four-well dishes by melting the bottom with heated steel rods. The WOWs were then rinsed, the wells were filled with medium, and the embryos were placed into the WOWs. To test the value of the WOW system a 3 x 3 factorial experiment was performed. Bovine...... presumptive zygotes were cultured from day 1 to day 7 (day 0: day of insemination) using three modules (single embryos, embryo groups of five, or single zona-digested embryos) and three different culture systems (400 microl medium, 200 microl drops, or WOWs). An additional control group consisted of 40 to 50...... embryos cultured in 400 microl medium. The WOW system resulted in higher blastocyst/oocyte rates for all three modules (single: 59 group of five: 61 single zona-digested: 53 than the culture in drops or in wells (P WOWs per well...

  18. Transcription of ribosomal RNA genes is initiated in the third cell cycle of bovine embryos

    DEFF Research Database (Denmark)

    Jakobsen, Anne Sørig; Avery, Birthe; Dieleman, Steph J.

    2006-01-01

    Transcription from the embryos own ribosomal genes is initiated in most species at the same time as the maternal-embryonic transition. Recently data have indicated that a minor activation may take place during the third embryonic cell cycle in the bovine, one cell cycle before the major activatio...

  19. 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...

  20. 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 multiplication of genetically valuable donor embryos. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Supplementation with small-extracellular vesicles from ovarian follicular fluid during in vitro production modulates bovine embryo development

    Science.gov (United States)

    Andrade, Gabriella M.; del Collado, Maite; Sampaio, Rafael V.; Sangalli, Juliano R.; Silva, Luciano A.; Pinaffi, Fábio V. L.; Jardim, Izabelle B.; Cesar, Marcelo C.; Nogueira, Marcelo F. G.; Cesar, Aline S. M.; Coutinho, Luiz L.; Pereira, Rinaldo W.; Perecin, Felipe; Meirelles, Flávio V.

    2017-01-01

    Pregnancy success results from the interaction of multiple factors, among them are folliculogenesis and early embryonic development. Failure during these different processes can lead to difficulties in conception. Alternatives to overcome these problems are based on assisted reproductive techniques. Extracellular vesicles are cell-secreted vesicles present in different body fluids and contain bioactive materials, such as messenger RNA, microRNAs (miRNAs), and proteins. Thus, our hypothesis is that extracellular vesicles from follicular fluid from 3–6 mm ovarian follicles can modulate bovine embryo development in vitro. To test our hypothesis follicular fluid from bovine ovaries was aspirated and small-extracellular vesicles (extracellular vesicles (EVs) were utilized for functional experiments investigating their role in modulating messenger RNA, microRNA as well as global DNA methylation and hydroxymethylation levels of bovine blastocysts. EVs from 3–6 mm follicles were used for RNA-seq and miRNA analysis. Functional annotation analysis of the EVs transcripts revealed messages related to chromatin remodeling and transcriptional regulation. EVs treatment during oocyte maturation and embryo development causes changes in blastocyst rates, as well as changes in the transcription levels of genes related to embryonic metabolism and development. Supplementation with EVs from 3–6 mm follicles during oocyte maturation and early embryo development (until the 4-cell stage) increased the levels of bta-miR-631 (enriched in EVs from 3–6 mm follicles) in embryos. Interestingly, the addition of EVs from 3–6 mm follicles induced changes in global DNA methylation and hydroxymethylation levels compared to embryos produced by the standard in vitro production system. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of 3–6 mm follicles during oocyte maturation and early embryo development can partially modify

  2. Effects of oocyte quality, incubation time and maturation environment on the number of chromosomal abnormalities in IVF-derived early bovine embryos.

    Science.gov (United States)

    Demyda-Peyrás, Sebastian; Dorado, Jesus; Hidalgo, Manuel; Anter, Jaouad; De Luca, Leonardo; Genero, Enrique; Moreno-Millán, Miguel

    2013-01-01

    Chromosomal aberrations are one of the major causes of embryo developmental failures in mammals. The occurrence of these types of abnormalities is higher in in vitro-produced (IVP) embryos. The aim of the present study was to investigate the effect of oocyte morphology and maturation conditions on the rate of chromosomal abnormalities in bovine preimplantational embryos. To this end, 790 early cattle embryos derived from oocytes with different morphologies and matured under different conditions, including maturation period (24 v. 36h) and maturation media (five different serum supplements in TCM-199), were evaluated cytogenetically in three sequential experiments. The rates of normal diploidy and abnormal haploidy, polyploidy and aneuploidy were determined in each embryo. Throughout all the experiments, the rate of chromosomal abnormalities was significantly (P<0.05) affected by oocyte morphology and maturation conditions (maturation time and culture medium). Lower morphological quality was associated with a high rate of chromosome abnormalities (P<0.05). Moreover, polyploidy was associated with increased maturation time (P<0.01), whereas the maturation medium significantly (P<0.05) affected the rates of haploidy and polyploidy. In general, supplementing the maturation medium with oestrous cow serum or fetal calf serum resulted in higher rates of chromosomal aberrations (P<0.05) compared with the other serum supplements tested (bovine steer serum, anoestroues cow serum, bovine amniotic fluid and bovine serum albumin). On the basis of the results of the present study, we conclude that the morphological quality of oocytes and the maturation conditions affect the rate of chromosomal abnormalities in IVP bovine embryos.

  3. Cryo-survival and development of bovine blastocysts are enhanced by culture with recombinant albumin and hyaluronan.

    Science.gov (United States)

    Lane, Michelle; Maybach, Jeffrey M; Hooper, Kathy; Hasler, John F; Gardner, David K

    2003-01-01

    Recombinant albumin can be used to supplement culture medium for the maturation and fertilization of bovine oocytes and subsequent embryo development to the blastocyst stage. Recombinant albumin was able to support blastocyst development at rates equivalent to that of bovine serum albumin (BSA) supplemented media. Supplementation of media containing recombinant albumin and citrate stimulated blastocyst expansion. Culture with recombinant albumin and citrate significantly increased the ability of the resultant blastocysts to re-expand and hatch following cryopreservation. The further addition of the glycosaminoglycan hyaluronan to the culture medium containing either BSA or recombinant albumin also increased the ability of blastocysts to survive cryopreservation. Inclusion of recombinant albumin and hyaluronan in culture media facilitates the development of physiological defined culture conditions. For bovine embryos this has implications for both research and commercial applications where defined reproducible conditions are desirable. Copyright 2003 Wiley-Liss, Inc.

  4. L- carnitine and trans-10, cis-12 conjugated linoleic acid on in vitro bovine embryo production and cryopreservation

    OpenAIRE

    Zolini, Adriana Moreira

    2015-01-01

    High lipid content in embryo is associated with low freezing tolerance. This study assessed the effects of exogenous L-carnitine and trans-10, cis-12 (t10, c12) conjugated linoleic acid (CLA) on in-vitro development and cryotolerance of bovine embryos when added during different stages of in vitro production of embryos. For all experiments, embryos were produced in vitro using slaughterhouse cows oocytes. Cleavage rates on Day 3, blastocyst and advanced blastocyst (hatching/hatched blastocyst...

  5. Telomere-to-centromere ratio of bovine clones, embryos, gametes, fetal cells, and adult cells.

    Science.gov (United States)

    Meerdo, Lora N; Reed, William A; White, Kenneth L

    2005-01-01

    In 1997, Dolly, the first animal cloned from an adult cell, was born. It was announced in 1999 that Dolly might be aging faster than normal because her telomeres were shorter than age-matched control sheep. Telomeres, a repeated DNA sequence located at the ends of linear chromosomes, allow for base pair loss during DNA replication. Telomere shortening acts as a "mitotic clock," leading to replicative senescence. By using whole cell lysate and slot-blot analysis, we determined the telomere-to-centromere ratio (T/C) for bovine gametes, embryos, fetal tissues (brain, heart, lung, kidney, uterus, ovary, and skin), adult donor cells, and cloned embryos. Our data indicates a consistency in T/C among the various fetal tissues. The T/C of sperm is significantly lower than in oocytes. The T/C decreases from the oocyte to the 2-8-cell stage embryo, increases dramatically at the morula stage, and decreases at the blastocyst stage. Our data shows no significant difference in T/C between cloned embryos and in vitro fertilized (IVF) embryos, but there is a significant difference between cloned embryos and adult donor cells. In conclusion, the enucleated bovine oocyte has the ability to reestablish the telomere length of adult somatic cell donor nuclei.

  6. CanUreaplasma diversumbe transmitted from donor to recipient through the embryo? Two case reports outliningU. diversumlosses in bovine embryo pregnancies.

    Science.gov (United States)

    Crane, M Bronwyn; Hughes, Colleen A

    2018-01-01

    Two bovine embryo recovery results are outlined from different herds. Both cases involve significant late gestational loss from embryos relating back to a single donor. Ureaplasma diversum was confirmed in 3 of 4 cases submitted for postmortem examination. Natural infection originating from the donor and transmitted to the recipient has not previously been documented.

  7. Factors that affect the reproductive efficiency of the recipient within a bovine embryo transfer program

    Directory of Open Access Journals (Sweden)

    Arturo Duica A.

    2007-12-01

    Full Text Available The embryo transfer is a biotechnological technique that allows increasing the descendant of animals with high genetic value. The positive results, represented in pregnancy after the application of this technique, are affected by some factors that are inherent to the donor, the embryo, the technique, and the recipients which receive a strange embryo in the uterus allowing pregnancy. This review describes some factors affecting the reproductive efficiency of the recipients of bovine embryos within a program of embryo transfer. Its important to evaluate the parameters in this kind of recipients, as race, age, physiological status, health status, weight, reproductive tract integrity and management, and also too monitoring the ovarian structures while the estrus synchronization, and within previous and posterior stages in embryo transfer procedure. Therefore an optimum follicular development will be determinant to corpus luteum formation which generates enough serum progesterone concentrations to offer a right uterine environment allowing the optimum embryo development. Controlling the factors that affect the efficiency of the embryo transfer, it will obtain an increasing of positive results represented in pregnancies and births of individuals come from animals with high genetic value.

  8. Short-term and long-term effects of embryo culture in the surrogate sheep oviduct versus in vitro culture for different domestic species.

    Science.gov (United States)

    Lazzari, G; Colleoni, S; Lagutina, I; Crotti, G; Turini, P; Tessaro, I; Brunetti, D; Duchi, R; Galli, C

    2010-04-01

    The culture of early embryos in the surrogate xeno-oviduct was first developed in the early 1950s to allow transport of embryos at long distances. Later, it was applied to the study of culture requirements of the early embryo especially that of bovine origin. In this article, we review the data available on the culture of in vitro-matured and in vitro-fertilized embryos of Bos taurus, Sus scrofa, Equus caballus and Ovis aries in the surrogate sheep oviduct compared with data on in vitro culture in different media. Short-term and long-term cellular and molecular effects are described mainly for the bovine species where more extensive use of this technique has been made. A comparison with in vitro culture in various conditions and species indicate that embryos cultured in the sheep oviduct have close similarities to totally in vivo-derived embryos. The data provided demonstrate that the technique of in vivo culture in the surrogate sheep oviduct is versatile and allows a high rate of embryonic development in all species examined. 2010 Elsevier Inc. All rights reserved.

  9. Quality improvement of transgenic cloned bovine embryos using an aggregation method: Effects on cell number, cell ratio, embryo perimeter, mitochondrial distribution, and gene expression profile.

    Science.gov (United States)

    Bang, J I; Jin, J I; Ghanem, N; Choi, B H; Fakruzzaman, M; Ha, A N; Lee, K L; Uhm, S J; Ko, D H; Koo, B C; Lee, J G; Kong, I K

    2015-09-01

    The production of cloned embryos using conventional methods has extremely low success rates owing to low embryo quality. To improve the quality of cloned bovine embryos expressing enhanced green fluorescent protein (EGFP), we applied an aggregation culture method. The EGFP gene was transfected into bovine fetal fibroblasts using a retroviral vector system. Somatic cell nuclear transfer was performed using these cells, and the resulting embryos were cultured in aggregates or individually. Gene expression was analyzed by a microarray, and differentially expressed genes were validated by quantitative real-time polymerase chain reaction. The total number of cells per blastocyst and the ratio of inner cell mass cells to trophectoderm cells were higher in aggregated transgenic cloned blastocysts (agBL; 368.7 ± 109.6 and 1:4.8, respectively) than in in vitro-fertilized blastocysts (ivfBL; 189.8 ± 65.8 and 1:2.6, respectively) and nonaggregated transgenic cloned blastocysts (sBL; 113.1 ± 36.3 and 1:1.5, respectively; P fluorescence intensity was higher in the agBL group than in the ivfBL and sBL groups (P genes identified in the microarray belonged to 18 categories. Expression of the Krüppel-like factor 4 gene, which is associated with cell proliferation, development, and transcription, was 7.2-fold higher in the agBL group than in the ivfBL group (P  0.05). Expression of the heat shock 70-kDa protein 1A gene, which is associated with apoptosis, was 12-fold higher in the sBL group than in the ivfBL and agBL groups (P Expression of a stemness-related gene (octamer-binding transcription factor 4) and trophectoderm-specific genes (homeobox protein CDX2 and keratin 18) was higher in the agBL group than in the sBL group (P expression of the stemness gene homeobox protein NANOG did not differ among the groups (P > 0.05). Taken together, these data suggest that the aggregation method improves the quality of cloned embryos expressing EGFP and might be helpful

  10. New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system.

    Science.gov (United States)

    Vajta, G; Peura, T T; Holm, P; Páldi, A; Greve, T; Trounson, A O; Callesen, H

    2000-03-01

    Culture of mammalian zygotes individually and in small groups results in lower developmental rates than culture of large groups. Zona-free zygotes also have impaired developmental potential in current culture systems. This paper describes a new approach to resolve the problems, the Well of the Well (WOW) system. Small wells (WOWs) were formed in four-well dishes by melting the bottom with heated steel rods. The WOWs were then rinsed, the wells were filled with medium, and the embryos were placed into the WOWs. To test the value of the WOW system a 3 x 3 factorial experiment was performed. Bovine presumptive zygotes were cultured from day 1 to day 7 (day 0: day of insemination) using three modules (single embryos, embryo groups of five, or single zona-digested embryos) and three different culture systems (400 microl medium, 200 microl drops, or WOWs). An additional control group consisted of 40 to 50 embryos cultured in 400 microl medium. The WOW system resulted in higher blastocyst/oocyte rates for all three modules (single: 59%; group of five: 61%; single zona-digested: 53%) than the culture in drops or in wells (P WOWs per well. The cell number of blastocysts cultured in the WOW system did not differ from that of the controls. Apart from its theoretical value in revealing the role of different factors influencing embryo development in vitro, the WOW system may have immediate practical consequences in certain areas of mammalian embryo production. Copyright 2000 Wiley-Liss, Inc.

  11. Freezing of in vitro produced bovine embryos in animal protein-free medium containing vegetal peptones.

    Science.gov (United States)

    George, F; Vrancken, M; Verhaeghe, B; Verhoeye, F; Schneider, Y-J; Massip, A; Donnay, I

    2006-09-15

    Successful cryopreservation is essential for a large-scale dispersal of bovine in vitro produced (IVP) embryos that have been shown to be more sensitive to cryopreservation than their in vivo counterparts. On the other hand, the use of animal proteins in freezing media increases sanitary risks. We first replaced animal proteins, such as bovine serum albumin (BSA) in the freezing medium by plant-derived peptides (vegetal peptones). A batch of wheat peptones was selected after a preliminary experiment showing the absence of toxicity of concentrationsanimal protein-free freezing medium containing 1.8 mg/mL peptones. No beneficial effect of adding 1 mg/mL sodium hyaluronate or 100 microM beta-mercaptoethanol was observed on embryo survival or quality. In conclusion, we have demonstrated that vegetal peptones can replace BSA in freezing media without affecting blastocyst survival and quality.

  12. Study Progress on Tissue Culture of Maize Mature Embryo

    Science.gov (United States)

    Wang, Hongzhen; Cheng, Jun; Cheng, Yanping; Zhou, Xioafu

    It has been paid more and more attention on maize tissue culture as it is a basic work in maize genetic transformation, especially huge breakthrough has been made in maize tissue culture utilizing mature embryos as explants in the recent years. This paper reviewed the study progress on maize tissue culture and plant regeneration utilizing mature embryos as explants from callus induction, subculture, plant regeneration and browning reduction and so on.

  13. Silencing CENPF in bovine preimplantation embryo induces arrest at 8-cell stage

    Czech Academy of Sciences Publication Activity Database

    Toralová, Tereza; Šušor, Andrej; Němcová, Lucie; Kepková, Kateřina; Kaňka, Jiří

    2009-01-01

    Roč. 138, č. 5 (2009), s. 783-791 ISSN 1470-1626 R&D Projects: GA ČR GA523/06/1226; GA ČR(CZ) GD204/05/H023 Institutional research plan: CEZ:AV0Z50450515 Keywords : CENPF * bovine embryo * preimplantation development Subject RIV: GI - Animal Husbandry ; Breeding Impact factor: 2.579, year: 2009

  14. Differences in gene expression profiles between human preimplantation embryos cultured in two different IVF culture media

    NARCIS (Netherlands)

    Kleijkers, Sander H. M.; Eijssen, Lars M. T.; Coonen, Edith; Derhaag, Josien G.; Mantikou, Eleni; Jonker, Martijs J.; Mastenbroek, Sebastiaan; Repping, Sjoerd; Evers, Johannes L. H.; Dumoulin, John C. M.; van Montfoort, Aafke P. A.

    2015-01-01

    Is gene expression in human preimplantation embryos affected by the medium used for embryo culture in vitro during an IVF treatment? Six days of in vitro culture of human preimplantation embryos resulted in medium-dependent differences in expression level of genes involved in apoptosis, protein

  15. Differences in gene expression profiles between human preimplantation embryos cultured in two different IVF culture media

    NARCIS (Netherlands)

    Kleijkers, S.H.M.; Eijssen, L.M.T.; Coonen, E.; Derhaag, J.G.; Mantikou, E.; Jonker, M.J.; Mastenbroek, S.; Repping, S.; Evers, J.L.H.; Dumoulin, J.C.M.; van Montfoort, A.P.A.

    2015-01-01

    STUDY QUESTION: Is gene expression in human preimplantation embryos affected by the medium used for embryo culture in vitro during an IVF treatment? SUMMARY ANSWER: Six days of in vitro culture of human preimplantation embryos resulted in medium-dependent differences in expression level of genes

  16. Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos

    DEFF Research Database (Denmark)

    Alexopoulos, Natalie I.; Maddox-Hyttel, Poul; Tveden-Nyborg, Pernille Yde

    2008-01-01

    the application of new reproductive technologies such as somatic cell nuclear transfer (SCNT). In the present study, days 14 and 21 bovine embryos, generated by either in vitro-production (IVP) or SCNT, performed by either subzonal injection (SUZI) or handmade cloning (HMC), were compared by stereomicroscopy...... of VIM and TUBB3 was less distinct in SCNT embryos when compared with IVP embryos, indicating slower or compromised development. In conclusion, SCNT and to some degree, IVP embryos displayed a high rate of embryonic mortality before D14 and surviving embryos displayed reduced quality with respect...

  17. Survival of embryo irradiated with gamma rays by embryo culture in Brassica pekinensis Rupr

    International Nuclear Information System (INIS)

    Moue, T.

    1984-01-01

    The effect of irradiation on the survival rates and embryonic development of Brassica pekinensis RUPR. (Varieties; Kashin, Kohai 65 nichi and kairyochitose) was investigated. The purpose of this study was to seek ways of increasing the survival rates of embryos such as B.oleracea obtained through embryo culture techniques after irradiation doses affecting seed fertility and germination, for the purpose of increasing mutation rates. Embryos at different developmental stages ranging from the globular to the early heart stages were irradiated with 20 KR of gamma rays at the daily rate 0L 20 KR or 10 KR (Fig.1 and Table 1). The embryos were excised from ovules 4 to 10 days after irradiation and cultured on White's medium. The shooting and rooting rates on the 34th day of culture were higher at the dose of 10 KR/day than 20 KR/day and were lower when the materials were irradiated at the young embryonic stage (Table 3). Varietal differences in the shooting and rooting rates were also observed. The irradiated embryos survived mainly in the state of callus. It was concluded that the embryo culture technique was successful when applied to irradiated embryos excised at the young embryonic stage and that the technique affected B.pekinensis less than B.oleracea

  18. Radioactive marking of proteins in cultured mouse embryos

    International Nuclear Information System (INIS)

    Nowak, J.

    1984-01-01

    The purpose of this work was to build an in vitro test system, with which on the one hand postimplantation embryos of the mouse could be cultured without morphological of physiological damage and on the other hand their protein could be as highly marked as possible. With this radioactively marked proteins were to be won, which are optimally suited for a high separation by two-dimensional electrophoresis. In addition incubation and preparation methods were found for the ages of day 10, 11 and 12 of the embryonic development. With the use of 3 H-marked amino acids in the culture medium it was determined that embryos without embryonic membranes, so-called N-embryos, built in more radioactivity into their proteins than the embryos with embryonic membranes, the so-called DAO-embryos or the DO-embryos. On the contrary, the embryos with intact blood circulation (DO-embryos) showed an even distribution of radioactive marker in their bodies. Since an even distribution of the marker in the embryo is a necessary prerequisite for a representative presentation of the proteins by 2DE, the DO-preparation was considered the best suited method. In order to increase the amount of radioactivity incorporated into the proteins of the DO-embryos, the concentration of the used isotope or the incubation length could be increased. A combination of both proved to be the best method. A 14 C-marked amino acid mixture of 20 μCi/corresponds to 20 μl instead of the usual 150 μCi 3 H-marked amino acids in a culture medium proved to be equally suitable. Notable changes which would have indicated a damaging affect of the used radioactivity or the in vitro culturing were not observed. The achieved methodical conditions were used for the presentation of the embryo proteins by two-dimensional electrophoresis and fluorography. (orig./MG) [de

  19. Obtaining barley haploid embryos and seedlings using anther culture technique

    International Nuclear Information System (INIS)

    Arabi, M.I.E.; Al-Safadi, B.; Mir Ali, N.

    2000-01-01

    The effect of three barley genotypes (Igri, Arabi abiad, and Taqa 76), three irradiation doses (0, 5, and 10 Gy), and two media (FW, modified FW), on the number of formed embryos, and the ratio between regenerated embryos to green seedlings and albinos, were studied using anther culture. Also the study involved the compatibility between seedling morphology and chromosome number. results indicated significant differences among the genotypes, and media in callus and embryos formation and also in the ratio and albino seedlings. However, the effect of gamma rays dose was significant only on embryos regeneration. A high percentage of compatibility (90%) was obtained between the seedling morphology and chromosome number. (author)

  20. Functional analysis of bovine Nramp1 and production of transgenic cloned embryos in vitro.

    Science.gov (United States)

    Cheng, Xiang; Yu, Xiaoli; Liu, Yajun; Deng, Jie; Ma, Xiaoling; Wang, Huayan

    2015-02-01

    Natural resistance-associated macrophage protein 1 (Nramp1) plays an important role in restraining the growth of intracellular pathogens within macrophages. In this study, Nramp1 cDNA was cloned from Qinchuan cattle and its anti-bacterial activity was demonstrated as being able to significantly inhibit the growth of Salmonella abortusovis and Brucella abortus in macrophages. Calf fibroblasts stably transfected with pSP-NRAMP1-HA vector were used to reconstruct bovine embryos by somatic cell nuclear transfer (SCNT). Reconstructed embryos were maturated in vitro and the blastocyst formation rate (14.0%) was similar to that of control embryos (14.5%). Transgenic blastocysts were transplanted into 43 recipient cattle, of which 14 recipients became pregnant as evidenced by non-return estrus and by rectal palpation. One fetus was aborted after 6½ months of pregnancy and transgene integration was confirmed by semi-quantitative polymerase chain reaction. Together, this study showed that bovine Nramp1 retains biological function against the growth of intracellular bacteria and can be used to reconstruct embryos and produce Nramp1 transgenic cattle, which may benefit the animal and enhance their ability to prevent attack by intracellular pathogens.

  1. Effect of maternal methionine supplementation on the transcriptome of bovine preimplantation embryos.

    Directory of Open Access Journals (Sweden)

    Francisco Peñagaricano

    Full Text Available Maternal nutrition exclusively during the periconceptional period can induce remarkable effects on both oocyte maturation and early embryo development, which in turn can have lifelong consequences. The objective of this study was to evaluate the effect of maternal methionine supplementation on the transcriptome of bovine preimplantation embryos. Holstein cows were randomly assigned to one of two treatments differing in level of dietary methionine (1.89 Met vs. 2.43 Met % of metabolizable protein from calving until embryo flushing. High quality preimplantation embryos from individual cows were pooled and then analyzed by RNA sequencing. Remarkably, a subtle difference in methionine supplementation in maternal diet was sufficient to cause significant changes in the transcriptome of the embryos. A total of 276 genes out of 10,662 showed differential expression between treatments (FDR <0.10. Interestingly, several of the most significant genes are related to embryonic development (e.g., VIM, IFI6, BCL2A1, and TBX15 and immune response (e.g., NKG7, TYROBP, SLAMF7, LCP1, and BLA-DQB. Likewise, gene set enrichment analysis revealed that several Gene Ontology terms, InterPro entries, and KEGG pathways were enriched (FDR <0.05 with differentially expressed genes involved in embryo development and immune system. The expression of most genes was decreased by maternal methionine supplementation, consistent with reduced transcription of genes with increased methylation of specific genes by increased methionine. Overall, our findings provide evidence that supplementing methionine to dams prior to conception and during the preimplantation period can modulate gene expression in bovine blastocysts. The ramifications of the observed gene expression changes for subsequent development of the pregnancy and physiology of the offspring warrant further investigation in future studies.

  2. Lipoprotein receptors in cultured bovine endothelial cells

    International Nuclear Information System (INIS)

    Struempfer, A.E.M.

    1983-07-01

    In this study, receptors that may be involved in the uptake of low density lipoproteins (LDL) and low density lipoproteins which have been modified by acetylation (AcLDL), were characterized. Aortic epithelial cells were used and a cell culture system which closely resembled the in vivo monolayer was established. Endothelial cell and lipoprotein interactions were examined by incubating the cells with 125 l-labelled lipoproteins under various conditions. The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased by about 50%. However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form cholesteryl esters

  3. In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media.

    Science.gov (United States)

    Kelley, Rebecca L; Gardner, David K

    2017-05-01

    Single embryo culture is suboptimal compared with group culture, but necessary for embryo monitoring, and culture systems should be improved for single embryos. Pronucleate mouse embryos were used to assess the effect of culture conditions on single embryo development. Single culture either before or after compaction reduced cell numbers (112.2 ± 3.1; 110.2 ± 3.5) compared with group culture throughout (127.0 ± 3.4; P media volume from 20 µl to 2 µl increased blastocyst cell numbers in single embryos cultured in 5% oxygen (84.4 ± 3.2 versus 97.8 ± 2.8; P Culture in microwell plates for the EmbryoScope and Primo Vision time-lapse systems changed cleavage timings and increased inner cell mass cell number (24.1 ± 1.0; 23.4 ± 1.2) compared with a 2 µl microdrop (18.4 ± 1.0; P media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P culture before or after compaction is therefore detrimental; oxygen, media volume and microwells influence single embryo development; and embryo-conditioned media may substitute for group culture. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  4. Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek

    2009-01-01

    Abstract The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed......, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...... restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development....

  5. Transcriptomics analysis of retinoic acid embryotoxicity in rat postimplantation whole embryo culture.

    Science.gov (United States)

    Luijten, Mirjam; van Beelen, Vincent A; Verhoef, Aart; Renkens, Marc F J; van Herwijnen, Marcel H M; Westerman, Anja; van Schooten, Frederik-J; Pennings, Jeroen L A; Piersma, Aldert H

    2010-09-01

    Rodent postimplantation whole embryo culture (WEC) is a classical alternative test to study developmental toxicants. Here, we have successfully applied transcriptomics to monitor early responses in WEC after exposure to the embryotoxicant retinoic acid (RA). We demonstrated that RA exposures ranging from 2 to 24h affect RA-responsive genes in individual embryos. Furthermore, 2, 3 or 4 somite embryos gave similar responses, allowing combining embryos of these embryonic stages within the same analysis. Microarray analysis of embryonic gene expression after RA exposure revealed the regulation of many genes known to be RA responsive. Finally, use of a culture medium based on bovine serum instead of rat serum yielded similar gene expression responses after RA exposure. These findings support the robustness of the identified gene expression patterns and show the feasibility of detecting early gene expression changes in WEC after embryotoxic exposures. This approach may result in a more sensitive readout for detecting embryotoxicity in WEC. Copyright 2010 Elsevier Inc. All rights reserved.

  6. 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.

  7. Plant regeneration in wheat mature embryo culture

    African Journals Online (AJOL)

    Kamil Haliloğlu

    2011-11-09

    Nov 9, 2011 ... medium and same amount (8 mg/l) of 2,4-D and dicamba, respectively except for non-endosperm sup- ported mature embryos and hormones, there was no plant regeneration in method #12 in which dicamba was used as auxine. Filippov et al. (2006) obtained the best plant regeneration in 10 mg/l doses of ...

  8. 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.

  9. In vitro development competence of bovine nuclear transfer embryos derived from Nanog-overexpressing fibroblast cells

    Directory of Open Access Journals (Sweden)

    Xi-bang Zheng, Yan Yun, Yong-ce Hu, Yong Li, Hua-yan Wang, Xiao-ling Ma, Jin-qiang Sui, An-min Lei and Zhong-ying Dou

    2014-04-01

    Full Text Available The purpose of this study was to establish Nanog-expressing cell lines that can be used as donor cells to construct transgenic cloned embryos, and to investigate their in vitro development competence. By reverse transcription-polymerase chain reaction (RT-PCR, the cDNA of Nanog gene was cloned from fetal bovine primordial genital ridge tissues. The gene was inserted into PMD18-T vector using recombination techniques and then subcloned into vector pEGFP-C1. After confirmation by restrictive endonuclease digestion and sequencing, the recombinant plasmid pEGFP-Nanog was transfected into skin fibroblast cells. A stable transfected cell line was successfully established after two months of selection with neomycine (G418. Fluorescence microscopy, RT-PCR, and Western Blotting assays indicated that Nanog mRNA and EGFP-Nanog fusion protein were expressed in these cells. The EGFP-Nanog expressing fibroblast cells and the intact fibroblast cells (BEF422 were respectively used to construct cloned embryos. The results showed that the cleavage rate of recombinant embryos in BEF422 cells was significantly (P<0.05 higher than in EGFP-Nanog expressing cells (82.14 vs 40.38 %, but the blastocyst development rate in the latter was slightly higher than in the former (17.30 vs 14.29% (P<0.05, indicating that Nanog-overexpressed fibroblasts may be a better candidate of donor cells. To our knowledge, this is the first time that Nanog gene has been introduced into fibroblast cells to produce cloned embryos in bovine.

  10. In vitro culture of mouse embryos amniotic fluid ID human

    African Journals Online (AJOL)

    1989-07-15

    Jul 15, 1989 ... Because human amniotic fluid is a physiological, balanced ultrafiltrate, it has been considered as an inexpensive alternative culture medium in. IVF. A study of the development of mouse embryos in human amniotic fluid was undertaken to assess the suitability of this as an optional culture medium in human ...

  11. 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

  12. [Research progress of whole embryo culture tool and its application].

    Science.gov (United States)

    Han, Jiayin; Liang, Aihua

    2010-03-01

    Whole embryo culture (WEC) is an experimental tool, which is made use of embryos in vitro to replace whole animals to investigate the growth and development of early organs, the embryo toxicity of chemical materials and the mechanism of the occurrence of embryo toxicity. Compared with experiment with whole animals, WEC could reduce the number of experimental animals, shorten experimental time, decrease experimental expenses, eliminate disturbing factors and control dosage more exactly. So it is generally received that WEC tool is a good experimental method to match the principles of replacement, reduction, refinement and responsibility. This article is a review of the WEC tool of rat and mouse, including the development of this tool, announcements, and the application in the development of organs, the embryo toxicity of environmental pollution and heavy metal, safety evaluation of medicine and the embryo toxicity of traditional Chinese medicine and its mechanism. There is also a discussion of the application of this tool in the investigation of the embryo toxicity of traditional Chinese medicine.

  13. In vitro culture of coconut (Cocos nucifera L.) zygotic embryos.

    Science.gov (United States)

    Engelmann, Florent; Malaurie, Bernard; N'Nan, Oulo

    2011-01-01

    Coconut is a very important crop for millions of people in tropical countries. With coconut, in vitro culture protocols have been developed with two main objectives, viz. the large scale production of particular types of coconuts and the international exchange and conservation of coconut germplasm. The methods described in this chapter have been developed in the framework of collaborative activities between research institutes in Côte d'Ivoire and France. Two coconut embryo in vitro collecting protocols have been established, one consisting of storing the disinfected embryos in a KCl solution until they are brought back to the laboratory, where they are re-disinfected and inoculated in vitro under sterile conditions, and the other including in vitro inoculation of the embryos in the field. For international germplasm exchange, zygotic embryos inoculated in vitro in plastic test tubes or endosperm cylinders containing embryos in plastic bags are used. For in vitro culture, embryos are inoculated on semi-solid medium supplemented with sucrose and activated charcoal and placed in the dark, and then transferred to light conditions with the same (solid or liquid) medium once the first true leaf is visible and the root system has started developing.

  14. Successful whole embryo culture with commercially available reagents.

    Science.gov (United States)

    Glanville-Jones, Hannah C; Woo, Ngai; Arkell, Ruth M

    2013-01-01

    Since its development in the 1970's, whole embryo culture (WEC) has provided an important method of growing and observing murine embryos ex utero. During WEC, embryos are immersed in a combination of rat serum and cell culture media, and supplied with heat and appropriate mixtures of CO₂ and oxygen that mimic growth conditions in utero. One significant factor limiting the widespread use of WEC is the perception that commercially produced rat serum is inadequate to support normal rates of embryonic growth and development. Conversely, production of serum 'in-house' is technically demanding, time-consuming and expensive. The current study aimed to identify a WEC medium comprising commercially manufactured rat serum that would produce cultured embryos of comparable standard to those grown in utero. A mixed culture medium, composed of 50% commercial rat serum and 50% F12 Ham's cell culture medium with an N-2 neuronal cell growth supplement, was shown to support both a rate of growth, and the development of a range of features comparable to that which normally occur in vivo. Furthermore, the F12 (N-2) supplemented rat serum displayed a very low propensity to induce morphological abnormalities during the culture period. The study establishes a novel method of successful WEC using readily available commercial reagents and should enable the broader use of WEC.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Preparation of rat serum suitable for mammalian whole embryo culture.

    Science.gov (United States)

    Takahashi, Masanori; Makino, Sayaka; Kikkawa, Takako; Osumi, Noriko

    2014-08-03

    Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

  17. In vitro maturation and embryo development of bovine oocytes after meiosis blockage with MPF inhibitors

    Directory of Open Access Journals (Sweden)

    Mariana Groke Marques

    2011-12-01

    Full Text Available This study evaluated the bovine oocyte maturation and embryo development after in vitro fertilization. The maturation of the oocytes was blocked using Butyrolactone I and Roscovitine using pre-maturation medium supplemented with fetal calf serum (FCS. The ocytes were divided in four groups: Control 0 hour, Control (24 hours of maturation, Roscovitine (maturation blockage with 50mM Roscovitine during 24 hours followed by 24 hours of maturation, and Butyrolactone I (maturation blockage with 150mM Butyrolactone I during 24 hours followed by 24 hours of maturation. The oocytes were fixed and stained with aceto orcein to evaluate the nuclear maturation. After the maturation period, the remaining oocytes of the Control group, Roscovitine, and Butyrolactone I were fertilized in vitro. Embryo development was assessed by the cleavage rate (D3 and blastocysts formation (D7. The Butyrolactone I group had similar rates of germinal vesical stage oocytes during blockage, and Metaphase 2 after maturation, comparing to Control group at 0 hour and Control group, respectively. On the other hand, the Roscovitine group had lower rates of vesical stage oocytes during blockage, and Metaphase 2 after maturation comparing to Control groups. After in vitro fertilization, higher rates of cleavage were observed in Control and Butyrolactone I groups. For the blastocyst formation rate, the Control group showed better results than Roscovitine group. In summary, Butyrolactone I group had similar results to the Control group, and for this reason, is suitable for pre-maturation of bovine oocytes using FCS. In contrast, Roscovitine group had lower oocyte maturation and embryo development.

  18. Effects of Antioxidants on Development of In Vitro Fertilized Bovine Embryos

    OpenAIRE

    Anderson, Bret L.

    1995-01-01

    Free radicals are short-lived molecules that can cause decreased embryonic development in vitro. Antioxidants are molecules that block free radical formation or guard against their harmful effects. Many studies have linked exposure of media to light and culturing of embryos in high (20%) oxygen concentrations to free radical production. Some of the antioxidants used in culture media are superoxide dismutase (SOD), catalase, zinc (II), ethylenedinitrilo tetraacetic acid (EDTA), mannitol, vitam...

  19. Monitoring in-vitro bovine embryo development during the first days after fertilization (Conference Presentation)

    Science.gov (United States)

    Kandel, Mikhail E.; Rubessa, Marcello; Fernandes, Daniel; Nguyen, Tan H.; Wheeler, Matthew B.; Popescu, Gabriel

    2016-03-01

    Conventional label-based contrast enhancement techniques (e.g., fluorescence) frequently modify the genetic makeup of tagged cells, making them poor candidates for use in in-vitro fertilization applications. Instead, we choose a label-free form of contrast, based on interferometric imaging, sensitive to optical path length differences. Compared to, single HeLa cells, typical mammalian ova and embryos are more than an order of magnitude thicker. As a result, regions of large phase variation lead to phase wrapping and an overall reduction in signal intensity occurs due to multiple scattering. These effects manifest themselves in low-spatial frequencies (blurs), with the desired details buried in the background. We present a phase shifting interferometer that yields the derivative of the phase, a quantity whose value is particularly sensitive to local variations and fine details. We demonstrate that our new real-time imaging platform is valuable in measuring the multiday development of bovine embryos. Reconstructing the derivative of the image phase and amplitude, we characterize the motion of previously low-contrast structures, which are relevant for embryo viability tests.

  20. [Cultural diversity in gamete and embryos donation].

    Science.gov (United States)

    Epelboin, S

    2014-09-01

    Through gamete and embryo donation have successively emerged new ways of designing individuals who, in turn, have generated mutations in the concept of parenthood. A debate is open to the society, which often raises ideological cleavages. Indeed, donation practices mobilize the conflicting interests of donor of gametes, the recipient couple, child, whose origins are complex, although his filiation is legally clear. Its place in the family genealogy can be examined in relation to other societies, which admit plural concepts called "classificatory" kinship. They set up role partition between parents and educators. Setting anthropological perspective provides a broadening of the reflection to answer questions from the donation practices, including genealogical questions of revelation to the child of his conception, his incorporation in family and social group and the importance of compensation of donation. Copyright © 2014. Published by Elsevier SAS.

  1. In vitro culture of mouse embryos amniotic fluid ID human

    African Journals Online (AJOL)

    1989-07-15

    Jul 15, 1989 ... Human amniotic fluid was compared with Ham's F-10 culture medium as a possible alternative for use in in vitro fertilisation. The cleavage success of mouse embryos in human amniotic fluid (experimental group) was 92% compared with 86% in. Ham's F-10 medium. It is concluded that human amniotic.

  2. Multiple shoot induction from embryo derived callus cultures of ...

    African Journals Online (AJOL)

    Administrator

    The production of whole plant of cowpea (Vigna unguiculata) L. Walp from calli is an efficient, reliable ... practices on beans. Kobuyana et al. (1992) reported the use of embryo culture to generate mature plants in vitro from hybridization of three lines of the garden bean ... for the regeneration of shoots to fertile plants.

  3. Factors affecting the outcome of in vitro bovine embryo production using ovum pick-up-derived cumulus oocyte complexes

    NARCIS (Netherlands)

    Merton, J.S.

    2013-01-01

    Optimization of bovine ovum pick up (OPU) followed by in vitro embryo production (IVP) has been driven by the desire of both beef and dairy cattle breeders to enhance genetic improvement. The work presented in this thesis focuses on optimizing the efficiency and efficacy of the OPU-IVP program.

  4. Cellular and molecular deviations in bovine in vitro-produced embryos are related to the large offspring syndrome

    NARCIS (Netherlands)

    Lazzari, G.; Wrenzycki, C.; Herrmann, D.; Duchi, R.; Kruip, T.; Niemann, H.; Galli, C.

    2002-01-01

    The large offspring syndrome (LOS) is observed in bovine and ovine offspring following transfer of in vitro-produced (IVP) or cloned embryos and is characterized by a multitude of pathologic changes, of which extended gestation length and increased birthweight are predominant features. In the

  5. 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

  6. Consequences of endogenous and exogenous WNT signaling for development of the preimplantation bovine embryo.

    Science.gov (United States)

    Tribulo, Paula; Leão, Beatriz Caetano da Silva; Lehloenya, Khoboso C; Mingoti, Gisele Zoccal; Hansen, Peter J

    2017-06-01

    The specific role of WNT signaling during preimplantation development remains unclear. Here, we evaluated consequences of activation and inhibition of β-catenin (CTNNB1)-dependent and -independent WNT signaling in the bovine preimplantation embryo. Activation of CTNNB1-mediated WNT signaling by the agonist 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) and a glycogen synthase kinase 3 inhibitor reduced development to the blastocyst stage. Moreover, the antagonist of WNT signaling, dickkopf-related protein 1 (DKK1), alleviated the negative effect of AMBMP on development via reduction of CTNNB1. Based on labeling for phospho c-Jun N-terminal kinase, there was no evidence that DKK1 activated the planar cell polarity (PCP) pathway. Inhibition of secretion of endogenous WNTs did not affect development but increased number of cells in the inner cell mass (ICM). In contrast, DKK1 did not affect number of ICM or trophectoderm (TE) cells, suggesting that embryo-derived WNTs regulate ICM proliferation through a mechanism independent of CTNNB1. In addition, DKK1 did not affect the number of cells positive for the transcription factor yes-associated protein 1 (YAP1) involved in TE formation. In fact, DKK1 decreased YAP1. In contrast, exposure of embryos to WNT family member 7A (WNT7A) improved blastocyst development, inhibited the PCP pathway, and did not affect amounts of CTNNB1. Results indicate that embryo-derived WNTs are dispensable for blastocyst formation but participate in regulation of ICM proliferation, likely through a mechanism independent of CTNNB1. The response to AMBMP and WNT7A leads to the hypothesis that maternally derived WNTs can play a positive or negative role in regulation of preimplantation development. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction.

  7. The influence of the type of embryo culture medium on neonatal birthweight after single embryo transfer in IVF

    NARCIS (Netherlands)

    Vergouw, C.G.; Kostelijk, E.H.; Doejaaren, E.; Hompes, P.G.A.; Lambalk, C.B.; Schats, R.

    2012-01-01

    STUDY QUESTION Does the type of medium used to culture fresh and frozenthawed embryos influence neonatal birthweight after single embryo transfer (SET) in IVF? SUMMARY ANSWER A comparison of two commercially available culture media showed no significant influence on mean birthweight and mean

  8. Optimization of embryo culture conditions for increasing efficiency of cloning in buffalo (Bubalus bubalis) and generation of transgenic embryos via cloning.

    Science.gov (United States)

    Wadhwa, Neerja; Kunj, Neetu; Tiwari, Shuchita; Saraiya, Megha; Majumdar, Subeer S

    2009-09-01

    Cloning in bovine species is marred by low efficiency of blastocyst formation. Any increase in the efficiency of blastocyst formation upon nuclear transfer will greatly enhance the efficiency of cloning. In the present study, the effect of various media, protein sources, and growth factors on the development of cloned buffalo embryos was evaluated. Among various combinations tested, culture of cloned embryos in TCM-199 media on the feeder layer of Buffalo Oviductal Epithelial Cells (BOEC) in the presence of bovine serum albumin-free fatty acid (BSA-FFA) and leukemia inhibitory factor (LIF) provided most suitable environment for efficient development of cloned blastocysts. Under these conditions, we achieved a blastocyst formation rate of 43%, which is better than those reported previously. Because preimplantation embryonic development, in vivo, occurs in an environment of oviductal cells, the blastocysts generated by this method may presumably be more suitable for implantation and further development. Additionally, we generated green blastocysts from enucleated oocytes by transfer of nuclei from cells transfected with EGFP transgene, showing possibility of transgenesis via cloning in this species. To our knowledge, this is the first report regarding the production of transgenic cloned buffalo embryos and their developmental competence with respect to various media, cocultures, and supplements.

  9. Composition of commercial media used for human embryo culture.

    Science.gov (United States)

    Morbeck, Dean E; Krisher, Rebecca L; Herrick, Jason R; Baumann, Nikola A; Matern, Dietrich; Moyer, Thomas

    2014-09-01

    To determine the composition of commercially available culture media and test whether differences in composition are biologically relevant in a murine model. Experimental laboratory study. University-based laboratory. Cryopreserved hybrid mouse one-cell embryos were used in experiments. Amino acid, organic acid, ions, and metal content were determined for two different lots of media from Cook, In Vitro Care, Origio, Sage, Vitrolife, Irvine CSC, and Global. To determine whether differences in the composition of these media are biologically relevant, mouse one-cell embryos were thawed and cultured for 120 hours in each culture media at 5% and 20% oxygen in the presence or absence of protein in an EmbryoScope time-lapse incubator. The compositions of seven culture media were analyzed for concentrations of 39 individual amino acids, organic acids, ions, and elements. Blastocyst rates and cell cycle timings were calculated at 96 hours of culture, and the experiments were repeated in triplicate. Of the 39 analytes, concentrations of glucose, lactate, pyruvate, amino acids, phosphate, calcium, and magnesium were present in variable concentrations, likely reflecting differences in the interpretation of animal studies. Essential trace elements, such as copper and zinc, were not detected. Mouse embryos failed to develop in one culture medium and were differentially affected by oxygen in two other media. Culture media composition varies widely, with differences in pyruvate, lactate, and amino acids especially notable. Blastocyst development was culture media dependent and showed an interaction with oxygen concentration and presence of protein. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  10. Optimization of in vitro culture and transfection condition of bovine ...

    African Journals Online (AJOL)

    The present study aimed to optimize the in vitro culture and transfection efficiency of bovine primary spermatogonial stem cells (SSCs). To this end, SSCs were obtained from newborn Holstein bull calves by two-step enzymatic digestion. After enrichment and culture, SSCs were characterized by using alkaline phosphatase ...

  11. Early somatic embryo induction events in alfalfa callus cultures

    International Nuclear Information System (INIS)

    El-Bakry, A.A.; Hildebrand, D.F.

    1987-01-01

    High and low regenerating alfalfa Medicago sativa L. cv Regen S full sibs were isolated from a callus culture screen on modified Blaydes medium. The average number of embryos per ovary were thirty and zero for the high and low genotypes respectively after six weeks in culture. Proembryonic cell masses (4-8 celled) were observed after 4-5 days in culture and maximum meristematic activity was at 6-7 days in culture, for the high regenerating genotypes. Well formed globular embryos, both epidermal and subepidermal in origin, were observed after 2 weeks is culture. Samples in culture for 3, 6 and 14 days from the high and low regenerating genotypes were radiolabeled in vivo with 35 S-methionine and run both on one and two dimension gels. The results will be discussed in relation to differences in proteins between the high and low regenerating genotypes at the stage of maximum meristematic activity (day 6) and differences occurring relative to the appearance of globular stage embryos (day 14) will be presented

  12. Differences in gene expression profiles between human preimplantation embryos cultured in two different IVF culture media.

    Science.gov (United States)

    Kleijkers, Sander H M; Eijssen, Lars M T; Coonen, Edith; Derhaag, Josien G; Mantikou, Eleni; Jonker, Martijs J; Mastenbroek, Sebastiaan; Repping, Sjoerd; Evers, Johannes L H; Dumoulin, John C M; van Montfoort, Aafke P A

    2015-10-01

    Is gene expression in human preimplantation embryos affected by the medium used for embryo culture in vitro during an IVF treatment? Six days of in vitro culture of human preimplantation embryos resulted in medium-dependent differences in expression level of genes involved in apoptosis, protein degradation, metabolism and cell-cycle regulation. Several human studies have shown an effect of culture medium on embryo development, pregnancy outcome and birthweight. However, the underlying mechanisms in human embryos are still unknown. In animal models of human development, it has been demonstrated that culture of preimplantation embryos in vitro affects gene expression. In humans, it has been found that culture medium affects gene expression of cryopreserved embryos that, after thawing, were cultured in two different media for 2 more days. In a multicenter trial, women were randomly assigned to two culture medium groups [G5 and human tubal fluid (HTF)]. Data on embryonic development were collected for all embryos. In one center, embryos originating from two pronuclei (2PN) zygotes that were not selected for transfer or cryopreservation on Day 2 or 3 because of lower morphological quality, were cultured until Day 6 and used in this study, if couples consented. Ten blastocysts each from the G5 and HTF study groups, matched for fertilization method, maternal age and blastocyst quality, were selected and their mRNA was isolated and amplified. Embryos were examined individually for genome-wide gene expression using Agilent microarrays and PathVisio was used to identify the pathways that showed a culture medium-dependent activity. Expression of 951 genes differed significantly (P culture experiment until Day 6. This study shows that gene expression in human preimplantation embryos is altered by the culture medium used during IVF treatment and provides insight into the biological pathways that are affected. Whether these changes in gene expression have any long-term effects

  13. Can established cultured papilloma cells harbor bovine papillomavirus?

    Science.gov (United States)

    Campos, S R C; Trindade, C; Ferraz, O P; Giovanni, D N S; Lima, A A; Caetano, H V A; Carvalho, R F; Birgel, E H; Dagli, M L Z; Mori, E; Brandão, P E; Richtzenhain, L J; Beçak, W; Stocco, R C

    2008-10-21

    Papillomaviruses have been reported to be very difficult to grow in cell culture. Also, there are no descriptions of cell cultures from lesions of bovine cutaneous papillomatosis, with identification of different bovine papilloma virus (BPV) DNA sequences. In the present report, we describe primary cell cultures from samples of cutaneous lesions (warts). We investigated the simultaneous presence of different BPV DNA sequences, comparing the original lesion to different passages of the cell cultures and to peripheral blood. BPV 1, 2 and 4 DNA sequences were found in lesion samples, and respective cell cultures and peripheral blood, supporting our previous hypothesis of the possible activity of these sequences in different samples and now also showing how they can be maintained in different passages of cell cultures.

  14. The method of rodent whole embryo culture using the rotator-type bottle culture system.

    Science.gov (United States)

    Takahashi, Masanori; Osumi, Noriko

    2010-08-28

    Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology (1). Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat (2, 3, 4). In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed (1). Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively (1). In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft (5, 6), and introduce various applications of WEC technique for studies in mammalian developmental biology.

  15. A microfluidic system supports single mouse embryo culture leading to full-term development

    NARCIS (Netherlands)

    Esteves, Telma Cristina; van Rossem, F.; Nordhoff, Verena; Schlatt, Stefan; Boiani, Michele; le Gac, Severine

    2013-01-01

    The present study demonstrates the feasibility of application of a microfluidic system for in vitro culture of pre-implantation mouse embryos, with subsequent development to full-term upon embryo transfer. Specifically, embryos cultured in groups in nL volume chambers achieve pre-implantation

  16. [Toxicity of monocrotaline on in vitro cultured mouse embryos].

    Science.gov (United States)

    Han, Jiayin; Liang, Aihua; Yi, Yan; Gao, Shuangrong; Nilsen, Odd Georg

    2011-02-01

    To investigate the fetotoxicity of monocrotaline. Mouse whole embryo culture (WEC) was applied. Post-implantation (8.5 d) mouse embryos were isolated from their mothers and put into the medium of immediately centrifuged serum (ICS) prepared from rats. Different concentrations of monocrotaline (100, 50, 25, 12.5 mg x L(-1)) were added into the WEC. Development (yolk sac diameter, crown-rump length, head length, somite number) and organic morphodifferentiation (yolk sac circulation, allantois, embryonic flexion, heart, brain, optic-otic-olfactory organ, branchial arch, maxillary, mandible, bud) of embryos were observed at 48 h after treatment. Obvious fetotoxicity could be observed in various monocrotaline treatment groups in a dose-dependent manner. Development of embryos was delayed significantly at dose 12.5-100 mg x L(-1). Malformations were shown in all organic morphodifferentiation indice, especially in opti-otic organ, mandible and bud. Monocrotaline had obvious fetotoxicity in vitro WEC, indicating that exposure of pregnant mice to monocrotaline may have potential risk on fetus.

  17. The Necessity of OCT-4 and CDX2 for Early Development and Gene Expression Involved in Differentiation of Inner Cell Mass and Trophectoderm Lineages in Bovine Embryos.

    Science.gov (United States)

    Sakurai, Nobuyuki; Takahashi, Kazuki; Emura, Natsuko; Fujii, Takashi; Hirayama, Hiroki; Kageyama, Soichi; Hashizume, Tsutomu; Sawai, Ken

    2016-10-01

    The functions of POU class 5 transcription factor 1 (Oct-4) and caudal-type homeobox 2 (Cdx2) in the differentiation of the murine inner cell mass (ICM) and trophectoderm (TE) have been described in detail. However, little is known about the roles of OCT-4 and CDX2 in preimplantation bovine embryos. To elucidate their functions during early development in bovine embryos, we performed OCT-4 and CDX2 downregulation using RNA interference. We injected OCT-4- or CDX2-specific short interfering RNAs (siRNAs) into bovine zygotes. The rate of blastocyst development of OCT-4-downregulated embryos was lower compared with uninjected or control siRNA-injected embryos. Gene expression analysis revealed decreased CDX2 and fibroblast growth factor 4 expression in OCT-4-downregulated embryos. CDX2-downregulated embryos developed to the blastocyst stage; however, in most cases, blastocoel formation was delayed. Gene expression analysis revealed decreased GATA3 expression and elevated NANOG expression in CDX2-downregulated embryos. In conclusion, OCT-4 and CDX2 are essential for early development and gene expression involved in differentiation of ICM and TE lineages in bovine embryos.

  18. 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.

  19. Selection of bovine oocytes by brilliant cresyl blue staining: effect on meiosis progression, organelle distribution and embryo development.

    Science.gov (United States)

    Silva, D S; Rodriguez, P; Galuppo, A; Arruda, N S; Rodrigues, J L

    2013-08-01

    The selection of competent oocytes for in vitro maturation is still a major problem during bovine in vitro embryo production. Markers for in vitro cytoplasmic maturation, based on the organization of cortical granule and mitochondria, are lacking. We examined the pre-selection of immature bovine oocytes by brilliant cresyl blue stain (BCB test) based on glucose-6-phosphate dehydrogenase (G6PDH) activity during oocyte development. Oocytes were recovered from ovarian follicles exposed to 26 μM BCB stain and classified according to the aspect of their cytoplasm: BCB(+) (oocytes with blue cytoplasm) and BCB(-) (unstained cytoplasm) and then in vitro matured into a conventional in vitro maturation (IVM) medium and standard procedure. In Experiment 1, nuclear maturation was determined by polar body identification, while cytoplasmic maturation was based on cortical granule (CG) migration (peripheral) and mitochondria distribution (central). Evidence of polar body, cortical granule migration and of centrally located mitochondria was significantly (p BCB(+) oocytes than in BCB(-) (polar body present: 65% vs 20%; peripheral CG: 72% vs. 14%; and central mitochondria: 85% vs. 19%, respectively). In Experiment 2, the efficiency pre-selection of bovine oocytes by BCB on embryo development in vitro was assessed. Cleavage rates were similar (75%) among control, BCB(+) and BCB(-) groups, while blastocyst rates on D7 were (p BCB(+) vs BCB(-) (10%) or control (28%). We showed that the BCB test is efficient to identify competent immature bovine oocytes to undergo synchronous nuclear and cytoplasmic in vitro maturation thus yielding higher in vitro embryo development to blastocyst stage.

  20. An investigation into the feasibility of culturing rat embryos in media ...

    African Journals Online (AJOL)

    in the media during 48-h rat embryo culture. Embryos are very sensitive to changes in the culture medium environment and will, as a result, develop abnormally when conditions are less than optimal. Little is known about the influence of culture envi- ronment on the medium parameters of pH, PCOr, pOr, bicarbo- nate levels ...

  1. Plating efficiency for primary hamster embryo cells as an index of efficacy of fetal bovne serum for cell culture.

    Science.gov (United States)

    Goodheart, C R; Castro, B C; Giviers, A; Regnier, P R

    1973-10-01

    Attachment and growth of mammalian cells plated at low cell density require optimum conditions for the cells to form colonies. Reliability, reproducibility, and validity of the plating efficiency test for evaluating cell culture sera were determined by measuring the plating efficiency of 37 lots of fetal bovine serum obtained from 8 suppliers (5 lots from each of 7, 2 lots from 1 supplier), by using hamster embryo fibroblasts plated at low cell density. The test revealed considerable variation between lots of serum and between suppliers. The five lots from some suppliers had consistently high plating efficiencies, whereas one or more lots from other suppliers had quite low efficiencies. The results were reproducible in repeated tests, and control experiments indicated that the test measured the efficiency of the test serum independently of the efficiency of the serum used for the primary outgrowth of the hamster embryo cells.

  2. The influence of the type of embryo culture medium on neonatal birthweight after single embryo transfer in IVF.

    Science.gov (United States)

    Vergouw, Carlijn G; Kostelijk, E Hanna; Doejaaren, Els; Hompes, Peter G A; Lambalk, Cornelis B; Schats, Roel

    2012-09-01

    Does the type of medium used to culture fresh and frozen-thawed embryos influence neonatal birthweight after single embryo transfer (SET) in IVF? A comparison of two commercially available culture media showed no significant influence on mean birthweight and mean birthweight adjusted for gestational age, gender and parity (z-scores) of singletons born after a fresh or frozen-thawed SET. Furthermore, we show that embryo freezing and thawing cycles may lead to a significantly higher mean birthweight. Animal studies have shown that culture media constituents are responsible for changes in birthweight of offspring. In human IVF, there is still little knowledge of the effect of medium type on birthweight. Until now, only a small number of commercially available culture media have been investigated (Vitrolife, Cook(®) Medical and IVF online medium). Our study adds new information: it has a larger population of singleton births compared with the previously published studies, it includes outcomes of other media types (HTF and Sage(®)), not previously analysed, and it includes data on frozen-thawed SETs. This study was a retrospective analysis of birthweights of singleton newborns after fresh (Day 3) or frozen-thawed (Day 5) SET cycles, using embryos cultured in either of two different types of commercially available culture media, between 2008 and 2011. Before January 2009, a single-step culture medium was used: human tubal fluid (HTF) with 4 mg/ml human serum albumin. From January 2009 onwards, a commercially available sequential medium was introduced: Sage(®), Quinn's advantage protein plus medium. Singletons born after a fresh SET (99 embryos cultured in HTF and 259 in Sage(®)) and singletons born after a frozen-thawed SET (32 embryos cultured in HTF only, 41 in HTF and Sage(®) and 86 in Sage(®) only) were analysed. Only patients using autologous gametes without the use of a gestational carrier were considered. Also excluded were (vanishing) twins, triplets

  3. 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, G1culture in the Gyr breed.

  4. Characteristics of pregnancies and offspring following transfer of bovine in vivo embryos assessed by nanorespirometry

    DEFF Research Database (Denmark)

    Lopes, Ana Sofia; Madsen, S E; Greve, Torben

    2008-01-01

    .05), with quality I embryos associated with heavier calves when compared to quality III embryos. Furthermore, light traction was more frequently used to retrieve heavier animals (P male calves were 3.2 kg heavier than females (P

  5. Germination of somatic embryos of Swietenia macrophylla in semisolid culture media

    Directory of Open Access Journals (Sweden)

    Raúl Collado

    2005-01-01

    Full Text Available With the objective to establish a suitable culture medium and to determine the optimum culture time for the germination of somatic embryos of Swietenia macrophylla King, it was carried out these work. Somatic embryos at the cotyledonal stage were placed in a culture medium formed by the half of the MS salts for their germination . three concentrations of 6-BAP (0.2, 0.4 and 0.6 mg.l-1, two concentrations of AG (1.0 and 2.0 mg.l-1 and a culture medium control without growth regulators were studied. In the culture medium wher3e the best results were obtained, the effect of the culture time on the germination of somatic embryos (30, 60 and 90 days of culture was studied. The major number of somatic embryos with total germination was obtained in the culture medium without growth regulators, with significant differences to the rest of the treatments. The highest value of somatic embryos with secondary embryogenesis was presented when 0.6 mg.l-1 of 6-BAP were added to the culture medium. The increase of the culture time to 60 and 90 days increased significantly the total germination of the somatic embryos. These results allowed to define a culture medium for the germination of somatic embryos of Swietenia macrophylla King composed by the half of the MS salts without growth regulators with a culture time of 60 days. Key words: in vitro culture, Mahogany, regeneration, somatic embryogenesis

  6. Exogenous growth factors do not affect the development of individually cultured murine embryos.

    Science.gov (United States)

    Herrick, Jason R; Greene-Ermisch, Alison F; Schoolcraft, William B; Krisher, Rebecca L

    2017-12-21

    The objective of this study was to evaluate the effects of multiple growth factors on the development of individually cultured murine embryos. Embryos produced by in vitro fertilization using in vitro (IVM) or in vivo (IVO) matured oocytes from three strains of mice (CF1, Swiss Webster, B6D2F1) were cultured individually (10 μl) in the absence (control) or presence of growth factors (paf, epidermal growth factor [EGF], insulin-like growth factor 1 [IGF-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Blastocyst formation, hatching, and blastocyst cell numbers (trophectoderm, inner cell mass, and total) were evaluated on days 4 and 5 of culture. Post-hatching development of CF1 IVO embryos was also evaluated in vitro and in vivo. The presence of growth factors did not improve the proportion of embryos forming blastocysts or initiating hatching for any of the types of embryos tested. The only significant (P embryos that formed blastocysts by day 5 in CF1 IVM embryos. The presence of growth factors also did not affect blastocyst cell numbers. For CF1 IVO embryos, the presence of growth factors during culture did not affect the proportion of embryos that attached to fibronectin-coated dishes, the size of the resulting outgrowths, or in vivo development following transfer. Combinations of paf, EGF, GM-CSF, and IGF-1 did not improve development of murine embryos cultured individually in a sequential medium containing a defined protein source.

  7. Postimplantation Whole Embryo Culture Assay for Hamsters: An Alternative to Rat and Mouse

    Directory of Open Access Journals (Sweden)

    Bogdan Wlodarczyk

    2001-01-01

    Full Text Available Postimplantation whole embryo culture (WEC assay for rats and mice has been well established and introduced to many laboratories. Recently WEC technique for rabbits has been developed; however, information on culture of other species is very limited. Knowing the usefulness of hamsters in classical embryotoxicology, we reasoned that hamster WEC could be an alternative model for the most frequently used rat and mouse WEC. Previously we have optimized culture conditions for postimplantation hamster embryos. The aim of this study was to test the susceptibility of hamster embryos cultured in vitro to embryotoxic compounds and to compare our results with those reported by others on rat or mouse embryo culture. For that purpose we choose three known embryotoxic compounds�valproic acid, cadmium chloride, and diethylstilbestrol�and tested them using a postimplantation hamster whole embryo culture assay. Hamster embryos were cultured from 7.5 days gestation for 24 h in a medium consisting of 35% hamster serum and 65% synthetic culture medium (Iscove�s or McCoy 5A. At the end of the culture period, the embryos were examined morphologically, measured with the aid of a computer image analysis system, and total protein content was assessed. All three compounds exhibited dose-related embryotoxic and teratogenic effects in hamster embryos. The malformations observed were similar to those reported on rat and mouse embryos. Comparison of the results with data reported by other authors indicates that hamster embryos cultured in vitromight be more susceptible to embryotoxic stimuli than rat and mouse embryos.

  8. 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...... 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...

  9. Effect of container, vitrification volume and warming solution on cryosurvival of in vitro-produced bovine embryos.

    Science.gov (United States)

    Rios, G L; Mucci, N C; Kaiser, G G; Alberio, R H

    2010-03-01

    The aim of the present research was to develop a low cost and easy to perform vitrification method for in vitro-produced cattle embryos. Effect of container material was evaluated (plastic straw compared to glass capillary, experiment 1), two volume sample (1 compared to 0.5 microL, experiment 2) and warming solution composition medium (Tissue Culture Medium 199 (TCM-199) compared to phosphate buffered saline (PBS), experiment 3) as modifications of the open pulled straw (OPS) system in order to reduce embryo damage caused by exposure to cold. In all experiments, day 7 and expanded blastocysts of cattle were exposed to the vitrification solution 1 for 3 min and 30s in solution 2. After this, embryos were placed in a droplet and loaded in a narrow end container, and immediately submerged into liquid nitrogen. For warming, vitrified embryos were plunged into warming solution 1 for 3 min, and transferred into warming solution 2 for 1 min. Fresh embryos kept in culture were used as control group. Hatching rates were recorded in all cases at day 13. In experiment 1 there was no significant effect of container material on hatching rates. Postwarming survival rate of vitrified embryos was lower than control (27.5% plastic straws, 18.9% glass capillary and 80.5% control, Pstraw (OPS) procedure, and that PBS can replace TCM-199 in warming solutions, but lesser hatching rates should be expected.

  10. Effects of embryo culture media do not persist after implantation: a histological study in mice

    NARCIS (Netherlands)

    Hemkemeyer, Sandra A.; Schwarzer, Caroline; Boiani, Michele; Ehmcke, Jens; le Gac, Severine; Schlatt, Stefan; Nordhoff, Verena

    Is post-implantation embryonic development after blastocyst transfer affected by exposure to different assisted reproduction technology (ART) culture media? Fetal development and placental histology of ART embryos cultured in vitro in different ART media was not impaired compared with embryos grown

  11. Dose-response analysis of phthalate effects on gene expression in rat whole embryo culture

    NARCIS (Netherlands)

    Robinson, J.F.; Verhoef, A.; van Beelen, V.A.; Pennings, J.L.A.; Piersma, A.H.

    2012-01-01

    The rat postimplantation whole embryo culture (WEC) model serves as a potential screening tool for developmental toxicity. In this model, cultured rat embryos are exposed during early embryogenesis and evaluated for morphological effects. The integration of molecular-based markers may lead to

  12. Microfluidics for mammalian embryo culture and selection: where do we stand now?

    NARCIS (Netherlands)

    le Gac, Severine; Nordhoff, Verena

    2016-01-01

    The optimization of in-vitro culture conditions and the selection of the embryo(s) with the highest developmental competence are essential components in an ART program. Culture conditions are manifold and they underlie not always evidence-based research but also trends entering the IVF laboratory.

  13. Changes in the transcriptome of morula-stage bovine embryos caused by heat shock: relationship to developmental acquisition of thermotolerance

    Directory of Open Access Journals (Sweden)

    Sakatani Miki

    2013-01-01

    Full Text Available Abstract Background While initially sensitive to heat shock, the bovine embryo gains thermal resistance as it progresses through development so that physiological heat shock has little effect on development to the blastocyst stage by Day 5 after insemination. Here, experiments using 3’ tag digital gene expression (3’DGE and real-time PCR were conducted to determine changes in the transcriptome of morula-stage bovine embryos in response to heat shock (40 degrees C for 8 h that could be associated with thermotolerance. Results Using 3’DGE, expression of 173 genes were modified by heat shock, with 94 genes upregulated by heat shock and 79 genes downregulated by heat shock. A total of 38 differentially-regulated genes were associated with the ubiquitin protein, UBC. Heat shock increased expression of one heat shock protein gene, HSPB11, and one heat shock protein binding protein, HSPBP1, tended to increase expression of HSPA1A and HSPB1, but did not affect expression of 64 other genes encoding heat shock proteins, heat shock transcription factors or proteins interacting with heat shock proteins. Moreover, heat shock increased expression of five genes associated with oxidative stress (AKR7A2, CBR1, GGH, GSTA4, and MAP2K5, decreased expression of HIF3A, but did not affect expression of 42 other genes related to free radical metabolism. Heat shock also had little effect on genes involved in embryonic development. Effects of heat shock for 2, 4 and 8 h on selected heat shock protein and antioxidant genes were also evaluated by real-time PCR. Heat shock increased steady-state amounts of mRNA for HSPA1A (PHSP90AA1 (PSOD1 or CAT. Conclusions Changes in the transcriptome of the heat-shocked bovine morula indicate that the embryo is largely resistant to effects of heat shock. As a result, transcription of genes involved in thermal protection is muted and there is little disruption of gene networks involved in embryonic development. It is likely that

  14. Developmental potential of bovine hand-made clone embryos reconstructed by aggregation or fusion with distinct cytoplasmic volumes.

    Science.gov (United States)

    Ribeiro, Eduardo de Souza; Gerger, Renato Pereira da Costa; Ohlweiler, Lain Uriel; Ortigari, Ivens; Mezzalira, Joana Cláudia; Forell, Fabiana; Bertolini, Luciana Relly; Rodrigues, José Luiz; Ambrósio, Carlos Eduardo; Miglino, Maria Angélica; Mezzalira, Alceu; Bertolini, Marcelo

    2009-09-01

    Animal cloning has been associated with developmental abnormalities, with the level of heteroplasmy caused by the procedure being one of its potential limiting factors. The aim of this study was to determine the effect of the fusion of hemicytoplasts or aggregation of hemiembryos, varying the final cytoplasmic volume, on development and cell density of embryos produced by hand-made cloning (HMC), parthenogenesis or by in vitro fertilization (IVF). One or two enucleated hemicytoplasts were paired and fused with one skin somatic cell. Activated clone and zona-free parthenote embryos and hemiembryos were in vitro cultured in the well-of-the-well (WOW) system, being allocated to one of six experimental groups, on a per WOW basis: single clone or parthenote hemiembryos (1 x 50%); aggregation of two (2 x 50%), three (3 x 50%), or four (4 x 50%) clone or parthenote hemiembryos; single clone or parthenote embryos (1 x 100%); or aggregation of two clone or parthenote embryos (2 x 100%). Control zona-intact parthenote or IVF embryos were in vitro cultured in four-well dishes. Results indicated that the increase in the number of aggregated structures within each WOW was followed by a linear increase in cleavage, blastocyst rate, and cell density. The increase in cytoplasmic volume, either by fusion or by aggregation, had a positive effect on embryo development, supporting the establishment of pregnancies and the birth of a viable clone calf after transfer to recipients. However, embryo aggregation did not improve development on a hemicytoplast basis, except for the aggregation of two clone embryos.

  15. 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.

  16. No effect of embryo culture media on birthweight and length of newborns.

    Science.gov (United States)

    Lin, Shengli; Li, Ming; Lian, Ying; Chen, Lixue; Liu, Ping

    2013-07-01

    Does the type of media used to culture embryos for IVF influence the birthweight and length of neonates? No significant differences were observed in birthweight and length among the three embryo culture media used for in vitro embryo culture. Since the establishment of IVF as an assisted reproductive technology (ART), many different culture systems have been used for the development of human embryos. Some studies have shown that the types of culture media influence the newborn birthweight; however, other studies have shown no effect. To further explore this contradictory issue, we compared the birthweight and length of neonates born after the transfer of embryos cultured in one of three commercially available media. This retrospective analysis of birthweight and length of newborns included 1201 women who delivered singletons and 445 women who delivered twins. The following three commercially available culture media were used: G5™, Global and Quinn's advantage media. Women who underwent IVF-ET cycles between 2008 and 2010 were analyzed. Patients younger than 40 years of age with a body mass index (BMI) culture medium. Inter-twin mean birthweight and length disparities were analyzed, but were not shown to be significantly different. Multiple linear regression analysis showed that maternal weight, maternal height, gestational age and infant gender were significantly related to birthweight, and paternal height, gestational age and newborn complications were significantly associated with birth length. The current study showed that birthweight and length of newborns were not associated with the embryo culture medium. More research needs to be performed to analyze the effects of other culture medium formulations and to evaluate the long-term effects of embryo culture medium on the health of children conceived through ART. WIDER IMPLICATIONS OF THESE FINDINGS: Our retrospective study suggests that embryo culture medium does not influence neonatal birthweight and length

  17. Non-invasive metabolomic profiling of embryo culture media and morphology grading to predict implantation outcome in frozen-thawed embryo transfer cycles.

    Science.gov (United States)

    Li, Xiong; Xu, Yan; Fu, Jing; Zhang, Wen-Bi; Liu, Su-Ying; Sun, Xiao-Xi

    2015-11-01

    Assessment of embryo viability is a crucial component of in vitro fertilization and currently relies largely on embryo morphology and cleavage rate. Because morphological assessment remains highly subjective, it can be unreliable in predicting embryo viability. This study investigated the metabolomic profiling of embryo culture media using near-infrared (NIR) spectroscopy for predicting the implantation potential of human embryos in frozen-thawed embryo transfer (FET) cycles. Spent embryo culture media was collected on day 4 after thawed embryo transfer (n = 621) and analysed using NIR spectroscopy. Viability scores were calculated using a predictive multivariate algorithm of fresh embryos with known pregnancy outcomes. The mean viability indices of embryos resulting in clinical pregnancy following FET were significantly higher than those of non-implanted embryos and differed between the 0, 50, and 100 % implantation groups. Notably, the 0 % group index was significantly lower than the 100 % implantation group index (-0.787 ± 0.382 vs. 1.064 ± 0.331, P  0.05). NIR metabolomic profiling of thawed embryo culture media is independent of morphology and correlates with embryo implantation potential in FET cycles. The viability score alone or in conjunction with morphologic grading is a more objective marker for implantation outcome in FET cycles than morphology alone.

  18. 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...

  19. Influence of embryo culture medium (G5 and HTF) on pregnancy and perinatal outcome after IVF : a multicenter RCT

    NARCIS (Netherlands)

    Kleijkers, Sander H. M.; Mantikou, Eleni; Slappendel, Els; Consten, Dimitri; van Echten - Arends, Jannie; Wetzels, Alex M.; van Wely, Madelon; Smits, Luc J. M.; van Montfoort, Aafke P. A.; Repping, Sjoerd; Dumoulin, John C. M.; Mastenbroek, Sebastiaan

    2016-01-01

    Does embryo culture medium influence pregnancy and perinatal outcome in IVF? Embryo culture media used in IVF affect treatment efficacy and the birthweight of newborns. A wide variety of culture media for human preimplantation embryos in IVF/ICSI treatments currently exists. It is unknown which

  20. Influence of embryo culture medium (G5 and HTF) on pregnancy and perinatal outcome after IVF: a multicenter RCT

    NARCIS (Netherlands)

    Kleijkers, S.H.; Mantikou, E.; Slappendel, E.; Consten, D.; Echten-Arends, J. van; Wetzels, A.M.M.; Wely, M. van; Smits, L.J.; Montfoort, A.P. van; Repping, S.; Dumoulin, J.C.; Mastenbroek, S.

    2016-01-01

    STUDY QUESTION: Does embryo culture medium influence pregnancy and perinatal outcome in IVF? SUMMARY ANSWER: Embryo culture media used in IVF affect treatment efficacy and the birthweight of newborns. WHAT IS KNOWN ALREADY: A wide variety of culture media for human preimplantation embryos in

  1. Influence of embryo culture medium (G5 and HTF) on pregnancy and perinatal outcome after IVF: a multicenter RCT

    NARCIS (Netherlands)

    Kleijkers, Sander H. M.; Mantikou, Eleni; Slappendel, Els; Consten, Dimitri; van Echten-Arends, Jannie; Wetzels, Alex M.; van Wely, Madelon; Smits, Luc J. M.; van Montfoort, Aafke P. A.; Repping, Sjoerd; Dumoulin, John C. M.; Mastenbroek, Sebastiaan

    2016-01-01

    Does embryo culture medium influence pregnancy and perinatal outcome in IVF? Embryo culture media used in IVF affect treatment efficacy and the birthweight of newborns. A wide variety of culture media for human preimplantation embryos in IVF/ICSI treatments currently exists. It is unknown which

  2. 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

    Full Text Available Abstract Background Since processes in well-known model organisms have specific features different from those in Bos taurus, the organism under study, a good way to describe gene regulation in ruminant embryos would be a species-specific consideration of closely related species to cattle, sheep and pig. However, as highlighted by a recent report, gene dictionaries in pig are smaller than in cattle, bringing a risk to reduce the gene resources to be mined (and so for sheep dictionaries. Bioinformatics approaches that allow an integration of available information on gene function in model organisms, taking into account their specificity, are thus needed. Besides these closely related and biologically relevant species, there is indeed much more knowledge of (i trophoblast proliferation and differentiation or (ii embryogenesis in human and mouse species, which provides opportunities for reconstructing proliferation and/or differentiation processes in other mammalian embryos, including ruminants. The necessary knowledge can be obtained partly from (i stem cell or cancer research to supply useful information on molecular agents or molecular interactions at work in cell proliferation and (ii mouse embryogenesis to supply useful information on embryo differentiation. However, the total number of publications for all these topics and species is great and their manual processing would be tedious and time consuming. This is why we used text mining for automated text analysis and automated knowledge extraction. To evaluate the quality of this “mining”, we took advantage of studies that reported gene expression profiles during the elongation of bovine embryos and defined a list of transcription factors (or TF, n = 64 that we used as biological “gold standard”. When successful, the “mining” approach would identify them all, as well as novel ones. Methods To gain knowledge on molecular-genetic regulations in a non model organism, we offer an

  3. Studies on inactivation of pathogenic microorganisms in culture media and in bovine semen by photosensitive agents.

    Science.gov (United States)

    Eaglesome, M D; Bielanski, A; Hare, W C; Ruhnke, H L

    1994-01-01

    The application of three photosensitive agents for disinfection of bovine semen was investigated. Bovine microbial pathogens suspended in tissue culture medium and/or PBS and also added to bovine semen were exposed to the photosensitive agents followed by irradiation. Hematoporphyrin, hematoporphyrin derivative and thiopyronine were effective against bovine herpes virus-1, bovine viral diarrhoea virus, Mycoplasma bovigenitalium, Mycoplasma canadense, and Ureaplasma diversum in culture media. In addition, thiopyronine was effective against Leptospira pomona. Similar treatments were not effective against Leptospira hardjo, Mycoplasma bovis, or Campylobacter fetus subsp. venerealis. When microorganisms were added to bovine semen, only bovine herpes virus-1 was controlled by the photosensitive agents used at concentrations which did not appear harmful to sperm cells.

  4. 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.

  5. In vitro zygotic embryo culture of Pinus peuce Gris.: Optimization of culture conditions affecting germination and early seedling growth

    Directory of Open Access Journals (Sweden)

    Stojičić Dragana

    2012-01-01

    Full Text Available This study reports a protocol for the germination and early seedling growth of Pinus peuce Gris. using zygotic embryo culture. In order to overcome seed dormancy and optimize organogenesis, the effect of nutritional, plant growth regulatory and physical factors on in vitro germination and growth of isolated mature zygotic embryos of P. peuce were investigated.

  6. Individual demands of human embryos on IVF culture medium: influence on blastocyst development and pregnancy outcome.

    Science.gov (United States)

    Wirleitner, B; Vanderzwalmen, P; Stecher, A; Zech, M H; Zintz, M; Zech, N H

    2010-12-01

    The elucidation of the metabolic requirements of human embryos in vivo or in vitro remains, despite being intensively investigated, a work in progress. The adoption of extended embryo culture to the blastocyst stage during the last decade has entailed new challenges. With the increased attention to culture media formulations, more evidence on the sensitivity of embryos to their early environmental conditions is accumulating which might affect phenotype and developmental potential. A retrospective study was conducted that comprised 286 IVF cycles to evaluate the effect of two different culture media on blastocyst development and pregnancy outcome. Embryos were either cultured in a one step or a sequential medium. Higher fertilization rates and augmented blastocyst rates as well as higher implantation rates were observed when embryos were cultured in one step medium (Pcultured in either medium resulted in a significantly higher rate of twin pregnancies. Although multiple pregnancies should be avoided in assisted reproduction treatment to reduce risks for offspring and mother, this higher frequency of twin pregnancies resulting from the transfer of embryos derived from different culture media suggests that each embryo makes individual demands on its early environment. Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  7. In-vitro propagation of Picralima nitida (Stapf) through embryo culture

    African Journals Online (AJOL)

    Embryo abortion in wide crosses and seed dormancy has hampered the mass propagation of selected tree germplasm from the wild. An in vitro plant regeneration protocol was successfully established in Picralima nitida (Stapf), a medicinal tropical plant, by culturing excised embryo from mature seeds collected from the ...

  8. Technique of the 'in vitro' fertilization and the culture of mouse embryos at preimplantation

    International Nuclear Information System (INIS)

    Kikuchi, Olivia Kimiko; Yamada, Takeshi

    1993-03-01

    The mammal embryo is an intensive cellular proliferating system, very radiosensitive and therefore adequate to the study of the biological effects of ionizing radiation. The technique of the in vitro fertilization and the culture of mouse embryos at preimplantation period, modified by Yamada et al (1982) to improve the efficiency of more than 95% of blastocyst formation is described. (author)

  9. In vitro production of bovine embryos: cumulus/granulosa cell gene expression patterns point to early atresia as beneficial for oocyte competence

    DEFF Research Database (Denmark)

    Mazzoni, Gianluca; Razza, Eduardo; Pedersen, Hanne S.

    2017-01-01

    In vitro production (IW) of bovine embryos has become widespread technology implemented in cattle breeding and production. Here, we review novel data on cumulus/granulosa cell gene expression, as determined by RNAseq on cellular material from pooled follicular fluids at the single animal level...

  10. 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

  11. Long-distance transportation of primate embryos developing in culture: a preliminary study.

    Science.gov (United States)

    Nichols, Stephanie; Harvey, Alexandra; Gierbolini, Lynette; Gonzalez-Martinez, Janis; Brenner, Carol; Bavister, Barry

    2010-03-01

    Non-human primate embryos are invaluable for conducting research relevant to human infertility and stem cells, but their availability is restricted. In this preliminary study, rhesus monkey embryos were produced by IVF at the Caribbean Primate Research Centre and shipped in tubes of gassed culture medium within a battery-powered transport incubator by overnight courier to Wayne State University in Michigan. Upon arrival, the embryos were incubated in fresh culture medium to evaluate further development. In 11 shipments comprising 98 cleavage-stage embryos developing from oocytes that were mature (MII) upon collection, 51 (52%) reached advanced preimplantation stages (morula to hatched blastocyst) during prolonged culture following transportation. However, most embryos produced from oocytes that were immature (MI) at collection arrested and only 5/51 (10%) reached advanced stages of development. This study demonstrates that non-cryopreserved primate embryos can be routinely transported between distant sites without loss of developmental ability. In this way, the processes of production and study of non-cryopreserved primate embryos need not be restricted to the same or nearby laboratories. This will expand the use of these embryos for research and facilitate generation of translationally relevant information. Published by Elsevier Ltd.

  12. 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.

  13. A prospective randomized comparison of early embryo cleavage kinetics between two media culture systems

    Science.gov (United States)

    Zhang, Huan; Zheng, Yi; Wu, Yonggen; Ye, Danna; Huang, Xuefeng

    2016-01-01

    Objective: To investigate whether early embryo cleavage kinetics were affected by type of culture media. Methods: In this prospective sibling-split study, 620 oocytes from 37 patients were randomly allocated into two groups: Cook group and Vitrolife group. Oocytes/embryos in Cook group, would be cultured with Cook sequential culture medium, while oocytes/embryos in Vitrolife group, would be cultured with Vitrolife sequential culture medium. Time-lapse imaging technology was used to calculate exact timing of early embryo cleavage events which included time to 2PN breakdown, cleavage to 2-, 3-, 4-, 5- cell and the time duration in the 2-,3-cell stage. Then these timing of early embryo cleavage events were compared between Cook group and Vitrolife group. Moreover, fertilization rate, cleavage rate, high quality embryo rate, usable blastocyst rate, pregnancy rate and implantation rate of these two groups were also analyzed. Results: The results showed there were no differences in all timing of early embryo cleavage events between the two groups. In addition, the two groups were similar in fertilization rate (Cook 71.0% vs. Vitrolife 71.3%, P>0.05), cleavage rate (Cook 98.1% vs. Vitrolife 98.2%, P>0.05), high quality embryo rate (Cook 52.1% vs. Vitrolife 52.7%, P>0.05), usable blastocyst rate (Cook 29.7% vs. Vitrolife 28.0%, P>0.05), pregnancy rate (Cook 46.7% VS. Vitrolife 50.0%, P>0.05) and implantation rate (Cook 30.3% VS. Vitrolife 29.0%, P>0.05). Conclusions: Morphokinetics used for embryo selection are not affected by the two different culture media. PMID:28083029

  14. [Isolation culture and identification of human neural stem cells from human embryos].

    Science.gov (United States)

    Luo, Shu-Wei; Xie, Chang-Qing; Lu, Guang-Xiu

    2004-04-01

    To obtain and culture the human neural stem cells from the aborted human embryos. The neural stem cells were isolated and purified by the specific proliferous culture system of neural stem cells, and the molecular maker and multi-potency of the obtained neural stem cells were identified. Human neural stem cells, which were isolated from 8 - 10 month old aborted human embryos, could express nestin ( a kind of specified antigen of the neural stem cell), and it could be differentiate into neurons and glials. Neural stem cells can exist in human embryos, and it can be expanded in vitro.

  15. Homocysteine in embryo culture media as a predictor of pregnancy outcome in assisted reproductive technology.

    Science.gov (United States)

    Boyama, Burcu Aydin; Cepni, Ismail; Imamoglu, Metehan; Oncul, Mahmut; Tuten, Abdullah; Yuksel, Mehmet Aytac; Kervancioglu, Mehmet Ertan; Kaleli, Semih; Ocal, Pelin

    2016-01-01

    The aim of this study was to determine whether homocysteine (hcy) concentrations in embryo culture media correlate with pregnancy outcome in assisted reproductive technology (ART) cycles. Forty patients who underwent single embryo transfer at the infertility clinic of a tertiary care center were recruited for this case-control study. Spent embryo culture media from all patients were collected after single embryo transfer on day 3 (n = 40). Hcy concentrations in embryo culture media were analyzed by enzyme cycling method. Patients were grouped according to the diagnosis of a clinical pregnancy. Sixteen patients were pregnant while 24 patients failed to achieve conception. Mean Hcy levels in the culture media were significantly different between the groups (p < 0.003), as 4.58 ± 1.31 μmol/l in the non-pregnant group and 3.37 ± 0.92 μmol/l in the pregnant group. Receiver operator curve analysis for determining the diagnostic potential of Hcy for pregnancy revealed an area under the curve of 0.792 (confidence interval: 0.65-0.94; p < 0.05). A cut-off value of 3.53 μmol/l was determined with a sensitivity of 83.3%, and a specificity of 68.8%. Lower hcy levels were associated with a better chance of pregnancy and better embryo grades. Hcy may be introduced as an individual metabolomic profiling marker for embryos.

  16. Radiation effects on cultured mouse embryos in relation to cell division cycle

    International Nuclear Information System (INIS)

    Domon, M.

    1982-01-01

    The authors have worked with mouse embryos in vitro asking first, what are the suitable parameters to define the radiation sensitivity of embryos, and second what is a major factor determining it. The LD 50 was adopted as a parameter of the radiation sensitivity of a population in a mouse embryo system in culture. The fertilized ova were collected into Whitten's medium at various times during the pronuclear and 2-cell stages of development. They were irradiated in chambers with X-rays at doses of 0 to 800 rads. After the embryos were cultured, a set of the lethal fractions for various X-ray doses were obtained. Regarding the radiation sensitivity variation of the embryos, the LD 50 varied from 100 to 200 rads during the pronuclear stage and from 100 to 600 rads during the 2-cell stage. The embryos during the pronuclear stage were most radioresistant at early G 2 phase, followed by an increase in the sensitivity. The embryos during the 2-cell stage were also most radioresistant at early G 2 phase and were more sensitive when they got close to either the first or the second cleavage division. Furthermore, it seems that the factor 6 of the large variation was due to the extremely long G 2 period, 14 hrs for the 2-cell embryos. That is, the pooled 2-cell embryos were in a relative sense well synchronized with G 2 phase. In contrast, the synchrony was poor during the pronuclear stage, which led to less variation of the LD 50 for the pronuclear embryos. It is concluded that during the early cleavage stages of mice, radiosensitivity is mainly governed by the content of cells of various cell cycle ages in the embryo. (Namekawa, K.)

  17. Taurina no desenvolvimento de embriões bovinos fecundados in vitro Taurine on the development of in vitro fertilized bovine embryos

    Directory of Open Access Journals (Sweden)

    L.S.A. Camargo

    2002-08-01

    Full Text Available O objetivo deste trabalho foi avaliar o efeito de diferentes concentrações de taurina no desenvolvimento de embriões bovinos fecundados in vitro em meio de cultivo com diferentes fontes de soro. No experimento 1, zigotos (n=440 fecundados in vitro foram distribuídos aleatoriamente nos tratamentos com 0, 3, 7 ou 14 mM de taurina em meio de cultivo acrescido de 10% de soro fetal bovino (SFB e 3g/l de albumina sérica bovina (BSA. No experimento 2, os zigotos (n=940 foram divididos nos tratamentos com 0, 3 ou 14 mM de taurina em meio acrescido de 10% de SFB ou 3g/l de BSA. No experimento 3, os zigotos (n=191 foram divididos nos tratamentos com 0 ou 3 mM de taurina em meio de cultivo sem fonte de soro, porém adicionado de 3 g/l de álcool polivinil. Nos experimentos 1 e 2 não se observou diferença (P>0,05 na taxa de clivagem, na produção de blastocistos e no número de células entre as concentrações de taurina avaliadas. No experimento 3 encontraram-se maior (PThe effect of different taurine concentrations on bovine embryo development in medium supplemented with different serum sources was studied. In the first experiment, in vitro fertilized zygotes (n=440 were divided into treatments with 0, 3, 7 or 14 mM of taurine in culture medium supplemented with 10% of fetal calf serum (FCS and 3g/l of bovine serum albumin (BSA. In the second experiment, zygotes (n=940 were divided into treatments with 0, 3 or 14 mM of taurine in cultured medium supplemented with 10% of FCS or 3g/l of BSA. In the third experiment, zygotes (n=191 were divided into treatments with 0 or 3 mM of taurine in culture medium without serum source, even so supplemented with 3g/l of polyvinyl alcohol. In the first and second experiments no differences (P>0.05 in cleavage rate, blastocyst production and cells number among the concentrations of taurine were observed. In the third experiment, taurine increased (P<0.05 cleavage rate (68.5% vs. 16.9% and blastocysts production

  18. Mitochondrial-related consequences of heat stress exposure during bovine oocyte maturation persist in early embryo development.

    Science.gov (United States)

    Payton, Rebecca R; Rispoli, Louisa A; Nagle, Kimberly A; Gondro, Cedric; Saxton, Arnold M; Voy, Brynn H; Edwards, J Lannett

    2018-03-18

    Hyperthermia during estrus has direct consequences on the maturing oocyte that carries over to the resultant embryo to compromise its ability to continue in development. Because early embryonic development is reliant upon maternal transcripts and other ooplasmic components, we examined impact of heat stress on bovine oocyte transcripts using microarray. Oocytes were matured at 38.5ºC for 24 h or 41.0ºC for the first 12 h of in vitro maturation; 38.5ºC thereafter. Transcriptome profile was performed on total (adenylated + deadenylated) RNA and polyadenylated mRNA populations. Heat stress exposure altered the abundance of several transcripts important for mitochondrial function. The extent to which transcript differences are coincident with functional changes was evaluated by examining reactive oxygen species, ATP content, and glutathione levels. Mitochondrial reactive oxygen species levels were increased by 6 h exposure to 41.0ºC while cytoplasmic levels were reduced compared to controls (P stressed oocytes at 24 h (P 0.05). Collectively, results indicate that direct exposure of maturing oocytes to heat stress may alter oocyte mitochondrial processes/function, which is inherited by the early embryo after fertilization.

  19. STUDIES REGARDING CULTURE MEDIUM INFLUENCE ON IN VITRO REGENERATION FROM WHEAT IMATURE EMBRYOS

    Directory of Open Access Journals (Sweden)

    M. DANCI

    2008-05-01

    Full Text Available Surnamed „embryos’ saving method”, embryos culture is an in vitro technique used for over half of the century for saving the distant hybridization products, that would have degenerate in other conditions. Immature embryos culture is used for initiation of in vitro cultures imposed by the impossibility of using other explants for some of the plant species. Wheat is one of the crops that immature embryos culture technique is suitable for. This methods principle is based on aseptic embryos excision and their inoculation to an adequate culture medium. In vitro culture results depend in a greater manner of the basic culture medium and the hormonal balance used. Immature embryos isolated from two Romanian wheat cultivars – Dropia and Lovrin 41 – were inoculated for callus production on two types of basic media added with 2,4 D. The selected calluses were transferred on MS basic medium and several parameters were registered. Both cultivars emphasized a good callusing capacity, in a different percentage depending on the culture media used, such as 71,09 – 94,45%.. big differences between the cultivars regarding embriogenic callus frequency, shooting callus frequency and regenerated plants percentage were registered.

  20. 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.

  1. In vitro development of canine somatic cell nuclear transfer embryos in different culture media.

    Science.gov (United States)

    Kim, Dong-Hoon; No, Jin-Gu; Choi, Mi-Kyung; Yeom, Dong-Hyeon; Kim, Dong-Kyo; Yang, Byoung-Chul; Yoo, Jae Gyu; Kim, Min Kyu; Kim, Hong-Tea

    2015-01-01

    The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.

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

    Science.gov (United States)

    Yu, Dawei; Zhang, Shoufeng; Du, Weihua; Zhang, Jinxia; Fan, Zongxing; Hao, Haisheng; Liu, Yan; Zhao, Xueming; Qin, Tong; Zhu, Huabin

    2014-01-01

    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.

  3. 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.

  4. Absence of Sperm Factors as in the Parthenogenesis Does Not Interfere on Bovine Embryo Sensitiveness to Heat Shock at Pre-Implantation Stage.

    Science.gov (United States)

    Camargo, L S A; Paludo, F; Pereira, M M; Wohlres-Viana, S; Gioso, M M; Carvalho, B C; Quintao, C C R; Viana, J H M

    2016-02-01

    Oocyte has been considered the major contributor for embryo thermo-tolerance. However, it was shown that sperm factors can be transferred to the oocyte during fertilization, raising the question of whether the absence of such factors could interfere on embryo thermo-tolerance. In this study, we used parthenogenesis to generate bovine embryos without spermatozoa in order to test whether the absence of sperm factors could influence their thermo-sensitiveness at early stages. In vitro fertilized (IVF) and parthenogenetic (PA) embryos at 44 h post-insemination/chemical activation were exposed to 38.5°C (control) or 41°C (heat shock) for 12 h and then developed for 48 h and up to blastocyst stage. Apoptosis index and expression of PRDX1, GLUT1, GLUT5 and IGF1r genes in blastocysts derived from heat-shocked embryos were also evaluated. The heat shock decreased the blastocyst rate at day seven (p 0.05) between method of activation (IVF and PA) and temperature (38.5°C or 41.5°C) for all developmental parameters evaluated. Expression of GLUT1 gene was downregulated (p < 0.05) by heat shock in both IVF and PA blastocyst whereas expression of GLUT5 and IGF1r genes was downregulated (p < 0.05) by heat shock in PA blastocysts. Those data show that the heat shock affects negatively the embryo development towards blastocysts stage, increases the apoptotic index and disturbed the expression of some genes in both IVF and PA embryos, indicating that the presence or absence of sperm factors does not influence the sensitivity of the bovine embryo to heat shock. © 2015 Blackwell Verlag GmbH.

  5. Weight gain potential affects pregnancy rates in bovine embryo recipients raised under pasture conditions.

    Science.gov (United States)

    Fernandes, Carlos Antonio de Carvalho; Palhao, Miller Pereira; Figueiredo, Ana Cristina Silva; Ribeiro, Josiane Rossi; Fonseca e Silva, Fabyano; Viana, Joao Henrique Moreira

    2016-01-01

    The aim of the present study was to evaluate the effect of differences in body weight gain after embryo transfer on the pregnancy rates of crossbred heifers used as recipients and raised under a grazing system. The study was performed during the dry (April to September) and the rainy (October to March) seasons. The embryos transferred were produced by in vitro fertilization. The body weight of each recipient was measured immediately before the embryo transfer and 23 to 25 days later, when the diagnosis of pregnancy was performed by ultrasonography. The associations among initial body weight (IBW), daily body weight gain (DWG), season, and pregnancy rate were evaluated using a logistic procedure that included the effect of the IBW, season, and linear and quadratic effects of the DWG. Altogether, there was no effect of season and pregnancy rates did not change between the dry and rainy seasons (42.3 vs. 45.8%, respectively; P > 0.05). However, the pregnancy rate was greater in the recipients with daily body weight gains over 250 g/day, regardless of the season. In addition, the pregnancy rate of the recipients was better (P heifer to become pregnant according to DWG is explained by the follow equation: P(y = 1) = (Exp((-1.06703 + 0.0108 * DWG - 0.00002 * DWG ^ 2)))/(1 + Exp((-1.6703 + 0.0108 * DWG - 0.00002 * DWG ^ 2))). In conclusion, body weight gain potential is a critical factor for the pregnancy rates of in vitro embryo recipients managed under grazing systems.

  6. Association of the transcription profile of bovine oocytes and embryos with developmental potential

    Czech Academy of Sciences Publication Activity Database

    Kaňka, Jiří; Němcová, Lucie; Toralová, Tereza; Vodičková Kepková, Kateřina; Vodička, Petr; Jeseta, M.; Machatková, M.

    2012-01-01

    Roč. 134, 1-2 (2012), 29-35 ISSN 0378-4320. [Embryo Genomics Meeting /3./. Bonn, 20.08.2012-22.08.2012] R&D Projects: GA ČR GA523/09/1035; GA MZe QI91A018 Institutional support: RVO:67985904 Keywords : oocyte * in vitro maturation * pre-implantation development Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.897, year: 2012

  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. Determination of escin content in androgenic embryos and hairy root culture of Aesculus hippocastanum.

    Science.gov (United States)

    Calić-Dragosavac, Dusica; Zdravković-Korać, Snezana; Savikin-Fodulović, Katarina; Radojević, Ljiljana; Vinterhalter, Branka

    2010-05-01

    Escin, a group of chemically related triterpenic glycosides, is widely used in commercial preparations for the treatment of venous insufficiency. Since the zygotic embryo cotyledons accumulate the highest amount of escin, it is currently extracted from the seeds of horse chestnut, Aesculus hippocastanum L. (Hippocastanaceae), on a large scale. As this material is available during only short period of the year, we studied the possibility of using plant tissue culture to obtain escin. For this purpose, the content of escin in androgenic embryos and hairy root cultures of horse chestnut was studied. Escin content was found to be dependent on the stage of androgenic embryo development and the type of phytoregulator supplemented to the nutritive medium. In the absence of phytoregulators, androgenic embryos at the globular stage of development contained approximately four times less escin than those at the cotyledonary stage. Inclusion of various phytoregulators in the nutritive media stimulated escin production. Among them, 2,4-dichlorophenoxyacetic acid (2,4-D) showed the most pronounced effect, with escin content almost reaching that found in zygotic embryos (6.77% versus 6.96%). Two hairy root clones produced substantial amounts of escin (3.57% and 4.09%), less than zygotic embryos, but higher than cotyledonary embryos on phytoregulator-free medium.

  9. Growth and development of cultured carrot cells and embryos under spaceflight conditions

    Science.gov (United States)

    Krikorian, A. D.; Dutcher, F. R.; Quinn, C. E.; Steward, F. C.

    1981-01-01

    Morphogenetically competent proembryonic cells and well-developed somatic embryos of carrot at two levels of organization were exposed for 18.5 days to a hypogravity environment aboard the Soviet Biosatellite Cosmos 1129. It was confirmed that cultured totipotent cells of carrot can give rise to embryos with well-developed roots and minimally developed shoots. It was also shown that the space hypogravity environment could support the further growth of already-organized, later somatic embryonic stages and give rise to fully developed embryo-plantlets with roots and shoots.

  10. Teratogenic and cytotoxic effects of VOsalen complex on chicken embryos, hepatic and fibroblastic- cell cultures

    Directory of Open Access Journals (Sweden)

    Abdolmaleki A

    2013-04-01

    Full Text Available Background: Salen metal complexes are used successfully in a wide range of asymmet-ric reactions and important in the pharmaceutical and industry. On the toxicity of salen vanadium oxide (VOsalen on embryo and cell cultures, little information is available. In the present study, the toxic and teratogenic effects of VOsalen was evaluated against chicken embryos as a animal model and liver and fibroblast cell cultures which was derived from the embryo.Methods: The VOsalen compound was synthesized. The compound solution was inject-ed in triplicate examination, in the air sac of the eggs, at third day of incubation. Treat-ed and control eggs, on day 19 of incubation opened and embryos were weighted, then mortality rate was recorded. The liver and fibroblast cell culture were treated by this and survival fraction was recorded.Results: The survived fraction of the embryos depends on the compound concentration. In concentration of 300μM/egg, 36/32% of the embryos survived and the Lethal dose 50% (LD50 was 226/37 μM/egg. Morphological study of the treated embryos showed retarded growth, and skeletal staining showed the deletion of caudal vertebrate. The compound was inhibited liver and fibroblast cells growth with IC50 1047/25 and 1036/82μM respectively. The cytoplasm of treated cells became dense and their interco-nnections were loosed.Conclusion: The VOsalen compound had low toxic effects against the embryos and the cultured cells at the concentrations. Significant cytotoxic effect was not observed in the treated cells. However the proliferative cells were affected significantly in comparison with the cells which their growth was stopped. The effect of VOsalen compound against replication of liver cells were lower than fibroblast cells.

  11. A novel embryo culture media supplement that improves pregnancy rates in mice.

    Science.gov (United States)

    Highet, A R; Bianco-Miotto, T; Pringle, K G; Peura, A; Bent, S; Zhang, J; Nottle, M B; Thompson, J G; Roberts, C T

    2017-03-01

    The preimplantation embryo in vivo is exposed to numerous growth factors in the female reproductive tract, which are not recapitulated in embryo culture media in vitro The IGF2 and plasminogen activator systems facilitate blastocyst development. We hypothesized that the addition of IGF2 in combination with urokinase plasminogen activator (uPA) and plasminogen could improve rates of blastocyst hatching and implantation in mice. B6BcF1 and CBAB6F2 mouse embryos were divided into one of four supplemented culture media treatment groups: (1) control (media only); (2) 12.5 nM IGF2; (3) 10 µg/mL uPA and 5 µg/mL plasminogen; or (4) a combination of IGF2, uPA and plasminogen treatments. Embryo development to blastocyst stage and hatching were assessed before transfer to pseudopregnant recipient females and implantation, pregnancy rates and postnatal growth were assessed. After 90.5 h of culture, IGF2 + U + P treatment increased the percentage of B6BcF1 embryos that were hatching/hatched and percentage developing to blastocyst stage compared with controls (P culture, IGF2, uPA and plasminogen supplementation of culture media can improve pregnancy success, but the effect of treatment is dependent on the mouse strain. © 2017 Society for Reproduction and Fertility.

  12. Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified.

    Science.gov (United States)

    Hammond, Elizabeth R; McGillivray, Brent C; Wicker, Sophie M; Peek, John C; Shelling, Andrew N; Stone, Peter; Chamley, Larry W; Cree, Lynsey M

    2017-01-01

    To characterize nuclear and mitochondrial DNA (mtDNA) in spent culture media from normally developing blastocysts to determine whether it could be used for noninvasive genetic assessment. Prospective embryo cohort study. Academic center and private in vitro fertilization (IVF) clinic. Seventy patients undergoing intracytoplasmic sperm injection (ICSI) and 227 blastocysts. Culture media assessment, artificial blastocoele fluid collapse and DNA analysis using digital polymerase chain reaction (dPCR), long-range PCR, quantitative PCR (qPCR), and DNA fingerprinting. Presence of nuclear and mtDNA in three different commercial culture media from Vitrolife and Irvine Scientific, spent embryo media assessment at the cleavage and blastocyst stages of development, and analysis of the internal media controls for each patient that had been exposed to identical conditions as embryo media but did not come into contact with embryos. Higher levels of nuclear and mtDNA were observed in the culture media that had been exposed to embryos compared with the internal media controls. Nuclear DNA (∼4 copies) and mtDNA (∼600 copies) could be detected in spent media, and the levels increased at the blastocyst stage. No increase in DNA was detected after artificial blastocoele fluid collapse. Mixed sex chromosome DNA was detected. This originated from contamination in the culture media and from maternal (cumulus) cells. Due to the limited amount of template, the presence of embryonic nuclear DNA could not be confirmed by DNA fingerprinting analysis. Currently DNA from culture media cannot be used for genetic assessment because embryo-associated structures release DNA into the culture medium and the DNA is of mixed origin. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  13. NGS Analysis of Human Embryo Culture Media Reveals miRNAs of Extra Embryonic Origin.

    Science.gov (United States)

    Sánchez-Ribas, Immaculada; Diaz-Gimeno, Patricia; Quiñonero, Alicia; Ojeda, María; Larreategui, Zaloa; Ballesteros, Agustín; Domínguez, Francisco

    2018-01-01

    Our objective in this work was to isolate, identify, and compare micro-RNAs (miRNAs) found in spent culture media of euploid and aneuploid in vitro fertilization (IVF) embryos. Seventy-two embryos from 62 patients were collected, and their spent media were retained. A total of 108 spent conditioned media samples were analyzed (n = 36 day 3 euploid embryos, n = 36 day 3 aneuploid embryos, and n = 36 matched control media). Fifty hed-control media embryos were analyzed using next-generation sequencing (NGS) technology. We detected 53 known human miRNAs present in the spent conditioned media of euploid and aneuploid IVF embryos. miR-181b-5p and miR-191-5p were found the most represented. We validated our results by quantitative polymerase chain reaction (qPCR), but no significant results were obtained between the groups. In conclusion, we obtained the list of miRNAs present in the spent conditioned media from euploid and aneuploid IVF embryos, but our data suggest that these miRNAs could have a nonembryonic origin.

  14. Comparative evaluation of the teratogenicity of genistein and genistin using rat whole embryo culture and limbud micromass culture methods.

    Science.gov (United States)

    Zou, Peng; Xing, Lina; Tang, Qiuqiong; Liu, Ran; Hao, Weidong

    2012-08-01

    Genistein (GEN) is one kind of phytoestrogen. Several studies have demonstrated the teratogenic potential of GEN in vitro by postimplantation rat whole embryo culture (WEC) assay, but GEN showed no teratogenic effects in vivo even at a dose up to 1000 mg/kg bw/day. The mechanism of such discrepancy is still unclear. Because more than 80% of total genistein (free plus glycoside form) in circulation is its glycoside metabolite, genistin (GIN), we thus hypothesize that genistin is non-teratogenic. To prove this hypothesis, rat whole embryo culture (WEC) and limbud micromass culture methods were applied to compare the teratogenic effects of GEN and GIN on developing embryos in vitro. In WEC assay, we found that the development of embryos was affected by GEN treatment dose-dependently, while GIN-treated embryos displayed slight developmental defects only at the highest dose (222 μM). In micromass culture assay, the IC50 of cell proliferation and differentiation for GEN were 15.6 and 37.2 μM, respectively, while neither was influenced by GIN treatment up to 111 μM. Collectively, our study indicated that GEN showed no teratogenic effects in vivo probably due to its transformation to the non-teratogenic metabolite, GIN. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. MODELAGEM BIOECONÔMICA DA TRANSFERÊNCIA DE EMBRIÕES EM BOVINOS BIOECONOMIC MODEL IN BOVINE EMBRYO TRANSFER

    Directory of Open Access Journals (Sweden)

    Renato Travassos Beltrame

    2010-04-01

    Full Text Available

    O objetivo deste trabalho foi desenvolver um modelo matemático orientado a eventos de simulação, para auxiliar tomadas de decisão relativas à transferência de embriões em bovinos, considerando-se as dinâmicas de dois componentes da transferência de embriões: receptoras e embriões. Na simulação, não se avaliaram respostas individuais de doadoras a coletas consecutivas e eventos correspondentes na transferência de embriões. Simulou-se o mesmo protocolo para superovulação a todas as doadoras. Receptoras foram sincronizadas simulando-se o uso de prostaglandina. O número de embriões viáveis produzido por doadora e sua variabilidade tiveram como base um processo aleatório de simulação de Monte Carlo, que pressupôs uma distribuição exponencial negativa de densidade de probabilidade. Custos e receitas foram inseridos no modelo por meio de um cenário-base para calcular indicadores econômicos de rentabilidade. A análise sugeriu a impraticabilidade da atividade, se realizada diante do cenário proposto (VPL – R$: 57.596,69. A partir do cenário proposto, o custo médio estimado foi de R$ 1.178,19, e de R$ 980,03, para se obter uma prenhez a partir de uma situação otimizada, sugerida pelo modelo (5/100; 5/190.

    PALAVRAS-CHAVES: Otimização, receptoras, simulação, transferência de embriões, viabilidade econômica.

    A simulation model related to embryo transfer programs in bovine was carried out through a mathematical model directed to events, considering the dynamic of two resources: recipients and embryos. Individual answers of donors to consecutive collections and corresponding events in embryo transfer were not evaluated. The same protocol for superovulation was simulated for all the donor collections, using similar doses of hormones and drugs for all the animals. Recipients were synchronized using prostaglandin. Meantime, the number of viable embryos produced by donor and its variability were based at

  16. Buffalo (Bubalus bubalis in vitro embryo production in two different defined culture media

    Directory of Open Access Journals (Sweden)

    B. Gasparrini

    2011-03-01

    Full Text Available In vitro embryo production (IVEP is largely applied world wide to animal breeding. One of the principal steps of the IVEP is represented by embryo culture (Khurana and Niemann., 2000. In the past, embryos were grown in co-culture systems with other cells such as oviductal epithelial cells, cumulus cells, Buffalo rat liver (BRL and VERO cells (Duszewska et al., 2000. These cells are able to supply the nutrients for embryo development by their replication and metabolism. Nevertheless, the metabolic activity of these cells is also responsible of an early lowering of pH in the culture medium: that needs to be changed every two days. Furthermore, with this culture system it is impossible to standardize all the procedure: in fact the result is dependent from several variables, as the quality of the cells and their concentration in co-culture. The use of defined culture media is necessary to acquire a better comprehension of metabolism and biochemical requirements for IVEP........

  17. Impact of embryo co-culture with cumulus cells on pregnancy & implantation rate in patients undergoingin vitrofertilization using donor oocyte.

    Science.gov (United States)

    Bhadarka, Harsha K; Patel, Nayana H; Patel, Niket H; Patel, Molina; Patel, Kruti B; Sodagar, Nilofar R; Phatak, Ajay G; Patel, Jagdish S

    2017-09-01

    Cumulus cell co-culture of embryo had been found to be beneficial for achieving better pregnancy and implantation rate (IR). The present study was aimed to evaluate efficiency of cumulus co-culture technique over simple culture of embryo in terms of pregnancy rate (PR) and IR in patients undergoing treatment for infertility using donor oocytes fertilized by intracytoplasmic sperm injection. This was a quasi-experimental study between control and study groups. The primary endpoint was achievement of pregnancy. Control group included 508 women who underwent embryo development without cumulus cell co-culture and study group included 394 women who underwent embryo development with cumulus cell co-culture using donor's cumulus cells. The present study demonstrated a significant increase in the IR (37.2 vs 24.2%, Pculture technique was found to be more effective than simple culture technique for embryo development in women undergoing treatment for infertility using donor oocytes fertilized by intracytoplasmic sperm injection.

  18. A serum-free and defined medium for the culture of mammalian postimplantation embryos.

    Science.gov (United States)

    Drakou, Katerina; Georgiades, Pantelis

    2015-12-25

    Whole embryo culture (WEC) of postimplantation rodent embryos is widely used for the study of mammalian embryogenesis and developmental toxicity testing. Its major advantage is that it allows direct access to embryos for experimental manipulations and the monitoring of their consequences that would otherwise not be possible or technically difficult to perform in utero. However, a major drawback of mammalian WEC is that the culture media currently in use display batch variations and are undefined, as they contain serum or serum replacements of unknown composition. Moreover, these media possess cell-signalling activities important for embryogenesis. Therefore, reproducibility of mammalian postimplantation WEC results may be affected by batch variation and their interpretation is complicated because the experimenter is unsure whether the embryo response to experimental perturbations is solely due to their action, or modified as a result of influences from undefined substances/signaling activities present in culture media. To alleviate these problems we investigated whether N2B27, a serum-free and defined medium, can support the in vitro development of postimplantation mammalian embryos. We show that N2B27 allows pre-gastrulation mouse embryos isolated at embryonic day 5.5 to develop to advanced gastrulation, reaching the mid- and late primitive streak stages. This is the first demonstration that postimplantation mammalian embryos can develop in vitro in a defined medium in the absence of serum and provides a novel WEC system for studying developmental mechanisms and testing for developmental toxicity during the early postimplantation period. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Human embryos secrete microRNAs into culture media--a potential biomarker for implantation.

    Science.gov (United States)

    Rosenbluth, Evan M; Shelton, Dawne N; Wells, Lindsay M; Sparks, Amy E T; Van Voorhis, Bradley J

    2014-05-01

    To determine whether human blastocysts secrete microRNA (miRNAs) into culture media and whether these reflect embryonic ploidy status and can predict in vitro fertilization (IVF) outcomes. Experimental study of human embryos and IVF culture media. Academic IVF program. 91 donated, cryopreserved embryos that developed into 28 tested blastocysts, from 13 couples who had previously completed IVF cycles. None. Relative miRNA expression in IVF culture media. Blastocysts were assessed by chromosomal comparative genomic hybridization analysis, and the culture media from 55 single-embryo transfer cycles was tested for miRNA expression using an array-based quantitative real-time polymerase chain reaction analysis. The expression of the identified miRNA was correlated with pregnancy outcomes. Ten miRNA were identified in the culture media; two were specific to spent media (miR-191 and miR-372), and one was only present in media before the embryos had been cultured (miR-645). MicroRNA-191 was more highly concentrated in media from aneuploid embryos, and miR-191, miR-372, and miR-645 were more highly concentrated in media from failed IVF/non-intracytoplasmic sperm injection cycles. Additionally, miRNA were found to be more highly concentrated in ICSI and day-5 media samples when compared with regularly inseminated and day-4 samples, respectively. MicroRNA can be detected in IVF culture media. Some of these miRNA are differentially expressed according to the fertilization method, chromosomal status, and pregnancy outcome, which makes them potential biomarkers for predicting IVF success. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  20. Tritiated thymidine radiotoxicity in mice embryos in preimplantation phase, culture and in vitro fertilization

    International Nuclear Information System (INIS)

    Kikuchi, O.K.; Ohyama, H.; Yamamada, T.

    1992-01-01

    Mouse embryos have been used as a good biological system to study the effects of ionizing radiation on proliferating cells and on the mammal embryos development. In vitro fertilization is an ideal technique for irradiating or labelling many zygotes at the same developmental stage and with the the same developmental stage and with the security that it will not have any influence of maternal organism. In the present work different concentrations of methyl- 3 H-thymidine was added to the culture medium micro drops containing the zygotes at pro nuclear stage and the embryos were cultured in vitro at 37 0 C in a humidified atmosphere with 5% of CO 2 for four days up established end point to calculate the LD 50 , the 50% lethal dose. (author)

  1. Comparison of the acidification activities of commercial starter cultures in camel and bovine milk

    DEFF Research Database (Denmark)

    Berhe, Tesfemariam; Ipsen, Richard; Seifu, Eyassu

    2018-01-01

    Camel milk has been reported to be difficult to ferment due to anti-microbial properties. The present study tested eight commercial starter cultures for their ability to grow in camel milk. All investigated cultures were able to acidify camel milk and reached a final pH at a level similar to what...... was achieved in bovine milk, but the speed of acidification was generally lower in camel milk. This could be due to inhibitory substances in camel milk or due to reduced availability of nutrients. Experiments using mixtures of camel and bovine milk or supplementation with casein hydrolysates allowed us...... to distinguish between these possibilities. High acidification rates were obtained in camel milk mixed with bovine milk or supplemented with casein hydrolysate. This demonstrates that the cultures are not inhibited by camel milk and we conclude that the growth rates of these cultures in pure camel milk...

  2. Using CR1aa versus KSOM as the culture medium for in vitro embryo production of cattle

    Directory of Open Access Journals (Sweden)

    Endang Triwulaninngsih

    2002-03-01

    Full Text Available This research has been conducted at the laboratory of in vitro fertilization in the Department of Animal Science University of Wisconsin, USA. These embryos can be used for improving genetic value of Indonesian cattle. Oocytes were matured in TCM- 199 medium (in 5% CO2 incubator and at 390C enriched with follicle stimulating hormone (FSH 10 μl/ml, oestradiol 17 β 1μl/ml and 10% Fetal Calf Serum (FCS. The oocytes were fertilized in vitro with motile sperm and incubation between sperm and oocytes in fertilization medium Tyroide Albumin Lactate Pyruvate (TALP for 20 hours. All zygotes were cultured in CR1aa (n=1549 medium versus modification of protein-free pottasium simplex optimized medium (KSOM (n=675 up to blastocyst stage and were fed FCS 5 μl/50 μl medium on day 6, as treatment A and B respectively. Data were analyzed by completely randomized design with SAS program. Percentages of cleavage, morula, blastocyst, expanded blastocyst, unfertilized and degenerated ova in this study were 91.4% vs 75.6 %; 75.6% vs 58.9%; 61.5% vs 38.5%; 31.2% vs 5.1%, 8.6% vs 24.4%, 15.7% vs 8% which were significantly different (P<0.01 for treatment CR1aa and KSOM respectively. Based on this study, CR1aa medium is better culture medium than KSOM for efficient in vitro production (IVP of bovine embryos.

  3. Generation of primary cultures of bovine brain endothelial cells and setup of cocultures with rat astrocytes

    DEFF Research Database (Denmark)

    Helms, Hans C; Brodin, Birger

    2014-01-01

    -brain barrier. The present protocol describes the setup of an in vitro coculture model based on primary cultures of endothelial cells from bovine brain microvessels and primary cultures of rat astrocytes. The model displays a high electrical tightness and expresses blood-brain barrier marker proteins....

  4. Birthweight distribution in ART singletons resulting from embryo culture in two different culture media compared with the national population

    DEFF Research Database (Denmark)

    Lemmen, Josephine Gabriela; Pinborg, Anja; Rasmussen, S

    2014-01-01

    and calculated the mean birthweight for the Cook-d2, Medicult-d2 and Medicult-d3 groups and found no significant differences. LIMITATIONS, REASONS FOR CAUTION: The retrospective design and the inherent risk of confounding factors is a limitation. Selection bias cannot be excluded as the embryos cultured in Cook...

  5. A study of two sequential culture media - impact on embryo quality ...

    African Journals Online (AJOL)

    Abstract. Objective. A comparative study of embryo quality and pregnancy outcome between Sydney IVF medium and. Quinn's Advantage sequential culture media. Design. A prospective randomised controlled trial and a retrospective study. Setting. In vitro fertilisation clinic in an academic research environment. Patients.

  6. Transcriptional profiles of chicken embryo cell cultures following infection with infectious bursal disease virus

    DEFF Research Database (Denmark)

    Li, Yiping; Handberg, K.J.; Juul-Madsen, H.R.

    2007-01-01

    -host interaction, we measured steady-state levels of transcripts from 28 cellular genes of chicken embryo (CE) cell cultures infected with IBDV vaccine stain Bursine-2 during a 7-day infection course by use of the quantitative real-time RT-PCR SYBR green method. Of the genes tested, 21 genes (IRF-1, IFN 1...

  7. Factors affecting the gene expression of in vitro cultured human preimplantation embryos

    NARCIS (Netherlands)

    Mantikou, E.; Jonker, M. J.; Wong, K. M.; van Montfoort, A. P. A.; de Jong, M.; Breit, T. M.; Repping, S.; Mastenbroek, S.

    2016-01-01

    What is the relative effect of common environmental and biological factors on transcriptome changes during human preimplantation development? Developmental stage and maternal age had a larger effect on the global gene expression profile of human preimplantation embryos than the culture medium or

  8. Embryotoxicant-specific transcriptomic responses in rat postimplantation whole-embryo culture

    NARCIS (Netherlands)

    Robinson, J.F.; van Beelen, V.A.; Verhoef, A.; Renkens, M.F.J.; Luijten, M.; van Herwijnen, M.; Westerman, A.; Pennings, J.L.; Piersma, A.H.

    2010-01-01

    Rat postimplantation whole-embryo culture (WEC) is a promising alternative test for the assessment of developmental toxicity. Toxicogenomic-based approaches may improve the predictive ability of the WEC model by providing a means to identify compound-specific mechanistic responses associated with

  9. In vitro culture of mouse embryos in human amniotic fluid | Coetzee ...

    African Journals Online (AJOL)

    Human amniotic fluid was compared with Ham's F-10 culture medium as a possible alternative for use in in vitro fertilisation. The cleavage success of mouse embryos in human amniotic fluid (experimental group) was 92% compared with 86% in Ham's F-10 medium. It is concluded that human amniotic fluid is a viable ...

  10. Effect of in vitro culture of human embryos on birthweight of newborns

    NARCIS (Netherlands)

    Dumoulin, John C.; Land, Jolande A.; Van Montfoort, Aafke P.; Nelissen, Ewka C.; Coonen, Edith; Derhaag, Josien G.; Schreurs, Inge L.; Dunselman, Gerard A.; Kester, Arnold D.; Geraedts, Joep P.; Evers, Johannes L.

    In animal models, in vitro culture of preimplantation embryos has been shown to be a risk factor for abnormal fetal outcome, including high and low birthweight. In the human, mean birthweight of singletons after in vitro fertilization (IVF) is considerably lower than after natural conception, but it

  11. Further evidence that culture media affect perinatal outcome : findings after transfer of fresh and cryopreserved embryos

    NARCIS (Netherlands)

    Nelissen, Ewka C.; Van Montfoort, Aafke P.; Coonen, Edith; Derhaag, Josien G.; Geraedts, Joep P.; Smits, Luc J.; Land, Jolande A.; Evers, Johannes L.; Dumoulin, John C.

    We have previously shown that the medium used for culturing IVF embryos affects the birthweight of the resulting newborns. This observation with potentially far-reaching clinical consequences during later life, was made in singletons conceived during the first IVF treatment cycle after the transfer

  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. 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.

  14. Hand-made cloned buffalo (Bubalus bubalis) embryos: comparison of different media and culture systems.

    Science.gov (United States)

    Shah, Riaz A; George, Aman; Singh, Manoj K; Kumar, Dharmendra; Chauhan, Manmohan S; Manik, Radhaysham; Palta, Prabhat; Singla, Suresh K

    2008-12-01

    Hand-made cloning (HMC) has proved to be an efficient alternative to the conventional micromanipulator-based technique in some domestic animal species. This study reports the development of an effective culture system for in vitro culture of zona-free cloned buffalo (Bubalus bubalis) embryos reconstructed using adult skin fibroblast cells as nucleus donor. Cleavage and blastocyst rates observed were 52 and 0% in modified Charles Rosenkrans 2 (mCR2), 61 and 4.6% in modified Synthetic Oviductal Fluid (mSOF), and 82 and 40.3% in Research Vitro Cleave (RVCL; Cook, Australia) medium, respectively. Similarly, higher blastocyst rates (24.5 +/- 4.1%) were observed when zona-free parthenotes were cultured in RVCL medium. Culturing zona-free cloned buffalo embryos on flat surfaces (FS) yielded significantly higher (p WOW) or microdrops (MD). Furthermore, development in WOW was found to be significantly better than MD culture. The quality of HMC blastocysts was examined using differential staining. This study establishes the application of zona-free nuclear transfer procedures for the production of hand-made cloned buffalo embryos and the development of efficient culture system and appropriate media requirements for enhancing their preimplantation development.

  15. Culturing Embryos and Larvae of Marine Molluscs and Protochordates.

    Science.gov (United States)

    Healey, R.; Turner, S. C.

    1979-01-01

    Presents a description for maintaining adult forms of molluscs and protochordates in order to obtain gametes for laboratory studies of animal development. The methods also include those for culturing embryonic larvae forms in vitro. (Author/SA)

  16. Differentiation of somatic embryos from protoplasts isolated from embryogenic suspension cultures of Abies alba L.

    Science.gov (United States)

    Hartmann, S; Lang, H; Reuther, G

    1992-10-01

    Embryogenic suspension cultures of Abies alba were established using an embryogenic suspensor mass culture originating from the zygotic embryo in immature seed explants (Schuller et al. 1989). Protoplasts were isolated from the suspension material. The protoplasts were immobilized in alginate layers in order to follow the development of single protoplasts. During the first days of protoplast culture a modified Kao and Michayluk (1975) medium proved to be necessary for subsequent divisions. The formation of proembryos succeeded within 2-3 weeks when subcultured with a modified Schenk and Hildebrandt (1972) liquid medium. Light, enhanced sugar concentration, and the addition of abscisic acid led to the formation of slightly green "torpedo"-shaped somatic embryos after 6-8 weeks from protoplast isolation.

  17. Growth regulators and darkness increase efficiency in in vitro culture of immature embryos from peppers

    Directory of Open Access Journals (Sweden)

    Juan Pablo Manzur

    2014-12-01

    Full Text Available Common pepper (Capsicum annuum L. is one of the most important vegetables in the world, and extensive breeding efforts are being made to develop new improved strains of this species. In this regard, in vitro culture of immature embryos may help breeders accelerate breeding cycles and overcome interspecific barriers, among other applications. In this study, we have optimized a protocol for in vitro culture of immature embryos of C. annuum. Levels of indole-3-acetic acid (IAA and zeatin have been tested to improve the efficiency (germination rates of this technique in C. annuum embryos at the four main immature stages (i.e. globular, heart, torpedo, and early cotyledonary from four varietal types of this species (California Wonder, Piquillo, Guindilla, and Bola. The effect of 5-day initial incubation in the dark was also tested on the most efficient hormone formulation. On average, relatively low levels of both IAA and zeatin (0.01 mg L−¹ each (M1 provided the highest germination rates, particularly in the advanced stages (torpedo and cotyledonary. To a lesser extent, the lack of these growth regulators (M0 or high IAA (0.2 mg L−¹/low zeatin (0.01 mg L−¹ (M2 combination also had a positive response. On the contrary, high zeatin levels (0.2 mg L−¹ produced very low germination rates or callus development (efficiency 0-7 %. Different responses were also found between genotypes. Thus, considering the best media (M0, M1, M2, Bola embryos had the highest rates. M1 plus 5-days of initial dark incubation (M1-D improved the efficiency rates at all embryo stages, particularly in the earliest (globular embryos which increased from 3 % to > 20 %.

  18. Culture media for human pre-implantation embryos in assisted reproductive technology cycles.

    Science.gov (United States)

    Youssef, Mohamed M A; Mantikou, Eleni; van Wely, Madelon; Van der Veen, Fulco; Al-Inany, Hesham G; Repping, Sjoerd; Mastenbroek, Sebastiaan

    2015-11-20

    Many media are commercially available for culturing pre-implantation human embryos in assisted reproductive technology (ART) cycles. It is unknown which culture medium leads to the best success rates after ART. To evaluate the safety and effectiveness of different human pre-implantation embryo culture media in used for in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) cycles. We searched the Cochrane Menstrual Disorders and Subfertility Group's Trials Register, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, the National Research Register, the Medical Research Council's Clinical Trials Register and the NHS Center for Reviews and Dissemination databases from January 1985 to March 2015. We also examined the reference lists of all known primary studies, review articles, citation lists of relevant publications and abstracts of major scientific meetings. We included all randomised controlled trials which randomised women, oocytes or embryos and compared any two commercially available culture media for human pre-implantation embryos in an IVF or ICSI programme. Two review authors independently selected the studies, assessed their risk of bias and extracted data. We sought additional information from the authors if necessary. We assessed the quality of the evidence using Grades of Recommendation, Assessment, Development and Evaluation (GRADE) methods. The primary review outcome was live birth or ongoing pregnancy. We included 32 studies in this review. Seventeen studies randomised women (total 3666), three randomised cycles (total 1018) and twelve randomised oocytes (over 15,230). It was not possible to pool any of the data because each study compared different culture media.Only seven studies reported live birth or ongoing pregnancy. Four of these studies found no evidence of a difference between the media compared, for either day three or day five embryo transfer. The data from the fifth study did not appear reliable

  19. Induction of microspore-derived embryos by anther culture in ...

    African Journals Online (AJOL)

    ajl yemi

    2011-11-28

    Nov 28, 2011 ... obtained from A269, one of the genotypes tolerant to low temperature. The anthers cultured from April to May, gave the ... microspores in each anther and numerous haploid plants can be obtained from a single anther. ... in Turkey can be observed in several regions in both open fields and greenhouses.

  20. Developmental competence of HMC(TM) derived bovine cloned embryos obtained from somatic cell nuclear transfer of adult fibroblasts and granulosa cells.

    Science.gov (United States)

    Bhojwani, Sanjay; Vajta, Gabor; Callesen, Henrik; Roschlau, Knut; Kuwer, Andreas; Becker, Frank; Alm, Hannelore; Torner, Helmut; Kanitz, Wilhelm; Poehland, Ralf

    2005-08-01

    To enable us to handle a large number of oocytes at a given time and to have an increased throughput of cloned embryos, we attempted the Handmade cloning (HMC) technique, a zona-free method of bovine somatic cell nuclear transfer. Our objective was to study the developmental competence of the HMC derived embryos obtained using different types of somatic cells. A total of 6,874 cumulus-oocyte-complexes were used with either 7th or 11th passage fibroblasts (1st and 2nd groups, respectively), which were prepared from male animals, or granulosa cells (3rd group) as nuclei donors. The average cleavage rate was 65%, accompanied by a blastocyst rate of just 2% for the cleaved products and 5% for the >8-cell embryos, and there was no significant difference between the three groups. Out of 27 blastocysts recovered, 22 blastocysts were transferred to 22 recipients, resulting in two pregnancies. One pregnancy was lost after the fourth week while the other progressed to full term with the birth of a male calf. This first successful cloning of a male calf with the HMC technique in Europe indicates the successful adoption and establishment of this technique in our laboratory, and that this technique can be successful in producing viable embryos.

  1. The effects of embryo culture media on the birthweight of singletons via fresh or frozen-thawed embryo transfer: a large-scale retrospective study.

    Science.gov (United States)

    Gu, Fang; Deng, Mingfen; Gao, Jun; Wang, Zilian; Ding, Chenhui; Xu, Yanwen; Zhou, Canquan

    2016-09-19

    Embryo culture media used for IVF treatment might affect fetal growth and thus birthweight of the newborns. A retrospective study was conducted in South China using data from 2370 singleton neonates born after IVF/ICSI between 2009 and 2012. Two culture media, i.e., either Vitrolife or SAGE were used as embryo culture media during the study period. Neonates' birthweights were compared between the two embryo culture media groups. Among the 2370 singletons, 1755 cases came from fresh cleavage embryo transfer while 615 were from frozen-thawed cleavage embryo transfer. Within the fresh embryo transfer newborns, no statistical difference was observed in either birthweight (mean ± SD: 3196.0 ± 468.9 versus 3168.4 ± 462.0g, p > 0.05) or adjusted birthweight controlled for gestational age and gender (z-score mean ± SD: 0.11 ± 1.02 versus 0.11 ± 0.99 g, P > 0.05) between the Vitrolife (n = 419) and the SAGE group (n = 1336). Likewise within frozen embryo transfer neotates, no statistical difference of the birthweight (3300.6 ± 441.3 vs.3256.0 ± 466.7 g, P > 0.05) and adjusted birthweight (0.30 ± 0.99 g versus 0.29 ± 0.97 g, P > 0.05) was found between the Vitrolife (n = 202) and the SAGE group (n = 413). The sex ratio [OR1.17, 95 % CI (0.94-1.46)/OR1.1, 95 % CI (0.78-1.54)], rate of small for gestational age [OR1.14, 95 % CI (0.82-1.59)/OR1.06, 95 % CI (0.56-2.02)] and large for gestational age [OR1.07, 95 % CI (0.64-1.76)/OR0.98, 95 % CI (0.47-2.02)] in fresh and frozen-thawed subgourps are all comparable respectively between the two culture media. No group differences were found in the rate of low birthweight and macosomia. Multiple linear regression analysis demonstrated that maternal weight, gestational age, frozen-thawed embryo transfer and infant gender were significantly related to neonatal birthweight (P cultured in SAGE or Vitrolife media after fresh or frozen-thawed cleavage

  2. Establishment of primary bovine intestinal epithelial cell culture and clone method.

    Science.gov (United States)

    Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

    2017-01-01

    The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

  3. In Vitro Fertilization and Embryo Culture Strongly Impact the Placental Transcriptome in the Mouse Model

    Science.gov (United States)

    Fauque, Patricia; Mondon, Françoise; Letourneur, Franck; Ripoche, Marie-Anne; Journot, Laurent; Barbaux, Sandrine; Dandolo, Luisa; Patrat, Catherine; Wolf, Jean-Philippe; Jouannet, Pierre; Jammes, Hélène; Vaiman, Daniel

    2010-01-01

    Background Assisted Reproductive Technologies (ART) are increasingly used in humans; however, their impact is now questioned. At blastocyst stage, the trophectoderm is directly in contact with an artificial medium environment, which can impact placental development. This study was designed to carry out an in-depth analysis of the placental transcriptome after ART in mice. Methodology/Principal Findings Blastocysts were transferred either (1) after in vivo fertilization and development (control group) or (2) after in vitro fertilization and embryo culture. Placentas were then analyzed at E10.5. Six percent of transcripts were altered at the two-fold threshold in placentas of manipulated embryos, 2/3 of transcripts being down-regulated. Strikingly, the X-chromosome harbors 11% of altered genes, 2/3 being induced. Imprinted genes were modified similarly to the X. Promoter composition analysis indicates that FOXA transcription factors may be involved in the transcriptional deregulations. Conclusions For the first time, our study shows that in vitro fertilization associated with embryo culture strongly modify the placental expression profile, long after embryo manipulations, meaning that the stress of artificial environment is memorized after implantation. Expression of X and imprinted genes is also greatly modulated probably to adapt to adverse conditions. Our results highlight the importance of studying human placentas from ART. PMID:20169163

  4. In vitro fertilization and embryo culture strongly impact the placental transcriptome in the mouse model.

    Directory of Open Access Journals (Sweden)

    Patricia Fauque

    Full Text Available BACKGROUND: Assisted Reproductive Technologies (ART are increasingly used in humans; however, their impact is now questioned. At blastocyst stage, the trophectoderm is directly in contact with an artificial medium environment, which can impact placental development. This study was designed to carry out an in-depth analysis of the placental transcriptome after ART in mice. METHODOLOGY/PRINCIPAL FINDINGS: Blastocysts were transferred either (1 after in vivo fertilization and development (control group or (2 after in vitro fertilization and embryo culture. Placentas were then analyzed at E10.5. Six percent of transcripts were altered at the two-fold threshold in placentas of manipulated embryos, 2/3 of transcripts being down-regulated. Strikingly, the X-chromosome harbors 11% of altered genes, 2/3 being induced. Imprinted genes were modified similarly to the X. Promoter composition analysis indicates that FOXA transcription factors may be involved in the transcriptional deregulations. CONCLUSIONS: For the first time, our study shows that in vitro fertilization associated with embryo culture strongly modify the placental expression profile, long after embryo manipulations, meaning that the stress of artificial environment is memorized after implantation. Expression of X and imprinted genes is also greatly modulated probably to adapt to adverse conditions. Our results highlight the importance of studying human placentas from ART.

  5. Composition of single-step media used for human embryo culture.

    Science.gov (United States)

    Morbeck, Dean E; Baumann, Nikola A; Oglesbee, Devin

    2017-04-01

    To determine compositions of commercial single-step culture media and test with a murine model whether differences in composition are biologically relevant. Experimental laboratory study. University-based laboratory. Inbred female mice were superovulated and mated with outbred male mice. Amino acid, organic acid, and ions content were determined for single-step culture media: CSC, Global, G-TL, and 1-Step. To determine whether differences in composition of these media are biologically relevant, mouse one-cell embryos were cultured for 96 hours in each culture media at 5% and 20% oxygen in a time-lapse incubator. Compositions of four culture media were analyzed for concentrations of 30 amino acids, organic acids, and ions. Blastocysts at 96 hours of culture and cell cycle timings were calculated, and experiments were repeated in triplicate. Of the more than 30 analytes, concentrations of glucose, lactate, pyruvate, amino acids, phosphate, calcium, and magnesium varied in concentrations. Mouse embryos were differentially affected by oxygen in G-TL and 1-Step. Four single-step culture media have compositions that vary notably in pyruvate, lactate, and amino acids. Blastocyst development was affected by culture media and its interaction with oxygen concentration. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  6. In vitro equine embryo production using air-dried spermatozoa, with different activation protocols and culture systems.

    Science.gov (United States)

    Alonso, A; Baca Castex, C; Ferrante, A; Pinto, M; Castañeira, C; Trasorras, V; Gambarotta, M C; Losinno, L; Miragaya, M

    2015-05-01

    The aim of this work was to evaluate the use of air-dried spermatozoa for in vitro production of equine embryos and verify if sperm extract activation and in vivo culture improve in vitro embryo production. Cooled spermatozoa (control) and air-dried spermatozoa stored for 2, 14 or 28 days were used for ICSI sperm extract, or ionomycin was used for oocyte activation, and embryos were in vitro or in vivo (in mare's oviduct) cultured for 7 days. With in vitro culture, cleavage rate was higher when activating with sperm extract (P  0.05). Blastocysts were obtained with cooled spermatozoa, and morulae were achieved using in vivo culture with 28-day storage spermatozoa and ionomycin-activated oocytes. When in vivo culture was performed, sperm DNA fragmentation was assessed using the sperm chromatin dispersion test and did not show statistical correlation with cleavage nor embryo recovery rates. In conclusion, equine embryos can be produced using air-dried spermatozoa stored for several weeks. Sperm extract activation increased cleavage rates but did not improve embryo development. In vivo culture allowed intrauterine stage embryos to be achieved. © 2014 Blackwell Verlag GmbH.

  7. TERATOGENIC EFFECTS OF TRANSPLACENTAL TRANSFUSION OF HETEROLOGOUS ANTISERA SIMULATED IN AN EXPERIMENTAL-MODEL USING INVITRO WHOLE RAT EMBRYO CULTURE

    NARCIS (Netherlands)

    VANDERZEE, DC; POELMANN, RE; ZWIERSTRA, RP; MENTINK, MMT; VERMEIJKEERS, C

    1991-01-01

    The effects of the transplacental transfusion of heterologous rabbit-anti-rat antiserum (RAR antiserum) and subsequent immunological interaction on the development of 9-10 days old rat embryos (stages 8-10 somites) were studied using an in vitro whole rat embryo culture. Transplacental transfusion

  8. Minute changes to the culture environment of mouse pre-implantation embryos affect the health of the conceptus

    Directory of Open Access Journals (Sweden)

    George Koustas

    2016-07-01

    Conclusions: Exposing mouse pre-implantation embryos to ambient air at 37.0 °C, even for brief periods for routine micromanipulations is detrimental to normal embryonic development. Our results highlight the importance of how small alterations in the culture environment can have major consequences for the health of the embryo.

  9. Embryo quality and implantation rate in two different culture media: ISM1 versus Universal IVF Medium.

    Science.gov (United States)

    Xella, Susanna; Marsella, Tiziana; Tagliasacchi, Daniela; Giulini, Simone; La Marca, Antonio; Tirelli, Alessandra; Volpe, Annibale

    2010-04-01

    To compare the outcome of two different culture media marketed by the MediCult AS Company (Jyllinge, Denmark)-Universal IVF Medium and ISM1 Medium culture-which, in addition to glucose, pyruvate, and energy-providing components, also contain amino acids, nucleotides, vitamins, and cholesterol. Laboratory and retrospective clinical study. University teaching hospital. A total of 726 patients, undergoing IVF-intracytoplasmic sperm injection procedure, comparable in mean age range, oocyte retrieval, and infertility indication, were included in the study. Laboratory quality and standard procedures were maintained unaffected. Oocyte retrieval, different embryo culture media. Embryo quality, ongoing pregnancy, and implantation rate. The frequency of good-quality embryos (79% vs. 74%) and the percentages of ongoing pregnancy (27.5% vs. 18%) and implantation rate (15% vs. 10%) were significantly higher in the group treated with ISM1 Medium rather than Universal IVF Medium. ISM1 Medium culture seems to improve the performance of embryonic growth and development, as well as increasing the percentage of pregnancy. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  10. Volatiles identified from five stages of embryo development separated from a heterogeneous suspension culture of Daucus carota.

    Science.gov (United States)

    Kennedy, A H; Chamberlain, D; Wilson, G; Ryan, M F

    1991-11-01

    Five stages of embryo development were fractionated from a mature culture of Daucus carota (Gelbe Rheinsche), using a series of metal sieves. The composition of the population of embryos in each fraction was determined quantitatively from microscopic investigations. Volatiles from samples of tissue from six stages of development were trapped on activated charcoal cartridges. These volatiles, some of which may play a significant role in the interaction of the plant with the carrot root fly (Psila rosae), were analysed using gas chromatography/mass spectroscopy. The resulting chromatograms are arranged in order of embryo development. The progressive elaboration of the volatile profile reflects the increased biosynthetic capacity of the developing embryo.

  11. Evaluation of Quantity and Hyperhydricity of Cocoa Somatic Embryo Obtained from Solid Culture, Liquid Culture, and Sequence Subculture

    Directory of Open Access Journals (Sweden)

    Sulistyani Pancaningtyas

    2013-05-01

    Full Text Available Research  aimed  to  study  the  effect  of  solid  culture,  liquid  culture,  and sequence  subculture  on  quantity  and  hyperhydricity  of  somatic  embryo  wascarried  out  at  Laboratory  of  Biotechnology,  Indonesian  Coffee  and  Cocoa Research  Institute.  Materials  used  in  this  study  were  embryogenic  callitransferred  on  somatic  embryos  expression  both  in  solid  and  liquid  media with  the  same  media  composition,  namely  MS  medium  with  the  addition  ofAdenine  (0.025  mg/L.  Gelling  agent  used  in  solid  media  was  gelrite  (3  g/L. Clones used in this study was Sca 6. This research consisted of two trials, namely1  effect  of  medium  type  (solid  and  liquid,  and  2  sequence  subculture  (four subcultures.  This  results  showed  that  the  production  of  somatic  embryosin  liquid  medium  was  higher  than  in  the  solid  medium.  Regeneration  of somatic  embryos  on  solid  medium  culture  showed  the  highest  percentage  of abnormality  embryos  due  to  hyperhydricity  at  the  cotyledonary  phase  60%. Meanwhile,  the  regeneration  of  somatic  embryos  in  liquid  culture  showed the  highest  percentage  of  abnormality  embryos  due  to   hyperhydricity  at the  globular  and  cotyledonary  phase  37%.  Frequent  subculture  increased abnormal embryos  and  decreased  the  number of  somatic  embryos.Key words: Cacao, hyperhydricity,  somatic embryos,  solid  culture,  liquid  culture,  subculture,  in  vitro.

  12. Purine nucleotide synthesis in cultured rat embryos undergoing organogenesis

    International Nuclear Information System (INIS)

    Rowe, P.B.; Kalaizis, A.; McEwen, S.E.

    1986-01-01

    The authors show that de n ovo synthesis is the sole source of the purine nucleotides required for in vitro rat embryonic growth during organogenesis. The presence of high levels of activity of purine catabolic enzymes in the homologous serum essential for culture prohibits the salvage of purine. While the 3-carbon atom of serine is the major source of one carbon units for purine ring synthesis there is a significant contribution from the 2-ring carbon atom of tryptophan. The paper describes in detail the incorporation of (1-14C)glycine into the acid soluble phase and other processes connected with de novo purine synthesis

  13. Platelet-rich plasma can replace fetal bovine serum in human meniscus cell cultures

    NARCIS (Netherlands)

    Gonzales, V.K.; Mulder, E.L.W. de; Boer, T. den; Hannink, G.; Tienen, T.G. van; Heerde, W.L. van; Buma, P.

    2013-01-01

    Concerns over fetal bovine serum (FBS) limit the clinical application of cultured tissue-engineered constructs. Therefore, we investigated if platelet-rich plasma (PRP) can fully replace FBS for meniscus tissue engineering purposes. Human PRP and platelet-poor plasma (PPP) were isolated from three

  14. Growth and development of rabbit oocytes in vitro: effect of fetal bovine serum concentration on culture medium.

    Science.gov (United States)

    Sugimoto, H; Kida, Y; Miyamoto, Y; Kitada, K; Matsumoto, K; Saeki, K; Taniguchi, T; Hosoi, Y

    2012-09-15

    The objective was to develop a culture system that produced blastocyst stage embryos from rabbit oocytes grown in vitro. Two experiments were performed. First, various concentrations of fetal bovine serum (FBS, 0, 0.05, 0.5 and 5%) were used in the culture medium for in vitro growth (IVG) of oocytes recovered from follicles 200 to 299 μm in diameter. Intracytoplasmic sperm injection (ICSI) was performed on mature oocytes obtained after IVG for 8 days and in vitro maturation for 14 to 16 h. Rates of survival and pronuclear formation after ICSI were higher for oocytes grown in a medium with 0.05% FBS compared to oocytes grown in a medium lacking FBS (97.6 vs. 76.9%, 97.5 vs. 70%, P medium containing 0.05% FBS than in the medium lacking FBS (9.5 vs. 17.9%, P medium for IVG improved developmental competence of rabbit oocytes grown in vitro. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Toxicity of Polychlorinated Diphenyl Ethers in Hydra Attenuata and in Rat Whole Embryo Culture

    Science.gov (United States)

    1991-05-01

    teratogenicity using the Hydra attenuata system, a screening test for the rapid detection and prioritization of developmental toxins (Johnson et al...validate other delivery systems for use with the hydra assay. 67 Rat whole embryo culture provides the opportunity to study the effects of a toxin on a...DATE 3. REPORT TYPE AND DATES COVERED May 1991 THESIS/ &I 4. TITLE AND SUBTITLE 5. FUNDING NUMBERS Toxicity of Polychlorinated Diphenyl Ethers in Hydra

  16. Evaluation of the developmental toxicity of citrinin using Hydra attenuata and postimplantation rat whole embryo culture.

    Science.gov (United States)

    Yang, Y G; Mayura, K; Spainhour, C B; Edwards, J F; Phillips, T D

    1993-12-31

    Citrinin (a mycotoxin produced as a frequent contaminant of food and feed by numerous species of Aspergillus and Penicillium fungi) is embryo/fetotoxic and embryocidal in mice and rats. The present study was designed to examine whether the in vivo observed developmental toxicity of citrinin could be recapitulated using the Hydra attenuata (HA) bioassay and then be confirmed in rat whole embryo culture (WEC). Results from the HA assay indicated that the minimal affective concentrations of citrinin required to elicit a toxic response in the adult hydra (MACA) and in the regenerating hydra (MACD) were 30 mg/l and 20 mg/l, respectively. The Hydra developmental hazard index (A/D ratio) was equal to 1.5, classifying citrinin as a coaffective developmental toxin. In WEC, rat embryos were cultured in homologous (rat) serum containing citrinin at various concentrations ranging from 0.0 and 300 micrograms/ml for a period of 45 h. The results indicated a concentration-dependent reduction in yolk sac diameter, crown-rump length, somite number, protein and DNA contents. No embryonic dysmorphogenesis was observed in any treatment group. Histological examination revealed severe diffuse mesodermal and ectodermal necrosis in embryos treated with 250 micrograms/ml citrinin. At lower concentrations of citrinin, embryos were neither grossly nor histologically different from controls. Both the HA and WEC bioassays demonstrated that citrinin is not a primary developmental toxin. The use of HA and WEC bioassays in tandem may facilitate the rapid detection and ranking of the developmental hazards of food and feedborne mycotoxins.

  17. Different Donor Cell Culture Methods Can Influence the Developmental Ability of Cloned Sheep Embryos.

    Directory of Open Access Journals (Sweden)

    LiBing Ma

    Full Text Available It was proposed that arresting nuclear donor cells in G0/G1 phase facilitates the development of embryos that are derived from somatic cell nuclear transfer (SCNT. Full confluency or serum starvation is commonly used to arrest in vitro cultured somatic cells in G0/G1 phase. However, it is controversial as to whether these two methods have the same efficiency in arresting somatic cells in G0/G1 phase. Moreover, it is unclear whether the cloned embryos have comparable developmental ability after somatic cells are subjected to one of these methods and then used as nuclear donors in SCNT. In the present study, in vitro cultured sheep skin fibroblasts were divided into four groups: (1 cultured to 70-80% confluency (control group, (2 cultured to full confluency, (3 starved in low serum medium for 4 d, or (4 cultured to full confluency and then further starved for 4 d. Flow cytometry was used to assay the percentage of fibroblasts in G0/G1 phase, and cell counting was used to assay the viability of the fibroblasts. Then, real-time reverse transcription PCR was used to determine the levels of expression of several cell cycle-related genes. Subsequently, the four groups of fibroblasts were separately used as nuclear donors in SCNT, and the developmental ability and the quality of the cloned embryos were compared. The results showed that the percentage of fibroblasts in G0/G1 phase, the viability of fibroblasts, and the expression levels of cell cycle-related genes was different among the four groups of fibroblasts. Moreover, the quality of the cloned embryos was comparable after these four groups of fibroblasts were separately used as nuclear donors in SCNT. However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability. The results of the present study indicate that there are synergistic effects of full confluency and serum starvation on arresting fibroblasts in

  18. Different Donor Cell Culture Methods Can Influence the Developmental Ability of Cloned Sheep Embryos.

    Science.gov (United States)

    Ma, LiBing; Liu, XiYu; Wang, FengMei; He, XiaoYing; Chen, Shan; Li, WenDa

    2015-01-01

    It was proposed that arresting nuclear donor cells in G0/G1 phase facilitates the development of embryos that are derived from somatic cell nuclear transfer (SCNT). Full confluency or serum starvation is commonly used to arrest in vitro cultured somatic cells in G0/G1 phase. However, it is controversial as to whether these two methods have the same efficiency in arresting somatic cells in G0/G1 phase. Moreover, it is unclear whether the cloned embryos have comparable developmental ability after somatic cells are subjected to one of these methods and then used as nuclear donors in SCNT. In the present study, in vitro cultured sheep skin fibroblasts were divided into four groups: (1) cultured to 70-80% confluency (control group), (2) cultured to full confluency, (3) starved in low serum medium for 4 d, or (4) cultured to full confluency and then further starved for 4 d. Flow cytometry was used to assay the percentage of fibroblasts in G0/G1 phase, and cell counting was used to assay the viability of the fibroblasts. Then, real-time reverse transcription PCR was used to determine the levels of expression of several cell cycle-related genes. Subsequently, the four groups of fibroblasts were separately used as nuclear donors in SCNT, and the developmental ability and the quality of the cloned embryos were compared. The results showed that the percentage of fibroblasts in G0/G1 phase, the viability of fibroblasts, and the expression levels of cell cycle-related genes was different among the four groups of fibroblasts. Moreover, the quality of the cloned embryos was comparable after these four groups of fibroblasts were separately used as nuclear donors in SCNT. However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability. The results of the present study indicate that there are synergistic effects of full confluency and serum starvation on arresting fibroblasts in G0/G1 phase

  19. The role of matrix metalloproteinase-2 in the culture media in embryo implantation rate in normogonadotrophic cases undergoing ICSI

    Directory of Open Access Journals (Sweden)

    Yasser Ibrahim Orief

    2013-12-01

    Recommendations: Results of the present study suggest searching for other markers in the culture media for better embryo selection and for prediction of implantation in the normogonadotrophic cases undergoing ICSI.

  20. Effects of varying chamber construction and embryo pre-incubation age on survival and growth of chick embryos in shell-less culture.

    Science.gov (United States)

    Dunn, B E; Fitzharris, T P; Barnett, B D

    1981-01-01

    Shell-less culture involves culturing chick embryos with associated yolk and albumen outside of the eggshell and shell membranes. The technique allows direct access to and continuous observation of cultured embryos almost to the time of hatching. The plastic wrap/culture tripod technique described in this paper allows normal embryonic growth and differentiation from 48 hours (in ovo pre-incubation age) through at least 10 days of total incubation. As the duration of in ovo pre-incubation is decreased below 36 hours, there is a concomitant increase in the percentage of grossly abnormal embryos associated with decreased survival and retarded growth and differentiation. Survival of embryos pre-incubated for 72 hours through 13 and 18 days of total incubation is greater than 80% and 40%, respectively. The wrap/tripod technique allows substantially better survival and more normal development of cultured embryos than does the petri dish technique of Auerbach et al. (1974). Embryonic growth in 7.8-cm-diameter chambers is significantly greater than in either larger (10.6 cm) or smaller (5.2 cm) diameter chambers. Some gas exchange through the culture chamber walls appears necessary for optimal embryonic survival and growth. Suspending egg contents in either Safeway or Handi-Wrap plastic wrap (both of intermediate permeability) in 7.8-cm tripods resulted in superior growth and/or survival compared to suspension in Silastic sheeting (high permeability), Saran Wrap (low permeability), glass dishes (nonpermeable), or glass dishes lined with an inner layer of Safeway wrap(nonpermeable).

  1. Developmental regulation and modulation of apoptotic genes expression in sheep oocytes and embryos cultured in vitro with L-carnitine.

    Science.gov (United States)

    Mishra, A; Reddy, I J; Gupta, Psp; Mondal, S

    2016-12-01

    The objective of this study was to find out the impact of L-carnitine (10 mM) on developmental regulation of preimplantation sheep embryos cultured in vitro when supplemented in maturation medium and post-fertilization medium separately. Subsequent objective was to observe the L-carnitine-mediated alteration in expression of apoptotic genes (Bcl2, Bax, Casp3 and PCNA) in sheep oocytes and developing embryos produced in vitro. Oocytes matured with L-carnitine showed significantly (p embryos cultured with actinomycin D and TNFα showed developmental arrest with significant (p embryo development and supplementation of L-carnitine during IVM altered the expression of apoptotic genes in the developmental stages of embryos. © 2016 Blackwell Verlag GmbH.

  2. Whole embryo culture: a "New" technique that enabled decades of mechanistic discoveries.

    Science.gov (United States)

    Ellis-Hutchings, Robert G; Carney, Edward W

    2010-08-01

    Denis New's development of the rodent whole embryo culture (WEC) method in the early 1960s was a groundbreaking achievement that gave embryologists and teratologists an unprecedented degree of access to the developing postimplantation rodent embryo. In the five decades since its development, WEC has enabled detailed investigations into the regulation of normal embryo development as well as a plethora of research on mechanisms of teratogenesis as induced by a wide range of agents. In addition, WEC is one of the few techniques that has been validated for use in teratogenicity screening of drugs and chemicals. In this review, we retrace the steps leading to New's development of WEC, and highlight many examples in which WEC played a crucial role leading to important discoveries in teratological research. The impact of WEC on the field of teratology has been enormous, and it is anticipated that WEC will remain a preferred tool for teratologists and embryologists seeking to interrogate embryo development for many years to come. Copyright 2010 Wiley-Liss, Inc.

  3. 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...

  4. Use of cultured rat embryos to evaluate the teratogenic activity of serum: cadmium and cyclophosphamide. [Serum-based culture media for growing rat embryos is used to determine the teratogenicity of cadmium and cyclophosphamide

    Energy Technology Data Exchange (ETDEWEB)

    Klein, N. W.; Vogler, M. A.; Chatot, C. L.; Pierro, L. J.

    1979-01-01

    Head fold stage rat embryos were cultured for 48 hrs in vitro on serum taken at various intervals from rats that had been injected ip with either cadmium or cyclophosphamide. Their response was compared to that of embryos cultured for the same period on control serum to which these substances were added directly. One and 4 hr sera from cadmium injected rats (2.13 mg Cd/sup + +//kg) were lethal. Eight hr serum allowed survival but embryos were exencephalic and contained reduced amounts of protein and DNA. The response to direct cadmium was characteristically different and was related to dosage and the extent to which zero-time embryos had progressed through the head fold stage. At 1.6 ..mu..M, Cd/sup + +/-susceptible embryos were hemorrhagic but not exencephalic. One hr serum from rats given cyclophosphamide (180 mg/kg) was lethal. On 4 hr serum, embryos survived but were exencephalic and contained less protein and DNA than controls. Embryos were resistant to direct cyclophosphamide up to 800 ..mu..g per ml of medium. At this concentration, embryos appeared morphologically normal but contained reduced amounts of protein.

  5. Risk and prevention of bovine viral diarrhea virus (BVDV) transmission through embryo production via somatic cell nuclear transfer (SCNT) using oocytes from persistently infected donors.

    Science.gov (United States)

    Gregg, K; Riddell, K P; Chen, S H; Galik, P K; Xiang, T; Guerra, T; Marley, M S; Polejaeva, I; Givens, M D

    2010-07-01

    The objective was to assess the risk of transmission of bovine viral diarrhea virus (BVDV) through embryo production via somatic cell nuclear transfer (SCNT), with oocytes obtained from persistently infected (PI) donors. Using ultrasound-guided follicular aspiration following superstimulation, oocytes were obtained from five female beef cattle, including three that were PI and two that were negative for BVDV. In the three PI cattle, seven aspirations yielded 32 oocytes (PI-1: three aspirations yielding six oocytes; PI-2: two aspirations yielding 14 oocytes; and PI-3: two aspirations yielding 12 oocytes). The oocyte recovery rate was better in negative control cattle, with 32 oocytes obtained from the two cattle in a single superstimulation and aspiration session. Oocytes were processed individually for SCNT, evaluated, and tested for BVDV. Nearly all (31/32) oocytes from the three PI donors were positive for BVDV by PCR, with detected viral RNA copy number ranging from 1 to 1.1 x 10(5). The proportion of oocytes acceptable for SCNT embryo production (based on oocyte quality and maturation status) was only 16 to 35% from PI donors, but was 81% from control donors. Therefore, routine testing of unacceptable (discarded) oocytes could be an effective approach to identify batches that might contain infected oocytes from PI donors. Identification and removal of high-risk batches of oocytes would minimize the risk of BVDV transmission through SCNT embryo production.

  6. Birthweight distribution in ART singletons resulting from embryo culture in two different culture media compared with the national population.

    Science.gov (United States)

    Lemmen, J G; Pinborg, A; Rasmussen, S; Ziebe, S

    2014-10-10

    Is there a difference in birthweight distribution in ART singletons born after IVF culture in two different culture media? There is no effect of culture media on both crude and adjusted birthweight distributions in ART singletons from nulliparous mothers. Studies on human ART singletons have reported a difference in birthweight in singletons following IVF culture in different culture media. However, other studies comparing different culture media have not shown any significant differences in birthweight. This study was a retrospective comparison of birthweights in IVF/ICSI singletons conceived after fresh embryo transfer following embryo culture in Cook or Medicult medium and in a national cohort of naturally conceived singletons in nulliparous women. The study compares four independent groups consisting of singletons in nulliparous women from Cook-d2: 2-day culture in Cook medium at Rigshospitalet (n = 974), Medicult-d2: 2-day culture in Medicult EmbryoAssist medium at Rigshospitalet (n = 147), Medicult-d3: 3-day culture in Medicult EmbryoAssist medium with and without added GM-CSF (n = 204), and DK: pregnancies from the Danish birth registry (n = 106842). The study compares the birthweights of singletons from nulliparous women in the four independent groups mentioned above; Cook-d2: Medicult-d2: Medicult-d3: and DK. In addition, distributions of large and small for gestational age infants were compared between the groups and a multiple linear regression analysis was used to determine which factors determined birthweight. We found no significant difference in the crude birthweight distributions between singletons born after culture in Cook-d2 or Medicult-groups. Singleton girls from the Cook-d2 group weighed 3302 ± 28 g, versus 3252 ± 76 in the Medicult-d2 group (difference 50 g; P = 0.547). Singleton boys from the Cook-d2 group weighed 3430 ± 27 g, versus 3354 ± 56 in the Medicult-d2 group (difference 76 g; P = 0.279). In the background population, mean

  7. Maintenance ofMestimprinted methylation in blastocyst-stage mouse embryos is less stable than other imprinted loci following superovulation or embryo culture.

    Science.gov (United States)

    Velker, Brenna A M; Denomme, Michelle M; Krafty, Robert T; Mann, Mellissa R W

    2017-07-01

    Assisted reproductive technologies are fertility treatments used by subfertile couples to conceive their biological child. Although generally considered safe, these pregnancies have been linked to genomic imprinting disorders, including Beckwith-Wiedemann and Silver-Russell Syndromes. Silver-Russell Syndrome is a growth disorder characterized by pre- and post-natal growth retardation. The Mest imprinted domain is one candidate region on chromosome 7 implicated in Silver-Russell Syndrome. We have previously shown that maintenance of imprinted methylation was disrupted by superovulation or embryo culture during pre-implantation mouse development. For superovulation, this disruption did not originate in oogenesis as a methylation acquisition defect. However, in comparison to other genes, Mest exhibits late methylation acquisition kinetics, possibly making Mest more vulnerable to perturbation by environmental insult. In this study, we present a comprehensive evaluation of the effects of superovulation and in vitro culture on genomic imprinting at the Mest gene. Superovulation resulted in disruption of imprinted methylation at the maternal Mest allele in blastocysts with an equal frequency of embryos having methylation errors following low or high hormone treatment. This disruption was not due to a failure of imprinted methylation acquisition at Mest in oocytes. For cultured embryos, both the Fast and Slow culture groups experienced a significant loss of maternal Mest methylation compared to in vivo -derived controls. This loss of methylation was independent of development rates in culture. These results indicate that Mest is more susceptible to imprinted methylation maintenance errors compared to other imprinted genes.

  8. [Intravaginal culture and embryo transfer. A new method for the fertilization of human oocytes].

    Science.gov (United States)

    Ranoux, C; Dubuisson, J B; Foulot, H; Aubriot, F X

    1987-12-01

    This technique was developed at the University Clinic of Port-Royal. It corresponds to the intravaginal culture of embryos and their transfer into the uterus. After ovocyte stimulation, most often by Clomid HMG, the follicles are aspirated under laparoscopic or sonographic control 34 to 36 hours after HCG. After being collected, the ovocytes are placed, whatever their stage of maturity, in one or several 3 ml tubes completely filled with culture medium (B2 of pure Menezo). Up to 4 ovocytes per tube are thus fertilized with 10 to 20,000 mobile spermatozoids/ml, prepared in the usual dilution, centrifugation and migration. Then the tube(s) are placed in the posterior vaginal cul-de-sac, kept in place with a diaphragm where they will remain during the 44 to 48 hours of culture time. Following that time, the contents of the tube are examined in order to evaluate the occurrence and the stage of embryonic division. A first series of 100 aspirations has enabled to obtain 15 pregnancies, still evolving, including two births of healthy children. A randomized series is currently in progress to determine a possible difference in the rates of pregnancy between CIVETE and the classic technique. Beside its new psychological contribution, this technique has demonstrated that it was possible to culture human embryos in the absence of CO2; its extreme simplicity should lead to a broader expansion of this technique.

  9. Falha na sexagem por inibição do desenvolvimento de embri��es bovinos produzidos in vitro com anticorpos anti H-Y Failure of sexing by developmental arrest of bovine embryos in vitro produced with H-Y antisera

    Directory of Open Access Journals (Sweden)

    M.V. Resende

    2008-06-01

    Full Text Available Embriões bovinos produzidos in vitro, em estádio de mórula, foram cultivados em meio contendo anticorpos anti H-Y de alto título proveniente de ratos por 24h e, após este tempo, classificados em dois grupos: 1 embriões inibidos em estádio de mórula (classificados como machos e 2 embriões que se desenvolveram e formaram a blastocele (classificados como fêmeas. O sexo de 311 embriões, distribuídos em três grupos de concentração dos anticorpos, 3%, 5% ou 7%, foi identificado pela reação em cadeia da polimerase. Não houve desvio da proporção entre machos e fêmeas (P>0,05 nos grupos em que se utilizaram os anticorpos anti H-Y, quando comparadas ao grupo-controle, sem adição de anticorpos anti H-Y. Diferentemente dos resultados obtidos utilizando-se embriões bovinos produzidos in vivo, a sexagem com anticorpos anti H-Y de alto título em embriões produzidos in vitro não propiciou sucesso.In vitro produced bovine embryos at morula stage were cultured in medium containing high titer of rat H-Y antisera for 24h. The embryos were classified in two groups: 1 embryos arrested at morula stage (classified as males; and 2 embryos that developed and formed a blastocoele (classified as female. The sex of 311 embryos, divided in three groups of concentration of H-Y antisera, 3%, 5% or 7%, was identified by polimerase chain reaction. The results showed no difference (P>0.05 on sexual deviation in groups in which the H-Y antisera was added, in relation to control group, in which no H-Y antisera was added. In contrast with results obtained with in vivo produced bovine embryos, the sexing of in vitro produced bovine embryos with high H-Y antisera titer did not succed.

  10. Different effectiveness of closed embryo culture system with time-lapse imaging (EmbryoScope(TM)) in comparison to standard manual embryology in good and poor prognosis patients: a prospectively randomized pilot study.

    Science.gov (United States)

    Wu, Yan-Guang; Lazzaroni-Tealdi, Emanuela; Wang, Qi; Zhang, Lin; Barad, David H; Kushnir, Vitaly A; Darmon, Sarah K; Albertini, David F; Gleicher, Norbert

    2016-08-24

    Previously manual human embryology in many in vitro fertilization (IVF) centers is rapidly being replaced by closed embryo incubation systems with time-lapse imaging. Whether such systems perform comparably to manual embryology in different IVF patient populations has, however, never before been investigated. We, therefore, prospectively compared embryo quality following closed system culture with time-lapse photography (EmbryoScope™) and standard embryology. We performed a two-part prospectively randomized study in IVF (clinical trial # NCT92256309). Part A involved 31 infertile poor prognosis patients prospectively randomized to EmbryoScope™ and standard embryology. Part B involved embryos from 17 egg donor-recipient cycles resulting in large egg/embryo numbers, thus permitting prospectively alternative embryo assignments to EmbryoScope™ and standard embryology. We then compared pregnancy rates and embryo quality on day-3 after fertilization and embryologist time utilized per processed embryo. Part A revealed in poor prognosis patients no differences in day-3 embryo scores, implantation and clinical pregnancy rates between EmbryoScope™ and standard embryology. The EmbryoScope™, however, more than doubled embryology staff time (P embryology. Appropriately designed and powered prospectively randomized studies appear urgently needed in well-defined patient populations before the uncontrolled utilization of these instruments further expands. NCT02246309 Registered September 18, 2014.

  11. Improving Cotton Embryo Culture by Simulating In Ovulo Nutrient and Hormone Levels

    Energy Technology Data Exchange (ETDEWEB)

    Rodney Fuller; Vincent Liddiard; J. Hess; John Carman

    2011-06-01

    Plant ovules provide zygotes with a physicochemical environment that supports embryo differentiation, growth, and maturation. The exact nature of this embryogenesis-enabling environment is not well characterized, as evidenced by failed attempts to induce normal embryony from zygotes or proembryos (precotyledonary) on defined media. To identify factors required for cotton (Gossypium hirsutum L.) zygotic embryony in vitro, we previously performed chemical and dissolved oxygen tension analyses of cotton ovule fluids and tissues at multiple stages of embryony in situ. Based on these analyses, we report herein the development of procedures that normalize embryo differentiation, growth, maturation, and germination in vitro, starting with proembryos. Our medium differed from Murashige and Skoog (MS) medium as follows (percentage of MS): N (30%, mostly from ten amino acids), P (815%), K (237%), Mg (85%), Ca (267%), S (506%), Fe (88%), and myoinositol (883%). Levels of other MS nutrients and vitamins, except sucrose, were kept at MS levels. Additionally, we included 100 mg L-1 casein hydrolysate plus the following (mmol L-1): d-glucose (1.8), fructose (4.7), sucrose (62.0), arabinose (7.1), melibiose (3.5), malic acid (11.6), and citric acid (3.8). Mannitol was added to achieve a medium osmotic potential of -1.10 MPa, and an atmospheric O2 tension of 3.3 mol m-3 at the surface of embryos was maintained during culture. When cultured on medium containing 8.0 µmol L-1 indole-3-acetic acid, 80-90% of proembryos (as small as 100 cells) of cultivars HS-26 and B-27 increased four- to eightfold in surface area during the first 18 d in culture and germinated thereafter to produce viable plants. Increases in surface area of proembryos cultured on a modified MS medium previously used for somatic embryogenesis were from 0.2- to 0.6-fold. The described embryo culture medium should be useful for studying nutritional and molecular aspects of early embryony and possibly for plant zygote

  12. Comparative anatomy and morphology of Vitis vinifera (Vitaceae) somatic embryos from solid- and liquid-culture-derived proembryogenic masses.

    Science.gov (United States)

    Jayasankar, S; Bondada, Bhaskar R; Li, Zhijian; Gray, D J

    2003-07-01

    Ontogeny of somatic embryos of grapevine (Vitis vinifera) produced from solid- and liquid-culture-derived proembryogenic masses (PEM) was compared using light and scanning electron microscopy. Somatic embryos produced from solid-medium-derived PEM (SPEM) had large cotyledons, little or no visible suspensor structure, and a relatively undeveloped concave shoot apical meristem, whereas those from liquid-medium-derived PEM (LPEM) had smaller cotyledons, a distinct suspensor, and a flat-to-convex shoot apical meristem. The convex shoot apical meristem in LPEM-derived somatic embryos formed as early as the heart stage of development; it was 4-6 cell layers deep and rich in protein. Suspensors persisted in fully developed and mature LPEM-derived somatic embryos. The SPEM-derived somatic embryos exhibited dormancy, as do mature zygotic embryos, which also have a rudimentary suspensor, whereas LPEM-derived embryos were not dormant. We hypothesize that the presence of a persistent suspensor in LPEM-derived somatic embryos modulates development, ultimately resulting in rapid germination and a high plant-regeneration rate.

  13. Application of a metabolizing system as an adjunct to the rat whole embryo culture.

    Science.gov (United States)

    Luijten, Mirjam; Verhoef, Aart; Westerman, Anja; Piersma, Aldert H

    2008-08-01

    Rodent post-implantation whole embryo culture (WEC) is a validated and widely used method in both mechanistic studies and as a screening test for developmental toxicants. Since metabolism is lacking in the WEC because of the developmental stage of the embryo, pro-teratogenic compounds are not detected. We investigated the inclusion of an in vitro metabolizing system as a pre-incubation step in the existing WEC. We started by testing cyclophosphamide, a known pro-teratogen. Without metabolic activation, cyclophosphamide is not teratogenic to rat embryos, as evidenced by a lack of effect on either embryonic growth or on morphological development. After pre-incubation with Aroclor-induced hepatic microsomes, cyclophosphamide became highly embryotoxic in the WEC. We tested five additional compounds to prevalidate this method: valpromide, 2-acetylaminofluorene, 2-methoxyethanol, retinol, and benzo[a]pyrene. Results revealed that not all of these five compounds underwent (complete) metabolic activation. This is probably due to the fact that microsomes do not contain the full spectrum of hepatic enzymes. Attempts have been made to replace microsomes by S9 liver fractions, which do contain microsomal enzymes as well as a series of cytoplasmic enzymes. However, S9 appeared to be extremely toxic in the WEC. Future experiments have to be performed to explore alternative ways of complete metabolic activation.

  14. Comparative effects of sodium channel blockers in short term rat whole embryo culture

    Energy Technology Data Exchange (ETDEWEB)

    Nilsson, Mats F, E-mail: Mats.Nilsson@farmbio.uu.se [Department of Pharmaceutical Biosciences, Uppsala University (Sweden); Sköld, Anna-Carin; Ericson, Ann-Christin; Annas, Anita; Villar, Rodrigo Palma [AstraZeneca R and D Södertälje (Sweden); Cebers, Gvido [AstraZeneca R and D, iMed, 141 Portland Street, Cambridge, MA 02139 (United States); Hellmold, Heike; Gustafson, Anne-Lee [AstraZeneca R and D Södertälje (Sweden); Webster, William S [Department of Anatomy and Histology, University of Sydney (Australia)

    2013-10-15

    This study was undertaken to examine the effect on the rat embryonic heart of two experimental drugs (AZA and AZB) which are known to block the sodium channel Nav1.5, the hERG potassium channel and the L-type calcium channel. The sodium channel blockers bupivacaine, lidocaine, and the L-type calcium channel blocker nifedipine were used as reference substances. The experimental model was the gestational day (GD) 13 rat embryo cultured in vitro. In this model the embryonic heart activity can be directly observed, recorded and analyzed using computer assisted image analysis as it responds to the addition of test drugs. The effect on the heart was studied for a range of concentrations and for a duration up to 3 h. The results showed that AZA and AZB caused a concentration-dependent bradycardia of the embryonic heart and at high concentrations heart block. These effects were reversible on washout. In terms of potency to cause bradycardia the compounds were ranked AZB > bupivacaine > AZA > lidocaine > nifedipine. Comparison with results from previous studies with more specific ion channel blockers suggests that the primary effect of AZA and AZB was sodium channel blockage. The study shows that the short-term rat whole embryo culture (WEC) is a suitable system to detect substances hazardous to the embryonic heart. - Highlights: • Study of the effect of sodium channel blocking drugs on embryonic heart function • We used a modified method rat whole embryo culture with image analysis. • The drugs tested caused a concentration dependent bradycardia and heart block. • The effect of drugs acting on multiple ion channels is difficult to predict. • This method may be used to detect cardiotoxicity in prenatal development.

  15. Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture.

    Science.gov (United States)

    Miller-Pinsler, Lutfiya; Wells, Peter G

    2015-09-15

    Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat(b)/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug=GD 1), exposed for 24h to 2 or 4mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (pcatalase (PEG-cat) 8h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (pcatalase is a determinant of risk for EtOH embryopathies. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. 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.

  17. Effect of embryo culture media on percentage of males at birth.

    Science.gov (United States)

    Zhu, Jinliang; Zhuang, Xinjie; Chen, Lixue; Liu, Ping; Qiao, Jie

    2015-05-01

    Does embryo culture medium influence the percentage of males at birth? The percentage of males delivered after ICSI cycles using G5™ medium was statistically significantly higher than after cycles where Global, G5™ PLUS, and Quinn's Advantage Media were used. Male and female embryos have different physiologies during preimplantation development. Manipulating the energy substrate and adding growth factors have a differential impact on the development of male and female embryos. This was a retrospective analysis of the percentage of males at birth, and included 4411 singletons born from fresh embryo transfer cycles between January 2011 and August 2013 at the Center for Reproductive Medicine of Third Hospital Peking University. Only singleton gestations were included. Participants were excluded if preimplantation genetic diagnosis, donor oocytes and donor sperm were used. The database between January 2011 and August 2013 was searched with unique medical record number, all patients were present in the database with only one cycle. Demographics, cycle characteristics and the percentage of male babies in the four culture media groups were compared with analysis of variance or χ(2) tests. Multivariable logistic regression was done to determine the association between the sex at birth and culture media after adjusting for other confounding factors, including parental age, parental BMI, type of infertility, parity, number of embryos transferred, number of early gestational sacs, cycles with testicular sperm aspiration (TESA)/percutaneous epididymal sperm aspiration (PESA)/testicular sperm extraction (TESE), number of oocytes retrieved, cycles with blastocyst transfers, and gestational age within ICSI group. Within the IVF group, the percentage of males at birth for G5™, Global, Quinn's and G5™ PLUS media were comparable (P > 0.05); however, within the ICSI group, the percentage of male babies in cycles using G5™(56.1%) was statistically significantly higher than

  18. In vivo culture of bovine embryos and quality assesment of in vivo vs. in vitro produced embryos

    Czech Academy of Sciences Publication Activity Database

    Havlíček, V.; Lopatářová, M.; Čech, S.; Doležel, R.; Huber, T.; Pavlok, Antonín; Brem, G.; Besenfelder, U.

    2005-01-01

    Roč. 50, č. 4 (2005), s. 149-157 ISSN 0375-8427 R&D Projects: GA ČR(CZ) GA524/02/0674 Institutional research plan: CEZ:AV0Z50450515 Keywords : cattle Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.621, year: 2005

  19. Culture conditions and enzymatic passaging of bovine ESC-like cells.

    Science.gov (United States)

    Gong, Guochun; Roach, Marsha L; Jiang, Le; Yang, Xiangzhong; Tian, Xiuchun Cindy

    2010-04-01

    The goals of the current study were to (1) improve culture conditions and (2) chemical passaging of bovine embryonic stem cell-like (bESC-like) cells. Specifically, the effects of human leukemia inhibitory factor (hLIF), two types of feeders, mouse embryonic fibroblast (MEF) and bovine embryonic fibroblast (BEF), as well as three different enzymatic treatments including Trypsin-EDTA, TrypLE, and Liberase Blendzymes 3 were investigated. The addition of hLIF at 1000 U/mL to the culture medium (41.2 and 36.9%), and the use of either MEF or BEF feeders (40.3 and 38.1%) had no significant effect on the ability of inner cell masses (ICMs) to form primary cell colonies compared to controls. All bESC-like cells were first dissociated mechanically for three passages followed by enzymatic dissociation. The ability to maintain ESC morphology to passage 10 was compared among the three enzymes above. More bESC-like cell lines survived beyond passage 10 when treated with TrypLE compared to Trypson-EDTA (28.8 and 12.6%; p culture. When EBs were cultured on tissue culture plates, they differentiated into various cell types. In summary, we were able to culture bESC-like cells more than 10 passages by enzymatic dissociation, which is important in gene targeting, maintenance, and banking of bESC lines.

  20. Influence of embryo culture medium (G5 and HTF) on pregnancy and perinatal outcome after IVF: a multicenter RCT.

    Science.gov (United States)

    Kleijkers, Sander H M; Mantikou, Eleni; Slappendel, Els; Consten, Dimitri; van Echten-Arends, Jannie; Wetzels, Alex M; van Wely, Madelon; Smits, Luc J M; van Montfoort, Aafke P A; Repping, Sjoerd; Dumoulin, John C M; Mastenbroek, Sebastiaan

    2016-10-01

    Does embryo culture medium influence pregnancy and perinatal outcome in IVF? Embryo culture media used in IVF affect treatment efficacy and the birthweight of newborns. A wide variety of culture media for human preimplantation embryos in IVF/ICSI treatments currently exists. It is unknown which medium is best in terms of clinical outcomes. Furthermore, it has been suggested that the culture medium used for the in vitro culture of embryos affects birthweight, but this has never been demonstrated by large randomized trials. We conducted a multicenter, double-blind RCT comparing the use of HTF and G5 embryo culture media in IVF. Between July 2010 and May 2012, 836 couples (419 in the HTF group and 417 in the G5 group) were included. The allocated medium (1:1 allocation) was used in all treatment cycles a couple received within 1 year after randomization, including possible transfers with frozen-thawed embryos. The primary outcome was live birth rate. Couples that were scheduled for an IVF or an ICSI treatment at one of the six participating centers in the Netherlands or their affiliated clinics. The live birth rate was higher, albeit nonsignificantly, in couples assigned to G5 than in couples assigned to HTF (44.1% (184/417) versus 37.9% (159/419); RR: 1.2; 95% confidence interval (CI): 0.99-1.37; P = 0.08). Number of utilizable embryos per cycle (2.8 ± 2.3 versus 2.3 ± 1.8; P culture media on perinatal outcome remains to be determined. Embryo culture media used in IVF affect not only treatment efficacy but also perinatal outcome. This suggests that the millions of human embryos that are cultured in vitro each year are sensitive to their environment. These findings should lead to increased awareness, mechanistic studies and legislative adaptations to protect IVF offspring during the first few days of their existence. This project was partly funded by The NutsOhra foundation (Grant 1203-061) and March of Dimes (Grant 6-FY13-153). The authors declare no conflict of

  1. Identification of loci associated with embryo yield in microspore culture of Brassica rapa by segregation distortion analysis.

    Science.gov (United States)

    Kitashiba, Hiroyasu; Taguchi, Kumiko; Kaneko, Ikuyo; Inaba, Kiyofumi; Yokoi, Shuji; Takahata, Yoshihito; Nishio, Takeshi

    2016-10-01

    We identified three physical positions associated with embryo yield in microspore culture of Brassica rapa by segregation distortion analysis. We also confirmed their genetic effects on the embryo yield. Isolated microspore culture is well utilized for the production of haploid or doubled-haploid plants in Brassica crops. Brassica rapa cv. 'Ho Mei' is one of the most excellent cultivars in embryo yield of microspore culture. To identify the loci associated with microspore embryogenesis, segregation analysis of 154 DNA markers anchored to B. rapa chromosomes (A01-A10) was performed using a population of microspore-derived embryos obtained from an F1 hybrid between 'CR-Seiga', a low yield cultivar in microspore-derived embryos, and 'Ho Mei'. Three regions showing significant segregation distortion with increasing 'Ho Mei' alleles were detected on A05, A08 and A09, although these regions showed the expected Mendelian segregation ratio in an F2 population. The additive effect of alleles in these regions on embryo yield was confirmed in a BC3F1 population. One region on A08 containing Br071-5c had a higher effect than the other regions. Polymorphism of nucleotide sequences around the Br071-5c locus was investigated to find the gene possibly responsible for efficient embryogenesis from microspores.

  2. Antiviral effects of bovine interferons on bovine respiratory tract viruses.

    OpenAIRE

    Fulton, R W; Downing, M M; Cummins, J M

    1984-01-01

    The antiviral effects of bovine interferons on the replication of bovine respiratory tract viruses were studied. Bovine turbinate monolayer cultures were treated with bovine interferons and challenged with several bovine herpesvirus 1 strains, bovine viral diarrhea virus, parainfluenza type 3 virus, goat respiratory syncytial virus, bovine respiratory syncytial virus, bovine adenovirus type 7, or vesicular stomatitis virus. Treatment with bovine interferons reduced viral yield for each of the...

  3. Age of G-1 PLUS v5 embryo culture medium is inversely associated with birthweight of the newborn.

    Science.gov (United States)

    Kleijkers, Sander H M; van Montfoort, Aafke P A; Smits, Luc J M; Coonen, Edith; Derhaag, Josien G; Evers, Johannes L H; Dumoulin, John C M

    2015-06-01

    Does age of G-1 PLUS v5 embryo culture medium affect IVF outcome? Birthweight of singletons born after IVF showed an inverse association with age of the embryo culture medium, while no association was found between age of culture medium and fertilization rate, embryonic development or ongoing pregnancy. It has been reported that IVF culture media can deteriorate during storage, which suggests that the capacity of culture media to support optimal embryo development decreases over time. Some animal studies showed an effect of storage time on embryo development, in contrast to other studies, while the effect of aging culture medium on IVF outcome in humans is unknown. We used data on outcome of 1832 IVF/ICSI cycles with fresh embryo transfer, performed in the period 2008-2012 to evaluate the association of fertilization rate, embryonic development, ongoing pregnancy and birthweight of singletons with age of the culture medium (Vitrolife AB G-1 PLUS v5). Age of the culture medium was calculated by subtracting the production date from the date of ovum retrieval. Data analysis included linear regression and logistic regression on continuous and categorical outcomes, respectively. Age of the culture medium was not associated with fertilization rate (P = 0.543), early cleavage rate (P = 0.155), percentage of embryos containing four or more cells on Day 2 (P = 0.401), percentage of embryos containing eight or more cells on Day 3 (P = 0.175), percentage of embryos with multinucleated blastomeres (P = 0.527), or ongoing pregnancy (P = 0.729). However, birthweight of the newborn was inversely associated with age of the medium (β = -3.6 g, SE: 1.5 g, P = 0.021), after controlling for possible confounders (day of embryo transfer, number of transferred embryos, child's gender, gestational age at birth, parity, pregnancy complications, maternal smoking, height and weight, and paternal height and weight) and the association was not biased by year of treatment, time since first

  4. Does Embryo Culture Medium Influence the Health and Development of Children Born after In Vitro Fertilization?

    Science.gov (United States)

    Bouillon, Céline; Léandri, Roger; Desch, Laurent; Ernst, Alexandra; Bruno, Céline; Cerf, Charline; Chiron, Alexandra; Souchay, Céline; Burguet, Antoine; Jimenez, Clément; Sagot, Paul; Fauque, Patricia

    2016-01-01

    In animal studies, extensive data revealed the influence of culture medium on embryonic development, foetal growth and the behaviour of offspring. However, this impact has never been investigated in humans. For the first time, we investigated in depth the effects of embryo culture media on health, growth and development of infants conceived by In Vitro Fertilization until the age of 5 years old. This single-centre cohort study was based on an earlier randomized study. During six months, in vitro fertilization attempts (No. 371) were randomized according to two media (Single Step Medium—SSM group) or Global medium (Global group). This randomized study was stopped prematurely as significantly lower pregnancy and implantation rates were observed in the SSM group. Singletons (No. 73) conceived in the randomized study were included (42 for Global and 31 for SSM). The medical data for gestational, neonatal and early childhood periods were extracted from medical records and parental interviews (256 variables recorded). The developmental profiles of the children in eight domains (social, self-help, gross motor, fine motor, expressive language, language comprehension, letter knowledge and number knowledge – 270 items) were compared in relation to the culture medium. The delivery rate was significantly lower in the SSM group than in the Global group (p<0.05). The culture medium had no significant effect on birthweight, risk of malformation (minor and major), growth and the frequency of medical concerns. However, the children of the Global group were less likely than those of the SSM group to show developmental problems (p = 0.002), irrespective of the different domains. In conclusion, our findings showed that the embryo culture medium may have an impact on further development. PMID:27008092

  5. Altered methanol embryopathies in embryo culture with mutant catalase-deficient mice and transgenic mice expressing human catalase

    International Nuclear Information System (INIS)

    Miller, Lutfiya; Wells, Peter G.

    2011-01-01

    The mechanisms underlying the teratogenicity of methanol (MeOH) in rodents, unlike its acute toxicity in humans, are unclear, but may involve reactive oxygen species (ROS). Embryonic catalase, although expressed at about 5% of maternal activity, may protect the embryo by detoxifying ROS. This hypothesis was investigated in whole embryo culture to remove confounding maternal factors, including metabolism of MeOH by maternal catalase. C57BL/6 (C57) mouse embryos expressing human catalase (hCat) or their wild-type (C57 WT) controls, and C3Ga.Cg-Catb/J acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1), exposed for 24 h to 4 mg/ml MeOH or vehicle, and evaluated for functional and morphological changes. hCat and C57 WT vehicle-exposed embryos developed normally. MeOH was embryopathic in C57 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed and turning, whereas hCat embryos were protected. Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to C3H WT controls, suggesting that endogenous ROS are embryopathic. MeOH was more embryopathic in aCat embryos than WT controls, with reduced anterior neuropore closure and head length only in catalase-deficient embryos. These data suggest that ROS may be involved in the embryopathic mechanism of methanol, and that embryonic catalase activity may be a determinant of teratological risk.

  6. Male fertility status is associated with DNA methylation signatures in sperm and transcriptomic profiles of bovine preimplantation embryos.

    Science.gov (United States)

    Kropp, Jenna; Carrillo, José A; Namous, Hadjer; Daniels, Alyssa; Salih, Sana M; Song, Jiuzhou; Khatib, Hasan

    2017-04-05

    Infertility in dairy cattle is a concern where reduced fertilization rates and high embryonic loss are contributing factors. Studies of the paternal contribution to reproductive performance are limited. However, recent discoveries have shown that, in addition to DNA, sperm delivers transcription factors and epigenetic components that are required for fertilization and proper embryonic development. Hence, characterization of the paternal contribution at the time of fertilization is warranted. We hypothesized that sire fertility is associated with differences in DNA methylation patterns in sperm and that the embryonic transcriptomic profiles are influenced by the fertility status of the bull. Embryos were generated in vitro by fertilization with either a high or low fertility Holstein bull. Blastocysts derived from each high and low fertility bulls were evaluated for morphology, development, and transcriptomic analysis using RNA-Sequencing. Additionally, DNA methylation signatures of sperm from high and low fertility sires were characterized by performing whole-genome DNA methylation binding domain sequencing. Embryo morphology and developmental capacity did not differ between embryos generated from either a high or low fertility bull. However, RNA-Sequencing revealed 98 genes to be differentially expressed at a false discovery rate fertility bull derived embryos, and 33 genes were upregulated in low fertility derived embryos. Expression of the genes CYCS, EEA1, SLC16A7, MEPCE, and TFB2M was validated in three new pairs of biological replicates of embryos. The role of the differentially expressed gene TFB2M in embryonic development was further assessed through expression knockdown at the zygotic stage, which resulted in decreased development to the blastocyst stage. Assessment of the epigenetic signature of spermatozoa between high and low fertility bulls revealed 76 differentially methylated regions. Despite similar morphology and development to the blastocyst

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

    Directory of Open Access Journals (Sweden)

    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

  8. Proof of concept: preimplantation genetic screening without embryo biopsy through analysis of cell-free DNA in spent embryo culture media.

    Science.gov (United States)

    Shamonki, Mousa I; Jin, Helen; Haimowitz, Zachary; Liu, Lian

    2016-11-01

    To assess whether preimplantation genetic screening (PGS) is possible by testing for free embryonic DNA in spent IVF media from embryos undergoing trophectoderm biopsy. Prospective cohort analysis. Academic fertility center. Seven patients undergoing IVF and 57 embryos undergoing trophectoderm biopsy for PGS. On day 3 of development, each embryo was placed in a separate media droplet. All biopsied embryos received a PGS result by array comparative genomic hybridization. Preimplantation genetic screening was performed on amplified DNA extracted from media and results were compared with PGS results for the corresponding biopsy. [1] Presence of DNA in spent IVF culture media. [2] Correlation between genetic screening result from spent media and corresponding biopsy. Fifty-five samples had detectable DNA ranging from 2-642 ng/μL after a 2-hour amplification. Six samples with the highest DNA levels underwent PGS, rendering one result with a derivative log ratio SD (DLRSD) of media and a result that is consistent with trophectoderm biopsy. Improvements in DNA collection, amplification, and testing may allow for PGS without biopsy in the future. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  9. Dose-response analysis of phthalate effects on gene expression in rat whole embryo culture.

    Science.gov (United States)

    Robinson, Joshua F; Verhoef, Aart; van Beelen, Vincent A; Pennings, Jeroen L A; Piersma, Aldert H

    2012-10-01

    The rat postimplantation whole embryo culture (WEC) model serves as a potential screening tool for developmental toxicity. In this model, cultured rat embryos are exposed during early embryogenesis and evaluated for morphological effects. The integration of molecular-based markers may lead to improved objectivity, sensitivity and predictability of WEC in assessing developmental toxic properties of compounds. In this study, we investigated the concentration-dependent effects of two phthalates differing in potency, mono(2-ethylhexyl) phthalate (MEHP) and monomethyl phthalate (MMP, less toxic), on the transcriptome in WEC to examine gene expression in relation with dysmorphogenesis. MEHP was more potent than MMP in inducing gene expression changes as well as changes on morphology. MEHP induced significant enrichment of cholesterol/lipid/steroid (CLS) metabolism and apoptosis pathways which was associated with developmental toxicity. Regulation of genes within CLS metabolism pathways represented the most sensitive markers of MEHP exposure, more sensitive than classical morphological endpoints. As shown in direct comparisons with toxicogenomic in vivo studies, alterations in the regulation of CLS metabolism pathways has been previously identified to be associated with developmental toxicity due to phthalate exposure in utero. Our results support the application of WEC as a model to examine relative phthalate potency through gene expression and morphological responses. Additionally, our results further define the applicability domain of the WEC model for developmental toxicological investigations. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. 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...... from immature oocytes with or without serum supplementation, and cultured subsequently in defined culture medium (SOFaaci) with or without serum supplementation. Time-lapse images were recorded every 0.5 h throughout the culture period. More in vivo- than in vitro-derived zygotes developed...... at first cleavage (defined serum: 20-22% versus in vivo-derived: 8 P zygotes were 1-5 h shorter compared with those of in vitro-derived zygotes cultured under similar conditions (P

  11. Different in vitro culture systems affect the birth weight of lambs from vitrified ovine embryos.

    Science.gov (United States)

    Mara, L; Sanna, D; Dattena, M; Mayorga Muñoz, I M

    2015-02-01

    It has been reported that different in vitro culture systems affect the birth weight of lambs. The aim of this study was to test body weight and lambing rate of lambs born from five different in vitro culture systems after vitrification. Oocytes of Sarda sheep were matured in TCM-199 plus 0.4% bovine serum albumin (BSA) using systems: (i) 4 mg/ml fatty acid-free BSA (BSA4); (ii) 8 mg/ml fatty acid-free BSA (BSA8); (iii) BSA8-hyaluronan (BSA8-HA); (iv) BSA8-charcoal-stripped FBS (BSA8-CH); or (v) with 10% fetal bovine serum (FBS; serum) and fertilized with fresh semen. The presumptive zygotes were cultured up to the blastocyst stage with BSA8, BSA8-HA, BSA8-CH or serum or BSA4. In the third and fifth days of culture 5% charcoal-stripped FBS was added into BSA8-CH and serum, while 8 mg/ml or 4 mg/ml fatty acid-free BSA was added as BSA8, BSA8-HA and BSA4 respectively; 6 mg/ml HA was added to BSA8-HA. In total, 240 vitrified blastocysts were transferred into synchronized ewes. The lambing rate was not significant different between BSA groups or between serum groups (BSA8-CH and serum), while serum groups showed significant lower values when compared with BSA groups. Only BSA8 groups produced heavy lambs (≥4.5 kg) with a significant difference between BSA4 and BSA8 groups (P < 0.05).

  12. Desiccation-Enhanced Maturation and Germination of Date Palm Somatic Embryos Derived from Cell Suspension Culture.

    Science.gov (United States)

    Boufis, Nazim; Titouh, Khayreddine; Khelifi, Lakhdar

    2017-01-01

    In vitro plant regeneration via somatic embryogenesis is a powerful tool for rapid, large-scale production of healthy true-to-type plants. This approach is suitable to preserve existing natural genetic variability and propagation of variability generated from genetic improvement programs, including crossing, somaclonal variation, mutagenesis, and somatic hybridization. This chapter outlines a simplified protocol for date palm regeneration via somatic embryogenesis induced in cell suspension cultures. In this protocol, culture medium composition is manipulated, including plant growth regulators and solid (addition of agar) and liquid media to achieve reduction of production cycle of somatic embryogenesis, which increases the multiplication rate of embryogenic callus and improves the quantity and quality of somatic embryos.

  13. Oviduct-on-a-chip : Creating an in vitro oviduct to study bovine gamete interaction and early embryo development

    NARCIS (Netherlands)

    Ferraz, Marcia de Almeida Monteiro Melo

    2018-01-01

    The oviduct is host to the period in which the early embryo undergoes complete reprogramming of its (epi)genome in preparation for the reacquisition of epigenetic marks as differentiation proceeds. This reprogramming period is vulnerable to changes in environmental conditions, such as compromised

  14. Multiple-embryo transfer for studying very early maternal-embryo interactions in cattle.

    Science.gov (United States)

    Gómez, E; Muñoz, M

    2015-08-01

    In the present paper, we highlight the need to study very early maternal-embryo interactions and discuss how these interactions can be addressed. Bovine species normally carry one or, less frequently, two embryos to term; there are very rare cases of triplets or higher-order multiple pregnancies in which all the offspring are born alive. Multiple-embryo transfer (MET) in cattle allows for the detection of endometrial responses in scenarios where single-embryo transfer would not. Although MET is non-physiological, the present study shows that at the very early embryonic stages, a uterus carrying zona-enclosed embryos does not exhibit non-physiological reactions. On the contrary, MET should be considered the sum of multiple individual effects triggered by developing embryos. We provide arguments to support our hypothesis that describe a rationale for current work with MET, and we discuss alternative hypotheses. Using cattle as a model, we describe how technical approaches to analyzing zona-enclosed early embryo-maternal interactions (i.e., transcriptomics, proteomics, and endometrial cell culture) can help identify molecular changes that may be difficult to observe when only a single embryo is present. We conclude that MET can be used for studying very early maternal-embryo interactions in vivo in monotocous species. Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/150/2/R35/suppl/DC1. © 2015 Society for Reproduction and Fertility.

  15. Crossability studies and zygotic embryo culture in cassava (manihot esculenta crantz)

    International Nuclear Information System (INIS)

    Nunekpeku, W.

    2010-01-01

    Cassava (Manihot esculenta Crantz) germplasm in Ghana is mostly uncharacterized and includes a large collection of landraces variously suitable for specific end-uses at different locations across the country. None of the existing released varieties meets the requirements of an emerging local industry in starch production. In the absence of an active molecular genetic research group in the country to facilitate the incorporation of desired genes for high yield, high starch content and disease resistance into a single genotype, intra-specific hybridization remains a viable option in creating variability from which new varieties with a combination of the desired characteristics may be selected. Following a study of their phenological and reproductive characteristics, crosses were carried out among nine accessions of cassava (Megyewontem, Bamboo Akwetey, Ankra, BNARI Selection-1, Afisiafi, Security, Larbi, Asare and HO-008, abbreviated as ME, BA, AN, BS-1, AF, SE, LA, AS and HO-008 respectively). Flowering and fruiting characteristics differed significantly among the accessions. Percent crossability ranged from 0% (in AN x HO-008, AF x ME and LA x HO-008 crosses) to 88% (in AS x AF crosses). No clear relationship existed between seed set and embryo formation among the accessions. Fruit drop rate ranged from 11.7% to 83.3%. Zygotic embryos were harvested prior to seed maturity and cultured in vitro on phytohormone-free Murashige and Skoog medium to raise a collection of F 1 base population lines. In vitro germination rates of the hybrid embryos harvested at 45DAP ranged from 32.14% to 100%. Ex vitro acclimatization of 237 plantlets recovered from zygotic embryo cultures resulted in the survival of 35 hybrid progenies. These were grown for six months in a plant barn. Preliminary characterization of the hybrids with reference to above- and below-ground morphological traits, using IBPGR descriptors, revealed that they are generally similar in terms of pubescence of young

  16. Effects of embryo culture media do not persist after implantation: a histological study in mice.

    Science.gov (United States)

    Hemkemeyer, Sandra A; Schwarzer, Caroline; Boiani, Michele; Ehmcke, Jens; Le Gac, Séverine; Schlatt, Stefan; Nordhoff, Verena

    2014-02-01

    Is post-implantation embryonic development after blastocyst transfer affected by exposure to different assisted reproduction technology (ART) culture media? Fetal development and placental histology of ART embryos cultured in vitro in different ART media was not impaired compared with embryos grown in vivo. The application of different in vitro culture (IVC) media for human ART has an effect on birthweight of newborns. In the mouse model, differences in blastocyst formation were reported after culture in different ART media. Moreover, abnormalities in the liver and heart have been detected as a result of suboptimal IVC conditions. Fertilized oocytes from inbred and outbred breeding schemes were retrieved and either immediately transferred to foster mothers or incubated in control or human ART culture media up to the blastocyst stage prior to transfer. Placental and fetal anatomy and particularly bone development were evaluated. B6C3F1 female mice were used as oocyte donors after ovulation induction. C57Bl/6 and CD1 males were used for mating and CD1 females as foster mothers for embryo transfer. Fertilized oocytes were recovered from mated females and incubated in sequential human ART media (ISM1/ISM2 and HTF/Multiblast), in control media [KSOM(aa) and Whitten's medium] or grown in utero without IVC (zygote control). As in vivo, control B6C3F1 females were superovulated and left untreated. Fetuses and placentae were isolated by Caesarean section and analysed at 18.5 days post-coitum (dpc) for placenta composition and at 15.5 dpc for body weight, crown-rump length (CRL), fetal organ development, morphological development, total bone length and extent of bone ossification. No major differences in the number of implantation sites or in histological appearance of the placentae were detected. CRL of KSOM(aa) fetuses was higher compared with zygote control and Whitten's medium. Histological analysis of tissue sections revealed no gross morphological differences compared

  17. Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration

    DEFF Research Database (Denmark)

    Vajta, Gábor; Holm, Peter; Greve, Torben

    1996-01-01

    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...... and zona-dissected embryos did not differ (67 and 87%, respectively), and hatching was more frequent for blastocysts frozen in advanced developmental stages (34, 47 and 63% for early blastocysts, blastocysts and expanded blastocysts, respectively). The re-expansion rate of morulae was lower (10......%) 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...

  18. Efeito do citrato e taurina em meio CR2aa no desenvolvimento de embriões bovinos fecundados in vitro Effect of citrate and taurine added to CR2aa medium on the development of in vitro-fertilized bovine embryos

    Directory of Open Access Journals (Sweden)

    L.S.A. Camargo

    2009-02-01

    Full Text Available Avaliou-se o efeito do citrato em meio CR2aa suplementado com soro fetal bovino (SFB ou livre de proteínas séricas e sua associação com taurina no desenvolvimento de embriões bovinos fecundados in vitro. Embriões foram cultivados em CR2aa contendo 0, 0,5, 1,0 e 3,0mM citrato, suplementado com 10% SFB (experimento 1 ou com álcool polivinil (PVA; experimento 2. No terceiro experimento, embriões foram cultivados em meio com 0,5mM citrato, ou 7mM taurina, ou com a associação de ambos, suplementado com SFB. Os cultivos foram realizados com células do cumulus em ambiente a 38,8ºC com 5% de CO2 em ar atmosférico. Melhora no desenvolvimento embrionário foi observado no cultivo de embriões em CR2aa com 0,5 e 1,0mM citrato na ausência de SFB (P0,05 a produção de embriões ou o número de células. Citrato em meio CR2aa pode ser uma alternativa para cultivo embrionário em condições atmosféricas com 5% de CO2 em ar na ausência de proteína sérica.The effect of citrate added to CR2aa medium supplemented with fetal calf serum (FCS or serum-proteinfree and its association with taurine on the development of in vitro-fertilized bovine embryos was evaluated. Embryos were cultured with 0, 0.5, 1.0, and 3.0mM citrate, in CR2aa supplemented with 10% FCS (experiment 1, or polyvinyl alcohol (PVA; experiment 2. In experiment 3, embryos were cultured with 0.5mM citrate, 7.0mM taurine or with association of both, in medium supplemented with FCS. Embryo culture was performed with cumulus cells at 38.8ºC in 5% CO2 under air for all experiments. Positive effect on embryo development was only observed with 0.5 and 1.0mM citrate in FCS-free CR2aa (P0.05 embryo rate nor total cell number. Citrate in CR2aa medium can be an alternative for serumfree embryo culture under 5% CO2 in air, absence of serum protein.

  19. Peroxidized mineral oil increases the oxidant status of culture media and inhibits in vitro porcine embryo development.

    Science.gov (United States)

    Martinez, C A; Nohalez, A; Ceron, J J; Rubio, C P; Roca, J; Cuello, C; Rodriguez-Martinez, H; Martinez, E A; Gil, M A

    2017-11-01

    The use of oils with undetected alterations is a long-recognized problem for in vitro embryo production systems. Since peroxides in oils have been associated with reduced embryo production outcomes, our goals were (1) to evaluate the effects of a batch of mineral oil (MO) that was suspected to be altered on the in vitro production of pig embryos and (2) to determine oil peroxide values throughout culture and the transfer of oxidant agents from oil to culture media. Sunflower oil, which has a completely different chemical composition than MO but a higher oxidative status, and unaltered MO were used as controls. Oocyte maturation, fertilization and embryo development were affected differently depending on the oil overlay used. While the suspected MO was not able to sustain in vitro maturation and fertilization, the oocytes incubated in the presence of sunflower oil were matured and fertilized similarly to those of the unaltered MO group. Moreover, the cleavage rate of presumed zygotes cultured under the suspected MO was severely reduced compared with those cultured under the other oils, and none of the cleaved embryos developed to the blastocyst stage. Although the cleavage rates in the sunflower oil and unaltered MO groups were similar, embryos cultured under sunflower oil also failed to develop to the blastocyst stage. Our results revealed that the suspected MO and sunflower oil had similar levels of peroxides and that these levels were much higher than those of the unaltered MO. The total oxidant status was higher in media incubated under peroxidized oils than in fresh media or media incubated without an oil overlay or under unaltered MO, indicating that oxidant agents were transferred to the incubation media. However, unlike the sunflower oil group, the culture media incubated under the suspected MO had high levels of total oxidant status and low levels of hydrogen peroxide and reactive oxygen species, suggesting the presence of other unknown oxidant agents in

  20. Triazole induced concentration-related gene signatures in rat whole embryo culture.

    Science.gov (United States)

    Robinson, Joshua F; Tonk, Elisa C M; Verhoef, Aart; Piersma, Aldert H

    2012-09-01

    Commonly used as antifungal agents in agriculture and medicine, triazoles have been shown to cause teratogenicity in a diverse set of animal models. Here, we evaluated the dose-dependent impacts of flusilazole, cyproconazole and triadimefon, on global gene expression in relation to effects on embryonic development using the rat whole embryo culture (WEC) model. After 4 h exposure, we identified changes in gene expression due to triazole exposure which preceded morphological alterations observed at 48 h. In general, across the three triazoles, we observed similar directionality of regulation in gene expression and the magnitude of effects on gene expression correlated with the degree of induced developmental toxicity. Significantly regulated genes included key members of steroid/cholesterol and retinoic acid metabolism and hindbrain developmental pathways. Direct comparisons with previous studies suggest that triazole-gene signatures identified in the WEC overlap with zebrafish and mouse, and furthermore, triazoles impact gene expression in a similar manner as retinoic acid exposures in rat embryos. In summary, we further differentiate pathways underlying triazole-developmental toxicity using WEC and demonstrate the conservation of these response-pathways across model systems. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Incidence of Listeria species in bovine, ovine, caprine, camel and water buffalo milk using cultural method and the PCR assay

    OpenAIRE

    Rahimi, Ebrahim; Momtaz, Hassan; Behzadnia, Asma; Baghbadorani, Zeinab Torki

    2014-01-01

    Objective: To determine the prevalence rate of Listeria species in bovine, ovine, caprine, camel and water buffalo milk in Iran. Methods: From September 2010 to December 2011 a total of 260 bulk milk samples including 85 bovine, 37 camel, 34 water buffalo, 56 ovine and 48 caprine bulk milk samples were collected from commercial dairy herds, in Fars and Khuzestan provinces, Iran and were evaluated for the presence of Listeria species using cultural method and the PCR assay. R...

  2. Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture

    International Nuclear Information System (INIS)

    Miller-Pinsler, Lutfiya; Wells, Peter G.

    2015-01-01

    Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat b /J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1), exposed for 24 h to 2 or 4 mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p < 0.001). Maternal pretreatment of C57BL/6 WT dams with 50 kU/kg PEG-catalase (PEG-cat) 8 h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p < 0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p < 0.01), and trends for reduced anterior neuropore closure, turning and crown–rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p < 0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies. - Highlights: • Ethanol (EtOH) exposure causes structural embryopathies in embryo culture. • Genetically enhanced catalase (hCat) protects against EtOH embryopathies. • Genetically deficient catalase (aCat) exacerbates EtOH embryopathies. • Embryonic catalase is developmentally important. • EtOH developmental

  3. Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture

    Energy Technology Data Exchange (ETDEWEB)

    Miller-Pinsler, Lutfiya [Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Wells, Peter G., E-mail: pg.wells@utoronto.ca [Division of Biomolecular Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario (Canada); Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada)

    2015-09-15

    Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat{sup b}/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1), exposed for 24 h to 2 or 4 mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p < 0.001). Maternal pretreatment of C57BL/6 WT dams with 50 kU/kg PEG-catalase (PEG-cat) 8 h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p < 0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p < 0.01), and trends for reduced anterior neuropore closure, turning and crown–rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p < 0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies. - Highlights: • Ethanol (EtOH) exposure causes structural embryopathies in embryo culture. • Genetically enhanced catalase (hCat) protects against EtOH embryopathies. • Genetically deficient catalase (aCat) exacerbates EtOH embryopathies. • Embryonic catalase is developmentally important. • Et

  4. Growth inhibitory factors in bovine faeces impairs detection of Salmonella Dublin by conventional culture procedure

    DEFF Research Database (Denmark)

    Baggesen, Dorte Lau; Nielsen, L.R.; Sørensen, Gitte

    2007-01-01

    Aims: To analyse the relative importance of different biological and technical factors on the analytical sensitivity of conventional culture methods for detection of Salmonella Dublin in cattle faeces. Methods and Results: Faeces samples collected from six adult bovines from different salmonella...... novobiocin, followed by combinations of culture media (three types) and selective media (two types). The sensitivity of each combination and sources of variation in detection were determined by a generalized linear mixed model using a split-plot design. Conclusions: Biological factors, such as faecal origin...... and S. Dublin strain influenced the sensitivity more than technical factors. Overall, the modified semisolid Rappaport Vassiliadis (MSRV)-culture medium had the most reliable detection capability, whereas detection with selenite cystine broth and Mueller Kauffman tetrathionate broth combinations varied...

  5. A randomized clinical trial to evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium for in vitro fertilization

    DEFF Research Database (Denmark)

    Ziebe, Søren; Loft, Anne; Povlsen, Betina B

    2013-01-01

    To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium on ongoing implantation rate (OIR).......To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium on ongoing implantation rate (OIR)....

  6. A comparison of gene expression responses in rat whole embryo culture and in vivo: time-dependent retinoic acid-induced teratogenic response

    NARCIS (Netherlands)

    Robinson, J.F.; Verhoef, A.; Pennings, J.L.A.; Pronk, T.; Piersma, A.H.

    2012-01-01

    The whole embryo culture (WEC) model serves as a potential alternative for classical in vivo developmental toxicity testing. In the WEC, cultured rat embryos are exposed during neurulation and early organogenesis and evaluated for morphological effects. Toxicogenomic-based approaches may improve the

  7. Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos

    DEFF Research Database (Denmark)

    Tveden-Nyborg, Pernille Yde; Peura, T.T.; Hartwich, K.M.

    2005-01-01

    were produced by in vitro culture of in vivo fertilized zygotes. Most SCNT and IVC embryos were transferred to recipients on day 6 while some remained in culture for day 7 processing. In vivo embryos were collected as zygotes, transferred to intermediate recipients and retransferred to final recipients...

  8. [Effects of glucose concentration fluctuation on function of cultured bovine arterial endothelial cells].

    Science.gov (United States)

    Ye, Xi-yun; Tu, Qian; Tong, Zhi; Weng, Yu-jing; Wang, Yao-fa

    2010-03-01

    To explore the effects of glucose concentration fluctuation on function of cultured bovine arterial endothelial cells and underlying mechanism. The thoracic aorta of newborn calf was used for primary endothelial cells culture. Cells were divided into 3 groups and cultured for 48 h: control group (C, 5.5 mmol/L), constant high glucose group (HG, 30 mmol/L) and glucose fluctuation (GF, three circles of 2 h 30 mmol/L followed by 3 h 5.5 mmol/L, 30 mmol/L overnight, repeat the whole procedure on the following day) groups. The membranes fluidity of endothelial cells was detected by fluorescence polarization method. The contents of sorbierite, aldose reductase (AR), sorbitol dehydrogenase (SDH) and advanced glycation end products (AGEs) were measured. RAGE, eNOS and ET-1 mRNA expressions were detected by semi-quantitative RT-PCR. The membranes fluidity of endothelial cells in HG or GF group were significantly decreased compared with the control group (all P RAGE, eNOS and ET-1 were significantly upregulated compared with the control group (all P bovine arterial endothelial cells dysfunction than high glucose via activating polyols metabolic pathways, upregulating the expression of AGEs, eNOS and ET-1. Therefore, glucose concentration fluctuation might play a crucial role on macrovascular complications of diabetes.

  9. Phylogenetic characterization of bovine parainfluenza 3 from contaminated cell cultures and field isolates from Brazil

    OpenAIRE

    de Almeida Vaucher, Rodrigo; Dezen, Diogenes; Simonetti, Amauri Braga; Spilki, Fernando Rosado; Roehe, Paulo Michel

    2011-01-01

    Genomic fragments of the HN and L genes from Brazilian bovine parainfluenza 3 virus (bPIV-3) isolated as contaminants from cell cultures and clinical specimens were amplified by reverse transcription-polymerase chain reaction (RT-PCR), sequenced using specific degenerate primers and analyzed by phylogenetic comparison with reference strains of bPI3V. The Brazilian isolates revealed a high degree of genomic when compared to SF4/32 prototype strain, within the recently proposed genotype A of bP...

  10. Phylogenetic characterization of bovine parainfluenza 3 from contaminated cell cultures and field isolates from Brazil.

    Science.gov (United States)

    de Almeida Vaucher, Rodrigo; Dezen, Diogenes; Simonetti, Amauri Braga; Spilki, Fernando Rosado; Roehe, Paulo Michel

    2011-10-01

    Genomic fragments of the HN and L genes from Brazilian bovine parainfluenza 3 virus (bPIV-3) isolated as contaminants from cell cultures and clinical specimens were amplified by reverse transcription-polymerase chain reaction (RT-PCR), sequenced using specific degenerate primers and analyzed by phylogenetic comparison with reference strains of bPI3V. The Brazilian isolates revealed a high degree of genomic when compared to SF4/32 prototype strain, within the recently proposed genotype A of bPIV-3.

  11. Phylogenetic characterization of bovine parainfluenza 3 from contaminated cell cultures and field isolates from Brazil

    Directory of Open Access Journals (Sweden)

    Rodrigo de Almeida Vaucher

    2011-12-01

    Full Text Available Genomic fragments of the HN and L genes from Brazilian bovine parainfluenza 3 virus (bPIV-3 isolated as contaminants from cell cultures and clinical specimens were amplified by reverse transcription-polymerase chain reaction (RT-PCR, sequenced using specific degenerate primers and analyzed by phylogenetic comparison with reference strains of bPI3V. The Brazilian isolates revealed a high degree of genomic when compared to SF4/32 prototype strain, within the recently proposed genotype A of bPIV-3.

  12. Viral antigen production in cell cultures on microcarriers Bovine parainfluenza 3 virus and MDBK cells.

    Science.gov (United States)

    Conceição, M M; Tonso, A; Freitas, C B; Pereira, C A

    2007-11-07

    Viral antigens can be obtained from infected mammalian cells cultivated on microcarriers. We have worked out parameters for the production of bovine parainfluenza 3 (PI-3) virus by Mandin-Darby Bovine Kidney (MDBK) cells cultivated on Cytodex 1 microcarriers (MCs) in spinners flasks and bioreactor using fetal bovine serum (FBS) supplemented Eagle minimal essential medium (Eagle-MEM). Medium renewal during the cell culture was shown to be crucial for optimal MCs loading (>90% MCs with confluent cell monolayers) and cell growth (2.5 x 10(6)cells/mL and a micro(x) (h(-1)) 0.05). Since cell cultures performed with lower amount of MCs (1g/L), showed good performances in terms of cell loading, we designed batch experiments with a lower concentration of MCs in view of optimizing the cell growth and virus production. Studies of cell growth with lower concentrations of MCs (0.85 g/L) showed that an increase in the initial cell seeding (from 7 to 40 cells/MC) led to a different kinetic of initial cell growth but to comparable final cell concentrations ((8-10)x10(5)cells/mL at 120 h) and cell loading (210-270 cells/MC). Upon infection with PI-3 virus, cultures showed a decrease in cell growth and MC loading directly related to the multiplicity of infection (moi) used for virus infection. Infected cultures showed also a higher consumption of glucose and production of lactate. The PI-3 virus and PI-3 antigen production among the cultures was not significantly different and attained values ranging from, respectively, 7-9 log(10) TCID(50)/mL and 1.5-2.2 OD. The kinetics of PI-3 virus production showed a sharp increase during the first 24h and those of PI-3 antigen increased after 24h. The differential kinetics of PI-3 virus and PI-3 antigen can be explained by the virus sensitivity to temperature. In view of establishing a protocol of virus production and based on the previous experiments, MDBK cell cultures performed under medium perfusion in a bioreactor of 1.2L were infected

  13. Day-3 embryo metabolomics in the spent culture media is altered in obese women undergoing in vitro fertilization.

    Science.gov (United States)

    Bellver, José; De Los Santos, María J; Alamá, Pilar; Castelló, Damià; Privitera, Laura; Galliano, Daniela; Labarta, Elena; Vidal, Carmen; Pellicer, Antonio; Domínguez, Francisco

    2015-06-01

    To determine whether the global metabolomic profile of the spent culture media (SCM) of day-3 embryos is different in obese and normoweight women undergoing in vitro fertilization (IVF). Prospective cohort analysis. IVF clinic. Twenty-eight young, nonsmoking women with normoweight, nonsmoking male partners with mild/normal sperm factors undergoing a first IVF attempt for idiopathic infertility, tubal factor infertility, or failed ovulation induction: obese ovulatory women (n = 12); obese women with polycystic ovary syndrome (PCOS; n = 4); normoweight ovulatory women (n = 12). Fifty μl of SCM collected from two day-3 embryos of each cohort. Metabolomic profiling via ultrahigh performance liquid chromatography coupled to mass spectrometry of SCM from a total of 56 embryos. The untargeted metabolomic profile was different in obese and normoweight women. Partial least squares discriminant analysis resulted in a clear separation of samples when a total of 551 differential metabolites were considered. A prediction model was generated using the most consistent metabolites. Most of the metabolites identified were saturated fatty acids, which were detected in lower concentrations in the SCM of embryos from obese women. The metabolomic profile was similar in obese women with or without PCOS. The metabolomic profile in the SCM of day-3 embryos is different in normoweight and obese women. Saturated fatty acids seem to be reduced when embryos from obese patients are present. NCT01448863. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  14. The effect of supplementation of amino acids and taurine to modified KSOM culture medium on rat embryo development.

    Science.gov (United States)

    Nakamura, Kazuomi; Morimoto, Kayoko; Shima, Kaoru; Yoshimura, Yuki; Kazuki, Yasuhiro; Suzuki, Osamu; Matsuda, Junichiro; Ohbayashi, Tetsuya

    2016-11-01

    The rat is widely used as a laboratory animal for research. In particular, genetically engineered rats are essential for production of animal models of several diseases. Although embryo manipulation techniques are needed to produce them, such technology for rat preimplantation embryos is not as advanced as it is for mouse embryos. One reason is that in vitro culture systems for preimplantation embryos are limited in rats. Therefore, we intended to develop a new culture system for rat preimplantation embryos focusing on supplementation of amino acids as nutrition to the culture media. First, we found that taurine, glycine, glutamate, and alanine were abundant in the oviductal fluid of Wistar rats. The profile of taurine and these three amino acids was unchanged during the estrous cycle and from Days 0 to 3 of pregnancy (Day 0; vaginal plug was confirmed). Second, we assessed the effect of phosphate and phenol red on the development of rat zygotes and confirmed that they caused two-cell block. Third, we examined the effect of changing the medium on zygote development because addition of amino acids into culture medium causes ammonium accumulation, which is detrimental to embryo development. Blastocyst formation was suppressed in cultures with no medium change (P = 0.004; decreased to approximately one-fourth of that with medium change). Fourth, we examined the effect of supplementation of these three amino acids and taurine to modified potassium simplex optimized medium (KSOM). The zygote development rates were increased by the three amino acids and taurine in a concentration-dependent manner at 48, 72, and 96 hours (P = 0.001, 0.005, and 0.009, respectively) in culture. Finally, we confirmed that blastocysts cultured in modified KSOM had the capacity to develop to full term after implantation. These results showed that not only the supply of nutrients but also removal of wastes and toxicants is important for culture of rat preimplantation embryos. Copyright

  15. Dose–response analysis of phthalate effects on gene expression in rat whole embryo culture

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, Joshua F. [National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Department of Toxicogenomics, Maastricht University, Maastricht (Netherlands); Verhoef, Aart; Beelen, Vincent A. van; Pennings, Jeroen L.A. [National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Piersma, Aldert H., E-mail: aldert.piersma@rivm.nl [National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht (Netherlands)

    2012-10-01

    The rat postimplantation whole embryo culture (WEC) model serves as a potential screening tool for developmental toxicity. In this model, cultured rat embryos are exposed during early embryogenesis and evaluated for morphological effects. The integration of molecular-based markers may lead to improved objectivity, sensitivity and predictability of WEC in assessing developmental toxic properties of compounds. In this study, we investigated the concentration-dependent effects of two phthalates differing in potency, mono(2-ethylhexyl) phthalate (MEHP) and monomethyl phthalate (MMP, less toxic), on the transcriptome in WEC to examine gene expression in relation with dysmorphogenesis. MEHP was more potent than MMP in inducing gene expression changes as well as changes on morphology. MEHP induced significant enrichment of cholesterol/lipid/steroid (CLS) metabolism and apoptosis pathways which was associated with developmental toxicity. Regulation of genes within CLS metabolism pathways represented the most sensitive markers of MEHP exposure, more sensitive than classical morphological endpoints. As shown in direct comparisons with toxicogenomic in vivo studies, alterations in the regulation of CLS metabolism pathways has been previously identified to be associated with developmental toxicity due to phthalate exposure in utero. Our results support the application of WEC as a model to examine relative phthalate potency through gene expression and morphological responses. Additionally, our results further define the applicability domain of the WEC model for developmental toxicological investigations. -- Highlights: ► We examine the effect of two phthalates on gene expression and morphology in WEC. ► MEHP is more potent than MMP in inducing gene expression changes and dysmorphogenesis. ► MEHP significantly disrupts cholesterol metabolism pathways in a dose-dependent manner. ► Specific phthalate-related mechanisms in WEC are relevant to mechanisms in vivo.

  16. High frequency somatic embryogenesis and plant regeneration from zygotic embryo-derived callus cultures of three Allium species

    NARCIS (Netherlands)

    Valk, P. van der; Scholten, O.E.; Verstappen, F.; Jansen, R.C.; Dons, J.J.M.

    1992-01-01

    The plant regeneration ability of zygotic embryo-derived callus cultures was studied for 12 A. cepa varieties and accessions, two A. fistulosum varieties, one A. fistulosum × A. cepa interspecific hybrid and two A. porrum varieties. Compact embryogenic callus was induced on Murashige and Skoog (MS)

  17. Gene expression and DNA-methylation of bovine pretransfer endometrium depending on its receptivity after in vitro-produced embryo transfer.

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    Siriluck Ponsuksili

    Full Text Available Embryonic implantation to establish a pregnancy is a complex process that requires appropriate communication between the embryo and the maternal endometrium. Inadequate uterine receptivity may contribute to the majority of implantation failures. To provide a comprehensive inventory of genes and functional networks that represent the maternal input of the embryo-maternal cross-talk, a longitudinal, holistic study of the endometrial transcriptome in relation to the days of estrous and to the receptivity of the endometrium was performed in bovine. At day 3 of estrous, genes related to cell communication and mitochondrial energy metabolism were differentially expressed among high- and low-receptive endometria (HR, LR; at day 7, transcripts functioning in immune and inflammatory pathways, oxidative stress, and angiogenesis had different abundances. Additionally, temporal transcriptional changes between days 3 and 7 differed considerably among HR and LR. Further, several transcription factors were predicted as relevant for receptivity because they were either differentially expressed among HR and LR animals or are known to be associated with genes we detected to have differential expression. Finally, global DNA methylation varied according to the interaction of receptivity group and day of estrous, and a divergent trend, which correlated with abundance of DNMT1 transcript, was observed in LR and HR along the estrous cycle days. The study revealed that, even in early estrous, transcripts related to cell communication and response to exogenous stimuli, vascularization, and energy supply show divergent expression and longitudinal temporal regulation in HR and LR. Key components of these molecular pathways are known to be dependent on ovarian hormones that promote uterine receptivity.

  18. Human autologous serum as a substitute for fetal bovine serum in human Schwann cell culture.

    Directory of Open Access Journals (Sweden)

    Parisa Goodarzi

    2014-04-01

    Full Text Available Nowadays, cell -based and tissue engineered products have opened new horizons in treatment of incurable nervous system disorders. The number of studies on the role of Schwann cells (SC in treating nervous disorders is higher than other cell types. Different protocols have been suggested for isolation and expansion of SC which most of them have used multiple growth factors, mitogens and fetal bovine sera (FBS in culture medium. Because of potential hazards of animal-derived reagents, this study was designed to evaluate the effect of replacing FBS with human autologous serum (HAS on SC's yield and culture parameters. Samples from 10 peripheral nerve biopsies were retrieved and processed under aseptic condition. The isolated cells cultured in FBS (1st group or autologous serum (2nd group. After primary culture the cells were seeded at 10000 cell/cm2 in a 12 wells cell culture plate for each group. At 100% confluency, the cell culture parameters (count, viability, purity and culture duration of 2 groups were compared using paired t-test. The average donors' age was 35.80 (SD=13.35 and except for 1 sample the others cultured successfully. In first group, the averages of cell purity, viability and culture duration were 97% (SD=1.32, 97/33% (SD=1.22 and 11.77 (SD=2.58 days respectively. This parameters were 97.33% (SD=1.00, 97.55% (SD=1.33 and 10.33 days (SD=1.65 in second group. The difference of cell count, purity and viability were not significant between 2 groups (P>0.05. The cells of second group reached to 100% confluency in shorter period of time (P=0.03. The results of this study showed that autologous serum can be a good substitute for FBS in human SC culture. This can reduce the costs and improve the safety of cell product for clinical application.

  19. The influence of exogenous eicosanoids on the radiation response of cultured bovine aortic endothelial cells

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    Rubin, D.B.; Drab, E.A.; Stone, A.M.; Walden, T.L. Jr.; Hanson, W.R. (Rush-Presbyterian-St. Luke' s-Medical Center, Chicago, IL (USA))

    1991-01-01

    The radioprotection by several eicosanoids was investigated in cultures of bovine aortic endothelial cells. One hour before irradiation (0-500 cGy, 137Cs gamma rays) 10 micrograms/ml of PGD2, PGE1, PGI2, misoprostol (PGE1-analog), 16,16-dimethyl PGE2, PGA2, or 1 microgram/ml LTC4 was added. Radiation decreased incorporation of (3H)thymidine at 4 h, cell number/culture at 24 h, and cell survival as measured by colony formation. Under these conditions the eicosanoids were not radioprotective. Two eicosanoids, PGD2 and PGA2, appeared to be toxic. Because receptors might mediate eicosanoid-induced radioprotection, radioligand binding of PGE2 and LTC4 and levels of adenosine 3',5'-cyclic monophosphate (cAMP) were measured. Evidence for a receptor was equivocal; there was nonspecific binding and metabolism of LTC4. The level of cAMP was elevated by 16-16-dimethyl-PGE2 in the presence of isobutyl methylxanthine; however, this combination of the prostaglandin and the methylxanthine was not radioprotective. These investigations suggest that an elevated cAMP level alone does not lead to eicosanoid-induced radioprotection of bovine aortic endothelial cell monolayers in vitro.

  20. The influence of exogenous eicosanoids on the radiation response of cultured bovine aortic endothelial cells

    International Nuclear Information System (INIS)

    Rubin, D.B.; Drab, E.A.; Stone, A.M.; Walden, T.L. Jr.; Hanson, W.R.

    1991-01-01

    The radioprotection by several eicosanoids was investigated in cultures of bovine aortic endothelial cells. One hour before irradiation (0-500 cGy, 137Cs gamma rays) 10 micrograms/ml of PGD2, PGE1, PGI2, misoprostol (PGE1-analog), 16,16-dimethyl PGE2, PGA2, or 1 microgram/ml LTC4 was added. Radiation decreased incorporation of [3H]thymidine at 4 h, cell number/culture at 24 h, and cell survival as measured by colony formation. Under these conditions the eicosanoids were not radioprotective. Two eicosanoids, PGD2 and PGA2, appeared to be toxic. Because receptors might mediate eicosanoid-induced radioprotection, radioligand binding of PGE2 and LTC4 and levels of adenosine 3',5'-cyclic monophosphate (cAMP) were measured. Evidence for a receptor was equivocal; there was nonspecific binding and metabolism of LTC4. The level of cAMP was elevated by 16-16-dimethyl-PGE2 in the presence of isobutyl methylxanthine; however, this combination of the prostaglandin and the methylxanthine was not radioprotective. These investigations suggest that an elevated cAMP level alone does not lead to eicosanoid-induced radioprotection of bovine aortic endothelial cell monolayers in vitro

  1. Embryogenic culture initiation and somatic embryo development in hybrid firs (Abies alba x Abies cephalonica, and Abies alba x Abies numidica).

    Science.gov (United States)

    Salajova, T; Jasik, J; Kormutak, A; Salaj, J; Hakman, I

    1996-03-01

    Embryogenic cultures were established from silver fir (Abies alba Mill.) female megagametliophytes with developing embryos and from excised mature embryos after pollination with Abies cephalonica Lond. or Abies numidica DeLann pollea The frequency of embryogenic callus formation was dependent on genotype, collection time, medium and explants used. The embryogenic callus initiation potential of megagamethophytes with developing embryos in both hybrids was higher in early July and dropped as the zygotic embryos matured. Excised cotyledonary embryos were less suitable for induction of embryogenic cultures. SH medium supplemented with 1mg/l BAP was the most efficient for callus induction and maintenance. Cultures were composed of early somatic embryos with an embryonal mass formed of highly cytoplasmic cells, rich in cell organelles and a suspensor built up by vacuolated, strongly elongated cells. Maturation of embryos was detected with the formation of bipolar structures with shoot and root apices. Nutrition reserves were observed in cells of embryos cultured on DCR medium containing 1 or 10 mg/l ABA. Cotyledon formation, hypocotyl elongation and low frequency germination occured following transfer of the embryos to the same medium without ABA.

  2. Platelet-rich plasma can replace fetal bovine serum in human meniscus cell cultures.

    Science.gov (United States)

    Gonzales, Veronica K; de Mulder, Eric L W; de Boer, Trix; Hannink, Gerjon; van Tienen, Tony G; van Heerde, Waander L; Buma, Pieter

    2013-11-01

    Concerns over fetal bovine serum (FBS) limit the clinical application of cultured tissue-engineered constructs. Therefore, we investigated if platelet-rich plasma (PRP) can fully replace FBS for meniscus tissue engineering purposes. Human PRP and platelet-poor plasma (PPP) were isolated from three healthy adult donors. Human meniscal fibrochondrocytes (MFCs) were isolated from resected tissue after a partial meniscectomy on a young patient. Passage-4 MFCs were cultured in monolayer for 24 h, and 3 and 7 days. Six different culture media were used containing different amounts of either PRP or PPP and compared to a medium containing 10% FBS. dsDNA was quantified, and gene expression levels of collagen types I and II and aggrecan were measured at different time points with quantitative polymerase chain reaction in the cultured MFCs. After 7 days, the dsDNA quantity was significantly higher in MFCs cultured in 10% and 20% PRP compared to the other PRP and PPP conditions, but equal to 10% FBS. Collagen type I expression was lower in MFCs cultured with medium containing 5% PRP, 10% and 20% PPP compared to FBS. When medium with 10% PRP or 20% PRP was used, expressions were not significantly different from medium containing 10% FBS. Collagen type II expression was absent in all medium conditions. Aggrecan expression did not show differences between the different media used. However, after 7 days a higher aggrecan expression was measured in most culture conditions, except for 5% PRP, which was similar compared to FBS. Statistical significance was found between donors at various time points in DNA quantification and gene expression, but the same donors were not statistically different in all conditions. At 7 days cell cultured with 10% PRP and 20% PRP showed a higher density, with large areas of clusters, compared to other conditions. In an MFC culture medium, FBS can be replaced by 10% PRP or 20% PRP without altering proliferation and gene expression of human MFCs.

  3. SGO1 maintains bovine meiotic and mitotic centromeric cohesions of sister chromatids and directly affects embryo development.

    Directory of Open Access Journals (Sweden)

    Feng-Xia Yin

    Full Text Available Shugoshin (SGO is a critical factor that enforces cohesion from segregation of paired sister chromatids during mitosis and meiosis. It has been studied mainly in invertebrates. Knowledge of SGO(s in a mammalian system has only been reported in the mouse and Hela cells. In this study, the functions of SGO1 in bovine oocytes during meiotic maturation, early embryonic development and somatic cell mitosis were investigated. The results showed that SGO1 was expressed from germinal vesicle (GV to the metaphase II stage. SGO1 accumulated on condensed and scattered chromosomes from pre-metaphase I to metaphase II. The over-expression of SGO1 did not interfere with the process of homologous chromosome separation, although once separated they were unable to move to the opposing spindle poles. This often resulted in the formation of oocytes with 60 replicated chromosomes. Depletion of SGO1 in GV oocytes affected chromosomal separation resulting in abnormal chromosome alignment at a significantly higher proportion than in control oocytes. Knockdown of SGO1 expression significantly decreased the embryonic developmental rate and quality. To further confirm the function(s of SGO1 during mitosis, bovine embryonic fibroblast cells were transfected with SGO1 siRNAs. SGO1 depletion induced the premature dissociation of chromosomal cohesion at the centromere and along the chromosome arm giving rise to abnormal appearing mitotic patterns. The results of this study infer that SGO1 is involved in the centromeric cohesion of sister chromatids and chromosomal movement towards the spindle poles. Depletion of SGO1 causes arrestment of cell division in meiosis and mitosis.

  4. Cell-transforming activity and genotoxicity of phenolphthalein in cultured Syrian hamster embryo cells.

    Science.gov (United States)

    Tsutsui, T; Tamura, Y; Yagi, E; Hasegawa, K; Tanaka, Y; Uehama, A; Someya, T; Hamaguchi, F; Yamamoto, H; Barrett, J C

    1997-11-27

    Phenolphthalein is a cathartic agent widely used in non-prescription laxatives. For the simultaneous assessment of in vitro carcinogenicity and mutagenicity of phenolphthalein, the ability of this chemical to induce cell transformation and genetic effects was examined using the Syrian hamster embryo (SHE) cell model. Cell growth was reduced by treatment with phenolphthalein at 10-40 microM in a dose-related manner. Treatment with phenolphthalein for 48 hr induced a dose-dependent increase in morphological transformation of SHE cells. Over the dose range that resulted in cell transformation ( 10-40 microM), treatment of SHE cells with phenolphthalein induced gene mutations at the hprt locus but not at the Na+/K+ ATPase locus. A statistically significant level of chromosomal aberrations was elicited in SHE cells treated with phenolphthalein at the highest dose (40 microM). Meanwhile, neither numerical chromosomal changes nor DNA adduct formation, analyzed by the nuclease P1 enhancement version of 32P-post-labeling, were induced by treatment with phenolphthalein at any concentrations examined. We thus report cell-transforming activity and mutagenicity of phenolphthalein assessed with the same mammalian cells in culture. Our results provide evidence that phenolphthalein has cell-transforming and genotoxic activity in cultured mammalian cells. The mutagenic and clastogenic activities of phenolphthalein could be a causal mechanism for carcinogenicity in rodents.

  5. Silkworm (Bombyx mori) hemolymph unable to substitute fetal bovine serum in insect cell culture

    Science.gov (United States)

    Suparto, Irma H.; Khalam, Chandra Nur; Praira, Willy; Sajuthi, Dondin

    2014-03-01

    Fetal Bovine Serum (FBS) in animal cell culture media is an important source of nutrients for cell growth. However, the harvest and collection of FBS cause bioethical concerns. Efforts to reduce and preferably replace FBS with synthetic or other natural alternatives are continually being explored. Hemolymph silkworm (Bombyx mori) contains many nutrients needed for the process of metamorphosis. Therefore, there is possibility as an alternative nutritional supplement for cell culture to reduce the use of FBS. The objective of this study was to evaluate the macrocomponent of hemolymph and the possibility as medium supplement for Spodoptera fugiperda (Sf9) cell culture. Proximate analyses showed that hemolymph contains 89.76% of water, 2.52 mg/mL carbohydrate, 2.35% fat and 55.61 mg/mL protein. Further protein analysis, it consists of 15 fractions containing molecular weight of 22 - 152 kDa. The use of hemolymph as FBS substitution in Sf9 cell culture with various concentrations was unable to maintain and support cell growth. Further research still needed by prior adaptation of the tissue culture to minimal nutrition media before introduction of the hemolymph as supplement.

  6. Effects of reactive oxygen species levels in prepared culture media on embryo development: a comparison of two media.

    Science.gov (United States)

    Shih, Ying-Fu; Lee, Tsung-Hsien; Liu, Chung-Hsien; Tsao, Hui-Mei; Huang, Chun-Chia; Lee, Maw-Sheng

    2014-12-01

    This study determined the correlation between the levels of reactive oxygen species (ROS) in prepared culture media and the early development of human embryos. This was an autocontrolled comparison study. A total of 159 patients undergoing in vitro fertilization/intracytoplasmic sperm injection treatment were recruited in this study. The pH values, osmolarity pressures, and ROS levels of 15 batches of two culture media were measured. Sibling oocytes or embryos from individual patients were randomly assigned to two culture groups with Quinn's Advantage Cleavage and Blastocyst media (QAC/QAB) or GIII series cleavage and blastocyst media (G1.3/G2.3). The difference between the two culture groups was analyzed using one-sample t test. The QAC/QAB and G1.3/G2.3 media exhibited similar pH values and osmolarity pressures. However, the prepared QAC/QAB media were characterized to contain lower amounts of ROS than the G1.3/G2.3 media. Furthermore, the blastocysts that developed under the QAC/QAB media were morphologically superior to those that developed under the G1.3/G2.3 media. The elevated ROS levels in culture media were associated with poor development of blastocyst-stage embryos. Measurement of ROS levels may be a valuable process for medium selection or modification. Copyright © 2014. Published by Elsevier B.V.

  7. Unconditioned commercial embryo culture media contain a large variety of non-declared proteins: a comprehensive proteomics analysis.

    Science.gov (United States)

    Dyrlund, Thomas F; Kirkegaard, Kirstine; Poulsen, Ebbe Toftgaard; Sanggaard, Kristian W; Hindkjær, Johnny J; Kjems, Jørgen; Enghild, Jan J; Ingerslev, Hans Jakob

    2014-11-01

    Which non-declared proteins (proteins not listed on the composition list of the product data sheet) are present in unconditioned commercial embryo culture media? A total of 110 non-declared proteins were identified in unconditioned media and between 6 and 8 of these were quantifiable and therefore represent the majority of the total protein in the media samples. There are no data in the literature on what non-declared proteins are present in unconditioned (fresh media in which no embryos have been cultured) commercial embryo media. The following eight commercial embryo culture media were included in this study: G-1 PLUS and G-2 PLUS G5 Series from Vitrolife, Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium from Cook Medical and EmbryoAssist, BlastAssist, Sequential Cleav and Sequential Blast from ORIGIO. Two batches were analyzed from each of the Sydney IVF media and one batch from each of the other media. All embryo culture media are supplemented by the manufacturers with purified human serum albumin (HSA 5 mg/ml). The purified HSA (HSA-solution from Vitrolife) and the recombinant human albumin supplement (G-MM from Vitrolife) were also analyzed. For protein quantification, media samples were in-solution digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For in-depth protein identification, media were albumin depleted, dialyzed and concentrated before sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gel was cut into 14 slices followed by in-gel trypsin digestion, and analysis by LC-MS/MS. Proteins were further investigated using gene ontology (GO) terms analysis. Using advanced mass spectrometry and high confidence criteria for accepting proteins (P media when the albumin is added. GO analysis showed that many of these proteins have roles in defence pathways, for example 18 were associated with the innate immune response and 17 with inflammatory responses. Eight proteins have been reported

  8. In Vitro Selection of Peanut Somatic Embryos on Medium Containing Culture Filtrate of Sclerotium rolfsii and Plantlet Regeneration

    Directory of Open Access Journals (Sweden)

    YUSNITA

    2005-06-01

    Full Text Available Attempts to identify somaclonal variants of peanut with resistance to Sclerotium stem rot disease due to infection of S. rolfsii were conducted. The objectives of this study were to develop in vitro selection method using culture filtrates of S. rolfsii, identify culture filtrate-insensitive somatic embryo (SE of peanut after in vitro selection and regenerate peanut R0 lines originated from culture filtrate-insensitive SE. To achieve these objectives, peanut embryogenic tissues were cultured on selective medium containing various concentrations of S. rolfsii culture filtrates and sublethal concentration of the filtrates. Medium containing sublethal level of S. rolfsii culture filtrates was used to identify culture filtrate-insensitive SE of peanut. Subsequently, the selected SEs were germinated, plantlets were regenerated and preliminary tested against S. rolfsii. Results of the experiments showed that addition of S. rolfsii culture filtrates into medium for inducing peanut somatic embryos drastically reduced their growth and proliferation. S. rolfsii culture filtrates at 10% concentration has significantly reduced the number of proliferated SE per explant. However, sublethal level was achieved at 30% of culture filtrates concentration. Responses of five peanut cultivars against 30% of culture filtrates were similar, indicating they were similar in their susceptibility against S. rolfsii. A number of culture filtrate-insensitive SE were identified after culturing 1500 clumps of embryogenic tissue of peanut cv. Kelinci for three consecutive passages on medium containing 30% of culture filtrates. Germination of selected SE and regeneration of plantlet from culture filtrate-insensitive SE resulted in 50 peanut R0 lines. These lines have been grown in the plastic house and produced normal seeds for further evaluation. Results of S. rolfsii inoculation indicated the existence of chimera for insensitivity against S. rolfsii.

  9. Evaluation on the Effects of Culture Medium on Regeneration of Oil Palm Plantlets from Immature Embryos (IE)

    International Nuclear Information System (INIS)

    Wan Nur Syuhada, W.S.; Rasid, O.A.; Parveez, G.K.A

    2016-01-01

    Oil palm (Elaeis guineensis) tissue culture is slow, inefficient and genotype dependent. Therefore, efforts to improve the regeneration system are essential and thereby on-going. Immature embryos (IE) have been reported to be more responsive to in vitro culture as compared to other explant types. The objective of this study is to evaluate the most efficient culture media for the regeneration of whole plants from IE. IE were cultured on three media, namely N6, MY3 and MS +Y3. Initial observation showed that calli were induced as early as two weeks after culture. They started to produce friable calli around three to four months later. IE were shown to respond differently to the tested culture media. This initial result will be useful to identify the most responsive culture media for IE as explant. (author)

  10. Improved blastocyst development of single cow OPU-derived presumptive zygotes by group culture with agarose-embedded helper embryos

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    Dey Shukla

    2011-08-01

    Full Text Available Abstract Background The in vitro culture of presumed zygotes derived from single cow ovum pick-up (OPU is important for the production of quality blastocysts maintaining pedigree. The aim of the present study was to evaluate the agar chip-embedded helper embryo coculture system for single cow OPU-derived zygotes by assessing embryo quality. Methods Cumulus oocyte complexes (COCs were collected from Hanwoo cows with high genetic merit twice a week using the ultra-sound guided OPU technique and from slaughterhouse ovaries. The Hanwoo cow COCs and slaughterhouse ovaries were matured in vitro, fertilized in vitro with thawed Hanwoo sperm and cultured for 24 h. The presumed zygotes were subsequently placed in three different culture systems: (1 control OPU (controlOPU with single cow OPU-derived presumed zygotes (2~8; (2 agar chip-embedded slaughterhouse helper embryo coculture (agarOPU with ten presumed zygotes including all presumed zygotes from a cow (2~8 and the rest from agar chip-embedded slaughterhouse presumed zygotes (8~2; and (3 slaughterhouse in vitro embryo production (sIVP with ten slaughterhouse ovary-derived presumed zygotes, each in 50 μL droplets. Day 8 blastocysts were assayed for apoptosis and gene expression using real time PCR. Results The coculture system promoted higher blastocyst development in OPU zygotes compared to control OPU zygotes cultured alone (35.2 vs. 13.9%; P CD9, 0.4-fold; AKRAB1, 0.3-fold and in cocultured zygotes (CD9, 0.3-fold; AKRAB1, 0.3-fold compared to sIVP blastocysts (1.0-fold. Moreover, genes involved in implantation and/or normal calf delivery were up-regulated (P PGSH2, 5.0-fold; TXN, 4.3-fold; PLAU, 1.7-fold and cocultured zygotes (PGSH2, 14.5-fold; TXN, 3.2-fold; PLAU, 6.8-fold compared to sIVP (1.0-fold blastocysts. However, the expression of PLAC8, TGF-β1, ODC1, ATP5A1 and CASP3 did not differ between the three culture groups. Conclusions Results show that the agar chip-embedded helper embryo

  11. Transcriptomic comparison of primary bovine horn core carcinoma culture and parental tissue at early stage

    Directory of Open Access Journals (Sweden)

    Sharadindu Shil

    2017-01-01

    Full Text Available Aim: Squamous cell carcinoma or SCC of horn in bovines (bovine horn core carcinoma frequently observed in Bos indicus affecting almost 1% of cattle population. Freshly isolated primary epithelial cells may be closely related to the malignant epithelial cells of the tumor. Comparison of gene expression in between horn’s SCC tissue and its early passage primary culture using next generation sequencing was the aim of this study. Materials and Methods: Whole transcriptome sequencing of horn’s SCC tissue and its early passage cells using Ion Torrent PGM were done. Comparative expression and analysis of different genes and pathways related to cancer and biological processes associated with malignancy, proliferating capacity, differentiation, apoptosis, senescence, adhesion, cohesion, migration, invasion, angiogenesis, and metabolic pathways were identified. Results: Up-regulated genes in SCC of horn’s early passage cells were involved in transporter activity, catalytic activity, nucleic acid binding transcription factor activity, biogenesis, cellular processes, biological regulation and localization and the down-regulated genes mainly were involved in focal adhesion, extracellular matrix receptor interaction and spliceosome activity. Conclusion: The experiment revealed similar transcriptomic nature of horn’s SCC tissue and its early passage cells.

  12. Maternal age and in vitro culture affect mitochondrial number and function in equine oocytes and embryos

    NARCIS (Netherlands)

    Hendriks, W Karin; Colleoni, Silvia; Galli, Cesare; Paris, Damien B B P; Colenbrander, Ben; Roelen, Bernard A J; Stout, Tom A E

    2015-01-01

    Advanced maternal age and in vitro embryo production (IVP) predispose to pregnancy loss in horses. We investigated whether mare age and IVP were associated with alterations in mitochondrial (mt) DNA copy number or function that could compromise oocyte and embryo development. Effects of mare age

  13. Decisions for the IVF laboratory: comparative analysis of embryo culture incubators.

    Science.gov (United States)

    Swain, Jason E

    2014-05-01

    Incubators in the IVF laboratory play a pivotal role in providing a stable and appropriate culture environment required for optimizing embryo development and clinical outcomes. With technological advances, several types of incubators are now available and careful consideration is required for selection. Examination of variables, such as recovery/stabilization of temperature, gas atmosphere and humidity, as well as understanding various approaches utilized by each device to regulate these variables, is critical. Additionally, a comprehensive examination of clinical studies that compare various incubators may provide insight into their efficacy. Other factors, both technical and practical, must also be considered when selecting an incubator. Importantly, proper management, including patient volume and workflow, is paramount in optimizing function of any incubator, regardless of the technology incorporated. This review highlights incubator function and reviews key environmental variables controlled and the technology utilized in various units. Additionally, existing comparative studies focused on incubator recovery and clinical outcomes are critically analysed. Finally, strategies employed for incubator management, as well as future potential incubator improvements are discussed. While existing reports indicate that smaller benchtop/topload incubators provide faster recovery of environmental variables, there is no clear advantage of any particular incubator based on clinical outcomes. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  14. Purification of binder of sperm protein 1 (BSP1) and its effects on bovine in vitro embryo development after fertilization with ejaculated and epididymal sperm.

    Science.gov (United States)

    Rodríguez-Villamil, P; Hoyos-Marulanda, V; Martins, J A M; Oliveira, A N; Aguiar, L H; Moreno, F B; Velho, A L M C S; Monteiro-Moreira, A C; Moreira, R A; Vasconcelos, I M; Bertolini, M; Moura, A A

    2016-02-01

    The present study evaluated functional aspects of binder of sperm 1 (BSP1) in the bovine species. In a first experiment, cumulus-oocyte complexes (n = 1274) were incubated with frozen-thawed ejaculated sperm (18 hours) in Fert-TALP medium containing: heparin, 10, 20, or 40 μg/mL BSP1. Heparin followed by gelatin affinity chromatography was used for purification of BSP1 from bovine seminal vesicle fluid. With ejaculated sperm, cleavage rates were similar when Fert-TALP medium was incubated with heparin (74.1 ± 2.7%), 10 μg/mL BSP1 (77.8 ± 3.1%), or 20 μg/mL BSP1 (74 ± 2.0%). Day-7 blastocyst rates were equivalent after incubations with heparin (40.8 ± 5.0%) and 10 μg/mL BSP1 (34.1 ± 4.4%), but reduced after 20 μg/mL BSP1 (22.4 ± 2.9%) and 40 μg/mL BSP1 (19.3 ± 4.1%; P epididymal sperm (18 hours) in Fert-TALP medium containing: no heparin, heparin, 10, 20, or 40 μg/mL. Cleavage and blastocyst rates were similar after treatments with heparin (68.5 ± 1.3% and 24.7 ± 3.2%, respectively) or without heparin (65.5 ± 1.8% and 27.3 ± 1.6%, respectively). Cleavage was higher after treatment with any BSP1 concentrations (74.2 ± 2.7%-79.0 ± 1.1%) than without heparin (P epididymal sperm, as observed with ejaculated sperm. On the basis of immunocytochemistry, there was BSP1 binding to frozen-thawed ejaculated but not to epididymal sperm. Also, anti-BSP1 reaction remained on ejaculated sperm (as expected) and appeared on epididymal sperm after incubation with purified BSP1. Acrosome reaction of ejaculated and epididymal sperm was induced after incubation with purified BSP1 as well, indicating an effect of BSP1 on capacitation. In conclusion, purified BSP1 from bull seminal vesicles was able to bind to and induce capacitation of ejaculated and epididymal sperm. Also, BSP1 added to fertilization media and allowed proper cleavage and embryo development, with the effects being modulated by previous exposure or not of spermatozoa to seminal plasma. Copyright

  15. Obstetric and perinatal outcomes of pregnancies conceived with embryos cultured in a time-lapse monitoring system.

    Science.gov (United States)

    Insua, Maria Fernanda; Cobo, Ana Cristina; Larreategui, Zaloa; Ferrando, Marcos; Serra, Vicente; Meseguer, Marcos

    2017-09-01

    To compare obstetric and perinatal outcomes of singleton pregnancies resulting from embryos incubated in a time-lapse system (TLS) with those of embryos grown in standard IVF incubators (SI). Retrospective description of a cohort of patients who conceived during a randomized, controlled trial. Private university-affiliated IVF center. Of 856 randomized patients, 378 gave birth to a live-born infant: 216 of the deliveries originated from embryos incubated in TLS, and 162 deliveries were from embryos cultured in SI. Embryo incubation and selection in TLS. Delivery and neonatal outcomes. No significant differences were observed in the baseline characteristics of the study population. The delivery rate was 49.3% (TLS) vs. 40.0% (SI), and multiple deliveries were higher in the TLS group: 31.0% (67 of 216) vs. 24.7% (40 of 162) in the SI group. When singleton pregnancies were analyzed no differences were found between the two groups in the rate of obstetric problems with respect to weeks at delivery: 38.8 (95% confidence interval [CI] 38.4-39.1) (TLS) vs. 39.5 (95% CI 38.0-39.9) (SI); preterm births (outcomes such as birth weight: 3,163 g (95% CI 3,035-3,292 g) (TLS) vs. 3,074 (95% CI 2,913-3,236) (SI); low birth weight (obstetric and perinatal outcomes when a time-lapse incubator was used rather than a more widely used conventional incubator. As far as we know this is the first report from a randomized study of the neonatal outcomes of time-lapse monitoring. Our results suggest that this technology is an effective and safe alternative for embryo incubation, though trials of larger numbers of patients are required to further confirm our conclusions. NCT01549262. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  16. In vitro exposure of bovine morulae to Ureaplasma diversum.

    Science.gov (United States)

    Britton, A P; Ruhnke, H L; Miller, R B; Johnson, W H; Leslie, K E; Rosendal, S

    1987-04-01

    Ureaplasma diversum has been associated with infertility in the cow experimentally and in naturally occurring cases. However, the pathogenic mechanism is undetermined. The purpose of this study was to determine whether ureaplasmas are pathogenic for bovine morulae in vitro. Twenty-one morulae were recovered from three superovulated, mature, Holstein cows six or seven days postestrus. The embryos were divided into three groups (A,B,C) and incubated for 16 hours at 37 degrees C in humidified air with 10% CO2. Group A was incubated in embryo culture medium alone, Group B was incubated in culture medium with sterile ureaplasma broth added and Group C was incubated in culture medium containing 1.7 X 10(6) colony forming units Ureaplasma diversum strain 2312. After incubation, the morulae were examined using an electron microscope. Structures morphologically identical to U. diversum were present on the outer surface of the zonae pellucidae of all the morulae exposed to the organism and none were present on the unexposed control embryos. No other morphological differences were observed in either the ureaplasma-exposed embryos or the two groups of control embryos. Ureaplasma diversum was isolated from three of the five embryos incubated in culture medium with sterile ureaplasma broth added. These three embryos were recovered from one donor cow which cultured positive for U. diversum from the vulva and flush fluid. This finding suggests that the contaminating organisms entered the embryo culture wells either in the embryo collection medium or attached to the embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Radiotoxicity and incorporation of methyl-tritiated-thymidine on preimplantation mouse embryo. In vitro fertilization and culture

    International Nuclear Information System (INIS)

    Kikuchi, O.K.; Ohyama, H.; Yamada, T.

    1993-04-01

    In the present work different concentrations of methyl- 3 H-thymidine was added to the culture medium micro drops containing the mouse zygotes at pro nuclear stage and the embryos were cultured in vitro at 37 0 C in a humidified atmosphere with 5% of CO 2 for four days up to the expanded blastocyst stage, the established end point to calculate the L D 50 lethal dose. The blastocyst formation rate decreased with increasing concentration of tritium in medium and a value of 2.4 X 10 3 Bq/ml for L D 50 was obtained. The 3 H-Td R incorporation by the embryo during the preimplantation period was low at the beginning and increased quickly during the morula and the blastocyst development. (author)

  18. Culture of bovine articular chondrocytes in funnel-like collagen-PLGA hybrid sponges.

    Science.gov (United States)

    Lu, Hongxu; Ko, Young-Gwang; Kawazoe, Naoki; Chen, Guoping

    2011-08-01

    Three-dimensional porous scaffolds play an important role in tissue engineering and regenerative medicine. Structurally, these porous scaffolds should have an open and interconnected porous architecture to facilitate a homogeneous cell distribution. Moreover, the scaffolds should be mechanically strong to support new tissue formation. We developed a novel type of funnel-like collagen sponge using embossing ice particulates as a template. The funnel-like collagen sponges could promote the homogeneous cell distribution, ECM production and chondrogenesis. However, the funnel-like collagen sponges deformed during cell culture due to their weak mechanical strength. To solve this problem, we reinforced the funnel-like collagen sponges with a knitted poly(D,L-lactic-co-glycolic acid) (PLGA) mesh by hybridizing these two types of materials. The hybrid scaffolds were used to culture bovine articular chondrocytes. The cell adhesion, distribution, proliferation and chondrogenesis were investigated. The funnel-like structure promoted the even cell distribution and homogeneous ECM production. The PLGA knitted mesh protected the scaffold from deformation during cell culture. Histological and immunohistochemical staining and cartilaginous gene expression analyses revealed the cartilage-like properties of the cell/scaffold constructs after in vivo implantation. The hybrid scaffold, composed of a funnel-like collagen sponge and PLGA mesh, would be a useful tool for cartilage tissue engineering. © 2011 IOP Publishing Ltd

  19. Effect of anabolics on bovine granulosa-luteal cell primary cultures.

    Directory of Open Access Journals (Sweden)

    Bartolomeo Biolatti

    2007-10-01

    Full Text Available Granulosa cell tumours are observed with increased frequency among calves slaughtered in Northern Italy. The use of illegal anabolics in breeding was taken into account as a cause of this pathology. An in vitro approach was used to detect the possible alterations of cell proliferation induced by anabolics on primary cultures of bovine granulosa-luteal cells. Cultures were treated with different concentrations of substances illegally used in cattle (17beta-estradiol, clenbuterol and boldione. Cytotoxicity was determined by means of MTT test, to exclude toxic effects induced by anabolics and to determine the highest concentration to be tested. Morphological changes were evaluated by means of routine cytology, while PCNA expression was quantified in order to estimate cell proliferation. Cytotoxic effects were revealed at the highest concentrations. The only stimulating effect on cell proliferation was detected in boldione treated cultures: after 48 h treated cells, compared to controls, showed a doubled expression of PCNA. In clenbuterol and 17beta-estradiol treated cells PCNA expression was similar to controls or even decreased. As the data suggest an alteration in cell proliferation, boldione could have a role in the early stage of pathogenesis of granulosa cell tumour in cattle.

  20. Integration of single oocyte trapping, in vitro fertilization and embryo culture in a microwell-structured microfluidic device.

    Science.gov (United States)

    Han, Chao; Zhang, Qiufang; Ma, Rui; Xie, Lan; Qiu, Tian; Wang, Lei; Mitchelson, Keith; Wang, Jundong; Huang, Guoliang; Qiao, Jie; Cheng, Jing

    2010-11-07

    In vitro fertilization (IVF) therapy is an important treatment for human infertility. However, the methods for clinical IVF have only changed slightly over decades: culture medium is held in oil-covered drops in Petri dishes and manipulation occurs by manual pipetting. Here we report a novel microwell-structured microfluidic device that integrates single oocyte trapping, fertilization and subsequent embryo culture. A microwell array was used to capture and hold individual oocytes during the flow-through process of oocyte and sperm loading, medium substitution and debris cleaning. Different microwell depths were compared by computational modeling and flow washing experiments for their effectiveness in oocyte trapping and debris removal. Fertilization was achieved in the microfluidic devices with similar fertilization rates to standard oil-covered drops in Petri dishes. Embryos could be cultured to blastocyst stages in our devices with developmental status individually monitored and tracked. The results suggest that the microfluidic device may bring several advantages to IVF practices by simplifying oocyte handling and manipulation, allowing rapid and convenient medium changing, and enabling automated tracking of any single embryo development.

  1. Release of somatic embryogenic potential from excised zygotic embryos of carrot and maintenance of proembryonic cultures in hormone-free medium

    Science.gov (United States)

    Smith, D. L.; Krikorian, A. D.

    1989-01-01

    Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and

  2. Species-specificity of ethylene glycol-induced developmental toxicity: toxicokinetic and whole embryo culture studies in the rabbit.

    Science.gov (United States)

    Carney, Edward W; Tornesi, Belen; Markham, Daniel A; Rasoulpour, Reza J; Moore, Nigel

    2008-12-01

    High-dose gavage exposure to ethylene glycol (EG) is teratogenic in rats, but not rabbits. To investigate the reason for this species difference, toxicokinetic and whole embryo culture (WEC) studies were conducted in gestation day 9 New Zealand White rabbits, and the data compared to very similar data previously generated in pregnant rats. In the toxicokinetic study, maximal levels of unchanged EG in rabbits were comparable to those reported for rats. However, maximal levels of EG's teratogenic metabolite, glycolic acid (GA), in rabbit maternal blood and embryo were only 46% and 10% of the respective levels in rats. The toxicokinetic profile suggested that the lower GA levels in rabbits were due to a slower rate of maternal metabolism of EG to GA, slow uptake of GA into the yolk sac cavity fluid which surrounds the embryo, and negligible transfer via the visceral yolk sac (VYS) placenta. In the WEC study, exposure of rabbit conceptuses to high concentrations (WEC at > or = 3 mM. Overall, these data implicate toxicokinetics as an important factor underlying the species difference, although intrinsic insensitivity of the rabbit embryo might also be involved. Integration of these findings with published human data suggest that the rabbit is the more relevant model for human EG exposure, based on the negligible role of the rabbit VYS in placental transfer (humans lack a VYS) and similar rates of EG metabolism and extraembryonic fluid turnover.

  3. Mouse preimplantation embryo responses to culture medium osmolarity include increased expression of CCM2 and p38 MAPK activation

    Directory of Open Access Journals (Sweden)

    Watson Andrew J

    2007-01-01

    Full Text Available Abstract Background Mechanisms that confer an ability to respond positively to environmental osmolarity are fundamental to ensuring embryo survival during the preimplantation period. Activation of p38 mitogen-activated protein kinase (MAPK occurs following exposure to hyperosmotic treatment. Recently, a novel scaffolding protein called Osmosensing Scaffold for MEKK3 (OSM was linked to p38 MAPK activation in response to sorbitol-induced hypertonicity. The human ortholog of OSM is cerebral cavernous malformation 2 (CCM2. The present study was conducted to investigate whether CCM2 is expressed during mouse preimplantation development and to determine whether this scaffolding protein is associated with p38 MAPK activation following exposure of preimplantation embryos to hyperosmotic environments. Results Our results indicate that Ccm2 along with upstream p38 MAPK pathway constituents (Map3k3, Map2k3, Map2k6, and Map2k4 are expressed throughout mouse preimplantation development. CCM2, MAP3K3 and the phosphorylated forms of MAP2K3/MAP2K6 and MAP2K4 were also detected throughout preimplantation development. Embryo culture in hyperosmotic media increased p38 MAPK activity in conjunction with elevated CCM2 levels. Conclusion These results define the expression of upstream activators of p38 MAPK during preimplantation development and indicate that embryo responses to hyperosmotic environments include elevation of CCM2 and activation of p38 MAPK.

  4. Primary Bovine Extra-Embryonic Cultured Cells: A New Resource for the Study of In Vivo Peri-Implanting Phenotypes and Mesoderm Formation.

    Directory of Open Access Journals (Sweden)

    Isabelle Hue

    Full Text Available In addition to nourishing the embryo, extra-embryonic tissues (EETs contribute to early embryonic patterning, primitive hematopoiesis, and fetal health. These tissues are of major importance for human medicine, as well as for efforts to improve livestock efficiency, but they remain incompletely understood. In bovines, EETs are accessible easily, in large amounts, and prior to implantation. We took advantage of this system to describe, in vitro and in vivo, the cell types present in bovine EETs at Day 18 of development. Specifically, we characterized the gene expression patterns and phenotypes of bovine extra-embryonic ectoderm (or trophoblast; bTC, endoderm (bXEC, and mesoderm (bXMC cells in culture and compared them to their respective in vivo micro-dissected cells. After a week of culture, certain characteristics (e.g., gene expression of the in vitro cells were altered with respect to the in vivo cells, but we were able to identify "cores" of cell-type-specific (and substrate-independent genes that were shared between in vitro and in vivo samples. In addition, many cellular phenotypes were cell-type-specific with regard to extracellular adhesion. We evaluated the ability of individual bXMCs to migrate and spread on micro-patterns, and observed that they easily adapted to diverse environments, similar to in vivo EE mesoderm cells, which encounter different EE epithelia to form chorion, yolk sac, and allantois. With these tissue interactions, different functions arose that were detected in silico and corroborated in vivo at D21-D25. Moreover, analysis of bXMCs allowed us to identify the EE cell ring surrounding the embryonic disc (ED at D14-15 as mesoderm cells, which had been hypothesized but not shown prior to this study. We envision these data will serve as a major resource for the future in the analysis of peri-implanting phenotypes in response to the maternal metabolism and contribute to subsequent studies of placental/fetal development in

  5. In vitro Propagation of Citrus limonia Osbeck Through Nucellar Embryo Culture

    OpenAIRE

    Alka Jajoo

    2010-01-01

    Citrus limonia Osbeck is a promising rootsctock for commercial citrus species with sturdy anddisease and drought resistant characters. A n efficient and highly reproducible plant regeneration protocol hasbeen developed from nucellar embryo of Citrus limonia. Murashige and Skoogs medium was used for plantregeneration from nucellar embryos. It was noted that 6-benzylaminopurine at a concentration of 2.22 mMinduced highest number of multiple shoots as 18.26 shoots per explant. O n transfer of in...

  6. Embryotoxicant-specific transcriptomic responses in rat postimplantation whole-embryo culture.

    Science.gov (United States)

    Robinson, Joshua F; van Beelen, Vincent A; Verhoef, Aart; Renkens, Marc F J; Luijten, Mirjam; van Herwijnen, Marcel H M; Westerman, Anja; Pennings, Jeroen L A; Piersma, Aldert H

    2010-12-01

    Rat postimplantation whole-embryo culture (WEC) is a promising alternative test for the assessment of developmental toxicity. Toxicogenomic-based approaches may improve the predictive ability of the WEC model by providing a means to identify compound-specific mechanistic responses associated with embryotoxicity in vivo. Furthermore, alterations in gene expression may serve as a sensitive, objective, and robust marker, which precedes the observation of classical developmental toxicity endpoints in time. In this study, in combination with morphological developmental assessments, we studied transcriptomic responses associated with four distinct teratogens (caffeine [CAF], methylmercury [MM], monobutyl phthalate, and methoxyacetic acid) after 4 h of exposure, well before apparent embryotoxicity in WEC. We evaluated gene expression changes associated with similar levels of induced morphological embryotoxicity for each teratogen (as determined by total morphological score), evaluating for functional enrichment and quantitative changes in response. Concentrations selected for each of the four teratogens used induced a number of common effects on embryonic development (neural tube closure and optic/otic system). Despite inducing common morphological effects, our analysis suggests limited overlap in terms of toxicogenomic response at the gene expression level and at the level of biological processes across all four test chemicals. Many unique responses associated with each chemical correlated with previously hypothesized modes of developmental toxicity. For example, alterations in developmental signaling and cholesterol metabolism were observed with MM and CAF, respectively. This initial study suggests that distinct chemically induced toxicogenomic responses precede morphological effects in WEC and that these responses are relevant with mechanisms of toxicity previously observed in vivo.

  7. Development of a streamlined rat whole embryo culture assay for classifying teratogenic potential of pharmaceutical compounds.

    Science.gov (United States)

    Zhang, Cindy; Cao, June; Kenyon, James R; Panzica-Kelly, Julieta M; Gong, Lei; Augustine-Rauch, Karen

    2012-06-01

    This study describes a novel rat whole embryo culture (rWEC) teratogenicity assay that applies a simplified experimental design and statistical prediction model, resulting in reduced animal requirements and increased throughput with low prediction error rate for classifying teratogenic potential of compounds. A total of 70 compounds (38 teratogens and 32 nonteratogens) were evaluated, and the prediction model was generated from a dataset of 59 compounds. The rWEC assay requires only one test concentration (1μM) and three structural endpoints (group average morphological scores of spinal cord, heart, and number of somite pairs), which are used in a recursive partition model for classifying teratogenic liability. The model fitting concordance between the WEC assay and in vivo outcome was 83% with a standard deviation (SD) of 4.9%. The predictivity for future compounds was evaluated by using two statistical methods. Fivefold cross-validation estimated the predictivity of this model at 73% (SD 5.8%). A second estimation of predictivity was obtained from an independent test set of 11 compounds that were not used to build the prediction model and reached 82% (SD 11.6%). The overall estimate for prediction concordance is 74% (SD 5.2%). There is no statistically significant difference (p value > 0.50) in the predictivity between this model and the model supporting European Center for the Validation of Alternative Methods WEC assay with predictivity of 80% (SD 10.6%). Overall, the streamlined WEC assay is estimated to reduce animal use and operational costs by more than 50%. It substantially improves results turnaround with no loss of predictivity.

  8. Protein A gold identification of ureaplasmas on the bovine zona pellucida.

    Science.gov (United States)

    Britton, A P; Miller, R B; Ruhnke, H L; Johnson, W M

    1989-04-01

    The object of this study was to develop a prefixation protein A gold labelling technique for Ureaplasma diversum and to apply this to bovine embryos. Sixteen hour cultures of Ureaplasma diversum strain 2312 were incubated with either specific antiserum or nonimmune serum, followed by exposure to protein A gold and negative staining. The ureaplasmas which were incubated with specific antiserum were labelled with gold particles while those ureaplasmas which were incubated with nonimmune serum were not labelled. Twenty-three unhatched, day 7 bovine embryos were then incubated in either embryo culture medium (ECM) alone, ECM with sterile ureaplasma broth added or ECM with 1.7 X 10(6) colony forming units of Ureaplasma diversum strain 2312 per embryo. After 16 hours, the embryos were washed twice and incubated with either specific antiserum or nonimmune serum. The embryos were then incubated with medium containing protein A gold and examined by electron microscopy. No ureaplasmas were identified on the zona pellucida of the control embryos. Ureaplasmas were identified on the outer surface of the zona pellucida of 13 of the 17 embryos which had been exposed to the organism. Of these, the embryos which were incubated with specific antiserum had labelled ureaplasmas while the embryos which were incubated with nonimmune serum had unlabelled ureaplasmas on the zona pellucida. It was concluded that the protein A gold method was a suitable technique for the identification of ureaplasmas in EM preparations. The presence of ureaplasmas on the outer surface of the bovine zona pellucida following in vitro exposure to the organism was confirmed.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Nucleolar remodeling in nuclear transfer embryos

    DEFF Research Database (Denmark)

    Laurincik, Jozef; Maddox-Hyttel, Poul

    2007-01-01

    nucleoli are not apparent until the 5th cell cycle, whereas in somatic cell nuclear transfer embryos the functional nucleoli emerge already during the 3rd cell cycle. Intergeneric reconstructed embryos produced by the fusion of bovine differentiated somatic cell to a nonactivated ovine cytoplast fail...... the developmental potential of embryos originating from varied nuclear transfer protocols. In bovine in vivo developed embryos, functional ribosome-synthesizing nucleoli become structurally distinct toward the end of the 4th post-fertilization cell cycle. In embryonic cell nuclear transfer embryos, fully developed...... is completed toward the end of the 4th cell cycle. A substantial proportion of bovine embryos produced by nuclear transfer of embryonic or somatic cells to bovine ooplasts display aberrations in protein localization in one or more blastomers. This information is indicative of underlying aberrations in genomic...

  10. In vitro fertilization with rice gametes: production of zygotes and zygote and embryo culture.

    Science.gov (United States)

    Okamoto, Takashi

    2011-01-01

    In vitro fertilization (IVF) systems using isolated male and female gametes have been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development, and early embryogenesis, since the female gametophytes of plants are deeply embedded within ovaries. A rice IVF system was established to take advantage of the abundant resources stemming from rice research for investigations into the mechanisms of fertilization and early embryogenesis. Fusion of gametes can be performed using electrofusion and the fusion product, a zygote, forms a cell wall and an additional nucleolus. The zygote divides into an asymmetric two-celled embryo and develops into an early globular embryo, as in planta. The embryo further develops into irregularly shaped cell masses and fertile plants can be regenerated from the cell masses. This rice IVF system is a powerful tool for studying the molecular mechanisms involved in the early embryogenesis of angiosperms and for making new cultivars.

  11. Effects of putrescine, cadaverine, spermine, spermidine and beta-phenylethylamine on cultured bovine mammary epithelial cells

    DEFF Research Database (Denmark)

    Fusi, Eleonora; Baldi, Antonella; Cheli, Federica

    2008-01-01

    A bovine mammary epithelial cell line (BME-UV1) and three-dimensional collagen primary bovine organoids were used to evaluate the effects of cadaverine, putrescine, spermine, spermicline and beta-phenylethylamine on mammary epithelial cells. Each biogenic amine was diluted in several concentratio...

  12. Cellular distribution of inorganic mercury and its relation to cytotoxicity in bovine kidney cell cultures

    International Nuclear Information System (INIS)

    Bracken, W.M.; Sharma, R.P.; Bourcier, D.R.

    1984-01-01

    A bovine kidney cell culture system was used to assess what relationship mercuric chloride (HgCl 2 ) uptake and subcellular distribution had to cytotoxicity. Twenty-four-hour incubations with 0.05-50 μM HgCl 2 elicited a concentration-related cytotoxicity. Cellular accumulation of 203 Hg was also concentration-related, with 1.0 nmol/10 6 cells at the IC50. Measurement of Hg uptake over the 24-h exposure period revealed a multiphasic process. Peak accumulation was attained by 1 h and was followed by extrusion and plateauing of intracellular Hg levels. Least-squares regression analysis of the cytotoxicity and cellular uptake data indicated a potential relationship between the Hg uptake and cytotoxicity. However, the subcellular distribution of Hg was not concentration-related. Mitochondria and soluble protein fractions accounted for greater than 65% of the cell-associated Hg at all concentrations. The remaining Hg was distributed between the microsomal (6-10%) and nuclear and cell debris (11-22%) fractions at all concentrations tested. Less than 20% of the total cell-associated Hg was bound with metallothionein-like protein. 31 references, 4 figures, 3 tables

  13. Incidence of Listeria species in bovine, ovine, caprine, camel and water buffalo milk using cultural method and the PCR assay

    Directory of Open Access Journals (Sweden)

    Ebrahim Rahimi

    2014-02-01

    Full Text Available Objective: To determine the prevalence rate of Listeria species in bovine, ovine, caprine, camel and water buffalo milk in Iran. Methods: From September 2010 to December 2011 a total of 260 bulk milk samples including 85 bovine, 37 camel, 34 water buffalo, 56 ovine and 48 caprine bulk milk samples were collected from commercial dairy herds, in Fars and Khuzestan provinces, Iran and were evaluated for the presence of Listeria species using cultural method and the PCR assay. Results: Using cultural method, 19 samples (7.3% were positive for Listeria spp. The highest prevalence of Listeria was found in raw water buffalo milk (11.8%, followed by raw bovine milk (10.6%, raw ovine milk (7.1%, and raw caprine milk (4.2% samples. All 37 camel milk samples from 20 camel breeding farms were negative for Listeria spp. The overall prevalence of Listeria was 7.3%, in which Listeria innocua was the most recovered species (4.2%; the remaining isolates were Listeria monocytogenes (1.9%, Listeria ivanovii (0.08% and Listeria seeligari (0.04%. The PCR assay could identify 8 Listeria-contaminated milk samples that were negative using the cultural method. Conclusions: The results presented in this study indicate the potential risk of infection with Listeria in people consuming raw and unpasteurized milk.

  14. Inhibitory effects of organochlorine pesticides on intercellular transfer of Lucifer Yellow in cultured bovine oviductal cells.

    Science.gov (United States)

    Tiemann, U; Pöhland, R

    1999-01-01

    The present study investigated the effects of dichlorodiphenyltrichloroethane (DDT), methoxychlor (MXC), and gamma-hexachlorocyclohexane (gammaHCH, lindane) on gap junctional intercellular communication (GJIC) in cultured bovine oviductal cells. GJIC was evaluated by microinjecting fluorescent dye Lucifer Yellow and observing the inhibition of the spreading of dye into adjacent cells. After incubation for 1 h at 37 degrees C, a dose-dependent inhibition of GJIC was observed over a concentration range of 16 to 128 microM DDT, MXC, or gammaHCH compared with nonexposed controls. A significant inhibition began at 32 microM DDT, MXC, or gammaHCH. After incubation for 5 h, a dose-dependent inhibition of GJIC was obtained in the concentration range from 8 to 64 microM of the pesticides. The first significant inhibitory effect on GJIC was caused by 8 microM DDT, 16 microM MXC, and 32 microM gammaHCH. The 128 microM concentration of the pesticides was toxic. At pesticide concentration of 64 microM, the decrease in dye-coupling observed was not due to lethal cell injury, as is indicated by the use of trypan blue dye exclusion. After removal of 64 microM DDT from the culture medium, intercellular communication was reestablished within 3 h. Measurement of cytosolic free Ca2+ concentration [Ca2+]i in fura-2/AM-loaded oviductal cells showed that the inhibition of GJIC by addition of DDT, MXC, or gammaHCH was not associated with a detectable increase in [Ca2+]i. Coincubation of the DDT with dibutyryl-cAMP prevented the 64 microM DDT-induced inhibition of intercellular communication in adherent oviduct cells. It is suggested that organochlorine pesticides can influence cells responsible for reproduction.

  15. A bovine mammary endothelial/epithelial cell culture model of the blood/milk barrier.

    Science.gov (United States)

    Guidry, A J; O'Brien, C N; Douglass, L W

    1998-04-01

    The complex nature of the mammary gland has hampered in-depth studies of the relationship of the circulatory system to cells lining the teat ducts and alveoli of the gland. This study reports an in vitro model of endothelial and epithelial cells separated by a subcellular matrix that simulates the blood milk barrier of the bovine mammary gland. Dual chamber culture dishes with a porous membrane separating the upper and lower chamber were used. Endothelial and epithelial cells were cultured on opposite sides of the porous membrane. A collagen and fibroblast subcellular matrix, separating the 2 cell layers, simulated the in vivo interstitial tissue. Changes in surface binding of anti-bodies to polymorphonuclear neutrophils (PMN) following their migration from the upper to the lower chamber simulated the passage of PMN from blood to milk. Changes in the binding of antibodies to PMN agreed with results observed following the migration of PMN from blood to milk in vivo. This gives credence to the model's potential value for studies where more direct observation of the blood/milk barrier is required. The model will be further tested for its usefulness as an assay for determining: 1) antibiotic diffusion from milk to blood and from blood to milk, 2) cytotoxicity of prophylactic and therapeutic mammary infusion products, 3) factors affecting bacterial adhesion and penetration of mammary epithelial tissue, 4) effectiveness of antibodies present in lacteal secretions in preventing bacterial adhesion, and 5) the feasibility of gene constructs to induce synthesis and secretion of mastitis-preventing compounds and prophylactic and therapeutic compounds for treatment of human disorders.

  16. Mitochondrial DNA dynamics during in vitro culture and pluripotency induction of a bovine Rho0 cell line.

    Science.gov (United States)

    Pessôa, L V F; Bressan, F F; Chiaratti, M R; Pires, P R L; Perecin, F; Smith, L C; Meirelles, F V

    2015-10-30

    Large number of cellular changes and diseases are related to mutations in the mitochondrial DNA copy number. Cell culture in the presence of ethidium bromide is a known way of depleting mitochondrial DNA and is a useful model for studying such conditions. Interestingly, the morphology of these depleted cells resembles that of pluripotent cells, as they present larger and fragmented mitochondria with poorly developed cristae. Herein, we aimed to study the mechanisms responsible for the control of mitochondrial DNA replication during mitochondrial DNA depletion mediated by ethidium bromide and during the in vitro induction of cellular pluripotency with exogenous transcription factor expression in a bovine model. This article reports the generation of a bovine Rho0 mesenchymal cell line and describes the analysis of mitochondrial DNA copy number in a time-dependent manner. The expression of apoptosis and mitochondrial-related genes in the cells during mitochondrial DNA repletion were also analyzed. The dynamics of mitochondrial DNA during both the depletion process and in vitro reprogramming are discussed. It was possible to obtain bovine mesenchymal cells almost completely depleted of their mitochondrial DNA content (over 90%). However, the production of induced pluripotent stem cells from the transduction of both control and Rho0 bovine mesenchymal cells with human reprograming factors was not successful.

  17. Pock forming ability of fowl pox virus isolated from layer chicken and its adaptation in chicken embryo fibroblast cell culture

    OpenAIRE

    Gilhare, Varsha Rani; Hirpurkar, S. D.; Kumar, Ashish; Naik, Surendra Kumar; Sahu, Tarini

    2015-01-01

    Aim: The objective of the present study was to examine pock forming ability of field strain and vaccine strain of fowl pox virus (FPV) in chorioallantoic membrane (CAM) of embryonated chicken eggs and its adaptation in chicken embryo fibroblast (CEF) cell culture. Materials and Methods: Dry scabs were collected from 25 affected birds in glycerin-saline and preserved at 4°C until processed. Virus was isolated in 10-day-old embryonated chicken eggs by dropped CAM method. The identity of the ...

  18. Biotechnological characteristics of callusogenesis in maize immature embryo culture under the influence of abscisic acid and 6-benzylaminopurine

    Directory of Open Access Journals (Sweden)

    O. E. Abraimova

    2010-02-01

    Full Text Available The effect of abscisic acid and 6-benzylaminopurine on the induction of callus tissue in maize immature embryo culture was studied. For the majority of investigated genotypes abscisic acid stimulated, but 6-benzylaminopurine inhibited the formation of morphogenic calli in induction medium. It was noted that genotype appeared to be an important factor that determined the character of the influence of phytohormonal composition of the medium on the induction of the specific types of calli. Using of 0.04-0.10 mg/l abscisic acid is recommended for biotechnological production of morphogenic callus tissue in dependence of donor plant genotype.

  19. Proteomic analysis of bovine blastocoel fluid and blastocyst cells

    DEFF Research Database (Denmark)

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

    2014-01-01

    development, little information is available about the protein complement of early embryos. Modern, sensitive proteomic technology (nano HPLC tandem mass spectrometry) allowed us to describe the proteome of the scarce blastocoel fluid and cell material of expanded bovine blastocysts isolated......Abstract The understanding of the early mammalian development is a prerequisite for the advancement of in vitro fertilization and improvement of derivation and culturing of embryonic stem cells. While, whole genome transcriptomic analysis on bovine blastocysts has identified genes active in early...... 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....

  20. Comparing 36.5°C with 37°C for human embryo culture: a prospective randomized controlled trial.

    Science.gov (United States)

    Fawzy, Mohamed; Emad, Mai; Gad, Mostafa A; Sabry, Mohamed; Kasem, Hesham; Mahmoud, Manar; Bedaiwy, Mohamed A

    2018-03-27

    This prospective, double-blind, randomized controlled trial was designed to evaluate the efficacy of a culture temperature of 36.5°C versus 37°C on human embryo development in vitro. A total of 412 women undergoing IVF were randomized to two groups: the oocytes and embryos of the intervention group were cultured at 36.5°C; those of the control group were cultured at 37°C. Although no significant effect of culture temperature was observed on pregnancy or implantation rates, differences were found in embryo development. Embryo culture at 36.5°C was associated with a significantly higher cleavage rate (OR 1.6, 95% CI 1.03 to 2.51), but a lower fertilization rate, fewer high-quality embryos on day 3, a lower blastocyst formation rate on day 5, and fewer high-quality and cryopreserved blastocysts (OR 0.87, 95% CI 0.78 to 0.98), (OR 0.60, 95% CI 0.53 to 0.69), (OR 0.85, 95% CI 0.75 to 0.97), (OR 0.5, 95% CI 0.44 to 0.56) and (OR 0.77, 95% CI 0.68 to 0.88), respectively, compared with 37°C. On the basis of these results, and in the absence of data on the optimal temperature for each stage of embryo development in vitro, we recommend continuation of the use of 37°C for human embryo culture. Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  1. Developmental Toxicity Assay for Food Additive Tartrazine Using Zebrafish ( Danio rerio) Embryo Cultures.

    Science.gov (United States)

    Joshi, Vani; Katti, Pancharatna

    Tartrazine (TTZ) is an azo dye used as a colorant in food products, drugs, and cosmetics. The present study evaluates the impacts of TTZ on embryonic development of zebrafish ( Danio rerio). Laboratory-raised D. rerio embryos (n = 20/concentration) were exposed to graded dilutions of TTZ (0, 0.1, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 75, and 100 mM) from gastrulation stage (5.25 hours postfertilization [hpf]) until hatching and developmental trajectory was traced up to day 7. The no observed effect concentration (NOEC), median lethal concentration (LC 50 ), median effective concentration (EC 50 ), and teratogenic index (TI) were calculated. Exposure of embryos to effects; 20 to 30 mM TTZ caused tail bending, cardiac and yolk sac edema in 50% of larvae; in 30 to 50 mM TTZ-exposed embryos the heart rates declined along with the above mentioned deformities, causing mortality within 96 to 144 hpf; development ceased completely at 75 to 100 mM concentration. The NOEC and LC 50 were recorded at 5 and 29.4 mM dose, respectively. The EC 50 values for heart rate, cardiac edema, tail bending, and hatching success were at 59.60, 53.81, 98.28, and 58.97 mM with TI quotient 0.49, 0.54, 0.29, and 0.49, respectively. We conclude that TTZ is not embryo toxic/teratogenic for zebrafish embryos up to a dose level of 10 mM concentration.

  2. In vitro culture and characterization of putative porcine embryonic germ cells derived from domestic breeds and yucatan mini pig embryos at days 20-24 of gestation

    DEFF Research Database (Denmark)

    Petkov, Stoyan Gueorguiev; Marks, Hendrik; Klein, Tino

    2011-01-01

    Embryonic germ cells (EGC) are cultured pluripotent cells derived from primordial germ cells (PGC). This study explored the possibility of establishing porcine EGC from domestic breeds and Yucatan mini pigs using embryos at Days 17-24 of gestation. In vitro culture of PGC from both pooled...

  3. Effects of Moraxella (Branhamella) ovis culture filtrates on bovine erythrocytes, peripheral mononuclear cells, and corneal epithelial cells.

    Science.gov (United States)

    Cerny, Henry E; Rogers, Douglas G; Gray, Jeffrey T; Smith, David R; Hinkley, Susanne

    2006-03-01

    Infectious bovine keratoconjunctivitis (IBK) is a highly contagious ocular disease that affects cattle of all ages and that occurs worldwide. Piliated hemolytic Moraxella bovis is recognized as the etiologic agent of IBK. According to data from the Nebraska Veterinary Diagnostic Laboratory System, however, Moraxella (Branhamella) ovis has been isolated with increasing frequency from cattle affected with IBK. The objective of this study was, therefore, to examine M. ovis field isolates for the presence of the putative virulence factors of M. bovis. Culture filtrates from selected M. ovis field isolates demonstrated hemolytic activity on bovine erythrocytes and cytotoxic activity on bovine peripheral blood mononuclear cells and corneal epithelial cells. The hemolytic activity of the culture filtrates was attenuated after heat treatment. Polyclonal antibodies raised against the M. bovis hemolysin-cytotoxin also recognized a protein of approximately 98 kDa in a Western blot assay. These data indicate that the M. ovis field isolates examined produce one or more heat-labile exotoxins and may suggest that M. ovis plays a role in the pathogenesis of IBK.

  4. Effects of Moraxella (Branhamella) ovis Culture Filtrates on Bovine Erythrocytes, Peripheral Mononuclear Cells, and Corneal Epithelial Cells†

    Science.gov (United States)

    Cerny, Henry E.; Rogers, Douglas G.; Gray, Jeffrey T.; Smith, David R.; Hinkley, Susanne

    2006-01-01

    Infectious bovine keratoconjunctivitis (IBK) is a highly contagious ocular disease that affects cattle of all ages and that occurs worldwide. Piliated hemolytic Moraxella bovis is recognized as the etiologic agent of IBK. According to data from the Nebraska Veterinary Diagnostic Laboratory System, however, Moraxella (Branhamella) ovis has been isolated with increasing frequency from cattle affected with IBK. The objective of this study was, therefore, to examine M. ovis field isolates for the presence of the putative virulence factors of M. bovis. Culture filtrates from selected M. ovis field isolates demonstrated hemolytic activity on bovine erythrocytes and cytotoxic activity on bovine peripheral blood mononuclear cells and corneal epithelial cells. The hemolytic activity of the culture filtrates was attenuated after heat treatment. Polyclonal antibodies raised against the M. bovis hemolysin-cytotoxin also recognized a protein of approximately 98 kDa in a Western blot assay. These data indicate that the M. ovis field isolates examined produce one or more heat-labile exotoxins and may suggest that M. ovis plays a role in the pathogenesis of IBK. PMID:16517853

  5. Morphological changes in cultured bovine lymphoid cell lines associated with bovine viral diarrhea virus (BVDV) single and dual infections with bovine leukemia virus (BLV)

    Science.gov (United States)

    Currently, American Type Culture Collection (ATCC) makes available two cell lines derived from the same lymphoblast-like suspension cell that have been confirmed by next-generation sequencing and RT-PCR to have either a single contaminate of BVDV2a (CRL-8037) or dual contaminates of both BVDV and BL...

  6. Genotoxicity determinations of coriander drop and extract of Coriander Sativum cultured fibroblast of rat embryo by comet assay

    International Nuclear Information System (INIS)

    Heibatullah, K.; Marzieh, P.; Arefeh, I.; Ebrahim, M.

    2008-01-01

    The single cell gel electrophoresis (SCGE) or comet assay is a quick, simple and sensitive technique for measuring DNA damage in cell nucleus. It is well known that medicinal herbs play an important role in the life of human beings, thus it is essential to determine their safety as public health is concerned. In this study the genotoxicity of Coriander drop, herbal pharmaceutical product, and the extract of Coriander sativum were examined in cultured fibroblast of rat embryo using comet assay. The thirteen to fifteen days old rat embryos were lysed with tripsin and after certain steps it was centrifuged and then cultured. After three to five passages, different concentrations of each product were applied to the fibroblasts. Lysing, electrophoresis, neutralization and staining were carried out. Finally the slides were analyzed with fluorescence microscope. In the test groups the results indicated that coriander drop at different doses showed some fragmentation of DNA but this damage as a result was deemed to be not significant. However, in the case of Coriander sativum extract the results showed no mutagenic effects in comparison with the positive control group (p<0.05). In conclusion, these herbal products did not show any magnetic effect according to our test, but further genotoxicity assays are recommended. (author)

  7. Global gene transcription patterns in in vitro-cultured fertilized embryos and diploid and haploid murine parthenotes

    International Nuclear Information System (INIS)

    Cui Xiangshun; Li Xingyu; Kim, Nam-Hyung

    2007-01-01

    To gain insights into the roles the paternal genome and chromosome number play in pre-implantation development, we cultured fertilized embryos and diploid and haploid parthenotes (DPs and HPs, respectively), and compared their development and gene expression patterns. The DPs and fertilized embryos did not differ in developmental ability but HPs development was slower and characterized by impaired compaction and blastocoel formation. Microarray analysis revealed that fertilized blastocysts expressed several genes at higher levels than DP blastocysts; these included the Y-chromosome-specific gene eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked (Eif2s3y) and the imprinting gene U2 small nuclear ribonucleoprotein auxiliary factor 1, related sequence 1 (U2af1-rs1). We also found that when DPs and HPs were both harvested at 44 and 58 h of culture, they differed in the expression of 38 and 665 genes, respectively. However, when DPs and HPs were harvested at the midpoints of 4-cell stage (44 and 49 h, respectively), no differences in expression was observed. Similarly, when the DPs and HPs were harvested when they became blastocysts (102 and 138 h, respectively), only 15 genes showed disparate expression. These results suggest that while transcripts needed for early development are delayed in HPs, it does progress sufficiently for the generation of the various developmental stages despite the lack of genetic components

  8. On the Future Advances in Engineering and In-Vitro Culture of Human Embryos

    OpenAIRE

    Anton, Roman

    2016-01-01

    The far-reaching socio-economic, the censored methodologic-scientific, and the forgotten moral-ethical prospects of the lately emerging "human engineering techniques" are newly discussed here. The unanimously unmentioned future "human embryo-based (embryogenic) transplantation methods" are first claimed here as a potential and very powerful biomedical strategy that could now become enabled in the future due to these recent new protocols that could cause an unseen political and societal dilemm...

  9. Human Sperm Bioassay for Reprotoxicity Testing in Embryo Culture Media: Some Practical Considerations in Reducing the Assay Time

    Directory of Open Access Journals (Sweden)

    Amjad Hossain

    2010-01-01

    Full Text Available Human sperm assay (HSA is a preferred in house quality control and proficiency test (PT practiced in fertility laboratories. HSA is performed over varying durations, apparently without following set criteria. To better understand the assay time required for reprotoxicity testing in embryo culture media, we compared American-Association-of-Bioanalysts-(AAB- administered HSA data to our own assay performed using PT samples obtained from AAB. Participating laboratories were required to culture sperm for 48 hours to determine media acceptability. Conclusions drawn from 48- and 24-hour observations were the same, suggesting that HSA could identify reprotoxic media in less time than required by AAB. Our assay revealed that changes in motility grade in adulterated media are significantly different from those in control media. Furthermore, grade changes can be identified earlier than differences in motility loss between samples. Analyzing motility and motility quality together provides a method for establishing an optimal time for HSA.

  10. Effect of the well of the well (WOW) system on in vitro culture for porcine embryos after intracytoplasmic sperm injection.

    Science.gov (United States)

    Taka, Mikiko; Iwayama, Hiroshi; Fukui, Yutaka

    2005-08-01

    For developmental competence of porcine embryos in vitro, it is important to improve the culture environment. The present study was performed to evaluate four different culture systems for in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI); drop, well and two sizes of the well of the well (WOW) systems (500 and 1,000 microm in diameter). The cleavage rate on Day 2 and the mean cell number in blastocysts on Day 6 were not significantly different among the four treatments. However, the 1,000 microm WOW (24.6%) resulted in a significantly higher (PWOW, respectively). The present study indicates that the microenvironment created by the 1,000 microm diameter WOW improves blastocyst production of in vitro matured porcine oocytes after ICSI, and that the effectiveness of the WOW system is dependent on the size (diameter) of the WOW.

  11. Effects of ulipristal acetate on human embryo attachment and endometrial cell gene expression in an in vitro co-culture system.

    Science.gov (United States)

    Berger, C; Boggavarapu, N R; Menezes, J; Lalitkumar, P G L; Gemzell-Danielsson, K

    2015-04-01

    Does ulipristal acetate (UPA) used for emergency contraception (EC) interfere with the human embryo implantation process? UPA, at the dosage used for EC, does not affect human embryo implantation process, in vitro. A single pre-ovulatory dose of UPA (30 mg) acts by delaying or inhibiting ovulation and is recommended as first choice among emergency contraceptive pills due to its efficacy. The compound has also been demonstrated to have a dose-dependent effect on the endometrium, which theoretically could impair endometrial receptivity but its direct action on human embryo implantation has not yet been studied. Effect of UPA on embryo implantation process was studied in an in vitro endometrial construct. Human embryos were randomly added to the cultures and cultured for 5 more days with UPA (n = 10) or with vehicle alone (n = 10) to record the attachment of embryos. Endometrial biopsies were obtained from healthy, fertile women on cycle day LH+4 and stromal and epithelial cells were isolated. A three-dimensional in vitro endometrial co-culture system was constructed by mixing stromal cells with collagen covered with a layer of epithelial cells and cultured in progesterone containing medium until confluence. The treatment group received 200 ng/ml of UPA. Healthy, viable human embryos were placed on both control and treatment cultures. Five days later the cultures were tested for the attachment of embryos and the 3D endometrial constructs were analysed for endometrial receptivity markers by real-time PCR. There was no significant difference in the embryo attachment rate between the UPA treated group and the control group as 5 out of 10 human embryos exposed to UPA and 7 out of 10 embryos in the control group attached to the endometrial cell surface (P = 0.650). Out of 17 known receptivity genes studied here, only 2 genes, HBEGF (P = 0.009) and IL6 (P = 0.025) had a significant up-regulation and 4 genes, namely HAND2 (P = 0.003), OPN (P = 0.003), CALCR (P = 0.016) and

  12. Addition of L-ascorbic acid to culture and vitrification media of IVF porcine blastocysts improves survival and reduces HSPA1A levels of vitrified embryos.

    Science.gov (United States)

    Castillo-Martín, Miriam; Yeste, Marc; Soler, Albert; Morató, Roser; Bonet, Sergi

    2015-09-01

    The aim of the present study was to determine the effect of L-ascorbic acid on embryo quality and gene expression of porcine blastocysts after supplementations of in vitro culture medium and/or vitrification-warming media. Embryo quality, in terms of total cell number (TCN), DNA fragmentation and peroxide levels, together with the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), POU class 5 homeobox 1 (POU5F1) and heat shock protein 70 (HSPA1A), was analysed. In Experiment 1, gene expression and embryo quality of fresh blastocysts were evaluated after culture with or without L-ascorbic acid; no significant differences were observed between the groups. In Experiment 2, blastocysts cultured with or without L-ascorbic acid were vitrified using two different vitrification solutions, supplemented or not with L-ascorbic acid. Supplementation of culture and vitrification media significantly enhanced survival rates and reduced peroxide levels. No significant differences in TCN, DNA fragmentation and BAX, BCL2L1 and POU5F1 expression were found in vitrified blastocysts among experimental groups. Vitrification procedures increase HSPA1A transcript abundance, but this increase was significantly lower in embryos cultured and/or vitrified with L-ascorbic acid. Thus, supplementing culture and/or vitrification media with L-ascorbic acid enhances survival rates of porcine blastocysts, suggesting a relationship with HSPA1A expression.

  13. Embryotoxic and pharmacologic potency ranking of six azoles in the rat whole embryo culture by morphological and transcriptomic analysis.

    Science.gov (United States)

    Dimopoulou, Myrto; Verhoef, Aart; Pennings, Jeroen L A; van Ravenzwaay, Bennard; Rietjens, Ivonne M C M; Piersma, Aldert H

    2017-05-01

    Differential gene expression analysis in the rat whole embryo culture (WEC) assay provides mechanistic insight into the embryotoxicity of test compounds. In our study, we hypothesized that comparative analysis of the transcriptomes of rat embryos exposed to six azoles (flusilazole, triadimefon, ketoconazole, miconazole, difenoconazole and prothioconazole) could lead to a better mechanism-based understanding of their embryotoxicity and pharmacological action. For evaluating embryotoxicity, we applied the total morphological scoring system (TMS) in embryos exposed for 48h. The compounds tested showed embryotoxicity in a dose-response fashion. Functional analysis of differential gene expression after 4h exposure at the ID 10 (effective dose for 10% decreased TMS), revealed the sterol biosynthesis pathway and embryonic development genes, dominated by genes in the retinoic acid (RA) pathway, albeit in a differential way. Flusilazole, ketoconazole and triadimefon were the most potent compounds affecting the RA pathway, while in terms of regulation of sterol function, difenoconazole and ketoconazole showed the most pronounced effects. Dose-dependent analysis of the effects of flusilazole revealed that the RA pathway related genes were already differentially expressed at low dose levels while the sterol pathway showed strong regulation at higher embryotoxic doses, suggesting that this pathway is less predictive for the observed embryotoxicity. A similar analysis at the 24-hour time point indicated an additional time-dependent difference in the aforementioned pathways regulated by flusilazole. In summary, the rat WEC assay in combination with transcriptomics could add a mechanistic insight into the embryotoxic potency ranking and pharmacological mode of action of the tested compounds. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Non-invasive assessment of culture media from goat cloned embryos associated with subjective morphology by gas chromatography - mass spectroscopy-based metabolomic analysis.

    Science.gov (United States)

    Zhang, Yan-Li; Zhang, Guo-Min; Jia, Ruo-Xin; Wan, Yong-Jie; Yang, Hua; Sun, Ling-Wei; Han, Le; Wang, Feng

    2018-01-01

    Pre-implantation embryo metabolism demonstrates distinctive characteristics associated with the development potential of embryos. We aim to determine if metabolic differences correlate with embryo morphology. In this study, gas chromatography - mass spectroscopy (GC-MS)-based metabolomics was used to assess the culture media of goat cloned embryos collected from high-quality (HQ) and low-quality (LQ) groups based on morphology. Expression levels of amino acid transport genes were further examined by quantitative real-time PCR. Results showed that the HQ group presented higher percentages of blastocysts compared with the LQ counterparts (P culture media of the HQ group showed lower levels of valin, lysine, glutamine, mannose and acetol, and higher levels of glucose, phytosphingosine and phosphate than those of the LQ group. Additionally, expression levels of amino acid transport genes SLC1A5 and SLC3A2 were significantly lower in the HQ group than the LQ group (P culture media. The biochemical profiles may help to select the most in vitro viable embryos. © 2017 Japanese Society of Animal Science.

  15. Cytokinins and coconut water promoted abnormalities in zygotic embryo culture of oil palm

    Directory of Open Access Journals (Sweden)

    Suhaimine Chaemalee

    2011-12-01

    Full Text Available Induction of adventitious shoot formation from mature zygotic embryo of oil palm was carried out in liquid MS mediumsupplemented with various types of cytokinins. Kinetin (KN alone at concentration of 0.5 mg/l gave the highest adventitiousshoot formation at 13.4%. However, abnormal shoots in form of inflorescence-like structure (ILS were obtained in 5mg/l KN containing medium. For coconut water a big ILSs were formed at 10.6%. Histological studied revealed that thoseinflorescences had no clear floral organs.

  16. A Functional Assay for Putative Mouse and Human Definitive Endoderm using Chick Whole-Embryo Cultures

    DEFF Research Database (Denmark)

    Johannesson, Martina; Semb, Tor Henrik; Serup, Palle

    2012-01-01

    Introduction: Embryonic stem cells (ESCs) represent a prospective cell source for treating degenerative diseases such as diabetes. Several studies have addressed the generation of definitive endoderm (DE) from this cell source by attempting to recapitulate the signaling events occurring during....... Thus, the purpose of this study is to describe a method whereby the in vivo functionality of DE derived from ESCs can be assessed. Methods: By directed differentiation, putative DE was derived from human and mouse ESCs. This putative DE was subsequently transplanted into the endoderm of chick embryos...

  17. 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.

  18. Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes.

    Science.gov (United States)

    Matsukawa, Kazutsugu; Turco, Margherita Yayoi; Scapolo, Pier Augusto; Reynolds, Lawrence; Ptak, Grazyna; Loi, Pasqualino

    2007-01-01

    Androgenetic embryos are useful model for investigating the contribution of the paternal genome to embryonic development. Little work has been done with androgenetic embryo production in domestic animals. The aim of this study was the production of diploid androgenetic sheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro; parthenogenetic and normally fertilized embryos were also produced as a control. Fifteen hours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored for the number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid, and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportion of oocytes with polyspermic fertilization increased linearly with increasing sperm concentration. After IVF, there was no significant difference in early cleavage and morula formation rates between the groups, while there was a significant difference on blastocyst development between IVF, parthenogenetic, and androgenetic embryos, the last ones displaying poor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploid androgenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgenetic embryonic development, we produced diploid androgenetic embryos through pronuclear transfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploid embryos and transferred into the perivitelline space of other haploid embryos, and the zygotes were reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfer significantly increased blastocyst development (IVF, parthenogenetic, androgenetic: Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly, the Haploid + Diploid group showed the highest development to blastocyst stage. The main findings of our study are: (1) sheep androgenetic embryos display poor developmental ability compared with

  19. Commonly used fertility drugs, a diet supplement, and stress force AMPK-dependent block of stemness and development in cultured mammalian embryos.

    Science.gov (United States)

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

    2016-08-01

    The purpose of the present study is to test whether metformin, aspirin, or diet supplement (DS) BioResponse-3,3'-Diindolylmethane (BR-DIM) can induce AMP-activated protein kinase (AMPK)-dependent potency loss in cultured embryos and whether metformin (Met) + Aspirin (Asa) or BR-DIM causes an AMPK-dependent decrease in embryonic development. The methods used were as follows: culture post-thaw mouse zygotes to the two-cell embryo stage and test effects after 1-h AMPK agonists' (e.g., Met, Asa, BR-DIM, control hyperosmotic stress) exposure on AMPK-dependent loss of Oct4 and/or Rex1 nuclear potency factors, confirm AMPK dependence by reversing potency loss in two-cell-stage embryos with AMPK inhibitor compound C (CC), test whether Met + Asa (i.e., co-added) or DS BR-DIM decreases development of two-cell to blastocyst stage in an AMPK-dependent (CC-sensitive) manner, and evaluate the level of Rex1 and Oct4 nuclear fluorescence in two-cell-stage embryos and rate of two-cell-stage embryo development to blastocysts. Met, Asa, BR-DIM, or hyperosmotic sorbitol stress induces rapid ~50-85 % Rex1 and/or Oct4 protein loss in two-cell embryos. This loss is ~60-90 % reversible by co-culture with AMPK inhibitor CC. Embryo development from two-cell to blastocyst stage is decreased in culture with either Met + Asa or BR-DIM, and this is either >90 or ~60 % reversible with CC, respectively. These experimental designs here showed that Met-, Asa-, BR-DIM-, or sorbitol stress-induced rapid potency loss in two-cell embryos is AMPK dependent as suggested by inhibition of Rex1 and/or Oct4 protein loss with an AMPK inhibitor. The DS BR-DIM or fertility drugs (e.g., Met + Asa) that are used to enhance maternal metabolism to support fertility can also chronically slow embryo growth and block development in an AMPK-dependent manner.

  20. Embryotoxic potential of N-methyl-pyrrolidone (NMP) and three of its metabolites using the rat whole embryo culture system.

    Science.gov (United States)

    Flick, Burkhard; Talsness, Chris E; Jäckh, Rudolf; Buesen, Roland; Klug, Stephan

    2009-06-01

    N-methyl-2-pyrrolidone (NMP), which undergoes extensive biotransformation, has been shown in vivo to cause developmental toxicity and, especially after oral treatment, malformations in rats and rabbits. Data are lacking as to whether the original compound or one of its main metabolites is responsible for the toxic effects observed. Therefore, the relative embryotoxicity of the parent compound and its metabolites was evaluated using rat whole embryo culture (WEC) and the balb/c 3T3 cytotoxicity test. The resulting data were evaluated using two strategies; namely, one based on using all endpoints determined in the WEC and the other including endpoints from both the WEC and the cytotoxicity test. On basis of the first analysis, the substance with the highest embryotoxic potential is NMP, followed by 5-hydroxy-N-methyl-pyrrolidone (5-HNMP), 2-hydroxy-N-methylsuccinimide (2-HMSI) and N-methylsuccinimide (MSI). Specific dysmorphogeneses induced by NMP and 5-HNMP were aberrations in the head region of the embryos, abnormal development of the second visceral arches and open neural pores. The second evaluation strategy used only two endpoints of the WEC, i.e. the no observed adverse effect concentration (NOAEC(WEC)) and the lowest concentration leading to dysmorphogenesis in 100% of the cultured embryos (IC(Max WEC)). In addition to these WEC endpoints the IC(50 3T3) from the cytotoxicity test (balb/c 3T3 fibroblasts) was included in the evaluation scheme. These three endpoints were applied to a prediction model developed during a validation study of the European Centre for the Validation of Alternative Methods (ECVAM) allowing the classification of the embryotoxic potential of each compound into three classes (non-, weakly- and strongly embryotoxic). Consistent results from both evaluation strategies were observed, whereby NMP and its metabolites revealed a direct embryotoxic potential. Hereby, only NMP and 5-HNMP induced specific embryotoxic effects and were classified as

  1. Efeito do ibuprofeno administrado uma hora antes da inovulação de embriões bovinos Effect of ibuprofen administered one hour before the bovine embryo transfer

    Directory of Open Access Journals (Sweden)

    H.J. Narváez

    2010-06-01

    Full Text Available Avaliou-se o efeito do ibuprofeno administrado uma hora antes da inovulação de embriões bovinos, com o objetivo de melhorar a taxa de prenhez. Após a avaliação da resposta ao protocolo de sincronização do estro, 76 fêmeas selecionadas como receptoras de embriões foram distribuídas em três grupos (G experimentais: G1 (n=25 receptoras usadas como controle, G2 (n=30 receptoras que receberam ibuprofeno 5mg/kg, I.M, uma hora antes da inovulação dos embriões, e G3 (n=21 receptoras que receberam uma matriz polimérica de liberação controlada de ibuprofeno administrado por via subcutânea. As taxas de prenhez foram de 16% (4/25, 43,3% (13/30 e 14,2% (3/21, para G1, G2 e G3, respectivamente. Observou-se diferença (PThe effect of the administered ibuprofen was evaluated one hour before the embryo transfer of bovine embryos in order to improve pregnancy rates. After evaluating the response to protocol synchronization of estrus, 76 Females selected as the recipients of embryos were distributed into three experimental groups: G1 (n = 25 surrogate cows used as control, G2 (n = 30 surrogate cows that received 5mg/kg ibuprofen, IM, one hour before the embryo transfer, and G3 (n = 20 surrogate cows that received an array polymeric release of controlled ibuprofen subcutaneously administered. The pregnancy rates were 16% (4/25, 43.3% (13/30, and 14.2% (3/21 for G1, G2, and G3, respectively. There was statistical difference (P<0.024 on pregnancy rate of G2, in comparison with those of G1 and G3. The administration of ibuprofen intramuscularly one hour before the embryo transfer resulted in better pregnancy rate in Nellore surrogate cows.

  2. DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

    International Nuclear Information System (INIS)

    Yaki, T.

    1982-01-01

    DNA repair in mouse cells derived from embryos of 3 inbred strains were investigated in comparison with that in human cells. The levels of unscheduled DNA synthesis after UV irradiation appeared to change at different passages, but capacities of host-cell reactivation of UV-irradiated herpes simplex virus were always reduced to the same levels as those in xeroderma pigmentosum cells. This implied that mouse cells are reduced in excision-repair capacities and that the apparently high levels of unscheduled DNA synthesis at certain passages are not quantitatively related to high levels of cell survival. Essentially no differences in DNA repair were noted among 3 strains - BALB/c, C3H/He and C57BL/10. (orig.)

  3. A comparison of the embryonic stem cell test and whole embryo culture assay combined with the BeWo placental passage model for predicting the embryotoxicity of azoles.

    NARCIS (Netherlands)

    Dimopoulou, Myrto; Verhoef, Aart; Gomes, Caroline A; van Dongen, Catharina W; Rietjens, Ivonne M C M; Piersma, Aldert H; van Ravenzwaay, Bennard

    2018-01-01

    In the present study, we show the value of combining toxico-dynamic and -kinetic in vitro approaches for embryotoxicity testing of azoles. Both the whole embryo culture (WEC) and the embryonic stem cells test (EST) predicted the in vivo potency ranking of twelve tested azoles with moderate accuracy.

  4. A comparison of the embryonic stem cell test and whole embryo culture assay combined with the BeWo placental passage model for predicting the embryotoxicity of azoles

    NARCIS (Netherlands)

    Dimopoulou, Myrto; Verhoef, Aart; Gomes, Caroline A.; Dongen, van Catharina W.; Rietjens, Ivonne M.C.M.; Piersma, Aldert H.; Ravenzwaay, van Bennard

    2018-01-01

    In the present study, we show the value of combining toxico-dynamic and -kinetic in vitro approaches for embryotoxicity testing of azoles. Both the whole embryo culture (WEC) and the embryonic stem cells test (EST) predicted the in vivo potency ranking of twelve tested azoles with moderate accuracy.

  5. Effect of arachidonic acid supplementation and cyclooxygenase/lipoxygenase inhibition on the development of early bovine embryos Influência do ácido araquidónico e da inibição da ciclo-oxigenase ou lipo-oxigenase no desenvolvimento inicial de embriões bovinos

    Directory of Open Access Journals (Sweden)

    Rosa Maria Pereira

    2006-04-01

    Full Text Available The effect of arachidonic acid (AA cascade on bovine embryo development in a granulosa cell co-culture system was studied. Arachidonic acid (100 µM was supplemented from 1-cell to 8-16 cell block stage (first three days of co-culture and from 1-cell to hatching. Specific cyclooxygenase (indomethacin, 28 µM and lipoxygenase (nordihydroguaiaretic acid - NDGA, 28 µM inhibitors were used from 1-cell to 8-16 cell block stage with AA. Embryo development was assessed by cleavage, day 7-day 8 and hatched embryo rates and by measuring growth rates through development stages found in days 7-10 of culture (day 0 = insemination day. Embryo quality was scored at day 8. A 6.5-10.4% increase on cleavage rate after AA supplementation was found. This AA supplementation from 1-cell to hatching delayed embryo growth rate beyond day 7 and a reduction on hatching rate was detected. When AA supplementation was restricted to the first three days of co-culture those negative effects were overcome. Also, indomethacin and NDGA prevented the positive effect of AA and induced a significant reduction on cleavage, respectively. NDGA further decreased day 7 embryo rate and quality. Results suggest that AA has a two-phase action on bovine embryos, promoting early development and impairing embryo growth from day 7 onwards and hatching rates. Both cyclooxygenase and lipoxygenase were found to be important pathways to promote cleavage.Estudou-se a influência da cascata do ácido araquidónico (AA no desenvolvimento de embriões bovinos produzidos in vitro em co-cultura com células da granulosa. Os embriões foram suplementados com AA (100 µM desde o estádio de 1 célula até 8-16 células (primeiros três dias de co-cultura ou até a eclosão. Introduziram-se inibidores específicos da ciclo-oxigenase (indometacina, 28 µM e da lipo-oxigenase (ácido nordihidroguaiarético - NDGA, 28 µM, juntamente com o ácido araquidónico, desde o estádio de 1 célula até 8-16 c

  6. Uso da uréia como suplemento protéico na dieta de doadoras e receptoras de embriões bovinos Urea as a protein supplementation in the diet of bovine embryo donors and recipients

    Directory of Open Access Journals (Sweden)

    Amílcar Gasperin Barreto

    2003-02-01

    degenerated embryos (0.5, 1.0 and 1.83, as well as in vitro eclosion rate (81.48, 78.57 and 84.62%, for the groups S, S+U and U, respectively. The 66 recipients were kept on Braquiaria decumbens pasture with 1.25kg of concentrate supplements for the S, S+U and U groups. Frozen embryos were thawed and transferred after 37 days. There was no significant statistical difference in pregnancy rates at 30 days (25, 28 and 28.57%, and 60 days of pregnancy (16.67, 28 and 25%. It may be concluded that urea can replace the soybean meal in concentrated rations for supplementation of bovine embryo donors and recipients since there were no negative effects in embryo quality, eclosion rate and recipient fertility.

  7. Paternal breed effects on expression of IGF-II, BAK1 and BCL2-L1 in bovine preimplantation embryos

    DEFF Research Database (Denmark)

    Valleh, Mehdi Vafaye; Tahmoorespur, Mojtaba; Joupari, Morteza Daliri

    2015-01-01

    Summary The effects of the paternal breed on early embryo and later pre- and postnatal development are well documented. Several recent studies have suggested that such paternal effects may be mediated by the paternally induced epigenetic modifications during early embryogenesis. The objective of ...

  8. An investigation into the feasibility of culturing rat embryos in media ...

    African Journals Online (AJOL)

    than optimal. Little is known about the influence of culture envi- ronment on the medium parameters of pH, PCOr, pOr, bicarbo- nate levels, glucose content and osmolarity during 48-h culture. .... regression curves, various growth rates were determined relative ..... method with continuous replacement of the gas phase.

  9. Embryo splitting

    OpenAIRE

    Karl Illmensee; Mike Levanduski

    2010-01-01

    Mammalian embryo splitting has successfully been established in farm animals. Embryo splitting is safely and efficiently used for assisted reproduction in several livestock species. In the mouse, efficient embryo splitting as well as single blastomere cloning have been developed in this animal system. In nonhuman primates embryo splitting has resulted in several pregnancies. Human embryo splitting has been reported recently. Microsurgical embryo splitting under Institutional Review Board appr...

  10. An investigation into the possibility of bluetongue virus transmission by transfer of infected ovine embryos

    Directory of Open Access Journals (Sweden)

    Estelle H. Venter

    2011-02-01

    Full Text Available Bluetongue (BT, a disease that affects mainly sheep, causes economic losses owing to not only its deleterious effects on animals but also its associated impact on the restriction of movement of livestock and livestock germplasm. The causative agent, bluetongue virus (BTV, can occur in the semen of rams and bulls at the time of peak viraemia and be transferred to a developing foetus. The risk of the transmission of BTV by bovine embryos is negligible if the embryos are washed according to the International Embryo Transfer Society (IETS protocol. Two experiments were undertaken to determine whether this holds for ovine embryos that had been exposed to BTV. Firstly, the oestrus cycles of 12 ewes were synchronised and the 59 embryos that were obtained were exposed in vitro to BTV-2 and BTV-4 at a dilution of 1 x 102.88 and 1 x 103.5 respectively. In the second experiment, embryos were recovered from sheep at the peak of viraemia. A total of 96 embryos were collected from BTV-infected sheep 21 days after infection. In both experiments half the embryos were washed and treated with trypsin according to the IETS protocol while the remaining embryos were neither washed nor treated. All were tested for the presence of BTV using cell culture techniques. The virus was detected after three passages in BHK-21 cells only in one wash bath in the first experiment and two unwashed embryos exposed to BTV-4 at a titre of 1 x 103.5. No embryos or uterine flush fluids obtained from viraemic donors used in the second experiment were positive for BTV after the standard washing procedure had been followed. The washing procedure of the IETS protocol can thus clear sheep embryos infected with BTV either in vitro or in vivo.

  11. Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

    International Nuclear Information System (INIS)

    Vasconcelos, R.B.; Salles, L.P.; Silva, I. Oliveira e; Gulart, L.V.M.; Souza, D.K.; Torres, F.A.G.; Bocca, A.L.; Silva, A.A.M. Rosa e

    2013-01-01

    Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E 2 ) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P 4 ) and E 2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P 4 throughout the culture period; however, P 4 concentration was significantly higher in NDM. In both media, E 2 concentration was increased at 24 h, followed by a decrease at 48 h. The E 2 :P 4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E 2 :P 4 ratio in FWS cultures

  12. Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

    Energy Technology Data Exchange (ETDEWEB)

    Vasconcelos, R.B. [Laboratório de Biotecnologia da Reprodução, Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF (Brazil); Salles, L.P. [Laboratório de Biologia Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF (Brazil); Silva, I. Oliveira e; Gulart, L.V.M. [Laboratório de Biotecnologia da Reprodução, Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF (Brazil); Souza, D.K. [Laboratório de Biotecnologia da Reprodução, Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF (Brazil); Faculdade de Ceilândia, Universidade de Brasília, Ceilândia, DF (Brazil); Torres, F.A.G. [Laboratório de Biologia Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF (Brazil); Bocca, A.L. [Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF (Brazil); Silva, A.A.M. Rosa e [Laboratório de Biotecnologia da Reprodução, Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF (Brazil)

    2013-08-16

    Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E{sub 2}) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P{sub 4}) and E{sub 2} concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P{sub 4} throughout the culture period; however, P{sub 4} concentration was significantly higher in NDM. In both media, E{sub 2} concentration was increased at 24 h, followed by a decrease at 48 h. The E{sub 2}:P{sub 4} ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E{sub 2}:P{sub 4} ratio in FWS cultures.

  13. Early embryo achievement through isolated microspore culture in Citrus clementina Hort. ex Tan., cvs. ‘Monreal Rosso’ and ‘Nules’

    Directory of Open Access Journals (Sweden)

    Benedetta eChiancone

    2015-06-01

    Full Text Available Microspore embryogenesis is a method of achieving complete homozygosity from plants. It is particularly useful for woody species, like Citrus, characterized by long juvenility, a high degree of heterozygosity and often self-incompatibility. Anther culture is currently the method of choice for microspore embryogenesis in many crops. However, isolated microspore culture is a better way to investigate the processes at the cellular, physiological, biochemical and molecular levels as it avoids the influence of somatic anther tissue. To exploit the potential of this technique, it is important to separate the key factors affecting the process and, among them, culture medium composition and particularly the plant growth regulators and their concentration, as they can greatly enhance regeneration efficiency. To our knowledge, the ability of meta-Topolin, a naturally occurring aromatic cytokinin, to induce gametic embryogenesis in isolated microspores of Citrus has never been investigated. In this study, the effect of two concentrations of meta-Topolin instead of benzyladenine or zeatin in the culture medium was investigated in isolated microspore culture of two genotypes of Citrus. After eleven months of isolated microspore culture, for both genotypes and for all the four tested media, the microspore reprogramming and their sporophytic development was observed by the presence of multinucleated calli and microspore-derived embryos at different stages. Microsatellite analysis of parental and embryo samples was performed to determine the embryo alleles constitution of early embryos produced in all tested media, confirming their origin from microspores.To our knowledge, this is the first successful report of Citrus microspore embryogenesis with isolated microspore culture in Citrus, and in particular in Citrus clementina Hort. ex Tan, cvs. ‘Monreal Rosso’ and ‘Nules’.

  14. The protein source in embryo culture media influences birthweight: a comparative study between G1 v5 and G1-PLUS v5.

    Science.gov (United States)

    Zhu, Jinliang; Li, Ming; Chen, Lixue; Liu, Ping; Qiao, Jie

    2014-07-01

    Does protein source or human serum albumin (HSA) in embryo culture media influence the subsequent birthweight? A significant difference was observed in gestational age- and gender-adjusted birthweight (Z scores) and the proportion of large-for-gestational age (LGA) babies between embryos cultured in G1 v5 and those cultured in G1-PLUS v5 media. It has been reported that the birthweights of singletons born from embryos cultured in Vitrolife are significantly higher than those cultured in the Cook group of media, and that G1-PLUS (Vitrolife, Gothenburg, Sweden) is associated with increased birth and placenta weights compared with Medicult ISMI. This study was a retrospective analysis of neonatal birthweights, and included 1097 singletons born from fresh embryo transfer cycles at the Center for Reproductive Medicine of Peking University Third Hospital between January 2011 and August 2012. The number of singletons born from G1 v5 culture media was 489, and the number of singletons born from G1-PLUS v5 media was 608. Patients media groups. The absolute birthweights for singletons resulting from G1-PLUS v5 were not different from singletons resulting from G1 v5 (3375.9 ± 479.6 g versus 3333.2 ± 491.6 g, respectively; P = 0.14). However the Z scores for singletons from embryos cultured in G1-PLUS v5 were significantly higher than for singletons cultured in G1 v5 (0.28 ± 1.12 versus 0.09 ± 1.15, respectively; P = 0.04), and more LGA babies were born from G1-PLUS v5 culture compared with G1 v5 (16.8 versus 12.1%, respectively; P = 0.03) culture. Finally, multiple linear regression analysis suggested that female weight (P = 0.00), male height (P = 0.04), gestational age at birth (P = 0.00), infant gender (P = 0.00) and culture media (P = 0.04) all had significant effects on the birthweights of singleton newborns. This study was limited by its retrospective design. Our study suggests that protein source/HSA has a significant effect on birthweights of singleton newborns

  15. Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes

    Directory of Open Access Journals (Sweden)

    Wertheimer Michael R

    2011-01-01

    Full Text Available Abstract Background Recent evidence indicates that osteoarthritis (OA may be a systemic disease since mesenchymal stem cells (MSCs from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification. We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.% of N. Methods In the present study, we examined the expression of type X collagen in fetal bovine growth plate chondrocytes (containing hypertrophic chondrocytes cultured on PPE:N. We also studied the effect of PPE:N on the expression of matrix molecules such as type II collagen and aggrecan, as well as on proteases (matrix metalloproteinase-13 (MMP-13 and molecules implicated in cell division (cyclin B2. Two other culture surfaces, "hydrophilic" polystyrene (PS, regular culture dishes and nitrogen-containing cation polystyrene (Primaria®, were also investigated for comparison. Results Results showed that type X collagen mRNA levels were suppressed when cultured for 4 days on PPE:N, suggesting that type X collagen is regulated similarly in hypertrophic chondrocytes and in human MSCs from OA patients. However, the levels of type X collagen mRNA almost returned to control value after 20 days in culture on these surfaces. Culture on the various surfaces had no significant effects on type II collagen, aggrecan, MMP-13, and cyclin B2 mRNA levels. Conclusion Hypertrophy is diminished by culturing growth plate chondrocytes on nitrogen-rich surfaces, a mechanism that is beneficial for MSC chondrogenesis. Furthermore, one major advantage of such "intelligent surfaces" over recombinant growth factors for tissue engineering and cartilage repair is potentially large cost-saving.

  16. Embryo splitting

    Directory of Open Access Journals (Sweden)

    Karl Illmensee

    2010-04-01

    Full Text Available Mammalian embryo splitting has successfully been established in farm animals. Embryo splitting is safely and efficiently used for assisted reproduction in several livestock species. In the mouse, efficient embryo splitting as well as single blastomere cloning have been developed in this animal system. In nonhuman primates embryo splitting has resulted in several pregnancies. Human embryo splitting has been reported recently. Microsurgical embryo splitting under Institutional Review Board approval has been carried out to determine its efficiency for blastocyst development. Embryo splitting at the 6–8 cell stage provided a much higher developmental efficiency compared to splitting at the 2–5 cell stage. Embryo splitting may be advantageous for providing additional embryos to be cryopreserved and for patients with low response to hormonal stimulation in assisted reproduction programs. Social and ethical issues concerning embryo splitting are included regarding ethics committee guidelines. Prognostic perspectives are presented for human embryo splitting in reproductive medicine.

  17. Chromosomal abnormality rate in human pre-embryos derived from in vitro fertilization cycles cultured in the presence of Follicular-Fluid Meiosis Activating Sterol (FF-MAS).

    Science.gov (United States)

    Bergh, Christina; Loft, Anne; Lundin, Kersti; Ziebe, Sören; Nilsson, Lars; Wikland, Matts; Gröndahl, Christian; Arce, J-C

    2004-09-01

    The objective of the study was to investigate the effect of Follicular-Fluid Meiosis Activating Sterol (FF-MAS) when added to the culture media on the incidence of chromosomal abnormalities and pre-embryo development in human pre-embryos. 243 women undergoing IVF/ICSI treatment donated 353 oocytes in a multicentre, prospective, randomized, double blind, four-arm, controlled trial performed at Danish and Swedish public and private IVF centers. Metaphase II oocytes were randomly assigned to: FF-MAS 5 microM, FF-MAS 20 microM, ethanol 0.2% (vehicle control) or water for injection (inert control). The exposure regimen of FF-MAS to the human oocytes was 4 h prior to fertilization by ICSI and 20 h exposure post ICSI. The primary endpoint was the incidence of numerical chromosomal abnormalities. Secondary endpoints were cleavage rate and pre-embryo quality. On the pre-embryo level, no significant differences in chromosomal abnormality rate were observed among the four groups. However, the percentage of uniformly normal pre-embryos was significantly lower in the pooled FF-MAS group (5 microM: 12% and 20 microM: 17%) than in the pooled control group (inert control 32% and vehicle control 42%). A high level of mosaicism (41-60%) was found in all groups. At the blastomere level, the percentage of blastomeres categorized as normal was significantly lower in the FF-MAS 5 microM group (41%) and the FF-MAS 20 microM (29%) group versus the inert (52%) and the vehicle (61%) groups. Significantly reduced cleavage and good quality pre-embryo rates were found in both FF-MAS groups. FF-MAS increased the rate of aneuploidy and had detrimental effects on cleavage and pre-embryo development, when exposed both before and after fertilization.

  18. Isolation and culture of porcine neural progenitor cells from embryos and pluripotent stem cells

    DEFF Research Database (Denmark)

    Rasmussen, Mikkel Aabech; Hall, Vanessa Jane; Hyttel, Poul

    2013-01-01

    therapy. The pig has become recognized as an important large animal model and establishment of in vitro-derived porcine NPCs would allow for preclinical safety testing by transplantation in a porcine biomedical model. In this chapter, a detailed method for isolation and in vitro culture of porcine NPCs......The isolation and culture of neural progenitor cells (NPCs) from pluripotent stem cells has facilitated in vitro mechanistic studies of diseases related to the nervous system, as well as discovery of new medicine. In addition, NPCs are envisioned to play a crucial role in future cell replacement....... The cells have the potential of long-term culture and the ability to differentiate into neural and glial cells....

  19. Toward a feline-optimized culture medium: impact of ions, carbohydrates, essential amino acids, vitamins, and serum on development and metabolism of in vitro fertilization-derived feline embryos relative to embryos grown in vivo.

    Science.gov (United States)

    Herrick, Jason R; Bond, Jennifer B; Magarey, Genevieve M; Bateman, Helen L; Krisher, Rebecca L; Dunford, Susan A; Swanson, William F

    2007-05-01

    The objective of this study was to define the physiologic needs of domestic cat embryos to facilitate development of a feline-specific culture medium. In a series of factorial experiments, in vivo-matured oocytes (n = 2040) from gonadotropin-treated domestic cats were inseminated in vitro to generate embryos (n = 1464) for culture. In the initial study, concentrations of NaCl (100.0 vs. 120.0 mM), KCl (4.0 vs. 8.0 mM), KH(2)PO(4) (0.25 vs. 1.0 mM), and the ratio of CaCl(2) to MgSO(4)-7H(2)O (1.0:2.0 mM vs. 2.0:1.0 mM) in the medium were evaluated during Days 1-6 (Day 0: oocyte recovery and in vitro fertilization [IVF]) of culture. Subsequent experiments assessed the effects of varying concentrations of carbohydrate (glucose, 1.5, 3.0, or 6.0 mM; l-lactate, 3.0, 6.0, or 12.0 mM; and pyruvate, 0.1 or 1.0 mM) and essential amino acids (EAAs; 0, 0.5, or 1.0x) in the medium during Days 1-3 and Days 3-6 of culture. Inclusion of vitamins (0 vs. 1.0x) and fetal calf serum (FCS; 0 vs. 5% [v/v]) in the medium also was evaluated during Days 3-6. Development and metabolism of IVF embryos on Day 3 or Day 6 were compared to age-matched in vivo embryos recovered from naturally mated queens. A feline-optimized culture medium (FOCM) was formulated based on these results (100.0 mM NaCl, 8.0 mM KCl, 1.0 mM KH(2)PO(4), 2.0 mM CaCl(2), 1.0 mM MgSO(4), 1.5 mM glucose, 6.0 mM L-lactate, 0.1 mM pyruvate, and 0x EAAs with 25.0 mM NaHCO(3), 1.0 mM alanyl-glutamine, 0.1 mM taurine, and 1.0x nonessential amino acids) with 0.4% (w/v) BSA from Days 0-3 and 5% FCS from Days 3-6. Using this medium, ~70% of cleaved embryos developed into blastocysts with profiles of carbohydrate metabolism similar to in vivo embryos. Our results suggest that feline embryos have stage-specific responses to carbohydrates and are sensitive to EAAs but are still reliant on one or more unidentified components of FCS for optimal blastocyst development.

  20. Simultaneous presence of bovine papillomavirus in blood and in short-term lymphocyte cultures from dairy cattle in Pernambuco, Brazil.

    Science.gov (United States)

    Diniz, N; Melo, T C; Santos, J F; Mori, E; Brandão, P E; Richtzenhain, L J; Freitas, A C; Beçak, W; Carvalho, R F; Stocco, R C

    2009-12-15

    Bovine papillomaviruses (BPV) are the causal agents of benign and malignant lesions; they can cause dramatic economic losses in cattle. Although 10 virus types have been described, three types are most common in tumors, namely BPV-1, -2 and -4. Previous studies have reported BPV in blood cells and the possibility of blood acting as a latent virus site and/or transmission agent of virus dissemination. We studied a Holstein dairy herd in Pernambuco, Brazil, in which several animals showed severe cutaneous papillomatosis, without previous determination of BPV types. Blood samples and short-term lymphocyte cultures were collected from 54 cows. We compared the BPV types detected in peripheral blood to those identified in the respective lymphocyte cultures: BPV-1 was detected in 74% and BPV-2 in 87% of the whole blood samples. Simultaneous virus presence (BPV-1 and BPV-2) was found in 65% of the blood samples. BPV-1 or BPV-2 were detected in the lymphocyte cultures in 93% of the samples, and both in 89%. The detection of viral DNA in whole blood and in lymphocyte cultures is evidence that this virus is carried by lymphocytes.

  1. 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

  2. Non-cytotoxic Thymus capitata extracts prevent Bovine herpesvirus-1 infection in cell cultures.

    Science.gov (United States)

    Boubaker-Elandalousi, Ramzi; Mekni-Toujani, Marwa; Kaabi, Belhassen; Larbi, Imen; Diouani, Mohamed-Fethi; Gharbi, Mohamed; Akkari, Hafidh; B'chir, Fatma; Ghram, Abdeljelil

    2014-09-27

    Bovine herpes virus type 1 (BHV-1) still causes great economic loss to the livestock industry and trade because there aren't any available drugs that proved to be fully effective against it. In this study, the cytotoxicity and the antiviral activities of the Thymus capitata extracts were evaluated for the development of new, non toxic and specific anti-herpesvirus drug. Aqueous extracts (AE), ethanolic extracts (EE) and essential oil (EO) of the aerial parts of Thymus capitata were analyzed to determine their chemical compositions by gas chromatography, and high performance liquid chromatography combined with mass spectrometry. Their cytotoxicity and antiviral activities against Bovine Herpesvirus type 1 (BHV-1) were evaluated by quantifying the reduction of the viral cytopathic effect using Madin-Darby Bovine Kidney cell line with colorimetric assay. T. capitata extracts were added at different stages of the viral infection to investigate and better quantify their potential inhibitory effects. Polyphenols and flavonoids were the major compounds found in T. capitata EO, EE and AE. The cytotoxic concentrations at 50% were 48.70, 189 and 289 μg ml(-1) for EO, EE and AE, respectively. The inhibitor concentrations at 50% for the EO, EE and AE, were 3.36, 47.80 and 164 μg ml(-1), respectively. The selectivity index anti-BHV-1 values were 14.49, 3.95 and 1.81 for EO, EE and AE, respectively. Thus, the EO extracts were the most efficient antiviral compounds. T. capitata extracts affect mainly the adsorption of BHV-1 virus to host cells. T. capitata extracts inhibit the viral replication by interfering with the early stages of viral adsorption and replication. Thus, T. capitata is a potential candidate for anti-herpesvirus treatment.

  3. Future aspects of micromanipualtion with embryos for

    African Journals Online (AJOL)

    Embryo micromanipulation techniques and their potential genetic impact in dairy cattle are discussed. In addition, some aspects of gene transfer are mentioned. Only the technique of splitting bovine embryos and the subsequent transfer of halfembryos has reached a stage which might make its application to cattle breeding ...

  4. Pock forming ability of fowl pox virus isolated from layer chicken and its adaptation in chicken embryo fibroblast cell culture

    Directory of Open Access Journals (Sweden)

    Varsha Rani Gilhare

    2015-03-01

    Full Text Available Aim: The objective of the present study was to examine pock forming ability of field strain and vaccine strain of fowl pox virus (FPV in chorioallantoic membrane (CAM of embryonated chicken eggs and its adaptation in chicken embryo fibroblast (CEF cell culture. Materials and Methods: Dry scabs were collected from 25 affected birds in glycerin-saline and preserved at 4°C until processed. Virus was isolated in 10-day-old embryonated chicken eggs by dropped CAM method. The identity of the virus is confirmed by clinical findings of affected birds, pock morphology and histopathology of infected CAM. In addition one field isolate and vaccine strain of FPV was adapted to CEF cell culture. CEF cell culture was prepared from 9-day-old embryonated chicken eggs. Result: Clinical symptoms observed in affected birds include pox lesion on comb, wattle, eyelids and legs, no internal lesions were observed. All field isolates produced similar findings in CAM. Pocks produced by field isolates ranged from 3 mm to 5 mm at the third passage while initial passages edematous thickening and necrosis of CAM was observed. Pocks formed by lyophilized strain were ranges from 0.5 mm to 2.5 mm in diameter scattered all over the membrane at the first passage. Intra-cytoplasmic inclusion bodies are found on histopathology of CAM. At third passage level, the CEF inoculated with FPV showed characteristic cytopathic effect (CPE included aggregation of cells, syncytia and plaque formation. Conclusion: FPV field isolates and vaccine strain produced distinct pock lesions on CAMs. Infected CAM showed intracytoplasmic inclusion bodies. The CEF inoculated with FPV field isolate as well as a vaccine strain showed characteristic CPE at third passage level.

  5. Characterization of secreted proteins of 2-cell mouse embryos cultured in vitro to the blastocyst stage with and without protein supplementation.

    Science.gov (United States)

    Burch, Tanya; Yu, Liang; Nyalwidhe, Julius; Horcajadas, Jose A; Bocca, Silvina; Swanson, R James; Oehninger, Sergio

    2014-06-01

    To identify the secreted proteins of murine embryos grown in vitro. Two-cell mouse embryos (n=432) were randomly allocated to culture to the blastocyst stage in protein-free and in protein-supplemented (3 % BSA) media. Proteins were separated by SDS-PAGE; bands were visualized by coomassie staining, followed by in-gel trypsin digestion and liquid chromatography-tandem mass spectrometry. RT-PCR and confocal microscopy were used to confirm gene/protein expression in blastocysts. Of all individually identified proteins, 34 and 23 were found in embryos cultured without and with BSA, respectively, and 20 were common. Identified proteins having an N-terminal secretory sequence or transmembrane domains located on the extracellular backbone were postulated as secreted proteins. Gene and protein expression for two selected molecules were confirmed. Functional analysis revealed over-represented processes related to lipid metabolism, cyclase activity, and cell adhesion/membrane functions. This study provided evidence to further characterize secreted proteins by mouse embryos grown from the 2-cell to the blastocyst stage in vitro. Because of homology between murine and human, these results may provide information to be translated to the clinical setting.

  6. Promising System for Selecting Healthy In Vitro–Fertilized Embryos in Cattle

    Science.gov (United States)

    Sugimura, Satoshi; Akai, Tomonori; Hashiyada, Yutaka; Somfai, Tamás; Inaba, Yasushi; Hirayama, Muneyuki; Yamanouchi, Tadayuki; Matsuda, Hideo; Kobayashi, Shuji; Aikawa, Yoshio; Ohtake, Masaki; Kobayashi, Eiji; Konishi, Kazuyuki; Imai, Kei

    2012-01-01

    Conventionally, in vitro–fertilized (IVF) bovine embryos are morphologically evaluated at the time of embryo transfer to select those that are likely to establish a pregnancy. This method is, however, subjective and results in unreliable selection. Here we describe a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse cinematography in our developed microwell culture dish and analyzes embryonic metabolism. The system can noninvasively identify prognostic factors that reflect not only blastocyst qualities detected with histological, cytogenetic, and molecular analysis but also viability after transfer. By assessing a combination of identified prognostic factors—(i) timing of the first cleavage; (ii) number of blastomeres at the end of the first cleavage; (iii) presence or absence of multiple fragments at the end of the first cleavage; (iv) number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle; and (v) oxygen consumption at the blastocyst stage—pregnancy success could be accurately predicted (78.9%). The conventional method or individual prognostic factors could not accurately predict pregnancy. No newborn calves showed neonatal overgrowth or death. Our results demonstrate that these five predictors and our system could provide objective and reliable selection of healthy IVF bovine embryos. PMID:22590579

  7. Synthesis of collagen by bovine chondrocytes cultured in alginate; posttranslational modifications and cell-matrix interaction

    NARCIS (Netherlands)

    Beekman, B.; Verzijl, N.; Bank, R.A.; Von Der Mark, K.; TeKoppele, J.M.

    1997-01-01

    The extracellular matrix synthesized by articular chondrocytes cultured in alginate beads was investigated. Collagen levels increased sigmoidally with time and remained constant after 2 weeks of culture. The presence of cartilage-specific type II collagen was confirmed immunohistochemically.

  8. Technique of the `in vitro` fertilization and the culture of mouse embryos at preimplantation; Tecnica de fertilizacao `in vitro` e cultura de embrioes de camundongo durante a pre-implantacao

    Energy Technology Data Exchange (ETDEWEB)

    Kikuchi, Olivia Kimiko [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil); Yamada, Takeshi [National Inst. of Radiological Sciences, Chiba (Japan)

    1993-03-01

    The mammal embryo is an intensive cellular proliferating system, very radiosensitive and therefore adequate to the study of the biological effects of ionizing radiation. The technique of the in vitro fertilization and the culture of mouse embryos at preimplantation period, modified by Yamada et al (1982) to improve the efficiency of more than 95% of blastocyst formation is described. (author) 2 refs., 7 figs.

  9. Obesity does not aggravate vitrification injury in mouse embryos: a prospective study

    Directory of Open Access Journals (Sweden)

    Ma Wenhong

    2012-08-01

    Full Text Available Abstract Background Obesity is associated with poor reproductive outcomes, but few reports have examined thawed embryo transfer in obese women. Many studies have shown that increased lipid accumulation aggravates vitrification injury in porcine and bovine embryos, but oocytes of these species have high lipid contents (63 ng and 161 ng, respectively. Almost nothing is known about lipids in human oocytes except that these cells are anecdotally known to be relatively lipid poor. In this regard, human oocytes are considered to be similar to those of the mouse, which contain approximately 4 ng total lipids/oocyte. To date, no available data show the impact of obesity on vitrification in mouse embryos. The aim of this study was to establish a murine model of maternal diet-induced obesity and to characterize the effect of obesity on vitrification by investigating the survival rate and embryo developmental competence after thawing. Methods Prospective comparisons were performed between six–eight-cell embryos from obese and normal-weight mice and between fresh and vitrified embryos. Female C57BL/6 mice were fed standard rodent chow (normal-weight group or a high-fat diet (obese group for 6 weeks. The mice were mated, zygotes were collected from oviducts and cultured for 3 days, and six–eight-cell embryos were then selected to assess lipid content in fresh embryos and to evaluate differences in apoptosis, survival, and development rates in response to vitrification. Results In fresh embryos from obese mice, the lipid content (0.044 vs 0.030, Pvs.9.3%, Pvs. 93.1%, P Conclusions This study demonstrated that differences in survival and developmental rates between embryos from obese and normal-weight mice were eliminated after vitrification. Thus, maternal obesity does not aggravate vitrification injury, but obesity alone greatly impairs pre-implantation embryo survival and development.

  10. A randomized clinical trial comparing embryo culture in a conventional incubator with a time-lapse incubator

    DEFF Research Database (Denmark)

    Kirkegaard, Kirstine; Hindkjaer, Johnny Juhl; Grøndahl, Marie Louise

    2012-01-01

    Time-lapse monitoring allows for a flexible embryo evaluation and potentially provides new dynamic markers of embryo competence. Before introducing time-lapse monitoring in a clinical setting, the safety of the instrument must be properly documented. Accordingly, the aim of this study...... was to evaluate the safety of a commercially available time-lapse incubator....

  11. A randomized clinical trial comparing embryo culture in a conventional incubator with a time-lapse incubator

    DEFF Research Database (Denmark)

    Kirkegaard, Kirstine; Hindkjaer, Johnny Juhl; Grøndahl, Marie Louise

    2012-01-01

    Time-lapse monitoring allows for a flexible embryo evaluation and potentially provides new dynamic markers of embryo competence. Before introducing time-lapse monitoring in a clinical setting, the safety of the instrument must be properly documented. Accordingly, the aim of this study was to eval...

  12. A transcriptomic approach for evaluating the relative potency and mechanism of action of azoles in the rat Whole Embryo Culture.

    Science.gov (United States)

    Dimopoulou, Myrto; Verhoef, Aart; Pennings, Jeroen L A; van Ravenzwaay, Bennard; Rietjens, Ivonne M C M; Piersma, Aldert H

    2017-12-01

    We evaluated the effect of six azoles on embryonic development in the rat whole embryo culture (WEC). Using the total morphological scoring system (TMS), we calculated the ID 10 concentration (effective dose for 10% decrease in TMS). For evaluating gene specific responses, we combined previously and newly collected transcriptomics data of rat WEC exposed to a total of twelve azoles at their ID 10 for 4h. Results revealed shared expressions responses in genes involved in the retinoic acid (RA) and sterol biosynthesis pathways, which are respectively representatives of developmental toxicity and targeted fungicidal action of the azoles. Azoles with more pronounced effects on the regulation of RA-associated genes were generally characterized as more potent embryotoxicants. Overall, compounds with strong sterol biosynthesis related responses and low RA related responses were considered as more favourable candidates, as they specifically regulated genes related to a desired target response. Among the identified sterol associated genes, we detected that methylsterol monooxygenase 1 (Msmo1) was more sensitively induced compared to Cyp51, a classical biomarker of this pathway. Therefore, we suggest that Msmo1 could be a better biomarker for screening the fungicidal value of azoles. In summary, we conclude that the embryonic regulation of RA and sterol metabolic pathways could be indicators for ranking azoles as embryotoxicants and determining their drug efficacy. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. 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.

  14. Evaluation of a simple Theileria annulata culture protocol from experimentally infected bovine whole blood

    Directory of Open Access Journals (Sweden)

    Gharbi M.

    2012-08-01

    Full Text Available We have evaluated a new simple technique using whole blood from experimentally infected cattle for the isolation and cultivation of Theileria annulata. The study was carried out on 20 Holstein-Frisian bovines that had been experimentally infected with a virulent lethal dose of Theileria annulata. This technique has been compared to the classical peripheral blood monocyte isolation with Ficoll carried out on 22 experimentally infected Holstein-Friesian calves. The effectiveness of the reference technique was estimated to 86.4%, whilst the effectiveness of the new technique was 100%. Moreover, this new technique leads to time and money saving estimated to € 3.06 per sample. It decreases the contamination risks by reducing the steps of sample manipulation.

  15. Evaluation of a simple Theileria annulata culture protocol from experimentally infected bovine whole blood

    Science.gov (United States)

    Gharbi, M.; Latrach, R.; Sassi, L.; Darghouth, M.A.

    2012-01-01

    We have evaluated a new simple technique using whole blood from experimentally infected cattle for the isolation and cultivation of Theileria annulata. The study was carried out on 20 Holstein-Frisian bovines that had been experimentally infected with a virulent lethal dose of Theileria annulata. This technique has been compared to the classical peripheral blood monocyte isolation with Ficoll carried out on 22 experimentally infected Holstein-Friesian calves. The effectiveness of the reference technique was estimated to 86.4%, whilst the effectiveness of the new technique was 100%. Moreover, this new technique leads to time and money saving estimated to € 3.06 per sample. It decreases the contamination risks by reducing the steps of sample manipulation. PMID:22910672

  16. Noninferiority, randomized, controlled trial comparing embryo development using media developed for sequential or undisturbed culture in a time-lapse setup.

    Science.gov (United States)

    Hardarson, Thorir; Bungum, Mona; Conaghan, Joe; Meintjes, Marius; Chantilis, Samuel J; Molnar, Laszlo; Gunnarsson, Kristina; Wikland, Matts

    2015-12-01

    To study whether a culture medium that allows undisturbed culture supports human embryo development to the blastocyst stage equivalently to a well-established sequential media. Randomized, double-blinded sibling trial. Independent in vitro fertilization (IVF) clinics. One hundred twenty-eight patients, with 1,356 zygotes randomized into two study arms. Embryos randomly allocated into two study arms to compare embryo development on a time-lapse system using a single-step medium or sequential media. Percentage of good-quality blastocysts on day 5. Percentage of day 5 good-quality blastocysts was 21.1% (standard deviation [SD] ± 21.6%) and 22.2% (SD ± 22.1%) in the single-step time-lapse medium (G-TL) and the sequential media (G-1/G-2) groups, respectively. The mean difference (-1.2; 95% CI, -6.0; 3.6) between the two media systems for the primary end point was less than the noninferiority margin of -8%. There was a statistically significantly lower number of good-quality embryos on day 3 in the G-TL group [50.7% (SD ± 30.6%) vs. 60.8% (SD ± 30.7%)]. Four out of the 11 measured morphokinetic parameters were statistically significantly different for the two media used. The mean levels of ammonium concentration in the media at the end of the culture period was statistically significantly lower in the G-TL group as compared with the G-2 group. We have shown that a single-step culture medium supports blastocyst development equivalently to established sequential media. The ammonium concentrations were lower in the single-step media, and the measured morphokinetic parameters were modified somewhat. NCT01939626. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  17. The impact of Cu treatment on phenolic and polyamine levels in plant material regenerated from embryos obtained in anther culture of carrot

    Czech Academy of Sciences Publication Activity Database

    Górecka, K.; Cvikrová, Milena; Kowalska, U.; Eder, Josef; Szafrańska, K.; Górecki, R.; Janas, K. M.

    2007-01-01

    Roč. 45, č. 1 (2007), s. 54-61 ISSN 0981-9428 R&D Projects: GA MŠk 1P05OC052 Institutional research plan: CEZ:AV0Z50380511 Source of funding: V - iné verejné zdroje Keywords : carrot culture * copper ions * embryo regeneration Subject RIV: GE - Plant Breeding Impact factor: 1.669, year: 2007

  18. Insulin and branched-chain amino acid depletion during mouse preimplantation embryo culture programmes body weight gain and raised blood pressure during early postnatal life.

    Science.gov (United States)

    Velazquez, Miguel A; Sheth, Bhavwanti; Smith, Stephanie J; Eckert, Judith J; Osmond, Clive; Fleming, Tom P

    2018-02-01

    Mouse maternal low protein diet exclusively during preimplantation development (Emb-LPD) is sufficient to programme altered growth and cardiovascular dysfunction in offspring. Here, we use an in vitro model comprising preimplantation culture in medium depleted in insulin and branched-chain amino acids (BCAA), two proposed embryo programming inductive factors from Emb-LPD studies, to examine the consequences for blastocyst organisation and, after embryo transfer (ET), postnatal disease origin. Two-cell embryos were cultured to blastocyst stage in defined KSOM medium supplemented with four combinations of insulin and BCAA concentrations. Control medium contained serum insulin and uterine luminal fluid amino acid concentrations (including BCAA) found in control mothers from the maternal diet model (N-insulin+N-bcaa). Experimental medium (three groups) contained 50% reduction in insulin and/or BCAA (L-insulin+N-bcaa, N-insulin+L-bcaa, and L-insulin+N-bcaa). Lineage-specific cell numbers of resultant blastocysts were not affected by treatment. Following ET, a combined depletion of insulin and BCAA during embryo culture induced a non sex-specific increase in birth weight and weight gain during early postnatal life. Furthermore, male offspring displayed relative hypertension and female offspring reduced heart/body weight, both characteristics of Emb-LPD offspring. Combined depletion of metabolites also resulted in a strong positive correlation between body weight and glucose metabolism that was absent in the control group. Our results support the notion that composition of preimplantation culture medium can programme development and associate with disease origin affecting postnatal growth and cardiovascular phenotypes and implicate two important nutritional mediators in the inductive mechanism. Our data also have implications for human assisted reproductive treatment (ART) practice. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  19. SREBP-1c Gene Silencing can Decrease Lipid Deposits in Bovine Hepatocytes Cultured in Vitro

    Directory of Open Access Journals (Sweden)

    Qinghua Deng

    2014-05-01

    Full Text Available Background: Fatty liver is a major metabolic disorder that occurs during early lactation in high-producing dairy cows. Sterol regulatory element-binding protein-1c (SREBP-1c is an important transcription factor that regulates lipid synthesis by regulating the expression of lipid metabolism genes. Methods: In this study, we reduced the expression of SREBP-1c by adenovirus-mediated SREBP-1c with a low expression vector (AD-GFP-SREBP-1c to study the effects of SREBP-1c on lipid deposits in bovine hepatocytes. The expression levels and enzyme activities of SERBP-1c and its target genes were determined by real-time PCR, western blot, and ELISA. Results: These results showed that Ad-GFP-SREBP-1c could inhibit SREBP-1c expression. The expression of the lipid synthesis enzyme acetyl-CoA carboxylase (ACC was down-regulated. The expression levels of the lipid oxidation enzymes long-chain fatty acyl-COA synthetase (ACSL-1, carnitine palmitoyltransferase І (CPT-І, carnitine palmitoyltransferase II (CPT- II, and β-hydroxyacyl-CoA-DH (HADH were significantly elevated. Furthermore, the expression levels of factors involved in the assembly and transport of very low-density lipoproteins (VLDLs, such as apolipoprotein B100 (ApoB, apolipoprotein E (ApoE, and microsomal triglyceride transfer protein (MTTP were decreased comparison with the negative control and the blank control groups, but the low-density lipoprotein receptor (LDLR was elevated. The concentrations of TG (triglyceride and VLDL were also reduced. Conclusion: These data suggest that low SREBP-1c expression can decrease lipid synthesis, increase lipid oxidation, and decrease the TG and VLDL content in bovine hepatocytes.

  20. Assessment of the toxic and potential teratogenic effects of four glycol ethers and two derivatives using the hydra regeneration assay and rat whole embryo culture.

    Science.gov (United States)

    Bowden, H C; Wilby, O K; Botham, C A; Adam, P J; Ross, F W

    1995-10-01

    The widespread use of the glycol ethers as solvents in manufacturing industries presents a vast potential for occupational exposure. In the present study the potential hazards of four glycol ethers and two derivatives were assessed using two in vitro tests, rat whole embryo culture and the hydra regeneration assay. Concentrations used ranged from 0.3 to 1.0 mg/ml in embryo culture and from 0.03 to 80.0 mg/ml in the hydra assay. The embryotoxic potential of the ethylene glycol mono-alkyl ethers was shown to increase with the length of the alkyl chain. This is in contrast to in vivo data but can be explained by the lack of maternal metabolism in the in vitro systems. However, the teratogenic hazard ratings obtained in the hydra assay and the types of malformations observed in embryo culture support in vivo data. Results obtained for diethylene glycol monoethyl ether are in agreement with in vivo data. Results of both assays suggest that ethylene glycol monosalicylate presents a significant potential teratogenic hazard and that ethylene glycol tetra-acetic acid presents toxic and teratogenic potentials. When the effects of maternal toxicity and metabolism are considered, the overall picture presented by the present results is one of general agreement with in vivo data.

  1. Hot topic: Bovine milk samples yielding negative or nonspecific results in bacterial culturing--the possible role of PCR-single strand conformation polymorphism in mastitis diagnosis.

    Science.gov (United States)

    Schwaiger, K; Wimmer, M; Huber-Schlenstedt, R; Fehlings, K; Hölzel, C S; Bauer, J

    2012-01-01

    A large proportion of mastitis milk samples yield negative or nonspecific results (i.e., no mastitis pathogen can be identified) in bacterial culturing. Therefore, the culture-independent PCR-single strand conformation polymorphism method was applied to the investigation of bovine mastitis milk samples. In addition to the known mastitis pathogens, the method was suitable for the detection of fastidious bacteria such as Mycoplasma spp., which are often missed by conventional culturing methods. The detection of Helcococcus ovis in 4 samples might indicate an involvement of this species in pathogenesis of bovine mastitis. In conclusion, PCR-single-strand conformation polymorphism is a promising tool for gaining new insights into the bacteriological etiology of mastitis. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  2. Metabolism of excised embryos of Lupinus luteus L. VI. An electrophoretic analysis of some dehydrogenases in cultured embryos as compared with the normal seedling axes

    Directory of Open Access Journals (Sweden)

    J. Czosnowski

    2015-01-01

    Full Text Available The electrophoretic patterns (disc electrophoresis of the studied dehydrogenases: glucose-6-phosphate - (A, malate - (B, glutamate - (C, alcohol - (D and lactate dehydrogenase (E, in the axial organs of isolated Lupinus luteus embryos and seedlings cultivated over 12 days are characterized by great similarities. With time, after the third day of cultivation the patterns begin to become less deyeloped. Analyses performed during the first 10 hours of imbibition of seed parts indicate that the maximal development of isozyme patterns occurs during the third hour after which the patterns become poorer. The most uniform type of pattern. and the lowest number of isozymes was shown by glutamate dehydrogenase, the richest pattern was shown by malate dehydrogenase. No band common for a 11 the 27 experimental elements was found.

  3. Effect of environmental particulates on cultured human and bovine endothelium. Cellular injury via an oxidant-dependent pathway

    International Nuclear Information System (INIS)

    Garcia, J.G.; Dodson, R.F.; Callahan, K.S.

    1989-01-01

    The effects of respirable environmental fibers on cultures of human umbilical vein and bovine pulmonary artery endothelial cell monolayers were studied. Interaction among endothelial cell monolayers and amosite and chrysotile asbestos, attapulgite, fiberglass, or latex beads resulted in rapid phagocytosis of the particulates. A gradient of time-dependent and concentration-dependent endothelial cell injury (measured by specific 51Cr release) was observed with amosite and attapulgite being markedly toxic. Chrysotile and fiberglass were much less toxic, and latex beads were not significantly injurious at any time or dose examined. Responses of bovine pulmonary artery and human endothelial vein endothelial cells to fiber phagocytosis and fiber-induced injury were similar. In human umbilical cell monolayers, fiber-mediated stimulation of the arachidonate metabolite prostacyclin paralleled endothelial cell injury; i.e. amosite and attapulgite were stimulatory, whereas fiberglass (0-500 micrograms/ml) and latex beads (10(9) beads/ml) did not significantly increase prostacyclin generation. Although chrysotile was only weakly cytotoxic, significant stimulation of prostacyclin was observed at the highest dose tested (500 micrograms/ml). To investigate whether toxic oxygen species may be involved in fiber-induced cytotoxicity, oxidant scavengers or inhibitors were used in injury studies. Both superoxide dismutase (a scavenger of O2-) and catalase (an inhibitor of H2O2) produced significant protection against fiber-mediated endothelial cell injury. In addition, chelation by deferoxamine of elemental Fe present in the fiber preparations was also protective, suggesting Fe, via the modified Haber-Weiss reaction, may promote hydroxyl radical formation and contribute to endothelial cell injury induced by these particulates

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Single and double polymerase chain reaction for detection of bovine viral diarrhea virus in tissue culture and sera.

    Science.gov (United States)

    Alansari, H; Brock, K V; Potgieter, L N

    1993-04-01

    Bovine viral diarrhea virus (BVDV) is an ubiquitous pathogen of cattle and has been reported in other ruminants. It is also frequently present in laboratory and biological materials as an adventitious agent. This virus is difficult to detect in some specimens, especially in the presence of specific antibody and when the virus is present in low concentrations. In this paper, we describe a single polymerase chain reaction (PCR) to amplify virus sequences from infected cell culture and a nested double PCR to detect small concentrations of several virus strains in sera. Total cellular RNA was extracted from cell cultures infected with the cytopathic strain 72 and noncytopathic strain 2724 of BVDV. Ten different genomic sequences along the length of the viral RNA ranging in size from 397 to 1,016 base pairs (bp) were successfully amplified by PCR. A 404-bp probe made from amplified product from the 3' end hybridized specifically with the RNA of several BVDV strains blotted on nylon filters. Viral RNA was extracted from serum and amplified using 2 sets of degenerate nested primers designed from the 3' end of the viral genome in a double PCR protocol. Double amplification of the viral sequences greatly enhanced the sensitivity of the detection of many strains present in serum. Advantages of using double PCR over single PCR and virus isolation is discussed.

  6. Noninvasive metabolomic profiling as an adjunct to morphology for noninvasive embryo assessment in women undergoing single embryo transfer

    NARCIS (Netherlands)

    Seli, E.; Vergouw, C.G.; Morita, H.; Botros, L.; Roos, P.; Lambalk, C.B.; Yamashita, N.; Kato, O.; Sakkas, D.

    2010-01-01

    Objective: To determine whether metabolomic profiling of spent embryo culture media correlates with reproductive potential of human embryos. Design: Retrospective study. Setting: Academic and a private assisted reproductive technology (ART) programs. Patient(s): Women undergoing single embryo

  7. Nucleolar remodeling in nuclear transfer embryos

    DEFF Research Database (Denmark)

    Laurincik, Jozef; Maddox-Hyttel, Poul

    2007-01-01

    the developmental potential of embryos originating from varied nuclear transfer protocols. In bovine in vivo developed embryos, functional ribosome-synthesizing nucleoli become structurally distinct toward the end of the 4th post-fertilization cell cycle. In embryonic cell nuclear transfer embryos, fully developed...... to develop fully functional nucleoli. In bovine in vivo developed embryos, a range of important nucleolar proteins (e.g., topoisomerase I, upstream binding factor and RNA polymerase I, fibrillarin, nucleophosmin and nucleolin) become localized to the nucleolar anlage over several cell cycles. This relocation......Transcription of the ribosomal RNA (rRNA) genes occurs in the nucleolus and results in ribosome biogenesis. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus serve to evaluate...

  8. Somatic embryogenesis and plant regeneration from embryo rescue ...

    African Journals Online (AJOL)

    The application of tissue culture techniques, particularly in the area of embryo rescue, has had a major impact on the maintenance and development of hybrid embryo from wide crosses. Embryo rescue techniques are directed towards obtaining more efficient survival of embryos in situations where very immature embryos ...

  9. 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.

  10. [Influence of the activator of transcription GAL4 on growth and development of embryos and embryonic cells in primary cultures of sand dollar].

    Science.gov (United States)

    Odintsova, N A; Kiselev, K V; Bulgakov, V P; Kol'tsova, E A; Iakovlev, K V

    2003-01-01

    In order to solve many tasks of biotechnology, constant lines of the cells of marine invertebrates with a high growth potential are required, which are absent at present. We used the universal activator of transcription gal4 to change the degree of expression of genes of growth factors in embryonic sea urchin cells and, thereby, increase their proliferative activity. The fertilized sea urchin eggs and dissociated embryonic cells at the blastula stage were treated with plasmids containing both the functional gene gal4 and the gene devoid of the regions encoding the activator domain. The transfection of embryonic sea urchin eggs with the functional gene led to cell dedifferentiation and formation of tumor-like structures in the embryos or increased number of embryonic cells in culture. In the cells obtained from the transfected embryos, the pigments were found within two months of cultivation, whose absorption spectrum coincided with that of echinochrome.

  11. Ammonium accumulation in commercially available embryo culture media and protein supplements during storage at 2-8°C and during incubation at 37°C.

    Science.gov (United States)

    Kleijkers, Sander H M; van Montfoort, Aafke P A; Bekers, Otto; Coonen, Edith; Derhaag, Josien G; Evers, Johannes L H; Dumoulin, John C M

    2016-06-01

    Does ammonium accumulate in commercially available culture media and protein supplements used for in vitro development of human pre-implantation embryos during storage and incubation? Ammonium accumulates in ready-to-use in vitro fertilization (IVF) culture media during storage at 2-8°C and in ready-to-use IVF culture media and protein supplements during incubation at 37°C. Both animal and human studies have shown that the presence of ammonium in culture medium has detrimental effects on embryonic development and pregnancy rate. It is, therefore, important to assess the amount of ammonium accumulation in ready-to-use IVF culture media under conditions that are common in daily practice. Ammonium accumulation was investigated in 15 ready-to-use media, 11 protein-free media and 8 protein supplements. Ammonium was measured by the use of an enzymatic method with glutamate dehydrogenase. To simulate the storage and incubation conditions during IVF treatments, ammonium concentrations were measured at different time-points during storage at 2-8°C for 6 weeks and during incubation at 37°C for 4 days. All ready-to-use, i.e. protein supplemented, culture media showed ammonium accumulation during storage for 6 weeks (ranging from 9.2 to 99.8 µM) and during incubation for 4 days (ranging from 8.4 to 138.6 µM), resulting in levels that might affect embryo development. The protein supplements also showed ammonium accumulation, while the culture media without protein supplementation did not. The main sources of ammonium buildup in ready-to-use culture media were unstable glutamine and the protein supplements. No additional ammonium buildup was found during incubation when using an oil overlay or with the presence of an embryo in the culture droplet. In addition to the unstable glutamine and the protein supplements, other free amino acids might contribute to the ammonium buildup. We did not investigate the deterioration of other components in the media. Break-down of

  12. Optimization of chemically defined cell culture media - Replacing fetal bovine serum in mammalian in vitro methods

    DEFF Research Database (Denmark)

    van der Valk, J; Brunner, D; De Smet, K

    2010-01-01

    Quality assurance is becoming increasingly important. Good laboratory practice (GLP) and good manufacturing practice (GMP) are now established standards. The biomedical field aims at an increasing reliance on the use of in vitro methods. Cell and tissue culture methods are generally fast, cheap, ...

  13. Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

    Science.gov (United States)

    Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

    2016-01-01

    In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

  14. Bovine serum albumin contained in culture medium used in artificial insemination is an important anaphylaxis risk factor.

    Science.gov (United States)

    Pagán, Juan A; Postigo, Idoia; Rodríguez-Pacheco, Jorge R; Peña, Maribel; Guisantes, Jorge A; Martínez, Jorge

    2008-11-01

    To analyze the cause of the anaphylactic reaction after a standard artificial insemination process in a patient diagnosed with asthma. Case report. Residencia Sanitaria Virgen de la Arrixaca (Murcia, Spain) and University of the Basque Country (Vitoria, Spain). A 30-year-old woman with a previous medical history compatible with respiratory allergy who suffered an anaphylactic reaction after an artificial insemination with spermatozoids in capable medium (Upgraded B2 INRA medium; Laboratories CCD, Paris, France). Cutaneous tests and specific IgE levels to inhalant allergens, grass and Olea pollens, and insemination medium were performed. Specific IgE levels to mammal epithelia and bovine serum albumin (BSA). Skin prick tests were positive for inhalant allergens such as mites, cat, dog, horse, and rabbit epithelia, grasses and Olea pollens, and the insemination medium. The beta-lactamic tests were negative. The determination of specific IgE demonstrated positive values to mammal epithelia and mammal serum albumins including BSA. We report a case of an anaphylactic reaction to the BSA included in the insemination culture medium induced by a subclinical sensitivity to serum albumins of mammal epithelia. A previous testing with the medium is recommended and specific testing might be needed in women who have a history of animal epithelium allergies.

  15. Pertussis toxin treatment does not block inhibition by atrial natriuretic factor of aldosterone secretion in cultured bovine zona glomerulosa cells

    International Nuclear Information System (INIS)

    De Lean, A.; Cantin, M.

    1986-01-01

    The authors have previously reported that atrial natriuretic factor (ANF) potently inhibits PGE or forskolin-stimulation aldosterone secretion in bovine zona glomerulosa (ZG) by acting through specific high affinity receptors. In order to evaluate the functional role of the regulatory protein N/sub i/ and the inhibition of adenylate cyclase activity (AC) in ZG, the authors have studied the effect of treatment with PT on inhibition by ANF of aldosterone production. Primary cultures of ZG were treated for 18 hours in serum-free F12 medium with (0-100 ng/ml PT). No effect of PT pretreatment was observed either on basal, PGE-stimulated or ANF-inhibited levels of steroidogenesis. When membranes prepared from control ZG were ADP-ribosylated with [ 32 P] NAD in the presence of PT, two toxin-specific bands with 39 Kd and 41 Kd were documented on SDS gel. Cell pretreatment with as low as 1 ng/ml drastically reduced further labelling of these two bands while higher doses completely abolished them. Since PT treatment covalently modifies completely the toxin substrate without altering ANF inhibition of adrenal steroidogenesis, the authors conclude that N/sub i/ is not involved in the mode of action of ANF on aldosterone production

  16. Oxidized LDL binding to LOX-1 upregulates VEGF expression in cultured bovine chondrocytes through activation of PPAR-γ

    International Nuclear Information System (INIS)

    Kanata, Sohya; Akagi, Masao; Nishimura, Shunji; Hayakawa, Sumio; Yoshida, Kohji; Sawamura, Tatsuya; Munakata, Hiroshi; Hamanishi, Chiaki

    2006-01-01

    It has been reported that vascular endothelial growth factor (VEGF) and its receptors play an important role in the destruction of articular cartilage in osteoarthritis through increased production of matrix metalloproteinases. We investigated whether the oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) upregulates VEGF expression in cultured bovine articular chondrocytes (BACs). Ox-LDL markedly increased VEGF mRNA expression and protein release in time- and dose-dependent manners, which was significantly suppressed by anti-LOX-1 antibody pretreatment. Activation of peroxisome proliferator-activated receptor (PPAR)-γ was evident in BACs with ox-LDL addition and was attenuated by anti-LOX-1 antibody. The specific PPAR-γ inhibitor GW9662 suppressed ox-LDL-induced VEGF expression. These results suggest that the ox-LDL/LOX-1 system upregulates VEGF expression in articular cartilage, at least in part, through activation of PPAR-γ and supports the hypothesis that ox-LDL is involved in cartilage degradation via LOX-1

  17. 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

  18. , , , , , and Gene Expression in Single- and Co-cultured Bovine Satellite Cells and Intramuscular Preadipocytes Treated with Palmitic, Stearic, Oleic, and Linoleic Acid

    Directory of Open Access Journals (Sweden)

    S. H. Choi

    2015-03-01

    Full Text Available We previously demonstrated that bovine subcutaneous preadipocytes promote adipogenic gene expression in muscle satellite cells in a co-culture system. Herein we hypothesize that saturated fatty acids would promote adipogenic/lipogenic gene expression, whereas mono- and polyunsaturated fatty acids would have the opposite effect. Bovine semimembranosus satellite cells (BSC and intramuscular preadipocytes (IPA were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS/Dulbecco’s Modified Eagle Medium (DMEM and 1% antibiotics during the 3-d proliferation period. After proliferation, cells were treated for 3 d with 3% horse serum/DMEM (BSC or 5% FBS/DMEM (IPA with antibiotics. Media also contained 10 μg/mL insulin and 10 μg/mL pioglitazone. Subsequently, differentiating BSC and IPA were cultured in their respective media with 40 μM palmitic, stearic, oleic, or linoleic acid for 4 d. Finally, BSC and IPA were single- or co-cultured for an additional 2 h. All fatty acid treatments increased (p = 0.001 carnitine palmitoyltransferase-1 beta (CPT1β gene expression, but the increase in CPT1β gene expression was especially pronounced in IPA incubated with palmitic and stearic acid (6- to 17- fold increases. Oleic and linoleic acid decreased (p = 0.001 stearoyl-CoA desaturase (SCD gene expression over 80% in both BSC and IPA. Conversely, palmitic and stearic acid increased SCD gene expression three fold in co-cultured in IPA, and stearic acid increased AMPKα gene expression in single- and co-cultured BSC and IPA. Consistent with our hypothesis, saturated fatty acids, especially stearic acid, promoted adipogenic and lipogenic gene expression, whereas unsaturated fatty acids decreased expression of those genes associated with fatty acid metabolism.

  19. Development of embryo-like structures in the suspension cultures of flax coincides with secretion of chitinase-like proteins

    Czech Academy of Sciences Publication Activity Database

    Petrovská, Beáta; Salaj, T.; Moravčíková, J.; Libantová, J.; Salaj, J.

    2010-01-01

    Roč. 32, č. 4 (2010), s. 651-656 ISSN 0137-5881 Institutional research plan: CEZ:AV0Z50380511 Keywords : Conditioned media * Embryo-like structures * Linum usitatissimum L Subject RIV: EF - Botanics Impact factor: 1.344, year: 2010

  20. Structure and development of somatic embryos formed in Arabidopsis thaliana pt mutant callus cultures derived from seedlings

    NARCIS (Netherlands)

    Recklinghausen, von I.R.; Iwanowska, A.; Kieft, H.; Mordhorst, A.P.; Schel, J.H.N.; Lammeren, van A.A.M.

    2000-01-01

    Seeds of the Arabidopsis thaliana mutant primordia timing (pt) were germinated in 2,4-dichlorophenoxyacetic acid- containing liquid medium. The seedlings formed somatic embryos and nonembryogenic and embryogenic callus in vitro in a time period of approximately two to three weeks. Embryogenesis and

  1. Anaplasma Marginale isolation from infected bovine erythrocytes or from its floating culture

    International Nuclear Information System (INIS)

    Canon Q, Y.

    1986-01-01

    Isolation of Anaplasma Marginale is of great importance because this is the cause of anaplasmosis in cattle. Anaplasmosis is a mortal disease and spreads easily. To isolate the anaplasm, four different experiments were developed; in the first experiment, the parasite was isolated from parasitic blood, by means of three methods; osmotic shock, sonic vibration and treatment with hemolisine. In the second experiment, the parasite source was the top of the bacteriologic culture of anaplasma marginale obtained by means of slow centrifugation. In the third experiment, it was used parasitic blood diluted in PBS and liquid nitrogen criopreserved. In the fourth experiment.it was used parasitic blood which was separated by means of sonic oscillation. This method was more adequate to free the parasite from the host cell. Differential centrifugation was the best method to separate parasite of the stroma and ghost cells

  2. A comparison of gene expression responses in rat whole embryo culture and in vivo: time-dependent retinoic acid-induced teratogenic response.

    Science.gov (United States)

    Robinson, Joshua F; Verhoef, Aart; Pennings, Jeroen L A; Pronk, Tessa E; Piersma, Aldert H

    2012-03-01

    The whole embryo culture (WEC) model serves as a potential alternative for classical in vivo developmental toxicity testing. In the WEC, cultured rat embryos are exposed during neurulation and early organogenesis and evaluated for morphological effects. Toxicogenomic-based approaches may improve the predictive ability of WEC by providing molecular-based markers associated with chemical exposure, which can be compared across multiple parameters (e.g., exposure duration, developmental time, experimental model). Additionally, comparisons between in vitro and in vivo models may identify objective relevant molecular responses linked with developmental toxicity endpoints in vivo. In this study, using a transcriptomic approach, we compared all-trans retinoic acid (RA)-exposed and nonexposed Wistar rat embryos derived using WEC (RA, 0.5 μg/ml) or in vivo (RA, 50 mg/kg, oral gavage) to identify overlapping and nonoverlapping effects of RA on RNA expression in parallel with morphological changes. Across six time points (gestational day 10 + 2-48 h), we observed strong similarities in RA response at the gene (directionality, significance) and functional (e.g., embryonic development, cell differentiation) level which associated with RA-induced adverse morphological effects, including growth reduction as well as alterations in neural tube, limb, branchial, and mandible development. We observed differences between models in the timing of RA-induced effects on genes related to embryonic development and RA metabolism. These observations on the gene expression level were associated with specific differential morphological outcomes. This study supports the use of WEC to examine compound-induced molecular responses relative to in vivo and, furthermore, assists in defining the applicability domain of the WEC in determining complementary windows of sensitivity for developmental toxicological investigations.

  3. Improving embryo quality in assisted reproduction

    NARCIS (Netherlands)

    Mantikou, E.

    2013-01-01

    The goal of this thesis was to improve embryo quality in assisted reproductive technologies by gaining more insight into human preimplantation embryo development and by improving in vitro culture conditions. To do so, we investigated an intriguing feature of the human preimplantation embryo, i.e.

  4. Influence of bradykinin on diacylglycerol and phosphatidic acid accumulation in cultured bovine adrenal chromaffin cells.

    Science.gov (United States)

    Owen, P J; Boarder, M R

    1991-09-01

    Earlier studies have shown that bradykinin stimulated release of catecholamines from chromaffin cells by an influx of calcium through dihydropyridine-insensitive channels, and also that bradykinin stimulated (poly)phosphoinositide hydrolysis. To investigate membrane-bound second messengers in chromaffin cells, and to elucidate any role these may play in stimulus-secretion coupling, we have studied the influence of bradykinin on diacylglycerol and phosphatidic acid (PA). Using equilibrium labelling of primary cultures of chromaffin cells with [3H]arachidonic acid or [3H]glycerol, we found no influence of bradykinin (10 nM) on labelled diacylglycerol formation, either in the presence or absence of inhibitors of diacylglycerol lipase or kinase. However, when we used cells prelabelled with 32Pi for 2.5 h, we found that bradykinin produced a substantial stimulation of label found in PA, with an EC50 value of about 1 nM. This bradykinin stimulation of [32P]PA formation was only partially dependent on extracellular calcium, in contrast to the smaller response to nicotine, which was completely dependent on extracellular calcium. Short (10 min) pretreatment with tetradecanoylphorbol acetate (TPA) almost completely eliminated the bradykinin-stimulated formation of inositol phosphates, but failed to affect bradykinin stimulation of label in PA, suggesting that PA production in response to bradykinin is not downstream of phospholipase C activation. TPA alone failed to stimulate [32P]PA substantially, whereas long-term (24 or 48 h) treatment with TPA failed to attenuate the response to bradykinin. Diacylglycerol kinase inhibitors were also without effect on the bradykinin stimulation of [32P]PA. These results suggest that bradykinin stimulates PA production by a mechanism independent of the activation of protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Development of rabbit embryos during a 96-h period of in vitro culture after superovulatory treatment under conditions of elevated ambient temperature.

    Science.gov (United States)

    Cheng, H; Dooley, M P; Hopkins, S M; Anderson, L L; Yibchok-anun, S; Hsu, W H

    1999-08-16

    The effects of elevated ambient temperature on the response to exogenous gonadotropins were evaluated in female New Zealand White rabbits exposed to 33+/-1 degrees C (mean +/- SE) and 10-30% relative humidity (8 h/day) during a 5-day period. Does were treated with pFSH (0.3 mg/0.3 ml Standard Armour) twice daily during three consecutive days with a minimum interval of 8 h between injections. Six hours after the last FSH injection all does were removed from the experimental chamber, given hCG (25 IU/kg) and paired overnight. Nineteen hours after pairing, embryos were flushed from the reproductive tracts, evaluated, and subjected to in vitro culture during a 96-h period. The ovulatory responses to exogenous gonadotropins and fertilization rates did not differ significantly under conditions of elevated ambient temperature, whereas fewer blastocysts and increased number of degenerate embryos were observed after culture. We conclude that although hyperthermia was induced during exposure to elevated ambient temperature, it did not alter the ovulatory responses to gonadotropin treatment and plasma concentrations of FSH and LH compared with does in a thermoneutral environment. Exposure of donor rabbits to elevated ambient temperature before mating, however, increased embryonic degeneration.

  6. Biology and culture of the clown loach Chromobotia macracanthus (Cypriniformes, Cobitidae) : 4- Thermal biology of embryos and larvae

    OpenAIRE

    Baras, Etienne; Ginanjar, R.; Ahmad, M.; Permana, A.; Priyadi, A.; Legendre, Marc; Pouyaud, Laurent; Slembrouck, Jacques

    2012-01-01

    International audience; The knowledge of how fish survive and grow at different temperatures, and how these traits vary between life stages, is essential to evaluate the effects of climate change on wild fish and implement effective strategies in aquaculture. These issues are addressed in this study through a series of experiments that evaluate the effect of temperature (23-34 degrees C) on the embryos and larvae of clown loach, Chromobotia macracanthus. This species is endemic to the rivers ...

  7. 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...

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

    Science.gov (United States)

    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.

  9. Diferentes suplementos no cultivo in vitro de embriões de pinhão-manso Different supplements for in vitro embryo culture of physic nut

    Directory of Open Access Journals (Sweden)

    Claudinéia Ferreira Nunes

    2008-01-01

    days. Only the zygotic embryos from immature fruits needed sucrose supplementation in the culture medium to sustain germination. Better embryos development occurs in MS medium supplemented with 60 g L-1 of sucrose. The association of activated carbon and coconut water provides better growth of seedlings derived from embryos extracted from seeds of dry fruits.

  10. 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.

  11. Manipulating early pig embryos.

    Science.gov (United States)

    Niemann, H; Reichelt, B

    1993-01-01

    On the basis of established surgical procedures for embryo recovery and transfer, the early pig embryo can be subjected to various manipulations aimed at a long-term preservation of genetic material, the generation of identical multiplets, the early determination of sex or the alteration of the genetic make-up. Most of these procedures are still at an experimental stage and despite recent considerable progress are far from practical application. Normal piglets have been obtained after cryopreservation of pig blastocysts hatched in vitro, whereas all attempts to freeze embryos with intact zona pellucida have been unsuccessful. Pig embryos at the morula and blastocyst stage can be bisected microsurgically and the resulting demi-embryos possess a high developmental potential in vitro, whereas their development in vivo is impaired. Pregnancy rates are similar (80%) but litter size is reduced compared with intact embryos and twinning rate is approximately 2%. Pig blastomeres isolated from embryos up to the 16-cell stage can be grown in culture and result in normal blastocysts. Normal piglets have been born upon transfer of blastocysts derived from isolated eight-cell blastomeres, clearly underlining the totipotency of this developmental stage. Upon nuclear transfer the developmental capacity of reconstituted pig embryos is low and culture. Sex determination can be achieved either by separation of X and Y chromosome bearing spermatozoa by flow cytometry or by analysing the expression of the HY antigen in pig embryos from the eight-cell to morula stage. Microinjection of foreign DNA has been successfully used to alter growth and development of transgenic pigs, and to produce foreign proteins in the mammary gland or in the bloodstream, indicating that pigs can be used as donors for valuable human pharmaceutical proteins. Another promising area of gene transfer is the increase of disease resistance in transgenic lines of pigs. Approximately 30% of pig spermatozoa bind

  12. Detection of genes associated with developmental competence of bovine oocytes

    Czech Academy of Sciences Publication Activity Database

    Němcová, Lucie; Jansová, Denisa; Vodičková Kepková, Kateřina; Vodička, Petr; Jeseta, M.; Machatková, M.; Kaňka, Jiří

    2016-01-01

    Roč. 166, č. 1 (2016), s. 58-71 ISSN 0378-4320 Institutional support: RVO:67985904 Keywords : oocyte * embryo * bovine * developmental competence * transcription Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.605, year: 2016

  13. [Philosophical aspects. Why the embryo is a issue].

    Science.gov (United States)

    Grassin, Marc

    2018-03-07

    How raising the embryo's issue today ? Far from evidences and certainties, we have to face the confrontations to understand them. Embryo's issue crystalise all cultural and human tensions. Beyond ontological disagrement on the embryo being what can we expect for mankind today and tomorrow ? Through an anthropological thinking, embryo's issue challenges us in our common capability of decision.

  14. Relative potency of albendazole and its sulfoxide metabolite in two in vitro tests for developmental toxicity: the rat whole embryo culture and the mouse embryonic stem cell test.

    Science.gov (United States)

    Eckardt, Kathrin; Kaltenhäuser, Johanna; Kilb, Caroline; Seiler, Andrea; Stahlmann, Ralf

    2012-11-01

    The benzimidazole carbamate albendazole (ABZ), a potent anthelmintic, is a teratogenic compound in rats. At present it is unclear to which degree this effect is caused by the parent compound or its major metabolite, albendazole sulfoxide (ASO). Both substances were studied separately and in combinations to mimic incomplete bioactivation in two in vitro tests: mouse embryonic stem cell test (EST) and rat whole embryo culture (WEC). In both assays, ABZ and mixtures with ASO induced detrimental effects at lower concentrations compared to ASO alone. While ABZ caused half-maximal effects on cardiomyocyte differentiation at a mean concentration of 0.26 μM (EST) and dysmorphogenic development of rat embryos at 3.7 μM (WEC), effective concentrations of ASO were similar in both assays (10-13 μM). By using WEC and EST we demonstrate that ABZ exhibits stronger inherent embryotoxic potency although ASO might be the proximate teratogen in vivo because of higher plasma concentrations. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Replacement of serum with sericin in in vitro maturation and culture media: Effects on embryonic developmental competence of Sanjabi sheep embryo during breeding season.

    Science.gov (United States)

    Hajarian, H; Aghaz, F; Karami Shabankareh, H

    2017-04-01

    Sericin is a water-soluble component of silk and has been used as a biomaterial due to its antibacterial and ultraviolet radiation-resistant properties. This study was designed to evaluate the effect of sericin supplementation, as a serum replacement, in maturation and culture media on the meiotic competence of oocytes or in vitro culture of ovine embryos. In experiment 1, oocytes were matured in the presence of 10% fetal ovine serum (FOS), 0.1% polyvinyl alcohol (PVA) and different concentrations of sericin (0.1, 0.5, 1 and 2.5%), for 24 h. The addition of 0.5% sericin to maturation medium increased the rates of maturation to metaphase II of oocytes compared with those in cultures with 0.1% PVA. Following fertilization, blastocyst development was higher for oocytes matured with 0.5% of sericin compared with 0.1% PVA. However, the rates of nuclear maturation of oocytes and blastocyst development under FOS and 0.5% sericin were not significantly different. In experiment 2, presumptive zygotes were cultured in the presence of 10% FOS, 0.1% PVA and different concentrations of sericin (0.1, 0.5, 1 and 2.5%), for 7-8 days. The addition of 0.5% sericin to culture medium increased the blastocyst rate compared with those in cultures without sericin or addition of 0.1% PVA and 10% FOS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM and IVC in ovine oocytes and zygotes. Copyright © 2016. Published by Elsevier Inc.

  16. Toxic effects of the mycotoxin zearalenone and its derivatives on in vitro maturation of bovine oocytes and 17 beta-estradiol levels in mural granulosa cell cultures.

    Science.gov (United States)

    Minervini, F; Dell'Aquila, M E; Maritato, F; Minoia, P; Visconti, A

    2001-01-01

    Moulds parasites of livestock foodstuffs alter the quality of grains by synthesizing mycotoxins. Zearalenone (ZEA) and its derivatives (alpha- and beta-zearalenol, zeranol, taleranol and zearalanone) are produced by fungi of the genus Fusarium and, after ingestion via contaminated cereals, may lead to fertility disturbances and other reproductive pathologies. Zearalenone, alpha-zearalenol and zearalanone were tested, at levels ranging from 0.3 to 30 microg/ml, in order to evaluate the effect on the in vitro maturation (IVM) rate of bovine oocytes and on the formation of 17 beta-estradiol in supernatants of mural granulosa cells (GC) cultures. These compounds induced dose-dependent oocyte maturation delay and chromatin abnormalities. Maturation of oocytes to metaphase II (M II) was inhibited in oocytes cultured in the presence of 30 microg/ml ZEA, alpha-zearalenol or zearalanone, with a significant increase in chromatin abnormalities occurring in the presence of ZEA (Pzearalenol (Pzearalenol (mean value 1.6 ng/ml) with respect to ZEA and zearalanone (mean estradiol concentrations of 0.06 and 0.5 ng/ml, respectively). These data demonstrate a negative effect of ZEA and its derivatives on meiotic progression of bovine oocytes, possibly attributable to a toxic mechanism not related to the binding affinity of these compounds to estrogen receptor sites, and support previous observations that alpha-zearalenol acts as a stronger estrogenic inducer than the original molecule (ZEA).

  17. Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments

    Directory of Open Access Journals (Sweden)

    Sondergaard Bodil-Cecilie

    2009-12-01

    Full Text Available Abstract Background Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release ex vivo of MMP- and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fragments with the activity of proteolytic enzymes using inhibitors. Findings Bovine cartilage explants were cultured in the presence or absence of the catabolic cytokines oncostatin M (OSM and tumor necrosis factor alpha (TNFα. In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3. Fragments released into the supernatant were determined using a range of neo-epitope specific immunoassays; (1 sandwich 342FFGVG-G2 ELISA, (2 competition NITEGE373ELISA (3 sandwich G1-NITEGE373 ELISA (4 competition 374ARGSV ELISA, and (5 sandwich 374ARGSV-G2 ELISA all detecting aggrecan fragments, and (6 sandwich CTX-II ELISA, detecting C-telopeptides of type II collagen. We found that (1 aggrecanase-derived aggrecan fragments are released in the early (day 2-7 and mid phase (day 9-14 into the supernatant from bovine explants cultures stimulated with catabolic cytokines, (2 the release of NITEGE373 neo-epitopes are delayed compared to the corresponding 374ARGSV fragments, (3 the MMP inhibitor GM6001 did not reduce the release of aggrecanase-derived fragment, but induced a further delay in the release of these fragments, and finally (4 the MMP-derived aggrecan and type II collagen fragments were released in the late phase (day 16-21 only. Conclusion Our data support the model, that aggrecanases and MMPs act independently in the processing of the aggrecan molecules, and furthermore that suppression of MMP-activity had little if

  18. The Periconceptional Environment and Cardiovascular Disease: Does In Vitro Embryo Culture and Transfer Influence Cardiovascular Development and Health?

    Directory of Open Access Journals (Sweden)

    Monalisa Padhee

    2015-02-01

    Full Text Available Assisted Reproductive Technologies (ARTs have revolutionised reproductive medicine; however, reports assessing the effects of ARTs have raised concerns about the immediate and long-term health outcomes of the children conceived through ARTs. ARTs include manipulations during the periconceptional period, which coincides with an environmentally sensitive period of gamete/embryo development and as such may alter cardiovascular development and health of the offspring in postnatal life. In order to identify the association between ARTs and cardiovascular health outcomes, it is important to understand the events that occur during the periconceptional period and how they are affected by procedures involved in ARTs. This review will highlight the emerging evidence implicating adverse cardiovascular outcomes before and after birth in offspring conceived through ARTs in both human and animal studies. In addition, it will identify the potential underlying causes and molecular mechanisms responsible for the congenital and adult cardiovascular dysfunctions in offspring whom were conceived through ARTs.

  19. Production of transgenic canine embryos using interspecies somatic cell nuclear transfer.

    Science.gov (United States)

    Hong, So Gun; Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Kim, Geon A; Koo, Ok Jae; Jang, Goo; Lee, Byeong Chun

    2012-02-01

    Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry 'foreign' DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8-16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8-16-cell stages without mosaicism. In summary, our results demonstrated that

  20. Transcriptomic analysis of neurulation and early organogenesis in rat embryos: an in vivo and ex vivo comparison

    NARCIS (Netherlands)

    Robinson, J.F.; Verhoef, A.; Piersma, A.H.

    2012-01-01

    Cultured embryos mimic the morphological developmental progression of embryos (in vivo) undergoing neurulation and early organogenesis. Using available genomics technologies, comparative molecular-based assessments between cultured embryos and in vivo models may further clarify commonalities and

  1. Effects of Moraxella (Branhamella) ovis Culture Filtrates on Bovine Erythrocytes, Peripheral Mononuclear Cells, and Corneal Epithelial Cells†

    OpenAIRE

    Cerny, Henry E.; Rogers, Douglas G.; Gray, Jeffrey T.; Smith, David R.; Hinkley, Susanne

    2006-01-01

    Infectious bovine keratoconjunctivitis (IBK) is a highly contagious ocular disease that affects cattle of all ages and that occurs worldwide. Piliated hemolytic Moraxella bovis is recognized as the etiologic agent of IBK. According to data from the Nebraska Veterinary Diagnostic Laboratory System, however, Moraxella (Branhamella) ovis has been isolated with increasing frequency from cattle affected with IBK. The objective of this study was, therefore, to examine M. ovis field isolates for the...

  2. Sensitivity, specificity and predictive probability values of serum agglutination test titres for the diagnosis of Salmonella Dublin culture-positive bovine abortion and stillbirth.

    Science.gov (United States)

    Sánchez-Miguel, C; Crilly, J; Grant, J; Mee, J F

    2017-12-12

    The objective of this study was to determine the diagnostic value of maternal serology for the diagnosis of Salmonella Dublin bovine abortion and stillbirth. A retrospective, unmatched, case-control study was carried out using twenty year's data (1989-2009) from bovine foetal submissions to an Irish government veterinary laboratory. Cases (n = 214) were defined as submissions with a S. Dublin culture-positive foetus from a S. Dublin unvaccinated dam where results of maternal S. Dublin serology were available. Controls (n = 415) were defined as submissions where an alternative diagnosis other than S. Dublin was made in a foetus from an S. Dublin unvaccinated dam where the results of maternal S. Dublin serology were available. A logistic regression model was fitted to the data: the dichotomous dependent variable was the S. Dublin foetal culture result, and the independent variables were the maternal serum agglutination test (SAT) titre results. Salmonella serology correctly classified 87% of S. Dublin culture-positive foetuses at a predicted probability threshold of 0.44 (cut-off at which sensitivity and specificity are at a maximum, J = 0.67). The sensitivity of the SAT at the same threshold was 73.8% (95% CI: 67.4%-79.5%), and the specificity was 93.2% (95% CI: 90.3%-95.4%). The positive and negative predictive values were 84.9% (95% CI: 79.3%-88.6%) and 87.3% (95% CI: 83.5%-91.3%), respectively. This study illustrates that the use of predicted probability values, rather than the traditional arbitrary breakpoints of negative, inconclusive and positive, increases the diagnostic value of the maternal SAT. Veterinary laboratory diagnosticians and veterinary practitioners can recover from the test results, information previously categorized, particularly from those results declared to be inconclusive. © 2017 Blackwell Verlag GmbH.

  3. Assessing embryo development using swept source optical coherence tomography

    Science.gov (United States)

    Caujolle, S.; Cernat, R.; Silvestri, G.; Marques, M. J.; Bradu, A.; Feuchter, T.; Robinson, G.; Griffin, D.; Podoleanu, A.

    2018-03-01

    A detailed assessment of embryo development would assist biologists with selecting the most suitable embryos for transfer leading to higher pregnancy rates. Currently, only low resolution microscopy is employed to perform this assessment. Although this method delivers some information on the embryo surface morphology, no specific details are shown related to its inner structure. Using a Master-Slave Swept-Source Optical Coherence Tomography (SS-OCT), images of bovine embryos from day 7 after fertilization were collected from different depths. The dynamic changes inside the embryos were examined, in detail and in real-time from several depths. To prove our ability to characterize the morphology, a single embryo was imaged over 26 hours. The embryo was deprived of its life support environment, leading to its death. Over this period, clear morphological changes were observed.

  4. Comparative embryotoxicity of different antimalarial peroxides: in vitro study using the rat whole embryo culture model (WEC).

    Science.gov (United States)

    Longo, Monica; Zanoncelli, Sara; Brughera, Marco; Colombo, Paolo; Wittlin, Sergio; Vennerstrom, Jonathan L; Moehrle, Joerg; Craft, J Carl

    2010-12-01

    Three groups of compounds: (i) active peroxides (artemisinin and arterolene), (ii) inactive non-peroxidic derivatives (deoxyartemisinin and carbaOZ277) and (iii) inactive peroxide (OZ381) were tested by WEC system to provide insights into the relationship between chemical structure and embryotoxic potential, and to assess the relationship between embryotoxicity and antimalarial activity. Deoxyartemisinin, OZ381 and carbaOZ277 did not affect rat embryonic development. Artemisinin and arterolane affected primarily nucleated red blood cells (RBCs), inducing anemia and subsequent tissue damage in rat embryos, with NOELs for RBC damage at 0.1 and 0.175μg/mL, respectively. These data support the idea that only active antimalarial peroxides are able to interfere with normal embryonic development. In an attempt to establish whether and to what extent activity as antimalarials and embryotoxicity can be divorced, IC(50)s for activity in Plasmodium falciparum strains and the NOELs for RBCs were compared. From this comparison, arterolane showed a better safety margin than artemisinin. Copyright © 2010 Elsevier Inc. All rights reserved.

  5. Eight-Shaped Hatching Increases the Risk of Inner Cell Mass Splitting in Extended Mouse Embryo Culture.

    Science.gov (United States)

    Yan, Zheng; Liang, Hongxing; Deng, Li; Long, Hui; Chen, Hong; Chai, Weiran; Suo, Lun; Xu, Chen; Kuang, Yanping; Wu, Lingqian; Lu, Shengsheng; Lyu, Qifeng

    2015-01-01

    Increased risk of monozygotic twinning (MZT) has been shown to be associated with assisted reproduction techniques, particularly blastocyst culture. Interestingly, inner cell mass (ICM) splitting in human '8'-shaped hatching blastocysts that resulted in MZT was reported. However, the underlying cause of MZT is not known. In this study, we investigated in a mouse model whether in vitro culture leads to ICM splitting and its association with hatching types. Blastocyst hatching was observed in: (i) in vivo developed blastocysts and (ii-iii) in vitro cultured blastocysts following in vivo or in vitro fertilization. We found that '8'-shaped hatching occurred with significantly higher frequency in the two groups of in vitro cultured blastocysts than in the group of in vivo developed blastocysts (24.4% and 20.4% versus 0.8%, respectively; n = 805, P cultured groups. Furthermore, we found more double OCT4-positive masses, suggestive of increased ICM splitting in '8'-shaped hatching and hatched blastocysts than in 'U'-shaped hatching and hatched blastocysts (12.5% versus 1.9%, respectively; n = 838, P culture can cause high frequencies of '8'-shaped hatching, and '8'-shaped hatching that may disturb ICM herniation leading to increased risk of ICM splitting in mouse blastocysts. These results may provide insights into the increased risk of human MZT after in vitro fertilization and blastocyst transfer.

  6. Cultura de embriões in vitro para o melhoramento de pessegueiros precoces In vitro embryo culture for early ripening peach breeding

    Directory of Open Access Journals (Sweden)

    Wilson Barbosa

    1985-01-01

    Full Text Available Efetuou-se o experimento de germinação das sementes de quinze seleções de pêssegos e nectarinas, a maioria de maturação bem precoce, através do método da cultura in vitro, de embriões imaturos, em confronto com a germinação obtida pelo processo de estratificação prévia das amêndoas a frio úmido, em substrato de algodão. Para a cultura in vitro, utilizaram-se embriões acompanhados de cotilédones, extraídos de frutos semimaduros, que foram "semeados" em meio de cultura, compostos de macro e microelementos de Murashige e Skoog, acrescido de tiamina a 0,1mg/litro, ácido nicotínico a 0,5mg/litro, inositol a 100mg/litro, glicina a 20mg/litro„ácido giberélico a 0,1mg/litro, glicoce a 3% e ágar a 0,7%. Os resultados mostraram que houve excelente desenvolvimento dos embriões in vitro para a maioria das seleções de ciclo bem precoce, e os "seedlings" resultantes tiveram crescimento normal. Ao contrário, o processo de estratificação prévia para as mesmas seleções proporcionou porcentagem de germinação mediana, quando as sementes foram estratificadas logo que extraídas dos frutos, e bastante baixa ou nula quando elas ficaram expostas ao ambiente natural por 96 horas antes da estratificação. Tais resultados indicam que o método de cultura in vitro de embriões imaturos constitui prática indispensável ao programa de melhoramento de pessegueiro, com vista à obtenção de cultivares cada vez mais precoces.This papar compares the results of an embryo culture with those of the usual seed stratification process. One hundred seeds obtained from early ripening peach were stratified either immediately after extraction from fruits (50 seeds or after exposure to room temperature for 96 hours (50 seeds. Embryos from mature fruits were cultured aseptically in vitro on nutrient media containing the macro and microelements of Murashige & Skoog, inusitol, thiamine, nicotinic acid, glycine, GA3, sucrose and agar at 5(0C for 50

  7. Ikarisoside A inhibits acetylcholine-induced catecholamine secretion and synthesis by suppressing nicotinic acetylcholine receptor-ion channels in cultured bovine adrenal medullary cells.

    Science.gov (United States)

    Li, Xiaojia; Toyohira, Yumiko; Horisita, Takafumi; Satoh, Noriaki; Takahashi, Keita; Zhang, Han; Iinuma, Munekazu; Yoshinaga, Yukari; Ueno, Susumu; Tsutsui, Masato; Sata, Takeyoshi; Yanagihara, Nobuyuki

    2015-12-01

    Ikarisoside A is a natural flavonol glycoside derived from plants of the genus Epimedium, which have been used in Traditional Chinese Medicine as tonics, antirheumatics, and aphrodisiacs. Here, we report the effects of ikarisoside A and three other flavonol glycosides on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. We found that ikarisoside A (1-100 μM), but not icariin, epimedin C, or epimedoside A, concentration-dependently inhibited the secretion of catecholamines induced by acetylcholine, a physiological secretagogue and agonist of nicotinic acetylcholine receptors. Ikarisoside A had little effect on catecholamine secretion induced by veratridine and 56 mM K(+). Ikarisoside A (1-100 μM) also inhibited (22)Na(+) influx and (45)Ca(2+) influx induced by acetylcholine in a concentration-dependent manner similar to that of catecholamine secretion. In Xenopus oocytes expressing α3β4 nicotinic acetylcholine receptors, ikarisoside A (0.1-100 μM) directly inhibited the current evoked by acetylcholine. It also suppressed (14)C-catecholamine synthesis and tyrosine hydroxylase activity induced by acetylcholine at 1-100 μM and 10-100 μM, respectively. The present findings suggest that ikarisoside A inhibits acetylcholine-induced catecholamine secretion and synthesis by suppression of nicotinic acetylcholine receptor-ion channels in bovine adrenal medullary cells.

  8. Stimulatory effect of nobiletin, a citrus polymethoxy flavone, on catecholamine synthesis through Ser19 and Ser40 phosphorylation of tyrosine hydroxylase in cultured bovine adrenal medullary cells.

    Science.gov (United States)

    Zhang, Han; Yanagihara, Nobuyuki; Toyohira, Yumiko; Takahashi, Keita; Inagaki, Hirohide; Satoh, Noriaki; Li, Xiaoja; Goa, Xiumei; Tsutsui, Masato; Takahaishi, Kojiro

    2014-01-01

    We previously reported the dual effects of nobiletin, a compound of polymethoxy flavones found in citrus fruits, on catecholamine secretion in cultured bovine adrenal medullary cells. Here, we report the effects of nobiletin on catecholamine synthesis in the cells. Nobiletin increased the synthesis of (14)C-catecholamines from [(14)C]tyrosine in a time (20-30 min)- and concentration (1.0-100 μM)-dependent manner. Nobiletin (10-100 μM) also activated tyrosine hydroxylase activity. The stimulatory effect of nobiletin on (14)C-catecholamine synthesis was not observed when extracellular Ca(2+) was not present in the incubation medium. Protein kinase inhibitors including H-89, an inhibitor of cyclic AMP-dependent protein kinase, and KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinase II, suppressed the stimulatory effects of nobiletin on catecholamine synthesis as well as tyrosine hydroxylase activity. Nobiletin also induced the phosphorylation of tyrosine hydroxylase at Ser(19) and Ser(40). Nobiletin (1.0-100 μM) inhibited (14)C-catecholamine synthesis induced by acetylcholine. The present findings suggest that nobiletin, by itself, stimulates catecholamine synthesis through tyrosine hydroxylase phosphorylation at Ser(19) and Ser(40), whereas it inhibits catecholamine synthesis induced by acetylcholine in bovine adrenal medulla.

  9. A comparison of the embryonic stem cell test and whole embryo culture assay combined with the BeWo placental passage model for predicting the embryotoxicity of azoles.

    Science.gov (United States)

    Dimopoulou, Myrto; Verhoef, Aart; Gomes, Caroline A; van Dongen, Catharina W; Rietjens, Ivonne M C M; Piersma, Aldert H; van Ravenzwaay, Bennard

    2018-04-01

    In the present study, we show the value of combining toxico-dynamic and -kinetic in vitro approaches for embryotoxicity testing of azoles. Both the whole embryo culture (WEC) and the embryonic stem cells test (EST) predicted the in vivo potency ranking of twelve tested azoles with moderate accuracy. Combining these results with relative placental transfer rates (Papp values) as determined in the BeWo cell culture model, increased the predictability of both WEC and EST, with R 2 values increasing from 0.51 to 0.87 and from 0.35 to 0.60, respectively. The comparison of these in vitro systems correlated well (R 2  = 0.67), correctly identifying the in vivo strong and weak embryotoxicants. Evaluating also specific gene responses related with the retinoic acid and sterol biosynthesis pathways, which represent the toxicological and fungicidal mode of action of azoles respectively in the WEC and EST, we observed that the differential regulation of Dhrs3 and Msmo1 reached higher magnitudes in both systems compared to Cyp26a1 and Cyp51. Establishing sensitive biomarkers across the in vitro systems for studying the underlying mechanism of action of chemicals, such as azoles, is valuable for comparing alternative in vitro models and for improving insight in the mechanism of developmental toxicity of chemicals. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

  10. Selection of the In Vitro Culture Media Influences mRNA Expression of Hedgehog Genes, Il-6, and Important Genes regarding Reactive Oxygen Species in Single Murine Preimplantation Embryos

    Directory of Open Access Journals (Sweden)

    N. Pfeifer

    2012-01-01

    Full Text Available Background. The aim of this paper was to determine the influence of different in vitro culture media on mRNA expression of Hedgehog genes, il-6, and important genes regarding reactive oxygen species in single mouse embryos. Methods. Reverse transcription of single embryos either cultured in vitro from day 0.5 until 3.5 (COOK’s Cleavage medium or Vitrolife’s G-1 PLUS medium or in vivo until day 3.5 post coitum. PCR was carried out for β-actin followed by nested-PCR for shh, ihh, il-6, nox, gpx4, gpx1, and prdx2. Results. The number of murine blastocysts cultured in COOK medium which expressed il-6, gpx4, gpx1, and prdx2 mRNA differed significantly compared to the in vivo group. Except for nox, the mRNA profile of the Vitrolife media group embryos varied significantly from the in vivo ones regarding the number of blastocysts expressing the mRNA of shh, ihh, il-6, gpx4, gpx1 and prdx2. Conclusions. The present study shows that different in vitro culture media lead to different mRNA expression profiles during early development. Even the newly developed in vitro culture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies.

  11. Multilineage Potential Research of Bovine Amniotic Fluid Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Yuhua Gao

    2014-02-01

    Full Text Available The use of amnion and amniotic fluid (AF are abundant sources of mesenchymal stem cells (MSCs that can be harvested at low cost and do not pose ethical conflicts. In human and veterinary research, stem cells derived from these tissues are promising candidates for disease treatment, specifically for their plasticity, their reduced immunogenicity, and high anti-inflammatory potential. This work aimed to obtain and characterize bovine amniotic fluid mesenchymal stem cells (AFMSC. The bovine AF from the amniotic cavity of pregnant gilts in the early stages of gestation (3- and 4-m-old bovine embryos was collected. AFMSCs exhibit a fibroblastic-like morphology only starting from the fourth passage, being heterogeneous during the primary culture. Immunofluorescence results showed that AFMSCs were positive for β-integrin, CD44, CD73 and CD166, but negative for CD34, CD45. Meanwhile, AFMSCs expressed ES cell markers, such as Oct4, and when appropriately induced, are capable of differentiating into ectodermal and mesodermal lineages. This study reinforces the emerging importance of these cells as ideal tools in veterinary medicine; future studies aimed at a deeper evaluation of their immunological properties will allow a better understanding of their role in cellular therapy.

  12. Eight-Shaped Hatching Increases the Risk of Inner Cell Mass Splitting in Extended Mouse Embryo Culture.

    Directory of Open Access Journals (Sweden)

    Zheng Yan

    Full Text Available Increased risk of monozygotic twinning (MZT has been shown to be associated with assisted reproduction techniques, particularly blastocyst culture. Interestingly, inner cell mass (ICM splitting in human '8'-shaped hatching blastocysts that resulted in MZT was reported. However, the underlying cause of MZT is not known. In this study, we investigated in a mouse model whether in vitro culture leads to ICM splitting and its association with hatching types. Blastocyst hatching was observed in: (i in vivo developed blastocysts and (ii-iii in vitro cultured blastocysts following in vivo or in vitro fertilization. We found that '8'-shaped hatching occurred with significantly higher frequency in the two groups of in vitro cultured blastocysts than in the group of in vivo developed blastocysts (24.4% and 20.4% versus 0.8%, respectively; n = 805, P < 0.01. Moreover, Oct4 immunofluorescence staining was performed to identify the ICM in the hatching and hatched blastocysts. Scattered and split distribution of ICM cells was observed around the small zona opening of '8'-shaped hatching blastocysts. This occurred at a high frequency in the in vitro cultured groups. Furthermore, we found more double OCT4-positive masses, suggestive of increased ICM splitting in '8'-shaped hatching and hatched blastocysts than in 'U'-shaped hatching and hatched blastocysts (12.5% versus 1.9%, respectively; n = 838, P < 0.01. Therefore, our results demonstrate that extended in vitro culture can cause high frequencies of '8'-shaped hatching, and '8'-shaped hatching that may disturb ICM herniation leading to increased risk of ICM splitting in mouse blastocysts. These results may provide insights into the increased risk of human MZT after in vitro fertilization and blastocyst transfer.

  13. Effects of cell culture techniques on gene expression and cholesterol efflux in primary bovine mammary epithelial cells derived from milk and tissue.

    Science.gov (United States)

    Sorg, D; Potzel, A; Beck, M; Meyer, H H D; Viturro, E; Kliem, H

    2012-10-01

    Primary bovine mammary epithelial cells (pbMEC) are often used in cell culture to study metabolic and inflammatory processes in the udder of dairy cows. The most common source is udder tissue from biopsy or after slaughter. However, it is also possible to culture them from milk, which is non-invasive, repeatable and yields less contamination with fibroblasts. Generally, not much is known about the influence of cell origin and cell culture techniques such as cryopreservation on pbMEC functionality. Cells were extracted from milk and udder tissue to evaluate if milk-derived pbMEC are a suitable alternative to tissue-derived pbMEC and to test what influence cryopreservation has. The cells were cultivated for three passages and stored in liquid nitrogen. The relative gene expression of the five target genes kappa-casein, lingual antimicrobial peptide (LAP), lactoferrin, lysozyme (LYZ1) and the prolactin receptor normalised with keratin 8 showed a tendency to decrease in the tissue cultures, but not in the milk-derived cultures, suggesting a greater influence of the cultivation process on tissue-derived cells, freezing lowered expression levels in both cultures. Overall expression of LAP and LYZ1 tended to be higher in milk cells. Cholesterol efflux was measured to compare passages one to seven in milk-derived cells. Passage number did not alter the efflux rate (p ≤ 0.05). We showed for the first time that the extraction of pbMEC from milk can be a suitable alternative to tissue extraction.

  14. 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

  15. Removal of transmissible spongiform encephalopathy prion from large volumes of cell culture media supplemented with fetal bovine serum by using hollow fiber anion-exchange membrane chromatography.

    Science.gov (United States)

    Chou, Ming Li; Bailey, Andy; Avory, Tiffany; Tanimoto, Junji; Burnouf, Thierry

    2015-01-01

    Cases of variant Creutzfeldt-Jakob disease in people who had consumed contaminated meat products from cattle with bovine spongiform encephalopathy emphasize the need for measures aimed at preventing the transmission of the pathogenic prion protein (PrPSc) from materials derived from cattle. Highly stringent scrutiny is required for fetal bovine serum (FBS), a growth-medium supplement used in the production of parenteral vaccines and therapeutic recombinant proteins and in the ex vivo expansion of stem cells for transplantation. One such approach is the implementation of manufacturing steps dedicated to removing PrPSc from materials containing FBS. We evaluated the use of the QyuSpeed D (QSD) adsorbent hollow-fiber anion-exchange chromatographic column (Asahi Kasei Medical, Tokyo, Japan) for the removal of PrPSc from cell culture media supplemented with FBS. We first established that QSD filtration had no adverse effect on the chemical composition of various types of culture media supplemented with 10% FBS or the growth and viability characteristics of human embryonic kidney (HEK293) cells, baby hamster kidney (BHK-21) cells, African green monkey kidney (Vero) cells, and Chinese hamster ovary (CHO-k1) cells propagated in the various culture-medium filtrates. We used a 0.6-mL QSD column for removing PrPSc from up to 1000 mL of Dulbecco's modified Eagle's medium containing 10% FBS previously spiked with the 263K strain of hamster-adapted scrapie. The Western blot analysis, validated alongside an infectivity assay, revealed that the level of PrPSc in the initial 200mL flow-through was reduced by 2.5 to > 3 log10, compared with that of the starting material. These results indicate that QSD filtration removes PrPSc from cell culture media containing 10% FBS, and demonstrate the ease with which QSD filtration can be implemented in at industrial-scale to improve the safety of vaccines, therapeutic recombinant proteins, and ex vivo expanded stem cells produced using growth

  16. Removal of transmissible spongiform encephalopathy prion from large volumes of cell culture media supplemented with fetal bovine serum by using hollow fiber anion-exchange membrane chromatography.

    Directory of Open Access Journals (Sweden)

    Ming Li Chou

    Full Text Available Cases of variant Creutzfeldt-Jakob disease in people who had consumed contaminated meat products from cattle with bovine spongiform encephalopathy emphasize the need for measures aimed at preventing the transmission of the pathogenic prion protein (PrPSc from materials derived from cattle. Highly stringent scrutiny is required for fetal bovine serum (FBS, a growth-medium supplement used in the production of parenteral vaccines and therapeutic recombinant proteins and in the ex vivo expansion of stem cells for transplantation. One such approach is the implementation of manufacturing steps dedicated to removing PrPSc from materials containing FBS. We evaluated the use of the QyuSpeed D (QSD adsorbent hollow-fiber anion-exchange chromatographic column (Asahi Kasei Medical, Tokyo, Japan for the removal of PrPSc from cell culture media supplemented with FBS. We first established that QSD filtration had no adverse effect on the chemical composition of various types of culture media supplemented with 10% FBS or the growth and viability characteristics of human embryonic kidney (HEK293 cells, baby hamster kidney (BHK-21 cells, African green monkey kidney (Vero cells, and Chinese hamster ovary (CHO-k1 cells propagated in the various culture-medium filtrates. We used a 0.6-mL QSD column for removing PrPSc from up to 1000 mL of Dulbecco's modified Eagle's medium containing 10% FBS previously spiked with the 263K strain of hamster-adapted scrapie. The Western blot analysis, validated alongside an infectivity assay, revealed that the level of PrPSc in the initial 200mL flow-through was reduced by 2.5 to > 3 log10, compared with that of the starting material. These results indicate that QSD filtration removes PrPSc from cell culture media containing 10% FBS, and demonstrate the ease with which QSD filtration can be implemented in at industrial-scale to improve the safety of vaccines, therapeutic recombinant proteins, and ex vivo expanded stem cells produced

  17. Comparison of the diagnostic performance of bacterial culture of nasopharyngeal swab and bronchoalveolar lavage fluid samples obtained from calves with bovine respiratory disease.

    Science.gov (United States)

    Capik, Sarah F; White, Brad J; Lubbers, Brian V; Apley, Michael D; DeDonder, Keith D; Larson, Robert L; Harhay, Greg P; Chitko-McKown, Carol G; Harhay, Dayna M; Kalbfleisch, Ted S; Schuller, Gennie; Clawson, Michael L

    2017-03-01

    OBJECTIVE To compare predictive values, extent of agreement, and gamithromycin susceptibility between bacterial culture results of nasopharyngeal swab (NPS) and bronchoalveolar lavage fluid (BALF) samples obtained from calves with bovine respiratory disease (BRD). ANIMALS 28 beef calves with clinical BRD. PROCEDURES Pooled bilateral NPS samples and BALF samples were obtained for bacterial culture from calves immediately before and at various times during the 5 days after gamithromycin (6 mg/kg, SC, once) administration. For each culture-positive sample, up to 12 Mannheimia haemolytica, 6 Pasteurella multocida, and 6 Histophilus somni colonies underwent gamithromycin susceptibility testing. Whole-genome sequencing was performed on all M haemolytica isolates. For paired NPS and BALF samples collected 5 days after gamithromycin administration, the positive and negative predictive values for culture results of NPS samples relative to those of BALF samples and the extent of agreement between the sampling methods were determined. RESULTS Positive and negative predictive values of NPS samples were 67% and 100% for M haemolytica, 75% and 100% for P multocida, and 100% and 96% for H somni. Extent of agreement between results for NPS and BALF samples was substantial for M haemolytica (κ, 0.71) and H somni (κ, 0.78) and almost perfect for P multocida (κ, 0.81). Gamithromycin susceptibility varied within the same sample and between paired NPS and BALF samples. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated culture results of NPS and BALF samples from calves with BRD should be interpreted cautiously considering disease prevalence within the population, sample collection relative to antimicrobial administration, and limitations of diagnostic testing methods.

  18. Uncoupling of attenuated myo-[3H]inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells

    International Nuclear Information System (INIS)

    Cammarata, P.R.; Tse, D.; Yorio, T.

    1991-01-01

    Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-[3H]inositol was lowered after 20 h of incubation in galactose and remained below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-[3H]inositol uptake. No significant difference in the rates of passive efflux of myo-[3H]inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia

  19. Uncoupling of attenuated myo-(3H)inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Cammarata, P.R.; Tse, D.; Yorio, T. (Department of Anatomy, Texas College of Osteopathic Medicine/University of North Texas, Fort Worth (USA))

    1991-06-01

    Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-(3H)inositol was lowered after 20 h of incubation in galactose and remained below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-(3H)inositol uptake. No significant difference in the rates of passive efflux of myo-(3H)inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.

  20. Study of callus initiation and growth criteria at different concentrations of 2,4-D and kinetin in Taxus baccata L. embryo culture

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    Ramin Karimian

    2015-03-01

    Full Text Available Taxus baccata L. is the main and almost the sole source of paclitaxel, which is an anti neoplastic agent and is used as a cure for treatment of breast and ovary cancer. Since production of this drug is almost a destructive process and yew is an endangered plant, it is required to optimize its tissue culture and micropropagation system. In order to produce embryo derived callus 16 combinations of two plant growth regulators 2,4-D (0, 0.5, 1 and 1.5 mg/L and Kinetin (0, 0.1, 0.5 and 1 mg/L were used to study its effects on callus formation and growth in Taxus baccata. It was determined that 1.5 mg/L 2,4-D + 0.1 mg/L kinetin was the best combination for callus growth. Increasing 2,4-D concentration, however, promoted callogenesis while kinetin only in a narrow range had positive effect on callus initiation.

  1. Methanol as a cryoprotectant for equine embryos.

    Science.gov (United States)

    Bass, L D; Denniston, D J; Maclellan, L J; McCue, P M; Seidel, G E; Squires, E L

    2004-09-15

    Equine embryos (n=43) were recovered nonsurgically 7-8 days after ovulation and randomly assigned to be cryopreserved in one of two cryoprotectants: 48% (15M) methanol (n=22) or 10% (136 M) glycerol (n=21). Embryos (300-1000 microm) were measured at five intervals after exposure to glycerol (0, 2, 5, 10 and 15 min) or methanol (0, 15, 35, 75 and 10 min) to determine changes (%) in diameter over time (+/-S.D.). Embryos were loaded into 0.25-ml plastic straws, sealed, placed in a programmable cell freezer and cooled from room temperature (22 degrees C) to -6 degrees C. Straws were then seeded, held at -6 degrees C for 10 min and then cooled to -33 degrees C before being plunged into liquid nitrogen. Two or three embryos within a treatment group were thawed and assigned to be either cultured for 12 h prior to transfer or immediately nonsurgically transferred to a single mare. Embryo diameter decreased in all embryos upon initial exposure to cryoprotectant. Embryos in methanol shrank and recovered slightly to 76+/-8 % of their original diameter; however, embryos in glycerol continued to shrink, reaching 57+/-6 % of their original diameter prior to cryopreservation. Survival rates of embryos through Day 16 of pregnancy were 38 and 23%, respectively (P>0.05) for embryos cryopreserved in the presence of glycerol or methanol. There was no difference in pregnancy rates of mares receiving embryos that were cultured prior to transfer or not cultured (P>0.05). Preliminary experiments indicated that 48% methanol was not toxic to fresh equine embryos but methanol provided no advantage over glycerol as a cryoprotectant for equine blastocysts.

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

    Directory of Open Access Journals (Sweden)

    Van Zeveren Alex

    2007-06-01

    vitro produced embryos, reflecting the influence of the in vitro culture system on the embryonic gene expression. Both RNA and protein expression analysis demonstrated that KRT18, FN1 and MYL6 are differentially expressed during preimplantation embryo development and those genes can be considered as markers for bovine blastocyst formation.

  3. Revisiting bovine pyometra-New insights into the disease using a culture-independent deep sequencing approach

    DEFF Research Database (Denmark)

    Knudsen, Lif Rødtness Vesterby; Karstrup, Cecilia Christensen; Pedersen, Hanne Gervi

    2015-01-01

    -independent studies have demonstrated that the bacterial diversity in most environments is underestimated in culture-based studies. Consequently, fastidious pyometra-associated pathogens may have been overlooked. Therefore, the primary purpose of this study was to investigate the diversity of bacteria in the uterus......The bacteria present in the uterus during pyometra have previously been studied using bacteriological culturing. These studies identified Fusobacterium necrophorum and Trueperella pyogenes as the major contributors to the pathogenesis of pyometra. However, an increasing number of culture...... of cows with pyometra by using culture-independent 16S rRNA PCR combined with next generation sequencing. We investigated the microbial composition in the uterus of 21 cows with pyometra, which were obtained from a Danish slaughterhouse. Similar to the observations from the culture studies...

  4. CULTURA IN VITRO DE EMBRIÕES ZIGÓTICOS DE AÇAIZEIRO IN VITRO CULTURE OF ASSAI PALM ZYGOTIC EMBRYOS

    Directory of Open Access Journals (Sweden)

    ANA DA SILVA LEDO

    2001-12-01

    average, larger formation of normal seedlings when compared with 5,37 muM NAA. The presence of 2,68 muM NAA plus 1,11; 1,55 and 2,22 muM BAP was induced largest shoot length. Significant differences were not verified between NAA and BAP concentrations for the number of roots/seedlings. The obtained results allowed verifying that an addition of NAA and BAP in the culture medium was necessary to the conversion of mature zygotic embryos in both vigorous and normal seedlings.

  5. Cytogenetics and immature embryo culture at Embrapa Trigo breeding program: transfer of disease resistance from related species by artificial resynthesis of hexaploid wheat (Triticum aestivum L. em. Thell

    Directory of Open Access Journals (Sweden)

    Maria Irene Baggio de Moraes Fernandes

    2000-12-01

    to facilitate gene flow between wheat and related species. Since the environment at the center of origin of wheat in Southern Asia is quite different from subtropical environments, Brazilian breeding programs overcome more challenges to adapt wheat crop to biotic and abiotic stresses than some other countries. The germplasm bank of Embrapa Trigo has about 1000 registered entries of Triticum relatives, Aegilops, Secale and Agropyron species supplied from several germplasm banks distributed over the world which were multiplied and/or selected for naturally occurring or artificially inoculated fungal diseases. Since Aegilops squarrosa L. entries showed very good performance, the genetic variability observed in this species was firstly exploited. It is reported here the strategy used for transferring useful genes from Ae. squarrosa (DD, 2n = 14: crossing with tetraploid species (AABB, 2n = 28, rescue and in vitro culture of immature embryos for regeneration of the trihaploid (ABD, 2n = 21 hybrid, and colchicine treatment for genome duplication resulting in the artificial synthesis of hexaploid wheat lines (AABBDD, 2n = 42. Results of 10,739 artificial pollinations involving 28 cross combinations amongst eight T. durum L., T. dicoccum and T. cartlicum tetraploid entries used as female parents and ten selected Ae. squarrosa sources of resistance as male parents are presented here. Immature embryos from 18 cross combinations were recovered and cultured in vitro. Green plantlets from 13 combinations were regenerated. Fertile amphiploids were recovered only from crosses among entries of tetraploid T. durum and diploid Ae. squarrosa. They originated 11 fertile synthetic amphiploid lines from seven different combinations. Useful stem and leaf rust as well as powdery mildew resistance for future use in breeding programs were obtained.

  6. Repair of wounded monolayers of cultured bovine aortic endothelial cells is inhibited by calcium spirulan, a novel sulfated polysaccharide isolated from Spirulina platensis.

    Science.gov (United States)

    Kaji, Toshiyuki; Fujiwara, Yasuyuki; Inomata, Yuki; Hamada, Chieko; Yamamoto, Chika; Shimada, Satomi; Lee, Jung-Bum; Hayashi, Toshimitsu

    2002-03-08

    Calcium spirulan (Ca-SP) is a novel sulfated polysaccharide isolated from a blue-green alga Spirulina platensis. Ca-SP inhibits thrombin by activation of heparin cofactor II. Therefore, it could serve as an origin of anti-atherogenic medicines. Since maintenance of vascular endothelial cell monolayers is important for prevention of vascular lesions such as atherosclerosis, the effect of Ca-SP at 20 microg/ml or less on the repair of wounded bovine aortic endothelial cell monolayers in culture was investigated in the present study. When the monolayers were wounded and cultured in the presence of Ca-SP, the polysaccharide inhibited the appearance of the cells in the wounded area. The inhibition was also observed even when the repair was promoted by excess basic fibroblast growth factor, which is one of the autocrine growth factors that are involved in the endothelial cell monolayer maintenance. On the other hand, Ca-SP inhibited the cell growth and the incorporation of [3H]thymidine into the acid-insoluble fraction of proliferating endothelial cells, suggesting that Ca-SP inhibits endothelial cell proliferation. From these results, it is concluded that Ca-SP may retard the repair process of damaged vascular endothelium through inhibition of vascular endothelial cell proliferation by induction of a lower ability to respond to stimulation by endogenous basic fibroblast growth factor.

  7. Endogenous folates and single-carbon metabolism in the ovarian follicle, oocyte and pre-implantation embryo.

    Science.gov (United States)

    Kwong, W Y; Adamiak, S J; Gwynn, A; Singh, R; Sinclair, K D

    2010-04-01

    Maternal B-vitamin status at conception can affect fertility and the health of offspring. This study details transcript expression for genes encoding key enzymes in the linked methionine/folate cycles in the bovine oocyte, somatic cells of the ovarian follicle and pre-implantation embryo. Transcripts for all 12 enzymes that were studied and for the two folate receptors (FOLR1 and FOLR2) and reduced folate carrier (SLC19A1) were expressed in liver cells, but transcripts for betaine-homocysteine methyltransferase and methionine adenosyl transferase 1A were absent in all ovarian cells, and transcripts for FOLR2 were absent in embryonic cells. Transcripts for glycine methyltransferase were also absent/weak in cumulus and granulosa cells. The absence of these enzymes could have a profound effect on single-carbon metabolism within the ovary and pre-implantation embryo. Immunocytochemical analysis revealed SLC19A1 protein expression on the plasma and basal-lateral membranes of the pre-implantation embryo. The folate antagonist methotrexate (MTX) enters the cell via SLC19A1, and in the current study, MTX inclusion in bovine/ovine culture media at either 1 or 10 microM from the 1-cell stage inhibited embryo development beyond the 8-cell stage. Hypoxanthine and thymidine (100 microM) increased the proportion of embryos that developed to blastocysts, but the cell number was reduced by 20%. The reduced uptake of [(35)S] methionine into intra-cellular S-adenosylmethionine and S-adenosylhomocysteine pools, together with reduced uptake of glutamate and tryptophan, was consistent with depleted intra-cellular pools of reduced folates. These data provide an insight into the importance of maternal dietary folate/B-vitamin status during the peri-conceptional period.

  8. Superovulatory response and embryonic progressive in Iranian Qezel ewes treated with two different concentrations of bovine somatotropin

    Directory of Open Access Journals (Sweden)

    Amir Hossein Asgari Safdar

    2016-05-01

    Conclusions: The treatment 50 mg bovine somatotropins enhance the ratio and growth of the transferable embryos. Embryos of bST-50 treatment indicated an improved embryonic development but bST did not affect the pregnancy rates of transferred embryos.