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Sample records for bovine embryo transfer

  1. Nucleolar ultrastructure in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

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

    1999-01-01

    (nonactivated) or S phase (activated) cytoplasts. Control embryos were fixed at the two-, four-, early eight- and late eight-cell stages; nuclear transfer embryos were fixed at 1 and 3 hr post fusion and at the two-, four-, and eight-cell stages. Control embryos possessed a nucleolar precursor body throughout...... at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary...... 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...

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

    DEFF Research Database (Denmark)

    Smith, Steven D.; Soloy, Eva; Kanka, Jiri

    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...... relies upon maternally derived RNA transcripts up to the 8-cell stage, at which time it begins to transcribe its own RNA. In this experiment, RNA synthesis was detected in nucleus transfer embryos (NTE) and control embryos by pulsing with 3H-uridine, fixation, and autoradiography on semithin sections....... NTE were produced using either a MII phase (nonactivated) cytoplasts at 32 hr of maturation or S-phase (activated) cytoplasts activated with calcium ionophore A23187 and cycloheximide treatment approximately 8 hr prior to fusion with a blastomere from an in-vitro-produced morula stage embryo at 32 hr...

  3. 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.

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

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    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.

  5. 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....

  6. In vitro assessment of a direct transfer vitrification procedure for bovine embryos.

    Science.gov (United States)

    Campos-Chillòn, L F; Walker, D J; de la Torre-Sanchez, J F; Seidel, G E

    2006-04-01

    We developed a simple vitrification technique for bovine embryos that could permit direct transfer. Embryos were produced in-vitro by standard procedures. The base medium for cryopreservation was a chemically defined medium similar to SOF + 25 mM Hepes and 0.25% fatty acid free bovine serum albumin (FAF-BSA) (HCDM2). In experiment 1, embryos were first exposed to 3.5M ethylene glycol (V1) for 1, 2 or 3 min at room temperature (20-24 degrees C), and then moved to 7 M ethylene glycol (V2) at 4 or 20-24 degrees C and loaded in 0.25-mL straws. After 45 s in 7 M ethylene glycol, straws were placed in liquid nitrogen. Embryos that were loaded at 20-24 degrees C had higher survival rates than those loaded at 4 degrees C (Pembryos survived well after 15 min in straws if warmed at 37 degrees C. In experiment 3, ethylene glycol concentration (3, 4 or 5 M) and exposure time (0.5 or 1 min) during two-step addition of cryoprotectant were studied for bovine morulae. In experiment 4, morulae were exposed to V2 for 30 or 45 s in HCDM2 or Vigro holding medium and then held in 22-24 degrees C air or 37 degrees C water post-warming. Experiment 5 was like experiment 4 except blastocysts were used. Overall survival rates of blastocysts in experiment 5 averaged 80% of non-vitrified controls after 48 h culture. The survival rates with in vitro-produced morulae in experiments 1, 3 and 4 were unacceptable. Vitrification solutions based on Vigro tended to result in higher survival than HCDM2 for blastocysts, but not morulae. In experiment 6, the survival rate in vitro of in vivo-produced morulae and blastocysts after two-step vitrification was nearly 100%. Our vitrification technique was very effective for in vitro produced blastocysts, but not for in vitro-produced morulae.

  7. Effect of addition of hyaluronan to embryo culture medium on survival of bovine embryos in vitro following vitrification and establishment of pregnancy after transfer to recipients.

    Science.gov (United States)

    Block, J; Bonilla, L; Hansen, P J

    2009-04-15

    Two experiments were conducted to determine whether addition of hyaluronan to culture medium could improve survival of bovine embryos after vitrification or following embryo transfer. In Experiment 1, embryos were produced in vitro and cultured for 7 days in modified synthetic oviductal fluid (SOF) containing one of four concentrations of hyaluronan (0, 0.1, 0.5, or 1mg/mL), with or without 4 mg/mL of bovine serum albumin (BSA). On Day 7 after insemination, blastocysts and expanded blastocysts were vitrified using open-pulled straws. At a concentration of 1mg/mL, hyaluronan increased (Pembryo hatching rate at 24 and 72 h. Treatment with BSA caused a slight reduction in cleavage rate (Pembryos were produced in vitro and cultured in modified SOF containing 4 mg/mL BSA, with or without 1mg/mL hyaluronan. At 159-162 h after insemination, grade 1 morula, blastocysts and expanded blastocysts were harvested for embryo transfer. Harvested embryos were transferred individually to lactating Holstein recipients with a palpable corpus luteum on Day 7 after presumptive ovulation. There was an interaction (Pembryo stage on pregnancy rate. Recipients that received morula and blastocyst stage embryos treated with hyaluronan had a higher pregnancy rate than recipients that received control embryos of the same stage. There was no effect of hyaluronan on pregnancy rates of recipients that received expanded blastocysts. In conclusion, addition of hyaluronan to embryo culture enhanced blastocyst yield, improved survival following vitrification, and enhanced the post-transfer survival of fresh morula and blastocyst stage embryos.

  8. A SIMPLE AND EFFICIENT VITRIFICATION METHOD FOR IN-STRAW DILUTION AND DIRECT TRANSFER OF BOVINE EMBRYOS.

    Science.gov (United States)

    Zhang, Youwen; Fu, Xiangwei; Chen, Long; Feng, Chuntao; Bi, Jianghua; Mo, Xianhong; Cheng, Keren; Zhang, Rina; Li, Shujing; Zhu, Shien

    2015-01-01

    An easy and user friendly protocol that produces consistent results will facilitate the commercial application of embryo vitrification technology in the field. This study was designed to develop a simple and efficient vitrification, in-straw dilution and direct transfer method for bovine embryos. After being vitrified and in-straw thawed, in vivo-derived and in vitro-produced bovine embryos were subjected to in vitro culture or embryo transplantation. There were no significant differences (P > 0.05) in survival rates (100.0% vs. 93.9%) and expansion rates (93.8% vs. 87.5%) between in vivo-derived and in vitro-produced blastocysts after vitrification and in-straw dilution. And there was also no significant difference (P > 0.05) in conception rates (56.5% vs. 58.8%) after ET between cryopreserved and fresh in vivo-derived blastocysts. Vitrification using EG-based vitrification solution and in-straw dilution with PBS-based diluent is a simple and efficient method for cryopreservation and direct transfer of bovine embryos.

  9. Cloned embryos from semen. Part 2: Intergeneric nuclear transfer of semen-derived eland (Taurotragus oryx) epithelial cells into bovine oocytes

    Science.gov (United States)

    Nel-Themaat, L.; Gomez, M.C.; Pope, C.E.; Lopez, M.; Wirtu, G.; Jenkins, J.A.; Cole, A.; Dresser, B.L.; Bondioli, K.R.; Godke, R.A.

    2008-01-01

    The production of cloned offspring by nuclear transfer (NT) of semen-derived somatic cells holds considerable potential for the incorporation of novel genes into endangered species populations. Because oocytes from endangered species are scarce, domestic species oocytes are often used as cytoplasts for interspecies NT. In the present study, epithelial cells isolated from eland semen were used for intergeneric transfer (IgNT) into enucleated bovine oocytes and compared with bovine NT embryos. Cleavage rates of bovine NT and eland IgNT embryos were similar (80 vs. 83%, respectively; p > 0.05); however, development to the morula and blastocyst stage was higher for bovine NT embryos (38 and 21%, respectively; p progress through the early cleavage stage arrest can (a) synthesize DNA, (b) progress through subsequent cell cycles, and (c) may have the potential to develop further. ?? 2008 Mary Ann Liebert, Inc.

  10. 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

    It has been speculated whether the metabolism of the pre-implantation embryo may be reflected on the pregnancy and characteristics of the newborn animal. The present study investigated whether respiration rates of individual embryos were correlated with gestation length, type of parturition, birth......, III), stage of development, and diameter and were subsequently transferred individually (n = 43) to synchronized recipients. Gestation length of the recipients (n = 22) was calculated and the type of parturition (no assistance, light traction, heavy traction, or caesarean section) recorded. Sex...... as the interaction of weight at birth and type of parturition (P type of parturition, which was not affected by sex. When embryos were divided into two even-sized categories according to their respiration rates...

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

    African Journals Online (AJOL)

    Mimi

    ... of the Kenyan livestock farmers and boost food security. It discusses the technical aspects of the procedures involved in the in-vitro production of bovine embryos and embryo transfer, with special reference to the application of the techniques in Kenya. MATERIALS AND METHODS. Production of embryos in the laboratory ...

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

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    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

  13. Carbon-activated gas filtration during in vitro culture increased pregnancy rate following transfer of in vitro-produced bovine embryos.

    Science.gov (United States)

    Merton, J S; Vermeulen, Z L; Otter, T; Mullaart, E; de Ruigh, L; Hasler, J F

    2007-04-15

    Many environmental conditions for in vitro embryo production (IVP) systems for cattle have been relatively standardised, e.g. media composition, temperature, pH, water quality, and atmospheric composition. However, little attention has been paid to the quality of ambient laboratory air and the gas environment in incubators. Although a few studies have examined the effects of chemical air contamination on IVP of human embryos, there are no published accounts for domestic animal embryos. Therefore, this study investigated the effects of an intra-incubator carbon-activated air filtration system (CODA) during in vitro culture (IVC) on embryonic development and subsequent pregnancy rate of bovine embryos. Immature cumulus-oocyte-complexes (COCs) were obtained twice-weekly by ultrasonic-guided transvaginal oocyte aspiration. The COCs were matured in TCM199/FCS/LH/FSH, fertilized with frozen-thawed Percoll-separated semen, and subsequently cultured for 7 day in SOFaaBSA. Day 7 embryos were transferred either fresh or frozen/thawed. The experimental design was a 2 x 2 factorial; presumptive zygotes were placed either in a conventional CO(2)-O(2)-N(2) incubator (Control group) or in an identical CO(2)-O(2)-N(2) incubator with a CODA intra-incubator air purification unit (CODA group) for IVC. The embryo production rate at Day 7 was not affected by the CODA air purification unit (23.4 and 24.7% morulae and blastocysts per oocyte for control and CODA, respectively) nor was there any significant effect on embryo stage or quality. However, the pregnancy rate was improved (P=0.043) for both fresh (46.3% versus 41.0%) and frozen/thawed embryos (40.8% versus 35.6%). In conclusion, atmospheric purification by the CODA intra-incubator air purification unit significantly increased pregnancy rate following transfer of in vitro-produced bovine embryos.

  14. Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development.

    Science.gov (United States)

    Park, Hyo-Jin; Min, Sung-Hun; Choi, Hoonsung; Park, Junghyung; Kim, Sun-Uk; Lee, Seunghoon; Lee, Sang-Rae; Kong, Il-Keun; Chang, Kyu-Tae; Koo, Deog-Bon; Lee, Dong-Seok

    2016-09-01

    Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos.

  15. Effects of vitrification medium composition on the survival of bovine in vitro produced embryos, following in straw-dilution, in vitro and in vivo following transfer.

    Science.gov (United States)

    Pugh, P A; Tervit, H R; Niemann, H

    2000-02-28

    This study examined the effects of adding a macromolecule, polyvinylpyrrolidone (10% PVP) and a sugar (0.3 M trehalose) to vitrification solutions (VS) containing either one (40% ethylene glycol [EG], two (25% EG+25% DMSO) or three (20% EG+20% DMSO+10% 1, 3-butanediol [BD]) permeable cryoprotectants on the survival and hatching of IVP bovine embryos, following vitrification, warming and in-straw cryoprotectant dilution. Grade 1 and 2 compact morulae and blastocysts were selected on Day 7 (Day 0=IVF) of culture in SOFaaBSA and equilibrated for 10 min at room temperature in 10% EG. Following exposure, for up to 1 min at 4 degrees C, to one of the above VS (with or without PVP+trehalose), the embryos were loaded into straws and immersed in liquid nitrogen. Following warming and in-straw cryoprotectant dilution, the embryos were cultured for 48 h to assess hatching. There was no effect of VS on the survival of embryos after 24 h, however fewer compact morulae than blastocysts survived after 24 h (24% vs. 75%; Pvitrification (fresh vs. vitrified; 1/5 [20%] vs. 3/18 [17]). These data demonstrate that a VS comprising three cryoprotectants, rather than one, enables more embryos to hatch during post-thaw culture and that the survival, following direct transfer of these vitrified embryos, is not different to non-vitrified embryos.

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

    DEFF Research Database (Denmark)

    Booth, P J; Holm, P; Vajta, G

    2001-01-01

    .7 +/- 5.1 vs. 31.4 +/- 4.5 [mean +/- SEM]). In contrast, nuclear transfer blastocyst rates per fused embryo were lower (P rates using day 3, 4, and 5 donors and using CHX and DMAP activation treatments were 31.9 +/- 5.0, 31.7 +/- 6.2, 20.......4 +/- 7.3 and 27.8 +/- 4.7, 20.1 +/- 7.5, 12.7 +/- 8.3, respectively. Blastocyst rate per fused embryo was negatively correlated (P = 0.0091) with the total number of blastomeres per donor embryo. Despite this inverse relationship, the calculated potential blastocyst yield per donor embryo was positively...

  17. Preimplantation death of xenomitochondrial mouse embryo harbouring bovine mitochondria

    Science.gov (United States)

    Kawahara, Manabu; Koyama, Shiori; Iimura, Satomi; Yamazaki, Wataru; Tanaka, Aiko; Kohri, Nanami; Sasaki, Keisuke; Takahashi, Masashi

    2015-01-01

    Mitochondria, cellular organelles playing essential roles in eukaryotic cell metabolism, are thought to have evolved from bacteria. The organization of mtDNA is remarkably uniform across species, reflecting its vital and conserved role in oxidative phosphorylation (OXPHOS). Our objectives were to evaluate the compatibility of xenogeneic mitochondria in the development of preimplantation embryos in mammals. Mouse embryos harbouring bovine mitochondria (mtB-M embryos) were prepared by the cell-fusion technique employing the haemagglutinating virus of Japan (HVJ). The mtB-M embryos showed developmental delay at embryonic days (E) 3.5 after insemination. Furthermore, none of the mtB-M embryos could implant into the maternal uterus after embryo transfer, whereas control mouse embryos into which mitochondria from another mouse had been transferred developed as well as did non-manipulated embryos. When we performed quantitative PCR (qPCR) of mouse and bovine ND5, we found that the mtB-M embryos contained 8.3% of bovine mitochondria at the blastocyst stage. Thus, contamination with mitochondria from another species induces embryonic lethality prior to implantation into the maternal uterus. The heteroplasmic state of these xenogeneic mitochondria could have detrimental effects on preimplantation development, leading to preservation of species-specific mitochondrial integrity in mammals. PMID:26416548

  18. 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.

  19. Nucleolar remodeling in nuclear transfer embryos

    DEFF Research Database (Denmark)

    Laurincik, Jozef; Maddox-Hyttel, Poul

    2007-01-01

    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 the devel...... reprogramming and may help to explain the abnormalities observed in a proportion of fetuses and offspring derived from nuclear transfer embryos....... 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...... 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...

  20. 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.

  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. 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

    , immunohistochemistry, and transmission electron microscopy to establish in vivo developmental milestones. Following morphological examination, samples were characterized for the presence of epiblast (POU5F1), mesoderm (VIM), and neuroectoderm (TUBB3). On D14, only 25, 15, and 7% of IVP, SUZI, and HMC embryos were...

  3. 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.

  4. 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.

  5. 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.

  6. Nucleolar changes in bovine nucleotransferred embryos.

    Science.gov (United States)

    Baran, V; Vignon, X; LeBourhis, D; Renard, J P; Fléchon, J E

    2002-02-01

    This study focused on nucleolar changes in bovine embryos reconstructed from enucleated mature oocytes fused with blastomeres of morulae or with cultured, serum unstarved bovine fetal skin fibroblasts (embryonic vs. somatic cloning). The nucleotransferred (NT) embryos were collected and fixed at time intervals of 1-2 h (early 1-cell stage), 10-15 h (late 1-cell stage), 22-24 h (2-cell stage), 37-38 h (4-cell stage), 40-41 h (early 8-cell stage), 47-48 h (late 8-cell stage), and 55 h (16-cell stage) after fusion. Immunocytochemistry by light and electron microscopy was used for structure-function characterization of nucleolar components. Antibodies against RNA, protein B23, protein C23, and fibrillarin were applied. In addition, DNA was localized by the terminal deoxynucleotidyl transferase (TdT) technique, and the functional organization of chromatin was determined with the nick-translation immunogold approach. The results show that fully reticulated (active) nucleoli observed in donor cells immediately before fusion as well as in the early 1-cell stage after fusion were progressively transformed into nucleolar bodies displaying decreasing numbers of vacuoles from the 2- to 4-cell stage in both types of reconstructed embryos. At the late 8-cell stage, morphological signs of resuming nucleolar activity were detected. Numerous new small vacuoles appeared, and chromatin blocks reassociated with the nucleolar body. During this period, nick-translation technique revealed numerous active DNA sites in the periphery of chromatin blocks associated with the nucleolar body. Fully reticulated nucleoli were again observed as early as the 16-cell stage of embryonic cloned embryos. In comparison, the embryos obtained by fetal cloning displayed a lower tendency to develop, mainly during the first cell cycle and during the period of presumed reactivation. Correlatively, the changes in nucleolar morphology (desegregation and rebuilding) were at least delayed in many somatic NT

  7. Vitrification of early-stage bovine and equine embryos.

    Science.gov (United States)

    Campos-Chillòn, L F; Suh, T K; Barcelo-Fimbres, M; Seidel, G E; Carnevale, E M

    2009-01-15

    The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5M ethylene glycol (EG) for 5min, 7M ethylene glycol and 0.6M galactose for 30s, loaded in an OPS, and plunged into liquid nitrogen. In Method 2 (EG-DMSO), embryos were exposed to 1.1M ethylene glycol and 1.1M dimethyl sulfoxide (DMSO) for 3min, 2.5M ethylene glycol, 2.5M DMSO and 0.5M galactose for 30s, and loaded and plunged as for EG-O. Cryoprotectants were removed after warming in three steps. One- and eight-cell bovine embryos were cultured for 7 and 4.5 d, respectively, after warming, and control embryos were cultured without vitrification. Cleavage rates of 1-cell embryos were similar (P>0.05) for vitrified and control embryos, although the blastocyst rates for EG-O and control embryos were similar and higher (Pvitrification and warming. In summary, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to establish equine pregnancies after vitrification and warming of 2- to 8-cell embryos produced by ICSI.

  8. In-straw cryoprotectant dilution for bovine embryos vitrified using Cryotop.

    Science.gov (United States)

    Inaba, Yasushi; Aikawa, Yoshio; Hirai, Tomokazu; Hashiyada, Yutaka; Yamanouchi, Tadayuki; Misumi, Koji; Ohtake, Masaki; Somfai, Tamas; Kobayashi, Shuji; Saito, Norio; Matoba, Satoko; Konishi, Kazuyuki; Imai, Kei

    2011-09-01

    The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrification-dilution (ISVD) and conventional slow freezing, outside dilution of straw (SFODS) methods. In Experiment 1, in vitro-produced (IVP) embryos cryopreserved using the CVSD method were diluted, warmed and exposed to the dilution solution at various times. When vitrified IVP embryos were exposed to the dilution solution for 30 min after warming, the rates of embryos developing to the hatched blastocyst stage after 72 h of culture (62.0-72.5%) were significantly lower (Pembryos exposed to the solution for 5 and 10 min (82.4-94.3%), irrespective of supplementation with 0.3 M sucrose in the dilution solution. In Experiment 2, the rate of embryos developing to the hatching blastocyst stage after 48 h of culture in IVP embryos cryopreserved using the SFODS method (75.0%) was significantly (Pembryos cryopreserved using the CVSD and ISVD methods (93.2 and 97.3%, respectively). In Experiment 3, when in vivo-produced embryos that had been cryopreserved using the CVSD, ISVD and SFODS methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception and delivery rates among groups. In Experiment 4, when IVP embryos derived from oocytes collected by ovum pick-up that had been cryopreserved using the CVSD and ISVD methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception rates among groups. Our results indicate that this simplified regimen of warming and diluting Cryotop-vitrified embryos may enable 1-step bovine embryo transfer without the requirement of a microscope or other laboratory equipment.

  9. Vitrification of in vitro produced bovine embryos: in vitro and in vivo evaluations.

    Science.gov (United States)

    Martínez, A G; Valcárcel, A; de las Heras, M A; de Matos, D G; Furnus, C; Brogliatti, G

    2002-09-16

    The efficacy of different vitrification solutions to cryopreserve in vitro produced bovine blastocysts was evaluated based upon in vitro development of embryos in culture and on in vivo development of embryos transferred into recipients. In the first experiment, ethylene glycol + glycerol (Eg + Gly) + different sucrose concentrations were evaluated. There were no significant differences in development rates among solutions. As for hatching, the Eg + Gly + 0.1 M sucrose group had a greater rate as compared with Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in the evaluations of Day 6, Day 7 and Day 6 + Day 7 embryos; and, Eg + Gly + 0.3 M sucrose group had a greater rate as compared with the Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in evaluations of Day 6 and Day 6 + Day 7 embryos. There were no significant differences in development and hatching rates between Day 6 and 7 in in vitro produced bovine embryos within each treatment group. There were significant differences in nuclei number after vitrification between Eg + Gly + 0.1 M and Eg + Gly + 0 M sucrose groups and the Eg + Gly + 0.5 M sucrose group. Pregnancy after 60 days of transfer and calving rates showed a difference between in vivo produced embryos freshly transferred and in vitro produced embryos vitrified with Eg + Gly + 0.3 M. There were no significant differences in gestation length and sex ratio between treatments. As for birth weight, there were significant differences between fresh in vivo produced embryos and all treatments of in vitro produced embryos. There were significant differences in dystocial parturition between in vivo produced embryos and all treatments with in vitro produced embryos. These results demonstrate that vitrification can be used successfully in the cryopreservation of in vitro produced bovine embryos, and that it might be considered for use in commercial programs. Copyright 2002 Elsevier Science B.V.

  10. In vitro production of bovine embryos derived from individual donors in the Corral® dish.

    Science.gov (United States)

    Catteeuw, Maaike; Wydooghe, Eline; Mullaart, Erik; Knijn, Hiemke M; Van Soom, Ann

    2017-06-15

    Since the identity of the embryo is of outmost importance during commercial in vitro embryo production, bovine oocytes and embryos have to be cultured strictly per donor. Due to the rather low yield of oocytes collected after ovum pick-up (OPU) per individual cow, oocyte maturation and embryo culture take place in small groups, which is often associated with inferior embryo development. The objective of this study was to improve embryonic development in small donor groups by using the Corral® dish. This commercial dish is designed for human embryo production. It contains two central wells that are divided into quadrants by a semi-permeable wall. In human embryo culture, one embryo is placed per quadrant, allowing individual follow-up while embryos are exposed to a common medium. In our study, small groups of oocytes and subsequently embryos of different bovine donors were placed in the Corral® dish, each donor group in a separate quadrant. In two experiments, the Corral® dish was evaluated during in vitro maturation (IVM) and/or in vitro culture (IVC) by grouping oocytes and embryos of individual bovine donors per quadrant. At day 7, a significantly higher blastocyst rate was noted in the Corral® dish used during IVM and IVC than when only used during IVM (12.9% ± 2.10 versus 22.8% ± 2.67) (P vitro embryo production (IVP) in cattle; allowing to allocate oocytes and/or embryos per donor. As fresh embryo transfers on day 7 have higher pregnancy outcomes, the Corral® dish offers an added value for commercial OPU/IVP, since a higher blastocyst development at day 7 is obtained when the Corral® dish is used during IVM and IVC.

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

    African Journals Online (AJOL)

    Yomi

    2012-03-06

    Mar 6, 2012 ... can also be applied to human embryos, using different primers, designed for human DNA. Key words: sexing, embryo, PCR, bovine. INTRODUCTION. In vitro fertilization represents nowadays a modern assisted reproductive technology that can be applied to couples with fertility problems that make natural ...

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

    NARCIS (Netherlands)

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

    1995-01-01

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

  13. PreImplantation Factor (PIF correlates with early mammalian embryo development-bovine and murine models

    Directory of Open Access Journals (Sweden)

    Coulam Carolyn B

    2011-05-01

    Full Text Available Abstract Background PreImplantation Factor (PIF, a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo. Methods Determine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy. Results PIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01. In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control (P = 0.01 and at day 7 were higher than day 3 (P = 0.03. In non-cleaving embryos culture medium was similar to medium alone (control. Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01 as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control. Conclusions PIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the

  14. 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.

  15. Phosphorylated H2AX in parthenogenetically activated, in vitro fertilized and cloned bovine embryos.

    Science.gov (United States)

    Pereira, A F; Melo, L M; Freitas, V J F; Salamone, D F

    2015-08-01

    In vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylated (γH2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring γH2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of γH2AX foci (606.1 ± 103.2) and greater area of γH2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P DNA damage was comparable among those embryos developing to the blastocyst stage among different methods for in vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development.

  16. Evaluation of the Cryotech Vitrification Kit for bovine embryos.

    Science.gov (United States)

    Gutnisky, C; Alvarez, G M; Cetica, P D; Dalvit, G C

    2013-12-01

    The purpose of this work was to assess commercially available Cryotech Vitrification Kit, in terms of survival, in vitro development and pregnancy rate for bovine embryos. Cumulus-oocyte complexes (COCs) were recovered from ovaries obtained from slaughtered cows and then matured in vitro for 22 h. COCs were fertilized by sex-sorted sperm in IVF-mSOF and cultured in IVC-mSOF for 7 days to the blastocyst stage. Blastocysts were vitrified with the Cryotech Vitrification Kit(®) and then either warmed to check viability or transferred to synchronized heifers. We observed 100% survival of the in vitro produced blastocysts and obtained the same pregnancy rate (46.8%) as that obtained using fresh in vitro produced blastocysts. We thus conclude that the Cryotech vitrification method is a valid alternative to other vitrification or slow-cooling methods in the bovine species and that it is ready for livestock production. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm

    OpenAIRE

    Trigal, B.; Gómez, E.; Caamaño, J.N. (José); Muñoz, M.; Moreno, Javier; Carrocera, S.; Martín, D.; Díez, C.

    2013-01-01

    In this work we analyzed the effects of three culture systems on developmental ability of bovine embryos in vitro produced with sexed sperm, the survival to vitrification (cryologic vitrification method) of such blastocysts, and their pregnancy rates after embryo transfer to recipients, both as fresh and after vitrification/warming. Finally, we measured the accuracy of the sorting protocol by a polymerase chain reaction-based method to validate the embryo sex at blastocyst stages. We confirme...

  18. Blocking connexin channels improves embryo development of vitrified bovine blastocysts

    OpenAIRE

    Ortiz Escribano, Nerea; Szymanska, Katarzyna; BOL, MELISSA; Vandenberghe, Lynn; Decrock, Elke; Van Poucke, Mario; Peelman, Luc; Van den Abbeel, Etienne; Soom, Ann Van; Leybaert, Luc

    2017-01-01

    Connexins (Cxs) are required for normal embryo development and implantation. They form gap junctions (GJs) connecting the cytoplasm of adjacent cells and hemichannels (HCs), which are normally closed but open in response to stress conditions. Excessive HC opening is detrimental for cell function and may lead to cell death. We found that hatching of in vitro-produced bovine embryos, matured in serum-containing conditions, was significantly improved when vitrification/warming was done in the pr...

  19. Embryo transfer in domestic South American camelids.

    Science.gov (United States)

    Sumar, Julio B

    2013-01-10

    Intraspecific and interspecific embryo transfer in domestic South American camelids is developing into a well-established technique. Reports reveal many benefits of using reproductive biotechnologies to allow rapid propagation of alpacas and llamas of high genetic merit (e.g., high fiber quality, preserve color variation). The objective of this review is to provide up-to-date information about embryo transfer in domestic South American camelids. Specific information is provided on criteria for male selection, donor and recipient synchronization, the practice of single- vs. super-ovulation protocols, embryo recovery and transfer techniques, advances in cryopreservation of embryos, results of intra- and inter-specific transfer, and the future of the embryo transfer in domestic South American camelids. Copyright © 2012. Published by Elsevier B.V.

  20. Cryopreservation and sexing of in vivo- and in vitro-produced bovine embryos for their practical use.

    Science.gov (United States)

    Tominaga, Keiichiro

    2004-02-01

    My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3

  1. Embryo transfer using cryopreserved Boer goat blastocysts ...

    African Journals Online (AJOL)

    The aim of this trial was to evaluate the effect of embryo cryopreservation techniques on the survivability of embryos and fertility following transfer to Boer goat does. The oestrous cycles of 27 mature recipients Boer goat does were synchronised using controlled internal drug release dispensers (CIDR's) for 16 days. At CIDR ...

  2. 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 (PTrolox had no effect (P>0.05). Whereas lipoproteins reduced zygote development to blastocysts (P=0.03), Trolox facilitated increased development (PTrolox (P>0.05) 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, PTrolox. Placentome numbers were greater in SOF and SOFLPT compared with SOFLP and SOFT (interaction, P=0.002); superior embryo 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.

  3. In vitro production of bovine embryos

    DEFF Research Database (Denmark)

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

    2015-01-01

    be improved, and aspects related to the oocyte donor, oocyte maturation and the recipients are addressed in the following. Also, some of the future aspects of genomic selection and systems biology are addressed with particular focus on the Brazilian-Danish collaboration in the so-called GIFT-project.......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 still...

  4. Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification.

    Science.gov (United States)

    Apparicio, M; Ruggeri, E; Luvoni, G C

    2013-04-01

    The aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n = 72) using a vitrification kit for bovine embryo or slow frozen (n = 69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48 h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n = 92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing/warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p vitrification compared to 60.9% of those submitted to slow freezing procedure (p bovine embryos retain their capacity to resume meiosis after warming and culture, albeit at lower rates than slow frozen oocytes. Vitrification and slow freezing methods show similar proportions of oocytes with normal morphology after culture, which demonstrate that thawed and warmed oocytes that resist to cryodamage have the same chances to maintain their integrity after 48 h of culture. © 2012 Blackwell Verlag GmbH.

  5. Serum free embryo culture medium improves in vitro survival of bovine blastocysts to vitrification.

    Science.gov (United States)

    Gómez, E; Rodríguez, A; Muñoz, M; Caamaño, J N; Hidalgo, C O; Morán, E; Facal, N; Díez, C

    2008-05-01

    The aim of this study was to examine the effects of co-culture with Vero cells during the in vitro maturation (IVM) and three culture media, B2+5% fetal calf serum (FCS) on Vero cells, synthetic oviduct fluid (SOF)+5% FCS, and SOF+20 gL(-1) bovine serum albumin (BSA), on the developmental competence of the embryos and their ability to survive vitrification/warming. We also tested the effect of morphological quality and the age of the embryo on its sensitivity to vitrification. The IVM system neither affects the embryo development up to Day 7 nor survival rates after vitrification. The culture of embryos in SOF+FCS and in Vero cells+B2 allowed obtaining more Day 6 and Day 7 blastocysts, and a higher % of Day 7 blastocysts vitrified than culture in SOF+BSA. Contrarily, on Day 8, more blastocysts were vitrified in SOF+BSA than in SOF+FCS. Blastocysts quality affected development after vitrification/warming, and Day 7 embryos showed higher survival rates than their Day 8 counterparts. Day 7 blastocysts produced in Vero cells or in SOF+BSA survived at higher rates than those produced in SOF+FCS at 24 and 48 h after warming. Embryo culture with BSA allows obtaining hatching rates after vitrification/warming higher than those obtained after co-culture with Vero cells in B2 and FCS. Moreover, this system provides hatching rates from Day 8 blastocysts comparable to those obtained on Day 7 in Vero cells. Further studies, including embryo transfer to recipients, are needed to clarify factors affecting the freezability of in vitro produced bovine embryos.

  6. Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification.

    Science.gov (United States)

    Sommerfeld, V; Niemann, H

    1999-03-01

    In this study, the cryoprotectant ethylene glycol (EG) was tested for its ability to improve and facilitate the cryopreservation of in vitro produced (IVP) bovine embryos. Embryos were cryopreserved in EG solutions supplemented with either newborn calf serum (NBCS) or polyvinyl alcohol (PVA). To assess EG toxicity, the embryos were equilibrated in EG concentrations from 1.8 to 8.9 M at room temperature for 10 min and then cultured for 72 h on a cumulus cell monolayer. The hatching rate was highest for day 7 blastocysts frozen in 3.6 M EG (98%) and was not different from the control group (85%). The controlled freezing (0.3 degrees C/min to -35 degrees C) of expanded day 7 blastocysts resulted in a hatching rate of 81%, which was similar to that of the nonfrozen controls (76%). Differential staining revealed only very few degenerate blastomeres attributed to freezing and thawing. Upon direct nonsurgical transfer of day 7 expanded blastocysts frozen in 3.6 M EG, a pregnancy rate of 43% was achieved, while the pregnancy rate after transfer of other developmental stages was significantly lower (22% with expanded day 8 blastocysts). When bovine IVP embryos were incubated at room temperature in 7.2 M EG preceded by preequilibration in 3.6 M EG, the hatching rate of day 7 expanded blastocysts reached 93%. Upon vitrification of IVP day 7 and day 8 blastocysts and expanded blastocysts in 7.2 M EG, the latter showed a higher hatching rate (42%) than blastocysts (12%). Overall, PVA as supplement to the basic freezing solution instead of NBCS had deleterious effects on survival after controlled freezing or vitrification. The simple cryopreservation protocol employed in this study and the low toxicity of ethylene glycol highlight the usefulness of this approach for controlled freezing of IVP embryos. However, further experiments are needed to improve the pregnancy rate following embryo transfer and to enhance survival after vitrification. Copyright 1999 Academic Press.

  7. In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm.

    Science.gov (United States)

    Trigal, B; Gómez, E; Caamaño, J N; Muñoz, M; Moreno, J; Carrocera, S; Martín, D; Diez, C

    2012-10-15

    In this work we analyzed the effects of three culture systems on developmental ability of bovine embryos in vitro produced with sexed sperm, the survival to vitrification (cryologic vitrification method) of such blastocysts, and their pregnancy rates after embryo transfer to recipients, both as fresh and after vitrification/warming. Finally, we measured the accuracy of the sorting protocol by a polymerase chain reaction-based method to validate the embryo sex at blastocyst stages. We confirmed an individual effect of the bull as well as development rates of embryos produced with sorted sperm lower than embryos with unsorted sperm, independent of the culture system used. The cryoresistance to vitrification of embryos produced with sexed sperm did not differ from that of conventionally produced embryos (re-expansion rates at 24 and 48 h: 74.6% vs. 75.5%, and 64.5% vs. 68.1% for embryos produced with conventional and sorted sperm, respectively; hatching rates at 48 h: 63.55% vs. 55.5% for embryos produced with conventional and sorted sperm, respectively). Finally, no significant differences were found in pregnancy rates after the embryo transfer of fresh and vitrified/warmed blastocysts (52.8% vs. 42.0%, respectively; P > 0.05). Male and female embryos produced with sorted sperm showed the same quality in terms of developmental ability, cryoresistance, and pregnancy rates after transfer. Our culture system, coupled with the vitrification in fiber plugs, provides good quality sex-known embryos which survive vitrification at similar rates than embryos produced with conventional unsorted sperm; also it produces good pregnancy rates after transfer of sexed embryos both fresh and after vitrification and warming. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. To transfer fresh or thawed embryos?

    DEFF Research Database (Denmark)

    Pinborg, Anja

    2012-01-01

    and multiple pregnancies, thereby increasing the safety for mother and child. Finally the article describes the accumulating literature on perinatal and long-term child outcome after transfer of frozen/thawed embryos, including a discussion on the concerns regarding cryo techniques and their possible roles...... and cons of FER versus fresh-embryo transfer with regard to both single-cycle and cumulative pregnancy and delivery rates. The review discusses the obvious advantages of FER: minimizing the proportion of pharmacological and surgical treatments, and lowering the risk of ovarian hyperstimulation syndrome...

  9. The effect of biopsy during precompacted morula stage on post vitrification development of blastocyst derived bovine embryos.

    Science.gov (United States)

    Shirazi, Abolfazl; Borjian, Sara; Ahmadi, Ebrahim; Nazari, Hassan; Heidari, Banafsheh

    2010-04-01

    Improvements on embryo micromanipulation techniques led to the use of embryo biopsy in commercial embryo transfer programs for genetic analysis of preimplantation bovine embryos. The aim of this study was to evaluate the quality of bovine blastocyst derived from embryos biopsied at different pre-compacted morulae stages by assessment of cryosurvivability of the resulting blastocysts. The in vitro produced bovine embryos were subjected to biopsy at days 2, 3, and 4 post-insemination with different cell numbers (4 to 16-cells). Embryo cell biopsy was carried out in a 100 µl drop of H-SOF following pronase drilling by aspiration of one blastomere. The biopsied embryos were then cultured in SOFaaBSA co-cultured with oviduct cells-monolayer until blastocyst formation. The blastocysts were cryopreserved at room temperature after exposure of equilibration (glycerol 1.4 M for 5 min and then glycerol 1.4 M and ethylene glycol 3.6 M for 5 min) and vitrification solutions (3.4 M glycerol and 4.6 M ethylene glycol). The blastocysts were loaded into the center of 0.25 ml straws separated by air bubbles from 2 columns of sucrose 0.5 M and plunged immediately into liquid nitrogen. There was no significant difference in cryosurvivability of vitrified-warmed blastocysts derived form biopsied embryos at different pre-compacted morula stages. The quality of biopsy derived blastocysts was identical to that of non-biopsy derived ones in terms of post vitrifcation survival and hatching rates. In conclusion there was no preference between different times of embryo biopsy at precompacted morula stages in term of cryosurvivability of biopsy derived bovine blastocysts.

  10. A simple and efficient method to transfect small interference RNA into bovine SCNT embryos.

    Science.gov (United States)

    Zhang, Hui; Wang, LiJun; Li, WenZhe; Mao, QingFu; Wang, YongSheng; Li, Qian; Hua, Song; Zhang, Yong

    2015-10-01

    RNA interference is an important tool to study the gene function. Microinjection and electroporation are usually used to transfer DNA, small interference RNA (siRNA), morpholinos, and protein into oocytes or embryos. This study used a simple and effective method to transfect siRNA into bovine somatic cell nuclear transfer (SCNT) embryos. In this method, siRNA transfection and electrofusion of SCNT were combined. A pair of platinum microelectrodes was used during SCNT to complete electrofusion. A CY3-labeled siRNA-targeted DNA methyltransferase-1 (DNMT1) was chosen to verify the siRNA transfection efficiency of this approach. First, a suitable concentration of siRNA was mixed with Zimmermann's fusion medium. Reconstructed embryos were then added into the microdrops of the mixed fusion medium to simultaneously transfect the siRNA and electrofuse the SCNT embryos. Our results showed that transfecting DNMT1 siRNA via the proposed method caused obvious CY3 fluorescence and significant downregulation of DNMT1 messenger RNA, DNMT1 protein, and global DNA methylation levels in the SCNT embryos. Meanwhile, the survival rate after electrofusion (90.4% vs. 89.4% vs. 89.1%, P > 0.05) and developmental rates of the SCNT embryos (72.8% vs. 74.9% vs. 72.4%, P > 0.05; 29.7% vs. 31.7% vs. 29.7%, P > 0.05) were not significantly affected. In summary, siRNAs were effectively transfected into the SCNT embryos via the proposed method and exert their functions, and the normal development of preimplantation SCNT embryos was not affected by the method used. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Economic optimization of the number of recipients in bovine embryo transfer programs Otimização econômica do número de receptoras em programas de transferência de embriões em bovinos

    Directory of Open Access Journals (Sweden)

    Renato Travassos Beltrame

    2007-06-01

    Full Text Available Purchase and maintenance of recipient females account for a large proportion of the costs and determine the number of calves that can be produced in an embryo transfer program. However, the large variability of embryo production by the donors and the need to purchase and synchronize the recipients before knowing the number of embryos collected make it difficult for the decision maker to identify the ideal number of recipient females to allocate. An ex-ante evaluation to determine the optimal number of recipient females was carried out through a sensitivity analysis for the ratio between the number of recipients and donors in a simulation model. The variability for the number of embryos collected was accounted for by applying the Monte Carlo simulation technique, assuming normal distribution and known values for mean and variance. The simulation considered monthly intervals between collections, during a 24 months program. The effect of embryo freezing on the number of pregnancies was considered by introducing a stock of frozen embryos into the mathematical model. Optimal recipient/donor ratio and the cost per pregnancy were compared for three recipient synchronization protocols (prostaglandin, progesterone - P4 and Ovsynch, based on the expected performance for synchronization, conception and transfer/treated rates for each protocol. Stochastic simulation associated with sensitivity analysis was effective in identifying the optimal donor to recipient ratio. Freezing embryos is effective to reduce the operational costs per pregnancy. The estimated optimal recipient/donor ratio was 20 for prostaglandin and 16.7 for the other protocols. The P4 protocol, although the most expensive, resulted in the lowest pregnancy cost estimation followed by prostaglandin and Ovsynch.A aquisição e manutenção de receptoras representam grande proporção dos custos e determinam o número de produtos gerados em um programa de transferência de embriões. Entretanto

  12. [Ectopic pregnancy following in vitro fertilization and embryo transfer].

    Science.gov (United States)

    Sudik, R; Fliess, F R; Bernt, W D; Meissner, J; Kunkel, S

    1984-01-01

    A report is given about one case of ectopic pregnancy after in vitro fertilization and transfer of three embryos. Possible causes of ectopic pregnancies following embryo transfer and conclusions are discussed.

  13. In vitro evaluation and pregnancy rates after vitrification of in vitro produced bovine embryos.

    Science.gov (United States)

    Martínez, A G; de Matos, D G; Furnus, C C; Brogliatti, G M

    1998-10-01

    The efficacy of different vitrification solutions to cryopreserve in vitro-produced bovine blastocysts was evaluated based on in vitro development of embryos in culture and on in vivo development of embryos transferred into recipients. In the first experiment, 2 vitrification solutions were compared: propylene glycol + glycerol (Pg + Gly) and ethylene glycol + Ficoll + sucrose (EFS). Differences in the overall development and hatching rates in favor of EFS were found (56.4 vs 33.3% and 35.4 vs 13.3%; P vitrification solutions were compared: EFS, modified EFS (EFSm) and ethylene glycol + glycerol (Eg + Gly). The vitrification solutions EFSm and Eg + Gly yield higher hatching rates than did EFS (57.7 vs 59.6 vs 35.7%; P vitrification solutions: EFSm and Eg + Gly. There were no differences between them based on the results obtained after transfer (35.2 vs 43.7%). The vitrification solutions EFSm and Eg + Gly have resulted in good pregnancy rates. These results demonstrated that vitrification can be used successfully in the cryopreservation of in-vitro produced bovine embryos, and it might be considered for use in commercial programs.

  14. Retrograde tubal transfer of human embryos.

    Science.gov (United States)

    Risquez, F; Boyer, P; Rolet, F; Magnani, M; Guichard, A; Cedard, L; Zorn, J R

    1990-02-01

    This preliminary study was designed to evaluate retrograde cannulation of the Fallopian tubes up to the isthmo-interstitial junction using the new technique of tubal embryo stage transfer (TEST). Follicular aspiration was performed under the guidance of a vaginal ultrasound probe in 51 women treated with GnRH + HMG. The oocytes retrieved were inseminated in vitro with 50,000 motile spermatozoa and kept in Menezo B2 medium without serum, at 37 degrees C, in an atmosphere of air + 5% CO2. The eggs were checked 24 and 36 h after insemination. No fertilization occurred in 23 patients. Cleaved embryos were obtained in the 28 other patients. One to seven embryos at the 2-4-cell stage were transferred with the 'Baudelocque Black Catheter' (BBC) into one tube and spare embryos were frozen. Five pregnancies occurred after retrograde TEST, for a pregnancy rate of 9.8% per cycle and 17.9% per transfer. One patient has given birth to a normal full-term baby. One singleton and one twin pregnancy are ongoing (8 months in June 1989). The other two pregnancies were ectopic.

  15. Reprogramming of fibroblast nuclei after transfer into bovine oocytes.

    Science.gov (United States)

    De Sousa, P A; Winger, Q; Hill, J R; Jones, K; Watson, A J; Westhusin, M E

    1999-01-01

    Recent landmark achievements in animal cloning have demonstrated that the events of cell differentiation can, in principle, be reversed. This reversal necessarily requires large-scale genetic reprogramming, of which little is known. In the present study we characterized the extent to which blastocyst stage-specific mRNA expression would be conserved in bovine embryos produced by nuclear transfer (NT) using fetal fibroblasts as nuclei donors (FF NT). The mRNA pool of FF NT embryos was compared with that of NT embryos reconstructed from embryonic blastomeres (Emb NT), with embryos produced under in vivo or in vitro conditions, and finally with fibroblast cells. Embryo/cell-specific mRNA pools were contrasted using differential display methodology. Random oligonucleotide primer pair combinations were used to subfractionate mRNA populations and represent individual mRNAs as copy DNA (cDNA) bands ranging in size from 100 to 800 base pairs. Regardless of whether bovine blastocysts developed in vivo or in vitro, or were derived after nuclear transplantation with embryonic blastomeres or fetal fibroblasts, their mRNA profile was highly conserved and distinct from that of fetal fibroblast cells. There was approximately 95% conservation in cDNA banding patterns between FF NT, Emb NT, and in vivo derived blastocysts, when compared with in vitro derived blastocysts. In contrast, the cDNA banding in fibroblasts was only 67% conserved with in vitro derived blastocysts (p types to serve as nuclear donors for embryo reconstruction and provide information that can be used to improve the efficiency of cloning animals by nuclear transplantation.

  16. Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells

    Directory of Open Access Journals (Sweden)

    Han Yong-Mahn

    2009-07-01

    Full Text Available Abstract Background Interspecies somatic cell nuclear transfer (iSCNT has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. Results No apparent difference in cleavage rate was found among IVF, monkey-bovine (MB-iSCNT, and bovine-bovine (BB-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM, we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively. Conclusion The down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

  17. Efficient introgression of allelic variants by embryo-mediated editing of the bovine genome.

    Science.gov (United States)

    Wei, Jingwei; Wagner, Stefan; Lu, Dan; Maclean, Paul; Carlson, Daniel F; Fahrenkrug, Scott C; Laible, Götz

    2015-07-09

    The recent development of designer nucleases allows for the efficient and precise introduction of genetic change into livestock genomes. Most studies so far have focused on the introduction of random mutations in cultured cells and the use of nuclear transfer to generate animals with edited genotypes. To circumvent the intrinsic uncertainties of random mutations and the inefficiencies of nuclear transfer we directed our efforts to the introduction of specific genetic changes by homology-driven repair directly in in vitro produced embryos. Initially, we injected zinc finger nuclease (ZFN)-encoding mRNA or DNA into bovine zygotes to verify cleavage activity at their target site within the gene for beta-lactoglobulin (LGB) and detected ZFN-induced random mutations in 30% to 80% of embryos. Next, to precisely change the LGB sequence, we co-injected ZFNs or transcription activator-like effector nucleases (TALENs) with DNA oligonucleotides (ODNs). Analysis of co-injected embryos showed targeted changes in up to 33% (ZFNs) and 46% (TALENs) of blastocysts. Deep sequence analysis of selected embryos revealed contributions of the targeted LGB allele can reach 100% which implies that genome editing by zygote injections can facilitate the one-step generation of non-mosaic livestock animals with pre-designed biallelic modifications.

  18. Survival and developmental competence of bovine embryos at different developmental stages and separated blastomeres after vitrification in different solutions.

    Science.gov (United States)

    Juanpanich, Theesit; Suttirojpattana, Tayita; Takayama, Mari; Liang, Yuanyuan; Dochi, Osamu; Parnpai, Rangsun; Imai, Kei

    2018-01-01

    Generating techniques to enhance the success of blastomere separation is important for bovine economy, because it increases the number of transferable embryos. This study aimed to identify the optimum cryoprotectants for the vitrification of bovine embryos and the separation of blastomeres at different stages. In experiment 1, expanded blastocysts were vitrified in two different vitrification solutions, either (1) ethylene glycol (EG) + propylene glycol (PG) or (2) EG. The survival rate of blastocysts in the EG + PG was higher than that of the EG. In experiment 2, intact two-cell and eight-cell stage embryos were vitrified in the same solutions used in experiment 1. The EG + PG produced more dead embryos than the EG (P < 0.05). In the EG, the rate of blastocyst formation was similar for the vitrified two- and eight-cell embryos and the non-vitrified ywo-cell embryos. In experiment 3, separated blastomeres of two- and eight-cell embryos were vitrified in EG. There was no difference in the rate of blastocyst formation and total number of cells between the two vitrified groups. In summary, at the blastocyst stage, EG + PG was superior, based on both survival rates and cell numbers; however, at the 2-8 cell stage, the use of EG alone was better than the other groups. © 2017 Japanese Society of Animal Science.

  19. Endometrial preparation methods in frozen-thawed embryo transfer

    NARCIS (Netherlands)

    Groenewoud, E.R.

    2017-01-01

    One in six couples suffer from infertility, and many undergo treatment with in-vitro fertilization (IVF). Given that IVF often results in more embryos than can be transferred during one embryo transfer cryopreservation of the supernumerary embryos has been an important addition to IVF. In recent

  20. 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.

  1. Comparison between Conventional Blind Embryo Transfer and Embryo Transfer Based on Previously Measured Uterine Length

    Directory of Open Access Journals (Sweden)

    Nasrin Saharkhiz

    2014-11-01

    Full Text Available Background: Embryo transfer (ET is one of the most important steps in assisted reproductive technology (ART cycles and affected by many factors namely the depth of embryo deposition in uterus. In this study, the outcomes of intracytoplasmic sperm injection (ICSI cycles after blind embryo transfer and embryo transfer based on previously measured uterine length using vaginal ultrasound were compared. Materials and Methods: This prospective randomised clinical trial included one hundred and forty non-donor fresh embryo transfers during January 2010 to June 2011. In group I, ET was performed using conventional (blind method at 5-6cm from the external os, and in group II, ET was done at a depth of 1-1.5 cm from the uterine fundus based on previously measured uterine length using vaginal sonography. Appropriate statistical analysis was performed using Student’s t test and Chi-square or Fisher’s exact test. The software that we used was PASW statistics version 18. A p value <0.05 was considered statistically significant. Results: Chemical pregnancy rate was 28.7% in group I and 42.1% in group II, while the difference was not statistically significant (p=0.105. Clinical pregnancy, ongoing pregnancy and implantation rates for group I were 21.2%, 17.7%, and 12.8%, while for group II were 33.9%, 33.9%, and 22.1, respectively. In group I and group II, abortion rates were 34.7% and 0%, respectively, indicating a statistically significant difference (p<0.005. No ectopic pregnancy occurred in two groups. Conclusion: The use of uterine length measurement during treatment cycle in order to place embryos at depth of 1-1.5cm from fundus significantly increases clinical and ongoing pregnancy and implantation rates, while leads to a decrease in abortion rate (Registration Number: IRCT2014032512494N1.

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

    National Research Council Canada - National Science Library

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

    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...

  3. Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system.

    Science.gov (United States)

    Pedersen, Mona E; Øzdas, Øzen Banu; Farstad, Wenche; Tverdal, Aage; Olsaker, Ingrid

    2005-01-01

    In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), (2)-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription-polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

  4. The effect of the number of transferred embryos, the interval between nuclear transfer and embryo transfer, and the transfer pattern on pig cloning efficiency.

    Science.gov (United States)

    Rim, Chol Ho; Fu, Zhixin; Bao, Lei; Chen, Haide; Zhang, Dan; Luo, Qiong; Ri, Hak Chol; Huang, Hefeng; Luan, Zhidong; Zhang, Yan; Cui, Chun; Xiao, Lei; Jong, Ui Myong

    2013-12-01

    To improve the efficiency of producing cloned pigs, we investigated the influence of the number of transferred embryos, the culturing interval between nuclear transfer (NT) and embryo transfer, and the transfer pattern (single oviduct or double oviduct) on cloning efficiency. The results demonstrated that transfer of either 150-200 or more than 200NT embryos compared to transfer of 100-150 embryos resulted in a significantly higher pregnancy rate (48 ± 16, 50 ± 16 vs. 29 ± 5%, pcloning efficiency is achieved by adjusting the number and in vitro culture time of reconstructed embryos as well as the embryo transfer pattern. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. 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.

  6. Closed pulled straw vitrification of in vitro-produced and in vivo-produced bovine embryos.

    Science.gov (United States)

    Yu, X L; Deng, W; Liu, F J; Li, Y H; Li, X X; Zhang, Y L; Zan, L S

    2010-03-01

    The objective of this study was to evaluate the efficiency of the closed pulled straw (CPS) method for cryopreserving in vitro-produced and in vivo-produced bovine (Bos taurus) embryos. Based on the open pulled straw (OPS) protocol, the top end of a CPS was closed by tweezers (heated in a flame) to prevent the cryoprotectant medium containing embryos from contacting the liquid nitrogen. Bovine in vitro or in vivo morulae and early blastocyst embryos were frozen by slow cryopreservation, OPS vitrification, or CPS vitrification. Morphology of postthawed embryos was evaluated, and normal embryos were used for successive culture for 72h. There were no significant differences between OPS and CPS freezing groups in postthawed in vitro-produced embryos with respect to rates of morphologically normal embryos (mean+/-SD, 87.9+/-5.2% vs. 85.4+/-4.9%), survival at 24h (58.0+/-6.8% vs. 56.3+/-4.4%), and survival at 72h (35.2+/-6.0% vs. 34.9+/-6.7%). However, both OPS and CPS vitrification resulted in higher postthaw rates of morphologically normal embryo and survival at 24 and 72h than those of the slow-freezing method (Pvitrification was a feasible method to cryopreserve both in vitro-derived and in vivo-derived bovine embryos. This method not only eliminated the risk of embryo contamination by preventing contact with liquid nitrogen but also retained the advantages of the OPS vitrification method. Copyright 2010. Published by Elsevier Inc.

  7. Embryo transfer and related technologies in sheep reproduction

    OpenAIRE

    Loi, Pasqualino; Ptak, Grazyna; Dattena, Maria; Ledda, Sergio; Naitana, Salvatore; Cappai, Pietro

    1998-01-01

    This paper reviews the status of embryo transfer and the major technologies applied to preimplantation of embryos in sheep. Embryo production from superovulated ewes is hindered by an unpredictable response to hormonal treatment. Progress in this area should be expected by an appropriated control of follicular development with gonadotropin-releasing hormone (GnRH) agonist or antagonist prior to gonadotrophin administration. Simple protocols for the cryopreservation of sheep embryos by vitrifi...

  8. New device for the vitrification and in-straw warming of in vitro produced bovine embryos.

    Science.gov (United States)

    Morató, Roser; Mogas, Teresa

    2014-04-01

    Two experiments were designed to test the use of a new device designed to vitrify and in-straw warm in vitro produced (IVP) embryos, which can potentially be used for their direct transfer to recipient females in field conditions. In experiment 1, IVP embryos from both prepubertal and adult animals were vitrified on cryotops and warmed in steps (1, 0.5 and 0M sucrose; protocol W3) or directly in 0.5M (protocol W1/0.5) or 0M sucrose (protocol W1/0). Similar survival rates were recorded 24h after warming for calf embryos irrespective of the warming procedure (W3: 79.2%, W1/0.5: 62.5%, W1/0: 66.7%). For cow embryos, survival rates at 24h post-warming were significantly higher when embryos were warmed using the W3 (85.7%) or W1/0.5 (89.1%) protocols compared to the W1/0 protocol (70.5%). In experiment 2, IVP embryos were vitrified on the new designed device followed by their in-straw cryoprotectant (0.5M sucrose) dilution/warming and different warming temperatures (45, 50, 60 and 70°C) were tested. When warming solution passed through the new vitrification/warming device at 45°C, 61.5% of blastocysts were fully re-expanded or hatched at 24h post-warming, being not significantly different to the control (65%). Other warming temperatures triggered significantly lower survival rates at 24h post-warming. No significant differences were detected in total cell numbers and blastocyst apoptosis indices in response to vitrification followed by warming at 45°C respect to the control. Our findings indicate that the new device allows vitrification and in-straw warming of IVP bovine embryos, being a useful option for their direct transfer in field conditions. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Effect of breed and corpus luteum on pregnancy rate of bovine embryo recipients

    Directory of Open Access Journals (Sweden)

    Ériklis Nogueira

    2012-09-01

    Full Text Available The objective of this study was to evaluate pregnancy rates of recipients of different breed groups (Nellore and crossbreed, as well as the effects of size and type of the corpus luteum (CL on plasmatic concentrations of progesterone and pregnancy rates of embryo recipients. A total of 152 heifers were synchronized with progesterone implants and on the day of embryo transfer, previously obtained by superovulation and frozen in ethylene glycol, the diameter and type of the corpus luteum (cavitary and compact was measured and blood was collected for progesterone measurement. The pregnancy rate was 44.1%, with a diameter of corpus luteum higher in recipients that became pregnant (2.03±0.41 compared with non-pregnant ones (1.86±0.34 cm. Plasmatic concentrations of progesterone did not differ between pregnant (1.50±1.05 and non-pregnant (1.31±0.91 ng/mL animals. The type of corpus luteum did not influence the pregnancy rates. Only Angus and crossbred Marchigiana differ among themselves in pregnancy rates (33.3 and 59.2%, respectively. The pregnancy probability was affected only by CL diameter, but not by P4 plasmatic concentration. Selection of the corpus luteum size at the time of embryo transfer is an important factor to increase pregnancy rates in recipients, and compact and cavitary corpora lutea do not influence the pregnancy rates of bovine embryo recipients. Nellore recipients have pregnancy rates that are satisfactory and comparable to crossbred (Bos taurus × Bos indicus recipients.

  10. Cryopreservation of Equine Embryos and First Report of a Native Colombian Breed Born by Transfer of an Equine Vitrified Embryo

    Directory of Open Access Journals (Sweden)

    Nadya Nathalie Martínez

    2014-06-01

    Full Text Available The aim of this paper is to report on the success of a cryopreservation procedure of equine embryos to achieve a viable pregnancy. Equine embryos were collected on day 6-6.5 (<300 μm, n = 24 and subjected to two cryopreservation techniques: group 1 (n = 12, vitrified, exposing them to a VS1 (Gli [1.4 M] 5 min, VS2 (Gli [1.4 M] + EG [3.6 M] and VS3 (Gli [3.4M] + EG [4.6 M] 1 min solution. They were packed in 0.25 ml straws and immersed in liquid nitrogen; group 2 (n = 12, slow freezing: exposed to a freezing solution (1.8 M EG + 0.1 M sucrose for 10 minutes, packed into 0.25 ml straws, brought to the embryos freezer, exposed to a freezing curve and immersed in liquid nitrogen. Following defrosting, cryoprotectants were removed from the 24 embryos in one step; they were submerged in culture medium DMEM/F12 + 10% of fetal bovine serum (FBS and incubated under controlled atmosphere (5% CO2, 5% N2, 90% O2 for 48 h. Embryonic development was evaluated in 75% of the vitrified embryos (n = 4; 20% of the embryos were subjected to slow freezing (n = 1. No significant difference was observed in the groups regarding embryonic development, but a greater survival tendency on the vitrified embryos was noted. Also, one of these vitrified embryos was transferred to a receiver, achieving a viable pregnancy and the birth of a living foal.

  11. 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

    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....... The IVP embryos allow us to estimate the effect of in vitro procedures and the analysis of parthenogenetic embryos provides provisional information on maternal genomic imprinting. Among the 8 genes investigated, only Mest-1 showed differential expression in Day 21 parthenogenetic embryos compared...... to in vivo and IVP counterparts, indicating maternal imprinting of this gene. In addition, our expression analysis of single embryos revealed a more heterogeneous gene expression in IVP than in in vivo developed embryos, adding further to the hypothesis of transcriptional dysregulation induced by in vitro...

  12. Factors affecting conception rates in cattle following embryo transfer ...

    African Journals Online (AJOL)

    Embryo Transfer Technology (ETT) plays an important role in improving productivity of dairy cattle (Bos indicus). Embryo Transfer Technology allows top quality female livestock to improve a herd or flock in much the same way that artificial insemination has allowed greater use of superior sires. The technology hastens ...

  13. Successful pregnancy following trans-myometrial embryo transfer ...

    African Journals Online (AJOL)

    ... factor infertility (poor motility). Routine mock embryo transfer indicated severe cervical stenosis which was confirmed at hysteroscopy. She subsequently had trans-myometrial embryo transfer. Blood pregnancy test at 2 weeks was positive and interval transvaginal ultrasound confirmed 2 viable intrauterine foetal poles.

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

    Energy Technology Data Exchange (ETDEWEB)

    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 {sup 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.

  15. 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 activation...... bovine embryos were investigated to allow comparison of transcription initiation. Signs of active transcription of rRNA were observed in the third cell cycle in 29% of the in vitro produced embryos (n=35) and in 58% of the in vivo developed embryos (n=11). Signs of active transcription of rRNA were...... not apparent in the early phase of the fourth cell cycle but restarted later on. All embryos in the fifth or later cell cycles were all transcribing rRNA. The signs of rRNA synthesis during the third and fourth embryonic cell cycles could be blocked by actinomycin D, which is a strong inhibitor of RNA...

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jenna eKropp

    2014-04-01

    Full Text Available 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.

  18. The current status and future of commercial embryo transfer in cattle.

    Science.gov (United States)

    Hasler, John F

    2003-12-15

    A commercially viable cattle embryo transfer (ET) industry was established in North America during the early 1970s, approximately 80 years after the first successful embryo transfer was reported in a mammal. Initially, techniques for recovering and transferring cattle embryos were exclusively surgical. However, by the late 1970s, most embryos were recovered and transferred nonsurgically. Successful cryopreservation of embryos was widespread by the early 1980s, followed by the introduction of embryo splitting, in vitro procedures, direct transfer of frozen embryos and sexing of embryos. The wide spread adoption of ethylene glycol as a cryoprotectant has simplified the thaw-transfer procedures for frozen embryos. The number of embryos recovered annually has not grown appreciably over the last 10 years in North America and Europe; however, there has been significant growth of commercial ET in South America. Within North America, ET activity has been relatively constant in Holstein cattle, whereas there has been a large ET increase in the Angus breed and a concomitant ET decrease in some other beef breeds. Although a number of new technologies have been adopted within the ET industry in the last decade, the basic procedure of superovulation of donor cattle has undergone little improvement over the last 20 years. The export-import of frozen cattle embryos has become a well-established industry, governed by specific health regulations. The international movement of embryos is subject to sudden and dramatic disturbances, as exemplified by the 2001 outbreak of foot and mouth disease in Great Britain. It is probable that there will be an increased influence of animal rights issues on the ET industry in the future. Several companies in North America are currently commercially producing cloned cattle. The sexing of bovine semen with the use of flow cytometry is extremely accurate and moderate pregnancy rates in heifers have been achieved in field trials, but sexed semen

  19. Increasing of blastocyst rate and gene expression in co-culture of bovine embryos with adult adipose tissue-derived mesenchymal stem cells.

    Science.gov (United States)

    Miranda, Moysés S; Nascimento, Hamilton S; Costa, Mayra P R; Costa, Nathália N; Brito, Karynne N L; Lopes, Cinthia T A; Santos, Simone S D; Cordeiro, Marcela S; Ohashi, Otávio M

    2016-10-01

    Despite advances in the composition of defined embryo culture media, co-culture with somatic cells is still used for bovine in vitro embryo production (IVEP) in many laboratories worldwide. Granulosa cells are most often used for this purpose, although recent work suggests that co-culture with stem cells of adult or embryonic origin or their derived biomaterials may improve mouse, cattle, and pig embryo development. In experiment 1, in vitro produced bovine embryos were co-cultured in the presence of two concentrations of bovine adipose tissue-derived mesenchymal cells (b-ATMSCs; 103 and 104 cells/mL), in b-ATMSC preconditioned medium (SOF-Cond), or SOF alone (control). In experiment 2, co-culture with 104 b-ATMSCs/mL was compared to the traditional granulosa cell co-culture system (Gran). In experiment 1, co-culture with 104 b-ATMSCs/mL improved blastocyst rates in comparison to conditioned and control media (p culture with 104 b-ATMSCs/mL improved not only blastocyst rates but also quality as assessed by increased total cell numbers and mRNA expression levels for POU5F1 and G6PDH (p culture of bovine embryos with b-ATMSCs was more beneficial than the traditional co-culture system with granulosa cells. We speculate that the microenvironmental modulatory potential of MSCs, by means of soluble substances and exosome secretions, could be responsible for the positive effects observed. Further experiments must be done to evaluate if this beneficial effect in vitro also translates to an increase in offspring following embryo transfer. Moreover, this study provides an interesting platform to study the basic requirements during preimplantation embryo development, which, in turn, may aid the improvement of embryo culture protocols in bovine and other species.

  20. Evaluation of developmental changes in bovine in vitro produced embryos following exposure to bovine Herpesvirus type 5

    Directory of Open Access Journals (Sweden)

    Brenner Mariana PC

    2012-07-01

    Full Text Available Abstract Background Bovine Herpesvirus type-5 (BoHV-5 is a neurovirulent α-Herpesvirus which is potentially pathogenic for cows and suspected to be associated with reproductive disorders. Interestingly, natural transmission of BoHV-5 by contaminated semen was recently described in Australia. Additionally, BoHV-5 was also isolated from the semen of a healthy bull in the same country and incriminated in a natural outbreak of reproductive disease after artificial insemination. In contrast with BoHV-1, experimental exposure of in vitro produced bovine embryos to BoHV-5 does not affect embryo viability and seems to inhibit some pathways of apoptosis. However, the mechanisms responsible for these phenomena are poorly understood. In this study, we examined mitochondrial activity, antioxidant protection, stress response and developmental rates of in vitro produced bovine embryos that were exposed and unexposed to BoHV-5. Methods For this purpose, bovine embryos produced in vitro were assayed for cell markers after experimental infection of oocytes (n = 30; five repetitions, in vitro fertilization and development. The indirect immunofluorescence was employed to measure the expression of superoxide dismutase 1 (SOD1, anti-oxidant like protein 1 (AOP-1, heat shock protein 70.1 (Hsp 70.1 and also viral antigens in embryos derived from BoHV-5 exposed and unexposed oocytes. The determination of gene transcripts of mitochondrial activity (SOD1, antioxidant protection (AOP-1 and stress response (Hsp70.1 were evaluated using the reverse transcriptase polymerase chain reaction (RT-PCR. MitoTracker Green FM, JC-1 and Hoechst 33342-staining were used to evaluate mitochondrial distribution, segregation patterns and embryos morphology. The intensity of labeling was graded semi-quantitatively and embryos considered intensively marked were used for statistical analysis. Results The quality of the produced embryos was not affected by exposure to BoHV-5. Of the 357

  1. Low serum concentration in bovine embryo culture enhances early blastocyst rates on Day-6 with quality traits in the expanded blastocyst stage similar to BSA-cultured embryos.

    Science.gov (United States)

    Murillo, A; Muñoz, M; Martín-González, D; Carrocera, S; Martínez-Nistal, A; Gómez, E

    2017-06-01

    In bovine, single in vitro embryo culture in protein-free medium from Day-6 to Day-7 leads to expanded blastocyst (XB) with improved pregnancy and birth rates after cryopreservation. Under these conditions, early blastocysts (EB) progress to the XB stage at higher rates than morulae (M). However, embryo production with BSA in culture prior to Day-6 leads to low EB rates. We investigated whether a very low FCS concentration (0.1%) in culture from Day-1 to Day-6 would improve EB rates and, subsequently, increase XB rates on Day-7 after single culture in protein-free medium. The quality of embryos produced was evaluated in terms of survival to cryopreservation, apoptosis percentage, lipid accumulation and transfer to recipients. On Day-6, EB rates from embryos cultured with FCS were higher than with BSA (P=0.022). On Day-7, XB rates were higher in embryos from Day-6 EB than from Day-6M, both with and without FCS (Pvitrification/warming of Day-7 XB, 100% embryos survived at 24h in all treatments, and total cell number and apoptosis percentage were not affected by the presence of FCS or embryonic stage on Day-6. Cryopreserved and fresh embryos produced with FCS until Day-6, and then deprived of protein and cultured individually, led to pregnancies after ET. In conclusion, minute FCS concentration improves EB rates on Day-6 leading, after one-day single culture without protein, to more XBs. The quality of XB produced with FCS compares well with XB produced with BSA in terms of apoptosis, lipid accumulation and pregnancy. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  2. Vitrification of in vitro produced bovine embryos: effect of embryonic block and developmental kinetics.

    Science.gov (United States)

    Asgari, V; Hosseini, S M; Forouzanfar, M; Hajian, M; Nasr-Esfahani, M H

    2012-12-01

    In order to investigate whether the kinetics and stage of embryo development affect cryosurvival of in vitro produced bovine embryos, cleaved embryos were categorized in six groups based on their developmental kinetics regarding the stage of embryonic block in bovine (8-16 cell stage): I and II--early (day 2) and late (day 3) 5-8 cell, III and IV--early (day 3) and late (day 4) 8-16 cell, and V and VI--early (day 4) and late (day 5) morula. The cryosurvival and developmental competence of these embryos were compared with each other and also with the corresponding control groups. The potential of 5-8 cell stage embryos to survive vitrification and further develop towards blastocyst stage was significantly lower than vitrified and un-vitrified 8-16 cell and morula stage embryos. These results suggest that, the survival rate and potential of embryos to develop towards blastocyst stage might be affected by the kinetic of the embryo development. Moreover, the results of this study indicated that the optimal stages of early embryo vitrification are post-embryonic block. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Air bubble migration is a random event post embryo transfer.

    Science.gov (United States)

    Confino, E; Zhang, J; Risquez, F

    2007-06-01

    Air bubble location following embryo transfer (ET) is the presumable placement spot of embryos. The purpose of this study was to document endometrial air bubble position and migration following embryo transfer. Multicenter prospective case study. Eighty-eight embryo transfers were performed under abdominal ultrasound guidance in two countries by two authors. A single or double air bubble was loaded with the embryos using a soft, coaxial, end opened catheters. The embryos were slowly injected 10-20 mm from the fundus. Air bubble position was recorded immediately, 30 minutes later and when the patient stood up. Bubble marker location analysis revealed a random distribution without visible gravity effect when the patients stood up. The bubble markers demonstrated splitting, moving in all directions and dispersion. Air bubbles move and split frequently post ET with the patient in the horizontal position, suggestive of active uterine contractions. Bubble migration analysis supports a rather random movement of the bubbles and possibly the embryos. Standing up changed somewhat bubble configuration and distribution in the uterine cavity. Gravity related bubble motion was uncommon, suggesting that horizontal rest post ET may not be necessary. This report challenges the common belief that a very accurate ultrasound guided embryo placement is mandatory. The very random bubble movement observed in this two-center study suggests that a large "window" of embryo placement maybe present.

  4. Developmental kinetics of in vitro-produced bovine embryos: An aid for making decisions.

    Science.gov (United States)

    Carrocera, S; Caamaño, J N; Trigal, B; Martín, D; Díez, C

    2016-03-15

    Embryo developmental kinetics and embryo survival after cryopreservation have been correlated with embryo quality and viability. The main objectives of this work were to analyze developmental ability and quality of in vitro-produced bovine embryos in relation to their kinetics and to establish a criterion of quality to predict further viability. Embryos were classified and grouped by their specific stage of development (2, 3-4, or ≥ 5 cells) at 44 hours post insemination (hpi) and cultured separately up to Day 8. On Days 7 and 8, good quality expanded blastocysts were vitrified or frozen. Cryopreserved surviving hatched embryos were stained for cell counts. Embryos at a more advanced stage (3-4 cells, and ≥5 cells) developed to morulae (P embryos that had cleaved once by 44 hpi. Vitrification improved the hatching rates of blastocysts at 48 hours (P embryos (3-4 cells and ≥5 cells). After vitrification/warming, blastocysts coming from 3- to 4-cell embryos had higher hatching rates at 48 hours than those that came from ≥5-cell embryos. With regard to differential cell counts, no effect of the initial developmental stage was observed after warming/thawing. However, trophectoderm and total cells were higher in vitrified/warmed than in the frozen/thawed embryos (P embryos at 44 hpi, after the evaluation of their in vitro embryo development, could be used as noninvasive markers of embryo developmental competence and may help to select IVF embryos that would be more suitable for cryopreservation. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Solid-surface vitrification and in-straw dilution after warming of in vitro-produced bovine embryos.

    Science.gov (United States)

    Rodriguez-Villamil, P; Ongaratto, F L; Fernandez Taranco, M; Bó, G A

    2014-02-01

    Three experiments were designed to test a solid-surface vitrification system for bovine in vitro-produced embryos and to develop a simple method of in-straw dilution after warming, which can be potentially used for direct transfer in the field. Experiment 1 evaluated embryo survival rates (i.e. re-expansion and hatching) after vitrification and warming in three different solutions: VS1 (20% ethylene glycol (EG) + 20% propanediol (PROH) + 0.25 m trehalose (Tr)), VS2 (20% EG + 1M Tr) or VS3 (30% EG + 0.75 m Tr). Re-expansion and hatching rates were higher (p vitrification: glass micropipettes or solid surface, using the VS1 or VS3 solutions. No significant differences were detected between the two methods; but re-expansion and hatching rates were higher (p vitrification using simplified EG-based solutions and in-straw dilution with holding media may be a practical alternative for cryopreservation and direct transfer of in vitro-produced bovine embryos. © 2013 Blackwell Verlag GmbH.

  6. Production, Preservation, and Transfer of South American Camelid Embryos

    Directory of Open Access Journals (Sweden)

    Virginia L. Trasorras

    2017-11-01

    Full Text Available The current review summarizes progress in the field of in vitro and in vivo production of South American Camelid embryos. Both methods require ovarian superstimulation (with FSH and eCG to obtain multiple ovulations (in vivo embryo production or to induce follicle growth for oocyte collection (in vitro embryo production. Moreover, superstimulation entails prior administration of hormones that inhibit follicular growth (progesterone, progestagens, and estrogens. Cumulus-oocyte complexes obtained must mature in vivo (buserelin administration or in vitro to then be subjected to in vitro fertilization or intracytoplasmic sperm injection. All these techniques also require morphologically normal, motile spermatozoa to achieve fertilization. Methods used to decrease semen viscosity and to select the best spermatozoa (Percoll®; Androcoll-ETM are described. Additionally, nuclear transfer or cloning has been applied in llamas. Up to now, embryo deep-freezing and vitrification have progressed slowly but are at the height of development. Embryos that are obtained by any of these techniques, either in vivo or in vitro, need to be transferred to synchronized recipient females. The best results are achieved after transfer to the left uterine horn with an ipsilateral ovulation. No live offspring have been obtained after the transfer of cryopreserved embryos. Applying reproductive biotechnologies, such as those described, will permit the expansion of genetically selected animals in the population and also that of wild camelid species, vicunas, and guanacos, whose embryos could then be transferred to the uterus of domestic species.

  7. Embryo transfer from cattle infected with bluetongue virus.

    Science.gov (United States)

    Bowen, R A; Howard, T H; Elsden, R P; Seidel, G E

    1983-09-01

    Embryos recovered nonsurgically from donor cattle during the peak of bluetongue viremia were surgically transferred to seronegative recipients 7 to 8 or 10 to 11 days after the onset of donor estrus. Virus was isolated from the uterine flushing medium recovered from 11 of the 20 donors. Bluetongue virus was not isolated from the blood of any of 39 recipients, nor did any recipient seroconvert to the virus following transfer. The number of recipients that became pregnant after transfer of embryos from infected donors (21 of 39) was not significantly different from contemporary controls. Virus antigen was not detected by immunofluorescence in any of 63 embryos and oocytes recovered from viremic donors. These results indicate that under standard embryo transfer conditions, transmission of bluetongue virus from infected donors to uninfected recipients is unlikely to occur.

  8. Forced collapse of the blastocoel enhances survival of cryotop vitrified bovine hatching/hatched blastocysts derived from in vitro fertilization and somatic cell nuclear transfer.

    Science.gov (United States)

    Min, Sung-Hun; Lee, Enok; Son, Hyeong-Hoon; Yeon, Ji-Yeong; Koo, Deog-Bon

    2013-04-01

    Freezing of bovine blastocysts has been widely used to improve the feasibility of cattle production by the embryo transfer technique. However, the low survival of vitrified-warmed embryos and their further development are crucial problems. Particularly, the production of offspring in vitrified-warmed bovine hatching/hatched blastocysts derived from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) is very low. Thus, we examined the effects of forced blastocoel collapse (FBC) before vitrification of bovine IVF- and SCNT-derived hatching/hatched embryos on the survival rate and apoptosis index after warming. Under optimal conditions, the overall survival rates in vitrified-warmed bovine IVF- and SCNT-derived hatching/hatched blastocysts were higher in FBC groups than in non-FBC groups (pvitrification of bovine IVF- and SCNT-derived hatching/hatched blastocysts. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. The factors affecting the outcome of frozen–thawed embryo transfer cycle

    Directory of Open Access Journals (Sweden)

    Mahnaz Ashrafi

    2011-06-01

    Conclusion: Protocol type, gonadotrophin preparations, fresh-cycle outcome, endometrial thickness and the numbers of obtained oocytes, embryos, and high-quality thawed embryos transferred are the factors affecting pregnancy outcome of frozen–thawed embryo transfer.

  10. Embryo development and embryo transfer in the European mink (Mustela lutreola), an endangered mustelid species.

    Science.gov (United States)

    Amstislavsky, S; Kizilova, E; Ternovskaya, Y; Zudova, G; Lindeberg, H; Aalto, J; Valtonen, M

    2006-01-01

    The European mink is an endangered Mustelidae species and thus requires effective conservation measures, although little is known about reproduction in this species. In particular, preimplantation development has not been studied and, therefore, embryonic development and the growth of embryos was documented in the present study for European mink using light and fluorescent microscopy. Embryos develop in the oviducts and then migrate into the uterus on Day 6 post coitum (p.c.) at the morula stage. Embryos expanded as blastocysts from Day 7 until implantation on Day 12 p.c. Based on these findings, the use of embryo transfer for a conservation programme for the European mink was evaluated. Embryos were flushed from European mink resource females and transferred into the uterine horns of recipient hybrid females (honoriks and nohoriks). These hybrids were obtained by mating European polecat males with European mink females and vice versa. A total of 40 embryos was transferred and 20 live kits were born. The rates of pre- and postnatal survival were 50% and 70%, respectively. Both male and female offspring were lighter at birth in the embryo transfer group compared with naturally born controls, but there was no difference at 3 months of age.

  11. Cryopreservation affects the quality of in vitro produced bovine embryos at the molecular level.

    Science.gov (United States)

    Stinshoff, H; Wilkening, S; Hanstedt, A; Brüning, K; Wrenzycki, C

    2011-11-01

    There are still immense differences in the quality of in vitro produced embryos compared to their in vivo generated counterparts. These differences include a higher sensitivity of in vitro produced embryos towards cryopreservation. The quality of such embryos has been evaluated by morphological examination as well as the assessment of total cell numbers, pregnancy rates and in few cases through the analysis of their gene expression. The aim of the present study was to determine whether different cryopreservation methods have an influence on the quality of in vitro produced embryos after thawing. Bovine blastocysts were produced in a standard culture system (SOFaa). Having reached the stage of an expanding blastocyst on day 7, embryos were randomly either vitrified (n = 106) or cryopreserved conventionally (n = 131). Reexpansion (24h post thawing; 86/106 [81.1 ± 20.3%] of the vitrified embryos, 104/131 [79.4 ± 16.5%] of the conventionally cryopreserved embryos respectively) and hatching rates (48 h post thawing; 67/106 [63.2 ± 24.5%] of the vitrified embryos, 80/131 [61.1 ± 29.3%] of the conventionally cryopreserved embryos respectively) were similar. No significant differences in total cell numbers as well as live-dead cell ratio could be seen in hatched blastocysts of either cryopreservation group as well as in a control group (embryos cultured up to the stage of a hatched blastocyst and not cryopreserved by either method). Additionally, RT-qPCR was used to assess the relative abundance of eight developmentally important genes (HSPA1A, SLC2A1, SLC2A3, DSC2, CDH1, TJP1, DNMT3A, IFNT2) in hatched embryos of all groups. The results revealed significant differences in 4 of 8 (HSPA1A, SLC2A1, TJP1, DSC2) gene transcripts when comparing vitrified embryos to embryos of the control group and in 6 of 8 gene transcripts (HSPA1A, SLC2A1, TJP1, SLC2A3, DNMT3A, IFNT2) when comparing slow frozen embryos to embryos of the control group. Furthermore, the comparison of

  12. Bovine ooplasm partially remodels primate somatic nuclei following somatic cell nuclear transfer.

    Science.gov (United States)

    Wang, Kai; Beyhan, Zeki; Rodriguez, Ramon M; Ross, Pablo J; Iager, Amy E; Kaiser, German G; Chen, Ying; Cibelli, Jose B

    2009-03-01

    Interspecies somatic cell nuclear transfer (iSCNT) has the potential to become a useful tool to address basic questions about the nucleus-cytoplasm interactions between species. It has also been proposed as an alternative for the preservation of endangered species and to derive autologous embryonic stem cells. Using chimpanzee/ bovine iSCNT as our experimental model we studied the early epigenetic events that take place soon after cell fusion until embryonic genome activation (EGA). Our analysis suggested partial EGA in iSCNT embryos at the eight-cell stage, as indicated by Br-UTP incorporation and expression of chimpanzee embryonic genes. Oct4, Stella, Crabp1, CCNE2, CXCL6, PTGER4, H2AFZ, c-MYC, KLF4, and GAPDH transcripts were expressed, while Nanog, Glut1, DSC2, USF2, Adrbk1, and Lin28 failed to be activated. Although development of iSCNT embryos did not progress beyond the 8- to 16-cell stage, chromatin remodeling events, monitored by H3K27 methylation, H4K5 acetylation, and global DNA methylation, were similar in both intra- and interspecies SCNT embryos. However, bisulfite sequencing indicated incomplete demethylation of Oct4 and Nanog promoters in eight-cell iSCNT embryos. ATP production levels were significantly higher in bovine SCNT embryos than in iSCNT embryos, TUNEL assays did not reveal any difference in the apoptotic status of the nuclei from both types of embryos. Collectively, our results suggest that bovine ooplasm can partially remodel chimpanzee somatic nuclei, and provides insight into some of the current barriers iSCNT must overcome if further embryonic development is to be expected.

  13. Isolation of bovine herpesvirus-1 (BHV-1) and bovine viral diarrhea virus (BVDV) in association with the in vitro production of bovine embryos.

    Science.gov (United States)

    Bielanski, A; Loewen, K S; Del Campo, M R; Sirard, M A; Willadsen, S

    1993-09-01

    The purpose of this study was to determine whether oocytes obtained from bovine ovaries collected at commercial abattoirs for use in in vitro fertilization programs would be contaminated with bovine herpesvirus-1 (BHV-1) and/or bovine viral diarrhea virus (BVDV). In total, of 85 samples tested containing 759 embryos produced by in vitro fertilization, 2 (2.4%) were positive for BHV-1 while none were positive for BVDV. The follicular fluid collected during oocyte aspiration tested positive in 11.8% for BVH-1 and in 4.7% for BVDV. Oviductal cells used to co-culture zygotes/embryos tested positive for BHV-1 and BVDV in 6.2% and 1.2% samples respectively.

  14. Effects of steroidal glycoalkaloids from potatoes (Solanum tuberosum) on in vitro bovine embryo development.

    Science.gov (United States)

    Wang, S; Panter, K E; Gaffield, W; Evans, R C; Bunch, T D

    2005-02-01

    alpha-Solanine and alpha-chaconine are two naturally occurring steroidal glycoalkaloids in potatoes (Solanum tuberosum), and solanidine-N-oxide is a corresponding steroidal aglycone. The objective of this research was to screen potential cyto-toxicity of these potato glycoalkaloids using bovine oocyte maturation, in vitro fertilization techniques and subsequent embryonic development as the in vitro model. A randomized complete block design with four in vitro oocyte maturation (IVM) treatments (Experiment 1) and four in vitro embryo culture (IVC) treatments (Experiment 2) was used. In Experiment 1, bovine oocytes (n=2506) were matured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVM medium only. The in vitro matured oocytes were then subject to routine IVF and IVC procedures. Results indicated that exposure of bovine oocytes to the steroidal glycoalkaloids during in vitro maturation inhibited subsequent pre-implantation embryo development. Potency of the embryo-toxicity varied between these steroidal glycoalkaloids. In Experiment 2, IVM/IVF derived bovine embryos (n=2370) were cultured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVC medium only. The results showed that the pre-implantation embryo development is inhibited by exposure to these glycoalkaloids. This effect is significant during the later pre-implantation embryo development period as indicated by fewer numbers of expanded and hatched blastocysts produced in the media containing these alkaloids. Therefore, we conclude that in vitro exposure of oocytes and fertilized ova to the steroidal glycoalkaloids from potatoes inhibits pre-implantation embryo development. Furthermore, we suggest that ingestion of Solanum species containing toxic amounts of glycoalkaloids may have negative effects on pre-implantation embryonic survival.

  15. Ovarian hyperstimulation syndrome and prophylactic human embryo cryopreservation: analysis of reproductive outcome following thawed embryo transfer

    Directory of Open Access Journals (Sweden)

    Sills Eric

    2008-11-01

    Full Text Available Abstract Objective To review utilisation of elective embryo cryopreservation in the expectant management of patients at risk for developing ovarian hyperstimulation syndrome (OHSS, and report on reproductive outcome following transfer of thawed embryos. Materials and methods Medical records were reviewed for patients undergoing IVF from 2000–2008 to identify cases at risk for OHSS where cryopreservation was electively performed on all embryos at the 2 pn stage. Patient age, total number of oocytes retrieved, number of 2 pn embryos cryopreserved, interval between retrieval and thaw/transfer, number (and developmental stage of embryos transferred (ET, and delivery rate after IVF were recorded for all patients. Results From a total of 2892 IVF cycles undertaken during the study period, 51 IVF cases (1.8% were noted where follicle number exceeded 20 and pelvic fluid collection was present. Elective embryo freeze was performed as OHSS prophylaxis in each instance. Mean (± SD age of these patients was 32 ± 3.8 yrs. Average number of oocytes retrieved in this group was 23 ± 8.7, which after fertilisation yielded an average of 14 ± 5.7 embryos cryopreserved per patient. Thaw and ET was performed an average of 115 ± 65 d (range 30–377 d after oocyte retrieval with a mean of 2 ± 0.6 embryos transferred. Grow-out to blastocyst stage was achieved in 88.2% of cases. Delivery/livebirth rate was 33.3% per initiated cycle and 43.6% per transfer. Non-transferred blastocysts remained in cryostorage for 24 of 51 patients (46.1% after ET, with an average of 3 ± 3 blastocysts refrozen per patient. Conclusion OHSS prophylaxis was used in 1.8% of IVF cycles at this institution; no serious OHSS complications were encountered during the study period. Management based on elective 2 pn embryo cryopreservation with subsequent thaw and grow-out to blastocyst stage for transfer did not appear to compromise embryo viability or overall reproductive outcome. For

  16. 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.

  17. Sex determination and milk protein genotyping of preimplantation stage bovine embryos using multiplex PCR.

    Science.gov (United States)

    Agrawala, P L; Wagner, V A; Geldermann, H

    1992-11-01

    A method for determining the sex and milk protein genotypes (RFLPs) of preimplantation stage bovine embryos using multiplex polymerase chain reaction (PCR) is described. Day 6 to 7 embryos were micromanipulated to isolate 5 to 6 cells. These cells were then dried in reaction tubes for transport to the laboratory. Subsequently, two sets of PCRs were performed using Y chromosome, k-casein and beta-lactoglobulin gene specific primers, followed by electrophoretic analysis of the PCR products. The presence or absence of the Y chromosome was ascertained in 90 of 92 embryos. Moreover, the k-casein specific fragment was amplified and detected in all these embryos. The PCR products were digested in order to genotype the k-casein gene. In 70% of the embryos, the beta-lactoglobulin specific fragment was amplified, although together with some unspecific fragments.

  18. Inhibition of apoptosis by caspase inhibitor Z-VAD-FMK improves cryotolerance of in vitro derived bovine embryos.

    Science.gov (United States)

    Pero, Maria Elena; Zullo, Gianluigi; Esposito, Luigi; Iannuzzi, Alessandra; Lombardi, Pietro; De Canditiis, Carolina; Neglia, Gianluca; Gasparrini, Bianca

    2017-12-02

    The aim of this work was to evaluate whether the treatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) during cryopreservation and post-warming in vitro culture improves cryotolerance of bovine in vitro produced (IVP) embryos. Abattoir derived bovine oocytes were in vitro matured, fertilized and cultured according to standard procedure. On Day 7, embryo yields were assessed and blastocysts randomly divided in 2 groups: vitrification and post-warming culture in the absence (n = 184) or presence (n = 156) of 20 μM Z-VAD-FMK. Resistance to cryopreservation was evaluated post-warming culture by assessing the survival rate and hatching rate. Differential staining combined with in situ terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) technique was performed to evaluate total cells number, cell allocation into inner cell mass (ICM) and trophectoderm (TE) lineages, as well as the DNA fragmentation rate of vitrified blastocysts, while immunohystochemical staining was used to assess the level of cleaved-caspase 3. It was demonstrated that inhibition of caspase activity by Z-VAD-FMK increases embryo cryotolerance, as indicated by higher survival (76.1 vs 51.1%; P vitrification/warming and post-warming culture partially inhibits cryopreservation-induced apoptosis by reducing the level of active caspase 3, suggesting a potential use as an additive to ameliorate the efficiency of embryo cryopreservation in cattle, critical for a further diffusion of IVEP technology in the field. Further studies are though needed to evaluate the effect of Z-VAD-FMK on post-transfer embryo development before considering a commercial application. Copyright © 2017. Published by Elsevier Inc.

  19. Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification.

    Science.gov (United States)

    Nedambale, T L; Dinnyés, A; Groen, W; Dobrinsky, J R; Tian, X C; Yang, X

    2004-08-01

    The objectives of this study were to identify an improved in vitro cell-free embryo culture system and to compare post-warming development of in vitro produced (IVP) bovine embryos following vitrification versus slow freezing. In Experiment 1, non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture: (1) SOF + 5% FCS for 9 days; (2) KSOM + 0.1% BSA for 4 days and then KSOM + 1% BSA to Day 9; (3) SOF + 5% FCS for 4 days and then KSOM + 1% BSA to Day 9; and (4) KSOM + 0.1% BSA for 4 days and then SOF + 5% FCS to Day 9. Treatment 4 (sequential KSOM-SOF culture system) improved (P > 0.05) morulae (47%), early blastocysts (26%), Day-7 blastocysts (36%), cell numbers, as well as total hatching rate (79%) compared to KSOM alone (Treatment 2). Embryos cultured in KSOM + BSA alone developed slowly and most of them hatched late on Day 9, compared to other treatments. In Experiment 2, the sequential KSOM-SOF culture system was used and Day-7 blastocysts were subjected to following cryopreservation comparison: (1) vitrification (VS3a, 6.5 M glycerol); or (2) slow freezing (1.36 M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rates (P vitrification at 6h (71%), 24h (64%), and 48h (60%) post-warming compared to slow freezing (48, 40, and 31%, respectively). Following transfer of vitrified embryos to synchronized recipients, a 30% pregnancy rate was obtained. In conclusion, replacing KSOM with SOF after 4 days of culture produced better quality blastocysts. Vitrification using VS3a may be used more effectively to cryopreserve in vitro produced embryos than the conventional slow freezing method.

  20. 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...... ≥6 mm always gave a higher blastocyst rate than oocytes from follicles...

  1. Birth of healthy calves after intra-follicular transfer (IFOT) of slaughterhouse derived immature bovine oocytes.

    Science.gov (United States)

    Hoelker, M; Kassens, A; Salilew-Wondim, D; Sieme, H; Wrenzycki, C; Tesfaye, D; Neuhoff, C; Schellander, K; Held-Hoelker, E

    2017-07-15

    To circumvent the negative impacts of in vitro culture on bovine embryos, we have recently established a new method, the so called intra-follicular oocyte transfer (IFOT), enabling in vivo fertilization and in vivo development of in vitro matured oocytes up to the blastocyst stage as well as to term. In this study, we raised the question whether immature bovine oocytes could also be transferred into a pre-ovulatory follicle to support in vivo maturation prior to subsequent in vivo fertilization, in vivo development as well as to term. To unravel that question, a total of 791 immature oocytes were transferred in groups of ∼50 into pre-ovulatory follicles of 16 recipient heifers. Consequently, we were able to recollect a total of 306 structures 8 days thereafter (38.5%). All in all, 12 heifers (75%) gave embryos developed to the morula or blastocyst stage in addition to the expected native embryos. Among all recollected structures, 40.1% had developed to the morula and/or blastocyst stage, meaning a total efficiency of 17.3% based on all transferred oocytes. Of impact, IFOT-embryos reached significantly higher developmental rates to the Morula and/or blastocyst stage until day 7 compared to in vitro cultured control embryos, despite being derived from the same charge of slaughterhouse ovaries (40.1 vs. 29.3%). This implicates a beneficial effect of the follicular environment for the intrinsic quality of the fertilized embryos during maturation and for subsequent developmental rates up to the blastocyst stage. Finally, the birth of two healthy calves after transfer of frozen-thawed IFOT-derived blastocysts to final recipients established the first proof of principle that IFOT of immature bovine oocytes generates bovine blastocysts bearing developmental capacity to term. Likewise, to the best of our knowledge, these calves are the first calves derived from full in vivo development of immature slaughterhouse derived oocytes. Thus, the results of the present

  2. Ploidy of Bovine Nuclear Transfer Blastocysts Blastomere Donors

    DEFF Research Database (Denmark)

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

    2000-01-01

    The higher rate of embryonic loss in nuclear transfer compared to in vitro produced embryos may be due to chromosome abnormalities that occur during preimplantation in vitro devel- opment. Because little is known about ploidy errors in nuclear transfer embryos, this was ex- amined using embryos...... cultured until day 7 at which time blastocyst nuclei were extracted and chromosome abnormalities were evaluated by fluorescent in situ hybridization using two probes that bind to the subcentromeric regions on chromosomes 6 and 7. In 16 nuclear transfer blastocysts generated from 5 donor embryos, 53.8 6 20...... fertil- ization but the distribution of chromosome abnormalities was different (p 5 0.0002). In conclusion, nuclear transfer embryos reconstructed using blastomere cells can produce over 50% blastocysts with a diploid chromosome complement. However, the contribution of chro- mosome abnormalities...

  3. Debating Elective Single Embryo Transfer after in vitro Fertilization ...

    African Journals Online (AJOL)

    Marqueta J, Pareja A, et al. Impact of the Spanish Fertility. Society guidelines on the number of embryos to transfer. Reprod Biomed Online 2010;21:667‑75. 21. Ryan GL, Sparks AE, Sipe CS, Syrop CH, Dokras A, Van. Voorhis BJ. A mandatory single blastocyst transfer policy with educational campaign in a United States ...

  4. Factors affecting conception rates in cattle following embryo transfer

    African Journals Online (AJOL)

    ACSS

    . The transfer gun/ insemination rod was carefully passed through the cervix. The embryo was then deposited in the uterine horn epsilateral to ... Observe heat. 24. 8.00 AM-5.00 PM. Transfer. CIDR = (Controlled Internal Drug Release device) ...

  5. Screening of biotechnical parameters for production of bovine inter-subspecies embryonic chimeras by the aggregation of tetraploid Bos indicus and diploid crossbred Bos taurus embryos.

    Science.gov (United States)

    Razza, Eduardo M; Satrapa, Rafael A; Emanuelli, Isabele P; Barros, Ciro M; Nogueira, Marcelo F G

    2016-03-01

    The aggregation of a tetraploid zebu embryo (Bos indicus, a thermotolerant breed) with a diploid taurine embryo (Bos taurus, a thermosensitive breed) should create a complete taurine fetus, whose extra-embryonic components, e.g., the chorion, is derived mainly from the zebu embryo. These zebu-derived extra-embryonic components may interact positively with the taurine embryo/fetus during pregnancy in a tropical environment. We tested different parameters for the production of tetraploid Nelore (Bos indicus) embryos to be combined via aggregation with crossbred Bos taurus (diploid) embryos in order to produce viable chimeric blastocysts. Bovine (Bos indicus or crossbred Bos taurus) embryos were produced in vitro according to standard procedures. Two-cell Bos indicus embryos were submitted to electrofusion with varying numbers of pulses (1 or 2), voltages (0.4, 0.5, 0.75, 1.0, 1.4 and 5.0 kV/cm) and time (20, 25, 50 and 60 μs) to produce tetraploid embryos. Electrofused embryos were cultured with crossbred non-fused embryos to form chimeras that developed until the blastocyst stage. The best fusion parameter was 0.75 kV/cm for 60 μs. Four chimeric blastocysts (tetraploid Nelore with diploid crossbred Holstein) were formed after 31 attempts in 4 replicates (13%). We established an optimal procedure for the production of tetraploid Bos indicus (4n) embryos and embryonic chimeras by aggregation of crossbred Bos taurus (2n) with Bos indicus (4n) embryos. This technique would be valid in applied research, by producing exclusively taurine calves, but with placental elements from the Bos indicus breed, following transfer of these chimeras into recipient cows. Copyright © 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Daniel R. Arnold

    2015-07-01

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

  7. Different co-culture systems have the same impact on bovine embryo transcriptome.

    Science.gov (United States)

    Carvalho, A Vitorino; Canon, E; Jouneau, L; Archilla, C; Laffont, L; Moroldo, M; Ruffini, S; Corbin, E; Mermillod, P; Duranthon, V

    2017-11-01

    During the last few years, several co-culture systems using either BOEC or VERO feeder cells have been developed to improve bovine embryo development and these systems give better results at high oxygen concentration (20%). In parallel, the SOF medium, used at 5% O2, has been developed to mimic the oviduct fluid. Since 2010s, the SOF medium has become popular in improving bovine embryo development and authors have started to associate this medium to co-culture systems. Nevertheless, little is known about the putative benefit of this association on early development. To address this question, we have compared embryo transcriptomes in four different culture conditions: SOF with BOEC or VERO at 20% O2, and SOF without feeders at 5% or 20% O2 Embryos have been analyzed at 16-cell and blastocyst stages. Co-culture systems did not improve the developmental rate when compared to 5% O2 Direct comparison of the two co-culture systems failed to highlight major differences in embryo transcriptome at both developmental stages. Both feeder cell types appear to regulate the same cytokines and growth factors pathways, and thus to influence embryo physiology in the same way. In blastocysts, when compared to culture in SOF at 5% O2, BOEC or VERO seems to reduce cell survival and differentiation by, at least, negatively regulating STAT3 and STAT5 pathways. Collectively, in SOF medium both blastocysts rate and embryo transcriptome suggest no influence of feeder origin on bovine early development and no beneficial impact of co-culture systems when compared to 5% O2. © 2017 Society for Reproduction and Fertility.

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

    Directory of Open Access Journals (Sweden)

    B. Gasparrini

    2010-02-01

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

  9. [Influence of patient age and the number of good-quality-embryos transferred on multiple gestation in in vitro fertilization and embryo transfer].

    Science.gov (United States)

    Zhang, Shun-Ji; Gong, Fei; Lin, Ge; Lu, Chang-Fu; Xiao, Hong-Mei; Lu, Guang-Xiu

    2008-08-01

    To observe the influence of patient's age, and the number of transferred-good-quality-embryos on multiple gestation rates in in vitro fertilization and embryo transfer (IVF-ET) cycles. In this retrospective study, a total of 4,395 patients who transferred fresh embryo between Jan 2004 and Nov 2006 was analyzed. According to the age, the patients were divided into 2 groups: aged or= 35 (953 cycles). We regularly transferred 2 - 3 embryos. If the patients had only one embryo, one was transferred. And those patients who had only 2 embryos, even if they were more than 35 years old or it would be the second time for them to transfer, were transferred 2 embryos. The influence of female age and the number of good quality embryos transferred on the multiple gestation rates in IVF-ET cycle was analyzed. (1) The multiple gestation rate of the groups of 1 good quality embryo, 2 good quality embryos, or 3 good quality embryos transferred were 21.08% (35/166), 31.41% (413/1315), and 42.37% (75/177), respectively in women aged good quality embryos transferred group and 3 good quality embryos transferred group. (2) The multiple gestation rates of the groups of 1 good quality embryo, 2 good quality embryos, or 3 good quality embryos transferred were 19.51% (8/41), 20.65% (19/92), and 40.66% (74/182), respectively, in women aged >or= 35; there were no significant differences between 1 good quality embryo transferred group and 2 good quality embryos transferred group. The pregnancy rates of these groups were 19.07% (41/215), 33.70% (92/273), and 39.14% (182/465), respectively; there were no significant differences between 2 good quality embryos transferred group and 3 good quality embryos transferred group. (3) The pregnancy rate of the patients aged or= 35 [33.05% (315/953)]. The transfer of 2 good quality embryos results in similar pregnancy rates and significantly reduced multiple gestation rates when compared to the transfer of 3 good quality embryos in women regardless of their

  10. Vitrification of bovine embryos followed by in vitro hatching and expansion.

    Science.gov (United States)

    Souza, J F; Oliveira, C M; Lienou, L L; Cavalcante, T V; Alexandrino, E; Santos, R R; Rodrigues, A P R; Campello, C C; Figueiredo, J R; Dias, F E F

    2017-12-18

    The objective of this study was to assess the effects of bovine embryo vitrification by applying three different vitrification solutions containing ethylene glycol (EG) and dimethylsulphoxide (DMSO) at different concentrations (10, 20 or 25% each) combined with 1.0 M glucose or 1.0 M sucrose, on the in vitro hatching and expansion rates. Healthy oocytes were selected for in vitro maturation and fertilization from 200 bovine ovaries, and subsequently cultured up to the blastocyst stage (n = 800). Control (n = 200) and vitrified cells (n = 100 per treatment; 600 in total) were cultured for an extra 24 or 48 h to evaluate hatching and expansion, respectively. Vitrification significantly decreased embryonic re-expansion and hatching rates independently of the tested solution when compared with control embryos, but solutions with 25% EG + 25% DMSO resulted in the highest re-expansion (75%) and hatching (70%) rates, independently of the added sugar. The addition of sucrose resulted in higher rates of re-expanded and hatched embryos when compared with glucose addition. We concluded that the combination of 25% EG + 25% DMSO and 1.0 M sucrose allowed hatching and expansion of vitrified-warmed bovine embryos produced in vitro.

  11. RECYCLING OF CATHETER FOR EMBRYO RECOVERY: A TOOL FOR COSTS REDUCTION IN EQUINE EMBRYO TRANSFER

    Directory of Open Access Journals (Sweden)

    Alberto Lopes Gusmao

    2010-06-01

    Full Text Available The embryo transfer is becoming a widespread practice.Most embryos are collected from spontaneous single ovulatingmares and result in 50% of embryo recovery, increasing the costsof production. To illustrate, the price of a catheter for embryosrecovery range from US$ 194.00 to US$ 250.00 (R$ 350.00 to R$450.00. Therefore, the aim of this work was to verify if catheterwith damaged balloon can be recuperated and reused withoutaltering its efficiency. For this study, two groups were used: acontrol group (GI, n=10, on which the nonsurgical recovery of theembryos of mares was performed with the catheter with originalballoon; and another group (GII, n=20, in which a restored catheterwas utilized. The mares of GI had an embryo recovery rate of60%, and GII mares had an embryo recovery rate of 55%. Therewas not statistical difference between groups I and II (P>0.05.Considering that the material used to restore the catheter costsUS$16.66 (R$30.00, this data show that the recuperation of thecatheters for embryo recovery in mares may reduce costs withoutcompromising the rates of embryo recovery.

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

    Directory of Open Access Journals (Sweden)

    Tatiane Almeida Drummond Tetzner

    2011-10-01

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

  13. Melatonin inhibits paraquat-induced cell death in bovine preimplantation embryos.

    Science.gov (United States)

    Pang, Yun-Wei; Sun, Ye-Qing; Sun, Wei-Jun; Du, Wei-Hua; Hao, Hai-Sheng; Zhao, Shan-Jiang; Zhu, Hua-Bin

    2016-03-01

    Preimplantation embryos are sensitive to oxidative stress-induced damage that can be caused by reactive oxygen species (ROS) originating from normal embryonic metabolism and/or the external surroundings. Paraquat (PQ), a commonly used pesticide and potent ROS generator, can induce embryotoxicity. The present study aimed to investigate the effects of melatonin on PQ-induced damage during embryonic development in bovine preimplantation embryos. PQ treatment significantly reduced the ability of bovine embryos to develop to the blastocyst stage, and the addition of melatonin markedly reversed the developmental failure caused by PQ (20.9% versus 14.3%). Apoptotic assay showed that melatonin pretreatment did not change the total cell number in blastocysts, but the incidence of apoptotic nuclei and the release of cytochrome c were significantly decreased. Using real-time quantitative polymerase chain reaction analysis, we found that melatonin pre-incubation significantly altered the expression levels of genes associated with redox signaling, particularly by attenuating the transcript level of Txnip and reinforcing the expression of Trx. Furthermore, melatonin pretreatment significantly reduced the expression of the pro-apoptotic caspase-3 and Bax, while the expression of the anti-apoptotic Bcl-2 and XIAP was unaffected. Western blot analysis showed that melatonin protected bovine embryos from PQ-induced damage in a p38-dependent manner, but extracellular signal-regulated kinase (ERK) and c-JUN N-terminal kinase (JNK) did not appear to be involved. Together, these results identify an underlying mechanism by which melatonin enhances the developmental potential of bovine preimplantation embryos under oxidative stress conditions. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Effect of vitrification using the Cryotop method on the gene expression profile of in vitro-produced bovine embryos.

    Science.gov (United States)

    de Oliveira Leme, Ligiane; Dufort, Isabelle; Spricigo, José Felipe Warmling; Braga, Thiago Felipe; Sirard, Marc-André; Franco, Maurício Machaim; Dode, Margot Alves Nunes

    2016-03-01

    The present study analyzed the changes in gene expression induced by the Cryotop vitrification technique in bovine blastocyst-stage embryos, using Agilent EmbryoGENE microarray slides. Bovine in vitro-produced embryos were vitrified and compared with nonvitrified (control) embryos. After vitrification, embryos were warmed and cultured for an additional 4 hours. Survived embryos were used for microarray analysis and quantitative polymerase chain reaction (qPCR) quantification. Survival rates were higher (P vitrification seems to be the activation of the apoptosis pathway in some cells. Indeed, FOSL1 is part of the activating protein 1 transcription factor complex and is implicated in a variety of cellular processes, including proliferation, differentiation, and apoptosis. Therefore, our results suggest that a limited increase in the rate of apoptosis was the only detectable response of the embryos to vitrification stress. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Embryo transfer and related technologies in sheep reproduction.

    Science.gov (United States)

    Loi, P; Ptak, G; Dattena, M; Ledda, S; Naitana, S; Cappai, P

    1998-01-01

    This paper reviews the status of embryo transfer and the major technologies applied to preimplantation of embryos in sheep. Embryo production from superovulated ewes is hindered by an unpredictable response to hormonal treatment. Progress in this area should be expected by an appropriated control of follicular development with gonadotropin-releasing hormone (GnRH) agonist or antagonist prior to gonadotrophin administration. Simple protocols for the cryopreservation of sheep embryos by vitrification are already available and the development of frozen-thawed blastocysts to term is close to the fresh ones. Further research is required to identify factors able to promote the maturation in vitro of oocytes, namely those obtained from prepubertal animals. Semen and embryo sexing procedures are available in cattle although much less attention was paid to their application to sheep. Among all the reproductive technologies, cloning with embryonic and foetal cells has progressed dramatically in sheep and nuclear transfer has been used to produce transgenic animals as an alternative to pronuclear injection. The production of the first lamb cloned from a somatic cell opened new opportunities in animal breeding as well as exciting lines of basic research. The overall conclusions are that, apart from superovulation, the application of in vitro technologies is likely to evolve rapidly and once applied, a great impact on traditional and new animal productions should be expected. However, a better understanding of the changes in gene expression, induced in embryos by different in vitro manipulation procedures, is necessary to prevent abnormal foetal development.

  16. Improved cryopreservation of in vitro-produced bovine embryos using a chemically defined freezing medium.

    Science.gov (United States)

    Bruyère, P; Baudot, A; Guyader-Joly, C; Guérin, P; Louis, G; Buff, S

    2012-10-01

    This study evaluates a new synthetic substitute (CRYO3, Ref. 5617, Stem Alpha, France) for animal-based products in bovine embryo cryopreservation solutions. During the experiment, fetal calf serum (FCS) and bovine serum albumin (BSA) were used as references. A combination of a thermodynamic approach using differential scanning calorimetry and a biological approach using in vitro-produced bovine embryo slow-freezing was used to characterize cryopreservation solutions containing CRYO3, FCS and BSA. The CRYO3 and fetal calf serum (FCS) slow-freezing solutions were made from Dulbecco's phosphate-buffered saline containing 1.5 m ethylene glycol, 0.1 m sucrose and 20% (v.v(-1)) of CRYO3 or FCS. The bovine serum albumin (BSA) solution was made by adding 0.1 m sucrose to a commercial solution containing 1.5 m ethylene glycol and 4 g L(-1) BSA. These solutions were evaluated using three characteristics: the end of melting temperature, the enthalpy of crystallization (thermodynamic approach) and the embryo survival and hatching rates after in vitro culture (biological approach). The CRYO3 and FCS solutions had similar thermodynamic properties. In contrast, the thermodynamic characteristics of the BSA solution were different from those of the FCS and CRYO3 solutions. Nevertheless, the embryo survival and hatching rates obtained with the BSA and FCS solutions were not different. Similar biological properties can thus be obtained with slow freezing solutions that have different physical properties within a defined range. The embryo survival rate after 48 h of in vitro culture obtained with the CRYO3 solution (81.5%) was higher than that obtained with the BSA (42.2%, P = 0.000 12) and FCS solutions (58%, P = 0.016). Similarly, the embryo hatching rate after 72 h of in vitro culture was higher with the CRYO3 solution (61.1%) than with the BSA (31.1%, P = 0.0055) and FCS solutions (36%, P = 0.018). We conclude that CRYO3 can be used as a chemically defined substitute for animal

  17. Developmental, qualitative, and ultrastructural differences between ovine and bovine embryos produced in vivo or in vitro.

    Science.gov (United States)

    Rizos, Dimitrios; Fair, Trudee; Papadopoulos, Serafeim; Boland, Maurice P; Lonergan, Patrick

    2002-07-01

    The objective of this study was to compare bovine and ovine oocytes in terms of (1) developmental rates following maturation, fertilization, and culture in vitro, (2) the quality of blastocysts produced in vitro, assessed in terms of their ability to undergo cryopreservation, and (3) the ultrastructural morphology of these blastocysts. In vitro blastocysts were produced following oocyte maturation/fertilization and culture of presumptive zygotes in synthetic oviduct fluid. In vivo blastocysts were used as a control from both species. In Experiment 1, the cleavage rate of bovine oocytes was significantly higher than that of ovine oocytes (78.3% vs. 58.0%, respectively, P bovine and ovine, respectively, P vitrification, there was no difference in survival between in vivo produced bovine and ovine blastocysts (72 hr: 85.7% vs. 75.0%). However, IVP ovine blastocysts survived at significantly higher rates than IVP bovine blastocysts at all time points (72 hr: 47.4% vs. 18.1%, P bovine IVP blastocysts, which also displayed electron-lucent mitochondria and large intercellular cavities. These observations may in part explain the species differences observed in terms of cryotolerance. In conclusion, the quality of ovine blastocysts was significantly higher than their bovine counterparts produced under identical in vitro conditions suggesting inherent species differences between these two groups affecting embryo quality. Copyright 2002 Wiley-Liss, Inc.

  18. High survival and hatching rates following vitrification of embryos at blastocyst stage: a bovine model study.

    Science.gov (United States)

    Huang, Jack Y J; Chung, Jin-Tae; Tan, Seang Lin; Chian, Ri-Cheng

    2007-04-01

    Cryopreservation of embryos at the blastocyst stage may provide an effective method to increase the cumulative pregnancy rate for each treatment cycle of ovarian-stimulated IVF. The objective of this study was to evaluate the survival rate and hatching rate of bovine blastocysts following vitrification using a method designed for oocytes, with a view to introducing this methodology into human assisted reproduction technology and reproductive medicine. Bovine blastocysts were produced from abattoir materials subjected to in-vitro maturation and in-vitro fertilization. Survival rate of the bovine blastocysts was 100% (94/94) following vitrification using a method designed for oocyte cryopreservation. There was no difference in the hatching rate of the bovine blastocysts between control (62.5%: 60/96) and vitrified (61.7%: 58/94) groups. The number of dead cells in the blastocysts was not significantly different between control (5.0 +/- 2.9) and vitrified (9.5 +/- 4.0) groups. In conclusion, the results of this study indicate that bovine blastocysts can be vitrified successfully using a procedure designed for oocyte cryopreservation. It is possible that this method may also be successful for the cryopreservation of human embryos. A further study into this is currently being organized.

  19. Serum free embryo culture medium improves in vitro survival of bovine blastocysts to vitrification

    OpenAIRE

    Gómez, E.; Rodríguez, A; Muñoz, M.; Caamaño, J.N. (José); Hidalgo, C.O. (Carlos); Morán, E.; Facal, Nieves; Díez, C.

    2013-01-01

    The aim of this study was to examine the effects of co-culture with Vero cells during the in vitro maturation (IVM) and three culture media, B2+5% fetal calf serum (FCS) on Vero cells, synthetic oviduct fluid (SOF)+5% FCS, and SOF+20 gL(-1) bovine serum albumin (BSA), on the developmental competence of the embryos and their ability to survive vitrification/warming. We also tested the effect of morphological quality and the age of the embryo on its sensitivity to vitrification. The IVM system ...

  20. Transfer of European mink (Mustela lutreola) embryos into hybrid recipients.

    Science.gov (United States)

    Amstislavsky, Sergei; Aalto, Jussi; Järvinen, Mikko; Lindeberg, Heli; Valtonen, Maija; Zudova, Galina; Ternovskaya, Yulia

    2004-08-01

    The European mink is considered as a highly endangered Mustelidae species. The objective of this study was to explore the intriguing possibility of embryo transfer from European mink to closely related Mustelidae recipient females. To overcome interspecies pregnancy failure, embryos of European mink (Mustela lutreola) were transferred into hybrid females obtained after mating of European polecat (Mustela putorius) males and European mink (M. lutreola) females and vice versa. A total of 32 blastocysts were surgically flushed from the uteri of nine European mink donors and surgically transferred into six pseudopregnant hybrid recipients. One of the recipients received a single embryo and did not whelp. The remaining five recipients each received five to eight embryos and delivered kits. The overall success rate was 50% (16 kits/32 transferred embryos). For both male and female offspring, the average birth weight was lower in ET group when compared with naturally bred control population of European mink. The postnatal mortality rate was significantly higher in ET group as compared to controls: only 9 of 16 kits survived past the first week. At 10 days of age, the average weight for male offspring from the ET and control groups did not differ, although differences still persisted at this age for female offspring. At 3 months of age, the weight of male and female offspring in the ET group did not differ from European minks born after natural mating. We propose that transfer of European mink embryos to hybrid recipients be considered as a new experimental tool within the framework of ex situ approach conservation of this aboriginal European mustelid.

  1. Survival of vitrified in vitro-produced bovine embryos after a one-step warming in-straw cryoprotectant dilution procedure.

    Science.gov (United States)

    Caamaño, J N; Gómez, E; Trigal, B; Muñoz, M; Carrocera, S; Martín, D; Díez, C

    2015-03-15

    Vitrification is an alternative to slow-rate freezing for cryopreserving bovine embryos. However, this technology requires simplification if it is to be used under field conditions. The main objective of this work was to develop a new system for the direct transfer of vitrified embryos to be used under farm conditions. For this, three objectives were set: (1) to compare the effect of vitrification, using the cryologic vitrification method (CVM), and slow-rate freezing on bovine embryo development and quality; (2) to develop a one-step warming procedure for bovine in vitro-produced (IVP) vitrified (by CVM) embryos; and (3) to assess the effects on embryo survival of a new method for the direct transfer of vitrified IVP bovine blastocysts. In vitro-produced blastocysts were initially either vitrified by CVM or subjected to slow freezing to compare embryo survival and quality (experiment 1). No differences were detected between these cryopreservation techniques in terms of the survival and quality variables at 24 hours or in terms of the proteins expressed. However, at 48 hours the vitrified embryos showed higher hatching rates, greater total cell numbers, and lower apoptotic indices (P < 0.05). In experiment 2, CVM-vitrified IVP blastocysts were warmed by the conventional two-step or one-step warming procedure by incubating them at 41 °C in 0.25 M sucrose for 10 minutes, 0.15 M sucrose for 10 minutes, or 0.25 M sucrose for 5 minutes. In addition, embryo transfer (ET) was performed using vitrified embryos warmed by the one-step procedure in 0.25 M sucrose solution for 5 minutes. As a control group, IVP fresh embryos were transferred to recipient females. No differences were observed in embryo survival or total cell number between any of the warming procedures. Moreover, no significant differences for pregnancy at 60 days were found between the ET groups. In experiment 3, expanded IVP blastocysts were then either vitrified using a conventional or a

  2. 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.

  3. 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.

  4. 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 mRNA...... 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...... 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...

  5. TWIN PREGNANCY AFTER THE TRANSFER OF TWO EMBRYOS

    Directory of Open Access Journals (Sweden)

    Lili Bačer Kermavner

    2003-12-01

    Full Text Available Background. Twin pregnancy rates following the transfer of two embryos in in vitro fertilization (IVF treatment are high. Therefore, the aim of this study was to evaluate twin pregnancy rates in regard to the developmental stage of the embryo, patient age, and cause of infertility.Methods. A retrospective analysis involved 1222 IVF procedures. Overall pregnancy rates and twin pregnancy rates following the transfer of two blastocysts or two cleavage-stage embryos were assessed. The blastocyst group was divided into four age subgroups, and into four subgroups by the cause of infertility.Results. After the transfer of two blastocysts or two cleavagestage embryos the overall pregnancy rates (45% vs. 9% and the twin pregnancy rates (18% vs. 5% were significantly higher in the blastocyst group. The effects of patient age and the transfer of two blastocysts on the overall pregnancy and twin pregnancy rates were significantly greater in the subgroups of patients aged ≤ 30 years (21% and 31–34 years (20% than in older patients (35–38 years = 15.7%; ≥ 39 years = 9%. In the blastocyst group the twin pregnancy rate was significantly higher in patients with tubal (21% and endocrinological causes of infertility (24.5% than in patients with endometriosis (7% and uterine malformations (12.5%.Conclusions. After the transfer of two blastocysts twin pregnancy rates are significantly higher in patients with tubal factor or endocrinological causes of infertility younger than 34 years. In this population of women the risk of twin pregnancy can be avoided by the transfer of a single embryo – most developed blastocyst.

  6. Differential glycolytic and glycogenogenic transduction pathways in male and female bovine embryos produced in vitro.

    Science.gov (United States)

    Garcia-Herreros, M; Aparicio, I M; Rath, D; Fair, T; Lonergan, P

    2012-01-01

    Previous studies have shown that developmental kinetic rates following IVF are lower in female than in male blastocysts and that this may be related to differences in glucose metabolism. In addition, an inhibition of phosphatidylinositol 3-kinase (PI3-K) inhibits glucose uptake in murine blastocysts. Therefore, the aim of this study was to identify and compare the expression of proteins involved in glucose metabolism (hexokinase-I, HK-I; phosphofructokinase-1, PFK-1; pyruvate kinase 1/2, PK1/2; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; glucose transporter-1, GLUT-1; and glycogen synthase kinase-3, GSK-3) in male and female bovine blastocysts to determine whether PI3-K has a role in the regulation of the expression of these proteins. Hexokinase-I, PFK-1, PK1/2, GAPDH and GLUT-1 were present in bovine embryos. Protein expression of these proteins and GSK-3 was significantly higher in male compared with female blastocysts. Inhibition of PI3-K with LY294002 significantly decreased the expression of HK-I, PFK-1, GAPDH, GSK-3A/B and GLUT-1. Results showed that the expression of glycolytic proteins HK-I, PFK-1, GAPDH and PK1/2, and the transporters GLUT-1 and GSK-3 is regulated by PI3-K in bovine blastocysts. Moreover, the differential protein expression observed between male and female blastocysts might explain the faster developmental kinetics seen in males, as the expression of main proteins involved in glycolysis and glycogenogenesis was significantly higher in male than female bovine embryos and also could explain the sensitivity of male embryos to a high concentration of glucose, as a positive correlation between GLUT-1 expression and glucose uptake in embryos has been demonstrated.

  7. Debating Elective Single Embryo Transfer after in vitro Fertilization ...

    African Journals Online (AJOL)

    Assisted reproductive technology (ART) has restored hope to millions of infertile couples globally, with In vitro fertilization (IVF) and embryo transfer offered in virtually all. ART units. This development in medicine lead to the delivery of the first test tube baby named Louise Brown on July 25,. 1978 in Oldham, England after a ...

  8. Debating elective single embryo transfer after in vitro fertilization: a ...

    African Journals Online (AJOL)

    The number of embryos transferred after in vitro fertilization (IVF) have been a topic of debate for over a decade now. Due to the risk associated with multiple pregnancy, there has been a global effort at reducing the multiple pregnancy rates to a minimum while maintaining an acceptable level of successful IVF pregnancy ...

  9. Patients' Preference for Number of Embryos Transferred During IVF ...

    African Journals Online (AJOL)

    Objective: To determine the number of embryos patients' attending a fertility clinic in Nigeria, would prefer transferred during IVF/ICSI. Materials and Methods: Fifty four consecutive female patients who underwent IVF/ICSI procedures between May 2006 and April 2007 at the Port Harcourt Fertility Centre, Rivers State were ...

  10. 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.

  11. Embryo transfer in competition horses: Managing mares and expectations

    Science.gov (United States)

    Campbell, M L H

    2014-01-01

    Embryo transfer (ET) is an accepted and successful technique for obtaining foals from mares without interrupting their competition careers. Recent research, however, suggests that the potential of factors including heat, exercise, repeated embryo flushing and repeated manipulation of the reproductive cycle using exogenous hormones to have a negative impact on fertility may have been underestimated. This paper reviews the evidence base for involvement of these factors in repeated failures to recover embryos from nongeriatric competition mares without obvious clinical or pathological indications of reproductive abnormalities. It concludes that, for some mares at least, a cessation of exercise for the periovulatory period and the period between ovulation and embryo flushing, combined with careful management of flushing-induced endometritis, and minimal hormonal manipulation of the reproductive cycle, may be necessary to optimise embryo recovery rates. Mare owners may have been encouraged to request ET for their mares following high-profile examples in the media of elite mares that have produced foals by ET whilst competing. The veterinarian should educate mare owners about the multiple factors that may affect the chances of recovering an embryo from their mares, and should manage the expectations of mare owners so that they do not approach ET programmes in the expectation that there will be no disruption to their training and competition plans. PMID:25977596

  12. Evaluation of different culture systems on the in vitro production of bovine embryos.

    Science.gov (United States)

    Pereira, Daniela Costa; Dode, Margot Alves Nunes; Rumpf, Rodolfo

    2005-03-01

    The objective of this study was to determine the development potential and quality of in vitro produced bovine embryos cultured individually or in groups. After IVM and IVF, presumptive zygotes were cultured in groups or individually, either in drops or in the modified "well of the well" (mWOW) system. In Experiment 1, four culture systems were utilized: T1: drop in group (control); T2: mWOW in groups; T3: mWOW individually; and T4: drop individually. Cleavage and blastocyst rates at Days 6, 7 and 8 and total cell number of Day 6 blastocysts were similar (P > 0.05) for all treatments. However, in Day 7 blastocysts, total cell number was lower (P embryos cultured individually in a small drop than those cultured in the mWOW. In Experiment 2, blastocysts of T1, T2 and T3 were allocated into two groups, control and vitrified. After warming, the vitrified embryos were cultured for 72 h. At 48 h, the development of the Days 6 and 7 embryos was similar (P > 0.05) for all treatments in the control group. For the vitrified embryos, lower hatching rates (P embryos cultured in groups in the mWOW system had the same blastocyst rates but better quality (measured by their survival after vitrification) than those cultured individually in the mWOW system.

  13. Crucial surviving aspects for vitrified in vitro-produced bovine embryos.

    Science.gov (United States)

    Sudano, Mateus José; Paschoal, Daniela Martins; da Silva Rascado, Tatiana; Crocomo, Letícia Ferrari; Magalhães, Luis Carlos Oña; Junior, Alício Martins; Machado, Rui; da Cruz Landim-Alvarenga, Fernanda

    2014-05-01

    The objective of the present study was to correlate some parameters (cleavage, blastocyst production, quality degree score, total cell number, fresh apoptosis and lipid content) with embryo survival after cryopreservation. A total of 1727 in vitro-produced bovine blastocysts were used to establish the parameters (mean ± standard error of the mean (SEM)) for cleavage (85.6 ± 0.8), blastocyst production (39.9 ± 1.4), quality degree score (1.6 ± 0.1), total cell number (140.1 ± 2.9), fresh apoptosis (20.8 ± 1.1) and lipid content (21.3 ± 0.8 droplets). On the same way 1316 blastocysts were vitrified for the determination of post-cryopreservation embryo survival (49.4 ± 1.9). Fresh apoptosis rate and total lipid droplets value were correlated (P embryo survival after cryopreservation (r = 0.91 and r = 0.59; respectively). However, cleavage, blastocyst production, quality degree score and total cell number were not correlated (P > 0.05) with embryo cryotolerance (r = 0.23, r = 0.38, r = 0.22 and r = 0.28; respectively). Therefore, the increased lipid content was moderately correlated with apoptosis in vitrified blastocysts. On the other hand, increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts, which indicated that the apoptosis rate in fresh embryos was a better parameter than the lipid content to predict post-vitrification embryo survival.

  14. Role of ooplasm in nuclear and nucleolar remodeling of intergeneric somatic cell nuclear transfer embryos during the first cell cycle

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, Frantisek; Petrovicova, Ida

    2011-01-01

    Initially, development of the zygote is under control of the oocyte ooplasm. However, it is presently unknown if and to what extent is the ooplasm able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). Here, one-cell...... intergeneric SCNT embryos were compared to their parthenogenetic counterparts to assess the effects of the introduced somatic cell. Despite the absence of morphological remodeling (premature chromatin condensation, nuclear envelope breakdown), reconstructed embryos showed nuclear and nucleolar precursor body...... (NPB) morphology similar to the host ooplasm, which, together with detected posttranslational activity of somatic cell introduced into the bovine ooplasm, suggests a universal function of ooplasmic factors. However, the lack of distinct UBF localization in intergeneric embryos indicates failures...

  15. Kinetics data from bovine sex-specific embryo development from three different bulls

    Directory of Open Access Journals (Sweden)

    C.S. Oliveira

    2016-06-01

    Full Text Available Here we present kinetics data from bovine sex-specific embryo development. Embryos were originated using sex-sorted semen from three different Nelore bulls, and semen from the same batch was used for X-and Y-chromosome spermatozoa sorting. Data was obtained for six time points (24, 48, 96, 120, and 144 h.p.i.. Analyses for each bull׳s embryos (1, 2 and 3 is presented for female and male groups separately. Also, grouped data analysis, considering bull and sex interaction, is shown. For further interpretation and discussion, see "Cell death is involved in sexual dimorphism during preimplantation development" (Oliveira et al., 2015 [1].

  16. 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.

  17. Gene transfer into mouse prepancreatic endoderm by whole embryo electroporation.

    Science.gov (United States)

    Pierreux, Christophe E; Poll, Aurélie V; Jacquemin, Patrick; Lemaigre, Frédéric P; Rousseau, Guy G

    2005-03-10

    Understanding gene function in the developing pancreas is a major issue for pancreatic cell therapy. The in vivo analysis of gene function has essentially been performed by modulating gene expression in transgenesis. A faster and easier method is electroporation of mouse embryos. This technique, coupled with whole embryo culture, enables one to deliver genes and analyze their effects in a spatially and temporally regulated manner. We wanted to adapt the electroporation technique for gene transfer of whole e8.5 mouse embryos into the endoderm to allow expression of transgenes in the pancreas or liver. Using two platinum plate electrodes, low voltage and a precise positioning of the embryo in the electroporation cuvette we could target and express DNA constructs in the prepancreatic or prehepatic territories, identified with cell markers. We also demonstrated that this technique is a valuable tool in the study of transcriptional regulation in the developing endoderm. Targeted electroporation of whole embryos is a useful method of characterizing the gene network which controls pancreatic development.

  18. The association between embryo quality and perinatal outcome of singletons born after single embryo transfers: a pilot study.

    Science.gov (United States)

    Oron, Galia; Son, Weon-Young; Buckett, William; Tulandi, Togas; Holzer, Hananel

    2014-07-01

    Does the quality of a single transferred embryo have an effect on the pregnancy outcome? After adjusting for confounding maternal variables, poor embryo quality was not associated with adverse obstetric or perinatal outcome in this small pilot study. Embryo quality is a major predictor of the success of in vitro fertilization treatment and studies have demonstrated a strong association between embryo morphology, implantation and clinical pregnancy rates. However, the association with obstetric and perinatal outcomes has not been evaluated. This single center, retrospective cohort study included 1541 fresh single embryo transfers (SETs) using non-donor oocytes in women ≤40 years between December 2008 and 2012. We compared the cycle outcome and singleton live births resulting from the transfer of a single fresh good quality (Grade 2) embryo with those resulting from the transfer of a single poor quality (fair, Grade 3 or poor, Grade 4) embryo in the cleavage or blastocyst stages. The cycle outcome parameters were biochemical pregnancy and clinical intrauterine pregnancy. The pregnancy outcomes were live birth, miscarriages and stillbirths after 20 weeks of gestation. Among the live births, perinatal outcome parameters included birthweight, small for gestational age, preterm delivery, pre-eclampsia, placental abruption and neonatal complications. Covariates were maternal age, body mass index, smoking status, parity and gender of the baby. There were 1193 good quality SETs and 348 poor quality embryo transfers. SETs performed during the study period resulted in 563 pregnancies and 440 singleton births. There was a higher clinical pregnancy rate (41.5%) and live birth rate (32.3%) in the good quality embryo transfer group compared with that in the poor quality transfer group (19.2 and 15.5%, respectively; P quality embryo. Multivariable logistic regression analyses for pregnancy complications revealed no increased risk of maternal or neonatal complications with the

  19. 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

    The bovine blastocyst hatches 8 to 9 days after fertilization, and this is followed by several days of preimplantation development during which the embryo transforms from a spherical over an ovoid to an elongated shape. As the spherical embryo enlarges, the cells of the inner cell mass differenti......The bovine blastocyst hatches 8 to 9 days after fertilization, and this is followed by several days of preimplantation development during which the embryo transforms from a spherical over an ovoid to an elongated shape. As the spherical embryo enlarges, the cells of the inner cell mass...... fluid and cellular components were isolated from 12 ovoid and three elongated embryos and using nano-high-performance liquid chromatography, tandem mass spectrometry, and isobaric tag for relative and absolute quantitation proteomic analysis, a total of 9652 unique proteins were identified. We performed...

  20. Adherence compounds in embryo transfer media for assisted reproductive technologies.

    Science.gov (United States)

    Bontekoe, Stephan; Heineman, Maas Jan; Johnson, Neil; Blake, Debbie

    2014-02-25

    This is an update of a Cochrane review first published in The Cochrane Library (2010, Issue 7).To increase the success rate of assisted reproductive technologies (ART), adherence compounds such as hyaluronic acid (HA) and fibrin sealant have been introduced into subfertility management. Adherence compounds are added to the embryo transfer medium to increase the likelihood of embryo implantation, with the potential for higher clinical pregnancy and live birth rates. To determine whether embryo transfer media containing adherence compounds improved live birth and pregnancy rates in ART cycles. The Menstrual Disorders and Subfertility Group Trials Register, the Cochrane Central Register of Controlled Trials (CENTRAL) and MEDLINE, EMBASE and PsycINFO electronic databases were searched (up to 13 November 2013) to look for publications that described randomised controlled trials on the addition of adherence compounds to embryo transfer media. Furthermore, reference lists of all obtained studies were checked, and conference abstracts were handsearched. Only truly randomised controlled trials comparing embryo transfer media containing functional (e.g. 0.5 mg/ml HA) concentrations of adherence compounds versus transfer media containing low or no concentrations of adherence compounds were included. The adherence compounds that were identified for evaluation were HA and fibrin sealant. Two review authors selected trials for inclusion according to the above criteria, after which two review authors independently extracted the data for subsequent analysis. Statistical analysis was performed in accordance with the guidelines developed by The Cochrane Collaboration. Seventeen studies with a total of 3898 participants were analysed. One studied fibrin sealant, and the other 16 studied HA. No evidence was found of a treatment effect of fibrin sealant as an adherence compound. For HA, evidence of a positive treatment effect was identified in the six trials that reported live birth

  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. Relation between physical properties of the zona pellucida and viability of bovine embryos after slow-freezing and vitrification.

    Science.gov (United States)

    Moreira da Silva, F; Metelo, R

    2005-06-01

    In vitro-produced bovine morulae/blastocyst embryos (n = 119) were slow-frozen and vitrified and the physical alterations of the zona pellucida (ZP) was observed by scanning electron microscopy (SEM) to find an explanation for the loss of developmental capacity of the embryos after freezing/thawing. A control group was provided, in which embryos (n = 38) were neither frozen nor vitrified. Embryos were in vitro-cultured in a standard CO2 Heraeus incubator and their viability was assessed 24 and 48 h after the start of culture, evaluating their morphological aspect. After 24 h of culture, embryo survival rate for slow-freezing/thawed (n = 23), vitrified/thawed (n = 20) and control embryos (n = 20) was 39, 27 and 90%, and 35, 14 and 65% after 48 h of culture, respectively. For evaluation of physical changes occurring in ZP, 20 embryos were slow-frozen, 18 were vitrified and 18 were used as control. All embryos were fixed, dried and examined under an SEM. Embryo's diameter, as well as the number of pores and their diameter was measured in squares of 6.4 microm width. We observed that, on average, the diameter of the embryos (92.26 +/- 10.15 microm) did not differ significantly among all embryos. As far as the diameter of the pores in the outer surface of the ZP is concerned, the results revealed a significant difference (P embryos. For the number of pores, statistical differences (p embryos (45.4 +/- 7.3 vs 38.2 +/- 8.2). It is possible that ZP functions as a barrier which is positive when dealing with pathogens, but is harmful when nutrients were supplied from the outside, especially at 48 h of culture. Results indicate that the steps of cryopreservation cause alterations in ZP, with irreversible damage on the further developmental competence of bovine embryos.

  3. Forskolin effect on the cryosurvival of in vitro-produced bovine embryos in the presence or absence of fetal calf serum.

    Science.gov (United States)

    Paschoal, Daniela Martins; Sudano, Mateus José; Guastali, Midyan Daroz; Dias Maziero, Rosiára Rosária; Crocomo, Letícia Ferrari; Oña Magalhães, Luis Carlos; da Silva Rascado, Tatiana; Martins, Alicio; da Cruz Landim-Alvarenga, Fernanda

    2014-05-01

    The objective of this study was to assess the viability and cryotolerance of zebu embryos produced in vitro with or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos produced in vivo were used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 μM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared with in vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P vitrification.

  4. Cellular alteration after dilution of cryoprotective solutions used for the vitrification of in vitro-produced bovine embryos.

    Science.gov (United States)

    Kaidi, S; Van Langendonckt, A; Massip, A; Dessy, F; Donnay, I

    1999-08-01

    Embryo quality of in vitro-produced bovine blastocysts was assessed at several steps of a vitrification procedure in which glycerol and ethylene glycol were used as cryoprotectants (3-step equilibration with cryoprotectants followed by vitrification, dilution of the cryoprotectants in 0.85 M galactose then in embryo transfer freezing medium [ETF], and finally co-culture for periods). To visualize cell membrane alterations, double staining was performed using a cell permeant fluorochrome (bisbenzimide--BIS) and a nonpermeant one (propidium iodide--PI). In Experiment 1, the effect of the vitrification procedure on the hatching rate and total cell number was assessed 72 h after treatment. Hatching rate and the number of stained nuclei were decreased in comparison with untreated embryos when blastocysts were exposed to the whole procedure with or without vitrification (respectively 42 and 53% vs 76% for hatching and 128 +/- 17 and 141 +/- 17 vs 226 +/- 13 for stained nuclei). In Experiment 2, the effect of cryoprotectants and their dilution was evaluated on membrane permeability and total cell numbers at various steps of the vitrification procedure. Blastocysts exposed only to cryoprotectant solutions and stained immediately after dilution in galactose showed no modification. After dilution in ETF, the total number of stained nuclei decreased, and the number of blastomeres showing membrane permeabilization (PI-stained) increased (respectively, 74 +/- 5 vs 110 +/- 5 and 32 +/- 2% vs 0.1 +/- 1.8%). In Experiment 3, we demonstrated that the total number of stained nuclei after ethanol fixation (membrane permeabilization) was higher when embryos treated up to dilution in ETF were stained with PI than when the same embryos were stained with BIS. This suggests that, for unknown reasons, some nuclei of the treated embryos were not stained with BIS. Membrane permeabilization and inability of BIS to stain some nuclei were the most obvious alterations probably induced by osmotic

  5. Vitrification of in vitro-produced bovine embryos matured inmodified TCM-199 medium

    OpenAIRE

    SANDAL, ASİYE İZEM; ÖZDAŞ, Özen Banu

    2015-01-01

    In vitro-produced bovine embryos matured in modified Tissue Culture Medium 199 (TCM-199) were vitrified at the 7th and 8th days of culture, and development at 48 h after warming was recorded. Ovaries were obtained from a local abattoir, and the oocytes were recovered by slicing method and divided into two main groups. The first group included cysteamine in the TCM-199 (Group A) and the second did not (Group B). They were incubated for 24 h at 38.8 °C in an atmosphere of 5% CO2 in humidified a...

  6. Effect of bovine oviductal extracellular vesicles on embryo development and quality in vitro.

    Science.gov (United States)

    Lopera-Vasquez, Ricaurte; Hamdi, Meriem; Maillo, Veronica; Gutierrez-Adan, Alfonso; Bermejo-Alvarez, Pablo; Ramírez, Miguel Ángel; Yáñez-Mó, María; Rizos, Dimitrios

    2017-04-01

    The aim of this study was to evaluate the effect of extracellular vesicles (EV) from oviductal fluid (OF), either from the ampulla or isthmus, on the development and quality of in vitro-cultured bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF + 3 mg/mL BSA) without calf serum (C- group), in the presence of 3 × 105 EV/mL from ampullary or isthmic OF at either 1 × 104 g (10 K) or 1 × 105 g (100 K), and compared with SOF + 5% FCS (C+ group). OF-EV size and concentration were assessed by electron microscopy and nanotracking analysis system. Embryo development was recorded on Days 7-9, and blastocyst quality was assessed through cryotolerance and gene expression analysis. Lower blastocyst yield was observed on Day 7 in the C- and OF-EV groups (12.0-14.3%) compared with C+ (20.6%); however, these differences were compensated at Days 8 and 9 (Day 9: 28.5-30.8%). Importantly, the survival rate of blastocysts produced with isthmic 100 K OF-EV was higher than that of C+ and C- group at 72 h after vitrification and warming (80.1 vs 34.5 and 50.5% respectively, P produced in the presence of 100 K isthmic OF-EV upregulated the water channel AQP3 and DNMT3A and SNRPN transcripts compared with the C+, with the expression in C- being intermediate. The lipid receptor LDLR was downregulated in C+ compared with all other groups. In conclusion, the addition of oviductal fluid extracellular vesicles from isthmus, to in vitro culture of bovine embryos in the absence of serum improves the development and quality of the embryos produced. © 2017 Society for Reproduction and Fertility.

  7. Expression and distribution of cell adhesion-related proteins in bovine parthenogenetic embryos: The effects of oocyte vitrification.

    Science.gov (United States)

    Zeng, Yan; Fu, Xiangwei; Zhou, Guangbin; Yue, Mingxing; Zhou, Yanhua; Zhu, Shien

    2013-07-01

    The objective was to investigate expression of cell adhesion-related proteins (E-cadherin, β-catenin, and the cytoskeletal protein F-actin) in bovine parthenogenetic embryos derived from vitrified-warmed oocytes. Bovine oocytes at metaphase II were randomly allocated into three groups: (1) untreated (control); (2) exposed to vitrification solution without freezing (toxicity); and (3) vitrified and warmed by the open-pulled straw method (vitrification). After parthenogenetic activation, in the vitrification group compared with the control, the timing of compaction was delayed in (108-120 vs. 96-108 hours, respectively), and the percentage of blastocysts that developed from eight-cell embryos was lower (32.08% vs. 61.03%; P vitrification delayed embryo compaction by affecting adhesion junction formation and function, immunostaining and quantitative reverse transcription polymerase chain reaction were done to characterize distribution patterns (E-cadherin, β-catenin, and the cytoskeletal protein F-actin) and expression levels of cell adhesion-related proteins (β-catenin). Distribution of β-catenin in eight-cell embryos from the vitrification group changed dramatically compared with the control and toxicity groups. Relative expression of β-catenin at the mRNA and protein levels was lower (P bovine parthenogenetic eight-cell embryos derived from vitrified-warmed oocytes were associated with embryo compaction and reduced competence for subsequent embryo development. Copyright © 2013 Elsevier Inc. All rights reserved.

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

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    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.

  9. 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 vitro than in embryos derived in 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 vitro procedures exacerbate chromosomal abnormalities during early embryo development. Hence, the present study highlights that IVF treatment compromises embryo viability and should be

  10. Influence of embryo handling and transfer method on pig cloning efficiency.

    Science.gov (United States)

    Shi, Junsong; Zhou, Rong; Luo, Lvhua; Mai, Ranbiao; Zeng, Haiyu; He, Xiaoyan; Liu, Dewu; Zeng, Fang; Cai, Gengyuan; Ji, Hongmei; Tang, Fei; Wang, Qinglai; Wu, Zhenfang; Li, Zicong

    2015-03-01

    The somatic cell nuclear transfer (SCNT) technique could be used to produce genetically superior or genetically engineered cloned pigs that have wide application in agriculture and bioscience research. However, the efficiency of porcine SCNT currently is very low. Embryo transfer (ET) is a key step for the success of SCNT. In this study, the effects of several ET-related factors, including cloned embryo culture time, recipient's ovulation status, co-transferred helper embryos and ET position, on the success rate of pig cloning were investigated. The results indicated that transfer of cloned embryos cultured for a longer time (22-24h vs. 4-6h) into pre-ovulatory sows decreased recipient's pregnancy rate and farrowing rate, and use of pre-ovulatory and post-ovulatory sows as recipients for SCNT embryos cultured for 22-24h resulted in a similar porcine SCNT efficiency. Use of insemination-produced in vivo fertilized, parthenogenetically activated and in vitro fertilized embryos as helper embryos to establish and/or maintain pregnancy of SCNT embryos recipients could not improve the success rate of porcine SCNT. Transfer of cloned embryos into double oviducts of surrogates significantly increased pregnancy rate as well as farrowing rate of recipients, and the developmental rate of transferred cloned embryos, as compared to unilateral oviduct transfer. This study provided useful information for optimization of the embryo handling and transfer protocol, which will help to improve the ability to generate cloned pigs. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Effects of flunixin meglumine and prostaglandin F2 α treatments on the development and quality of bovine embryos in vitro.

    Science.gov (United States)

    Kim, S-S; Bang, J-I; Fakruzzaman, M; Lee, K-L; Ko, D-H; Ghanem, N; Wang, Z; Kong, I-K

    2014-12-01

    Assisted reproduction procedures, such as embryo transfer (ET) and artificial insemination (AI), in cattle could induce the secretion of prostaglandin F2 -alpha (PGF2 α) from uterine horns which may in turn interrupt embryo development and implantation. This study investigated the effect of flunixin meglumine (FM), prostaglandin F2 alpha (PGF2α) and FM combined with PGF2α supplementation in culture medium (IVC-II) on the development and quality of in vitro produced bovine embryos. The development rate of embryos was significantly higher in the FM group (33.3%) than in control (24.3%), PGF2 α (23.9%) and FM + PGF2 α groups (24.5%). The percentage of hatched blastocysts was also higher (p < 0.05) in the FM group (41.2%) than in the control (27.8%) and PGF2 α groups (19.8%). While, there was no significant difference in total cell number in all experimental groups, the number of apoptotic cells was significantly higher in the PGF2 α group (8.2 ± 6.6) than in the control (4.7 ± 3.2), FM (4.7 ± 2.5) and FM + PGF2 α (4.9 ± 3.4) groups. Detected by real-time PCR, secreted vesicle seminal protein 1 (SSLP1) and prostaglandin G/H synthase 2 (PTGS2) gene expression decreased (p < 0.05) in the PGF2 α group. However, SSLP1 and PTGS2 gene expression in the FM + PGF2 α group returned to their baseline levels, similar to the control and FM groups. Caspase 3 (CAPS3) gene expression increased in the PGF2 α group compared with other groups (p < 0.05). In conclusion, addition of FM in vitro culture significantly improved embryo development as well as alleviated the negative impact of PGF2 α. © 2014 Blackwell Verlag GmbH.

  12. Calves born after direct transfer of vitrified bovine in vitro-produced blastocysts derived from vitrified immature oocytes.

    Science.gov (United States)

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

    2008-06-01

    Vitrification has been the method of choice for the cryopreservation of bovine oocytes, as rapid cooling decreases chilling sensitivity. The aim of this study was to determine the in vitro and in vivo survival and the viability of immature oocytes vitrified using super-cooled liquid nitrogen. Immature oocytes were randomly allocated to three groups: (i) non-vitrified control group, (ii) vitrified in normal (-196 degrees C) liquid nitrogen (LN(2)) and (iii) vitrified in super-cooled LN(2) (vitrification containers. Immature oocytes were in vitro-matured, fertilized and cultured to the blastocyst stage. In vitro viability was assessed by cleavage and blastocyst rates on days 2 and 7 of culture respectively. Vitrified blastocysts derived from the immature vitrified oocytes were directly transferred to synchronous recipients. The in vitro embryo development of vitrified immature oocytes was not influenced by the LN(2) state. After direct transfer (one embryo per recipient) of 16 embryos obtained from immature vitrified oocytes (eight from each vitrified group), two healthy calves were born in each group. These results indicated that vitrification of immature bovine oocytes using glass micropipettes under normal or super-cooled LN(2), resulted in viable blastocysts and live calves following in vitro embryo production.

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Paulo B.D. Gonçalves

    2010-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Melissa Meneghel

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

  16. Osmotic challenge and expression of aquaporin 3 and Na/K ATPase genes in bovine embryos produced in vitro.

    Science.gov (United States)

    Camargo, Luiz Sergio Almeida; Boite, Mariana Cortes; Wohlres-Viana, Sabine; Mota, Gustavo Bruno; Serapiao, Raquel Varela; Sa, Wanderlei Ferreira; Viana, Joao Henrique Moreira; Nogueira, Luiz Altamiro Garcia

    2011-12-01

    The aim of this study was to evaluate the effect of culture media and stage of development in the osmotic ability of in vitro-fertilized bovine embryos and the expression of aquaporin 3 (Aqp3) and Na/K ATPase isoform 1 (ATPAse1) genes in embryos (i) with different ability to undergo rehydration and (ii) following vitrification. In experiment 1, in vitro fertilized presumptive zygotes were co-cultured in SOFaac or modified CR2aa medium and embryos at blastocyst and expanded blastocyst stages at day 7 post-insemination were exposed to NaCl hypertonic medium (900 mOsm) for 5 min following 120 min of culture in isotonic medium in order to evaluate dehydration and rehydration, respectively. No difference (P>0.05) on blastocyst rate was found between CR2aa and SOFaac medium but embryos co-cultured in SOFaac medium underwent greater (PEmbryos at expanded blastocyst stage underwent greater dehydration but slower rehydration than embryos at blastocysts stage (P0.05) on relative amount of transcripts was found in either genes. In the experiment 3, expanded blastocysts produced in a co-culture system were vitrified, warmed and then cultured for 72 h for analysis of embryo survival and amount of Aqp3 and ATPase1 transcripts. Lower (Pembryo survival rate was found for vitrified-warmed embryos (57.9%) than for their fresh counterparts (84.6%). There was no difference on expression of ATPase1 gene but lower (Pembryos. In conclusion, embryo ability to undergo shrinkage and swelling is influenced by medium used in a co-culture system and by embryo stage. Rehydrating ability of embryos after exposure to NaCl hypertonic medium is not associated with variations on expression of Aqp3 and ATPase1 genes, but the vitrification can alter gene expression of in vitro-fertilized bovine embryos produced in a co-culture system. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Effects of oocyte vitrification on epigenetic status in early bovine embryos.

    Science.gov (United States)

    Chen, Huanhuan; Zhang, Lei; Deng, Tengfei; Zou, Pengda; Wang, Yongsheng; Quan, Fusheng; Zhang, Yong

    2016-08-01

    Oocyte cryopreservation has a great impact on subsequent embryonic development. Currently, several studies have primarily focused on the consequences of vitrification and the development potential of cellular structures. This study determined whether oocyte vitrification caused epigenetic instabilities of bovine embryos. The effects of oocyte vitrification on DNA methylation, histone modifications, and putative imprinted genes' expression in early embryos derived by intracytoplasmic sperm injection were examined. Results showed that oocyte vitrification did not affect zygote cleavage rates (67.0% vs. 73.8% control, P > 0.05) but reduced the blastocyst rate (9.6% vs. 23.0%, P vitrification group during the early cleavage phases. No differences were observed for DNA methylation, H3K9me3, and acH3K9 in the inner cell mass of blastocysts, whereas decreased levels of DNA methylation and acH3K9 (P vitrification. The expression of putative-imprinted genes PEG10, XIST, and KCNQ1O1T was upregulated in blastocysts. These epigenetic abnormalities may be partially explained by altered expression of genes associated with epigenetic regulations. DNA methylation and H3K9 modification suggest that oocyte vitrification may excessively relax the chromosomes of oocytes and early cleavage embryos. In conclusion, these epigenetic indexes could be used as damage markers of oocyte vitrification during early embryonic development, which offers a new insight to assess oocyte vitrification. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Embryo quality is the main factor affecting cumulative live birth rate after elective single embryo transfer in fresh stimulation cycles.

    Science.gov (United States)

    Niinimäki, Maarit; Veleva, Zdravka; Martikainen, Hannu

    2015-11-01

    The study was aimed to evaluate which factors affect the cumulative live birth rate after elective single embryo transfer in women younger than 36 years. Additionally, number of children in women with more than one delivery per ovum pick-up after fresh elective single embryo transfer and subsequent frozen embryo transfers was assessed. Retrospective cohort study analysing data of a university hospital's infertility clinic in 2001-2010. A total of 739 IVF/ICSI cycles with elective single embryo transfer were included. Analyses were made per ovum pick-up including fresh and subsequent frozen embryo transfers. Factors affecting cumulative live birth rates were examined in uni- and multivariate analyses. A secondary endpoint was the number of children born after all treatments. In the fresh cycles, the live birth rate was 29.2% and the cumulative live birth rate was 51.3%, with a twin rate of 3.4%. In the multivariate analysis, having two (odds ratio (OR) 1.73; 95% confidence interval (CI) 1.12-2.67) or ≥3 top embryos (OR 2.66; 95% CI 1.79-3.95) was associated with higher odds for live birth after fresh and frozen embryo cycles. Age, body mass index, duration of infertility, diagnosis or total gonadotropin dose were not associated with the cumulative live birth rate. In cycles with one top embryo, the cumulative live birth rate was 40.2%, whereas it was 64.1% in those with at least three top embryos. Of women who had a live birth in the fresh cycle, 20.4% had more than one child after all frozen embryo transfers. Among women with three or more top embryos after ovum pick-up, 16.1% gave birth to more than one child. The cumulative live birth rate in this age group varies from 40% to 64% and is dependent on the quality of embryos. Women with three or more top embryos have good chance of having more than one child per ovum pick-up without elevated risk of multiple pregnancies. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

    OpenAIRE

    B. Gasparrini; G. Campanile; D. Vecchio; L. Boccia; L. Attanasio; De Rosa, A.

    2010-01-01

    The aim of this study was to evaluate the possibility to vitrify in vitro produced (IVP) buffalo and bovine embryos at different stages of development by an advanced version of the “minimal volume approaches”: the Cryotop method. In both experiments, the embryos were vitrified at the tight morula (TM), early blastocyst (eBl), blastocyst (Bl), expanded blastocyst (xBl) and, only for buffalo, at the hatched blastocyst (hBl) stage. After warming, the embryos were cultured in vitro fo...

  20. Vitrification of in vitro produced bovine embryos at different ages using one- and three-step addition of cryoprotective additives.

    Science.gov (United States)

    Saha, S; Suzuki, T

    1997-01-01

    The effect of embryo age on development and ratio of live: dead cells after vitrification and warming was examined. One-step and three-step addition of cryoprotectants in vitrification solution (40% ethylene glycol, 0.3 M trehalose and 12% polyvinylpyrrolidone) were compared using in vitro produced (IVP) bovine blastocysts and expanded blastocysts. Rates of development and hatching were 74.2% and 41.9% for Day 7, 57.8% and 23.8% for Day 8, 33.7% and 6.1% for Day 9 embryos with one-step addition. In three-step addition, those rates were 86.2% and 77.3% for Day 7, 72.3% and 39.0% for Day 8, 47.3% and 10.5% for Day 9 embryos. Day 7 embryos showed highest (P embryos with three-step addition was higher (P embryos (P bovine embryos against vitrification and the potential for three-step addition of cryoprotectants to yield a higher survival rate after warming than with one-step addition.

  1. Multiple ovulation and embryo transfer (MOET in camels: An overview

    Directory of Open Access Journals (Sweden)

    Binoy S. Vettical

    2016-04-01

    Full Text Available Unlike in other domestic animal species like cattle, reproductive biotechnologies like Artificial Insemination (AI and Embryo Transfer (ET are not well developed and thus are not being used as routine breeding procedures in camels. One of the important objectives of this manuscript is to focus on analyzing the present status of Multiple Ovulation and Embryo Transfer (MOET in camels and its future perspectives. Camels are induced ovulators, thus require hormonal treatment to induce ovulation and control the follicular cycles, which is the main reason why protocols used in other domestic animal species cannot be directly used in this species. The review suggests that the best method for super stimulation of ovaries in camels is use of a combination of Equine Chorionic Gonadotropin (eCG and Follicle Stimulating Hormone (FSH at any stage after elimination of dominant follicle if any or at the early stage of the follicular wave and ovulation of the developed multiple follicles can be achieved by mating donors. The review highlights that a better pregnancy rate is achieved with recipients who ovulate 24 h after the donor.

  2. 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.

  3. Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification.

    Science.gov (United States)

    Mucci, N; Aller, J; Kaiser, G G; Hozbor, F; Cabodevila, J; Alberio, R H

    2006-05-01

    The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest post-cryopreservation survival rates, regardless of the presence of serum in the culture medium.

  4. Consequences of transfer of an in vitro-produced embryo for the dam and resultant calf.

    Science.gov (United States)

    Bonilla, L; Block, J; Denicol, A C; Hansen, P J

    2014-01-01

    No reports exist on consequences of in vitro production (IVP) of embryos for the postnatal development of the calf or on postparturient function of the dam of the calf. Three hypotheses were evaluated: calves born as a result of transfer of an IVP embryo have reduced neonatal survival and altered postnatal growth, fertility, and milk yield compared with artificial insemination (AI) calves; cows giving birth to IVP calves have lower milk yield and fertility and higher incidence of postparturient disease than cows giving birth to AI calves; and the medium used for IVP affects the incidence of developmental abnormalities. In the first experiment, calves were produced by AI using conventional semen or by embryo transfer (ET) using a fresh or vitrified embryo produced in vitro with X-sorted semen. Gestation length was longer for cows receiving a vitrified embryo than for cows receiving a fresh embryo or AI. The percentage of dams experiencing calving difficulty was higher for ET than AI. We observed a tendency for incidence of retained placenta to be higher for ET than AI but found no significant effect of treatment on incidence of prolapse or metritis, pregnancy rate at first service, services per conception, or any measured characteristic of milk production in the subsequent lactation. Among Holstein heifers produced by AI or ET, treatment had no effect on birth weight but the variance tended to be greater in the ET groups. More Holstein heifer calves tended to be born dead, died, or were euthanized within the first 20d of life for the ET groups than for AI. Similarly, the proportion of Holstein heifer calves that either died or were culled for poor health after 20d of age was greater for the ET groups than for AI. We observed no effect of ET compared with AI on age at first service or on the percentage of heifers pregnant at first service, calf growth, or milk yield or composition in the first 120d in milk of the first lactation. In a second experiment, embryos were

  5. Does the transfer of a poor quality embryo together with a good quality embryo affect the In Vitro Fertilization (IVF) outcome?

    Science.gov (United States)

    Wintner, Eliana Muskin; Hershko-Klement, Anat; Tzadikevitch, Keren; Ghetler, Yehudith; Gonen, Ofer; Wintner, Oren; Shulman, Adrian; Wiser, Amir

    2017-01-13

    IVF cycles which result in only one good quality embryo, and a second poor quality embryo present a dilemma when the decision involves transferring two embryos. The aim of this study was to evaluate whether a poor quality embryo has a negative effect on a good quality embryo when transferred along with a good quality embryo. We retrospectively evaluated in vitro fertilization (IVF) cycles involving single embryo transfers (SET) and double embryo transfers (DET). Embryo quality was divided into poor "P" and good "G" quality. The main outcome measures were: live birth, implantation rate, miscarriage rate, clinical pregnancy rate and multiple pregnancy ratio. Six hundred three women were included. The study group consisted of 180 (29.9%) patients who had a double embryo transfer (DET) with one poor quality embryo and one good quality embryo (P + G). Control 1 group included 303 (50.2%) patients who had DET with two good quality embryos (G + G), and control 2 group consisted of 120 (19.9%) patients who had a single embryo transfer (SET) with one good quality embryo (G). Live birth rates were not significantly different when compared between study groups: 30.8% in the SET group (G), 27.2% in the (G + P) group and 33.7% in the (G + G) group. The SET group had the highest implantation rate (33.9%) compared to the DET groups (21.8% (G + P), 25.4% (G + G)) (P =0.022). The clinical pregnancy rate was 33.3% in the SET group (G), 33.3% in the (G + P) group, and 39.3% in the (G + G) group (P =0.39). The miscarriage rate was comparable in all groups. A poor quality embryo does not negatively affect a good quality embryo, when transferred together in a double embryo transfer.

  6. Impact of the assessment of early cleavage in a single embryo transfer policy.

    Science.gov (United States)

    Emiliani, Serena; Fasano, Giovanna; Vandamme, Brigitte; Vannin, Anne-Sophie; Verdoodt, Miranda; Biramane, Jamila; Delbaere, Anne; Englert, Yvon; Devreker, Fabienne

    2006-08-01

    The policy of single embryo transfer (SET) adopted for women groups. In the first group, early cleavage was assessed (ECA) 25 h after insemination. The embryo with the best score that cleaved early, if present, was selected for transfer. In the second group, early cleavage was not assessed (ECNA) and embryo selection was based solely on the embryo score. Ninety-eight cycles were allocated in the ECA and ECNA group respectively. Early cleavage occurred in 64% of cycles and 32.2% of embryos. Patient population and embryo morphology were similar between the two groups, and similar delivery rates were observed (27.6 versus 24.5% respectively in the ECA and ECNA groups). The assessment of early cleavage as additional parameter did not improve the delivery rate in the single embryo transfer policy.

  7. Quality of embryos transferred and progesterone levels are the most important predictors of live birth after fresh embryo transfer: a retrospective cohort study.

    Science.gov (United States)

    Cai, Qianfang; Wan, Fei; Appleby, Dina; Hu, Linli; Zhang, Hanwang

    2014-02-01

    To identify the independent predictors of live birth following IVF, and to assess the role of cohort-specific parameters, including antral follicle count (AFC), the number of oocytes retrieved, the total number of embryos, and the total number of good-quality embryos, in fresh IVF cycles. A retrospective cohort study of 2,525 infertile women undergoing IVF between 2002 and 2007. The hypothesis that the number and quality of embryos transferred capture the effects previously attributed to cohort-specific variables was examined using mediation analysis and spline analysis. Independent predictors were identified by a bootstrap algorithm. Multivariable logistic regression was performed and the proportion of explained variation was measured to compare the relative importance of transfer-specific vs. cohort-specific predictors. The number of good-quality embryos transferred and progesterone level on the day of hCG administration ranked as the two most important predictors of live birth. Prospects of pregnancy started to decrease after progesterone level exceeded 0.6 ng/ml. The achievement of live birth in a fresh IVF cycle is primarily determined by the number and quality of embryos transferred, rather than by embryo cohort-specific variables. The associations between cohort-specific variables and live birth in a fresh IVF cycle are completely mediated by the quality of embryos transferred. Progesterone level on the day of hCG administration is an independent predictor of pregnancy and merits further investigation.

  8. IVF and embryo transfer: historical origin and development.

    Science.gov (United States)

    Biggers, John D

    2012-08-01

    IVF and embryo transfer for the treatment of human infertility has now resulted in the birth of over 4 million babies. The technique did not arise as a quantum event but was built on the efforts of many earlier workers in the fields of reproductive endocrinology and development. One should remember the famous saying of Isaac Newton: 'If I have seen further than most, it is because I have stood on the shoulder's of giants'. Ethical and moral issues have always arisen when investigators study early mammalian development, particularly human development. This paper documents these earlier studies and also draws attention to the ethical and moral arguments that inevitably arose. Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  9. Successful pregnancy rates achieved with day 4 embryo transfers.

    Science.gov (United States)

    Skorupski, Josh C; Stein, Daniel E; Acholonu, Uchenna; Field, Heather; Keltz, Martin

    2007-04-01

    To assess the success of day 4 embryo transfers (ETs) following IVF at one institution. Retrospective analysis. A university hospital IVF program. Two hundred nondonor, fresh IVF cycles. None. Outcomes of IVF. Outcome assessments after day 4 ETs included rates of implantation, clinical pregnancy, and singleton and multiple live births. The overall live-birth rate was 54.4%. Implantation rates were highest in younger age groups, and similar in patients 35-40 years of age. Pregnancy and live-birth rates were similar across all age groups up to age 40 years. Multiple gestations were highest in women < or =40 years of age. Acceptable pregnancy rates can be achieved with day 4 ETs.

  10. Case Report of Ectopic Ovarian Pregnancy Following Fresh Embryo Transfer.

    Science.gov (United States)

    Samara, Nivin; Bentov, Yaakov

    2016-01-01

    Ovarian pregnancy is a rare and challenging clinical phenomenon. Recent studies have identified assisted reproductive treatments and infertility as risk factors. However, neither a definite mechanism nor clear risk factors were identified and therefore prevention strategies are yet unavailable. In this article, we present a case of ovarian pregnancy occurring following in vitro fertilization treatment and a fresh embryo transfer. The couple was diagnosed with unexplained infertility and no identifiable risk factors for extrauterine pregnancy. The diagnosis of ovarian pregnancy was made during explorative laparoscopy performed due to suspected extrauterine pregnancy. The patient had normal intra- and postoperative course. Ovarian pregnancy is an infrequent and a challenging diagnosis. Yet, late diagnosis and lack of appropriate intervention may have long-term implications. Several mechanisms and risk factors are proposed, and their acknowledgment may improve early diagnosis and prevention of complications.

  11. Oocyte pre-IVM with caffeine improves bovine embryo survival after vitrification.

    Science.gov (United States)

    Bernal-Ulloa, Sandra Milena; Lucas-Hahn, Andrea; Herrmann, Doris; Hadeler, Klaus-Gerd; Aldag, Patrick; Baulain, Ulrich; Niemann, Heiner

    2016-09-15

    Cryopreservation of in vitro produced bovine embryos is associated with significantly reduced survival rates, mainly due to insufficient quality of the embryos. Caffeine supplementation during IVM has been used to delay meiotic resumption and concomitantly also increased embryo quality. Here, we investigated the influence of pre-IVM with caffeine on oocyte maturation, intraoocyte cAMP concentration, developmental competence after IVF, and blastocyst cryotolerance. Oocytes were obtained by slicing of ovaries and were submitted to either 2 hours culture before IVM with or without caffeine (0, 1, 5, 10, 20, 30 mM), or standard IVM (no pre-IVM). Oocytes were in vitro matured and fertilized and zygotes were cultured under standard in vitro conditions until Day 8. Expanded blastocysts derived from either standard control or the 10-mM caffeine treatments were submitted to vitrification. Caffeine delayed meiotic resumption after 9-hour IVM in a concentration-dependent manner. The cAMP levels were similar before and after IVM. Matured oocytes, cleavage, and blastocyst rates were reduced in the 30-mM caffeine concentration and were similar among the other treatment groups. Number and proportion of inner cell mass and trophectoderm cells in blastocysts did not differ among treatments. Forty-eight hours after thawing, hatching rates were higher in the 10-mM caffeine group (73.8%) compared with the standard control (59.7%). Reexpansion rates and total number of cells after 48 hours were similar in both treatments. The ratio of live/total cells was higher in the caffeine treatment. These results suggest that caffeine supplementation before IVM delayed meiotic resumption and improved blastocyst quality shown in higher cryotolerance. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Economic evaluations of single- versus double-embryo transfer in IVF

    NARCIS (Netherlands)

    Fiddelers, A. A. A.; Severens, J. L.; Dirksen, C. D.; Dumoulin, J. C. M.; Land, J. A.; Evers, J. L. H.

    2007-01-01

    Multiple pregnancies lead to complications and induce high costs. The most successful way to decrease multiple pregnancies in IVF is to transfer only one embryo, which might reduce the efficacy of treatment. The objective of this review is to determine which embryo-transfer policy is most

  13. In situ DNA transfer to chicken embryos by biolistics

    Directory of Open Access Journals (Sweden)

    Luciana A. Ribeiro

    1999-12-01

    Full Text Available Fertilized chicken eggs were bombarded with a biolistic device. Transient expression of the lacZ gene under the control of a human cytomegalovirus (CMV promoter was assessed after in situ gene transfer using this approach. The influence of different pressures, vacuum levels and particles was tested. Survival rate improved as particle velocity decreased, but resulted in lower levels of expression. The best survival and expression were obtained with gold particles, a helium gas pressure of 600 psi and a vacuum of 600 mmHg. Under these conditions, all bombarded embryos showed b-galactosidase activity, indicating that this was an effective method for transformation of chicken embryos.Ovos fertilizados de galinha foram bombardeados através da técnica de biobalística. A expressão transiente do gene lacZ, sob o controle do promotor humano citomegalovírus, foi verificada após a transferência in situ. Diferentes níveis de pressão de gás hélio, vácuo e tipos de partículas foram testados. A taxa de sobrevivência aumentou à medida que a velocidade das partículas diminuíram, entretanto, o nível de expressão foi menor. Os melhores resultados, combinando taxa de sobrevivência e expressão, foram obtidos com partículas de ouro, 600 libras por polegada ao quadrado de hélio e 600 mmHg de vácuo. Nestas condições, todos os embriões bombardeados apresentaram atividade da b-galactosidase, indicando que esta técnica é eficiente para a transformação de embriões de galinhas.

  14. Does the number of oocytes retrieved influence pregnancy after fresh embryo transfer?

    Directory of Open Access Journals (Sweden)

    Qianfang Cai

    Full Text Available BACKGROUND: The nature of the association between the number of oocytes retrieved and in vitro fertilization (IVF outcomes after fresh embryo transfer remains unclear because of conflicting results reported in the studies on this subject. In addition, the influence of the quality of the embryos transferred is usually neglected. The objective of this study is to assess the relationships of the number of oocytes retrieved, the number and quality of embryos transferred, and the prospects of pregnancy after fresh embryo transfer. METHODS: The data on 3131 infertile women undergoing their first IVF treatment cycle between January 2009 and December 2010 were collected retrospectively. Restricted cubic splines and stratified analyses were used to explore the relationships between the number of oocytes retrieved, the number and quality of embryos transferred, and the IVF outcomes. RESULTS: When stratified by the number and quality of transferred embryos, no significant differences in the chances for clinical pregnancy and live birth were found in three groups of oocytes yielded (≤6, 7-14, or ≥15. The relationship between the number of oocytes retrieved and pregnancy is nearly a reflection of the pattern of the relationship between the number of oocytes retrieved and the probability of having two good-quality embryos transferred. The patients with the "optimal" number of oocytes were not only younger but also had the highest probability of having two good-quality embryos replaced. CONCLUSIONS: Similarly aged patients have similar pregnancy prospects after fresh embryo transfer when the same number and quality of embryos are replaced, irrespective of their number of oocytes. Selecting the desired number of good-quality embryos for transfer is the key to IVF success. Thus, aiming at retrieving an optimal number of oocytes to maximize IVF outcomes in a fresh cycle could place undue stress on the patients and may not be the best medical decision.

  15. Vesicular transfer of membrane components to bovine epididymal spermatozoa.

    Science.gov (United States)

    Schwarz, A; Wennemuth, G; Post, H; Brandenburger, T; Aumüller, G; Wilhelm, B

    2013-09-01

    Epididymosomes (apocrine secreted epididymal vesicles) are assumed to play a crucial role in sperm maturation. Our aim has been to analyze the fusogenic properties of bovine epididymosomes and their involvement in the transfer of membrane components (lipids, proteins, plasma membrane Ca(2+)-ATPase 4 [PMCA4]) into bovine sperm. The fusogenic properties of epididymosomes with spermatozoa were investigated in vitro by using octadecyl rhodamine-B (R18)-labeled epididymosomes. Spermatozoa isolated from the epididymal caput showed a higher fusion rate than those taken from the cauda. The fusion rate was dependent on pH and time. Furthermore, the lipid and protein content in spermatozoa changed during epididymal transit and after in vitro fusion with epididymosomes. Following the in vitro fusion of caput spermatozoa with epididymosomes, the cholesterol/total phospholipid ratio of the sperm plasma membrane decreased. The effect was comparable with the cholesterol/total phospholipid ratio of native cauda spermatozoa. Co-incubation experiments of spermatozoa with biotinylated epididymosomes additionally revealed that proteins were transferred from epididymosomes to sperm. To examine the potential transfer of epididymis-derived PMCA4 to spermatozoa, immunofluorescence analysis and Ca(2+)-ATPase activity assays were performed. In caput spermatozoa, the PMCA4 fluorescence signal was slightly raised and Ca(2+)-ATPase activity increased after in vitro fusion. Thus, our experiments indicate significant changes in the lipid and protein composition of epididymal sperm following interaction with epididymosomes. Moreover, our results substantiate the presumption that PMCA4 is transferred to spermatozoa via epididymosomes.

  16. Vitrification of in vitro-produced bovine embryos by addition of ethylene glycol in one-step.

    Science.gov (United States)

    Walker, D J; Campos-Chillon, L F; Seidel, G E

    2006-10-01

    The objective of this study was to simplify two-step addition of cryoprotectant for vitrification of bovine embryos by developing a one-step procedure. Survival was calculated as a percentage of non-vitrified controls developed from the same batch of oocytes. In experiment 1, bovine blastocysts were vitrified following one- or two-step addition of cryoprotectant. Exposure of embryos to cryoprotectant in one-step resulted in survival rates not significantly lower (p > 0.1) than those obtained by two-step addition (85% vs 98%, respectively). Based on these results, experiments 2-4 were designed to test one-step addition of cryoprotectant more rigorously. Experiment 2 exposed day 7 blastocysts to 6, 7 or 8 M ethylene glycol for 2.5 or 3.5 min. At 24 h post-vitrification, survival of embryos was similar, irrespective of ethylene glycol concentration or exposure time (6 M 38%, 7 M 51%, 8 M 59%; 2.5 min 54%, 3.5 min 45%). In experiment 3, blastocysts were exposed to 7 M ethylene glycol for shorter times (30 or 60 s); 30 s exposure resulted in decreased survival (8% vs 31%, p bovine morulae, exposed to 7 M ethylene glycol for 1 or 1.5 min. There was no difference in survival between exposure times of 1 or 1.5 min (28% vs 45%, respectively; p > 0.1). It is unclear why many embryos survive vitrification with one-step addition of cryoprotectant, but others do not. Although, one-step addition of cryoprotectant simplifies the vitrification procedure, survival rates were inadequate for routine cryopreservation of in vitro-produced bovine embryos.

  17. L-ergothioneine supplementation during culture improves quality of bovine in vitro-produced embryos.

    Science.gov (United States)

    Zullo, G; Albero, G; Neglia, G; De Canditiis, C; Bifulco, G; Campanile, G; Gasparrini, B

    2016-03-01

    The aim of this study was to evaluate whether supplementation of bovine culture medium with the natural antioxidant L-ergothioneine (LE), improves in vitro blastocyst development and quality, assessed as resistance to cryopreservation, total cells number, cellular differentiation, and apoptosis index. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid with 0, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM of LE (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On the basis of the results of this dose-response trial, the range of concentrations to test was reduced in experiment 2, in which presumptive zygotes were cultured with 0, 0.05 mM, and 0.1 mM of LE. On Day 7, embryo yields were assessed, and the blastocysts (BL) were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO and 0.5 M sucrose. Finally, BL produced on Day 8 in the absence (control) and presence of 0.1 mM LE were used for transferase-mediated dUTP nick end labeling and differential staining to evaluate, respectively the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm lineages (experiment 3). Despite similar blastocyst yields, supplementation of culture medium with 0.1 mM LE improved the cryotolerance of in vitro-produced (IVP) embryos compared to the control group, as indicated by higher (P culture (48.5%, 50.0%, and 63.8%, respectively with 0, 0.05, and 0.1 mM LE). Interestingly, when embryos were cultured in the presence of 0.1 mM LE, the percentage of BL with the most physiological ICM:total cells ratio (20%-40%) increased (85.1 vs. 66.0%, P culture medium with 0.1 mM LE improves embryo quality, as indicated by the improved cryotolerance, the lower apoptotic rate, and the higher percentage of BL with the most physiological ICM:total cells ratio. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Is a Blanket Elective Single Embryo Transfer Policy Defensible?

    Directory of Open Access Journals (Sweden)

    Eli Y. Adashi

    2017-04-01

    Full Text Available For the purpose of reducing maternal and neonatal morbidity, elective single transfer (eSET in in vitro fertilization (IVF was first proposed in 1999. The purpose of this review is to summarize recent oral debate between a proponent and an opponent of expanded eSET utilization in an attempt to determine whether a blanket eSET policy, as is increasingly considered, is defensible. While eSET is preferable when possible, and agreed upon by provider and patient, selective double embryo transfer (DET must be seriously entertained if deemed more appropriate or is desired by the patient. Patient autonomy, let alone prolonged infertility and advancing age, demand nothing less. Importantly, IVF-generated twins represent only 15.7% of the national twin birth rate in the United States. Non-IVF fertility treatments have been identified as the main cause of all multiple births for quite some time. However, educational and regulatory efforts over the last decade, paradoxically, have exclusively only been directed at the practice of IVF, although IVF patient populations are rapidly aging. It is difficult to understand why non-IVF fertility treatments, usually applied to younger women, have so far escaped attention. This debate on eSET utilization in association with IVF may contribute to a redirection of priorities.

  19. Embryo transfer: a comparative biosecurity advantage in international movements of germplasm.

    Science.gov (United States)

    Thibier, M

    2011-04-01

    This paper uses cattle as a model to provide an overview of the hazards involved in the transfer of in vivo-derived and in vitro-produced embryos. While scientific studies in recent decades have led to the identification of pathogens that may be associated with both in vivo- and in vitro-derived embryos, those studies have also been the basis of appropriate disease control measures to reduce the risks to a negligible level. These disease control measures have been identified and assessed by the International Embryo Transfer Society's (lETS) Health and Safety Advisory Committee, the expert body that advises the World Organisation for Animal Health (OIE) on matters related to the safety of embryo transfer. Through the OIE's processes for developing and adopting international standards, the disease control measures identified by the IETS have been incorporated into the Terrestrial Animal Health Code. The basic principles rely on the crucial ethical roles of the embryo collection team and embryo transfer team, under the leadership of approved veterinarians. Decades of experience, with nearly 10 million embryos transferred, have demonstrated the very significant biosecurity advantage that embryo transfer technology has when moving germplasm internationally, provided that the international standards developed by the IETS and adopted by the OIE are strictly followed.

  20. Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos.

    Science.gov (United States)

    Nicacio, Alessandra Corallo; Simões, Renata; de Paula-Lopes, Fabiola Freitas; de Barros, Flavia Regina Oliveira; Peres, Maria Angelica; Assumpção, Mayra Elena Ortiz D'Avila; Visintin, Jose Antonio

    2012-05-01

    The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

  1. Effect of cysteamine supplementation of in vitro matured bovine oocytes on chilling sensitivity and development of embryos.

    Science.gov (United States)

    Balasubramanian, S; Rho, Gyu-Jin

    2007-04-01

    In vitro techniques for production of bovine embryos including in vitro oocyte maturation (IVM), fertilization (IVF) and culture (IVC) are becoming increasingly employed for a variety of research purposes. However, decreased viability following cryopreservation by conventional methods has limited commercial applications of these technologies. A practical alternative to facilitate transport would be to arrest development by chilling without freezing. The present research was undertaken to evaluate chilling sensitivity of IVM-IVF embryos at different stages of development, and to determine possible beneficial effects of cysteamine treatment during IVM, previously shown to enhance embryo development in culture, on survival following chilling at different stages. Embryos produced by standard IVM-IVF-IVC methods were chilled to 0 degrees C for 30 min at 2-cell (30-34 h post-insemination, hpi), 8-cell (48-52 hpi) or blastocyst (166-170 hpi) stages. Viability after chilling was assessed by IVC with development to expanded blastocyst stage determined on days 7 and 8 post-insemination (pi) and hatching blastocyst stage determined on days 9 and 10 pi. Control embryos at the same stages were handled similarly, but without chilling, and development during culture similarly assessed. The effect of cysteamine supplementation (100 microM) of the IVM medium was determined for both chilled and non-chilled (control) embryos. Cysteamine supplementation during IVM had no significant effect on oocyte maturation or fertilization, but increased the proportions of oocytes developing to blastocyst stage by day 7 (13.7+/-0.9% versus 7.2+/-0.9%; Pchilling of blastocysts produced by IVM-IVF of oocytes matured in media supplemented with cysteamine offers promise for applications requiring short-term storage to facilitate transport of in vitro produced bovine embryos.

  2. Clinical effectiveness of elective single versus double embryo transfer: meta-analysis of individual patient data from randomised trials

    NARCIS (Netherlands)

    McLernon, D.J.; Harrild, K.; Bergh, C.; Davies, M.J.; de Neubourg, D.; Dumoulin, J.C.M.; Gerris, J.; Kremer, J.A.M.; Martikainen, H.; Mol, B.W.; Norman, R.J.; Thurin-Kjellberg, A.; Tiitinen, A.; van Montfoort, A.P.A.; van Peperstraten, A.M.; van Royen, E.; Bhattacharya, S.

    2010-01-01

    Objective To compare the effectiveness of elective single embryo transfer versus double embryo transfer on the outcomes of live birth, multiple live birth, miscarriage, preterm birth, term singleton birth, and low birth weight after fresh embryo transfer, and on the outcomes of cumulative live birth

  3. A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions : a comparative analysis

    OpenAIRE

    Brinkhof, Bas; Helena T. A. van Tol; Groot Koerkamp, Marian J A; Riemers, Frank M; IJzer, Sascha G; Mashayekhi, Kaveh; Haagsman, Henk P; Roelen, Bernard A. J.; Holstege, FCP

    2015-01-01

    BACKGROUND: Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm a...

  4. A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis.

    OpenAIRE

    Brinkhof, B.; van Tol, H.T.; Groot Koerkamp, M J; Riemers, F M; IJzer, S.G.; Mashayekhi, K.; Haagsman, H P; Roelen, B.A.

    2015-01-01

    Background Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm an...

  5. Embryo transfer simulation improves pregnancy rates and decreases time to proficiency in Reproductive Endocrinology and Infertility fellow embryo transfers.

    Science.gov (United States)

    Heitmann, Ryan J; Hill, Micah J; Csokmay, John M; Pilgrim, Justin; DeCherney, Alan H; Deering, Shad

    2017-05-01

    To design and evaluate an ET simulator to train Reproductive Endocrinology and Infertility (REI) fellows' techniques of ET. Simulation model development and retrospective cohort analysis. Not applicable. Patients undergoing IVF. Simulation model evaluation and implementation of ET simulation training. Pregnancy rates. The REI fellow and faculty evaluation responses (n = 19/21 [90%]) of the model demonstrated realistic characteristics, with evaluators concluding the model was suitable for training in almost all evaluated areas. A total of 12 REI fellows who performed ET were analyzed: 6 before ET trainer and 6 after ET trainer. Pregnancy rates were 31% in the initial 10 ETs per fellow before simulator vs. 46% after simulator. One of six pre-ET trainer fellows (17%) had pregnancy rates ≥40% in their first 10 ETs; whereas four of six post-ET trainer fellows had pregnancy rates ≥40% in their first 10 ETs. The average number of ETs to obtain >40% pregnancy efficiency was 27 ETs before trainer vs. 15 ETs after trainer. Pregnancy rates were similar in the two groups after 20 ETs, and collective terminal pregnancy rates were >50% after 40 ETs. Embryo transfer simulation improved REI fellow pregnancy rates in their first 10 transfers and led to a more rapid ET proficiency. These data suggest potential value in adopting ET simulation, even in programs with a robust history of live ET in fellowship training. Published by Elsevier Inc.

  6. Production of female bovine embryos with sex-sorted sperm using intracytoplasmic sperm injection: efficiency and in vitro developmental competence.

    Science.gov (United States)

    Jo, Hyun-Tae; Bang, Jae-Il; Kim, Seong-Su; Choi, Byung-Hyun; Jin, Jong-In; Kim, Heyng-Lyool; Jung, In-Suk; Suh, Tae-Kwang; Ghanem, Nasser; Wang, Zhongde; Kong, Il-Keun

    2014-03-15

    The production of embryos with a preselected sex sperm is important in the livestock industry. In this study, we examined the efficiency of producing female embryos by intracytoplasmic sperm injection (ICSI) with flow cytometry sorted (ssICSI) and unsorted (usICSI) bovine sperm, and their developmental competence in vitro. For comparison, bovine embryos were also produced by in vitro fertilization (IVF) with sorted (ssIVF) and unsorted (usIVF) bovine sperm. The semen used in this study was from a bull selected for its high fertility and blastocyst developmental competence among four bulls. We first examined and compared pronuclear (PN) formation and cleavage rates of the produced embryos among the treatment groups. Our results demonstrated that PN formation rates (judged by two pronucleus [2PN]) and cleavage rates in ssIVF group (23.1% and 43.6%) were lower than those in the usIVF (71.1% and 71.6%), usICSI (73.1% and 92.8%) and ssICSI (75% and 79.1%) groups, respectively (P sex-sorting. Of note, we achieved a blastocyst formation rate in the ssICSI group to be comparable with the usIVF group. We then examined embryo quality by counting the number of normal and apoptotic cells in blastocysts. It was found that, despite the fact that blastocyst formation rate in the ssIVF group was significantly lower than those in the usIVF, usICSI and ssICSI groups, there was no difference in total and apoptotic cell numbers among these groups (P > 0.05). Finally, karyotyping analysis demonstrated that the proportion of female embryos in the ssICSI and ssIVF groups was 100%, whereas it was 58.8% and 57.8% in the usIVF and usICSI groups, respectively. In conclusion, ICSI with flow cytometry sorted bovine sperm provides an alternative approach to produce embryos with predetermined sex. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. In vitro maturation supplements affect developmental competence of bovine cumulus-oocyte complexes and embryo quality after vitrification.

    Science.gov (United States)

    Räty, Mervi; Ketoja, Elise; Pitkänen, Timo; Ahola, Virpi; Kananen, Kirsi; Peippo, Jaana

    2011-12-01

    Oocyte quality affects subsequent embryo development and quality. We examined the impact of bovine oocyte in vitro maturation (IVM) conditions on subsequent embryo yield, quality and cryosurvival. Cumulus-oocyte complexes (COCs) were sampled for cytological and gene expression analysis after IVM in TCM199 supplemented with 10% fetal bovine serum (FBS), 4 mg/ml of fatty-acid-free bovine serum albumin (FAFBSA), 4 mg/ml of polyvinylpyrrolidone (PVP), FAFBSA with epidermal growth factor (EGF, 100 ng/ml) and insulin-like growth factor 1 (IGF-I, 100 ng/ml) (FAFBSAGF), PVP with EGF and IGF-I (PVPGF) or PVP with single strength BME and MEM amino acids (PVPAA). The remaining COCs were fertilized. On day 7 (IVF=day 0) quality 1 blastocysts were vitrified or analyzed for glucose transporter 1 (Glut-1) expression levels. The remaining blastocysts (days 7-9) were evaluated for morphology and total cell counts. After warming, survival and hatching rates were evaluated followed by total cell counts and Glut-1 expression levels. Only PVPGF IVM resulted in embryo production rates comparable to those recorded with FBS IVM. Growth factors with FAFBSA and amino acids with PVP reduced embryo production rates whereas the effect of the growth factors with PVP was negligible. Insulin-like growth factor 2 binding protein 3 (IGF2BP3) and beta cell translocation gene 4 (BTG4) were revealed as potential candidates for oocyte developmental competence, and secreted protein, acidic and rich in cysteine (SPARC) for cumulus cell expansion. There were no differences among treatments in hatching rates of vitrified embryos after warming. However, total cell numbers and Glut-1 expression levels at 72 h were affected. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Early zygotes are suitable recipients for bovine somatic nuclear transfer and result in cloned offspring.

    Science.gov (United States)

    Schurmann, Anita; Wells, David N; Oback, Björn

    2006-12-01

    Cloning by somatic cell nuclear transfer (SCNT) subverts sperm-mediated fertilization that normally leads to physiological activation of the oocyte. Therefore, artificial activation is required and it is presently unclear what developmental consequences this has. In this study, we aimed to improve cattle cloning efficiency by utilizing a more physiological method of activating SCNT reconstructs. We carried out in vitro fertilization (IVF) of zona-intact bovine oocytes before SCNT. We removed the zona pellucida 4 h after insemination, stained the fertilized eggs with Hoechst 33342 and mechanically removed both male and female chromatin. The enucleated pre-activated cytoplasts were fused with male adult ear skin fibroblasts ("IVF-NT" group). Chemically activated SCNT embryos, produced according to our standard operating procedure for zona-free SCNT, served as controls. After 7 days, in vitro development to blastocysts of morphological grade 1-3 or grade 1-2 was very similar in both groups (39 vs 40% and 20 vs 21% respectively). However, post-implantation development was improved after sperm-mediated activation. Across four replicate runs, pregnancy establishment at day 35 was significantly higher for IVF-NT than for control SCNT embryos (30/49 = 61 vs 17/41 = 42% respectively; P calves at term or weaning was also higher in the IVF-NT group compared with control SCNT (9/49 = 18 vs 3/41 = 7% and 6/49 = 12 vs 3/41 = 7%; P = 0.11 and 0.34 respectively).

  9. Surgical transfer of in vivo produced farmed European polecat (Mustela putorius) embryos.

    Science.gov (United States)

    Lindeberg, Heli; Amstislavsky, Sergei; Järvinen, Mikko; Aalto, Jussi; Valtonen, Maija

    2002-06-01

    Surgical embryo transfer of farmed European polecat (Mustela putorius) was investigated as part of an ex situ preservation project. The long-term objective of the project is to develop effective technology for ex situ conservation of the European mink (Mustela lutreola), which is a highly endangered aboriginal European species. Twenty European polecat females, which served as a model species for the European mink, were humanely killed 4-9 days after first mating and embryos were recovered from oviducts and uteri. Donor-recipient pairs (n = 16) were generated by mating the donors (n = 20) once a day for 2 consecutive days with fertile males and by mating the corresponding recipients (n = 16) on the same days with vasectomized males. An embryo recovery rate of 70% (200 recovered embryos/284 corpora lutea) was achieved from 20 donors. Morulae and blastocysts were recovered between Days 5 and 9 after first mating and were regarded as the best developmental stages for uterine embryo transfer. A total of 172 embryos were transferred surgically under general anaesthesia into the ovarian third of the left uterine horn of 16 recipients with a thin glass capillary. Eleven recipients (69%) produced 72 pups equivalent to an average success rate of 42% (72 pups/172 transferred embryos). The average litter size was 4.5 (range 0-9). These results with this model species, farmed European polecat, demonstrate the potential of embryo transfer as an effective method for the preservation of the endangered European mink (M. lutreola). These species are closely related and have a similar reproductive physiology. However, success of applying embryo transfer in conserving European mink is still dependent on further studies both into its reproductive physiology and developing of improved flushing techniques for anaesthetized donors and the successful transfer of frozen-thawed embryos.

  10. Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the oosight imaging system.

    Science.gov (United States)

    Kim, Eun Young; Park, Min Jee; Park, Hyo Young; Noh, Eun Ji; Noh, Eun Hyung; Park, Kyoung Sik; Lee, Jun Beom; Jeong, Chang Jin; Riu, Key Zung; Park, Se Pill

    2012-08-01

    In somatic cell nuclear transfer (SCNT) procedures, exquisite enucleation of the recipient oocyte is critical to cloning efficiency. The purpose of this study was to compare the effects of two enucleation systems, Hoechst staining and UV irradiation (hereafter, irradiation group) and Oosight imaging (hereafter, Oosight group), on the in vitro production of bovine SCNT embryos. In the Oosight group, the apoptotic index (2.8 ± 0.5 vs. 7.3 ± 1.2) was lower, and the fusion rate (75.6% vs. 62.9%), cleavage rate (78.0% vs. 63.7%), blastocyst rate (40.2% vs. 29.2%), and total cell number (128.3±4.8 vs. 112.2 ± 7.6) were higher than those in the irradiation group (all p<0.05). The overall efficiency after SCNT was twice as high in the Oosight group as that in the irradiation group (p<0.05). The relative mRNA expression levels of Oct4, Nanog, Interferon-tau, and Dnmt3A were higher and those of Caspase-3 and Hsp70 were lower in the Oosight group compared with the irradiation group (p<0.05). This is the first report to show the positive effect of the Oosight imaging system on molecular gene expression in the SCNT embryo. The Oosight imaging system may become the preferred choice for enucleation because it is less detrimental to the developmental potential of bovine SCNT embryos.

  11. 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.

  12. Effects of Differences in Dietary Protein and Varying the Interval from Collection of Bovine Embryos to Freezing on Embryo Quality and Viability

    OpenAIRE

    Jousan, Frank Dean

    2002-01-01

    High levels of dietary protein may be detrimental to reproductive performance in cattle. The objective of Exp. 1 was to determine the effects of differences in dietary protein on the production and quality of bovine embryos collected from superovulated donors. Angus cows were randomly assigned to receive one of three experimental diets: a daily ration of 5.7 kg poultry litter, 2.0 kg hay, 3.1 kg corn, and 0.5 kg peanut hulls (LITTER; n = 15); a daily ration of 6.2 kg peanut hulls, 2.2 k...

  13. The Holstein cow in embryo transfer today as compared to 20 years ago.

    Science.gov (United States)

    Hasler, J F

    2006-01-07

    Embryo transfer practice and results were examined over a 20-year period in Holstein cows and heifers within four commercial embryo transfer programs located in different areas of North America. Mean embryo production per collection decreased (P cows entering embryo transfer programs, the number of times they were superstimulated and changes in the brands of gonadotropins used for superstimulation all complicated the analysis of embryo production over time. Data reveal higher pregnancy rates (P cows. It is not clear whether pregnancy rates have decreased over time as a result of the change from surgical to non-surgical embryo transfer. In the two programs in which pregnancy rates were analyzed, there was a decrease (P < 0.001) when non-surgical transfers were adopted in one program, while no change occurred in the other. One of the biggest changes in all programs was that more than 50% of embryos recovered from donors are now frozen after collection, whereas the majority were transferred fresh 20 years ago.

  14. Single top quality embryo transfer as a model for prediction of early pregnancy outcome.

    Science.gov (United States)

    De Neubourg, D; Gerris, J; Mangelschots, K; Van Royen, E; Vercruyssen, M; Elseviers, M

    2004-06-01

    Single embryo transfer (particularly of a top quality embryo) is an excellent model to correlate embryo quality in terms of morphological criteria to early pregnancy. We investigated whether this model could provide us with more information on what happens after implantation in the first trimester of pregnancy. The outcome of 370 consecutive single top quality embryo transfers in patients younger than 38 years was analysed for pregnancy and first-trimester pregnancy loss (FTPL) before 13 weeks of gestation. Analysis was done on each cohort of embryos from which the transferred top quality embryo was selected. Serum HCG levels were measured on day 8 and day 12 after day 3 embryo transfer. The HCG index was calculated as the level of HCG on day 12/HCG on day 8. The pregnancy rate after single top quality embryo transfer was 51.9%. This was independent of the patients' age. FTPL, however, appeared to be age dependent: 15.4% for the whole group, 9% in patients younger than 30 years and 19% in patients above 30 years. The pregnancy rate was 50% in IVF cycles and 52% in ICSI cycles; FTPL was 19% in IVF cycles and 10% in ICSI cycles. Multiple regression analysis showed that these differences originated from age differences between both populations rather than from technique-related factors. An HCG level >or=45 IU/l on day 12 was predictive for ongoing pregnancy with 75.6% sensitivity and 100% specificity; an HCG index >or=3.5 similarly predicted ongoing pregnancy with 72.3% sensitivity and 100% specificity. These data show that embryo selection for transfer on day 3 can be used as an excellent tool for prediction of pregnancy but not for prediction of FTPL. The pregnancy rate of a single top quality embryo is not related to age, whereas FTPL is age dependent.

  15. Use of versapoint to refashion the cervical canal to overcome unusually difficult embryo transfers and improve in-vitro fertilization-embryo transfer outcome: A case series

    Directory of Open Access Journals (Sweden)

    Nalini Mahajan

    2011-01-01

    Full Text Available Background : Smooth atraumatic embryo transfer is paramount for the success of in-vitro fertilization (IVF. In difficult cases, cervical canal manipulation may be required. Aim : To see if surgical correction of the cervical canal or cervical canal refashioning could improve ease of embryo transfer. Setting : Private infertility and IVF hospital. Design : Prospective study. Materials and Methods : Patients: 11 women with failed 1-3 IVF cycles with history of extremely difficult embryo transfers (ETs despite undergoing cervical dilatation in the cycle prior to IVF. Interventions : Operative hysteroscopy using Versapoint for refashioning of the cervical canal. Main Outcome Measures : Ease of ET in the subsequent IVF cycle. Secondary outcome measure was to assess reproductive outcome. Results : Easy and atraumatic ET in the IVF cycle after procedure in 100% patients. PR was 46.5%. Conclusions : Use of Versapoint for refashioning the cervical canal can improve the quality of ET and PR.

  16. 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.

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

    Science.gov (United States)

    Wang, Feng; Tian, XiuZhi; Zhou, YanHua; Tan, DunXian; Zhu, ShiEn; Dai, YunPing; Liu, GuoShi

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Feng Wang

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

  19. Uterine peristalsis exerts control over fluid migration after mock embryo transfer.

    Science.gov (United States)

    Zhu, L; Xiao, L; Che, H S; Li, Y P; Liao, J T

    2014-02-01

    What is the effect of uterine peristalsis on fluid migration after mock embryo transfer? Uterine peristaltic wave frequency was positively correlated with the distance that fluid moved after it was deposited in the uterine cavity. Embryos have been found outside the uterine cavity after embryo transfer. It has been suggested that uterine contractions expelled these embryos. A prospective cohort study of a total of 112 infertile women was conducted between March 2013 and May 2013. Uterine peristaltic activity was assessed before and after a mock embryo transfer, in which 20 µl of ultrasound contrast agent was placed in the uterine lumen 3 days after ovulation in a natural cycle. The movement of this fluid was measured by ultrasound at 0, 15 and 30 min after placement. The uterine peristaltic wave frequency was significantly higher after than before mock transfer (3.06 ± 0.99 versus 2.24 ± 0.74, P uterine peristaltic wave frequencies before (r = 0.518, P uterine peristaltic wave frequency was significantly higher both before and after embryo transfer in cases where the fluid was extruded. Mock embryo transfer was performed in the luteal phase of a natural cycle instead of a controlled ovarian stimulation cycle. The endometrial environment and uterine peristalsis may be different in a stimulated cycle. Uterine peristalsis exerts control over embryo migration and could adversely affect the chances of pregnancy if the wave frequency is too high. It could be used as a predictor of uterine irritability before embryo transfer. The authors declare that they have not received any particular study funding and do not have competing interests in this study.

  20. Non-invasive assessment of in-vitro embryo quality to improve transfer success

    DEFF Research Database (Denmark)

    Højbøge, Tina Rødgaard; Heegaard, Peter M. H.; Callesen, Henrik

    2015-01-01

    embryos before the transfer to a recipient still remains challenging. Presently, the predominant non-invasive technique for selecting viable embryos is based on morphology, where parameters such as rates of cleavage and blastocyst formation as well as developmental kinetics are evaluated mostly...... subjectively. The simple morphological approach is, however, inadequate for the prediction of embryo quality, and several studies have focused on developing new non-invasive methods using molecular approaches based particularly on proteomics, metabolomics and most recently small non-coding RNA, including micro......RNA. This review outlines the potential of several non-invasive in-vitro methods based on analysis of spent embryo culture medium....

  1. 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

  2. Large baby syndrome in singletons born after frozen embryo transfer (FET)

    DEFF Research Database (Denmark)

    Pinborg, Anja; Henningsen, AA; Loft, A

    2013-01-01

    Are singletons born after frozen embryo transfer (FET) at increased risk of being born large for gestational age (LGA) and if so, is this caused by intrinsic maternal factors or related to the freezing/thawing procedures?...

  3. Reducing twin pregnancy rates after IVF--elective single embryo transfer (eSET).

    LENUS (Irish Health Repository)

    Milne, P

    2010-01-01

    Multiple pregnancy is a major complication of IVF and is associated with increased maternal, fetal and neonatal morbidity. Elective single embryo transfer (eSET) during IVF, rather than the more standard transfer of two embryos (double embryo transfer or DET), has been shown to significantly reduce the multiple pregnancy rate associated with IVF, while maintaining acceptable pregnancy rates. Couples undergoing IVF in 2008 who met good prognostic criteria had eSET performed. Pregnancy and twinning rates were compared with those for similar couples in 2007 who had DET. Couples unsuccessful with a fresh cycle of treatment had subsequent frozen embryo transfer cycles with DET. The cumulative pregnancy rate was similar for each group. However there were no multiple pregnancies in the eSET group, compared to 4 twins of 5 pregnancies in the DET group. 96% of eligible couples agreed to eSET. ESET is successful in and acceptable to good prognosis Irish couples undergoing IVF.

  4. 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

  5. Application of reproductive biotechnology for the recovery of endangered breeds: birth of the first calf of Murciana-Levantina bovine breed derived by OPU, in vitro production and embryo vitrification.

    Science.gov (United States)

    Ruiz, S; Romero-Aguirregomezcorta, J; Astiz, S; Peinado, B; Almela, L; Poto, A

    2013-12-01

    In a conservation project, reproductive biotechnology was implemented for the recovery and conservation of an endangered bovine breed in Spain. The breed Murciana-Levantina, declared to be worthy of special protection status (http://www.fao.org/newsroom/en/news/2006/1000464/index.html), is of great interest because of its hardness, longevity, docility and disease resistance. This contribution describes the birth of the first calf of this breed obtained by reproductive biotechnology, using ultrasound-guided punction and aspiration of ovarian follicles, in vitro embryo production, vitrification of embryos by a cryotop device and, finally, the transfer of cryopreserved embryos to recipient heifers of a commercial dairy herd. © 2013 Blackwell Verlag GmbH.

  6. Repeated embryo collection and interspecies transfer in alpacas and llamas during non-breeding season

    Directory of Open Access Journals (Sweden)

    Pacheco J

    2015-08-01

    Full Text Available Sexual behavior evaluation was evaluated, collecting and interspecies embryo transfer inter species in llamas and alpacas during non-breeding season, 10 and 10 donor alpacas llamas, alpacas and 20 receiving 20 llamas, 5 alpacas and 5 llamas males were used. Sexual behavior by libido in males and acceptance of female to male in the presence of a dominant follicle was evaluated, the collection of embryos simple ovulation by non-surgical technique was performed and the fresh embryos are transferred directly into the horn left. It was observed that only 40% of alpaca accept the male and female in all cases had to use two males for mating, but all llama males mounted on the first attempt and accepted all females breeding. Embryos were collected at 25 and 60% of alpacas and llamas washes respectively, all were grade 1 embryos transferable; the embryo transfer fertility evaluated by ultrasound at 25 days was 100 and 41.6% respectively for donor alpaca and llama, however ultrasound evaluation at 60 days fertility was 50 and 25% respectively for donor alpaca and llama. We conclude that there is greater reproductive seasonality in alpaca regard to llamas, all were grade 1 embryos collected, fertility evaluated by ultrasound 25 days down to 60 days, demonstrating embryonic mortality, possibly due to the non-breeding season of both species.

  7. Ultra-Structural Alterations in In Vitro Produced Four-Cell Bovine Embryos Following Controlled Slow Freezing or Vitrification.

    Science.gov (United States)

    Cavusoglu, T; Popken, J; Guengoer, T; Yilmaz, O; Uyanikgil, Y; Ates, U; Baka, M; Oztas, E; Zakhartchenko, V

    2016-08-01

    Cryopreservation is the process of freezing and preserving cells and tissues at low temperatures. Controlled slow freezing and vitrification have successfully been used for cryopreservation of mammalian embryos. We investigated the effect of these two cryopreservation methods on in vitro produced four-cell stage bovine embryos which were classified according to their quality and separated into three groups. The first group was maintained as untreated controls (n = 350). Embryos of the second (n = 385) and the third (n = 385) groups were cryopreserved either by controlled slow freezing or by vitrification. Embryos in groups 2 and 3 were thawed after 1 day. Hundred embryos were randomly selected from the control group, and 100 morphologically intact embryos from the second and third group were thawed after 1 day and cultured to observe the development up to the blastocyst stage. The blastocyst development rate was 22% in the control group, 1% in the slow-freezing group and 3% in the vitrification group. Remaining embryos of all three groups were examined by light microscopy, transmission electron microscopy and immunofluorescence confocal microscopy with subsequent histological staining procedures. Cryopreservation caused degenerative changes at the ultra-structural level. Compared with vitrification, slow freezing caused an increased mitochondrial degeneration, cytoplasmic vacuolization, disruption of the nuclear and plasma membrane integrity, organelle disintegration, cytoskeletal damage, a reduced thickness of the zona pellucida and a formation of fractures in the zona pellucida. Further studies are required to understand and decrease the harmful effects of cryopreservation. © 2015 Blackwell Verlag GmbH.

  8. Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer.

    Science.gov (United States)

    Boss, R; Cosandey, A; Luini, M; Artursson, K; Bardiau, M; Breitenwieser, F; Hehenberger, E; Lam, Th; Mansfeld, M; Michel, A; Mösslacher, G; Naskova, J; Nelson, S; Podpečan, O; Raemy, A; Ryan, E; Salat, O; Zangerl, P; Steiner, A; Graber, H U

    2016-01-01

    Staphylococcus aureus is globally one of the most important pathogens causing contagious mastitis in cattle. Previous studies using ribosomal spacer (RS)-PCR, however, demonstrated in Swiss cows that Staph. aureus isolated from bovine intramammary infections are genetically heterogeneous, with Staph. aureus genotype B (GTB) and GTC being the most prominent genotypes. Furthermore, Staph. aureus GTB was found to be contagious, whereas Staph. aureus GTC and all the remaining genotypes were involved in individual cow disease. In addition to RS-PCR, other methods for subtyping Staph. aureus are known, including spa typing and multilocus sequence typing (MLST). They are based on sequencing the spa and various housekeeping genes, respectively. The aim of the present study was to compare the 3 analytic methods using 456 strains of Staph. aureus isolated from milk of bovine intramammary infections and bulk tanks obtained from 12 European countries. Furthermore, the phylogeny of animal Staph. aureus was inferred and the zoonotic transfer of Staph. aureus between cattle and humans was studied. The analyzed strains could be grouped into 6 genotypic clusters, with CLB, CLC, and CLR being the most prominent ones. Comparing the 3 subtyping methods, RS-PCR showed the highest resolution, followed by spa typing and MLST. We found associations among the methods but in many cases they were unsatisfactory except for CLB and CLC. Cluster CLB was positive for clonal complex (CC)8 in 99% of the cases and typically positive for t2953; it is the cattle-adapted form of CC8. Cluster CLC was always positive for tbl 2645 and typically positive for CC705. For CLR and the remaining subtypes, links among the 3 methods were generally poor. Bovine Staph. aureus is highly clonal and a few clones predominate. Animal Staph. aureus always evolve from human strains, such that every human strain may be the ancestor of a novel animal-adapted strain. The zoonotic transfer of IMI- and milk-associated strains

  9. Effect of hyaluronic acid-enriched transfer medium on frozen-thawed embryo transfer outcomes.

    Science.gov (United States)

    Fu, Wei; Yu, Min; Zhang, Xiao-Jin

    2018-02-14

    To determine if hyaluronic acid-enriched transfer medium (HETM) affects the implantation rate (IR) and clinical pregnancy rate (PR) in women undergoing frozen-thawed embryo transfer (FET). The records of women who underwent FET from May 2014 to October 2014 were retrospectively reviewed. Outcome measures were IR and PR. In all 1721 cycles of 1632 patients were included in this study. HETM was used for 347 cycles of 342 patients, and standard medium for 1374 cycles of 1290 patients. Overall, FET outcomes were similar between the groups. For patients undergoing their first FET attempt, the IR (24.3% vs 31.6%, P = 0.042) and clinical PR (34.3% vs 50.1%, P = 0.004) were lower in the HETM group. For patients undergoing their second FET attempt, pregnancy outcomes were similar between the groups. For patients undergoing their third or more FET attempt, HETM was associated with a higher IR (33.3% vs 16.4%, P < 0.001) and higher PR (52.2% vs 27.4%, P < 0.001). HETM can improve the embryo IR and clinical PR in patients with repeated implantation failure in the third or more FET attempt. However, the use of HETM for first and second FET should be done with caution. © 2018 Japan Society of Obstetrics and Gynecology.

  10. Factors affecting the efficiency of embryo transfer in the domestic ferret (Mustela putorius furo).

    Science.gov (United States)

    Li, Ziyi; Sun, Xingshen; Chen, Juan; Leno, Gregory H; Engelhardt, John F

    2006-07-15

    Embryo transfer (ET) to recipient females is a foundational strategy for a number of assisted reproductive technologies, including cloning by somatic cell nuclear transfer. In an attempt to develop efficient ET in domestic ferrets, factors affecting development of transferred embryo were investigated. Unilateral and bilateral transfer of zygotes or blastocysts in the oviduct or uterus was evaluated in recipient nulliparous or primiparous females. Developing fetuses were collected from recipient animals 21 days post-copulation and examined. The percentage of fetal formation was different (PEuropean mink.

  11. Oocyte maturation, embryo development and gene expression following two different methods of bovine cumulus-oocyte complexes vitrification.

    Science.gov (United States)

    Azari, Mehdi; Kafi, Mojtaba; Ebrahimi, Bita; Fatehi, Roya; Jamalzadeh, Mahboobeh

    2017-03-01

    To examine the maturational competence, embryo development and expression of genes involved in oocyte maturation and cumulus expansion (GDF9, BMP15, HAS2, TNFAIP6, FGF17 and FSHr) following two standard methods of bovine COCs vitrification. Bovine cumulus-oocyte complexes (COCs) were aspirated from slaughtered ovaries and then distributed into three groups: non-vitrified COCs (control), vitrification 1 group (V1); vitrification was performed by 15% ethylene glycol (EG) and 15% DMSO in holding media (TCM-199 with 20% FCS); and vitrification 2 group (V2); vitrification was performed by 40% EG in holding media. After vitrification, COCs were warmed in two steps and cultured and then evaluated for nuclear maturation, embryo development and gene expressions. The mean (±SD) percentages of nuclear maturation and blastocyst/cleaved were higher in control group (79.5 ± 8.0 and 31.0 ± 5.1%) than the V1 (34.8 ± 9.1 and 4.4 ± 5.1%) and V2 (47.8 ± 11.7 and 7.1 ± 5.8%) groups (P vitrification groups (P vitrification procedure and conditions. Using EG alone for vitrification of bovine immature COCs, resulted in higher expression of GDF9, BMP15 and production of more in vitro matured and cleaved oocytes.

  12. Validating reference microRNAs for normalizing qRT-PCR data in bovine oocytes and preimplantation embryos.

    Science.gov (United States)

    Mahdipour, Mahdi; van Tol, Helena T A; Stout, Tom A E; Roelen, Bernard A J

    2015-06-12

    MicroRNAs (miRNAs) are small noncoding RNAs that act as post-transcriptional regulators of gene targets. Accurate quantification of miRNA expression using validated internal controls should aid in the understanding of their role in epigenetic modification of genome function. To date, most studies that have examined miRNA expression levels have used the global mean expression of all expressed genes or the expression of reference mRNAs or nuclear RNAs for normalization. We analyzed the suitability of a number of miRNAs as potential expression normalizers in bovine oocytes and early embryos, and porcine oocytes. The stages examined were bovine oocytes at the germinal vesicle (GV) and metaphase II stages, bovine zygotes, 2, 4 and 8 cell embryos, morulae and blastocysts, as well as porcine cumulus oocyte complexes, GV, metaphase I and II oocytes. qRT-PCR was performed to quantify expression of miR-93, miR-103, miR-26a, miR-191, miR-23b, Let-7a and U6 for bovine samples and miR-21, miR-26a, miR-93, miR-103, miR-148a, miR-182 and miR-191 for porcine oocytes. The average starting material for each sample was determined using specific standard curves for each primer set. Subsequently, geNorm and BestKeeper software were used to identify a set of stably expressed miRNAs. Stepwise removal to determine the optimum number of reference miRNAs identified miR-93 and miR-103 as the most stably expressed in bovine samples and miR-26a, miR-191 and miR-93 in porcine samples. The combination of miR-93 and miR-103 is optimal for normalizing miRNA expression for qPCR experiments on bovine oocytes and preimplantation embryos; the preferred combination for porcine oocytes is miR-26a, miR-191 and miR-93.

  13. 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.

  14. Confirmed dioestrus in pseudopregnant mice using vaginal exfoliative cytology improves embryo transfer implantation rate.

    Science.gov (United States)

    Mamrot, Jared; Pangestu, Mulyoto; Walker, David; Gardner, David K; Dickinson, Hayley

    2015-10-01

    Embryo transfer is a commonly performed surgical technique. In mice, protocols typically specify pairing recipient females with vasectomized males to induce a receptive uterine environment for embryo implantation. However, this induced receptive state is not always maintained until implantation occurs. The use of a well-characterized correlation between oestrous state and exfoliative vaginal cytology was therefore evaluated to assess uterine receptivity immediately before embryo transfer. Eight- to 12-week-old virgin female CD1 mice (n = 22) were paired overnight with vasectomized males and successfully mated, indicated by the presence of a vaginal plug. These dams underwent embryo transfer 3 days later with embryos obtained from superovulated 4-week-old F1 (C57BL/6 × CBA) females. Non-invasive vaginal lavage was conducted immediately before transfer. Dams were killed 6 days after transfer and the uterus collected for histological analysis. Embryo implantation rate in mice was 96% when cytological analysis of the lavage samples signified dioestrus (n = 6), whereas the implantation rate was cytology signified other stages of oestrous. This simple, quick, non-invasive measure of receptivity was accurate and easily adopted and, when applied prospectively, will avoid unnecessary surgery and subsequent culling of non-suitable recipients, while maximizing the implantation potential of each recipient female. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  15. First Irish delivery following sequential, two-stage embryo and blastocyst transfer.

    OpenAIRE

    Hayrinen, L. H.; Sills, Eric Scott; Fogarty, Alicia O; Walsh, David J; Lutsyk, Alexander D; Walsh, Anthony PH

    2012-01-01

    BACKGROUND: The timing of embryo transfer (ET) after in vitro fertilisation (IVF) remains controversial, and there are no reliable guidelines available to prospectively identify which patients would benefit from either day-3 or blastocyst transfer. While blastocyst transfer is generally favoured over day-3 transfers, very few IVF patients get both in the same treatment cycle. CASE DESCRIPTION: We report on a 35.5-year-old female with tubal factor infertility who underwent IVF, which includ...

  16. Effect of maternal age on the ratio of cleavage and mitochondrial DNA copy number in early developmental stage bovine embryos.

    Science.gov (United States)

    Takeo, Shun; Goto, Hiroya; Kuwayama, Takehito; Monji, Yasunori; Iwata, Hisataka

    2013-01-01

    Age-associated deterioration in both the quality and quantity of mitochondria occurs in older women. The main aim of this study was to examine the effect of age on mitochondrial DNA copy number (mtDNA number) in early developmental stage bovine embryos as well as the dynamics of mtDNA number during early embryo development. Real-time PCR was used to determine mtDNA number. In vitro-produced embryos 48 h after insemination derived from Japanese black cows, ranging in age from 25 to 209 months were categorized based on their cleavage status. There was an overall negative relationship between the age of the cow and cleavage status, to the extent that the ratio of embryos cleaved over the 4-cell stage was greater in younger cows. The mtDNA number did not differ among the cleaved status of embryos. In the next experiment, oocytes collected from each donor cow were divided into 2 groups containing 10 oocytes each, in order to compare the mtDNA number of mature oocytes and early developmental stage embryos within individuals. Upon comparing the mtDNA number between oocytes at the M2 stage and early developmental stage 48 h post insemination, mtDNA number was found to decrease in most cows, but was found to increase in some cows. In conclusion, age affects the cleaving ability of oocytes, and very old cows (> 180 months) tend to have lower mtDNA numbers in their oocytes. The change in mtDNA number during early development varied among individual cows, although overall, it showed a tendency to decrease.

  17. Application of hollow fiber vitrification for cryopreservation of bovine early cleavage stage embryos and porcine morula-blastomeres.

    Science.gov (United States)

    Uchikura, Ayuko; Matsunari, Hitomi; Nakano, Kazuaki; Hatae, Shota; Nagashima, Hiroshi

    2016-04-22

    A novel hollow fiber vitrification (HFV) method was applied to materials that have previously been difficult to cryopreserve, thereby expanding the potential application of this method. The results showed that zona-free porcine morulae and their isolated blastomeres remained viable even after vitrification. The rate of development to blastocysts after vitrification was similar for zona-free and zona-intact morulae (21/23, 91.3% for both). Vitrified blastomeres had a developmental potential equal to that of non-vitrified blastomeres (blastocyst formation rate after reaggregation: 16/17, 94.1% for both). The HFV method was also effective for the cryopreservation of in vitro matured/fertilized bovine embryos at the 2- to 4-cell, 8- to 16-cell and morula stages. The blastocyst formation rates of vitrified embryos (66.1-82.5%) were similar to those of non-vitrified embryos (74.5-82.5%). These results indicate that this novel HFV method is an effective tool for embryo cryopreservation that can enhance current practices in reproductive biology.

  18. Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification.

    Science.gov (United States)

    Sudano, Mateus José; Paschoal, Daniela Martins; Rascado, Tatiana da Silva; Magalhães, Luis Carlos Oña; Crocomo, Letícia Ferrari; de Lima-Neto, João Ferreira; Landim-Alvarenga, Fernanda da Cruz

    2011-04-15

    The objective was to evaluate supplementation of fetal calf serum (FCS) and phenazine ethosulfate (PES), a metabolic regulator that inhibits fatty acid synthesis, in culture media during in vitro production (IVP) of bovine embryos. Taking oocyte fertilization (n = 4,320) as Day 0, four concentrations of FCS (0, 2.5, 5, and 10%) and three periods of exposure to PES (without addition-CONTROL; after 60 h-PES Day 2.5 of embryo culture; and after 96 h-PES Day 4) were evaluated. Increasing FCS concentration in the culture media enhanced lipid accumulation (P vitrification (2.5%: 81.6 ± 2.5 vs 10%: 67.3 ± 3.5, P vitrification ( 72.0 ± 3.0 vs PES Day 2.5: 79.9 ± 2.8 or PES Day 4: 86.2 ± 2.4, P vitrification. Moreover, embryo quality, represented by the fresh apoptosis rate, was better than lipid content for predicting embryo survival after vitrification. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Effect of resveratrol supplementation during culture on the quality and cryotolerance of bovine in vitro produced embryos.

    Science.gov (United States)

    Salzano, A; Albero, G; Zullo, G; Neglia, G; Abdel-Wahab, A; Bifulco, G; Zicarelli, L; Gasparrini, B

    2014-12-30

    The aim of the study was to evaluate whether resveratrol supplementation of bovine culture medium improves in vitro blastocyst development, embryo cryotolerance and cell numbers. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, zygotes were cultured in SOF medium, supplemented with 0 (control, n=439), 0.25μM (n=422), 0.5μM (n=447) and 1μM resveratrol (n=416). On Day 7 (IVF=Day 0) blastocysts were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide and 0.5M sucrose. Development rate, i.e. the percentage of embryos resuming development to reach a more advanced stage, and hatching rate were evaluated after 24 and 48h culture. Blastocysts cultured with (0.5μM) and without resveratrol underwent differential staining to count inner cell mass (ICM) and trophectoderm (TE) cells. Resveratrol during culture did not increase blastocyst yields (57.1, 57.7, 59.2 and 46.6%, respectively in 0, 0.25, 0.5 and 1μM resveratrol). However, 0.5μM resveratrol improved embryo cryotolerance compared to the control, as indicated by higher development rates (67.3% vs 50.3%, respectively; Pculture. Blastocysts produced in the control and in 0.5μM resveratrol groups had similar numbers of ICM (34.1 and 36.4, respectively), TE (88.1 and 85.3, respectively) and total (122.2 and 121.7, respectively) cells. In conclusion, low levels of resveratrol during in vitro culture improve the quality of IVP bovine embryos, as indicated by their increased resistance to cryopreservation. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Reproduction results and offspring performance after non-surgical embryo transfer in pigs

    NARCIS (Netherlands)

    Ducro-Steverink, D.W.B.; Peters, C.G.W.; Maters, C.C.; Hazeleger, W.; Merks, J.W.M.

    2004-01-01

    Increased interest in transfer of valuable genetic material around the world with minimal health risks has stimulated the development of non-surgical embryo transfer (nsET) technologies in pigs. Experimental evidence shows that nsET without sedation of the recipients is now feasible. The goal of

  1. Phosphatidylcholine transfer protein from bovine liver contains highly unsaturated phosphatidylcholine species

    NARCIS (Netherlands)

    Geijtenbeek, T. B.; Westerman, J.; Heerma, W.; Wirtz, K. W.

    1996-01-01

    The phosphatidylcholine transfer protein (PC-TP) from bovine liver contains one molecule of non-covalently bound PC. In order to gain more insight into the physiological function of PC-TP, PC was extracted from bovine liver PC-TP and its molecular species composition identified by fast atom

  2. Increasing twinning rate in beef cattle through embryo transfer ...

    African Journals Online (AJOL)

    Thirty-nine Limousin X Friesian heifers and forty-five Limousin X Friesian cows were randomly allocated to three treatments in a twin-induction breeding program. Animals were inseminated twice with Limousin semen (treatment A) or inseminated twice also with Hereford semen and each implanted with one embryo 7 days ...

  3. 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.

  4. 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, P; Shukri, N.N; Vajta, Gabor

    1998-01-01

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

  5. Embryo transfer practices in the United States: a survey of clinics registered with the Society for Assisted Reproductive Technology.

    Science.gov (United States)

    Jungheim, Emily S; Ryan, Ginny L; Levens, Eric D; Cunningham, Alexandra F; Macones, George A; Carson, Kenneth R; Beltsos, Angeline N; Odem, Randall R

    2010-09-01

    To gain a better understanding of factors influencing clinicians' embryo transfer practices. Cross-sectional survey. Web-based survey conducted in December 2008 of individuals practicing IVF in centers registered with the Society for Assisted Reproductive Technology (SART). None. None. Prevalence of clinicians reporting following embryo transfer guidelines recommended by the American Society for Reproductive Medicine (ASRM), prevalence among these clinicians to deviate from ASRM guidelines in commonly encountered clinical scenarios, and practice patterns related to single embryo transfer. Six percent of respondents reported following their own, independent guidelines for the number of embryos to transfer after IVF. Of the 94% of respondents who reported routinely following ASRM embryo transfer guidelines, 52% would deviate from these guidelines for patient request, 51% for cycles involving the transfer of frozen embryos, and 70% for patients with previously failed IVF cycles. All respondents reported routinely discussing the risks of multiple gestations associated with standard embryo transfer practices, whereas only 34% reported routinely discussing single embryo transfer with all patients. Although the majority of clinicians responding to our survey reported following ASRM embryo transfer guidelines, at least half would deviate from these guidelines in a number of different situations. Copyright (c) 2010 American Society for Reproductive Medicine. All rights reserved.

  6. [Association of fertilization strategy and embryo transfer time with the incidence of ectopic pregnancy].

    Science.gov (United States)

    Li, Ming-zhao; Zhao, Wan-qiu; Ren, An-qi; Shi, Juan-zi

    2015-10-01

    To investigate the correlation of the fertilization strategy and embryo transfer (ET) time with the incidence of ectopic pregnancy. We selected 3,331 fresh and 2,706 frozen-thawed ET cycles for the patients undergoing in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). The fresh transfers included 2 546 IVF-ET and 785 ICSI-ET cycles and 2,220 day-3 embryo and 1,111 day-5 blastocyst transfers, while the frozen-thawed transfers included 2,080 IVF-ET and 626 ICSI-ET cycles and 741 day-3 embryo and 1 965 day-5 or -6 blastocyst transfers. We compared the incidence rate of ectopic pregnancy associated with different fertilization strategies and ET time. The incidence rate of ectopic pregnancy was 1. 41% (36/2 546) in the IVF-ET cycles and 3.44% (27/785) in the ICSI-ET cycles of the fresh transfers, significantly lower in the IVF-ET than in the ICSI-ET cycles (P 0.05). The IVF-ET and ICSI-ET cycles included 2,220 fresh day-3 (F-D3) embryos, 1,111 F-D5 blastocysts, 741 frozen-thawed day-3 (T-D3) embryos, and 1,965 T-D5/6 blastocysts. The incidence rate of ectopic pregnancy was 1.71% (n = 38) in the F-D3, 2.25% (n = 25) in the F-D5, 1.35% (n = 10) in the T-D3, and 0.81% (n = 16) in the T-D5/6 group, respectively, significantly lower in the T-D5/6 than in the other three groups (P ectopic pregnancy is associated with fertilization strategies, which is significantly lower in frozen-thawed than in fresh embryo transfers.

  7. Inositol and In Vitro Fertilization with Embryo Transfer

    Directory of Open Access Journals (Sweden)

    G. Simi

    2017-01-01

    Full Text Available Recently, studies on inositol supplementation during in vitro fertilization program (IVF have gained particular importance due to the effect of this molecule on reducing insulin resistance improving ovarian function, oocyte quality, and embryo and pregnancy rates and reducing gonadotropin amount during stimulation. Inositol and its isoforms, especially myoinositol (MYO, are often used as prestimulation therapy in infertile patients undergoing IVF cycle. Inositol supplementation started three months before ovarian stimulation, resulting in significant improvements in hormonal responses, reducing the amount of FSH necessary for optimal follicle development and serum levels of 17beta-estradiol measured the day of hCG injection. As shown by growing number of trials, MYO supplementation improves oocyte quality by reducing the number of degenerated and immature oocytes, in this way increasing the quality of embryos produced. Inositol can also improve the quality of sperm parameters in those patients affected by oligoasthenoteratozoospermia.

  8. Increasing vaginal progesterone gel supplementation after frozen-thawed embryo transfer significantly increases the delivery rate

    DEFF Research Database (Denmark)

    Alsbjerg, Birgit; Polyzos, Nikolaos P; Elbaek, Helle Olesen

    2013-01-01

    The aim of this study was to evaluate the reproductive outcome in patients receiving frozen-thawed embryo transfer before and after doubling of the vaginal progesterone gel supplementation. The study was a retrospective study performed in The Fertility Clinic, Skive Regional Hospital, Denmark....... A total of 346 infertility patients with oligoamenorrhoea undergoing frozen-thawed embryo transfer after priming with oestradiol and vaginal progesterone gel were included. The vaginal progesterone dose was changed from 90mg (Crinone) once a day to twice a day and the reproductive outcome during the two...... rate (8.7% versus 20.5%, respectively; P=0.002). Doubling of the vaginal progesterone gel supplementation during frozen-thawed embryo transfer cycles decreased the early pregnancy loss rate, resulting in a significantly higher delivery rate. This study evaluated the reproductive outcome of 346 women...

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

    Science.gov (United States)

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

    2011-09-15

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

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

    Science.gov (United States)

    Turenne, Nicolas; Tiys, Evgeniy; Ivanisenko, Vladimir; Yudin, Nikolay; Ignatieva, Elena; Valour, Damien; Degrelle, Séverine A; Hue, Isabelle

    2012-08-29

    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. To gain knowledge on molecular-genetic regulations in a non model organism, we offer an approach based on literature-mining and score

  11. 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

  12. Acupuncture on the day of embryo transfer: a randomized controlled trial of 635 patients

    DEFF Research Database (Denmark)

    Andersen, Dorthe; Løssl, Kristine; Nyboe Andersen, Anders

    2010-01-01

    This prospective, randomized, controlled and double-blinded trial studied whether acupuncture in relation to embryo transfer could increase the ongoing pregnancy rates and live birth rates in women undergoing assisted reproductive therapy. A total of 635 patients undergoing IVF or intracytoplasmic...... sperm injection (ICSI) were included. In 314 patients, embryo transfer was accompanied by acupuncture according to the principles of traditional Chinese medicine. In the control group, 321 patients received placebo acupuncture using a validated placebo needle. In the acupuncture group and the placebo...

  13. Cell apoptosis and lipid content of in vitro-produced, vitrified bovine embryos treated with forskolin.

    Science.gov (United States)

    Paschoal, Daniela Martins; Sudano, Mateus José; Schwarz, Kátia Regina Lancellotti; Maziero, Rosiára Rosário Dias; Guastali, Midyan Daroz; Crocomo, Letícia Ferrari; Magalhães, Luis Carlos Oña; Martins, Alício; Leal, Claudia Lima Verde; Landim-Alvarenga, Fernanda da Cruz

    2017-01-01

    The presence of fetal calf serum in culture medium influences embryo quality, causing a reduction in postcryopreservation survival. Forskolin has been used to induce lipolysis and increase cryotolerance, functioning as an activator of adenylate cyclase and elevating cAMP levels. In the present experiment, bovine zygotes were cultured in synthetic oviduct fluid with amino acid plus 2.5% fetal calf serum for 6 days, when forskolin was added in three concentrations: 2.5, 5, and 10 μM. Treatment with forskolin lasted for 24 hours. Blastocyst formation rate, quantification of lipid granules, total cell numbers, and apoptosis rate were evaluated. In a second assessment, embryos were vitrified, and warming, re-expansion rate, total cell numbers, and apoptosis rate were also evaluated. There was no difference due to forskolin in blastocyst formation or re-expansion rates after vitrification. However, lipid measurements were lower (control: 136.8 and F 2.5 μM: 128.5; P < 0.05), and number of cells per embryo higher (control: 140.1 and F 2.5 μM: 173.5; P < 0.05) than controls for 2.5 μM forskolin but not for higher forskolin concentrations. The number of intact cells per embryo was higher, and the rate of apoptosis was lower in fresh than in vitrified embryos (number of cells of warmed embryos, control: 104.1, F 2.5 μM: 101.3, F 5 μM: 115.4, F 10 μM: 95.1; apoptotic of fresh cells, control: 12.1%, F 2.5 μM: 16.7%, F 5 μM: 11.1%, F 10 μM: 14.2%; and apoptotic warmed embryos, control: 22.3%, F 2.5 μM: 37.3%, F 5 μM: 33.2%, F 10 μM: 30.3%; P < 0.05). It was concluded that forskolin is an effective lipolytic agent even at low concentrations, leading to formation of blastocysts with a comparatively larger number of cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Hyperspectral microscopy can detect metabolic heterogeneity within bovine post-compaction embryos incubated under two oxygen concentrations (7% versus 20%).

    Science.gov (United States)

    Sutton-McDowall, Melanie L; Gosnell, Martin; Anwer, Ayad G; White, Melissa; Purdey, Malcolm; Abell, Andrew D; Goldys, Ewa M; Thompson, Jeremy G

    2017-10-01

    (P heterogeneity was evident within the cleavage stage (i.e. arrested) embryos exposed to fluorophores, there were no detectable difference in fluorescence intensity and pattern localisation in morula exposed to the two different oxygen concentrations (P > 0.05). While there were no significant differences in two-channel autofluorescent profiles of morula exposed to 7% and 20% oxygen (main effect, P > 0.05), morula that subsequently progressed to the blastocyst stage had significantly higher levels of FAD and NAD(P)H fluorescence compared to arrested morula (P success. Advanced autofluorescence imaging techniques, such as hyperspectral microscopy, may provide clinics with additional tools to improve the assessment of embryos prior to transfer. This study was funded by the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premier's Science and Research Fund. The authors declare there are no conflict of interest.

  15. Surgical recovery and successful surgical transfer of conventionally frozen-thawed embryos in the farmed European polecat (Mustela putorius).

    Science.gov (United States)

    Lindeberg, Heli; Aalto, Jussi; Amstislavsky, Sergei; Piltti, Katja; Järvinen, Mikko; Valtonen, Maija

    2003-11-01

    Surgical transfer of in vivo produced conventionally frozen-thawed embryos of farmed European polecat (Mustela putorius) was investigated as a part of an ex-situ preservation program which has the long-term aim of developing a genome resource bank for the endangered European mink (Mustela lutreola). Eighteen oestrous yearling European polecat donors were mated once daily on two consecutive days using 13 fertile males. The donors were surgically flushed for embryos 8-9 days after the first mating. The embryo recovery rate was 60% (116 embryos/193 corpora lutea). The embryos were cryopreserved with 1.5 M ethylene glycol in a programmable freezer using a conventional slow freezing protocol. The thawed embryos were surgically transferred either after dilution with 0.5 M sucrose or directly without removal of ethylene glycol. To induce ovulation, eight recipient females were mated once daily on two consecutive days with vasectomized males starting 7 or 8 days before embryo transfer. The recipients received 7-11 embryos each and three recipients delivered a total of nine pups after a gestation length of 44-46 days. The embryo survival rate was 10% (9 pups/93 frozen embryos). This report describes the first successful cryopreservation of embryos in the Mustelidae family resulting in viable offspring. The low embryo survival rate, however, indicates that the freezing-thawing protocol needs to be improved.

  16. 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.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  18. 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

  19. 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.

  20. Transferring two grades I cleavage-stage embryo might not be a good protocol.

    Science.gov (United States)

    Li, Mingzhao; Wang, Hui; Ma, Chun; Shi, Juanzi

    2017-07-01

    The aim of this study was to explore whether transferring two grades I cleavage-stage embryo was suitable for the patients in the first fresh transfer. This study included 202 single grades I cleavage-stage, 229 single grades III cleavage-stage, 743 single excellent blastocyst, 522 double grades I cleavage-stage, and 596 double grades III cleavage-stage embryo transfers. Main clinical outcomes: clinical pregnancy and twin-pregnancy rate. Among single excellent blastocyst, single grades I and single grades III group, the clinical pregnancy rate was significantly higher in single excellent blastocyst group than single grades I and grades III group (67.16% versus 42.08% versus 23.97%; p grades I cleavage-stage embryos, the clinical pregnancy rate reached 68.20% which was no significant difference compared with the single excellent blastocyst group (67.16%). However, the twin-pregnancy rate was significantly higher in double grades I group than double grades III and single excellent blastocyst group (43.26% versus 26.70% versus 0.60%; p grades I cleavage-stage embryo might not be a good protocol. Extended culture to blastocyst-stage could be considered for the patient with only two grades I cleavage-stage embryos.

  1. Evidence of Placental Autophagy during Early Pregnancy after Transfer of In Vitro Produced (IVP Sheep Embryos.

    Directory of Open Access Journals (Sweden)

    Paola Toschi

    Full Text Available Pregnancies obtained by Assisted Reproductive Technologies (ART are associated with limited maternal nutrient uptake. Our previous studies shown that in vitro culture of sheep embryos is associated with vascularization defects in their placentae and consequent reduction of embryo growth. Autophagy is a pro-survival cellular mechanism triggered by nutrient insufficiency. Therefore, the goal of our present study was to determine if autophagy is involved in early placental development after transfer of in vitro produced (IVP embryos. To do this, placentae obtained following transfer of IVP sheep embryos were compared with placentae obtained after natural mating (control-CTR. The placentae were collected on day 20 post-fertilization and post-mating, respectively, and were analyzed using molecular (qPCR, ultrastructural and histological/immunological approaches. Our results show drastically increased autophagy in IVP placentae: high levels of expression (p<0.05 of canonical markers of cellular autophagy and a high proportion of autophagic cells (35.08%; p<0.001 were observed. We conclude that high autophagic activity in IVP placentae can be a successful temporary counterbalance to the retarded vasculogenesis and the reduction of foetal growth observed in pregnancies after transfer of IVP embryos.

  2. Piglets produced by transfer of vitrified porcine embryos after stepwise dilution of cryoprotectants.

    Science.gov (United States)

    Kobayashi, S; Takei, M; Kano, M; Tomita, M; Leibo, S P

    1998-02-01

    A total of 498 porcine embryos at various stages of development collected from superovulated gilts was used to investigate cryopreservation. First, blastocysts (BL), expanded blastocysts (ExB), and hatched blastocysts (HB) were used to determine the effect of exposure to concentrated solutions of ethylene glycol as cryoprotective additives (CPAs) on embryo survival. Then, survival of other embryos after vitrification by rapid cooling was determined. Based on their development after 48 h in culture, embryos were not injured by being exposed to 2.0 M ethylene glycol (EG) for 15 min or to 2.0 M EG for 5 min and then to a solution of 8.0 M EG in 7% polyvinylpyrrolidone (PVP) for 1 min. The CPAs were removed from the embryos by diluting them with 1.7 M galactose. To vitrify the embryos, they were exposed to 2.0 M EG for 5 min and then were pipetted directly into short columns of 8.0 M EG-PVP contained within (1.25-ml plastic straws and separated from long columns of 1.7 M galactose by an air bubble. The straws were plunged directly into LN2. After the straws were warmed rapidly in a 25 degrees C water bath, the embryos were immediately mixed with galactose within the straws by shaking them vigorously to mix the contents. In sequential experiments, three methods were used to dilute the CPA solutions. Method 1: Embryos in the EG-PVP-galactose mixture were expelled from the straws and rinsed and cultured in modified CZB medium (mCZB). Method II: Embryos in the mixture were placed briefly into 1.5 M EG and then rinsed and cultured in mCZB. Method III: Embryos in the mixture were rinsed in 1.0 M EG and then in 0.5 M EG and finally rinsed with mCZB and cultured. After 48 h in culture, the respective percentages of survival of embryos vitrified as BL, ExB, or HB were: Method I, 21, 32, and 13%; Method II, 9, 40, and 24%; Method III, 35, 85, and 71%. Of 20 additional ExB vitrified embryos diluted by Method III and transferred into a recipient, four developed into live piglets

  3. Improved bovine embryo production in an oviduct-on-a-chip system : prevention of poly-spermic fertilization and parthenogenic activation

    NARCIS (Netherlands)

    Ferraz, Marcia A.M.M.; Henning, Heiko H.W.; Costa, Pedro F.; Malda, Jos; Melchels, Ferry P.W.; Wubbolts, Richard; Stout, Tom A.E.; Vos, Peter L.A.M.; Gadella, Bart M.

    2017-01-01

    The oviduct provides the natural micro-environment for gamete interaction, fertilization and early embryo development in mammals, such as the cow. In conventional culture systems, bovine oviduct epithelial cells (BOEC) undergo a rapid loss of essential differentiated cell properties; we aimed to

  4. 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, a...

  5. 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.

  6. Banteng (Bos javanicus) embryos and pregnancies produced by interspecies nuclear transfer.

    Science.gov (United States)

    Sansinena, M J; Hylan, D; Hebert, K; Denniston, R S; Godke, R A

    2005-03-01

    The banteng (Bos javanicus), a member of the bovidae family, is currently listed as threatened by the IUCN Red List and it is estimated the total world population is banteng fibroblasts and a total of 103 bovine oocytes were parthenogenically activated as a control (Treatment C). There was no significant difference in fusion rate (68 versus 77%) between Treatments A and B. Of fused couplets, those in Treatment A had greater (P banteng karyoplasts, and was capable of reprogramming the nucleus to achieve blastocyst stage embryos and pregnancies in exotic bovids.

  7. Assisted reproduction technique outcomes for fresh versus deferred cryopreserved day-2 embryo transfer: a retrospective matched cohort study.

    Science.gov (United States)

    Bourdon, Mathilde; Santulli, Pietro; Gayet, Vanessa; Maignien, Chloé; Marcellin, Louis; Pocate-Cheriet, Khaled; Chapron, Charles

    2017-03-01

    Ovarian stimulation could adversely affect endometrial receptivity and consequently embryo implantation. One emerging strategy is the 'freeze-all' approach. Most studies have focused on blastocyst transfers, with limited research on day-2 deferred cryopreserved embryo transfers. In this large retrospective cohort study, outcomes were compared between day-2 fresh versus deferred cryopreserved embryo transfers. After matching by age and number of previous cycles, 325 cycles were included in the fresh group and 325 in the deferred cryopreserved embryo transfers group: no significant differences were found between groups in implantation (0.20 ± 0.33 versus 0.17 ± 0.31, respectively) and ongoing pregnancy rates (21.85% versus 18.46%). Independent predictors for ongoing pregnancy after a multiple logistic regression analysis were the women's age (OR = 0.92; 95% CI 0.88 to 0.97), body mass index (OR = 0.94; 95% CI 0.89 to 0.99), the number of two pronuclei embryos (OR = 1.19; 95% CI 1.04 to 1.40) and at least one grade 1 embryo transferred (OR = 1.97; 95% CI 1.26 to 3.05). In the case of a day-2 embryo transfer, outcomes after treatment with assisted reproduction techniques are similar for fresh versus deferred cryopreserved embryo transfers when pre-transfer progesterone exposures are similar in the two groups. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  8. Which set of embryo variables is most predictive for live birth? A prospective study in 6252 single embryo transfers to construct an embryo score for the ranking and selection of embryos.

    Science.gov (United States)

    Rhenman, A; Berglund, L; Brodin, T; Olovsson, M; Milton, K; Hadziosmanovic, N; Holte, J

    2015-01-01

    Which embryo score variables are most powerful for predicting live birth after single embryo transfer (SET) at the early cleavage stage? This large prospective study of visual embryo scoring variables shows that blastomere number (BL), the proportion of mononucleated blastomeres (NU) and the degree of fragmentation (FR) have independent prognostic power to predict live birth. Other studies suggest prognostic power, at least univariately and for implantation potential, for all five variables. A previous study from the same centre on double embryo transfers with implantation as the end-point resulted in the integrated morphology cleavage (IMC) score, which incorporates BL, NU and EQ. A prospective cohort study of IVF/ICSI SET on Day 2 (n = 6252) during a 6-year period (2006-2012). The five variables (BL NU, FR, EQ and symmetry of cleavage (SY)) were scored in 3- to 5-step scales and subsequently related to clinical pregnancy and LBR. A total of 4304 women undergoing IVF/ICSI in a university-affiliated private fertility clinic were included. Generalized estimating equation models evaluated live birth (yes/no) as primary outcome using the embryo variables as predictors. Odds ratios with 95% confidence intervals and P-values were presented for each predictor. The C statistic (i.e. area under receiver operating characteristic curve) was calculated for each model. Model calibration was assessed with the Hosmer-Lemeshow test. A shrinkage method was applied to remove bias in c statistics due to over-fitting. LBR was 27.1% (1693/6252). BL, NU, FR and EQ were univariately highly significantly associated with LBR. In a multivariate model, BL, NU and FR were independently significant, with c statistic 0.579 (age-adjusted c statistic 0.637). EQ did not retain significance in the multivariate model. Prediction model calibration was good for both pregnancy and live birth. We present a ranking tree with combinations of values of the BL, NU and FR embryo variables for optimal

  9. Effect of insulin-like growth factor-1 during in vitro oocyte maturation and in vitro culture of bovine embryos

    Directory of Open Access Journals (Sweden)

    Quetglas M.D.

    2001-01-01

    Full Text Available The effects of insulin-like growth factor-I (IGF-I on in vitro maturation (IVM (experiment I and on in vitro embryo development (experiment II of bovine oocytes fertilized in vitro, were evaluated in terms of cleavage (CR, blastocyst (BR and hatching (HR rates. For IVM, immature cumulus-oocyte complexes were cultured in TCM-199 medium supplemented with Hepes, sodium bicarbonate, sodium pyruvate, additives, fetal calf serum (B-199 medium and gonadotropins (14 U/ml PMSG and 7 U/ml hCG. For embryo development, the oocytes/zygotes were cultured in B-199 medium with bovine oviduct epithelial cells in suspension under silicon oil. Treatments for in vitro culture conditions for both experiments were: 1- B-199 + 200 ng/ml IGF-I; 2- B-199 + 100 ng/ml IGF-I; 3- B-199 + 50 ng/ml IGF-I; 4- B-199 + 10 ng/ml IGF-I; 5- B-199 + 0 ng/ml IGF-I. All cultures were performed at 38.5ºC in 5% CO2 in air and the data were analyzed by chi-square test. In experiment I, there were no differences (P>0.05 among treatments for CR, BR or HR. In experiment II, the addition of IGF-I to the embryo culture medium (ECM resulted in a significant increase in CR while for BR and HR this effect was not observed. The addition of 200 ng/ml IGF-I to ECM increased CR (71.1% when compared to 100 ng/ml IGF-I (57.6% or control (56.7% groups, however, there were no differences when compared to 50 (69.4% or 10 ng/ml (73.1% groups. There was no beneficial effect of the addition of IGF-I in the IVM or ECM media on the in vitro development of embryos produced from oocytes matured and fertilized in vitro.

  10. Improvement of bovine in vitro embryo production by ovarian follicular wave synchronization prior to ovum pick-up.

    Science.gov (United States)

    Cavalieri, F L B; Morotti, F; Seneda, M M; Colombo, A H B; Andreazzi, M A; Emanuelli, I P; Rigolon, L P

    2017-11-26

    This study evaluated the effects of the synchronization of ovarian follicular wave emergence on the efficiency of in vitro embryo production. Bos indicus cows (n = 20) were divided into two groups (control vs. synchronization) and subjected to repeated ovum pick-up (OPU) sessions (8 replicates each, with an interval of 21 days in a 2 × 2 crossover design) and subsequent in vitro embryo production. Cows in the control group (n = 10) were submitted to OPU procedures without any stimulation every 21 days. Animals in the synchronization group received a protocol-based progesterone implant, estradiol benzoate and prostaglandin on a random day of the estrus cycle (Day 0) and the OPU was performed on Day 5. After in vitro production, embryos were transferred to recipients synchronized at a fixed time and the diagnosis was performed 60 days later. An evaluation of the parameters for each OPU session revealed that donors that received the synchronization protocol pre-OPU showed a greater number of embryos (5.9 ± 0.5 vs. 4.5 ± 0.4; P = 0.037), higher rate of embryo production (45.8% vs. 38.5%; P = 0.001) and higher mean number of conceptions per group (2.2 ± 0.2 vs. 1.6 ± 0.2; P = 0.07) in relation to the group that did not receive hormonal treatment. We concluded that synchronization of the follicular wave prior to OPU showed positive effects on in vitro embryo production as well as on pregnancy rates. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Application of in vitro production-embryo transfer in the protection ...

    African Journals Online (AJOL)

    Yomi

    2012-01-12

    Jan 12, 2012 ... The advances of assisted reproductive technology (ART) can effectively protect and multiply the superior genes of lactation and reproduction within the dairy farms. Key words: In vitro production, embryo transfer, genetics potential, dairy cattle. INTRODUCTION. Since the birth of the first calf derived from in ...

  12. Re-Thinking Elective Single Embryo Transfer: Increased Risk of Monochorionic Twinning - A Systematic Review.

    Science.gov (United States)

    Dziadosz, Margaret; Evans, Mark I

    2017-01-01

    Multiple pregnancies have tripled in the United States over the past 3 decades. Attributed to increasing maternal age at delivery but more so assisted reproductive technological advances, an effort has been made to decrease twinning through elective single embryo transfer. We sought to review and evaluate risks of monochorionic twinning as a predictable consequence of increasing utilization of elective single embryo transfer on perinatal outcomes. Primary outcomes included twinning rates, fetal anomalies, growth, preterm birth, and mortality. Secondary outcomes included neurological and pulmonary disability, intrauterine growth restriction, and congenital cardiac anomalies and twin-twin transfusion syndrome. PubMed and Embase. A total of 106 studies identified by systematic search met the inclusion criteria. The trend for lower numbers of embryos transferred has inadvertently led to an increase in monochorionic twinning. This is associated with worse outcomes compared to dichorionic twinning and singleton gestations for all outcomes studied. Of great concern for monochorionic twins is the risk profile of significant morbidity and mortality. Transfer of 2 embryos should be considered to avoid higher risks inherent to the shared placental phenomena related to monochorionic twins. © 2017 S. Karger AG, Basel.

  13. Term delivery following pyometra after in vitro fertilization and embryo transfer

    OpenAIRE

    Sathya Balasubramanyam; Thankam R Varma

    2014-01-01

    A 30 year old woman presented 12 days after embryo transfer with lower abdominal pain and orange vaginal discharge. She was diagnosed to have pyometra. A conservative management with drainage of the pyometra was followed by an uneventful pregnancy and term delivery. Conservative management in a case of pregnancy with pyometra needs close supervision to ensure maternal and fetal well being.

  14. Term delivery following pyometra after in vitro fertilization and embryo transfer

    Directory of Open Access Journals (Sweden)

    Sathya Balasubramanyam

    2014-01-01

    Full Text Available A 30 year old woman presented 12 days after embryo transfer with lower abdominal pain and orange vaginal discharge. She was diagnosed to have pyometra. A conservative management with drainage of the pyometra was followed by an uneventful pregnancy and term delivery. Conservative management in a case of pregnancy with pyometra needs close supervision to ensure maternal and fetal well being.

  15. Artificial cryopreserved embryo transfer cycle success depends on blastocyst developmental rate and progesterone timing

    DEFF Research Database (Denmark)

    Ozgur, Kemal; Bulut, Hasan; Berkkanoglu, Murat

    2018-01-01

    This retrospective cohort analysis compared the developmental competence of cryopreserved day-4 and 5 blastocysts, and investigated the effect of progesterone administration duration on the success of artificial frozen embryo transfers. Between October 2015 and March 2016, 868 intracytoplasmic....... The retrospective analysis and lack of adjustment for all known confounding variables limit the study....

  16. Factors associated with dizygotic twinning after IVF treatment with double embryo transfer

    NARCIS (Netherlands)

    Groeneveld, E.; Lambers, M.J.; Stakelbeek, M.E.; Mooij, T.M.; Belt-Dusebout, A.W. van den; Heymans, M.W.; Schats, R.; Hompes, P.G.; Hoek, A.; Burger, C.W.; Leeuwen, F.E. van; Lambalk, C.B.; Kortman, M.; Laven, J.S.E.; Jansen, C.A; Helmerhorst, F.M.; Cohlen, B.J.; Braat, D.D.M.; Smeenk, J.M.; Simons, A.H.; Veen, F. van der; Evers, J.L.; Dop, P.A. van

    2012-01-01

    BACKGROUND: Dizygotic twin pregnancies after IVF treatment are the result of multiple embryos transferred into the uterine cavity, followed by successful double implantation. Factors that increase the chance of multiple implantation after IVF are relatively unknown. The present study aimed to

  17. Factors associated with dizygotic twinning after IVF treatment with double embryo transfer

    NARCIS (Netherlands)

    Groeneveld, E.; Lambers, M. J.; Stakelbeek, M. E. F.; Mooij, T. M.; van den Belt-Dusebout, A. W.; Heymans, M. W.; Schats, R.; Hompes, P. G. A.; Hoek, A.; Burger, C. W.; van Leeuwen, F. E.; Lambalk, C. B.

    2012-01-01

    Dizygotic twin pregnancies after IVF treatment are the result of multiple embryos transferred into the uterine cavity, followed by successful double implantation. Factors that increase the chance of multiple implantation after IVF are relatively unknown. The present study aimed to investigate

  18. [Analysis of pregnancy outcomes of polycystic ovary syndrome patients after frozen embryo transfer].

    Science.gov (United States)

    Lyu, X D; Qiao, J

    2018-01-25

    Objective: To investigate pregnancy outcomes of the patients with polycystic ovary syndrome (PCOS) after frozen embryo transfer (FET) . Methods: Data of 2 367 PCOS patients received in vitro fertilization-embryo transfer [including fresh embryo transfer (fET) and FET] from January 2009 to December 2015 in Peking University Third Hospital were evaluated retrospectively. The basal characteristics, pregnancy complications and outcomes were analyzed, then identified the relative factors followed. Results: Totally 2 367 patients received in vitro fertilization-embryo transfer: 1 106 were treated with fET, and the rest 1 261 cases were treated with FET. The incidence of gestational diabetes mellitus (GDM) was lower in FET group [4.04%(51/1 261) versus 6.15%(68/1 106)], the difference was statistically significant ( Ppregnancy complications between the two groups (all P> 0.05). fET was an independent risk factor for GDM (adjusted OR= 1.570, 95% CI: 1.075-2.294). Conclusion: Compared with fET, FET could decrease the risk of GDM and receive better neonatal outcomes in patients with PCOS.

  19. Randomized trial of harp therapy during in vitro fertilization-embryo transfer.

    Science.gov (United States)

    Murphy, Erin M; Nichols, Jennifer; Somkuti, Steve G; Sobel, Michael; Braverman, Andrea; Barmat, Larry I

    2014-04-01

    This study evaluated whether harp therapy reduces levels of stress and improves clinical outcomes in patients undergoing embryo transfer. This prospective randomized trial enrolled 181 women undergoing embryo transfer, who were randomized to harp therapy during embryo transfer or standard treatment. Patients underwent standardized psychological testing and physiologic assessment of stress. The study was conducted in a reproductive medicine practice. No statistically significant differences were found in the heart and respiratory rates, nor was there a significant difference in event-based anxiety at baseline. Harp therapy had a significantly larger decrease in state anxiety from pre- to post-embryo transfer. Clinical pregnancy was 53% versus 48% for the harp therapy and standard treatment groups, respectively. Harp therapy decreases state, or event-based, anxiety, significantly lowering state scores posttransfer and having a positive effect on acute levels of stress. There was an increased pregnancy rate, but larger sample sizes are needed to evaluate whether harp therapy has an effect on clinical outcomes.

  20. Numerical chromosome errors in day 7 somatic nuclear transfer bovine blastocysts

    DEFF Research Database (Denmark)

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

    2003-01-01

    families, consisting of 112 blastocysts reconstructed from five different primary granulosa cell cultures, were examined. Overall, the mean chromosome complement within embryos was 86.9 +/- 3.7% (mean +/- SEM) diploid, 2.6 +/- 0.5% triploid, 10.0 +/- 3.1% tetraploid, and 0.5 +/- 0.2% pentaploid or greater......-free manipulation method: half-cytoplasts were made from zona-free oocytes by bisection, after which two half-oocytes and one granulosa cell (serum-starved primary culture) were fused together and activated. The NT embryos were cultured in modified synthetic oviductal fluid containing essential and nonessential...... amino acids, myoinositol, sodium citrate, and 5% cattle serum in microwells for 7 days, at which time nuclei from all blastocysts were extracted and chromosome aberrations were evaluated using dual-color fluorescent in situ hybridization with bovine chromosome 6- and 7-specific probes. Five embryo clone...

  1. Morphological assessment on day 4 and its prognostic power in selecting viable embryos for transfer.

    Science.gov (United States)

    Fabozzi, Gemma; Alteri, Alessandra; Rega, Emilia; Starita, Maria Flavia; Piscitelli, Claudio; Giannini, Pierluigi; Colicchia, Antonio

    2016-08-01

    The aim of this study was to describe a system for embryo morphology scoring at the morula stage and to determine the efficiency of this model in selecting viable embryos for transfer. In total, 519 embryos from 122 patients undergoing intracytoplasmic sperm injection (ICSI) were scored retrospectively on day 4 according to the grading system proposed in this article. Two separate quality scores were assigned to each embryo in relation to the grade of compaction and fragmentation and their developmental fate was then observed on days 5 and 6. Secondly, the prediction value of this scoring system was compared with the prediction value of the traditional scoring system adopted on day 3. Morulas classified as grade A showed a significant higher blastocyst formation rate (87.2%) compared with grades B, C and D (63.8, 41.3 and 15.0%, respectively), (P < 0.001). Furthermore, the ability to form top quality blastocysts was significantly higher for grade A morulas with respect to grades B, and C and D (37.8% vs. 22.4% vs. 11.1%), (P < 0.001). Finally, the morula scoring system showed more prediction power with respect to the embryo scoring a value of 1 [Akaike information criterion (AIC) index 16.4 vs. 635.3 and Bayesian information criterion (BIC) index -68.8 vs. -30.0 for morulas and embryos respectively]. In conclusion, results demonstrated that the presented scoring system allows for the evaluation of eligible embryos for transfer as a significant correlation between the grade of morula, blastulation rate and blastocyst quality was observed. Furthermore, the morula scoring system was shown to be the best predictive model when compared with the traditional scoring system performed on day 3.

  2. Supplementation of l-carnitine during in vitro maturation improves embryo development from less competent bovine oocytes.

    Science.gov (United States)

    Knitlova, Drahomira; Hulinska, Pavlina; Jeseta, Michal; Hanzalova, Katerina; Kempisty, Bartosz; Machatkova, Marie

    2017-10-15

    The present study was designed to define the impact of l-carnitine, supplemented during maturation, on bovine oocytes with different meiotic competence in terms of their IVF outcomes. Meiotically more competent (MMC) and less competent (MLC) oocytes were obtained separately from differently sized follicles at selected phases of folliculogenesis. The oocytes were matured with or without l-carnitine, fertilized and cultured to the blastocyst stage. The oocytes were examined for nuclear maturation, mitochondrial cluster formation, lipid consumption, fertilization and embryo development. The proportion of oocytes at metaphase II was significantly higher in the l-carnitine-treated MMC than that in the l-carnitine-treated MLC oocytes. However in comparison with the untreated controls, the proportion of MII oocytes with mitochondrial clusters was significantly higher only in the l-carnitine-treated MLC oocytes, which also showed a significantly lower mean lipid content. The l-carnitine-treated MLC oocytes showed significantly higher fertilization and syngamy rates than the untreated MLC oocytes. On the other hand, in the l-carnitine-treated MMC oocytes, the fertilization rate was similar to that of the untreated controls and the syngamy rate was significantly delayed. Although no significant differences in cleavage on Day 2 were found among all oocyte categories, l-carnitine treatment resulted in a significantly higher blastocyst yield in the MLC oocytes on Day 7 and Day 8 and a significantly higher proportion of expanded blastocysts in relation to the total number of blastocysts in MMC oocytes on Day 8. It can be concluded that l-carnitine supplementation during maturation improves the development of bovine embryos from meiotically less competent oocytes and accelerates blastocyst formation from more competent oocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Correlation of technical difficulty during embryo transfer with rate of clinical pregnancy

    Directory of Open Access Journals (Sweden)

    Neeta Singh

    2012-01-01

    Full Text Available Aim: To correlate the ease or difficulty of embryo transfer and blood at catheter tip with pregnancy rate when embryo transfer (ET was performed by the same operator using soft catheter. Materials and Methods: A retrospective analysis of 342 patients who underwent in vitro fertilization or ICSI cycle from January 2008 to December 2010 in a single centre was done. The type of transfer was divided into two groups: ′easy′ or ′difficult′. Transfer was considered difficult when additional instrumentation was required or firmer catheter was used or required changing of catheter. Patients undergoing cryo-preserved ET were excluded from the study. Results: On the day of transfer in 284 (83% patients, ET was easy and difficulty was encountered in 58 (17% patients. Blood at catheter was seen in 101 (29.53% patients. In the group of 58 difficult transfers, 10 pregnancies resulted with a clinical pregnancy rate of 17.2%, while 67 pregnancies resulted in 284 cycles of easy transfer with clinical pregnancy rate of 23.6% (P value = 0.045. While no significant difference was seen in pregnancies with blood on outer catheter and blood less transfer, there was significant reduction in pregnancy rate when blood was present on catheter tip compared to bloodless transfer (13.3% v/s 24.1; P value = 0.032. Conclusion: Reduction in clinical pregnancy rate is seen with difficult ETs, more when blood is present at the catheter tip.

  4. Increasing The Number of Embryos Transferred from Two to Three, Does not Increase Pregnancy Rates in Good Prognosis Patients

    Directory of Open Access Journals (Sweden)

    Mahnaz Ashrafi

    2015-10-01

    Full Text Available Background: To compare the pregnancy outcomes after two embryos versus three embryos transfers (ETs in women undergoing in vitro fertilization (IVF/intracytoplasmic sperm injection (ICSI cycles. Materials and Methods: This retrospective study was performed on three hundred eighty seven women with primary infertility and with at least one fresh embryo in good quality in order to transfer at each IVF/ICSI cycle, from September 2006 to June 2010. Patients were categorized into two groups according to the number of ET as follows: ET2 and ET3 groups, indicating two and three embryos were respectively transferred. Pregnancy outcomes were compared between ET2 and ET3 groups. Chi square and student t tests were used for data analysis. Results: Clinical pregnancy and live birth rates were similar between two groups. The rates of multiple pregnancies were 27 and 45.2% in ET2 and ET3 groups, respectively. The rate of multiple pregnancies in young women was significantly increased when triple instead of double embryos were transferred. Logistic regression analysis indicated two significant prognostic variables for live birth that included number and quality of transferred embryos; it means that the chance of live birth following ICSI treatment increased 3.2-fold when the embryo with top quality (grade A was transferred, but the number of ET had an inverse relationship with live birth rate; it means that probability of live birth in women with transfer of two embryos was three times greater than those who had three ET. Conclusion: Due to the difficulty of implementation of the elective single-ET technique in some infertility centers in the world, we suggest transfer of double instead of triple embryos when at least one good quality embryo is available for transfer in women aged 39 years or younger. However, to reduce the rate of multiple pregnancies, it is recommended to consider the elective single ET strategy.

  5. Effects of fetal calf serum, phenazine ethosulfate and either glucose or fructose during in vitro culture of bovine embryos on embryonic development after cryopreservation.

    Science.gov (United States)

    Barceló-Fimbres, M; Seidel, G E

    2007-11-01

    This study investigated effects of hexoses, fetal calf serum (FCS), and phenazine ethosulfate (PES) during the culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. The basal, control medium was chemically defined (CDM) plus 0.5% fatty acid-free BSA. In vitro-produced bovine zygotes were cultured in CDM-1 with 0.5 mM glucose; after 60 hr, 8-cell embryos were cultured 4.5 days in CDM-2. The 8-cell embryos were randomly allocated to a 2 x 3 x 2 x 3 factorial experimental design with two energy substrates (2 mM glucose or fructose); three additives (0.3 microM PES, 10% FCS, and control); two cryopreservation methods using no animal products (conventional slow freezing or vitrification); and semen from three bulls with two replicates for each bull. A total of 1,107 blastocysts were produced. Fructose resulted in 13% more blastocysts per oocyte than glucose (37.2% vs. 32.9%), and per 8-cell embryo (51.3% vs. 45.3%; P 0.1) control, FCS, or PES for blastocysts per oocyte or per 8-cell embryo. There was a significant interaction (P embryos with PES, which reduces cytoplasmic lipid content, improved cryotolerance of bovine embryos; post-cryopreservation survival of blastocysts averaged over vitrification and slow freezing (between which there was no difference) was 91.9%, 84.9%, and 60.2% of unfrozen controls (P < 0.01) for PES, control, and FCS groups, respectively. (c) 2007 Wiley-Liss, Inc.

  6. Predictive value of plasma human chorionic gonadotropin measured 14 days after Day-2 single embryo transfer

    DEFF Research Database (Denmark)

    Løssl, Kristine; Oldenburg, Anna; Toftager, Mette

    2017-01-01

    Introduction: Prediction of pregnancy outcome after in vitro fertilization is important for patients and clinicians. Early plasma human chorionic gonadotropin (p-hCG) levels are the best known predictor of pregnancy outcome, but no studies have been restricted to single embryo transfer (SET) of Day......-2 embryos. The aim of the present study was to investigate the predictive value of p-hCG measured exactly 14 days after the most commonly used Day-2 SET on pregnancy, delivery, and perinatal outcome. Material and methods: A retrospective analysis of prospectively collected data on 466 women who had...

  7. [In vitro fertilization and embryo transfer. Personal experiences in the introduction of laboratory methodology].

    Science.gov (United States)

    Fliess, F R; Sudik, R

    1984-01-01

    Our laboratory technique includes recovery and handling with gametes outside the body, in vitro fertilization, culture of embryos, and finally embryo-transfer. Time-table, culture media and technique of the cultivation as well as laboratory devices are described and than compared with informations about methods from other teams. The necessity of transport of the follicle fluids from operating theatre to laboratory is given caused by local conditions. All dates about number of oocytes (73), quantities of preovulatory oocytes (47) and cleavage rates (55%) are descended from 30 laparoscopic oocyte recoveries of the 2. in vitro fertilization program of the university woman hospital in Rostock, January and February 1984.

  8. The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

    DEFF Research Database (Denmark)

    Morovic, Martin; Murin, Matej; Strejcek, Frantisek

    2016-01-01

    One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription....... In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly...... influenced by the ooplasmic environment....

  9. Reduction in cytoplasmic lipid content in bovine embryos cultured in vitro with linoleic acid in semi-defined medium is correlated with increases in cryotolerance.

    Science.gov (United States)

    Accorsi, Mônica F; Leão, Beatriz Caetano da Silva; Rocha-Frigoni, Nathália Alves de Souza; Perri, Silvia Helena Venturoli; Mingoti, Gisele Zoccal

    2016-08-01

    We examined whether culturing embryos with linoleic acid (LA) in semi-defined medium reduces lipid accumulation and improves cryosurvival after vitrification. Embryos were cultured with LA (100 μM) and a semi-defined medium was used during in vitro culture (IVC), in which the fetal calf serum was substituted by bovine serum albumin (BSA). There was a reduction (P embryos stained with the fluorescent dye Nile Red), consequently increasing (P = 0.0490) the embryo survival after 24h of culture post-warming ( 50.0% versus LA: 71.7%). The results question the criteria used to evaluate the efficiency of an in vitro production system specifically with relation to the maximum number of blastocysts produced and suggest that might be more appropriate to improve the desired characteristics of embryos generated in accordance with the specific purpose of in vitro embryo production, commercial or scientific. In conclusion, supplying LA to serum-free culture medium was found to adversely affect the rates of embryo development to the blastocyst stage, but significantly reduced embryo lipid accumulation and improved cryopreservation survival.

  10. Effect of Adding Human Chorionic Gonadotropin to The Endometrial Preparation Protocol in Frozen Embryo Transfer Cycles

    Directory of Open Access Journals (Sweden)

    Maryam Eftekhar

    2012-01-01

    Full Text Available Background: Human chorionic gonadotropin (HCG, one of the initial embryonic signals, isprobably a major regulator of the embryo-endometrial relationship. This study aims to assess theadvantage of HCG supplementation during the secretory phase of hormonally prepared cycles forthe transfer of cryopreserved-thawed embryos.Materials and Methods: This study was a randomized clinical trial. Infertile women who werecandidates for frozen-thawed embryo transfers entered the study and were divided into two groups,HCG and control. The endometrial preparation method was similar in both groups: all women receivedestradiol valerate (6 mg po per day from the second day of the menstrual cycle and progesteronein oil (100 mg intramuscular (I.M. when the endometrial thickness reached 8 mm. Estradiol andprogesterone were continued until the tenth week of gestation. In the HCG group, patients received anHCG 5000 IU injection on the first day of progesterone administration and the day of embryo transfer.Results: In this study, 130 couples participated: 65 in the HCG group and 65 in the control group.There was no statistically significant difference between groups regarding basic characteristics.Implantation rate, chemical pregnancy, clinical pregnancy, ongoing pregnancy, and abortion rateswere similar in both groups.Conclusion: Although HCG has some advantages in assisted reproductive technology (ARTcycles, our study did not show any benefit of HCG supplementation during the secretory phase offrozen cycles (Registration Number: IRCT201107266420N4.

  11. Effect of Temporary Meiotic Attenuation of Oocytes with Butyrolactone I and Roscovitine in Resistance to Bovine Embryos on Vitrification.

    Science.gov (United States)

    Maziero, R R D; Guaitolini, C R F; Paschoal, D M; Kievitsbosch, T; Guastali, M D; Moraes, C N; Landim-Alvarenga, F C

    2016-04-01

    This study aimed to produce in vitro bovine embryos by the addition of two drugs, which is responsible for oocyte meiosis inhibition: roscovitine (ROS) and butyrolactone I (BL-I). Oocytes were recovered from slaughtered cows and matured in a commercial medium and maintained in a 5% CO2 atmosphere. Oocytes were maintained for 6 h in an in vitro maturation (IVM) medium containing ROS (12.5 μm), BL-I (50 μm) and association of drugs (ROS 6.25 μm and BL-I 25 μm). Oocytes were cultured for 18 h in an agent-free medium for the resumption of meiosis. After 24 h of maturation, oocytes were inseminated in the commercial in vitro fertilization (IVF) medium. Presumptive zygotes were cultured in SOFaa medium in a 5% CO2 atmosphere. On day 3, rate of cleavage was evaluated and on days 6 and 7, rate of blastocyst formation. BL-I and its association with the ROS increased the rates of cleavage and blastocyst formation (p vitrification process, presenting a higher rate of embryonic re-expansion (p < 0.05). In conclusion, block of meiosis using BL-I or its association with ROS increased the rate of blastocyst formation, and the association of ROS+BL-I resulted in a better resistance to the embryo cryopreservation process. © 2016 Blackwell Verlag GmbH.

  12. 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.

  13. Correlation between the cryosurvival, cell number and diameter in bovine in vitro produced embryos.

    Science.gov (United States)

    Kocyigit, Alper; Cevik, Mesut

    2016-10-01

    The selection of quality embryos is a prerequisite of cryopreservation process. Present study was conducted to examine the correlation between the diameter and cryotolerance, on the cell number of the cryopreserved embryos. The blastocyst stage embryos were collected at culture days 7-9, evaluated morphologically under a microscope and divided according to the diameter into three groups: Group 1; (larger than 150 μm), Group 2; (diameter of 100-150 μm), Group 3; (smaller than 100 μm). Blastocysts were vitrified-thawed using the classical vitrification method and then cultured in SOF medium drops at 24 h. Blastocysts were considered viable if they re-expanded or hatched from the zona pellucidae. Finally re-expanded blastocysts from the Group 1 and Group 2 to determine the differential count of cells in the ICM and TE. The re-expansion ability of blastocysts 100-150 μm in diameter (69.56%) was significantly higher than other groups (52.17 and 47.36%). The value of the correlation coefficient between the re-expansion rate and cell number of blastocysts in the group 2 (r = 0.784) tended to be higher than that in the group 1 (r = 0.512) and group 3 (r = 0.491) (p < 0.05). For ICM/total cell ratio yield group 2 embryos showed higher rate (0.28), compared to the other groups (0.19 and 0.16). In conclusion, the present study demonstrates that the correlation between diameter of embryos and their cryosurvival based on re-expansion ability and cell allocation. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Comparison of different fertilisation media for an in vitro maturation?fertilisation?culture system using flow-cytometrically sorted X chromosome-bearing spermatozoa for bovine embryo production.

    Science.gov (United States)

    Ferré, Luis B; Bogliotti, Yanina; Chitwood, James L; Fresno, Cristóbal; Ortega, Hugo H; Kjelland, Michael E; Ross, Pablo J

    2015-05-13

    High demand exists among commercial cattle producers for in vitro-derived bovine embryos fertilised with female sex-sorted spermatozoa from high-value breeding stock. The aim of this study was to evaluate three fertilisation media, namely M199, synthetic oviductal fluid (SOF) and Tyrode's albumin-lactate-pyruvate (TALP), on IVF performance using female sex-sorted spermatozoa. In all, 1143, 1220 and 1041 cumulus-oocyte complexes were fertilised in M199, SOF and TALP, respectively. There were significant differences among fertilisation media (P fertilisation medium. Embryos derived from SOF and TALP fertilisation media had higher numbers of ICM, TE and total cells than those fertilised in M199. In conclusion, fertilisation media affected cleavage rate, as well as subsequent embryo development, quality and hatching ability. SOF and TALP fertilisation media produced significantly more embryos of higher quality than M199.

  15. Ascorbic acid increases demethylation in somatic cell nuclear transfer embryos of the pig (Sus scrofa).

    Science.gov (United States)

    Zhao, Minghui; Hur, Tai-Young; No, Jingu; Nam, Yoonseok; Kim, Hyeunkyu; Im, Gi-Sun; Lee, Seunghoon

    2017-07-01

    Investigated the effect and mechanism of ascorbic acid on the development of porcine embryos produced by somatic cell nuclear transfer (SCNT). Porcine embryos were produced by SCNT and cultured in the presence or absence of ascorbic acid. Ten-eleven translocation 3 (TET3) in oocytes was knocked down by siRNA injection. After ascorbic acid treatment, reprogramming genes were analyzed by realtime reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, relative 5-methylcytosine and 5-hydroxymethylcytosine content in pronucleus were detected by realtime PCR. Ascorbic acid significantly increased the development of porcine embryos produced by SCNT. After SCNT, transcript levels of reprogramming genes, Pou5f1 , Sox2 , and Klf were significantly increased in blastocysts. Furthermore, ascorbic acid reduced 5-methylcytosine content in pronuclear embryos compared with the control group. Knock down of TET3 in porcine oocytes significantly prevents the demethylation of somatic cell nucleus after SCNT, even if in the presence of ascorbic acid. Ascorbic acid enhanced the development of porcine SCNT embryos via the increased TET3 mediated demethylation of somatic nucleus.

  16. Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology.

    Science.gov (United States)

    Glujovsky, Demián; Blake, Debbie; Farquhar, Cindy; Bardach, Ariel

    2012-07-11

    Advances in cell culture media have led to a shift in in vitro fertilization (IVF) practice from early cleavage embryo transfer to blastocyst stage transfer. The rationale for blastocyst culture is to improve both uterine and embryonic synchronicity and enable self selection of viable embryos thus resulting in higher implantation rates. To determine if blastocyst stage (Day 5 to 6) embryo transfers (ETs) improve live birth rate and other associated outcomes compared with cleavage stage (Day 2 to 3) ETs. Cochrane Menstrual Disorders and Subfertility Group Specialised Register of controlled trials, Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library), MEDLINE, EMBASE and Bio extracts. The last search date was 21 February 2012. Trials were included if they were randomised and compared the effectiveness of early cleavage versus blastocyst stage transfers. Of the 50 trials that were identified, 23 randomised controlled trials (RCTs) met the inclusion criteria and were reviewed (five new studies were added in this update). The primary outcome was rate of live birth. Secondary outcomes were rates per couple of clinical pregnancy, cumulative clinical pregnancy, multiple pregnancy, high order pregnancy, miscarriage, failure to transfer embryos and cryopreservation. Quality assessment, data extraction and meta-analysis were performed following Cochrane guidelines. Twelve RCTs reported live birth rates and there was evidence of a significant difference in live birth rate per couple favouring blastocyst culture (1510 women, Peto OR 1.40, 95% CI 1.13 to 1.74) (Day 2 to 3: 31%; Day 5 to 6: 38.8%, I(2) = 40%). This means that for a typical rate of 31% in clinics that use early cleavage stage cycles, the rate of live births would increase to 32% to 42% if clinics used blastocyst transfer.There was no difference in clinical pregnancy rate between early cleavage and blastocyst transfer in the 23 RCTs (Peto OR 1.14, 95% CI 0.99 to 1.32) (Day 2 to 3: 38

  17. Disparities in reproductive outcomes according to the endometrial preparation protocol in frozen embryo transfer : The risk of early pregnancy loss in frozen embryo transfer cycles.

    Science.gov (United States)

    Hatoum, I; Bellon, L; Swierkowski, N; Ouazana, M; Bouba, S; Fathallah, K; Paillusson, B; Bailly, M; Boitrelle, F; Alter, L; Bergère, M; Selva, J; Wainer, R

    2017-11-06

    The purpose of this study was to determine the effect of stimulated and artificial endometrial preparation protocols on reproductive outcomes in frozen embryo transfer (FET) cycles. We performed a retrospective study of 1926 FET cycles over a 3.5-year period in the Fertility Unit at a University Hospital. Stimulated and artificial protocols were used for endometrial preparation. The embryos for FET were obtained from either in vitro fertilization or intracytoplasmic sperm injection cycles. Live birth rate and early pregnancy loss rates were retrospectively compared. In artificial protocols, oral or vaginal administration of oestradiol 2 mg two or three times a day was followed by vaginal supplementation with progesterone 200 mg two or three times a day. In stimulated protocols, recombinant follicle-stimulating hormone was administered from day 4 onward. Vaginal ultrasound was used for endometrial and ovarian monitoring. A pregnancy test was performed 14 days after FET. If it was positive, oestradiol and progesterone were administered up until the 12th week of gestation in artificial cycles. We defined early pregnancy losses as biochemical pregnancies (preclinical losses) and miscarriages. Data on 865 artificial cycles (45% of the total) and 1061 stimulated cycles (55%) were collected. Early pregnancy loss rate was significantly lower for stimulated cycles (34.2%) than for artificial cycles (56.9%), and the live birth rate was significantly higher for stimulated cycles (59.7%) than for artificial cycles (29.1%). In frozen embryo transfer, artificial cycles were associated with more early pregnancy loss and lower live birth rate than stimulated cycles.

  18. Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Yao, Chaogang; Zhou, Yang; Zhu, Jianguo; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

    2011-12-01

    Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.

  19. The influence of season and sire on the results of superovulation and embryo transfer

    Directory of Open Access Journals (Sweden)

    Zdeňka Hegedűšová

    2006-01-01

    Full Text Available The aim of the study was evaluate the influence of season and sires on profit and quality of embryos after superovulated treatment. Next we evaluated the conception rate after transfer of fresh and frozen embryos.In 1991–2004 there were used the beef cattle. Into the basic statistic evaluation it was involved 487 realised embryo recoveries and 2008 realised transfers in 1991–2004. Data for database were obtaining from ETprotocols – ET team Research Institute for Cattle Breeding, Ltd., Rapotin, prof. Říha. The data processing was carried out by means of the common variation-statistical methods.The best results were achieved in summer (suitable 3.68 ± 3.65; the ratio of the suitable and total: 59.3% and in autumn (suitable 3.54 ± 3.80; the ratio: 54.48% and the good results, little different from the summer and autumn results, were achieved in spring.The average number of the recovered ova of the chosen breeds sires were variable (from 6.60 ± 6.17 in Blonde d´Aquitaine to 17.16 ± 6.66 in Charolais. The most of the suitable embryos was recovered in the donors inseminated by the Hereford breed sires (7.15 ± 6.42. It was evaluated the above-average conception in the Simmental breed (63.43 %.

  20. 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.

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

    Directory of Open Access Journals (Sweden)

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

    2014-02-01

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

  2. Influences of somatic donor cell sex on and embryo development following somatic cell nuclear transfer in pigs

    Directory of Open Access Journals (Sweden)

    Jae-Gyu Yoo

    2017-04-01

    Full Text Available Objective The present study investigates pre- and post-implantation developmental competence of nuclear-transferred porcine embryos derived from male and female fetal fibroblasts. Methods Male and female fetal fibroblasts were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence of reconstructed embryos was investigated. And, a total of 6,789 female fibroblast nuclear-transferred embryos were surgically transferred into 41 surrogate gilts and 4,746 male fibroblast nuclear-transferred embryos were surgically transferred into 25 surrogate gilts. Results The competence to develop into blastocysts was not significantly different between the sexes. The mean cell number of female and male cloned blastocysts obtained by in vivo culture (143.8±10.5 to 159.2±14.8 was higher than that of in vitro culture of somatic cell nuclear transfer (SCNT groups (31.4±8.3 to 33.4±11.1. After embryo transfer, 5 pregnant gilts from each treatment delivered 15 female and 22 male piglets. The average birth weight of the cloned piglets, gestation length, and the postnatal survival rates were not significantly different (p<0.05 between sexes. Conclusion The present study found that the sex difference of the nuclear donor does not affect the developmental rate of porcine SCNT embryos. Furthermore, postnatal survivability of the cloned piglets was not affected by the sex of the donor cell.

  3. Acupuncture on the day of embryo transfer: a randomized controlled trial of 635 patients

    DEFF Research Database (Denmark)

    Andersen, Dorota; Løssl, Kristine; Nyboe Andersen, Anders

    2010-01-01

    This prospective, randomized, controlled and double-blinded trial studied whether acupuncture in relation to embryo transfer could increase the ongoing pregnancy rates and live birth rates in women undergoing assisted reproductive therapy. A total of 635 patients undergoing IVF or intracytoplasmic...... sperm injection (ICSI) were included. In 314 patients, embryo transfer was accompanied by acupuncture according to the principles of traditional Chinese medicine. In the control group, 321 patients received placebo acupuncture using a validated placebo needle. In the acupuncture group and the placebo...... group, the ongoing pregnancy rates were 27% (95% CI 22-32) and 32% (95% CI 27-37), respectively. Live birth rates were 25% (95% CI 20-30) in the acupuncture group and 30% (95% CI 25-30) in the placebo group. The differences were not statistically significant. These results suggest that acupuncture...

  4. 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

  5. Comparison of neonatal outcomes following progesterone use during ovarian stimulation with frozen-thawed embryo transfer

    OpenAIRE

    Zhu, Xiuxian; Ye, Hongjuan; Fu, Yonglun

    2017-01-01

    Progesterone soft capsules (brand name: Utrogestan) were demonstrated to be an effective oral alternative to prevent premature LH surges both in normal-ovulatory and polycystic ovarian syndrome (PCOS) patients. However, its safety in terms of neonatal outcomes is unclear. To evaluate whether Utrogestan use increase the risk of adverse neonatal outcomes compared with short protocol in patients undergoing IVF/ICSI treatments in combination with frozen-thawed embryo transfer (FET), we performed ...

  6. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Pang, Daxin, E-mail: pdx@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Ouyang, Hongsheng, E-mail: ouyh@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China)

    2011-07-29

    Highlights: {yields} Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. {yields} The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. {yields} A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 {mu}g/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  7. Genetic variation in resistance of the preimplantation bovine embryo to heat shock.

    Science.gov (United States)

    Hansen, Peter J

    2014-12-01

    Reproduction is among the physiological functions in mammals most susceptible to disruption by hyperthermia. Many of the effects of heat stress on function of the oocyte and embryo involve direct effects of elevated temperature (i.e. heat shock) on cellular function. Mammals limit the effects of heat shock by tightly regulating body temperature. This ability is genetically controlled: lines of domestic animals have been developed with superior ability to regulate body temperature during heat stress. Through experimentation in cattle, it is also evident that there is genetic variation in the resistance of cells to the deleterious effects of elevated temperature. Several breeds that were developed in hot climates, including Bos indicus (Brahman, Gir, Nelore and Sahiwal) and Bos taurus (Romosinuano and Senepol) are more resistant to the effects of elevated temperature on cellular function than breeds that evolved in cooler climates (Angus, Holstein and Jersey). Genetic differences are expressed in the preimplantation embryo by Day 4-5 of development (after embryonic genome activation). It is not clear whether genetic differences are expressed in cells in which transcription is repressed (oocytes >100 µm in diameter or embryos at stages before embryonic genome activation). The molecular basis for cellular thermotolerance has also not been established, although there is some suggestion for involvement of heat shock protein 90 and the insulin-like growth factor 1 system. Given the availability of genomic tools for genetic selection, identification of genes controlling cellular resistance to elevated temperature could be followed by progress in selection for those genes within the populations in which they exist. It could also be possible to introduce genes from thermotolerant breeds into thermally sensitive breeds. The ability to edit the genome makes it possible to design new genes that confer protection of cells from stresses like heat shock.

  8. Activation of ribosomal RNA genes in porcine embryos produced in vitro or by somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Pedersen, Hanne Gervi; Jakobsen, Anne Sørig

    2007-01-01

    The onset of ribosomal RNA (rRNA) synthesis occurs during the second half of the third cell cycle, that is, at the four-cell stage, in porcine embryos developed in vivo. In the present study the onset of rRNA synthesis was investigated in porcine embryos produced in vitro (IVP) or by somatic cell...... nuclear transfer (SCNT) using fluorescence in situ hybridization (FISH) with an rDNA probe and subsequent visualization of the nucleolar proteins by silver staining. In the 205 IVP embryos investigated, all two-cell embryos (n = 34) were categorized as transcriptionally inactive. At the late four...

  9. Reduced progesterone concentration during growth of the first follicular wave affects embryo quality but has no effect on embryo survival post transfer in lactating dairy cows.

    Science.gov (United States)

    Rivera, Fernando A; Mendonça, Luís G D; Lopes, Gláucio; Santos, José E P; Perez, Rolando V; Amstalden, Marcel; Correa-Calderón, Abelardo; Chebel, Ricardo C

    2011-03-01

    Fertility of lactating dairy cows is associated with reduced progesterone (P(4)) concentration compared with nonlactating animals. The objective of the current study was to determine whether P(4) during growth of the first follicular wave (FFW) affects embryo quality. Lactating Holstein cows at 33±3 days post partum were allocated to one of three treatments. Cows in the FFW and FFW with P(4) (FFWP) treatments started the superstimulation protocol on day 1 of the estrous cycle and second follicular wave (SFW) cows started the superstimulation protocol on estrous cycle day 7. Cows were superstimulated with 400  mg of NIH-FSH-P1 (FSH) given twice daily for 5 days, two prostaglandin F(2α) (PGF(2α)) injections given with the ninth and tenth injections of FSH, GNRH given 48  h after the first PGF(2α) injection, and timed insemination 12 and 24  h after the GNRH injection. Cows in the FFWP treatment received two intravaginal P(4) inserts during the superstimulation. Embryos were recovered 6.5 days after artificial insemination and excellent/good and fair embryos were frozen and transferred. Blood was sampled daily from estrous cycle day 0 until insemination from donor cows. During the superstimulation protocol, P(4) was (P<0.01) greatest for SFW cows followed by FFWP and FFW cows respectively. The percentage of embryos-oocytes from SFW and FFWP cows classified as excellent/good and fair embryos was (P=0.02) greater than those of FFW cows. Pregnancy per embryo transfer was not (P≥0.73) affected by embryo donor treatment. Reduced embryo quality of cows induced to ovulate the follicles from the first follicular wave is a consequence of reduced P(4) during follicle growth.

  10. Membrane lipid profile monitored by mass spectrometry detected differences between fresh and vitrified in vitro-produced bovine embryos.

    Science.gov (United States)

    Leão, Beatriz C S; Rocha-Frigoni, Nathália A S; Cabral, Elaine C; Franco, Marcos F; Ferreira, Christina R; Eberlin, Marcos N; Filgueiras, Paulo R; Mingoti, Gisele Z

    2015-10-01

    This study aimed to evaluate the impact of vitrification on membrane lipid profile obtained by mass spectrometry (MS) of in vitro-produced bovine embryos. Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) has been used to obtain individual embryo membrane lipid profiles. Due to conditions of analysis, mainly membrane lipids, most favorably phosphatidylcholines (PCs) and sphingomyelins (SMs) have been detected. The following ions described by their mass-to-charge ratio (m/z) and respective attribution presented increased relative abundance (1.2-20×) in the vitrified group: 703.5 [SM (16:0) + H]+; 722.5 [PC (40:3) + Na]+; 758.5 [PC (34:2) + H]+; 762.5 [PC (34:0) + H]+; 790.5 [PC (36:0) + H]+ and 810.5 [PC (38:4) + H]+ and/or [PC (36:1) + Na]+. The ion with a m/z 744.5 [PCp (34:1) and/or PCe (34:2)] was 3.4-fold more abundant in the fresh group. Interestingly, ions with m/z 722.5 or 744.5 indicate the presence of lipid species, which are more resistant to enzymatic degradation as they contain fatty acyl residues linked through ether type bonds (alkyl ether or plasmalogens, indicated by the lowercase 'e' and 'p', respectively) to the glycerol structure. The results indicate that cryopreservation impacts the membrane lipid profile, and that these alterations can be properly monitored by MALDI-MS. Membrane lipids can therefore be evaluated by MALDI-MS to monitor the effect of cryopreservation on membrane lipids, and to investigate changes in lipid profile that may reflect the metabolic response to the cryopreservation stress or changes in the environmental conditions.

  11. Detection of early cleavage embryos improves pregnancy and delivery rates of Day 3 embryo transfer during in vitro fertilization.

    Science.gov (United States)

    Lee, Chun-I; Lee, Tsung-Hsien; Huang, Chun-Chia; Chen, Hsiu-Hui; Liu, Chung-Hsien; Lee, Maw-Sheng

    2016-08-01

    This study established a simple criterion for improving the pregnancy and delivery rates of Day 3 embryo transfer for in vitro fertilization (IVF) by assessing the early cleavage of two-cell stage embryos. In total, 258 cycle patients undergoing an IVF and Day 3 embryo transfer program were recruited. All cycles were divided into four groups containing viable Day 3 embryos and those (A) with distinct early cleavage (equal-sized blastomeres and ≤10% fragmentation: ECA grade); (B) with indistinct early cleavage (equal sized blastomeres, >2 blastomeres, or >10% fragmentation: ECB grade); (C) without early cleavage [no early cleavage (NEC grade)]; or (D) without early cleavage being assessed (control) at 25-27 after insemination. The percentage of viable Day 3 embryos from ECA grade (75.1%, 507/675) was significantly higher than that from ECB grade (19.2%, 151/403) or NEC grade (27.1%, 127/469) embryos (p ECA group [65.7% (65/990) and 48.5% (48/990), respectively] were significantly higher than those in the ECB group [30.8% (4/13) and 7.7% (1/13), respectively] or NEC group [36.8% (14/38) and 23.7% (9/38), respectively; all p ECA group (32.3%, 129/400) was higher than those in the ECB (6.8%, 4/59) and NEC (13.0%, 18/136) groups (p < 0.01). Simple selection using the early cleavage morphology may improve the pregnancy and delivery rates of Day 3 embryo transfer programs. Copyright © 2016. Published by Elsevier B.V.

  12. Simplification of bovine somatic cell nuclear transfer by application of a zona-free manipulation technique

    DEFF Research Database (Denmark)

    Booth, P J; Tan, S J; Reipurth, R

    2001-01-01

    Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods.......8% of cultured oocytes). Subsequent application of the optimized technique for nuclear transfer using nine different granulosa cell primary cultures (cultured in 0.5% serum for 5-12 days) generated 37.6 +/- 3.9% (11 replicates; range, 16.4-58.1 blastocysts per successfully fused and surviving reconstructed...... embryo (after activation), and 33.6 +/- 3.7% blastocysts per attempted reconstructed embryo. Mean day 7 total blastocyst cell numbers from 5 clone families was 128.1 +/- 15.3. The ongoing pregnancy rate of recipients each receiving two nuclear transfer blastocysts is 3/13 (23.1 recipients pregnant at 5...

  13. Comparison of ectopic pregnancy risk among transfers of embryos vitrified on day 3, day 5, and day 6.

    Science.gov (United States)

    Du, Tong; Chen, Hong; Fu, Rong; Chen, Qiuju; Wang, Yun; Mol, Ben W; Kuang, Yanping; Lyu, Qifeng

    2017-07-01

    To compare ectopic pregnancy risk among transfers of embryos vitrified on day 3, day 5, and day 6. Retrospective cohort study. Academic tertiary-care medical center. A total of 10,736 pregnancies after 23,730 frozen-thawed embryo transfer (FET) cycles of in vitro fertilization/intracytoplasmic sperm injection from March 2003 to May 2015. The ectopic pregnancy rate was compared among pregnancies resulting from transfers of embryos vitrified on day 3, day 5, and day 6. Generalized estimation equation regression models were used to calculate unadjusted and adjusted odds ratios and 95% confidence intervals for the association between ectopic pregnancy and selected patient and treatment characteristics. We studied this association in both the group that achieved pregnancy and the group that underwent an FET cycle. Odds of ectopic pregnancy. The overall rate of ectopic pregnancy was 2.8% (304/10,736). Ectopic pregnancy rates after day-3, day-5, and day-6 vitrified embryo transfers were 3.1% (287/9,224), 2.0% (11/562), and 0.6% (6/950), respectively. After adjusting for confounders, the risks of ectopic pregnancy in day-3 and day-5 vitrified embryo transfers were both significantly higher than in day-6 vitrified embryo transfers. The associations were similar when we did calculations per cycle. In women undergoing FET, day-6 vitrified embryo transfer is associated with a significantly lower risk of ectopic pregnancy than both day-3 and day-5 vitrified embryo transfers. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  14. Elective single-embryo transfer: persuasive communication strategies can affect choice in a young British population.

    Science.gov (United States)

    van den Akker, O B A; Purewal, S

    2011-12-01

    This study tested the effectiveness of the framing effect and fear appeals to inform young people about the risks of multiple births and the option of selecting elective single-embryo transfer (eSET). A non-patient student sample (age (mean±SD) 23±5.5 years; n=321) were randomly allocated to one of seven groups: (1) framing effect: (1a) gain and (1b) loss frame; (2) fear appeal: (2a) high, (2b) medium and (2c) low fear; or (3) a control group: (3a) education and (3b) non-education. The primary outcome measure was the Attitudes towards Single Embryo Transfer questionnaire, before exposure to the messages (time 1) and immediately afterwards (time 2). Results revealed participants in the high fear, medium fear and gain condition demonstrated the most positive and significant differences (Pmessages. The results demonstrate that the use of complex persuasive communication techniques on a student population to promote immediate and hypothetical eSET preferences is more successful at promoting eSET than merely reporting educational content. Future research should investigate its application in a clinical population. A multiple pregnancy is a health risk to both infant and mother following IVF treatment. The aims of this study were to test the effectiveness of two persuasive communication techniques (the framing effect and fear appeals) to inform young people about the risks of multiple births and the hypothetical option of selecting elective single-embryo transfer (eSET) (i.e., only one embryo is transferred to the uterus using IVF treatment). A total of 321 non-patient student sample (mean age 23) were randomly allocated to read a message from one of seven groups: (1) framing effect: (1a) gain and (1b) loss frame; (2) fear appeal: (2a) high, (2b) medium and (2c) low fear; or (3) a control group: education (3a) and (3b) non-education. Participants completed the Attitudes towards Single Embryo Transfer questionnaire, before exposure to the messages (time 1) and

  15. Use of purified FSH and LH for embryo production, cryopreservation by conventional freezing or vitrification and transfer of embryos in dairy ewes

    Directory of Open Access Journals (Sweden)

    Giovanni Martemucci

    2010-01-01

    Full Text Available Three experiments were carried out with the aim of evaluating the efficiency of techniques of in vivo production, storageand transfer of embryos in dairy sheep. Experiment I - For embryo production, thirty-one ewes were synchronized withFGA (vaginal sponges, 40 mg, 9 d and PGF2α (ICI; 50 μg, 7th d, and subdivided into three groups corresponding to thefollowing superovulatory treatments over 3 days with purified gonadotrophic preparations: A control, FSH/LH ratio = 1(250 IU p-FSH : 250 UI p-LH; B FSH/LH ratio = 2 (250 IU p-FSH : 125 IU p-LH and daily FSH/LH ratio of 3.4 – 1.7 –0.8 in the 3 days of treatment, respectively; C FSH/LH ratio = 2 (250 IU p-FSH : 125 IU p-LH and daily FSH/LH ratioof 5.0 – 1.0 – 0.3. On the 7th day after oestrus and mating, ovarian response and embryo production were evaluated.Experiment II – Three freezing methods were evaluated based upon post-thaw embryo quality: CF conventional slowfreezing by 1.5 M ethylene glycol (EG; V-1 one-step vitrification based on exposure of the embryos to one solution (EG7.15 M + ficoll 2.5 mM; V-3 vitrification in three steps, corresponding to three solutions at increasing concentration ofglycerol (GLY and EG (GLY 1.4 M; GLY 3.4 M + EG 1.4 M; GLY 4.6 M + EG 3.4 M. V-1 and V-3 frozen embryos weredirectly plunged in liquid nitrogen. At thawing, embryo viability was evaluated on the basis of morphological features.Experiment III – For embryo transfer, a total of 26 recipient ewes were synchronized with donors. On the 7th d fromoestrus, 11 recipient ewes received fresh embryos (Group FE – control and 15 recipients received vitrified-thawedembryos (Group VTE. Each recipient received 2 embryos. Superovulatory treatment B significantly advanced the onsetof oestrus compared to the control (27.3 vs 34.7 h; P10.8. Transferable embryos in Group B (7.2 resulted similar to Group A (5.3 and significantly (Pcompared to Group C (3.2. V3-method resulted in the highest (PCF- and V1-methods

  16. [Pregnancy and obstetric outcomes of fresh embryo transfer versus frozen-thawed embryo transfer in women below 35 years of age].

    Science.gov (United States)

    Sun, Ling; Chen, Zhi-Heng; Yin, Min-Na; Deng, Yu

    2017-07-20

    To compare the obstetric and perinatal outcomes between fresh embryo transfer (ET) and frozen-thawed ET (the "freeze-all" strategy) and evaluate the benefits of the "freeze-all" embryo strategy for young patients. We reviewed a total of 2091 ET cycles performed between January, 2011 and December, 2015 in women aged 20-35 years, including 1295 fresh ET cycles and 796 frozen-thawed ET cycles. The demographic characteristics, ovarian stimulation syndrome, clinical pregnancy rates, live birth rate and the obstetric outcomes (gestational age, preterm delivery rate and mean birth weight) were compared between the two groups. The mean age of the patients receiving frozen-thawed ET cycles had a significantly younger age than those having fresh ET cycles (29.5 vs 30.2 years, P0.05), live birth rate (50.3% vs 47.0%; P>0.05), mean birth weight or gestational age between the two groups. The freeze-all policy produces similar pregnancy and obstetric outcomes with those of fresh ET. Our results support the hypothesis that the freeze-all strategy help to prevent OHSS with a good pregnancy rate.

  17. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  18. Effects of fatty acid-free bovine serum albumin and fetal calf serum supplementing repair cultures on pre- and post-warm viability of biopsied bovine embryos produced in vitro.

    Science.gov (United States)

    Yotsushima, Kenji; Sakaguchi, Minoru; Shimizu, Manabu; Okimura, Tomoko; Izaike, Yoshiaki

    2004-08-01

    The objective of this study was to investigate the influence of fatty acid-free bovine serum albumin (BSA) or fetal calf serum (FCS) on the re-expansion of biopsied blastocysts and post-warm viability of subsequently vitrified embryos. Firstly, blastocysts produced in vitro were biopsied at Day 7 and cultured to allow repair in TCM199 with 0.3% BSA or 5% FCS for 24 h. The re-expansion rates and mean total numbers of cells of the re-expanded embryos after the repair culture with BSA were almost the same as that with FCS. Secondly, after biopsied embryos were similarly cultured for repair with BSA or FCS, re-expanded embryos were selected for vitrification. After warming and exposure to 0.5 M sucrose with 20% FCS in mPBS, the embryos were cultured in TCM199 with 5% FCS for 24 h. The re-expansion rate and mean total number of cells in re-expanded blastocysts in the BSA treatment group (97.4 +/- 2.9% and 106 +/- 42) was significantly higher than that in the FCS treatment group (51.6 +/- 9.1% and 61 +/- 38), respectively (Pbovine biopsied blastocysts; but, compared with BSA supplementation, FCS supplementation during repair culture reduces the post-warm viability of biopsied and subsequently vitrified embryos.

  19. Embryo transfer day does not affect the initial maternal serum β-hCG levels: A retrospective cohort study.

    Science.gov (United States)

    Dahiya, Mona; Rupani, Karishma; Yu, Su Ling; Fook-Chong, Stephanie M C; Siew Fui, Diana Chia; Rajesh, Hemashree

    2017-05-01

    The aim of this study is to compare the serum β-hCG values post transfer of a cleavage stage embryo versus a blastocyst stage embryo at equal time intervals post oocyte retrieval (OR) in clinically pregnant patients, and to ascertain a β-hCG value to predict pregnancy outcomes. This is a retrospective cohort study of 560 women with clinical pregnancy who underwent an embryo transfer performed at either the cleavage stage or the blastocyst stage of embryo development between January 2003 and June 2014 at the Center for Assisted Reproduction (CARE), Singapore General Hospital. The serum β-hCG level was measured on day 17 post OR. The β-hCG values were not significantly different in the cleavage stage versus the blastocyst stage embryos (mean±SD: 387±486IU/L D3 vs. 352±268IU/L D5, p=0.96, median value 297 in both groups). Our study suggests that the initial maternal serum β-hCG values were not affected by the day of transfer of the embryos since assessing the β-hCG at equivalent points after transfer should not lead to a significant difference assuming the progress and development of the embryos occurred as expected. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Effect of frozen/thawed embryo transfer on birthweight, macrosomia, and low birthweight rates in US singleton infants.

    Science.gov (United States)

    Litzky, Julia F; Boulet, Sheree L; Esfandiari, Navid; Zhang, Yujia; Kissin, Dmitry M; Theiler, Regan N; Marsit, Carmen J

    2017-12-29

    Singleton infants conceived using assisted reproductive technology have lower average birthweights than naturally conceived infants and are more likely to be born low birthweight (macrosomia (>4000 g) and low birthweight (macrosomia and low birthweight were evaluated using multivariable predicted marginal proportions from logistic regression models. In total, 180,184 singleton, term infants were included, with 55,898 (31.02%) having been conceived from frozen/thawed embryos. Frozen/thawed embryo transfer was associated with, on average, a 142 g increase in birthweight compared with infants born after fresh embryo transfer (P macrosomia following frozen/thawed embryo transfer was greater than that following fresh embryo transfer, but the risk of low birthweight among frozen/thawed embryo transfer infants was significantly decreased in comparison with fresh embryo transfer infants. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Clinical predictive criteria associated with live birth following elective single embryo transfer.

    Science.gov (United States)

    Sifer, Christophe; Herbemont, Charlène; Adda-Herzog, Elodie; Sermondade, Nathalie; Dupont, Charlotte; Cedrin-Durnerin, Isabelle; Poncelet, Christophe; Levy, Rachel; Grynberg, Michael; Hugues, Jean-Noël

    2014-10-01

    We aimed to define clinical criteria from the patients related to the occurrence of live birth in case of elective single embryo transfer (eSET). We analyzed retrospectively 409 eSET at day 2/3 between March 2005 and July 2012, proposed in case of (i) woman's age quality embryos obtained (3-5/6-10 blastomeres at day 2/3 and live births (30.3%) were obtained, separating patients into groups of women who had birth or not. Different clinical parameters of interest were compared between each group, using appropriate statistical tests at plive birth. BMI appears to be the only clinical parameter statistically associated with delivery following eSET strategy in a good prognosis infertile population. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  2. [Uterine contractile activity at embryo transfer--as a new pharmacotherapeutic target in assisted reproduction].

    Science.gov (United States)

    Pierzyński, Piotr; Zbucka-Kretowska, Monika

    2014-08-01

    Uterine contractile activity plays an important role in the reproduction of mammals, influencing sperm transport in the genital tract and positioning of the implanting embryo within the uterine cavity In humans, apart from the time of menses, the activity of a non-pregnant uterus is usually not perceived, and it is also not a subject of any routine clinical testing. Major contractile factors in non-gravid uteri are oxytocin and prostaglandins, locally produced within the endometrium. Oxytocin synthesis and expression of its receptors is gradually increasing in the follicular phase, following an increase in estrogen levels, and reaches its peak in the periovulatory period. In stimulated cycles, where supraphysiological estradiol concentrations are present, uterine contractile activity can be elevated. Exaggerated uterine contractions before embryo transfer are observed in one third of women undergoing controlled ovarian stimulation. Detection of such patients could enable their qualification for pharmacologic treatment. Evaluation of uterine contractions in such cases should be done non-invasively in order to avoid any endometrial trauma. Ultrasound evaluation of the movements of endometrial interface can be applied. Pharmacologic treatment of elevated uterine contractility before embryo transfer could improve the success rates of fertility treatments. So far application of beta mimetics or non-steroid anti-inflammatory drugs has not been associated with any progress. Oxytocin receptor system in the myometrium and the endometrium is a potential target for new class of medications aiming to improve implantation rates. This review summarizes up-to-date knowledge on the significance of uterine contractile activity in embryo implantation and describes the emerging new treatment targets in assisted reproduction.

  3. bovine

    African Journals Online (AJOL)

    of various breeds under local conditions of management. (Hale, 1974b). AdditionaIly, this procedure has been used to assess the production of LH by the bovine anterior pituitary in vitro and to study the relationships between this production and the activity of the pineal- hypothalamic axis (Hayes, Knight & Symington, 1974;.

  4. Effect of culture system on survival rate of vitrified bovine embryos produced in vitro.

    Science.gov (United States)

    Shirazi, A; Nazari, H; Ahmadi, E; Heidari, B; Shams-Esfandabadi, N

    2009-12-01

    This study was designed to evaluate the effect of in vitro culture system on bovine blastocyst yield and quality after vitrification. In Experiment 1, IVM/IVF zygotes were allocated to three culture conditions: (I) Oviductal cells-SOF (OCM-SOF); (II) Oviductal cells-TCM (OCM-TCM); and (III) SOF for 8 days. There was no significant difference between blastocyst rates among groups. In Experiment 2, the IVP-blastocysts in three above culture conditions were vitrified within groups segregated according to age (Day 7 and 8) and blastocoelic cavity size (early and expanded blastocysts). A trend of higher survival rate was obtained in vitrified/warmed early blastocysts compared with expanded ones, so that the difference in OCM-TCM group was significant (P<0.001). Higher survival and hatching rates (P<0.001) were obtained in OCM-SOF and OCM-TCM groups (co-culture) compared with SOF group and the age of blastocyst had no effect on post-thaw survival and hatching rates. In Experiment 3, after staining of blastocysts, in fresh blastocysts the highest number of trophectoderm cells was observed in OCM-TCM group and the number of inner cell mass (ICM) was higher in co-culture groups than SOF group (P<0.001). In vitrified/warmed blastocysts the number of ICM and trophectoderm cells in co-culture groups was higher than SOF group (P<0.001) except for the ICM of expanded blastocysts. In conclusion, in our culture conditions, the blastocyst yield is not influenced by culture system, while the cryotolerance of IVP-blastocysts is positively influenced by the presence of somatic cells. Moreover, the expanded blastocysts are more susceptible to cryoinjury than early blastocysts.

  5. The effects of macromolecular and serum supplements and oxygen tension during bovine in vitro procedures on kinetics of oocyte maturation and embryo development.

    Science.gov (United States)

    Mingoti, Gisele Zoccal; Castro, Viviane Sggobi Dias Caiado; Méo, Simone Cristina; Sá Barretto, Letícia Siqueira; Garcia, Joaquim Mansano

    2011-06-01

    Aiming to standardize in vitro production of bovine embryos and to obtain supplements to replace serum in culture media, this study evaluated the nuclear maturation kinetics and embryonic development in bovine after in vitro maturation (IVM) and culture (IVC) with several macromolecules (animal origin: bovine serum albumin (BSA), fetal calf serum (FCS); synthetic: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), Ficoll, and Knockout) at two oxygen tensions (20% and 5% O(2)). Regarding nuclear kinetics, neither the presence of the expected stage (metaphase I, transition anaphase to telophase, and metaphase II) at each evaluation moment (6, 18, and 24 h after IVM, respectively) nor the accelerated polar body emission (at 18 h after IVM) related developmental competence to blastocyst stage when different supplements were compared. Independently of supplement, cleavage rates at 20% O(2) (61.6-79.2%) were higher than at 5% O(2) (38.9-58.7%). At 20% O(2), higher blastocyst and hatching rates, respectively, were obtained in treatments BSA, FCS, Knockout, and control group (IVM with FCS and IVC with BSA + FCS, 14.0-23.5% and 6.8-15.4%) in comparison to PVA, PVP, and Ficoll (0%). The same was observed at 5% O(2) for blastocyst rates with BSA, FCS, Knockout, and control (5.4-16.8%) and for hatching rates with BSA, FCS, and control (2.0-11.1%). We can conclude that producing bovine embryos at 20% O(2) during the entire IVP process resulted in higher developmental rates than at 5% O(2). In addition, while defined macromolecules PVA, PVP, and Ficoll were not suitable for embryonic development, the synthetic serum Knockout was able to replace serum and albumin for IVP in bovine at 20% O(2).

  6. Ratio of proliferating cell nuclear antigen-positive nuclei to total cell number is higher in day 7 than in day 8 vitrified in vitro-produced bovine embryos.

    Science.gov (United States)

    Markkula, M; Räty, M; Jauhiainen, L; Paranko, J; Raula, J; Makarevich, A

    2001-07-01

    The aim of the present study was to find a reliable functional criterion for the evaluation of the proliferation potential of bovine in vitro-produced embryos. We used immunocytochemical detection of proliferating cell nuclear antigen (PCNA) combined with propidium iodide (PI) staining and subsequent confocal laser scanning microscopy together with routine morphological evaluation under a stereomicroscope to study fresh Day 7, 8, and 9, and cryopreserved Day 7 and 8 embryos. The ratio of PCNA/PI-positive nuclei was equal in fresh Day 7 and Day 8 embryos and significantly lower in Day 9 embryos. In general, Day 7 embryos tolerated the cryopreservation treatments better than Day 8 embryos. Vitrification in normal straws was especially detrimental to Day 8 embryos. In fresh Day 7 and 8 embryos, the PCNA results were in agreement with stereomicroscopic evaluation. However, in Day 9 fresh and in Day 7 and 8 treated embryos, the missing PCNA revealed disorders that were not observed under morphological evaluation. PCNA immunocytochemistry is an effective method to obtain information about the functional state of nuclei. The ratio of PCNA-positive nuclei can provide more information and numerical data about the developmental potential of bovine embryos after cryopreservation.

  7. Assisted reproductive technology use, embryo transfer practices, and birth outcomes after infertility insurance mandates: New Jersey and Connecticut.

    Science.gov (United States)

    Crawford, Sara; Boulet, Sheree L; Jamieson, Denise J; Stone, Carol; Mullen, Jewel; Kissin, Dmitry M

    2016-02-01

    To explore whether recently enacted infertility mandates including coverage for assisted reproductive technology (ART) treatment in New Jersey (2001) and Connecticut (2005) increased ART use, improved embryo transfer practices, and decreased multiple birth rates. Retrospective cohort study using data from the National ART Surveillance System. We explored trends in ART use, embryo transfer practices and birth outcomes, and compared changes in practices and outcomes during a 2-year period before and after passing the mandate between mandate and non-mandate states. Not applicable. Cycles of ART performed in the United States between 1996 and 2013. Infertility insurance mandates including coverage for ART treatment passed in New Jersey (2001) and Connecticut (2005). Number of ART cycles performed, number of embryos transferred, multiple live birth rates. Both New Jersey and Connecticut experienced an increase in ART use greater than the non-mandate states. The mean number of embryos transferred decreased significantly in New Jersey and Connecticut; however, the magnitudes were not significantly different from non-mandate states. There was no significant change in ART birth outcomes in either mandate state except for an increase in live births in Connecticut; the magnitude was not different from non-mandate states. The infertility insurance mandates passed in New Jersey and Connecticut were associated with increased ART treatment use but not a decrease in the number of embryos transferred or the rate of multiples; however, applicability of the mandates was limited. Published by Elsevier Inc.

  8. IVF outcomes in average- and poor-prognosis infertile women according to the number of embryos transferred.

    Science.gov (United States)

    Vega, Mario G; Gleicher, Norbert; Darmon, Sarah K; Weghofer, Andrea; Wu, Yan-Guang; Wang, Qi; Zhang, Lin; Albertini, David F; Barad, David H; Kushnir, Vitaly A

    2016-09-01

    Outcome measures of IVF success, which account for effectiveness of IVF and perinatal outcome risks, have recently been described. The association between number of embryos transferred in average and poor-prognosis IVF patients, and the chances of having good or poor IVF and perinatal outcomes, was investigated. Good IVF and perinatal outcome was defined as the birth of a live, term, normal-weight infant (≥2500 g). Poor IVF and perinatal outcome was defined as no live birth or birth of a very low weight neonate (IVF cycles in 504 average and poor-prognosis patients from January 2010 to December 2013 were identified. The odds of having good IVF and perinatal outcomes increased by 28% for each additional embryo transferred. The odds of poor IVF and perinatal outcome decreased by 32% with an additional embryo transferred. The likelihood of live birth with good perinatal outcome in average- and poor-prognosis patients after IVF increases with additional embryos being transferred. These data add to recently reported evidence in favour of multiple embryo transfer in older women and those with average or poor IVF prognosis. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  9. Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning.

    Science.gov (United States)

    Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan; Wu, Zhenfang

    2013-02-01

    Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 ± 0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 ± 0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 ± 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a

  10. CD9-positive microvesicles mediate the transfer of molecules to Bovine Spermatozoa during epididymal maturation.

    Directory of Open Access Journals (Sweden)

    Julieta N Caballero

    Full Text Available Acquisition of fertilization ability by spermatozoa during epididymal transit occurs in part by the transfer of molecules from membranous vesicles called epididymosomes. Epididymosomes are heterogeneous in terms of both size and molecular composition. Exosomes and other related small membranous vesicles (30-120 nm containing tetraspanin proteins on their surface are found in many biological fluids. In this study, we demonstrate that these vesicles are present in bovine cauda epididymal fluid as a subpopulation of epididymosomes. They contain tetraspanin CD9 in addition to other proteins involved in sperm maturation such as P25b, GliPr1L1, and MIF. In order to study the mechanism of protein transfer to sperm, DilC12-labeled unfractionated epididymosomes or CD9-positive microvesicles were coincubated with epididymal spermatozoa, and their transfer was evaluated by flow cytometry. CD9-positive microvesicles from epididymal fluid specifically transferred molecules to spermatozoa, whereas those prepared from blood were unable to do so. The CD9-positive microvesicles transferred molecules to the same sperm regions (acrosome and midpiece as epididymosomes, with the same kinetics; however, the molecules were preferentially transferred to live sperm and, in contrast to epididymosomes, Zn(2+ did not demonstrate potentiated transfer. Tetraspanin CD9 was associated with other proteins on the membrane surface of CD9-positive microvesicles according to coimmunoprecipitation experiments. CD26 cooperated with CD9 in the molecular transfer to sperm since the amount of molecules transferred was significantly reduced in the presence of specific antibodies. In conclusion, CD9-positive microvesicles are present in bovine cauda epididymal fluid and transfer molecules to live maturing sperm in a tissue-specific manner that involves CD9 and CD26.

  11. CD9-positive microvesicles mediate the transfer of molecules to Bovine Spermatozoa during epididymal maturation.

    Science.gov (United States)

    Caballero, Julieta N; Frenette, Gilles; Belleannée, Clémence; Sullivan, Robert

    2013-01-01

    Acquisition of fertilization ability by spermatozoa during epididymal transit occurs in part by the transfer of molecules from membranous vesicles called epididymosomes. Epididymosomes are heterogeneous in terms of both size and molecular composition. Exosomes and other related small membranous vesicles (30-120 nm) containing tetraspanin proteins on their surface are found in many biological fluids. In this study, we demonstrate that these vesicles are present in bovine cauda epididymal fluid as a subpopulation of epididymosomes. They contain tetraspanin CD9 in addition to other proteins involved in sperm maturation such as P25b, GliPr1L1, and MIF. In order to study the mechanism of protein transfer to sperm, DilC12-labeled unfractionated epididymosomes or CD9-positive microvesicles were coincubated with epididymal spermatozoa, and their transfer was evaluated by flow cytometry. CD9-positive microvesicles from epididymal fluid specifically transferred molecules to spermatozoa, whereas those prepared from blood were unable to do so. The CD9-positive microvesicles transferred molecules to the same sperm regions (acrosome and midpiece) as epididymosomes, with the same kinetics; however, the molecules were preferentially transferred to live sperm and, in contrast to epididymosomes, Zn(2+) did not demonstrate potentiated transfer. Tetraspanin CD9 was associated with other proteins on the membrane surface of CD9-positive microvesicles according to coimmunoprecipitation experiments. CD26 cooperated with CD9 in the molecular transfer to sperm since the amount of molecules transferred was significantly reduced in the presence of specific antibodies. In conclusion, CD9-positive microvesicles are present in bovine cauda epididymal fluid and transfer molecules to live maturing sperm in a tissue-specific manner that involves CD9 and CD26.

  12. Success of frozen embryo transfer: Does the type of gonadotropin influence the outcome?

    Directory of Open Access Journals (Sweden)

    Hesham Al-Inany

    2010-05-01

    Full Text Available Hesham Al-Inany1, Pieter van Gelder21Egyptian IVF-ET Center, Maadi, Egypt; 2PSCT BV, Den Haag, The NetherlandsObjectives: To determine whether there is a difference in outcome between different ovulationinduced cycles after frozen-thawed embryo transfer (FET.Methods: We searched the Cochrane Menstrual Disorders and Subfertility Group’s trials register in May 2009, the Cochrane Central Register of Controlled Trials (Cochrane Library, Issue 1, 2008, ISI Web of Knowledge (1985 to August 2009, and reference lists of articles. Relevant conference proceedings were hand-searched and researchers in the field were contacted. Randomized controlled trials and retrospective studies were included, comparing the various cycle regimens and different methods during FET in assisted reproductive technology, ie, in vitro fertilization and intracytoplasmic sperm injection.Results: Using the agonist long protocol for downregulation, five trials provided extractable data for live-birth rates, ongoing pregnancy, and clinical pregnancy rates following FET. One trial provided extractable data for clinical pregnancy rate. There was no evidence of a significant difference in any outcome between the users of urinary gonadotropins versus recombinant follicle-stimulating hormone. Data on implantation and miscarriage rates following FET were not available for analysis.Conclusions: It seems that clinical pregnancy rate after FET is not influenced by the type of gonadotropins used. Research should be directed towards improving freezing and thawing techniques.Keywords: infertility, assisted reproductive technology, frozen embryo transfer, gonadotropins

  13. Pregnancy loss after frozen-embryo transfer--a comparison of three protocols

    DEFF Research Database (Denmark)

    Tomás, Candido; Alsbjerg, Birgit; Martikainen, Hannu

    2012-01-01

    of embryos and ET. MAIN OUTCOME MEASURE(S): Pregnancy test rate, clinical pregnancy rate, and pregnancy loss rate. RESULT(S): The natural cycle followed by P (NC + P) was used in 26% of cycles, the natural cycle with hCG (NC + hCG) in 10%, and the substituted cycle with estrogen and P (E + P) in 64......% of cycles. The rate of transfers after thawing was similar in all groups (87.2%, 73.9%, and 87.2%, respectively). There was a significantly higher positive pregnancy test rate in the E + P (34.3%) and NC + hCG (35.5%) cycles as compared with the NC + P cycles (26.7%). However, the clinical pregnancy rate......, irregular cycles, endometrial thickness, number, and quality of embryos transferred did not correlate to pregnancy loss. CONCLUSION(S): A higher positive pregnancy test rate was obtained in E + P frozen ET cycles in comparison with other protocols; however, due to an increased preclinical and clinical...

  14. Screening somatic cell nuclear transfer parameters for generation of transgenic cloned cattle with intragenomic integration of additional gene copies that encode bovine adipocyte-type fatty acid-binding protein (A-FABP).

    Science.gov (United States)

    Guo, Yong; Li, Hejuan; Wang, Ying; Yan, Xingrong; Sheng, Xihui; Chang, Di; Qi, Xiaolong; Wang, Xiangguo; Liu, Yunhai; Li, Junya; Ni, Hemin

    2017-02-01

    Somatic cell nuclear transfer (SCNT) is frequently used to produce transgenic cloned livestock, but it is still associated with low success rates. To our knowledge, we are the first to report successful production of transgenic cattle that overexpress bovine adipocyte-type fatty acid binding proteins (A-FABPs) with the aid of SCNT. Intragenomic integration of additional A-FABP gene copies has been found to be positively correlated with the intramuscular fat content in different farm livestock species. First, we optimized the cloning parameters to produce bovine embryos integrated with A-FABP by SCNT, such as applied voltage field strength and pulse duration for electrofusion, morphology and size of donor cells, and number of donor cells passages. Then, bovine fibroblast cells from Qinchuan cattle were transfected with A-FABP and used as donor cells for SCNT. Hybrids of Simmental and Luxi local cattle were selected as the recipient females for A-FABP transgenic SCNT-derived embryos. The results showed that a field strength of 2.5 kV/cm with two 10-μs duration electrical pulses was ideal for electrofusion, and 4-6th generation circular smooth type donor cells with diameters of 15-25 μm were optimal for producing transgenic bovine embryos by SCNT, and resulted in higher fusion (80%), cleavage (73%), and blastocyst (27%) rates. In addition, we obtained two transgenic cloned calves that expressed additional bovine A-FABP gene copies, as detected by PCR-amplified cDNA sequencing. We proposed a set of optimal protocols to produce transgenic SCNT-derived cattle with intragenomic integration of ectopic A-FABP-inherited exon sequences.

  15. Frozen embryo transfer can be performed in the cycle immediately following the freeze-all cycle.

    Science.gov (United States)

    Ozgur, Kemal; Bulut, Hasan; Berkkanoglu, Murat; Humaidan, Peter; Coetzee, Kevin

    2018-01-01

    In this study, we investigated whether the time interval between oocyte retrieval and frozen embryo transfer (FET) affected the live birth (LB) rates of human segmented-IVF cycles. A total of 1338 ICSI freeze-all cycles were performed between February 2015 and January 2016, with 1121 FET cycles being retrospectively analyzed. All vitrified-warmed blastocyst transfers were performed in artificial FET cycles, using gonadotropin-releasing hormone (GnRH) agonist downregulation and oral estrogen endometrial preparation. The primary outcome measure was LB. Cycles were investigated in oocyte retrieval-to-FET interval groups of 32-46, 47-61, 62-76, 77-91, and ≥ 92 days, with the 47-61-day group used as the reference group. There were no significant differences in LB rates between the groups in the overall analysis, as well as, in sub-analyses investigating LB in terms of single blastocyst transfer (SBT), trigger type (GnRH agonist, triggers including hCG), oocyte number (≤ 5 and ≥ 15), and maternal age (> 35 years). The present study showed that it is feasible to perform transfers 36 days after oocyte retrieval and that delaying FET in freeze-all beyond the cycle immediately following oocyte retrieval does not increase LB rates.

  16. The effects of conjugated linoleic acid isomers cis-9,trans-11 and trans-10,cis-12 on in vitro bovine embryo production and cryopreservation.

    Science.gov (United States)

    Absalón-Medina, V A; Bedford-Guaus, S J; Gilbert, R O; Siqueira, L C; Esposito, G; Schneider, A; Cheong, S H; Butler, W R

    2014-10-01

    Conjugated linoleic acid (CLA) isomers can affect the lipid profile and signaling of cells and thereby alter their function. A total of 5,700 bovine oocytes were used in a structured series of experiments to test the effects of CLA cis-9,trans-11 and CLA trans-10,cis-12 in vitro. In experiment 1, high doses of each CLA isomer during in vitro maturation (IVM) were compared with high or low doses during the entire in vitro culture (IVC) of parthenogenetic embryos. High doses of the CLA isomers ranged from 50 to 200 μM and low doses were 15 and 25 μM. In experiment 2, the low doses of each CLA isomer were tested during IVM/IVC on embryos produced by in vitro fertilization (IVF). Experiment 3 compared the effects of 15 μM doses of each CLA isomer during IVM or IVC of IVF embryos. In experiment 4, post-rewarming survival rates and blastomere counts were assessed for embryos supplemented with each CLA isomer during IVM or for 36 h before vitrification. In experiment 1, when either CLA isomer was provided only during IVM, we observed no effects on overall rates of maturation, cleavage, or blastocysts (92.2 ± 1.6%, 78.3 ± 4.1%, and 28.9 ± 5.1%, respectively). However, high doses of each CLA isomer, but not low doses, during the entire embryo culture period decreased blastocyst rates (5-20%) in a dose-dependent manner. Cleavage rates improved with 15 or 50 μM CLA trans-10,cis-12. Progesterone concentrations in maturation media were significantly increased by high doses of each CLA isomer compared with control, but low doses of CLA isomers had no effect. In experiment 2 with IVF embryos, low doses of each CLA isomer did not alter cleavage rates (average 84.9 ± 1.9%) and only 25 μM CLA trans-10,cis-12 during IVC reduced blastocyst rates below those of controls (25.5 ± 2.1 vs. 38.2 ± 2.3%). The lipid content of embryos was increased and relative expression of the BIRC5 (baculoviral IAP repeat containing 5) gene was depressed by CLA trans-10,cis-12. In experiment 3

  17. Embryo selection in IVF

    National Research Council Canada - National Science Library

    Mastenbroek, Sebastiaan; van der Veen, Fulco; Aflatoonian, Abbas; Shapiro, Bruce; Bossuyt, Patrick; Repping, Sjoerd

    2011-01-01

    To optimize success rates of IVF, selection of the most viable embryo(s) for transfer has always been essential, as embryos that are cryopreserved are thought to have a reduced chance of implanting after thawing...

  18. Effect of mating between the donor cow and bull (Holstein versus Gyr on the in vitro production of bovine embryos

    Directory of Open Access Journals (Sweden)

    Ana Paula Toledo Barbosa da Silva

    2015-03-01

    Full Text Available The objective of this study was to evaluate the effect of the breed of the oocyte donor cow and bull (Holstein versus Gyr on in vitro production (IVP parameters of bovine embryos comparing the mean number of recovered oocytes and oocytes suitable for culture, the rate of suitable oocytes, and cleavage and blastocyst rates. Data from 1,000 follicular aspiration sessions (OPU, including 500 in donor cows of the Holstein breed and 500 of the Gyr breed, were collected. The results were analyzed by the unpaired Student t-test and chi-square test, adopting a level of significance of 5%. The mean number and standard deviation of recovered oocytes and oocytes suitable for culture were 15.1±13.0 and 8.7±7.6 for the Holstein breed and 15.5±11.9 and 9.1±7.9 for the Gyr breed. The rates of suitable oocytes were 57.7% and 58.5% for Holstein and Gyr breeds, respectively. A significant difference between breeds was observed for the number of oocytes suitable for culture (P<0.05, but not for the number of recovered oocytes or rates of suitable oocytes (P>0.05. Similarly, the breed of the oocyte donor cow and bull influenced cleavage and blastocyst rates (P<0.05. The cleavage rates were 65.7, 60.3, 59.6 and 56.5% for the combinations (donor breed x bull breed Holstein x Holstein (G1, Holstein x Gyr (G2, Gyr x Holstein (G3 and Gyr x Gyr (G4, respectively, with G1>G2, G1>G3, G1>G4, G2=G3, G2>G4, and G3>G4. The blastocyst rates were 28.1, 33.3, 26.8 and 31.0%, respectively, with G1>G2, G1=G3, G1

  19. Effects of Two Types of Melatonin-Loaded Nanocapsules with Distinct Supramolecular Structures: Polymeric (NC) and Lipid-Core Nanocapsules (LNC) on Bovine Embryo Culture Model.

    Science.gov (United States)

    Komninou, Eliza Rossi; Remião, Mariana Härter; Lucas, Caroline Gomes; Domingues, William Borges; Basso, Andrea Cristina; Jornada, Denise Soledade; Deschamps, João Carlos; Beck, Ruy Carlos Ruver; Pohlmann, Adriana Raffin; Bordignon, Vilceu; Seixas, Fabiana Kömmling; Campos, Vinicius Farias; Guterres, Silvia Stanisçuaski; Collares, Tiago

    2016-01-01

    Melatonin has been used as a supplement in culture medium to improve the efficiency of in vitro produced mammalian embryos. Through its ability to scavenge toxic oxygen derivatives and regulate cellular mRNA levels for antioxidant enzymes, this molecule has been shown to play a protective role against damage by free radicals, to which in vitro cultured embryos are exposed during early development. In vivo and in vitro studies have been performed showing that the use of nanocapsules as active substances carriers increases stability, bioavailability and biodistribution of drugs, such as melatonin, to the cells and tissues, improving their antioxidant properties. These properties can be modulated through the manipulation of formula composition, especially in relation to the supramolecular structures of the nanocapsule core and the surface area that greatly influences drug release mechanisms in biological environments. This study aimed to evaluate the effects of two types of melatonin-loaded nanocapsules with distinct supramolecular structures, polymeric (NC) and lipid-core (LNC) nanocapsules, on in vitro cultured bovine embryos. Embryonic development, apoptosis, reactive oxygen species (ROS) production, and mRNA levels of genes involved in cell apoptosis, ROS and cell pluripotency were evaluated after supplementation of culture medium with non-encapsulated melatonin (Mel), melatonin-loaded polymeric nanocapsules (Mel-NC) and melatonin-loaded lipid-core nanocapsules (Mel-LNC) at 10-6, 10-9, and 10-12 M drug concentrations. The highest hatching rate was observed in embryos treated with 10-9 M Mel-LNC. When compared to Mel and Mel-NC treatments at the same concentration (10-9 M), Mel-LNC increased embryo cell number, decreased cell apoptosis and ROS levels, down-regulated mRNA levels of BAX, CASP3, and SHC1 genes, and up-regulated mRNA levels of CAT and SOD2 genes. These findings indicate that nanoencapsulation with LNC increases the protective effects of melatonin

  20. Current Status of Comprehensive Chromosome Screening for Elective Single-Embryo Transfer

    Directory of Open Access Journals (Sweden)

    Ming-Yih Wu

    2014-01-01

    Full Text Available Most in vitro fertilization (IVF experts and infertility patients agree that the most ideal assisted reproductive technology (ART outcome is to have a healthy, full-term singleton born. To this end, the most reliable policy is the single-embryo transfer (SET. However, unsatisfactory results in IVF may result from plenty of factors, in which aneuploidy associated with advanced maternal age is a major hurdle. Throughout the past few years, we have got a big leap in advancement of the genetic screening of embryos on aneuploidy, translocation, or mutations. This facilitates a higher success rate in IVF accompanied by the policy of elective SET (eSET. As the cost is lowering while the scale of genome characterization continues to be up over the recent years, the contemporary technologies on trophectoderm biopsy and freezing-thaw, comprehensive chromosome screening (CCS with eSET appear to be getting more and more popular for modern IVF centers. Furthermore, evidence has showen that, by these avant-garde techniques (trophectoderm biopsy, vitrification, and CCS, older infertile women with the help of eSET may have an opportunity to increase the success of their live birth rates approaching those reported in younger infertility patients.

  1. Embryo transfer by reproductive endocrinology fellows vs attending physicians: are live birth rates comparable?

    Science.gov (United States)

    Eaton, Jennifer L; Zhang, Xingqi; Barnes, Randall B

    2014-11-01

    To compare live birth rates following ultrasound-guided embryo transfer (ET) by reproductive endocrinology and infertility fellows versus attending physicians. Women who underwent their first day-3, fresh, nondonor ET between Oct. 1, 2005, and April 1, 2011, at our academic center were included in this retrospective cohort study. Embryos were designated high quality if they had 8 cells, less than 10% fragmentation, and no asymmetry. ET was performed with the afterload technique under ultrasound guidance. Categorical variables were evaluated with the χ(2) test and continuous variables with the Student t test. Logistic regression was performed to assess the relationship between ET physician and live birth rate while adjusting for potential confounders. Seven hundred sixty women underwent ET by an attending physician, and 104 by a fellow. Baseline characteristics were similar between the groups. The live birth rate was 31% following ET by an attending physician, compared with 34% following ET by a fellow (P = .65). Logistic regression adjusting for potential confounders demonstrated no significant association between ET physician and live birth rate. This retrospective study demonstrated no significant difference in live birth rates following ultrasound-guided ET by fellows vs attending physicians at our institution. These data suggest that academic practices using the afterload method and ultrasound guidance can train fellows to perform ET without compromising success rates. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. A Comparison of Success Rates of Embryo Transfer on Weekdays and Weekends

    Directory of Open Access Journals (Sweden)

    Pinar Solmaz Hasdemir

    2016-05-01

    Full Text Available Background: The aim of this study is to examine the effect of the embryo transfer (ET day on clinical pregnancy success rates in in vitro fertilization-ET (IVF-ET cycles. Materials and Methods: In this retrospective study, we divided patients with infertility who underwent IVF-ET with fresh embryos into two groups depending on whether the ET was performed on weekdays or weekends. The main outcome measure was to compare the clinical pregnancy rates of patients with similar demographic and clinical characteristics who underwent ET on weekdays or weekends. Results: A total of 188 patients underwent IVF-ET on weekdays (n=156 or weekends (n=32. Both groups had similar demographic and cycle characteristics. The overall pregnancy rate was 42.8%. Among the study groups, the weekday group had a 40.2% ET success rate and the weekend group had a 54.8% success rate (P=0.517. Although no statistically significant difference existed between the two groups, we observed an absolute 14.6% increase in pregnancy rate for ETs performed during weekends compared to those performed on weekdays, with a 35% statistical power. Conclusion: ETs performed during weekends were more successful than ETs performed during weekdays with an absolute 14.6% increase in clinical pregnancy rate. This finding should be confirmed by conducting further studies with larger groups of patients.

  3. Comparison of neonatal outcomes following progesterone use during ovarian stimulation with frozen-thawed embryo transfer.

    Science.gov (United States)

    Zhu, Xiuxian; Ye, Hongjuan; Fu, Yonglun

    2017-08-10

    Progesterone soft capsules (brand name: Utrogestan) were demonstrated to be an effective oral alternative to prevent premature LH surges both in normal-ovulatory and polycystic ovarian syndrome (PCOS) patients. However, its safety in terms of neonatal outcomes is unclear. To evaluate whether Utrogestan use increase the risk of adverse neonatal outcomes compared with short protocol in patients undergoing IVF/ICSI treatments in combination with frozen-thawed embryo transfer (FET), we performed a retrospective analysis including 1008 FET cycles, with embryos originated from either Utrogestan + hMG protocol (n = 499), or short protocol (n = 509), which led to 546 live-born infants. The neonatal characteristics regarding preterm birth (PTB), low birth weight (LBW), gestational age and mode of delivery were comparable in the two groups. The incidence of live-birth defect was 0.68% (2/293) in the Utrogestan + hMG protocol compared with 0.79% (2/253) in the short protocol. No early neonatal death or intrauterine death were recorded in either group. To date, the data do not indicate an elevated rate of abnormality at birth after progesterone use during ovarian stimulation but further study with larger populations is needed to confirm these results.

  4. "Finnish embryo transfer breeding program ""ASMO"": description of the goals and a summary of the results of initial selection"

    Directory of Open Access Journals (Sweden)

    E. A. MÄNTYSAARI

    2008-12-01

    Full Text Available In 1990 the organizations responsible for Finnish dairy breeding established an open nucleus multiple ovulation and embryo transfer (MOET breeding program called ASMO. The aim was, besides to test effectiveness of MOET, to improve the protein to fat ratio in milk produced by Finnish Ayrshires but without sacrificing the progress in protein yield. The relative weights of traits were such that equal importance was assigned to protein % and protein yield. Negative weight was assigned to fat % to ensure it remained unaltered. The MOET work continued until 1994 after which the performance of selected animals has been monitored. During the five years the scheme operated, 276 cows were flushed for embryos, and 2751 embryos were recovered, of which 1810 were transferable. More than 1600 embryos were transferred to recipients, and 813 calves were born. Eighty bull calves were sold for the artificial insemination test scheme. In December 1995 the first 125 ET daughters were evaluated with the national animal model program. Their mean estimated breeding values (EBVs were +0.13 for protein % and -0.18 for fat % compared with the genetic base of progeny tested sires born in 1986-1988, and the protein yield EBVs were 12 kg above the genetic base. Despite the efficiency of selection, the program was discontinued in 1994. Due to the difficulty of maintaining sufficient control over donor animals, there were fewer than expected embryos per flush and also too few flushes per donor.;

  5. [Correlation between polyspermy and the outcome of in vitro fertilization-embryo transfer

    Science.gov (United States)

    Ye, Ying-Hui; Xing, Lan-Feng; Jin, Fan; Xu, Chen-Ming

    2002-06-01

    OBJECTIVE: To explore the influence of polyspermy on IVF outcomes in in vitro fertilization and embryo transfer(IVF-ET). METHODS: The data from 496 IVF-ET cycles and 5349 oocytes were analyzed retrospectively. A comparison of a number of fertility parameters with and without polyspermy was done. The fertility parameters were the number of oocytes retrieved, percentage of mature oocytes, fertilization rate, cleavage rate, occytes for ET, pregnancy rate. RESULTS: The percentage of mature occytes, fertilization rate, cleavage rate was 67.0 %,76.7 %and 95.6 %, respectively( Ppolyspermy(23.6 %),but with no statistical significance ( P>0.05). CONCLUSION: Polyspermic fertilization is correlated with improved oocyte receptibility to sperm and could be considered as an encouraging sign for the success of IVF.

  6. 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

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

  7. Spontaneous LH surges prior to HCG administration in unstimulated-cycle frozen-thawed embryo transfer do not influence pregnancy rates

    NARCIS (Netherlands)

    Groenewoud, E. R.; Kollen, B. J.; Macklon, N. S.; Cohlen, B. J.

    LH surges are the start of a period of optimal endometrial receptivity. Missing these surges in an unstimulated-cycle frozen-thawed embryo transfer (FET) based on ultrasound alone might lead to incorrect timing of embryo transfer. This prospective, non-randomized trial established the incidence and

  8. Preferences of subfertile women regarding elective single embryo transfer : additional in vitro fertilization cycles are acceptable, lower pregnancy rates are not

    NARCIS (Netherlands)

    Twisk, Moniek; van der Veen, Fulco; Repping, Sjoerd; Heineman, Maas-Jan; Korevaar, Johanna C.; Bossuyt, Patrick M. M.

    2007-01-01

    With identical pregnancy rates after elective single embryo transfer (ET) and double ET strategies consisting of three cycles of IVF or intracytoplasmic sperm injection (ICSI) plus transfers of thawed/frozen embryos if available, 46% of the women undergoing IVF/ICSI favor elective single ET. If

  9. First-principles molecular dynamics study of proton transfer mechanism in bovine cytochrome c oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Kamiya, Katsumasa [Center for Computational Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577 (Japan); Boero, Mauro [Center for Computational Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577 (Japan); Tateno, Masaru [Center for Computational Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577 (Japan); Shiraishi, Kenji [CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012 (Japan); Oshiyama, Atsushi [Center for Computational Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577 (Japan)

    2007-09-12

    Density functional based first-principles molecular dynamics calculations, performed on a model system extracted from the bovine cytochrome c oxidase, have been performed in an attempt to inspect the proton transfer mechanism across a peptide group. Our model system includes the specific Tyr440-Ser441 peptide group involved in a novel proton transfer path and shows that the Y440-S441 enol peptide group [-C(OH) = N-], which is a structural isomer of a keto form [-CO-NH-], is the product of the deprotonation of an imidic acid [-C(OH)-NH-] occurring in the vicinity of the deprotonated aspartic acid residue. For the subsequent enol-to-keto tautomerization, a direct H{sup +} transfer path in the Y440-S441 peptide group has been identified, in which the transition state takes a distorted four-membered ring structure.

  10. Misdiagnosis and delayed diagnosis for ectopic and heterotopic pregnancies after in vitro fertilization and embryo transfer.

    Science.gov (United States)

    Wang, Lin-lin; Chen, Xin; Ye, De-sheng; Liu, Yu-dong; He, Yu-xia; Guo, Wei; Chen, Shi-ling

    2014-02-01

    This study examined the misdiagnosis and delayed diagnosis factors for ectopic pregnancy (EP) and heterotopic pregnancy (HP) after in vitro fertilization and embryo transfer (IVF-ET) in an attempt to reduce the diagnostic error. Clinical data of patients who underwent IVF-ET treatment and had clinical pregnancy from 12463 cycles were retrospectively analyzed. Their findings of serum β-hCG test and transvaginal ultrasonography were also obtained during follow-up. These patients were divided into two groups according to the diagnosis accuracy of EP/HP: early diagnosis and misdiagnosis/delayed diagnosis. The results showed that the incidence of EP and HP was 3.8% (125/3286) and 0.8% (27/3286) respectively for IVF/ICSI-ET cycle, and 3.8% (55/1431) and 0.7% (10/1431) respectively for frozen- thawed embryo transfer (FET) cycle. Ruptured EP occurred in 28 patients due to initial misdiagnosis or delayed diagnosis. Related factors fell in 3 categories: (1) clinician factors: misunderstanding of patients' medical history, insufficient training in ultrasonography and unawareness of EP and HP; (2) patient factors: noncompliance with medical orders and lack of communication with clinicians; (3) complicated conditions of EP: atypical symptoms, delayed elevation of serum β-hCG level, early rupture of cornual EP, asymptomatic in early gestation and pregnancy of unknown location. All the factors were interwoven, contributing to the occurrence of EP and HP. It was concluded that complicated conditions are more likely to affect the diagnosis accuracy of EP/HP after IVF-ET. Transvaginal ultrasonography should be performed at 5 weeks of gestation. Intensive follow-up including repeated ultrasonography and serial serum β-hCG tests should be performed in patients with a suspicious diagnosis at admission.

  11. Should intrauterine human chorionic gonadotropin infusions ever be used prior to embryo transfer?

    Science.gov (United States)

    Volovsky, Michelle; Healey, Martin; MacLachlan, Vivien; Vollenhoven, Beverley J

    2017-09-25

    The aim of this study was to explore the factors that influence the outcome of intrauterine human chorionic gonadotropin (hCG) infusion at the time of embryo transfer (ET), in particular, the effect of hCG infusions on fresh and frozen embryo transfers (FETs) and whether prior recurrent implantation failure (RIF) impacts upon outcomes. This was a case-control study based on a standardized database from a multi-site in vitro fertilization clinic. The analysis contains 458 cases and 749 matched controls, with an intervention group of those given intrauterine hCG prior to ET and a control group of patients receiving no hCG infusion. Outcomes were defined as clinical pregnancy and live birth rates. Two analyses were performed. The first separated FETs (cases n = 224, controls n = 325) and fresh ETs (cases n = 234, controls n = 424), with outcomes calculated in each group. The second analysis divided patients into those with RIF (cases n = 149, controls n = 200) and those without (cases n = 309, controls n = 549). Results in fresh ETs demonstrated a 5.8% reduction (adjusted odds ratio (AOR) = 0.60, p = 0.041) in clinical pregnancy rates with the use of intrauterine hCG. In those without defined RIF, clinical pregnancy rates were reduced by 8.1% (AOR = 0.61, p = 0.023) and live birth rates by 7.2% (AOR = 0.56, p = 0.32) with intrauterine hCG use. There were no significant differences in outcomes in FETs and in the RIF cohort. Intrauterine hCG at the time of ET not only seems to have no benefit, but rather a negative effect in fresh ETs and those without RIF.

  12. Embryo technologies and animal health - consequences for the animal following ovum pick-up, in vitro embryo production and somatic cell nuclear transfer.

    Science.gov (United States)

    McEvoy, T G; Alink, F M; Moreira, V C; Watt, R G; Powell, K A

    2006-03-15

    Mammalian reproductive technologies that aim either to complement or to transcend conventional livestock breeding options have contributed to some of the most remarkable achievements in the field of reproductive biology in recent decades. In so doing they have extended our horizons in two distinct dimensions, the first concerning what it is technically possible to achieve and the second relating to the time-frame within which an individual's life-long developmental capability is initially established and ultimately realized or undermined. Our impressions of the benefits and values, or otherwise, of technologies such as in vitro embryo production and nuclear transfer are rightly influenced by the extent to which they impinge on the health of animals either subjected to or derived from them. Here, we consider some of the health implications of oocyte/embryo-centric technologies applied to farm livestock.

  13. The role of angiogenic markers in adverse perinatal outcomes: fresh versus frozen embryo transfers.

    Science.gov (United States)

    Woo, Irene; Chan, Yen; Sriprasert, Intira; Louie, Kristin; Ingles, Sue; Stanczyk, Frank; McGinnis, Lynda K; Chung, Karine

    2017-12-01

    We aimed to investigate the angiogenic balance in fresh compared to frozen embryo transfers, and among neonates with adverse perinatal outcomes. This was a retrospective cohort study. All IVF cycles resulting in a singleton live birth at a university academic fertility center from January 1, 2011, to December 31, 2013, were examined. Concentrations of sFLT-1 and PlGF were measured in previously frozen serum specimens collected during early gestation at approximately 5 weeks gestation. Patients completed an electronic survey to detail perinatal outcome. We identified 152 singleton live births (103 fresh, 49 frozen). Demographic characteristics were similar between the two groups. Ratios of sFlt-1:PlGF were not different between fresh and frozen transfers. Neonates from fresh cycles had a mean birth weight 202 g lighter (p = 0.01) than frozen cycles, after adjusting for gestational age. Among babies born with poor perinatal outcomes, there was a difference in sFlt-1:PlGF ratios after adjusting for race. In non-Asians, infants born small for gestational age (SGA) (< 10th percentile) had significantly higher sFLT-1:PLGF ratio, median ratio (0.21 vs 0.12, p = 0.016). Fresh transfers were associated with lower birth weight infants compared to frozen transfers. While there was no difference in sFlt-1:PlGF ratios between fresh and frozen transfers, these ratios were significantly lower in SGA infants, suggesting an imbalance in angiogenic markers during placentation.

  14. Abnormal human chorionic gonadotropin (hCG) trends after transfer of multiple embryos resulting in viable singleton pregnancies.

    Science.gov (United States)

    Brady, Paula C; Farland, Leslie V; Missmer, Stacey A; Racowsky, Catherine; Fox, Janis H

    2017-12-19

    The purpose of this study is to investigate whether abnormal hCG trends occur at a higher incidence among women conceiving singleton pregnancies following transfer of multiple (two or more) embryos (MET), as compared to those having a single embryo transfer (SET). Retrospective cohort study was performed of women who conceived singleton pregnancies following fresh or frozen autologous IVF/ICSI cycles with day 3 or day 5 embryo transfers between 2007 and 2014 at a single academic medical center. Cycles resulting in one gestational sac on ultrasound followed by singleton live birth beyond 24 weeks of gestation were included. Logistic regression models adjusted a priori for patient age at oocyte retrieval and day of embryo transfer were used to estimate the Odds Ratio of having an abnormal hCG rise (defined as a rise or hCG rises between the first and second measurements, compared to 2.7% (n = 17) of patients undergoing SET (OR 2.16, 95% CI 1.26-3.71). Among patients with initially abnormal hCG rises who had a third level checked (89%), three-quarters had normal hCG rises between the second and third measurements. Patients who deliver singletons following MET were more likely to have suboptimal initial hCG rises, potentially due to transient implantation of other non-viable embryo(s). While useful for counseling, these findings should not change standard management of abnormal hCG rises following IVF. The third hCG measurements may clarify pregnancy prognosis.

  15. The function of the milk-clotting enzymes bovine and camel chymosin studied by a fluorescence resonance energy transfer assay.

    Science.gov (United States)

    Jensen, Jesper Langholm; Jacobsen, Jonas; Moss, Marcia L; Rasmussen, Fred; Qvist, Karsten Bruun; Larsen, Sine; van den Brink, Johannes M

    2015-05-01

    Enzymatic coagulation of bovine milk can be divided in 2 steps: an enzymatic step, in which the Phe105-Met106 bond of the milk protein bovine κ-casein is cleaved, and an aggregation step. The aspartic peptidases bovine and camel chymosin (EC 3.4.23.4) are typically used to catalyze the enzymatic step. The most commonly used method to study chymosin activity is the relative milk-clotting activity test that measures the end point of the enzymatic and aggregation step. This method showed that camel chymosin has a 2-fold higher milk-clotting activity toward bovine milk than bovine chymosin. To enable a study of the enzymatic step independent of the aggregation step, a fluorescence resonance energy transfer assay has been developed using a peptide substrate derived from the 98-108 sequence of bovine κ-casein. This assay and Michaelis-Menten kinetics were employed to determine the enzymatic activity of camel and bovine chymosin under milk clotting-like conditions (pH 6.65, ionic strength 80 mM). The results obtained show that the catalytic efficiency of camel chymosin is 3-fold higher than bovine chymosin. The substrate affinity and catalytic activity of bovine and camel chymosin increase at lower pH (6.00 and 5.50). The glycosylation of bovine and camel chymosin did not affect binding of the fluorescence resonance energy transfer substrate, but doubly glycosylated camel chymosin seems to have slightly higher catalytic efficiency. In the characterization of the enzymes, the developed assay is easier and faster to use than the traditionally used relative milk-clotting activity test method. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  16. On the relationship between the dual specificity of the bovine brain phosphatidylinositol transfer protein and membrane phosphatidylinositol levels

    NARCIS (Netherlands)

    Paridon, P.A. van; Gadella, T.W.J.; Somerharju, P.J.; Wirtz, K.W.A.

    1987-01-01

    The phosphatidylinositol transfer protein from bovine brain has a remarkable specificity pattern with a distinct preference for phosphatidylinositol (PI) and a low affinity for phosphatidylcholine (PC). In this study we have determined the affinity of PI-transfer protein for PI relative to that for

  17. Evaluation of genetic components in traits related to superovulation, in vitro fertilization, and embryo transfer in Holstein cattle

    Science.gov (United States)

    The objectives of this study were to estimate variance components and identify regions of the genome associated with traits related to embryo transfer in Holsteins. Reproductive technologies are used in the dairy industry to increase the reproductive rate of superior females. A drawback of these met...

  18. Evaluating the interaction between progesterone, TNF alpha and cortisol on early loss of transferred embryos in beef cows

    Science.gov (United States)

    Fifty-eight non-lactating cows previously synchronized for estrus were assigned to two treatments to assess the effects of progesterone supplementation and its correlation with TNF-a and cortisol on the survival of the transferred embryos. On day 7 after exhibiting estrus (day 0), cows in both group...

  19. State-of-the-art production, conservation and transfer of in-vitro-produced embryos in small ruminants.

    Science.gov (United States)

    Cognié, Yves; Poulin, Nati; Locatelli, Yann; Mermillod, Pascal

    2004-01-01

    Today, although not efficient enough to replace multiple ovulation and embryo transfer, in vitro embryo production for small ruminants is a platform for new reproductive technologies, such as embryo sexing, transgenesis and cloning. The in vitro embryo-production system developed for sheep and goats is more efficient now than 15 years ago, but could still be improved. Laparoscopic collection of oocytes in live animals treated with gonadotrophin indicates a promising future for the application of this technology to genetic improvement programmes. Oocyte maturation in defined medium with epidermal growth factor and cysteamine appears as efficient as oocyte maturation in follicular fluid-supplemented medium and allows future study of the effect of other factors involved in the cytoplasmic maturation of oocytes from these species. Further efforts have to be made to standardise the semen-capacitating process and to improve the quality and freezability of in-vitro-produced (IVP) embryos. The optimisation of IVP procedures for deer species has required the study of the seasonal variation of oocyte competence and the development of a specific methodology to allow the culture of embryos up to the blastocyst stage.

  20. Pluripotency maintenance in mouse somatic cell nuclear transfer embryos and its improvement by treatment with the histone deacetylase inhibitor TSA.

    Science.gov (United States)

    Hai, Tang; Hao, Jie; Wang, Liu; Jouneau, Alice; Zhou, Qi

    2011-02-01

    Reprogramming of somatic cells to pluripotency can be achieved by nuclear transfer into enucleated oocytes (SCNT). A key event of this process is the demethylation of the Oct4 gene and its temporally and spatially regulated expression. Different studies have shown that it occurs abnormally in some SCNT embryos. TSA is a histone deacetylase inhibitor known to increase the efficiency of development to term of SCNT embryos, but its impact on the developmental features of SCNT embryos is poorly understood. Here, we have followed the fate of the pluripotent cells within SCNT embryos, from the late blastocyst to the early epiblast prior to gastrulation. Our data show a delay in development correlated with a defect in forming and maintaining a correct number of Oct4 expressing ICM and epiblast cells in SCNT embryos. As a consequence, during the outgrowth phase of embryonic stem cell derivation as well as during diapause in vivo, part of the SCNT blastocysts completely lose their ICM cells. Meanwhile, the others display a correctly reprogrammed ICM compatible with the derivation of ES cells and development of the epiblast. Our data also indicate that TSA favors the establishment of pluripotency in SCNT embryos.

  1. Optimizing the number of cleavage stage embryos to transfer on day 3 in women 38 years of age and older: a Society for Assisted Reproductive Technology database study.

    Science.gov (United States)

    Stern, Judy E; Goldman, Marlene B; Hatasaka, Harry; MacKenzie, Todd A; Surrey, Eric S; Racowsky, Catherine

    2009-03-01

    To determine the optimal number of day 3 embryos to transfer in women >or=38 years by conducting an evidence-based evaluation. Retrospective analysis of 2000-2004 national SART data. National writing group. A total of 36,103 day 3 embryo transfers in women >or=38 years undergoing their first assisted reproductive technology cycle. None. Logistic regression was used to model the probability of pregnancy, delivery, and multiple births (twin or high order) based on age- and cycle-specific parameters. Pregnancy rates, delivery rates, and multiple rates increased up to transfer of three embryos in 38-year-olds and four in 39-year-olds; beyond this number, only multiple rates increased. In women >or=40 years, delivery rates and multiple rates climbed steadily with increasing numbers transferred. Multivariate analysis confirmed the statistically significant effect of age, number of oocytes retrieved, and embryo cryopreservation on delivery and multiple rates. Maximum FSH level was not an independent predictor by multivariate analysis. Use of intracytoplasmic sperm injection was associated with lowered delivery rate. No more than three or four embryos should be transferred in 38- and 39-year-olds, respectively, whereas up to five embryos could be transferred in >or=40-year-olds. Numbers of embryos to transfer should be adjusted according to number of oocytes retrieved and availability of excess embryos for cryopreservation.

  2. Number of supernumerary vitrified blastocysts is positively correlated with implantation and live birth in single-blastocyst embryo transfers.

    Science.gov (United States)

    Hill, Micah J; Richter, Kevin S; Heitmann, Ryan J; Lewis, Terrance D; DeCherney, Alan H; Graham, James R; Widra, Eric; Levy, Michael J

    2013-05-01

    To estimate whether live birth in single-blastocyst transfers is correlated with the number of sibling supernumerary vitrified blastocysts (embryos not transferred) generated from that same cycle. Retrospective cohort study. A large academic assisted reproduction clinic. All single-blastocyst transfers in 2010 graded as "good" embryos by Society for Assisted Reproductive Technologies (SART) criteria. None. Implantation and live birth. Of the 655 single-blastocyst transfers that met inclusion criteria, implantation occurred in 65% and live birth in 54% of cycles. In chi-square analysis, patients with supernumerary vitrified blastocysts had a statistically higher implantation rate (65% versus 50%) and live-birth rate (56% versus 41%) when compared with patients without supernumerary blastocysts. Univariate logistic regression demonstrated an increase in implantation (OR 1.09; 95% CI, 1.03-1.15) and live birth (OR 1.06; 95% CI, 1.02-1.09) with increasing number of supernumerary blastocysts. Multivariate logistic regression analysis demonstrated that patient age and the number of supernumerary blastocysts were statistically significantly associated with implantation and live birth. The number of supernumerary vitrified blastocysts correlated positively with the odds of implantation and live birth in good quality single-blastocyst transfers. Patients with supernumerary blastocysts are good candidates for single-embryo transfer. Published by Elsevier Inc.

  3. DOT1L inhibitor improves early development of porcine somatic cell nuclear transfer embryos

    DEFF Research Database (Denmark)

    Tao, Jia; Zhang, Yu; Zuo, Xiaoyuan

    2017-01-01

    for 12 or 24 h significantly enhanced the blastocyst rate of SCNT embryos and dramatically reduced the level of H3K79me2 during SCNT 1-cell embryonic development. Additionally, H3K79me2 level in the EPZ-treated SCNT embryos was similar to that in in vitro fertilized embryos, suggesting that DOT1L...

  4. Abdominal ectopic pregnancy after in vitro fertilization and single embryo transfer: a case report and systematic review.

    Science.gov (United States)

    Yoder, Nicole; Tal, Reshef; Martin, J Ryan

    2016-10-19

    Ectopic pregnancy is the leading cause of maternal morbidity and mortality during the first trimester and the incidence increases dramatically with assisted-reproductive technology (ART), occurring in approximately 1.5-2.1 % of patients undergoing in-vitro fertilization (IVF). Abdominal ectopic pregnancy is a rare yet clinically significant form of ectopic pregnancy due to potentially high maternal morbidity. While risk factors for ectopic pregnancy after IVF have been studied, very little is known about risk factors specific for abdominal ectopic pregnancy. We present a case of a 30 year-old woman who had an abdominal ectopic pregnancy following IVF and elective single embryo transfer, which was diagnosed and managed by laparoscopy. We performed a systematic literature search to identify case reports of abdominal or heterotopic abdominal ectopic pregnancies after IVF. A total of 28 cases were identified. Patients' ages ranged from 23 to 38 (Mean 33.2, S.D. = 3.2). Infertility causes included tubal factor (46 %), endometriosis (14 %), male factor (14 %), pelvic adhesive disease (7 %), structural/DES exposure (7 %), and unexplained infertility (14 %). A history of ectopic pregnancy was identified in 39 % of cases. A history of tubal surgery was identified in 50 % of cases, 32 % cases having had bilateral salpingectomy. Transfer of two embryos or more (79 %) and fresh embryo transfer (71 %) were reported in the majority of cases. Heterotopic abdominal pregnancy occurred in 46 % of cases while 54 % were abdominal ectopic pregnancies. Our systematic review has revealed several trends in reported cases of abdominal ectopic pregnancy after IVF including tubal factor infertility, history of tubal ectopic and tubal surgery, higher number of embryos transferred, and fresh embryo transfers. These are consistent with known risk factors for ectopic pregnancy following IVF. Further research focusing on more homogenous population may help in better characterizing

  5. Association between ABO blood type and live-birth outcomes in single-embryo transfer cycles.

    Science.gov (United States)

    Pereira, Nigel; Patel, Hency H; Stone, Logan D; Christos, Paul J; Elias, Rony T; Spandorfer, Steven D; Rosenwaks, Zev

    2017-11-01

    To investigate the association between ABO blood type and live-birth outcomes in patients undergoing IVF with day 5 single-embryo transfer (SET). Retrospective cohort study. University-affiliated center. Normal responders, Live-birth rate was the primary outcome. Secondary outcomes were birth weight and gestational age at delivery. Univariate and multivariable logistic regression was used to examine the association between blood type and live birth, while controlling for confounders. Odds ratios (OR) with 95% confidence intervals (CI) for live birth were estimated. A total of 2,329 patients were included. The mean age of the study cohort was 34.6 ± 4.78 years. The distribution of blood types was as follows: A = 897 (38.5%); B = 397 (17.0%); AB = 120 (5.2%); and, O = 1,915 (39.3%) patients. There was no difference in the baseline demographics, ovarian stimulation, or embryo quality parameters between the blood types. The unadjusted ORs for live birth when comparing blood type A (referent) with blood types B, AB, and O were 0.96 (95% CI, 0.6-1.7), 0.72 (95% CI, 0.4-1.2), and 0.96 (95% CI. 0.6-1.7), respectively. The adjusted ORs for live birth remained not significant when comparing blood type A to blood types B, AB, and O individually. No difference in birth weight or gestational age at delivery was noted among the four blood types. Our findings suggest that ABO blood type is not associated with live-birth rate, birth weight, or gestational age at delivery in patients undergoing IVF with day 5 SET. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  6. Isolation and characterization of mesenchymal stem cells derived from bovine Wharton's jelly and their potential for use in cloning by nuclear transfer

    Directory of Open Access Journals (Sweden)

    Carolina Gonzales da Silva

    Full Text Available ABSTRACT: Wharton's jelly is a source of mesenchymal stem cells (MSCs that had not yet been tested for bovine embryo production by nuclear transfer (NT. Thus, the objective of this study was to isolate, characterize and test MSCs derived from Wharton's jelly for embryo and pregnancy production by NT in cattle. The umbilical cord was collected during calving and cells derived from Wharton's jelly (WJCs were isolated by explant and cultured in Dulbecco's Modified Eagle Medium. Skin Fibroblasts (FB were isolated after 6 months of life. Morphological analysis was performed by bright field and scanning electron microscopy (SEM during cell culture. Phenotypic and genotypic characterization by flow cytometry, immunocytochemistry, RT-PCR and differentiation induction in cell lineages were performed for WJC. In the NT procedure, oocytes at the arrested metaphase II stage were enucleated using micromanipulators, fused with WJCs or FB and later activated artificially. SEM micrographs revealed that WJCs have variable shape under culture. Mesenchymal markers of MSCs (CD29+, CD73+, CD90+ and CD105+ were expressed in bovine-derived WJC cultures, as evidenced by flow cytometry, immunocytochemistry and RT-PCR. When induced, these cells differentiated into osteocytes, chondrocytes and adipocytes. After classification, the WJCs were used in NT. Blastocyst formation rate by NT with WJCs at day 7 was 25.80±0.03%, similar to blatocyst rate with NT using skin fibroblasts (19.00±0.07%. Pregnancies were obtained and showed that WJCs constitute a new cell type for use in animal cloning.

  7. 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.

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

    Science.gov (United States)

    Vajta, G; Holm, P; Greve, T; Callesen, H

    1996-12-16

    The aim of this work was to investigate the possibilities of simplification, and to outline the limits of application, of a vitrification method for cow embryos. Morulae and blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed bull semen, and subsequent culture on a granulosa cell monolayer. Vitrification was performed by equilibration of embryos with 12.5% ethylene glycol and 12.5% dimethylsulphoxide at 20-22 degrees C for 60 s, then with 25% ethylene glycol and 25% dimethylsulphoxide at 4 degrees C for another 60 s. Embryos were then loaded in straws, placed in liquid nitrogen vapour for 2 min, and then plunged. Straws were thawed in a 22 degrees C water-bath, the embryos were directly rehydrated and further incubated in straw, and were then expelled and cultured in vitro for 72 h. In the first experiment, embryos of different age 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 zone 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 TCM199+calf serum as holding media. No differences in re-expansion and hatching rates were seen. However, when incubation with the concentrated cryoprotectant solution was performed at 20-22 degrees C, the embryo survival rate decreased (PBS+albumin) or no embryo survived (TCM199+calf serum) the vitrification procedure. In

  9. Impact of equine assisted reproductive technologies (standard embryo transfer or intracytoplasmic sperm injection (ICSI) with in vitro culture and embryo transfer) on placenta and foal morphometry and placental gene expression.

    Science.gov (United States)

    Valenzuela, Orlando A; Couturier-Tarrade, Anne; Choi, Young-Ho; Aubrière, Marie-Christine; Ritthaler, Justin; Chavatte-Palmer, Pascale; Hinrichs, Katrin

    2017-07-24

    Assisted reproductive technologies (ARTs) such as intracytoplasmic sperm injection (ICSI), in vitro embryo culture and embryo transfer (ET) may be associated with alterations in fetal and placental development. In horses, ET has been used for decades. More recently, in vitro embryo production by ICSI and in vitro culture, followed by embryo transfer (ICSI-C) has become an accepted method for clinical foal production. However, no information is available on the effects of ICSI-C or even of standard ET itself on placental and neonatal parameters in horses. We therefore evaluated placental and neonatal morphology and placental gene expression in reining- and cutting-type American Quarter Horse foals produced using different technologies. Thirty foals and placentas (naturally conceived (NC), ET and ICSI-C; 10 in each group) were examined morphometrically. The only parameter that differed significantly between groups was the length of the foal upper hindlimb, which was longer in ET and ICSI-C than in NC foals. Evaluation of placental mRNA expression for 17 genes related to growth and vascularisation showed no difference in gene expression between groups. These data indicate that within this population, use of ARTs was not associated with meaningful changes in foal or placental morphometry or in expression of the placental genes evaluated.

  10. In vitro produced and cloned embryos: Effects on pregnancy, parturition and offspring

    NARCIS (Netherlands)

    Kruip, T.A.M.; Daas, den J.H.G.

    1997-01-01

    Earlier reports have indicated that the transfers of bovine and ovine embryos produced by in vitro procedures (IVP) or by nuclear transfer (NT) have resulted in the birth of heavy offspring. The present paper presents summary information from 30 data sets obtained worldwide (WW) on IVP and NT in

  11. Optimal parameters for determining the LH surge in natural cycle frozen-thawed embryo transfers.

    Science.gov (United States)

    Irani, Mohamad; Robles, Alex; Gunnala, Vinay; Reichman, David; Rosenwaks, Zev

    2017-10-16

    There is no consensus on the exact parameters that define the LH surge for natural cycle frozen-thawed embryo transfers (NC-FET). Accurately determining the LH surge would affect the timing, and subsequently the success rates, of embryo transfer. Therefore, the aim of this study was to delineate the optimal levels and relationship for luteinizing hormone (LH) and estradiol in an effort to optimally identify the LH surge in NC-FET. It is a retrospective study that was performed in an academic medical center. Patients who underwent blastocyst NC-FET who either had preimplantation genetic screening (PGS) or were surge was defined as the first attainment of LH ≥ 17 IU/L during the follicular phase with a ≥30% drop in estradiol levels the following day; group B encompassed patients whose LH level continued to rise and the surge was defined as the highest serum LH level occurring a day after LH ≥ 17 IU/L despite a ≥ 30% drop in estradiol levels. The main outcomes measures were implantation and live birth rates. Four hundred-seven non-PGS and 284 PGS NC-FET were included. Among non-PGS cycles, group A was associated with significantly higher implantation rates (48.7% vs. 38.1%) and live birth rates (52.9% vs. 40.1%) compared to group B. In contrast, group A and B had comparable live birth rates among PGS cycles. Among non-PGS cycles, measuring LH and estradiol levels the day after an LH ≥ 17 IU/L and defining the surge as the first day of LH ≥ 17 IU/L in the context of a ≥ 30% drop in estradiol the following day was associated with better NC-FET outcomes than defining the surge as the day representing the highest serum LH level despite a ≥30% drop in estradiol levels.

  12. Pregnancies and piglets from large white sow recipients after two transfer methods  of cloned  and trangenic embryos of different pig breeds

    DEFF Research Database (Denmark)

    Schmidt, Mette; Kragh, Peter Michael; Li, Juan

    2010-01-01

    The aim of this study was to report from a larger study with pregnancy and delivery results after transfer of cloned transgenic/non-transgenic Large White or minipig embryos to Large White sow recipients. The effect of both total numbers of transferred embryos as well as site of their deposition ...... (uni- vs. bi-lateral) was studied. Four to five days after natural heat, 85 Large White (LW) sows received Day 5 or 6 handmade cloned embryos. Large White embryos were non-transgenic and were transferred to 36 recipients, while 49 recipients each received Minipig embryos, either non......%) and mean litter size (6.1 ± 0.7 vs. 4.2 ± 0.6) than the unicornual. The mean rate of piglets/transferred embryos was 7.3 ± 0.6% while the mean rate of piglets/reconstructed embryos was 179/18,000 = 1% with no difference between breeds or number of embryos transferred. The overall perinatal mortality rate...... was 49%, and it was significantly lower in LW piglets (20/59 = 34%) than in Minipiglets (67/120 = 56%) (vs. 10-15% in normal piglets at the farm) and the total rate of piglets with one or more malformation was 22%, and lower in LW (12%) than in Minipiglets (28%). This study demonstrate that although...

  13. Initial maternal serum human chorionic gonadotropin levels in pregnancies achieved after assisted reproductive technology are higher after preimplantation genetic screening and after frozen embryo transfer: a retrospective cohort.

    Science.gov (United States)

    Hobeika, Elie; Singh, Sonali; Malik, Shaveta; Knochenhauer, Eric S; Traub, Michael L

    2017-10-01

    Few published articles have compared initial hCG values across all different types of ART cycles, including cycles with fresh or frozen embryo transfer. No articles have compared initial hCG values in cycles utilizing preimplantation genetic screening (PGS). The purpose of this study is to compare initial hCG values after fresh embryo transfer, frozen embryo transfer, and after PGS. This was a single-center retrospective cohort study at an academically affiliated private IVF center. All fresh and frozen embryo transfers between January 2013 and December 31, 2015 were included. We compared mean initial serum hCG values 14 days after oocyte retrieval for fresh cycles and 9 days after frozen embryo transfer. We examined cycles of single embryo transfer (SET) and double embryo transfer (DET). Two hundred elven IVF (fresh embryo transfer), 128 FET (frozen embryo transfer cycles, no PGS), and 111 PGS cycles (ovarian stimulation with embryo cryopreservation, PGS, and frozen transfer in a subsequent estrogen-primed cycle) with initial positive hCG values were analyzed. In patients achieving a positive hCG after SET, initial hCG values were higher after PGS compared to FET (182.4 versus 124.0 mIU/mL, p = 0.02) and IVF (182.4 versus 87.1 mIU/mL, p < 0.001) as well as FET compared to IVF (124.0 versus 87.1 mIU/mL, p < 0.01). After DET, initial hCG values were higher after PGS (222.8 mIU/mL) compared to FET (182.1 mIU/mL, p = 0.02) and IVF (131.1 mIU/mL, p = 0.001). Our study suggests that initial serum hCG values are higher after using PGS and higher after the transfer of a frozen embryo compared to a fresh embryo. This suggests that initial hCG values relate to the chromosomal status of embryos. Initial hCG values may help determine intervention and monitoring later in pregnancy.

  14. Characterization of microRNA in bovine in vitro culture media associated with embryo quality and development.

    Science.gov (United States)

    Kropp, Jenna; Khatib, Hasan

    2015-09-01

    Dairy cattle fertility has declined over time due to factors including reduced fertilization and early embryonic loss. To counter fertility problems and better study preimplantation embryonic development, in vitro production systems have been developed. These systems largely assess embryos based on their morphology, which is not a strong indicator of developmental potential. Currently, no biomarkers can be used to noninvasively survey an embryo's potential in terms of its development and ability to establish a pregnancy. Thus, the objective of this study was to characterize and identify microRNA (miRNA) in culture media of embryos of differing developmental competence for future development as noninvasive biomarkers of embryo quality. The MiRNA sequencing of media conditioned by blastocyst and degenerate (those that failed to develop from the morula to blastocyst stage) embryos, revealed 11 differentially expressed miRNA; all were higher in concentration in degenerate conditioned media. Differential expression of mature microRNA (miR)-24, miR-191, and miR-148a was further validated using quantitative real-time PCR. Functional analysis of miR-24 revealed that addition of a mimic miRNA to culture media of morulae embryos resulted in a 27.3% decrease in development to the blastocyst stage. Furthermore, expression of miR-24 was 44.29-fold higher in blastocysts cultured with a miR-24 mimic compared with control blastocysts. Interestingly, the expression of CDKN1b, a target gene of miR-24 was repressed in embryos grown in the presence of the miRNA mimic. Mimic supplementation experiments suggest that miRNA are taken up by the embryo and that extracellular miRNA affect embryonic development. Overall, identification of a rich extracellular milieu in conditioned media sets the framework for future studies to determine the long-term predictive ability of embryo-based miRNA biomarkers on pregnancy outcome. Copyright © 2015 American Dairy Science Association. Published by

  15. Intravenous intralipid therapy is not beneficial in having a live delivery in women aged 40-42 years with a previous history of miscarriage or failure to conceive despite embryo transfer undergoing in vitro fertilization-embryo transfer.

    Science.gov (United States)

    Check, J H; Check, D L

    2016-01-01

    To evaluate the efficacy of intralipid intravenous infusion in achieving a live pregnancy following IVF--embryo transfer in women of advanced reproductive age (40-42 years). A matched control was performed. Women aged 40-42 with a previous history of miscarriage or who failed to conceive despite previous embryo transfer who entered an IVF program were offered intravenous intralipid therapy (four ml of 20% liposyn II in 100 ml normal saline over one hour) during the mid-follicular phase. Clinical pregnancy rates (eight weeks with viable gestation) and live delivered pregnancy rates were then determined and compared. The results were evaluated after ten matched cycles. There were no clinical pregnancies in those receiving intralipid vs. a 40% clinical and a 30% live delivered pregnancy rate in the untreated controls (p = 0.087, Fisher's exact test). The study was terminated because of these preliminary data. In the test tube, adding intralipid to natural killer cells can inhibit their cytolytic action. However, the use of intravenous intralipid to suppress natural killer cell activity does not seem to improve the chance of a live delivery in women aged 40-42 years with a previous history of miscarriage. In fact this therapy may actually be detrimental in this age group. Since efficacy of this therapy was not found in a group of advanced reproductive age, it is not clear why this should be effective for a younger population. A controlled study for the younger group is needed. Perhaps such a study could be limited to only those with miscarriage rather than also concluding failure to conceive despite embryo transfer. Intralipid failed to improve live delivered pregnancy rates in women with prior miscarriage or previous failure with embryo transfer.

  16. Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system.

    Science.gov (United States)

    Bevacqua, R J; Fernandez-Martín, R; Savy, V; Canel, N G; Gismondi, M I; Kues, W A; Carlson, D F; Fahrenkrug, S C; Niemann, H; Taboga, O A; Ferraris, S; Salamone, D F

    2016-11-01

    The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was

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

    Directory of Open Access Journals (Sweden)

    Luciana Simões Rafagnin Marinho

    2015-12-01

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

  18. Vitrification of immature bovine cumulus-oocyte complexes: effects of cryoprotectants, the vitrification procedure and warming time on cleavage and embryo development.

    Science.gov (United States)

    Prentice-Biensch, Jennifer R; Singh, Jaswant; Mapletoft, Reuben J; Anzar, Muhammad

    2012-09-06

    The present studies evaluated the effects of cryoprotectants, the vitrification procedure and time in the warming solution containing sucrose on cleavage and embryo development of immature (GV stage) bovine cumulus-oocyte complexes (COCs). Two experiments were conducted. In Experiment 1, COCs (n = 420) were randomly assigned to four groups: 1) CONTROL GROUP: no treatment; 2) VS1 group: COCs were exposed to vitrification solution 1 (VS1) containing 7.5% ethylene glycol [EG] + 7.5% dimethyl sulfoxide [DMSO] + 20% calf serum [CS] in TCM-199 at 37 C for 5 min; 3) VS1 + VS2 group: COCs were exposed to VS1 for 5 min followed by VS2 (15% EG + 15% DMSO + 17.1% sucrose + 20% CS) at 37 C for 45-60 sec; and 4) Vitrified group: COCs were exposed to VS1 and VS2, loaded on cryotops, vitrified in liquid nitrogen and then warmed in TCM-199 + 17.1% sucrose + 20% CS at 37 C for 1 min. In Experiment 2, COCs (n = 581) were assigned to the same groups, but those in VS1, VS1 + VS2 and Vitrified groups were sub-divided and exposed to the warming solution for either 1 or 5 min. After treatment and/or warming, all COCs in both experiments underwent in vitro maturation, in vitro fertilization and in vitro culture. Cleavage and blastocyst rates did not differ among Control, VS1 and VS1 + VS2 groups in either experiment. In Experiment 2, there was no effect of time in the warming solution.However, both cleavage and blastocyst rates were lower (P bovine COCs. However, cleavage rate and early embryo development were reduced following the vitrification and warming.

  19. Maintenance of meiotic arrest in bovine oocytes using the S-enantiomer of roscovitine: effects on maturation, fertilization and subsequent embryo development in vitro.

    Science.gov (United States)

    Coy, Pilar; Romar, Raquel; Payton, Rebecca R; McCann, Lisa; Saxton, Arnold M; Edwards, J Lannett

    2005-01-01

    The overall objective was to evaluate the effectiveness of the S-enantiomer of roscovitine (inhibitor of p34cdc2/cyclin B kinase) to maintain bovine cumulus-oocyte complexes at the germinal vesicle (GV) stage for extended times after removal from antral follicles without compromising subsequent maturation, fertilization and embryo development. Oocytes were cultured in 0, 12.5, 25 or 50 micromol/l S-roscovitine for 24 h. Hoechst staining showed that 50 micromol/l S-roscovitine maintained >90% of oocytes at the GV stage and inhibited gonadotropin-induced cumulus expansion. Fewer oocytes underwent nuclear maturation after in vitro maturation (Hoechst staining) when cultured in 50 micromol/l S-roscovitine for 66 versus 21 or 42 h. Zona pellucida (ZP) hardening (pronase resistance), cortical granule types (lens culinaris agglutinin-fluorescein isothiocyanate), nuclear maturation and fertilization with frozen-thawed spermatozoa (Hoechst staining) were assessed after culture of oocytes in 50 micromol/l S-roscovitine for 0, 24 or 48 h. Neither ZP hardening, nor nuclear maturation nor fertilization were altered by roscovitine culture for 48 h. A higher proportion of oocytes had a type III cortical granule pattern (premature translocation to the oolemma) after roscovitine culture for 48 h. However, embryo development was not compromised as cleavage, development to 8-16 cell and blastocyst stages were at least comparable in control and roscovitine-treated oocytes. In conclusion, the studies have shown that S-roscovitine reversibly maintained bovine oocytes at the GV stage for 48 h. However, maintenance of oocytes in static culture for 48 h was not sufficient to improve development above non-treated controls.

  20. Efficacy of mechanical micro-vibration in the development of bovine embryos during in vitro maturation and culture.

    Science.gov (United States)

    Takahashi, Masahiro; Honda, Tatsutoshi; Hatoya, Shingo; Inaba, Toshio; Kawate, Noritoshi; Tamada, Hiromichi

    2018-02-06

    It is currently unclear how mechanical micro-vibration affects the in vitro culture of embryos in Japanese Black cow. In the experimental groups, immature oocytes and fertilized embryos were cultured using the micro-vibration culture system with the vibration set for 5 sec at intervals of 60 min and frequency of 20, 40, or 80 Hz, respectively, during in vitro maturation and in vitro development. Compared with the control group, the rate of blastocyst development significantly increased in the 40 Hz group. In addition, the number of blastocyst cells reduced significantly in the 80 Hz group. In conclusion, the development of blastocysts in cows is facilitated by providing moderate mechanical micro-vibration to immature oocytes and embryos during the in vitro maturation and in vitro development.

  1. 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

    was lacking and clustering of nucleolar proteins was hampered. In conclusion, rDNA transcription is not required for targeting of rRNA processing proteins, rRNA is maternally inherited and target to rDNA independent of transcription, and de novo transcription is required for proper nucleologenesis in cattle.......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......, Ad 0.2 µg/ml; total transcriptional inhibition, AD 2.0 µg/ml). Late four-cell embryos were cultured to late eight-cell stage in 0.2 µg/ml AD (MTA prevention, ADLT (long-term total transcriptional inhibition group). Embryos were processed for autoradiography, transmission electron microscopy...

  2. Assisted hatching and live births in first-cycle frozen embryo transfers.

    Science.gov (United States)

    Knudtson, Jennifer F; Failor, Courtney M; Gelfond, Jonathan A; Goros, Martin W; Chang, Tiencheng Arthur; Schenken, Robert S; Robinson, Randal D

    2017-10-01

    To assess the effect of assisted hatching (AH) on live-birth rates in a retrospective cohort of patients undergoing first-cycle, autologous frozen embryo transfer (FET). Longitudinal cohort using cycles reported to the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System between 2004 and 2013. Not applicable. Women who underwent first-cycle, autologous FET with (n = 70,738) and without (n = 80,795) AH reported from 2004 to 2013. None. Live births. Propensity matching was used to account for confounding covariates, and a logistic regression model was constructed to identify the predictors of live-birth rates in relationship to AH. In all first-cycle FETs, there was a slight but statistically significant decrease in the live-birth rate with AH compared with no AH (34.2% vs. 35.4%). In older patients and in the years 2012-2013 AH was associated with decreased live births. Live-birth rates and the number of AH cycles performed before FET vary by the geographic location of clinics. Assisted hatching slightly decreases the live-birth rate in first-cycle, autologous FET. Its use should be carefully considered, especially in patients 38 years old and older. Prospective, clinical studies are needed to improve our knowledge of the impact of AH. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  3. [IVF surrogacy after embryo transfer abroad. Dilemmas of pregnancy follow-up].

    Science.gov (United States)

    Winkel, Esther; Roumen, Frans J M E; Dermout, Sylvia M

    2010-01-01

    A 43-year-old female, gravida 3, para 2, who was 9 weeks pregnant, presented herself as a surrogate mother for a 33-year-old couple at our outpatient clinic in Heerlen, the Netherlands, for pregnancy follow-up. As she had not passed the selection procedure in the Netherlands (VU University Medical Center, Amsterdam), IVF using the gametes of the prospective parents and embryo transfer was performed in Belgium. We discussed the management of possible problems and complications during pregnancy and delivery. After an uneventful pregnancy and delivery a healthy boy was taken home by the donor couple. In the Netherlands, high-tech surrogate motherhood under strict non-commercial conditions has been accepted by law since 1997. Since the inclusion criteria are very strict, some couples seem to find a way to have their wish fulfilled abroad. Uniformity of the IVF surrogacy legislation in Europe is necessary to discourage this practice. When this situation occurs nevertheless, it is important that doctors involved know how to handle the (often unknown) medical, ethical, legal, emotional and psychosocial aspects associated with high-tech IVF-surrogacy.

  4. 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

  5. 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

  6. Fluorescent fatty acid transfer from bovine serum albumin to phospholipid vesicles: collision or diffusion mediated uptake.

    Science.gov (United States)

    Elmadhoun, Bassam M; Swairjo, Manal A; Burczynski, Frank J

    2012-01-01

    The extent of palmitate uptake by hepatocytes is dependent upon the surface charge of the extracellular binding protein. Specifically, hepatocyte uptake is greater when palmitate is bound to cationic binding proteins than when it is bound to anionic proteins. To further understand the role of protein surface charge on the uptake process of protein-bound ligands, we examined the rate of transfer of fluorescent anthroyloxy palmitic acid (AOPA) in the presence of anionic and cationic extracellular proteins to model membranes containing different surface charged groups. AOPA transfer rate in the presence of bovine serum albumin (ALB; isoelectric point pI = 4.8-5.0) or modified ALB (ALBe; pI = 7.0-7.5) to negative, positive and neutral lipid vesicles was investigated using a fluorescence resonance energy transfer assay. The rate of AOPA transfer from both proteins was decreased when ionic strength was increased; directly dependent on the concentration of acceptor lipid vesicles; and was affected by both the lipid membrane surface charge and protein-bound concentration. The data support the notion that AOPA transfer from binding proteins to lipid membranes occurred through two concomitant processes, aqueous diffusion of the unbound ligand (diffusion-mediated process) and a collisional interaction between the protein-ligand complex and acceptor membrane. The contribution of diffusional mediated transfer to the overall uptake process was determined to be 3 to 4 times less than the contribution of a collisional interaction. This study strengthened the hypothesis that charged amino acid residues on proteins are important for effective collisional interaction between protein-ligand complexes and cell membranes through which more free ligand could be supplied for the uptake process.

  7. Effective Oocyte Vitrification and Survival Techniques for Bovine Somatic Cell Nuclear Transfer.

    Science.gov (United States)

    Park, Min Jee; Lee, Seung Eun; Kim, Eun Young; Lee, Jun Beom; Jeong, Chang Jin; Park, Se Pill

    2015-06-01

    Bovine somatic cell nuclear transfer (SCNT) using vitrified-thawed (VT) oocytes has been studied; however, the cloning efficiency of these oocytes is not comparable with that of nonvitrified (non-V) fresh oocytes. This study sought to optimize the survival and cryopreservation of VT oocytes for SCNT. Co-culture with feeder cells that had been preincubated for 15 h significantly improved the survival of VT oocytes and their in vitro developmental potential following SCNT in comparison to co-culture with feeder cells that had been preincubated for 2, 5, or 24 h (pvitrification groups [enucleated-vitrified-thawed (EVT) group, 13.7%; VT group, 15.0%; pvitrification groups (pvitrification groups than in the non-V group (pvitrification groups, blastocysts in the EAVT group had the best developmental potential, as judged by their high mRNA expression of developmental potential-related genes (POU5f1, Interferon-tau, and SLC2A5) and their low expression of proapoptotic (CASP3) and stress (Hsp70) genes. This study demonstrates that SCNT using bovine frozen-thawed oocytes can be successfully achieved using optimized vitrification and co-culture techniques.

  8. The correlation between endometrial thickness and outcome of in vitro fertilization and embryo transfer (IVF-ET) outcome

    OpenAIRE

    Al-Rejjal Rafat; Al-Hassan Saad; Coskun Serdar; Al-Ghamdi Ahlam; Awartani Khalid

    2008-01-01

    Abstract Background To evaluate the relationship between endometrial thickness on day of human chorionic gonadotrophin administration (hCG) and pregnancy outcome in a large number of consecutive in vitro fertilization and embryo transfer (IVF-ET) cycles. Methods A retrospective cohort study including all patients who had IVF-ET from January 2003–December 2005 conducted at a tertiary center. Results A total of 2464 cycles were analysed. Pregnancy rate (PR) was 35.8%. PR increased linearly (r =...

  9. Effect of urea during in vitro maturation on nuclear maturation and embryo development of bovine cumulus-oocyte-complexes

    NARCIS (Netherlands)

    Wit, de A.A.C.; Cesar, M.L.F.; Kruip, T.A.M.

    2001-01-01

    High concentrations of urea in reproductive tract fluids are detrimental to bovine reproduction. Therefore, in experiment 1, the effect of 6 mM urea on nuclear maturation of cumulus-oocyte-complexes (COC) collected from abattoir ovaries was studied. After 4, 8, 12, 16, 20, and 24 h of in vitro

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

    Science.gov (United States)

    Chacón, Liliana; Gómez, Martha C; Jenkins, Jill A; Leibo, Stanley P; Wirtu, Gemechu; Dresser, Betsy L; Pope, C Earle

    2009-11-01

    SummaryUsually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 degrees C/min in a low-temperature (-80 degrees 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 degrees 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.

  11. The changing pattern of uterine contractions before and after fresh embryo transfer and its relation to clinical outcome.

    Science.gov (United States)

    Chung, Cathy Hoi Sze; Wong, Alice Wai Yee; Chan, Carol Pui Shan; Saravelos, Sotirios H; Kong, Grace Wing Shan; Cheung, Lai Ping; Chung, Jacqueline Pui Wah; Li, Tin Chiu

    2017-03-01

    In this prospective cohort study of 286 women undergoing fresh embryo transfer after IVF, uterine contraction frequency and direction were measured before (-5 min), 5 min after (+5 min) and 60 min after (+60 min) embryo transfer. Mean ± SD uterine contraction frequency at -5 min was 1.8 ± 1.1 contractions per min, increasing significantly (P contractions were 33%, 44%, 17% and 6%; at +5 min, 40%, 42%, 13% and 5%, and at +60 min, 42%, 38%, 14% and 6%. No significant change was observed in the proportion of direction at these three time points. Logistic regression analysis showed live birth rate was significantly reduced in older women (P = 0.035) and in those with higher uterine contraction frequency at +5 min (P = 0.006). Frequency of uterine contraction immediately after embryo transfer (+5 min) seemed to be a significant predictor of IVF outcome and may help to identify women who could benefit from the use of muscle relaxant therapy to improve outcome. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  12. The integrated HIV-1 provirus in patient sperm chromosome and its transfer into the early embryo by fertilization.

    Directory of Open Access Journals (Sweden)

    Dian Wang

    Full Text Available Complete understanding of the route of HIV-1 transmission is an important prerequisite for curbing the HIV/AIDS pandemic. So far, the known routes of HIV-1 transmission include sexual contact, needle sharing, puncture, transfusion and mother-to-child transmission. Whether HIV can be vertically transmitted from human sperm to embryo by fertilization is largely undetermined. Direct research on embryo derived from infected human sperm and healthy human ova have been difficult because of ethical issues and problems in the collection of ova. However, the use of inter-specific in vitro fertilization (IVF between human sperm and hamster ova can avoid both of these problems. Combined with molecular, cytogenetical and immunological techniques such as the preparation of human sperm chromosomes, fluorescent in situ hybridization (FISH, and immunofluorescence assay (IFA, this study mainly explored whether any integrated HIV provirus were present in the chromosomes of infected patients' sperm, and whether that provirus could be transferred into early embryos by fertilization and maintain its function of replication and expression. Evidence showed that HIV-1 nucleic acid was present in the spermatozoa of HIV/AIDS patients, that HIV-1 provirus is present on the patient sperm chromosome, that the integrated provirus could be transferred into early embryo chromosomally integrated by fertilization, and that it could replicate alongside the embryonic genome and subsequently express its protein in the embryo. These findings indicate the possibility of vertical transmission of HIV-1 from the sperm genome to the embryonic genome by fertilization. This study also offers a platform for the research into this new mode of transmission for other viruses, especially sexually transmitted viruses.

  13. Impact of whole-genome amplification on the reliability of pre-transfer cattle embryo breeding value estimates.

    Science.gov (United States)

    Shojaei Saadi, Habib A; Vigneault, Christian; Sargolzaei, Mehdi; Gagné, Dominic; Fournier, Éric; de Montera, Béatrice; Chesnais, Jacques; Blondin, Patrick; Robert, Claude

    2014-10-12

    Genome-wide profiling of single-nucleotide polymorphisms is receiving increasing attention as a method of pre-implantation genetic diagnosis in humans and of commercial genotyping of pre-transfer embryos in cattle. However, the very small quantity of genomic DNA in biopsy material from early embryos poses daunting technical challenges. A reliable whole-genome amplification (WGA) procedure would greatly facilitate the procedure. Several PCR-based and non-PCR based WGA technologies, namely multiple displacement amplification, quasi-random primed library synthesis followed by PCR, ligation-mediated PCR, and single-primer isothermal amplification were tested in combination with different DNA extractions protocols for various quantities of genomic DNA inputs. The efficiency of each method was evaluated by comparing the genotypes obtained from 15 cultured cells (representative of an embryonic biopsy) to unamplified reference gDNA. The gDNA input, gDNA extraction method and amplification technology were all found to be critical for successful genome-wide genotyping. The selected WGA platform was then tested on embryo biopsies (n = 226), comparing their results to that of biopsies collected after birth. Although WGA inevitably leads to a random loss of information and to the introduction of erroneous genotypes, following genomic imputation the resulting genetic index of both sources of DNA were highly correlated (r = 0.99, P<0.001). It is possible to generate high-quality DNA in sufficient quantities for successful genome-wide genotyping starting from an early embryo biopsy. However, imputation from parental and population genotypes is a requirement for completing and correcting genotypic data. Judicious selection of the WGA platform, careful handling of the samples and genomic imputation together, make it possible to perform extremely reliable genomic evaluations for pre-transfer embryos.

  14. OPTIMISATION OF TOTAL RNA EXTRACTION FROM BOVINE OOCYTES AND EMBRYOS FOR GENE EXPRESSION STUDIES AND EFFECTS OF CRYOPROTECTANTS ON TOTAL RNA EXTRACTION.

    Science.gov (United States)

    Pavani, K C; Baron, E E; Faheem, M; Chaveiro, A; Da Silva, F Moreira

    2015-01-01

    Gene expression is required for understanding bovine oocytes meiotic maturation as well as the potential of embryonic development. In the present study a standardized reagent protocol for total RNA extraction was designed for bovine oocytes and embryos, which is considered specific and less expensive. For such purpose oocytes (n = 795) recovered from about 80 ovaries were divided in three groups: Group 1 modified Trizol (MTP, n = 355); Group 2 Guanidinium thiocyanate protocol (GNTC, n = 140) and Group 3 Commercial Kit protocol (CKP, n = 60). Oocytes belonging to group 1 (n = 100) and 3 (n = 20) were subjected to vitrification using two cryoprotectants 1,2 propandiol (PROH) or Dimethylsulfoxide (DMSO). The 240 remaining oocytes were divided into 3 groups in which 100 were used, in fresh, for in vitro fertilization, and 140 oocytes were vitrified using PROH (n = 70) and DMSO (n = 70) as cryoprotectants, being then fertilized in vitro after thawing. Embryos were used nine days after fertilization. Gene amplification (SDHA, (GAPDH and DNMT1) was performed in oocytes, and gene quantification (DNMT1) in in vitro produced embryos at the stage of blastocyst (n = 10). Efficiency of the extraction was further compared. The purity of all samples to different protocols ranged from 1.10 to 1.25 for GNTC protocol; from 2.05 to 2.63 for the CKP and from 1.50 to 2.11 for the developed MTP, being the last one nearest to the expected purity levels for RNA samples (1.7 to 2.0). On average, for 30 fresh oocytes, from spectrophotometer readings, total RNA concentration was 127.8 ± 9.3 ng μl(-1) for MTP, against 46.4 ± 9.5 ng μl(-1) from CKP and 476 ± 12.9 ng μl(-1) for GNTC protocol. Using the MTP to evaluate RNA in 30 vitrified/thawed oocytes, resulted in a total RNA concentration of 61.3 ± 3.3 ng μl(-1) and 40.0 μ 12.4 ng μ(-1), respectively for DMSO and PROH. Regarding total RNA concentration and purity, in blastocyst stage, more purity was observed in DMSO as compared to

  15. Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer.

    Science.gov (United States)

    Arias, María Elena; Sánchez-Villalba, Esther; Delgado, Andrea; Felmer, Ricardo

    2017-02-01

    Sperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

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

    Directory of Open Access Journals (Sweden)

    Yi-Lin Chen

    2011-03-01

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

  17. Factors related to clinical pregnancy after vitrified-warmed embryo transfer: a retrospective and multivariate logistic regression analysis of 2313 transfer cycles.

    Science.gov (United States)

    Shi, Wenhao; Zhang, Silin; Zhao, Wanqiu; Xia, Xue; Wang, Min; Wang, Hui; Bai, Haiyan; Shi, Juanzi

    2013-07-01

    What factors does multivariate logistic regression show to be significantly associated with the likelihood of clinical pregnancy in vitrified-warmed embryo transfer (VET) cycles? Assisted hatching (AH) and if the reason to freeze embryos was to avoid the risk of ovarian hyperstimulation syndrome (OHSS) were significantly positively associated with a greater likelihood of clinical pregnancy. Single factor analysis has shown AH, number of embryos transferred and the reason of freezing for OHSS to be positively and damaged blastomere to be negatively significantly associated with the chance of clinical pregnancy after VET. It remains unclear what factors would be significant after multivariate analysis. The study was a retrospective analysis of 2313 VET cycles from 1481 patients performed between January 2008 and April 2012. A multivariate logistic regression analysis was performed to identify the factors to affect clinical pregnancy outcome of VET. There were 22 candidate variables selected based on clinical experiences and the literature. With the thresholds of α entry = α removal= 0.05 for both variable entry and variable removal, eight variables were chosen to contribute the multivariable model by the bootstrap stepwise variable selection algorithm (n = 1000). Eight variables were age at controlled ovarian hyperstimulation (COH), reason for freezing, AH, endometrial thickness, damaged blastomere, number of embryos transferred, number of good-quality embryos, and blood presence on transfer catheter. A descriptive comparison of the relative importance was accomplished by the proportion of explained variation (PEV). Among the reasons for freezing, the OHSS group showed a higher OR than the surplus embryo group when compared with other reasons for VET groups (OHSS versus Other, OR: 2.145; CI: 1.4-3.286; Surplus embryos versus Other, OR: 1.152; CI: 0.761-1.743) and high PEV (marginal 2.77%, P = 0.2911; partial 1.68%; CI of area under receptor operator characteristic

  18. A biolistic process for in vitro gene transfer into chicken embryos

    Directory of Open Access Journals (Sweden)

    L.A. Ribeiro

    2001-09-01

    Full Text Available Chicken embryos kept in culture medium were bombarded using a high helium gas pressure biolistic device. To optimize the factors that affect transformation efficiency, the lacZ gene under control of the human cytomegalovirus immediate early enhancer/promoter was used as a reporter gene. There was an inverse relationship between survival rate and transformation efficiency. The best conditions obtained for high embryo survival and high transformation efficiency were achieved with 800 psi helium gas pressure, 500 mmHg vacuum, gold particles, an 8 cm DNA-coated microparticle flying distance to the embryo and embryo placement 0.5 cm from the center of the particle dispersion cone. Under these conditions, transformation efficiency was 100%, survival rate 25% and the number of expression units in the embryo body cells ranged from 100 to 1,000. Expression of green fluorescent protein was also detected in embryos bombarded under optimal conditions. Based on the results obtained, the biolistic process can be considered an efficient method for the transformation of chicken embryos and therefore can be used as a model system to study transient gene expression and tissue-specific promoters.

  19. Transfer of Dicamba Tolerance from Sinapis arvensis to Brassica napus via Embryo Rescue and Recurrent Backcross Breeding.

    Science.gov (United States)

    Jugulam, M; Ziauddin, Asma; So, Kenny K Y; Chen, Shu; Hall, J Christopher

    2015-01-01

    Auxinic herbicides (e.g. dicamba) are extensively used in agriculture to selectively control broadleaf weeds. Although cultivated species of Brassicaceae (e.g. Canola) are susceptible to auxinic herbicides, some biotypes of Sinapis arvensis (wild mustard) were found dicamba resistant in Canada. In this research, dicamba tolerance from wild mustard was introgressed into canola through embryo rescue followed by conventional breeding. Intergeneric hybrids between S. arvensis (2n = 18) and B. napus (2n = 38) were produced through embryo rescue. Embryo formation and hybrid plant regeneration was achieved. Transfer of dicamba tolerance from S. arvensis into the hybrid plants was determined by molecular analysis and at the whole plant level. Dicamba tolerance was introgressed into B. napus by backcrossing for seven generations. Homozygous dicamba-tolerant B. napus lines were identified. The ploidy of the hybrid progeny was assessed by flow cytometry. Finally, introgression of the piece of DNA possibly containing the dicamba tolerance gene into B. napus was confirmed using florescence in situ hybridization (FISH). This research demonstrates for the first time stable introgression of dicamba tolerance from S. arvensis into B. napus via in vitro embryo rescue followed by repeated backcross breeding. Creation of dicamba-tolerant B. napus varieties by this approach may have potential to provide options to growers to choose a desirable herbicide-tolerant technology. Furthermore, adoption of such technology facilitates effective weed control, less tillage, and possibly minimize evolution of herbicide resistant weeds.

  20. 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...

  1. Propensity score-matched study and meta-analysis of cumulative outcomes of day 2/3 versus day 5/6 embryo transfers.

    Science.gov (United States)

    Yin, Ye; Chen, Ge; Li, Kezhen; Liao, Qiuyue; Zhang, Sijia; Ma, Nieying; Chen, Jing; Zhang, Yan; Ai, Jihui

    2017-12-01

    The superiority of the cumulative outcomes of day 5/6 embryo transfer to those of day 2/3 embryo transfer in infertile couples has been debated. This retrospective study included data collected from 1051 patients from July 2011 to June 2014. Multiple maternal baseline covariates were subjected to propensity score matching analysis, and each day 5/6 group woman was matched to one day 2/3 group woman. A systematic meta-analysis was conducted to validate the results. After matching was completed, 217 patients on the day 2/3 group were matched with those on the day 5/6 group, and no significant differences in the baseline characteristics were observed between the two groups. The cumulative pregnancy rate (57.14% vs. 53.46%, OR 1.16, 95% CI 0.79-1.70) and cumulative live birth rate (53.00% vs. 49.77%, OR 1.14, 95% CI 0.78-1.66) of day 5/6 embryo transfers were higher than those of day 2/3 embryo transfers, but this difference was not significant. The mean cycles per live birth and mean days per live birth in the day 5/6 group were significantly lower than those in the day 2/3 group. This study demonstrated that day 5/6 embryo transfer is a more cost-effective and time-efficient policy than day 2/3 embryo transfer to produce a live baby.

  2. Association between growth dynamics, morphological parameters, the chromosomal status of the blastocysts, and clinical outcomes in IVF PGS cycles with single embryo transfer.

    Science.gov (United States)

    Barash, Oleksii O; Ivani, Kristen A; Willman, Susan P; Rosenbluth, Evan M; Wachs, Deborah S; Hinckley, Mary D; Pittenger Reid, Sara; Weckstein, Louis N

    2017-08-01

    The purpose of the present study is to examine interconnection between speed of embryo development, the genetic status of the blastocysts, and clinical outcomes in IVF preimplantation genetic screening (PGS) cycles with single embryo transfer (SET). The retrospective comparative study has been performed between January 2013 and January 2016. Seven hundred thirty-seven cycles of IVF treatment with PGS, followed by 503 SETs, were included in the study. Normally fertilized oocytes were hatched on day 3, were cultured to the blastocyst stage, and were biopsied only when at least three to seven cells were herniating from zona pellucida on the morning of day 5 (≤118 h) or day 6 (≥139 h). A total of 3705 embryos were analyzed for euploidy rates and blastocyst morphology. All embryos were vitrified after the biopsy, and selected embryos were subsequently thawed for a hormone replacement frozen embryo transfer cycle. The euploidy rate was significantly higher among embryos biopsied on day 5 versus day 6: 59.44 ± 4.1 and 48.19 ± 3.8, respectively, p < 0.05. The difference in euploidy rates between embryos biopsied on day 5 versus day 6 in matched age groups increased from 5.83 to 25.46% with advancing maternal age. Our data demonstrated no statistically significant difference in euploidy rates between good-quality embryos biopsied on day 5 in the group of patients <38 years old and embryos in PGS cycles using donor oocytes: 71.12% (336/472) and 75.68% (221/292), respectively, p = 0.174, χ (2) = 1.848. In 270 out of 503 SETs, transferred embryos were biopsied on day 5 (ongoing pregnancy rate was 64.6% in a group of patients <38 years old, and in a group of patients ≥38 years old, ongoing PR was 64.2%). In 233 out of 503 cycles, transferred embryos were biopsied on day 6 (ongoing PR was 46.6% in a group of patients <38 years old, and in a group of patients ≥38 years old, ongoing PR was 50.8%). In all study groups, the ongoing pregnancy rate was

  3. Tiger, Bengal and Domestic Cat Embryos Produced by Homospecific and Interspecific Zona-Free Nuclear Transfer.

    Science.gov (United States)

    Moro, L N; Jarazo, J; Buemo, C; Hiriart, M I; Sestelo, A; Salamone, D F

    2015-10-01

    The aim of this study was to evaluate three different cloning strategies in the domestic cat (Felis silvestris) and to use the most efficient to generate wild felid embryos by interspecific cloning (iSCNT) using Bengal (a hybrid formed by the cross of Felis silvestris and Prionailurus bengalensis) and tiger (Panthera tigris) donor cells. In experiment 1, zona-free (ZP-free) cloning resulted in higher fusion and expanded blastocyst rates with respect to zona included cloning techniques that involved fusion or injection of the donor cell. In experiment 2, ZP-free iSCNT and embryo aggregation (2X) were assessed. Division velocity and blastocyst rates were increased by embryo aggregation in the three species. Despite fewer tiger embryos than Bengal and cat embryos reached the blastocyst stage, Tiger 2X group increased the percentage of blastocysts with respect to Tiger 1X group (3.2% vs 12.1%, respectively). Moreover, blastocyst cell number was almost duplicated in aggregated embryos with respect to non-aggregated ones within Bengal and tiger groups (278.3 ± 61.9 vs 516.8 ± 103.6 for Bengal 1X and Bengal 2X groups, respectively; 41 vs 220 ± 60 for Tiger 1X and Tiger 2X groups, respectively). OCT4 analysis also revealed that tiger blastocysts had higher proportion of OCT4-positive cells with respect to Bengal blastocysts and cat intracytoplasmic sperm injection blastocysts. In conclusion, ZP-free cloning has improved the quality of cat embryos with respect to the other cloning techniques evaluated and was successfully applied in iSCNT complemented with embryo aggregation. © 2015 Blackwell Verlag GmbH.

  4. Post-fusion treatment with MG132 increases transcription factor expression in somatic cell nuclear transfer embryos in pigs.

    Science.gov (United States)

    You, Jinyoung; Lee, Joohyeong; Kim, Jinyoung; Park, Junhong; Lee, Eunsong

    2010-02-01

    The objective of this study was to examine the effect of post-fusion treatment of somatic cell nuclear transfer (SCNT) oocytes with the proteasomal inhibitor MG132 on maturation promoting factor (MPF) activity, nuclear remodeling, embryonic development, and gene expression of cloned pig embryos. Immediately after electrofusion, SCNT oocytes were treated with MG132 and/or caffeine for 2 hr, vanadate for 0.5 hr, or vanadate for 0.5 hr followed by MG132 for 1.5 hr. Of the MG132 concentrations tested (0-5 microM), the 1 microM concentration showed a higher rate of blastocyst formation (25.9%) than 0 (14.2%), 0.5 (16.9%), and 5 microM (16.9%). Post-fusion treatment with MG132, caffeine, and both MG132 and caffeine improved blastocyst formation (22.1%, 21.4%, and 24.4%, respectively), whereas vanadate treatment inhibited blastocyst formation (6.5%) compared to the control (11.1%). When examined 2 hr after fusion and 1 hr after activation, MPF activity remained at a higher (P fusion with caffeine and/or MG132, but it was decreased by vanadate. The rate of oocytes showing premature chromosome condensation was not altered by MG132 but was decreased by vanadate treatment. In addition, formation of single pronuclei was increased by MG132 compared to control and vanadate treatment. MG132-treated embryos showed increased expression of POU5F1, DPPA2, DPPA3, DPPA5, and NDP52l1 genes compared to control embryos. Our results demonstrate that post-fusion treatment of SCNT oocytes with MG132 prevents MPF degradation and increases expression of transcription factors in SCNT embryos, which are necessary for normal development of SCNT embryos. (c) 2009 Wiley-Liss, Inc.

  5. High gonadotropin dosage does not affect euploidy and pregnancy rates in IVF PGS cycles with single embryo transfer.

    Science.gov (United States)

    Barash, Oleksii O; Hinckley, Mary D; Rosenbluth, Evan M; Ivani, Kristen A; Weckstein, Louis N

    2017-11-01

    Does high gonadotropin dosage affect euploidy and pregnancy rates in PGS cycles with single embryo transfer? High gonadotropin dosage does NOT affect euploidy and pregnancy rates in PGS cycles with single embryo transfer. PGS has been proven to be the most effective and reliable method for embryo selection in IVF cycles. Euploidy and blastulation rates decrease significantly with advancing maternal age. In order to recruit an adequate number of follicles, the average dosage of gonadotropins administered during controlled ovarian stimulation in IVF cycles often increases significantly with advancing maternal age. A retrospective study of SNP (Single Nucleotide Polymorphism) PGS outcome data from blastocysts biopsied on day 5 or day 6 was conducted to identify differences in euploidy and clinical pregnancy rates. Seven hundred and ninety four cycles of IVF treatment with PGS between January 2013 and January 2017 followed by 651 frozen embryo transfers were included in the study (506 patients, maternal age (y.o.) - 37.2 ± 4.31). A total of 4034 embryos were analyzed (5.1 ± 3.76 per case) for euploidy status. All embryos were vitrified after biopsy, and selected embryos were subsequently thawed for a hormone replacement frozen embryo transfer cycle. All cycles were analyzed by total gonadotropin dosage (5000 IU), by number of eggs retrieved (1-5, 5-10, 10-15 and >15 eggs) and patient's age (IVF cycles) euploidy rates ranged from 62.3% (IVF cycle) to 67.5% (>5000 IU were used in the IVF cycle) (OR = 0.862, 95% CI 0.687-1.082, P = 0.2) and from 69.5% (1-5 eggs retrieved) to 60.0% (>15 eggs retrieved) (OR = 0.658, 95% CI 0.405-1.071, P = 0.09). Similar data were obtained in the oldest group of patients (≥41 y.o. - 189 IVF cycles): euploidy rates ranged from 30.7 to 26.4% (OR = 0.811, 95% CI 0.452-1.454, P = 0.481) when analyzed by total dosage of gonadotropins used in the IVF cycle and from 40.0 to 30.7% (OR = 0.531, 95% CI 0.204-1.384, P = 0.19), when assessed by the

  6. 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

  7. Cryosurvival of in vitro produced bovine embryos supplemented with l-Carnitine and concurrent reduction of fatty acids.

    Science.gov (United States)

    Held-Hoelker, E; Klein, S L; Rings, F; Salilew-Wondim, D; Saeed-Zidane, M; Neuhoff, C; Tesfaye, D; Schellander, K; Hoelker, M

    2017-07-01

    Lipid accumulation is associated with reduced embryonic quality, causing limited survival after cryopreservation. Therefore, in the present study we aimed to reveal the effects of supplementation of a lipid reducing agent, l-carnitine and the removal of fatty acids during in vitro culture on the morphological as well as on the molecular level. To accomplish that, presumptive zygotes were cultured in 4 contrasting groups: namely SOFaa medium supplemented with BSA, (BSA), SOFaa medium supplemented with fatty acid free BSA (FAF), SOFaa medium supplemented with BSA as well as l-Carnitine (BSA + LC) and SOFaa medium concurrently supplemented with fatty acid free BSA and l-Carnitine (FAF + LC). Considering the developmental rates, no impact of different treatments was observed. Conversely, treatment groups clearly affected lipid content, with the lowest amounts detected in embryos derived from FAF and BSA + LC groups, implicating that both removal of fatty acids and supplementation of LC reduces lipid content effectively. Importantly, survival rates after cryopreservation show that LC significantly affects the kinetics of re-expansion, with the highest hatching rates detected for embryos cultured in FAF + LC (p < 0.05). Noteworthy, the highest cryotolerance did not go along with lowest lipid contents. Finally, metabolic alterations between the groups were reflected in different abundances of selected candidate genes related to lipid metabolism and oxidative stress response, like AMPKA1, ACC and PGC1 α or KEAP1 and SOD1. All in all, highly beneficial effects on survival rates after cryopreservation have been detected when embryos were cultured in absence of fatty acids and concurrent presence of l-Carnitine. Highest cryotolerance, however, did not correlate with lowest lipid contents. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. In vitro production of sexed embryos for gender preselection: high-speed sorting of X-chromosome-bearing sperm to produce pigs after embryo transfer.

    Science.gov (United States)

    Rath, D; Long, C R; Dobrinsky, J R; Welch, G R; Schreier, L L; Johnson, L A

    1999-12-01

    The objectives for the present experiments were to apply sperm sexing technology to an in vitro production system with porcine oocytes obtained from slaughterhouse material. On six experimental days, ovaries were obtained from an abattoir, and cumulus-oocyte-complexes were matured in vitro. Semen was collected from mature boars of proven fertility and was sorted for X-chromosome-bearing sperm, using the Beltsville Sperm Sexing Technology incorporating the use of high-speed sorting. A total of 5,378 oocytes were submitted for in vitro fertilization (IVF). Of these, 559 ova were stained for cytogenetic analysis 18 h after IVF. From the remaining 4,819 ova, 1,595 cleaved, and 1,300 of the cleaved embryos were transferred into 26 synchronized recipients (5 control gilts for unsorted sperm, 21 gilts for X-sorted sperm). In a test of two fertilization media (FERT-A vs FERT-B) higher cleavage rates (Psexed sperm (X-sorted) produced 33 females (97%) and one male. Three litters from control transfers produced 23 pigs, 11 of which were female (48%). The sex ratio of the offspring was predicted based on the sort reanalysis of the sorted sperm for DNA content.

  9. Vitrification of immature bovine cumulus-oocyte complexes: effects of cryoprotectants, the vitrification procedure and warming time on cleavage and embryo development

    Directory of Open Access Journals (Sweden)

    Prentice-Biensch Jennifer R

    2012-09-01

    Full Text Available Abstract Background The present studies evaluated the effects of cryoprotectants, the vitrification procedure and time in the warming solution containing sucrose on cleavage and embryo development of immature (GV stage bovine cumulus-oocyte complexes (COCs. Methods Two experiments were conducted. In Experiment 1, COCs (n = 420 were randomly assigned to four groups: 1 Control group: no treatment; 2 VS1 group: COCs were exposed to vitrification solution 1 (VS1 containing 7.5% ethylene glycol [EG] + 7.5% dimethyl sulfoxide [DMSO] + 20% calf serum [CS] in TCM-199 at 37 C for 5 min; 3 VS1 + VS2 group: COCs were exposed to VS1 for 5 min followed by VS2 (15% EG + 15% DMSO + 17.1% sucrose + 20% CS at 37 C for 45–60 sec; and 4 Vitrified group: COCs were exposed to VS1 and VS2, loaded on cryotops, vitrified in liquid nitrogen and then warmed in TCM-199 + 17.1% sucrose + 20% CS at 37 C for 1 min. In Experiment 2, COCs (n = 581 were assigned to the same groups, but those in VS1, VS1 + VS2 and Vitrified groups were sub-divided and exposed to the warming solution for either 1 or 5 min. After treatment and/or warming, all COCs in both experiments underwent in vitro maturation, in vitro fertilization and in vitro culture. Results Cleavage and blastocyst rates did not differ among Control, VS1 and VS1 + VS2 groups in either experiment. In Experiment 2, there was no effect of time in the warming solution. However, both cleavage and blastocyst rates were lower (P  Conclusions The permeating cryoprotectants (EG and DMSO present in VS1 and VS2 solutions and the time in the warming solution containing sucrose had no adverse effects on cleavage and blastocyst rates of immature bovine COCs. However, cleavage rate and early embryo development were reduced following the vitrification and warming.

  10. Actions of activin A, connective tissue growth factor, hepatocyte growth factor and teratocarcinoma-derived growth factor 1 on the development of the bovine preimplantation embryo.

    Science.gov (United States)

    Kannampuzha-Francis, Jasmine; Tribulo, Paula; Hansen, Peter J

    2017-07-01

    The reproductive tract secretes bioactive molecules collectively known as embryokines that can regulate embryonic growth and development. In the present study we tested four growth factors expressed in the endometrium for their ability to modify the development of the bovine embryo to the blastocyst stage and alter the expression of genes found to be upregulated (bone morphogenetic protein 15 (BMP15) and keratin 8, type II (KRT8)) or downregulated (NADH dehydrogenase 1 (ND1) and S100 calcium binding protein A10 (S100A10)) in embryos competent to develop to term. Zygotes were treated at Day 5 with 0.01, 0.1 or 1.0nM growth factor. The highest concentration of activin A increased the percentage of putative zygotes that developed to the blastocyst stage. Connective tissue growth factor (CTGF) increased the number of cells in the inner cell mass (ICM), decreased the trophectoderm:ICM ratio and increased blastocyst expression of KRT8 and ND1. The lowest concentration of hepatocyte growth factor (HGF) reduced the percentage of putative zygotes becoming blastocysts. Teratocarcinoma-derived growth factor 1 increased total cell number at 0.01nM and expression of S100A10 at 1.0nM, but otherwise had no effects. Results confirm the prodevelopmental actions of activin A and indicate that CTGF may also function as an embryokine by regulating the number of ICM cells in the blastocyst and altering gene expression. Low concentrations of HGF were inhibitory to development.

  11. The association between polycystic ovary syndrome and ectopic pregnancy after in vitro fertilization and embryo transfer.

    Science.gov (United States)

    Wang, Jing; Wei, Yongyue; Diao, Feiyang; Cui, Yugui; Mao, Yundong; Wang, Wei; Liu, Jiayin

    2013-08-01

    We sought to assess the association between polycystic ovary syndrome (PCOS) and ectopic pregnancy after in vitro fertilization-embryo transfer (ET). In this retrospective cohort study, we included 5339 women who had clinical pregnancies after in vitro fertilization treatment (PCOS, 205 women; non-PCOS, 5134 women) at Nanjing Medical University (China) between 2007 and 2011. Fresh and cryo-thawed ET cycles were analyzed respectively. The primary outcome measure was the occurrence of ectopic pregnancy. Multivariate logistic regression analysis was used to adjust for important confounders. In fresh ET cycles of women who were undergoing controlled ovarian hyperstimulation (COH; n = 3303), women with PCOS had 3.06 times higher risk of ectopic pregnancy compared with those without PCOS (7.0% vs 2.4%; adjusted odds ratio [aOR], 3.06; 95% confidence interval [CI], 1.34-6.96). In the stratified analysis, for women without PCOS, the high estradiol group (>4085 pg/mL) had higher ectopic pregnancy rates compared with the low estradiol group (≤4085 pg/mL; 3.4% vs 2.0%; aOR, 1.99; 95% CI, 1.19-3.35); however, for women with PCOS, both high and low estradiol groups had high ectopic pregnancy rates (5.6% vs 7.7%; aOR, 0.92; 95% CI, 0.15-5.67). In cryo-thawed ET cycles without COH (n = 2036), the ectopic rates between women with and without PCOS were similar (2.2% vs 2.0%; aOR, 0.94; 95% CI, 0.22-4.07). PCOS was associated with an increased risk of ectopic pregnancy after COH in fresh ET cycles, but not in cryo-thawed ET cycles. A possible explanation is that, compared with women without PCOS, women with PCOS appear to hold a lower threshold of hyperphysiologic estradiol level that triggers the occurrence of ectopic pregnancy after COH. Copyright © 2013 Mosby, Inc. All rights reserved.

  12. Endometrial thickness as a predictor of the reproductive outcomes in fresh and frozen embryo transfer cycles

    Science.gov (United States)

    Zhang, Tao; Li, Zhou; Ren, Xinling; Huang, Bo; Zhu, Guijin; Yang, Wei; Jin, Lei

    2018-01-01

    Abstract To evaluate the relationship between endometrial thickness during fresh in vitro fertilization (IVF) cycles and the clinical outcomes of subsequent frozen embryo transfer (FET) cycles. FET cycles using at least one morphological good-quality blastocyst conducted between 2012 and 2013 at a university-based reproductive center were reviewed retrospectively. Endometrial ultrasonographic characteristics were recorded both on the oocyte retrieval day and on the day of progesterone supplementation in FET cycles. Clinical pregnancy rate, spontaneous abortion rate, and live birth rate were analyzed. One thousand five hundred twelve FET cycles was included. The results showed that significant difference in endometrial thickness on day of oocyte retrieval (P = .03) was observed between the live birth group (n = 844) and no live birth group (n = 668), while no significant difference in FET endometrial thickness was found (P = .261) between the live birth group and no live birth group. For endometrial thickness on oocyte retrieval day, clinical pregnancy rate ranged from 50.0% among patients with an endometrial thickness of ≤6 mm to 84.2% among patients with an endometrial thickness of >16 mm, with live birth rate from 33.3% to 63.2%. Multiple logistic regression analysis of factors related to live birth indicated endometrial thickness on oocyte retrieval day was associated with improved live birth rate (OR was 1.069, 95% CI: 1.011–1.130, P = .019), while FET endometrial thickness did not contribute significantly to pregnancy outcomes following FET cycles. The ROC curves revealed the cut-off points of endometrial thickness on oocyte retrieval day was 8.75 mm for live birth. Endometrial thickness during fresh IVF cycles was a better predictor of endometrial receptivity in subsequent FET cycles than FET cycle endometrial thickness. For those females with thin endometrium in fresh cycles, additional estradiol stimulation might be helpful for

  13. The effect of a multifaceted empowerment strategy on decision making about the number of embryos transferred in in vitro fertilisation: randomised controlled trial.

    Science.gov (United States)

    van Peperstraten, Arno; Nelen, Willianne; Grol, Richard; Zielhuis, Gerhard; Adang, Eddy; Stalmeier, Peep; Hermens, Rosella; Kremer, Jan

    2010-09-30

    To evaluate the effects of a multifaceted empowerment strategy on the actual use of single embryo transfer after in vitro fertilisation. Randomised controlled trial. Five in vitro fertilisation clinics in the Netherlands. 308 couples (women aged fertilisation cycle. The multifaceted strategy aimed to empower couples in deciding how many embryos should be transferred. The strategy consisted of a decision aid, support of a nurse specialising in in vitro fertilisation, and the offer of reimbursement by way of an extra treatment cycle. The control group received standard care for in vitro fertilisation. Use of single embryo transfer in the first and second treatment cycles as well as decision making variables and costs of the empowerment strategy. After the first treatment cycle, single embryo transfer was used by 43% (65/152) of couples in the intervention group and 32% (50/156) in the control group (difference 11%, 95% confidence interval 0% to 22%; P=0.05). After the second treatment cycle, single embryo transfer was used by 26% (14/154) of couples in the intervention group compared with 16% (8/51) in the control group (difference 10%, -6% to 26%; P=0.20). Compared with couples receiving standard care, those receiving the empowerment strategy had significantly higher empowerment and knowledge levels but no differences in anxiety levels. Mean total savings per couple in the intervention group were calculated to be €169.75 (£146.77; $219.12). A multifaceted empowerment strategy encouraged use of single embryo transfer, increased patients' knowledge, reduced costs, and had no effect on levels of anxiety or depression. This strategy could therefore be an important tool to reduce the twin pregnancy rate after in vitro fertilisation. This trial did not, however, demonstrate the anticipated 25% difference in use of single embryo transfer of the power calculation. ClinicalTrials.gov NCT00315029.

  14. Efficacy of dehydroepiandrosterone (DHEA) supplementation for in vitro fertilization and embryo transfer cycles: a systematic review and meta-analysis.

    Science.gov (United States)

    Liu, Yaofang; Hu, Lina; Fan, Lingye; Wang, Fang

    2018-03-01

    Dehydroepiandrosterone (DHEA) supplementation might hold some promise in vitro fertilization and embryo transfer cycles. However, the results remain controversial. We conducted a systematic review and meta-analysis to evaluate the efficacy of DHEA in patients for in vitro fertilization. PubMed, EMbase, Web of science, EBSCO and Cochrane library databases were systematically searched. Randomized controlled trials (RCTs) assessing the effect of DHEA versus placebo on in vitro fertilization were included. Two investigators independently searched articles, extracted data and assessed the quality of included studies. The primary outcomes were clinical pregnancy and live birth rate. Meta-analysis was performed using random-effect model. Six RCTs involving 745 patients were included in the meta-analysis. Overall, compared with placebo, DHEA supplementation was associated with the significant increase in clinical pregnancy (OR = 1.45; 95% CI = 1.04-2.03; p = .03), live birth rate (OR = 2.70; 95% CI = 1.24-5.85; p = .01) and endometrial thickness (Std. mean difference = 0.67; 95% CI = 0.02-1.32; p = .04) but showed no influence on E 2 on hCG day (Std. mean difference = 0.69; 95% CI =  -0.46 to 1.85; p = .24), embryos transferred (Std. mean difference = 0.42; 95% CI =  -0.04 to 0.88; p = .07) and miscarriage rate (OR = 0.43; 95% CI = 0.03-6.66; p = .55). DHEA supplementation could significantly improve clinical pregnancy, live birth rate, endometrial thickness and retrieved oocytes but failed to alter E 2 on hCG day, embryos transferred and miscarriage rate.

  15. Reduced levels of intracellular reactive oxygen species and apoptotic status are not correlated with increases in cryotolerance of bovine embryos produced in vitro in the presence of antioxidants.

    Science.gov (United States)

    Rocha-Frigoni, Nathália A S; Leão, Beatriz C S; Nogueira, Ériklis; Accorsi, Mônica F; Mingoti, Gisele Z

    2014-01-01

    The effects of intracellular (cysteine and β-mercaptoethanol) and extracellular (catalase) antioxidant supplementation at different times during in vitro production (IVM and/or in vitro culture (IVC)) on bovine embryo development, intracellular reactive oxygen species (ROS) levels, apoptosis and re-expansion rates after a vitrification-thawing process were examined. Blastocyst frequencies were not affected by either antioxidant supplementation (40.5%-56.4%) or the timing of supplementation (41.7%-55.4%) compared with control (48.7%; P>0.05). Similarly, antioxidants and the moment of supplementation did not affect (P>0.05) the total number of blastomeres (86.2-90.5 and 84.4-90.5, respectively) compared with control (85.7). However, the percentage of apoptotic cells was reduced (P0.05) from that in the control group (1.00). Re-expansion rates were not affected (P>0.05) by the treatments (50.0%-93.0%). In conclusion, antioxidant supplementation during IVM and/or IVC reduces intracellular ROS and the rate of apoptosis; however, supplementation does not increase embryonic development and survival after vitrification.

  16. 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.

  17. Improved bovine embryo production in an oviduct-on-a-chip system: prevention of poly-spermic fertilization and parthenogenic activation.

    Science.gov (United States)

    Ferraz, Marcia A M M; Henning, Heiko H W; Costa, Pedro F; Malda, Jos; Melchels, Ferry P; Wubbolts, R; Stout, Tom A E; Vos, Peter L A M; Gadella, Bart M

    2017-02-28

    The oviduct provides the natural micro-environment for gamete interaction, fertilization and early embryo development in mammals, such as the cow. In conventional culture systems, bovine oviduct epithelial cells (BOEC) undergo a rapid loss of essential differentiated cell properties; we aimed to develop a more physiological in vitro oviduct culture system capable of supporting fertilization. U-shaped chambers were produced using stereo-lithography and mounted with polycarbonate membranes, which were used as culture inserts for primary BOECs. Cells were grown to confluence and cultured at an air-liquid interface for 4 to 6 weeks and subsequently either fixed for immune staining, incubated with sperm cells for live-cell imaging, or used in an oocyte penetration study. Confluent BOEC cultures maintained polarization and differentiation status for at least 6 weeks. When sperm and oocytes were introduced into the system, the BOECs supported oocyte penetration in the absence of artificial sperm capacitation factors while also preventing polyspermy and parthenogenic activation, both of which occur in classical in vitro fertilization systems. Moreover, this "oviduct-on-a-chip" allowed live imaging of sperm-oviduct epithelium binding and release. Taken together, we describe for the first time the use of 3D-printing as a step further on bio-mimicking the oviduct, with polarized and differentiated BOECs in a tubular shape that can be perfused or manipulated, which is suitable for live imaging and supports in vitro fertilization.

  18. Reproductive efficiency of asymptomatic Theileria equi carriers mares submitted to an embryo transfer program

    Directory of Open Access Journals (Sweden)

    Luciana L. Bezerra

    2015-03-01

    Full Text Available This study aimed to assess and evaluate the effects of Theileria equi infection on embryonic recovery, gestation and early embryonic loss. Thirteen Mangalarga Marchador Theileria equi positive donors (diagnosed through nested-PCR and 40 embryos receptors were used. Donors were submitted to two embryo collections in two consecutive estrous cycles (GId; after, the same mares were treated with imidocarb dipropionate (1.2mg/kg IM. in order to collect more embryos in two more estrous cycles (GIId. Receptors were divided into two groups (control and with treated with 20 animals each, where one group was the control (GIr and the other one (GIIr treated with 1.2mg/kg IM of imidocarb dipropionate assessing the gestation rate at 15, 30, 45 and 60 days. After 52 embryo collections, the embryonic recovery rates were 53.84% (14/26 and 65.38% (17/26 (p> 0.05 for GId and GIId, respectively. The gestation rate was 70% (14/20 (p>0.05 at 15, 30, 45 and 60 days in group GIr and for GIIr was 85% (17/20 (p>0.05 at 15 days, 80% (16/20 (p>0.05 at 30, 45 and 60 days. The treatment with imidocarb dipropionate did not cause significant improvement in the reproductive efficiency at an ET program.

  19. Application of in vitro production-embryo transfer in the protection ...

    African Journals Online (AJOL)

    Yomi

    2012-01-12

    Jan 12, 2012 ... reproductive technology (ART) can effectively protect and multiply the superior genes of lactation and reproduction within the dairy farms. Key words: In vitro production, embryo ..... In this context, generator cows at the end of their first year of life are prepared for artificial insemination and pregnancy. Also, it.

  20. Development of a new clinically applicable device for embryo evaluation which measures embryo oxygen consumption.

    Science.gov (United States)

    Kurosawa, Hiroki; Utsunomiya, Hiroki; Shiga, Naomi; Takahashi, Aiko; Ihara, Motomasa; Ishibashi, Masumi; Nishimoto, Mitsuo; Watanabe, Zen; Abe, Hiroyuki; Kumagai, Jin; Terada, Yukihiro; Igarashi, Hideki; Takahashi, Toshifumi; Fukui, Atsushi; Suganuma, Ryota; Tachibana, Masahito; Yaegashi, Nobuo

    2016-10-01

    . Furthermore, the developed blastocysts were scored using the blastocyst quality score (BQS), and the correlation with oxygen consumption rate was also assessed. The device enabled the oxygen consumption rate of an embryo to be measured automatically within a minute. The oxygen concentration gradient profile showed excellent linearity in a distance-dependent change. A close correlation in the oxygen consumption rates of bovine embryos was observed between the SECM measuring system and CERMs, with a determination coefficient of 0.8203 (P = 0.0008). Oxygen consumption rates of human embryos that have reached the blastocyst stage were significantly higher than those of arrested embryos at 48, 72 and 96 h after thawing (P = 0.039, 0.004 and 0.049, respectively). Thus, in vitro development of frozen-thawed human embryos to the blastocyst stage would be predicted at 48 h after thawing (day 4) by measuring the oxygen consumption using CERMs. Although a positive linear relationship between BQS and the oxygen consumption rate was observed [the determination coefficient was R(2) = 0.6537 (P = 0.008)], two blastocysts exhibited low oxygen consumption rates considering their relatively high BQS. This suggests that morphology and metabolism in human embryos might not correlate consistently. Transfer of the embryo and pregnancy evaluation was not performed. Thus, a correlation between oxygen consumption and the in vivo viability of embryos remains unknown. Clinical trials, including embryo transfer, would be desirable to determine a threshold value to elect clinically relevant, quality embryos for transfer. We utilized frozen-thawed human embryos in this study. The effect of these manipulations on the respiratory activity of the embryo is also unknown. Selection of quality embryos, especially in a single embryo transfer cycle, by CERMs may have an impact on obtaining better clinical outcomes, albeit with clinical trials being required. Furthermore, the early determination of quality

  1. Holding immature bovine oocytes in a commercial embryo holding medium: High developmental competence for up to 10 h at room temperature.

    Science.gov (United States)

    Pascottini, Osvaldo Bogado; Catteeuw, Maaike; Van Soom, Ann; Opsomer, Geert

    2017-11-04

    Bovine in vitro embryo production (IVP) following Ovum Pick Up (OPU) is all too often hampered by a large time gap between the harvest of oocytes of the first and last OPU session of the day. Immediately after retrieval, oocyte maturation is initiated, resulting in oocytes maturing at different time points which necessitates laborious scheduling of the IVP process. In this study, the potential of a commercial embryo holding medium (EHM; Syngro, Bioniche Inc.) to hold immature bovine oocytes was validated. We assessed the effect of holding time and temperature on (1) oocytes' maturation; (2) blastocyst development and quality at day 8 post insemination; and (3) blastocyst yield in small groups of oocytes/zygotes simulating OPU settings. Oocytes, harvested from slaughterhouse ovaries, were held for 6 h (either at 4 °C, room temperature [RT; 22-25 °C], or 38.5 °C), for 10 h (at 4 °C or RT), and for 14 h (only at RT) in 1 mL sterile glass osmometer tubes filled with EHM prior to standard maturation (22 h at 38.5 °C) and subsequent IVP. Results were compared with controls in which no prior holding was applied. Differences between the treated and control groups were assessed by generalized mixed-effects models and considered significant at P h in EHM at different temperatures remained at the germinal vesicle stage. Holding immature oocytes in EHM for 6 h at 38.5 °C and for 10 h at 4 °C significantly decreased maturation (57.1 ± 4.1% VS 80.9 ± 3.2% and 68.6 ± 3.5% VS 80.7 ± 2.9%; respectively), and development (11.0 ± 1.8% VS 36.2 ± 2.8% and 20.1 ± 3.3% VS 40.6 ± 4.6%) (P h at RT, did not affect the maturation rates (83.2 ± 2.9% and 78.9 ± 3.2%) nor day 8 blastocyst rates (35.2 ± 2.7% and 40.2 ± 4.5%). Prolonging holding time to 14 h in RT decreased maturation and day 8 blastocyst yield (71.9 ± 3.5% VS 84.5 ± 2.7% and 25.7 ± 2.5% VS 39.5 ± 2.8%, respectively) (P h at RT. When subsequently

  2. Monochorionic-triamniotic triplet pregnancy after intracytoplasmic sperm injection, assisted hatching, and two-embryo transfer: first reported case following IVF

    Directory of Open Access Journals (Sweden)

    Eller Daniel P

    2003-08-01

    Full Text Available Abstract Background We present a case of monochorionic-triamniotic pregnancy that developed after embryo transfer following in vitro fertilization (IVF. Methods After controlled ovarian hyperstimulation and transvaginal retrieval of 22 metaphase II oocytes, fertilization was accomplished with intracytoplasmic sperm injection (ICSI. Assisted embryo hatching was performed, and two embryos were transferred in utero. One non-transferred blastocyst was cryopreserved. Results Fourteen days post-transfer, serum hCG level was 423 mIU/ml and subsequent transvaginal ultrasound revealed a single intrauterine gestational sac with three separate amnion compartments. Three distinct foci of cardiac motion were detected and the diagnosis was revised to monochorionic-triamniotic triplet pregnancy. Antenatal management included cerclage placement at 19 weeks gestation and hospital admission at 28 weeks gestation due to mild preeclampsia. Three viable female infants were delivered via cesarean at 30 5/7 weeks gestation. Conclusions The incidence of triplet delivery in humans is approximately 1:6400, and such pregnancies are classified as high-risk for reasons described in this report. We also outline an obstetric management strategy designed to optimize outcomes. The roles of IVF, ICSI, assisted embryo hatching and associated laboratory culture conditions on the subsequent development of monozygotic/monochorionic pregnancy remain controversial. As demonstrated here, even when two-embryo transfer is employed after IVF the statistical probability of monozygotic multiple gestation cannot be reduced to zero. We encourage discussion of this possibility during informed consent for the advanced reproductive technologies.

  3. Ectopic pregnancy following in vitro fertilization with embryo transfer: A single-center experience during 15 years.

    Science.gov (United States)

    Cheng, Ling-Yun; Lin, Pin-Yao; Huang, Fu-Jen; Kung, Fu-Tsai; Chiang, Hsin-Ju; Lin, Yu-Ju; Lan, Kuo-Chung

    2015-10-01

    Ectopic pregnancy is an obstetrical disease that is potentially associated with maternal death in the first trimester. It is one of the well-known complications following in vitro fertilization (IVF) with embryo transfer (ET). The incidence of ectopic pregnancy is estimated to be 2.1-8.6% of clinical pregnancy after IVF-ET, which is higher than natural conceptions (incidence rate 2%). This study aimed to re-evaluate the ectopic pregnancy rate in patients undergoing IVF-ET and to investigate the effects of embryo stage and frozen-thawed blastocyst transfer and ET during full bladder distention on ectopic pregnancy rate. This retrospective study reviewed women who achieved a clinical pregnancy after IVF-ET at the Department of Obstetrics and Gynecology, Kaohsiung Chang Gung Memorial Hospital between 1999 and 2013. We compared ectopic pregnancy rate following Day 3 ET with Day 5 ET, and after fresh ET with thawed ET. Besides, multivariate analysis was used to clarify the factors affecting ectopic pregnancy after IVF-ET. Of the total 1213 clinical pregnancies after fresh ET, 18 (1.5%) were verified as ectopic, which is similar to the rate following natural conception. The ectopic pregnancy rates were similar for Day 3 (1.2%) and Day 5 (1.7%) ETs. The incidence of ectopic pregnancy in thawed ET cycles (0.6%) was not significantly reduced than fresh ET cycles (1.5%). Tubal ET (TET) and ET under full bladder distention had a significant effect on ectopic pregnancy. Thawed ET was not associated with a lower incidence of ectopic pregnancy than fresh ET, and embryo stage did not affect the rate of ectopic pregnancy. In addition, TET and ET under conditions of full bladder distention may increase the ectopic pregnancy rate. Copyright © 2015. Published by Elsevier B.V.

  4. IVF policy and global/local politics: the making of multiple-embryo transfer regulation in Taiwan.

    Science.gov (United States)

    Wu, Chia-Ling

    2012-08-01

    This paper analyzes the regulatory trajectory of multiple-embryo transfer in in-vitro fertilization (IVF) in Taiwan. Taking a latecomer to policy-making as the case, it argues the importance of conceptualizing the global/local dynamics in policy-making for assisted reproductive technology (ART). The conceptual framework is built upon recent literature on standardization, science policy, and global assemblage. I propose three interrelated features that reveal the "global in the local": (1) the power relationships among stakeholders, (2) the selected global form that involved actors drew upon, and (3) the re-contextualized assemblage made of local networks. Data included archives, interviews, and participant observation. In different historical periods the specific stakeholders selected different preferred global forms for Taiwan, such as Britain's code of ethics in the 1990s, the American guideline in the early 2000s, and the European trend in the mid-2000s. The global is heterogeneous. The failure to transfer the British regulation, the revision of the American guideline by adding one more embryo than it specified, and the gap between the cited European trend and the "no more than four" in Taiwan's 2007 Human Reproduction Law all show that the local network further transforms the selected global form, confining it to rhetoric only or tailoring it to local needs. Overall, Taiwanese practitioners successfully maintained their medical autonomy to build a 'flexible standardization'. Multiple pregnancy remains the most common health risk of IVF in Taiwan. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. GnRH agonist versus GnRH antagonist in in vitro fertilization and embryo transfer (IVF/ET

    Directory of Open Access Journals (Sweden)

    Depalo Raffaella

    2012-04-01

    Full Text Available Abstract Several protocols are actually available for in Vitro Fertilization and Embryo Transfer. The review summarizes the main differences and the clinic characteristics of the protocols in use with GnRH agonists and GnRH antagonists by emphasizing the major outcomes and hormonal changes associated with each protocol. The majority of randomized clinical trials clearly shows that in “in Vitro” Fertilization and Embryo Transfer, the combination of exogenous Gonadotropin plus a Gonadotropin Releasing Hormone (GnRH agonist, which is able to suppress pituitary FSH and LH secretion, is associated with increased pregnancy rate as compared with the use of gonadotropins without a GnRH agonist. Protocols with GnRH antagonists are effective in preventing a premature rise of LH and induce a shorter and more cost-effective ovarian stimulation compared to the long agonist protocol. However, a different synchronization of follicular recruitment and growth occurs with GnRH agonists than with GnRH antagonists. Future developments have to be focused on timing of the administration of GnRH antagonists, by giving a great attention to new strategies of stimulation in patients in which radio-chemotherapy cycles are needed.

  6. Embryo transfer in no cycling Crioula and Quarter horse breeds treated with estradiol cipionate and long-acting progesterone

    Directory of Open Access Journals (Sweden)

    Fernanda Kaercher

    2011-12-01

    Full Text Available The aim of this work was to prepare the mares for embryo transfer. In group 1 (G1,treated, n=15, recipient mares in anoestrus or in a transition period were treated with 5.0, 3.0 and 2.0 mg of estradiol cipionate at the days 0, 1 and 2 respectively, beginning at the day of ovulation (DO. From the fourth day on, the mares this group received long-acting progesterone weekly, up to the 120 day of gestation. At D8, the embryo was collected from the donor and transferred to the recipient. At D12, the ultrasonographyc diagnosis of pregnancy was carried out. The control group (G2, not treated, n=20 was formed by cycling recipient mares, displaying ovulation at each 2 to 3 days after the donors mare ovulation. The pregnancy rate was higher (p<0.05 in the mares from G2 (85.0% than from G1 (53.3%. Thus, it could be concluded that the treated mares although showed lesser pregnancy rate than the cycling mare, were satisfactory alternative to be used mainly when there is no available cycling recipient.

  7. The Effects of Perioperative Analgesia on Litter Size in Crl:CD1(ICR) Mice Undergoing Embryo Transfer

    Science.gov (United States)

    Goulding, David R; Myers, Page H; Goulding, Eugenia H; Blankenship, Terry L; Grant, Mary F; Forsythe, Diane B

    2010-01-01

    The objective of this study was to evaluate the effect on litter size of 2 analgesics used perioperatively during mouse embryo transfer surgery. Day 2.5 pseudopregnant CD1 mice (n = 96) were divided equally into 2 analgesic treatment groups and a saline control group. Each mouse received a single, subcutaneous dose of buprenorphine hydrochloride (0.1 mg/kg), flunixin meglumine (2.5 mg/kg), or saline immediately after induction of anesthesia with 2.5% isoflurane. Each mouse then was prepared for aseptic surgery. Blastocysts had previously been collected from C57BL/6NCrl female mice that were synchronized and superovulated by using pregnant mare serum gonadotropin and human chorionic gonadotropin and mated with C57BL/6NTac male mice 3.5 d before collection. Viable blastocysts were pooled, and 8 were selected arbitrarily and transplanted into the right uterine horn of each pseudopregnant CD1 mouse. Mice were monitored throughout pregnancy, and the number of pups at birth was documented. No statistically significant difference was found between the 3 groups. These results indicate that perioperative analgesic treatment with buprenorphine or flunixin in the CD1 mouse undergoing embryo transfer is not associated with increased embryonic loss. PMID:20819387

  8. Association Between Endometrial/Subendometrial Vasculature and Embryo Transfer Outcome: A Meta-analysis and Subgroup Analysis.

    Science.gov (United States)

    Wang, Jianing; Xia, Fei; Zhou, Ying; Wei, Xuedong; Zhuang, Yanyan; Huang, Yingxue

    2018-01-01

    To examine the association between endometrial/subendometrial vasculature and in vitro fertilization-embryo transfer (IVF-ET) and frozen embryo transfer (FET) outcomes. A meta-analysis of studies using endometrial/subendometrial 3-dimensional ultrasound and power Doppler angiography was performed to examine the vascularization index (VI), flow index (FI), and vascularization-flow index (VFI) in pregnant and nonpregnant women. Ten articles were analyzed, including 895 pregnant women and 882 nonpregnant women. A subgroup analysis of the measuring time showed that the endometrial VI (standardized mean difference [SMD], 0.57; 95% confidence interval [CI], 0.40, 0.74; P endometrial VI (SMD, 0.52; 95% CI, 0.30, 0.74; P endometrial VI, FI, and VFI on the ET day are potentially associated with pregnancy occurrence during IVF-ET. The endometrial VI, FI, and VFI could help identify appropriate timing for FET. However, the accuracy of these indices in predicting pregnancy occurrence must be further evaluated in additional large-scale studies. © 2017 by the American Institute of Ultrasound in Medicine.

  9. Review: Placental perturbations induce the developmental abnormalities often observed in bovine somatic cell nuclear transfer.

    Science.gov (United States)

    Chavatte-Palmer, P; Camous, S; Jammes, H; Le Cleac'h, N; Guillomot, M; Lee, R S F

    2012-02-01

    Since the first success in cloning sheep, the production of viable animals by somatic cell nuclear transfer (SCNT) has developed significantly. Cattle are by far the most successfully cloned species but, despite this, the technique is still associated with a high incidence of pregnancy failure and accompanying placental and fetal pathologies. Pre- and early post-implantation losses can affect up to 70% of the pregnancies. In the surviving pregnancies, placentomegaly and fetal overgrowth are commonly observed, but the incidence varies widely, depending on the genotype of the nuclear donor cell and differences in SCNT procedures. In all cases, the placenta is central to the onset of the pathologies. Although cellular organisation of the SCNT placenta appears normal, placental vascularisation is modified and fetal-to-maternal tissue ratios are slightly increased in the SCNT placentomes. In terms of functionality, steroidogenesis is perturbed and abnormal estrogen production and metabolism probably play an important part in the increased gestation length and lack of preparation for parturition observed in SCNT recipients. Maternal plasma concentrations of pregnancy-associated glycoproteins are increased, mostly due to a reduction in turnover rate rather than increased placental production. Placental glucose transport and fructose synthesis appear to be modified and hyperfructosemia has been observed in neonatal SCNT calves. Gene expression analyses of the bovine SCNT placenta show that multiple pathways and functions are affected. Abnormal epigenetic re-programming appears to be a key component of the observed pathologies, as shown by studies on the expression of imprinted genes in SCNT placenta. Copyright © 2012. Published by Elsevier Ltd.

  10. 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

  11. Ongoing and cumulative pregnancy rate after cleavage-stage versus blastocyst-stage embryo transfer using vitrification for cryopreservation: impact of age on the results.

    Science.gov (United States)

    Fernández-Shaw, S; Cercas, R; Braña, C; Villas, C; Pons, I

    2015-02-01

    To determine if blastocyst transfer increases the ongoing and cumulative pregnancy rates, compared with day 3 embryo transfer, in women of all ages when at least 4 zygotes are obtained. Prospective study including patients undergoing a first IVF/ICSI treatment and assigned to cleavage stage (n = 46) or blastocyst (n = 58) embryo transfer. Supernumerary embryos were vitrified and patients failing to achieve an ongoing pregnancy after fresh embryo transfer would go through cryopreserved cycles. The main outcome measure was the ongoing pregnancy rate after the fresh IVF/ICSI transfer and the cumulative ongoing pregnancy rate. Results were also analyzed according to age (under 35 and 35 or older). A majority of patients (96.6 %) had a blastocyst transfer when at least 4 zygotes were obtained. The ongoing pregnancy rate was significantly higher in the day-5 group compared with the day-3 group (43.1 % vs. 24 %, p = 0.041). The cumulative ongoing pregnancy rate was higher (but not significantly) with blastocyst than with cleavage stage embryos (56.8 % vs. 43.4 %, p = 0.174). When analysed by age, patients 35 or older showed significantly higher ongoing pregnancy rate (48.4 % vs. 19.3 %, p = 0.016) and cumulative ongoing pregnancy rate (58 % vs. 25.8 %, p = 0.01) in the day-5 group compared to the day-3 group, while no such differences were observed in women under 35. Blastocyst transfer can be suggested whenever there are at least 4 zygotes. While there are no differences in women under 35, the benefit of this option over cleavage stage transfer could be significant in women 35 or older.

  12. No Peri- and Postnatal Effects on Calves Born After Transfer of in Vitro Produced Embryos Vitrified by the Open Pulled Straw (OPS Method

    Directory of Open Access Journals (Sweden)

    Callesen H

    2003-06-01

    Full Text Available The general objective of this study was to perform follow-up studies including selected peri- and postnatal characteristics on calves born after transfer of in vitro produced (IVP embryos vitrified by the 'Open Pulled Straw' (OPS method. An overall pregnancy rate of 16% after transfer of the OPS-vitrified IVP embryos was achieved and resulted in birth of 9 calves, with 11 AI calves serving as controls. There were no immediate or long-term effects on these calves with respect to birth weight, gestation length, perinatal mortality, growth rate, disease susceptibility and reproductive performance.

  13. No peri- and postnatal effects on calves born after transfer of in vitro produced embryos vitrified by the open pulled straw (OPS) method

    DEFF Research Database (Denmark)

    Jacobsen, H.; Holm, P.; Schmidt, M.

    2003-01-01

    The general objective of this study was to perform follow-up studies including selected peri- and postnatal characteristics on calves born after transfer of in vitro produced (IVP) embryos vitrified by the 'Open Pulled Straw' (OPS) method. An overall pregnancy rate of 16% after transfer of the OPS......-vitrified IVP embryos was achieved and resulted in birth of 9 calves, with 11 AI calves serving as controls. There were no immediate or long-term effects on these calves with respect to birth weight, gestation length, perinatal mortality, growth rate, disease susceptibility and reproductive performance....

  14. Effect of histone acetylation modification with MGCD0103, a histone deacetylase inhibitor, on nuclear reprogramming and the developmental competence of porcine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Jin, Long; Zhu, Hai-Ying; Guo, Qing; Li, Xiao-Chen; Zhang, Yu-Chen; Cui, Cheng-Du; Li, Wen-Xue; Cui, Zheng-Yun; Yin, Xi-Jun; Kang, Jin-Dan

    2017-01-01

    Cloning remains as an important technique to enhance the reconstitution and distribution of animal population with high-genetic merit. One of the major detrimental factors of this technique is the abnormal epigenetic modifications. MGCD0103 is known as a histone deacetylase inhibitor. In this study, we investigated the effect of MGCD0103 on the in vitro blastocyst formation rate in porcine somatic cell nuclear transferred (SCNT) embryos and expression in acetylation of the histone H3 lysine 9 and histone H4 lysine 12. We compared the in vitro embryonic development of SCNT embryos treated with different concentrations of MGCD0103 for 24 hours. Our results reported that treating with 0.2-μM MGCD0103 for 24 hours effectively improved the development of SCNT embryos, in comparison to the control group (blastocyst formation rate, 25.5 vs. 10.7%, P MGCD0103 for various intervals after activation. Treatment for 6 hours significantly improved the development of pig SCNT embryos, compared with the control group (blastocyst formation rate, 21.2 vs. 10.5%, P MGCD0103 supplementation significantly (P MGCD0103-treated SCNT embryos were transferred into two surrogate sows, one of whom became pregnant and three fetuses developed. These results suggest that MGCD0103 can enhance the nuclear reprogramming and improve in vitro developmental potential of porcine SCNT embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Correlation analysis between ultrasound findings and abnormal karyotypes in the embryos from early pregnancy loss after in vitro fertilization-embryo transfer.

    Science.gov (United States)

    Li, Xihong; Ouyang, Yan; Yi, Yan; Tan, Yueqiu; Lu, Guangxiu

    2017-01-01

    The purpose of the study is to evaluate the correlation between ultrasound findings and abnormal karyotypes in early pregnancy losses (EPLs) after in vitro fertilization-embryo transfer (IVF-ET). This retrospective analysis assessed 2172 cases of EPL after IVF-ET occurring between January 2008 and December 2013. The cases were examined via transvaginal ultrasonography (TVS). Embryonic tissue karyotyping following miscarriage was performed using a comparative genomic hybridization (CGH) analysis with fluorescence in situ hybridization (FISH). The correlations between the ultrasound findings and the karyotypes were evaluated. Six categories of ultrasound findings were observed: normal ultrasound, empty sac, yolk sac only, small gestational sac, small embryonic pole, and early symmetrical arrested growth. The overall rate of abnormal karyotypes was 44.9 % (976/2172), and the rate of abnormal karyotypes associated with a normal ultrasound, empty sac, yolk sac only, small gestational sac, small embryonic pole, and early symmetrical arrested growth was 49.5 % (218/440), 28.1 % (138/491), 43.4 % (197/454), 50.0 % (43/86), 49.8 % (155/311), and 57.7 % (225/390), respectively. Compared with the other groups, the prevalence of chromosomal abnormalities was significantly higher in the early symmetrical arrested growth group but was markedly lower in the empty sac group in all cases and when cases of 46,XX were excluded (p early symmetrical arrested growth groups. In the empty sac, small gestational sac and normal ultrasound groups, monosomy X was the most frequent abnormality. Chromosomal anomalies may be associated with specific types of ultrasound findings in EPLs after IVF-ET.

  16. Reduced Ectopic Pregnancy Rate on Day 5 Embryo Transfer Compared with Day 3: A Meta-Analysis.

    Science.gov (United States)

    Zhang, Bingqian; Cui, Linlin; Tang, Rong; Ding, Lingling; Yan, Lei; Chen, Zi-Jiang

    2017-01-01

    To compare the risk of ectopic pregnancy (EP) after embryo transfer on day 3(D3-ET) and day 5(D5-ET). Meta-analysis. Women with pregnancy resulting from in vitro undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Twenty-two studies were identified through research conducted using the PubMed, Embase, and Cochrane databases and ClinicalTrials.gov. All studies were conducted prior to October 2016. Adding the reproductive data from our center, a total of 143 643 pregnancies were reviewed(D3-ET: n = 62027,D5-ET:n = 81616). A lower EP rate was found in women undergoing D5-ET than in those undergoing D3-ET [relative risk (RR), 0.67;95% confidence interval (CI), 0.54-0.85;143643 pregnancies in 23 studies; I2 = 67%]. These results were validated in subgroups of fresh embryo-transfer (Fre-ET) cycles [RR, 0.78; 95%CI, 0.69-0.88; 91 871 pregnancies in 21 studies; I2 = 29%] and frozen-thawed embryo-transfer (Fro-ET) cycles [RR, 0.43; 95%CI, 0.36-0.51; 51 772 pregnancies in 10 studies; I2 = 33%]. After separating out the randomized controlled trials (RCTs), a significant difference was found in the retrospective studies in both subgroups [both Fre-ET (RR,0.78;95% CI 0.69-0.88);91182 pregnancies in 14 studies; I2 = 45%] and Fro-ET(RR,0.43;95% CI 0.36-0.51; 51751pregnancies in 9 studies;I2 = 33%)], while the RCTs showed no statistical significance for Fre-ET cycles[RR,0.86;95% CI 0.32-2.26); 689 pregnancies in 7 studies; I2 = 0%]. The present study indicates that D5-ET reduces the risk for EP in cycles that use IVF or ICSI, compared with D3-ET. It suggests that D5-ET may be a better choice for decreasing the EP rate in assisted reproductive technology. Further high-quality randomized controlled trials are anticipated.

  17. Influence of temperature and humidity in a equine embryo transfer program, in the Baixada Fluminense, Rio de Janeiro

    Directory of Open Access Journals (Sweden)

    Jhonnatha Paulo Oliveira

    2015-06-01

    Full Text Available ABSTRACT. Oliveira J.P., Jacob J.C.F., Jesus V.L.T. & Silva P.C.A. [Influence of temperature and humidity in a equine embryo transfer program, in the Baixada Fluminense, Rio de Janeiro.] Influência da temperatura e umidade ambiente em um programa de transferência de embriões equinos, na Baixada Fluminense, Rio de Janeiro. Revista Brasileira de Medicina Veterinária, 37(2:158- 162, 2015. Departamento de Reprodução e Avaliação Animal, Universidade Federal Rural do Rio de Janeiro, BR 465, Km 7, Seropédica, RJ 23890-970, Brasil. E-mail: juliorep@ufrrj.br This study aimed to evaluate the relationship between high environment temperature and humidity and reproductive rates in a equine embryo transfer program in Baixada Fluminense RJ. We evaluated the reproductive history of 60 donor mares and 111 recipient mares during summer of breeding seasons of 2008/2009, 2009/2010 and 2010/2011. Daily climatics data of environmental temperature (°C and relative humidity (% for the each breeding seasons were obtained from the web site of the National Institute of Meteorology (INMET, based on these data we calculated the temperature x humidity index (TUI wich measures the thermal comfort zone. The reproductive parameters assessed were embryo recovery rate (RR and pregnancy rate (PR. After the computation of reproductive and climatic data, these were compared to establish relationships between high temperatures and humidity on reproductive rates. There was a negative relationship between RR and high environmental temperatures, especially in the summer time, greater RR at 26°C (71% and lower RR at 27°C (51.4% (p <0.05. To PR there was a negative relationship to high environmental temperatures, the higher PR was obtained at 24°C (81.5% and lowest PG (35% at 27°C (p <0.05. We conclude that there are relationships between environmental variables and ET success.

  18. EFSA Panel on Biological Hazards (BIOHAZ); Scientific Opinion on the risk of transmission of classical scrapie via in vivo derived embryo transfer in ovine animals

    DEFF Research Database (Denmark)

    Hald, Tine; Baggesen, Dorte Lau

    . Under natural exposure conditions, animals that are heterozygous or homozygous A136R154R171 display respectively a low or negligible risk of being infected. The genetic control of the susceptibility to classical scrapie is also likely to impact on the risk of transmitting the disease via embryo transfer......The risk of transmission of classical scrapie via the transfer of in vivo derived embryo in ovines was assessed, taking into account the scientific information made available since the last EFSA opinion on this topic (2010) (see http://www.efsa.europa.eu/en/efsajournal/pub/1429.htm). The potential...... impact of PrP genotype of the embryo and/or of the ram and donor ewe on this risk was also assessed. The new data made available over the last three years further reinforce the view that classical scrapie could be vertically transmitted in sheep. Since the possibility of such vertical transmission...

  19. Embryo transfer techniques: an American Society for Reproductive Medicine survey of current Society for Assisted Reproductive Technology practices.

    Science.gov (United States)

    Toth, Thomas L; Lee, Malinda S; Bendikson, Kristin A; Reindollar, Richard H

    2017-04-01

    To better understand practice patterns and opportunities for standardization of ET. Cross-sectional survey. Not applicable. Not applicable. An anonymous 82-question survey was emailed to the medical directors of 286 Society for Assisted Reproductive Technology member IVF practices. A follow-up survey composed of three questions specific to ET technique was emailed to the same medical directors. Descriptive statistics of the results were compiled. The survey assessed policies, protocols, restrictions, and specifics pertinent to the technique of ET. There were 117 (41%) responses; 32% practice in academic settings and 68% in private practice. Responders were experienced clinicians, half of whom had performed technique. Multiple steps in the ET process were identified as "highly conserved;" others demonstrated discordance. ET technique is divided among [1] trial transfer followed immediately with ET (40%); [2] afterload transfer (30%); and [3] direct transfer without prior trial or afterload (27%). Embryos are discharged in the upper (66%) and middle thirds (29%) of the endometrial cavity and not closer than 1-1.5 cm from fundus (87%). Details of each step were reported and allowed the development of a "common" practice ET procedure. ET training and practices vary widely. Improved training and standardization based on outcomes data and best practices are warranted. A common practice procedure is suggested for validation by a systematic literature review. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  20. Cryo-thawed embryo transfer: natural versus artificial cycle. A non-inferiority trial.(ANTARCTICA trial

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    Groenewoud Eva R

    2012-09-01

    Full Text Available Abstract Background Frozen thawed embryo transfer (FET is a cost- effective adjunct to IVF or IVF-ICSI treatment. In order to optimize treatment outcome, FET should be carried out during a period of optimal endometrial receptivity. To optimize implantation several methods for endometrium preparation have been proposed. In natural cycle FET (NC-FET, the endometrium develops under endogenous hormonal stimulation. The development of the dominant follicle and endometrium is monitored by ultrasound and FET is timed after triggering ovulation induction or determination of the spontaneous LH surge. In an artificial cycle FET (AC-FET estrogens and progesterone are administered to prepare the endometrium for implantation. While the currently available data show no significant difference in pregnancy rates between these methods, well designed randomized controlled trials are lacking. Moreover there is little literature on difference in cancellation rates, cost-efficiency and adverse events. Methods and design In this randomized, multi-centre, non-inferiority trial we aim to test the hypothesis that there is no significant difference in live birth rates between patients undergoing NC-FET versus AC-FET. The primary outcome will be live birth rate per embryo transfer procedure. Secondary outcomes will be ongoing and clinical pregnancy rate, cancellation rate, (serious adverse events and cost-efficiency. Based on a live birth rate of 20% and a minimal clinical important difference of 7,5% (one-sided alpha 2,5%, beta 20% a total of 1150 patients will be needed. Analyzes will be performed using both per protocol as well as intention to treat analyses. Discussion This prospective, randomized, non –inferiority trial aims to address the hypothesis that there is no significant difference in live birth rates between patients undergoing NC-FET versus patients undergoing AC-FET. Moreover it addresses cost-efficiency as well as the perceived burden of both treatments

  1. Elevated serum estradiol levels in artificial autologous frozen embryo transfer cycles negatively impact ongoing pregnancy and live birth rates.

    Science.gov (United States)

    Fritz, Rani; Jindal, Sangita; Feil, Heather; Buyuk, Erkan

    2017-08-19

    The aim of this study is to evaluate the correlation between serum estradiol (E2) levels during artificial autologous frozen embryo transfer (FET) cycles and ongoing pregnancy/live birth rates (OP/LB). A historical cohort study was conducted in an academic setting in order to correlate peak and average estradiol levels with ongoing pregnancy/live birth rates for all autologous artificial frozen embryo transfer cycles performed from 1/2011 to 12/2014. Average and peak E2 levels from 110 autologous artificial FET cycles from 95 patients were analyzed. Average E2 levels were significantly lower in cycles resulting in OP/LB compared to those that did not (234.1 ± 16.6 pg/ml vs. 315 ± 24.8 pg/ml, respectively, p = 0.04). Although peak E2 levels were not significantly different between cycles resulting in OP/LB compared with those that did not (366.9 ± 27.7 pg/ml vs. 459.1 ± 32.3 pg/ml, respectively, p = 0.19), correlation analysis revealed a statistically significant (p = 0.02) downward trend in OP/LB rates with increasing peak E2 levels. This study suggests that elevated E2 levels in artificial autologous FET cycles are associated with lower OP/LB rates. Estradiol levels should be monitored during artificial FET cycles.

  2. Ultrastructure of bovine in vitro-produced blastocysts cryopreserved by vitrification.

    Science.gov (United States)

    Ohboshi, S; Fujihara, N; Yoshida, T; Tomagane, H

    1998-02-01

    The objective of this study was to examine ultrastructural aspects of bovine in vitro-produced blastocysts associated with cryopreservation by vitrification. Morphologically good embryos were used and treated with ethylene-glycol-based vitrification solution (VS). The untreated embryos had conventional fine structure. The post-warming embryos treated with direct exposure to VS (one-step procedure) showed cellular damage structurally by cryopreservation, which included loss of microvilli, disruption of the plasma membrane, mitochondrial changes and swelling of the endoplasmic reticulum. However, nuclei and junctional regions seemed to be resistant to cryoinjury. In contrast, the post-warming embryos pre-equilibrated with 10% ethylene glycol for 5 min and subsequent exposure to VS (two-step procedure) showed less damage than those treated by the one-step procedure. Post-warming embryos treated by the two-step procedure were cultured in vitro for 18 h. Some embryos survived and their structures re-formed to the former state, while other embryos showed serious injuries and could not reconstitute the blastocoele. Three post-warming embryos treated by the two-step procedure that survived after in vitro culture were transferred to three recipients and one of these resulted in pregnancy. These results indicate that cryopreservation by vitrification can damage membranous structures of the cells of bovine embryos, the extent and nature of this damage being dependent on the vitrification procedure.

  3. Sperm-mediated DNA transfer to cells of the uterus and embryo.

    Science.gov (United States)

    Chan, P J

    2000-06-01

    There is a paucity of information about sperm-mediated transmission of exogenous DNA to implanting embryos and cells of the reproductive tract. Preliminary experiments established that sperm has the capacity to actively take in exogenous DNA derived from HPV. In addition, blastocysts also take up exogenous HPV DNA, but in contrast to sperm, the process appears passive. DNA-carrying sperm migrating in an artificial glass tube or excised mouse bicornuate uteri transfected the blastocysts at the remote position using a flip-flop mechanism. There were preferential transmission of the types of HPV DNA but this was not attributed to the gene sequence or the size of the DNA fragments. The internalized DNA became undetectable unless continuous sperm bombardment or pricking took place. Mycoplasma vectors offer a novel way to enhance the transfection of blastocyst with exogenous DNA.

  4. Detection of endometrial and subendometrial vasculature on the day of embryo transfer and prediction of pregnancy during fresh in vitro fertilization cycles

    Directory of Open Access Journals (Sweden)

    Ari Kim

    2014-09-01

    Conclusion: Three dimensional PD-US was a useful and effective method for assessing endometrial blood flow in IVF cycles. Good endometrial blood flow on the day of embryo transfer might be associated with high pregnancy success with a GnRH long protocol, because this is indicative of endometrial receptivity in fresh IVF cycles.

  5. Mosaic embryo transfer after oocyte in vitro maturation in combination with non-invasive prenatal testing (NIPT)-first report of a euploid live birth.

    Science.gov (United States)

    Inoue, Naomi; Lopez, Rosmary; Delgado, Andrea; Nuñez, Denisse; Portella, Jimmy; Noriega-Hoces, Luis; Guzmán, Luis

    2017-06-24

    The purpose of this study is to describe a healthy life birth after a mosaic embryo transfer in oocyte in vitro maturation (IVM). Patient received minimal stimulation, starting on day 3 after menstrual period. No hCG trigger was administered. Oocyte retrieval was performed and oocytes were matured for 30 h. After denuding, mature oocytes were inseminated by ICSI. Embryos were cultured until blastocyst stage and biopsied. One euploid embryo after array comprehensive genome hybridization (aCGH) was diagnostic. However, the next-generation sequencing (NGS) re-analysis showed that embryo was a mosaic for chromosome 13 and 21. Nevertheless, pregnancy ultrasound scans and non-invasive prenatal test (NIPT-Verifi-Illumina) indicated a normal fetus development. Finally, a healthy baby was born after 38 weeks. Its weight was 4480 g, head circumference 36 cm, and total length of 51 cm. To confirm that the baby was chromosomically normal, an NGS test was performed in buccal cells, a normal profile was obtained. Our finding confirmed that mosaic embryo transfer would bring a healthy offspring.

  6. Determining the status of non-transferred embryos in Ireland: a conspectus of case law and implications for clinical IVF practice.

    LENUS (Irish Health Repository)

    Sills, Eric Scott

    2009-01-01

    The development of in vitro fertilisation (IVF) as a treatment for human infertilty was among the most controversial medical achievements of the modern era. In Ireland, the fate and status of supranumary (non-transferred) embryos derived from IVF brings challenges both for clinical practice and public health policy because there is no judicial or legislative framework in place to address the medical, scientific, or ethical uncertainties. Complex legal issues exist regarding informed consent and ownership of embryos, particularly the use of non-transferred embryos if a couple separates or divorces. But since case law is only beginning to emerge from outside Ireland and because legislation on IVF and human embryo status is entirely absent here, this matter is poised to raise contractual, constitutional and property law issues at the highest level. Our analysis examines this medico-legal challenge in an Irish context, and summarises key decisions on this issue rendered from other jurisdictions. The contractual issues raised by the Roche case regarding informed consent and the implications the initial judgment may have for future disputes over embryos are also discussed. Our research also considers a putative Constitutional \\'right to procreate\\' and the implications EU law may have for an Irish case concerning the fate of frozen embryos. Since current Medical Council guidelines are insufficient to ensure appropriate regulation of the advanced reproductive technologies in Ireland, the report of the Commission on Assisted Human Reproduction is most likely to influence embryo custody disputes. Public policy requires the establishment and implementation of a more comprehensive legislative framework within which assisted reproductive medical services are offered.

  7. Determining the status of non-transferred embryos in Ireland: a conspectus of case law and implications for clinical IVF practice

    Directory of Open Access Journals (Sweden)

    Sills Eric

    2009-07-01

    Full Text Available Abstract The development of in vitro fertilisation (IVF as a treatment for human infertilty was among the most controversial medical achievements of the modern era. In Ireland, the fate and status of supranumary (non-transferred embryos derived from IVF brings challenges both for clinical practice and public health policy because there is no judicial or legislative framework in place to address the medical, scientific, or ethical uncertainties. Complex legal issues exist regarding informed consent and ownership of embryos, particularly the use of non-transferred embryos if a couple separates or divorces. But since case law is only beginning to emerge from outside Ireland and because legislation on IVF and human embryo status is entirely absent here, this matter is poised to raise contractual, constitutional and property law issues at the highest level. Our analysis examines this medico-legal challenge in an Irish context, and summarises key decisions on this issue rendered from other jurisdictions. The contractual issues raised by the Roche case regarding informed consent and the implications the initial judgment may have for future disputes over embryos are also discussed. Our research also considers a putative Constitutional 'right to procreate' and the implications EU law may have for an Irish case concerning the fate of frozen embryos. Since current Medical Council guidelines are insufficient to ensure appropriate regulation of the advanced reproductive technologies in Ireland, the report of the Commission on Assisted Human Reproduction is most likely to influence embryo custody disputes. Public policy requires the establishment and implementation of a more comprehensive legislative framework within which assisted reproductive medical services are offered.

  8. Are endometrial parameters by three-dimensional ultrasound and power Doppler angiography related to in vitro fertilization/embryo transfer outcome?

    Science.gov (United States)

    Mercé, Luis T; Barco, María J; Bau, Santiago; Troyano, Juan

    2008-01-01

    To evaluate whether endometrial parameters by three-dimensional ultrasonography and power Doppler angiography (3D US-PDA) can predict in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcome. Prospective clinical study. Assisted reproduction unit in a referral hospital. Eighty women who underwent IVF cycles. Endometrial 3D US-PDA evaluated by VOCAL software (plane C and 9 degrees of rotational steps). Endometrial pattern, endometrial thickness (ET), endometrial volume (EV), and PDA indexes of vascularization index (VI), flow index (FI), and vascularization flow index (VFI) were measured on the day of human chorionic gonadotropin (hCG) administration. These measurements were related to IVF/ICSI and embryo transfer outcome. In the pregnant group, EV, VI, FI, and FVI but not triple-line pattern and ET were statistically significantly higher. The area under receiver operating characteristic (ROC) curve was statistically significant for EV (0.746), VI (0.724), FI (0.828), and VFI (0.800) when no grade 1 embryos or only one were transferred (43 cycles, 14 pregnancies) but not when two or three grade 1 embryos were transferred. Moreover, these parameters were statistically significant in predicting a normal pregnancy outcome (no early pregnancy loss) but were not related to multiple pregnancies. In IVF/ICSI cycles, 3D US-PDA is useful for evaluating endometrial receptivity. Endometrial volume and 3D power Doppler indexes are statistically significant in predicting the cycle outcome when one grade 1 or no grade 1 embryos are transferred, which could be helpful data in a single-embryo transfer policy.

  9. Differences in cumulus cell gene expression indicate the benefit of a pre-maturation step to improve in-vitro bovine embryo production.

    Science.gov (United States)

    Dieci, Cecilia; Lodde, Valentina; Labreque, Rémi; Dufort, Isabelle; Tessaro, Irene; Sirard, Marc-André; Luciano, Alberto M

    2016-12-01

    Does the gene expression profile of cumulus cells (CC) accompanying oocytes with different degrees of chromatin compaction within the germinal vesicle (GV) reflect the oocyte's quality and response in culture during in-vitro embryo production (IVP). The transcriptomic profile of the CC is related to oocyte competence, setting the stage for the development of customized pre-maturation strategies to improve IVP. Oocytes complete the acquisition of their competence during antral follicle development. During this period, the chromatin configuration within the GV changes dynamically and is indicative of oocyte's developmental potential. The interactions between somatic and germ cells modulate chromatin morphology and function and are critical for acquisition of oocyte competence. Bovine cumulus-oocyte complexes (COC) were isolated from 0.5 to 6 mm antral follicles. Surrounding CC were separated from the oocyte and classified as GV0, GV1, GV2 and GV3 according to the degree of the oocyte's chromatin compaction. RNA extracted from CC of each group was amplified and hybridized on a bovine embryo-specific 44 K Agilent slide. The CC_GV1, CC_GV2 and CC_GV3 classes were each hybridized against the CC_GV0 class, representing an early oocyte differentiation stage with poor development competence. The data were normalized and fold changes of the differentially expressed genes were determined. Microarray data were validated using quantitative RT-PCR on selected targets. Microarray data were further analyzed through: (i) between-group analysis (BGA), which classifies the samples according to their transcriptomic profiles; (ii) cluster analysis according to the expression profile of each gene; and (iii) Ingenuity Pathway Analysis (IPA) to study gene regulation patterns and predicted functions. Furthermore, CC of each GV group were cultured and apoptotic cells were assessed after 3 h by caspase analysis. Finally, based on the analysis of CC transcriptomic profiles and the

  10. Decreased Sperm Motility Retarded ICSI Fertilization Rate in Severe Oligozoospermia but Good-Quality Embryo Transfer Had Achieved the Prospective Clinical Outcomes.

    Science.gov (United States)

    Zheng, Jufeng; Lu, Yongning; Qu, Xianqin; Wang, Peng; Zhao, Luiwen; Gao, Minzhi; Shi, Huijuan; Jin, Xingliang

    Spermatozoa motility is the critical parameter to affect the treatment outcomes during assisted reproductive technologies (ART), but its reproductive capability remains a little informed in condition of severe male factor infertility. This retrospective cohort study aimed to evaluate the effects of reduced sperm motility on the embryological and clinical outcomes in intra-cytoplasmic sperm injection (ICSI) treatment of severe oligozoospermia. 966 cycles (812 couples) of severe oligozoospermia diagnosed by spermatozoa count ≤ 5 × 106/mL and motile spermatozoa ≤ 2 × 106/mL were divided into four groups in according to the number of motile spermatozoa in one ejaculate on the day of oocyte retrieval (Group B-E). The control (Group A) was 188 cycles of moderate oligozoospermia with spermatozoa count > 5 × 106/mL and motile spermatozoa > 2 × 106/mL. All female partners were younger than 35 years of age. Logistic regression analyzed embryological outcomes (the rates of fertilization, cleavage and good-quality embryo) and clinical outcomes (the rates of pregnancy, implantation, early miscarriage and live birth). Quality of embryo transfer (ET) was divided into three classes as continuous factor to test the effects of embryo quality on clinical outcomes. The reduction in the number of motile sperm in four groups of severe oligozoospermia gave rise to comparable inability of the fertilization (p good-quality embryo at Day 3 (p good-quality embryo. Obtaining and transfer of good-quality embryos was the good prognostic to achieve prospective clinical outcomes regardless of the severity of oligozoospermia.

  11. Pregnancy outcomes of day 5 embryo transfer in patients at high risk of developing ovarian hyperstimulation syndrome and analysis of factors affecting blastocyst formation.

    Science.gov (United States)

    Xin, Zhi-min; Zhu, Hong; Jin, Hai-xia; Song, Wen-yan; Sun, Ying-pu

    2013-08-01

    To investigate the effects of day 5 embryo transfer (D5ET) compared with day 3 embryo transfer (D3ET) in patients at high risk of developing ovarian hyperstimulation syndrome (OHSS); to analyse factors affecting blastocyst formation. Patients at high risk of developing OHSS underwent either D3ET or D5ET. A total of 253 patients received D3ET; 263 received D5ET. The number of embryos transferred was lower in the D5ET group than in the D3ET group. There were no between-group differences in pregnancy or live birth rates. Implantation rate was higher, and multifetation rate lower, in the D5ET group compared with the D3ET group. In addition, the incidence of moderate or severe OHSS was lower in the D5ET group than in the D3ET group. The woman's age, gonadotrophin dosage and insemination method were associated with the quality of blastocyst formation. In patients with a high risk of developing OHSS, compared with D3ET, D5ET decreased the multifetation rate and the incidence of moderate or severe OHSS, but did not affect the pregnancy or live birth rate. Women of a younger age, who have had an appropriate gonadotrophin dose and insemination by in vitro fertilization, are suitable candidates for blastocyst transfer.

  12. Should we consider day-2 and day-3 embryo morphology before day-5 transfer when blastocysts reach a similar good quality?

    Science.gov (United States)

    Herbemont, Charlene; Sarandi, Solmaz; Boujenah, Jeremy; Cedrin-Durnerin, Isabelle; Sermondade, Nathalie; Vivot, Alexandre; Poncelet, Christophe; Grynberg, Michael; Sifer, Christophe

    2017-11-01

    Clinical outcomes of 291 day-5 blastocyst transfers carried out between January 2012 and March 2016 were retrospectively compared according to their quality at day 2 and 3. Inclusion criteria were female age younger than 37 years; first or second IVF and intracytoplasmic sperm injection cycle; quality of the transferred blastocyst: blastocoele B3 or higher; inner-cell-mass A/B; trophectoderm A/B; and known implantation outcome for each transferred blastocyst. Blastocysts were classified into good-quality and poor-quality embryo groups at day 2 and 3. Implantation (38.7% versus 41.4), clinical pregnancy (40.3% versus 45.9%), miscarriage (22.2% versus 26.7%;) and live birth rates (37.4% versus 38.8%) were comparable in day 2 good and poor-quality embryo groups. No signficiant differences in morphology of transferred blastocysts at day 3 were found. Multivariable analysis highlighted that poor or good embryo quality at day 2 and day 3 were not predictive of the implantation of good-quality blastocysts (at day 2: adjusted odds ratio = 0.82 CI 95% 0.49 to 1.38; at day 3: adjusted odds ratio = 1.39; CI 95% 0.77 to 2.52). Good-quality blastocyst transfer should, therefore, be carried out irrespective of embryo quality at cleavage stage, as it may not compromise success rates in a good-prognosis population. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  13. Viable offspring after successful non-surgical embryo transfer in goats

    Directory of Open Access Journals (Sweden)

    J.F. Fonseca

    2014-04-01

    Full Text Available O objetivo do presente estudo foi avaliar a viabilidade da técnica de transferência não cirúrgica em cabras. Quatro cabras não-lactantes pluríparas da raça Toggenburg foram utilizadas como receptoras de embriões, sendo que duas receberam um embriões e duas receberam dois embriões coletados não cirurgicamente cabras doadoras. Os corpos lúteos das receptoras foram detectados um dia antes da transferência de embriões por ultrassonografia transretal. Uma seringa de 5mL contendo 2mL de meio holding foi acoplada em um cateter tomcat, no qual os embriões foram aspirados em uma coluna central a duas outras colunas. Um espéculo Colin número 2 foi inserido na vulva e na vagina, e com o uso de uma fonte de luz, a cerviz foi localizada e imobilizada com uma pinça de Allis. Um cateter uretral número seis acoplado a um mandril e lubrificado com meio PBS foi inserido na cérvix, e assim os aneis cervicais foram gradualmente transpostos. Após perder a resistência, o cateter uretral foi movido lateralmente para o corno uterino desejado. O mandril e a pinça de Allis foram retirados e o conjunto seringa e tomcat foi acoplado ao cateter uretral e o conteúdo injetado no corno uterino ipsilateral ao corpo lúteo com posterior retirada do cateter. Cabras que ovularam em apenas um ovário foram usadas para testar a eficiência da deposição do embrião. O tempo gasto entre a inserção do espéculo e a sua remoção foi inferior a três minutos. O tempo para transpor a cérvix foi inferior a um minuto. A ultrassonografia revelou a deposição de líquido no corno desejado. Receptoras que receberam dois embriões tornaram-se gestantes e pariram três crias. Estes primeiros resultados encorajam a técnica e demonstram que a transferência de embriões em caprinos pode ser feita totalmente por procedimentos não cirúrgicos.

  14. Hypothesis: Co-transfer of genuine embryos and implantation-promoting compounds via artificial containers improve endometrium receptivity.

    Science.gov (United States)

    Celik, Onder; Acet, Mustafa; Celik, Sudenaz; Sahin, Levent; Koc, Onder; Celik, Nilufer

    2017-06-01

    As with other organs endometrial functions are altered with the advancing age. Age related decrease in reproductive functions leads to decline in the number of oocytes retrieved and the synthesis of endometrial receptivity molecules. Despite the significant improvement in assisted reproductive technologies we do not have so many options to enhance endometrial receptivity. Due to lack of drugs having endometrium receptivity enhancement properties, oocyte donation seems to be the only solution for women with implantation failure. The euploid oocytes come from young and healthy donors may overcome age associated endometrial receptivity defect. Nevertheless, many reasons restrict us from using oocyte donation in women with implantation failure. We, therefore, hypothesized that by mimicking a young blastocyst's effect on endometrium, the transfer of genuine embryos and implantation-promoting compounds together might be the new treatment option for infertile women with recurrent implantation failure. Artificial beads, MI or GV oocytes, and empty zona can be used as a container for intrauterine replacement of implantation-promoting compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Effect of chromosomal polymorphisms of different genders on fertilization rate of fresh IVF-ICSI embryo transfer cycles.

    Science.gov (United States)

    Liang, Ji; Zhang, Yongsheng; Yu, Yang; Sun, Wentao; Jing, Jili; Liu, Ruizhi

    2014-10-01

    To explore whether chromosomal polymorphisms of different genders affect outcomes of fresh IVF and intracytoplasmic sperm injection (ICSI) embryo transfer cycles differently, 37 couples with chromosomal polymorphisms were identified out of 614 infertile couples undergoing IVF-ICSI treatments. Group 1 included 20 couples in which only the male carried chromosomal polymorphisms; group 2 included 17 couples with female carriers only; group 3 included 19 infertile couples with normal karyotypes randomly selected as controls. A significantly lower fertilization rate was found in group 1 compared with groups 2 and 3 (56.68% in Group 1, 78.02% in group 2 and 71.74% in group 3; group 1 versus group 2, P < 0.001; group 1 versus group 3, P = 0.001; respectively). When stratified according to fertilization method, the fertilization rate in IVF cycles of group 1 was significantly lower than group 3 (50.00% in Group 1, 73.89% in Group 3, P < 0.001). Fertilization rates in ICSI cycles between groups 1 and 3 were not significantly different. This study suggests that male chromosomal polymorphisms adversely influence fertilization rates of IVF cycles. The use of ICSI may improve the success of infertility treatment by increasing the fertilization rate for men with chromosomal polymorphisms. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  16. Evaluation of the effect of oral ritodrine on implantation rate in in-vitro fertilization-embryo transfer cycles

    Directory of Open Access Journals (Sweden)

    Maryam Ahmadi

    2011-01-01

    Full Text Available Background: Pregnancy rate with IVF cycle is almost 22%. Many investigations perform to increase this rate in IVF. Various factors affect the result of IVF cycles. One of these factors could be uterine contractions that expel transferred embryo. Ritodrine is a beta mimetic agent that can block and decrease uterine contractions.Objective: The objective of this study was to determine ritodrine effectiveness for increasing the implantation rate in IVF cycles, and its probable mechanisms in decreasing uterine contractions as well.Materials and Methods: A total of 100 patients of IVF-ET cycles were divided randomly in two groups in a university hospital, Hamadan, Iran. The case group were prescribed ritodrine 10 mg / bid orally after oocyte retrieval until 10 days. The control group didn’t received ridotrine.Results: In ritodrine group 14% of patients and in control group 16% had positive β-hCG test (p-value>0.5.Conclusion: Ritodrine did not improve the implantation rate in IVF-ET cycles

  17. Estimating transfer of bovine virus-diarrhoea virus in Danish cattle by use of register data

    DEFF Research Database (Denmark)

    Alban, L.; Stryhn, H.; Kjeldsen, A.M.

    2001-01-01

    To study how routinely recorded data (also called "register data") might be used in disease monitoring on a regional or national level, a database for bovine virus-diarrhoea virus (BVDV) was made from existing databases, covering the period January 1995-November 1999. This paper includes a general...... description of the database, including basic statistics for selected variables. Information was largely complete for cattle herds in the milk-recording scheme (MRS), but only partly available for other herds. A methodology was developed to identify when and how a herd initially was infected. For most herds...

  18. Transfer, loss and physical processing of water in hit-and-run collisions of planetary embryos

    Science.gov (United States)

    Burger, C.; Maindl, T. I.; Schäfer, C. M.

    2018-01-01

    Collisions between large, similar-sized bodies are believed to shape the final characteristics and composition of terrestrial planets. Their inventories of volatiles such as water are either delivered or at least significantly modified by such events. Besides the transition from accretion to erosion with increasing impact velocity, similar-sized collisions can also result in hit-and-run outcomes for sufficiently oblique impact angles and large enough projectile-to-target mass ratios. We study volatile transfer and loss focusing on hit-and-run encounters by means of smooth particle hydrodynamics simulations, including all main parameters: impact velocity, impact angle, mass ratio and also the total colliding mass. We find a broad range of overall water losses, up to 75% in the most energetic hit-and-run events, and confirm the much more severe consequences for the smaller body also for stripping of volatile layers. Transfer of water between projectile and target inventories is found to be mostly rather inefficient, and final water contents are dominated by pre-collision inventories reduced by impact losses, for similar pre-collision water mass fractions. Comparison with our numerical results shows that current collision outcome models are not accurate enough to reliably predict these composition changes in hit-and-run events. To also account for non-mechanical losses, we estimate the amount of collisionally vaporized water over a broad range of masses and find that these contributions are particularly important in collisions of ˜ Mars-sized bodies, with sufficiently high impact energies, but still relatively low gravity. Our results clearly indicate that the cumulative effect of several (hit-and-run) collisions can efficiently strip protoplanets of their volatile layers, especially the smaller body, as it might be common, e.g., for Earth-mass planets in systems with Super-Earths. An accurate model for stripping of volatiles that can be included in future planet

  19. Co-cultura de embriões humanos em células vero e transferência em fase de blastocisto Human embryo coculture in vero cells and blastocyst stage transfer

    Directory of Open Access Journals (Sweden)

    Carlos Gilberto Almodin

    1999-08-01

    Full Text Available Objetivos: determinar se o dia da transferência ou o estágio em que o embrião é transferido interferem nas taxas de gravidez e implantação. Métodos: cento e sete pacientes tiveram seus oócitos aspirados e submetidos à fertilização in vitro. Os embriões foram co-cultivados em células Vero e transferidos no dia 3 ou dia 5 pós-fertilização, após avaliação morfológica. Resultados: a taxa de implantação dos embriões transferidos no dia 5 foi significativamente maior que quando os embriões eram transferidos no dia 3, mas as taxas de gravidez não variaram. Observou-se uma diferença significativa nas taxas de gravidez quando se comparou o estágio em que o embrião era transferido, obtendo-se 70,6% de gravidez quando se transferiam blastocistos expandidos e 20,0% e 10,5% quando eram transferidos blastocistos iniciais ou mórulas, respectivamente. Conclusões: as taxas de implantação e gravidez são significativamente aumentadas quando se transferem embriões em estágio de blastocisto expandido, mas os meios e condições de cultura de que dispomos no momento ainda são insuficientes para nos fornecer uma taxa satisfatória de embriões neste estágio.Purpose: to determine whether the transfer day or the stage that the embryo is transferred interferes in pregnancy and implantation rates. Methods: oocytes we recovered from 107 patients and submitted to in vitro fertilization. The embryos were cocultured on Vero cells and transferred on day 3 or day 5 post-fertilization, after morphological assessment. Results: the implantation rate of the transferred embryos on day 5 was significantly higher than when the embryos were transferred on day 3, but the pregnacy rates did not change. However, a significant difference was observed in the pregnancy rates for embryos transferred at the expanded blastocyst stage (70.6% of pregnancy when compared to 20.0% and 10.5% at the earlier blastocyst and morula stages, respectively. Conclusions

  20. Occurrence of Campylobacter in the genitals of teaser bulls maintained at an embryo transfer center

    Directory of Open Access Journals (Sweden)

    Modolo J.R.

    2000-01-01

    Full Text Available Em central de transferência de embriões, após os procedimentos de reconhecimento do cio em 37 vacas receptoras, através de quatro rufiões vasectomizados, observou-se que 83% delas apresentavam retorno ao cio e algum corrimento serofibrinoso. Nos exames bacteriológicos realizados nos lavados prepuciais dos rufiões foi isolado o Campylobacter fetus subsp. venerealis em todos, fato que, analisado associadamente com o retorno ao cio das vacas receptoras, é indicativo da ocorrência de campilobacteriose no plantel. Essa ocorrência demonstra a necessidade de medidas eficazes de planejamento de saúde animal, pela utilização de rufiões com desvio lateral do pênis. Uma vez impossibilitado o contato sexual, seria impedida a transmissão do agente durante o coito. Torna-se imperioso consignar que a prática da prevenção racional de enfermidades continua sendo o procedimento mais econômico para uma produtividade animal mais rentável.

  1. The Effect of Intercourse around Embryo Transfer on Pregnancy Rate in Assisted Reproductive Technology Cycles

    Directory of Open Access Journals (Sweden)

    Nasim Tabibnejad

    2009-01-01

    Full Text Available Background: Implantation failure is the most important cause of recurrent in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI failure. Several reports suggest that intercourse during theperitransfer period might improve pregnancy rates. This study is designed to determine whetherintercourse during the peritransfer period will improve pregnancy and implantation rates in patientsundergoing IVF or ICSI.Materials and Methods: In a randomized control trial study, 390 women with at least five yearsinfertility were evaluated. In the study group, 195 patients had intercourse at least once 12 hours afterembryo transfer. Implantation and clinical pregnancy rates were compared with 195 patients in thecontrol group who had no intercourse for the entire assisted reproductive technology (ART cycle.Results: Implantation rate in the study group was 6.5% in comparison with 5.5% for the controlgroup. Clinical pregnancy rates were not significantly higher in study patients when compared tothe control group (14.2% and 11.7% respectively.Conclusion: The results showed that intercourse during the peritransfer period can not increasepregnancy outcome.

  2. Developmental stage on day-5 and fragmentation rate on day-3 can influence the implantation potential of top-quality blastocysts in IVF cycles with single embryo transfer

    Directory of Open Access Journals (Sweden)

    Devroey Paul

    2007-01-01

    Full Text Available Abstract Background In IVF-ICSI cycles with single embryo transfer (SET, embryo selection for transfer is of crucial importance. The present study aimed to define which embryo parameters might be related to the implantation potential of advanced blastocysts. Methods Overall, in 203 cycles with SET, developmental characteristics of 93 implanted (group A and 110 non-implanted (group B advanced blastocysts of good quality were compared. The following developmental parameters were assessed in the two groups: normal fertilization, developmental stage on day 5, number of blastomeres on day 2 and on day 3, fragmentation rate on day 3, compaction on day 4 and cleavage pattern on day 2 and day 3. Results Expanded blastocysts compared to full blastocysts have higher implantation potential (56.5% vs. 29.3%, p 10–50% fragments on day 3 showed a significant lower implantation (29.7% than those with ≤ 10%fragments (49.4%, P = 0.03. All the other parameters analysed were comparable for the two groups. Conclusion Developmental stage on day 5 and fragmentation rate on day 3 were related to the implantation potential of advanced blastocysts and should also be taken into account in the selection of the best advanced blastocyst for transfer.

  3. Intrauterine insemination of cultured peripheral blood mononuclear cells prior to embryo transfer improves clinical outcome for patients with repeated implantation failures.

    Science.gov (United States)

    Madkour, Aicha; Bouamoud, Nouzha; Louanjli, Noureddine; Kaarouch, Ismail; Copin, Henri; Benkhalifa, Moncef; Sefrioui, Omar

    2016-02-01

    Implantation failure is a major limiting factor in assisted reproduction improvement. Dysfunction of embryo-maternal immuno-tolerance pathways may be responsible for repeated implantation failures. This fact is supported by immunotropic theory stipulating that maternal immune cells, essentially uterine CD56+ natural killer cells, are determinants of implantation success. In order to test this hypothesis, we applied endometrium immuno-modulation prior to fresh embryo transfer for patients with repeated implantation failures. Peripheral blood mononuclear cells were isolated from repeated implantation failure patients undergoing assisted reproductive technology cycles. On the day of ovulation induction, cells were isolated and then cultured for 3 days and transferred into the endometrium cavity prior to fresh embryo transfer. This immunotherapy was performed on 27 patients with repeated implantation failures and compared with another 27 patients who served as controls. Implantation and clinical pregnancy were increased significantly in the peripheral blood mononuclear cell test versus control (21.54, 44.44 vs. 8.62, 14.81%). This finding suggests a clear role for endometrium immuno-modulation and the inflammation process in implantation success. Our study showed the feasibility of intrauterine administration of autologous peripheral blood mononuclear cells as an effective therapy to improve clinical outcomes for patients with repeated implantation failures and who are undergoing in vitro fertilization cycles.

  4. No peri- and postnatal effects on calves born after transfer of in vitro produced embryos vitrified by the open pulled straw (OPS) method

    DEFF Research Database (Denmark)

    Jacobsen, Helene; Holm, Peter; Schmidt, Mette

    2003-01-01

    The general objective of this study was to perform follow-up studies including selected peri- and postnatal characteristics on calves born after transfer of in vitro produced (IVP) em- bryos vitrified by the ’Open Pulled Straw’ (OPS) method. An overall pregnancy rate of 16% after transfer...... of the OPS-vitrified IVP embryos was achieved and resulted in birth of 9 calves, with 11 AI calves serving as controls. There were no immediate or long- term effects on these calves with respect to birth weight, gestation length, perinatal mor- tality, growth rate, disease susceptibility and reproductive...

  5. Attachment of Coxiella burnetii to the zona pellucida of in vitro produced goat embryos.

    Science.gov (United States)

    Pellerin, J L; Alsaleh, A; Mermillod, P; Souza-Fabjan, J M G; Rodolakis, A; Rousset, E; Dubreil, L; Bruyas, J F; Roux, C; Fieni, F

    2018-01-15

    Previous work demonstrated that after infection of in vivo derived caprine embryos, Coxiella burnetti (C. burnetii) showed a strong tendency to adhere to the zona pellicida (ZP). To investigate the risk of C. burnetii transmission via embryo transfer of in vitro-produced goat embryos the aim of this study was, (i) to evaluate the ability of C. burnetii to adhere to the intact zona pellicida of in vitro-produced goat embryos and to determine by confocal microscopy the location of the bacteria, (ii) to test the efficacy of IETS recommended rules for the washing of bovine embryos to eliminate C. burnetii. One hundred ZP-intact caprine embryos, produced in vitro, at the 8 to 16 cell stage, were randomly divided into 11 batches of eight to nine embryos. Nine batches were incubated for 18 h with 109Coxiella/ml of CbB1 strain (IASP, INRA Tours). The embryos then were recovered and washed in batches in 10 successive baths following the IETS guidelines. In parallel, two batches of embryos were subjected to similar procedures but without exposure to C. burnetii, to serve as the control group. One of the nine batches of infected embryos and one of the two non-infected control batch