In vitro fertilization of in vitro-matured equine oocytes: Effect of maturation medium, duration of maturation, and sperm calcium ionophore treatment, and comparison with rates of fertilization in vivo after oviductal transfer
ABSTRACT Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P < 0.05). There was no significant difference in fertilization rate among 3 sperm treatments utilizing 7.14 microM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40-44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P < 0.05). We conclude that in vitro-matured horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.
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ABSTRACT: Oviductal environment affects preparation of gametes for fertilization, fertilization itself, and the subsequent embryo development. The aim of this study is to evaluate the effect of oviductal fluid and the possible involvement of Deleted in Malignant Brain Tumours 1 (DMBT1) on in vitro fertilization (IVF) in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase of the monospermic fertilization rate, and that this effect is cancelled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase of the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the oestrus cycle was shown by Western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus-oocyte complexes was shown by Western blot analysis, and the localisation of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatics and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization.Reproduction 05/2013; DOI:10.1530/REP-13-0007
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ABSTRACT: The development of methods to produce embryos in vitro in the horse has been delayed compared with other domestic species. Oocytes can be collected from excised ovaries or from the small or preovulatory follicles of live mares. Intracytoplasmic sperm injection is the only reliable method to fertilize equine oocytes in vitro. Intracytoplasmic sperm injection-produced embryos can be transferred into the oviducts of recipient mares or cultured to the morula or blastocyst stage of development for nonsurgical embryo transfers into recipients' uteri. Embryos cultured in vitro have some morphological differences compared with embryos collected from the mares' uteri. Most notably, the embryonic capsule does not form in culture, and the zona pellucida fails to expand completely. However, embryo produced in vitro can result in viable pregnancies and healthy offspring.Journal of Equine Veterinary Science 07/2012; 32(7):367-371. DOI:10.1016/j.jevs.2012.05.054 · 0.89 Impact Factor
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ABSTRACT: Developments in assisted reproduction have provided valuable tools for sub-fertility treatment and for selective breeding in animals. In horses, techniques such as artificial insemination and embryo transfer are used successfully to aid genetic progress but the commercial application of other assisted reproductive techniques, such as in vitro production of embryos (IVP), has been severely restricted by their low efficiency. The development of culture systems that can support embryo development in vitro to a stage suitable for transfer to the uterus of recipient mares has been slow and the state-of-the-art far behind that in other species. This thesis has focused on the complex cellular events that take place in the oocyte during maturation and fertilization and, later on, during early embryo development that may help to define and solve the problems that have thus far hampered IVP. The first objective was to investigate why in oocytes matured in vitro (IVM) have a lower developmental capacity their in vivo counterparts and how to improve the quality of cytoplasmic maturation and its synchrony with nuclear maturation. The control of oocyte maturation in vivo is dictated primarily by its follicular and the maternal endocrinological environment and, using this rationale, we demonstrated that the follicular wall, and in particular the theca cells, play an important role in maintaining the horse oocytes in meiotic arrest during culture. Such culture conditions may enhance cytoplasmic maturation improving the developmental competence of in vitro matured oocytes (IVM). The complex interdependency of nuclear and cytoplasmic maturation was also demonstrated by describing the cytoskeletal restructuring that accompany the process of chromosomal alignment and segregation during meiosis in the horse oocyte. Poor fertilization rates obtained with conventional IVF remain the greatest obstacle to large-scale horse IVP. The interaction between sperm and oocyte during IVF was investigated using confocal laser scanning microscopy showing that sperm was able to bound to but not penetrate the zona pellucida of both in vivo and in vitro matured oocytes. The bound sperm did not undergo acrosome reaction, indicating that failed oocyte penetration is most probably due to inadequate sperm activation. The problems of conventional IVF can be overcome by ICSI and, in this study, the nuclear and cytoskeletal events that occur in horse oocytes fertilized by ICSI were described, with special attention to the stages at which fertilization or zygote development fails. Sperm incorporation and fusion of the parental genomes were shown to involve a complex series of cytoskeletal changes, and comparison of zygotes and parthenotes showed that both gametes contribute to the formation of the zygotic centrosome. Even after successful fertilization, however, the rate of blastocyst production in vitro is low, presumably because of inadequacies in culture conditions. The structural and cellular characteristics of horse embryos produced totally in vitro or by temporary transfer to the oviduct of surrogate sheep, were compared with those of in vivo produced embryos. IVP embryos were smaller, had fewer cells, high rates of apoptosis and disrupted microfilaments patterns than in vivo embryos. In addition, IVP embryos secreted capsular material but failed to coalescence into a complete capsule enveloping the embryo. Finally the organization of the chromatin and cytoskeleton of cloned horse embryos constructed using adult or fetal fibroblasts was correlated with the success of nuclear reprogramming and the degree of subsequent developmental disturbance.