In vitro production of horse embryos: fundamental aspects

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