From sperm selection to embryo development: New insights into key assisted reproductive technologies in the horse.

Since the early 1990s, sport horse breeding has increasingly adopted assisted reproductive technologies (ARTs), starting with artificial insemination (AI) and followed by embryo transfer and in vitro embryo production (IVP). AI is preferentially performed with chilled transported semen, which is limited by optimal storage times of 24-48 hours. We investigated the impact of prolonged cold storage (96 hours at 5 °C) on sperm quality and found time-related increases in plasma membrane fluidity and intra-cellular Ca2+, indicators of premature capacitation, that probably explain diminishing fertility. While IVP by intracytoplasmic sperm injection (ICSI) dramatically reduces the number of ‘fertile’ spermatozoa required, it bypasses natural sperm selection processes that prevent sperm with damaged DNA fertilizing the oocyte. We developed a 3-layer density-gradient centrifugation technique that selects a population of motile, morphologically normal spermatozoa with intact DNA better suited for ICSI. We also investigated the impact of IVP on early cell lineage segregation and found IVP blastocysts to contain fewer, more widely dispersed epiblast cells. Epiblast cell dispersal may explain why IVP embryos are less viable than in vivo embryos, but more likely to give rise to monozygotic twins. Fortunately, transferring IVP blastocysts into a mare's uterus triggered rapid epiblast compaction. Whereas IVP embryos are routinely cryopreserved, freezing larger (>300 µm diameter) in vivo-derived blastocysts has proven challenging. Recently, it transpired that puncturing large blastocysts and aspirating the blastocoel fluid improves their freezability; we further demonstrated that punctured embryos survive vitrification better than slow-freezing. Overall, this thesis highlights areas for improvement within equine ART.