Hyperactivation and Tyrosine Phosphorylation of Cryopreserved Stallion Spermatozoa in Relation to Conventional In Vitro Fertilization Blastocyst Production

BACKGROUND: Challenges in establishing clinical conventional in vitro fertilization (IVF) in horses include variation in sperm and oocyte quality, which can be affected by mare age. Extended preincubation of stallion spermatozoa has resulted in successful IVF. However, there is limited research on the impact of preincubation on sperm characteristics and IVF success. OBJECTIVES: Responses of frozen-thawed stallion spermatozoa over time under IVF preincubation conditions were evaluated, and samples based on specific responses were tested for their ability to fertilize oocytes, including those from older mares, using conventional IVF. MATERIALS AND METHODS: Frozen-thawed spermatozoa were sorted, incubated for 10 h in capacitating conditions, and evaluated for motility, membrane integrity, mitochondrial function, and protein tyrosine phosphorylation (PY). Sperm samples for IVF were selected based on motion and PY patterns using oocytes from younger and older mares. Accessory spermatozoa binding to the zona pellucida (ZP), fertilization, cleavage, and blastocyst rates were assessed. RESULTS: Sperm motility, velocities, linearity, and straightness were significantly reduced during incubation, while the amplitude of lateral head displacement increased. Plasma membrane integrity and mitochondrial activity were also significantly reduced over time. After 5 h of incubation, an increase in tyrosine-phosphorylated proteins was observed, occurring primarily in the equatorial segment, but also including the midpiece or tail. The response of sperm samples to capacitating conditions varied. Sperm samples selected for IVF were successful at fertilizing oocytes from younger and older mares, with more accessory spermatozoa bound to ZP in oocytes from younger than older mares, although cleavage and blastocyst rates did not differ. DISCUSSION AND CONCLUSION: We concluded that changes in sperm motion and PY patterns were indicative of hyperactivation and capacitation, although wide variation occurred among stallions. Cryopreserved spermatozoa can successfully fertilize oocytes using conventional IVF. Age of the mare oocyte donor does not appear to affect fertilization after IVF.