Equine in vitro fertilization with frozen–thawed semen is associated with shortened pre-incubation time and modified capacitation-related changes

We recently reported successful equine in vitro fertilization using fresh semen pre-incubated for a prolonged period (22 h) before co-culture with oocytes. In this study, we evaluated the feasibility of equine in vitro fertilization with frozen-thawed sperm and evaluated capacitation-related changes in these sperm over the pre-incubation period. Sperm selected via a commercial sperm separation device yielded significantly higher fertilization than did sperm selected by swim-up or by colloid centrifugation. Using the sperm separation device method, fertilization rates with sperm pre-incubated for 15 min, 3, 6, and 9 h were 7.1, 22.2, 38.5, and 73.3% respectively (9 h vs. 15 min or 3 h, P < 0.05). Fertilization rates differed significantly (45.9% vs. 85.5%) between freezing extenders. Blastocysts were produced using frozen-thawed semen from each of three stallions and transfer of nine vitrified-warmed blastocysts to mares yielded seven embryonic vesicles. Anti-protein tyrosine phosphorylation staining of the entire sperm tail increased over pre-incubation, and sperm both with and without staining in the tail bound to the oocyte cumulus after co-incubation. Using the stain DiSC3(5) and flow cytometric analysis, a population of apparently hyperpolarized sperm was identified at 22 h in fresh sperm that was not seen at any time in frozen-thawed sperm. We conclude that frozen-thawed equine sperm can successfully fertilize oocytes after a shortened pre-incubation time of 9 h, suggesting that the freeze-thawing process induces capacitation-related changes. Our findings on evaluation of pre-incubated sperm indicate that the mechanisms by which frozen-thawed sperm become capable of fertilization may differ from those found in fresh sperm.