Effects of equine sperm incubation under capacitation-supporting conditions on homologous and heterologous ICSI outcomes.

Intracytoplasmic sperm injection (ICSI) in horses has become sufficiently efficient to be considered for clinical and commercial application; however, several factors influencing its efficiency remain poorly understood. This study evaluated whether incubating equine sperm under capacitating conditions induces capacitation-associated changes and affects embryo development following homologous equine ICSI, as well as oocyte activation, early cleavage, and 5-methylcytosine (5 mC) and 5-hydroxymethylcytosine (5hmC) levels following heterologous ICSI using bovine oocytes. Equine spermatozoa were incubated for 45 min in either unsupplemented Tyrode's albumin lactate pyruvate (TALP), representing non-capacitating conditions (NC), or TALP supplemented with bicarbonate and polyvinyl alcohol to support capacitating conditions (CAP), before being used for ICSI. Quantitative data were analyzed using a paired t-test, whereas binomial data were analyzed using Fisher's exact test. Differences were considered significant at P < 0.05. Following equine ICSI, cleavage rates, blastocyst rates, and total cell number were comparable between treatments. Similarly, after heterologous ICSI, pronuclear formation and cleavage did not differ between groups. However, levels of 5-methylcytosine at the 8-cell stage were significantly higher in embryos derived from CAP sperm compared with NC sperm. In conclusion, the capacitation conditions evaluated in this study induced capacitation-associated events in equine sperm but did not affect the in vitro developmental competence of equine embryos produced by ICSI. Further studies are warranted to investigate the biological significance of differential DNA methylation levels during early embryo development after sperm incubation. Overall, these findings provide new insight into male factors influencing the efficiency of ICSI in horses.