Effect of refreezing extender on stallion sperm quality and embryo production after intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) is a valuable assisted reproduction technology in clinical practice, especially when semen availability is limited. Since the number of sperm required per ICSI cycle is much less than the number of sperm available in a standard straw of frozen semen, refreezing semen at lower sperm concentrations could yield multiple straws for ICSI use. However, there is little data on the effect of sperm refreezing on ICSI outcomes, especially on the effect of extender used for refreezing. The objective of the present study was to evaluate the effect of refreezing extender on stallion sperm quality and embryo production after ICSI. Semen was frozen in a low egg yolk/glycerol/amide extender (Extender 1), then thawed, re-extended, and refrozen in each of three extenders. When sperm were refrozen in Extender 1, the cleavage rate was lower (P < 0.05) and the blastocyst development rate tended to be lower (P = 0.06) than for the once-frozen sperm. In contrast, when sperm were refrozen in high egg yolk/glycerol (Extender 2) or low egg yolk/milk/glycerol (Extender 3) extenders, cleavage and blastocyst development rates did not differ significantly from those for the once-frozen semen. Notably, sperm refrozen in Extender 1, which yielded the lowest ICSI outcomes, showed the highest sperm motility and viability, demonstrating that traditional measures of sperm quality were inadequate to assess the suitability of refrozen sperm for ICSI. In a follow-up experiment conducted to evaluate the effects of Extenders 1 and 3 when used for once-frozen semen, cleavage and blastocyst rates did not differ between extenders. In conclusion, the extender used to initially freeze stallion sperm may not significantly affect ICSI outcomes; however, the extender used for refreezing can significantly affect embryo production. Refrozen stallion semen can be effectively used for ICSI when low egg yolk/milk/glycerol extender is used for refreezing. Until further research is available, use of extenders without amides is recommended when refreezing stallion semen for ICSI.