Enhancing Stallion Semen Cryopreservation: Selected Antioxidant Extracts and Sperm Freezability

Cryopreservation of equine semen remains challenging due to pronounced individual variability in cryotolerance. Because freezing induces oxidative stress and spermatozoa are particularly susceptible to such damage, this study aimed to comparatively evaluate the effects of natural extracts from nutraceutical compounds with high antioxidant activity, specifically matcha, spirulina, and horseradish, as well as quercetin, a well-known antioxidant molecule. These compounds were added to the freezing extender, and semen from 12 Salernitano stallions (48 ejaculates in total) was analyzed. Several parameters were assessed, including sperm kinetics, bioenergetics, oxidative and nitrosative stress markers, and the sperm DNA fragmentation index, both before and after cryopreservation. Neither the natural extracts nor quercetin significantly improved sperm freezability, likely due to the high degree of inter-individual variability. Stallion age also had a significant effect on nearly all the parameters evaluated, although no significant interactions were observed between age and treatment for any of the sperm quality traits. In conclusion, supplementation of the freezing extender with matcha, spirulina, horseradish extracts, or quercetin did not significantly enhance stallion semen cryopreservation outcomes. Conversely, stallion age and individual variability had a marked effect on sperm cryotolerance, highlighting the need for customized and holistic strategies to optimize cryotolerance in individual stallions.