Assessment of the cryopreservation of equine spermatozoa in the presence of enzyme scavengers and antioxidants

OBJECTIVE: To evaluate the effect of the addition of enzyme scavengers and antioxidants to the cryopreservation extender on characteristics of equine spermatozoa after freezing and thawing. SAMPLE POPULATION: 2 ejaculates collected from each of 5 stallions. PROCEDURE: Equine spermatozoa were cryopreserved in freezing extender alone (control samples) or with the addition of catalase (200 U/mL), superoxide dismutase (200 U/mL), reduced glutathione (10 mM), ascorbic acid (10 mM), alpha-tocopherol (25, 50, 100, or 500 microM or 1 mM), or the vehicle for alpha-tocopherol (0.5% ethanol). After thawing, spermatozoal motility was assessed via computer-assisted analysis and DNA fragmentation was assessed via the comet assay. Spermatozoal mitochondrial membrane potential, acrosomal integrity, and viability were determined by use of various specific staining techniques and flow cytometry. RESULTS: The addition of enzyme scavengers or antioxidants to cryopreservation extender did not improve spermatozoal motility, DNA fragmentation, acrosomal integrity, viability, or mitochondrial membrane potential after thawing. Superoxide dismutase increased DNA fragmentation, likely because of the additional oxidative stress caused by the generation of hydrogen peroxide by this enzyme. Interestingly, the addition of the vehicle for alpha-tocopherol resulted in a significant decrease in live acrosome-intact spermatozoa. CONCLUSIONS AND CLINICAL RELEVANCE: The addition of antioxidants to the cryopreservation extender did not improve the quality of equine spermatozoa after thawing, which suggests that the role of oxidative stress in cryopreservation-induced damage of equine spermatozoa requires further investigation. Our data suggest that solubilizing alpha-tocopherol in ethanol may affect spermatozoal viability; consequently, water-soluble analogues of alpha-tocopherol may be preferred for future investigations.