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.


METHODS
2 ejaculates collected from each of 5 stallions.


METHODS
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
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.
Baumber
Julie
J
Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
Ball
Barry A
BA
Linfor
Jennifer J
JJ
D000818:Animals; D000975:Antioxidants; D002374:Catalase; D015925:Cryopreservation / Q000662:veterinary*; D004305:Dose-Response Relationship, Drug; D016166:Free Radical Scavengers; D006736:Horses; D008297:Male; D018384:Oxidative Stress; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology*; D013482:Superoxide Dismutase
D000975:Antioxidants; D016166:Free Radical Scavengers; D002374:Catalase; D013482:Superoxide Dismutase