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

Julie BaumberDepartment of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616.

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Barry A. BallDepartment of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616.

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Jennifer J. LinforDepartments of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616.

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Abstract

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 (10mM), ascorbic acid (10mM), α-tocopherol (25, 50, 100, or 500µM or 1mM), or the vehicle for α-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 α-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 α-tocopherol in ethanol may affect spermatozoal viability; consequently, water-soluble analogues of α-tocopherol may be preferred for future investigations. (Am J Vet Res 2005;66:772–779)

Abstract

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 (10mM), ascorbic acid (10mM), α-tocopherol (25, 50, 100, or 500µM or 1mM), or the vehicle for α-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 α-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 α-tocopherol in ethanol may affect spermatozoal viability; consequently, water-soluble analogues of α-tocopherol may be preferred for future investigations. (Am J Vet Res 2005;66:772–779)