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Abstract

Objective—To determine glutathione peroxidase (GPX) and superoxide dismutase (SOD)-like activities in spermatozoa, seminal plasma, and reproductive tissues (ie, testis, epididymis, bulbourethral gland, prostate, vesicular gland, and ampulla) in horses.

Sample Population—Seminal plasma from 17 stallions, spermatozoa from 5 stallions, and reproductive tissues from 3 stallions.

Procedure—Activity of GPX was determined by use of assays measuring oxidation of NADPH in the presence of exogenous glutathione, cumene hydroperoxide, and glutathione reductase. Activity of SOD-like enzymes was determined by use of the nitroblue tetrazolium assay.

Results—Mean GPX and SOD-like activities in seminal plasma were 1.3 ± 0.1 nmol of NADPH oxidized/ min/mg of protein and 29.2 ± 6.6 U/mg of protein, respectively. Mean GPX activities in spermatozoa separated from seminal plasma by centrifugation and via Percoll gradient were 2.2 ± 0.3 nmol and 6.1 ± 1.3 nmol of NADPH oxidized/min/mg of protein, respectively. Mean SOD-like activity of spermatozoa separated by centrifugation was 58.6 ± 22.3 U/mg of protein; SOD-like activity was not detected in Percollseparated spermatozoa. Among reproductive tissues, the ampulla and prostate had the highest SOD-like activity, although this was not significantly different from activity in other tissues. Testes and spermatozoa from the cauda epididymis contained significantly more GPX activity than other tissues.

Conclusions and Clinical Relevance—Results suggest that although equine seminal plasma contains high SOD-like enzyme activity, spermatozoa have limited GPX and SOD-like activity. Enzymatic antioxidant activity in equine spermatozoa appears to be predominantly derived from seminal plasma adsorbed onto the plasma membrane. Removal of seminal plasma during semen processing may increase oxidative stress in equine spermatozoa. (Am J Vet Res 2005;66:1415–1419)

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in American Journal of Veterinary Research

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)

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in American Journal of Veterinary Research

Abstract

Objective—To characterize generation of reactive oxygen species (ROS) by equine spermatozoa.

Sample Population—Multiple semen samples collected from 9 stallions.

Procedure—Equine spermatozoa were separated from seminal plasma on a discontinuous polyvinylpyrrolidone (PVP)-coated silica gradient and resuspended in a modified Tyrode albumin-lactate-pyruvate medium. Amount of hydrogen peroxide (H2O2) generated was assayed by use of a 1-step fluorometric assay, using 10-acetyl-3,7-dihydroxyphenoxazine as a probe for detection of H2O2 in a microplate assay format. Concentration of H2O2 was determined by use of a fluorescence microplate reader.

Results—Amount of H2O2 generated increased significantly with time and spermatozoa concentration for live and flash-frozen spermatozoa, and amount of H2O2 generated was significantly greater for flash-frozen than for live spermatozoa. Addition of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) significantly increased generation of H2O2 by live and flash-frozen spermatozoa. Addition of a calcium ionophore also significantly increased the amount of H2O2 generated by live spermatozoa but did not have an effect on amount of H2O2 generated by flash-frozen spermatozoa. Abnormal equine spermatozoa generated significantly greater amounts of H2O2 than did normal spermatozoa.

Conclusion and Clinical Relevance—Equine spermatozoa generate ROS in vitro, possibly via a NADPH-oxidase reaction. Spermatozoa damaged during flash-freezing or morphologically abnormal spermatozoa generated significantly greater amounts of ROS than did live or morphologically normal spermatozoa. Damaged and abnormal spermatozoa generate greater amounts of ROS that may contribute to reduced fertility or problems related to semen preservation. (Am J Vet Res 2001;62:508–515)

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in American Journal of Veterinary Research

Abstract

Objective—To characterize the activity of catalase in equine semen.

Animals—15 stallions of known and unknown reproductive history.

Procedure—Seminal plasma was collected from raw equine semen by centrifugation, and samples of seminal plasma were frozen prior to assay for catalase activity. Tissue samples (n = 3 stallions) from the bulbourethral gland, prostate gland, vesicular gland, and testis were homogenized, and cauda epididymal fluid was collected for determination of catalase activity. Catalase activity was determined as an enzyme kinetic assay by the disappearance of H2O2 as measured by ultraviolet spectrophotometry.

Results—Catalase activity in equine seminal plasma was 989.3 ± 167.8 U/ml (mean ± SEM), and the specific activity of catalase in equine seminal plasma was 98.7 ± 29.2 U/mg of protein. Specific activity of catalase in tissue homogenates was significantly higher in the prostate gland (954 ± 270 U/mg of protein) than in the ampulla (59 ± 5 U/mg of protein), bulbourethral gland (54 ± 11 U/mg of protein), vesicular gland (39 ± 3 U/mg of protein), cauda epididymal fluid (11 ± 3 U/mg protein), or testis (54 ± 6 U/mg of protein).

Conclusions and Clinical Relevance—Equine seminal plasma contains a high activity of catalase that is derived primarily from prostatic secretions. Procedures such as semen cryopreservation that remove most seminal plasma from semen may reduce the ability to scavenge H2O2 and thereby increase the susceptibility of spermatozoa to oxidative stress. (Am J Vet Res 2000;61:1026–1030)

Full access
in American Journal of Veterinary Research