Determination of glutathione peroxidase and superoxide dismutase-like activities in equine spermatozoa, seminal plasma, and reproductive tissues

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 Percoll-separated 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.