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The generation of hydrogen peroxide (H2O2) by ram spermatozoa (spz) was measured using a flurometric assay with 10-acetyl-3,7-dihydroxyphenoxazine agent as a probe for H2O2 detection. The kinetics of H2O2 production from both live and dead spz at 1 × 10 6, 3 × 106, and 6 × 106 spz/well concentrations were assessed in the tyrode albumin lactate (TAL) medium every 15 min for 120 min. An increase in H2O2 production from both live and dead spz was noted with a significant difference (P < 0.05) between the 1 × 106 and 6 × 106 spz/well concentrations. Although dead sperm generated higher amounts of H2O2 than live ones, no significant differences (P > 0.05) were observed between the two types of sperm for the three different concentrations. The generation of H2O2 by ram spz was also compared in the presence and absence of nicotinamide adenine dinucleotide phosphate (NADPH) and phenylalanine, substrates of the two specific oxidases. The supplementation with these substrates significantly (P < 0.05) increased the amounts of H2O2 generated from both live and dead spz, but for the two substrates, the increase was higher with dead than with live spz especially when phenylalanine was added. Addition of the antioxidant catalase significantly (P < 0.05) decreased the generation of H2O2 by live and dead spz with no significant differences (P > 0.05) between the two types of sperm before or after the antioxidant addition. This study showed the ability of live and dead ram spz to generate H2O2 in TAL medium. This ability was significantly influenced by the addition of NADPH and phenylalanine and also by the supplementation of the antioxidant catalase.

Kinetics of hydrogen peroxide generated from live and dead ram spermatozoa and the effects of catalase and oxidase substrates addition