Generation of reactive oxygen species by equine spermatozoa

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. Conclusions 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.