Reactive oxygen species in stallion semen can be affected by season and colloid centrifugation

There are anecdotal reports that equine fertility may decline towards the end of the breeding season. Previous studies have examined differences in sperm quality between the breeding season and non-breeding season but few studies have investigated the proportions of superoxide or peroxide containing spermatozoa at different times during the breeding season. The purpose of this study was to measure the content of these reactive oxygen species (ROS) at the beginning and end of the Swedish breeding season, using flow cytometric analysis of the fluorescence produced after staining with hydroethidium and dichlorodihydrofluorescein diacetate. In addition, the effects of a new method of selecting good quality spermatozoa by colloid centrifugation, known as Single Layer Centrifugation (SLC), on ROS-content were investigated. Superoxide production by stallion spermatozoa was found to be higher at the start than at the end of the breeding season in Sweden (22 ± 16% versus 9 ± 6%, P < 0.05), whereas sperm motility was lower (total motility 80 ± 9% versus 90 ± 6%, P < 0.01; progressive motility 55 ± 12% versus 60 ± 8%, P < 0.05, at the beginning and end of the breeding season respectively). The mean values of the other parameters of sperm quality measured did not differ with time within the breeding season although differences did occur for individual stallions. SLC was found to select motile spermatozoa that contained less superoxide (16 ± 14% versus 23 ± 18%, P < 0.01) and less peroxide (0.3 ± 0.8 versus 1 ± 2%, P < 0.01) than uncentrifuged controls, although they were capable of producing ROS when stimulated with menadione. This reduced peroxide production may contribute to the enhanced sperm survival (retention of motility) seen in the SLC samples during storage.