Reactive oxygen species and equine sperm function

The use of cryopreserved equine spermatozoa has increased; however, the fertility of frozen semen is not comparable to fresh spermatozoa and there is a significant variation between stallions. An improved understanding of equine sperm physiology and the cell changes occurring during the freeze-thaw process is required. This dissertation was directed towards understanding the impact of oxidative stress on equine spermatozoa. Previous studies indicate that equine spermatozoa generate reactive oxygen species (ROS). In this study I have demonstrated that equine neutrophils are an additional source of ROS. The addition of activated equine neutrophils to equine spermatozoa in vitro resulted in a significant (P < 0.01) increase in hydrogen peroxide generation and a significant (P < 0.001) decrease in sperm motility that could be counteracted by the addition of the antioxidant, catalase.; The generation of ROS by equine spermatozoa suggests a physiological role for ROS in sperm function. I report that low-level generation of ROS resulted in a significant increase in tyrosine phosphorylation (P < 0.005) and capacitation (P < 0.0001) in equine spermatozoa, prevented by the addition of catalase; implicating hydrogen peroxide as the causative ROS. Therefore, ROS generation by equine spermatozoa may be involved in the signaling pathways underlying sperm capacitation. In contrast to the physiological effect of low-level ROS generation, high levels of ROS generation were associated with a significant (P < 0.005) increase in DNA fragmentation of equine spermatozoa; counteracted by the addition of catalase or reduced glutathione. Cryopreservation of equine spermatozoa was also associated with a significant (P < 0.01) increase in DNA fragmentation.; Equine seminal plasma contains a relatively high activity of enzyme scavengers; however, cryopreservation is associated with the removal of seminal plasma and consequently the predominant source of antioxidants protection. Since, the addition of antioxidants in vitro counteracted the detrimental effects of oxidative stress on equine spermatozoa, I investigated whether antioxidant addition to the cryopreservation extender could improve post-thaw sperm parameters. The results of this study show no improvement in post-thaw equine sperm motility, DNA fragmentation, viability, acrosomal integrity or mitochondrial membrane potential with the addition of enzyme scavengers or antioxidants. The role of oxidative stress in cryopreservation-induced damage to equine spermatozoa requires further investigation.