Effects of different concentrations of N-acetylcysteine on the sperm quality, antioxidant enzyme activity, and antioxidant gene expression of cryopreserved goat semen

Abstract

During cryopreservation, spermatozoa produce excess reactive oxygen species (ROS), which attack the plasma membrane, disrupt the physiological structure of the sperm, and ultimately decrease semen quality. This study investigated the effects of different N-acetylcysteine (NAC) concentrations on the cryopreservation of semen from Qianbei Ma goats. Semen samples were collected from five bucks with motility rates above 80 %. The treatment groups were diluted 20-fold in extenders containing 3 or 9 mM NAC and cryopreserved in liquid nitrogen, whereas the control group did not include NAC. After thawing, the sperm motility, antioxidant gene expression, enzyme activity, and cell structure were analysed. The NAC-treated groups showed improved post-thaw sperm motility. The 9 mM NAC group presented the highest catalase (CAT) and glutathione peroxidase activities, lowest ROS levels, and fewest apoptotic sperms. Moreover, the 3 mM NAC group presented the highest superoxide dismutase activity and L-cysteine levels and the lowest malondialdehyde levels. Additionally, sperm membrane integrity and mitochondrial membrane potential were significantly higher in the NAC-treated group than that in the control. Further analysis of antioxidant and apoptotic gene expression in the treated sperm revealed that the 9 mM NAC group presented significantly greater CAT and GPX4 expression than the control and 3 mM NAC groups, whereas the apoptotic genes BAX and Caspase3 were elevated in the control group compared to both the NAC groups. In summary, adding NAC to semen extenders enhanced antioxidant gene expression, increased enzyme activity, and improved post-thaw semen quality, with the 9 mM NAC treatment showing the optimal effects.