Abstract
Sensitivity to freezing remains a critical issue in stallion semen cryopreservation procedures. To explore this topic in-depth, semen was collected from ten stallions, diluted with three different extenders, transported to the laboratory, and then centrifuged and frozen with four different extenders. We conducted analyses of sperm kinetics, mitochondrial membrane potential (MMP), and hydrogen peroxide content both before and after freezing. Additionally, we assessed antioxidant activity using the ABTS and FRAP methods and measured nitric oxide stable metabolites (NOx) in the blank extenders, seminal plasma, and extenders conditioned by spermatozoa before and after freezing. We found significant variability in the antioxidant activity and NOx content of the blank extenders and the seminal plasma. In the seminal plasma, ABTS-based antioxidant activity and NOx values were correlated with some sperm kinematic parameters and MMP in refrigerated semen, while no correlation was observed in frozen sperm parameters. Sperm function varied significantly between stallions but not between extenders, either before or after freezing. However, significant differences in antioxidant activities and NOx values were found among extenders conditioned following freezing. These results provide new insights into the factors contributing to the variability in individual stallions' tolerance to sperm freezing.