Repeatability of Selected Parameters Related to Stallion Sperm Quality and Cryotolerance

This study evaluated the repeatability of selected sperm parameters in Salernitano stallions housed on the same farm. Semen was collected weekly for four weeks, and sperm kinetics, mitochondrial activity, and oxidative/nitrosative status were assessed before and after freezing the sperm with HF-20 and INRA Freeze. Pre-freezing, significant individual variability was observed, with low repeatability for semen volume (r = 0.32), total motility (r = 0.38), curvilinear velocity (r = 0.32), and lipoperoxidation (r = 0.36). Post-thaw, sperm frozen with INRA Freeze showed significant inter-stallion differences and low-to-moderate repeatability across kinematic parameters, mitochondrial membrane potential, nitric oxide, and lipoperoxidation, whereas those frozen with HF-20 showed repeatability only for progressive motility and intracellular H₂O₂. An assessment of freezability revealed significant inter-stallion variability and low-to-moderate repeatability for most kinematic traits in sperm frozen with INRA Freeze. Age influenced specific parameters in both fresh and frozen-thawed semen. Kinematic traits were strongly intercorrelated and associated with mitochondrial activity, as well as with lipoperoxidation, the latter being significantly related to H₂O₂ and nitric oxide levels. Although the overall post-thaw differences between extenders were not statistically significant, INRA Freeze enabled clearer discrimination among stallions. The generally low-to-moderate repeatability observed in this study suggests that extender choice can influence cryopreservation outcomes, and supports the need for tailored protocols.