Antioxidant Activity of Stallion Spermatozoa After Cryopreservation with Natural Antioxidant-Supplemented Extenders

Cryopreservation of stallion semen is associated with oxidative stress (OS), which can impair sperm function and fertility. This study evaluated antioxidant activities in seminal plasma and sperm cytosols and investigated their relationships with selected sperm functional parameters following cryopreservation, with or without antioxidant supplementation. Semen was collected from ten fertile stallions and processed using a split-ejaculate design, including fresh semen and six freezing treatments: HF-20 extender alone; HF-20 supplemented with matcha, spirulina, horseradish, or quercetin; and a commercial extender (INRA Freeze). Total antioxidant capacity (FRAP) and enzymatic activities (superoxide dismutase, SOD; catalase, CAT; and glutathione reductase, GR) were measured in seminal plasma and sperm lysates. Linear regression analyses revealed significant associations between seminal plasma and fresh spermatozoa with respect to SOD and GR activities. In frozen-thawed semen, FRAP and CAT activities differed between samples cryopreserved with and without antioxidant supplementation. Significant correlations were observed among antioxidant activities, sperm kinetics, OS markers, and DNA fragmentation indices. Principal component analysis provided an exploratory overview of multidimensional patterns of covariation among sperm kinetics, redox balance, and nuclear fragmentation, explaining for 73% of the total variance. Overall, the results suggest complex associations between the antioxidant system and sperm quality and indicate that antioxidant supplementation of freezing extenders may modulate the redox status of stallion sperm after thawing.