Synergistic protection of chilled canine spermatozoa by apigenin and N-acetylcysteine amide through enhanced antioxidant defense and mitochondrial function

OBJECTIVES: Semen preservation is a critical aspect of assisted reproductive technologies in canine breeding programs; however, oxidative stress during chilled storage negatively affects sperm quality by impairing motility, membrane integrity, and mitochondrial function. This study investigated the protective effects of apigenin and N-acetylcysteine amide (NACA), individually and in combination, on the quality, antioxidant defense system, and mitochondrial activity of canine spermatozoa stored at 5 °C for 72 h. METHODS: Semen samples collected from five healthy mixed-breed dogs with proven fertility were pooled and diluted in a Tris-egg yolk extender supplemented with apigenin (0.2 or 0.4 mM), NACA (0.5 or 1 mM), or a combination of both (0.4 mM apigenin + 1 mM NACA). Sperm motility, membrane functionality, viability, DNA integrity, oxidative/antioxidant biomarkers, and mitochondrial parameters were evaluated at 0, 24, 48, and 72 h. RESULTS: Both antioxidants significantly improved all parameters compared with the control, with the combined treatment exhibiting the most pronounced and synergistic effects: motility and kinematic parameters approximately twofold higher, antioxidant enzyme activities markedly enhanced, lipid/protein oxidation reduced, and mitochondrial integrity and ATP production preserved after 72 h. CONCLUSIONS: In conclusion, the synergistic combination of apigenin and N-acetylcysteine amide significantly improves the quality and longevity of chilled canine spermatozoa, offering a promising strategy for optimizing semen extenders in canine breeding programs.