Dose-Dependent Effects of Protocatechuic Acid on Motility, Redox Balance, and DNA Integrity of Frozen-Thawed Ram Spermatozoa.

This study investigated the effects of supplementing Tris semen extender with protocatechuic acid (PCA) at doses of 25, 50, and 100 µg/mL on the quality of ram spermatozoa after cryopreservation. We evaluated motility, kinematic parameters, DNA integrity, redox status, plasma membrane-acrosome integrity (PMAI), viability, and high mitochondrial membrane potential (HMMP). PCA supplementation produced concentration-dependent and parameter-specific responses. The 25 µg/mL PCA group exhibited the most favorable functional profile, demonstrating improved progressive and total motility alongside reduced DNA damage. This protective effect was evidenced by lower tail length, tail DNA percentage, and tail moment in the comet assay. Conversely, treatment with 100 µg/mL PCA appeared to exert a notable effect on the redox balance, indicated by lower malondialdehyde, total oxidant status, and oxidative stress index levels, alongside an elevated total antioxidant status (p < 0.05). However, this higher concentration (100 µg/mL) did not provide parallel DNA protection and was associated with increased comet assay indices. PMAI, viability, and HMMP were not significantly affected by PCA supplementation (p > 0.05). These findings indicate that PCA exerts dose-dependent but parameter-specific effects on frozen-thawed ram spermatozoa. Low-dose PCA, particularly 25 µg/mL, appears more suitable for preserving motility and nuclear integrity, whereas 100 µg/mL mainly improves oxidative balance.