Dose‐Dependent Effects of Clinoptilolite Supplementation on Sperm Quality and Oxidative Stress During Canine Semen Cryopreservation

BACKGROUND: Cryopreservation induces oxidative stress, leading to reduced semen quality in canine semen. Clinoptilolite (CLP), a natural zeolite, has potential antioxidant properties that may improve post-thaw sperm parameters. OBJECTIVES: This study aimed to evaluate the dose-dependent effects of CLP supplementation in semen extenders on sperm quality and oxidative stress in dogs. METHODS: Semen was collected from four adult dogs by manual manipulation, divided into five equal aliquots, and diluted at a ratio of 1:3 (v/v) with a Tris-citrate extender containing 0% (control), 1%, 2%, 3%, or 4% CLP. Samples were cooled to 5°C within 45 min, equilibrated for 2 h in 0.25 mL straws, frozen in liquid nitrogen vapour, and stored in liquid nitrogen. Samples were thawed at 37°C for 30 s. Post-thaw sperm quality was evaluated together with oxidative stress markers, including total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI). RESULTS: The 2% CLP group showed the highest post-thaw sperm quality, whereas the lowest performance was observed in the 4% CLP group. Additionally, the 2% CLP group exhibited a more favourable oxidative profile, with higher TAS and lower TOS and OSI values than all other groups. CONCLUSIONS: CLP supplementation exerted dose-dependent effects on sperm quality and oxidative balance in canine semen cryopreservation. A 2% CLP concentration appears to be the most effective for improving post-thaw sperm quality.