Pectin supplementation improves post-thaw cryotolerance of Zandi ram spermatozoa: associations with mitochondrial function and redox status

The present study evaluated the effect of pectin supplementation in a freezing extender on the post-thaw quality of Zandi ram sperm. Semen was collected from mature Zandi rams using an artificial vagina and diluted in an extender supplemented with pectin at 400, 450, 500, or 550 μg/mL after initial semen-quality assessment. A non-supplemented extender was used as the control. Motility and kinematic parameters, plasma membrane integrity, abnormal morphology, viability, apoptosis-like changes, mitochondrial activity, ATP content, reactive oxygen species (ROS), and antioxidant status were evaluated after freezing and thawing. The results showed that pectin significantly affected most post-thaw sperm-quality traits, with 450 μg/mL being the most effective concentration. This treatment increased total and progressive motility, sperm velocity indices, plasma membrane integrity, mitochondrial activity, ATP content, and viability while reducing the proportion of abnormal sperm, apoptosis-like changes, and ROS production. It also enhanced antioxidant status, particularly total antioxidant capacity and glutathione peroxidase activity. The beneficial responses were not sustained at 550 μg/mL, indicating a concentration-dependent effect. Overall, the most effective concentration of pectin was 450 μg/mL. These findings suggest that pectin may be a promising natural polysaccharide-based additive for ram semen cryopreservation.