Influence of antifreeze protein III on canine sperm cryopreservation

Sperm cryopreservation is crucial in reproductive biotechnology; however, the longevity of frozen and thawed semen is limited by the deterioration of sperm cell integrity. This study aimed to examine the effects of adding antifreeze protein III (AFP III) to the diluent, using samples from eight healthy mature dogs. The ejaculates were divided into aliquots and diluted with a standard Tris-fructose-egg yolk extender containing AFP III at concentrations of 0, 0.75, 1.0, and 2.0 μg/ml. After thawing, the samples were analyzed for kinematic parameters, membrane Integrity, lipid peroxidation, viability, acrosome integrity, intracellular hydrogen peroxide, mitochondrial membrane potential and apoptotic metrics. The results show that while motility and velocity were not significantly different between the treated and control groups (p > 0.05), the treated groups generally performed better. Specifically, the 0.75 and 1.0 μg/ml groups exhibited better movement compared to the 2.0 μg/ml group. Additionally, there was a significant difference (p < 0.05) in membrane integrity between the control and treated groups, though no differences were observed among the treated groups. Significant differences (p < 0.05) were also observed in viability and acrosome integrity, with the 0.75 and 1.0 μg/ml groups outperforming the control and 2.0 μg/ml groups. There were no significant variations (p > 0.05) in phosphatidylserine translocation, lipid peroxidation, mitochondrial membrane potential, or hydrogen peroxide levels. However, the 0.75 and 1.0 μg/ml groups demonstrated superior effects compared to both the control and the 2.0 μg/ml groups. These results suggest that the addition of antifreeze proteins, specifically AFP III, markedly improves the protection of canine sperm during cryopreservation. This enhancement is evident in various parameters, underscoring the beneficial effects of AFP III in maintaining sperm quality.