Effects of Various Antifreeze Protein Type III Concentrations on Post-Thawed Dog Sperm Quality and Protein Expression

Introduction: The development of ice crystals during the freezing process can be detrimental to the viability and fertilization capacity of frozen–thawed spermatozoa. The unique properties of antifreeze proteins allow them to inhibit the formation of ice crystals during cell cryopreservation. Objective: Previous studies have assessed the general sperm quality parameters, reactive oxygen species (ROS) level, lipid peroxidation, and sperm apoptosis after using antifreeze protein III (AFP III) for cryopreservation. However, the data regarding changes in protein expression and their relation to sperm quality after thawing are still lacking. Therefore, this work addresses associated proteomic changes in post-thawed dog sperm. Methods: Two experiments were conducted using high (Experiment I) and low (Experiment II) concentrations of AFP III. Semen samples from four dogs were divided into aliquots and diluted with Tris-egg yolk extender supplemented with 0 (control), 1, 5, 10, or 15 µg/mL AFP III (Experiment I) or 0, 1, 2, 3, or 4 µg/mL AFP III (Experiment II) based on a previous literature review. After being frozen in LN 2 and thawed, sperm motility parameters, viability, acrosome integrity, and apoptosis were evaluated. Furthermore, the AKAP4, ATP1B1, and HSP70 proteins, which are associated with good sperm quality and freezability, were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting to assess their expression and levels. Results: In the high-concentration experiment, AFP III at 1 µg/mL significantly increased ( p < 0.05) total and moderate-progressive motility compared with the control and other AFP III concentrations. Moreover, the 1 µg/mL group had higher HSP70 and AKAP4 protein levels than the control and other concentration groups, though the differences were not significant. Conclusion: These findings suggest that adding 1 µg/mL AFP III to the semen extender enhances the motility of post-thawed dog sperm and suggest the potential use of HSP70 and AKAP4 proteins as biomarkers for good semen quality.