Dog Sperm Cryopreservation in Glucose-fructose or Sucrose Supplemented Glycerol-free Tris: Effect of Post-thaw Incubation Time on Gene Expression Related to Apoptosis and Motility

OBJECTIVE: The study evaluated the effects of glucose-fructose or sucrose supplementation in glycerol-free Tris (GFT) solution on motility, viability, the level of reactive oxygen species (ROS), as well as the level of apoptosis (BAX and BCL2) and motility (SMCP)-related gene expression of dog spermatozoa. MATERIALS AND METHODS: Spermatozoa (5×10⁷ sperm/ml) were cryopreserved in GFT containing 86 mM glucose and 86 mM fructose (GF-GFT) or 100 mM sucrose (S-GFT). Progressive motility, viability, ROS (H₂O₂) level and mRNA gene expression of spermatozoa were evaluated 0 h, 3 h or 6 h post-thaw at 24°C. RESULTS: The motility of spermatozoa cryopreserved in GF-GFT was increased throughout the post-thaw incubation time. The motility of spermatozoa cryopreserved in S-GFT was increased at 3 h of post-thaw incubation. The sperm ROS level in the GF-GFT group was inconsistent during the post-thaw incubation time; however, the ROS level in the S-GFT group was gradually increased with progression of the post-thaw incubation period. The post-thaw incubation had no substantial effect on the mRNA expression of the BAX, BCL2, and SMCP genes of spermatozoa in both the GF-GFT and S-GFT groups. CONCLUSION: The supplementation of glucose and fructose improves progressive sperm motility during 6 h of post-thaw incubation while maintaining similar sperm viability. The addition of GF to GFT for cryopreservation and post-thaw incubation would yield more functional spermatozoa for future assisted reproduction practices.