In the present study, we evaluated the effect of glucose-fructose and sucrose supplementation in glycerol-free tris (GFT) on sperm motility, viability, ROS level, apoptosis (BAX and BCL2) and motility (SMCP) related gene expression of dog sperm according to different post-thaw incubation time. The spermatozoa collected from five dogs were resuspended (5×107 cell/ml) with GFT containing 86 mM glucose and 86 mM fructose (GF-GFT) or 100 mM sucrose (S-GFT). The sperm (500 μl) were loaded in straws, cooled for 50 min at 4℃, frozen using liquid nitrogen (LN2) vapor for 20 min and plunged in LN2. The progressive motility, viability, ROS (H2O2) level and mRNA expression of spermatozoa were evaluated according to post-thaw incubation time (0 h, 3 h and 6 h) at 24℃. ROS was assessed using H2DCFDA stain by flow cytometry. The relative abundances of BAX, BCL2 and SMCP were assessed using quantitative real-time polymerase chain reaction (RT-PCR). 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. Whereas, the sperm ROS level in GF-GFT group was decreased at 6 h of post-thaw incubation. However, the ROS level in the group S-GFT was gradually increased with the progress of post-thaw incubation period. The post-thaw incubation had no substantial effect on mRNA expression of BAX, BCL2 and SMCP genes of dog spermatozoa in both the GF-GFT and S-GFT groups. These results indicate that GF supplementation in GFT improves the progressive sperm motility during the 6 h of post-thaw incubation with maintaining similar sperm viability and is more efficient in reducing ROS after 3 h of post-thaw incubation. The addition of GF in GFT for the cryopreservation of dog spermatozoa and post-thaw incubation would open an option to achieve more functioning spermatozoa for future assisted reproduction practices.

The objective of the present study was to evaluate the effect of disaccharides supplementation in glycerol-free tris (GFT) on dog sperm cryopreservation with respect to pH adjustment of extender and post-thaw incubation. The spermatozoa collected from five dogs were resuspended (5×107 cell/ml) with GFT containing 100 mM of lactose (L), trehalose (T) or sucrose (S) or pH adjusted (6.85) 100 mM of lactose (LP), trehalose (TP) or sucrose (SP). The sperm (500 μl) were loaded in straws, cooled for 50 min at 4℃, frozen using liquid nitrogen (LN2) vapor for 20 min and plunged in LN2. After thawing at 37℃ for 25 s in a water bath, the spermatozoa were incubated at 24℃ for 30 min. The progressive motility, viability, mitochondrial membrane potential (MMP) and mRNA expression of SMCP gene were then assessed. The MMP was evaluated by combined JC-1 plus PI staining. The relative abundance of SMCP was assessed using quantitative real-time polymerase chain reaction (RT-PCR). Adjustment of pH in GFT extender supplemented with disaccharides did not improve sperrm motility and viability. In general, post-thaw incubation increased the progressive motility of spermatozoa. The sperm motility in the group S was significantly (P<0.05) higher than other groups regardless of post-thaw incubation time. Similarly, the sperm viability in the group S was significantly (P<0.05) higher following post-thaw incubation. The higher sperm motility in the group S was also supported with the significantly (P<0.05) higher live sperm having high MMP. There was no significant difference in mRNA expression of SMCP gene among the experimental groups. These results indicate that cryopreservation of dog sperm in GFT supplemented with S and 30 min post-thaw incubation at 24℃ could provide better freezability of dog spermatozoa with improved motility and higher MMP.