The present study was undertaken to evaluate the effect of trisaccharides supplementation in glycerol-free tris (GFT) for the cryopreservation of dog spermatozoa. In the first experiment (E1), dog spermatozoa were resuspended with 50, 75, 100 or 125 mM of raffinose, melezitose or maltotriose and cooled at 4 ℃ for 10 min. To determine the effect of different cooling time, the spermatozoa resuspended with 100 mM of raffinose, melezitose or maltotriose were cooled during 10, 20, 30 or 40 min at 4 ℃ (second experiment; E2). The straws were then aligned horizontally for 10 min on the rack and then plunged into LN2. In the third experiment (E3), to determine the effect of different vapor freezing time, the spermatozoa resuspended with 100 mM raffinose were cooled at 4 ℃ for 20 min and frozen in LN2 for 5, 10, 15 or 20 min and then plunged into LN2. In the fourth experiment (E4), to compare different freezing methods [cooling plus vapor freezing (CV), cooling plus step-down freezing (CS) and direct step-down freezing (SD)], the spermatozoa resuspended with 100 mM raffinose were cooled for 20 min and frozen in LN2 vapor for 5 min in case of CV method. In case of CS method, spermatozoa were cooled for 20 min at 4℃ and then frozen by the step-down freezing method. The straws were then aligned horizontally at 18, 15, 5, and 2 cm respectively from the surface of LN2 for 1, 1, 1.4, and 5 min, respectively in an L shaped straw holder and then plunged into LN2. For SD method, the straws were directly aligned horizontally at the same levels as CS from the surface of LN2 for 1, 1, 1.9, and 5 min, respectively and then plunged into LN2. After thawing at 37℃ for 25 sec, the spermatozoa were then incubated for 30 min in the freezing extender (E1) or in the 50 mM sucrose supplemented GFT (E2, E3, and E4) at 24℃. Following post-thaw incubation, sperm progressive motility and viability were assessed in E1, E2, E3, and E4. In addition, acrosome integrity, and gene expression related to apoptosis (BAX, BCL2, and Caspase10) and sperm motility (SMCP) were evaluated in E4. The results demonstrated that, in E1, using 75 mM trisaccharides resulted in significantly (p<0.05) higher sperm motility in all sugar groups. Using 100 mM melezitose significantly (p<0.05) improved the post-thaw viability than the 100 mM raffinose. The viability in 100 mM maltotriose was similar with 100 mM raffinose and melezitose group. In E2, the different cooling time has no significant effect on post-thaw sperm progressive motility in all the sugar types. In addition, the viability was variable among the different groups. In E3, liquid nitrogen vapor freezing for 5 min resulted in improved motility and viability. The sperm progressive motility was significantly (p<0.05) higher in CV and SD group compared to CS group and the sperm viability was significantly (p<0.05) higher in CV group compared to the other groups in E4. However, the acrosomal integrity of spermatozoa in the group CV was significantly (p<0.05) higher than the group CS and SD. In addition, the expression of SMCP gene was significantly (p<0.05) higher in the CV group than the CS group. In contrast, the expression of Caspase10 significantly (p<0.05) lower in the group CV and SD than the group CS. Furthermore, the ratio of gene expression of BAX and BCL2 was significantly (p<0.05) lower in the group CV than the group CS. Therefore, cryopreservation of dog spermatozoa in 100 mM of raffinose supplemented GFT cooled for 20 min and vapor freezing for 5 min provides better progressive sperm motility, viability, and acrosome integrity with higher expression of SMCP gene and lower expression of caspase10 and BAX/BCL2 ratio following post-thaw incubation in 50 mM sucrose supplemented GFT for 30 min at 24℃.

The aim of this study was to develop a chemically defined extender for dog sperm cryopreservation by supplementation of essential and non-essential amino acids solution in EY-free PVA extender. Spermatozoa collected from mature dogs (1 ｘ 108 cell/ml) were frozen with EY-free extender supplemented with 0 (control), 1, 2, 4 % essential amino acids (EAAs) or 1, 2, 4 % non-essential amino acids (NEAAs). Sperm progressive motility, viability and acrosome integrity were evaluated immediately after thawing at 37 ℃ for 25 s and post-thaw incubation at room temperature for 20 min. In addition, to evaluate the synergistic effect of EAAs and NEAAs, spermatozoa were frozen with 0, 0.5, 1 or 2 % EAAs-NEAAs mixture (v:v). Sperm progressive motility, viability and acrosome integrity were evaluated immediately after thawing and post-thaw incubation. Additionally, spermatozoa were frozen using EY-free PVA extender supplemented with 2 % EAAs, 2 % NEAAs or 0.5 % EAAs-NEAAs mixture. The ROS level and phosphatidylserine (PS) translocation (Annexin V-FITC assay) were assessed using flow cytometry. In addition, gene expression level for SMCP (motility-related), apoptosis-related BCL2 and BAX was measured after freezing-thawing. The progressive motility of spermatozoa cryopreserved in EAAs or NEAAs significantly increased (P < 0.05) in all groups compared to the control group regardless of thawing conditions. In addition, 1 % NEAAs significantly protected the acrosome membrane of spermatozoa after freezing-thawing (P < 0.05). However, EAAs has shown no significant effect on viability and acrosome membrane integrity of spermatozoa. On the other hand, addition of EAAs-NEAAs mixture to EY-free PVA extender significantly (P < 0.05) increased sperm progressive motility without any effect on viability. Supplementation of 0.5 % EAAs-NEAAs mixture significantly (P < 0.05) increased the expression level of SMCP, BCL2 and BAX compared to control without significant effect on PS translocation and ROS level. We conclude that essential and non-essential amino acids solution can be effectively used in EY-free extender to improve sperm motility, acrosome integrity and gene expression of SMCP and BCL2 in dog sperm cryopreservation.