Cryopreservation adversely affects the acrosomal integrity and post-thawing survival of frozen-thawed ram spermatozoa. The objectives of this study were to evaluate the effect of thawing and thermal resistance test on acrosomal integrity of ram spermatozoa frozen under controlled (Group 1) and uncontrolled (Group 2) cooling rates prior to programmable freezing. Semen samples obtained from adult Malpura rams were diluted with Egg yolk-Test-glycerol extender and packaged in 0.25 ml straws. Straws representing Group 1 were cooled in a programmable cell freezer from 25° to 5°C at the rate of 0.15°C per min followed by a holding time of 2 h for equilibration, while straws of Group 2 were allowed to slowly cool up to 5°C and equilibrate for 2 h in the cold cabinet. After equilibration, straws of Group 2 were also loaded in the cell freezer for freezing straws of both the treatment groups from 5° to - 125°C at the rate of 25°C per min. In the first experiment, straws of each group were thawed individually at 50°C for 10 s and dried semen smear was prepared. In the second experiment 4 frozen straws were thawed collectively at 50°C for 10 s per sample and subjected to post-thawing thermal resistance test. Samples of each group were incubated at 37°C for 4h and dried semen smear was prepared at an hourly interval. Acrosomal integrity was evaluated after staining the dried smears by Giemsa stain. Thawing had a significant (P<0.05) effect on acrosomal status of frozen-thawed ram spermatozoa. The percentage of spermatozoa with normal acrosomes was significantly (P<0.05) higher while the spermatozoa with acrosomal damage were significantly (P<0.05) lower in Group 1 compared to Group 2. Post-thawing incubation had a significant (P<0.05) effect on acrosomal integrity of ram spermatozoa. The percentage of spermatozoa with intact acrosome declined significantly (P<0.05) on incubation. However, the percentage of spermatozoa with intact acrosomes was significantly (P<0.05) higher in Group 1 than in Group 2. The results indicate that the acrosomal status of ram spermatozoa after thawing and thermal resistance test was better in samples processed by controlled-rate cooling compared to uncontrolled cooling prior to programmable freezing of ram semen in straws.
Acrosomal integrity, Controlled-rate cooling and freezing, Semen, Sheep
