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One of the most important factors contributing to poor quality semen post thaw has been reported to be oxidative stress. Continued interaction of spermatozoa with dissolved oxygen in extender renders detrimental effect by generating reactive oxygen species during processing. This formed the basis to investigate the effects of removal of oxygen from the Tris-Glycerol-Egg Yolk (TG-EY) extender on the post-thaw semen quality. Semen ejaculates (n=26) collected from three cross bred bulls with mass activity of ≥3 and progressive motility of ≥70% were split in two fractions (control and treatment) to obtain 80 x 10 6 progressively motile sperm/mL of extended semen. The TG-EY extender composed of 3.028 g of Tris, 1.675 g of citric acid, 1.25 g of fructose, 7.0 mL of glycerol, 10 mL of egg yolk, 10 5 IU Penicillin G Sodium and 10 5 µg Streptomycin for 100 mL of deionized water for control and degasified (freeze-pumpthaw cycling method using liquid nitrogen) for treatment. Semen was processed and filled in 0.25 mL polyvinyl chloride straws, sealed and cryopreserved for at least 2 week before evaluation. Assessment of postthaw motility, viability, acrosome and plasma membrane integrity of spermatozoa were evaluated after thawing at 37°C for 30s. Study revealed a significant (viability and acrosome integrity, p<0.05) and highly significant (post-thaw motility and plasma membrane integrity, p<0.01) improvement in all the semen quality parameters in degasified as compared to control group. This study showed that degasification of extender prior to their use can serve to improve semen quality to highly acceptable levels.This study shows importance of degasification of extender in cryopreservation protocol. The assays used to evaluate effect of degasification were currently in vogue. Fewer studies were available to evaluate the effect of degasification in cattle sperm cryopreservation. The paper contributes by showing the importance of degasification on sperm cryosurvival.

Aim: The study was conducted to investigate the effect of cholesterol loaded cyclodextrin (CLC) on freezability of buffalo spermatozoa. Materials and Methods:Murrah buffalo bull semen samples with progressive motility of 70% and greater were used. After the evaluation of motility and livability, four equal fractions of semen samples were made. Group I was kept as control and diluted with Tris, whereas Group II, III and IV were treated with CLC solution at the rate of 2.0, 3.0 and 4.0 mg/ml respectively to obtain 120 × 10 6 sperm/ml as final spermatozoa concentration. The aliquots of all the groups were incubated for action of CLC, followed by dilution and freezing. Evaluation at pre-freeze and post-thaw stage of progressive motility, viability and level of cholesterol and phospholipid was done. Results:The mean cholesterol content (μg/100 × 10 6 spermatozoa) of Group I, II, III and IV at pre-freeze stage was 21.55±0.63, 49.56±1.38, 55.67±0.45 and 47.79±1.01 and at post-thaw stage were 13.18±0.45, 34.27±0.71, 36.21±0.48 and 33.68±0.56, respectively. At pre-freeze stage, cholesterol content was significantly (p<0.01) higher in Group III in comparison to other groups. The mean cholesterol and phospholipids content of fresh sperm was 24.14±0.58 and 51.13±0.66 μg/100 × 10 6 sperm cells, respectively, and C/P ratio of spermatozoa at fresh stage was 0.47±0.067. Conclusion:CLC treatment maintains the C/P ratio and plays an important role in maintaining membrane architecture of spermatozoa. Hence, addition of CLC may be helpful in increasing freezability of buffalo spermatozoa by increasing the C/P ratio of spermatozoa.

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