Cryopreservation of Water Buffalo, Bubalus bubalis (L.) Spermatozoa Using Ethylene Glycol: Effects of Equilibration Time, Thawing Temperature and Time on Post-thaw Motility Rate

The study was conducted to determine the optimal equilibration time, thawing temperature and. thawing time on the post-thaw motility of buffalo spermatozoa frozen using ethylene glycol as the cryoprotectant in the diluent. Collected semen samples from three different bulls were all diluted with a Tris-raffinose-egg yolk extender with 7% ethylene glycol. The samples were then subjected to three different equilibration times (i.e., 20, 40 and 60 min) at 5 degrees C. After the equilibration time has been reached, the straws were suspended in liquid nitrogen vapor for 7 min. (-126 degrees C) and then stored in liquid nitrogen (-196 degrees C) for 7 to 14 days until thawed. The thawed samples were then subjected to different thawing temperatures and thawing times. The study showed that the semen subjected to 20-min equilibration time had a comparable level of post-thaw motility with those equilibrated for 60 min. However, those subjected to 40-min equilibration time had a significantly lower (p<0.05) motility rate as compared to semen samples subjected to 20 and 60 min equilibration times. Results further showed that there were no significant differences in survival rate of samples thawed under different temperatures, and that frozen semen thawed for 17 sec had a significantly higher motility (p<0.05) compared to those thawed for 12 sec. Overall, the study demonstrates that semen equilibrated for 60 min, frozen and thawed at 45 degrees C for 12 or 17 sec yielded higher post-thaw motility rates than the other treatment groups. However, the cryopreservation of bubaline spermatozoa using 7% ethylene glycol in the diluent should be further optimized and evaluated in fertility trials.