THE EFFECT OF EXTENDER, pH, CRYOPROTECTANT AND EQUILIBRATION TIME ON THE MOTILITY OF CRYOPRESERVED TOR KHUDREE (SYKES) SPERMATOZOA

摘要

Milt samples of Torkhudree (Cyprinidae), pooled from different males, were diluted with two extenders (E_1 and E_2) having djmethylsulphoxide (DMSO) as a cryoprotectant. The optimum pH levels were decided based on preliminary trials carried out at a pH range of 7.0 to 8.5 for both the extenders and DMSO was tested at three levels viz. 5, 10 and 15%. The diluted samples were subjected to equilibration periods of 0, 30, 60 and 90 min, stored in 500 μl plastic straws, cooled over liquid nitrogen (LN_2)and subsequently kept immersed in LN_2 for 10 weeks. The milt samples were thawed at 37 ± 1 °C for 5-10 sec and the motility of fresh and thawed spermatozoa was estimated on an arbitrary scale of 0-10 using an inverted microscope. Both the extenders at a ratio of 1:10 (spermiextender) were found to be good at the tested pH levels in the presence of DMSO, with E_1 being slightly better than E_2. The spermatozoa showed motility rates as high as 95% after 10 weeks of storage in LN_2. Compared to 5 or 15%, samples containing 10% DMSO, equilibrated for 0-90 min, gave higher rates of post-thaw motility. The post-thaw motility rate was significantly higher (P < 0.05) for DMSO when compared with methanol and no post-thaw motility was observed with other cryoprotectants. The spermatozoa count in fresh milt ranged between 4.98 to 7.45 x 10~6 per mm~3 and the spermatocrit value was 1:4 to 5 (fluid:packed sperm cells). The results indicate that T. khudreespermatozoa could be successfully cryopreserved using either extender E_1 at a pH of 7.48 or extender E_2 at a pH of 7.75 in the presence of DMSO at an equilibration period of 0-90 min.