Partial Deoxygenation of Semen Extender Minimizes Post-thaw Damages and Improves Freezability of Crossbred Bull Spermatozoa

Abstract

Background: Oxidative stress occurs when oxygen or oxygen derived oxidants exceed antioxidants and become responsible for poor post-thaw semen quality during the cryopreservation process. Therefore, the present study was designed to investigate the effects of dissolved oxygen (DO) levels (4, 6 and 8 ppm) in semen extender on freezability of crossbred bull spermatozoa. As the gap between 4 and 8 ppm existed in earlier studies, DO of 6 ppm in semen extender was further standardized and the effects were compared with other respective groups to contemplate any improvement in post-thaw semen quality.Methods: For the experiment, Tris-egg Yolk-Glycerol (TYG) extender was partially deoxygenated by nitrogen gassing @ 2-3 bubbles/sec at 34°C for 0, 16, 12 and 9 minutes to obtain DO levels of 11.7 ppm (Group-I/control), 4 ppm (Group-II), 6 ppm (Group-III) and 8 ppm (Group IV), respectively and collected semen samples were diluted with these extender groups to have 80×106 spermatozoa/ ml of the extender. Semen samples were evaluated for individual progressive motility (IPM), lipid peroxidation (LPO), total antioxidant capacity (TAC), plasma membrane integrity, acrosomal integrity and apoptotic changes at different stages of cryopreservation.Result: At the post-thaw stage, progressive motility was greater (p less than 0.05) in Group II compared to Group I and the least reduction from the post-dilution to the post-thaw stage was observed in Group II. In comparison to Group I, Groups II, III and IV showed lesser (p less than 0.05) MDA production with Group II having greater (p less than 0.05) TAC concentration than other groups at the post-thaw stage. A declining trend was observed in membrane integrity as DO levels increased from 4 ppm to 11.7 ppm. Acrosomal integrity did not differ among treatment groups, but, found to be higher (p less than 0.05) than the control group. Per cent viable spermatozoa was greater (p less than 0.05) in Group II than Group I and vice versa for necrotic spermatozoa as assessed by Annexin VFITC/PI staining. In conclusion, reducing the DO level to 4 ppm before cryopreservation improved the freezability by reducing oxidative stress and apoptotic changes while, above 4 ppm tended to lower it. An appreciable improvement in freezability can be seen at 6 ppm of DO, but, not up to that extent as observed at 4 ppm.