Abstract
Antioxidants are known to prevent the reactive oxygen species (ROS)-mediated peroxidative damage to the membrane lipids during hypothermic storage of mammalian spermatozoa. We hypothesized here that ROS also affect the lipid-protein interactions, thereby diminishing the membrane's integrity and proteins' anchorage to the bilayer. Antioxidants prevent these damages by scavenging the ROS. Ejaculates from Patanwadi rams were pooled after subjective evaluation and centrifuged using Percoll® . Sperm pellet was resuspended in soya lecithin-Tris-fructose diluent (400×106 cells/ml) containing either antioxidants (100IU/ml catalase+10mM reduced glutathione) or no antioxidant. Aliquots were chilled to 5°C in a cabinet and stored in a refrigerator at 3-5°C for 72hr. Sperm motility, viability, lipid peroxidation (LPO) and hypo-osmotic swelling test (HOST) were performed at 0, 24, 48 and 72hr. Sperm proteins extracted with 0.5% Triton X-100 were resolved by SDS-PAGE and quantified using Quantity One software (Bio-Rad, USA). The rapid motility, linearity and straight-line velocity (VSL) were found significantly (p<.05) higher in the antioxidant-treated group compared to the control at 48hr of storage. Sperm viability was found comparable between the groups. Higher HOST response and lower LPO were found in the antioxidant-treated sample compared to the control both at 48 and at 72hr. Overall, the proteins P1 (106.09kDa), P2 (87.00kDa) and P4 (51.14kDa) were lower (p<.05) in the sperm extract of antioxidant-treated group compared to the control. The content of P4 (51.14kDa) in sperm extract was found to increase (p<.05) earlier (48 vs. 72hr) in the control group compared to the antioxidant-treated group. Altogether, the results suggested that antioxidants reduced LPO in spermatozoa, resulting in higher sperm motility, plasma membrane integrity and protection of proteins' anchorage to the plasma membrane at 48 and 72hr of storage.
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