Effect of Mito-TEMPO Incorporated Semen Extender on Physico-morphological Attributes and Functional Membrane Integrity of Frozen Thawed Buffalo Spermatozoa.

BACKGROUND Sperm mitochondria are the major site of reactive oxygen species (ROS) production and excess production during freezing-thawing process inflicts oxidative damages to spermatozoa. Buffalo spermatozoa are more prone to oxidative damage due to inherently more polyunsaturated fatty acids and low cholesterol to phospholipids ratio in the plasma membrane. A mitochondrial targeted antioxidant, Mito-TEMPO was used in this study. OBJECTIVE To study the effect of Mito-TEMPO incorporated semen extender on the post-thaw semen quality in buffalo. MATERIALS AND METHODS A total of 18 ejaculates from three murrah buffalo bulls with ≥70% individual progressive motility were utilized for the study. Each semen sample was equally divided and extended with five groups: Group I (Control, without Mito-TEMPO addition); Group II (10 µM Mito-TEMPO); Group III (50 µM Mito-TEMPO); Group IV (100 µM Mito-TEMPO); Group V (500 µM Mito-TEMPO) to have 80×106 progressive motile sperm/mL of extender, filled and sealed in French mini straws (0.25 mL) and frozen following equilibration. The effect of Mito-TEMPO was assessed at fresh/post-dilution and post-thaw stages by evaluating physico-morphological attributes and functional membrane integrity such as hypo-osmotic swelling test (HOST). RESULTS Initial progressive motility, viability, acrosomal integrity and HOS response was significantly (p<0.05) improved and sperm abnormality was significantly (p<0.05) reduced in extended semen with Mito-TEMPO (50 µM) compared to control at post-thaw stage, although improvement was also observed at 10 and 100 µM in post-thaw samples. CONCLUSION Mito-TEMPO incorporated semen extender at 50 µM concentration, could be part of a rationale for improving post-thaw semen quality in buffalo.