LAUSR

BACKGROUND Oxidative stress is one of the leading factors responsible for poor post-thaw semen quality due to overproduction of reactive oxygen species (ROS) over neutralizing antioxidants present in semen. Mainly two ROS generation sites are present in spermatozoa i.e. Mitochondria and plasma membrane. Therefore, the idea of targeting these specific sites for minimization of ROS production with the compounds having known mechanism of actions was built up as a core for this research. OBJECTIVE Present study was done to investigate the effects of Mito TEMPO and Acetovanillone individually and in combination on freezability of buffalo spermatozoa. MATERIALS AND METHODS For the experiment, semen extender was supplemented with Mito TEMPO (50μM), Acetovanillone (50μM) and a combination of Mito TEMPO + Acetovanillone (50μM+50μM), designated as Group II, Group III and Group IV, respectively. Control group without any supplementation was designated as Group I. Total 24 ejaculates with individual progressive motility (IPM) of ≥ 70% were selected for the study. After final dilution, filling-sealing of straws, equilibration, and freezing was done as per standard procedure. Semen samples were evaluated for IPM, plasma membrane integrity, LPO, TAC, and C: P ratio at both fresh and post-thaw stages. Evaluation of ROS, MMP, capacitation status (CTC assay) and in vitro fertility potential were conducted only on frozen-thawed samples. RESULTS Addition of Mito TEMPO (50 μM) and Acetovanillone (50 μM) individually and in combination significantly (p<0.05) improved post-thaw semen quality in terms of IPM, plasma membrane integrity, TAC, Cholesterol content, C/P ratio, MMP, CTC- F pattern and zona binding ability of buffalo spermatozoa, while significantly (p<0.05) reduced ROS production, lipid peroxidation, and capacitation like changes as compared to control group. DISCUSSION As Mito TEMPO acts as SOD mimetic and also detoxifies ferrous iron at the mitochondria level, it aids in neutralization of excessive ROS production and minimizes oxidative stress related damages which enhances the antioxidant potential of sperm mitochondria. Earlier studies also indicated improved post-thaw semen quality in 50 μM supplemented group. Improvement observed in Acetovanillone (50 μM) group might be due to inhibition of NADPH oxidase as this enzyme activation by various physical/chemical inducers during cryopreservation process leads to activation of CatSper channel resulting calcium influx, premature capacitation and acrosomal reaction like changes through activation of adenylate cyclase, cAMP/PKA mediated tyrosine phosphorylation of sperm proteins. Acetovanillone also prevents NADPH oxidase mediated inhibition of glutathione reductase activity which has vital role in protecting structural and functional integrity of sperm plasma membrane. CONCLUSION Results indicated beneficial effects of supplementation of Mito TEMPO and Acetovanillone on sperm freezability and individual supplementation was as efficient as the combination group for sustaining post-thaw semen quality. This article is protected by copyright. All rights reserved.