Supplementation of Mito TEMPO and acetovanillone in semen extender improves freezability of buffalo spermatozoa

Background Oxidative stress is one of the leading factors responsible for poor post‐thaw semen quality because of overproduction of reactive oxygen species (ROS) over neutralizing antioxidants present in semen. Mainly two ROS generation sites are present in spermatozoa, that is, 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 (50 μM), acetovanillone (50 μM), and a combination of Mito TEMPO + acetovanillone (50 μM+ 50 μM), designated as Group II, Group III, and Group IV, respectively. Control group without any supplementation was designated as Group I. A total of 24 ejaculates with individual progressive motility (IPM) of ≥70% were selected for the study. After final dilution, filling‐sealing of straws, equilibration, and freezing were done as per the standard procedure. Semen samples were evaluated for IPM, plasma membrane integrity, lipid peroxidation, total antioxidant capacity (TAC), and cholesterol to phospholipids (C/P) ratio at both fresh and post‐thaw stages. Evaluation of ROS, mitochondrial membrane potential (MMP), capacitation status (CTC assay), and in vitro fertility potential were conducted only on frozen‐thawed samples. Results The addition of Mito TEMPO (50 μM) and acetovanillone (50 μM) individually and in combination significantly (p