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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 < 0.05) improved post‐thaw semen quality in terms of IPM, plasma membrane integrity, TAC, cholesterol content, C/P ratio, MMP, Chlortetracycline (CTC)‐Full (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 the control group. Discussion: As Mito TEMPO acts as an 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 that enhances the antioxidant potential of sperm mitochondria. Earlier studies also indicated improved post‐thaw semen quality in 50 μM supplemented group. The improvement observed in acetovanillone (50 μM) group might be because of inhibition of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as this enzyme activation by various physical/chemical inducers during cryopreservation process leads to activation of CatSper channel resulting in calcium influx, premature capacitation, and acrosomal reaction like changes through activation of adenylate cyclase and cAMP/PKA‐mediated tyrosine phosphorylation of sperm proteins. Acetovanillone also prevents NADPH oxidase–mediated inhibition of glutathione reductase activity, which has a vital role in protecting the 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. [ABSTRACT FROM AUTHOR]

Background: The excessive reactive oxygen species produced during semen‐freezing and ‐thawing damage the macromolecules resulting in impairment of cellular functions. Proteins are the primary targets of oxidative damage, wherein methionine residues are more prone to oxidation and get converted into methionine sulfoxide, thus affecting the protein function. The methionine sulfoxide reductase A (MsrA) catalyzes the conversion of methionine sulfoxide to methionine and restores the functionality of defective proteins. Objectives: To establish the expression of MsrA in male reproductive organs, including semen and its effect on quality of cryopreserved semen upon exogenous supplementation, taking buffalo semen as a model. Materials and methods: The expression of MsrA was established by immunohistochemistry, PCR, and Western blots. Further, the effect of recombinant MsrA (rMsrA) supplementation on the quality of cryopreserved spermatozoa was assessed in three treatment groups containing 1.0, 1.5, and 2.0 µg of rMsrA/50 million spermatozoa in egg yolk glycerol extender along with a control group; wherein the post‐thaw progressive motility, viability, membrane integrity, and zona binding ability of cryopreserved spermatozoa were studied. Results: The MsrA was expressed in buffalo testis, epididymis, accessory sex glands, and spermatozoa except in seminal plasma. In group 2, the supplementation has resulted in a significant (p < 0.05) improvement as compared to the control group in mean progressive motility (47.50 ± 2.50 vs. 36.25 ± 2.63), viability (56.47 ± 1.85 vs. 48.05 ± 2.42), HOST (50.76 ± 1.73 vs. 44.29 ± 1.29), and zona binding ability of spermatozoa (149.50 ± 8.39 vs. 29.50 ± 2.85). Discussion and conclusion: In the absence of native MsrA of seminal plasma, the supplementations of rMsrA may repair the oxidatively damaged seminal plasma proteins and exposed sperm plasma membrane proteins resulting in better quality with a fivefold increase in fertilizability of frozen‐thawed spermatozoa. The findings can be extended to other species to improve the semen quality with the variation in the amounts of rMsrA supplementation. [ABSTRACT FROM AUTHOR]