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The quality of spermatozoa at post-thaw stage depends on many factors including the level of lipid per-oxidation (LPO) and the antioxidant profile of the ejaculates at fresh stage. Thus, the present investigation was done to evaluate discard rate of semen ejaculates at fresh and post-thaw stages in association with various semen quality parameters in Murrah buffalo. A total of 79 ejaculates from seven Murrah buffalo bulls formed part of the study. Following application of rigorous prescribed selection criteria it was observed that 17.72% (14/79) ejaculates were discarded at fresh stage due to poor quality. The initially selected ejaculates were processed for cryopreservation as per the minimum standard protocols. The study show satisfactory results for 31 (47.69%) samples for inclusion in the further AI programme (discard rate of 52.31% (34/65) at post thaw. The mean post-thaw motility (PTM; %) of the passed ejaculates was found to be 44.2±1.0. In conclusion, study results show efforts to minimize LPO and enhance total antioxidant capacity (TAC) at various processing stages shall play a vital role in reducing discard rate of the semen samples in buffalo bulls. [ABSTRACT FROM AUTHOR]

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]

Despite recent advances in technique of spermatozoa cryopreservation, there are still ejaculates present that fail to meet strict quality standard; mainly due to detrimental effect of imbalance of free radicals. The omnipresence of dead/defective spermatozoa in ejaculates of eutherian species is a major source of excessive free radicals. Though sperm-selection techniques, as well as addition of antioxidants addressed the problem to a certain extent, the major source of free radicals in the semen remained, causing much damage. This study attempts to remove dead/damaged spermatozoa using negative fertility-marker. The effect is unraveled by Hypo-osmotic (HOS), and fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) assay, further confirmed by Ca2+-regulating mechanisms and depolarization of sperm membrane potential, reduction in concentration of free radicals and finally by in vitro fertility assay. The study involved functionalization of iron oxide nanoparticles (IONPs) with silane followed by bio-conjugation with anti -ubiquitin antibodies. The nano-purification of semen using anti -ubiquitin conjugated iron oxide nanoparticles (IONPs) (antibody concentrations 0.5, 1.0 and 2.0 μg/ml) was attempted. The efficiency of nano-purification was 18.1%–43.8% in the study. The results revealed greater (P ≤ 0.05) spermatozoa population with intact plasma membrane, acrosome integrity, high mitochondrial membrane potential and pattern-F (least intracellular Ca2+), evidence of low lipid peroxidation and higher total antioxidant capacity in nano-purified groups. More number of spermatozoa were bound to zona pellucida of matured oocytes from nano-depleted than non-depleted group. The findings demonstrate antibody concentration of 1.0 μg/ml bio-conjugated with IONPs as most efficient in enriching the ejaculate with functional spermatozoa with the highest percentage of zona binding. • Nano-purification improves semen quality in buffalo. • Removal of dead and damaged spermatozoa reduces the oxidative stress level in semen. • Nano-purification improves the fertility of semen. [ABSTRACT FROM AUTHOR]