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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]

The current study intended to optimize the concentration of Oxyrase in the semen dilutor and to evaluate its effect on freezability of spermatozoa of Sahiwal bulls. Supplementation of Oxyrase at 0.125 IU/mL concentration significantly reduced dissolved oxygen (DO) in the dilutor to 4 ppm in 16–18 min at 35 °C. For supplementation studies, a total of 24 ejaculates were categorized into poor and good ejaculates categories (n = 12 each) based on their initial progressive motility. Each ejaculate was further divided into two aliquotes. The first aliquote was diluted with tris-egg yolk extender without Oxyrase (control group) whereas, in the treatment group, Oxyrase was supplemented at the concentration of 0.125 IU/mL of extender. The parameters evaluated include cholesterol and plasma membrane phospholipids (PMP) at fresh, while IPM, acrosomal and plasma membrane integrity, cholesterol, PMP and oxidative stress parameters like lipid peroxidation (LPO), total antioxidant capacity (TAC) and reactive oxygen species (ROS) were evaluated at pre-freeze and post-thaw stages. The IPM and acrosomal intactness were higher (p < 0.05) in treatment group at post-thaw stage in good ejaculates. Oxyrase supplementation resulted in lower (p < 0.05) cholesterol leakage in both categories and lower (p < 0.05) LPO in good ejaculates at post-thaw stage. No statistical difference in ROS was observed between control and treatment groups at all stages whereas, level of TAC was higher (p < 0.05) in the treatment group compared to control group at post-thaw stage of both categories. Therefore, Oxyrase as an oxygen scavenging agent could preserve the post-thaw quality of Sahiwal bull spermatozoa. [ABSTRACT FROM AUTHOR]