Nano-depletion of morbid spermatozoa up-regulate Ca2+ channel, depolarization of membrane potential and fertility in buffalo

Abstract

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.