Synthesis of green zinc‐oxide nanoparticles and its dose‐dependent beneficial effect on spermatozoa during preservation: sperm functional integrity, fertility and antimicrobial activity

Abstract

Introduction: The development of an effective extender is important for semen preservation and the artificial insemination (AI) industry. This study demonstrates the beneficial effect of zinc oxide nanoparticles (ZnO-NPs) as an additive to semen extenders to improve semen quality, fertility, and antibacterial activity during liquid preservation in a boar model.Methods: Initially, to find out the safe concentration of ZnO-NPs in sperm cells, a wide range of ZnO-NP concentrations (0, 5, 10, 50, 100, 500, and 1,000 μM) were co-incubated with sperm at 37°C for a cytotoxic study. These NP concentrations were compared to their salt control zinc acetate (ZA) at the same concentrations and to a control group. The effect of the different concentrations of ZnO-NPs on sperm motility, membrane integrity, mitochondrial membrane potential (MMP), and apoptosis was assessed. Accordingly, the non-toxic dose was selected and supplemented in MODENA extender to determine its beneficial effect on the boar semen parameters mentioned and the lipid peroxidation (LPO) levels during liquid preservation at 16°C for 6 days. The non-cytotoxic dosage was subsequently chosen for AI, fertility investigations, and the evaluation of the antibacterial efficacy of ZnO-NPs during preservation hours. An antibacterial study of ZnO-NPs and its salt control at doses of 10 μM and 50 μM was carried out by the colony forming unit (CFU) method.Results and discussion: The cytotoxic study revealed that 5, 10, and 50 μM of ZnO-NPs are safe. Consequently, semen preserved in the MODENA extender, incorporating the non-toxic dose, exhibited 10 and 50 μM ZnO-NPs as the optimal concentrations for beneficial outcomes during liquid preservation at 16°C. ZnO-NPs of 10 μM concentration resulted in a significantly (p < 0.05) improved conception rate of 86.95% compared to the control of 73.13%. ZnO-NPs of 10 and 50 μM concentrations exhibit potent antimicrobial action by reducing the number of colonies formed with days of preservation in comparison to the negative control. The investigation concluded that the incorporation of 10 μM ZnO-NPs led to enhancements in sperm motility, membrane integrity, and MMP, attributed to a reduction in the malondialdehyde (MDA) levels. This improvement was accompanied by a concurrent increase in fertility rates, including farrowing rate and litter size, during the liquid preservation process. Furthermore, ZnO-NPs exhibited an antimicrobial effect, resulting in decreased bacterial growth while preserving boar semen at 16°C for 6 days. These findings suggest that ZnO-NPs could serve as a viable alternative to antibiotics, potentially mitigating antibiotic resistance concerns within the food chain.