The Effect of Adding Green Zinc Oxide Nanoparticles to Ram Semen Dilution Medium and Its Effects on Sperm Quality and Microbial Load of Frozen Semen

Introduction: Maintaining sperm quality and reducing contaminants will ensure the success of fertilization. The presence of bacterial contamination in the reproductive system of animals and their transfer to the semen causes a decrease in sperm quality and problems of disease transmission through inseminated. During the process of freezing and thawing, cold shock as well as environmental microbial pollution causes sperm quality to decrease. Zinc oxide nanoparticles, in addition to increasing the antioxidant capacity, also have antibacterial effects. The sperm membrane of ruminants are rich in unsaturated fatty acids and Zinc is as a cofactor of biological antioxidant biomolecules that suggested zinc increases protecting cell membrane and other inside organelles. Conventional chemical methods are expensive and require the use of chemical compounds/ solvents as reducing agents, which are toxic and as a risk factor for environment. Green chemistry reduces the risk of pollution at the source level, and instead of producing waste, it can use waste materials as a source of producing nanoparticles. This technology focuses on selecting reactions (for example biological molecules such as polyphenols, flavonoids, carbonyl and protein compounds) that are compatible with the environment.Materials and Methods: In this research, green zinc oxide (gZnO) nanoparticles are produced from saffron petals. Sperms were collected from 6 Afshari rams and after checking and confirming the quality, the sperms were mixed together in a 4x4 factorial design with 4 levels of antibiotics (0, 50, 75 and 100) recommended percentage and 4 levels of gZnO (0, 7.5, 10 and 12.5) μg/ml were added to the each diluent and entered to the freezing process. In order to check the quality of sperm after freezing CASA system were used. Membrane functionality was measured using osmotic method. The total microbial load was estimated using the colony count on blood-agar medium method.Results and Discussion: The results showed that 12.5 µg/ml level of gZnO nanoparticles becomes destructive and reduced quality of sperm, but the lower levels of gZnO significantly (P<0.05) increased the quality of sperm motility.  Rapid progressive motility, progressive motility and total motility were significantly increased at 7.5 and 10 μg/L levels compared to other levels (P<0.05).  Also, the addition of 7.5 μg/ml of gZnO significantly improved the membrane integrity performance compared to other levels (P<0.05). Antibiotic and gZnO significantly decreased microbial loaded (P<0.01). The strong positive correlation between zinc and both total motility and progressive motility is attributed to zinc's high antioxidant power. Zinc reduces the production of reactive oxygen species and lipid peroxidation, thereby protecting sperm cells from damage. Zinc nanoparticles stabilize the peroxidation of membrane lipids and increase the mitochondrial and functional activity of sperm without having a negative effect on sperm motility parameters. Concentrations higher than 10 μg/ml of gZnO nanoparticles had toxic effects on sperm, which has been noted by other researchers. The use of zinc oxide nanoparticles reduces the amount of antibiotics needed in the production and processing of frozen sperm, reduces the cytotoxicity of both substances, greatly reduces the amount of antibiotic consumption, and increases the antimicrobial effects. The synergistic effects of zinc oxide nanoparticles and antibiotics have recently been given great importance, and in various researches, conjugated antibiotics have been invented. It was shown that the combination of zinc oxide nanoparticles and ampicillin increased the antibiotic power six times and greatly reduced the need for antibiotics.The addition of zinc oxide nanoparticles to the diluent caused a significant increase in the quality of frozen-thawed sperm, so that the levels of 7.5 and 10 mg/μL had the greatest improvement in quality after freezing and thawing. The use of concentrations of 7.5 and 10 μg/ml of gZnO nanoparticles along with reducing the consumption of antibiotics to half of the recommended amount has increased the quality of frozen sperm after thawing, reduced antibiotic resistance and finally reduced purchase cost of antibiotics in sperm production centers. studies show that the use of nanoparticles and antibiotics together not only reduced the toxicity of both substances on human cells, but also increased the effectiveness of antibiotics effects. Even the simultaneous use of nanoparticles and antibiotics caused the reversal of antibiotic resistance. Nanoparticles increase the antibiotic concentration at the site of antibiotic-bacterial activity and increase the binding of the antibiotic with the microorganism.