Zinc oxide nanoparticles alter the membrane potential of mitochondria from post-thawed ram spermatozoa

Abstract The objective of this study was to evaluate how the addition of zinc oxide nanoparticles (nano-ZnO) to freezing extender affect the characteristics of post-thawed ram semen. For this research, seminal pools from three Santa Ines rams were used. Each seminal pool was diluted in Tris-egg yolk (5 % glycerol) extender, supplemented with nano-ZnO (0, 10, 50, 100 or 200 μg/mL), at the final concentration of 200 × 106 spermatozoa/mL. Subsequently, samples were filled in 0.25-mL straws, frozen in an automated system and stored in liquid nitrogen (−196 °C). At the time of analyses, semen samples were thawed in a water bath at 37 °C for 30 s and processed for evaluation. All samples were evaluated for sperm kinetics by computer-assisted sperm analysis (CASA), as well as assessed for mitochondrial membrane potential (MMP), plasma membrane integrity (PMi) and acrosomal membrane integrity (ACi), by epifluorescence microcopy. The sperm kinetics results showed that when adding nano-ZnO (10, 50, 100 and 200 μg/mL) to freezing extender, there were no differences (P > 0.05) in post-thawed ram semen between control and treatment groups, nor among the treated groups themselves. It is emphasised that all groups treated with nano-ZnO presented more gametes (P ≤ 0.05) with high MMP than the control group. However, no differences were observed (P > 0.05) in the percentage of sperm with PMi between control and treatment groups (nano-ZnO). In conclusion, the addition of nano-ZnO (10, 50, 100 or 200 μg/mL) to freezing extender increases MMP but does not alter the kinetics or protect membranes of ram spermatozoa after the freezing-thawing process.