Quercetin-coated CuO–ZnO nanoparticles enhance post-thaw quality of ram semen

Excessive production of reactive oxygen species (ROS) during freezing and thawing negatively impacts sperm quality and subsequent fertilization capacity. The properties of nanoparticles (with antioxidant properties) have recently gained considerable attention in livestock due to their ability to enhance sperm longevity and improve male fertility. Therefore, this study aimedto investigate the effect of adding Quercetin (Quer), a combination of copper chloride and zinc chloride, CuO-NPs doped with ZnO-NPs (NCu-do-NZn), and CuO-NPs doped with ZnO-NPs coated with Quercetin (NCu-do-NZn-co-Quer), on ram sperm quality under post-thaw conditions. Doped nanoparticles were purchased, and Quer was loaded onto them in the laboratory. The characteristics of the nanoparticles were determined using EDX, FE-SEM, UV-visible, FT-IR, and zeta potential. Mixed ejaculates from four rams were diluted with a cryopreservation extender. Different concentrations of treatments (1, 5, 25, and 125 µg/mL) were added to the ram sperm freezing extender. The control group was considered the group without any treatment. The semen, diluted and enriched with the above treatments, was gradually cooled to 4 °C over 4 h, then drawn into 0.25 mL straws and frozen in liquid nitrogen for storage. Sperm parameters, such as viability, total motility, membrane and DNA integrity, total abnormalities, and malondialdehyde levels, were assessed in the different groups. The results showed that adding 5 µg/mL NCu-do-NZn-co-Quer significantly improved sperm viability (74.86 ± 0.75), total motility (71.79 ± 1.07), membrane functionality (72.99 ± 1.17), and DNA integrity (95.79 ± 0.98), and reduced malondialdehyde levels (21.89 ± 0.17) compared to all other groups (p < 0.05). The addition of 5 µg/mL NCu-do-NZn-co-Quer (11.83 ± 0.71) also significantly reduced abnormalities compared to all other groups, except for the 25 µg/mL Quer group (p < 0.05). Therefore, adding 5 µg/mL NCu-do-NZn-co-Quer as an antioxidant to the ram semen extender can improve sperm tolerance to freezing and enhance sperm stability after thawing.