Razi University

   The freeze–thaw process of ram sperm, despite its numerous advantages, can induce oxidative stress, leading to damage to the sperm plasma membrane and DNA and consequently reducing post-thaw sperm quality. The use of antioxidants and nanotechnology has been proposed as an effective strategy to reduce cryopreservation-induced sperm damage. The aim of the present study was to investigate the effects of different concentrations of manganese-doped zinc oxide nanoparticles, either alone or coated with vanillic acid, on the post-thaw quality parameters of Sanjabi ram sperm. Manganese-doped zinc oxide nanoparticles were synthesized at a Zn:Mn ratio of 90:10 using barberry fruit extract through a green synthesis method and were subsequently coated with vanillic acid at a 1:1 ratio. The nanoparticles were characterized using Fourier-transform infrared spectroscopy (FTIR), energy-dispersive X-ray spectroscopy (EDX), field-emission scanning electron microscopy (FE-SEM), dynamic light scattering (DLS), and zeta potential analysis. The antioxidant activity of the materials was also evaluated using the DPPH assay. Following preliminary screening of different concentrations based on sperm motility and viability at 4 and 37°C, vanillic acid at 1 and 10 µg/mL, manganese-doped zinc oxide nanoparticles at 0.1 and 1 µg/mL, and vanillic acid-coated manganese-doped zinc oxide nanoparticles at 0.01 and 0.1 µg/mL were selected for the freezing experiment. Semen samples were diluted with a Tris–egg yolk extender, packaged in 0.25-mL straws, and subsequently frozen in liquid nitrogen. After thawing, sperm motility, viability, plasma membrane integrity, morphological abnormalities, DNA integrity, and malondialdehyde (MDA) levels were evaluated. Data were analyzed in a completely randomized design using    software, and treatment means were compared using Duncan’s multiple range test at a significance level of 5%. The results showed that 0.1 µg/mL vanillic acid-coated manganese-doped zinc oxide nanoparticles provided the best overall performance among the tested treatments. This treatment significantly increased sperm motility, viability, and plasma membrane integrity and reduced sperm abnormalities compared with the control group. DNA integrity was also improved compared with the control group and some treatments; however, no significant difference was observed between this treatment and some of the nanoparticle-treated groups. MDA levels were significantly reduced in this treatment compared with the control group and most treatment groups, although no significant difference was observed compared with 10 µg/mL vanillic acid. Vanillic acid at 10 µg/mL and manganese-doped zinc oxide nanoparticles at 0.1 µg/mL also improved several sperm quality parameters. In contrast, the concentration of 100 µg/mL was associated with reduced sperm motility and viability and showed the greatest adverse effects. Based on the results of this study, the use of vanillic acid-coated manganese-doped zinc oxide nanoparticles at 0.1 µg/mL may be considered a potential treatment for reducing freeze–thaw-induced damage in ram sperm. However, further studies are required to determine the precise mechanisms underlying these effects, including the roles of antioxidant systems and molecular pathways. Keywords: Manganese-doped zinc oxide nanoparticles, vanillic acid, freeze–thawing, ram sperm