Effects of Zinc Oxide Nanoparticles on Post-Thaw Functional, Biochemical and Ultrastructural Characteristics of Cryopreserved Beetal Buck (Capra hircus) Spermatozoa.

Cryopreservation of buck semen is associated with excessive reactive oxygen species generation, oxidative stress and cellular damage, leading to reduced post-thaw sperm quality. This study evaluated the effects of zinc oxide nanoparticles (ZnO-NPs) added to a Tris-egg yolk-citric acid extender on the post-thaw functional, biochemical and ultrastructural characteristics of Beetal buck spermatozoa. Ten independent pooled ejaculates were equally divided among four treatment groups containing 0 (control), 0.05, 0.10 and 0.20 mg/mL ZnO-NPs before cryopreservation. Post-thaw sperm quality was evaluated using conventional and fluorescence-based assays, including motility, plasma membrane and acrosomal integrity, viability, DNA integrity, mitochondrial membrane potential, superoxide dismutase (SOD) activity and malondialdehyde (MDA) concentration. Transmission electron microscopy (TEM) was used to assess sperm ultrastructure in the control and 0.10 mg/mL ZnO-NP groups. ZnO-NP supplementation significantly improved post-thaw sperm motility, plasma membrane functionality, viability, acrosomal integrity, SOD activity, DNA integrity and mitochondrial membrane potential, while reducing MDA concentration compared with the control (p < 0.01). Sperm abnormalities were not significantly affected by ZnO-NP supplementation. Among the tested concentrations, 0.10 mg/mL ZnO-NPs showed the most favourable numerical profile across the evaluated parameters. TEM further indicated better preservation of the plasma membrane, acrosome, mitochondrial sheath and axonemal organization in the 0.10 mg/mL group than in the control. Overall, ZnO-NP supplementation was associated with improved post-thaw functional and ultrastructural sperm quality and a more favourable oxidative status in cryopreserved Beetal buck semen.