Effects of Zinc oxide nanoparticles on llama sperm cryopreservation

Zinc oxide nanoparticles (ZnO-NPs) have been reported to enhance sperm cryopreservation in several species, but their effect on frozen llama semen remains unexplored. This study aims to evaluate the effect of ZnO-NPs on sperm parameters in frozen/thawed llama semen. Fourteen ejaculates, obtained from seven male llamas, were each divided into three equal aliquots. Each aliquot was diluted with 1. AndroMed® with 20 % egg yolk and no ZnO-NPs (AM-EY0, Control); 2. AM-EY supplemented with 50 μg/ml of ZnO-NPs (AM-EY50) and 3. AM-EY supplemented with 100 μg/ml of ZnO-NPs (AM-EY100). Freezing was performed using an automatic machine. Evaluations were carried out on raw semen, immediately after dilution (0 h), and following the freezing/thawing process. Additionally, frozen/thawed samples were incubated at 37 °C and assessed at 15, 90, and 180 min. Data were analysed using Friedman tests, conventional ANOVA, or split-plot design models. The results showed no significant differences in sperm motility patterns, live sperm with intact acrosomes, membrane function, lipid peroxidation, sperm morphology, or DNA integrity among frozen/thawed groups (P > 0.05). A trend toward higher sperm vigour was detected in frozen/thawed samples cryopreserved with 50 and 100 μg/ml of ZnO-NPs (P = 0.08). Moreover, ZnO-NPs did not enhance sperm survival during post-thaw incubation at 37 °C for up to 180 min. In conclusion, supplementation of the AM-EY extender with 50 or 100 μg/ml ZnO-NPs did not provide broad protection against cryodamage in llama sperm. Further studies testing a wider range of concentrations are needed to assess their potential benefits for sperm cryopreservation in this species.