Andrea Romina di Fonzo

Background: There are no reports evaluating the effect of antioxidant supplementation during semen cooling in llamas. This study evaluates the impact of zinc oxide nanoparticles (ZnO-NPs) on sperm ultrastructure and characteristics in cooled llama semen. Methods: Eighteen ejaculates obtained from six male llamas were divided into three aliquots and diluted with: AndroMed®-20% egg-yolk (AM-EY); AM-EY with 50 μg/ml of ZnO-NPs; and AM-EY with 100 μg/ml of ZnO-NPs. Samples were cooled to 5°C in an Equitainer®. Evaluations were carried out in raw semen, after dilution (pre-cooling; 0 h), and after 24 and 48 h of storage. Additionally, cooled samples were evaluated after 15, 90, and 180 min of incubation at 37°C. Data were analyzed using Friedman tests, conventional ANOVA, or split-plot design models, depending on the structure and distribution of the data. Results: Oscillatory sperm motility remained unchanged after 24 or 48 h of cooling (P > 0.05). Progressive sperm motility (PM) was similar between all samples cooled for 24 h and pre-cooling samples. A significant decrease in PM was observed in all cooled samples for 48 h compared to pre-cooling samples with ZnO-NPs. Total sperm motility also decreased in cooled samples, particularly after 48 h of storage. No significant differences were found in sperm membrane functionality, morphology, or DNA integrity between raw semen and samples cooled for 24 and 48 h. Regarding viability and acrosomal integrity and intact DNA, no changes were observed among the cooled samples at any of the evaluated storage times (24 and 48 h), nor compared to pre-cooling samples. Live lipoperoxidation-negative sperm decreased in all cooled semen samples compared to raw semen, showing no differences among cooled samples. Plasma membrane damage was evident in all cooled samples, more clearly seen with transmission electron microscopy. Finally, ZnO-NPs did not improve the preservation of cooled samples at 37°C for 180 min. Conclusions: The addition of ZnO-NPs at 50 or 100 μg/ml to AM-EY extenders does not fully protect llama sperm from cooling damage. Further evaluation using other concentrations may be necessary to determine the optimal dose of ZnO-NPs for cooling llama semen. Exploring other nanoparticles or antioxidants could be beneficial. Electron microscopy shows notable structural damage in all samples (with and without ZnO-NPs), highlighting the sensitivity of llama spermatozoa to cooling.