The objective of this study was to evaluate how the addition of zinc oxide nanoparticles (nano-ZnO) to freezing extender affect the characteristics of post-thawed ram semen. For this research, seminal pools from three Santa Inês rams were used. Each seminal pool was diluted in Tris-egg yolk (5 % glycerol) extender, supplemented with nano-ZnO (0, 10, 50, 100 or 200 μg/mL), at the final concentration of 200 × 10⁶ spermatozoa/mL. Subsequently, samples were filled in 0.25-mL straws, frozen in an automated system and stored in liquid nitrogen (−196 °C). At the time of analyses, semen samples were thawed in a water bath at 37 °C for 30 s and processed for evaluation. All samples were evaluated for sperm kinetics by computer-assisted sperm analysis (CASA), as well as assessed for mitochondrial membrane potential (MMP), plasma membrane integrity (PMi) and acrosomal membrane integrity (ACi), by epifluorescence microcopy. The sperm kinetics results showed that when adding nano-ZnO (10, 50, 100 and 200 μg/mL) to freezing extender, there were no differences (P > 0.05) in post-thawed ram semen between control and treatment groups, nor among the treated groups themselves. It is emphasised that all groups treated with nano-ZnO presented more gametes (P ≤ 0.05) with high MMP than the control group. However, no differences were observed (P > 0.05) in the percentage of sperm with PMi between control and treatment groups (nano-ZnO). In conclusion, the addition of nano-ZnO (10, 50, 100 or 200 μg/mL) to freezing extender increases MMP but does not alter the kinetics or protect membranes of ram spermatozoa after the freezing-thawing process.

本研究的目的是评估向冷冻填充剂中添加氧化锌纳米颗粒（纳米 ZnO）如何影响解冻后的公羊精液的特性。在这项研究中，使用了来自三只 Santa Inês 公羊的精液池。每个精液池用 Tris-蛋黄（5% 甘油）稀释剂稀释，并添加纳米 ZnO（0、10、50、100 或 200 μg/mL），终浓度为 200 × 10 ⁶精子/毫升。随后，将样品装入 0.25 毫升的吸管中，在自动化系统中冷冻并储存在液氮 (-196 °C) 中。在分析时，将精液样品在 37°C 的水浴中解冻 30 秒并进行处理以进行评估。通过计算机辅助精子分析 (CASA) 评估所有样本的精子动力学，并通过荧光显微镜评估线粒体膜电位 (MMP)、质膜完整性 (PMi) 和顶体膜完整性 (ACi)。精子动力学结果表明，在冷冻补充剂中加入纳米ZnO（10、50、100和200 μg/mL）后，对照组和治疗组之间以及在解冻后的公羊精液中均无差异（P > 0.05）。治疗组本身。需要强调的是，所有用纳米 ZnO 处理的组都呈现出更多的配子（P ≤ 0. 05) MMP 比对照组高。然而，在对照组和治疗组（纳米氧化锌）之间，PMi 的精子百分比没有观察到差异（P > 0.05）。总之，将纳米 ZnO（10、50、100 或 200 μg/mL）添加到冷冻补充剂中会增加 MMP，但不会改变冻融过程后公羊精子的动力学或保护膜。