The objective of this study was to compare the effects of different concentrations of two different cryoprotectants (glycerol, G and ethylene glycol, EG) and trehalose (T), added to the semen extender, on post-thaw ram sperm parameters. Ejaculates, collected from 6 Merino rams, were pooled and evaluated at 37 °C. The pooled samples were divided into six equal aliquots, and diluted in Tris-based extenders containing 5% G, 3% G + 60 Mm T, 1.5% G + 100 Mm T, 5% EG, 3% EG + 60 mM T, and 1.5% EG + 100 Mm T. Subsequently, the samples were cooled to 5 °C, frozen in 0.25-ml French straws, and stored in liquid nitrogen (LN2). Frozen samples were thawed individually, at 37 °C for 25 s in a water bath, for evaluation. Sperm motility was assessed using a phase-contrast microscope with a warm stage maintained at 37 °C. Acrosome integrity (FITC/PNA-PI), sperm viability (SYBR-14/PI), mitochondrial activity (JC-1/PI), DNA damage (COMET assay) and DNA fragmentation (TUNEL test) were determined. The group of samples diluted in an extender containing 5% of glycerol (Group 5% G) displayed higher percentages of subjective motility, viability and mitochondrial activity of sperm, compared to the other groups (P < 0.05). On the other hand, Group 3% G + 60 mM T yielded the second-best results for subjective motility, viability and mitochondrial activity of sperm, when compared to the other groups. The post-thaw sperm parameters of Group 3% G + 60 Mm T did not show any statistically significant difference from those of Group 5% G. There were no statistically significant differences between the groups for acrosome integrity (P > 0.05). The results of the COMET assay showed that the use of low concentrations of cryoprotectants in combination with trehalose decreased sperm DNA damage. Accordingly, Group 1.5% G + 100 mM T and Group 3% EG + 60 mM T benefited from a significantly stronger cryoprotective effect on DNA integrity, in comparison to Group 5% G (P < 0.05). According to the results of the TUNEL test, the combined use of low concentrations of cryoprotectants with trehalose decreased sperm DNA damage, when compared to the use of 5% glycerol, but this difference was statistically insignificant (P > 0.05). In conclusion, G and EG concentrations can be reduced by adding various amounts of T (60 mM, 100 mM) to the semen extender. The addition of 5% of glycerol and 3% G + 60 mM T to the semen extender did not yield statistically different post-thaw sperm parameters, when compared for protection against cryoinjury. Post-thaw sperm parameters can be improved by the supplementation of the semen extender with 3% G + 60 mM T. Thus, we recommend the use of freezing extenders containing low cryoprotectant concentrations (3% G) combined with trehalose to avoid the high level of toxic and osmotic damage caused by 5% G.

中文翻译：

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海藻糖和低浓度冷冻保护剂的协同作用可改善解冻后公羊精子参数

本研究的目的是比较不同浓度的两种不同冷冻保护剂（甘油，G 和乙二醇，EG）和海藻糖 (T)，添加到精液补充剂中，对解冻后公羊精子参数的影响。从 6 只美利奴公羊收集的精液在 37 °C 下汇集并评估。将合并的样品分成六等分，并在含有 5% G、3% G + 60 Mm T、1.5% G + 100 Mm T、5% EG、3% EG + 60 mM T 的基于 Tris 的增量剂中稀释，和 1.5% EG + 100 Mm T。随后，将样品冷却至 5°C，在 0.25-ml 法国吸管中冷冻，并储存在液氮 (LN2) 中。冷冻样品在水浴中于 37 °C 下单独解冻 25 秒，以进行评估。使用相位对比显微镜评估精子活力，温台保持在 37°C。顶体完整性 (FITC/PNA-PI)，测定了精子活力 (SYBR-14/PI)、线粒体活性 (JC-1/PI)、DNA 损伤（COMET 试验）和 DNA 断裂（TUNEL 试验）。与其他组相比，在含有 5% 甘油的稀释剂中稀释的样品组（组 5% G）显示出更高百分比的精子主观运动性、活力和线粒体活性（P < 0.05）。另一方面，与其他组相比，3% G + 60 mM T 组在精子的主观运动性、活力和线粒体活性方面产生了第二好的结果。3% G + 60 Mm T组的解冻后精子参数与5% G组的精子参数没有显示出任何统计学上的显着差异。在顶体完整性方面，各组之间没有统计学上的显着差异（P > 0.05）。COMET 分析的结果表明，低浓度冷冻保护剂与海藻糖的结合使用可减少精子 DNA 损伤。因此，与 5% G 组相比，1.5% G + 100 mM T 组和 3% EG + 60 mM T 组受益于对 DNA 完整性的显着更强的冷冻保护作用（P < 0.05）。根据 TUNEL 试验结果，与使用 5% 甘油相比，低浓度冷冻保护剂与海藻糖联合使用可减少精子 DNA 损伤，但这种差异无统计学意义（P > 0.05）。总之，G 和 EG 的浓度可以通过向精液补充剂中添加不同量的 T（60 mM、100 mM）来降低。向精液稀释剂中添加 5% 的甘油和 3% G + 60 mM T 不会产生统计学上不同的解冻后精子参数，与防止冷冻损伤相比。解冻后精子参数可以通过补充 3% G + 60 mM T 的精液稀释剂来改善。 因此，我们建议使用含有低冷冻保护剂浓度 (3% G) 的冷冻稀释剂与海藻糖相结合，以避免高水平5% G 引起的毒性和渗透性损害。