Abstract The aim of this study was to evaluate the effect of ascorbic acid (Vitamin C) and 2-mercaptoethanol (ME) added to buffalo semen samples after thawing for 1 h and 2 h incubation. Ejaculates (n = 20) from eight Bulgarian Murrah buffaloes were frozen in pellets and straws at a final sperm concentration of 80 × 106 mL. We investigated the effect of supplementations on the motility, morphology of spermatozoa at 1 h and 2 h after thawing, sperm plasma membrane status and mitochondrial trans-membrane potential. The comparative CASA analysis of the motility and velocity parameters of spermatozoa showed significant differences in the progressive motility and in the VCL values in favour of control vs. samples with additives (p < 0.05). The molecular changes in PM phospholipid asymmetry were higher after thawing (p < 0.05). The semen treated with Vit C showed significant (p < 0.05) lower number of apoptotic spermatozoa (6CF+/Ann V Cy3+) in comparison with samples incubated with ME. The numbers of dead spermatozoa (6CF-/Ann V Cy3+) in samples with ME were significantly higher than other groups. The percentage of spermatozoa with well preserved and functioning mitochondria in control semen was higher (p < 0.05) than that in the experimental group with ME. The results obtained revealed that addition of Vit C (5 µmol/L) and ME (50 µmol/L) has no positive effect on post-thaw semen quality of buffalo sperm, because of the impaired integrity of PM and mitochondrial trans-membrane potential.

中文翻译：

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用抗氧化剂维生素 C 和 2-巯基乙醇对冷冻水牛精液进行解冻后处理

摘要 本研究的目的是评估抗坏血酸（维生素 C）和 2-巯基乙醇（ME）在解冻 1 小时和 2 小时温育后添加到水牛精液样品中的效果。来自八头保加利亚墨拉水牛的精液（n = 20）被冷冻在颗粒和吸管中，最终精子浓度为 80 × 106 mL。我们研究了补充剂对解冻后 1 小时和 2 小时精子的运动性、形态、精子质膜状态和线粒体跨膜电位的影响。精子运动性和速度参数的比较 CASA 分析表明，在渐进运动性和 VCL 值方面有显着差异，有利于对照与含有添加剂的样品 (p < 0.05)。PM 磷脂不对称的分子变化在解冻后更高（p < 0.05）。与用 ME 孵育的样品相比，用 Vit C 处理的精液显示出显着 (p < 0.05) 较低的凋亡精子数量 (6CF+/Ann V Cy3+)。ME样本中死亡精子数（6CF-/Ann V Cy3+）显着高于其他组。对照精液中线粒体保存完好且功能正常的精子百分比高于 ME 实验组（p < 0.05）。所得结果表明，添加 Vit C (5 µmol/L) 和 ME (50 µmol/L) 对水牛精子解冻后精液质量没有积极影响，因为 PM 的完整性和线粒体跨膜电位受损. ME样本中死亡精子数（6CF-/Ann V Cy3+）显着高于其他组。对照精液中线粒体保存完好且功能正常的精子百分比高于 ME 实验组（p < 0.05）。所得结果表明，添加 Vit C (5 µmol/L) 和 ME (50 µmol/L) 对水牛精子解冻后精液质量没有积极影响，因为 PM 的完整性和线粒体跨膜电位受损. ME样本中死亡精子数（6CF-/Ann V Cy3+）显着高于其他组。对照精液中线粒体保存完好且功能正常的精子百分比高于 ME 实验组（p < 0.05）。所得结果表明，添加 Vit C (5 µmol/L) 和 ME (50 µmol/L) 对水牛精子解冻后精液质量没有积极影响，因为 PM 的完整性和线粒体跨膜电位受损.