海藻糖替代部分甘油对延边黄牛冻融精子质量的影响

doi:10.16431/j.cnki.1671-7236.2022.01.028

摘要/Abstract

摘要： [目的] 探讨是否可以通过补充海藻糖来降低冷冻保护剂中甘油的浓度，从而提高冻融精子的质量。[方法] 分别用6%甘油（Ⅰ组）及3%甘油+0、50、100和150 mmol/L海藻糖（Ⅱ、Ⅲ、Ⅳ和Ⅴ组）处理精子，用精子-微生物动（静）态图像检测分析系统（CASA）检测冻融精子的活力、质膜完整性和顶体完整性及动力学相关参数，筛选出最佳海藻糖处理浓度用于后续试验。通过Western blotting方法检测精子蛋白酪氨酸磷酸化水平评价精子获能状态，Hoechst 33342/PI/JC-1联合染色法检测精子的活率及线粒体膜电位，色霉素A₃（CMA₃）染色法检测精子DNA的完整性，部花青540（M540）和Yo-Pro-1染色检测精子膜脂质紊乱水平。[结果] 与Ⅰ组相比，Ⅱ组精子的活力、质膜完整性、顶体完整性以及曲线速度（VCL）和直线率（STR）均显著下降（P<0.05），Ⅲ、Ⅳ和Ⅴ组组精子的活力、VCL、直线速度（VSL）、平均路径速度（VAP）、直线性运动（LIN）和STR均显著升高（P<0.05）。因此，后续试验选用100 mmol/L海藻糖处理精子。与新鲜精子相比，冻融精子获能后其蛋白酪氨酸磷酸化的条带显著减少（P<0.05），冻融精子的活率和线粒体膜电位均显著降低（P<0.05）；冻融活精子和死精子的高度膜脂质紊乱水平均显著升高（P<0.05），100 mmol/L海藻糖可显著降低高度膜脂质紊乱水平（P<0.05）；冻融精子的鱼精蛋白缺失率显著增加（P<0.05），且Ⅱ组和100 mmol/L海藻糖组精子的鱼精蛋白缺失率显著低于Ⅰ组（P<0.05）。[结论] 海藻糖可作为一种新型冷冻保护剂降低甘油毒性，用100 mmol/L海藻糖替代部分甘油可显著改善延边黄牛冻融精子的质量。

关键词: 延边黄牛; 冻融; 海藻糖; 甘油; 精子质量

Abstract: [Objective] This study was aimed to explore whether the concentration of glycerol in cryoprotectant could be reduced by supplementing trehalose, so as to improve the quality of frozen-thawed sperm. [Method] Sperm were treated with 6% glycerol (group Ⅰ) and 3% glycerol+0, 50, 100 and 150 mmol/L trehalose (Ⅱ, Ⅲ, Ⅳ and Ⅴ groups), respectively. The motility, plasma membrane integrity, acrosome integrity and dynamic parameters of frozen-thawed sperms were detected by the dynamic (static) image detection system (CASA), and the best trehalose treatment concentration was selected for subsequent experiments. Tyrosine phosphorylation of sperm protein was detected by Western blotting, the viability and mitochondrial membrane potential of sperm were detected by Hoechst 33342/PI/JC-1 combined staining, sperm DNA integrity was detected by chromomycin A₃ (CMA₃) staining, and the level of lipid disorder in sperm membrane was detected by cyanine 540 (M540) and Yo-Pro-1 staining. [Result] Compared with Ⅰ group, sperm motility, plasma membrane and acrosomal membrane integrity, VCL (curvilinear velocity) and STR (straightness) of Ⅱ group were significantly decreased (P<0.05), the sperm motility, VCL, VSL (straight line velocity), VAP (average path velocity), LIN (linearity) and STR of Ⅲ, Ⅳ and Ⅴ groups were significantly increased (P<0.05). Therefore, 100 mmol/L trehalose was used to treat sperm in the follow-up test. Compared with fresh sperm, the protein tyrosine phosphorylation bands of frozen-thawed sperm was decreased significantly after capacitation (P<0.05), and the viability and mitochondrial membrane potential of frozen-thawed sperm was decreased significantly (P<0.05). The level of high membrane lipid disorder of frozen-thawed live sperm and dead sperm was increased significantly (P<0.05), and 100 mmol/L trehalose could significantly reduce the level of high membrane lipid disorder (P<0.05). The protamine deletion rate of frozen-thawed sperm was significantly increased (P<0.05), and the protamine deletion rate of Ⅱ and 100 mmol/L trehalose groups was significantly lower than that of Ⅰ group (P<0.05). [Conclusion] Trehalose could be used as a new cryoprotectant to reduce the toxicity of glycerol. Replacing part of glycerol with 100 mmol/L trehalose could significantly improve the sperm quality of Yanbian Yellow cattle after freezing and thawing.

Key words: Yanbian Yellow cattle; frozen-thawed; trehalose; glycerol; sperm quality

中图分类号:

S823.8

陈璇, 吕艳秋, 韩越, 徐一超, 金一. 海藻糖替代部分甘油对延边黄牛冻融精子质量的影响[J]. 中国畜牧兽医, 2022, 49(1): 256-264.

CHEN Xuan, LYU Yanqiu, HAN Yue, XU Yichao, JIN Yi. Effect of Trehalose Replacing Partial Glycerol on Frozen-thawed Sperm Quality of Yanbian Yellow Cattle[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 256-264.

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