冷冻保存对公猪精子功能的影响

doi:10.16431/j.cnki.1671-7236.2021.05.021

摘要/Abstract

摘要： 试验旨在探究公猪精液冷冻保存对其精子功能的影响。取长白猪的鲜精和优质冻精，用精子分析仪检测精子的运动能力，台盼蓝染色检测精子活率，体外受精（IVF）试验检测卵裂率与囊胚率，采用不同功能检测试剂盒检测冻精和鲜精的顶体完整率、线粒体膜通道孔（MPTP）活性、线粒体膜电位（MMP）、线粒体活性、线粒体氧化应激活性氧（ROS）以及精子DNA完整性，实时荧光定量PCR检测弱精子症相关蛋白基因SMCP、TEKT3、DNAH1、TCTE3的表达。结果表明，与猪鲜精相比，猪冻精的活率及活力均显著降低（P<0.05），冻精的顶体完整率也明显下降（P<0.05）；冻精的卵裂率和囊胚率显著低于鲜精（P<0.05）；精子线粒体功能分析结果显示，冻精的MPTP相对荧光单位值（RFU）、线粒体膜电位荧光比率以及线粒体活性光密度（OD）值均显著低于鲜精（P<0.05）；精子线粒体ROS检测发现，冻精的RFU值显著高于鲜精（P<0.05）；精子DNA完整性检测结果显示，冻精拖尾率显著高于鲜精（P<0.05）；而弱精子症相关蛋白基因的表达与鲜精相比，差异不显著（P>0.05）。综上所述，冷冻导致猪精子活率、活力、线粒体功能、DNA完整性下降，最终使得冷冻精液精子的受精能力降低。

关键词: 公猪; 冷冻精液; 精子线粒体; 精子DNA完整性; 受精能力

Abstract: To investigate the effect of cryopreservation on function of landrace sperm,fresh and frozen semen were selected as research materials.Sperm motility was detected by sperm analyzer.Sperm viability was detected by Trypan blue staining.The cleavage rate and blastocyst rate were measured by in vitro fertilization (IVF).The acrosome integrity rate,mitochondrial permeability transition pore (MPTP) activity,mitochondrial membrane potential (MMP),mitochondrial activity and reactive oxygen species (ROS),and integrity of sperm were measured using different kits.Expression levels of asthenospermia related genes SMCP, TEKT3,DNAH1 and TCTE3 were measured by Real-time quantitative PCR.The results showed that the motility,viability and acrosome integrity rate of frozen sperm were significantly lower than that of fresh sperm (P<0.05).The cleavage rate and blastocyst rate of frozen sperm decreased significantly compared with fresh sperm (P<0.05).The results of mitochondrial function analysis showed that the relative fluorescence units (RFU) value of MPTP,optical density (OD) value of mitochondrial activity and mitochondrial fluorescence ratio of MMP in frozen sperm were significantly lower than that of fresh sperm (P<0.05).The detection of ROS in sperm mitochondria showed that the RUF value of ROS in frozen sperm was significantly higher than that of fresh sperm (P<0.05).DNA integrity test results showed that the sperm tail dragging rate of frozen sperm increased significantly compared with fresh sperm (P<0.05).However,the genes expression of asthenospermia related proteins had no significant difference compared with fresh sperm (P>0.05).In conclusion,cryopreservation decreased the motility,viability,mitochondrial function and DNA integrity of boar sperm,resulting in the decrease of the fertilization ability of frozen sperm.

Key words: boar; frozen semen; sperm mitochondria; sperm DNA integrity; fertilization ability

中图分类号:

S814

郝肖琼, 武占营, 崔艳颖, 史秀杰. 冷冻保存对公猪精子功能的影响[J]. 中国畜牧兽医, 2021, 48(5): 1699-1706.

HAO Xiaoqiong, WU Zhanying, CUI Yanying, SHI Xiujie. Effects of Cryopreservation on the Function of Boar Sperm[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(5): 1699-1706.

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