鞣花酸对猪精液常温保存效果的研究

发布时间：2018-03-01 20:06

  本文关键词： 猪精液 鞣花酸 常温保存 氧自由基 冷打击　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：猪的人工授精技术是猪规模化养殖的关键技术之一,而精液保存质量则是保证人工授精成功率与母猪产仔数的关键因素之一。猪精液体外保存的方式有三种:即常温保存、低温和冷冻保存。由于猪精子细胞膜的胆固醇/磷脂较低,精子对于温度相对敏感,对低温的耐受能力较差,因此,在生产实践中多采用猪精液常温保存技术保存精液。然而,在常温保存过程中精子细胞内会产生过量的氧自由基(ROS),对精子质膜和顶体完整性造成氧化损伤,使精子的运动能力和活力降低,导致精液保存品质和人工授精成功率降低。为了降低ROS对于精子的损伤,提高精液保存质量,为此本研究向猪精液常温保存稀释液Modena中添加了一种天然植物提取物抗氧化剂——鞣花酸。本研究利用计算机辅助精子质量分析仪(CASA)、荧光显微镜、多功能酶标仪等仪器,检测鞣花酸对大白猪精液常温保存效果的影响并探究其作用途径;同时研究了鞣花酸对于猪精子冷打击的保护作用;以期减缓猪精液保存过程中产生的氧化应激、延长精液有效保存时间,同时为我国精液保存抗氧化剂的研发提供科学资料。本研究获得如下重要结果:1.将Modena稀释液分为6组分别添加不同浓度的鞣花酸,1:1稀释精液(鞣花酸终浓度为:0、15、25、35、50、75μmol/L),并检测保存1、3、5、7、9 d的精子活、质膜及顶体的完整情况,结果发现:15、25μmol/L的鞣花酸均能提高精子的保存质量;而且,当鞣花酸添加浓度为25μmol/L时,保存7d的精子活力为66.63%,质膜完整率为84.05%、顶体完整率为87.07%,均显著优于其他处理组(P0.05);精子的快速前进行性运动率(PRO)、平均直线运动速率(VSL)和精子平均路径速度(VAP)显著高于对照组(P0.05);此外,精子平均曲线运动速率(VCL)与对照组相比无显著差异(P0.05)。这些结果表明,鞣花酸对于猪精液常温保存具有保护作用,最佳添加浓度为25μmol/L。2.检测常温保存7d后精子细胞内ROS、脂质过氧化水平和总抗氧化能力,发现25μmol/L鞣花酸处理组的ROS和脂质过氧化水平最低,总抗氧化能力最高,且与对照组相比结果显著(P0.05)。提示鞣花酸能够通过清除精子细胞内ROS、降低脂质过氧化水平和提高总抗氧化能力的途径来保护精子,缓解常温保存过程中精子的氧化损伤。3.在猪精子由体温快速降温至5℃的过程中,发现猪精子活力大幅下降,在精子遭受冷打击时,鞣花酸对精子活力的影响与对照组相比结果不显著,表明鞣花酸保护猪精子免受冷打击损伤的作用不显著。综上,本研究证明在猪精液常温保存过程中添加鞣花酸能够通过清除精子细胞内的活性氧自由基,降低脂质过氧化水平和提高总抗氧化能力等作用途径,来提高大白猪精液常温保存的质量,且在猪精液稀释液Modena的最佳添加浓度为25μmol/L。此外,当精子遭受冷打击时,鞣花酸对其保护结果不显著。
[Abstract]:Artificial insemination is one of the key techniques in large-scale pig breeding, and the quality of semen preservation is one of the key factors to ensure the success rate of artificial insemination and the litter size of sows. Cryopreservation and cryopreservation. Because of the low cholesterol / phospholipid ratio of porcine sperm cell membrane, the sperm is sensitive to temperature and has poor tolerance to low temperature. During normal temperature preservation, excessive oxygen free radicals will be produced in sperm cells, which will cause oxidative damage to the integrity of sperm plasma membrane and acrosome, and decrease the motility and motility of spermatozoa. In order to reduce the damage of ROS to sperm and improve the quality of semen preservation, In this study, a natural plant extract antioxidant, tannic acid, was added to porcine semen at room temperature preservation diluent (Modena). A computer-assisted sperm quality analyzer (CASAA), fluorescence microscope, and multifunction enzyme marker were used in this study. The effects of tannic acid on cryopreservation of pig semen at room temperature and the protective effect of tannic acid on cold attack of pig sperm were studied in order to slow down the oxidative stress during the preservation of pig semen. Prolong the effective preservation time of semen, At the same time, it provided scientific data for the research and development of antioxidants for semen preservation in China. In this study, the following important results were obtained: 1: 1.The diluents of Modena were divided into 6 groups, which were diluted with different concentrations of tannic acid and 1: 1 respectively. The sperm motility was detected after the storage of 1 ~ (3) C ~ (5) C _ (7) C ~ (2 +) for 9 days. The integrity of plasma membrane and acrosome was obtained. The results showed that tannic acid at 25 渭 mol/L could improve the quality of spermatozoa, and when the concentration of tannic acid was 25 渭 mol/L, the quality of spermatozoa could be improved when the concentration of tannic acid was 25 渭 mol/L. The sperm motility, plasma membrane integrity and acrosomal integrity were 66.63%, 87.07%, 87.07% and 87.07%, respectively, which were significantly better than those of other treatment groups (P 0.05%), and the rate of progressive sperm motility before rapid progression was significantly higher than that of the other treatment groups (P < 0.05). The average linear motion rate (VSLL) and the average sperm path velocity (VAPs) were significantly higher than those in the other treatment groups. In addition, in the control group, P0.05, There was no significant difference in sperm mean curve motility rate (VCLL) compared with the control group (P 0.05). These results showed that tannic acid had protective effect on the preservation of porcine semen at room temperature. The best addition concentration was 25 渭 mol / L ~ (-2). After 7 days of storage at room temperature, ros, lipid peroxidation and total antioxidant capacity of spermatozoa were measured. The results showed that 25 渭 mol/L tannic acid treatment group had the lowest level of ROS and lipid peroxidation, and the highest total antioxidant capacity. Compared with the control group, the results showed that tannin could protect spermatozoa by scavenging ROSin, decreasing lipid peroxidation and increasing total antioxidant capacity. Alleviating oxidative damage of spermatozoa during normal temperature preservation. During the rapid cooling of porcine spermatozoa from body temperature to 5 鈩，

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