【作者】 刘斌；

【导师】 杨公社；

【作者基本信息】 西北农林科技大学 ， 农业推广硕士（专业学位）， 2017， 硕士

【摘要】 随着规模化养猪业发展,猪人工授精技术的普及面不断扩大,而精液保存质量的优劣决定生产效率高低,其中稀释剂发挥着至关重要的作用。精液常温保存过程中需要进行稀释,会减弱精液原有抗氧化作用,使过多活性氧无法及时清除,质膜易受被损伤,导致精子质量下降。同时,微生物的存在也会影响精液保存质量。为同时解决抗菌和抗氧化的问题,本研究在稀释液中添加原花青素(Oligomeric Proantho Cyanidins,OPC)和乳酸链球菌素(Nisin),检测精液保存质量、渗透压、总抗氧化能力、丙二醛含量、酸性磷酸酶活性和抑菌效果等,以期为OPC和Nisin在精液稀释液中的联合添加提供一些理论参考。本研究主要获得以下结果:1.猪精液稀释液中添加不同浓度(10、20、30、40、50、60、70μg/mL)OPC溶液,可优化精液保存效果。其中,稀释液中添加50μg/mL OPC效果最好,常温保存第5d的精子活率61.63%、质膜完整率41.88%、顶体完整率75.88%,显著高于对照组(P<0.05)。常温保存第5d时精子T-AOC为1.43 U/mL,高于对照组,二者差异不显著(P>0.05);MDA含量为2.37 nmol/mL,显著低于对照组(P<0.05)。另外,稀释液中添加OPC对稀释液渗透压无显著影响(P>0.05)。2.稀释液中添加不同浓度(10、20、30、40、50μg/mL)Nisin到稀释液中,能够在提高精液保存质量同时抑制细菌繁殖。添加40μg/mL Nisin组的常温保存效果最好,保存到第5d精子活率能达到60.12%,精子质膜完整率为44.98%,顶体完整率为71.78%,显著高于对照组(P<0.05)。稀释液中添加30μg/mL Nisin抑制金黄色葡萄球菌效果显著优于庆大霉素(P<0.05),抑制鲁菲不动杆菌效果与乳酸链球菌素相当(P>0.05);稀释液中添加40μg/mL Nisin抑制大肠杆菌效果显著低于庆大霉素(P<0.05)。3.猪精液稀释液中联合添加50μg/mL OPC和20μg/mL Nisin的常温保存效果优于单独添加两种物质组和对照组(P<0.05),常温保存到第5 d精子活率为62.07%,精子质膜、顶体完整率分别为56.00%和78.64%,精子线粒体活性为60.46%。第5 dOPC:Nisin=50:20组精子T-AOC为1.32 U/mL,酸性磷酸酶活性为573.34 U/L,均显著高于对照组和单独添加药物组(P<0.05);MDA含量最少,为2.36 nmol/mL,显著低于其余各组(P<0.05)。4.稀释液中添加OPC-Nisin可显著抑制金黄色葡萄球菌生长(P<0.05),且作用效果优于庆大霉素,但对大肠杆菌和鲁菲不动杆菌的抑制效果欠佳(P>0.05)。综上所述,添加原花青素和乳酸链球菌素能提高精液保存质量并延长保存时间。原花青素最适添加浓度为50μg/mL,乳酸链球菌素最适添加浓度为40μg/mL,联合添加最适浓度为50μg/mL原花青素和20μg/mL乳酸链球菌素。更多还原

【Abstract】 With the development of large-scale pig,popularization of artificial insemination techniques continues to expand,and the preservation of semen quality determines the level of production efficiency,which plays a vital role.The diluent must be diluted semen stored at room temperature in the process of weakening the original antioxidant effect of semen,excessive ROS can not be cleared in time,easy membrane by the injury,resulting in the decline in sperm quality.At the same time,the presence of microorganisms will affect the semen quality.At the same time to solve the problem of antibacterial and antioxidant,this study added procyanidins in dilute solution(Oligomeric Proantho Cyanidins,OPC)and Nisin,semen quality of preservation,osmotic pressure,total antioxidant capacity,MDA content,acid phosphatase activity and antibacterial effect,in order to OPC and Nisin in semen dilution combined with medium added to provide some theoretical reference.The main results are as follows:1.Adding different concentrations of pig semen dilution(10,20,30,40,50,60,70 μg/mL)OPC solution,which can optimize the semen preservation effect.The diluent adding 50 μg/mL OPC best,stored at room temperature fifth day sperm live rate and sperm membrane integrity rate of 61.63%,41.88%,acrosome integrity rate was 75.88%,significantly higher than the control group(P<0.05).Room temperature preservation of fifth day sperm T-AOC was 1.43 U/mL,higher than the two control group,no significant difference(P>0.05);the content of MDA was 2.37 nmol/mL,significantly lower than the control group(P<0.05).In addition,adding OPC to dilution dilution osmotic pressure had no significant effect(P>0.05).2.Different concentration dilution(10,20,30,40,50 μg/mL)Nisin to dilute solution,can improve the semen quality and inhibit bacterial reproduction.Added 40 μg/mL Nisin group normal temperature preservation effect is the best,to save fifth day sperm live rate can reach 60.12%,sperm plasma membrane integrity rate was 44.98%,acrosome integrity rate was 71.78%,significantly higher than the control group(P<0.05).Added 30 μg/mL Nisin dilution in inhibition of Staphylococcus aureus was better than gentamicin(P<0.05),Acinetobacter inhibition effect and nisin equivalent(P>0.05);rather add 40 μg/mL Nisin dilution the effect of inhibition to Escherichia coli was significantly lower than gentamicin(P<0.05).3.With the addition of 50 μg/mL OPC and 20 μg/mL Nisin pig semen dilution in the normal temperature preservation effect is better than that adding two kinds of substances group and the control group(P<0.05),stored at room temperature to fifth day sperm survival rate was 62.07% and the sperm plasma membrane,acrosome integrity rate were 56% and 78.64%,sperm mitochondrial activity 60.46 %.Fifth day OPC: Nisin=50: 20 sperm group T-AOC 1.32 U/mL,acid phosphatase activity was 573.34 U/L,significantly higher than that of control group and alone added drug group(P<0.05);MDA was the least,was 2.36 nmol/mL,significantly less than the other groups(P<0.05).4.Adding OPC-Nisin dilution can significantly inhibit the growth of Staphylococcus aureus,and the effect is better than that of gentamicin(P<0.05),but the inhibitory effect on Escherichia coli and Acinetobacter is poor(P>0.05).In conclusion,adding procyanidins and nisin can improve the semen quality of p-reservation and extend the preservationtime.The optimum concentration of proanthoc-y anidins was 50 μg/mL,the optimum concentration of nisin was 40 μg/mL,with optimum concentration was 50 μg/mL procyanidins and 20 μg/mL nisin.更多还原