D-异抗坏血酸钠和溶菌酶对猪精液常温保存效果的研究

发布时间：2018-02-16 11:48

本文关键词： 猪精液常温保存 D-异抗坏血酸钠溶菌酶精液保存效果　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】：精液稀释和保存是人工授精技术的关键步骤,在猪精液常温保存过程中,随着保存时间的延长,过量活性氧(ROS)在精液中开始积累,而猪精子的特殊膜结构使其更容易受到自由基的氧化损伤,最终导致精子活率下降、顶体受损甚至受精能力丧失。此外,精液中的细菌和一些病原微生物不仅会直接和精子竞争营养物质,其代谢活动的产物还会损伤精子的正常生理功能,这是精液质量下降、人工授精效果降低的重要原因。本试验通过测定精子活率、质膜完整率、顶体完整率、总抗氧化能力(T-AOC)、丙二醛(MDA)含量、活性氧(ROS)水平、精子运动性能和精液中细菌含量等指标,探究了D-异抗坏血酸钠和溶菌酶对猪精液常温保存效果的影响,并分析筛选了两种物质在猪精液常温保存基础稀释粉中的最适添加浓度,为猪精液常温保存稀释粉配方的改良提供了参考。以下为本研究主要结果:1.将不同浓度(0、0.002、0.004、0.006、0.008、0.010 mg/mL)的D-异抗坏血酸钠添加到猪精液常温保存基础稀释液中可以不同程度地改善精液保存效果。随着D-异抗坏血酸钠浓度的增加,精液的保存效果呈现先增后减的趋势。当D-异抗坏血酸钠的浓度为0.006 mg/mL时,精液有效保存时间为5d,且精子活率、质膜完整率、顶体完整率和精液总抗氧化能力(T-AOC)显著高于对照组和其他试验组(P0.05),丙二醛(MDA)含量和活性氧(ROS)水平显著低于其他组。保存第3d时,0.006 mg/mL试验组的精子运动性能参数最好,且显著优于对照组和低浓度组(0.002 mg/mL)。当D-异抗坏血酸钠的浓度为0.008 mg/mL时,精液各项指标虽然显著高于对照组(P0.05),但相比于0.006mg/mL试验组却有明显的下降趋势,所以,添加0.006 mg/mL D-异抗坏血酸钠时,精液常温保存效果最好。2.在猪精液常温保存基础稀释粉中添加不同浓度(0、0.5、1.0、1.5、2.0、2.5 mg/mL)的溶菌酶,对于猪精液的常温保存有一定的改善作用。当溶菌酶的浓度为2.0 mg/mL时,保存各天的精子活率、质膜完整率和顶体完整率都显著高于其他各组(P0.05)。保存的第1 d、第3 d和第5 d,同一天的精液中的细菌含量随着溶菌酶浓度的增加而减少,到第5d时,各组间细菌含量差异显著,当溶菌酶的浓度为2.5 mg/mL时,精液中的细菌含量显著低于其他各组(P0.05)。保存第3d,当溶菌酶浓度为2.0 mg/mL时,VSL(直线速度)、VAP(路径速度)和ALH(侧摆幅度)3项运动性能参数显著高于对照组(P0.05),CMS(曲线运动)和BCF(鞭打频率)则显著低于对照组(P0.05),其他各试验组间无显著性差异。综合对比结果表明,2.0 mg/mL为溶菌酶在猪精液常温保存稀释粉中的最适添加浓度。3.在交叉混合试验中,将0.006 mg/mL的D-异抗坏血酸钠和1.5 mg/mL的溶菌酶联合添加到猪精液常温保存基础稀释粉中能够显著提升保存后猪精子的活率和质膜完整率,且效果显著好于对照组和两种物质单独使用的试验组(P0.05)。综上,D-异抗坏血酸钠和溶菌酶对于猪精液常温保存的效果有一定的提高作用,能够有效保护精子性能,提升精液保存质量,其最适添加浓度分别为0.006 mg/mL和2.0mg/mL,联合添加的最适浓度组合为0.006 mg/mL的D-异抗坏血酸钠和1.5 mg/mL的溶菌酶。
[Abstract]:Semen dilution and preservation is a key step in artificial insemination, pig semen preservation at room temperature in the process, with the increase of the storage time, the excess reactive oxygen species (ROS) accumulation in semen, and the special membrane structure of pig sperm to make it easier to oxidative damage by free radicals, resulting in decreased sperm motility, top body damage or even loss of ability to fertilization. In addition, semen bacteria and some pathogenic microorganisms will not only directly and sperm competition of nutrients, the normal physiological function of the product will damage the sperm metabolic activities, the semen quality decline, the important reason to reduce the effects of artificial insemination. In this experiment, through the determination of sperm motility, membrane integrity rate of acrosome integrity rate, total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, reactive oxygen species (ROS) level, bacteria content in semen and sperm motility index performance, explores the D- and sodium erythorbate Effect of preservation effect of lysozyme on boar semen of normal temperature, and the analysis of the screening of two kinds of material stored in the powder based diluted the optimum concentration in normal porcine semen, provides a reference for improvement of preservation of diluted powder formula for boar semen of normal temperature. The following is the main results of this study: 1. different concentrations (0,0.002,0.004,0.006,0.008,0.010 mg/mL) D- ISO sodium ascorbate was added to the normal pig semen preservation based diluent can improve the semen preservation effect. With the increase of D- concentration of sodium erythorbate, preservation of semen showed the trend of first increase and then decrease. When D- concentration of sodium D-isoascorbate was 0.006 mg/mL, semen effective preservation time is 5D, and sperm the survival rate, plasma membrane integrity, acrosome integrity and sperm total antioxidant capacity (T-AOC) was significantly higher than the control group and the other experimental groups (P0.05), malondialdehyde (MDA) content and reactive oxygen species (ROS) The level is significantly lower than other groups. The retention of article 3D, the best sperm motion parameters of 0.006 mg/mL in the experimental group, and significantly better than the control group and low concentration group (0.002 mg/mL). When the D- concentration of sodium D-isoascorbate was 0.008 mg/mL, while the semen indexes were significantly higher than the control group (P0.05), but compared to 0.006mg/mL the test group was significantly decreased, so, adding 0.006 mg/mL D- sodium erythorbate, adding different concentrations of semen stored at room temperature the best preserved in.2. Based Diluted pig semen powder at room temperature (0,0.5,1.0,1.5,2.0,2.5 mg/mL) of lysozyme, for boar semen stored at room temperature to a certain extent. When the concentration of lysozyme was 2 mg/mL when the day of the preservation of sperm motility, plasma membrane integrity and acrosome integrity rate were significantly higher than other groups (P0.05). The preservation of first D, third D and fifth D, the same day in semen containing bacteria The amount decreases with the increasing of lysozyme concentration at 5D, the difference between groups was significant when the bacterial content, lysozyme concentration was 2.5 mg/mL, the bacterial content in semen was significantly lower than that of the other groups (P0.05). The preservation of 3D, when the concentration of lysozyme was 2 mg/mL, VSL (straight line speed (VAP). The path and ALH (speed) side swing) 3 sports performance parameters were significantly higher than the control group (P0.05), CMS (curve movement) and BCF (BCF) is significantly lower than the control group (P0.05), the other was no significant difference between each experimental group. Comparison results show that 2 mg/mL lysozyme in room temperature preservation of boar sperm dilution powder in the optimum concentration of.3. in the cross hybrid test, 0.006 mg/mL D- d-vic sodium and 1.5 mg/mL lysozyme and added to the room temperature preservation of boar sperm dilution based powder can significantly improve the preservation of pig sperm motility and membrane integrity Rate, and the effect was significantly better than the control group and the experimental group of two substances used alone (P0.05). In conclusion, D- of sodium erythorbate and lysozyme has a certain improving effect on pig semen of normal temperature preservation effect, can effectively protect the sperm performance, enhance the quality of semen preservation, the optimal concentration was 0.006 mg/mL and 2.0mg/mL, in combination with the optimal concentration combination of 0.006 mg/mL D- sodium erythorbate and 1.5 mg/mL respectively.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

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