猪精子在液态保存过程中果糖代谢的研究
发布时间：2018-07-30 08:50
【摘要】：用液态稀释液延长猪精液的保存时间对猪人工授精的推广及猪优良种畜的繁育非常关键,但是用稀释液保存精子的过程中会遇到一些问题:稀释液中添加成分及添加量的多样性;精子在保稀释液中存活时间较短;受精率低等问题。解决精液液态保存过程中保存效果不佳可从两个方面出发,一是精子在代谢过程中的能量供应,二是及时清除掉精子在代谢过程中生成的不利物质。然而,在猪精液的液态保存过程中关于射精精子在体外长时间培养期间的糖代谢研究尚少,不同哺乳动物精子PPP途径是否存在,三羧酸循环是否必需,并没有明确的结论。因此,对猪精液液态保存过程中精子果糖代谢途径的研究无论对于丰富精子能量代谢基础理论,还是对于提高精液液态保存时间、提高精子活力、扩大猪人工授精的推广,提供了有利的理论支撑和重要的实践意义。本试验以不含任何能量物质的磷酸缓冲盐溶液(PBS)为保存稀释液,通过添加果糖和糖代谢中间产物以及糖酵解、PPP途径和电子传递的抑制剂,在15℃下液态保存猪精液不同时间检测精子活力、活率、顶体完整率、DNA损伤率、抗氧化能力与线粒体膜电位高低的变化等指标,研究猪精子在稀释液中液态保存过程中对果糖的利用情况和代谢途径,并以此为基础,研制改进精子能量代谢的保存体系,延长猪精液液态保存时间。结果发现:(1)在15℃下,在PBS保存稀释液中添加3mM的果糖对精子的保存最有利,精子活力保持30%以上的保存时间达到5天。(2)在15℃下,在PBS保存稀释液中添加5mM的丙酮酸对精子的保存最有利,精子活力保持30%以上的保存时间达到5天。(3)在15℃下,在PBS保存稀释液中添加3mM果糖作为对照组,对比组在此基础上添加碘醋酸钠(糖酵解途径抑制剂),发现对比组精子活力显著降低、精子保存时间显著缩短、精子线粒体功能完整性显著下降。(4)在15℃下,在PBS保存稀释液中添加3mM果糖作为对照组,对比组在此基础上添加6-氨基烟酰胺(磷酸戊糖途径抑制剂),发现对比组精子活力与保存时间无显著变化,但精子的抗氧化能力却显著下降。(5)在PBS保存稀释液中加入3mM果糖作为对照组,对比组在此基础上添加鱼藤酮(电子传递链的抑制剂),精子活力、保存时间显著下降通过以上实验结果本研究得出以下结论:1)在15℃下,PBS保存稀释液中添加3mM的果糖有利于猪精子的存活,添加过量的果糖会减少猪精子的体外保存时间2)猪精子不但可以利用果糖进行代谢,还能利用糖代谢过程中的中间产物用丙酮酸。3)糖酵解途径是维持猪精子活力、保持线粒体功能不受影响的重要保证。4)磷酸戊糖途径不影响精子活力,但可增加精子的抗氧化能力。5)有氧氧化途径有助于猪精子活力的维持。
[Abstract]:Prolonging the preservation time of porcine semen with liquid diluent is very important for the popularization of artificial insemination and breeding of fine breeds of pigs. However, there are some problems in the process of sperm preservation with diluent, such as the diversity of the added components and amount in the diluent, the short survival time of sperm in the diluent, the low fertilization rate, and so on. There are two ways to solve the problem, one is the energy supply in the process of sperm metabolism, the other is to remove the unfavorable substances produced by the sperm in the course of metabolism in time. However, there are few studies on glucose metabolism of ejaculated sperm during the liquid preservation of porcine semen. There is no clear conclusion as to whether PPP pathway exists in different mammalian sperm and whether tricarboxylic acid cycle is necessary. Therefore, the study on the pathway of fructose metabolism in the process of porcine semen liquid preservation is not only for enriching the basic theory of sperm energy metabolism, but also for increasing the time of liquid sperm preservation, improving sperm motility and expanding the popularization of artificial insemination in pigs. It provides favorable theoretical support and important practical significance. In this experiment, the phosphate buffer solution (PBS), which contains no energy substance, was used as the diluent, and the inhibitors of fructose and carbohydrate metabolites, glycolysis and electron transport were added. Sperm motility, acrosome integrity, DNA damage rate, antioxidant capacity and mitochondrial membrane potential were measured at 15 鈩，
本文编号：2154468
[Abstract]:Prolonging the preservation time of porcine semen with liquid diluent is very important for the popularization of artificial insemination and breeding of fine breeds of pigs. However, there are some problems in the process of sperm preservation with diluent, such as the diversity of the added components and amount in the diluent, the short survival time of sperm in the diluent, the low fertilization rate, and so on. There are two ways to solve the problem, one is the energy supply in the process of sperm metabolism, the other is to remove the unfavorable substances produced by the sperm in the course of metabolism in time. However, there are few studies on glucose metabolism of ejaculated sperm during the liquid preservation of porcine semen. There is no clear conclusion as to whether PPP pathway exists in different mammalian sperm and whether tricarboxylic acid cycle is necessary. Therefore, the study on the pathway of fructose metabolism in the process of porcine semen liquid preservation is not only for enriching the basic theory of sperm energy metabolism, but also for increasing the time of liquid sperm preservation, improving sperm motility and expanding the popularization of artificial insemination in pigs. It provides favorable theoretical support and important practical significance. In this experiment, the phosphate buffer solution (PBS), which contains no energy substance, was used as the diluent, and the inhibitors of fructose and carbohydrate metabolites, glycolysis and electron transport were added. Sperm motility, acrosome integrity, DNA damage rate, antioxidant capacity and mitochondrial membrane potential were measured at 15 鈩，
本文编号：2154468
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