三种抗氧化剂对猪精子冷冻保存效果的影响

发布时间：2018-05-24 10:51

本文选题：猪 + 精子　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：为了提高养猪的效益,猪的人工授精技术得到了广泛的应用。但是由于猪精液在冷冻保存过程中容易受到低温打击,造成解冻后猪精子的活率不甚理想,从而影响繁殖率,因此找到一些冷冻保护剂成为了亟需解决的问题。猪精子质膜中含有较多的不饱和脂肪酸,冷冻保存及冷冻解冻过程中容易被氧化,从而破坏了精子质膜完整性。而竹叶黄酮、异抗坏血酸钠和丁基羟基茴香醚作为食品添加剂中几种常见的高效抗氧化剂,在食品抗氧化方面的研究较多,但是其对猪精液冷冻保存的影响还未见相关报道。本试验以竹叶黄酮、异抗坏血酸钠和丁基羟基茴香醚为冷冻保护剂,分别以不同的浓度加入猪精液冷冻稀释液,通过显微镜观察、碘化丙啶(PI)和罗丹明123联合染色、低渗肿胀实验(HOST)以及异硫氰酸荧光素连接的花生凝集素(PITC-PNA)分别检测了解冻后猪精子的活力、活率、线粒体膜完整性、质膜完整性和顶体完整性,评价几种抗氧化剂对猪精液冷冻保存的保护作用。同时,还利用酶活性检测试剂盒检测了冷冻-解冻后猪精子的丙二醛(MDA)水平、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)等酶的活性,得出了以下几个结论:1.将不同浓度的竹叶黄酮添加到猪精液冷冻稀释液中,可以显著提高冷冻解冻后猪精子的活力、活率、线粒体膜完整率、质膜完整率和顶体完整率(P0.05)。当竹叶黄酮的浓度为5 mg/mL时,冷冻解冻后猪精子的活力、活率、线粒体膜完整率、质膜完整率和顶体完整率最高。2.在猪精液冷冻稀释液中添加不同浓度的异抗坏血酸钠,可以显著提高冷冻解冻后猪精子的活力、活率、线粒体膜完整率、质膜完整率和顶体完整率(P0.05)。当异抗坏血酸钠的浓度为6 mg/mL时,冷冻解冻后猪精子的活力、活率、线粒体膜完整率、质膜完整率和顶体完整率最高。3.在猪精液冷冻稀释液中添加不同浓度的丁基羟基茴香醚,可以显著提高冷冻解冻后猪精子的活力、活率、线粒体膜完整率、质膜完整率和顶体完整率(P0.05)。当丁基羟基茴香醚的浓度为0.2 mg/mL时,冷冻解冻后猪精子的活力、活率、质膜完整率和顶体完整率最高,而当丁基羟基茴香醚的浓度为0.15 mg/mL时,线粒体膜完整率最高。4.在猪精液冷冻稀释液中添加2 mg/mL~6 mg/mL的竹叶黄酮,与对照组相比,MDA水平的降低、SOD酶活力和GSH-Px酶活力的升高具有显著性差异(P0.05)。当竹叶黄酮浓度为4 mg/mL和5 mg/mL时,MDA水平降低最多;浓度为5 mg/mL时,SOD酶活力效果最好;浓度为4 mg/mL时,GSH-Px酶活力最佳。冷冻稀释液中添加6 mg/mL异抗坏血酸钠时,MDA水平下降最明显;添加8 mg/mL时,SOD酶活力最高;而异抗坏血酸钠浓度为6 mg/mL~10 mg/mL时,GSH-Px酶活力比其他组差异显著。在冷冻稀释液中添加不同浓度的丁基羟基茴香醚时,MDA水平、SOD酶活力和GSH-Px酶活力比对照组差异显著(P0.05),且浓度为0.2 mg/mL时,效果均为最佳。5.两种抗氧化剂联合作用能显著提高解冻后猪精子的活力、活率和膜完整性(P0.05)。其中5 mg/mL竹叶黄酮和6 mg/mL的异抗坏血酸钠配伍作用后的效果最好。
[Abstract]:In order to improve the benefit of pig raising, pig artificial insemination technology has been widely used. However, because the pig semen is vulnerable to cryopreservation during cryopreservation, the survival rate of pig sperm is not ideal after thawing, thus affecting the reproductive rate. Therefore, finding some cryopreservation agents has become an urgent problem to be solved. More unsaturated fatty acids, frozen storage and freezing thawing are easily oxidized, thus destroying the integrity of sperm plasma membrane. However, bamboo leaf flavonoids, sodium isoascorbate and butyl hydroxy anisole are used as some common high effective antioxidants in food additives, but they have more research on the antioxidation of food products, but they have a good effect on pig sperm. The effect of liquid cryopreservation has not been reported. In this experiment, the freeze protectant of bamboo leaf flavone, sodium isoascorbate and butyl hydroxy anisole was added to the cryopreservation agent at different concentrations, respectively, by microscope observation, PI and Luo Danming 123 combined dyeing, hypotonic swelling test (HOST) and isothiocyanate. Peanut agglutinin (PITC-PNA) was used to detect the viability, viability, membrane integrity, membrane integrity and acrosome integrity of the porcine sperm after thawing, respectively, to evaluate the protective effect of several antioxidants on the cryopreservation of porcine semen. At the same time, the enzyme activity detection kit was used to detect the malondialdehyde (MD) of frozen thawing sperm. A) levels, the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and other enzymes, and the following conclusions were obtained: 1. adding different concentrations of bamboo leaf flavonoids to the cryopreservation diluents of porcine semen can significantly improve the viability, survival rate, mitochondrial membrane integrity, membrane integrity and acrosome integrity after freezing and thawing. P0.05) when the concentration of the bamboo leaf flavonoids was 5 mg/mL, the vitality, the survival rate, the membrane integrity of the mitochondria, the integrity of the plasma membrane and the highest acrosome rate of.2. after the freezing and thawing of the pig sperm were added with different concentrations of sodium isoascorbate in the cryopreservation solution of the pig's semen, which could significantly improve the viability, the survival rate and the complete mitochondrial membrane after the freezing thawing. Rate, plasma membrane integrity and acrosome integrity (P0.05). When the concentration of sodium isoascorbate is 6 mg/mL, the vitality, viability, membrane integrity, integrity of plasma membrane and the highest acrosome integrity after freezing thawing can be added to the freezing thawing solution of the pig semen with different concentrations of butyl hydroxy anisole, which can significantly improve the freezing thawing. The viability, survival rate, mitochondrial membrane integrity, membrane integrity and acrosome integrity (P0.05). When the concentration of butyl hydroxy anisole was 0.2 mg/mL, the viability, viability, plasma membrane integrity and acrosome integrity were the highest after freezing thawing, and the mitochondrial membrane integrity was the highest when the concentration of Ding Jiqiang anisole was 0.15 mg/mL High.4. added 2 mg/mL~6 mg/mL of bamboo leaf flavonoids in the frozen semen of pigs. Compared with the control group, the MDA level decreased, the activity of SOD enzyme and the activity of GSH-Px enzyme increased significantly (P0.05). When the concentration of the bamboo leaf flavonoids was 4 mg/mL and 5 mg/mL, the MDA level decreased most; when the concentration was 5 mg/mL, the activity of the SOD enzyme was the best; concentration was the best; concentration was the best; concentration was the best; concentration was the best; concentration was the best; concentration was the best; concentration was the best; concentration was the best; concentration was the best; concentration was the best; concentration was the best; concentration was the best; concentration was the best; concentration was the best; concentration was the best; concentration At 4 mg/mL, GSH-Px enzyme activity was the best. When 6 mg/mL isoascorbate was added to the frozen diluent, the level of MDA decreased most obviously; when adding 8 mg/mL, the activity of SOD enzyme was the highest; and when the concentration of sodium ascorbate was 6 mg/mL~10 mg/mL, the activity of GSH-Px enzyme was significantly different. When aromatic ether, MDA level, SOD enzyme activity and GSH-Px enzyme activity were significantly different than those of the control group (P0.05). When the concentration was 0.2 mg/mL, the combined effect of the best.5. and two kinds of antioxidants could significantly improve the viability, viability and membrane integrity (P0.05) of the porcine sperm after thawing. The compatibility of 5 mg/mL bamboo leaf flavonoids and 6 mg/mL isoascorbate sodium. The latter is the best.
【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】：2015
【分类号】：S828

【参考文献】