不同抗氧化剂对地中海水牛精液冷冻保存效果的影响研究

发布时间：2018-03-20 09:15

本文选题：地中海水牛　切入点：精液冷冻　出处：《广西大学》2017年硕士论文　论文类型：学位论文

【摘要】：意大利地中海水牛因其高产奶量和优良的奶质被公认为是目前世界上河流型水牛中乳用性能最好的品种之一。广西于2014年引进该品种水牛,受冷冻保存配方、冷冻程序和个体差异等因素影响,目前的种公牛精液冷冻保存效果不甚理想,这极大地影响了地中海水牛的繁殖效率。因此,探讨抗氧化剂等对精液冷冻保存效果的影响,研发针对意大利地中海水牛的精液稀释液配方,对提高种公牛利用率有着重大意义。为此,本研究在传统精液稀释液基础上,首先重新比较有效成分、调整其比例、同时引入新的保护剂、拟出新配方,随后应用改进的配方探讨添加褪黑素、谷胱甘肽和维生素E等不同抗氧化剂对地中海水牛精液冷冻保存的效果影响。研究结果如下。1.地中海水牛精液冷冻稀释液配方的改进研究在水牛精液常用冷冻稀释液的基础上,首先对配方中的能量物质、抗冻物质和抗菌物质分别进行效果比较。结果发现,稀释液成分中卵黄的冷冻保存效果优于鲜全奶;3%甘油和2%乙二醇联合作用效果优于5%的甘油、5%的乙二醇和5%的DMSO三者单独作用时的效果;青霉素和庆大霉素的抑菌效果比较明显;其次,对于新引入的保护剂牛血清白蛋白(BSA),和未添加组相比,0.9 mg/mL的BSA显著提高了水牛精子的活率,降低其畸形率(P0.05);改进后的地中海水牛精液稀释液配方为:0.10 g/mL葡萄糖、0.0006 g/mL柠檬酸钠、0.0011 g/mL青霉素、0.0006 g/mL庆大霉素、0.9 mg/mLBSA、3%甘油、2%乙二醇、20%卵黄、75%蒸馏水。2.褪黑素、谷胱甘肽和维生素E对地中海水牛精液冷冻保存效果的影响在冷冻稀释液中添加褪黑素(MLT)时发现,添加0.Img/mLMLT显著提高了受精卵分裂率和GSH-Px酶活性(P0.05),对精子活率、畸形率、运动性能等变化无显著差异(P0.05);添加0.2mg/mL、0.4mg/mL MLT明显提高了冻后精子活率、顶体完整率、GSH-Px酶活性、受精卵分裂率和囊胚率(P0.05),添加0.2mg/mLMLT还显著降低了精子畸形率(P0.05)。添加0.8mg/mLMLT明显降低了精子活率(P0.05),对精子运动性能、受精卵分裂率、囊胚率等均没有显著影响(P0.05)。添加谷胱甘肽(GSH)处理时发现,添加0.1mMGSH显著提高精子前向性和质膜完整率,降低了精子畸形率(P0.05),对精子活率和顶体完整率无明显影响(P0.05)。添加0.2 mMGSH明显了提高精子活率、运动性能、质膜完整率、GSH-Px酶活性和囊胚率(P0.05),显著降低精子畸形率和MDA水平(P0.05)。0.4mMGSH增加了精子畸形率,显著提高GSH-Px酶活性、受精率分裂率、囊胚孵化率(P0.05),对顶体完整率、SOD酶活性等无明显影响(P0.05)。添加0.8 mMGSH增加了精子畸形率(P0.05),对精子抗氧化物酶活性和囊胚率等无显著性影响(P0.05)。添加维生素E(VE)处理时发现,与未添加组相比,0.1 mg/mL和0.2 mg/mLVE对精子活率无明显影响(P0.05)。0.2mg/mL、0.4mg/mLVE明显提高精子的GSH-Px酶活性、顶体完整率、质膜完整率、受精卵分裂率、囊胚率和囊胚孵化率,显著降低了精子MDA水平(P0.05)。0.4mg/mL、0.8mg/mLVE显著提高了精子活率(P0.05),其中0.4mg/mL时精子活率最大,0.4mg/mL VE显著降低了精子畸形率,0.8mg/mLVE导致畸形率增加(P0.05),0.8mg/mLVE明显提高受精卵分裂率(P0.05),对囊胚率和囊胚孵化率无显著影响(P0.05)。3.三种抗氧化剂配伍作用对地中海水牛精液冷冻保存效果的影响为了解两种或多种抗氧化剂配伍添加的协同效果,根据单独添加MLT、GSH和VE时的作用效果确定各抗氧化剂的配伍浓度,分别为MLT(0.2 mg/mL、0.4 mg/mL)、GSH(0.1 mM、0.2 mM)、VE(0.2 mg/mL、0.4 mg/mL),将其两两组合,共形成12个配伍组合。结果发现,与未添加组相比,0.2 mg/mL VE+O.1 mM GSH、0.4 mg/mL VE+0.2 mg/mL MLT、0.2 mg/mL MLT+0.1 mM GSH组合均能在一定程度上提高了精子活率,降低精子畸形率,其中 0.2 mg/mL MLT+0.1 mM GSH 和 0.4 mg/mL VE+0.2 mg/mL MLT 表现较突出。三个配伍组合均显著提高了精子运动性能、顶体完整率和质膜完整率(P0.05),其中尤以0.2mg/mLMLT+0.ImMGSH组合提升效果最好。三个配伍组合均显著提高了受精卵分裂率(P0.05),0.4 mg/mL VE+0.2 mg/mL MLT和 0.2 mg/mL MLT+0.1 mM GSH组合对囊胚率的提高有显著差异(P0.05),0.4 mg/mL VE+0.2 mg/mL MLT和0.2 mg/mL VE+0.1 mM GSH显著提高了囊胚孵化率(P0.05)。综合考虑,0.2 mg/mL MLT+0.1 mM GSH 和 0.4 mg/mL VE+0.2 mg/mL MLT 组合优于 0.2 mg/mL VE+0.1 mM GSH。以上结果表明,添加 0.2 mg/mL MLT、0.2 mM GSH 或 0.04 mg/mL VE可提高水牛精液的冷冻保存效果较好,采用0.4mg/mLVE+0.2mg/mL MLT或0.2 mg/mL MLT+0.1 mM GSH的组合添加剂对地中海水牛精液冷冻保存可取得较好效果。
[Abstract]:Italy Mediterranean buffalo because of its high milk production and excellent milk quality is recognized as the world river buffalo milk with one of the best varieties performance. Guangxi in 2014 to introduce the varieties of buffalo, the cryopreservation formula, factors affecting the freezing procedure and individual differences, the bull semen cryopreservation effect ideal, which greatly affects the reproductive efficiency of the Mediterranean buffalo. Therefore, to investigate the effect of antioxidants on the preservation effect of frozen semen in Italy, Mediterranean semen dilution formula Buffalo Research, is of great significance to improve the utilization rate of male cattle. Therefore, this study based on traditional semen dilution, first re comparison of effective components, adjust the proportion, while the introduction of new protective agents, a new formula, then using the improved formula to investigate the effects of melatonin, glutathione and vitamin E etc. Effect of antioxidants on the preservation of Mediterranean buffalo semen cryopreservation effect. The results are as follows.1. Mediterranean buffalo frozen semen dilution formula improvement based on buffalo common semen freezing diluent on the first energy formula, and antibacterial substances of antifreeze material respectively compared the effects. The results showed that the preservation effect is better than that of fresh whole milk yolk diluent ingredient in freezing; 3% glycerol and 2% ethylene glycol combination is better than 5% glycerol, 5% ethylene glycol and 5% DMSO three respectively. The effect of anti bacteria effect; penicillin and gentamicin obviously; secondly, the protective agent of bovine serum albumin (BSA), the newly introduced compared and without group, 0.9 mg/mL BSA significantly increased the survival rate of buffalo sperm, reduce the deformity rate (P0.05); the buffalo semen dilution formula of the improved: 0.10 g/mL glucose, 0. 0006 g/mL sodium citrate, 0.0011 g/mL penicillin, gentamicin 0.0006 g/mL, 0.9 mg/mLBSA, 3% glycerol, 2% ethylene glycol, 20% egg yolk, 75%.2. distilled water preservation effect of melatonin, glutathione and vitamin E on buffalo in the Mediterranean frozen semen freezing dilution liquid added melatonin (MLT) found that the addition of 0.Img/mLMLT significantly increased cleavage rate and enzyme activity of GSH-Px (P0.05), survival rate, the rate of sperm deformity, no significant difference in movement performance changes (P0.05); adding 0.2mg/mL, 0.4mg/mL MLT increased obviously after freezing sperm motility, acrosome integrity rate, GSH-Px activity, cleavage rate and blastocyst rate (P0.05) add 0.2mg/mLMLT, also significantly reduced the rate of sperm deformity (P0.05). Adding 0.8mg/mLMLT significantly reduced sperm motility, sperm motility (P0.05) on performance, cleavage rate, blastocyst rate did not significantly affect (P0.05) added Gu Guanggan. Peptide (GSH) treatment was added prior to 0.1mMGSH significantly increased and sperm membrane integrity, reduced sperm deformity rate (P0.05), the sperm motility and acrosome integrity rate had no significant effect (P0.05). Adding 0.2 mMGSH significantly improve sperm motility, exercise performance, membrane integrity rate the activity of GSH-Px and blastocyst rate (P0.05), significantly reduced the rate of sperm deformity and the level of MDA (P0.05).0.4mMGSH increased the rate of sperm deformity, increased the GSH-Px activity, the fertilization rate cleavage rate, blastocyst hatching rate (P0.05), the rate of intact acrosome, had no significant effect on the activity of SOD (P0.05) added. 0.8 mMGSH increased the rate of sperm deformity of sperm (P0.05), antioxidant enzyme activity and blastocyst rate had no significant effect (P0.05). The addition of vitamin E (VE) treatment was found, compared with not adding group, 0.1 mg/mL and 0.2 mg/mLVE of sperm survival rate had no significant effect (P0.05).0.2mg/mL, 0.4mg/mLVE obviously To improve the GSH-Px activity of sperm, acrosome, plasma membrane integrity rate, cleavage rate, blastocyst rate and blastocyst hatching rate, significantly reduced the level of sperm MDA (P0.05).0.4mg/mL, 0.8mg/mLVE significantly increased the survival rate of sperm (P0.05), the 0.4mg/mL 0.4mg/mL VE maximum motility significantly reduced sperm. The deformity rate, deformity rate increased due to 0.8mg/mLVE (P0.05), 0.8mg/mLVE significantly increased the cleavage rate (P0.05), had no significant effect on blastocyst rate and blastocyst hatching rate (P0.05) of.3. three compatible effect of antioxidant effect on preservation of Mediterranean buffalo frozen semen for understanding the synergistic effect of two or more antioxidants added compatibility. According to the addition of MLT, the effect of GSH and VE to determine the concentration of each combination of antioxidants, respectively MLT (0.2 mg/mL, 0.4 mg/mL), GSH (0.1 mM, 0.2 mM), VE (0.2 mg/mL, 0.4 mg/mL), the combination of 22, A total of 12 combinations. The results showed that compared with not adding group, 0.2 mg/mL VE+O.1 mM GSH mg/mL VE+0.2 mg/mL, 0.4 MLT, 0.2 mg/mL MLT+0.1 mM GSH combination can to some extent improve sperm motility, reduce the rate of sperm deformity, including 0.2 mg/mL MLT+0.1 mM GSH VE+0.2 mg/mL MLT and 0.4 mg/mL more prominent. Three combinations significantly increased sperm movement performance, acrosome and plasma membrane integrity rate (P0.05), especially 0.2mg/mLMLT+0.ImMGSH combination to enhance the effect of three. The best combination can significantly increase the fertilized egg split rate (P0.05), 0.4 mg/mL VE+0.2 mg/mL MLT and MLT+0.1 mM GSH 0.2 mg/mL the combination has significant difference to improve the blastocyst rate (P0.05), 0.4 mg/mL VE+0.2 mg/mL MLT mg/mL VE+0.1 mM and 0.2 GSH significantly increased the blastocyst hatching rate (P0.05). Considering the 0.2 mg/mL and 0.4 mg/m MLT+0.1 mM GSH L VE+0.2 mg/mL MLT mg/mL VE+0.1 mM is better than that of combination of above 0.2 GSH. results showed that the addition of 0.2 mg/mL MLT, 0.2 mM GSH or 0.04 mg/mL buffalo VE can improve the sperm cryopreservation effect is good, the sea buffalo semen cryopreservation can achieve good effect with the combination of 0.4mg/mLVE+0.2mg/mL MLT or mg/mL MLT+0.1 0.2 additive mM GSH.

【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823.83

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