大黄鱼和鮸鱼精子的超低温冷冻保存研究

科研成果详情

题名	大黄鱼和鮸鱼精子的超低温冷冻保存研究
其他题名	Cryopreservation of Spermatozoa of large yellow croaker and brown croaker
作者	戴同仁
学位类型	硕士
导师	董巧香
答辩日期	2014-05-27
学位授予单位	温州医科大学
学位专业	遗传学
关键词	大黄鱼，鮸鱼，精子，超低温冷冻保存，线粒体膜电位，ATP，膜完整性，活性氧，人工受精，流式细胞术
摘要	大黄鱼是我国的四大渔业之一，在我国渔业中有着很重要经济地位。大黄鱼和鮸鱼都是我国重要的海洋经济鱼类。但是由于近年海洋生态的破坏和种质资源的下降，使得大黄鱼的生物多样性下降，种质资源退化，品质下降，对病害和环境胁迫的防御能力下降，病害发生日趋严重，造成了巨大的经济损失。超低温冷冻保存作为一种保护种质资源的新技术，已成功的运用到海洋鱼类的精子冷冻保存中。本文主要研究了大黄鱼和鮸鱼的精子密度、活力的生理特性；研究了抗冻剂和冷冷冻速率对大黄鱼和鮸鱼精子超低温冷冻的影响，从而建立一种高效且快速的精子超低温冷冻方案；同时我们还对冷冻前后大黄鱼精子的线粒体膜电位、ATP水平、活性氧、膜完整性等进行检测，对大黄鱼精子超低温冷得出一个比较全面的精子质量评价；同时检测了鮸鱼精子线粒体膜电位、活性氧、膜完整性和受精实验，从而来评价鮸鱼精子超低温冷冻的效果。在大黄鱼精子超低温冷冻保存研究中，我们采用了挤压腹部的方法收集精子，采集到的精子呈乳白色，比较粘稠。精子密度为（3.06±0.79）×1011个精子/ml。在大黄鱼精子激活实验中，两种溶液在渗透压低于300mOsm/kg时均不能激活，在渗透压为300mOsm/kg时只能轻微激活，精子激活后的活力随着两种溶液渗透压的增高而呈“倒U”字行变化，均在渗透压为600mOsm/kg时达到最大。精子的运动时间在400mOsm/kg时最大，之后随着渗透压的逐渐增高，精子的运动时间逐渐降低。鲜精的短期保存实验中，大黄鱼的精子使用Free组、Pyr组、Lac组和Glc组处理后，在4℃冰箱内保存16小时精子活力未出现明显的下降。Free组在保存48小时后，精子活力下降为0，而其他三组的活力均大于60%。在保存72小时后Pyr组、Lac组和Glc组的精子活力也降为0。Pyr组的精子运动时间明显比其他几组要长。说明外源能量物质在精子离体保存有着很重要的作用。在大黄鱼精子超低温冷冻方案筛选中，我们选用了三种抗冻剂：DMSO、Gly和EG；每种抗冻剂选择4个浓度：5%、10%、15%和20%；精子的冷冻采用了四个不同高度的泡沫船 1、3.4、5、7cm来控制得到四种冷冻速率。使用DMSO作为抗冻剂时，在5%和10%的浓度时，都获得活力＞75%的效果；在DMSO浓度较高时（15-20%），5cm和7cm高度泡沫船的冷冻效果明显差与1cm和3.4高度；在10%的浓度1cm泡沫船高度下，获得了86.7±2.9%的最高活力。精子的运动时间随着泡沫船高度的增高而呈明显的降低趋势，在10%的浓度1cm高度时获得了229±14.8s的最长运动时间。使用EG作为抗冻剂时，20%浓度的精子活力显著低于5%组、10%和15%三组；1cm、3.4cm和5cm都获得活力≥80%，都显著高于7cm组；在5%的浓度1cm高度时获得了93.3±2.9%的最高活力；随着泡沫船高度的增加，精子的运动时间也呈现逐渐降低的趋势，在15%的浓度1cm高度时获得了281.5±20.5s的最长运动时间。使用Gly作为抗冻剂时，随着浓度的增加，精子解冻后的活力都逐渐降低；随着泡沫船高度的增高，精子活力呈逐渐减低的趋势；使用1cm高度的泡沫船，5%Gly进行冷冻取得了最高的活力86.7±2.9%；并没有表现出很好的规律和趋势。在15%Gly，5cm高度的泡沫船冷冻时获得了最高的精子运动时间246.5±43.1s，但与5%浓度1cm高度并没有显著性差异（P=0.284）。总之，5%DMSO、5%EG和5%Gly在1cm高度快速冷冻大黄鱼的精子均获得了很理想的效果。本文对大黄鱼的精子冷冻前后进行了全面的评价，发现冷冻导致了大黄鱼精子线粒体膜电位的和细胞内的ATP含量明显降低，并且冷冻过程造成大量精子质膜的破裂，诱导细胞内ROS的大量产生。但是DMSO与EG对精子的保护，在不同方面都有他们各自的优势，DMSO对冷冻过程ATP的保护和防止ROS的生成都要好于EG，但对MMP的保护EG则更好，对MI的保护两者都相差无几。对鮸鱼精子超低温冷冻的研究中，使用了与大黄鱼同样的精子采集方法，CASA检测了6条鮸鱼的鲜精精子密度，平均密度为（6.39±2.23×1010）个精子/ml。本文根据实验室前辈的研究结果，选择了两种效果比较好的抗冻剂：DMSO和EG；每种抗冻剂选择4个浓度：5%、10%、15%和20%；精子的冷冻采用了四个不同高度的泡沫船 1、3.4、5、7cm来控制得到四种冷冻速率。使用DMSO作为抗冻剂时，在5%-10%的浓度是都获得了大于70%的精子活力，10%DMSO的冷冻—解冻后的精子活力显著高于其他三组。在1cm高度，5%的浓度下获得了85%的最高的精子活力。使用EG作为抗冻剂时，1cm泡沫船的冷冻后精子活力明显高于其他三组，在5%的浓度是获得了83.3±2.9最高的精子活力。鮸鱼的受精实验我们发现，随着精卵比的减少，受精率也呈下降的趋势，在精卵比为107：1，鲜精和冻精的受精率没有明显的差别。当精卵比为106：1时，DMSO组的受精率高于鲜精组和EG组，DMSO组和EG组之间没有显著性差异。在精卵比为105：1时，EG组显著低于其他两组，两组之间没有显著性差异。说明DMSO的效果要好于EG。这与后面的精子质量评价的结果相一致，DMSO对线粒体膜电位和质膜的保护都要好于EG。精子的受精能力和精子质量评价的结果有一定的正相关。
其他摘要	The large yellow croaker（Pseudosiaena crocea） is one of four big fishery in our country, which has very important economic status. The large yellow croaker and brown croaker (Miichthys miiuy) are two important marine economy fish. But because of the marine ecological destruction and the decline of the germplasm resources of large yellow croaker, loss of biodiversity, germplasm resource degradation, declining quality, disease and environmental stress on defensive ability drops, to the more serious diseases, caused a great economic loss in recent years. Cryopreservation of germplasm resources as a new technology, has been successfμlly applied to marine fish sperm cryopreservation. This paper mainly studies large yellow croaker and brown croaker fish sperm density,and dynamic analysis; study the effect of cryoprote ctants and frozen rate of large yellow croaker and brown croaker sperm cryopreservation, so as to establish an efficient and rapid sperm cryopreservation programme; at the same time, we also testing mitochondrial membrane potential, the level of ATP, reactive oxygen species, membrane integrity of large yellow croaker spermatozoa before and after freezing, in order to comprehensive evaluation of sperm quality. The study of brown croaker, we detected in the mitochondrial membrane potential, reactive oxygen species, membrane integrity and fertilization experiment, to evaluate sperm cryopreservation effect.In large yellow croaker spermatozoa cryopreservation study, we used a squeezing the abdomen for collection the sperm, the collected sperm is milky white, relatively viscous. Sperm density was 3.06±0.79×1011 sperm/ ml. In large yellow croaker spermatozoa activation experiments, results showed that CF-HBSS and Glc solution canactivate the large yellow croaker spermatozoa, two kinds of solution are lower than 300mOsm / kg are can not activate the sperm. With two kinds of solution osmotic pressure increasing, Sperm motility was changed as "pour U". When the osmotic pressure at 600mOsm/kg the sperm motility reached maximum. The osmotic pressure at 400mOsm/kg, the sperm movement time of those two group were reached maximum. In short-term preservation experiment, the results showed the sperm treatment with Free group, Pyr group, Lac group and Glc group treatment, in the refrigerator 4℃ for 16 hours without a noticeable decline in sperm motility. The Free group preservation after 48 hours, sperm was dead, while the other three groups were greater than 60%. After 72 hours, Pyr group, Lac group and Glc group were dead. It is suggest that exogenous energy has a very important role in sperm vitro preservation. In large yellow croaker fish sperm cryopreservation, cryoprotectants and the freezing rate are key factors. In large yellow croaker spermatozoa cryopreservation study, we choose three kinds of cryoprotectants: DMSO, Gly and EG; each kind of cryoprotectants selection in 4 concentrations: 5%, 10%, 15% and 20%; sperm cryopreservation using four different height of the bubble boat (1, 3.4, 5, 7cm) to control freezing rate. DMSO as the cryoprotectant, the concentration from 5% to 10%, have achieved good results, the sperm motility>75%; At the higher concentration, 5cm and 7cm have worse result than 1cm and 3.4cm. When the concentration of DMSO at 10% and 1cm foam board, obtained the maximum sperm motility 86.7±2.9%. And sperm movement time decreased with bubble boat height increasing significantly, when the concentration of DMSO at 10% and 1cm foam board, obtained 229± 14.8s the longest movement time. The EG as cryoprotectant, when the concentration at 20%, sperm motility was significantly lower than 5%, 10% and 15%; 1cm, 3.4cm and 5cm have achieved good results, sperm motility > 80%, were significantly higher than 7cm; When the concentration of EG at 5% and 1cm foam board, the maximum sperm motility 93.3±2.9%; sperm movement time decreased with bubble boat height increasing significantly. When the concentration of EG at 15% and 1cm foam board, obtained longest movement time 281.5±20.5s. The Gly as cryoprotectant, with the increased of concentration, sperm motility decreased; When the concentration of Gly at 5% and 1cm foam board, the maximum sperm motility 86.7±2.9%; sperm motility in time did not show good regularity and trend. When the concentration of DMSO at 15% and 1cm foam board, obtained longest movement time 246.5±43.1s, but have no significant difference with he concentration of Gly at 5% and 1cm foam board,(P=0.284). In short, 5%DMSO, 5%EG and 5%Gly in 1cm foam board rapid freezing of large yellow croaker spermatozoa are obtained very ideal result.The large yellow croaker spermatozoa before and after freezing conducted a comprehensive evaluation, found frozen resulted in a large yellow croaker mitochondrial membrane potential and cellular ATP content decreased obviously, and the freezing process resulted in a large number of sperm plasma membrane rupture, induced intracellular ROS Substantial increase. But DMSO and EG on sperm protection in different aspects, have their respective advantages, DMSO on the freezing process of ATP protection and prevent the generation of ROS is better than EG, but to protect MMP EG is better, to protect MI are not much difference between the two.The brown croaker sperm cryopreservation studies, and uses the same sperm acquisition method of large yellow croaker, we detecte 6 fish fresh semen density by CASA, average density is 6.39±2.23×1010 sperm /ml. Based on the laboratory of advanced research resμlts, choose two kinds of cryoprotectants: DMSO and EG; each kind of cryoprotectants selection in 4 concentrations: 5%, 10%, 15% and 20%; sperm cryopreservation using four different height of the bubble boat (1, 3.4, 5, 7cm) to control freezing rate. The use of DMSO as cryoprotectants, at the concentration of 5%-10% obtained more than 70% sperm motility, 10%DMSO frozen - thawed sperm motility was significantly higher than the other three groups (P=0.005; P=0.004; P= 0.001). Freezing rate, the four rate was no significant difference between frozen. In 1cm height, 5% DMSO were obtained the highest sperm motility 85%. The use of EG as cryoprotectant, 1cm foam boat frozen sperm motility was significantly higher than the other three groups, at 5% concentration are obtained the highest sperm motility 83.3±2.9%. Artificial insemination resμlts show, with sperm-egg ratio decreases, the fertilization rate also showed a decreasing trend, at 107:1 sperm-egg ratio, the fertilization rate of fresh semen and frozen semen has no obvious difference. When the sperm-egg ratio is 106 : 1, DMSO group is higher than EG group. When the sperm-egg ratio is 105 : 1, EG group is significantly lower than the other two groups, those had no significant difference. Description of DMSO better than EG. This and the subsequent sperm quality evaluation result is consistent, DMSO on mitochondrial membrane potential and membrane protection are better than that of EG. Fertilization ability of sperm and sperm quality evaluation results have some positive correlation
语种	中文
学号	2009010027
发布年限	2010-05-27
毕业论文分类号	0R03
原始专题	环境与公共卫生学院
学位论文研究方向	低温繁殖生物学
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全文文件名	2009010027戴同仁2012遗传学.pdf\|2009010027戴同仁2012遗传学.pdf
文献类型	学位论文
条目标识符	https://kms.wmu.edu.cn/handle/3ETUA0LF/117925
专题	温州医科大学
作者单位	溫州医科大学环境与公共卫生学院
推荐引用方式 GB/T 7714	戴同仁. 大黄鱼和鮸鱼精子的超低温冷冻保存研究[D]. 温州医科大学,2014.