猕猴精子冷冻保存方法的改进及活率检测
文献类型：学位论文
| 作者 | 采克俊 |
| 学位类别 | 博士 |
| 答辩日期 | 2005-01 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 季维智 |
| 关键词 | 猕猴 精子 冷冻保存 化学成分确定稀释液 流式细胞术 |
| 其他题名 | Improved Methods for Cryopreservation of Rhesus Monkey Spermatozoa and Their |
| 中文摘要 | 精液的冷冻保存对家畜繁殖、人类辅助生殖以及濒危物种保护有着重要意义。然而目前精子冷冻的研究很大程度上是经验性的，并且要使用含卵黄、脱脂乳等复杂成分的稀释液。这些复杂成分不仅是潜在的污染源，也使精子冷冻中的质量控制及分析特定物质的功能变得困难。即便如此也仅有约50％的精子能够存活下来，而且存活精子的受精能力也有所下降。称猴是生物医学研究中使用广泛的灵长类动物，开展赫猴精子的冷冻保存有利于该物种的繁殖并促进基础胚胎学的研究，成功的称猴精子冷冻方法还可以为其它珍稀濒危灵长类动物所借鉴。但是目前成功的称猴精子冷冻的报道仍然很少，且精子质量检测方法并不完善。因此本文致力于建立简单有效的称猴精子冷冻方法和活率检测方法。主要结果如下：1）建立了一种用单一紫外激光同时激发Hoechst33342和碘化丙锭结合流式细胞术检测称猴精子活率的有效方法，并将此方法用于称猴精子冷冻研究。2）发现甘油的一步加入或去除与分步加入或去除对称猴精子冷冻效果没有明显影响，在此基础上发现各种糖类稀释液之间的作用差异很小，但不含糖的化学成分确定的TT稀释液对精子冷冻保护作用显著高于糖类稀释液。这可能是因为TT的渗透压（138mOsm/kg）较好地适合于猫猴精子的冻存，高渗毒害作用可能是导致称猴精子用TEST冷冻存活率低的重要原因。3）以TT为基础稀释液，探讨了精子冷冻中一些关键因素，以期进一步改进冷冻方法。结果表明精液一步稀释于含5％甘油的一倍浓度TT，平衡0.5h可得到最佳效果，总体上复苏精子的运动度可达到55％以上，质膜完整性能达到6D％左右。这种方法与传统的以含有卵黄的TTE为稀释液的称猴精子冷冻方法效果相当，复苏精子还能够成功地进行体外受精。另外，用液化精液1：2直接稀释于防冻液还能进一步提高冷冻效果。本研究建立的用化学成分确定稀释液冷冻称猴精子的简单方法，有利于提高称猴精子的冻存效率，并有助于分析特定成分在精子冷冻中的作用。而Hoechst33342和碘化丙锭双染方法，是一种简单有效的精子活率检测方法，并在多参数精子功能的流式检测中有很高应用价值。 |
| 英文摘要 | Semen cryopreservation has been of great benefit to livestock breeding, human infertility treatment and the conservation of endangered animals. However, the study of spermatozoa cryopreservation has been done empirically, using the complex extender containing egg yolk or skim milk. Besides being a potential source of infectious agent, these undefined components make it difficult to undertake proper quality control and to analyze the roles of a particular compound plays in sperm cryopreservation. Even so, only about 50% spermatozoa can survive during the freeze-thaw process, and the survivors' fertility is reduced. The rhesus monkey, one species of nonhuman primate, has been extensively used in biomedical research for long history. Cryopreservation of rhesus monkey spermatozoa would facilitate the breeding of this species and basic embryological research. Success with rhesus spermatozoa could also be useful in developing protocols for freezing sperm from other rare or endangered nonhuman primates. However, only a few reports have been published on cryopreservation of rhesus monkey spermatozoa, and effective methods for their quality evaluation are required. Thus, this investigation was devoted to establishing simple and effective methods for rhesus monkey spermatozoa cryopreservation and viability assessment. The main results were as follows. 1 An effective method for evaluating rhesus monkey sperm viability by flow cytometry using Hoechst 33342 and propidium iodide dual staining excited by a single UV laser was established and applied to the study of spermatozoa cryopreservation. 2 It was found that glycerol can be added or removed in a single or fractionated manner, but a single manner is easier and more practical Using the single manner, further experiments showed that extenders containing different sugars gave similar results, whereas TT extender with no sugar was more effective than sugar extenders in protecting sperm function after freezing and thawing. The reason was that the osmolalities of extenders were crucial to the cryoprotection of rhesus monkey spermatozoa. The osmolality of TT (138mOsm/kg) was favorable for the cryopreservation of rhesus monkey spermatozoa and the hyperosmotic stress of TEST accounts for the lower survival of rhesus monkey spermatozoa frozen in this medium. 3 In order to further optimize the freezing procedure, several key factors were investigated using TT as the basic extender. The results showed that the best sperm survival rate was obtained after freezing spermatozoa in 1 * TT containing 5% glycerol with 0.5h equilibration. Overall, post-thaw sperm motililty and viability were about 55% and 60%, respectively. The optimized method using TT yielded similar post-thaw results as did the conventional freezing method using the egg yolk based TTE extender, and the post-thaw sperm can be used successfully for in vitro fertilization. Furthermore, sperm ryopreservation efficacy could be further improved by diluting the liquefied semen directly with freezing medium at a rate of 1:2. (v/v). The establishment of the simple method using chemically defined medium should prove useful in future investigations to analyze the actual cryoprotecting potential of different reagents. The H342/PI dual staining technique, was simple and effective in assessing sperm viability and would be valuable for multiparameter flow cytometric analysis of sperm function. |
| 语种 | 中文 |
| 公开日期 | 2010-10-15 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6193]  |
| 专题 | 昆明动物研究所_生殖与发育生物学 |
| 推荐引用方式 GB/T 7714 | 采克俊. 猕猴精子冷冻保存方法的改进及活率检测[D]. 北京. 中国科学院研究生院. 2005. |
入库方式： OAI收割
来源：昆明动物研究所
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