CRYOPRESERVATION OF SPERM

精子保存是保存转基因的合理途径 老鼠基因为动物群体提供灾难保险，预防 遗传漂移和未使用品系的维护，直到需要为止。当前 小鼠精子的冷冻保存技术效率低下，并导致 精子存活率低，受精率低。这项研究的目标是 致： 1)开发评估多种属性的实验室分析方法 以提供种群中单个小鼠精子的估计 受精潜力；(提高精子效率所必需的 超低温保存)， 2)开发有效的冷冻保存小鼠精子的方法，以及 3)开发简单的技术，如人工授精，使用 冷冻保存小鼠精子以产生可存活的后代。 为了实现这些目标，设计了用于开发分析的实验 基于流式细胞术、荧光法、计算机运动的小鼠精子 分析，和体外受精；数据将进行比较，以确定 三种品系小鼠精子的受精能力。后续 旨在通过以下方式改进冷冻保存技术的实验 使用选定的添加剂研究冷冻介质的添加剂 由于预期的精子浆或顶体膜的保护。 此外，脂质体和亲脂化合物将用于修饰 冷冻前精子膜的组成，从而促进细胞 冷藏--生存。最后，我们将测试获得鼠标的可行性 后代不需要杀死雌性老鼠或使用先进的实验室 设备。这些实验将检验收集的可行性， 冷冻解冻精子在人工授精中的应用 而不是昂贵和技术要求高的体外受精 繁殖小鼠的计划。 通过将几个小鼠品系的使用纳入到这个实验中 项目、开发的技术不应依赖于应变，并且结果 应采用高效采集法(S)进行采集、超低温保存 和利用冷冻解冻的小鼠精子，允许实际储存 来自不同转基因系小鼠的精子。

Storage of spermatozoa is a logical approach for preserving transgenic mouse genes as insurance against disaster to animal colonies, prevention of genetic drift and maintenance of unused lines until needed. Current cryopreservation techniques for mouse sperm are inefficient and result in poor sperm survival and low fertility. The objectives of this research are to: 1) develop laboratory assays which evaluate multiple attributes of individual mouse sperm in a population to provide an estimate of fertilizing potential; (necessary to improve the efficiency of sperm cryopreservation), 2) develop effective methodologies to cryopreserve mouse sperm, and 3) develop simple techniques, such as artificial insemination, using cryopreserved mouse sperm to produce viable offspring. To accomplish these goals, experiments are designed to develop analyses for mouse sperm based on flow cytometry, fluorometry, computerized motion analysis, and in vitro fertilization; data will be compared to determine fertilizing potential of sperm from three strains of mice. Subsequent experiments are designed to improve cryopreservation techniques by investigating additives for freezing media, using additives selected because of anticipated protection of sperm plasma or acrosomal membranes. In addition, liposomes and lipophilic compounds will be used to modify composition of sperm membranes before freezing, thereby facilitating cell cryo-survival. Finally, we will test feasibility of obtaining mouse offspring without killing female mice or using sophisticated laboratory equipment. These experiments will test the feasibility of collecting, freezing and using frozen-thawed sperm in artificial insemination programs rather than costly and technically demanding in vitro fertilization programs to propagate mice. By incorporating use of several mouse strains into the experiments of this project, techniques developed should not be strain dependent, and results should yield efficient protocol(s) for the collection, cryopreservation and utilization of frozen-thawed mouse sperm, permitting practical storage of sperm from diverse transgenic lines of mice.