CRYOPRESERVATION OF SPERM

精子的保存是保存转基因动物的合理方法 老鼠基因作为动物群体抵御灾难的保险， 遗传漂变和维持未使用的品系直到需要。电流 小鼠精子的冷冻保存技术效率低， 精子存活率低和生育能力低。本研究的目的是 收件人： 1)开发实验室检测方法，评估 群体中的单个小鼠精子，以提供 潜在的;（必要的提高精子的效率 冷冻保存）， 2)开发有效的方法来冷冻保存小鼠精子， 3)发展简单的技术，如人工授精， 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，设计了实验来开发分析 基于流式细胞术、荧光测定法、计算机化运动 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精子活力。后续 设计实验以改进低温保存技术， 研究冷冻介质的添加剂，使用选定的添加剂 因为预期的保护精子的血浆或顶体膜。 此外，脂质体和亲脂性化合物将用于修饰 冷冻前精子膜的组成，从而促进细胞 低温生存最后，我们将测试获得小鼠的可行性， 不杀死雌性小鼠或使用复杂的实验室 设备.这些实验将测试收集的可行性， 在人工授精程序中冷冻和使用冷冻解冻的精子 而不是昂贵和技术要求高的体外受精 繁殖老鼠的程序。 通过将几种小鼠品系的使用纳入本实验， 项目，开发的技术不应依赖于应变，结果 应该产生有效的收集、冷冻保存 和利用冷冻解冻的小鼠精子，允许实际储存 不同转基因小鼠品系的精子。

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.