Rat sperm cryobanking for genetic resources

描述(申请人提供)：种质冷藏库是一种无限期保存科学、医学、经济和环境方面重要基因储备的有效方法。虽然大鼠是模拟人类疾病的重要系统，但目前还没有成熟的种质超低温保存方案，可以对大鼠遗传资源进行经济有效的管理。收集附睾精子是保存遗传学以备将来使用的最简单和最经济的方法。然而，到目前为止，只有一种方案显示解冻后的能动性只有10%。因此，迫切需要了解影响大鼠精子的生理和低温生物学因素，以提高大鼠精子的冷冻存活率。这项应用的目标是开发冷冻附睾大鼠精子的方案，使其能够通过使用标准的人工授精和体外受精方法重建遗传学。在过去的几年里，我们的实验室已经生成了大量与最佳精子处理、Percoll分离、冷冻延长器以及大鼠精子的冷暖条件有关的数据。这些将有助于减少物理、低温、渗透压力和致命冰层形成造成的伤害，并最终产生可接受的融化后恢复率。我们实验室收集的数据与本申请中提出的冷冻生物学研究相结合，将使我们能够开发出有效和可靠的附睾鼠精子冷冻保存方案。 公共卫生相关性(由申请人提供)：这项建议旨在开发高效的大鼠精子冷冻保存方法，用于通过常用的辅助生殖技术，如人工授精或体外受精，用于重建具有重要遗传意义的大鼠品系。因此，拟议的研究将面向生物医学界，为大鼠精子的冷冻提供可靠的方案，以避免连续的活体动物收容所，这对美国国立卫生研究院及其研究人员来说是一个沉重的负担。

DESCRIPTION (provided by applicant): Germplasm cryobanking is an effective way of preserving scientifically, medically, economically and environmentally important genetic stocks indefinitely. Although rats are important system for modeling human disease, there is no established germplasm cryopreservation protocol which would allow cost-effective management of rat genetic resources. Collection of epididymal sperm is the simplest and most cost-effective method of archiving genetics for future use. However, to date, there is only one protocol showing only 10% post-thaw motility. Thus, there is an urgent need for understanding the physiological and cryobiological factors affecting rat sperm in order to improve the rat sperm cryosurvival. The goal of this application is to develop freezing protocols for epididymal rat sperm which would allow reconstitution of genetics by using standard artificial insemination and in-vitro fertilization methods. Over the last few years, our laboratory has generated ample amount of data related with optimal sperm handling, Percoll separation, freezing extender and cooling and warming conditions for the rat sperm. These would help reduce the injuries caused by physical, hypothermic, osmotic stress and lethal ice formation, and ultimately yield acceptable post-thaw recovery rate. The data which have been collected in our laboratory in combination with proposed cryobiologic studies outlined in this application will allow us to develop efficient and reliable cryopreservation protocol for epididymal rat sperm. PUBLIC HEALTH RELEVANCE (provided by applicant): This proposal aims at developing efficient cryopreservation method for rat sperm which would be used to reconstitute genetically important rat strains through commonly used assisted reproductive technologies such as artificial insemination or in-vitro fertilization if needed. The proposed studies will therefore address to the biomedical community by providing a reliable protocol for rat sperm cryopreservation in order to avoid continuous live animal housing which is a significant burden for National Institutes of Health and its investigators around the nation.