Preservation of Nonhuman Primate Spermatozoa by Lyophilization

项目摘要 俄勒冈州国家灵长类动物研究中心（ONPRC）是七个国家灵长类动物研究中心之一 （NPRCs）由NIH赞助。它由P51核心补助金（P51-OD 011092）支持，并提供 专业知识，技术和资源，为尖端研究人员提供非人类 灵长类动物（NHP）进行生物医学研究。ONPRC维持着一个大约4900只 和研究NHP，其中大多数是恒河猴。目前，全国缺乏 恒河猴可用于研究，这限制了保护遗传动物的尝试 罕见或不常见疾病的携带者。成功地保存配子是一条重要的道路， 确保未来的研究项目能够获得这些动物模型。此外，还有一个 NHP模型开发的巨大转变，从活体动物中的组织特异性诱导表型， 在单细胞胚胎中诱导疾病特异性基因型，后来用于衍生成年动物。因为这些基因- 编辑的动物很难实现，保存配子成为构建的关键组成部分 疾病模型群组。随着越来越强调配子保存作为NHP模型程序的一部分， 长期配子储存的替代品，不需要液氮和昂贵的储存或运输 因此，在NHP辅助生殖技术（ART）中，新的战略已成为新的焦点。 在本补充建议，以家长赠款，我们将测试的假设，1）现代冷冻干燥（FD） 设备和单纯形优化将推导出新鲜采集恒河猴冻干的最佳条件 猕猴精子，用于在普通冷藏温度下长期储存;和2）精子 从睾丸、附睾和输精管收集的死后组织可以进行冷冻干燥和长期保存。 食蟹猴、猪尾猴、狒狒在常用冷藏温度下储存， 绒猴物种。为了检验这些假设，将使用以下方法进行FD培养基的单纯形优化： 以前出版的公式尝试作为起点。培养基成分、pH值、 冷冻形式和干燥时间将通过已在本研究中进行的标准精子质量测定进行评估。 ONPRC艺术核心。其他NHP种属的尸检组织将从华盛顿获得， 德克萨斯州和威斯康星州NPRC通过其组织分配计划，并评价组织差异 来源冻干耐受性。我们的研究结果将为NHP配子存储的替代方案提供基准 并建立一个基础，继续努力完善和最大限度地保护 宝贵的基因。

PROJECT SUMMARY The Oregon National Primate Research Center (ONPRC) is one of seven National Primate Research Centers (NPRCs) sponsored by the NIH. It is supported by the P51 Core Grant (P51-OD011092) and provides the expertise, technology, and resources required to deliver cutting-edge researchers access to nonhuman primates (NHPs) for biomedical investigation. The ONPRC maintains a colony of approximately 4900 breeding and research NHPs, the majority of which are rhesus macaques. Currently there is a national shortage of rhesus monkeys available for research, which is constraining attempts to preserve animals that are genetic carriers of rare or uncommon diseases. Successful preservation of gametes is a critical path forward to ensuring access to these animal models for future research programs. Additionally, there has been a tremendous shift in NHP model development from tissue-specific induced phenotypes in live animals to induced disease-specific genotypes in single-cell embryos later used to derive adult animals. As these gene- edited animals are difficult to achieve, preservation of gametes becomes a critical component to building disease model cohorts. With an increasing emphasis on gamete preservation as part of NHP model programs, alternatives to long-term gamete storage that does not require liquid nitrogen and costly storage or shipping strategies have become the new focus in NHP assisted reproductive technology (ART). In this supplement proposal to the parent grant, we will test the hypotheses that: 1) Modern freeze-drying (FD) equipment and simplex optimization will deduce optimal conditions for lyophilizing fresh collected rhesus macaque spermatozoa for long-term storage at common refrigeration temperatures; and 2) Spermatozoa collected post-mortem from testis, epididymis, and vas deferens can undergo lyophilization and long-term storage at common refrigeration temperatures for cynomolgus macaque, pigtailed macaque, baboon, and marmoset species. To test these hypotheses, simplex optimization of FD medium will be performed using previous published formulation attempts as a starting point. Elements such as medium components, pH, freezing format, and drying times will be assessed by standard sperm quality assays already performed in the ONPRC ART Core. Necropsy tissues from additional NHP species will be obtained from the Washington, Texas, and Wisconsin NPRCs through their tissue distribution programs and evaluated for differences in tissue origin lyophilization tolerance. Our findings will provide a benchmark for alternatives to NHP gamete storage and establish a foundation from which to continue efforts to refine and maximize opportunities for preserving valuable genetics.