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.

PROJECT NARRATIVE Cryopreservation of nonhuman primate (NHP) spermatozoa and long-term storage in liquid nitrogen is currently the only method utilized for preserving specimens with valuable genetics. These methods require skilled technicians to perform and costly storage vessels that are susceptible to damage and catastrophic sample loss. We propose to develop a standardized method of NHP semen freeze-drying (FD) that can be easily performed by most laboratory personnel and supports long-term storage at common refrigeration temperatures.