PROJECT SUMMARY/ABSTRACT The NIH-funded aquatic model stock centers have expressed concerns over growing needs for them and their associated research communities to use germplasm repositories in preservation and sharing of genetic resources. Because of a lack of comprehensive repository capabilities, the National Xenopus Resource (NXR), Ambystoma Genetic Resource Center (AGSC), and National Resource for Aplysia (NRA) face many challenges such as the risk of losing valuable genetic lines. In the ongoing R24 project (2020-2024) we have developed or improved practical approaches for sperm cryopreservation for NXR and AGSC, and we are making breakthrough progress in embryo and larvae cryopreservation for NRA. For Xenopus, we developed a standardizable, streamlined pathway which is scalable and substantially improves throughput and quality assurance. For Ambystoma we developed a de novo cryopreservation protocol and expanded it into a functional pathway which produced a 50% fertilization rate with thawed sperm. For Aplysia, we have recently achieved short-term (>10 hr) post-thaw survival of free-swimming larvae by vitrification. During this project, we have collaborated closely with the stock centers and responded to their needs through hundreds of interactions including site visits, training visits, phone calls, remote meetings, and workshops. In addition, we have developed a central “Hub” at the Aquatic Germplasm and Genetic Resource Center (AGGRC) that supports resource sharing among the stock centers, the USDA National Animal Germplasm Program, and user communities leading to the creation of a NIH Aquatic Biomedical Germplasm Repository Network. In sum, the ongoing Hub R24 project has developed basic reproductive and cryopreservation protocols and customized these based on the specific resources and needs of the stock centers. Based on this success, the overall goal of this proposed renewal R24 Hub work is to advance the capabilities at the resource centers from feasibility establishment to operational repositories with high- throughput production and routine quality management programs. Proposed translational research will focus on building practical capabilities at the stock-center level for amphibians; expanded fundamental research and characterization will proceed at the AGGRC for Aplysia, and resource sharing platforms will be established at the stock center and research community levels. To facilitate resource sharing and distribution, we will advance the NIH Aquatic Biomedical Germplasm Repository Network from proof of concept to establishment of formal operations. This will ensure that research communities will have access to valuable genetic lines that may otherwise be at risk of being lost. The specific aims are: 1) Develop high-throughput sperm cryopreservation pathways and quality management programs for operational repository capabilities at NXR and AGSC; 2) Develop operational repository capabilities for early life stages of Aplysia ...