There is an immediate need to improve preservation of genetic resources from aquatic model organisms such as zebrafish and medaka. The availability of research lines through cryopreservation will profoundly influence the advancement of biomedical research in toxicology, embryology, genetics, drug development, and human disease. In the past 15 years, laboratories around the world have produced thousands of mutant, transgenic and wild-type fish lines. However, live maintenance of these lines is expensive, risky, and beyond the current capacity of even the largest stock centers. Although cryopreservation is a proven method for long-term maintenance of genetic material, current protocols for fish are not standardized, yield inconsistent results, and threaten the efficacy of large-scale genetic screening. For sperm cryopreservation to become a reliable, cost-effective tool for genetic banking of fish, the overall cryopreservation process needs to be improved and integrated into an efficient large-scale platform that links with genetic and biological databases, archival storage capabilities, inventory management, and sample distribution pathways. Our Project Goal is to establish high-throughput cryopreservation of zebrafish and medaka sperm for stock center application and for research laboratory use by optimizing, streamlining, and standardizing procedures, and by applying basic cryobiological and physiological parameters. We will evaluate the effects of: 1) animal conditioning, 2) sample collection, 3) sperm concentration, 4) extender composition, 5) additives, 6) refrigerated storage and transport, 7) cryoprotectant toxicity, 8) interactions of cooling rate and cryoprotectants, 9) thawing, 10) post-thaw amendments, and 11) fertilization procedures. We will establish high-throughput sample management of: 1) straw-filling and sealing, 2) labeling, 3) inventory management, 4) shipping reliability, and 5) adoption of automated processing of high-security plastic straws. We will define terms and standardize procedures for quality control and biosecurity at stock centers and donor laboratories. Germplasm cryopreservation will greatly enhance development, management, and distribution of NIH resources by: 1) reducing size and production costs of facilities, 2) protecting genetic diversity and reducing inbreeding, 3) maximizing efficiency of holding live animals, and 3) facilitating global, regional, and institutional transport of genetic material.
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