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.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Resource-Related Research Projects (R24)
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National Center for Research Resources Initial Review Group (RIRG)
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Chang, Michael
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Louisiana State University Agricultural Center
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Baton Rouge
United States
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Hu, E; Childress, William; Tiersch, Terrence R (2017) 3-D printing provides a novel approach for standardization and reproducibility of freezing devices. Cryobiology 76:34-40
Torres, Leticia; Liu, Yue; Guitreau, Amy et al. (2017) Challenges in Development of Sperm Repositories for Biomedical Fishes: Quality Control in Small-Bodied Species. Zebrafish 14:552-560
Cuevas-Uribe, Rafael; Hu, E; Daniels, Harry et al. (2017) Vitrification as an Alternative Approach for Sperm Cryopreservation in Marine Fishes. N Am J Aquac 79:187-196
Tiersch, Nolan J; Tiersch, Terrence R (2017) Standardized Assessment of Thin-film Vitrification for Aquatic Species. N Am J Aquac 79:283-288
Tiersch, Terrence R; Monroe, William T (2016) Three-dimensional printing with polylactic acid (PLA) thermoplastic offers new opportunities for cryobiology. Cryobiology 73:396-398
Torres, Leticia; Tiersch, Terrence R (2016) Amine reactive dyes: an alternative to estimate membrane integrity in fish sperm cells. Aquaculture 463:71-78
Yang, Huiping; Daly, Jonathan; Carmichael, Carrie et al. (2016) A Procedure-Spanning Analysis of Plasma Membrane Integrity for Assessment of Cell Viability in Sperm Cryopreservation of Zebrafish Danio rerio. Zebrafish 13:144-51
Torres, Leticia; Hu, E; Tiersch, Terrence R (2016) Cryopreservation in fish: current status and pathways to quality assurance and quality control in repository development. Reprod Fertil Dev :
Park, Daniel S; Egnatchik, Robert A; Bordelon, Hali et al. (2012) Microfluidic mixing for sperm activation and motility analysis of pearl Danio zebrafish. Theriogenology 78:334-44
Scherr, Thomas; Quitadamo, Christian; Tesvich, Preston et al. (2012) A Planar Microfluidic Mixer Based on Logarithmic Spirals. J Micromech Microeng 22:55019

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