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.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Resource-Related Research Projects (R24)
Project #
5R24RR023998-02
Application #
7680084
Study Section
National Center for Research Resources Initial Review Group (RIRG)
Program Officer
Chang, Michael
Project Start
2008-09-01
Project End
2011-05-31
Budget Start
2009-06-01
Budget End
2010-05-31
Support Year
2
Fiscal Year
2009
Total Cost
$520,343
Indirect Cost
Name
Louisiana State University Agricultural Center
Department
Type
Organized Research Units
DUNS #
783201833
City
Baton Rouge
State
LA
Country
United States
Zip Code
70803
Yang, Huiping; Hu, E; Buchanan, John T et al. (2018) A Strategy for Sperm Cryopreservation of Atlantic Salmon, Salmo salar, for Remote Commercial-scale High-throughput Processing. Journal of the World Aquaculture Society 49:96-112
Matthews, Jennifer L; Murphy, Joy M; Carmichael, Carrie et al. (2018) Changes to Extender, Cryoprotective Medium, and In Vitro Fertilization Improve Zebrafish Sperm Cryopreservation. Zebrafish 15:279-290
Liu, Yue; Torres, Leticia; Tiersch, Terrence R (2018) Cryopreservation of sperm bundles (spermatozeugmata) from endangered livebearing goodeids. Cryobiology 82:49-56
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
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 :
Torres, Leticia; Tiersch, Terrence R (2016) Amine reactive dyes: an alternative to estimate membrane integrity in fish sperm cells. Aquaculture 463:71-78

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