In the past decade, laboratories around the world have produced tens of thousands of mutant, transgenic, and wild-type fish lines. Maintaining these valuable genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. One of the major recommendations of the 2007 NCRR workshop 'Achieving High-Throughput Repositories for Biomedical Germplasm Preservation'was for performance of detailed studies to improve sperm cryopreservation for important aquarium fish models. Pursuant to this we propose to establish high-throughput processing of zebrafish sperm for stock centers and laboratories by extending a comprehensive cryopreservation pathway, and by intensifying efficiency and output to enable implementation of germplasm repositories.
The Specific Aims are to: 1) broaden the pathway to improve quality and provide multiple access points for samples produced by current approaches, 2) intensify the pathway for automated processing and repository development including use of industrial modeling and decision-making tools, and 3) implement two scalable cryopreservation protocols;the large-scale high throughput method for Resource Center use and, reverse-engineered from it, a small to intermediate scale application for other research laboratories to back up their own lines or submit them to the Resource Center. This will improve quality, reduce variability, and increase efficiency. We propose to improve broodstock conditioning, gamete quality, fertilization and reproduction, and to accommodate the variety of approaches, levels of application, and current and future needs of the research community. This will yield two complementary approaches: one at an industrial scale using automated equipment and pooling of samples to yield the highest level of intensity and efficiency;the other, less intensive but highly reliable, designed or dissemination to other laboratories. The materials generated from this external pathway will feed into the central pathway and will provide an additional form of throughput based on the aggregate production from multiple locations. A high level of standardization will be necessary for both approaches to merge efficiently to deliver high-quality samples into large comprehensive germplasm repositories. Thus, we envision this renewal as a funnel to improve and adapt the wide breadth of technical approaches available for zebrafish cryopreservation, and to focus these activities for repository development that can be eventually coupled with commercially available high-throughput equipment, supplies, and reagents specifically optimized for aquarium fish. This approach is practical and extremely feasible. This renewal proposal builds directly on the success of our established pathway and looks to couple efficiently with future advances.

Public Health Relevance

Aquarium fish models are rapidly transforming genetic research of disease. Thousands of new research models become available each year, and we must develop the abilities and resources necessary to preserve them in repositories for biomedical research and improvement of human health. The most significant problems constraining repository development for these fish are the lack of automated, high-throughput cryopreservation capabilities, and the lack of standardized protocols and terminology. These deficits prevent the coupling and integration of activities among different laboratories and stock centers.

National Institute of Health (NIH)
Office of The Director, National Institutes of Health (OD)
Resource-Related Research Projects (R24)
Project #
Application #
Study Section
National Center for Research Resources Initial Review Group (RIRG)
Program Officer
Chang, Michael
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Louisiana State University Agricultural Center
Organized Research Units
Baton Rouge
United States
Zip Code
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
Martínez-Páramo, Sonia; Horváth, Ákos; Labbé, Catherine et al. (2017) Cryobanking of aquatic species. Aquaculture 472:156-177
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
Yang, Huiping; Daly, Jonathan; Tiersch, Terrence R (2016) Determination of sperm concentration using flow cytometry with simultaneous analysis of sperm plasma membrane integrity in zebrafish Danio rerio. Cytometry A 89:350-6
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 :
Hu, E; Liao, T W; Tiersch, T R (2015) Simulation modeling of high-throughput cryopreservation of aquatic germplasm: a case study of blue catfish sperm processing. Aquac Res 46:432-445

Showing the most recent 10 out of 29 publications