The African clawed frog (Xenopus laevis) is used in laboratories worldwide as a tool to investigate biochemistry, genetics, cell biology and development biology in normal and disease states. The number of transgenic and mutant lines is rapidly increasing and maintenance as live organisms is costly and risky. Storage of cryopreserved germplasm in repositories can provide a way to protect lines and reduce the number of live animals. In collaboration with the National Xenopus Resource (NXR, Marine Biological Laboratory, MA), we aim to develop a cryopreservation pathway for this species. Currently, the NXR cryopreserves sperm, but routine quality evaluation of samples is not performed, and a high variability among males in fertilizing capacity with frozen sperm has been found, with 30% failing to produce enough embryos to recover a line. We developed methods for evaluation of sperm viability and motility in this species (Figure 1). We tested the effect of cryoprotectants (DMFA, DMSO, methanol), concentration (5%, 10% and 20%), addition of sugar (sucrose), and cooling rate (5, 10 and 20 ºC/min) on sperm quality. Also, the fertilizing capacity of testicular sperm frozen at different concentrations (30, 50 and 100 × 106 cells/mL) was assessed. This protocol will help in reducing post-thaw variability among X. laevis males and in setting standards for samples to be accepted into the repository at the NXR. In addition, we are using a center-based approach for application of this protocol to develop a high-throughput cryopreservation pathway that is scaleable and generalizable, integrating processing and quality management to establish repository capabilities to serve the needs of research communities. This repository would be integrated into a larger aquatic species repository network developed in cooperation with the USDA National Animal Germplasm Program located in Ft. Collins, CO and the AGGRC. A major design consideration is that the practices and operations at this center will be in accordance with those of other centers to facilitate network activities. Thus, this collaborative initiative will consider repository development in a multi-level approach that addresses more than protocol development.