Cryopreservation ofHydractinia symbiolongicarpussperm to support community-based repository development for preservation of genetic resources

Hydractinia symbiolongicarpus is an emerging model organism in which cutting-edge genomic tools and resources are being developed for use in a growing number of research fields. However, one limitation of this model system is the lack of long-term storage for genetic resources. Our goal in this study was to establish a generalizable approach to sperm cryopreservation that would support future repository development and could be applied to many species according to available resources. Our approach was to: 1) Assess sperm characteristics and standardize collection and processing; 2) Assess acute toxicity to cryoprotectants, and 3) Evaluate and refine freezing conditions to permit post-thaw fertilization and produce viable offspring. By following this approach, we found that Hydractinia sperm incubated in 5% DMSO, equilibrated at 4°C for 20 min, and cooled at a rate of 20°C/min to - 80°C at a cell concentration of 10 8 -10 9 /mL in 0.25-mL aliquots were able to fertilize 150-300 eggs which yielded offspring that could metamorphose into juvenile polyps. In addition, improvements were made for processing sperm using a customized 3-D printed collection system. Other opportunities for improvement include optimizing the volumetric sperm-to-egg ratio for fertilization. Establishing repository capabilities for the Hydractinia research community will be essential for future development, maintenance, protection, and distribution of genetic resources. More broadly, this application-based approach highlights the long-term value of establishing repository-level resources that can be expanded to fit community needs.