Optimizing Sperm Collection Procedures in Zebrafish.

Zebrafish are a highly-valued model organism used for developmental biology research. Zebrafish can be used for genetic manipulation and hence, many mutant and transgenic lines exist. It is impractical to maintain lines of adult zebrafish, due to resource constraints and the need to continuously produce new generations. Therefore, a practical way to preserve zebrafish lines is to freeze sperm and retrieve lines using in vitro fertilization of fresh eggs. Most existing in vitro protocols used by research labs have a wide variety of fertilization rates (ranging from 0% to >90%). Due to this variability, lines may be at risk of not being regenerated, and may be permanently lost. For this project, aspects of existing published sperm collection protocols were tested and modified, with the goal of improving the proportion of males giving quality ejaculate. Males were tested for production of ejaculate by housing fish either in groups or in separate, individual tanks the night before sperm collection. The effect of age of male zebrafish and genetic background (5D and AB lines) on production of quality ejaculate was also tested. Isolating males before sperm collection significantly increased the proportion of individuals producing quality ejaculate. The proportion of fish that gave quality ejaculate samples did not co-vary with age between 17-68 weeks. Overall, AB fish were significantly more likely to give quality ejaculate samples compared to 5D fish. Based on this study, we strongly recommend separating male fish before sperm collection to improve the likelihood of obtaining samples. Our results indicate that AB fish give proportionately better samples than 5D fish, and this does not vary with age between 17-68 weeks.