Modeling of cryopreservation pathway operation at an aquatic biomedical stock center for zebrafish.

Aquatic biomedical model organisms play a substantial role in advancing our understanding of human health, however, comparably little work has been directed towards developing dependable, high-throughput storage programs for valuable genetic resources. The Zebrafish International Resource Center (ZIRC) has developed a standardized cryopreservation pathway and stored thousands of genetic lines in their repository for use by the biomedical research community. This has yet to be replicated in other facilities, and an overall repository-level pathway has never been analyzed for aquatic species. To encourage repository development for other biomedical models and to improve the ZIRC storage process and system, this study used discrete-event simulation modeling to systematically analyze the cryopreservation pathway for efficiency, and to identify improvements. The models reflected "real-world" working conditions and were used to simulate key outputs, such as production capacity over time (throughput) and steps in the process that limit production (bottlenecks). With these models, recommendations were identified to eliminate waiting times and increase efficiency. These included following proper husbandry protocols because male quality significantly affected production time, and the use of part-time operators to assist with steps that had longer Waiting Times (i.e., time samples spent in a queue) to increase production capacity. Simulation process modeling is a powerful tool that can improve the operations of existing repositories. It can also support repository development at other biomedical stock centers, and at other facilities devoted to aquatic species such as research, conservation, and aquaculture production hatcheries.

A Simple Method for Inducing Masculinization of Zebrafish Stocks Using 17α-Methyltestosterone.

Severely skewed sex ratios in zebrafish stocks can pose significant hurdles for line propagation and sperm cryopreservation. To overcome female-biased sex ratios in stocks derived from imported sperm samples, the Zebrafish International Resource Center has implemented routine supplementation of larval food with 17α-methyltestosterone to skew gonadal sex differentiation toward masculinization. Resulting stocks averaged 80% males.

Changes to Extender, Cryoprotective Medium, and In Vitro Fertilization Improve Zebrafish Sperm Cryopreservation.

Sperm cryopreservation is a highly efficient method for preserving genetic resources. It extends the reproductive period of males and significantly reduces costs normally associated with maintenance of live animal colonies. However, previous zebrafish (Danio rerio) cryopreservation methods have produced variable outcomes and low post-thaw fertilization rates. To improve post-thaw fertilization rates after cryopreservation, we developed a new extender and cryoprotective medium (CPM), introduced quality assessment (QA), determined the optimal cooling rate, and improved the post-thaw in vitro fertilization process. We found that the hypertonic extender E400 preserved motility of sperm held on ice for at least 6 h. We implemented QA by measuring sperm cell densities with a NanoDrop spectrophotometer and sperm motility with computer-assisted sperm analysis (CASA). We developed a CPM, RMMB, which contains raffinose, skim milk, methanol, and bicine buffer. Post-thaw motility indicated that the optimal cooling rate in two types of cryogenic vials was between 10 and 15°C/min. Test thaws from this method produced average motility of 20% ± 13% and an average post-thaw fertilization rate of 68% ± 16%.

Parallel genetic basis for repeated evolution of armor loss in Alaskan threespine stickleback populations.

Most adaptation is thought to occur through the fixation of numerous alleles at many different loci. Consequently, the independent evolution of similar phenotypes is predicted to occur through different genetic mechanisms. The genetic basis of adaptation is still largely unknown, however, and it is unclear whether adaptation to new environments utilizes ubiquitous small-effect polygenic variation or large-effect alleles at a small number of loci. To address this question, we examined the genetic basis of bony armor loss in three freshwater populations of Alaskan threespine stickleback, Gasterosteus aculeatus, that evolved from fully armored anadromous populations in the last 14,000 years. Crosses between complete-armor and low-armor populations revealed that a single Mendelian factor governed the formation of all but the most anterior lateral plates, and another independently segregating factor largely determined pelvic armor. Genetic mapping localized the Mendelian genes to different chromosomal regions, and crosses among these same three widely separated populations showed that both bony plates and pelvic armor failed to fully complement, implicating the same Mendelian armor reduction genes. Thus, rapid and repeated armor loss in Alaskan stickleback populations appears to be occurring through the fixation of large-effect variants in the same genes.