Development of a simplified and standardized protocol with potential for high-throughput for sperm cryopreservation in zebrafish Danio rerio.

Sperm cryopreservation offers potential for long-term storage of genetic resources. However, the current protocols for zebrafish Danio rerio are cumbersome and poorly reproducible. Our objective was to facilitate adoption of cryopreservation by streamlining methods from sperm collection through thawing and use. First, sperm activation was evaluated, and motility was completely inhibited when osmolality of the extender was >/=295-300mOsmol/kg. To evaluate cryoprotectant toxicity, sperm were incubated with dimethyl sulfoxide (DMSO), N,N-dimethyl acetamide (DMA), methanol, or glycerol at 5, 10, and 15% concentrations. Based on motility, DMSO, DMA, and methanol (</=10%) were less toxic; therefore, sperm were cryopreserved using these cryoprotectants at cooling rates of 10 and 20 degrees C/min. The highest motility (mean+/-S.D.) (35+/-23%; P</=0.0001) and fertility (13+/-8%; P</=0.001) in thawed sperm were obtained with the combination of 8% methanol and a cooling rate of 10 degrees C/min. Further evaluations of 8% methanol and 10 degrees C/min were performed with males from populations with high (2.05+/-0.24) and low (1.18+/-0.12) body condition (P=0.0001). Motility of thawed sperm from the two populations was 38+/-16% (range, 10 to 60%) and 78+/-10% (50 to 90%) (P=0.0001), and fertilization was 6+/-6% (0 to 18%) and 33+/-20% (5 to 81%) (P=0.0001). These values were positively related with body condition factor. Overall, this study simplified and standardized sperm cryopreservation, and established a protocol using French straws as a freezing container and an extender without powdered milk. This protocol can be readily adapted for high-throughput application using automated equipment, and motility and fertility comparable to previous reports were obtained. Male variability and sperm quality remain important considerations for future work, especially in mutant and inbred lines.