Zebrafish sperm cryopreservation with N,N-dimethylacetamide.

High fecundity, rapid generation time, and external development of optically clear embryos make the zebrafish (Danio rerio) a convenient vertebrate model for genetic, developmental, and disease studies. Efficient sperm cryopreservation enhances the zebrafish model system by optimizing productive use of facility space, extending the reproductive lifetime of males, providing an alternative to live stocks for strain recovery, and ensuring the survival of valuable mutant lines. Here we identify a cryoprotective medium, 10% N,N-dimethylacetamide (DMA) (v/v) diluted in buffered sperm motility-inhibiting solution (BSMIS), as well as parameters for zebrafish sperm cryopreservation that enhance cryopreservation efficiency and significantly increase the yield of live embryos from archived stocks. Our experiments emphasize the effect of the ratio of sperm and medium volume and the use of large egg clutches to maximize the recovery of viable embryos.