Abstract
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 (