Factors affecting chilled storage of zebrafish (Danio rerio) embryos

Chilled storage of zebrafish embryos was investigated at a temperature that arrests embryonic development as this technique might offer interesting practical applications. Five parameters played an important role for chilled storage: (a) storage temperature, (b) development stage of embryos, (c) storage solution (extender), (d) postchilling treatment, and (e) inhibition of growth of microorganisms by antibiotics. The optimal chilling temperature was 8 °C. Prim-5 stage (24 h postfertilization hpf]) and prim-25 stage (36 hpf) embryos had similar high chilling resistance and could be chilled for 33 h without a loss in viability. Five-somite stage (12 hpf) embryos had a lower chilling resistance and could be chilled only for 14 h without a loss in viability. After longer incubation periods, the viability started to decrease. Under these conditions, chilling in physiologic saline solutions was superior to that in water. Fifty percent of the prim-5 stage and prim-25 stage embryos survived for 41 h at 8 °C in water but for 46 h in physiologic saline solution. A similar effect was observed for 5-somite stage embryos (50% survival rate in water, 28 h; 50% survival rate in physiologic saline solution, 35 h). When embryos were incubated in physiologic saline solution instead of water in the postchilling phase, the embryo viability was positively affected, too. Also, supplementation of the storage solution with antibiotics (penicillin and streptomycin) increased the viability of chilled embryos. In summary, the current study shows that chilled storage of zebrafish embryos is possible for sufficiently long periods to synchronize the development of embryos deriving from different spawning dates or to delay the development for experimental purposes. To prolong the storage periods, further development and standardization of the methodology is necessary.