Suitability of cryoprotectants and impregnation protocols for embryos of Japanese whiting Sillago japonica

Abstract

The purpose of this study was to examine the suitability of cryoprotectant agent (CPA) impregnation protocols for the embryos of Japanese whiting ( Sillago japonica), a small-sized, easy-to-rear, and prolific marine fish which may constitute a suitable experimental material for the development of cryopreservation methods for fish embryos. Our immediate goals were to assess the toxicity and permeability of various CPAs to whiting embryos of different developmental stages. Exposure of gastrula, somites, tail elongation, and pre-hatching embryos to 10%, 15%, and 20% solutions of propylene glycol (PG), methanol (MeOH), dimethyl sulfoxide (Me 2SO), dimethylformamide (DFA), ethylene glycol (EG), and glycerol (Gly) in artificial sea water (ASW; 33 psu) for 20 min revealed that CPA toxicity for whiting embryos increased in the order of PG < Me 2SO < DFA < EG < MeOH < Gly. Relative CPA permeability, estimated by proton nuclear magnetic resonance spectroscopy, showed the same trend as toxicity except for Gly, which was highly toxic but showed only moderate permeability. There were no marked differences in CPA tolerance between developmental stages except for a slight decrease in pre-hatching embryos. Stepwise (20% × 5 steps) impregnation with CPA mixtures of 20–25% PG with 10–15% DFA, Me 2SO, MeOH, or EG in ASW were well tolerated by the embryos. Overall, the results of toxicity and permeability suggest that PG, MeOH, and EG could be useful for the development of CPA solutions for whiting embryos.