Studies on limiting factors relating to the cryopreservation of fish embryos
Cryopreservation of fish embryos has proven to be a difficult problem in cryobiology. Three main difficulties have been identified or suspected: 1) embryo membrane permeability barriers to cryoprotectants and water; 2) high chilling sensitivity of the embryos; and 3) the twocompartment nature of the embryos with a large yolk. Using the zebrafish embryo as a model system, these limiting factors and possible approaches to overcoming them were investigated with a view to developing an effective procedure for fish embryo cryopreservation. Compared to previous studies, vitrification of zebrafish embryos on gold electron microscope grids using methanol as the cryoprotectant resulted in improved morphological survival, being -50% for early stage (I-cell and 64-cell) and ~ 80% for late stage (50%epiboly, 6-somite and prim-6) embryos, but no embryo showed viability. Poor cryoprotectant permeation and embryo dehydration, and consequently intraembryonic ice formation, remained as the main problem for vitrification. Embryo chilling sensitivity studies suggested that later stage zebrafish embryos were sensitive to cold shock injury arising from rapid cooling followed by being held for an extended exposure period (1 h) at 0 or -5°C. Studies on embryo developmental arrest by anoxia showed that chilling injury in zebrafish embryos was probably not associated with their high development rate. However, the chilling sensitivity of zebrafish embryos was found to be related to the amount of yolk present. Yolk-reduced embryos at prim-6 and high-pec stages became less sensitive to chilling at O°C. Differential scanning calorimetry studies on the depression of intraembyonic nucleation temperatures by cryoprotectants revealed that multi-punctured embryos at 6-somite and prim-6 stages became significantly more permeable to methanol and propylene glycol when compared with their nonpunctured controls. Puncturing of the yolk-sac of fish embryos to reduce yolk content, and the increased permeability to cryoprotectants that this promotes, may offer a new approach to surmounting the difficulties confronting the cryopreservation of fish embryos.