Effects of different vitrificant solutions on the embryos of the Chinese mitten crab Eriocheir sinensis (Decapoda, Brachyura)

Abstract

Vitrification could provide a promising tool for the cryopreservation of embryos. However, in order to achieve a vitrifiable medium, a high concentration of permeable cryoprotectants must be used, and the incorporation of high-molecular-weight compounds should also be considered. In the present study, the toxicity of six vitrificant solutions was determined in Eriocheir sinensis H. Milne Edwards, 1853 embryos at five developmental stages (cleavage, blastula, gastrula, eyed stage and heart beating stage). Six vitrifying solutions were prepared with seawater of salinity 15 and the cryoprotectants propylene glycol (PG), methanol (MeOH), dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) in different proportions (A: 30% PG + 20% DMF; B: 30% MeOH + 20% DMF; C: 30% PG + 20% MeOH; D: 30% PG + 10% MeOH + 10% DMF; E: 20% DMSO + 30% PG; F: 20% DMSO + 30% MeOH). The impact of each of these solutions on E. sinensis embryos was then assessed and compared. Vitrifying solution “A” produced the highest survival rate, albeit lower than that of the control. A five-step equilibration of embryos in all six vitrifying solutions resulted in higher survival rates than equilibration in 3 or 2 steps. The tolerance of E. sinensis embryos to the vitrifying solutions increased with embryonic maturity, cleavage and blastula stage embryos being very sensitive to vitrifying solutions, and a two-step equilibration of embryos in the six vitrifying solutions resulted in no survival at all. Embryos in the eyed stage and heart beating stage showed a higher tolerance to vitrifying solutions as compared to the other stages, with the survival rates achieved over 50% and 60%, respectively. Cleavage stage and gastrula stage embryos did not survive after cryopreservation, although we acquired a percentage of transparent embryos (generally an indication of successful cryopreservation). Fifteen viable embryos were eventually recovered from 361 freeze-thawed embryos, all from eyed stage and heart beating stage embryos. One larva with apparently normal morphology hatched successfully from those 15 embryos.