EFFECTS OF VITRIFICATION PROTOCOL ON THE LACTATE DEHYDROGENASE AND TOTAL ATPASE ACTIVITIES OF CHINESE MITTEN CRAB Eriocheir sinensis EMBRYOS

摘要

BACKGROUND: Vitrification is the most promising option for the cryopreservation of fish embryos but requires high concentrations of potentially toxic cryoprotectants that can also cause cell injury, and affect cell division, enzymatic activities and cell metabolism. The effect of cryopreservation on the enzyme activity in crustacean embryos has not been reported. OBJECTIVES: The aim of this study was to investigate the effects of a vitrification protocol on the enzyme activity of different stages of Eriocheir sinensis embryos. The goal was to select an appropriate stage and vitrifying solution for the cryopreservation of embryos from this crustacean. MATERIALS AND METHODS: Embryos from E.sinensis at five development stages (cleavage, blastula, gastrula, eyed stage and heart beating stage) were submitted to six kinds of cryoprotectant incorporation protocol with a five stepwise method. Six vitrifying solutions were prepared by combining cryoprotectants PG, Me0H, Me2SO and DMF in different proportions (A: 20% PG + 20% DMF; B: 20%MeOH+20% DMF; C: 20%PG+20%MeOH; D:20% PG+10% MeOH+10% DMF; E:20% Me2SO+20% PG;F:20% Me2SO+20% MeOH). After incubation in the six kinds of vitrification solutions, embryos were loaded into cryo-tubes and plunged into liquid nitrogen. The activities of two cytoplasmic enzymes, LDH and total ATPase were analyzed in control embryos, those subjected to the cryoprotectant solutions and in frozen/thawed embryos. RESULTS: The cryoprotectant incorporation protocol had important effects on the enzymatic activities, and different vitrifying solutions had distinct effects on the enzymatic activities. The early stage embryo was sensitive to the toxic effect of the cryoprotectants, with a significant drop in total ATPase in comparison with fresh, control embryos. Enzymatic activities dropped significantly after vitrification, indicating cell damage and loss of cytoplasmic enzymes. CONCLUSION: The composition of vitrifying solutions had different effects on the loss of the enzyme activity, and the later stage embryo was more resistant to the effect of vitrification.