Cryopreservation of Chinese mitten crab, Eriocheir sinensis H. Milne Edwards, 1853 (Decapoda, Brachyura), embryos by vitrification

in Crustaceana
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Though cryopreservation of some invertebrate embryos was successful, the cryopreservation of crustacean embryos has not yet been reported. On the basis of previous study, a single vitrifying cocktail (Code A: 30% propanediol and 20% dimethyl formamide) was selected as a cryoprotectant from six kinds of vitrifying solutions to cryopreserve E. sinensis embryos. The cleavage stage, the gastrula, the pre-nauplius, and the first zoea stage embryos were serially acclimated in 25, 33, 50, 67 and 100% vitrifying solution A for 6 min. Embryos were acclimated for 1 min on the 20 cm liquid nitrogen (LN2) layer, and then plunged into liquid nitrogen. After storage for several minutes in LN2, straws containing frozen embryos were acclimated for 1 min on the 20 cm liquid nitrogen (LN2) layer, then quickly removed from LN2. For thawing, straws were immersed quickly for 1 min in a water bath at 37°C, then carefully washed with 0.25 mol/l sucrose and were then incubated with 15 ppt seawater in a Petri dish at 25°C. The survival rate in the vitrifying solution differed for embryos in different stages of development, and the survival rate of different stage embryos declined with the increase of acclimation time in code A vitrifying solution. The survival rate of pre-nauplius stage embryos did not significantly differ when the embryos were washed for 5, 10, 15, or 20 min with 0.25 mol/l sucrose after thawing. There was no survival after either cleavage stage embryos, or gastrula stage embryos were frozen. Eight pre-nauplius stage embryos survived and the survival rate was 9.3 ± 2.5%, but subsequently the embryos died at the fourth day. Seven first zoea stage embryos survived, and the survival rate was 11.3 ± 3.6%, one frozen-thawed embryo hatched at the seventh day. We conclude that cryopreservation of E. sinensis embryos by vitrification is feasible.

Crustaceana

International Journal of Crustacean Research

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References

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Figures

  • The effects (mean ± S.E.M., n=3) of acclimation time on the survival rates of Eriocheir sinensis H. Milne Edwards, 1853 embryos at different stages.

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  • The effects (mean ± S.E.M., n=3) of washing time on the survival rate of Eriocheir sinensis H. Milne Edwards, 1853 stage embryos.

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  • Results of vitrificable cryopreservation of Eriocheir sinensis H. Milne Edwards, 1853 embryos. Different stage embryos were acclimated in vitrifying solution A for 30, 40, 50 and 60 min, and the transparent embryos rate (TM) and survival rate (SR) (mean ± S.E.M, n=5) were determined.

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  • Embryonic development of Eriocheir sinensis H. Milne Edwards, 1853 after cryopreservation. A, the second day of pre-nauplius stage embryo after washing. It was acclimated for 40 min in code vitrifying solution A and frozen for 40 min in liquid nitrogen; B, the third day of frozen pre-nauplius stage embryo; C, the fourth day of frozen pre-nauplius stage embryo; D, the second day of first zoea stage embryo after washing. It was acclimated for 40 min in vitrifying solution A and frozen for 35 min in liquid nitrogen; E: the third day of frozen first zoea stage embryo; F, the fourth day of frozen first zoea stage embryo; G, the fifth day of frozen first zoea stage embryo; H, the sixth day of frozen first zoea stage embryo, the tail curved; I, on the seventh day of frozen first zoea stage embryo, the embryo hatched.

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