Feasibility studies on vitrification of intact zebrafish (Brachydanio rerio) embryos

Authors: Zhang, T. and Rawson, D.M.

Journal: Cryobiology

Volume: 33

Pages: 1-13

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Authors: Zhang, T. and Rawson, D.M.

Journal: Cryobiology

Volume: 33

Issue: 1

Pages: 1-13

ISSN: 0011-2240

DOI: 10.1006/cryo.1996.0001

The feasibility of cryopreservation by vitrification of intact zebrafish (Brachydanio rerio) embryos at the 6-somite (12-h) and heartbeat (27-h) developmental stages was investigated. The vitrification characteristics and the toxicity of cryoprotectants methanol, Me2SO, ethanediol, glycerol, acetamide, propane-1,2-diol, and butane-2,3-diol, and their mixtures (supplemented with polyethylene glycol 400) were examined prior to embryo vitrification studies. The studies on vitrification characteristics of cryoprotectants in straws in the absence of embryos showed that butane-2,3-diol vitrified at the lowest concentration of 3 M, and cryoprotectant mixtures containing Me2SO (2 M), propane-1,2-diol (3 M), and polyethylene glycol 400 (0.5 M); methanol (2 M), propane-1,2-diol (5 M), and polythylene glycol 400 (0.15 M or 6% w/v); and butane-2,3-diol (2 M), propane-1,2-diol (3 M), and polyethylene glycol 400 (6%) vitrified in straws during cooling and showed no observable ice formation during warming. Cryoprotectant toxicity studies showed propane-1,2-diol and methanol to be the least toxic to zebrafish embryos, with a maximum no observed effect concentration of 3 and 5 M at 22 and 0°C, respectively, after 30-min exposure. A cryoprotectant mixture containing butane-2,3-diol (2 M), propane-1,2-diol (3 M), and polyethylene glycol 400 (6%) was found to be the least toxic to both 6-somite and heartbeat stage embryos at 22°C. Osmotic responses of both intact and dechorionated embryos during equilibration in vitrification solutions were also studied. The results demonstrated that although the chorion of the embryos was permeable to water and cryoprotectants, the vitelline membrane permeability to cryoprotectant was low. Vitrification of zebrafish embryos in straws showed that some embryos remained morphologically intact (up to 32%) although embryos were mostly ruptured after vitrification procedures. Embryos were observed to become temporarily opaque during warming, which was an indication of intraembryonic ice formation, especially following cracking of the glass. The attempts at vitrification of zebrafish embryos failed to result in embryo viability. The low degree of cryoprotectant permeability of the embryos is considered to be directly responsible for embryo injury during vitrification procedures. © 1996 Academic Press, Inc.

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