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Published Online: 20 January 2015

Short-Term Chilled Storage of Zebrafish (Danio rerio) Embryos in Cryoprotectant As an Alternative to Cryopreservation

Publication: Zebrafish
Volume 12, Issue Number 1


As zebrafish embryos have never been cryopreserved, we developed a protocol to store zebrafish embryos (50% epiboly—5.3 hour post fertilization) for up to 18 h at 0°C. Initial experiments to optimize the cryoprotectant (CPA) solution demonstrated improved embryo hatching rate following chilling at 0°C for 18 h with 1 M MeOH+0.1 M sucrose (56±5%) compared with other combinations of methanol (0.2–0.5 M) and sucrose (0.05–0.1 M). This combination of CPAs that protects against chilling injury was further tested to assess its impact on sox gene and protein expression. Significant decreases in sox3 gene expression were observed in hatched embryos that had been chilled for 18 h in 1 M MeOH+0.1 sucrose compared with non-chilled controls, however the expression of both sox2 and sox3 proteins was unaffected. Significant decreases in sox2 protein expression were, however, observed in embryos that had been chilled without CPAs and these embryos also had lower hatching rates than those chilled with the optimal CPA solution. We, therefore, conclude that the CPA combination of 1 M MeOH+0.1 M sucrose facilitates chilled storage of early stage (50% epiboly) zebrafish embryos for up to 18 h without compromising transcriptional response.

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Information & Authors


Published In

cover image Zebrafish
Volume 12Issue Number 1February 2015
Pages: 111 - 120
PubMed: 25545702


Published in print: February 2015
Published online: 20 January 2015
Published ahead of print: 29 December 2014


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Kunjan Desai
Department of Neuroscience and Regenerative Medicine, Georgia Regents University, Augusta, Georgia.
Institute of Biomedical Science and Environmental Science and Technology, University of Bedfordshire, Luton, United Kingdom.
Emma Spikings
Institute of Biomedical Science and Environmental Science and Technology, University of Bedfordshire, Luton, United Kingdom.
Tiantian Zhang
Institute of Biomedical Science and Environmental Science and Technology, University of Bedfordshire, Luton, United Kingdom.
School of Applied Sciences, Bournemouth University, Poole, United Kingdom.


Address correspondence to:Tiantian Zhang, PhDSchool of Applied SciencesBournemouth UniversityPoole BH12 5BBUnited Kingdom
E-mail: [email protected]

Author's Contributions

K.D. performed all the experiments and wrote the first draft of the article. E.S., contributed to data analysis and interpretation, codesigned the experiments, helped with the qPCR experiments, and the article preparation and revision. T.Z., participated in experimental design and article preparation. All authors approved the current version of the article.

Disclosure Statement

No competing financial interests exist.

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