Developing chick embryos are a classical research tool in developmental biology. The whole embryo culture technique can be applied to various fields, such as embryo manipulation, toxicology, tumorigenesis, and basic research in regenerative medicine. When used for the generation of transgenic chickens, a high hatchability of genetically engineered embryos is essential to support normal embryonic development during culture. In this study, calcium carbonate, which is the main component of eggshells, was added as a calcium source in shell-less chick embryo cultures using a transparent plastic film as a culture vessel. In the absence of a calcium source in the shell-less culture system, embryogenesis ceased during culture, resulting in failed embryonic hatching. We found that the direct addition of calcium carbonate to the chorioallantoic membrane of the developing embryo was effective for the hatching of cultured chick embryos. The amount, timing, and location of calcium carbonate addition were investigated to maximize the hatchability of cultured embryos. Starting from the time of calcium carbonate supplementation, >40% hatchability was obtained with the optimal condition. This established method of shell-less chick embryo culture provides a useful tool in basic and applied fields of chick embryo manipulation.
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