The development of shell-less culture methods for bird embryos with high hatchability would be useful for the efficient generation of transgenic chickens, embryo manipulations, tissue engineering, and basic studies in regenerative medicine. To date, studies of culture methods for bird embryos include the whole embryo culture using narrow windowed eggshells, surrogate eggshells, and an artificial vessel using a gas-permeable membrane. However, there are no reports achieving high hatchability of ＞50% using completely artificial vessels. To establish a simple method for culturing chick embryos with high hatchability, we examined various culture conditions, including methods for calcium supplementation and oxygen aeration. In the embryo cultures where the embryos were transferred to the culture vessel after 55-56 h incubation, more than 90% of embryos survived until day 17 when a polymethylpentene film was used as a culture vessel with calcium lactate and distilled water supplementations. The aeration of pure oxygen to the surviving embryos from day 17 yielded a hatchability of 57.1% (8 out of 14). Thus, we successfully achieved a high hatchability with this method in chicken embryo culture using an artificial vessel.

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Ex ovo culture of avian embryos can be applied not only to embryology but also to various fields of basic research such as embryo manipulation, toxicology, and regenerative medicine. The windowing method, which facilitates various manipulations and observations by opening a hole in one part of the eggshell, and culture systems using surrogate eggshells, are widely used. Despite this, biology lessons in high schools cover shell-less culture systems, which involve the development of avian embryos in artificial vessels, such as rice bowls, without using surrogate eggshells. However, as embryo development stops at its early stages in this method, it is not possible to continuously observe the development of the embryo. This led to attempts to develop an embryo culture method using a complete artificial culture vessel that does not use surrogate eggshells, and Kamihira et al. (1998) succeeded in hatching quail embryos in an artificial culture vessel using polytetrafluoroethylene membranes. In addition, Tahara succeeded in hatching chick embryos in artificial culture vessels that used cling film made of polymethylpentene and reported their detailed methodology ( Tahara and Obara, 2014 ). These technologies are being applied not only to school education but also to various fields of research.

This study examined the effects of calcium lactate on the development of chicken embryos in a shell-less culture system (cSLCS) up to the seventeenth day of incubation.In the presence of calcium lactate, a significant reduction in embryo viability was observed during the first week of incubation in cSLCS. On day 17 of embryo development, no significant difference was observed in the blood plasma calcium concentration or tibia bone density between cSLCS and intact control embryos, whereas the tibia length was significantly shorter in cSLCS embryos than in the intact control. These results suggest that calcium lactate supplementation in cSLCS supports bone formation in developing chicken embryos, but has adverse effects on the viability of embryos, particularly during the first week of embryo development.

The effects of oxygen gas injection starting on day 17 of incubation (D17) in a chick shell-less culture system (cSLC) on the subsequent embryo development were examined on day 19 of incubation (D19). On D19 of cSLC, the plasma phosphorus and total cholesterol concentrations of the embryos were significantly higher (P < 0.05), while the plasma calcium concentrations were significantly lower (P < 0.05) than those in the intact control (IC) group. However, no significant differences in embryo viability and other major blood component levels were observed among the experimental groups (P > 0.05). The percutaneous oxygen saturation was lower in D17-cSLC embryos before oxygen gas supplementation than in the IC (P < 0.05) embryos. Severe renal tubular degeneration of the metanephros was observed in D19-cSLC embryos despite oxygen gas injection starting from D17. These results indicate that D19-cSLC embryos are hypoxia even after injecting oxygen gas starting on D17.