Shell-less Culture Methods for Bird Embryos with High Hatchability

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. The 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 to achieve high hatchability of >50% using completely artificial vessels. So, our group established 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 8days incubation, more than 90% of embryos survived until day 12 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 8 yielded until day 14 with no hatchability. Only one egg survives until the 14 days. Mostly, die at day 10. Only 2 days survive after the first experiment where the egg was in the day 8 development. Thus, our lab not successfully achieved a high hatchability with this method in chicken embryo culture using an artificial vessel.

Fertileze hens’ eggs, incubated in room air, were examined at 8, 9, 10, 11, 12, 13 and 14 days of incubation and compared with eggs incubated in 60 % oxygen. Eggs incubated in high oxygen had lower diffusing capacities for carbon monoxide, lower rates of oxygen consumption per gram of embryo, heavier embryos and more advanced embryonic morphological development than eggs incubated for the same time in room air. The lower rates of oxygen consumption per gram of embryo are consistent with an increased rate of growth, because oxygen consumption (per gram of embryo) decreases as weight increases; the lower diffusing capacities of eggs incubated in 60% oxygen are interpreted as due to either a decreased chorioallantoic capillary surface area, or an increased thickness of the diffusion barrier between the chick blood and air, or both.

In order to investigate the influence of the egg shell on the process of shell calcium mobilization by the chorioallantoic membrane (CAM), chick embryos were maintained in long-term cultures in vitro without the shells. The shell-less embryos were severely calcium deficient and showed signs of retarded development and anomalous skeletal calcification. Throughout development, calcium transport and calcium-binding protein (CaBP) activities were diminished in the CAM of shell-less embryos as compared to those of control embryos which developed in ovo. The levels of developmentally expressed carbonic anhydrase activity remained, however, similar. By means of a single radial immunodiffusion assay of CaBP using a specific anti-CaBP antiserum, the level of immunoreactive CaBP was found to be significantly increased in the CAM of the shell-less embryos. These studies indicate that the CAM of chick embryos cultured under shell-less conditions is defective in calcium transport, probably as a result of the expression of an inactive form of the CaBP.