Shell-Less Culture System Lets Scientists Watch Chicken Embryos Hatch

Birds are everywhere: they chirp, glide, paddle and waddle, with feathers, beaks and claws. These extraordinarily varied creatures are the nearest living relatives of the mighty dinosaurs, and have adapted to life in every part of the planet.

Yet one stage of their reproduction has remained difficult to study. Developing chicks are concealed within opaque shells, changing from a zygote into a hatchling before completing their journey to adulthood under the care of attentive parents.

Scientists have now achieved a long-standing ambition in avian developmental biology. They have created an egg-culture system that substitutes the opaque shell of domestic White Leghorn chickens (Gallus domesticus), making it possible to watch embryonic development almost from start to finish.

The shell-less culture system worked well enough for several chicks to hatch after the standard incubation period. Those chicks subsequently grew into normal chickens.

Shell-less culture system for chicken embryos

For decades, researchers have sought a shell-less culture system, or SLCS, that would allow them to view a developing bird embryo. One promising approach, first reported in 2014, allowed embryos to remain in the egg for their first three days, a period necessary for survival. They were then transferred into a culture vessel covered with a sheet of polymethylpentene kitchen cling film.

That technique had an important shortcoming, however. The chicks could not develop normally, which severely limited the hatching success rate.

Veterinary scientist Katsuya Obara of Okayama University of Science, who helped pioneer the earlier technique, led a team with colleague Chizuka Obara to address one of the barriers to normal development and hatching.

The researchers found that the yolk vitelline membrane, which protects the cell layer around the embryo’s outer edge called the blastoderm, was becoming dry inside the culture vessel. They suspected this drying could be disrupting ordinary embryonic development.

Testing movement and oxygen during development

To keep the membrane moist, the researchers placed the culture vessel on a rotary shaker, a device normally used to provide continuous movement for mixing. Its top plate was positioned at a 7-degree angle, and the team tested speeds of 6 rotations per minute, 10 rotations per minute and 28 rotations per minute.

The steady movement maintained a continuous flow of albumen and stopped the membrane from drying out, although development and survival through 10 days differed between the test conditions.

At 6 rotations per minute, survival was at its highest, but every embryo displayed developmental delays.

At 10 rotations per minute, survival was a little lower and some embryos developed abnormalities. Even so, the survival rate among normal embryos was high.

At 28 rotations per minute, every embryo had developed abnormalities incompatible with survival by day 10.

Since 10 rotations per minute offered the best balance of survival and development, the team then tested the addition of oxygen at several points after day 10. Supplemental oxygen was required, but the precise point at which it was introduced did not appear to affect embryo development.

Finally, the researchers repeated the procedure at 10 rotations per minute, adding supplemental oxygen and a dusting of calcium carbonate powder, the substance from which eggshells are made. This time, they also manually wiggled the culture vessel back and forth three to five times every 3 to 10 hours during the first 24 to 48 hours of shell-less culture.

Improved hatching rates in domestic White Leghorn chickens

This approach delivered the best hatching rate for normal chicks. With oxygen at 10 rotations per minute, the hatch rate was 3.3 percent. Manual back-and-forth shaking raised it to 10.5 percent.

One hatched chicken was allowed to live for a year before being euthanised and dissected. It was entirely normal. The successful system therefore provides a literal viewing window onto chicken development until hatching.

"The development of visible-cSLCSs has opened new possibilities by facilitating omnidirectional real-time monitoring of the phenotypic outcomes of experimental treatments through a transparent plastic film from blastoderm stage embryos until hatching," the researchers write in their paper.

"This system will serve as a powerful tool in various disciplines, such as toxicology, stem cell research, bioimaging, and regenerative medicine."

The team’s findings were published in Scientific Reports.