A Pseudo-4D Study of Embryonic Skeletal Development in the Domestic Chicken, and New Insights into the 3D Structure of the Avian Eggshell Membrane

The avian embryo develops inside a perfect incubation chamber - the egg. In this study, we used X-ray microscopy to image the entire chick embryo inside intact fertilized eggs at 11 (the onset of mineralization), 13, 15 and 17 days post-fertilization. We applied a pre-trained neural network for segmentation of the embryonic skeleton in 3D, followed by quantitative morphometric analysis. A comparison between two genetic chicken strains (a layer strain bred for table eggs, and a broiler strain bred for meat) illustrates the robust and reproducible trajectories of bird development as refined by millions of years of evolutionary adaptation, and minor changes occurring between strains. A thin fibrous layer called the eggshell membrane intervenes between the contents of the egg and its calcitic shell, and this we have also characterized by X-ray microscopy, and how it attaches to the shell.

Key Learnings:

• The non-destructive nature of X-ray imaging allows visualization of chicks as they develop inside eggs
• Comparisons between embryo strains can be made by imaging and subsequently segmenting the skeleton in 3D prior to morphological analysis
• Different parts of the egg can also be characterized using X-ray microscopy, including the shell and eggshell membrane