Egg Windowing: A Method of Opening Fertilized Chicken Egg Shell to Expose Chorioallantoic Membrane Vasculature

On developmental day 7 or 8, when the CAM has fully developed, turn off the egg rotator and place approximately 1 to 3 eggs into the egg rack in the biosafety cabinet. With the lights turned off, place an egg candler against the eggshell to identify and mark the location of the air cell of one egg. Then, move the egg candler over the shell to find a large blood vessel network, rotating the egg as necessary.

An ideal vasculature will branch near the middle of the egg. Use a marker to trace the vasculature to be used for implantation. After turning back on the light in the hood, use a cordless rotary tool fitted with a silicon carbide grinding stone to drill a small hole in the shell directly over the center of the air cell. When most of the shell has been removed, but with white, inner membrane intact, drill another small hole where the vascular window will be opened as just demonstrated. When the second hole is ready, use an 18 gauge needle to gently pierce the white inner membrane over the air cell and vasculature, taking care that the white, inner membrane, but not the CAM, is disrupted throughout the entirety of the drilled area.

With the hood light off again, use the egg candler to verify that the air cell has been transferred from the end of the egg to the area over the vasculature. If necessary, place vacuum tubing inserted into a pipette controller around the hole over the original air cell and gently apply suction in short bursts to move the air cell. Mark the outline of the new location of the air cell, approximately 0.5 centimeters inside the air-CAM boundary, and fix a piece of packing tape just big enough to cover the hole over the new air cell.

After preparing the other two eggs as just demonstrated, return the eggs to the incubator and open any additional eggs for the experiment in groups of 1 to 3 as demonstrated. When all of the eggs have been opened, transfer 1 to 3 eggs with relocated air cells to the egg rack in the biosafety cabinet, and use a cordless rotary tool fitted with a circular cutting wheel to cut a small line over the air cell boundary completely through the cell without disrupting the CAM or vasculature. Using curved scissors, cut around the remaining air cell to create a window in the shell and verify the viability of the embryo.

Viable embryos will display an extensive vasculature, clear albumen, embryo movement, and/or a visible heartbeat. In non-viable embryos, the CAM may appear opaque with few, if any, vessels present and the embryos may be small or absent without movement or a heartbeat. Using Semken forceps, pull small pieces of cotton from a sterile cotton ball and gently blot the CAM surface of the viable embryos to remove any shell dust and debris. Then, cover the shell opening with a one-quarter piece of 6 by 7 centimeter transparent film dressing without touching the CAM, and return the eggs to the incubator with the opened window facing up. Use a piece of egg rack, the edge of the egg rotator, or another suitable item to prop any eggs that keep rolling.