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

All procedures involving chicken egg embryos have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparing the eggs

1. Before receiving the eggs, assemble and equilibrate the egg incubator to 37.8 °C (100 °F) with 60-70% humidity following the manufacturer's instructions.
   NOTE: To minimize contamination risks, autoclaved water may be used to control the humidity.
2. Upon receiving fertilized Rhode Island Red chicken eggs from a certified laboratory-grade egg supplier, dry wipe the surface of the shells with paper towels. A lightly dampened paper towel may be used to remove adhered material. Dry immediately.
   NOTE: Wetting the shell with liquid below 43.3 °C can introduce bacteria into the egg. If one chooses to wash or disinfect the eggs, the temperature of the liquid must be between 43.3-48.9 °C. Higher temperatures can boil the eggs. The choice of disinfectant should be made based on the microorganisms causing concern.
3. Use a pencil or marker to label the date on the egg. This is considered development day 0.
4. Place the eggs into the egg incubator and incubate for at least 7 days with rotation to permit CAM development. An automatic rotator may be used, or the eggs may be rotated 180° 2-3x per day.

2. Opening the eggs

NOTE: Opening of the eggs should be done when the CAM has fully developed. This is typically on development day 7 or 8.

1. Disinfect a biosafety cabinet with 70% ethanol. Similarly, disinfect and place into the biosafety cabinet an egg rack, egg candler, marker, cordless rotary tool with a silicon carbide grinding stone and circular cutting wheel, 18 G needle, pipette controller with quarter-inch vacuum tubing, packing tape, office scissors, curved scissors, Semken (or similar) forceps, cotton balls, and 6 x 7 cm transparent film dressing. Whenever possible, use sterile, disposable, or autoclaved tools.
2. Turn off the egg rotator. Place approximately 1-3 eggs into the biosafety cabinet on the egg rack. While in the dark, place the egg candler against the eggshell to identify the air cell. Mark the location of the air cell.
   NOTE: The intensity of illumination from the egg candler is crucial for visualizing the interior of the egg. If the air cell or vasculature is consistently difficult to see, try replacing the egg candler batteries.
3. Move the egg candler over the shell to find a large blood vessel network. Rotate the egg if necessary. An ideal vasculature will be branching near the middle of the egg. Use a marker to draw over the vasculature to be used for implantation.
4. Turn on the light in the hood. Using a cordless rotary tool fitted with a silicon carbide grinding stone, drill a small hole in the shell directly over the center of the air cell. Drill until most of the shell has been removed, but the white, inner membrane is intact.
   NOTE: Drilling over the air cell before targeting the vasculature permits determining the thickness of that particular eggshell over the air cell rather than over the CAM and vasculature. If the CAM or vasculature is disrupted, then the egg is not usable for implantation.
5. Drill and open another small hole where the vascular window will be opened as was done for the air cell (step 2.4).
6. Using an 18 G needle, gently pierce through the white, inner membrane over the air cell and vasculature. If unable to penetrate the shell, then carefully drill a little more. Ensure that the white, inner membrane (but not the CAM) is disrupted throughout the entirety of the drilled area. Removal of the membrane piece is not necessary.
   NOTE: If the CAM or vasculature is disrupted during this step, discard the egg. Damage is evident if there is blood or albumin leaking from a drilled hole.
7. Turn off the hood light. Using the egg candler, verify that the air cell was transferred from the end of the egg to the area over the vasculature. If necessary, place the 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.
8. Use a marker to outline the new location of the air cell approximately 0.5 cm inside the air-CAM boundary. Affix a piece of packing tape just over the new air cell. If necessary, use standard office scissors to trim the tape to an appropriate size.
   NOTE: Tape can disrupt airflow through the shell and should not be larger than necessary to fully cover the air cell.
9. Return the eggs to the incubator to warm without rotation with the new air cell facing up. Repeat steps 2.2-2.9 for the remaining eggs to be opened.
   NOTE: The protocol may be paused here. If unsure of the quality of CAM development, a small number of eggs should proceed through step 2.16 prior to opening the remaining eggs.
10. Place approximately 1-3 eggs with relocated air cells onto the egg rack in the biosafety cabinet.
    NOTE: Care should be taken to avoid introducing contamination into the opened egg. Therefore, always open eggs inside a biosafety cabinet, and use sterile tools and equipment whenever possible.
11. Using a cordless rotary tool fitted with a circular cutting wheel, cut a small line over the air cell boundary drawn in step 2.8. Ensure that this is approximately 0.5 cm inside the actual air-CAM boundary to avoid disrupting the CAM or vasculature. Cut completely through the shell but be careful not to penetrate deeply enough to disrupt the CAM or vasculature.
12. Using curved scissors, cut around the remaining air cell to create a window in the shell.
    NOTE: If blood or membrane is present on the removed shell, then the egg is not suitable for implantation. If a high proportion of the eggs are not cleanly opened, consider waiting at least 1 additional day to open the remaining eggs to permit better CAM formation. If the shells of the remaining eggs have not been pierced, rotation of the eggs within the incubator should be resumed.
13. Verify the viability of the embryo. Viable embryos will display extensive vasculature, clear albumin, embryo movement, or a visible heartbeat.
14. Using Semken forceps, pull small pieces of cotton from a sterile cotton ball. Gently blot the CAM surface to remove the shell dust and debris.
    NOTE: The removal of debris must be performed on the same day that the eggs are opened.
15. Cover the shell opening with a one-quarter piece of 6 x 7 cm transparent film dressing.
16. Return the eggs to the incubator. Ensure that the egg sits securely with the opened window facing up and the CAM not touching the transparent film dressing. Use a piece of egg rack, the edge of the egg rotator, or another suitable item to prop any eggs that keep rolling.
17. Repeat steps 2.10-2.16 for all remaining eggs to be opened.
    NOTE: The opening of the eggs does not need to be completed on the same day as implantation. Waiting at least 1 day can help eliminate eggs whose viability was compromised by opening the shell.