Oxygen consumption was measured independently at the air
cell (V · O2lung) and the rest of the egg (V · O2CAM) in internally pipped
chicken embryos. Eggs were considered internally pipped if we
were able to see the embryo's beak in the air cell when candling
the egg. To isolate the air cell, we candled the egg and outlined the
air cell with a sharpie marker. Two small holes were made in the
shell above the air cell using an 18 gauge needle and short pieces
of PE190 tubing were inserted into the holes. The entire shell
above the air cell was then painted with Kerr DentalWax, sealing
the PE190 tubing into place. This allowed us to use the air cell as a
mini respirometer and regulate the O2 and CO2 content flowing
through the air cell. The egg was sealed in a respirometer jar
(236 mL) with ports for gas flow into and out of the chamber. The
PE190 tubing coming from the air cell was attached to 18 gauge
needles that were glued into the top of the jar allowing for flow of
the mixed gas through the air cell, independent of the rest of the
egg. The gas content of the air flowing into the air cell and the egg
chamber were controlled with two mass flow control systems
(Porter Mass Flow and MFC-2, Sable Systems). The initial gas
flowing though the air cell was composed of 12% O2, 5% CO2,
and 83% N2 and was considered to be normoxic for air cell gas
(Wangensteen and Rahn, 1970/71). The flowrate through the egg
respirometer and the air cell was set to approximately 50 mL
min−1 and was measured with a Flowbar-1 Mass Flow Meter
(Sable Systems).