Author: Jason Raynor
First, a bit of explanation on how CO2 actually kills. CO2 is a natural by-product of respiration. This is what you exhale when you breath. The brain detects the CO2 concentration in your body and triggers the diaphragm to exhale. When using a CO2 chamber you are replacing the Oxygen in the chamber with CO2 (CO2 is heavier than Oxygen and forces it out of the chamber through the vent tube). Since the mice’s brains detect the increased CO2 levels in their bodies it causes hyperventilation (which just speeds up unconsciousness because the Oxygen in the chamber is being replaced with CO2). Though this looks bad, more often than not they are usually unconscious by this point. CO2 binds to the hemoglobin in their blood and causes unconsciousness very quickly.
All in all this is probably the most humane way to euthanize rodents besides cervical dislocation. The problem with Cervical Dislocation is that it can go badly if the rodent squirms at the last second (I’ve experienced this and it wasn’t pleasant).
After much research on different methods of building a CO2 chamber, I decided to go with a CO2 chamber with the CO2 provided by a paintball canister. After research and discussions with other feeder breeders I decided that the baking soda method was to inaccurate. If your baking soda isn’t fresh, the reaction will not be efficient and might not produce enough CO2.
Here is the parts list for the system I have built:
- 1 Paintball CO2 Canister: ~ $20 or so
- 1 CO2 Fill Adapter Remote On/Off 3000psi Gauge: ~ $10 on ebay
A word of warning: the valve with the gauge is kind of counter intuitive. The wheel on the top needs to be totally UP before you attach the valve to the tank. Screwed UP is OFF for this valve, screwed down is ON. I learned that the hard way. 
- 1 Watts Compression Needle Valve 1/4 in OD x 1/8 in MIP
- 2 Watts Hose Barb 1/8″ x 1/4″
- 10′ 1/8″ Clear Tubing
- Small Plastic Tub
- Window / Door Rubber Weather Stripping (optional)
- Silicone
- Teflon Plumbers Tape (for the threads of the various components)
All these parts can be found at a big chain hardware store except for the paintball CO2 canister & the valve / gauge. You will also need an adjustable wrench to tighten all the components to the valve. Once everything is together, put in a bathtub full of water and turn it on slightly to check for leaks.
Below is a short video on how it all goes together:
https://www.youtube.com/watch?v=U_5Jj6yYgT8
The CO2 chamber worked really well. The needle valve was instrumental in being able to easy control the flow of CO2. Slow at first to knock them out and then turned up to finish the job. There was no pee or poop to indicate fear. The just lay down and that was it. Their lungs were working hard but that is a natural reaction because of the limited oxygen. All in all, I was very happy with how humane the process was.
WARNING: GRAPHIC VIDEO BELOW – EUTHANASIA OF RODENTS (Click link for video)