Instructions: Carbon Dioxide Euthanasia

CO₂ Euthanasia Procedure 10-2013 


“Euthanasia” is a term meaning “good death”.

The goal of euthanasia is to provide a rapid, painless, stress-free death. Carbon dioxide (CO2) overdose causes rapid unconsciousness followed by death. The CO₂ flow rate should displace 10% to 30% of the cage volume per minute. Exposure to CO2 without removing animals from their home cage is a rapid and humane method of euthanasia, because the animals are not stressed by handling or being moved to a new environment.

Exposure to CO2 can cause deep narcosis that can appear to be, but is not, death. In such cases, animals that superficially appear to be dead may eventually awaken; this arousal can occur after the disposal of carcasses into refrigerators or freezers. Presumed death after exposure to carbon dioxide must be confirmed based on careful assessment of the animal for unambiguous signs of death, such as cardiac arrest or fixed, dilated pupils.  CO₂ Narcosis must be followed with secondary method of euthanasia (i.e. cervical dislocation, thoracotomy, etc.)


            Euthanasia – derived from the Greek terms eu meaning good and thanatos meaning death.  A good death is the humane termination of an animal’s life.

            Bilateral thoracotomy – Incision of both sides of the chest cavity to cause the lungs to collapse

            Cervical dislocation- method that severs the spinal cord in the neck

            Exsanguination –method of withdrawing blood from the body

            Decapitation – method of using a guillotine or sharp pair of scissors to severe the head at the atlas/axis joint of the neck

Procedures and Instructions

Standard Size Caging for Medical Campus

  Cage size Flow rate


Mouse 7” x 11” x 5” 1.3 Liters/minute


Mouse 8” x 13” x 5” 1.7 Liters/minute


Rat 10” x 19” x 9” 5.6 Liters/minute

If a standard size cage is not used the flow rate needs to be calculated using the formula below.

 To Calculate flow rate:

Length x width x height of cage in inches = total cubic inches

1 liter = 61.02 cubic inches

Divide total cubic inches by 61.02 cubic inches = liters

Multiply cage size in liters by 20 percent = flow rate 

  1. Euthanasia apparatus are found in the procedure rooms, specific animal rooms, satellite labs, or necropsy rooms of each facility. Bring the animals, in their home cages, to the CO₂ euthanasia apparatus found in the room.
  2. Do not overload the cage. The Housing and Cage Density Policy must be followed at all times. Five mice per cage maximum and for other species, only 2 per cage under 500 grams and 1 per cage for animals larger than 500 grams.  Animals from different cages should not be combined – this causes extreme stress and is at odds with the goal of euthanasia. In some cases, the animals may need to be placed into a standard size cage to be euthanized.  If the home cage is not used, the chamber should be emptied and cleaned between uses. 
  3. Remove the filter top from the cage, and cover the cage with the stainless steel euthanasia lid.
  4. Ensure that the tubing from the CO2 tank is properly attached to the lid via the Quick-connect valve. The quick-connect works by pulling back on the ring round the valve, not by screwing it on. 
  5. Turn the large knob (H tank) or the key (E tank) on top of the CO2 tank to turn on the CO2 flow. 
  6. Turn on the flow of CO2 to the cage by using the flow meter attached to the pressure regulator. Adjust flow meter to correct setting according to the cage size. 
  7. Expose the animals to CO₂until complete cessation of breathing is observed for a minimum of 2 minutes (a total of approximately 5 to 10 minutes is usually required). Visually inspect the animals for the absence of movement and respiration.
  8. Death must be assured by subsequent use of a secondary form of euthanasia.  The following methods as secondary methods to ensure euthanasia after CO2 asphyxiation: cervical dislocation (under 200 grams), bilateral thoracotomy, removal of multiple organs for tissue procurement, exsanguinations. Quickly decapitate all pups with sharp scissors. (Note: Newborn pups (up to 7 days old) are highly resistant to CO2, and require at least 4-5 minutes of exposure followed by a physical means such as decapitation to ensure death). 
  9. At this point, if you have another cage to euthanize, repeat steps 3-8.
  10.  When all cages of animals are euthanized, turn the flow meter to zero and the tank control knob off, and return the lids to their racks.
  11. Be sure to check again that all animals have been euthanized completely before securely double-bagging them in plastic bags. Excess bedding is not to be placed in the bag.
  12. Place the carcass bag in the facility cooler. Return the empty dirty cages to the dirty cage wash area.


Guide for the Care and Use of Laboratory Animals 8th edition, 2011, National Academy Press, Washington, D. C.

 AVMA Guidelines for the Euthanasia of Animal,  2013 edition, American Veterinary Medical Association, Schaumburg, IL