Laboratory Animal Science Center (BUMC)

CO2 Euthanasia of Rats and Mice

Background

“Euthanasia" is a term meaning “good death". The goal of euthanasia is to provide a rapid, painless, stress-free death. Carbon dioxide overdose causes rapid unconsciousness followed by death. In adult mice and rats, unconsciousness occurs within 10 seconds and death occurs within 1-2 minutes of exposure to 100% CO2. (Note: Newborn animals and pups are highly resistant to CO2, and require at least 10 minutes of exposure to ensure death.) Exposure of mice and rats to CO2 without removing them 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 carbon dioxide (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. BUMC IACUC policy states that when CO2 is used to euthanize small rodents, death must be assured by subsequent use of a secondary form of euthanasia. The IACUC recommends the following methods as secondary methods to ensure euthanasia after CO2asphyxiation:

  • Bilateral thoracotomy
  • Cervical dislocation
  • Removal of multiple organs for tissue procurement
  • Exsanguination

  1. Euthanasia apparatus are found in the procedure rooms of each facility. Bring the animals, in their home cages, to the procedure room.

  2. Do not overload the cage. The Mouse/Rat Housing and Cage Density Policy must be followed at all times. Mice and rats from different cages should not be combined- this causes extreme stress and is at odds with the goal of euthanasia.

  3. Remove the filter top from the cage, and cover the cage with the stainless steel euthanasia lid.
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  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 around the valve, not by screwing it on.
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  5. Turn the large knob (A) on top of the CO2 tank to turn on the CO2 flow.
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  6. Important: the pressure gauge (B) should read 15 psi; if it does not, adjust it using the small handle below it.
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  7. Turn on the flow of CO2 to the cage by pressing the red lever (C) until it is vertical. Wait 30 seconds, then return the lever to the horizontal off position. BUMC IACUC policy states that when CO2 is used to euthanize small rodents, death must be assured by subsequent use of a secondary form of euthanasia. The IACUC recommends the following methods as secondary methods to ensure euthanasia after CO2 asphyxiation: bilateral thoracotomy, cervical dislocation, removal of multiple organs for tissue procurement, exsanguinations.

  8. At this point, if you have another cage to euthanize, disconnect the quick-connect valve, connect it to ANOTHER lid (each procedure room contains at least two), and repeat step 5. You must leave the original lid in place on the first cage for at least two minutes.

  9. Wait two minutes, and visually inspect the animals for the absence of movement and respiration. If there is any question that an animal is dead, check for a heartbeat, if necessary.

  10. When all cages of animals are euthanized, turn the main tank control knob (A) 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 plastic bags. Excess bedding is not to be placed in the bag. A glove can be used to bag a single mouse.

  12. Place the carcass bag in the facility cooler. Return the empty dirty cages in the dirty cage wash