Carbon Dioxide Euthanasia of Laboratory Rodents


Euthanasia is defined as the act of killing an animal by a method that induces rapid unconsciousness and death without pain or distress. In order to comply with federal regulations (USDA and PHS) and the Guide for the Care and Use of Laboratory Animals, the UConn Health’s Institutional Animal Care and Use Committee has established a policy on the use of CO2 for euthanasia.


  1. Ideally, animals should be euthanized in their home cage.  If this is not possible (e.g., because an investigator is culling animals from multiple cages for euthanasia), the following will apply for transport of animals to the euthanasia station:
    1. The maximum cage density for euthanasia in a standard mouse cage will be 250 g/cage (approximately 8-10 mice)
    2. For cage densities greater than 125 g/cage in a standard mouse cage, animals must be euthanized within 30 minutes of being placed at these cage densities.
  2. Compressed CO2 gas will be allowed to flow into an uncharged cage bottom or container.  Pre-filling of the chamber with CO2 is not allowed. As high concentrations of CO2 may be distressful to some species, gradual displacement of room air with the CO2 should allow for the anesthetic effect of CO2 to take place prior to asphyxiation which will result in the least amount of pain and/or distress to the animals.
  3. For successive euthanasia procedures, the euthanasia chamber must be filled with room air before flooding with CO2 again.
  4. To comply with the current AVMA Guidelines on Euthanasia, the flow rate into the euthanasia chamber must be at a rate of 30-70% of the total volume of the chamber per minute. To this end, a regulator must be used. The Euthanex® system is recommended by the IACUC; however, any commercial regulator system may be used as long as it meets the following criteria:
    1. The system must be AVMA compliant;
    2. The system must regulate a precise control of CO2 flow;
    3. The system must be calibrated specifically for CO2 gas; and
    4. The flow rate must be set by the factory and not be alterable by the user.
  5. Death must be verified after euthanasia and prior to disposal. This typically should be done by visual examination (cessation of respiration and heartbeat), cervical dislocation, or thoracic trans-section.
  6. CO2 euthanasia must be performed by individuals who are skilled in this method for the species in question and it must be performed in a professional and compassionate manner.
  7. Unintended recovery of the animals after apparent death from CO2 is a documented occurrence. These incidents constitute serious non-compliance with PHS Policy and deviations from the provisions of the Guide.


1. Conlee, KM, Stephens, ML, Rowan, AN, and LA King. “Carbon Dioxide for Euthanasia: Concerns Regarding Pain and Distress, with Special Reference to Mice and Rats.” Laboratory Animals (2005) 30, 137-61.

2. Hawkins, P, Playle, L, Golledge, H, Leach, M, Banzett, R, Coenen, A, Coooper, J, Dannemann, P, Flecknell P, Kirkden, R, Niel, L, and M. Raj. Report of the Newcastle Consensus Meeting on Carbon Dioxide Euthanasia of Animals held on 2/27-28/06, University of Newcastle upon Tyne, UK, August 9, 2006.

3. PHS Policy on Humane Care and Use of Laboratory Animals Clarification Regarding Use of Carbon Dioxide for Euthanasia of Small Laboratory Animals. NIH Notice NOT-OD-02-062. July 17, 2002.

4. The Guide for the Care and Use of Laboratory Animals. National Research Council, 2011.

Effective Dates: June 2, 2022 through June 30, 2025