CO2 Euthanasia

Guidelines for Euthanasia of Rodents Using Carbon Dioxide

Rodents must be euthanized by trained personnel using appropriate techniques, equipment and agents. This is necessary to ensure a painless death that satisfies research requirements. Death should be induced as painlessly and quickly as possible. Upon completion of the procedure, death must be confirmed by an appropriate method, such as ascertaining cardiac and respiratory arrest or noting an animal's fixed and dilated pupils. Euthanasia should not be performed in the animal room. The euthanasia method must be appropriate to the species, approved in the animal study proposal and conform to the most recent Report of the AVMA Panel on Euthanasia.

  1. The euthanasia chamber should allow ready visibility of the animals. Do not overcrowd the chamber: all animals in the chamber must be able to make normal postural adjustments.
  2. "Compressed CO2 gas in cylinders is the only recommended source of carbon dioxide because the inflow to the chamber can be regulated precisely. Carbon dioxide generated by other methods such as from dry ice, fire extinguishers, or chemical means (eg, antacids) is unacceptable. Species should be separated and chambers should not be overcrowded. With an animal in the chamber, an optimal flow rate should displace at least 20% of the chamber volume per minute. Gas flow should be maintained for at least 1 minute after apparent clinical death. It is important to verify that an animal is dead before removing it from the chamber. If an animal is not dead, CO2 narcosis must be followed with another method of euthanasia." From the AVMA Guidelines on Euthanasia 2007
  3. Animals should be left in the container until clinical death has been ensured.
  4. Neonatal animals (up to 14 days of age) are resistant to the effects of CO2, therefore, alternative methods are recommended.3 Carbon dioxide may be used for narcosis of neonatal animals provided it is followed by another method of euthanasia (i.e. decapitation using sharp blades).
  5. If an animal is not dead following CO2 exposure, another approved method of euthanasia (e.g. decapitation) must be added while the animal is under CO2 narcosis to assure death. Please refer to Appendixes 1 and 2 of the 2007 Report of the AVMA Panel on Euthanasia for additional recommended methods.


  1. Danneman PJ, Stein S, Walshaw SO. Humane and practical implications of using carbon dioxide mixed with oxygen for anesthesia or euthanasia of rats. Lab Anim Sci 1997, 47:376-385.
  2. AVMA Panel on Euthanasia. 2007 Report of the AVMA Panel on Euthanasia
  3. Guidelines for the Euthanasia of Mouse and Rat Fetuses and Neonates. NIH Animal Research Advisory Committee, 2001.