Carbon dioxide (CO2) asphyxiation is an approved method of euthanasia for rodents, but must be performed properly to be effective and humane. The following procedures are designed to assure that CO2 euthanasia is performed properly. In all instances, a secondary method must be described in the approved ACUP and performed to ensure death. The procedures described are for adult animals. Neonatal and fetal rodents are very resistant to CO2 euthanasia and special procedures are required.

  • CO2 euthanasia can only be performed when approved as part of an animal use protocol approved by the OSU IACUC.
  • The source of CO2 should be from a compressed gas cylinder. Other options for generating CO2 gas are not approved for use.
  • Personnel performing CO2 euthanasia must be trained to do so.
  • Animals may be exposed to CO2 in their home cage or by placing the animals in a secondary container that is more easily sanitized. Either procedure will make it easier to keep the euthanasia apparatus clean and free of debris.
  • Animals may be euthanized singly or in groups. Each animal should have space to stand on all four feet and have sufficient space to turn around.
  • Pre-filling the chamber is not an approved step for this method of euthanasia.
  • The gas flow rate should provide a balance between the time to unconsciousness and the excitement caused by the noise or high-velocity air movement from high flow rates. A fill rate that gradually increases to replacing 20% of the chamber volume per minute is recommended.
  • CO2 is heavier than air. Excess gas must be allowed to exit from the top of the chamber.
  • The euthanasia chamber and/or lid should be transparent. You must be able to see animals without opening the chamber.
  •  Animals undergoing CO2 euthanasia should not be left unattended until death is assured. Gas should remain on for 1 minute after observation of clinical death.
  •  You must perform the secondary method approved by the IACUC in your ACUP.
  • Euthanasia chambers must be cleaned after use.

IACUC approved 5-4-11