CO2 Euthanasia of Rodents

IACUC Policy: 034-06, Effective Date: 06/01/2009, Last Revision: 03/18/2026

Overview/Purpose

Carbon dioxide (CO2) gas is frequently used to euthanize rodents in a research setting. CO2 is introduced into a cage or space in a controlled manner to induce unconsciousness followed by respiratory and cardiac arrest. When using CO2 as a euthanasia method, care needs to be taken to follow the recommendations of the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals to ensure pain and distress are minimized. Improper euthanasia is considered noncompliance.

The purpose of this policy is to outline the appropriate steps for the use of CO2 to euthanize adult rodents. For neonatal or fetal rodents, see the "Euthanasia of Rodent Fetuses and Neonates" Policy as neonates/fetuses are resistant to hypoxia and require additional considerations.

Definitions

• Rodents: mammals of the order Rodentia which includes species such as mice, rats, hamsters, and guinea pigs.

Requirements

• Personnel must be appropriately trained to perform euthanasia. Training is available free of charge by University Laboratory Animal Resources (ULAR).
• CO2 must be supplied from a commercial compressed gas cylinder that allows for precise regulation of the flow rate.
• The flow rate for CO2 must be set to ensure 30–70% displacement of the chamber volume/min.
• Carbon dioxide flow should be maintained for at least 1 minute after respiratory arrest.
• Chambers cannot be prefilled with CO2 and should be inverted and cleaned between groups.
• Personnel must remain in the room during the entire euthanasia process, as animals must be observed during euthanasia to ensure that death is humane. Cages or chambers used for euthanasia must be clear or have a viewing port to allow visualization of the animals.
• Care should be taken to minimize stress prior to euthanasia. When possible, animals should be maintained in familiar groups in their home cage for euthanasia.
• If unfamiliar animals are combined for euthanasia, they:
  • Must be compatible.
  • Must be of the same species.
  • Should not exceed recommended densities:
    • 8 mice in a small rodent cage.
    • 15 mice in a large rodent cage.
    • 2 (>250 gm) or 4 (<250 gm) rats or guinea pigs in a large rodent cage.
• Confirmation of death is required prior to disposal. The IACUC requires a secondary physical method of euthanasia following euthanasia from an inhaled agent and confirmed lack of consciousness. Acceptable secondary methods include creation of a bilateral pneumothorax, removal of vital organs, decapitation, exsanguination, or cervical dislocation. Cervical dislocation must not be performed in rats >200 gm.
• Disposal bags must be labeled with the date, personnel initials, and name of the Principal Investigator (PI).