CO2 Euthanasia of Rodents

IACUC Policy: 034-06, Effective Date: 06/01/2009, Last Revision: 03/17/2023

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 ensure that the rate for introduction of CO₂ follows 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 with the Public Health Service Policy on Humane Care and Use of Laboratory Animals (PHS Policy) and the Guide for the Care and Use of Laboratory Animals and is reportable to the Office of Laboratory Animal Welfare (OLAW).

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 the “Euthanasia of Rodent Fetuses and Neonates” Policy should be referenced as neonates / fetal animals are resistant to hypoxia and require additional considerations.

Definitions

• Rodents: mammals of the order Rodentia which includes 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 commercial compressed gas cylinder that allows for precise regulation of the flow rate. The flow rate for CO2 should be set to ensure 30-70% displacement of the chamber volume/min. Chambers cannot be prefilled with CO₂ and should be inverted and cleaned between groups. Carbon dioxide flow must be maintained for at least 1 minute after respiratory arrest. Dry ice as a source of CO₂ is unacceptable.
• 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 their home cage for euthanasia. If non-familiar animals are combined for euthanasia, they must be compatible (e.g. multiple foreign males should not be placed together for euthanasia); animals must be of the same species; and the total number of animals in a single cage should not exceed 8 in a small rodent cage or 15 in a large rodent cage for mice, or 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 Ohio State University Institutional Animal Care and Use Committee (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, initials, and name of the Principal Investigator (PI).

Applicable Regulations

• Animal Welfare Act (AWA, Public Law 89-544, 7 U.S.C.)
• Animal Welfare Act Regulations (AWAR, 9 CFR, Chapter 1, Subchapter A)
• Health Research Extension Act of 1985 and Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals
• National Research Council Guide for the Care and Use of Laboratory Animals, Eighth Edition. National Academy of Sciences, 2011
• AVMA Guidelines for the Euthanasia of Animals

Additional Information/Guidance

• None