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SOP 7.5.2 - Humane Killing of Mice with CO2

University Animal Care Committee Standard Operating Procedure

Document No: 7.5.2

Subject: Humane Killing of Mice with CO2

Date Issued: August 21, 2012

Revision: 2

Location: Queen鈥檚 University

Responsibility: Principal Investigators (PI), Research Staff, Veterinary Staff

Purpose: The purpose of this Standard Operating Procedure (SOP) is to describe the procedure for the humane killing of adult rodents using CO2 asphyxiation.

1. Introduction and Definitions:

As per the UACC policy on humane killing of animals used in science, CO2 asphyxiation is a conditionally acceptable method of humane killing for rodents when conducted properly (current Canadian Council on Animal Care guidelines). Principal Investigators will be required to justify its use (within their animal use protocol). Following humane killing with CO2 asphyxiation, a secondary method must be used to confirm death.

Abbreviations:

Animal Care Services ACS, Principal Investigator PI, subcutaneous SC, intravenous IV, intraperitoneal IP, intramuscular IM, per os PO, per rectum PR

2. Materials:

  • CO2 source (facility outlet, or tank and regulator)
  • Flow meter
  • Appropriately sized lid for the rodent cage (custom lids for CO2 humane killing)
  • Instruments (for secondary method of humane killing)
  • Body bags

3. Procedures:

  • In order to minimize stress, animals should be humanely killed in their home cage with a maximum of:
    • Five adult mice or one litter per cage (do not pool mice from different cages).
    • Neonatal mice (up to 10 days of age) are resistant to the hypoxia induced by CO2, therefore, alternative methods are recommended (e.g. decapitation using sharp blades). See SOP 7.5.5 Euthanasia Method for Rodent Neonates and Fetuses.
  • Place the appropriately sized CO2 lid on the animal cage (remove cage lid first). Lid holes must be unobstructed and over the body of the cage.
  • Ensure the regulator hose is connected to the lid fitting.
  • Do not pre-charge the chamber.
  • Open the CO2 tank valve (if using).
  • Set the regulator to 3.44 LPM
    • Mouse cage (5鈥 x 8鈥 x 15鈥): 3.44 LPM (liters per minute)
    • Cages of different dimensions: contact Veterinarians
  • After the animals have become unconscious, the flow rate can be increased to reduce the time to death. The time required for humane killing can be several minutes.
  • Maintain the CO2 flow until the animal has stopped breathing.
  • Close the flow meter, or the valve on the tank (if using tank system).
  • Leave the animals in contact with loaded CO2 cage for a minimum of 2 additional minutes.
  • To check for death, verify: no chest movement, poor mucous membrane colour, and colour change or opacity of the eyes.
  • To confirm death, verify: no palpable heartbeat and no response to toe pinch.
  • Once the cage is empty and the session is complete, ensure the tank is closed and flush the line (if using this system).
  •  A secondary method of humane killing such as cervical dislocation or bilateral pneumothorax is required (unless justified in the animal use protocol) before disposal to ensure death.

, Volume 45, Number 1, January 2006, pp. 98-105(8) James Artwohl, Patricia Brown, Brian Corning and Susan Stein Report of the ACLAM Task Force on Rodent Euthanasia

Date New Version
08/21/2012 SOP Created
02/28/2019 Triennial Review
01/22/2025 Triennial Review - changed format, updated wording, removed fetus and neonate section, and updated to Humane Killing

 

SOP 7.5.2 - Humane Killing of Mice with CO2

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