Guidelines for Mouse and Rat Euthanasia Procedures for Investigative Personnel

Unit for Laboratory Animal Medicine
Jul 20, 2020 12:00 am

To establish acceptable methods of euthanasia for investigative personnel to perform. The current procedure is composed of two parts; a primary method followed by a secondary method to ensure death and/or prevent revival.

  • Responsibility

    1. Investigative Personnel
  • Glossary Definitions

    American Veterinary Medical Association (AVMA)

    An association representing veterinarians across the United States focusing on advancing the science and practice of veterinary medicine to improve animal and human health.


    Term used to describe ending the life of an individual animal in a way that minimizes or eliminates pain and distress.

    Euthanasia Chamber

    Used for exposure to euthanasia-inducing gases and may be either the animal’s home cage or a chamber specifically designed for the purpose of euthanasia. The euthanasia chamber or the euthanasia chamber lid must be transparent and allow an unobstructed view of the animal during the euthanasia process.

    Institutional Animal Care & Use Committee (IACUC)

    Institutional Animal Care & Use Committee. The IACUC is ultimately responsible for approving, requiring modification of, or prohibiting the use of vertebrate animals in research, teaching, and/or testing activities at the University of Michigan. This includes supervision, coordination, training, guidance, and review of every project proposed to include the use of vertebrate animals to ensure that the project has appropriate scientific merit. 

    Laminar Flow Hood (LFH)

    An enclosure in which room air is drawn through a HEPA filter and the blown in a very smooth and vertical, laminar flow so as to prevent contamination of animals by airborne organisms.

  • Procedures

    1. General Procedures

    1. Methods of euthanasia must be in accordance with an IACUC approved animal use protocol.
    2. Deviations from the AVMA guidelines require scientific justification and approval by the IACUC.
    3. Methods described in this document are the most common forms of euthanasia. Contact Training Core for training on all euthanasia methods,

    2. Animal Consolidation Procedure for Inhalant Euthanasia

    It is preferable to use the home cage during CO2 euthanasia. However, if it is necessary to consolidate rodents for CO2 euthanasia the following limits should apply to ensure brief grouping of compatible animals in acceptable densities to reduce potential for stress during euthanasia.

    1. Euthanize animal’s in the home cage to reduce stress whenever possible.
    2. Consolidate compatible animals, when necessary, in the LFH or BSC within the animal room.
      1. Do not combine incompatible animals.
    3. Provide ample space for animals to move and assume normal postures.
      1. Maximum of 10 mice in a standard mouse shoe box cage.
      2. Maximum of 25 mice in a large mouse or small rat shoe box cage.
    4. Euthanize animals immediately, within 10 minutes, upon the start of consolidation to reduce animal stress and injury.
    5. Follow standard housing densities when consolidating other rodents (e.g. rats) for euthanasia.

    3. Primary Euthanasia Method

    1. Employ euthanasia methods according to the IACUC approved animal care and use protocol.
    2. Refer to the CO2 Step by Step Euthanasia Procedures posted within the euthanasia room for guidance.
      1. See Animals Administered a Hazardous Substance Requiring Containment for additional information regarding hazardous animal euthanasia.
    3. Calibrate the carbon dioxide (CO2) station to the optimal flow rate. See Appendix C if rate is not currently calibrated.
    4. Ensure the euthanasia chamber is NOT pre-filled with CO2 prior to placement of animals into the chamber.
      1. CO2 must be in compressed gas form (dry ice is not acceptable).
    5. Observe and monitor animals continuously until death (breathing ceases) for all animals in the euthanasia chamber.
      1. It may take animals several minutes to stop breathing during euthanasia.
    6. Ensure animals have stopped breathing during euthanasia before turning the CO2 tank off.
    7. Euthanize rodents 10 days of age and older or if pregnant with CO2.
      1. This method causes death to embryos and fetuses.
    8. Perform a secondary method of euthanasia (see Procedures section 4).
    9. Euthanize neonates <10 days old, by decapitation. A secondary method is not required when utilizing the decapitation method for neonates (pinkies).
      1. Exposure to CO2 is not as effective in neonates and is not recommended as a primary method of euthanasia.
        1. CO2 exposure or inhalation anesthetics can be used as an induction method prior to decapitation if necessary.
      2. Perform a decapitation if CO2 exposure or inhalation anesthetics are utilized in neonates.
    10. Clean or disinfect the euthanasia chamber:
      1. Between each group (e.g. cage) of animals.
      2. After the euthanasia process is complete.

    4. Secondary Euthanasia Method

    1. Procedures must be employed to ensure death so that the animal cannot revive and must be in accordance with the IACUC approved animal care and use protocol.
    2. Secondary methods of euthanasia include the following:
      1. Bilateral pneumothorax
      2. Decapitation
      3. Cervical dislocation
      4. Exsanguination
      5. Removal of a vital organ
    3. The most commonly used secondary method is bilateral pneumothorax.
      1. Puncture the diaphragm and/or each lung through the rodent’s chest cavity using a scalpel blade.

    5. ULAM Fee for Service

    1. Laboratories may request ULAM to euthanize animals by:
      1. Flipping the original cage card over and denoting on the back “ULAM sac/euth” and date, or by providing the following information on the back of a replacement cage card:
        1. PI name
        2. Protocol number
        3. Account number or shortcode number
        4. Laboratory contact name and phone number
        5. ULAM sac/euth
        6. Date
      2. Ensure animals have adequate food and water.
      3. Per diem is not charged past the date of request.
      4. Cannot be used for animals that require immediate attention, e.g. moribund.
      5. All animals in the cage are euthanized when utilizing this service from ULAM.
  • Appendix A: (Quick Reference Sheet) Guidelines for Rats & Mice Euthanasia Procedures

  • Appendix B: CO2 Step by Step Rodent Euthanasia Instructions (Inhouse)

  • Appendix C: Calculate CO2 Flow Rate

  • Appendix D: CO2 Euthanasia Station Ordering Information

Species: Mice Rats
Topic: Euthanasia

Questions or concerns about the content of this document should be directed to the Unit for Laboratory Animal Medicine (ULAM) at (734) 764-0277 or Contact Training Core for training on all euthanasia methods,