Guidelines for Mouse and Rat Euthanasia Procedures for Investigative Personnel

Unit for Laboratory Animal Medicine
Jul 1, 2018 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


    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.

  • Procedures

    1. General Procedures

    1. Methods of euthanasia must be in accordance with an IACUC approved animal use protocol.
    2. Deviations from this Guideline 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. Consolidate compatible animals within the animal room.
    2. 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.
    3. Euthanize animals immediately upon consolidation.
      1. Start the euthanasia process no more than 10 minutes from the start of animal consolidation.
    4. 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 use protocol.
    2. Follow the CO2 Step by Step Euthanasia Procedures posted within the euthanasia room to euthanize animals.
      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.
    5. Observe and monitor animals continuously until death.
      1. It can take animals five, or more, minutes to cease breathing from the start of CO2 exposure.
    6. Ensure animals have stopped breathing during euthanasia before turning the CO2 station off.
      1. The most common method of adult and adolescent rodent (> 10 days old) euthanasia is exposure to CO2 in compressed gas form (dry ice is not acceptable).
      2. Euthanize pregnant rodents with CO2. This method causes death to embryos and fetuses.
      3. Perform a secondary method of euthanasia (see Procedures section 4).
      4. Neonates (pinkies) <10 days old, are typically euthanized by decapitation. A secondary method is not required when utilizing the decapitation method for neonates (pinkies).
        1. CO2 euthanasia is not effective in neonates. Inhalation anesthetics or CO2 exposure may be used as an induction method prior to decapitation but is not required.
        2. Perform a decapitation if inhalant anesthetics or CO2 exposure is utilized in neonates.
    7. Clean or disinfect the euthanasia chambers, between each group of animals euthanized and 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 use protocol.
    2. Secondary methods of euthanasia include the following: bilateral pneumothorax, decapitation, cervical dislocation, exsanguination, or removal of a vital organ.
    3. The most commonly used secondary method is bilateral pneumothorax.
      1. Using a scalpel blade, an incision is made in both sides of the rodent's chest cavity or through the diaphragm.

    5. ULAM Fee for Service

    1. Laboratories may request ULAM to euthanize animals by:
      1. Flippin​g 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. Animals under 21 days of age cannot be removed from the dam’s cage.
      6. All animals in the cage are euthanized when utilizing this service from ULAM.
  • Appendix A: CO2 Step by Step Rodent Euthanasia Instructions

  • Appendix B: CO2 Step by Step Rodent Euthanasia Instructions (Brehm / BSRB)

  • 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,