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, [email protected].

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

1. Euthanize animals in the home cage to reduce stress whenever possible.
2. Do not consolidate animals with previously demonstrated aggression.
3. Consolidation of female mice and non-weaned pups from different cages is permitted, when necessary, utilizing the LFH or BSC within the animal housing room or at the euthanasia station.
   1. Do not consolidate weaned male mice within the animal housing room.
   2. Euthanize animals within 10 minutes upon the start of consolidation to reduce animal stress and potential for injury.
4. Consolidation of weaned male mice from different cages is permitted, when necessary, at the euthanasia station ONLY.
   1. Do not consolidate weaned male mice within the animal housing room.
   2. Euthanize weaned male mice immediately upon consolidation to reduce animal stress and injury.
5. 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.

1. Follow standard housing densities when consolidating rat (Rattus norvegicus) for euthanasia.
2. Euthanize USDA-regulated species per cage without consolidation.
3. Perform a secondary method of euthanasia (see Procedures section 4).
4. 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.
5. Calibrate the carbon dioxide (CO₂) station to the optimal flow rate. See Appendix C if rate is not currently calibrated.
6. Ensure the euthanasia chamber is made of a material that:
   1. Allows the animals to be visible during the entire procedure.
   2. Can be sanitized.
7. 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).
8. Observe and monitor animals continuously until death (breathing ceases) for all animals in the euthanasia chamber.
   1. It may take several minutes for animals to stop breathing during euthanasia.
9. Ensure animals have stopped breathing during euthanasia before turning the CO2 tank off.
10. The CO2 method for primary euthanasia is effective in:
    1. Weaned rodents.
    2. Non-weaned rodents between 10-21 days old.
    3. Pregnant rodents.
       1. The CO2 method causes death to embryos and fetuses.
11. The decapitation method for primary euthanasia is effective and recommended for rodents less than 10 days old.
    1. Exposure to CO2 is not as effective in rodents less than 10 days age 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 as a secondary method of euthanasia if CO2 exposure or inhalation anesthetics are utilized in neonates.
12. Clean or disinfect the euthanasia chamber:
    1. Between each group (e.g., cage) of animals.
    2. After the euthanasia process is complete.

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.

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.

1. See Documenting, Disposal and Reporting Animal Deaths.