Guidelines for Rodent Euthanasia Procedures for Investigative Personnel

1. Investigative Personnel

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 the ULAM Training Core ([email protected]) for training on all euthanasia methods.

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 small mouse cage.
   2. Maximum of 25 mice in a large mouse or small rat cage.

1. Follow standard housing densities when consolidating rat (Rattus norvegicus) for euthanasia.
2. Euthanize USDA-regulated rodent species as housed per cage without consolidation.
3. Common primary methods of euthanasia include, but are not limited to:
   1. Carbon dioxide (CO2) overdose
   2. Anesthetic overdose
4. Perform a secondary method of euthanasia after the primary method of euthanasia is performed (see Procedures Section 4).
5. 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.
6. Steps to operate the ULAM CO2 euthanasia station:
   1. Ensure the euthanasia chamber is:
      1. Made of a material that:
         1. Allows the animals to be visible during the entire procedure.
         2. Can be sanitized.
         3. NOT pre-filled with CO₂ prior to placement of animals into the chamber.
         4. CO₂ must be in compressed gas form (dry ice is not acceptable).
   2. Remove the cage lid, feeder, water bottle (if present),
      1. Ensure all animals are visible during the euthanasia process, e.g., pull apart the nest so that animals are not hiding from view.
   3. Place the euthanasia lid over the top of the cage.
      1. The CO2 hose can be inserted through the water bottle grommet or water valve opening if there is no euthanasia lid available.
   4. Open the CO2 tank by turning the valve counterclockwise and ensure the needle rises on the pressure gauge.
   5. Adjust the flowmeter by turning the valve counterclockwise until the gauge reaches the desired setting.
      1. Pre-calibrated settings are:
         1. Black for small RAT cages.
         2. Green for LARGE mouse cages.
         3. Blue for SMALL mouse cages.
      2. Calculate the optimal flow rate if there are no pre-calibrated flow rates on the flowmeter. See Appendix C.
   6. 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.
      2. Do NOT leave the animals unattended at any point during the euthanasia process.
      3. Ensure animals have stopped breathing during euthanasia before turning the CO₂ tank off.
   7. Close the CO2 tank by turning the valve clockwise until snug.
   8. Observe the pressure gauge dropping to zero, followed by the flowmeter ball dropping to zero.
   9. Close the flowmeter by turning the valve clockwise until snug. Do not overtighten.
7. 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.
8. 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.
9. Clean or disinfect the euthanasia chamber:
   1. Between each group (e.g., cage) of animals.
   2. After the euthanasia process is complete.

1. Perform a secondary, physical method of euthanasia on all animals to ensure death and that the animal cannot revive.
2. Secondary methods of euthanasia include the following:
   1. Bilateral pneumothorax
      1. Puncturing the diaphragm and/or each lung through the rodent’s chest cavity using a scalpel blade or other approved sharp instrument.
   2. Decapitation
      1. Removal of the head from the body using an approved sharp instrument.
   3. Cervical dislocation
      1. Severing the cervical (neck) vertebrae by applying the appropriate amount of pressure. There are different methods on how to apply the pressure needed to correctly sever the cervical vertebrae.
   4. Exsanguination
      1. Removal of a large volume of blood to prevent adequate circulation in order to prevent resuscitation.
   5. Removal of a vital organ
      1. Opening the animals body cavity and removing the brain, heart, or lungs entirely.
3. Seek additional training on all methods of euthanasia as needed by contacting the ULAM Training Core at [email protected].

1. See Documenting, Disposal and Reporting Animal Deaths SOP

1. a. Investigative personnel can request ULAM to euthanize animals as needed 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.