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]) or the ULAM veterinarians ([email protected]) for training or additional questions regarding euthanasia methods.

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

1. Mice (Mus musculus)
   1. If mice are consolidated into fewer cages for euthanasia, do so at the euthanasia station whenever possible. Do not consolidate mice which have previously been separated for fighting.
      1. Euthanize male mice immediately upon consolidation to reduce animal stress and the potential for fighting or injury.
      2. If consolidation must happen in the housing room:
         1. Use the LFH or BSC and micro-isolation technique.
         2. Consolidate only female mice or non-weaned pups.
         3. Do not consolidate weaned male mice within the animal room.
         4. Euthanize mice within 10 minutes of beginning consolidation to reduce animal stress and the potential for fighting or injury.
      3. Provide ample space for mice 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.
2. Rats: follow standard housing densities when consolidating rats (Rattus norvegicus) for euthanasia.
3. USDA-regulated rodents: do not consolidate USDA-regulated rodent species. Euthanize these rodents as housed, per cage.
   

   	Euthanize in Home Cage	Consolidate at Euthanasia Station	Consolidate in Housing Room
   Female or Neonatal Mice	Preferred	Permitted if consolidation is necessary	Permitted if consolidation is necessary
   Male Mice	Preferred	Permitted if consolidation is necessary	Not permitted
   Rats	Preferred	Permitted if consolidation is necessary	Not permitted
   USDA-Regulated Species	Preferred	Not permitted	Not permitted

1. Common primary methods of euthanasia include, but are not limited to:
   1. Carbon dioxide (CO₂) overdose
   2. Anesthetic overdose
   3. Decapitation (for neonatal mice and rats less than 10 days old)
2. The CO₂ overdose method for primary euthanasia is effective in:
   1. Weaned rodents
   2. Non-weaned rodents between 10-21 days old
   3. Pregnant rodents
3. 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 old 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 decapitation as a secondary method of euthanasia if CO₂ exposure or inhalation anesthetics are utilized in neonates.
4. If animals have received a hazard, see Animals Administered a Hazardous Substance Requiring Containment for additional information regarding euthanasia.

See Appendix A: CO₂ Step-by-Step Rodent Euthanasia Instructions and Appendix B: CO₂ Step by Step Rodent Euthanasia Instructions for Building-Supplied CO₂ for abbreviated versions of these instructions that include photos):

1. 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
   3. Has NOT been pre-filled with CO₂ prior to placement of the 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 next so that animals are not hidden from view.
3. Place the euthanasia lid over the top of the cage if available. If no euthanasia lid is present, insert the CO₂ hose through the water bottle grommet or water valve opening.
4. Open the CO₂ tank by turning the valve on the tank counterclockwise. Ensure the needles rises on the pressure gauge.
5. Adjust the flowmeter by turning the valve on the flowmeter counterclockwise until the gauge reaches the desired setting.
   1. Pre-calibrated settings are marked:
      1. RAT (in black) for small RAT cages.
      2. LARGE (in green) for LARGE mouse cages.
      3. SMALL (in blue) for SMALL mouse cages.
   2. If the flowmeter does not have pre-calibrated markings, calculate the flow rate and set the flowmeter accordingly. See Appendix C.
6. Observe and monitor animals continuously until all animals in the euthanasia chamber have stopped breathing.
   1. It may take several minutes for animals to stop breathing during euthanasia.
   2. Do NOT leave animals unattended at any point during the euthanasia process.
7. Once all animals have stopped breathing, close the CO₂ tank by turning its valve clockwise until snug.
8. Watch the pressure gauge drop to zero, followed by the flowmeter ball dropping to zero.
9. Close the flowmeter by turning its valve clockwise until snug. Do not overtighten.
10. If a euthanasia chamber is being used for more than one group of animals, clean or disinfect the euthanasia chamber between each group (cage) of animals and once 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: Puncturing the diaphragm and/or each lung through the rodent’s chest cavity using a scalpel blade or other approved sharp instrument.
   2. Decapitation: Removal of the head from the body using an approved sharp instrument.
   3. Cervical dislocation: Severing the spinal cord by applying the appropriate amount of pressure to the cervical (neck) vertebrae. There are different methods by which to apply the pressure needed to correctly sever the spinal cord.
   4. Exsanguination: Removal of a large volume of blood to prevent adequate circulation in order to prevent resuscitation.
   5. Removal of a vital organ: Opening the animals body cavity and removing the brain, heart, or lungs entirely.
3. For additional training on all primary or secondary methods of euthanasia, contact the ULAM Training Core at [email protected].

1. See Documenting, Disposal and Reporting Animal Deaths SOP

1. Investigative personnel can request ULAM to euthanize animals.
   1. To request all animals in an existing cage to be euthanized, flip the original cage card over and write “ULAM sac/euth” and the date on the back.
   2. To request a subset of animals in a cage to be euthanized, separate the animals into a new cage and provide the following information on the back of a replacement cage card:
      1. PI name
      2. Protocol number
      3. Account number or shortcode
      4. Laboratory contact name and phone number
      5. “ULAM sac/euth”
      6. Date
   3. Ensure all animals that have been requested to be euthanized have adequate food and water.
   4. Per diem is not charged past the date of the request.
   5. This service cannot be used for animals that require immediate euthanasia, for example moribund animals.

1. See Documenting, Disposal and Reporting Animal Deaths.