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Policy#: 529-350

Title: ANIMAL USE GUIDANCE: Guidelines for Rodent Euthanasia

Effective: 6/21/2021



These guidelines for rodent euthanasia are intended to provide direction in complying with the 2020 edition of the AVMA Guidelines for the Euthanasia of Animals. UCR, as an PHS-funded institution, must comply with the most recent published AVMA Guidelines.  AUP-specific methods other than the acceptable methods listed in the AVMA Guidelines must be scientifically justified and approved by the IACUC.

Related Policies and Guidance

  •  Policy 529-227: Agents for Anesthesia, Analgesia and Euthanasia
  • Policy 529-340: Non-pharmaceutical grade substances in animals
  • Guidance 529-268: Guidelines for Compounding and Secondary Container Use for Injectable Drugs



  1. It is the responsibility of the Principal Investigator (PI) to ensure that personnel performing euthanasia are appropriately trained in the method listed in the Animal Use Protocol (AUP). 
  2.  All euthanasia methods must be described in the approved AUP.
  3. Euthanasia should not be performed in the animal room and/or within sight/sound/smell of other living rodents.
  4. Death of each animal must be confirmed prior to carcass disposal. 


Injectable anesthetics

Injectable anesthetic overdose using barbiturates, barbiturate derivatives, or dissociative agent combinations are acceptable methods of euthanasia.
  1. Pentobarbital is the most commonly used barbiturate for laboratory rodents.  The euthanasia dose is typically 3 times the anesthetic dose.
  2. Ketamine and similar dissociative agents should be used in combination with an a2-adrenergic receptor agonist such as xylazine or benzodiazepines such as diazepam.  At least 4 times the anesthetic dose should be used for euthanasia.
  3. After drug administration, a physical method must be used to confirm euthanasia (see Confirmation of Death section below).

It is expected that all compounds used in animals are pharmaceutical grade. See Policy 529-340: “Non-pharmaceutical grade substances in animals” for information and exceptions.


Carbon Dioxide (CO2)

CO2, the most common means of euthanasia for rodents, is considered an acceptable agent of euthanasia with conditions.  

  1. CO2 should ONLY come from a compressed gas source equipped with a regulator and flow meter to adjust the flow rate of 30% to 70% of the chamber or cage volume/minute. 
  2. CO2 from other sources (e.g., dry ice, fire extinguishers, etc.) is UNACCEPTABLE because gas flow cannot be regulated.
  3. Chambers pre-filled with CO2 are not acceptable.
  4. Whenever possible, animals should be euthanized in their home cages to minimize distress. Otherwise, the euthanasia chamber must be thoroughly cleaned between each use and at the end of the day to remove debris and pheromones.
  5. Euthanasia cages or chambers must not be overcrowded and should not be mixed with unfamiliar or incompatible animals. Different species should never be mixed, and dead animals should NEVER be mixed with live animals.
  6. It usually takes 2-3 minutes for an animal to become unconscious. The animal should be left in the chamber at the current flow rate for at least one additional minute after the complete cessation of respiration. 


Inhalant anesthetics

Overdose with inhalant anesthetics (e.g., isoflurane) is acceptable for euthanasia of rodents with conditions.

  1. When used as a sole euthanasia agent delivered via vaporizer, animals may need to be exposed for pro­longed time periods to ensure death. 
  2. When using the open-drop technique, care must be taken to ensure that the rodent does not come in direct contact with the anesthetic or anesthetic soaked cotton or gauze materials. The jar must not be overcrowded.
  3. Adequate scavenging systems should be in place to prevent personnel exposure (see Policy 529-303 for more information).


Euthanasia of Neonatal Rodents (< 10 days of age)

Neonates may be euthanized by the following methods:

  1. Injection of chemical agents (e.g., pentobarbital)
  2. Decapitation (preferred method)
  3. Carbon Dioxide (CO2)
    • Neonates are resistant to hypoxia, requiring prolonged exposure to CO2 (up to 50 minutes).  Therefore, CO2 is not recommended as the sole method of euthanasia for neonates. 
  4. Hypothermia
    •  Gradual cooling of pups < 6 days of age is acceptable with conditions. 
    • It should be followed with a secondary method following loss of movement. 
    • The animals should not come in direct contact with ice or precooled surfaces
  5. Immersion in liquid nitrogen
    • Rapid freezing in liquid nitrogen is acceptable for neo­nates < 5 days of age only if preceded by anesthesia.


Confirmation of Death

Death must be confirmed prior to carcass disposal.

  1. Confirmation of death must be done by one of the following methods:
    • 10 minutes of careful observation of multiple signs that signify death (i.e., loss of bladder control, lack of rear limb withdrawal reflex, absence of a palpable apical heartbeat when palpated over the rib cage, faced eye color, absence of respirations). Confirmation of death must not be based on a single sign such as absence of respirations. 
    • Physical methods:
      1. Thoracotomy (opening the chest cavity)
      2. Cervical dislocation* – rodents < 200g only
      3. Decapitation*
      4. Removal of vital organs (i.e., heart, lungs, brain)
      5. Exsanguination
      6. Perfusion
  2. One of the above physical methods must be performed after rodents have lost consciousness from CO2 inhalation or drug overdose.
  3. Failure to ensure death of animals after euthanasia procedures is a significant non-compliance that is reportable to the appropriate regulatory and accrediting agencies.
*These methods may not be performed in conscious animals, without specific IACUC approval.
Please see Policy 529-331: Cervical Dislocation and Policy 529-332: Decapitation, respectively.



  1. AVMA Guidelines for the Euthanasia of Animals, 2020.
  2. The Guide for the Care and Use of Laboratory Animals, 8th edition, 2010.
  3. NOT-OD-05-034: Guidance on Prompt Reporting to OLAW under the PHS Policy on Humane Care and Use of Laboratory Animals.  2005
  4. Report of the ACLAM Task Force on Rodent Euthanasia. August 2005.
  5. Shomer, NH, et, al. Review of Rodent Euthanasia Methods. JAALAS. Vol 59, No 3. 2020.