Euthanasia of Rodents

Euthanasia of Rodents

Approved December 18, 2003, Revised April 18, 2013   (Download) 

Background: The AVMA Guidelines for the Euthanasia of Animals: 2013 Edition, states that acceptable methods of euthanasia of rodents include injection with barbiturate and barbituric acid derivatives and dissociative agent combinations such as ketamine and xylazine.  In addition, the following are classified as acceptable if certain conditions are met: euthanasia utilizing certain inhaled anesthetics and carbon dioxide (CO2); injection of tribromoethanol; and physical methods such as cervical dislocation and decapitation.  This IACUC policy outlines the conditions required for each of the above euthanasia methods.  In all cases, the method of euthanasia used must be described in the appropriate IACUC approved protocol.

The methods listed below primarily apply to mice and rats, although most are applicable to many other rodents.  For species differences, please consult with veterinary services.


A. Confirming death:

  1. For all types of euthanasia, it is the responsibility of the investigator to confirm death has occurred, by utilizing reliable indicators such as the lack of heartbeat and respirations, lack of corneal reflex, lack of response to toe pinch, and graying of mucous membranes.  One sign alone is not sufficient.  For all inhalation agents, secondary methods of euthanasia are required and must be listed in the IACUC-approved protocol.

B. Injectable anesthetics:

  1. Pentobarbital.  Three times the anesthetic dose is the minimum dose required for euthanasia with pentobarbital, whether pentobarbital is used alone or used in combination with phenytoin (Euthasol®) (IP in mice, 270 mg/kg; IP in rats, 150 mg/kg).  Dosing is based on the pentobarbital component.
  2. Ketamine/xylazine combinations (mice and rats) and tribromoethanol (mice only) can be utilized for euthanasia at 4 times the anesthetic dose by the normal route.  Proper preparation and storage of tribromoethanol is required (see Animal Use Training Mouse Lab Handout for details).

C. Inhalation agents:

  1. General conditions for all inhalation agents:
    1. Secondary methods of euthanasia are required for all inhalation agents.  For mice, secondary euthanasia methods include placing the animal in a tightly knotted bag filled with CO2 (if CO2 is used as the original euthanasia method), cervical dislocation, exsanguination, thoracotomy, decapitation, or anesthetic overdose (e.g., sodium pentobarbital, etc).  For rats, secondary methods include all of the above, except that cervical dislocation can only be performed on rats weighing less than 200 grams.
    2. Animals must be transported and euthanized in a method that minimizes stress.  Inhaled agents should be administered in the home cage of rodents when possible.  If animals need to be combined, they should be combined only with compatible cohorts of the same species.  Chambers must not be overloaded and must be kept clean (including being cleaned between animals).  Euthanasia should always be done in cohorts (live animals must not be placed in the chamber with dead animals).
  2. Inhalation anesthetics:
    1. The order of preference of commercially available agents from most preferable to least preferable is isoflurane, sevoflurane, enflurane, desflurane.
    2. All machinery used must be in good working order with no leaks and noise and cold drafts minimized.
  3. CO2:
    1. CO2 must be administered from compressed commercial cylinders utilizing a flow meter to deliver 10-30% of the chamber volume/min.  Pre-filling of the chamber with CO2 is not acceptable and CO2 within the chamber needs to be removed between groups.
    2. Total gas exposure must be at least 5 minutes for mice and 10 minutes for rats. Gas flow must be maintained for at least 1 minute after apparent clinical death. A timer must be used to ensure adequate length of exposure. It is important to confirm that an animal is dead after removing it from the chamber.
    3. Neonates: The time period for euthanasia utilizing CO2 is substantially prolonged in neonatal rodents that are 10 days of age or younger. Due to their inherent resistance to hypoxia, CO2narcosis in neonates must be followed by decapitation.
    4. Animals must not be left unattended during CO2 exposure.

 D. Physical Methods:

  1. Physical methods of euthanasia usually require certification of competency (see below).  Competency must be demonstrated utilizing the same species and conditions in which the method will be performed.  Granting of certification is at the discretion of the attending veterinarian or designee who observes the demonstration.
  2.  Cervical dislocation:
  1. Cervical dislocation is an acceptable method of euthanasia for mice and for rats that are <200g.
  2. Certification is required for utilization of cervical dislocation as a method of euthanasia in awake or anesthetized rodents.

3.    Decapitation

  1. Decapitation of neonatal mice or rats ≤14 days of age does not require certification.
  2. Decapitation of unanesthetized neonates (≥ 14 days of age), juvenile, and adult rodents requires both certification of the individual doing the procedure and scientific justification for its use.
  3. The equipment used to perform decapitation must be maintained in good working order and records demonstrating service of the equipment on a regular basis must be maintained.

Additional Information – CO2 Delivery Information