Revised on
12/07/17 06:28pm

Guidelines for Euthanasia of Research and Teaching Animals


To provide guidance regarding humane euthanasia of animals used in research or teaching.


According to the Guide for the Care and Use of Laboratory AnimalsPHS Policy on the Humane Care and Use of Laboratory Animals, and the Animal Welfare Regulations, methods of euthanasia should be consistent with the American Veterinary Medical Association Guidelines for Euthanasia of Animals (AVMA Guidelines), unless a deviation is justified for scientific or medical reasons. In addition, methods must be specified and approved in Animal Care and Use Protocols. Methods are chosen to minimize pain and distress to the animals as well as meet the needs of the research or teaching protocol.

Policy Statement

The method of euthanasia must be an acceptable method as outlined in the AVMA Guidelines; deviations from the AVMA Guidelines require IACUC approval prior to implementation. If unique circumstances arise that require deviation from these guidelines the Principal Investigator must consult with Campus Veterinary Services (530-752-0514).

Chemical methods of euthanasia (CO2 chamber, inhalant anesthetics, Sodium Pentobarbital) may be followed with a physical method (cervical dislocation, bilateral thoracotomy, or exsanguination) to ensure death unless the effectiveness of the chemical method (e.g., specific chamber and process) has been appropriately validated. Regardless of which method of euthanasia is performed, personnel must ensure that death has occurred. Assurance of death may include ascertaining the absence of heartbeat and respiration.

Personnel performing euthanasia must be trained, knowledgeable, and proficient in the chosen techniques, trained on how to ensure that animals are no longer viable, and training must be documented. Personnel using physical meth­ods of euthanasia must be well trained and have demonstrated proficiency for each type of physical method used to ensure euthanasia is conducted appropriately.

Individuals performing cervical dislocation, and/or decapitation in adult animals, without anesthesia, in a research or teaching setting must be certified to perform these techniques.  They must contact Campus Veterinary Services (CVS) or the Institutional Animal Care and Use Committee (IACUC) office, or designated trainers (designated trainers will provide training and assessment of the participant’s skill level and verify that person’s proficiency in use of the technique). Once the person has been certified as proficient in the techniques, they may be listed as certified to perform these techniques on an approved Animal Care and Use Protocol.  A list of certified trainers is available in CVS and the IACUC Office.

Ideally, euthanasia should be performed in procedure rooms or laboratory space away from other animals. Death must be confirmed prior to bagging the carcass for disposal.

NOTE: Please refer to the AVMA Guidelines for Euthanasia of Animals: 2013 for information on other species and additional euthanasia methods not covered in this policy. 

Euthanasia of Mice and Rat Fetuses and Neonates

A.  Fetuses:

Rodent fetuses along with other mammals are unconscious in utero and hypoxia does not evoke a response. Therefore, it is unnecessary to remove fetuses for euthanasia after the dam is euthanized, if fetuses are not collected.

  1. If fetuses are collected:
  •  Fetuses up to 14 days of gestation: Euthanasia of the dam or removal of the fetus results in rapid fetal death since they cannot survive outside of the uterus.
  •  Fetuses from 15 days of gestation to birth: An injection of a chemical anesthetic overdose is an acceptable chemical method. Decapitation with surgical scissors, hypothermia, or cervical dislocation are acceptable physical methods. 

B.  Neonates:

  1. Neonates less than 5 days of age: Altricial neonates less than 5 days of age do not have sufficient nervous system development to perceive pain and may be quickly euthanized by rapidly freezing in liquid nitrogen (N2).
  2. Neonates up to or less than 7 days of age:
  • Hypothermia may be used as a method of euthanasia of altricial neonates up to 7 days old and provided they are not placed directly on the frozen surface (i.e., place in a latex glove, plastic bag or on a cloth).
  • Decapitation using scissors or a sharp blade is acceptable for altricial neonates less than 7 days of age.

   3. Neonates 8-14 days of age:

  •  Injection of an overdose of chemical anesthetics should be used whenever possible.
  •  Cervical dislocation is an acceptable method of euthanasia with appropriate training and demonstrated proficiency.
  •  Decapitation is an acceptable method of euthanisa with documented training. Depending on the developmental stage of animals, this technique may require the use of a guillotine.
Neonatal rodents are resistant to the effects of CO2, thus an adjunctive method (e.g., cervical dislocation or decapitation) should be performed after a neonate is nonresponsive to painful stimuli.  The use of CO2 as a sole method of euthanasia in neonates is strongly discouraged and must be approved in an IACUC protocol.

Mice and rats older than 14 days should be euthanized following the guidelines for adult rodents.

Other Euthanasia Methods

A.  Carbon dioxide (CO2):

A CO2 chamber is the most common method of euthanasia for small rodents. The chamber must allow viewing of the animal during euthanasia. Proper technique must be followed to ensure a humane death, as CO2 has noxious properties that can cause unnecessary pain and suffering. 

  1. Euthanasia in the home cage is recommended. If euthanasia cannot be conducted in the home cage, chambers should be emptied and cleaned between uses.  Do not overcrowd the chamber; all animals in the chamber must be able to make normal postural adjustments. If combining animals from multiple cages for euthanasia, euthanasia must be performed immediately to prevent animals from fighting.
  2. The flow rate for CO2 euthanasia systems should displace 10% to 30% of the chamber or cage volume/min. The higher range is recommended and should be continued at least one minute after respiratory arrest.
  3. Prefilled chambers are unacceptable.
  4. An appropriate pressure-reducing regulator and flow meter or equivalent equipment with demonstrated capability for generating the recommended displacement rates for the size container utilized are required.
  5. Remove the animal from the chamber and confirm the absence of respiration. It is important to verify that an animal is dead after exposure to CO2. Death must be confirmed by physical examination, and may be ensured by an adjunctive physical method (i.e., bilateral thoracotomy or decapitation).
  6. Clean the chamber with disinfectant to remove all urine, feces, and fur.
  7. CO2 generated from dry ice is NOT an acceptable method of euthanasia.

B.   Potassium Chloride (KCl) saturated solution:

Personnel performing this technique must be trained and knowledgeable in anesthetic techniques, and be competent in assessing the level of unconsciousness that is required for ad­ministration of potassium chloride solution IV. Administration of potassium chloride solution intravenously (IV) requires animals to be at a surgical plane of anesthesia characterized by loss of consciousness, loss of reflex muscle response, and loss of response to noxious stimuli.

C.   Cervical dislocation (without anesthesia) of rodents, small rabbits, chickens, and other birds:

  1. Cervical dislocation is acceptable for mice and rats <200 g. Personnel must be trained on anesthetized and/or dead animals to demonstrate proficiency.
  2. The use of cervical dislocation for euthanasia is lim­ited to small birds (<200 g), chickens, mice, immature rats (< 200 g), and rabbits (< 1 kg).
  3. All users approved to perform cervical dislocation (without anesthesia) as the    method of euthanasia must be certified by one of the approved campus trainers.

D.   Decapitation (without anesthesia) of rodents, small rabbits, poultry, and other birds:

  1. Decapitation is acceptable for mice and rats. Personnel must be trained on anesthetized and/or dead animals to demonstrate pro­ficiency.
  2. Decapitation is justified for studies where un­damaged and uncontaminated brain tissue is required. The equipment used to perform decapitation must be maintained in good working order and serviced on a regular basis to ensure it is effective.
  3. All users approved to perform decapitation (without anesthesia) as the method of euthanasia must be certified by one of the approved campus trainers.

E.   Euthanasia of amphibians, reptiles and fish: 

  1. Pithing of amphibians:  Pithing (destroying both the brainstem and spinal cord tissue) can be used as a second-step euthanasia method in unconscious animals when performed by properly trained individuals.
  2. Decapitation of amphibians and reptiles: After an animal has been anesthetized decapitation must be followed by pithing or another method to destroy brain tissue. Decapitation can only be used as part of a 3-step euthanasia process (anesthesia, decapitation, pithing).
  3. Buffered Tricaine Methanesulfonate (MS222) in amphibians and fish:The solution must be buffered with sodium bicarbonate resulting in a pH between 7.0-7.5. Due to species differences in response to MS222, a secondary method of euthanasia is recommend in some finfish and amphibians to ensure death.    
  4. Amphibians MS222 dose = 500 mg/L (immersion for at least one hour). If immersion time is less a secondary euthanasia method must be used. NOTE: Overdose of MS222 in frogs must be followed by pithing with or without decapitation.
  5. Finfish MS222 dose = 500 mg/L (immersion for at least 10 minutes).
  6. Rapid chilling (hypothermic shock) in finfish: Rapid chilling is acceptable for small-bodied (3.8-cm-long or smaller) tropical and subtropical stenothermic finfish, for which the lower lethal temperature range is above 4°C. Because it is often difficult to confirm that an amphibian or reptile is dead, the application of two or more euthanasia procedures is usually recommended.

F. Equipment Maintenance:

In accordance with the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition “The equipment used to perform decapitation should be maintained in good working order and serviced on a regular bases to ensure sharpness of blades.”

  1. Decapitation may be accomplished by use of a commercial guillotine, dedicated scissors or razor/scalpel blades.
  • Scissors, razors or scalpel blades may only be used for neonatal rodents (altricial neonates less than 7 days of age) and small amphibians/fish. 
  • Dedicated scissors must be clean, in good condition, sharp and move freely. 
  • Razors and scalpel blades should be new.
  • Guillotine: 
  1. Guillotines used to perform decapitation must be maintained in good working condition, serviced on a regular basis to ensure sharpness of blades and cleaned after each use. 
  2. Before each use of a guillotine, it should be checked for rust, lack of visible nicks or other damage to the cutting edges and cleanliness. The operator should ensure that the action is smooth with no perceptible binding or resistance.
  3. A record certifying maintenance/sharpening of a guillotine must be maintained. The IACUC recommends sharpening at least annually, however the species involved and the number of animals will dictate how often the blades need to be sharpened.
  4. Professional blade sharpening services may be used or a work order can be placed through CVS to request sharpening (
  5. If lubrication of the guillotine is necessary, the use of a Teflon or Silicone containing compound is recommended.
  6. Old guillotine blades that are no longer serviceable must be discarded in the sharps container.
  7. The responsibility for sharpening the guillotine rests with the Principal Investigator.
  8. Guillotines and their maintenance records will be inspected as part of the IACUC semi-annual inspections.
  9. The IACUC requires that all individuals using guillotines be trained and certified on the proper use.  

NOTE: Please refer to the AVMA Guidelines for Euthanasia of Animals: 2013 for information on additional euthanasia methods

G. Animal Disposal

After death has been ensured, place the carcass in a disposable waterproof bag. Seal the bag and place the bagged carcass in one of the barrels found inside the cold rooms or freezers designated for animal disposal. 

Carcasses that are radioactive must be disposed of according to the procedures stated in the Principal Investigator’s Radioactive Use Authorization. Carcasses that are infectious must be disposed according to the procedures stated in the Principal Investigator’s Biological Use Authorization. Carcasses that have chemical contamination must be disposed of according to procedure established during review of the protocol by the Chemical Hygiene Officer.


    1. AVMA Guidelines for the Euthanasia of Animals: 2013 Edition
    2. Guide for the Care and Use of Laboratory Animals, Eight Edition


    Procedure: IACUC-37
    Date: February 9, 2017
    Enabled By: Guide, AVMA
    Supersedes: October 20, 2016