Euthanasia of Research Animals

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Definitions

Euthanasia - the act of inducing humane death in an animal by a method that induces rapid loss of consciousness and death with a minimum of pain, discomfort or distress.

“Acceptable method” - As defined by the American Veterinary Medical Association, an acceptable method of euthanasia renders an animal unconscious and insensitive to pain and distress as quickly as possible, followed by cessation of all respiratory and circulatory functions and brain activity.

“Acceptable method with Conditions” - These methods are considered “Acceptable” (see above) only if specific conditions are met, as indicated in the AVMA Guidelines for the Euthanasia of Animals: 2020 Edition.  For example, neonatal rodents and reptiles may be resistant to the effects of CO2 inhalation – therefore, a secondary physical method to confirm death is required.  On the other hand, the preparation for some physical methods (e.g., decapitation) may cause stress in the animals, so administration of a sedative or anesthesia is required.  Once the specified conditions are met, these methods are considered fully acceptable under the Guidelines.

Policy

To prevent/alleviate animal suffering, all laboratory animals must be euthanized in a timely manner, either as described in the approved protocol according to established timepoints, or as soon as necessary if established criteria or humane endpoints are reached.  While every effort will be made to contact a member of the study team in the event that an animal requires early or unscheduled euthanasia due to unrelieved pain/distress, the IACUC has empowered ULAR veterinarians to immediately euthanize any animal that is found near death or suffering from intractable pain.  Researchers are advised to ensure that Veterinary Services personnel can always contact someone with authority to make decisions about sick or injured animals.

All IACUC protocols must include a description of the method(s) that will be used to euthanize animals as well as a description of how death will be confirmed.  The method(s) to be used must be consistent with the AVMA Guidelines for the Euthanasia of Animals: 2020 Edition.

An anesthetized animal in deep narcosis following administration of an injectable or inhalant agent may appear dead but may eventually recover.

  • For inhalant agents, the IACUC requires that an approved secondary physical method of euthanasia (e.g. decapitation, cervical dislocation, exsanguination or removal of vital organs) be performed prior to carcass disposal.  Failure to perform both the primary and secondary methods as described in the protocol will be treated as serious protocol non-compliance.
  • For injectable agents, death must be confirmed using criteria appropriate for the species and euthanasia method, as described in the approved protocol. Confirmation methods may include assessment for cessation of vital signs and/or other scientifically and veterinary medically accepted indicates of death. See “Confirmation of Death in Euthanized Animals” section below.

Unintended recovery of animals after apparent death from CO2 or other euthanasia agents constitutes serious regulatory noncompliance.

Common Euthanasia Methods

  • Inhalation of anesthesia gas – acceptable with conditions for rodents and in certain other small animals (< 7 kg).  Typically used as part of a two-step process with a secondary physical method of euthanasia such as decapitation or cervical dislocation.
  • Inhalation of CO2 – acceptable with conditions, including the special considerations listed below.
  • Immersion agents – e.g., MS-222/Tricaine.  Acceptable for aquatic species, followed by a secondary physical method.
  • Cervical Dislocation – acceptable for small birds, mice and immature rats.  Requires training and should be performed under anesthesia unless specifically approved by the IACUC.
  • Decapitation – acceptable for rodents and small rabbits.  Requires training; anesthesia is strongly recommended unless approved by the IACUC with strong scientific justification.  Guillotines must be sharpened and adjusted as needed to ensure proper performance.
  • Injectable barbiturate agents – e.g., sodium pentobarbital, Euthasol®, Eutha 6®, Fatal Plus® - acceptable for most species.
  • Exsanguination/Cardiac Perfusion – acceptable with conditions; animals must be anesthetized.  The IACUC strongly recommends the use of injectable anesthetic agents (sodium pentobarbital, ketamine/xylazine, telazol, etc.) prior to cardiac perfusion.

CO2 (Carbon Dioxide Gas) - Special Considerations for the use of CO2 as a euthanasia agent:

The following additional guidelines must be followed when using CO2:

  • CO2 must be delivered from compressed gas canister only. Gas should be delivered using a gradual fill method- a displacement rate from 30% to 70% of the chamber volume per minute is recommended. Use of a flowmeter is strongly recommended.
  • Use of dry ice to deliver CO2 gas is unacceptable.
  • High concentrations of CO2 have been determined to cause pain and distress. The practice of immersion, where conscious animals are placed directly into a container filled with 100% CO2, is unacceptable.
  • Use of the rodents’ home cage is recommended as it minimizes stress in the animals.
  • Chambers used for CO2 euthanasia must not be overcrowded. Overcrowding in this situation is defined as less than one half the normal housing space normally required for the animals – no more than 10 adult mice per typical cage.
  • If animals are removed from their home cages prior to euthanasia, the container used to transport animals between the housing area and procedure room/euthanasia chamber must not be overcrowded.
  • Male mice from different cages should not be mixed in transport cages or the euthanasia chamber to prevent distress and/or fighting.
  • Detailed instructions for the use of CO2 euthanasia are posted in all vivarium procedure rooms and can be reviewed on the ULAR website.

Confirmation of Death in Euthanized Animals

All research personnel must receive training in all methods of euthanasia employed in the laboratory, including evaluation of vital signs used to confirm death. Measurements such as lack of movement or visible lack of breathing are not precise enough to declare that a euthanized animal is dead.

Assessment Criteria for Confirmation of Death:

A combination of criteria is most reliable in confirming death because no sign alone, except rigor mortis, confirms death.

Absence of the following criteria for 5 minutes can be used to verify death:

  • Heartbeat – by palpation, auscultation or ECG
  • Pulse – by palpation or pulse oximetry
  • Respiration – by auscultation or observation of thoracic or opercular movements
  • Corneal reflex (assuming that no neuromuscular blockers have been used)
  • Pupillary response to light – by observation (note that certain drugs can cause pupillary dilation)

If heartbeat cannot be auscultated or palpated in very small animals, a needle and syringe can be placed into the heart percutaneously and blood aspirated to confirm placement. Lack of movement of the needle/syringe verifies lack of cardiac movement.

Rodent Euthanasia - Special Considerations

Some rodents, especially neonates, are relatively resistant to overdose of inhaled agents (e.g., CO2, isoflurane), and injectable agents may be difficult to perform in rodent neonates. Therefore, IACUC requires a secondary physical method of euthanasia when inhalant euthanasia agents are used (and recommends it for all rodent euthanasia), performed after the animal has lost consciousness, stopped breathing, and is non-responsive to noxious stimuli (e.g., hind paw pinch).

Acceptable physical euthanasia methods for adult and neonatal rodents after exposure to inhalant euthanasia agents include:

  • Decapitation
  • Removal of vital organs (e.g., heart, lungs, brain)
  • Opening of the chest cavity to induce pneumothorax
  • Cutting the major blood vessels to induce exsanguination (e.g., aorta, vena cava)
  • Cervical dislocation – formal training is recommended (contact Veterinary Services for details)

Note: The above physical methods may not be performed in conscious animals, without specific IACUC approval.

Ectothermic Vertebrates - Confirmation of Death

Additional care must be taken to ensure death following euthanasia in ectothermic vertebrates such as fish, reptiles and amphibians. Such animals may normally exhibit very low heart rates, and the heart and brain are very tolerant to hypoxia. Many ectotherms can temporarily cease respiration for an hour or more. Absence of heart rate and/or breathing will not necessarily provide confirmation of death in these animals.

  • For reptiles and amphibians (such as Xenopus frogs),
  • For small, tropical fish such as zebrafish, rapid chilling in ice slurry with immersion for at least 20 minutes can be used as a primary euthanasia method with no additional physical method necessary to confirm death.

References

For more detailed information and training in acceptable euthanasia methods, please contact Veterinary Services (949) 824-7788.