Methods of Euthanasia

BACKGROUND

This Standard Operating Procedure (SOP) outlines approved methods of euthanasia. The Guide for the Care and Use of Laboratory Animals (the Guide, NRC 2011) states: "Unless a deviation is justified for scientific or medical reasons, methods should be consistent with the AVMA Guidelines on Euthanasia (AVMA 2007 or later editions)." (p. 123)

The AVMA Guidelines for the Euthanasia of Animals: 2013 Edition states: "Euthanasia methods are classified in the Guidelines as acceptable, acceptable with conditions, and unacceptable. Acceptable methods are those that consistently produce a humane death when used as the sole means of euthanasia. Methods acceptable with conditions are those techniques that may require certain conditions to be met to consistently produce humane death, may have greater potential for operator error or safety hazard, are not well documented in the scientific literature, or may require a secondary method to ensure death. Methods acceptable with conditions are equivalent to acceptable methods when all criteria for application of a method can be met." (p.10)

IACUC Procedure

The tables on the subsequent pages outline both acceptable methods of euthanasia and methods that are acceptable with conditions. If the use of a euthanasia method that is acceptable with conditions is proposed in an IACUC protocol, the Principal Investigator must review the conditions that must be met and assure the IACUC that they will be followed.

Laboratory rodents should be euthanized in their home cage, and must not be placed in unfamiliar groups. Activities that contribute to distress in rodents include transport, handling (in animals not accustomed to it), disruption of compatible groups, and elimination of established scent marks. While eliminating all sources of distress may not be practical or possible, the selected method of euthanizing rodents must minimize these sources of potential distress. Methods of euthanasia likely to elicit distress vocalizations or pheromones that other animals could hear or smell should be performed in another location, if transportation distress can be minimized.

Rodents Method Condition

Acceptable Method(s)

  • Injected barbiturates and barbiturate combinations
  • Injected dissociative agent combinations
  • Adjunctive method under anesthesia (exsanguination, bilateral pneumothorax, perfusion with fixative)
 

Acceptable Method(s) With Conditions

Carbon dioxide (CO2)

  • CO2exposure using a gradual fill method with a displacement rate from 10% to 30% of the chamber volume/min is recommended.
  • CO2must be supplied in a precisely regulated and purified form without contaminants or adulterants, typically from a commercially supplied cylinder or tank. An appropriate pressure-reducing regulator and flow meter is absolutely necessary.
  • CO2 flow should be maintained for at least 1 minute after respiratory arrest.
  • Rodents should be kept in their home cage with familiar cagemates during CO2 administration.
  • The practice of immersion, where conscious animals are placed directly into a container prefilled with 100% CO2, is unacceptable.
  • Rat and mouse neonates < 10 days must not be euthanized with CO2.*
Acceptable Method(s) With Conditions

Cervical dislocation

  • Cervical dislocation is acceptable with conditions for mice and rats < 200 g.
  • It may be used in research settings when required by the experimental design (scientific justification must be provided in the IACUC protocol).
  • Those responsible for use of this method must ensure that personnel who perform cervical dislocation have been properly trained using anesthetized or dead animals to demonstrate proficiency , and consistently apply it humanely and effectively.
Acceptable Method(s) With Conditions

Decapitation**

  • Decapitation is acceptable with conditions for mice and rats.
  • It may be used in research settings when required by the experimental design (scientific justification must be provided in the IACUC protocol).
  • Specialized rodent guillotines are available and must be kept clean, in good condition with sharp blades**.
  • The use of plastic cones to restrain animals is required unless justified in the protocol.
  • Those responsible for use of this method must ensure that personnel who perform decapitation have been properly trained using anesthetized or dead animals to demonstrate proficiency, and are subsequently monitored for competence.
  • Decapitation of rat or mouse fetuses and neonates should be done using very sharp scissors.
Acceptable Method(s) With Conditions

Tribromoethanol

  • Tribromoethanol is acceptable with conditions as a method for euthanasia of laboratory rodents when approved by the IACUC and prepared, stored, and administered at the appropriate dosage.

*Refer to Euthanasia of Rodent Rat Fetuses and Neonates Guideline

**Refer to Use of Guillotines Policy

Rabbits

Method Conditions

Acceptable Method(s)

  • Intravenous or intraperitoneal barbiturates
  • Adjunctive method under anesthesia (exsanguination, bilateral pneumothorax, perfusion with fixative
 
Acceptable Method(s) With Conditions

Barbiturates (alternate routes of administration)

  • Intracardiac injections must only be used if the animal is anesthetized.

 

Cats and Dogs Method Conditions
Acceptable Method(s)
  • Intravenous barbiturates
  • Injected anesthetic overdose
 
Acceptable Method(s) With Conditions

Barbiturates (alternate routes of administration)

  • Intraperitoneal injection may be used in situations when an IV injection is difficult due to small patient size. Intracardiac injections must only be used if the animal is anesthetized.
Acceptable Method(s) With Conditions

Inhaled anesthetic followed by adjunctive method

  • Inhaled anesthetics can be administered as part of a 2-step euthanasia process, in which animals are first rendered unconscious through inhaled anesthetic agent exposure, and then a secondary method subsequently applied, such as bilateral thoracotomy. Effective procedures should be in place to reduce animal worker exposure to anesthetic vapors.

 

Small Ruminants and Swine

Method Conditions
Acceptable Method(s)

Intravenous injected barbiturates

 

Acceptable Method(s) With Conditions

Barbiturates (alternate routes of administration

  • Intraperitoneal injection may be used in situations when an IV injection is difficult due to small patient size. Intracardiac injections must only be used if the animal is anesthetized.
Acceptable Method(s) With Conditions

Intravenous potassium chloride under anesthesia

  • 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 solutions IV.
  • Administration of potassium chloride solutions IV requires animals to be in a surgical plane of anesthesia characterized by loss of consciousness, loss of reflex muscle response, and loss of response to noxious stimuli.

 

Zebrafish Method Conditions

Acceptable Method(s)

  • Immersion in buffered benzocaine or benzocaine hydrochloride, isoflurane, sevoflurane, quinaldine sulfate, buffered tricaine methanesulfonate (MS-222), 2-phenoxyethanol
  • Injected pentobarbital (IP/IC, IM)
  • Rapid chilling at 2 - 4◦C (must be > 3dpf)
  • Embryos < 3dpf: MS-222 immersion or rapid chilling followed by immersion in sodium or calcium hypochlorite

Note: keep fish in an immersion euthanasia solution for at least 10 minutes after cessation of opercular movements.

Acceptable Method(s) With Conditions

Decapitation followed by pithing

  • Decapitation followed by pithing (2 step). Rapid severance of the head and brain from the spinal cord, followed by pithing of the brain, will cause rapid death and unconsciousness. Decapitation alone is not considered a humane approach to euthanasia, especially for species that may be particularly tolerant of low O2 concentrations. Pithing helps ensure rapid loss of brain function and death for those species.

 

Approved: May 2016

Revisions Approved: 6/2018, 2/2019