Background

This Standard Operating Procedure (SOP) outlines approved methods of euthanasia. This policy has been created to ensure that euthanasia complies with the requirements of the Animal Welfare Act and Regulations, the Guide for the Care and Use of Laboratory Animals, 8th Edition (the Guide), and the AVMA Guidelines on Euthanasia of Animals (AVMA Guidelines).

General

In order to minimize animal suffering, laboratory animals must be euthanized either as described in the protocol at established endpoints, or expeditiously if criteria for humane endpoints have been reached. Animals must be continually observed and never be left unattended during the euthanasia procedure. All methods used must result in the confirmed death of the animal; for several methods this requires a secondary physical method after the primary chemical method to ensure death. Animal carcasses and tissues must be properly disposed of after euthanasia.

Rodent Euthanasia

Laboratory rodents should be euthanized in their home cage and must not be placed or recombined 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 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. Rodent fetuses are in a state of unconsciousness during pregnancy, so euthanasia of the dam is considered sufficient to euthanize fetuses if they remain in the uterus. However, if fetuses must be removed from the uterus for tissue collection or experimentation, they should be treated as altricial neonates. 

1. Chemical Methods
   1. Carbon Dioxide Inhalation (≥10 days old)
   • CO₂ exposure using a gradual fill method with a displacement rate from 30% to 70% of the chamber volume/min is recommended.
   • CO₂ must 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.

2. Injectable Anesthetic Overdose
   • Intraperitoneal injection of at least 200 mg/kg sodium pentobarbital is recommended.
   • Sodium pentobarbital containing solutions can be viscous and are best diluted to a concentration of no more than 60 mg/ml.
   • Other injectable anesthetics may be approved and delivered at an overdose.
   • Tribromoethanol is acceptable with conditions as a method for euthanasia of laboratory rodents. It should not be used as a sole agent and must accompany a physical method of euthanasia. Its use requires scientific justification.
3. Inhalant Anesthetic Overdose
   • Isoflurane inhalation at an overdose may be utilized as a method of euthanasia, preferably by precision vaporizer.

2. Physical Methods
   1. As Secondary Methods
      • Chemical methods must be followed by a confirmatory physical method.
      • Decapitation, exsanguination, cervical dislocation, bilateral thoracotomy, tissue perfusion, or dissecting of a vital organ must occur after the animal has been determined to be non-responsive to noxious stimuli.
   2. As Primary Methods (>10 days of age)
      • When animals are fully anesthetized as at the end of a non-survival surgery, methods such as bilateral thoracotomy, exsanguination, removal of a vital organ or perfusion are acceptable
      • Physical-only methods of euthanasia such as decapitation or cervical dislocation of un-anesthetized animals must be approved by the IACUC with appropriate scientific justification in the IACUC protocol.
        1. Those responsible for use of this method must ensure that personnel who perform decapitation or cervical dislocation have been properly trained using anesthetized or dead animals to demonstrate proficiency, and consistently apply it humanely and effectively.
        2. Specialized rodent guillotines are available and must be kept clean, in good condition, with sharp blades.

3. Neonates (<10 days old)
   1. Anesthetic overdose, as listed in the chemical methods above, can be used.
   2. Decapitation using scissors or sharp blades is acceptable as a sole means of euthanasia for most protocols. VA-protocols that decapitate rodent neonates without anesthesia require scientific justification.

Non-Rodent Mammalian Euthanasia

Use of an anesthetic agent for euthanasia must be at an overdose, not an anesthetic dose. To confirm death, the administration of any chemical agent used for euthanasia must be followed by a physical method from which the animal cannot recover, such as bilateral thoracotomy or fixative perfusion. The animal must be completely non-responsive to noxious stimuli before any physical method is performed.

1. Rabbits
   1. Intravenous or intraperitoneal injection of barbiturates (i.e., pentobarbital) is recommended. Intracardiac injections must only be used if the animal is anesthetized.
   2. When animals are fully anesthetized (e.g., at the end of non-survival surgery), methods such as bilateral thoracotomy, exsanguination, or perfusion are acceptable.

2. Cats/Dogs
   1. Intravenous injection of barbiturates (i.e., pentobarbital) is recommended. 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.
   2. When animals are fully anesthetized (e.g., at the end of non-survival surgery), methods such as bilateral thoracotomy, exsanguination, or perfusion are acceptable.

3. Small Ruminants/Swine
   1. Intravenous injection of barbiturates (i.e., pentobarbital) is recommended. 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.
   2. Intravenous administration of potassium chloride is acceptable only if animal is fully anesthetized characterized by loss of consciousness, loss of reflex muscle response, and loss of response to noxious stimuli. 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.

Zebrafish Euthanasia

Approved methods for zebrafish vary by age and agent.

1. Adults and Fry (>3 days post fertilization and older)
   1. Tricaine methanesulfate (MS-222).
      • Immerse fish in a solution of pharmaceutical grade MS-222 (i.e. Syncaine.) The solution must be buffered with sodium bicarbonate to a pH of 7.0-7.5.
      • Keep fish in an immersion euthanasia solution for at least 30 minutes after cessation of opercular movements when using MS-222.
      • If a secondary method of euthanasia/confirmatory method (exsanguination, decapitation, etc.) is performed after cessation of opercular movement is noted, the 30-minute wait time is not necessary.
   2. Other solutions inclusive of eugenol, isoeugenol and clove oil require fish to remain in the solution for at least 10 minutes after cessation of opercular movements. Other solutions require fish to remain in the solution for at least 30 minutes after cessation of opercular movements. If animals are too young to observe opercular movement, keep immersed for 30 minutes.
   3. Rapid chilling
      • Submerge fish in 2-4°C water for 10 minutes for zebrafish >7 days post fertilization and older and 20 minutes for zebrafish 4-7 days post fertilization.
   4. Sodium or calcium hypochlorite may be used as a single agent on zebrafish 4-7 days post fertilization. Animals should be immersed for at least 5 minutes..
2. Embryos (<3 days post fertilization)
   1. Tricaine, other agents, or rapid chilling may be used as above, but embryos <3 days post fertilization should be followed with an adjunctive method.
   • Adjunctive methods include immersion in sodium or calcium hypochlorite.
   2. Sodium or calcium hypochlorite may be used as a single agent on zebrafish embryos <3 days post fertilization. Animals should be immersed for at least 5 minutes. 

Approved: May 2023

Edited: January 2024