Rodent Euthanasia

The Public Health Service Policy on Humane Care and Use of Laboratory Animals, Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals require the IACUC to review and approve the proposed methods of euthanasia. The proposed methods must be consistent with the recommendations of the 2000 Report of the American Veterinary Medical Association (AVMA) Panel on Euthanasia, or succeeding revised editions, unless there are scientific justifications for alternative methods. The AVMA Panel categorizes each method of euthanasia as acceptable (methods which consistently produce a humane death when used as the sole means of euthanasia), conditionally acceptable (methods which by the nature of the technique or because of greater potential for operator error or safety hazards might not consistently produce humane death or are methods not well documented in the scientific literature) and unacceptable (methods deemed inhumane under any conditions or that the panel found posed a substantial risk to the human applying the technique).

Euthanasia is the act of humanely killing animals by methods that induce rapid unconsciousness and death without pain or distress. In evaluating the appropriateness of methods, some of the criteria that should be considered are ability to induce loss of consciousness and death while minimizing animal pain, distress, or anxiety; reliability; irreversibility; time required to induce unconsciousness; species and age limitations; compatibility with research objectives; and safety of and emotional effect on personnel.

Euthanasia should be carried out in a manner that avoids animal distress. Depending on the species involved, some animals being euthanized may vocalize, release pheromones or behave in a manner which may be distressing to other animals. For those reasons, animals should not be euthanized while crowded or in the presence of animals not being euthanized.

It is essential that euthanasia be performed by personnel who are skilled in methods for the species in question and that it be performed in a professional and compassionate manner. It is imperative that death be verified after euthanasia and before disposal of the animal. Animals in deep narcosis following exposure to euthanasia agents may appear dead, but may eventually recover. Death must be confirmed by examining the animal for cessation of vital signs with consideration given to the animal species and method of euthanasia when determining the criteria for confirming death.

If conditionally acceptable techniques are planned, they must be scientifically justified and approved by the IACUC prior to implementing. Principal Investigators are responsible for ensuring all personnel performing animal euthanasia have been properly trained to consistently apply the technique(s) in a humane and effective manner.

Carbon dioxide (Acceptable)

Carbon dioxide has a rapid depressant, analgesic and anesthetic effect. Carbon dioxide is nonflammable, nonexplosive, and poses minimal hazard to personnel when used with properly designed equipment. Because CO₂ is heavier than air, incomplete filling of a chamber may permit animals to climb or raise their heads above the higher concentrations and avoid exposure. Compressed CO₂ gas in cylinders is the only recommended source of carbon dioxide because the inflow to the chamber can be regulated precisely. Carbon dioxide generated by other methods such as from dry ice, fire extinguishers, or chemical means (i.e. antacids) is unacceptable. During euthanasia, species should be separated and chambers should not be overcrowded. With an animal in the chamber, an optimal flow rate should displace at least 20% of the chamber volume per minute. Loss of consciousness may be induced more rapidly by exposing animals to a CO₂ concentration of 70% or more by prefilling the chamber. Gas flow should be maintained for at least 1 minute after apparent clinical death. It is important to verify that an animal is dead when removing it from the chamber.

Inhalant anesthetics (Acceptable)

Inhalant anesthetics are particularly valuable for euthanasia of smaller animals or for animals in which venipuncture may be difficult. Although acceptable, these agents are generally not used in larger animals because of their cost and difficulty of administration. When using inhalant anesthetics, chronic exposure to anesthetic gases by personnel or animals should be avoided. Also, since the liquid state of most inhalant anesthetics is irritating, animals should be exposed only to vapors and prevented from contacting the anesthetic agent in its liquid form. In order of preference, halothane, enflurane, isoflurane, sevoflurane, and desflurane, with or without nitrous oxide, are generally acceptable for euthanasia of small animals (< 7 kg).

Halothane induces anesthesia rapidly and is the most effective inhalant anesthetic for euthanasia.
Enflurane is similar to halothane in rate of induction but may induce seizure at deep planes of anesthesia.
Isoflurane should induce anesthesia more rapidly than halothane, however it has a slightly pungent odor and animals may hold their breath, thus delaying onset of loss of consciousness.
Sevoflurane does not have an objectionable odor and anesthetic concentrations can be rapidly achieved.
Desflurane is quite pungent and may be slow to induce anesthesia and subsequent euthanasia. Desflurane also could displace oxygen and induce hypoxemia during induction if supplemental O₂ is not provided.
Nitrous oxide (N₂O) is not flammable, however it will support combustion. It may be used with other inhalants to speed the onset of anesthesia, but alone it does not induce anesthesia in animals. When used alone, N₂O produces hypoxemia before respiratory or cardiac arrest and can result in animal distress prior to loss of consciousness. Therefore, nitrous oxide should not be used alone. Also, there can be a potential for human abuse to N₂O.

Carbon monoxide (Acceptable)

Carbon monoxide induces loss of consciousness without pain and with minimal discernible discomfort. Hypoxemia induced by CO is insidious, so that the animal appears to be unaware. Death occurs rapidly if concentrations of 4-6% are used. Safeguards must be taken to prevent exposure to personnel. Any electrical equipment exposed to CO must be explosion proof. When using carbon monoxide for euthanasia, only compressed CO from cylinders should be used and all applicable safety precautions must be taken.

Inhalant Anesthetics (Conditionally Acceptable)

Methoxyflurane has a slow induction rate and may be accompanied by agitation. Induction with methoxyflurane is unacceptably slow in some species.
Ether is irritating to the mucous membranes and poses serious risks associated with its flammability and explosiveness. Explosions have occurred when animals, euthanatized with ether, were placed in an ordinary (not explosion proof) refrigerator or freezer and when bagged animals were placed in an incinerator. Ether should only be used after IACUC approval in carefully controlled situations in compliance with all applicable safety policies and regulations.

Nitrogen, Argon (Conditionally Acceptable)

Hazards to personnel are minimal, however loss of consciousness is preceded by hypoxemia and ventilatory stimulation, which may be distressful to some species (i.e. rats). This technique is conditionally acceptable only if O₂ concentrations <2% are rapidly achieved and the animals are heavily sedated or anesthetized. Although N₂ and Ar are effective, other methods of euthanasia are preferable.

Barbiturates (Acceptable)

A primary advantage of barbiturates is speed of action, which depends on the dose, concentration, route, and rate of injection. Barbiturates induce euthanasia smoothly, with minimal discomfort to the animal. Intravenous injection is the preferred route of administration, however intraperitoneal injections may be used in situations when intravenous injections would be distressful or impractical. Intracardiac injection must only be used if the animal is heavily sedated, unconscious, or anesthetized. Barbiturates may be administered intraperitoneally and induce rapid, smooth euthanasia with minimal animal discomfort. As with all controlled substances, barbiturate usage requires having appropriate licensure and registration, ensuring secure storage and maintaining accurate drug accountability.

Potassium chloride in conjunction with general anesthesia (Acceptable)

Although unacceptable when used in unanesthetized animals, the use of potassium chloride administered intravenously or intracardially in animals under general anesthesia is an acceptable method of euthanasia. It is important for personnel performing this method of euthanasia to be trained and knowledgeable in anesthetic techniques, and competent in assessing anesthetic depth. Administration of potassium chloride 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.

Physical methods of euthanasia when properly used by skilled personnel with well maintained equipment, may result in less fear and anxiety and be more rapid, painless, humane, and practical than other forms of animal euthanasia. Exsanguination and stunning are not recommended as sole means of euthanasia, but should be considered adjuncts to other agents or methods. Personnel performing physical methods of euthanasia must be well trained and monitored for each type of physical euthanasia technique performed. Since most physical methods involve trauma, there is inherent risk for animals and humans, therefore extreme care and caution should be used. Methods not performed correctly can result in animal and personnel injuries. Inexperienced persons should be trained by experienced persons and should practice on carcasses or anesthetized animals to be euthanatized until they are proficient in performing the method properly and humanely.

Cervical Dislocation (Conditionally Acceptable)

Cervical dislocation is a technique that is rapidly accomplished and can induce rapid loss of consciousness without chemically contaminating tissue but requires technical proficiency. Manual cervical dislocation is a humane technique for euthanasia of rodents weighing < 200 g when performed correctly. In lieu of demonstrated technical competency, animals must be sedated or anesthetized prior to cervical dislocation.

Decapitation (Conditionally Acceptable)

Decapitation is a technique that is rapidly accomplished and appears to induce rapid loss of consciousness without chemically contaminating tissues. Personnel performing this technique should recognize the inherent danger of the guillotine and take adequate precautions to prevent personal injury. This method of euthanasia should be used only when its use is required by the experimental design and approved by the IACUC. The equipment used to perform decapitation should be maintained in good working order and serviced on a regular basis to ensure sharpness of blades. The use of plastic cones to restrain animals appears to reduce distress from handling, minimizes the chance of injury to personnel, and improves positioning of the animal in the guillotine. Those responsible for the use of this technique must ensure that personnel who perform decapitation techniques have been properly trained to do so.

Microwave irradiation (Acceptable)

Microwave irradiation is a humane method for euthanatizing small laboratory rodents. Loss of consciousness is achieved in < 100 ms, and death in less than 1 second. Only instruments designed for euthanasia purposes and having appropriate power and microwave distribution should be used. Microwave ovens designed for domestic and institutional kitchens are absolutely unacceptable for euthanasia. Instruments designed to accommodate mice and rats are commercially available.

Air embolism - Air embolism may be accompanied by convulsions, opisthotomus, and vocalization. If used, it should be done only in anesthetized animals.

Blow to the head - Unacceptable for most species.

Burning - Chemical or thermal burning of an animal is not an acceptable method of euthanasia.

Chloral hydrate - Unacceptable in small mammals.

Chloroform - Chloroform is a known hepatotoxin and suspected carcinogen and, therefore, is extremely hazardous to personnel.

Cyanide - Cyanide poses an extreme danger to personnel and the manner of death is aesthetically objectionable.

Decompression - Decompression is unacceptable for euthanasia because of numerous disadvantages including:

• Many chambers are designed to produce decompression at a rate 15 to 60 times faster than that recommended as optimum for animals, resulting in pain and distress attributable to expanding gases trapped in body cavities.
• Immature animals are tolerant of hypoxia, and longer periods of decompression are required before respiration ceases.
• Accidental recompression, with recovery of injured animals, can occur.
• Bleeding, vomiting, convulsions, urination, and defecation, which are aesthetically unpleasant, may develop in unconscious animals.

Drowning - Drowning is not a means of euthanasia and is inhumane.

Exsanguination - Because of the anxiety associated with extreme hypovolemia, exsanguination should be done only in sedated, stunned, or anesthetized animals.

Formalin - Direct immersion of an animal into formalin, as a means of euthanasia, is inhumane.

Household products and solvents - Acetone, quaternary compounds (including CCl₄), laxatives, clove oil, dimethylketone, quaternary ammonium products (i.e. Roccal D Plus), antacids, and other commercial and household products or solvents are not acceptable agents for euthanasia.

Hypothermia - Hypothermia is not an appropriate method of euthanasia.

Neuromuscular blocking agents (nicotine, magnesium sulfate, potassium chloride, all curariform agents) - When used alone, these drugs all cause respiratory arrest before loss of consciousness, so the animal may perceive pain and distress after it is immobilized.

Rapid Freezing - Rapid freezing as a sole means of euthanasia is not considered to be humane. If used, animals should be anesthetized prior to freezing.

Strychnine - Strychnine causes violent convulsions and painful muscle contraction.

Stunning - Stunning may render an animal unconscious, but it is not a method of euthanasia (except for neonatal animals with thin craniums). If used, it must be immediately followed by a method that ensures death.