Guidelines for Euthanasia |
Creighton University IACUC |
The following information on euthanasia is based on regulations and information from the USDA, PHS, the 2000 Report of the AVMA Panel on Euthanasia and other articles cited in the references, as interpreted by the Creighton University Institutional Animal Care and Use Committee.
Euthanasia is the act of killing an animal by a method that induces rapid loss of consciousness and death without pain or distress. The procedure(s) chosen to sacrifice animals as part of an IACUC should meet the following objectives:
Only trained personnel should perform euthanasia. It is the responsibility of the principal investigator to assure that personnel performing euthanasia have been trained to perform the procedure used. ARP offers training on an as needed basis.
Methods that meet the objectives above have been outlines by the 2000 Report of the AVMA Panel on Euthanasia. Based on this publication, the IACUC recognizes three categories of procedures that bring about death in animals:
In addition, the IACUC recognizes adjunctive methods of euthanasia. These are methods that cannot be used as the sole means of euthanasia, but may be used in conjunction with anesthesia or other acceptable methods to ensure death.
Many protocols involve the euthanasia of study animals at a predetermined end point, when the animals often are still clinically healthy. However, in the event animals become ill or debilitated, either as a result of spontaneous disease or as a result of research, the criteria below must be utilized in the decision to provide euthanasia. Fulfillment of one criterion can constitute grounds for euthanasia. If veterinary or ARF personnel identify animals showing one or more of these symptoms, an attempt will be made to contact the primary investigator or an alternate responsible person. If a responsible person can not be located, the Veterinarian in Charge will authorize euthanasia of severely debilitated or moribund animal. Exceptions to these euthanasia guidelines are permitted only if the clinical signs listed below are expected as part of the experiment and appropriate measures are taken to minimize pain or discomfort in the animals. Such exceptions must be approved by the IACUC as part of the protocol review process.
Criteria for euthanasia include:.
Some methods of euthanasia (e.g. decapitation) are considered definitive and do not require verification of death. However, other methods (e.g.overdose of anesthetics, carbon dioxide) require confirmation of death as part of euthanasia. Creighton's IACUC generally requires one of the following observations or methods be used in such cases.
If any doubt occurs concerning the fact that death has occurred, a additional technique should be used to ensure the death of the animal.
The most commonly used AVMA approved methods of euthanasia are discussed in this section. Other methods, as well as specific information on dosage and techniques for the methods below, are detailed under species listings.
Barbiturates act to depress the central nervous system. At lower doses, they have anesthetic effects; at higher doses, they bring about the cessation of respiration, which is followed by cardiac arrest. All barbituric acid derivatives may be used for euthanasia when given by intravenous injection; in some species, intraperitoneal injection may also be used. Pain is limited to that associated with the injection and is thus minimal. The most commonly used barbiturate is sodium pentobarbital, although other barbiturates are also acceptable.
Barbiturates are DEA Schedule II drugs in unadulterated form, but pentobarbital combinations (with added local anesthetic agents or agents that metabolize to pentobarbital) are Schedule III, making them somewhat simpler to obtain, store, and administer.
Inhalant anesthetics are an effective means of euthanasia in many species. The most commonly used forms for euthanasia are:
With inhalant anesthetics, the animal can be placed in a closed receptacle containing cotton or gauze soaked with an appropriate amount of the anesthetic, or the anesthetic can be introduced from a vaporizer. The latter method may be associated with a longer induction time. Vapors are inhaled until respiration ceases and death ensues. Because the liquid state of most inhalant anesthetics is irritating, animals should be exposed only to vapors. Also, sufficient air or O2 must be provided during the induction period to prevent hypoxemia. Animals placed together in chambers should be of the same species, and chambers should not be overloaded. If needed, animals should be restrained so that they will not hurt themselves or others.
Occupational exposure to inhalant anesthetics constitutes a human health hazard. Therefore, effective procedures must be used to protect personnel from anesthetic vapors, such as conducting the euthanasia procedures in a biosafety cabinet.
Carbon dioxide is acceptable for euthanasia in appropriate species. Inhalation of CO2 at a concentration of 7.5% increases the pain threshold, and higher concentrations of CO2 have a rapid anesthetic effect. Concentrations of 70% CO2 generally lead to unconsciousness within one minute and death within five minutes in all adult laboratory mammals and birds. Time to loss of consciousness will be longer if the concentration is increased slowly rather than immersing the animal in the full concentration immediately. Neonates and species with physiologic mechanisms for coping with hypercapnia may require higher CO2 concentrations and/or longer exposure times for euthanasia.
Compressed CO2 gas in cylinders is the only acceptable source of carbon dioxide, because the inflow to the chamber can be regulated precisely. 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 for species in which this has not been shown to cause distress. Gas flow should be maintained for at least 1 minute after apparent clinical death. It is important to verify that an animal is dead before disposal. If an animal is not dead, CO2 narcosis must be followed with another method of euthanasia.
The NIH Office of Laboratory Animal Welfare (OLAW) recently issued a Public Health Service Policy guidance clarifying current requirements for the use of CO2 as a euthanasia agent.
Cervical dislocation is a technique that has been used for many years and, when performed by well-trained individuals, appears to be humane. However, there are few scientific studies to confirm this observation. Data suggest that electrical activity in the brain persists for 13 seconds following cervical dislocation, and unlike decapitation, rapid exsanguination does not contribute to loss of consciousness.
Manual cervical dislocation may be performed on conscious mice, rats weighing under 200 g, rabbits weighing under 1 kg, and small birds, when scientifically justified and performed by individuals with a demonstrated high degree of technical proficiency. Cervical dislocation may be used on anesthetized animals without specific justification.
Decapitation can be used to euthanatize rodents and small rabbits if scientifically justified. It provides a means to recover tissues and body fluids that are chemically uncontaminated; it also provides a means of obtaining anatomically undamaged brain tissue for study. Recent studies and reports indicate that loss of consciousness develops rapidly following decapitation.
Decapitation of amphibians, fish, and reptiles is acceptable, but must be followed by pithing due to the high tolerance of the nervous system in these species to hypoxia and hypotensive conditions.
Guillotines that are designed to accomplish decapitation in adult rodents and small rabbits in a uniformly instantaneous manner are commercially available. 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. Guillotines are not commercially available for neonatal rodents, but sharp blades can be used for this purpose. Equipment used to perform decapitations should be maintained in good working order and serviced on a regular basis to ensure sharpness of blades. Those responsible for the use of this technique must ensure that personnel who perform decapitation techniques have been properly trained to do so.
Adjunctive methods may be used to euthanize animals already anesthetized by other means. They may not be used on conscious animals. When used as adjunctive methods of euthanasia, cervical dislocation and decapitation do not require special justification.
Exsanguination can be used to ensure death in unconscious animals. Because anxiety is associated with extreme hypovolemia, exsanguination must not be used as a sole means of euthanasia. Animals may be exsanguinated to obtain blood products, but only when they are sedated or anesthetized.
Although unacceptable and condemned when used in unanaesthetized animals, the use of a supersaturated solution of potassium chloride (KCl) injected intravenously or intracardially in an animal under general anesthesia is an acceptable method to produce cardiac arrest and death. The potassium ion is cardiotoxic, and rapid intravenous or intracardiac administration of 1 to 2 mmol/kg of body weight will cause cardiac arrest. One disadvantage of potassium chloride as a euthanasia agent is that rippling of muscle tissue and clonic spasms may occur on or shortly after injection.
It is of utmost importance that personnel performing this technique are trained and knowledgeable in anesthetic techniques, and are competent in assessing anesthetic depth appropriate for administration of potassium chloride intravenously. Administration of potassium chloride intravenously 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.
The injection of fixative into the bloodstream of a living animal is an effective way to preserve tissues before they are effected by hypoxia or other factors associated with the cessation of respiration and circulation. It is also effective in bringing about the death of the animal, and thus can be used as an adjuctive method of euthanasia following anesthetization. As with potassium chloride, care must be taken to ensure that the animal is in a surgical plane of anesthesia before perfusion takes place.
The information in this section is provided to assist investigators in selecting acceptable euthanasia methods for common laboratory species. The list of methods is not exhaustive, and the 2000 Report of the AVMA Panel on Euthanasia should be considered a more complete reference. Questions or concerns about these methods, alternatives methods, or methods suitable for species not listed can be directed to the Veterinarian in Charge or the Animal Resource Facility Manager.
These methods are generally applicable to any mammal under 1 kg.
Carbon dioxide is considered an acceptable euthanasia agent for laboratory rodents when properly administered. At a CO2 concentration of 70% or more, an exposure time of 5 minutes appears generally adequate to kill adult animals. Note that in some animals, particularly under deep carbon dioxide anesthesia, heartbeat can be maintained after visible respiration has ceased, and the animal might eventually recover. Gas flow should be maintained for at least one minute after apparent clinical death (cessation of respiration), and animals should be observed for at least ten minutes after removal from the chamber to ensure that unintended recovery does not occur. Alternatively, after CO2 exposure cervical dislocation, decapitation, exsanguination, or bilateral thoracotomy (a stab incision into the chest cavity on both sides) may be performed.
A CO2 euthanasia chamber is available in the Animal Resource Facility.
Barbiturates may be injected intraperitoneally as an acceptable method of euthanizing rodents. Sodium pentobarbital at 150 mg/kg is suitable for mice, rats, hamsters and gerbils.
Animals are typically placed in a sealed container such as a bell jar containing gauze soaked with the anesthetic agent; animals should be separated from the anesthetic soaked gauze by a false bottom or other method to prevent direct animal contact with the liquid anesthetic. The animal should be left in the chamber for at least one minute after apparent clinical death (cessation of respiration). Animals should be observed for at least five minutes after removal from the chamber to ensure that unintended recovery does not occur. Alternatively, after anesthetic exposure cervical dislocation, decapitation, exsanguination, or bilateral thoracotomy (a stab incision into the chest cavity on both sides) may be performed.
Cervical dislocation and decapitation are each conditionally acceptable method of euthanasia, which may be used under the following conditions:
Investigators are responsible for determining that personnel performing cervical dislocation or decapitation are properly trained to do so. In these animals, cervical dislocation involves holding the animal prostrate with the thumb and fore-finger firmly squeezing the neck, while the free hand pulls the quarters caudally. The need for technical competency is greater in heavy rats, in which the large muscle mass in the cervical region makes manual cervical dislocation physically more difficult.
Exsanguination, potassium chloride infusion, and perfusion with fixative may all be used to bring about death in anesthetized animals when scientifically justified.
The AVMA panel does not provide specific guidelines for the euthanasia of neonatal rodents. The IACUC recommends one of the following methods for use in rodents under 14 days of age.
To minimize the period of breathlessness guinea pigs should be placed in a container pre-filled with carbon dioxide, rather than filling the container with CO2 once the guinea pigs have been placed inside.
Barbiturates are an acceptable means of euthanasia when administered intraperitoneally at a dose rate of 100 mg/Kg.
A concentration of 4% halothane can produce cardiac arrest in 90 seconds.
Cervical dislocation may be used to euthanize young guinea pigs when scientifically justified The technique must only be performed by skilled operators. Training should take place by practicing on dead or anaesthetized animals. Dislocation is performed by holding the animal with the fingers of one hand around the back of the neck with the elbow flexed while restraining the hindquarters with the other. The elbow at the back of the neck is extended and the wrist flicked backwards as the elbow becomes straight.
Exsanguination, potassium chloride infusion, and perfusion with fixative may all be used to bring about death in anesthetized animals when scientifically justified.
Sodium pentathol can be administered i.v. via the ear vein at a dose of 60 mg/kg. Dilation of the ear vein to facilitate administration may be achieved by warming the ear. Intraperitoneal injection is conditionally acceptable and must be justified.
Halothane or isoflurane may be used as an acceptable means of euthanasia. Exposure should be maintained for at least one minute after apparent clinical death (cessation of respiration). Vital signs should be checked for at least five minutes after exposure to ensure that unintended recovery does not occur. Alternatively, after anesthetic exposure cervical dislocation, decapitation, exsanguination, or bilateral thoracotomy (a stab incision into the chest cavity on both sides) may be performed.
Cervical dislocation and decapitation are each conditionally acceptable method of euthanasia, which may be used on rabbits under 1 kg under the following conditions:
Investigators are responsible for determining that personnel performing cervical dislocation or decapitation are properly trained to do so. In rabbits, cervical dislocation is performed by holding the head in one hand and the hind limbs in the other. The animal is stretched and the neck is hyperextended and dorsally twisted to separate the first cervical vertebra from the skull. The need for technical competency is greater than in mice, since the large muscle mass in the cervical region makes manual cervical dislocation physically more difficult.
Use of a captive bolt may be an option when the use of an anesthetic is not acceptable within the scientific aims of the study. Full justification must be provided, and operators need training in this technique. The captive bolt is applied to the head between the ears and aimed downwards so as to parallel the vertical plane of the head. Performed correctly it results in immediate unconsciousness and immediate loss of reflexes and respiration.
Exsanguination, potassium chloride infusion, and perfusion with fixative may all be used to bring about death in anesthetized animals when scientifically justified.
Sodium pentathol can be administered i.v. via the ear vein at a dose of 150 mg/kg. Dilation of the ear vein to facilitate administration may be achieved by warming the ear.
The captive bolt should be placed at the intersection of lines drawn from each eye to the opposite ear. The operator must ensure that the captive bolt is of an appropriate size with appropriate sized cartridges. A cattle captive bolt pistol is appropriate for larger pigs.
Sodium pentobarbital is normally given at a dose rate of 60 mg/kg by the i.v. route, using the jugular vein with the animal restrained in a sitting position and the head laterally inclined to make the jugular apparent. In sheep, wool may need to be removed to enable the vein to be seen.
This method is acceptable in the hands of a trained operator but reflex movements following euthanasia may be aesthetically objectionable. Experience would indicate with horned and hornless animals the best site of placement is 1cm behind the nuchal crest and aimed vertically downward towards the gullet.
By far the most acceptable technique for euthanasia of dogs and cats is i.v. administration of sodium pentobarbital at a dose of 150 mg/kg, with almost immediate unconsciousness followed by respiratory depression.
Halothane or isoflurane may be used as an acceptable means of euthanasia. Exposure should be maintained for at least one minute after apparent clinical death (cessation of respiration). Vital signs should be checked for at least five minutes after exposure to ensure that unintended recovery does not occur.
Use sodium pentobarbital at a dose rate of at least 80 mg/kg given i.p.
Carbon dioxide is recommended for chicks only. Chicks should remain in the CO2 chamber for at least ten minutes. Carbon dioxide is conditionally acceptable for older birds, but the chamber should be filled with carbon dioxide before putting birds in. Older birds can show excessive flapping following loss of consciousness, which may be aesthetically unpleasant.
Halothane or isoflurane may be used as an acceptable means of euthanasia, but care should be taken that birds do not injure themselves when initially exposed to anesthetic vapors. Exposure should be maintained for at least one minute after apparent clinical death (cessation of respiration). Vital signs should be checked for at least five minutes after exposure to ensure that unintended recovery does not occur.
Cervical dislocation or decapitation may be used to euthanize smaller birds when scientifically justified. The technique must only be performed by skilled operators.
Exsanguination, potassium chloride infusion, and perfusion with fixative may all be used to bring about death in anesthetized animals when scientifically justified.
Sodium pentobarbital (60-100 mg/kg) can be administered intravenously or intraperitoneally.
Use of inhalant agents for euthanasia is acceptable for lizards and snakes, but not recommended for chelonians (turtles) as many of these species can hold their breath for extended periods. A physical method of euthanasia must be used after apparent clinical death to ensure that death has in fact occurred
Decapitation alone is not considered an acceptable means of euthanasia in reptiles because of the high tolerance of nervous tissue to hypoxia. Double pithing, or decapitation followed by double pithing, is required to ensure brain death. Ideally these methods should follow anesthesia, but stunning prior to decapitation or pithing is acceptable when the operator is well-trained and when scientific justification is provided. Pithing may not be performed on conscious animals.
Like inhalant agents, carbon dioxide may be used in lizards and snakes but not cheloneans. Loss of consciousness usually develops rapidly, but exposure times required for euthanasia are prolonged. Exposure must be followed by a physical method of euthanasia.
Perfusion with fixative may be used to bring about death in anesthetized animals when scientifically justified.
Cooling may be used to decrease activity in animals prior to euthanasia, but the temperature must be no lower than that normally experienced by the species. Freezing a conscious animal is not permissible, except when small animals are flash frozen in liquid nitrogen.
Chemical means are the preferred method of euthanasia in amphibians, but these methods should always be followed by double pithing (with or without decapitation) or freezing below -10°C to ensure death.
Sodium pentobarbital is effective at a dose of 60 to 100 mg/kg. The intravenous route is recommended where possible, especially when using concentrated euthanasia solutions. Otherwise the i.p. route may be used using the 60mg/ml solution usually used for anesthetic purposes.
Animals can be placed in a 2-3 mm deep anesthetic solution, which is then absorbed through the ventral skin. Commonly used anesthetics are:
Decapitation alone is not considered an acceptable means of euthanasia in amphibians because of the high tolerance of nervous tissue to hypoxia. Double pithing, or decapitation followed by double pithing, is required to ensure brain death. Ideally these methods should follow anesthesia, but stunning prior to decapitation or pithing is acceptable when the operator is well-trained and when scientific justification is provided. Pithing may not be performed on conscious animals.
Carbon dioxide can be used for euthanasia in amphibians, but care must be taken due to the relatively high tolerance of some species to hypoxia and hypercapnia. Loss of consciousness usually develops rapidly, but exposure times required for euthanasia are prolonged. As with anesthetics, pithing or freezing should be used to ensure euthanasia.
Perfusion with fixative may be used to bring about death in anesthetized animals when scientifically justified.
Cooling may be used to decrease activity in animals prior to euthanasia, but the temperature must be no lower than that normally experienced by the species. Freezing a conscious animal is not permissible, except when small animals are flash frozen in liquid nitrogen.
Chemical means are the preferred method of euthanasia in fish. In species with high tolerance to hypoxia, these methods should always be followed by decapitation and pithing or freezing below -10°C to ensure death.
Sodium pentobarbital is effective at a dose of 60 to 100 mg/kg and can be administered intravenously, intraabdominally or intrapleuroperitoneally.
Animals can be placed in an anesthetic solution, which is then absorbed through the gills. Animals should be left in the solution for at least 10 minutes following cessation of respiratory movement. Commonly used anesthetics are:
Decapitation alone is not considered an acceptable means of euthanasia in fish because of the potential tolerance of nervous tissue to hypoxia. Subsequent pithing is required to ensure brain death. Ideally these methods should follow anesthesia, but stunning prior to decapitation is acceptable when the operator is well-trained and when scientific justification is provided.
Perfusion with fixative may all be used to bring about death in anesthetized animals when scientifically justified.
Cooling may be used to decrease activity in animals prior to euthanasia, but the temperature must be no lower than that normally experienced by the species. Freezing a conscious animal is not permissible, except when small animals are flash frozen in liquid nitrogen.
ARAC [National Institutes of Health Animal Research Advisory Committee]. 2002. Guidelines for the Euthanasia of Mouse and Rat Fetuses and Neonates.
ARAC [National Institutes of Health Animal Research Advisory Committee]. 2002 .Guidelines for Euthanasia of Rodents Using Carbon Dioxide.
AVMA [American Veterinary Medical Association]. 2000. Report of the AVMA Panel on Euthanasia. JAVMA 218:675-685.
DeNardo, D. 1995. Amphibians as laboratory animals. ILAR Journal 37:173-181.
Detolla L.J., Srinivas S., Whitaker B.R., Andrews C., Hecker B., Kane A.S., Reimschuessel R. 1995. Guidelines for the care and use of fish in research. ILAR Journal 37:159-173.
Greene, H.W. 1995. Nonavian Reptiles as Laboratory Animals. ILAR Journal 37:182-186.