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CONTENTS
A. Introduction
B. The Obligation to Provide Euthanasia
C. Euthanasia as Animals Experience It
D. Euthanasia From the Investigator's Perspective
E. The Controversy Concerning Decapitation and Cervical
Dislocation
F. Choosing a Humane Method
G. Acceptable Methods of Euthanasia
H. Methods of
Euthanasia for Poikilotherms
I. Unacceptable methods of Euthanasia
1993 Report of the AVMA Panel on Euthanasia
Euthanasia: The word can serve as its own guideline.
Though sometimes regarded as a euphemism for "killing," the word euthanasia actually means "good death." It is derived from the Greek terms eu for "good" and thanatos for "death." For purposes of this manual, euthanasia is the act of inducing a death without pain or stress. To carry out the act is to euthanize the animal.
Most investigators who use animals must eventually face the need to bring about their deaths. In some cases, the animal must die so that its tissues can be recovered for analysis. In others, the animal's scientific usefulness has ended, and it must be disposed of. In either case, the investigator is obligated to carry out euthanasia; he or she must provide the animal a good death.
In the simplest terms, a good death is one in which the animal experiences no pain, no fear, and no other significant stress before dying. Usually, this means that death occurs instantly or that the animal is first rendered fully unconscious through painless methods.
If postmortem tissues are needed for research, euthanasia must leave those tissues free of exogenous substances or alterations that could confound the collection or interpretation of data.
This chapter discusses appropriate and inappropriate methods of euthanasia. Researchers are also encouraged to consult the 1993 Report of the AVMA Panel on Euthanasia.
B. The Obligation to Provide Euthanasia
The obligation to provide laboratory animals with stress-free and painless death emerges from both good experimental design (data from a stressed animal or from biochemically altered animal tissues may, for example, be invalid) and humane considerations. Moreover, the obligation is backed by federal law and regulations and supported by guidelines established by the U.S. Public Health Service and promulgated through the National Institutes of Health (NIH). These guidelines, in turn, stipulate that methods of euthanasia should follow the recommendations formulated and updated periodically by the American Veterinary Medical Association (AVMA).
In purely practical terms, every investigator should understand that grant applications to NIH are required to set forth the euthanasia methods proposed, and that these methods should conform to the NIH and AVMA, and University guidelines. Further, the proposal must have the approval of the Animal Care and Facilities Committee.
If, in the investigator's judgement, scientific considerations dictate the use of a method of euthanasia not recommended by the AVMA panel, then he or she will need to explain these scientific considerations in writing. This written statement must satisfy the ACFC before it will approve the proposed use of a method not recommended by the AVMA.
The following sections of this chapter discuss the various considerations that enter into a wise and humane selection of euthanasia methods.
C. Euthanasia as Animals Experience It
Because painlessness is the chief ingredient of euthanasia, investigators need to understand the nature of pain and its manifestation in animals. Such an understanding can guide the intelligent choice of euthanasia methods. Those who misunderstand may conclude that an animal exhibiting spectacular muscular reflexes is in pain when it is not and that a quiet, unresponsive animal feels no pain when, in fact, it may be suffering greatly.
Pain, neurologists generally agree, involves a perception that results when impulses traveling in the nociceptive (pain transmitting) pathways reach a conscious cerebral cortex. Nociceptive pathways also carry signals to the neural network in the CNS that governs unconscious muscular reflexes. Thus, nociceptive impulses may stimulate muscle reflexes without being consciously perceived as pain, or they may be perceived as pain without stimulating reflexes.
The investigator's choice of a euthanasia method and his or her skill in carrying it out will determine which neural networks are activated, if any. Light surgical anesthesia, as anesthesiologists know, will block pain perception but not muscle reflexes. On the other hand, muscle-immobilizing agents (such as curare, succinylcholine, gallamine, pancuronium, nicotine, and decamethonium) block reflex action but do not affect pain perception.
The use of muscle-immobilizing agents for euthanasia is "absolutely condemned" by the AVMA panel (and is illegal for euthanasia in New Jersey). Human patients given curare-like drugs have reported feelings of extreme anxiety as a result of being totally paralyzed but fully conscious. Even massive doses given to dogs, though producing a variety of cardiac and respiratory abnormalities, failed to depress consciousness. In some experiments, the dogs retained a conscious EEG for as long as seven minutes after the drugs had paralyzed respiratory muscles.
Deep anesthesia, as used in most forms of general anesthesia for surgery on humans, blocks both nociceptive pathways, preventing reflex actions and rendering the animal unconscious. Still deeper doses are well known to depress vital centers of the brain, bringing on death.
Among physical methods of euthanasia, for example, stunning an animal (a method strongly discouraged under AVMA guidelines, except when carried out with the requisite skill) has been found to block pain perception by rendering the animal unconscious and unable to perceive pain. Stunning must then be followed by some other method, such as exsanguination, that ensures death before the animal can regain consciousness.
Methods that cause significant degrees of fear or other conscious stress are considered inhumane. Such stress can be caused by rough handling, sudden removal to strange surrounding, struggling against physical restraints, overcrowding in a gas chamber, and pain and fear induced by euthanasia methods that are improperly or inexpertly carried out.
Fear and panic, according to the AVMA panel, can even be induced in animals not undergoing euthanasia simply because they see, hear, or smell animals being killed, especially when members of their own species are involved. Some animals being euthanized may emit distress vocalizations or stress pheromones, even when unconscious and feeling no pain, that trigger stress reactions in other animals. The stress of one rat witnessing the death of another has been found to produce a fivefold increase in the level of plasma corticosterone. Many experienced investigators say they make it a point never to carry out euthanasia in a room where other animals are present.
Euthanasia is not to be performed in animal rooms at Rutgers.
Among the behavioral states that have been elicited by noxious stimuli in animals undergoing euthanasia are distress vocalizations, struggling, aggression, salivation, urination, defecation, emptying of anal sacs, pupillary dilation, tachycardia, sweating, and reflexive contractions of skeletal muscles causing shivering, tremors, or spasms. Some of these responses have been found to occur whether the noxious stimuli are consciously perceived (as pain) or not.
Complete immobility and unresponsiveness should not be taken as evidence that the animal is unconscious or without pain. Some species, particularly rabbits and chickens, may "freeze," becoming temporarily immobile when reacting to noxious stimuli.
Perhaps the most severe inducer of pain and panic among laboratory animals undergoing euthanasia is that elicited by the use of inappropriate or inexpertly executed methods. All methods require training and skill, but some are especially liable to being executed inhumanely because they produce severe physical trauma. To prevent the lethal trauma from causing pain, the method must be executed in a way that induces unconsciousness instantly. This requires considerable skill based on correct training and practice.
D. Euthanasia From the Investigator's Perspective
However humane, no euthanasia method is likely to be carried out unless it is compatible with the investigator's research needs and aesthetically acceptable to the persons who must execute it. In addition, a good euthanasia method must be safe for people.
The investigator's first task is to choose a method that does not interfere with the scientific objectives of the project. If the animals have already made their contribution and are simply to be dispatched humanely, any of the methods approved for the species will do. If, however, the dead animal's tissues are required for analysis, then the euthanasia method needs to be one that will not significantly alter the tissues.
The researcher who wants to study neurochemistry, for example, needs to avoid anesthetic agents that modify brain chemistry. The use of anesthesia in euthanasia also has been shown to alter liver metabolites. Even seemingly inoffensive chemical methods, such as carbon dioxide or nitrogen inhalation, can confound pulmonary studies. Both agents can alter the cellular architecture of lung tissue. Clearly, investigators need to familiarize themselves with the detailed effects of various euthanasia methods before they can make an intelligent choice.
Aesthetic considerations should also figure in an investigator's choice of euthanasia methods. Some quite painless methods may stimulate various unconscious vocalizations, muscle twitchings, and other behaviors that an observer might find unpleasant to witness. Even methods that produce no visible response from the animal have been found to elicit feelings of distaste and revulsion from workers who must spend much of their time killing animals, however humanely.
Euthanizing experimental animals can be a major source of stress for some laboratory workers, who may find euthanasia so distasteful that they actually increase an animal's suffering. Such workers may, for example, try to stun an animal with too light a blow. Investigators should be sensitive to the feelings of personnel in their laboratory.
Of equal concern to the investigator selecting a method is its safety to the workers carrying it out. Many euthanizing chemicals are potentially hazardous to humans. Some euthanasia methods pose physical dangers. Fingers, for example, have been lost in guillotines by workers trying to hold an animal in position.
Personnel who do not work with animals, such as employees, guests and the general public, may find euthanasia unpleasant to watch even if an acceptable method is performed properly. Euthanasia should not be performed where a casual observer might be able to view the procedure through a window or open door. Animal carcasses should be handled carefully and should not be left where they might be observed by the public. Carcasses should be placed in a closed, opaque container or bag for transportation and disposal.
E. The Controversy Concerning Decapitation and Cervical Dislocation
The National Picture
Members of the biomedical research community, veterinarians, and ethicists specializing in questions of humane animal care generally agree about which euthanasia methods are acceptable and which are not. They disagree, however, about the acceptability of euthanizing small animals by means of decapitation without prior anesthesia.
Researchers who needed to avoid the tissue-altering effects of chemical anesthesia had long used decapitation on small rodents, usually with the aid of a small guillotine. In one cut, it was believed, the blade would open the main blood vessel to the brain, causing so swift an exsanguination that the animal would go into immediate shock and unconsciousness. The guillotine, long held to be a humane, though visually shocking, way of executing human criminals, seemed an ideal method for animals.
In 1975, however, doubts emerged. Electroencephalographic studies of rats being decapitated showed that certain brain waves continued for about 14 seconds after the head was severed. The waves were of a type known to accompany a state of alertness and arousal and would be expected to appear if the brain were experiencing pain. Brain death, as indicated by a flat EEG, did not occur until 27 seconds after decapitation.
On the basis of this one report, the 1986 revision, the 1986 Report of the AVMA Panel on Euthanasia ruled that decapitation alone was not a humane procedure. (The panel also included cervical dislocation, a kind of internal decapitation, in this ruling.) Much to the dissatisfaction of neurochemists and other specialists, the panel held that before being guillotined, animals should be chemically sedated or anesthetized.
Fearing the loss of a necessary research tool, but not wishing to treat their animals inhumanely, neurochemists and others in the animal research community have reexamined the interpretation of the 1975 EEG study. One new analysis of the issue points out that while the particular EEG waves detected in the disembodied rat brains (low wave, fast activity) were typical of an alert brain, they were also present in the sleeping brain and can be produced by hypoxia, precisely the lethal condition induced by decapitation. Moreover, an old French study had already shown that exactly the same waves are produced by an isolated cerebral cortex, surgically separated from any sensory input.
Examination of the issue by many in the biomedical research community, including the American Physiological Society's Committee on Animal Care and Experimentation, has led to a widely shared feeling that the lone 1975 study is not persuasive. Many of these researchers and professional societies dissent from the AVMA's revised position and have requested that the revision be rescinded.
The recommendation of the AVMA in 1986 read in part, "Until additional information is available to better ascertain whether guillotined animals perceive pain, the technique should be used only after the animal has been sedated or lightly anesthetized [emphasis added], unless the head will be immediately frozen in liquid nitrogen subsequent to severing." In the 1993 version, this recommendation was changed to omit the requirement for the use of sedation or anesthesia, but states that "this technique should be used in research settings only when scientifically justified by the user and approved by the [ACFC]".
Rutgers Policy on Rodent Euthanasia
The Animal Care and Facilities Committee (ACFC) has issued a position on several methods of euthanasia for rodents. These recommendations affect most investigators using rodents and should be studied carefully. The new recommendations are described.
Review of animal use protocols ("protocol review") as we know it today, has been in place at Rutgers and other NIH grantee institutions since January 1986. At that same time, the American Veterinary Medical Association revised the Report of the AVMA Panel on Euthanasia. One of the key changes in the 1986 Report was language that discouraged the use of two commonly used procedures for the euthanasia of rodents: cervical dislocation and decapitation. Since that time, the ACFC has discouraged the use of these techniques as a "routine" method of euthanasia, but allowed them when scientifically justified, in writing, in the protocol review form.
Although other methods are approved, one of the most common acceptable methods of euthanasia for rodents is CO2 asphyxiation.
Since 1986 then, the committee’s approach to this issue has been to routinely approve CO2 asphyxiation, or other methods recommended by the AVMA Panel, while discouraging cervical dislocation and decapitation, but approving them for specific projects when justified in writing.
Based on recent reports, including one co-authored by Dr. Buzsaki of Rutgers, Newark, the committee feels that decapitation may in fact be preferred and more humane as compared to CO2. The original concern about decapitation arises from a study which showed electrical activity persisting in the brain for 14 seconds after decapitation. Further analysis of these studies discounts this electrical activity as evidencing consciousness. The effectiveness of decapitation is attributed largely to the rapid loss of blood flow. Since this does not occur following cervical dislocation, one should not extrapolate findings about decapitation to cervical dislocation. Furthermore, the committee is concerned that CO2 asphyxiation may be stressful prior to unconsciousness.
The result of this interpretation is that the ACFC will no longer discourage decapitation. Investigators are in fact encouraged to consider it as method of euthanasia. However, it’s not that simple!
Rutgers Policy on Decapitation of Small Rodents
Decapitation is an acceptable method of euthanasia for small rodents. Decapitation is not without risk to the operator and can be aesthetically unpleasant. It may be stressful for technicians who have to perform it. It should only be performed by properly trained individuals. LAS can provide training for faculty and staff.
To the extent that Rutgers’ Policy has been set by the ACFC, it is not inconsistent with PHS Policy. However, grant applicants should realize that decapitation is still generally discouraged at most institutions and adherence to guidelines which defer to the AVMA panel report (PHS Policy, the Guide, AAALAC, USDA, AWA regulations) still requires written justification. Its use should be explained and defended in grant applications. A suggested statement to include in the required description of the method of euthanasia would be, "The Rutgers Animal Care and Facilities Committee has determined that decapitation is an acceptable and preferred method of euthanasia for rodents."
In order to comply with PHS Policy and the NIH "Guide", investigators should still include a written justification for use of decapitation in the protocol review form. The suggested statement in the previous paragraph should be included in the response to the question about anesthesia methods on the Rutgers protocol review form.
Rutgers Policy on the Use of CO2 for Euthanasia of Small Rodents
CO2 asphyxia is an acceptable method of euthanasia for small rodents at Rutgers. The Rutgers ACFC has determined that CO2 asphyxia may be stressful to rodents, and that other methods, including decapitation by properly trained individuals, may be preferable. CO2 is probably most appropriate when larger numbers of animals must be euthanized at one time, making decapitation time-consuming and stressful to those performing the procedure.
Rutgers Policy on the Use of Cervical Dislocation for Euthanasia of Small Rodents
Cervical dislocation is an acceptable method of euthanasia for mice and rats less than 200 g. However, unless used with prior sedation, it should only be used when it has specific advantages for the research project, and such use must be justified in writing as part of the animal use protocol.
F. Choosing a Humane Method
In a time when evidence of humane animal care is being examined ever more closely by both federal granting agencies and the general public, it behooves every scientist to reexamine his or her chosen euthanasia methods in light of these questions:
_ Do the methods interfere with the scientific goals? Are there alternatives that would leave the tissues unaltered?
_ Are the methods safe for laboratory workers? Are there safer alternatives?
_ Are the euthanasia methods a source of job stress for laboratory workers? Can less distressing methods be used?
_ Do the euthanasia methods provide the animals a stress-free and painless death? Is there another method more likely to provide the animals - the living beings that we study precisely because they are our biological kin - a good death?
G. Acceptable Methods of Euthanasia
The Rutgers Animal Care and Facilities Committee carefully reviews the proposed method(s) of euthanasia for each project. A change in method of euthanasia constitutes a significant change and should be reviewed by the ACFC. The committee uses the 1993 Report of the AVMA Panel on Euthanasia as the authority on acceptable methods of euthanasia. The methods listed below are generally acceptable when used as described.
Most of the comments are extracted from the AVMA Report. Links following each section will take you to the relevant section of the AVMA Panel Report. The report has an extensive list of references.
1. Inhalant Anesthetics
Advantages - (1) Inhalant anesthetics are particularly valuable for euthanasia of smaller animals (<about 7 kg) or in animals in which venipuncture may be difficult. (2) Halothane, enflurane, isoflurane, methoxyflurane, and nitrous oxide (N2O) are nonflammable and nonexplosive under ordinary environmental conditions.
Disadvantages - (1) Struggling and anxiety may develop during induction of anesthesia because anesthetic vapors may be irritating and can induce excitement. (2) Ether is flammable and explosive and should not be used near an open flame or other ignition sources. 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. (3) Nitrous oxide will support combustion. (4) Personnel and animals can be injured by exposure to these agents. (5) There is a potential for human abuse of some of these drugs, especially nitrous oxide.
AVMA Recommendations - In order of preference, halothane, enflurane, isoflurane, methoxyflurane, and ether, with or without nitrous oxide, are acceptable for euthanasia of small animals (< about 7 kg). Nitrous oxide should not be used alone, pending further scientific studies on its suitability for animal euthanasia. Although acceptable, these agents are generally not used in larger animals because of their cost and difficulty of administration.
Note: The use of ether as an anesthetic is strongly discouraged by LAS.
Note: All volatile anesthetics must be properly vented. Open containers, euthanasia chambers, and open anesthetic systems must be used in a fume hood certified by REHS. The hood location must be specified in the Animal Use Protocol Review Form. Anesthetic machine waste gases must be properly exhausted.
2. Carbon Dioxide (CO2)
Rutgers Policy on the Use of CO2 for Euthanasia of Small Rodents: CO2 asphyxia is an acceptable method of euthanasia for small rodents at Rutgers. The Rutgers ACFC has determined that CO2 asphyxia may be stressful to rodents, and that other methods, including decapitation by properly trained individuals, may be preferable. CO2 is probably most appropriate when larger numbers of animals must be euthanized at one time, making decapitation time-consuming and stressful to those performing the procedure. See also Rutgers Policy on Rodent Euthanasia.
Advantages (AVMA) - (1) The rapid depressant and anesthetic effects of CO2 are well established. (2) Carbon dioxide may be purchased in cylinders or in solid state as "dry ice." (3) Carbon dioxide is inexpensive, nonflammable, and nonexplosive, and poses minimal hazard to personnel when used with properly designed equipment. (4) Carbon dioxide does not result in accumulation of tissue residues in food producing animals. (5) Carbon dioxide euthanasia does not distort cellular architecture.
Disadvantages (AVMA) - (1) Because CO2 is heavier than air, incomplete filling of a chamber may permit tall or climbing animals to avoid exposure and to survive. This appears to be very distressful to the animals. (2) Some species may have extraordinary tolerance for CO2.
AVMA Recommendations - Carbon dioxide is acceptable for euthanasia. Compressed CO2 gas in cylinders is preferable to dry ice because the inflow to the chamber can be regulated precisely. If dry ice is used, animal contact must be avoided to prevent freezing or chilling. Carbon dioxide generated by other methods such as from a fire extinguisher or from chemical means (e.g., Alka-Seltzer) are (sic) unacceptable. With an animal in the chamber, an optimal flow rate should displace at least 20% of the chamber volume per minute. Unconsciousness may be induced more rapidly by exposing animals to a CO2 concentration of 70% or more by prefilling the chamber. It is important to verify that an animal is dead before removing it from the chamber. If an animal is not dead, CO2 narcosis must be followed with another method of euthanasia. Larger animals, such as rabbits, cats, and swine, appear to be more distressed by CO2 euthanasia; therefore, other methods of euthanasia are preferable.
Note: LAS maintains CO2 euthanasia chambers in most animal facilities.
Note: A fume hood is not required for CO2.
3. Barbituric Acid Derivatives
Advantages - (1) A primary advantage of barbiturates is speed of action. This effect depends on the dose, concentration, and rate of injection. (2) Barbiturates induce euthanasia smoothly, with minimal discomfort to the animal. (3) Barbiturates are less expensive than many other euthanasia agents are.
Disadvantages - (1) Intravenous injection is necessary for best results, necessitating trained personnel. (2) Each animal must be restrained. (3) Current federal drug regulations require strict accounting for the barbiturates and these must be used under the supervision of personnel registered with the US Drug Enforcement Administration (DEA) and with the New Jersey Department of Consumer Affairs. (4) An aesthetically objectional (sic) terminal gasp may occur in unconscious animals.
AVMA Recommendations - The advantages of using barbiturates for euthanasia in small animals far outweigh the disadvantages. The intravenous injection of a barbituric acid derivative is the preferred method for euthanasia of dogs, cats, other small animals, and horses. Intraperitoneal injection may be used in situations wherein these approaches would cause less distress than intravenous injection.
Note: Federal and state controlled drug registrations are required to obtain and store controlled substances. LAS cannot dispense more than one day’s supply of these drugs to faculty.
4. Pentobarbital Combinations
Several euthanasia products are formulated to include a barbituric acid derivative (usually sodium pentobarbital), with added local anesthetic agents or agents that metabolize to pentobarbital. Although some of these additives are slowly cardiotoxic, this pharmacologic effect is inconsequential.
These combination products are listed by the DEA as Schedule III drugs, making them somewhat simpler to obtain, store, and administer than Schedule II drugs such as sodium pentobarbital.
The pharmacologic properties and recommended usage of combination products presently available (which combine sodium pentobarbital with lidocaine or phenytoin) are interchangeable with those of pure barbituric acid derivatives. A combination of pentobarbital with a neuromuscular blocking agent is not an acceptable euthanasia agent.
A dosage rate of 2 to 3 times the usual anesthetic dose of pentobarbital is generally adequate for euthanasia. For rabbits and small rodents, 150-200 mg/kg is adequate.
Note: Federal and state controlled drug registrations are required to obtain and store barbiturates. LAS cannot dispense more than one day’s supply of these drugs to faculty.
Note: Pentobarbital is available for use as an anesthetic and also as a product made specifically for euthanasia. The euthanasia products generally have a much higher concentration of drug. Products intended for euthanasia should not be used as anesthetics.
5. Combination of Chloral Hydrate, Magnesium Sulfate, and Sodium Pentobarbital
A commercially available combination of chloral hydrate, magnesium sulfate, and sodium pentobarbital has been used for anesthesia of large animals, and is an acceptable large animal euthanasia agent when an overdose is administered intravenously.
AVMA Recommendations--This mixture is an acceptable large animal euthanizing agent when administered intravenously. Chloral hydrate alone is not acceptable except for large farm animal species under certain circumstances.
6. Magnesium Sulfate (MgSO4) or Potassium Chloride (KCl), with General Anesthesia
AVMA Recommendations--Magnesium or potassium salts must not be used alone for euthanasia because of the lack of analgesic or anesthetic effect. Potassium chloride may be administered to an anesthetized animal as an efficient and inexpensive way to cause cardiac arrest and death.
7. Captive Bolt Pistol (Livestock)
Advantage - The penetrating captive bolt is an effective method for use in slaughterhouses and in research facilities when the use of drugs is inappropriate.
Disadvantages - (1) It is aesthetically displeasing. (2) Death may not occur.
AVMA Recommendations - Use of the penetrating captive bolt is a practical method of euthanasia for horses, ruminants, and swine when chemical agents cannot be used. It is strongly recommended that other adjunctive measures (e.g., exsanguination) be used to ensure rapid death. Except for unusual circumstances, there are more acceptable methods of euthanasia for dogs and rabbits. The nonpenetrating captive bolt is not recommended as a method of euthanasia.
CAUTION: Captive bolt pistols should be treated like a firearm. They are extremely powerful and can cause serious injury to personnel.
Note: Development of a captive bolt pistol for small laboratory animals has been discussed, but one is not available at present.
8. Cervical Dislocation
Rutgers Policy on the Use of Cervical Dislocation for Euthanasia of Small Rodents: Cervical dislocation is an acceptable method of euthanasia for mice and rats less than 200 g. However, unless used with prior sedation, it should only be used when it has specific advantages for the research project, and such use must be justified in writing as part of the animal use protocol. See also Rutgers Policy on Rodent Euthanasia.
Advantages (AVMA) - (1) Cervical dislocation is a technique that may induce rapid unconsciousness. (2) It does not chemically contaminate tissue. (3) It is rapidly accomplished.
Disadvantages (AVMA) - (1) Cervical dislocation may be aesthetically displeasing to personnel. (2) Data suggest that electrical activity in the brain persists for 13 seconds following cervical dislocation. (3) Its use is limited to poultry, other small birds, mice, and immature rats and rabbits.
AVMA Recommendations - When properly executed, manual cervical dislocation is a humane technique for euthanasia of poultry, other small birds, mice, rats weighing <200 g, and rabbits weighing <1 kg. In heavier rats and rabbits, the greater muscle mass in the cervical region makes manual cervical dislocation physically more difficult; accordingly, it should be performed only with mechanical dislocators or by individuals who have demonstrated proficiency euthanizing heavier animals. Until additional information is available to better define the nature of the persistent EEG activity, this technique should be used in research settings only when scientifically justified by the user and approved by the Animal Care and Facilities Committee (ACFC). Those responsible for the use of this technique must determine that personnel who perform cervical dislocation techniques have been properly trained to do so.
The ACFC must determine that personnel who perform cervical dislocation techniques have been properly trained.
9. Decapitation with Guillotine
Rutgers Policy on Decapitation of Small Rodents: Decapitation is an acceptable method of euthanasia for small rodents. Decapitation is not without risk to the operator and can be aesthetically unpleasant. It may be stressful for technicians who have to perform it. It should only be performed by properly trained individuals. LAS can provide training for faculty and staff.
To the extent that Rutgers’ Policy has been set by the ACFC, it is not inconsistent with PHS Policy. However, grant applicants should realize that decapitation is still generally discouraged at most institutions and adherence to guidelines which defer to the AVMA panel report (PHS Policy, the Guide, AAAALC, USDA, AWA regulations) still requires written justification. Its use should be explained and defended in grant applications. A suggested statement to include in the required description of the method of euthanasia would be, "The Rutgers Animal Care and Facilities Committee has determined that decapitation is an acceptable and preferred method of euthanasia for rodents."
In order to comply with PHS Policy and the NIH "Guide", investigators should still include a written justification for use of decapitation in the protocol review form. The suggested statement in the previous paragraph should be included in the response to question about anesthesia methods on the Rutgers protocol request form. See also Rutgers Policy on Rodent Euthanasia.
Advantages (AVMA) - (1) Decapitation is a technique that may induce rapid unconsciousness. (2) It does not chemically contaminate tissues. (3) It is rapidly accomplished.
Disadvantages (AVMA) - (1) The handling and restraint required to perform this technique may be distressful to animals. (2) Data suggest that electrical activity in the brain persists for 13-14 seconds following decapitation. (3) Personnel performing this technique should recognize the inherent danger of the guillotine and take adequate precautions to prevent personal injury. (4) Decapitation may be aesthetically displeasing to personnel performing or observing the technique.
AVMA Recommendations-- Until additional information is available to better define the nature of the persistent EEG activity, this technique should be used in research settings only when scientifically justified by the user and approved by the Animal Care and Facilities Committee. Decapitation of amphibians fish, and reptiles is addressed elsewhere …. Those responsible for the use of this technique must determine that personnel who perform decapitation techniques have been properly trained to do so.
Exsanguination can be used to ensure death subsequent to stunning, electrical stunning, or in otherwise 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, stunned, or anesthetized.
Investigators should always specify the method of anesthesia to be used in conjunction with exsanguination.
11. Microwave Irradiation
Heating by microwave irradiation is used primarily by neurobiologists to fix brain metabolites in vivo while maintaining the anatomic integrity of the brain. Microwave instruments have been specifically designed or modified for use in euthanasia of laboratory mice and rats. The instruments differ in design from kitchen units and may vary in the maximal power output from 1.3 to 10 kw. All units direct their microwave energy to the head of the animal. The power required to rapidly halt brain enzyme activity depends on the efficiency of the unit, the ability to tune the resonant cavity and the size of the rodent head. There is considerable variation among instruments in the time required to induce unconsciousness and euthanasia. A 10-kw, 2,450-MHz instrument operated at a power of 9 kw will increase the brain temperature of 18- to 28-g mice to 79 degrees C in 330 msec, and the brain temperature of 250- to 420-g rats to 94 degrees C in 800 msec.
Advantages - (1) Unconsciousness is achieved in less than 100 msec, and death in less than one second. (2) This is the most effective method to fix brain tissue in vivo for subsequent assay of enzymatically labile chemicals.
Disadvantages - (1) Instruments are expensive. (2) Only animals the size of mice and rats can be euthanatized with commercial instruments that are currently available.
AVMA Recommendations - Microwave irradiation is a humane method to euthanize small laboratory rodents if instruments that induce rapid unconsciousness are used. Only instruments that are designed for this use and have appropriate power and microwave distribution can be used. Microwave ovens designed for domestic and institutional kitchens are absolutely unacceptable for euthanasia.
Microwave ovens designed for domestic and institutional kitchens are absolutely condemned for euthanasia use.
H. Methods of Euthanasia for Poikilotherms
Euthanasia of poikilotherms ("cold-blooded animals") poses special challenges. The 1993 Report of the AVMA Panel on Euthanasia expanded on recommendations for fish, amphibians and reptiles.
Rutgers Policy on Pithing as a Method OF Euthanasia for Frogs
Pithing is an acceptable method of euthanasia for amphibious frogs such as Leopard Frogs. The following guidelines should be followed. Procedures other than those recommended below must be approved by the Animal Care and Facilities Committee.
Comments on Pithing Policy
The following sources were consulted:
Reference 3 defers to reference 1, with the caution that "AVMA recommendations cannot be taken too rigidly for ectotherms...". Otherwise, it is not too helpful.
The AVMA panel considers DOUBLE pithing (both brain and spinal cord) to be an acceptable method of euthanasia. "Pithing", by which they mean single pithing or not-double pithing, is conditionally acceptable. It is assumed that single pithing refers to spinal cord only, but this is not clear. The "condition" as best it can be determined, is that the pithing should be double pithing. The Canadian guide states that pithing and other physical methods (i.e. a blow on the head, crushing the skull) can be used if chemical anesthetics (e.g. MS-222) cannot be used, implying that chemical euthanasia is preferred. The Canadian guide goes on to say, "It is important that the proper procedure for pithing should be learned in advance by the study of skeletal preparations and practice on freshly killed or preserved specimens. The pithing needle should be inserted into the foramen magnum and the brain destroyed quickly by lateral and vertical movement of the needle tip inside the brain case." Both the AVMA Panel and Canadian guide address the use of chilling the amphibian to 4 degrees C prior to pithing. The Canadian guide says "preferably", the AVMA Panel states, "It has been suggested that, when using physical methods of euthanasia in poikilothermic species, cooling to 4 C will decrease metabolism and facilitate handling, but there is no evidence that it raises the pain threshold."
The AVMA panel also cautions that "the anatomic features of some species preclude effective use of this method." The ACLAM text states that pithing should not be used for Xenopus as it is difficult to bend the head forward to expose the atlanto-occipital space and other methods should be used.
The ACLAM text states that pithing, which it defines as "severing the spinal cord behind the head" is "rapid and effective, but then does not list it as one the "recommended" procedures.
I. Unacceptable Methods of Euthanasia
1. Chloroform
Recommendations--Chloroform is a known potent hepatotoxin and is a suspected carcinogen. Although chloroform is nonexplosive, its use in the presence of a flame may result in the production of phosgene gas. Because of its significant hazards to human beings, chloroform is not permitted for euthanasia at Rutgers.
2. Nitrogen
Recommendations--Not an acceptable method at Rutgers
3. Hydrogen Cyanide Gas
Advantages--(1) Hydrogen cyanide gas induces rapid death, and (2) it can be used under field conditions where other agents are impractical.
Disadvantages--(1) The animal manifests violent convulsive seizures and opisthotonos prior to death; (2) the responses are disagreeable to most observers; (3) the gas is very irritating to the respiratory mucosa; and (4) there is extreme danger to personnel.
Recommendations--Not an acceptable method at Rutgers. Although the effect of hydrogen cyanide gas is rapid, reliable, and irreversible, it endangers the operator, and the manner of death is aesthetically objectionable.
4. Carbon Monoxide
Advantages - (1) Carbon monoxide induces unconsciousness without pain and with minimal discernible discomfort. (2) Hypoxemia induced by CO is insidious, so that the animal appears to be unaware. (3) Death occurs rapidly if concentrations of 4-6% are used.
Disadvantages - (1) Safeguards must be taken to prevent exposure of personnel. (2) Any electrical equipment exposed to CO (e.g., lights and fans) must be explosion proof.
AVMA Recommendations - Carbon monoxide used for individual animal or mass euthanasia is acceptable for small animals, including dogs and cats, provided that commercially compressed CO is used and the following precautions are taken: (1) Personnel using CO must be instructed thoroughly in its use and must understand its hazards and limitations. (2) The CO source and chamber must be located in a well-ventilated environment, preferably out of doors. (3) The chamber must be well lit and have viewports that allow personnel direct observation of the animals. (4) The CO flow rate should be adequate to rapidly achieve a uniform CO concentration of at least 6% after animals are placed in the chamber, although some species (e.g., neonatal pigs) are less likely to become agitated with a gradual rise in CO concentration. (5) If the chamber is inside a room, CO monitors must be placed in the room to warn personnel of hazardous concentrations.
5. T-61
T-61 is not legal for euthanasia of animals in New Jersey and so should not be used. T-61 is an injectable nonbarbiturate, non-narcotic mixture of three drugs used for euthanasia. These drugs provide a combination of general anesthetic, curariform, and local anesthetic actions. T-61 has been withdrawn from the market and is no longer manufactured or commercially available in the United States.
6. Chloral Hydrate
AVMA Recommendations--Chloral Hydrate is not recommended for euthanasia of dogs, cats, and other small animals because the associated signs may be severe and are aesthetically objectionable.
7. Strychnine
AVMA Recommendations--Strychnine is absolutely condemned for euthanasia.
8. Nicotine
AVMA Recommendations--Because nicotine sulfate is an extremely dangerous drug for personnel, is inhumane for animals, and induces serious side effects prior to death, it is absolutely condemned for euthanasia. Not lawful in New Jersey for euthanasia.
9. Curariform Drugs
Recommendations--Not lawful in New Jersey for euthanasia.
10. Ketamine and Xylazine
Recommendations--These drugs are anesthetics and should not be used alone for euthanasia. High doses may cause convulsions before death.
11. Air Embolism
Recommendations--Intravenous injection of 5 to 50 ml/kg of air induces rapid death in rabbits. However, it may be accompanied by convulsions, opisthotonos, and vocalization. It is an acceptable method only in anesthetized animals.