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Weichbrod RH, Thompson GAH, Norton JN, editors. Management of Animal Care and Use Programs in Research, Education, and Testing. 2nd edition. Boca Raton (FL): CRC Press/Taylor & Francis; 2018. doi: 10.1201/9781315152189-35

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Management of Animal Care and Use Programs in Research, Education, and Testing. 2nd edition.

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Chapter 35 Euthanasia

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Background and Perspective


The word euthanasia comes from the Greek terms eu (good) and thanatos (death). In the medical field, it is often defined as the act of painlessly killing an individual who is suffering from an incurable or painful disease. The Animal Welfare Act (AWA) and Animal Welfare Regulations (AWR), 9 CFR Part 1, §1.1 (U.S. Department of Agriculture 2002), define euthanasia as the humane destruction of an animal accomplished by a method that produces rapid unconsciousness and subsequent death without evidence of pain or distress, or a method that utilizes anesthesia produced by an agent that causes painless loss of consciousness. Although commonly used in the field of laboratory animal science to describe the killing of research animals at the end of an experiment, some assert that this is a misuse of the word (Pavlovic et al. 2011). According to this view, since animal experimentation is not in the best interest of the animal, the act of killing the animal cannot be considered euthanasia, and is instead part of the experimental process. Despite this criticism, the term euthanasia continues to be used in animal care and use programs in research, teaching, and testing.

Ethical Considerations

There is frequent discussion regarding the ethical considerations pertaining to the use of animals in research. Many points of view exist, from those who are vehemently opposed to those in favor, and discussions can lead to significant emotional responses (Gnadt and Leland 2002). Public perception of conditions under which animals used in research are kept is often unfavorable, and concerns regarding euthanasia of research animals are a specific area of debate on this topic. There is concern about not only the potential pain and suffering of animals while undergoing experimental procedures, but also how an animal’s life ends. As biomedical research involving animals continues, society’s awareness of animal welfare issues is increasing as well (Rollin 2009). According to Rollin (2009), it is important that the summation of their lives not involve fear, horror, pain, or suffering. Euthanasia of healthy research animals at the end of the experiment is a serious concern for the public (AVMA Panel on Euthanasia 2013). Recently, legislation has been passed in some states requiring that healthy dogs and cats used in research be made available for adoption rather than euthanized at the end of the experiment (Simmons 2014). While the adoption of some animals following the research project is possible, for others it is not. Often, postmortem analysis or sample collection must be performed to verify study results. Those who work in the laboratory animal field should be aware of the public’s ethical concerns surrounding the use and euthanasia of research animals.

Veterinarians and technicians working in the laboratory animal field may face unique ethical challenges. Like all veterinarians, those in the laboratory field have taken an oath to use their “scientific knowledge and skills for the benefit of society through the protection of animal health and welfare, the prevention and relief of animal suffering, the conservation of animal resources, the promotion of public health, and the advancement of medical knowledge” (Nolen 2011). According to the American Veterinary Medical Association’s (AVMA) Animal Welfare Principles, the responsible use of animals for human purposes, including research conducted for the benefit of both humans and animals, is consistent with the Veterinarian’s Oath (AVMA 2015a). Additionally, veterinarians are expected to adhere to the AVMA’s Principles of Veterinary Medical Ethics (AVMA 2015b). The first principle states that a veterinarian shall be dedicated to providing competent veterinary medical care, with compassion and respect for animal welfare and human health. Under this principle, humane euthanasia of animals is described as an ethical veterinary procedure. In some cases, however, the decision regarding if and how an animal should be euthanized may be complicated by other factors, such as the greater public good, as with euthanasia of animals used in medical research (AVMA Panel on Euthanasia 2013). Veterinarians and technicians should use their experience and knowledge to ensure the ethical care and use of research animals, promote humane euthanasia practices, and encourage refinement of procedures consistent with guidelines and regulations. They are also in a position to lend an important perspective to the ethical debate regarding animal experimentation.

Ethical concerns are frequently encountered in the laboratory animal facility and often involve animal care personnel, facility managers, veterinarians, principal investigators, and their staff (Gnadt and Leland 2002). Concerns involving endpoints for animals on study can be particularly emotional. In these cases, the veterinarian is generally called on to investigate the concern by reviewing the animal use protocol for information regarding approved endpoints. Follow-up and resolution may include discussion with the investigator or laboratory staff regarding following approved endpoints, and in some cases, Institutional Animal Care and Use Committee (IACUC) involvement may be required to resolve the issue. In all cases, the veterinarian or manager should follow up with the animal care personnel to communicate the outcome. Depending on the information discovered during investigation, this could involve a discussion about the specific study and why the approved endpoints are appropriate. Although that may not completely resolve the ethical concern for the employee, understanding why that endpoint is important to fulfill the scientific objectives of that particular study may relieve some of the concern.

Regulatory Expectations

There are several laws, regulations, and guidelines that provide guidance to the research community regarding the appropriate care and use of animals. Many of these outline specific expectations for adhering to humane endpoints and euthanasia procedures. IACUCs have the responsibility of evaluating animal care and use protocols as they relate to the welfare and use of animals within the framework of these regulations and guidelines (Committee for the Update of the Guide for the Care and Use of Laboratory Animals 2011). Special consideration should be given to protocols that involve the potential for unrelieved pain and distress, and the committee should encourage methods of replacement, refinement, and reduction and ensure a humane death (AVMA Panel on Euthanasia 2013).

Animal Welfare Act and Regulations

The AWA was initially enacted in 1966 and has been amended several times. The 1985 amendments included specific changes promoting laboratory animal welfare. Some of these changes included establishing an IACUC and the minimization of pain and distress. The importance of appropriate endpoints and euthanasia methods is highlighted in the AWR. They state that during the protocol review process, the IACUC shall determine that the proposed activities or significant changes in ongoing activities meet several requirements. One of the requirements is that animals that would otherwise experience severe or chronic pain or distress that cannot be relieved will be painlessly euthanized at the end of the procedure or, if appropriate, during the procedure. Additionally, the regulations state that methods of euthanasia used must be in accordance with the AWR definition unless a deviation is justified for scientific reasons (9 CFR Part 1, §2.31). The USDA Animal Care Policy Manual also addresses euthanasia of covered species, indicating that euthanasia should be in accordance with the current AVMA Guidelines for the Euthanasia of Animals (USDA 2016).

U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training

The Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals, administered by the Office of Laboratory Animal Welfare (OLAW), implements the U.S Government Principles (Interagency Research Animal Committee 1985). There are nine principles that must be followed when U.S. government agencies perform or sponsor procedures involving the use of vertebrate animals. Like the requirements described in the AWR, Principle VI addresses appropriate humane endpoints and states that animals that would otherwise suffer severe or chronic pain or distress that cannot be relieved should be painlessly killed at the end of the procedure or, if appropriate, during the procedure. Principle VIII pertains to personnel training and indicates that all shall be appropriately qualified and experienced for conducting experiments, and that adequate arrangements should be made for in-service training, including the proper and humane care and use of laboratory animals. As euthanasia is often a component of experiments involving animals, training on proper technique when performing euthanasia procedures is also important.

Guide for the Care and Use of Laboratory Animals

The Guide for the Care and Use of Laboratory Animals (Guide) is endorsed by the Institute for Laboratory Animal Research and the National Academy of Sciences. It establishes the minimum ethical, practice, and care standards for researchers and their institutions (Committee for the Update of the Guide for the Care and Use of Laboratory Animals 2011). Regarding euthanasia of research animals, the Guide states that methods should be consistent with the AVMA Guidelines for the Euthanasia of Animals, and that it should be carried out in a manner that avoids animal distress. The Guide also addresses the importance of training and proficiency when performing euthanasia.

Reasons for Euthanasia

Although proper performance results in the humane death of the animal, there exist varied reasons for euthanasia. In some instances, euthanasia is performed out of concerns for the well-being of the animal, while in others, it is undertaken as part of the experimental design of studies in which the animals are being used. In either case, the reasons for euthanasia should be clearly understood by all involved personnel.

Experimental Endpoints

Many studies have included in the experimental design specified points at which animals are euthanized so that samples or other data can be collected that would otherwise not be obtainable, that is, the point at which the scientific objectives of the project are reached. In some cases, animals may need to be euthanized when the research requires that tissue samples from sites such as the heart, lung, liver, or brain be collected in a way or quantity that would not allow the animal to survive. In other cases, the research may involve surgical implantation of materials or devices that must be retrieved for further evaluation, thus necessitating euthanasia of the animal. Typically, the point at which the animal is euthanized for the purposes of harvesting tissue or implanted materials is defined by elapsed time after experimental manipulation. For example, animals might be euthanized minutes, hours, days, or longer after initial manipulation, and these time points are designed to permit the investigator to study the biological response to some manipulation over time. Interestingly, changes in tissue induced by the hypoxia associated with euthanasia has led some to argue that collection on anesthetized, but alive, animals allows superior outcome (Overmyer et al. 2015). Other experimental criteria used to define the time of euthanasia are occasionally based on other criteria related to a biological response, such as a defined tumor volume (Yapp et al. 1998).

Although not strictly an experimental endpoint, circumstances sometimes arise in which animals are euthanized because they are no longer needed for the research, for example, at the conclusion of studies that do not require euthanasia as part of the experimental design or in the case of breeding colonies that generate animals of a genotype that are not needed for the research. In the spirit of reducing the number of animals used in or produced for research, the manager should encourage oversight of breeding colonies in such a way that the number of animals of the required genotype produced closely matches the number needed. In some cases, animals that are not needed for research can be transferred to another project after approval by the IACUC to do so, assuming that the animals have not already been used in research that would preclude additional use, such as an additional survival surgical procedure. It is also possible that animals not needed for research might be adopted out as pets. In such cases, the institution should ensure that the animal is healthy and that the adopter can responsibly provide for the needs of the animal. It would be important that legal counsel for the institution review the planned process for adoption and that the policy meets all state and local regulations. Some institutions make arrangements with bird sanctuaries or zoological parks to use naïve euthanized animals as a food source.

Experimental Humane Endpoints

Sometimes, animals must be euthanized because they are ill. In some cases, this is the result of spontaneous and unexpected disease, while in other cases, illness may be associated with experimental use, age, or phenotype. In the case of the former, decisions to euthanize an animal are usually based on the professional judgment of the veterinarian and made in consultation with the research team. With respect to the latter, defined endpoints are often stated within the protocol approved by the IACUC so that personnel have a predetermined point of reference from which to make decisions when animals decline clinically. Such defined endpoints generally reflect the sort of sound veterinary judgment that guides the decision to euthanize an animal due to illness unrelated to experimentation and is based on specified clinical criteria. In any case, the overall goal is to mitigate the pain or distress of the animal that would be otherwise unrelieved.

The precise endpoints that are defined for a study may depend on the type of animal model used and the goals of the research. Identifying acceptable humane endpoints is especially important for those studies in which animals may experience significant and unrelieved pain or distress, such as those involving tumor growth, infectious disease, and toxicology. The endpoints agreed to by the IACUC, the investigator, and the veterinary team should address the needs of the animals, yet allow reasonable room for conduct of the study. It is important that all personnel involved with the care and use of the animals clearly understand up front what these endpoints are and what happens when they are reached. In addition, a plan or system that aids rapid communication between personnel and facilitates decision making as endpoint criteria are reached is essential.

It is sometimes difficult to determine the exact point at which pain or distress changes from tolerable to intolerable; thus, humane endpoints are somewhat subjective but should be based on sound professional judgment with input from the veterinary team. Some typical parameters that might be used as endpoint criteria include

  1. Level of activity. Animals that are lethargic may be experiencing pain or distress. For example, an animal that is in pain could be unwilling to move. Similarly, animals that are severely ill due to various forms of metabolic disease may demonstrate lethargy. Many times, failure to groom will result in an unkempt appearance. In addition, vocalization can sometimes indicate the presence of pain or distress, such as the whining of a dog. Further, lack of vocalization, such as the absence of singing in a songbird, may also suggest that the animal is experiencing pain or distress.
  2. Decreased food and water consumption. Careful attention should be paid to the amount of food and water consumed by research animals, since decreases may signal significant pain and/or distress. It is important, therefore, that the amount normally consumed be known.
  3. Body weight. Along with decreased food and water consumption, loss in body weight can signal a decline in the animal’s health. If the animal has a tumor (spontaneous or induced), body weight may not be as useful for determining health status, as the tumor can artificially alter the animal’s true weight. In this case, other criteria should be considered. For example, body condition scoring has been used to evaluate health status in mice (Ullman-Cullere and Foltz 1999).
  4. Body temperature. A persistent or precipitous rise or decrease in body temperature can indicate significant illness and can be used to define humane endpoints. In particular, hypothermia is a good indicator of impending death, and it is common to use a decrease in body temperature below a specific point as an endpoint (Toth 2000; Cates et al. 2014; Dellavalle et al. 2014).
  5. Behavior and alertness. Listlessness, aggressiveness, or otherwise abnormal behavior can be signs of significant pain and distress in animals, and behavior and activity levels can be used as criteria in euthanasia decisions (Warren et al. 2014). As a result, it is important that personnel clearly understand what the expected normal behavior of a given species of animal is so that they may accurately note the occurrence of behaviors that are not normal.
  6. Evidence of infection. Infection may be associated with surgical wounds or result from opportunistic microbes associated with a debilitated animal. Signs of infection vary but may include discharge from wounds, fever, and listlessness.
  7. Presence of wounds or tumors. Unexpected traumatic wounds or nonhealing surgical wounds may be severe and merit euthanasia of the animal. Likewise, tumors that occur spontaneously or are the result of experimental manipulation may grow very large and abrade to the point of being an open wound, become necrotic, interfere with locomotion or normal function of a tissue, or significantly impact the health and well-being of the animal in other ways.
  8. Clinical pathology. Parameters related to hematology and clinical chemistry can be used to weigh humane endpoint decisions. For example, in Ebola virus–infected nonhuman primates, extreme alterations to several commonly monitored serum parameters were related to survival outcome and could therefore be used to reasonably establish endpoint criteria, particularly when evaluated in conjunction with measurements of body temperature and activity levels (Warren et al. 2014).

The above are examples of endpoint criteria that might be used. Of course, there are others, and in all cases the specific point at which an animal would need to be euthanized should be defined before the study begins, and it should be specific. For example, it may be determined that animals on a study should be euthanized when they lose 15% or 20% of their body weight (compared with age-matched controls) or when a tumor reaches a certain measurable size. In some cases, endpoints are reached when a combination of factors are considered. In this regard, scoring systems have been developed that consider clinical observational data as a means to identify humane endpoints (Nunamaker et al. 2013; Warren et al. 2014).

Veterinary Medical Conditions

Illness that is not caused by experimental manipulation can occur in animals as a result of their genotype, or it may be spontaneous. With respect to genotype, illness may be expected in animals whose genome has been altered as a way to create an animal model of a disease, although sometimes genomic alterations result in phenotypes that are unexpected and may be deleterious to the health of the animal. In such cases, the IACUC should be informed of the unexpected phenotype-associated illness.

Similar to humans and pet animals, laboratory animals may develop spontaneous illness, such as tumors, diabetes, renal disease, and others. This is especially true in aged animals. Laboratory animals may also experience illness associated with trauma, for example, that associated with fighting.

Any animal experiencing illness should be reported to the veterinary staff. Animals with severe illness due to spontaneous conditions or phenotype should be evaluated the same as those with illness due to experimental manipulation; that is, illness that is of clinical severity associated with poor outcome should be euthanized. In this regard, it is essential to invoke the opinion of the veterinarian.

Methods of Euthanasia

The AVMA has published guidelines for the euthanasia of animals, and those guidelines are widely considered to be the authoritative document on the subject (AVMA Panel on Euthanasia 2013). The AVMA guidelines consider various methods of euthanasia as being acceptable, acceptable with conditions, or unacceptable. Acceptable methods reliably result in the humane death of the animal, methods that are acceptable with conditions are those that require some additional conditions to be met in order for humane death to reliably result, and unacceptable methods are those that are regarded as inhumane.

With respect to research animals, selection of a euthanasia method should ensure humane death, as well as meet the experimental needs of the study (Angus et al. 2008; Karmarkar et al. 2010; Hazzard et al. 2014). With respect to the former, it is common to confirm death by means of an adjunctive (secondary) method, such as induction of pneumothorax, cervical dislocation (in rodents), or exsanguination. The method chosen should also consider the impact on personnel, in terms of both safety and emotional load, as individuals may perceive and respond differently to performing or witnessing euthanasia. In addition, it is good practice to euthanize animals out of the sensory range of other animals in order to minimize distress to those nearby.

The euthanasia methods used can be categorized with respect to whether they utilize pharmacological or chemical agents versus physical methods. The methods described here are not an exhaustive list of all possible methods, but rather represent methods that are commonly used for euthanasia of research animals.

Pharmacological and Chemical Methods

Methods for euthanasia that employ either drugs or chemicals generally involve exposure of the animal to the agent via inhalation or other routes of administration, such as intravenous or, in the case of aquatic species, immersion. When such agents are used, it is advisable to use pharmaceutical-grade compounds to ensure a smooth and rapid death of the animal.

Methods Involving Inhaled Agents

Inhaled agents are generally easy to use and can be used to euthanize larger numbers of animals at a single time. This approach is very commonly used to euthanize rodents. The most common agents used include carbon dioxide (CO2) and halogenated fluorocarbon anesthetics, such as isoflurane.

Carbon Dioxide

CO2 is an odorless, colorless gas that rapidly induces narcosis, progressing to death when used properly. Most commonly, CO2 is administered to animals contained within a chamber into which animals, or the home cage housing the animals, are placed. Animals placed in a chamber should not be crowded; thus, the typical chamber size accommodates rodents but not larger species. Animals to be euthanized as a group should be compatible and of the same species. A compressed gas cylinder equipped with a regulator and flowmeter is the preferred source of CO2. Although some have historically used dry ice as a CO2 source, it is widely regarded as unacceptable because of the inability to closely control CO2 concentration, as well as the risk for pain and distress to the animals resulting from thermal injury.

CO2 should be administered to chambers containing animals to be euthanized at a displacement rate of 10%–30% chamber volume per minute (AVMA Panel on Euthanasia 2013). It has been determined that this rate allows for rapid euthanasia of animals. A flowmeter attached downstream of the regulator is useful to consistently achieve flow rates that have been calculated to meet the 10%–30% displacement goal. Animals are usually left in the chamber until visible movement has ceased, and it is recommended that a method to confirm death is used. Because immature animals tolerate low oxygen levels better than adults, they require extended exposure time to CO2 for death to result (Klaunberg et al. 2004).

Because CO2 is heavier than atmospheric air, it is important to purge the chamber of remaining CO2 between groups so that it does not build up to levels that would cause pain and distress to the animals (Djoufack-Momo et al. 2014). Commercially available systems exist that automatically regulate the flow of CO2 and purge the chamber following a period that allows for death of the animals. Some such systems involve a single chamber, while others allow for multiple cages placed on a rack to be euthanized simultaneously.

Halogenated Fluorocarbon Anesthetics

The class of halogenated fluorocarbons includes several agents that are commonly used as anesthetics for surgery, including isoflurane, sevoflurane, halothane, and enflurane. Of these, isoflurane is the most commonly used. When administered as an overdose, these agents can be used for euthanasia of animals. Note that ether is sometimes used for anesthesia and euthanasia; however, its use is discouraged due to risks associated with its high flammability and explosive potential (including carcasses of animals euthanized by ether), as well as respiratory tract irritation following inadvertent inhalation by personnel.

Inhaled anesthetics are sometimes administered to rodents by means of placing an anesthetic-soaked cotton ball in a bell jar that includes a platform on which the animals may stand to avoid direct contact with the anesthetic. This method does not allow control of the anesthetic concentration, the buildup of high anesthetic concentrations may be distressful to animals, and the risk of personnel exposure to waste gas exists. The preferred method for administration of inhalant anesthetics is via precision vaporizer, a tool that carries specified concentrations of anesthetic, along with oxygen, or another carrier gas, to the animal or the chamber in which the animal is placed. The chamber should be gradually filled. Commercial systems are available that feed a specified concentration of anesthetic into a chamber at a constant rate. Any system using halogenated fluorocarbons for euthanasia should be equipped with a means to scavenge waste gas away from personnel. Another option is to initially anesthetize the animal with isoflurane and then use CO2 for euthanasia.

In general, inhaled anesthetics are used for euthanasia of small animals weighing less than 7 kg (AVMA Panel on Euthanasia 2013). It is recommended that once voluntary movement of the animal has ceased, adjunctive procedures be followed to confirm death.

Methods Involving Noninhaled Agents

Some agents used for euthanasia are administered to animals by means other than inhalation. For the most part, such agents are administered intravenously in nonaquatic species, and via immersion in aquatic species. Although many anesthetics could be used to induce death by overdose, some of the common noninhaled agents are described here.

Euthanasia Solutions

Solutions containing compounds such as sodium pentobarbital and phenytoin are commercially available for euthanasia of animals. Although pentobarbital is classified by the U.S. Drug Enforcement Agency as a Schedule II drug, euthanasia solutions containing pentobarbital are typically classified as Schedule III drugs. These solutions require special storage and documentation requirements for their use. For purposes of euthanasia, solutions are administered as an intravenous overdose and induce profound depression of the central nervous system, resulting in deep anesthesia, followed by death. Although quick, it is important to ensure death so that recovery from very deep anesthesia does not occur.

Potassium Chloride

Solutions of potassium chloride cause cardiac arrest when administered intravenously or intracardially and have been used to induce euthanasia, typically in larger mammals such as cattle and swine. Because of the distress likely to occur in conjunction with cardiac arrest, animals need to be deeply anesthetized prior to administration of potassium chloride. Pharmaceutical-grade preparations of potassium chloride at concentrations needed for euthanasia are generally not available commercially.

Tricaine Methanesulfonate

Tricaine methanesulfonate (MS-222) is commonly used for euthanasia of aquatic species. Animals are typically immersed in solutions that are pH buffered with sodium bicarbonate. Specific concentrations used are typically 200–300 mg/L. Immersed animals progress from deep anesthesia to death; however, it is standard practice to ensure death by means of a secondary method. Pharmaceutical-grade preparations of MS-222 are available for temporary immobilization of aquatic species (U.S. Food and Drug Administration 2015).

Clove Oil

A primary component of clove oil is eugenol, a compound with anesthetic properties. Similar to MS-222, aquatic species can be euthanized by immersion in a clove oil solution, followed by a secondary method to ensure death.

For zebrafish fry, it has been demonstrated that hypothermic shock is a more effective means for euthanasia than immersion in MS-222 or eugenol, although extended exposure times are needed (Strykowski and Schech 2015).

Physical Methods of Euthanasia

A variety of physical methods can be employed for rapid euthanasia of animals, particularly small rodents. When a secondary method to ensure death is appropriate, a physical technique is most commonly employed. Some of the more common physical methods are described here. Although true for any method of euthanasia, it is particularly important for those using physical methods to be well trained and skilled. For training of individuals to properly use these methods, it is suggested that animals that have already been euthanized by other means or are deeply anesthetized be used. Although common physical methods for the euthanasia of rodents and small birds are covered here, one may wish to note that physical methods such as the captive bolt and electrocution are sometimes used for euthanasia of livestock, primarily in meat production circumstances.

Cervical Dislocation

Used primarily for euthanasia of small mammals weighing less than 200 g and small birds, the objective of cervical dislocation is to separate the first cervical vertebra from the skull, thereby disrupting essential brain function and resulting in death. Briefly, the skull is firmly held in place and the trunk of the animal quickly and firmly pulled in the reverse direction so as to separate the cervical vertebra from the skull. Devices designed for cervical dislocation of small rodents are available commercially (Stoelting Co., Wood Dale, Illinois). The method can be aesthetically displeasing to some individuals, and so consideration should be given to alternative methods when appropriate. It is preferable to use cervical dislocation for euthanasia in combination with deep anesthesia when possible.


Primarily used for small animals such as rodents, decapitation is sometimes used in instances in which tissues or body fluids must be recovered quickly after euthanasia. Although it is recommended that animals first be anesthetized, scientific reasons sometimes require that decapitation be performed on the unanesthetized animal. Guillotines are commercially available for the purpose of decapitation, and it is important that a program be in place for routine maintenance of the guillotine to ensure the instrument remains sharp and operational. Following use, it is important that equipment be sanitized. Neonatal rodents are sometimes euthanized in this way, and since an appropriate-size guillotine is not available, sharp scissors can be used. In all cases, it is important that the animal be immobilized so that decapitation occurs quickly and precisely, resulting in death of the animal rather than severe injury. For rodents, a disposable, flexible plastic restrainer is available that provides sufficient immobilization (DecapiCone®, Braintree Scientific, Inc., Braintree, Massachusetts). As with cervical dislocation, this method can be aesthetically displeasing to some individuals, and so consideration should be given to alternative methods when appropriate. It is preferable to use decapitation for euthanasia when combined with anesthesia. Special care needs to be exercised, due to the increased personnel hazard associated with a guillotine.

Thoracic Compression

Sometimes used for euthanasia of small mammals and birds, thoracic compression is of value in field study circumstances where it is not practical to carry equipment or compounds needed for some of the other methods of euthanasia already mentioned. Briefly, the method is performed by placing quick, firm pressure on the upper chest, resulting in respiratory compromise and relatively quick death. Although this method is advocated by some for use in field circumstances (Sikes et al. 2011), the most recent recommendation of the AVMA (2013) regards the technique as unacceptable in unanesthetized animals due to presumed distress.

Confirmation of Death

The objective of euthanasia is, of course, death of the animal, and although the methods described here are designed to achieve that objective, it is essential that death be confirmed. This is especially true of pharmacological methods. Typically, death is confirmed either through close observation of the animal or by use of adjunctive techniques. Both approaches are described here.


Very close observation can be used to confirm death of animals, but it must be undertaken with care to ensure that animals are not merely deeply anesthetized or unconscious at a point from which they might revive. Toward this end, it is helpful, in a larger animal, to listen with a stethoscope for the heartbeat. Once an extended period of time (generally10 minutes or longer) has elapsed after the euthanasia method has been performed, animals can be assessed for any movement, including respiratory movement of the chest. With enough time, the muscles of a dead animal will stiffen (rigor mortis) and observation of such can be taken as evidence of death.

Adjunctive Methods

Because confirmation of death by observation relies on the somewhat subjective skill and interpretation by personnel, the potential for error is greater compared with assurance of death by means of additional physical methods. Any of the physical euthanasia methods described earlier in this chapter can be employed after the presumptive demise of the animal to further ensure death. Typically, these methods would only be done following induction of deep anesthesia by use of a pharmacological agent. In addition to the physical methods already described, several additional techniques can be employed to ensure death:


Withdrawal of a large blood volume can be used to ensure the death of the animal and prevent resuscitation. Typically, this would require removal of at least 25% of the animal’s calculated blood volume.


This method involves insertion of a sharp tool just beneath the base of the skull in an attempt to destroy the brainstem, thereby disrupting brain function essential for survival. This is sometimes performed in amphibians and requires extensive training.


For some studies, it is important to clear the vasculature of blood and instill a fixative, such as paraformaldehyde. This should be performed only on deeply anesthetized animals. Usually, intravascular access is established by means of a catheter or sometimes, in the case of small rodents, a needle and syringe. Saline is first administered slowly and at a volume sufficient to replace the blood. Next, the fixative is perfused in the same manner. Death quickly results from this method. It is important to seek the advice of institutional safety experts to establish practices for working with fixatives.

Personnel Training and Oversight

Training Personnel to Perform Euthanasia

The importance of proper training on euthanasia techniques is stressed in many regulatory and guidance documents (National Institutes of Health 1986; Committee for the Update of the Guide for the Care and Use of Laboratory Animals 2011; AVMA Panel on Euthanasia 2013). Proficiency in euthanasia techniques, as well as identifying humane endpoints and procedures that minimize pain and distress, promotes animal welfare and ensures the safety of personnel. Training programs should include information on the methods of euthanasia personnel are expected to utilize, depending on the species they work with, and should provide information regarding how the chosen technique induces loss of consciousness and death (AVMA Panel on Euthanasia 2013). When selecting a euthanasia method, consideration should be given to minimizing pain and fear experienced by the animal (Artwohl et al. 2006). Techniques for minimizing pain and distress may vary by species and can include euthanizing animals in their home cage, using proper methods of handling and restraint, and providing animals with a familiar and quiet environment.

Research and animal facility staff must also be trained on techniques used to verify death. This is generally accomplished by confirming cessation of vital signs, mainly the heartbeat. It should be stressed to staff during training that some animals may appear to be dead following euthanasia but may recover following disposal if death has not been confirmed. Additionally, it has been shown that neonatal rodents are more resistant to hypoxia, and a prolonged exposure to CO2 is necessary for euthanasia by this method (Klaunberg et al. 2004; Pritchett et al. 2005). Exposure times of 10–60 minutes have been recommended for neonatal mice based on age (Pritchett et al. 2005). For these reasons, it may be necessary to require increased exposure times or include a secondary method of euthanasia, such as bilateral thoracotomy, cervical dislocation, or decapitation, following CO2 euthanasia of neonatal rodents. In either case, the amount of time spent by staff performing euthanasia may increase, but is necessary to ensure complete and humane euthanasia in accordance with the guidelines.

Euthanasia training programs should take into account the potential for significant variation in learning styles among those who work in laboratory animal facilities. Age, cultural or socioeconomic differences, educational levels, and English as a second language may affect the way an individual processes and retains information (Kennedy 2002). Presenting information in multiple formats may be useful during training sessions. These may include verbal presentation, providing written materials, demonstrations by the trainer, and hands-on practice by trainees. This will allow the manager or trainer to confirm proficiency prior to allowing individuals to perform the procedure without observation. This is particularly important when training personnel on procedures that have a direct impact on animal welfare, such as euthanasia techniques.

Even with significant training on proper technique, performing euthanasia can be difficult for some animal facility personnel. They may find it difficult to euthanize specific animals for which they have cared or may be uneasy performing certain euthanasia methods. It is recommended that the chosen methods of euthanasia take into account the effects of the method on personnel (Artwohl et al. 2006; AVMA Panel on Euthanasia 2013). The use of CO2 for the euthanasia of rodents may be less distressful to personnel than physical methods (Pritchett et al. 2005), such as decapitation or thoracotomy. The manager should discuss concerns regarding euthanasia practices with personnel on a case-by-case base in order to find an acceptable resolution. For instance, animal care technicians or technologists who routinely perform CO2 euthanasia of rodents but are uncomfortable performing a secondary method per facility or institutional requirements may need to work with another member of the animal care or veterinary team to complete these procedures. While performing euthanasia is generally a job requirement of the animal care position, making it mandatory for those with a significant aversion to a specific technique may result in the loss of high-quality animal care personnel. It is recommended that managers discuss animal use protocols in detail with facility personnel, including the necessity for euthanasia (Wolfle 1985; Overhulse 2002). Principal investigators or their research staff may also be requested to speak to animal facility personnel regarding the goals of their research and the importance of the animal caretakers to their work. Being involved in larger discussions about research projects may alleviate some of their concern and facilitate their participation in euthanasia procedures, as well as instill a sense of pride in being involved in scientific discoveries that could lead to treatments for human or animal disease and ease the suffering of both (Herzog 2002).

Psychological Impact of Euthanasia on Personnel

Many animal caregivers and other animal facility personnel chose these jobs due to their affinity for animals and a desire to provide for their care and well-being. Often, bonds form between animals and those who care for them (Arluke 1996; Bayne 2002; Herzog 2002; Overhulse 2002). Being involved in the euthanasia of animals, especially those they have worked closely with, can be difficult for personnel. The killing–caring paradox describes situations in which individuals must euthanize the animals for which they provided care (Arluke 1994). Similarly, Rollin uses the term moral stress to describe the stress experienced by individuals who must euthanize animals for reasons that do not directly benefit the animal; it stems from a conflict between an individual’s reasons for working in an animal-related job and having to euthanize the animals in their care (Rollin 1986, 2011).

Multiple studies have been conducted to evaluate the psychological impact of euthanasia on personnel from animal shelters, research facilities, and veterinary clinics. Reeve et al. (2005) sought to determine the extent of euthanasia-related strain among shelter workers and its effects on overall employee well-being. The results showed evidence that animal euthanasia was an important source of job strain for employees and was associated with increased levels of general job stress, work and family conflict, and somatic complaints. In another study, Rohlf and Bennet (2005) explored possible perpetration-induced traumatic stress (PITS) among veterinary clinic, research laboratory, and animal shelter employees whose jobs required the euthanasia of animals. PITS is similar to posttraumatic stress but differs in that those with PITS actively participate in traumatic events. They found that 11% of participants reported moderate levels of traumatic symptoms, and that PITS is a valid avenue of study in animal workers. Additionally, the results revealed that only one-quarter of the participants had received grief counseling or stress management, leading the authors to recommend that education of employees in these areas be implemented.

As some level of psychological impact is expected among those who work in animal facilities, it is important that managers acknowledge the potential stress that employees can experience due to the formation of bonds with research animals (Herzog 2002). It is also necessary for managers to recognize the impact that performing euthanasia may have on employees and provide resources for those that need additional support (Chang and Hart 2002). The American Association for Laboratory Animal Science (2015) has recommended several additional steps that managers can take to support employees involved in the euthanasia of animals:

  1. Learn and recognize stressors to personnel related to euthanasia.
  2. Institute an open-door policy with supervisors and administrators.
  3. Provide a pleasant work environment.
  4. Supply a comfortable break area.
  5. Offer education relative to humane care and use and ethics.
  6. Recruit investigators to conduct informational seminars for the research team highlighting the various aspects of their particular study (especially desired benefits and outcomes).
  7. Recruit investigators to detail the significance of specific endpoints of the experimentation.
  8. Encourage group support meetings among laboratory personnel, and enlist the aid of an outside professional to facilitate therapeutic sessions.
  9. Ensure that individuals are properly trained in the procedures of euthanasia, including mechanisms of action and how each contributes to ensuring a humane death.
  10. Initiate policies that do not require the technician caring for long-term animals to participate in the euthanasia of those animals.
  11. Honor the request of an individual to be excused from euthanizing an animal to which he or she is particularly attached.
  12. Allow homes to be found for research animals suitable for adoption (after soliciting institutional and IACUC approval).


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© 2018 by Taylor & Francis Group, LLC.
Bookshelf ID: NBK500441PMID: 29787219DOI: 10.1201/9781315152189-35


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