AVMA Guidelines for Euthanasia of Animals 2020 PDF

**AVMA Guidelines for the Euthanasia of Animals: 2020 Edition** Part I---Introduction and General Comments I1 Preface Animal issues are no longer socially invisible, and increasingly, greater attention is being devoted to understanding the moral significance of experiences of animals and to taking into consideration the welfare of animals. During the past half-century, efforts to ensure the respectful and humane treatment of animals have garnered global attention. Concern for the welfare of animals is reflected in the growth of animal welfare science and ethics. The former is evident in the emergence of academic programs, establishment of specialty colleges, implementation of curricular changes in veterinary colleges, proliferation of scientific journal articles, and development of funding streams committed either partially or exclusively to the study of how animals are impacted by various environments and human interventions. The latter has seen the application of numerous ethical approaches (eg, rights-based theories, utilitarianism, virtue ethics, contractarianism, pragmatic ethics) to assessing the moral value of animals and the nature of the human-animal relationship. The proliferation of interest in animal use and care, at the national and international levels, is also apparent in recent protections accorded to animals in new and amended laws and regulations, institutional and corporate policies, and purchasing and trade agreements. Changing societal attitudes toward animal care and use have inspired scrutiny of some traditional and contemporary practices applied in the management of animals used for agriculture, research and teaching, companionship, and recreation or entertainment and of animals encountered in the wild. Attention has also been focused on conservation and the impact of human interventions on terrestrial and aquatic wildlife and the environment. Within these contexts, veterinarians provide leadership on how to care well for animals, including how to relieve unnecessary pain and suffering. In creating the 2020 and 2013 edition of the AVMA Guidelines for the Euthanasia of Animals (Guidelines), the POE made every effort to identify and apply the best research and empirical information available. As new research is conducted and more practical experience gained, recommended methods of euthanasia may change. As such, the AVMA and its POE have made a commitment to ensure the Guidelines reflect an expectation and paradigm of continuous improvement that is consistent with the obligations of the Veterinarian's Oath. As for other editions of the document, modifications of previous recommendations are also informed by continued professional and public sensitivity to the ethical care of animals. While some euthanasia methods may be utilized in slaughter (which refers to humane killing of animals destined for human consumption) or harvest and depopulation, recommendations related to humane slaughter and depopulation fall outside the purview of the Guidelines and are addressed by separate documents. The Guidelines set criteria for euthanasia, specify appropriate euthanasia methods and agents, and are intended to assist veterinarians in their exercise of professional judgment. The Guidelines acknowledge that euthanasia is a process involving more than just what happens to an animal at the time of its death. Apart from delineating appropriate methods and agents, these Guidelines also recognize the importance of considering and applying appropriate pre-euthanasia (eg, sedation) and animal handling practices, as well as attention to disposal of animals' remains. I2 Historical Context and Current Edition I2.1 HISTORY OF THE PANEL ON EUTHANASIA Since 1963 the AVMA has convened a POE to evaluate methods and potential methods of euthanasia for the purpose of creating guidelines for veterinarians who carry out or oversee the euthanasia of animals. The scope of the 1963 edition was limited to methods and recommendations applicable to dogs, cats, and other small mammals. Subsequent editions published in 1972 and 1978 encompassed more methods and species (laboratory animals and food animals, respectively), and included additional information about animals' physiologic and behavioral responses to euthanasia (specifically, pain, stress, and distress), euthanasia's effects on observers, and the economic feasibility and environmental impacts of various approaches. In 1986 information on poikilothermic, aquatic, and fur-bearing wildlife was introduced; in 1993 recommendations for horses and wildlife were added; and in 2000 an update acknowledged a need for more research on approaches suitable for depopulation. An interim revision by the AVMA Animal Welfare Committee in 2007 incorporated information derived from an existing, but separate, AVMA policy on the use of maceration to euthanize day-old chicks, poults, and pipped eggs, and the name of the report was changed to the AVMA Guidelines on Euthanasia. In 2013 the process for compiling the POE's report was substantially changed to include more breadth and depth of expertise in the affected species and environments in which euthanasia is performed. More than 3 years of deliberation by more than 60 individuals, including veterinarians, animal scientists, behaviorists, psychologists, and an animal ethicist, resulted in robust commentary and recommendations. A comment period allowed AVMA members an opportunity to provide input and share their experiences directly with POE members. The 2020 iteration of the Guidelines constitutes the ninth edition of the POE's report. The process for compiling this edition was similar to that of the 2013 edition. Two years of review, discussion, and revision by the POE culminated in this edition. A comment period was held and the input from AVMA members helps ensure the resulting document is not only scientifically robust, but practically sound. I2.2 SUBSTANTIVE CHANGES SINCE THE LAST EDITION In this interim update of the Guidelines, methods, techniques, and agents of euthanasia have been updated and detailed descriptions have been included to assist veterinarians in applying their professional judgment. Species-specific sections have been expanded or added to include more guidance for terrestrial and aquatic species kept for a variety of purposes and under different conditions. Where possible, appropriate flowcharts, illustrations, tables, and appendices have been used to clarify recommendations. Appendices 1 and 2 may be useful as a quick reference guide, but should never be used in lieu of the full text of the document by those performing euthanasia. All illustrations and figures have been moved to Appendix 3 of the document. Some of the more significant changes are as follows: Language was added to clarify the distinction between sedation and anesthesia. Specifically, animals under sedation may be aroused to a conscious state with sufficient stimulation. Recognizing this is critical when categorizing the effects of agents and distinguishing even deep states of sedation from unconsciousness. The conditions for the use of CO2 with rodents in the laboratory have changed from a recommended 10% to 30% of the chamber or cage volume/min to a recommended 30% to 70% of the chamber or cage volume/min. The extensive literature used to make this recommendation is cited and the AVMA appreciates the proactive efforts made by the international research community to provide the evidence needed to make this determination. Euthanasia techniques appropriate for use with rabbits raised for meat are categorized and described. This material is located in the Laboratory Animals section to place them with other techniques used with these species. The Animals Farmed for Food and Fiber section has been expanded to include American bison, water buffalo, camelids, and cervids. Updates to the application of captive bolt in several species have been made and new illustrations are available to assist veterinarians in proper usage. In the Avians section the recommendation for when avian embryos achieve the potential for perception has been amended from 50% to 80% of incubation for all avian eggs. This recommendation should be applied across avians with consideration for species-specific differences in development and using the best available data. I2.3 STATEMENT OF USE The Guidelines are designed for use by members of the veterinary profession who carry out or oversee the euthanasia of animals. As such, they are intended to apply only to nonhuman species. The species addressed by the practice of veterinary medicine are diverse. A veterinarian experienced with the species of interest should be consulted when choosing a method of euthanasia, particularly when little species-specific research on euthanasia has been conducted. Methods and agents selected will often be situation specific, as a means of minimizing potential risks to the animal's welfare and personnel safety. Given the complexity of issues that euthanasia presents, references on anatomy, physiology, natural history, husbandry, and other disciplines may assist in understanding how various methods may impact an animal during the euthanasia process. Veterinarians performing or overseeing euthanasia must assess the potential for animal distress due to physical discomfort, abnormal social settings, novel physical surroundings, pheromones or odors from nearby or previously euthanized animals, the presence of humans, or other factors (including impact on the environment and other animals). In addition, human safety and perceptions, availability of trained personnel, potential infectious disease concerns, conservation or other animal population objectives, regulatory oversight that may be species specific, available equipment and facilities, options for disposal, potential secondary toxicity, and other factors must be considered. Human safety is of utmost importance, and appropriate safety equipment, protocols, and knowledge must be available before animals are handled. Advance preparation includes protocols and supplies for addressing personnel injury due to animal handling or exposure to drugs and equipment used during the process. Once euthanasia has been carried out, death must be carefully verified. All laws and regulations pertaining to the species being euthanized, the methods employed, and the disposal of the animal's remains and/or any water containing pharmaceuticals used for euthanasia must be followed. The POE's objective in creating the Guidelines is to provide guidance for veterinarians about how to prevent and/or relieve the pain and suffering of animals that are to be euthanized. While every effort has been made to identify and recommend appropriate approaches for common species encountered under common conditions, the POE recognized there will be less than perfect situations in which a recommended method of euthanasia may not be possible and a method or agent that is best under the circumstances will need to be applied. For this reason, although the Guidelines may be interpreted and understood by a broad segment of the general population, a veterinarian should be consulted in their application. I3 What Is Euthanasia? Euthanasia is derived from the Greek terms eu meaning good and thanatos meaning death. The term is usually used to describe ending the life of an individual animal in a way that minimizes or eliminates pain and distress. A good death is tantamount to the humane termination of an animal's life. In the context of these Guidelines, the veterinarian's prima facie duty in carrying out euthanasia includes, but is not limited to, (1) their humane disposition to induce death in a manner that is in accord with an animal's interest and/or because it is a matter of welfare, and (2) the use of humane techniques to induce the most rapid and painless and distress-free death possible. These conditions, while separate, are not mutually exclusive and are codependent. Debate exists about whether euthanasia appropriately describes the killing of some animals at the end of biological experiments11 and of unwanted shelter animals. The Panel believes that evaluating the social acceptability of various uses of animals and/or the rationale for inducing death in these cases is beyond its purview; however, current AVMA policy supports the use of animals for various human purposes, and also recognizes the need to euthanize animals that are unwanted or unfit for adoption. Whenever animals are used by humans, good animal care practices should be implemented and adherence to those good practices should be enforced. When evaluating our responsibilities toward animals, it is important to be sensitive to the context and the practical realities of the various types of human-animal relationships. Impacts on animals may not always be the center of the valuation process, and there is disagreement on how to account for conflicting interspecific interests. The Panel recognizes these are complex issues since how to bring about a "good death" for animals is regarded as "essentially contested" (morally and conceptually), raising concerns across a large number of domains, including scientific, ethical, economic, environmental, political, and social. I3.1 A GOOD DEATH AS A MATTER OF HUMANE DISPOSITION Humane disposition reflects the veterinarian's desire to do what is best for the animal and serves to bring about the best possible outcome for the animal. Thus, euthanasia as a matter of humane disposition can be either intent or outcome based. Euthanasia as a matter of humane disposition occurs when death is a welcome event and continued existence is not an attractive option for the animal as perceived by the owner and veterinarian. When animals are plagued by disease that produces insurmountable suffering, it can be argued that continuing to live is worse for the animal than death or that the animal no longer has an interest in living. The humane disposition is to act for the sake of the animal or its interests, because the animal will not be harmed by the loss of life. Instead, there is consensus that the animal will be relieved of an unbearable burden. As an example, when treating a companion animal that is suffering severely at the end of life due to a debilitating terminal illness, a veterinarian may recommend euthanasia, because the loss of life (and attendant natural decline in physical and psychological faculties) to the animal is not relatively worse compared with a continued existence that is filled with prolonged illness, suffering, and duress. In this case, euthanasia does not deprive the animal of the opportunity to enjoy more goods of life (ie, to have more satisfactions fulfilled or enjoy more pleasurable experiences). And, these opportunities or experiences are much fewer or lesser in intensity than the presence or intensity of negative states or affect. Death, in this case, may be a welcome event and euthanasia helps to bring this about, because the animal's life is not worth living but, rather, is worth avoiding. Veterinarians may also be motivated to bring about the best outcome for the animal. Often, veterinarians face the difficult question of trying to decide (or helping the animal's owner to decide) when euthanasia would be a good outcome. In making this decision many veterinarians appeal to indices of welfare or quality of life. Scientists have described welfare as having 3 components: that the animal functions well, feels well, and has the capacity to perform behaviors that are innate or species-specific adaptations (an alternative view is also available18). An animal has good welfare if, overall, its life has positive value for it. When an animal no longer continues to enjoy good welfare (when it no longer has a life worth living because, on balance, its life no longer has positive value for it, or will shortly be overcome by negative states), the humane thing to do is to give it a good death. Euthanasia relieves the animal's suffering, which is the desired outcome. I3.2 A GOOD DEATH AS A MATTER OF HUMANE TECHNIQUE When the decision has been made to euthanize and the goal is to minimize pain, distress, and negative effect to the animal, the humaneness of the technique (ie, how we bring about the death of animals) is also an important ethical issue. As veterinarians and human beings it is our responsibility to ensure that if an animal's life is to be taken, it is done with the highest degree of respect, and with an emphasis on making the death as painless and distress free as possible. When euthanasia is the preferred option, the technique employed should result in rapid loss of consciousness followed by cardiac or respiratory arrest and, ultimately, a loss of brain function. In addition, animal handling and the euthanasia technique should minimize distress experienced by the animal prior to loss of consciousness. The POE recognized that complete absence of pain and distress cannot always be achieved. The Guidelines attempt to balance the ideal of minimal pain and distress with the reality of the many environments in which euthanasia is performed. While recommendations are made, it is important for those utilizing these recommendations to understand that, in some instances, agents and methods of euthanasia identified as appropriate for a particular species may not be available or may become less than an ideal choice due to differences in circumstances. Conversely, when settings are atypical, methods normally not considered appropriate may become the method of choice. Under such conditions, the humaneness (or perceived lack thereof) of the method used to bring about the death of an animal may be distinguished from the intent or outcome associated with an act of killing. Following this reasoning, it may still be an act of euthanasia to kill an animal in a manner that is not perfectly humane or that would not be considered appropriate in other contexts. For example, due to lack of control over free-ranging wildlife and the stress associated with close human contact, use of a firearm may be the most appropriate means of euthanasia. Also, shooting a suffering animal that is in extremis, instead of catching and transporting it to a clinic to euthanize it using a method normally considered to be appropriate (eg, barbiturates), is consistent with one interpretation of a good death. The former method promotes the animal's overall interests by ending its misery quickly, even though the latter technique may be considered to be more acceptable under normal conditions. Neither of these examples, however, absolves the individual from their responsibility to ensure that recommended methods and agents of euthanasia are preferentially used. I4 Euthanasia and Veterinary Medical Ethics The AVMA has worked to ensure that veterinarians remain educated about public discourse around animal ethics and animal welfare issues and that they are able to participate in meaningful ways. While an essential ingredient in public discourses about animals, sound science is by itself inadequate to address questions of ethics and values that surround the appropriate treatment of animals, especially as they relate to end-of-life issues. Since the 2013 edition, a number of authors20,21 have probed in greater depth the issue of a good death for animals in both philosophical and ethical terms. To this end, and consistent with its charge, the POE hopes to provide veterinarians, those under their supervision, and the public with well-informed and credible arguments on how to approach the ethically important and sometimes complex issue of the death of an animal. In so doing, it hopes to promote greater understanding regarding the contexts or settings involving euthanasia and the complexity of end-of-life issues involving animals. While not a regulatory body, the AVMA also hopes to offer guidance to those who may apply these Guidelines as part of regulatory structures designed to protect the welfare of animals used for human purposes. By creating and maintaining these Guidelines, the AVMA hopes to ensure that when a veterinarian or other professional intentionally kills an animal under their charge, it is done with respect for the interests of the animal and that the process is as humane as possible (ie, that it minimizes pain and distress to the animal and that death occurs as rapidly as possible). The AVMA does not take the death of nonhuman animals lightly and attempts to provide guidance for its members on both the morality and practical necessity of the intentional killing of animals. Veterinarians, in carrying out the tenets of their Oath, may be compelled to bring about the intentional death of animals for a variety of reasons. The finality of death is, in part, what makes it an ethically important issue; death forever cuts off future positive states, benefits, or opportunities. In cases where an animal no longer has a good life, however, its death also extinguishes permanently any and all future harms associated with poor welfare or quality of life. What constitutes a good life and what counts as an impoverished life, or one that has limited quality such that the death of the animal is the most humane option, are research areas in need of further study by the veterinary and ethics communities. Animal scientists and veterinarians are also investigating the processes by which an animal dies during the antemortem period and euthanasia methods and techniques that mitigate harmful effects. Further research is also needed regarding the different contexts within which euthanasia occurs, so that improvements in the performance and outcomes of euthanasia can be made. The intentional killing of healthy animals, as well as those that are impaired, is a serious concern for the public. When animals must be killed and veterinarians are called upon to assist, the AVMA encourages careful consideration of the decision to euthanize and the method(s) used. This is also true for euthanasia carried out during the course of disease control or protection of public health, as a means of domestic or wild animal population control, in conjunction with animal use in biomedical research, and in the process of food and fiber production. Killing of healthy animals under such circumstances, while unpleasant and morally challenging, is a practical necessity. The AVMA recognizes such actions as acceptable if those carrying out euthanasia adhere to strict policies, guidelines, and applicable regulations. In thinking seriously about veterinary medical ethics, veterinarians should familiarize themselves with the plurality of public moral views surrounding animal issues and also be cognizant of personal views and complicating factors that may impact their own ethical decision-making. While the Veterinarian's Oath, Principles of Veterinary Medical Ethics of the AVMA, state veterinary practice acts, and other guidance emanating from veterinary professional organizations and regulatory bodies provide direction for how veterinarians should interact with clients and their animals, different veterinarians may have different personal ethical values1,30 and this may impact their recommendations. In their capacity as animal advocate and client advisor, the precision and credibility of advice provided by veterinarians will help to advance client compliance. In many instances when veterinarians are called upon to benefit society through their scientific knowledge, practical experience, and understanding of how animals are benefited and harmed, straightforward answers may not be forthcoming. In such cases, veterinarians and animal welfare scientists may have to facilitate conscientious decision-making by promoting ethical dialogue. As advisor and conduit for information (and while respecting the autonomy of their clients to make decisions on behalf of their animals), veterinarians should advance pertinent scientific knowledge and ethical concerns related to practices and procedures so that their clients and/or society can make informed decisions. Veterinarians who are committed to a broad understanding of the "do no harm" principle may have to determine whether an animal's life is worth living, especially when there is no consensus on when it is appropriate to let that life go. While welfare or quality of life is typically adopted as part of the assessment of an animal's interests, what is in an animal's interest need not be singularly identified with its welfare, especially if welfare is defined narrowly and if the animal is harmed more by its continued life than its death. For example, if welfare is defined solely in terms of an animal's subjective experience, euthanasia may be warranted even if the animal is not showing signs of suffering at the present time and if there is some commitment to avoid harm. Euthanasia may be considered to be the right course to spare the animal from what is to come (in conjunction with a more holistic or objective account of what is in an animal's interest), if medical intervention would only prolong a terminal condition, or if current health conditions cannot be successfully mitigated. In these instances, intentional killing need not be motivated by narrow welfare-based interests35 but may be connected to the overall value of death to the animal. That some animals are subjects-of-a-life, and that human caretakers have moral responsibilities to their animals and do not want to see them endure continued harm, may be factors in deciding whether death is in an animal's interest. (A subject-of-a-life is a being that is regarded as having inherent value and should not be treated as a mere means to an end. It is a being that possesses an internal existence and has needs, desires, preferences, and a psychosocial identity that extends through time.) In some cases (eg, animals used for research), intentional killing of the animal to minimize harm to it may be trumped by more pressing ends. Here, the decision to kill an animal and how to do so will be complicated by external factors, such as productivity, the greater public and general good, economics, and concern for other animals. In human-animal relationships there usually are other mitigating factors that are relevant besides ones pertaining only to animal welfare or the animal's interest(s). In laboratory situations, for example, where animals are employed as research subjects and death may be a terminal point, animal welfare considerations are balanced against the merits of the experimental design and merits of the research. In such cases, ensuring the respectful and humane treatment of research animals will be largely up to IACUCs. These committees must apply the principles of refinement, replacement, and reduction, and ensure a respectful death for research animals. The decision to induce death may also involve whether replacements can be created for the animals that are killed. These other factors might justify killing an animal, despite the fact that the animal might otherwise have had a life worth living. For example, killing may be justified for disease control or public health purposes, population control, biomedical research, or slaughter for food and/or fiber. In other instances, keeping an animal alive that does not have a life worth living can be justified (eg, research circumstances where it would be impractical to kill the animal or when ensuring its survival would promote a greater good). There may be instances in which the decision to kill an animal is questionable, especially if the animal is predicted to have a life worth living if it is not killed. One example is the healthy companion animal whose owner wants to euthanize it because keeping it in the home is no longer possible or convenient. In this case, the veterinarian, as advisor and animal advocate, should be able to speak frankly about the animal's condition and suggest alternatives to euthanasia. Prima facie, it is the ethical responsibility of veterinarians to direct animal owners toward euthanasia as a compassionate treatment option when the alternative is prolonged and unrelenting suffering. However, accommodating a pluralism of values, interests, and duties in animal ethics is challenging. This underscores the need for veterinarians to consider the broader context in thinking about what animal care she or he will prescribe. There are no easy reductionist formulas to which to appeal. In many cases, advice will need to be responsive to the needs at hand. Attention must be given to how the welfare and suffering of the animal are understood within the context of its whole life and in light of socially acceptable ways in which humans and animals interact in different environments. Because veterinarians are committed to improving animal and human health and welfare, and because they work tirelessly to discover causes and cures for animal diseases and promote good animal management, some may feel a sense of disquiet or defeat when euthanasia becomes the better course of action. The POE hopes that these Guidelines and other AVMA policies will assist veterinarians who may be struggling with what may seem to be gratuitous euthanasia, the acceptability of certain procedures, and the sometimes routine nature of performing euthanasia. Toward that end, the decision aids in Figures 1 and 2a are offered as a resource. I5 Evaluating Euthanasia Methods In evaluating methods of euthanasia, the POE considered the following criteria: (1) ability to induce loss of consciousness and death with a minimum of pain and distress; (2) time required to induce loss of consciousness; (3) reliability; (4) safety of personnel; (5) irreversibility; (6) compatibility with intended animal use and purpose; (7) documented emotional effect on observers or operators; (8) compatibility with subsequent evaluation, examination, or use of tissue; (9) drug availability and human abuse potential; (10) compatibility with species, age, and health status; (11) ability to maintain equipment in proper working order; (12) safety for predators or scavengers should the animal's remains be consumed; (13) legal requirements; and (14) environmental impacts of the method or disposition of the animal's remains. Euthanasia methods are classified in the Guidelines as acceptable, acceptable with conditions, and unacceptable. Acceptable methods are those that consistently produce a humane death when used as the sole means of euthanasia. Methods acceptable with conditions are those techniques that may require certain conditions to be met to consistently produce humane death, may have greater potential for operator error or safety hazard, are not well documented in the scientific literature, or may require a secondary method to ensure death. Methods acceptable with conditions are equivalent to acceptable methods when all criteria for application of a method can be met. Unacceptable techniques are those methods deemed inhumane under any conditions or that the POE found posed a substantial risk to the human applying the technique. The Guidelines also include information about adjunctive methods, which are those that should not be used as a sole method of euthanasia, but that can be used in conjunction with other methods to bring about euthanasia. The POE recognized there will be less-than-perfect situations in which a method of euthanasia that is listed as acceptable or acceptable with conditions may not be possible, and a method or agent that is the best under the circumstances will need to be applied. As with many other procedures involving animals, some methods of euthanasia require physical handling of the animal. The amount of control and kind of restraint required will be determined by the species, breed, and size of animal involved; the degree of domestication, tolerance to humans, level of excitement, and prior handling experience of the animal; the presence of painful injury or disease; the animal's social environment; and the method of euthanasia and competence of the person(s) performing the euthanasia. Proper handling is vital to minimize pain and distress in animals, to ensure the safety of the person performing euthanasia, and, often, to protect other people and animals. Handling animals that are not accustomed to humans or that are severely injured or otherwise compromised may not be possible without inducing stress, so some latitude in the means of euthanasia is needed in some situations. The POE discussed the criteria for euthanasia used in the Guidelines as they apply to circumstances when the degree of control over the animal makes it difficult to ensure death without pain and distress. Premedication with the intent of providing anxiolysis, analgesia, somnolence for easier and safer IV access, and reduction of stage II or postmortem activity that could be distressing to personnel is strongly encouraged to reduce animal distress and improve personnel safety. This is particularly important for prey species, nondomesticated species, and animals enduring painful conditions. Personnel who perform euthanasia must demonstrate proficiency in the use of the technique in a closely supervised environment. Each facility or institution where euthanasia is performed (whether a clinic, laboratory, or other setting) is responsible for training its personnel adequately to ensure the facility or institution operates in compliance with federal, state, and local laws. Furthermore, experience in the humane restraint of the species of animal to be euthanized is important and should be expected, to ensure that animal pain and distress are minimized. Training and experience should include familiarity with the normal behavior of the species being euthanized, an appreciation of how handling and restraint affect that behavior, and an understanding of the mechanism by which the selected technique induces loss of consciousness and death. Euthanasia should only be attempted when the necessary drugs and supplies are available to ensure a smooth procedure. Selection of the most appropriate method of euthanasia in any given situation depends on the species and number of animals involved, available means of animal restraint, skill of personnel, and other considerations. Information in the scientific literature and available from practical experience focuses primarily on domesticated animals, but the same general considerations should be applied to all species. Euthanasia must be performed in accord with applicable federal, state, and local laws governing drug acquisition and storage, occupational safety, and methods used for euthanasia and disposal of animals, with special attention to species requirements where possible. The AVMA encourages those responsible for performing euthanasia of nonhuman animals to review current federal, state, and local regulations. If drugs have been used, careful consideration must be given to appropriate disposal of the animal's remains and steps should be taken to avoid environmental contamination or harm to other animals. Circumstances may arise that are not clearly covered by the Guidelines. Whenever such situations arise, a veterinarian experienced with the species should apply professional judgment, knowledge of clinically acceptable techniques, professional ethos, and social conscience in selecting an appropriate technique for ending an animal's life. It is imperative that death be verified after euthanasia and before disposal of the animal. An animal in deep narcosis following administration of an injectable or inhalant agent may appear to be dead, but might eventually recover. Death must be confirmed by examining the animal for cessation of vital signs. Consideration should be given to the animal species and method of euthanasia when determining appropriate criteria for confirming death. Safe handling and disposal of the resulting animal remains are also critically important when the presence of zoonotic disease, foreign animal diseases, or other diseases of concern to population health is suspected. Appropriate diagnostic samples should be collected for testing, pertinent regulatory authorities should be notified, and the animal's body should be incinerated, if possible. Use of personal protective equipment and precautions for handling biohazardous materials are recommended. Animals that have injured humans may require specific actions to be taken depending on local and state laws. I5.1 CONSCIOUSNESS AND UNCONSCIOUSNESS Consciousness refers to the subjective or inner qualitative experience of an animal in question. In humans, consciousness is common during both sleep and anesthesia, as evidenced by dreaming. One defining feature of dreaming is that, even while conscious, we do not experience our environment---we are disconnected from it. Ideally, general anesthesia prevents the experience of surgery and pain (connected consciousness), as well as producing behavioral unresponsiveness, either by inducing unconsciousness or by disconnecting consciousness from the environment. Unconsciousness, defined as loss of individual awareness, occurs when the brain's ability to integrate information is blocked or disrupted. In humans, onset of anesthetic-induced unconsciousness has been functionally defined by loss of appropriate response to verbal command; in animals, by loss of the righting reflex. This definition, introduced with the discovery of general anesthesia more than 160 years ago, is still useful because it is an easily observable, integrated whole-animal response. Anesthetics produce unconsciousness either by preventing integration (blocking interactions among specialized brain regions) or by reducing information (shrinking the number of activity patterns available to cortical networks) received by the cerebral cortex or equivalent structure(s). Further, the abrupt loss of consciousness that occurs at a critical concentration of anesthetic implies that the integrated repertoire of neural states underlying consciousness may collapse nonlinearly. Cross-species data suggest that memory and awareness are abolished with less than half the concentration required to abolish movement. Thus, an anesthetic state (unconsciousness and amnesia) can be produced at concentrations of anesthetic that do not prevent physical movements. Measurements of brain electrical function have been used to objectively quantify the unconscious state. At some level between behavioral unresponsiveness and the induction of a flat EEG (indicating the cessation of the brain's electrical activity and brain death), consciousness must vanish. However, EEG data cannot provide definitive answers as to onset of unconsciousness. Brain function monitors based on EEG are limited in their ability to directly indicate presence or absence of unconsciousness, especially around the transition point; also, it is not always clear which EEG patterns are indicators of activation by stress or pain. Physical methods that destroy or render nonfunctional the brain regions responsible for cortical integration (eg, gunshot, captive bolt, cerebral electrocution, blunt force trauma, maceration) produce instantaneous unconsciousness. When physical methods directly destroy the brain, signs of unconsciousness include immediate collapse and a several-second period of tetanic spasm, followed by slow hind limb movements of increasing frequency in cattle; however, there is species variability in this response. The corneal reflex will be absent. Signs of effective electrocution are loss of righting reflex, loss of eyeblink and moving object tracking, extension of the limbs, opisthotonos, downward rotation of the eyeballs, and tonic spasm changing to clonic spasm, with eventual muscle flaccidity. Decapitation and cervical dislocation as physical methods of euthanasia require separate comment. The interpretation of brain electrical activity, which can persist for up to 30 seconds following these methods,55--58 has been controversial.59 As indicated previously, EEG methods cannot provide definitive answers as to onset of unconsciousness. Other studies60--63 indicate such activity does not imply the ability to perceive pain and conclude that loss of consciousness develops rapidly. Once loss of consciousness occurs (ie, there is no longer an inner qualitative experience) subsequently observed activities, such as convulsions, vocalization, reflex struggling, breath holding, and tachypnea, can be attributed to the second stage of anesthesia, which by definition lasts from loss of consciousness to the onset of a regular breathing pattern.64,65 Thus, events observed following loss of the righting reflex are likely not consciously perceived. Some agents may induce convulsions, but these generally follow loss of consciousness. Agents inducing convulsions prior to loss of consciousness are unacceptable for euthanasia. I5.1.1 A Review Sedatives and immobilizing agents should not be confused with anesthetics, since animals are not necessarily rendered unconscious by the former 2 agents. Sedated and immobilized animals may still be aware of their environment. During anesthesia, consciousness is not necessarily associated with connectedness, responsiveness, or even recall. The concept of a transition zone between consciousness and unconsciousness has been discussed by Terlouw et al. This is especially true as it pertains to animals in slaughter plants. When animals are exsanguinated without stunning,68 EEG studies show that a corneal reflex in response to touch can occur in unconscious animals. To clarify assessment of unconsciousness and consciousness, it is recommended to separate signs of definite consciousness from signs of unconsciousness or death. Following this paragraph is a list of 6 signs that an animal is definitely conscious67; the subsequent paragraph is followed by a list of 3 signs that an animal is unconscious or (brain) dead. Consciousness likely depends on integrity of the corticothalamic networks. Spontaneous responsiveness may depend on subcortical and spinal cord networks and connectedness (namely, an awareness of one's environment) and may depend on continued information integration in corticothalamic circuits and unperturbed norepinephrinergic signaling. According to Terlouw et al, terrestrial animals are definitely conscious when they exhibit any 1 of these 6 indicators: standing posture, head or body righting reflex, voluntary vocalization, spontaneous blinking (no touching), eye pursuit, and response to threat or menace test (no touching). Some modification of these indicators may be required on the basis of factors such as species and developmental stage. A terrestrial animal that is unconscious and brain-dead will not have corneal reflex, eyelash reflex (in response to touch), or rhythmic breathing.67 Determining similar indicators for other species of animals is desired, and research into them is highly encouraged to help practitioners distinguish between animals that are brain-dead, unconscious (by anesthesia), immobilized, or sedated. Following are the 6 indicators of definite consciousness, in list form: Standing posture. Head or body righting reflex. Voluntary vocalization. Spontaneous blinking (no touching). Eye pursuit. Response to threat or menace test (no touching). Before carcass disposal or invasive dressing procedures occur at a slaughter plant, it should be confirmed that an animal is unconscious or brain-dead. Ensuring that an animal is unconscious or brain-dead requires all 3 of the following indicators: Absence of corneal reflex. Absence of eyelash reflex (response to touch). Absence of rhythmic breathing.67 I5.2 PAIN AND ITS PERCEPTION Criteria for painless death can be established only after the mechanisms of pain are understood. The perception of pain is defined as a conscious experience. The International Association for the Study of Pain (IASP) describes pain as "\[a\]n unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage. Activity induced in the nociceptor and nociceptive pathways by a noxious stimulus is not pain, which is always a psychological state, even though we may well appreciate that pain most often has a proximate physical cause." The perception of pain based on mammalian models requires nerve impulses from peripheral nociceptors to reach a functioning conscious cerebral cortex and the associated subcortical brain structures. Noxious stimulation that threatens to damage or destroy tissue produces activity in primary nociceptors and other sensory nerve endings. In addition to mechanical and thermal stimulation, a variety of endogenous substances can generate nociceptive impulses, including prostaglandins, hydrogen ions, potassium ions, substance P, purines, histamine, bradykinin, and leukotrienes, as can electrical currents. Nociceptive impulses are conducted by nociceptor primary afferent fibers to either the spinal cord or the brainstem and 2 general sets of neural networks. Reflex withdrawal and flexion in response to nociceptive input are mediated at the spinal level while ascending nociceptive pathways carry impulses to the reticular formation, hypothalamus, thalamus, and cerebral cortex (somatosensory cortex and limbic system) for sensory processing and spatial localization. Thus, movement observed in response to nociception can be due to spinally mediated reflex activity, cerebral cortical and subcortical processing, or a combination of the two. For example, it is well recognized clinically that spinally mediated nociceptive reflexes may remain intact distal to a compressive spinal lesion or complete spinal transaction that blocks the ascending nociceptive pathways. In contrast, administration of a local anesthetic into the epidural space suppresses both spinally mediated nociceptive reflexes and ascending nociceptive pathways; in either case, noxious stimuli are not perceived as pain in conscious human or nonhuman animals because activity in the ascending pathways, and thus access to the higher cortical centers, is suppressed or blocked. It is therefore incorrect to substitute the term pain for stimuli, receptors, reflexes, or pathways because the term implies higher sensory processing associated with conscious perception. Consequently, the choice of a euthanasia agent or method is less critical if it is to be used on an animal that is anesthetized or unconscious, provided that the animal does not regain consciousness prior to death. Pain is subjective in the sense that individuals can differ in their perceptions of pain intensity as well as in their physical and behavioral responses to it. Pain can be broadly categorized as sensory-discriminative, where the origin and the stimulus causing pain are determined, or as motivational-affective, where the severity of the stimulus is perceived and a response to it determined. Sensory-discriminative nociceptive processing occurs within cortical and subcortical structures using mechanisms similar to those used to process other sensory-discriminatory input and provides information on stimulus intensity, duration, location, and quality. Motivational-affective processing involves the ascending reticular formation for behavioral and cortical arousal, as well as thalamic input to the forebrain and limbic system for perception of discomfort, fear, anxiety, and depression. Motivational-affective neural networks also provide strong inputs to the limbic system, hypothalamus, and autonomic nervous system for reflex activation of the cardiovascular, pulmonary, and pituitary-adrenal systems. Although the perception of pain requires a conscious experience, defining consciousness, and therefore the ability to perceive pain, across many species is quite difficult. Previously it was thought that fish, amphibians, reptiles, and invertebrates lacked the anatomic structures necessary to perceive pain as we understand it in birds and mammals. For example, the invertebrate taxa include animals with no nervous system (eg, sponges) and nervous systems with no ganglionation or minimal ganglionation (eg, starfish). However, there are also invertebrate taxa with well-developed brains and/or complex behaviors that include the ability to analyze and respond to complex environmental cues (eg, octopus, cuttlefish, spiders, honeybees, butterflies, ants). Most invertebrates do respond to noxious stimuli and many have endogenous opioids. Amphibians and reptiles also represent taxa with a diverse range of anatomic and physiologic characteristics such that it is often difficult to ascertain that an amphibian or reptile is, in fact, dead. Although amphibians and reptiles respond to noxious stimuli and are presumed to feel pain, our understanding of their nociception and response to stimuli is incomplete. Nevertheless, there is increasing taxa-specific evidence of the efficacy of analgesics to minimize the impact of noxious stimuli on these species. Consequently, euthanasia techniques that result in "rapid loss of consciousness" and "minimize pain and distress" should be strived for, even where it is difficult to determine that these criteria have been met. Compelling recent evidence indicates finfish possess the components of nociceptive processing systems similar to those found in terrestrial vertebrates, though debate continues based on questions of the impact of quantitative differences in numbers of specific components such as unmyelinated C fibers in major nerve bundles. Suggestions that fish responses to pain merely represent simple reflexes have been refuted by studies demonstrating forebrain and midbrain electrical activity in response to stimulation and differing with type of nociceptor stimulation. Learning and memory consolidation in trials where finfish are taught to avoid noxious stimuli have moved the issue of fish cognition and sentience forward to the point where the preponderance of accumulated evidence supports the position that finfish should be accorded the same considerations as terrestrial vertebrates in regard to relief from pain. The POE was not able to identify similar studies of Chondrichthyes (cartilaginous fish), amphibians, reptiles, and invertebrates, but believes that available information suggests that efforts to relieve pain and distress for these taxa are warranted, unless further investigation disproves a capacity to feel pain or distress. While there is ongoing debate about fishes', amphibians', reptiles', and invertebrate animals' ability to feel pain or otherwise experience compromised welfare, they do respond to noxious stimuli. Consequently, the Guidelines assume that a conservative and humane approach to the care of any creature is warranted, justifiable, and expected by society. Euthanasia methods should be employed that minimize the potential for distress or pain in all animal taxa, and these methods should be modified as new taxa-specific knowledge of their physiology and anatomy is acquired. I5.3 STRESS AND DISTRESS An understanding of the continuum that represents stress and distress is essential for evaluating techniques that minimize any distress experienced by an animal being euthanized. Stress has been defined as the effect of physical, physiologic, or emotional factors (stressors) that induce an alteration in an animal's homeostasis or adaptive state. The response of an animal to stress represents the adaptive process that is necessary to restore the baseline mental and physiologic state. These responses may involve changes in an animal's neuroendocrinologic system, autonomic nervous system, and mental status that may result in overt behavioral changes. An animal's response varies according to its experience, age, species, breed, and current physiologic and psychological state, as well as handling, social environment, and other factors. Stress and the resulting responses have been divided into 3 phases. Eustress results when harmless stimuli initiate adaptive responses that are beneficial to the animal. Neutral stress results when the animal's response to stimuli causes neither harmful nor beneficial effects to the animal. Distress results when an animal's response to stimuli interferes with its well-being and comfort.89 To avoid distress, veterinarians should strive to euthanize animals within the animals' physical and behavioral comfort zones (eg, preferred temperatures, natural habitat, home) and, when possible, prepare a calming environment. I5.4 ANIMAL BEHAVIOR Although evaluations of euthanasia methods in the veterinary context are driven by science, clinical considerations and expectations from the public that high ethical standards will be observed may, in some cases, also play a role. When addressing euthanasia, veterinarians may disagree about what constitutes humane measures and a compassionate outcome for an animal or group of animals. This is reflective of the complexity or messiness of real-world situations veterinarians can sometimes find themselves in, where difficult decisions must be made involving euthanasia, and the multifaceted nature of animal welfare. In the latter case, conceptions of animal welfare are linked to varying normative approaches to how an animal is doing as described by different human assessors. Here, this disagreement may not necessarily involve disagreements about empirical information or clinical measures but instead may be due to a values-based disagreement about what constitutes good animal welfare90 or how an animal may be harmed or distressed by a particular clinical option. So, while the core issue concerning euthanasia is how to bring about a good death for an animal, a disagreement may persist among veterinarians about how to weigh or weight various social and clinical trade-offs. For example, there may be disagreement over whether a quick death with some short-lived but acute distress, aversion, or suffering is preferable to one where the animal becomes unconscious over a longer period of time but does not demonstrate much behavioral aversion. More specifically, veterinarians in the laboratory context may debate which type of inhalant to use or its optimal flow rate to get rodents quicker to death or which can be anxiety producing and may not create a desired anesthetic state in the animal. Furthermore, depending on which conception of welfare is emphasized, behavioral aversion as an indicator of poor animal welfare may be viewed as problematic by some but not others if, for example, more weight is given to the intensity of negative states experienced by an animal instead of the duration of exposure to a noxious agent. Measures designed to minimize pain or distress before animals become unconscious will likely achieve widespread support only if veterinarians are sensitive to the variety of conceptions of animal welfare and are willing to engage openly about how animals may be impacted by various alternatives. In the context of laboratory animals, for example, resolution of a disagreement in emphasis or interpretation regarding affective states, basic functioning, and evidence of frustration, anxiety, or fear will likely be influenced by programmatic policies and practices that have been identified by the institution's IACUC as ensuring high animal welfare standards. The need to minimize animal distress, including negative affective or experientially based states like fear, aversion, anxiety, and apprehension, must be considered in determining the method of euthanasia. Ethologists and animal welfare scientists are getting better at discerning the nature and content of these states. Veterinarians and other personnel involved in performing euthanasia should familiarize themselves with pre-euthanasia protocols and be attentive to species and individual variability. For virtually all animals, being placed in a novel environment is stressful; therefore, a euthanasia approach that can be applied in familiar surroundings may help reduce stress. For animals accustomed to human contact, gentle restraint (preferably in a familiar and safe environment), careful handling, and talking during euthanasia often have a calming effect and may also be effective coping strategies for personnel. Sedation and/or anesthesia may assist in achieving the best conditions for euthanasia. It must be recognized that sedatives or anesthetics given at this stage that change circulation may delay the onset of the euthanasia agent. Animals that are in social groups of conspecifics or that are wild, feral, injured, or already distressed from disease pose another challenge. For example, mammals and birds that are not used to being handled have higher corticosteroid levels during handling and restraint compared with animals accustomed to frequent handling by people. For example, beef cattle that are extensively raised on pasture or range have higher corticosteroid levels when restrained in a squeeze chute compared with intensively raised dairy cattle that are always in close association with people, and being placed in a new cage has been shown to be stressful for rodents. Because handling may be a stressor for animals less accustomed to human contact (eg, wildlife, feral species, zoo animals, and some laboratory animals), the methods of handling and degree of restraint (including none, such as for gunshot) required to perform euthanasia should be considered when evaluating various methods.86 When handling such animals, calming may be accomplished by retaining them (as much as possible) in familiar environments, and by minimizing visual, auditory, and tactile stimulation. When struggling during capture or restraint may cause pain, injury, or anxiety to the animal or danger to the operator, the use of tranquilizers, analgesics, and/or anesthetics may be necessary. A method of administration should be chosen that causes the least distress in the animal for which euthanasia must be performed. Various techniques for oral delivery of sedatives to dogs and cats have been described that may be useful under these circumstances. Expressions and body postures that indicate various emotional states of animals have been described for some species. Behavioral responses to noxious stimuli in conscious animals include distress vocalization, struggling, attempts to escape, and defensive or redirected aggression. In cattle and pigs, vocalization during handling or painful procedures is associated with physiologic indicators of stress. Vocalization is associated with excessive pressure applied by a restraint device. Salivation, urination, defecation, evacuation of anal sacs, pupillary dilatation, tachycardia, sweating, and reflex skeletal muscle contractions causing shivering, tremors, or other muscular spasms may occur in unconscious as well as conscious animals. Fear can cause immobility or playing dead in certain species, particularly rabbits and chickens. This immobility response should not be interpreted as loss of consciousness when the animal is, in fact, conscious. Distress vocalizations, fearful behavior, and release of certain odors or pheromones by a frightened animal may cause anxiety and apprehension in other animals. Therefore, for sensitive species, it is desirable that other animals not be present when individual animal euthanasia is performed. Often, simple environmental modifications can help reduce agitation and stress, such as providing a nonslip floor for the animals to stand on, reducing noise, blocking the animal's vision with a blindfold or a barrier, or removing distracting stimuli that cause animals to become agitated. I5.5 HUMAN BEHAVIOR The depth of the emotional attachment between animals and their owners or caretakers requires an additional layer of professional respect and care beyond the ethical obligation to provide a good death for the animal. Human concerns associated with the euthanasia of healthy and unwanted animals can be particularly challenging, as can situations where the health interests of groups of animals and/or the health interests of people conflict with the welfare of individual animals (eg, animal health emergencies). The human-animal relationship should be respected by discussing euthanasia openly, providing an appropriate place to conduct the process, offering the opportunity for animal owners and/or caretakers to be present when at all possible (consistent with the best interests of the animal and the owners and caretakers), fully informing those present about what they will see (including possible unpleasant side effects), and giving emotional support and information about grief counseling as needed. Regardless of the euthanasia method chosen, it is important to consider the level of understanding and perceptions of those in attendance as they witness euthanasia. When death has been achieved and verified, owners and caretakers should be verbally notified. Owners and caretakers are not the only people affected by the euthanasia of animals. Veterinarians and their staffs may also become attached to patients and struggle with the ethics of the caring-killing paradox, particularly when they must end the lives of animals they have known and treated for many years. Repeating this scenario regularly may lead to emotional burnout, or compassion fatigue. The various ways in which veterinarians cope with euthanasia have been discussed elsewhere. There are 6 settings in which the Panel was most aware of the potential for substantive psychological impacts of animal euthanasia on people. The first setting is the veterinary clinical setting (clinics and hospitals or mobile veterinary practices) where owners have to make decisions about whether and when to euthanize. Although many owners rely heavily on their veterinarian's judgment, others may have misgivings about making a decision. This is particularly likely if an owner feels responsible for an animal's medical or behavioral problem. Owners choose euthanasia for their animals for a variety of reasons, including prevention of suffering from a terminal illness, their inability to care for the animal, the impact of the animal's condition on other animals or people, and/or financial considerations. The decision to euthanize often carries strong feelings of emotion such as guilt, sadness, shock, and disbelief. As society continues to pay more attention to questions about the moral status of animals, loss of animal life should be handled with the utmost respect and compassion by all animal care staff. The ability to communicate well is crucial to helping owners make end-of-life decisions for their animals and is a learned skill that requires training. Almost 80% of clients who recently experienced the death of a pet (87% by euthanasia) reported a positive correlation between support from the veterinarian and staff and their ability to handle the grief associated with their pet's death. Owners should be given the opportunity to be present during euthanasia, when feasible, and they should be prepared for what to expect. What drugs are being used and how the animal could respond should be discussed. Behaviors such as vocalization, agonal breaths, muscle twitches, failure of the eyelids to close, urination, or defecation can be distressing to owners. Counseling services for owners having difficulty coping with animal death are available in some communities, and veterinarians are encouraged to seek grief support training to assist their clients. While good euthanasia practices (ie, client communication and education, compassionate species-appropriate handling and selection of technique, pre-euthanasia sedatives or anesthetics as needed to minimize anxiety and facilitate safe restraint, and careful confirmation of death) are often applied in the euthanasia of dogs and cats, they should also be followed for other species that are kept as pets, including small mammals, birds, reptiles, farm animals, and aquatic animals. The second setting is in animal care and control facilities where unwanted, homeless, diseased, and injured animals must be euthanized in large numbers. The person performing euthanasia must be technically proficient (including the use of humane handling methods and familiarity with the method of euthanasia being employed), and must be able to understand and communicate to others the reasons for euthanasia and why a particular approach was selected. This requires organizational commitment to provide ongoing professional training on the latest methods, techniques, and materials available for euthanasia. Distress may develop among personnel directly involved in performing euthanasia repeatedly, and may include a psychological state characterized by a strong sense of work dissatisfaction or alienation, which may be expressed by absenteeism, belligerence, or careless and callous handling of animals. The impact on personnel may be worse when euthanasia is conducted in frequent, shorter sessions compared with fewer, longer sessions. In addition, animal shelter personnel have been shown to have more difficulty dealing emotionally with the euthanasia of healthy, unwanted animals than those that are old, sick, injured, or wild. Specific coping strategies that can make the task more tolerable include adequate training programs so that euthanasia is performed competently, rotation of duties and shared responsibilities for staff performing euthanasia, peer support in the workplace, professional support as necessary, focusing on animals that are successfully adopted or returned to owners, devoting some work time to educational activities, and providing time off when workers feel distressed. Management should be aware of potential personnel problems related to animal euthanasia and determine whether it is necessary to institute a program to prevent, decrease, or eliminate this problem. The third setting is the laboratory. Researchers, technicians, and students may become attached to animals that must be euthanized in laboratory settings, even though the animals are often purpose-bred for research. The human--research animal bond positively impacts quality of life for a variety of research animals, but those caring for the animals often experience euthanasia- related stress symptoms comparable to those encountered in veterinary clinics and animal shelters. The same considerations afforded pet owners or shelter employees should be provided to those working in laboratories, particularly the provision of training to promote grief coping skills. The fourth setting is wildlife conservation and management. Wildlife biologists, wildlife managers, and wildlife health professionals are often responsible for euthanizing animals that are injured, diseased, or in excessive number or those that threaten property or human safety. Although relocation of some animals may be appropriate and attempted, relocation is often only a temporary solution and may be insufficient to address a larger problem. People who must deal with these animals, especially under public pressure to save the animals rather than destroy them, can experience extreme distress and anxiety. In addition, the perceptions of not only the wildlife professionals, but of onlookers, need to be considered when selecting a euthanasia method. The fifth setting is livestock and poultry production. As for shelter and laboratory animal workers, on-farm euthanasia of individual animals by farm workers charged with nurturing and raising production animals can take a heavy toll on employees both physically and emotionally. The sixth setting is that in which there is broad public exposure. Because euthanasia of zoo animals, animals involved in roadside or racetrack accidents, stranded marine animals, and nuisance or injured wildlife can draw public attention, human attitudes and responses must be considered whenever these animals are euthanized. Natural disasters and foreign animal disease programs also present public challenges. Attention to public perceptions, however, should not outweigh the primary responsibility of doing what is in the animal's best interest under the circumstances (ie, using the most appropriate and painless euthanasia method possible). In addition to ensuring good care of animals during euthanasia and considering the psychological well-being of human participants, the physical safety of personnel handling the animals and performing euthanasia needs to be protected. The safe use of controlled substances and diversion control to prevent abuse is also part of the responsibility of those using such substances in the performance of euthanasia. I5.6 SEDATION VERSUS ANESTHESIA A distinction must be made between the terms sedation, tranquilization, and anesthesia as utilized in these Guidelines. A common characteristic of both sedatives and tranquilizers is that arousal to a conscious state can occur with sufficient stimulation, such that animals sedated or immobilized with these agents may still be consciously aware of, and connected to, their environment. Unlike properly applied physical euthanasia methods where loss of consciousness is instantaneous and unambiguous (eg, captive bolt, gunshot, electrocution), application of other approved euthanasia methods requires animals be first rendered fully unconscious (eg, intracardiac pentobarbital, IV MgSO4 or KCl, exsanguination). While sedatives, hypnotics, and tranquilizers, when administered in sufficient quantity, can produce a sleep-like state, humans may recall connected awareness of their environment, and the same is likely true for animals. Indeed, humans experienced connected awareness of their environment during sedation with dexmedetomidine sufficient to lose responsiveness,144 and a state of surgical anesthesia could not be produced even when xylazine was administered at 55 to 88 times the usual dose (0.1 mg/kg \[0.05 mg/lb\]) required to produce recumbency in cattle. Immobilizing, tranquilizing, or sedative agents should not be relied on to produce a truly unresponsive, disconnected unconscious state, regardless of the dose administered. Instead, an effective dose of a general anesthetic should be used when performing euthanasia with methods causing distress or noxious stimulation prior to loss of consciousness. **I6 Mechanisms of Euthanasia** Euthanizing agents cause death by 3 basic mechanisms: (1) direct depression of neurons necessary for life function, (2) hypoxia, and (3) physical disruption of brain activity. The euthanasia process should minimize or eliminate pain, anxiety, and distress prior to loss of consciousness. As loss of consciousness resulting from these mechanisms can occur at different rates, the suitability of a particular agent or method will depend on whether an animal experiences distress prior to loss of consciousness. Unconsciousness, defined as loss of individual awareness, occurs when the brain's ability to integrate information is blocked or disrupted (see comments on unconsciousness for additional information). Ideally, euthanasia methods should result in rapid loss of consciousness, followed by cardiac or respiratory arrest and the subsequent loss of brain function. Loss of consciousness should precede loss of muscle movement. Agents and methods that prevent movement through muscle paralysis, but that do not block or disrupt the cerebral cortex or equivalent structures (eg, succinylcholine, strychnine, curare, nicotine, potassium, or magnesium salts), are not acceptable as sole agents for euthanasia of vertebrates because they result in distress and conscious perception of pain prior to death. In contrast, magnesium salts are acceptable as the sole agent for euthanasia in many invertebrates due to the absence of evidence for cerebral activity in some members of these taxa, and there is evidence that the magnesium ion acts centrally in suppressing neural activity of cephalopods. Depression of the cortical neural system causes loss of consciousness followed by death. Depending on the speed of onset of the particular agent or method used, release of inhibition of motor activity may be observed accompanied by vocalization and muscle contraction similar to that seen in the initial stages of anesthesia. Although distressing to observers, these responses do not appear to be purposeful. Once ataxia and loss of righting reflex occur, subsequent observed motor activity, such as convulsions, vocalization, and reflex struggling, can be attributed to the second stage of anesthesia, which by definition lasts from the loss of consciousness to the onset of a regular breathing pattern. Hypoxia is commonly achieved by exposing animals to high concentrations of gases that displace oxygen (O2), such as carbon dioxide (CO2), nitrogen (N2), or argon (Ar), or by exposure to carbon monoxide (CO) to block uptake of O2 by RBCs. Exsanguination, an adjunctive method, is another method of inducing hypoxia, albeit indirectly, and can be a way to ensure death in an already unconscious or moribund animal. As with other euthanasia methods, some animals may exhibit motor activity or convulsions following loss of consciousness due to hypoxia; however, this is reflex activity and is not consciously perceived by the animal. In addition, methods based on hypoxia will not be appropriate for species that are tolerant of prolonged periods of hypoxemia. Physical disruption of brain activity can be produced through a blow to the skull resulting in concussive stunning; through direct destruction of the brain with a captive bolt, bullet, or pithing rod; or through depolarization of brain neurons following electrocution. Death quickly follows when the midbrain centers controlling respiration and cardiac activity fail. Convulsions and exaggerated muscle activity can follow loss of consciousness. Physical disruption methods are often followed by exsanguination. These methods are inexpensive, humane, and painless if performed properly, and leave no drug residues in the animal's remains. Furthermore, animals presumably experience less fear and anxiety with methods that require little preparatory handling. However, physical methods usually require a more direct association of the operator with the animals to be euthanized, which can be offensive to, and upsetting for, the operator. Physical methods must be skillfully executed to ensure a quick and humane death, because failure to do so can cause substantial suffering. In summary, the cerebral cortex or equivalent structure(s) and associated subcortical structures must be functional for pain to be perceived. If the cerebral cortex is nonfunctional because of neuronal depression, hypoxia, or physical disruption, pain is not experienced. Reflex motor activity that may occur following loss of consciousness, although distressing to observers, is not perceived by the animal as pain or distress. Given that we are limited to applying euthanasia methods based on these 3 basic mechanisms, efforts should be directed toward educating individuals involved in the euthanasia process, achieving technical proficiency, and refining the application of existing methods. **I7 Confirmation of Death** Death must be confirmed before disposal of any animal remains. A combination of criteria is most reliable in confirming death, including lack of pulse, breathing, corneal reflex, and response to firm toe pinch; inability to hear respiratory sounds and heart beat by use of a stethoscope; graying of the mucous membranes; and rigor mortis. None of these signs alone, except rigor mortis, confirms death. In small animals, particularly in animal shelter settings, verification of death may be supplemented by percutaneous cardiac puncture after the animal is unconscious. Failure of the needle and attached syringe to move after insertion into the heart (aspiration of blood provides evidence of correct location) indicates lack of cardiac muscle movement and death. I8 Disposal of Animal Remains Regardless of the euthanasia method chosen, animal remains must be handled appropriately and in accord with state and local law. Regulations apply not only to the disposition of the animal's remains (eg, burial, incineration, rendering), but also to the management of chemical residues (eg, pharmaceuticals \[including but not limited to barbiturates, such as pentobarbital\] and other residues, such as lead) that may adversely affect scavengers or result in the adulteration of rendered products used for animal feed. Use of pentobarbital invokes legal responsibilities for veterinarians, animal shelters, and animal owners to properly dispose of animal remains after death. Animal remains containing pentobarbital are potentially poisonous for scavenging wildlife, including birds (eg, bald and golden eagles, vultures, hawk species, gulls, crows, ravens), carnivorous mammals (eg, bears, coyotes, martens, fishers, foxes, lynxes, bobcats, cougars), and domestic dogs.150 Federal laws protecting many of these species apply to secondary poisoning from animal remains containing pentobarbital. The Migratory Bird Treaty Act, the Endangered Species Act, and the Bald and Golden Eagle Protection Act may carry civil and criminal penalties, with fines in civil cases up to \$25,000 and in criminal cases up to \$500,000 and incarceration for up to 2 years.150 Serious repercussions may occur when veterinary health professionals who should be well-informed about the necessity for proper disposal of animal remains fail to provide it, or fail to inform their clients how to provide it, whether there was intent to cause harm or not.151,152 Cases of suspected wildlife death from animal remains containing pentobarbital are investigated by the regional US Fish and Wildlife Service law enforcement office. Recommendations by the US Fish and Wildlife Service for prevention of secondary poisoning from pentobarbital are to (1) incinerate or cremate animal remains whenever possible, (2) immediately bury deeply according to local laws and regulations, (3) securely cover or store animal remains if the ground is frozen until such time as deep burial is practical, (4) review and modify local landfill practices to prevent access of scavengers to legally disposed animal remains, (5) educate clients about proper disposal, (6) include a warning regarding disposal of animal remains on the euthanasia consent form, and (7) tag animal remains and outer bags or containers with prominent poison tags. Rendering is an important means of disposal of dead livestock and horses, and since many horses are euthanized with barbiturates, related residues can be hazardous. Rendered protein is used in animal feed for cattle, swine, poultry, fish, and companion animals, but products rendered from ruminants are prohibited by law for use in ruminant feed. Many pet food manufacturers have lowered their acceptance thresholds for barbiturate concentrations in rendered product. Advances in analytical chemistry have spawned increasingly sensitive assays, and pet food manufacturers are using these techniques to ensure the purity of the rendered protein incorporated in their products. Accordingly, increased analytical sensitivity has led many renderers to reconsider accepting horses euthanized using barbiturates. This places renderers and those wishing to employ rendering as a means of disposal for animals euthanized using pentobarbital in a difficult position, and may result in renderers being reluctant to accept more animal remains than they can reasonably manage without creating residue concerns. Alternatives for disposal of animal remains must be considered in advance, in case the renderer cannot or will not accept animal remains containing barbiturate residues. Composting is another means of disposing of animal remains that is becoming increasingly common. Studies examining the persistence of barbiturate residues in composted animal remains are few, but those that do exist suggest the persistence of the drugs in composted material. While the implications of this are still unclear, it does raise questions about potential environmental impacts in the case of animal health emergencies or mass mortality events. Alternatives to the use of pentobarbital that may reduce the risk of secondary toxicity include general anesthesia followed by nontoxic injectable agents such as potassium chloride, or the application of physical methods such as PCB or gunshot. These alternatives, however, are not risk free. For example, pharmaceutical residues in animal remains other than barbiturates (eg, xylazine) may affect scavengers and can reduce the acceptability of the animal remains for renderers. Unfortunately, specific guidance from regulators regarding the use of such alternatives is limited. The persistence of antimicrobials in animal remains presents parallel concerns, particularly for animal remains that will be rendered. While many antimicrobials may be inactivated or destroyed through the rendering process, public health concerns associated with antimicrobial resistance, coupled with the enhanced sensitivity of chemical assays and limited regulatory guidance for renderers, further complicate veterinarians' responsibilities for safe remediation. Safe handling and disposal of the resulting animal remains are also critically important when zoonotic diseases, foreign animal diseases, or diseases of concern to population health are suspected. Appropriate diagnostic samples should be collected for testing, regulatory authorities must be contacted, and the animal remains must be incinerated (if possible). Personal protective equipment and precautions for handling biohazardous materials are recommended. Animals that have injured humans may require specific actions to be taken depending on local and state laws. Part II---Methods of Euthanasia M1 Inhaled Agents M1.1 COMMON CONSIDERATIONS Inhaled vapors and gases require a critical concentration within the alveoli and blood for effect; thus, all inhaled methods have the potential to adversely affect animal welfare because onset of unconsciousness is not immediate. Distress may be created by properties of the agent (eg, pungency, hypoxia, hypercarbia) or by the conditions under which the agent is administered (eg, home cage or dedicated chamber, gradual displacement or prefilling of the container), and may manifest itself behaviorally (eg, overt escape behaviors, approach-avoidance preferences \[aversion\]) or physiologically (eg, changes in heart rate, SNS activity, HPA activity). Although SNS and HPA activation are well accepted as markers of a stress response, these systems are activated in response to both physical and psychological stressors and are not necessarily associated with higher-order CNS processing and conscious experience by the animal. Furthermore, use of SNS and HPA activation to assess distress during inhalation of euthanasia agents is complicated by continued exposure to the agents during the period between loss of consciousness and death. Distress during administration of inhaled agents has been evaluated by means of both behavioral assessment and aversion testing. While overt behavioral signs of distress have been reported in some studies, others have not consistently found these effects. Through preference and approach-avoidance testing, all inhaled agents currently used for euthanasia have been identified as being aversive to varying degrees. Aversion is a measure of preference, and while aversion does not necessarily imply that the experience is painful, forcing animals into aversive situations creates distress. The conditions of exposure used for aversion studies, however, may differ from those used for stunning or killing. In addition, agents identified as being less aversive (eg, Ar or N2 gas mixtures, inhaled anesthetics) can still produce overt signs of behavioral distress (eg, open-mouth breathing) in some species under certain conditions of administration (eg, gradual displacement). As previously noted in the section on consciousness, one of the characteristics of anesthesia in people is feeling as if one is having an out-of-body experience, suggesting a disconnection between one's sense of self and one's awareness of time and space. Although we cannot know for certain the subjective experiences of animals, one can speculate similar feelings of disorientation may contribute to the observed signs of distress. As for physical methods, the conditions under which inhaled agents are administered for euthanasia can have profound effects on an animal's response and, thus, agent suitability. Simply placing Sprague-Dawley rats into an unfamiliar exposure chamber containing room air produces arousal, if not distress. Pigs are social animals and prefer not to be isolated from one another; consequently, moving them to the CO2 stunning box in groups, rather than lining them up single file as needed for electric stunning, improves voluntary forward movement, reduces handling stress and electric prod use, and improves meat quality. That inhaled agents can produce distress and aversion in people raises concerns for their use in animals, in that the US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training state, "Unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings may cause pain or distress in other animals." Interestingly, more than 40% of human children 2 to 10 years old display distress behaviors during sevoflurane induction, with 17% displaying significant distress and more than 30% physically resisting during induction. Fear in children undergoing anesthesia may be due to odor, feel of the mask, or a true phobia of the mask. Despite evidence of distress and aversion, inhaled anesthetics continue to be administered because the benefits associated with their use greatly outweigh any distress and/or aversion they may cause. The suitability of any particular inhaled agent for euthanasia therefore depends largely on distress and/or pain experienced prior to loss of consciousness. Distress can be caused by handling, specific agent properties, or method of administration, such that a 1-size-fits-all approach cannot be easily applied. Suffering can be conceptualized as the product of severity, incidence, and duration. As a general rule, a gentle death that takes longer is preferable to a rapid, but more distressing death7; however, in some species and under some circumstances, the most humane and pragmatic option may be exposure to an aversive agent or condition that results in rapid unconsciousness with few or no outward signs of distress. Our goal is to identify best practices for administering inhaled agents, defining the optimal conditions for transport, handling, and agent selection and delivery to produce the least aversive and distressing experience for each species. The following contingencies are common to all inhaled euthanasia agents: \(1) Time to unconsciousness with inhaled agents is dependent on the displacement rate, container volume, and concentration. An understanding of the principles governing delivery of gases or vapors into enclosed spaces is necessary for appropriate application of both prefill and gradual displacement methods. The desired final concentration will be achieved more quickly by using a greater displacement rate (see M1.2). \(2) Loss of consciousness will be more rapid if animals are initially exposed to a high concentration of the agent. However, for many agents and species, forced exposure to high concentrations can be aversive and distressing, such that gradual exposure may be the most pragmatic and humane option. \(3) Inhaled agents must be supplied in purified form without contaminants or adulterants, typically from a commercially supplied source, cylinder, or tank, such that an effective displacement rate and/or concentration can be readily quantified. The direct application of products of combustion or sublimation is not acceptable due to unreliable or undesirable composition and/or displacement rate. \(4) The equipment used to deliver and maintain inhaled agents must be in good working order and in compliance with state and federal regulations. Leaky or faulty equipment may lead to slow, distressful death and may be hazardous to other animals and to personnel. \(5) Most inhaled agents are hazardous to animal workers because of the risk of explosions (eg, ether, CO), narcosis (eg, halocarbon anesthetics, nitrous oxide \[N2O\], CO2, asphyxiating gases), hypoxia (eg, asphyxiating gases, CO), addiction or physical abuse (eg, N2O, halocarbon anesthetics), or health effects resulting from chronic exposure (eg, N2O, CO, possibly halocarbon anesthetics). \(6) In sick or depressed animals where ventilation is decreased, agitation during induction is more likely because the rise in alveolar gas concentration is delayed. A similar delayed rise in alveolar gas concentration can be observed in excited animals having increased cardiac output. Suitable premedication or noninhaled methods of euthanasia should be considered for such animals. \(7) Neonatal animals appear to be resistant to hypoxia, and because all inhaled agents ultimately cause hypoxia, neonatal animals take longer to die than adults. Inhaled agents can be used alone in unweaned animals to induce loss of consciousness, but prolonged exposure time or a secondary method may be required to kill the unconscious animal. \(8) Reptiles, amphibians, and diving birds and mammals have a great capacity for holding their breath and for anaerobic metabolism. Therefore, induction of anesthesia and time to loss of consciousness when inhaled agents are used may be greatly prolonged. Noninhaled methods of euthanasia should be considered for these species and a secondary method is required to kill the unconscious animal. \(9) Rapid gas flows can produce noise or cold drafts leading to animal fright and escape behaviors. If high flows are required, equipment should be designed to minimize noise and gas streams blowing directly on the animals. \(10) When possible, inhaled agents should be administered under conditions where animals are most comfortable (eg, for rodents, in a darkened home cage; for pigs, in small groups). If animals need to be combined, they should be of the same species and compatible cohorts, and, if needed, restrained or separated so that they will not hurt themselves or others. Chambers should not be overloaded and need to be kept clean to minimize odors that might cause distress in animals subsequently euthanized. \(11) Because some inhaled agents may be lighter or heavier than air, layering or loss of agent may permit animals to avoid exposure. Mixing can be maximized by ensuring incoming gas or vapor flow rates are sufficient. Chambers and containers should be as leak free as possible. \(12) Death must be verified following administration of inhaled agents. This can be done either by examination of individual animals or by adherence to validated exposure processes proven to result in death. If an animal is not dead, exposure must be repeated or followed with another method of euthanasia. M1.2 PRINCIPLES GOVERNING ADMINISTRATION Changes in gas concentration within any enclosed space involve 2 physical processes: 1) wash- in of new gas (or wash-out of existing gas) and 2) the time constant required for that change to occur within the container for a known flow rate. These processes are commonly combined in the practice of anesthesia to predict how quickly a change in concentration of an inhaled anesthetic will occur within a circle rebreathing circuit. An understanding of how these processes work together is critical for the appropriate application of both gradual displacement and prefill immersion euthanasia methods. The rate of change of gas concentration within any enclosed space is a special form of nonlinear change known as an exponential process, and as such can be derived from the wash-in and wash-out exponential functions. Briefly, for the wash-in exponential function the quantity under consideration rises toward a limiting value, at a rate that progressively decreases in proportion to the distance it still has to rise. In theory, the quantity approaches, but never reaches, 100%. Conversely, for the wash-out exponential function the quantity under consideration falls at a rate that progressively decreases in proportion to the distance it still has to fall. Again, in theory, the quantity approaches, but never reaches, zero. The exponential wash-in and wash-out equations are used to derive the time constant (τ) for an enclosed volume or space. This constant is mathematically equal to the enclosed volume or space undergoing wash-in or wash-out divided by the rate of flow, or displacement, into that space, where τ = volume/ flow rate. Thus, the time constant represents the time at which the wash-in or wash-out process would have been complete had the initial rate of change continued as a linear function rather than an exponential function. As such, the time constant is similar in concept to the half-life, although they are neither identical nor interchangeable. For the wash-in function, 1(τ) is required for the concentration of the inflowing gas to rise to 63.2% of the inflowing gas concentration, 2(τ) are required for the concentration to rise to 86.5%, and 3(τ) are required for the concentration to rise to 95%, with ∞(τ) required for the gas concentration within the container to equal the inflowing gas concentration. Conversely, for the wash-out function, 1(τ) is required for the remaining gas concentration to fall to 36.8% of the original value, 2(τ) are required for gas concentration to fall to 13.5%, 3(τ) are required for gas concentration to fall to 5%, with ∞(τ) required for gas concentration to fall to 0% (Figure 3). The flow, or displacement rate, therefore determines the time constant for any given enclosed volume, such that increasing the flow rate will result in a proportional reduction of the wash-in and wash-out time constants for any size chamber (and vice versa). Based on Figure 3, it can be shown that a gradual inflow or displacement rate of 20% of the chamber volume/minute represents a time constant (τ) value of 5 minutes (1 divided by 0.2/min) regardless of chamber volume. For example, CO2 displacement rate equivalent to 20% of the chamber volume/min, as recommended by Hornett and Haynes15 and Smith and Harrap,16 is predicted to increase CO2 concentration from zero to 63.2% in 5 minutes (1τ), to 86.5% in 10 minutes (2τ), and to 95% in 15 minutes (3τ). An examination of the published experimental data of Smith and Harrap confirms this, where CO2 supplied at a displacement rate of 22% of chamber volume increased the CO2 concentration to approximately 64% in 4.5 minutes (1τ for their chamber). Similarly, Niel and Weary17 reported 65% after 340 seconds (1τ) and 87% after 600 seconds (2τ) for a CO2 displacement rate of 17.5% of chamber volume/min. Prefill methods will require displacement rates of 3τ to attain 95% of the inflow gas concentration within the chamber. Thus, gas displacement rate is critical to the humane application of inhaled methods, such that an appropriate pressure-reducing regulator and flow meter combination or equivalent equipment with demonstrated capability for generating the recommended displacement rate for the size container being utilized is absolutely necessary when compressed gases are used for euthanasia. Nitrogen, Ar, and CO are all commercially supplied in cylinders under high pressure, but CO2 is unique in that it is supplied as a liquefied gas under high pressure. By reducing high pressure at the cylinder valve, gas flow is made constant to the flow meter as cylinder pressure decreases during use. With CO2, the regulator also acts to prevent high gas flow rates that can lead to delivery of freezing gas and dry ice snow to the animals as well as regulator icing and cylinder freezing. A distinction must be made between immersion, where animals are directly placed into a gas or vapor contained within a container, and the process of CAS as employed for the commercial stunning of poultry and hogs. Although a complete description of the operation of the commercial CAS systems currently in use is beyond the scope of this document, typically the entry point is open to the atmosphere with negligible concentrations of stunning gas present. Unlike immersion, animals are introduced at a controlled rate into a tightly controlled stunning atmospheric gradient, such that CAS can be considered to be a gradual displacement method. M1.3 INHALED ANESTHETICS Overdoses of inhaled anesthetics (eg, ether, halothane, methoxyflurane, isoflurane, sevoflurane, desflurane, enflurane) have been used to euthanize many species. Presently, only isoflurane, enflurane, sevoflurane, and desflurane are clinically available in the United States, although halothane and methoxyflurane are still available elsewhere in the world. Halothane induces anesthesia rapidly and is an effective inhaled agent for euthanasia. Enflurane is less soluble in blood than halothane, but, because of its lower vapor pressure and lower potency, induction rates may be similar to those for halothane. At deep anesthetic planes, convulsions may occur. Enflurane is an effective agent for euthanasia, but the associated seizure activity may be disturbing to personnel. Isoflurane is less soluble than halothane, and it induces anesthesia more rapidly. However, it has a pungent odor and onset of unconsciousness may be delayed due to breath holding. Due to lower potency, isoflurane also may require more drug to kill an animal, compared with halothane. Sevoflurane is less potent than either isoflurane or halothane and has a lower vapor pressure. Anesthetic concentrations can be achieved and maintained rapidly but more drug will be required to kill the animal. Although sevoflurane is reported to possess less of an objectionable odor than isoflurane, some species may struggle violently and experience apnea when sevoflurane is administered by face mask or induction chamber. Like enflurane, sevoflurane induces epileptiform electrocortical activity. Desflurane is currently the least soluble potent inhaled anesthetic, but the vapor is quite pungent, which may slow induction. This drug is so volatile that it could displace O2 and induce hypoxemia during induction if supplemental O2 is not provided. Both diethyl ether and methoxyflurane are highly soluble, and may be accompanied by agitation because anesthetic induction is quite slow. Diethyl ether is irritating to the eyes, nose, and respiratory airways; poses serious risks due to flammability and explosiveness; and has been used to create a model for stress. Although inhaled anesthetics are routinely used to produce general anesthesia in humans and animals, these agents may be aversive and distressful under certain conditions. Flecknell et al reported violent struggling accompanied by apnea and bradycardia in rabbits administered isoflurane, halothane, and sevoflurane by mask or induction chamber, and concluded these agents were aversive and should be avoided whenever possible. Leach et al found inhaled anesthetic vapors to be associated with some degree of aversion in laboratory rodents, with increasing aversion noted as concentration increased; halothane was least aversive for rats, while halothane and enflurane were least a

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