Animal Welfare

Animal Welfare

C H A P T E R 39 Animal Welfare Marilyn J. Brown, DVM, MS, DACLAM, DECLAMa and Christina Winnicker, DVM, MPH, DACLAMb a Global Animal Welfare, Depar...

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C H A P T E R

39 Animal Welfare Marilyn J. Brown, DVM, MS, DACLAM, DECLAMa and Christina Winnicker, DVM, MPH, DACLAMb a

Global Animal Welfare, Department of Animal Welfare, Charles River Laboratories, Wilmington, MA, USA bEnrichment & Behavioral Medicine, Department of Animal Welfare, Charles River Laboratories, Wilmington, MA, USA

O U T L I N E   I. Introduction

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  II. Animal Welfare as a Key Component in Research, Teaching, and Testing Using Animals 1654 III. Animal Welfare – A Historical and Philosophical Perspective

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IV. Guidelines and Principles A. Replacement B. Reduction C. Refinement

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  V. Strategies to Optimize Animal Welfare

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I. INTRODUCTION The term ‘animal welfare,’ in both the lay and scientific community, is often used to refer to a concept. In this context, positive animal welfare may be substituted with the term ‘well-being.’ Animal welfare serves as a cornerstone, or foundation, for laboratory animal medicine and the use of animals in research. ‘Animal welfare’ also refers to a measurable state in an animal which may be related to the adequacy of an animal’s ability to cope with its environment. Animal welfare is a branch of science which looks at these measurable states in almost all areas Laboratory Animal Medicine, Third Edition DOI: http://dx.doi.org/10.1016/B978-0-12-409527-4.00039-0

  VI. Examples of Challenging Research and Opportunities for Animal Welfare Optimization 1664 A. Genetically Altered Research Models 1664 B. Cancer Research 1665 C. Neuroscience and Behavioral Research 1666   VII. Science of Animal Welfare A. Introduction B. Modern Animal Welfare Science C. Assessing Animal Welfare

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VIII. Conclusion and Summary

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References 1669

of our interaction with animals – agriculture, entertainment, companionship, research, and others. This chapter will highlight the significant emphasis on animal welfare in the field of laboratory animal science and medicine, beginning with some of the history, philosophies, ethics and events which have shaped the impact of animal welfare on the use of animals in research. Laws and regulations will only be briefly mentioned as they are covered elsewhere in this book; however, guidelines and principles, such as the 3Rs, will be covered in more detail. Using examples of reduction and refinement, strategies for optimizing laboratory animal welfare in general and

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2015 Elsevier Inc. All rights reserved. © 2012

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in several specific areas of research will be discussed. Animal welfare as a science, measuring the animal’s perception of their state of well-being and going beyond just physiologic measurement of health and production, will be discussed, with particular focus on the use of behavioral monitoring to measure welfare.

II.  ANIMAL WELFARE AS A KEY COMPONENT IN RESEARCH, TEACHING, AND TESTING USING ANIMALS A discussion of animal welfare should begin with defining the term. There are many definitions of animal welfare and only a few are provided here to highlight the similarities and also some of the nuanced differences. The reader is referred to the American College of Animal Welfare (ACAW) website (http://www.acaw. org/animal_welfare_principles.html) and the various references in this chapter for additional definitions. Many of the definitions and principles of animal welfare focus upon how animal welfare is influenced by humans and thus may be looked upon as what we ‘owe’ animals. Welfare is a broad term which includes the many elements that contribute to an animal’s quality of life, including those referred to in the ‘five freedoms’ (freedom from hunger, thirst and malnutrition; freedom from fear and distress; freedom from physical and thermal discomfort; freedom from pain, injury and disease; and freedom to express normal patterns of behaviour). (OIE Animal Welfare Guidelines, 2005) …animal welfare is concerned with assuring humane treatment of animals; maintaining good health, minimizing negative states such as pain, enhancing positive states, and giving animals freedom to behave in ways that are natural to that species. (Gilbert et al., 2012) Animal Welfare … “is the degree of pleasure that an animal obtains from performing a behavior or obtaining a valued resource, rather than the amount of suffering caused by the inability to perform the behaviour or the absence of the resource. (Appleby et al., 2011)

Signs of the growing recognition of the importance of animal welfare are the inclusion of animal welfare as a strategic imperative for the American Veterinary Medical Association (AVMA) for the second consecutive planning period, creation of a Division of Animal Welfare within the AVMA, and a standing Animal Welfare Committee of the AVMA. Although the concept of animal welfare is not new to the field of veterinary medicine, the increased focus on animal welfare can be demonstrated in the newly created American College of Animal Welfare (ACAW) incorporated in 2010 and recognized by the AVMA in 2012. Many readers of this book will be veterinarians so it is apt to begin the discussion

of animal welfare with reference to the AVMA Animal Welfare Principles. (Although the AVMA is the largest veterinary medical association, it should be noted that they are by no means the only veterinary association to have principles and guidelines pertaining to animal welfare so readers are encouraged to look at guidelines from their own professional associations.) The AVMA Animal Welfare Principles state: The responsible use of animals for human purposes, such as companionship, food, fiber, recreation, work, education, exhibition, and research conducted for the benefit of both humans and animals, is consistent with the Veterinarian’s Oath. ● Decisions regarding animal care, use, and welfare shall be made by balancing scientific knowledge and professional judgment with consideration of ethical and societal values ● Animals must be provided with water, food, proper handling, health care, and an environment appropriate to their care and use, with thoughtful consideration for their species-typical biology and behavior. ● Animals should be cared for in ways that minimize fear, pain, stress, and suffering. ● Procedures related to animal housing, management, care, and use should be continuously evaluated, and when indicated, refined, or replaced. ● Conservation and management of animal populations should be humane, socially responsible, and scientifically prudent. ● Animals shall be treated with respect and dignity throughout their lives and, when necessary, provided a humane death. ● The veterinary profession shall continually strive to improve animal health and welfare through scientific research, education, collaboration, advocacy, and the development of legislation and regulations (https:// www.avma.org/KB/Policies/Pages/AVMA-AnimalWelfare-Principles.aspx Accessed May 11, 2015). ●

Animal welfare, as a key component in research, teaching and testing involving animals, can be found in the very title of the primary law impacting animal research in the United States – The Animal Welfare Act (AWA) and the subsequent Animal Welfare Regulations which provide details for implementation of the AWA. Research supported by Public Health Service funds, must comply with the Office of Laboratory Animal Welfare (OLAW). Researchers and Institutional Animal Care and Use Committees (IACUCs) are urged to use resources provided by the Animal Welfare Information Center of the National Agriculture Library to search for alternatives during the planning and execution of their studies. In The Guide for the Care and Use of Laboratory Animals, the word ‘welfare’ occurs 178 times and ‘well-being’ occurs 68 times (NRC, 2011). Organizations providing support to the research

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industry include groups such as the Laboratory Animal Welfare Exchange (LAWTE); Scientist Center for Animal Welfare (SCAW); United Federation for Animal Welfare (UFAW), to name just a few. Clearly, welfare is an important consideration of veterinary medicine as a whole, but particularly in the areas of research, teaching and testing. Animal welfare is a key component in research, teaching and testing involving animals, but how did we get here?

III.  ANIMAL WELFARE – A HISTORICAL AND PHILOSOPHICAL PERSPECTIVE Our relationship with animals began when man only hunted animals for food and clothing or feared animals for man’s safety. It has evolved into domestication of animals for many human uses including food and clothing, transportation, companionship, entertainment and research. We moved away from humans as the ‘shepherds’ or stewards over animals during the industrial revolutions where populations moved from rural to urban settings. Organizations such as the British Society for the Protection of Animals (1866) and the American Humane Association (1874) were formed as society became increasingly concerned about animal welfare (Patterson-Kane and Golab, 2013). At the same time, shelters for stray animals were also being established. The impetus for these developments was concern for horses and later dogs and cats. The first legislation which represented a concern for animals in research was the Cruelty to Animals Act (1876) in Great Britain. This legislation licensed researchers using animals with a focus on painful research and the use of anesthesia. Only three prosecutions resulted from this Act (Patterson-Kane and Golab, 2013). The period after World War II saw an awakening in awareness of, and concern for, animal welfare in the research setting. Within the research community, organizations such as the American Association for Laboratory Animal Science (AALAS) was formed in 1950 followed closely by the American College of Laboratory Animal Medicine (ACLAM) in 1958 and the American Association for the Accreditation of Laboratory Animal Care (AAALAC) in 1965. These organizations had a common goal of providing better care for animals used in research, teaching and testing through better facilities and development of formal qualifications for those involved in laboratory animal science and medicine. Another key event impacting laboratory animal welfare included the publication of the first Guide for the Care and Use of Laboratory Animals in 1963 (hereafter referred to as the Guide). For the public, two pivotal events raised concerns about animals used in research, particularly how pets could be acquired by random source animal dealers and how these animals were kept in some situations. The first was an article in

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Sports Illustrated in 1965 which highlighted the story of Pepper, a dog that was stolen from a home and who ended up in a research laboratory (Phinizy, 1965). The other was in Life Magazine in 1966 where poor conditions at vendor were exposed (Wayman, 1966). The public outcry led to the passing of the first Animal Welfare Act in the United States in 1966. Concurrently, in agriculture, public concern for animal welfare increased after the publication of Animal Machines by Ruth Harrison in 1964 highlighting the plight of agricultural animals in postindustrial farming (Harrison, 1964). Later the Brambell Report (Brambell, 1965) described guidelines, referred to as the Five Freedoms, in response to concerns about the welfare of agricultural animals in the United Kingdom. These guidelines are also applicable to laboratory animals and will be elaborated upon later in this chapter. Although animal research oversight committees had been in place at some institutions, the requirement for such committees only became mandatory in 1979 (Patterson-Kane and Golab, 2013). With subsequent revisions of the AWA in 1985, 1990, 2002, 2007, and 2008, passage of the Health Research Extension Act of 1985 and subsequent Public Health Service Policy on Humane Care and Use of Laboratory Animals, as well as revisions of the Guide in 1978, 1985, 1996, and most recently in 2011, the scope and functions of the animal welfare oversight committee, called the Institutional Animal Care and Use Committee (IACUC) in the United States, has continued to be defined and expanded. The Guide defines the animal care and use program as comprised of “all activities conducted by and at an institution that have a direct impact on the well-being of animals…” and places the responsibility for the oversight of the program in the hands of the IACUC (NRC, 2011). While it is not the purpose of this chapter to focus on philosophy and ethics, some mention of philosophies such as the moral standing of animals and basic ethical theories helps to put animal welfare into a philosophical context. Readers interested in exploring this further, might seek additional references listed (Mill, 1863; Frey, 1988; Regan and Singer, 1989; Tannenbaum, 1991; Carruthers, 1992; Cohen and Regan, 2001; Olsson et al., 2011; Rollin, 2011; Gilbert et al., 2012; Brown, 2013). The moral status of animals has been considered by some as “the key question in veterinary ethics” (Rollin, 2011). The question of the moral status of animals is not the same as the question if animals matter (Carruthers, 1992). Carruthers says “common sense morality” indicates that animals have partial moral standing because their lives and experiences have direct moral significance, but much less than humans. Criteria for ascribing moral status can use a ‘uni-criterial approach’ which uses a key property of life such as capacity to feel pain, language, ability to reason, or personhood. Alternatively, a ‘multicriterial approach’ where more than one valid criterion

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and more than one type of moral status with different types imposing obligations on moral agents can be used (Hill, 1992; Warren, 1997). Ethical or moral theories provide a framework for reaching decisions when faced with an ethical or moral dilemma (Brown, 2013). Some useful qualities of an adequate theory include: universal validity (ability to apply it in all similar circumstances and by all comparable individuals); evident to reason; ease of determination if theory applies to a given situation; provision of a basis for moral motivation and the ability to provide guidance in the face of needed decisions. Ethical theories generally fall into two major categories, deontological, which are based upon the idea of right and wrong or teleological, which are consequential, by evaluating the results of actions. Deontological theories vary based upon what is considered right or wrong and often deal with concepts of duty and moral obligation. Utilitarianism is the most prominent of the teleological theories. Utilitarianism seeks to provide the greatest good (or least harm) to the greatest number of individuals. Challenges with this theory include determination of who ‘counts’ as an individual (the question of moral standing) and balance of the theory with commonly held beliefs about the ‘rights’ of an individual – the principle of equal consideration protects lives, liberty and well-being of individuals only if it maximizes overall utility. Utilitarianism has been used to both support the use of animals in research (Russow, 1990) and to conclude a vast majority of animal research is immoral (Singer, 1975). The harm:benefit analysis of a proposed study by an IACUC is an example of a utilitarian calculation in which the moral significance of humans is usually considered greater than that of animals. A well known exception to this approach is the concept of ‘speciesism,’ first proposed by Ryder in 1975 (Ryder, 1975) but popularized by Singer in 1975 (Singer, 1975). Finally, as we look at how some historical events have impact upon views about animal welfare, it is also important to note other cultural influences that impact on how society views animal welfare and our obligations toward animals. Today, many people have grown up around pets and often pets are referred to as ‘members of the family.’ Spending on pets is reported to be in the billions of dollars each year as estimated by the American Pet Products Association (http://www.americanpetproducts.org/press_industrytrends.asp). Of the people who refused to relocate during Hurricane Katrina in New Orleans, 44% gave their reason for refusing relocation as wanting to stay near their pet. Disney studios made a mouse an American icon – who can say they have not heard of Mickey Mouse? Animals have been ‘humanized’ in TV and film – Mr. Ed, the talking horse of the 1950s; the animals in Charlotte’s web in 1972; Stuart Little in 1991; and a rat as the star of a popular film, Ratatouille in 2007.

Concern about animal welfare is not just based upon anthropomorphic images in the media. As science helps us better understand both animal behavior and physiology and as discussions of animals’ emotional/mental states and feelings become more mainstream, concern for animal welfare will likely continue to grow. The laboratory animal science community have the opportunity to be leaders in optimizing animal welfare.

IV.  GUIDELINES AND PRINCIPLES As mentioned earlier, in 1965 a committee was convened in London to look at concerns with the welfare of agricultural animals. This committee developed guidelines referred to as the Five Freedoms which include: (1) Freedom from Hunger and Thirst – by ready access to fresh water and a diet to maintain full health and vigor; (2) Freedom from Discomfort – by providing an appropriate environment including shelter and a comfortable resting area; (3) Freedom from Pain, Injury, or Disease – by prevention or rapid diagnosis and treatment; (4) Freedom to Express Normal Behavior – by providing sufficient space, proper facilities and company of the animal’s own kind; (5) Freedom from Fear and Distress – by ensuring conditions and treatment which avoid mental suffering (Brambell, 1965). Between 1982 and 1984, international interdisciplinary consultations were undertaken by the Council for Inter­ national Organizations of Medical Sciences (CIOMS) to create the International Guiding Principles for Biomedical Research Involving Animals (CIOMS, 1985). These principles were fully endorsed by the European Medical Research Councils (EMRC) and World Health Organ­ ization Advisory Committee on Medical Research in 1984 and served as a foundation for the US National Institutes of Health, US Interagency Research Animal Committee (IRAC), and the US Government Principles for the Utilization and Care of Vertebrate Animals used in Testing, Research, and Training in 1985 (Table 39.1). The CIOMS Principles were revised in 2013 and are found in Table 39.2. Both sets of Principles can be found in the 2011 edition of the Guide (NRC, 2011) and the IRAC Principles can also be found on the cover of the PHS Policy (OLAW, 2002). Although the principles and guidelines listed above had significant impact on animal welfare in the laboratory, perhaps the simplest and the one with greatest impact on animal research today is the Three Rs of Russell and Burch (Russell and Burch, 1959). The CIOMS Principles previously mentioned include reference to the 3Rs. The Statement of Task to the revision committee of the latest Guide begins with “The use of laboratory animals for biomedical research, testing and education is guided by the principles of the 3Rs…” (NRC, 2011). The term ‘the three (3) Rs’ or the word alternatives, replacement,

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TABLE 39.1  IRAC US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training The development of knowledge necessary for the improvement of the health and well-being of humans as well as other animals requires in vivo experimentation with a wide variety of animal species. Whenever U.S. Government agencies develop requirements for testing, research, or training procedures involving the use of vertebrate animals, the following principles shall be considered; and whenever these agencies actually perform or sponsor such procedures, the responsible Institutional Official shall ensure that these principles are adhered to: I. The transportation, care, and use of animals should be in accordance with the Animal Welfare Act (7 U.S.C. 2131 et. seq.) and other applicable Federal laws, guidelines, and policies. II. Procedures involving animals should be designed and performed with due consideration of their relevance to human or animal health, the advancement of knowledge, or the good of society. III. The animals selected for a procedure should be of an appropriate species and quality and the minimum number required to obtain valid results. Methods such as mathematical models, computer simulation, and in vitro biological systems should be considered. IV. Proper use of animals, including the avoidance or minimization of discomfort, distress, and pain when consistent with sound scientific practices, is imperative. 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. V. Procedures with animals that may cause more than momentary or slight pain or distress should be performed with appropriate sedation, analgesia, or anesthesia. Surgical or other painful procedures should not be performed on unanesthetized animals paralyzed by chemical agents. VI. Animals that would otherwise suffer severe or chronic pain or distress that cannot be relieved should be painlessly killed at the end of the procedure or, if appropriate, during the procedure. VII. The living conditions of animals should be appropriate for their species and contribute to their health and comfort. Normally, the housing, feeding, and care of all animals used for biomedical purposes must be directed by a veterinarian or other scientist trained and experienced in the proper care, handling, and use of the species being maintained or studied. In any case, veterinary care shall be provided as indicated. VIII. Investigators and other personnel shall be appropriately qualified and experienced for conducting procedures on living animals. Adequate arrangements shall be made for their in-service training, including the proper and humane care and use of laboratory animals. IX. Where exceptions are required in relation to the provisions of these Principles, the decisions should not rest with the investigators directly concerned but should be made, with due regard to Principle II, by an appropriate review group such as an institutional animal care and use committee. Such exceptions should not be made solely for the purposes of teaching or demonstration. IRAC (1985).

reduction or refinement is found 131 times in the Guide (NRC, 2011). The AWA refers to the concept of the 3Rs and alternatives throughout the Regulations (CFR, rev. 1998). Likewise, the EU Directive states “The care and use of live animals for scientific purposes is governed by internationally established principles of replacement, reduction and refinement…” (Anonymous, 2010). These three words, or the words 3Rs or alternatives are found 90 times in the EU Directive. Inclusion of the concept of the 3Rs in multinational laws, regulations and guidelines has given these concepts significant influence over how global animal research is conducted today. The 3Rs are a common theme in the Guide which states “Throughout the Guide, scientists and institutions are encouraged to give careful and deliberate thought to the decision to use animals taking into consideration the contribution that such use will make to new knowledge, ethical concerns, and the availability of alternatives to animal use. A practical strategy for decision making, [is] the “Three Rs” (Replacement, Reduction, and Refinement) approach…” (NRC, 2011). The concept of the 3Rs is also infused in US regulations covering research using animals. Although the US

Animal Welfare Act (AWA) regulations do not include the word ‘alternatives’ in its section of definitions, the term is used several times in the regulations themselves (Brown and Smiler, 2012). For example, in the section on IACUC review of protocols the regulations state protocols must indicate that (i) Procedures involving animals will avoid or minimize discomfort, distress, and pain to the animals; (ii) The principle investigator has considered alternatives to procedures that may cause more than momentary or slight pain or distress to the animals, and has provided a written narrative description of the methods and sources, e.g. The Animal Welfare Information Center, used to determine that alternatives were not available (AWA [Animal Welfare Act], 1990). The focus of USDA inspectors on adherence to this section of the regulations can be appreciated when looking at the USDA Research Facility Inspection Guide which instructs inspectors several times to evaluate institutional compliance in this area (USDA, 2009). In addition, the requirement for a search for alternatives is the subject of an Animal Care Policy – Policy 12 (APHIS, 2000). Strategies to enhance search efficiency using a search filter for PubMed have been published (Hooijmans et al.,

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TABLE 39.2  International Guiding Principles for Biomedical Research Involving Animals The following principles should be used for the international scientific community to guide the responsible use of vertebrate animals in scientific and/or educational activities. I. The advancement of scientific knowledge is important for improvement of human and animal health and welfare, conservation of the environment, and the good of society. Animals play a vital role in these scientific activities and good animal welfare is integral to achieving scientific and educational goals. Decisions regarding the welfare care and use of animals should be guided by scientific knowledge and professional judgment, reflect ethical and societal values and consider the potential benefits and the impact on the well-being of the animals involved. II. The use of animals for scientific and/or educational purposes is a privilege that carries with it moral obligations and responsibilities for institutions and individuals to ensure the welfare of these animals to the greatest extent possible. This is best achieved in an institution with a culture of care and conscience in which individuals working with animals willingly, deliberately, and consistently act in an ethical, humane and compliant way. Institutions and individuals using animals have an obligation to demonstrate respect for animals, to be responsible and accountable for their decisions and actions pertaining to animal welfare, care and use, and to ensure that the highest standards of scientific integrity prevail. III. Animal should be used only when necessary and only when their use is scientifically and ethically justified. The principles of the Three Rs – Replacement, Reduction and Refinement – should be incorporated into the design and conduct of scientific and/or educational activities that involve animals. Scientifically sound results and avoidance of; unnecessary duplication of animal-based experimental design. When no alternative methods, such as mathematical models, computer simulation, in vitro biological systems, or other non-animals (adjunct) approaches are available to replace the use of live animals, the minimum number of animals should be used to achieve the scientific or educational goals. Cost and convenience must not take precedence over these principles. IV. Animals selected for the activity should be suitable for the purpose and of an appropriate species and genetic background to ensure scientific validity and reproducibility. The nutritional, microbiological, and general health status as well as the physiological and behavioral characteristics of the animals should be appropriate to the planned use as determined by scientific and veterinary medical experts and/or the scientific literature. V. The health and welfare of animals should be primary considerations in decisions regarding the program of veterinary medical care to include animal acquisition and/or production, transportation, husbandry and management, housing, restraint and final disposition of animals, whether euthanasia, rehoming or release. Measures should be taken to ensure that the animal’s environment and management are appropriate for the species and contribute the animal’s well-being. VI. The welfare, care and use of animals should be under the supervision of a veterinarian or scientists trained and experienced in the health, welfare, proper handling, and use of the species being maintained or studied. L The individual or team responsible for animal welfare, care and use should be involved in the development and maintenance of all aspects of the program. Animal health and welfare should be continuously monitored and assessed with measures to ensure that indicators of potential suffering are promptly detected and managed. Appropriate veterinary care should always be available and provided as necessary by a veterinarian. VII. Investigators should assume that procedures that would cause pain or distress in human beings cause pain or distress in animals, unless there is evidence to the contrary. Thus, there is a moral imperative to prevent or minimize stress, distress, discomfort and pain in animals, consistent with sound scientific or veterinary medical practice. Taking into account the research and educational, goals, more than momentary or minimal pain and/or distress in animals should be managed by refinement of experimental techniques and/or appropriate sedation, analgesia, anesthesia, non-pharmacologic interventions, and/or other palliative measures developed in consultation with a qualified veterinarian or scientist. Surgical or other painful procedures should not be performed on unanesthetized animals. VIII. Endpoints and timely interventions should be established for both humane and experimental reasons. Humane endpoints and/or interventions should be established before animal use begins, should be assessed throughout the course of the study, and should be applied as early as possible to prevent, ameliorate, or minimize unnecessary and/or unintended pain and/or distress. Animals that would otherwise suffer severe or chronic pain, distress or discomfort that cannot be relieved and is not part of the experimental design, should be removed from the study and/or euthanized using a procedure appropriate for the species and condition of the animal. IX. It is the responsibility of the institution to ensure that personnel responsible for the welfare, care and use of animals are appropriately qualified and competent through training and experience for the procedures they perform. Adequate opportunities should be provided for on-going training and education in the humane and responsible treatment of animals. Institutions also are responsible for supervision of personnel to ensure proficiency and the use of appropriate procedures. X. While implementation of these Principles may vary from country to country according to cultural, economic, religious and social factors, a system of animal use oversight that verifies commitment to the Principles should be implemented in each country. This system should include a mechanism for authorization (such as licensing or registering of institutions, scientist and/or projects) and oversight which may be assessed at the institutional, regional, and/or national level. The oversight framework should encompass both ethical review of animal use as well as considerations related to animal welfare and care. It should promote a harm benefit analysis for animal use, balancing the benefits derived from the research or educational activity with the potential for pain and/or distress experienced by the animal. Accurate records should be maintained to document a system of sound program management, research oversight, and adequate veterinary care.

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2010a). A “Gold Standard Publication Checklist” has been proposed to help fully integrate the 3Rs into systematic reviews of the literature (Hooijmans et al., 2010b). In the section on personnel qualifications, the AWA says that the institution should ensure adequate training and qualifications and that this is fulfilled in part through the provision of training and instruction on the “concept, availability, and use of research or testing methods that limit the use of animals [Reduction] or minimize animal distress [Refinement]” (AWA [Animal Welfare Act], 1990). The AWA further indicates that research staff should be trained on the “utilization of services (e.g., National Agriculture Library of Medicine) available to find information:… (ii) On alternatives to the use of live animals in research [Replacement];…” (AWA [Animal Welfare Act], 1990). In addition to the specific references above, the AWA regulations also refer to the use of anesthetics, analgesics and sedatives, the availability of appropriate veterinary care, the use of appropriate housing; and timely, appropriate euthanasia – all of which demonstrate the concept of ‘refinement’ (Brown and Smiler, 2012). The PHS Policy contains similar language regarding minimizing discomfort, distress, pain, use of appropriate anesthesia, and the use of humane endpoints – all examples of refinement. In addition, the PHS Policy refers to the IRAC Principles, requiring institutions receiving PHS funds to use the Guide as a basis for their animal care and use programs. National and international agencies and organizations such as the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) (http:// iccvam.niehs.nih.gov/), the European Centre for the Validation of Alternative Methods (ECVAM) (http:// ecvam.jrc.ec.europa.eu/), and the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) (http://www.nc3rs.org.uk/) are charged with helping to find and promote the use of alternatives (Brown and Smiler, 2012). With so much ‘official’ emphasis on the 3Rs, why is there a perception that the concept is not being adequately implemented in practice? It has been suggested that scientists and IACUCs do not fully understand the concepts of the 3Rs (Graham, 2002; Schuppli and Fraser, 2005). In addition to incomplete understanding of the concepts, factors believed to negatively influence the full implementation of the 3Rs included a belief that the scientists themselves would implement the 3Rs; funding agencies reviewed the use of the 3Rs when reviewing proposals; sample size, rather than study design, was the important criteria for reduction; and focusing upon potential harm from procedures without considering potential distress from husbandry and housing was appropriate. Although such conclusions were based upon very small groups (4 and 3 IACUCs, respectively),

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these are troubling observations and indicate the need for greater emphasis on the 3Rs in training programs for scientists and IACUCs (Brown and Smiler, 2012). Others suggest that the extent of the 3Rs implementation is “substantially underestimated” due to the lack of recognition by the scientist or protocol review committee that a proposed procedure will result in a 3Rs outcome (Mellor et al., 2007). It would be disingenuous to imply that, although well established, the concept of the 3Rs is universally accepted. In an article titled Time to Abandon the Three Rs, Derbyshire wrote that the 3Rs “draw attention away from the value of experimentation and toward the importance of animal welfare” (Derbyshire, 2006). Although the article supports the concept of reducing animal stress for the sake of science, this article clearly does not recognize the opportunity to balance between facilitating science and, at the same time, applying the 3Rs.

A. Replacement Replacement refers to methods that avoid using animals. The term includes absolute replacements (i.e., replacing animals with inanimate systems such as computer programs) as well as relative replacements (i.e., replacing animals, such as vertebrates, with animals that are lower on the phylogenic scale) (NRC, 2011). Relative replacement may be controversial to some people as it implies ‘speciesism’ (Singer, 1975). Like many of the ethical considerations relating to animal use, this concept can also be considered on a continuum. Society, in general, often differentiates between humans and non human animals. However, when we consider the animal world, different opinions exist about our obligations to some species versus others. For example, nonhuman primates and animals commonly kept as pets such as horses, dogs, and cats seem to be considered differently than rats and mice, which are considered differently from fruit flies, worms, and so on. This is consistent with what Patterson-Kane terms the ethics of care. This philosophy “honors the human–animal bond as a morally significant and ethically acceptable attachment that creates a duty to care for and protect a specific animal regardless of the objective status of the animal” (Patterson-Kane and Golab, 2013). Development of fully validated and accepted replacement alternatives can be a frustratingly slow process, but scientists and regulatory agencies must ensure that products are non hazardous (or appropriately labeled if hazardous). However, there are significant examples of successful replacement of live animals (Brown and Smiler, 2012). Perhaps one of the most criticized uses of animals for toxicity testing is the Draize test in rabbits. This test was developed to determine ocular toxicity caused by products and chemicals. Ocular toxicity tests represent one of the four most commonly conducted product safety

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tests (ICCVAM, 2010). The 3Rs were implemented by the development of validated and accepted replacements for screening products – the bovine corneal opacity and permeability test using an isolated cow eye or an isolated chicken eye (both by-products of the meat industry), and the cytosensor microphysiometer. One refinement, a balanced preemptive pain-management plan for rabbit Draize tests which still are required, has also been validated and accepted (ICCVAM, 2010). A second example of implementation of the 3Rs involves the replacement of rabbits in the rabbit pyrogen test by using an in vitro Limulus Ameobecyte Lysate (LAL) Test. In this safety test, blood of horseshoe crabs is collected and the animals are returned, unharmed, back to the ocean. A component of the horseshoe crab’s blood reacts with bacterial endotoxin or lipopolysacchride, a membrane component of gram-negative bacteria, allowing detection of bacterial contamination. Previous tests required the injection of drugs, biologics, medical devices, or raw materials into rabbits to look for a febrile response to indicate contamination with endotoxins. Now the majority of these products are chemically tested using LAL instead of being injected into rabbits (Brown and Smiler, 2012).

B. Reduction “Reduction includes strategies for obtaining comparable levels of information from the use of fewer animals or for maximizing the information obtained from any given number of animals (without increasing pain or distress) to ultimately require fewer animals to acquire the same scientific information. This approach relies on an analysis of experimental design, applications of newer technologies, the use of appropriate statistical methods, and control of environmentally related variability in animal housing and study areas” (NRC, 2011). Strategies to reduce the numbers of animals needed include improved experimental design, by formulation of a good experimental question and logical development of a hypothesis (Frey, 2014), appropriate statistical design of a study (Dell et al., 2002), and improved selection of an animal model, including selection of animals with the most appropriate health and genetic status. Control of the genetic status is an advantage of using rats and mice. The use of inbred strains of rats and mice allows scientists to control and investigate genetic variation, and to evaluate responses to treatments on specific areas of interest (Festing, 2004). The use of animals without confounding disease or genetic variation results in less variation and requires fewer animals to determine a treatment effect. Individuals involved with study design, study review, or as a member of the research team have the ethical imperative to ensure studies use the minimum numbers of animals necessary to achieve the scientific

objective. Scientists should design studies with particular attention to methodology, statistics and choice of model. Veterinarians and facility staff should collaborate to minimize non-experimental variables in animal care. IACUCs should be diligent during review of the protocol, semiannual program and facility evaluations, and review of post approval monitoring to ensure that the appropriate number of animals have been used. Having a statistician on the IACUC is one strategy that may be helpful (Brown and Smiler, 2012).

C. Refinement “Refinement refers to modifications of husbandry or experimental procedures to enhance animal well-being and to minimize or eliminate pain and distress” (NRC, 2011). In the authors’ opinion, refinement is commonly employed by scientists in ongoing efforts to improve their science – better animal welfare leading to betterquality science, particularly in cases where the impacts of animal distress would be a confounding variable. Many scientists do not recognize this as utilization of ‘alternatives,’ even though it clearly falls within the 3Rs (Mellor et  al., 2007). However, this is also an area where scientists, veterinarians and IACUCs can make significant strides to enhance animal welfare (Brown and Smiler, 2012). Use of less invasive procedures (e.g,. the use of a blood pressure cuff versus a catheter) is one method of refining a study. However, there are also situations where an invasive procedure, such as implantation of telemetry, can result in much less stressful data collection (Stephens et  al., 2002). Other general refinements utilized in experimental procedures include microsampling to decrease the amount of blood needed (which also leads to a reduction of the animals needed) and imaging which allows non-invasive collection of data over time, rather than euthanizing animals at multiple time-points to follow progression of a disease. Examples of other refinements which would often be most influenced by the laboratory animal medicine professional would be more accurate recognition of pain and the use of analgesics and supportive care, implementation of humane endpoints and enhanced housing and husbandry. These will be discussed in more detail as we review strategies to optimize animal welfare.

V.  STRATEGIES TO OPTIMIZE ANIMAL WELFARE Refinement and reduction are two key areas where the laboratory animal professional can optimize animal welfare. Strategies which can aid in optimizing animal welfare include: input into experimental design to minimize the number of animals that need to be used and

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facilitating sharing of animal resources when possible; recognition of signs of pain, distress, and other negative welfare states; pharmacologic and non-pharmacologic interventions to minimize pain, distress, and other negative welfare states; pre-study agreement on humane endpoints and providing input during research actively modify those endpoints if needed; acclimatizing animals to potentially painful or distressful situations; and training animals to cooperate in experimental procedures and provision of appropriate environmental enrichment. Carbone and Garnett, in a chapter on Ethical Issues in Anesthesia and Analgesia in Laboratory Animals state, “… the prime ethical concerns in laboratory animal welfare are what animals consciously experience: their pain, distress, fear, boredom, happiness and psychological well being” (Carbone and Garnett, 2008). The emotional dimension of pain, a characteristic of suffering, requires pain pathways to extend to higher levels of the cortex which has been reported as being unique to humans and some other primates (such as apes) (Nuffield Council on Bioethics, 2005). “However, the absence of analogous structures cannot necessarily be taken to mean that they [animals] are incapable of experiencing pain, suffering or distress or any other higher order states of conscious experience” (Nuffield Council on Bioethics, 2005). “Fundamental to the relief of pain in animals is the ability to recognize its clinical signs in specific species” (NRC, 2011). Thus it is imperative that a program of animal care and use includes adequate training of all personnel with animal responsibilities. It has been suggested that some animals (particularly prey species) may try to mask pain to avoid displaying abnormal activity and increasing their risk of predation (Roughan and Flecknell, 2000). In addition, many of these animals are most active during the dark cycle, when observations are more difficult. Since clinical indices of pain may be very subtle, it is important to be able to recognize a departure from normal behavior and appearance (Table 39.3) (NRC, 2003). A short list of general signs and measurements includes: animals vigorously seeking to escape;

changes in biological characteristics such as food and water consumption and body weight; blood levels of hormones and glucose; adrenal gland mass; and speciesspecific appearance, posture, and behavior (Moberg, 1985, 2000). Behavioral indicators of pain in mice and rats have also been described (Flecknell, 1999; Roughan and Flecknell, 2000, 2001, 2003; Karas, 2003; Kohn et al., 2007). Guidelines for the assessment and management of pain in rodents and rabbits have been published by the ACLAM (ACLAM, 2006). The potential for pain-relieving medications to interfere with research objectives/results should be considered. Many studies have been carried out investigating analgesic effect on a wide variety of parameters (e.g., litter size, body weight, behavior, and hemodynamic parameters) (Lamon et  al., 2008; Valentim et  al., 2008; McBrier, et  al., 2009; Bourque et al., 2010; Goulding et al., 2010). Instead of assuming that analgesics cannot be given, literature searches should be conducted to determine if studies have been done validating the effect on the variables of concern, and investigators should be required to provide scientific justification for withholding analgesia. If data does not exist, consideration should be given for conducting and publishing an appropriate study to indicate which analgesics are, or are not, a viable scientific option for future experiments. In addition to considering the potential impact of pain-relieving drugs on research, the impact of not relieving pain must also be considered as such states can result in adverse physiologic and behavioral consequences such as weight loss, compromised immune function, and tumor progression (NRC, 2009). It has been suggested that administering analgesics to both control and test animals is a way to ensure appropriate comparisons (Stokes and Marsman, 2014). If it is determined that a study must involve unrelieved pain, there should be well defined criteria for removal of an animal from study and clearly defined procedures for how such decisions will be made (Stokes and Marsman, 2014). There is a growing understanding in neurology of the similarities of emotional pain, such as the pain of social separation,

TABLE 39.3  Indicators of Pain in Several Common Laboratory Animals Species

General behavior

Appearance

Other

Rodents

Decreased activity; excessive licking and scratching; self-mutilation; may be unusually aggressive; abnormal locomotion (stumbling, falling); writhing; does not make nest; hiding

Piloerection; rough/stained haircoat; abnormal stance or arched back; porphyrin staining (rats)

Rapid, shallow respiration; decreased food/water consumption; tremors

Rabbit

Head pressing; teeth grinding; may become more aggressive; increased vocalizations; excessive licking and scratching; reluctant to locomote

Excessive salivation; hunched posture

Rapid, shallow respiration; decreased food/water consumption

NRC (2003). No single observation is sufficiently reliable to indicate pain; rather several signs, taken in the context of the animal’s situation should be evaluated. The signs of pain may vary with the type of procedure (e.g., orthopedic versus abdominal pain).

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to physical pain (Panksepp et al., 1997; Kross et al., 2011; McMillan, 2014). In the past, the alleviation of pain has been limited to the alleviation of physical pain. As our understanding of emotional pain increases, the quantification and alleviation of distress from emotional pain will also increase. Defining distress in animals has proven to be a difficult task. The ILAR Committee on the Recognition and Alleviation of Distress in Laboratory Animals stated “However, the absence of analogous structures cannot necessarily be taken to mean that they [animals] are incapable of experiencing pain, suffering or distress or any other higher order states of conscious experience” (NRC, 2008). Stress has been defined as “the biological response an animal exhibits in an attempt to cope with threats to its homeostasis” (Stokes, 2000). This response can involve immunologic, metabolic, autonomic, neuroendocrine, and behavioral changes (Moberg, 2000; Moberg and Mench, 2000). The type, pattern, and level of the response depend upon the strength, severity, intensity, and duration of the stressor(s). The aversive state of distress results when an animal is unable to adapt to the stressor(s). By limiting the frequency, strength, severity, intensity, and/or duration of the stressor(s), it may be possible to limit the level of distress in the animal. One method is to reduce the cumulative stress an animal experiences (e.g., allowing recovery or adaptation to a given stressful situation before adding additional stressors) or refining practices and procedures to make the individual stressors less severe or shorter (Brown and Smiler, 2012). Non-pharmacological approaches to minimizing pain and distress should also be considered. Interventions should allow the animal to maintain good hydration and nutritional status and may include moistening food, provision of food and water in a more readily accessible location, supplemental food and liquid which may be more highly palatable, or provision of parenteral fluids and/or nutrition. Making the animal as physically comfortable as possible, by providing additional bedding or nesting material, removing items from the cage which may cause injury in the animal’s debilitated state, and adding or removing social partners as appropriate can also be strategies to improve welfare. Humane endpoints are a refinement of experimental endpoints that result in more severe animal pain and distress. Scientific or experimental endpoints are defined as occurring when the objectives of the study have been reached. Humane endpoints occur at the point at which pain or distress is prevented, terminated, or relieved in an experimental animal, and are used to reduce the severity and/or duration of an animal’s pain and/or distress (Stokes and Marsman, 2014). The scientific and humane endpoints can occur at the same time, in other words, the scientific endpoint occurs prior to the development of pain or distress. Studies that may result in severe or

chronic pain or significant alterations in the animal’s ability to maintain normal physiology, or adequately respond to stressors, should contain descriptions of appropriate humane endpoints or provide science-based justification as to why a particular, accepted humane endpoint cannot be employed (Brown and Smiler, 2012). Veterinary consultation must occur when pain or distress is beyond the level anticipated in the protocol description or when interventional control is not possible (NRC, 2011). Most of the ethical principles guiding humane animal research mention the use of humane endpoints, (Bankowski, 1985; Council for International Organizations of Medical Sciences (CIOMS), 1985; IRAC, 1985) and clinical signs, physiologic parameters, biochemical measurements and other parameters that can potentially serve as early biomarkers for such endpoints (Stokes, 2002; Stokes and Marsman, 2014). Stokes provides an overview and reviews specific situations where endpoints are utilized (Stokes, 2000, 2002). The need, criteria and timing for humane endpoints should be part of pre-study planning which is best done as a research team of scientists, technicians, and veterinarians (CCAC, 1998; OECD, 2000; NCR, 2003). Because endpoints are an important element of IACUC protocol review, it is essential that a protocol contains all appropriate information regarding the criteria for humane endpoints, observation schedules and training of personnel to adequately observe for the agreed upon criteria. Approved pilot studies may be useful for gathering this information if it is not known at the time of protocol submission. In addition, the criteria and other details may need to be modified when unexpected adverse events occur. The IACUC should be notified when this happens and the protocol amended as needed (Brown and Smiler, 2012). Development of criteria for humane endpoints may be general and applicable to any study, such as when an institution adopts standards or policies that cover situations when study specific endpoints have not been determined. These documents often appear as Standard Operating Procedures (SOPs) or guidelines, and may be developed and instituted by the IACUC, the veterinary staff, the institutional administration, or any collaboration of the above. The policies should encompass generic clinical or behavioral conditions that potentially are associated with pain and distress, and should be widely recognized and accepted by the research staff (Brown and Smiler, 2012). General clinical signs which may be monitored include: weight loss, inability to ambulate adequately to obtain food and/or water, and body condition scores (Figure 39.1) (Hickman and Swan, 2010). Anorexia, or lack of appetite, is a significant observation since parenteral supplementation in rodents is not commonly used. In some cases in these species, clinical observations are only obvious when advanced illness,

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BC 1 Rat is emaciated • Segmentation of vertebral column prominent if not visible. • Little or no flesh cover over dorsal pelvis. Pins prominent if not visible. • Segmentation of caudal vertebrae prominent. BC 2 Rat is under conditioned • Segmentation of vertebral column prominent. • Thin flesh cover over dorsal pelvis, little subcutaneous fat. Pins easily palpable. • Thin flesh cover over caudal vertebrae, segmentation palpable with slight pressure. BC 3 Rat is well-conditioned • Segmentation of vertebral column easily palpable. • Moderate subcutaneous fat store over pelvis. Pins easily palpable with slight pressure. • Moderate fat store around tail base, caudal vertebrae may be palpable but not segmented. BC 4 Rat is overconditioned • Segmentation of vertebral column palpable with slight pressure. • Thick subcutaneous fat store over dorsal pelvis. Pins of pelvis palpable with firm pressure. • Thick fat store over tail base, caudal vertebrae not palpable. BC 5 Rat is obese • Segmentation of vertebral column palpable with slight pressure; may be a continuous column. • Thick subcutaneous fat store over dorsal pelvis. Pins of pelvis palpable with firm pressure. • Thick fat store over tail base, caudal vertebrae not palpable.

FIGURE 39.1  Body condition scoring scale for the rat. From Hickman and Swan (2010).

toxicity, or impending death (e.g., a moribund state) are reached (Toth, 2000). Toth described various clinical signs indicative of the moribund state including impaired ambulation which prevents animals from reaching food or water; excessive weight loss and extreme emaciation; lack of physical and mental alertness; difficult labored breathing; and inability to remain upright (Toth, 2000). It is important to note that using the moribund state as an endpoint does not eliminate pain and distress that an animal experiences progressing to that state (Stokes and Marsman, 2014). Ongoing refinements, using objective data-based criteria, that is predictive of impending death avoids animals being found dead and allows for collection of tissues and other biological specimens that may otherwise be wasted (Stokes and Marsman, 2014).

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In some cases, humane endpoints may be developed for a specific type of study (Montgomery, 1987; Olfert, 1996; Dennis, 2000; Olfert and Godson, 2000; Sass, 2000; Workman et  al., 2010) or a specific individual study (Hickman and Swan, 2010; Singh et  al., 2010). The use of scoring systems has been described and usually utilizes multiple observations which, in total, identify the humane endpoint (Lloyd and Wolfensohn, 1998; Morton, 2000, 2003; Medina, 2004). Other systematic approaches have also been described (Morton, 2000; Medina, 2004). There are excellent references available in the Guide that review the establishment and use of humane endpoints to avoid death as an end point (NRC, 2011). To be prepared for situations when unanticipated clinical observations of pain and distress may occur, the institution should have sufficient veterinary oversight in place to advise when alleviation of negative consequences from experimental procedures should be addressed. This may result in animals receiving veterinary medical care, dosing holidays, removal from the experiment, or euthanasia to best align with the scientific objectives of the research. These decisions are ideally made through a collaborative discussion including animal care, scientific and veterinary staff. However, in cases where the animals are significantly compromised, the veterinarian is obligated to take whatever actions are necessary for animal welfare (AWA [Animal Welfare Act], 1990; OECD, 2000). When multiple animals receiving the same treatment exhibit severe adverse effects, consideration should be given to adjusting the endpoint in the remaining animals, stopping the study and restoring the health of the animals, or to euthanizing an entire treatment group if experimental objectives can no longer be achieved. Euthanasia means ‘good death.’ It is important to consider in discussions about laboratory animal welfare because a majority of laboratory animals are euthanized (Carbone, 2014). Euthanasia is often one of the only remaining options to implement humane endpoints – when death best meets the animal’s needs. Carbone has identified three key areas where animal welfare issues are related to euthanasia: “decisions about when and whether to euthanize an animal; potential animal pain and distress in the minutes to hours preceding the euthanasia process; and pain and distress of the euthanasia process itself” (Carbone, 2014). Of the criteria for judging euthanasia methods, Carbone identifies the following as having the greatest impact on animal welfare: ability to induce loss of consciousness and death with a minimum of pain and distress; time required to induce loss of consciousness; reliability; irreversibility; compatibility with species, age, and health status; and ability to maintain equipment in proper working order (Carbone, 2014). Different euthanasia methods score higher or lower than others in each of these criteria so one method may be the best in some circumstances but completely inappropriate

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in others. In the US, the definitive guide on euthanasia is the AVMA Guidelines for the Euthanasia of Animals (AVMA Panel on Euthanasia, 2013) which was the result of two years of work by a group of veterinary and nonveterinary scientists, using a multidisciplinary approach. Methods are listed as acceptable, acceptable with conditions, or unacceptable. Those listed as acceptable with conditions are considered to be equivalent to those listed as acceptable when all criteria for their application have been met (Carbone, 2014). There are three main reasons a decision may be made to euthanize an animal. As previously mentioned, an animal may be euthanized for humane reasons. Animals may also be euthanized as an integral part of the study to collect tissues and samples, or animals may be euthanized as a default because there is no present or planned future use for the animal. It is this third reason which presents the greatest concern for animal welfare. Although philosophical debate exists about whether death is, in fact, a harm to animals and if they have insufficient cognition and self-awareness of their future (Cigman, 1989; Regan, 1989; DeGrazia, 1996; Kaldewaig, 2008; Harman, 2011; Carbone, 2014), reuse or rehoming should be a considered as an alternative to euthanasia where practical and legal (Carbone, 2014). In the situation where euthanasia is being performed for humane reasons, prompt action is necessary. This can be facilitated by clear understanding of the criteria needed to make the decision to terminate the animal’s life and a well-defined delineation of personnel responsibilities. Alternatively, there may be situations where euthanasia is not urgent. In these cases, husbandry considerations such as presence of food, water, bedding, and housing with compatible conspecifics remain important considerations, as well as euthanizing in the home cage of the animal when possible. Care should also be taken to use the minimal amount of restraint necessary to ensure that the animal and operator are both safe and secure. Although the general principles of euthanasia apply internationally, the actual accepted methods of euthanasia may differ in various reports and regulations (Anonymous, 2010; AVMA Panel on Euthanasia, 2013). It is beyond the scope of this chapter to provide a review of the significant body of literature on the subject, so it is necessary for laboratory animal professionals and IACUCs to develop a mechanism to remain current regarding new developments in euthanasia methods and how they impact animal welfare. Research on euthanasia is complex, making it difficult for any single study to be able to compare or rank various techniques scientifically. Studies look at behavioral observations, physiology (neurophysiology and stress endocrinology), and even extrapolate from the experiences of human volunteers. “No single study can compare and rank different techniques which complicates evidence based

euthanasia updates” (Carbone, 2014), but as a procedure which will be performed on a vast majority of laboratory animals, researchers must continuously review the literature for best welfare recommendations. Acclimatization, or the process by which an animal can adjust to a gradual change in their environment, can also be a refinement technique. The gradual acclimatization of animals to procedures, particularly procedures that may be distressful, allows the animal to become familiarized with the procedure. Predictability reduces stress and distress, and has even been shown to decrease perceived pain in both animals and humans (Bolles and Fanselow, 1980; Carlsson et al., 2006). Operant conditioning can be used to train animals in techniques that elicit voluntary cooperation to research procedures. While the training of dogs to sit for exams and nonhuman primates to extend an arm or leg for blood collection which are the most common examples, all species can be trained to cooperate in the laboratory. Hurst and West developed a method of handling mice with either an open hand or in a tunnel structure instead of the common tail-base method of restraint. Studies found that the mice were less anxious in the elevated plus maze test, and they displayed increased voluntary interaction with the handler (Hurst and West, 2010). The ability to express normal patterns of behavior is also a refinement to standard laboratory housing, as well as one of the Five Freedoms. The ability of an animal to display a normal behavioral repertoire frequently requires either the provision of some substrate (e.g., appropriate nesting material) or complex environment (e.g., visual blocks for socially housed nonhuman primates). Understanding what substrates or environmental complexities an animal desires requires an understanding of their natural behavioral repertoire, behavioral motivations, and the complexities of their social structures and interactions, as well as an appreciation for how their natural behavioral repertoire may have been shifted due to domestication.

VI.  EXAMPLES OF CHALLENGING RESEARCH AND OPPORTUNITIES FOR ANIMAL WELFARE OPTIMIZATION A.  Genetically Altered Research Models The use of genetically altered (GA) animals has seen a 10-fold increase in the past decade. This increased use is driven by many factors such as the desire to study gene function and to create new models of disease (Medina and Hawkins, 2014). The use of GA animals offers some unique opportunities to enhance animal welfare and the 3Rs since careful phenotyping can reveal early indicators of disease that may replace the need to develop more

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serious clinical disease (Brown and Murray, 2006). At the same time animal welfare concerns exist with the creation, maintenance and use of GA animals (Brown and Murray, 2006; Wells et al., 2006; NRC, 2011). When lines are created, there are significant numbers of animals that do not have the genes of interest and are used for other purposes or euthanized. To determine if the animal has incorporated the desired genetic modification, the animal must be tissue typed, usually by taking blood, ear, or tail tissue (Hamann et  al., 2010; Ravine and Suthers, 2012; Bonaparte et al., 2013). In addition to expressing the desired phenotype under study, unforeseen traits may be expressed, thus making close monitoring of the colony a critical concern (Rose, 2011). Using a harmonized list of Mouse Welfare Terms (http://mousewelfareterms. org) can help to ensure that animals are monitored and described in a consistent manner. Creation and use of a mouse passport, a document that describes phenotype, husbandry considerations, and special needs, can help reduce the welfare impact of transport and acclimatization to new facilities if they include details of husbandry refinements that can alleviate welfare problems. To maximize the use of created lines, sharing of established lines of interest is encouraged using searchable databases for registering GA lines such as International Mouse Strain Resource (http://www.findmice.org). Creation of GA lines is a time-consuming and expensive process, both in terms of resources and appropriate animal care. Once a line is created, cryopreserving GA lines can minimize the numbers of mice which must be maintained (Medina and Hawkins, 2014). An additional discussion of animal welfare concerns in the use of GA lines can be found in The Ethics of Research Involving Animals by the Nuffield Council on Bioethics (Nuffield Council on Bioethics, 2005).

B.  Cancer Research The use of animals continues to be essential to understand the fundamental mechanisms of malignancy and to discover improved methods to prevent, diagnose, and treat cancer. An excellent summary of standards and recommendations for animal care and use in cancer research will serve as a basis for this section (Workman et al., 2010). The recommendations include study design, statistics, and pilot studies; choice of tumor models (e.g., genetically engineered, orthotopic, and metastatic); therapy (including drugs and radiation); imaging humane endpoints (including tumor burden and site); and publication of best practice. As with other examples of studies with animal welfare challenges, when designing studies with unknown tumors, pilot tumor growth studies using small numbers of animals can establish patterns of local and metastatic growth. They also highlight adverse effects associated with tumor progression, allowing the development of

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observation and treatment strategies and identification of humane endpoints (Brown and Smiler, 2012). For metastatic models, pilot experiments should define the extent and time course of dissemination to internal organs if such information is not already available. Early endpoints reduce non-specific systemic effects and may increase the precision of the results obtained (Workman et al., 2010). Aseptic technique, anesthetics and post-implantation analgesia are recommended for the subcutaneous implantation of tumor material. For injection of cell suspensions, the minimum number of cells in the smallest volume should be used, consistent with the properties of the tumor. Implantation of tumor material into the muscle requires special justification as it is more likely to affect mobility and cause pain. The choice of site for solid tumors will influence the maximum acceptable tumor load and the appropriate humane endpoints. Sites such as the footpad, tail, eye, or bone are likely to be painful or distressing and require special justification and earlier endpoints. Footpad injection, which has been traditionally used to potentiate lymphatic dissemination, is unacceptable without exceptional scientific justification and should then only involve a single paw (Workman et al., 2010). Similarly, tumors that metastasize to sensitive sites need great care (Workman et al., 2010). If brain tumors can be justified, body weight loss is reportedly a sensitive endpoint (Redgate et al., 1991) and MRI or bioluminescent imaging (BLI) techniques can be very useful (van Furth et al., 2003; Ragel et al., 2008; McCann et al., 2009). Tumor size and burden is an important consideration in determining endpoints. When procedures are used to improve tumor take rate, for example, moderate doses of whole-body irradiation, the added potential impact on animal welfare should be considered. The use of biomarkers, imaging, and measurement of circulating tumor cells can facilitate humane endpoints in these studies (Glinskii et al., 2003; Komatsubara et al., 2005). Intravital microscopic imaging uses a wide variety of optical imaging techniques, often incorporating fluorescent or bioluminescent genetic reporters or markers, including nanoparticles (Hoffman, 2005). This type of imaging has particular animal welfare issues because it involves surgery to provide optical clarity and visualization on a microscope stage or using fiberoptic light guides (Weissleder and Pittet, 2008). Surgical implantation of ‘window’ chambers for tumor implantation enables imaging to be performed over days to weeks (Dewhirst et  al., 1987; Lehr et  al., 1993; Brown et  al., 2001; Reyes-Aldasoro et  al., 2008). Here, general anesthesia is only essential for the initial surgery and imaging may be performed with restrained animals. Acclimation to restraint techniques should be used to decrease stress associated with restraint and positive reinforcement training techniques to elicit animal cooperation with the restraint techniques should be

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implemented wherever possible. Strict aseptic technique and good post-operative care and analgesia are essential (Richardson and Flecknell, 2005; Flecknell, 2008).

C.  Neuroscience and Behavioral Research While data collection in behavioral research is observational, the study design frequently requires the denial of some behavior in order to measure the motivation for it, or motivate animals to perform tasks. Many behavioral research protocols, for example, require feed or water restriction in order to motivate animals to perform tasks (Toth and Gardiner, 2000). The deprivation of food and water is considered to be a procedure that may cause pain and distress (APHIS, 1997), and the use of a highly preferred food or the least restriction necessary is encouraged (NRC, 2011). Specific strategies can be employed, however, to decrease the potential distress associated with food and water restriction when it is necessary for the study. Animals should be acclimated to the restriction regime, and feeding or watering protocols should take into account both potential nutritional imbalances and the timing of natural feeding behaviors and incorporate strategies accordingly to accommodate both. A well-defined monitoring program should be in place, to document and have defined endpoints or intervention strategies for criteria such as body weight, food and water intake, general physical appearance and activity, and hydration status (Brown and Smiler, 2012). Neurobehavioral measurements may require invasive devices or long-term tethering or other restrictions in order to gain the required data measurements. If learned behavior is going to be required of the animal, conditioning or training should be carried out prior to the surgery in order to preemptively eliminate animals that will not train from being surgical candidates (NRC, 2003). For protocols or procedures that will require restricted movement or the tolerance of a long-term, indwelling device, animals need to be acclimated to the restraint or device, the restraint should be of the minimum duration necessary to achieve the scientific endpoints, and procedures need to be scientifically justified (Brown and Smiler, 2012). Animal models of neurodegenerative, neural trauma, or psychoses can in and of themselves cause detriment to the animals’ welfare, but may be necessary in order to study the condition. The confirmation of sterile technique if surgeries are to be performed is vital. It is also critical to anticipate and plan for the supportive care of the created condition or surgical complications. Supportive care measures such as easier access to food or water for neurologically impaired animals, the addition of additional or softer bedding for animals where ambulation or activity is affected, and the development of humane endpoints can also improve welfare in these models (Brown and Smiler, 2012).

Neuroscience and behavioral testing are likely the most vulnerable research paradigms to being confounded by many of the refinements previously discussed. Efforts to decrease anxiety, through environmental enrichment or behavioral management techniques like acclimation, could adversely affect results, and yet, the behavioral research paradigms that measure anxiety (e.g., the light–dark box, elevated plus maze, open-field testing, and forced swim test) must, by design, induce anxiety. In general, aversive stimuli are only acceptable when well tolerated by the animal and produce neither maladaptive behaviors (Brown and Smiler, 2012) nor excessive escape–avoidance activity (NRC, 2003). A delicate balance between the welfare of the animals in these types of research and the value of the research question to be answered by the work is often needed.

VII.  SCIENCE OF ANIMAL WELFARE A. Introduction Taking the assumption that using animals in research has a negative impact on their welfare, implementation of replacement and reduction strategies of the 3Rs, such as those described previously, are obvious improvements in animal welfare. This leaves the third R, refinement, as the remaining strategy we can take to improve animal welfare. The aforementioned reduction of pain and distress strategies and the implementation of humane endpoints are clear examples of refinements. For more subtle refinements, scientific evaluation of the procedure is required to determine whether the refinements are actually improving welfare. While examples of research have shown how animal welfare has been improved with certain refinements, there is certainly much more to be learned about the welfare state of the animals in our care and how to accurately assess whether the behavioral management and environmental enrichment programs we are providing are indeed supporting good welfare. Our aim is to help the reader to understand the science of animal welfare, including objective measures that can be used to assess welfare, as well as to stimulate you to think about what you might do to add to the body of information to increase the proper objective evaluation of refinements made that are intended to improve animal welfare. Historically, the improvement of animal welfare via refinement concentrated on the reduction of negative welfare states. Examples would include: the recognition of pain via vigilant monitoring (particularly in prey species) (Roughan and Flecknell, 2006; Langford, 2010; Keating et  al., 2012), the judicious use of anesthesia, analgesia, and supportive care measures, and

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the development of humane endpoints where pain and distress can be prevented, relieved, or minimized. For prey species, simply being moved or handled can be a stressor (Gartner et al., 1980; Burn et al., 2006). As previously described, habituation to handling, particularly when paired with positive reinforcement (Jezierski and Konecka, 1996; Csatadi et  al., 2005; Meijer et  al., 2006) or prefaced by being put into a positive affective state (Cloutier and Newberry, 2009) can decrease the distress associated with common research handling. Operant conditioning and complex environments with ethologically relevant environmental enrichment provide animals with choice and control and are likely to provide a positive welfare state for the animals and not just alleviate the negative. Some enrichment, such as ‘tickling’ in rats (Cloutier and Newberry, 2008; Cloutier et  al., 2010) has been shown to induce a positive effective state. With improved understanding of behavior and refined welfare assessments, it is the opinion of the authors that the provision of good animal welfare will go beyond just the alleviation of the negative, and be the promotion of positive experiences, as suggested by Weary (Weary, 2011).

B.  Modern Animal Welfare Science By the mid-20th century, the disciplines of ethology and neuroscience were starting to garner widespread acceptance in the scientific community, and were contributing to our understanding of the animal experience. The appendix to the Brambell Report proposed various ways to research stress, pain, discomfort, fear, and other aspects of welfare, tying animal behavior to welfare for the first time. The period shortly after World War II was dominated by efforts in animal protection, like the Animal Welfare Act, rather than assessment of animal welfare. The scientific discipline through the 1970s and 1980s consisted of developments in ethology (the study of behavior) and the understanding of behavioral motivations. Preference testing measured how hard an animal would work to obtain a resource, and comparisons of the behavior of free-living and captive animals were used to measure what was ‘good’ for animals: behaviors that they naturally displayed or were motivated to work for. By the mid 1980s, the definition of welfare included behavioral and biological needs that were being measured in these ways, and the reader is referred to further works referenced for detail (Dawkins, 1980; Broom, 1988, 2009; Duncan, 1993, 1996; Fraser, 1999). Animal welfare as a scientific discipline developed around 1990. The discipline, however, stands on the shoulders of aforementioned decades of work in the veterinary, behavioral, nutritional, physiological, and other animal-based sciences. The difference is this animal welfare as an independent discipline is focused

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on the measurement of the animal’s perception in addition to their physical state and behavior. In other words, animal welfare is measurable and therefore a scientific discipline in its own right. Debate continues between those that supported welfare as a purely emotional state (Dawkins, 1980, 1990; Sandoe and Simonsen, 1992), and those who tend to present welfare as a purely physical state of health and normal physiological function. The current position is that welfare involves aspects of all three areas: the contribution of physical state, including health and physiology, the behaviors, and the emotional state of the animal. The mental state of the animal cannot be directly measured like the physiology and behavior, so it has to be inferred through the other two measures. Cortisol is frequently used to measure stress and/or welfare states of animals, but its measurement alone is not analogous to measuring stress, in that both eustress and distress can elevate cortisol, and that cortisol can be decreased in situations of chronic stress (Moberg and Mench, 2000). A constellation of such measures, such as body weight and food consumption; general clinical signs such as hunched posture, and rough hair coat; and behavior, assessed together, frequently is the best overall measure of welfare. As the measurement of objective physiologic parameters and observation of clinical signs are better covered in standard veterinary texts on the various species, we will discuss the observation and quantification of behavior as it relates to the measurement of welfare in more detail.

C.  Assessing Animal Welfare 1.  Behavior Assessment The study and understanding of a species’ natural behavior can predict its reaction to different stimuli and give an indication of the state of the animal in captivity. However, most animals can, and do adapt their behavior to the environment and situation, some differences may just illustrate the adaptability of the animal and be no reflection of welfare, so comparisons between wild or free-living animal behavior, or ‘naturalness,’ has been criticized as a measure of animal welfare (Dawkins, 2008). Domestication does not always change behavior; it may instead change behavior quantitatively not qualitatively. For example, domesticated guinea pigs show higher levels of sociopositive behavior than their wild cavy counterparts (Sachser et al., 2004). An ethogram, or a list of observable behaviors, is an integral tool to measuring behavior. In order to be most useful, the behaviors in an ethogram should be meaningful, both to the paradigm being measured and the species being observed; in other words, as non-subjective as possible. Clear, well-described, non-overlapping, unambiguous definitions of the behaviors listed in the

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ethogram that have been discussed and agreed upon by all observers ensure better behavioral observation and assessment accuracy. Continuous recording of behaviors allows for the documentation of the occurrence and duration of every behavior, and true calculations of frequency, duration, latency, sequence, as well as true time budgets. It’s also almost prohibitively time consuming, unless you’re a behaviorist, a grad student, or a naturalist. In most laboratory animal settings, interval scanning is the most useful and common method of recording behavior. As a general rule, frequent behaviors can have long intervals between observations; quick or infrequent behaviors need to have shorter intervals. For rare behaviors, a one– zero or yes–no technique of recording during focal scan sessions, or watching behaviors for a defined period of time and documenting whether or not the behavior occurred in that interval, may be most useful. For further instruction on data collection methods, the reader is referred to works by Martin and Bateson or Altmann for further detail (Altmann, 1974; Martin and Bateson, 2004) and to Beaver and Bayne (Beaver and Bayne, 2014) for an excellent overview of further resources. In order to use behavior to indicate the state of the animal, however, the ethogram must contain not just the behaviors of the animal but also the temporal, environmental and social context in which the behavior is occurring (Appleby et  al., 2011). The first question you can answer with behavioral observation is “what do they do.” Because you can never actually know the “why” a behavior is done, the context can provide the all-important clues. Any one set of behavioral responses rarely indicates reduced welfare, though changes in the frequencies of individual and social behaviors or the suppression of behaviors can suggest that there are welfare problems. For example, play or exploratory behavior decreases with decreased welfare (Arnsten et  al., 1985; Krachun et al., 2010) and may thus be “luxuries” that are dispensed with during periods of stress. Behavior that occurs out of context may also indicate disturbance. A thorough understanding of the natural behavioral repertoire of the species being evaluated is vital to putting welfare states into context. 2.  Avoidance and Anxiety Over the years, a variety of tests have been developed to equate or quantify the amount of avoidance and relate it to the animals’ level of anxiety based on fearful reactions or situational avoidance. Examples of these assessments include the light/dark box test, where a rodent is allowed choice between a brightly lit section and a darkened section of an environment. Based on a rodent’s instinct as a prey species to avoid open, lit, exposed places, an animal that spends a greater amount of time in that area is interpreted to be ‘less anxious.’ Likewise,

the open-field or elevated plus maze are designed to assess the animals’ willingness to enter an open area as a measure of anxiety. The issue with these types of tests is that they are measuring ‘state’ anxiety, or anxiety induced by a particular situation, in a particular time and place. Welfare, however, requires more a measure of ‘trait’ anxiety, an assessment of the animals’ general being over time, which does not vary from moment to moment but reflects the mental state, as opposed to the physical state that the animal is in. The usefulness, shortcomings, and suggestions for refinements of these types of tests are more thoroughly covered in works by Bourin and Rodgers (Rodgers, 1997; Rodgers et al., 1997; Bourin et al., 2007), and the reader is referenced to those sources for further detail. 3.  Preference Testing Observing an animal’s preference can give us an indication of what might improve an animal’s welfare. This testing paradigm can be set up in a few slightly different ways. One is to set up a complex environment with multiple options and measure where or with what the animal spends its time. A simpler setup is to allow the animal to choose between two options, with all other variables held constant. Interpretation of preference testing is fraught with complexity, however, as it can be influenced by the state of the animal (e.g., swine chose bedded or bare floors based on room temperature [Fraser, 1985]) or previous experience (e.g., rodents made better nests with materials with which they had prior exposure [Van Loo and Baumans, 2004]). It must also be understood that animals may not always choose what is best for them, so choice tests alone may not be adequate in determining what is best for the animal and its welfare. As an example, rabbits have evolved to graze on low-caloric-density forage material but when provided with high-calorie or free-choice high-quality food, they will eat to obesity. In addition, choice testing does not infer any sort of importance to the resource or environment chosen. Using two choices only tells which of the two choices is preferred, even if neither is particularly good for the animal. Another consideration is that just because an animal spends a long period of time performing a particular behavior or interacting with a particular enrichment, does not necessarily mean that item holds high value or is very important for welfare. For instance, a diabetic will likely spend a large portion of their day doing things other than insulin injection, however they would work very hard to get to the insulin should its access be restricted, due to its importance to their welfare. 4.  Consumer Demand In order to better assess the strength of a preference, testing paradigms that infer a cost to the access of the

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enrichment can quantify the preference. Some sort of barrier, such as a weighted door (Ågren et  al., 1989) or longer distance (Guerra and Ades, 2002) can be placed between the animal and the desired resource. The weight can be sequentially increased to determine the ‘cost’ that the animal will endure for access to the resource. Alternatively, the preference for a resource can be compared to another known desired or aversive resource, giving each a relative value. 5.  Cognitive Bias Assessment Cognitive bias measures of animals’ emotional or ‘affective’ state by measuring the way that an animal perceives incoming information and responds to it. This concept for animals is based on work that shows that people in negative emotional states will show increased attention to negative stimuli and are more likely to make pessimistic interpretations of ambiguous situations. These paradigms have been applied to animals in attempts to assess their cognitive bias. Harding trained rats to press a lever to either receive a food reward when one tone was heard or avoid a negative (white noise) stimulus when a different tone was heard. When the rats were placed in an environment of chronic stress (constant unpredictable disturbances in their housing conditions) the rats responded to ambiguous tones as though they expected the negative stimulus more often than rats in a non-stressful environment (Harding et al., 2004). Conversely, a different researcher found that rats raised in enriched environments were more likely to expect a positive reward (Brydges et al., 2011). While this kind of work is still in its relative infancy, it is possible that with further refinements of this type of testing, it might be possible to assess the emotional state of our animals more directly or to make general statements on the types of environmental enrichments or behavioral management strategies that positively or negatively affect the laboratory animal species. 6.  Coming Together While preference, consumer demand, and cognitive bias testing may seem to be advanced techniques for the everyday application in the assessment of laboratory animal welfare, they have the potential to forward our efforts to support animal welfare in the laboratory environment. Their application displays an opportunity for collaborative work on applying science to refine procedures in the laboratory for the benefit the animals. While the authors acknowledge that there are real challenges of resources and practicality, we hope that we have illuminated areas where these disciplines can bring together the concept of animal welfare with the science of animal welfare to develop practical and scientifically sound methods to measure the welfare of animals under our care.

VIII.  CONCLUSION AND SUMMARY The science, study and philosophical concept of animal welfare have been the culmination of an ethical journey. Starting with philosophy and based on our relationship with animals, progressing through a series of ethical questions and obligations that directed the development of laws, regulations, and guidelines to ensure our moral obligations in looking after animal welfare, the current field in which we now scientifically assess and support strategies to measure an animals’ welfare has come to be. Whenever possible, subjective data should be used to develop assessment parameters, husbandry and enrichment conditions, and to determine humane endpoints. There is a moral obligation to ensure good welfare for all animals, including those in the laboratory setting. The challenge is to meld the philosophy and the science into a practical application that positively affects the animal’s welfare. While we acknowledge that the sheer numbers of animals (in particular rodents), economic constraints and resource availability are real challenges to our ability to develop and implement welfare supportive programs, we would purport that these are not insurmountable obstacles. The provision of good welfare is not a static program: as the science in this field continues to progress, our understanding and abilities will grow, and occasional review of the entire program, with adjustments as indicated, will be essential.

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