Session 2
Assessment of Animal Housing Needs in the Research Setting—Peer-reviewed Literature Approach



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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop Session 2 Assessment of Animal Housing Needs in the Research Setting—Peer-reviewed Literature Approach

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop Disruption of Laboratory Experiments Due to Leaching of Bisphenol A from Polycarbonate Cages and Bottles and Uncontrolled Variability in Components of Animal Feed Frederick S. vom Saal, Catherine A. Richter, Rachel R. Ruhlen, Susan C. Nagel, and Wade V. Welshons Mammalian embryonic development is epigenetic in that hormonal signals not only control the timing of gene expression but also set the activity of genes and thus the functioning of organs and homeostatic systems for the remainder of life. Variation in endogenous hormones (e.g., estradiol and testosterone), which regulate the development of organs (vom Saal 1989), or disruption of the activity of these hormones during development by chemicals can lead to permanent changes in organ structure and function. Adult exposure to endocrine-disrupting chemicals can lead to transient changes in organ function that can disrupt experiments. Polycarbonate cages and water bottles are manufactured by polymerizing the chemical bisphenol A, which was initially considered for use as an estrogenic drug before being used to manufacture polycarbonate in the 1950s (Dodds and Lawson 1936). More than 50 published studies have shown effects of developmental as well as adult exposure to bisphenol A on a wide variety of traits in mollusks, insects, fish, frogs, rats, and mice. Polycarbonate cages have been commonly used to house rodents and aquatic animals in laboratory experiments. What was not appreciated by scientists using these cages until recently is that after repeated washings the rate of leaching of bisphenol A increases dramatically and can reach levels that can alter traits in animals. Howdeshell and coworkers reported that a small but detectable amount of bisphenol A leached out of new polycarbonate animal cages into water at room temperature, and the rate of leaching was more than 1000 times greater (> 300 µg/L) in old, visibly

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop worn (scratched and discolored) polycarbonate cages (Howdeshell and others 2003). Hunt and colleagues reported that an adverse effect of exposure to very low doses of bisphenol A leaching from polycarbonate animal cages and water bottles is profound disruption of chromosomes during meiosis in oocytes in female mice. Specifically, there was a dramatic increase in the incidence of abnormal alignment of chromosomes during the first meiotic division in oocytes, which was caused by the leaching of bisphenol A from the polycarbonate cages washed with a harsh detergent (Hunt and others 2003; Koehler and others 2003). Abnormal alignment of chromosomes results in aneuploidy, or abnormal numbers of chromosomes in oocytes, which can lead to abnormal development such as occurs in Down’s syndrome. These authors thus refer to bisphenol A as a “potent meiotic aneugen.” Aneuploidy is thought to be a major cause of embryonic mortality in humans. Hunt and coworkers reported that severe oocyte chromosome abnormalities increased in peripubertal female mice in the following proportions: from a baseline frequency of 1.8% in control animals (not housed in damaged cages) to 20% due to housing females in damaged polycarbonate cages; 30% due to the use of damaged polycarbonate water bottles; and 41% due to combined use of both damaged cages and water bottles. In a subsequent experiment, the researchers intentionally accelerated the normal aging process associated with repeated washing of polycarbonate cages and water bottles by washing them different numbers of times in a harsh detergent. The polycarbonate water bottles were found by gas chromatography/mass spectrometry analysis to release between 100 (mild damage) and 260 µg/liter (severe damage) of free bisphenol A into water placed into the bottles, resulting in daily exposure of the female mice ranging between 15 and 72 µg/kg. When peripubertal female mice housed in undamaged new cages were fed bisphenol A once daily at the very low doses of 20, 40, and 100 µg/kg to simulate exposure within the range released by the polycarbonate, there was a significant dose-related increase in the incidence of chromosomal damage beginning even at the lowest dose. Based on studies in which bisphenol A was found to have limited binding to the plasma proteins that serve as a barrier to the movement of estrogen from blood into tissues (Nagel and others 1997), we had predicted that doses of bisphenol A as low as 20 µg/kg/day would disrupt development in mice. This dose is below the predicted “safe” or reference dose for human exposure of 50 µg/kg/day, which was calculated based on old studies that examined only very high doses of bisphenol A, when the lowest dose administered (50 mg/kg/day) had resulted in adverse effects (IRIS 2002). Taken together, the large number of independent findings concerning adverse effects of very low doses of bisphenol A suggest that the use of

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop polycarbonate to manufacture animal cages and water bottles can alter the results of laboratory animal research. In fact, due to greater resistance to heat and alkaline detergents, many facilities have switched from polycarbonate to polysulfone animal cages. The ether bond in the polysulfone co-polymer is more resistant to heat and alkaline detergents relative to the ester bond in polycarbonate. We have always used polypropylene cages and glass water bottles, because these cages do not leach biologically active amounts of estrogenic chemicals (Howdeshell and others 2003). Some components of feed used in laboratory experiments (e.g., phytoestrogens and mycotoxins) have hormonal activity that can interfere with experiments involving outcomes that are sensitive to these hormone-mimicking chemicals. There is wide variation in phytoestrogen content in different types of commercial rodent feed. Both the amount of phytoestrogens and metabolizable energy in different feeds were sources of phenotypic variation (specifically body weight, uterine growth, and age at vaginal opening) in prepubertal CD-1 female mice (Thigpen and others 2003). Thigpen and colleagues selected one soy-based commercial feed (Purina 5002) and examined the consequences of using five batches of this diet with different mill dates. They first measured the amounts of the soy phytoestrogens genistein and daidzein in the five different batches, which ranged from 159 to 431 µg/g. It is well known that one of the effects of the estrogenic drug diethylstilbestrol (DES) is to accelerate vaginal opening, and although a 4-ppb dose of DES accelerated vaginal opening in female CD-1 mice fed the batch of feed with 159 µg/g of genistein and daidzein, there was no accelerating effect of DES in females being fed the batch of feed with 431 µg/g of genistein and daidzein. Administration of even this potent estrogenic drug could not further accelerate this process (Thigpen and others 2003). A similar finding had previously been reported for prepubertal female rats fed different batches of feed produced by another feed manufacturer (Boettger-Tong and others 1998). Together, these studies reveal that the issue of variation in phytoestrogen content in batches of feeds is one that is a general problem and not just restricted to Purina 5002 feed, because any closed-formula diet can contain variable amounts of phytoestrogens due to the source as well as the amount of soy isoflavones. It is important to emphasize that the isoflavones genistein and daidzein are only two of many naturally occurring compounds that could be sources of estrogenic activity in feed, and even casein-based feeds show variation in total estrogenic activity (Thigpen and others 2003). For example, we have found significant variation in estrogenic activities in different batches of casein-based feeds, and none of these estrogens were genistein or daidzein (unpublished observation). Simply screening for these two isoflavones will thus not guarantee the lack of variability in other potential endocrine-disrupting contaminants in a feed. Feed

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop manufacturers need to develop new approaches to reduce variability in endocrine-disrupting activity in different batches of feeds to attain levels that will not disrupt research results. We have also found that there are components of some batches of commercial mouse feeds, such as soy-based Purina 5002 certified diet, that, relative to other feeds (Purina 5008 soy-based pregnancy diet), dramatically increase endogenous estradiol in CD-1 mouse fetuses (unpublished observation). This increase is associated during later life in both males and females fed Purina 5002 throughout life with an increase in postnatal rate of growth, accelerated onset of puberty in females, and an increase in the amount of abdominal fat. Male mice fed Purina 5002 diet also evidenced differences in reproductive organs, such as an increase in prostate size and a decrease in daily sperm production, relative to males whose mothers were fed Purina 5008 during pregnancy and lactation, followed by soy-based Purina 5001 after weaning. An interesting additional finding is that oral administration of DES to pregnant mice of a low dose (0.1 µg/kg/day) and a high dose (50 µg/kg/day) resulted in a dose-related decrease in daily sperm production in adult male offspring on the Purina 5008/5001 regimen, whereas males from the Purina 5002 regimen showed no effect of DES, even at the high dose (unpublished observation). Most investigators are aware of the marked effects that different types of feed can have on the phenotype of their animals. Of great concern, however, is that variability between batches of some feeds is a potential source of uncontrolled variability in research results. Another variable of concern in laboratory studies is water quality. Copper pipes are used inside the building that houses our mice, and water is provided to the mice in glass bottles. We purify the water by ion exchange and a series of carbon filters. These measures are important to remove contaminants such as phthalates, which can enter water from PVC water pipes and herbicides. These potential contaminants, as well as other solutes that can affect an animal’s physiology, are an issue particularly in agricultural areas. ACKNOWLEDGMENTS Support was provided during the preparation of this paper from grants to F.vS. from the National Institute of Environmental Health Sciences (NIEHS) (ES11283), C.A.R. from NIEHS (ES-11549), S.C.N. from the National Institute of Diabetes and Digestive and Kidney Diseases (DK60567), and W.V.W. from the National Cancer Institute (CA50354) and the University of Missouri (VMFC0018).

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop REFERENCES Boettger-Tong, H., Murthy, L., Chiappetta, C., Kirkland, J.L., Goodwin, B., Adlercreutz, H., Stancel, G.M., Makela, S. 1998. A case of a laboratory animal feed with high estrogenic activity and its impact on in vivo responses to exogenously administered estrogens. Environ Health Perspect 106:369-373. Dodds, E.C., and Lawson, W. 1936. Synthetic oestrogenic agents without the phenanthrene nucleus. Nature 137:996. Howdeshell, K.L., Peterman, P.H., Judy, B.M., Taylor, J.A., Orazio, C.E., Ruhlen, R.L., vom Saal, F.S., Welshons, W.V. 2003. Bisphenol A is released from used polycarbonate animal cages into water at room temperature. Environ Health Perspect 111:1180-1187. Hunt, P.A., Koehler, K.E., Susiarjo, M., Hodges, C.A., Hagan, A., Voigt, R.C., Thomas, S., Thomas, B.F., and Hassold, T.J. 2003. Bisphenol A causes meiotic aneuploidy in the female mouse. Current Biol 13:546-553. IRIS, Bisphenol, A. (CASRN 80-05-7), US-EPA Integrated Risk Information System (IRIS). Substance file. http://www.epa.gov/iris/subst/0356.htm. Accessed January 2002. Koehler, K.E., Voigt, R.C., Thomas, S., Lamb, B., Urban, C., Hassold, T.J., Hunt, P.A. 2003. When disaster strikes: Rethinking caging materials. Lab Anim 32:32-35. Nagel, S.C., vom Saal, F.S., Thayer, K.A., Dhar, M.G., Boechler, M., Welshons, W.V. 1997. Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A and octylphenol. Environ Health Perspect 105:70-76. Thigpen, J.E., Haseman, J.K., Saunders, H.E., Setchell, K.D.R., Grant, M.G., Forsythe, D.B. 2003. Dietary phytoestrogens accelerate the time of vaginal opening in immature CD-1 mice. Comp Med 53:477-485. vom Saal, F.S. 1989. Sexual differentiation in litter-bearing mammals: influence of sex of adjacent fetuses in utero. J Anim Sci 67:1824-1840.

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop Assessment of Animal Housing Needs in the Research Setting Using a Peer-reviewed Literature Approach: Dogs and Cats Graham Moore INTRODUCTION A previous presentation (De Leeuw 2004) provided an outline of the Council of Europe (CoE) and the background of its process to revise Appendix A (guidelines for accommodation and care) of Convention ETS 123 (European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes) of 1986. In this presentation, I will describe how the Council of Europe’s requirements have been applied to the preparation of draft guidance on the accommodation and care of dogs and cats. COUNCIL OF EUROPE PROCESS FOR THE REVISION OF APPENDIX A In 1997, the CoE adopted a resolution on accommodation and care of vertebrate animals used for experimental and other scientific purposes (Council of Europe 1997). It had been generally agreed by the CoE that the existing “Guidelines for accommodation and care of animals” presented in Appendix A of Convention ETS 123 had proved very useful and had been applied widely within Europe. However, it was also acknowledged that scientific knowledge and experience had progressed since 1986 and

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop the entry into force of the Convention, such that a review of the guidelines was necessary. The resolution stipulated that the new proposals should be divided into General and Species-Specific recommendations and also indicated key areas to which attention should be given. It further identified the way in which guidance should be prepared. Expert Groups, with representation from nominees of observer nongovernmental organizations of the Council of Europe but not of the national authorities, were to be set up to prepare proposals on the main groups of species covered by the Convention. These proposals would then be submitted to a Working Party for comment, amendment, and endorsement. Membership of the Working Party comprised representatives of the national authorities of the CoE member states, together with observers from a wide range of concerned nongovernmental organizations. A Drafting Group assisted in the work of the Expert Groups and of the Working Party. Once the Working Party agreed on all proposals from the Expert Groups, they would be received by a CoE Multilateral Consultation for any further discussion and approval, before being submitted to the Committee of Ministers for final approval. It is important to note that the status of Appendix A is “guidance” and the guidelines are not mandatory. However, it was generally considered by the Expert Groups that these should be regarded as minimum requirements. CURRENT STATUS OF THE REVISION Initially, only four Expert Groups had been established, on (1) Rodents and Rabbits, (2) Dogs and Cats, (3) Nonhuman Primates, and (4) Pigs and Minipigs. These four groups were given the task of preparing Species-Specific proposals, with the General Part of the new proposals, including provisions common to all species covered, being drafted with input from all four groups. The Working Party later decided to add additional species covered by the Convention to the list of those already to be covered by the revision; thus, the number of groups grew from four to eight. Furthermore, the Pigs and Minipigs group was expanded to cover all farm animal species, and ferrets were added to the Dogs and Cats group. Currently, the General Section and Species-Specific proposals for Rodents, Rabbits, Dogs, Cats, and Ferrets have been finalized by the Working Party. Those for other species have not yet been finalized, although those for Nonhuman Primates, Birds, and Amphibians are at a very advanced stage.

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop MEMBERSHIP OF EXPERT GROUP ON DOGS AND CATS AND MODUS OPERANDI As with all of the Expert Groups, there was a broad-based representation drawn from the observer nongovernmental organizations of the CoE. The membership of the Expert Group on Dogs and Cats comprised one representative from each of the following: the Eurogroup for Animal Welfare, the European Federation of Pharmaceutical Industries and Associations (EFPIA), the Federation of Laboratory Animal Breeders Associations (FELABA), the Federation of Veterinarians in Europe (FVE), and the International Society for Applied Ethology (ISAE). Meetings were coordinated and chaired by a representative of the Federation of European Laboratory Animal Science Associations (FELASA). A second ISAE representative was subsequently added because more input on cat ethology was deemed necessary. This membership was thought to provide a broad spread of expertise and opinion, which would result in the formulation of an expert view on minimum standards for these species. It was decided at an early stage that the group would work primarily on the basis of face-to-face meetings (held in London or Brussels), with e-mail communication between meetings. Additional input would be sought as necessary from within represented organizations or from other experts. The Coordinator of the Group, together with one or more members, attended all meetings of the Working Party in Strasbourg to present the Group’s proposals, discuss their content and answer questions, and refer matters back to the Group as appropriate. BASIS FOR DOG AND CAT RECOMMENDATIONS The CoE stipulated the provision of proposals for a General Section and for Species-Specific Sections (called Part A). It also requested a supporting explanatory and referenced text (Part B) for each of the sections. Groups were directed to pay special attention to enrichment of the environment, particularly in relation to social interactions, activity-related use of the space, and provision of appropriate stimuli and materials. Proposals were to be based on science-based information when it was available, and otherwise on practical experience and good or “best” practice. Where appropriate, Expert Groups were given the task of identifying areas in which additional research would be desirable. The Expert Group on Dogs and Cats considered these areas and paid attention to existing guidance documents, such as the current Appendix A, UK Home Office guidance (1989, 1995), and the ILAR Guide for the Care and Use of Laboratory Animals (NRC 1996). Some significant variations were found in these recommendations, particularly those for space

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop requirements and for some environmental parameters. It was therefore decided that where robust science-based information was not available, proposals would be based primarily on an examination of the animals’ physiological and ethological needs, taking into account the current views on good/best practice and the inevitable constraints of a research environment. Species-Specific Section—Subject Headings The species-specific sections for dogs and cats covered the following: Preamble Introduction The environment and its control Ventilation Temperature Humidity Lighting Noise Alarm systems Health Housing and enrichment Housing Early socialization with conspecifics and humans Enrichment Animal enclosures Outside runs (dogs only) Dimensions Flooring Feeding Watering Substrate, litter, bedding, and nesting material Cleaning Handling (cats only) Humane killing Records Identification For subject headings that were common to all species-specific sections, the Group decided no proposals were necessary other than those already contained in the General Section. These headings are shown in italics in the list above. Additional headings in the General Section were Definitions; Physical Facilities; Education and Training; Care (incorporating

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop other adolescent males before attempting to gain entry to a new troop. It has been estimated that at least one-third of the males do not survive this change (Berard 1989; Dittus 1979). Social interactions within the troop are based in part on dominance, wherein some animals have priority of access to incentives. Two features of the dominance hierarchy are redirection of aggression and recruitment of agonistic aid. Threats and aggression can cascade down the hierarchy, with low-ranking animals receiving more “bystander” aggression than higher-ranking animals. Animals threatened or attacked by others frequently attempt to “recruit” others to their defense by screaming at the attacker or by rapidly alternating their gaze between friends and foe (Gouzoules and others 1998). From an ecological perspective, rhesus monkeys live in a wide variety of different habitats. They have been observed in remote forests, agricultural regions, and many urban areas (Teas and others 1980). Unlike some other primate species, rhesus monkeys appear to thrive in areas of deforestation, and they have been termed “weed” macaques because of this versatility (Richard and others 1989). Rhesus monkeys spend nearly 50% of their time moving to food sites and foraging for food (Goldstein and Richard 1989; Teas and others 1980). They subsist on the fruits and shoots of well over 100 species of plants, and they occasionally supplement their food with eggs, insects, and small animals. This widely varied diet may contribute to their ability to flourish in very different environments. In addition to these general features, individual monkeys differ with respect to reactive and impulsive temperaments (Suomi 2000). Approximately 20% of the rhesus monkey population appears to be quite reactive to novel events. This reactivity is manifested by heightened and prolonged activation of the hypothalamic-pituitary-adrenal (HPA) axis and by behavioral responses including fear and withdrawal. In contrast, the remaining members of the population show only mild activation of the HPA axis and only brief responses of wariness or caution in response to novel stimuli (Suomi 1991). In nature, a high reactive temperament is associated with heightened emotional responses to maternal disruption (Berman and others 1994), and in males, with later emigration from the natal troop (Suomi and others 1992). Individual rhesus monkeys also vary with respect to impulsivity. Some male monkeys (~5%) are highly aggressive and do not appear to moderate their aggressiveness with appeasement behavior. This trait is also associated with the presence of low levels of serotonin in the brain as measured by the metabolite 5-hydroxyindoleacetic acid (5-HIAA). In nature, low levels of CSF 5-HIAA in male rhesus monkeys are associated with extreme aggression, earlier emigration from their natal troop com-

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop pared with other adolescent males (Mehlman and others 1995), and greatly increased risk of mortality (Higley and others 1996). Knowledge of how monkeys behave in nature can inform how we house and enrich the environments of captive primates. From a social housing perspective, free-ranging monkeys live in complex social groups. Although it is not really possible to duplicate troop life in the laboratory, some form of social housing may be crucial for maintaining well-being. However, there are distinctions that must be considered along with potential costs and benefits. For example, males and females may be affected differently by the presence or absence of partners. Females live in large kin groups throughout their lives, whereas males emigrate and occasionally become solitary. Furthermore, some groupings or pairings will not necessarily be amicable. In nature, rhesus monkey troops are “closed societies,” and troop members react aggressively to strangers. Furthermore, social housing may not be optimal or even desirable for certain individuals. In nature, male monkeys with low central nervous system serotonin levels show extreme aggression and are ultimately forced out of their natal troop. In addition to their complex social environment, rhesus monkeys exist in habitats where they must forage for food and find suitable resting/ sleeping sites. Movement and exploration are therefore crucial for survival. Exposure to novel stimuli or foraging devices (i.e., environmental enrichment) would appear to be essential for housing monkeys in captivity; however, this view must be adjusted to account for differences in temperament. Reactive monkeys may show heightened stress responses to enrichment. LABORATORY FINDINGS Both social housing and environmental enrichment are considered important regulatory requirements for promoting psychological well-being in captive primates. The logic of this view for rhesus monkeys is derived in part from their life history. However, there are also laboratory studies in which the effectiveness of social housing and environmental enrichment have been examined, and the emerging picture from this work suggests that there are both benefits and costs, depending on the research objectives. Social Housing Scientific evidence suggests that there are a number of potential benefits to social housing, the most obvious of which is the ability to groom and affiliate with other monkeys. Companions may also serve as a buffer

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop to stressful events (Winslow and others 2003). Other potential benefits of companionship include increased disease resistance (Shively and others 1989) and improved immune response (Lilly and others 1999; Schapiro and others 2000). However, these relationships are more complex than these descriptions imply. For example, in Shively and colleagues’ (1989) study, socially housed female macaques showed less coronary artery disease than singly housed monkeys, but this difference was evident only for dominant socially housed females. Another important benefit of social housing is that there is greater correspondence to the human situation. In a recent study, intracerebroventricular infusions of corticotropin-releasing factor caused depressive-like symptoms, but only in socially housed monkeys (Strome and others 2002). Social housing is not without cost, and one of the most significant costs is the development of aggression and competition. Rhesus monkeys do not always coexist amicably. Even in stable social groups, aggression can escalate and lead to violent outcomes (Hird and others 1975). From a research perspective, there may be circumstances in which social housing increases experimental variability. For example, moving monkeys from individual cage housing to social housing led to an increase in the availability of dopamine D2 receptors in dominant, but not subordinate, monkeys (Morgan and others 2002). Social housing may also minimize the effects of certain manipulations (e.g., coronary artery disease) or introduce other variables that may mask the effects of manipulations. For example, removal from companions for testing may induce stress reactions as a consequence of separation (see Lyons and others 1998 for squirrel monkeys). Environmental Enrichment There are many different methods to enrich the environment of captive primates, ranging from the provision of objects, foraging devices, or videotapes to the redesign of the cage environment (see various commercial cage vendors). As with social housing, the emerging picture suggests that there are both costs and benefits. The most obvious benefit is that environmental enrichment promotes species-typical behavior in the form of exploration. Thus, most monkeys spend some time using foraging devices (Lutz and Novak 1995) and manipulating objects (Novak and others 1993). Some monkeys also appear to watch videotapes (Platt and Novak 1997). The benefits of enrichment may extend beyond mere exploration to include a reduction in stress levels (Boinski and others 1999; Byrne and Suomi 1991) and a decrease in stereotypic behavior (Bayne and others 1991). However, enrichment has not been shown to reduce severe

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop forms of abnormal behavior, such as self-injurious behavior (Novak and others 1998). Enrichment efforts also incur costs to the animal and to the research enterprise. Rotation of enrichment devices through the colony can increase the risk of disease transmission (Bayne and others 1993). The provision of foraging devices can lead to increased body weight (Brent 1995). Plastic and rubber objects are typically gnawed and chewed, and in some cases can result in injury from foreign material in the intestine (Hahn and others 2000). Developing optimal housing strategies for rhesus monkeys requires balancing two different but interconnected needs: promoting primate well-being and achieving research objectives. A strong case can be made that both social housing and environmental enrichment foster well-being. However, there are also risks to housing monkeys in social groups and to enriching the environment. Furthermore, the costs and benefits are often relative. What may be a benefit under some conditions can become a cost under other conditions. A thorough review of life history patterns and a careful cost-benefit analysis may provide guidance in designing housing strategies for particular research programs. REFERENCES Bayne, K.A., Dexter, S.L., Hurst, J.K., Strange, G.M., Hill, E.E. 1993. Kong toys for laboratory primates: Are they really an enrichment or just fomites? Lab Anim Sci 43:78-85. Bayne, K., Mainzer, H., Dexter, S., Campbell, G., Yamada, F., Suomi, S.J. 1991. The reduction of abnormal behaviors in individually housed rhesus monkeys (Macaca mulatta) with a foraging/grooming board. Am J Primatol 23:23-35. Berard, J.D. 1989. Life histories of male Cayo Santiago macaques. Puerto Rico Health Sci J 8:61-64. Berman, C.M., Rasmussen, K.L.R., Suomi, S.J. 1994. Responses of free-ranging rhesus monkeys to a natural form of social separation: I. Parallels with mother-infant separation in captivity. Child Dev 65:1028-1041. Boinski, S., Swing, S.P., Gross, T.S., Davis, J.K. 1999. Environmental enrichment of brown capuchins (Cebus apella): Behavioral and plasma and fecal cortisol measures of effectiveness. Am J Primatol 48:49-68. Brent, L. 1995. Feeding enrichment and body weight in captive chimpanzees. J Med Primatol 24:12-16. Byrne, G.D., and Suomi, S.J. 1991. Effects of woodchips and buried food on behavior patterns and psychological well-being of captive rhesus monkeys. Am J Primatol 23:141-151. Dittus, W.P.J. 1979. The evolution of behavior regulating density and age-specific sex ratios in a primate population. Behaviour 69:265-302. Goldstein, S.J., and Richard, A.F. 1989. Ecology of rhesus macaques (Macaca mulatta) in Northwest Pakistan. Int J Primatol 10:531-567. Gouzoules, H., Gouzoules, S., Tomaszycki, M. 1998. Agonistic screams and the classification of dominance relationships: Are monkeys fuzzy logicians? Anim Behav 55:51-60.

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop Hahn, N.E., Lau, D., Eckert, K., Markowitz, H. 2000. Environmental enrichment-related injury in a macaque (Macaca fascicularis): Intestinal linear foreign body. Comp Med 50:556-558. Higley, J.D., Mehlman, P.T., Higley, S.B., Ferrnald, B., Vickers, J., Lindell, S.G., Taub, D.M., Suomi, S.J., Linnoila, M. 1996. Excessive mortality in young free-ranging male nonhuman primates with low cerebrospinal fluid 5-hydroxyindoleacetic acid concentrations. Arch Gen Psychiatry 53:537-543. Hird, D.W., Henrickson, R.V., Hendrickx, A.G. 1975. Infant mortality in Macaca mulatta: Neonatal and postnatal mortality at the California Primate Research Center, 1968-1972. A retrospective study. J Med Primatol 4:4-22. Lilly, A.A., Mehlman, P.T., Higley, J.D. 1999. Trait-like immunological and hematological measures in female rhesus across varied environmental conditions. Am J Primatol 48:197-223. Lindburg, D.G. 1971. The rhesus monkey in North India: An ecological and behavioral study. In: Rosenblum, L.A., ed. Primate Behavior Developments in Field and Laboratory Research. New York: Academic Press. p. 1-106. Lutz, C.K., and Novak, M.A. 1995. The use of foraging racks and shavings as enrichment tools for social groups of rhesus monkeys (Macaca mulatta). Zoo Biol 14:463-474. Lyons, D.M., Kim, S., Schatzberg, A.F., Levine, S. 1998. Postnatal foraging demands alter adrenocortical activity and psychosocial development. Dev Psychobiol 32:285-291. Mehlman, P.T., Higley, J.D., Faucher, I., Lilly, A.A., Taub, D.M., Vickers, J., Suomi, S.J., Linnoila, M. 1995. Correlation of CSF 5-HIAA concentration with sociality and the timing of emigration in free-ranging primates. Am J Psychiatry 152:907-913. Morgan, D., Grant, K.A., Gage, H.D., Mach, R.H., Kaplan, J.R., Prioleau, O., Nader, S.H., Buchheimer, N., Ehrenkaufer, R.L., Nader, M.A. 2002. Social dominance in monkeys: Dopamine D2 receptors and cocaine self-administration. Nature Neurosci 5:169-174. Novak, M.A., Kinsey, J.H., Jorgensen, M.J., Hazen, T.J. 1998. The effects of puzzle feeders on pathological behavior in individually housed rhesus monkeys. Am J Primatol 46:213-227. Novak, M.A., Musante, A., Munroe, H., O’Neill, P.L., Price, C., Suomi, S.J. 1993. Old socially housed rhesus monkeys show sustained interest in objects. Zoo Biol 12:285-298. Platt, D.M., and Novak, M.A. 1997. Video-stimulation as enrichment for captive rhesus monkeys (Macaca mulatta). J App Anim Behav Sci 52:139-155. Richard, A.F., Goldstein, S.J., Dewar, R.E. 1989. Weed macaques: The evolutionary implications of macaque feeding ecology. Int J Primatol 10:569-594. Schapiro, S.J., Nehete, P.N., Perlman, J.E., Sastry, K.J. 2000. A comparison of cell-mediated immune responses in rhesus macaques housed singly, in pairs, or in groups. Appl Anim Behav Sci 68:67-84. Shively, C.A., Clarkson, T.B., Kaplan, J.R. 1989. Social deprivation and coronary artery atherosclerosis in female cynomolgus monkeys. Atherosclerosis 77:69-76. Southwick, C.H., Beg, M.A., Siddiqi, M.R. 1965. Rhesus monkeys in North India. In: DeVore, I., ed. Primate Behavior Field Studies of Monkeys and Apes. New York: Holt, Rinehart and Winston. p. 111-159. Southwick, C.H., Siddiqi, M.F., Farooqui, M.Y., Pal, B.C. 1974. Xenophobia among free-ranging rhesus groups in India. In: Holloway, R., ed. Primate Aggression, Territoriality, and Xenophobia: A Comparative Perspective. New York: Academic Press. p. 185-209. Strome, E.M., Wheler, G.H., Higley, J.D., Loriaux, D.L., Suomi, S.J., Doudet, D.J. 2002. Intracerebroventricular corticotropin-releasing factor increases limbic glucose metabolism and has social context-dependent behavioral effects in nonhuman primates. Proc Natl Acad Sci U S A 99:15749-15754.

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop Suomi, S.J., 1991. Up-tight and laid-back monkeys: Individual differences in the response to social challenges. In: S. Brauth, W. Hall, and R. Dooling, eds. Plasticity of Development. Cambridge: MIT Press. p. 27-56. Suomi, S.J., 2000. Behavioral inhibition and impulsive aggressiveness: Insights from studies with rhesus monkeys. In: L. Balter, C. Tamis-Lamode, eds. Child Psychology: A Handbook of Contemporary Issues. New York: Taylor and Francis. p. 510-525. Suomi, S.J., Rasmussen, K.L.R., Higley, J.D. 1992. Primate models of behavioral and physiological change in adolescence. In: E.R. McAnamey, R.E. Kriepe, D.P. Orr, and G.D. Comerci, eds. Textbook of Adolescent Medicine. Philadelphia: Saunders. p. 135-139. Teas, J., Richie, T., Taylor, H., Southwick, C. 1980. Population patterns and behavioral ecology of rhesus monkeys (Macaca mulatta) in Nepal. In: Lindburg, D., ed. The Macaques Studies in Ecology, Behavior and Evolution. New York: Van Nostrand Reinhold Company. p. 247-262. Winslow, J.T., Noble, P.L., Lyons, C.K., Sterk, S.M., Insel, T.R. 2003. Rearing effects on cerebrospinal fluid oxytocin concentration and social buffering in rhesus monkeys. Neuropsychopharmacology 28:910-918.

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop Assessment of Animal Housing Standards for Rabbits in a Research Setting Markus Stauffacher and Vera Baumans INTRODUCTION Laboratory animals such as rabbits are bred and housed for experimental use. The living conditions, housing, and husbandry are often more obstructive and more stressful for the animals than the experimental procedure itself. Therefore, the potential negative effects of an experiment on a laboratory animal’s well-being are not restricted to the experiment itself but instead cover the whole life span of the animal. Discussions of welfare requirements and their practical implementation could be improved substantially if decision makers would bear in mind that the desire to protect animals in captivity is based on ethical considerations of humans. However, in contrast to this perspective, the true well-being of captive animals should be based on a biological understanding that relates to the specific needs of the respective species and strains. In 1986, the current housing standards for laboratory rabbits were established in Article 5 of the Council of Europe’s Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (CoE 1986): “Any animal used or intended for use in a procedure shall be provided with accommodation, an environment, at least a minimum degree of freedom of movement, food, water and care, appropriate to its health and well-being. Any restriction on the extent to which an animal can satisfy its physiological and ethological needs shall be limited as far as practicable. In the implementation of this provision, regard

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop should be paid to the guidelines for accommodation and care of animals set out in Appendix A to this Convention….” Cages and pens “should be designed for the well-being of the species” and “should permit the satisfaction of certain ethological needs (for example the need to climb, hide or shelter temporarily)…” (CoE 1986, Appendix A, paragraph 3.6.3). However, on the species-specific level, the guidelines are restricted to recommendations on minimum cage dimensions and stocking densities. Regulations set up by a political body do not define an optimum but instead set limits and minimum standards. All concepts of animal protection are composed of conventions and assessments that are inevitably linked to those individuals who prepare and make the decisions. Working out minimum requirements with respect to animal welfare (ethical) and to the supposed well-being of laboratory animals (biological) is, last but not least, a political (mostly economical) question. Nevertheless, the decision-making process must be based first and overall on sound arguments concerning the biology of species and strains in question. The environment of a rabbit kept in captivity—as a pet, for fattening, or in the laboratory—has a considerable impact on its well-being and functioning. Important environmental factors include not only climate (e.g., light cycle, temperature, relative humidity, ammonia concentration, and ventilation) but also hygiene, food and water supply, housing, and the presence of conspecifics. In this presentation, we focus on the housing environment. ESTABLISHED SPACE REQUIREMENTS The minimum space requirements of the European Convention (CoE 1986) are based on a mathematical calculation model with arbitrarily set constant factors, slope, and starting point. The slopes represent weight-bands and allow the space requirements for a given number of rabbits to be calculated. The heavier the rabbits, the fewer square centimers of space are required per weight unit. The calculation model refers only to body weight and does not make a distinction between strains, sex, and age. As a result, this model does not adequately reflect the fact that young, growing animals need much more space in relation to their body weight than adults. The minimum space requirements of the European Convention ETS 123, Appendix A, 1986, apply to a medium-sized (< 4 kg) rabbit such as the New Zealand White rabbit: 2500 cm2 with a height of 35 cm. However, in the Sixth Edition of the Guide for the Care and Use of Laboratory Animals (the Guide) (NRC 1985), the requirement applies to a rabbit of 2700 cm2 with a height of 35 cm. When we consider the current standard housing of laboratory rabbits

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop from a human point of view, single-housed animals are easy to control and handle. Animal staff can easily handle a cage size of approximately 2500 cm2 with a height of 35 cm and can easily disinfect the metal and plastic walls. The slatted or perforated floors allow automatic cleaning, and the pelleted food and water bottles can be controlled. All of these factors are consistent with Good Laboratory Practice. In contrast, from the rabbit’s perspective, the current housing practices provide the following characteristics: limited freedom of movement, a barren cage environment with restricted challenges and possibilities for occupation, no social partners, and an artificial open nest-box. All of these characteristics afford the rabbit hardly any possibility of performing species-specific behavior. A series of studies have shown that in cages that comply with the minimum dimensions required in Appendix A of the European Convention (CoE 1986), the welfare of laboratory rabbits is impaired (Stauffacher 2000). The consequences of limited freedom of movement are changes in locomotor patterns and sequences (e.g., inability to hop), which result in skeletal damage in, for example, the femur proximalis and the vertebral column (Bigler 1995; Drescher 1993). The barren cage environment with a severe lack of stimulation leads to behavioral disorders such as wiregnawing and excessive wall-pawing, as well as to panic reactions and signs of “boredom” (Lidfors 1997). During breeding, an open nest-box and poor quality and quantity of nesting material do not permit the natural behavior of the doe (e.g., closing up the nest entrance when triggered by odor cues of the litter). In addition, these conditions do not allow the doe any chance to withdraw from the litter, which can result in behavioral disorders in the mother and in significant rearing losses (Stauffacher 2000). In 1997, the Multilateral Consultation of the Council of Europe adopted a resolution on the accommodation and care of laboratory animals, which specified that “young and female rabbits should be housed in socially harmonious groups…” and that “pens, as well as cages, should include enrichment material e.g. roughage, sticks, an area for withdrawal and nesting material.” As a consequence of that resolution, an international expert group was set up in 1998 to devise science-based proposals for a revision of Appendix A. RABBIT BEHAVIOR AND NEED FOR SPACE Rabbits do not use space per se; they use resources and structures within an area for specific behaviors. Appropriate structuring of the cage/ pen environment may be more beneficial than provision of a larger floor area; however, a minimum floor area is needed to provide a structured

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop space (e.g., blinds, shelters, and platforms) that includes withdrawal areas and vantage points. Rabbits tend to be highly motivated, to make use of enrichment based on food items, and to satisfy their need for roughage (hay, straw) and gnawing (soft wood chew sticks). With respect to the social environment of rabbits, the domestic rabbit with its wild ancestor the European wild rabbit (Oryctolagus cuniculi L.) is a highly social animal that lives in the wild in stable groups of one male, three to five females, and their offspring in a home territory or “warren.” They establish a linear rank order (males and females), and subadult males must leave the warren. A social partner always creates new and unpredictable situations to which a rabbit must react. Such situations lead to an increase of alertness and exploratory behavior, and provide diversion, occupation, and probably also some feelings of “security.” References to these situations appear in the following documents: (1) The Council of Europe Multilateral Consultation on the Revision of the Convention for the Protection of Animals Used for Scientific or Other Purposes, ETS 123, Part II, Appendix A, 1997: “Young and female rabbits should be housed in socially harmonious groups unless the experimental procedure or veterinary reasons make this impossible.” (2) The Seventh Edition of the Guide (NRC 1996): “Animals should be housed with a goal of maximizing species-specific behaviors and minimizing stress-induced behaviors. For social species, this normally requires housing in compatible pairs or groups.” The crucial question, however, is how to assess minimum recommendations. To determine the minimum recommendations for sizes of primary enclosures (cages, pens) for laboratory rabbits, it is necessary to consider both the quantity and the quality of space. The crucial point is the interaction between the space—the structure of the cage, the rabbits, and the type and quantity of enrichment provided. These variables must be based on experimental results and scientific papers, good/best practice, and experimental constraints. Although good scientific arguments may exist regarding why limits should be set in particular cases, the exact numeric values for minimum cage sizes and heights as well as for maximum stocking densities can never be scientifically evaluated and “proved.” RECOMMENDATIONS AND ANTICIPATED REVISIONS In the proposal for the revision of Appendix A of the European Convention ETS 123, the Expert Group on Rodents and Rabbits recommends that medium-sized (< 4 kg) rabbits such as New Zealand White rabbits should be housed in cages with a floor area of 4200 cm2 and a height of

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The Development of Science-Based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop 45 cm, including a raised area of approximately 55 × 30 cm where one rabbit or two compatible rabbits can be housed. Without a raised area, floor space should be 5600 cm2 for one rabbit and 6700 cm2 for two rabbits. According to Swiss law, one rabbit or two compatible rabbits can be housed in a cage with a floor that measures 4200 cm2 + 1800 cm2 with a height of 60 cm (SOAP 1991). In the process of refinement of housing standards, the following points should be taken into account: Biological facts and scientific evidence (related to the animals); Experimental tasks and constraints (related to the research goals); Practical experience (related to the debating subjects); Ethical principles (related to animal protection); and Assessment of economical and political reasonableness (related to human societies). REFERENCES Bigler, L. 1995. Zusammenfassung der Ergebnisse radiologischer Untersuchungen an Zuchtzibben-Wirbelsäulen und Mastkaninchen-Femurknochen in der Schweiz von 1984-1995 [in German]. Bern: Bericht z.Hd. Bundesamt für Veterinärwesen. CoE [Council of Europe]. 1986. European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (ETS 123). Strasbourg: Council of Europe. Drescher, B. 1993. Zusammenfassende Betrachtung über den Einfluss unterschiedlicher Haltungsverfahren auf die Fitness von Versuchs- und Fleischkaninchen [in German]. Tierärztl Umschau 48:72-78. Lidfors L. 1997. Behavioural effects of environmental enrichment for individually caged rabbits. Appl Anim Behav Sci 52:157-169. NRC [National Research Council]. 1985. Guide for the Care and Use of Laboratory Animals. 6th Ed. Washington, DC: National Academy Press. NRC [National Research Council]. 1996. Guide for the Care and Use of Laboratory Animals. 7th Ed. Washington, DC: National Academy Press. SOAP [Swiss Ordinance on Animal Protection]. 1991. Schweiz. Tierschutzverprdnung vom 27. Mai 1081, Änderung vom 23. Oktober 1991. Bern: Eidgenössische Daten-und Materialzentrale, 1991. Stauffacher, M. 2000. Refinement in rabbit housing and husbandry. In: M. Balls, A.-M. Van Zeller, M.E. Halder, eds. Progress in the Reduction, Refinement and Replacement of Animal Experimentation. Amsterdam: Elsevier Science B.V. Publ. p 1269-1277.