4.2 C HAPTER Mouse Enrichment Kathryn Bayne AAALAC International, Frederick, Maryland, USA Hanno Würbel Veterinary Public Health Institute, University of Berne, Switzerland Introduction It is tempting to consider the laboratory mouse simply as the common house mouse, Mus musculus, and therefore a homogeneous animal for which enrichment can easily be provided. The labora- tory mouse is a ubiquitously used research subject whose genetics, anatomy, physiology, immu- nology and behaviour have been studied in detail for generations. Thus, it might seem that providing a housing environment that is species- appropriate would be a simple matter. However, it would be a serious mistake to approach mouse enrichment as a one-size-fits-all husbandry procedure. The laboratory mouse is still consid- ered behaviourally similar to wild mice in many ways [1], though it differs somewhat from the wild-type ancestor in its behaviour, with running behaviour, open-field freezing behaviour and a generally higher level of activity more evident in wild-type mice than in laboratory-bred animals [2]. Over decades of purposeful breeding, a variety of characteristics (e.g. ease of handling) have been either deliberately or inad- vertently introduced into the behaviour profile of the laboratory mouse. Today, the increasing trend in the use of transgenic mice has only amplified the diversity of traits being bred for, and thus the potential exists for both extensive and subtle differences in mouse behaviour and physiology as well as their response to their environment. This behavioural breadth of the species may help to account for the fact that the literature is replete with contradictory findings and diverse conclusions about the potential benefits and unexpected consequences from providing enrichment to laboratory mice. Although such contradictions may be frustrating for laboratory managers who wish to provide meaningful and practical enrichment to the mice in their colonies, they clearly signal both inadequate objective information regarding the behaviour of the The Laboratory Mouse Ó 2012 Elsevier Ltd. All rights reserved. ISBN 978-0-12-382008-2 DOI: 10.1016/B978-0-12-382008-2.00023-4 ENRICHMENT 547 HUSBANDRY AND MAINTENANCE
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C H A P T E R
4.2 Mouse Enrichment
ENRICHMEN
Kathryn Bayne
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AAALAC International, Frederick, Maryland, USA
Hanno Würbel
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Veterinary Public Health Institute, University of Berne,Switzerland
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IntroductionIt is tempting to consider the laboratory mousesimply as the common house mouse,Mus musculus,and therefore a homogeneous animal for whichenrichment can easily be provided. The labora-tory mouse is a ubiquitously used research subjectwhose genetics, anatomy, physiology, immu-nology and behaviour have been studied in detailfor generations. Thus, it might seem thatproviding a housing environment that is species-appropriate would be a simple matter. However,it would be a serious mistake to approach mouseenrichment as a one-size-fits-all husbandryprocedure. The laboratory mouse is still consid-ered behaviourally similar to wild mice in manyways [1], though it differs somewhat from thewild-type ancestor in its behaviour, with runningbehaviour, open-field freezing behaviour anda generally higher level of activity more evidentin wild-type mice than in laboratory-bred
The Laboratory Mouse� 2012 Elsevier Ltd. All rights reserved.ISBN 978-0-12-382008-2
animals [2]. Over decades of purposefulbreeding, a variety of characteristics (e.g. ease ofhandling) have been either deliberately or inad-vertently introduced into the behaviour profileof the laboratory mouse. Today, the increasingtrend in the use of transgenic mice has onlyamplified the diversity of traits being bred for,and thus the potential exists for both extensiveand subtle differences in mouse behaviour andphysiology as well as their response to theirenvironment.
This behavioural breadth of the species mayhelp to account for the fact that the literature isreplete with contradictory findings and diverseconclusions about the potential benefits andunexpected consequences from providingenrichment to laboratory mice. Although suchcontradictions may be frustrating for laboratorymanagers who wish to provide meaningful andpractical enrichment to the mice in their colonies,they clearly signal both inadequate objectiveinformation regarding the behaviour of the
DOI: 10.1016/B978-0-12-382008-2.00023-4
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mice we use in research and the need for addi-tional basic studies to better characterize theanimal model as its genome is modified. Due tothe unpredictability of the effect of manyenrichment techniques, the reality is that mouseenrichment programmes are complex, must bethoroughly researched, and implemented onthe basis of input from investigators, veterinar-ians and husbandry personnel.
For these reasons, this chapter cannot providea roadmap to designing a single perfect enrich-ment programme for all laboratory mice. Rather,the more general landscape of commonapproaches to providing enrichment to mice,issues of cage space, effects of enrichment onboth the animal and the research, assessing thevalue of enrichment and pragmatic approachesto providing enrichment will be described. Thegoal, then, is to provide the reader with tools tocritically evaluate the types and relative meritsof enrichment available and to consider ways toenhance the welfare of themice without compro-mising their research purpose.
ImplementingenvironmentalenrichmentConceptual issuesThe goal of enrichment
Environmental enrichment has been variouslydefined, but generally includes the goal ofimproving the welfare of the animal throughthe thoughtful inclusion of meaningful featuresto the cage environment. More than 10 years ago,enrichment was described as:
any modification in the environment of the captiveanimals that seeks to enhance its physical andpsychological well-being by providing stimuli meetingthe animals’ species-specific needs [3].
More recently, the aim of enrichment hasbeen described as:
a method to enhance animal well-being by providinganimals with sensory and motor stimulation, throughstructures and resources that facilitate the expression
of species-typical behaviours and promote psycholog-ical well-being through physical exercise, manipulativeactivities and cognitive challenges according tospecies-specific characteristics [4].
In some cases, the objective of enrichment isto increase the expression of certain behaviourswhile, in other cases, reduction of specific behav-iours is intended. For example, reduction in theexpression of stereotypic behaviours may bea goal, which is achieved by providing resourcessuch as a shelter [5]. In all instances, the provisionof enrichment should not negatively impacteither the health and safety of the animal, or itsutility for research.
There are several general characteristics ofnon-social enrichments that are desired andwhich may drive the selection process amongenrichment choices. Primary among these isthat ideally there should be demonstrable valuederived from the enrichment technique inenhancing the welfare of the animal. Optimally,this evidence should be contained in the peer-reviewed literature and it should be possibleto extrapolate the results of the publisheddata to the specific context of the institutionconsidering implementing that type of enrich-ment. It is worthy to note that enrichment istypically intended to improve animal welfareover some established baseline. Often, the wildcounterpart of the laboratory animal is heldup as the standard for comparison. However,as noted above, this comparison may be inade-quate because of the potential for changes tohave occurred in the biology of the laboratorymouse following generations of targetedbreeding. The question thus becomes what stan-dard should be used as the benchmark formouse well-being. If one considers thata C57BL/6 or p53 knockout mouse should serveas its own control for characterizing the welfareof the entire strain, then it becomes a monu-mental task to accomplish this for each strainand transgenic line created. Clearly, the logis-tics, time investment and cost of such anapproach are prohibitive. Relying on past expe-rience alone and retrospectively assessing howthe welfare of various mouse strains and linesis affected by the housing environment entailsthe potential for welfare to have been uninten-tionally compromised before the error isdetected, if it is detected at all.
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One reasonable approach is to base welfareassessments on a composite of types of micethat evidence similar behaviours, responses toexperimental challenges, or fragility. In thismanner, groupings of strains or lines of micewould be made and common approaches toassessing welfare could be applied. Needless tosay, the success of such a strategy would bedependent on the accuracy of the groupingsand, of necessity, would rely on the availabilityof information to make these judgements. Insome circumstances, scoring systems for behav-ioural phenotyping facilitate the description ofbehaviours of transgenic and knockout mice.These systems typically involve analysis ofa battery of responses to stimuli and resting activ-ities [6], as well as physical characteristics (e.g. baldpatches in the hair coat). This tool may aid in thegrouping of mice by determining when ananimal differs from its prototype, which couldbe an indicator of altered welfare. In the absenceof an obvious metric for assessing the welfare ofthe diverse range of mice used in research, theremay be an inclination to rely on the wild-typemouse or on an inappropriate laboratory strainor line as the basis for comparison. And, althoughprogress has been made in identifying pain anddistress in micedsuch as the mouse grimace scale[7], changes in activity [8] and changes in behav-iours such as flinching, writhing, rear leg liftand press [9]dstrain differences continue toplague us, making some of these strategiesbroadly utilitarian [9]. Also, the value of systemsbased primarily on behaviour change foranimals in a prolonged state of compromisedwelfare (e.g. chronic pain) has not been deter-mined [10].
Enrichment and refinement
The ‘Three Rs’ of Russell and Burch [11]dReplacement, Reduction and Refinementdhavebecome mainstream principles for the care anduse of laboratory animals. In their discussion ofthe Three Rs, the authors define refinement asa strategy whose ‘object is simply to reduce toan absolute minimum the amount of distressimposed on those animals that are still used.’ Asthe publication of their thesis regarding theThree Rs predated notions of providing environ-mental enrichment to laboratory animals, it is not
surprising that, as noted by Buchanan-Smith andcolleagues, the concept of refinement haschanged substantially since it was first proposed[12]. Over time, as the tenets of the Three Rshave gained in international stature and havebeen referenced in several pivotal guidancedocuments, the general consensus that environ-mental enrichment is a prominent method ofrefinement has also gained momentum. Forexample, both enrichment and the Three Rs arereferenced in the Guide [4], an internationallyaccepted standard for the treatment of researchanimals, and more recently both concepts havebeen adopted into the International GuidingPrinciples for Biomedical Research InvolvingAnimals of the Council of International Organi-zations of Medical Science (CIOMS) which isused by the international scientific communityto guide the responsible use of vertebrateanimals in scientific and educational activities[13]. The 2012 version of the Principles is explicitin stating that the ‘tenets of the Three RsdRe-placement, Reduction and Refinementdshouldbe incorporated in the design and conduct ofscientific and/or educational activities thatinvolve animals,’ and that ‘Measures should betaken to ensure that the animals’ environmentand management are appropriate for the speciesand contribute to the animals’ well-being.’ Inaddition, the World Organisation for AnimalHealth (OIE) cites the Three Rs in its TerrestrialAnimal Code and encourages the use of environ-mental enrichment as a:
means of increasing the complexity (e.g. with toys,cage furniture, foraging opportunities, social housing,etc.) in a captive animal’s environment to foster theexpression of non-injurious species-typical behavioursand reduce the expression of maladaptive behaviours,as well as provide cognitive stimulation [14].
These sources reflect the widely held notionthat refinement strategies often entail attentionto the animal’s housing environment andconcomitant enrichment techniques.
This view is evident in the United Kingdom’sNational Centre for the Replacement, Refine-ment and Reduction of Animals in Research(http://www.nc3rs.org.uk/) whose mission is touse the Three Rs to support science, innovationand animal welfare in the biosciences. TheNC3Rs supported a special initiative to fund
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research projects that would develop refinementsin rodent husbandry, care and procedures, andthe organization’s website includes recommenda-tions for housing elements (http://www.nc3rs.org.uk/category.asp?catID¼39), many of whichare complexities aimed at enhancing mousewelfare, such as something to gnaw on, tubes,nesting material, nest boxes and bedding. In addi-tion, the Canadian Council on Animal Care(CCAC) notes in its policy statement on ‘Ethicsof animal investigation’ [15] that investigatoradherence to the Three Rs is required, and theCCAC website includes a Three Rs microsite(http://threers.ccac.ca/en/alternatives/intro.html) as a resource for the scientific community.It is here that the CCAC defines refinement asthe ‘modification of husbandry or experimentalprocedures to minimize pain and distress, andto enhance the welfare of an animal used inscience from the time it is born until its death,’thus clearly linking enrichment with refinement.Buchanan-Smith et al. [12] underscore the impor-tance of the relationship between enrichmentand refinement in their recommendation thata proactive approach to refinement should betaken which includes enhancing animal well-being through environmental enrichmenttechniques.
Caging characteristicsThe characteristics of the caging provided tomice can significantly impact the welfare of theanimals. Caging, properly designed and con-structed, can support species-appropriate behav-iours, and more specifically a variety ofmeaningful enrichment strategies. Considerationshould be given to the material from which thecage is manufactured; the floor space, cageheight, and thus cage volume; the quality of thecage space; and the accessibility of key resources.
Cage size
Perhaps the most contentious topic regardinghousing is the amount of cage space that shouldbe provided for mice. A very cursory scan ofthe literature pertaining to this subject revealsquite different recommendations as to what isconsidered adequate. A key factor contributingto this debate is the fact that different strains
and lines of mice respond quite differently toenvironmental conditions. The mouse is perhapsthe most genetically manipulated laboratoryanimal that we study. As a result, mice can evinceboth subtle and obvious differences in behav-iour, reinforcing the notion that environmentalconditions that may be optimal for one type ofmouse may be inadequate for another type.Further confounding resolution of this questionis the fact that different parameters aremeasured across studies, or different methodsare used to achieve the differences in floor spaceafforded the subject animals. Depending on thestudy, animal responses to cage size might beassessed based on weight gain/body weight[16–22], food and water consumption [17–19],immunological parameters [16–18], behaviour[21, 23], emotionality of the animals [24], impacton first- or second-generation pups derivedfrom litters of dams raised with different avail-able cage space [25], and mortality [16–19]. Cageenvironmental conditions such as ammonia,carbon dioxide, temperature and relativehumidity [18, 20, 26] have also been evaluated asa function of animal density and the impact ofincreasing animal density on the health of thecage occupants. Two primary methods of modi-fying the cage space available to an individualanimal are typically used. In some studies (e.g.[16, 17]) the number of animals in the cage spaceremains the same while the cage size is reduced.In other studies (e.g. [18, 19]), cage density isincreased while the actual cage size remainsstatic. A criticism of the latter approach is thepotential confounding effect on the data bysocial interactions and pressures that occur withcrowding of animals, but such studies also usecommercially available cage equipment ratherthan customized caging of incrementally differentsizes, thereby perhaps facilitating extrapolation offindings into typical animal housing units.
Several recommendations have been maderegarding what constitutes adequate cage space.These range from a performance-basedapproach, such as providing sufficient space forexercise and normal social behaviour and theinclusion of enrichment items [27], to varyingthe amount of cage space based on the strain,number of animals in the cage, age of theanimals, reproductive status, familiarity of theanimals with each other and the work being
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done with them [28]. While it has been postulatedthat the quality of the cage space is more impor-tant than simply a larger floor area [29], otherwork has shown that animals evaluated usinga consumer demand paradigm will work to gainaccess to additional cage space that is emptyexcept for bedding [30]. Still others have sug-gested that the amount of cage space is important,in and of itself, as it can become the limitingfactor for the provision of enrichment [28]. Ofinterest is the finding in female C57BL-derivedmice that the motivation to access additionalcage space did not depend on the amount ofadditional space accessed [30]. In this study,a single mouse would move away from its socialgroup, food, water and nesting material to accessadditional space (a bedded cage of varyingdimensions). The test mouse had been acquaintedwith the additional cage space tomitigate possiblenovelty effects. Despite increasing demands(number of presses on a switch) to access theadditional space, mice demonstrated strongmotivation to do so. The author suggests thatthis sustained response could represent a drivefor exploration, territorial monitoring or escapefrom cagemates. However, it may be arguedthat the additional space offered, which promp-ted the expression of exploratory behaviour,does not equate to enhanced welfare.
Jennings et al. [28] propose that in the contextof a lack of concurrence regarding what consti-tutes ‘optimum’ cage space, the dynamic resultingfrom the space, design and construction of thecage, the animals, and the enrichment shouldserve as a guide for establishing space require-ments. However, the element missing from thisrecommendation is that the scientific use of theanimal should also be considered, as the cageenvironment should not only foster animalwelfare but also facilitate high quality science.While there may not be absolute concurrenceregarding an optimum cage size, the scientificliterature provides many examples of studiesdemonstrating that some strains can be housedat higher densities with no adverse effect (e.g.,[16–19, 23, 31, 32]), with the strains evaluatedincluding C57BL/6 lines, BALB/c lines, NOD/LtJ, FVB/NJ and MF1. Several of these studiesalso show, however, that while mice appear tobe able to be housed at densities greater thanthe cage space recommendations in the Guide
[4], there is a limit to the increase [33, 34]. Smithand Corrow [35] postulate that the reason forthe reduced aggression observed in mice housedat higher densities (e.g. [23]) is because there is lessdefensible territory with less floor space. Thishypothesis is supported by findings in wildmice, which are strongly territorial when popula-tion density is low or moderate [36].
Informative summaries of the measuredeffects of different cage sizes on various physio-logical and behavioural parameters for differentstrains of mice are available in the literature[18, 20]. The significant point derived from thesesummaries is that cage size effects simply cannotbe extrapolated from one strain of mouse toanother, as the response may be quite different(reduced, unaffected or increased). Thus, thedilemma faced by colony managers housingmouse strains, or lines for which there is no objec-tive, peer-reviewed data regarding the animals’response to different cage sizes, is what standardto use in determining the most appropriate cagesize and housing density. For two common strainsof mice, C57BL/6J and BALB/cJ, Nicolson et al.[20] suggest that the most reliable metrics fordetermining appropriate cage size are bodyweight gain, adrenal gland size and percentageadrenal cortex (the corticosterone-producingportion of the gland), faecal corticosteronemetabolites, in-cage telemetry of activity andheart rate, behaviours such as barbering,whisker-picking and fighting, and formal testsof anxiety. Clearly, experience with the animalswill provide practical information upon whichto base cage size determinations, albeit in a posthoc manner. However, a potential flaw with thisscheme is that until the post hoc analysis has beendone, animals may not be housed in an appropri-ately sized cage (e.g. more available cage spacewith resulting negative consequences such asincreased aggression [23] or lower body weight[22]; alternatively, less available cage space withnegative consequences such as increased serumcorticosterone [16, 35] or gastritis [33].
Structural enrichmentNests
Nesting behaviour appears to be an activity thatis well preserved from wild-type progenitor
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mice [1]. The provision of nesting material tocaged mice has received widespread supportbecause there appears to be a strong motivationfor mice to build nests (even among non-breeding mice). It can enhance pup surviv-ability, it is a behaviour that is commonlyperformed by numerous strains of mice and itoffers the opportunity for mice to better ther-moregulate in their environment [37, 38].Numerous studies have assessed the relativemerits of different kinds of nesting material,including commercially available Nestlets,paper strips, tissue or paper towel, cotton string,wood wool and wood shavings. The value of thenesting material to the mouse has also been crit-ically evaluated, using the complexity or archi-tecture of the nest as a metric for the qualityof the nesting material provided [39, 40] orthe mouse’s willingness to work to access nest-ing material [41]. Of significant welfare benefitis the finding that some kinds of nesting mate-rial (e.g. corn husks) reduce aggressive behav-iour in a line of BALB/c mice, as indicated byobserved decreased wounding of the animals[42], possibly due to the availability of areas toescape aggressive animals. Clearly the type ofnesting material impacts this welfare benefit,as aggression was decreased in 7 week oldmale BALB/c type mice provided with tissuetorn into strips [43], though intracage fightingwas not reduced by providing wood wool asthe nesting material to BALB/c and C57BL/6Jmice [44] and actually increased fighting inNIH/S male mice [45]. Yet, there is evidencethat some strains of mice, such as BALB/cand CD-1 mice, show reduced signs of stress,including lower urine corticosterone levels andheavier thymuses, if they are provided withnesting material and if the nest is transferredduring cage cleaning procedures [46, 47].Although there are contradictions in the litera-ture regarding optimal nesting material (e.g.paper strips [40] versus tissue or paper towel[48]), an important consideration is the planneduse of the mice. As Pasalic and colleagues [49]determined, tissue nesting material can be a con-founding variable for studies of allergic asthmain BALB/c mice, resulting in increased total cellnumber, eosinophil number and IL-13 concen-tration in bronchoalveolar lavage fluid ascompared with non-enriched control animals.
Also, warnings have been given with regard tosome types of nest-building material that canentangle the limbs of pups [28].
Nest boxes/shelters
As a prey species, wild-type mice will attempt toflee and hide from predators, and it has beenpostulated that laboratory mice retain this fearresponse behaviour. For example, laboratorymice may exhibit aggression to handlers if star-tled or fearful, and thus the provision of sheltershas been suggested as a means of reducing themouse’s fear response [50]. The inclusion of shel-ters or nest boxes has been evaluated as a singleenrichment and in combination with otherenrichments (e.g. nesting material, runningwheels). As has been demonstrated by investiga-tions into other forms of enrichment, varyingresults have been obtained on the merits ofproviding shelters, depending on the strain ofmouse, whether nestingmaterial was also present,the number of openings in the nest box and thematerial from which the nest box or shelter wasconstructed (e.g. metal, plastic, wood, paperproduct). In fact, the material of which the nestbox is constructed has been proposed as a signifi-cant factor in preferences expressed by mice [51].
In some cases, the shelter provided is a tube(perforated along the sides of the tube or non-perforated), while in others it is designed to func-tion more specifically for nesting. Baumans et al.[29] suggest that partitioning the cage space withstructures like shelters allowsmice to use separateareas for feeding, resting and urination/defaeca-tion and that shelters aid mice in controlling theirenvironment, such as exposure to illumination.The relative value of the shelter may vary withits location in the cage or if other enrichmentsare also available. A tube-shaped shelter withinthe cage may be located either directly on thecage floor or suspended from the cage wall,though animals appear to use the tube sheltermore frequently if it is located on the cage floor.Even the location of the nest box or shelter on thecage floor will be adjusted bymice in individuallyventilated cages, with animals moving the shelterto a position under the food hopper [52]. Sherwin[53] determined that individually housed maleTO mice would not use a tube for sleeping ifsawdust was provided as bedding in the cage;
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rather, these mice used the tube for refuge and asa latrine.
Recently, it has been shown that the numberof days of survival of Tabby jimpy (Ta-jp) mice(TaPlp1jp/þ) resulting from the breeding ofheterozygous females (TaPlpNjp/þþ) with white-bellied agouti males (B6CBACa-Ww-J/A) wasincreased in those animals provided with a nestbox constructed of paper boxes [54]. Theseanimals also had a higher weaning rate, hada statistically significant higher weaning weightand developed few abnormal jumping behav-iours. Because the nest box was fabricated froma paper product, the dams could create additionalholes in the shelter and use the shredded paper asa component in their nest-building activity,thereby adding value to this type of shelter.Male BALB/c mice also had increased longevityif they had access to a shelter [55]. Mice living incages containing a nest box, nesting material,chew blocks and a running wheel consumed lessfeed than mice housed in standard cages thatwere allowed to self-administer an anxiolytic,and spent less time performing bar-relatedbehaviours and bar-circling stereotypies [56].Another dimension to the complex picture ofshelter use is provided by a third reportregarding C57BL/6J mouse use of a polyvinylchloride (PVC) nest box. Animals were alsoprovided with a wooden chew block, a cardboardtube and nesting material [57]. The authors deter-mined that in this arrangement, use of the shelterincreased between 4 and 8 weeks of age, but theanimals used the shelter as a nesting/sleepingsite only at 4 weeks of age, and at an older agegenerally preferred to use the nesting materialfor sleeping. Of note is the finding that, forsome strains of mice, inclusion of a nest box orshelter has been implicated in increased levelsof aggression in animals [43], though this is notalways the case [55, 58].
Wheel-running
Although wheel-running has been described asan artefact of captivity [59], there is generalagreement that it is a highly robust behaviouramong mice that are afforded the opportunityto engage in the activity. Reports of mice running2–5 km over a 24 h period (mostly during the darkcycle) emphasize the motivation of mice to use
this type of apparatus [60, 61]. The question ofwhether wheel-running actually benefits thewelfare of the mouse, and thus can be consideredan enrichment strategy, has been debated.Wheel-running has variously been described assubstitute activity for other kinds of behaviours[60] and an incentive-induced motivated behav-iour [61]. Sherwin [59] offers a detailed and well-balanced review of the sometimes conflictingdata regarding causal factors and impacts.
Wheel-running does not reduce the expres-sion of stereotypic motor behaviour in deermice [62] and it can alter the time budget forother activities [61]. Specifically, female C57BL/6mice provided with a wheel for running hadaltered hourly patterns of movement andreduced time in a shelter, though they had over-all higher levels of activity. Much of the change inmotor activity was reflected in less time movingon the cage floor. The authors concluded thatwheel-running should not be considered simplyan extended opportunity for locomotion becauseit actually reorganizes daily behaviour, withstrain differences apparent between DBA/2and C57BL/6 mice [63]. Female C57BL/6 micehoused with a wheel also exhibited angiogenesis,enhanced motor coordination and some behav-ioural changes such as improved water mazeperformance [64], which led the authors tocaution use of a running wheel as a standardenrichment component due to the need tocontrol for its effect for some types of studies.The potential for wheel-running to effect exper-imental data is of wide concern. Recently, Zajacand colleagues [65] documented that wheel-running significantly increased total brain-derived neurotrophic factor (BDNF). Disruptionof BDNF gene expression is critical to the devel-opment of symptoms in Huntington’s disease(HD). Wheel-running in HD mice results ina delay in the onset of motor deficits acrossseveral tests [66]. In addition to physical changesassociated with mice using a running wheel,behavioural effects have also been observed.For example, intermittent individual housing offemale BALB/c mice typically affects open-fieldbehaviour, but these differences can bemitigatedby access to a running wheel [67]. Wheel-runningmay also be a stereotypy [68]. Perhaps the mostsignificant behavioural effect associated withthe running wheel is the evidence of increased
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aggression associated with its presence in thecage, as well as data suggesting that the wheelmay be disrupting social organization in thecage [69].
Cage or shelter colour
Contradictions in optimal characteristics of thehousing environment are as fundamental as thecolour of the cage itself. Rodents are consideredto be dichromats, able to perceive colours in thegreen–yellow region of the spectrum and in theultraviolet range, but with red appearing dark[70]. Sherwin and Glen [71] evaluated the choiceof cage colour for female CBA mice by prefer-ence test. The authors housed the mice ina home cage painted either black, red, green orwhite. Five weeks later, the mouse was allowedto choose the colour of its home cage amongthese same colour types. All mice showed a statis-tically significant preference for the white cages,with red being the least preferred colour.However, such findings must be approachedwith caution as the authors did not specify thesubstrain of the mouse, and CBA/J carries thePde6brd1 mutation, which can cause blindness bythe age of weaning [72].
This finding led to an assessment ofa commercially available mouse shelter con-structed of red transparent thermoplastic mate-rial and a shelter made of a paper-basedproduct [73]. When given a choice of a cagewith the red thermoplastic shelter or the papershelter, female mice of BALB/c, C3H andC57BL/6J lines chose the shelter fabricatedfrom the paper product significantly more often.The authors considered the red colour of thethermoplastic shelter, the shelter material (i.e.the thermoplastic could not be modified bychewing on it, whereas the paper product couldbe modified by the mice), and the fact that thepaper product was sufficiently lightweight tomove around the cage, as possible factors drivingthe preference toward the paper-based shelter.However, a subsequent study of male mice ofBALB/c, C57BL/6, CBA and NMRI linesprovided with only a red thermoplastic cage,plus bedding and nesting material, showeddifferences among the strains regarding thetime spent in the shelter and number of entriesinto the shelter, though the shelter was routinely
used [74]. Thus, there appears to be some vari-ability in the literature regardingmouse responseto shelters; however, these preliminary datasuggest that mice will rank different types ofshelter if offered a choice, but that if no choiceis offered, the shelter will certainly be used bythe animals to varying degrees.
Effects ofenrichmentEffects on the animalsOne of the challenges associated with cata-loguing the effects of environmental enrichmenton mice is that reports of effects from studiesusing ‘enrichment’ may be confounded by thefact that the items provided in the cage toincrease structural complexity were not objectsthat actually enhanced the welfare of theanimals. Clearly, semantics plays a role in thisproblem, as any addition to the cage environmentseems to be automatically labelled as an enrich-ment, whether the actual definition of enrich-ment is achieved or not. As described above,many preconceived notions about the benefitsof certain cage structures must be discarded asevidence mounts regarding their value as trueenrichments. Further complicating the pictureis the variability among strains of mice in termsof responses to enrichment items or structuraladditions to the cage environment. For example,Hutchinson et al. [1] describe the differences inthe number of litters produced by female miceliving in enriched or standard housing. In thesestudies, the enriched cages included a ladderand jar with nesting material, while the standardcage had bedding. BALB/c and Swiss-Websterfemales produced significantly fewer litters(p <0.001) and had fewer pups per litter whenhoused in the enriched cage as compared to thestandard cage. However, CB17-Prkdcscid, B6D2F2and ICR mice did not show any difference innumber of litters or pups per litter when housedin standard or enriched cages. The authors alsodetail striking gender differences for BALB/cand Swiss-Webster mice. Specifically, females ofthese two strains (but not males) demonstrated
significantly lower levels of thymocytes whenliving in an enriched cage as compared to thestandard cage.
An understanding of the effects of providingan enriched, or stimulating, environment torodents has its roots in studies done with rats andassessing effects of handling and maze trainingon brain chemistry and anatomy [75]. Since then,the body of information regarding the influenceof cage complexities on the mouse has grownconsiderably and new findings continue to bepublished.These findings can generally be catego-rized into effects on the behaviour or biology ofthe animals, often described in the context ofchanges in a specific animal model.
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Behaviour
The standard cage provides limited scope for theexpression of species-appropriate behaviours inthe laboratory mouse. Signs of this deficiencyinclude abnormal behaviours such as stereotypies(e.g. bar-mouthing, jumping, circling [76]) andcompulsive behaviours such as barbering [77],elevated stress hormone levels, fearful andanxiety-like behaviour [78] and impaired thermo-regulation [79]. It is therefore not unexpectedthat the addition of complexities to the cage envi-ronment can evoke a change in behaviour.General activity level, which in some studies isdissected into the more specific behaviours ofexploration and locomotion, as well as sleep,stress or anxiety-related behaviours (sometimesreferred to as emotionality), social behaviours,appetitive behaviour and grooming are amongthe parameters evaluated when one or moreobjects is introduced to the cage environment.Results vary among strains, gender and type ofobject(s) introduced. But, the data converge indemonstrating reduced stereotypies [80],increased exploratory behaviour [81], at leastinitially; increases in aggression between animals,with many, though not all, types of enrichment[43, 82]; alterations in the open-field test behav-iours [83]; and general ‘use’ of the variety ofenrichments.
Neurological effects
Although initial studies regarding the influenceof enrichment on the central nervous system
were conducted with rats, there are similarlynumerous reports of neurological effects ofenrichment on mice and some excellent reviewshave been published on this subject [84, 85]. Ashas been observed in rats, exposure of adultCBA/B6 hybrid mice to enrichment inducesstructural changes in the brain [86, 87]. Forexample, mice placed in a large box containingtoys, wooden blocks, a running wheel and sheltersfor 3 h/day had altered mRNA levels of genesassociated with structural changes in the brain.The authors identified an upregulation of dynac-tin, a cytoskeletal protein involved in retrogradeaxonal transport, which is thought to havea role in neuronal growth and synaptogenesis.The protein cortactin, which is involved insynaptic formation and plasticity, was also upre-gulated in the enriched animals. The authorsalso determined that the protein known asdefender against cell death 1 (DAD1) was upregu-lated after exposure to enrichment. Theyconcluded that environmental enrichment influ-ences the expression of several genes linked toneuronal structure, synaptic signalling and plas-ticity which have a role in learning, memoryand age-related memory deficits. Mitigatingeffects of enrichment on age-related memorydeficits has also been demonstrated by Frickand colleagues [87] using C57BL/6 mice. In thisresearch, the effect of enrichment on the spatialmemory of ‘middle-aged’ (7 months) male andfemale mice was evaluated in a Morris watermaze. Enrichment, consisting of a running wheeland various shaped toys, was provided in thehome cage for 25–29 days. Data indicated thatenrichment provided to middle-aged micereduced age-related spatial reference memorydeficits (both spatial task acquisition and reten-tion) as compared to socially housed controlanimals.
Much of the research into the effect ofenrichment on neural structure has focused onthe hippocampus due to evidence that enrich-ment enhances hippocampal function, includinglong-term potentiation, neurogenesis, dendriticspine growth and neurotrophin mRNA expres-sion [88], which in the hippocampus are impli-cated in learning and memory functions.Indeed, the hippocampus appears to be one ofthe more susceptible areas of the brain to theinfluence of enrichment [89]. Diabetic mice
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show structural alterations in the hippocampus,including reduced neurogenesis in the dentategyrus, decreased dendritic complexity andreduced vascularization of the dentate gyrus.However, an exposure of 10 days’ duration to16 week old C57BL/6 mice that had streptozoto-cin-induced diabetes resulted in neural cellproliferation, differentiation and retention,vascularization of the dentate gyrus andenhanced dendritic complexity of hippocampalneurons [90]. The mechanism for this activitywas not definitively determined, although anincrease in BDNF or a modulating effect ofenrichment on the hypothalamic–pituitary–adrenal axis were postulated as possible path-ways. A relevant point is that Zhu et al. [89]observed increased levels of BDNF in C57BL/6male and female mice that lived in an enrichedcage environment for 4months, thus lendingfurther support to the hypothesis that neurotro-pin levels may be modulated by external stimuli.Increased levels of BDNF have also been detectedin the retina of enrichedmice, with a concomitantincrease in retinal ganglion cell (RGC) dendrites,even preventing the typical suppression ofdendritic branching caused by dark rearingwhich suggests that visual stimulation alone isnot the modulating factor for RGC dendriticenhancement [91].
The effect of enrichment (includinga running wheel) on mice used to study severalneurological disorders has recently beenreviewed [84, 85]. The modulating effect ofenrichment has been consistently demonstratedin several mouse models of neurological disease.For example, results in a delayed onset andprogression of the motor deficits in miceafflicted with HD [92–94]; a reduction of amyloiddeposition in transgenic mice used to study Alz-heimer’s disease; increased resistance to MPTP,used to induce Parkinson’s disease and enhancedrecovery of motor function [95]; extended life-span and enhanced Rotor-Rod performance inmice with amyotrophic lateral sclerosis [96, 97];increased dendritic branching and spine densityin mice with fragile X syndrome [98]; delayedand reduced neurological deficits, includingmotor impairment and anxiety behaviours, inmice lacking Mecp2 and used as a model for Rettsyndrome [99]; reduced central nervous systemcell infiltration by Piry virus (used to induce
encephalitis) and more rapid viral clearance,with reduced microgliosis in infected mice [100];and enhanced spatial memory acquisition infemale Ts65Dn mice (a model for Down’ssyndrome), but, of note, reduced spatial memoryacquisition in the trisomic male mice [101].
Effects on researchBeneficial effects or confounding variable?
The provision of environmental enrichment mayresult in unintended consequences for bothanimals and research results [102]. However,studies specifically examining the effects of envi-ronmental enrichment on the variation of exper-imental results have demonstrated mixed results.Van de Weerd and colleagues [103] evaluatedboth behavioural and physiological parametersin mice used for potency testing for tetanusvaccine and stress-induced hyperthermia.Although they observed some differences inbody weight and open-field testing responsesbetween control and experimental animals, therewas no increase in variability among the twogroups, and for some measures there was lessvariability for the experimental animals. Simi-larly, Augustsson et al. [104] determined that thegreatest effect on variation for C57BL/6 andBALB/c mice used in a light/dark test using diaz-epam as an anxiolytic agent was the strain ofmouse rather than the presence of enrichment.
Clearly, the welfare of laboratory mice maybe seriously altered by housing them in inappro-priately designed and insufficiently complexcages. Attenuating these adverse effects throughadequate environmental enrichment is likely toimprove not only the animals’ well-being, butalso the scientific validity of a wide range ofexperiments conducted with them. As Baumans[50] has suggested, with the provision of enrich-ment, the mouse can exhibit more species-appropriate behaviour, and thus may be able tobetter cope with unexpected changes to its envi-ronment and respond more uniformly todifferent challenges. Abnormal behaviour, stress,fear and anxiety, and impaired thermoregulationare potentially confounding variables that mayadversely affect the outcome of animal experi-ments and consequently increase variation inthe data. Therefore, it is a reasonable conclusion
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that the most appropriate enrichment is in thebest interest of both the animals and the research.
Nevertheless, environmental enrichment isstill far from being a standard operational proce-dure inmost mouse facilities, though one study of22 animal facilities indicated that 73% providedstructural enrichment to their mice, with 20 ofthe facilities using nestingmaterial [1]. One reasonfor the reluctance to provide enrichment is theconcern that environmental enrichment itselfcould be a confounding variable that adverselyaffects the scientific validity of animal experi-ments. In particular, it has been argued thatenvironmental enrichment might disrupt envi-ronmental standardization in ways that are detri-mental to the precision and reproducibility ofresults from animal experiments. If true, thiswould mean that environmental enrichmentmay create a conflict between the welfare of theanimals and the validity of the research, andthat the benefits of enrichment in terms of betteranimal welfare need to be gauged against its costsin terms of poorer scientific validity.
Environmental standardizationStandardization in animal experimentation andits implications for the precision, reproducibilityand validity of animal experiments has typicallybeen the backdrop for the conduct of research.According to textbooks on laboratory animalscience, standardization refers to ‘the definingof the properties of any given animal (or animalpopulation) and its environment’ [105]. Likegenetic standardization, environmental standard-ization essentially serves two distinct goals. First,by defining environmental conditions andexposing all animals used in an experiment tothe same defined conditions, environmentalstandardization is aimed at reducing within-experiment variation, that is, variation due toindividual differences resulting from individualanimals being exposed to different environ-mental conditions. Although the ‘defining ofthe properties’ does not necessarily implicateidentical environmental conditions for all theanimals of an experiment, environmental stan-dardization is generally equated with such envi-ronmental homogenization. Thus, environmentalstandardization renders animals within experi-ments more homogeneous. The intention behind
such uniformity is to minimize within-experi-ment variation in order to maximize test sensi-tivity. Higher test sensitivity means that a givendifference between the means of two popula-tions is statistically more significant, or that thesame level of statistical significance is achievedwith fewer animals. Therefore, environmentalstandardization has the effect, both economicallyand ethically, of reducing the number of animalsneeded per experiment. However, this benefit isachieved with an increasingly narrow spectrumof phenotypes. Therefore, the external validityof experimental results obtained from sucha homogeneous population may be limited toa narrow range of environmental conditions[106]. This has important implications for thereproducibility of the results, as discussed below.
Second, by using the same defined environ-mental conditions across different experiments,environmental standardization is aimed atreducing between-experiment variation, that is,variation in the outcome of an experiment result-ing from experimental populations being exposedto different environmental conditions. The inten-tion behind such harmonization of environmentalconditions across experiments is to minimizebetween-experiment variation in order to maxi-mize the reproducibility of results. Reproducibilityis a cornerstone of all scientific research and isparticularly crucial in animal research where thelives of the animals are highly valuable. Forexample, in the USA animal care and use regula-tions require scientists not to ‘unnecessarily dupli-cate previous experiments’ [107]. This requirementcritically depends on the results of animal experi-ments being reproducible bothwithin andbetweenlaboratories, and harmonization of environmentalconditions across experiments and laboratoriesaims to guarantee such reproducibility.
Concerns that environmental enrichmentmight disrupt environmental standardization relateto both of these aspects. Thus, it has been arguedthat enrichment might increase either within-experiment variation or between-experimentvariation or both (for example, see [108]).
Enrichment and within-experimentvariation
Concerns that enrichment would increase within-experiment variation rest on the hypothesis that
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a more complex environment produces a greaterdiversity of phenotypes among the animals ofa study population. On the one hand, a morecomplex environment might create more oppor-tunities for individuation, for example, byproviding different niches within an environ-ment so that the different animals within a cageare exposed to different environmental condi-tions. On the other hand, an unenriched environ-ment might increase individual differences, asindicated by the occurrence of abnormal behav-iours such as stereotypies, resulting in variableand individual coping responses. Whetherphenotypic diversity is a function of environ-mental complexity, and whether this relationshipis positive or negative, are empirical questionsthat have never been systematically addressed.However, several studies have examined theeffects of various enrichment protocols onwithin-experiment variation in physiologicaland behavioural measures [96, 97, 109]. None ofthem identified evidence that enrichment wouldaffect within-experiment variation consistentlyin one or the other direction.
Present evidence therefore suggests that thewelfare of mice can be improved by provisionof suitable environmental enrichment withoutincreasing within-experiment variation, pro-vided the enrichment is adequate for the animalsand does not itself constitute a stressor, in whichcase variation in experimental results mayindeed be enhanced.
Enrichment and between-experimentvariation
The hypothesis that enrichment might increasewithin-experiment variation is also used to arguethat enrichment might compromise the repro-ducibility of experimental results. However,reproducibility is not determined by variationwithin experiments but by variation betweenexperiments, and a recent multilaboratory studyshowed that even extensive enrichment hadno adverse effect on between-experiment varia-tion, demonstrating that a more complex envi-ronment does not compromise reproducibility[109, 110].
Others are concerned that more complexhousing conditions would inevitably lead togreater differences in the environmental
conditions between laboratories, becausedifferent institutions would choose differentenrichment items, use different products,arrange them differently within cages and differin how often they replace them. However, besidesthe possibility to standardize all of these aspects, itis unlikely that they represent a significantproblem, given the variation in environmentalconditions that exists anyway between differentlaboratories. Thus, there are many environ-mental factors that simply cannot be standard-ized (including staff, room architecture, noise,smells, air exchange rates, illumination levelsand vibrations). It is therefore unavoidable thatdifferent laboratories standardize to differentlocal environmental conditions, and enrichmentis one more factor that varies across laboratories.Given that environmental variation amonglaboratories (and even between experimentswithin the same laboratory) is a matter offact, results will only be reproducible if theygeneralize to at least the range of conditionscovered by these inherent laboratory differences.Therefore, reproducibility primarily dependson the external validity of the results, which ispartly determined by the treatment and themeasured response (some treatment effects arefairly robust against variation of conditions),and partly by the experimental design.
Enrichment and scientific validity
Validity in the context of animal experimentsrefers to whether an experiment or a measuredresult is scientifically meaningful. There isa range of different concepts of validity,including internal vs external validity, conver-gent vs discriminant validity, and face vsconstruct vs predictive validity. The latter formsof validity all depend on experimental design,types of measurements and measured outcomes,and are independent of whether or not theanimals’ cages are enriched. Validity in themore general sense, however, may well beaffected by environmental enrichment. Thus,scientific validity in this general sense encom-passes the rationale of the study and all aspectsof the methodology, and thereby determineswhether the results obtained meet all of therequirements of good scientific practice. Anyaspect that violates scientific principles or
principles of Good Laboratory Practice willcompromise the integrity of the results.
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Assessing the valueof enrichmentFor rodent enrichment to be considered a mean-ingful addition to standard animal care practices,a harm–benefit analysis should be undertaken.This analysis should be inclusive of effects onthe animal and on the research. Clearly, sometypes of research will be less impacted by theoften subtle impact on the animal, while otherswill be exquisitely sensitive to small changes inthe animal’s physiology. In many instances, theinclusion of enrichment in a mouse’s cage hasled to intriguing discoveries regarding the effectof environmental complexity on a particularanimal model of human disease, leading to newtheories of pathogenesis as well as potential treat-ment adjuncts.
Several fundamental questions bracket theimplementation of mouse enrichment. Themost basic of these is whether the animal ‘uses’the enrichment. Animal use of the enrichmentcan be described as moving the object around,making contact with it (e.g. climbing, resting orrunning on it), entering it (e.g. a shelter), orchanging its configuration (e.g. nesting material).Next, it should be determined how the animal isusing the enrichment. For example, if the animalis defending the enrichment to the point ofincreasing its aggressive behaviour towards otheranimals, an alternative form of enrichment, orpossibly other approaches to improving theanimals’ welfare, and their value as a researchsubject, should be considered. Finally, and impor-tantly, is the need for a sound understanding ofthe potential ramifications of the enrichmenttechnique on the animal’s biology and whetherthis may have consequences for the intendedresearch use of the animal. The scientific litera-ture is rich in studies that have evaluated thistopic, although much remains to be done, whichcan give direction to this determination.
As already described, enrichment offeredwith the best of intentions can have a negativeimpact on the animal. The mouse’s response
appears to depend on the type of enrichment;strain, sex and age of mouse; individual or socialhousing; and whether the enrichment is providedin the home cage or the animal is moved to a sepa-rate enrichment space. These responses caninclude stress, fear and anxiety; aggression; injurydue to the enrichment itself and potentially theintroduction of contaminants into the mouseenvironment. Yet, the evidence is clear thata sterile cage environment results in mice withdeficits in brain development and exhibitingabnormal behaviour [79], which is certainlya welfare concern, but also presents doubtsregarding the validity of the animal as a researchsubject. Because not providing an adequatehousing environment for mice has both welfareand scientific implications, and becauseproviding mice with inappropriate enrichmentalso has both welfare and scientific repercussions,the approach to optimizing mouse cage environ-ments should be a measured one. In addition toestablishing a team of professionals at the institu-tion to assess the specific circumstances anddetermine the proper strategy for housing themice in question, consideration should be givento implementing enrichment in an incrementalmanner, if possible, so that the impact on theanimals and the research can be reviewed andany necessary adjustments to the programmemade accordingly. While preference testing hasbeen conducted to evaluate the relative impor-tance of enrichments to mice (e.g. [72, 111]), itshould be recognized that this method of testinghas limitations and does not address the suit-ability of the enrichment for the specific mousestrain, sex or age.
Keeping mouseenrichment practicalEase of husbandryFor an enrichment technique to be widelyaccepted within an institution, it should notperceptibly impede the work of the animal carestaff. Husbandry procedures may be facilitatedif consideration is given to the sanitizability ofthe enrichment item(s), or alternatively if the
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item is sufficiently inexpensive that it can bedisposed of when degraded or soiled. It is veryhelpful to have husbandry staff participate inthe selection, planning and implementation ofthe mouse enrichment programme because oftheir detailed knowledge of, and experiencewith, the cage equipment and routine feedingand cleaning procedures. Concerns regardingenrichment in mouse cages tend to cluster intofive general themes: (i) the enrichment precludesadequate visualization of the animals for dailyobservation of health status, (ii) the enrichmenttakes up so much space that the animals arecrowded, (iii) the enrichment item is difficult tosanitize or complicates routine cage change oper-ations, (iv) the enrichment impedes removing theanimals from the cage, and (v) less frequentlyreported, though a significant potential problem,the enrichment can accidentally bump up againstthe automatic watering device or water bottle,resulting in flooding of the cage.
The impact of husbandry procedures andconditions onmice can be significant. Tomitigatesome of the effects of the routine cage changeprocedures and the potential social disruptionthat may ensue due to the sudden absence offamiliar odours, the transfer of nesting materialduring cage changing has been recommendedas one method to reduce aggression in maleBALB/c mice [46], though this has not been repli-cated in SJL/J mice [35] and aggression in micehas been linked to caging with a high ventilationrate alone [112]. It is also suggested that nests andshelters can bemanipulated by themice tomodu-late the ventilation and illumination levels expe-rienced by the animals [52] and so, while theymay impede daily observation of the mice unlessthey are moved by the care staff, these itemsfoster an ability in the animals to better controlenvironmental conditions. The approach of‘super-enrichment’ described by Hutchinsonet al. [1] entails providing a variety of enrichmentseither simultaneously or through a rotationschedule. If provided simultaneously, there isthe possibility that the cage becomes too crowdedfor the animals to move around freely or forhusbandry and research staff to easily accessthe animals, and items may ‘wick’ water into thecage from the watering system. Therefore,although there is evidence that super-enrichmenthas positive effects on neural plasticity, care
should be exercised to not excessively crowdthe cage with enrichment devices.
Although much of the evidence pertaining tothe effect of enrichment on efficient husbandryis anecdotal and experiential, Moons andcolleagues [113] objectively evaluated the timenecessary to catch mice from a cage containingtwo PVC tubes as compared to mice housed incages without the tubes. The study animals weremale, 10 week old FVB and NMRI mice. Theydetermined that the PVC tubes decreased thetime to catch the NMRI mice and had no signifi-cant effect on catching the FVBmice. In addition,the presence of the tubes did not make theanimals harder to handle, as assessed by therebeing no difference in resistance to being heldfor a sham injection procedure between thecontrol and enriched mice. In addition, a recentstudy has shown that picking mice up out of theircage using a tube, rather than manually by thetail, resulted in a low anxiety, voluntary approachof the mice to the experimenter, and greateracceptance of physical restraint [114].
Budgetary and otherconsiderationsAnimal per diem costs are a constant concern toresearchers and facility managers alike. Thus,the type and design of caging system used shouldsupport and, optimally, enhance the animal’shealth and welfare so that the most refinedanimal model is available for the planned scien-tific enquiry. To that end, the enrichmentselected should meet the criteria of fosteringanimal well-being and enhancing the value ofthe animal for the experiment, and it should beeconomical. It should be recognized that theremay be an initial investment in different typesof enrichment, with the selection of the types ofenrichment based on information from thescientific literature, to assess their utility in thecontext of the individual institutional animalcare and use programme. Following this selectionprocess, the enrichments should be included inthe budget in the same manner as other recur-ring expenses, such as bedding.
A mechanism should be established to ensureadequate communication between the personnelwho are implementing the mouse enrichment
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programme and the research team whoseanimals are impacted by those actions. Thiscommunication should occur before enrich-ments are put in the cages of animals that havejust been obtained for a study, and shouldcontinue regularly to allow for feedbackregarding the enrichment programme and toupdate research teams about any plannedchanges in the programme. Husbandrypersonnel should be trained to be sensitive tomouse behaviour and able to interpret changesrelative to the enrichment programme. Staffshould be alert both to positive effects resultingfrom the programme (e.g. animals that are easierto handle) as well as possible concerns. Theselatter outcomes should be reported andaddressed in a timely manner.
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ConclusionsWhile there is general consensus that an unen-riched cage environment is not recommendedbecause of the detrimental effects on the animals,and the concomitant potential for the quality ofthe research data to be negatively impacted, it isequally clear that a ‘cookie-cutter’ approach tomouse enrichment is naive and possibly harmfulboth to the animal and to the research. Whilemouse enrichment is a highly complex topic,and one that certainly merits further investiga-tion, studies on the effects of enrichment haveyielded surprising results that offer intriguingglimpses into biological mechanisms and suggestfascinating new paths of research.
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