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Abstract

In the majority of countries where there are legislative requirements pertaining to the use of animals in research, figures are quoted for minimum cage sizes or space allocation to be provided per animal. These figures are generally based on professional judgement and are in common usage. However, there is a growing trend and expectation that welfare science should inform regulatory decision-making. Given the importance of the potential welfare influences of cage size on the animals themselves, this paper presents the latest scientific knowledge on this topic in one of the most commonly used animals in research, the mouse. A comprehensive review of studies in laboratory mice was undertaken, examining the effects of space allocation per animal and animal density on established welfare indicators. To date, animal density studies have predominated, and the effects of space allocation per se are still relatively unclear. This information will guide those involved in facility management or legislative review, and provide a more solid foundation for further studies into the effects of routine husbandry practices on animal welfare.

Keywords

Space density mice welfare refinement

In most countries of the world where there are legislation or guidelines pertaining to laboratory animal care and use, figures are quoted for minimum cage sizes for mice and/or space to be provided per animal (Table 1). The intent of these publications is to set minimum standards for space allocation which attempt to balance optimal animal wellbeing while meeting the needs of the scientific community. Cage size or density must minimize experimental variation, support study design, be practical in the context of vivaria space and design, and not be cost prohibitive to research. Additionally, wellbeing of the animal should not be adversely affected and it is indeed preferable that housing encourages positive welfare outcomes.^1,2 This is not only of importance in consideration of the animal as a sentient being, but also reduces the possibility of confounding experimental results by causing alterations in physiological parameters. Additionally, use of evidence-based recommendations assists in reassuring the public regarding the humane use of animals in science. These considerations are important for regulators since decisions should be based on sound animal welfare science. However, the current regulatory requirements for several countries note the complexity of this issue which not only involves an identification of effects of space allocation but also includes consideration of vertical height, quality of space, and effects of group size and social interaction.^1,3,4 Since cage floor area and space allocation logically provide an underpinning foundation to research activities in all of these areas, a closer examination of the scientific literature relating to these factors is required to propose avenues for further improvement. Recognition that objective data are lacking has been noted by multiple regulatory authorities, perhaps best reflected by the Resolution on Accommodation and Care of Laboratory Animals⁵ and the Federation of European Laboratory Animal Science Associations (FELASA) Rodent Refinement Working Party.⁶ Laboratory animal professional organizations and funding agencies (e.g. National Centre for the 3Rs [NC3Rs], American College of Laboratory Animal Medicine [ACLAM] Foundation, American Association for Laboratory Animal Science [AALAS] and Universities Federation for Animal Welfare [UFAW]) have also identified this knowledge gap and developed funding mechanisms to support the appropriate research directions.

Table 1
Comparison of space recommendations for laboratory mice in different jurisdictions

Reference Animals Weight (g) Floor area/animal cm² (in²) Height cm (in) Comments

EC Directive 2010/63/EU (2010)² Mice in groups <2020–2525–30>30 607080100 12121212

Code of Practice for the Housing and Care of Animals used in Scientific Procedures (UK,1989)⁷ Mice in groups <30≥30 60100 12

Mice singly housed 200 12

Code of Practice for the Housing of Animals in Designated Breeding and Supplying Establishments (UK,1995)⁸ Monogamous pair 300 12 For each additional female plus litter an additional 180 cm² should be added

Trio 300 12 For each additional female plus litter an additional 180 cm² should be added

The Guide (USA, 2011)¹ Mice in groups <10Up to 15Up to 25>25 (6) 38.7(8) 51.6(12) 77.4≥(15) 96.7 (5) 12.7(5) 12.7(5) 12.7(5) 12.7 Larger animals may require more space

Female +litter (51) 330 (space for the housing group) (5) 12.7 Space may vary due to other considerations such as age and size of litter

NHMRC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (2004)³ No specific values given (NB some States and Territories provide their own guidelines)

Canadian Council on Animal Care Guide (1993)⁹ Mice in groups <20>20 65100 1315

Female+litter 160

Mice singly housed 200 12

Guidelines for Proper Conduct of Animal Experiments (Japan, 2006)⁴ No specific values given

Guidelines on the Care and Use of Animals for Scientific Purposes (Singapore, 2004)¹⁰ Mice in groups <10Up to 15Up to 25>25 385177>96 12121212

Note: Figures quoted for all jurisdictions are listed as minimum standards

A comparison between Guidelines of the European Union, UK, USA, Canada and Singapore (Table 1) shows similar maximal space allowances per mouse of approximately 100 cm², but some variation in the intermediate values and the animal weight at which certain space allowances are required.^1–4,7,9,10 These guidelines also make the assumption that, as group size increases, the space per animal decreases. For example, a singly-housed animal would require a larger space allowance than individual assignment in a group-housed situation. For practical reasons, these guidelines cannot include suggested values for different stocks and strains. However, it is reasonable to assume that variation in space needs may exist between strains, and that this difference might be an important consideration for those responsible for animal care.

This review presents a summary of the published research investigating aspects of cage floor area with respect to animal wellbeing. It also considers the influence that animal variables, such as strain or sex, may have on recommendations for space allowances. An issue when drawing conclusions from the available data is the confounding of space allocation per animal with increasing group size, and therefore potential influence of animal social structure. To facilitate reader understanding, this review has been divided into sections on space allocation per se, and animal density. The terms welfare and wellbeing have been used synonymously throughout to refer to ‘the welfare state of the animal’.

Animal welfare assessment

Assessment of welfare in animals is complex and often controversial. Welfare scientists generally make use of three different approaches to resolve welfare questions. These include: the ‘teleological or nature of the species’, the ‘biological function’ and the ‘feelings-based’ approaches.¹¹ The biological function approach has been widely used in welfare science investigations into the subject at hand. This approach requires measurement of parameters that include stress physiology, behaviour, mortality, health and productivity.

Criteria used to assess welfare rely on measuring some degree of change, either as a result of the stress response, i.e. a physiological indicator^12,13 or a behavioural change from the normal repertoire seen.¹⁴ Traditionally, behaviour has been evaluated to determine negative welfare states. For example, investigating indicators suggestive of pain or distress, aggression, and boredom, or abnormal behaviours (redirected behaviours or stereotypies).¹⁵ More recently, there has been some interest in behaviour that focuses on positive outcomes (a paradigm shift from purely the absence of negative behaviours).¹⁶ Such behaviours might include vocalization,¹⁷ play¹⁸ and facial expressions.¹⁹ Quantification of normal behaviour has also been suggested as a potential welfare indicator,²⁰ for example, sleep behaviour may provide a reliable, non-invasive indicator of stress and welfare in rats.²¹ It is self-evident that causes of reduced health impact negatively on welfare.²² Commonly used health indicators include: disease, injury and immune function measures. Productivity measures are controversial as a welfare indicator, but since indicators such as growth and reproductive function are frequently related to the presence of a chronic physiological stress response then their use can probably be scientifically justified.²²

However, since change itself is an important component of an animal's normal homeostatic mechanisms, the major debate arises over interpretation of these changes. Determining what degree of change indicates that welfare is at risk, and the role that the animal's compensatory adaptations to the environment play are two such complications.²³ Another problem in animal welfare science investigations stems from the multidimensional nature of welfare where scoring highly in one criterion is unlikely to compensate for poor scores in another (e.g. good health cannot counteract behavioural restriction). In order to make an overall assessment of welfare there needs to be an aggregation of the individual measures with some assessment of their relative contribution to welfare. Individual measures might include health, expression of normal behaviour, thermal comfort and a number of other principles appropriate to the species concerned. An approach being investigated in farm animals as part of the EU Welfare Quality project uses mathematical methods to follow a hierarchical aggregation process to go from animal or farm measures to an overall assessment score.^24,25 This index system endeavours to identify issues that matter to the animal so achieving stakeholder consensus is a key challenge.^26,27 Such a method could be similarly applied to laboratory animal housing scenarios although it appears not to have been performed at this point in time.

Another consideration relevant to welfare is the debate surrounding the use of engineering- versus performance-based standards in legislative instruments. Control of variability in the housing environment of the animal is essential to ensure that experiments are reproducible. Traditionally, this has been achieved by prescriptive statements (engineering-based) relating to husbandry inputs, for example, the number of air changes per hour. Performance-based standards focus on outcomes as a result of an environmental input. In the case of air changes, this might be ammonia levels in the animal room. Such standards provide flexibility to use science to achieve suggested outcomes rather than rely on what may be arbitrary input stipulations. It has been suggested that such an approach reduces regulatory burden and is a more cost-effective and beneficial way of improving animal welfare.²⁸ In the current discussion such an approach might invalidate the need for specification of housing area but rely on quantification of welfare outcomes based on some of the parameters discussed earlier. However, in order to use performance standards as a reliable tool, the desired outcome must be well defined and contain specific criteria for assessment. Professional input and judgement are also essential.²⁹

Range in wild Mus musculus

As a prelude to discussions on the current understanding of space requirements for laboratory mice, the literature on territories and related behaviours of free-living Mus musculus was examined. This is an important consideration for those who take a teleological approach to the assessment of welfare, since it allows comparison of behaviour between the wild species and its domesticated counterpart. In addition, regulators are increasingly charged with stipulating housing requirements that allow animals to express their normal behavioural repertoire. Understanding species biology has also been deemed to be important for good husbandry.³⁰

Mice generally display thigmotaxis (following barriers and walls) especially when in novel environments³¹ and this behavioural characteristic has been widely used in psychological research as a measure of anxiety.^32,33 In laboratory mice, this movement has been shown to be broken up by pauses, with wall rearing, or reaches forward with sniffing. In between explorations, mice return to a familiar safe area using visual and olfactory landmarks for navigation.^34,35 Despite this caution, dispersing mice have been shown to travel large distances sometimes covering kilometres daily.^36,37

Home range shows considerable variation, with food availability and population density as the main determining factors. When food is in abundance, range is relatively small with mice staying within a few metres of their nests.³⁸ Range area may also be affected by both season and animal gender. During the breeding season, animals have a smaller home range, but this may increase 10-fold following the end of the season when they revert to a nomadic lifestyle.³⁹ Adult males consistently have larger territories than females.^39,40 Values for these ranges have been estimated to be up to 365 m² in fields,⁴¹ around 6000 m² in forest habitats⁴² and up to 80,000 m² in the Australian wheatlands.⁴³ Not surprisingly, given their susceptibility to predation, mice show preference for nest sites in close proximity to walls that are easily accessible from overhead cover. Enclosure areas with little ground level structure and no overhead cover are less frequently used.⁴⁴

In comparing captive and wild populations, at the current time we can only hypothesize about the effects that this large difference in territory size might have on animal welfare. It seems reasonable to assume that it will lead to differences in behaviour and some negative welfare consequences^31,45 Indeed, it has been demonstrated that in a number of species of carnivores, those naturally wide-ranging species are more likely to exhibit captive infant mortality or stereotypical pacing behaviours.⁴⁶ Further research needs to be undertaken to determine whether this holds true for rodents. In the latter case a key consideration needs to be the behavioural adaptation that has occurred in laboratory mouse strains as a result of controlled selection and breeding.⁴⁵

Animal space use

Qualitative and quantitative space requirements have been proposed. Quantitative space is the immediate environment that the animal physically occupies and hence is simple to calculate. Qualitative space is the space required for performance of normal activities such as feeding, exploring, carrying out social behaviour or for animals to remove themselves from visual contact with others. This implies a need for each animal to have an area of empty space around it to avoid continuous physical contact with others, and to be able to defend this territory against invasion from conspecifics.⁴⁷ This value is more difficult to ascertain and is the subject of significant research. Ecological research has demonstrated that space use by animals increases with increasing body size and mechanistic models have been derived that predict the frequency of interaction, spatial overlap and resource loss to neighbours.⁴⁸ In terms of allocation in captivity it has been suggested that there are no specific spatial needs, only behavioural needs that, in order to be met, require a certain amount and quality of space.^49,50 Housing design, therefore, requires careful consideration of not only absolute floor area but also perimeter length and perimeter length-to-floor area ratio, length-to-width ratio and position of resources.⁵⁰ To date, our understanding of all these elements and their interactions across species is poor. Quality of space also varies with cage shape, enrichment provision and environment, as well as population density and composition.⁵⁰ There have been numerous studies on the addition of enrichment material and devices to mouse caging (reviewed in ref. 51); however, many of the other aspects of quality of space appear to have not been studied in laboratory rodents. A particularly interesting research question, given the mouse's thigmotaxic tendency, surrounds the use of space in cages with increased numbers of walls or perimeter length. The ethological needs of mice include resting, grooming, exploration, gnawing, nesting, hiding and social interaction, so at a minimum, consideration should at least be given to providing cage design or furniture which allows performance of these behaviours.⁵² Given the complexity of this issue, legislators tend to use a necessarily simplistic approach of specifying minimum cage floor areas with no specific mention of which behaviours can be achieved in such a space. Use of this method makes the scientific data available relating to floor area important, although our knowledge in this area is incomplete.

Space allocation per animal

The question of the effects of space per animal on welfare indicators is probably most pertinent to legislators, given the wording of, and emphasis placed on this in current regulatory documents.⁶ However, this appears to be the least researched aspect of cage size. A handful of studies have maintained group size to assess the effects of floor space alone on parameters which include behaviour, the hypothalamic–pituitary–adrenal (HPA) axis, and measures of biological function. Adrenal cortex activity produces substrates for fright and flight, such as amino and fatty acids, mediated through the production of glucocorticoids such as corticosterone.⁵³ As such, it can be a useful objective indicator of welfare state provided care is taken over interpretation.²³ Whitaker et al. carried out studies investigating reproductive indices with varying space allocations. In this study, breeding trios of C57BL/6Tac mice were housed in small (55 cm²) or large (77 cm²) individually ventilated cages and reproductive data were compared. Parameters included; litter size, litter survival to weaning, inter-birth interval and pup weight. No significant differences were found in any of the measured indices, with cage size, although there was a slight trend towards lower body weights and longer inter-birth interval in the larger cages.⁵⁴ In a later study, with a larger sample size, the previous trend towards lower body weight of pups in larger cages attained significance. Two theories were suggested for this: that females showed increased activity in larger cages, and therefore spent less time nursing, or that pups showed more activity and therefore spent less time nursing or had greater energy expenditure. These studies also involved behavioural testing of the pups and found no differences related to housing conditions.⁵⁵ Davidson et al. also chose to focus on behavioural testing of pups reared in different sized cages and failed to find any consistent link between space and anxiety behaviour in Swiss Webster (Cr:SW) mice. Mice were assessed using the open field, elevated plus maze and light–dark exploration tests.⁵⁶

In a study researching interactions between both space and ambient temperature on aggression, the authors used an aggression score method (based on number of bites inflicted on another animal) to characterize this aspect of behaviour in groups of eight C57BL/10J and A/J male mice. No significant interaction was found between aggression and cage size. This study did however use space allowances much larger than the laboratory setting, with a range from 900 to 3800 cm².⁵⁷ However, one study evaluated both animal density and space effects in male BALB/c AnNCrlBr mice, in space allocations more likely to be employed in standard caging systems (Table 2). Consistent correlations were found between space and aggression, namely that duration of agonistic behaviour and the number of wounds increased as space increased.⁵⁸ This may have been due to a crowding effect at the lower population densities, with a possible curvilinear relationship between density and aggression. Initially, as space decreases, aggression will increase as an individual's territory is invaded more frequently. However, as crowding continues the space may become too small for the dominant mouse to form a defendable territory thus decreasing aggression.⁵⁸

Table 2
Summary of published effects of housing density on indicators of wellbeing in mice

Reference Mouse strain/stock (sex) Space allocation per mouse in² (cm²) Mice per cage Type of cage Enrichment Study duration Endpoints Result with increased cage density

Christian⁵⁹ NMRI, house mice (M) 142.5, 36,18, 9, 4.5142.5, 47.5, 36, 18, 15.8, 8.3 1, 4, 8, 16, 32 NMRI1, 3, 4, 6, 8, 9, 17 house mice Metal Unknown 1 week Adrenal weightsBody weightThymusPreputial gland weightSeminal vesicles lengthTestes weight/length IncreasedNo effectNo effectDecreasedDecreasedDecreased

Bronson^60,61 C57BL/10J and Peromyscus (M) – 1, 2, 4, 8 Unknown Unknown 1 week Adrenal weightsAbscorbic acid content adrenals Increased (no effect in Peromyscus)Decreased (no effect in Peromyscus)

Southwick⁶² CFW (M) 66.5, 38, 19, 9.5 1, 4, 8, 16 Metal Shredded paper 1 week Adrenal weightsBody weightSeminal vesicle weightsTestes weight No effectNo effectDecreasedNo effect

Chvedoff⁶³ Crl, CDl, ICR, BR (M, F) (567), (283.5), (141.8), (70.9) 1, 2, 4, 8 Makrolon solid-floored with wire top None 18 months Food consumptionBody weightBody weight varianceIncidence of gastritisTumour incidence DecreasedDecreasedDecreasedIncreasedNo effect

Peters⁶⁴ BALB/c, MF1 (M, F) (55), (33) 6, 10 Standard plastic (M2 and MB1: North Kent plastics) None 6 weeks Body weightAdrenal weight No effect (strain effects though)No effect (strain effects though)

O'Malley⁶⁵ ICR (F) 65 (419.25) Dam + intact litter or Dam + 6 pups IVC (Tecniplast) Cotton nestlets or wire grid insert with absorbent paper below (first generation animals) Three generations of breeding Dam body weight (delivery)Number of pups weanedGrowth rate of pupsFaecal corticosteroneSubsequent reproductive performance No effectNo effectNo effect (following adjustment for pups weaned)No effectNo effect

Brain⁶⁶ TT (M) (588), (294), (147), (73.5), (36.75) 1, 2, 4, 8, 16 Makrolon Unknown 3 weeks Plasma corticosteroneAdrenal weightTestes weightProstate weight Increased (pairs exception)Increased (pairs exception)DecreasedDecreased

Nicholson⁶⁷ C57BL/6J, BALB/cJ (M, F) 12.9, 8.6, 6.5 4, 6, 8 IVC duplex cage (Thoren) None 16 weeks (BL/6), 17 weeks (BALB/c) Faecal corticosteroneAnxiety/barbering Growth rateAdrenal gland sizeBody composition and bone density (DEXA)FSH, LH, testosteroneAutonomic response (telemetry) IncreasedIncreasedReducedIncreasedNo effectNo effectNo effect on heart rate or body temperature (increased activity intermediate densities)

Laber⁶⁸ C57BL/6, BALB/c (F) 37.5, 15, 7.5 2, 5, 10 IVC (Lab Products) Cotton nestlets 70 days Plasma corticosteroneWeight gainOpen field locomotor behaviourHelper T-cell subpopulations Increased Decreased (not BL6)DecreasedDecreased (not BL6)

Peng⁶⁹ BALB/c (M) (195), (97.5), (49) 2, 4, 8 Stainless steel None 2 weeks Plasma corticosteroneLymphocyte numbersLymphocyte subpopulations Increased DecreasedNo effect

Ortiz⁷⁰ OF1 (M) (132), (66), (22) 4, 8, 24 Unknown Unknown 30 days Body weight gainPlasma corticosteroneAdrenal weightExploratory activity DecreasedNo effectNo effectNo effect

Anton⁷¹ DUB/ICR (M, F) 73.5, 10.5, 5.25, 2.6 1, 7, 14, 28 Metal Unknown 1 week (females), 4 weeks (males) Adrenal weightsBody weightTissue catecholaminesBehaviour No effectNo effectNo effectIncreased physical contact and activity

Van Loo⁵⁸ BALB/c AnNCrlBr (M) (80) or (125) (factorial design) 3, 5, 8 Perspex solid-floored with wire top None 14 weeks Aggressive behaviourUrine corticosteroneTestosterone levelsOrgan weightsTyrosine hydroxylase IncreasedNo effectNo effectNo effectNo effect

Reiss⁷² 129, BALB/c ByJ, C57BL/6J (M) (578), (82.5) 1, 7 IVC (M.I.C.E type) None 13 weeks Open fieldElevated plus mazeAcoustic startleNociceptin transmit expression Reduced locomotor activity and access to openDecreased % open arms timeExaggerated startle responseIncreased

FSH: follicle-stimulating hormone; LH: luteinizing hormone

A study by Fullwood et al. examined four space allowances (ranging from approximately 30 to 129 cm²) in groups of three, male 4–5-week-old C57BL/6 mice. Measures included plasma corticosterone levels, mortality and immune measures (adrenal and spleen weights, natural killer (NK) cell activity, mitogen-induced lymphocyte proliferation and differential blood cell counts). Results were somewhat contradictory, with adrenal responses being greater in conditions of reduced space, while lymphocyte proliferation and NK cell activity was greater in mice given less space, implying that immune responsiveness was improved at lower space allowance. Mortality, due to aggression, was also higher with increased space allowance. The authors reconciled the results by concluding that limited space promotes improved mouse welfare, with the caveat that more studies in this area were needed before drawing firm conclusions.⁷³ In a different inbred strain (BALB/c), a severe restriction of space (at approximately one-third of the recommended level) largely failed to cause any differences in body weight gains, feed and water consumption or immunological measures in both sexes.⁷⁴

One behavioural test commonly applied in welfare science investigations is the preference test. Traditional tests provide the animal with a choice between two conditions favourable to the same behaviour. Motivation testing using consumer demand theory compares motivation to work for different resources, one of which is usually highly valued, for example, food.⁷⁵ Use of preference testing in this area of research does not appear to have been applied to any great extent. However, Sherwin used the consumer demand theory approach to determine whether C57BL females were prepared to work for additional space of 319, 777 or 1600 cm². Cost of the visits was increased by increasing the number of lever presses needed to gain access to the additional space. As the cost of visits increased, the mice continued to work for access to the added space, although the numbers of visits and the time in the space were shown to decrease. The author concluded that mice were highly motivated to work to gain access to this additional space with elasticity coefficients being similar to those reported for other essential resources such as social contact and water. However, elasticity coefficients for all space values were similar indicating that despite the high strength of motivation for additional space, the mice did not differentiate between the differing values available.⁷⁶

Animal density

A number of studies have been performed to investigate the effects of animal density on a broad range of indicators suggestive of ‘ability to cope’ (summarized in Table 2). These include measures of stress hormone activation, behaviour, immune function and biological function, such as reproductive indices and growth rate.

Some of the early studies in this area concentrated on the concept of ‘crowding’. This idea has been introduced to describe movement or activity restriction caused by the physical presence of others.⁷⁷ Early studies in this area were performed by Christian who compared males of the albino NMRI strain with laboratory-raised house mice housed in a fixed cage size at varying population densities. Group numbers ranged from singly-housed animals to 32 animals in a single cage. Measures taken included weights of a number of organs on postmortem: adrenal glands, thymus, testes and accessory sex glands, as well as mouse body weight. The author commented on fighting behaviour in the groups but did not analyse this as part of the study. In both genetic backgrounds, it was shown that weight of the adrenal glands increased with crowding (although this reached a peak at intermediate animal levels and then started to decline). This decline was hypothesized to be due to a breakdown in social organization due to a failure of individual animal recognition. Other significant results included: a correlation between crowding and decreased preputial weight – presumably attributable to a decline in androgenic activity, and a decrease in length of the seminal vesicles and testes. The author concluded that crowding animals to the extent performed here produced a physiological stress response and had negative effects on reproduction.⁵⁹ Similarly, Bronson showed crowding to increase adrenal gland weights in male C57BL/10J mice^60,61 and decrease ascorbic acid content of the adrenals (another index of adrenal function). These values were not statistically altered in Peromyscus. These experiments were designed to evaluate differences in adrenal stress response in two species known to differ genetically in aggressiveness. The results support the conclusion that crowding elicits greater response in the more aggressive animals, i.e. C57. Conversely a study done by Southwick, reproducing the study of Christian almost exactly, found no effect of crowding on adrenal gland weight or testicular weight.⁶² Peters similarly reported no effect of cage density on adrenal weights. However, the addition of females to one experiment did show that there was an increase in fertility (as evidenced by pregnancy rates) with intermediate housing densities.⁶⁴

A number of studies have examined density effects on measures of biological function such as reproduction or growth rates. While the use of such parameters alone is often controversial as an indicator of wellbeing (since many animals may continue to grow well and reproduce under conditions assumed to be stressful, such as in poor health or with supportive evidence of physiological responses to stress), they can provide some useful data when examined in conjunction with other welfare indices. One study using relatively large space allowances per animal showed an inverse relationship between increasing density and body weight, food consumption and body weight variance across a number of strains and both sexes. Interestingly, despite the decrease in food consumption at higher densities, the corresponding decrease in body weight was relatively small. This was hypothesized to be due to the ability of more densely housed animals to huddle, thereby reducing heat loss and caloric expenditure. Another negative effect was the increase in gastritis lesions with crowding. This was presumed to be due to the well-described effects of stress on gastric inflammation and ulceration.⁶³ Housing of lactating females and their pups was examined to evaluate measures of reproductive performance, such as pup growth rate and numbers weaned. The authors subsequently examined these measures in the second generation. Animal numbers were obtained by either keeping a dam with her intact litter, or culling pups to form a group consisting of the dam and six pups. No significant differences were found in any of the parameters measured, suggesting that density (in females at least) had no effect on reproductive efficiency.⁶⁵

A number of other studies have explored the effects of housing density on the hypothalamic–pituitary axis, generally determined by measurement of the steroid hormone corticosterone, either in free form or excreted as a metabolite via faeces or urine. A number of these studies have recorded behavioural observations or carried out behavioural tests in conjunction with this physiological measure. The findings of Brain et al. concurred with those of Christian. As animal density increased, there was an increase in size of those organs related to the stress response, and a corresponding decrease in reproductive organ weights in males. In addition, plasma corticosterone measurements were taken and these also showed an upward trend with increased density. Interestingly, this study showed differences in pair-housed animals compared with all other treatment groups with increased adrenocortical activity and a greater decline in gonadal activity compared with all other groups. It was postulated that this was due to the subordinate animal being subject to all the social stresses imposed by the dominant animal in pair groups, as opposed to a greater distribution of stress between subordinates in larger groups. The author also commented on the difficulty in attributing the increased stress in larger groups to be a result of space alone, given there were likely to also be a higher number of social interactions.⁶⁶ Results from a number of other studies agree with this finding that adrenocortical axis activity is increased as group size grows.^67–69 However, evaluation of hormonal products has produced some conflicting results, with one study showing no effect of density on plasma corticosterone⁷⁰ and another showing no effect of crowding on measured catecholamines, e.g. epinephrine and norepinephrine (another sensitive indicator of acute stress).⁷¹ However, Ortiz took the study one step further and showed that crowded male animals did show an increased adrenocortical response to induced stress such as that caused by noise and forced swimming. An adrenocorticotropic hormone stimulation test was performed and the production of similar levels of corticosterone in both housing densities supported the author's hypothesis that a pituitary–adrenal hyper-reactivity occurred in crowded groups as a result of the chronic stress elicited.⁷⁰

Some studies have investigated various subsets of behaviour to ascertain emotional state. Aggression has been a focus of a few studies in mice. This area obviously needs care in interpretation due to the wide phenotypic variation in performance of this according to strain but it is rightly assumed to be a major cause of poor welfare in animals when they are on the receiving end of aggressive acts. However, evidence in this area is contradictory. Van Loo et al. studied male BALB/c AnNCrlBr mice in two space allocations and with three group sizes. This was a well-controlled study, which fully considered the complex interaction between space and group size, which commonly leads to confounding of data. A factorial study design, making use of space and group size as input factors, was used to overcome this issue.⁵⁸ An assessment was made of injury scores and both incidence and duration of various behaviours interpreted as agonistic (such as chasing and biting) and defensive (flee and defensive posture adoption). Increased group size was found to be significantly positively correlated with behavioural acts of aggression and increased injury to animals. However, at odds with this finding; organ size measures, testosterone levels and urinary corticosterone showed no correlation with group size. This implied that that there was no measured physiological mediator as a precursor, or result of these behavioural events. Two studies found no effect of density on aggression in males and females of C57BL/6J,⁷⁸ BALB/cJ, NOD/LtJ and FVB/NJ strains.⁷⁹ There were, however, changes in some microenvironmental gases, such as ammonia and carbon dioxide, but these did not stray above recommended guidelines and appeared to have no effect on the animals based on histopathological examination of the eyes and nasal passageways.

Anxiety behaviour has been assessed using a battery of tests to determine whether there were any effects of crowding on this important research and animal wellbeing measure. Such tests include open field, elevated plus maze and acoustic startle. In general, anxiety behaviour increased at higher densities in both sexes, as shown by reduced exposure to open space or increased startle. This was influenced somewhat by strain and no intermediate group sizes were measured (only singly housed and 7 per group) which makes it difficult to extrapolate this to everyday husbandry recommendations.⁷² Single housing leads to marked behavioural and physiological effects alone, which mask effects attributable to space allowance.^80,81 Nevertheless, this provides interesting evidence for the effect that group size might have, not only on the animals themselves, but on possible effects on research using such behavioural assessment tools.⁷²

Conclusion

Interpretation of the results underpinning the current regulatory framework is challenging. The complexity of experimental designs used and the conflicting nature of some of the results contribute to the lack of clear outcomes. Some studies have also used space allocations or group compositions which are significantly different from those being used routinely in laboratory animal management. Thus, applying the findings to the latter situation is problematic. Broadly speaking, scientific evidence suggests that space per se has little effect on reproductive parameters^54,55 and that increasing individual space allowance may actually increase aggressive encounters at the values typically used in the laboratory setting.^58,73 Thus, in terms of the biological function approach to animal welfare, reduced space allocation may have little impact on animal wellbeing. However, evidence from the single study evaluating space from a ‘feelings-based’ viewpoint suggests that mice are highly motivated to work for increased space. Many welfare scientists would suggest that this finding alone provides the greatest justification for provision of increased space as a husbandry input. The confounding of space with density, and the relative lack of literature on space allocation, means we probably still lack an answer to the question of whether floor area actually has any effect on mouse wellbeing. Additionally, there is a dearth of studies that have assessed positive emotion or mouse preference and this is a priority area for the focus of further studies.

Studies examining the effects of animal density on welfare parameters are more numerous, and while results are still contradictory, inferring trends is simpler. Increased density of animals leads to effects on the HPA axis, including a stress response and reduced immune function,^63,66–69 increases aggressive behaviour⁵⁸ and may have effects on reproductive parameters particularly in male animals.^59,62,66 The varying study designs of the research to date do not allow any conclusions to be drawn on the effects of sex, age or strain on space requirements. Given the diversity of stocks and strains used in research and the well-known differing predispositions to aggression among them, strain may be a far greater influence on space inputs than size, weight or age of the animal.

Additional well-controlled experiments in this area are needed. These studies should consider the confounding effects of increasing group size on studies into space allocation, and make provisions to control this in the experimental design by ensuring group size remains constant. Other confounding variables that need to be controlled include the use of environmental enrichment and growing animals. Given that cage complexity is likely to affect space needs, standardization or preferably removal of enrichment will allow for more accurate interpretation of experimental results. As a priority, further study into the effects of floor area in mice is needed as this will provide a basis for further studies on group size, social dynamics and aspects of quality of space. This work should take into consideration the effects of sex, age and strain on provision of space. Further to this, study into mouse preference using the ‘feelings-based’ approach to welfare study would add an interesting dimension and give weight to any recommendations arising as a result. These investigations could include further use of operant testing to determine animals’ propensities to ‘work for’ extra space, when compared with a highly valued commodity such as food. The use of a recently developed test measuring cognitive basis, i.e. the animal's ‘positivity or negativity’ based on their emotional state could also be applied to studies in this area.⁸² This strategy has the potential to give real insight into husbandry effects on mouse welfare, based on the animal's perception of the world.

As alluded to by the regulatory documents, the scientific literature suggests that area as an input is probably of much less concern than the quality of space and the ability of the animal to use the space available to carry out a full behavioural repertoire, especially in this thigmotaxic prey species. While it would be ideal to include guidelines relating to the former in regulatory documents, areas such as these are more difficult to be prescriptive about in comparison with cage area inputs. Having performed preliminary investigations into space requirements, scientists will be in a much better position to inform legislation, and to investigate aspects of quality of space such as: cage shape, cage furniture and other enrichment forms. Long-term outcomes of this research should include the provision of modified housing for laboratory animals based on science-based evaluation and with consideration for the animal's behavioural biology.

Reference	Animals	Weight (g)	Floor area/animal cm² (in²)	Height cm (in)	Comments
EC Directive 2010/63/EU (2010)²	Mice in groups	<2020–2525–30>30	607080100	12121212
Code of Practice for the Housing and Care of Animals used in Scientific Procedures (UK,1989)⁷	Mice in groups	<30≥30	60100	12
	Mice singly housed		200	12
Code of Practice for the Housing of Animals in Designated Breeding and Supplying Establishments (UK,1995)⁸	Monogamous pair		300	12	For each additional female plus litter an additional 180 cm² should be added
	Trio		300	12	For each additional female plus litter an additional 180 cm² should be added
The Guide (USA, 2011)¹	Mice in groups	<10Up to 15Up to 25>25	(6) 38.7(8) 51.6(12) 77.4≥(15) 96.7	(5) 12.7(5) 12.7(5) 12.7(5) 12.7	Larger animals may require more space
	Female +litter		(51) 330 (space for the housing group)	(5) 12.7	Space may vary due to other considerations such as age and size of litter
NHMRC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (2004)³	No specific values given (NB some States and Territories provide their own guidelines)
Canadian Council on Animal Care Guide (1993)⁹	Mice in groups	<20>20	65100	1315
	Female+litter		160
	Mice singly housed		200	12
Guidelines for Proper Conduct of Animal Experiments (Japan, 2006)⁴	No specific values given
Guidelines on the Care and Use of Animals for Scientific Purposes (Singapore, 2004)¹⁰	Mice in groups	<10Up to 15Up to 25>25	385177>96	12121212

Reference	Mouse strain/stock (sex)	Space allocation per mouse in² (cm²)	Mice per cage	Type of cage	Enrichment	Study duration	Endpoints	Result with increased cage density
Christian⁵⁹	NMRI, house mice (M)	142.5, 36,18, 9, 4.5142.5, 47.5, 36, 18, 15.8, 8.3	1, 4, 8, 16, 32 NMRI1, 3, 4, 6, 8, 9, 17 house mice	Metal	Unknown	1 week	Adrenal weightsBody weightThymusPreputial gland weightSeminal vesicles lengthTestes weight/length	IncreasedNo effectNo effectDecreasedDecreasedDecreased
Bronson^60,61	C57BL/10J and Peromyscus (M)	–	1, 2, 4, 8	Unknown	Unknown	1 week	Adrenal weightsAbscorbic acid content adrenals	Increased (no effect in Peromyscus)Decreased (no effect in Peromyscus)
Southwick⁶²	CFW (M)	66.5, 38, 19, 9.5	1, 4, 8, 16	Metal	Shredded paper	1 week	Adrenal weightsBody weightSeminal vesicle weightsTestes weight	No effectNo effectDecreasedNo effect
Chvedoff⁶³	Crl, CDl, ICR, BR (M, F)	(567), (283.5), (141.8), (70.9)	1, 2, 4, 8	Makrolon solid-floored with wire top	None	18 months	Food consumptionBody weightBody weight varianceIncidence of gastritisTumour incidence	DecreasedDecreasedDecreasedIncreasedNo effect
Peters⁶⁴	BALB/c, MF1 (M, F)	(55), (33)	6, 10	Standard plastic (M2 and MB1: North Kent plastics)	None	6 weeks	Body weightAdrenal weight	No effect (strain effects though)No effect (strain effects though)
O'Malley⁶⁵	ICR (F)	65 (419.25)	Dam + intact litter or Dam + 6 pups	IVC (Tecniplast)	Cotton nestlets or wire grid insert with absorbent paper below (first generation animals)	Three generations of breeding	Dam body weight (delivery)Number of pups weanedGrowth rate of pupsFaecal corticosteroneSubsequent reproductive performance	No effectNo effectNo effect (following adjustment for pups weaned)No effectNo effect
Brain⁶⁶	TT (M)	(588), (294), (147), (73.5), (36.75)	1, 2, 4, 8, 16	Makrolon	Unknown	3 weeks	Plasma corticosteroneAdrenal weightTestes weightProstate weight	Increased (pairs exception)Increased (pairs exception)DecreasedDecreased
Nicholson⁶⁷	C57BL/6J, BALB/cJ (M, F)	12.9, 8.6, 6.5	4, 6, 8	IVC duplex cage (Thoren)	None	16 weeks (BL/6), 17 weeks (BALB/c)	Faecal corticosteroneAnxiety/barbering Growth rateAdrenal gland sizeBody composition and bone density (DEXA)FSH, LH, testosteroneAutonomic response (telemetry)	IncreasedIncreasedReducedIncreasedNo effectNo effectNo effect on heart rate or body temperature (increased activity intermediate densities)
Laber⁶⁸	C57BL/6, BALB/c (F)	37.5, 15, 7.5	2, 5, 10	IVC (Lab Products)	Cotton nestlets	70 days	Plasma corticosteroneWeight gainOpen field locomotor behaviourHelper T-cell subpopulations	Increased Decreased (not BL6)DecreasedDecreased (not BL6)
Peng⁶⁹	BALB/c (M)	(195), (97.5), (49)	2, 4, 8	Stainless steel	None	2 weeks	Plasma corticosteroneLymphocyte numbersLymphocyte subpopulations	Increased DecreasedNo effect
Ortiz⁷⁰	OF1 (M)	(132), (66), (22)	4, 8, 24	Unknown	Unknown	30 days	Body weight gainPlasma corticosteroneAdrenal weightExploratory activity	DecreasedNo effectNo effectNo effect
Anton⁷¹	DUB/ICR (M, F)	73.5, 10.5, 5.25, 2.6	1, 7, 14, 28	Metal	Unknown	1 week (females), 4 weeks (males)	Adrenal weightsBody weightTissue catecholaminesBehaviour	No effectNo effectNo effectIncreased physical contact and activity
Van Loo⁵⁸	BALB/c AnNCrlBr (M)	(80) or (125) (factorial design)	3, 5, 8	Perspex solid-floored with wire top	None	14 weeks	Aggressive behaviourUrine corticosteroneTestosterone levelsOrgan weightsTyrosine hydroxylase	IncreasedNo effectNo effectNo effectNo effect
Reiss⁷²	129, BALB/c ByJ, C57BL/6J (M)	(578), (82.5)	1, 7	IVC (M.I.C.E type)	None	13 weeks	Open fieldElevated plus mazeAcoustic startleNociceptin transmit expression	Reduced locomotor activity and access to openDecreased % open arms timeExaggerated startle responseIncreased

Footnotes

Acknowledgement

Professor Gordon S Howarth is supported by the Sally Birch Cancer Council Australia Senior Research Fellowship in Cancer Control.

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Effects of space allocation and housing density on measures of wellbeing in laboratory mice: a review

Abstract

Keywords

Animal welfare assessment

Range in wild Mus musculus

Animal space use

Space allocation per animal

Animal density

Conclusion

Footnotes

Acknowledgement

References