The effect of early life experience, environment, and genetic factors on spontaneous home-cage aggression-related wounding in male C57BL/6 mice

Abstract

Aggression is a major welfare issue in mice, particularly when mice unfamiliar to each other are first placed in cages, as happens on receipt from a vendor, and following cage cleaning. Injuries from aggression are the second leading cause of unplanned euthanasia in mice, following ulcerative dermatitis. Commonly employed strategies for reducing aggression-related injury are largely anecdotal, and may even be counterproductive. Here we report a series of experiments testing potential explanations and interventions for post-shipping aggression-related injuries in C57BL/6 mice. First, we examined the effects of weaning: testing whether manipulating weaning age reduced aggression-related injuries, and if repeated mixing of weaned mice before shipping increased these injuries. Contrary to our predictions, repeated mixing did not increase post-shipping injurious aggression, and early weaning reduced aggression-related injuries. Second, we examined potential post-shipping interventions: testing whether lavender essential oil applied to the cage reduced aggression-related injuries, and whether a variety of enrichments decreased injurious aggression. Again, contrary to predictions, lavender increased wounding, and none of the enrichments reduced it. However, consistent with the effects of weaning age in the first experiment, cages with higher mean body weight showed elevated levels of aggression-related wounding. Finally, we tested whether C57BL/6 substrains and identification methods affected levels of intra-cage wounding from aggression. We found no effect of strain, but cages where mice were ear-notched for identification showed higher levels of wounding than cages where mice were tail-tattooed. Overall, these results emphasize the multifactorial nature of home-cage injurious aggression, and the importance of testing received wisdom when it comes to managing complex behavioral and welfare problems. In terms of practical recommendations to reduce aggressive wounding in the home cage, tail tattooing is recommended over ear notching and late weaning should be avoided.

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Mice are the most commonly used mammal in biomedical research,
and are typically group-housed for good reasons. Group housing is
important for the welfare of social animals such as mice and is man-
dated by law in many countries. Furthermore, external predictive
validity (to humans) and internal construct validity1 require that
animal experiments be performed on a background of good general
physical and mental health2. For instance, if a human patient has
a stable environment and abundant social support, then an animal
patient should receive the same. Besides its other welfare benefits,
support from conspecifics markedly improves health outcomes, and
therefore model quality, in mice3–5. However, social housing often
gives rise to aggression, one of the most serious welfare concerns in
laboratory mouse husbandry6.
Severe fighting can lead to pain, injury and death. Daily visual
inspections may fail to detect aggression problems until they have
become quite severe, as mice can inflict extensive wounds rapidly.
Both injured and very aggressive mice are generally separated and
may be euthanized. These problems could be avoided by housing all
mice singly7,8, although this is clearly not optimal for welfare either.
Ultimately, injuries, deaths and social isolation directly conflict with
the 3Rs goals of reduction and refinement.
Aggression is not only an animal welfare concern, but may also
compromise the scientific process. First, aggression can inflate the
number of animals required to achieve sufficient statistical power.
Deaths or separations as a result of aggression can undermine exper-
imental design by altering the numbers of groups and of animals per
cage. Aggression, pain and social isolation can also change several
physiological parameters, particularly immune function9,10. Thus,
working with animals that are severely socially stressed, wounded or
singly-housed as a result of aggression creates additional variability
that can reduce statistical power and may reduce external valid-
ity11. Second, fighting injuries and risks of aggression may compli-
cate or render unfeasible certain research procedures. Third, some
researchers may only use females in an attempt to avoid aggres-
sion12. This can lead to sex differences being overlooked, and runs
counter to US federal policy that now requires that studies include
both sexes where relevant13.
Group housing without severe aggression is therefore the ideal
from welfare and scientific standpoints. However, problematic
aggression persists despite some general recommendations produced
by the few studies attempting to find solutions to the problem14,15.
We begin by examining the contexts in which aggression has been
1Stanford University, Department of Comparative Medicine, Stanford, California, USA. 2Purdue University, Animal Science Department, West Lafayette, Indiana, USA.
3Harvard University Faculty of Arts and Sciences, Oce of Animal Resources, Cambridge, Massachusetts, USA. 4University of Washington, Department of Comparative
Medicine, Seattle, Washington, USA. 5(By courtesy) Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, California, USA. Correspondence
should be addressed to E.M.W. (elweber@stanford.edu).
Aggression in group-housed laboratory mice: why
can’t we solve the problem?
Elin M. Weber1, Jamie Ahloy Dallaire1, Brianna N. Gaskill2, Kathleen R. Pritchett-Corning3,4 & Joseph P. Garner1,5
Group housing is highly important for social animals. However, it can also give rise to aggression, one of
the most serious welfare concerns in laboratory mouse husbandry. Severe fighting can lead to pain, injury
and even death. In addition, working with animals that are severely socially stressed, wounded or singly-
housed as a result of aggression may compromise scientific validity. Some general recommendations on
how to minimize aggression exist, but the problem persists. Thus far, studies attempting to find solutions
have mainly focused on social dominance and territorial behavior, but many other aspects of routine
housing and husbandry that might influence aggressive behavior have been overlooked. The present
way of housing laboratory mice is highly unnatural: mice are prevented from performing many species-
typical behaviors and are routinely subjected to painful and aversive stimuli. Giving animals control over
their environment is an important aspect of improving animal welfare and has been well-studied in the
field of animal welfare science. How control over the environment influences aggression in laboratory
mice, however, has not been closely examined. In this article, we challenge current ways of thinking and
propose alternative perspectives that we hope will lead to an enhanced understanding of aggression in
laboratory mice.
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158 Volume 46, No. 4 | APRIL 2017
studied and then summarize what is currently known about the
causes of aggression before turning to remaining areas of uncer-
tainty. We end by discussing directions for future research. We hope
to challenge current ways of thinking and propose alternative per-
spectives that will lead to an enhanced understanding of aggression
in laboratory mice.
How aggression has been studied in mice
Our current understanding of mouse aggression is based on three
distinct areas of the ethological literature. First, social interac-
tions and relationships have been studied in free-ranging mice
and mice housed in naturalistic settings. Second, aggression and
social defeat have been studied using staged encounters between
unfamiliar mice. Finally, dominance and aggression have been
studied in social groups of caged mice, which represent typical labo-
ratory conditions.
In the wild, the social organization of mice varies depending
on local resource availability and distribution. Studies conducted
in semi-natural enclosures have identified social organizations
ranging from situations in which males individually defend estab-
lished territories and attack male intruders (females may move
freely between males’ territories), to those in which multiple males
occupy the same area and maintain dominant-subordinate rela-
tionships, and others in which a minority of males defend territo-
ries while males that do not have territories co-exist more or less
peacefully in a ‘no-man’s land’ between defended territories16–18.
Thus, the nature and severity of aggression in wild and feral mice
seems to depend on their social organization. Dominance requires
the establishment and maintenance of social relationships, which
are characterized by animals recognizing and consistently behav-
ing differently and adaptively toward different individuals. In con-
trast, territorial aggression need not involve social relationships,
with mice using olfactory and behavioral signals (for example,
tail rattling) indiscriminately toward all intruders. Each context
can involve mediated aggression (threats that are terminated by
submissive behavior or fleeing, without physical damage) and esca-
lated aggression (actual attacks or retaliation involving biting)19,
although the exact behaviors and signals may differ by context.
Mice are also used extensively as a model species to study
aggression and social defeat. This literature has focused on terri-
torial aggression, exemplified by the resident-intruder test, which
measures the latency for a ‘resident’ mouse to attack an unfamiliar
‘intruder’ mouse introduced into the resident’s home cage20. This
reliably induces aggression, at least in males, specifically because
it exploits ethological principles of territory ownership, defense
and territorial aggression. Other protocols pair unfamiliar mice in
a neutral testing chamber, often adding stimuli known to induce
aggression (for example, shock-induced aggression)21. Because the
chamber is neutral, it is assumed that ownership has not been estab-
lished and territorial aggression is unlikely. Instead, these protocols
are designed to induce stress, emotional conflict and frustration;
aggression is often used as a readout of these states, rather than as
the focus of the study.
Finally, some researchers have studied aggression in long-term
group-housed animals, which is the context of this article (for exam-
ple, see refs. 7,8,22,23). In the wild, the smallest mouse territories
are seen when mice live commensally in human dwellings; these
territories typically measure approximately 2 m2 (ref. 24). Mice do
not use this space evenly, however. Only some parts of a defensible
territory will be used regularly, such as a sleeping area. A standard
mouse cage housing 4–5 same-sex adults provides around 0.0525 m2
of floor space. We do not know whether mice perceive this environ-
ment as sharing 2.6% of the minimum space they would inhabit in
the wild, or individually occupying as little as 0.5% of that space.
Their behavioral and genetic flexibility and adaptability is one of
the primary reasons mice have become the most commonly used
animal in research, and laboratory mice have been selected that
reproduce and survive under these conditions. However, we do not
know whether they can respond to such extreme space limitations
without becoming fundamentally abnormal.
Because mice are not free to spatially disperse in the laboratory
setting, it is difficult to establish what kind of social organization
they maintain. Mice rarely meet unfamiliar mice in their home
cage, suggesting that territorial aggression is not a major factor.
One way to cope with living together in a cage might be to form a
dominance hierarchy; a breakdown of which could be one cause
of injurious escalated aggression19. However, it is unclear whether
cases in which aggression does break out represent a failure of
dominance relationships to mediate aggression (for example, lack
of appropriate submissive behavior or recognition thereof, lead-
ing to escalated aggression), whether this is more closely related
to territorial aggression (for example, one mouse establishing a
territory and perceiving its cage mates as trespassers), or if it is
a result of frustration or pain (for example, different animal ID
methods, such as ear tags, trigger different risks of aggression25).
Furthermore, we caution against the simplistic view of a fixed
pecking order: real animals have complex social structures that are
not linear, transitive or consistent across resources, context or time,
which makes measuring dominance a non-trivial and sometimes
irrelevant task, as the outcome of such measures might not accu-
rately reflect the social dynamics in a group of undisturbed mice26.
Finally, the social organization of lab mice could potentially be
closer to that of males living in the no-man’s land between other
males’ defended territories18: a type of social organization that
has been universally overlooked in the mouse housing literature.
These mice are in poor health and physical condition and live in
a state of constant scramble competition, but do crowd into nest
sites at densities similar to the lab environment. If this is how lab
mice perceive their housing, then aggression might result from
scramble competition for territory or other resources cued by
salient changes in the environment (such as cage change), and if
their physiology is affected as well, this raises additional welfare
and quality-of-science concerns.
What we have learned about aggression in laboratory mice
Group composition modulates aggression in mice under normal
husbandry conditions. Most studies of group composition manipu-
late stocking density, without considering the different influences
of group size and cage size. Only one study has properly teased
out these two effects to show that aggression is affected by group
size and not by cage size23. Thus, aggression levels increase with
group size, particularly in excess of three individuals in a standard
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LabAnimal Volume 46, No. 4 | APRIL 2017 159
shoebox cage23. Establishing stable groups, such as keeping sib-
lings or familiar mice together from weaning, generally decreases
aggression7,27. Large differences in aggression also exist between
strains, both in the resident-intruder test28 and in the home cage29,
indicating that there is a possible genetic component.
In terms of the physical environment, temperature and bedding
material may be important. Mice in the wild suppress territorial
aggression in cold months18, and mice show marked increases in
aggression in the lab as temperatures increase from 20 to 25 °C29.
Considering bedding material, mice show preferences for certain
types of bedding30, which may affect physical comfort; bedding may
also have unexpected endocrine effects—corncob bedding contains
estrogen disruptors that increase aggression in resident-intruder
tests in Peromyscus californicus31.
In terms of enrichment, transferring nesting material at cage
cleaning decreases aggression, whereas transferring soiled bedding
increases it14. However, the literature on structural enrichments,
such as shelters, is mixed32, with studies reporting both increases
and decreases in aggression with structural enrichments. This topic
is plagued with untested received wisdom, particularly the idea that
shelters with multiple entry points do not cause aggression. In fact,
providing a single multi-entrance shelter per cage can cause large
increases in escalated aggression19.
Given these findings, recommendations for minimizing aggres-
sion can be organized in a hierarchy of ease of implementation and
strength of evidence. Minimum standard practice, supported clearly
by empirical evidence, should be: ensuring that the nest site, but
not soiled bedding, is transferred during cage change; maintain-
ing cages at 20–22 °C and providing sufficient nesting material for
mice to thermoregulate; avoiding mixing unfamiliar males, and
keeping littermates together whenever possible.
We also strongly recommend some practices with a solid evi-
dence base, but some logistical difficulty. First, group size should
be limited to three animals in a standard cage. This may seem
impractical, but aggression is so common that many four- or five-
animal cages are split. This adds variability and disrupts experi-
mental designs, as discussed earlier. Furthermore, the fecal output
in a three-mouse cage is much less, and the per diem cost of a small
group size is potentially offset by lengthening the cage change inter-
val. Second, shelter enrichments should not be provided in circum-
stances in which these are known to increase aggression19 (which
is best assessed on a case-by-case basis in each facility). Note that
providing multiple shelters per cage or providing shelters to smaller
groups may have beneficial effects on aggression33, although this has
not been studied in males or in isolation from other factors.
Finally, evidence extrapolated from non-home cage data and/or
other species suggests other housing and husbandry practices that
may mitigate aggression: ensuring physical comfort, providing
adequate pain control, using handling and identification methods
that minimize stress and pain, and avoiding exposure to potential
endocrine disruptors.
But mice still fight: possible explanations that have not
been investigated
The environment of laboratory mice is extremely far removed
from their ecological niche, and as such they are exposed to highly
unnatural stimuli and prevented from performing many species-
typical behaviors. In addition to the obvious spatial restriction,
laboratory cages also lack the complexity of a natural environ-
ment. Wild mice spend a large amount of time searching for and
hoarding food, but food is easily accessed and provided ad libitum in the
laboratory. Mice burrow, but are typically kept on a minimal amount
of unsuitable substrate that discourages burrowing. They are highly
motivated to build nests, but are often kept with such small amounts
of nesting material that formation of a fully enclosed nest is
impossible, despite ambient temperatures being set below the mice’s
thermoneutral zone. These examples clearly illustrate that labo-
ratory mice are routinely exposed to stressors and prevented
from performing a wide range of their natural behaviors.
Captive environments elicit natural behaviors that are attempts
by the animal to gain control over their situation, such as foraging
if hungry, hiding if scared or nesting if cold. Animals that cannot
perform highly motivated behaviors or control their environment
can experience frustration, stress and boredom, which can lead to
disturbed social behavior and increased aggression34,35.
Furthermore, laboratory mice lack social control; they cannot
choose their social group or escape from conspecifics. Mice may
form dominance hierarchies as a way to cope with enforced proxim-
ity with conspecifics and avoid escalated aggression36. Formation
of dominance hierarchies is predicated on submission, which may
involve submissive postures, exiting line of sight of the mouse
displaying dominant behavior or outright fleeing. The last two of
these three methods to show submission are impossible in non-
enriched laboratory cages. The remaining response to a threat might
then be for the mouse to fight or to be attacked for failing to leave.
An animal’s control over its environment and how the lack thereof
might contribute to aggression has not been examined.
Disturbance, pain and other aversive stimuli. Disturbing mice for
experimental or management procedures may contribute to aggres-
sion. For example, mice are nocturnal and light sensitive, but are
kept in brightly lit vivariums and are typically handled or otherwise
used in experiments during the light phase, when they should be
asleep37. A disturbed sleeping pattern can lead to stress, frustra-
tion and aggressive behavior38. Although cage cleaning is known
to lead to flare-ups in aggression22,39, the effects of other forms of
disturbance have not been studied.
Aversive stimuli may also induce aggression. Mice sometimes turn
and bite when held by forceps40 or by the tail, and electric shocks can
cause one mouse to attack another41. Fearful animals may become
aggressive when they cannot escape, but fear may also suppress
aggression, at least toward intruders42. Painful, frightening and aver-
sive stimuli are routinely imposed on laboratory mice. Even standard
procedures, such as ear marking, can be painful to mice43, and mice
lifted by the tail (a routine handling technique) avoid contact with
humans and are more anxious when compared with mice lifted by
using a tube44. Still, the effects of such common procedures on aggres-
sion under normal husbandry have not been investigated.
Effect of resource distribution. Environmental enrichment is
potentially a powerful means of ameliorating many of the prob-
lems listed above. Additional cage furnishings, such as running
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160 Volume 46, No. 4 | APRIL 2017
wheels, shelters, nesting materials and burrowing substrates could
allow captive mice to engage in many highly motivated behaviors
and to exercise some control over their environment and their
exposure to aversive stimuli. However, as mentioned above, several
studies have found that enrichment can increase within-cage aggres-
sion in male mice19,45,46.
It is possible that insufficient enrichment is to blame. Only a
small number of enrichment items fit inside a conventional mouse
cage. Scarce, but important, resources are highly valued, and animals
will compete over resources that they can monopolize. However, if
resources are provided in abundance and/or spread out, one animal
may not need to or be able to defend all of them. The extent to which
food resources are clumped and defendable has already been shown
to affect social organization in free-ranging mice17 and many other
species47. Given that most cages used in laboratory environments
are otherwise barren, the enrichments provided in past experiments
may have been very valuable and thus defended. Female mice fight
less if enrichment items are dispersed34, but the effects of providing
multiple, dispersed enrichments on within-cage aggression have
not been investigated in male laboratory mice.
Are lab mice deficient in social communication? Laboratory mice
are usually weaned abruptly and at an unnaturally early age48. Early
weaned mice may lack certain communication skills, and this could
conceivably influence aggressive behavior49. In addition, some
strains of laboratory mice are blind or severely visually impaired50
and others have hearing deficiencies51, both of which may affect
certain components of communication, such as the perception of
submissive displays.
However, even if early weaned mice are perfectly capable in
social communication, does their social and physical environment
allow them to communicate effectively? Mice use olfactory cues
for communication and individual recognition52; however, the
majority of mice used in biomedical research are inbred, and such
genetically identical individuals may be difficult to distinguish by
olfactory signals alone. Lack of individual recognition could poten-
tially prevent or disrupt the formation and maintenance of domi-
nance hierarchies, but has also been suggested to lower aggression
between males of the same strain, as they are recognized as close
kin53. The short-term effects of cage cleaning on aggression also
seem to be modulated, at least in part, by removal or disruption of
these odor cues. In addition to olfactory cues, the laboratory envi-
ronment may also disrupt some visual cues. The visual range of mice
is shifted toward short wavelengths, relative to that of humans, and
includes the ultraviolet range54. Given that mouse urinary cues are
visible in the ultraviolet, the lack of ultraviolet light sources in the
laboratory may impede some forms of communication55.
Discussion and conclusion
Laboratory mice live a fundamentally unnatural existence, with
a housing environment unlike anything most would experience
in the wild. For some social groups, these circumstances may be
essentially incompatible with peaceful coexistence, but it is difficult to
establish which components of captive life contribute to aggression.
Instead of focusing on solving aggression as an isolated issue, the
ultimate way forward might be to consider alternative ways to house,
handle and experiment on mice that will take their natural behavior
into account and give them opportunities to control social interac-
tions with conspecifics.
A major part of the effort to make captivity more suitable for
mice will be providing environmental enrichment. In a way, ani-
mals fighting over enrichment is a good sign: it shows that the
resource provided was highly valued as a result of its rarity. The
solution to fighting over a rare resource is to increase its abundance;
providing more resources may decrease aggression34. Multiple
resources may not only reduce competition, but also have other
independent effects on aggression. For instance, although it is not
possible to escape from the cage, shelters or visual barriers could
allow mice to hide from a threatening conspecific.
Van Loo et al.14 described abnormal levels of aggression. But
how do we define what is abnormal? Everything we know about
wild mice suggests that it is in fact the lack of aggression that is
abnormal. This might explain why aggression is so unpredictable.
We may have unwittingly created a perfect storm of unnatural cues
that combine to suppress aggression when it would normally occur.
Thus, changes in husbandry that appear benign to us may disrupt
this fragile equilibrium with aggression, resulting in the resumption
of normal mouse behavior.
Alternatively, the levels of aggression seen in the laboratory
environment might be viewed as normal reactions to an unnatu-
ral situation in which the animal’s ability to successfully cope is
constantly challenged. In this scenario, we have managed to house
mice in conditions adequate to suppress aggression, but again, a
small perturbation may be the straw that breaks the camel’s back. If
so, there may be large-scale changes that further reduce the stress
on the animals and give them enough resiliency to effectively con-
trol aggression. Although this is the scenario implicitly assumed in
most existing work, it has been markedly ineffective at finding a
reliable solution; to our frustration, we generally find ways of mak-
ing aggression worse, not better.
Whichever scenario turns out to be correct, it may be impossible
to completely remove aggression between mice while at the same
time continuing with business as usual. It might be the case that
fighting will persist if mice are housed and treated the way they
are in laboratories, as they are not given the possibility to express
a full repertoire of natural behaviors that would include those that
would normally reduce fighting. If this is the case, truly prioritizing
animal welfare and scientific validity may mean we must seriously
reconsider the present way of housing laboratory mice.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Received 14 November 2016; accepted 25 January 2017
Published online at http://www.nature.com/laban
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