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



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. (
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.
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
PERSPECTIVE Focus on Reproducibility
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-mans 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
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
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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.
The authors declare no competing financial interests.
Received 14 November 2016; accepted 25 January 2017
Published online at
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... From all articles that studied enrichment, there were 13 observations of decreased aggression [21,23,24,[27][28][29][30][31], 32 where the enrichment did not have any effect [20][21][22][23]29,[32][33][34][35][36][37][38][39][40][41] and 15 observations of increased aggression [18,19,22,32,34,37,[42][43][44][45][46][47]. In addition, there were 4 observations with different outcomes depending on the method used to measure aggression [48][49][50][51] (Supplementary Table S4). ...
... In one case there was a decrease in aggression, but increased aggression was never observed (Supplementary Table S4). From all articles that studied enrichment, there were 13 observations of decreased aggression [21,23,24,[27][28][29][30][31], 32 where the enrichment did not have any effect [20][21][22][23]29,[32][33][34][35][36][37][38][39][40][41] and 15 observations of increased aggression [18,19,22,32,34,37,[42][43][44][45][46][47]. In addition, there were 4 observations with different outcomes depending on the method used to measure aggression [48][49][50][51] (Supplementary Table S4). ...
... Bartolomucci et al. (2004) studied group formation from non-sibling CD-1 mice at weaning or adolescence and observed increased aggression in groups formed from juvenile CD-1 mice [93]. The effects of other early life events, such as weaning age [41,94] and nesting condition (standard or communal) [95], were also studied. The two articles studying weaning age showed contrary results; one observed increased aggression after early weaning [94] while the other observed increased aggression after late weaning [41]. ...
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Aggression among group-housed male mice is a major animal welfare concern often observed at animal facilities. Studies designed to understand the causes of male mice aggression have used different methodological approaches and have been heterogeneous, using different strains, environmental enrichments, housing conditions, group formations and durations. By conducting a systematic literature review based on 198 observed conclusions from 90 articles, we showed that the methodological approach used to study aggression was relevant for the outcome and suggested that home cage observations were better when studying home cage aggression than tests provoking aggression outside the home cage. The study further revealed that aggression is a complex problem; one solution will not be appropriate for all animal facilities and all research projects. Recommendations were provided on promising tools to minimize aggression, based on the results, which included what type of environmental enrichments could be appropriate and which strains of male mice were less likely to be aggressive.
... Over the longer term, aggression between ear-notched male C57BL/6 mice was higher than in tail-tattooed ones, as measured via post-mortem lesion scores [12]. Taken together, this literature therefore suggests that there is scope for refining ear-biopsy procedures to reduce pain and/or stress. ...
... We also measured longer lasting anxiety via an EPM [19,21,25]; a hyponeophagia test [34,35]; reduced voluntary interaction with the handler [8]. Finally, we observed social effects including aggression and wounding [12,36], allogrooming, and sniffing of the cagemate [31]. ...
... We found no significant effect of ear-punching on aggression, unlike Gaskill et al. [12], because we saw almost no signs of aggression. This could be due to BALB/c siblings being used here, or because we used brief live scoring of aggression and wounding, rather than post-mortem pelt scores. ...
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Identification marking mice commonly involves ear-punching with or without anaesthetic, or tail-marking with ink. To identify which is most humane, we marked weanling male BALB/c mice using ear-punching (EP), ear-punching with anaesthetic EMLATM cream (EP+A), or permanent marker pen (MP). We compared marked mice, unmarked cagemates, and control mice (n = 12–13/group) for 5 weeks, reapplying MP weekly. Treatment-blind observations following marking showed that EP and EP+A mice were allogroomed (p < 0.001) and sniffed (p < 0.001) by their cagemates more than MP and control mice were. EP+A mice groomed themselves (p < 0.001) and their ears (p < 0.001) ~5 times more than most other mice; their cagemates also increased self-grooming (p < 0.001). Unmarked MP cagemates (p = 0.001), and possibly EP+A mice (p = 0.034; a nonsignificant trend), grimaced the most. The following day, half the EP+A mice showed hyponeophagia versus no MP and control mice (p = 0.001). Over the 5 weeks, EP mice approached the handler significantly less than unmarked cagemates did (p < 0.001). Across weeks, defecation during marking of MP mice decreased (p < 0.001). Treatment showed no effects on immediate responses during marking, aggression, bodyweight, plus-maze behaviour or corticosterone. MP mice showed no differences from controls, whilst EP and EP+A mice showed altered behaviour, so ink-marking may be the more humane identification method.
... Although reliable alternatives, like tail tattooing, have emerged, ear-notching remains one of the most used methods due to its simplicity, permanence, and the possibility of using the removed tissue for genotyping [135]. Additionally, there is still controversy regarding the suitability and welfare cost of both, being tail tattooing also stressful and painful to mice [135,136]. Alternatively, if identification is meant for short-term purposes, using non-toxic fur dyes is considered a refined alternative to the methods discussed [137]. ...
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Patients with cirrhosis present multiple physiological and immunological alterations that play a very important role in the development of clinically relevant secondary complications to the disease. Experimentation in animal models is essential to understand the pathogenesis of human diseases and, considering the high prevalence of liver disease worldwide, to understand the pathophysiology of disease progression and the molecular pathways involved, due to the complexity of the liver as an organ and its relationship with the rest of the organism. However, today there is a growing awareness about the sensitivity and suffering of animals, causing opposition to animal research among a minority in society and some scientists, but also about the attention to the welfare of laboratory animals since this has been built into regulations in most nations that conduct animal research. In 1959, Russell and Burch published the book “The Principles of Humane Experimental Technique”, proposing that in those experiments where animals were necessary, everything possible should be done to try to replace them with non-sentient alternatives, to reduce to a minimum their number, and to refine experiments that are essential so that they caused the least amount of pain and distress. In this review, a comprehensive summary of the most widely used techniques to replace, reduce, and refine in experimental liver research is offered, to assess the advantages and weaknesses of available experimental liver disease models for researchers who are planning to perform animal studies in the near future.
... 25,93 However, new approaches should be carefully evaluated for each species, especially because some scents that humans find pleasant (for example, lavender) can result in aggression in some species. 40 Based on our survey results and published research, implementation of sensory enrichment is beneficial for some species 20,85 and should be promoted. Ultimately, before implementing any new enrichment, consideration should be given to relevance to the species and to evaluating its effects on individual animals. ...
Enrichment is important for animal welfare and data quality. Provision of enrichment opportunities varies between species and enrichment category. However, data benchmarking these differences does not exist. Our objective was to characterize enrichment provision and associated factors across species in the US and Canada. Personnel who work with research animals ( n = 1098) in the US and Canada voluntarily responded to online promotions and completed a survey about enrichment used for the species they worked with most, their control of and wish for more enrichment, stress or pain in the animals they worked the most with, and demographics. All participants (except those working with rats) received the same questionnaire regardless of species to allow objectivity, as the effects of many enrichment items on some species have not yet been determined. The questionnaire asked about enrichments that were beneficial to at least one species. The provision of enrichment was allocated into 2 outcome variables: diversity and frequency per enrichment category. Results showed a significant interaction between enrichment category and species. Generally, physical, nutritional, and sensory enrichments were provided less often than social enrichment. In addition, nonhuman primates received more diverse and more frequent enrichment than did other species (twice as much as rats and mice). Enrichment was provided less frequently by personnel who wished they could do more than the status quo. Both enrichment frequency and diversity were higher in respondents from Canada, those who had more control over provision, and those who had been in the field longer. While our results cannot be used to determine the quality of enrichment provided to various species, they do provide information on current enrichment practices in the US and Canada and identify differences in implementation by species and enrichment category. The data also indicate provision of enrichment is influenced by factors such as country and individual control over enrichment. This information can also be used to identify areas for greater enrichment efforts for some species (for example, rats and mice) and categories, with the ultimate goal of improving animal welfare.
... We were able to examine neural responses to freely behaving animals interacting with one another, whereas the experiments described above in mice (Dolen et al., 2013) and hamsters (Song et al., 2016) used social CPP assays where neural metrics were not obtained when two animals were freely interacting. This is likely because Syrian hamsters and C57BL/6 mice are quite aggressive with same-sex conspecifics (Elidio et al., 2021;Gaskill et al., 2017;Grifols et al., 2020;Weber et al., 2017). The preference to spend time in the socially conditioned chamber in a CPP assay may reflect social investigation or territoriality, and not necessarily prosocial (i.e., positive social) behavior. ...
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We investigated whether nonreproductive social interactions may be rewarding for colonial, but not non-colonial, species. We found that the colonial spiny mouse (Acomys cahirinus) is significantly more gregarious, more prosocial, and less aggressive than its non-colonial relative, the Mongolian gerbil (Meriones unguiculatus). In an immediate early gene study, we examined oxytocin (OT) and tyrosine hydroxylase (TH) neural responses to interactions with a novel, same-sex conspecific or a novel object. The paraventricular nucleus of the hypothalamus (PVN) OT cell group was more responsive to interactions with a conspecific compared to a novel object in both species. However, the ventral tegmental area (VTA) TH cell group showed differential responses only in spiny mice. Further, PVN OT and VTA TH neural responses positively correlated in spiny mice, suggesting functional connectivity. These results suggest that colonial species may have evolved neural mechanisms associated with reward in novel, nonreproductive social contexts to promote large group-living.
Exposure to acute and chronic stress has significant effects on the basic mechanisms of associative learning and memory. Stress can both impair and enhance associative learning depending on type, intensity, and persistence of the stressor, the subject's sex, the context that the stress and behavior is experienced in, and the type of associative learning taking place. In some cases, stress can cause or exacerbate the maladaptive behavior that underlies numerous psychiatric conditions including anxiety disorders, obsessive-compulsive disorder, post-traumatic stress disorder, substance use disorder, and others. Therefore, it is critical to understand how the varied effects of stress, which may normally facilitate adaptive behavior, can also become maladaptive and even harmful. In this review, we highlight several findings of associative learning and decision-making processes that are affected by stress in both human and non-human subjects and how they are related to one another. An emerging theme from this work is that stress biases behavior towards less flexible strategies that may reflect a cautious insensitivity to changing contingencies. We consider how this inflexibility has been observed in different associative learning procedures and suggest that a goal for the field should be to clarify how factors such as sex and previous experience influence this inflexibility.
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Housing conditions can affect the well-being of laboratory animals and thereby affect the outcomes of experiments. The appropriate environment is essential for the expression of natural behavior in animals. Here, we compared survival rates in four inbred mouse strains maintained under three different environmental conditions. Three mouse strains (C57BL/6J, C3H/HeN, and DBA/2J) housed under environmental enrichment (EE) conditions showed improved survival; however, EE did not alter the survival rate of the fourth strain, BALB/c. None of the strains showed significant differences in body weights or plasma corticosterone levels in the three environmental conditions. For BALB/c mice, the rates of debility were higher in the EE group. Interestingly, for C57BL/6J and C3H/HeN mice, the incidence of animals with alopecia was significantly lower in the EE groups than in the control group. It is possible that the enriched environment provided greater opportunities for sheltering in a secure location in which to avoid interactions with other mice. The cloth mat flooring used for the EE group was bitten and chewed by the mice. Our findings suggest that depending on the mouse strains different responses to EE are caused with regard to health and survival rates. The results of this study provide basic data for further studies on EE.
In Edition 2 of the Animal Welfare Assessment Topic, we showcased a collection of 13 peer reviewed articles which highlight advancements in animal welfare assessment methods across animal production systems. It includes works of animal welfare experts, veterinarians, animal physiologists and animal managers that will generate a healthy discussion and showcase latest studies working toward finding the harmony between animal performance, health and welfare.
Even after a century or more in captivity and being perpetuated under stringent confinement and controlled conditions, the rodents and rabbits still retain the behaviour of their counterparts living in natural habitats. Ethology, the study of behaviour makes use of the observed behaviour enlisted in detail known as ethograms, enabling the analysis of the normal behavioural repertoire of each species and its deviations. Providing an environment that simulates and promotes the expression of natural behavioural patterns in the wild to its closest degree can play a positive role in the care and welfare of animals in confinement. Behavioural studies enable scientists to “impart a culture of care” by generating data to improve animal care and use programs and hastening the process of finding better housing standards. Behavioural analysis in animals also finds its use in drug discovery research and also in studying specific pathologies of diseases especially in the area of neurobehavioral research. This chapter reveals the secrets of measuring the animal’s mental state from a practical point of view for the benefit of both laboratory animal species and mankind.
Rotational outbred adult rats, phenotypically selected to prefer drinking alcohol (“P” rats) frequently present with selfinflicted wounds and ulcerative dermatitis, similar to that seen in C57BL/6 mice. Historically, veterinary interventions used to address this clinical condition have included triple antibiotic ointment (TABO), Columbia wound powder (CPW), nailtrims, or plastic tubes that allow affected animals to hide. More recent studies have suggested that nail trims are the mostsuccessful intervention in mice, but this has not been evaluated previously in rats. In this study, we evaluated nail trims in rats and also tested whether placing a pumice stone in the cage would reduce the need for nail trims to reduce self-inflicted wounds. Our hypothesis was that interacting with the pumice stone would dull/trim the rats’ nails without causing stress or illness and allow the wounds time to heal. We used 66 P rats that were assigned to 1 of 6 treatment groups (pumice stone, TABO, CWP, huts, nail trims, and an untreated control group) of 11 rats each. Rats were transferred to this study from a colony of experimentally naïve animals that had evidence of dermatitis. The wounds were photographed and measured for12 wk at 2 wk intervals. At the end of the study,representative skin samples from the site of the wound were collected forhistopathologic evaluation of inflammation. Our data showed no significant differences in the inflammation scores. The ratstreated with nail trims healed significantly more often than did all of the other treatment groups. This suggests that nail trims are the most effective intervention for treating self-inflicted wounds in P rats.
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The white-footed mouse (Peromyscus leucopus) is an important host of the deer tick (Ixodes dammini), and the principle reservoir for the spirochete (Borrelia burgdorferi) known to cause Lyme disease. In summer and autumn 1991, we uniquely marked small rodents, including P. leucopus, with metal ear tags. The presence of ear tags increased rates of infestation by larval ticks on mice by 50 to 100%, probably because the tags reduced grooming efficiency. Because larval deer ticks acquire the Lyme disease spirochete more efficiently from P. leucopus than from other mammalian and avian hosts, increasing the numbers of ticks parasitizing mice may cause a higher percentage of ticks to carry Lyme disease.
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In nature, mice live in burrows with nest chambers where they breed and may hide from predators. In the laboratory, a shelter or refuge is an easily applicable form of enrichment which may enhance the welfare of laboratory mice by giving them more control over their environment. Six nest boxes made of different materials were evaluated in a preference test with male and female mice of two strains (C57BL/6J and BALB/c). In general, mice showed a preference for cages with a nest box made of grid metal as compared to clear or white perspex nest boxes, or no nest box. They also showed a preference for a cage with a nest box of perforated metal as compared to nest boxes made of grey PVC or sheet metal, or no nest box. When offered a nest box with one open side or a nest box with two open sides, most mice preferred the nest box with one open side and were observed to lie in it with their heads directed towards the opening. The results of this study show that nest boxes may be used for enrichment purposes in caged mice, although it is not yet entirely clear what are the main features influencing the animals’ preferences. When providing nest boxes as shelters, the structure and design of this type of enrichment should be taken into account, because these may have an effect on the social structure of groups of mice.
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Mice are housed at temperatures (20-26°C) that increase their basal metabolic rates and impose high energy demands to maintain core temperatures. Therefore, energy must be reallocated from other biological processes to increase heat production to offset heat loss. Supplying laboratory mice with nesting material may provide sufficient insulation to reduce heat loss and improve both feed conversion and breeding performance. Naïve C57BL/6, BALB/c, and CD-1breeding pairs were provided with bedding alone, or bedding supplemented with either 8g of Enviro-Dri, 8g of Nestlets, for 6 months. Mice provided with either nesting material built more dome-like nests than controls. Nesting material improved feed efficiency per pup weaned as well as pup weaning weight. The breeding index (pups weaned/dam/week) was higher when either nesting material was provided. Thus, the sparing of energy for thermoregulation of mice given additional nesting material may have been responsible for the improved breeding and growth of offspring.
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The present study investigated the effects of lavender oil, which has been used for aromatherapy, using the Geller type and the Vogel type conflict tests in mice. The results showed that lavender oil produced apparent anti-conflict effects in the two tests, suggesting that lavender oil possesses anti-anxiety effect. Analysis of the oil using GC/MS revealed that lavender oil contains alpha-pinene, camphene, beta-myrcene, p-cymene, limonene, cineol, linalool, borneol, terpinen-4-ol, linalyl acetate, geranyl acetate and caryophyllene. Then effects of these constituents were examined using two kinds of conflict tests. Linalool produced significant anti-conflict effects in the two tests, showing that linalool is a major pharmacologically active constituent of lavender oil.
Agonistic behavior in group-housed male mice is a recurring problem in many animal research facilities. Common management procedures, such as the removal of aggressors, are moderately successful but often fail, owing to recurrence of aggressive behavior among cagemates. Studies have incorporated enrichment devices to attenuate aggression, but such devices have had mixed results. However, these studies did not include research manipulations when assessing the benefits of various enrichment devices. We obtained 100 male athymic nude mice and studied the efficacy of various enrichment devices, including cotton squares, paper rolls, shredded paper, nylon bones, and a mouse house and wheel combination in the reduction of fighting during an ongoing study that involved randomization followed by prostate and intratibial injections. Groups were evaluated according to a numerical grading system for wound assessment. Examination of the data revealed that the enrichment devices had no effect on the presence of wounds, thus none of the devices tested affected fighting in nude mice. However, when mice began experimental use, fight wounds increased significantly at cage change and after randomization, reflecting a disruption of existing social hierarchies. Therefore, in the context of an actual research study that involves common manipulations, the specific enrichment device had less effect on aggression in male nude mice than did the destruction and reconstruction of social structures within each group.
1. This study of the population changes of Peromyscus maniculatus was made in Douglas fir forest, Point Grey, Vancouver, British Columbia from March 1962 to October 1963. 2. Monthly live trappings confirmed previous reports that populations remain relatively stable through the summer, increase suddenly to a peak in autumn, and then decline through the winter. In some of the populations there were sudden drops in numbers. 3. Male Peromyscus caught each month were subjected to behaviour tests which showed a rise in aggression during the breeding season. The sudden drops in numbers coincided with increases in aggression of the males sampled. Thus, the drops may have been caused by behavioural upsets in the populations as the breeding season began. 4. Considerable juvenile mortality occurred during the summer breeding season and juvenile survival was found to be much better on an area from which adults had been removed. 5. This behavioural relationship between adult and juvenile Peromyscus was investigated in a laboratory maze. It was shown that resident adults were usually extremely antagonistic to intrusive juveniles, which either died or were restricted to small areas of the maze. 6. It was concluded that changes in survival and recruitment of young during the breeding season could be explained by changes in aggressive behaviour of the adults.