The Impact of Social and Behavioral Factors on Reproducibility in Terrestrial Vertebrate Models

Abstract

The use of animal models remains critical in preclinical and translational research. The reliability of the animal models and aspects of their validity is likely key to effective translation of findings to medicine. However, despite considerable uniformity in animal models brought about by control of genetics, there remain a number of social as well as innate and acquired behavioral characteristics of laboratory animals that may impact on research outcomes. These include the effects of strain and genetics, age and development, sex, personality and affective states, and social factors largely brought about by housing and husbandry. In addition, aspects of the testing environment may also influence research findings. A number of considerations resulting from the animals' innate and acquired behavioral characteristics as well as their social structures are described. Suggestions for minimizing the impact of these factors on research are provided.

Full text

Received: March 12, 2019. Revised: January 30, 2020. Accepted: February 7, 2020
© The Author(s) 2020. Published by Oxford University Press on behalf of the National Academies of Sciences, Engineering, and Medicine.
All rights reserved. For permissions, please email: journals.permissions@oup.com
252
ILAR Journal, 2019, Vol. 60, No. 2, 252–269
doi: 10.1093/ilar/ilaa005
Advance Access Publication Date: 28 July 2020
Review
The Impact of Social and Behavioral Factors on
Reproducibility in Terrestrial Vertebrate Models
Alexandra L. Whittaker1and Debra L. Hickman2,*
1School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, South Australia,
Australia; and 2Laboratory Animal Resource Center, Indiana University, Indianapolis, Indiana.
*Corresponding Author: D. L. Hickman, DVM, Laboratory Animal Resource Center School of Medicine Indiana University 975 W Walnut St (IB008)
Indianapolis, IN 46202 hickmand@iupui.edu.
Abstract
The use of animal models remains critical in preclinical and translational research. The reliability of the animal models and
aspects of their validity is likely key to effective translation of findings to medicine. However, despite considerable
uniformity in animal models brought about by control of genetics, there remain a number of social as well as innate and
acquired behavioral characteristics of laboratory animals that may impact on research outcomes. These include the effects
of strain and genetics, age and development, sex, personality and affective states, and social factors largely brought about by
housing and husbandry. In addition, aspects of the testing environment may also inf luence research findings. A number of
considerations resulting from the animals’ innate and acquired behavioral characteristics as well as their social structures
are described. Suggestions for minimizing the impact of these factors on research are provided.
Key words: animal model reproducibility; biology; behavior; experimental confounding; social factors; translation
Introduction
Animal models are one of the critical components of research,
providing insights into the whole animal that cannot be obtained
from cell and tissue cultures or computer simulations (eg,
[1–3]). Likewise, the animal model provides advantages that
cannot be obtained when using humans, such as genetic
uniformity [4–6], controlled environments, and relatively
shorter lifespans. Genetically modified animals, predominantly
mice and zebrafish, can also provide invaluable information
not otherwise available using well-established techniques
(eg, [7,8]).
Despite genetic uniformity, there are a number of social
as well as innate and acquired behavioral characteristics that
may impact research outcomes. In particular, this may occur in
studies where aspects of behavior, physiology, and/or immune
function are assessed. Recognition of these characteristics and
the careful consideration of experimental design to control for
these possible confounders are critical to address translata-
bility and reproducibility between laboratories. Tools such as
the ARRIVE Guidelines and experimental design checklists have
been proposed to help improve reporting details regarding ani-
mal, environmental, and procedural data in preclinical studies,
but these are inconsistently applied across journals, leading to
gaps in information [9–12].
Social and behavioral considerations are multi-factorial and
influenced by the interactions of a variety of effectors. In this
manuscript, we will focus on the effects of strain and genetics,
age and development, sex, personality and affective states, and
social factors. We will also address how components of the test-
ing environment, including the experimenter and experimental
design, can influence the reproducibility and translatability of
research results. There are other factors that can inf luence
the behavior of laboratory rodents that will be covered else-
where in this issue and will not be discussed in this manuscript.
These include the effect of circadian rhythm and sleep cycles
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

ILAR Journal, 2019, Vol. 60, No. 2 253
[13], nutritional status [14], housing systems [15,16], and micro-
biota [17] on animal behavior and physiology.
Impact of Genetics on Animal Models
Genetics are considered the foundational building blocks of
the phenotypic expression for an individual organism. Pheno-
typic subgroups within a particular genus (eg, a mouse strain)
are driven by differences in alleles that have arisen naturally
through spontaneous mutations [18,19] or have been induced
through genetic modification techniques (such as transgene
insertion or knocking out or in particular genes) [20]. This diver-
sity is widely recognized in the laboratory mouse with over 500
inbred [21] and outbred strains [22], plus hundreds of thousands
of different genetically engineered mutants [23](www.findmi
ce.org).
Species-Specific Considerations
In general, the selection of an animal model based on genetics
is primarily a concern for rodents where there have been
intensive efforts to create models with specific genotypes for
detailed study [20,24,25]. This is echoed in other genetically
modified species, such as zebrafish and pigs [26–29]. However,
consideration of genetics is of importance in other species used
in research. Breeds of dogs and cats tend to be homogeneous
populations that can serve as valuable animal models [30,31].
For example, boxers with a variant in chromosome 17 are
predisposed to development of spontaneous arrhythmogenic
right ventricular cardiomyopathy, while the Golden Retriever
with muscular dystrophy is a valuable animal model for the
human condition [32]. Likewise, although pigs show many
similarities to humans in their skin anatomy and physiol-
ogy, selection of breed must be considered. An example is
the use of the Red Duroc pig as a model of hypertrophic
scarring [33].
Summary
The research question under consideration needs to be
specifically defined to ensure selection of the appropriate
genetic model. Selection of genetically modified and transgenic
animal models allow for a detailed evaluation of specific
mechanisms of action while removing confounding effects
(for examples of the critical need for appropriate genetic
model selection, see [34–37]). Additionally, selection of inbred
animals reduces genetic variation and potential confounding
variables, but outbred rodent stocks may better represent
heterogeneous human populations. Festing provides consid-
erations regarding the appropriateness of inbred vs outbred
animals in experimental design [38]. The introduction of human
genes can create humanized mouse models that can facilitate
the exploration of human clinical conditions in a mouse
model [39–41].
When utilizing genetically defined or modified animals, it is
important to be aware that spontaneous mutations and genetic
drift can occur [42]. Because selection of an animal model
is usually based on a particular genotype and its associated
phenotype, it is critical to perform genetic testing for quality
assurance. This allows the scientist to more confidently assure
that the expressed phenotype under study is caused by the
genotype.
Impact of Age and Development on Animal Models
Behavioral expression changes extensively over an animal’s life-
time. For example, early behavioral profiles include play, which
allows the animal to learn and navigate species appropriate
social interactions, though these behaviors are less critical com-
ponents of the behavioral profiles of older animals [43–45]. Cog-
nition and neural processing change with age as various factors
affect developing neurons (examples [46–49]).These changes can
be influenced by a myriad of epigenetic factors [49–51],including
immune modulation [52,53], the microbiome [46,52], and the
environment [54,55].
Species-Specific Considerations
There is a significant body of literature regarding the effects and
purpose of play in species such as rats [43,56–58], dogs [59–62],
and nonhuman primates [63–66]. The presence or absence of
play behavior in young animals has been proposed as a potential
metric for the assessment of animal welfare [67,68]. Engaging in
heterospecific play (eg, tickling rats or playing with dogs) can
improve the well-being of animals in addition to fostering a posi-
tive human-animal bond [69–72]. Characteristics of the microen-
vironment have also been implicated in the development of
laboratory animals, including rodents and nonhuman primates.
In general, the addition of environmental enrichment to increase
the complexity of the living environment and handling tech-
niques improves cognitive and behavioral responses of develop-
ing animals and should be provided to stimulate development
[73–81], resulting in the expression of more natural behaviors
and improved well-being [82–84]. Likewise, there is a compelling
argument that the use of individually ventilated caging and the
practice of autoclaving all of the materials that come in contact
with the animals to prevent the entry of nondesirable organisms
from the laboratory animal environment has led to models with
underdeveloped immune systems [75,85].
Summary
When designing studies, careful selection and standardization
of age is critical to minimize age-associated effects of behavior
and physiology. All factors of the environment and its potential
positive or deleterious effects on cognitive development should
also be carefully considered. However, it should be noted that the
historical tendency to house laboratory rodents in relatively bar-
ren environments with micro-isolation techniques to minimize
potential contamination by undesirable organisms can result in
deficiencies in development, including immune and cognitive
function [73–75,85–87].
Effect of Sex on Animal Models
The effect of sex on the outcome of research studies has long
been recognized. However, this recognition historically led to
the selection of males in order to remove the presumptive con-
founding factors of female reproductive status and estrus cycle.
The selection of males has potentially contributed to reported
reproducibility and translatability issues, especially when eval-
uating potential interventions for women. A plan to correct this
deficiency was implemented in 2015 by the National Institutes
of Health [88–90]. This topic has been discussed in detail exten-
sively, so the reader is directed to review relevant references for
additional information [91].
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

254 Whittaker and Hickman
Summary
When designing experiments, consideration should be given to
the potential for differences in responses to the experimen-
tal stimuli between the sexes. It should also be noted that
the presence of members of the opposite sex can trigger vari-
ous behavioral and physiological responses in multiple species
[92–95]. Depending on the scientific question under study, sepa-
ration by sex may be desirable.
Effect of Personality as an Individual
Modifier of Behavior
Genetically homogeneous animals express individual variations
in behavior that are shaped by epigenetics and by their micro-
biome and early learning environment [96,97]. These consistent
inter-individual behavioral differences that persist over time
are referred to as animal personalities. These include behav-
ioral traits such as boldness, activity, and aggression [96,97].
Interpretation of these behaviors requires understanding the
adaptive advantage they provide to individual animals faced
with varying environmental states. This nascent area of research
will continue to shape ongoing evaluations of animal well-being
and proposed interventions to improve husbandry and care. For
an introduction to this topic, readers are directed to current
literature reviews (see, eg, [96,98–101]).
Summary
People who work closely with animals recognize that their ani-
mals have unique personalities just as humans do.Although the
understanding of what shapes these personalities is not well-
characterized, personnel working with animals in research can
use the inherent understanding that individual animals may
respond to a particular stimulus differently even if they are
genetically homogeneous, requiring the experimenter to be open
to adapting their technical approach to minimize the potential
for animal pain or distress. However, it should be noted that
altering animal handling can confound experimental design,
although details remain uncharacterized. Therefore, it is recom-
mended that as much detail as possible regardingthe techniques
used be provided when reporting scientific findings (eg, did
a variety of treats need to be employed to meet the training
personality and preference profiles of the research subjects?)
to facilitate the ability of other laboratories to reproduce the
reported results.
Effect of Human–Animal Interaction
on Animal Models
The effect of the human–animal interaction should not be
neglected in experimental design. Familiarity of the handler
can directly affect the behavior expressed by an animal subject.
Dogs demonstrate different behavioral responses with different
handlers [102,103]asdootherspeciessuchasmice[104–106],
rats [78,78,107], and others [108,109].
Sorge et al published one of the earliest studies suggesting
that the experimenter’s sex can influence behavioral studies
[110]. Similar findings have been reported by other laboratories
with differences in behavioral responses affected by experi-
menter sex or their comfort and experience handling animals,
suggesting that the experimenter should be considered a poten-
tial confounder [78,105,111].
Summary
Although the effect of the handler on an animal has been
recognized in neuroscience and other studies with significant
behavioral components [105,111], recognition of how the stress
response can modulate other physiologic responses, such as
immune function, is critical to designing experiments with
minimal confounders [110]. Thoughtful consideration of who
will handle the animals and how animals will be handled is
necessary to minimize potential confounding stressors.
Interspecies Interactions
In general, it is recommended that animals be separated by
species to prevent unintended confounders associated with
interspecies interactions. However, the effect of interspecies
interactions is not well characterized. For decades, rats have
been characterized as muricidal [112–114], with mice and other
rodents displaying strong aversive responses to predator scents,
especially after primary exposure to the predator [115–117].
However, more recent studies suggested that this response is
adaptive, as mice that have been co-housed in rooms with
rats do not demonstrate aversive behavior to rat scents [118]
or decreases in reproductive success [119], but the mechanism
for these interspecies responses is not well characterized [120].
Summary
It is recommended that unless a study is specifically evaluat-
ing interactions between species, different species should be
separated from one another to minimize potential confounding
effects on interspecies interactions.
Effect of Pain or Distress on Animal Models
Unalleviated pain or distress can negatively affect the repro-
ducibility of research studies. This has led to the expectation
that experimental methods should be used that minimize their
potential [121]. Strategies to accomplish this goal range from
pharmacological, such as the provision of analgesics (exam-
ples include [122–124]) to modification of the techniques used
for manipulation [125], dosing [126,127], and sample collection
[128,129]. There is a significant challenge associated with the
identification of these affective states in rodents because there is
a strong evolutionary drive to hide pain or distressfrom potential
predators (such as humans) [130]. This phenomenon is further
complicated by the inability of a human to accurately assess
the pain or distress that another human being is experienc-
ing (eg, [131–133]), as well as the fact that there is strong evi-
dence of social modulation of the perception of pain or distress
[134]; additionally, it has been demonstrated that rodents display
behaviors consistent with those indicating empathy in humans
[135–137].Thepresenceofpainordistresscanmodifybiolog-
ical function, with effects that range from failure to grow and
reduction in reproductive success to the expression of abnormal
behaviors, including stereotypies [138–140].
One widely proposed strategy to identify pain is the use of
a “grimace scale” [141]. These scales have been validated to
correlate facial expression with other physiological and behav-
ioral assessments to determine potential pain, especially fol-
lowing surgeries [110,141–144]. Although automated systems are
under development, one challenge to the implementation of
these techniques for routine assessment of pain is that they
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

ILAR Journal, 2019, Vol. 60, No. 2 255
may require remote digital recording of the animal followed by
assessment of the recording (or stills from the recording) to score
the individual animal. However, they do serve an important role
in the assessment of pain and possibly distress. Other options
under development include methods that evaluate the time to
incorporate nesting material [145–147] or ultrasonic vocaliza-
tions [93].
The assessment of distress has been more challenging. As
stated above, alterations in behavior and reproductive success
can be signs of distress as can evidence of overt clinical diseases
and disorders, but these indicators are not usually expressed
until there is severe and chronic distress. Evaluations such as
judgement bias assessments can provide invaluable information
about the affective state of an animal [148]. These assays have
been developed for multiple species used in the laboratory and
provide insight into the alleviation of potential distress in hus-
bandry and techniques [148–151].
Social Factors
Single-housing. Most laboratory animal species are social. Posi-
tive social interactions provide a range of benefits, which include
providing emotional well-being, allowing for healthy neural and
behavioral development, and supporting reproductive success
[152]. However, it is not unusual for animals to be singly housed
in the vivarium for scientific or practical reasons, for example,
the need to measure food intake and excretions or to prevent
access to implanted devices or sutures by other animals, or
because of aggressive behavior in the animals themselves (typi-
cally males).
Although the effect of social isolation on biology is generally
consistent across species, some literature suggests inconsistent
findings in animal response to isolation, as described below.
These differences may be reconciled, in part,by considering that
effects are modulated by age of onset and duration of isola-
tion, type of deprivation, and procedural testing variables [153–
155]. Furthermore, group housing scenarios are more likely to
resemble natural group compositions in females than in males,
and hence it would be expected that housing conditions affect
behavior and physiology differently between the sexes [156]. It is
also important to consider that while individual housing causes
social isolation, most laboratory housing scenarios maintain
some form of contact with conspecifics through auditory, olfac-
tory, or visual stimulation so absolute isolation is not occurring.
It is speculated, however, that the increased use of individually
ventilated cages in rodent facilities may be reducing these other
forms of sensory contact [157]. The majority of studies in the
laboratory animal literature in this area have focused on rats,
mice, and primates. The following provides a brief overview.
Readers are encouraged to consult reviews on the topic for
further detail (see, eg, [158–161]).
Species-Specific Considerations
Individual housing has been found to induce both anxiolytic
and anxiogenic-type behaviors in male mice. Using the ele-
vated plus-maze as a measure of anxiety, single-housed DBA/2,
C57BL/6 J, and Swiss mice in 2 studies were found to be less
anxious than group-housed animals [162,163], yet Ferrari et al
reported opposite effects in Swiss mice [164]. Increased anxiety-
like responses have also been exhibited by isolated rodents in
the forced swim test and sucrose preference tests [165,166].
Alternatively, a number of studies failed to find any differences
in anxiety behaviors between group-housed and individually
housed males and females utilizing the elevated plus-maze and
modified hole-board tests [155,156].
Isolation may also impact the affective state generally, result-
ing in behaviors indicative of negative affective state responses
during judgment bias testing, a test gaining importance in the
study of mood disorders [167]. Judgment bias methodologies vary
widely, and the reader should consult comprehensive reviews
on the topic for more detail [168,169]. As an overview, animals
are generally trained to associate 1 set of cues with a positive
event and another set of cues, such as an auditory tone, with a
negative or less positive event.Once the discrimination has been
learned, a nonreinforced cue is introduced, such as an auditory
tone intermediate in frequency between the tones previously
experienced: the ambiguous stimulus [170]. The presumptive
basis of the technique is that animals in a negative affective
state will respond to an ambiguous stimulus more negatively
than animals in a positive state, that is, perform the behavior
associated with the negative event [171]. Isolation has been
shown to cause a negative affective state in rats [166,172]and
chicks [173,174] but not in pigs and laying hens [149,175,176].
The impact of habituation to the testing conditions may have
brought about these species’ differences in response,but further
evaluation of the effects of isolation on affective state are needed
since these assessment techniques are in their infancy.
Isolation has also been shown to impact mouse locomotor
exploratory activity in the open field test. However, sex
differences in this response are apparent, with isolated females
increasing their activity [177] and males generally showing
reductions [178–180], with some exceptions [181]. Procedural
elements may contribute to this apparent inconsistency since
it has been shown that habituation to the arena, for example
after repeat testing, may increase exploratory activity [180].
The effects of isolation on open field test responses in rats are
similarly variable (see, eg, [182,183]).
There are also effects of individual housing on social behavior
and the behavioral tests that measure it. Many studies conclude
that single-housing increases aggressiveness in male mice that
are later paired or during tests such as the resident–intruder test
[184–186]. However, rarely in these studies were the effects of
social rank in group-housed mice considered [187]. In rats, envi-
ronmental stressors tend to decrease interaction in the social
interaction test [188], whereas isolation increases positive social
interaction [189–192]. This difference may reflect the intrinsic
need of social species to find companions to reduce anxiety [193].
Numerous rodent studies have investigated the impact of
individual housing on the hypothalamic-pituitary-adrenal axis
(HPA) stress response through measurement of corticosterone
(see [158] for review). In a similar vein to studies evaluating
behavioral outputs, findings have been inconsistent, with lack of
an HPA axis response [166,177,180,194–196],an elevated response
[197–199], and lower responses [200] seen after housing rats and
mice for periods of 1 week to a few months. However, the type of
housing may be important since metabolic cage housing, which
introduces a number of stressors in addition to the isolation,
has consistently been shown to increase corticosterone levels
afterafewweeksofhousing[200,202]. Furthermore, interpret-
ing results across experiments is challenging due to different
lengths of housing, stocking densities, and possible inf luences
of husbandry practice and disease status.
Given the well-documented impact of the stress response on
theimmunesystem[203] and the interaction between nervous,
immune, and endocrine functions (Figure 1), it is feasible that
longer-term effects on neuro-endocrine immune function
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

256 Whittaker and Hickman
Figure 1: The interactions between nervous, endocrine, and immune systems. Adapted from Szalach et al, 2019. Archivum Immunologiae et Therapiae Experimentalis
2019;67(7):143–151. doi: 10.1007/s00005-019- 00543-8. Reproduced under the terms of the Creative Commons Attribution-Non Commercial-Share Alike License (CC BY
NC SA).
may also occur after single housing [203]. However, individual
housing was not shown to induce any major neuro-endocrine
effects on immune function in mice assessed through lym-
phocyte proliferation and production of various cytokines
[204]. However, individually housed mice were more reactive
to stressor challenge, as indicated by higher basal blood
corticosterone levels, lower IL2 and IL4 cytokine production,
and reduced splenocyte proliferation [204]. These findings
indicate individually housed animals had a less effective coping
response. [204]
There are a range of other potential effects of individual
housing that may impact research. Individual housing has been
shown under some circumstances to reduce barbiturate sleep
time (see, eg, [205–207]). However, in 1 study, time spent in iso-
lation impacted this finding, manifested as a lack of difference
between group and individually caged rats, with respect to barbi-
turate sleep time after 6 weeks [205]. This finding implied adap-
tation had occurred [205]. While it is postulated that reduced bar-
biturate sleep time results from altered pharmacokinetics [207],
contradictory evidence exists: Watanabe et al found increased
hepatic drug metabolism in isolated rats [207], but Dairman and
Balazs failed to demonstrate increased liver microsomal activity
or a change in liver to bodyweight ratios [205]. This finding
could have profound implications for pharmacokinetic research
but needs further characterization. Alterations to neurochem-
istry may also occur, leading to decreased opioid responses
and increased pain thresholds, possibly resulting from changes
to dopaminergic activity in the brain [208]. Brain-derived neu-
rotrophic factor, which is involved in neuronal survival and is
reduced in the face of stress or a depressive disorder [209],
was also decreased in socially isolated male mice [165]. Finally,
bodyweight and growth may be impacted by single housing due
to reduced competition for food. The literature is conflicting in
this regard (see [159] for review). However, it remains important
for researchers to consider the potential impact of weight and
caloric intake on other pathologies, such as tumors, with the
suggestion that obesity may increase tumor incidence and alter
tumor kinetics and metastasis (see, eg, [210]).
In primates, individual housing reduces species-typical
behaviors and increases abnormal behaviors. Injurious self-
biting is elevated in individually housed primates, leading to
tissue damage and potentially triggering a range of inflam-
matory responses with the potential to influence research
outcomes (reviewed in [211]). In contrast to rodents, rearing
of primates in isolation leads to less exploratory behavior [212].
There may also be an impact on parental competence, although
this needs further investigation [160]. Schapiro, in a review of
their own work on cell-mediated immune (CMI) response to
social isolation, concluded that in most studies differences
in CMI between singly and socially housed subjects were
present [213]. Many, although not all, of their studies implied
that single caging had negative immunological consequences.
ThemagnitudeoftheCMIresponsemayhavearelationship
with disease susceptibility. Interestingly, in 1 study, pair-
housed macaques produced higher proliferation responses to
2 common diarrhea-inducing pathogens than both single- and
group-housed animals [214]. This result suggests that while
isolation may be detrimental to welfare and study outcomes,the
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

ILAR Journal, 2019, Vol. 60, No. 2 257
alternative of group housing may also evoke similar responses,
and therefore finding a suitable group size and establishing
stable social relationships may be of greater value.
Summary
While the impact of individual housing on research parameters
is not uniform and fully characterized, it is clear that there is suf-
ficient evidence to indicate that there may be research impact.
This is especially true for scientists in neuroscience conducting
behavioral tests or assessing neurobiological processes. Given
this finding, it is wise to consider ways of minimizing such
impact.
Whereas individual housing may be necessary to minimize
the effects of aggression, it is worth considering whether all
options for group housing have been exhausted. The achieve-
ment of harmonious groups through manipulation of the social
and micro-environment is 1 strategy to be trialed. This can
be done by optimizing group sizes to reduce aggression (see
below) or by transferring nesting material at cage change [215].
Other options include co-housing males with neutered members
of the opposite [216]orsamesex[217,218] to maintain social
relationships with reduced aggression.
Additionally, procedural modifications or training may allow
group housing where individual housing was previously neces-
sary, for example, using clips or buried sutures to reduce the risk
of wound interaction by conspecifics [219] or training to allow
control over individual food intake in primates [220].
If social isolation is required and it is not consistent across
groups/sexes, it would be worth assessing its impact when per-
forming statistical analyses. The inclusion of “cage”as a factor in
analysis would identify significant changes arising from single
housing and allow full consideration of its effects on the data
presented.
Group Housing
While group housing provides the opportunity for social behav-
ior and enhances animal welfare, it may have experimental
impact. If groups are not stable, social stresses impact health,
behavior, and physiology. This is of particular concern in male
laboratory animals where dominance hierarchies need to be
established and on occasion reestablished [221]. However,
even positive social interactions can impact behavior and
stress responses and consequently need to be considered and
accounted for by scientists. This impact is the so-called “social
buffering” effect (see below). Since most research in this area
is rodent focused, rats and mice predominate the following
discussion.
Species-Specific Considerations. In agonistic encounters, effects
on animals and consequently on physiology depend on the
social status of the animals involved. Victory in mice leads to a
rewarding experience for the victor, generating a positive affec-
tive state [158] and operant learning and reinforcing the aggres-
sive behavior [222]. Neurobiological conditioning results in cat-
echolaminergic [223] and HPA axis activation [224]. Reduced
serotonergic activity and modulation of the Gamma aminobu-
tyric acidA(GABAA) receptor complex are also thought to con-
tribute to the heightened aggression [225]. Despite their success,
these mice can develop abnormal locomotor and exploratory
behaviors in the open-field test, exhibited through hyperactivity
[226], anxiety [227], disturbances in motivation and cognitive
behaviors, and impairments of sociability [228]. These effects
resemble those seen in psychopathies such as bipolar disorder
[226].
For defeated animals, injury and mortality are obvious
consequences of aggression. The impact of mortality on
research is self-evident. The process of wounding triggers a
complex process involving inf lammation, proliferation, and
remodeling (reviewed in [229]). This includes the production
of pro-inflammatory cytokines, growth factors, and similar
molecules that are often of interest in research studies [229]. The
additional stress caused by further aggressive encounters may
additionally impair wound healing [230] and cytokine expression
[231] through stress-induced increases in corticosterone. This
mechanism can also lead to exacerbation of neuronal death
following stroke and cardiac arrest and therefore influence
recovery from these events when modeled [232].
Even in the absence of physical trauma, a range of studies
has demonstrated that social defeat and subordination leads to
changes in immune function, metabolism, and behavior [233].
Subordinate rats showed anxiety-like behaviors in the open-
field, social interaction, novel object recognition, and judgment
bias tests [234] and learning impairment in the passive avoid-
ance task [235] and water-maze test [236,237]. However, the
converse has been seen in mice with subordinates showing
less anxiety [238], although learning was impaired [239]. Fur-
thermore, the bladders of subordinate male mice have been
shown to be fuller than dominant animals due to differences in
cage-marking [240]. This finding could have health implications
[240]. Conversely, it has been suggested there are no differ-
ences between dominants and subordinates in physiological and
behavioral responses in stable social groups [241,242]. However,
these studies involved familiar sibling cage-mates.
In rabbits, while aggression in group-housed animals can
result in significant clinical issues, limited studies have not
demonstrated differences in humoral and delayed hypersen-
sitivity responses, feed intake, growth rates, and physiologi-
cal stress responses (corticosterone and lymphocyte counts)
between single- and group-housed rabbits [243,244]. However,
in an agricultural setting, bone thickness of meat rabbits was
increased when group housed, presumably due to increased
activity [245], although this activity may have resulted from
conspecifics f leeing agonistic encounters [245]. Since rabbits are
common orthopedic research models, this may be an important
finding. Wounding has consistently been shown to increase in
group-housed animals [246,247] with the accompanying inflam-
matory, proliferation, and remodeling responses (reviewed in
[229]).
A range of beneficial effects of group housing has been
described in nonhuman primates. These include a reduction in
coronary artery atherosclerosis in cynomolgus macaques [248]
as well as reduced stress hormone concentrations and plasma
prolactin [249]. However, social status may moderate these
responses. For example, subordinate females had increased
atherosclerosis compared with dominant females [250][248],
and dopamine receptor availability was increased in dominant
compared with subordinate cynomolgus macaques [251–253].
Consideration also must be given to the effect of removal from
groups for testing on research parameters, since this may induce
stress [254]. As in other species, aggression is common and may
lead to significant injury [255].
Summary. Although group housing can impact research
parameters, it is generally desirable in adherence with current
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

258 Whittaker and Hickman
Figure 2: Schematic demonstrating the types of stressors that may impact on an individual and the factors that may modify the resulting effects. Reproduced from
Beery and Kaufer, 2015, Neurobiology of Stress 2015;1:116–127 doi: 10.1016/j.ynstr.2014.10.004. Reproduced under the terms of the Creative Commons Attribution-Non
Commercial-Share Alike License (CC BY NC SA).
regulations and best practice based on animal welfare benefits
(see, eg, [256]). Data from rodent studies imply that a sudden
change in housing method is more likely to induce stress than
the method itself [257]. Familiarity as a moderator of social
tension has been examined in rodents [258] and primates [259].
These relationships are complex and likely subject to effects
of social status. Therefore, consideration should be given to
mixing unfamiliar animals as well as other individual factors
such as the animals’ age. In primates, immature and aged
animals are more likely to integrate into an established male
macaque group than adult males [160]. There are methods to
prepare the environment prior to establishing new groups—
for example, by providing a new territory or a complex
enriched environment [160], removing the odor of females when
grouping males [260], bedding transfer [215], and perhaps using
noncontact familiarization in macaques [160], although the
latter is controversial [261]—that may improve the likelihood
of establishing a new group. The literature should be consulted
to determine the optimal group size based on the species, sex,
and strain of interest (see eg, [262,263]). Finally, given that social
status may impact experimental results, the social status of
individual animals should be ascertained through behavioral
observation (see eg, [264,265]) and accounted for when analyzing
experimental outcomes.
Social Buffering
Negative social interactions can adversely affect animal welfare
and impact research. However, social interaction can also moder-
ate stressful experiences by buffering adverse impact and build-
ing resilience [266]. This phenomenon, called social buffering,
has been documented in rodents, birds, and nonhuman primates
(see Figure 2 [267]).
Research has shown that there is considerable inter-
individual variability in the behavioral response to stressful
events. For example, while chronic social defeat stress in mice
leads to a range of outcomes, including impaired neurogenesis,
altered fear acquisition, anhedonia, and changes in neural
circuitry and transmission [264], there is bimodal segregation
of animals into affected and resistant individuals. Resistant
animals remain motivated for social contact; that is, they
demonstrate resilience [265].
Although stress affects future social interaction, the inter-
action itself buffers stressor effects. In humans, positive social
relationships have been shown to improve immune function,
cardiovascular health, and other health indicators [270]. Lifespan
hasalsobeenshowntoincreaseasaresultofpositiveandstable
relationships in a range of species, including humans [271],
baboons [272], and rats [273]. Furthermore, a range of studies
has demonstrated that social support has a variety of effects on
behavior, the endocrine response, and neurobiology (reviewed
in [267]). Pertinent findings, which may impact research, are
discussed.
Species-Specific Considerations. A growing body of research has
demonstrated the effects of social buffering on pain tolerance
(reviewedin [134]). The mechanisms contributing to these effects
are likely diverse and include interplay between various neuro-
transmitters, their receptors, and neurotrophic factors. Tabl e 1
provides a summary of some of the major neurotransmitters
of interest. Fanselow (1985) found that naïve rats exposed to
stressed rats exhibited less pain behavior after injection of an
inflammatory agent, a form of contagious analgesia mediated
by endogenous opioids [274]. Mice that witnessed other mice
being attacked by biting flies themselves demonstrated analge-
sia when later exposed to nonbiting flies [275]. Visual cues are
considered to play a primary role in mediating “pain communi-
cation” [130]. Langford et al (2006) demonstrated that pain status
could be transmitted between cage-mates by facial expression
or grimace, causing pain hypersensitivity [130]. However, other
sensory modalities beyond vision are also suspected to play
a role in the social communication of the pain response. For
example, naïve mice exposed to olfactory cues on bedding from
mice that displayed hypersensitivity due to ethanol withdrawal
also demonstrated mechanical hypersensitivity [276]. Investiga-
tions in this area have been principally directed to the study of
“empathy” using animal models. The social sharing of emotion,
known as emotional contagion, is described in the rodent lit-
erature [277]. It increases the probability of similar behavior in
others and a quick adaptation to challenge [278]. Several studies
in rodents have demonstrated that rodents can share negative
emotional states such as pain, fear, and anxiety (see eg, [130,279–
282]). Information about positive affective states may also be able
to be communicated similarly, but this is a difficult area to study
[283].
Studies investigating social buffering effects have tended to
focus on the HPA axis.Rats exposed to a novel environment with
partner rats had a lower corticosterone response than solitary
animals exposed to the same environment [284]. Similarly, fol-
lowing hormonal stimulation of the HPA axis via corticotrophin
releasing factor,group-housed rats had lower corticosterone and
adrenocorticotrophic hormone (ACTH) responses than singly-
housed males [285]. Male guinea pigs also exhibited reduced
HPA axis responses when entering a novel environment in the
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

ILAR Journal, 2019, Vol. 60, No. 2 259
Tab le 1 Summary of Major Transmitters Involved In Regulation of Emotion
Neurotransmitter Function Effect of Deficit Effect of Surplus Excitatory or
Inhibitory
Receptor Binding
Acetylcholine Regulates sleep cycle,
essential for muscle
functioning
Lack of muscle
movement and
control
Severe muscle
spasms
Excitatory (except in
heart)
Nicotinic ACh
receptors
mAChRs
Glutamate Most common CNS
neurotransmitter
regulates CNS
excitability, learning
process, and memory,
ensures homeostasis
with GABA, aids efficient
synaptic transmission
Cognitive and
affective disorders
when balance
disrupted
Excess with too little
GABA leads to
epileptic seizures
Strongly excitatory
•NMDA
•AMPA
•Kainate
•Metabotropic
G-protein
coupled
receptors
Norepinephrine Increases alertness and
wakefulness, stimulates
bodily processes such as
adrenaline production
and consequent
fight/f light response
Depression and
anxiety
Impaired sleep cycle Excitatory Adrenergic
receptors (αand ß
Dopamine Pleasurable sensation,
inhibits unnecessary
movement, role in
motivation or desire to
complete a task,
stimulates reward center
(hypothalamus)
Parkinson’s Disease
(failure to inhibit
un- necessary
movement)
Anxiety, memory
impairment,
attention deficit
Confusion and
inability to focus
Excitatory and
inhibitory depending
on type of receptor
bound to
Dopamine receptors
of 5 subtypes
GABA Most common CNS
inhibitory
neurotransmitter offsets
excitatory messages in
homeostasis with
glutamate
Anxiety, seizures,
tremors, insomnia
Sleep disorders Strongly inhibitory
•GABA A
receptors:
ionotropic
•GABA B
receptors:
metabotropic
Endorphins Pain perception and
positive emotions,
opioid-like. Suppresses
GABA to produce
dopamine
Experience of pain Artificial “highs,”
change in behavior in
response to pain to
prevent injury
exacerbation may
not occur
Inhibitory Opioid receptors:
mu-receptors
(primarily)
delta-receptors
kappa-receptors
nociceptin receptors
Serotonin Regulates body
temperature, perception
of pain, emotions, and
sleep cycle
Depression and
mood disorders,
decreased immune
system function
Mania Inhibitory 5-HT (serotonin)
receptors of a range
of subtypes [304]
Oxytocin Circulating hormone that
can act as
neurotransmitter and
modulator, involved in
social behavior and
bonding, inhibits fear
responses in amygdala,
hence coining “trust
hormone,” may enhance
social memory, inhibits
stress-induced activity in
hypothalamic-pituitary
axis [305]
Autism spectrum
disorders,
decreased empathy,
mood and anxiety
disorders
Possible emotional
oversensitivity [306]
Excitatory and
inhibitory dependent
on brain region, cell
type and receptor
coupling, modulates
GABA and glutamate
synaptic actions to
exerteffects[307]
Oxytocin receptor: G
protein-linked [308]
Compiled from [311–311]. mAChRs = muscarinic ACh receptors; GABA = gamma aminobutyric acid; CNS = central nervous system; ACh = acetylcholine; NMDA =
N-Methyl- d-aspartate; AMPA = α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; 5-HT = 5-hydroxytryptamine.
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

260 Whittaker and Hickman
Figure 3: Proposed mechanisms for social buffering. ACTH = adrenocorticotrophic hormone; CRF = corticotrophin-releasing factor; DA= dopamine; OPD = opioid;
OXY = oxytocin. Reproduced from Kikusui et al. Philosophical transactions of the Royal Society of London. Series B, Biological sciences 2006;361(1476):2215–2228 doi:
10.1098/rstb.2006.1941. Reproduced with permission of the Royal Society in the format republish in a journal via Copyright Clearance Center.
presence of a female [286]. Of interest in this study is that cortisol
levels were reduced and similar in either the presence of the
biological mother or an unfamiliar adult female. This finding is
in contrast with much of the literature that implies that strength
of affiliation or attachment enhances social buffering effects
(see [267] for review).
Social buffering effects on the endocrine system are likely
mediated through increased neural activity in the paraventric-
ular nucleus (PVN) with a role for oxytocin. Kiyokawa et al (2004)
fear-conditioned rats to a shock box and then reexposed the
animals to the box with or without a partner. Reexposure to
the box caused an increase in freezing behavior and a decrease
in activity in conjunction with increased PVN c-Fos immunore-
activity. These responses were reduced when accompanied by
a partner on re-exposure [287]. Direct administration of oxy-
tocin to the PVN in prairie voles also reduced corticosterone
stressor responses [288], which was supported by findings from
humans where oxytocin and social support reduced cortisol
responses [289].
The opioid system also plays an essential role in the neu-
robiology of social attachment. Opioids ameliorate separation-
induced anxiety behavior [290] and are produced in response
to social contact [291,292]. There are clearly overlaps between
the role of the neural opioid system in reward and facilitation
of social bonding and their analgesia-inducing effects, as previ-
ously discussed. Additionally, a complex relationship between
the oxytocin and the opioid systems is likely to exist and is
involved in the neural mechanisms of social buffering [267].
Figure 3 illustrates the range of interrelationships in the CNS and
their effects on social buffering. This figure illustrates that the
potential effects of social buffering on research outcomes are
wide-ranging and extensive.
Social buffering has also been well-documented in primate
species (see [293] for review). Early studies that considered
the role of the maternal relationship in modulating HPA axis
responses showed that the presence of the mother reduced
stress responses [294–296]. The nature of the cues needed to
elicit buffering has also been investigated; maternal separation
with visual and auditory access to the mother still led to a
bufferingresponse[297]. This study also suggests that the
group may act as a social buffer in young, maternally separated
rhesus monkeys. Although socially stable, positive relationships
may have value [298], it appears that the early mother–infant
relationship is critical in the later ability of primates to benefit
from social buffering [299]. Social buffering has also been found
in adult squirrel monkeys [300], marmosets [301], and macaques
[302,303].
Summary. The impact of social buffering, the nature of social
relationships, and the practical realities of group housing in
laboratory animals, as discussed, are clearly linked in relation
to their impact on research parameters. The possibility of emo-
tional contagion of pain or distress, resulting in measurable
physiological outcomes, provokes interesting questions about
the reliability of research findings when healthy animals are
co-housed with those experiencing adverse effects. The impact
is difficult to control for in research studies. An understanding
of the nature and strength of social relationships in different
species and strain is necessary since not all social interac-
tions are equal and buffering effects may differ by familiar-
ity, sex, age, and affective state [266]. Using this understand-
ing to guide housing practices and attempting to standardize
the types of relationships experienced by study animals—for
instance, reducing early maternal separation and maintaining
animals in familiar groups—is perhaps the best that scientists
can do.
Conclusion
A variety of factors may influence the reproducibility of stud-
ies utilizing animal models and thus impact the translation
of research resulting from behavior, whether it be innate or
acquired or as a result of social groupings. In many cases, these
factors may be hard to control due to their inherent nature
or the practicalities of the vivarium environment. However, an
awareness of the factors is important since their impact could
possibly be reduced through careful experimental design and
their consideration in data analyses. In many cases, a general
awareness that all groups of animals should be treated the
same throughout the study, and that changes to social groupings
should be minimized, will reduce variation.
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

ILAR Journal, 2019, Vol. 60, No. 2 261
References
1. Sneddon LU, Halsey LG, Bury NR. Considering aspects of the
3Rs principles within experimental animal biology. J. Exp.
Biol. 2017; 220:3007–3016. doi: 10.1242/jeb.147058.
2. Pridgeon CS et al. Innovative organotypic in vitro models
for safety assessment: aligning with regulatory require-
ments and understanding models of the heart, skin, and
liver as paradigms. Arch. Toxicol. 2018; 92:557–569. doi:
10.1007/s00204-018-2152-9.
3. Freires IA, Sardi JC, de Castro RD et al. Alternative animal
and non-animal models for drug discovery and develop-
ment: bonus or burden? Pharm. Res. 2017; 34:681–686. doi:
10.1007/s11095-016-2069-z.
4. Loos M et al. Within-strain variation in behavior differs
consistently between common inbred strains of mice. Mam-
malian genome : official journal of the International Mammalian
Genome Society 2015; 26:348–354. doi: 10.1007/s00335-015-
9578-7.
5. Moy SS et al. Mouse behavioral tasks relevant to autism:
phenotypes of 10 inbred strains. Behav. Brain Res. 2007;
176:4–20. doi: 10.1016/j.bbr.2006.07.030.
6. Phillips TJ et al. Harnessing the mouse to unravel the genet-
ics of human disease. Genes Brain Behav. 2002; 1:14–26.
7. Rafferty SA, Quinn TA.A beginner’s guide to understanding
and implementing the genetic modification of zebrafish.
Prog. Biophys. Mol. Biol. 2018; 138:3–19. doi: 10.1016/j.
pbiomolbio.2018.07.005.
8. Delerue F, Ittner LM. Generation of genetically modified
mice through the microinjection of oocytes. Journal of visu-
alized experiments : JoVE 2017. doi: 10.3791/55765.
9. Nam M-H, Chun M-S, Seong J-K et al. Ensuring reproducibil-
ity and ethics in animal experiments reporting in Korea
using the ARRIVE guideline. Lab. Anim. Res. 2018; 34:11–19.
doi: 10.5625/lar.2018.34.1.11.
10. Han S et al. A checklist is associated with increased qual-
ity of reporting preclinical biomedical research: a sys-
tematic review. PLoS One 2017; 12:e0183591. doi: 10.1371/
journal.pone.0183591.
11. NC3R.org. ARRIVE Guidelines.https://www.nc3rs.org.uk/arri
ve-guidelines (2019).
12. Checklists work to improve science. Nature 2018;
556:273–274. doi: 10.1038/d41586-018-04590-7.
13. Hanifin JP, Dauchy RT, Blask DE, Hill SM, Brainard GC. Rele-
vance of Electrical Light on Circadian, Neuroendocrine, and
Neurobehavioral Regulation in Laboratory Animal Facili-
ties. ILAR Journal 2020; 1–9. doi: 10.1093/ilar/ilaa010.
14. Kurtz DM, Feeney WP. The Inf luence of Feed and Drinking
Water on Terrestrial Animal Research and Study Replicabil-
ity. ILAR Journal 2020; 1–22. doi: 10.1093/ilar/ilaa012.
15. Hasenau JJ. Reproducibility and Comparative aspects of
Terrestrial Housing Systems and Husbandry Procedures in
Animal Research Facilities on Study Data. ILAR Journal 2020;
1–9. doi: 10.1093/ilar/ilz021.
16. Sundberg JP, Schofield PN. Living inside the box: environ-
mental effects on mouse models of human disease. Dis.
Model. Mech. 2018; 11. doi: 10.1242/dmm.035360.
17. Franklin CL, Ericsson AC. Complex Microbiota in Lab-
oratory Animal RodCentralized mouse repositoriesents:
Management Considerations. ILAR Journal 2020; 1–9. doi:
10.1093/ilar/ilaa011.
18. Casane D, Policarpo M, Laurenti P. Why the mutation rate
never reaches zero? Medecine sciences : M/S 2019; 35:245–251.
doi: 10.1051/medsci/2019030.
19. Lynch M et al. Genetic drift, selection and the evolution
of the mutation rate. Nat. Rev. Genet. 2016; 17:704–714. doi:
10.1038/nrg.2016.104.
20. Kumar TR, Larson M, Wang H et al. Transgenic
mouse technology: principles and methods. Methods
in molecular biology (Clifton, N.J.) 2009; 590:335–362. doi:
10.1007/978-1-60327-378-7_22.
21. Beck JA et al. Genealogies of mouse inbred strains. Nat.
Genet. 2000; 24:23–25. doi: 10.1038/71641.
22. Chia R, Achilli F, Festing MF et al. The origins and uses of
mouse outbred stocks. Nat. Genet. 2005; 37:1181–1186. doi:
10.1038/ng1665.
23. Bult CJ, Eppig JT, Blake JA et al. Mouse genome database
2016. Nucleic Acids Res. 2016; 44:D840–D847. doi: 10.1093/nar/
gkv1211.
24. Lau CH, Suh Y. In vivo epigenome editing and transcrip-
tional modulation using CRISPR technology. Transgenic Res.
2018; 27:489–509. doi: 10.1007/s11248-018-0096-8.
25. Lampreht Tratar U, Horvat S, Cemazar M. Transgenic mouse
models in cancer research. Front. Oncol. 2018; 8:268. doi:
10.3389/fonc.2018.00268.
26. Ito Y, Noguchi K, Morishima Y et al. Generation and charac-
terization of tissue-type plasminogen activator transgenic
rats. J. Thromb. Thrombolysis 2018; 45:77–87. doi: 10.1007/
s11239-017-1582-1.
27. Nohmi T, Masumura K, Toyoda-Hokaiwado N. Transgenic
rat models for mutagenesis and carcinogenesis. Genes and
environment : the official journal of the Japanese Environmen-
tal Mutagen Society 2017; 39:11. doi: 10.1186/s41021-016-
0072-6.
28. Chen X, Gays D, Santoro MM. Transgenic Zebrafish. Methods
in molecular biology (Clifton, N.J.) 2016; 1464:107–114. doi:
10.1007/978-1-4939-3999-2_10.
29. Lee O, Green JM, Tyler CR. Transgenic fish systems and
their application in ecotoxicology. Crit. Rev. Toxicol. 2015;
45:124–141. doi: 10.3109/10408444.2014.965805.
30. Stern JA, Ueda Y. Inherited cardiomyopathies in veterinary
medicine. Pflugers Archiv : European journal of physiology 2019;
471:745–753. doi: 10.1007/s00424-018-2209-x.
31. Gurda BL, Bradbury AM, Vite CH. Canine and feline mod-
els of human genetic diseases and their contributions to
advancing clinical therapies. The Yale journal of biology and
medicine 2017; 90:417–431.
32. Camacho P, Fan H, Liu Z et al. Large mammalian animal
models of heart disease. Journal of cardiovascular development
and disease 2016; 3. doi: 10.3390/jcdd3040030.
33. Seaton M, Hocking A, Gibran NS. Porcine models of
cutaneous wound healing. ILAR J. 2015; 56:127–138. doi:
10.1093/ilar/ilv016.
34. Overgaard NH et al. Of mice, dogs, pigs, and men: choosing
the appropriate model for immuno-oncology research. ILAR
J. 2018; 59:247–262. doi: 10.1093/ilar/ily014.
35. Muller AMS et al. Modeling chronic graft-versus-host dis-
ease in MHC-matched mouse strains: genetics, graft com-
position, and tissue targets. Biology of blood and marrow trans-
plantation : journal of the American Society for Blood and Marrow
Transplantation 2019. doi: 10.1016/j.bbmt.2019.08.001.
36. Wodarz D, Komarova N. Towards predictive computational
models of oncolytic virus therapy: basis for experimental
validation and model selection. PLoS One 2009; 4:e4271. doi:
10.1371/journal.pone.0004271.
37. Pfefferle AD et al. Transcriptomic classification of geneti-
cally engineered mouse models of breast cancer identifies
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

262 Whittaker and Hickman
human subtype counterparts. Genome Biol. 2013; 14:R125.
doi: 10.1186/gb-2013-14-11-r125.
38. Festing MF. Evidence should trump intuition by preferring
inbred strains to outbred stocks in preclinical research. ILAR
J. 2014; 55:399–404. doi: 10.1093/ilar/ilu036.
39. Walsh NC et al. Humanized mouse models of clinical
disease. Annu. Rev. Pathol. 2017; 12:187–215. doi: 10.1146/
annurev-pathol-052016-100332.
40. Brehm MA, Shultz LD, Greiner DL. Humanized mouse mod-
els to study human diseases. Current opinion in endocrinol-
ogy, diabetes, and obesity 2010; 17:120–125. doi: 10.1097/
MED.0b013e328337282f.
41. Shultz LD, Brehm MA, Garcia-Martinez JV et al. Humanized
mice for immune system investigation: progress, promise
and challenges. Nat. Rev. Immunol. 2012; 12:786–798. doi:
10.1038/nri3311.
42. Foster PL. Methods for determining spontaneous
mutation rates. Methods Enzymol. 2006; 409:195–213. doi:
10.1016/S0076-6879(05)09012-9.
43. Pellis SM, Pellis VC. Play f ighting of rats in comparative per-
spective: a schema for neurobehavioral analyses. Neurosci.
Biobehav. Rev. 1998; 23:87–101.
44. Pellis SM, Pellis VC. What is play fighting and what
is it good for? Learn. Behav. 2017; 45:355–366. doi:
10.3758/s13420-017-0264-3.
45. Palagi E et al. Rough-and-tumble play as a window on
animal communication. Biol. Rev. Camb. Philos. Soc. 2016;
91:311–327. doi: 10.1111/brv.12172.
46. Caracciolo B, Xu W, Collins S et al. Cognitive decline, dietary
factors and gut-brain interactions. Mech. Ageing Dev. 2014;
136-137:59–69. doi: 10.1016/j.mad.2013.11.011.
47. Blusztajn JK, Slack BE, Mellott TJ. Neuroprotective
actions of dietary choline. Nutrients 2017; 9. doi: 10.3390/
nu9080815.
48. Obri A, Khrimian L, Karsenty G et al. Osteocalcin in the
brain: from embryonic development to age-related decline
in cognition. Nat. Rev. Endocrinol. 2018; 14:174–182. doi:
10.1038/nrendo.2017.181.
49. Spiegel AM, Sewal AS, Rapp PR. Epigenetic contributions
to cognitive aging: disentangling mindspan and lifespan.
Learning & memory (Cold Spring Harbor, N.Y.) 2014; 21:569–574.
doi: 10.1101/lm.033506.113.
50. Weinhold B. Epigenetics: the science of change.
Environ. Health Perspect. 2006; 114:A160–A167. doi:
10.1289/ehp.114-a160.
51. Szyf M, Weaver I, Meaney M. Maternal care, the epigenome
and phenotypic differences in behavior. Reproductive toxi-
cology (Elmsford, N.Y.) 2007; 24:9–19. doi: 10.1016/j.reprotox.
2007.05.001.
52. Gareau M, Cognitive G. Function and the microbiome. Int.
Rev. Neurobiol. 2016; 131:227–246. doi: 10.1016/bs.irn.2016.
08.001.
53. Bilbo SD, Schwarz JM. The immune system and
developmental programming of brain and behavior. Front.
Neuroendocrinol. 2012; 33:267–286. doi: 10.1016/j.yfrne.
2012.08.006.
54. Ziegler-Waldkirch S et al. Seed-induced Abeta deposition is
modulated by microglia under environmental enrichment
in a mouse model of Alzheimer’s disease. EMBO J. 2018;
37:167–182. doi: 10.15252/embj.201797021.
55. Stuart KE et al. Mid-life environmental enrichment
increases synaptic density in CA1 in a mouse model of
Abeta-associated pathology and positively influences
synaptic and cognitive health in healthy ageing. J. Comp.
Neurol. 2017; 525:1797–1810. doi: 10.1002/cne.24156.
56. Vanderschuren LJ, Trezza V. What the laboratory rat has
taught us about social play behavior: role in behavioral
development and neural mechanisms. Curr.Top.Behav.Neu-
rosci. 2014; 16:189–212. doi: 10.1007/7854_2013_268.
57. Hori M et al. Tickling during adolescence alters fear-related
and cognitive behaviors in rats after prolonged isola-
tion. Physiol. Behav. 2014; 131:62–67. doi: 10.1016/j.physbeh.
2014.04.008.
58. LaFollette MR, O’Haire ME, Cloutier S et al. Rat tick-
ling: a systematic review of applications, outcomes, and
moderators. PLoS One 2017; 12:e0175320. doi: 10.1371/jour-
nal.pone.0175320.
59. Byosiere SE, Espinosa J, Smuts B. Investigating the func-
tion of play bows in adult pet dogs (Canis lupus famil-
iaris). Behav. Process. 2016; 125:106–113. doi: 10.1016/j.beproc.
2016.02.007.
60. Byosiere SE, Espinosa J, Marshall-Pescini S et al. Investigat-
ing the function of play bows in dog and wolf puppies (Canis
lupus familiaris, Canis lupus occidentalis). PLoS One 2016;
11:e0168570. doi: 10.1371/journal.pone.0168570.
61. Bradshaw JW, Pullen AJ, Rooney NJ. Why do adult dogs
’play’? Behav. Process. 2015; 110:82–87. doi: 10.1016/j.beproc.
2014.09.023.
62. Mehrkam LR, Hall NJ, Haitz C et al. The influence of breed
and environmental factors on social and solitary play in
dogs (Canis lupus familiaris). Learn. Behav. 2017; 45:367–377.
doi: 10.3758/s13420-017-0283-0.
63. Leca JB et al. A multidisciplinary view on cultural prima-
tology: behavioral innovations and traditions in Japanese
macaques. Primates; journal of primatology 2016; 57:333–338.
doi: 10.1007/s10329-016-0518-2.
64. Shimada M, Sueur C. Social play among juvenile wild
Japanese macaques (Macaca fuscata) strengthens their
social bonds. Am. J. Primatol. 2018; 80. doi: 10.1002/ajp.22728.
65. Ballesta S, Reymond G, Pozzobon M et al. Compete to
play: trade-off with social contact in long-tailed macaques
(Macaca fascicularis). PLoS One 2014; 9:e115965. doi: 10.1371/
journal.pone.0115965.
66. Yanagi A, Berman CM. Body signals during social play in
free-ranging rhesus macaques (Macaca mulatta): a sys-
tematic analysis. Am.J.Primatol.2014; 76:168–179. doi:
10.1002/ajp.22219.
67. Ahloy-Dallaire J, Espinosa J, Mason G. Play and optimal
welfare: does play indicate the presence of positive affec-
tive states? Behav. Process. 2018; 156:3–15. doi: 10.1016/
j.beproc.2017.11.011.
68. Cloutier S, LaFollette MR, Gaskill BN et al. Tickling, a tech-
nique for inducing positive affect when handling rats. Jour-
nal of visualized experiments :JoVE 2018. doi: 10.3791/57190.
69. LaFollette MR, O’Haire ME, Cloutier S et al. Practical rat
tickling: determining an efficient and effective dosage of
heterospecific play. Appl. Anim. Behav. Sci. 2018; 208:82–91.
70. Mitchell RW. A critique and empirical assessment of
Alexandra Horowitz and Julie Hecht’s "examining dog-
human play: the characteristics, affect, and vocalizations
of a unique interspecific interaction". Anim. Cogn. 2017;
20:553–565. doi: 10.1007/s10071-017-1075-9.
71. Horowitz A, Hecht J. Examining dog-human play: the char-
acteristics, affect, and vocalizations of a unique interspe-
cific interaction. Anim. Cogn. 2016; 19:779–788. doi: 10.1007/
s10071-016-0976-3.
72. Herwijnen IRV, van der Borg JAM, Naguib M et al. Dog own-
ership satisfaction determinants in the owner-dog relation-
ship and the dog’s behaviour. PLoS One 2018; 13:e0204592.
doi: 10.1371/journal.pone.0204592.
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

ILAR Journal, 2019, Vol. 60, No. 2 263
73. Benefiel AC, Dong WK, Greenough WT. Mandatory
“enriched” housing of laboratory animals: the need for
evidence-based evaluation. ILAR J. 2005; 46:95–105. doi:
10.1093/ilar.46.2.95.
74. Bailoo JD et al. Effects of cage enrichment on behavior.
Welfare and outcome variability in female mice. Frontiers in
behavioral neuroscience 2018; 12:232–232. doi: 10.3389/fn-
beh.2018.00232.
75. Masopust D, Sivula CP, Jameson SC. Of mice, dirty mice, and
men: using mice to understand human immunology. Jour-
nal of immunology (Baltimore, Md. : 1950) 2017; 199:383–388.
doi: 10.4049/jimmunol.1700453.
76. van der Horst FC, van der Veer R. Loneliness in infancy:
Harry Harlow. John Bowlby and issues of separation.
Integrative psychological & behavioral science 2008; 42:325–335.
doi: 10.1007/s12124-008-9071-x.
77. Murthy S, Gould E. Early life stress in rodents: animal
models of illness or resilience? Front. Behav. Neurosci. 2018;
12:157–157. doi: 10.3389/fnbeh.2018.00157.
78. Chapillon P, Patin V, Roy V et al. Effects of pre- and post-
natal stimulation on developmental, emotional, and cog-
nitive aspects in rodents: a review. Dev. Psychobiol. 2002;
41:373–387. doi: 10.1002/dev.10066.
79. Plescia F et al. Early handling effect on female rat spatial
and non-spatial learning and memory. Behav. Process. 2014;
103:9–16. doi: 10.1016/j.beproc.2013.10.011.
80. Pritchard LM, Van Kempen TA, Zimmerberg B. Behavioral
effects of repeated handling differ in rats reared in social
isolation and environmental enrichment. Neurosci. Lett.
2013; 536:47–51. doi: 10.1016/j.neulet.2012.12.048.
81. Siviy SM. Effects of neonatal handling on play and anxiety
in F344 and Lewis rats. Dev. Psychobiol. 2018; 60:458–467. doi:
10.1002/dev.21622.
82. Sampedro-Piquero P, Begega A. Environmental enrichment
as a positive Behavioral intervention across the lifespan.
Curr. Neuropharmacol. 2017; 15:459–470. doi: 10.2174/157015
9x14666160325115909.
83. Queen NJ et al. Environmental enrichment improves
metabolic and behavioral health in the BTBR mouse model
of autism. Psychoneuroendocrinology 2019; 111:104476. doi:
10.1016/j.psyneuen.2019.104476.
84. Bayne K. Environmental enrichment and mouse mod-
els: current perspectives. Animal models and experimental
medicine 2018; 1:82–90. doi: 10.1002/ame2.12015.
85. Dobson GP, Letson HL, Biros E et al. Specific pathogen-free
(SPF) animal status as a variable in biomedical research:
Have we come full circle? EBioMedicine 2019; 41:42–43. doi:
10.1016/j.ebiom.2019.02.038.
86. Mo C, Renoir T, Hannan AJ. What’s wrong with my mouse
cage? Methodological considerations for modeling lifestyle
factors and gene-environment interactions in mice. J. Neu-
rosci. Methods 2016; 265:99–108. doi: 10.1016/j.jneumeth.
2015.08.008.
87. Cutuli D et al. Pre-reproductive parental enriching expe-
riences influence Progeny’s developmental trajectories.
Front. Behav. Neurosci. 2018; 12:254. doi: 10.3389/fnbeh.2018.
00254.
88. NIH. Consideration of sex as a biological variable in NIH-
funded research,https://grants.nih.gov/grants/guide/notice-
files/NOT-OD-15-102.html (2015).
89. Clayton JA, Collins FS. Policy: NIH to balance sex in cell and
animal studies. Nature 2014; 509:282–283.
90. Clayton JA. Studying both sexes: a guiding principle for
biomedicine. FASEB journal: official publication of the Fed-
eration of American Societies for Experimental Biology 2016;
30:519–524. doi: 10.1096/fj.15-279554.
91. Swanborg RH. Experimental autoimmune encephalomyeli-
tis in the rat: lessons in T-cell immunology and autoreac-
tivity. Immunol. Rev. 2001; 184:129–135.
92. Hammerschmidt K, Radyushkin K, Ehrenreich H et al. The
structure and usage of female and male mouse ultrasonic
vocalizations reveal only minor differences. PLoS One 2012;
7:e41133. doi: 10.1371/journal.pone.0041133.
93. Smith BJ, Bruner KEP, Kendall LV. Female- and intruder-
induced ultrasonic vocalizations in C57BL/6J mice as proxy
indicators for animal wellbeing. Comp Med 2019; 69:374–383.
doi: 10.30802/aalas-cm-18-000147.
94. Crouse KN, Miller CM, Wilson ML. New approaches to
modeling primate socioecology: does small female group
size BEGET loyal males? J. Hum. Evol. 2019; 137:102671. doi:
10.1016/j.jhevol.2019.102671.
95. Rodriguez-De Lara R et al. Controlled doe exposure as
biostimulation of buck rabbits. Anim. Reprod. Sci. 2010;
122:270–275. doi: 10.1016/j.anireprosci.2010.09.002.
96. Carter AJ, Feeney WE, Marshall HH et al. Animal personality:
what are behavioural ecologists measuring? Biol. Rev. Camb.
Philos. Soc. 2013; 88:465–475. doi: 10.1111/brv.12007.
97. Biro PA, Stamps JA. Are animal personality traits linked to
life-history productivity? Trends Ecol. Evol. 2008; 23:361–368.
doi: 10.1016/j.tree.2008.04.003.
98. Sih A et al. Animal personality and state-behaviour feed-
backs: a review and guide for empiricists. Tr en ds Ec ol. Evo l.
2015; 30:50–60. doi: 10.1016/j.tree.2014.11.004.
99. Wolf M, Weissing FJ. An explanatory framework for adap-
tive personality differences. Philosophical transactions of
the Royal Society of London. Series B. Biological sciences 2010;
365:3959–3968. doi: 10.1098/rstb.2010.0215.
100. Jandt JM et al. Behavioural syndromes and social insects:
personality at multiple levels. Biol. Rev. Camb. Philos. Soc.
2014; 89:48–67. doi: 10.1111/brv.12042.
101. Webster MM, Ward AJ. Personality and social context.
Biol. Rev. Camb. Philos. Soc. 2011; 86:759–773. doi:
10.1111/j.1469-185X.2010.00169.x.
102. Jamieson TJ, Baxter GS, Murray PJ. You are not my han-
dler! Impact of changing handlers on dogs’ behaviours and
detection performance. Animals : an open access journal from
MDPI 2018; 8. doi: 10.3390/ani8100176.
103. Berns GS, Brooks AM, Spivak M. Scent of the familiar: an
fMRI study of canine brain responses to familiar and unfa-
miliar human and dog odors. Behav. Process.2015; 110:37–46.
doi: 10.1016/j.beproc.2014.02.011.
104. Wahlsten D et al. Different data from different labs: lessons
from studies of gene-environment interaction. J. Neurobiol.
2003; 54:283–311. doi: 10.1002/neu.10173.
105. Bohlen M et al. Experimenter effects on behavioral test
scores of eight inbred mouse strains under the influ-
ence of ethanol. Behav. Brain Res. 2014; 272:46–54. doi:
10.1016/j.bbr.2014.06.017.
106. Neely C, Lane C, Torres J et al. The effect of gentle handling
on depressive-like behavior in adult male mice: considera-
tions for human and rodent interactions in the laboratory.
Behav. Neurol. 2018; 2018:7. doi: 10.1155/2018/2976014.
107. Wheeler R, Hickman DL. Measuring wellbeing: tickling
makes rats Happy. LAS Pro 2014; March:53–56.
108. Pongracz P, Altbacker V, Fenes D. Human handling might
interfere with conspecific recognition in the European
rabbit (Oryctolagus cuniculus). Dev. Psychobiol. 2001; 39:
53–62.
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

264 Whittaker and Hickman
109. Swennes AG et al. Human handling promotes compliant
behavior in adult laboratory rabbits. Journal of the Ameri-
can Association for Laboratory Animal Science : JAALAS 2011;
50:41–45.
110. Sorge RE et al. Olfactory exposure to males, including men,
causes stress and related analgesia in rodents. Nat. Methods
2014; 11:629–632. doi: 10.1038/nmeth.2935.
111. van Driel KS, Talling JC. Familiarity increases consistency
in animal tests. Behav. Brain Res. 2005; 159:243–5. doi:
10.1016/j.bbr.2004.11.005.
112. Albert DJ, Walsh ML, Ryan J et al. Mouse killing in rats: a
comparison of spontaneous killers and rats with lesions
of the medial hypothalamus or the medial accumbens
nucleus. Physiol. Behav. 1982; 29:989–994.
113. M, O. B. The rat as a predator. Psychol. Bull. 1975; 82:
460–462.
114. Malick JB. Effects of age and food deprivation on the devel-
opment of muricidal behavior in rats. Physiol. Behav. 1975;
14:171–175.
115. Adamec R, Walling S, Burton P. Long-lasting, selective,
anxiogenic effects of feline predator stress in mice.
Physiol. Behav. 2004; 83:401–410. doi: 10.1016/j.physbeh.
2004.08.029.
116. Albrechet-Souza L, Gilpin NW. The predator odor avoidance
model of post-traumatic stress disorder in rats. Behav. Phar-
macol. 2019; 30:105–114. doi: 10.1097/fbp.0000000000000460.
117. Voznessenskaya VV. In C. Mucignat-Caretta, ed., Neurobiol-
ogy of Chemical Communication. CRC Press/Taylor & Francis
(c) 2014 by Taylor & Francis Group, LLC; 2014.
118. Liu YJ et al. Chronic co-species housing mice and rats
increased the competitiveness of male mice. Chem. Senses
2017; 42:247–257. doi: 10.1093/chemse/bjw164.
119. Pritchett-Corning KR, Chang FT, Festing MF. Breeding and
housing laboratory rats and mice in the same room does
not affect the growth or reproduction of either species. Jour-
nal of the American Association for Laboratory Animal Science :
JAALAS 2009; 48:492–498.
120. Apfelbach R, Blanchard CD, Blanchard RJ et al. The effects
of predator odors in mammalian prey species: a review of
field and laboratory studies. Ne u ro s ci. B iob e hav. R ev. 2005;
29:1123–1144. doi: 10.1016/j.neubiorev.2005.05.005.
121. Russell, W. M. S. & Burch, R. L. The Principles of Humane Exper-
imental Technique,http://altweb.jhsph.edu/pubs/books/hu
mane_exp/het-toc (1959).
122. Jeger V, Hauffe T, Nicholls-Vuille F et al. Analgesia in clin-
ically relevant rodent models of sepsis. Lab. Anim. 2016;
50:418–426. doi: 10.1177/0023677216675009.
123. Redaelli, V. et al. A refinement approach in a mouse model
of rehabilitation research. Analgesia strategy, reduction
approach and infrared thermography in spinal cord injury.
PLoS One 14:e0224337, doi:10.1371/journal.pone.0224337
(2019).
124. Mundt S, Groettrup M, Basler M. Analgesia in mice
with experimental meningitis reduces pain without alter-
ing immune parameters. ALTEX 2015; 32:183–189. doi:
10.14573/altex.1502021.
125. Gouveia K, Hurst JL. Reducing mouse anxiety during han-
dling: effect of experience with handling tunnels. PLoS One
2013; 8:e66401. doi: 10.1371/journal.pone.0066401.
126. Coutant T, Laniesse D, Sykes JMT. Advances in thera-
peutics and delayed drug release. The veterinary clinics of
North America. Exotic animal practice 2019; 22:501–520. doi:
10.1016/j.cvex.2019.05.006.
127. Keraliya RA et al. Osmotic drug delivery system as a part
of modified release dosage form. ISRN pharmaceutics 2012;
528079:2012. doi: 10.5402/2012/528079.
128. Palme R, Rettenbacher S, Touma C et al. Stress hormones
in mammals and birds: comparative aspects regarding
metabolism, excretion, and noninvasive measurement in
fecal samples. Ann. N. Y. Acad. Sci. 2005; 1040:162–171. doi:
10.1196/annals.1327.021.
129. Meyer JS, Novak MA. Minireview: hair cortisol: a novel
biomarker of hypothalamic-pituitary-adrenocortical activ-
ity. Endocrinology 2012; 153:4120–4127. doi: 10.1210/en.2012-
1226.
130. Langford DJ et al. Social modulation of pain as evidence for
empathy in mice. Science 2006; 312:1967–1970.
131. Baxt C, Kassam-Adams N, Nance ML et al. Assessment of
pain after injury in the pediatric patient: child and parent
perceptions. J. Pediatr. Surg. 2004; 39:979, 979–983. Discus-
sion, 983.
132. Kelly AM, Powell CV, Williams A. Parent visual analogue
scale ratings of children’s pain do not reliably reflect pain
reported by child. Pediatr. Emerg. Care 2002; 18:159–162.
133. Vervoort T, Caes L, Trost Z, Notebaert L, Goubert L.
Parental attention to their child’s pain is modulated by
threat-value of pain. Health Psychol.2012;31:623–631. doi:
10.1037/a0029292.
134. Mogil JS. Social modulation of and by pain in humans and
rodents. Pain 2015; 156(Suppl 1):S35–S41. doi: 10.1097/01.j.
pain.0000460341.62094.77.
135. Chen J. Empathy for distress in humans and rodents. Neu-
rosci. Bull. 2018; 34:216–236. doi: 10.1007/s12264-017-0135-0.
136. Panksepp JB, Lahvis GP. Rodent empathy and affective neu-
roscience. N eur osc i . Bio beh av. Re v. 2011; 35:1864–1875. doi:
10.1016/j.neubiorev.2011.05.013.
137. Keum S, Shin HS. Rodent models for studying empathy.
Neurobiol. Learn. Mem. 2016; 135:22–26. doi: 10.1016/j.nlm.
2016.07.022.
138. Crawley JN. Mouse behavioral assays relevant to the symp-
toms of autism. Brain pathology (Zurich, Switzerland) 2007;
17:448–459. doi: 10.1111/j.1750-3639.2007.00096.x.
139. Pekow C. Defining, measuring, and interpreting stress
in laboratory animals. Contemp. Top. Lab. Anim. S ci. 2005;
44:41–45.
140. Sharp JL, Zammit TG, Azar TA et al. Stress-like responses
to common procedures in male rats housed alone or with
other rats. Contemp. Top. Lab. An im. Sci. 2002; 41:8–14.
141. Matsumiya LC et al. Using the mouse grimace scale to
reevaluate the efficacy of postoperative analgesics in lab-
oratory mice. J. Am. Assoc . Lab. Anim. S ci. 2012; 51:42–49.
142. Hager C et al. The sheep grimace scale as an indicator of
post-operative distress and pain in laboratory sheep. PLoS
One 2017; 12:e0175839. doi: 10.1371/journal.pone.0175839.
143. Hampshire V, Robertson S. Using the facial grimace scale
to evaluate rabbit wellness in post-procedural monitoring.
Lab animal 2015; 44:259–260. doi: 10.1038/laban.806.
144. Viscardi AV, Hunniford M, Lawlis P et al. Development
of a piglet grimace scale to evaluate piglet pain using
facial expressions following castration and tail docking:
a pilot study. Frontiers in veterinary science 2017; 4:51. doi:
10.3389/fvets.2017.00051.
145. Oliver VL, Thurston SE, Lofgren JL. Using cageside measures
to evaluate analgesic efficacy in mice (Mus musculus) after
surgery. Journal of the American Association for Laboratory
Animal Science : JAALAS 2018; 57:186–201.
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

ILAR Journal, 2019, Vol. 60, No. 2 265
146. Rock ML et al. The time-to-integrate-to-nest test as an
indicator of wellbeing in laboratory mice. Journal of the
American Association for Laboratory Animal Science : JAALAS
2014; 53:24–28.
147. Hager C et al. Time to integrate to nest test evaluation in a
mouse DSS-colitis model. PLoS One 2015; 10:e0143824. doi:
10.1371/journal.pone.0143824.
148. Brydges NM, Hall L. A shortened protocol for assessing
cognitive bias in rats. J. Ne uros ci. Methods 2017; 286:1–5. doi:
10.1016/j.jneumeth.2017.05.015.
149. Düpjan S, Ramp C, Kanitz E et al. A design for studies
on cognitive bias in the domestic pig. Journal of Veterinary
Behavior: Clinical Applications and Research 2013; 8:485–489.
150. Barker T, Howarth G, Whittaker A. The effects of metabolic
cage housing and sex on cognitive bias expression in rats.
Appl. Anim. Behav. Sci. 2016; 177:70–76.
151. Wheeler RR, Swan MP, Hickman DL. Effect of multilevel
laboratory rat caging system on the well-being of the
singly-housed Sprague Dawley rat. Lab. Anim. 2015;
49:10–19. doi: 10.1177/0023677214547404.
152. Trezza V, Campolongo P, Vanderschuren LJMJ. Evaluat-
ing the rewarding nature of social interactions in labo-
ratory animals. Developmental Cognitive Neuroscience 2011;
1:444–458. doi: 10.1016/j.dcn.2011.05.007.
153. Mason WA. Effects of social interaction on well-being:
development aspects. Lab. Anim. Sci. 1991; 41:323–328.
154. Hall FS. Social deprivation of neonatal, adolescent, and
adult rats has distinct neurochemical and behavioral con-
sequences. Crit.Rev.Neurobiol.1998; 12.
155. Rodgers RJ, Cole JC. Influence of social isolation, gender,
strain, and prior novelty on plus-maze behaviour in mice.
Physiol. Behav. 1993; 54:729–736.
156. Arndt SS et al. Individual housing of mice—impact
on behaviour and stress responses. Physiol. Behav. 2009;
97:385–393.
157. Hawkins P et al. Individually ventilated cages and rodent
welfare: report of the 2002 RSPCA/UFAW rodent welfare
group meeting. Anim Technol Welf 2003; 2:23–34.
158. Kappel S, Hawkins P, Mendl MT. To group or not to group?
Good practice for housing male laboratory mice. Animals
2017; 7:88.
159. Krohn TC, Sørensen DB, Ottesen JL et al. The effects of
individual housing on mice and rats: a review. Anim. Welf.
2006; 15:343–352.
160. Olsson IAS, Westlund K. More than numbers matter: the
effect of social factors on behaviour and welfare of labo-
ratory rodents and non-human primates. Appl. Anim. Behav.
Sci. 2007; 103:229–254. doi: 10.1016/j.applanim.2006.05.022.
161. Arakawa H. Ethological approach to social isolation effects
in behavioral studies of laboratory rodents. Behav. Brain Res.
2018; 341:98–108. doi: 10.1016/j.bbr.2017.12.022.
162. Voikar V, Polus A, Vasar E et al. Long-term individual hous-
ing in C57BL/6J and DBA/2 mice: assessment of behavioral
consequences. Genes Brain Behav. 2005; 4:240–252.
163. Hilakivi LA, Ota M, Lister R. Effect of isolation on brain
monoamines and the behavior of mice in tests of explo-
ration, locomotion, anxiety and behavioral ‘despair’. Phar-
macol. Biochem. Behav. 1989; 33:371–374.
164. Ferrari P, Palanza P, Parmigiani S et al. Interindividual
variability in Swiss male mice: relationship between social
factors, aggression, and anxiety. Physiol. Behav. 1998; 63:
821–827.
165. Berry A et al. Social deprivation stress is a triggering
factor for the emergence of anxiety-and depression-like
behaviours and leads to reduced brain BDNF levels in
C57BL/6J mice. Psychoneuroendocrinology 2012; 37:762–772.
166. Barker TH, Bobrovskaya L, Howarth GS et al. Female
rats display fewer optimistic responses in a judgment
bias test in the absence of a physiological stress
response. Physiol. Behav. 2017; 173:124–131. doi:
10.1016/j.physbeh.2017.02.006.
167. Stuart SA, Butler P, Munafò MR et al. A translational
rodent assay of affective biases in depression and antide-
pressant therapy. Neuropsychopharmacology 1625, 2013; 38.
doi: 10.1038/npp.2013.69.https://www.nature.com/articles/
npp201369#supplementary-information.
168. Roelofs S, Boleij H, Nordquist RE et al. Making decisions
under ambiguity: judgment bias tasks for assessing emo-
tional state in animals. Front. Behav. Neurosci. 2016; 10. doi:
10.3389/fnbeh.2016.00119.
169. Mendl M, Burman OHP, Parker RMA et al. Cognitive bias
as an indicator of animal emotion and welfare: emerg-
ing evidence and underlying mechanisms. Appl. Anim.
Behav. Sci. 2009; 118:161–181. doi: 10.1016/j.applanim.2009.
02.023.
170. Harding EJ, Paul ES, Mendl M. Cognitive bias and affective
state. Nature 2004; 427:312–312. doi: 10.1038/427312a.
171. Clegg IL, Cognitive K. Bias in zoo animals: an optimistic
outlook for welfare assessment. Animals (Basel) 2018; 8:104.
doi: 10.3390/ani8070104.
172. Barker T, Howarth G, Whittaker A. The effects of metabolic
cage housing and sex on cognitive bias expression in rats.
Appl. Anim. Behav. Sci. 2016; 177:70–76.
173. Hymel KA, Sufka KJ. Pharmacological reversal of cognitive
bias in the chick anxiety-depression model. Neuropharma-
cology 2012; 62:161–166.
174. Salmeto AL et al. Cognitive bias in the chick anxiety–
depression model. Brain Res. 2011; 1373:124–130.
175. Hernandez CE, Hinch G, Lea J et al. Acute stress enhances
sensitivity to a highly attractive food reward without affect-
ing judgement bias in laying hens. Appl. Anim. Behav. Sci.
2015; 163:135–143.
176. Murphy E, Nordquist RE, van der Staay FJ. Responses of
conventional pigs and Göttingen miniature pigs in an active
choice judgement bias task. Appl. Anim. Behav. Sci. 2013;
148:64–76.
177. Benton D, Brain PF. Behavioral and adrenocortical
reactivity in female mice following individual or group
housing. Dev. Psychol. 1981; 14:101–107. doi:
10.1002/dev.420140203.
178. Benton, D. & Brain, P. F. Behavioural comparisons of isolated,
dominant and subordinate mice. Behav. Process. 4, 211–219,
doi: 10.1016/0376-6357(79)90002-0 (1979).
179. Thiessen D. Varying sensitivity of C57BL/Crgl mice to group-
ing. Science 1963; 141:827–828.
180. Goldsmith, J. F., Brain, P. F. & Benton, D. Effects of the
duration of individual or group housing on behavioural and
adrenocortical reactivity in male mice. Physiol. Behav. 21,
757–760, doi: 10.1016/0031-9384(78)90015-X (1978).
181. Faggin BM, Palermo-Neto J. Differential alterations in
brain sensitivity to amphetamine and pentylenetetra-
zol in socially deprived mice. Gen. Pharmacol. 1985; 16:
299–302.
182. File SE. Exploration, distraction, and habituation in rats
reared in isolation. Dev. Psychobiol. 1978; 11:73–81. doi:
10.1002/dev.420110111.
183. Einon DF, Humphreys AP, Chivers SM et al. Isolation has
permanent effects upon the behavior of the rat, but not
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

266 Whittaker and Hickman
the mouse, gerbil, or Guinea pig. Dev. Psychobiol. 1981;
14:343–355. doi: 10.1002/dev.420140407.
184. Goldsmith JF, Brain PF, Benton D. Effects of age at
differential housing and the duration of individual
housing/group on intermale fighting behavior and
adrenocortical activity in TO strain mice. Aggress. Behav.
1976; 2:307–323. doi: 10.1002/1098-2337(1976)2:4<307::AID-
AB2480020407>3.0.CO;2-I.
185. Cairns RB, Nakelski JS. On fighting in mice: ontogenetic
and experiential determinants. J Comp Physiol Psychol 1971;
74:354.
186. Crawley J, Schleidt WM, Contrera JF. Does social environ-
ment decrease propensity to fight in male mice? Behav. Biol.
1975; 15:73–83.
187. Koyama S. Isolation effect in mice (Mus musculus): (ii)
variance in aggression. J. Ethol. 1993; 11:131–140. doi:
10.1007/BF02350046.
188. File, S. E. & Seth, P. A review of 25 years of the social
interaction test. Eur. J. Pharmacol. 2003; 463, 35–53, doi: htt
ps://doi.org/10.1016/S0014-2999(03)01273-1.
189. Douglas LA, Varlinskaya EI, Spear LP. Rewarding properties
of social interactions in adolescent and adult male and
female rats: impact of social versus isolate housing of
subjects and partners. Dev. Psychobiol. 2004; 45:153–162. doi:
10.1002/dev.20025.
190. Whittaker AL, Lymn KA, Howarth GS. Effects of metabolic
cage housing on rat behavior and performance in the social
interaction test. J. Appl. Anim. Welf. Sci. 2016; 19:363–374.
191. Varlinskaya EI, Spear LP, Spear NE. Social behavior and
social motivation in adolescent rats: role of housing condi-
tions and partner’s activity. Physiol. Behav. 1999; 67:475–482.
doi: 10.1016/S0031-9384(98)00285-6.
192. Niesink RJ, van Ree JM. Short-term isolation increases social
interactions of male rats: a parametric analysis. Physiol.
Behav. 1982; 29:819–825.
193. Taylor SE. Tend and befriend: biobehavioral bases of affili-
ation under stress. Curr.Dir.Psychol.Sci.2006; 15:273–277.
194. Gentsch C, Lichtsteiner M, Feer H. Locomotor activity, defe-
cation score and corticosterone levels during an openfield
exposure: a comparison among individually and group-
housed rats, and genetically selected rat lines.Physiol. Behav.
1981; 27:183–186.
195. Stern J, Winokur G, Eisenstein A et al. The effect of
group vs. individual housing on behavior and physiologi-
cal responses to stress in the albino rat. J. Psychosom. Res.
1960;4:185–190.
196. Viveros M, Hernandez R, Martinez I et al. Effects of social
isolation and crowding upon adrenocortical reactivity and
behavior in the rat. Rev. Esp. Fisiol. 1988; 44:315–321.
197. Weltman A, Sackler A, Schwartz R et al. Effects of iso-
lation stress on female albino mice. Lab Anim Care 1968;
18:426–435.
198. Plaut S, Grota L. Effects of differential housing on adreno-
cortical reactivity. Neuroendocrinology 1971; 7:348–360.
199. Gamallo A, Villanua A, Trancho G et al. Stress adaptation
and adrenal activity in isolated and crowded rats. Physiol.
Behav. 1986; 36:217–221.
200. Hurst JL, Barnard CJ, Nevison CM et al. Housing and wel-
fare in laboratory rats: welfare implications of isolation
and social contact among caged males. Anim. Welf. 1997;
6:329–347.
201. Gomez-Sanchez EP, Gomez-Sanchez CE. 19-
Nordeoxycorticosterone, aldosterone, and corticosterone
excretion in sequential urine samples from male and
female rats. Steroids 1991; 56:451–454.
202. Kalliokoski O et al. Mice do not habituate to metabolism
cage housing–a three week study of male BALB/c mice.PLoS
One 2013; 8:e58460.
203. Dhabhar FS. Effects of stress on immune function: the good,
the bad, and the beautiful. Immunol. Res. 2014; 58:193–210.
doi: 10.1007/s12026-014-8517-0.
204. Bartolomucci A et al. Individual housing induces altered
immuno-endocrine responses to psychological stress in
male mice. Psychoneuroendocrinology 2003; 28:540–558.
205. Dairman W, Balazs T. Comparison of liver microsome
enzyme systems and barbiturate sleep times in rats
caged individually or communally. Biochem. Pharmacol. 1970;
19:951–955.
206. Einon D, Stewart J, Atkinson S et al. Effect of isolation on
barbiturate anaesthesia in the rat. Psychopharmacology 1976;
50:85–88. doi: 10.1007/BF00634160.
207. Watanabe H, Ohdo S, Ishikawa M et al. Effects of social
isolation on pentobarbital activity in mice: relationship to
racemate levels and enantiomer levels in brain. J. Pharmacol.
Exp. Ther. 1992; 263:1036.
208. Puglisi-Allegra S, Oliverio A. Social isolation: effects on pain
thresholds and stress-induced analgesia. Pharmacology.Bio-
chemistry and Behavior 1983; 19:679–681. doi: 10.1016/0091-
3057(83)90344-1.
209. Lee B-H, Kim Y-K. The roles of BDNF in the pathophysiol-
ogy of major depression and in antidepressant treatment.
Psychiatry Investig. 2010; 7:231–235. doi: 10.4306/pi.2010.
7.4.231.
210. Martin B, Ji S, Maudsley S et al. “Control” laboratory rodents
are metabolically morbid: why it matters. Proc. Natl. Acad.
Sci. 2010; 107:6127–6133. doi: 10.1073/pnas.0912955107.
211. Reinhardt V, Rossell M. Self-biting in caged macaques:
cause, effect, and treatment. J. Appl. Anim. Welf. Sci. 2001;
4:285–294. doi: 10.1207/S15327604JAWS0404_05.
212. Harlow HF, Zimmermann RR. Affectional responses in the
infant monkey. Science 1959; 130:421–432.
213. Schapiro SJ. Effects of social manipulations and environ-
mental enrichment on behavior and cell-mediated immune
responses in rhesus macaques. Pharmacol. Biochem. Behav.
2002; 73:271–278.
214. Schapiro SJ, Nehete PN, Perlman JE et al. A comparison
of cell-mediated immune responses in rhesus macaques
housed singly, in pairs, or in groups. Appl. Anim. Behav. Sci.
2000; 68:67–84.
215. Van Loo P, Kruitwagen C, Van Zutphen L et al. Modulation of
aggression in male mice: influence of cage cleaning regime
and scent marks. Anim. Welf. 2000; 9:281–295.
216. Späni D, Arras M, König B et al. Higher heart rate of labora-
tory mice housed individually vs in pairs. Lab. Anim. 2003;
37:54–62.
217. Vaughan LM, Dawson JS, Porter PR et al. Castration pro-
motes welfare in group-housed male Swiss outbred mice
maintained in educational institutions. Journal of the Amer-
ican Association for Laboratory Animal Science : JAALAS 2014;
53:38–43.
218. Lofgren JL et al. Castration eliminates conspecific aggres-
sion in group-housed CD1 male surveillance mice (Mus
musculus). J. Am. Assoc. Lab. Anim. Sci. 2012; 51:594–599.
219. Hoogstraten-Miller SL, Brown PA. Techniques in aseptic
rodent surgery. Curr. Protoc. Immunol. 2008;82(Chapter 1-
1.12. 1-1):12–14. doi: 10.1002/0471142735.im0112s82.
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

ILAR Journal, 2019, Vol. 60, No. 2 267
220. Schapiro SJ, Bloomsmith MA, Laule GE. Positive reinforce-
ment training as a technique to alter nonhuman primate
behavior: quantitative assessments of effectiveness. J. Appl.
Anim. Welf. Sci. 2003; 6:175–187.
221. Poole TB, Morgan HD. Differences in aggressive behaviour
between male mice (Mus musculus L.) in colonies of differ-
ent sizes. Anim. Behav. 1973; 21:788–795.
222. Falkner AL, Grosenick L, Davidson TJ et al. Hypothalamic
control of male aggression-seeking behavior. Nat. Neurosci.
2016; 19:596. doi: 10.1038/nn.4264.https://www.nature.co
m/articles/nn.4264#supplementary-information.
223. Roitman MF, Stuber GD, Phillips PE et al. Dopamine operates
as a subsecond modulator of food seeking. J. Neurosci. 2004;
24:1265–1271.
224. Piazza PV, Le Moal M. Glucocorticoids as a biological
substrate of reward: physiological and pathophysiological
implications. Brain Res. Rev. 1997; 25:359–372.
225. Miczek K, Weerts E, Vivian J et al. Aggression, anxiety and
vocalizations in animals: GABA a and 5-HT anxiolytics.
Psychopharmacology 1995; 121:38–56.
226. Kovalenko IL, Galyamina AG, Smagin DA et al. Hyperactivity
and abnormal exploratory activity developing in CD-1 male
mice under chronic experience of aggression and social
defeats. JBehavBrainSci2015; 5:478.
227. Kudryavtseva NN, Bondar NP, Avgustinovich DF. Effects
of repeated experience of aggression on the aggressive
motivation and development of anxiety in male mice. Neu-
rosci. Behav. Physiol. 2004; 34:721–730. doi: 10.1023/B:NEAB.
0000036013.11705.25.
228. Kudryavtseva NN, Smagin DA, Kovalenko IL et al. Repeated
positive fighting experience in male inbred mice. Nat. Protoc.
2014; 9:2705–2717. doi: 10.1038/nprot.2014.156.
229. Barrientos S, Stojadinovic O, Golinko MS et al. Growth fac-
tors and cytokines in wound healing. Wound Repair Regen.
2008; 16:585–601.
230. Padgett DA, Marucha PT, Sheridan JF. Restraint stress slows
cutaneous wound healing in mice. Brain Behav. Immun. 1998;
12:64–73.
231. Mercado AM, Padgett DA, Sheridan JF et al. Altered kinet-
ics of IL-1α,IL-1β, and KGF-1 gene expression in early
wounds of restrained mice. Brain Behav. Immun. 2002; 16:
150–162.
232. DeVries AC, Craft TK, Glasper ER et al. 2006 Curt
P. Richter award winner: social inf luences on stress
responses and health. Psychoneuroendocrinology 2007; 32:
587–603.
233. Bartolomucci A et al. Social factors and individual vulner-
ability to chronic stress exposure. N eur osc i. Bi o beh av. Re v.
2005; 29:67–81. doi: 10.1016/j.neubiorev.2004.06.009.
234. Barker TH, George RP, Howarth GS et al. Assessment of
housing density, space allocation and social hierarchy of
laboratory rats on behavioural measures of welfare. PLoS
One 2017; 12:e0185135.
235. Colas-Zelin D et al. The imposition of, but not the propen-
sity for, social subordination impairs exploratory behav-
iors and general cognitive abilities. Behav. Brain Res. 2012;
232:294–305.
236. Touyarot K, Venero C, Sandi C. Spatial learning impair-
ment induced by chronic stress is related to individ-
ual differences in novelty reactivity: search for neu-
robiological correlates. Psychoneuroendocrinology 2004; 29:
290–305.
237. Alzoubi KH et al. Adverse effect of combination of chronic
psychosocial stress and high fat diet on hippocampus-
dependent memory in rats. Behav. Brain Res. 2009;
204:117–123. doi: 10.1016/j.bbr.2009.05.025.
238. Ferrari PF, Palanza P, Parmigiani S et al. Interindividual
variability in Swiss male mice: relationship between social
factors, aggression, and anxiety. Physiol. Behav. 1998; 63(97):
821, 00544–00547, 00541. doi: 10.1016/S0031-9384.
239. Fitchett AE, Barnard CJ, Cassaday HJ. There’s no place
like home: cage odours and place preference in subordi-
nate CD-1 male mice. Physiol. Behav. 2006; 87:955–962. doi:
10.1016/j.physbeh.2006.02.010.
240. Desjardins C, Maruniak JA, Bronson FH. Social rank in house
mice: differentiation revealed by ultraviolet visualization of
urinary marking patterns. Science 1973; 182:939–941.
241. Bartolomucci, A. et al. Social status in mice: behavioral,
endocrine and immune changes are context dependent.Phys-
iol. Behav. 73, 401–410, doi: 10.1016/S0031-9384(01)00453-X
(2001).
242. Bartolomucci A, Palanza P, Parmigiani S. Group housed
mice: are they really stressed? Ethol. Ecol. Evol. 2002;
14:341–350. doi: 10.1080/08927014.2002.9522735.
243. Whary M, Peper R, Borkowski G et al. The effects of group
housing on the research use of the laboratory rabbit. Lab.
Anim. 1993; 27:330–341.
244. Bell DJ, Bray G. Effects of single-and mixed-sex caging on
postweaning development in the rabbit. Lab. Anim. 1984;
18:267–270.
245. Buijs S, Hermans K, Maertens L et al. Effects of semi-
group housing and floor type on pododermatitis, spinal
deformation and bone quality in rabbit does. Animal 2014;
8:1728–1734.
246. Buijs S, Maertens L, Hermans K et al. Behaviour, wounds,
weight loss and adrenal weight of rabbit does as affected by
semi-group housing. Appl. Anim. Behav. Sci. 2015; 172:44–51.
247. Andrist CA, van den Borne BHP, Bigler LM et al. Epidemio-
logic survey in Swiss group-housed breeding rabbits: extent
of lesions and potential risk factors. Prev Vet Med 2013;
108:218–224. doi: 10.1016/j.prevetmed.2012.07.015.
248. Shively CA, Clarkson TB, Kaplan JR. Social deprivation
and coronary artery atherosclerosis in female cynomolgus
monkeys. Atherosclerosis 1989; 77:69–76.
249. Lilly AA, Mehlman PT, Higley JD. Trait-like immunologi-
cal and hematological measures in female rhesus across
varied environmental conditions. Am.J.Primatol.1999;
48:197–223. doi: 10.1002/(sici)1098-2345(1999)48:3<197::aid-
ajp3>3.0.co;2-y.
250. Kaplan JR, Adams MR, Clarkson TB et al. Psychosocial inf lu-
ences on female ‘protection’among cynomolgus macaques.
Atherosclerosis 1984; 53:283–295.
251. Nader MA et al. Social dominance in female monkeys:
dopamine receptor function and cocaine reinforcement.
Biol. Psychiatry 2012; 72:414–421. doi: 10.1016/j.biopsych.
2012.03.002.
252. Morgan D et al. Social dominance in monkeys: dopamine
D2 receptors and cocaine self-administration. Nat. Neurosci.
2002; 5:169–174. doi: 10.1038/nn798.
253. Czoty PW, Gould RW, Gage HD et al. Effects of social
reorganization on dopamine D2/D3 receptor availabil-
ity and cocaine self-administration in male cynomol-
gus monkeys. Psychopharmacology 2017; 234:2673–2682. doi:
10.1007/s00213-017-4658-x.
254. Aghajani M et al. Effects of dominant/subordinate social
status on formalin-induced pain and changes in serum
Proinflammatory cytokine concentrations in mice. PLoS One
e80650, 2013; 8. doi: 10.1371/journal.pone.0080650.
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

268 Whittaker and Hickman
255. Hird DW, Henrickson RV, Hendrickx AG. Infant mortality
in Macaca mulatta: neonatal and post-neonatal mortality
at the California primate research Center, 1968-1972. A
retrospective study. J Med Primatol 1975; 4:4–22.
256. Reinhardt V. Social enrichment for laboratory primates: a
critical review. Laboratory Primate Newsletter 1990; 29:7–11.
257. Benton D, Brain PF, Goldsmith JF. Effects of prior housing
on endocrine responses to differential caging in male TO-
strain mice. Physiol. Psychol. 1979; 7:89–92.
258. Van Loo PL, de Groot AC, Van Zutphen BF et al. Do male
mice prefer or avoid each other’s company? Influence of
hierarchy, kinship, and familiarity. J. Appl. Anim. Welf. Sci.
2001; 4:91–103.
259. Schino G, Maestripieri D, Scucchi S et al. Social tension
in familiar and unfamiliar pairs of long-tailed macaques.
Behaviour 1990; 264–272.
260. Hurst J. Making sense of scents: reducing aggression and
uncontrolled variation in laboratory mice. National Centre
for the Replacement, Refinement and Reduction of Animals in
Research: London, UK 2005; 1–8.
261. Bernstein I. Social housing of monkeys and apes: group
formations. Lab. Anim. Sci. 1991; 41:329.
262. Van Loo PLP, Mol JA, Koolhaas JM et al. Modulation of aggres-
sion in male mice: influence of group size and cage size.
Physiol. Behav. 2001; 72:675–683. doi: 10.1016/S0031-9384(01
)00425-5.
263. National Research Council. The development of science-
based guidelines for laboratory animal care: proceedings of the
November 2003 international workshop.NationalAcademies
Press; 2004.
264. Hurst J, Barnard C, Tolladay U et al. Housing and wel-
fare in laboratory rats: effects of cage stocking density
and behavioural predictors of welfare. Anim. Behav. 1999;
58:563–586.
265. Lee W et al. Social status in mouse social hierarchies is
associated with variation in oxytocin and vasopressin 1a
receptor densities. bioRxiv 2019; 566067.
266. Beery AK, Kaufer D. Stress, social behavior, and resilience:
insights from rodents. Neurobiology of Stress 2015; 1:116–127.
doi: 10.1016/j.ynstr.2014.10.004.
267. Kikusui T, Winslow JT, Mori Y. Social buffering: relief from
stress and anxiety. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci.
2006; 361:2215–2228. doi: 10.1098/rstb.2006.1941.
268. Donahue RJ, Muschamp JW, Russo SJ et al. Effects of stri-
atal FosB overexpression and ketamine on social defeat
stress–induced anhedonia in mice. Biol. Psychiatry 2014;
76:550–558.
269. Krishnan V et al. Molecular adaptations underlying sus-
ceptibility and resistance to social defeat in brain reward
regions. Cell 2007; 131:391–404.
270. Kawachi I, Berkman L. Social cohesion, social capital, and
health. Soc Epidemiol 2000; 174:190.
271. Holt-Lunstad J, Smith TB, Layton JB. Social relationships
and mortality risk: a meta-analytic review. PLoS Med. 2010;
7:e1000316.
272. Silk JB et al. Strong and consistent social bonds enhance the
longevity of female baboons. Curr. Biol. 2010; 20:1359–1361.
273. Yee JR, Cavigelli SA, Delgado B et al. Reciprocal affiliation
among adolescent rats during a mild group stressor pre-
dicts mammary tumors and lifespan. Psychosom. Med. 2008;
70:1050.
274. Fanselow MS. Odors released by stressed rats produce
opioid analgesia in unstressed rats. Behav. Neurosci. 1985;
99:589.
275. Kavaliers M, Choleris E, Colwell D. Learning from others
to cope with biting flies: social learning of fear-induced
conditioned analgesia and active avoidance. Behav. Neurosci.
2001; 115:661.
276. Smith ML, Hostetler CM, Heinricher MM et al. Social transfer
of pain in mice. Sci. Adv. 2016; 2:e1600855. doi: 10.1126/
sciadv.1600855.
277. Meyza K, Knapska E. What can rodents teach us about
empathy? Curr. Opin. Psychol. 2018; 24:15–20. doi: 10.1016/j.
copsyc.2018.03.002.
278. Hatfield E, Cacioppo JT, Rapson RL. Emotional contagion.
Curr.Dir.Psychol.Sci.1993; 2:96–100.
279. Martin LJ et al. Reducing social stress elicits emotional
contagion of pain in mouse and human strangers. Curr. Biol.
2015; 25:326–332.
280. Mikosz M, Nowak A, Werka T et al. Sex differences in social
modulation of learning in rats. Sci. Rep. 2015; 5:18114.
281. Jeon D et al. Observational fear learning involves affective
pain system and Ca v 1.2 Ca 2+channels in ACC. Nat.
Neurosci. 2010; 13(482).
282. Keum S et al. Variability in empathic fear response among
11 inbred strains of mice. Genes Brain Behav. 2016; 15:
231–242.
283. Meyza KZ, Bartal IB-A, Monf ils MH et al. The roots of empa-
thy: through the lens of rodent models. Neurosci. Biobehav.
Rev. 2017; 76:216–234. doi: 10.1016/j.neubiorev.2016.10.028.
284. Armario A, Luna G, Balasch J. The effect of conspecifics
on corticoadrenal response of rats to a novel environment.
Behav. Neural Biol. 1983; 37:332–337. doi: 10.1016/S0163- 1047
(83)91425-5.
285. Ruis MAW et al. Housing familiar male wildtype rats
together reduces the long-term adverse behavioural and
physiological effects of social defeat. Psychoneuroendocrinol-
ogy 1999; 24:285–300. doi: 10.1016/S0306-4530(98)00050-X.
286. Hennessy MB, Maken DS, Graves FC. Consequences of
the presence of the mother or unfamiliar adult female
on cortisol, ACTH, testosterone and behavioral responses
of periadolescent Guinea pigs during exposure to nov-
elty. Psychoneuroendocrinology 2000; 25:619–632. doi: 10.1016/
S0306-4530(00)00014-7.
287. Kiyokawa Y, Kikusui T, Takeuchi Y et al. Partner’s stress
status influences social buffering effects in rats. Behav. Neu-
rosci. 2004; 118:798–804. doi: 10.1037/0735-7044.118.4.798.
288. Smith AS, Wang Z. Hypothalamic oxytocin mediates social
buffering of the stress response. Biol. Psychiatry 2014;
76:281–288. doi: 10.1016/j.biopsych.2013.09.017.
289. Heinrichs M, Baumgartner T, Kirschbaum C et al. Social
support and oxytocin interact to suppress cortisol and
subjective responses to psychosocial stress. Biol. Psychiatry
2003; 54:1389–1398. doi: 10.1016/S0006- 3223 (03)00465-7.
290. Panksepp J, Herman B, Conner R et al. The biology of social
attachments: opiates alleviate separation distress. Biol. Psy-
chiatry 1978; 13:607–618.
291. Panksepp J, Bishop P. An autoradiographic map of
(3H)diprenorphine binding in rat brain: effects of social
interaction. Brain Res. Bull. 1981; 7:405–410.
292. Nelson EE, Panksepp J. Brain substrates of infant-mother
attachment: contributions of opioids, oxytocin, and nore-
pinephrine. Neurosci. Biobehav. Rev. 1998; 22:437–452.
293. Sanchez MM, McCormack KM, Howell BR. Social buffer-
ing of stress responses in nonhuman primates: mater-
nal regulation of the development of emotional regu-
latory brain circuits. Soc. Neurosci. 2015; 10:512–526. doi:
10.1080/17470919.2015.1087426.
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021

ILAR Journal, 2019, Vol. 60, No. 2 269
294. Hill SD, McCormack SA, Mason WA. Effects of artifi-
cial mothers and visual experience on adrenal respon-
siveness of infant monkeys. Dev. Psychobiol. 1973; 6:
421–429.
295. Mendoza SP, Smotherman WP, Miner MT et al. Pituitary-
adrenal response to separation in mother and infant squir-
rel monkeys. Developmental Psychobiology: The Journal of the
International Society for Developmental Psychobiology 1978;
11:169–175.
296. Coe CL, Mendoza SP, Smotherman WP et al. Mother-infant
attachment in the squirrel monkey: adrenal response to
separation. Behav. Biol. 1978; 22:256–263.
297. Bayart F, Hayashi KT, Faull KF et al. Inf luence of mater-
nal proximity on behavioral and physiological responses
to separation in infant rhesus monkeys (Macaca mulatta).
Behav. Neurosci. 1990; 104:98.
298. COE CL. Endocrine and immune responses to separation
and maternal loss in nonhuman primates. The psychobiology
of attachment and separation 1985.
299. Winslow JT, Noble PL, Lyons CK et al. Rearing effects
on cerebrospinal fluid oxytocin concentration and social
buffering in rhesus monkeys. Neuropsychopharmacology
2003; 28:910.
300. Coe CL, Franklin D, Smith ER et al. Hormonal responses
accompanying fear and agitation in the squirrel monkey.
Physiol. Behav. 1982; 29:1051–1057.
301. Smith TE, McGreer-Whitworth B, French JA. Close proximity
of the heterosexual partner reduces the physiological and
behavioral consequences of novel-cage housing in black
tufted-ear marmosets (Callithrix kuhli). Horm. Behav. 1998;
34:211–222.
302. Smith TE, McGreer-Whitworth B, French JA. Close proximity
of the heterosexual partner reduces the physiological and
behavioral consequences of novel-cage housing in black
tufted-ear marmosets (Callithrix kuhli). Horm. Behav. 1998;
34:211–222.
303. Young C, Majolo B, Heistermann M et al. Responses to social
and environmental stress are attenuated by strong male
bonds in wild macaques. Proc. Natl. Acad. Sci. U. S. A. 2014;
111:18195–18200.
304. Frazer A & JG H. in In: Siegel GJ, Agranoff BW, Albers
RW, et al., eds. Basic Neurochemistry: Molecular, Cellular
and Medical Aspects. Philadelphia PA: Lippincott-Raven;
1999).
305. Cochran DM, Fallon D, Hill M et al. The role of oxytocin in
psychiatric disorders: a review of biological and therapeutic
research findings. Harv Rev Psychiatry 2013; 21:219–247. doi:
10.1097/HRP.0b013e3182a75b7d.
306. Cardoso C, Ellenbogen MA, Linnen AM. The effect of
intranasal oxytocin on perceiving and understanding
emotion on the Mayer-Salovey-Caruso emotional
intelligence test (MSCEIT). Emotion 2014; 14:43–50. doi:
10.1037/a0034314.
307. Bakos J, Srancikova A, Havranek T et al. Molecular
mechanisms of oxytocin signaling at the synaptic
connection. Neural Plasticity 2018; 2018:9. doi: 10.1155/
2018/4864107.
308. Galasko, G. T. In: Frank J. Dowd, Barton S. Johnson, & Angelo
J. Mariotti, eds. Pharmacology and Therapeutics for Dentistry.
7th ed. Mosby; 2017: 417–428.
309. Chaudhry SR, Kum B. Biochemistry, Endorphin.https://www.
ncbi.nlm.nih.gov/books/NBK470306/>.
310. Abel, P. W. & Piascik, M. T. In F J. Dowd, B-
 S. Johnson, and A J. Mariotti eds. Pharmacol-
ogy and Therapeutics for Dentistry. 7th ed. Mosby; 2017:
71–81.
311. Hall, J. E. & Guyton, A. C. Textbook of Medical Physiology. 11th
ed. Saunders Elsevier; 2011.
Downloaded from https://academic.oup.com/ilarjournal/article/60/2/252/5877149 by guest on 03 May 2021