Access to this full-text is provided by Frontiers.
Content available from Frontiers in Behavioral Neuroscience
This content is subject to copyright.
SYSTEMATIC REVIEW
published: 20 January 2021
doi: 10.3389/fnbeh.2020.624036
Frontiers in Behavioral Neuroscience | www.frontiersin.org 1January 2021 | Volume 14 | Article 624036
Edited by:
Tim Karl,
Western Sydney University, Australia
Reviewed by:
Aki Takahashi,
University of Tsukuba, Japan
Thiago C. Moulin,
Uppsala University, Sweden
*Correspondence:
Justin A. Varholick
j.a.varholick@gmail.com
Specialty section:
This article was submitted to
Pathological Conditions,
a section of the journal
Frontiers in Behavioral Neuroscience
Received: 30 October 2020
Accepted: 15 December 2020
Published: 20 January 2021
Citation:
Varholick JA, Bailoo JD, Jenkins A,
Voelkl B and Würbel H (2021) A
Systematic Review and Meta-Analysis
of the Relationship Between Social
Dominance Status and Common
Behavioral Phenotypes in Male
Laboratory Mice.
Front. Behav. Neurosci. 14:624036.
doi: 10.3389/fnbeh.2020.624036
A Systematic Review and
Meta-Analysis of the Relationship
Between Social Dominance Status
and Common Behavioral Phenotypes
in Male Laboratory Mice
Justin A. Varholick 1,2
*, Jeremy D. Bailoo 2,3,4 , Ashley Jenkins 5, Bernhard Voelkl 2and
Hanno Würbel 2
1Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL,
United States, 2Division of Animal Welfare, Veterinary Public Health Institute, Universität Bern, Bern, Switzerland,
3Department of Cell Biology and Biochemistry, School of Medicine, Texas Tech University Health Sciences Center, Lubbock,
TX, United States, 4Department of Civil, Environmental, and Construction Engineering, Texas Tech University, Lubbock, TX,
United States, 5Department of Biology, College of Liberal Arts and Sciences, University of Florida, Gainesville, FL,
United States
Background: Social dominance status (e.g., dominant or subordinate) is often
associated with individual differences in behavior and physiology but is largely neglected
in experimental designs and statistical analysis plans in biomedical animal research.
In fact, the extent to which social dominance status affects common experimental
outcomes is virtually unknown. Given the pervasive use of laboratory mice and
culminating evidence of issues with reproducibility, understanding the role of social
dominance status on common behavioral measures used in research may be of
paramount importance.
Methods: To determine whether social dominance status—one facet of the social
environment—contributes in a systematic way to standard measures of behavior in
biomedical science, we conducted a systematic review of the existing literature searching
the databases of PubMed, Embase, and Web of Science. Experiments were divided into
several domains of behavior: exploration, anxiety, learned helplessness, cognition, social,
and sensory behavior. Meta-analyses between experiments were conducted for the open
field, elevated plus-maze, and Porsolt forced swim test.
Results: Of the 696 publications identified, a total of 55 experiments from 20 published
studies met our pre-specified criteria. Study characteristics and reported results were
highly heterogeneous across studies. A systematic review and meta-analyses, where
possible, with these studies revealed little evidence for systematic phenotypic differences
between dominant and subordinate male mice.
Conclusion: This finding contradicts the notion that social dominance status impacts
behavior in significant ways, although the lack of an observed relationship may
be attributable to study heterogeneity concerning strain, group-size, age, housing
Varholick et al. Social Dominance and Behavior
and husbandry conditions, and dominance assessment method. Therefore, further
research considering these secondary sources of variation may be necessary to
determine if social dominance generally impacts treatment effects in substantive ways.
Keywords: social dominance, behavior, systematic review, meta-analysis, reproducibility, preclinical, experimental
design
INTRODUCTION
AROUND the mid-20th century, scientists began to document
and to understand that wild or laboratory mice residing
within groups could be categorized individually by their social
dominance status (Uhrich, 1937; Crowcroft, 1966; Scott, 1966;
Desjardins et al., 1973). In the time since, growing evidence
has suggested that social dominance status is associated with
variability in behavior and physiology, where dominant mice
within a hierarchy have markedly different phenotypic traits
than subordinate cage-mates—despite similarity in genetics and
cage-context (Lathe, 2004; Freund et al., 2013; Wang et al.,
2014; Williamson et al., 2016a; Lee et al., 2018; Varholick et al.,
2018, 2019). Given the pervasive use of mice throughout animal
research and that they are commonly housed in groups to
account for their social needs (National Research Council, 2011;
Bailoo et al., 2018), understanding the biological differences
between dominant and subordinate cage-mates is of significant
interest (Lathe, 2004). Moreover, neglecting social dominance
status in experimental designs and statistical analysis plans may
inadvertently lead to the masking of treatment effects and/or
contribute to idiosyncratic patterns of experimental results and
in turn, poor reproducibility, if social dominance status interacts
with the treatment of interest (Würbel, 2002; Bailoo et al., 2014;
Varholick et al., 2018, 2019; Voelkl et al., 2020).
Social dominance relationships are often determined by
observing predictable patterns regarding which animal retreats
(i.e., subordinate) or chases (i.e., dominant) during social
interactions (Drews, 1993). These predictable patterns may then
be organized in a hierarchical fashion depending on the number
of animals in the group and their dominance relationships with
each other. Although different organizations of relationships can
be defined, it is thought that one of the greatest differences in
social dominance experience within the cage and in phenotypic
traits is between the most dominant and most subordinate cage-
mates, especially in male mice (Bernstein, 1981; Williamson et al.,
2017a; Lee et al., 2019b). This sex difference is likely because
dominant and subordinate males often engage in more overt
forms of agonistic behavior (e.g., chase, lunge, bite, flee) (Lee
et al., 2019a) while female laboratory mice engage in more covert
forms of agonistic behavior (e.g., side-push, or over-climbing)
(Schuhr, 1987)—albeit males are also studied more often than
females. To determine whether dominance behavior in the home-
cage is linked to differences in other phenotypic traits, scientists
typically measure individual social dominance status using the
gold-standard of home-cage observation or with a variety of
correlated assays (e.g., tube-test or urine marking assay) (Wang
et al., 2014). Scientists may then make comparisons between
cage-mates of different social rank on common measures of
behavior used for the screening of phenotypes (e.g., open field
or elevated plus-maze) (Wahlsten, 2011).
To date, a number of studies have reported significant
behavioral differences between dominant and subordinate mouse
cage-mates (Hilakivi et al., 1989; Hilakivi-Clarke and Lister,
1992; Ferrari et al., 1998; Vekovishcheva and Sukhotina, 2000;
Bartolomucci et al., 2001, 2004; Palanza et al., 2001; Fitchett
et al., 2005a, 2009; Sá-Rocha et al., 2006; Saldívar-González et al.,
2007; Wang et al., 2011; Colas-Zelin et al., 2012; Horii et al.,
2017; Larrieu et al., 2017; Zhou et al., 2017; Kunkel and Wang,
2018; Pallé et al., 2019; Varholick et al., 2019), but whether such
differences generalize to male laboratory mice used in biomedical
research remains unknown. To provide an initial evaluation of
the relationship between social dominance status and behavioral
phenotype, we conducted a systematic review and ran meta-
analyses when sufficient data were available. We discuss these
findings in relation to the heterogeneity in the methods for
measuring dominance across experiments and with respect to
risk of bias.
METHODS
Search Strategy
Using pre-specified inclusion and exclusion criteria we identified
all publications reporting relevant experiments (see below) by
searching three electronic databases (PubMed, ISI Web of
Science, and EMBASE) using the search strategy “(anxiety OR
arousal OR learned helplessness OR explor∗OR choice OR learn∗
OR cognition OR preference OR motor OR pain OR maze)
AND (‘social status’ OR ‘social rank’ OR ‘social dominance’ OR
‘dominance hierarchy’ OR ‘social hierarchy’ OR submiss∗) AND
(mouse OR mus OR mice),” with search results limited to title
and abstracts. The cut-off date for our search was on September
20, 2019. Further details on the search strategy can be found
in the supplement (Supplementary Text 1). This study is in
accordance with PRISMA guidelines and the Systematic Review
Center for Laboratory Animal Experimentation (SYRCLE)
(Hooijmans et al., 2014); the checklist can be found in the
Supplementary Information section.
Inclusion and Exclusion Criteria
One investigator (JAV) retrieved and reviewed all publications.
First the titles and abstracts of 30 randomly selected papers from
the 696 (∼4.3%) were screened to develop the key exclusion
criteria. From this screening process we set the following
exclusion criteria: studies were excluded if they: (i) did not
include mice; (ii) used mice housed singly; (iii) did not report
measures of social interactions between cage-mates; (iv) were
part of a symposium/conference proceedings or review; or
Frontiers in Behavioral Neuroscience | www.frontiersin.org 2January 2021 | Volume 14 | Article 624036
Varholick et al. Social Dominance and Behavior
(v) were written in a language other than English. In the
next screening step only studies were included that housed
mice in static groups/pairs for 2 weeks or more, reported
methods for measuring social dominance behavior, and reported
behavioral tests from the following domains: anxiety, arousal,
learned helplessness, exploration, preference, learning/cognition,
motor, pain, or social, outlined and described in, “Mouse
Behavioral Testing: How to use mice in behavioral neuroscience”
(Wahlsten, 2011), page 40. Studies or data were excluded if:
(i) dominance relationships were measured between non-cage-
mates; (ii) treatments were administered in addition to behavioral
phenotyping (only control groups from these studies were used);
or (iii) data from the same study were published more than
once (no studies met this criterion). A detailed study protocol
and flow diagram of the search and exclusion process can
be found in the supplementary (Supplementary Text 1 and
Supplementary Figure 1).
Risks of Bias and Quality Assessment
Assessment of risk of bias and study quality were conducted
independently by two reviewers (JAV and AJ) using a modified
SYRCLE Risk of Bias Tool (Hooijmans et al., 2014) with the
inclusion of sample size calculation (see Supplementary Table 1
for more details) for each of the 20 studies that met the
prespecified inclusion/exclusion criteria. Any disagreements
were resolved by consensus—these were low (<6%).
Data Extraction
After compiling a final list of the 20 included studies, two
reviewers (JAV and AJ) independently extracted the sample
sizes, means, and standard deviations for each dominant
and subordinate comparison (i.e., experiment) made within
each study. For example, if a study compared dominant and
subordinate mice on exploration in the open field; the sample
size, mean value of exploration, and standard deviation of
exploration was extracted for dominant male mice and the
respective values for subordinate male mice were also extracted.
This was done for each metric of each experiment within a
study (a total of 99 metrics, across 55 experiments, across 20
studies). When studies housed more than two mice per cage,
only the most dominant and most subordinate rankings were
considered—intermediate rank assigned mice were excluded.
Data were either copied directly from tables, calculated from
data provided by the respective corresponding author (Larrieu
et al., 2017; Varholick et al., 2018, 2019) or extracted using “Web
Plot Digitizer” (Rohatgi, 2018). If data were not reported because
there were null effects, the unreported data with a null effect was
noted but not included in the meta-analyses (specifics provided
in results). Once all data were collected by the two reviewers
(JAV and AJ), a mean value for each mean and each standard
deviation extracted was calculated and rounded to the nearest
hundredths place. These mean values calculated between the two
reviewers were used in the meta-analyses and reported tables.
Both reviewers agreed on all sample sizes across the experiments.
Statistical Analyses
All meta-analyses were calculated using jamovi (Jamovi. jamovi,
2020) and the MAJOR module (Hamilton, 2018). Jamovi is a
Graphical User Interface (GUI) version of R, and MAJOR is
based on the commonly used R package, Metafor (Viechtbauer,
2010). Separate meta-analyses were run for behavioral tests
that had been used by 5 or more studies—the open field,
elevated-plus maze, and Porsolt forced swim test. Because various
outcome measures were often reported across studies for the
same behavioral test (e.g., time spent in open arms, total distance
traveled, number of open arm entries), we used the most
frequently reported measure across studies for the meta-analysis.
If a study did not use the most frequently reported measure, then
we used the second most frequently used measure, and so forth.
For example, the most frequently used measure for the open field
was total distance traveled, followed by number of crossings on
a grid, then velocity (cm traveled per second). More specifics can
be found in each respective results subsection and all data can be
found in the supplement.
Due to the high degree of heterogeneity between studies,
meta-analyses were run by fitting a random-effects model
using the standardized mean difference between dominant and
subordinate mice for each respective outcome measure for
each study. The sample sizes for each respective dominance
status group were used for calculating each standardized mean
difference for the meta-analysis. Sample sizes were determined
by the number of animals per each dominance status group,
the number of dominants and the number of subordinates,
separately (intermediate rank assigned mice were excluded). A
restricted maximum-likelihood (REML) estimation was used for
calculating the heterogeneity statistic Tau2. No moderator was
used. Behavioral tests (i.e., experiments) for which fewer than 5
studies were available, were categorized within their respective
domain described in, “Mouse Behavioral Testing: How to use mice
in behavioral neuroscience” (Wahlsten, 2011), page 40. Hedge’s g
was then calculated for the metric with the largest effect size for
each experiment. General comparisons (e.g., smaller vs. larger
effect sizes) were made between studies within their domain
foregoing any further statistical testing.
RESULTS
Study Characteristics
By electronic search we identified 20 studies (i.e., published
manuscripts) (Hilakivi et al., 1989; Hilakivi-Clarke and Lister,
1992; Ferrari et al., 1998; Vekovishcheva and Sukhotina, 2000;
Bartolomucci et al., 2001, 2004; Palanza et al., 2001; Fitchett
et al., 2005a, 2009; Sá-Rocha et al., 2006; Saldívar-González et al.,
2007; Wang et al., 2011; Colas-Zelin et al., 2012; Horii et al.,
2017; Larrieu et al., 2017; Zhou et al., 2017; Kunkel and Wang,
2018; Varholick et al., 2018, 2019; Pallé et al., 2019) divided
into 55 separate experiments that met our pre-specified inclusion
criteria (Methods and Supplementary Text 1). These studies
varied concerning strain, supplier, group-size, and whether
littermates were housed together (Table 1). Almost half of the
studies used outbred mice of various strains (n=9/20), one
study used inbred Balb/cJ mice, while the remainder used sub-
strains of the C57BL/6 mouse (n=9/20). Group-sizes varied
from 2 to 9 mice per cage and only two studies housed
litter-mates together.
Frontiers in Behavioral Neuroscience | www.frontiersin.org 3January 2021 | Volume 14 | Article 624036
Varholick et al. Social Dominance and Behavior
TABLE 1 | Heterogeneity in strain, group-size, and littermates for studies meeting pre-specified criteria.
Study Strain Supplier Group-size (# mice
per cage)
Bartolomucci et al. (2001) Swiss CD-1∧Charles River, Italy 3
Bartolomucci et al. (2004) Swiss CD-1 Charles River, Italy 3
Colas-Zelin et al. (2012) CD-1 Harlan Sprague Dawley Inc., Indianapolis, IN 3
Ferrari et al. (1998) Swiss-Webster Banting & Kingman, UK 5 to 6
Fitchett et al. (2005a) CD-1 Harlan, UK 2
Fitchett et al. (2009) CD-1 Harlan, UK 2
Hilakivi et al. (1989) NIH Swiss – 4 to 5
Hilakivi-Clarke and Lister (1992) NIH Swiss – 5
Horii et al. (2017) C57BL/6NCrSlc Japan SLC Inc., Shizuoka 4
Kunkel and Wang (2018) C57BL/6 – 3 to 5*
Larrieu et al. (2017) C57BL/6J Charles River 4
Palanza et al. (2001) Swiss CD-1∧Charles River, Italy 3*
Pallé et al. (2019) C57BL/6J Envigo, UK 4
Saldívar-González et al. (2007) BALB/cJ∧– 3 and 9
Sá-Rocha et al. (2006) C57BL/6 – 2
Varholick et al. (2018) C57BL/6ByJ Charles River, France 4 to 5
Varholick et al. (2019) RjOrl:SWISS Janvier Labs, France 3
Vekovishcheva and Sukhotina (2000) White Outbred Rappolovo, Russia 3
Wang et al. (2011) C57BL/6 – 4
Zhou et al. (2017) C57BL/6J Shanghai laboratory animal center 4
∧denotes mice bred in house; *denotes litter-mates, – denotes information not available. For the “group-size” column the conjunction “and” denotes that both group sizes were studied,
where the conjunction “to” denotes that range of group sizes that were studied but not systematically varied.
FIGURE 1 | Stacked bar chart of risk of bias assessment. Each row
represents a type of bias assessed for the studies. Further details can be
found in Supplementary Table 1.
Risk of Bias and Quality Assessment
The risk of bias evaluation of the 20 included articles in this
review is reported in Figure 1 and Supplementary Table 3. All
20 studies had a low risk of bias concerning three indices:
baseline characteristics, incomplete outcome data, and random
housing. A total of 19 studies had a low risk of bias for
sequence generation, the remaining article had an unclear risk
of bias because it did not explicitly describe the method for
determining dominance. Another combination of 19 articles had
low risk of bias from allocation concealment, meaning that the
social dominance status of the animal was concealed during
the dominance assessment method. Only one study reported
a sample size calculation. The other indices had more varied
distributions of low, unclear, or high risk of bias. For example,
seven articles expressly stated random outcome assessment
reflecting a low risk of bias, while the other 13 had unclear
risk of bias. Notably, 16 and 11 of the studies had a low
risk of bias from investigator blinding and outcome assessor
blinding, respectively; many of the other articles had unclear
risk of bias from blinding as they did not expressly describe
the blinding process. An example of high risk of bias for both
investigator blinding and outcome assessor blinding would be
that the subordinate ranked animals always had bite-marks
while dominant ranked animals had none. Importantly, seven
articles had a high risk of bias from excluding cage-groups that
had an unstable dominance organization or ranking, this was
consistently found as “other sources of bias.” In summary, all
but one study (Varholick et al., 2019) had at least one unclear
(19 out of 20) and/or at least one high risk of bias (12 out of 20)
(Supplementary Table 3).
Frontiers in Behavioral Neuroscience | www.frontiersin.org 4January 2021 | Volume 14 | Article 624036
Varholick et al. Social Dominance and Behavior
Heterogeneity in Dominance Assessment
Method
The collected studies differed in their method of dominance
assessment, the frequency of measuring dominance, age of
animals at grouping, and the age of animals when assessing
dominance—not all studies reported these metrics (Table 2).
Several studies used multiple methods to determine social
dominance status (Vekovishcheva and Sukhotina, 2000; Wang
et al., 2011; Larrieu et al., 2017; Zhou et al., 2017).
The most common method used in the assessment of social
dominance status was home-cage behavior observation and
scoring for three or more consecutive days before behavioral
testing (12 out of 20) (Ferrari et al., 1998; Vekovishcheva and
Sukhotina, 2000; Bartolomucci et al., 2001, 2004; Palanza et al.,
2001; Fitchett et al., 2005a, 2009; Sá-Rocha et al., 2006; Saldívar-
González et al., 2007; Wang et al., 2011; Horii et al., 2017; Larrieu
et al., 2017). Mice that engaged in more offensive behavior (e.g.,
attack, chase, mount, bite) compared to defensive behavior (e.g.,
flee, freeze, supine posture) were rated dominant, while those
that showed more defensive behavior than offensive behavior
were ranked subordinate. A total of four studies of these 12
also considered bite-wounds as a sign of dominance where the
subordinate incurred bite-wounds and the dominant had none
(Ferrari et al., 1998; Bartolomucci et al., 2001, 2004; Colas-Zelin
et al., 2012). This method of identifying dominance was used in
two other studies without the provision of home-cage behavior
(Hilakivi et al., 1989; Hilakivi-Clarke and Lister, 1992). Notably,
one study measured home-cage behavior but only for a single
TABLE 2 | Heterogeneity in dominance assessment method for studies meeting pre-specified criteria.
Study Dominance test Freq. measure dom. Age at
grouping
Age at
testing
Time together
before dom. test
Bartolomucci et al.
(2001)
Home-cage observation |
Bite-marks
3 consec. days | 60 min
observation
90 90 0
Bartolomucci et al.
(2004)
Home-cage observation |
Bite-marks
Daily | 60 min
observation
100 100 0
Colas-Zelin et al. (2012) Home-cage observation |
Bite-marks
1 day | three 10 min
observations
67 68 1
Ferrari et al. (1998) Home-cage observation |
Bite-marks
3 consec. Days | three
10 min observations
56 77 21
Fitchett et al. (2005a) Home-cage observation – 77 – –
Fitchett et al. (2009) Home-cage observation 14 consec. Days |
30 min observation
77 77 0
Hilakivi et al. (1989) Presence/Absence of Bite-marks – – – –
Hilakivi-Clarke and
Lister (1992)
Presence/Absence of Bite-marks – – – –
Horii et al. (2017) Home-cage observation 4 consec. Days |
20 min after first attack
35 56 21
Kunkel and Wang
(2018)
Tube-test 4 consec. Days 0 90 90
Larrieu et al. (2017) Tube-test | Urine Marking |
Home-cage observation
8 consec. days | One
20 min home-cage
observation
35 70 35
Palanza et al. (2001) Home-cage observation >1 consec. Day |
Unclear duration
0 90 90
Pallé et al. (2019) Tube-test 3–7 days (until stable
ranks)
56 70 21
Saldívar-González et al.
(2007)
Home-cage observation 5 consec. Days | 10
25–30 s
– 98 –
Sá-Rocha et al. (2006) 7 min Isolation, then Home-cage
observation
3 consec. Days |
15 min observation
21 90 69
Varholick et al. (2018) Tube-test 3 weekly tests 21 180 159
Varholick et al. (2019) Tube-test 3 weekly tests 21 70 49
Vekovishcheva and
Sukhotina (2000)
Intruder | Home-cage
observation
2 days separated by
2–3 days |
predetermined
dominance
– – 7
Wang et al. (2011) Tube-test | Urine Marking |
Home-cage observation |
Ultrasonic Vocalization
6 consec. days | One
20 min home-cage
observation
70 84 14
Zhou et al. (2017) Tube-test | Warm-spot
competition
4 consec. days 60 74 14
Frontiers in Behavioral Neuroscience | www.frontiersin.org 5January 2021 | Volume 14 | Article 624036
Varholick et al. Social Dominance and Behavior
day before testing general behavior (Colas-Zelin et al., 2012)—
while all other home-cage behavior studies measured dominance
for at least three consecutive days. The studies using home-
cage observation had very inconsistent methods regarding the
frequency of measuring dominance (Range =1–14 consecutive
days, Media n=3 days), time spent observing dominance for
each test day (5–60 min, see Table 2), the age of the animals
at grouping (Range =0–100 days of age, Median=68.5
days of age), and the time the animals were housed together
before recording dominance (Range =0–90 days, Median=
10.5 days). Of the studies that evaluated home-cage behavior,
three of them discarded groups where cage-mates did not have
unique dominance ranks (Vekovishcheva and Sukhotina, 2000;
Bartolomucci et al., 2001; Wang et al., 2011). Notably, the
study by Vekovishcheva and Sukhotina (2000) experimentally
formed groups with linear hierarchies by identifying the primary
aggressor in a group of eight, then identifying the secondary
aggressors by consecutively removing the group aggressor every
3 days until a final submissive animal that showed no aggressive
behavior was left. The groups of eight were then reduced to
groups of three composed of a primary aggressor, secondary
aggressor, and the final submissive animal.
The next most common method for the assessment of
dominance behavior involved the use of the tube-test (or
competitive exclusion test) (seven out of 20) (Wang et al.,
2011; Larrieu et al., 2017; Zhou et al., 2017; Kunkel and Wang,
2018; Varholick et al., 2018, 2019; Pallé et al., 2019). For
this task, cage-mates are simultaneously placed on opposite
ends of a long-narrow tube to impose a face-to-face conflict
terminating when one cage-mate retreats backwards to their
starting point. The retreating cage-mate is assigned a “loss” and
its partner is assigned a “win.” These dyadic encounters are
usually organized in a predetermined and random round-robin
tournament. The total number of “losses” with the respective
pairing compose the dominance hierarchy for each cage. Studies
indicate dominance in the tube-test significantly correlates with
home-cage observation (Wang et al., 2014), although some have
questioned the utility of the test; namely that it doesn’t always
correlate with home-cage observation, animals adapt to the
test over time, and it only measures a single facet of social
dominance behavior (Wilson, 1968; Syme, 1974; Miczek and
Barry, 1975; Benton et al., 1980; Curley, 2011; Varholick, 2019).
Again, methodologies greatly varied between studies regarding
the frequency of measuring dominance, the age of the animals,
and the time the animals spent together before testing (see
Table 2). For example, some studies measured dominance for 1
day every week for 3 weeks (Varholick et al., 2018, 2019), while
others measured dominance across four or more consecutive
days (Wang et al., 2011; Larrieu et al., 2017; Zhou et al., 2017;
Kunkel and Wang, 2018), or every day until a group of cage-
mates attained stable ranks (Pallé et al., 2019). Several studies
discarded groups that had unstable hierarchies across their
predetermined number of study days (Wang et al., 2011; Zhou
et al., 2017).
Other methods of assessing dominance like the urine marking
assay, ultrasonic vocalization, and warm-spot competition were
always used in conjunction with either home-cage behavior or
the tube-test but were rarely used in general [i.e., urine marking
assay two out of 20 (Wang et al., 2011; Larrieu et al., 2017),
ultrasonic vocalization one out of 20 (Wang et al., 2011), and
warm spot competition one out of 20 (Zhou et al., 2017)]. In
all cases dominance measured in these tests correlated with
assessments in the home-cage or tube-test for the respective
study. The urine marking assay was used for two studies (Wang
et al., 2011; Larrieu et al., 2017), and involves placing two cage-
mates in a novel empty cage separated by a mesh barrier with
filter paper flooring. The dominant cage-mate will leave urine
marks throughout their partitioned area while the subordinate
will leave a pool of urine in a corner. The ultrasonic vocalization
test was used once (Wang et al., 2011) in conjunction with the
tube-test and home-cage observation. Here, separated males are
presented with a female and 70 kHz vocalizations are recorded
where the most dominant vocalizes for the longest and the
subordinate often does not vocalize at all (Nyby et al., 1976).
The warm-spot competition test was also used once (Zhou et al.,
2017) with the tube-test, and involved presenting a group of cage-
mates with a cold floored cage with a single warm-spot in the
corner. Cage-mates that spent the longest time on the warm-spot
were considered most dominant, with subordinate cage-mates
spending the least amount of time on the warm-spot. Additional
explanations of each dominance assessment method can be found
in the Supplementary Text 2.
General Composition of Behavioral Tests
The 55 individual experiments from the 20 studies were
categorized into several behavioral outcome assessment domains
according to Wahlsten (2011) and the studies’ method sections;
Exploration (n=17) (Hilakivi et al., 1989; Hilakivi-Clarke and
Lister, 1992; Vekovishcheva and Sukhotina, 2000; Bartolomucci
et al., 2001, 2004; Palanza et al., 2001; Sá-Rocha et al., 2006;
Saldívar-González et al., 2007; Wang et al., 2011; Colas-Zelin
et al., 2012; Horii et al., 2017; Larrieu et al., 2017; Zhou et al.,
2017; Varholick et al., 2018, 2019), Anxiety (n=17) (Hilakivi
et al., 1989; Hilakivi-Clarke and Lister, 1992; Ferrari et al., 1998;
Vekovishcheva and Sukhotina, 2000; Bartolomucci et al., 2004;
Sá-Rocha et al., 2006; Saldívar-González et al., 2007; Colas-Zelin
et al., 2012; Horii et al., 2017; Larrieu et al., 2017; Varholick
et al., 2018, 2019; Pallé et al., 2019), Learned Helplessness (n=5)
(Hilakivi et al., 1989; Hilakivi-Clarke and Lister, 1992; Saldívar-
González et al., 2007; Horii et al., 2017), Cognitive (n=8)
(Fitchett et al., 2005a, 2009; Colas-Zelin et al., 2012; Varholick
et al., 2018, 2019), Social (n=2) (Zhou et al., 2017; Kunkel and
Wang, 2018), and Sensory (n=6) (Colas-Zelin et al., 2012; Zhou
et al., 2017). Because tests measuring exploration or anxiety were
most frequent, they were further sub-divided into a specific test
and other category: for exploration, Open Field (n=11) and
Other Exploration tests (n=6); and for anxiety, Elevated Plus-
Maze (n=13), and Other Anxiety tests (n=4). Several studies
met all inclusion and exclusion criteria, yet reported null effects
without the provision of data: Exploration (n=3/16) (Wang
et al., 2011; Colas-Zelin et al., 2012; Zhou et al., 2017), Anxiety
(n=4/17) (Bartolomucci et al., 2004; Colas-Zelin et al., 2012)
Cognitive (n=4/8) (Fitchett et al., 2009; Colas-Zelin et al., 2012),
Frontiers in Behavioral Neuroscience | www.frontiersin.org 6January 2021 | Volume 14 | Article 624036
Varholick et al. Social Dominance and Behavior
Social (n=1/2) (Zhou et al., 2017), Sensory (n=5/6) (Colas-
Zelin et al., 2012; Zhou et al., 2017). These studies are included in
the tables but are excluded from the meta-analyses (Bartolomucci
et al., 2004; Wang et al., 2011; Zhou et al., 2017). Only 2 studies
used males and females (Varholick et al., 2018, 2019), while the
remaining 18 studies exclusively studied males.
Analysis of Exploration Behavior
Meta-analyses for exploration behavior were divided into
two separate analyses for behavior in the open field and
other exploration tests. The category designated as “other” in
exploration behavior was not used in meta-analyses due to
high heterogeneity in paradigm methodologies between studies
(e.g., novel object exploration, hole-board crossings, and activity
meter). For the meta-analysis that was run, open field behavior
had heterogeneous metrics reported between studies. Thus, we
prioritized common metrics for the data analysis. The most
common metric for the open-field was total distance traveled,
followed by number of crossings, then velocity; and for general
exploration the number of crossings was most common followed
by total distance.
The meta-analysis on exploration in the open field estimated
a medium effect size of 0.484 (k =9, se =0.273) that was
not statistically significant (p=0.077, 95% CI = −0.05, 1.02,
Figure 2A), with experiments generally finding a small and
statistically non-significant effect with dominant mice exploring
more than subordinates (7/9). The other two experiments found
a large statistically significant effect of dominants exploring more
than subordinates (Saldívar-González et al., 2007) or a small
non-significant effect in the opposite direction (Larrieu et al.,
2017). There was significant between-study heterogeneity (Tau2
=0.466, se =0.332, df =8, p=0.002). For the experiments
categorized as “other” within the exploration domain, the mean
differences between dominant and subordinate mice, the pooled
standard deviations, and Hedge’s g values were calculated for each
study (Table 3).
Analysis of Anxiety Behavior
As in the exploration domain, analyses in the anxiety behavior
domain were sub-divided into elevated plus-maze and other
anxiety tests. A meta-analysis was performed for the elevated
plus-maze, while tests in the “other” category were not
used in meta-analyses due to high heterogeneity in paradigm
methodologies between studies (e.g., light/dark box, defensive
burying, shuttlebox). The elevated plus-maze had heterogeneous
metrics reported between studies, thus we prioritized common
metrics; percent entries in open arms was most common,
followed by percent duration in open arms, and finally number
of open arm entries. Notably, not all studies directly reported
“percent” but provided enough information to calculate the
percentage (e.g., number of open arm entries divided by total
entries), allowing more studies to have more comparable metrics.
The meta-analysis on elevated plus-maze behavior yielded a
small effect size of 0.132 (k =10, se =0.206) that was
not statistically significant (p=0.522, 95% CI= −0.27, 0.54,
Figure 2B). With about an equal number of experiments finding
a small and non-significant effect in contradicting directions (4
indicating increased anxiety for dominants and three increased
anxiety for subordinates), another two with significant effects in
contradicting directions (Horii et al., 2017; Larrieu et al., 2017),
and one study finding virtually no effect (Varholick et al., 2018).
There was, again, significant between-study heterogeneity (Tau2
=0.256, se =0.197, df =9, p=0.003). For the experiments
categorized as “other” within the anxiety domain, the mean
differences between dominant and subordinate mice, the pooled
standard deviations, and Hedge’s g values were calculated for each
study (Table 3).
Analysis of Learned Helplessness Behavior
The only tests that measured learned helplessness in this
review were those using the Porsolt forced swim test. Five
studies conducted this test in relation to dominance and thus
satisfied our criteria for inclusion. All studies reported the same
metric, duration immobile, thus the meta-analysis was limited
to comparing duration immobile between studies. The meta-
analysis on the Porsolt forced swim test estimated a small effect
size of 0.0480 (k =5, se =0.872) that was not statistically
significant (p=0.956, 95% CI = −1.66, 1.76, Figure 2C). This
was attributable to contradictory statistically significant findings
across experiments with three reporting subordinates spend
more time immobile (Hilakivi et al., 1989; Hilakivi-Clarke and
Lister, 1992; Horii et al., 2017) and two reporting dominants
spend more time immobile—albeit the latter experiments were
from the same study (Saldívar-González et al., 2007). There was
again substantial between-study heterogeneity (Tau2=3.574, se
=2.691, p=0.001).
A secondary set of forest-plots for open field, elevated
plus-maze, and Porsolt forced swim test focusing on
relevant study characteristics (i.e., strain, group-size, and
dominance assessment method) can be found in the supplement
(Supplementary Figure 2).
Analysis of Cognitive, Social, and Sensory
Behavior
Given a high degree of heterogeneity across behavioral outcome
assessments and inconsistent reporting between studies within
the separate domains; cognitive, social, and sensory, the data
were insufficient for meta-analyses. However, for comparison
to the previous meta-analyses and further discussion, the mean
differences, pooled standard variations, and Hedge’s g values
are reported in Table 4. Experiments marked with a “=” sign
in the “Dom vs. Sub” column did not report values in their
study, they just reported that there was no significant difference
between groups.
Similar to the meta-analyses for exploration and anxiety
behavior, experiments generally reported no large effects between
dominant and subordinate mice for cognitive, social, or sensory
behavior. Regarding cognitive behavior, most experiments
reported small effect sizes with no statistical significance (n=
6/8), albeit five of those experiments did not report the data
(Fitchett et al., 2009; Colas-Zelin et al., 2012). Only two studies
measured social behavior, beyond dominance, with opposing
Frontiers in Behavioral Neuroscience | www.frontiersin.org 7January 2021 | Volume 14 | Article 624036
Varholick et al. Social Dominance and Behavior
FIGURE 2 | Forest plots of meta-analyses. (A) Open field, (B) Elevated plus-maze, (C) Porsolt forced swim test. Observed effect sizes and 95% confidence intervals
are provided in the right column. Negative effect sizes represent increased values for subordinate mice (e.g., increased exploration in the open field), while positive
effects represent increased values for dominants. The overall effect size is denoted by the diamond symbol. The study by Saldívar-González et al. (2007) is marked
with 3*and 9*to denote the effects of the separate group-sizes of 3 and 9.
Frontiers in Behavioral Neuroscience | www.frontiersin.org 8January 2021 | Volume 14 | Article 624036
Varholick et al. Social Dominance and Behavior
TABLE 3 | Heterogeneity in effect sizes and directionality of effect for studies meeting pre-specified criteria for exploratory and anxiety domains.
Domain Study Paradigm Dom Sub Mean Diff. Pooled SD g Dom v. Sub
Exploration Pallé et al., 2019 General Exploration 5 5 72.5 76.20 0.95 <
Varholick et al., 2018 Object Exploration 10 7 298.03 1288.46 0.23 <
Bartolomucci et al., 2004 Free Exploration 8 8 – – – =
Hilakivi et al., 1989 Hole-board 18 22 9.11 18.66 0.49 >
Hilakivi-Clarke and Lister,
1992
Hole-board 10 23 19.44 19.50 1.0 >
Vekovishcheva and
Sukhotina, 2000
Actometer 20 20 32.10 28.78 1.12 >
Anxiety Colas-Zelin et al., 2012 Light/Dark Box 8 8 – – – =
Saldívar-González et al.,
2007 (9*)
Defensive Burying 8 8 5.44 25.77 0.22 >
Vekovishcheva and
Sukhotina, 2000
Shuttle box 20 20 6.3 11.46 0.55 >
Saldívar-González et al.,
2007 (3*)
Defensive Burying 8 8 19.24 22.59 0.85 >
The directionality in the dominance subordinance relationship is noted in the “Dom vs. Sub” column and does not necessarily reflect statistical significance. Also, studies marked with
a “=” sign in the ‘Dom vs. Sub’ column did not report values in their study but reported that there was no significant difference between groups. A Hedge’s g value of more than 1
indicates a difference >1 standard deviation. Data ordered by direction of effect and ascending Hedge’s g, similar to forest plots. The study by Saldívar-González et al. (2007) is marked
with 3*and 9*to denote the effects of the separate group-sizes of 3 and 9.
TABLE 4 | Heterogeneity in effect sizes and directionality of effect for studies meeting pre-specified criteria for cognitive, social, and sensory domains.
Domain Study Paradigm Dom Sub Mean Diff. Pooled SD g Dom v. Sub
Cognitive Varholick et al., 2019 Novel object discrimination 26 26 0.11 0.12 0.88 <
Varholick et al., 2018 Novel object discrimination 10 7 0.02 0.21 0.07 <
Fitchett et al., 2009 T-Maze 9 9 – – – =
Colas-Zelin et al., 2012 Fear conditioning 8 8 – – – =
Colas-Zelin et al., 2012 Morris water maze 8 8 – – – =
Colas-Zelin et al., 2012 Lashley III Maze 8 8 – – – =
Colas-Zelin et al., 2012 Odor Discrimination 8 8 – – – =
Fitchett et al., 2005a T-Maze 6 6 0.41 0.63 0.65 >
Social Zhou et al., 2017 Social memory – – – – – =
Kunkel and Wang, 2018 Social memory 8 8 15.14 11.38 1.39 >
Sensory Vekovishcheva and
Sukhotina, 2000
Hot-plate 6 6 0 1.86 0 =
Colas-Zelin et al., 2012 Hot-plate 8 8 – – – =
Colas-Zelin et al., 2012 Balance beam 8 8 – – – =
Colas-Zelin et al., 2012 Grip strength 8 8 – – – =
Zhou et al., 2017 Grip strength – – – – – =
Colas-Zelin et al., 2012 Balance pole 8 8 – – – =
Data ordered by direction of effect and ascending Hedge’s g, similar to forest plots. The directionality in the dominance subordinance relationship is noted in the “Dom vs. Sub” column
and does not necessarily reflect statistical significance. Also, studies marked with a “=” sign in the “Dom vs. Sub” column did not report values in their study but reported that there
was no significant difference between groups. A Hedge’s g-value of more than 1 indicates a difference >1 standard deviation.
results; one with no effect and no reported data (Zhou et al.,
2017) and another with a large effect indicating dominant mice
had increased social memory compared to subordinate mice
(Kunkel and Wang, 2018). Finally, regarding sensory behavior
all experiments found virtually no effect between dominant mice
and subordinate mice, with 5 out of 6 not reporting the data.
DISCUSSION
This systematic review and meta-analysis revealed limited
evidence to support the notion that a clear difference exists
between dominant and subordinate male laboratory mice on
standard measures of behavior commonly used in biomedical
Frontiers in Behavioral Neuroscience | www.frontiersin.org 9January 2021 | Volume 14 | Article 624036
Varholick et al. Social Dominance and Behavior
research. The 55 experiments from 20 published papers used
to inform this review were heterogeneous concerning strain,
group-size, age of testing dominance, and their methods for
assessing dominance. Such heterogeneity likely increased the
generalizability of our assessment, but the unsystematic nature
of this heterogeneity may have also clouded our understanding
on which genetic, environmental, and developmental factors
might be most important when considering dominance and
behavior. Most studies (12 out of 20) had at least one high risk
of bias, and only a single study (Varholick et al., 2019) had
neither high nor unclear risks of bias. A number of studies failed
to report experimental data and/or exclusively studied groups
of mice with stable dominance hierarchies thereby excluding
other dominance organizations (e.g., despotic or unclear).
Studies were also quite heterogenous regarding the domains of
behavior measured; exploration, anxiety, learned helplessness,
cognitive, social, and sensory behavior domains. With the
domains of exploration, anxiety, and learned helplessness being
the most frequent outcomes, we were able to conduct meta-
analyses finding that dominant and subordinate mice tend to
have small to medium effect size differences in exploratory
behavior in the open field and elevated plus maze, but none
of the summary effect sizes reached statistical significance.
Systematic review and meta-analyses concerning the Porsolt
forced swim test (i.e., learned helplessness) found extremely
large and paradoxical patterns of differences between dominant
and subordinate mice across studies, which overall led to a
non-significant summary effect size. Comparison of Hedge’s
g values for the other behavioral domains which were too
heterogeneous to consider in meta-analyses yielded a similar
pattern of results found in exploration, anxiety, and learned
helplessness behavior.
Our overall assessment of risks of bias highlighted potential
issues which precludes us from drawing firm conclusions about
the relationships between social dominance status and common
measures of behavior. Five domains of bias considered in this
review were (i) selection bias, (ii) performance bias, (iii) detection
bias, (iv) reporting bias, and (v) other bias (Hooijmans et al.,
2014). Risk from (i) selection bias was generally low and was
assessed by baseline characteristics, sequence generation, and
allocation concealment. All studies had low risk of bias from
baseline characteristics since animals were randomly distributed
across housing and then dominance was assessed. However,
one study (Fitchett et al., 2005a) did not explicitly specify how
dominance was determined, making the assessment of selection
bias unclear for sequence generation and allocation concealment.
Specifically, the study was a brief report that failed to describe
any methods but cited a publication (Fitchett et al., 2005b)
when referring to “further details” of their urinary corticosterone
assay. The cited publication also measured social dominance, but
whether the included study and the cited publication used the
exact dominance method was unclear. Another study determined
dominance on the first day of assessment and then confirmed
dominance each subsequent day (Bartolomucci et al., 2004),
which increased the risk of bias for allocation concealment.
Risk from (ii) performance bias was also generally low and was
assessed by random housing and blinding of dominance rank
to housing and husbandry staff. All studies randomly allocated
mice to cages upon arrival to their lab or the start of the
experiment. Notably, one study (Vekovishcheva and Sukhotina,
2000) randomly housed mice in groups of 8, assessed dominance
rank, and then reduced groups to 3 composed of a dominant,
sub-dominant, and subordinate. This could be considered a
risk of bias from non-random housing but was categorized
as “other bias” since animals were randomly housed prior to
dominance assessment. Several instances of high risks of bias
from determining dominance solely by bite-marks (Hilakivi et al.,
1989; Hilakivi-Clarke and Lister, 1992) were concerning since
all individuals handling the mice would immediately recognize
whether they were dominant or subordinate and might handle
them differently. This source of bias may be unavoidable due
to the nature of social dominance, however, including other
methods of dominance assessment beyond bite-wounds could
reduce the risk. Risk from (iii) detection bias was mostly unclear
throughout studies with several instances of high risk of bias,
and was assessed by randomization of outcome assessment, and
investigator blinding during outcome assessment. Most studies
(13 out of 20) (Hilakivi-Clarke and Lister, 1992; Ferrari et al.,
1998; Vekovishcheva and Sukhotina, 2000; Palanza et al., 2001;
Saldívar-González et al., 2007; Fitchett et al., 2009; Wang et al.,
2011; Colas-Zelin et al., 2012; Horii et al., 2017; Larrieu et al.,
2017; Zhou et al., 2017; Kunkel and Wang, 2018; Pallé et al., 2019)
did not report whether dominance rank was counterbalanced
or considered in the order of testing, making randomization of
outcome assessment unclear. More than a third of studies (seven
out of 20) (Vekovishcheva and Sukhotina, 2000; Bartolomucci
et al., 2004; Fitchett et al., 2005a, 2009; Saldívar-González et al.,
2007; Zhou et al., 2017; Kunkel and Wang, 2018) also did
not explicitly state whether the investigator assessing outcome
measures was blinded to the dominance rank of the animals
tested (if this was possible). Risk from (iv) reporting bias occurred
in a quarter of the studies (five out of 20) (Bartolomucci
et al., 2004; Fitchett et al., 2009; Wang et al., 2011; Colas-
Zelin et al., 2012; Zhou et al., 2017), which selectively reported
statistically significant differences and just reported “no statistical
difference” for non-significant effects without the provision of
data. This prevented us from determining the effect size for these
outcome measures. One (v) “other bias” that was common across
studies was discarding social groups that did not form clear
dominance hierarchies (seven out of 20 studies) (Vekovishcheva
and Sukhotina, 2000; Bartolomucci et al., 2001; Palanza et al.,
2001; Sá-Rocha et al., 2006; Wang et al., 2011; Horii et al.,
2017; Zhou et al., 2017). Several studies have estimated that
male laboratory mice form stable groups with unique ranks 60%
of the time, while unstable or despotic structures are formed
in the other 40% (Wang et al., 2011, 2014; Varholick et al.,
2019). Thus, discarding unstable or despotic structures would
bias our understanding of the general relationship between social
dominance and behavioral tests. Finally, only one study reported
conducting a sample size calculation (Varholick et al., 2019). This
is concerning because it is unclear whether studies considered
effect sizes before designing and conducting the experiment
Frontiers in Behavioral Neuroscience | www.frontiersin.org 10 January 2021 | Volume 14 | Article 624036
Varholick et al. Social Dominance and Behavior
(Carneiro et al., 2018). Moreover, the study that did conduct
the sample size calculation (Varholick et al., 2019) considered
the likely distribution of stable (estimated at 60%) and unstable
groups (estimated at 40%) of social dominance (Wang et al.,
2011; Colas-Zelin et al., 2012; Varholick et al., 2018). This
method decreases the risk of bias from the stability of dominance,
something few studies considered throughout this review.
The general finding of non-significant and relatively small
differences is contrary to the theoretical and individual
experimental findings that social status shapes behavior
and physiology across life-histories in male laboratory mice
(Williamson et al., 2016a, 2017a,b; Lee et al., 2018, 2019b;
Williamson et al., 2019), and the large effects of the social
environment reported in other species (Snyder-Mackler et al.,
2020). Over the years, researchers have reported many large
effects associated with dominance in male laboratory mice for
measures of the hypothalamic-pituitary-adrenal (HPA) and
hypothalamic-pituitary-gonadotropic (HPG) axes (Louch and
Higginbotham, 1967; Bronson, 1973; Ely and Henry, 1978;
Williamson et al., 2017a,b), the mesolimbic dopaminergic
pathway (Balog et al., 2014; Larrieu et al., 2017; Papilloud et al.,
2020), and exploratory behavior (Sloan Wilson et al., 1994). Some
of these studies have used a more ethological approach when
studying social behavior with large group sizes (>10) in complex
housing with additional structures and space (Williamson et al.,
2016b)—albeit larger groups and complex housing may not
generalize to common laboratory conditions or experimental
designs in biomedical science. To better understand these effects,
individual experiments could consider how larger group sizes
or complex housing are related to dominance in comparison to
more standard laboratory conditions.
It is likely that the discrepancy between the findings of this
systematic review and the aforementioned literature is partially
due to study heterogeneity and the sensitivity of dominance
structures to differences in genetics and the environment. That is,
some combinations are more likely to have more divergent social
dominance statuses with consequent effects than others. Studies
included in the meta-analyses of this review greatly varied in the
reported factors of strain, group-size, age, housing and husbandry
conditions, and even method of assessing dominance (see
Supplementary Text 2 for a brief description of each method
from this systematic review). They also greatly varied in reported
effect sizes ranging from small to extremely large effects. These
inconsistencies highlight the potential sensitivity of dominance
to other variables across a “reaction norm” and development
(Woltereck, 1909; Wahlsten, 2010; Voelkl and Würbel, 2016;
Voelkl et al., 2020). Indeed, previous studies considering multiple
strains (Mondragón et al., 1987), group-sizes (Schuhr, 1987;
Saldívar-González et al., 2007; Williamson et al., 2017a), and ages
(Bartolomucci et al., 2004) have all shown robust interactions
with dominance relationships—albeit not directly for the metrics
reviewed in the current study. This is surprising, however, it is
possible that different dominance assessment methods highlight
specific facets of social dominance more than others, thereby
increasing the chance of finding seemingly congruent effects
(Bernstein, 1981; Varholick et al., 2019).
Apart from differences in experimental design and
assessment, between-study variation might be due to tests
measuring facets of anxiety that are sensitive to common
laboratory environmental variables like the familiarity of
the experimenter, position of the home-cage on the rack, or
arousal state of the animal immediately before the test (Izídio
et al., 2005). No studies in this review explicitly reported
controlling for these variables. Some researchers have suggested
that differences between studies can be, in part, evaluated by
authorship heterogeneity since related co-authors can publish
multiple papers with similar methods and effect sizes (Moulin
and Amaral, 2020). However, this rarely occurred in the current
review. Besides the same author publishing two studies with the
Porsolt forced swim test (Hilakivi et al., 1989; Hilakivi-Clarke
and Lister, 1992), there was one occurrence in the open field
analysis across 8 studies (Bartolomucci et al., 2001; Palanza
et al., 2001) and two for the elevated-plus maze across 10
studies (Hilakivi et al., 1989; Hilakivi-Clarke and Lister, 1992;
Varholick et al., 2018, 2019). Given the low occurrence of
authorship relatedness, that most studies reported no significant
differences, and study characteristics greatly differed; we posit
that authorship relatedness cannot accurately capture a lab effect
and future empirical research is necessary.
Given this perspective, a logical next step to disentangle
idiosyncratic results would be to replicate the experiments
involving the elevated plus-maze and Porsolt forced swim test—
where individual studies reported large effect sizes—while also
considering different strains, group-sizes, ages, and dominance
methodologies as heterogenization factors to further explore
these effects, followed up by appropriately powered experiments
for hypothesis testing (Voelkl et al., 2020). Consideration for
other dominance organizations (e.g., despotic, double-dominant,
double-subordinate, or open) may also be helpful as such
evaluations were explicitly performed in two of the included
studies (Horii et al., 2017; Varholick et al., 2019). This will allow
these other variables to be considered against the backdrop of
reproducibility while providing mechanistic insight into which
combinations of genetics, environments, and developmental
phases may be relevant to dominance relationships and thus,
require further investigation. Such an understanding may also
provide a platform for increasing the chance of finding an effect if
one exists concerning the domains of behavior evaluated here—
cognitive, social, and sensory—but where too few studies and
inconsistency in the pattern of results of behavior precluded
firm conclusions. Moreover, these studies could be beneficial in
the formulation of studies considering dominance relationships
in female laboratory mice—which this review found to be
critically lacking.
DATA AVAILABILITY STATEMENT
The datasets extracted from publications and used for the
meta-analyses for this study can be found in the figshare at:
doi: 10.6084/m9.figshare.13313258.
Frontiers in Behavioral Neuroscience | www.frontiersin.org 11 January 2021 | Volume 14 | Article 624036
Varholick et al. Social Dominance and Behavior
AUTHOR CONTRIBUTIONS
This research was conceptualized by JV, JB, BV, and HW.
Methodology by JV, JB, BV, and AJ. Investigation by JV
and AJ. Risk of bias sections by AJ and JV. Formal
analysis and original draft by JV. Reviewing and editing by
all authors.
FUNDING
This research was funded by the European Research Council
(ERC Advanced Grant REFINE, grant agreement no. 322576)
awarded to HW.
ACKNOWLEDGMENTS
We are gratefully acknowledge the authors of the evaluated
studies, particularly Dr. Thomas Larrieu who graciously provided
all necessary data from the included experiment. The research
leading to these results has received funding from the European
Union’s Seventh Framework Programme (FP7/2007–2013).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fnbeh.
2020.624036/full#supplementary-material
REFERENCES
Bailoo, J. D., Murphy, E., Varholick, J. A., Novak, J., Palme, R., and Würbel,
H. (2018). Evaluation of the effects of space allowance on measures of
animal welfare in laboratory mice. Sci. Rep. 8:713. doi: 10.1038/s41598-017-
18493-6
Bailoo, J. D., Reichlin, T. S., and Würbel, H. (2014). Refinement of experimental
design and conduct in laboratory animal research. ILAR J. Natl. Res. Counc.
Inst. Lab. Anim. Resour. 55, 383–391. doi: 10.1093/ilar/ilu037
Balog, J., Matthies, U., Naumann, L., Voget, M., Winter, C., and Lehmann,
K. (2014). Social experience modulates ocular dominance plasticity
differentially in adult male and female mice. Neuroimage 103, 454–61.
doi: 10.1016/j.neuroimage.2014.08.040
Bartolomucci, A., Chirieleison, A., Gioiosa, L., Ceresini, G., Parmigiani, S.,
and Palanza, P. (2004). Age at group formation alters behavior and
physiology in male but not female CD-1 mice. Physiol. Behav. 82, 425–434.
doi: 10.1016/j.physbeh.2004.04.011
Bartolomucci, A., Palanza, P., Gaspani, L., Limiroli, E., Panerai, A. E.,
Ceresini, G., et al. (2001). Social status in mice: behavioral, endocrine
and immune changes are context dependent. Physiol. Behav. 73, 401–410.
doi: 10.1016/S0031-9384(01)00453-X
Benton, D., Dalrymple-Alford, J. C., and Brain, P. F. (1980). Comparisons of
measures of dominance in the laboratory mouse. Anim. Behav. 28, 1274–9.
doi: 10.1016/S0003-3472(80)80115-1
Bernstein, I. (1981). Dominance: the baby and the bathwater. Behav. Brain Sci. 4,
419–457. doi: 10.1017/S0140525X00009614
Bronson, F. H. (1973). Establishment of social rank among grouped male mice:
relative effects on circulating FSH, LH, and corticosterone. Physiol. Behav. 10,
947–51. doi: 10.1016/0031-9384(73)90065-6
Carneiro, C. F. D., Moulin, T. C., Macleod, M. R., and Amaral, O. B. (2018).
Effect size and statistical power in the rodent fear conditioning literature
– a systematic review. PLoS ONE 13:e0196258. doi: 10.1371/journal.pone.
0196258
Colas-Zelin, D., Light, K. R., Kolata, S., Wass, C., Denman-Brice, A., Rios, C.,
et al. (2012). The imposition of, but not the propensity for, social subordination
impairs exploratory behaviors and general cognitive abilities. Behav. Brain Res.
232, 294–305. doi: 10.1016/j.bbr.2012.04.017
Crowcroft, P. (1966). Mice All Over. London: GT. Foulis.
Curley, J. P. (2011). Is there a genomically imprinted social brain? BioEssays 33,
662–8. doi: 10.1002/bies.201100060
Desjardins, C., Maruniak, J. A., and Bronson, F. H. (1973). Social rank in house
mice: differentiation revealed by ultraviolet visualization of urinary marking
patterns. Science 182, 939–941. doi: 10.1126/science.182.4115.939
Drews, C. (1993). The concept and definition of dominance in animal behaviour.
Behaviour 125, 283–313. doi: 10.1163/156853993X00290
Ely, D. L., and Henry, J. P. (1978). Neuroendocrine response patterns
in dominant and subordinate mice. Horm Behav. 10, 156–69.
doi: 10.1016/0018-506X(78)90005-3
Ferrari, P. F., Palanza, P., Parmigiani, S., and Rodgers, R. J. (1998). Interindividual
variability in Swiss male mice: relationship between social factors, aggression,
and anxiety. Physiol. Behav. 63, 821–827. doi: 10.1016/S0031-9384(97)00544-1
Fitchett, A. E., Barnard C. J., and Cassaday, H. J. (2009). Corticosterone differences
rather than social housing predict performance of T-maze alternation in male
CD-1 mice. Anim. Welf. 18, 21–31.
Fitchett, A. E., Collins S. A., Barnard, C. J., and Cassaday, H. J. (2005a).
Subordinate male mice show long-lasting differences in spatial learning
that persist when housed alone. Neurobiol. Learn. Mem. 84, 247–251.
doi: 10.1016/j.nlm.2005.08.004
Fitchett, A. E., Collins S. A., Mason, H., Barnard, C. J., and Cassaday, H. J. (2005b).
Urinary corticosterone measures: effects of strain and social rank in BKW and
CD-1 mice. Behav. Process. 70, 168–76. doi: 10.1016/j.beproc.2005.06.006
Freund, J., Brandmaier, A. M., Lewejohann, L., Kirste, I., Kritzler, M., Krüger, A.,
et al. (2013). Emergence of individuality in genetically identical mice. Science
340, 756–759. doi: 10.1126/science.1235294
Hamilton, K. (2018). MAJOR - Meta-Analysis. Available online at: https://github.
com/kylehamilton/MAJOR#major-meta-analysis- jamovi-r
Hilakivi, L. A., Lister, R. G., Durcan, M. J., Ota, M., Eskay, R. L., Mefford, I., et al.
(1989). Behavioral, hormonal and neurochemical characteristics of aggressive
α-mice. Brain Res. 502, 158–166. doi: 10.1016/0006-8993(89)90471-X
Hilakivi-Clarke, L. A., and Lister, R. G. (1992). Are there preexisting behavioral
characteristics that predict the dominant status of male NIH Swiss mice (Mus
musculus)? J. Comp. Psychol. 106, 184–189. doi: 10.1037/0735-7036.106.2.184
Hooijmans, C. R., Rovers, M. M., de Vries, R. B., Leenaars, M., Ritskes-Hoitinga,
M., and Langendam, M. W. (2014). SYRCLE’s risk of bias tool for animal
studies. BMC Med. Res. Methodol. 14:43. doi: 10.1186/1471-2288-14-43
Horii, Y., Nagasawa, T., Sakakibara, H., Takahashi, A., Tanave, A., Matsumoto,
Y., et al. (2017). Hierarchy in the home cage affects behaviour and
gene expression in group-housed C57BL/6 male mice. Sci. Rep. 7:6991.
doi: 10.1038/s41598-017-07233-5
Izídio, G. S., Lopes, D. M., Spricigo, L., and Ramos, A. (2005). Common variations
in the pretest environment influence genotypic comparisons in models of
anxiety. Genes Brain Behav. 4, 412–9. doi: 10.1111/j.1601-183X.2005.00121.x
Kunkel, T., and Wang, H. (2018). Socially dominant mice in C57BL6
background show increased social motivation. Behav. Brain Res. 336, 173–176.
doi: 10.1016/j.bbr.2017.08.038
Larrieu, T., Cherix, A., Duque, A., Rodrigues, J., Lei, H., Gruetter, R., et al.
(2017). Hierarchical status predicts behavioral vulnerability and nucleus
accumbens metabolic profile following chronic social defeat stress. Curr. Biol.
27, 2202–2210. doi: 10.1016/j.cub.2017.06.027
Lathe, R. (2004). The individuality of mice. Genes Brain Behav. 3, 317–327.
doi: 10.1111/j.1601-183X.2004.00083.x
Lee, W., Fu, J., Bouwman, N., Farago, P., and Curley, J. P. (2019a). Temporal
microstructure of dyadic social behavior during relationship formation in mice.
PLoS ONE 14:e0220596. doi: 10.1371/journal.pone.0220596
Lee, W., Hiura, L. C., Yang, E., Broekman, K. A., Ophir, A. G., and Curley, J. P.
(2019b). Social status in mouse social hierarchies is associated with variation
Frontiers in Behavioral Neuroscience | www.frontiersin.org 12 January 2021 | Volume 14 | Article 624036
Varholick et al. Social Dominance and Behavior
in oxytocin and vasopressin 1a receptor densities. Horm. Behav. 114:104551.
doi: 10.1016/j.yhbeh.2019.06.015
Lee, W., Yang, E., and Curley, J. P. (2018). Foraging dynamics are associated
with social status and context in mouse social hierarchies. PeerJ. 6:e5617.
doi: 10.7717/peerj.5617
Louch, C. D., and Higginbotham, M. (1967). The relation between social rank
and plasma corticosterone levels in mice. Gen. Comp. Endocrinol. 8, 441–4.
doi: 10.1016/S0016-6480(67)80006-6
Miczek, K. A., and Barry, H. (1975). What does the tube test measure? Behav. Biol.
13, 537–9. doi: 10.1016/S0091-6773(75)91249-3
Mondragón, R., Mayagoitia, L., López-Luján, A., and Díaz, J. L. (1987).
Social structure features in three inbred strains of mice, C57Bl/6J,
Balb/cj, and NIH: a comparative study. Behav. Neural Biol. 47, 384–91.
doi: 10.1016/S0163-1047(87)90500-0
Moulin, T. C., and Amaral, O. B. (2020). Using collaboration networks to
identify authorship dependence in meta-analysis results. Res. Synth. Methods
11, 655–68. doi: 10.1002/jrsm.1430
National Research Council (2011). Guide for the Care and Use of Laboratory
Animals.8th ed. National Academies Press: Washington, D.C. Available online
at: https://www.ncbi.nlm.nih.gov/books/NBK54050/
Nyby, J., Dizinno, G. A., and Whitney, G. (1976). Social status and
ultrasonic vocalizations of male mice. Behav. Biol. 18, 285–9.
doi: 10.1016/S0091-6773(76)92198-2
Palanza, P., Gioiosa, L., and Parmigiani, S. (2001). Social stress in mice: gender
differences and effects of estrous cycle and social dominance. Physiol. Behav.
73, 411–420. doi: 10.1016/S0031-9384(01)00494-2
Pallé A., Zorzo, C., Luskey, V. E., McGreevy, K. R., Fernández, S., and Trejo, J.
L. (2019). Social dominance differentially alters gene expression in the medial
prefrontal cortex without affecting adult hippocampal neurogenesis or stress
and anxiety-like behavior. FASEB J. 33, 6995–7008. doi: 10.1096/fj.201801600R
Papilloud, A., Weger, M., Bacq, A., Zalachoras, I., Hollis, F., Larrieu, T.,
et al. (2020). The glucocorticoid receptor in the nucleus accumbens plays a
crucial role in social rank attainment in rodents. Psychoneuroendocrinology
112:104538. doi: 10.1016/j.psyneuen.2019.104538
Rohatgi, A. (2018). WebPlotDigitizer. Austin, Texas, USA. Available online
at: https://automeris.io/WebPlotDigitizer
Saldívar-González, J. A., Posadas-Andrews, A., Rojas, J. A., Yoldi-Negrete, M.,
Alvarez-Sekely, A., Flores-Hernandez, V., et al. (2007). Effect of imipramine
and electro convulsive stimulation in mice under social stress conditions. Curr.
Top. Pharmacol. 11, 57–70.
Sá-Rocha, V. M., Sá-Rocha, L. C., and Palermo-Neto, J. (2006). Variations in
behavior, innate immunity and host resistance to B16F10 melanoma growth in
mice that present social stable hierarchical ranks. Physiol. Behav. 88, 108–115.
doi: 10.1016/j.physbeh.2006.03.015
Schuhr, B. (1987). Social structure and plasma corticosterone level in female albino
mice. Physiol. Behav. 40, 689–693. doi: 10.1016/0031-9384(87)90269-1
Scott, J. P. (1966). Agonistic behavior of mice and rats: a review. Am. Zool. 6,
683–701. doi: 10.1093/icb/6.4.683
Sloan Wilson, D., Clark, A. B., Coleman, K., and Dearstyne, T. (1994). Shyness
and boldness in humans and other animals. Trends Ecol. Evol. 9, 442–6.
doi: 10.1016/0169-5347(94)90134-1
Snyder-Mackler, N., Burger, J. R., Gaydosh, L., Belsky, D. W., Noppert, G. A.,
Campos, F. A., et al. (2020). Social determinants of health and survival
in humans and other animals. Science 368:eaax9553. doi: 10.1126/science.
aax9553
Syme, G. J. (1974). The approach response and performance in the dominance
tube. Aust. J. Psychol. 26, 31–6. doi: 10.1080/00049537408254633
The Jamovi project (2020). *jamovi* (Version 1.2) (Computer Software). Retrieved
from: https://www.jamovi.org
Uhrich, J. (1937). The social hierarchy in albino mice. J. Comp. Psychol. 25,
373–413. doi: 10.1037/h0056350
Varholick, J. A. (2019). “Competitive exclusion,” in Encyclopedia of Animal
Cognition and Behavior, eds J. Vonk J., and T. Shackelford (Cham: Springer).
doi: 10.1007/978-3-319-47829-6_707-1
Varholick, J. A., Bailoo, J. D., Palme, R., and Würbel, H. (2018). Phenotypic
variability between social dominance ranks in laboratory mice. Sci Rep. 8:6593.
doi: 10.1038/s41598-018-24624-4
Varholick, J. A., Pontiggia, A., Murphy, E., Daniele, V., Palme, R., Voelkl,
B., et al. (2019). Social dominance hierarchy type and rank contribute to
phenotypic variation within cages of laboratory mice. Sci Rep. 9:13650.
doi: 10.1038/s41598-019-49612-0
Vekovishcheva, O. Y., and Sukhotina, I. A. (2000). Co-housing in a
stable hierarchical group is not aversive for dominant and subordinate
individuals. Neurosci. Behav. Physiol. 30, 195–200. doi: 10.1007/BF024
63158
Viechtbauer, W. (2010). Conducting meta-analyses in R with the metafor package.
J. Stat. Softw. 36, 1–48. doi: 10.18637/jss.v036.i03
Voelkl, B., Altman, N. S., Forsman, A., Forstmeier, W., Gurevitch, J., Jaric, I., et al.
(2020). Reproducibility of animal research in light of biological variation. Nat.
Rev. Neurosci. 21, 384–393. doi: 10.1038/s41583-020-0313-3
Voelkl, B., and Würbel, H. (2016). Reproducibility crisis: are we ignoring reaction
norms? Trends Pharmacol. Sci. 37, 509–10. doi: 10.1016/j.tips.2016.05.003
Wahlsten, D. (2011). Mouse Behavioral Testing: How to Use Mice in
Behavioral Neuroscience.1st ed. University of Alberta, Canada: Elsevier
Inc. doi: 10.1016/B978-0-12-375674-9.10002-3
Wahlsten, D. (2010). “Probabilistic epigenesis and modern behavioral and neural
genetics,” in Handbook of Developmental Science, Behavior, and Genetics. First,
eds K. E. Hood, C. T. Halpern, G. Greenberg, and R. M. Lerner (London:
Blackwell Publishing), 110–22. doi: 10.1002/9781444327632.ch5
Wang, F., Kessels, H. W., and Hu, H. (2014). The mouse that roared:
neural mechanisms of social hierarchy. Trends Neurosci. 37, 674–682.
doi: 10.1016/j.tins.2014.07.005
Wang, F., Zhu, J., Zhu, H., Zhang, Q., Lin, Z., and Hu, H. (2011). Bidirectional
control of social hierarchy by synaptic efficacy in medial prefrontal cortex.
Science 334, 693–697. doi: 10.1126/science.1209951
Williamson, C. M., Franks, B., and Curley, J. P. (2016a). Mouse social network
dynamics and community structure are associated with plasticity-related brain
gene expression. Front. Behav. Neurosci. 10:152. doi: 10.3389/fnbeh.2016.00152
Williamson, C. M., Lee, W., and Curley, J. P. (2016b). Temporal dynamics of social
hierarchy formation and maintenance in male mice. Anim. Behav. 115, 259–72.
doi: 10.1016/j.anbehav.2016.03.004
Williamson, C. M., Lee, W., Decasien, A. R., Lanham, A., Romeo, R. D., and
Curley, J. P. (2019). Social hierarchy position in female mice is associated
with plasma corticosterone levels and hypothalamic gene expression. Sci. Rep.
9:7324. doi: 10.1038/s41598-019-43747-w
Williamson, C. M., Lee, W., Romeo, R. D., and Curley, J. P. (2017a). Social context-
dependent relationships between mouse dominance rank and plasma hormone
levels. Physiol. Behav. 171, 110–119. doi: 10.1016/j.physbeh.2016.12.038
Williamson, C. M., Romeo, R. D., and Curley, J. P. (2017b). Dynamic changes in
social dominance and mPOA GnRH expression in male mice following social
opportunity. Horm. Behav. 87, 80–8. doi: 10.1016/j.yhbeh.2016.11.001
Wilson, W. J. (1968). Adaptation to the dominance tube. Psychon. Sci. 10, 119–20.
doi: 10.3758/BF03331437
Woltereck, R. (1909). Weitere experimentelle untersuchungen über
artveränderung, speziel über das wesen quantitativer artunterschiede bei
daphniden. Verhandlungen Dtsch. Zool. Ges. 1909, 110–72.
Würbel, H. (2002). Behavioral phenotyping enhanced–beyond
(environmental) standardization. Genes Brain Behav. 1, 3–8.
doi: 10.1046/j.1601-1848.2001.00006.x
Zhou, T., Zhu, H., Fan, Z., Wang, F., Chen, Y., Liang, H., et al. (2017). History of
winning remodels thalamo-PFC circuit to reinforce social dominance. Science
357, 162–168. doi: 10.1126/science.aak9726
Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2021 Varholick, Bailoo, Jenkins, Voelkl and Würbel. This is an open-
access article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) and the copyright owner(s) are credited and that the
original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply
with these terms.
Frontiers in Behavioral Neuroscience | www.frontiersin.org 13 January 2021 | Volume 14 | Article 624036
Available via license: CC BY
Content may be subject to copyright.
