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Citation: Koyasu, H.; Takahashi, H.;
Sasao, I.; Takagi, S.; Nagasawa, M.;
Kikusui, T. Sociality of Cats toward
Humans Can Be Influenced by
Hormonal and Socio-Environmental
Factors: Pilot Study. Animals 2023,13,
146. https://doi.org/10.3390/
ani13010146
Academic Editor: Mandy Paterson
Received: 28 October 2022
Revised: 29 December 2022
Accepted: 29 December 2022
Published: 30 December 2022
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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4.0/).
animals
Article
Sociality of Cats toward Humans Can Be Influenced by
Hormonal and Socio-Environmental Factors: Pilot Study
Hikari Koyasu 1, Hironobu Takahashi 1, Ikuto Sasao 1, Saho Takagi 1,2, Miho Nagasawa 1, *
and Takefumi Kikusui 1
1Laboratory of Human-Animal Interaction and Reciprocity, Azabu University, Sagamihara 252-5201, Japan
2Japan Society for the Promotion of Science, Tokyo 102-8471, Japan
*Correspondence: nagasawa@carazabu.com
Simple Summary:
Cats are the most widely kept companion animal in the world. Various factors
influence the sociality of cats. Here, we investigated whether the hormonal status of cats, and the
age at which they began living with a human, affected their behaviors toward humans. The results
showed that male cats that began living with a human earlier had more contact with humans. In
addition, males with lower testosterone levels had more contact with humans. The results of this
pilot study suggest that testosterone levels and the timing of when cats begin living with humans
modulate affinity behavior of male cats toward humans.
Abstract:
Individual differences in the sociality of cats are influenced by inherited and environmental
factors. We recently revealed that hormones can make a difference in intraspecies social behavior. It
remains unclear whether cat behavior toward humans is modulated by hormones. Therefore, we
analyzed the relationship between cat behavior and their basal hormone concentrations after spending
time together with human experimenters. In addition, we analyzed the relationship between cat
behavior and the timing of when the individual cats began living with a human because the sociality
of cats could be dependent on their developmental experiences. The results showed that male cats
that began living with humans earlier had more contact with an experimenter. In addition, individual
male cats with low testosterone levels were more likely to interact with an experimenter. These
findings of this pilot study suggest that the sociality of male cats toward humans is affected by
testosterone and the age at which they begin to live with humans.
Keywords:
cat behavior; animal socialization; cat-human interaction; testosterone; cortisol; oxytocin
1. Introduction
Animals live in relationships with other members of their species, as well as with
different species. Cats, in particular, live in the same space with humans and have a
close relationship through their interactions based on various senses [
1
]. Cats have many
natural instincts that promote human interaction [
1
]. In vocal communication, for example,
meowing has changed to communicate more effectively with humans. Although meowing
is typically only used by mothers and kittens among cats [
2
], they also meow to attract
human attention, even after they become adult animals [
3
]. Visual communication has
also developed between cats and humans [
1
]. Cats follow human gaze in food selection
tasks for referential information [
4
], and eyeblink synchronization, an indicator of smooth
communication, has also been observed [5–7].
Although cats have evolved various behaviors toward humans, there is large indi-
vidual variability. Although cats have evolved various behaviors toward humans, there
is large individual variability owing to selection preference that favors appearance over
sociality [
8
]. These differences in behavior are influenced by genetic and environmental fac-
tors. The question of whether genetic factors influence animal behavior has been analyzed
Animals 2023,13, 146. https://doi.org/10.3390/ani13010146 https://www.mdpi.com/journal/animals
Animals 2023,13, 146 2 of 10
by investigating behavioral differences between breeds, and supported by the responses
to questionnaires [9,10]. In addition, coat color, which is genetically determined, has been
studied in relationship to behavior in cats [11–13].
Furthermore, genetic variation can lead to differences in the development of the
hormone system in cats and the amount of basal hormone secretion, which could influence
cat behavior. Another factor that influences cat behavior toward humans, such as early
childhood experiences [
14
–
20
]. Cats receiving 30–40 min of handling per day when they are
kittens display a greater affinity towards humans [
16
]. In addition, the number of handlers
that kittens are exposed to affects their affinity towards humans as adult cats [
16
,
17
]. We
previously showed that intraspecific behavior in cats is modulated by hormones, and it is
possible that their behavior toward humans is also modulated by hormones. Carlstead et al.
showed that cat urinary cortisol is negatively correlated with hiding from human behaviors.
In addition, concentrations of urinary cortisol [
21
] and oxytocin [
22
] have been shown to
change depending on how humans care for cats. Finally, aggressive behavior decreases in
cats after castration [23,24], which may be due to decreased testosterone concentration.
Although this was a pilot study due to the limited number of sample size, we aimed
to clarify whether the behavior of cats toward humans is influenced by the age at which
cats begin living with humans and/or by cat hormone levels. We recorded the age at
which the cats began to live with humans, their behavior while spending time with human
experimenters, and measured their hormone levels. We hypothesized that (1) the younger
a cat begins to live with humans, the greater the affinity they would have with humans
as adult cats, (2) individual cats with lower testosterone concentrations would avoid
experimenters less than that of cats with higher testosterone concentrations, (3) individual
cats with lower cortisol concentrations would avoid experimenters less than that of cats with
higher cortisol concentrations, and (4) individual cats with lower oxytocin concentrations
would exhibit lower group boundaries, interact with others, and develop more friendly
interactions with experimenters.
2. Materials and Methods
2.1. Cat Subjects
Cats living in a cat caféor shelter participated in this experiment, and all of the cats
were rescued cats looking for adoptive homes. There were 4 male and 11 female cats aged
20.0
±
12.6 months of age; all of the male cats were castrated. Detailed information about
each cat is shown in Table S1.
2.2. Experimental Environment
For the cafécats, the experiment was conducted in an 8.6 m
2
room of the cat café.
Because the shelter cats had been kept at Azabu University for 3 months for another
experiment, we used a 10 m
2
room at the Companion Dog Laboratory of Azabu University
as the experimental room for the shelter cats. Within each room, there was a sofa for the
human experimenters and a cat bed or cat tower for cats to choose where they wanted to
rest. The environment of the two rooms was almost the same and both rooms were new
places for the cats.
2.3. Behavioral Observation Flow-Chart
We investigated the behavior of the cats toward the experimenter over a period of
2 h. A flow-chart of the experiment is shown in Figure 1. First, the cat was introduced to
the experimental room with the experimenter petting and talking to the cat for
10–20 min
to get used to the room. Second, after the cat walked freely and explored the room, the
experimenter left the room and the cat spent 5 min alone. Third, the experimenter re-
entered the room and spent 2 h with the cat. During this time, the experimenter read a
book or worked on a computer and was not allowed any interaction with the cat, such
as petting, talking to, or looking at the cat. The experimenters included two males and
two females who had met the subject cat at least five times, but less than ten times. One
Animals 2023,13, 146 3 of 10
recording camera (HDR-AS50R, SONY) was installed on the ceiling of each room and a
second camera was used in the case of blind areas.
Animals 2023, 13, x 3 of 10
talking to, or looking at the cat. The experimenters included two males and two females
who had met the subject cat at least five times, but less than ten times. One recording
camera (HDR-AS50R, SONY) was installed on the ceiling of each room and a second cam-
era was used in the case of blind areas.
Figure 1. Experimental procedure.
2.4. Behavioral Analysis
The behavior of cats toward experimenters was recorded. The following behaviors
were analyzed: rubbing against experimenter, being on the same sofa with experimenter,
touching, meowing, sniffing, staying near the door, greeting score, and following score.
Greeting and following were scored based on the Strange Situation Test [25]. The follow-
ing score was calculated when the experimenter left the room once after the familiariza-
tion period, and the greeting score was calculated when the experimenter re-entered the
room. Other behaviors during the reunion were recorded by continuous sampling. The
definition of each behavior is listed in Table S2.
2.5. Hormone Assay
Cat urine samples were collected with cotton immediately after urination using a
two-layer toilet between 7:00 am and 10:00 am. A total of 101 urine samples (6.7 ± 3.6
samples/cat) were collected, and the experimenter was able to determine which cat ex-
creted the urine in each case. Urine samples were collected during the month before and
after the experiment. Basal hormone concentrations for each individual cat were obtained
by averaging samples.
2.5.1. Cortisol Concentrations
Cortisol concentrations were measured using an enzyme-linked immunosorbent as-
say (ELISA). We prepared the ELISA-plate using a mouse IgG-Fc fragment antibody (A90-
131A, Bethyl Laboratories). The secondary antibody was diluted 500-fold and dispensed
in 100 µL aliquots. After overnight incubation at 22–25 °C, the liquid in each well was
discarded. Subsequently, a phosphate buffer containing 0.1% bovine serum albumin
(BSA) was dispensed in 200 µL aliquots to all wells. After incubation for 30 min at 22–25
°C, the plates were stored at 4 °C in the dark until used in the assay.
Undiluted urine samples were dispensed into the wells of the ELISA-plate. An anti-
cortisol antibody (ab1949; Abcam) diluted 200,000-fold was used as a primary antibody.
Cortisol-3-CMO-HRP (FKA403, COSMO) diluted 10,000-fold was used as a horseradish
peroxidase (HRP). 15 µL standard samples, 15 µL urine samples and 100 µL primary and
Figure 1. Experimental procedure.
2.4. Behavioral Analysis
The behavior of cats toward experimenters was recorded. The following behaviors
were analyzed: rubbing against experimenter, being on the same sofa with experimenter,
touching, meowing, sniffing, staying near the door, greeting score, and following score.
Greeting and following were scored based on the Strange Situation Test [
25
]. The following
score was calculated when the experimenter left the room once after the familiarization
period, and the greeting score was calculated when the experimenter re-entered the room.
Other behaviors during the reunion were recorded by continuous sampling. The definition
of each behavior is listed in Table S2.
2.5. Hormone Assay
Cat urine samples were collected with cotton immediately after urination using a two-layer
toilet between 7:00 a.m. and 10:00 a.m. A total of 101 urine samples (
6.7 ±3.6 samples/cat
)
were collected, and the experimenter was able to determine which cat excreted the urine in each
case. Urine samples were collected during the month before and after the experiment. Basal
hormone concentrations for each individual cat were obtained by averaging samples.
2.5.1. Cortisol Concentrations
Cortisol concentrations were measured using an enzyme-linked immunosorbent assay
(ELISA). We prepared the ELISA-plate using a mouse IgG-Fc fragment antibody (A90-131A,
Bethyl Laboratories, Montgomery, TX, USA). The secondary antibody was diluted 500-fold
and dispensed in 100
µ
L aliquots. After overnight incubation at 22–25
◦
C, the liquid in
each well was discarded. Subsequently, a phosphate buffer containing 0.1% bovine serum
albumin (BSA) was dispensed in 200
µ
L aliquots to all wells. After incubation for 30 min at
22–25 ◦C, the plates were stored at 4 ◦C in the dark until used in the assay.
Undiluted urine samples were dispensed into the wells of the ELISA-plate. An anti-
cortisol antibody (ab1949; Abcam, Cambridge, UK) diluted 200,000-fold was used as
a primary antibody. Cortisol-3-CMO-HRP (FKA403, COSMO, Dublin, Ireland) diluted
10,000-fold
was used as a horseradish peroxidase (HRP). 15
µ
L standard samples, 15
µ
L
urine samples and 100
µ
L primary and 100
µ
L secondary antibodies were dispensed into
each well. After incubation for at least 6 h, the liquid in each well was discarded and the
plate was washed four times using a plate washer. Thereafter, 150
µ
L of substrate buffer
was dispensed into all wells. The reaction was stopped by adding 50
µ
L of 4 N H
2
SO
4
.
Animals 2023,13, 146 4 of 10
Absorbance was measured at a wavelength of 450 nm using a microplate reader (Model
680XR, Bio-Rad Laboratories, Inc., Hercules, CA, USA).
2.5.2. Testosterone Concentrations
Testosterone concentrations were also measured using ELISA and the same plates as
used for cortisol assay. After washing the plate three times, urine samples were dispensed
into the wells. Urine samples with higher concentrations were diluted 2-fold with a
phosphate buffer containing 0.1% BSA before dispensing. An anti-testosterone 3 CMO
antibody (ab35878, Abcam) diluted 25,000-fold was used as a primary antibody, and a
mouse IgG-Fc fragment antibody (A90-131A, Bethyl Laboratories) diluted 500-fold was
used as secondary antibody. The HRP used in this process was Testosterone-3-CMO-HRP
(FKA101, COSMO) that was diluted 10,000-fold. 25
µ
L standard samples and 25
µ
L urine
samples were dispensed in each well. Then, 100
µ
L primary antibody, 100
µ
L secondary
antibody and 100
µ
L HRP were dispensed. After incubation for at least 6 h, the liquid in the
plate was discarded and the plates were washed four times using a plate washer. Thereafter,
150
µ
L of substrate buffer was dispensed into all wells. The reaction was stopped by
adding 50
µ
L of 4 N H
2
SO
4
. Absorbance was measured at a wavelength of 450 nm using a
microplate reader.
2.5.3. Oxytocin Concentrations
We used a commercially available oxytocin ELISA kit (ADI-901-153A-0001, ENZO,
New York, NY, USA) for the assay. Urine samples diluted 50-fold with the assay buffer
in the kit were dispensed into the wells of the ELISA-plates. 15
µ
L standard samples and
15
µ
L urine samples were dispensed in each well. A primary antibody and HRP were
dispensed in 50
µ
L aliquots in each well. After incubation at 4
◦
C for 18–24 h, the substrate
solution was dispensed in 200
µ
L aliquots in all wells. After incubation at 22–25
◦
C for 1 h,
50
µ
L of stop solution was added to each well. Absorbance was measured at a wavelength
of 405 nm using a microplate reader.
2.5.4. Creatinine Concentration
A creatinine standard samples and the urine samples diluted 100-fold with distilled
water were dispensed into a 96-well microplate (AS ONE Co., Ltd., Osaka, Japan) at 100
µ
L
each, followed by 50
µ
L of 1 M NaOH and 50
µ
L of 1 g/dL trinitrophenol. The absorbance
was measured at a wavelength of 490 nm using a microplate reader after the plate was left
at room temperature (22–25
◦
C) for 20 min. The samples were used undiluted. To adjust
for variations in urine concentration, all hormone concentrations were indexed against the
creatinine concentration.
2.6. Statistical Analysis
All statistical analyses were conducted using R version 3.5.1. Linear regression model
and correlation analysis were conducted to investigate the relationship between cat be-
havior toward humans and their age in months and hormone concentrations. Each social
behavior towards humans was analyzed using a linear model (LM) and the lmer function
in the lme4 package version 1.1.10. Testosterone concentration, cortisol concentration,
oxytocin concentration, age at experiment, and age at which cats began to live with humans
were entered as fixed factors. Correlation analysis was conducted separately for males
and females since an analysis including interactions between sex and other factors could
not be performed due to the lack of samples. In the correlation analysis, spearman’s rank
correlation coefficient was used. The significance level was adjusted because the analysis
was repeated five times with months and hormones for a behavior (significance was set at
p< 0.01).
Animals 2023,13, 146 5 of 10
3. Results
3.1. Effects of Age and Hormones on Cats’ Behaviors toward Humans
None of the behaviors examined was affected by age or hormone concentrations
(Table S3).
3.2. Correlation of Male Cats’ Behavior toward Humans with Age and Hormones
We then examined correlations between behavior, age, and hormone concentrations
separately by sex. As a result, the age at which male cats began living with humans corre-
lated negatively with sharing sofa (Figure 2A; rs =
−
1.000, p< 0.001), contact (Figure 2B;
rs =
−
1.000, p< 0.001), rubbing (Figure 2C; rs =
−
1.000, p< 0.001), tail up (Figure 2D;
rs =−1.000
,p< 0.001), and following (Figure 2E; rs =
−
1.000, p< 0.001). In males, testos-
terone concentrations correlated negatively with sharing sofa (Figure 3A; rs =
−
1.000,
p< 0.001
), contact (Figure 3B; rs =
−
1.000, p< 0.001), rubbing (Figure 3C; rs =
−
1.000,
p< 0.001
). In addition, there was a positive correlation between the age at which the cats
began living with humans and testosterone concentrations (Figure S1; rs = 1.000, p< 0.001).
The results of the correlation between all behaviors, hormones, and age in months are
shown in Tables S4 and S5.
Animals 2023, 13, x 5 of 10
was repeated five times with months and hormones for a behavior (significance was set
at p < 0.01).
3. Results
3.1. Effects of Age and Hormones on Cats’ Behaviors toward Humans
None of the behaviors examined was affected by age or hormone concentrations (Ta-
ble S3).
3.2. Correlation of Male Cats’ Behavior toward Humans with Age and Hormones
We then examined correlations between behavior, age, and hormone concentrations
separately by sex. As a result, the age at which male cats began living with humans corre-
lated negatively with sharing sofa (Figure 2A; rs = −1.000, p < 0.001), contact (Figure 2B; rs =
−1.000, p < 0.001), rubbing (Figure 2C; rs = −1.000, p < 0.001), tail up (Figure 2D; rs = −1.000, p
< 0.001), and following (Figure 2E; rs = −1.000, p < 0.001). In males, testosterone concentra-
tions correlated negatively with sharing sofa (Figure 3A; rs = −1.000, p < 0.001), contact (Fig-
ure 3B; rs = −1.000, p < 0.001), rubbing (Figure 3C; rs = −1.000, p < 0.001). In addition, there
was a positive correlation between the age at which the cats began living with humans and
testosterone concentrations (Figure S1; rs = 1.000, p < 0.001). The results of the correlation
between all behaviors, hormones, and age in months are shown in Tables S4 and S5.
Figure 2. Correlation between behavior towards experimenters and age at keeping with humans
(months). (A–E) indicate the relationship between age at beginning to live with humans and the
following behaviors, (A) Sharing sofa, (B) Contact, (C) Rubbing, (D) Tail up, (E) Following.
Figure 2.
Correlation between behavior towards experimenters and age at keeping with humans
(months). (
A
–
E
) indicate the relationship between age at beginning to live with humans and the
following behaviors, (A) Sharing sofa, (B) Contact, (C) Rubbing, (D) Tail up, (E) Following.
Animals 2023,13, 146 6 of 10
Animals 2023, 13, x 6 of 10
Figure 3. Correlation between behavior towards experimenters and testosterone concentrations. (A–
C) indicate the relationship between testosterone concentration and the following behaviors, (A)
Sharing sofa, (B) Contact, (C) Rubbing.
3.3. Correlation of Female Cats’ Behavior toward Humans with Age and Hormones
In females, there was no correlation between behaviors towards humans and age or
hormone concentrations.
4. Discussion
In this study, we investigated the relationship between the behavior of cats toward
humans and their age and hormone concentrations to determine whether behavior to-
ward humans is influenced by the age at which cats begin living with humans and/or their
hormone levels. We found that the earlier a male cat began living with humans and the
lower the testosterone level, the more likely the cats were to interact with experimenters.
Only the results in males were consistent with our original hypothesis.
A decrease in testosterone leads to a decrease in aggression. The relationship has
been reported in various species, including rodents, e.g., [26–28], primates, e.g., [29–32],
and canidae, e.g., [33]. Lower testosterone levels may have made the cats more tolerant of
other individual cats and increased their contact with humans. This association was only
observed in males, which may have been influenced by sexual differentiation of the brain.
For example, the brain of rats is sexually undifferentiated until about one week after birth.
Androgen action during the perinatal period results in masculinization and defeminiza-
tion of their brain [34–38]. Individuals that develop masculine brains during this period
depend on testosterone for their behavior even after growth [39]. Additionally, in cats, the
sexual differentiation of the brain during fetal life might have resulted in different out-
comes for males and females. In addition, androgen precursors are also secreted from the
adrenal glands in both sexes and converted to testosterone in the peripheral tissues [40].
Although all of the male cats were castrated, the testosterone produced by their adrenal
glands could have affected their behavior. In addition, it has been reported that neutered
Figure 3.
Correlation between behavior towards experimenters and testosterone concentrations.
(
A
–
C
) indicate the relationship between testosterone concentration and the following behaviors,
(A) Sharing sofa, (B) Contact, (C) Rubbing.
3.3. Correlation of Female Cats’ Behavior toward Humans with Age and Hormones
In females, there was no correlation between behaviors towards humans and age or
hormone concentrations.
4. Discussion
In this study, we investigated the relationship between the behavior of cats toward
humans and their age and hormone concentrations to determine whether behavior toward
humans is influenced by the age at which cats begin living with humans and/or their
hormone levels. We found that the earlier a male cat began living with humans and the
lower the testosterone level, the more likely the cats were to interact with experimenters.
Only the results in males were consistent with our original hypothesis.
A decrease in testosterone leads to a decrease in aggression. The relationship has
been reported in various species, including rodents, e.g., [
26
–
28
], primates, e.g., [
29
–
32
],
and canidae, e.g., [
33
]. Lower testosterone levels may have made the cats more tolerant of
other individual cats and increased their contact with humans. This association was only
observed in males, which may have been influenced by sexual differentiation of the brain.
For example, the brain of rats is sexually undifferentiated until about one week after birth.
Androgen action during the perinatal period results in masculinization and defeminization
of their brain [
34
–
38
]. Individuals that develop masculine brains during this period depend
on testosterone for their behavior even after growth [
39
]. Additionally, in cats, the sexual
differentiation of the brain during fetal life might have resulted in different outcomes for
males and females. In addition, androgen precursors are also secreted from the adrenal
glands in both sexes and converted to testosterone in the peripheral tissues [
40
]. Although
all of the male cats were castrated, the testosterone produced by their adrenal glands could
have affected their behavior. In addition, it has been reported that neutered males are
Animals 2023,13, 146 7 of 10
friendlier to humans than neutered female [
41
]. The sex differences in hormone-behavior
associations shown in this study may help us understand sex differences in behavior
toward humans.
Individual male cats that began to live with a human later in life spent more time
at the door, avoiding humans, which is consistent with previous studies. Early handling
has shown positive effects on behavior in several studies of cat behavior towards hu-
mans
[16,18–20]
. The results of this study support these studies that the timing of first
human contact during development is an important factor in a cat’s socialization with
humans. There is another possibility that the cats beginning to live with humans early in
their lives had lower testosterone baselines, which may have affected the cats’ behavior
toward humans, since there was also a relationship between the age at which they began to
live with humans and their testosterone levels.
Since cortisol and oxytocin are related to the behavior of cats toward other cats [
42
,
43
],
we predicted that these hormones would also affect the behavior of cats toward humans.
Our results did not support the prediction. Cortisol is also associated with anxiety and
aggression in cats [
43
,
44
], but none of the individuals in this study showed excessively
anxiety or aggression toward humans. In a previous study examining the relationship
between fear responses to humans and cortisol, there was no relationship between them [
45
].
These may be the reasons why there was no correlation between cortisol and social behavior.
In addition, oxytocin acts on a specific individual, especially the owner, and its effects
depend on whom it interacts with. In dogs, oxytocin is increased by interaction with owners
and familiar humans [
46
–
51
]. In this experiment, the experimenter met more than five
times with each individual cat. It is possible that the effect of oxytocin is not present in these
experimenters or in the entire category of humans. In addition, the first human encounter
for cats is important for the subsequent relationship because it makes clear the other’s
motivations and behavioral strategies. Because some differences in cat behavior occur
based on the attributes of humans [
3
], investigating the behavior of cats toward a variety of
humans, including strangers and familiar people, will lead to further understanding of cat
social behaviors.
Most rarely a single hormone influences behavior; multiple hormones influence be-
havior integratedly. For example, testosterone secretion in the testis is affected by cortisol in
an
in vitro
study of cats [
52
]. Other hormones, such as serotonin, also influence cat behav-
ior [53], but the relationships between serotonin, oxytocin and testosterone are unknown.
In previous study, sociality with humans can be explained by paternal inheritance
[54,55]
.
McCune examined how childhood experiences of interaction with humans influence later
behavior, and whether these influences are affected by paternity [
53
]. As a result, individual
cats that had a father who was friendly to humans, and experienced human contact from an
early age, approached humans earlier and spent more time with humans than those who had
not experienced human contact or had an unfriendly father [
54
]. These observations indicate
that cat sociality toward humans is influenced by both environmental and inherited factors.
Therefore, in the future, more profound insights into the sociality of cats with humans may be
gained by examining the genetic background of individual cats and their behavior.
Although it has been shown that testosterone concentrations and the age at which they
began living with humans may influence the behaviors of males toward humans, there
are some limitations of this pilot study. One of the most concerns is the small sample size.
In particular, only four males were sampled, which is not a sufficient number of data to
account for all cats. Additional experiments need to be conducted to confirm reproducibility
with more samples. Second, it is possible that the indicator of when cats began to live
with humans is not appropriate. Although we used the age in months when the cats
were rescued as an indicator of when they began interacting with humans, they might
have been interacting with humans even before they were rescued. It will be necessary to
conduct experiments on individual cats so that we can accurately determine when they
start interacting with humans.
Animals 2023,13, 146 8 of 10
5. Conclusions
In this pilot study, there was no relationship between hormones or age and behaviors
toward humans in male and female cats overall. In only male cats, the lower testosterone
and the earlier they began to live with humans, the more interactions they had with
humans. These results suggest one possibility that socialization of male cats with humans
is influenced by both experience in kittenhood and testosterone, although reproducibility
needs to be confirmed. Research on the relationship between cat behavior and endocrine
status could be key to revealing the evolution of cat sociality.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ani13010146/s1, Figure S1: Correlation between age at beginning
to live with humans and testosterone levels; Table S1: Information of cats; Table S2: Analyzed
behaviors and their definitions; Table S3: Relationship between behavior toward humans, the age in
months, and hormones. SS: Sum of Squares, df: degree of freedom; Table S4 Correlation between
behaviors towards humans, age in months, and hormones in female; Table S5: Correlation between
behaviors towards humans, age in months, and hormones in male.
Author Contributions:
Conceptualization, H.K., S.T., T.K. and M.N.; data curation, H.K., H.T. and
I.S.; formal analysis, H.K. and S.T.; funding acquisition, H.K., S.T., T.K. and M.N.; investigation, H.K.,
H.T. and I.S.; methodology, H.K., T.K. and M.N.; project administration, T.K. and M.N.; supervision,
T.K. and M.N.; visualization, H.K.; writing—original draft, H.K., S.T., T.K. and M.N. All authors have
read and agreed to the published version of the manuscript.
Funding:
This research was funded by the Japan Society for the Promotion of Science, and Grants-in-
Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology
of Japan, grant numbers 20J14760 (H.K.), 19J01485 and 22J40175 (S.T.), 18H02489 and 19K22823 (M.N.),
and 19H00972 (T.K.).
Institutional Review Board Statement:
The animal study protocol was approved by the Animal
Experiment Committee of Azabu University (No. 180410-1 and No. 210325-12).
Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement:
The data presented in this study are available within the article and in
the Supplementary Materials.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
References
1.
Koyasu, H.; Kikusui, T.; Takagi, S.; Nagasawa, M. The Gaze Communications Between Dogs/Cats and Humans: Recent Research
Review and Future Directions. Front. Psychol. 2020,11, 613512. [CrossRef] [PubMed]
2.
Bradshaw, J.; Cameron-Beaumont, C. The Signalling Repertoire of the Domestic Cat and Its Undomesticated Relatives. In The
Domestic Cat: The Biology of Its Behaviour, 2nd ed.; Cambridge University Press: Cambridge, UK, 2000; pp. 67–93.
3.
Mertens, C.; Turner, D.C. Experimental Analysis of Human-Cat Interactions during First Encounters. Anthrozoös
1988
,2, 83–97.
[CrossRef]
4.
Pongrácz, P.; Szapu, J.S.; Faragó, T. Cats (Felis Silvestris Catus) Read Human Gaze for Referential Information. Intelligence
2019
,
74, 43–52. [CrossRef]
5.
Humphrey, T.; Proops, L.; Forman, J.; Spooner, R.; McComb, K. The Role of Cat Eye Narrowing Movements in Cat-Human
Communication. Sci. Rep. 2020,10, 16503. [CrossRef]
6.
Humphrey, T.; Stringer, F.; Proops, L.; McComb, K. Slow Blink Eye Closure in Shelter Cats Is Related to Quicker Adoption.
Animals 2020,10, 2256. [CrossRef] [PubMed]
7.
Koyasu, H.; Goto, R.; Takagi, S.; Nagasawa, M.; Nakano, T.; Kikusui, T. Mutual Synchronization of Eyeblinks between Dogs/cats
and Humans. Curr. Zool. 2021,68, 229–232. [CrossRef] [PubMed]
8.
Finka, L.R. Conspecific and Human Sociality in the Domestic Cat: Consideration of Proximate Mechanisms, Human Selection
and Implications for Cat Welfare. Animals 2022,12, 298. [CrossRef]
9.
Hart, B.L. Selecting the Best Companion Animal: Breed and Gender Specific Behavioral Profiles. In Pet Connection: Its Influence
on Our Health and Quality of Life; Center to Study Human-Animal Relationships and Environments; University of Minnesota:
Minneapolis, MN, USA, 1984; pp. 180–193.
Animals 2023,13, 146 9 of 10
10. Fogle, B. The Cats Mind-Understanding Your Cats Behaviour; Howell Books: Toronto, ON, Canada, 1991.
11.
Ledger, R.; O’Farrell, V. Factors Influencing the Reactions of Cats to Humans and Novel Objects. In Proceedings of the 30th
International Congress of the International Society for Applied Ethology, Guelph, ON, Canada, 14–17 August 1996; Colonel KL
Campbell Centre for the Study of Animal Welfare: Guelph, ON, Canada, 1996.
12.
Stelow, E.A.; Bain, M.J.; Kass, P.H. The Relationship Between Coat Color and Aggressive Behaviors in the Domestic Cat. J. Appl.
Anim. Welf. Sci. 2016,19, 1–15. [CrossRef]
13.
Wilhelmy, J.; Serpell, J.; Brown, D.; Siracusa, C. Behavioral Associations with Breed, Coat Type, and Eye Color in Single-Breed
Cats. J. Vet. Behav. 2016,13, 80–87. [CrossRef]
14.
Wilson, M.; Warren, J.M.; Abbott, L. Infantile Stimulation, Activity, and Learning by Cats. Child Dev.
1965
,36, 843–853. [CrossRef]
15.
Mendl, M.T. Effects of Litter Size and Sex of Young on Behavioural Development in Domestic Cats. Ph.D. Thesis, University of
Cambridge, Cambridge, UK, 1986.
16.
Karsh, E.B. The Effects of Early and Late Handling on the Attachment of Cats to People. In The Pet Connection; Globe Press: New
York, NY, USA, 1983.
17.
Collard, R.R. Fear of Strangers and Play Behavior in Kittens with Varied Social Experience. Child Dev.
1967
,38, 877–891. [CrossRef]
[PubMed]
18.
Karsh, E.B.; Turner, D.C. The Human-Cat Relationship. In The Domestic Cat: The Biology of Its Behaviour; Cambridge University
Press: Cambridge, UK, 1988; pp. 159–177.
19.
Casey, R.A.; Bradshaw, J.W.S. The Effects of Additional Socialisation for Kittens in a Rescue Centre on Their Behaviour and
Suitability as a Pet. Appl. Anim. Behav. Sci. 2008,114, 196–205. [CrossRef]
20.
Turner, D.C. A Review of over Three Decades of Research on Cat-Human and Human-Cat Interactions and Relationships. Behav.
Process. 2017,141, 297–304. [CrossRef] [PubMed]
21.
Carlstead, K.; Brown, J.L.; Strawn, W. Behavioral and Physiological Correlates of Stress in Laboratory Cats. Appl. Anim. Behav. Sci.
1993,38, 143–158. [CrossRef]
22.
Nagasawa, T.; Ohta, M.; Uchiyama, H. The Urinary Hormonal State of Cats Associated with Social Interaction with Humans.
Front. Vet. Sci. 2021,8, 680843. [CrossRef]
23.
Hart, B.L.; Barrett, R.E. Effects of Castration on Fighting, Roaming, and Urine Spraying in Adult Male Cats. J. Am. Vet. Med.
Assoc. 1973,163, 290–292.
24.
Tarttelin, M.F.; Hendriks, W.H.; Moughan, P.J. Relationship between Plasma Testosterone and Urinary Felinine in the Growing
Kitten. Physiol. Behav. 1998,65, 83–87. [CrossRef]
25.
Topál, J.; Gácsi, M.; Miklósi, Á.; Virányi, Z.; Kubinyi, E.; Csányi, V. Attachment to Humans: A Comparative Study on Hand-Reared
Wolves and Differently Socialized Dog Puppies. Anim. Behav. 2005,70, 1367–1375. [CrossRef]
26.
Lumia, A.R.; Thorner, K.M.; McGinnis, M.Y. Effects of Chronically High Doses of the Anabolic Androgenic Steroid, Testosterone,
on Intermale Aggression and Sexual Behavior in Male Rats. Physiol. Behav. 1994,55, 331–335. [CrossRef]
27.
Motelica-Heino, I.; Edwards, D.A.; Roffi, J. Intermale Aggression in Mice: Does Hour of Castration after Birth Influence Adult
Behavior? Physiol. Behav. 1993,53, 1017–1019. [CrossRef]
28.
Melloni, R.H., Jr.; Connor, D.F.; Hang, P.T.; Harrison, R.J.; Ferris, C.F. Anabolic-Androgenic Steroid Exposure during Adolescence
and Aggressive Behavior in Golden Hamsters. Physiol. Behav. 1997,61, 359–364. [CrossRef] [PubMed]
29.
Higley, J.D.; Mehlman, P.T.; Higley, S.B.; Fernald, B.; Vickers, J.; Lindell, S.G.; Taub, D.M.; Suomi, S.J.; Linnoila, M. Exces-
sive Mortality in Young Free-Ranging Male Nonhuman Primates with Low Cerebrospinal Fluid 5-Hydroxyindoleacetic Acid
Concentrations. Arch. Gen. Psychiatry 1996,53, 537–543. [CrossRef] [PubMed]
30.
Mehlman, P.T.; Higley, J.D.; Fernald, B.J.; Sallee, F.R.; Suomi, S.J.; Linnoila, M. CSF 5-HIAA, Testosterone, and Sociosexual
Behaviors in Free-Ranging Male Rhesus Macaques in the Mating Season. Psychiatry Res. 1997,72, 89–102. [CrossRef] [PubMed]
31. Kalin, N.H. Primate Models to Understand Human Aggression. J. Clin. Psychiatry 1999,60 (Suppl. 15), 29–32.
32.
Rejeski, W.J.; Brubaker, P.H.; Herb, R.A.; Kaplan, J.R.; Koritnik, D. Anabolic Steroids and Aggressive Behavior in Cynomolgus
Monkeys. J. Behav. Med. 1988,11, 95–105. [CrossRef]
33.
Creel, S.; Creel, N.M.; Mills, M.G.L.; Monfort, S.L. Rank and Reproduction in Cooperatively Breeding African Wild Dogs:
Behavioral and Endocrine Correlates. Behav. Ecol. 1997,8, 298–306. [CrossRef]
34.
Meaney, M.J.; Stewart, J.; Poulin, P.; McEwen, B.S. Sexual Differentiation of Social Play in Rat Pups Is Mediated by the Neonatal
Androgen-Receptor System. Neuroendocrinology 1983,37, 85–90. [CrossRef]
35.
Isgor, C.; Sengelaub, D.R. Effects of Neonatal Gonadal Steroids on Adult CA3 Pyramidal Neuron Dendritic Morphology and
Spatial Memory in Rats. J. Neurobiol. 2003,55, 179–190. [CrossRef]
36.
Isgor, C.; Sengelaub, D.R. Prenatal Gonadal Steroids Affect Adult Spatial Behavior, CA1 and CA3 Pyramidal Cell Morphology in
Rats. Horm. Behav. 1998,34, 183–198. [CrossRef]
37.
Joseph, R.; Hess, S.; Birecree, E. Effects of Hormone Manipulations and Exploration on Sex Differences in Maze Learning. Behav.
Biol. 1978,24, 364–377. [CrossRef]
38.
Zuloaga, D.G.; Puts, D.A.; Jordan, C.L.; Breedlove, S.M. The Role of Androgen Receptors in the Masculinization of Brain and
Behavior: What We’ve Learned from the Testicular Feminization Mutation. Horm. Behav.
2008
,53, 613–626. [CrossRef] [PubMed]
Animals 2023,13, 146 10 of 10
39.
Kikusui, T. The Role of Sex Spectrum Differences in Reproductive Strategies and the Endocrine Mechanisms Underlying It.
In Spectrum of Sex: The Molecular Bases that Induce Various Sexual Phenotypes; Tanaka, M., Tachibana, M., Eds.; Springer Nature:
Singapore, 2022; pp. 165–179. ISBN 9789811953590.
40. Shea, J.L.; Wong, P.-Y.; Chen, Y. Free Testosterone: Clinical Utility and Important Analytical Aspects of Measurement. Adv. Clin.
Chem. 2014,63, 59–84. [CrossRef] [PubMed]
41.
Hart, B.L.; Hart, L.A. Your Ideal Cat: Insights into Breed and Gender Differences in Cat Behavior; Purdue University Press: Lafayette,
IN, USA, 2013.
42.
Koyasu, H.; Takahashi, H.; Yoneda, M.; Naba, S.; Sakawa, N.; Sasao, I.; Nagasawa, M.; Kikusui, T. Correlations between Behavior
and Hormone Concentrations or Gut Microbiome Imply That Domestic Cats (Felis Silvestris Catus) Living in a Group Are Not
like “groupmates”. PLoS ONE 2022,17, e0269589. [CrossRef] [PubMed]
43.
Finkler, H.; Terkel, J. Cortisol Levels and Aggression in Neutered and Intact Free-Roaming Female Cats Living in Urban Social
Groups. Physiol. Behav. 2010,99, 343–347. [CrossRef]
44.
Gourkow, N.; LaVoy, A.; Dean, G.A.; Phillips, C.J.C. Associations of Behaviour with Secretory Immunoglobulin A and Cortisol in
Domestic Cats during Their First Week in an Animal Shelter. Appl. Anim. Behav. Sci. 2014,150, 55–64. [CrossRef]
45.
Siegford, J.M.; Walshaw, S.O.; Brunner, P.; Zanella, A.J. Validation of a Temperament Test for Domestic Cats. Anthrozoös
2003
,
16, 332–351. [CrossRef]
46.
Odendaal, J.S.; Meintjes, R.A. Neurophysiological Correlates of Affiliative Behaviour between Humans and Dogs. Vet. J.
2003
,
165, 296–301. [CrossRef]
47.
Rehn, T.; Handlin, L.; Uvnäs-Moberg, K.; Keeling, L.J. Dogs’ Endocrine and Behavioural Responses at Reunion Are Affected by
How the Human Initiates Contact. Physiol. Behav. 2014,124, 45–53. [CrossRef]
48.
Hritcu, L.D.; Horhogea, C.; Ciobica, A.; Spataru, M.C.; Spataru, C.; Kis, A. Conceptual Replication of Canine Serum Oxytocin
Increase Following a Positive Dog-Human Interaction. Rev. Chim. 2019,70, 1579–1581. [CrossRef]
49.
MacLean, E.L.; Gesquiere, L.R.; Gee, N.R.; Levy, K.; Martin, W.L.; Carter, C.S. Effects of Affiliative Human–Animal Interaction on
Dog Salivary and Plasma Oxytocin and Vasopressin. Front. Psychol. 2017,8, 1606. [CrossRef]
50.
Mitsui, S.; Yamamoto, M.; Nagasawa, M.; Mogi, K.; Kikusui, T.; Ohtani, N.; Ohta, M. Urinary Oxytocin as a Noninvasive
Biomarker of Positive Emotion in Dogs. Horm. Behav. 2011,60, 239–243. [CrossRef] [PubMed]
51.
Handlin, L.; Hydbring-Sandberg, E.; Nilsson, A.; Ejdebäck, M.; Jansson, A.; Uvnäs-Moberg, K. Short-Term Interaction between
Dogs and Their Owners: Effects on Oxytocin, Cortisol, Insulin and Heart Rate—An Exploratory Study. Anthrozoös
2011
,
24, 301–315. [CrossRef]
52.
Genaro, G.; Franci, C.R. Cortisol Influence on Testicular Testosterone Secretion in Domestic Cat: An in Vitro Study. Pesqui. Vet.
Bras. 2010,30, 887–890. [CrossRef]
53.
Cools, A.R. Serotonin: A Behaviorally Active Compound in the Caudate Nucleus of Cats. Isr. J. Med. Sci.
1973
,9, 5–16. [PubMed]
54.
McCune, S. The Impact of Paternity and Early Socialisation on the Development of Cats’ Behaviour to People and Novel Objects.
Appl. Anim. Behav. Sci. 1995,45, 109–124. [CrossRef]
55.
Reisner, I.R.; Houpt, K.A.; Erb, H.N.; Quimby, F.W. Friendliness to Humans and Defensive Aggression in Cats: The Influence of
Handling and Paternity. Physiol. Behav. 1994,55, 1119–1124. [CrossRef]
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