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ORIGINAL RESEARCH
published: 31 May 2019
doi: 10.3389/fvets.2019.00162
Frontiers in Veterinary Science | www.frontiersin.org 1May 2019 | Volume 6 | Article 162
Edited by:
Sabine G. Gebhardt-Henrich,
University of Bern, Switzerland
Reviewed by:
Carissa Wickens,
University of Florida, United States
Katherine Albro Houpt,
Cornell University, United States
*Correspondence:
Sabrina Briefer Freymond
sabrina.briefer@agroscope.admin.ch
Elodie F. Briefer
elodie.briefer@usys.ethz.ch
Specialty section:
This article was submitted to
Animal Behavior and Welfare,
a section of the journal
Frontiers in Veterinary Science
Received: 17 December 2018
Accepted: 10 May 2019
Published: 31 May 2019
Citation:
Briefer Freymond S, Bardou D,
Beuret S, Bachmann I, Zuberbühler K
and Briefer EF (2019) Elevated
Sensitivity to Tactile Stimuli in
Stereotypic Horses.
Front. Vet. Sci. 6:162.
doi: 10.3389/fvets.2019.00162
Elevated Sensitivity to Tactile Stimuli
in Stereotypic Horses
Sabrina Briefer Freymond 1
*, Déborah Bardou 1, Sandrine Beuret 2, Iris Bachmann 1,
Klaus Zuberbühler 2,3 and Elodie F. Briefer 4
*
1Agroscope, Swiss National Stud Farm, Avenches, Switzerland, 2Faculty of Science, Institute of Biology, University of
Neuchâtel, Neuchâtel, Switzerland, 3School of Psychology and Neuroscience, University of St. Andrews, St. Andrews,
Scotland, 4Institute of Agricultural Sciences, ETH Zürich, Zurich, Switzerland
Although stereotypic behaviors are a common problem in captive animals, why certain
individuals are more prone to develop them remains elusive. In horses, individuals show
considerable differences in how they perceive and react to external events, suggesting
that this may partially account for the emergence of stereotypies in this species. In this
study, we focused on crib-biting, the most common stereotypy displayed by horses.
We compared how established crib-biters (“CB” =19) and normal controls (“C” =18)
differed in response to a standard “personality” assessment test battery, i.e., reactivity to
humans, tactile sensitivity, social reactivity, locomotor activity, and curiosity vs. fearfulness
(both in novel and suddenness situations). Our analyses showed that crib-biters only
differed from control horses in their tactile sensitivity, suggesting an elevated sensitivity
to tactile stimuli. We suggest that this higher tactile sensitivity could be due to altered
dopamine or endogenous opioid physiology, resulting from chronic stress exposition.
We discuss these findings in relation to the hypothesis that there may be a genetic
predisposition for stereotypic behavior in horses, and in relation to current animal
husbandry and management practices.
Keywords: personality, crib-biting horses, stereotypies, coping styles, βendorphin
INTRODUCTION
Stereotypies are defined as repetitive and invariant behaviors, which are thought to be a
consequence of suboptimal environmental or housing conditions. Stereotypic behaviors are often
described as abnormal and with no obvious goal or function (1), and are sometimes compared to
human developmental, neurological, or psychiatric disorders, such as autism, obsessive compulsive
disorders or schizophrenia (2). In animals, stereotypies include locomotor (e.g., “pacing”) and
oral (e.g., “sham chewing”; “crib-biting”) behavioral abnormalities, which can be debilitating for
individuals, especially if they are expressed extensively.
The causal factors and neurobiological mechanisms underlying stereotypic behaviors are only
partially understood (2). A recurrent hypothesis is that sustained “stress” or chronic stress, mainly
in the form of restricted and suboptimal living conditions, can lead to the development of
stereotypic behaviors in animals (2). At the neurobiological level, the idea is that if animals are
prevented from executing some behaviors, then this can facilitate the development of alternative
behaviors such as stereotypies, via sensitization of the underlying neural systems involved (3).
Indeed, exposition to chronic stress is supposed to trigger the release of βendorphin in the brain,
stimulating simultaneously dopamine release in the striatum and activating some part of the basal
ganglia (4). The basal ganglia are thought to constitute the location where neural alterations might
Briefer Freymond et al. Elevated Sensitivity in Crib-Biters
take place, and hence to play a key role in the development of
stereotypies, particularly within the dopaminergic system (5,6).
Yet, it is still largely unclear why only certain individuals develop
stereotypic behaviors, while others remain unaffected (7).
One possible explanation for the susceptibility of some
individuals but not others to develop stereotypies despite being
exposed to similar environments is the existence of individual
differences, or personality. Personality and its various sub-traits,
such as temperament, refers to between-individual differences
in behavior that are relatively stable across various kinds of
situations and over the course of time (8–10). This means
that differences between individuals are largely maintained,
although behavioral attitudes can evolve with age or with the
environment (11). Individual differences (i.e., personality) are
thought to result from a combination of nature and nurture
influences, that is, from an interaction between neural, genetically
inherited system (i.e., temperament) and specific environmental
influences linked to current and previous experiences during
ontogeny (12,13). Quantifying individual differences (called
phenotypes) is usually done via multivariate analyses, which
allow behavioral traits to be grouped into larger categories (e.g.,
“fearfulness” is defined by a range of behavioral reactions to
different fear-inducing situations) (14,15). The more general
goal of personality assessments is thus to establish categories that
reflect how these animals behave, perceive, and react to the world
beyond individual stimuli or specific situations.
One aspect of personality that might affect predispositions to
develop stereotypies are individual differences in motivation to
perform specific behaviors. Indeed, stereotypies often develop
following the prevention of highly motivated behaviors, such
as consummatory acts (16). In captivity, the performance
of some highly motivated consummatory behaviors may be
impossible. This can result in frustration-related stress and, if
sustained or repeated, in stereotypies (17). A classic example
is carnivores, which are highly motivated to hunt. In captivity,
however, individuals are usually prevented from hunting and,
according to the stress-by-frustration hypothesis, are thus
prone to develop locomotor stereotypies (18). Another classic
example is ungulates, which are highly motivated to engage
in food processing over long periods of time. Since this is
usually not possible in captivity, it frequently results in oral
stereotypies (19,20). Similarly, within each of these systems,
individual differences in the propensity to develop certain
types of stereotypies could exist. Specifically, in carnivores
and rodents, more active individuals could be more prone to
develop locomotor stereotypies (21), while in ungulates, more
explorative individuals could be more prone to develop food-
related stereotypies (22,23).
Chronic stress, which can occur when animals face aversive
situations over prolonged periods of time (24), is another
potential precursor of stereotypies. Individual differences in
response to chronic stress could thus also affect propensities to
develop stereotypies (25). In particular, a distinction has been
made between animals responding proactively or reactively when
facing an aversive stimulus (26). Proactively coping individuals
tend to escape from or remove aversive stimuli (fight-or-
flight), whereas reactively coping individuals show no obvious
reactions in similar situations (conservation—withdrawal) (13).
In addition, proactive individuals are generally characterized by
higher levels of mobility, aggression, exploration, and persistence
than reactive individuals (27). These individual differences in
coping styles are also frequently related to underlying physiology
(26). Proactive individuals tend to have a lower reactivity
of the hypothalamo-pituitary-adrenocortical axis (HPA) but
a higher reactivity of the sympatho-adreno-medullary (SAM)
axis compared to reactive individuals (26). Because proactive
animals act to exert control over their environment, they might
be more prone to form routines and, by extension, to fall
into stereotypies (7). This hypothesis, however, has not been
supported by the physiological results of our previous study,
which revealed higher HPA-axis reactivity in stereotyped (crib-
biters) compared to control horses, which is more characteristic
of reactive individuals than proactive ones (28). In sum, the
issue of whether stereotypic behavior can be linked to individual
differences, and particularly coping styles in response to chronic
stress, has not been resolved.
The domesticated horse is an interesting model to study
stereotypies, because horses are often confined individually with
limited movement for extended periods of time, and with
restricted time to forage (19,29,30), which makes them prone
to develop stereotypies. Other factors, such as sex, age, breed,
type of work (dressage), type of diet, and early experiences (e.g.,
weaning time and start of training) have been associated with
the development of stereotypies in this species (31–37). Horses
can express different forms of stereotypies, such as weaving,
box walking and crib-biting (38,39). Crib-biting behavior is
the most common form of stereotypy in this species (34). It is
an oral stereotypy that consists in grasping a fixed object with
the incisors, pulling back and drawing air into the esophagus.
Crib-biting is related to another common stereotypic behavior,
windsucking, which consists of the same behavioral elements,
but without grasping an object. The initiation of these behaviors
are thought to be associated with diet and time spent foraging
(40,41). Indeed, crib-biting has been shown to increase on low-
forage and high-concentrated diet (19,37,42,43). Horses are
adapted to eat forage and chew for the majority of the time (29).
Because chewing gives the opportunity to moisten food with
alkaline saliva essential for digestion, it has been proposed that
wood-chewing may precede the development of crib-biting, as a
redirected movement in attempts to stimulate saliva production
and reduce the acidity of the stomach (40). Despite the fact
that no specific genes have been linked to stereotypies in
horses, this behavior has been reported more predominantly in
certain pedigrees (44–47). Since genetic differences could imply
differences in personality, we suggest that individual variation
in behavioral responses could predispose horses to develop crib-
biting (25).
Even if several studies have aimed at assessing the personality
of horses (48–55), investigations of the personality of stereotypic
horses are scarce. The evidence so far seems to suggest
that crib-biters are less anxious and show no difference in
trainability compared to non-crib-biters (56). However, this
low level of anxiety in crib-biting horses might be preceded
by an initial increase in anxiety, as revealed by an elevation
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Briefer Freymond et al. Elevated Sensitivity in Crib-Biters
TABLE 1 | Characteristics of the horses used in the experiment.
Horses Sex Crib-biters or control Age
(years)
Breed Housing Place
1 M CB 6 Warmblood Box paddock c
2 M CB 22 Criollo Box g
3 M CB 16 Franches-Montagnes Box y
4 M CB 9 Hispano-Arabian Box paddock b
5 M CB 5 Quarter horse Box s
6 M CB 9 Paint horse Box r
7 M CB 5 Paint horse Box paddock k
8 G CB 9 Franches-Montagnes Box d
9 G CB 11 Warmblood Box g
10 G CB 23 Franches-Montagnes Box paddock n
11 G CB 11 Franches-Montagnes Box bo
12 S CB 17 Franches-Montagnes Box h
13 S CB 15 Franches-Montagnes Box h
14 M CB 5 Franches-Montagnes Box paddock m
15 G CB 19 Haflinger Box paddock se
16 G CB 18 Warmblood Box a
17 G CB 7 Unknown origin Box paddock v
18 G CB 10 English thoroughbred Paddock d
19 S CB 11 Franches-Montagnes Box h
20 M C 7 Quarter horse Box paddock s
21 M C 20 Franches-Montagnes Box y
22 M C 14 Warmblood Loose housing h
23 M C 18 Camargue Box paddock b
24 M C 14 Warmblood Loose housing h
25 M C 16 Trotter Box h
26 M C 18 Franches-Montagnes Loose housing h
27 M C 10 Warmblood Box g
28 G C 4 Friso-Arabian box Paddock n
29 G C 24 Unknown origin box Paddock v
30 G C 22 English thoroughbred Paddock d
31 G C 7 Quarter horse Loose housing k
32 G C 6 Franches-Montagnes box Paddock di
33 G C 8 Franches-Montagnes Box d
34 G C 15 Warmblood Loose housing h
35 G C 11 Warmblood Box h
36 S C 17 Franches-Montagnes Box h
37 S C 7 Franches-Montagnes Box h
Sex (M, mare; G, gelding, S, stallion), group (CB, crib-biters; C, controls), age, breed, housing (loose housing, paddock, box), and place (each letter refers to a given farm).
of dopamine, until the stereotypy is fully-established (57). A
recent study did also find a relationship between oral abnormal
behaviors (crib-biting and lip-twisting) and the “personality
traits” intelligence, cooperation, curiousness, equability, and
playfulness (58). Overall, a more thorough investigation of crib-
biters’ personality is required to investigate if a relationship
between horse personality and crib-biting might exist.
The aim of this study was to investigate if certain personality
traits could be associated with crib-biting behavior. Our current
knowledge of crib-biters’ personality is limited to the use of
questionnaires (56–58). Here, we aimed to obtain more objective
measures, by comparing crib-biting and control horses along
five “personality” traits following a previously validated model of
tests, relevant for equitation practice [reactivity to humans, tactile
sensitivity, social reactivity, locomotor activity, and curiosity
vs. fearfulness (both in novel and suddenness situations)] (14,
59). These traits have been shown to appear early in life and
remain relatively stable across time and situations (60–63). Any
possible links between the five traits and crib-biting behavior
could, in the long term, help to rapidly identify horses that are
more prone to develop stereotypic behavior. According to our
previous results on crib-biters’ physiology (28), we predicted
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Briefer Freymond et al. Elevated Sensitivity in Crib-Biters
FIGURE 1 | Experimental procedure for the personality tests. The black dotted lines indicate the time at which each period started and ended. The personality tests
are indicated (1–5). The different tests are Test 1, passive presence test (i.e., reactivity to human); Test 2, tactile sensitivity test (i.e., tactile sensitivity); Test 3, novel
object test (i.e., curiosity/ fearfulness); Test 4, playback test (i.e., social reactivity); Test 5, umbrella opening test (i.e., curiosity/ fearfulness). The trait locomotor activity
of the horses, was scored as the propensity to demonstrate locomotor activity during the passive human test, the social motivation test, and the novel object test. The
learning tests that are indicated are part of another study (Briefer Freymond et al., submitted).
these horses to show behavioral characteristics of reactive coping
individuals, and hence to be generally less anxious (56,57), or
less prone to express their emotions (26), compared to control
horses. We therefore also expected them to interact less with
unfamiliar humans (i.e., less bold), to show less locomotor and
less exploratory behavior (64,65). Regarding social reactivity,
it has been shown in pigs that reactive coping individuals are
more social (66). We thus expected, if the same applies to horses,
that crib-biters would show more social reactions. Regarding
tactile sensitivity, because low responsiveness to external stimuli
has been reported in humans and animals after experiencing
chronic stress situations [human (67), horses (68)], we expected
crib-biters to display a lower tactile sensitivity. Alternatively,
because crib-biting behavior has previously been associated with
a decrease in nociceptive threshold and therefore potentially
enhanced responses to external stimuli (69), the opposite could
be predicted, i.e., higher responses in crib-biters to tactile
stimulation compared to non-stereotypic horses.
MATERIALS AND METHODS
Subject and Management Conditions
The present study was carried out on 19 crib-biters and 18
control horses (total =37 horses) of various breeds, sex (mares,
geldings, and stallions) and ages (4–24 years old; Table 1),
housed in 19 different farms in Switzerland between September
2013 and February 2014. Except for one control, all horses
participated beforehand in a study (performed between April and
July 2013) aimed at testing the physiological reaction of crib-
biters and non-stereotypic horses in a standard ACTH challenge
test (28). Twenty-six horses were privately owned, and 11 were
obtained from the Swiss National Stud Farm. All the horses
had been at their respective farms for at least 1 year. To be
eligible for inclusion in the study, crib-biters were required to
have demonstrated crib-biting behavior, according to the horse
owners, for a minimum of 1 year. The control group was made up
of horses that had never been observed crib-biting or performing
other stereotypies by their owners [i.e., weaving, box walking,
head tossing nodding (38)]. This grouping was verified later on
during the first study (28) and during this study (i.e., crib-biters
were all observed crib biting, while control horses were not seen
displaying any stereotypy). For each crib-biting horse, we tried to
find a control horse that was of similar breed, sex and age, and
that was housed in the same conditions (i.e., if possible, on the
same farm, Table 1). Horses were housed, either individually or
in groups, in single boxes or in boxes with paddocks (Table 1).
Routine care of the study animals was provided by the farm/horse
owners. All these horses were ridden or had been ridden in the
past. The study was approved by the Swiss Federal Veterinary
Office (approval number VD 2677 bis; Switzerland). The owners
of the horses were provided with a detailed written description of
the experiment to be conducted and agreed to the research being
carried out on their animals.
Experimental Procedure
The content of this paper is the first part of a study. The
other part, aimed at characterizing the learning capabilities of
crib-biters, is being prepared for submission (Briefer Freymond
et al., submitted).
Horses were tested at their home farm in a standardized way.
Each horse was subjected to a total of five “personality” tests. The
tests were divided in two sessions (Figure 1). Between the two
sessions, the horses were returned to their home pen for a break
of about 1 h. During this time, in the farms where two horses were
tested, the second horse took part in the experiment (Table 1).
The procedure, based on preliminary tests performed with 20
pilot horses (different horses as those used in this study), was as
follows; at the start of the experiment, the subject was led to and
then released by one experimenter (experimenter 1) in a 8 ×10 m
delimited arena that was familiar and at the same time, a second
experimenter (experimenter 2) started to record the behavior
with a camera, Sony Handycam HDR-CX700. After 15 min of
habituation to the experimental arena, the first session started
and the horse was subjected to four personality tests (about
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Briefer Freymond et al. Elevated Sensitivity in Crib-Biters
FIGURE 2 | Scheme of the different “personality” tests. The camera is represented at top left. In Test 1 (passive presence test), Experimenter 2 is in the middle of the
arena. In Test 2 (tactile sensitivity test), the Experimenter 2 holds the horse and applies the different filament von Frey. The white triangle in Test 3, (novel object test),
designates the unknown object. In Test 4 (playback test), the loudspeaker is represented at the top left, next to the camera. In Test 5 (umbrella opening test),
Experimenter 2, squatted down, holds the umbrella at 1 m from the bucket and 1 m above the ground, the dotted box designates the bucket with food. The black line
in the arena designates the arena divided into four sectors to assess locomotor activity (See Behavioral analyses).
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Briefer Freymond et al. Elevated Sensitivity in Crib-Biters
15 min duration in total, Figure 1). Directly after these tests,
another set of learning tests were carried out (Briefer Freymond
et al., submitted). A final personality test (about 3 min duration)
was performed at the end of the second part (after the learning
tests, Figure 1). During the experiment, experimenter 1 recorded
the tests with the video and was in charge of preparing the arena
for the next tests; whereas experimenter 2 was performing the
tests (see details below, in “Personality tests”).
“Personality” Tests
The horses performed five “personality” tests adapted from
Lansade and Bouissou (60) and Lansade et al. (61–63) (Figures 1,
2). The tests were always conducted in the same order. They
are presented in the order in which they were conducted. The
behavioral measures, based on (60–63), which were scored from
a video later on are detailed for each test (see also Table 2). Only
those for which the inter-observer reliability (“ICC”) were high
and that were expressed by at least 40% of the horses (i.e., 15
horses) are reported. Such cut-off of 40% allowed us to exclude
behaviors that were performed by very few animals, and which
were hence not representative of the responses of the subjects to
our tests (70).
Test 1: Passive Presence Test
This test assesses the propensity of a horse to react to a passive
human, i.e., “reactivity to humans.” An unknown experimenter
(always the same person; experimenter 2) entered the test pen
and settled motionless in the middle of the arena. The horse had
the possibility to interact with the motionless person for 3 min
(Figure 2). We scored the following behaviors related to the trait
“reactivity to humans”; the time the horses spent interacting with
the unknown experimenter (“Conth”), the time spent standing
attentive (“Att”), the time spent standing resting (“SR”), the time
spent standing while exploring (“Sexpl”), the time spent walking
active (“Movact”) and the time spent close (0–1 m, “CAT1”) or
far (>1 m, “CAT2”) from the unknown person (Table 2).
Test 2: Tactile Sensitivity Test
This test assesses the propensity of a horse to react to a
greater or lesser extent to tactile stimuli, i.e., “tactile sensitivity.”
Experimenter 2 held the horse and applied a “filament von
Frey” on its skin (Figure 2). These filaments consist of a hard
plastic body connected to a nylon thread, and are calibrated
to exert a specific magnitude of force on the skin, ranging
from 0.008 to 300 g. Such filaments are commonly used to
measure mechanical sensory thresholds in people (through
verbal responses) and animals (through behavioral responses)
(71). Five different forces were applied, always in the same
random arrangement (300, 0.6, 0.02, 0.008, 1 g) perpendicularly
to the animal’s skin at wither’s height, until the nylon thread
started to bend (i.e., for the exact location of the application
of the filaments, see (68). The interval between the application
of each filament was about 30 s. Experimenter 1 recorded
directly in binary form the following behavior related to the
trait “tactile sensitivity”; trembling of platisma muscle [behavior
used by horses to drive away flies, (72); trembling or not,
“React”; Table 2].
Test 3: Novel Object Test
This test assesses the propensity of a horse to react with
fear or curiosity when exposed to a novel situation, i.e.,
“curiosity/fearfulness.” A novel object (i.e., transparent hose fixed
with colorful string) was placed in the middle of the arena,
in front of the horse held by experimenter 2. The horse was
then released for a duration of 3 min, during which it had the
possibility to explore the object (Figure 2). We analyzed the
following behaviors related to the trait “curiosity/fearfulness”;
the time the horse spent interacting with the novel object
(“Conto”), the time spent standing attentive (“Att”), the time
spent standing while exploring the ground (“Sexpl”), the time
spent walking active (“Movact”), the time spent walking while
exploring (“Movexpl”), and the time spent close (0–1 m, “CAT1”)
or far (>1 m, “CAT2”) from the novel object (Table 2).
Test 4: Playback Test
This test assesses the propensity of a horse to react to a
conspecific, i.e., “social reactivity.” This test was adapted from
a study including a playback procedure (73). It consisted in
measuring the reactions of the subjects to the vocalizations of
conspecifics. We used a loudspeaker located next to the camera,
on one side of the arena, and played one 2-s whinny from an
unknown mare, one 3-s whinny from an unknown gelding, and a
control sound (15 s of skylark song, Alauda arvensis) (Figure 2).
All the sounds were played at similar amplitude, estimated to
be normal for the horses (85.2 ±2.4 dB measured at 1 m using
a sound level meter, C weighting; SoundTest-Master, Laserliner,
UK). All the horses received the same three sounds, played in
a random order, with 10-s silence interval. We analyzed the
following behaviors related to the trait “social reactivity”; the
vocal response of the horses to each sound (“Whin”), the time
spent walking active (“Movact”) and the time spent standing
attentive (“Att”) (Table 2).
Test 5: Umbrella Opening Test
This test was carried out after a battery of learning tests (Briefer
Freymond et al., submitted). It assesses the propensity of a horse
to react with fear or curiosity to a sudden situation (umbrella
opening), i.e., “curiosity/fearfulness.” This test, in a similar way
to Test 3 (novel object test), measures fear reactions but this time
in situations involving suddenness. A bucket of pellets was placed
next to the entrance, with a closed umbrella held at 1 m from the
bucket and 1 m above the ground by experimenter 2, who was
visible to the horse. Experimenter 1 released the horse and it was
free to go eat from the bucket. When the animal was eating with
its head in the bucket for more than 3 s, experimenter 2 suddenly
opened the umbrella and the chronometer started (Figure 2). The
test stopped when the horse resumed eating. The time it took for
the horses to come back to eat in the bucket after the umbrella
opened was directly recorded (“Time”). A maximum of 300 s was
allocated. In case the horse did not come back to eat, the time was
fixed at 300 s (Time =300 s). We scored the following behaviors
related to the trait “fearfulness”; the intensity of the reaction using
a scale (“React”) (Table 2) and the estimated flight distance of the
horses after opening the umbrella (“Flight”) (Table 2). Because of
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Briefer Freymond et al. Elevated Sensitivity in Crib-Biters
TABLE 2 | Definitions of the parameters and the behaviors recorded during the different personality tests (1–5) and to measure the locomotor activity.
Abbreviation
behaviors
Definition Tests/personality
trait
“Att” (c) Proportion of time spent in an attentive state; horse stands still with raised neck and ears pointing
toward different stimuli situated outside or inside of the arena (experimenter (Test 1), object (Test 3),
camera or outside)
Test 1, 3, 4
“Sr” (c) Proportion of time spent resting; horse stands still, usually supported by only three legs, with the
neck low, the muscles relaxed, the ears to the side, the eyelids closed or half closed, and the lips
getting droopy
Test 1
“Sexpl” (c) Proportion of time spent standing while exploring; horse stands still, with low neck exploring toward
different stimuli situated inside of the arena (experimenter (Test 1), object (Test 3), the ground).
Test 1, 3
“Movact” (c) Proportion of time spent walking active; horse walks with raised neck and ears pointing toward
different stimuli situated outside or inside of the arena (experimenter (Test 1), object (Test 3), camera
or outside)
Test 1, 3, 4
“Movexpl” (c) Proportion of time spent walking while exploring; horse walks with low neck exploring toward
different stimuli situated inside of the arena (experimenter (Test 1), object (Test 3), the ground)
Test 3
Contact with human, “Conth” or
object, “Conto”
Proportion of time spent sniffing, licking or nibbling the experimenter or object, moving the object
with the foreleg or the mouth
Test 1, 3
“Whin” (c) Number of whinnies produced; longest, loudest and most common horse vocalization Test 4
Parameters
“CAT1” (0–1 m), “CAT2” (>1 m) (s) Proportion of time spent at (0–1 m) and (>1 m), respectively; estimated distance between the horse
and the object or human
Test 1, 3
“React” (b) Reaction to the tactile filament—trembling or not Test 2
“Locom” (n) Sectors entered; number of sectors entered during the Tests 1, 3, and 4 Locom
“Reacum” Intensity of the reaction to the opened umbrella; nothing (“A”), raises head (“B”), steps back (“C”),
jumps back and looks at the umbrella (“D”), jumps back and looks outside (“E”), jumps back and
canter (“F”)
Test 5
“Flight” Flight distance from the opened umbrella; how far the horse moves away from the food bucket (“A”
=0 m; “B” =0–1 m, “C” =1–2 m, “D” ≥2 m)
Test 5
“Time” Time until eating in seconds; time to resume eating in the bucket after the umbrella was opened Test 5
Control factors
“Force” Five different filament forces (“F”) applied to the skin (“F1”=300 g, “F2”=0.6 g, “F3”=0.02 g,
“F4”=0.008 g, “F5”=3 g)
Test 2
“Sound” Sound treatments played to the horses; whinny from a mare, whinny from a gelding, and a control
sound (skylark song)
Test 4
“Order” Order in which the sound treatments were played to the horses Test 4
C, continuously recorded or as duration; s, instantaneous time sampling; n, number; b, binary.
The crosses indicate which parameters or response variables were recorded for each personality trait. The different tests (i.e., personality trait) are Test 1, passive presence test (i.e.,
reactivity to human); Test 2, tactile sensitivity test (i.e., tactile sensitivity); Test 3, novel object test (i.e., curiosity/fearfulness); Test 4, playback test (i.e., social reactivity); Test 5, umbrella
opening test (i.e., curiosity/fearfulness); Locomotor activity (“Locom”).
a technical problem with the cameras, we were not able to score
the behaviors React and Flight of two control horses in this test.
Behavioral Analyses
All tests were video recorded by experimenter 1, who was located
outside of the arena (Figure 2), using a Sony Handycam HDR-
CX700. From the video of the tests, two different observers
(experimenter 2 and another observer, who was blind to the
group of the horses since she had not participated in the
experiment and scored the videos after renaming them with a
code) scored for each test (i.e., all the videos) the behaviors
either as occurrence using an instantaneous time sampling
method every 10 s (for “CAT1” and CAT2 ; “Point Events”), or
continuously as duration (for other behaviors; “State Events”)
using the Observer software XT v.11 (Noldus). We then
calculated the frequency of occurrence for the Point Events, and
the proportion of the total time spent performing the behavior
for State Events (Table 2). The last personality trait assessed
in the study, locomotor activity of the horses, was scored as
the propensity to demonstrate locomotor activity during the
passive human test, the social motivation test, and the novel
object test. In order to record this personality trait, the arena
was divided into four sectors of equal size using tracks made in
the sand beforehand (Figure 2). To assess the locomotor activity,
experimenter 1 recorded directly the number of times the horses
changed sectors (“Locom”) (Table 2). Because of a problem, we
were not able to score the activity level of three control horses in
this test.
Statistical Analysis
Inter-observer Reliability (ICC)
Inter-observer reliability between the two observers scoring
the videos continuously was assessed by intraclass correlation
coefficients (ICC). ICC were calculated using a two-way mixed
design to assess the absolute agreement between the scores of the
two observers (74,75). ICCs range from 0 to 1, with 0 indicating
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Briefer Freymond et al. Elevated Sensitivity in Crib-Biters
no agreement and 1 indicating full agreement. Generally, ICCs
≤0.40 are considered as poor, those between 0.40 and 0.59 as
fair, those between 0.60 and 0.74 as good, and those between
0.75 and 1.00 as excellent (76). We kept for the analysis only the
behaviors for which ICCs revealed fair to excellent agreements
between the scores of the two observers. To this aim, the time
spent at 1–2 m and the time spent at more than 2 m (Table 2),
which obtained low ICC (ICC: range =0.35–0.55) were grouped
into one category (CAT2; ICC: range =0.90–0.93).
Behavioral Measures
The statistical analyses were carried out on the behavioral
parameters for which the inter-observer reliability (ICC) between
the two observers was fair to excellent (ICC: mean ±SD =0.84
±0.14; range =0.55–0.98). The behavior of the crib-biters (CB)
was compared to the behavior of the control horses (C) for each
test separately (Tables 2,3), using linear mixed-effects models
(LMM; lmer function, lme4 library), generalized linear mixed
models [GLMM; glmer function; lme4 library; multcomp library;
(77)], or cumulative link mixed models [CLMM, clmm function
in R 3.0.2 (78)]. The different models included as a response
variable the behavioral parameters scored (Tables 2,3). The fixed
and control factors are described in Tables 2,3. To control for
repeated measurements of the same subjects, the identity of the
horses nested within the farms where they were housed (“Farms”)
was included as a random factor for Tests 2 and 4. For Test 1, Test
5, and for the locomotor activity, only Farms was included as a
random factor, as there was only one behavioral value for each
horse. When significant interaction effects between fixed and/or
control factors were found, further post-hoc analysis were carried
out using further LMMs and GLMMs. Bonferroni corrections
were applied to these post-hoc tests accordingly.
The residuals were checked graphically for normal
distribution and homoscedasticity. To satisfy the model
assumptions, we used log transformation for “Att” and “Movact”
in the presence passive test, “Movact” and “Sexpl” in the unknown
object test, and for “Time” in the umbrella test (Table 2). All
the parameters satisfying model assumptions were then input
into LMMs (lmer function). Some parameters did not meet the
statistical assumptions despite transformation. They were thus
transformed to binomial data as follows; behavior occurred =1
or did not occur =0 for “CAT1” in novel object test, “Movact” ,
“Sexpl,” and “Sr” in presence passive test, and “Movact” in
playback test (Table 2); and value equal or higher than median
=1 or value lower than median =0, for “Att,” “Conth,” “CAT2”
in novel object test, “Att” in playback test and “Locom” for
locomotor activity (Table 2). The parameters scored as binomial
(“React” and “Whin”), as well as parameters transformed to
binomial data, were input into GLMMs fit with binomial family
distribution and logit link function (glmer function). In the
umbrella opening test, in order to compare the intensity of
the reaction after opening the umbrella (“Reacum”) and the
distance of flight from the open umbrella (“Flight”) (Table 2),
we used CLMM (clmm function) (79). To this aim, Reacum
was transformed in six distinct categories and Flight in four
categories (Table 2).
TABLE 3 | Response variables, as well as fixed and control factors used in the
different model (LMM and GLMM).
Response variables Test 1 Test 2 Test 3 Test 4 Test 5 Locom
Att x x x
Sr x
Sexpl x x
Movact x x x
Movexpl x
Conth x
Conto x
Whin x
CAT1 x x
CAT2 x x
React x
Locom x
Reacum x
Flight x
Time x
Control and fixed factors
Group x x x x x x
Force * Group x
Sound * Group x
Order * Group x
Sex x x x x x x
Age x x x x x x
Arena x x x x x x
Farm x x x x x x
Force x
Order x
Sound x
The abbreviations are described in Table 2. The crosses (“x”) indicate which response
variables or factors were recorded in the different personality tests (1–5) and to assess
the locomotor activity “Locom,” The different tests are Test 1, passive presence test; Test
2, tactile sensitivity test; Test 3, novel object test; Test 4, playback test; Test 5, umbrella
opening test. The fixed parameters are the “Group” (“CB”=crib-biters and “C”=controls),
the interaction term “*” between the filament forces “Force” and Group CB-C (Test 2), the
interaction term between sound treatment “Sound” and Group CB-C and between the
order of the sound “Order” and Group CB-C (Test 4). The other parameters are control
parameters: the sex, age, and farm where the horses were housed (Table 1), whether the
arena where the horses were tested was situated outside or indoor “Arena”, Force, Order,
and Sound (Table 2).
For the LMMs and GLMMs, a standard model simplification
procedure was used to remove each non-significant term until
the deletion caused a reduction in goodness of fit (in this case,
the term was left in the model). P-values were calculated based
on Satterthwaite’s approximations (anova function, lmerTest
package in R). The significance level was set at α=0.05. Only the
results of the fixed factors are described in details in the results.
RESULTS
Passive Presence Test
There were no differences between groups CB and C in their
time spent interacting with the person (“Conth”), standing
attentive (“Att”), standing while resting (“Sr”) and standing while
exploring the ground (“Sexpl”) (LMM: effect of Group CB-C
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Briefer Freymond et al. Elevated Sensitivity in Crib-Biters
FIGURE 3 | Responses to the different filaments von Frey. Proportion of
controls (C, white, N=18) and crib-biters (CB, gray, N=19), respectively, that
responded to each Filament von Frey (300, 0.6, 0.02, 0.008, 1 gr.).
on Conth, X2
1=1.71, p=0.19; effect of Group CB-C on Att,
X2
1=1.66, p=0.20; GLMM: effect of Group CB-C on Sr, X2
1=
0.26, p =0.87; effect of Group CB-C on Sexpl, X2
1=0.27, p
=0.60). There were also no group differences neither in the
time spent walking active (“Movact”), nor in the time spent close
(“CAT1”) or far (“CAT2”) from the unknown person (LMM:
effect of Group CB-C on Movact, X2
1=0.11, p=0.74; LMM:
effect of Group CB-C on CAT1, X2
1=0.89, p=0.35; LMM: effect
of Group CB-C on CAT2, X2
1=0.89, p=0.35).
Tactile Sensitivity Test
A greater proportion of crib-biters reacted to the filament von
Frey (“React”) than the control horses (GLMM: effect of Group
CB-C on React, X2
1=8.14, p=0.004, Figure 3).
Novel Object Test
There were no differences between groups CB and C in their
time spent interacting with the unknown object (“Conto”),
standing attentive (“Att”), and standing while exploring the
ground (“Sexpl”) (GLMM: effect of Group CB-C on Conto, X2
1
=0.30, p=0.58; effect of Group CB-C on Att, X2
1=0.67, p
=0.41; LMM: effect of Group CB-C on Sexpl, X2
1=0.67, p
=0.41). There were also no group differences, neither in their
time spent walking active (“Movact”), in walking while exploring
(“Movexpl”), nor very close (“CAT1”) or very far (“CAT2”) from
the object (LMM: effect of Group CB-C on Movact, X2
1=0.49, p
=0.48; GLMM: effect of Group CB-C on Movexpl, X2
1=0.41, p
=0.52; effect of Group CB-C on CAT 1, X2
1=0.005, p=0.94;
effect of Group CB-C on CAT 2, X2
1=0.02, p=0.89).
Playback Test
There were no differences between groups CB and C in their vocal
responses to the playbacks (“Whin”) and in the time they spent
standing attentive (“Att”) (GLMM: effect of the Group CB-C on
Whin, X2
1=2.77, p=0.10; effect of the Group CB-C on Att, X2
1
=0.016, p=0.73). There was also no group effect on the time
spent walking active (“Movact”) during this test (GLMM: effect
of the Group CB-C on Movact, X2
1=2.06, p=0.15).
Locomotor Activity
There were no differences between groups CB and C in the
number of sectors they entered, which reflects locomotor activity
(“Locom”) (GLMM: effect of the group CB-C on Locom,
X2
1=1.74, p=0.18).
Umbrella Test
There were no differences between groups CB and C in their
time taken to resume eating in the bucket after the umbrella
was suddenly opened (“Time”) (LMM: effect of the Group
CB-C on Time, X2
1=0.69, p=0.41). There were also no
differences between groups in the intensity of their reaction
after the umbrella was opened (“Reacum”) (CLMM: effect of
the Group CB-C on Reacum, X2
1=0.366, p=0.55). Finally,
there was no group effect on the flight distance (“Flight”)
(CLMM: effect of the Group CB-C on Flight, X2
1=1.065,
p=0.30).
Control Factors
The type of arena (indoor or outdoor, “Arena”) had a significant
effect on Reacum in the umbrella test, (CLMM: effect of Arena on
Reacum, X2
1=4.2, p=0.04), and on React in the tactile sensitivity
test (GLMM: effect of Arena on React, X2
1=5.36, p=0.02). The
age of the horses (“Age”) had a significant effect in the passive
presence test on CAT1 and CAT2 (GLMM: effect of Age on the
CAT1, X2
1=6.09, p=0.01; effect of Age on CAT2, X2
1=6.09,
p=0.01) and on Att (LMM: effect of Age on Att, X2
1=9.15,
p=0.002). In the Novel object test, Age had an effect on CAT2
(GLMM: effect of Age on CAT2, X2
1=9.90, p=0.002). The sex
of the horses (“Sex”) had a significant effect in the playback test
on Movact and on Att (GLMM: effect of Sex on Movact, X2
2=
6.20, p=0.05; GLMM: effect of Sex on Att, X2
2=5.99, p=
0.05). The strength of the filaments (“Force”) had an effect in the
tactile sensitivity test on React (GLMM: effect of Force on React,
X2
4=13.78, p=0.008). The order in which the sound treatments
were played to the horses in the playback test (“Order”) had an
effect on Movact (GLMM: effect of Order on Movact, X2
1=
6.76, p=0.009). The control factors not mentioned above were
not significant and were thus removed from the models during
model selection.
DISCUSSION
Environmental causes responsible for the development of
stereotypies are partially known (80), but little is known
about why some individuals develop stereotypies and others
do not, despite being exposed to the same environmental
conditions. Here, we tested the hypothesis that predispositions to
stereotypies might be linked to individual differences in behavior
(“personalities”), which are in part genetically determined. To
this aim, we compared how stereotypic and non-stereotypic
horses (controls), responded to a standardized test battery
commonly used to assess individual differences in horses (60–
63). Based on previous findings (26,28,56,64,65,68,69), we
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Briefer Freymond et al. Elevated Sensitivity in Crib-Biters
expected crib-biters to show behavioral characteristics of reactive
coping individuals, namely to be less anxious (e.g., fearful), to
interact less with unfamiliar humans, to be less active, to show
less exploratory behaviors, and to be more social compared to
control horses. However, contrary to our expectations, we did
not find any differences in these traits between stereotypic and
control horses. Since reactive coping strategies are characterized
by freeze responses and unresponsiveness, it might be more
difficult to detect fear in these animals (81). Surprisingly,
however, we found that a greater proportion of crib-biters
reacted to the tactile filaments compared with control horses,
suggesting a higher tactile sensitivity in crib-biters, which to
our knowledge has never been reported before. We suggest that
this higher tactile sensitivity could be due to altered dopamine
or endogenous opioid physiology, resulting from chronic stress
exposition. We conclude that it might be valuable to conduct
further investigations to assess the personality of stereotypic
horses, as it could help to identify genetic loci associated
with stereotypies.
Reactivity to Humans
We did not find any difference between crib-biters and
controls in their propensity to react to a passive human.
Because this trait has been previously related to boldness, a
characteristic of proactive individuals (64,65), we expected
crib-biters, as potential reactive individuals, to interact less
with unfamiliar humans. Since how animals react to humans
is known to be heavily influenced by the environment (e.g.,
previous human handling) (82), genetic predisposition acting
on this trait might be difficult to detect. We thus suggest that
environmental components could have influenced previously
existing differences between stereotyped and control horses in
our study.
Tactile Sensitivity
In our tactile sensitivity test, a greater percentage of crib-biters
reacted to the tactile filaments compared with the control horses.
This suggests that crib-biters might be highly sensitive to tactile
stimulation. Tactile stimuli stimulate skin receptors also called
mechanoreceptors. This information is then transmitted via
the spinal cord to the thalamus and on to cortical sensory
areas. Tactile information is mapped onto the primary and
secondary somatosensory cortex. This cortex shows a somatotopic
organization, with the most sensitive parts of the body occupying
the most cortical territory (83). Difference in sensitivity to
tactile stimuli has been reported in some human developmental
disorders, such as autism (83). For instance, autistic people with
Asperger syndrome are often described as being easily disturbed
by their environment because they perceive external stimuli with
higher intensities than other people. Some studies also report
hypersensitivity to senses, such as touch, smell, and taste in these
people (84). Existing theories suggest that this hypersensitivity is
due to enhanced processing of stimuli details in the secondary
somatosensory cortex, or impairment of top-down modulation
of incoming stimuli (85,86). In other mood disorders such
as “depression.” on the other hand, “unresponsiveness” to
environmental stimuli (tactile or visual) have been reported
in both human (67) and animals [monkeys (87), horses (68)].
Tactile sensitivity might therefore be an important indicator of
developmental or mood disorders.
A distinction can be made between sensory processing and
sensory sensitivity, since individuals can perceive stimuli and not
respond to them. Therefore, the hypersensitivity that we observed
in crib-biters could be explained firstly by the fact that some
horses might feel a tactile stimulation without responding to
it. We could hence suggest that control horses might have felt
the stimulation, while being less disturbed by it than crib-biters.
On the other hand, crib-biters, because of their higher stress
sensitivity reported in our previous study (28), might be easily
irritated by tactile stimuli and may took a longer time to habituate
to them than the controls, as suggested for hypersensitive people
(88). It would be interesting to conduct further experiments
testing the sensitivity of crib-biters within other senses [e.g.,
gustato-olfactory, auditory and visual sensitivity; (62)].
The hypersensitivity found in crib-biters could otherwise
be explained by neural differences between these horses and
controls. We could suggest that exposition to chronic stress
may cause alteration of dopaminergic systems, not only in the
mesoaccumbens dopamine system as reported in stereotypic
animals (2,89), but also in dopaminergic nerve cells implicated in
sensory sensitivity (90). Therefore, the dopaminergic modulation
impairment that crib-biters potentially suffer from could also be
implicated in their sensory hypersensitivity (91). We could hence
suggest that the hypersensitivity that we found in crib-biters is
explained by differences between stereotypic and non-stereotypic
horses in their neural processing of tactile stimuli or in dopamine
modulation (5,57).
An object pressed against the skin can produce various
kinds of perception, such as “pain,” “tickle,” or “touch” (72).
Similarly, the application of different forces on the skin using von
Frey filaments could produce different sensations. Although the
exact sensation produced by these filaments remains unknown
(71), von Frey filaments are considered as a good method
for assessing nociceptive thresholds [in rats (92), in horses
(93,94)]. Differences in β-endorphin physiology has been
reported to be implicated in the causal and/ or functional
aspect of stereotypic behaviors. Indeed, administration of µ
opioid receptor antagonists to different species (dogs, pigs,
cats, chickens, horses, and bank voles) has been shown to
reduce the performance of stereotypies (4,95,96), Even if
measurements of plasma βendorphin in crib-biting horses
has produced conflicting results (69,97,98), a recent study
aimed at reassessing opioid physiology in these horses found
an upregulation of µopioid receptors in some part of the
mesoaccumbens pathway (99). Because endorphins play a role in
assessing pain and analgesia, we could hence suppose that the
hypersensitivity we found in crib-biters is related to differences
in endorphin modulation between the two groups. In alignment
with this hypothesis, crib-biting behavior has previously been
associated with a decrease in nociceptive (thermal) threshold
during crib-biting periods (69). It would be interesting in future
studies to investigate opioid receptor sensitivity in stereotypic
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Briefer Freymond et al. Elevated Sensitivity in Crib-Biters
and normal horses. To summarize, we could thus suggest that
the hypersensitivity that we found in crib-biters is related to
differences between stereotypic and non-stereotypic horses in
their neural processing of pain.
Social Reactivity
We did not find any difference between crib-biters and controls
in their social reactivity, suggesting that crib-biters are not more
aroused than control horses when hearing unknown horses,
according to our hypothesis. If crib-biters indeed display a
reactive coping strategy, these results might contradict findings
in pigs, which showed that reactive pigs could be more
social (66).
Locomotor Activity
The data used to score the “locomotor activity” in the passive
human test, the social motivation test, and the novel object test
did not reveal any difference between crib-biters and controls
in this trait. According to previous studies, we expected crib-
biters, if they indeed behave as reactive individuals, to show
less locomotor behavior than control horses (26,64). On the
other hand, in stereotypic animals of other species [e.g., mice
(25,100) and rhesus macaques (22)], higher incidence of
stereotypy development have been associated with more activity.
Yet, discrepancies between these studies and ours could be
explained by differences in the type of stereotypy displayed
(locomotor stereotypies vs. oral stereotypy in our case), by species
or experimental protocol differences. Indeed, the measured
phenotype of individuals will depend on the initial definition
and use of each trait and on the terminology used to define
personality, which varies widely between studies (14,64).
Curiosity/Fearfulness
According to our previous results showing that crib-biters
display physiological characteristics of reactive individuals, i.e.,
high HPA-axis reactivity (28) and to the results of Nagy et al.
(56), we expected stereotypic horses to also display behavioral
characteristics of reactive individuals, such as being less fearful
(or anxious) than control horses. However, we did not find any
difference between our two groups in their fear reaction to the
sudden opening of the umbrella. Previous studies showed that
crib-biters seem to be less reactive while restrained with a lip-
twitch, but to react more strongly to a rapidly inflating balloon
compared to non-stereotypic horses (101). Our results did not
confirm these results. Discrepancies between these studies and
ours could be explained, once more, by experimental protocol
differences (102).
We could also suggest that there might exist a difference in
fearfulness between crib-biters and non-stereotypic horses, but
that the behavioral indicators that are generally used to assess
fearfulness are not appropriate to detect such differences between
reactive and proactive individuals. Indeed, behavioral reactions
to fear-induced reactions might be less strongly expressed (e.g.,
characterized by freeze responses and unresponsiveness) in those
individuals compared to proactive ones (26,81). Therefore, if
crib-biters are really more reactive than other horses, it is possible
that, despite a stronger physiological reaction to the opening of
the umbrella, their behavior did not change (81). It would thus be
useful, in further studies investigating differences in fearfulness
between proactive and reactive animals, to measure other types
of fear indicators [e.g., physiological responses, Equine Facial
Action Coding Systems (FACS)] (103) in addition, in order to
increase the accuracy of fear assessments.
In the same way as for the reaction to the opening of the
umbrella, we did not find any difference in reaction toward the
novel object between crib-biters and controls. Links between
reactions to novel objects and stereotypies have also been tested
in species other than horses. Unlike in our study, stereotypic mice
show greater reactivity, quicker time to approach novel objects
and increased object manipulation, suggesting less fearfulness
or higher levels of curiosity compared to control mice (25).
Similarly, rhesus macaques that express a higher rate of motor
stereotypic behavior in captivity are characterized by more
frequent contacts with a novel object, indicating higher levels
of curiosity than other monkeys (22). Differences between these
studies and ours might be due to the type of stereotypies
investigated in mice and rhesus macaques, which was, unlike
in ours, locomotor (22,25). We suggest that some similarities
with these studies could be found in weaving more than crib-
biting horses.
CONCLUSION
Our results suggest that crib-biters are more sensitive to tactile
stimulation than non-stereotypic horses. This suggest that this
higher tactile sensitivity could be also one of the underlying
causes of their higher stress sensitivity (28), which might result
in the development of stereotypic behavior in these individuals.
We also suggested that this higher tactile sensitivity could be
due to altered dopamine or endogenous opioids physiology,
resulting from chronic stress exposition. On the other hand,
we did not find any personality traits that are characteristic of
reactive coping individuals in crib-biters, as we had expected
(i.e., to be less fearful, to interact less with unfamiliar human,
to be less active, to show less exploratory behaviors and to
be more social). We suggest that further studies investigating
differences in fearfulness between proactive and reactive animals,
which in our case were expected to reflect differences between
control and stereotyped horses, should include further behavioral
and particularly physiological measures. Indeed, this might to
help detect differences between proactive and reactive coping
strategies, since fear-induced behavioral reactions might be less
strongly expressed in reactive individuals compared to proactive
ones (26), as recently suggested by Squibb et al. (81). We conclude
that further investigations are required to fully characterize the
personality of stereotypic horses. This could allow an early
detection of individuals prone to develop stereotypies, and hence
might help to prevent them to develop this abnormal behavior.
ETHICS STATEMENT
This study was carried out in accordance with the
recommendations of the Swiss Federal Veterinary Office.
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Briefer Freymond et al. Elevated Sensitivity in Crib-Biters
The protocol was approved by the Swiss Federal Veterinary
Office (approval number VD 2677 bis; Switzerland).
AUTHOR CONTRIBUTIONS
SBF and SB carried out the experiment. DB scored the videos.
SBF scored the videos, wrote the manuscript and performed the
statistical analyses. EB, KZ, and IB participated to design an
edited the manuscript. EB supervised the project.
ACKNOWLEDGMENTS
We are grateful to all the owners of the horses, who offered to
participate in this study.
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Conflict of Interest Statement: The authors declare that the research was
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