https://doi.org/10.52973/rcfcv-e34493
Received: 18/07/2024 Accepted: 10/09/2024 Published: 16/12/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34493
ABSTRACT
Stress response of the horse may be related to the behavioral and
physiologic factors e.g., stress hormones. The aim of this study
was to evaluate the different rider contact on the stress hormones
of the horses that are used for javelin sport in a local riding club.
Seven Arabian horses were ridden by familiar riders with the horses
in the rst and second weeks and by additional unfamiliar riders
with the horses in the third and fourth weeks. Cortisol, oxytocin and
beta–endorphin levels in sera samples and cortisol levels in saliva
were measured before and after the riding. There was no statistical
difference in serum cortisol and β–endorphin and salivary cortisol
values between the groups with regard to the familiarity (P>0.05).
Behavioral scoring did not differ between the groups. However, there
was an increase in the oxytocin level of the horses ridden by the
familiar riders in the second week compared with the rst week
(P<0.05). The horses did not respond stressfully associated with the
hormone levels and behavioral changes; besides, they responded well
to the familiar riders by elevating the oxytocin level. In conclusion,
the horses used herein responded well to the familiar and unfamiliar
riders with regard to potential stress factors. It has been therefore
suggested that the familiarity towards the interaction between the
human and the horse may not alter the physiological stress of the
horses that are regularly ridden by various people in a riding club.
Key words: Horse; β–endorphin; cortisol; oxytocin; stress
RESUMEN
La respuesta al estrés del caballo puede estar relacionada con factores
siológicos y de comportamiento, por ejemplo, las hormonas del
estrés. El objetivo de este estudio fue evaluar los diferentes contactos
de los jinetes sobre las hormonas del estrés de los caballos que se
utilizan para el deporte de jabalina en un club de equitación local. Siete
caballos árabes fueron montados por jinetes familiarizados con los
caballos en la primera y segunda semanas y por jinetes desconocidos
adicionales con los caballos en la tercera y cuarta semanas. Se
midieron los niveles de cortisol, oxitocina y betaendorna en muestras
de suero y los niveles de cortisol en saliva antes y después de montar.
No hubo diferencias estadísticas en los valores de cortisol sérico
y β–endorna y cortisol salival entre los grupos con respecto a la
familiaridad (P>0,05). La puntuación conductual no dirió entre los
grupos. Sin embargo, hubo un aumento en el nivel de oxitocina de
los caballos montados por jinetes familiares en la segunda semana
en comparación con la primera semana (P<0,05). Los caballos no
respondieron al estrés asociado con los niveles hormonales y los
cambios de comportamiento; Además, respondieron bien a los
jinetes familiares elevando el nivel de oxitocina. En conclusión, los
caballos utilizados aquí respondieron bien a los jinetes familiares y
desconocidos con respecto a los posibles factores de estrés. Por lo
tanto, se ha sugerido que la familiaridad con la interacción entre el
ser humano y el caballo no puede alterar el estrés siológico de los
caballos que regularmente son montados por varias personas en un
club de equitación.
Palabras clave: Caballo; β–endorna; cortisol; oxitocina; estrés
Effect of familiar and unfamiliar riders on Cortisol, Oxytocin and
Beta–endorphin levels in horses
Efecto de los jinetes familiares y no familiares sobre los niveles
de Cortisol, Oxitocina y beta–endorna en caballos
Nergis Ulas
1
* , Omer Aydin
1
, Sumeyye Baysal
1
, Mustafa Ileriturk
2
, Omer Eltas
3
1
Atatürk University, Faculty of Veterinary Medicine, Department of Internal Medicine. Erzurum, Türkiye.
2
Atatürk University, Horasan Vocational Collage, Department of Animal Science. Erzurum, Türkiye.
3
Atatürk University, Faculty of Veterinary Medicine, Department of Zootechny. Erzurum, Türkiye.
*Corresponding author: nergisulas@atauni.edu.tr
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INTRODUCTION
Since ancient times, the horse (Equus caballus) has been the closest
animal to humans with many activities such as transport, chevalier
and sports. Equestrian sport has been adopted as an ancestral
sport in modern culture including physiotherapy and psychotherapy
associated with the horse and human interaction. Currently, horses
are used in the elds of racing, riding, and therapy (hippotherapy)
as well as equestrian sports (javelin, etc.). Emotional or physical
interaction depends on the relationship between the horse and the
human. Although there are previous reports about the value of the
horse for human health [1], there is limited information on the stress
response and well–being of the horse. With the increased number of
horses Equine–Assisted Activities and Therapies (EAAT) programs
and the growing concern for animal welfare, it has become important
to understand the effects of these programs on the stress levels,
stress–related disorders, and quality of horse life. Also, stress in
the horse can negatively affect the horse–rider couple and increase
the risk of accidents. The measurement of pain and stress level as a
result of stress exposure depends directly on observations with regard
to behavioral and physiologic changes including circulating stress
hormones. Behavioral and physiological indicators for assessing
the emotional state and well–being of the horse should include both
positive and negative outcomes [2]. Behavioral scoring is an objective,
non–invasive and easy to assess for welfare in animals [3, 4].
Stress can be dened as a condition in which an animal “must make
abnormal or extreme adjustments to its physiology or behavior to cope
with the negative aspects of its environment and management” [5].
Responses to stressful stimuli involve behavioral changes, decreased
immunity, and activation of the Hypothalamic–Pituitary–Adrenal (HPA)
axis and Autonomic Nervous System [6, 7]. Adrenocorticotropic
hormone (ACTH) is released into the systemic circulation from
the pituitary gland following the activation of HPA axis through
hypothalamic integration induced by signals from the peripheral
and central nervous system (CNS). Thus, ACTH further stimulates
the synthesis and secretion of stress hormone cortisol from the
adrenal gland [8]. Stress may cause daily changes in heart rates and
endocrinological changes such as plasma cortisol, beta–endorphins,
and catecholamines in racing or training horses [5] as well as in other
animals under stress with metabolic adaptation [9] and cardiotoxicity
[10]. Corticotropic hormones, serotonin and catecholamines are of the
stress hormones in order to assess the level of response in the horse
[11, 12]. Adrenal gland secretes catecholamines and glucocorticoid
cortisol hormone after exposure of physical and/or psychological
stressor. When stress is evaluated, it is more useful and appropriate
to measure free cortisol rather than total cortisol in serum [13]. Since
cortisol diffuses rapidly into saliva, salivary cortisol concentrations
reect changes in cortisol concentrations in blood plasma. Thus,
salivary cortisol concentrations are used as an index of serum–free
cortisol [14]. Blood cortisol secretion follows a circadian rhythm with
the highest concentrations in the morning and lowest in the afternoon
and evening [15, 16] in horses, as in humans, and other species [17].
Therefore, samples were taken at the same time of the day.
The activation of oxytocin neurons causes an increase in oxytocin
secretion not only after reproductive stimuli but also after stressful
stimuli [18]. Oxytocin can further modulate response of the body against
stressful fear, panic, and extreme exercise. Besides, oxytocin is released
during pleasant social activities and has antistress or antidepressant
effects by reducing cortisol due to an increased parasympathetic
function [19]. Previous studies in human–beings indicate that animal
interaction stimulates the production of circulating oxytocin resulting
in a calming effect and relaxation [20, 21].
Beta–endorphins, opiate–like peptides of pituitary origin, play roles
in learning and memory, feeding behavior, thermoregulation, blood
pressure regulation, and reproductive behavior [11, 21]. Beta endorphins
can also modulate excitability of CNS by activating motor function and
pain perception during exercise–induced catecholamine secretion and
lactic acid accumulation [5]. The release of beta–endorphins into the
blood is particularly evident in horses during stress reactions [22].
In the present study, behavioral scoring and the levels of cortisol,
serum oxytocin and beta–endorphin levels in saliva and serum were
investigated in the horses before and after riding to compare the weekly
stress response in between the riders who were familiar with the horses
and the additional riders who were not familiar with the horses.
MATERIALS AND METHODS
Animals
This study was approved by the Local Ethics Committee for Animal
Experiments of Ataturk University (Protocol No.: 2020/170). The
horses used in this study, with permission from their owners, were
from a local private equestrian sports club where they were kept for
sporting activities (e.g., javelin riding). The horse and human interaction
during the sportive activity has been illustrated in FIG.1. The study was
conducted in 7 clinically healthy intact male 4–16–year–old Arabian
horses. The weight of the horses ranged from 400 to 500 kg. All horses
were fed with a standard horse ration in the same housing conditions.
Clinical health status of the horses was evaluated by anamnesis and
full clinical examinations of respiratory system, digestive system,
circulatory system, lymph nodes, mucous membranes, secretion, and
excretion ndings and musculo–skeletal motor functions. Complete
blood count was also performed to evaluate the hematological status of
the horses. The horses that represent normal clinical and hematological
ndings were included in the study.
Behavioral assessment
The behavioral scoring system was selected based on the previous
reports [3, 23, 24]. Parameters were determined as 1: no stress, 2:
low stress, 3: medium stress and 4: severe stress, depending on
the horse’s general attitude, neck position, tail movements, ear and
mouth position. On the days of the study, each horse was observed
for 15 min during the preparation for riding, contact with the rider and
going for a ride. After the ride, the horses were also observed for 15
min when they entered the barn.
Study design
All tests were carried out in the equestrian area where the horses
are routinely housed at all times. The method of this study was a
measurement design repeated at weekly intervals over four weeks
on horses ridden at a slow walk by licensed riders on sampling days.
The horse’s gait is shown in FIG 2. On the remaining d of the week, the
horses were allowed to continue their daily routine activities. On the
sampling days in the rst and second weeks, the horses were ridden
for one–hour by two riders who were familiar with the horses, i.e., the
people who had often ridden these horses. On the sampling days in
the third and fourth weeks, the same horses were ridden for one–hour
by two additional riders who were not familiar with the horses, i.e.,
the people who had never ridden these horses.
FIGURE 1. Illustrations for the horse and human interaction during javelin sport
(A) and sportive activity (B). The illustrations have been provided here courtesy
of the rider, the owner and the riding club
FIGURE 2. The horse’s gait with the familiar rider
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Sample Collection
Samples were taken gently by an experienced veterinarian without
stressing the horse. To evaluate hematological status of the horses
before the study initiation, blood was drawn using 18G needle from
jugular vein into spray–coated vacutainers with anti–coagulant K2EDTA
(Becton Dickinson Co., USA). Complete blood count was performed in a
veterinary hematology analyzer (Abacus Junior Vet5, Diatron, Hungary)
at the beginning of the study to check the health status of the animals.
Sampling procedure of the repeated measurement was performed
before and after the riding of all horses on the days of sampling weekly
for four weeks. Weekly blood and saliva samples were obtained from
the horses before contacting the riders (pre–riding) and just after the
riding (post–riding) at the time period of the d (15:00 to 16:00) to account
for the same daily rhythm of hormone levels [25]. The saliva samples
were taken before blood sampling to refrain from possible effects
of blood drawing. Saliva was sampled by using Salivettes (Sarstedt,
Nümbrecht, Germany) without restraining the horse. A swab was held
inside the mouth of the horse above and below the tongue for one
minute with a metal clamp and inserted into the Salivates. The samples
were immediately stored at +4°C during the activity and taken to the
laboratory at the end of each study. The saliva samples were centrifuged
at 1,500 G for 10 min and saliva was aspirated then placed in godets
and stored at -20°C (Vestel, SC47011, Türkiye) until analysis [25, 26].
Blood was also drawn from jugular vein on the days of sampling (pre–
riding and post–riding) into spray–coated silica vacutainers containing
polymer gel without anti–coagulant (Becton Dickinson Co., USA) for
hormone analysis to obtain serum samples weekly for four weeks after
the collection of the saliva. Following coagulation, the blood samples
was centrifuged at 4,000 G for 10 min (Beckman Coulter, Allegra X–30R,
USA) and sera samples were allocated into sterile tubes and stored at
-80°C until analysis.
Cortisol, β–endorphin and oxytocin analysis
Saliva and serum cortisol (Horse cortisol, BTLaB, China), serum
β–endorphin (Horse Beta–Endorphin, BTLaB, China), and serum
oxytocin (Horse Oxytocine, Sunred, China) hormone levels were
measured by using horse–specic ELISA test kits according to the
manufacturers’ instructions.
Statistical analysis
Data comparison was made in a model to evaluate both effects of
riding and period on hormone levels. The data showed normal distribution
in the Shapiro–Wilk test. The Paired–samples T test and repeated
measures design were used to compare the hormone levels in the
SPSS 18 package software program for statistical analysis. Chi Square
analysis was used to compare behavioral scoring between groups.
RESULTS AND DISCUSSION
The owners of the horses no complaint for the anamnesis of
the horses. There were no abnormalities in physical examination
findings with regard to full clinical examination of respiratory
system, digestive system, circulatory system, lymph nodes, mucous
membranes, secretion, and excretion ndings and musculo–skeletal
motor functions. Erythrocyte, leukocyte and platelet parameters were
within normal ranges related with complete blood count analysis of
the blood samples. FIG. 3 shows the blood serum level of oxytocin (A),
cortisol (B) and β–endorphin (C) and saliva level of cortisol (D) at the
pre–riding and post–riding time points. The levels of serum oxytocin
Week 1 Week 2 Week 3 Week 4
Pre-Riding Post-Riding Pre-Riding Post-Riding Pre-Riding Post-Riding Pre-Riding Post-Riding
Familiar Riders Unfamiliar Riders
0
5
10
15
Oxytocin (pg·mL
-1
)
Pre-Riding Post-Riding
a
a
b
b
ab
ab
a
a
Week 1 Week 2 Week 3 Week 4
Pre-Riding Post-Riding Pre-Riding Post-Riding Pre-Riding Post-Riding Pre-Riding Post-Riding
Familiar Riders Unfamiliar Riders
0
10
20
30
40
β-Endorphin (ng·L
-1
)
Pre-Riding Post-Riding
Week 1 Week 2 Week 3 Week 4
Pre-Riding Post-Riding Pre-Riding Post-Riding Pre-Riding Post-Riding Pre-Riding Post-Riding
Familiar Riders Unfamiliar Riders
0
20
40
60
80
Cortisol (ng·L
-1
)
Pre-Riding Post-Riding
Week 1 Week 2 Week 3 Week 4
Pre-Riding Post-Riding Pre-Riding Post-Riding Pre-Riding Post-Riding Pre-Riding Post-Riding
Familiar Riders Unfamiliar Riders
0
50
100
150
Cortisol (ng·L
-1
)
Pre-Riding Post-Riding
A B
C D
FIGURE 3. The pre–riding and post–riding levels of oxytocin (A), cortisol (B) and β–endorphin (C) in blood serum and levels of cortisol in saliva (D) of the horses (n=7)
for the days with a one–week interval. Note that oxytocin levels are higher on the day of the second week than on the day of the rst week in the horses ridden by
familiar riders (P<0.05). Dierent letters in the same graph indicate a statistical dierence (P<0.05) and no letters in the same graph indicate no statistical dierence
(P>0.05) between mean levels. The data are presented as mean ± standard deviation
Effects of familiarity on stress in horses / Ulas et al. ________________________________________________________________________________
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hormone levels increased on the day in the second week at pre–riding
and post–riding time points compared with the levels on the day in the
rst week (P<0.05) in the horses ridden by familiar riders. The increased
oxytocin levels were gradually decreased on the fourth week (P<0.05)
in the horses that were ridden by unfamiliar riders. There were no
statistical changes for serum β–endorphin, serum cortisol, and salivary
cortisol levels during the study (P>0.05) in the horses that were ridden
by familiar riders for two weeks and by unfamiliar riders for additional
two weeks. It was also noted that there was a tendency to decrease
in cortisol level in saliva samples between pre–riding and post riding
periods on the day in the rst week (P=0.096).
The mean values of behavioral scoring were 1.6 ± 0.12 in the familiar
rider group and 1.7 ± 0.18 in the unfamiliar rider group. There was no
statistical difference in between behavioral stress scoring of the
riding groups (P>0.05).
The stress response is dened by physiological changes to short
– or long–term unpredictable changes in environmental conditions
that cause a redirection of resources to vital processes and disrupt
(or threaten to disrupt) homeostasis [27]. Stress response varies
according to the stimulus causing special consequences. Responses
to stimuli (e.g., stress) include behavioral and hormonal changes [28].
Beta–endorphin [29], ACTH, cortisol, total and free iodothyronines
[11], or reproductive hormones and oxytocin are used to evaluate
the response to the stress [19]. The horse should be refrained from
the stress to perform better in the equestrian sport or not to harm
the riders in EAAT studies. Stress may also affect the performance
of the horse in javelin sport or any other competitions depending
on the physiological and hormonal mechanisms. Thus, the results
of this study evaluated the effect of the different rider factor on
stress hormone levels and behavioral changes in the horses that are
regularly ridden by various people in an equestrian club.
The measurement of pain and stress level as a result of stress
exposure depends directly on observations with regard to behavioral
and physiologic changes including circulating stress hormones in
animals [2]. The previous assessments are most likely to have some
misinterpretation due to an over – or under–measurement of the pain
and stress [8]. Behavioral scoring is an objective, non–invasive and
easy to assess for welfare in animals [3, 30]. In this study, there was
no statistical difference in between behavioral stress scoring of the
riding groups. Potier and Louzier [24] investigated the stress levels
of eight healthy horses during hippotherapy in two riding sessions
on separate days, one with disabled riders and one with beginners.
They found no signicant difference between stress scores as seen
in this study. Hovey et al. [23] studied differences in behavior and
serum cortisol concentrations in horses used in a therapeutic riding
program. They reported that overall behaviour scores were relatively
low in the horses in the two groups included in the study similarly.
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In animal welfare studies, glucocorticoids are used widely as an
animal–based welfare evaluation [19]. Cortisol measurement is an
indicator of Hypothalamus–Pituitary–Adrenocortical Axis (HPA) activity,
which reects the physiological response to acute or prolonged stress
[31, 32]. The release of cortisol is stimulated by the sympathetic
nervous system, and cortisol maintains the homeostasis of biochemical
processes by mobilizing energy during physical and psychological
stress [33]. In horses, as in humans and other species, blood cortisol
secretion is in a circadian rhythm, with the highest concentrations in
the morning and lowest in the afternoon and evening [15]. The samples
were therefore taken at the same time period in the afternoon to ensure
uniformity and to prevent hormonal uctuations in this study.
Serum cortisol concentration is used to evaluate the type of stress
level in horses [14] associated with transport [34, 35, 36], rival [33],
training and exercises [37, 38]. The serum cortisol level mainly
increases during acute stress associated with the free cortisol level.
Free cortisol measurement is more useful to evaluate the stress rather
than total cortisol in serum [13, 32]. Salivary cortisol values have been
shown to correspond to serum cortisol levels, and many studies to
assess the acute stress response in horses measure salivary cortisol
concentration [25]. Salivary cortisol, by the passive diffusion to
the salivary glands, provides information about the free cortisol
concentration [14]. It is widely used as a biomarker in mental, physical
diseases or psychological stress [13].
Previous studies reported the association of blood and saliva
cortisol concentrations suggesting the saliva samples can be used
as a non–invasive technique for cortisol level in the horse [14]. Salivary
cortisol concentrations have been previously measured in horses
during therapeutic riding, conventional riding, and resting conditions,
and no signicant difference has been detected in delta cortisol
values between riding conditions [39]. Smith et al. [40] measured
blood cortisol values before and after transport in horses and
reported that cortisol values increase after transport. Horses used
in therapeutic riding have presented lower cortisol concentrations
5 and 30 min after the session, and the therapeutic riding session
had minor effects on HPA response [41].
Arfuso et al. [42], investigated the effects of environmental
temperature and work on cortisol in tourism carriage horses in
Italy. They emphasized that there was no change in cortisol levels
because the horses were used to the work they were doing. Cortisol
has been evaluated as acute stress in this study. Similar to the study
by Arfuso et al. [42] blood and saliva cortisol values did not change
in between different rider groups before and after one–hour riding
period suggesting that the horses regularly used for riding purposes
with various people adopted themselves well to the stress factors
arise from the rider contact in an equestrian sport facility.
Emotional contagion is also thought to occur between animals
and humans, and horses are also thought to be sensitive to human
emotions [43]. The activation of oxytocin neurons causes increased
oxytocin secretion not only after reproductive stimuli but also after
various stressful stimuli [18]. Recent studies have shown that oxytocin
levels are associated with positive human–animal interactions [44].
Oxytocin released by positive social interaction causes anti–stress
effects by reducing glucocorticoid stress hormones in humans and
animals and is associated with increased parasympathetic function
[19]. In a previous study, oxytocin level was found to be unchanged
in the horses after the exposure to audio–visual and unfamiliar eld
stimuli [45]. The change in oxytocin value is dicult to determine
due to the short half–life of oxytocin [19]. Indeed, plasma oxytocin
level increases in humans and dogs and plasma cortisol decreases
in humans with relational interaction [21].
In this study, oxytocin values have not been differed signicantly
except for the second week. Oxytocin values may not change with
constant contact with humans because riding horses may clear it
from the bloodstream within an hour of riding [19]. The increase
on the second week is considered to be a neurophysiological
consequence of relational behavior in the horse–human contact
with the familiar riders.
The release of beta–endorphins from the pituitary gland in horses
is synchronized with the rst stage of the stress reaction, reducing
the negative effects of cortisol on the organism [11, 21]. The release
of beta–endorphins into the blood is a particular evident during
stress reactions in the horse [22]. Potential physical and emotional
stress factors might occur commonly in horses with the elevations
of plasma beta–endorphin concentrations with severe abdominal
pain [46]. However, horses with painful but chronic lameness have
a plasma beta–endorphin concentration similar to that of normal
horses. The similarity of beta–endorphin values of the horses before
and after riding in the present study indicates the horses are not
physically or emotionally under stress. The similarities during riding
with familiar and unfamiliar riders may be related to the adapted
horses accustoming to the riders. The similar stress parameters
in present the horses might be the adaptation of the horses to the
potential stressful factors caused by different rider contact.
CONCLUSIONS
Stress hormone levels of serum and saliva cortisol and serum beta–
endorphin values in familiar and unfamiliar rider groups were found
similar in the horses that are regularly ridden by different people in
a javelin sport club conforming with the steady behavioral scoring.
The increase in the oxytocin values on the second week in the horses
ridden by the familiar riders could be related with the well response to
the riders indicating positive social interaction and a familiarity of the
people and the horses. Therefore, the riding the horses by familiar or
unfamiliar certied horse riders may not cause physiological stress
on the horses that are regularly ridden by different people for hobby
and sportive activities.
ACKNOWLEDGEMENTS
This work was supported by the Ataturk University Scientific
Research Unit under TSA–2021–8740. Additionally, we would like to
thank the special Uzmanlar Equestrian Sports Club and the riders
for their kind support.
Authors’ contributions
NU and OA contributed to the design and process of the study. NU,
OA and SB were including sample collection and processing materials,
and data analysis and presentation. All the authors made substantial
inputs in drafting and editing the nal manuscript. MI contributed to the
biochemical analysis of the samples and OE contributed to the statistical
analysis of the data. All authors read and approved the nal manuscript.
Conict of Interest
The authors declare that they have no conict of interest.
Effects of familiarity on stress in horses / Ulas et al. ________________________________________________________________________________
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