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Review

Role of Cortisol in Horse’s Welfare and Health

by
Urszula Sikorska
1,
Małgorzata Maśko
1,*,
Anna Ciesielska
1,
Łukasz Zdrojkowski
2,* and
Małgorzata Domino
2
1
Department of Animal Breeding, Institute of Animal Science, Warsaw University of Life Sciences (WULS–SGGW), 02-787 Warsaw, Poland
2
Department of Large Animal Diseases and Clinic, Institute of Veterinary Medicine, Warsaw University of Life Sciences (WULS–SGGW), 02-787 Warsaw, Poland
*
Authors to whom correspondence should be addressed.
Agriculture 2023, 13(12), 2219; https://doi.org/10.3390/agriculture13122219
Submission received: 19 October 2023 / Revised: 28 November 2023 / Accepted: 28 November 2023 / Published: 30 November 2023
(This article belongs to the Section Farm Animal Production)
Versions Notes

Abstract

:
This review describes the physiological role of cortisol in the horses’ body and the usefulness of cortisol concentration measurements in assessing welfare and diagnosing diseases. Cortisol is examined in terms of its production and functions, along with the concept of circadian cortisol rhythms and potential disruptions in these patterns. The significance of cortisol concentration is emphasized, as it provides insights into stress levels, and the connection between cortisol and stereotypical horse behaviors, raising the question of whether these behaviors signify stress or serve as coping mechanisms. Moreover, cortisol concentration helps in diagnosing various health conditions such as Cushing’s Disease, inflammation, and metabolic imbalances. As cortisol concentration is considered a stress indicator that may be affected by the sampling protocol, the matrices for cortisol sampling and their practical applications are summarized. In this review, three major cortisol sampling methods: plasma, salivary, and hair cortisol, are described in detail. Plasma cortisol measurements offer acute stress insights, while salivary cortisol analysis provides a non-invasive method for continuous stress monitoring. Hair cortisol, on the other hand, offers an assessment of long-term stress levels. This text underscores the importance of cortisol control in safeguarding the welfare and health of horses.

1. Introduction

Since domestication, horses have been subjected to a wide array of management techniques and have been utilized in various roles [1]. These practices encompass human handling, single housing in stalls, and controlled meal feeding, all which can induce stress in these animals, diverting them from their natural habitat [2]. That is why domestic horses during everyday life and use are exposed to many external stressors. Among the stressors that are commonly accepted and considered in everyday routine are those related to use, namely physical exertion, transportation, competitive events, and veterinary procedures [3,4,5,6]. Equally frequently considered stressors concern the housing and maintenance of horses and include social isolation [6], heat stress [7], and the effect of poor environmental conditions [8]. However, a group that is much less aware, but no less important, are stressors related to nutrition. One may observe that unbalanced diets and fattening conditions are considered potential stressor [9,10,11,12], which should be taken into account when assessing the welfare of horses and carrying out corrective actions to improve the well-being of these sensitive and social animals. Each of these stressors holds the potential to evoke emotional excitement, elevate heart rate, and cause the release of stress hormones [13,14,15,16,17]. Thus, stressed animals may exhibit atypical and aggressive behaviors [2,18,19,20], an array of health issues [2,18], heightened hormone secretion [18], or diminished performance [19]. Consequently, the assessment and regulation of stress are indispensable for ensuring the safety and welfare of horses [2].
The equine stress response serves as a vital supplementary homeostatic system, mobilizing a spectrum of neurological and hormonal pathways to enhance immune, cardiovascular, musculoskeletal, and metabolic processes when the body confronts disturbance or threat [21]. In response to stress, horses activate the hypothalamic-pituitary-adrenal (HPA) axis, initiating the release of a range of chemicals. These include mineralocorticoids such as aldosterone, glucocorticoids such as cortisol, and adrenal androgens such as dehydroepiandrosterone (DHEA), all of which belong to the class of steroid hormones known as corticosteroids. These corticosteroids play pivotal roles in various physiological processes, encompassing the regulation of fluid and electrolyte balance, maintenance of cardiovascular homeostasis, metabolism of carbohydrates, proteins, and lipids, regulation of immunological and inflammatory responses, and the facilitation of sexual maturation and reproductive functions [22].
The equine stress response, the HPA axis, regulates the body’s fundamental physiological functions [3,23]. The anterior pituitary gland responds to the secretion of corticotropin-releasing factor (CRF) by the hypothalamus by producing increased levels of adrenocorticotropic hormone (ACTH), also known as corticotropin. The ACTH circulates in the bloodstream, binding to the adrenal cortex, thereby raising the production and release of glucocorticoids. It is worth noting that any form of stress, whether physical, emotional, environmental, or feeding, can trigger the release of glucocorticoids in horses [24].
The primary glucocorticoid released by horses is cortisol [25,26]. Cortisol belongs to the group of steroid hormones, with a molecular weight of approximately 362.46 g/mol and a chemical formula of C21H30O5 [3,27]. Cortisol significantly influences the metabolism of carbohydrates, proteins, and lipids. It actively promotes gluconeogenesis, the synthesis of glucose from non-carbohydrate sources, thereby ensuring a consistent supply of energy substrates, particularly during periods of fasting or stress [28]. The cortisol concentration increases rapidly in circulating blood after stressor, and can be accurately detected in various collected material [29,30,31,32]. It is worth noting that blood [13,33,34,35,36,37,38,39] and partially salivary [13,25,32,39,40,41] cortisol samples reflect an acute stress, whereas salivary [41,42,43], hair [44,45,46,47,48,49] and feces [8,17,38,50,51,52,53] cortisol samples show a chronic stress. This is why cortisol is considered a biomarker for assessing both psychological and physiological stress, which can be used in the identification, treatment, and prevention of stress-related disorders.
Therefore, this review describes the physiological role of cortisol in the horses’ body and the usefulness of cortisol concentration measurements in assessing welfare and diagnosing diseases. As cortisol concentration is considered a stress indicator that may be affected by the sampling protocol, the matrices for cortisol sampling and their practical applications are summarized in detail. We believe that by underlining the importance of cortisol management, this review contributes to a better understanding of equine welfare and health.

2. Cortisol in Horse’s Welfare and Health

While cortisol is indispensable for survival and adaptation, chronically elevated concentration can yield detrimental effects [54]. High cortisol concentration can negatively impact heart rate, causing it to increase, although simultaneously, the organism may be stimulated to combat dehydration [55]. Numerous studies have demonstrated a positive correlation between cortisol concentration and various physiological stress indicators in horses, including heart rate, respiratory rate, rectal temperature, eye temperature, and blood lactic acid [13,33,56,57,58,59]. Similarly, numerous studies point to the positive correlation between cortisol concentration and stereotypical behavior occurrence [8,60,61,62]. However, the question is still raised whether such behavior is an indication of stress or a coping mechanism. One may observe that higher plasma cortisol concentration was noted before stereotypical behaviors, such as weaving and crib-biting than following them, hinting at stereotypy as a potential coping mechanism [60]. On the other hand, crib-biting behavior did not have a noticeable impact on the horses’ plasma cortisol, beta-endorphin, and adrenocorticotropic hormone concentrations [62]. No differences in plasma cortisol concentration were found between horses displaying stereotypical behaviors and those that do not exhibit such behaviors [61]. Moreover, no differences in plasma cortisol concentration and fecal cortisol metabolites concentration were found in stereotypic and non-stereotypic horses, which confirms the possibility of stress assessment and the relationship between stress and welfare indicators using less invasive, and thus less biased fecal sampling [8].
Cortisol serves as an important diagnostic indicator for various diseases and health conditions in horses, such as stress-related conditions [29,30], equine Cushing’s disease (ECD) (also known as Pituitary Pars Intermedia Dysfunction (PPID)) [63,64], inflammatory and infectious diseases [65,66,67], as well as metabolic imbalances, dehydration, and electrolyte imbalances [55]. The ECD is the most prevalent endocrine condition affecting 20–25% of elderly horses over the age of 15 years [34,54,63,68,69,70]. ECD is characterized by a long-term elevation in circulating glucocorticoids [63], however, plasma cortisol concentration is typically within the normal range or even lower [71]. Some research suggests that the paradox of normal plasma cortisol concentration in horses with ECD may be best explained by alterations in cortisol metabolism [28]. According to a recent study, the free plasma cortisol fraction was found to be higher in ECD horses than in normal horses of the same age and season [72]. The increased free plasma cortisol fraction could be attributed to a decline in the binding affinity of cortisol-binding globulin. Apart from the obvious role of cortisol in the pathogenesis of ECD, this hormone contributes to the body’s stress response to serious illness and is instrumental in regulating the inflammatory reaction [65,67]. One may observe that in relative adrenal insufficiency (RAI) or critical illness-related corticosteroid insufficiency (CIRCI), the cortisol response to illness is inadequate. This can result in cardiovascular collapse and an extensive, uncontrolled systemic inflammatory response, which may lead to serious decrease of general condition or death [66,67].
It is worth emphasizing that cortisol concentration in horses may be affected by factors other than stress or illness, including physical activity and the time of day. Horses exhibit a circadian rhythm in cortisol release, akin to other species such as humans [73], dogs [74], and rhesus monkeys [75], with peak levels in the morning and troughs in the late afternoon and evening. This circadian rhythm is crucial for maintaining the horse’s physiological balance [31,76,77], therefore, even minor disruptions in this pattern can diminish daily oscillations and elevate cortisol concentration [31,32]. Therefore, technique and time of sample collection and storage, analytical method used, and variables beyond stress, such as physical activity, health status, and the time of day should be considered when interpreting equine cortisol concentration.

3. Matrices for Cortisol Sampling

3.1. Blood Cortisol

Blood cortisol concentration offer valuable insights into the horse’s acute stress response, serving as a snapshot of their immediate state. An elevation in blood cortisol concentration indicates recent stress or activation of the horse’s HPA axis. Thus, blood cortisol concentration is often used in clinical settings to assess acute stress responses [33], to assess welfare [8,38], to diagnose cortisol-related diseases [37], and to evaluate the effectiveness of treatment protocols [36,40]. While measurement of blood cortisol is a useful tool for assessing acute stress responses, it may not provide a complete picture. It is essential to recognize that blood cortisol concentration may not offer a comprehensive assessment of chronic stress, as the pulsatile production of glucocorticoids have to be considered for the evaluation of blood samples [8,78,79].
The blood cortisol concentration is typically measured in plasma or serum. For this reason, a blood sample is obtained most commonly through venipuncture. However, this method is invasive and potentially stressful for the horse, especially if they are anxious or uncooperative during the process. The handling of the animals necessary to obtain blood samples can introduce additional stress, which could have an impact on the final results [8]. The serum or plasma cortisol concentration is measured using laboratory methods such as radioimmunoassay (RIA) [8,35,39], enzyme-linked immunosorbent assay (ELISA) [13], rapid enzyme immunoassay (EIA) [33], or liquid chromatography–tandem mass spectrometry (LC-MS/MS) [36].
The nominal or basal value of plasma cortisol concentration in horses typically falls within a specific range, although it can vary based on factors such as the horse’s age, health status, and circadian rhythm. The literature offers a reference range for basal cortisol concentration in equines, so that according to available reports, healthy adult horses exhibit resting basal cortisol concentration that typically range from 30 to 395 nmol/L [22,35,80,81]. The reference range provided offers a guideline for what is considered normal or nominal basal cortisol concentration in adult, healthy horses. However, it is important to note that individual variations and the clinical context should also be considered when interpreting cortisol values in specific cases. Some of the publications on basal blood cortisol concentrations are shown in Table 1.

3.2. Salivary Cortisol

The traditional approach to assessing HPA axis activity in horses has primarily involved measuring cortisol concentration in blood. However, in recent years, there has been a growing trend toward utilizing salivary cortisol analysis for this purpose [82,83]. Salivary cortisol is considered the biologically active form of the hormone [78], and there is substantial evidence of a strong correlation between cortisol levels in blood and saliva in horses [13,39]. Total blood cortisol concentration could account for approximately 80% of the variations in salivary cortisol concentration, and vice versa [39]. However, the use of salivary cortisol concentration measurement for the evaluation of acute physiological stress in horses may be limited, as free cortisol is excreted from the salivary gland during oral movements, which makes it available [61]. Prolonged elevations in salivary cortisol concentration may be linked to chronic stress, which, in turn, can contribute to multiple health disorders, including weakened immune systems and metabolic aberrations [42]. Thus, salivary cortisol concentration was proposed in clinical settings to assess stress levels, particularly in exercised horses [25,41,42,43], during competition [40], during transportation, under environmental pressure [41], and in order to identify horses that have been exposed to chronic stress and require special care to maintain their welfare [42].
The measurement of salivary cortisol concentration offers a non-invasive and convenient method for repeated sampling without causing disruption to the animal [40,84]. This is particularly advantageous in situations where collecting blood samples may be challenging or unfeasible. Additionally, compared to blood sampling, saliva collection does not require the involvement of highly trained and certified personnel [40,84]. Due to ethical concerns, earlier research on stress often focused on evaluating cortisol concentration in saliva, since methods involving blood serum collection may induce fear and stress in animals [40,85]. As a result, the practical and noninvasive technique of measuring cortisol concentration in saliva has been increasingly applied to reduce stress and ensure the welfare of horses and other animals [3,37,40,85]. Various techniques are employed to collect saliva samples from horses. One popular approach involves the use of special sponges or cotton balls that are placed in the horse’s mouth for a brief period and then subsequently analyzed for cortisol content [86]. Cortisol in saliva can be measured using a variety of techniques, including RIA [38,40], ELISA [13,25], or EIA [32,41], which make it possible to measure this material precisely. In addition, salivary cortisol research also uses gas chromatography and other mass spectrometry techniques, which ensure the accuracy of the results [32].
Studies reporting salivary cortisol concentrations in horses are summarized in Table 2.

3.3. Blood and Salivary Cortisol in Stimulation or Suppresion Tests

The baseline cortisol measurement [4,31,36,87], the ACTH-stimulation test [4,33,36,39,88,89,90], and the dexamethasone-suppression test [44,90,91,92,93,94,95] are commonly used to assess blood cortisol concentration in horses, each serving specific diagnostic or research purposes.
The baseline cortisol measurement involves the collection of a single blood sample from a resting horse to determine plasma or serum cortisol concentration at a specific moment. This test provides insights into the horse’s current cortisol status [4,36]. Typically, the blood is obtained through venipuncture, commonly from the jugular vein, using standard aseptic techniques [87]. The timing of sample collection is crucial. Ideally, the blood sample should be taken from a resting horse in the morning or early afternoon when cortisol concentration tends to be relatively stable [31]. This minimizes the influence of diurnal variations. Reducing stress during sample collection is essential to obtain accurate baseline cortisol concentration. Gentle handling and restraint techniques are employed to prevent the elevation of cortisol concentration due to stress during the procedure.
It is worth noting that while baseline cortisol measurement offers a snapshot of cortisol concentration at a specific moment, other dynamic tests, such as the ACTH-stimulation test or dexamethasone-suppression test, may be required for a more comprehensive evaluation of the horse’s endocrine function and stress response.
The ACTH-stimulation test evaluates the horse’s ability to respond to stress by administering synthetic ACTH and measuring the subsequent rise in cortisol concentration [4]. This test is valuable for diagnosing conditions such as ECD [36,96]. ACTH-stimulation test can be performed using synthetic ACTH1-24 (Synacthen tetracosactidum) [88,89,90]. While sometimes a high dosage is used, 1 μg/kg BW, it was found, that in horses maximal increase in secretion rate occurs after administration of only 0.1 μg/kg ACTH. Increased dosage only prolongs cortisol secretion, while its levels remain stable [88,90]. Thus, slightly different time points for sampling are recommended, with 12 h in 1 μg/kg, and in around 9 h in 0.1 μg/kg. Peeters et al. (2011) found changes in cortisol concentration from 189 ± 52 to 357 ± 55 nmol/L after ACTH stimulation [39]. Due to diurnal variations in cortisol secretion, tests should be performed in the afternoon hours, as the concentration variability decreases [89,90]. Low-dose stimulation test, with the administration of 0.01–0.02 μg/kg, is also described as a useful tool in accurately diagnosing RAI/CIRCI, as in some patients adrenal glands fail to secrete an adequate amount of cortisol with higher dosage [22]. Studies evaluating ACTH stimulation test are presented in Table 3.
The dexamethasone-suppression test is used to evaluate the decrease in cortisol levels, caused by suppression of the HPA axis by exogenous corticosteroids (dexamethasone) [91]. While it initially was considered as highly specific and sensitive, further studies proved it to be less reliable [100]. Dexamethasone is administered by intravenous or intramuscular injection. Dexamethasone under normal circumstances should suppress the release of ACTH from the pituitary gland and subsequently reduce cortisol production by the adrenal glands [44,92,93]. There are variations of the dexamethasone-suppression test, including the nighttime testing, which differ in the duration of dexamethasone delivery and the interval before cortisol testing. The administered intramuscular dose is around 0.04 mg/kg BW of dexamethasone [90]. Sojka et al. (1993) showed concentrations of cortisol before dexamethasone administration to be 155 ± 8.6 nmol/L, after 12 h it dropped to 1.9 ± 1.4 nmol/L, while after 24 h the concentration was 30.4 ± 16 nmol/L [94]. It is advised, that using dexamethasone suppression test sampling should be around 19 h after administration (up to 24 h), and levels below 27.6 nmol/L are considered normal [90,95]. These tests are typically designed as single-point observation tests, hence the dexamethasone-suppression test data may show the existence of ECD in healthy horses because cortisol production has a circadian rhythm in addition to inter-individual variability [101]. In horses with early ECD, results are reliable when sampling is performed 24 h after dexamethasone administration, as they have a limited capacity for proper reaction [95]. False positive results may be found in the fall when the production of ACTH is increased. However, individuals with cortisol below 5.5 nmol/L after dexamethasone administration in the fall can be assigned as healthy [95].

3.4. Hair Cortisol

Blood cortisol concentration can exhibit fluctuations when horses are subjected to either short- or long-term confinement [102]. Whereas, measuring cortisol concentration in hair is a potential technique for assessing long-term stress [103,104]. One may observe that analyzing hair cortisol concentration provides a more reliable assessment of long-term stress compared to saliva or serum measurements [105]. This is attributed to the belief that hair cortisol concentration serves as an indicator of cortisol secretion and the stress experienced over extended periods [106]. Moreover, horses with higher concentration of hair cortisol tend to exhibit lower compliance in training or work. This means that horses that have experienced higher levels of stress in the past may be less cooperative and more resistant to training. This finding carries significant practical implications for trainers and horse owners. Thus, hair cortisol concentration was proposed in clinical settings to assess stress levels in working horses and horses’ ability to perform tasks and achieve success in various areas [107].
Cortisol in the blood circulation is gradually absorbed into growing hair, effectively creating a historical chronology of cortisol concentrations [108]. Extensive examination has been carried out on the advantages and drawbacks of different matrices used for determining glucocorticoid concentrations [108,109,110]. These matrices, including blood serum, saliva, urine, feces, hair, and claws or fingernails, exhibit variations in several characteristics. However, the most significant distinction lies in the time frame that the hormones recovered from them represent. Blood, saliva, urine, and feces primarily reflect recent circulating hormone concentration and are often influenced by daily rhythms or other unpredictable events. In contrast, slow-growing matrices such as hair, claws, or fingernails offer a reflection of glucocorticoid accumulation over extended time periods, providing a more comprehensive view of stress levels in horses [46,107]. Hair stands out as a favorable matrix due to its minimally invasive collection process, resilience under different storage conditions and exposures [46,111,112], and its capacity to provide an extended retrospective perspective on an individual’s HPA axis activity [46,108,113]. Each cycle of hair development is comprised of three distinct phases: active growth (anagen), transition (catagen), and rest (telogen) [114]. It is during the anagen phase that systemic cortisol can passively diffuse from blood vessels into the developing hair shaft [105,109]. Additionally, hair cortisol concentration may detect temporal variations in circulating cortisol concentration in longer hairs where cortisol can be measured in several segments [115,116,117,118]. Hair is a stable substrate for measuring hormones and allows for storage for extended periods, which are additional benefits of using hair to monitor cortisol concentration [115,119,120]. In studies evaluating cortisol concentration, hair sampling was performed by shaving the neck or abdomen region, tail hair, and mane hair with bulbs or cut with scissors [45,46,47,48,49,104]. Cortisol in hair can be measured using a variety of techniques, including RIA [48], ELISA [45,47] or EIA [46,49], LC-HRMS/MS [45], and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLCMS/MS) method [44]. Recent studies have utilized hair cortisol as a measure of the health and stress levels of horses, making it a valuable tool in equine welfare research (Table 4).

3.5. Fecal Cortisol

Since the positive correlation between the plasma cortisol and fecal cortisol metabolite concentrations was noted [8], fecal measurements were used for the identification of past potentially stressful situations [121]. It should be noted that after secretion, circulating steroids reach the liver, where they undergo catabolic changes. Then, metabolites are excreted with bile and reach the gastrointestinal tract. Further changes are caused by bacterial enzymes, which may also act outside of the gastrointestinal tract, influencing the result depending on the time of sampling [121,122]. Samples can be obtained from the rectum, or within a short time after defecation, from bedding or pasture [8,38,50,51,52,53]. Feces samples, if they cannot be tested straightaway, should be frozen in order to inhibit changes in the concentration of metabolites for reliability, as in some cases bacterial activity increases metabolite concentration, while sometimes the concentration decreases, as further metabolic processes also take place [50,122,123]. Thus, a separate method, focused on metabolites, must be used instead of evaluating the intact cortisol [51]. In most cases, 11,17-dioxoandrostanes (11,17-DOA) levels are measured with enzyme immunoassay, although antibody selection is influential on the quality of results.
Cortisol in feces can be measured using an EIA method [8,17,38,51,52]. The 3α,11-oxo-A EIA method seems to be more valuable than Extr-DOA EIA, as it cross-reacts with some other metabolites, is more stable at room temperature, and ACTH stimulation showed the higher amplitude of metabolite concentration between baseline value and following ACTH administration. The throughput of this method is also higher than in previously developed methods [8]. This method has been validated and can be effectively used to evaluate the concentration of fecal metabolites of cortisol [124].
Fecal measurements of cortisol metabolites are informative regarding the situation starting from 24 h from sampling, up to 48, or even 72 h [51,53,121]. Merl et al. (2000) found the peak of concentration occurring 36 h after a stressful event (castration) [122]. It is the time necessary for cortisol release, circulation, catabolism, excretion, and passage through the gastrointestinal tract. Thus, in the identification of potentially stressful situations, samples can be collected the day after their occurrence, giving an opportunity to evaluate stress post factum [121]. While blood and saliva testing provides data about rapid reaction and instant cortisol increase, feces concentration changes are rather delayed. Another important issue regarding fecal metabolites is that they are less variable, as they are a consequence of the overall stress reaction in a unit of time, rather than ongoing fluctuations of concentration, as it smoothens the pulsatility of cortisol secretion [43,53,123]. Recent studies have utilized fecal cortisol as an important animal welfare indicator considering animal keeping and day-long stress (Table 5).

3.6. Other Cortisol Matrices

In addition to the methods mentioned above, cortisol can also be obtained from various other matrices such as urine, tears, or hooves. Urine cortisol concentration provides insights into the horse’s stress response over a slightly longer duration than blood cortisol, making it valuable for assessing stress associated with specific situations [125], whereas cortisol concentration in tears was reported to rise concurrently with blood cortisol after ACTH stimulation [71]. These studies may often involve a limited number of animals, but the results are frequently reported in the context of the coping theory, focusing on stereotypical behavior as a potential sign of poor welfare. Further research is required to identify whether endogenous cortisol in tears plays a role in ocular disease [71]. Interestingly, it is thought that cortisol is not fully recognized as a primary biomarker for stress in horses [21]. In general, more markers should be evaluated, also to assess a range of stressful stimulus, such as glucose and catecholamines. Such an approach would allow to evaluate the physiological effect of stress reaction. Generally, in stress evaluation more than one stress marker should be measured, as adrenal glands can be stimulated with exercise and excitement, not only with stress reactions [121].

4. Conclusions

Cortisol plays a pivotal role in assessing the welfare and health of horses, as it is closely tied to their physiological responses to stress in various conditions. The extensive body of research demonstrates the strong correlations between cortisol levels and physiological stress indicators, offering valuable insights into the horse’s overall state, exercise effort, and health. While cortisol is crucial for adaptation and survival, chronically elevated concentration can have detrimental effects, making it a critical indicator of specific diseases. Additionally, studying cortisol concentration in different matrices such as blood, saliva, and hair provides versatile tools for monitoring acute and long-term stress, offering non-invasive and retrospective perspectives on equine health and welfare.

Author Contributions

Conceptualization, U.S. and M.M.; writing—original draft preparation, U.S., M.M., A.C., Ł.Z. and M.D.; writing—review and editing U.S., M.M., A.C., Ł.Z. and M.D.; supervision, M.M.; project administration, M.M.; funding acquisition, M.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Centre for Research and Development as part of the POIR 2014–2020 project number POIR.01.01.01-00-1001/20.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

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Table 1. Studies reporting basal blood cortisol concentrations in horses.
Table 1. Studies reporting basal blood cortisol concentrations in horses.
ObjectivesDemographic DataTime of Day and MethodResultsReference
Correlates of oral and motor stereotypic behaviors and glucocorticoid concentration55 horses
(41 geldings, 14 mares)
5 to 20 years old		Blood sampling between 18:00 and 19:00
RIA method		Plasma cortisol concentration varied from 7 to 112 nmol/L[8]
The use of plasma sampling for the determination of cortisol concentration in relation to the intensity of exercise in horses during race training12 horses
(6 stallions, 6 mares)
2 to 3 years old		Blood sampling between 7:00 and 9:00
ELISA method		Plasma cortisol concentration varied from 249 nmol/L at rest,
335 nmol/L immediately after exercise, and 281 nmol/L 30 min after exercise		[13]
Plasma cortisol and ACTH concentrations in the warmblood horse in response to standardized treadmill exercise test10 horses
(10 geldings)
3 years old		Time not reported
EIA method		Plasma cortisol concentration before exercise varied
from 41 to 185 nmol/L		[33]
Database for steroid reference values for domestic Mongolian horses123 horses
(18 colts, 34 stallions, 25 geldings, 17 fillies, 29 mares)
2 months to 17 years old		Blood sampling twice on 10:00 and 14:00
RIA method		Serum cortisol
concentration varied in groups, with 64 nmol/L for colts,
158 nmol/L for stallions,
193 nmol/L for geldings,
102 nmol/L for fillies,
171 nmol/L for mares,
140 nmol/L for all horses		[35]
Steroid profiles in equine plasma before ACTH-stimulation test11 horses
(8 geldings, 3 mares)
6 to 14 years old		Time not reported
LC-MS/MS method		Plasma cortisol concentration was
138 ± 54 nmol/L
before the ACTH-stimulation test		[36]
Determine the use of plasma cortisol concentrations in the diagnosis of the ECD7 horses
(5 geldings, 2 mares)
5 to 15 years old		Blood sampling twice on 06:00 and 18:00
RIA method		Plasma cortisol concentration varied from 251 nmol/L at 06:00 and 142 nmol/L at 18:00[37]
Relation between compromised welfare, such as chronic pain and hematological anomalies, and cortisol concentration in domestic horses49 horses
(44 geldings, 15 mares)
5 to 20 years old		Blood sampling twice a day in the morning (between 08:00 and 09:00)
and in the evening
(between 18:00 and 19:00)
RIA method		Plasma cortisol
concentration varied from 80 nmol/L in the morning,
42 nmol/L in the evening (day of work), and 35 nmol/L in the evening (day of rest)		[38]
Develop a sensitive and specific RIA to measure cortisol concentration precisely in serum5 horses
(2 geldings, 3 mares)
9 to 17 years old		Time not reported
RIA method		Serum cortisol concentration varied from 137.84–256.92 nmol/L at rest and 146–222 nmol/L during catheter placement[39]
Table 2. Studies reporting salivary cortisol concentrations in horses.
Table 2. Studies reporting salivary cortisol concentrations in horses.
ObjectivesDemographic DataTime of Day and MethodResultsReference
The use of saliva sampling for the determination of cortisol concentration in relation to the intensity of exercise in horses during race training12 horses
(6 stallions, 6 mares)
2 to 3 years old		Saliva sampling between 7:00 and 9:00
ELISA method		Salivary cortisol concentration varied from 1.63 nmol/L at rest,
2.57 nmol/L immediately after exercise, and 3.82 nmol/L 30 min after exercise.		[13]
Evaluation of salivary cortisol concentration for association with strongyle-type egg shedding levels200 horses
(132 geldings, 3 stallions, 65 mares)
1 to 30 years old		Saliva sampling between 11:00 and 13:00
ELISA method		Salivary cortisol concentration was 2.7 nmol/L[25]
Estimation of the change of stress level in horses based on saliva cortisol concentration61 horses
(28 geldings, 33 mares)
5 to 20 years old		Saliva sampling at 7:00, 11:00, 14:00, and 16:00
EIA method		Salivary cortisol concentration varied from 2.44 nmol/L in tourist riding group, 3.07 nmol/L in resting group, and 2.76 nmol/L in education horse-riding group.[32]
Develop a sensitive and specific RIA to measure cortisol concentration precisely in saliva5 horses
(2 geldings, 3 mares)
9 to 17 years old		Time not reported
RIA method		Salivary cortisol concentration varied from 0.58–1.77 nmol/L at rest and 0.59–2.21 nmol/L during catheter placement[39]
Quantification of the stress levels in competition using salivary cortisol concentration23 horses
(11 geldings, 12 mares)
6 to 11 years old		Saliva sampling at 9:00
and just before competition, and 20, 40, and 60 min after
RIA method		Salivary cortisol concentration varied from 1.01 nmol/L at rest to
1.56 nmol/L after whole day of competition		[40]
Analyze the salivary cortisol concentration in horses related to stressors14 horses
(9 geldings, 3 stallions, 2 mares)
3 to 7 years old		Saliva sampling at 15:00
EIA method		Salivary cortisol concentration varied from 9 nmol/L to 9.5 nmol/L[41]
Table 3. Studies reporting blood and salivary cortisol concentrations in horses after ACTH stimulation.
Table 3. Studies reporting blood and salivary cortisol concentrations in horses after ACTH stimulation.
ACTH Dose and Route of AdministrationTimelineChanges in Concentrations Over TimeReference
IV, 1 μg/kg BW of ACTH (Synacthen tetracosactidum 0.25 mg/mL equivalent to 25 IU/mL)Tests performed from 1 p.m. to 4 p.m.
Saliva samples were collected before, and 30, 60, 90, 120, 150, and 180 min after ACTH administtration		Salivary baseline 0.83–6.34 nmol/L (2.2 ± 0.83 nmol/L)
significant increase after 30 min
the peak reached after 122 ± 22 min
mean percentage increase: 1620% ± 760		[4]
IV, bolus injection of
250 µg ACTH per horse 		Net plasma cortisol measurement every 10 min before administration, starting from 60 min prior; control after 30 min from administration Plasma baseline: not reported
cortisol net increases above 275.86 nmol/L plasma were observed in six out of seven horses.
mean percentage increase: not calculable %		[33]
IV, 1 μg/kg BW of ACTH
(Synacthen tetracosactidum 0.25 mg/mL equivalent to 25 IU/mL)		Blood collection directly before ACTH administration, and 60 min afterPlasma baseline: 138 ± 54 nmol/L
mean percentage increase 60 min after: 196%		[36]
IV, 1 μg/kg BW of ACTH (Synacthen tetracosactidum 0.25 mg/mL)Collection 30 and 15 min before ACTH injection and the following at 10, 20, 30, 40, 60, 80, 100,120, 140, 160, 180, 200, 220, 240, 280, 320, 260, 400 and 500 min after ACTH injectionSerum baselline: 125–224 nmol/L
(148.14 ± 42.69 nmol/L)
Significant increase after 10 min
The peak reached after 96 ± 16.7 min
Return to baseline after 280 min
400 min after ACTH administration concentration dropped to 62.39 ± 16.14 nmol/L
Mean percentage increase in serum: 225%
Salivary baseline: 0.46–3.14 nmol/L (1.23 ± 1.08 nmol/L)
Significant increase after 30 and 40 min
(20–30 min later than in serum)
The peak reached after 124 ± 8.9 min
Return to baseline after 180 min
(100 min earlier than in serum)
Mean percentage increase in saliva: 2150%		[39]
IV, 10 μg and 100 μg of ACTH (cosyntropin)Collection before 10 μg ACTH injection, 30 min later, 90 min later followed by 100 μg ACTH injection, 30 min and 90 min after second ACTH injection, protocol performed at birth, 12–24 h, 36–48 h, 5–7 daysPlasma baseline at birth: 281.38 ± 63.45 nmol/L, 12–24 h: 99.31 ± 44.14 nmol/L, 36–48 h: 71.72 ± 27.59 nmol/L, 5–7 days: 55.17 ± 22.07 nmol/L
Mean percentage increase in plasma after 10 μg of ACTH at birth: 140% ± 20, 12–24 h: 290% ± 100, 36–48 h: 280% ± 160%, 5–7 days: 180% ± 50, 30 min after injection
Mean percentage increase in plasma after 100 μg of ACTH at birth: 160% ± 40, 12–24 h: 400% ± 170, 36–48 h: 450% ± 280%, 5–7 days: 320% ± 150, 90 min after injection		[97]
IV, 0.1 μg/kg BW of ACTH (cosyntropin 250 μg/mL)Collection before ACTH injection, 30 min and 60 min after, performed at birth, 3, 5, 7, 10, 14, 21, 28, 42, 56, and 84 daysAverage plasma baseline: 75.31 nmol/L
Average 30 min after: 114.21 nmol/L
Average 60 min after: 79.72 nmol/L
Mean percentage increase in plasma at birth: 192.5% ± 63.1
Mean percentage increase in plasma 3–56 days old: 37.7% ± 9.4
Mean percentage increase in plasma 84 days old: 52.5% ± 18.7		[98]
IV, 0.02, 0.1, 0.25, and 0.5 μg/kg (cosyntropin)Collection before ACTH injection, 30, 60, 90, 120, 180, and 240 min afterPlasma baseline 76 to 264 nmol/L (mean ± SD, 172.4 ± 44.8 nmol/L)
Peak percentage increase after 0.02 μg/kg ACTH: 150% 30 min after injection
Peak percentage increase after 0.1 μg/kg ACTH: 190% 30 min after injection
Peak percentage increase after 0.25 μg/kg ACTH: 200% 90 min after injection
Mean percentage increase after 0.5 μg/kg ACTH: 230% 90 min after injection		[99]
Table 4. Studies reporting hair cortisol concentrations in horses.
Table 4. Studies reporting hair cortisol concentrations in horses.
ObjectivesDemographic DataTime of Day and MethodResultsReference
Determination of hair cortisol concentration in horses 47 horses
(9 geldings, 38 mares)
age not reported		Time not reported
Mane hair with bulbs from mid-neck region
ELISA and LC-HRMS/MS methods		Hair cortisol concentration varied from 2.0 to 17.9 pg/mg by ELISA and 1.3 to 8.8 pg/mg by LC-HRMS/MS[45]
Determination of hair cortisol concentrations in feral horses282 horses
(135 stallions, 113 mares)
known-age up to 6 years old and a unknown-age		Time not reported
Hair collected from tail by hair snags
EIA method		Hair cortisol concentration varied from 2.17 pg/mg in stallions to 1.64 pg/mg in mares[46]
Monitoring of hair cortisol concentrations during a one-year time-lapse47 horses
(26 geldings, 21 mares)
6 to 20 years old		Time not reported
Hair collected from neck region by shaving
ELISA method		Hair cortisol concentration varied from 290 pg/mg in feale to 230 pg/mg in gelding male[47]
Verification of the reliability of a hair cortisol assay. Measuring of hair cortisol at birth and at 30 and 60 days old204 horses
(102 foals, 102 mares)
birth day to 17 years old		Time not reported
hair collected from withers by shaving
RIA method		Hair cortisol concentration varied from 18.02 to 156.51 pg/mg at birth to 40.56 pg/mg in the first quartile to 62.88 pg/mg in the third quartile[48]
Determination of the effect of a relocation period and the multiple factors associated with a rest period on hair cortisol concentrations in horses13 horses
(13 stallions)
5 to 13 years old		Time not reported
hair collected from ventral abdomen area by shaving
EIA method		Hair cortisol concentration varied from 5.34 pg/mg in control group to 3.09 pg/mg in relocated horses[49]
Determination of the mane hair and body hair cortisol concentration in horses153 horses
3 to 28 years old		Time not reported
Mane hair cut with scissors, hair from above the scapula obtained with clippers from the left side
UHPLCMS/MS method		Hair cortisol concentration varied from 6.0 pg/mg in the mane to 6.1 pg/mg in the body hair[104]
Table 5. Studies reporting fecal cortisol concentrations in horses.
Table 5. Studies reporting fecal cortisol concentrations in horses.
ObjectivesDemographic DataTime of Day and MethodResultsReference
Correlates of oral and motor stereotypic behaviors and glucocorticoid concentration55 horses
(41 geldings, 14 mares)
5 to 20 years old		Feces sampling
between 12:00 and 13:00, from bedding, directly after defecation
EIA method		Fecal cortisol metabolite concentration varied from 2.4 to 37.6 ng/g[8]
Evaluation of the influence of reproductive status and reproductive procedures on cortisol secretion50 horses
(50 mares)
6 to 16 years old		Feces sampling three times a day at 0:00, 8:00, 16:00
sampling method not reported
EIA methods		Fecal 3α,11-oxo-A concentration varied from 3.8 ± 0.6 ng/g in teaching mares to 12.7 ± 4.0 ng/g in maiden mares[17]
Relation between compromised welfare, such as chronic pain and hematological anomalies, and cortisol concentration in domestic horses49 horses
(44 geldings, 15 mares)
5 to 20 years old		Feces sampling
between 12:00 and 13:00, from bedding, directly after defecation
EIA method		Fecal cortisol metabolite concentration varied from 5.0 ± 0.3 ng/g in the working day, 4.9 ± 0.4 ng/g in the day of rest.[38]
Evaluation of the influence of insects on stress reaction39 horses
(8 geldings, 1 stallion, 30 mares)
1 to 21 years old		Feces sampling
between 12:00 and 14:00
or between 14:00 and 16:00, from pasture, immediately after defecation
EIA method		Fecal 3α,11-oxo-A concentration varied from 38.1 ± 2.8 ng/g
to 45.6 ± 2.7 ng/g		[50]
Validation of method for glucocorticoid metabolites measurement in feces10 horses
(5 stallions, 5 mares)
3 to 14 years old		In the morning (hour not reported) and
in the evening (hour not reported), feces removed from the rectum
EIA method		Fecal 3α,11-oxo-A concentration baseline was 49.91 ± 21.13 ng/g
fecal Extr-DOA baseline was 3.48 ± 1.65 ng/g		[51]
Evaluation of exercise and psychological endocrine responses to a new environment and initial training40 horses
(12 geldings, 9 stallions, 19 mares)
2 years old		Fecal sampling twice a day in the morning (between 07:00 and 09:00)
and in the evening
(between 16:00 and 19:00), from bedding, within 30 min after defecation EIA method		Fecal 11-oxoetiocholanolone concentration varied from 1.3 and 20.1 ng/g[52]
Quantification of the stress response of transrectal palpation36 horses
(36 mares)
5 to 14 years old		Time not reported
Feces removed from the rectum
EIA method		Fecal 3α,11-oxo-A concentration was
25.5 ng/g with ranges
16.3–121.4 ng/g		[53]
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Sikorska, U.; Maśko, M.; Ciesielska, A.; Zdrojkowski, Ł.; Domino, M. Role of Cortisol in Horse’s Welfare and Health. Agriculture 2023, 13, 2219. https://doi.org/10.3390/agriculture13122219

AMA Style

Sikorska U, Maśko M, Ciesielska A, Zdrojkowski Ł, Domino M. Role of Cortisol in Horse’s Welfare and Health. Agriculture. 2023; 13(12):2219. https://doi.org/10.3390/agriculture13122219

Chicago/Turabian Style

Sikorska, Urszula, Małgorzata Maśko, Anna Ciesielska, Łukasz Zdrojkowski, and Małgorzata Domino. 2023. "Role of Cortisol in Horse’s Welfare and Health" Agriculture 13, no. 12: 2219. https://doi.org/10.3390/agriculture13122219

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