Open access peer-reviewed chapter

Equine Stress: Neuroendocrine Physiology and Pathophysiology

Written By

Milomir Kovac, Tatiana Vladimirovna Ippolitova, Sergey Pozyabin, Ruslan Aliev, Viktoria Lobanova, Nevena Drakul and Catrin S. Rutland

Submitted: 26 July 2021 Reviewed: 25 April 2022 Published: 09 June 2022

DOI: 10.5772/intechopen.105045

From the Edited Volume

Updates on Veterinary Anatomy and Physiology

Edited by Catrin Sian Rutland and Samir A.A. El-Gendy

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Abstract

This review presents new aspects to understanding the neuroendocrine regulation of equine stress responses, and their influences on the physiological, pathophysiological, and behavioral processes. Horse management, in essence, is more frequently confirmed by external and internal stress factors, than in other domestic animals. Regardless of the nature of the stimulus, the equine stress response is an effective and highly conservative set of interconnected relationships designed to maintain physiological integrity even in the most challenging circumstances (e.g., orthopedic injuries, abdominal pain, transport, competitions, weaning, surgery, and inflammation). The equine stress response is commonly a complementary homeostatic mechanism that provides protection (not an adaptation) when the body is disturbed or threatened. It activates numerous neural and hormonal networks to optimize metabolic, cardiovascular, musculoskeletal, and immunological functions. This review looks into the various mechanisms involved in stress responses, stress-related diseases, and assessment, prevention or control, and management of these diseases and stress. Stress-related diseases can not only be identified and assessed better, given the latest research and techniques but also prevented or controlled.

Keywords

  • equine stress
  • physiology
  • pathophysiology
  • neuroendocrine regulation

1. Introduction

Despite approximately 100 years of intensive research (and more than 1 million citations in PubMed), stress, due to its multidimensional characteristics, remains a problematic concept not only in equine but also in human medicine; even today there is not a consensus on the question of what is stress? Stress may be defined as a relationship between an organism and external or internal factors that act to disrupt homeostasis. It has been suggested that “Living beings have evolved various specific and nespecidic reactions and pathways to mitigate the detrimental effects of stress to restore homeostasis” [1]. Thus, common acute stress responses can be evaluated as a process of constant flow moving around a homeostatic point, which is the optimal state for the existence of living beings (including horses). However, this definition also leads to a broader concept of stress, since it can include all temporary physiological adaptations to any change in the environment. All living organisms, from the bacterium, to the horse or human, live (and lived) in potentially dangerous conditions, and in the process of evolution, they have developed specific protection mechanisms to survive before leaving offspring. There is not a consensus whether the highly variable stress environment promotes adaptation and the process of evolution itself, or only contributes to reflex defense [2]. In any case, stress responses in living organisms are constitutional—genetically programmed and are constantly modulated by environmental factors in the form of the gene–environment interactions [3, 4]. Thus, equine temperament traits (e.g., neophobic and neurotic behavior) that are known to be heritable strongly influence the intensity of the animal’s stress response [4, 5]. Various specific equine genes influencing behavior have been identified [6]. For example, a frustration-related stress behavior in stabled horses is linked to an A–G substitution in the DRD4 (dopamine receptor) gene [7]. Additionally, it was found that an “A” variation of the G292A version of the DRD4 gene contributes to decreased curiosity and increased vigilance, and was more prevalent in Thoroughbreds compared with native breeds [8].

In the stress-induced disturbance of hemostasis or a possible threat to it, it is commonly noticed that a nonspecific multisystem three-stage body response occurs, which the famous Canadian endocrinologist Dr. Hans Selye (1907–1982) first termed the General Adaptation Syndrome (GAS) [9]. An alarm is the first stage or wave, where animals through a high concentration of catecholamines and activation primarily of cardiovascular, respiratory, and locomotory systems, and large energy consumption, trying to cope with adverse, threatening situations, or to escape from them (“fight-flight-or-freeze” response). In free nature, horses show usual a proactive response—flight (fearful behavior), rather than fight (or aggressive-dominant behavior), and this is considered a fundamental natural survival mechanism that increases protection in a threat environment. Logically, without this alarm phase developing through evolution, the horses would have no chance of escaping predators and ensuring their survival and continuation of the species. Occasionally, in “human controlled” environments, horses in the alarm phase show a passive response that involves behavioral inhibition, with lower locomotion, immobility, or withdrawal, but with focused attention. Which reaction horses show depends on the stress factors, and the reactivity of the neuroendocrine and sympathetic nervous systems. In this first wave of the stress response, within seconds there is an increased release of catecholamines, and it has been noticed that there is also increased secretion of corticotropin-releasing hormone (CRF), prolactin, growth hormone, and glucagon, and a decrease in the release of hypothalamic gonadotropin-releasing hormone (GnRH).

If the stressful situation is not resolved, the horse’s body uses its additional energy resources and activates other physiological systems to protect or adapt to the stressful condition. This is the resistance stage. In this phase or the second wave, an increase in the concentration of glucocorticoids is mainly noticed. If the physiological compensatory mechanisms have succeeded in overcoming the stress, the recovery stage is entered. In contrast, if the animal body has used up its resources and is unable to maintain normal homeostasis that leads to the stage of exhaustion with various pathophysiological changes occurring.

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2. Equine stress factors

There is wide consensus, that horses, by the nature of their use and management, are more likely to be exposed to different stressor factors compared to other domesticated animals. There are numerous equine stress factors, these can roughly be divided into internal and external, acute and chronic, physical and psychological, but more often horses are exposed to numerous stress factors at the same time. From a pathophysiological and clinical point of view, strangulation intestinal obstruction causes the strongest equine stress response, as it is accompanied by several stress factors at the same time. Firstly, there is severe abdominal pain (through tissue damage and intestinal distension) [10]. Secondly, there is the occurrence of intestinal dysbacteriosis with the release of LPS, which triggers an increased concentration of proinflammatory cytokine and the development of severe endotoxic shock [11]. The treatment of such horses in equine clinics and the associated changes in environmental conditions (other stables, unknown people, and other horses) significantly enhance the stressful response [12]. If such horses undergo abdominal surgery and general anesthesia, this will increase stress exposure placed on the animals [13]. Numerous other equine stress factors also exist, for example for used horses in sports and competition there are associated transport conditions, the novelty of their surroundings, exposure to a noisy public, and physical overload [14, 15]. Physical exercise is a stress condition solicited in the organism creating a new dynamic equilibrium that requires adaptive responses. Exercise-induced stress is often proportional to the horse’s competition level [16]. Water or food deprivation (after intestinal surgery), metabolic disorders by various equine diseases (acidosis, hypovolemia, electrolyte imbalance, and hypoglycemia), and inflammation also cause a stress response [1718]. Temporarily limited but very intensive psychological loads are exposed to foals at weaning [19, 20]. The lack of activity, for example constantly staying in a stall, stabling, and isolation without social contact with other horses, causes most horses to undergo chronic stress responses [21]. It should be taken into account that different horses may show stress in specific ways, and some horses respond better to stressful situations than others. The adaptive response of each horse to stress is determined by a multiplicity of genetic, environmental, and developmental factors. Equine stress response in horses is also dependent on the animal’s perception of the situation. It is considered in human and equine medicine that the crucial factor that determines if a psychological stressor has a negative, neutral, or even positive outcome, is whether the central nervous system (CNS) perceives to be in control of the situation or not [22]. Certainly, it has an important role has the experience of the horse. Frequently, most horses appear to fear novel situations, and these are perceived as being threatening. In addition to all of the above, nervous riders or veterinary personnel may cause a horse to behave more reactively because they present as ambiguous stimuli [23]. Undoubtedly, nervous people also transmit their fear to horses, which enhances the equine stress response. It is well known that horses recognize angry human faces and interpret them as negative [24].

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3. Neuroendocrine regulation of equine stress reaction

In contrast to rodents or humans, horses are not as well studied with regards to the neuroendocrine regulation of stress response, especially at the acellular and molecular level in brain structures, therefore rodent and human results will be partially extrapolated to horses in this review. For a long time, researchers suggested that the sympathetic-adrenal medullary (SAM) and hypothalamic–pituitary–adrenal (HPA) axis play the main role in stress responses [25, 26, 27]. This theory, up to the present, seems to limit or oversimplify the weight aspect of the animal’s stress response. There is almost no tissue or cell in animals (including microbiota) that, directly or indirectly, does not play some role in the maintenance of homeostasis during the acute or chronic stress response [28]. In a simplified version, the stress systems in horses have two essential components—controllers and effectors. The controllers or receptors of sensory systems monitor the value of the regulated homeostatic parameter which they are customized, compare it to the reference value and generate a neural (or hormonal) signal that is proportional to the absolute value of the difference. If the sensory signal is interpreted in the CNS as a threat, this will be acted upon through various pathways affecting different effectors, which creates physiological reactions into or out of the system in order to bring the controlled variable closer to the reference value [29]. Equine homeostasis is usually maintained through complex, coordinated mechanisms of self-regulation, among which feedback plays an important, but not determinative role.

The first step in the stress response is the perception of a stressor through the sensory system, which is composed of sensory receptor cells, neural and blood pathways, and parts of the brain involved in sensory perception. The brain interprets them as either a real or a potential threat, which triggers nonspecific and specific stress responses that are commensurate with the nature of the stimulus (Figure 1) [30]. Thus, the physical stressors which are well studied, for example, pain and blood loss, require an immediate “systemic” reflex reaction. On the other hand, the equine brain also responds to non-physical or “psychogenic” stressors (for example transport or weaning) based on prior experience [20, 31].

Figure 1.

Schematic representation of the central and peripheral components with regulatory pathways involved in the equine stress response caused by colic disease. LC/NE, locus coeruleus/norepinephrine system; SNS, sympathetic nervous system; PVN, paraventricular nucleus; BST, bed nucleus of the stria terminalis; POMC, proopiomelanocortin; CRH, corticotropin-releasing hormone; VP, vasopressin; GABA, γ-aminobutyric acid; ACTH, adrenocorticotropic hormone; NPY, neuropeptide Y; SP, substance P; Ach, acetylcholine; PACAP, pituitary adenylate cyclase-activating polypeptide; LPS, lipopolysaccharide; NA, norepinephrine; E, epinephrine; DA, dopamine. Activation is represented by green lines, inhibition by red lines and auto regulatory feedback loop by red dashed lines.

Sensory systems code for four aspects of different stress stimuli—type, intensity, location, and duration [32]. There are different receptors monitoring equine homeostasis, and among them, the nociceptors have the most important role in the induction of the acute stress reaction. As a reaction to physical trauma or another noxious stimulus, nociceptors send sensory stimulation primarily through to the preganglionic sympathetic neurons in the intermediolateral cell column of the thoracolumbar spinal cord, and from there further through the neospinothalamic, paleospinothalamic and archispinothalamic tract to different thalamic “relay” neurons [33]. Then, the thalamus sends the noxious signal to other brain structures, which initiate, spread, memorize, and cessation in an equine stress reaction [34]. As a reaction to homeostatic imbalance or inflammation, the brainstem is also able to generate rapid stress responses via direct projections of neurons in the paraventricular nucleus of the hypothalamus (PVN) or to preganglionic autonomic neurons [35]. It is considered that information on blood volume or oxygenation is communicated via baro- and chemoreceptors to the nucleus of the tractus solitarius (NTS), which then send direct noradrenergic projections to the PVN, ensuring a rapid HPA axis response [31]. The forebrain limbic regions (which mediate psychogenic stressors) have no direct connections with the HPA axis or the SAM, and thus require intervening synapses (primarily to the locus coreleus, amygdala, and bed nucleus of the stria terminalis), prior to initiating a stress response [35, 36].

Nociceptors, interneurons, and “relay” neurons of the thalamus release a variety of excitation (pain) neurotransmitters, primarily, the substance P (SP), neurokinin A, Glutamat, calcitonin-gene-related peptide (CGRP), and cholecystokinin [37]. Recently, in the equine thalamic reticular neurons (TRN neurons), unique dopaminergic projections to the thalamic relay neurons were found, whereas in primates this input arises from a variety of dopaminergic neurons within the classically defined catecholaminergic system [38]. This possibly novel, potentially dopaminergic, projection upon thalamic relay neurons within the equids may play a modulatory role in the output of thalamic relay neurons to other structures of the brain [38]. It is considered, that these neurons have a strong influence on the processing of neural information, potentially providing the equid cerebral cortex with neural information that has a lower signal-to-noise ratio, making the extraction of salient neural information more precise than observed in other mammals [39]. The presence of catecholamines in the TRN neurons modifies stress behavior and may play a role in the various aspects of sleep observed in equids. It is well known that sleep in equids appears to be unusual, as they are short sleepers (around 2.9–3.3 hours per day), and have brief sleep cycles of around 15 min, with a non-rapid eye movement (REM) phase followed by a brief REM phase (less than 30 s), mostly while standing [40]. Undoubtedly, this evolutionary trait in horses on a daily basis supports a quick and effective alarm response to various acute stress factors.

After receiving a threat sensory information, most researchers argued that two areas of the brain have distinctive important roles in the stress reaction—the catecholaminergic neurons in the locus coeruleus (LC-NA system), which are mainly responsible for activation of the SAM axis, and the hypothalamus, which is responsible for activation of HPA axis (Figure 1) [41, 42, 43]. Furthermore, other brain circuits modulate and fine-tune the adaptive or protective stress responses, including the amygdala-hippocampus complex, the mesocortical and mesolimbic components of the dopaminergic system, the noradrenergic cell group A2/C2 in the solitary nucleus, the A1/C1 cell groups in the ventrolateral medulla, the cuneiform nucleus and dorsal raphe nucleus, the parabrachial nucleus, and the bed nucleus of the stria terminalis [44, 45]. These structures are responsible for releasing various excitatory and inhibitory neurotransmitters, via overlapping brain circuits, in accordance with stressor modality or intensity. In addition, an alteration of the parasympathetic nervous system (PNS) with attenuation of the “vagal tone” of the heart and lungs occurs to help control the duration of activation of the SAM axis. Reconciliation of the PNS response to stress is mediated via the nucleus ambiguus and dorsal motor nucleus of the vagus nerve, possibly via input from the nucleus of the solitary tract [45].

3.1 LC/NE-sympathetic systems in the equine stress reaction

The locus coeruleus (LC) with noradrenergic neurons have been expressly implicated in the initiation and speed of acute physiological and behavioral stress changes in rodents and humans (likely in horses) [41, 42, 46, 47]. Extrapolation of results from other species to a horse should be performed with caution, to obtain a remarkable difference between equid and other animals because catecholamine metabolites are mostly glucuroconjugated and not sulfoconjugated [48]. LC receives inputs, not only from the spinal cord and thalamus, but also from the hypothalamus, medial prefrontal cortex, nucleus prepositus hypoglossi, and nucleus paragigantocellularis [49]. LPS (endotoxin) associated release proinflammatory cytokines (IL-1β, IL-6, and TNF-α) also facilitate norepinephrine (NA) release in LC [50]. Likely, this endotoxic activation of LC is especially important in horses with strangulation intestinal obstruction. After input activation, the noradrenergic neurons in the LC send then excitatory signals to different areas of the brain and to the spinal cord that are accompanied primarily by the alarm phase of the acute stress response [49]. Amplification of LC activity leads to increased signs of alertness in electroencephalographic (EEG) analysis [51]. The LC-NA system, through projections to the sympathetic preganglionic neurons in the spinal cord with activation of α1-adrenoceptors, increases sympathetic activity and reduces parasympathetic activity, via the activation of α2-adrenoceptors on preganglionic parasympathetic neurons [49, 52, 53]. Therefore, activation of the LC-NA system within seconds leads to the activation of the equine adrenal medulla (SAM), with a distant release of norepinephrine (NA) and other catecholamines—epinephrine (E) and dopamine (Figure 1). Consequently, this chain process in the alarm stress phase is more correctly denoted as activation of the LC-NA-sympathetic system, instead of activation of the SAM. In addition to NA release, the sympathetic nerve fibers also secrete adenosine triphosphate (ATP) and the neuropeptide Y (NPY), which enhance the systemic action of the catecholamines [54]. Accompanying the LC-NA-sympathetic system, the synthesis of E in the adrenal medulla partially stimulates the ACTH, cortisol, and pituitary adenylate cyclase-activating polypeptide (PACAP) [55].

3.1.1 Concentrations of equine epinephrine

The concentration of equine E in the blood depends on the tone of the sympathetic system, as it is associated with active escape, attack, and fear. In nonstress horses, blood concentrations of E show a circadian rhythm. The mean plasma E concentrations were highest in the morning (~ 30 pg./ml at 8:00 hr), with a significant nadir in the sleeping phase at 04:00 hr. (~18 ng/ml) [56]. The concentrations of E increase up to 21 times higher during severe acute stress (fear, trauma) and intense physical activity [57]. The circadian rhythm of NA also exists, at 08:00 in the nonstressed horse, it was found to be 70–80 pg./ml, with the nadir observed at night (~50 pg./ml) [56]. In equine physical exercise stress, the NA concentration increases approximately 13-fold [58]. Thus, the response plasma levels of E and NA during exercise or other forms of acute stress in the horse is considerably greater than in people. The difference between horses and humans in SAM activity may help explain the superiority of the athletic performance of equine athletes compared to that of human athletes [59]. In horses during exercise, the increase in plasma NA is almost linearly proportional to exercise intensity, being higher after brief maximal exercise than after an endurance ride [58]. On the contrary, a marked increase in plasma E only occurs during strenuous exercise, especially if it is accompanied by psychogenic stress.

3.1.2 Roles of equine catecholamines

Basically, the equine catecholamines regulate many biochemical processes involved in energy metabolism, as well as the physical homeostasis adaptation associated with acute and rapid stress responses. Equine catecholamines have a strong impact on the bone marrow and spleen to enable the mobilization of additional blood. It has long been known that during the resting phase of horses, approximately 50–60% of blood (i.e., more than 20 L) is kept within reservoirs, in comparison to dogs (20–25%) and humans (12%) [60]. Consequently, during the alarm phase of the equine stress response, the splenic contraction under adrenergic control, ejects reservoir blood into the circulation, following significant increases in the hematocrit levels and concentrations of the erythrocytes, leucocytes, thrombocytes, hemoglobin, and plasma protein. The normal human spleen, unlike the horse spleen, does not contain many smooth muscle adrenergic receptors; therefore, it cannot strongly contract. Thus, the equine splenic contractile response to the various stress factors is more sensitive than that of any other species [60].

An increase in catecholamine release within the alarm phase leads to significant magnification of equine cardiac function. The cardiovascular system in horses is more stress sensitive than that of any other domestic species, as the increase in heart rate (HR), stroke volume (SV), cardiac output (CO), and blood pressure (BP) is enormous during an acute alarm stress reaction [61]. Independent of stress factors and its intensity, and through catecholamine binding to β1 receptors in adult thoroughbreds (TB) horses, it was noticed that increasing the HR to 240 beats per minute (at rest 30–40 beats per minute), the SV to 1200 ml (at rest 900 ml), the BP to 250/120 mmHg (at rest 155/110 mmHg), the CO reached 240–340 l/min [62]. In nonstress horses, the CO is about 40 l/min, that is, by intense acute stress in horses is possible a 7–8 fold increase in cardiac function, in contrast to human athletes at approximately 2-fold [63, 64]. Commonly, these changes in cardiovascular parameters by acute stress horses are for a short time (5–60 sec), are intensively energy-consuming and straining, and the catecholamine concentrations quickly return to their resting levels [65]. It should also be remembered that these equine cardiovascular changes in acute stress are not only related to the increasing activities of the SNS and catecholamines but are also controlled by other vasoactive hormones, for example through plasma renin activity, atrial natriuretic peptide, endothelin-1 and vasopressin in the renin–angiotensin–aldosterone system.

3.1.3 Physiological changes during equine stress

Horses have a normal resting respiratory rate (RR) of 12–20 breaths per minute. During the onset of acute severe stress, in accordance with the body’s need for oxygen, the RR rises as high as 180 breaths per minute [66]. In an adult TB horse at rest, the tidal volume (TV) is about 4–7 liters, rising to a maximum of about 10 liters during intense stress exercise [67]. When breathing at rest the dead space accounts for about 70% of the TV and the alveolar volume is around 30%. With exercise stress, there is a large increase in alveolar volume and a small increase in dead space. In adult nonstressed horses, the amount of air passing in and out of the lungs per minute (MV) is approximately 100 l/min. At maximal stress exercise, the MV reached 1500 l/min (due to a 7-fold increase in RR and a 2-fold increase in TV) [66]. Rate and depth of breathing are controlled in part by chemoreceptors in the blood vessels which respond to changes in pH, arterial oxygen, and carbon dioxide tension. When in gallop (stress flight) the RR is coupled with stride rate and so the mechanics of locomotion override the chemical control of breathing. This unique equine phenomenon is known as locomotor-respiratory coupling [68]. When exercise ceases, the RR decreases due to the cessation of the locomotor forces that drive respiration. These equine physiological and anatomic adaptations allow an extremely high maximal rate of use per minute of O2 consumption (VO2 max). By strenuous physical (stress) horse activity the VO2 max reaches up to 200 ml/kg/min [66].

In addition, via stress activation of the LC-NA-sympathetic system, it has been noticed in horses (through contraction of the m. iris dilator) mydriasis with the appearance of tunnel vision (i.e., loss of peripheral vision) and increased body temperature, following sweating and suppression of secretion of the lacrimal and saliva glands can occur. In stressed animals, it was also noticed that decreases in gastrointestinal mobility, blood flow, and secretion could happen [69]. In the alarm phase of the stress response through catecholamines (but also through glucocorticoids), the horse’s blood clotting function is accelerated to prevent excessive blood loss in the event of an injury sustained during the potential “stress fight response” [70, 71]. In the alarm phase of the equine stress response, it was noted that different immune functions were enhanced, through catecholamines binding to β-2 adrenergic receptors on immune cells (primarily on the NK cells), as well as through blood mobilization and direct sympathetic innervation of lymphoid organs (Figure 1) [72, 73, 74].

3.1.4 Catecholamines during stress responses

Catecholamines in an alarm phase of stress response vie β-2 adrenergic receptors induce significant lipolysis with increasing concentrations of blood fatty acids, that can be used directly as energy sources primarily by the locomotor system [75]. In different animal studies, it has been found that catecholamines, through α-adrenergic receptor binding, provoke inhibition of insulin secretion and significantly increased concentrations of glucagon (mediated through binding to the β-adrenergic receptor), and glucose, as a result of increasing either glycogenolysis or by gluconeogenesis [76, 77]. Additionally, catecholamine stimulation also releases ACTH, cortisol, and renin following the retention of sodium in the bloodstream [78].

Numerous other physiological reactions of catecholamines have also been noticed, which lead to the equine body producing additional speed and strength. For example, via the binding of alpha-1 adrenergic receptors by NA, it was noted that vasoconstriction of most blood vessels occurred in the skin, digestive tract, and kidneys [78]. These receptors are inhibited and counterbalanced by beta-2 adrenergic receptors (stimulated by E release from the adrenal glands) in the skeletal muscles, heart, lungs, and the brain during a SAM response. At rest, in horses, only about 15% of the circulating blood is delivered to the muscles, but this increases to as much as 85% during strenuous stress exercise [66]. In other words, in the alarm phase of the stress response, through the activation of the LC-NA-sympathetic system, oxygen, and nutrient delivery is directed toward the CNS and areas of the body (primarily the cardiovascular and respiratory systems), where they are most needed to cope and escape from threat factors. In contrast, during severe acute stress responses other energy-consuming functions, such as digestion, reproduction, and growth, are temporally suppressed [59, 79, 80]. These changes are conditional and strongly depend on the type, intensity, and frequency of the stress factor. Thus, during equine stress exercise, a different picture of the hormonal background is observed in horses.

3.2 HPA axis in the equine stress reaction

In the second phase of the stress response (the stage of resistance), it is commonly observed that a strong activation of the HPA axis and the renin–angiotensin system (RAS) occurs, although a strict distinction between these stress phases is difficult to conclusively make as the reactions are dependent on the stress factor [81]. The intensity and frequency of the stressor is a major factor in determining the overall trajectory of the HPA axis response, with significantly increasing concentrations of the corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), vasopressin (VP), and cortisol. Stressors of a presumptive “lesser” severity (for example the horse having 5 min exposure to a novel open field) produce lower peaks in HPA activation, in contrast to severe abdominal pain following, for example, an intestinal obstruction. The main goal of strongly activating the HPA axis during a stress reaction is the reinforcement of the homeostatic mechanisms and to provide additional energy through enhanced glycogenolysis, gluconeogenesis, and lipolysis [82, 83].

Usually, activation of the HPA axis is slightly slower than the activation of the LA-NA–sympathetic system. Commonly, after the onset of stress factor, the concentration of CRF rises immediately (as do concentrations of the NA), but the peak secretion of pituitary ACTH occurs around 5–15 s later, followed by the peak levels of cortisol 15 and 60 min later [84, 85]. There are also differences here, which are dependent on the stress factor. Commonly, various inflammatory stimuli cause prolonged HPA axis activation (2–3 hours after onset) commensurate with the need to limit immune responses [86].

3.2.1 Equine CRF

Equine CRF is a 41-amino acid peptide identical in structure to human and rat CRF. CRH is produced primarily by parvocellular neuroendocrine cells within the PVN, but this neuropeptide and specific CRH receptors have been identified in numerous extra hypothalamic regions of the brain, including the pituitary and adrenal glands [81, 87]. In addition to stimulating the secretion of the ACTH, CRH coordinates various physiological and behavioral responses, for example, induced anorectic effect, stereotyped behaviors, and enhancing the activity of the SNS [88]. Thus, one of the central actions of CRF is to appropriately facilitate “fight or flight” responses [89]. Besides this, CRF during stress responses inhibits, particularly, the secretion of GnRH, LH, testosterone, and estrogen, and through the stimulation of somatostatin secretion, it also inhibits the secretion of GH, TRH, and TSH [81].

Equine CRF concentrations in pituitary venous blood are lower compared to other species. In nonstress horses, the CRF concentration ranges from 0.25 to 0.8 pmol/l, but is very changeable day-to-day [90]. The regulation of CRF and VP secretion is complex. CRH and VP neurons in the PVN have dense connections with various structures in the brain. In the rodent and humans, the LC and other NE-synthesizing cell groups belonging to the medulla and pons have reciprocal reverberatory neural connections with the CRH neurons in the PVN and stimulate the secretion of each other through CRH receptor-1 (CRH-R1) and the α1-noradrenergic receptors, respectively (Figure 1) [91, 92]. It was also found that auto regulatory ultrashort negative feedback loops exist in both the PVN CRH and the catecholaminergic neurons of the LC, with collateral fibers inhibiting CRH and catecholamine secretion respectively, via inhibition of the corresponding presynaptic CRH- and α2-noradrenergic receptors [93, 94].

In addition, multiple other regulatory central pathways exist, since both CRH and the catecholaminergic neurons receive stimulatory (stress-excitatory) innervation from different brain structures through various neurotransmitters, among which is the especially important pituitary adenylate cyclase-activating polypeptide (PACAP; Figure 1). PACAP is a key emergency neuropeptide, mediating central and peripheral components of the stress axes [95]. This neurotransmitter is primarily expressed in the CNS and also within the sympathetic nervous system including the sympathetic preganglionic neurons that innervate the adrenal gland [96]. Thus, PACAP participates in stimulating the secretion of various hormones and neurotransmitters, including ACTH, VP, epinephrine, insulin, melatonin, prolactin, MSH, brain natriuretic peptide, follistatin, and the enkephalins [95, 97].

Serotonin, cytokines, and other inflammatory factors (e.g., nitric oxide) also participate in CRF secretion in the PVN in horses, as also seen in other mammals [98, 99]. Interestingly, NPY has multiple regulatory central pathways as it stimulates CRH neurons, whereas it inhibits the LC (Figure 1) [100]. On the other hand, SP, as the first responder to most noxious/extreme stimuli, has reciprocal actions to those of NPY, since it inhibits CRH neurons, whereas it activates the CA-NA system (Figure 1) [101, 102].

CRH and AVP neurons have reciprocal reverberatory neural connections to the pro-opiomelanocortin (POMC)-containing neurons in the arcuate nucleus (AN) of the hypothalamus. POMC-containing neurons primarily secrete β-endorphin [103]. Information on horse energy balance appears to access the PVN via projections namely, from neurons in the AC, which process circulating signals relevant to metabolic status (for example, glucose and fatty acid blood concentration) [104].

The neurons of the suprachiasmatic nucleus (SCN) of the hypothalamus also have several direct projections to the CRH neurons. The SCN is known to be the main (but not the only one) coordinator of biological circadian rhythms in mammals, described as the “CLOCK system” [105]. It is well known that the SCN neurons, through photoreception, have important roles in the basal daily and seasonal variation, not only on the equine HPA axis, but also on the hypothalamic–pituitary–gonadal axis, melatonin, insulin, grelin and adinopectin secretion, body temperature, and other factors [105, 106]. Thus, in resting horse conditions, through SCN input the HPA axis activity has circadian and ultradian variations. In the stress-free condition, there is a pulsatile secretion of equine CRF, VP, ACTH, and glucocorticoids (one per hour), with greater amplitudes in the morning (upon waking up) than at night [107, 108]. The circadian rhythm secretion of these hormones is disrupted under equine stressful conditions, as with other animals [109].

3.2.2 Equine vasopressin

Equine vasopressin (VP) is a nonapeptide, which is primarily produced in the magnocellular neurons of the PVN, its main effect is on the regulation of the blood pressure and ACTH secretion. VP is also expressed in other structures of the CNS, with functions in behavior, stress analgesia, and the regulation of circadian rhythms [110]. Normally, plasma vasopressin concentration in nonstress horses is less than 15 pg./mL [111]. An increase in equine VP concentrations is correlated with both the duration and intensity of the stress factor [112, 113, 114]. The basic physiological stimulus for a 5–10 fold increase in the secretion of VP is increased osmolality of the plasma, as well as the presence of hypotension due to hemorrhage or endotoxemia, for example in horses with colic [111]. Although VP has a short half-life (16–24 min), after a 3-day event endurance test, equine VP was elevated for 6 h [80].

3.2.3 Equine ACTH

Equine ACTH is a 39-amino acid peptide derived from pro-opiomelanocortin (POMC). POMC is the widespread archetypal polypeptide precursor of various hormones and neuropeptides with different functions, including several distinct melanotropins (β-MSH, α-MSH, γ-MSH), lipotropins, and endorphins (β-endorphin and met-enkephalin), corticotropin-like intermediate peptide (CLIP), that are contained within the adrenocorticotrophin and β-lipotropin peptides [115]. POMC is synthesized not only in corticotroph cells in the anterior pituitary gland, but also in the intermediate lobe of the pituitary gland, in neurons within the dorsomedial hypothalamus and brainstem, and as mentioned above it is also in the neurons within the AC of the hypothalamus. The first 18 amino acids of ACTH have the full biological activity of the whole molecule, and the first 24 amino acids are the same in all species of animals. Thus, the primary structure of equine ACTH is identical to that of the human hormone, as such, it has been suggested that they have the same biological activity [90].

CRF and VP act synergistically via specific receptors (CRF1 and V1B receptor, respectively) to trigger the release of ACTH from corticotroph cells, into the systemic circulation [116]. It has been suggested that CRF and VP mobilize different pools of pituitary ACTH. The equine pituitary gland has specific anatomical and functional features. The pars intermedia is particularly well developed in horses, and the equine pars distalis encloses the pars intermedia in a thin adherent layer as horses lack a clear hypophysial cleft [117].

ACTH production and secretion are indirectly influenced, not only by CRF, but also by the LC-NA-sympathetic system, PACAP, angiotensin II, vasoactive intestinal polypeptide, lipid mediators of inflammation, and cytokines, including TNF, IL-1β, and IL-6 [31, 81, 97]. Furthermore, endocannabinoids and endogenous opioid peptides appear to negatively regulate basal and stimulated ACTH release at multiple levels of the HPA axis (Figure 1) [115, 118].

The gradient in equine ACTH concentrations between pituitary effluent and jugular plasma can have an over 30-fold difference, with mean jugular plasma ACTH concentrations significantly higher in healthy horses (approximately 41 pmol/L), than in ponies [119]. Interestingly, a circadian rhythm in equine ACTH release is often undetectable. There is a disassociation between ACTH and corticosteroids during the circadian cycle suggesting a diurnal variation in the adrenal sensitivity to ACTH, with higher responsiveness during the peak phase of glucocorticoid secretion [120].

ACTH via binding specific receptors, namely type 2 melanocortin receptors (MC2-R) is the key regulator of glucocorticoid secretion (GCc) from the adrenal cortex [121122]. Currently, the expression of this subtype of melanocortin receptor in the equine adrenal cortex has not been characterized, but it is presumed to be similar to that described in humans. It has not been a significant relationship found between equine plasma ACTH and cortisol concentrations during exercise stress, and the maximum concentration of cortisol had no correlation with maximum ACTH concentrations [80].

3.2.4 Equine cortisol

Equine cortisol (EC) is a steroid hormone synthesized from cholesterol. EC is released into the circulation under the influence primarily of ACTH, but notably, existing evidence shows that cortisol secretion is further regulated by other hormones and/or cytokines coming from the adrenal medulla or the systemic circulation, and by neuronal signals via the autonomic innervation of the adrenal cortex (for example, as discussed above, through neuromediator PACAP) [95].

EC secretion rates are similar to humans and independent of various physiological factors, such as race, age, circadian rhythm, seasonality, exercise, and pregnancy [123]. Consequently, establishing a reference interval for the basal EC is difficult. In healthy adult horses at rest, the plasma EC levels range from 12 to 68 ng/ml or 33–187 nmol/l (total cortisol) or 10–23 nmol/l (free cortisol) [123, 124]. Under basal (nonstress) conditions, the equine adrenal gland produces cortisol at about 1 mg/kg body weight, with a pronounced pulsating rhythm in regular bursts (more as 10 per day) [124]. The highest daily value is reached shortly after waking up in the morning, before feeding and the minimum levels are observed between 6:00 and 9:00 pm [123]. The circadian rhythm of EC can be affected by various factors, such as exercise, mating, feeding, training, sleep patterns, individual activities, and especially during acute or chronic stress [125]. Plasma EC concentrations during stress responses are directly dependent on the stress factors, their duration, and frequency. Based on our unpublished studies, the total plasma EC concentration in horses with strangulated intestinal obstruction increases rapidly. We found that in colic horses independent of the degree of pain and endotoxic shock, upon admission into the clinic and before treatment commenced, there was wide individual variation in EC level (between 190 and 625 nmol/L), that is, 2–5 fold increases in comparison to the concentrations that are usually present in horses under resting conditions. We have also noticed that horses with a larger colon volvulus, hernia foraminis omentalis and inguinal hernia have, on average, higher EC concentrations than horses with right or left dorsal displacement of the large colon. The EC concentrations were significantly decreased after an abdominal surgery had been performed and steroid and non-steroidal antiphlogistics had been administered, but levels still remain more elevated than normal even when these horses had been discharged home (i.e., 10 days post-surgery). These findings are also supported by previous studies [10, 126, 127]. There is little doubt that EC levels in horses with colic are higher than those seen following transport stress [128, 129]. EC levels in transport horses correlate positively with transport duration and its conditions, but are also dependent on the individuals and their hormonal backgrounds, in this case on the stages of the estrous cycle and gestation [130, 131]. EC is frequently used to assess stress levels induced by exercise. In stress-induced exercise, a marked increase in EC levels was attributed to exercise duration and not to intensity [123]. In addition, the secretion of EC depends on the animal’s prior experiences in competitions and the horse’s character.

3.2.5 Interactions in the HPA axis response during equine stress

It is well known that a depletion of cortisol stores is noticed when animals are chronically stressed and thus the EC concentrations in stressed animals vary widely within the literature. Therefore, it is considered that cortisol levels are not always reliable indicators of chronic stress in horses. On the one hand, high cortisol levels can be a sign of positive stress that promotes higher performance; on the other hand, low cortisol does not necessarily mean the absence of stress. Usually, peak cortisol levels are reached 10–20 min after the onset of acute stress when transporting horses [128]. However, the ability of the adrenal glands to produce cortisol was preserved during transportation and did not decrease, and the pulsation from the transportation of horses after traveling 100–300 km persists [128]. In contrast, an elevation in ACTH concentration gradually decreases after transportation at increasing distances, and these changes were not directly associated with changes in cortisol levels. Similar to this, although an initial rise in EC levels follows a large spike in ACTH levels, if prolonged inflammatory stress occurs, ACTH levels return to near basal levels, while cortisol levels remain elevated as a result of adrenal hypersensitivity [131].

In plasma, EC binds to approximately 90% of a specific a1-glycoprotein named cortisol-binding globulin (CBG) and particularly to albumin. An inverse relationship between CBG Bmax and CBG affinity was demonstrated in mammals including the equine species [132]. The CBG maximal capacity (Bmax) was 0.22 in horses equivalent to 59% plasma cortisol concentration [123]. Equine CBG content at birth was the lowest of any species studied [132]. On the other hand, CBG concentration increased with age, whereas in other species it decreases, and the plasma of the newborn foal has a binding protein that has not been reported in other species, which binds as much cortisol as CBG does [132]. In studies on horses and other species, it has been shown that different stressors can influence CBG levels either by increasing or decreasing them in response to acute or chronic stress [133].

It is well known, those cortisol receptors are located in the cytoplasm of steroid-sensitive cells, and only the free portion of circulating cortisol is available to enter the cells by diffusion through the plasma membrane and binds to these intracellular glucocorticoid receptors (GR) [66, 81]. The steroid-sensitive cells are located in any organ and tissue of the equine body, and for this, the EC performs over a hundred different functions. This hormone has strong metabolic effects on carbohydrate balance (promoting glucose production in the liver), lipid metabolism (promoting the lipolytic effects of E and NA), protein catabolism (promoting amino acid mobilization), electrolyte and fluid balance, cardiovascular and respiratory homeostasis, sexual development and reproduction [22, 59, 73, 83, 88]. Thus, EC is critical for energy mobilization and distribution, and is needed to assure energy availability during, but also in the absence of, stress responses. EC exert their permissive effects on catecholamine release and take action in both vascular and cardiac tissue, as well as in the lungs. It has been noticed that glucocorticoids enhance cardiovascular sensitivity to catecholamines by increasing the binding capacity and affinity of β-adrenergic receptors in arterial smooth muscle cells, receptor-G protein coupling, and catecholamine-induced cAMP synthesis [134]. In addition, glucocorticoids prolong catecholamine actions in neuromuscular junctions by inhibiting catecholamine reuptake and decreasing peripheral levels of catechol-O-methyltransferase and monoamine oxidase [135]. There is not always such an unambiguous effect of cortisol with catecholamines. Sometimes, glucocorticoids can also inhibit a few features of sympathetic function and catecholamine release in response to some stressors in rodents [136]. Along with this, glucocorticoids working through negative feedback also inhibit stress-induced NA in the PVN (Figure 1) [88]. Nonetheless, in acute stress the glucocorticoids facilitate sympathetic interactions, causing changes in the dopamine (DA) and noradrenaline (NA) systems, and their overall physiological effects are to permissively augment cardiovascular, respiratory, and locomotor activation. EC through inhibiting prostaglandin synthesis at basal levels blocks their vasodilatory effects. This is without doubt the central pathway by which cortisol causes increased blood pressure and the onset of laminitis in equine Cushing’s syndrome (PPID) [137].

In general, glucocorticoids are powerful inhibitors of the immune system, primarily through inhibition of leukocyte traffic, secretion of cytokines by macrophages, and the production of antibodies (Figure 1) [138]. Due to evidence surrounding the immunosuppressive effects of cortisol, it has been proposed that a physiological function of strong stress-induced increases in this hormone is used to protect not against the source of stress itself, but against the normal defense reactions that are activated by stress. Thus, EC accomplishes this function by turning off those defense reactions, thus preventing them from overshooting themselves and threatening homeostasis [85].

Any trauma-induced hemorrhage causes a robust stress response in horses, along with the enhanced secretion of VP and renin, inducing water retention and vasoconstriction. Interestingly, EC through negative feedback inhibits the release of VP (by restoring the actions of inotropic and vasoconstrictive hormones), increases glomerular filtration rates, and increases the secretion and efficacy of atrial natriuretic polypeptide, all of which enhance water excretion [139]. From the point of view of homeostasis, the importance of suppression by EC in response to hemorrhage is that it prevents the organism from being injured or killed by its own defense mechanisms.

Cortisol also raises insulin concentrations in horses, but EC actions generally oppose but sometimes synergize with those of insulin [140]. For example, EC and insulin have opposite actions on blood glucose levels, as well as on appetite, gluconeogenesis, glucose transport, protein synthesis, muscle wastage, lipolysis, lipogenesis, and fat deposition in adipose tissue [141, 142]. Suppression of insulin and maintenance of blood glucose concentration has been related to the prevention of the onset of the central mechanism of fatigue [80]. EC also stimulates appetite over days in horses. In considering the criterion of homeostasis, it has been suggested that it aids recovery from the anorectic facet of the stress response caused by CRF. It has long been known that EC through negative feedback has indirect inhibitory effects on the CRH neuron, pituitary ACTH secretion, and POMC transcription (Figure 1) [43]. For energy homeostasis in stress conditions, humoral factors and neural afferents from the gastrointestinal tract communicate information to the brain to regulate energy intake and expenditure. Integrating these responses is a very important role carried out by prolactin-releasing peptide (PrRP), which is synthesized in discrete neuronal populations in the hypothalamus and brainstem [81]. Additionally, EC has a potentially disruptive effect on the reproductive function of horses through a number of mechanisms, for example, it decreases hypothalamic GnRH release and basal or GnRH-stimulated release of LH from the pituitary gland, but also through direct effects on the equine spermatogenesis and folliculogenesis in the gonads [143]. Prolonged activation of the HPA axis leads to decreased synthesis of thyroid-stimulating hormone (TSH) due to increased concentrations of CRH-induced somatostatin, which in turn suppresses both thyroid-releasing hormone (TRH) and TSH.

3.3 Endogenous opioid system in the equine stress response

It is well known that a prolonged phase of resistance is an energetically “disadvantageous” process of the metabolic load and with time the body’s reserve will be depleted. Naturally, when the threatening challenge has passed, the equine body will try to shut off the stress response through various physiological mechanisms, for example, the organism trying to get into the recovery phase (or sanogenesis process). Endogenous mechanisms that oppose the stress response can determine the vulnerability or resilience of animals to the pathological consequences of stress. Turning off the equine stress reaction leads to a return to the baseline concentrations of CRF, VP, ACTH, EC, and catecholamines, and this normally happens when the danger has passed and/or the infection has been contained.

Numerous neural, endocrine, and paracrine mechanisms of physiological processes are involved in attenuating or mimicking stress responses, but one of the central roles is played by the endogenous opioids system (EOS). Thus, the EOS plays a dominant role in the third stage of GAS, just as catecholamines are the “main conductors” in the first stage, and glucocorticoids are essential in the second stage. Almost every acute stressor directly or indirectly causes the release of opioid peptides within seconds, but their action is commonly later than other stress hormones [144]. The EOS and its receptors are widely distributed throughout the CNS, and present in various organ systems and glands, such as the pituitary and adrenal glands [145]. There are three major endogenous opioid peptide families, preproopiomelanocortin (POMC), preproenkephalin, and preprodynorphin, which are cleaved active peptides, primarily endorphins, enkephalins, and dynorphin. These produce their effects through actions on μ-, δ and κ- G-protein coupled receptors, respectively [146].

The density reciprocal innervation between POMC-producing opioid peptide neurons of the hypothalamic arcuate nucleus and both the CRH/VP-producing and LC/NE-noradrenergic neurons is indicated inFigure 1 [92]. Opiate peptides (enkephalins, dynorphins) and μ-opiate receptors are highly concentrated directly within the LC [49]. Additionally, it has been shown that μ-opiate receptors are co-localized with α2-adrenoceptors in the LC, and their activation results in cellular inhibition via a shared potassium channel [49]. On the other hand, it has been shown that the LC plays an important role in the processes underlying opiate withdrawal [147]. Furthermore, endogenous opioid peptides (EOPs) strongly inhibit HPA axis activity [145].

In horses, in normal and in stressful conditions, the POMC acts as a precursor central endogenous opioid peptide β-endorphin (β-EP) and is primarily produced in the pars intermedia of the pituitary gland. Stress system activation also stimulates hypothalamus release of POMC-derived peptides, which reciprocally inhibits the activity of central stress system components [148]. The EOS performs various physiological functions, for example, it modifies the excitability of the CNS and induces control of various functional mechanisms, such as pain control, motor activity, lethargic stereotypical behaviors, feeding, immunity, thermoregulation, reproduction, antioxidation, ACTH secretion, and others [149].

3.3.1 The roles of β-EP and ACTH

Under nonstress conditions, equine plasma β-EP concentrations were recorded as 5.71–22.4 pmol/l [150, 151]. The daily rhythm of β-EP secretion is similar to that of ACTH and EC. The highest values of this opioid were noted in blood samples taken in the morning. The application of an upper lip twitch resulted in a doubling of plasma β-EP concentration after 5 min [151]. It was found that the rise of equine β-EP was dependent on the type, intensity, and duration of stress physical exercise, modulates fatigue catecholamine secretion, and causes impairment of performance [152, 153]. During incremental exercise tests, plasma β-EP concentrations were positively correlated with exercise speed and intensity [154]. The critical threshold intensity of ≤60% VO2max for significant increases in β-EP concentrations has been also recorded [154]. Prolonged air transportation also resulted in a sustained elevation of plasma β-EP concentrations compared to values measured at rest on the ground during the same day, but short-term road transport (i.e., for 1 hour) did not alter circulating equine plasma β-EP concentrations [151]. Other investigations noticed that concentrations of β-EP were raised when compared to the basic level only after a distance of 100 kilometers. After the ensuing 100 and 200 kilometers, a decrease was observed. Simultaneously in these horses, increases in the levels of circulating ACTH were observed after traveling distances of 100 and 200 kilometers, and levels of cortisol were higher after traversing distances of 100, 200, and 300 kilometers [150]. Horses with intestinal strangulation obstruction showed 5–10-fold elevations in plasma β-EP concentrations, which may have contributed to endotoxic shock and severe pain [151]. In contrast, horses with painful, but chronic lameness, had plasma concentrations of β-EP similar to those of normal horses, which may suggest an effect of negative feedback signaling or other factors, for example, cellular depletion of this hormone.

It may thus be assumed that upon analysis of the levels of β-EP and ACTH, the release of the opioid into the blood occurs maximally 1 hr after the appearance of the stressor [150]. The authors suggest that β-EP modulates the HPA axis activity. It may also be stated that β-EP release from the equine pituitary gland is synchronized with the initial phase of the stress reaction and may, in this way, mitigate the negative results of catecholamines and cortisol on the organism. Especially high concentrations of β-EP are found in horses with stereotypical behaviors and pituitary pars intermedia dysfunction (PPID). According to Millington et al., [155], this concentration is 60 times higher in the plasma and 120 times higher in the cerebrospinal fluid of PIPD affected animals. Equine stereotypical behaviors under chronically stressful conditions help horses cope with stressors in the domesticated environment through their reputed self-rewarding effect. This behavior is highly likely to be associated with the EOS, as the use of opioid antagonists significantly reduces this equine behavioral appearance [156].

3.3.2 Stress-induced analgesia and the EOS

It has long been known that with acute stress there exists a phenomenon known as stress-induced analgesia (SIA) with a significantly increased pain threshold. From an evolutionary perspective, SIA can be viewed as a component of the predator–prey interactions, helping the survival of animals in the wild. A painful injury will not contribute toward the survival of the animal if there is a threat of further injury or death. In the process of SIA, a central role is played by the EOS [119]. SIA is primarily mediated through the binding of the μ-opioid receptor, this receptor shows greater selectivity for the β-EP, endomorphin, and enkephalins. Pharmacological studies have demonstrated that along with EOS, and a large number of other neurotransmitters and neuropeptides involved in the formation of the SIA, for example, GABA, serotonin, norepinephrine, dopamine, acetylcholine, glycine, oxytocin, vasopressin and neurotensin [92]. SIA is influenced by age, gender, and previous experiences with stressful, painful, or other environmental stimuli. Commonly, the SIA lasts for a certain amount of time. However, when the equine body is no longer in danger, increased nociception, which manifests after the stress factor disappears, can be beneficial, since normal behavior can exacerbate the trauma.

3.3.3 Other roles of EOPs

EOP (including β-EP) also performs other functions, and one of these is transferring the body into hypobiosis, or the maximum energy-saving mode. It is characterized by heart and respiration rate decreases, changes in blood pressure, and temperature decline [144]. It turns out that hypobiosis is caused not so much by the depletion of the other two regulatory systems (SAM and HPA axis), via activation of the EOS. The shock (state of exhaustion) is an extreme degree of the stress response, in which the EOS has a very important role. Recently, it has been shown that a natural mechanism for recovering from abimal shock has “small peptides”, or metabolic substances originating from the EOS, for example, the FMRFamide-related peptides (FaRPs) [157, 158]. These metabolic substances from endogenous opioids have an effect that is the opposite to them (supporting autoregulatory negative feedback). The first thing that happens during stress is the depletion of the adrenal apparatus. To return to its normal state, it must be activated in some way. FaRPs through binding of β-adrenergic receptors perform this function, thus bringing the body out of a state of exhaustion. This indicates that self-healing of stress (natural sanogenesis) is particularly associated with this peptide origin from the EOS.

3.3.4 EOS attenuation of stress

Unaccompanied, the EOS is not able to attenuate or shut off the stress response. In addition to the EOS, there are other neuromediators and hormones that have been proposed which protect against the effects of stress. Undoubtedly, to attenuate the equine stress response, it is necessary to activate the parasympathetic nervous system (PNS) with an increased tone of the vagus nerve and release of the neurotransmitter acetylcholine. Activation of PNS is mediated, among other things, via the neurons from the double reticular nucleus and the neurons from the nucleus of the solitary pathway of the medulla oblongata [81]. But this is not enough to completely “shut off” the animal’s stress reaction. Numerous animal studies have shown that the hippocampus inhibits the activation of the CRH neurons in the PVN and LA through GABAergic and endocannabinoid neurotransmitters [31]. PVN neurons in the hypothalamus produce numerous neuropeptides that may contribute to activation via local paracrine actions, either by recurrent collaterals or dendritic release [159]. As mentioned above, due to mutual reverberant neural connections a gradually decreasing intensity or autoregulatory negative feedback loops exist between the CRF and the noradrenergic neurons, as in the initial stage they stimulate each other [93, 94].

Certainly, a pivotal role in attenuation of the stress response is also played via the mechanism of negative glucocorticoid feedback. Glucocorticoids have direct and indirect pathways to negative feedback to the limbic system, hypothalamus, and pituitary gland. This attenuates or decreases the primary release of CRF and ACTH. In the PVN, the binding of glucocorticoids to its receptor causes rapid synthesis and release of endocannabinoids. The released endocannabinoids bind to CB1 receptors on presynaptic terminals, inhibiting glutamate release and thereby reducing the drive to CRH neurons [160]. The effect of negative glucocorticoid feedback on equine VP secretion is less pronounced than that for CRF because VP secreting neurons are less sensitive to glucocorticoids. Glucocorticoids may also provide positive feedback in some brain structures, particularly under chronic stress conditions [161]. In addition, neuropeptides associated with CRF, such as urocortin, also play an important role in suppressing (or activating) the function of the HPA axis during the stress response [162]. Numerous other hormones directly or indirectly attenuate deleterious effects of prolonged activation of the LA-NA-sympathetic and HPA axis in horses, foremost in that are the hormones melatonin and insulin, and therefore they are sometimes referred to anti-stress hormones [163, 164].

Through all of the physiological processes listed above, gradual decreases in the activities of the LA-NA-sympathetic axis and the HPA-axis have commonly been noticed. Depending on the stress factors (as well as their intensity and duration) a decrease in stress hormone levels in horses does not occur immediately. Commonly, it may take several hours or days for stress hormones to drop back to their baseline levels. It has also been noticed that in horses, repeated exposure to the same stressor (novelty stress) can result in habituation of the HPA axis response, characterized by decreasing glucocorticoid responses over time [165, 166]. Habituation appears to be mediated, at least in part, by the paraventricular thalamic nucleus [167]. But, it must also be noted that not all stressors cause response habituation in horses. Responses to more ‘severe’ stressors (e.g., pain) are maintained, therefore here it is possibly not an adaptation but only protection that comes into play.

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4. Pathophysiology of equine stress

From the outset of stress research, it has been observed that not all stress reactions and the consequences are equal (despite the stereotypical neuroendocrine changes), and Selye introduced the terms “distress” (pathological) and “eustress” (physiological). Physiological stress accompanies long-term positive biological adaptation of the animal (for example, physical training, increases in horse muscle mass). The acute or chronic stress of severe intensity, which is not resolved by the adaptation of the animal, is considered distress. However, the clear contours and boundaries between eustress and distress, acute or chronic stress are difficult to define in equine practice, support for its existence is ambiguous, and there are still efforts in this area to clearly differentiate between normal homeostatic and pathophysiological responses. In other words, the chronic stress phenotype is not clearly defined [92]. Further complicating toward a clearer understanding is that equine stress responses are context-dependent and may reflect differences in the environment, timing (e.g., time of day, season), history of previous stressors, and huge among-individual variations [168]. There is widespread recognition that the effects of stress on the equine body are associated with the characteristics of the stress factor (the strength of the action, its duration, and frequency, predictability, controllability, avoidability), the breed and age, as well as some features of the stressful experience (previous contact with the same or other stress stimuli) [80, 112]. Thus, young horses and foals are much more stress reactive with more pathophysiological responses in comparison to adult animals.

The importance of acknowledging the protective, as well as the potentially damaging effects of stress reaction, has led to the introduction of different terms, for example, allostasis, allostatic load, or allostatic overload [169]. In response to a threat, allostasis maintains stability through adaptive dynamic activation of neuroendocrine, autonomic, cardiovascular, and immune systems, principally aiming to produce the new optimal level of various physiological parameters. For example, physical exercise in horses leads to increased heart rate and blood pressure to provide optimal oxygen concentration to the vital organs. By focusing on neuroendocrine responses, allostasis involves a feed-forward mechanism rather than the negative feedback mechanisms used in homeostasis, with a continuous re-evaluation of need and continuous readjustment of all parameters toward new set points [170]. Using this theory allows us to explain the positive effects of stress on the animal body, for example, an increase in resistance to adverse factors and survival in extreme conditions. As mentioned above, the allostatic mechanism in horses must quickly disconnect once the threat has passed, as it impossible to keep a high level for a long time, due to depletion of reserve capabilities. Straining of the body supporting homeostasis under frequent or prolonged stress is called allostatic loading. It is also defined as the wear and tear associated with chronic hyperactivity or inadequate responses [171]. As allostatic load is characterized by an unstable functioning of the body, if it lasts for a long time or often, it causes the appearance of pathological changes (due to the cumulative effect), which are called allostatic overload.

Usually, under chronic stress, pathological changes are noticed simultaneously in many organs and tissues, primarily stress hormones cause the production of reactive oxygen species (ROS) or free radicals. It especially produces catecholamines, which as phenolic compounds easily undergo oxidation via a one-electron pathway involving several toxic products, such as semiquinones, quinones, and ROS, independently of the oxidation promotors. Almost any chronic stress through ROS has been shown to be responsible for the depletion of several free radical detoxifying enzymes, such as glutathione peroxidase, catalase, and superoxide dismutase [172]. It results in oxidative overloading, which has been implicated in the pathogenesis of different stress-associated pathologies, as well as in the occurrence of various mutations [173]. It is known that mutations accumulate as a result of DNA damage and imperfect DNA repair mechanisms. In animals (including horses) the accumulation of mutations is limited in two primary ways: through p53-mediated programmed cell death and cellular senescence mediated by telomeres at the end of chromosomes [174]. Telomeres shorten at every cell division and cells stop dividing once the shortest telomere reaches a critical length [175]. Cellular stress shortens the length of telomeres, and therefore it indirectly records the history of stress exposure. As a result, as biomarkers of cellular aging, telomere length and telomerase activity, have been considered for investigating the effects of chronic stress in human medicine [176]. This aspect of stress has not been adequately researched in horses to date.

There are numerous equine pathologies that are directly or indirectly associated with stress factors. According to our clinical experience, due to stress exposure, the most commonly noticed diseases in horses are—gastric ulcers, proximal enteritis, acute colitis, and pleuropneumonia [177, 178, 179]. It is important to note that for the occurrence of these diseases along with stress, other pathogenomonic factors have also had a significant impact. For example, for the occurrence of paralytic ileus or colitis, a high concentration of endotoxins in the blood also plays a very important role [177, 178]. Noxious gases (e.g., NH, NO, and CO) in the transport environment may be partially responsible for transport-related equine pleuropneumonia [179]. It is known that equine transport causes strong psychological stress reactions as it often combines the effects of neophobia, claustrophobia, social separation, and balancing. During equine transport, the physiological and endocrine stress changes prepare the horse’s body for the “fight or flight” reaction, however, these actions do not actually follow, and therefore the mobilized energy is not used. Without a doubt in horses this leads to more often pathophysiological consequences, in comparison to stress physical overload, the neuroendocrine changes are accompanied by locomotion thus providing an outlet for the mobilized energy. The reason for this phenomenon is poorly understood. It is possible, that physical activity in horses, as well as in humans, leads to the release of higher concentrations of endogenous opioids and other protective mediators against the destructive effects of stress hormones. It is also possible that the mobilized, but “unused” energy will lead to higher production of ROS.

Interestingly, of the four most common stress pathologies in the clinic in horses, three are associated with the gastrointestinal tract. The reasons for this appearance are multiple. Primarily, stress has strong adverse effects affecting the normal function of the GI tract, for example on the absorption process, mucus and stomach acid secretion, functioning of ion channels, and peristalsis [178, 180]. Stress induces increased intestinal permeability, allowing bacterial antigens (including LPS) to cross the epithelial barrier which activates a mucosal immune response, which in turn alters the composition of the microbiome and leads to enhanced activation of the neuroendocrine HPA axis. In other words, the equine intestinal microbiota also has been implicated in a variety of stress-pathophysiological responsiveness, but also in healthy horses, it clearly modulates the function of the immune and neuroendocrine systems, as well as various metabolic processes. The routes of communication between equine microbiota and CNS are slowly being unraveled, and include the microbial metabolites such as short-chain fatty acids, the vagus nerve, intestinal hormone signaling, and tryptophan metabolism.

Whether the horse will get sick or die from these and other stress-associated diseases depend primarily on immune systems and individual resistance. The individual characteristics of equine stress resistance are determined by the type of the nervous system, lability, or dominance of the parasympathetic or sympathetic nervous systems [168]. Based on our experience, horses with a high parasympathetic tone are much less likely to die from the stress-associated disease than animals with a high sympathetic tone (neurotic horses). Equine neuroticism is also linked to a low pain threshold, indicating such horses were more likely to be stressed by pain [181]. The effect of stress is also dependent on the initial level of hormones, which in turn depends on various factors, including the phase of the light cycle [92], but to date, there has not been a detailed investigation as to which period of the day horses are more tolerant toward stress-induced disease.

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5. Indicators of equine stress

Information on equine stress levels is important when evaluating many aspects of horse management, training, and the treatment of different diseases. It is apparent that great effort is being made to improve recognition and quantitative evaluation of stress in horses. Various testing techniques are used to measure stress responses in horses, which can be roughly divided into visual, clinical and laboratory, or noninvasive and invasive methods [182]. Behavioral indicators of stress responses are very different, and very well described [183, 184]. In an attempt to quantify visual changes equine stress, the The Horse Grimace Scale” (HGS) has recently been used, which is fine-tuned for detecting equine pain, but no validated grimace scale for detecting fear or anxiety exists yet [184].

5.1 Clinical signs of equine stress

Clinical signs of disturbance of the sympathovagal balance can be used to assess acute equine stress which primarily includes changes in cardiovascular parameters, for example, increased heart rate (i.e., presence of sinus tachycardia) and heart rate variability (i.e., presence of nonrespiratory sinus arrhythmia) [62, 182, 185, 186]. According to our recent investigations, sinus tachycardia and sinus arrhythmia are the most frequently diagnosed arrhythmia in equine clinic praxis [187]. Other clinical signs of acute stress are altered respiratory rate and increased body/eye temperatures [66, 182]. For studying the functional state of the brain, to test its bioelectric activity it is possible to use electroencephalographic (EEG) analysis in horses with or without stress conditions. We improved the methods for recording multichannel EEG in horses with specific six unipolar leads using overhead electrodes (Ippolitova/Gauss method) (Figure 2) [188]. Comparative evaluation of electroencephalographic patterns in sporting horses with different types of higher nervous activity, as well as taking account for the age and training level was recently conducted for the first time. It allows the determination of an organism’s potential capabilities, its resistance to stress, and thus the expected performance in competitions [188].

Figure 2.

Equine electroencephalographic recording of brain activity.

5.2 Blood parameters during equine stress

Various specific blood parameters can be used to assess the degree of stress activation. Commonly used were performance analyses of equine blood concentrations of E, NA, ACTH, cortisol, and β-EP [57, 111, 123, 125, 150]. However, E has a short half-life of only a few minutes, making this substance an impractical parameter for studies under field conditions [56]. Secretion of alpha-amylase from the salivary glands is controlled by autonomic nervous signals, and several studies have revealed that salivary alpha-amylase is correlated with SNS activity under stress conditions [189]. For ease of accessibility reasons equine cortisol is most often measured as a biomarker of the stress response, not only in blood but also in saliva, feces, and hair [123]. Interestingly cortisol concentration increases are noticed in saliva with a delay of approximately 20–30 min before the same observation in blood. However, it is becoming clear that relying on glucocorticoids to define stress is incomplete, and there is no current consensus that glucocorticoids should serve as the primary biomarker for defining the stress phenotype.

5.3 Pathological changes during equine stress

Stress-induced pathological changes also confirm the presence of chronic stress in the horse, for example, the occurrence of gastric ulcers. Parameters such as altered metabolism or suppressed immune function may have the potential to provide information on the long-term effects of stress, especially those which are related to blood chemistry (for example plasma or blood lactate levels, prolactin, iodothyronine, estradiol-17β, serum creatine kinase activity, IL-1, TNF). But these biochemical parameters are not specific for the measurement of stress in animals. Therefore, to date, there have been significant difficulties in measuring stress biomarkers in horses and their pathological effects, especially at the genetic, molecular, and cellular levels. In recent times in humans, different damage markers (e.g., lipid peroxidation, protein oxidation, stress-associated proteins, or oxidative stress mediators, as well length of telomeres) have been used to better reflect how people have coped with stress exposure [190].

Finally, it is necessary to take into account all these listed methods in the present day, not only in equine, but also in human medicine, and also to highlight that we have deficiencies in our abilities to analyze chronic stress before it becomes pathological. Thus far, there is a lack of understanding relating to the stress threshold, in analyses of the cumulative impact of multiple stressors over time, as well as the role of individual equine variation in reaction to stress and timing (e.g., time of day, season). Therefore, equine medicine requires improved contact sensing technology development that will allow for long-term, dynamic, noninvasive, multifactorial measurements of sets of stress mediators, as well as improvements in the diagnosis of stress damage at the cellular and genetic levels.

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6. Conclusions

This review presents new aspects toward understanding neuroendocrine regulation of equine stress, and its influences on the physiological and pathophysiological processes. Horse management, in essence, is more frequently confirmed by external and internal stress factors, in comparison to other domestic animals.

In the last few decades, the initial concepts of stress have been revised. New studies have expanded the basis of stress responses to signals from various sensory receptors, through complex interactions of at least three systems that are activated in a time sequence: the locus coeruleus-norepinephrine (LC-NE)-sympathetic system, the HPA axis, and the endogenous opioid system. The interconnection of these systems provides control of norepinephrine, epinephrine, and cortisol release, but also other stress mediators, such as corticotropin-releasing hormone, adrenocorticotropin, vasopressin, β-endorphin, dopamine, neuropeptide Y, serotonin, oxytocin, cytokines (IL-1β, IL-6), pituitary adenylate cyclase-activating polypeptide, FMRF-amide-related peptides (FaRPs), prolactin-releasing peptide (PrRP), and others. Linked into this interconnection network are other organ systems affected in one or several stages of the stress response. Commonly, these reactions in the horse are related to short-term stress responses leading to mobilization of the body’s reserves in a process of constant flow around the homeostatic point. In situations of allostatic overload, a dynamic change from physiological to pathological response can be expected in horses, with the induction of primarily stomach ulcers, paralytic ileus, colitis, and pleuropneumonia. The change from equine physiological to pathological stress response depends not only on whether the stress is acute or chronic but also on the physical characteristics of the stress signals (intensity), as well as on the initial hormone levels, which in turn depend on various factors, including photoperiod sensitivity.

The development of stress-related diseases in horses dictates how long the interaction of sanogenesis with pathogenic factors will last. These diseases can often be prevented or controlled by keeping risk factors minimal, and by assessing stress levels using a variety of testing techniques. Regardless of the nature of the stimulus, the equine stress response is an effective and highly conservative set of interconnected relationships designed to maintain physiological integrity even in the most challenging circumstances. It activates numerous neural and hormonal networks to optimize metabolic, cardiovascular, respiratory, locomotory, and immunological functions.

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Conflict of interest

The authors declare no conflict of interest.

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Written By

Milomir Kovac, Tatiana Vladimirovna Ippolitova, Sergey Pozyabin, Ruslan Aliev, Viktoria Lobanova, Nevena Drakul and Catrin S. Rutland

Submitted: 26 July 2021 Reviewed: 25 April 2022 Published: 09 June 2022