*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 [121, 122]. 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 steroidsensitive 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 in **Figure 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.
