Chronic stress and pituitary-adrenal function in female pigs

IntroductionThe main purpose of the studies described in this thesis was to gain more insight in the regulation of the hypothalamic-pituitary-adrenocorticaI (HPA) system and the mechanisms underlying adaptation to chronic stress in female pigs. The function of the HPA axis, which coordinates multiple neuroendocrine and metabolic responses to stressors, has been subject of extensive basic and clinical research. HPA-activation by stressful stimuli results in an increase in circulating adrenocorticotropic hormone (ACTH) and consequently of glucocorticoid hormones. A brief review of stress and HPA functions is given in Chapter 1.It has been demonstrated in a variety of species that exposure to chronic or repeated stress may induce long-term changes in the regulation of the HPA axis. These changes may occur at the following levels: 1) extrahypothalarnic centers modulating the activity of neurons in the paraventricular nucleus of the hypothalamus that secrete corticotropin-releasing hormone (CRH) and/or other ACTH secretagogues; 2) hypothalamic sites releasing ACTH secretagogues; 3) ACTH-secreting cells in the anterior lobe of the pituitary; 4) glucocorticoid secreting cells in the adrenal cortex.This project was mainly focussed on the regulation of pituitary-adrenocortical function during chronic stress. Long-term restraint of female pigs by a neck-tether was used as a chronic stress paradigm. Previous studies have demonstrated that tethered pigs may develop behavioural, hormonal, and cardiovascular disturbances characteristic for chronic stress. In Chapters 2 and 3 of this thesis we showed that prolonged tethered housing leads to a sustained elevation of basal plasma cortisol concentrations. Chapters 4, 5 and 6 provide evidence that chronic stress may induce long-term changes at the level of the adrenal cortex, resulting in increased steroidogenic capacity and sensitivity for ACTH. No change was found in the sensitivity of the pituitary for CRH or vasopressin. There was no indication for an increase in basal ACTH levels, but the ACTH response to a superimposed acute stress appeared to be reduced during chronic stress. The hypersecretion of cortisol may therefore well be a consequence of stress- induced changes in adrenocortical function. Moreover, chronic stress leads to an increase in the activity of- opioid systems that inhibit pituitary-adrenal responses to additional acute stressors. These alterations in opioid systems may be of adaptive value in that they prevent excessive reactions of the pituitary-adrenal system during chronic stress. This may also underlie the transient nature of the hypercortisolaemia.Pituitary-Adrenocortical Function after Chronic StressHypercortisolaemiaThere is ample evidence, particularly from studies in the rat, that repeated exposure to stressors may produce an increase in the activity of the adrenocortical system, as evidenced by increased circulating corticosteroid concentrations andlor adrenocortical hypertrophy and increased adrenal weight Indeed, in female pigs we found that chronic stress induces long-term elevated basal cortisol levels (Chapters 2 and 3). This hypercortisolaemia, which develops within the first weeks of tethered housing, is evident in every phase of the oestrous cycle of the pigs. It is particularly obvious during the luteal phase of the oestrous cycle when cortisol levels remain rather stable (Chapter 2). In addition, our data reveal circadian differences in the effect of chronic stress on HPA- regulation. Elevated cortisol concentrations are most pronounced in the evening, i.e. during the nadir of cortisol secretion, and lead to a flattened diurnal rhythm of cortisol secretion during chronic restraint stress (Chapter 3). These findings correspond with observations in rats and in man, that chronic stressinduced corticosteroid levels are increased during the trough of circadian adrenocortical activity Determination of cortisol over a 24-hour period demonstrated that elevated evening cortisol concentrations during chronic stress are not the result of a stress-induced shift in the phase of the cortisol rhythm (Chapter 3).Chronic stress may conceivably cause "facilitation" of the HPA system, either by increasing its sensitivity to stimulus input or by decreasing its sensitivity to negative glucocorticoid feedback, or both, so that elevated corticosteroid concentrations are maintained throughout the period of stress. It is well-known that ACTH has a trophic effect on the adrenal cortex. Prolonged ACTH stimulation (e.g., during chronic stress) may therefore lead to hypertrophic enlargement of the adrenal gland, resulting in increased output of glucocorticoids in response to this peptide with time. Furthermore, in rats it has been reported that chronic stress may result in a persistent decrease in feedback sensitivity to corticosteroids. Akana and co-workers have suggested that there is a high sensitivity to glucocorticoid feedback during the trough of the circadian corticosteroid rhythm, accompanied by a diminished (or absent) circadian-dependent drive to CRH secretion in rats. Moreover, they suggested that the circadian rise in plasma corticocosteroid levels is a result of stimulated CRH secretion as a consequence of diminished sensitivity to steroid feedback. One may speculate that the increase in evening, but not in morning plasma cortisol levels during chronic stress, described in Chapter 3, is in part the result of stress-induced changes in feedback sensitivity to circulating cortisol. In other words, the hypothalamus remains insensitive to cortisol, resulting in increased cortisol levels throughout the day.Increased Sensitivity and Capacity of the Adrenal CortexWe found that the chronic hypersecretion of cortisol in tethered pigs does not coincide with an increase in plasma levels of β-endorphin (Chapter 2), a peptide co-secreted with ACTH from the pituitary corticotroph cell. It may therefore be inferred that the hypercortisolaemia is not a reflection of a sustained increase in ACTH release from the pituitary, although basal plasma ACTH levels were not measured in those experiments. Several reports in rats, showing that elevated glucocorticoid levels during exposure to chronic stress are not necessarily associated with significant increases in plasma ACTH, support this contention.Our data (Chapters 4, 5 and 6) provide evidence for stress-induced changes in HPA- regulation. In pigs tethered for a period of 10-11 weeks, the ACTH response to a superimposed stressor (acute nose-sling stress) appears to be reduced as compared with loose housing, whereas the cortisol response remains unaltered (Chapter 5). These data indicate that the sensitivity of the adrenal cortex to circulating ACTH increases as a result of chronic stress. This finding is supported by the study described in Chapter 6, in which the cortisol/ACTH ratio after CRH treatment in tethered pigs shows a two-fold increase when compared with loose housed pigs. Moreover, challenge with exogenous ACTH(1-24) reveals that the capacity of the adrenal cortex to secrete cortisol increases during chronic stress (Chapter 4), which is in line with findings of other studies in pigs1 and other species. This increase in adrenocortical capacity may result from a hypertrophic enlargement of the adrenal gland. All in all, our observations point to the adrenal cortex as one of the dominant sites where functional changes occur during chronic stress.We speculate that these changes in adrenocortical function may well underlie the hypercortisolaemia in tethered pigs (Chapters 2 and 3). Although ACTH is generally considered to be the most important factor in the control of glucocorticoid secretion from the adrenal cortex, there is increasing evidence that steroidogenesis is also under control of neural inputs at the level of the adrenal gland, which can modulate adrenocortical sensitivity to ACTH and thereby control the secretory activity of the adrenal cortex. In rats it has been reported that locally secreted CRH is likely to mediate such an increase in adrenal sensitivity to ACTH by stimulating the blood flow through the adrenal gland, although, to our knowledge, no such mechanism has (yet) been demonstrated in the pig. In this context, it is interesting to note that a challenge with vasopressin in our study produced an approximately five-fold greater cortisol/ACTH ratio than a challenge with CRH (Chapter 6). This implies that in the pig, vasopressin can stimulate corticosteroidogenesis beyond that induced by vasopressin-stimulated ACTH release, possibly by a direct effect on the adrenal gland. In addition, our data indicate a synergism between vasopressin and CRH in their cortisol releasing effects. which may also result from a vasopressin-induced increase in the responsiveness of the adrenal cortex to ACTH.Effects of Housing Conditions on Adrenocortical CapacityIt was interesting to find in our study that housing factors different from the physical restraint of tethered housing per se play a role in the development of adaptive changes in the steroidogenic capacity of the adrenal cortex during chronic stress. We showed that the increase in the cortisol response to challenge with exogenous ACTH(1-24) is considerably more pronounced and persistent in tethered pigs deprived of visual and tactile contacts with conspecifics and with very limited visual control over the environment, than in tethered pigs that have such possibilities, albeit to a limited degree (Chapter 4). The least restricted pigs were separated by horizontal bars, thus allowing social contacts with neighbouring pigs and visual control over the environment. In the most restricted pigs, closed partitions between the pens precluded social interactions and severely limited their visual range. It seems likely that the relative lack of visual information from the environment reduces the predictability of environmental changes and increases uncertainty for those animals, thereby contributing to the stress experienced by the animals. As has been discussed in Chapter 1, low predictability or uncertainty is generally recognized as a characteristic of stressful situations. Furthermore, it has been demonstrated that lack of social interactions with conspecifics may affect stress responses, especially with respect to the HPA axis. Thus, both reduced visual control and social restriction are likely to be important factors contributing to the changes in adrenocortical function observed earlier. These findings underscore the notion that psychological factors are important activators of the HPA axis.Sensitivity of the PituitarySo far, effects of chronic stress on adrenocortical function have been emphasized. Studies in rats have provided evidence that chronic stress may also lead to changes in the sensitivity of the pituitary for hypothalamic peptides regulating ACTH secretion, possibly as a consequence of changes in expression and secretion of these secretagogues. Repeated activation of the HPA system may lead to plastic changes in hypothalamic CRH neurons, resulting in increased expression of vasopressin in CRH- containing neurons and increased vasopressinergic stores in vesicles in the median eminence, leading to an increased ratio of vasopressin/CRH that is secreted.Hashimoto et al. found that the pituitary-adrenocortical response to vasopressin was enhanced in rats that were chronically immobilized as compared with unstressed controls, whereas responses to exogenous CRH remained unaltered in these animals. They suggested that chronic stress caused a hypersensitivity of the pituitary to vasopressin. In pigs tethered for a 10 to 13week period, the absolute ACTH and cortisol responses to exogenous CRH, vasopressin, or a combination of these two peptides (Chapter 6) are not significantly altered, as compared with loose housed control animals. The cortisol/ACTH ratio after CRH treatment, however, is significantly higher in tethered than in loose pigs.In summary, chronic stress leads to an increase in the sensitivity and capacity of the adrenal cortex to circulating ACTH, whereas the sensitivity of the pituitary to stimulation with CRH and/or vasopressin in pigs remains unaltered. These findings again point to adaptive changes in adrenocortical function as a consequence of stress, possibly mediated by mechanisms modulating adrenocortical sensitivity to ACTH.Adaptation to Chronic StressActivation of stress systems results in behavioural and physiological changes which allow the organism to adapt. In general, adaptive responses to stress involve a redirection of both behaviour and energy. Simultaneously, digestion and anabolic processes, such as growth, reproduction and immune function are suppressed. It appears that the ability to regulate the stress response appropriately may be as important as the ability to initiate it. Containment of the stress response is crucial to avoid detrimental consequences of excessive mobilization of resources and behavioural responses.As stated earlier, chronically stressed pigs develop changes in adrenocortical function so that further responsiveness of the adrenal system is maintained, despite elevated glucocorticoid levels. The hypersecretion of cortisol -is maintained for at least three complete oestrous cycles after tethering (approximately 9 weeks) and thereafter it gradually disappears (Chapter 3). This suggests that adaptive changes occur during chronic stress affecting HPA-activity and leading to normalization of adrenocortical output. Nevertheless, the fact that both the sensitivity and the capacity of the adrenal cortex are increased during the same period (Chapters 4, 5, and 6), suggests that adrenocortical function has chronically changed. Moreover, the apparent adaptation of cortisol levels during chronic stress does not imply normalization of brain mechanisms controlling cortisol concentrations in response to challenges or stressors (Chapter 5). Presumably, adaptation consists in changing certain "set-points" in order to meet the new demands.The mechanisms responsible for these adaptive changes during chronic stress likely include an increase in the activity of endogenous opioid systems. It has been demonstrated that tethered sows may develop stereotypies associated with and dependent on the activation of opioid systems. Administration of naloxone, a specific opiate receptor antagonist, has shown to block or reduce the occurrence of this invariant behaviour. Although we did not quantify behaviour in our experiments, we also observed performance of stereotypies such as bar or chain biting and sham chewing in tethered gilts. Brain opioid systems, however, are not only involved in the neurochemical control of behaviour, e.g., of stereotypies, but have also been implicated in the regulation of the activity of several hormonal systems, including the HPA axis. Thus, chronic stress of tethered housing leads to increased activity of endogenous opioid systems and may therefore affect the HPA axis by means of opioids. In view of this hypothesis, we subjected both tethered and loose housed female pigs to an acute nose-sling challenge. The enhanced and prolonged ACTH and cortisol responses to nose-sling challenge after pretreatment with naloxone (Chapter 5) point to an activation of both the HPA system and the endogenous opioid systems. This naloxone-dependent increment in ACTH and the cortisol responses are significantly greater in animals tethered for a 10 to 11-week period than in loose animals, indicating that the opioidmediated suppression of the pituitary-adrenocortical response is increased during chronic stress (Chapter 5). It may be speculated that the gradual reduction of the hypercortisolaemia, observed during the same period of tethered housing is also a consequence of an increase in the impact of endogenous opioid systems. Based on our findings and on literature data, we postulate the hypothalamus to be a key central site for the mediation of this effect of opioids (Chapter 5). All in all, these data indicate that chronic stress of tethered housing leads to adaptive changes in opioid systems modifying behavioural as well as endocrine reactions.Individual Differences in Pituitary-Adrenocortical ResponsesA major focus of the stress and coping literature has been on individual differences in reactivity to stressors under challenging conditions. There is ample evidence that not all individuals of the same species experience the same situation as stressful, and, vice versa, that the same stressors do not necessarily result in identical behavioural and peripheral responses. As mentioned in the general introduction, differences between individuals in coping with stressful situations appear to be related to genetic constitution, as well as to prior (particularly: early life) experiences, and the actual physiological and psychological state of the organism. Important in this respect is the way in which an individual estimates the situation and its possibilities to cope with the situation.In the present project, we obtained evidence that pigs express individual variability in the (re)activity of the pituitary-adrenocortical system in adapting to the chronic stress of tethered housing. There are, for example, great differences in. basal cortisol levels between individual pigs during tethered housing (Chapter 3), particularly with respect to the moment of disappearance of the stress-induced hypercortisolaemia. In addition, as described in the experiment in Chapter 4, considerable differences are found between individual animals in their cortisol responses to challenge with exogenous ACTH. Within the same pigs, however, the cortisol response to repeated challenge with ACTH (measured in the loose housed control groups) is consistent during an 18-week period of loose housing. This is in good agreement with findings of others in pigs, indicating that the adrenocortical reactivity is an individual characteristic that remains stable in time. The interanimal differences in the adrenocortical reactivity pattern are particularly evident during exposure to chronic stress (Chapter 4). It is noteworthy, that the greatest individual differences are observed in animals that are tethered housed under the most restricted conditions (i.e. lacking possibilities for visual and tactile contacts with conspecifics and with very limited visual control over the environment).It may be argued that variability among individuals in their ability to adapt to conditions inducing stress responses reflects differences in neuroendocrine susceptibility to the stressful situation and the degree of control sensed by the individuals. Hessing demonstrated that types of pigs can be identified based on individual behavioural characteristics and that those characteristics are related to different physiological strategies displayed in response to stressors. In addition, Schouten and collaborators not only showed individual differences in performance of stress-induced stereotypies between tethered pigs, but also in feeding- induced cardiovascular responses during prolonged tethered housing. It has been suggested that these differences in behavioural and physiological reactions may represent individual coping characteristics of the animals involved.Practical ImplicationsConsequences of Chronic Stress and HypercortisolaemiaIn the present study, prolonged tethered housing of female pigs was used as a chronic stress paradigm. Although this husbandry system, which is used in intensive pig farming, may be advantageous from an economical point of view, the well-being of the animals is lost sight of. Tethered pigs are housed in a barren environment with physical restraint and social restriction, which may have consequences for the animal's behavioural and physiological performance.We showed that tethered pigs develop a hypercortisolaemia. Chronic elevation of cortisol levels may have profound physiological consequences. As has been discussed in Chapter 1, chronic activation of the catabolic stress response may ultimately lead to various pathophysiological states. The systems responsible for reproduction, growth and immunity are directly linked to the stress system, and each is profoundly influenced by the effectors of the stress response. It has been reported that tethered housing of pigs may induce reproductive disorders, such as a reduced rate of oestrous detection (e.g., occurrence of silent oestrus) and a reduced pregnancy rate. The present findings show that 3-6 weeks of tethered housing results in significantly decreased levels of plasma progesterone throughout the oestrous cycle (Chapter 2). No effects of 3-6 weeks of chronic stress were found on plasma LH concentrations, oestrous behaviour or on the length of the oestrous cycle. Nonetheless, it is well possible that more prolonged tethered housing may disrupt LH secretion or ovarian function and thereby adversely affect reproductive performance in the pig. Indeed, Helmond, Soede and Kemp recently showed that in tethered sows the duration of oestrous behaviour was significantly shorter as compared with loose housed sows (to be published). Moreover, the pulsatile LH release in that study seemed to be more "chaotic" in the tethered animals.Besides suppression of reproductive performance, long-term activation of the HPA axis and consequently elevated cortisol levels may have metabolic (myopathy, fatigue, changes in glycemia) and cardiovascular consequences (hypertension). Furthermore, compromised growth and tissue repair, and peptic ulceration may occur, as well as immunosuppression. In this respect, parallels have been drawn between the chronically stressed animal model and several human diseases. It has been demonstrated in clinical studies that some common psychiatric disorders, such as depression, panic disorder, anxiety and anorexia nervosa, may represent disorders occurring in response to stress. Hypersecretion of glucocorticoids and resistance to glucocorticoid negative feedback have been shown to occur in people with anorexia nervosa and major depression.In addition to changes in HPA regulation, long-term exposure of pigs to tethered housing may lead to development of stereotypies and persisting disturbances in the hormonal, cardiovascular, and immune systems. Von Borell and Ladewig showed that growth rate was reduced in tethered sows as compared with loose housed sows, despite the fact that both groups had similar food intake. This reduction in growth rate indicates disturbed body function, possibly caused by stress related catabolic processes.As described previously, we also found that chronic stress leads to an increase in the impact of endogenous opioid systems that mitigate HPA responses to additional acute stressors. Besides analgesic effects, activation of endogenous opioid systems may have a broader adaptive function. It has been demonstrated that rats which can turn off shock stimulation show no opioid activation, whereas yoked animals, which experience the same electric shock without being able to control its offset, give evidence of stress-activated opioids. These data indicate that not the physically painful stimulation per se, but rather the psychological stress of its uncontrollability seems to be the key factor in opioid activation. By blunting the aversive impact of stressors, endogenous opioids enable the individual to deal more effectively with distressing environmental events. This contention is in line with the idea that stereotypies, displayed when loss of control is experienced and associated with activation of opioid systems, may serve a de-arousal purpose. Schouten et al, showed that the feeding-induced cardiovascular response is decreased and shortened in tethered pigs performing stereotypies, and that this effect is opioid-dependent. The adaptive alterations in opioid systems in tethered pigs may thus be a means of coping with the chronic stress conditions, thereby preventing excessive stress responses (e.g., of the hormonal and cardiovascular systems). However, all of the above mentioned adaptations in behavioural and physiological performance have to be recognized as symptoms of chronic stress. Therefore, they indicate that welfare and health of tethered pigs are compromised.Concluding RemarksThe experiments described in this thesis provide evidence that long-term tethered housing of female pigs induces adaptive changes in adrenocortical function and in activity of endogenous opioid systems. Important in this respect is the finding that loss of control and predictability by factors, such as lack of (visual) information and restriction of social contact, play an important role in the development of these changes. It should be kept in mind, that the adaptive behavioural and physiological changes, observed in tethered pigs, are in fact the symptoms of their "struggle" in order to withstand the environmental demands, and thus indicate compromised welfare. Some of these changes may lead to increased vulnerability of the animals for diseases. Therefore, it is likely that the stress of tethered housing contributes to the high incidence of health problems observed in tethered sows.