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The effect of transportation on energy metabolism in young calves was assessed at two feeding levels by indirect calorimetry during a 5·5-day experimental period. Twenty-six Holstein-Friesian male calves were assigned in a 2 × 2 factorial arrangement of treatments. Calves were fed below or near the maintenance requirements (270 or 420 k) metabolizable energy per kg M0·75 per day). At 5 days of age, 13 calves were transported. The applied transportation procedure was aimed to mimic common transport of young calves. The other 13 calves were controls. Metabolizability of dietary energy was low (0·808), but similar for control and transported calves. Despite the similar salivary cortisol concentrations after transportation, heat production was higher by 23 kJ/kg M0·75 per day in transported than in control calves. Calves were not in a steady-state regarding their energy metabolism. Heat production decreased with time. Both transportation and feeding level influenced the decline in heat production with time. Only during the first 3 days after transportation was heat production enhanced, suggesting a relatively short-term effect of transportation in young calves. The impact of transportation on heat production and time related alterations in heat production were not related to the calfs activity.

Day-11 spherical and Day-13 filamentous blastocysts were cultured in vitro without supplementation of oestrogen precursors for 2 consecutive periods of 24 h, with or without endometrial tissue. Spherical Day-11 blastocysts flushed from one uterine horn (5.5 +/- 0.8 blastocysts; 5.3 +/- 0.4 mm in diameter; mean +/- s.d.) did not show gross morphological changes during the culture period. Free and conjugated oestrone (E1) and oestradiol-17 beta (E2) were released during the first and second 24 h culture period and was maximal during the first 24 h. In the presence of endometrial tissue the release of oestrogens from the blastocysts was reduced to 30-50%. Clewed Day-13 filamentous blastocysts, recovered after flushing, formed trophospheres and also in these cultures both free and conjugated E1 and E2 were released. In this case the addition of endometrial tissue did not affect the release of oestrogens from the blastocysts. Single Day-13 filamentous blastocysts cultured without endometrial tissue (n = 33, from 4 gilts), developed trophospheres and all blastocysts released E1 and E2. During the first 24 h on average per blastocyst 2.6 ng E1 (range 1.6-4.0) and 9.7 ng E2 (range 4.3-15.3) were released. In all cultures of spherical and filamentous blastocysts E2 release exceeded E1 release.These data show that Day 11-13 blastocysts are able to release both free and conjugated E1 and E2 during in vitro culture from endogenous precursors. The addition of endometrial tissue reduced the release of oestrogens from Day-11 but not Day-13 blastocysts. Whether this is due to an altered oestrogen release of the blastocysts in the presence of endometrium on Day-11 or due, to an increased uptake of oestrogens by the endometrium on Day-11 remains to be investigated.

In the 1st of 2 experiments, 5 gilts were injected with saline at the onset of standing oestrus (control oestrus), and at the next cycle the same gilts were injected with 100 mu g GnRH analogue. The LH and FSH peaks in the GnRH-treated oestrus were not significantly different from those in the control oestrus, but both peaks were sharper in the GnRH-treated oestrus. In the 2nd experiment, 49 gilts were injected with 100 mu g GnRH at the onset of standing oestrus or were not treated (controls), and slaughtered on day 4, 11, 13 or 35 of pregnancy. For animals slaughtered on day 4, 11 or 13, compared with controls, treated animals had significantly more corpora lutea (16.3+or-2.3 vs. 14.8+or-1.5) and embryos (15.0+or-3.0 vs. 12.1+or-3.4). Embryonic mortality on day 35 was 24.7% in the treated group vs. 11.3% in controls. The av. number of corpora lutea for gilts slaughtered on all 4 days was greater in treated gilts than in controls (16.4+or-2.3 vs. 14.3+or-3.3), but the difference was not significant. A similar trend was observed for number of embryos (15.4+or-2.0 vs. 13.0+or-3.3).

Progesterone profiles of 7 cycling gilts and those of 6 gilts inseminated on day 1 (1 day after the onset of oestrus) were studied. The results showed that plasma progesterone concentration in the inseminated group was higher (P

Effects of rearing condition on behavioural and physiological reaction to social confrontations and to social and non-social stressors were studied in female pigs. The pigs were reared under either poor (the standard farrowing crate) or enriched (group of free-ranging sows with piglets) conditions. At the age of 14-17 weeks, the pigs were exposed to a series of social confrontations where an intruder was introduced into the home pen of a resident. The results show the presence of a clear difference in terms of aggressive behaviour between residents and intruders from enriched but not from poor rearing conditions. Furthermore pigs reared under poor conditions inflicted more wounds on each other. We suggest that this reflects a difficulty in establishing a dominance relationship in poorly reared pigs, caused by impaired development of social skills in these pigs. Subsequently, reaction to novel object, non-social and social stress was measured in adult age, showing that the effects of rearing conditions are long-lasting, and give rise to differences in reaction to challenges in that pigs from enriched rearing conditions showed more avoidance behaviour than pigs from poor rearing conditions.

Effects of rearing conditions on the development of social relationships were investigated in female pigs. For that purpose, one group of piglets was reared in a poor environment (i.e., a commercially used indoor 4.2-m2 standard farrowing crate) and the other in an enriched environment (i.e., 0.5-ha outdoor pasture with half-open farrowing crates). After weaning, all piglets were housed in pairs under standard conditions. Dominant and subordinate individuals were distinguished within pairs and social relationships between members of a pair were studied in adulthood. The results show that pairs reared in a poor environment behave more aggresively. The subordinates of these pairs also develop symptoms indicative of chronic social stress exposure: i.e., they show a delayed onset of puberty, a smaller daily weight gain, and elevated basal cortisol levels that are also higher 5 h after an acute restraint stressor (tethering stress). No such deviations were found in subordinates reared in an enriched environment. It is concluded that rearing piglets in a poor environment may facilitate the development of social stress in adult (subordinate) pigs. Two underlying mechanisms may be proposed: a) deteriorated social skills lead to increased social stress, or b) a failure to cope with stressors in general, and the stress of being subordinate in particular, occurs in animals reared under poor conditions.

The influence of tethered housing (a condition of chronic stress) on morning and evening basal plasma cortisol levels was investigated in a longitudinal study in cyclic female nulliparous pigs (gilts). After a period of loose housing in individual pens ("nonstress" estrous cycles), six cannulated gilts were tethered by a neck chain and housed for a period of 20 wk (chronic stress estrous cycles). Blood was sampled twice daily (1000 and 1800 hr) for cortisol determination. Plasma cortisol levels showed a diurnal rhythm with significantly higher levels at 1000 hr than at 1800 hr. Tethered housing induced a significant increase in the 1800-hr plasma cortisol concentrations during the first three estrous cycles after tethering, whereas the 1000-hr plasma cortisol concentrations did not change throughout the experimental period. During the period of increased 1800-hr levels, cortisol was still released in a circadian fashion, albeit, the rhythm was flattened. In control gilts, housed loose during the entire experimental period, plasma cortisol concentrations at 1000 hr and at 1800 hr remained unaltered and 1000-hr cortisol concentrations were significantly higher than the 1800-hr concentrations during the experimental period. Therefore, possible effects of the experimental procedure or age-related effects could be excluded. These data indicate that, in tethered gilts, the chronic stress-induced hypercortisolemia is of transient nature, suggesting adaptive changes in the regulation of the hypothalamic-pituitary-adrenocortical axis. In addition, the data reveal circadian differences in the effect of chronic stress on hypothalamic-pituitary- adrenocortical function.

Cytosolic oestrogen receptors (ER(c)) were determined with a Dextran-coated charcoal assay and Scatchard plot analysis in endometrial tissue of non-pregnant and pregnant gilts on Days 10-13 after standing oestrus. The Kd of the ER(c) was 0.40 +/- 0.04 nM (mean +/- SEM) and was not affected by day or reproductive state. The endometrial ER(c) concentration was affected by day and reproductive state (P

Long-term tethered housing of pigs, which is not unusual in modern pig breeding farms, largely impairs the animals in their movements and thus represents a condition of chronic restraint stress. Some tethered pigs display stereotypies, that can be blocked by the opiate receptor antagonist naloxone, suggesnng increased activity of endogenous opioid systems in these animals. We have used the tethered pig as a model to further investigate the role of opioids in changes in hormonal and physiological regulation induced by chronic stress

The timing between the onset of oestrus and the preovulatory Luteinizing hormone (LH) peak in gilts was investigated. The LH peak was most frequently observed approximately 1Oh after the onset of oestrus, although large variations from 20h before to more than 24h after the onset of oestrus were noticed. The time interval between the LH peak and ovulation, however, was rather constant and so a large variation with regard to the time interval between the onset of oestrus and ovulation was observed. Insemination of pigs at a fixed time interval after the onset of oestrus resulted in conceptions with sperm of variable age. The results suggest a higher incidence of embryonic mortality with an increasing interval between the time of insemination and ovulation. The rate of development of pig embryos between day 3 and 7 of pregnancy was studied with respect to the time of ovulation. The results indicate that pig embryos double their number of cells every 11 hours.

Blastocysts were flushed out of both uterine horns of gilts on Days 10, 11, 12 or 13. In mated non-pregnant gilts flushing had no effect on progesterone profile or cycle length (20.8 +/- 0.4 versus 20.6 +/- 0.6 days in the preflush cycle, N = 6, mean +/- s.e.m.). Flushing the blastocysts out of the uterine horns on Day 10 resulted in a cycle with a normal progesterone profile and a normal length (21.2 +/- 0.4 days, N = 5). Flushing on Days 11, 12 or 13 resulted in a normal cycle or in maintenance of the CL for 3-13 days as indicated by elevated progesterone concentrations and an increased interoestrous interval of, respectively, 22.0 +/- 1.2 versus 19.8 +/- 0.6 days (Day 11; N = 6), 24.8 +/- 1.4 versus 21.0 +/- 0.6 days (Day 12; N = 5; P less than 0.05) and 26.3 +/- 2.3 versus 20.5 +/- 0.4 days (Day 13; N = 6; P less than 0.05). There was a positive relationship between the change in interoestrous interval and the interval between the first observed standing oestrus and flushing of the blastocysts (rs = 0.350; n = 22; P less than 0.1). There was a large variation in the diameter of the blastocysts flushed on the same day. Only in those gilts in which the blastocysts were greater than or equal to 8 mm or filamentous were the CL maintained for 3 or more days. These results indicate that a first signal for maternal recognition of pregnancy is generated on Day 12 and that blastocysts greater than or equal to 8 mm are required for prolongation of CL function for 3 or more days. Since CL function is only extended for a maximum of 13 days (mean 7.4 +/- 1.0), a second signal seems necessary to maintain the CL for the whole period of pregnancy.

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 ofβ-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.

A vital link in a complex of physiological processes occurring during early pregnancy is the so-called maternal recognition of pregnancy: the prolongation of ovarian luteal function for continuation of progesterone secretion by an anti-luteolytic action of the developing embryos. Progesterone is crucial for the secretion of histotrophe, which in turn is essential for conceptus development and inhibition of uterine contraction activity. In the pig the corpora lutea (CL) remain the major source of progesterone throughout pregnancy.To establish the time of maternal recognition of pregnancy in the pig (a) progesterone profiles of pregnant and cyclic gilts were compared (chapter III) and (b) blastocysts were flushed out of the uterine horns of gilts on Days 10-13 of pregnancy (chapter IV).From Day 14, the progesterone concentration in pregnant gilts was higher than in cyclic gilts. This indicates that rescue of the CL during pregnancy occurs before Day 14.Flushing of blastocysts out of both uterine horns on Day 10 of pregnancy resulted in a cycle with a normal progesterone profile and a normal length. Flushing of blastocysts on Days 11, 12 or 13 resulted in the maintenance of the CL for 3-13 days in those gilts in which at least some of the blastocysts were≥8 mm or filamentous. Since CL function is only extended for on average 7 days, a second signal seems necessary to maintain the CL throughout pregnancy.At the time that a first signal for maternal recognition of pregnancy is generated, the blastocysts start to secrete appreciable amounts of proteins, prostaglandins and oestrogens. So far, no anti- luteolytic role of proteins has been demonstrated in the pig, and data concerning an anti-luteolytic action of prostaglandin E2 are contradictory. Oestrogens exert an anti-luteolytic effect as indicated by CL maintenance in cyclic gilts after i.m. injection of large doses of oestrogens.The ability of blastocysts to synthesize oestrogens has been demonstrated by conversion of labelled precursors into both oestrone and oestradiol-17βduring in vitro culture. The conversion of androgens to oestrogens by aromatase activity has first been detected in litters with spherical Day-11 blastocysts.To gain a clear understanding of the participation of each blastocyst in the initiation of oestrogen production, aromatase activity was determined in individual Day-11 blastocysts (chapter V). The oestrogen-synthesis capacity of blastocysts during the period that a first signal for maternal recognition of pregnancy is generated, was determined by measurement of oestrogen release by spherical and filamentous blastocysts during in vitro culture without supplementation of precursors (chapter VI).Blastocysts flushed out of both uterine horns of 10 gilts on Day 11 of pregnancy varied in diameter and aromatase activity. The variation in aromatase activity reflected a difference between and within gilts. Of the total variation between all blastocysts, 33% was due to differences within gilts. A positive exponential relationship between blastocyst diameter and aromatase activity was determined, but this relationship differed between gilts.Day-11 spherical blastocysts and clewed Day-13 filamentous blastocysts released both free and conjugated oestrone and oestradiol-17βduring 2 successive periods of 24 h in vitro culture. Individually collected and cultured Day-13 filamentous blastocysts developed trophospheres, and differences existed in the release of oestrone and oestradiol-17βbetween and within gilts. Individual Day-13 filamentous blastocysts released on average 2.6 ng oestrone and 9.8 ng oestradiol-17βper 24 h. In all cultures of spherical and filamentous blastocysts, the oestradiol-17βrelease was significantly larger than the oestrone release. The data indicate that Day-11 and Day-13 blastocysts are able to synthesize oestrogens from endogenous precursors.Assuming that CL function in cyclic gilts can be maintained by i.m. injection of large doses Of oestrogens, several mechanisms have been proposed to explain the anti-luteolytic action of oestrogens in the pig. Oestrogens released by blastocysts, however, are supposed to exert their anti-luteolytic effect at the level of the uterine endometrial epithelium. Large doses of intramuscularly injected oestrogens, however, may act anti- luteolytica1ly via a direct or indirect (pituitary) effect on the CL.To investigate the influence of oestrogens at the level of the uterine endometrial epithelium, the concentration of endometrial oestradiol receptors at the expected time of maternal recognition of pregnancy in cyclic and pregnant gilts was determined (chapter VII).Cytosolic oestradiol receptor concentrations did not change between Days 10-13 in cyclic gilts, but it decreased in pregnant gilts. The cytosolic oestradiol receptor concentration on Day 12 of pregnancy was higher in gilts with spherical blastocysts than in gilts with filamentous blastocysts.To investigate the effect of a locally administered physiological dose of oestrogens, oestradiol-17βwas administered intrauterine in non-pregnant gilts and gilts pregnant up to Day 10 (chapter VIII).Injection of 380 ng oestradiol-17βevery 6 h from Day 11 to Day 15 into both uterine horns resulted in normal cycles in non-pregnant gilts and gilts Pregnant up to Day 10. Implantation of three oestradiol-17βreleasing micropellets into each uterine horn of non-pregnant gilts on Day 10 of the cycle resulted also in normal oestrous cycles. The results indicate that administration of oestradiol-17βin physiological doses into the uterus lumen, the presumed site of action in the maternal recognition of pregnancy, does not result in CL maintenance in non-pregnant gilts.From this study it may be concluded that a first signal for CL maintenance is generated in gilts in which at least some of the blastocysts were≥8 mm or filamentous, but a second signal is necessary to maintain CL function throughout pregnancy. Already before the time of generating a first signal for CL maintenance, the blastocysts start to synthesize both oestradiol-17βand oestrone. The oestrogens exert an effect on the uterine endometrium, which is indicated by a change in endometrial oestradiol receptor concentration. However, since intrauterine oestradiol-17βadministration does not mimic the effect of the blastocysts, the first signal for maternal recognition of pregnancy is not a simple, merely oestrogenmediated mechanism.