Hair cortisol concentration in postpartum dairy cows and its association with parameters of milk production.

Hair cortisol concentration (HCC) is considered as an indicator for a minimally invasive assessment of long-term stress. In dairy cows, in addition to stress influences, changing physiological conditions during gestation and lactation (eg, due to varying energy requirements or fluctuating milk yield) may affect HCCs. Thus, the aim of our study was to investigate HCCs of dairy cows during different stages of lactation and to determine the relationship between milk production traits and hair cortisol levels. Samples of natural hair and regrown hair were collected from 41 multiparous Holstein Friesian cows at 100-d intervals from parturition to 300 d postpartum. All samples were analyzed for cortisol concentration and the association of HCC with milk productions traits was evaluated. Our results show that cortisol concentration in natural hair increased after parturition and was highest 200 d postpartum. Cumulative milk yield from parturition to 300 d showed moderate and positive correlation with HCC in natural hair at 300 d. There was a positive correlation between urea concentration in milk and cortisol levels in regrown hair at 200 d, and between somatic cell count in milk and HCC in natural and regrown hairs 200 d postpartum. Together, these findings suggest that physiological loads during lactation, eg, caused by metabolic stress and/or inflammation, may be associated with increased HCC levels. In addition, the results on hair color confirm previous findings in cattle that black hair has higher cortisol concentrations than white hair. Black hair therefore appears to be more suitable for hair cortisol analysis as it provides higher protection against photodegradation.

Early-life maternal deprivation affects the mother-offspring relationship in domestic pigs, as well as the neuroendocrine development and coping behavior of piglets.

Early-life adversity may have programming effects on the psychological and physiological development of offspring. Domestic pigs (Sus scrofa) are an excellent model species for studying these effects because of their many physiological similarities to humans. Piglets from 10 sows were subjected to daily 2-h maternal deprivation on postnatal days (PND) 2-15 alone (DA) or in a group of littermates (DG). Control piglets (C) from 10 sows stayed with their mothers. Mother-offspring interaction, milk oxytocin, and cortisol were analyzed. An open-field/novel-object (OF/NO) test was performed with piglets on PNDs 16 and 40. Plasma cortisol and immune parameters were determined on PND 5 and 16. Two piglets from each group and sow were sacrificed on PND 20 and stress-related gene expression in the limbic system and prefrontal cortex (PFC), as well as splenic lymphocyte proliferative abilities, were examined. The milk cortisol of sows increased during the first separation of mother and offspring on the second day of lactation, whereas milk oxytocin did not change. The increase in cortisol by the OF/NO test on PND 16 was greater in C piglets than in DA and DG ones. DA piglets showed less agitated behavior than DG and C piglets in the OF/NO test at PND 16, but appeared more fearful. On PND 40, DA piglets showed more arousal than DG and C piglets in the OF/NO test. Neither plasma IgA nor N/L ratios in blood nor mitogen-induced proliferation of spleen lymphocytes were affected by deprivation. We found a higher mRNA expression of CRHR1 in the hypothalamus and a higher expression of MR in the hippocampus in DA piglets than in DG ones. The expression of GR, MR, and CRHR1 genes in the PFC was reduced by maternal deprivation, however, the expression of arginine vasopressin and oxytocin receptors was not affected. Repeated maternal deprivation induces sustained effects on stress reactivity and behavior of domestic piglets. Some of these effects were buffered by the presence of littermates. In addition, we found sex-specific differences in behavior and gene expression.

Effects of 2 liquid feeding rates over the first 3 months of life on whole-body energy metabolism and energy use efficiency of dairy calves up to 5 months.

Intensified milk replacer (MR) feeding in calves has nutritional long-term effects and is suggested to increase milk production later in life. However, the underlying mechanisms are not completely understood. The aim of our study was to investigate whether MR feeding intensity has long-term effects on energy metabolism and energy use efficiency of dairy calves. Newborn female Holstein calves (n = 28) were randomly assigned to 2 liquid feeding groups offered daily either 10% of body weight (BW) colostrum followed by 10% of BW MR (10%-MR) or 12% of BW colostrum followed by 20% of BW MR (20%-MR). Calves were housed individually. Weaning was completed by the end of wk 12. Hay and calf starter were fed from d 1 until the end of wk 14 and 16, respectively. A total mixed ration was fed from wk 11 onward, and the metabolizable energy intake (MEI) was determined daily. Energy metabolism of calves was measured in respiratory chambers before weaning in wk 6 and 9, and after weaning in wk 14 and 22. The MEI/BW0.75 was higher before weaning but lower during and shortly after weaning in 20%-MR calves. During the preweaning period, the 20%-MR animals had higher average daily gain, BW, back fat thickness and muscle diameter, but lower plasma ß-hydroxybutyrate concentrations. The group difference in average daily gain ceased in wk 9, differences in back fat thickness and muscle diameter ceased after weaning, whereas difference in BW0.75 persisted until wk 23. The energy conversion ratio (BW gain/MEI) was not different before weaning, but was lower during and after weaning in 20%-MR calves. The higher MEI and BW0.75 in 20%-MR calves resulted in higher heat production (HP), as well as in higher carbohydrate oxidation (COX) and fat oxidation during the preweaning period. Gas exchange variables normalized to BW0.75 or MEI differed between groups only during preweaning. The energy balance was lower in 10%-MR calves in wk 6 and 9. The HP/BW0.75 and COX/BW0.75 were higher, whereas HP/MEI was lower in 20%-MR calves in wk 6. When normalized to BW0.75 and MEI, HP in wk 6 and 9, and COX in wk 9 was lower in 20%-MR calves. In conclusion, 20%-MR calves showed greater efficiency estimates preweaning, but this effect did not occur after weaning, suggesting that energy use efficiency does not persist until later stages in life.

A natural Ala610Val substitution causing glucocorticoid receptor hypersensitivity aggravates consequences of endotoxemia.

Despite the crucial role of glucocorticoid receptor (GR) in proper immune responses, the effect of GR hypersensitivity on inflammation is rarely reported. To fill this knowledge gap, we exploited the natural gain-of-function substitution in the porcine glucocorticoid receptor (GRAla610Val) and challenged pigs carrying normal or hypersensitive GR using 50 µg/kg lipopolysaccharide (LPS) following pretreatment with either saline or single bolus of 60 µg/kg dexamethasone (DEX). The GRAla610Val substitution reduced baseline cortisol, adrenocorticotropic hormone (ACTH), and triglyceride concentration and granulocyte proportion whereas baseline platelet counts were elevated. Val-carriers, i.e. AlaVal as well as ValVal pigs, showed less LPS-induced cortisol rise but the cortisol fold change was similar in all genotypes. Differently, ACTH response to LPS was most significant in GRAla610Val heterozygotes (AlaVal). LPS-induced disorders, including sickness behaviors, anorexia, thrombocytopenia, cytokine production, and metabolic alterations were more intense in Val-carriers. On the other hand, Val-carriers were more sensitive to DEX effect than wild types (AlaAla) during endotoxemia, but not under unchallenged conditions. This is the first report revealing aggravated responses to endotoxemia by GR gain-of-function. Together, these results imply that GR hypersensitivity is difficult to diagnose but may represent a risk factor for endotoxemia and sepsis.

Early-Life Maternal Deprivation Predicts Stronger Sickness Behaviour and Reduced Immune Responses to Acute Endotoxaemia in a Pig Model.

Early-life adversity may have programming effects on neuroendocrine and immune adaptation mechanisms in humans and socially living animals. Using a pig model, we investigated the effect of daily 2-h maternal and littermate deprivation from postnatal days 2-15, either alone (DA) or in a group of littermates (DG) on the neuroendocrine, immunological and behavioural responses of piglets challenged with the bacterial endotoxin lipopolysaccharide (LPS) on day 42. LPS increased plasma concentrations of cortisol, tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10) and induced typical signs of sickness in all piglets. DA+DG piglets showed stronger signs of sickness compared to control (C) piglets. Plasma TNF-α concentrations were significantly lower in DA+DG males. In addition, the TNF-α/IL-10 ratio was significantly lower in DA than in DG and C males. Gene expression analyses showed lower hypothalamic TNF-α mRNA expression and diminished mRNA expression of the mineralocorticoid receptor (MR) and IL-10 in the amygdala of DA+DG piglets in response to LPS. Interestingly, males showed a higher MR- and a lower IL-10 mRNA expression in the amygdala than females. The present data suggest that repeated maternal deprivation during early life may alter neuroendocrine and immune responses to acute endotoxaemia in a sex-specific manner.

Is it getting in the hair? - Cortisol concentrations in native, regrown and segmented hairs of cattle and pigs after repeated ACTH administrations.

Adrenocorticotropic hormone (ACTH) is part of the hypothalamic-pituitary-adrenal axis response to stress and induces the release of cortisol, which is commonly used as an indicator in stress and animal welfare research. In recent years, hair cortisol concentration (HCC) gained increasing importance as a promising retrospective indicator for stress in animals. Thus, the aim of our study was to validate HCC as a potential indicator of increased endogenous cortisol release in cattle and pigs by repeated ACTH administrations followed by cortisol analysis in different hair types. For this purpose, 34 cattle and 38 gilts were treated either with repeated i.m. injections of ACTH or saline every second day over a period of 4 weeks. Saliva samples were taken before and after injections once a week from selected animals to verify the endogenous cortisol response. At the end of the treatment (week 4) and after 8 and 12 weeks, samples of natural and regrown hair were taken from the caudo-dorsal region of the back and analyzed for cortisol concentrations. In addition, natural hair was sampled after 12 weeks and cut into segments prior to analysis. Treatment with ACTH revealed a significant increase in salivary cortisol after application in both species, although this increase was attenuated in pigs compared to cattle. In week 4, HCCs were significantly elevated in natural and regrown hair of ACTH-treated animals. In cattle, HCCs significantly increased after ACTH treatment in natural, regrown and segmental hair compared with control animals, indicating that HCC may be a promising indicator of stress, as cortisol levels in all hair types reflected the preceding period with increased cortisol release. In pigs, there were no differences in HCCs between treatments. This may be caused by the lower systemic cortisol response in pigs, but seasonally reduced hair growth and external cross-contamination of hair by saliva and urine under commercial husbandry conditions may also interfere with the validity of HCC in this species.

It's getting hairy - External contamination may affect the validity of hair cortisol as an indicator of stress in pigs and cattle.

Hair cortisol concentration (HCC) is increasingly used for the assessment of enhanced hypothalamic-pituitary-adrenal (HPA) axis activity, e.g., caused by repeated or long-term stressful conditions or pathologies. However, there is still a lack of knowledge regarding the mechanisms and sources of cortisol incorporation into the hair and possible confounding factors, especially in non-human animals. Farm animals are usually kept under confined housing conditions, have close contact with each other and with soiled environments and may thus be exposed to contamination with urine, feces and saliva, which are known to contain substantial concentrations of cortisol or its metabolites. Therefore, the aim of our study was to investigate the impact of contamination with urine, feces and saliva on the cortisol concentration in the hair of pigs and cattle. In an in vitro experiment, hair strands of 12 pigs and 12 cattle were repeatedly contaminated with urine and saliva, containing either low or high cortisol concentrations, or with the feces of the respective species and were compared with hair treated with water or untreated hair. Contamination was performed over 20 days for two hours daily. Thereafter, all samples were washed, ground, extracted and analyzed for HCCs following the same protocol. Our results showed that contamination with urine caused a considerable and concentration-dependent increase in HCCs in both species. Saliva had a comparable effect only in cattle. In addition, the treatment with water led to a reduction in the cortisol concentration of porcine hair, whereas contamination with feces caused an increase in HCC only in cattle. Our findings provide evidence that contamination of hair with cortisol-containing body fluids causes incorporation of cortisol into the hair shaft, probably via diffusion depending on the concentration gradient. In that case, cortisol in hair derived from contamination cannot be distinguished from cortisol originating from blood. Thus, contamination may affect the validity of hair cortisol as an indicator of HPA axis activity and cannot be prevented by decontamination protocols prior to analysis.

Within a hair's breadth - Factors influencing hair cortisol levels in pigs and cattle.

During the last two decades, hair cortisol concentration (HCC) has proven to be a promising marker for the evaluation of increased hypothalamic-pituitary-adrenal axis activity caused by repeated or long-term stressful conditions. A minimally invasive sampling procedure, simple storage and the retrospective characteristic of one hair sample are reasons why HCC is increasingly used not only in human medicine but also in animal welfare research. However, before applying HCC as a reliable indicator for stress, it is important to investigate potential influencing factors in addition to stressors in the species of interest. Thus, the aim of our study was to elucidate the impact of age, sex, hair color, body region, age of hair segments and season of hair sampling on HCC in pigs and cattle. Hair samples were taken by electric clippers and analyzed by ELISA after extraction. Our results show similar effects of influencing factors in both species. Significantly increased HCCs were found in young animals after birth compared with older age groups. In addition, HCCs were significantly higher in samples obtained from the tail tip in comparison with samples from the shoulder, neck and back regions, in black hair compared with white hair and in distal hair segments. Season had an impact on HCC only in cattle, which exhibited higher levels in winter than in summer. In conclusion, age, body region, hair color, hair segment and season affect hair cortisol concentrations and should be considered and controlled for when HCC is applied as a potential stress indicator in pigs and cattle. In addition, further research is necessary to elucidate the mechanisms by which cortisol is incorporated into the hair shaft.

Assessing animal individuality: links between personality and laterality in pigs.

Animal individuality is challenging to explain because individual differences are regulated by multiple selective forces that lead to unique combinations of characteristics. For instance, the study of personality, a core aspect of individuality, may benefit from integrating other factors underlying individual differences, such as lateralized cerebral processing. Indeed, the approach-withdrawal hypothesis (the left hemisphere controls approach behavior, the right hemisphere controls withdrawal behavior), may account for differences in boldness or exploration between left and right hemispheric dominant individuals. To analyze the relationships between personality and laterality we tested 80 male piglets with established laterality patterns for 2 motor functions (tail curling direction and the side of the snout used for manipulation) and a combined classification integrating both motor functions using cluster analysis. We analyzed basal salivary testosterone and cortisol along with their behavior in standardized tests as pre-established indicators of different personality traits (Boldness, Exploration, Activity, Sociability, and Coping). We found that the direction of the single motor biases showed significant associations with few personality traits. However, the combined laterality classification showed more, and more robust, significant associations with different personality traits compared with the single motor biases. These results supported the approach-withdrawal hypothesis because right-biased pigs were bolder and more explorative in a context of novelty. Additionally, right-biased pigs were more sociable than left-biased pigs. Therefore, the present study indicates that personality is indeed related to lateralized cerebral processing and provides insight into the multifactorial nature of individuality.

Coping Style of Pigs Is Associated With Different Behavioral, Neurobiological and Immune Responses to Stressful Challenges.

Based on the animal's reaction to environmental challenges, consistent but different coping styles can be identified, which in turn may have consequences for health and welfare. Therefore, profound knowledge of the complex interrelationships between individual behavioral response patterns, underlying neurobiological mechanisms and immunological effects is required. The aim of this study was to examine whether pigs with different coping styles exhibit distinct behavioral, neurobiological and immune responses to stressful situations. Therefore, pigs (n = 40) were classified as proactive, reactive or intermediate animals according to a repeatedly-performed backtest, and behavioral, neuroendocrine and immune alterations were analyzed without any stress before weaning on day 28 and after a stress treatment on day 32. Our results show that the behavioral responses in an open-field/novel-object test characterized proactive pigs as more active. There were no significant differences in adrenocorticotropic hormone and cortisol concentrations between pigs with different coping characteristics. However, we found that proactive pigs displayed significantly increased plasma noradrenaline levels in response to stress, which may reflect a higher sympathetic reactivity of these animals. Furthermore, the present study revealed coping style differences in mRNA expression of mineralocorticoid, glucocorticoid, oxytocin and arginine vasopressin receptors and the immediate early gene c-fos in stress-related brain regions. While proactive pigs responded to stress with higher mRNA expression of arginine vasopressin, mineralocorticoid and glucocorticoid receptors, reactive pigs displayed higher oxytocin receptor and c-fos mRNA expression, indicating different neurobiological mechanisms of distinct coping styles in response to stressful challenges. Moreover, we also found humoral immune differences between proactive, intermediate and reactive animals. Proactive pigs had a higher total serum IgA concentration before and after stress treatment, with a significant increase in response to stress compared to reactive and intermediate pigs. In contrast, stress-induced IgM concentrations only increased in reactive and intermediate animals, suggesting that the effects of coping style on humoral immunity may differ depending on the specific function of the immunoglobulin classes. In conclusion, this multidisciplinary study expands the concept of coping style in farm animals, particularly in terms of individual stress reactivity and disease susceptibility, and thus contributes to the understanding of the biology of animal welfare.

Kinetics of Physiological and Behavioural Responses in Endotoxemic Pigs with or without Dexamethasone Treatment.

Although dexamethasone (DEX) is a widely used immunoregulatory agent, knowledge about its pharmacological properties in farm animals, especially pigs, is insufficient. Previous studies suggest that compared to other species, pigs are less sensitive to the immunosuppression conferred by DEX and more sensitive to the threat of bacterial endotoxins. However, there is a paucity of studies examining DEX immunomodulation in endotoxemia in this species. In this study, a porcine endotoxemia model was established by lipopolysaccharide (LPS) and the effect of DEX-pretreatment on the magnitude and kinetics of neuroendocrine, metabolic, hematologic, inflammatory, and behavioural responses were examined. DEX decreased cortisol, adrenocorticotropic hormone (ACTH), red blood cell, hemoglobin, hematocrit, and lymphocyte whereas glucose concentration was increased under both normal and endotoxemic conditions. By contrast, DEX decreased triglyceride, lactate, and IL-6 concentrations and increased platelet count only under an endotoxemic condition. DEX also reduced the frequency of sickness behaviour following LPS challenge. PCA showed that glucose and triglyceride metabolism together with red blood cell count mainly contributed to the separation of clusters during DEX treatment. Our study demonstrates that DEX protects pigs from inflammation and morbidity in endotoxemia, in spite of their less sensitivity to DEX. Moreover, its considerable role in the regulation of the metabolic and hematologic responses in endotoxemic pigs is revealed for the first time.

The use of hair cortisol for the assessment of stress in animals.

The hair cortisol concentration (HCC) is assumed to be a retrospective marker of integrated cortisol secretion and stress over longer periods of time. Its quantification is increasingly used in psychoneuroendocrinological studies in humans, but also in animal stress and welfare research. The measurement of HCCs for the assessment of stress offers many considerable benefits for use in domesticated and wild animals, especially due to the easy and minimally invasive sampling procedure and the representation of longer time periods in one sample. This review aims to outline the different fields of application and to assess the applicability and validity of HCC as an indicator for chronic stress or long-term activity of the hypothalamic-pituitary-adrenal axis in wild and domesticated animals. Specific hair characteristics are presented and the advantages and limitations of using HCC are discussed. An overview of findings on the impact of stress- and health-related factors on HCCs and of diverse influencing factors causing variation in hair cortisol levels in different species is given. Recommendations for the use of hair cortisol analysis are proposed and potential fields of future research are pointed out. The studies indicate an effect of age and pregnancy on HCCs, and cortisol incorporation into hair was also found to depend on hair colour, body region, sex and season of year, but these results are less consistent. Furthermore, the results in animals show that a wide array of stressors and pathological conditions alters the cortisol concentrations in hair and that HCC thereby provides a reliable and valid reflection of long-term cortisol secretion in many species. However, more research is necessary to investigate the underlying mechanisms of cortisol incorporation into the hair and to explore the hair growth characteristics in the species of interest. To overcome confounding influences, the use of standardized sampling protocols is strongly advised.

Effects of 1-Methyltryptophan on Immune Responses and the Kynurenine Pathway after Lipopolysaccharide Challenge in Pigs.

An enhanced indoleamine 2,3-dioxygenase 1 (IDO1) activity is associated with an increased mortality risk in sepsis patients. Thus, the preventive inhibition of IDO1 activity may be a promising strategy to attenuate the severity of septic shock. 1-methyltryptophan (1-MT) is currently in the interest of research due to its potential inhibitory effects on IDO1 and immunomodulatory properties. The present study aims to investigate the protective and immunomodulatory effects of 1-methyltryptophan against endotoxin-induced shock in a porcine in vivo model. Effects of 1-MT were determined on lipopolysaccharide (LPS)-induced tryptophan (TRP) degradation, immune response and sickness behaviour. 1-MT increased TRP and its metabolite kynurenic acid (KYNA) in plasma and tissues, suppressed the LPS-induced maturation of neutrophils and increased inactivity of the animals. 1-MT did not inhibit the LPS-induced degradation of TRP to kynurenine (KYN)-a marker for IDO1 activity-although the increase in KYNA indicates that degradation to one branch of the KYN pathway is facilitated. In conclusion, our findings provide no evidence for IDO1 inhibition but reveal the side effects of 1-MT that may result from the proven interference of KYNA and 1-MT with aryl hydrocarbon receptor signalling. These effects should be considered for therapeutic applications of 1-MT.

Psychosocial Stress and Immunity-What Can We Learn From Pig Studies?

Psychosocial stress may impair immune functions and provoke the development of pathologies. The underlying communication between the brain and the immune system is being studied predominantly in rodents. However, pigs offer several advantages as preclinical models for humans because pigs are more similar to humans than rodents in many anatomical and physiological characteristics. Unlike in rodents, the main stress-induced glucocorticoid in humans and pigs is cortisol with a similar circadian rhythm. In this study, we summarize data on short-term and long-term effects of social stress in pigs for their immunity and neuroendocrine regulation with consequences for their health and well-being. As typical social stressors, regrouping, crowding, social isolation, and maternal deprivation have been studied. Psychosocial stress in pigs may affect various reactions of innate and adaptive immunity, such as leukocyte distribution, cytokine secretion, lymphocyte proliferation, and antibody production as well as immune responses to viral infection or vaccination. Furthermore, social stress may induce or promote gastrointestinal diseases through dysregulation of inflammatory processes. In piglets, psychosocial stress may also result in glucocorticoid resistance of lymphocytes, which has been discussed as a cause of allergic asthma in humans. Stress-related neuroendocrine alterations in the cortico-limbic structures, such as the prefrontal cortex, amygdala, hippocampus and hypothalamus, have been demonstrated in pigs at different ages. Based on these data, we propose using pigs as models for psychosocial stress in humans to study the mechanisms of brain-to-immune and immune-to-brain communication from the systemic level down to the cellular and subcellular levels.

Sea Buckthorn Pomace Supplementation in the Diet of Growing Pigs-Effects on Fatty Acid Metabolism, HPA Activity and Immune Status.

There is evidence that sea buckthorn, as a source of n-3 polyunsaturated fatty acids (n-3 PUFA), possesses health-enhancing properties and may modulate neuroendocrine and immune functions. In the present study, we investigated the effect of sea buckthorn pomace (SBP) supplementation in the diet of growing German Landrace pigs on fatty acids in the blood and hypothalamus, peripheral immune parameters and mRNA expression of corticotropin-releasing hormone (CRH), mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in the hypothalamus and spleen. Pigs were fed diets supplemented with 12% of dried SBP or 0% SBP (control group) over an intervention period of eight weeks. The fatty acid profiles in blood plasma were significantly affected by SBP supplementation only for C18:2n-6 and n-6/n-3 PUFA ratio compared with the control group. SBP supplementation did not significantly affect the fatty acid concentrations in the hypothalamus. Furthermore, there were no significant differences in mRNA expression of CRH, MR and GR in the hypothalamus or of GR mRNA expression in the spleen. Concerning the immune status, the plasma IgG levels tended to be higher in SBP pigs, whereas the leukocyte distribution, mitogen-stimulated lymphocyte proliferation, and serum IgM levels remained unchanged. In conclusion, the SBP supplementation of the diet only caused moderate effects on fatty acid metabolism, but no significant effects on hypothalamic-pituitary-adrenal (HPA) activity and immunity in growing pigs. It seems that a beneficial effect of dietary n-3 PUFA on health and welfare is more likely to be expected during stressful situations.

Psychosocial stress sensitizes neuroendocrine and inflammatory responses to Escherichia coli challenge in domestic piglets.

Exposure to psychosocial stress can have a profound impact on immune reactivity and health mediated by hypothalamic-pituitaryadrenal (HPA) axis activation. However, current knowledge regarding the mechanisms involved in cross-sensitization between stress and the immune system is limited. Here, we investigated the effects of a single social isolation followed by repeated oral Escherichia coli (E. coli) applications on cortisol, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), haptoglobin and C-reactive protein (CRP) concentrations in the blood; on clinical signs of disease; and on mRNA expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11ß-hydroxysteroid dehydrogenase 1 and 2 (11ß-HSD1 and 11ß-HSD2), TNF-α and IL-6 in the hypothalamus, prefrontal cortex (PFC) and spleen of 7-, 21- and 35-day-old piglets. Additionally, the protein levels of splenic TNF-α and IL-6 were analyzed. Non-isolated, E. coli-challenged piglets served as a control. Social isolation for 4 h induced a rise in the plasma cortisol concentrations immediately after social treatment and after repeated E. coli applications in isolated compared to non-isolated piglets. The circulating TNF-α concentration was not affected by social treatment. Furthermore, previously isolated piglets showed a higher frequency of signs of disease in response to E. coli challenge than non-isolated piglets, while the haptoglobin and CRP concentrations did not significantly differ between social treatments. In the brain, 11ß-HSD1, 11ß-HSD2 and IL-6 mRNA expression in the hypothalamus and GR, and 11ß-HSD1 and 11ß-HSD2 mRNA expression in the PFC were higher in isolated, E. coli-challenged piglets than in the corresponding controls. Moreover, isolated piglets also displayed higher MR, 11ß-HSD1 and IL-6 mRNA expression levels and TNF-α concentrations in the spleen. The stress-induced alterations in the hypothalamus and spleen were particularly pronounced in younger piglets. The present findings may contribute to a better understanding of the complex interplay between early psychological stress and an increased risk of disease and might also have implications on aspects of the health and welfare of farm animals and humans.

Interference of stress with the somatotropic axis in pigs - lights on new biomarkers.

The acceptance of animal products is increasingly associated with standardized animal welfare, which relates to appropriate animal husbandry from birth to slaughter. In particular, shipment to the slaughterhouse is considered as a critical process exposing the animals to a number of, in part severe, stressors. New biomarkers may be useful for the assessment of animal welfare. The IGF-system has been assessed in a commercial pig transport in conjunction with established markers of stress response. Furthermore, the effect of repeated restraint as an experimental model for repeated acute stress was investigated. During shipment from farm to slaughterhouse, plasma concentrations of IGFBP-3 and IGFBP-2 were significantly reduced (p < 0.01). After shipment, the plasma concentrations of IGFBP-5, glucocorticoids and IL-2 increased but decreased after lairage (p < 0.05) whereas IGF-1 decreased after shipment (p < 0.01). Repeated acute stress increased concentrations of IGFBP-3 and IGF-1 in exsanguination blood (p < 0.05). Differential IGF- signatures can indicate altered endocrine or metabolic control and thus contain complex animal-related information. The somatotropic axis may be of particular interest when established biomarkers such as cortisol, glucose, or lactate cannot be used for the assessment of animal stress or welfare.

Social Support Modulates Stress-Related Gene Expression in Various Brain Regions of Piglets.

The presence of an affiliative conspecific may alleviate an individual's stress response in threatening conditions. However, the mechanisms and neural circuitry underlying the process of social buffering have not yet been elucidated. Using the domestic pig as an animal model, we examined the effect of a 4-h maternal and littermate deprivation on stress hormones and on mRNA expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11ß-hydroxysteroid dehydrogenase (11ß-HSD) types 1 and 2 and the immediate early gene c-fos in various brain regions of 7-, 21- and 35-day old piglets. The deprivation occurred either alone or with a familiar or unfamiliar age-matched piglet. Compared to piglets deprived alone, the presence of a conspecific animal significantly reduced free plasma cortisol concentrations and altered the MR/GR balance and 11ß-HSD2 and c-fos mRNA expression in the prefrontal cortex (PFC), amygdala and hypothalamus, but not in the hippocampus. The alterations in brain mRNA expression were particularly found in 21- or 35-day old piglets, which may reflect the species-specific postnatal ontogeny of the investigated brain regions. The buffering effects of social support were most pronounced in the amygdala, indicating its significance both for the assessment of social conspecifics as biologically relevant stimuli and for the processing of emotional states. In conclusion, the present findings provide further evidence for the importance of the cortico-limbic network underlying the abilities of individuals to cope with social stress and strongly emphasize the benefits of social partners in livestock with respect to positive welfare and health.

A Natural Mutation in Helix 5 of the Ligand Binding Domain of Glucocorticoid Receptor Enhances Receptor-Ligand Interaction.

The glucocorticoid receptor (GR) is a central player in the neuroendocrine stress response; it mediates feedback regulation of the hypothalamus-pituitary-adrenal (HPA) axis and physiological actions of glucocorticoids in the periphery. Despite intensive investigations of GR in the context of receptor-ligand interaction, only recently the first naturally occurring gain-of-function substitution, Ala610Val, of the ligand binding domain was identified in mammals. We showed that this mutation underlies a major quantitative trait locus for HPA axis activity in pigs, reducing cortisol production by about 40-50 percent. To unravel the molecular mechanisms behind this gain of function, receptor-ligand interactions were evaluated in silico, in vitro and in vivo. In accordance with previously observed phenotypic effects, the mutant Val610 GR showed significantly increased activation in response to glucocorticoid and non-glucocorticoid steroids, and, as revealed by GR-binding studies in vitro and in pituitary glands, enhanced ligand binding. Concordantly, the protein structure prediction depicted reduced binding distances between the receptor and ligand, and altered interactions in the ligand binding pocket. Consequently, the Ala610Val substitution opens up new structural information for the design of potent GR ligands and to examine effects of the enhanced GR responsiveness to glucocorticoids on the entire organism.

Effects of social support on glucocorticoid sensitivity of lymphocytes in socially deprived piglets.

There is growing evidence that social support given by a conspecific attenuates stress responses of a socially deprived animal. We hypothesized that the presence of a familiar social partner modulates the effectiveness of social buffering as assessed by an altered glucocorticoid sensitivity of immune cells. The current study investigated the effects of a 4-h social deprivation procedure on stress hormone responses and immune cell functions in 7-, 21- and 35-day-old piglets (52 males and 56 females). Within each of the three age categories, nine piglets were deprived of their mother and littermates either alone or with a familiar or unfamiliar age-matched piglet. Compared to non-deprived controls, piglets that were alone displayed significant increases in plasma adrenocorticotropic hormone and cortisol concentrations, and all socially deprived piglets showed a greater in vitro proliferative response of peripheral blood mononuclear cells (PBMCs) to lipopolysaccharide (LPS)-stimulation than controls. Concanavalin A (ConA)-induced in vitro proliferation was not affected by social treatment. Additionally, both the ConA- and LPS-stimulated PBMCs from piglets that experienced any of the deprivation treatments were more resistant to the inhibitory effects of cortisol than PBMCs from the controls in each of the three age categories. Irrespective of the mitogen used, the presence of an age-matched conspecific during deprivation attenuated the dose-dependent cortisol resistance. Here, familiarity between the piglets clearly improved the effectiveness of social support in an age-dependent manner. Collectively, our findings emphasize the benefits of social partners regarding stress coping abilities, with positive implications for animal welfare and health.