Ellen Kanitz

Consequences of repeated early isolation in domestic piglets (Sus scrofa) on their behavioural, neuroendocrine, and immunological responses.

Stress in the form of intermittent maternal deprivation and social isolation during early postnatal life in rats and monkeys produces persistent changes in physiology and behaviour. In farm animals physiological consequences of disrupting mother-infant interactions with respect to health and animal welfare are relatively unknown. Therefore, the aim of the present study was to investigate the behavioural, neuroendocrine and immunological consequences of a 2 h daily social isolation from day 3 to day 11 of age in domestic piglets as well as potential long-term effects on the brain-endocrine-immune regulation. Repeated social isolation resulted in significantly decreased open-field activity (locomotion, vocalization) during the isolation period, increased basal cortisol concentrations and decreased lymphocyte proliferation in response to concanavalin A and pokeweed mitogen one day after the isolation. There was also a significant increase of interleukin-1beta (IL-1beta) concentration in hippocampus in isolated piglets compared to controls at this time. Six weeks after isolation significant enhanced basal ACTH concentrations as well as higher IL-1beta content and glucocorticoid receptor (GR) binding in hippocampus were found. These endocrine and immune responses were associated with decreased CRH levels in the hypothalamus and increased CRH content in the amygdala. The present data indicate that early social isolation in pigs may cause changes in behavioural, neuroendocrine, and immune regulation and produce long-term effects not only on the activity of the hypothalamic-pituitary-adrenal (HPA) system, but also on the immune-brain circuitry with possible negative consequences in health and welfare of commercial pigs. Using the pig as a suitable animal model, the finding of this study may also have some implications for the etiology of anxiety and depression in humans.

Effects of social status after mixing on immune, metabolic, and endocrine responses in pigs

The effects of social rank on immune, metabolic, and endocrine responses were studied in 10 newly mixed groups of German Landrace pigs (9 individuals each) at an age of 12 weeks. Immediately after mixing, the agonistic interactions (AI) of all group members were continuously recorded over 3 days (10 h daily). An individual dominance value (DV) was calculated by the number of wins minus defeats in relation to all decisive fights (DV < or = 0, subordinate; DV > 0, dominant). Blood samples were taken 24 h before and 3 days after mixing. The data showed that the social status had a significant effect on lymphocyte proliferation in responses to different mitogens: socially dominant pigs had higher proliferative response than subordinate pigs. In addition, during the observation period the lymphocyte activation by mitogens increased in the dominant animals and decreased in the subordinate animals with increasing number of agonistic interactions. The rise in total serum IgG concentration 3 days after mixing was higher in dominant pigs compared with subordinates. The dominance status did not significantly affect plasma metabolic levels nor cortisol concentrations. However, mixing appeared to increase glucose and total protein values and to decrease alkaline phosphatase and cortisol levels in both, dominant and subordinate pigs. In conclusion, mitogen induced cell proliferation seems to be a valuable marker for acute social stress in pigs.

Variation in fat mobilization during early lactation differently affects feed intake, body condition, and lipid and glucose metabolism in high-yielding dairy cows

Fat mobilization to meet energy requirements during early lactation is inevitable because of insufficient feed intake, but differs greatly among high-yielding dairy cows. Therefore, we studied milk production, feed intake, and body condition as well as metabolic and endocrine changes in high-yielding dairy cows to identify variable strategies in metabolic and endocrine adaptation to overcome postpartum metabolic load attributable to milk production. Cows used in this study varied in fat mobilization around calving, as classified by mean total liver fat concentrations (LFC) postpartum. German Holstein cows (n=27) were studied from dry off until d 63 postpartum in their third lactation. All cows were fed the same total mixed rations ad libitum during the dry period and lactation. Plasma concentrations of metabolites and hormones were measured in blood samples taken at d 56, 28, 15, and 5 before expected calving and at d 1 and once weekly up to d 63 postpartum. Liver biopsies were taken on d 56 and 15 before calving, and on d 1, 14, 28, and 49 postpartum to measure LFC and glycogen concentrations. Cows were grouped accordingly to mean total LFC on d 1, 14, and 28 in high, medium, and low fat-mobilizing cows. Mean LFC (±SEM) differed among groups and were 351±14, 250±10, and 159±9 mg/g of dry matter for high, medium, and low fat-mobilizing cows, respectively, whereas hepatic glycogen concentrations postpartum were the highest in low fat-mobilizing cows. Cows in the low group showed the highest dry matter intake and the least negative energy balance postpartum, but energy-corrected milk yield was similar among groups. The decrease in body weight postpartum was greatest in high fat-mobilizing cows, but the decrease in backfat thickness was greatest in medium fat-mobilizing cows. Plasma concentrations of nonesterified fatty acids and β-hydroxybutyrate were highest around calving in high fat-mobilizing cows. Plasma triglycerides were highest in the medium group and plasma cholesterol concentrations were lowest in the high group at calving. During early lactation, the decrease in plasma glucose concentrations was greatest in the high group, and plasma insulin concentrations postpartum were highest in the low group. The revised quantitative insulin sensitivity check index values decreased during the transition period and postpartum, and were highest in the medium group. Plasma cortisol concentrations during the transition period and postpartum period and plasma leptin concentrations were highest in the medium group. In conclusion, cows adapted differently to the metabolic load and used variable strategies for homeorhetic regulation of milk production. Differences in fat mobilization were part of these strategies and contributed to the individual adaptation of energy metabolism to milk production.

Dissecting the effects of mtDNA variations on complex traits using mouse conplastic strains

Previous reports have demonstrated that the mtDNA of mouse common inbred strains (CIS) originated from a single female ancestor and that mtDNA mutations occurred during CIS establishment. This situation provides a unique opportunity to investigate the impact of individual mtDNA variations on complex traits in mammals. In this study, we compiled the complete mtDNA sequences of 52 mouse CIS. Phylogenetic analysis demonstrated that 50 of the 52 CIS descended from a single female Mus musculus domesticus mouse, and mtDNA mutations have accumulated in 26 of the CIS. We then generated conplastic strains on the C57BL/6J background for 12 mtDNA variants with one to three functional mtDNA mutations. We also generated conplastic strains for mtDNA variants of the four M. musculus subspecies, each of which contains hundreds of mtDNA variations. In total, a panel of conplastic strains was generated for 16 mtDNA variants. Phenotypic analysis of the conplastic strains demonstrated that mtDNA variations affect susceptibility to experimental autoimmune encephalomyelitis and anxiety-related behavior, which confirms that mtDNA variations affect complex traits. Thus, we have developed a unique genetic resource that will facilitate exploration of the biochemical and physiological roles of mitochondria in complex traits.

Effects of postnatal social isolation on hormonal and immune responses of pigs to an acute endotoxin challenge

Social stress during early postnatal life often results in long-term effects on neuroendocrine and immune adaptation mechanisms. Therefore, the aim of the present study was to determine the influence of a 2-h daily social isolation from Day 3 to Day 11 on the acute and long-term proinflammatory and neuroendocrine responses of piglets challenged with the bacterial endotoxin lipopolysaccharide (LPS; 100 microg/kg body weight). Peripheral LPS administration significantly increased plasma concentrations of tumor necrosis factor-alpha (TNF-alpha), ACTH and cortisol in isolated and control pigs. However, the activity of the hypothalamic-pituitary-adrenal (HPA) axis after LPS stimulation was not significantly affected by isolation treatment, whereas the prior social isolation diminished the plasma TNF-alpha response to LPS 1 day as well as 45 days after the isolation period. The hippocampal TNF-alpha concentration in response to LPS was also reduced in priorly isolated pigs compared to control animals. Furthermore, the significant increase of TNF-alpha in the spleen caused by LPS was associated with a dramatic decrease in glucocorticoid receptor (GR) binding. The GR binding in hippocampus was increased in isolated pigs and was significantly decreased after LPS injection. In addition, the repeated isolation stressor was shown to increase hippocampal levels of interleukin-1beta (IL-1beta). The present results indicate that repeated social isolation of neonatal pigs may cause long-term effects on proinflammatory regulation at the periphery and in the brain following immune challenge with particular importance of TNF-alpha in mediating these interactions.

Agonistic interactions and physiological reactions of top- and bottom-ranking pigs confronted with a familiar and an unfamiliar group: Preliminary results

Abstract Four trials were carried out, where in each trial during a selection test two groups of growing pigs (12 weeks old) with nine animals each were randomly formed. After a week, the top (TR) and bottom (BR) ranking pig from each group was isolated and kept under single housing conditions. At the age of 15 to 16 weeks, the test animals were confronted once with the familiar and once with the unfamiliar group. During the 10-h social confrontation test, agonistic behaviour (agonistic interactions, individual dominance values) and physiological stress reactions (epinephrine, norepinephrine, cortisol, heart rate) were investigated simultaneously. The social confrontation of a pig with a familiar or an unfamiliar group caused very frequent agonistic interactions during the first 30 min. This behaviour was accompanied by a rapid increase of the heart rate and the plasma catecholamine concentrations and a delayed increase of cortisol. TR pigs elicited more agonistic interactions during the first 30 min and displayed higher plasma catecholamine concentrations. Additionally, TR animals showed significantly higher dominance values during the 10-h testing situation compared to the BR pigs. The confrontation of an animal with the unfamiliar group resulted in a more pronounced adrenocortical reaction compared to the confrontation with the familiar group. In particular, the TR animals showed a distinct increase of plasma cortisol during the first hour and a later bradycardia when they were confronted with the unfamiliar group. The preliminary results presented in this paper indicate that pigs show different, but inconsistent behavioural and physiological reactions when they are confronted with a social group. The agonistic behaviour and the physiological stress reactions during a social confrontation test are still influenced by the former rank of the animal, even when the animal was isolated under single housing conditions in the meantime for 2 to 3 weeks.

Neuroendocrine and Immune Responses to Acute Endotoxemia in Suckling and Weaned Piglets

The objective of this study was to characterize effects of weaning stress on behavioural, endocrine and immune responses to acute peripheral lipopolysaccharide (LPS) challenge in neonatal pigs. Weaning in 28-day-old piglets was accompanied by a significant increase in ACTH concentrations (p = 0.0378) and an increase in basal cortisol level (p = 0.0135). There was also a significant suppressive effect on lymphocyte proliferation in response to concanavalin A (p = 0.0048) in newly weaned piglets. Peripheral administration of LPS induced vomiting, diarrhoea and somnolence in both suckling and weaned piglets. The frequency of these signs of sickness was significantly higher in weaned piglets compared with suckling piglets (p = 0.0049). Additionally, LPS significantly increased plasma concentrations of TNF-α, cortisol and ACTH. While weaned piglets reacted to LPS with a higher release of ACTH (p = 0.0239) and cortisol (p = 0.0015) than suckling piglets there was no significant effect of weaning on the magnitude of TNF-α. The present data indicate that weaning suppresses the lymphocyte function, causes changes in endocrine regulation and has a substantial effect on the behavioural and endocrine response to an acute peripheral LPS challenge; consequently it could increase disease susceptibility.

A single exposure to social isolation in domestic piglets activates behavioural arousal, neuroendocrine stress hormones, and stress-related gene expression in the brain

Stressful early life events can have short- and long-term effects on neuroendocrine and behavioural mechanisms of adaptation. Here, we investigated the effects of a single social isolation (4 h) of domestic piglets on both behavioural alterations in open-field tests and modifications in the expression of genes regulating glucocorticoid response in stress-related brain regions at 7, 21 or 35 days of age. The mRNAs of glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11ss-hydroxysteroid dehydrogenase 1 and 2 (11ss-HSD1 and 11ss-HSD2) and c-fos were analysed by real-time RT-PCR in the hypothalamus, hippocampus and amygdala. The social isolation caused both elevated stress hormone concentrations (e.g. cortisol) and open-field reactivity (e.g. locomotion, vocalisation) compared to control piglets. The enhanced behavioural and neuroendocrine activity was associated with distinct changes in gene expression in the limbic system. The hypothalamic GR, MR and 11ss-HSD1 mRNA expressions and the hippocampal 11ss-HSD1 mRNA was significantly higher in isolated piglets, whereas in the amygdala social isolation caused a significant decrease in MR mRNA expression. Isolated piglets also displayed significantly higher c-fos mRNA expression, an estimate of neuronal activation, in hypothalamus and amygdala. The mRNA alterations as well as the behavioural and hormonal pattern show an effect of social isolation on days 7 and 21, but no effect on day 35. In conclusion, a single social isolation in piglets caused age-dependent neuroendocrine and behavioural changes that indicate increased arousal and experienced distress. The present results also suggest that psychosocial stress effects should be considered for the assessment of livestock handling practices with respect to health and welfare.

Effects of weaning and restraint stress on glucocorticoid receptor binding capacity in limbic areas of domestic pigs

Changes in glucocorticoid receptor (GR) binding in different brain areas were investigated in neonatal and adult pigs exposed to psychological stress (weaning) and a physical stressor (repeated snaring). The GR binding was significantly decreased 4 days after weaning in both the hippocampus and the amygdala, but there were no changes in the hypothalamus. Repeated snaring of adult pigs resulted in a significant diminished GR binding only in the hippocampus.

Physiological and behavioral effects of different success during social confrontation in pigs with prior dominance experience

The impact of a 10-h social confrontation on behavior, plasma adrenaline, noradrenaline and cortisol was studied in 14 domestic pigs (eight castrated males and six females) with prior dominance experience. Prior to the experiments, animal groups, each consisting of nine growing pigs (12 weeks old) from different litters, were composed randomly. After 5 days, the pig with the highest rank from each group was removed, provided with a jugular vein catheter and kept in single housing for 2-3 weeks. After this period, each test animal was returned into its familiar group for a 10-h social confrontation. The reintroduction of the test animals into the familiar groups caused frequent agonistic interactions during the first 30 min. Seven animals succeeded to win most of their encounters during the confrontation test and were classified as High Success (HS) animals, whereas seven other animals lost most of their encounters and were classified as Low Success (LS) animals. The reintroduction of the test animals into the groups provoked also marked changes in behavioral and physiological measures. The frequent fighting behavior during the first 30 min was accompanied by a rapid increase of plasma catecholamines and a delayed increase of cortisol. Immediately after introduction, LS pigs tended to show higher plasma adrenaline and noradrenaline concentrations than HS pigs. There was also a tendency for a sustained increase of noradrenaline/adrenaline ratios in HS pigs, whereas the ratios of LS pigs remained nearly unchanged. No significant differences between both groups were found in cortisol levels and in the frequency of agonistic interactions. However, LS animals showed less locomotion, more lying and spent less time exploring the pen or other animals. These results show that during a social confrontation the experience of defeats for formerly high-ranking pigs is accompanied by increased submissive or passive behavior and a higher sympathoneural and adrenomedullary reaction, which may indicate more emotional distress and fear compared to successful animals.