Animal welfare is a major concern among livestock producers in the U.S. Heat stress, particularly, compromises animal welfare and productivity, causing >$2B annual economic loss to producers. The presence of toxic alkaloids within tall fescue (Schedonorus phoenix syn Lolium arundinaceum, syn Festuca arundinacea) plants induces a complex of disorders collectively known as fescue toxicosis. Summer slump, a reduction in animal performance due to heat stress, is common and evident of the symptoms. Various techniques for assessing animal physiological and behavioral responses to heat stress were explored in this dissertation, with particular emphasis on fescue toxicosis and its mitigation strategies. In the first study, the physiological and behavioral responses of heifers was evaluated when grazing either toxic tall fescue with wildtype endophyte (WE) or tall fescue with a novel endophyte (NE). Heifers (n = 24) were assigned to either WE or NE pastures for a 56-d grazing study during the summers of 2020 and 2021. Heifer average daily gain (ADG), intravaginal temperature, and degree of hair shedding were recorded during the grazing period. Blood samples were collected through coccygeal venipuncture and hair was collected from the left rump for cortisol analysis. Ear, tail, and hoof temperatures were collected for each heifer using an infrared thermal imaging camera. In 2020, heifers that grazed NE tall fescue had greater (P = 0.0160) ADG over the season (0.22 vs. 0.12 kg day-1). Hair retention score was greater (P = 0.0029) for heifers that grazed WE tall fescue compared to heifers that grazed NE tall fescue across both years. Heifers that grazed WE tall fescue had decreased ear skin temperature (P = 0.0001), tail skin temperature (P = 0.0058), and hoof surface temperature (P = 0.0075) compared to heifers that grazed NE. Heifers that grazed WE had 0.3-0.9 ºC hotter intravaginal temperatures than heifers that grazed NE, especially during daytime. Hair cortisol levels of heifers that grazed WE tall fescue were greater (P < 0.0001) compared to hair cortisol levels of heifers that grazed NE tall fescue. From 1200h-1700h each day, heifers that grazed WE tall fescue spent 1.5 more (P = 0.0003) hours standing and 0.9 fewer (P = 0.0402) hours lying down compared to heifers that grazed NE tall fescue. These results suggest that heat stress and other physiological changes in heifers grazing WE tall fescue could be mitigated by renovating pastures with NE tall fescue that does not impose any negative effects on grazing animals. The second study explored the potential benefits of consuming condensed tannins as a means of negating the effects of toxic alkaloids in tall fescue. Twelve fall-born steers were assigned to one of the two diet supplement treatments - sericea lespedeza pellets (LES) or sericea lespedeza pellets mixed with polyethylene glycol (LES+PEG) for 12 weeks during the summers of 2020 and 2021. The LES+PEG treatment served as a positive control since polyethylene glycol binds tannins, rendering them inactive in the gastrointestinal system. Animal body weight (BW), hair retention score (HRS), rectal temperature, and thermographic images were collected every 4 weeks. Hair and blood samples were also collected for cortisol analysis. Fecal and urine samples were collected and analyzed for total ergot alkaloid (TEA) concentration. Steers on LES had greater (P = 0.0033) ADG compared to steers on LES+PEG in 2021, but not in 2020 (P = 0.8707). In 2021, HRS was greater (P < 0.05) for steers fed LES+PEG compared to steers fed LES. In 2020, ear skin temperature (P < 0.0001) and hoof surface temperature (P = 0.0382) was greater in steers on LES compared to steers on LES+PEG. Rectal temperatures were lower (P = 0.02905) for steers fed LES compared to steers fed LES+PEG in 2021. Plasma cortisol levels did not differ (P ≥ 0.1566) between LES and LES+PEG treatments for both years. In 2020, hair cortisol levels did not differ (P = 0.8295) between treatments while in 2021, the hair cortisol level of steers on LES+PEG was greater (P = 0.0221) compared to hair cortisol levels of steers on LES. This study indicated some changes in animal physiology in response to dietary supplements containing condensed tannins, but results were inconsistent and further studies are needed to better understand the potential benefits of tannins in reducing the effects of toxic alkaloids consumption. In the third study, behavioral and physiological responses of ewes that grazed either mid-stage hardwood silvopastures or open pasture (OP) were compared. The study site consists of 0.27-ha of black walnut (Juglans nigra; BSP) and honeylocust (Gleditsia triacanthos; HSP) silvopastures and open pasture treatments, each replicated three times. During the summers of 2020 and 2021, thirty-six Katahdin ewes were assigned to one of the treatments for a 6-week summer grazing trial. Ewe weights were recorded for two consecutive days at the beginning and end of the study and intravaginal temperatures were recorded for two consecutive days at every 3-week intervals. Trail cameras captured animal behavior. Blood was collected via jugular venipuncture. Hair grown during the trial was collected from the loin region. Blood and hair cortisol concentrations were determined by ELISA. Ewe ADG was greater in HSP compared to OP (P = 0.0456) but did not differ with BSP (P = 0.4686) across both years. Ewes on OP had hotter (P ≤ 0.0343) intravaginal temperatures than ewes on both silvopasture treatments between 1100h-1700h. Plasma cortisol level was lower in ewes on BSP compared to ewes on OP (P = 0.0400) but did not differ with ewes on HSP (P = 0.6954) across both years. Ewes that grazed OP had greater hair cortisol levels compared to ewes on silvopasture treatments both in 2020 and 2021 (P < 0.0001). In 2020, ewes on BSP spent about 20% more time grazing than ewes on OP (P = 0.0054) while in 2021 ewes on BSP spent about 36% more time grazing than ewes on HSP (P = 0.0014). Ewes on OP spent 400% more time standing than ewes on BSP (P < 0.0001) and 750% more time standing than ewes on HSP (P < 0.0001). Ewe on OP spent 20% less time lying down compared to ewes on BSP (P < 0.0001) and 33% less time lying down compared to ewes on HSP (P < 0.0001). Hair cortisol measures and intravaginal temperature sensors can be utilized as reliable and relatively non-invasive techniques for measuring heat stress response in livestock managed in extensive grazing systems. / Doctor of Philosophy / Heat stress compromises animal welfare and productivity, causing > $2B annual economic loss to producers in the U.S. In the southeastern U.S, heat stress in livestock is largely due both to an environmental condition and to the effect of fescue toxicosis caused by toxic alkaloids. Various management strategies to mitigate the effect of heat stress in livestock due to environmental conditions or due to the effect of tall fescue toxicosis are explored in this dissertation. In the first study, behavioral and physiological responses of heifers that grazed either toxic (WE) or novel (NE) endophyte-infected tall fescue was compared. Heifers that grazed WE tall fescue had lower gains, rough hair coat, higher hair cortisol level (stress measure), cooler extremity temperature, and hotter intravaginal temperature compared to heifers that grazed NE tall fescue. These results suggest that heat stress and other physiological changes in heifers grazing WE tall fescue could be mitigated by renovating pastures with NE tall fescue, a forage cultivar that does not impose any negative physiological effects on grazing animals. In the second study, we tested the effect of condensed tannins in reducing the severity of fescue toxicosis in steers. We fed sericea lespedeza pellets to steers in a study to test this effect. All steers were fed sericea pellets, but pellets for half (6) of the steers were treated with polyethylene glycol, which inactivates the condensed tannins in the sericea lespedeza. In our study, we found that the steers fed the sericea lespedeza had higher temperatures at their extremities (ears and tail), but lower rectal temperatures than the control steers. Interestingly, the steers fed the sericea lespedeza pellets also had slicker hair coats and lower levels of hair cortisol (indicating reduced stress) in the second year compared to the control steers. This study showed some potential of condensed tannins in reducing the effects of fescue toxicosis, but needs further study to better understand the potential benefits of tannins in reducing the post-ingestive effects of fescue toxicosis. In the third study, we compared behavioral and physiological (temperature, hair, and blood cortisol) responses of ewes that grazed either mid-stage hardwood silvopastures or open pastures (OP). Ewes on OP showed signs of heat stress with higher hair cortisol levels and hotter intravaginal temperatures during the daytime compared to ewes on silvopasture treatments. Ewes on OP spent 400% more time standing compared to ewes on BSP and 750% more time standing than ewes on HSP. Ewe on OP spent 20% less time lying down compared to ewes on BSP and 33% less time lying down compared to ewes on HSP. Ewes on silvopasture treatments spent 70% less time drinking water compared to ewes on OP. Trees within the silvopasture systems can alter the micro climatic condition thus creating a suitable environment for livestock. This can ultimately reduce stress and improve overall behavioral and physiological responses of livestock in silvopastures compared to open pastures.

No description available.

La schizophrénie est un trouble psychiatrique répandu qui affecte grandement la vie de l’individu touché. Même si certains facteurs ont été identifiés comme causes de cette maladie, notamment des facteurs génétiques, ceux-ci n’expliquent pas entièrement les bases biologiques qui la sous- tendent. Certaines causes infectieuses ont été soulevées, dont la « toxoplasmose ». Celle-ci réfère à une infection par un parasite, Toxoplasma gondii (TG), qui touche environ le tiers de la population humaine mondiale. Des études animales ont montré que ce parasite peut manipuler le comportement de son hôte en menant à une diminution de la peur et à une augmentation de la prise de risque. Les résultats d’autres études portent à croire que ces types de comportements pourraient également être affectés par ce parasite chez l’humain, et ce, peut-être de manière plus importante chez les personnes souffrant de schizophrénie. La peur et la prise de risque étant toutes deux fortement associées aux réponses de stress, nous avons émis l’hypothèse que TG pourrait être relié à des différences au niveau de la réponse de stress chez l’humain, particulièrement chez la personne souffrant de schizophrénie. Nous avons testé si les biomarqueurs de stress (cortisol salivaire et des cheveux) diffèrent chez des personnes avec un diagnostic de schizophrénie et des contrôles en fonction de l’infection par TG. Nous avons aussi vérifié si TG était relié à des symptômes psychotiques spécifiques et/ou à de l’impulsivité (comme marqueur proxy des comportements de prise de risque). Dans cette étude, nous avons utilisé des échantillons de sang, de salive et de cheveux pour mesurer les anticorps TG, les niveaux de cortisol circulant et la sécrétion de cortisol à long terme, respectivement. Les résultats montrent que les personnes avec une schizophrénie infectées par TG présentent des concentrations de cortisol des cheveux significativement plus élevées que les personnes avec une schizophrénie non infectées, un effet qui n'a pas été trouvé chez les participants contrôles. Aucune différence de cortisol salivaire n'a été observée entre les participants infectés par TG et non infectés, autant dans le groupe schizophrène que contrôle. De plus, il n'y a aucune association entre TG et les symptômes psychotiques positifs ou entre TG et l'impulsivité. Ces résultats suggèrent que l’infection par TG est associée à des différences dans la production de cortisol à long terme chez les personnes avec une schizophrénie, mais pas dans la sécrétion de cortisol à court terme. / Schizophrenia is an important psychiatric disorder that greatly affects the life of the affected individual. Although some factors have been identified as causes of this disease, including genetic factors, these do not fully explain the biological basis behind it. Some infectious causes have been raised, including the “toxoplasmosis”. It refers to an infection by a parasite, Toxoplasma gondii (TG), which affects approximately one third of the world's human population. Animal studies have shown that this parasite can manipulate its host's behavior leading to decreased fear and increased risk taking. The results of other studies suggest that these types of behaviors could also be affected by this parasite in humans, and perhaps to a greater extent in people with schizophrenia. Since fear and risk-taking are both strongly associated with stress responses, we hypothesized that TG might be related to differences in the stress response in humans, particularly in people suffering from schizophrenia. We tested whether stress biomarkers (salivary and hair cortisol) differ in people with schizophrenia and controls depending on TG infection. We also checked whether TG was related to specific psychotic symptoms and/or impulsivity (as a proxy marker of risk-taking behaviors). In this study, we used blood, saliva, and hair samples to measure TG antibodies, circulating cortisol levels, and long-term cortisol secretion, respectively. Results show that TG-infected people with schizophrenia had significantly higher hair cortisol concentrations than uninfected people with schizophrenia, an effect that was not found in control participants. No difference in salivary cortisol was observed between TG- infected and uninfected participants, neither in the schizophrenic group nor in the control group. Moreover, there was no association between TG and positive psychotic symptoms or between TG and impulsivity. These results suggests that TG infection is linked to differences in long-term cortisol secretion in people with schizophrenia, but not in short-term cortisol secretion.

No description available.

The stress system comprises the hypothalamic-pituitary-adrenal (HPA) and the sympathetic-adrenal-medullary (SAM) axes. The two operate synergistically to regulate metabolic and biological processes, maintain homeostasis, and manage physiological responses towards various environmental challenges, known as stressors. Numerous studies have observed impaired HPA response among White breast cancer survivors followed by an eventual normalization of the HPA axis, but little is known about SAM functioning, the complementary relationship between physiological and psychological stress, and the influence of culturally related factors. This dissertation aims to address these gaps in the literature via three studies. In Study 1, we examined the diurnal and reactive patterns of salivary alpha-amylase, a SAM biomarker, in a sample of predominantly White women with and without a prior diagnosis of breast cancer. The purpose was to establish an initial understanding of SAM functioning in relation to the participants’ HPA activity as measured by cortisol responses (complementary to a previously published study in the laboratory). Results of Study 1 revealed no abnormal response to stress. Virtually identical alpha-amylase slopes were observed between breast cancer survivors and control participants, except that breast cancer survivors had elevated basal levels of alpha-amylase, thus suggesting a “heightened sympathetic tone”. In Study 2 acute and diurnal cortisol profiles and their accompanying psychological stress responses were examined in a sample of Chinese and White women with and without a prior diagnosis of breast cancer. In the same participants, we also examined chronic stress levels via hair cortisol concentrations which was the subject of Study 3. In both studies 2 and 3, we assessed the potential influences of cultural orientation and ethnocultural group membership on physiological and psychological stress patterns. For the most part, Study 2 supported previous findings from the literature and our laboratory: Breast cancer survivors displayed a blunted cortisol response and their diurnal profile was comparable to that of control participants. But contrary to our hypotheses, ethnocultural membership and cultural orientation did not influence physiological patterns of stress. However, an interaction between ethnocultural group, health status, and time (i.e., from baseline to one hour after stress induction) was observed in the subjective appraisal of an acute stressor. Specifically, White breast cancer survivors reported significantly lower levels of perceived stress than the other three groups. These results suggest that health-related stress may supersede the effects of culturally related stress and indicate the potential presence of posttraumatic growth among our sample of White breast cancer survivors only. Study 3 revealed no differences in hair cortisol concentrations between breast cancer survivors and control participants, nor between Chinese and White breast cancer survivors. However, it was observed that healthy Chinese women exhibited significantly higher levels of hair cortisol concentrations than their Western counterpart. Further analyses revealed that health status and cultural orientation did not significantly predict the observed patterns of physiological or psychological stress. Although non-significant, Study 3 results offered preliminary evidence that higher orientation towards both the dominant and non-dominant cultures is associated with higher levels of chronic physiological and psychological stress. Collectively, our studies may provide support for the long-term recovery of the HPA axis via the examination of acute, diurnal, and chronic patterns of cortisol, but further research will be required. The dissertation also highlights several important key points regarding culturally related factors, health, and stress: (1) The perception of stress is influenced by one’s degree of cultural orientation and (2) effects of health-related stress may supersede those of culturally related stress, but (3) among healthy women, ethnocultural minorities may have more stressful encounters than their Western counterpart, thus having important clinical implications for ethnocultural minorities who are newly diagnosed with a chronic condition. Together, results of the studies highlight the importance of further investigating the enduring and acute implications of psychosocial variables – particularly the influence of cultural orientation – on the experience and perception of stress.

Human-caused ecological change negatively affects the sustainability of many wildlife populations but may be especially challenging for large carnivores and ungulates. Long-term physiological stress may be an important mechanism linking ecological change with impaired health and reduced population performance in these groups. The determination of hair cortisol concentration (HCC) has recently demonstrated potential as a biomarker of long-term stress in humans and domestic animals, and may also represent a practical technique for use in free-ranging wildlife. The objectives of this research program were to: 1) develop and apply an accurate and reliable method for measuring cortisol levels in hair collected opportunistically or remotely from free-ranging grizzly bears (Ursus arctos), polar bears (Ursus maritimus), and caribou (Rangifer tarandus sp.), and 2) to evaluate the utility of HCC as a biomarker of long-term stress (and thus potentially useful conservation tool) in these threatened species. An enzyme-immunoassay (EIA) based technique for measuring HCC in non-human primates was successfully modified for use with small quantities (5-100 mg) of hair representative of samples which may be obtained through opportunistic (e.g. hunting, research captures, archives) or remote (e.g. barb wire snagging) methods in each species. HCC was determined in 151 free-ranging grizzly bears from Alberta, Canada (mean 2.84 pg/mg, range 0.62-43.33 pg/mg); 185 free-ranging polar bears from southern Hudson Bay, Canada (mean 0.48 pg/mg range, 0.16-2.26 pg/mg); in 12 captive Alaskan caribou (R. t. granti) (mean 2.31 pg/mg, range, 1.57-3.86 pg/mg) and 12 captive reindeer (R .t. tarandus) (mean 2.88 pg/mg, range 2.21-3.40 pg/mg) injected either with adrenocorticotropic hormone (ACTH) or saline; and in 94 free-ranging caribou (R. t. groenlandicus) from West Greenland (mean 2.21 pg/mg, range 0.60-6.90 pg/mg). Factors influencing HCC in each species were then explored including: 1) technical considerations for the prudent use of HCC analysis and 2) potential relationships between HCC, biological traits, health, and prevailing environmental conditions. Evidence revealed in this study suggests that, with further research, this technique may show potential as a practical conservation tool for use in free-ranging grizzly bear, polar bear, and caribou populations.

A primary focus of wildlife research is to understand how habitat conditions and human activities impact the health of wild animals. External factors, both natural and anthropogenic that impact the ability of an animal to acquire food and build energy reserves have important implications for reproductive success, avoidance of predators, and the ability to withstand disease, and periods of food scarcity. In the analyses presented here, I quantify the impacts of habitat quality and anthropogenic disturbance on indicators of health for individuals in a threatened grizzly bear population in Alberta, Canada. The first analysis relates spatial patterns of hair cortisol concentrations, a promising indicator of long-term stress in mammals, measured from 304 grizzly bears to a variety of continuous environmental variables representative of habitat quality (e.g., crown closure, landcover, and vegetation productivity), topographic conditions (e.g., elevation and terrain ruggedness), and anthropogenic disturbances (e.g., roads, forest harvest blocks, and oil and gas well-sites). Hair cortisol concentration point data were integrated with continuous variables by creating a stress surface for male and female bears using kernel density estimation validated through bootstrapping. The relationships between hair cortisol concentrations for males and females and environmental variables were quantified using random forests, and landscape scale stress levels for both genders was predicted based on observed relationships. Low female stress levels were found to correspond with regions with high levels of anthropogenic disturbance and activity. High female stress levels were associated primarily with high-elevation parks and protected areas. Conversely, low male stress levels were found to correspond with parks and protected areas and spatially limited moderate to high stress levels were found in regions with greater anthropogenic disturbance. Of particular concern for conservation is the observed relationship between low female stress and sink habitats which have high mortality rates and high energetic costs. Extending the first analysis, the second portion of this research examined the impacts of scale-specific habitat selection and relationships between biology, habitat quality, and anthropogenic disturbance on body condition in 85 grizzly bears represented using a body condition index. Habitat quality and anthropogenic variables were represented at multiple scales using isopleths of a utilization distribution calculated using kernel density estimation for each bear. Several hypotheses regarding the influence of biology, habitat quality, and anthropogenic disturbance on body condition quantified using linear mixed-effects models were evaluated at each habitat selection scale using the small sample Aikake Information Criterion. Biological factors were influential at all scales as males had higher body condition than females, and body condition increased with age for both genders. At the scale of most concentrated habitat selection, the biology and habitat quality hypothesis had the greatest support and had a positive effect on body condition. A component of biology, the influence of long-term stress, which had a negative impact on body condition, was most pronounced within the biology and habitat quality hypothesis at this scale. As the scale of habitat selection was represented more broadly, support for the biology and anthropogenic disturbance hypothesis increased. Anthropogenic variables of particular importance were distance decay to roads, density of secondary linear features, and density of forest harvest areas which had a negative relationship with body condition. Management efforts aimed to promote landscape conditions beneficial to grizzly bear health should focus on promoting habitat quality in core habitat and limiting anthropogenic disturbance within larger grizzly bear home ranges. / Graduate / 0768 / 0463 / 0478 / mathieub@uvic.ca

No description available.

Nutztiere sind aufgrund ihrer Haltungsbedingungen diversen Stressoren ausgesetzt, welche ihre physische und psychische Gesundheit beeinträchtigen können. Im Rahmen von Animal Welfare Monitoring besteht daher ein großes Interesse an der Entwicklung und dem Einsatz minimal-invasiver Methoden und tierbezogener Belastungsindikatoren. Haarcortisol-konzentrationen (HCCs) könnten hierfür ein vielversprechender, retrospektiver Stressindikator sein, da sie die durchschnittlichen systemischen Cortisolkonzentrationen der letzten Wochen bis Monate in nur einer Probe widerspiegeln. Bevor HCCs jedoch als zuverlässiger Indikator eingesetzt werden können, müssen potenzielle Einflussfaktoren darauf ermittelt werden. Das generelle Ziel der vorliegenden Arbeit ist die Untersuchung von Einflussfaktoren auf Haarcortisol und seine Eignung für die Beurteilung von Langzeitstress bei Rindern und Schweinen. Daher zielten die vorliegenden Studien darauf ab, (1) Wissenslücken in der Forschung zu identifizieren, (2) die Wirkung potenzieller Einfluss- und Störfaktoren zu evaluieren und (3) zu untersuchen, ob und wann erhöhte systemische Cortisollevel durch HCCs nachweisbar sind. Alle Haarproben wurde von Holstein-Rindern, Landrasse- und Sattelschweinen bzw. Kreuzungstieren entnommen. Aufgrund der Literaturrecherche (Studie 1) wurden potenzielle Einflussfaktoren identifiziert und in den nachfolgenden Studien untersucht. Für die Evaluierung nicht-stressbedingter Faktoren (Studie 2), wurden insgesamt 614 Tiere verwendet. Hierfür wurden Haarproben in verschiedenen Altersstufen (Neugeborene bis Erwachsene), von beiden Geschlechtern und während der Sommer- und Wintersaison entnommen. Außerdem wurden schwarze und weiße Haarproben, unterschiedliche Körperregionen (Nacken/Schulter, Rücken, Schwanzspitze) und verschiedene Haarsegmente untersucht. Der Einfluss von Kontaminationen auf HCCs wurde in einer in-vitro-Studie (Studie 4) mit Haarproben von 12 Kühen und 12 Sauen evaluiert. Diese Proben wurden für vier Wochen täglich mit Urin, Speichel, Kot oder Wasser behandelt oder blieben unbehandelt. Um die Wirkung von Langzeitstress auf HCCs zu untersuchen (Studie 3), wurde bei 34 Rindern und 38 Jungsauen über vier Wochen jeden zweiten Tag ACTH- oder Kochsalzlösung appliziert. Vor, sowie zu drei Zeitpunkten nach Ende der Behandlung, wurden native und neu gewachsene Haare sowie zusätzlich Haarsegmente entnommen. Alle Haarproben wurden zweimal mit Isopropanol gewaschen, mit einer Kugelmühle gemahlen und Cortisol nach Extraktion mit Methanol mittels ELISA nachgewiesen. Die statistische Auswertung erfolgte mittels SAS/STAT-Software unter Anwendung von ANOVA und paarweisen Vergleichen durch Tukey-Kramer Tests. Die Ergebnisse der Studie 2 zeigen signifikant höhere HCCs bei neugeborenen Kälbern im Vergleich zu Jungrindern, Färsen und Kühen (p < 0,001). Ebenso wiesen 2 Wochen alte Ferkel höhere HCCs auf als Schweine im Alter von 10 oder 27 Wochen oder Sauen (p < 0,001). Das Geschlecht hatte keinen Einfluss auf die HCCs beider Tierarten. Jedoch waren bei beiden Spezies die HCCs in Schwanzhaaren, im Vergleich zu den Schulter-, Nacken- und Rückenhaaren signifikant erhöht (p < 0,001), ebenso in schwarzen Haaren im Vergleich zu weißen Haaren (p < 0,05) und in distalen im Vergleich zu proximalen Haarsegmenten (p < 0,001). Außerdem wiesen Rinder im Winter höhere HCCs als im Sommer auf (p < 0,001). Die Ergebnisse der Studie 4 zeigen, dass die Kontamination mit Urin bei beiden Spezies eine konzentrationsabhängige Zunahme der HCCs bewirkt. Auch die Kontamination mit Speichel und Kot erhöhte die HCCs, jedoch nur bei Rindern (alle p < 0,05). Die Behandlung mit Wasser führte zu einer Auswaschung von Cortisol aus Haaren vom Schwein, jedoch nicht vom Rind. Die Ergebnisse der ACTH-Studie (Studie 3) zeigen signifikant erhöhte HCCs bei ACTH-Tieren zum Ende der Behandlung in nativen Haaren (p < 0,001), nachgewachsenen Haaren (p < 0,01) und in Haarsegmenten (p < 0,05). Die höchsten HCCs wurden innerhalb von vier Wochen nach Behandlungsende gefunden. Bei Schweinen wurde ein Anstieg der HCCs sowohl in ACTH- als auch in Kontrolltieren beobachtet ohne dass Unterschiede zwischen den Behandlungen auftraten. Die Ergebnisse zeigen, dass die Cortisolkonzentrationen im Haar bei Schweinen und Rindern durch Alter, Körperregion, Haarfarbe, Haarsegment und Jahreszeit beeinflusst werden. Es gibt erste Belege, dass die Kontamination von Schweine- und Rinderhaaren mit cortisolhaltigen Körperflüssigkeiten, wie Urin und Speichel, die Aufnahme von externem Cortisol in den Haarschaft verursacht. Bei der Verwendung von HCC als Stressindikator sollten daher diese Einflussfaktoren standardisiert und Kontaminationseffekte vermieden werden, z.B. durch die Verwendung von Aufwuchsproben oder nur proximaler Haarsegmente. Darüber hinaus konnte gezeigt werden, dass Langzeitstress durch wiederholte Aktivierung der HPA-Achse zu erhöhten Cortisolkonzentrationen im Haar führt. Bei Rindern zeigt sich dies in den HCCs verschiedener Haarprobentypen. Insgesamt erweist sich die Analyse von Cortisol im Haar als eine geeignete Methode zum Nachweis von Langzeitstress bei Rindern und Schweinen und könnte daher eine wichtige Komponente bei der Beurteilung von Animal Welfare sein.:1 General introduction 2 Review of the literature 2.1 The relevance of stress assessment in animal welfare 2.2 Stress response in mammals 2.2.1 Structure and function of the hypothalamic-pituitary-adrenal axis 2.2.2 Characteristics and effects of cortisol 2.2.3 Conventional biological matrices for cortisol analysis 2.3 Hair as a matrix for cortisol analysis 2.3.1 Hair structure and hair types 2.3.2 Hair growth cycle 2.3.3 Incorporation of cortisol into the hair 2.3.3.1 Passive diffusion 2.3.3.2 Multi-compartment model 2.3.4 Elimination of cortisol from the hair 2.3.5 Specific characteristics and applications of hair cortisol 3 Research focuses and aims 4 Results 4.1 Study 1: Hair cortisol for the assessment of stress (review) 4.2 Study 2: Effects of animal-based, seasonal and hair-specific factors on hair cortisol concentrations 4.3 Study 3: Effects of long-term stress on hair cortisol concentrations 4.4 Study 4: Effects of contamination and elimination on hair cortisol concentrations 5 General discussion 5.1 Influencing factors on hair cortisol concentrations in cattle and pigs 5.1.1 Impact of animal-based, seasonal and hair-specific factors 5.1.2 Impact of contamination and elimination by washout 5.1.3 Implications 5.2 Hair cortisol concentration as an indicator of long-term stress in cattle and pigs 5.2.1 Model for the increased release of systemic cortisol 5.2.2 Models for the time course of cortisol incorporation into the hair shaft 5.2.3 Impact of hair sample type and sampling time 5.2.4 Implications Table of contents 5.3 Future perspectives 5.4 Conclusions 6 Summary 7 Zusammenfassung 8 References 9 Danksagung / Farm animals can be exposed to various stressors due to their husbandry conditions, which can impair their health and welfare. Thus, there is interest in the use of minimally invasive methods and animal-based stress indicators as part of welfare assessment. Cortisol in hair is a promising retrospective stress indicator, as a sample reflects systemic cortisol levels of the past weeks or months. Previous studies have shown that long-term stress with elevated cortisol release can be related to increased cortisol incorporation into the hair shaft. However, potential influencing factors that may affect hair cortisol concentrations (HCCs) must be determined before HCC can be applied as a reliable indicator of stress. The general objectives of this thesis are to investigate influencing factors on HCC, and to examine the potential of hair cortisol concentration as an indicator of long-term stress in cattle and pigs. Thus, the present studies aimed to (1) identify knowledge gaps in hair cortisol research, (2) evaluate the impact of animal-based, seasonal and hair-specific factors as well as contamination and elimination on HCC, and (3) investigate whether and when long-term increased systemic cortisol levels are reflected in elevated HCCs. Hairs were sampled from Holstein Friesian cattle, Landrace or Saddleback pigs and crossbreeds. The findings of the literature review (Study 1) identified potential animal-based, seasonal, hair-specific and stress-related factors on HCCs, which our experimental studies considered. To examine the impact of influencing factors (Study 2), a total of 614 animals were used. Hair samples were taken at different ages (newborn to adult), from different sexes and during both summer and winter. Variations by hair-specific factors were determined by studying black and white hair samples, varying body regions (neck/shoulder, back and tail tip) and different hair segments. In general, female animals were used. The effect of contamination on HCCs was examined in an in vitro study (Study 4) using hair samples from 12 cows and 12 sows. Samples were treated daily with urine, saliva, faeces or water for four weeks or remained untreated. To investigate long-term stress (Study 3), 34 cattle and 38 gilts were injected intramuscularly either with ACTH solution or saline every second day for four weeks. Natural and regrown hair samples were taken before and three times after the end of treatment, and hair segments were collected. All the hair samples were shaved with electric clippers, washed twice with isopropanol and ground with a ball mill. Cortisol was detected by ELISA after extraction with methanol. Statistical analyses were performed using ANOVA and pairwise comparisons of the least square means by Tukey-Kramer tests with the MIXED procedure in SAS/STAT software. The results of Study 2 showed significantly higher HCCs in newborn calves than in young cattle, heifers and cows (p < 0.001). Likewise, 2-week-old piglets had higher HCCs than pigs aged 10 or 27 weeks and sows (p < 0.001). Sex had no effect on HCCs in pigs or cattle. In both species, HCCs were also significantly higher in samples obtained from the tail tip than from the shoulder, neck and back regions (p < 0.001), in black hair than in white hair (p < 0.05) and in distal hair than in proximal hair segments (p < 0.001). Season had an impact on HCC only in cattle, which exhibited higher levels in winter than in summer (p < 0.001). The results of Study 4 showed that contamination with urine caused a considerable concentration-dependent increase in HCCs in both species. Contamination with saliva and faeces also raised HCCs, but only in cattle (all p < 0.05). Treatment with water washed cortisol out from porcine hair but not from bovine hair. In cattle, repeated ACTH application (Study 3) revealed significantly higher HCCs after the end of treatment in natural hair (up to eight weeks, p < 0.001), regrown hair (up to four weeks, p < 0.01) and segmental hair (eight weeks, p < 0.05) than in the control animals. The highest HCCs were found four weeks after the end of treatment. In pigs, elevated HCCs were observed in both ACTH and control animals in all hair sample types after the application period, with no differences between treatments. These results show that hair cortisol concentrations in pigs and cattle are affected by age, body region, hair colour, hair segment and season. There is first evidence that contamination of porcine and bovine hair with cortisol-containing body fluids, such as urine and saliva, may cause the incorporation of external cortisol into the hair shaft. Thus, when using HCC as a potential stress indicator, these influencing factors should be standardised and contamination effects should be avoided, such as by using the shave-reshave procedure, clean sampling regions and only the most proximal hair segments. The results also demonstrated that long-term stress by repeated activation of the HPA axis increases hair cortisol concentrations. In cattle, HCCs in different hair sample types reliably reflected the preceding period with increased systemic cortisol levels. In conclusion, the analysis of HCC appears to be a suitable method to evaluate long-term stress in cattle and pigs and can therefore be an important component in the assessment of animal welfare.:1 General introduction 2 Review of the literature 2.1 The relevance of stress assessment in animal welfare 2.2 Stress response in mammals 2.2.1 Structure and function of the hypothalamic-pituitary-adrenal axis 2.2.2 Characteristics and effects of cortisol 2.2.3 Conventional biological matrices for cortisol analysis 2.3 Hair as a matrix for cortisol analysis 2.3.1 Hair structure and hair types 2.3.2 Hair growth cycle 2.3.3 Incorporation of cortisol into the hair 2.3.3.1 Passive diffusion 2.3.3.2 Multi-compartment model 2.3.4 Elimination of cortisol from the hair 2.3.5 Specific characteristics and applications of hair cortisol 3 Research focuses and aims 4 Results 4.1 Study 1: Hair cortisol for the assessment of stress (review) 4.2 Study 2: Effects of animal-based, seasonal and hair-specific factors on hair cortisol concentrations 4.3 Study 3: Effects of long-term stress on hair cortisol concentrations 4.4 Study 4: Effects of contamination and elimination on hair cortisol concentrations 5 General discussion 5.1 Influencing factors on hair cortisol concentrations in cattle and pigs 5.1.1 Impact of animal-based, seasonal and hair-specific factors 5.1.2 Impact of contamination and elimination by washout 5.1.3 Implications 5.2 Hair cortisol concentration as an indicator of long-term stress in cattle and pigs 5.2.1 Model for the increased release of systemic cortisol 5.2.2 Models for the time course of cortisol incorporation into the hair shaft 5.2.3 Impact of hair sample type and sampling time 5.2.4 Implications Table of contents 5.3 Future perspectives 5.4 Conclusions 6 Summary 7 Zusammenfassung 8 References 9 Danksagung

Hintergrund: Anorexia nervosa (AN), allgemein als Magersucht bezeichnet, gehört zu den häufigsten psychischen Erkrankungen im Kindes- und Jugendalter. Dabei kommt es aufgrund einer gestörten Wahrnehmung der eigenen Figur zu einem erheblichen, selbst herbeigeführten Gewichtsverlust. Die Mehrheit der Patientinnen muss aufgrund der Schwere der Erkrankung mindestens einmal stationär behandelt werden. Cortisol ist ein lebenswichtiges Steroidhormon, das dafür sorgt, dass der menschliche Körper länger andauernde Belastungen bewältigen kann. Halten diese Stresssituationen jedoch zu lange an, kann es erhebliche Störungen des Stoffwechsels, des Wachstums, der Reproduktion und der Immunkompetenz verursachen. Cortisol und seine Stoffwechselprodukte werden überwiegend mit dem Urin ausgeschieden. Die Bestimmung der Urincortisolkonzentration (UCC) gehört daher zur klinischen Standarddiagnostik. In der Forschung hat sich die UCC als zuverlässige Methode zur Untersuchung des Stresshormons bei gesunden Probanden und psychischen Erkrankungen bewährt. Auch im Haar kann das Stresshormon nachgewiesen werden. Hauptsächlich gelangt es aus dem Blut in die Zellen des wachsenden Haarschaftes. Haaranalysen werden seit langer Zeit als zuverlässige Methode in der Forensik und Toxikologie angewandt. Mittlerweile hat sich die Analyse von Haarproben zur Bestimmung der Haarcortisolkonzentration (HCC) auch in der Forschung als valide Methode etabliert. Dabei bietet diese nicht-invasive und leicht durchführbare Methode einen retrospektiven Blick auf die Langzeitausschüttung von Cortisol. In zahlreichen Studien wurde bei AN eine Erhöhung des Stresshormons Cortisol nachgewiesen (sog. Hypercortisolismus). Dabei scheinen sich der Hypercortisolismus, die körperlichen Symptome und die psychischen Veränderungen in einer komplexen Krankheitspathologie wechselseitig zu beeinflussen. Mehrere Studien konnten bei AN bereits eine erhöhte Cortisolausscheidung im Urin nachweisen, die mit zunehmender Gewichtsnormalisierung rückläufig ist. Die HCC wurde bei AN bisher nur in einer Querschnittstudie untersucht. Entgegen des bekannten Hypercortisolismus konnte hier eine signifikant niedrige HCC nachgewiesen werden. Fragestellung: Die vorliegende Studie untersucht die HCC bei Patientinnen mit AN zum ersten Mal in einer Längsschnittstudie. Durch Beobachtung von Patientinnen über den Verlauf einer stationären, krankheitsbildspezifischen Therapie und zusätzlicher Erhebung der UCC als Vergleichsvariable sollte die Anwendbarkeit der HCC bei AN untersucht werden. Material und Methoden: Neben der Anwendung psychopathologischer Testverfahren (EDI-2) und regelmäßiger Bestimmung des BMI wurde wöchentlich durch einen 24-h-Sammelurin die UCC bestimmt. Eine Substichprobe gab am Ende des durchschnittlich 13-wöchigen Therapiezeitraumes eine Haarprobe ab, anhand derer eine monatliche HCC bestimmt wurde. Die Analyse der Urinproben erfolgte mittels immunochemischer Verfahren, die Haarproben wurden mittels Hochleistungsflüssigkeitschromatographie mit Tandem-Massenspektrometrie analysiert. Durch individuelle Zuordnung einer medianen UCC zu jeder HCC wurden für jede Patientin schließlich drei HCC-UCC-Wertepaare erzeugt, die den Anfang, die Mitte und das Ende der stationären Therapie abbildeten. Ergebnisse: Insgesamt nahmen 33 Patientinnen des Zentrums für Essstörungen der Klinik und Poliklinik für Kinder- und Jugendpsychiatrie und -psychotherapie teil (Stichprobe 1), wobei 16 Patientinnen der Entnahme einer Haarprobe zustimmten (Stichprobe 1a). Während der stationären Therapie kam es zu einer leitliniengerechten Gewichtsrehabilitation. Die UCC war deutlich erhöht und fiel während des Therapieverlauf signifikant ab. Die HCC zeigte hingegen keine Veränderung. Bei der Korrelation der HCC mit der medianen UCC konnte lediglich ein Trendeffekt nachgewiesen werden. Separate Analysen der einzelnen Zeitabschnitte ergaben eine signifikante Korrelation von HCC und UCC am Ende der stationären Therapie. Bei Analyse des EDI-2 korrelierte die Unzufriedenheit mit dem eigenen Körper zu Beginn und am Ende der Therapie mit der UCC, jedoch nicht mit der HCC. Haareigenschaften und Haarbehandlungen hatten keinen Einfluss auf die HCC. Schlussfolgerungen: Insgesamt konnte die UCC den bekannten Hypercortisolismus abbilden und bildete eine valide Grundlage für die Überprüfung der HCC bei AN. Trotz ihres Absinkens blieb die UCC nach erfolgreicher Gewichtsrehabilitation erhöht. Zudem konnte eine Korrelation der UCC mit einer zentralen psychopathologischen Veränderung bei AN nachvollzogen werden. Dies bestätigt die Hypothese einer komplexen Krankheitspathologie mit somatischen und psychiatrischen Einflussfaktoren auf den Hypercortisolismus. Bei einem Literaturvergleich der HCC mit gesunden Probanden konnte kein Hypercortisolismus nachvollzogen werden. Insgesamt konnte die HCC die UCC insbesondere in der Phase der akuten Erkrankung und beginnenden Gewichtsrehabilitation nicht zuverlässig abbilden. Nach Gewichtsnormalisierung schienen sich HCC und UCC jedoch wieder dem bei gesunden Probanden bestätigten Verhältnis anzunähern. Als Ursache für diese fehlende Korrelation kommen trophische Störungen des Haarwachstums bei AN in Betracht. So ist aufgrund eines verminderten Haarwachstums eine gestörte Einlagerung des Hormons in den Haarschaft möglich. Auch enzymatische Störungen des Cortisolabbaus werden diskutiert. Ein gestörtes Gleichgewicht des Enzyms 11-β-HSD, welches die Umwandlung von Cortisol zu Cortison (und umgekehrt) katalysiert, scheint ebenfalls die Einlagerung in das Haar zu beeinflussen. Insgesamt ergaben sich zahlreiche Hinweise, dass die HCC entgegen den Studienergebnissen bei gesunden Probanden und anderen stressassoziierten Zuständen bzw. Erkrankungen nicht zur Untersuchung des Hypercortisolismus bei AN im akuten Erkrankungszustand und im Verlauf einer stationären Therapie angewendet werden kann. Eine Nutzung dieser Methode zur Untersuchung gewichtsrehabilitierter Patientinnen bleibt offen und bedarf weiterer Forschungen. / Background: Anorexia nervosa (AN) is one of the most common mental disorders in children and adolescents. Disturbances in the way in which one´s body shape is experienced lead to a severe self-inflicted weight loss. Majority of patients need to be treated in hospital at least once in a lifetime. Cortisol is an essential adrenal cortical hormone, which enables human organism to cope with long-lasting stress. However, perseverative stress can cause abnormalities in metabolism, growth, reproduction and the immune system. Cortisol and its metabolites are excreted renal predominantly. Therefore, analyzing urinary cortisol concentration (UCC) is part of good clinical practice and proved of value in research. Recently, the assessment of long-term cortisol concentration via hair samples has been shown to be a sensitive cortisol parameter too. According to the multicompartment model diffusion of cortisol from blood capillaries into the growing hair cells of the follicle represents the main pathway of cortisol incorporation into the hair. Hair cortisol concentrations (HCC) offer a retrospective view that is easily accessible. Numerous studies have used hair cortisol successfully to examine changes in long-term cortisol levels in a number of psychiatric diseases. Underweight patients with AN are known to have increased cortisol levels (hypercortisolism). Cortisol levels were found to correlate with both physical and mental symptoms in a complex interplay. Most studies reported a urinary hypercortisolism in AN and a significant decrease in cortisol levels during weight gain. So far, the only other study that examined HCC in AN found lower HCC compared to healthy controls, which is contradictory to the existing literature. Aim: The aim of this study was to investigate whether HCC in acutely ill AN patients reflects a well-established classic cortisol measure (UCC) using a longitudinal study design over the course of inpatient weight-restoration treatment. Methods: Therefore, after admission every patient provided a weekly 24-h-urine sample. The corresponding body weight was measured on each day of collection. After the last urine collection, a hair sample was taken for analyzing a monthly HCC. Additionally, eating disorder symptoms were measured by EDI-2. Hair samples were analyzed by a LC-MS/MS-based method. The determination of cortisol concentration in urine was performed by using a competitive immuneenzymatic colorimetric method. As urinary cortisol was measured weekly while hair cortisol values were reflecting cortisol levels over one month, we calculated the median urinary cortisol corresponding to each monthly hair cortisol value individually. In sum, we matched three pairs of median UCC and HCC for each patient, representing the beginning, middle and end of the therapy. Results: The cohort in our longitudinal analysis consisted of a total of 33 patients who participated for 13 weeks on average. A subsample of 16 patients provided a hair sample after inpatient treatment. UCC was increased and revealed the expected decrease during weight gain while HCC showed no significant change. Tested for associations between HCC and UCC there was a trend effect for UCC on HCC. Separate correlation analyses per time-period showed a significant correlation between UCC and HCC for the third time period at the end of the therapy. Disaffection with the own body correlated with UCC, but not with HCC at the beginning and end of inpatient therapy. Hair treatment had no effect on HCC. Discussion: In summary UCC proved to be a reliable cortisol measure in our sample. Despite reduction, UCC remained increased after weight-restoration and correlated with eating disorder symptoms. This confirms the supposed complex interplay of hypercortisolism with physical and mental symptoms. Compared to cortisol levels of previously reported healthy females HCC was normal in AN and did not decline significantly during weight-restoration. Overall HCC did not reflect UCC, particularly with regard to the phase of acute starvation and incipient weight gain. However, association of HCC and UCC seemed to approximate healthy conditions after weight restoration. One possible explanation for lack of correlation might be trophic disturbances of hair in AN. Studies reported that scalp hair in AN show a higher percentage of hair in the telogen (resting) phase, which implies no further hair follicle activity, consequently less hair growth, and less storage of hormones like cortisol in hair. Furthermore, disturbances of the activity of 11-β-HSD-enzymes, converting cortisol in cortisone and vice versa, might influence the incorporation of cortisol in hair too. Altogether we suggest that incorporation of cortisol into the hair might be impaired due to factors associated with underweight and undernutrition in AN. Our data demonstrate that HCC does not reflect hypercortisolism in individuals suffering from acute AN, while after some weight gain it may proof to be a useful measurement of long-term cortisol levels.