Parents of children with a chronic disease are usually highly involved in their child’s treatment and may be affected by the heavy demands and constant stress. This can increase the risk of developing burnout, which is an individual reaction to long-term stress consisting of symptoms associated with emotional exhaustion, as well as physical and cognitive fatigue. The overall aim was to estimate the prevalence of burnout in parents of children with Type 1 Diabetes Mellitus (T1DM) and inflammatory bowel disease (IBD) (paper I), identify the risk factors associated with parenting a child with T1DM (paper II), explore how mothers suffering from burnout describe their mothering of a child with diabetes, with special focus on their need for control and Performance-based self-esteem (PBSE) (paper IV), and to evaluate the effect of a group intervention aimed at reducing stress-related symptoms (paper III). A total of 251 parents of children with T1DM, 38 parents of children with IBD and 124 parents of healthy children participated in a population-based study (I, II). The validated Shirom-Melamed Burnout Questionnaire (SMBQ) was used to assess burnout. 16 parents (SMBQ ≥3.75) participated in a group intervention and were evaluated for changes in SMBQ and PBSE (III). A total of 21 mothers of children with T1DM who scored for clinical burnout (SMBQ) participated in a qualitative study. Semi-structured interviews were conducted and Inductive content analysis was used (IV). In the study group 36.0% parents of children with a chronic disease scored for clinical burnout (SMBQ ≥3.75) compared to 20.2% of the reference parents (p=0.001) with a preponderance of mothers compared to fathers, 42% vs. 20.5% (p=0.001), respectively (I). Less support from the social network, sleep disturbances and lack of personal leisure time and recovery seem to be important risk factors for clinical burnout in parents of children with T1DM, especially mothers (II). Mothers’ experiences of mothering a child with T1DM were interpreted as one theme; Mission impossible, illustrating the extremely difficult circumstances under which they bring up the child with diabetes to adulthood (IV). Parents’ subjective evaluation of the intervention group was mainly positive and SMBQ (p=0.01) and PBSE scale (p= 0.04) measurements were significantly reduced 6 months after completion of the intervention (III). It is important to pay attention to how parents and especially mothers experience their daily life in order to support those who are at risk of developing burnout.

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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.

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

Humans and dogs have been living close for over 15,000 years, resulting in a unique relationship. The dog-owner relationship can affect stress, and could be affected by contact-seeking behaviour, personalities, and behavioural synchronization. Due to Covid-19, an increase in registered dogs could be observed. This could lead to more dogs being rehomed once the restrictions are eased and people go back to their workplaces, hence the importance of more knowledge concerning rehomed dogs and their behaviour, stress, and the relationship to its new owner. Thirty rehomed dogs (26 owners) and 31 control dogs (28 owners) participated. Dogs and their owners were tested in an unsolvable problem task to observe contact-seeking behaviour, and a behavioural synchronization test was conducted. Afterwards, dogs were equipped with activity collars to measure physical activity and dog-hair was collected to assess long-term stress. In addition, owners completed three questionnaires to assess the dog-owner relationship, and the personalities of the dogs and their owners. The results revealed no significant differences in behavioural synchronization, contact-seeking behaviour, stress, physical activity, or the dog-owner relationship between rehomed and control dogs. However, owners owning a rehomed dog potentially scored higher on the personality trait “agreeableness”, and rehomed dogs were potentially scored as more fearful of handling and less playful than control dogs. In conclusion, there is potentially a difference in personality traits in both dogs and owners between the groups, however, rehomed dogs are similar to control dogs in terms of behavioural synchronization, contact-seeking behaviour, long-term stress, and the dog-owner relationship.