Abstract: Reasons for performing study: In horses, serum cortisol concentration is considered to provide an indirect measurement of stress. However, it includes both free and bound fractions. The sampling method is also invasive and often stressful. This is not the case for salivary cortisol, which is collected using a more welfare-friendly method and represents a part of the free cortisol fraction, which is the biologically active form. Objectives: To compare salivary and serum cortisol assays in horses, in a wide range of concentrations, using an adrenocorticotropic hormone (ACTH) stimulation test, in order to validate salivary cortisol for stress assessment in horse. Methods: In 5 horses, blood samples were drawn using an i.v. catheter. Saliva samples were taken using swabs. Cortisol was assayed by radioimmunoassay. All data were treated with a regression method, which pools and analyses data from multiple subjects for linear analysis. Results: Mean ± s.d. cortisol concentrations measured at rest were 188.81 ± 51.46 nmol/l in serum and 1.19 ± 0.54 nmol/l in saliva. They started increasing immediately after ACTH injection and peaks were reached after 96 ± 16.7 min in serum (356.98 ± 55.29 nmol/l) and after 124 ± 8.9 min in saliva (21.79 ± 7.74 nmol/l, P<0.05). Discharge percentages were also different (225% in serum and 2150% in saliva, P<0.05). Correlation between serum and salivary cortisol concentrations showed an adjusted r2= 0.80 (P<0.001). The strong link between serum and salivary cortisol concentrations was also estimated by a regression analysis. Conclusions: The reliability of both RIAs and regression found between serum and salivary cortisol concentrations permits the validation of saliva-sampling as a noninvasive technique for cortisol level assessment in horses.

Abstract: Horses are a highly social species living in complex social systems which should require them to memorise and generalise social experiences and distinguish between familiar and unfamiliar conspecifics. In the main part of my thesis I concentrated on the specific conflict situation of a horse being introduced into a new social group, and investigated its behaviour and stress level. Horses were either introduced (1) immediately, (2) after an observation period, or (3) together with an integration horse after an observation period. Additionally, in the second part of my thesis I arranged several experiments to elaborate additional aspects which could affect the behaviour of horses during introductions. In this study I could describe a simplified method for measuring stress through the analysis of faecal GCMs in horses. An enzyme immunoassay (EIA) for 11-oxoaetiocholanolone using 11-oxoaetiocholanolone-17-CMO: BSA (3?,11-oxo-A EIA) as antigen showed high amounts of immunoreactive substances. The new assay increases the accuracy of the test and lowers the expenses per sample; also storing of samples at room temperature after collection is less critical. This is a big advantage both in the field of wildlife management of equids and in the field of equestrian sports (chapter 1). Comparing the different introduction techniques, the introduction with an integration horse led to significantly less total interactions and lower levels of aggression than the introduction of single horses, both immediately and after several days of observing the new group. Additionally, by observing the behaviour of the horses during everyday sociality I could develop a formula describing the interrelationship between expected aggression level and enclosure size per horse. The curve takes an exponential shape. Starting from a space allowance of 300 m2 and more per horse, the amount of aggressions per hour approaches zero. For the reduction of aggression levels and injury risks in socially kept horses I recommend an enclosure size of at least 300 m2 per horse (chapter 2). I further investigated the stress level of the introduced animals. Horses which were immediately introduced did not show elevated faecal GCMs. In contrast, horses which were introduced after an observation period had slightly elevated values 2 and 3 days after the introduction. For horses introduced together with an integration horse faecal GCMs were significantly above the baseline value on the day of introduction and 1 day after it. These differences between introduction techniques indicate that the introduction event itself is not as stressful as previously assumed. Rather standing together with an integration horse and not being able to integrate immediately into the complete group elicits stress in horses (chapter 3). In the commentary of chapter 4 several studies are discussed which failed to demonstrate social learning in horses. It is argued that they did not consider important aspects which could have an influence, such as the dominance status or the social background of the horses (chapter 4). In chapter 5 a social feeding situation was investigated. The social rank as well as the position of conspecifics affected the feeding strategy of horses. Domestic horses used social cognition and strategic decision making in order to decide where to feed. When possible they tended to return to the same, continuously supplied feeding site and switched to an ?avoidance tendency? in the presence of dominant horses or when another horse was already feeding there (chapter 5). One possibility to recognize group members is through olfactory recognition. In chapter 6 it is shown that horses are able to distinguish their own from their conspecifics? faeces. In addition, they paid most attention to the faeces of those group members from which they received the highest amount of aggressive behaviour (chapter 6). Horses show cognitive abilities because they are able to use humans as local enhancement cues when searching for food, independently of their body posture or gaze consistency when the persons face them. Moreover, they seem to orientate on the attention of familiar persons more than of unfamiliar persons (chapter 7). Altogether, the results of this thesis provide further support for the view that horses show good conflict resolution strategies. They are perfectly able to deal with the conflict situation of being introduced to new group members, and the introduction event itself is not as stressful as previously assumed. It is rather suggested that standing together with an integration horse and not being able to integrate immediately into the complete group elicits stress in horses. All additional experimental set-ups could demonstrate that horses are well capable of social cognition.

Abstract: A multitude of endocrine mechanisms are involved in coping with challenges. Glucocorticoids, secreted by the adrenal glands, are in the front line of the battle to overcome stressful situations. They are usually measured in plasma samples as parameters of adrenal activity and thus of disturbance. Unfortunately, collecting blood samples itself can disturb an animal. Thus, non-invasive methods for the determination of glucocorticoids or their metabolites have become increasingly popular. The pros and cons of various non-invasive sample materials (saliva, excreta, milk, hair/feathers and eggs) for glucocorticoid determination are given. Above all, faecal samples offer the
advantage that they can be collected easily. In faecal samples, circulating hormone levels are integrated over a certain period of time and represent the cumulative secretion of hormones. Thus, the levels are less affected by short fluctuations or the pulse-like nature of hormone secretion. However, using this technique to assess an animal’s adrenocortical activity is not especially simple. Whether frequent sampling is necessary or single samples will suffice depends upon the study’s aim (whether one is examining the impact of acute or chronic stressors). Background knowledge of the metabolism and excretion of cortisol/corticosterone metabolites is required and a careful validation for each species and sex investigated is obligatory. The present review also addresses analytical issues regarding sample storage, extraction procedures and immunoassays and includes a comprehensive list of published studies (up to 2011) describing the use of such methods in farmed animals. Applied properly, non-invasive techniques to monitor glucocorticoid metabolites in faecal samples of various species are a useful tool for welfare assessment, especially as they are easily applied at farm or group level.

Abstract: In dieser Untersuchung sollte überprüft werden, ob die für die Stressmessung beim Menschen angewandte „Chronopsychobiologische Regulationsdiagnostik“ (CRD) mithilfe des smardwatch®-Messsystems auch beim Pferd psychische Belastungssituationen aussagekräftig widerspiegelt. Menschen können anhand variierender physiologischer und ethologischer Reaktionen auf Stressoren aus der Umwelt in Stresstypen eingeteilt werden. Auch Pferde reagieren in identischen Situationen ganz unterschiedlich. Es stellte sich daher die Frage, ob Pferde ebenfalls bestimmten Stresstypen zugeordnet werden können.
Mit 26 dreijährigen Hengsten des Landgestüts Celle wurde ein Verhaltenstest durchgeführt. Auf der Grundlage wissenschaftlich dokumentierter und modifizierter Testsituationen (Open-Field-Test, Novel-Object-Test, Startling-Test Objekt/Geräusch, Mensch-Pferd-Interaktion, Ressourcenkontrolle) wurde das Erkundungs- und Fluchtverhalten untersucht (Goslar 2011). Während dieser Belastungssituationen wurden mit dem smardwatch®-Messgerät die elektrophysiologischen Parameter Hautwiderstand, Hautpotential und Muskelaktivität erfasst. Durch eine Zeitreihenmessung dieser physiologischen Parameter konnten die Reaktionen des übergeordneten Reglers (vegetatives Nervensystem) dargestellt werden. Der Hautwiderstand spiegelt die vegetativ-emotionalen Reaktionen wider, das Hautpotential die vegetativ-nervalen und somit laut Balzer (2009) die kognitiven Verarbeitungsweisen, und durch das Elektromyogramm werden die typischen muskulär-motorischen Reaktionen aufgezeichnet. Die vorliegenden Datenzeitreihen wurden mit Hilfe einer biorhythmometrischen Zeitreihenanalyse nach Balzer und Hecht (Hecht 2001, Balzer 2009) ausgewertet. Als Ergebnis wurden chronobiologische Regulationszustände definiert, die dann gemäß dem „Periodischen System der Regulationszustände“ (PSR) (Balzer 2000) eine Beurteilung der psychischen Belastungssituation erlauben. Zur Bestimmung von Stresstypen wurde die Untersuchung nach dem Reiz-Reaktions-Prinzip in drei Phasen unterteilt: Ruhephase, Stressreizphase und Stressverarbeitungsphase. Der Verhaltenstest wurde nach einer Woche zur gleichen Tageszeit mit jedem Hengst einmal wiederholt. Als physiologische Vergleichsparameter wurden vor, während und nach der Belastung insgesamt zehn Speichelproben zur Cortisolbestimmung entnommen.
Die smardwatch®-Messtechnik ist prinzipiell geeignet die physiologischen Parameter Hautwiderstand, Hautpotential und Muskelaktivität auf der Haut des Pferdes aufzunehmen. Durch die biorhythmometrische Zeitreihenanalyse konnten chronobiologische Regulationszustände definiert werden.
Der Verhaltenstest führte bei allen Pferden zu einem signifikanten Anstieg der Cortisolwerte. Ein ebenfalls statistisch gesicherter Unterschied der aktivierten Cortisolwerte zwischen der ersten und zweiten Testwoche zeigt eine geringere Stressreaktion und weist auf einen Lerneffekt der Pferde hin:
Entsprechend der relativen Instabilitäten der Regulation der Körperfunktionen in Messphase 2 und 3 konnten die Pferde in vier Stressregulationstypen (nach Balzer u. Hecht 1996) eingeteilt werden: Stressbeherrscher, -bewältiger und kompensierer sowie Stressnichtbewältiger.
Anhand der Aktivierung von Sympathikus und Parasympathikus konnten zusätzlich vier verschiedene Vegetative Stresstypen unterschieden werden: Sympathikotoniker, Amphotoniker, Indifferenter Typ und Vagotoniker.
Die Cortisolreaktion der Hengste war individuell sehr unterschiedlich. Um die maximalen Anstiege vergleichen zu können, wurde der Trend der jeweiligen Cortisolverlaufskurve bestimmt. Anhand der Trend-korrigierten Cortisolkurven konnten einerseits die relativen Maxima der Cortisolreaktion besser verglichen werden, anderseits konnten die Pferde mittels des unterschiedlichen Trendes in drei Cortisolverlaufsgruppen eingeteilt werden: Tendenz fallend, gleichbleibend oder steigend.
Ein statistischer Nachweis für eine Abhängigkeit zwischen den verschiedenen Stresstypenklassifizierungen konnte anhand der geringen Stichprobenzahl nicht erbracht werden. Jedoch lassen sich Pferde, wie Menschen, unterschiedlichen Stresstypen zuordnen. Schwierig bleibt aber die objektive Beurteilung von Befindlichkeiten, da Empfindungen und Gefühle nur subjektiv wahrnehmbare Qualitäten sind, die von einer Reihe innerer und äußerer Faktoren abhängen. Die Verhaltenszuordnung emotionaler Zustände durch die „Chronopsychobiologische Regulationsdiagnostik“ (CRD) kann nicht ohne die Basis weiterer vergleichender Studien vom Mensch auf das Pferd übertragen werden.
Die CRD-Methode könnte allerdings einen interdisziplinären Ansatz ermöglichen und zukünftig neben den klassischen deskriptiven Verhaltensbeobachtungen bei der Beurteilung von Haltungs- und Umgangssituationen von Tieren wertvolle Aufschlüsse über die Fähigkeit zur Stressbewältigung und deren Konsequenzen für das Wohlbefinden der Tiere geben.
In this study we examined, whether the “Chronopsychobiological regulation diagnosis” (CRD) with the smardwatch®-system which is used to assess specific strain in humans, is also able to reflect convincingly specific strain in horses. Humans can be categorized into so-called stress types, because they react differently in physiology and behaviour to environmental stimuli. Concerning horses, it is also known that individuals react differently in identical situations. The question to be answered is, if it is possible to categorize horses into certain stress types as well.
We carried out a behavioural test with 26 three-year-old stallions of the State Stud of Celle, involving different test situations. On the basis of test situations, well known in scientific literature including slight modifications (open-field-test, novel-object-test, startling-test object/sound, human-horse-interaction, resource control) the explorative and flight behaviour of horses were examined (Goslar 2011). During these situations of strain the system smardwatch® measured the electro-physiological parameters skin resistance, skin potential and muscle activity. With time series analyses of these physiological parameters the reactions of the vegetative nervous system as superior control could be shown. The skin resistance reflects the vegetative-emotional, the skin potential the vegetative-nervous hence according to Balzer (2009) cognitive reactions and the electromyogram shows the motorized reactions. The time series of measured data was analyzed using the biorhythmometrical time series analysis of Balzer and Hecht (Hecht 2001, Balzer 2009). As a result of this, typical states of chronobiological regulation were defined. With the help of the “periodic system of regulatory states” (PSR) (Balzer 2000) these led to a classification of mental stress situations. To categorize horses into stress types the study was devided into three phases according to the stimulus-response principle: phase 1 (relaxing), phase 2 (situation of strain) and phase 3 (stimulus processing phase). The behavioural test was repeated once with each stallion exactly one week later. In addition ten samples of saliva were taken before, during and after the situations of strain from which we determined the cortisol concentration to be compared with the chronopsychobiological parameters.
The smardwatch®-measurement technology is fundamentally suitable to measure the physiological parameters skin resistance, skin potential and electrical muscle activity of horses. Using the biorhythmometrical time series analysis, chronobiological regulatory states could be defined for horses as well.
For each horse the cortisol value increased significantly during the behavioural test. A remarkable difference of the cortisol values assessed in the first and second test, indicates a learning effect: The behavioural test led to a significantly lower stress reaction in the second week.
Depending on the proportions of unstable regulation processes during and after the situations of strain, four types of regulation (Balzer u. Hecht 1996) can be defined: the Control-, Cope-, Compensate- and Non-cope-type.
Using the activation of the sympathetic and parasympathetic nervous system four vegetative stress types could be determined: Sympathicotonic, Amphotonic, Indifferent Type and Vagotonic.
The stress reaction of the stallions in terms of cortisol level was quite individual. To compare the maximum increase the trend of each cortisol trajectory was analyzed. On the one hand correcting for the trend allowed a comparison of relative maxima, on the other hand the horses could be categorized into groups, according to the evolution of their cortisol level (cortisol progression groups): Tendency falling, stable or rising.
The number of samples was not considered to be sufficient in order to statistically assess some dependence among the different classifications of stress types. But to our point of view horses can be assigned to different stress types like humans. But it’s still difficult to judge the emotions of animals, because emotions are subjectively perceptible qualities dependent on many internal and external factors. The assignment of emotional behaviour to the chronopsychobiological regulation diagnosis (CRD) can’t be transferred from human to horse without further studies.
However, the CRD method could enable an interdisciplinary approach. Besides classic descriptive observations of behavior, the CRD could give further information about the coping capacity and the consequences for animal welfare in the assessment of stressful situations.