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: 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.

Abstract: In order to improve the housing conditions of stallions in individual boxes by offering a possibility to have more social contact, the Swiss national stud farm tested a new box system for horses, allowing increased physical contact with the neighbouring stallion. The aim of this part of the study was to investigate whether this type of housing system (named “social box”) potentially induces a change in stress reactions in stallions compared to conventional boxes. Therefore faecal cortisol metabolite (FCM) concentration was measured as a non-invasive parameter to assess endocrine responses related to this new environment.
Four groups each consisting of eight adult Freiberger breeding stallions were included in the test design. Every stallion spent three weeks in a conventional box and in a social box respectively (cross-over design). The conventional box consisted of a separation wall with a lower opaque part and an upper part with vertical barriers (5 cm between barriers), allowing visual and olfactory contact but strongly limiting tactile contact. The separating wall of the social box consisted of two lateral sections, one part being opaque to the ceiling and the second part consisting of vertical barriers (30 cm between barriers), allowing the horse to have physical contact with its neighbour or to avoid it.
In horses, FCM concentration reflects an average level of circulating cortisol over a period of approximatively 24h. Faecal samples were collected the day following integration in social / conventional boxes, reflecting the potential stress induced by increased social interactions during the integration. In order to asses potential chronical stress, faeces samples were also collected in week one, two and three after the integration into the social / conventional box (in total: 4 samples per horse and housing system). The samples were immediately stored at -20°C until they were analysed. The samples were not analysed in the laboratory until the end of the experiment, therefore the duration of conservation in the freezer varied from 40 to 429 days.
A considerable percentage of data from groups 1 and 2 was below the detection limit (<0.8 ng/g) (Tab. 1). Thus the statistical analysis was conducted with the FCM concentration from groups 3 and 4 (n horses = 16) which contained no values below the detection limit.
Tab. 1: Details about FCM values and storage time for the 4 groups of stallions
Group Storage duration [d] Proportion of data below the detection limit (<0.8 ng/g) Mean [ng/g] Median [ng/g]
Group 1 384-429 55.6 % 2.2 0
Group 2 315-360 25.5 % 5.8 6.3
Group 3 41-79 0.0 % 8.7 8.0
Group 4 40-85 0.0 % 5.8 5.4
Despite the impressive social interactions observed between the stallions directly after being introduced into the social boxes, we did not find any differences in FCM concentration between the stallions being introduced into the conventional box and the social box on the day of integration (social box: n samples = 16, mean±SD: 6.9±4.7 ng/g; conventional box: n samples = 16, mean±SD: 9.0±11.2 ng/g; Wilcoxon signed rank test V = 70, p = 0.94).
Overall the samples taken during integration and in week one, two and three did not show evidence of changes in FCM concentration in either housing system over a longer period of time (social box: n samples = 64, mean±SD: 7.9±6.2 ng/g; conventional box: n samples = 64, mean±SD: 6.6±3.4 ng/g; Linear mixed model (LMM), p = 0.56).
Our results suggest that the possibility of having physical contact with a conspecific does not induce changes in FCM concentration in breeding stallions. The considerable percentage of values below the detection limit in groups 1 and 2 seemed to correlate with the increasing duration of storage before analysis. During the IESM Network Meeting 2015, we would like to discuss possible methodological issues and the possibilities to correctly integrate these low values in the statistical analysis.

Abstract: Naturally, horses live in groups in which all individuals are long-term acquainted with each other and a stable hierarchical system is established. In conservation management, introduction of horses into foreign groups is often required but will lead to fights, stress and increased health risks for the animals. We investigated the integration process of four Przewalski mares from the Zoo Leipzig into the herd of five Przewalski horses of the semireserve Liebenthal (Brandenburg, Germany). We observed changes in social hierarchy as well as a higher stress level (expressed by disturbed activity pattern and lower synchronization with environmental conditions) especially in the introduced horses (from Zoo Leipzig). We investigated the animals continuously over two years to detect what time is needed for a successful integration. Finally, we give some advice for the integration of Przewalski horses into a new herd to reduce the stress of the animals substantially.

Abstract: The first month of training of a young horse is suspected to be stressful, but the endocrine responses to initial training are unknown. Therefore in our study a total of 40 Quarter Horses (QH), all at the age of almost 2 years, were followed during the first 30 days of their training. During this time faecal samples were collected twice daily and faecal cortisol metabolites (FCM) were measured. Baseline values of FCM ranged between 1.3 and 20.1 (median: 6.7) ng/g faeces. No differences in FCM values between days of training were found. Mares showed the highest values. Significant diurnal variations were observed in mares (p=0.035) and stallions (p=0.003), but not in geldings (p=0.282). As in this study adrenocortical activity was not increased during initial training, horses seem to cope very well with this new situation. The results of our large-scale study provide basic physiological data about initial training. This gives additional input in an emotional debate about animal welfare aspects of first time handling and training of horses.
Abbreviations: 11,17-DOA = 11,17-dioxoandrostanes; EIA = Enyzme Immunoassay; FCM = faecal cortisol metabolites; GC = glucocorticoids; HPA-axis = hypothalamic-pituitary-adrenocortical-axis; QH = Quarter Horses
[Das Einreiten eines jungen Pferdes steht unter Verdacht belastend zu sein. Bisher gibt es aber keine Veröffentlichungen über endokrine Vorgänge während dieser Phase. Mit der vorliegenden Studie wurde überprüft, ob Pferde aufgrund physischer und psychischer Belastungen während des Trainings höhere Konzentrationen an Kortisolmetaboliten im Kot (FCM) aufweisen. Es wurden dazu 40 Quarter Horses im Alter von 2 Jahren während der ersten 30 Tage der Grundausbildung des Westernreitens beobachtet und ihre FCM Werte gemessen. Während dieser Zeitspanne wurden täglich morgens und abends Kotproben der Pferde genommen. Die Basalwerte der FCM Konzentration variierten zwischen 1,3 und 20,1 (Median: 6,7) ng/g Kot, wobei Stuten die höchsten Werte hatten. Signifikante Unterschiede während der einzelnen Trainingstage konnten nicht festgestellt werden. In der Tagesrhythmik wurden signifikante Unterschiede bei Stuten (p=0,035) und bei Hengsten (p=0,003), jedoch nicht bei Wallachen (p=0,282) ermittelt. In dieser Studie konnte keine erhöhte Aktivität der Nebennierenrinde im Verlauf der Grundausbildung eines Pferdes im Westernreitstil festgestellt werden. Das legt nahe, dass Pferde mit dieser neuen, zeitlich kurz andauernden Situationen gut zurechtkommen. Unsere Studie wurde an einer großen Anzahl von Tieren unter Feldbedingungen durchgeführt. Sie bietet daher eine gute Datenbasis über Belastungen während des Einreitens. Damit liefert sie einen zusätzlichen Beitrag zu einer mitunter emotional geführten Debatte über tierschutzrelevante Aspekte bei der Grundausbildung von Pferden.]