Abstract: Non-invasive techniques to monitor stress hormones in small animals like mice offer several advantages and are highly demanded in laboratory as well as in field research. Since knowledge about the species-specific metabolism and excretion of glucocorticoids is essential to develop such a technique, we conducted radiometabolism studies in mice (Mus musculus f. domesticus, strain C57BL/6J). Each mouse was injected intraperitoneally with 740 kBq of 3H-labelled corticosterone and all voided urine and fecal samples were collected for five days. In a first experiment 16 animals (eight of each sex) received the injection at 9 a.m., while eight mice (four of each sex) were injected at 9 p.m. in a second experiment. In both experiments radioactive metabolites were recovered predominantly in the feces, although males excreted significantly higher proportions via the feces (about 73%) than females (about 53%). Peak radioactivity in the urine was detected within about 2h after injection, while in the feces peak concentrations were observed later (depending on the time of injection: about 10h postinjection in experiment 1 and about 4h postinjection in experiment 2, thus proving an effect of the time of day). The number and relative abundance of fecal [3H]corticosterone metabolites was determined by high performance liquid chromatography (HPLC). The HPLC separations revealed that corticosterone was extensively metabolized mainly to more polar substances. Regarding the types of metabolites formed, significant differences were found between males and females, but not between the experiments. Additionally, the immunoreactivity of these metabolites was assessed by screening the HPLC fractions with four enzyme immunoassays (EIA). However, only a newly established EIA for 5alpha-pregnane-3beta,11beta,21-triol-20-one (measuring corticosterone metabolites with a 5alpha-3beta,11beta-diol structure) detected several peaks of radioactive metabolites with high intensity in both sexes, while the other EIAs showed only minor immunoreactivity. Thus, our study for the first time provides substantial information about metabolism and excretion of corticosterone in urine and feces of mice and is the first demonstrating a significant impact of the animals' sex and the time of day. Based on these data it should be possible to monitor adrenocortical activity non-invasively in this species by measuring fecal corticosterone metabolites with the newly developed EIA. Since mice are extensively used in research world-wide, this could open new perspectives in various fields from ecology to behavioral endocrinology.

Abstract: Crib-biting (wind-sucking) might be a coping response of the horses to the challenges of
uncontrolled environmental events. Prevention of this stereotypic behaviour evokes
physiological responses consistent with increased stress. Reducing the incidence of cribbiting,
however, is important in order to prevent undesirable physical and behavioural
consequences (tooth erosion, altered gut function, gastric inflammation/ulceration, colic, etc.).
Common treatment of crib-biting is the application of a cribbing collar, which limits the
flexion of the neck making this stereotypic movement uncomfortable and difficult. Another
method, the modified Forssell"s operation, is becoming more and more popular amongst the
horse owners. It is based on the removal of the muscles used in crib-biting (m.omohyoideus,
m.sternohyoideus, m.sternothyrohyoideus) and the ventral branches of the spinal accessory
nerves. Surveys on the success of this surgical procedure have revealed inconsistent results,
and, contrary to the cribbing collar, its effect on the stress level have not been studied either.
The aim of our study was to determine whether the modified Forssell"s procedure is superior
to the cribbing collar treatment.
Differences in stress management was tested by a crib-biting provoking test, in which
surgically treated horses, crib-biting horses, crib-biting horses with cribbing collar, and
normal horses (those showing no stereotypies), altogether 56 horses were compared. In this
test, a food bucket had been placed out of the reach of the animal, from which titbits were
given 3 times. Behaviour and heart rate variability (HRV) of the horses were recorded and
analysed throughout the test. Hypotheses were tested by linear mixed model.
According to our results, both prevention methods (collar or surgery) inhibited crib-biting
successfully though not totally. Regarding behaviour and heart rate variability, horses
prevented from crib-biting (by collar or surgery) differed significantly from crib-biting and
normal horses but not from each other.
Normal horses were usually trying to reach the food-bucket while present and were standing
still afterwards, whereas the other three groups had not really made efforts to reach the
bucket, spent less time with resting, and performed or tried crib-biting. During the stress-test,
normal and crib-biting horses had shown good stress-adaptation to the challenge since their
HRV, after an initial increase, returned to the basal value by the end. On the contrary, HRV of
the two prevented groups remained elevated and showed large oscillations throughout. They
had not found a successful coping behaviour either.
Our results suggest that since prevention may significantly increase distress, the treatment in
itself, without changing the motivation of the horse to perform the replacement behaviour – it
seems to be unsatisfactory and insufficient. After prevention the motivation of the horse to
perform crib-biting should be addressed. In addition, considering that prevention by collar and
surgery had not resulted in any significant behavioural or physiological differences, the
superiority of the modified Forssell"s procedure might be questioned. However, the surgery
might be recommended if treatment with collar is ineffective.

Abstract: The ability and ease of horses to habituate to frightening stimuli greatly increases safety in the horse-human relationship. Several different techniques have been suggested for habituation training of horses and under certain conditions, preventing animals from avoidance reactions during exposure to frightening stimuli is believed to facilitate habituation. Response prevention does, however, lead to a loss of control, which is a known stress inducer in both animals and humans. This experiment investigated whether horses show increased stress responses when negatively reinforced to approach a mildly frightening stimulus, compared to horses allowed to voluntarily explore the same stimulus. We further investigated whether the prevention of avoidance responses in horses that are negatively reinforced to approach the stimulus, facilitates habituation to the stimulus. Twenty-two 2-3 years old Danish warmblood geldings were included in the study. Half of the horses (NR group) were negatively reinforced (through halter and rope pressure) by a familiar human handler to approach a collection of frightening objects (six open and colourful umbrellas) placed in a semi-circle in a familiar test arena. The other half of the horses were released in the arena and were free to avoid or explore the objects (VOL group). On the next day, all horses were exposed to the objects again without a human to investigate the rate of habituation. Behavioural and heart rate responses were recorded on both days. Data were analysed in a two way repeated measures ANOVA and post hoc analysed via the Holm-Sidak method. In the VOL group, all horses initially chose to avoid the unknown objects, whereas the handler managed to get all horses in the NR group to approach and stand next to the objects within the first 2-min session. As expected, horses in the NR group had a significantly longer duration of alertness (sec, mean ± se: NR: 23 ± 4.1 vs. VOL: 16 ± 4.7, P=0.026) and a higher max HR in the first session (bpm, mean ± se: NR: 106 ± 5.2 vs. VOL: 88 ± 4.4, P=0.004). On the next day, however, the NR horses spent significantly less time investigating the objects (sec, mean ± se: NR: 13 ± 4.1 vs. VOL: 24 ± 6.0, P=0.005) and had a shorter latency to approach a feed container, placed next to the objects (sec, mean ± se: NR: 25 ± 3.9 vs. VOL: 47 ± 16.2, P=0.031), indicating increased habituation. In conclusion, negatively reinforced approach to mildly frightening objects appears to increase stress responses during the initial exposure, but also to facilitate habituation in young horses.

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: We developed a biorhythmical method to assess behaviour patterns and to evaluate living conditions of animals. All kinds of continuous and equidistant long-term recordings of behaviour are suitable for this method. As simple behavioural parameters, such as motor activity, can be conveniently recorded by telemetry from wild animals now, it is possible to investigate stressors by analysing its biorhythmic structure. It is the purpose of this report to describe the basic idea, and the procedure, and to give some examples of application measured on Przewalski horses in an Semireserve.