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: For free-living or feral horses weaning takes place naturally at around 8-9 months [Gill, E.L., 1988. Factors affecting body condition of New Forest Ponies. Ph.D. Thesis. Department of Biology, University of Southampton]. Some mares will continue to suckle their foal until shortly before the arrival of their next foal, gestation being approximately 342 days depending upon the breed of the horse [Ropiha, R.T., Mathews, G., Butterfield, R.M., 1969. The duration of pregnancy in Thoroughbred mares. Vet. Rec. 84, 552-555]. Under domestic conditions, weaning tends to take place earlier, typically between 4 and 6 months of age. The weaning process has been identified as associated with potential psychological, physical and nutritional stressors that are of welfare concern. Following a review of the literature it is evident that there is a need for detailed research into what should constitute best practice with respect to foal and mare welfare. In addition, there is a need to understand the potential long-term impact of weaning on, for example, trainability and later maternal behaviour, and whether the stresses associated with early weaning have detrimental effects on the performance horse. There is also a lack of clear information concerning the most frequently observed weaning practices and the reasons why certain weaning methods are chosen. Some variables should be closely managed during weaning in order to minimise stress responses. These include: early creep feeding to familiarise the young animal with the food it will be exposed to during weaning, feeding a high fibre diet and keeping the animal in extensive conditions using a gradual approach to weaning. However, we conclude that there may not be one best method for weaning, since the chosen method must take into account a number of factors including: available resources, the housing environment, the individual foal's stage of development, the strength of the mare-foal attachment, the foal's ability to cope with changes in social conditions and the ability of the horse owner to implement the chosen method. We do however suggest that the fewest stress responses appear to occur where foals are weaned gradually and allowed to have social contact either with other foals or with older horses.

Abstract: Veterinary and management procedures often are aversive to the animals, resulting in physiological and behavioural stress reactions, which increase the risk of accidents and might lower performance. We investigated the effects of previous positive handling and of gentle interactions during the procedure on behaviour and heart rate in dairy cows during rectal palpation with sham insemination. Twenty cows were allocated randomly into two groups of 10 animals: handling, received additional positive handling over a period of four weeks by one person (handler); control, only routine handling by different caretakers. The week after the handling period, tests lasting 9 min, including 4 min rectal palpation, were carried out with each animal on four successive days in four situations in a balanced order: cow is alone during the test, with the handler, with an usual caretaker, or with an unknown person. Behaviour and heart rate were recorded. Previously handled animals had lower heart rate during tests (P<=0.05, n=19), kicked less when alone (P<=0.05, n=19) and tended to show less restless behaviour (P<=0.1, n=19). Cows were further calmed by gentle interactions during the test, but people differed remarkably. Cows showed less restless behaviour when gentled by the handler, both in the 4 min of rectal palpation and in the 9 min test period (each: P<=0.001, n=19). No significant stress reducing effect was found for the other two persons. In conclusion, stress reactions of cows during rectal palpation/insemination can be reduced by previous positive handling as well as by a person providing positive, gentle interactions during the procedure. The results underline the importance of positive, gentle interactions with the animals to enhance animal welfare and reduce the risk of accidents. They also show that people differ in the success to calm down the animals in aversive situations and indicate the need to investigate the characteristics responsible for the differences and identify the preconditions for a stress reducing effect in future research.

Abstract: Measuring HPA axis activity is the standard approach to the study of stress and welfare in farm animals. Although the reference technique is the use of blood plasma to measure glucocorticoid hormones (cortisol or corticosterone), several alternative methods such as the measurement of corticosteroids in saliva, urine or faeces have been developed to overcome the stress induced by blood sampling itself. In chronic stress situations, as is frequently the case in studies about farm animal welfare, hormonal secretions are usually unchanged but dynamic testing allows the demonstration of functional changes at several levels of the system, including the sensitization of the adrenal cortex to ACTH and the resistance of the axis to feedback inhibition by corticosteroids (dexamethasone suppression test). Beyond these procedural aspects, the main pitfall in the use of HPA axis activity is in the interpretation of experimental data. The large variability of the system has to be taken into consideration, since corticosteroid hormone secretion is usually pulsatile, follows diurnal and seasonal rhythms, is influenced by feed intake and environmental factors such as temperature and humidity, age and physiological state, just to cite the main sources of variation. The corresponding changes reflect the important role of glucocorticoid hormones in a number of basic physiological processes such as energy metabolism and central nervous system functioning. Furthermore, large differences have been found across species, breeds and individuals, which reflect the contribution of genetic factors and environmental influences, especially during development, in HPA axis functioning. Usually, these results will be integrated with data from behavioral observation, production and pathology records in a comprehensive approach of farm animal welfare.

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.