Abstract: Behaviour scores (BS) offer non-invasive, objective and easy to use ways of assessing welfare in animals. Their development has, however, largely focused on behavioural reactions to stressful events (often induced), and little use of physiological measures has been made to underpin and validate the behavioural measures. This study aimed to develop a physiologically validated scale of behavioural indicators of stress for the purpose of welfare assessment in stabled domestic horses. To achieve this, behavioural and physiological data were collected from 32 horses that underwent routine husbandry procedures. Principal component analysis (PCA) of the behavioural and physiological data revealed three meaningful components that were used as the basis of the scale. Analysis of video clips of the horses’ responses to the husbandry procedures was undertaken by a panel of equestrian industry professionals using a free choice profiling (FCP) methodology. These results were added to the scale along with key definitions from relevant literature. Salivary cortisol levels were significantly correlated with the BS confirming the scale was meaningful and reflected physiological stress. The scale offers an easy to use ‘tool’ for rapid, reliable non-invasive welfare assessment in horses, and reduces the need for potentially invasive physiological measures.

Abstract: We studied influences of dental care, food and storage on the reproducibility of salivary steroid levels. Cortisol (F), 17OH-progesterone (17OHP) and Progesterone (P) were measured using adapted commercial radioimmunoassays. Saliva samples of healthy adults (n = 15; m:8; f:7) were collected directly before and after dental care, and directly before and after breakfast with various foodstuffs. A second experiment investigated stability of steroids under different storage conditions. Four series of identical saliva portions (I: Native saliva; II: Centrifuged saliva; III: Saliva with trifluor acetate (TFA); IV: Saliva with 0.5% NaN3) were stored at room temperature and at 4°C for up to three weeks. To demonstrate influences of repeated thawing and re-freezing of saliva on steroid values, saliva samples (n = 15) were divided into identical portions. These portions were frozen and re-thawed up to 5 times before measurement. Neither dental care nor intake of bread or milk effected the reproducibility of F, 170HP, and P. Steroid levels decreased significantly in the course of three weeks under different storage conditions (P < 0.001). This decrease was clinically relevant from the second week onward, with exception of NaN3 treated samples. After repeated freezing and re-thawing 17OHP and P decreased slightly (about 5%). Only F decreased significantly after the third thawing (P < 0.001). The results show the usefulness of standardized handling of saliva samples for improving reproducibility and reliability of salivary steroid measurements.

Abstract: Initial equestrian training and especially first mounting of a rider are stressful challenges for young horses (1). Most young horses are raised in groups but, in association with equestrian training, they are commonly transferred to individual stabling in loose boxes. Although, in most stables, visual contact with horses in adjacent boxes is possible, separation from the herd might be an additional stressor. We have studied physiological stress parameters, in 3-year-old sport horse mares (n=8), transferred from a group stable with access to a paddock to individual boxes without paddock. Once stabled in the individual boxes, mares underwent a standard training for young horses. Horses had been accustomed to lunging and tolerating a rider on their back several weeks before the study. Mares were studied from 5 days before to 5 days after changing the stable. Cortisol concentration in saliva, locomotion activity (ALT pedometers), heart rate (HR) and HR variability (RMSSD: root mean square of successive beat-to-beat intervals) were determined. We hypothesized that the change of the stable increases cortisol release and is associated with changes in HR and RMSSD and reduced locomotion. Before mares were moved to individual boxes, cortisol concentration showed a pronounced diurnal rhythm with values around 0.6 ng/ ml in the morning and a continuous decrease throughout the day. When the mares were moved to individual boxes, cortisol concentration increased to 1.8±0.2 ng/ml and did not return to baseline values within 6 h (p<0.05 over time). On subsequent days, a diurnal rhythm was re-established but shifted to a higher level than before. Locomotion activity determined by ALT pedometers was increased for some minutes only after mares has been placed in individual boxes but was only slightly higher than during the time mares spent with the group in a paddock. On days 2-5 in individual boxes, locomotion activity was reduced compared to the group stable. HR increased and the HRV variable RMSSD decreased when mares were separated. In conclusion, separating horses during initial training from their group is an additional stressor, although the stress is less pronounced than induced by other social challenges, e.g. weaning of foals (2). When stabled in individual boxes, mares move less than when kept as a group. Horses kept in a group thus appear to exercise themselves freely, such an effect is absent when the animals are kept individually.

Abstract: Based on plasma cortisol concentrations it is widely accepted that transport is stressful to horses. So far, cortisol release during transport has not been evaluated in depth by non-invasive techniques such as analysis of salivary cortisol and faecal cortisol metabolites. Transport also causes changes in heart rate and heart rate variability (HRV). In this study, salivary cortisol, faecal cortisol metabolites, heart rate and HRV in horses transported by road for short (one and 3.5 h) and medium duration (8 h) were determined. With the onset of transport, salivary cortisol increased immediately but highest concentrations were measured towards the end of transport (4.1 ± 1.6, 4.5 ± 2.6, 6.5 ± 1.8 ng/ml in horses transported for one, 3.5 and 8 h, respectively). Faecal cortisol metabolite concentrations did not change during transport, but 1 day after transport for 3.5 and 8 h had increased significantly (p < 0.01), reflecting intestinal passage time. Compared to salivary cortisol, changes in faecal cortisol metabolites were less pronounced. Heart rate increased and beat-to-beat (RR) interval decreased (p < 0.05) with the onset of transport. Standard deviation of heart rate increased while root mean square of successive RR differences (RMSSD) decreased in horses transported for 3.5 (from 74 ± 5 to 45 ± 6 ms) and 8 h (from 89.7 ± 7 to 59 ± 7 ms), indicating a reduction in vagal tone. In conclusion, transport of horses over short and medium distances leads to increased cortisol release and changes in heart rate and HRV indicative of stress. The degree of these changes is related to the duration of transport. Salivary cortisol is a sensitive parameter to detect transient changes in cortisol release.

Abstract: The degree of lateralization (LI) indicates both the direction and strength of a paw preference. Here, a positive value is indicative of a right paw bias, and a negative value of a left paw bias. Higher numbers on the positive side of the scale and lower numbers on the negative side of the scale indicate a greater strength of that lateralization. The strength of motor lateralization (|LI|) is the absolute value of the LI. The use of absolute value removes directionality (i.e., does not indicate left or right paw bias) and instead indicates only the strength of the paw preference. Both LI and |LI| have been associated with behavioral differences in a range of species. The assessment of motor lateralization in the dog can be conducted by observing the paw used to perform motor tasks. Elevated cortisol concentrations have been associated with fearfulness in many species. Additionally, fearfulness and boldness can be assessed in response to so-called temperament tests. Consequently, in this study we examine the relationship between lateralization, temperament test results, and cortisol concentrations in 43 potential guide dogs, of which 38 were Labrador retrievers and 5 were golden retrievers. Over a 14-month period, the current study assessed motor lateralization and salivary cortisol concentrations 3 times (approximately 6 months of age, 14 months of age, and after the dogs' performance in the guide dog program had been determined) and behavior twice (approximately 6 and 14 months of age). This study is the first to examine the relationship between behavior, lateralization, and cortisol concentrations in dogs. It implemented an objective and quantifiable assessment of behavior that may be of use to a variety of dog-focused stakeholders. Findings show that during the Juvenile testing period (6 months of age), dogs with higher cortisol concentrations were typically less able to rest when exposed to the unfamiliar testing room. Results from both Juvenile and Adult Test (14 months of age) periods showed that a greater |LI| and LI were associated with more confident and relaxed behavior when dogs were exposed to novel stimuli and unfamiliar environments. Significant elevations of cortisol concentrations were found at the completion of guide dog training when compared with results from the 2 prior test periods. This finding may reflect maturation or the effect of the prolonged kenneling which occurred during this period.