Abstract: Abstract Analysis of salivary cortisol is increasingly used to assess stress responses in horses. Since spontaneous or experimentally induced increases in cortisol concentrations are often relatively small for stress studies proper controls are needed. This requires an understanding of factors affecting salivary cortisol over longer times. In this study, we have analysed salivary cortisol concentration over 6 mo in horses (n = 94) differing in age, sex, reproductive state and housing. Salivary cortisol followed a diurnal rhythm with highest concentrations in the morning and a decrease throughout the day (P < 0.001). This rhythm was disrupted in individual groups on individual days; however, alterations remained within the range of diurnal changes. Comparison between months showed highest cortisol concentrations in December (P < 0.001). Cortisol concentrations increased in breeding stallions during the breeding season (P < 0.001). No differences in salivary cortisol concentrations between non-pregnant mares with and without a corpus luteum existed. In stallions, mean daily salivary cortisol and plasma testosterone concentration were weakly correlated (r = 0.251, P < 0.01). No differences in salivary cortisol between female and male young horses and no consistent differences between horses of different age existed. Group housing and individual stabling did not affect salivary cortisol. In conclusion, salivary cortisol concentrations in horses follow a diurnal rhythm and are increased in active breeding sires. Time of the day and reproductive state of the horses are thus important for experiments that include analysis of cortisol in saliva.

Abstract: A behavioral assessment of horses who were being used and not used in therapeutic riding programs was conducted to help determine useful methods of selecting horses for use in therapeutic riding programs. A total of 103 horses (76 horses from five therapeutic riding centers and 27 non-therapeutic riding horses from four sites) were used. Temperament survey for each horse were completed by three riding instructors at each therapeutic riding center or by the individual most knowledgeable about the horse at the other sites. Twenty personality traits from the survey were used to quantify temperament. Concentrations of plasma cortisol, norepinephrine and epinephrine were also measured in each horse. A reactivity test was then conducted which involved introducing three novel stimuli: a walking and vocalizing toy pig placed on a cardboard surface in front of the horse for 20 s; popping a balloon near the horse's flank area; and suddenly opening an umbrella and holding it open in front of the horse for 20 s. Reactions (expressions, vocalizations and movement) to each of the stimuli were scored and used to calculate an average reactivity score for each horse. The therapeutic riding instructors did not often agree on the temperament of their center's horses. The personality trait ratings made by the therapeutic riding instructors at each center were on average significantly correlated (P<0.01, r>0.52) for only 37.8% of the horses for any two instructors and 7.8% for three instructors. No significant correlations were found between temperament, reactivity, and the hormone concentrations (r<0.19), but regression analysis indicated a possibility of predicting temperament from the reactivity score and hormone concentrations (P<0.08). There was also a tendency for relationships between extremes in temperament (desirable vs. undesirable) and the hormone concentrations (P<0.09), and between extremes in reactivity (low vs. high) and the hormone concentrations (P=0.08). The difference in ratings among riding instructors indicates a need for more collaboration between instructors when evaluating horse temperament. This study also indicates that it was very difficult to objectively determine the suitability of horses for therapeutic riding programs regarding their temperament and reactivity, probably because other traits (e.g., smoothness of gait) are also considered very important.

Abstract: Cortisol levels in hair samples were examined in fourteen domestic dogs and related to the dogs’ responses to different acoustic stimuli. Stimuli were playbacks of species-typical vocalizations recorded during three different situations (“disturbance”, “isolation” and “play” barks) and the sounds of a thunderstorm. Hair samples were collected at 9:00 h and 17:00 h two weeks after the behavioural tests. Results showed that behavioural reactivity to playback of the various stimuli correlates with cortisol levels in hair samples collected at 9:00 h, and the same was the case for the separate measures of behaviour (i.e. hiding, running away, seeking attention from the tester, panting and lowering of the body posture). Hence, levels of cortisol in hair appear to reflect the dog’s chronic state of emotional reactivity, or temperament.

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: 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.