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: Horses kept in stalls are deprived of opportunities for social interactions, and the performance of natural behaviors is limited. Inadequate environmental conditions may compromise behavioral development. Initial training is a complex process and it is likely that the responses of horses may be affected by housing conditions. Sixteen 2-year-old Arabian horses were kept on pasture (P) (n=8) or in individual stalls (S) (n=8). Twelve horses (six P and six S) were subjected to a standardized training procedure, carried out by two trainers in a round pen, and 4 horses (two P and two S) were introduced to the round pen but were not trained (C; control). On sample collection day 0, 7, 21 and 28, behavior observations were carried out, blood samples were drawn and heart rates were monitored. Total training time for the stalled horses was significantly higher than total time for the pastured horses (S: 26.4+/-1.5 min; P: 19.7+/-1.1; P=0.032). The stalled group required more time to habituate to the activities occurring from the start of training to mounting (S: 11.4+/-0.96; P: 7.3+/-0.75 min; P=0.007). Frequency of unwanted behavior was higher in the stalled horses (S: 8.0+/-2.0; P: 2.2+/-1.0; P=0.020). Pastured horses tended to have higher basal heart rates on day 0 (S: 74.7+/-4.8; P: 81.8+/-5.3 bpm; P=0.0771). While the physiological data failed to identify differences between housing groups, the behavioral data suggest that pasture-kept horses adapt more easily to training than stalled horses.

Abstract: All feces produced during 24 h were collected from five pigs and cortisol and immunoreactive cortisol metabolites (CICM), and IgA were quantified. Within pigs, the concentrations of CICM and IgA varied extensively between random samples obtained from a single fecal dropping, and deviated in most cases significantly from the true concentration measured in total fecal output (CV 6.7–130%). The CICM and IgA contents varied considerably (CV 8.1–114%) within and between individual fecal droppings from the same pig compared to the total fecal excretion. In conclusion, single random samples could not be used to reliably quantify the total fecal concentration or excretion of CICM or IgA in pigs. Analyses of all feces collected during shorter periods than 24 h did not provide an accurate estimate of the daily excretion of CICM. Thus, the concentration of stress sensitive molecules in random single fecal samples as an indicator of animal welfare should be interpreted with prudence.

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: Animal management often involves procedures that, while unlikely to cause physical pain, still cause aversive responses. The domestic horse (Equus caballus) regularly has excessive hair clipped off to facilitate its use as a riding/driving animal and this procedure causes adverse behavioral responses in some animals. The aim of this study was to compare behavioral and physiological measures to assess the aversive effect of this procedure. Ten horses were selected on the basis of being either compliant (C: n=5) or non-compliant (NC: n=5) during this procedure. The horses were subjected to a sham clipping procedure (SC: where the blades had been removed from the clippers) for a period of ten minutes. Measures were taken pre, during and post SC (-10min to +30min) and mean values calculated for ALL horses and for C and NC separately. Behavioral activity was scored (scale 1-5) by twenty students from video footage in (phase/group-blind scoring). Heart rate (HR), salivary cortisol and eye temperature were monitored throughout the procedure. The NC horses were found to be significantly more behaviorally active/less relaxed throughout the trial than C horses (p<0.05) with the greatest difference occurring during the SC procedure (p<0.01). NC horses were more active/less relaxed during, compared with pre or post SC (p<0.05), but showed no behavioral difference pre and post SC. HR of the NC horses was higher than that of the C horses throughout the trial but only significantly so after 10min of SC (p<0.01). ALL horses showed a significant increase in HR between +5 and +10min into the procedure (p<0.05). There was a significant increase in salivary cortisol concentration in ALL horses post procedure (p<0.01) with levels peaking at 20minute post SC. No significant differences in salivary cortisol concentration between C and NC were found at any stage of the trial. Eye temperature increased significantly in ALL horses during SC, peaking at +10min into the procedure (p<0.05) and then decreased substantially when SC had ceased (p<0.01). Although no significant differences were found between C and NC per se, there was a significant interaction between group and phase of trial (p<0.05) with the NC group showing a greater decrease in eye temperature post SC. There was a significant positive correlation between changes in salivary cortisol concentration and eye temperature (p<0.01) but no correlation between any of the other measures. Although the behavioral response of C and NC to this procedure was significantly different the physiological responses indicated that ALL horses found the procedure aversive. Eye temperature could be used as an objective and immediate measure of how an animal is responding to a specific situation in order to evaluate management procedures and adapt them where appropriate to reduce the negative impact on animal health and welfare.