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: Abstract Stress is a generic concept describing the body's reaction to external stimuli, including both physiological and psychological factors. Therefore, by definition, the assessment of psychological stress in the exercising horse encompasses the problem of teasing apart the psychological and physiological factors both of which result in stress responses. The present study reviews the existing literature on various measures of stress taken specifically in the context of equitation science. Particular attention has been paid to short-term effects, and commonly used measurements of short-term stress include heart rate, a number of heart rate variability parameters, blood or saliva cortisol levels, eye temperature, and various behaviour parameters including in particular behaviour patterns presumably indicative of conflict with the rider's/trainer's aids. Inspection of the individual studies' results revealed that disagreement between these different measures of stress is commonplace. For physiological parameters, the largest proportion of agreement (i.e. both parameters simultaneously indicated either higher, insignificant or lower stress compared to a control treatment) was found for heart rate and heart rate variability parameters, while generally limited agreement was found for cortisol. It appears that cortisol levels may not be particularly useful for assessing/assessment of the valence of a situation in the exercising horse as cortisol levels are predominantly linked to activation and exercise levels. Although heart rate variability parameters reflect in theory more closely sympathovagal balance compared to cortisol levels, great care has to be taken regarding the use of appropriate time-frames, appropriate raw data correction methods as well as the use of appropriate equipment. In spite of its wide-spread and apparently successful use, popular equipment may in fact not be accurate enough under field conditions. Eye temperature is another promising parameter for assessment of psychological stress, but the technique is likewise susceptible to application errors. Given the high susceptibility of physiological parameters to errors at various experimental stages, behavioural rather than physiological parameters may in fact provide more accurate measures of valence when conducting experiments in the exercising horse. Behavioural parameters that appear to be particularly practical in assessing stress in ridden horses' behaviour are associated with frequencies of behaviour indicative of conflict. However, while increased frequencies of are a good indicator of stress, the absence of conflict behaviour does not provide proof of the absence of stress due to the possible occurrence of conditions such as Learned Helplessness. In future studies, the above issues should be taken into consideration when designing experiments to assess psychological stress in ridden horses.

Abstract: Group turnout provides important socializing opportunities for horses, particularly those that are primarily stalled. A high percentage of equine injuries occur during group turnout, which could partly be due to the physical constraints of fencing. To investigate appropriate paddock sizes for group turnouts, horses (n = 12) from a single herd were divided into groups of 4, stalled for 24 hours, and then turned out for 1 hour into one of three differently sized pens: 342, 263, and 184 m2 per horse. Groups rotated through pens across 3 days, receiving one treatment per day. Blood was sampled for cortisol concentrations at 08:00 hours each morning, and then at 15 and 60 minutes into the turn out sessions, and 60 minutes after return to individual stalls. Groups rotated through three turnout times: 09:00, 12:00, 14:00 hours. Counts of agonistic behaviors (chasing, contact biting, and kicking) and low-level threats (pinned ears, tail swishing, bite and kick threats) were recorded. When turned out in pens that provided 342 m2 per horse, horses exhibited reduced plasma cortisol concentrations by 15 minutes after turnout and at 1 hour after return to their stalls (P < .05). Horses in pens providing 184 m2 per horse exhibited greater agonistic (P < .001) and low-level threat (P < .01) behaviors than horses in larger pens. These data provide insight into appropriate pen sizes for horses from established herds. Providing at least 342 m2 per horse may reduce the chance of injury in horses accustomed to group turnout.