Cortisol concentration in the saliva of horses subjected to different kinds of exercise

The aim of this study was to characterize the stress level in sport horses subjected to exercise by measuring the cortisol concentration in their saliva. The experiment was conducted on 5 groups of horses (49 animals): one control and four groups undergoing different types of exercises. The saliva samples were collected 3 × a day: late at night (between 20:00 and 23:00 h), early in the morning (between 5:00 and 8:00 h) and directly after the exercise. The concentration of cortisol was measured by the enzyme-immunoassay programme. The highest cortisol concentration was observed for the horses participating in 3-day events (8.93 nmol/dm3), whereas in the control group it was only 0.28 nmol/dm3. When compared to the cortisol concentration of other horse groups, this suggests that the stress level as measured by the cortisol concentration in saliva increases with increasing exercise intensity and its duration. The obtained results do not confirm the existence of a day rhythm of the cortisol concentration in horses. Moreover, the results may help in choosing the appropriate system of training and in improving horse welfare during competitions. Effort, equestrian disciplines, stress, sport horses Stress is related to biochemical reactions leading to the formation of substances that are commonly described as indicators of the stress level. These indicators are responsible for the restoration of homeostasis. One of the main substances that indicate stress level is cortisol which is a natural glucocorticoid hormone produced by the adrenal cortex. Cortisol has many general as well as metabolic functions, including a significant influence on the metabolism of carbohydrates, proteins and fats. The concentration of cortisol depends on the natural circadian rhythm, which can be disturbed by physical as well as mental stress. The cortisol concentration in blood serum is the highest between 6:00 and 8:00 h and the lowest between 23:00 and 4.00 h (Hoff is et al.1970; Lindner et al. 1990). However, cortisol concentration can increase even ten-fold as a result of stress (Ślebodzinski 1979; Hartwig 1989). Cortisol can be found not only in the blood serum but also in other body fluids including body excrements (Most l and Palme 2002). Cortisol secretion and its concentration in plasma depend strictly on adrenocorticotrophin which significantly increases when the sympathetic nervous system is stimulated especially under stress or in inflammatory states. Its catabolic function leads to an increase in the turnover of skeleton muscle protein. It does this by stimulating the disintegration and slowing down the synthesis, which can result in losing muscle tissue. Catabolic function also boosts the glucose production from the amino acids coming from protein disintegration. Cortisol can enhance and prolong the effects of other hormones including the adrenergic ones. However, the determination of cortisol concentration in animal serum is not always possible and taking blood samples causes additional stress. For this reason, easier and less invasive methods have been invented that make use of saliva, urine and faeces. ACTA VET. BRNO 2011, 80: 101–105; doi:10.2754/avb201180010101 Address for correspondence: Katarzyna Strzelec Department of Horse Breeding and Use Faculty of Animal Biology and Breeding University of Life Science in Lublin, Akademicka 13 20-950 Lublin, Poland Phone: +4881 445 65 75 Fax: +4881 533 37 52 E-mail: kasia.strzelec@poczta.fm http://www.vfu. cz/acta-vet/actavet.htm The most effective way of determining cortisol concentration in animals seems to be by using their saliva. This is because taking saliva samples is a rather simple, non-invasive and low-stress procedure (Aardal and Holm 1995). Moreover, the rate of balancing the cortisol concentration in saliva and blood is rapid: the whole process takes less than 5 min. For these reasons, determining the level of cortisol using saliva is a more advantageous way of examining the adrenal cortex functions than using blood serum (Vining et al. 1983). Therefore, the saliva testing is selected as the method of choice for sport horses. Such horses must often undergo testing during their competition periods in stressful conditions. Their saliva sampling is convenient as it does not require any special treatment and the samples can be stored at room temperature for up to 7 days (Aadral and Holm 1995; Clements and Parker 1998). Sport horses are used in a variety of sport disciplines which require different preliminary physical work and exercise during competition, and hence impose different stress intensities. These disciplines can be grouped by how intense the work is, for example: dressage, endurance, show jumping, three day event and races. However, we need a relationship between such different stress intensities and the cortisol concentration in horse saliva. The aim of this study, therefore, is to estimate the intensity of the stress level in sport horses as a function of exercise intensity by measuring the cortisol concentration in their saliva. Materials and Methods This study was based on 5 groups of horses (n = 49) doing different types of exercise. The first group was the control group of 10 horses (5 of the Malopolska breed, 3 of the Polish noble half breed, 1 Wielkopolska breed and 1 pony) aged from 4 to 20 years. The control group of horses spent time in the paddock doing no special exercise. The second group of 9 horses (4 of the Malopolska breed, 3 of the Polish noble half breed, 1 Wielkopolska breed and 1 pony) aged from 4 to 16 years was exposed to light exercise (walk, trot) for 4 to 6 h a day. The other three groups (each of 10 animals) represented different disciplines such as dressage, show jumping and three days events. Within each group there were animals of different breeds but approximately the same age (5 years). These 3 groups took part in the 2008 Polish Horses Championships For Young Horses in Ksiąz, Łąck and Bialy Bor. Their saliva samples were collected 3 × a day: late at night (between 20:00 and 23:00 h), early in the morning (between 5:00 and 8:00 h) and right after the exercise. In the control group the samples were collected four times a day. All the saliva samples were collected with a small piece of sponge (2 cm × 2 cm) soaked in 1% acetic acid and dried at room temperature (as described by Pel l and McGreevy 1999). Using metal tweezers, the sponge was inserted into the horse’s mouth and rubbed against the animal’s tongue, inner cheeks and palate. The sponge soaked in saliva was then placed in a centrifugation tube. Each tube had a 2 cm long piece of plastic straw preventing the sponge from touching the bottom of the tube and absorbing the saliva during the centrifugation. Next, all the samples were frozen. This allowed their storage in an unchanged physical and biological state. Before examination the sample was melted, warmed up to room temperature and centrifuged in MPW 211 at 500 × g for 15 min at room temperature. Next, the sponge and the straw were removed and the saliva was transferred to Eppendorf test tubes. The coritsol level measurement was conducted with the enzyme-immunoassay using the CORTISOL EIA kit (for saliva) DSL_10-67100 made by Diagnostic System Laboratories Inc., USA. The absorbance level measurement was conducted at 450 and 620 nm wavelength using a Multiscan reader (Labsystems, Helsinki) equipped with a GENESIS V 3.00 programme. The results were expressed in nmol/dm3. Statistical analysis The cortisol concentration determinations were duplicated. The results were processed using a STATISTICA 6.0 package based on the one-way analysis of variance. The differences between the group averages and the circadian rhythm data within each group were estimated from Fischer’s LSD. Results and Discussion The control group of horses was not under the influence of any stress factors that could significantly change the cortisol concentration in their saliva. This was confirmed by all the data taken at different times of the day (20:00 h, 7:00 h, 17:00 h, 22:00 h), namely the mean cortisol concentration usually did not exceed 0.45 nmol/dm3 (Table 1) and for the majority 102