Salivary cortisol levels in horses and their junior riders during show jumping

Katarzyna STRZELEC, Sławomir PIETRZAK, Tomasz PRÓCHNIAK*, Iwona JANCZAREK, Anna CYWIŃSKA, Andrzej BEREZNOWSKI Department of Horse Breeding and Use, Faculty of Animal Sciences and Bioeconomy, University of Life Sciences in Lublin, Lublin, Poland Institute of Biological Basis of Animal Production, Faculty of Animal Sciences and Bioeconomy, University of Life Sciences in Lublin, Lublin, Poland Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Science, Warsaw, Poland Laboratory of Veterinary Epidemiology and Economics, Warsaw University of Life Science, Warsaw, Poland


Introduction
One of the main substances that indicate stress level is cortisol [1]. The concentration of cortisol depends on the natural circadian rhythm, which can be disturbed by physical as well as mental stress. The most effective way of determining cortisol concentration in animals seems to use their saliva. This is because taking saliva samples is a noninvasive and a low-stress procedure [2]. Therefore, the saliva testing is selected as the method of choice for sport horses and riders too. In humans, mental stress caused by participation in sport competitions was found to increase salivary cortisol level substantially [3]. Additionally, in horses, the salivary cortisol levels were found to reflect the stress level not only during various equestrian competition events [4,5] but also during training and road transport [6,7]. However it is known that not only stress, but also physical activity is a factor, which increases the hypothalamus-pituitary-adrenal axis and cortisol release [8].
Jumping round is not too long effort but it is really intensive for a horse and a rider because of turns, number of fences, character and size of obstacles, and speed etc. [9].
The aim of the study was to compare the salivary cortisol levels in jumping horses and their junior riders during jumping competition.

Material and methods
Horses and riders: the study took place during international show jumping (Cavaliada Tour 2016 Lublin) for juniors (height of fences up to 130 cm) in Poland. Twenty two horses (10 Polish noble half-breed, 4 Hanoverian breed, 4 Wielkopolski breed, 2 Dutch warm-blood, 2 Westphalian breed) were taken for the study. The horses (6 stallions, 6 mares, 10 geldings) were aged from 7 to 17 years, while the riders (12 female and 10 male) were aged from 15 to 18 years. None of the horses had any clinical symptoms of health disorders. Each rider had trained and competed with his or her horse for at least 10 months prior to the study.
Saliva sampling from each horse and rider, 2 saliva samples were collected: 1/before warm up and start (in a stable), 2/immediately after the end of a jumping round. The saliva samples were collected with a small piece of sponge, which was inserted into mouth of the horse with the use of tweezers. The riders were instructed to place and hold the sponge in their mouths until it became saturated. After soaking in saliva, the sponge was placed in a plastic tube, as described previously [10], and frozen. All procedures were performed in respect of the legislation on Animal Care (EU Directive 2010/63/EU) and the Internal Rules of University of Life Science in Lublin.

Analyses
Before the examination, the saliva samples were melted, warmed up to room temperature, and centrifuged at 500 × g for 15 min at room temperature. Afterwards, the sponge was removed and the saliva was transferred into test tubes. The concentration of cortisol in saliva samples was measured by the enzyme-immunoassay method [11] using the Cortisol ELISA kit SLV-4635 (DRG Instruments Company, Marburg,Germany). All assays were based on polyclonal rabbit antibody directed towards an antigenic site on the cortisol molecule. The analytical sensitivity of the assay was 0.012 ng/mL. The absorbance was measured using a Multiscan reader (Labsystem, Vantaa, Finland) equipped with a GENESIS V 3.00 program. The results were expressed as nmol/L. The minimal detectable concentration was 0.3 nmol/L.

Statistics
The results including standard deviation (SD) were analysed using the common SAS software (Version 9.4, 2013) and the ANOVA-GLM method for analyzing repeated measures and Tukey test. The following factors and effects were considered: 1/ rider-age group and sex 2/ horse-age group, sex, and breed. The Pearson correlation coefficient was also assessed to compare the analysed data. The statistical significance was accepted at 2 levels of P ≤ 0.05 and P ≤ 0.01.

Results
The results of the salivary cortisol concentration in the studied riders are shown in Table 1. The results obtained for the samples before the start of women (14.20 nmol/L) were significantly higher than those obtained by men (9.98 nmol/L). The obtained data did not differ statistically for men and women immediately after finishing a jumping round. However, the differences between the level of salivary cortisol concentration (before starting and after finishing the round) in both cases (men and women) were statistically significant. In women's case, this level increased more than twice that of men's case (respectively woman 14.20-31.33; man 9.98-24.31 nmol/L).
Interesting data are shown in Table 2, which presents an average salivary cortisol concentration in 2age groups of riders (15-16 and 17-18). In the group of the younger juniors (15)(16), the average salivary cortisol concentration measured before the start was at the level of 9.87 but in the older one it was higher (16.50 nmol/L). The differences were not statistically significant. In the group of younger juniors, after the end of jumping rounds, cortisol concentration was nearly 3 times higher in comparison to the level before the start. The differences were statistically significant. Table 3 includes average cortisol concentration of studied horses within their sex. The level of mares' cortisol concentration was higher than stallions' and geldings' not only after exercise but also before it. The differences were statistically significant between mares and other gender groups only after effort. All studied horses were divided into 2groups by their age (up to 10 years old and older than 10) to see the differences of saliva cortisol concentration (Table 4). There were no significant statistical differences between the age groups before the effort and after it as well, but they were present in both groups between the level of values before and after a jumping round. Data included in Table 5 show that there are no significant statistical differences between the studied horse breeds due to cortisol concentration before the start of a jumping competition and after it. A statistically important correlation was found between the level of cortisol concentration in riders before and after Averages with different letters (a, b in columns, x, y in rows) differ significantly at P ≤ 0.05. the start (0,41) Table 6. For the horses it was not found a similar dependence. Negative statistically significant correlation coefficient (-0.70) between the level of cortisol concentration, measured before the start of a jumping competition and after it, was found in the group of male juniors (Table 7). In the case of age, statistically significant difference was found for younger juniors (0.64). Another statistically significant correlation coefficient between cortisol concentration in horses before the start and after it, was found in the group of stallions (0.95) Table 8. The same situation was observed in the group of older horses (0.72) as well as in the group of polish half-breed horses (0.72). High value of correlation coefficient (0.80) was noticed also for Hanoverian horses, but due to the low number, it was not considered as statistically significant.

Discussion
Results obtained in this study proved a significant influence of the analysed factors (sex and age of riders and also sex, age, and breed of horses) on saliva cortisol concentration before and after exercise. Significantly higher cortisol concentration in the group of women, before the start of a jumping competition, can prove their higher emotional sensitivity, when compared to men in show jumping. This observation is not consistent with the results of other studies, in which males showed greater cortisol response to stress than females [12]. Similar results to Franco et al. [12] were shown by Strzelec et al. [13], where females Averages with different letters (a, b in columns, x, y in rows) differ significantly at P ≤ 0.05. Averages with different letters (a, b in columns, x, y in rows) differ significantly at P ≤ 0.05. Averages with different letters (a, b in columns, x, y in rows) differ significantly at P ≤ 0.05. participating in eventing had significantly lower level of cortisol concentration than males. Significant differences between cortisol concentration before and after the effort found in the group of men are interesting. About 3 times increase of level of cortisol concentration after finishing a jumping round was probably an effect of mental stress and also physical effort. This observation is consistent with the results of Garwin et al. [14], Franco et al. [12], and Strzelec et al. [13], in which males showed greater activity of the hypothalamuspituitary-adrenal axis and higher cortisol release than females.
Significant differences were not found between cortisol concentrations before exercise in both age groups of riders. Higher level of older juniors can be influenced by the atmosphere of rivalry before competition as well as after jumping round because of a bigger emotional engagement of more experienced athletes [13].
The effect of horse's sex was the highest in the group of mares. Average cortisol concentration in this group was higher than those of stallions and geldings. The obtained results are similar to the other authors' [1,6,7,9]. According to Strzelec [15], this situation can be influenced by physical effort of jumping and the atmosphere of the show, but also of mares' sex hormones activity. Different results were found by Fazio et al. [16], where the level of cortisol concentration of stallions was higher than that of mares.
Age of horses was not a factor which determined saliva cortisol concentration. Slightly lower level of cortisol after exercise suggests that older horses have more experience and release of the hormone is influenced mostly by physical effort. According to Nogueira et al. [17], the proper training of sport horses can decrease stress during the competition. After coming to the show, horses feel the atmosphere of rivalry so the level of cortisol concentration measured before the start shows their mental stress [1,18].
In the studied horses, participation in the jumping competition caused cortisol release; however, between the horses' breeds, the changes in the mean salivary cortisol concentration did not reach the level of statistical significance. Probably, the relatively small number of horses, and the high individual variability of salivary cortisol levels from horse to horse, which is expected, decreased the possibility of observing statistically significant differences. This observation is not consistent with the results of Strzelec [15] in which German warmblood breeds of horses (very well known in world equestrianism) were characterized by higher level of cortisol concentration in comparison to the other breeds. High variability of salivary cortisol level from horse to horse was also observed by other researchers [4,10].
There was a significant correlation between the level of cortisol concentration before the start and after it in the group of riders but not in the horses. It may suggest that young riders are more stressed before the start than horses. It could be explained by their age and lack of experience. Similar results were shown by Peeters et al. [4] and Strzelec et al. [13]. Significant negative correlation was found in the sex group of male but positive significant correlation in the group of younger juniors. Therefore, the age of the riders cannot be influenced by saliva cortisol concentration. According to the other researches this situation depends more on the level of physical effort and individual immunity to stress of riders and horses [3,19].
High significant correlations were found due to the influence of sex, age, and breed of horses on salivary cortisol concentration, but only for stallions and mares. It means that high level of cortisol concentration before exercise in the case of stallions and mares is also high after exercise. Similar results were shown by Schmidt et al. [6,7] and Strzelec [15] where high cortisol concentration before effort, especially in the group of mares, was present also after exercise. These researches explain this fact by the excessive excitability of stallions and specific psychic traits of mares which can influence their behaviors during performance tests.
In conclusion, individual horses are found to vary greatly in their intensity of cortisol release in response to participation in jumping competition. In the context of such individual variability, the horse's response to a jumping round is found to be repetitive. Therefore, the salivary cortisol test seems to be a useful and efficient method to evaluate the horse's response to competitions which include the influence of the rider.