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Animal Science Journal (2014) 85, 313–317
doi: 10.1111/asj.12146
ORIGINAL ARTICLE Changes in salivary and plasma cortisol levels in Purebred Arabian horses during race training session Witold KE˛DZIERSKI,1 Anna CYWIN´SKA,3 Katarzyna STRZELEC2 and Sylwester KOWALIK1 Departments of 1Animal Biochemistry and Physiology, 2Horse Breeding and Use, University of Life Sciences in Lublin, Lublin, and 3Department of Pathology and Veterinary Diagnostic, Warsaw University of Life Sciences – SGGW, Warszawa, Poland
ABSTRACT Physical activity and stress both cause an increase in cortisol release ratio. The aim of this study was to evaluate the use of saliva samples for the determination of cortisol concentrations indicating the work-load level in horses during race training. Twelve Purebred Arabian horses aged 3–5 years were studied during the routine training session. After the warm-up, the horses galloped on the 800 m sand track at a speed of 12.8 m/s. Three saliva samples, and three blood samples were collected from each horse. Both types of samples were taken at rest, immediately after return from the track and after 30 min restitution. The concentrations of blood lactic acid (LA), and cortisol in saliva and plasma samples were measured and analyzed. Blood LA, plasma and salivary cortisol levels increased significantly after exercise (P < 0.05). Salivary cortisol concentration determined 30 min after the exercise correlated significantly with plasma cortisol level obtained immediately after exercise (P < 0.05) as well as measured 30 min after the end of exercise (P < 0.05). The determination of cortisol concentration in saliva samples taken from racehorses 30 min after the end of exercise can be recommended to use in field conditions to estimate the work-load in racehorses.
Key words: cortisol, exercise, racehorses, saliva, stress.
INTRODUCTION Cortisol is a natural glucocorticoid hormone produced by the adrenal cortex. Its secretion is stimulated by the sympathetic nervous system activity. The main function of this hormone is energy mobilization to protect the homeostasis of vital biochemical processes during effort and stressful situations. In horses, the amount of cortisol in blood increased in response to various types of exercise (Lassourd et al. 1996) and is believed to depend on the duration rather than the intensity of exercise (Valberg et al. 1989; Desmecht et al. 1996; Nagata et al. 1999). The measurement of cortisol release rate can also be used for the estimation of horse performance. Mircean et al. (2007) stated that during exercise tests, serum cortisol levels were higher in horses with a low performance level than in welltrained ones. The study by Marc et al. (2000) showed that plasma cortisol response to standardized exercise tests can be a useful marker in the evaluation of performance during the training process. Moreover, Malinowski et al. (2006) found that the cortisol response to acute exercise was altered by the age of horses and was significantly higher in younger than in © 2013 Japanese Society of Animal Science
older ones. Blood cortisol concentration reflects not only the influence of physical effort but also the physiological reaction to road transport (Fazio et al. 2008a, 2013) and other stressful stimuli (Colborn et al. 1991). Generally, at the beginning of the training process, young horses are exposed to various types of stress stimuli (Alexander & Irvine 1998; Cayado et al. 2006; Janczarek & Ke˛dzierski 2011). It was stated that the cortisol level following stressful situations was higher in inexperienced horses than in animals that were used to such conditions (Cayado et al. 2006; Fazio et al. 2008b). Therefore, the estimation of blood plasma cortisol levels in horses can be useful not only in the evaluation of horse performance but also can help to improve the training process. The determination of
Correspondence: Witold Ke˛dzierski, Department of Animal Biochemistry and Physiology, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Akademicka 12, 20-033 Lublin, Poland. (Email: witold.kedzierski@up .lublin.pl) Received 19 April 2013; accepted for publication 15 August 2013.
314 W. KE˛DZIERSKI et al.
cortisol concentration in plasma requires taking blood samples that can cause additional stress and may not be well accepted by the owners. Therefore, in recent years, more attention has been devoted to the use of saliva for diagnostic tests. Saliva sampling is certainly less stressful for horses than taking blood samples (Peeters et al. 2011). The high correlation coefficient, which has been found between blood plasma and salivary cortisol concentrations in horses, permitted the validation of salivasampling as a technique for the measurement of cortisol release (van der Kolk et al. 2001; Schmidt et al. 2009; Peeters et al. 2011). The level of plasma cortisol partially correlates with that in saliva, also across a 24-h period (Bohák et al. 2013). Salivary cortisol level was successfully used as the indicator of stress levels in horses in response to stabling (Harewood & McGowan 2005) and during road transport (Schmidt et al. 2010a,b,c). Also the initial training of horses, especially mounting by a rider, resulted in significant increase in salivary cortisol levels (Schmidt et al. 2010d). The determination of cortisol concentration in saliva was useful also for assessing stress levels in horses during various equestrian competitions (Peeters et al. 2010; Strzelec et al. 2011). Finally, it was found that salivary cortisol concentrations could be useful markers of relative intensity of exercise in thoroughbred racehorses (Ke˛dzierski et al. 2012). According to our best knowledge, Purebred Arabian racehorses have not been examined in this context. During race training, Purebred Arabian horses should be submitted to less intensive exertion than thoroughbreds (Ke˛dzierski & Bergero 2006). It is not known if the effort which is routinely applied to Arabian racehorses will be sufficient to involve significant increase in salivary cortisol levels, especially because cortisol release is delayed in time in response to a stimulating factor (Thompson et al. 1988; Peeters et al. 2011). The purpose of this study was to evaluate the use of saliva sampling for the measurement of cortisol concentration and to detect work-load levels in young Purebred Arabian horses during race training session.
MATERIAL AND METHODS Horses This study was performed in Poland at the Sluzewiec (Warsaw) racetrack. Twelve Purebred Arabian horses aged 3–5 years and comprising an equal number of stallions and mares were examined. The study took place in the middle of the racing season in Poland. The horses included in the study competed in official races at least 2 weeks before. All the horses were trained by one trainer and maintained in one stable. They were fed with typical fodder used as a standard diet for racehorses. Medium body weight, withers height and girth circumference of stallions amounted to 464 ± 55.0 kg, 156 ± 4.15 cm and 178 ± 5.65 cm, respectively. Measurement of mares amounted to, respectively, 413 ± 12.0 kg, © 2013 Japanese Society of Animal Science
148 ± 1.02 cm and 173 ± 1.72 cm. Prior to the study, the horses were trained 5 days a week. The study was accepted by the Local Ethics Review Committee for Animal Experimentation and conducted according to the European Community regulations concerning the protection rules of experimental animals.
Exercise protocol The horses were examined during their training session which was a part of the routine training. They were in the last phase of preparation to a start in races. At the day of the study, the training session was performed in the morning, from 07.00 to 09.00 hours. Mares were exercised first, then the stallions. All horses had a 10 min warm-up trot and canter with a rider. Then they galloped on the 800 m sand track at a speed amounting to 12.8 m/s. Finally, they returned trotting to the stable. After unsaddling, the horses were cooled down in an automatic horse walker for about 20 min. Three saliva samples and three blood samples were collected from each horse. All horses were sampled by the same operators: one person sampled saliva and the other one collected blood samples. Both types of samples were taken according to the following protocol: (i) at rest; (ii) immediately after return from the track; and (iii) after 30 min restitution. In each case, saliva was sampled first and then blood was taken. The saliva samples were collected with a small piece of sponge which was inserted into the horse’s mouth and then, after soaking in saliva, it was placed in a plastic tube, as previously described (Strzelec et al. 2011). Blood samples were collected by jugular venipuncture to ethylenediaminetetraacetic acid (EDTA) K3 tubes. Blood lactic acid (LA) concentrations were determined immediately using Dr Lange’s enzymatic cuvette test (Berlin, Germany) and expressed in mmol/L. The remaining blood was immediately centrifuged at 2000 × g for 10 min and plasma was stored at −20°C until assayed.
Laboratory analysis Before laboratory analysis, the saliva samples were centrifuged at 500 × g for 15 min at room temperature. Next, the sponge with the straw was removed and the saliva was transferred to test tubes. The concentrations of cortisol in saliva samples were measured by enzyme-immunoassay method using the CORTISOL EIA kit DSL (Diagnostic System Laboratories Inc., Webster, TX, USA). For plasma cortisol determination, the CORTISOL ELISA kit (DRG International Inc., Mountainside, NJ, USA) was used. All samples were analyzed in duplicate. The absorbance was measured by Multiscan reader (Labsystem, Helsinki, Finland) using a GENESIS V 3.00 software program (VWR International Ltd., Lutterworth, UK). The intra- and interassay coefficients of variation for salivary cortisol determined in the laboratory amounted to 9% and 11%, and for plasma cortisol concentration 5% and 8%, respectively. The results were expressed in nmol/L.
Statistical analysis The results are presented as means ± standard deviation (SD). Statistical analyses were performed using the statistical software package GraphPad PrismTM (Graph Pad Software, La Jolla, CA, USA). Comparisons between the results obtained before the training session, immediately after and 30 min after the end of exercise in the blood and saliva samples were Animal Science Journal (2014) 85, 313–317
SALIVARY CORTISOL LEVEL IN TRAINED HORSES
315
Table 1 Blood lactic acid and plasma and salivary cortisol levels determined in Purebred Arabian horses (n = 12) at a training session (means± SD)
Sample:
At rest
Immediately after the exercise
30 min after the exercise
Blood lactic acid (mmol/L) Plasma cortisol (nmol/L) Salivary cortisol (nmol/L)
0.92 ± 0.15a 79.3 ± 18.1a 1.06 ± 0.64a
4.64 ± 3.14b 118 ± 19.1b 5.45 ± 4.80b
1.23 ± 0.31a 103 ± 18.6b 5.06 ± 4.38b
a,b
Means in rows with different superscripts differ significantly at P < 0.05.
Figure 1 Correlation of salivary cortisol concentration determined 30 min after the end of exercise (ordinate; nmol/L) and plasma cortisol level determined (a) immediately after exercise and (b) 30 min after the end of exercise (abscissa; nmol/L).
made by the Tukey test (analysis of varaince). The coefficient correlation to compare the above data was assessed by Pearson’s test. Statistical significance was accepted at P < 0.05.
RESULTS The results of LA and cortisol measurements are presented in Table 1. Blood LA and plasma cortisol levels increased significantly after exercise (P < 0.05). The mean value of plasma cortisol concentration determined after the 30 min of restitution was lower than measured immediately after the exercise but did not reach the levels at rest (P < 0.05). Salivary cortisol concentrations determined immediately after exercise and after 30 min restitution were significantly higher (P < 0.05) than values obtained at rest. A significant correlation coefficient was found between plasma cortisol concentration determined immediately after exercise and salivary cortisol levels obtained 30 min after the exercise (r = 0.55, P < 0.05) and also between plasma cortisol concentrations determined 30 min after the exercise and salivary levels of cortisol measured after the 30 min of restitution (r = 0.67, P < 0.05; Fig. 1).
DISCUSSION Blood LA level was determined in the study to indicate the relative intensity of performed exercise (Guhl et al. 1996; Ke˛dzierski & Bergero 2006). The mean blood LA level determined immediately after exercise reached 4.64 mmol/L. This value is typical for exercises applied to the horses in routine race training (Podolak et al. 2006; Ke˛dzierski et al. 2009). It is believed that LA production is far more related to the intensity of exercise than cortisol secretion. The Animal Science Journal (2014) 85, 313–317
results of other studies indicate that plasma cortisol concentrations reflected rather the duration of exercise (Desmecht et al. 1996) or stress level (Berghold et al. 2007; Mircean et al. 2007). Spectacularly high values of plasma cortisol were noted after endurance exercises (Snow & Rose 1981; Lassourd et al. 1996). Two-time increase in plasma cortisol level was induced by participation in cross-country (Snow 1990; Linden et al. 1991) or draught work (Ke˛dzierski & Pluta 2013). The horses investigated in this study performed a short exercise of moderate intensity. They galloped only for few minutes covering the distance of 800 m; however, in response to this exercise, the plasma cortisol concentration increased significantly in comparison to the level at rest. This indicates that such kind of work-load was sufficient to increase the hypothalamic-pituitaryadrenocortical axis. An increase in plasma cortisol level was stated also in other studies in response to gallops (Desmecht et al. 1996; Mircean et al. 2007; Ke˛dzierski et al. 2012). The measurement of plasma cortisol concentration was also used for the evaluation of horse performance. The cortisol release rate in response to a standardized exercise test was higher in untrained horses than in well-trained horses (Marc et al. 2000). The facts mentioned above indicated that short and moderate intensive exercises in horses are able to induce cortisol release, the rate of which is proportional to an individual, relative work load. In the present study, salivary cortisol levels also significantly increased under the influence of exercise. The means of salivary cortisol concentrations were characterized by high SD values. High variability of salivary cortisol levels was also stated by other researchers (Peeters et al. 2010; 2011; Strzelec et al. 2011). Also in our previous work, each studied horse © 2013 Japanese Society of Animal Science
316 W. KE˛DZIERSKI et al.
was characterized by individual intensity of cortisol release in response to each competition during 3-day events (Strzelec et al. 2013). This phenomenon can be explained partially by the genetic conditioning of the horse and partially by their interaction with their rider. The statistically significant correlations were stated between salivary cortisol levels determined 30 min after the end of exercise and plasma cortisol concentrations. This indicated that salivary cortisol levels measured 30 min after exercise clearly reflected the exercise-induced cortisol release in studied horses. The positive correlation between plasma and salivary cortisol concentrations have been described previously in horses submitted to different stressors (Schmidt et al. 2009). The increase in both salivary and plasma cortisol was also stated during the crosscountry round (Peeters et al. 2010). On the other hand, Elsaesser et al. (2001) have shown that the correlation between salivary and plasma cortisol levels in trained warm-blooded sports horses was low and insufficient to use salivary cortisol measurements for diagnostic purposes. However, in that study, cortisol levels were measured at distinct points of time after adrenocorticotropic hormone (ACTH) injection and not after exercise. In fact, cortisol release from the adrenal cortex is stimulated by ACTH, which is released from the pituitary gland under the influence of corticotrophin-releasing hormone (CRH) produced by the hypothalamus. Moreover, in exercise conditions, the role of CRH is replaced by vasopressin (Alexander et al. 1991). Also, the increase in plasma cortisol level is the result of complicated and timedependent processes. For example, short-term stress induced in pony mares caused an increase in plasma cortisol concentration about 20 min later (Thompson et al. 1988). Similar results were achieved after CRH injection (Reijerkerk et al. 2009). ACTH administration also increased plasma and salivary cortisol concentrations over time (van der Kolk et al. 2001). The detailed studies performed by Peeters et al. (2011) indicated that salivary cortisol peak level appeared 30 min later than found in plasma following an ACTH stimulation test. A similar situation can be observed in the present study, in which only the salivary cortisol level determined 30 min after the end of exercise reflected the cortisol concentration in plasma. In conclusion, the high correlation between plasma and salivary cortisol levels found in this study indicated that measuring the cortisol level in saliva, especially after restitution, 30 min after the end of an effort, can pose as a useful marker of exercise-induced work load in racehorses. Therefore, the sampling of saliva 30 min after the end of exercise can be recommended to use in practice for the estimation of cortisol release rate in Purebred Arabian horses during race training sessions in field conditions. © 2013 Japanese Society of Animal Science
REFERENCES Alexander S, Irvine CHG. 1998. Stress in the race horse: coping vs not coping. Journal of Equine Science 9, 77–81. Alexander SL, Irvine CHG, Ellis MJ, Donald RA. 1991. The effect of acute exercise on the secretion of corticotropinreleasing factor, arginine vasopressin, and adrenocorticotropin as measured in pituitary venous blood from the horse. Endocrinology 128, 65–72. Berghold P, Möstl E, Aurich C. 2007. Effects of reproductive status and management on cortisol secretion and fertility of oestrous horse mares. Animal Reproduction Science 102, 276–285. Bohák Z, Szabó F, Beckers JF, Melo de Sousa N, Kutasi O, Nagy K, Szenci O. 2013. Monitoring the circadian rhythm of serum and salivary cortisol concentrations in the horse. Domestic Animal Endocrinology 45, 38–42. Cayado P, Munˇoz-Escassi B, Dominguez C, Manley W, Olabarri B, Sánchez de la Muela M, Castejon F, Maranˇon G, Vara E. 2006. Hormone response to training and competition in athletic horses. Equine Veterinary Journal Supplement 36, 274–278. Colborn DR, Thompson DL Jr, Roth TL, Capehart JS, White KL. 1991. Responses of cortisol and prolactin to sexual excitement and stress in stallions and geldings. Journal of Animal Science 69, 2556–2562. Desmecht D, Linden A, Amory H, Art T, Lekeux P. 1996. Relationship of plasma lactate production to cortisol release following completion of different types of sporting events in horses. Veterinary Research Communications 20, 371–379. Elsaesser F, Klobasa F, Ellendorff F. 2001. ACTH stimulation test for the determination of salivary cortisol and of cortisol responses as markers of the training states/fitness of warm-blooded sports horses. Deutsche Tierärztliche Wochenschrift 108, 31–36. Fazio B, Medica P, Aronica V, Grasso L, Ferlazzo A. 2008a. Circulating β-endorphin, adrenocorticotrophic hormone and cortisol levels of stallions before and after short road transport: stress effect of different distances. Acta Veterinaria Scandinavica 3, 50–56. Fazio B, Medica P, Cravana C, Aveni F, Ferlazzo A. 2013. Comparative endocrinal responses to short transportation of Equidae (Equus asinus and Equus caballus). Animal Science Journal 84, 258–263. Fazio B, Medica P, Cravana C, Ferlazzo A. 2008b. Effects of competition experience and transportation on the adrenocortical and thyroid responses of horses. Veterinary Record 163, 713–716. Guhl A, Linder A, von Wittke P. 1996. Use of relationship between blood lactate and running speed to determine the exercise intensity of horses. Veterinary Record 139, 108–110. Harewood EJ, McGowan CM, 2005. Behavioral and physiological responses to stabling in naïve horses. Journal of Equine Veterinary Science 25, 164–170. Janczarek I, Ke˛dzierski W. 2011. Emotional response of young race horses to a transfer from a familiar to an unfamiliar environment. Animal Science Papers and Reports 29, 205–212. Ke˛dzierski W, Bergero D. 2006. Comparison of plasma biochemical parameters in Thoroughbred and Purebred Arabian horses during the same-intensity exercise. Polish Journal of Veterinary Sciences 9, 233–238. Ke˛dzierski W, Bergero D, Assenza A. 2009. Trends of hematological and biochemical values in blood of young Animal Science Journal (2014) 85, 313–317
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race horses during standardized field exercise test. Acta Veterinaria (Beograd) 59, 457–466. Ke˛dzierski W, Pluta M. 2013. The welfare of young Polish Konik horses subjected to agricultural workload. Journal of Applied Animal Welfare Science 16, 35–46. Ke˛dzierski W, Strzelec K, Cywin´ska A, Kowalik S. 2012. Plasma and salivary cortisol levels as the indicators of stress and fatigue during field exercise test in Thoroughbred horses. Wien Tierärztliche Monatsschrift Supplement 1, 63. Lassourd V, Gayrard V, Laroute V, Alvinerie M, Benard P, Courtot D, Toutain PL. 1996. Cortisol disposition and production rate in horses during rest and exercise. American Journal of Physiology 271, R25–R33. Linden A, Art T, Amory H, Massart AM, Burvenisch C, Lekeux P. 1991. Quantitative buffy coat analysis related to adrenocortical function in horses during a three-day event competition. Journal of Veterinary Medicine. A, Physiology, Pathology, Clinical Medicine 38, 376–382. Malinowski K, Shock EJ, Rochelle P, Kearns CF, Guirnalda PD, McKeever KH. 2006. Plasma beta-endorphin, cortisol and immune responses to acute exercise are altered by age and exercise training in horses. Equine Veterinary Journal Supplement 36, 267–273. Marc M, Parvizi N, Ellendorff F, Kallweit E, Elsaesser F. 2000. Plasma cortisol and ACTH concentrations in the warmblood horse in response to a standardized treadmill exercise test as physiological markers for evaluation of training status. Journal of Animal Science 78, 1936–1946. Mircean M, Giurgiu G, Mircean V, Zinveliu E. 2007. Serum cortisol variation of sport horses in relation with the level of training and effort intensity. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca 64, 488–492. Nagata S, Takeda F, Kurosawa M, Mima K, Hiraga A, Kai M, Taya K. 1999. Plasma adrenocorticotropin, cortisol and catecholamines response to various exercises. Equine Veterinary Journal Supplement 30, 570–574. Peeters M, Sulon J, Beckers JF, Ledoux D, Vandeheede M. 2011. Comparison between blood serum and salivary cortisol concentrations in horses using an adrenocorticotropic hormone challenge. Equine Veterinary Journal 43, 487–493. Peeters M, Sulon J, Serteyn D, Vandeheede M. 2010. Assessment of stress level in horses during competition using salivary cortisol: preliminary studies. Journal of Veterinary Behavior: Clinical Application and Research 5, 216. Podolak M, Ke˛dzierski W, Bergero D. 2006. Comparison of the blood plasma catecholamines level in Thoroughbred and Arabian horses during the same-intensity exercise. Polish Journal of Veterinary Sciences 9, 71–73. Reijerkerk EP, Visser EK, van Reenen CG, van der Kolk JH. 2009. Effects of various doses of ovine corticotrophin-
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releasing hormone on plasma and salivary cortisol concentrations in horses. American Journal of Veterinary Research 70, 361–364. Schmidt A, Aurich C, Neuhauser S, Aurich J, Möstl E. 2009. Comparison of cortisol levels in blood plasma, saliva and faeces of horses submitted to different stressors or treated with ACTH. Proceedings, 5th Internationla Symposium Equitation Science, Sydney, July 2009, 53. Schmidt A, Aurich J, Möstl E, Muller J, Aurich C. 2010d. Changes in cortisol release and heart rate and heart rate variability during the initial training of 3-year-old sport horses. Hormones and Behavior 58, 628–636. Schmidt A, Biau S, Möstl E, Becker-Birck M, Morillon B, Aurich J, Faure JM, Aurich C. 2010a. Changes in cortisol release and heart rate variability in sport horses during long distance-road transport. Domestic Animal Endocrinology 38, 179–189. Schmidt A, Hodl S, Möstl E, Aurich J, Muller J, Aurich C. 2010b. Cortisol release, heart rate, and heart rate variability in transport-naïve horses during repeated road transport. Domestic Animal Endocrinology 39, 205–213. Schmidt A, Möstl E, Wehnert C, Aurich J, Muller J, Aurich C. 2010c. Cortisol release and heart rate variability in horses during road transport. Hormones and Behavior 57, 209– 215. Snow DH, Rose RJ. 1981. Hormonal changes associated with long distance exercise. Equine Veterinary Journal 13, 185– 197. Snow HD. 1990. Haematological, biochemical and physiological changes in horses and ponies during the cross country stage of driving trial competitions. Veterinary Record 126, 233–239. Strzelec K, Kankofer M, Pietrzak S. 2011. Cortisol concentration in the saliva of horses subjected to different kinds of exercise. Acta Veterinaria Brno 80, 101–105. Strzelec K, Ke˛dzierski W, Bereznowski A, Janczarek I, Bocian K, Radosz A. 2013. Salivary cortisol levels in horses and their riders during three-day-events. Bulletin of the Veterinary Institute in Pulawy 57, 237–241. Thompson DL Jr, Garza F Jr, Mitchell PS, St George RL. 1988. Effects of short-term stress, xylazine tranquilization with xylazine plus ketamine on plasma concentrations of cortisol, luteinizing hormone, follicle stimulating hormone and prolactin in ovatiectomized pony mares. Theriogenology 30, 937–946. Valberg S, Gustavsson BE, Lindholm A, Persson SGB. 1989. Blood chemistry and skeletal muscle metabolic responses during and after different speeds and durations of trotting. Equine Veterinary Journal 21, 91–95. van der Kolk JH, Nachreiner RF, Schott HC, Refsal KR, Zanella AJ. 2001. Salivary and plasma concentration of cortisol in normal horses and horses with Cushing’s disease. Equine Veterinary Journal 33, 211–213.
© 2013 Japanese Society of Animal Science
Animal Science Journal (2014) 85, 313–317
doi: 10.1111/asj.12146
ORIGINAL ARTICLE Changes in salivary and plasma cortisol levels in Purebred Arabian horses during race training session Witold KE˛DZIERSKI,1 Anna CYWIN´SKA,3 Katarzyna STRZELEC2 and Sylwester KOWALIK1 Departments of 1Animal Biochemistry and Physiology, 2Horse Breeding and Use, University of Life Sciences in Lublin, Lublin, and 3Department of Pathology and Veterinary Diagnostic, Warsaw University of Life Sciences – SGGW, Warszawa, Poland
ABSTRACT Physical activity and stress both cause an increase in cortisol release ratio. The aim of this study was to evaluate the use of saliva samples for the determination of cortisol concentrations indicating the work-load level in horses during race training. Twelve Purebred Arabian horses aged 3–5 years were studied during the routine training session. After the warm-up, the horses galloped on the 800 m sand track at a speed of 12.8 m/s. Three saliva samples, and three blood samples were collected from each horse. Both types of samples were taken at rest, immediately after return from the track and after 30 min restitution. The concentrations of blood lactic acid (LA), and cortisol in saliva and plasma samples were measured and analyzed. Blood LA, plasma and salivary cortisol levels increased significantly after exercise (P < 0.05). Salivary cortisol concentration determined 30 min after the exercise correlated significantly with plasma cortisol level obtained immediately after exercise (P < 0.05) as well as measured 30 min after the end of exercise (P < 0.05). The determination of cortisol concentration in saliva samples taken from racehorses 30 min after the end of exercise can be recommended to use in field conditions to estimate the work-load in racehorses.
Key words: cortisol, exercise, racehorses, saliva, stress.
INTRODUCTION Cortisol is a natural glucocorticoid hormone produced by the adrenal cortex. Its secretion is stimulated by the sympathetic nervous system activity. The main function of this hormone is energy mobilization to protect the homeostasis of vital biochemical processes during effort and stressful situations. In horses, the amount of cortisol in blood increased in response to various types of exercise (Lassourd et al. 1996) and is believed to depend on the duration rather than the intensity of exercise (Valberg et al. 1989; Desmecht et al. 1996; Nagata et al. 1999). The measurement of cortisol release rate can also be used for the estimation of horse performance. Mircean et al. (2007) stated that during exercise tests, serum cortisol levels were higher in horses with a low performance level than in welltrained ones. The study by Marc et al. (2000) showed that plasma cortisol response to standardized exercise tests can be a useful marker in the evaluation of performance during the training process. Moreover, Malinowski et al. (2006) found that the cortisol response to acute exercise was altered by the age of horses and was significantly higher in younger than in © 2013 Japanese Society of Animal Science
older ones. Blood cortisol concentration reflects not only the influence of physical effort but also the physiological reaction to road transport (Fazio et al. 2008a, 2013) and other stressful stimuli (Colborn et al. 1991). Generally, at the beginning of the training process, young horses are exposed to various types of stress stimuli (Alexander & Irvine 1998; Cayado et al. 2006; Janczarek & Ke˛dzierski 2011). It was stated that the cortisol level following stressful situations was higher in inexperienced horses than in animals that were used to such conditions (Cayado et al. 2006; Fazio et al. 2008b). Therefore, the estimation of blood plasma cortisol levels in horses can be useful not only in the evaluation of horse performance but also can help to improve the training process. The determination of
Correspondence: Witold Ke˛dzierski, Department of Animal Biochemistry and Physiology, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Akademicka 12, 20-033 Lublin, Poland. (Email: witold.kedzierski@up .lublin.pl) Received 19 April 2013; accepted for publication 15 August 2013.
314 W. KE˛DZIERSKI et al.
cortisol concentration in plasma requires taking blood samples that can cause additional stress and may not be well accepted by the owners. Therefore, in recent years, more attention has been devoted to the use of saliva for diagnostic tests. Saliva sampling is certainly less stressful for horses than taking blood samples (Peeters et al. 2011). The high correlation coefficient, which has been found between blood plasma and salivary cortisol concentrations in horses, permitted the validation of salivasampling as a technique for the measurement of cortisol release (van der Kolk et al. 2001; Schmidt et al. 2009; Peeters et al. 2011). The level of plasma cortisol partially correlates with that in saliva, also across a 24-h period (Bohák et al. 2013). Salivary cortisol level was successfully used as the indicator of stress levels in horses in response to stabling (Harewood & McGowan 2005) and during road transport (Schmidt et al. 2010a,b,c). Also the initial training of horses, especially mounting by a rider, resulted in significant increase in salivary cortisol levels (Schmidt et al. 2010d). The determination of cortisol concentration in saliva was useful also for assessing stress levels in horses during various equestrian competitions (Peeters et al. 2010; Strzelec et al. 2011). Finally, it was found that salivary cortisol concentrations could be useful markers of relative intensity of exercise in thoroughbred racehorses (Ke˛dzierski et al. 2012). According to our best knowledge, Purebred Arabian racehorses have not been examined in this context. During race training, Purebred Arabian horses should be submitted to less intensive exertion than thoroughbreds (Ke˛dzierski & Bergero 2006). It is not known if the effort which is routinely applied to Arabian racehorses will be sufficient to involve significant increase in salivary cortisol levels, especially because cortisol release is delayed in time in response to a stimulating factor (Thompson et al. 1988; Peeters et al. 2011). The purpose of this study was to evaluate the use of saliva sampling for the measurement of cortisol concentration and to detect work-load levels in young Purebred Arabian horses during race training session.
MATERIAL AND METHODS Horses This study was performed in Poland at the Sluzewiec (Warsaw) racetrack. Twelve Purebred Arabian horses aged 3–5 years and comprising an equal number of stallions and mares were examined. The study took place in the middle of the racing season in Poland. The horses included in the study competed in official races at least 2 weeks before. All the horses were trained by one trainer and maintained in one stable. They were fed with typical fodder used as a standard diet for racehorses. Medium body weight, withers height and girth circumference of stallions amounted to 464 ± 55.0 kg, 156 ± 4.15 cm and 178 ± 5.65 cm, respectively. Measurement of mares amounted to, respectively, 413 ± 12.0 kg, © 2013 Japanese Society of Animal Science
148 ± 1.02 cm and 173 ± 1.72 cm. Prior to the study, the horses were trained 5 days a week. The study was accepted by the Local Ethics Review Committee for Animal Experimentation and conducted according to the European Community regulations concerning the protection rules of experimental animals.
Exercise protocol The horses were examined during their training session which was a part of the routine training. They were in the last phase of preparation to a start in races. At the day of the study, the training session was performed in the morning, from 07.00 to 09.00 hours. Mares were exercised first, then the stallions. All horses had a 10 min warm-up trot and canter with a rider. Then they galloped on the 800 m sand track at a speed amounting to 12.8 m/s. Finally, they returned trotting to the stable. After unsaddling, the horses were cooled down in an automatic horse walker for about 20 min. Three saliva samples and three blood samples were collected from each horse. All horses were sampled by the same operators: one person sampled saliva and the other one collected blood samples. Both types of samples were taken according to the following protocol: (i) at rest; (ii) immediately after return from the track; and (iii) after 30 min restitution. In each case, saliva was sampled first and then blood was taken. The saliva samples were collected with a small piece of sponge which was inserted into the horse’s mouth and then, after soaking in saliva, it was placed in a plastic tube, as previously described (Strzelec et al. 2011). Blood samples were collected by jugular venipuncture to ethylenediaminetetraacetic acid (EDTA) K3 tubes. Blood lactic acid (LA) concentrations were determined immediately using Dr Lange’s enzymatic cuvette test (Berlin, Germany) and expressed in mmol/L. The remaining blood was immediately centrifuged at 2000 × g for 10 min and plasma was stored at −20°C until assayed.
Laboratory analysis Before laboratory analysis, the saliva samples were centrifuged at 500 × g for 15 min at room temperature. Next, the sponge with the straw was removed and the saliva was transferred to test tubes. The concentrations of cortisol in saliva samples were measured by enzyme-immunoassay method using the CORTISOL EIA kit DSL (Diagnostic System Laboratories Inc., Webster, TX, USA). For plasma cortisol determination, the CORTISOL ELISA kit (DRG International Inc., Mountainside, NJ, USA) was used. All samples were analyzed in duplicate. The absorbance was measured by Multiscan reader (Labsystem, Helsinki, Finland) using a GENESIS V 3.00 software program (VWR International Ltd., Lutterworth, UK). The intra- and interassay coefficients of variation for salivary cortisol determined in the laboratory amounted to 9% and 11%, and for plasma cortisol concentration 5% and 8%, respectively. The results were expressed in nmol/L.
Statistical analysis The results are presented as means ± standard deviation (SD). Statistical analyses were performed using the statistical software package GraphPad PrismTM (Graph Pad Software, La Jolla, CA, USA). Comparisons between the results obtained before the training session, immediately after and 30 min after the end of exercise in the blood and saliva samples were Animal Science Journal (2014) 85, 313–317
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Table 1 Blood lactic acid and plasma and salivary cortisol levels determined in Purebred Arabian horses (n = 12) at a training session (means± SD)
Sample:
At rest
Immediately after the exercise
30 min after the exercise
Blood lactic acid (mmol/L) Plasma cortisol (nmol/L) Salivary cortisol (nmol/L)
0.92 ± 0.15a 79.3 ± 18.1a 1.06 ± 0.64a
4.64 ± 3.14b 118 ± 19.1b 5.45 ± 4.80b
1.23 ± 0.31a 103 ± 18.6b 5.06 ± 4.38b
a,b
Means in rows with different superscripts differ significantly at P < 0.05.
Figure 1 Correlation of salivary cortisol concentration determined 30 min after the end of exercise (ordinate; nmol/L) and plasma cortisol level determined (a) immediately after exercise and (b) 30 min after the end of exercise (abscissa; nmol/L).
made by the Tukey test (analysis of varaince). The coefficient correlation to compare the above data was assessed by Pearson’s test. Statistical significance was accepted at P < 0.05.
RESULTS The results of LA and cortisol measurements are presented in Table 1. Blood LA and plasma cortisol levels increased significantly after exercise (P < 0.05). The mean value of plasma cortisol concentration determined after the 30 min of restitution was lower than measured immediately after the exercise but did not reach the levels at rest (P < 0.05). Salivary cortisol concentrations determined immediately after exercise and after 30 min restitution were significantly higher (P < 0.05) than values obtained at rest. A significant correlation coefficient was found between plasma cortisol concentration determined immediately after exercise and salivary cortisol levels obtained 30 min after the exercise (r = 0.55, P < 0.05) and also between plasma cortisol concentrations determined 30 min after the exercise and salivary levels of cortisol measured after the 30 min of restitution (r = 0.67, P < 0.05; Fig. 1).
DISCUSSION Blood LA level was determined in the study to indicate the relative intensity of performed exercise (Guhl et al. 1996; Ke˛dzierski & Bergero 2006). The mean blood LA level determined immediately after exercise reached 4.64 mmol/L. This value is typical for exercises applied to the horses in routine race training (Podolak et al. 2006; Ke˛dzierski et al. 2009). It is believed that LA production is far more related to the intensity of exercise than cortisol secretion. The Animal Science Journal (2014) 85, 313–317
results of other studies indicate that plasma cortisol concentrations reflected rather the duration of exercise (Desmecht et al. 1996) or stress level (Berghold et al. 2007; Mircean et al. 2007). Spectacularly high values of plasma cortisol were noted after endurance exercises (Snow & Rose 1981; Lassourd et al. 1996). Two-time increase in plasma cortisol level was induced by participation in cross-country (Snow 1990; Linden et al. 1991) or draught work (Ke˛dzierski & Pluta 2013). The horses investigated in this study performed a short exercise of moderate intensity. They galloped only for few minutes covering the distance of 800 m; however, in response to this exercise, the plasma cortisol concentration increased significantly in comparison to the level at rest. This indicates that such kind of work-load was sufficient to increase the hypothalamic-pituitaryadrenocortical axis. An increase in plasma cortisol level was stated also in other studies in response to gallops (Desmecht et al. 1996; Mircean et al. 2007; Ke˛dzierski et al. 2012). The measurement of plasma cortisol concentration was also used for the evaluation of horse performance. The cortisol release rate in response to a standardized exercise test was higher in untrained horses than in well-trained horses (Marc et al. 2000). The facts mentioned above indicated that short and moderate intensive exercises in horses are able to induce cortisol release, the rate of which is proportional to an individual, relative work load. In the present study, salivary cortisol levels also significantly increased under the influence of exercise. The means of salivary cortisol concentrations were characterized by high SD values. High variability of salivary cortisol levels was also stated by other researchers (Peeters et al. 2010; 2011; Strzelec et al. 2011). Also in our previous work, each studied horse © 2013 Japanese Society of Animal Science
316 W. KE˛DZIERSKI et al.
was characterized by individual intensity of cortisol release in response to each competition during 3-day events (Strzelec et al. 2013). This phenomenon can be explained partially by the genetic conditioning of the horse and partially by their interaction with their rider. The statistically significant correlations were stated between salivary cortisol levels determined 30 min after the end of exercise and plasma cortisol concentrations. This indicated that salivary cortisol levels measured 30 min after exercise clearly reflected the exercise-induced cortisol release in studied horses. The positive correlation between plasma and salivary cortisol concentrations have been described previously in horses submitted to different stressors (Schmidt et al. 2009). The increase in both salivary and plasma cortisol was also stated during the crosscountry round (Peeters et al. 2010). On the other hand, Elsaesser et al. (2001) have shown that the correlation between salivary and plasma cortisol levels in trained warm-blooded sports horses was low and insufficient to use salivary cortisol measurements for diagnostic purposes. However, in that study, cortisol levels were measured at distinct points of time after adrenocorticotropic hormone (ACTH) injection and not after exercise. In fact, cortisol release from the adrenal cortex is stimulated by ACTH, which is released from the pituitary gland under the influence of corticotrophin-releasing hormone (CRH) produced by the hypothalamus. Moreover, in exercise conditions, the role of CRH is replaced by vasopressin (Alexander et al. 1991). Also, the increase in plasma cortisol level is the result of complicated and timedependent processes. For example, short-term stress induced in pony mares caused an increase in plasma cortisol concentration about 20 min later (Thompson et al. 1988). Similar results were achieved after CRH injection (Reijerkerk et al. 2009). ACTH administration also increased plasma and salivary cortisol concentrations over time (van der Kolk et al. 2001). The detailed studies performed by Peeters et al. (2011) indicated that salivary cortisol peak level appeared 30 min later than found in plasma following an ACTH stimulation test. A similar situation can be observed in the present study, in which only the salivary cortisol level determined 30 min after the end of exercise reflected the cortisol concentration in plasma. In conclusion, the high correlation between plasma and salivary cortisol levels found in this study indicated that measuring the cortisol level in saliva, especially after restitution, 30 min after the end of an effort, can pose as a useful marker of exercise-induced work load in racehorses. Therefore, the sampling of saliva 30 min after the end of exercise can be recommended to use in practice for the estimation of cortisol release rate in Purebred Arabian horses during race training sessions in field conditions. © 2013 Japanese Society of Animal Science
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