Racing Induces Changes in the Blood Concentration of Serum ...

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Oct 9, 2015 - a Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Warsaw, ...
Journal of Equine Veterinary Science 36 (2016) 15–18

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Original Research

Racing Induces Changes in the Blood Concentration of Serum Amyloid A in Thoroughbred Racehorses  ska a, Michał Czopowicz b, Lucjan Witkowski b, Agnieszka Turło a, *, Anna Cywin a Anna Jaskiewicz , Anna Winnicka a a b

Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Warsaw, Poland Laboratory of Veterinary Epidemiology and Economics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Warsaw, Poland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 August 2015 Received in revised form 13 September 2015 Accepted 18 September 2015 Available online 9 October 2015

Intensive exercise results in the increased blood concentration of the acute phase proteins in horses competing in some sport disciplines. In this study, the blood level of serum amyloid A (SAA) was analyzed in Thoroughbred racehorses during 5 days after completion of the race. Samples were collected from 25 healthy Thoroughbred horses beginning with 4 hours after the race and repeated daily up to the fifth day after the race. Serum amyloid A analysis was performed using commercial enzyme-linked immunosorbent assay kit, and the results were presented as median, interquartile range (IQR), and range. Data were analyzed using Friedman’s nonparametric analysis of variance. The acute phase response (APR) was reflected by an increased SAA level after the race, reaching significantly higher concentrations on days 1 (P < .001) and 2 (P ¼ .005) and falling below the level of the first sample on day 5 (P ¼ .006). The median peak concentration observed on day 1 after the race was 3.84 mg/L (IQR, 2.32 to 8.86). Racing induces minute changes in SAA concentration typical for the exercise-induced APR; however, the significance of this reaction in the context of horse health and fitness remains unclear. Ó 2015 Elsevier Inc. All rights reserved.

Keywords: Serum amyloid A Acute phase response Racehorse Exercise

1. Introduction Strenuous exercise causes the release of the proinflammatory agents such as cytokines, prostaglandins, and acute phase proteins (APPs) into the bloodstream in horses, dogs, and humans [1–7]. Acute phase proteins are a group of blood proteins whose concentrations decrease or increase in animals subjected to external or internal challenges. The origin of acute phase response (APR) can be attributable to infection, inflammation, surgical trauma, or other causes and the purpose of the response is to restore homeostasis and to remove the cause of its disturbance [8]. The main APP in horses is serum amyloid A (SAA), the blood

* Corresponding author at: Agnieszka Turło, Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Warsaw, Poland. E-mail address: [email protected] (A. Turło). 0737-0806/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2015.09.008

protein whose concentration can increase up to a 1,000 times in response to tissue-damaging factors [9–15]. Owing to its fast and explicit reaction to inflammatory stimuli, SAA is considered a reliable tool for health assessment in horses, more sensitive than the total white blood cell count [16]. Recognizing the effect of different types of exercise on the SAA is extremely important for the correct analysis of this biomarker, especially in sport horses regularly subjected to training. The first reports on changes in SAA blood concentration induced by heavy exercise came from endurance horses participating in the long distance rides (120 and 160 km) [5]. These findings stood in line with observation previously made in human ultramarathon and triathlon competitors [1,2]. Moreover, high-intensity exercise of moderate length, such as sled racing in Alaskan dogs and 2-day eventing in sport horses, did also result in minor but significant increase of the C-reactive protein and SAA,

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respectively [4,17]. Occurrence of a similar SAA reaction has not been confirmed in racehorses after training or racing [18,19], even though elevated expression of the proinflammatory cytokine genes was reported in 2-year-old Thoroughbred racehorses after the training session [6]. The results of our preliminary study [20] suggested that the change in SAA level in Thoroughbred racehorses can be detected in 24 to 48 hours after the race and, therefore, could not have been observed in the studies examining SAA level immediately after exercise [18,19]. However, the other study comparing the concentrations of APPs in standardbred trotters before and 2 days after the race did not indicate the SAA response [21]. In this study, we aimed to test the hypothesis that racing induces changes in blood SAA concentration in Thoroughbred racehorses within 5 days after completion of the race. Determining the exact time frame of this reaction could facilitate the interpretation of control SAA measurements in racehorses during the racing season.

assumed. The analysis was carried out in Statistica 10 (StatSoft Inc). 3. Results The results were reported as median, interquartile range, and range (i.e., min–max). The significant changes in the mean SAA concentration were noted in the first 5 days after the race (c2(5) ¼ 71.5, P < .001). Compared with the first measurement obtained 4 hours after the effort, the serum SAA level increased on the day 1 (P < .001) and was still significantly higher on the day 2 (P ¼ .005), then returned to the level comparable with the initial level on the days 3 (P ¼ .999) and 4 (P ¼ .262) and fell below the initial level on the day 5 (P ¼ .006; Fig. 1). The median peak SAA concentration was observed at day 1 (3.84 mg/L [2.32–8.86]) with the highest individual SAA concentration of 10.15 mg/L. 4. Discussion

2. Materials and Methods Twenty five privately owned Thoroughbred racehorses aged 2 to 6 years (mean, 2.8 years) stabled in one horse racing facility were used in this study. All horses were trained and competed in flat racing throughout the whole racing season (April to November). The study was performed on the last day of the season, in a dry, cold weather (environmental temperature 3 C), with track elasticity rate 3.8 (in a 1- to 5-point scale). Racing distances varied from 1,200 to 1,600 m and were selected appropriately to the age and fitness of the horse by the horse trainers. Collection of blood samples was approved by the Local Ethic Committee, the trainers, and the owners of the horses. Samples were collected by jugular venipuncture into tubes with no additives (Vacutainer Systems; Becton Dickinson, France) starting 4 hours after completion of the race (day 0). The procedure was repeated daily for the next five consecutive days. In this period, horses were subjected to light exercise in the horse walker. With each sample collection, horses underwent thorough clinical examination performed by the equine veterinarian. All horses recovered well after the race, and none of them showed clinical signs of disease or injury during the time of the study. Blood samples were transported immediately after collection to the laboratory, where they were centrifuged at 4,380g for 5 minutes. The collected serum was divided in aliquots, frozen, and stored at 20 C for SAA analysis. Serum amyloid A concentration was measured with the commercial enzyme-linked immunosorbent assay kit (PHASE SAA; Tridelta Ltd, Ireland) previously validated for use in the equine studies [5,11,17,19,20]. The analysis was performed using Friedman’s nonparametric analysis of variance. Changes of SAA concentration compared to the first measurement 4 hours after the race were evaluated using Wilcoxon signed-rank tests with a Bonferroni correction applied (given that five mutual comparisons were performed; P value from each pairwise comparison was multiplied by five to control for the increased possibility of type I error associated with the multiple comparisons). An overall alfa level of 0.05 was

The change between the highest and the lowest median SAA concentration, observed in horses on days 1 and 5 after race, was approximately sevenfold. The highest individual concentration was still relatively low comparing with those reported in horses with acute inflammatory diseases of respiratory or gastrointestinal system [13–15]. Such small changes are not likely to be of much relevance in the assessment of horse health based on the interpretation of serum SAA level. The SAA concentration range in healthy horses may differ depending on the population and the method used [5,11,13–15,17,20,22]. The highest SAA level reported of healthy horses was 20 mg/L [22]. The postexercise changes in horses described in the recent and the previous studies remain far below this level and could not be interpreted as indicative of serious health conditions. The similar minute difference has been previously reported in endurance and eventing horses tested for SAA concentration before and shortly after the competition or training [5,17,19]. The limitation of our study is the lack of

Fig. 1. Median concentration of serum amyloid A in 5 days after the race (boxes stand for the interquartile range (IQR), and whiskers stand for the range). *SAA concentrations significantly different from the baseline (P < .05). SAA, serum amyloid A.

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the prerace measurement because of the reluctance of horse trainers to allow blood sampling close to the race. The first measurement was performed 4 hours after finishing the race. At this time point of the APR, SAA blood concentration begins to increase to reach the maximal value 24 to 48 hours after induction of the response [9]. The previous studies showed no difference between SAA level measured before and up to 2 hours after exercise in Thoroughbred and Arabian racehorses [18,19]. We hypothesized that, regarding to the typical SAA reaction timeline, no major changes in its concentration would occur within the first 4 hours after the race. The results showed that the most radical increase in SAA concentration appeared between 4 hours and 1 day after racing, which fits the typical SAA pattern reported in the APR. However, the concentration drop observed on the day 5 suggests that at 4 hours after the race, the SAA concentration might have already risen slightly and that beginning of the reaction may have been missed due to the lack of the prerace sample. On the other hand, SAA may increase during the intensive training when horses are being prepared to the race and decrease to the true rest value after 5 days of light exercise on the horse walker. Release of the inflammatory markers after strenuous exercise has been proposed to result from the stressinduced damage to musculoskeletal system in horse and human studies [2,6,23–25]. This relation was supported by increase in serum concentration of muscle enzymesd creatinine kinase (CK) and aspartate transaminase (AST) and the occurrence of delayed onset muscle soreness accompanying secretion of proinflammatory cytokines in human after exercise [2,23,24]. Conversely, the lack of APR in standardbred trotters after competing on the sprinting distances was reported with the unaltered activity of AST and CK [21]. Our previous study showed that SAA level is higher in the racehorses diagnosed after race with the stress-related orthopedic disorders than in the control horses subjected to racing [20]. The robust SAA response (up to 160 mg/L) was also reported in Arabian endurance horses affected with exertional rhabdomyolysis [26]. These findings may suggest that clinical injury of musculoskeletal system results in the well-detectable APR, whereas the exercise-induced microdamage is reflected only by the minute changes in SAA concentration. The exercise-related APR could be involved in monitoring of the response to training with special regard to recognizing overtraining syndrome (OTS). Overtraining syndrome is a complex condition frequently associated with the excessive training with insufficient recovery and involves a large variety of signs and symptoms [27]. The main sign of overtraining is deterioration in the performance. However, this could not always be objectively measured, and it is possible that other symptoms of OTS are detectable before decline in performance occurs. These symptoms might include changes in hematological, biochemical, and immunologic markers detectable in the blood [28–30]. Excessive training is associated with accumulation of repetitive tissue trauma, especially within the musculoskeletal system. Muscle enzymes are among the proposed markers of overtraining in horses and humans, with muscle soreness being one of the main

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clinical signs of OTS [27–32]. As previously mentioned, muscle damage is likely to result in the induction of APR and consequently in altered blood concentration of cytokines and APPs [27,31]. Therefore, evaluation of the postexercise changes in APPs could be further investigated as indicator of horses’ response to training [33]. 5. Conclusions In conclusion, this study shows that in healthy Thoroughbred racehorses, small changes in SAA concentration occur in the 5 days after the race. This response is not likely to interfere with the interpretation of SAA level as a health marker. Its potential significance in assessment of the horses’ reaction to training load needs to be further investigated. Acknowledgments This research study was funded with statutory sources. References [1] Fallon KE. The acute phase response and exercise: the ultramarathon as prototype exercise. Clin J Sport Med 2001;11:38–43. [2] Suzuki K, Peake J, Nosaka K, Okutsu M, Abbiss CR, Surriano R, et al. Changes in markers of muscle damage, inflammation and HSP70 after an Ironman triathlon race. Eur J Appl Physiol 2006;98:525–34. [3] Donovan DC, Jackson CA, Colahan PT, Norton N, Hurley DJ. Exerciseinduced alterations in pro-inflammatory cytokines and prostaglandin F2a in horses. Vet Immunol Immunopathol 2007;118:263–9. [4] Wakshlag JJ, Stokol T, Geske SM, Greger CE, Angle CT, Gillette RL. Evaluation of exercise-induced changes in concentrations of C-reactive protein and serum biochemical values in sled dogs completing a long-distance endurance race. Am J Vet Res 2010;71:1207–13.  ska A, Szarska E, Górecka R, Witkowski L, Hecold M, [5] Cywin Bereznowski A, et al. Acute phase protein concentrations after limited distance and long distance endurance rides in horses. Res Vet Sci 2012;93:1402–6. [6] Horohov DW, Sinatra ST, Chopra RK, Jankowitz S, Betancourt A, Bloomer RJ. The effect of exercise and nutritional supplementation on proinflammatory cytokine expression in young racehorses during training. J Equine Vet Sci 2012;32:805–15. [7] Casella S, Fazio F, Russo C, Giudice E, Piccione G. Acute phase proteins response in hunting dogs. J Vet Diagn Invest 2013;25:577–80. [8] Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 1999;340:448–54. [9] Jacobsen S, Adersen PH. The acute phase protein serum amyloid A (SAA) as a marker of inflammation in horses. Equine Vet Educ 2007;19:38–46. [10] Casella S, Fazio F, Giannetto C, Giudice E, Piccione G. Influence of transportation on serum concentrations of acute phase proteins in horse. Res Vet Sci 2012;93:914–7. [11] Pollock PJ, Prendergast M, Schumacher J, Bellenger CR. Effects of surgery on the acute phase response in clinically normal and diseased horses. Vet Rec 2005;156:538–42. [12] Jacobsen S, Nielsen JV, Kjelgaard-Hansen M, Toelboell T, Fjeldborg J, Halling-Thomsen M, et al. Acute phase response to surgery of varying intensity in horses: a preliminary study. Vet Surg 2009;38:762–9. [13] Pihl TH, Andersen PH, Kjelgaard-Hansen M, Mørck NB, Jacobsen S. Serum amyloid A and haptoglobin concentrations in serum and peritoneal fluid of healthy horses and horses with acute abdominal pain. Vet Clin Pathol 2013;42:177–83. [14] Hobo S, Niwa H, Anzai T. Evaluation of serum amyloid A and surfactant protein D in sera for identification of the clinical condition of horses with bacterial pneumonia. J Vet Med Sci 2007;69:827–30. [15] Vandenplas ML, Moore JN, Barton MH, Ruossel AJ, Cohen ND. Concentrations of serum amyloid A and lipopolysaccharide-binding protein in horses with colic. Am J Vet Res 2005;66:1509–16. [16] Anhold H, Candon R, Chan DS, Amos W. A comparison of elevated blood parameter values in a population of thoroughbred racehorses. J Equine Vet Sci 2014;34:651–5.

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