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УДК 636.1:591.111
HEMATOLOGICAL AND BIOCHEMICAL CHANGES IN THE BLOOD OF DRESSAGE HORSES DURING EXERCISE
A. V. Andriichuk, H. M. Tkachenko, I. V. Tkachova
ГЕМАТОЛОГИЧЕСКИЕ И БИОХИМИЧЕСКИЕ ИЗМЕНЕНИЯ В КРОВИ ВЫЕЗДКОВЫХ ЛОШАДЕЙ ПОД ВЛИЯНИЕМ ФИЗИЧЕСКИХ НАГРУЗОК
А. В. Андрийчук, Г. М. Ткаченко, И. В. Ткачова
Physical exercise for athletic horses induces a series of normal physiological and biochemical adaptations. Dressage is a combination of mental and physical prowess to perform practiced movements to exacting standards with harmony between a horse and its rider. Many articles focus on exercise-induced changes of metabolic and biochemical parameters of horses involved in jumping, eventing and other equine athletic disciplines. But a scientific review to assess the hematological and some biochemical changes in horses involved in dressage still requires investigation and discussion. The objective of the present study was to investigate alterations of hematological parameters [haematocrit (HCT), hemoglobin concentration (HGB), the count of red blood cells (RBC), white blood cells (WBC), platelets (PLT), leucogram, mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red cell distribution width (RDW) and platelets distribution width (PDW)], as well as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) activities, and lactate and pyruvate concentrations in sport horses involved in dressage before and after exercise. The results of the present study showed a significant increase of WBC and granulocytes count, as well as RBC, HGB and HCT levels after exercise. Activity of AST reduced after exercise that may reflect adaptation related to regular dressage training.
dressage, horses, hematological parameters, aminotransferases
К лошадям, используемым в выездке, предъявляются определенные требования, но, к сожалению, в научных работах немного исследований, связанных с анализом гематологических и биохимических показателей. Учитывая актуальность данной проблемы, мы поставили перед собой цель проанализировать количественные изменения гематологических показателей и некоторых биохимических маркеров у выездковых лошадей до и после физических нагрузок. Объектом исследований были девять спортивных лошадей. Изучали следующие гематологические параметры: количество эритроцитов (RBC), средний объем эритроцитов (MCV), индекс анизоцитоза (RDWc), содержание гемоглобина (HGB), среднее содержание гемоглобина в эритроците (MCH), средняя концентрация гемоглобина в эритроцитах (МСНС), гематокрит (НСТ), количество тромбоцитов (PLT), тромбо-крит (РСТ%), средний объем тромбоцитов (MPV), ширина распределения тромбоцитов (PDWc), количество лейкоцитов (WBC) с дифференцировкой на три
субпопуляции - лимфоциты (LYM), гранулоциты (GRA), моноциты, эозинофилы, базофилы и их предшественники (MID). Также проанализировали изменения активности аминотрансфераз и лактатдегидрогеназы, содержание лактата и пирува-та в крови лошадей в динамике физических нагрузок. После физических нагрузок установлено существенное увеличение количества лейкоцитов и гранулоцитов, что свидетельствует об адаптационных реакциях организма к систематическим физическим нагрузкам через активацию фагоцитарного звена иммунной защиты. Существенное увеличение общего количества эритроцитов, гемоглобина и гема-токрита после физических нагрузок связано с выходом эритроцитов из депо, что необходимо для обеспечения поставок кислорода к работающим мышцам и органам. Снижение активности аспартатаминотрансферазы у лошадей, используемых в выездке, указывает на адаптационные изменения метаболических процессов к физическим нагрузкам и на высокий уровень тренированности этих лошадей.
выездка, лошади, гематологические показатели, аминотрасферазы
INTRODUCTION
Hematological parameters provides relatively simple but reliable methods of establishing physiological status by giving insight in various processes in a horse organism [1, 2]. In addition, blood hematological and biochemical parameters can be good indicators of the response to treatment, the severity and the systemic effects of a disease, as well as horse welfare, health and fitness levels of horses [2-4]. Despite the extended use of hematology in equine medicine, interpretation may be a challenge in some cases, because it can be significantly influenced by a great number of factors. Hematological parameters may vary according to a breed, sex, age, reproductive status, fitness and training levels, exercise, feeding, circadian variations, handling procedure of animals during blood withdrawal, degree of excitement and health state [2, 4, 5]. Among these factors, hematological adaptation is necessary to provide an appropriate blood supply during exercise.
Dressage is a combination of mental and physical prowess to perform practiced movements to exacting standards with harmony between a horse and its rider. Despite many claims that a dressage horse does not require a high level of physical fitness, in fact, collected dressage movements benefit from a much more sustained physical fitness as compared to many other equine athletic disciplines, especially in preparation for the advanced levels of the sport [6]. For this reason, certain cardiovascular and haematolog-ical adaptations are necessary to guarantee correct oxygen and blood borne substrates supply to active muscles during exercise and release of metabolites. These systems could act as limiting factors to the aerobic potential and, thereby, could limit physical performance of dressage horses [1].
Recent studies of equine exercise physiology have focused mainly on determining the usefulness of biochemical and hematological parameters for evaluating physiological capacity and adaptation to increasing loads in horses of different breeds [4, 7, 8]. It is important to understand the biochemical changes produced by various types of exercises, because they reflect changes in the functions of different systems and in the type of energy utilized [3, 7, 8]. Many articles focus on exercise-induced changes of metabolic and biochemical parameters in horses involved in jumping, eventing and other equine athletic disciplines [1, 4, 7, 8]. But a scientific review to assess the hematolog-
ical and some biochemical changes in horses involved in dressage still require investigation and discussion. Therefore, the aim of the present study was to determine hemato-logical and biochemical parameters of horses involved in dressage before and after exercises.
MATERIALS AND METHODS Horses and training. Nine well trained Ukrainian Warmblood horses (3 mares, 5 geldings, 2 stallions) aged 8.3±1.6 years, involved in dressage were used in this study. Horses were housed in the same environment. They had the same vaccinate status and no signs of clinical disease. The females were non-pregnant. The diets were composed of grass hay and concentrated forage, fed three times per day. All horses had been in regular training for several years.
Exercise test. The study design consisted of one exercise test according to training program. This test consist of a number of dressage movements of average intensity including collected and extended movements in trot and canter. The exercise test consisted with the walk - 5 min; the trot - 10 min; the walk - 5 min; the collected trot with working in passage - 10 min; the walk - 10 min; the extended trot with half-pass diagonal - 10 min; the walk - 10 min; the collected and extended canter with flying changes in sequence - 10 min; the walk - 15 min. The duration of training was 1.25 hour.
Blood samples. Blood was drawn from jugular veins of the animals in the morning, 90 minutes after feeding, while the horses were in the stables (between 8:30 and 10 AM), and immediately after endurance race (between 11:00 AM and 2:00 PM). Blood was stored into tubes with K-EDTA and held on ice until centrifugation at 3,000g for 15 minutes. The plasma was removed. The erythrocytes' suspension (one volume) was washed with five volumes of saline solution three times and centrifuged at 3,000g for 15 minutes. Plasma aliquots were frozen and stored at -25°C until analyzed.
Hematological assays. Routine hematological parameters [haematocrit (HCT), haemoglobin concentration (HGB), the count of red blood cells (RBC), white blood cells (WBC), platelets (PLT), leucogram, mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red cell distribution width (RDW) and platelets distribution width (PDW)] were measured and counted with an automated hematology analyzer (Abakus Junior Vet, Austria).
Biochemical assays. Alanine aminotransferase (ALT, E.C. 2.6.1.2) and Aspartate aminotransferase (AST, E.C. 2.6.1.1) activities were analyzed spectrophotometri-cally by standard enzymatic method [9]. The colorimetric method of Sevela and Tova-rek (1959) was used for the determination of lactate dehydrogenase (LDH, E.C. 1.1.1.27) activity [10]. Lactate and pyruvate concentrations were measured according to the procedure described by Herasimov and Plaksina (2000) [11].
Statistical analysis. Results are expressed as mean ± S.E.M. All variables were tested for normal distribution using the Kolmogorov-Smirnov test (p>0.05). In order to find significant differences (significance level, p<0.05) between states before and after exercise, Wilcoxon signed-rank test was applied to the data [12]. All statistical analyses were performed using STATISTICA 10.0 software (StatSoft, Poland).
RESULTS AND DISCUSSION Exercise-induced physiological processes are reflected in changes in blood he-matological parameters. In our study, all hematological parameters of horses were with-
in the reference values (Table 1). However, significant changes levels of leukocytes (WBC), granulocytes (GRA), erythrocytes (RBC), hemoglobin (HCT), hematocrit (HCT), and red cell distribution width (RDWc) in horses after exercise test were observed. In particular, WBC and GRA levels were increased by 25% and 25% (p<0.05), respectively. The results showed increased RBC, HGB HCT and RDWc values by 26.9%, 29.5%, 27%, and 2.6% (p<0.05), respectively (Table 1).
Table 1. Hematological indices of horses before and after exercise
Таблица 1. Гематологические показатели лошадей до и после физической нагрузки
Hematological indices Before exercise After exercise The reference values
Leukocyte count, WBC [• 109/L] 6.83±0.49 8.55±0.74* 5.4-14.3* 5.5-12.0«
Lymphocyte count, LYM [• 109/L] 2.14±0.29 2.50±0.39 1.5-7.7*
Monocytes and some eosinophils count, MID [109/L] 0.14±0.03 0.33±0.11 0-1.5*
Granulocytes (neutrophils, eosinophils and basophils) count, GRA [• 109/L] 4.55±0.36 5.72±0.46* 2.3-9.5*
Percentage of lymphocytes, LY% 31.09±3.15 28.60±3.33 17-68*
Percentage of monocytes and some eosinophils, MID% 2.08±0.46 3.71±1.14 0-14*
Percentage of granulocytes, GR% 66.83±3.35 67.70±3.52 22-80*
Erythrocyte count, RBC [• 109/L] 7.47±0.39 9.48±0.60* 6.8-12.9* 5.5-10.0**
Hemoglobin level, HGB [g/dL] 11.67±0.52 15.12±1.0* 11-19* 8-18"
Hematocrit, HCT [%] 33.82±0.98 43.19±2.95* 32-53* 24-52**
Mean Corpuscular Volume, MCV [fL] 45.56±1.30 45.83±1.30 34-58* 35-58**
Mean Corpuscular Hemoglobin, MCH [pg] 15.68±0.20 15.94±0.23 12.3-19.7* 10-20**
Mean Corpuscular Hemoglobin Concentration, MCHC [g/dL] 34.42±0.76 34.95±0.70 31-39* 31-37**
Red Blood Cell Distribution Width, RDW [%] 20.31±0.21 20.85±0.22* 11-17*
Platelet count, PLT [• 109/L] 159.78±8.19 180.41±11.71 100-400* 150-400**
Mean platelet volume, MPV 8.18±0.25 8.92±0.46 9.7-12.8*
Platelet distribution width, PDW 33.27±0.73 33.42±0.47
Legend: • - reference values according to the Operating Instructions Hematolo-
gy Analyzer Abacus Junior Vet;
•• - reference values according to A. Winnicka (2004) (13);
* - statistical significance (p<0.05) between means in groups of horses before and after exercise.
* - реферативные значения гематологического анализатора Abacus Junior Vet; •• - реферативные значения согласно А. Винницкой (2004) [13];
* - статистически существенные изменения (р <0,05) между показателями, полученными до и после физической нагрузки.
There are two main WBC responses, physiological leukocytosis and stress leukocytosis. Physiological leukocytosis refers to changes in circulating WBC associated with the intervention of the sympathetic-adrenal axis resulting from splenic contraction in cases of fear, excitement, or high intensity exercise. Stress leukocytosis is associated with cortisol release under certain stressful situations [2]. Regular exercise is considered a physical stressor and as such has been shown to have profound effect on the immune response of many species [2, 14]. Mechanisms responsible for change of leukocyte values and function following exercise are believed to be multifactorial and complex; although, alterations in neuroendocrine hormones, in particular, catecholamines and corti-costeroids, in response to an exercise challenge have been associated with transient immunosuppression [15]. In general, moderate training is considered to have beneficial effects on host defense mechanism [14], whereas prolonged periods of high-intensity training may lead to slight impairment of several immune parameters [2]. Krumrych (2006) investigated the effect of routine training exercise on hematological indices in jumping horses. As a result of training, a significant increase of WBC, RBC, HGB and cell volume counts were noted [4].
Our results showed significant increase granulocytes count in dressage horses after exercise (Table 1). Research studies examining granulocyte and monocyte function reported varying of acute exercise on phagocytic activity in horses [14, 15]. Moderate exercise was found to either have no effect on granulocyte and monocyte oxidative burst capacity or to increase these parameters [2]. Most studies show that of all subsets of circulating leukocytes, mainly neutrophils and lymphocytes, increase immediately after exercise [2, 4, 14, 16]. In summary, exercise increase granulocytes level and may reflect an appropriate response to exercise-induced stress rather than an impaired im-munocompromised state [2, 14].
In our study was established that dressage horses had significantly higher values of all RBC, HGB, HCT, RDWc after exercise test. The most common finding for horses undergoing high-intensity training has been significant increases in the resting red blood cell count, hematocrit, and hemoglobin concentration [1, 3-5]. The increased of red blood cells indices after exercise test, observed in the present study, caused by mobilization of splenic erythrocytes and, therefore, increases the oxygen transport capacity [2, 4]. Increase of RDW levels in horses after exercise test were observed (Table 1). Red blood cell distribution width (RDW) is a simple and inexpensive parameter, routinely reported by automated hematological analyzers, and conventionally used as a measure of anisocytosis [17]. A previous study showed that RDW values are not affected by endurance running in altitude [18]. Our results agreed with Ellis and co-authors which have shown increased RDW values in healthy males after exhaustive running exercise [16].
Measurement of values of liver (AST, ALT) and muscle damage indicator (LDH), followed by a variety of training programs, can help to better understand the acute and chronic effects of resistance training [7, 8]. The results of the biochemical parameters in the two groups of examined horses before and after exercise are presented in Table 2.
Table 2. Biochemical parameters in the blood of dressage horses before and after exercise
Таблица 2. Биохимические показатели выездковых лошадей до и после физических нагрузок_
Parameters Before exercise After exercise
ALT, mmolh-1L-1 2.83±0.11 2.85±0.10
AST, mmolh-1L-1 4.33±0.14 4.05±0.11*
LDH, mmolh-1L-1 6.45±0.52 6.85±0.71
Lactate, mmolL-1 4.59±0.77 5.13±0.78
Pyruvate, mmolL-1 3.08±0.62 2.76±0.47
* - statistical significance (p<0.05) between means in groups of horses before and after exercise.
* - статистически существенные изменения (р <0,05) между показателями, полученными до и после физической нагрузки.
Significant decrease activities of aspartate aminotransferase by 6% (p<0.05) after exercise test was observed. The extent of changes depends on several factors: type of exercise, intensity of work (strength, duration and frequency) and individual variation [19]. Physiological increases of ALT and AST have been shown to occur without any tissue destruction [19]. AST catalyzes the transfer of an amino group from L-aspartate to a-ketoglutarate, the products of this reversible transamination reaction are oxaloace-tate and glutamate [20]. AST is a muscle-derived enzyme and as such is commonly used as indicator of muscle damage in the human athlete or athletic horses [21]. Physical training prepares the equine athlete for competition by inducing the physiological adaptations necessary to performance at a high level with minimal risk of injury, and by providing the appropriate behavioral and psychological factors essential for effective competition [8]. Fazio et al. (2011) reported a gradual and significant increase of AST and creatine kinase activities until the 60th day of training and a decrease at the end of training period (80th day) in Thoroughbred horses [8]. This increased could be explained to higher response of glycolytic metabolism that caused permeability changes in muscle fiber membranes [8]. These findings can be useful to assess the status of horses' performance and the degree of its training adaptability [8]. An increase in muscle mass and an increased activity of specific metabolic enzymes in concert with changes in the expression of structural proteins are known to contribute to changes in muscle and metabolism. Bouwman et al. (2010) investigated training-induced differential expression of equine muscle biopsy proteins [22]. Despite the poor resolution of the equine genome and proteome, they were able to identify the proteins of 20 differential spots representing 16 different proteins [22]. Evaluation of those proteins, including expression of mitochondrial AST, complies with adaptation of the skeletal muscle after normal training involving structural changes towards a higher oxidative capacity, an increased capacity to take up long-chain fatty acids, and to store energy in the form of glycogen [22]. Consequently, our findings are in agreement with a previous reports [8] where serum enzymatic activities increased in the early stages of training and decreased in the later stages of training.
CONCLUSION
1. Exercise lead to significant increase of leukocytes and granulocytes values. These changes most likely occurred due the external factors, such as regular training and could improve the immune function of horses involved in dressage.
2. The increased of red blood cells indices after exercise test observed in the present study, caused by mobilization of splenic erythrocytes and, therefore, increases the oxygen transport capacity.
3. Significant decrease of aspartate aminotransferase activity in horses after exercise could reflect adaptations related to regular training for dressage.
4. It is important to understand the hematological and biochemical changes caused by various physical efforts because they reflect changes in the functions of different body systems. It can be used for health control and diagnosis of diseases and allow the evaluating the level of sport performance the accuracy of training and physiological condition of equine athletes.
This study was supported by Polish National Commission for UNESCO.
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ИНФОРМАЦИЯ ОБ АВТОРАХ
Андрийчук Анастасия Васильевна - Институт животноводства Национальной академии аграрных наук Украины, пгт Кулиничи, Харьковский район, Харьковская область; аспирант; E-mail: anastasia.pohlyad@gmail.com
Andriichuk Anastasiia - Institute of Animal Science, National Academy of Agrarian Sciences of Ukraine, urban-type settlement Kulinichi, the Kharkov region, Postgraduate student; E-mail: anastasia.pohlyad@gmail.com
Ткаченко Галина Михайловна - Институт биологии и охраны среды, Поморская Академия, г. Слупск, Польша; кафедра зоологии и физиологии животных; кандидат биологических наук, докторант; E-mail: tkachenko@apsl.edu.pl
Tkachenko Halyna - Institute of Biology and Environmental Protection, Pomeranian University, Slupsk, Poland; Department of Zoology and Animal Physiology; Candidate of Biological Sciences, Assistant Professor; E-mail: tkachenko@apsl.edu.pl
Ткачева Ирина Владимировна - Институт животноводства Национальной академии аграрных наук Украины, пгт Кулиничи, Харьковский район, Харьковская область; зав. отделом коневодства; E-mail: i-tkachova@yandex.ru
Tkachova Irina - Institute of Animal Science, National Academy of Agrarian Sciences of Ukraine, urban-type settlement Kulinichi, the Kharkov region; Head of the Horse-breeding Department; E-mail: i-tkachova@yandex.ru
