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Раздел 4. ЧАСТНАЯ ЗООТЕХНИЯ И ТЕХНОЛОГИЯ ПРОИЗВОДСТВА ПРОДУКЦИИ ЖИВОТНОВОДСТВА, ПРОМЫШЛЕННОЕ РЫБОВОДСТВО
UDC. 636.1.083.38:591.1
INFLUENCE OF ENDURANCE RACE ON BLOOD BIOCHEMICAL PARAMETERS OF HORSES
A. V. Andriichuk1, H. M. Tkachenko2, I. V. Tkachova1
1Institute of Animal Science, National Academy of Agrarian Sciences of Ukraine, Kharkiv, Ukraine, 61026
2Institute of Biology and Environmental Protection, Pomeranian University in Slupsk, Poland, 76-200
(Поступила в редакцию 04.01.2016)
Резюме. В статье изучены биохимические показатели (содержание ТБК-активных продуктов, активность аминотрансфераз и лактатдегидрогеназы, содержание лак-тата и пирувата) в крови лошадей, используемых в дистанционных пробегах на 32 км в динамике физических нагрузок. Установлены специфические изменения метаболических процессов у лошадей после дистанционного пробега в 32 км. В крови лошадей после пробегов обнаружено повышение содержания маркеров перекисного окисления липидов, что указывает на развитие окислительного стресса, вызванного физической нагрузкой большого объема и интенсивности. Незначительные колебания активности аминотрансфераз вместе с несущественными изменениями концентраций лактата и пирува-та в динамике физических нагрузок указывает на хороший уровень тренированности и адаптационных возможностей лошадей используемых в дистанционных пробегах. Проведенный нами корреляционный анализ между содержанием маркеров окислительного стресса показал зависимости в протекании процессов окислительного стресса с аэробно-анаэробными механизмами энергообеспечения мышечной деятельности, о чем свидетельствуют корреляционные связи между сожержанием ТБК-активных продуктов и содержанием пирувата в крови лошадей как в состоянии покоя, так и после пробегов. Таким образом, установленные корреляционные зависимости между маркером перекис-ного окисления липидов в крови, активностью аспартатаминотрансферазы и уровнем пирувата свидетельствуют о взаимосвязи протекания окислительного стресса с метаболическими реакциями, контролирующими ключевые пути обновления энергетических субстратов при физических нагрузках.
Ключевые слова: окислительный стресс, аминотрансферазы, лактатдегидрогена-зы, лактат, пируват, метаболизм.
Summary. The aim of the present study was to examine the influence of endurance race on some biochemical parameters (thiobarbituric acid reactive substances (TBARS) level, ami-notransferases, lactate dehydrogenase, lactate, pyruvate) in horses involved in endurance race. Our results suggest that 32 km endurance race caused different consequences on oxidative stress biomarkers in the blood, plasma, and erythrocytes of horses. It led to significant increase of TBARS level in the blood, while in erythrocytes and plasma does not. The significant increase of TBARS level in the blood is a result of exercise-induced oxidative stress. This difference in TBARS level between rest and training periods most likely is a consequence of differing levels of oxidative stress occurring in the tissues and the blood. No significant changes of aminotransferases activities, lactate and pyruvate levels were noted. This may indicate a normal course of aerobic-anaerobic glycolysis in horses during distance race. Dependencies
between oxidative stress and metabolic enzymes activity, as well as lactate and pyruvate level in the blood of horses before endurance race confirm a main role of pyruvate level to assess the adaptive reaction of organism to exercise-induced oxidative stress in horses. Correlations between TBARS level, transaminases, as well as pyruvate concentration in the blood of horses indicate the relationship between oxidative stress and metabolic reactions that control key ways of renovation energy substrates during physical activity.
Key words: oxidative stress, aminotransferases, lactate dehydrogenase, lactate, pyruvate, metabolism.
Introduction. Exercises remains a key aspect of improving of endurance and performance for horses involved to recreational riding and endurance race (Hinchcliff and Geor, 2008). Endurance competitions are extremely difficult from a metabolic point of view, and for this reason, they are subjected to very strict veterinary controls to spare the horse's health. In overview of 7117 starts in international (Eldric) races, only 50 % of the subjects completed the ride, and 30 % were eliminated: 63 % because of lameness, 24 % for metabolic reasons, and 13 % for other causes (Bargero et al., 2005). For this reason, the correct metabolic management of the endurance horse is of the utmost importance, together with the correct prevention and treatment of disease (Bargero et al., 2005). Exercise induces a multitude of physiological and biochemical changes in blood that may ultimately induce of oxidative stress (Urso, 2003). 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 (Urso, 2003; Hinchcliff and Geor, 2008). For this reason, the correct interpretation of the metabolic changes after endurance exercise of horses is required, because it can help the veterinarian, trainer, or owner to choose appropriate training and post-exercise recovery. Thus, the aim of this study was to appraise the alterations in some biochemical parameters of endurance horses after 32 km distance race.
Material and methods of research. Seven horses in Crimean region (Bilohirsk, Crimean region, Ukraine) were involved in our study. All horses participate in endurance race. Horses were subjected of herd maintenance with feeding (hay and oat) provided twice a day and water available ad libitum. All horses were thoroughly examined clinically and screened for hematological, biochemical and vital parameters, which were within reference ranges. All horses were participated in endurance 32 km. Blood samples were collected from the jugular vein of horses into EDTA tubes at 2 time points: baseline at rest and after endurance race. The level of lipid peroxidation was determined by quantifying the concentration of 2-thiobarbituric acid reacting substances (TBARS) with the Kamyshnikov (2004) method for determining the malondialdehyde (MDA) concentration.
Assays of Alanine aminotransferase (ALT, E.C. 2.6.1.2) and Aspartate aminotransferase (AST, E.C. 2.6.1.1) activities was analyzed spectrophoto-
metrically by standard enzymatic method (Reitman and Frankel, 1957). The colorimetric method of Sevela and Tovarek (1959) was used for the determination of lactate dehydrogenase (LDH, E.C. 1.1.1.27). Lactate and py-ruvate concentrations were measured according to the procedure described by Herasimov and Plaksina (2000).
Results were 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 (Zar, 1999). All statistical analyses were performed using STATISTICA 8.0 software (StatSoft, Poland).
Results and Discussion. Lipid peroxidation is a complex phenomenon involving the generation of many products. However, the content of MDA, one of most important end-products of lipid peroxidation, in the tissues is usually accepted as an index of lipid peroxidation intensity (Urso and Clarkson, 2003). The lipid peroxide before and after 32 km endurance race was measured through analysis of the TBARS level and shown in Fig. 1.
F i g. 1. The level of lipid peroxidation determined by quantifying of 2-thiobarbituric acid reactive substrates (TBARS) concentration (^mol MDAL-1) in the blood, erythrocytes and plasma of horses before and after 32 km distance race
* The level of significance is set at p<0.05, paired samples by Wilcoxon signed-rank test.
TBARS level in the blood of horses showed a significant increase (by 88 %, p<0.05) immediately after exercise as compared to period before riding. The increase in blood TBARS level in endurance horses after 32 km distance race could be attributed to oxidative damage owing to free radicals being produced as a consequence of exercise. Our results are in agreement with a previous studies (Chiaradia et al., 1998), where TBARS level in blood of horses was higher after exhaustive or strong acute exercise. Gondim et al.
(2009) reported significant rise of TBARS level in the blood of horses after multiday 210-km endurance race. However, TBARS level remained at the same levels throughout 3 days of competition (Gondim et al., 2009).
Measurement of values of liver biomarkers (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 (Hinchcliff and Geor, 2008). The results of the biochemical parameters in examined horses are presented in Fig. 2.
F i g. 2. Biochemical parameters in the blood of endurance horses before and after 32 km distance race
In our research, non-significant differences of AST, ALT and LDH activities in endurance horses horse after race were observed (Fig. 2). The regular training lead to adaptive processes which provoke to changes in haematological and biochemical indices. The extent of changes depends on several factors: type of exercise, intensity of work (strength, duration and frequency) and individual variation (Hinchcliff and Geor, 2008). Physiological increases of ALT and AST have been shown to occur without any tissue destruction (Hinchcliff and Geor, 2008). Aspartate aminotransferase is a cytoplasmic and mitochondrial enzyme that catalyzes the deamination of aspartate to form oxaloacetate, which can enter the Krebs cycle (Andrews, 1995). Increases in plasma AST activity may be due to hepatocyte damage, muscle damage, or in vitro hemolysis (Andrews, 1995). ALT is a pyridoxal-dependent enzyme that catalyzes the reversible transamination of L-alanine and a-ketoglutarate to form pyruvate and L-glutamate (Hinchcliff and Geor, 2008). The extent of changes in AST, ALT and LDH activities depends on the nature of the exercise (Hinchcliff and Geor, 2008). Bashiri et al. (2010) found that resistance training for two months leads to non-significant increase in serum ALT and AST levels in non-athlete students (Bashiri et al.,
2010). Therefore, non-significant changes in ALT and AST suggests a special form of adaptation, and if exercise stress is followed by proper recovery, it will prevent more damage to the liver and muscles (Bashiri et al., 2010). LDH catalyzes a redox reaction, the reversible conversion between pyruvate and L-lactate. L-lactate formation consumes reduced nicotinamide adenine dinucleotide (NADH2) and generates oxidized nicotinamide adenine dinucleotide (NAD+), whereas NADH2 is produced during the oxidation of L-lactate to pyruvate. LDH converts pyruvate, the final product of glycolysis to lactate when oxygen is absent or in short supply, and it performs the reverse reaction during the Cori cycle in the liver (Andrews, 1995). In our study, non-significant changes in LDH activity in endurance horses after race were noted (Fig. 2). This may indicate a normal course of aerobic-anaerobic glycolysis in horses under the influence of distance race.
Lactate is widely used to examine the effects of training and diagnose positive performance and to assess the level of fitness in sport horses (Hinchcliff and Geor, 2008). Pyruvate can improve exercise endurance capacity, effectively reduce cholesterol, and serves as a potent antioxidant. Therefore, the measurement of pyruvate concentration can give the valuable information to progress of specific biochemical reactions (Olek et al., 2014). The lactate and pyruvate concentrations in the blood of endurance horses are presented in Fig. 3.
F i g. 3. The levels of lactate and pyruvate in the blood of endurance horses before and after 32 km distance race
Insignificantly decrease of pyruvate level by 28 % (p>0.05) after endurance race were observed (Fig. 3). Pyruvate is a key intermediate in cellular metabolic pathways (Olek et al., 2014). It is produced from glucose in the cytoplasm of skeletal muscle cells by the glycolytic pathway. Pyruvate is either metabolized into acetyl-CoA, or is converted into lactate, depending
on the cytosolic redox state. Pyruvate also interacts with the amino acids alanine, glutamine, and glutamate and the decline in pyruvate production could affect tricarboxylic acid cycle flux as well as gluconeogenesis (Olek et al., 2014). Therefore, pyruvate participates in several various reactions and plays a key role in energy metabolism. Insignificantly decrease of py-ruvate concentration after the endurance race indicates about the level of anaerobic glycolysis contribution to the total energy supply of muscle activity in horses involved in racing.
Correlative dependencies between oxidative stress and biochemical parameters, as well as metabolic enzymes activity in the blood of endurance horses before and after distance rice are presented in Figs 4 and 5. Before endurance rice, blood TBARS level is determined by lactate (r=0.925, p=0.003) and pyruvate concentration (r=0.841, p=0.018) (Fig. 4).
TBARS (blood):Lactate: y = -0.052 + 0.033*x; r = 0.925; p = 0.003; r2 = 0.856 TBARS (blood):Pyruvate: y = -0.084 + 0.i33*x; r = 0.841; p = 0.018; r2 = 0.708
F i g. 4. Correlative dependencies between TBARS level and concentration of lactate and pyruvate in the blood of horses before endurance rice
After endurance rice, plasma TBARS level is determined by AST activity (r=0.835, p=0.019) and lower pyruvate level (r=-0.760, p=0.047) (Fig. 5A). Pyruvate level is determined by TBARS level in erythrocytes (r=-0.884, p=0.008) and plasma (r=-0.760, p=0.047) (Fig. 5B).
Dependencies between oxidative stress and metabolic enzymes activity, as well as, lactate and pyruvate level in the blood of horses before and endurance race confirm a main role of pyruvate to assess the adaptive reaction of organism to exercise-induced oxidative stress in horses.
TBARS (p!asma):AST: y = 1.131 + o.4o6*x; r = 0.835; P = 0.019; r2 = 0.698 TBARS (plasma):Pyruvate: y = 4.561 - 0.412X; r = -0.760; p = 0.047; r2 = 0.578
A
Pyruvate:TBARS (Erythrocytes): y = 51,415 - I0,0 2*x; r = -0,884; P = 0,008; r2 = 0,782 Pyruvate:TBARS (Plasma): y = 8.782 - L403*x; r = -0.760; p = 0.047; r2 = 0.578
P yruvate
B
F i g. 5. Correlative dependencies between plasma TBARS level, AST activity and pyruvate concentration (A), between pyruvate concentration and TBARS level in erythro-cytes and plasma after endurance rice (B)
Therefore, the pyruvate participates in several various reactions and plays a key role in energy metabolism. Pyruvate acts as indicator and a measure of aerobic conditions (based on the presence of oxygen) in the liver
and muscle tissues as in both tissues, it may operate according to physiological requirements and presence of molecular oxygen in both aerobic and anaerobic conditions.
Conclusions. Our results suggest that endurance race leads to different consequences on oxidative stress biomarkers in the blood, plasma, and erythrocytes of horses. The increase in blood TBARS level in horses after distance race could be attributed to oxidative damage owing to free radicals being produced as a consequence of endurance exercise. Based on these results, it is concluded that the endurance exercises lead to specific metabolic changes accompanied by a redistribution of energy supply for improving of resistance to exercises and athletic performance of horses.
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