Bull Vet Inst Pulawy 53, 97-103, 2009
EFFECT OF PHYSICAL EXERCISE ON OXYGEN METABOLISM OF NEUTROPHILS IN HORSES WIESŁAW KRUMRYCH Department of Pathophysiology of Reproduction and Mammary Gland, National Veterinary Research Institute, 85-090 Bydgoszcz, Poland
[email protected] Received for publication October 24, 2008
Abstract The study included 114 clinically healthy horses representing different groups: breeding horses (27), recreation horses (22), and sport horses (65). The group of sport horses consisted of racehorses (11), trotters (15), jumping horses (25), and driving horses (14). The peripheral blood samples collected three times: before exercise, immediately after exercise, and after 30-min rest were examined for the activity of oxygen metabolism of neutrophils using chemiluminescence (CL). The study demonstrated a temporary post-exercise intensification of free radical processes in sport horses. The intensity of this reaction depended on the intensity and duration of the exercise workload, which was confirmed by the changes in the heart rate and breathing. The analysis of the results of pre-exercise examination demonstrated clearly higher CL values of neutrophils in horses trained regularly and intensively than in animals of small physical activity. This result proves a positive influence of regular training on oxygen-dependent bactericidal activity of neutrophils.
Key words: horses, physical exercise, neutrophils, oxygen metabolism, respiratory burst. Physical exercise is a commonly recognised stress factor, which can modify the immunological response. Numerous studies, carried out mainly in humans, demonstrate that moderate physical activity stimulates defence mechanisms whereas excessive effort decreases their effectiveness as a result of the change of the number of immunocompetent cells in blood and also impairs their functional activity (21, 24). It is believed that these reactions may result from an increase in neurohormonal factors (cortisol, catecholamines, βendorfins, growth hormone, sex steroids) and changes in the concentration of metabolic factors (glutamine, glucose, lipids) in blood (11, 12, 20, 24). Despite significant differences in the scope of effort changes taking place in humans and horses, it seems that immunomodulatory influence of physical effort is similar. The greatest attention in this respect was devoted to the evaluation of the values of immunological factors in horses subjected to intensive and exhausting exercise e.g. during long distance endurance rallies. Their effect is a significant postexercise leukocytosis and the so-called “stress leukogram” characterised by an increase in the number of neutrophils and lymphopoenia determined mainly by lower participation of T CD4+ and NK cells (13, 29). These reactions are accompanied by the impairment of the function of cells involved in the immune system that manifests by a temporary decrease in phagocytic and bactericidal activity of neutrophils, monocytes, and pulmonary macrophages, lower values of chemotactic
index of neutrophils, impairment of lymphocyte proliferation and cytotoxic activity of NK cells, and also lower titre of antibodies after vaccination against influenza (8, 11, 13, 14, 27, 29). A moderate physical effort was the subject of relatively few studies in horses. However, the obtained results entitle one to state that small exercise workload may induce a stimulating effect in the number and functional activity of immune system cells (14, 27). Previous studies on effort induced immunomodulation were carried out in the course of experimental effort tests and participation of horses in sport events. It seems, however, that these loads do not fully reflect the everyday work of these animals. What is more, the presented results were limited to only one type of horses, which in combination with different laboratory methods used made it difficult if not impossible to compare them. The aim of the study was to evaluate the oxygen metabolism of neutrophils during every day, standard physical exercise of recreation and competitive horses representing different sports.
Material and Methods The study was conducted on 114 horses aged 2 to 16 years and included: competitive sport horses (S) representing different sports (races – Ra, trotters races – T, obstacle jumps – J, and one-horse cart driving – D), recreation horses (R), and breeding horses (B). A more
98 detailed characteristic of the horses is presented in Table 1. The elimination of potential influence of endogenous factors such as breed, age, and sex was not possible due to the specificity of the use of particular groups of the horses. The animals came from seven training centres, two recreation centres, and one breeding farm. The data collected during an interview directly before the beginning of the research procedure, clinical examination (rectal temperature, heart and breathing rates, appearance of the mucous membranes, and filling time of capillary vessels), as well as results of haematological and biochemical examinations obtained ex post indicated that the animals were healthy. They were subject to regular prophylactic treatment including deworming and vaccination against influenza and tetanus. The horses’ diet was balanced in respect of energy-protein, minerals, and vitamins according to dietary norms prepared for these animals (19). The horses had a permanent access to mineral supplements and water. The conditions in all studied centres were similar and fulfilled the requirements concerning their welfare. The horses participated in a daily training programme from 6 months (T) to several years (J and D), which depended on the type of sport discipline. Recreation animals were used in the spring-summer season on a rather irregular basis. An average time of their daily work was 2-3 h during which the horses were ridden mainly by young people learning to horse ride. The physical activity of control horses (B) was much more limited and consisted mainly in spending time on
Groups of horses (n) B (27) R (22)
S (65)
pastures and paddocks for 8-10 h a day. The realisation of the subject of the study was based on the exercise workload the form, time, and intensity of which depended on the trainers. The interference in their work was minimised and came down to running clinical tests and collecting blood samples. The general characteristic of the physical effort of the horses representing different groups is presented in Table 2. The study was carried out in summer (from June to September) in the horses’ permanent place of living. The average air temperature during the study was 21.8 ±4.2°C whereas humidity, atmospheric pressure, and wind were 40-75%, 998-1016 hPa, and 3-5.5 m/s, respectively. The horses exercised on an even and sandy ground. The measurements of the heart and breathing rates were used to evaluate the intensity of the effort. Blood samples were collected from the external jugular vein using Vacuette system three times: before exercise – I (in the stable, before saddling or harnessing and at least an hour after feeding), immediately after exercise – II, and after 30-min rest (III), which included relaxation of the horses. Every time, the blood samples were collected into two plastic tubes: 9 ml tubes containing lithium heparin and 4 ml containing potassium versatate (K2EDTA). In order to eliminate the influence of the daily rhythm on the neurohormonal activity, among other blood cell count and their function, the first blood samples were collected at the same time (9:00 ±30 min) and then stored at 4°C. Laboratory assay was performed no later than 5 h after the collection of the blood.
Table 1 The characteristics of the horses according to their use Subgroups of Age Sex Breed horses (n) (years) (n) (n) 6.41 Mares (17) Half-breed (27) (3-16) Stallions (10) Wielkopolska (14) 8,36 Mares (9) Welsh pony (5) (4-15) Geldings (13) Haflinger (3) Mares (4) Ra 4.09 English Thoroughbred (11) Stallions (5) (11) (3-5) Geldings (2) T 2.13 Mares (9) Standard bred (15) (15) (2-3) Stallions (6) KWPN (15) Mares (5) Hanoverian (6) J 8.44 Stallions (14) Oldenburg (3) (25) (6-14) Geldings (6) Selle Francais (1) Mares (2) Śląska (11) D 7.50 Stallions (4) KWPN (3) (14) (4-11) Geldings (8)
Explanation: n – number of horses; B – breeding horses; R – recreation horses; S – sport horses; Ra – racehorses; T - trotters; J – jumping horses; D – driving horses; KWPN – Dutch Warmblood (Koninkliijke Vereniging Warmbloed Paardenstamboek Nederland)
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Horses R Ra T S J D
Table 2 General characteristics of standard exercise of horses from different groups Time (min) Type of physical exercise A B Walk (10 min), trot alternated with walk (25-30 min), gallop (5 min), 10-15 55-60 trot (5 min), walk (10 min) Walk (1 min), trot (5 min), gallop (200 m at the pace 13-14 m/s), trot 2-3 12-15 (5 min) The training of driving horses included: walk (200 m), trot (3,000 m at 7 m/s), walk (400 m), trot (1,400 m at 7 m/s), trot (400 m at 11 12-18 20-30 m/s), trot (1,200 m at 7 m/s), trot (400 m at 11 m/s), trot (300 m at 7 m/s) and walk (200 m) The training included initial phase: walk, trot and gallop alternated 20-30 50-60 with trot (for 10 min) and intensive phase (30-40 jumps over obstacles 120-140 cm high during 20-30 min) Driving one-horse carts: walk (500 m), trot (5,000 m at 4.2 m/s), 30-40 40-50 walk (1,000 m), trot with three obstacles (3,000 m at 3.9 m/s)
Explanation: A – intensive phase; B – whole exercise session
The evaluation of oxygen metabolism of neutrophils was done in whole blood (collected into tubes containing lithium heparin) using chemiluminescence (CL) with luminol (5-amino-2,3dihydro-1,4-phthalazinedione) diluted in 0.4% solution of NaOH to a concentration of 28 µmol/mL. The assessment was performed using Luminometr BioOrbit 1251 (Pharmacia LKB, Finland) by kinetic method for 40 min at 38.0 ±0.1°C measuring CL at 5-min intervals. The results were presented as the value of CL integration that is area under the curve of emission against the function of time (mV/min). Both spontaneous luminol dependent (without stimulation – WS) and stimulated chemiluminescence was determined. The following CL stimulators were used: - opsonised zymosan (OZ): 100 mg of zymosan suspended in PBS (10 ml) and equine plasma (10 ml), - N-formyl-methionyl-leucyl-phenylalanine (fMLP): 5 mg of fMLP dissolved in 4.56 ml of dimethyl sulphoxide (DMSO) and diluted in PBS to 2×10-6 mol/L, and - phorbol myristate acetate (PMA): 5 mg of PMA dissolved in 5ml of 95% ethanol and then diluted in PBS to 0.32 µmol/L. The examined sample contained 200 µl of PBS or 100 µl of PBS+100 µl of stimulator, 100 µl of luminol and 150 µl of whole blood collected into a tube with heparin. The tests started immediately after the addition of blood to the prepared reagents. All measurements were repeated three times during one test and arithmetic mean value was calculated. Due to the fact that CL value is inversely proportional to the concentration of haemoglobin (Hb), whose spectrum of light absorption weakens chemiluminescence, and directly proportional to the number of neutrophils (N) in the sample, the obtained results were corrected relating CL value to 1,000 cells. In the blood collected into tubes containing K2EDTA, Hb concentration was determined as well as white blood cells count (WBC) and percentage of N. The assay of
Hb and WBC was performed by half automatic method using haematological analyser Sysmex F800 (TOA Medical Electronics Co. Ltd, Japan) whereas the percentage of N was determined by microscopic analysis of blood smears using May-Grünwald-Giemsa stain. Optimisation of the results was done according to the formula taking into account the volume of the blood sample (15): CL calculated =
CL measured × Hb
( WBC × N × 150 ) / 100 Hb – haemoglobin content in % (in relation to the mean value calculated for the particular group of horses), WBC (×103/µl) – absolute value of white blood cells count, N – percentage of neutrophils in the white blood cell picture, 150 – volume of blood in µl. The obtained results are presented as arithmetic mean ( x ) and standard deviation (±SD). The significance of the differences between mean values was verified using Tukey test assuming the differences to be significant if their probability was below 5%. All statistical analyses were performed using Statistica v. 6.0 StatSoft software.
Results The results of the measurements of the heart and breathing rates are presented in Table 3. Their analysis demonstrated post-exercise increase of the values of the two indices in all groups of the animals. Only in the group of recreation horses (R) the heart rate was not confirmed statistically. The biggest increase of the values of these indices was found in driving horses (D), which seemed to have the highest workload (Table 2). The observed changes were temporary and shortlived because after 30-min rest there was a considerable decrease in the heart and breathing rates towards the values before the effort. It is worth noting that the difference between I and III test for the above indices was statistically significant only in driving horses (D).
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Table 3 Mean values ( x ± sd) of the heart rate and breath rate in the course of physical exercise of different groups of horses Heart rate Breath rate Horses Sampling ( /min) ( /min) Breeding 15.44 ± 4.06 36.59 ± 4.96 (n=27) I 34.36 ± 5.51 16.36 a ± 4.03 Recreation II 43.45 ± 6.21 35.18 b ± 10.32 (n=22) III 19.55 a ± 3.32 37.55 ± 6.47 a I 36.73 ± 8.78 17.64 a ± 4.37 Ra b II 82.73 ± 15.50 67.82 b ± 10.60 (n=11) a III 38.73 ± 5.68 28.18 a ± 5.96 a I 35.87 ± 5.78 19.33 a ± 5.43 S T b II 88.27 ± 11.83 62.40 b ± 13.25 p (n=15) a III 39.20 ± 6.13 21.73 a ± 5.28 o a r I 34.56 ± 7.31 16.24 a ± 4.67 J b t II 72.08 ± 13.54 83.44 b ± 22.25 (n=25) a III 38.80 ± 7.51 22.56 a ± 7.20 a I 33.57 ± 7.32 14.86 a ± 2.80 D b II 104.86 ± 20.85 109.57 b ± 22.05 (n=14) c III 48.57 ± 6.20 30.43 c ± 5.88 Explanation: x - arithmetic mean; sd – standard deviation; n – number of horses; Ra – racehorses; T - trotters; J – jumping horses; D – driving horses; I, II, III – time of blood collection (before, immediately after exercise and after 30 min rest); a,b – mean values differ significantly for P
