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Keywords:

  • horse;
  • aldosterone;
  • angiotensin;
  • exercise;
  • exhaustion;
  • renin;
  • vasopressin

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Conflicts of interest
  10. Manufacturers' addresses
  11. References

Reason for performing study: Limited information exists concerning the defence of homeostasis during endurance competitions and the relationship with performance.

Objectives: This research analysed renin (REN), angiotensin II (ANG), aldosterone (ALD) and vasopressin (AVP) in horses covering different distances, assesses differences between successful and eliminated horses and evaluates correlations between hydration status, renal function, electrolytes, REN, ANG, ALD and AVP.

Methods: Packed cell volume (PCV), velocity and serum concentrations of REN, ANG, ALD, AVP, Na, K, Cl, Ca, Mg, P, creatine kinase, aspartate aminotransferase, lactate dehydrogenase, total proteins (TSP), albumin (ALB), serum ureic nitrogen (SUN), creatinine (CREAT) and lactate were analysed in both successful horses (SH) and in horses eliminated due to metabolic problems (MH). Two types of competition were studied: 91 km in one day (Competition A: 20 SH, 9 MH) and 166 km in 2 days, 83 km/day (Competition B: 10 SH and 5 MH).

Results: Research analysed renin was not affected by exercise, whereas ANG, ALD and AVP increased. In the SH group, resting ALD and AVP concentrations at the beginning of the second day of Competition B were higher than preride values. Vasopressin did not change during the second day of Competition B, whereas ALD progressively increased. Metabolic problems of both groups showed more evident dehydration (higher PCV, TSP, ALB, SUN and CREAT) and electrolyte alterations (more intense decreases of Na and Cl) than SH at the different sampling times. Metabolic problems presented higher ALD and AVP concentrations. Angiotensin II was higher at certain sampling times in the horses.

Conclusions: Endurance horses with dehydration and electrolyte disturbances showed a more intense activation of the REN-ANG-ALD-AVP axis.

Potential relevance: The study of the response of the REN-ANG-ALD-AVP axis during prolonged exercise in horses with different performance will aid to minimise the risk of metabolic diseases during competitions.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Conflicts of interest
  10. Manufacturers' addresses
  11. References

Endurance horses are able to perform prolonged submaximal exercise by increasing their metabolic rate by 10- to 20-fold, with the production of important amounts of energy. Approximately 70–80% of this energy is eliminated in the form of heat to avoid substantial core body temperature increases. The main mechanisms of thermolysis in the horse are sweat evaporation from the skin (65%) and respiratory evaporation (25%) (Hodgson et al. 1993), with sweating rates of 10–15 l/h in hot environmental conditions. As a result of sweating, water is lost mainly from the extracellular fluid, resulting in decreases in blood and plasma volumes (Hyyppa et al. 1996; Flaminio and Rush 1998). Therefore, clinical and laboratory evidence of a mild to moderate dehydration are common in successful endurance horses, although more intense dehydration might have negative consequences on both performance and health. Hypovolaemia reduces perfusion to skeletal muscles and other vital organs, with inefficient oxygen and substrate utilisation. Additionally, severe dehydration compromises heat dissipation. In these situations, if exercise is continued, the thermoregulatory processes will be overwhelmed with severe consequences for the health of the equine athlete. Another consequence of sustained sweating is electrolyte loss, as equine sweat is isotonic or slightly hypertonic in relation to plasma and contains high concentrations of Na, K and Cl and some Ca and Mg (Flaminio and Rush 1998). Prolonged sweating will cause significant electrolyte deficits promoting weakness, muscle cramps, acid-base imbalances, heart arrhythmias, decreased performance and eventually exhaustion (Foreman 1998).

All these factors would result in significant changes in internal homeostasis, blood volume, mean arterial pressure and plasma tonicity. There are several neuroendocrine mechanisms involved in the acute and chronic defence of internal homeostasis, which act to ensure an adequate blood flow to the working muscles and other vital tissues, together with the provision of a proper fluid volume for sweating and thermoregulation. The changes in blood flow lead to an increase in renin (REN) release from the juxtaglomerular apparatus of the kidney (McKeever et al. 1991, 1992). This enzyme acts on circulating angiotensinogen, which is converted to angiotensin I and then to angiotensin II (ANG), which stimulates the production of aldosterone (ALD) from the adrenal cortex. ALDacts in the conservation of Na in the extracellular fluid and enhances Na and Cl absorption from the colon (Clarke et al. 1992). Moreover, the antidiuretic hormone or arginine vasopressin (AVP), secreted by the posterior pituitary gland, is an important determinant of water excretion, as it induces the kidney to retain water. Furthermore, ANG, ALD and AVP promote vasoconstriction and thereby elevate blood pressure (McKeever et al. 1991, 1992; McKeever 1998; Muñoz et al. 2010a,b).

The role of these neuroendocrine factors during different types of exercise has been investigated. McKeever et al. (1991) reported significant increases in plasma REN activity, ALD and AVP during a one hour steady-state treadmill submaximal exercise. Similarly, significant increases in REN, ALD and AVP were detected during an incremental maximal exercise test (McKeever et al. 1992). Equine endurance competitions represent a challenge for the internal homeostasis and therefore, increased concentrations of REN, ANG, ALD and AVP are expected. However, to the authors' knowledge, the evolution of these parameters during an endurance competition has not been studied yet, with exception of ALD (Schott et al. 1997).

This study has been designed with 3 main objectives: 1) to describe the changes in serum REN, ANG, ALD and AVP concentrations in endurance horses covering different distances; 2) to assess whether there are significant differences in these neuroendocrine factors when comparing successful horses with those eliminated due to exhaustion, dehydration and/or lack of heart rate (HR) recovery; and 3) to evaluate the differences in laboratory parameters according to performance and to analyse the associations between laboratory markers of dehydration, renal function, electrolytes and concentrations of REN, ANG, ALD and AVP.

Previous studies have demonstrated that changes in electrolytes and hydration markers are similar in successful endurance horses covering different distances (Barton et al. 2003; Castejón et al. 2006). On the other hand, if the hypothalamus-hypophysis-adrenal axis is activated in face of a dehydrated state, it is plausible to think that the exhausted horses would experience a more intense release of REN, ANG, ALD and AVP. Additionally, it has been shown that the administration of desmopressin, an AVP analogue, would lead to prolonged running performance in hot and humid environments in human athletes (Ftaitli et al. 2001). Taking into account these ideas, we hypothesised that, firstly, no significant differences will exist in REN, ANG, ALD and AVP concentrations in endurance horses competing over different distances; secondly, the endurance horses eliminated due to dehydration and/or lack of HR recovery would experience a greater release of these neuroendocrine factors and thirdly, horses with metabolic disorders would present higher dehydration and significant correlations would exist between laboratory markers of hydration, renal function and electrolytes and the concentrations of REN, ANG, ALD and AVP.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Conflicts of interest
  10. Manufacturers' addresses
  11. References

Horses and competitions

Data were obtained from horses that took part in 2 types of endurance competitions: A (91 km/day; n = 29) and B (166 km in 2 days; 83 km/day; n = 15). According to their performance, the animals were divided into 2 groups: successful horses (SH), when they finished the competitions and were considered fit in the last vet-gate or veterinary inspection and metabolic horses (MH), when they were eliminated from competition due to exhaustion, dehydration and/or lack of HR recovery. From Competition A, 20 were SH and 9 were MH and from Competition B, 10 were SH and 5 were MH. The horses were of different breeds, mainly Arabian and cross-Arabian, aged 6–14 years and of both sexes. All were in good health and no significant problems were found in the physical examination at rest, before the competition. These animals had a broad range of previous competitive experience and were subjected to different diets and protocols of electrolyte supplementation, both during training and competition. Consent was obtained from the owners or riders of the horses before enrolling in the research.

The studied races followed FEI rules and were held in the south of Spain. Competition A consisted of 3 loops of 35, 28 and 28 km and were carried out on a flat, firm terrain of good quality. Competition B was 3 loops each day, of 27, 35 and 21 km. The terrain was variable, with some muddy and some firm places and slight elevation changes (± 300 m). Environmental conditions during the competitions varied, with mean temperatures of 22.5°C (range 12.50–26.5°C) and mean relative humidity of 73.20% (range 52–96%). These data were obtained from the closest meteorological station to the event venue. Maximum HRs allowed were 56 and 64 beats/min for Competitions A and B, respectively.

The veterinary examinations were performed the day before the competitions and after the completion of each loop. These inspections followed the ‘vet-gate’ method and consisted of HR, metabolic and locomotor examinations. After each vet-gate, the horses had a mandatory rest period of 30 min.

Data, blood sampling and management

Speed during each loop was recorded for each horse. The HR recovery time (HRrt), i.e. the period between the completion of each loop, and the recovery of HR and presentation to the vet-gates was also recorded.

Jugular venous blood samples were taken at rest, the day before the competitions (R) and after each loop, immediately after the vet-gates (PH1, PH2 and PH3). Additionally, in Competition B, blood samples were also extracted during the second day, following the sample temporal protocol (R2, PH4, PH5 and PH6). A total volume of 10 ml was withdrawn in each sampling time. Immediately after extraction, blood was poured into tubes with heparin-lithium and plain tubes. From the tube with heparin-lithium, a microhaematocrit tube was filled and centrifuged and packed cell volume (PCV, %) obtained in the field. The plain tubes were centrifuged and serum harvested and kept under refrigeration (4–8°C) for approximately 24 h, during the competitions and transport. In the laboratory, the samples were frozen at −20°C until analysis, which was performed within 2 months after the events.

Laboratory analysis

The following parameters were determined by spectrophotometry (Spectrophotometer Termo Spectronic)1, using specific reagents1: total serum proteins (TSP, g/l), albumin (ALB, g/l), serum ureic nitrogen (SUN, mg/l), creatinine (CREAT, mg/l), creatin kinase (CK, iu/l), aspartate aminotransferase (AST, iu/l), lactate dehydrogenase (LDH, iu/l), total Ca (mg/l), P (mg/l), Mg (mg/l) and lactate (LA, mmol/l) concentrations. Serum Na, K and Cl concentrations (mmol/l) were measured with an analyser with selective electrodes for each electrolyte (Vetlyte)2.

Serum REN concentrations (pg/ml) were measured with a sandwich immune-enzyme analysis for serum active REN. This assay was highly specific for REN, with percentages of recovery of known quantities of REN of 95–100%. The sensitivity of the technique was of 1 pg/ml, the intra-assay coefficient of variation (CV) <5% and the interassay CV <15%. Serum ANG concentrations (ng/ml) were determined with a competitive ELISA based on the biotin-streptavidinase-peroxidase system. The percentage of known quantities of ANG was 98.5% and the technique had a sensitivity of 100 ng/ml, with intra- and interassay CVs <5 and 14%, respectively. Serum ALD concentrations (pg/ml) were obtained with a competitive ELISA, with a percentage of recovery of known quantities of ALD of 97.6%. This method showed cross-reactions with 11-deoxicorticosterone (1.1%) and cortisone, corticosterone, 11-deoxicortisol and 21-deoxicortisol (<0.1%). The sensitivity of the assay for ALD was 15 pg/ml and the intra- and interassay CVs were 4.7–6.4% and 8.5–9.6%, respectively. Serum AVP concentrations (pg/ml) were measured with a competitive ELISA based on the biotin-streptavidinase-peroxidase system. The assay was specific for AVP (cross-reactivity with AVP, 100%; lysine vasopressin <0.1%; vasotocin <0.1% and desmopressin, DDAVP <0.1%). The sensitivity of the assay was 1.3 pg/ml, with interassay CVs ranging 3.7–10% and intra-assay CVs of 6.9%. The extraction recovery of known quantities of AVP was 100.1%. All of these techniques have been validated for horses.

Statistic procedures

Data are presented as mean ± s.e. and the level of significance was fixed at P<0.05. The normality of the parameters was checked with a Shapiro-Wilk's test and normal probability plots were also evaluated in order to assess outlier values. Serum concentrations of Cl, CK, AST, LDH, LA, ALD and AVP were not normally distributed so they were log-transformed (CK, AST, LDH, LA and ALD) and sine-transformed (Cl and AVP). The effect of the covered distance in the studied parameters for SH horses in both types of competition was analysed with a repeated-measures ANOVA. When significant differences were found between the different distances, a post hoc comparison of means (Tukey test) was made. The influence of the performance, i.e. the differences between SH and MH in each sampling time and for each competition was studied with a t test. The correlations between V, HRrt and the laboratory parameters were studied with a linear correlation (Pearson product moment correlation). The statistical software package Statistica3 was used.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Conflicts of interest
  10. Manufacturers' addresses
  11. References

Fourteen of the studied horses were disqualified due to metabolic problems, exhaustion and/or lack of HR recovery: 9 in Competition A (4 at PH1, one at PH2 and 4 at PH3) and 5 in Competition B (one at PH2, 2 at PH3, one at PH5 and one at PH6).

Effects of the distance in the SH group

Significant differences were not found when comparing values at R in Competitions A and B. On the contrary, horses in B had: in PH1 higher ALD, CK and LDH and lower AVP; in PH2, higher ALD, CK, AST and LDH; and in PH3, higher CK, AST and LDH. Because of these differences, the data of Competition A and the first day of Competition B were not processed together. Differences in speed and HRrt were not detected when comparing Competition A and the first day of B.

The effect of the exercise in both types of competitions for SH is presented in Tables 1 and 2. The changes of the 4 neuroendocrine factors in both types of competition are presented in Figures 1 and 2.

Table 1. Mean ± s.e. values for 20 endurance horses that successfully finished a 91 km competition
ParameterRPH1PH2PH3
  1. Statistical significance at P<0.05. Values with different superscripts are significantly different from each other; indicated in bold. HRrt = heart rate recovery time; PCV = packed cell volume; TSP = total serum protein; ALB = albumin; SUN = serum ureic nitrogen; CREAT = creatinine; LA = lactate; CK = creatine kinase; AST = aspartate transaminase; LDH = lactate dehyrogenase.

Speed (km/h) 15.27 ± 0.560A14.46 ± 0.673A12.75 ± 0.459B
HRrt (s) 338.2 ± 35.10A373.3 ± 63.34A542.9 ± 41.02B
Na (mmol/l)146.2 ± 0.871A143.8 ± 0.672A142.1 ± 0.766B141.6 ± 0.659B
K (mmol/l)4.147 ± 0.094A3.916 ± 0.069A3.900 ± 0.065A3.845 ± 0.093A
Cl (mmol/l)107.6 ± 0.589A104.8 ± 0.669A102.0 ± 0.762B98.85 ± 0.559B
Ca (mg/l)108.9 ± 3.72A106.8 ± 3.75A114.8 ± 3.94A113.1 ± 4.46A
P (mg/l)26.09 ± 2.27A28.23 ± 1.58A31.03 ± 2.33A34.01 ± 1.88B
Mg (mg/l)24.70 ± 2.18A26.79 ± 2.82A30.21 ± 2.73A32.39 ± 2.79A
PCV (%)37.33 ± 1.130A44.89 ± 2.037B46.44 ± 0.394B47.00 ± 1.980B
TSP (g/l)64.64 ± 0.85A70.96 ± 1.14B73.48 ± 1.19B74.59 ± 0.81B
ALB (g/l)26.33 ± 0.86A29.94 ± 1.04B30.87 ± 0.91B31.45 ± 0.97B
SUN (mg/l)252.8 ± 11.62A320.3 ± 1.405374.0 ± 18.03B427.7 ± 11.63C
CREAT (mg/l)13.80 ± 0.50A16.97 ± 0.76B18.57 ± 1.07B21.00 ± 0.76C
LA (mmol/l)0.684 ± 0.082A2.173 ± 0.287B2.191 ± 0.189B2.125 ± 0.175B
CK (iu/l)131.1 ± 7.022A175.8 ± 10.91A269.7 ± 22.10A613.9 ± 29.73B
AST (iu/l)166.4 ± 10.09A202.1 ± 13.18A218.0 ± 16.44B248.8 ± 12.09B
LDH (iu/l)342.8 ± 23.71A465.5 ± 34.02A572.0 ± 45.67B724.4 ± 55.99C
Table 2. Mean ± s.e. values for 10 endurance horses that successfully finished a 166 km competition in 2 days, 83 km/day
ParameterRPH1PH2PH3R2PH4PH5PH6
  1. Statistical significance at P<0.05. *in bold indicates significant differences with successful horses at each sampling time. For abbreviations see Table 1.

Speed (km/h) 16.82 ± 0.520A14.30 ± 1.132A14.38 ± 1.761A 15.49 ± 0.894A15.52 ± 1.130A15.52 ± 1.646A
HRrt (s) 362.5 ± 55.86A371.0 ± 24.10A488.7 ± 90.77B 352.3 ± 69.73A285.9 ± 37.66C329.4 ± 80.80A
Na (mmol/l)144.9 ± 1.545A143.0 ± 1.468A142.9 ± 0.795A141.7 ± 1.257B145.6 ± 1.842A144.6 ± 0.447A141.4 ± 1.784B139.9 ± 1.394B
K (mmol/l)3.950 ± 0.154A3.760 ± 0.114A3.830 ± 0.204A3.810 ± 0.196A4.033 ± 0.151A3.970 ± 0.162A3.720 ± 0.207A3.590 ± 0.155A
Cl (mmol/l)107.9 ± 1.370A104.7 ± 0.184A102.0 ± 1.125B99.30 ± 1.001B107.4 ± 1.573A106.2 ± 1.133A102.7 ± 0.932B98.30 ± 0.943B
Ca (mg/l)101.0 ± 4.87A103.1 ± 5.53A103.7 ± 5.35A102.9 ± 3.34A94.38 ± 7.78A95.00 ± 3.82A89.90 ± 3.25A87.60 ± 4.53A
P (mg/l)26.74 ± 2.40A36.14 ± 2.34B38.61 ± 3.06B40.27 ± 3.34B30.00 ± 1.87A30.49 ± 3.30A30.32 ± 2.97A33.63 ± 1.77A
Mg (mg/l)22.47 ± 2.73A22.37 ± 1.46A28.21 ± 3.61A24.38 ± 1.87A20.55 ± 1.84A21.62 ± 1.23A21.21 ± 1.12A20.48 ± 1.64A
PCV (%)35.00 ± 1.000A45.00 ± 4.000B41.00 ± 1.000C46.50 ± 1.000B39.00 ± 0.978A41.50 ± 1.500C42.00 ± 1.000C48.50 ± 7.060B
TSP (g/l)64.69 ± 0.88A72.41 ± 1.19B75.37 ± 0.88C72.11 ± 1.49B64.66 ± 1.24A67.36 ± 1.80A67.61 ± 1.69A68.77 ± 1.52A
ALB (g/l)28.45 ± 2.00A32.70 ± 1.63B32.92 ± 1.47B33.66 ± 2.17B26.73 ± 2.02A28.66 ± 2.63A31.05 ± 2.41A31.48 ± 2.48B
SUN (mg/l)250.6 ± 19.00A340.0 ± 22.70A379.3 ± 22.07B451.1 ± 25.98C319.7 ± 17.39A379.8 ± 36.20B400.3 ± 37.13B434.3 ± 41.08C
CREAT (mg/l)12.56 ± 0.28A16.25 ± 0.93A21.50 ± 1.83B21.57 ± 1.74B14.19 ± 0.97A16.71 ± 1.02A18.63 ± 1.23A20.29 ± 2.20B
LA (mmol/l)1.143 ± 0.258A1.852 ± 0.347B1.812 ± 0.329B1.988 ± 0.326B0.996 ± 0.136A1.096 ± 0.116A1.266 ± 0.148A1.681 ± 0.346B
CK (iu/l)155.2 ± 18.43A893.4 ± 534.9B2098 ± 1517C2432 ± 1590D408.6 ± 157.3E585.6 ± 117.6F1087 ± 258.0B1309 ± 290.7G
AST (iu/l)195.1 ± 18.40A266.9 ± 27.51A388.5 ± 88.07B403.4 ± 90.31B330.5 ± 82.98A336.6 ± 45.91A437.3 ± 77.19B435.8 ± 92.33B
LDH (iu/l)346.0 ± 37.85A646.7 ± 67.57B906.2 ± 236.2C1035 ± 166.4D613.4 ± 71.55B692.8 ± 63.48B861.2 ± 78.01C970.5 ± 80.75C
image

Figure 1. Mean ± s.e. values of renin, angiotensin, aldosterone and arginine vasopressin concentrations in endurance horses during a 91 km competition (SH: successful horses, n = 20; MH: horses disqualified because of exhaustion, dehydration or lack of recovery of heart rate; at R, n = 9; at PH1, n = 9; at PH2, n = 5; at PH3, n = 4) (values with the same superscripts are not significantly different from each other; capital letters: SH; lower case letter: MH) (* significant differences between SH and MH in each sampling time). Statistical significance at P<0.05.

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image

Figure 2. Mean ± s.e. values of renin, angiotensin, aldosterone and arginine vasopressin concentrations in endurance horses during a 166 km competition in 2 days, 83 km/day (SH: successful horses, n = 10; MH: horses disqualified because of exhaustion, dehydration or lack of recovery of heart rate; at R, n = 5; at PH1, n = 5; at PH2, n = 5; at PH3, n = 4; at R2, n = 2; at PH4, n = 2; at PH5, n = 2; at PH6, n = 1) (values with the same superscripts are not significantly different from each other; capital letters: SH; lower case letter: MH) (* significant differences between SH and MH in each sampling time). Statistical significance at P<0.05.

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Differences between SH and MH

The data for the MH in both types of competition are presented in Tables 3 and 4, indicating the differences with SH. The concentrations of REN, ANG, ALD and AVP in endurance horses with different physical performance (SH vs. MH) for both competition types are presented in Figures 1 and 2.

Table 3. Mean ± s.e. values for 9 endurance horses that were disqualified during a 91 km competition because of exhaustion, dehydration or lack of recovery of heart rate
ParameterR (n = 9)PH1 (n = 9)PH2 (n = 5)PH3 (n = 4)
  1. Statistical significance at P<0.05. *in bold indicates significant differences with successful horses at each sampling time. For abbreviations see Table 1.

Speed (km/h) 14.75 ± 0.84114.71 ± 0.59812.77 ± 0.330
HRrt (s) 383.6 ± 88.62357.5 ± 25.40900.0 ± 85.40*
Na (mmol/l)149.1 ± 0.676*146.0 ± 2.958145.6 ± 1.568*144.5 ± 1.190
K (mmol/l)4.400 ± 0.1203.889 ± 0.9863.800 ± 0.0953.525 ± 0.225
Cl (mmol/l)105.8 ± 0.722104.2 ± 0.142101.6 ± 0.748100.0 ± 1.000
Ca (mg/l)114.4 ± 10.05100.6 ± 9.69115.0 ± 10.25110.5 ± 9.61
P (mg/l)22.01 ± 1.4324.46 ± 6.5531.04 ± 3.8535.58 ± 3.74
Mg (mg/l)24.75 ± 2.2520.71 ± 2.2325.41 ± 1.8728.80 ± 4.17*
PCV (%)42.00 ± 3.24051.50 ± 3.841*51.00 ± 4.000*55.00 ± 3.000*
TSP (g/l)64.64 ± 1.7576.36 ± 1.47*76.12 ± 3.3185.05 ± 0.70*
ALB (g/l)24.92 ± 1.1732.33 ± 2.7132.28 ± 2.3435.85 ± 3.24
SUN (mg/l)245.9 ± 21.71416.3 ± 2.99*459.0 ± 34.77*544.9 ± 21.12*
CREAT (mg/l)15.59 ± 0.7225.22 ± 33.35*25.40 ± 2.23*34.10 ± 1.53*
LA (mmol/l)1.013 ± 0.1142.477 ± 0.2342.144 ± 0.2733.329 ± 0.394*
CK (iu/l)163.0 ± 10.45*267.6 ± 58.37*541.0 ± 70.86*802.8 ± 408.4*
AST (iu/l)187.6 ± 32.31221.1 ± 24.30226.6 ± 12.15287.5 ± 34.92
LDH (iu/l)391.0 ± 37.16591.3 ± 108.2*626.0 ± 71.28579.5 ± 75.07
Table 4. Mean ± s.e. values for 5 endurance horses disqualified during a 166 km competition in 2 days, 83 km/day, because of exhaustion, dehydration or lack of recovery of heart rate
ParameterR (n = 5)PH1 (n = 5)PH2 (n = 5)PH3 (n = 4)R2 (n = 2)PH4 (n = 2)PH5 (n = 2)PH6 (n = 1)
  1. Statistical significance at P<0.05. *in bold indicates significant differences with successful horses at each sampling time. For abbreviations see Table 1.

Speed (km/h) 16.52 ± 0.38514.83 ± 1.46111.52 ± 2.530 15.00 ± 1.29011.80 ± 0.870*9.400
HRrt (s) 390.7 ± 105.9682.3 ± 178.4*1160 ± 99.02* 408.0 ± 157.01010 ± 730.0329.0
Na (mmol/l)153.5 ± 0.500*152.3 ± 2.848*148.8 ± 2.500*146.5 ± 1.453*146.3 ± 1.500143.5 ± 2.121143.0 ± 1.000140.0
K (mmol/l)3.500 ± 0.4004.300 ± 0.208*3.633 ± 0.0883.733 ± 0.1763.733 ± 0.5504.350 ± 0.7784.000 ± 0.5003.600
Cl (mmol/l)106.5 ± 1.500104.7 ± 1.202101.3 ± 2.00093.00 ± 1.202*99.00 ± 1.500104.5 ± 2.121101.5 ± 2.50096.00
Ca (mg/l)92.75 ± 7.53113.5 ± 5.33119.3 ± 19.79129.3 ± 14.01119.0 ± 5.00107.0 ± 2.1083.00 ± 10.9092.40
P (mg/l)26.25 ± 1.6227.88 ± 2.1538.60 ± 9.4941.40 ± 8.7235.90 ± 3.3029.10 ± 11.9233.30 ± 9.8734.59
Mg (mg/l)19.60 ± 1.27*22.68 ± 1.3629.68 ± 2.3238.33 ± 4.99*40.00 ± 0.56*36.30 ± 3.24*25.80 ± 0.8728.49
PCV (%)38.20 ± 1.00047.00 ± 1.23043.20 ± 1.34552.20 ± 2.34045.00 ± 0.64049.10 ± 2.31050.01 ± 0.15053.00
TSP (g/l)67.15 ± 1.4575.47 ± 2.7871.57 ± 2.3780.23 ± 4.40*73.20 ± 2.10*74.40 ± 2.9781.25 ± 6.65*83.00
ALB (g/l)24.70 ± 3.1034.90 ± 3.0932.63 ± 0.5839.43 ± 2.95*33.30 ± 1.90*34.20 ± 2.6937.75 ± 2.35*37.00
SUN (mg/l)232.5 ± 33.50390.0 ± 76.45441.7 ± 15.68570.0 ± 47.51*491.3 ± 8.00423.0 ± 11.31*518.5 ± 0.50*579.0
CREAT (mg/l)13.75 ± 2.5518.23 ± 3.5625.03 ± 3.0731.43 ± 1.16*28.60 ± 4.70*29.00 ± 6.05*32.75 ± 1.95*40.80
LA (mmol/l)1.209 ± 0.2541.942 ± 0.1832.186 ± 0.5054.414 ± 0.306*2.236 ± 0.727*2.103 ± 1.207*2.366 ± 0.275*1.750
CK (iu/l)115.5 ± 12.50170.0 ± 25.59*271.0 ± 74.39*370.0 ± 57.98*2022 ± 2856*3053 ± 4039*5039 ± 4742*12281
AST (iu/l)187.5 ± 9.500191.0 ± 35.10264.3 ± 33.56282.0 ± 25.26318.7 ± 208.5426.5 ± 292.4480.0 ± 256.01200
LDH (iu/l)243.0 ± 88.00379.7 ± 25.10*461.3 ± 68.96*649.3 ± 67.58*806.0 ± 11491648 ± 1625*1710 ± 1046*3120

Correlations between neuroendocrine factors and markers of hydration, renal function and electrolytes

The correlations between REN, ANG, ALD and AVP concentrations and the different indicators of hydration, renal function and electrolytes are presented in Table 5.

Table 5. Correlations between renin, angiotensin, aldosterone and arginine vasopressin concentrations in endurance horses and different markers of renal function, hydration and electrolyte balance
 RENANGALDAVPVHRrtPCVNaKClCaPMgTSPALBSUNCREATLACKAST
  1. Statistical significance at P<0.05 indicated in bold.

ANG0.230                   
ALD0.3400.710                  
AVP0.2200.7700.680                 
V0.3400.0900.2100.450                
HRrt0.4500.0500.7600.7700.700               
PCV0.120−0.1500.7800.6700.6700.760              
Na0.2300.390−0.7400.560−0.4500.250−0.400             
K0.3400.1900.1200.1200.4500.3200.3300.050            
Cl0.120−0.0500.3200.1300.4200.220−0.2000.4000.110           
Ca0.2100.0800.2000.1200.1500.2600.3600.1700.4500.290          
P0.2300.0700.2200.1300.1300.2500.120−0.1000.2700.4500.680         
Mg0.2100.2100.1200.2100.1800.2300.1700.1600.3700.2500.7000.740        
TSP0.100−0.0700.6500.4500.1500.2200.5800.3100.250−0.5800.6700.5100.420       
ALB0.120−0.1170.6400.4600.5000.5500.5900.2600.350−0.6000.8100.7400.5000.720      
SUN0.3200.1400.7200.4700.4800.5400.6500.2100.140−0.6400.2500.1700.0900.6800.500     
CREAT0.3400.7200.7300.4700.2300.5500.7600.2200.100−0.7200.4500.3800.1700.7000.6200.760    
LA0.2300.0100.4500.2500.2300.1200.5000.3400.060−0.6600.2300.2500.1900.3300.3200.2300.370   
CK0.1300.1400.1000.1300.6600.740−0.1200.0400.360−0.6400.5700.7500.6200.3200.4200.0100.2100.150  
AST0.2100.1300.1200.1500.5500.5500.1500.0100.240−0.3900.5100.7700.7600.4000.4800.0300.3600.1300.810 
LDH0.2000.1700.1300.1500.6000.6900.1600.0300.480−0.5800.5400.7600.7500.4300.4200.2400.3500.3600.96000760

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Conflicts of interest
  10. Manufacturers' addresses
  11. References

Endurance horses are subjected to substantial modifications in their internal homeostasis due to prolonged exercise and water and electrolyte losses. In our experience, these losses are more intense in horses eliminated from competition due to dehydration, metabolic disturbances and/or lack of HR recovery. Consequently, it would be interesting to examine the concentrations of the main neuroendocrine factors involved in the control of blood volume, osmolality and electrolyte balance. This might represent a step towards understanding why despite a proper training protocol, a well designed competition strategy, including electrolyte supplementation and acclimation to weather and terrain conditions, some horses undergo life-threatening metabolic disturbances or loss of performance. The main aim of this study was to obtain practical applications in order to increase performance and minimise the risk of metabolic problems in competition in this type of equine athlete.

This study has limitations inherent to field conditions. One of them is the time of blood withdrawal. Although a precise standardisation while taking the samples was followed, the duration of the vet-gate was not controlled. The MH probably had longer HRrt and vet-gates because of their more compromised status. Secondly, the duration of transportation before the competition was not recorded. Barton et al. (2003) described that as time spent in transit to the competition place increased, horses were more likely to be withdrawn from the race. Nevertheless, when the horses were presented to the veterinary examinations the day before the competitions, the physical examination showed that they were fully recovered from transport. Finally, the amount, time of food and water intake and electrolyte supplementations were not recorded, as endurance horses are trained to eat and to drink water in ponds and creeks during competitions.

Effect of the distance on the circulating concentrations of REN, ANG, ALD and AVP in successful endurance horses

It has been shown that REN activity increased from 2–4 ng/ml/h during a 60 min treadmill exercise (McKeever et al. 1991). However, in the present study, plasma REN concentrations were not significantly altered at the different sampling times. McKeever et al. (1991) reported REN activity, whereas we determined REN concentrations. Both activity and concentration are valid ways to measure REN and the same factors influenced its release in response to exercise: renal nerve stimulation via increased sympathetic drive, changes in renal flow and pressure in association with juxtaglomerular function and changes in electrolyte concentrations at the juxtaglomerular apparatus of the kidney (Sealey and Laragh 1990; McKeever et al. 1991). McKeever et al. (1991) indicated that a major contributing factor to the increased REN activity during the first 40 min of exercise was an increase in sympathetic drive. The first blood sample extracted in our study was made after 27–35 km. Another mechanism involved in the elevation of REN is the decrease of Cl concentrations, as the juxtaglomerular apparatus is more sensitive to Cl than to Na (McKeever et al. 1991). It was found that both serum Na (4.6 mmol/l in A and 3.2 and 5.7 mmol/l on the 2 days of B) and Cl (8.7 mmol/l in A and 8.6 and 8.1 in the 2 days of B) decreased. However, serum REN were not affected by the endurance exercise and no significant correlations with Cl and Na were found. A possible reason could be the different amounts and protocols of electrolyte supplementation during the competitions.

Angiotensin II is a potent vasoconstrictor, which also stimulates Na reabsorption in the kidneys, and regulates the release of ALD and AVP (Mulrow 1999; A. Muñoz, C. Riber, P. Trigo and F.M. Castejón, unpublished), hence the positive correlations found between ANG, ALD and AVP in the present research. The significant increase in ANG in both types of competitions was probably a response to the exercise-induced dehydration, as increases in PCV, TSP, ALB, SUN and CREAT were also found.

Different researchers concluded that increased plasma K concentration was the strongest stimulus for the release of ALD in the exercising horse (McKeever et al. 1991, 1992). However, we did not find significant changes in K concentrations, and a significant correlation between K and ALD concentrations, was not observed. K is an intracellular ion and furthermore, circulating K in endurance horses is related to the time of blood extraction after cessation of exercise, and dependant on the activity of the muscle Na-K ATP-ase pump (Hess et al. 2006). These characteristics of K balance might explain our results. According to the data from the current study, it seems that the main determinants of ALD release in endurance competitions are the decrease in the natraemia and the activation of the REN-ANG-ALD axis, as confirmed by the correlations between ALD, Na and ANG. Although other authors reported that faster endurance horses had higher ALD concentrations (Schott et al. 1997), a positive correlation between ALD and speed was not found in the current study.

The lack of recovery of serum ALD concentrations at the beginning of the second day of competition B supports the results of Schott et al. (1997). These findings might indicate that fluid and electrolyte deficits persist for >12 h after competition, despite the return of other hydration markers and electrolytes to preride values. These results are interesting, as ALD concentrations appear to be a better marker of hydration than others used more frequently (TSP, ALB, SUN and CREAT), although these parameters are easier to monitor.

Vasopressin is another hormone involved in the control of homeostasis during exercise, with important functions, including vasoconstriction and decreased free water clearance (McKeever et al. 1991, 1992; McKeever 1998; Muñoz et al. 2010). Its antidiuretic actions would be important during prolonged exercise, and in fact, a significant increase in serum AVP was found in both types of endurance competitions. The positive correlation between HRrt, AVP and ALD concentrations seems to indicate that the more dehydrated horses need longer periods of time to recover their HR. This fact reflects an increase in HR in order to maintain cardiac output when stroke volume is limited because of hypovolaemia. Similarly to what happened with ALD, serum AVP concentrations remained elevated the second day of Competition B in comparison with the preride values. However, AVP did not change during this second day, whereas ALD concentrations increased progressively.

Differences in REN, ANG, ALD, AVP, electrolytes, and markers of hydration and renal function between successful and metabolic endurance horses

Our study revealed that significant differences existed between SH and MH before the competitions, with MH having highest ALD, AVP and Na. It would be interesting to investigate the effect of hydration and electrolyte balance before exercise on these parameters. It has been demonstrated both in man and horses that hyperhydration or dehydration before exercise significantly affects resting AVP (Wade and Freund 1990; Muñoz et al. 2010). According to our data, it seems that some MH started the competitions with an altered hydration and/or electrolyte status, not detected in the physical examination. However, no significant differences in the other parameters were found at R between both performance groups. Another hypothesis could be that some horses received a greater electrolyte supplementation some hours before competition, resulting in increased osmolality and release of ALD and AVP.

Metabolic horses mainly presented higher ANG, ALD and AVP at the different sampling times, even though the most consistent finding was the higher ALD and AVP, reaching means >2000 pg/ml and 200 pg/ml, respectively. This finding is in line with the more intense dehydration of MH, as indicated by the higher PCV, TSP, ALB, SUN and CREAT. Therefore, our results suggested a more intense activation of the REN-ANG-ALD-AVP axis in the MH.

Both SH and MH presented a progressive reduction of Na, although MH had higher natraemia than SH. Because of equine sweat composition (McCutcheon and Geor 2000), a reduction in Cl was found in both types of competition. Metabolic problems loss >13 mmol/l of Cl during the first day of Competition B, indicating a greater sweat rate, consistent with the higher concentrations of the hydration markers (TSP, ALB, SUN and CREAT). Fielding et al. (2009) also indicated that MHs that require medical treatment present lower Cl concentrations. Decreased K concentration after a ride is a common finding (Lindinger and Ecker 1995; Schott et al. 1997, 2006; Barton et al. 2003), associated with loss in the sweat and increased activity of the Na-K-ATPase pumps during recovery. In our study, K was not modified by the distance and significant differences between SH and MH were not found.

In contradiction to previous studies (Foreman et al. 2004; Schott et al. 2006), total Ca concentrations were not modified by endurance exercise and no differences were found between SH and MH. The increased P concentrations were attributed to a combination of dehydration and use of intrafibrillar ATP and phosphocreatine. The higher Mg concentrations in the MH in some sampling times could be justified by the greater haemoconcentration.

Haemoconcentration appeared early in SH and after that, hydration status did not show evident changes. In addition, a parallel change was found between PCV and TSP. We have recently found that horses which experience nonproportional changes of PCV and TSP are at risk of disqualification for metabolic problems during the next vet-gate (Trigo et al. 2010). Metabolic problems in our study, generally showed PCV >50%, TSP >80 g/l, SUN >410 mg/l and CREAT >25 mg/l (maximum mean values for these parameters: 55%, 85 g/l, 550 mg/l and 40 mg/l, respectively).

Lactate in SH did not increase significantly, probably because of the aerobic characteristics of this type of competition, although metabolic acidosis has been reported in endurance horses exercising at fast speeds and is presumably associated with lactic acidosis (Barton et al. 2003). In the present study, a positive correlation between LA and speed was not found. The higher lactacidaemia of MH therefore can not be associated with a greater V, as MH had lower speed during some laps, although it could be linked to the degree of haemodynamic compromise, as indicated by the correlations.

Serum muscle enzymes increased in SH, with higher rises in the longer competition, as reported previously (Noakes 1987). Competition B was held over a muddy terrain that might have increased muscle effort. Exertional rhabdomyolysis cannot be ruled out in these horses, although clinical signs were not detected during the vet-gates. Metabolic problems presented higher CK and LDH activities at different times. This difference can not be associated with exercise duration, as speed was not different between SH and MH, except at PH5. These data suggest that a mild to moderate rhabdomyolysis existed in MH and higher serum muscle enzymes were associated with a longer HRrt, although it was not clear if the rhabdomyolysis was one of the causes implied in the exhaustion and/or lack of HR recovery. On the other hand, it is possible that fatigue would lead to the use of different neuromuscular groups and the development of muscle injury.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Conflicts of interest
  10. Manufacturers' addresses
  11. References

The current research was designed in order to investigate the changes in REN, ANG, ALD and AVP concentrations during endurance competitions, the differences between horses according to their performance and in relation to other laboratory parameters determined commonly in endurance horses in competition. It was found that REN concentrations were not affected by the distance, whereas ANG, ALD and AVP increased. Angiotensin II recovered after rest, whereas ALD and AVP persisted elevated overnight and therefore horses that started a second day of endurance competitions had increased ALD and AVP. Horses eliminated due to metabolic problems were more dehydrated, and they underwent a greater activation of the REN-ANG-ALD-AVP axis.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Conflicts of interest
  10. Manufacturers' addresses
  11. References

The authors want to express their gratitude to the owners and riders of the horses, as well as the organisers of the endurance competitions, who kindly agreed to participate in the research.

Manufacturers' addresses

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Conflicts of interest
  10. Manufacturers' addresses
  11. References

1 Biosystems, Barcelona, Spain.

2 Idexx Laboratories Inc., Westbrook, Maine, USA.

3 Statsoft Inc, Tulsa, Oklahoma, USA.

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  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Conflicts of interest
  10. Manufacturers' addresses
  11. References
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