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Keywords:

  • horse;
  • fence height;
  • showjumping;
  • total and free iodothyronines

Summary

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

Reasons for performing study: Involvement of thyroid function on performance warrants further investigation as limited data exists on the effects of showjumping on the dynamics of total and free iodothyronines.

Objectives: To investigate the response of circulating total and free iodothyronines in horses to experimental showjumping sessions and compare with the effects normally induced by competition and determine if fence height has any effect.

Materials and methods: Using a randomised crossover study design 6 trained horses were studied during experimental showjumping sessions over 10 fences of different height: 1.00 m (Session 1), 1.10 m (Session 2), 1.20 m (Session 3). Hormone levels were recorded before, after warm-up, 5 and 30 min post exercise. T3, T4, fT3, fT4 concentrations were analysed by ELISA/competition using streptavidin technology. RM-ANOVA was applied to test for any differences in basal and warm-up values of different sessions. Two way RM-ANOVA was applied to test for any effects of interaction between fence height and time. The differences between individual means over time were assessed using a post hoc multiple comparison test (Bonferroni).

Results: Basal T4 changes over the sessions (P<0.05) were recorded. After warm-up, T4 concentration results were lower than basal in Session 1 (P<0.05). Higher than basal values were recorded 30 min post exercise for T3 (P<0.001), T4 and fT4 (P<0.01) in Session 2 and for T4 (P<0.05) and fT4 (P<0.01) in Session 3. The interaction fence height/time results were significant on T3 (P<0.03) and fT4 (P<0.03); sampling time on T3 (P<0.0007), T4 (P<0.001) and fT4 (P<0.002) post exercise changes.

Conclusion: Showjumping over the highest fences induced a release of T3 from skeletal muscle, probably due to 5′-desiodase activity and increase of fT4, due to thyroid stimulation and/or changes in capacity to bind iodothyronines.


Introduction

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

The successful showjumper combines a unique blend of power, precision and speed (Clayton 1994), supported by biochemical and functional adaptation to exercise and motivation to competition. The neuroendocrine system is directly involved in the context of physical effort and emotional tone and in jumper horses, as well as in sportsmen with different types of motor ability and it could have a significance in performance due also to the arousal of motor and cognitive processes and neuromuscular coordination related to motor ability.

In horses the problem of relating exercise capability to thyroid function and iodothyronine levels and the involvement of the hypothalamus-pituitary-thyroid axis in physical performance warrants further investigation. Nevertheless, an enhanced responsiveness to endogenous catecholamines caused by an increase in adrenergic receptor in the presence of thyroid hormones has been postulated in several models (Gonzáles et al. 1998). Moreover, in man, the role of thyroid hormones in the degree of brain arousal has been discussed by Tucker et al. (1984) and in other species the regulatory role of deiodinases in active thyroid hormone content in brain tissue has also been reported (Rudas et al. 2005).

Exercise-induced changes in serum iodothyronine concentrations have been previously recorded in horses (Garcia and Beech, 1986; Ferlazzo and Fazio 1997; Gonzáles et al. 1998; Graves et al. 2006; Ferlazzo et al. 2007) as well as in different species (Terjung and Winder 1975; Sander and Rocker 1988; Hesse et al. 1989; Hackney and Gulledge 1994; Sander and Rocker 1988; Panciera et al. 2003; Kanaka-Gantenbein 2005) and are mainly affected by the severity of exercise.

Moreover, previous investigations on jumper horses have described that, as well as experience (Clayton 1994), fence height influences post exercise cortisol changes, whilst circulating ACTH and β-endorphin concentrations are not affected (Ferlazzo et al. 1998, 2010; Fazio et al. 2000). To the contrary, after experimental show jumping sessions, endocrine changes were not found to be dependent on fence height (Ferlazzo et al. 2009).

The hypothesis of this study was to offer further data on thyroid response to showjumping by determining whether the pattern of total and free iodothyronine changes differed in horses undergoing differing levels of difficulty of jumping sessions in comparison with changes usually induced by emotive stress arising from a competition environment. On this basis, the study investigated the response of circulating total and free iodothyronine levels of healthy trained horses to 3 showjumping sessions with different fence heights during experimental conditions.

Materials and methods

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

Horses and experimental design

All methods and procedures used in this experiment were reviewed and approved by the Messina University Institutional Board for the Care and Use of Animals.

Six trained sport jumper horses (3 Sella Italiano, 1 Belgian Warmblood, 1 Dutch Warmblood, 1 Selle Français), 3 geldings, 2 mares and 1 stallion, mean ± s.d. 10.3 ± 3.4 years (range 6–13) and weight 580 ± 50 kg were used in the study. All horses were housed individually in 4 × 4 stables at the Equine Research Facility where the test was carried out.

Each horse was fed a daily ration of 8 kg of alfalfa and grass hay and 4 kg of a commercially available hay cube ration (split into 2 feeds). Water and a salt/mineral block were provided in the boxes ad libitum.

The horses in the study were able to cope with fences up to 1.30 m high and all had previous experience of showjumping competition at the same level.

The horses were in regular training and considered by the referring veterinarian to be clinically healthy before entering the study. The horses and rider were usually trained together approximately 1–2 h per day, performing low level jumping exercises approximately twice per week and were familiar with the jumping exercises required.

Before exercise the horses were equipped with a heart rate meter (Polar Electro Europe BV)1; one electrode was placed under the girth behind the left elbow, the other under the saddle at the withers. Heart rate was monitored at the beginning of the warm-up and recorded at 15 intervals during warm-up and during the courses and until 30 min after completing the courses.

The experimental study was designed to exclude the emotive stress arising from a competition environment and the influence of differences in horse competition experience and rider. With this in mind and as the horses were all trained for the competition season to cope with fences 1.30 m high, the need to have a control group excluding jumping was not considered in order to assess the workload of jumping fences. Heart rate was adopted as an internal quantitative measure to assess workloads of different jumping exercises.

Using a randomised crossover study design, the horses performed the 3 experimental jumping sessions over fences of different heights: 1.00 m (Session 1), 1.10 m (Session 2), 1.20 m (Session 3) over 3 days. The jumping sessions were performed after a warm-up period which consisted of 10 min walk, trot and quiet canter. Before tests, the horses jumped 2 low fences. After the warm-up, the horses walked for 10 min on average until heart rate decreased to <50 beats/min within 30 min.

The horses performed the 3 different sessions with the same rider. All tests were performed before the morning feed in an outdoor arena when mean ambient temperature was 12°C (range 10–15°C). The horses cantered a course of 800 m and the circuit design was differentiated exclusively on the basis of fence height. Five upright and 5 cross-pole fences were utilised in each session.

The jumping sessions were cantered at an average speed of approximately 350 m/min and were scored by time and knockdowns on the courses. Time for completion of the courses and thus the speed of each course were registered. Fault and refusal of each horse were also registered.

Blood samples before and after the tests were taken in the morning always between 07.00 and 09.00 h in order to minimise eventual effects of diurnal rhythms.

Blood samples (10 ml) were collected by jugular venipuncture using evacuated tubes2, while horses were stalled at rest, daily at 07.00 h, before feeding and before exercise by the same operator under quiet conditions and they were not restrained during the procedure. All other samples were collected after warm-up, then 5 and 30 min after the end of showjumping sessions.

Serum samples were harvested after centrifugation at 3000 g for 15 min at 4°C and stored at -20°C until the day of analysis.

Serum total and free iodothyronines (T3, T4, fT3, fT4) concentrations were analysed in duplicate by ES300 Testing Procedure based on ELISA/competition to polyclonal biotinylated ovine iodothyronines-antibodies using streptavidin technology3, suitable for equine use (Ferlazzo et al. 1996). The respective intra- and interassay CVs were as follows: 11.4% and 7.3% for T3, 5.7% and 2.3% for T4, 11.9% and 4.2% for fT3, 9.6% and 6.6% for fT4.

Statistical analysis

Data are presented as mean ± standard deviation (s.d.) and results analysed using a statistical analysis software programme4. The coefficient of variations of total and free iodothyronines within a group and within a horse for each sampling time was calculated dividing the standard deviation through the mean and multiplying by 100. A one-way analysis of variance for repeated measures (RM-ANOVA) was applied to test for any differences in the basal values of the 3 experimental days and for any differences in values after warm-up recorded on the 3 experimental days. A 2-way analysis of variance for repeated measures (2-way RM-ANOVA) was applied to test for the effects of interaction between different fence heights and sampling times. When the F statistic was significant, the differences between individual means over time were then assessed using a post hoc multiple comparison test (Bonferroni). The percentage differences (Δ%) of changes in hormone concentrations were also calculated by comparing the different post exercise times with basal and warm-up concentrations. The ratios of total iodothyronines (T4/T3), free iodothyronines (fT4/fT3) and respective percentage of free iodothyronines to total iodothyronines (fT3/T3%; fT4/T4%) in the different sessions before and post exercise were also calculated. The changes in heart rate were analysed by a one-way analysis of variance for repeated measures (RM-ANOVA) and a paired Student's t test. Statistical significance was set at P<0.05.

Results

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

Result of performances

In Session 1, 5 horses performed a clear round and one horse performed a penalty round. In Session 2, 5 horses performed a clear round and one horse performed a penalty round. In Session 3, 4 horses performed a clear round and 2 horses performed a penalty round. There were no refusals.

Speed

The mean speed in Session 1 was 5.6 ± 0.1 m/s, Session 2 5.7 ± 0.3 m/s and Session 3 5.8. ± 0.4 m/s. The mean duration in Session1 was 81.2 ± 0.1 s, Session 2 was 90.0 ± 0.1 s and Session 3 was 93.4 ± 0.1 m/s.

Heart rate

The horses had a mean heart rate of 93.7 ± 6.3 beats/min at the end of warm-up, which decreased to a mean of 48.2 ± 2.3 beats/min in average 10 min. A progressive significant rise in heart rate was recorded from the onset to the end of exercises, with peak values at approximately 60 s (Session 1: P<0.05; Session 2: P<0.01; Session 3: P<0.01), afterwards falling during recovery. The respective mean peak heart rates during the sessions were: 175.2 ± 15.1 in Session 1, 182.4 ± 10.3 in Session 2 and 185.6 ± 18.3 beats/min in Session 3. During recovery, heart rate decreased to a mean of 70.5 ± 9.5 beats/min after 5 min (P<0.05) and was 44.5 ± 2.2 beats/min after 30 min. To ensure that peak heart rates were not overestimated by the heart rate meter, the mean of the 3 highest values of each session were compared.

Total and free iodothyronines

Data recorded from horses before exercise, after warm-up and at different times after showjumping in the 3 experimental sessions are presented in Figures 1a–d.

image

Figure 1. Serum total and free iodothyronine levels of horses after show jumping in 3 experimental sessions. Asterisks indicate significant (*P<0.05; **P<0.01; ***P<0.001) differences in average hormone concentration vs. basal in each session.

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Variability in values of basal and post exercise within a group (Table 1) for each sampling time was large, especially for T3 and fT3. There was a large variability within a horse (Table 2) for each sampling time. The greatest variations on T3 and fT3 values were observed in only one of the horses studied.

Table 1. Coefficient of variance (%) of total and free iodothyronines within a group of horses for each sampling time during Session 1, Session 2 and Session 3
Fences heightT3T4
BasalWarm-up5 min30 minBasalWarm-up5 min30 min
1.0062.340.143.930.019.029.925.414.2
1.1017.919.720.236.124.121.628.210.7
1.207.714.427.320.521.620.228.815.1
 fT3fT4
1.0064.935.151.640.721.035.318.323.5
1.1017.423.615.538.039.621.021.429.2
1.2039.518.227.046.133.252.636.425.5
Table 2. Coefficient of variance (%) of total and free iodothyronines within a horse for each sampling time
HorseT3T4
BasalWarm-up5 min30 minBasalWarm-up5 min30 min
115.439.517.121.723.926.13.5030.0
218.623.610.916.623.844.522.819.2
329.318.125.011.817.717.629.120.1
423.616.47.937.529.337.033.914.2
560.643.156.513.225.128.423.713.0
612.55.828.624.115.421.16.220.3
 fT3fT4
120.439.323.955.549.244.435.814.9
210.86.921.426.313.013.134.814.4
312.020.632.554.84.311.35.030.9
429.415.413.441.014.928.315.327.2
568.943.554.332.120.017.717.831.0
615.29.320.133.226.345.530.840.0

Basal mean T4 concentrations showed changes over the 3 sessions (F = 5.31; P<0.05) and were higher (P<0.05) in Session 3 than in Session 1. Ratio of T4/T3 was equal to 32.3:1 in Session 1, 41.6:1 in Session 2 and 44.5:1 in Session 3. Ratio of fT4/fT3 was equal to 2.58:1 in Session 1, 3.32:1 in Session 2 and 3.01:1 in Session 3.

The percentage of fT4/T4 was equal to 0.030 ± 0.03 in Session 1, 0.029 ± 0.04 in Session 2 and 0.019 ± 0.02 in Session 3. The percentage of fT3/T3 was equal to 0.378 ± 0.39 in Session 1, 0.367 ± 0.35 in Session 2 and 0.284 ± 1.4 in Session 3.

T4 concentrations decreased after warm-up exclusively in Session 1 (P<0.05). Higher values than basal have been recorded 30 min post exercise for T3 (P<0.001), T4 (P<0.01) and fT4 (P<0.01) concentrations in Session 2 and for T4 (P<0.05) and fT4 (P<0.01) concentrations in Session 3.

T4/T3, fT4/fT3 and the percentages of fT4/T4 and fT3/T3 showed no significant post exercise changes in the different sessions.

The interaction fence height/time results were statistically significant for T3 (P<0.007) and fT4 (P<0.02) changes post exercise. Sampling time significantly affected T3 (P<0.002), T4 (P<0.0001) and fT4 (P<0.002) changes post exercise.

Discussion and conclusions

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

Basal total and free iodothyronines (T4, T3, fT4, fT3) values and their ratios were in the physiological ranges previously recorded for horses (Anderson et al. 1988). Individual variations in hormone concentrations were recorded and the results were lower than in racing Thoroughbreds (Bayly et al. 1996). Differences in basal T4 concentrations were recorded between Session 1 and Session 3 and, although a control study on a sedentary group at rest and not jumping was not performed, they seem likely to be not dependent on diurnal variations, as blood sampling was constantly carried out in the same time period between 07.00 and 09.00 h. In fact, in horses no clear-cut evidence of circadian rhythmicity of iodothyronines was recorded (Bayly et al. 1996). Nevertheless, in Thoroughbreds, basal T4 concentrations had a marked variability within a horse (Ferlazzo et al. 1996) and in racehorses actively training and competing appeared to be susceptible to the effects of a variety of exogenous stimuli and to show extreme fluctuations, with differences between 06.00 and 18.00 h (Bayly et al. 1996). It is not easy to explain this result, which is probably caused by random variations, because the horses being studied had the same feeding regimen, did not show any clinical reason and were not on medication during the experimental period. The coefficient of variation of basal T4 values was between 19.0 and 24.1%, although lower than racing subjects (Bayly et al. 1996). Thus, although the horses studied were trained, it cannot be excluded that the highest T4 values recorded in Session 3 could also be the consequence of an increase of T4 induced in individual horses by the previous jumping sessions they had performed.

During exercise and recovery, the heart rate data was not influenced by excitement or extrinsic factors, as all horses were tested in an outdoor environment that was quiet and familiar to them. The increase in heart rate after showjumping was comparable to the results obtained after showjumping in different studies, in elite jumper horses during competition (Lekeux et al. 1991) and riding-school horses during jumping (Sloet van Oldruitenborgh-Oosterbaan et al. 2006). Heart rate values showed a trend to be higher in Session 2 and Session 3 than in Session 1, probably due to different course speed as well as to unequal fitness and athletic ability of horses (Kingston et al. 2006).It is supposed that this is due to a greater exercise intensity with higher fences during jumping. It is known that during showjumping there appears to be a direct correlation between heart rate and fence height, which may be related to a faster speed approaching larger fences (Barrey and Valette 1992).

In this study, the pattern of post exercise total and free iodothyronine concentrations showed that show jumping induced significant increased values at 30 min for T3 and T4 in Session 2 and for T4 and fT4 in Session 3. The iodothyronine increases were recorded in only 2 sessions and exclusively at 30 min. Showjumping is considered not to induce a consistent and overall iodothyronine increase in the 30 min after exercise in trained horses during experimental conditions, although failure to demonstrate differences in values was possibly due to a large coefficient of variation in hormone levels.

Nevertheless, in this study, a significant effect of the interaction fence height/time was demonstrated on T3 and fT4 post exercise changes.

Exercise-induced changes in serum iodothyronines concentrations have been previously reported in horses, man and dogs (Irvine 1967, Terjung and Winder 1975; Garcia and Beech 1986; Ferlazzo and Fazio 1997; Panciera et al. 2003; Ferlazzo et al. 2007) and previous work has found considerable variability in iodothyronine response to acute short-term and more prolonged exercise. The most physiological relevant factors that influence circulating thyroid hormone concentrations with exercise appear to be frequency and intensity of exercise and total amount of work performed. In previous studies performed in horses after show jumping, results demonstrated only concentrations of free iodothyronine significantly increased, both during competition and training (Ferlazzo and Fazio 1997; Ferlazzo et al., 2007). In Thoroughbreds, short-term high-intensity exercise during competition induced an increment in T4 and T3 concentrations, with parallel changes in plasma catecholamines (Gonzáles et al. 1998). A slight, but statistically significant increase, in T3 and T4 concentrations was observed in horses 1 h after swimming for 1–20 min (Garcia and Beech 1986). Endurance exercise resulted in transient decreases of serum iodothyronine concentrations and longer distance endurance exercise resulted in a more prolonged post exercise decrease in serum iodothyronines and a greater decline in T4 in comparison to T3 (Graves et al. 2006). In controlled experimental conditions (standardised exercise tests on treadmill) performed in Thoroughbred horses (Ferlazzo and Fazio 1997), plasma T3 and T4 concentrations did not change after a 2 step exercise test (V2,5 and V12), while fT4 concentrations increased significantly in the 45th and 60th min and fT3 concentrations augmented significantly after warm-up but before exercise and remained constant thereafter. A multiple-step exercise test only induced fT3 values significantly higher than basal, while T3, T4 and fT4 were unchanged during and after the test.

In sportsmen, a significant increment in T4 plasma concentrations was found both after aerobic and anaerobic exercise, while T3 increased transiently only after aerobic exercise (Hackney and Gulledge 1994). In marathon runners with better performances, prolonged endurance exercise produced inconsistent changes in thyroid hormone concentrations (Hesse et al. 1989) and decreased in dogs (Panciera et al. 2003).

The percentage of post exercise increases of total and free iodothyronines was not equivalent for the different variables and the different sessions and therefore an influence of haemoconcentration could not explain the results obtained. In the horse, the relationships between the equilibrium among the different forms of thyroid hormones and the differentiated contribution of thyroid gland and extrathyroidal tissues (e.g. active muscle) to circulating hormone levels during physical exercise has not been completely explained. Usually, all the iodothyronine forms maintain equilibrium and any change regarding uptake rate, intracellular handling, metabolism and deiodination rate may influence hormone levels. In this study, hormone ratio and percentage changes did not undergo significant changes among the different sessions. Showjumping is a short-term exercise and it works at a slower average speed than other sport activities, as shown by the evidence that in showjumpers cortisol increase after exercise is less than in racehorses and showjumping exercise requires the use of anaerobic metabolism (Lekeux et al. 1991; Linden et al. 1991). T3 represents the active form of iodothyronines and appears to be less susceptible to exogenous influences (Bayly et al. 1996). Therefore, showjumping with higher fences probably could induce in horses a supply of T3 from skeletal muscle in response to exercise, as a consequence of the improved circulation (Art et al. 1990) and the increase of 5-desiodase activity in muscle for increased tissue hormone utilisation. This could be supported by the increased post exercise fT4 concentrations, probably due to thyroid stimulation and/or changes in capacity to bind iodothyronines. Although the data is not available in this study, total plasma protein concentrations are usually increased after showjumping (Lekeux et al. 1991; Sloet van Oldruitenborgh-Oosterbaan et al. 2006). On the other hand, the major involvement of fT4 during the recovery phase could support the hypothesis of a stimulated activity of thyroid gland when the homeostatic response to jumping exercise is in progress, inducing increasing circulating concentrations of total iodothyronines and fT4 ready for intracellular utilisation.

This study was carried out under experimental conditions, so the emotional component of the exercise was probably kept under control and adaptation to the physiological exercise stress was adequately working out even after jumping with the highest fences during the recovery phase. Statistical analysis of data showed that the time of blood sampling significantly affected T3, T4 and fT4 post exercise changes and therefore could have influenced the pattern of iodothyronine changes after showjumping. Nevertheless, in the same horses, increased cortisol concentrations were recorded 5 min post exercise only after performances over fence heights of 1.10–1.20 m (Session 2 and Session 3) and declining concentrations at 30 min (Ferlazzo et al. 2009).

The total amount of work in the 3 jumping sessions was possibly similar but intensity slightly greater over the highest fences. In fact, although the horses were trained to cope with fences up to 1.30 m, heart rate showed differences between the different sessions and the greater intensity of jumping could have influenced T3 and fT4 post exercise changes.

No definite conclusions can be made from this study leading to the question of physiological relevance concerning the effects of exercise over fences of varying height on iodothyronine changes after showjumping. Nevertheless, these data suggest the opportunity to carry out further investigations, supported by more essential control studies, may result in a better understanding of the role of different iodothyronines either on motivational or muscular activity in supporting the physical and psychical requirements evoked by showjumping.

Acknowledgements

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

This research was supported by MIUR grants and by the University of Messina Research Program.

Manufacturers' addresses

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

1 Polar Electro Europe BV, Fleurier, Switzerland.

2 Terumo®, Leuven, Belgium.

3 Roche Diagnostics GmbH, Mannheim, Germany.

4 SAS/STAT, Version 8.0; SAS Institute Inc., Cary, NC, USA.

References

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