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Wednesday, 6 July 2016

Genetic study of stress assessed with infrared thermography during dressage competitions in the Pura Raza Español horse

Applied Animal Behaviour Science
Volume 174, January 2016, Pages 58–65

doi:10.1016/j.applanim.2015.11.006
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Highlights

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A selection made to improve ET values would improve Dressage performance.
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ET appears to be measuring physiological stress rather than emotional stress.
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ET is suitable for assessing stress, despite being influenced by many environmental factors.
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This is the first study to determine the heritability of stress assessed with ET.
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h2 0.14–0.50 supported the assumption that horses inherit susceptibility to stress.

Abstract

Despite the fact that physiological parameters in dressage are important because of their close connection to human–horse accidents, performance and welfare, these parameters are only rarely included in horse breeding programs. In Spain, the Pura Raza Español horse (PRE) Breeding Program focuses mainly on the selection of horses for satisfactory dressage ability. Studying the genetic parameters of eye temperature (ET) using infrared thermography (IR) as an indicator of stress in horses could highlight the suitability of this parameter to be included as a selection criterion in the PRE Breeding Program. The aim of this study was to investigate the heritability of ET measured with IR in Young Horse Dressage tests (YHDT) from the Breeding Program. Accordingly, 343 PRE were measured during 3 YHDT final competitions held in Spain. From these animals, 1746 ET measurements were taken on 3 different moments during each competition: 3 h before the competition (ET_B), just after the competition (<5 min after the dressage exercise) (ET_JA), and 3 h after the competition, when the animal was resting (ET_A). Moreover, 6 different dressage score records were taken for each animal during the competitions: walk, trot, canter, general impression, submission and total score. The genetic parameters were estimated using a Bayesian procedure. The environmental effects (Age, Stud, Trip, Training, Event-Year and Rider) that were statistically significant (p < 0.05) for each temperature trait were used in the genetic models. The pedigree file included a total of 3350 PRE. The correlations between the ET and expected breeding values for dressage (EBV), as well as the coincidence between the animals’ genetic rankings by their EBV, were also studied. Heritabilities for temperature traits ranged from 0.14 (ET_JA) to 0.50 (ET_A), while heritabilities for performance traits ranged from 0.44 (walk) to 0.37 (submission) and repeatability ranged from 0.25 (ET_JA) to 0.79 (walk). The dressage EBV and the ET EBV just after exercise showed statistically significant correlations with r2 = 0.21. Finally, the range of matching animals was higher (from 17% to 40%) when the top 25% of the animals for both parameters was selected. Our findings indicate that the selection of the best animals for dressage performance would also involve selecting the animals with higher ET values, showing a higher level of physiological stress. Hence, despite selection using ET in horses is achievable, more evidence about its validity, sensitivity and specificity is still needed in order to include ET as an additional selection criteria in Breeding Programs.

Keywords

  • Breeding value;
  • Equine;
  • Eye temperature;
  • Heritability;
  • Stress

1. Introduction

Animal welfare science is increasingly concerned with the use of animals in sports, yet little is known about how to measure this. The dressage discipline consists of a horse guided by a rider, having to demonstrate its gaits at walk, trot and canter, and the changes between these gaits. To do this successfully, there needs to be a good understanding between the ride and the horse. This relation is influenced by the level of experience of both rider and horse and by the individual behavior of the horse (Visser et al., 2008). It is widely accepted that this interaction, together with other factors such as handling, transport or weather conditions etc, affects the horse's performance and can even act as a stress factor (Bartolomé et al., 2013 and Stewart et al., 2011). In horses, stress related to the response to new external stimuli is expressed by changes in homeostatic, behavioral and physiological parameters (Stephens, 1980), that are developed when a stimulus is perceived as a potential threat, in order to alleviate the effects of the perceived stressor. Stress can have positive as well as negative effects on the body, helping the animal to cope with routine short-term stressors (Moberg, 2000). Hence, stress could be divided in “physiological stress” and “emotional stress”. During the former, the biological costs of the stress response are lower than the animal's biological reserves to cope with them, hence the animal is unaware of having to develop a homeostatic response to it thus the horse would be developing a proactive response to cope with the stressful situation. Besides, during the “emotional stress” or “distress”, the biological costs are higher than the biological reserves, implying that the animal is consciously experiencing a negative emotional state (Cockram, 2004), thus affecting its welfare as it is not copying with the stressful situation.
Changes in behavioral and physiological parameters in dressage are important in relation to human–horse accidents, performance and the welfare of horses (McLean and McGreevy, 2010 and Peeters et al., 2010). In spite of this, the stress factor is often not taken into account in horse breeding programs.
In Spain, the genetic evaluation of dressage includes only one factor which estimates the effect of stress (stress related to traveling), but to date, no studies have been published about the individual stress levels of each horse during dressage competitions, their heritability or their influence on the horse's dressage performance. To measure this influence, a precise, reliable method for stress assessment during competitions is needed. Infrared thermography (IR) is the recording of the infrared radiation emitted by a body surface using a thermography camera. In particular, the measurement of eye temperature (ET) using IR has been shown to be a reliable indicator of stress in different species (Ludwig et al., 2007 and Stewart et al., 2007). The non-invasive nature, easy interpretation and fast application under field conditions of the IR, make it a suitable technique to evaluate the stress of horses during equestrian competitions (Valera et al., 2012). However, the usefulness of this method has been studied only for the Show Jumping discipline: it may also be relevant in other equestrian disciplines, such as dressage competitions.
In Spain, the Pura Raza Español horse (PRE) is the most important horse breed participating in the dressage discipline and its Breeding Program focuses mainly on the selection of good dressage ability.
Studying the genetic parameters of ET measured by IR could demonstrate the suitability of this parameter to be included as a selection criterion in the PRE Breeding Program. The aims of the present study were therefore (1) to study how environmental effects influence dressage and ET traits, (2) to investigate the heritability of ET measured with IR in a practical situation related to the Breeding Program of PRE participating in dressage competitions, (3) to estimate the correlation between dressage and ET traits.

2. Materials and methods

2.1. Collecting the temperature data

A total of 343 different PRE stallions, aged from 4 to 6 years old, were analyzed, with an average of 5.09 repeated records per horse (a total of 1746 records). Records were taken during three final dressage competitions for young horses in Spain held in the same equestrian center during the years 2012, 2013 and 2014. The competitions were all held in October, under similar weather conditions. During this period, the animals were housed in stall boxes (measuring 3 m2) at the equestrian center and were fed with hay, concentrate and water ad libitum, thus providing also standardized environmental and housing conditions. The stress levels of the participating animals were assessed with ET measurement. ET samples were collected three times during each competition day (at three stages of the competition): 3 h before the competition (ET_B), just after the competition (<5 min after the dressage exercise) (ET_JA), and 3 h after the competition (ET_A), when the animal was resting. In addition, the temperature difference between ET collected 3 h before the competition and just after the competition (ET_B_JA); and the temperature difference between ET collected just after the competition and 3 h after it (ET_JA_A), were also estimated. ET images were taken with a FLIR i7 camera, following the indications of Bartolomé et al. (2013).
All the procedures used in this study complied with the animal ethical guidelines published by the International Society for Applied Ethology and met the International Guiding Principles for Biomedical Research Involving Animals.

2.2. Performance and competition data

In order to evaluate the relationship between the temperature data measured for these animals (ET measurements) and their sport performance, dressage results were obtained from the competitions where the ET data was collected. Performance score records were awarded points on a scale of 1–10 by a panel of judges. The parameters evaluated were:
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Walk (W): Rhythm, relaxation, activity, ground cover.
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Trot (T): Rhythm, suppleness, elasticity, impulsion, swinging back, ground cover, ability to collect.
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Canter (C): Rhythm, suppleness, elasticity, natural balance, impulsion, ground cover, uphill tendency.
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Submission (S): Contact, straightness, obedience, including flying changes, shoulder-in and half pass.
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General impression (GI): Potential as a dressage horse, standard of training according to age.
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Total Store (TS): The sum of the previous five scores.

2.3. Statistical procedure

Both temperature and performance parameters (ET and Dressage measurements, respectively) satisfied assumptions of normal distribution.
Six environmental factors were analyzed using a factorial General Linear Model:
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The age of the participating animals: 4 years old (140 horses, 589 records), 5 years old (118 horses, 582 records and 6 years old (111 horses, 575 records), with 26 horses that performed at at least two different ages.
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The stud (56 classes).
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The journey to the competition center (20 classes), including the combination of the following factors: ‘type of transport to the event’ (walking or by trailer/box), ‘journey duration to the event’ (<30 min, 30 min to 2 h, 2–4 h, 4–6 h, 6– 8 h and >8 h) and ‘arrival time before the beginning of the event’ (<6 h before, 6–12 h before, 12–24 h before and >24 h before).
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The training of the horse (18 classes), defined as the combination of the factors ‘number of previous events in which the animal has participated’ (<5 competitions, 5– 10, 10–20 and >20), ‘daily hours of training’ (<3 h, 3– 6 h, 6–10 h and >10 h) and ‘length of time for which the horse has been trained’ (<6 months, 6–12, 12–24 and >24 months).
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The event (including the year of the competition) (3 classes).
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The rider (207 classes).

The trip and training information was collected via a survey completed by the horse's trainer, and for the journey effect, veterinary travel guides were also checked. Data was checked to ensure that the distribution of records for training and journey effect was independent.

2.4. Genetic parameters

The genetic parameters of all the temperature and performance traits were estimated using a Bayesian procedure, which was carried out using the TM software (Legarra, 2008). A univariate linear model was developed to study the genetic value of the variables used to measure eye temperature (ET_B, ET_JA, ET_A, ET_B_JA and ET_JA_A) and those used to assess sport performance (W, T, C, S, GI and TS). The pedigree file included 3350 PRE. The environmental effects that were used in the genetic models were those that were considered statistically significant (p < 0.05) by the previous GLM analysis: age (except for ET_B_JA, ET_JA_A, C, S and GI), journey to the competition center, the horse's training, the stud and the event as systematic effects, and the rider (except for ET_B and ET_A) and a permanent environmental effect as random effects. This last effect referred to the environmental effects related to dressage competitions not included in the systematic effects and which can permanently influence an individual's performance and/or temperature traits.
Thus, the general genetic model used for each of the temperature and performance traits considered (considering all of the possible random effects), was:
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where y was the vector of observations, X the incidence matrix of systematic effects, Z the incidence matrix of animal genetic effects, W the incidence matrix of permanent environmental effects, Q the incidence matrix of the rider effect, b the vector of systematic effects, u the vector of direct animal genetic effects, p the vector of permanent environmental effects, r the vector of rider effects and e the vector of residual effects.
Marginal posterior distributions of all parameters were estimated using the Gibbs sampling algorithm. Prior distributions for systematic effects were assigned as bounded uniform prior distribution and the variance components were scaled using inverted chi-squared distributions (v = 2 and S = 0) to perform a flat prior distribution. Total Gibbs chain lengths of 1 000 000 samples for each analysis were defined, with a burn-in period of 100 000 and a thinning interval of 100.
The repeatability of each temperature and performance trait was calculated as the sum of the heritability and the ratio between the environmental permanent effect and the phenotypic variance. Furthermore, in order to assess the relative importance of the rider on these genetic models, a “rider ratio” was calculated as the ratio between the variance of the rider random effect and the phenotypic variance.
In order to study if there was a relationship between temperature and performance traits, Pearson correlations between performance and ET breeding values obtained for each parameter and its significance (p < 0.05) were assessed using Statistica software 8.0 (StatSoft Inc., 2007).
Finally, the coincidence between the animals’ genetic rankings by their breeding values was calculated using the percentage of coincidence for the top 25% of the animals in the genetic ranking of ET breeding evaluation, compared with the top 25% of the animals in the genetic ranking of dressage performance and vice versa.

3. Results

3.1. Temperature data and dressage performance: environmental effects

In this study, dressage horses obtained a mean of 35.2 °C ET_B, 36.3 °C ET_JA r and 35.9 °C t ET_A. The environmental effects that could influence temperature and performance traits the most were studied before carrying out the genetic evaluation (Table 1). According to age, statistically significant differences were found between ET values with an increasing tendency of the means with age, except for average temperature differentials (ET_B_JA and ET_JA_A) which showed a decreasing tendency with age. As regards the performance traits in walk, trot, canter and total score, points awarded were between 6.3 (submission score in 4-year-old animals) and 6.8 (walk score in 6 year-old animals). The average score obtained in 4-year-old animals was always the lowest (63.5 points). Both temperature and performance traits showed statistically significant differences due to the environmental factors studied (stud, journey duration, type of training and rider). As for the event, the average ET value in 2014 (event 3) was significantly lower than that of 2013 and 2012, except for ET_JA_A. As far as the performance traits and the event were concerned, the PRE showed a significantly worse average score in 2012 (event 1) than that shown in 2013 and 2014, for all the dressage parameters studied.
Table 1. General Lineal Model and post-hoc LSM test analysis of the environmental effects in thermography and Dressage performance variables in Pura Raza Español breed.
Traits
StudTripTrainingRiderAge (LSMean)
Event (LSMean)
4
years		5
years		6
years		Event 1Event
2		Event
3
Temperature traitsET_B*****–35.4a35.2ab35.0b35.4a35.5a34.9b
ET_JA************36.6a36.4 a35.9b36.9a36.6a35.5b
ET_A*********–36.4a35.8b35.6b36.2a36.6a35.3b
ET_B_JA********0.57a0.44a0.50a0.58ab0.64 a0.27b
ET_JA_A**********−0.24a−0.58a−0.32a−0.74a−0.10b−0.12b

Performance traitsWalk Score************6.4b6.7a6.8a6.1b6.7a6.8a
Trot Score************6.4b6.7a6.7ab6.2b6.8a6.9a
Canter Score************6.6a6.7a6.7a6.2b6.9a6.9a
Submission Score************6.3a6.5a6.6a6.1b6.7a6.7a
General Impression Score************6.4a6.7a6.6a6.1b6.7a6.8a
Total Dressage Score************63.5b66.5a65.9ab60.4b67.3a68.2a
ET_B = eye temperature taken 3 h before the competition; ET_JA = eye temperature taken just after the competition (<5 min after the dressage exercise); ET_A = eye temperature taken 3 h after the competition, when the animal was resting; ET_B_JA = difference between eye temperature taken 3 h before the competition and eye temperature taken just after the competition (<5 min after the dressage exercise) and ET_JA_A = difference between eye temperature taken just after the competition (<5 min after the dressage exercise) and eye temperature taken 3 h after the competition, when the animal was resting. abcValues within a row with different superscripts differ significantly at *p < 0.05; **p < 0.01; ***p < 0.001
Table options

3.2. Genetic parameters

Heritabilities, repeatability values and the rider ratio, are shown in Table 2 for both performance and temperature traits. The values are accompanied by the standard deviation of the marginal posterior distribution. It should be noted that these are not standard errors of estimates since a Bayesian analysis has been performed, and the standard deviation of their marginal posterior distribution usually tends to be much higher than the standard error. Heritabilities for temperature traits ranged from 0.14 (ET_JA) to 0.50 (ET_A), while heritabilities for dressage traits ranged from 0.37 (submission score) to 0.44 (walk score). The repeatability for temperature traits ranged from 0.25 (ET_JA) to 0.77 (ET_A), whereas repeatabilities for dressage traits ranged from 0.71 (submission score) to 0.79 (walk score). The ratio for the rider was highlighted in the IRT measured just after the competition, and was three times higher than the heritability.
Table 2. Mean (and standard deviation) of the marginal posterior distributions means for heritabilities, repeatability and the rider ratio, for all the traits analyzed.
Trait
HeritabilitiesRepeatabilityRider ratio
Temperature traitsET_B0.38(0.173)0.76(0.343)
ET_JA0.14(0.082)0.25(0.153)0.45(0.127)
ET_A0.50(0.216)0.77(0.431)
ET_B_JA0.17(0.113)0.30(0.204)0.24(0.161)
ET_JA_A0.31(0.180)0.59(0.389)0.30(0.243)

Performance traitsWalk Score0.44(0.183)0.79(0.370)0.19(0.119)
Trot Score0.40(0.187)0.76(0.370)0.22(0.117)
Canter Score0.43(0.176)0.78(0.352)0.20(0.113)
Submission Score0.37(0.163)0.71(0.326)0.28(0.123)
General Impression Score0.40(0.168)0.73(0.335)0.26(0.127)
Total Dressage Score0.42(0.179)0.73(0.350)0.24(0.121)
ET_B = eye temperature taken 3 h before the competition; ET_JA = eye temperature taken just after the competition (<5 min after the dressage exercise); ET_A = eye temperature taken 3 h after the competition, when the animal was resting; ET_B_JA = difference between eye temperature taken 3 h before the competition and eye temperature taken just after the competition (<5 min after the dressage exercise) and ET_JA_A = difference between eye temperature taken just after the competition (<5 min after the dressage exercise) and eye temperature taken 3 h after the competition, when the animal was resting.
Table options
To ascertain any possible relation between ET and sport performance results, Pearson correlations between the ET and dressage EBV were made (shown in Table 3). The dressage EBV and the ET EVB just after the exercise (ET_JA) were all low, but significantly correlated (except for the walk), ranging from 0.19 (trot) to 0.28 (general impression).
Table 3. Correlations between eye temperature and performance breeding values.
Trait
Breeding value of temperature traits
ET_BET_JAET_AET_B_JAET_JA_A
Breeding value of performance traitsWalk Score−0.000.070.07−0.10−0.13
Trot Score0.020.19*0.120.00−0.05
Canter Score0.070.22*0.14−0.040.02
Submission Score0.000.22*0.11−0.03−0.00
General Impression Score0.020.28*0.10−0.060.02
Total Dressage Score−0.000.21*0.09−0.03−0.03
ET_B = eye temperature taken 3 h before the competition; ET_JA = eye temperature taken just after the competition (<5 min after the dressage exercise) ET_A = eye temperature taken 3 h after the competition, when the animal was resting; ET_B_JA = Difference between eye temperature taken 3 h before the competition and eye temperature taken just after the competition (<5 min after the dressage exercise) and ET_JA_A = Difference between eye temperature taken just after the competition (<5 min after the dressage exercise) and eye temperature taken 3 h after the competition, when the animal was resting. *p < 0.05; **p < 0.01; ***p < 0.001.
Table options
To study a possible interaction when making genetic selection for dressage performance and for ET parameters, two different coincidences were calculated: the coincidence of the top 25% animals with the best EBV (higher scores) with the top 25% animals obtaining the highest EBV (more stressed animals) for ET parameters and the coincidence of the bottom 25% animals with the worst EBV (lower scores) for dressage performance with the bottom 25% animals obtaining the lowest EBV for ET values (less stressed animals) (see Table 4). When we analyzed the bottom 25% of the animals for temperature traits and performance in dressage, the percentage of matching animals ranged from 14.29% (between ET_JA_A and walk) to 37.14% (for ET_JA with submission score and trot score). When the top 25% of the animals for both parameters (temperature and performance parameters in dressage) was selected, we observed that there was a match that ranged from 17.14% (for ET_B_JA with walk, canter, general impression and total dressage score and for ET_JA_A with walk) to 40.00% (for ET_A with canter score).
Table 4. Percentage of coincidence of the animals ranked by their breeding values (EBV), comparing the bottom 25% EBV (above, in bold) with the top 25% EBV (below) obtained between eye temperature and dressage performance, for every analyzed trait.
% match
Breeding value of temperature traits
(bottom 25% EBV; top 25% EBV)
ET_BET_JAET_AET_B_JAET_JA_A
Breeding value of performance traits
(bottom 25% EBV;top 25% EBV)		Walk Score22.86%
28.57%		20.00%
28.57%		22.86%
28.57%		20.00%
17.14%		14.29%
17.14%
Trot Score17.14%
22.86%		37.14%
37.14%		22.86%
34.28%		28.57%
25.71%		22.86%
25.71%
Canter Score25.71%
28.57%		31.43%
31.43%		25.71%
40.00%		28.57%
17.14%		22.86%
31.43%
Submission Score22.86%
25.71%		37.14%
37.14%		25.71%
37.14%		25.71%
20.00%		20.00%
28.57%
General Impression Score25.71%
28.57%		31.43%
25.71%		25.71%
34.28%		31.43%
17.14%		20.00%
22.86%
Total Dressage Score22.86%
31.42%		34.29%
25.71%		25.71%
31.42%		28.57%
17.14%		22.86%
22.86%
ET_B = eye temperature taken 3 h before the competition; ET_JA = eye temperature taken just after the competition (<5 min after the dressage exercise); ET_A = eye temperature taken 3 h after the competition, when the animal was resting; ET_B_JA = difference between eye temperature taken 3 h before the competition and eye temperature taken just after the competition (<5 min after the dressage exercise) and ET_JA_A = difference between eye temperature taken just after the competition (<5 min after the dressage exercise) and eye temperature taken 3 h after the competition, when the animal was resting. BV = breeding value.
Table options

4. Discussion

The present study is the first to provide heritabilities and repeatabilities of ET assessed with IR in sport horses, analyzing the potential for this temperature trait to be included in horse performance tests for routine evaluations and for its later inclusion in Breeding Programs for sport horse breeds.

4.1. Temperature data and dressage performance: environmental effects

The fear and anxiety response is associated with a liberation of Adrenaline and Noradrenaline (Kvetňanský, 1973) by the adrenal medulla, as a result of the activation of an acute stress response (Fell et al., 1985), facilitating the activation of the Hypothalamic-Pituitary-Adrenal (HPA) axis (Dinan, 1996). These findings supported previous studies in horses that related changes in ET with activation of the HPA axis (Cook et al., 2001 and Valera et al., 2012), and both had a similar physiological basis. In this study, dressage horses obtained a mean ET value similar to those found by Dai et al. (2015) in animals of different breeds and ages, measured before and after a Novel Object Test, highlighting a fear response associated with the increase of ET.
During exercise, several environmental factors can affect either horse performance or the stress perceived by the animal, biasing its results. In fact, the six environmental factors analyzed in this study (age, stud, trip stress, training, event and rider) all produced statistically significant differences in both temperature and performance traits.
As regards age, in general, the ET corrected means by age were lower than those shown by Spanish Sport Horses (CDE) in Show Jumping competitions (Bartolomé et al., 2013). This could be due to a number of different factors: First, Dressage submits the animal to less potentially frightening stimuli than Show Jumping. The former focuses more on the concentration and precision of the movements performed in a “reprise” in an empty arena, whereas the latter takes place on a track full of colorful obstacles, with the occasional pool of water, all of which are challenging, stressful objects (Hausberger et al., 2004), especially for young horses (Visser et al., 2002 and Visser et al., 2003), hence producing a greater stress response. Secondly, Show Jumping exercises involve greater physical effort, which naturally increases the physiological parameters. The third factor is the horse breed used for these studies. For the Show Jumping study, a cross-breed horse (CDE) (Bartolomé et al., 2011) was used, whereas for the Dressage, the PRE was used. As suggested by different authors (Hausberger and Muller, 2002 and Lloyd et al., 2008), the different breeds show differences in temperament and reactivity, with the CDE more reactive and temperamental than the PRE, which is a pure-bred horse, bred to be noble and docile, as stated in the official PRE Studbook regulations (ANCCE, 2012).
On the other hand, the descending trend observed in ET and the ascending trend observed in performance traits from 4 to 6 years old animals for every parameter measured, may be due, first, to the presence of an underlying learning component that would imply a habituation of the animal to the potential stressors that it may have encountered during the dressage events (Hall et al., 2011) and, secondly, to the improvement of their proficiency in specific skills, experience and stability, thus giving them the ability to perform better. König et al. (2012) indicated that horses easily got used to new stimuli, even when tests were spaced several weeks apart and when no particular training was undertaken meanwhile. Moreover, growth with age also brings an improvement of sport endurance, due to the progressive and continuous training received during the years to meet the physical demands of dressage (Evans, 2008). These findings were in accordance to those found in CDE during Show Jumping competitions (Bartolomé et al., 2013).
Other effects that were statistically significant for all temperature and performance traits were the “stud”, “trip stress” and “training”. These effects were considered together as they all reflect the breeder/owner characteristics in some way. The “stud”, because it reflects the genetic background of the animal, which could influence the inherited temperament, reactivity and fear of the animal (Dai et al., 2015 and Oki et al., 2007) and hence its tendency to become stressed over new stimuli, together with its ability to perform in dressage (Sánchez Guerrero et al., 2014).
The “trip stress” factor combines effects of the journey, all of which are well known for producing stress in the horse (Padalino, 2015), which could bias both its sensibility to new environmental conditions and its later sport performance. And finally the “training” effect, which is also a combination of effects, including an owner effect, since it reflects the owner's preferences for training. Recent studies have highlighted the major influence that the training method and routine used in horses has, not only on their performance development, but also on their character, behavior and sensitivity to becoming stressed by environmental stimuli (McGreevy et al., 2012 and Visser et al., 2009). As regards the “event” effect, in general, older animals showed the lowest ET and ET interval values, and the highest performance scores, which highlights an underlying learning component in these animals that affects the animal, first by improving the psychological adaptation to new environmental stimuli and hence diminishing the associated stress response (Mengoli et al., 2014), and second, by improving the physical adaptation, the sports skills and resistance of the horse to performing the type of exercise required in dressage (Sánchez Guerrero et al., 2014), hence improving their sports results.
Finally, the “rider” in this study influenced all temperature and performance characters, as expected, since Dressage can only be performed well when horse and rider are in perfect communication in order to perform the required figures and transitions in the “reprise” (Van Erck-Westergren and Foreman, 2014). Furthermore, recent studies have also reported how this combination could strongly influence the behavior and reactivity of the horse during competitions and hence its sensitivity to developing a stress response that could affect its sport performance (Munsters et al., 2012 and Wolframm and Meulenbroek, 2012). In fact, differences in stress assessed with ET were also found in sport horses for this rider–horse relation (Bartolomé et al., 2013 and Hall et al., 2014). Finally, this factor has also been demonstrated as a key effect as regards the genetic evaluations of sport horses to improve Dressage performance (Sánchez Guerrero et al., 2014 and Vicente et al., 2014), which was also found to be essential for the estimation of genetic parameters for the temperature parameter evaluated in this study.
However, from our results it would be more accurate to just infer that the ET may be considered an approximate marker of arousal and stress, considering this stress as the physiological response of the horse that tries to restore homeostasis after a threatening stimulus, rather than as emotional distress that could be related to a fear response of the horse.

4.2. Genetic parameters

As regards the genetic analysis, the authors are aware that the small sample size of the horses used in this study is a limitation. Nevertheless, the estimated heritability lies in the range found in other genetic studies for stress related to reactivity in horses (Rothmann et al., 2014) and slightly higher than the estimated heritabilities of behavioral responses to the inspections of conjunctiva, auscultation and blood sampling in the Japanese Thoroughbred (Oki et al., 2007). Taking into consideration that ET is measuring the arousal secondary to the stress response, it could be measuring either the stress related to fear or anxiety (expressed through a behavioral response), the stress related to a great physical effort, which is accomplish during the dressage discipline, or a combination of both. Regarding the genetic variation of behavioral responses in horses, Hausberger et al. (2004) reported that stallions could share similar genetic factors and influence neurotic reactions in their progeny. On the other hand, the repeatabilities found in this study for ET_B and ET_A were higher than those found by König et al. (2012) for temperament scores in a similar number of horses, which in turn were higher than those found for ET measured just after exercise and for both the intervals calculated. All in all, they were all considerably lower than the repeatability values found by Oki et al. (2007) in racing horses, probably due to the huge number of animals used for that study in comparison with the number used in this one.
In addition, heritability estimates found in the recent literature for walk, trot, canter and the final dressage score showed similar values to ours (Ducro et al., 2007, Sánchez Guerrero et al., 2014 and Vicente et al., 2014). Despite this, the heritabilities could be overestimated because we were unable to include the rider–horse interaction as a random factor in this study due to the small number of animals and riders studied.
Furthermore, the ratio for the rider effect was almost three times higher than the heritability for temperature parameters, despite the fact that it represented almost half the heritability of all performance traits. This highlights the far greater importance of the rider effect for temperature parameters of the horse than for performance, as the rider influences considerably the horse's perception of the exercise they have to carry out and hence its perception of the new stimuli associated.
When analyzing the correlations between the EBV calculated for each performance and temperature trait, only those between performance traits and ET_JA were statistically significant with medium values, which confirms previous studies supporting the suitability of this temperature parameter to assess stress related to sport performance in horses (Bartolomé et al., 2013 and Valera et al., 2012). However, the positive sign for correlations found here was contrary to that found in the studies referred to above. We have to take into account that the performance and temperature traits correlated in this study were breeding values, unlike those found by Bartolomé et al. (2013), which were rough performance and temperature results obtained during competitions. A breeding value is a more accurate indication of an animal's genetic ability for a certain trait, because all available information (genealogical, environmental, individual results, etc.) is used to obtain an EBV, not only the animal's own results (Cassady and Wayne Robinson, 2002). Hence, accounting for the genetic correlation between EBV for performance and temperature traits would indicate the strength of the genetic relationship between these traits. Thus the positive correlation found in this study between stress measured by ET and dressage sport performance would comprise that selection made to improve one trait would improve the other. Besides, taking into consideration that an increase in ET implies also increasing the sympathetic arousal with an associated liberation of catecholamines into the blood stream, it could be associated with a more proactive response of the horse to the environmental stimuli, rather than with a distress response. That is, horses with a higher ET EBV would tend to transmit the ability to take an action and perform when their stress response is highly activated, while horses with a lower ET EBV would respond to the stressful context with a more reactive/emotional response that impedes them to perform well, hence obtaining worse results.
On the other hand, Górecka-Bruzda et al. (2014) found that Dressage and Show Jumping disciplines exert different pressure or stressful influences on the horses: dressage horses may have to cope with multidimensional, over-complex or somewhat ambiguous cues or signals from the rider, whereas show jumping horses tend to display resistance to elements of restraint. Besides, in dressage horses, the physical activity performed by the horse during the exercise may also influence its ET, thus increasing its value as a performer and thus supporting our findings, with animals with higher ET values being a consequence of a higher physical activity and a more proactive attitude when performing. Furthermore, as PRE horses are known to have a calm and docile temperament and seem to be preferred by the judges as “the perfect dressage performers”, it could hence be inferred that if more temperamental PRE animals performed these exercises, the judges would perceive a more energetic and powerful performance and would assign them higher scores.
The coincidence between the highest 25% EBV and lowest 25% EBV for Dressage ability and the highest 25% EBV and lowest 25% EBV animals for ET measurements showed that the coincidence between the animals selected as parents of the next generation (best BV values for both parameters) was higher than between the animals that were discarded for selection (worst BV values for both parameters) within the population. Hence, when selecting for the best dressage horses, we would select also the animals with highest ET values, or more proactively stressed animals, thus supporting the previous correlation results we obtained. This could be due to the fact that, for Dressage, more proactive animals are desirable in the case of PRE due to the psychological and physical efforts they have to make, since the dressage exercise requires considerable effort on the part of the horse to cope with the challenge of the rider's demands, besides the fact that the physical activity performed by the horse during the exercise might increase its ET.
When breeding sport horses, breeders have to take into consideration different factors, including parameters related to conformation, sport ability, gaits, health, fertility and even psychological traits (Koenen et al., 2004). However, none of these include sport-related stress as a possible parameter to be implemented or even to be accounted for. Despite the fact that behavior could also be described as a measurement of stress, as it is the expression of the horse's reaction when confronting a new stimuli (Moberg, 2000), the methodologies used to measure it have not been sufficiently standardized and parameterized to obtain valuable, objective, physiological and psychological information, despite being considered in general as an extremely relevant breeding parameter by sport horse breeders (Koenen et al., 2004). Hence, including this easily measurable parameter (ET assessed with IR) which would be easy to use in sport horse competitions, could prove to be of great interest in obtaining valuable information about the stress of horses competing in Dressage. However, more evidence about its validity, sensitivity and specificity is still to be proved before including this parameter in a Breeding Program as an additional selection criterion for improving Dressage performance in horses.

5. Conclusion

The results obtained in this study highlight the fact that dressage performance was influenced by the level of physiological stress developed by the animals participating in competitions.
ET was therefore seen to be a suitable method for assessing stress in sport horses competing in dressage competitions, despite the fact that this measure was influenced by different environmental factors such as the stud, the stress related to the journey, the previous training of the horse, the event and the age of the animal or the rider. Thus, calculation of EBVs developed in this study corrected the effect of all of them on the performance and temperature traits considered.
Low-medium heritability values of ET were obtained when ET was assessed during the final dressage championship in young horses. These findings indicate that it is possible to assess selection for stress with ET in horses. Furthermore, correlations between performance and ET parameters highlighted that a selection made to improve ET values would also improve Dressage performance. However, despite ET has been shown to be a valuable tool for stress assessment in horses during Dressage competitions, more evidence about its validity, sensitivity and specificity is still needed in order to include ET in sport competitions as an additional selection criterion in horses. The positive medium correlations found between ET and performance breeding values suggested that horses with higher ET values were better dressage performers, appearing to be related with a more proactive and energetic response of the horse to the environmental stimuli and hence, to physiological stress rather than emotional stress (or “distress”).
Finally, a selection of the best animals for dressage performance would also involve selecting for the animals with highest ET values, or more proactive animals. This study highlights the potentially valuable role of ET as a tool for selecting Dressage horses according to their physiological stress response. However, further research is needed to support the conclusions found here.

Conflict of interest

None.

Acknowledgement

The authors wish to thank the National Association of Pura Raza Español Horse Breeders (ANCCE) for providing the data used in this study.

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