Journal of Equine Veterinary Science 75 (2019) 1e8

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Journal of Equine Veterinary Science

journal homepage: www.j-evs.com

Original Research

Eye Surface Temperature as a Potential Indicator of Physical Fitness in

Ranch Horses
Pedro Henrique Esteves Trindade a, Guilherme de Camargo Ferraz b,
~o d, Mateus J.R. Paranhos da Costa e, *
~o Alberto Negra
Maria Lúcia Pereira Lima c, Joa
a
UNESP, Universidade Estadual Paulista, Programa de Po s-Graduaça~o em Zootecnia, Faculdade de Ci^ rias e Veterina
encias Agra rias, Jaboticabal, Sa~o Paulo,
Brazil
b
UNESP, Universidade Estadual Paulista, Departamento de Morfologia e Fisiologia Animal, Faculdade de Ci^ encias Agrarias e Veterina rias, Jaboticabal, Sa
~o
Paulo, Brazil
c ~ozinho, Sa
Instituto de Zootecnia (IZ), Serta ~o Paulo, Brazil
d
USP, Universidade de Sa~o Paulo, Departamento de Ci^ sicas, Faculdade de Zootecnia e Engenharia de Alimentos, Pirassununga, Sa
encias Ba ~o Paulo, Brazil
e
UNESP, Universidade Estadual Paulista, Departamento de Zootecnia, Faculdade de Ci^ encias Agrarias e Veterina
rias, Jaboticabal, Sa~o Paulo, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: This study aimed to investigate the use of maximum eye surface temperature (ESTmax), recorded by infrared
Received 12 April 2018 thermography, as an indicator of physical fitness in ranch horses. Data collection was carried out with 16
Received in revised form horses by recording six physical fitness biomarkers (ESTmax, respiratory rate, creatine kinase activity [CK], and
18 October 2018
concentrations of total serum protein, plasmatic cortisol, and lactate) before and after a regular working day.
Accepted 26 November 2018
Available online 21 December 2018
We also recorded heart rate during the exercise, exercise duration, average velocity, distance traveled, and air
temperature and humidity. The variation between the sampling times was compared by using repeated
measures ANOVA or Wilcoxon paired test. The t-test was used to compare ESTmax means of dehydrated and
Keywords:
Animal welfare
nondehydrated horses. The differences (DIF-) between each variable's sampling times were calculated, and
Creatine kinase activity the Spearman's correlation coefficients between DIF-ESTmax and DIF- of other variables were estimated.
Infrared thermography A hierarchical cluster analysis was used to classify the horses according to their physical fitness. All biomarkers
Equine increased after the workday, and the ESTmax means of dehydrated and nondehydrated horses did not differ
Muscle damage (P < .05). DIF-CK and DIF-ESTmax were positively correlated (rs ¼ 0.56, P < .05). The cluster analysis defined two
groups of horses (LOW and HIGH), with DIF-ESTmax and DIF-CK differing statistically between them. HIGH
horses showed CK higher than 350 U/L, indicating some degree of muscle damage. These results suggest that
ESTmax has potential to predict changes in CK and, thus could be a useful indicator of physical fitness in ranch
horses; although, its effectiveness has to be confirmed in future studies.
© 2018 Elsevier Inc. All rights reserved.

1. Introduction cattle farms, especially in countries with significant beef cattle

Around the world, horses perform various activities that involve
physical exercise, such as sports competitions, leisure, military, and
agricultural use. Horses are commonly used for working on beef
Conflict of interest statement: The authors declare that there is no conflict of in-
terest.
Animal welfare/ethical statement: This study was approved by the Ethics Com-
mittee on the Use of Animals of the Faculty of Agricultural and Veterinary Sciences,
UNESP, Jaboticabal-SP, Brazil (protocol number 13466/15).
* Corresponding author at: Mateus Jose  Rodrigues Paranhos da Costa, UNESP, Sa~o
Paulo State University, Rod. Prof. Paulo Donato Castellane, km 5 Departamento de
Zootecnia, FCAV-UNESP, 14884-900 Jaboticabal, S~ ao Paulo, Brazil.
E-mail address: mpcosta@fcav.unesp.br (M.J.R. Paranhos da Costa).
production, such as Australia, Brazil, Canada, and the United States
[1e4].
In tropical countries, physical exercise may affect horses' ther-
mal homeostasis, with potential negative impact on animals’ health
and physical fitness. Physical activity is a relatively inefficient
process, as a large amount of chemical energy is converted into
thermal energy instead of mechanical energy [5], which increases
the risk of stress and muscle disorders. Such conditions can be
identified through clinical diagnoses [6], or by monitoring specific
biomarkers: heart and respiratory rates, lactate and cortisol con-
centrations, and creatine kinase enzyme activity [7e10].
Physical exercise may result in momentary loss of homeostasis
[7,9]. After exercise, lactate increases due to anaerobic metabolism;
thus, lactate may be used to indicate aerobic capacity [11e16].

https://doi.org/10.1016/j.jevs.2018.11.015
0737-0806/© 2018 Elsevier Inc. All rights reserved.
2 P.H. Esteves Trindade et al. / Journal of Equine Veterinary Science 75 (2019) 1e8
Table 1
Chronological description of data collection for 16 stock horses on beef cattle farms, considering the assessments before and after the workday.
Periods Collection Times
Before Workday 14 hr before exercise (between 5:30
and 6:30 PM on the day before)
After Workday Immediately after exercise
(11:00 AM or 4:00 PM)
10e12 min after workday
10e12 min after workday
15 min after workday
30 min after workday
60 min after workday
6 hr after workday
12 hr after workday
Cortisol release during exercise results in energetic availability
through hepatic gluconeogenesis and the production of lipolysis
promoters [7,9]. Moreover, physical effort can cause microlesions in
the sarcolemma, increasing membrane permeability and allowing
extravasation of CK into the blood stream. Thus, CK can be used to
assess muscle damage after physical exercise [10,17e21].
During the last decades, scientists have been investigating the
use of infrared thermography (IRT) as a noninvasive method to
quantify body surface temperature [22e25]. A positive relationship
was found between increased maximum eye surface temperature
(ESTmax) and salivary cortisol concentrations after adrenocortico-
tropic hormone injection in horses [26]. The increased eye surface
temperature is probably related to ocular vasodilation, which leads
to higher heat dissipation (identified by IRT), specifically in the
lacrimal caruncle, which contains capillaries controlled by the
sympathetic nerves [27e29]. Other studies also reported a positive
relationship between increased ESTmax (measured by IRT) and
behavioral indicators of fear during novel object, voluntary and
forced approach tests [28], as well as with heart rate during
sporting competitions [30]. However, other researchers who tested
stress caused by visual and auditory stimuli during events [31e33]
observed increased ESTmax in horses after physical exercise but no
association between ESTmax levels and cortisol concentrations.
To the best of our knowledge, no studies to date have evaluated
the relationship between ESTmax and the effects of physical exercise
in horses. We hypothesize that there is a relationship between
ESTmax, workload variables, and physical fitness biomarkers in
horses. Thus, the aim of this study was to investigate the usefulness
of the ESTmax, as measured with IRT, as an indicator of physical
fitness in ranch horses.
2. Materials and Methods
This study was approved by the Ethics Committee on the Use of
Animals of the Faculty of Agricultural and Veterinary Sciences,
UNESP, Jaboticabal-SP, Brazil (protocol number 13466/15).
2.1. Animals and Handling
Sixteen healthy (evaluated by physical exam and hemogram, see
Supplementary File) ranch horses were assessed (15 castrated
males and 1 female; ages 6e27 years old, mean ± SD: 14 ± 6 years
old). The horses were kept on two commercial beef cattle farms:
farm 1 (F1; n ¼ 8) and farm 2 (F2; n ¼ 8) located in the municipality
aozinho-SP, Brazil (21.1380 S, 47.9901 W, and approxi-
of Sert~
mately 579 m altitude). The horses were commonly used for
Data Collection Evaluations
Respiratory rate Flank/nostril movements count for 15 s
Environmental information Air temperature and humidity
Infrared thermography Eye images
Blood sample Total protein, cortisol, lactate and creatine kinase
Respiratory rate Flank/nostrils movements count for 15 s
Blood sample Total protein, cortisol, lactate and creatine kinase
Environmental information Air temperature and humidity
Infrared thermography Eye images
Blood sample Lactate
Blood sample Lactate
Blood sample Lactate
Blood sample Creatine kinase
Blood sample Creatine kinase
herding cattle and moving around the farm to check fences, water
troughs and pastures conditions.
Horses were kept at pastures (minimum of two horses per
paddock) with free access to Cynodon sp. grass, water, and mineral
supplement, and on F1, each horse received a dietary supplement of
1 kg of corn, 1 kg of soybean meal, and 800 g of calcium carbonate
per day and on F2, 2 kg of commercial concentrate per day.
2.2. Data Collection and Measured Variables
Data were collected on seven noncontinuous days during
summer, from December 2015 to January 2016. Horses were always
assessed in pairs and the workday session started after a period of
26 hours without any forced exercise. Each pair of horses was
assessed before and after a regular working day at the farm and
each horse acted as its own control, as described in Table 1. To
minimize disturbances to the farms' routine, basal data (before
exercise) were collected 14 hours before exercise, at the end of the
stockpeople's workday (between 5:30 and 6:30 PM). In addition, to
avoid possible lesions in the smooth musculature of the jugular
vein and the adjacent skeletal muscle due horse response from
venipuncture blood samples were collected always alternating
between left and right sides of the horse’s neck [34]. Lactate and
creatine kinase sampling were conducted 15e30 minutes [35] and
6e12 hours [10,36] after exercise, respectively, to obtain peak
concentrations. Following hormonal circadian cycles, the post-
workday samples were collected around 11:30 AM to 12:30 PM or
4:30 PM to 5:30 PM, to be compared with the pre-exercise samples
(5:30 PM to 6:30 PM).
During the workday, horses were ridden by the stockperson
who usually worked with them. Ten horses worked half a day (al-
ways in the morning) and six horses worked the whole day
(morning and afternoon), according to the farm's routine. Horses
that worked all day had 1.0 ± 0.05 hours of interval during lunch-
time. During this period, they were kept in a paddock, without the
bridle, but with the saddle on their backs. There was no control of
water intake during the workday or the interval. However, when
kept in the paddock or in the stall (after the workday), horses had
free access to water and mineral supplementation and received
supplementary feeding according to their regular diet.
2.3. Infrared Thermography
We assessed horses' eye surface temperature by thermograms
captured with a digital thermographic camera (ThermaCam i50;
FLIR, Finland) with a 19,600-pixel resolution, thermal sensitivity of
< 0.1 C, and range of capturing temperatures between 20 C and
 P.H. Esteves Trindade et al. / Journal of Equine Veterinary Science 75 (2019) 1e8
350 C. All horses were kept in the shade before capturing the
infrared images to minimize the effect of direct solar radiation. We
waited for 10e12 minutes for animals’ acclimatization to the fa-
cility, where the infrared images were taken. The facility had open
sides and roof with ceramic tiles.
All horses were photographed on the left side, with the camera
positioned at a 90 angle to the head and approximately 0.5 m from
the animal, and emissivity of 0.98 mm (the emissivity for
mammalian skin) [37,38]. According to Hall et al. [39], horse eye
temperature measured by IRT does not vary between the left and
right eyes, so we chose to measure all animals on the left side.
To aid thermogram recording at a standard angle and distance,
we selected the “area” option for measurement analysis on the
thermal camera, which produced a sample box of 46  46 pixels,
clipped from the center of the screen. Before data collection, we
checked which anatomical region of the eye was delimited by the
box positioned in the center of the screen when the camera was
within 0.5 m measured by a digital laser beam (TLM99, Stanley,
Brazil). This thermal camera option is usually used to calculate in
real time the minimum or maximum temperature within the box
generated. However, we used this box to help frame the eye during
data collection.
Air temperature (TEMP) and humidity (HUMI) inside the facility
were recorded with a digital thermo-hygrometer (Datalogger
AK170 USB; AKSO, Brazil) at the time of thermographic image
capture. After data collection, we used only the thermograms that
showed sufficient focus to delimit the anatomical regions of the
eye. Next, we used a thermal imaging analysis software (FLIR Tools,
FLIR Systems, version 5.5.1) to determine the maximum (ESTmax)
and mean (ESTmean) eye surface temperature at the commissure
region of the lacrimal caruncle in a circular area with a 22-pixel
radius, as illustrated in Fig. 1. The distance at which the images were
recorded, as well as TEMP and HUMI, were included in the soft-
ware, which corrects for any environmental variations. We chose
Fig. 1. Example of the thermogram of the left eye of the horse captured with a 90 angle
to the head, at a distance of approximately 0.5 m, and emissivity of 0.98 mm. The red
triangle indicates the maximum temperature and the blue triangle indicates the mini-
mum temperature ( C) in the circular area (22-pixel radius) of the commissure region of
the lacrimal caruncle, from where the maximum surface temperature of the eye was
assessed using an image analysis software (FLIR Tools, FLIR Systems, version 5.5.1).
3
the region of the lacrimal caruncle commissure because it contains
less hair, thus minimizing any interference in the thermographic
measurement [26,28,30,33,39,40].
2.4. Habituation and Conditioning for Blood Sampling
Before data collection, we tested the horses’ reactions to blood
sampling and noticed that some horses on F1 showed signs of
avoidance or aggression (e.g., attempting to bite or kick, moving the
neck or body away from the person, or positioning ears back or
flattened). To minimize the risk of accidents, these horses under-
went a conditioning process using positive reinforcement, as
described by Trindade et al. [41]. None of the F2 farm horses
showed any avoidance or aggressive behavior during the pre-
liminary blood sampling and therefore were not trained.
2.5. Blood Samples and Respiratory Rate
Blood samples were collected by jugular venipuncture, always
alternating between the left and right jugulars veins. To determine
plasma lactate concentrations (LAC), we used 4 mL tubes containing
fluoride/EDTA; the blood used to assess the enzymatic activity of
creatine kinase (CK) and serum concentration of total protein (TP)
was collected in 10 mL tubes containing clot activator and separation
gel (SST II Advance gel); and to determine plasma cortisol concen-
tration (COR), we used 10 mL tubes containing sodium heparin. All
blood samples were stored in an ice cooler box (with recyclable ice)
which was kept in the shade until sent to the laboratory, where they
were centrifuged for 5 minutes at 560 G force. Next, the supernatant
from the samples was pipetted and transferred to Eppendorf tubes,
which were labeled and stored at 4 C.
TP assays were performed in duplicate (intra-assay coefficient of
variation was 4.7%) by using the biuret method, and the CK activity
tests were carried out by the kinetic method; these two assays were
conducted in a semiautomatic analyzer (Labquest, Labtest, Brazil)
using commercial kits (Labtest, Brazil). LAC quantification was
conducted via the electroenzymatic method using an automatic
bioanalyzer (YSL 2300 Sport, Yellow Springs). We repeated the first
three samples twice to evaluate the intra-assay coefficient of vari-
ation (CV ¼ 11%). Cortisol concentration was measured with a
commercial ELISA kit (Enzo Life Sciences Inc, Farmingdale, NY),
using the Mulstiscan reader (ThermoFisher Scientific, Waltham
MA), intra- and inter-assay coefficients of variation were 2.7% and
6.0%, respectively, and the sensitivity of the dosage was 0.15 ng/dL.
Cortisol concentration (immediately postworkday) and CK
(immediately and 12 hours postworkday) concentrations were
analyzed after correction to avoid the influence of hemoconcen-
tration, taking into account the changes in TP, according to
Cywinska et al. [42].
Respiratory rate (RR) was measured twice (14 hours before and
immediately after the workday) by counting the respiratory
movements in horses’ flanks or nostrils for 15 seconds and multi-
plying by four (movements/min).
2.6. Workload Variables
The workload was evaluated using a GPS-coupled frequency
meter (Garmim Forerunner 310XT, Garmin), which recorded dis-
tance traveled (DIS, km), exercise duration (DUR; h) and average
velocity (AV; km/h). An equine adapter kit (Equine Heart Rate
Monitor System, V-Max) was used to obtain the heart rate
(HR, bpm) and was coupled to the frequency meter.
One equine adapter kit electrode was positioned between the
third and sixth thoracic intercostal space, above the humeral
scapular joint on the left side of the animal's body, and the other
4 P.H. Esteves Trindade et al. / Journal of Equine Veterinary Science 75 (2019) 1e8

was positioned on the right side of the withers. Both were wet with A B
saline solution and conductive gel before their placement. Elec-

Maximum eye superficial temperature (°C)

P=0.003 P<0.000
trodes were attached to the animal's body with elastic tape [35].

35.5 36.0 36.5 37.0 37.5

We extracted the HR information from the frequency meter using

Plasmatic cortisol (ng/dL)

15
the equipment manufacturer's software (Garmin connect, Garmin).
Heart rate was recorded every 5 minutes during the workday, and

10
based on this information, we calculated the HR mean during ex-
ercise for each horse as an indicator physiological demand.

5
2.7. Statistical Analyses

0
14 h before Immediataly 14 h before Immediataly
All statistical analyses were performed using the software RStudio
workday after workday workday after workday
[43] (version 1.0.143). First, we estimated the Spearman's rank cor-
relation coefficients (P  .05, function “rcorr” of the R “Hmisc” pack-
age) between ESTmax and ESTmean, as well as DUR and DIS, to C P=0.033 D P<0.000
determine whether these variables were independent of each other.

20 40 60 80 100 120
10
Total serum protein (g/dL)
Statistical analyses were carried out in four steps: first, we tested

Respiratory rate (mpm)

whether the variables changed after the workday. We tested

9
normality with the ShapiroeWilk test (P .05; function “shapiro.test”
of the R “stats” package). Because ESTmax, LAC, and CK showed a

8
normal distribution, we used repeated measures analyses of variance
using mixed linear models for repeated measures (function “lmer” of

7
the R “lme4” package). For ESTmax, the model considered sampling

time as a fixed effect, and horse, age of horses, TEMP, and HUMID as
random effects. For LAC and CK, sampling time was also included as a
fixed effect, and horse and age of horses as random effects. The means
of the sampling times were compared using Tukey tests (P  .05;
functions “lsmeans” and “cld” of the R “lsmeans” package). Because
RR, TEMP, HUMID, COR, and TP did not show a normal distribution,
sampling times (14 hours before and immediately after the workday)
were analyzed with the paired Wilcoxon test (P  .05; function
“wilcox.test” of the R “stats” package).
Fifty percent of the horses showed high concentration of TP after
the workday, indicating that they were dehydrated. The postwork
ESTmax means of the two groups (dehydrated and nondehydrated
horses, n ¼ 8 each) were compared using t test (P  .05; function
“t.test” of the R “stats” package), assuming gaussian distribution
applying ShapiroeWilk test.
Next, we evaluated the relationships among all variables that
changed significantly after the workday, as well as the workload and
environmental variables. To do this, we calculated the differences in
ESTmax, RR, COR, TP, TEMP, and HUMID by subtracting the values
measured immediately after the workday from the values measured
14 hours before the workday. For variables with more than two
sampling times (i.e., LAC and CK), we extracted the differences using
the sampling times that presented the greatest significant difference
(considering the lowest P-value; i.e., 14 hours before and 30 minutes
after the workday for LAC and 14 hours before and 12 hours after the
workday for CK). Next, we estimated the Spearman's correlation
coefficients (P  .05) considering the following variables: DUR, AV,
HR mean during exercise, and the differences in ESTmax (DIF-ESTmax),
RR (DIF-RR), COR (DIF-COR), LAC (DIF-LAC), TP (DIF-TP), CK (DIF-CK),
TEMP (DIF-TEMP), and HUMI (DIF-HUMI).
Finally, we classified the horses according to their physical
fitness and subjected DIF-CK and DIF-ESTmax to a nonhierarchical
cluster analysis (function “dist” of the R “stats” package), following
the “ward.D” method using “gower” measurement matrix distance
(function “vegdist” of R's vegan package). We then applied the
paired Wilcoxon test (P  .05) to compare DIF-ESTmax and DIF-CK
values between the groups formed by the cluster analysis.
3. Results
DUR and DIS (4 ± 2 hours and 18 ± 4 km, respectively) were
positively correlated (rs ¼ 99; P < .000), as well as ESTmax (36.4 ± 0.4
6
14 h before Immediataly 14 h before Immediataly
workday after workday workday after workday
Fig. 2. Comparisons of the maximum eye surface temperature (A), plasma cortisol (B),
total serum protein (C), and respiratory rate (D) measured 14 hours before and imme-
diately after the workday in 16 ranch horses. Comparisons were made using the paired
Wilcoxon test (P  .05). The reference values for cortisol, total protein, and respiratory
rate are 23.91e35.73 ng/dL, 5.6e7.9 g/dL, and 18e20 mpm, respectively [44].
and 36.8 ± 0.4 C, 14 hours before and immediately after the
workday) and ESTmean (35.4 ± 0.5 and 35.9 ± 0.5, 14 hours before
and immediately after the workday; rs ¼ 0.90; P < .000). Owing to
these high correlation coefficients, we chose ESTmax and DUR
instead of ESTmean and DIS to represent the eye temperature and
the workload, as these variables had lower standard deviations. The
biomarkers (ESTmax, RR, CK, TP, COR, and LAC) increased after the
workday (Figs. 2 and 3). The postwork ESTmax means did not differ
(P ¼ .139) between dehydrated and nondehydrated horses (36.58 ±
0.55 and 37.14 ± 0.60 C, respectively).
The means of DUR, AV, and HR during exercise were 4 ± 2 hours,
5 ± 1 km/h, and 91 ± 12 bpm, respectively. TEMP 14 hours before
working was slightly (P ¼ .015) higher than immediately after the
workday (30 ± 1 and 29 ± 1 C, respectively), and HUMI did not vary
significantly (P > .05) between sampling times (65 ± 7% and 70 ± 6%
for 14 hours before and immediately after the workday,
respectively).
We found a significant coefficient of correlation between DIF-
ESTmax and DIF-CK (rs ¼ 0.56; P < .05; Table 2 and Fig. 4), and owing
to this we used only these two variables to classify horses’ physical
fitness by the nonhierarchical cluster analysis, which separated the
horses into two groups (LOW and HIGH), with DIF-ESTmax and DIF-
CK differing statistically between the groups (Fig. 5).
4. Discussion
To our knowledge, this is the first study investigating the
relationships between the maximum eye surface temperature
(ESTmax) as a biomarker of physical fitness, and workload variables
in ranch horses after a routine workday on beef cattle farms. Our
results suggest that ESTmax has potential to be used as an indicator
of horses’ physical fitness after a day of work.
 P.H. Esteves Trindade et al. / Journal of Equine Veterinary Science 75 (2019) 1e8 5

A P<0.000 B
P<0.000
P=0.004 P=0.005

Creatine kinase activity (U/L)

1.8

500 600
Plasmatic lactate (mmol/L)
1.4

400
1.0

300
0.6

200
14 h before Immediataly 15 min 30 min 60 min 14 h before Immediataly 6h 12 h
workday after workday after workday workday after workday after workday

Fig. 3. Comparisons between different collection times for lactate plasma concentration (A) and creatine kinase activity (B) measured in 16 ranch horses. Means comparisons were
made using the Tukey's test (P  .05). The reference values for lactate are 0.50e1.78 mmol/L; and values below 350 U/L for creatine kinase activity are considered normal [44].

As expected, all variables of ranch horse physical fitness (ESTmax,
RR, CK, TP, COR, and LAC) increased following work on beef cattle
farms, corroborating results from previous studies assessing the
physiological reactions of horses subjected to physical exercise
[30e33,36,45,46].
These results, together with the workload indicators (HR, DUR,
DIS, and AV), suggest that horses performed prolonged exercise at
low speeds, producing mostly aerobic energy, which is character-
istic of submaximal exercise [10,47,48]. In addition, the physical
effort during the workday, together with the high temperatures
typical of the Brazilian summer, resulted in slight dehydration (as
indicated by changes in TP) in eight of the 16 assessed horses;
which can be explained, at least partially, by the amount of water
lost through sweating, an important mechanism of heat loss in
horses [5]. Dehydration causes chances in the perineal blood flow
[49] and, owing to this, it could have an influence in the infrared
thermography measurement. However, this was not the case
because we did not find any difference between the ESTmax means
of dehydrated and nondehydrated ranch horses.
One could expect that the increase in ESTmax would be related to
the effects of physical exercise on intraocular pressure, as reported
in humans [50e52]. However, this was not confirmed in a study
conducted with horses [53], where there was no association be-
tween increased intraocular pressure and ESTmax in postexercise
horses. Another explanation for increased ESTmax after exercise
could be the heat produced by skeletal muscle contractions, as well
Table 2
Spearman's rank correlation coefficients between the difference in maximum eye
surface temperature (DIF-ESTmax) and the differences (DIFF) in the environmental
variables (TEMP ¼ air temperature, and HUMI ¼ air humidity) and a set of physical
fitness (RR ¼ respiratory rate, COR ¼ serum concentration of cortisol, LAC ¼ serum
as by increased blood flow in the muscles [54], both resulting from
the exercise [5], that may be dissipated by convection for peripheral
tissues.
It must also be considered that environmental factors (e.g., solar
radiation, air temperature, and humidity) can affect the superficial
body temperature of animals, mainly when assessed by infrared
thermography [24,25]. Nevertheless, this did not seem to be the
case in the present study because we did not find any correlation
between ESTmax and TEMP or HUMID, and the TEMP measured
14 hours before the workday was always higher than that regis-
tered immediately after the workday. Moreover, in our study, the
mean ESTmax values measured 14 hours before the workday were
comparable with temperatures recorded by other researchers
[30,33], indicating that ESTmax increased after exercise indepen-
dently of changes in TEMP and HUMID.
Another explanation for the increased ESTmax after the workday
would be the increase in serum cortisol concentrations due to
hypothalamic-pituitary-adrenal axis (HPA) activation, as reported
for horses that showed higher ESTmax after receiving an adreno-
corticotropic hormone injection [26]. We did not observe a corre-
lation between ESTmax and COR, but both variables increased
significantly after the workday, corroborating previous findings
with cattle [27] and horses competing in equestrian modalities
[31,33]. For the horses worked half day, there is another possibility
to explain the increase of COR. It could be explained in part by the
circadian rhythm because blood samples after workday were
collected between 11:30 AM and 12:30 AM, close to the peak of
serum cortisol concentration, which occurs around 10:00 AM [55];
while the preworkday blood samples were collected around 6:30
PM, when the COR is naturally lower.
Difference of the creatine kinase activity

concentration of lactate, TP ¼ total protein, CK ¼ creatine kinase activity, and HR ¼ r = 0.56

heart rate) and workload (DUR ¼ duration of the exercise, and AV ¼ average speed of P = 0.023
300

the exercise) indicators measured 14 hours before and immediately after the
workday in 16 ranch horses.

Pairs of Variables r-Values P-Values

100

DIF-ESTmax
DIF-RR 0.30 .260
DIF-COR 0.10 .711
0

DIF-LAC 0.43 .096

DIF-TP 0.17 .537
DIF-CK 0.65 .006 -0.5 0.0 0.5 1.0 1.5
DIF-TEMP 0.01 .956
DIF-HUMI 0.12 .645
Difference of the maximum eye surface temperature
HR 0.48 .060
DUR 0.11 .677 Fig. 4. Dispersion of the differences in maximum eye surface temperature (DIF-EST-
AV 0.14 .060 max) and serum creatine kinase activity (DIF-CK) collected immediately after and
14 hours before the workday, in 16 ranch horses, by Spearman's rank correlation
Bold ¼ significant r-value (P < .01).
coefficient.
6 P.H. Esteves Trindade et al. / Journal of Equine Veterinary Science 75 (2019) 1e8

A predefined distance and angle when taken the thermograms, and

recording the TEMP and HUMID for temperature corrections.
6

Smartphones can be equipped with technology for capturing

5

infrared rays and forming a thermographic image [57]. Although we

4
Height

LOW HIGH did not find studies with horses that used smartphones to capture
3

thermograms, we did find studies with humans in which smart-

2

phone thermograms were used to diagnose vasculitis and monitor

1

tissue transplantation (free flap) [58]; the authors considered the

0

increase in the portability of the thermographic cameras as a factor

M

K
A

H
N

O
B

L
E
F

J
I
that facilitated the use and economic viability of this technology.
An important consideration is that the number of horses per
B C stockperson on Brazilian beef cattle farms is usually three or less,
P<0.000 P=0.005
far below six, the number recommended by Lima and Cintra [59].
Difference of creatine

0 100 200 300 400

Difference of maximum
eye surface temperature

This may impoverish ranch horse welfare, as animals will be more

kinase activity
-0.5 0.0 0.5 1.0

susceptible to musculoskeletal injuries. In this sense, the IRT can be

a useful tool for horse riders, allowing them to identify horses at
risk for muscle damage. One practical action would be to determine
a suitable training program for each horse, based on the level of
LOW HIGH LOW HIGH physical fitness and target workload. Such actions could avoid some
of the consequences of excessive exercise, such as rhabdomyolysis,
Fig. 5. Dendrogram generated through hierarchical grouping analysis applied to the
acute physical exhaustion, and overtraining [60,61].
differences between the values of maximum eye surface temperature (DIF-ESTmax)
measured immediately after and 14 hours before the workday, and the creatine kinase In sum, we propose the use of ESTmax (assessed by infrared
activity (DIF-CK) measured 14 hours prior and 12 hours after the workday, showing the thermography in the commissure of the lacrimal caruncle) as a
formation of two clusters, LOW and HIGH (A). Medians of DIF-ESTmax (B) and DIF-CK potential indicator of physical fatigue in horses, which may result
(C) for each group (LOW and HIGH) were compared using the paired Wilcoxon test from inadequate exercise intensities [62]. This assessment can be
(P  .05).
applied to ranch horses as well as horses from other equestrian
modalities. Future studies should consider standardizing the
We did not find any association between HR and ESTmax, dis- workload and physical fitness level of horses and subjecting the
agreeing with the results reported by Bartolome  et al. [30] when same animals to different physical effort intensities (dose-response
assessed such association in horses during sports competitions. study) and measure ESTmax over time for the development of
Stressful stimuli can activate the sympathetic nervous system and physical fitness scales based on ESTmax. We also expect that the
the HPA axis, causing blood redistribution and possibly promoting ESTmax measured before exercise may be similar when collected the
dilation of the blood vessels of the periocular musculature, raising next day or a week after the exercise (in baseline values); however,
attention or visual orientation. This process can also contribute to further studies should investigate this possibility. Our findings may
heat dissipation in the orbital region, which can be measured by IRT also be applied to future studies that address the relationships
[27,29]. This is in line with Dai et al [28], who observed an associ- among muscle activity, physical exercise, and animal welfare.
ation between increased ESTmax and body language signs of fear
during the novel object and voluntary and forced approach tests. 5. Conclusions
Furthermore, some horses in our study presented CK levels above
the reference values (>350 U/L [44]), which suggests they were We conclude that the increase of ESTmax in our study was
suffering from some degree of muscle damage. Under such condi- probably determined by the physiological changes caused by the
tions, horses should have a resting period between work sessions [10] exercise, as indicated by the moderate positive correlation between
to recover from sarcolemma microinjuries and avoid further delete- ESTmax and CK activity, suggesting that ESTmax has potential to be
rious effects. In addition, we found a moderate and positive correla- used as an indicator of physical fitness of ranch horses; although,
tion coefficient between DIF-CK and DIF-ESTmax. This relationship there is still a need of further studies to prove its effectiveness.
was confirmed by the cluster analysis, which clearly distinguished
between two groups of horses: one with low and the other with high Acknowledgment
DIF-CK and DIF-ESTmax values. Based on these results, we propose the
use of ESTmax (assessed by infrared thermography in the commissure The authors express our gratitude to Karen Camile Rocha Go  is,
of the lacrimal caruncle) as a potential indicator of physical fatigue Thais Sgarbiero and Carmen Perez for their contribution to data
from submaximal exercise intensities in horses. collection, and to Joslaine N. S. G. Cyrillo (head of the Centro de
A recent study conducted with rats [56] showed that heat stress Pesquisa em Bovinos de Corte, Instituto of Zootecnia, Serta~ozinho-
can cause progressive elevations in CK in the muscles. From a SP), Frederico Simioni (Sa~o Geraldo Farm owner), who facilitated
practical perspective, we can assume that the horses from the HIGH our access to the horses and farm facilities. The support given by
group (CK > 350 U/L and ESTmax > 37.1 C) faced some degree of the staff and students from ETCO Group (Ethology and Animal
muscle damage. Therefore, one can use ESTmax to determine Ecology Research Group) and LAFEQ (Laboratory of Physiology of
whether the interval between physical activity sessions is sufficient Equine Exercise and Pharmacology), both from FCAV-UNESP,
for muscle recovery, thus preventing myopathies caused by Jaboticabal-SP, and from LAFA (Laboratory of Animal Physiology)
excessive physical effort. However, to generalize its applicability, from FZEA-USP, Pirassununga-SP, are also acknowledged.
this critical level of ESTmax should be tested in different contexts,
where horses are subjected to physical exercise. Financial Disclosures
We consider IRT to be a useful tool for assessing physical fitness in
horses in the field, given the following precautions are taken: This research was supported by the S~
ao Paulo Research Foun-
waiting for a period of acclimatization and capturing the thermo- dation (FAPESP, Brazil, grant numbers 2015/14421-3 and 2016/
graphic images with the horse in the shade, respecting the 15374-1).
 P.H. Esteves Trindade et al. / Journal of Equine Veterinary Science 75 (2019) 1e8
Supplementary Data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.jevs.2018.11.015.
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