animals
Article
Effect of Thermal Stress on Thermoregulation, Hematological
and Hormonal Characteristics of Caracu Beef Cattle
Natalya G. Abduch 1 , Bianca V. Pires 2 , Luana L. Souza 3 , Rogerio R. Vicentini 4 , Lenira El Faro Zadra 1 ,
Breno O. Fragomeni 5 , Rafael M. O. Silva 6 , Fernando Baldi 3 , Claudia C. P. Paz 1 and Nedenia B. Stafuzza 1, *
Citation: Abduch, N.G.; Pires, B.V.;
Souza, L.L.; Vicentini, R.R.;
Zadra, L.E.F.; Fragomeni, B.O.;
Silva, R.M.O.; Baldi, F.; Paz, C.C.P.;
Stafuzza, N.B. Effect of Thermal
Stress on Thermoregulation,
Hematological and Hormonal
Characteristics of Caracu Beef Cattle.
Animals 2022, 12, 3473. https://
doi.org/10.3390/ani12243473
Academic Editor: Mariangela
Caroprese
Received: 22 November 2022
Accepted: 6 December 2022
Published: 8 December 2022
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Copyright: © 2022 by the authors.
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This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Animals 2022, 12, 3473. https://doi.org/10.3390/ani12243473
1 Centro Avançado de Pesquisa e Desenvolvimento em Bovinos de Corte, Instituto de Zootecnia (IZ),
Sertãozinho 14174-000, SP, Brazil
2 Departamento de Genética, Faculdade de Medicina de Ribeirão Preto (FMRP),
Universidade de São Paulo (USP), Ribeirão Preto 14049-900, SP, Brazil
3 Departamento de Zootecnia, Faculdade de Ciências Agrárias e Veterinárias (FCAV),
Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Jaboticabal 14884-900, SP, Brazil
4 Departamento de Zoologia, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora 36036-900, MG, Brazil
5 Department of Animal Science, University of Connecticut, Storrs, CT 06269, USA
6 Zoetis, São Paulo 04710-230, SP, Brazil
* Correspondence: nedeniabs@gmail.com; Tel.: +55-16-3475-9163
Simple Summary: Heat stress impacts animal production, reproduction, health, and welfare, making
it profitable to raise animals adapted to the climate. In this context, Caracu (Bos taurus taurus)
is the large Creole breed adapted to the Brazilian tropical climate. Thus, this study investigated
the influence of environmental temperature on thermoregulation (surface temperature obtained by
infrared thermography and rectal temperature), hormonal (cortisol, cortisone, and progesterone),
and hematological characteristics of Caracu. The results revealed that heat stress changes several
physiological characteristics of Caracu cattle, although most of them remained within the regular
values observed for taurine Creole breeds. In addition, males and females exhibited differences
in their responses to heat stress. The decrease in animal production caused by heat stress is a
growing concern. Therefore, the identification and wide use of naturally resistant animals in terminal
crossbreeding schemes to explore heterosis and complementarity, and in pure herds, is important to
minimize the impact of heat stress on animal production.
Abstract: This study evaluated the influence of environmental temperature on thermoregulation,
hormonal, and hematological characteristics in Caracu cattle. Blood samples, hair length, coat and
muzzle colors, rectal (RT), and surface temperatures were collected from 48 males and 43 females
before (morning) and after sun exposure for eight hours (afternoon). Infrared thermography (IRT)
was used to identify superficial temperature that exhibits a high correlation with RT. Hematological
parameters, hormone concentrations, RT, and the superficial temperature obtained by IRT that
exhibited the highest correlation with RT were evaluated by variance analysis. Regarding IRT, the
lower left side of the body (LS) showed the highest correlation with the RT. Interaction between
period and sex was observed for LS, cortisol, and eosinophils. Cortisone, progesterone, and RT were
influenced by period and sex. Neutrophils and segmented neutrophils were influenced by the period,
which showed the highest concentrations after sun exposure. Platelets, leukocytes, lymphocytes,
and monocytes were influenced by sex. Heat stress changes several physiological characteristics
where males and females exhibited differences in their responses to heat stress. Furthermore, most
characteristics evaluated remained within the regular values observed for taurine Creole breeds,
showing that Caracu is adapted to tropical climates.
Keywords: adaptability; Bos taurus; hemogram; infrared thermography; thermotolerance
https://www.mdpi.com/journal/animals
Animals 2022, 12, 3473 2 of 17

1. Introduction
The increase in environmental temperature can directly affect the production and
reproduction of cattle since heat stress negatively impacts the physiological, immunological,
and performance characteristics of animals, such as feed intake, body weight gain, carcass
composition, and reproductive efficiency [1,2]. In this context, beef cattle are among the
main affected livestock species due to the extensive nature of production systems adopted
in most countries [3].
Although cattle can adapt to different environmental temperatures, the response
mechanisms that ensure their survival also affect their performance. The energy that would
be directed to production is redirected to the activation of thermoregulatory mechanisms
that will act on the animals’ metabolic energy balance, causing changes such as decreased
food intake and a decrease in weight gain [4,5].
Heat stress can lead to adaptive changes, resulting in variations observed between
animals to thermotolerance. These traits can be quantified by measuring physiological
responses such as rectal temperature and surface temperature [6]. Additionally, hematolog-
ical and hormonal parameters also can be modified by heat stress since they are directly
related to mechanisms of heat loss [7]. Factors that affect the caloric elimination capacity
from the skin surface, such as coat color, density, hair length, the thickness of the hair fibers,
and both hair and skin color, also appear to be related to heat stress adaptiveness providing
higher protection against sunlight and higher animal welfare [8–10]. Intrinsic factors such
as breed, age, sex, and metabolic rate can directly influence the animal’s response to heat
stress [11]. Heat stress can also reduce feed intake, ending up in production and health
losses [12]. Although zebu breeds (Bos taurus indicus) have higher heat tolerance than
taurine breeds (Bos taurus taurus) [10], taurine breeds can also be selected for tolerance to
tropical climatic conditions, as has been observed in Creole breeds [13,14].
The Caracu cattle breed (Bos taurus taurus) was formed after uncontrolled crosses be-
tween several Portuguese breeds, including Algariviano, Alentejana, Minhota, Arouquesa,
Barrosã, and Mirandesa, brought from the Iberian Peninsula to Brazil in 1534 by Portuguese
colonizers. The animals were submitted to the adversities of the tropical climate, as well
as the scarce feeding and the presence of parasites, contributing to their adaptation to the
tropical and subtropical climate [15]. At the beginning of the 20th century, the genetic
breeding program of Caracu was created, which established the breed pattern and pro-
moted its development, becoming 1940 one of the most expressive breeds of Brazilian
livestock [16]. However, the breed was almost extinct in the 60s due to the high inbreeding
and the introduction of Zebu breeds in Brazil. Currently, the Caracu breed has the largest
herd among the Brazilian Creole breeds, with a herd estimated at 30,000 animals [17].
The Caracu breed is well-known for its precocity, adaptability, rusticity, and good
productive and reproductive performances [15–21]. Nowadays, Caracu is widely used
in crossbreeding, mainly with zebu breeds, producing animals with a high degree of het-
erosis and good meat quality traits. Cyrillo et al. [22] observed the following means for
rump height, rump length, chest circumference, back length, and body length in Caracu:
137.17 ± 4.57 cm, 41.64 ± 4.04 cm, 192.08 ± 12.54 cm, 98.92 ± 7.90 cm, and 147.22 ± 10.62 cm,
respectively. The weight of adult males varies from 800 to 1000 kg and females 450 to 650 kg
in extensive breeding systems [22].
Given the difficulty in maintaining animal productivity at high levels and promoting
their welfare in a tropical climate, knowledge of the response of Creole breeds to thermal
stress is important to develop breeding strategies for these populations. Such strategies
would enable their use in terminal crossbreeding schemes to explore heterosis and com-
plementarity and in pure herds since the genetic material of Creole breeds can become
more productive in their own environments than exotic breeds. Therefore, the aims of this
study were to evaluate the use of infrared thermography as a tool for the assessment of
animal heat stress and to evaluate the adaptability to heat stress in Caracu animals through
thermoregulation, hormonal, hematological, skin, and hair characteristics.
Animals 2022, 12, 3473 3 of 17

2. Materials and Methods

2.1. Animals and Data Collection
All the experimental procedures involving animals were approved by the Institutional
Animal Care and Use Committee at the Animal Science Institute, Nova Odessa, São Paulo,
Brazil (protocol code CEUA Nº. 292-19, 7 October 2019), following guidelines for animal
welfare according to State Law No. 11.977 of the São Paulo state, Brazil.
A total of 104 Caracu steers (43 females with 335 ± 35.15 kg and 61 intact males
with 421 ± 45.07 kg), with an average age of 16 months, from the same herd located in
Sertãozinho, São Paulo, Brazil (21◦ 170 S and 48◦ 120 W) were used in this study. All steers
were certified for breed purity by [20]. All animals were raised in grazing conditions using
Brachiaria sp. forage and supplemented with mineral salt.
The experiment was performed in February 2019, a month characterized by high
temperatures in this region of Brazil, in three days when the black globe temperature was
higher than 45 ◦ C. The ITEG-500 (Incon Eletronica Ltd., São Paulo, Brazil) data logger was
installed at the paddock where the experiment was conducted for the measurement of
ambient temperature, black globe temperature, and relative air humidity in the environ-
ment. The outdoor wet bulb globe temperature (WBGT) index was used as an indicator of
thermal comfort, which is calculated using the following equation [23]:

WBTG = 0.7tnwb + 0.2tbg + 0.1tdb (1)

where tnwb is the natural wet bulb temperature obtained under sun and wind, tbg is the
black globe temperature measured, and tdb is the dry bulb temperature. Thus, the influence
of each temperature is weighted 70%, 20%, and 10%, respectively.
In order to minimize the handling-stress, all animals were brought from the pasture to
a paddock with water and food (60% sorghum silage, 25% ground corn, 13% soybean meal,
1.75% mineral salt, and 0.2% urea) ad libitum 21 days before the experiment. During this
adaptation period, all animals were taken to the corral twice a day (4:00 AM and 2:00 PM)
to simulate the experiment that would be performed later.
All animals were tested before and after sun exposure, hereby termed morning and
afternoon measurements. In the morning (4:00 to 6:00 AM), all animals were taken to
the open corral, where phenotypes (rectal and surface temperatures) and blood were
collected. The animals were then taken back to the paddock with water and food, where
they remained exposed to the sun without shade. In the afternoon (2:00 to 4:00 PM), all
animals were taken to the open corral again for phenotypes and blood collection.
Rectal temperature (RT) was measured by introducing the Animed model 6200.03
digital clinical thermometer (precision of ± 0.1 ◦ C, Incoterm, Porto Alegre, Brazil) into the
animal’s rectum up to ± 5 cm for 2 min. The heat tolerance index (HTI) was calculated
using the following equation [24]:

HTI = 10 − ( RT2 − RT1) (2)

where RT2 is the rectal temperature after heat stress, and RT1 is the rectal temperature
before heat stress. A value closer to 10 indicates a better-adapted animal.
A thermal imaging camera (FLIR TB420X, FLIR Systems Inc., Willsonville, OR, USA)
was used for the body surface temperature measurements, which were obtained from
four regions chosen to represent a complete view of the body: left side of the body (from
the neck to the rump), muzzle, loin, and left eye. Since the total infrared radiation of an
object captured through infrared images represents the sum of the emitted, transmitted,
and reflected radiation, the loin and the left side of the body were chosen to observe the
total amount of heat [25]. The eye was also measured because it is related to internal
body temperature, and it is not affected by environment temperature as other peripheral
regions [26]. Additionally, the muzzle is related to heat dissipation mechanisms through
respiration and was also measured by infrared thermography [25]. The emissivity value
was adjusted (0.98 ε), and the camera resolution was calibrated before each period (morning
Animals 2022, 12, 3473 4 of 17
Animals 2022, 12, x 4 of 17

and afternoon) according to local ambient temperature and humidity. A standard distance
afternoon) according
of 1 m between to localand
the animal ambient temperature
the camera and for
was used humidity. A standard distance of 1 m
image capture.
between the animal and the camera was used for image capture.
The Research IR 4 Software (FLIR Systems Inc., Willsonville, OR, USA) was used
The Research
to calculate IR 4 Software
minimum, mean, and (FLIRmaximum
Systems Inc., Willsonville,for
temperatures OR,each
USA) was usedregion.
measured to cal-
culate minimum, mean, and maximum temperatures for each measured
After preliminary analysis, the maximum temperature of each region was used in the region. After prelim-
inary analysis,
statistical the maximum
analyses to reducetemperature
environmental of each region was
interference withused
theininfrared
the statistical analyses
thermography
to reduce environmental interference with the infrared thermography
measurements, as described by [27]. On each image obtained, subregions of measurement measurements, as de-
scribed by [27]. On each image obtained, subregions of measurement were
were traced using the bendable line tool to detect the targeted subregions for temperature traced using the
bendable line tool to detect the targeted subregions for temperature determination
determination in a rainbow palette, as performed by [28] (Figure 1). Two subregions were in a rain-
bow
tracedpalette,
on theasleft
performed
side of thebybody:
[28] (Figure
upper 1). Two
(US) andsubregions
lower (LS); were
twotraced on thewere
subregions left side of
traced
the body:
on the upper (US)
muzzle: and(CM)
caudal lower (LS);
and two subregions
middle (MM); three were traced on the
subregions weremuzzle:
tracedcaudal
on the(CM)loin:
and middle
left (LL), (MM);(LM)
middle threeand
subregions were
right (LR); traced
two on the loin:
subregions wereleft (LL),inmiddle
traced (LM)
the left eye:and right
lacrimal
(LR);
canaltwo
(LC)subregions were(EG).
and eye globe traced in the left eye: lacrimal canal (LC) and eye globe (EG).

Figure
Figure 1.1. Thermographic
Thermographic images
images andand subregions
subregions traced
traced the
the left
left side
side of
of the
the body,
body, muzzle,
muzzle, loin,
loin, and
and
left
left eye
eye in
in Caracu.
Caracu. (A)
(A) The
The left
left side of the
side of the body:
body: ee =
= upper
upper (US),
(US), ff == lower
lower (LS);
(LS); (B)
(B) Muzzle:
Muzzle: gg == caudal
caudal
area
area of
of the
themuzzle
muzzle(CM),
(CM),hh==middle
middleofofthe muzzle
the muzzle (MM);
(MM); (C)(C)
Loin:
Loin:i = ileft (LL),
= left j = middle
(LL), (LM),
j = middle k=
(LM),
right (LR); (D) Left eye: l = lacrimal canal region (LC), m = eye globe (EG). Rainbow
k = right (LR); (D) Left eye: l = lacrimal canal region (LC), m = eye globe (EG). Rainbow palette: palette: light
colors indicate warmer temperatures (white) and, dark colors indicate cooler temperatures (dark
light colors indicate warmer temperatures (white) and, dark colors indicate cooler temperatures
blue), other colors indicate intermediate temperatures.
(dark blue), other colors indicate intermediate temperatures.
The
The animals
animals also
also had
had their
their coat
coat color
color classified
classified as
as cream,
cream, yellow,
yellow, orange,
orange, and
and red,
red, and
and
hair samples were collected from a central area of the shoulder on both sides using a plier
hair samples were collected from a central area of the shoulder on both sides using a plier and and
measured
measured using
using aa pachymeter.
pachymeter. The
Themuzzle
muzzlecolor
colorwas
wasalso
alsoclassified
classifiedas
aswhite,
white,black,
black,ororpied.
pied.

2.2.
2.2. Blood
Blood Sample
Sample Collections
Collections
The
The blood
blood samples
samples collected
collected from
from thethe jugular
jugular vein
vein ofof each
each animal
animal in in both
both periods
periods
(before and after
(before and aftersun
sunexposure)
exposure)werewereused
used to to perform
perform steroid
steroid hormones
hormones (cortisol,
(cortisol, corti-
cortisone,
sone, and progesterone)
and progesterone) quantification
quantification and the and the hemogram
hemogram analysis analysis (erythrocytes,
(erythrocytes, hemo-
hemoglobin,
globin, hematocrit,
hematocrit, mean corpuscular
mean corpuscular volume—MCV,
volume—MCV, mean corpuscular
mean corpuscular hemoglobin—MCH,
hemoglobin—MCH, mean
mean corpuscular
corpuscular hemoglobin
hemoglobin concentration—MCHC,
concentration—MCHC, leukocytes,
leukocytes, band neutrophils,
band neutrophils, segmentedseg-
mented neutrophils,
neutrophils, neutrophils,
neutrophils, eosinophils,
eosinophils, lymphocytes,
lymphocytes, monocytes,
monocytes, and platelets).
and platelets).
For
For hormone
hormoneanalysis,
analysis,blood
bloodsamples
samples were
were centrifugated
centrifugated (1500×
(1500g,×15g,min, 10 °C),
15 min, 10 and
◦ C),

200
andμL200plasma obtained
µL plasma werewere
obtained prepared in 1:1inmethanol
prepared (Merck,
1:1 methanol Darmstadt,
(Merck, Germany)
Darmstadt, Germany)and
stored at −80
and stored at°C.
−80The quantification of circulating hormones was performed using the high-
◦ C. The quantification of circulating hormones was performed using the

resolution multiple
high-resolution reaction
multiple monitoring
reaction (MRMHR)
monitoring (MRMHR)analysis in liquid
analysis chromatography-tan-
in liquid chromatography-
dem mass spectrometry (LC-MS/MS) usingusing
a TripleTOF 5600 (SCIEX, Foster Foster
City, CA, USA)
tandem mass spectrometry (LC-MS/MS) ®
a TripleTOF ® 5600 (SCIEX, City, CA,
equipment as described
USA) equipment by [29]. Identification
as described of the steroidofspecies
by [29]. Identification obtained
the steroid in theobtained
species LC-MS/MS in
analysis was performed
the LC-MS/MS analysis using
wasPeakView
performedv.2.1 using andPeakView
MultiQuant™ v.2.1 v.3.0.2 (SCIEX, Fosterv.3.0.2
and MultiQuant™ City,
CA, USA)
(SCIEX, software.
Foster City, CA, USA) software.

2.3. Statistical
2.3. Statistical Analysis
Analysis
The environmental conditions (wet
The environmental conditions (wet bulb,
bulb, dry
dry bulb,
bulb, black
black globe,
globe, and
and WBGT
WBGT index)
index)
were analyzed using the GLM procedure of SAS v. 9.2 (SAS Inst. Inc., Cary, NC, USA),
were analyzed using the GLM procedure of SAS v. 9.2 (SAS Inst. Inc., Cary, NC, USA),
considering periods before and after sun exposure as a fixed effect.
considering periods before and after sun exposure as a fixed effect.
Animals 2022, 12, 3473 5 of 17

Pearson correlation coefficients for each sex were estimated by the CORR procedure
of SAS v. 9.2 (SAS Inst. Inc., Cary, NC, USA) to examine the relationship between the
maximum temperature of each subregion of measurement obtained by infrared thermogra-
phy (US, LS, LL, LM, LR, CM, MM, LC, and EG) and RT. The subregion that exhibited the
highest correlation with RT was included in the analysis of variance as a covariate.
The hair length, coat color, and muzzle color were analyzed by the GENMOD proce-
dure of SAS v. 9.2 (SAS Inst. Inc., Cary, NC, USA), presuming gamma distribution to hair
length. The RT and the temperature obtained by infrared thermography that exhibited the
highest correlation with RT were used as covariates to evaluate the hair length, coat color,
and muzzle color data.
Hematological parameters (erythrocytes, hemoglobin, hematocrit, MCV, MCH, MCHC,
leukocytes, band neutrophils, segmented neutrophils, neutrophils, eosinophils, lympho-
cytes, monocytes, and platelets), hormone concentrations (cortisol, cortisone, and pro-
gesterone), RT and the temperature obtained by infrared thermography that exhibited
the highest correlation with RT were analyzed using the MIXED procedure of SAS v. 9.2
(SAS Inst. Inc., Cary, NC, USA), and the model included the fixed effects of the period
(morning and afternoon) and sex, as well as the interaction between all effects (p < 0.05).

3. Results
3.1. Environmental Conditions and HTI
All the environmental conditions evaluated showed significant differences (p < 0.0001)
between periods (Table 1), without differences observed among collection days. The
afternoon environment exhibited the highest temperatures (wet bulb, dry bulb, and black
globe) and WBGT index (Table 1). The black globe temperature was under 25 ◦ C in the
morning period and above 45 ◦ C in the afternoon period.

Table 1. Least squares means and standard errors (SE) from environmental conditions obtained at
the paddock before (morning) and after (afternoon) sun exposure (p < 0.0001).

Temperature (◦ C) Morning ± SE Afternoon ± SE Variation ± SE

Wet bulb 20.71 ± 0.10 36.35 ± 0.10 15.64 ± 0.14
Dry bulb 20.60 ± 0.09 35.93 ± 0.09 15.33 ± 0.12
Black globe 20.70 ± 0.21 49.15 ± 0.21 28.45 ± 0.30
WBGT index 20.69 ± 0.12 38.87 ± 0.12 18.18 ± 0.16

The high differences observed in environmental conditions between both periods

influenced the HTI of the animals, which ranged from 7.2 to 9.7 (mean of 8.5 ± 0.49, a
median of 8.6, and mode of 8.6). No significant difference was observed between HTI
observed in males and females.

3.2. Rectal Temperature and Infrared Thermography

The highest and lowest body surface temperatures obtained by infrared thermography
in the morning period were identified in LC and CM subregions, respectively, in both males
and females (Table 2). In the afternoon period, the LM and MM subregions exhibited the
highest and lowest temperatures, respectively, also in both sexes (Table 2).
The temperature of all nine subregions measured by infrared thermography from four
different regions of the animal’s body presented a high and significant correlation (p < 0.001)
with RT (Table 3). Females showed higher correlation values with RT in most subregions
evaluated (US, LS, MM, LL, LM, EG, and LC), while males exhibited higher correlation values
only in CM and LR subregions. The EG subregion had the lowest correlation with RT in both
males (0.740) and females (0.719), while the LS subregion exhibited the highest correlation
with RT in both males (0.911) and females (0.898). Among all subregions analyzed, LS was
used in the further analysis because it presented the highest correlation with RT.
Animals 2022, 12, 3473 6 of 17

Table 2. Mean and standard deviation of rectal temperature and body surface temperature measure-
ments obtained by infrared thermography.

Male Female
Subregion
Morning Afternoon Morning Afternoon
RT 38.09 ± 0.30 39.62 ± 0.51 37.99 ± 0.45 39.40 ± 0.41
US 34.76 ± 0.77 40.45 ± 1.22 33.64 ± 1.29 40.63 ± 0.92
LS 34.89 ± 0.86 39.28 ± 0.88 33.73 ± 1.14 39.59 ± 0.62
MM 34.05 ± 1.00 36.78 ± 0.79 31.77 ± 1.85 36.61 ± 0.68
CM 33.85 ± 1.52 39.06 ± 0.63 30.13 ± 2.75 39.04 ± 0.45
LL 34.75 ± 1.01 40.17 ± 1.21 33.40 ± 0.88 40.36 ± 1.06
LM 34.69 ± 1.04 41.66 ± 1.97 33.47 ± 1.52 41.32 ± 1.10
LR 34.53 ± 0.94 40.16 ± 1.21 33.39 ± 1.17 40.17 ± 1.17
EG 37.36 ± 0.51 38.74 ± 0.98 35.97 ± 1.44 38.47 ± 0.94
LC 38.25 ± 0.51 39.88 ± 0.96 37.60 ± 0.94 39.70 ± 0.50
RT: rectal temperature, US: upper left side of the body US, LS: lower left side of the body, MM: middle of the
muzzle, CM: caudal area of the muzzle, LL: loin left, LM: loin middle, LR: loin right, EG: eye globe, LC: lacrimal
canal region.

Table 3. Pearson correlation coefficients (p < 0.0001) estimated between the maximum temperature of
each subregion of body surface obtained by infrared thermography and rectal temperature (RT) in
males (top of the table) and females (bottom of the table).

Body Muzzle Loin Eye

Subregion RT
US LS MM CM LL LM LR EG LC
RT 1 0.893 0.911 0.813 0.853 0.889 0.870 0.863 0.740 0.874
US 0.861 1 0.968 0.854 0.913 0.966 0.950 0.958 0.708 0.879
LS 0.898 0.976 1 0.836 0.916 0.949 0.922 0.941 0.726 0.890
MM 0.813 0.877 0.868 1 0.904 0.828 0.843 0.821 0.705 0.838
CM 0.867 0.912 0.923 0.917 1 0.898 0.889 0.895 0.670 0.841
LL 0.872 0.975 0.965 0.851 0.903 1 0.957 0.970 0.707 0.839
LM 0.853 0.963 0.946 0.889 0.911 0.956 1 0.947 0.707 0.842
LR 0.877 0.965 0.963 0.863 0.909 0.962 0.938 1 0.695 0.835
EG 0.719 0.765 0.770 0.709 0.753 0.729 0.707 0.774 1 0.777
LC 0.775 0.798 0.815 0.685 0.737 0.814 0.787 0.819 0.586 1
US: upper left side of the body, LS: lower left side of the body, MM: middle of the muzzle, CM: caudal area of the
muzzle, LL: loin left, LM: loin middle, LR: loin right, EG: eye globe, LC: lacrimal canal region.

3.3. Temperatures, Hematological and Hormonal Traits

The descriptive statistical analysis of the hematological and hormonal traits, RT and
LS, can be seen in Table 4.
The interaction between period and sex (Figure 2) influenced the LS (p < 0.0001),
cortisol (p = 0.0304), and eosinophils (p = 0.0495). The highest LS was observed in females
after exposure to the sun (39.59 ◦ C), while the lowest LS was observed in females before
exposure to the sun (33.73 ◦ C). The highest and lowest cortisol concentration was observed
in females before sun exposure (7.61 nmol/L) and in males also before sun exposure
(3.57 nmol/L), respectively. The highest eosinophils concentration was observed in females
after sun exposure (0.782 g/L) and the lowest in males after sun exposure (0.562 g/L).
The RT, cortisone, progesterone, hemoglobin, hematocrit, and MCV were influenced
by sex and period, without interaction between them (Table 5). Males presented higher RT
(38.85 ◦ C) than females (38.70 ◦ C), and the RT observed after sun exposure (39.51 ◦ C) was
higher than before sun exposure (38.03 ◦ C). Females exhibited higher cortisone concentrations
(1.55 nmol/L) than males (1.26 nmol/L), and the samples collected after sun exposure had
higher cortisone concentrations (1.47 nmol/L) than those collected before sun exposure
(1.34 nmol/L). Regarding progesterone, hemoglobin, hematocrit, and MCV, females presented
higher means (1.33 nmol/L, 6.03 nmol/L, 0.31 L/L, and 42.08 fL, respectively) compared to
males (0.22 nmol/L, 5.73 nmol/L, 0.29 L/L, and 40.92 fL, respectively). In addition, the highest
Animals 2022, 12, 3473 7 of 17

estimated means of progesterone, hemoglobin, hematocrit, and MCV were observed before
sun exposure (0.86 nmol/L, 5.98 nmol/L, 0.30 L/L, and 41.60 fL, respectively) compared to
after sun exposure (0.69 nmol/L, 5.78 nmol/L, 0.29 L/L, and 41.40 fL, respectively).

Table 4. Descriptive statistics (N: number of observations, mean, SD: standard deviation, SE: standard
error, CV: coefficient of variation, minimum and maximum) of the physiological, hormonal and
hematological traits observed.

Trait N Mean SD SE CV Min. Max.

RT (◦ C) 182 38.78 0.86 0.063 2.21 37.10 41.00
LS (◦ C) 182 36.88 2.73 0.202 7.41 30.40 41.20
HTI 182 8.52 0.49 0.04 5.75 7.20 9.70
Cortisone (nmol/L) 179 1.40 0.48 0.036 33.99 0.58 3.01
Cortisol (nmol/L) 179 5.49 3.60 0.269 65.57 0.43 17.40
Progesterone (nmol/L) 179 0.76 1.34 0.100 176.66 0 6.77
Erythrocytes (T/L) 182 7.21 0.83 0.062 11.58 5.34 10.03
Hemoglobin (nmol/L) 182 5.87 0.67 0.049 11.39 4.47 7.94
Hematocrit (L/L) 182 0.30 0.04 0.003 12.32 0.22 0.41
MCV (fL) 182 41.47 2.22 0.165 5.35 36.86 47.33
MCHC (nmol/L) 182 19.68 0.58 0.043 2.97 17.51 21.74
MCH (pg) 182 0.82 0.03 0.002 4.24 0.75 0.93
Leukocytes (g/L) 182 13.27 2.22 0.164 16.72 8.60 18.90
Band neutrophils (g/L) 182 0.04 0.06 0.004 174.18 0 0.19
Segmented neutrophils (g/L) 182 3.50 0.95 0.701 27.26 1.62 6.64
Neutrophils (g/L) 182 3.54 0.97 0.702 27.47 1.64 6.80
Eosinophils (g/L) 182 0.66 0.44 0.03 66.76 0.10 2.77
Lymphocytes (g/L) 182 8.67 1.61 0.120 18.63 4.90 13.99
Monocytes (g/L) 182 0.40 0.12 0.009 30.65 0.19 0.76
Platelets (g/L) 182 527.74 182.21 13.507 34.53 190.00 928.00
RT: rectal temperature; LS: superficial temperature of the lower side of the body obtained by infrared ther-
mography; HTI: heat tolerance index, MCV: mean corpuscular volume; MCHC: mean corpuscular hemoglobin
concentration; MCH: mean corpuscular hemoglobin.

The erythrocytes, MCHC, neutrophils, and segmented neutrophils were influenced

only by the period (Table 5). The highest erythrocytes concentration was observed before
sun exposure (7.34 T/L) compared to after sun exposure (7.09 T/L). Regarding MCHC,
neutrophils, and segmented neutrophils, the highest concentrations were observed after
sun exposure (19.74 nmol/L, 3.63 g/L, and 3.59 g/L, respectively) in relation to before sun
exposure (19.60 nmol/L, 3.46 g/L, and 3.43 g/L, respectively).
The platelets, band neutrophils, leukocytes, lymphocytes, monocytes, and MCH
were influenced only by sex (Table 5). Males had higher concentrations of platelets
(569.33 g/L) than females (481.31 g/L), while females exhibited higher concentrations
of band neutrophils, leukocytes, lymphocytes, monocytes, and MCH than males (Table 5).

3.4. Hair and Skin Characteristics

The hair length ranged from 3 to 16 mm. The results showed that RT (p = 0.41) and LS
(p = 0.92) were not significantly influenced by hair length (Figure 3). In this herd, 7% of
the animals were classified as cream, 33% as yellow, 40% as orange, and 20% as red, which
exhibited RT means of 39.7 ◦ C, 39.6 ◦ C, 39.5 ◦ C, and 39.4 ◦ C, respectively, not differing from
each other (p = 0.95). Similar results were observed for LS, where the means were 39.1 ◦ C
for cream, 39.5 ◦ C for yellow, 39.4 ◦ C for orange, and 39.4 ◦ C for red, not differing from
each other (p = 0.69).
 The interaction between period and sex (Figure 2) influenced the LS (p < 0.0001), cor-
tisol (p = 0.0304), and eosinophils (p = 0.0495). The highest LS was observed in females
after exposure to the sun (39.59 °C), while the lowest LS was observed in females before
exposure to the sun (33.73 °C). The highest and lowest cortisol concentration was observed
in females before sun exposure (7.61 nmol/L) and in males also before sun exposure (3.57
Animals 2022, 12, 3473 8 of 17
nmol/L), respectively. The highest eosinophils concentration was observed in females af-
ter sun exposure (0.782 g/L) and the lowest in males after sun exposure (0.562 g/L).

Figure 2.
Figure 2. The
Theleast square
least means
square obtained
means for thefor
obtained superficial temperature
the superficial of the lower
temperature ofside
the of the side of
lower
body (LS) obtained by infrared thermography (p < 0.0001), cortisol (p < 0.05), and eosinophils (p <
the body (LS) obtained by infrared thermography (p < 0.0001), cortisol (p < 0.05), and eosinophils
0.05) according to sex and period interaction.
(p < 0.05) according to sex and period interaction.

Table 5. Least square means the physiological, hormonal, and hematological traits observed in male
and female Caracu cattle in the morning and in afternoon periods.

Period Sex
Trait
Morning Afternoon p-Value Male Female p-Value
RT (◦ C) 38.038 ± 0.044 39.512 ± 0.044 p < 0.0001 38.854 ± 0.050 38.696 ± 0.053 p = 0.0326
LS (◦ C) 34.311 ± 0.094 39.434 ± 0.094 p < 0.0001 37.083 ± 0.102 36.662 ± 0.108 p = 0.0058
Cortisone (nmol/L) 1.344 ± 0.048 1.470 ± 0.048 p = 0.0123 1.262 ± 0.057 1.552 ± 0.059 p = 0.0007
Cortisol (nmol/L) 5.591 ± 0.338 5.517 ± 0.340 p = 0.8775 3.911 ± 0.332 7.197 ± 0.345 p < 0.0001
Progesterone (nmol/L) 0.856 ± 0.128 0.698 ± 0.128 p = 0.0013 0.219 ± 0.173 1.335 ± 0.183 p < 0.0001
Erythrocytes (T/L) 7.341 ± 0.087 7.088 ± 0.087 p = 0.0003 7.107 ± 0.110 7.322 ± 0.116 p = 0.1818
Hemoglobin (nmol/L) 5.979 ± 0.068 5.784 ± 0.068 p = 0.0003 5.730 ± 0.087 6.033 ± 0.092 p = 0.0182
Hematocrit (L/L) 0.305 ± 0.004 0.293 ± 0.004 p < 0.0001 0.291 ± 0.005 0.308 ± 0.005 p = 0.0134
MCV (fL) 41.605 ± 0.225 41.403 ± 0.226 p = 0.0158 40.923 ± 0.306 42.085 ± 0.324 p = 0.0107
MCHC (nmol/L) 19.605 ± 0.061 19.743 ± 0.061 p = 0.0027 19.737 ± 0.078 19.610 ± 0.082 p = 0.2672
MCH (pg) 0.815 ± 0.003 0.817 ± 0.003 p = 0.1865 0.807 ± 0.005 0.825 ± 0.005 p = 0.0125
Leukocytes (g/L) 13.247 ± 0.220 13.372 ± 0.220 p = 0.4731 12.535 ± 0.278 14.084 ± 0.293 p = 0.0002
Band neutrophils (g/L) 0.031 ± 0.006 0.042 ± 0.006 p = 0.2119 0.025 ± 0.007 0.047 ± 0.007 p = 0.0310
Segmented neutrophils (g/L) 3.426 ± 0.099 3.589 ± 0.099 p = 0.0389 3.379 ± 0.126 3.636 ± 0.133 p = 0.1638
Neutrophils (g/L) 3.457 ± 0.101 3.631 ± 0.101 p = 0.0332 3.405 ± 0.128 3.684 ± 0.135 p = 0.1366
Eosinophils (g/L) 0.656 ± 0.046 0.672 ± 0.046 p = 0.6120 0.585 ± 0.059 0.742 ± 0.062 p = 0.0710
Lymphocytes (g/L) 8.740 ± 0.161 8.656 ± 0.161 p = 0.5320 8.172 ± 0.202 9.224 ± 0.213 p = 0.0006
Monocytes (g/L) 0.394 ± 0.013 0.414 ± 0.013 p = 0.1969 0.374 ± 0.014 0.434 ± 0.014 p = 0.0033
Platelets (g/L) 527.961 ± 18.712 522.686 ± 18.712 p = 0.3620 569.333 ± 25.420 481.314 ± 26.857 p = 0.0194
RT: rectal temperature; LS: superficial temperature of the lower side of the body obtained by infrared thermog-
raphy; MCV: mean corpuscular volume; MCHC: mean corpuscular hemoglobin concentration; MCH: mean
corpuscular hemoglobin.
Animals 2022,
Animals 12, xx
2022, 12, 99 of
of 17
17

exhibited RT
exhibited RT means
means of of 39.7
39.7 °C,
°C, 39.6
39.6 °C,
°C, 39.5
39.5 °C,
°C, and
and 39.4
39.4 °C,
°C, respectively,
respectively, not
not differing
differing
from each
from each other
other (p
(p == 0.95).
0.95). Similar
Similar results
results were
were observed
observed for
for LS,
LS, where
where the
the means
means were
were
Animals 2022, 12, 3473 9 of 17
39.1 °C
39.1 °C for
for cream,
cream, 39.5
39.5 °C
°C for
for yellow,
yellow, 39.4
39.4 °C
°C for
for orange,
orange, and
and 39.4
39.4 °C
°C for
for red,
red, not
not differing
differing
from each
from each other
other (p
(p == 0.69).
0.69).

Figure 3.
Figure
Figure 3. Hair
3. Hairlength
Hair lengthdistribution
length distributionaccording
distribution according
according to
toto rectal
rectal
rectal temperature
temperature
temperature (RT)
(RT)
(RT) and
andand the superficial
the the superficial
superficial temper-
temper-
temperature
ature
ature
of of
theof the lower
the
lower lower side
side ofside of
the of the (LS)
the
body bodyobtained
body (LS) obtained
(LS) obtained by infrared
by
by infraredinfrared thermography.
thermography.
thermography.

Regarding the
Regarding
Regarding the muzzle
the muzzle color,
muzzle color, 82%
color, 82% of
82% of the
of the animals
the animals were
animals were classified
were classified as
classified as white,
as white, 15%
white, 15% as
15% as pied,
as pied,
pied,
and 3%
and
and 3%as
3% asblack.
as black.The
black. TheRTRT
The RT means
means
means did
diddid not
notnot differ
differ
differ between
between
between animals
animals
animals with different
with different
with different muzzle
muzzlemuzzle
colors
colors
colors
(p (p ==being
(p
= 0.12), 0.12),39.6
0.12), being
being 39.6 °C
39.6 °C for
for white,
white, 39.4
39.4 °C
°C for
for pied,
pied, and
and 39.3
39.3 °C
°C for
for black.
black. Similar
Similar
◦ C for white, 39.4 ◦ C for pied, and 39.3 ◦ C for black. Similar results

results
were were
also also
observedobserved
for LS, for
withLS,
thewith the
followingfollowing
means: means:
39.4 39.4 °C for white, 39.3 °C for
for
◦ C for white, 39.3 ◦ C for pied,
results were also observed for LS, with the following means: 39.4 °C for white, 39.3 °C
pied, and 40.0 °C for black, not differing from each other (p = 0.57)
◦ C for black, not differing from each other (p = 0.57) (Figure 4).
(Figure
pied, and 40.0 °C for black, not differing from each other (p = 0.57) (Figure 4).
and 40.0 4).

Figure 4.
Figure 4. Coat
Coat color
color distribution
distribution according
according to
to rectal
rectal temperature
temperature (RT)
(RT) and
and the
the superficial
superficial tempera-
tempera-
Figure 4. Coat color distribution according to rectal temperature (RT) and the superficial temperature of
ture of
ture of the
the lower
lower side
side of
of the
the body
body (LS)
(LS) obtained
obtained by
by infrared
infrared thermography.
thermography. Coat
Coat color
color 1:
1: cream;
cream; Coat
Coat
the lower
color 2: side ofCoat
yellow; the body
color (LS)
3: obtained
orange; andbyCoat
infrared
colorthermography.
4: red. Coat color 1: cream; Coat color 2:
color 2: yellow; Coat color 3: orange; and Coat color 4: red.
yellow; Coat color 3: orange; and Coat color 4: red.

4. Discussion
4.1. Environmental Conditions and HTI
The increase in environmental temperature can induce the elevation of body temperature,
causing physiological disturbances. The environmental conditions evaluated demonstrated
the influence of the environment on the adaptive mechanisms of the Caracu animals, in which
differential responses were identified for the morning and afternoon periods. Differences
Animals 2022, 12, 3473 10 of 17

observed between the WBGT index in the morning and afternoon periods indicated thermal
comfort in the morning and thermal stress in the afternoon period.
The HTI showed that there is variability for thermotolerance to the tropical climate
within this herd, which may allow the selection of heat-tolerant animals. Environmental
differences during RT measurements directly impact the HTI obtained, which makes it
difficult to compare our results with the literature. This fact can be corroborated by [18],
which found an HTI mean of 9.7 studying the same Caracu herd used in the present study.
The divergence of results is due to the methodology proposed since the RT measurements
in the morning were performed by [18] in the presence of sunlight, while in the present
study, RT measurements in the morning were performed in the absence of sunlight.

4.2. Rectal Temperature and Infrared Thermography

Several tools have been arising as alternatives to assess the impact of environmental
factors on heat stress in animals, such as infrared thermography. This tool contributes
to animal welfare because it enables the detection of heat stress without contact with the
animal, avoiding handling stress [30]. Moreover, thermography enables the measurement
of the temperature radiated from different parts of the animal body, increasing the range of
possibilities and facilitating data collection management [31].
In our study, all nine subregions evaluated exhibited positive, high, and significant
correlations with RT. The US and LS body subregions exhibited the highest correlation with
RT among the infrared measurements in both sex and periods evaluated, which could be
attributed to the influence of ruminal activity. In cattle, the rumen occupies a large area
of the left half of the abdominal cavity. Moreover, heat is one of the products of ruminal
fermentation, and the temperature of this region is closely related to the internal core body
temperature [32,33]. In addition, several factors can influence infrared thermography, such
as environmental conditions, meteorological variables (e.g., wind speed and solar loading),
refractive emissivity, the distance between the equipment and the animal evaluated, and
breed characteristics [25].
For the muzzle region (CM and MM), lower temperature values are usually recorded.
However, the vast blood irrigation in the muzzle contributes to the relationship with
internal body temperature [25]. The loin subregions (LL, LM, and LR) were in direct
contact with sunlight and more susceptible to changes in weather. Thus, the correlations
between loin subregions and RT may be related to thermoregulatory mechanisms in search
of homeostasis [34].
It has been suggested that the eye is not affected by ambient temperature and has
been considered a useful indicator of core body temperature [35], which can be used for
detecting temperament [36], pain [37], or response to stress in cattle [38]. Studies with
several cattle breeds reported that the eye had high correlations with RT [9,26]. The LC
subregion is more innervated, has more superficial capillaries beds and could be related to
the internal core body temperature [27,35].
Although infrared thermography has been used in several studies encompassing many
breeds, there are some limitations of this technology, and many factors can influence the
thermal readings by the infrared device (e.g., anatomical and physiological characteristics of
each breed, environmental conditions, as well as differences in the measurement methodology
used) [25,28,30]. Differences observed in correlation values between sex could be a result
of those physiological and anatomical variables. The internal temperature of cows can vary
during the stages of the estrous cycle [12,28]. However, the animals’ reproductive aspects were
not evaluated, which may have affected part of our results. Additionally, there was a difference
in size between the animals. Males had greater body volume than females. It is known that
surface areas rather than body volumes limit metabolic activity such as thermoregulation [39].
Thereby, females may have greater heat dissipation and consequently higher temperatures
captured by thermography, reflecting also higher correlation values.
Animals 2022, 12, 3473 11 of 17

4.3. Temperatures, Hematological and Hormonal Traits

The sex and period interaction modified the LS, where the highest and lowest es-
timated mean was identified in females after sun exposure and before sun exposure,
respectively (Figure 2). Although infrared thermography has been used in several studies
encompassing many breeds, there are some limitations of this technology, and many factors
can influence the thermal readings by infrared devices, as described previously [25,28,30].
Differences observed between sex could be a result of physiological and anatomical par-
ticularities of each sex. In addition, the total infrared radiation captured through infrared
thermographic images represents the sum of the emitted, transmitted, and reflected radia-
tion [40]. It also can explain the higher correlation observed between LS and RT in relation
to other subregions located in the upper area of the animal’s body, such as US, LL, LR, and
LM, which are affected by a higher incidence of solar radiation and, therefore, exhibited
higher temperatures than RT and, consequently, lower correlations with RT.
Caracu females presented higher LS temperatures after sun exposure compared to
males. Authors reported a negative correlation between body surface area and body weight,
where animals with lower weight have higher body surface per unit of weight compared to
animals with higher weight [41]. It is known that surface areas rather than body volumes
limit metabolic activity such as thermoregulation [41]. In the present study, females had
lower body weight (335 ± 35.15 kg) than males (421 ± 45.07 kg), which may explain the
higher LS means observed in females after sun exposure when compared to males in the
same period since females have higher body surface per unit of weight. Thereby, females
may have greater heat dissipation and, consequently, higher temperatures captured by
thermography, resulting in higher correlation values.
A significant interaction between sex and period was also detected for serum cor-
tisol concentration (Figure 2), where the highest cortisol level was identified in females
before sun exposure, and the smallest cortisol level were identified in males also in the
morning period. Although several researchers have been studying the cortisol hormone
as a biomarker for heat stress, cortisol levels can be influenced by several factors such as
environment, management, adaptation period, and temperament of the animal, making it
difficult to use as an indicator of heat stress [42].
The highest cortisol levels in females, when compared to males, was also observed in
cattle [43] and goats [44], although some authors have not found differences in cortisol levels
between sex in cattle [45,46]. Factors such as breed, handling, behavioral patterns, body condi-
tion, and gonadal steroid metabolism can also influence cortisol levels [47,48]. Furthermore,
the concentration of plasma cortisol is directly related to the animal’s reactivity [47]. These
facts could help to explain the highest cortisol levels observed in females in the morning
period, once a higher reactivity was observed in females during all experiments and in our
previous study [21].
In this study, no significant differences in cortisol levels were observed between the
morning and afternoon periods. In the absence of stressful stimulation, corticotrophin-
releasing hormone (CRH) and vasopressin (VP) are released in greater amounts in the
early morning, causing an increase in cortisol levels in this period when compared to
the afternoon [47]. Differences observed in the literature can be explained by differences
in the methodology applied for data collection, as well as breed characteristics [49,50].
Furthermore, cortisol can remain in the animal’s body for 80 to 120 min [51], so the cortisol
levels can remain altered for up to two hours after the stressor action.
A significant interaction between sex and period was also detected for eosinophils con-
centration, where females, after sun exposure, exhibited higher eosinophils concentrations
while males presented lower concentrations of eosinophils in the same period. Eosinophils
are white blood cells that are analyzed together with other immune system cells to identify
systemic stress and acute or chronic inflammatory processes. In our study, heat stress
changed the eosinophil concentration, demonstrating a change in immunological patterns,
but it was not harmful to the animals’ health since the values observed are within those
found in healthy animals. According to [52], calves under heat environments presented
Animals 2022, 12, 3473 12 of 17

a significant decrease in eosinophil concentration. Although Caracu females presented

higher concentrations of eosinophils than males, there is no evidence in the literature of a
significant difference in eosinophils concentrations between sexes.
The RT is one of the most widely used physiological parameters to measure heat stress
in cattle because it indicates the internal temperature of the animal more accurately than
other parameters studied [6]. The RT measured after sun exposure (39.51 ◦ C) was higher
than obtained before sun exposure (38.04 ◦ C) in Caracu cattle, and males (38.85 ◦ C) showed
higher RT than females (38.70 ◦ C). In our study, all animals maintained their RT within the
expected temperatures for healthy animals (37.1 to 41.0 ◦ C), which may indicate adaptation
to the tropical environment.
Changes in serum cortisone levels act to suppress productive and reproductive func-
tions to support animal maintenance and survival [53]. Cortisone levels were higher in
animals after sun exposure than before sun exposure, and higher cortisone levels were
identified in Caracu females when compared to males. This result can be explained by the
tissue’s ability to increase or decrease cortisol concentration through 11ß-hydroxysteroid
dehydrogenase (11ßHSD) enzymes, which convert cortisol into hormonally inactive cor-
tisone [54]. Thus, the increase in cortisone levels after sun exposure and the consequent
decrease in cortisol levels in the same period was observed in the present study. Although
cortisone is not widely used as a heat stress marker, previous studies indicated that corti-
sone could be a useful biomarker for this purpose [55,56]. Therefore, additional studies to
validate this glucocorticoid as a marker of heat stress are needed.
The highest means of plasmatic progesterone levels was observed in animals before
sun exposure when compared to after sun exposure. Regarding sex, females exhibited
higher progesterone levels when compared to males. It was expected because it is known
that females produce higher concentrations of this hormone [57]. The heat stress effect on
plasmatic progesterone levels is controversial. Studies have reported that the progesterone
concentration is increased during heat stress [12,58], decreased [59], or did not change [60]
in cattle. These differences observed in the literature probably is due to the methodology
used or the variation in other factors that influence blood progesterone concentrations, such
as the kind of stress (acute or chronic) [53]. Furthermore, progesterone concentrations are
determined by differences between the production and the hepatic metabolism, and both
are affected by changes in dry matter intake [53], which can explain the highest means of
plasmatic progesterone levels observed in Caracu before sun exposure when compared to
after sun exposure. Furthermore, glucocorticoids act in several tissues, including the repro-
ductive system, provoking a decrease in hormone production, including progesterone [61].
Therefore, the alteration in the concentration of glucocorticoids due to heat stress observed
in this study may have affected the concentration of progesterone.
Clinical and hematological parameters can be used to assess animal welfare [62].
Hematocrit indicates the percentage of erythrocytes in the blood, which are cells rich in
hemoglobin, a metalloprotein responsible for transporting oxygen to the body’s tissues.
According to [52], animals lose metabolic intermediates in the blood when exposed to
chronic heat stress, resulting in decreased concentration of hemoglobin and hematocrit. In
the present study, the hemoglobin concentration and hematocrit percentage in Caracu were
higher in animals before sun exposure when compared to after sun exposure, and females
had the highest averages when compared to males. These results showed that sun exposure
could negatively influence the hemoglobin and hematocrit, which were also observed
by [50]. According to [62], cattle under prolonged thermal stress exhibit a reduction in
the percentage of hematocrit, while the increase in this parameter in animals under shade
(absence of thermal stress) is directly related to erythrocytes increase [63].
Erythrocytes are responsible for tissue oxygenation, so the greater the number of
erythrocytes directly influences the tissue oxygenation capacity [62]. The concentration of
erythrocytes before exposure to the sun was higher than after exposure to the sun, suggest-
ing that exposure to heat stress can negatively influence the concentration of erythrocytes.
Animals 2022, 12, 3473 13 of 17

Band neutrophil are immature neutrophils usually found in the blood when there is
an immune response in the acute phase, while segmented neutrophils are the most mature
neutrophilic granulocyte cells abundant in the circulating blood of healthy organisms [64].
In the present study, the concentration of band neutrophils was influenced only by sex,
and females had higher concentrations than males. It was observed that the concentra-
tion of neutrophils increased in animals after sun exposure when compared to before sun
exposure, as well as segmented neutrophils also increased in animals after sun exposure
when compared to before sun exposure. According to [65], the concentration of neutrophils
is negatively correlated with the serum cortisol concentration, demonstrating that gluco-
corticoids produced under stress can provoke a reduction in neutrophils. In the present
study, a higher concentration of cortisol in animals was also observed in Caracu before sun
exposure, and consequently, a lower concentration of neutrophils was observed in the same
period. Furthermore, the increase in the neutrophils and segmented neutrophils after sun
exposure could be an organism response, which may have interpreted the heat exposure as
an inflammatory response [66]. According to [50], glucocorticosteroids released due to heat
stress often results in neutrophilia. In addition, the concentrations of neutrophils and seg-
mented neutrophils observed in Caracu, as well as remaining erythrocytes concentrations,
keep within the regular values, indicating that this herd is tolerant to heat stress under
tropical conditions.
Platelets are blood components that play an important role in blood coagulation [64].
As observed in Caracu, the sun exposure does not affect the concentration of platelets,
which was influenced only by sex, with males showing higher concentrations than females.
The lower concentration of platelets observed in females can be explained by hormonal
influence, as progesterone and estrogen act on the endocrine system, increasing the blood
volume and resulting in hemodilution [67]. In addition, testosterone and its metabolites,
which are normally found in higher concentrations in males, are important stimulators of
hematopoiesis. The number of platelets may be raised by androgens, and there may be
an effect on platelet function with increased expression of the thromboxane-A2 receptor,
which can promote platelet aggregation [68].
The concentrations of leukocytes, lymphocytes, and monocytes were influenced by sex
in Caracu, with females showing higher concentrations than males. Studies have reported
metabolic disorders and chronic and infectious diseases in cattle related to changes in
leukocyte [69,70], lymphocyte [69,71], and monocyte [51,69] concentrations. However, only
healthy animals were used in our study, without the detection of pathologies or infectious
diseases. Thus, the higher concentrations of all these cells observed in Caracu females can
be attributed as a consequence of the higher reactivity observed in females throughout the
experiment and in our previous study [21].
Due to few thermal stress studies performed with Creole cattle breeds, which are already
considered adapted to the tropical climate, it was difficult to find studies to corroborate our
findings. Furthermore, the diversity of methodologies applied both to subject the animals to
heat stress and to collect the phenotypes may be responsible for the discrepancy observed in
relation to the hematological and hormonal parameters observed in the literature.

4.4. Hair and Skin Characteristics

Although several studies have been reported that animals with dark hair color had higher
RT when exposed to high temperatures compared to animals with light hair color [6,72,73], our
results showed that hair length, coat color, and muzzle color did not influence the RT and LS
traits (p > 0.05). Nicolau et al. [74] also not observe the correlation between RT and hair length,
hair, or skin colors in Caracu cattle. As suggested by [75], adaptation to hot environments
leads to the animal’s resistance to heat stress and not the type of coat. In addition, there is a
thermal gradient from the inside to the outside of the animal’s body [76], where the internal
temperature is higher and decreases to its periphery (skin and hair), which may explain why
RT and LS were not influenced by hair and skin traits evaluated. Thus, our results suggest that
hair and skin traits measured herein are not fundamental for heat stress evaluation in Caracu
Animals 2022, 12, 3473 14 of 17

cattle which can be attributed to natural selection for adaptability to the tropical climate that
the Caracu breed has been undergoing since its formation.

5. Conclusions
Infrared thermography displayed an effective tool to detect the internal temperature of
animals according to the methodology proposed in this study, and its use could reduce the
stress caused by handling and contributing to animal welfare. In addition, although several
thermoregulations, hematological and hormonal characteristics were altered when the
animals were under heat stress, most of them remained within the regular values observed
for taurine cattle breeds adapted to the tropical climate, evidencing the phenotypic plasticity
and adaptability of Caracu to the tropical environment. The climatic changes expected
in the coming decades may influence the type of cattle raised in herds around the world
under conditions of extreme heat. For this reason, in addition to contributing to the breed’s
preservation, it is essential to investigate the thermoregulation physiological aspects of a
Creole cattle breed.

Author Contributions: Conceptualization, N.B.S.; methodology, N.G.A., R.R.V. and L.E.F.Z.; formal
analysis, B.V.P., R.R.V., R.M.O.S. and C.C.P.P.; investigation, N.G.A., B.V.P., L.L.S. and R.R.V.; data
curation, C.C.P.P. and F.B.; writing—original draft preparation, N.G.A.; writing—review and editing,
L.E.F.Z., B.O.F., R.M.O.S., F.B., C.C.P.P. and N.B.S.; visualization, B.O.F., F.B., C.C.P.P. and N.B.S.;
supervision, N.B.S.; project administration, N.B.S.; funding acquisition, N.B.S. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was funded by São Paulo Research Foundation (FAPESP, Brazil), grant
number #2018/19216-7. The authors also thank the FAPESP for the fellowships granted to N.G.A.
(#2019/17251-2 and #2020/03699-9), L.L.S. (#2019/11738-7), and N.B.S. (#2019/10438-0). C.C.P.P.
received a productivity fellowship from CNPq (#303972/2018-1).
Institutional Review Board Statement: The animal study protocol was approved by the Institutional
Ethics Committee of Animal Science Institute, Nova Odessa, SP, Brazil (protocol code CEUA Nº.
292-19, 7th October 2019) for studies involving animals.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to privacy or ethical restrictions, and
the data that support the findings of this study are available in this article.
Acknowledgments: The authors would like to thank the Animal Science Institute for providing the
animals used in this study. We also would like to thank all researchers and students that contributed
to phenotype collection.
Conflicts of Interest: The authors declare no conflict of interest.

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