Archivos Latinoamericanos de Producción Animal. 2024. 32 (1)
Hematological and biochemical reference values for
the Brazilian Pony breed
Recibido: 20220225. Revisado: 20220509. Aceptado: 20231226
1Corresponding author: crq@uenf.br
1
Miguel Alejandro Silva Rua
Abstract: The aim of this study was to characterize the hematological profile of the equine Brazilian Pony breed herds
in northern of Rio de Janeiro state, Brazil. Blood samples were separated in categories: adult stallions, pregnant mares
and nonpregnant mares. The red blood cells (RBC), packed cell volume (PCV), hemoglobin (Hgb), main corpuscular
volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white
blood cells (WBC) and differential count of basophils (Baso), eosinophils (Eos), band cells (Bands), segmented
neutrophils (Neut), lymphocytes (Lym), monocytes (Mono), and platelets (Plat) were evaluated. The biochemical
parameters of urea (Urea), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl
transpeptidase (GGT), and creatinine were measured in pregnant and nonpregnant mares. Analysis of variance was
carried out with difference of hematological parameters between males and females. Also, analysis of variance was
used to verify the effect of pregnancy on biochemical parameters. There was effect of sex on RBC, hemoglobin, VCM
and MHCH, but PVC and MHC were not affected by sex. Higher means were observed in females. There was an
effect of sex on WBC, eosinophils and neutrophils. There was effect of pregnancy on AST, but no effect on urea, GGT
and Creatinine. According to the results obtained in this study, it can be concluded that the analysis of the blood count
and the biochemical of the Brazilian Pony reveals specificities compared to other equine breeds. Thus, these results
could be used as biomarkers for the evaluation of the Brazilian Pony breed in Rio de Janeiro.
Keywords: blood, horse, leukogram, pony.
https://doi.org/10.53588/alpa.320101
Laboratory of Animal Reproduction and Genetic Improvement, Northern Rio de Janeiro State University
(Universidade Estadual do Norte Fluminense/UENF). Brazil.
Wilder Hernando Ortiz Vega
Valores de referencia hematológicos y bioquímicos para la raza Pony Brasileño
Hematología y bioquímica en Pony brasileño
Resumen. El objetivo de este estudio fue caracterizar el perfil hematológico de un plantel de equinos de la raza
Pony Brasilera en el norte del estado de Rio de Janeiro, Brasil. Muestras de sangre fueron separadas en categorías:
sementales adultos, yeguas preñadas y yeguas no preñadas. Fueron evaluados los glóbulos rojos (RBC), el volumen
globular (PCV), la hemoglobina (Hgb), el volumen corpuscular principal (MCV), la hemoglobina corpuscular media
(MCH), la concentración de hemoglobina corpuscular media (MCHC), los glóbulos blancos (WBC) y el contaje
diferencial de basófilos (Baso), eosinófilos (Eos), células en banda (Bands), neutrófilos segmentados (Neut),
linfocitos (Lym), monocitos (Mono) y plaquetas (Plat). Los parámetros bioquímicos de Urea (Urea), alanina
aminotransferase (ALT), aspartato aminotransferase (AST), gama glutamil transpeptidase (GGT) y creatinina fueron
medidos en yeguas gestantes y no gestantes. Fue realizado análisis de varianza para verificar diferencias entre
machos y hembras en los parámetros hematológicos y bioquímicos. Fue verificado efecto del sexo sobre RBC,
hemoglobina, VCM y MHCH, pero PVC y MHC no fueron afectados por el sexo. Medias mayores fueron
observadas en el sexo femenino. Hubo efecto del sexo sobre leucocitos, eosinófilos y neutrófilos. Hubo efecto de la
preñez en AST, pero ningún efecto en la Urea, GGT ni Creatinina. De acuerdo con los resultados obtenidos en este
estudio, se puede concluir que el análisis del hemograma y la bioquímica del Pony Brasileño revelan especificidades
en comparación con otras razas de equinos. Por lo tanto, estos resultados podrían ser utilizados como puntos de
referencia para la evaluación de la raza Pony Brasileño.
Palabras clave: equinos, leucograma, bioquímica, poni.
Aylton Bartholazzi Junior
Luís Fonseca Matos Celia Raquel Quirino1
2
Introduction
Silva Rua et al.
The Brazilian Pony breed is a recently created breed,
records began in 1970, with the founding of the
Brazilian Association of Piquira and Pony Horses
(ABCCPONEI, 2022). These horses have been selected
since then, and due to their characteristics it is
necessary to conservation of the breed, as well as an
exhaustive control of mating in order to avoid
problems related to inbreeding. The control of animals
from the point of view of health is fundamental in any
program of genetic improvement and conservation. In
association with this, demand is increasing for
information about these horses’ physiology to support
diagnosis of health based on hematological parameters.
The literature contains a large amount of information
about the hematological and biochemical profile of
horses (Zobba et al., 2011; Adamu et al., 2013; Rukavina
et al., 2017; Ono et al., 2019; Ata et al., 2020;), but none is
available regarding the Brazilian Pony breed.
Horse breed is the result of natural and artificial
selection, and animals of the same breed in different
regions can present peculiarities regarding their
physiological characteristics (Fonteque et al., 2016). The
population of the modern horse have particularity
comparing to locally adapted breeds of horses
subjected to periods of natural selection (Petersen et al.,
2013). The correct interpretation of hematological and
biochemical test results requires specific reference
values for studied populations, due to important
variations observed in the local environment.
The horse hematological profile reported in the
literature (Chiba et al., 2016; Kupczynski & Spitalniak,
2015; Wanderley et al., 2015) may not be fully applicable
under all conditions because it is influenced by multiple
factors, including breed, environment and differences in
management (Adamu et al., 2013; Paden et al., 2014).
The evaluation of blood cells and biochemical
hematological profile is part of the clinical assessment
routine of horses. Hematological and biochemical
blood parameters are important to identifying the
breed’s physiological variations and are used for
clinical diagnosis of infectious and parasitic diseases.
They are also used to provide information about the
metabolic situation of animals and to monitor recovery
in the case of health problems (Adamu et al., 2013).
Information about the physiological characteristics
of the Brazilian Pony horse breed is limited in the
literature. Thus, the aim of this study was to evaluate
hematological and biochemical characteristics in
clinically healthy stallions and mares of the Brazilian
Pony breed.
Valores de referência hematológicos e bioquímicos para a raça Pônei Brasileiro
Hematologia e bioquímica em pôneis brasileiros
Resumo. O objetivo neste estudo foi caracterizar o perfil hematológico de um rebanho de equinos da raça Pônei
Brasileiro no norte do estado do Rio de Janeiro, Brasil. As amostras de sangue foram separadas em categorias:
garanhões adultos, éguas gestantes e éguas não gestantes. Os parámetros avaliados foram glóbulos vermelhos
(RBC), volume globular (PCV), hemoglobina (Hgb), volume corpuscular principal (VCM), hemoglobina corpuscular
média (HCM), concentração de hemoglobina corpuscular média (CHCM), glóbulos brancos (WBC), e contagem
diferencial de basófilos (Baso), eosinófilos (Eos), células em banda (Bands), neutrófilos segmentados (Neut),
linfócitos (Lym), monócitos (Mono) e plaquetas (Plat). Os parâmetros bioquímicos de Uréia (Urea), alanina
aminotransferase (ALT), aspartato aminotransferase (AST), gama glutamil transpeptidase (GGT) e creatinina foram
mensurados em éguas gestantes e não gestantes. A análise de variância foi realizada para comparar parâmetros
hematológicos e bioquímicos de equinos machos e fêmeas. . Efeito significativo do sexo foi verificado sobre
hemácias, hemoglobina, VCM e MHCH (médias mais elevadas foram observadas em fêmeas), mas PVC e MHC
foram semelhantes entre animais machos e fêmeas. O fator sexo também afetou leucócitos, eosinófilos e neutrófilos.
A prenhes afetou AST, mas os resultados de Uréia, GGT ou Creatinina foram semelhantes. De acordo com os
resultados obtidos neste estudo, as análises do hemograma e da bioquímica do Pônei Brasileiro revelam
especificidades entre os fatores em estudo e em comparação com outras raças de cavalos. Assim, esses resultados
podem ser utilizados como valores de referência para a raça Pônei Brasileiro.
Palavraschave: cavalos, leucograma, bioquímica, pônei.
ISSNL 10221301. Archivos Latinoamericanos de Producción Animal. 2023. 32 (1): 17
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Hematological and biochemical reference values for the Brazilian Pony breed
Animals
The animals, the animals of the Brazilian Pony breed,
were raised in the municipality of Campos dos
Goytacazes, northern Rio de Janeiro state, Brazil (21o 45
´15´´ South latitude, 41o 19´28´´ West longitude, and 13
meters above sea level). The feeding of the animals was
the same for males and females. All animals were
maintained in pastures with Coast Cross grass
(Cynodon dactylon) throughout the experiment.
According to the scale of Köppen and Geiger, the
climate of this region is classified as Aw, with the mean
annual minimum temperature of 20° C and mean
annual maximum temperature of 36° C). The average
annual rainfall is 1,073 mm, with more rainfall in the
summer than winter. The precipitation varies 156 mm
between the driest month and the wettest month. This
experiment was carried out in the autumn period
(April to June).
Brazilian Pony animals were evaluated and separa
ted in the following categories: 1 stallions (N= 20,
aged 613 years); 2 pregnant mares (PM, N= 44, aged
619 years) (Figure 1). The animals were clinically
examined, performed by veterinarians or specialized
techniques, before the experiment and only healthy
ones were used. Clinical examination was performed
and the body condition score, cardiac frequency and
respiratory frequency were measured to determine
health. Normal ranges for hematology and plasma
chemistry consider the values published by
Southwood, L.L. 2013.
Sample collection and handling
A blood sample was taken from all subjects between
8:00 am and 10:00 am and were then taken once a month
between February and November for examination.
Blood samples were obtained from all animals by jugular
venipuncture and stored in Vacutainer tubes. For
hematological study blood was collected in sterile vial (5mL)
using potassium ethylenediaminetetraacetic acid (EDTA
Sigma Chemical Co, St. Louis, MO, USA) as anticoagulant
and for collection and preservation of serum 10 ml of blood
was collected from jugular vein of each animal in
heparinized test tubes. All material was processed in the
Clinical Pathology sector (UENF).
The tubes containing blood were kept in slanting position
and the blood was allowed to clot. After the blood got
clotted, the tubes were transferred to refrigerator at 4 degree
centigrade for 12 hours to allow maximum secretion of
serum from the clot. Then the serum was pipetted out from
the tubes and was centrifuged at 2,500 rpm for 5 minutes to
separate unlysed cells and other darts. The clear serum was
collected carefully into sterilized vials and stored in the
frozen chamber of the refrigerator. No preservatives were
added to the serum. Before using the frozen serum for
experiment, it was allowed to defrost at room temperature.
The first study compared the males and pregnant
mares regarding red blood cells (RBC), packed cell
volume (PCV), hemoglobin (Hgb), main corpuscular
volume (MCV), mean corpuscular hemoglobin (MCH),
mean corpuscular hemoglobin concentration (MCHC),
white blood cells (WBC) and differential count
basophils (Baso), eosinophils (Eos), band cells (Bands),
segmented neutrophils (Neut), lymphocytes (Lym),
monocytes (Mono) and platelets (Plat). Hematology
assessment was accomplished with an electronic cell
counter (MS4®) and the Instant prov (Panótico PA205
Newprov® dye).
The second study compared two stages: the pregnant
and nonpregnant mares, with biochemical analysis of
urea (Urea), alanine aminotransferase (ALP), aspartate
aminotransferase (AST), gamma glutamyl transpeptidase
(GGT), and creatinine (Creatinine). Were analyzed using
standard kits with the aid of a spectrophotometer.
Material and Methods
Figure 1. Brazilian Pony breed. Stallion (A). Pregnant mare (B).
ISSNL 10221301. Archivos Latinoamericanos de Producción Animal. 2023. 32 (1): 17
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Effects were detected of sex on RBC, hemoglobin,
VCM and MHCH (P < 0.05). Means and standard
deviations of hematological parameters in Brazilian
Pony horses are presented in Table 1. Only PCV and
MCH showed no effect regarding sex (P > 0.05).
Results and Discussion
Statistical analysis
The data were analyzed using the SAS® software
(Statistical Analysis System, University Edition) to
verify the mean, median, standard deviation, mean
standard error, and maximum and minimum values
for each variable. The assumption of normal
distribution of all variables was checked (Proc
Univariate), and the variables that were not normally
distributed (white blood cells, segmented neutrophils,
lymphocytes, eosinophils, monocytes) were
transformed with (log10+1). The means and standard
deviations are presented in the original scale.
In the first study involving hematological parameters,
analysis of variance of repeated measures was carried out
with the difference of hematological parameters between
males and females using the GLM procedure. Sex effects
were considered significant if P < 0.05.
In the second study, involving differences of
biochemical parameters between pregnant and non
pregnant mares and in the comparison between the
stages pregnant mares and nonpregnant mares (nPM),
the Tukey test was used at 5 % probability.
Table 1. Hematological values (Mean ± standarddesviation) in the Brazilian Pony breed according the sex.
Parameter Reference values Male(N= 20) Female pregnant(N= 44)
RBC (x106/µL) 6.210.2 6.1 ± 1.2a 5.9 ± 1.1b
PCV (%) 2445 31.7 ± 4.7a 31.4 ± 4.2a
Hgb (g/dL) 11.417.3 10.8 ± 1.6a 10.4 ± 1.4b
MCH (pg) 12.319.9 17.1 ± 3.5a 17.4 ± 3.4a
MCV (fL) 3758 52.1 ± 5.4b 53.6 ± 5.4a
MCHC (g/dL) 3138.6 33.4 ± 1.6a 32.8 ± 2.7b
N= number of animals in the study. Values with different letters in the same row are significantly different (P <0.05). Red blood cells (RBC); packed cell volume
(PCV); hemoglobin concentration (Hgb); mean corpuscular hemoglobin (MCH); volumetric index (MCV); mean corpuscular hemoglobin concentration (MCHC).
Effects were detected of sex on WBC, eosinophils
and neutrophils (P < 0.05). However, there were no
effects of sex on basophils, band cells, lymphocytes,
monocytes and platelets (P> 0.05). For the three
variables affected by sex (RBC, Hgb and MCHC), the
higher means were observed in females (6.1 ± 1.2, 10.8
± 1.6 and 33.4 ± 1.6 x 106 µ/L respectively) (Table 2).
Table 2. Leukogram values (Mean ± standarddesviation) in the Brazilian Pony breed according the sex.
Parameter Reference values Male(N= 20) Female pregnant(N= 44)
WBC (x109/L) 4.910.3 6.775 ± 1.778b 7.492± 3.756a
Baso (x109/L) 00.3 0.005 ± 0.018a 0.005 ± 0.022a
Eos (x109/L) 00.8 0.213 ± 0.182b 0.283 ± 0.447a
Bands (x109/L) 00.2 0.025 ± 0.055a 0.441 ± 0.147a
Neut (x109/L) 2.28.1 3.777 ± 1.224b 4.284 ± 1.794a
Lym (x109/L) 1.75.8 2.732 ± 1.324a 1.203 ± 0.001a
Mono (x109/L) 0.11.0 0.247 ± 0.318a 0.245 ± 0.216a
Plat (x109/L) 72183 251.945 ± 417.883a 230.781 ± 125.679a
Values with different letter in the same row are significantly different (P < 0.05).
Number of animals (N); white blood cells (WBC); basophils (Baso); eosinophils (Eos); band cells (Bands); segmented neutrophils (Neut); lymphocytes (Lym);
monocytes (Mono); platelets (Plat).
The blood biochemistry of mares was evaluated in
two categories (nonpregnant and pregnant mares) and
we observed effects of the category on ALT and AST (P
< 0.05). However, there were no effects of mare
category on urea, GGT and creatinine (P > 0.05). The
higher means for AST were observed in nonpregnant
mares (Table 3).
Silva Rua et al.
ISSNL 10221301. Archivos Latinoamericanos de Producción Animal. 2023. 32 (1): 17
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The means of RBC, hemoglobin and MCHC observed
in this study in the Brazilian Pony breed, for males and
females were different, where the males presented
higher means (Table 1). The male horses were all in
reproductive activity. Therefore, during the experiment
they presented more anxious behavior and excitement
most of time, mainly with the presence of mares,
different from the females, who behaved more calmly,
with lass movement than stallions. Thus, we can
suggest that higher frequency of exercise of males might
act to increase the release of RBC into the circulation as
result of splenic contraction. And the higher means of
Hgb and MCHC can be explained by the fact that the
increased activity induced higher oxygencarrying
capacity of the blood to the muscles in activity (Zobba et
al.,2011). The same authors reported that these
hematological variables vary with exercise intensity.
Comparison of the hematological parameters with
those reported in other studies showed that the means
of RBC, hemoglobin and PCV were lower comparing
to the results reported by Andrade et al. (2016)
studying Brazilian Pony horses from the municipality
of Orlando, São Paulo, Brazil. We can suggest these
differences were due to different regions where the
studies were conducted. The difference of altitude and
climate between the regions could cause differences in
hematological profiles of animals. The altitude of
Orlando is 695 m, higher than Campos dos
Goytacazes. According to Vivek et al. (2018),
hematological profiles of RBC, hemoglobin and PCV
tend to increase with the increase of altitude due to
greater splenic contraction. In comparison with the
study by Zobba et al. (2011), with Thoroughbred horses
in Italy, the means of RBC, Hgb and PCV of the
Brazilian Pony animals in this study were lower, and
the mean of MCVC was higher. The higher means of
MCHC of the animals we evaluated could be a
compensatory reaction to lower values of erythrocytes
and hematocrit. We found values of erythrocytes
lower than reported by Shawaf et al. (2018), who
evaluated hematological parameters of Shetland
ponies in Saudi Arabia. However, we found higher
means of hemoglobin, PCV, MCV, MHC and MCHC.
Regarding the leukogram analysis (Table 2), we
observed higher means of white blood cell count,
eosinophils and neutrophils in females. Some factors
can explain this result. Mares’ hemogram parameters
can be affected by hormonal concentration during
their estrous cycle, pregnancy and lactation (Faramarzi
et al., 2018; Mariella et al., 2014) and increase of
regulatory T cells after artificial insemination (Awan et
al., 2016). In contrast, stallions have rarely been found
to have eosinophils in the interstitial regions of the
testes (Brosnahan 2018).
We observed lower means of leukogram parameters
compared with the values presented by Andrade et al.
(2016), also studying the Brazilian Pony breed (10.7 ±
0.22 x 109/L of WBC, 0.626 ± 0.317 x 109/L of
basophils, 4.589 ± 0.697 x 109/L of neutrophils and
4.336 ± 0.701 x 109/L of lymphocytes). These
differences could be due to the local climates where
the studies were conducted. The present study was
performed in a region with a climate characterized by
high temperatures, reaching 34° C, and mean annual
temperature range from 25 to 28° C (Rua et al., 2018).
High temperature can cause stress on equines, and this
is followed by increased cortisol release (Fenner et al.,
2016; Rizzo et al., 2017).
The analysis of the blood count and the biochemical
of the Brazilian Pony reveals specificities compared to
other equine breeds. The data for Brazilian Pony in the
present study were obtained in Autumn. Is necessary
to consider that the season period can affect the
hematological and biochemical parameters. In
addition, reference values for young, growing horses
should be established separately. It is necessary to
further investigate the effects of feed, age, and season.
However, the results obtained in this study will be
utilized for veterinary practice and appropriate
management of Brazilian Pony breed.
Table 3. Biochemistry of nonpregnant and pregnant Brazilian Pony mares.
Mare stage
Parameter Reference values Nonpregnant(N= 18) Pregnant(N= 44)
Urea (mg/dL) 2656 34.6 ± 8.8a 33.7 ± 9.9a
ALT (UI/L) 410 5.0 ± 1.64a 3.78 ± 1.93b
AST (UI/L) 205555 391.3 ± 137.6a 237.9 ± 128.4b
GGT (UI/L) 1245 32.5 ± 55.6a 24.9 ± 36.9a
Creatinine (mg/dL) 0.61.8 1.4 ± 0.3a 1.3 ± 1.7a
Values with different letter in the same row are significantly different (P < 0.05).
Number of animals (N); Urea (Urea); alanine aminotransferase (ALT); Aspartate aminotransferase (AST); Gamma glutamyl transpeptidase (GGT).
Discussion
Conclusion
Hematological and biochemical reference values for the Brazilian Pony breed
ISSNL 10221301. Archivos Latinoamericanos de Producción Animal. 2023. 32 (1): 17
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Acknowledgement: To the owner and employees of pony Farm.
Conflict of interests: The authors declare there are no conflicts of interests in the present paper.
Ethics statement: This experiment was approved by the Committee on Ethical use of animals (CEUA) of Norte
Fluminense State University (UENF) of the state of Rio de Janeiro, according to the standards of the Brazilian Society
of Laboratory Animal Science/Brazilian College of Animal Experimentation (SBCAL/COBEA), under protocol
number 307.
Funding Information: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES), finance code 001. We are also thankful for the support of fellowship of the Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and CNE FAPERJ.
Author contributions: Miguel Alejandro Silva Rua and Celia Quirino Conceived or designed study. Aylton
Bartholazzi Junio and Miguel Alejandro Silva Rua, Performed research. Celia Quirino and Aylton Bartholazzi
Junior, Analyzed data. Aylton Bartholazzi Junior, Wilder Hernando Ortiz Vega and Luis Fonseca Matos. Wrote
the paper. Luis Fonseca Matos and Celia Quirino Supervision. Wilder Hernando Ortiz Vega. Reviewed manuscript.
Edited by: Julio Cesar de Souza
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Hematological and biochemical reference values for the Brazilian Pony breed
