Received: 12 November 2020 | Revised: 8 April 2021 | Accepted: 3 May 2021

DOI: 10.1111/vcp.13036

R E FE R E N CE I NTE RVA L S

Hematologic and biochemical reference intervals of

brown-­throated sloths (Bradypus variegatus)

Carolina Romeiro Fernandes Chagas1,2 | Caio Filipe da Motta Lima1 |

Irys Hany Lima Gonzalez1 | Paula Andrea Borges Salgado1,3 | Cauê Monticelli1 |
Patrícia Locosque Ramos1,3

1
São Paulo Zoological Park Foundation,
São Paulo, Brazil
2
Nature Research Centre, Vilnius,
Lithuania
3
Federal University of São Carlos –­
Post Graduation Program in Wildlife
Conservation, São Carlos, Brazil
Correspondence
Carolina Romeiro Fernandes Chagas,
Nature Research Centre, Vilnius,
Lithuania.
Email: crfchagas@gmail.com
Funding information
American Society for Veterinary Clinical
Pathology; Park Foundation
Abstract
The brown-­
throated sloth, Bradypus variegatus, is a common species endemic to
South and Central America. Nonetheless, maintaining these animals in captivity can
be challenging, and very few institutions manage to do so. The São Paulo Zoological
Park Foundation is in a remnant of the Atlantic rainforest in the middle of São Paulo,
the largest city in Brazil. This forest fragment has a population of B. variegatus that
is geographically isolated and yet to be studied. Assessing the health status of indi-
viduals remains difficult due to the lack of reference intervals (RIs) for hematologic
and biochemical variables for this species. We aimed to establish hematologic and
biochemical RIs in a population of wild B. variegatus living in the largest remnant of
Atlantic rainforest in São Paulo city, Brazil. Blood samples from 25 individuals of wild
B. variegatus were collected and analyzed for 20 hematologic and 21 biochemical vari-
ables, using standard laboratory techniques. Each variable was statistically analyzed
according to the American Society for Veterinary Clinical Pathology guidelines. The
results obtained for each variable were statically analyzed, making it possible to ob-
tain descriptive statistics for all hematologic and biochemical variables. RIs were de-
termined for 16 hematologic variables. During the microscopic analysis, we observed
anisocytosis, polychromatophils, Howell-­Jolly bodies, macroplatelets, and reactive
lymphocytes. The RIs and descriptive statistics described here establish important
baseline numbers that could be essential for the management and treatment of both
captive and wild B. variegatus sloths.

KEYWORDS
clinical analyses, Pilosa, reference interval, Urban Fauna, wildlife, Xenarthra

1 | I NTRO D U C TI O N Ecuador, Honduras, Nicaragua, Panama, Peru, and Venezuela.1

This species is listed as “least concern” by the IUCN (International
The brown-­throated sloth, Bradypus variegatus (Xenarthra: Pilosa), is Union for Conservation of Nature) Red List,1 and the species is not
one of the most common sloth species. It is endemic to South and listed as threatened of extinction by the Ministry of Environment
Central America, present in Brazil, Bolivia, Colombia, Costa Rica, in Brazil. This is mainly because B. variegatus has a wide geographic

© 2022 American Society for Veterinary Clinical Pathology

126 | 
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Chagas et al.
distribution and usually inhabits protected areas. 2 In addition, this
is a tree mammal, with camouflage fur, slow and silent movements,
making it very difficult to observe in their natural habitat, likely
explaining the limited knowledge regarding their behavior, genetic
diversity, ecology, and biology.3 Moreover, genus Bradypus' sloths
4
rarely survive for long periods under human care, mainly due to
them being essentially folivorous with a highly selective diet that is
difficult to replicate,5,6 further contributing to the limited knowledge
available.
Thus, to establish better conditions to maintain healthy B. varie-
gatus individuals in captivity, we must (i) have proper facilities pro-
viding all the environmental conditions required by the species;6 (ii)
provide biologists and veterinarians with the technical knowledge
to better manage and assess an animal’s health status. Indeed, this
assessment can be made much easier using reference intervals (RIs),
a critically important element for diagnosis and health management.
Because the variables used to determine RIs can vary due to several
environmental factors, biotic and abiotic, it is important to follow the
American Society for Veterinary Clinical Pathology (ASVCP) guide-
7
lines to establish RIs.
The goal of the present study was to describe RIs for hemato-
logic and biochemical variables in a wild Bradypus variegatus popu-
lation living in the largest remnant of the Atlantic rainforest in São
Paulo, Brazil. These RIs will likely provide important information for
veterinarians and biologists that manage these animals.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Study area and studied population
The study was conducted at Fontes do Ipiranga State Park (PEFI)
(23°39′05”S, 46°37′24”W) in São Paulo, the largest city in Brazil.
PEFI is a well-­preserved remnant of a native Atlantic rainforest sur-
rounded by a large urban area within the metropolitan region of São
Paulo (Figure 1). These large urban areas provide both genetic and
geographic isolation since the nearest fragment of forest is located
more than 20 km away, in Cantareira State Park.
Between March 2011 and August 2018, 25 brown-­throated
sloths were captured for clinical examination and sample collection.
The individuals were classified according to age-­
weight catego-
ries,8,9 consisting of 17 adults (>4000 g; >3 years old), five subadults
(2301–­4 000 g; between 1.5 and 3 years old), two juveniles (1000–­
2300 g; between 11 months and 1.5 years), and one individual of
unknown age. Of these, 13 were females, six were males, and six
were not sexed. All animals were caught manually after being visu-
ally located, and immediately transported to the Veterinary Division
for a more careful examination. Each brown-­
throated sloth was
gently restrained (no sedation or anesthetic drugs were used), and
blood samples were drawn. All animals received a transponder (mi-
crochips) placed between the shoulder blades for individual identi-
fication in case of recapture and were released within 60 minutes
or less after being captured. Official permission for capturing and
| 127
collecting blood samples was issued by the Brazilian Environment
Agency, SISBIO, under number 49627.
All brown-­throated sloths captured were physically examined
by a veterinarian. Animals were considered healthy if they had no
visible lesions, altered behavior, or any other symptom that would
be compatible with underlying pathologies. Individuals showing any
disease symptoms and juveniles were excluded from the study. Each
individual contributed a single sample.
2.2 | Blood collection and laboratory analyses
Three to 5 mL of blood was drawn from either the cephalic or
femoral vein, using disposable syringes and 20 × 0.55 mm needles.
Immediately after sample collection, a few drops of blood were used
to prepare thin blood smears that were air-­dried and stained with
Panótico (Laborclin), a type of Diff Quick stain for the morphologic
analysis of blood cells and stains leukocytes similarly to other mam-
mals.10 The remaining blood sample was stored in two different
tubes, one containing EDTA, used for hematologic analyses, and
a second one with lithium heparin used for biochemical analyses.
Samples were immediately taken to the Clinical Analyses Laboratory,
in the Applied Research Department from FPZSP, where they were
kept refrigerated between 4 and 8°C until processing. All samples
were processed within 2–­3 hours after collection.
The hematologic analyses consisted of determining red blood
cell (RBC) numbers, packed cell volumes (PCVs), hemoglobin con-
centrations, mean corpuscular volumes (MCVs), mean corpuscular
hemoglobin (MCH), mean corpuscular hemoglobin concentration
(MCHC), platelets, nucleated red blood cells (nRBCs), reticulocytes,
white blood cells (WBCs), and WBC differential counts (Table 1).
RBCs, WBCs, and platelets were manually counted in a Neubauer
type chamber;10 if platelet clumping occurred during the counting
process, the quantification was discontinued, and the measurement
was discarded. Hemoglobin concentration was determined by the
cyanmethemoglobin method using a commercial hemoglobin kit
(Labtest Diagnóstica S.A., Brazil). PCVs were determined using the
microhematocrit technique, using microcapillary tubes that were
centrifuged for 5 minutes at approximately 16 128 g. MCVs, MCH,
and MCHC were calculated according to RBC, hemoglobin, and
PCVs results.11 Reticulocytes were determined by mixing one part
blood with one part methylene blue. After 5 minutes, this mixture
was used to prepare a thin blood smear, which was microscopically
analyzed. The number of reticulocytes was determined in 1000
RBC.10 WBC differential counting was performed on 100 consecu-
tive WBCs using 1000× magnification. The counting was performed
in the monolayer part of the smear, and both the edges and center of
the blood smear were evaluated. When the counting was finished,
the absolute number of each WBC type was calculated.10 All micro-
scopic analyses were conducted by professionals with several years
of experience in wild animal hematology.
The biochemical analysis consisted of measuring 21 variables
(Table 2) and was performed with plasma, obtained after the
128 | Chagas et al.
F I G U R E 1 Study site map. The São Paulo Zoological Park Foundation is located in the biggest city of Brazil, São Paulo
centrifugation (5 minutes at 700 g) of blood samples containing lith-
ium heparin. Measurements were conducted using a Cobas c111
Analyzer + ion-­
selective electrode (ISE) Unit (Roche Instrument
Center). All kits used in these analyses are provided by the manufac-
turer, and the samples were processed according to the manufactur-
er’s instructions; except for globulin, which was indirectly determined
10
by subtracting albumin from total protein concentrations.
The instrument was calibrated following the manufacturer’s in-
structions when reagents were replaced, or quality controls were
out of the predefined interval. Quality control was performed
with two human controls (Precinorm U and Precipath U; Roche
Instrument Center) every other day, and each time the equipment
was calibrated.
2.3 | Statistical analyses
Statistical analysis followed guidelines of the ASVCP for determining
RIs, which considers that population-­based RIs encompass the cen-
tral 95% of reference values (RVs) and are bound by upper and lower
reference limits. The statistical method selected for the determina-
tion of reference limits is based on the number and distribution of
RVs.7 We prepared histograms and quantile-­quantile plots for each
variable quantified and examined the distributions. Variables with
less than 20 samples were included only as descriptive statistics and
were not used to determine RIs, as the sample size was too small to
determine the limits. For variables with more than 20 samples, we
used the Shapiro-­Wilk test to confirm normality. If the test P < 0.3,
the RV was judged as having a non-­
Gaussian distribution. This
threshold was chosen due to our small population size, as proposed
by Le Boedec.12 We also used Dixon’s Q test to detect possible outli-
ers. Outlier detection was repeated until there were no more out-
liers identified and considered for removal. RIs were calculated by
parametric (if normality could be established) or robust (distribution-­
independent) methods. To highlight the uncertainty inherent with
small sample sizes, 90% confidence intervals were calculated using a
bootstrap method. We used the ReferenceIntervals package in the R
software (R Development Core Team, 2019) for all statistical tests.
3 | R E S U LT S
A total of 23 individuals from B. variegatus were included in the
statistical analysis; 17 were adults, five were subadults, and one
 Chagas et al.
TA B L E 1 Descriptive statistics and reference intervals of hematologic measurands for wild Bradypus variegatus from Fontes do Ipiranga State Park, São Paulo, Brazil

Measurand Units N Mean SD Median Min Max p-­value Dist Method RI LRL (CI 90%) RI URL (CI 90%)

PCV % 22 35 5.5 34 25 49 0.52 G P 24 (20.6–­27.2) 46 (42.2–­48.8)

RBC count 106/μL 21 3 0.6 3 2.1 4.5 0.174 NG R 1.7 (1.3–­2.1) 4.2 (3.7–­4.6)
Hemoglobin g/dL 22 10 1.5 9.7 8.2 13.0 0.015 NG R 6.7 (6–­7.7) 13.1 (12.2–­14.3)
MCV fL 22 120 20.4 124.1 66.7 152.4 0.06 NG R 80.7 (63–­96.3) 168.7
(155.2–­184.6)
MCHC g/dL 22 29.6 2.8 29.6 24.8 36.5 0.798 G P 24 (22.3–­25.7) 35.1 (33.4–­36.8)
MCH pg 22 35.2 5.45 36.2 24.4 45.1 0.07 NG R 25.1 (21.3–­3 0.1) 48.3 (45.9–­51.8)
nRBC /100 WBC 22 1.4 1.3 1 0 5 0.005 NG R 0 4.1 (3.1–­5.2)
3
Platelet count 10 /μL 10 252.5 66.3 256.5 184 388 —­ —­ —­ —­ —­
Reticulocyte % 11 1.2 0.7 1.1 0.3 2.4 —­ —­ —­ —­ —­
WBC count 103/μL 21 8.7 2.8 8.1 4.2 15.4 0.092 NG R 2.5 (0.6–­4.0) 14.6 (12.9–­16.8)
a
Band Neutrophil % 22 0.82 1.9 0 0 9.0 —­ —­ —­ —­ —­
a 3
Band Neutrophil 10 /μL 22 0.08 0.2 0 0 0.7 —­ —­ —­ —­ —­
Neutrophil % 22 40 18.5 37.5 13 81 0.032 NG R 0 (0–­0.6) 7.2 (5.8–­8.9)
Neutrophil 103/μL 22 3.51 1.82 3.1 1.4 8.8 0.258 NG R 0 (0–­11.48) 78.19
(65.54–­92.77)
Lymphocyte % 22 51 17.8 53.5 19 78 0.296 NG R 13.7 (0.2–­24.4) 90.7 (80.6–­100.3)
Lymphocyte 103/μL 22 4.91 3.06 4.2 1.4 14 0.025 NG R 0 11.01
(8.62–­13.84)
Monocyte % 22 4 2.9 3.5 0 12 0.101 NG R 0 9.7 (7.2–­12.2)
Monocyte 103/μL 22 0.35 0.28 0.3 0 1 0.051 NG R 0 0.92 (0.71–­1.17)
Eosinophil % 22 4 3 3 0 11 0.142 NG R 0 9.9 (7.8–­12.4)
Eosinophil 103/μL 22 0.31 0.23 0.4 0 0.6 0.006 NG R 0 (0–­0.03) 0.84 (0.72–­0.96)

Abbreviations: Dist, distribution; G, Gaussian; LRL, lower reference limit; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean cell volume; N, number of
valid observations; NG, non-­Gaussian; P, nonparametric; PCV, packed cell volume; RBC, red blood cell; R, robust method; RI, Reference interval; SD, Standard deviation; URL, upper reference limit; WBC;
white blood cell.
a
Due to the presence of outliers, it was not possible to calculate the reference interval for this measurand.

|
129
130 | Chagas et al.

TA B L E 2 Descriptive statistics of biochemical measurands of wild Bradypus variegatus from Fontes do Ipiranga State Park, São Paulo,
Brazil

Measurand Units N Mean SD Median Min Max Analytical method

Sodium mEq/L 17 124.1 5.1 125 117 140 Ion-­selective electrode (ISE)
Potassium mEq/L 17 4.5 0.6 4.5 3.6 6.2 Ion-­selective electrode (ISE)
Chloride mEq/L 17 91.9 4.7 91 82 103 Ion-­selective electrode (ISE)
Calcium mg/dL 18 10.7 0.8 10.7 9.5 12.1 Enzymatic colorimetric
Phosphorus mg/dL 18 3.5 1.1 3.25 1.9 6.6 Endpoint with sample blanking
Uric acid mg/dL 16 1.3 0.5 1.3 0.3 2.2 Enzymatic colorimetric
Urea nitrogen mg/dL 20 38.2 8.8 37.8 22.8 53.2 Kinetic urease/glutamate dehydrogenase
Creatinine mg/dL 17 0.5 0.2 0.5 0.2 0.7 Jaffé method
AST U/L 18 342.4 99.1 336 156 559 L-­aspartate and α-­ketoglutaric as substrates
CK U/L 17 242.4 174.6 183 63 600 UV-­test
GGT U/L 16 42.9 40.2 31 15 186 Enzymatic colorimetric
ALP U/L 17 275.5 125.9 256 94 478 p-­nitrophenyl phosphate as the substrate
ALT U/L 16 5.8 5.2 4 2 23 L-­alanine and 2-­oxoglutarate as substrate
Magnesium mg/dL 16 3 0.7 2.89 2.14 4.53 Colorimetric with chlorophosphonazo III
Glucose mg/dL 20 91.8 16.7 93.5 49 127 Hexokinase
Amylase U/L 15 90.2 27.6 90 51 135 Enzymatic colorimetric
Triglyceride mg/dL 18 45.9 17.7 43 25 98 Enzymatic colorimetric
Cholesterol mg/dL 18 96.9 35.9 91 52 188 Enzymatic colorimetric
Total protein g/dL 18 7.5 0.7 7.45 5.9 8.5 Colorimetric with cupper
Albumin g/dL 18 3.3 0.5 3.2 2.7 4.6 Bromocresol green
Globulin g/dL 18 4.1 0.9 4.2 2.2 5.5 Calculated

Abbreviations: CK, creatine kinase; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, γ-­glutamyl
transferase; N, number of valid observations; SD, standard deviation.

had no known age. Of these, 12 were females, six were males; and
five were not sexed. Two individuals were excluded from the study
because the clinical and laboratory exams showed signs of illness.
Importantly, all animals were negative for blood parasites. In con-
trast, 20 individuals (out of 25) had ectoparasite infections (these
results are partially reported in Gonzalez et al13).
The remaining data were sufficient to establish brown-­throated
sloth Bradypus variegatus RIs for 16 of 20 hematologic variables
(Table 1). Due to the small number of individuals, it was not possible
to determine RIs for any of the biochemical variables, as well as a few
(4) hematologic variables. Nonetheless, we provide the descriptive
statistics for all variables quantified (Table 1).
For hematologic RI determinations, measurements from 22 in-
dividuals were statistically analyzed (17 were adults, four were sub-
adults, and one was of an undetermined age; 12 were females, six
were males, and four were not sexed). The RIs for platelet, reticulo-
cytes, band neutrophils could not be determined because the values
could not be determined in enough samples (Table 1). A similar situa-
tion occurred with the biochemical variables, in which samples from
21 individuals were analyzed (15 were adults, five were subadults,
and one was of undetermined age; 10 were females, six were males,
and five were not sexed). Unfortunately, we could not measure any
biochemical variables in >20 individuals (Table 2). As a result, we were
not able to calculate RIs for these variables. These values were used
to provide descriptive statistics instead, with maximum and minimum
values, means, standard deviations, and medians. Table 1 shows the
hematologic variables, and Table 2 shows the biochemical variables.
Histograms, quantile-­quantile plots, and boxplots for each measured
variable are available in the supplementary material (Data S1). In addi-
tion, despite the small number of individuals, we provided descriptive
statistics for females and males separately (Tables S1 and S2).
Of note, the morphologic analyses of peripheral blood smears
revealed anisocytosis, polychromatophils, and Howell-­Jolly bodies.
Some macroplatelets were also observed, as well as reactive lym-
phocytes (identified by increased basophilic cytoplasm). We did not
find basophils in the WBC differential counts.
4 | DISCUSSION
This is the first study that provides population-­based RI for Bradypus
variegatus. There are only a few reports of hematologic and plasma
11,14-­16
biochemical values in B. variegatus, all had smaller sample
sizes (10–­19 individuals) than in the present study. Importantly, none
of these prior studies calculated population-­based RIs using sta-
tistical analysis. Even though descriptive values are often the only
available tool to interpret laboratory results, they should be used
cautiously because they lack statistical validation and are prone to
Chagas et al.
bias. The present study addresses this problem not only by describ-
ing the hematologic and biochemical profile for a larger number of
individuals but also by proving the first population-­based hemato-
logic RIs for this species.
There is only one study with B. variegatus that used similar meth-
15
odologies. The main hematologic differences between our results
and the ones reported by Pereira et al15 was the percentage of
neutrophils and lymphocytes (Table 1). More specifically, we found
a higher relative neutrophil mean (40% vs 22.37% and 21.57%, in
each of the distinct study sites) and lower lymphocyte count (51%
vs 70.5 and 72%, in each of the distinct study sites). The absence
of basophils in the WBC differential count was also reported by
Pereira et al,15 which is not surprising since these cells are less fre-
10
quently observed in mammalian blood smears. Additionally, AST
(342.4 U/L) and ALP (275.5 U/L) were higher in our study than in
the results reported by Pereira et al.15 A possible explanation for the
differences observed here is that we had a higher number of females
15
compared with Pereira et al, which had a higher number of males.
Due to the few studies addressing the RIs for sloths, there is no con-
crete evidence on how sex would interfere with sloth RIs, which is a
known variable for other species.7 Thus, we provide descriptive sta-
tistics of females and males for all studied variables (Tables S1 and
S2), indicating sex differences in several parameters, including RBC,
WBCs, and ALP. However, due to the small number and unbalanced
nature of our sample (higher number of females), it is not possible
to establish firm conclusions about differences between sexes, and
further research is needed. Another reason could involve the envi-
ronmental habitat, as the study site was different.
A direct comparison of the descriptive values presented here
with previously reported numbers is challenging. There are many dif-
ferences between our study and previous reports: (i) the number of
individuals is often smaller; (ii) the geographic origin of the samples
is different with distinct biotic and abiotic factors (ie, captivity ani-
11,14-­16 11
mals); (iii) sample collection conditions differ (ie, anesthesia);
and finally, (iv) some of the samples were either analyzed by distinct
processes,15 or did not describe the methodology used.14 With so
many confounding factors and smaller sample sizes, it is impossible
to disambiguate real population differences from methodologic and
geographic differences. Altogether, this scenario reinforces the need
for further and more robust studies regarding hematologic and bio-
chemical RIs for this species.
A common practice in veterinary medicine is to use RIs from
closely related species when practitioners are faced with the ab-
sence of species-­specific RIs. However, this practice needs to be
applied with caution as the values can often vary from species to
species. Catenacci et al17 reported RIs for the maned sloth, Bradypus
torquatus, an endemic species in some regions of the Atlantic rain-
forest. Comparing their results with ours, one can notice that some
variables share similar values, including those for RBC, hemoglobin,
PCVs, WBCs, and relative segmented neutrophil and monocyte val-
ues. Yet, as has been alluded to previously, these comparisons should
be taken carefully, considering the distinct habitats and biologic and
physiologic particularities of each species.
| 131
According to ASVCP, ideally, a population-­based RI should be
determined using samples from at least 120 individuals. However,
there are instances in veterinary medicine when only a limited
number of reference samples are available, often the case for wild-­
caught species. In these situations, the ASVCP recommends that RIs
could be calculated with at least 20 individuals using specific statis-
tical methods.7 Even though the number of tested B. variegatus in the
present study was not ideal, it was enough to calculate population-­
based hematologic RIs and confidence intervals for 16 variables and
basic descriptive statistics for 45 variables. Thus, the current study is
not complete, but it represents an important starting point consider-
ing the limited information available for this species. It is also worth
mentioning that the present study was conducted with wild-­caught
animals living in an isolated area of approximately 340 ha. It rein-
forces the importance of our results and their applicability regarding
the Bradypus variegatus conservation actions in PEFI during veteri-
nary care and rehabilitation of these wild animals.
To improve the RI confidence, it is recommended that a review
be conducted periodically, adding new individuals to the analyses.
Increasing the sample size is also crucial for partitioning the analysis
into subclasses such as sex, age, environmental conditions, and sea-
sonality. Partitioning favors homogeneous subpopulations, decreas-
ing variability between individuals and narrowing the RI. However,
partitioning analyses require at least 40 individuals within each sub-
class.7 Another way of increasing the sample size would be adding
captive animals to the study. However, there is a limited number of
species and a small number of specimens available in zoos. This is, in
fact, the case for B. variegatus, and it is one of the biggest challenges
for the establishment of RIs in wild animals. Additionally, the RIs var-
ies according to the management protocol adopted by each institu-
tion and between wild and captive animals.7 Indeed, this might also
change depending on the population or time of year.7
It is also necessary to highlight that one limitation of this study
was conducting manual cell counts instead of using an automated
hematologic analyzer. It is known that manual cell counting can
result in lower accuracy, sensitivity, and higher variability.18 Yet,
when dealing with the hematologic analyses of wild animals, not all
commercial equipment available in the market can process these
samples as efficiently as they would process samples from domes-
tic and poultry animals. Some biologic variables are very different
depending on the species, which might also result in errors. For
example, Cervidae species have small RBCs (ie, Dama dama MCV
is 33–­56.6 fL), approximately the size of platelets, while Xenarthra
species have large RBCs (ie, Myrmecophaga tridactyla MCV is 119.3–­
179 fL).19 To increase accuracy and sensitivity and reduce variabil-
ity, these analyses must be conducted by experienced and skilled
professionals.
Additionally, establishing RIs for wild animals is further compli-
cated because health status cannot be readily assessed. Therefore,
RVs located at the extremities should be examined rigorously for
possible exclusion. In these cases, Dixon´s Q test is recommended
to detect outliers because it is more likely to exclude values.7 The
outliers should be carefully identified, and when multiple outliers
132 | Chagas et al.
are found, it is an indication that the whole set is compromised and
should be deemed not useful. In our case, we detected outliers in
four variables and only one outlier value in each of them. Therefore,
we decided to keep the dataset and exclude only the values consid-
ered outliers. Due to data consistencies, the risk of individual bias
affecting the RIs’ width is low.
Another issue that should be mentioned is the accuracy of differ-
ent methods for biochemical assays. For example, albumin using the
bromocresol purple works poorly for animals, whereas bromocresol
green works poorly in avian samples but can be used for the majority
of mammalian species, except for rabbits. 20,21 Thus, the most ade-
quate method to be used for the determination of albumin values
20,21
would be electrophoresis. Nevertheless, in the daily routine of
the veterinary laboratories, several tests are adapted from human
diagnostics, and it is challenging to know if the same methods have
the same performance for all animal species. Another issue is that
in the majority of available studies that propose to establish RIs in
animals do not describe the method used for biochemical analysis,
which is the case for B. variegatus.16 It is worth mentioning that the
methods used in the present study have not been specifically val-
idated in B. variegatus but have been used previously, and similar
species and are expected to perform adequately. Further studies ad-
dressing the comparison of different laboratory diagnostic methods
for biochemical analysis are encouraged.
Performing microscopic examination of blood smears is also an
essential part of hematologic analyses in wild animals. This is im-
portant because it allows for the observation of cell morphology and
morphologic variations, including alterations that might be present
in sick animals, such as toxic changes in neutrophils, inclusions in
WBCs, immature cells, and parasites. In our case, it was possible to
observe several morphologic features, such as anisocytosis, poly-
chromatophils, Howell-­
Jolly bodies, macroplatelets, and reactive
lymphocytes. It is important to emphasize that further studies are
necessary to determine if these features represent a pathologic con-
dition or are normal variants for this species.
Together, despite the small number of individual and potential
methodologic caveats, we presented the most robust study to date
of hematologic and biochemical values for B. variegatus. Our results
will provide critically important reference points essential for evalu-
ating the health and physiologic status of this species. Furthermore,
we hope it might contribute to this species' management and the con-
servation in both free-­living and captive conditions. Nevertheless,
we recognize that hematologic and biochemical RIs for Bradypus var-
iegatus remain incomplete for a wide variety of variables, and further
studies are needed.
AC K N OW L E D G M E N T S
We thank all biologists from the Mammalian Sector of São Paulo Zoo
for tracking and capturing the animals used in this study and the vet-
erinarians from the Veterinary Division of São Paulo Zoo for collect-
ing samples used in this study. The authors thank Paulo Magalhães
Bressan and Fátima A. Viveiros Valente (São Paulo Zoological
Foundation) for allowing this study to be carried out. We also thank
Species360 and Species360 members for making biologic and physi-
ologic information about wild animals available. We are thankful to
Joshua David Cameron for the English revision and for Dr. Tarciso
André Velho for the scientific contribution and English revision of
this manuscript.
ORCID
Carolina Romeiro Fernandes Chagas https://orcid.
org/0000-0002-7884-9085
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