Coad 001

Volume 11 • 2023 10.
1093/conphys/coad001
Research article
Haematology, biochemistry and morphological

features of peripheral blood cells in captive Boa
constrictor
Downloaded from https://academic.oup.com/conphys/article/11/1/coad001/7008708 by guest on 13 May 2024

E. Dervas1, *, E. Michalopoulou1 , A. Liesegang2 , M. Novacco3 , F. Schwarzenberger4 , U. Hetzel1 and A. Kipar1
1 Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 268, 8057, Zurich, Switzerland
2 Institute for Animal Nutrition and Dietetics, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 270, 8057, Zurich, Switzerland
3 Clinical Laboratory, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, 8057, Zurich, Switzerland
4 Unit of Physiology, Pathophysiology and Experimental Endocrinology, Department of Biomedical Sciences, University of Veterinary Medicine
Vienna, Veterinärplatz 1, 1210, Vienna, Austria

*Corresponding author: Institute of Veterinary Pathology (IVPZ), Winterthurerstrasse 268, CH-8057 Zürich. Tel. +41 44 635 85 80.
Fax: +41 44 635 89 34. Email: eva.dervas@uzh.ch
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The common boa (Boa constrictor) belongs to the family Boidae and represents one of the most popular traded and kept
snake species in captivity. The early diagnosis, prevention and prophylaxis of diseases in this species, and in reptiles in
general, still pose major challenges, also due to the lack of reliable reference values. This prompted us to conduct a study on
clinically healthy captive B. constrictor to assess their basic health parameters in the blood (haematological and biochemical
values, stress markers). Several parameters differed significantly between younger (<3 years) and older (≥3 years) boas; in
the latter, the percentages of eosinophils, the haemoglobin and haematocrit levels, as well as the albumin and total protein
levels, were higher. In male snakes, cholesterol levels were significantly higher than in females. Light and electron microscopy
as well as immunohistochemistry served to identify and determine the morphological features of peripheral blood cells,
that is, heterophils, basophils, eosinophils, azurophils, monocytes, lymphocytes, thrombocytes and erythrocytes. Leukocyte
subpopulations, that is, T and B cells and monocytes, were also identified based on specific marker expression. The study
provides data on haematological, biochemical and stress hormone levels, suitable as reference values, and on the blood cell
morphology of B. constrictor which can serve as a guideline for further research on this species.
Editor: Steven Cooke

Received 7 October 2022; Revised 21 December 2022; Editorial Decision 24 December 2022; Accepted 6 January 2023
Cite as: Dervas E, Michalopoulou E, Liesegang A, Novacco M, Schwarzenberger F, Hetzel U, Kipar A (2023) Haematology, biochemistry and
morphological features of peripheral blood cells in captive Boa constrictor. Conserv Physiol 11(1): coad001; doi:10.1093/conphys/coad001.
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Introduction 20 years or more (Divers, 1996). It is one of the snake species

most frequently traded as well as kept and bred in captivity
Boa constrictor, the sole species historically allocated to the (Montgomery et al., 2015). In most countries, the husbandry
monotypic genus Boa of the family Boidae, is a large non- of B. constrictor follows defined guidelines that, despite being
venomous viviparous snake indigenous in tropical forests closely adapted to the natural environment, subject the animal
of Central and South America. The B. constrictor is one to very ‘standardized’ housing conditions regarding environ-
of the longest-lived snakes with a life span in captivity of mental temperature and humidity, enclosure size etc., with
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© The Author(s) 2023. Published by Oxford University Press and the Society for Experimental Biology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/ 1
by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Research article Conservation Physiology • Volume 11 2023
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only little seasonal variation (van Waeyenberge et al., 2018). and focus mainly on wild animals and their seasonal varia-
Captivity in general has often been stated to expose rep- tions (Fonseca Sarmiento et al., 2018; Machado et al., 2006;
tiles to stress, favouring the development of immunosup- Quadrini et al., 2018). The present study aimed to establish
pression and, therefore, infectious diseases (Warwick et al., basic haematologic and biochemical parameters and to gather
2013; van Waeyenberge et al., 2018; Morgan and Trom- data on the ‘stress level’ of clinically healthy captive B.
borg, 2007; Schumacher, 2006). Another risk for infections constrictor, to obtain a diagnostic tool set for the assessment
comes from the mixing of species from different geographic of their health status.
regions, resulting in the potential exposure to pathogens
that the animals have previously not been in contact with
(Schumacher, 2006). B. constrictor are susceptible to a wide Material and Methods
range of viral, bacterial and parasitic infections. Important
viral agents reported in this species are reptarenaviruses (the Animals. The study was undertaken on blood samples col-

causative agents of Boid inclusion body disease, BIBD) (Hetzel lected from 79 clinically healthy B. constrictor originating
et al., 2013) and the ophidian paramyxovirus (Piewbang et al. from collections of Swiss (A, C-L) and German (M) breeders
2021). Parasitic infections are also of relevance; reported are, or shelters.
for example, arthropods (mites, e.g. Ophionyssus natricis),
intestinal parasites (e.g. Cryptosporidia) and blood parasites Twenty-one animals (B1-B21) originated from a reptile
(Hepatozoon sp.) (García-Márquez et al., 2019). shelter that takes over reptiles from private owners or breed-
ers who are unable to maintain their collection. The snakes
Although knowledge on appropriate maintenance and were euthanized as the shelter had not succeeded in finding
upbringing of captive snakes is growing, the early diagnosis, new owners for the animals within a prolonged period of
prevention and prophylaxis of infectious diseases still poses time. Euthanasia was performed at the Institute of Veterinary
major challenges. In general, establishing a definitive clinical Pathology, Vetsuisse Faculty, University of Zurich, followed
diagnosis is often difficult in reptiles, as the animals show only by blood collection and a full diagnostic post mortem exam-
few pathognomonic clinical signs and often have a chronic ination to exclude any disease and to obtain information on
disease course (Divers, 1996). In both human and veterinary the general health status in the facility. Information on the
medicine, haematological and biochemical parameters are exact origin of the snakes was not available.
important, widely used markers for assessment of the health In all animals, the blood sampling was undertaken upon
status, clinical diagnosis and prognosis of disease, and to the owners’ request and with their full consent, with the aim
monitor the response to treatment. They are also fundamental to diagnose or exclude BIBD and reptarenavirus infection, as
tools to monitor the health status of wild and captive reptiles a means to determine the infection status of the shelter and
(Christopher et al., 1999; Lisičić et al., 2013). However, in the collections, respectively. In live animals (A10-A28, C1-C7,
contrast to domestic animals, haematological and biochem- D1-2), it was performed onsite in the collection. All blood
ical data in reptiles are currently not as readily available sampling was undertaken on the basis of a project permit
because reference values are still lacking for most species (cantonal number ZH 195/2016 and ZH136/2020) from the
(Nardini et al., 2013). The latter may also be the reason why Cantonal Veterinary Office in Zurich.
haematologic and biochemical values in reptiles are often
interpreted in analogy to those of mammals (Grego et al., In the cases where euthanasia was performed, an ASPA
2006). Establishment of haematological reference intervals is (Animals Scientific Procedures Act 1986) schedule 1 (appro-
also hardened by the inconsistent classification of leukocyte priate methods of humane killing (http://www.legislation.gov.uk/
subpopulations in reptiles, as some authors characterized ukpga/1986/14/schedule/1)) procedure was applied.
peripheral blood cells based on their function (Carvalho et
al., 2017), others solely based on their morphology (Svoboda All animals were tested negative for BIBD by cytological
et al., 2006; Zimmerman et al., 2010). examination of a blood smear (see below).
Similarly, the determination of stress in reptiles, particu- Blood sampling

larly of chronic stress, also remains challenging. Corticos- Blood samples were collected from living animals by cardio-
terone levels are commonly used as biomarker for stress in centesis using a 22- or 25-gauge needle on a 3-ml syringe.
reptiles (van Waeyenberge et al., 2018), yet the interpretation The heart was visualized by a SonoTrax Vascular Doppler
of the results is difficult as, similar to blood parameters, (FS15575, EDAN, USA). A blood volume of 1–2 ml was col-
they are strongly dependent on several extrinsic parame- lected into heparinized tubes (Sarstedt, Germany), the volume
ters (eg. temperature, season, habitat, diet, disease, stress, depending on the size of each animal but not exceeding 5–8%
venipuncture site) as well as intrinsic factors (species, sex, age, of the total blood volume (Nardini et al., 2013).
physiologic status) (Joseph, 2015).
From dead animals, blood was extracted by cardiocentesis
Studies that provide reference values for haematological, using a 22- or 25-gauge needle on a 3-ml syringe immediately
biochemical and stress markers in boid snakes are sparse after euthanasia.
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Conservation Physiology • Volume 11 2023 Research article
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Blood cytology For the identification of B cells, RNA-ISH was performed

using the RNAscope® technology (Advanced Cell Diagnostics
Smears were prepared from all blood samples and stained (ACD), Silicon Valley, USA) and the RNAscope 2.5 Detection
with May–Grünwald–Giemsa. They were subjected to a cyto-
Reagent Kit (Brown) according to the manufacturer’s
logical examination to identify the morphological features of
protocol. All cases were first tested for the suitability of the
leukocyte subpopulations and to determine the BIBD-negative material (RNA preservation and quality) with an oligoprobe
status of the animals (no evidence of the pathognomonic cyto-
for mouse ubiquitin (MUC) (BLAST analysis (NCBI, BLAST)
plasmic inclusion bodies (IB) within blood cells), as previously
of the MUC oligoprobe sequence to the python bivitattus
described (Hetzel et al., 2013). genome (accession number XM_015890515.2) identified
87.99% as the highest percentage of identity). Those yielding
good MUC signals were then subjected to RNA-ISH with
Buffy coat preparation, processing and oligoprobes coding for B. constrictor CD20 (mRNA Sequence

staining, immunohistochemistry, RNA-ISH ID XM_007431414.3). Briefly, sections were heated to 60◦ C
and transmission electron microscopy for 1 h and subsequently deparaffinized. Permeabilization was
achieved by incubation in pretreatment solution 1 (RNAscope
In 13 animals, the blood volume was sufficient to also pre- Hydrogen Peroxide) for 10 min at RT. Afterwards, the
pare a buffy coat. Plain micro haematocrit capillary tubes sections were boiled in RNAscope 1× Target Retrieval
(BR749321, Merck, Germany) were filled with blood and Reagents solution at 100◦ C for 15 min, followed by washing
spun in a microhaemofuge (Heraeus Medical AG, Switzer- in distilled water and ethanol. After digestion with RNAscope
land) at 12 700 g for 5 min according to previously described Protease Plus for 30 min at 40◦ C, sections were hybridized
protocols (Fontes Pinto et al., 2018). Tubes were broken with the oligoprobes at 40◦ C in a humidity control tray for
at the plasma-buffy coat interface using a diamond pen, 2 h (HybEZ Oven, ACD). Thereafter, serial amplification
and the remaining portion of the capillary tubes (containing with different amplifying solutions (AMP1, AMP2, AMP3,
the buffy coat) immersed in either 4% formalin for 24 h AMP4: alternating 15 and 30 min at 40◦ C) was performed.
for light microscopy, or 2.5% glutaraldehyde for 24 h and Between each incubation step, slides were washed with
then buffered in 0.2 M cacodylic acid buffer, pH 7.3 for washing buffer. They were subsequently incubated with
transmission electron microscopy (TEM). AMP5, AMP6 and DAB at RT for 30 min and 15 min,
The formalin-fixed buffy coat pellets were paraffin wax respectively. Gill’s haematoxylin served to counterstain the
embedded. Consecutive sections (3–5 μm) were prepared, sections that were then dehydrated with graded alcohol and
stained with haematoxylin eosin (HE) and subjected to RNA xylene and coverslipped. Consecutive sections incubated
in situ-hybridization (RNA-ISH) and immunohistochemical accordingly but without including the hybridization step
staining, respectively. served as negative controls.
Immunohistochemistry served for the identification of T For TEM, glutaraldehyde buffy coat pellets from 3
cells (CD3+) and cells with monocytic morphology (mono- adult animals were routinely embedded in epoxy resin.
cytes and azurophils; Iba1+), applying cross-reacting anti- Toluidine blue-stained semithin sections (1.5 μm) were
bodies and the horseradish peroxidase method, and using a prepared to select areas of interest for the preparation of
Dako autostainer (Dako, Glostrup, Denmark). Briefly, after ultrathin sections (75 nm). The latter were contrasted with
deparaffinization, antigen retrieval was performed by incu- lead citrate and uranyl acetate and viewed with a Philips
bation of the slides with citrate buffer (pH 6) for Iba1 CM10, operating with a Gatan Orius Sc1000 digital camera
or EDTA buffer (pH 9) for CD3 at 98◦ C for 20 min in (Gatan Microscopical Suite, Digital Micrograph, Pleasanton,
a pressure cooker. After incubation with the primary anti- USA).
bodies, rabbit anti-human Iba1 (1:350, 019–19 741, Wako,
Osaka, Japan) overnight at 4◦ C and mouse anti-human CD3
(1:150, M725401, clone F7.2.38, Dako) for 1 h at room
Morphological identification of blood cells
temperature (RT), sections were incubated with the secondary For the identification of the blood cells and to determine their
antibodies (EnVision+ HRP Rabbit (Dako) for anti-Iba1, morphological features, previous literature was consulted.
EnVision+HRP Mouse (Dako) for anti-CD3) according to To identify the blood cells in cytologic smears, we referred
the manufacturer’s protocol. Endogenous peroxidase was to literature on other snake families/species, for example,
blocked by incubation with peroxidase blocking solution kingsnakes, kobras, blood pythons and pine snakes (Salakij
(Dako) for 10 min at RT. Sections were washed with TBS et al., 2002; Giori et al., 2020; Stacy et al., 2011) and/or
buffered saline (pH 7) between each incubation step. Finally, other reptile classes (Moller et al., 2016) as data on the
sections were counterstained with haematoxylin for 20 s and morphological features of blood cells in B. constrictor were
mounted. Slides incubated with non-immune serum from the not available. The ultrastructural identification of blood cells
species in which the primary antibody was raised instead of was also partially based on findings from previous studies on
the primary antibodies served as negative controls. Lymphoid other reptile species (Salakij et al., 2002; Moller et al., 2016).
tissue of B. constrictor served as a positive control. However, there was no literature on reptiles that could be
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3
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referred to for a clear differentiation and distinction of certain Clinical chemistry

cell types, for example, azurophils and monocytes. Therefore,
we used the ‘monocytic’ features known from mammals, The following biochemical parameters were measured in the
plasma: total protein (TP), cholesterol, aspartate transami-
that is, horses and dogs (Mehta and Sinha, 2018; Pereira et
nase (AST), albumin (ALB), lactate dehydrogenase (LDH),
al., 2019) to identify monocytes ultrastructurally. Azurophils
were identified based on their appearance and overall high uric acid and glucose. The tests were performed in 66 (TP,
cholesterol, AST, LDH) and 39 (uric acid and glucose) ani-
number in the cytological smear (Stacy et al., 2011); this
mals, respectively (Supplemental Table 1).
was then correlated with their location in the buffy coat and
the expression of Iba1, a monocyte marker expressed across All parameters were determined by colorimetry with an
different orders (Pierezan et al., 2014). autoanalyser (Cobas Mira Roche autoanalyser, Hoffmann-
La Roche Ltd, Basel, Switzerland), using commercially
An overall distinction between blood cells of the granulo-
available kits (AST, cholesterol, LDH, TP, ALB: Diatools
cytic and agranulocytic lineage as applied in some former rep-

tile studies (Hüseyin Arıkan, 2014; Jenkins-Perez, 2012) was AG, Villmergen, Switzerland; uric acid: AxonLab, Dättwil,
not found to be appropriate for the present study. This was Switzerland). The glucose concentration was measured using
due to the fact that monocytes, historically belonging to the a Roche Cobas c501 analyser (Roche Diagnostics, Schweiz
‘agranulocytes’ (McMillan and Harris RJ, 2018), exhibited AG, Rotkreuz, Switzerland).
structures in TEM that were ultrastructurally consistent with
intracytoplasmic granules; these have also been described in Corticosterone measurement
humans (Collin et al., 2016). In 22 animals (Supplementary Table 1), plasma samples were
analysed using a specific enzyme-immunoassay for corticos-
Haematology terone (Möstl et al., 2002). A 150-μl aliquot of plasma was
Haematological parameters (total leukocyte count, haema- extracted with 3 ml of diethyl ether. After vortexing for
tocrit, haemoglobin, mean cell haemoglobin concentration 30 min, the plasma ether mixture was frozen at −20◦ C. The
(MCHC), heterophil, azurophil, lymphocyte, eosinophil, ether was poured into a new vial and evaporated to dryness,
basophil and monocyte counts) were assessed in 49 indi- and then the residue was dissolved in 300-μl assay buffer. The
viduals (Supplemental Table 1). extracted samples were analysed in duplicates. Serial dilutions
of serum samples yielded parallel profiles with the standard
Blood smears were prepared and stained with an auto- curve.
mated staining instrument (HEMA-TEK 2000 slide stainer,
Bayer HealthCare AG, Berlin, Germany), using a modified Statistical analysis
Wright-Giemsa solution (Hematek® Stain Pak, Siemens
Healthcare Inc., New York, USA) within 6 h of sampling and Haematological, plasma biochemical and corticosterone val-
air dried. A 100 white blood cell (WBC) differential count was ues were analysed using Stata 13 (StataCorp. 2013. Stata
performed by two laboratory technicians with experience in Statistical Software: Release 13. College Station, TX: Stata-
reptilian haematology. Out of the two differential counts, Corp LP.). The level of significance testing was set with a P
the mean was calculated to obtain a percentage for each value of 0.05. Descriptive statistics were applied, and the data
cell type. were tested for normality by the Shapiro–Wilk W test. For
assessment of age-related differences, two age groups were
The packed cell volume (PCV) was determined by placing established, young animals (<3 years of age; prior to definite
blood into a plain glass microhaematocrit tube (Arnold sexual maturity and reproduction) and adult (≥3 years of age;
Bott AG, Glattbrugg, Switzerland) with one end sealed sexually mature, reproducing animals).
and spun at 12 000 g for 5 min using a microhaematocrit
centrifuge (Haematocrit 210 Centrifuge, Hettich, Bäch, Normally distributed data were analysed using t-test and
Switzerland). ANOVA; non-parametric tests (Wicoxon rank-sum/Mann–
Whitney) were used for data that were not normally dis-
The haemoglobin concentration was assessed with the tributed. Geometric and arithmetic mean, including confi-
cyanmethaemoglobin method and measured using a pho- dence intervals, were determined for logarithmically trans-
tometer (Photometer LP 400, Dr Lange, Switzerland) after formed and raw data, respectively. Medians were reported
lysis of the red blood cells and centrifugation of the lysate where non-parametric tests are being used. Linear regression
for 5 min at 12 000 g, followed by colorimetric measurement was used and linearity was confirmed using the qnorm plot
at 540 nm. The MCHC was calculated from the PCV and the and the Shapiro–Wilk W test on residuals.
haemoglobin concentration.
Variables where data were normally distributed include
Total WBC and red blood cell counts were obtained using the percentage of lymphocytes and azurophils, haematocrit,
a 1:200 blood dilution with Natt and Herrick solution and an haemoglobin, MCHC, total protein, albumin, and glucose.
improved Neubauer haemocytometer as previously reported Logarithmic transformation was applied on data for total
(Carisch et al., 2019). number of leukocytes, number of lymphocytes, eosinophils,
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percentage of heterophils, eosinophils, monocytes and P < 0.05 (female: haemoglobin = 9.647–1.078 + 0.089 × age;
basophils, LDH, AST, urea, cholesterol and corticosterone. male: haemoglobin = 9.647 + 0.089 × age).
Nonparametric tests were used for number of azurophils,
heterophils, monocytes and basophils and uric acid. All other haematological parameters did not vary signifi-
cantly with age and sex (Tables 1–3).
Results Identification and morphological features of

blood cells
Animals and disease/infection state
The light microscopic examination of the buffy coats revealed
Thirty-one animals were young (<3 years of age; juvenile no distinct layering of the leukocytes (Fig. 2), confirming
and subadult; Myers et al., 2022); the remaining 48 animals a previous report (Fontes Pinto et al., 2018). Apart from
were adult (≥3 years of age) (Table 1). Overall, the age heterophils, which could be identified due to their promi-

of the animals ranged from 3 months to 20 years, with nent intracytoplasmic granules (Fig. 2B), differentiation of
an average of 4.52 years. Both age groups comprised male the leukocyte subpopulations based on their morphology
and female animals in almost equal proportions; however, was not possible, as cell borders, nuclear and/or cytoplasmic
for 14 snakes, information on the sex either was not pro- features could not be fully discerned (Fig. 2A). However, the
vided or could not be determined due to their young age immunohistochemical staining and RNA-ISH for T and B
(Supplementary Table 1). cells and monocytes (Fig. 2B-D), the cytological examination
of the blood smears and the ultrastructural examinations of
the buffy coat pellets allowed further identification of the
Haematology different blood cell populations (Figs. 3-5).
Haematological data were collected from 49 animals, 22
young and 27 adult boas. The differential WBC counts for the Erythrocytes
two age groups and for male and female snakes are provided
The red blood cells formed a large aggregate below the buffy
in Tables 1–3.
coat (Fig. 2A). Together with occasional variably sized fibrin
Lymphocytes were the most abundant WBC in all animals, clots, they were also present as small groups throughout all
regardless of age and sex, followed by azurophils and layers (Fig. 2A). Cytologically, erythrocytes presented as large
heterophils; in contrast, the percentage of eosinophils, oval uniform cells of consistent size (length: 12–16 μm, width:
monocytes and basophils was always low and never exceeded 5–7 μm), with a centrally located oval nucleus (Fig. 3A).
3% (Fig. 1 A, B). The younger animals (<3 years) showed Ultrastructurally, they were characterized by their elongate
a significantly lower percentage of eosinophils (geometric shape and uniformly electron-dense cytoplasm. Nuclei were
mean = 2.64%, CI = 1.63–4.28) than the adult animals round and uniform, with clumped heterochromatin that was
(geometric mean = 1.18%, CI = 0.83–1.68, t = 2.895, df = 31, arranged peripherally, along the nuclear membrane (Fig. 3B).
P < 0.01) (Fig. 1A).
Thrombocytes
The percentage of lymphocytes showed a linear association
with age and sex: R2 = 0.2421, F(2.31) = 4.95, P < 0.05 Thrombocytes were found on the top of the buffy coat. In
(lymphocyte percentage for female animals = 43.38 + 9.62 + the cytological specimens, they were 8–10 μm in diameter
(−2.006 × age), and for male animals = 43.38 + (−2.006 × and varied in shape. Most were round with small cytoplasmic
age)) (Fig. 1C). The percentage of heterophils was sig- pseudopodia and a round central nucleus and often found
nificantly associated with the age group: R2 = 0.1809, in aggregates; others were elongate and exhibited a clear
F(2,33) = 3.64, P < 0.05, and higher in the adult boas. juxtanuclear cytoplasmic halo and an oval nucleus with dense
chromatin (Fig. 3C). At ultrastructural level, thrombocytes
Adult animals had a significantly higher haematocrit were characterized by small electron-dense vacuoles and elon-
(mean = 28.56, CI = 26.85–30.24) and haemoglobin (mean = gated to round electron loose structures (canalicular struc-
8.66, CI = 8.01–9.26) than the young animals (mean = 23.86, tures) in the cytoplasm (Israels SJ, 2007) and a round central
CI = 21.64–26.08 with t = −3.5, df = 41, P < 0.01 for haema- nucleus (Fig. 3D).
tocrit levels, and mean = 7.31, CI = 6.7–7.94 with t = 3.21,
df = 46, P < 0.01 for haemoglobin levels). Both parameters Heterophils represented the most abundant granulocytes
were significantly higher in males (mean = 29.79, CI = 27.52– (Fig. 1A). They could be readily identified in the cytological
30.05 for haematocrit, and mean = 9.1, CI = 8.35–9.85 for specimens, as round cells with an eccentric round nucleus
haemoglobin levels) than in females (mean = 26.28, CI = 24.2– and a high number of sometimes refringent cytoplasmic
28.35 with t = 2.42, df = 30, P < 0.05 for haematocrit, and granules with variable staining properties (Fig. 4A). Their size
mean = 7.81, CI = 7.18–8.42 with t = 2.85, df = 35, P < 0.01 ranged between 15 and 25 μm in diameter. In the buffy coats,
for haemoglobin levels). Haemoglobin levels also showed heterophils were mainly found in the bottom layer (Fig. 2B).
a linear association with sex: R2 = 0.2064, F2,31 = 40.03, TEM showed that the cytoplasm of heterophils is packed
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6
Table 1: Leukocyte numbers in the peripheral blood (per μl) of clinically healthy captive B. constrictor.
Research article
Cell type Female Male Test para meters Total

<3 years ≥3 years Total <3 years ≥3 years Total
(n) Mean∗ (n) Mean (n) Mean (n) Mean (n) Mean (n) Mean (n) Mean
(95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI)
Leukocytes(2) (3) 12.004 (16) 9.561 (20) 9.101 (6) 7.418 (11) 7.793 (17) 7.659 t = −0.6769, (49) 8.306
× 103 /μl (7.009– 20.558) (5.851– 15.624) (5.945– 13.932) (4.125– 13.338) (5.210– 11.657) (5.719– 10.257) df = 35, (6.768–10.193)
P = 0.5029
t = 0.4162, df = 17, P = 0.685 t = −0.1660, df = 15, P = 0.8704
(2)
Lymphocytes (3) 8.822 (16) 3.877 (20) 4.020 (6) 3.640 (11) 2.678 (17) 2.984 t = −0.9083, (49) 3.480
× 103 /μl (3.142– 24.765) (2.037– 7.381) (2.295– 7.043) (1.831– 7.238) (1.680– 4.270) (2.107– 4.227) df = 35, (2.679–4.522)
P = 0.3699
t = 1.1424, df = 17, P = 0.2691 t = 0.8873, df = 15, P = 0.3889
Azurophils(3) (3) 1.300 (16) 2.245 (20) 1.935 (6) 1.275 (11) 2.590 (17) 2.340 z = 0.411, (49) 1.880
× 103 /μl (0.150– 2.240) ∗∗ (1.222– 4.996) (0.637– 3.782) (0.185– 5.756) (1.344– 4.140) (1.084– 3.319) P = 0.6807 (1.475–2.499)
z = −1.230, P = 0.2188 z = −1.407, P = 0.1594
(3)
Heterophils (3) 1.250 (16) 2.090 (20) 1.900 (6) 1.390 (11) 2.290 (17) 1.740 z = −0.305, (49) 1.610
× 103 /μl (0.690– 1.460 ∗∗ (0.963– 3.255) (0.899– 2.776) (0.290– 2-280) (0.806– 3.777) (0.830– 2.399) P = 0.7605 (1.040– 2.132)
z = −1.342, P = 0.1797 z = −0.905, P = 0.3654
(2)
Eosinophils (3) 0.161 (10) 0.144 (14) 0.147 (3) 0.129 (7) 0.062 (10) 0.077 t = −1.3731, (34) 0.151
× 103 /μl (0.013– 2.010) (0.049– 0.421) (0.069– 0.314) (0.009– 1.804) (0.032– 0.117) (0.042– 0.140) df = 22, (0.098–0.231)
P = 0.1836
t = 0.1208, df = 11, P = 0.9060 t = 1.3394, df = 8, P = 0.2172
(3)
Monocytes (3) 0.100 (16) 0.180 (20) 0.165 (6) 0.360 (11) 0.210 (17) 0.220 z = 1.646, (49) 0. 210
× 103 /μl (0– 0.320) ∗∗ (0.056– 0.260) (0.043– 0.247) (0.111– 1.582) (0.121– 0.409) (0.130–0.429) P = 0.0997 (0.151–0.267)
z = −0.615, P = 0.5384 z = 0.804, P = 0.4214
(3)
Basophils (3) 0 (16) 0.220 (20) 0.220 (6) 0.065 (11) 0.250 (17) 0.220 z = −0.259, (49) 0.230
× 103 /μl (0– 0.270) ∗∗ (0.111– 0.324) (0.095– 0.269) (0.001– 0.287) (0.093– 0.409) (0.060– 0.260) P = 0.7953 (0.151–0.260)
z = −1.345, P = 0.1788 z = −1.208, P = 0.2269
(Continued)
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Conservation Physiology • Volume 11 2023
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Table 1: Continued
Cell type <3 years ≥3 years Total

Female Male Total Female Male Total
(n) Mean∗ (n) Mean (n) Mean (n) Mean (n) Mean (n) Mean Test para meters (n) Mean
(95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI)
Leukocytes(2) (3) 12.004 (6) 7.418 (21) 8.296 (16) 9.561 (11) 7.793 (27) 8.797 t = −0.2915, (49) 8.306
× 103 /μl (7.009–20.558) (4.125–13.338) (6.554–10.502) (5.851–15.624) (5.210–11.657) (6.413–12.068) df = 46, (6.768–10.193)
P = 0.7719
t = −1.3992, df = 7, P = 0.2045 t = −0.6459, df = 25, P = 0.5243
(2)
Lymphocytes (3) 8.822 (6) 3.640 (21) 3.974 (16) 3.877 (11) 2.678 (27) 3.335 t = 0.6734, (49) 3.480
× 103 /μl (3.142–24.765) (1.831–7.238) (2.916–5.414) (2.037–7.381) (1.680–4.270) (2.218–5.013) df = 46, (2.679–4.522)
P = 0.5041
t = −2.0990, df = 7, P = 0.0740 t = −0.9137, df = 25, P = 0. 3696
(3)
Azurophils (3) 1.300 (6) 1.275 (21) 1.630 (16) 2.245 (11) 2.590 (27) 2.500 z = −1.673, (49) 1.880
× 103 /μl (0.150–2.240)∗∗ (0.185–5.756) (1.035–2.122) (1.222–4.996) (1.344–4.140) (1.859–3.425) P = 0.0943 (1.475–2.499)
z = 0.258, P = 0.7963 z = 0.271, P = 0.7860
(3)
Heterophils (3) 1.250 (6) 1.390 (21) 1.270 (16) 2.090 (11) 2.290 (27) 2.230 z = −1.964, (49) 1.610
× 103 /μl (0.690–1.460∗∗ (0.290–2-280) (0.875–1.717) (0.963–3.255) (0.806–3.777) (1.029–2.812) P < 0.05 (1.040–2.132)
z = 0.519, P = 0.6041 z = −0.271, P = 0.7860
(2)
Eosinophils (3) 0.161 (3) 0.129 (16) 0.231 (10) 0.144 (7) 0.062 (17) 0.101 t = 1.9916, (34) 0.151
× 103 /μl (0.013–2.010) (0.009–1.804) (0.130–0.410) (0.049–0.421) (0.032–0.117) (0.053–0.196) df = 21, (0.098–0.231)
P = 0.0553
t = −0.2606, df = 4, P = 0.8072 t = −1.3863, df = 45, P = 0.1859
(3)
Monocytes (3) 0.100 (6) 0.360 (21) 0.230 (16) 0.180 (11) 0.210 (27) 0.190 z = 0.811, (49) 0. 210
× 103 /μl (0–0.320)∗∗ (0.111–1.582) (0.129–0.365) (0.056–0.260) (0.121–0.409) (0.149–0.251) P = 0.4175 (0.151–0.267)
z = 1.549, P = 0.1213 z = 0.667, P = 0.5050
(3)
Basophils (3) 0 (6) 0.065 (21) 0.220 (16) 0.220 (11) 0.250 (27) 0.230 z = −0.250, (49) 0.230
× 103 /μl (0–0.270)∗∗ (0.001–0.287) (0.044–0.339) (0.111–0.324) (0.093–0.409) (0.149–0.262) P = 0.8029 (0.151–0.260)
z = 0.788, P = 0.4308 z = 0.099, P = 0.9213
∗ The measures of position depend on data distribution: Arithmetic mean (1) for normally distributed data, geometric mean (2) for logarithmic transformation and median (3) for non-normal distribution.
∗∗ Lower (upper) confidence limit held at minimum (maximum) of sample.
A) Leukocyte concentrations in female and male B. constrictor, further divided according to the age groups (young: < 3 year; adult: ≥ 3 years). B) Leukocyte concentrations in young (< 3 years) and adult (≥ 3 years)
B. constrictor, further divided by sex.
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8
Research article
Table 2: Relative leukocyte percentages of clinically healthy captive B. constrictor.
Cell type Female Male Total

<3 years ≥3 years Total <3 years ≥3 years Total
(n) Mean∗ (n) Mean (n) Mean (n) Mean (n) Mean (n) Mean Test para meters (n) Mean
(95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI)
Lymphocytes(1) (3) 74.5% (16) 45.13% (20) 49.20% (6) 51.42% (11) 35.73% (17) 41.26% t = −1.3089, (49) 45.17%
(%) (35.70–100.00) (34.95–55.30) (39.44–58.96) (33.88–68.95) (28.34–43.12) (33.59–48.94) df = 35, (40.26–50.09)
P = 0.1991
t = 2.4938, df = 17, P < 0.05 t = 2.3456, df = 15, P < 0.05
Azurophils(1) (3) 11.50% (16) 26.38% (20) 22.98% (6) 24.83% (11) 31.27% (17) 29.00% t = 1.3813, (49) 25.95%
(%) (13.99–36.99) (18.61–34.14) (15.87–30.08) (9.46–40.20) (26.48–36.06) (23.64–34.36) df = 35, (22.21–29.69)
P = 0.1759
t = −1.6721, df = 17, P = 0.1128 t = −1.2361, df = 15, P = 0.2354
(2)
Heterophils (3) 9.00% (16) 18.32% (20) 17.02% (6) 14.28% (11) 23.13% (17) 19.51% t = 0.6696, (49) 17.76%
(%) (2.011–40.27) (13.17–25.47) (12.49–23.19) (7.11–28.68) (17.14–31.22) (14.60–26.09) df = 35, (15.08–20.91)
P = 0.5075
t = −1.8303, df = 17, P = 0.0848 t = −1.7953, df = 15, P = 0.0928
(2)
Eosinophils (3) 1.36% (10) 1.46% (14) 1.63% (3) 1.82% (7) 0.87% (10) 1.08% t = −1.2964, (34) 1.83%
(%) (0.16–11.84) (0.88–2.43) (1.01–2.61) (0.46–7.22) (0.51–1.47) (0.69–1.72) df = 22, (1.33–2.51)
P = 0.2083
t = −0.1540, df = 11, P = 0.8804 t = 1.8867, df = 8, P = 0.0959
(2)
Monocytes (2) 1.73% (15) 2.36% (18) 2.22% (6) 3.10% (11) 2.76% (17) 2.87% t = 1.2452, (47) 2.50%
(%) (0.002–100.00) (1.63–3.41) (1.61–3.07) (2.17–4.43) (1.76–4.30) (2.15–3.83) df = 33, (2.08–3.00)
P = 0.2218
t = −0.6110, df = 15, P = 0.5503 t = 0.4027. df = 15, P = 0.6929
(2)
Basophils (1) 2.5% (15) 2.27% (17) 2.54% (10) 3.05% (5) 1.47% (15) 2.39% t = −0.1818, (44) 2.61%
(%) (1.46–3.52) (1.63–3.94) (1.58–5.87) (0.38–5.72) (1.37–4.17) df = 30, (1.96–3.47)
P = 0.8569
N/A t = −1.3723, df = 13, P = 0.1932
(Continued)
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Table 2: Continued
Cell type <3 years ≥3 years Total

(n)Mean∗ (n) Mean (n) Mean (n) Mean (n) Mean (n) Mean Test para meters (n) Mean
(95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI)
Lymphocytes(1) (3) 74.5% (6) 51.42% (21) 50.48% (16) 45.13% (11) 35.73% (27) 41.30% t = 1.8759, (49) 45.17%
(%) (35.70–100.00) (33.88–68.95) (42.75–58.20) (34.95–55.30) (28.34–43.12) (34.69–47.90) df = 46, (40.26–50.09)
P = 0.0670
t = −1.9900, df = 7, P = 0.0869 t = −1.4678, df = 25, P = 0.1546

(1)
Azurophils (3) 11.50% (6) 24.83% (21) 23.93% (16) 26.38% (11) 31.27% (27) 28.37% t = −1.2062 (49) 25.95%
(%) (13.99–36.99) (9.46–40.20) (17.89–29.96) (18.61–34.14) (26.48–36.06) (23.56–33.19) df = 46, (22.21–29.69)
P = 0.2339
t = 1.3929, df = 0.2063 t = 1.0284, df = 25, P = 0.3136
Heterophils(2) (3) 9.00% (6) 14.28% (21) 14.61% (16) 18.32% (11) 23.13% (27) 20.14% t = −2.0114 (49) 17.76%
(%) (2.011–40.27) (7.11–28.68) (11.45–18.65) (13.17–25.47) (17.14–31.22) (16.15–25.12) df = 46, (15.08–20.91)
P = 0.0502
t = 1.0088, df = 7, P = 0.3467 t = 1.0713, df = 25, P = 032943
(2)
Eosinophils (3) 1.36% (3) 1.82% (16) 2.64% (10) 1.46% (7) 0.87% (17) 1.18% t = 2.8946, (34) 1.83%
(%) (0.16–11.84) (0.46–7.22) (1.63–4.28) (0.88–2.43) (0.51–1.47) (0.83–1.68) df = 31, (1.33–2.51)
P < 0.01
t = 0.4889, df = 4, P = 0.6505 t = −1.6073, df = 15, P = 0.1288
(2)
Monocytes (2) 1.73% (6) 3.10% (20) 2.53% (15) 2.36% (11) 2.76% (26) 2.52% t = 0.0224, (47) 2.50%
(%) (0.002–100.00) (2.17–4.43) (1.91–3.35) (1.63–3.41) (1.76–4.30) (1.93–3.28) df = 44, (2.08–3.00)
P = 0.9822
t = 1.6064, df = 6, P = 0.1593 t = 0.5859, df = 24, P = 0.5634
(2)
Basophils (1) 2.5% (10) 3.05% (18) 2.45% (15) 2.27% (5) 1.47% (25) 2.55% t = −0.1454 (44) 2.61%
(%) (1.58–5.87) (1.46–4.12) (1.46–3.52) (0.38–5.72) (1.81–3.61) df = 41, (1.96–3.47)
P = 0.8851
N/A t = 0.8581, df = 23, P = 0.3997
∗ The measures of position depend on data distribution: Arithmetic mean (1) for Normally distributed, Geometric mean (2) for logarithmic transformation and median (3) for non-normal distribution
A) Relative leukocyte percentages in female and male B. constrictor, further divided according to the age groups (young: < 3 year; adult: ≥ 3 years). B) Relative leukocyte percentages in young (< 3 years) and adult (≥
3 years) B. constrictor, further divided by sex.
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Table 3: Blood parameters (haematocrit, haemoglobin, MCHC) of clinically healthy captive B. constrictor, divided by sex (upper table) and age (lower table)
Research article
Female Male
Para meter <3 years ≥3 years Total <3 years ≥3 years Total Test para meters Total
(n)Mean∗ (n) Mean (n) Mean (n) Mean (n) Mean (n) Mean (n) Mean
(95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI)
Haematocrit(1) (3) 24.333 (14) 27.429 (18) 26.278 (6) 28.833 (8) 30.500 (14) 29.786 t = 2.4211, (44) 26.023
(10.208–38.459) (25.864–28.993) (24.204–28.351) (26.313–31.353) (26.478–34.522) (27.519–32.052) df = 30, (24.470–27.575)
P < 0.05
t = −1.4892, df = 15, P = 0.1572 t = −0.7739, df = 12, P = 0.4540
(1)
Haemoglobin (3) 7.033 (16) 8.101 (20) 7.805 (6) 8.417 (11) 9.473 (17) 9.100 t = 2.8486, (49) 8.012
(3.174–10.892) (7.518–8.694) (7.192–8.418) (7.848–8.985) (8.347–10.598) (8.352–9.848) df = 35, (7.546–8.479)
P < 0.01
t = −1.4631, df = 17, P = 0.1617 t = −1.4830, df = 17, P = 0.1588
(1)
MCHC (3) 29.333 I16) 29.250 (20) 29.450 (6) 29.333 (11) 31.091 (17) 30.471 t = 1.0088, (49) 30.429
(27.899–30.768) (27.593–30.907) (28.096–30.804) (27.166–31.501) (28.605–33.577) (28.794–32.148) df = 35, (29.567–31.290)
P = 0.3200
t = 0.0452, df = 17, P = 0.9644 t = −1.0662, df = 15, P = 0.3032
<3 years ≥3 years
(n) Mean∗ (n) Mean (n) Mean (n) Mean (n) Mean (n) Mean
(95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI)
Haematocrit(1) (3) 24.333 (6) 28.833 (21) 23.857 (14) 27.429 (8) 30.500 (22) 28.545 t = −3.5187 (44) 26.023
(10.208–38.459) (26.313–31.353) (21.635–26.079) (25.864–28.993) (26.478–34.522) (26.854–30.236) df = 41, P < 0.01 (24.470–27.575)
t = 1.7413, df = 7, P = 0.1252 t = 1.9316, df = 20, P = 0.677
(1)
Haemoglobin (3) 7.033 (6) 8.417 (21) 7.305 (16) 8.101 (11) 9.473 (27) 8.662 t = −3.2108 (49) 8.012
(3.174–10.892) (7.848–8.985) (6.671–7.938) (7.518–8.694) (8.347–10.598) (8.070–9.257) df = 46, (7.546–8.479)
P < 0.01
t = 2.0630, df = 7, P = 0.780 t = 2.5628, df = 25, P < 0.05
(1)
MCHC (3) 29.333 (6) 29.333 (21) 30.857 (16) 29.250 (11) 31.091 (27) 30.000 t = 0.9789, (49) 30.429
(27.899–30.768) (27.166–31.501) (29.777–31.937) (27.593–30.907) (28.605–33.577) (28.647–31.353) df = 46, (29.567–31.290)
P = 0.3327
t = 0.0000, df = 7, P = 1.000 t = 1.3997, df = 25, P = 0.1739
∗ The measures of position depend on data distribution: Arithmetic mean (1) for Normally distributed, Geometric mean (2) for logarithmic transformation and median (3) for non-normal distribution
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Figure 1: Age- and sex-associated differences in blood parameters of healthy B. constrictor. A, B. Distribution of leukocyte
subpopulations. A. Lymphocytes, azurophils and heterophils are the leukocyte subpopulations with the highest concentration in young
(<3 years) and adult (≥3 years) B. constrictor. The concentration of eosinophils is significantly higher in the young animals. Box and whisker
plots, ∗ P < 0.05. B. Lymphocytes, azurophils and heterophils are the dominant leukocyte subpopulations in both female and male B. constrictor.
Data provided as log-transformed values. C. Age associated differences. The percentage of lymphocytes shows a linear association and
decreases with age. D. Sex associated differences. Male snakes show higher cholesterol levels than female snakes in both age groups. Box and
whisker plots, ∗ P < 0.05.
with granules; these are heterogeneous in shape, about 0.5– density (Fig. 4D). Like in heterophils, the cytoplasm also
1 μm in diameter and of moderate to high electron density. contained a few distinct organelles such as mitochondria,
Some heterophils also exhibited electron lucent vacuoles and the nucleus was round, with marginalized clumps of
that contained irregularly shaped material (phagosomes/ heterochromatin.
phagolysosomes) (data not shown). A few distinct organelles
such as mitochondria were also observed. The nucleus Eosinophils were the smallest granulocytes (5–8 μm in
was round, with marginalized clumps of heterochromatin diameter) in the cytological specimens (Fig. 4E). They were
(Fig. 4B). also round. The cytoplasm was more dense than in heterophils
and contained abundant, variably distinct dark basophilic and
Basophils were also readily identified in the cytological eosinophilic granules. The nucleus was usually hyperchro-
specimens, due to their abundant cytoplasm filled with matic and located in the periphery. TEM revealed an eccentric,
numerous round, variably sized, intensely basophilic granules ovoid nucleus with a moderate amount of peripheral hete-
that occasionally masked the nucleus (Fig. 4C). They were rochromatin. The abundant round to elongate cytoplasmic
20–25 μm in diameter, round to oval, with a central to granules had a variably electron-dense homogenous content.
eccentric round nucleus. Ultrastructurally, the granules Again, a few distinct organelles such as mitochondria were
were 0.2–1 μm in diameter, round and of high electron also identified in the cytoplasm (Fig. 4F).
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11
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Figure 2: Buffy coat of peripheral blood of healthy B. constrictors after formalin fixation and paraffin embedding. A. HE-stained section
of a buffy coat. Top: The blood cells do not arrange in distinct layers. Erythrocytes accumulate as a large aggregate below the buffy coat
(asterisk). Bar = 100 μm. Bottom: The higher magnification shows the presence of small groups of erythrocytes (arrowhead) and small fibrinous
aggregates (arrows) between the leukocytes. Bar = 25 μm. B. Iba-1, a confirmed marker of monocytes across animal classes, is expressed by
numerous cells primarily at the bottom of the buffy coat (top, arrow). Due to their abundance, the Iba1-positive cells are interpreted as both
monocytes and azurophils. Bottom: Strongly Iba1-positive monocytes/azurophils interspersed with numerous Iba1-negative heterophils,
identified based on the presence of their distinct cytoplasmic granules (inset, arrow). Immunohistochemistry, haematoxylin counterstain.
Bars = 100 μm (top) and 10 μm (bottom and inset). C. CD3, a confirmed marker of T cells across animal classes, is expressed by numerous cells
primarily at the top of the buffy coat (top, arrow). Bottom: A higher magnification confirms the presence of abundant positive cells, interspersed
with some aggregates of thrombocytes (arrow). Immunohistochemistry, haematoxylin counterstain. Bars = 100 μm (top) and 10 μm (bottom).
D. RNA-ISH for CD20, a confirmed marker of B cells across animal classes, shows a signal in a low number of cells throughout the buffy coat,
either as individual (arrowheads) or small groups (arrow) of round cells. RNA-ISH, haematoxylin counterstain. Bar = 10 μm.
Azurophils and monocytes round and, different from basophils and eosinophils, centrally
located, with clumped chromatin (Fig. 5A). Monocytes pre-
While monocytes in reptiles are generally thought to be
sented as roundish cells, with a slightly broader size range
morphologically and functionally similar to their mammalian
(12–25 μm in diameter). They exhibited small pseudopodia
counterpart, azurophils appear to be unique to reptile species
and a pale basophilic and moderately granular cytoplasm
(Vickie Joseph, 2015; Stacy et al., 2011). Depending on the
with or without vacuoles. The nucleus was central or eccentric
reptile order, however, they are described as morphologi-
and sometimes indented (Fig. 5C). Azurophils were by far
cally (and possibly functionally) similar to both granulocytes
more abundant than monocytes in the cytological specimens
and monocytes (Stacy et al., 2011; Vickie Joseph, 2015).
and could readily be differentiated from the latter due to the
As previously described (Stacy et al., 2011), azurophils and
more eosinophilic cytoplasm (Fig. 5E).
monocytes could be readily discerned in the cytological spec-
imens (Fig. 5). Azurophils were round, 15–20 μm in diam- In HE-stained buffy coat sections, however, monocytes and
eter, with often brightly eosinophilic cytoplasm that con- azurophils could not readily be discerned (Fig. 2A). Staining
tained occasional clear vacuoles. The nucleus was generally for Iba1, a marker of monocytes in a wide range of animal
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12
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Figure 3: Morphological features of erythrocytes and thrombocytes in the peripheral blood of healthy B. constrictors. A, B.
Erythrocytes. A. Cytological specimen. Erythrocytes are large, oval cells with a central oval nucleus and homogenous eosinophilic cytoplasm.
B. TEM. Erythrocytes are elongated cells with uniformly electron-dense cytoplasm. The nucleus is round, with clumped heterochromatin that is
arranged peripherally, along the nuclear membrane. C, D. Thrombocytes. C. Cytological specimen. Aggregate of four round thrombocytes,
with small cytoplasmic pseudopodia and a round central nucleus (‘round’ form). There is also one thrombocyte with an ‘elongated’ form
(arrowhead), a clear juxtanuclear cytoplasmic halo (arrow) and an oval nucleus. D. TEM of three thrombocytes. The cells are dominated by a
central, irregularly outlined nucleus, a small amount of cytoplasm that contains small electron-dense vacuoles (arrowheads) and elongated to
round more electron-lucent structures (‘canalicular structures’, arrow). A, C: May–Grünwald–Giemsa stain, bars = 10 μm; B, D: TEM, bars = 5 μm
(C) and 2 μm (D).
classes/species (Pierezan et al., 2014) showed positive cells a significant amount of clumped heterochromatin. The sec-
primarily in the bottom layer, suggesting these to be mono- ond, less frequently encountered cells, hence interpreted as
cytes (Fig. 2B). However, considering the morphology of the monocytes, were 8–12 μm in diameter and had an eccen-
Iba1-positive cells and their number and proportion in the tric, often indented nucleus with a lesser amount of hete-
haematological assessment, it appears likely that these repre- rochromatin. Their cytoplasm formed thin projections and
sented both monocytes and azurophils (Fig. 2B). In contrast, contained a high number of organelles, that is, mitochondria,
heterophils, which could be readily discerned based on their rough endoplasmic reticulum and lysosomes, as well as abun-
distinct granules, were clearly Iba1 negative (Fig. 2B bottom, dant small electron-dense granules, and (Fig. 5D and F).
inset).
Lymphocytes
Also at ultrastructural level, monocytes and azurophils
could readily be discerned (Fig. 5). The more abundant cells In the cytological specimens, lymphocytes presented as small-
that were neither granulocytes nor lymphocytes, hence inter- to medium-sized cells (5–10 μm in diameter) with a roundish
preted as azurophils, were 8–10 μm in diameter, with small outline and a central, round, purple to basophilic nucleus con-
cytoplasmic projections and vacuoles of variable number and taining clumped chromatin (Fig. 5G). Immunohistochemical
size (0.5–2 μm) that contained finely granular material of low staining for CD 3 identified abundant T cells primarily at the
electron density (Fig. 5B and F). They occasionally contained top of the buffy coat (Fig. 2C). In contrast, B cells, identified
cytoplasmic vacuoles filled with irregularly shaped material based on the transcription of the B cell marker CD20 as
(phagosomes/phagolysosomes) and had a round nucleus with shown by RNA-ISH, were rare and found individually or in
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13
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Figure 4: Morphological features of heterophils, basophils and eosinophils in the peripheral blood of healthy B. constrictor. A, B.
Heterophils. A. Cytological specimen, showing a heterophil. The cytoplasm of the round cell is brightly eosinophilic and contains a moderate
number of variably distinct granules (arrow); the nucleus is oval and eccentric. B. TEM shows abundant round electron dense cytoplasmic
granules of variable size (arrows). The round nucleus is slightly peripherally located and shows marginalized clumps of heterochromatin. C, D.
Basophils. C. Cytological specimen showing a basophil. The cell is slightly ovoid and exhibits abundant cytoplasm that is entirely filled by
numerous round, distinct, intensely basophilic granules. D. Ultrastructurally, the granules are round and contain homogenous, highly
electron-dense material (arrows). A few mitochondria (arrowheads) are also obvious. The nucleus is located peripherally and contains clumped
heterochromatin. E, F. Eosinophils. E. Cytological specimen with an eosinophil. This round cell is smaller than heterophils and basophils, the
cytoplasm is entirely filled by small indistinct darkly basophilic and eosinophilic granules. The nucleus is located in the periphery and appears
slightly indented. F. TEM shows numerous round to elongated, variably sized granules in the cytoplasm filled with variably electron-dense
material (arrows). Some granules show a central core (C) and a matrix (M). The nucleus has an irregular outline and abundant clumped
heterochromatin. Arrowhead: mitochondrium. A, C, E: May–Grünwald–Giemsa stain, bars = 5 μm; B, D, F: TEM, bars = 2 μm.
small groups in the mid and upper layers of the buffy coat Adult animals had significantly higher total protein
(Fig. 2D). This suggests that T cells are far more abundant (mean = 53.41, CI = 49.82–57) and albumin (mean = 30.37,
than B cells in the peripheral blood of B. constrictor. The CI = 28.55–31.19) levels than the young snakes (mean = 41.03,
ultrastructural examination confirmed that lymphocytes have CI = 37.37–44.7 with t = −4.7, df = 64, P < 0.0001 for total
scant cytoplasm and few organelles, such as mitochondria, protein, and mean = 22.97, CI = 20.74–25.19 with t = −5.22,
and a round nucleus with large clumps of mainly peripheral df = 64, P < 0.0001 for albumin). The total protein level also
chromatin (Fig. 5F). showed an association with age (R2 = 0.1483, F2,48 = 4.18,
P < 0.05), as older snakes showed higher levels than young
Biochemical parameters and corticosteroid snakes.
levels Male snakes generally showed higher cholesterol levels
Biochemical parameters (total protein, albumin, cholesterol, than female snakes regardless of age (young animals: males
uric acid, glucose, urea, AST and LDH) were assessed in 66 geometric mean = 2.82, CI = 1.96–4.07, females geometric
animals, 27 young and 39 adult boas. The biochemical data mean = 1.56, CI = 0.858–2.85, with t = 2.33, df = 10, P < 0.05;
for the two age groups and for male and female snakes (26 adult animals: males geometric mean = 3.12, CI = 2.21–4.14,
males and 25 females) are provided in Table 4. females geometric mean = 1.94, CI = 1.4–2.66, with t = −2.13,
..........................................................................................................................................................
14
..........................................................................................................................................................

Figure 5: Morphological features of azurophils and monocytes in the peripheral blood of healthy B. constrictor. A, B. Azurophils.
A. Cytological specimen showing an azurophil, a round cell with abundant variably basophilic to intensely eosinophilic cytoplasm that contains
a few small clear vacuoles (arrow). The central nucleus is round, with clumped chromatin. B. Ultrastructure of an azurophil. The cell exhibits
small cytoplasmic projections (small arrow) and variably sized cytoplasmic vacuoles that contain finely granular material of low electron density
(arrow). C, D. Monocytes. C. Cytological specimen showing a monocyte. The round cell has abundant pale basophilic and moderately granular
cytoplasm with a variable number of small vacuoles (arrow) and small cytoplasmic protrusions. The nucleus is eccentric and slightly indented. D.
TEM image of a monocyte. There are several fine cytoplasmic protrusions (small arrows); the cytoplasm contains abundant organelles, amongst
them small electron-dense round to elongated granules (asterisk), mitochondria (arrowhead), rough endoplasmic reticulum and lysosomes
(arrows). The nucleus is eccentric and indented, with marginalized clumped chromatin and a distinct nucleolus. E, F. Direct comparison of
azurophils and monocytes. E. In the cytological specimens, azurophils (A) present a darker, more homogenous, largely bright eosinophilic
cytoplasm and a central, round nucleus. In contrast, the monocyte (M) has pale basophilic cytoplasm and an eccentric, indented nucleus.
F. Ultrastructurally, the cytoplasm of the azurophil (A) is rich in vacuoles with variable content (asterisk), whereas the cytoplasm of the monocyte
(M) is rich in organelles (mitochondria, rER, lysosomes) and contains abundant small electron dense granules. G, H. Lymphocytes.
G. Cytological specimen with a lymphocyte, presenting as a small round cell with a round nucleus containing highly clumped chromatin; the
cytoplasm is scant and basophilic. H. The cells exhibit scant cytoplasm with few mitochondria (arrow) and a round nucleus with large clumps of
mainly peripheral heterochromatin. A, C, E, G: May–Grünwald–Giemsa stain, bars = 5 μm; B, D, F, H: TEM, bars = 2 μm.
df = 37, P < 0.05); this is illustrated in Figure 1D. The well as the morphology of the peripheral blood cells were
association of cholesterol levels with sex was also confirmed investigated. An age and sex group distinction was applied
(R2 = 0.1416, F2,48 = 3.96, P < 0.05). due to the commonly known fact that regardless of animal
classes or species, total WBC concentration can differ signif-
All other biochemical parameters as well as the corti- icantly between young and adult animals and between sexes,
costerone levels did not vary significantly with age and sex the former possibly being related to sexual maturity (Wood,
(Table 4). 2014; Raskin et al., 2004). Multiple studies in mammals, birds
and fewer in reptiles have highlighted the importance of age-
Discussion related reference values for the evaluation of leukograms (Lee
et al., 2020; Fonseca Sarmiento et al., 2018; Trimboli et al.,
This study aimed to establish reference data for the peripheral 2020; Dolka et al., 2014). We chose 3 years as the cut-off,
blood of captive B. constrictor. A wide range of biochemical because captive B. constrictor are known to reach sexual
and haematologic variables, plasma corticosterone levels as maturity around 3 years of age. This does not necessarily
..........................................................................................................................................................
15
16
Table 4: Biochemical parameters and corticosterone levels of clinically healthy captive B. constrictor.
Female Male
Research article
Para-meter <3 years ≥3 years Total <3 years ≥3 years Total Test para meters Total
(n) Mean∗ (n) Mean (n) Mean (n) Mean (n) Mean (n) Mean (n) Mean
(95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI)
Total protein(1) (5) 38.680 (20) 53.030 (25) 50.160 (7) 44.843 (19) 53.468 (26) 51.146 t = 0.2809, df = 49, (66) 48.533
(20.048–57.312) (47.864–58.196) (44.805–55.515) (34.782–54.904) (47.713–59.224) (46.261–56.032) P = 0.7800 (45.577–51.489)
t = −2.4273, df = 23, P < 0.05 t = −1.6696, df = 24, P = 0.1080
(1)
Albumin (5) 21.860 (20) 29.900 (25) 28.292 (7) 25.971 (19) 30.416 (26) 29.219 t = 0.5115, df = 49, (66) 27.453
(12.622–31.098) (26.936–32.864) (25.323–31.261) (19.591–32.352) (28.077–32.755) (26.919–31.519) P = 0.6113 (25.815–29.091)
t = −2.4591, df = 23, P < 0.05 t = −1.8487, df = 24, P = 0.0769
(2)
LDH (5) 68.367 (20) 112.309 (25) 101.696 (7) 115.381 (19) 100.631 (26) 104.406 t = 0.1215, df = 49, (66) 105.845
(37.131–125.880) (75.385–167.319) (72.801–142.059) (58.512–227.522) (70.036–144.530) (77.585–140.497) P = 0.9038 (88.532–
126.543)
t = −1.2396, df = 23, P = 0.2276 t = 0.4138, df = 24, P = 0.6827
AST(2) (5) 18.969 (20) 16.619 (25) 17.065 (7) 19.770 (19) 19.258 (26) 19.394 t = 0.6081, df = 49, (66) 18.907
(4.994–72.056) (11.041–25.016) (11.797–24.685) (12.644–30.912) (14.241–26.042) (15.333–24.532) P = 0.5459 (15.821–22.597)
t = 0.2900, df = 23, P = 0.7744 t = 0.0999, df = 24, P = 0.9212
(2)
Cholesterol (5) 1.564 (20) 1.935 (25) 1.855 (7) 2.822 (19) 3.122 (26) 3.038 t = 2.7396, df = 49, (66) 2.375
(0.858–2.851) (1.406–2.664) (1.422–2.420) (1.958–4.067) (2.208–4.414) (2.345–3.937) P < 0.01 (2.030–2.779)
t = −0.6534, df = 23, P = 0.5200 t = −0.3500, df = 24, P = 0.7294
(2)
Urea (5) 0.214 (18) 0.244 (24) 0.216 (7) 0.153 (19) 0.217 (25) 0.214 t = −0.0395, df = 47, (61) 0.190
(0.111–0.411) (0.174–0.343) (0.159–0.293) (0.076–0.309) (0.149–0.315) (0.159–0.289) P = 0.9686 (0.155–0.232)
t = −0.0344, df = 22, P = 0.9729 t = −1.4983, df = 23, P = 0.1477
(3)
Uric acid (5) 0.111 (20) 0.244 (25) 0.242 (7) 0.212 (19) 0.180 (26) 0.189 z = −0.104, (66) 0.200
(0.068–0.369)∗∗ (0.167–0.279) (0.159–0.274) (0.087–0.446) (0.130–0.297) (0.137–0.296) P = 0.9175 (0.167–0.275)
z = −1.223, P = 0.2214 z = 0.434, P = 0.6646
(1)
Glucose (2) 3.100 (10) 1.940 (13) 2.062 (5) 3.300 (9) 2.044 (14) 2.493 t = 0.9948, df = 25, (39) 2.3
(0–17.077) (1.202–2.678) (1.385–2.738) (2.016–4.584) (1.311–2.778) (1.840–3.146) P = 0.3294 (1.947–2.653)
t = 1.3668, df = 10, P = 0.2016 t = 2.293, df = 12, P < 0.05
Corticosterone (2) (7) 3.52 (7) 2.980 (11) 3.246 (3) 2.427 (4) 3.769 (10) 3.150 t = −0.0913, df = 19, (22) 3.246
(1.881–6.594) (1.259–7.058) (1.833–5.749) (0.639–9.223) (1.068–13.304) (2.000–4.960) P = 0.9282 (2.356–4.471)
t = 0.4219, df = 9, P = 0.6830 t = −0.8349, df = 8, P = 0.4280
(Continued)
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..........................................................................................................................................................

Table 4: Continued
Para meter <3 years ≥3 years Total

Female Male Total Female Male Total Test para meters
(n) Mean∗ (n) Mean (n) Mean (n) Mean (n) Mean (n) Mean (n) Mean (95%
(95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) CI)
Total protein (1) (5) 38.680 (7) 44.843 (26) 41.031 (20) 53.030 (19) 53.468 (40) 53.410 t = −4.7042, (66) 48.533
(20.048–57.312) (34.782–54.904) (37.367–44.695) (47.864–58.196) (47.713–59.224) (49.815–57.005) df = 64, P < 0.0001 (45.577–51.489)
t = 0.8293, df = 10, P = 0.4263 t = 0.1191, df = 37, P = 0.9058
(1)
Albumin (5) 21.860 (7) 25.971 (26) 22.969 (20) 29.900 (19) 30.416 (40) 30.368 t = −5.2232, df = 64, (66) 27.453
(12.622–31.098) (19.591–32.352) (20.740–25.199) (26.936–32.864) (28.077–32.755) (28.549–32.186) P < 0.0001 (25.815–29.091)
t = 0.9862, df = 10, P = 0.3473 t = 0.2844, df = 37, P = 0.7777
(2)
LDH (5) 68.367 (7) 115.381 (26) 102.920 (20) 112.309 (19) 100.631 (40) 107.791 t = −0.2508, df = 64, (66) 105.845
(37.131–125.880) (58.512–227.522) (79.762–132.800) (75.385–167.319) (70.036–144.530) (83.733–138.761) P = 0.828 (88.532–
126.543)
t = 1.3790, df = 10, P = 0.1980 t = −0.4259, df = 37, P = 0.6727
(2)
AST (5) 18.969 (7) 19.770 (26) 20.016 (20) 16.619 (19) 19.258 (40) 18.220 t = 0.5118, df = 64, (66) 18.907
(4.994–72.056) (12.644–30.912) (15.197–26.363) (11.041–25.016) (14.241–26.042) (14.298–23.218) P = 0.6105 (15.821–22.597)
t = 0.0910, df = 10, P = 0.9293 t = 0.6025, df = 37, P = 0.5505
(2)
Cholesterol (5) 1.564 (7) 2.822 (26) 2.205 (20) 1.935 (19) 3.122 (40) 2.493 t = −0.7600, df = 64, (66) 2.375
(0.858–2.851) (1.958–4.067) (1.832–2.654) (1.406–2.664) (2.208–4.414) (1.971–3.153) P = 0.4500 (2.030–2.779)
t = 2.3296, df = 10, P < 0.05 t = 2.1302, df = 37, P < 0.05
(2)
Urea (5) 0.214 (7) 0.153 (23) 0.148 (18) 0.244 (19) 0.217 (38) 0.221 t = −1.9731, df = 59, (61) 0.190
(0.111–0.411) (0.076–0.309) (0.106–0.207) (0.174–0.343) (0.149–0.315) (0.172–0.283) P = 0.0532 (0.155–0.232)
t = −0.8491, df = 10, P = 0.4157 t = 0.5009, df = 35, P = 0.6196
Uric acid(3) (5) 0.111 (7) 0.212 (26) 0.239 (20) 0.244 (19) 0.180 (40) 0.208 z = 0.623, (66) 0.220
(0.068–0.369)∗∗ (0.087–0.446) (0.121–0.349) (0.167–0.279) (0.130–0.297) (0.160–0.275) P = 0.5330 (0.167–0.275)
z = 0.731, P = 0.4649 z = −0.604, P = 0.5457
Glucose (1) (2) 3.100 (5) 3.300 (19) 2.668 (10) 1.940 (9) 2.044 (19) 1.989 t = 1.9973, df = 36, (39) 2.3
(0–17.077) (2.016–4.584) (2.128–3.209) (1.202–2.678) (1.311–2.778) (1.522–2.457) P = 0.0534 (1.947–2.653)
t = 0.2065, df = 5, P = 0.8445 t = 0.2282, df = 17, P = 0.8222
(2)
Corticosterone (7) 3.52 (3) 2.427 (7) 3.121 (7) 2.980 (4) 3.769 (15) 3.306 t = −0.1692, df = 20, (22) 3.246
(1.881–6.594) (0.639–9.223) (1.661–5.867) (1.259–7.058) (1.068–13.304) (2.166–5.044) P = 0.8673 (2.356–4.471)
t = −0.8211, df = 5, P = 0.4489 t = 0.3830, df = 12, P = 0.7084
∗ The measures of position depend on data distribution: Arithmetic mean (1) for Normally distributed, Geometric mean (2) for logarithmic transformation and median (3) for non normal distribution
∗∗ Lower (upper) confidence limit held at minimum (maximum) of sample
(A) Biochemical parameters and corticosterone levels for young (< 3 years) and adult (≥ 3 years) B. constrictor, further divided by sex. (B) Biochemical parameters and corticosterone levels for young (< 3 years) and
adult (≥ 3 years) B.constrictor, further divided by sex.
..........................................................................................................................................................
17
Research article
..........................................................................................................................................................

..........................................................................................................................................................
apply to their wild counterparts, as here, the onset of repro- mammals (ruminants, dogs etc.) normally have higher
ductive activity is strongly dependent on the season (Bertona lymphocyte concentrations than older animals (Wood, 2014).
and Chiaraviglio, 2003). As it does not represent a pathologic process, this physiologic
variation is often termed ‘pseudolymphocytosis’ of young
In the wild, B. constrictor can be found in the tropical animals (Boes et al., 2017). In humans, the establishment
forests of Central and South America on altitudes between of the adaptive immune system is initiated after birth, and
0 and 1500 metres above sea level, where there are strong the juvenile haematopoiesis becomes lymphoid biassed,
seasonal changes in precipitation intensity (dry season: March which correlates with an increase in the total number of T
to November; wet season: December to February) and an lymphocytes by thymic expansion (Holcar et al., 2015). The
ambient temperature that does not fluctuate substantially. results of the present study, as well as those of previous studies
Hence, previous studies on the haematological and biochem- on other reptile species (Fonseca Sarmiento MVZ et al.,
ical parameters of wild reptiles comprized two sampling 2018; Page-Karjian et al., 2021; Casal et al., 2009), indicate a
time points (winter/summer season) (el Masri et al., 1995;

similar phenomenon also in the class reptilia. Interestingly, the
Holding et al., 2014; Hussein et al., 1979; Leceta et al., 1989; adult boas of the present study showed a higher lymphocyte
Leceta and Zapata, 1985; Silva et al., 2011; Machado et al., percentage than a cohort of captive amazon tree boas
2006). Such an approach was not taken in our study, which previously studies (Corallus hortulanus) (Quadrini et al.,
included samples collected at ad hoc time points during the 2018). Whether this reflects real differences between closely
year, as to our knowledge the housing conditions of captive related species remains unclear but would be suggested,
snakes in Switzerland and Germany do not mimic seasonal because all examined animals were clinically healthy, without
variations. While maintenance guidelines for B. constrictor evidence of inflammatory processes, infectious disease
are closely adapted to the conditions in the wild and often or ecdysis, which rules out most of the reported causes
recommend 50–80% humidity and an air temperature of 24– of lymphocytosis in reptiles (Stacy et al., 2011; Vickie
32◦ C (Hedley, 2022), personal communication with breeders Joseph, 2015).
indicates that B. constrictor is commonly held at the same
temperature throughout the year, with slight individual tem- In contrast to the lymphocyte percentages, we observed an
perature differences between day and night. increase in the percentage of heterophils with age. This finding
The haematological examination in the present study is in accordance with a study performed on Brazilian wild
yielded leukocyte subset concentrations that overall aligned B. constrictor (Fonseca Sarmiento et al., 2018). In humans,
with those reported for captive and wild boid snakes so haematopoiesis becomes myeloid biassed with increasing age
far (Machado et al., 2006; Fonseca Sarmiento et al., 2018; and sexual maturation, an effect that is regulated by the influ-
Jenkins-Perez, 2012). In comparison to previous reports on ence of extrinsic and intrinsic factors on the haematopoietic
captive and wild boas, our total blood leukocyte numbers stem cells and multipotent progenitor cells (Wang et al., 2022;
were relatively high (in the upper third of the values published Ho et al., 2019; Heo et al., 2015).
so far). However, the WBC numbers were similar to those
shown in the few other studies on captive B. constrictor in We observed significantly higher haematocrit and
which this ‘leukocytosis’ was speculated to be related to the haemoglobin levels in adult and in male snakes. In mammals,
stress induced by captivity (Machado et al., 2006; Brenner the haematocrit is also known to increase with age/maturity
et al., 2002). Interestingly, a study on wild boids (Corallus (Cornell et al., 2017; Eklom and Lill, 2006), a fact that
hortulanus) indeed reported notably lower WBC counts (in has been attributed to developmental changes in the
the lower third of the values published so far) (Quadrini et haematopoietic system (Sealander, 1965). Studies on humans
al., 2018). and other mammals have also reported higher haemoglobin
levels in male individuals; this is thought to be due to the
In the most common domestic mammal species, neu- stimulatory effect of androgens on the bone marrow (Wahed
trophils (dogs, cats, horses etc.) or lymphocytes (pigs, cows) and Dasgupta, 2015). Interestingly, lower haematocrit and
are the dominating leukocyte subpopulations in the periph- haemoglobin values were reported in adult compared
eral blood of healthy individuals, with monocytes, eosinophils to juvenile wild B. constrictor from Colombia (Fonseca
and basophils comprising a noticeably smaller proportion Sarmiento et al., 2018); yet, unfortunately, information on
(Wood, 2014). In reptiles, lymphocytes (comprising up to exact age and age group definition was not provided for these
80% of all leukocytes), heterophils and azurophils, the animals. There is an overall paucity of data on the age-related
latter representing a cell type unique to reptiles (Stacy changes of haematological variables in reptiles in general and
et al., 2011), have been described as the most frequent a direct comparison with mammals might be inappropriate,
leukocyte subpopulations. This was confirmed by the present due to differences in evolutionary development. As an
study that identified lymphocytes as the most abundant example, the overall life span of reptile erythrocytes has been
leukocyte subtype regardless of age and sex. We also observed stated to be 600–800 days, depending on the species (Vickie
a correlation of the lymphocyte proportion with age, as Joseph, 2015), whereas it is around 20–160 days in mammals
younger snakes presented a higher percentage of circulating (Vácha and Znojil, 1981). Therefore, more studies, with a
lymphocytes. This is not surprising considering that young variety of reptile species and larger any cohorts of different
..........................................................................................................................................................
18
..........................................................................................................................................................
age groups are needed to consolidate the findings of this and We used fixed and processed buffy coat preparations to
previous studies. gather detailed information on leukocyte subsets in boa con-
strictors, following a previously reported approach to the
Overall, the biochemical values obtained from the present examination of blood cells in several reptile species (Fontes
boa constrictor cohort were in line with those formerly Pinto et al., 2018). Our results confirm that a distinction
reported for this species (Chiodini and Sundberg, 1982). Also, of leukocyte subpopulations is not possible on HE stained
the significantly higher total protein levels in the older snakes sections prepared after paraffin embedding, confirming that
are consistent with the relative total protein increase with age cytological specimens obtained from blood smears are most
and weight that has been reported in turtles, tortoises and suited for this endeavour. However, using immunohistochem-
iguanas (Casal et al., 2009; Harr et al., 2001; Dickinson et istry and RNA-ISH on the buffy coat preparations, we were
al., 2002). We observed variable cholesterol levels observed in able to identify T and B cells for the first time. The results
our group of snakes. Similar to observations in Nile crocodiles show that T cells are markedly more numerous than B cells

(Crocodylus niloticus) and loggerhead sea turtles (Caretta in the peripheral blood of healthy boas. Since there are so
caretta), we found the these to be higher with age and in far no studies on lymphocyte subpopulations in reptiles, we
males (Rousselet et al., 2013; Morpurgo and Gelman, 1991). rely on the finding in other animal classes to put this result
In humans, decreased low density lipoprotein (LDL)-receptor into context. Indeed, also in birds, i.e. in chickens, the order
activity is responsible for the increase in total cholesterol and phylogenetically closest to reptiles, T cells are far more numer-
LDL levels observed with age, whereas the generally lower ous (> 20%) in the peripheral blood than B cells (maximally
levels in women are mostly due to the influence of oestrogen 10%) (Hao et al., 2020). Expression of the T cell marker
(Downer et al., 2014). CD3 was observed in approximately 12–24% (average) of the
lymphoid cells in the blood of chickens, with a dominance of
Circulating corticosterone levels are widely accepted as CD4+ T helper cells over CD8+ cytotoxic T cells (Fair et al.,
biomarkers for stress in vertebrates (Claunch et al., 2017), and 2008). In the present study, we decided against an attempt
plasma corticosterone levels are commonly used to quantify to further determine lymphocyte subsets due to the lack of
acute stress in snakes (Gangloff et al., 2017; Neuman-Lee antibodies for reptiles. More detailed examinations on the
et al., 2020); however, an elevation in baseline blood corti- haemolymphatic organs of B.constrictor are now needed to
costerone has also been stated to be an indicator of chronic determine the general distribution of lymphocyte subtypes.
stress in snakes (van Waeyenberge et al., 2018). There is also
the assumption that chronically stressed animals will show a We used a rabbit polyclonal antibody against human Iba1,
greater increase in stress hormones when subjected to acute a calcium binding protein that is upregulated during the
stimuli, such as blood sampling (Dantzer et al., 2014). The activation of macrophages, that we have previously shown
potential elevation of plasma corticosterone levels due to to cross react with snakes (Dervas et al., 2020), to detect
capture and handling (Herr et al., 2017) brings in a bias that monocytes in the boas. Interestingly, the marker appeared
needs to be considered also in our study, since handling of the to be expressed also by azurophils (indirect evidence from
snakes for blood sampling was within a time frame of ∼5 min their frequency and morphology in the blood smear), which,
(never exceeding 10 min). Interestingly though, the corticos- along with heterophils, thrombocytes and B cells, are con-
terone levels determined in our cohort were overall much sidered as phagocytic cells in reptiles (Vickie Joseph, 2015,
lower than those reported in wild, free-ranging snakes, such as Zimmerman et al., 2010). The literature on the classification
rattlesnakes, dice snakes and watersnakes at the time of blood of monocytes and azurophils in snakes is contradictory. Some
sampling (Lakušić et al., 2020; Claunch et al., 2017; McCallie authors state that azurophils should be counted separately
and Klukowski, 2022; Lind et al., 2018). This would indicate in snakes, as their staining properties (e.g. they are posi-
that captive snakes, which are at least used to handling, are tive in both the PAS and acid phosphatase reaction) render
not significantly stressed by the blood sampling. However, them more similar to mammalian neutrophils (Zimmerman
the interpretation of stress hormone levels is challenging, as et al., 2010; Campbell and Grant, 2022). However, because
they are influenced by a range of factors, such as age and their granulopoietic origin has not been confirmed, others
nutritional status, all well described in mammals (Reeder claim that monocytes and azurophils should not be looked
and Kramer, 2005), but can also be affected by numerous upon separately, as the azurophils might represent immature
other, less described factors in birds and reptiles, including monocytes (Vickie Joseph, 2015). Similar to other studies,
the type of environment, environmental temperature and we also found azurophils to be by far more abundant in
humidity, handling and the frequency of feeding (Jaffredo the differential blood count (second most common leukocyte
et al., 2006; Holding et al., 2014). Due to these limitations subtype) than monocytes (Nardini et al., 2013; Stacy et al.,
and the lack of similar studies in wild boa constrictors, it is 2011). It was possible to discern them from monocytes in the
difficult to comment to which extent the more ‘standardized’ peripheral blood smear as they showed a darker basophilic
housing conditions and the frequent handling by/habituation cytoplasm. The very limited literature on the ultrastructural
to humans of the captive snakes in this study may indeed have features of monocytes and azurophils (Martinez Silvestre
contributed to the observed low overall corticosterone and et al., 2005; Salakij et al., 2002), however, did so far not
therefore likely low stress levels. allow direct conclusions on the appearance of the two cell
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19
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types in Boa constrictor. The present study sheds light on (Boa constrictor occidentalis). J Herpetol 37: 510–516. https://doi.
these, identifying monocytes as cells with clear cytoplasmic org/10.1670/122-02A.
granules, similar to those described in human bone marrow
Boes KM, Durham AC, Zachary JF (2017) Bone marrow, blood cells,
promonocytes, blood monocytes and macrophages (Collin et
and the lymphoid/lymphatic system. Pathologic Basis of Veterinary
al., 2016). Azurophils, in contrast, showed intracytoplasmic
Disease (JF Zachary, Ed.), Elsevier, Amsterdam, Netherlands. https://
vacuoles and/or phagocytosed material (indicating phago-
doi.org/10.1016/B978-0-323-35775-3.00013-8.
cytic activity), but lacked intracytoplasmic granules. Taken
together, our findings suggest that azurophils do not represent Brenner D, Lewbart G, Stebbins M, Herman DW (2002) Health survey
an earlier/different developmental stage of a monocyte but of wild and captive bog turtles (Clemmys muhlenbergii) in North
constitute a distinct, fully differentiated, phagocytically active Carolina and Virginia. J Zoo Wildl Med 33: 311–316. https://doi.
blood cell type in B. constrictor that might indeed originate org/10.1638/1042-7260(2002)033[0311:HSOWAC]2.0.CO;2.
from the monocytic lineage. Further studies are required

to fully determine the origin and function of azurophils vs Wahed A, Dasgupta A (2015) Red blood cell disorders. In:
monocytes in boas and in snakes in general, and to determine Wahed A, Dasgupta A, eds. Hematology and coagulation, 2nd.
whether they are still apparent in inflammatory processes. Edition. Elsevier, San Diego, pp. 31–53, https://doi.org/10.1016/
B978-0-12-800241-4.00003-6.
In summary, the present study provides reference values for
relevant haematological and biochemical markers as well as Campbell TW, Grant KR (2022) Herptile Cytodiagnosis. In Campbell
stress hormone levels in healthy captive B. constrictor, taking TW (Ed). Exotic Animal Hematology and Cytology, 5th Edition. Wiley-
into account age- and sex-related differences. Because data Blackwell, Hoboken NJ, pp. 229–233
were obtained under ‘standardized’ captive conditions, they
Carisch L, Stirn M, Hatt JM, Federer K, Hofmann-Lehmann R, Riond B
should be useful for studies on diseased captive boas and to
(2019) White blood cell count in birds: evaluation of a commer-
compare captive and wild boa populations. The in-depth mor-
cially available method. BMC Vet Res 15: 93. https://doi.org/10.1186/
phological approach to characterize the cells of the peripheral
s12917-019-1834-8.
blood, using a combined cytologic, histologic, immunohisto-
chemical, RNA-ISH and ultrastructural approach can pave de Carvalho MPN, Queiroz-Hazarbassanov NGT, de Oliveira Massoco C,
the way for further research on leukocyte functions, the com- Sant’Anna SS, Lourenço MM, Levin G, Sogayar MC, Grego KF, Catão-
position of haemolymphatic tissues in boa constrictors which Dias JL (2017) Functional characterization of neotropical snakes
have, despite its large captive global population and distribu- peripheral blood leukocytes subsets: linking flow cytometry cell fea-
tion, so far not been elucidated. They can also serve towards tures, microscopy images and serum corticosterone levels. Dev Comp
studies on immunopathological processes in this species. Immunol 74: 144–153. https://doi.org/10.1016/j.dci.2017.04.007.
Casal AB, Camacho M, López-Jurado LF, Juste C, Orós J (2009) Com-

Data availability statement parative study of hematologic and plasma biochemical variables in
eastern Atlantic juvenile and adult nesting loggerhead sea turtles
Datasets are available on request: The raw data supporting (Caretta caretta). Vet Clin Pathol 38: 213–218. https://doi.org/10.1111/
the conclusions of this article will be made available by the j.1939-165X.2008.00106.x.
authors, without undue reservation.
Chiodini RJ, Sundberg JP (1982) Blood chemical values of the common
boa constrictor (constrictor constrictor). Am J Vet Res 43: 1701–1702.
Acknowledgements Christopher MM, Berry KH, Wallis IR, Nagy KA, Henen BT,
The authors wish to thank the technical staff of the Histology Peterson CC (1999) Reference intervals and physiologic alterations
Laboratory and the Electron Microscopy Unit of the Institute in hematologic and biochemical values of free-ranging desert
of Veterinary Pathology, the Veterinary Medical Laboratory tortoises in the Mojave Desert. J Wildl Dis 35: 212–238. https://doi.
and the Institute for Animal Nutrition and Dietetics, Vet- org/10.7589/0090-3558-35.2.212.
suisse Faculty, University of Zurich, for excellent technical
Claunch NM, Frazier JA, Escallón C, Vernasco BJ, Moore IT, Taylor EN
support. They are grateful to Dr Jussi Hepojoki, Institute of
(2017) Physiological and behavioral effects of exogenous corticos-
Veterinary Pathology, Vetsuisse Faculty, University of Zurich,
terone in a free-ranging ectotherm. Gen Comp Endocrinol 248: 87–96.
and Medicum, Department of Virology, Faculty of Medicine,
https://doi.org/10.1016/j.ygcen.2017.02.008.
University of Helsinki, for determination of the CD20 mRNA
sequence of Boa constrictor based on data generated by next Collin M, Hughes D, Plüddemann A, Gordon S (2016) Monocytes,
generation sequencing. macrophages, and dendritic cells monocytes, macrophages,
and dendritic cells. https://oncohemakey.com/monocytes-
macrophages-and-dendritic-cells (last accessed October 2016).
References
Cornell A, Gibson KF, Williams TD (2017) Physiological maturity at a
Bertona M, Chiaraviglio M (2003) Reproductive biology, mating aggre- critical life-history transition and flight ability at fledging. Funct Ecol
gations, and sexual dimorphism of the Argentine boa constrictor 31: 662–670. https://doi.org/10.1111/1365-2435.12777.
..........................................................................................................................................................
20
..........................................................................................................................................................
Dantzer B, Fletcher QE, Boonstra R, Sheriff MJ (2014) Measures of physio- García-Márquez LJ, Rodríguez-Vázquez A, León-Règagnon V, Osorio-
logical stress: a transparent or opaque window into the status, man- Sarabia D, García-Prieto L (2019) Parasites of boa constrictor (Squa-
agement and conservation of species? Conserv Physiol 2: cou023. mata: Boidae) captive in Colima, Mexico and their pathologi-
https://doi.org/10.1093/conphys/cou023. cal effects. Rex Mex Biodiv 90: e902687. https://doi.org/10.22201/
ib.20078706e.2019.90.2687.
Dervas E, Hepojoki J, Smura T, Prähauser B, Windbichler K, Blümich S,
Ramis A, Hetzel U, Kipar A (2020) Serpentoviruses: more than res- Giori L, Stacy NI, Ogle M, Nelson S, Fecteau KA, Cushing AC (2020)
piratory pathogens. J Virol 94: e00649-20. https://doi.org/10.1128/ Hematology, plasma biochemistry, and hormonal analysis of cap-
JVI.00649-20. tive Louisiana pine snakes (Pituophis ruthveni): effects of intrinsic
factors and analytical methodology. Comp Clin Pathol 29: 145–154.
Dickinson VM, Jarchow JL, Trueblood MH (2002) Hematology and https://doi.org/10.1007/s00580-019-03030-w.
plasma biochemistry reference range values for free-ranging
desert tortoises in Arizona. J Wildl Dis 38: 143–153. https://doi. Grego KF, Alves J, de Albuquerque LR, Fernandes W (2006) Referên-

org/10.7589/0090-3558-38.1.143. cias hematológicas Para a jararaca de rabo branco (Bothrops leu-
curus) recém capturadas da natureza. Arq Bras Med Vet Zootec 58:
Divers JS (1996) The captive husbandry of captive boa constrictor 1240–1243. https://doi.org/10.1590/S0102-09352006000600040.
(Boa constrictor spp.) In: Proceedings of the Association of Reptilian &
Amphibian Veterinarians, pp. 77–81, ARAV (Ed.). Hao X, Li S, Chen L, Dong M, Wang J, Hu J, Gu M, Wang X, Hu S, Peng
D et al. (2020) Establishing a multicolor flow cytometry to charac-
Dolka B, Włodarczyk R, Zbikowski A, Dolka I, Szeleszczuk P, Kluciński terize cellular immune response in chickens following H7N9 avian
W (2014) Hematological parameters in relation to age, sex and bio- influenza virus infection. Viruses 12: 1396. https://doi.org/10.3390/
chemical values for mute swans (Cygnus olor). Vet Res Commun 38: v12121396.
93–100. https://doi.org/10.1007/s11259-014-9589-y.
Harr KE, Alleman AR, Dennis PM, Maxwell LK, Lock BA, Bennett RA,
Jacobson ER (2001) Morphologic and cytochemical characteristics
Downer B, Estus S, Katsumata Y, Fardo DW (2014) Longitudinal tra-
of blood cells and hematologic and plasma biochemical refer-
jectories of cholesterol from midlife through late life according to
ence ranges in green iguanas. J Am Vet Med Assoc 218: 915–921.
apolipoprotein E allele status. Int J Environ Res Public Health 11:
https://doi.org/10.2460/javma.2001.218.915.
10663–10693. https://doi.org/10.3390/ijerph111010663.
Heo HR, Chen L, An B, Kim KS, Ji J, Hong SH (2015) Hormonal regulation
Eklom K, Lill A (2006) Development pattern of blood oxygen carrying of hematopoietic stem cells and their niche: a focus on estrogen. Int
capacity in rainbow bee-eater nestlings. Aust J Zool 54: 1. https://doi. J Stem Cells 8: 18–23. https://doi.org/10.15283/ijsc.2015.8.1.18.
org/10.1071/ZO05063.
Herr MW, Graham SP, Langkilde T (2017) Stressed snakes strike first:
El Masri M, Saad AH, Mansour MH, Badir N (1995) Seasonal distribution hormone levels and defensive behavior in free ranging cotton-
and hormonal modulation of reptilian T cells. Immunobiology 193: mouths (Agkistrodon piscivorus). Gen Comp Endocrinol 243: 89–95.
15–41. https://doi.org/10.1016/s0171-2985(11)80153-1. https://doi.org/10.1016/j.ygcen.2016.11.003.
Fair JM, Taylor-McCabe KJ, Shou Y, Marrone BL (2008) Immunophe- Hetzel U, Sironen T, Laurinmäki P, Liljeroos L, Patjas A, Henttonen H,
notyping of chicken peripheral blood lymphocyte subpopulations: Vaheri A, Artelt A, Kipar A, Butcher SJ et al. (2013) Isolation, iden-
individual variability and repeatability. Vet Immunol Immunopathol tification, and characterization of novel arenaviruses, the etiologi-
125: 268–273. https://doi.org/10.1016/j.vetimm.2008.05.012. cal agents of boid inclusion body disease. J Virol 87: 10918–10935.
https://doi.org/10.1128/JVI.01123-13.
Fonseca Sarmiento JC, Jaramillo GH, Eduardo Nieto Pico J (2018) Estab-
Ho Y-H, Del Toro R, Rivera-Torres J, Rak J, Korn C, García-García A,
lishment of hematological parameters in boas (boa constrictor) in
Macías D, González-Gómez C, Del Monte A, Wittner M et al. (2019)
the Centro de atencion y valoracion de fauna silvestre del Valle
Remodeling of bone marrow hematopoietic stem cell niches pro-
de aburra (Cav amva). Int J Avian Wildl Biol 3: 146–150. https://doi.
motes myeloid cell expansion during premature or physiologi-
org/10.15406/ijawb.2018.03.00076.
cal aging. Cell Stem Cell 25: 407–418.e6. https://doi.org/10.1016/j.
stem.2019.06.007.
Fontes Pinto F, Lopes C, Malhão F, Marcos R (2018) Unraveling avian and
reptilian hematology: An optical and electron microscopic study of Holcar M, Goropevšek A, Ihan A, Avčin T (2015) Age-related differences
the buffy coat. Vet Clin Pathol 47: 407–414. https://doi.org/10.1111/ in percentages of regulatory and effector T lymphocytes and their
vcp.12640. subsets in healthy individuals and characteristic STAT1/STAT5 signal-
ing response in helper T lymphocytes. J Immunol Res 2015: 1–13.
Gangloff EJ, Sparkman AM, Holden KG, Corwin CJ, Topf M, Bronikowski https://doi.org/10.1155/2015/352934.
AM (2017) Geographic variation and within-individual correlations
of physiological stress markers in a widespread reptile, the common Holding ML, Frazier JA, Dorr SW, Pollock NB, Muelleman PJ, Branske A,
garter snake (Thamnophis sirtalis). Comp Biochem Physiol A Mol Integr Henningsen SN, Eikenaar C, Escallón C, Montgomery CE et al. (2014)
Physiol 205: 68–76. https://doi.org/10.1016/j.cbpa.2016.12.019. Wet- and dry-season steroid hormone profiles and stress reactivity of
..........................................................................................................................................................
21
..........................................................................................................................................................
an insular dwarf snake, the Hog Island boa (boa constrictor imperator). tics of the blood cells of the giant lizard of El Hierro (Gallotia simonyi).
Physiol Biochem Zool 87: 363–373. https://doi.org/10.1086/675938. Res Vet Sci 78: 127–134. https://doi.org/10.1016/j.rvsc.2004.07.009.
Hedley J (2022) Boa constrictors, Barrier Animal Care Clinic, Independent McCallie KL, Klukowski M (2022) Corticosterone in three species of free-
Vetcare Ltd, Bristol, UK. https://www.barriervets.co.uk/exotics/boa- ranging watersnakes: testing for reproductive suppression and an
constrictor association with body condition. Comp Biochem Physiol A Mol Integr
Physiol 269: 111214. https://doi.org/10.1016/j.cbpa.2022.111214.
Arıkan H (2014) Haematology of amphibians and reptiles: a review.
Noth-West J Zool 10: 190–209. McMillan DB, Harris RJ (Eds.)(2018) Blood and Lymph. In: An Atlas of
Comparative Vertebrate Histology, Elsevier, United Kingdom, London,
Hussein MF, Badir N, El Ridi R, Akef M (1979) Lymphoid tissues of the
pp.171–201, https://doi.org/10.1016/B978-0-12-410424-2.00007-X.
snake, Spalerosophis Diadema, in the different seasons. Dev Comp
Immunol 3: 77–88. https://doi.org/10.1016/S0145-305X(79)80008-7. Mehta S, Sinha R (2018) Comparative transmission electron microscopic

studies on monocytes of horse and dog. Haryana Veterinarian 58:
Israels JS (2007) Platelet function of the newborn. In: Michelson AD.
205–206.
Platelets, 2nd Edition, Elsevier, Massachusetts, USA, pp. 431–442,
https://doi.org/10.1016/B978-012369367-9/50784-9 Moestl E, Maggs JL, Schrötter G, Besenfelder U, Palme R (2002) Measure-
ment of cortisol metabolites in faeces of ruminants. Vet Res Commun
Jaffredo T, Fellah JS, Dunon D (2006) Immunology of Birds and Reptiles.
26: 127–139. https://doi.org/10.1023/a:1014095618125.
In: Encyclopedia of Life Sciences, John Wiley & Sons (Ed.), Ltd, London,
New York, https://doi.org/10.1038/npg.els.0000521. Moller CA, Gaál T, Mills JN (2016) The hematology of captive bobtail
Jenkins-Perez J (2012) Hematologic evaluation of reptiles: a diagnostic lizards (Tiliqua rugosa): blood counts, light microscopy, cytochem-
mainstay. Vet Tec 33: 1–8. istry, and ultrastructure. Vet Clin Pathol 45: 634–647. https://doi.
org/10.1111/vcp.12425.
Lakušić M, Billy G, Bjelica V, Golubović A, And̄elković M, Bonnet X (2020)
Effect of capture, phenotype, and physiological status on blood glu- Montgomery CE, Boback SM, Reed RN, Frazier JA (eds) (2015) An assess-
cose and plasma corticosterone levels in free-ranging dice snakes. ment of the impact of the pet trade on five CITES-Appendix II case studies
Physiol Biochem Zool 93: 477–487. https://doi.org/10.1086/711958. - Boa constrictor imperator. International Union for the Conservation
of Nature, Honduras
Leceta J, Garrido E, Torroba M, Zapata AG (1989) Ultrastructural changes
in the thymus of the turtle Mauremys caspica in relation to the Morgan KN, Tromborg CT (2007) Sources of stress in captivity.
seasonal cycle. Cell Tissue Res 256: 213–219. https://doi.org/10.1007/ Appl Anim Behav Sci 102: 262–302. https://doi.org/10.1016/j.
BF00224736. applanim.2006.05.032.
Leceta J, Zapata A (1985) Seasonal changes in the thymus and Morpurgo B, Gelman A (1991) The effect of age, sex and diet on the
spleen of the turtle, Mauremys caspica. A morphometrical, light plasma cholesterol levels of the Nile crocodile, Crocodylus niloticus.
microscopical study. Dev Comp Immunol 9: 653–668. https://doi. Comp Biochem Physiol A Mol Integr Physiol 99: 687–689. https://doi.
org/10.1016/0145-305x(85)90030-8. org/10.1016/0300-9629(91)90151-2.
Lee SH, Kim JW, Lee BC, Oh HJ (2020) Age-specific variations in hema- Myers P, Espinosa R, Parr CD, Jones T, Hammond GS, Dewey TA
tological and biochemical parameters in middle- and large-sized of (2022) The Animal Diversity Web (online), Michigan, USA. https://
dogs. J Vet Sci 21: e7. https://doi.org/10.4142/jvs.2020.21.e7. animaldiversity.org
Lind C, Moore IT, Akçay Ç, Vernasco BJ, Lorch JM, Farrell TM (2018) Nardini G, Leopardi S, Bielli M (2013) Clinical Hematology in reptilian
Patterns of circulating corticosterone in a population of rattlesnakes species. Vet Clin North Am Exot Anim Pract 16: 1–30. https://doi.
afflicted with Snake fungal disease: stress hormones as a potential org/10.1016/j.cvex.2012.09.001.
mediator of seasonal cycles in disease severity and outcomes. Physiol
Neuman-Lee LA, Hudson SB, Webb AC, French SS (2019) Investigating
Biochem Zool 91: 765–775. https://doi.org/10.1086/695747.
the relationship between corticosterone and glucose in a reptile.
Lisičić D, Ðikić D, Benković V, Horvat Knežević A, Oršolić N, Tadić Z (2013) J Exp Biol 223: jeb203885. https://doi.org/10.1242/jeb.203885.
Biochemical and hematological profiles of a wild population of the
Pereira MA, Alexandre-Pires G, Câmara M, Santos M, Martins C, Rodrigues
nose-horned viper Vipera ammodytes (Serpentes: viperidae) during
A, Adriana J, Passero LFD, Da Pereira FI, Santos-Gomes G (2019)
autumn, with a morphological assessment of blood cells. Zool Stud
Canine neutrophils cooperate with macrophages in the early stages
52: 11. https://doi.org/10.1186/1810-522X-52-11.
of Leishmania infantum in vitro infection. Parasite Immunol 41:
Machado CC, Silva LFN, Ramos PRR, Takahira RK (2006) Seasonal e12617. https://doi.org/10.1111/pim.12617.
influence on hematologic values and hemoglobin electrophoresis
Pierezan F, Mansell J, Ambrus A, Hoffmann AR (2014) Immunohisto-
in Brazilian boa constrictor amarali. J Zoo Wildl Med 37: 487–491.
chemical expression of ionized calcium binding adapter molecule
https://doi.org/10.1638/05-124.1.
1 in cutaneous histiocytic proliferative, neoplastic and inflammatory
Martinez Silvestre A, Marco I, Domínguez MÁ, Lavin S, Cuenca R (2005) disorders of dogs and cats. J Comp Pathol 151: 347–351. https://doi.
Morphology, cytochemical staining, and ultrastructural characteris- org/10.1016/j.jcpa.2014.07.003.
..........................................................................................................................................................
22
..........................................................................................................................................................
Piewbang C, Wardhani SW, Poonsin P, Yostawonkul J, Chai-in P, Stacy NI, Alleman AR, Sayler KA (2011) Diagnostic hematology
Lacharoje S, Saengdet T, Vasaruchapong T, Boonrungsiman S, Kong- of reptiles. Clin Lab Med 31: 87–108. https://doi.org/10.1016/j.
makee P et al. (2021) Epizootic reptilian ferlavirus infection in individ- cll.2010.10.006.
ual and multiple snake colonies with additional evidence of the virus
Svoboda M, Knotek Z, Knotkova Z, Trnkova S (eds) (2006) Advances
in the male genital tract. Sci Rep 11: 12731. https://doi.org/10.1038/
in reptilian hematology and blood chemistry. In 31st World Small
s41598-021-92156-5.
Animal Veterinary Association World Congress, Elsevier, Prague, Czech
Quadrini AE, Garcia VC, Freire BC, Martins M (2018) Haematological Republic, 11–14 Oct
reference of snakes: Amazon tree boa (Corallus hortulanus, Lin-
Trimboli F, de Amicis I, Di Loria A, Ceniti C, Carluccio A (2020) Ref-
naeus, 1758) and Burmenese python (Python bivittatus, Kuhl, 1820)
erence ranges for hematological and biochemical profile of Mar-
in captive. Arq Bras Med Vet Zootec 70: 1172–1178. https://doi.
tina franca donkeys. Front vet sci 7: 602984. https://doi.org/10.3389/
org/10.1590/1678-4162-9865.
fvets.2020.602984.

Raskin RE (Ed.), Latimer KS, Tvedten H (Ed.) (2004) Leukocyte disor- Vácha J, Znojil V (1981) The allometric dependence of the life span of
ders. Small Animal Clinical Diagnosis by Laboratory Methods, Else- erythrocytes on body weight in mammals. Comp Biochem Physiol A
vier, Amsterdam, Netherlands, pp. 63–91. https://doi.org/10.1016/ 69: 357–362. https://doi.org/10.1016/0300-9629(81)92990-X.
B0-72-168903-5/50008-2.
Van Waeyenberge J, Aerts J, Hellebuyck T, Pasmans F, Martel A
Reeder DM, Kramer KM (2005) Stress in free-ranging mammals: integrat- (2018) Stress in wild and captive snakes: quantification, effects
ing physiology, ecology, and natural history. J Mammal 86: 225–235. and the importance of management. VDT 87: 59–65. https://doi.
https://doi.org/10.1644/BHE-003.1. org/10.21825/vdt.v87i2.16082.
Rousselet E, Stacy NI, LaVictoire K, Higgins BM, Tocidlowski ME, Joseph V (2015) Reptile clinical pathology. In ExoticsCon, San Antonio,
Flanagan JP, Godard-Codding CA (2013) Hematology and plasma Texas, USA, 29 Aug-2 Sep
biochemistry analytes in five age groups of immature, captive-reared
Wahed A, Dasgupta A (2015b) Red blood cell disorders. In:
Loggerhead Sea turtles (Caretta caretta). J Zoo Wildl Med 44: 859–874.
Wahed A, Dasgupta A, eds. Hematology and coagulation, 2nd.
https://doi.org/10.1638/2012-0162R1.1.
Edition. Elsevier, San Diego, pp. 31–53, https://doi.org/10.1016/
Salakij C, Salakij J, Suthunmapinunta P, Chanhome L (2002) Hematology, B978-0-12-800241-4.00003-6.
morphology and ultrastructure of blood cells and blood parasites
Wang D, Tanaka-Yano M, Meader E, Kinney MA, Morris V, Da Lum-
from puff-faced watersnakes (Homalopsis buccata). Kasetsart J 36:
mertz RE, Liu N, Liu T, Zhu Q, Orkin SH et al. (2022) Develop-
35–43.
mental maturation of the hematopoietic system controlled by a
Schumacher J (2006) Selected infectious diseases of wild reptiles and Lin28b-let-7-Cbx2 axis. Cell Rep 39: 110587. https://doi.org/10.1016/
amphibians. J Exot Pet Med 15: 18–24. https://doi.org/10.1053/j. j.celrep.2022.110587.
jepm.2005.11.004. Warwick C, Arena P, Lindley S, Jessop M, Steedman C (2013) Assess-
ing reptile welfare using behavioural criteria. In Pract 35: 123–131.
Sealander JA (1964) The influence of body size, season, sex, age and
https://doi.org/10.1136/inp.f1197.
other factors upon some blood parameters in small mammals.
J Mammal 45: 598. https://doi.org/10.2307/1377331. Wood RD (2014) Leukogram Abnormalities, Merck & Co., Inc., Rahway, NJ,
USA. https://www.msdvetmanual.com (last accessed March 2022).
Silva LF, Riani-Costa CC, Ramos PR, Takahira R (2011) Seasonal influ-
ence on biochemical profile and serum protein electrophoresis Zimmerman LM, Vogel LA, Edwards KA, Bowden RM (2010) Phagocytic
for Boa constrictor amarali in captivity. Braz J Biol 71: 517–520. B cells in a reptile. Biol Lett 6 (2): 270–273. https://doi.org/10.1098/
https://doi.org/10.1590/S1519-69842011000300023. rsbl.2009.0692.
..........................................................................................................................................................
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Volume 11 • 2023 10.

Haematology, biochemistry and morphological

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Vienna, Veterinärplatz 1, 1210, Vienna, Austria

Editor: Steven Cooke

Introduction 20 years or more (Divers, 1996). It is one of the snake species

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Similarly, the determination of stress in reptiles, particu- Blood sampling

Blood cytology For the identification of B cells, RNA-ISH was performed

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referred to for a clear differentiation and distinction of certain Clinical chemistry

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Results Identification and morphological features of

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Cell type Female Male Test para meters Total

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Cell type <3 years ≥3 years Total

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Table 2: Relative leukocyte percentages of clinically healthy captive B. constrictor.

Cell type Female Male Total

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Cell type <3 years ≥3 years Total

t = −1.9900, df = 7, P = 0.0869 t = −1.4678, df = 25, P = 0.1546

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Para meter <3 years ≥3 years Total

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Casal AB, Camacho M, López-Jurado LF, Juste C, Orós J (2009) Com-

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