REFERENCE INTERVALS FOR ERYTHROCYTE

SEDIMENTATION RATE, LACTATE, FIBRINOGEN,

HEMATOLOGY, AND PLASMA PROTEIN
ELECTROPHORESIS IN CLINICALLY HEALTHY
CAPTIVE GOPHER TORTOISES (GOPHERUS
POLYPHEMUS)
Author(s): Justin F. Rosenberg, D.V.M., James F.X. Wellehan, Jr.,D.V.M., M.S.,
Ph.D., Dipl. A.C.Z.M., Dipl. A.C.V.M., Sarah E. Crevasse, Carolyn Cray, Ph.D.,
and Nicole I. Stacy, D.V.M., Dr. Med. Vet., Dipl. A.C.V.P.
Source: Journal of Zoo and Wildlife Medicine, 49(3):520-527.
Published By: American Association of Zoo Veterinarians
https://doi.org/10.1638/2017-0183.1
URL: http://www.bioone.org/doi/full/10.1638/2017-0183.1

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 Journal of Zoo and Wildlife Medicine 49(2): 520–527, 2018
Copyright 2018 by American Association of Zoo Veterinarians

REFERENCE INTERVALS FOR ERYTHROCYTE SEDIMENTATION

RATE, LACTATE, FIBRINOGEN, HEMATOLOGY, AND PLASMA
PROTEIN ELECTROPHORESIS IN CLINICALLY HEALTHY
CAPTIVE GOPHER TORTOISES (GOPHERUS POLYPHEMUS)

Justin F. Rosenberg, D.V.M., James F.X. Wellehan, Jr., D.V.M., M.S., Ph.D., Dipl. A.C.Z.M., Dipl.
A.C.V.M., Sarah E. Crevasse, Carolyn Cray, Ph.D., and Nicole I. Stacy, D.V.M., Dr. Med. Vet., Dipl.
A.C.V.P.

Abstract: Currently available tests for the diagnosis of inflammatory disease in reptiles are limited and poorly
sensitive. However, a number of hematological and plasma biochemical analytes are validated in the diagnosis of
inflammation in mammals. The objective of this study was to establish reference intervals for erythrocyte
sedimentation rate, lactate, heat-precipitated fibrinogen, hematology, and plasma protein electrophoresis based
on total protein by biuret method in 23 clinically healthy, captive gopher tortoises (Gopherus polyphemus) after
successful rehabilitation and to determine differences by age, sex, and season. In order to investigate biological
differences, samples were collected in April, July, and November. There were no sex differences in any measured
analyte; however, there were significant differences by age and season. Immature animals (,2 kg) had significantly
higher total protein, albumin : globulin ratio, pre-albumin, albumin, and a-1 globulin than adults. Tortoises
sampled in the spring season had significantly higher total solids (refractometer) and lower eosinophils compared
with animals sampled in the summer. Further investigation is required to determine the clinical utility of these
analytes in the diagnosis of inflammation in this species.
Key words: Gopher tortoise, Gopherus polyphemus, hematology, inflammation, plasma protein electrophoresis,
reference intervals.

INTRODUCTION tests are nonspecific as they detect inflammatory

Hematology can be a useful tool for evaluating
systemic health in any species and can give the
clinician valuable information into the presence of
underlying inflammatory conditions. However,
hematology data may be challenging to interpret
in the reptilian patient, since reptiles may exhibit
a leukocytosis, leukopenia, or a normal leuko-
gram during times of infection and/or inflamma-
tion.36,39 Thus, there exists a need to develop more
sensitive methods for the diagnosis of inflamma-
tory conditions in reptilian species.
Diagnostic tests such as erythrocyte sedimen-
tation rate, lactate, fibrinogen, and plasma protein
electrophoresis can provide insight into inflam-
matory status in a multitude of species. These
From the Department of Comparative, Diagnostic, and
Population Medicine, College of Veterinary Medicine,
University of Florida, 2015 Southwest 16th Avenue,
Gainesville, Florida 32608, USA (Rosenberg, Wellehan,
Crevasse, Stacy); the Division of Comparative Pathology,
Department of Pathology & Laboratory Medicine, Uni-
versity of Miami Miller School of Medicine, 1600
Northwest 10th Avenue, Miami, Florida 33136, USA
(Cray). Correspondence should be directed to Dr. Rosen-
berg (rosenbergdvm@gmail.com).
changes from any etiology, but can provide useful
information regarding the presence and trends of
systemic inflammation in the individual patient.
The erythrocyte sedimentation rate (ESR) is a
relatively simple test that is used to identify the
presence of an inflammatory process and other
disorders.25 Its utility as a diagnostic indicator is
well established in human and marine mammal
medicine, but is not widely used outside of these
species.7 This test relies on acute-phase proteins,
primarily fibrinogen, to cause erythrocytes to
form rouleaux, and thus increases the rate at
which the red blood cells sediment under the
influence of gravity.25 A higher result, based on
mm of sedimentation per the standardized time of
60 min, suggests an underlying focus of inflam-
mation. However, inter- and intra-species varia-
tions in results and interpretation need to be
considered during times of both health and
disease.
Reptiles have a high capacity to support vigor-
ous activity using anaerobic metabolism.3 Due to
a relatively slow metabolism and unique methods
for lactate removal, there is potential for extreme-
ly high lactate values in reptiles following periods
of physical exertion or illness.3,20 Lactate has been

520
 ROSENBERG ET AL—GOPHER TORTOISE REFERENCE INTERVALS
evaluated in sea turtles, but studies in tortoises
are lacking.8,22,26
In mammals, elevated fibrinogen correlates
with active inflammation.38 There is evidence to
support that fibrinogen in reptilian species may be
more involved in inflammatory processes than
that of other species and that this acute-phase
protein may be highly conserved across spe-
cies.15,44 However, there have been variations by
species and methodology, which can influence the
usefulness of this test in healthy versus sick
animals.30,33 Specifically in reptiles, fibrinogen
was not influenced by ranavirus infection in red-
eared sliders (Trachemys scripta elegans).29
Plasma protein electrophoresis (EPH) quanti-
fies plasma protein fractions, which may provide
evidence for changes in health status.11,12 EPH has
been examined in a variety of chelonians includ-
ing radiated tortoises (Astrochelys radiata),43 pond
sliders (Trachemys scripta),19 eastern box turtles
(Terrapene carolina carolina),16 European pond
terrapin (Emys orbicularis),10 and loggerhead sea
turtles (Caretta caretta).18 These studies have
identified a need to determine reference intervals
for each species.
Gopher tortoises (Gopherus polyphemus) are a
keystone species in the southeastern United
States coastal plain habitats,9 but populations
have been rapidly declining.5,41 There are known
hematological reference intervals40 and known
seasonal hematological changes32 in wild popula-
tions. There has also been evaluation into the
seasonal changes in immune status based on
leukocyte counts.21 However, to date, there are
no known normal intervals for erythrocyte sedi-
mentation rate, lactate, heat-precipitated fibrino-
gen, or protein electrophoretogram fractions in
this species.
The objectives of this project were to establish
reference intervals for erythrocyte sedimentation
rate, lactate, heat-precipitated fibrinogen, hema-
tology, and plasma protein electrophoresis in
apparently healthy captive gopher tortoises and
to determine differences by sex, age, and season.
MATERIALS AND METHODS
Animals
Gopher tortoises housed at Santa Fe College
Teaching Zoo (SFTZ) in Gainesville, Florida,
were evaluated by thorough physical examination.
This study was approved by the University of
Florida’s Institutional Animal Care and Use
Committee (study number 201609305). Animals
were included in the study based on body weight
521
.600 g, normal physical examination, and lack of
ongoing medical conditions. All gopher tortoises
were previously treated at the University of
Florida Zoological Medicine Service (UFZMS)
for a variety of medical conditions, were deemed
healthy after completion of treatments, and were
transferred to SFTZ for long-term monitoring or
were nonreleasable. The average time between
animal transfer from UFZMS to SFTZ and the
date of sampling was 384 days (range: 6–1,141
days). For the purpose of this project, chronic
wound healing was not considered an active
medical condition; wounds that had keratinized
but not yet ossified did not exclude animals from
the study. Animals were excluded if their packed
cell volume (PCV) was ,15%. All animals were
housed in a natural, outdoor environment and
were maintained on a diet of fresh produce, fresh
greens, and natural grass.
Gender was determined based on external
characteristics. Animals ,2 kg were classified as
immature while those .2 kg were considered to
be adults. This weight cutoff was roughly based
on size estimates from a previous study.31
Sampling procedures
Physical examinations were performed imme-
diately prior to sample collection. A review of
complete medical records ensured that there were
no ongoing medical concerns and were deemed
‘‘healthy.’’ A random cohort of 8–10 animals,
determined by commercially available software
(www.randomizer.org), was sampled in April,
July, and November to span the spring, summer,
and autumn seasons, respectively.
Tortoises were restrained manually, without
anesthesia or sedation, during venipuncture from
the subcarapacial sinus. Blood (;3 ml; not to
exceed 0.5% body weight) was collected using a
heparinized 22-ga, 1.5-inch needle and 3-ml
syringe. Following sampling, whole blood was
placed into a lithium heparin container and
carefully inverted to ensure proper mixing of
blood and anticoagulant. The erythrocyte sedi-
mentation rate and lactate (Lactate Plus Analyzer,
Nova Biomedical, Waltham, Massachusetts
02454, USA) were processed within 10 min of
venipuncture. Packed cell volume, total solids
(TS) by refractometer, and plasma color were
recorded. Blood films were prepared for micro-
scopic evaluation. Fibrinogen was determined by
heat-precipitated protein methodology.28 The
erythrocyte sedimentation rate was determined
using the Wintrobe tube method that requires 1
ml of heparinized whole blood. It should be noted
522 JOURNAL OF ZOO AND WILDLIFE MEDICINE
that EDTA is the anticoagulant of choice for
performing ESR, but lithium heparin is the
anticoagulant of choice for reptilian blood as
EDTA can cause lysis and other hematological
changes.23
Two blood films were prepared and stained with
Wright Giemsa. The review of the blood films for
white blood cell (WBC) estimate, differential, and
morphology was performed by one author (NIS)
using blinded slide labels. This information in
addition to PCV, TS, and plasma color comprised
the hematological analysis.
The remaining whole blood was centrifuged
(StatSpin VT Centrifuge, IDEXX Laboratories,
Inc., Westbrook, Maine 04092, USA) for 90 sec at
12,000 g and aliquots of plasma were frozen at
208C until sample analysis.
Protein electrophoresis
Samples were shipped frozen for analysis and
were provided a blinded sample number. Total
protein (TP) was determined by biuret (Ortho
Vitros 250 analyzer, Ortho Clinical Diagnostics,
Rochester, New York 14626, USA). Plasma
samples were analyzed with the use of a commer-
cial electrophoresis system (SPIFE Split Beta
SPE System, Helena SPIFE 3000, Helena Labo-
ratories, Inc., Beaumont, Texas 77707, USA).
Fraction delimits were placed as previously
demonstrated for other tortoise species.43 Per-
centages for each fraction were determined using
this software and absolute concentrations (g/dl)
for each fraction were obtained by multiplying the
percentage by the TP concentration. The albu-
min : globulin (A : G) ratio was calculated by
dividing the sum of pre-albumin and albumin by
the sum of the globulin fractions.
Statistical analysis
The lactate analyzer reported samples ,0.3
mM/L as ‘‘low.’’ Thus, all samples with a ‘‘low’’
result were conservatively recorded as 0.3 mM/L
for statistical analysis. Likewise, standard fibrin-
ogen methodology was used to record results as a
multiple of 100 mg/dl; any value ,100 mg/dl was
recorded as 100 mg/dl for statistical analysis.
To determine reference intervals, data were
analyzed for normalcy using the Shapiro–Wilk
test. For data that were normally distributed,
parametric methods were used for analysis; non-
parametric methods were utilized for data that
were not normally distributed. All reference
intervals were calculated using the robust method
as previously described by the American Society
of Veterinary Clinical Pathology.17
Data were further categorized by gender, age,
and season. For independent variables with two
categories (age, gender), a t-test with Welch
correction was utilized for parametric data while
a Mann-Whitney test was performed for nonpara-
metric data. Since season had more than two
categories, a one-way analysis of variance (AN-
OVA) was utilized for parametric data while a
Kruskal-Wallis test was performed for nonpara-
metric data; a Dunn’s multiple comparisons post-
test was performed to identify sources of differ-
ence. A least-squares linear regression was used
to determine the correlation between TS and TP
values.
The protein variables examined in the statistical
analysis included total protein, pre-albumin, al-
bumin, a-1 globulin, a-2 globulin, b globulin, c
globulin, and A : G ratio. P , 0.05 was used to
determine statistical significance. Statistical soft-
ware (GraphPad InStat 3, La Jolla, California
92037, USA, and MedCalc version 17, Maria-
kerke, 8400 Ostend, Belgium) was used to analyze
the data.
RESULTS
Animals and blood samples
In total, 31 healthy gopher tortoises were
sampled during the study period. Seven animals
were excluded based on PCV , 15% or incom-
plete data sets (1 animal did not have plasma
available for electrophoresis).
Of the 23 healthy tortoises included, there were
11 males and 12 females. Nine were classified as
immature while 14 were adults. Six animals were
sampled in the spring, 8 in the summer, and 9 in
autumn. No samples were significantly hemo-
lyzed; however, 4 samples that were initially clear
exhibited very slight hemolysis after centrifuga-
tion, which may have had minimal effects, ie a
slight alteration of the A : G ratio by protein
electrophoresis and/or total solids reading by
refractometer.13 Four samples had a mild yellow
hue (presumably from dietary pigments) and the
remaining 15 were clear and straw colored.
Reference intervals
Descriptive statistics for hematology and elec-
trophoresis data are reported in Tables 1 and 2,
respectively. Analytes that were parametric have
the mean, standard deviation, median, minimum-
maximum, and 90% confidence intervals report-
ed. For nonparametric data, the mean, median,
 ROSENBERG ET AL—GOPHER TORTOISE REFERENCE INTERVALS 523

Table 1. Descriptive statistics for hematological analytes in n ¼ 23 healthy gopher tortoises (Gopherus
polyphemus) using the robust method for calculating reference intervals (RI). ESR, erythrocyte sedimentation rate;
P, parametric distribution; NP, nonparametric distribution; T, data transformed for analysis.

Lower Upper
reference reference
Conventional limit limit
Analytes units Mean SD Median Min. Max. RI 90% CI 90% CI Distribution

PCV % 22 4.6 22 15 34 12–31 10–15 28–33 P

Total solids g/dl 3.4 0.9 3.4 1.8 4.8 1.5–5.4 1.1–2.2 4.6–5.7 P
ESR mm/hr 5 — 5 2 7 3–8 2.4–3.3 6.5–9 NP, T
Lactate mM/L 0.4 — 0.3 0.3 1.3 0.3–1.3 NAa 0.7–1.3 NP, T
Fibrinogen mg/dl 130 — 100 100 400 100–380 NAb 226–369 NP, T
Total X 103/lL 10.3 — 8.3 5.8 30 2.83–27.7 2.12–4.09 17.7–40.7 NP, T
leukocytes
Heterophils X 103/lL 4.43 — 4.00 1.7 11 1.28–14.9 0.81–1.68 9.58–20.6 NP, T
Immature X 103/lL 0.60 — 0 0 9 NAc NAc NAc NP, T
heterophils
Lymphocytes X 103/lL 3.11 — 3.1 1.8 6.1 1.82–5.38 1.51–2.20 4.23–6.26 NP, T
Monocytes X 103/lL 0.96 — 0.88 0.26 4.5 0.19–4.14 0.12–0.31 2.41–6.78 NP, T
Eosinophils X 103/lL 0.60 — 0.47 0 4.9 0–4.09 0–0.14 2.05–7.87 NP, T
Basophils X 103/lL 0.43 — 0.44 0 2.3 0–3.01 0–0.11 1.67–4.50 NP, T
a
Unable to calculate lower reference limit 90% CI because 0.3 mM/L was lower cutoff of lactatometer; any value less than this
was denoted by ‘‘low’’ and was entered as 0.3 mM/L.
b
Unable to calculate lower reference limit 90% CI because 100 mg/dl was the lowest possible result; any value less than this
was denoted as 100 mg/dl and was entered as 100 mg/dl.
c
Unable to calculate reference intervals given n ¼ 13 animals with 0 immature heterophils.

and minimum-maximum values are reported.
Histograms were created and visually evaluated
for outliers, but are not reported here.
Gender differences
There were no statistically significant differenc-
es between males and females with regard to any
measured analyte. Normally distributed variables
included PCV, total solids (by refractometer),
total protein by biuret method, A : G ratio, pre-
albumin, albumin, a-1 globulin, c globulin, and
absolute lymphocytes.
Age differences
Immature (,2 kg) animals had significantly
higher total protein by biuret method (mean: 3.9
g/dl) (P ¼ 0.0475), A : G ratio (0.53) (P , 0.05),
pre-albumin (0.65 g/dl) (P , 0.001), albumin
(0.68 g/dl) (P , 0.01), and a-1 globulin (0.19 g/
dl) (P , 0.01) compared with adult (.2 kg)

Table 2. Descriptive statistics for plasma protein electrophoresis in n ¼ 23 healthy gopher tortoises (Gopherus
polyphemus) using the robust method for calculating reference intervals (RI). P, parametric distribution; NP,
nonparametric distribution; T, data transformed for analysis.

Lower Upper
reference reference
Conventional limit limit
Analytes units Mean SD Median Min. Max. RI 90% CI 90% CI Distribution

Total protein g/dl 3.44 0.91 3.5 1.8 5.1 1.50–5.38 1.02–2.10 4.82–5.87 P
by biuret
Albumin : — 0.45 — 0.46 0.33 0.81 0.29–0.73 0.26–0.34 0.61–0.79 NP, T
globulin ratio
Pre-albumin g/dl 0.52 0.16 0.53 0.23 0.85 0.18–0.86 0.09–0.28 0.76–0.96 P
Albumin g/dl 0.56 0.18 0.58 0.21 0.98 0.15–0.95 0.04–0.28 0.83–1.06 P
a-1 globulin g/dl 0.15 0.05 0.16 0.07 0.24 0.06–0.25 0.04–0.09 0.22–0.27 P
a-2 globulin g/dl 0.45 — 0.42 0.21 1.09 0.18–0.96 0.16–0.26 0.75–1.20 NP, T
b globulin g/dl 1.24 0.40 1.09 0.67 2.04 0.32–2.12 0.14–0.58 1.80–2.31 P
c globulin g/dl 0.49 0.12 0.47 0.32 0.75 0.23–0.74 0.14–0.29 0.65–0.81 P
524 JOURNAL OF ZOO AND WILDLIFE MEDICINE

Figure 1. Plasma total protein (biuret method) and total solids (refractometer) in healthy gopher tortoises
(Gopherus polyphemus) were significantly correlated (least-squares linear regression (n ¼ 23, R2 ¼ 0.70, P , 0.01).
The equation for the fitted line was TS ¼ (0.8312 3 TP) þ 0.5586 (in g/dl).

animals (3.14 g/dl, 0.42, 0.44 g/dl, 0.48 g/dl, 0.13
g/dl, respectively). There was no statistical dif-
ference in total solids by refractometer (P ¼
0.6882). No other variables had significant age
differences.
Seasonality
There were significant differences between
seasons for total solids by refractometer (P ,
0.05) and absolute eosinophils (P , 0.05). There
were no significant differences in total protein by
biuret method (P ¼ 0.1927), WBC estimate (P ¼
0.10), or with any other variable. Total solids were
significantly increased in spring (mean: 4.1 g/dl)
compared with summer (3.0 g/dl) (P , 0.05).
Eosinophils were significantly decreased in spring
(mean: 0.2 3 103/ll) compared with summer (1.6 3
103/lL) (P , 0.05).
TS and TP
TS and TP values were linearly related (R2 ¼
0.70, P , 0.01), and the equation for the fitted line
was TS ¼ (0.8312 3 TP) þ 0.5586, where TS and TP
are measured in g/dl (Fig. 1).
DISCUSSION
This study is the first to report reference
intervals of erythrocyte sedimentation rate, lac-
tate, heat-precipitated fibrinogen, and plasma
protein electrophoresis in healthy, captive gopher
tortoises. Understanding species-specific refer-
ence intervals for diagnostic tests is of the utmost
importance in the evaluation of clinically abnor-
mal animals.
Only one prior study has evaluated hemograms
for healthy gopher tortoises, based on 13 free-
ranging individuals.40 That study reported season-
al differences in total WBC and monocytes, both
higher in the spring compared with autumn.40
There were no differences in gender for any
analyte.40 The current study differs from this
previous report in that there is a lower total
WBC mean, characterized by lower absolute
lymphocytes and higher absolute heterophils.
The two studies are in agreement with respect to
PCV, monocytes, and basophils. It is known that
reptilian leukocytes can be influenced by factors
such as gender, age, location, and season;36
without knowing the demographics of the prior
study population, it is difficult to compare the
 ROSENBERG ET AL—GOPHER TORTOISE REFERENCE INTERVALS
data directly. However, it is possible that differing
methodology could account for the discrepancy in
total leukocyte counts and that WBC variations
may result from differences in environment and
diet (ie, wild vs captive).35
Regardless of which data set the clinician uses
for interpretation, clinical judgement is essential.
In the current study, several animals (n ¼ 4) had
WBC estimates between 22–30 3 103/ll; these are
likely not ‘‘healthy’’ animals. However, they all
met the criteria for ‘‘apparently healthy’’ based on
study parameters and physical examination and
were therefore included in the statistical analysis.
The wide range of total leukocytes encountered
herein further demonstrates that reptile hematol-
ogy can be limited in its clinical application and
difficult to interpret. Other tests and trend
monitoring for determining the presence and
severity of inflammation should be utilized, if
possible. It is also important to consider that the
study tortoises were under human care and thus in
an environment that may expose these animals to
some stressors, including higher population den-
sity than in the wild, stress from handling, or
other variables from diet and husbandry.
The use of ESR in a chelonian species is novel.
There is, however, one report of ESR utilized in a
grass snake, also from a heparinized blood
sample.42 That species had an ESR of 5.88 6
1.99 mm/hr in males and 5.49 6 2.09 mm/hr in
females.42 ESR in humans is known to be affected
by age and gender.34 However, there were no
significant sex or seasonal differences in captive
gopher tortoises in the current study.
Lactate data in the present study were lower
than those previously reported in other cheloni-
ans. The previous studies utilized captured sea
turtles that were restrained out of the water.8,22,26
Since approximately 50% of lactate is produced
within 30 sec of capture or physical exertion, and
nearly 90% is formed within the first 90 sec,3 this
could result in increased lactate compared with
the minimal restraint required for the terrestrial
species in this study. In addition, the study
animals were habituated to handling in compari-
son with wild animals of other species.
Fibrinogen in this study was measured by heat
precipitation, which has been shown to correlate
with fibrinogen measurements by different stan-
dard methodology.28 The heat-precipitation meth-
odology is semiquantitative and is often used in
species where methods of detecting inflammation
are less reliable.37 The reported range is relatively
consistent with accepted normal intervals of other
species, including red-eared sliders and ornate
525
box turtles (Terrapene ornata ornata).29,33 Those
studies, however, utilized a modified Jacobson
method or modified Clauss method, respectively,
for measurement of fibrinogen rather than the
heat-precipitation methods. Overall, heat-precip-
itated fibrinogen provides an insensitive method
for quantifying an inflammatory condition and
should be interpreted with caution, especially in
cases of hypofibrinogenemia.37
Given the fact that reptile plasma proteins can
vary greatly between both species and individuals,
direct comparison between protein fractions of
different species has limited value.27 There are a
variety of physical and physiological parameters
than can influence reptile protein fractions.1
Repeated measurement of protein fractions in
clinically normal and abnormal animals can prove
useful to the clinician in interpreting results of the
individual.
When evaluating for differences between gen-
der, age, and season, there were several analytes
with statistically significant differences. There
were no sex differences in any analyte, which is
consistent with hematology findings reported in
eastern box turtles.24 Both age and season showed
differences between the measurement of total
solids (refractometer) and total protein (biuret
method). Total solid measurement by refractom-
eter is an estimate of the refractive index of the
evaluated fluid, and the variations observed in
gopher tortoise plasma by age and season suggest
that several plasma components may vary. Fur-
ther evaluation is warranted to interpret the
clinical significance of these findings. The ob-
served correlation between TS (refractometer)
and TP (biuret method) shows that plasma TS
provide a rapid and inexpensive diagnostic tool in
the estimation of TP in healthy gopher tortoises.
With regard to seasonal differences, the study
group was sampled in early April, mid July, and
late November to account for the seasons expe-
rienced in north-central Florida. No samples were
collected in the coldest months of January or
February. However, based on historical clinical
case numbers, only a small number of animals are
presented during that time (likely due to winter
brumation of wild animals), which may prevent
meaningful statistical analysis.4 The data indicate
seasonal differences between spring (April) and
summer (July) with no differences during autumn
(November). The clinical significance of this is
unknown, although physiologic hormonal chang-
es may contribute to this seasonal variability in
plasma proteins, especially hormone-binding
plasma proteins, similar to that in birds.13
526 JOURNAL OF ZOO AND WILDLIFE MEDICINE
The subcarapacial sinus was the only site used
for sampling. It is known that sampling from the
subcarapacial sinus, among other reptile veni-
puncture locations, can result in lymphatic hemo-
dilution.6 To account for this possibility, animals
with PCV ,15% were excluded. It is suspected
that lymphatic hemodilution would falsely lower
PCV, elevate ESR, and affect the WBC differen-
tial; further evaluation would be indicated to
confirm these concerns and to determine the
possible effect on other analytes.
The reference intervals reported herein are
potentially biased given the relatively low sample
size as per definition by the ASVCP guidelines.17
However, the sample size is consistent with other
reports of reference ranges in chelonians.2,14,18,24,43
Establishing reference intervals has great value
for interpreting clinical findings in both captive
and free-ranging animals. Furthermore, this study
explored diagnostic tests not previously per-
formed in chelonians and that require a baseline
for comparison.
CONCLUSIONS
This study defines reference intervals for ESR,
lactate, heat-precipitated fibrinogen, hematology,
and protein electrophoresis in apparently healthy,
captive gopher tortoises. The reference intervals
presented herein provide a diagnostic tool to the
clinician when interpreting diagnostic data from
clinically abnormal animals. Further investigation
is required to determine the sensitivity and
specificity of these analytes in the diagnosis of
inflammation in this species.
Acknowledgments: The authors would like to
thank Kathy Russell and the students and staff at
Santa Fe College Teaching Zoo for providing
long-term care to the tortoises used in this study.
This project was funded by a resident research
grant through the University of Florida College of
Veterinary Medicine Research Committee.
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Accepted for publication 18 January 2018