ComparativeHaematologyInternational(1997) 1:109-112
1997 Springer-VerlagLondonLimited
Original Article
Haematological Reference Intervals of the South American Rattlesnake
(Crotalus durissus terrificus, Laurenti, 1768) in Captivity
J. C. Troiano 1, J. C. Vidal l, J. G o u l d 2 and E. G o u l d 2
1Area de Iologfa, Museo Argentino de Ciencias Naturales Bernardino Rivadavia, Av. Angel Gallardo 470, Buenos Aires,
Argentina; 2Fundaci6n de Estudios Biol6gicos, Gral. Urquiza 1942, Buenos Aires, Argentina
Abstract. Blood samples were taken from 180 healthy
specimens of South American rattlesnake (Crotalus
durissus terrificus) kept in captivity. The red blood cell
count (RBC), haematocrit value, haemoglobin con-
centration, mean corpuscular volume (MCV), mean
corpuscular haemoglobin (MCH) and mean corpuscular
haemoglobin concentration (MCHC) obtained were
within the normal values reported for other Crotalid,
Viperid and Elapid species. Total leucocyte (WBC)
counts were within the range described in other ophidian
species. No significant changes were observed as a
function of sex and age. The period of the year (seasonal
variation) was the only factor which had a significant
influence on the haematological parameters. RBC
counts, haematocrit, haemoglobin concentration, MCV,
MCH and MCHC were 17-30% higher during winter-
time (p<0.01), whereas the WBC and thrombocyte
counts were 15-20% higher (p<0.01) during the
summer.
Keywords: Crotalus durissus terrificus; Haematology;
Reference parameters; Seasonal variation
Introduction
Extensive information is available on comparative
haematological parameters, particularly in upper verte-
brates like the avian and mammalian species. Con-
versely, information regarding the haematological
parameters in reptilian species is scarce. This is
Correspondence and offprint requests to." J. C. Troiano, Area de
Iolo~a, Museo Argentino de Ciencias Naturales BernardinoRivada-
via, Av. Angel Gallardo 470, Buenos Aires, Argentina.
COMPARATIVE
HAEMATOLOGY
INTERNATIONAL
particularly true regarding South American snakes
(Leal-Prado 1945; Valle and Leal-Prado 1947; Sano-
Martins 1978). In previous studies, it has been pointed
out that the haematological parameters in snakes are
influenced by the age (Duguy 1970), the sex (Duguy
1970), and the period of the year in which the samples
are taken (Izard et al. 1961; Mutere 1965). In this paper
we report haematological reference intervals of an
autoctonous species, the South American rattlesnake
(Crotalus durissus terrificus) maintained in captivity.
The influence of the age and sex of the specimens, and
the seasonal variation in these parameters were explored.
Materials and Methods
Rattlesnakes and Care
A total of 180 healthy specimens of C. durissus terrificus
of different age and sex captured in the northwestern part
of Argentina were transported to the Fundaci6n de
Estudios Biol6gicos Serpentarium (Buenos Aires). All
animals were quarantined for a month. During this time
they were submitted to clinical examination and
deparasitised when eggs, larvae or adult parasites were
found in faeces. At the farm, the animals were housed in
individual wooden boxes at 22-28C and 40%-60%
humidity with natural light/dark periods corresponding
to the season.
All the snakes received filtered water ad libitum and
fed spontaneously on living prey. Specimens up to 2
years of age (born is the snake farm) were considered as
juvenile and were fed one newborn mouse (5-8 g) a
week. Older specimens were fed one adult mouse (20-25
g) a week.
110 J.c. Troiano et al.

Blood Sample Collection and Handling
In order to take the blood samples, the rattlesnakes were
removed from their boxes by means of a metallic hook
and the head and the rest of the body was firmly
restrained by two operators. The use of anaesthetics or
sedative agents, which are known to induce significant
alterations in the haematological parameters (Custer and
Bush 1980) were avoided. Blood (1.0 ml) was obtained
by a third operator by venipuncture of the caudal vein
(Bush and Smeller 1978) using a 21G-needle fitted to a
sterile disposable 2.5 ml syringe. A drop of blood was
immediately smeared on three clean microscopic slides
and the rest of the blood sample was transferred to a
clean glass tube containing heparin (Liquemine, Hoff-
man-La Roche 20 #l/ml of blood) as anticoagulant.
Haematocrit (PCV) and Haemoglobin Evaluation
Haematocrit (packed cell volume) was determined by
the microhaematocrit method. Blood was collected in a
capillary tube and centrifuged in a microhaematocrit
centrifuge at 12000 g for 5 min.
Haemoglobin was measured by mixing 20 #1 blood
with 2.5 ml of Drabkin solution (Wiener Laboratories,
Argentina) containing potassium ferricyanide (600 /~M)
and potassium cyanide (768 pM) in 1.0 mM phosphate
buffer, pH 7.2. After sedimentation of the nuclei,
formation of cyanmethaemoglobin was determined by
measuring the absorbance at 540 nm and compared to
that of a standard haemoglobin solution (Wiener
Laboratories, Argentina).
which reptilian leucocytes are classified as mononuclear
cells (lymphocytes, monocytes and azurophils) or
granulocytes (heterophils, basophils and eosinophils).
In brief, lymphocytes are characterised by their round
shape, central or slightly eccentric nuclei and clear
basophilic cytoplasm; monocytes by their basophilic
kidney-shaped nuclei with abundant, vacuolated cyto-
plasm; azuorphils by their round, eccentrically located
nuclei and a pale blue cytoplasm, sometimes with
granular appearance. Heterophils are round cells with
eccentrically placed lobulated nuclei and pale cytoplasm
filled with pleomorphic, eosincolour granules; basophils
are small, round cells with oval eccentrically positioned
nuclei. The cytoplasm is filled with round, homogeneous
basophilic granules that mask the nucleus. Finally,
eosinophils are round cells, with bilobed or round
nuclei, located eccentrically and spherical, homogeneous
eosinophilic granules in the cytoplasm.
However, ultrastructural and cytochemical studies (in
progress) might modify the differential leucocyte count
as described here.
Statistics
All data (Tables 1-3) are presented as mean _+ SD.
Student's t-test was employed to determine statistical
significance of differences between groups and ANOVA
test was employed to detect a significant difference
between groups means (SX35 software).
Table 1. Crotalusdurissus terriflcus: reference intervals as functionof
the age (mean _+ SD)

Juveniles (n=30) Adults (n=30)

Erythrocyte, Total Leucocyte, Total Thrombocyte
Count RBC count (109/1) 1520 134 1560 130
Haematocrit (%) 18.3 + 4.6 22.7 +__3.57
Red blood cells, total leucocyte and thrombocyte counts Haemoglobin (g/dl) 10_+4.5 11.5 _+4.3
were performed in a 1:200 dilution of blood in Natt and MCV (fl) 120 _+51 145 + 62
Herrick (1952) solution (0.1 g methyl violet 2B in MCH (pg) 66 13 74 q: 17
phosphate-buffered formaldehyde). The Natt and Her- MCHC (g/dl) 54 -+8 51 -~:6
rick procedure was used instead of automatic counting WBC count (109/1) 12000 3600 11400 -+3500
because it allows erythrocytes, total leucocytes and total Tbrombocytes (109/1) 7200_+1300 6900_+2400
thrombocyte to be counted in the same haemocytometer, Basophils 193.2_+84 171 _+9 t .2
and the measurement can be performed at the (1.61 _+0.70%) (1.5 _+0.8%)
serpentarium. When compared to electronic counting Eosinophils 1372 _+456 1482 _+342
methods, these advantages compensate the possible loss (11.4-+ 3.8%) (13_+3%)
of accuracy due to the use of manual diluting pipettes. Heterophils 873.6 _+456 684_+239
(7.28_+ 1.5%) (6_+2.1%)
Lymphocytes 6764.4 _+2280 6412 _+1396
(56.37 _+19%) (56.25 _+12.25%)
Differential Leucocyte Count Monocytes 660 _+144 593 206
(5.5_+ 1.2%) (5.2_+ 1.8%)
Blood smears were air dried and stained with a mixture Azurophils 2136 _+648 2057 _+799
of May-Grunwald and Giemsa stains in absolute (17.80 _+5.4%) (18.05 _+3.5%)
methanol (Rosenfeld 1947) and examined with a
Nikon EF100 optical microscope using the oil-immer- RBC, red blood cell; MCV, mean cell volume; MCH, mean cell
haemoglobin; MCHC, mean cell haemoglobin concentration; WBC,
sion lens. At least 200 cells were counted in each slide. white blood cell.
The leucocyte types described here follow the morpho- Values in parentheses represent the percentages of each leucocyte
logical nomenclature of Hawkey and Dennet (1994) in type.
Haematology of Crotalus durissus terrificus in Captivity 111

Results Table 3. Crotalus durissus terrificus: reference intervals as a function

of the season (mean SD)

Various haematologicaI parameters of C. durissus Winter (n=30) Summer (n=30)

terrificus are compared as a function of the age (Table
1) and sex (Table 2) of the specimens and in relation to RBC count (109/1) 1725 + 125 1440,+ 120
the seasonal variation (Table 3). Haematocrit (%) 23 _+4.3 18.3 -+4
Haemoglobin (g/dl) 19.1 ,+ 2.3 13.1 ,+ 3.2
Table 2. Crotalus durissus terrificus: reference intervals as a function MCV (fl) 133 ,+41 137 ,+ 37
of sex (mean -+ SD) MCH (pg) 76 ,+ 25 63 ,+ 14
MCHC (g/dl) 57 ,+4 50 ,+7
Males (n=30) Females (n=30) WBC count (109/1) 11550 _ 3800 13200 ,+ 3200
Thrombocytes (109/I) 6650 ,+ 1600 8530,+ 900
RBC count (109/1) 1535,+ 135 1530,+ 120 Basophils 185 ,+ 115 230-+ 145
Haematocrit (%) 20.9 ___4.1 22 ,+3.72 (1.6,+ 1%) (1.78,+ 1.1%)
Haemoglobin (g/dl) 11.1 ,+ 3.5 12,+ 3.2 Eosinophils 1271 ,+439 1498 ,+ 369
MCV (fl) 136,+37 143 __.41 (11 ,+ 3.8%) (11.35 ,+ 2.8%)
MCH (pg) 72___23 81 ,+ 17 Heterophils 693 ,+ 157 970 ,+ 303
MCHC (g/dl) 55 ,+ 7 51 ,+6 (6,+ 1.36%) (7.35 ,+ 2.3%)
WBC count (109/1) 12300,+ 3500 11000 ,+ 3200 Lymphocytes 6814 ,+ 1617 7392 2376
Thrombocytes (109/1) 7240___1500 6950-+ 1300 (59+ 14%) (56-+ 18%)
Basophils 204 ,+ 110 137 _+88 Monocytes 520 ,+ 182 737 + 183
(4.5 _+1.58%) (5.6__+1.35%)
(1.65 ,+0.9%) (1.25 0.8%)
Eosinophils 1396 ,+ 344 1402 _ 352 Azurophils 2067 ,+542 2374 ,+475
( 11.35,+ 2.8%) ( 12.75 ,+ 3.2%) (17.9_+4.7%) (18_+3.6%)
Heterophils 893 ,+ 184 793 ,+ 231
(7.25+ 1.5%) (7.2,+2.1%) RBC, red blood cell; MCV, mean cell volume; MCH, mean cell
haemoglobin; MCHC, mean cell haemoglobin concentration; WBC,
Lymphocytes 6919 ,+ 1845 6160 1602 white blood cell.
(56.25 ,+ 15%) (56-+ 14.57%) Values in parentheses represent the percentages of each leucocyte
Monocytes 676 ,+ 3147 550 ,+231 type.
(5.5 ,+ 1.2%) (5 ,+ 2.1%)
Azurophils 2214 ,+492 1458 385
(18,+4%) (17.8_+3.5%)

RBC, red blood cell; MCV, mean cell volume; MCH, mean cell
haemoglobin; MCHC, mean cell haemoglobin concentration; WBC,
white blood cell.
Values in parentheses represent the percentages of each leucocyte
type.
Discussion
This paper constitutes the first report of reference values
for the haematological parameters for C. durissus
terrificus from Argentina. The values of RBC count,
haematocrit, haemoglobin concentration, MCV, MCH
and MCHC, WBC and thrombocyte counts presented in
Tables 1, 2 and 3 are within the ranges reported for other
crotalid snakes, like Crotalus horridus (Carmichael and
Prechter 1945)and Bothropsjararaca (Leal-Prado 1945;
Valle and Leal Prado 1947). They are also similar to
those reported by Hattings and Willemse (1976), for the
viperid Bitis arietans although, for the same species, Otis
(1973) reported a slightly lower haemoglobin concentra-
tion. With another viperd, Causus rhombeatus, Pienaar
(1962) reported a lower total erythrocyte count. The
reference values for C. durissus terrificus (Tables 1-3)
are also within the ranges reported for the Australian
elapids Pseudonaja muchalis, Pseudechis prophyriacus,
Austerelaps superbus (Board and Shine 1977) and those
reported for other snakes (Duguy 1970).
As shown in Tables 1-3, the total leucocyte count is
within the range described for other Crotalus species
(MacMahon and Hummer 1975). The different leucocyte
types identified in the present study in the peripheral
blood of C. durissus terrificus were only those described
by Hawkey and Dennet (1994). No other cell types such
as 'neutrophils' described by Frye (1977) were observed.
On a morphological basis only, lymphocytes represented
more than 50% of the total WBC. This is within the
range described by Duguy (1970), Pienaar (1962) and by
Roskopf et al. (1982) for other ophidian species.
Conversely, Dotson et al. (1995) and Bounous et al.
(1996) reported that in rattlesnake and boa constrictor,
the predominat leucocyte is the azurophil, whereas in
some reptiles it is the heterophil.
As shown in Tables 1 and 2, the haematological
parameters of C. durissus terrificus specimens used in
this study, do not present any significant variation with
the age (Table 1, p > 0 . 3 ) or sex (Table 2, p > 0 . 1 ) in
contrast to the results reported by Duguy (1970). Table 3
shows that seasonal variation has a statistically sig-
nificant influence on the haematological parameters of
the C. durissus terrificus specimens. In fact, during
wintertime the RBC count, haematocrit, MCV, MCH,
MCHC and haemoglobin concentration were 17%-30%
112
higher (p<0.01) than the corresponding parameters
measured during the summertime. Conversely, during
summertime, the WBC and thrombocyte counts were
15%-20% higher (p<0.01) than the corresponding
values during wintertime. These results are in agreement
with previous reports (Duguy 1970; Izard et al. 1961;
Mutere 1965. On the other hand, in contrast to the results
reported by Duguy (1970), the seasonal variation in
haematological parameters is not accompanied by a
statistically significant change in the differential
leucocyte count (p > 0.1).
This paper is the first approach aimed at acquiring an
adequate knowledge of the haematological parameters of
C. durissus terrificus, and may provide the basis for
further studies, in particular with the snakes in their
natural environment.
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