Comparative Biochemistry and Physiology, Part C 139 (2004) 245 – 250
www.elsevier.com/locate/cbpc
Effects of different stressors in haematological variables
in cultured Oreochromis aureus S.
R. Silveira-Coffignya,*, A. Prieto-Trujilloa, F. Ascencio-Valleb
b
a
Fisheries Research Center, 5ta Ave y 246, Barlovento, Playa, Ciudad de la Habana, Cuba
Northwest Biological Research Center, Mar Bermejo 195, Colonia Playa Palo de Santa Rita, Baja California Sur, México
Received 24 July 2004; received in revised form 18 November 2004; accepted 19 November 2004
Abstract
Since haematological variables can be used to assess the health state in cultured fish, a haematological characterization of clinically
healthy Oreochromis aureus was done to establish the reference indices of this species. Fish were subjected to different stressed conditions
(bacterial infection, nitrite intoxication, malachite green overdose) to study the changes in the haematological indices and its relation with the
health condition. This species showed microcytic anaemia under experimental bacterial infection by Corynebacterium sp.; anaemia,
neutrophilia and erythrocytes deformation following nitrite intoxication and medication overdose with malachite green.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Anaemia; Corynebacterium sp.; Haematology; Malachite green; Nitrite; Oreochromis aureus; Reference indices; Stress
1. Introduction
The ecological damage of the environment caused by
anthropogenic factors as well as the presence of hazardous,
infectious and parasitizing agents may affect fish. Fish
peripherical blood analysis is used as a diagnosis to assess
its physiological state and the effect of hazardous substances,
to determine the nonspecific resistance of progenitors and its
descendants, to establish the quality of food, the genetic
variability and the effect of stress (Svobodová and Vysuková,
1991). Haematology is used as an index of fish health status
in a number of fish species to detect physiological changes
following different stress conditions like exposure to
pollutants, diseases, metals, hypoxia etc. (Blaxhall, 1972;
Duthie and Tort, 1985; Morgan and Iwama, 1997).
In order to use these blood parameters with a diagnostic
purpose, it is necessary to analyze healthy fish in the best
ambient conditions to establish the reference indices for the
species object of study, as well as to analyze fish under
different biological, physical and environmental stressed
* Corresponding author. Tel.: +53 7 2097851; fax: +53 7 2049827.
E-mail address: [email protected] (R. Silveira-Coffigny).
1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.cca.2004.11.009
conditions and establish specific variation in each parameter, compare it with the normal values and make a diagnosis.
(Hrubec et al., 2000) determined haematology and plasma
chemistry reference intervals for cultured tilapia hybrids.
The objective of the present research is to establish the
variation of haematological parameters in tilapia, starting
with the haematological characterization of Oreochromis
aureus to establish the blood normal values, as well as to
determine the behavior of these indicators in animals under
stress caused by pathogenic bacteria (Corynebacterium sp.),
nitrite toxicity, overdose of extensive used drugs (malachite
green).
2. Materials and methods
2.1. Experimental animals, sample taking and experimental
conditions
The species characterized haematologically was O.
aureus. All fish were obtained from semiintensive cultured
systems from the research facilities of Aquaculture Research
Center in Havana. They had a body length ranging from 16
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R. Silveira-Coffigny et al. / Comparative Biochemistry and Physiology, Part C 139 (2004) 245–250
to 24 cm and weighed from 75 to 275 g. Fish were raised
under the required oxygen (5–7 mg/l), pH (5.5–7.1),
temperature (25 8C–27 8C) and feeding (4% body mass
per day) conditions. These conditions were kept constant
during the assays.
The fish were kept 72 h without feeding before blood
sampling. Underyearlings O. aureus were sampled, and the
assays were carried at the Aquaculture Research Center
facilities in Havana City.
Blood samples were taken from 100 O. aureus, sampling
trips were made on a daily basis, 20 fish each day were
sampled from their rearing tanks to determine normal levels
of the measured variables in a controlled production
situation. Careful handling was implemented to minimize
stress. Fish were rapidly netted and anesthetized in 80 mg/L
aerated buffered tricaine methanesulfonate (MS-222).
Peripheral blood of each fish was taken from the caudal
artery using sterile heparinized syringe fitted with 21 G
needles. To compare the results, fish were grouped by length
(2-cm intervals) and weight classes (50-g intervals).
The experimental fish were transferred and acclimated in
the laboratory during 21 days before the extraction of blood,
maintaining the previously described culture conditions.
2.2. Haematological examination
The haematological variables analyzed were haemoglobin (Hb), haematocrit (Ht), red blood cells count (RBCC),
mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin
concentration (MCHC). Haemoglobin concentration was
determined by the cyanmethaemoglobin method. The blood
was analyzed using an electronic cell counter (Coulter
Counter Z2, Coulter, FL). Total RBC count was determined
manually with a Neubauer haematocytometer using Hayems
solution as a diluent stain (Shaperclaus, 1979). The
haematocrit (Ht) value was measured as fractional red cell
volume following centrifugation at 5000 g for 5 min.
Based on the standard formulas, the RBC, MCV, MCH,
MCHC were calculated. For the morphological studies, blood
smears were fixed in absolute methanol for 5 min and stained
with May–Grunwald and Giemsa stain. Blood smears were
examined by light microscopy at 100 magnification. One
hundred leucocytes were categorized based on their morphology (Doggett et al., 1987; Rowley, 1990).
Blood cell size was measured in 20 WBC of each type in
20 different fish to give range diameters for each cell type.
Thirty 1000 microscopic field of each stained blood smear
were examined, and the frequency of immature and deformed
erythrocytes were recorded (100 number of immature or
deformed erythrocytes/number of total erythrocytes).
2.3. Experimental inoculation with Corynebacterium sp:
Five replicate samples of five animals were inoculated
intraperitoneally with Corynebacterium sp. (0.2 mL/g body
mass); one sample was left without being inoculated, and
one sample was inoculated with sterile peptone saline
solution (0.2 mL/g body mass). The primary isolate
(Bacteriological Laboratory of Aquaculture Research Center
in Havana City) was obtained from tilapia O. aureus that
had died from natural exposure to the pathogen. Twentyfour-hour cells of Corynebacterium sp. were harvested from
human blood agar 5% plates transferred to sterile peptone–
saline (0.1% peptone and 1.5% saline) and centrifuged at
1200 g for 15 min. The concentration of the Corynebacterium sp. suspension was estimate from absorbance measurements made at 420 nm. The suspension was diluted, and
each fish received an injected dose of 218.72106 cell/mL.
Fish from five tanks were injected with the bacterial
suspension. Blood samples were taken 13 days after
inoculation.
2.4. Experimental contamination with nitrite
Groups of six fish distributed in glass fiber tanks were
used in the experiment. The concentration of NO2 used in
four tanks was 30 mg/L, leaving four tanks as control. The
desired nitrite concentration in the experimental tanks was
achieved by adding sodium nitrite NaNo2, and the obtained
concentration was checked at the beginning of the experiment by the sulfanilamide naftilendiamide method of Greiss
(Green et al., 1982). Six fish were removed from test tanks
and control tanks after 2, 6, 24 and 48 h of exposition.
2.5. Malachite green overdoses
Groups of six fish were placed in glass fiber tanks. The
experimental unit consisted of five test tanks and five
control tanks. Malachite green (p,p-benzylidenebis-N,Ndimethylaniline was dissolved in water and added to the
tanks at a concentration of 1.0 mg/L before fish were
Table 1
Blood normal values of Oreochromis aureus n=102, raised in semiintensive
production systems
Parameter
Hb (g/dL)
Ht (%)
RBCC (106/mm3)
MCV (fL)
MHC (pg)
MCHC (g/dL)
Monocytes (%)
Large lymphocytes (%)
Small lymphocytes (%)
Thrombocytes (%)
Neutrophils (%)
Blood normal
values meansFSEM
5F0.72
30.6F4.34
1.42F0.24
214.7F27.6
35.8F7.30
17.7F2.93
8F3.75
6.3F3.23
17.4F6.41
66.5F10.2
1.6F1.67
Corynebacterium
infection
Control
fish
means
Inoculated
fish
means
5.0a
29.0a
1.15
263.9a
43.5a
17.4a
3.6a
11a
26.1
58.9a
0.4
3.8b
21b
1.11
239.5b
33.2b
14.6 b
20.8b
27b
13.0
29.6b
9.6
Effect of Corynebacterium sp. on haematological parameters of Oreochromis aureus.
R. Silveira-Coffigny et al. / Comparative Biochemistry and Physiology, Part C 139 (2004) 245–250
stocked. Six fish were removed from the bottom test tanks
and control tanks after 2, 6, 24 and 48 h of exposition.
2.6. Statistical analysis
Statistical tests used were Kruskall–Wallis variance test
for PV0.05, and when a significant difference was found,
Scheffé test and one-way ANOVA were used, accepting
PV0.05 as significant. We used Duncan’s multiple comparison test to identify the values causing the significant
difference.
3. Results
The normal haematological parameters obtained for O.
aureus are shown in Table 1. No statistical differences were
found between the blood indices with respect to the weight
or length of the animals. A positive correlation was obtained
between haemoglobin and the erythrocyte count (r=0.215),
247
between haemoglobin and the haematocrit (r=0.364) and
between the erythrocyte counts and the haematocrit
(r=0.25).
In relation to the morphological elements of peripheral
blood of O. aureus, we identified the following cells:
erythrocytes, thrombocytes, small and large lymphocytes,
monocytes, neutrophils. Only a single eosinophil cell in one
occasion was found, therefore, it could not be expressed in
proportion.
The erythrocytes found in O. aureus were nucleated,
elliptical cells measuring 7.2513.05 Am in size. (Fig. 1a).
The nucleus was also elliptical and centrally located with
purple color and acidophilic cytoplasm.
The thrombocytes were 4.9 to 5.8 Am in diameter (Fig.
1a,c,d), elliptical, and their nuclei were also elliptical and
centrally located; thrombocyte cytoplasm was clear to
slightly basophilic, and some spherical shapes were found.
The small lymphocytes (Fig. 1b) ranged from 4.7 to 5.2 Am
in diameter, with irregular form, had a rounded red–violet
nucleus and blue cytoplasm. The large lymphocytes (Fig. 1b)
Fig. 1. Micrographs of blood smear of the peripheral blood in tilapia O. aureus. (a) Normal blood T—thrombocytes. (b) Normal blood LL—large lymphocytes,
SL—small lymphocytes, N—neutrophil. (c) Blood with abnormal mature erythrocytes (microcytes) N—neutrophil. (d) Blood with abnormal mature
erythrocytes and abnormal immature erythrocytes (open arrowheads). Filled arrowheads represent mitotic cells. (e) Blood monocytes (M). The insert depicts a
neutrophil with a bean-shaped nucleus. (f) Deformed erythrocytes (triangular erythrocytes, poikilocytosis and anisocytosis) in tilapia with malachite green
treatment, 48-h exposure. Bar=18 Am.
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R. Silveira-Coffigny et al. / Comparative Biochemistry and Physiology, Part C 139 (2004) 245–250
Table 2
Effect of nitrite on blood parameters of Oreochromis aureus
Parameters
Hb (g%)
Ht (%)
RBCC (106/mm3)
MCV (fL)
MHC (pg)
MCHC (g%)
Reference indices (meanFSD)
5F0.72
30.6F4.34
1.42F0.24
214.7F27.6
35.8F7.30
17.7F2.93
Control
a
5.34
25a
1.05
238.1a
50.9 a
21.63b,c
2h
6h
a
4.37
13.2b
0.9
151.9b
52.6a
32.4a
24 h
a
5.2
16c
1.12
148.3b
47.8a
32.7a
48 h
b
4.2
13.8b
0.9
149.4b
45.6b
30.2a
4.2b
17.6c
0.9
193.2c
45.9b
25.3b
Mean values depending on exposure times.
Values with differing superscripts are significantly different for PV0.05.
measured 5.8 to 6.7 Am in diameter, with red–violet nucleus
and a pale blue cytoplasm. The monocytes measured 9.5 to
10.9 Am in diameter (Fig. 1e), have round form with red–
violet nucleus, generally eccentric and with gray-smoke
color cytoplasm. The neutrophils (Fig. 1c,e) have rounded
form and a compact red–violet color nucleus, the cytoplasm
is grayish pink.
observed at 24 h with respect to the control (Table 2), but at
48 h, the haematocrit and MCV increased significantly; the
mean corpuscular haemoglobin and the concentration of
mean corpuscular haemoglobin also decreased.
3.1. Inoculation with Corynebacterium spp.
To date, little is known about the haematology of the
tilapia O. aureus, which is one of the most important species
in freshwater aquaculture worldwide; the results presented
above have revealed an interesting pattern of response on
the haematological variables in stressed fish. When we
compare O. aureus normal blood values with those obtained
previously for this species, we find that Hussein et al., 1996
reported lower values haematocrit (20%) and RBC count
(1.31106 cells/AL) but obtained slightly higher values for
haemoglobin concentration (6 g/dL). Hrubec et al., 2000
reported even higher haematocrit (33%), RBC count
(2.31106 cells/AL) and haemoglobin concentration (8.2
g/dL). These differences may be due to diverse strains used
in previous studies and different environmental and cultured
conditions.
Five types of white blood cells were identified in O.
aureus: thrombocytes, monocytes, neutrophiles, and small
and large lymphocytes. The morphological and staining
characteristics observed in this study were similar to those
described for an Oreochromis hybrid by Hrubec et al.
(2000), as well as those described for O. niloticus by Ueda
et al. (2001). Basophils and eosinophils have not been
observed from the blood of rainbow trout (Klontz, 1972)
and brown trout (Blaxhall and Daisley, 1973). Ellis (1977)
claimed that the entire literature concerning eosinophils in
fish is contradictory in that there have been reports of their
presence and absence in many fish species. Ellis (1976)
reported the absence of eosinophils and basophils from the
circulation in plaice (Pleuronectes platessa).
In the fish injected with Corynebacterium sp., a grampositive bacterium that causes great mortality in tilapia
culture systems, the experimental inoculation only provoked
a subclinical infection and no clinical signs of infectious
process were detected.
In accordance with the behavior of the corpuscular
constant and the detected findings in the erythrocytes of
blood smears, we can classify this anaemia as microcytic
In the experimental inoculation with Corynebacterium
spp., only two animals died after 10 days, and no typical
signs of the disease were observed in the inoculated fish. No
mortality or morbidity was observed in uninjected controls
or controls injected with sterile peptone–saline.
From the data on red blood cell count, haematocrit and
haemoglobin content of the blood, the mean corpuscular
volume (MCV), mean corpuscular haemoglobin concentration (MCHC) and mean corpuscular haemoglobin (MCH)
were calculated (Table 1). From these indices, an indication
of whether the animal was suffering from microcytic
hypochromic anaemia could be discerned. Haematocrit
and haemoglobin content in the blood are reduced below
normal, and the MCV, MCH and MCHC values were much
lower in relation to controls (Table 1), the monocytes and
neutrophils increased. The smears showed 7.14% immature
and 20% deformed erythrocytes and microcytes and 7%
erythrocytes with segmented nuclei (Fig. 1c,d).
3.2. Malachite green
The use of high doses of the therapeutic agent malachite
green promotes haematocrit (39.5%), and the neutrophil
counts (11%) increased 24 h after exposure with respect to the
control (Ht=30%, neutrophil=1.0%), besides abundance of
erythroblasts (10%) and the presence of 29.8% deformed
erythrocytes (triangular erythrocytes, poikilocytosis and
anisocytosis) was observed(Fig. 1f). There were no significant changes in the other haematology parameters measured.
3.3. Nitrite
During the course of the experiments, no mortalities were
recorded in the fishes exposed to nitrite. A decrease of the
haemoglobin, erythrocyte counts and haematocrit was
4. Discussion
R. Silveira-Coffigny et al. / Comparative Biochemistry and Physiology, Part C 139 (2004) 245–250
hypochromic. Nakayasu et al. (2002) described microcytic
hypochromic anaemia in Paralichthys olivaceus due to the
decrease of mean corpuscular volume, hypoglobulia and the
structural abnormality of the erythrocytes. A hypochromic
microcytic anaemia, where the haematocrit and haemoglobin content in the blood are reduced below normal, has been
observed in carp, Cyprinus carpio (Sakamoto and Yone,
1978), red sea bream, Chrysophrys major (Sakamoto and
Yone, 1977), and fingerling channel catfish, Ictalurus
punctatus (Gatlin and Wilson, 1985). In O. aureus blood
smears, we observed erythrocytes with segmented nuclei
and poikilocytosis. Such changes were characterized by
Takahashi et al. (1992) as a sign of recovery from infectious
disease in coho salmon (Oncorhynchus kisutch). It is
believed that neutrophils and monocytes have phagocytic
activity which might explain their increased percentage
during infectious situations.
Hlavek and Bulkley (1980) found a transient neutrophilia
in rainbow trout 24 h after treatment with malachite green,
but this decreased after 4 days. These authors stated that the
white cells changes in the trout exposed to malachite green
were the result of a stress syndrome not specific to
vertebrates and not due to leucocytotoxic effects of this
chemical compound in the fish. Darwish et al. (2001) also
found an increase of neutrophil counts in channel catfish
exposed to high doses of potassium permanganate. Changes
in the differential leucocyte count are one of the most
sensitive indicators of acute stress in fish (Wedemeyer et al.,
1990).
Ueda et al. (2001) saw erythroblast abundance as a result
of the acceleration of erythropoiesis in different physiological conditions, while Shaperclaus (1979) reported abnormal
forms of erythrocytes caused by haemolysis for intoxication
which is accompanied by observation of triangular erythrocytes. We observed these same abnormalities in our
tilapia.
Although tilapia are more tolerant to nitrite toxicity than
many freshwater species (Popma and Masser, 1999), nitrite
is toxic for many fish species because of its effects on
haemoglobin. The nitrite concentration in water induced a
macrocytic anaemia in our experimental fish due to the body
response to increase the erythrocyte size to compensate for
the haemoglobin decrease caused by the blood oxygen
decrease. This effect was also observed in I. punctatus
(Scotts and Rogers, 1981).
The decrease in blood oxygen is due to the nitritedependent transformation of haemoglobin in methaemoglobin, which cannot transport oxygen (Brown and McLeay,
1975; Watanabe and Yasutake, 1978; Tomasso, 1986).
Popma and Masser (1999) reported that for certain
conditions in tilapia, the LC50 for nitrite after 96 h is 89
mg/L, although the appropriate concentration for freshwater
culture is 27 mg/L. On the other hand, Palachek and
Tomasso (1984) stated a LC50 after 96 h of 16.2F2.3 mg/L
for Tilapia aurea. Atwood et al. (2001) established that the
nitrate tolerance for tilapia depends on fish size, since in fish
249
with a mass of 4.4 g, the LC50 after 96 h is 81 mg/L, and for
90-g animals, the LC50 is 8 mg/L.
Our results establish that, for cultured O. aureus, the
bacterial infection caused by Corynebacterium sp. provoked
microcytic anaemia and increased the titers for monocytes
and neutrophils. An overdose with malachite green and
nitrate contamination result in anaemia, neutrophilia and
erythrocyte deformations.
Acknowledgements
We are grateful to the Consejo Nacional de Ciencia y
Tecnologia (CONACYT) of Mexico for financing this
project.
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