Aquaculture International

https://doi.org/10.1007/s10499-021-00668-8

Comparative study of hematological parameters

of Colossoma macropomum anesthetized
with benzocaine and eugenol by using
different anticoagulants

Andria Gama Sousa 1 & Acácio H. B. Pacheco 1 & Gilson A. Siqueira-Pinto 1 &
Gleika T. J. dos Reis 2 & Michelle M. S. Fugimura 2 & Luciano J. Vaz 2 &
Paulo F. Marcusso 3 & Fernando C. Ramos-Espinoza 4 & Gustavo da Silva Claudiano 1

Received: 2 September 2020 / Accepted: 9 February 2021/

# The Author(s), under exclusive licence to Springer Nature Switzerland AG part of Springer Nature 2021

Abstract
The objective of the present study was to evaluate the action of benzocaine and eugenol
over anesthesia stages and the use of three anticoagulants (ethylenediamine tetraacetic
acid, sodium citrate, and sodium heparin) over hematological and biochemical parameters
and erythrocyte osmotic fragility in tambaquis Colossoma macropomum. For that, fish
were distributed into two experimental groups: G1: fish anesthetized with benzocaine and
G2: fish anesthetized with eugenol. Induction and recovery times of anesthesia were
evaluated and blood samples with different anticoagulants were collected for hematolog-
ical and biochemical analysis and osmotic erythrocyte osmotic fragility. Fish anesthetized
with eugenol showed a longer period of anesthesia, shorter induction time, and longer
recovery time. In addition, fish anesthetized with benzocaine by using sodium citrate
showed leukopenia and lymphopenia and morphological alterations when compared to
the other anticoagulants. Furthermore, fish anesthetized with eugenol by using sodium
citrate showed significantly lower values (P < 0.05) of hematocrit and hemoglobin when
compared to other anticoagulants and there were significant differences in erythrocyte
osmotic fragility between anticoagulants in fish anesthetized with eugenol. The results
observed in this study indicate that eugenol is a good alternative as anesthetics for
tambaquis when compared to benzocaine because of its rapid anesthesia induction and
few alterations in blood parameters. In addition, the use of EDTA facilitates the erythro-
cyte conservation and cause few blood alterations compared to other anticoagulants.

Keywords Ethylenediamine tetraacetic acid . Sodium heparin . Sodium citrate . Tambaqui .

Anesthesia

Handling Editor: Gavin Burnell

* Gustavo da Silva Claudiano

claudianovet@yahoo.com.br

Extended author information available on the last page of the article

 Aquaculture International

Introduction

The use of anesthetics during fish management is important in order to preserve integrity and
reduce adverse effects such as stress and mortality (Hasan and Bart 2007; Pádua et al. 2013).
However, there is no regulation about anesthetics for aquaculture in Brazil. Consequently,
some anesthetics are used by taking into account some recommendations of the Food and
Drugs Administration (FDA). Currently, benzocaine and eugenol are the most used anesthetics
in fish (Delbon and Paiva 2012; Cosenza et al. 2014) and usually administered by immersion
(Priborsky and Velisek 2018).
Controversy exists in the literature regarding the use of anesthetics because of some effects
over physiological parameters and proper use according to species, age, sex, and size, as it is
seen in other vertebrates (Sladky et al. 2001; Roubach et al. 2005; Okamoto et al. 2009; Inoue
et al. 2011). So, it is important to evaluate the anesthetics in each species, with a focus on
anesthesia induction, recovery time, and physiological parameters in order to determine which
anesthetic is safer or convenient (Delbon and Paiva 2012; Cosenza et al. 2014). The hemato-
logical parameters are important tools for determining physiopathological alterations
(Claudiano et al. 2019), immunological and nutritional status (Barros et al. 2009; Claudiano
et al. 2019), vaccine efficacy (Salvador et al. 2013), and safety studies (Pádua et al. 2013;
Cosenza et al. 2014; Yunis-Aguinaga et al. 2016).
Furthermore, there is no standardization in fish anesthetics due to the variety of fish species
and the different effects on blood cells (Walencik and Witeska 2007; Lătărełu et al. 2013). In
addition, some anticoagulants used in teleost fish cause alterations in blood cells during
sampling (Mafuvadze and Erlwanger 2007; Walencik and Witeska 2007), hemolysis
(Walencik and Witeska 2007), erythrocyte volume increase (Mafuvadze and Erlwanger
2007), alterations in storage time (Castro et al. 2019), which can influence on hematological
results and diagnostic.
Tambaqui is one of the main native species cultured in Brazil and several other countries in
South and Central America (Woynárovich and Van Anrooy 2019). In addition, this species has
been selected as a model to establish studies with anesthetics (Roubach et al. 2005). The
objective of the present study was to evaluate the action of benzocaine and eugenol over
anesthesia stages and the use of three anticoagulants (ethylenediamine tetraacetic acid, sodium
citrate, and sodium heparin) over hematological and biochemical parameters and erythrocyte
osmotic fragility in tambaquis.

Material and methods

Fish and experimental conditions

Tambaqui, Colossoma macropomum, used during the study were obtained from a commercial
fish farm, transported to the Federal University of Western Pará facilities and placed in 250-L
polypropylene tanks supplied with constant aeration, and continuous water flow (1 L·min−1).
Fish were fed with commercial fish feed pellets (28% crude protein, 4000 kcal·kg−1) at 3% of
body weight throughout the whole study.
Fish were adapted for 7 days before the experiment. The water quality conditions were kept
within the optimal range for the species: dissolved oxygen 4.63 ± 0.6 mg L−1 and temperature
27.53 ± 0.5 °C (YSI Model 55, USA); pH 6.51 ± 0.08 and conductivity 136.48 ± 3.3 μS/cm
Aquaculture International

(YSI 63 Model, USA). The present study was according to the ethical principles for animal
experimentation of Brazilian laws of the National Council for the Control of Animal Exper-
imentation of Brazil (CONCEA) and was approved by the Ethics Committee on Animal Use
of Federal University of Western Pará (protocol number 720180034).

Experimental design

Following the acclimation period, 20 tambaquis, Colossoma macropomum (403.1 ± 51.8 g,

and mean length 27.7 ± 1.0 cm), were randomly assigned into two experimental groups (N =
10 fish per tank), each animal was used as an experimental unit. Distribution was as follows:
group 1: eugenol, and group 2: benzocaine. Fish were captured by a hand net, transferred to
50-L glass aquariums and monitored during anesthesia induction corresponding to stages I, II,
and III (Table 1).
After anesthesia induction (eugenol: 100 mg L−1 and benzocaine: 100 mg L−1), fish
were transferred to aquariums with constant aeration without anesthetic and monitored
during recovery time corresponding to stages IV and V (Table 1). After that, mortality
was monitored for 24 h. Before anesthesia induction and after recovery, water quality
parameters such as temperature, oxygen, pH, and conductivity were kept as stated
above.

Blood sample collection

A total of 10 fish per group after reaching stage III, described as a surgical anesthesia, were
used for blood sampling. For blood analysis, 3 mL of blood was collected by caudal
venipuncture per fish (N = 10 / group), using syringes (3 mL) with 26-G needles containing
EDTA, sodium heparin, or sodium citrate. Firstly, blood samples were transferred to
microtubes without anticoagulants, allowed to clot for 30 min at room temperature, and later
centrifuged at 5000 rpm for 5 min for serum collection and stored at −80 °C until further
biochemical analysis (alanine aminotransferase, iron, lactate, glucose, serum total protein,
albumin, globulin, and albumin/globulin ratio). In addition, blood samples were transferred to
microtubes with the three anticoagulants (EDTA 10%—2.0 mg mL−1, sodium heparin—15 UI
mL−1, and sodium citrate 3.2%—1:9 sodium citrate/blood) for use in the hematological
analysis and erythrocyte osmotic fragility.

Table 1 Description of the stages of anesthesia in fish (modified from Ross and Ross (2008) and Hikasa et al.
(1986))

Stage Description Behavioral response in fish

I Deep sedation Total loss of reactivity to stimuli except to strong pressure

Normal equilibrium; reduced opercular movements
II Deep anesthesia Total loss of muscular tone; total loss of equilibrium
Reduced and regular opercular movements
III Surgical anesthesia Total loss of stimuli; slow and irregular opercular movements
Slow heart rate; total loss of reflexes
IV Return of movements Total recovery of equilibrium; slow reactivity stimuli
V Return of swimming Total recovery of equilibrium and swimming activity
 Aquaculture International

Biochemical parameters

Alanine aminotransferase (ALT) and lactate were determined by IFCC UV kinetic method and
iron by ferrozine method by using a commercial kit (Analisa Diagnóstica, Minas Gerais,
Brazil). Glucose was determined by enzymatic colorimetric test following the GOD–POD
method (Trinder 1969) by using commercial kit (Labtest Diagnóstica, Brazil).
Serum total proteins and albumin were determined by biuret and bromocresol method,
respectively, by using a commercial kit (Labtest, Minas Gerais, Brazil). The globulin content
was estimated by subtracting the albumin content from total protein content (Claudiano et al.
2019).

Hematological parameters

The total red blood cell (RBC, 106 cells/μL) counts were performed in a Neubauer chamber
using the Natt and Herrik diluent (1:200) (Ishikawa et al. 2008). Aliquots were used for the
determination of hematocrit (Htc %) (Goldenfarb et al. 1971); hemoglobin concentration
(Drabkin 1935). Hematimetric indexes: mean corpuscular volume (MCV), mean corpuscular
hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were also
assessed (Cosenza et al. 2014).
Additionally, blood smears were stained with rapid panoptic staining for total throm-
bocyte, total leukocyte, and differential leukocyte counts (neutrophils, monocytes, and
lymphocytes) by light microscopy using an indirect method (Tavares-Dias et al. 2008;
Ishikawa et al. 2008). In this method, 2000 erythrocytes were counted in blood extensions
for determination of total leukocytes and thrombocytes according to the formula: total
leukocytes (μL) = No. de leukocytes in extension × No. de erythrocytes count in Neubauer
chamber/2000 erythrocytes in extension; total thrombocytes (μL) = No. de thrombocytes
in extension × No. de erythrocytes count in Neubauer chamber (μL)/2000 erythrocytes in
extension. Morphological alterations in red and white blood cells were analyzed according
to Palmer et al. (2015).

Erythrocyte osmotic fragility

The erythrocyte osmotic fragility was measured according to Ishikawa et al. (2010).
Briefly, blood aliquots were mixed with a 10.5% buffered saline stock solution (pH 7.4)
in different serial concentrations (0.9; 0.7; 0.5; 0.3; and 0.10% NaCl-PO4). Then, blood
samples were diluted in each serial concentration (1:100) and kept at room temperature
and homogenized three times for 10 min. After 30 min, the solutions were centrifuged at
2000 rpm and the supernatant was subjected to a hemoglobin test (Drabkin and Austin
1935).

Statistical analysis

All data were analyzed using the R Software (R version 3.6.3). First, data were tested for
normality (Shapiro–Wilk test). Statistical analysis was performed using two-way
ANOVA and Tukey’s multiple comparisons to identify statistical differences between
experimental groups. Data were expressed as mean ± SD. The significance level was
defined as P < 0.05.
Aquaculture International

Results

Anesthesia induction and recovery

No alterations were observed in water quality parameters before and after anesthesia for each
anesthetic (P > 0.05). Also, no mortality was observed during the anesthesia period and the
following 24 h after anesthesia. In addition, the study revealed that fish anesthetized with
eugenol reach stages II and III of anesthesia in a shorter time than benzocaine (P < 0.05).
However, the recovery stages IV and V showed a significantly shorter time in fish anesthetized
with benzocaine when compared to those anesthetized with eugenol (Fig. 1).

Biochemical parameters

The biochemical analysis (ALT, iron, lactate, glucose, serum total protein, albumin, globulin,
and albumin/globulin ratio) did not show significant differences (P > 0.05) between fish
anesthetized with benzocaine and eugenol (Table 2).

Hematological parameters

The leukogram (Table 3) revealed leukopenia and lymphopenia (P < 0.05) in fish anesthetized
with benzocaine using sodium citrate as anticoagulant. In addition, monocytes and neutrophils
did not show significant differences (P > 0.05) between anesthetics groups and also between
the three anticoagulants. Finally, thrombocyte values in fish anesthetized with benzocaine
using sodium citrate and sodium heparin were significantly lower when compared to the group
using EDTA.
The erythrogram (Table 3) revealed a decrease in erythrocytes and hematocrit values (P <
0.05) in fish anesthetized with benzocaine using sodium citrate. Fish anesthetized with
benzocaine showed significantly higher MCV values when compared to the fish anesthetized
with eugenol for the three anticoagulants. Also, hematocrit and hemoglobin values in fish
anesthetized with eugenol using sodium citrate showed significantly lower values when
compared to the other anticoagulants.

a 300 A
b
400

A
Time (seconds)

300
Time (seconds)

200
B
A 200
B
A
100 B
A A 100 B

0 0
Benzocaine Eugenol Benzocaine Eugenol

Fig. 1 Duration of anesthesia stages in Colossoma macropomum anesthetized with eugenol (100 mg L-1) and
benzocaine (100 mg L-1). a Induction period (light gray bar: stage I; dark gray bar: stage II; and black bar: stage
III). b Recovery period (light gray bar: stages IV and dark gray bar: stages V). Different uppercase letters indicate
significantly different values (P<0.5) between different anesthetics
 Aquaculture International

Table 2 Biochemical parameters (mean ± standard deviation) of tambaqui anesthetized with benzocaine and
eugenol

Parameters Groups

Eugenol Benzocaine

Glucose (mg/dL) 63.6 ± 3.20 A 66.3 ± 5.10 A

ALT (U/L) 137.1 ± 48.39 A 194.2 ± 98.20 A
Iron (g/dL) 178.7 ± 65.59 A 224.6 ± 80.43 A
Lactate (mg/dL) 31.6 ± 7.32 A 36.1 ± 11.74 A
Total protein (g/dL) 4.2 ± 0.47 A 4.3 ± 0.47 A
Albumin (g/dL) 2.1 ± 0.39 A 1.6 ± 0.48 A
Globulin (g/dL) 2.1 ± 0.43 A 2.6 ± 0.37 A
A:G ratio 1.0 ± 0.50 A 0.6 ± 0.25 A

Different uppercase letters indicate significantly different values (P<0.5) between differentt anesthetics. ALT,
alanine aminotransferase; A:G ratio, albumin:globulin ratio

Evaluation of blood smears showed a size increase in erythrocytes for fish anesthetized with
benzocaine when compared to fish anesthetized with eugenol and morphological alterations
such as macrocytes, anisocytosis, and equinocytes in fish anesthetized with benzocaine.
The interaction effect between anesthetic and anticoagulants was significant (P < 0.05) for
leukocytes, lymphocytes, thrombocytes, and hematocrit when used with sodium citrate,
regardless of the anesthetic used (Table 3). Moreover, a significant interaction effect between
anesthetics and hematocrit was observed when used with sodium heparin. Finally, the results
showed no interaction effects between anesthetic and anticoagulant for any parameters when
used with EDTA.

Erythrocyte osmotic fragility

Erythrocyte osmotic fragility (Fig. 2) revealed that total hemolysis occurred at 0.3% of NaCl-
PO4 in all groups, independently of the anesthetic and anticoagulants. Also, there were no
significant differences (P > 0.05) in total hemolysis percentages between the three anticoag-
ulants at different concentrations of NaCl-PO4 in fish anesthetized with benzocaine. On the
other hand, fish anesthetized with eugenol by using sodium heparin showed a significantly
higher susceptibility to hemolysis (P < 0.05) when compared to the samples with EDTA at
0.7% and 0.9% of NaCl-PO4. Finally, there were no significant differences between benzo-
caine and eugenol at different concentrations of NaCl-PO4.

Discussion

Benzocaine and eugenol showed to be effective and clinically safe when used in
tambaquis, which was evidenced by mortality absence and slight alterations in blood
parameters and few variations in water quality parameters. Similar to other studies, the
fish showed a hyperactivity reaction on contact with anesthetics, verified by a rapid
movement in the aquariums, which was decreasing after anesthetic action (Grush et al.
2004; Vidal et al. 2006; Vidal et al. 2007). Both anesthetics stopped the reaction caused by
handling and all fish reached the stage V of anesthesia, similar to the reported in Nile
tilapia Oreochromis niloticus (Cosenza et al. 2014), lambari Astyanax altiparanae (Gimbo
 Aquaculture International

Table 3 Hematologial parameters (mean ± standard deviation) of tambaqui anesthetized with benzocaine and eugenol using different anticoagulants

Parameters EDTA Sodium citrate Sodium heparin

Eugenol Benzocaine Eugenol Benzocaine Eugenol Benzocaine

WBCs (x103μL-1) 22.3 ± 9.69 Aa 14.5 ± 6.42 Aa 20.0 ± 8.29 Aa* 11.4 ± 3.85 Bb* 28.6 ± 12.23 Aa 27.4 ± 10.53 Aa
Neutrophils (x103μL-1) 4.5 ± 1.79 Aa 3.0 ± 1.9 Aa 2.5 ± 1.58 Aa 3.5 ± 1.9 Aa 6.5 ± 3.42 Aa 4.9 ± 2.68 Aa
Lymphocytes (x103μL-1) 12.1 ± 5.69 Aa 8.1 ± 4.2 Aa 16.0 ± 8.13 Aa* 5.8 ± 3.54 Ab* 15.2 ± 5.64 Aa 17.2 ± 7.32 Aa
Monocytes (x103μL-1) 5.7 ± 2.06 Aa 3.4 ± 1.75 Aa 1.5 ± 3.45 Aa 2.1 ± 1.17 Aa 6.9 ± 3.09 Aa 5.3 ± 2.17 Aa
Thrombocytes (x103μL-1) 9.8 ± 4.37 Aa 12.1 ± 8.54 Aa 1.8 ± 2.25 Aa* 3.5 ± 1.72 Bb* 4.8 ± 4.29 Aa 5.3 ± 2.38 Ba
Erythrocytes (x10 6μL-1) 8.2 ± 3.31 Aa 6.8 ± 2.19 Aa 7.5 ± 2.25 Aa 4.8 ± 2.68 Bb 9.1 ± 3.16 Aa 6.8 ± 3.08 Aa
Hematocrit (%) 32.2 ± 2.18 ABb 39.0 ± 4.85 Aa 31.1 ± 2.34 Ba* 28.0 ± 2.22 Bb* 35.2 ± 1.66 Ab* 39.1 ± 3.29 Aa*
Hemoglobin (g dL-1) 7.5 ± 0.7 Ab 8.1 ± 0.71 Aa 5.9 ± 0.6 Ba 7.1 ± 1.75 Aa 7.9 ± 1.6 Ab 9.0 ± 2.32 Aa
MCV (fL) 46.7 ± 22.60 Ab 63.4 ± 22.95 Aa 41.1 ± 13.52 Ab 89.6 ± 54.39 Aa 44.6 ± 17.79 Ab 82.6 ± 64.96 Aa
MCH (g dL-1) 10.7 ± 4.27 Aa 13.4 ± 5.00 Aa 8.7 ± 2.94 Aa 21.0 ± 13.34 Aa 9.9 ± 3.60 Aa 20.9 ± 19.68 Aa
MCHC (%) 23.5 ± 2.93 Aa 21.2 ± 3.11 Aa 21.4 ± 1.72 Aa 22.5 ± 4.9 Aa 22.6 ± 3.87 Aa 22.9 ± 5.17 Aa

Different uppercase letters indicate significant differences between anticoagulants and lowercase letters indicate significant differences between anesthetics (P<0.05)
*Indicate P <0.05 for interaction anesthetic-anticoagulant
WBCs, total leukocytes; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration
 Aquaculture International

a 120 b 120 EDTA

A A A A A A A A A A A A Sodium citrate
A A A AB B A
Sodium heparin
90 90

Hemolysis (%)
Hemolysis (%)

A
A A
60 60
A B
A B A
A A B AB
30 30

0 0
0.1 0.3 0.5 0.7 0.9 0.1 0.3 0.5 0.7 0.9
NaCl-PO4 concentration (%) NaCl-PO4 concentration (%)

Fig. 2 Benzocaine (a), eugenol (b). Median values of osmotic fragility using different anticoagulants and
anesthetized with benzocaine or eugenol. Different uppercase letters in columns of NaCl-PO4 concentrations
indicate significantly different values (P<0.5) between anticoagulants

et al. 2008), Atlantic salmon Salmo salar (Iversen et al. 2003), snook Centropomus
parallelus (Souza et al. 2018), and tambaqui (Pádua et al. 2013).
However, tambaquis exhibited a more rapid anesthesia induction with eugenol. On the
contrary, the recovery stage was faster with benzocaine. The shorter effect of benzocaine can
be explained due to the action mechanism which blocks calcium channels and reduction of
membrane potential (Holloway et al. 2004), with a lower effect on residual metabolism in fish
and faster recovery (Cosenza et al. 2014). The shorter times of recovery also were verified in
other teleost species (Souza et al. 2018; Cosenza et al. 2014), evidencing that there is no
relation with a particular species. A longer period of anesthesia can be favorable for handling
activities in aquaculture (Prince and Powell 2000; Souza et al. 2018; Pádua et al. 2013).
The use of anesthetics is taking into account some considerations such as price, availability,
product efficiency, and necessity. According to the results, eugenol shows a better cost-benefit
relationship, because of its price, availability, and similar action in tambaquis when compared
to benzocaine (Vidal et al. 2008; Souza et al. 2018). Furthermore, anesthetics derived from
essential oils demonstrated advantages when used as sedatives or anesthetics compared to
synthetic compounds due to their ability to be non-aversive to fish (Souza et al. 2019).
However, some studies showed that some essential oils should be carefully evaluated and
used with caution because of some adverse effects (Aydın and Barbas 2020). These data
evidence the need of regulation for anesthetics in Brazilian aquaculture.
Fish anesthetized with benzocaine showed an increase of alterations in hematological
parameters, which could be related to the longer induction period of this anesthetic when
compared to those anesthetized with eugenol. However, biochemical parameters did not show
significant differences, including glucose values, between the two anesthetics, which suggests
that these alterations seen in other farmed fish species (Claudiano et al. 2019; Costa et al. 2020)
could be due to physiological alterations caused by stress (Fazio 2019).
The lower thrombocyte counts by using sodium citrate and sodium heparin and the lower
leukocyte counts by using sodium citrate in fish anesthetized with benzocaine are opposite to
the studies in Nile tilapia (Delbon and Paiva 2012) and roach Rutilus rutilus (Sudagara et al.
2000) anesthetized with benzocaine which showed no alterations in leukocytes and
thrombocytes counts. In addition, Pádua et al. (2013) reported an increase of thrombocytes
and leukocytes in tambaquis with the same anesthetic. The opposite results could be due to
different methods used for thrombocytes and leukocyte counts. Some studies in fish showed a
higher accuracy in total leucocyte counts by using the indirect method (Tavares-Dias et al.
Aquaculture International

2008; Ishikawa et al. 2010). On the other hand, the direct method could lead to identification
errors between leukocytes and thrombocytes in the hemocytometer (Tavares-Dias et al. 2008).
In addition, lower erythrocyte and hematocrit values in fish anesthetized with benzocaine
could be attributed to hemolysis due to its pharmacodynamics, which blocks sodium channels
and reduces the membrane potential (Holloway et al. 2004). Furthermore, the sodium citrate
could bind free calcium forming insoluble salts and affecting its biological availability (Baynes
and Dominiczark 2005), which may have led to a decrease of erythrocyte and thrombocytes.
The higher MCV values in fish anesthetized with benzocaine by using the three anticoagulants
showed that benzocaine caused hematological alterations in tambaquis. Similar results were
reported in Oreochromis aureus (Allen 1994), Oreochromis mossambicus (Nussey et al.
1995), and O. niloticus (Bittencourt et al. 2003).
During the evaluation of blood smears, the erythrocytes in fish anesthetized with benzo-
caine showed some alterations such as macrocytes, anisocytosis, and equinocytes, which could
indicate osmotic stress or hypoxemia (Nussey et al. 1995).
The absence of a significant interaction effect between anesthetic and anticoagulant when
used with EDTA suggests that its use shows stable results in relation to hematological
parameters when compared to the other anticoagulants tested.
Erythrocyte osmotic fragility is a test which evaluates the resistance of erythrocytes to
osmotic stress (Sarkar et al. 1999). The differences in erythrocyte osmotic fragility between
anticoagulants in fish anesthetized with eugenol could be related to alterations in permeabil-
ity, which was similar to other studies in matrinxã Brycon cephalus and tambaquis (Inoue et al.
2005; Tavares-Dias and Sandrin 1998).
The EDTA showed a significantly lower hemolysis in fish anesthetized with eugenol when
compared to the other anticoagulants. These results are opposite from those found in other
species using the same anticoagulant (Van Vliet et al. 1985; Walencik and Witeska 2007).
Also, calcium chelation causes variation in erythrocyte membranes (Walencik and Witeska
2007). However, it is necessary to investigate more about the role of anticoagulants in the
increase of erythrocyte fragility taking into account fish species and intrinsic and extrinsic
factors and also some erythrocyte characteristics such as volume, size, hemoglobin quantity,
composition chemical, viscosity, and elasticity of cell membrane (Parpart et al. 1947).
The results of this study demonstrated that eugenol (100 mg L−1) is a good alternative as
anesthetic for tambaquis when compared to benzocaine (100 mg L−1) because of its rapid
anesthesia induction, few alterations in blood parameters, and the best benefit-cost for
aquaculture. In addition, the use of EDTA facilitates the erythrocyte conservation and causes
few blood alterations compared to other anticoagulants and shows no interaction effects when
used with benzocaine and eugenol in tambaquis.

Availability of data and materials Not applicable

Code availability Not applicable

Author contribution Andria Gama Sousa: Formal analysis, investigation, methodology, project administration,
writing—original draft preparation
Acácio H. B. Pacheco: Investigation, methodology, writing—review and editing
Gilson A. Siqueira-Pinto: Investigation, visualization, writing—review and editing
Gleika T. J. dos Reis: Investigation, resources, writing—review and editing
 Aquaculture International

Michelle M. S. Fugimura: Investigation, methodology, validation, writing—review and editing

Luciano J. Vaz: Investigation, methodology, validation
Paulo F. Marcusso: Conceptualization, funding acquisition, supervision, writing—review and editing
Fernando C. Ramos-Espinoza: Conceptualization, funding acquisition, supervision, writing—review and
editing
Gustavo S. Claudiano: Conceptualization, funding acquisition, supervision, validation, writing—review and
editing

Funding The authors are grateful for the support of the Federal University of Western Pará –UFOPA (10/2018
PROPPIT/UFOPA).

Declarations

Conflict of interest The authors declare no competing interests.

References

Allen P (1994) Changes in the hematological profile of the cichlid Oreochromis aureus (Steindachner) during
acute inorganic Mercury intoxication. Comp Biochem Physiol C Toxicol Pharmacol 108:117–121. https://
doi.org/10.1016/1367-8280(94)90097-3
Aydın B, Barbas LAL (2020) Sedative and anesthetic properties of essential oils and their active compounds in
fish: a review. Aquaculture 520:734999. https://doi.org/10.1016/j.aquaculture.2020.734999
Barros MM, Ranzani Paiva MJT, Pezzato LE (2009) Hematological response and growth performance of Nile
tilapia fed diets containing folic acid. Aquaculture Research 40:95–98. https://doi.org/10.1111/j.1365-2109.
2009.02175.x
Baynes JW, Dominiczark MH (2005) Bioquímica Médica. Mosby, Elsevier
Bittencourt NLR, Molinari LM, Scoaris DO, Pedroso RB, Nakamura CV, Nakamura TU, Abreu Filho BA,
Diasfilho BP (2003) Hematological and biochemical values for Nile tilapia Oreochromis niloticus cultured
in semi-intensive system. Acta Scientiarum 25:385–389
Castro KS, Sousa AG, Fugimura MMS, Vaz LJ, Marcusso PF, Claudiano GS (2019) Estabilidade das variáveis
hematológicas em sangue de Colossoma macropomum armazenado com diferentes anticoagulantes. Ragros
11:171–180. https://doi.org/10.18542/ragros.v11i2.8963
Claudiano GS, Yunis-Aguinaga J, Marinho-Neto FA, Miranda RL, Martins IM, Otani FS, Mundim AV,
Marzocchi-Machado CM, Moraes JRE, Moraes FR (2019) Hematological and immune changes in
Piaractus mesopotamicus in the sepsis induced by Aeromonas hydrophila. Fish Shellfish Immunol 88:
259–265. https://doi.org/10.1016/j.fsi.2019.01.044
Cosenza GR, Claudiano GS, Marcusso PF, Eto SF, Manrique WG, Loureiro BA, Shimada MT, Salvador R,
Moraes JRE, Moraes FR (2014) Influence of glyceryl guaiacolate ether on anesthetics in tilapia compared to
benzocaine and eugenol. Rev. MVZ Córdoba 19:3944–3953. https://doi.org/10.21897/rmvz.114
Costa LFA, Claudiano G.S, Ramos-Espinoza FC, Marinho-Neto FA, Marcusso PF, Yunis-Aguinaga J, Moraes
FR; Moraes JRE (2020). Hematological and glycemic changes in Oreochromis niloticus subjected to acute
stress by syphoning. J Vet Sc Public Health 7: 97-101. https://doi.org/10.4025/rcvsp.v7i2.46639
Delbon MCE, Paiva MJTR (2012) Eugenol em juvenis de tilápia do Nilo: concentrações e administrações
sucessivas. Bol Inst Pesca 38:43–52
Drabkin DL, Austin JH (1935) Spectrophotometric studies II. Preparations from washed blood cells; nitric oxide
hemoglobin and sulfhemoglobin. J Biol Chem 112(1):51–65
Fazio F (2019) Fish hematology analysis as an important tool of aquaculture: a review. Aquaculture 500:237–
242. https://doi.org/10.1016/j.aquaculture.2018.10.030
Gimbo RY, Saita MV, Gonçalves AFN, Takahashi LS (2008) Diferentes concentrações de benzocaína na
indução anestésica do lambari-do raboamarelo (Astyanax altiparanae). Rev Brasa Saúde Prod Anim 9(2):
350–357
Goldenfarb PB, Bowyer FP, Hall E, Brosious E (1971) Reproducibility in the hematology laboratory: the
microhematocrit determination. Am J Clin Pathol 56:35–39
Grush J, Noakes DLG, Moccia RD (2004) The efficacy of clove oil as an anesthetic for the zebrafish, Danio rerio
(Hamilton). Zebrafish 1:46–53. https://doi.org/10.1089/154585404774101671
Aquaculture International

Hasan M, Bart AN (2007) Improved survival of rohu, Labeo rohita (Hamilton-Buchanan) and silver carp,
Hypophthalmichthys molitrix (Valenciennes) fingerlings using low-dose quinaldine and benzocaine during
transport. Aquac Res 38:50–58. https://doi.org/10.1111/j.1365-2109.2006.01628.x
Hikasa Y, Takase K, Ogasawara T, Ogasawara S, Ogasawara T, Ogasawara S (1986) Anaesthesia and recovery
with tricain methansulfonate, eugenol and thiopental sodium in carp, Cyprinus carpio. Jpn J Sci 48(2):341–
351
Holloway AC, Keene JL, Noakes DG, Moccia RD (2004) Effects of clove oil and MS-222 on blood hormone
profiles in rainbow trout Oncorhynchus mykiss. Aquac Res 35:1025–1030. https://doi.org/10.1111/j.1365-
2109.2004.01108.x
Inoue L, Afonso L, Iwama G, Moraes G (2005) Effects of clove oil on the stress response of matrinxã (Brycon
cephalus) subjected to transport. Acta Amazon 35:145–151. https://doi.org/10.1590/S0044-
59672005000200018
Inoue LAKA, Boijink CL, Ribeiro PT, Silva AMD, Affonso EG (2011) Avaliação de respostas metabólicas do
tambaqui exposto ao eugenol em banhos anestésicos. Acta Amazon 41(2):327–332. https://doi.org/10.1590/
S0044-59672011000200020
Ishikawa NM, Ranzani-Paiva MJT, Lombardi JV (2008) Total leukocyte counts methods in fish, Oreochromis
niloticus. Arch Vet Sci 13(1):54–63
Ishikawa NM, Pádua SB, Satake F, Hisano H, Jerônimo GT, Martins ML (2010) Heparina e Na2 EDTA como
anticoagulantes para surubim híbrido (Pseudoplatystoma reticulatum × P. corruscan): eficácia e alterações
hematológicas. Cienc Rural 40:1557–1561. https://doi.org/10.1590/S0103-84782010005000113
Iversen M, Finstad B, Mckinley RS, Eliassen RA (2003) The efficacy of metomidate, clove oil, Aqui-STM and
Benzoak® as anaesthetics in Atlantic salmon (Salmo salar L.) smolts, and their potential stress-reducing
capacity. Aquaculture 221(1-4):549–566. https://doi.org/10.1016/S0044-8486(03)00111-X
Lătărełu A, Teusdea V, Furnaris F, Bunea R, Mitrănescu E (2013) Comparative study of the effect of different
anticoagulants on blood cell morphology in common carp (C. carpio) and rainbow trout (O. mykiss). Bull
UASVM Anim Sci Biotechnol 70(2):284–288. https://doi.org/10.15835/BUASVMCN-ASB:70:2:9473
Mafuvadze B, Erlwanger KH (2007) The effect of EDTA, Heparin and storage on the erythrocyte osmotic
fragility, plasma osmolality and hematocrit of adult ostriches (Struthio camelus). Arch Vet Sci 77:427–434
Nussey G, Van Vuren JHJ, Du Preez HH (1995) Effects of copper on the haematological and osmoregulation of
the Mozambique tilapia. Oreochromis mossambicus (Cichlidae). Comp Biochem Physiol C Toxicol
Pharmacol 111:369–380. https://doi.org/10.1016/0742-8413(95)00063-1
Okamoto MH, Tesser MB, Louzada LR, Santos RA, Sampaio LA (2009) Benzocaína e eugenol como
anestésicos para juvenis do pampo Trachinotus marginatus. Ciência Rural 39:866–870. https://doi.org/10.
1590/S0103-84782008005000100
Pádua SB, Neto JD, Sakabe R, Claudiano GS, Chaga EC, Pilarski F (2013) Variáveis hematológicas em
tambaquis anestesiados com óleo de cravo e benzocaína. Pesq Agrop Bras 48(8):1171–1174. https://doi.
org/10.1590/S0100-204X2013000800056
Palmer L, Briggs C, Mcfadden S, Zini G, Burthem J, Rozenberg G, Proytcheva M, Machin SJ (2015) ICSH
recommendations for the standardization of nomenclature and grading of peripheral blood cell morpholog-
ical features. Int Jnl Lab Hem 37:287–303. https://doi.org/10.1111/ijlh.12327
Parpart AK, Lorenz PB, Parpart ER, Gregg JR, Chase AM (1947) The osmotic resistance (fragility) of human red
cells. J Clin Invest Ann Arbor 26:636–640. https://doi.org/10.1172/JCI101847
Priborsky J, Velisek J (2018) A review of three commonly used fish anesthetics. Rev Fish Sci Aqua 26(4):417–
442. https://doi.org/10.1080/23308249.2018.1442812
Prince A, Powell C (2000) Clove oil as an anesthetic for invasive field procedures on adult rainbow trout. N Am J
Fish Manag 20(4):1029–1032. https://doi.org/10.1577/1548-8675(2000)0202.0.CO;2
Ross LG, Ross BR (2008) Anaesthetic and sedative techniques for aquatic animals. Blackwell Science, Oxford
Roubach R, Gomes LC, Leão Fonseca FA, Val AL (2005) Eugenol as an efficacious anaesthetic for tambaqui,
Colossoma macropomum (Cuvier). Aquac Res 36(11):1056–1061. https://doi.org/10.1111/j.1365-2109.
2005.01319.x
Salvador R, Claudiano GS, Loureiro BA, Marcusso PF, Eto SF, Pilarski F, Toazza CS, Moraes JRE, Moraes FR
(2013) Performance and hematological profile of Nile tilapia fed with Saccharomyces cerevisiae and
vaccinated against Streptococcus agalactiae. Pesq Agropec Bras 48(8):892–898. https://doi.org/10.1590/
S0100-204X2013000800012
Sarkar M, Barari SK, Mandal DB, Nandankar UA, Basu A, Mohanty TK, Ray S (1999) The effect of anti-
coagulants on the osmotic fragility of erythrocytes in the yak (Poephagus grunniens). Vet J 157:91–93.
https://doi.org/10.1053/tvjl.1998.0257
Sladky KK, Swanson CR, Stoskopf MK, Loomis MR, Lewbart GA (2001) Comparative efficacy of tricaine
methanesulfonate and clove oil for use as anesthetics in red pacu (Piaractus brachypomis). Amer J Vet Res
62:337–342. https://doi.org/10.2460/ajvr.2001.62.337
 Aquaculture International

Souza RAR, Carvalho CVA, Nunes FF, Scopel BR, Guarizi JD, Tsuzuki MY (2018) Comparative effect of
benzocaine, menthol and eugenol as anesthetics for juvenile fat snook. Bol Inst Pesca 38(3):247–255
Souza C, Baldissera M, Baldisserotto B, Heinzmann B, Martos-Sitcha JÁ, Mancera JM (2019) Essential oils as
stress-reducing agents for fish aquaculture: a review. Front Physiol 10:785. https://doi.org/10.3389/fphys.
2019.00785
Sudagara M, Mohammadizarejabada A, Mazandarania R, Pooralimotlagha S (2000) The efficacy of clove
powder as an anesthetic and its effects on hematological parameters on roach (Rutilus rutilus). Journal of
Aquaculture Feed Science and Nutrition 1:1–5
Tavares-Dias M, Sandrin EFS (1998) Influence of anticoagulants and blood storage on hematological values in
tambaqui, Colossoma macropomum. Acta Sci. 20:151–155. https://doi.org/10.4025/actascibiolsci.v20i0.
4465
Tavares-Dias M, Moraes FR, Imoto ME (2008) Hematological parameters in two neotropical freshwater teleost,
Leporinus macrocephalus (Anostomidae) and Prochilodus lineatus (Prochilodontidae). Biosci J 24(3):96–
101
Trinder P (1969) Determination of blood glucose using 4-amino phenazone as oxygen acceptor. J Clin Pathol
22(2):246. https://doi.org/10.1136/jcp.22.2.246-b
Van Vliet KJ, Smit GL, Pieterse JJ, Schoonbee HJ, Van Vuren JHJ (1985) The effects of generally used
anticoagulants on the hemolysis of fish erythrocytes. Water SA, Pretoria 2:87–92. https://doi.org/10.1016/j.
cbpc.2007.04.004
Vidal LVO, Albinati RCB, Albinati ACL, Mecêdo GR (2006) Utilização do eugenol como anestésico para o
manejo de juvenis de pintado (Pseudoplatystoma corruscans). Acta Scientiarum. Biological Sciences 28:
275–279. https://doi.org/10.4025/actascibiolsci.v28i3.400
Vidal LVO, Furuya WM, Graciano TS, Schamber CR, da Silva LCR, dos Santos LD, de Souza SR (2007)
Eugenol como anestésico para juvenis de matrinxã (Brycon cephalus). Rev Bras Saúde Prod Anim 8:335–
342
Vidal LVO, Albinati RCV, Albinati ACL (2008) Eugenol como anestésico para a tilápia-do-nilo. Pesq Agropec
Bras 43:1069–1074. https://doi.org/10.1590/S0100-204X2008000800017
Walencik J, Witeska M (2007) The effects of anticoagulants on hematological índices and blood cell morphology
of common carp (Cyprinus carpio L.). Comp Biochem Physiol C Toxicol Pharmacol 146:331–335. https://
doi.org/10.1016/j.cbpc.2007.04.004
Woynárovich A, Van Anrooy R (2019) Field guide to the culture of tambaqui (Colossoma macropomum, Cuvier,
1816). FAO Fisheries and Aquaculture Technical Paper N°. 624. FAO, Rome 132 p
Yunis-Aguinaga J, Fernandes DC, Eto SF, Claudiano GS, Marcusso PF, Marinho-Neto FA, Fernandes JB,
Moraes FR, Moraes JRE (2016) Dietary camu camu, Myrciaria dubia, enhances immunological response in
Nile tilapia. Fish Shellfish Immunol 58:284–291. https://doi.org/10.1016/j.fsi.2016.08.030

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Affiliations

Andria Gama Sousa 1 & Acácio H. B. Pacheco 1 & Gilson A. Siqueira-Pinto 1 & Gleika T. J.
dos Reis 2 & Michelle M. S. Fugimura 2 & Luciano J. Vaz 2 & Paulo F. Marcusso 3 & Fernando
C. Ramos-Espinoza 4 & Gustavo da Silva Claudiano 1
1
Institute of Biodiversity and Forests, Federal University of Western Pará, UFOPA, Rua Vera Paz, s/n
(Unidade Tapajós), Santarém, PA 68040-255, Brazil
2
Laboratory Multiple for the Production of Aquatic Organisms, LAMPOA, Federal University of Western
Pará, UFOPA, Pará, Brazil
3
Institute of Agricultural Sciences, Federal University of Jequitinhonha and Mucuri Valleys, Minas Gerais,
Brazil
4
Aquaculture Center of UNESP, Caunesp, Jaboticabal, São Paulo, Brazil