Content uploaded by Carlos Eduardo Copatti
Author content
All content in this area was uploaded by Carlos Eduardo Copatti on Oct 17, 2017
Content may be subject to copyright.
Essential oil of Aloysia triphylla in Nile tilapia:
anaesthesia, stress parameters and sensory
evaluation of fillets
Rafael Rivas Teixeira
1
, Renilde Cordeiro de Souza
1
, Artur Cedraz Sena
2
,
Bernardo Baldisserotto
3
, Berta Maria Heinzmann
4
, Ricardo David Couto
5
&
Carlos Eduardo Copatti
1,2
1
Programa de P
os Graduac
ß
~
ao em Zootecnia, Universidade Federal da Bahia, Salvador, BA, Brazil
2
Programa de P
os Graduac
ß
~
ao em Diversidade Animal, Universidade Federal da Bahia, Salvador, BA, Brazil
3
Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
4
Departamento de Farm
acia Industrial, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
5
Departamento de An
alises Cl
ınicas e Toxicol
ogicas, Faculdade de Farm
acia, Universidade Federal da Bahia, Salvador,
BA, Brazil
Correspondence: C E Copatti, Programa de P
os Graduac
ß
~
ao em Zootecnia, Universidade Federal da Bahia, 40170-110 Salvador,
BA, Brazil. E-mail: carloseduardocopatti@yahoo.com.br
Abstract
The optimum concentration of anaesthetic used to
facilitate fish handling can be determined based on
induction and recovery time, among other factors.
This study aimed to evaluate the effectiveness of
the essential oil of Aloysia triphylla (EOAT) as an
anaesthetic in the handling of Nile tilapia juve-
niles. Nile tilapias exemplars were immersed at 0
(control group), 10, 20, 30, 40, 80, 150, 300 and
450 lLL
1
EOAT to evaluate sedation, anaesthe-
sia and recovery time. The stress response was
evaluated by the determination of plasma glucose,
cortisol, lactate and paraoxonase levels in anaes-
thetized (300 lLL
1
EOAT) and non-anaesthe-
tized fish. Fillets from both groups were evaluated
for organoleptic characteristics (taste and odour).
Concentrations of 80–450 lLL
1
EOAT were
effective in inducing anaesthesia. There was a sig-
nificant reduction in plasma cortisol levels one
hour after handling in individuals anaesthetized
with 300 lLL
1
EOAT compared to the control
group. Tasters did not detect differences in fillet
taste and odour between fish exposed and not
exposed to EOAT. A concentration of 300 lLL
1
EOAT is recommended for handling Nile tilapia
because it presents good anaesthetic induction and
recovery times, causes a reduction in plasma corti-
sol levels one hour after handling and does not
affect the taste or odour of the fillets.
Keywords: sedation, recovery, handling, corti-
sol, organoleptic characteristics
Introduction
Aloysia triphylla (L’Herit.) Britton (Verbenaceae) is
a herbal species native to South America, also cul-
tivated in Southern Europe and North Africa
(Valent~
ao, Fernandes, Carvalho, Andrade, Seabra
& Bastos 2002; Paulus, Valmorbida, Toffoli, Nava
& Paulus 2013). It is rich in highly volatile com-
pounds that have aromatic and sedative properties
(Schwerz, Caron, Manfron, Schmidt & Elli 2015).
The commercial importance of the essential oil of
A. triphylla (EOAT) also depends on its two main
components, geranial and neral, that are directly
related to its functional properties (Tabatabaie &
Nazari 2007). The use of EOAT as an anaesthetic
has been studied in white shrimp (Litopenaeus van-
namei) (Parodi, Cunha, Heldwein, De Souza, Mar-
tins, Garcia, Junior, Monserrat, Schmidt, Caron,
Heinzmann & Baldisserotto 2012) and silver cat-
fish (Rhamdia quelen) (Parodi, Cunha, Becker, Zep-
penfeld, Martins, Koakoski, Barcellos, Heinzmann
& Baldisserotto 2014; Zeppenfeld, Toni, Becker,
Miron, Parodi, Heinzmann, Barcellos, Koakoski,
Rosa, Loro, Cunha & Baldisserotto 2014). In addi-
tion, it has been shown to be more efficient in
reducing stress providing oxidative protection than
MS-222 in silver catfish (Gressler, Riffel, Parodi,
©2016 John Wiley & Sons Ltd 3383
Aquaculture Research, 2017, 48, 3383–3392 doi:10.1111/are.13165
Saccol, Koakoski, Costa, Pavanato, Heinzmann,
Car
on, Schmidt, Llesuy, Bascellos & Baldisserotto
2014). Recently, EOAT was patented as a sedative
and anaesthetic for aquatic animals (PI0904839-1
A2).
The use of anaesthetics to reduce stress is com-
mon in fish farming and is thought to improve fish
welfare and facilitate handling (Coyle, Durborow &
Tidwell 2004; Sim~
oes & Gomes 2009) by depress-
ing their central and peripheral nervous system
activities (Summerfelt & Smith 1990). Demand for
natural anaesthetics such as EOAT as an alterna-
tive to synthetic anaesthetics has increased
because synthetic anaesthetics have side effects,
such as changes to the cardiovascular and endo-
crine systems as well as to osmoregulation and ion
regulation (Carter, Woodley & Brown 2011).
An increase in plasma cortisol levels is one of
the most frequent physiological alterations in fish
submitted to handling and can be used as an indi-
cator of the condition of individuals exposed to
stress (Baker, Gobush & Vynne 2013). The capture
and exposure of individuals to air prior to han-
dling generates stress responses that can be char-
acterized by elevated plasma levels of glucose
resulting from the release of catecholamines (Hos-
hiba, Gonc
ßalves & Urbinati 2009). Catecholamines
and cortisol are associated with high energy con-
sumption, gluconeogenesis and lipolysis, which
may influence the performance of the fish after
stress (Wendelaar Bonga 1997).
Nile tilapia (Oreochromis niloticus) is one of the
most important species of freshwater fish for global
aquaculture, primarily because of its known hardi-
ness and good adaptation to captive conditions
(Hohlenwerger, Copatti, Sena, Couto, Baldisserotto,
Heinzmann, Caron & Schimdt 2016). So this work
aimed to determine the efficiency of EOAT for the
induction of and recovery from anaesthesia in Nile
tilapia. Additionally, we evaluated the effect of
EOAT during handling stress via plasma parame-
ters (cortisol, glucose, lactate and paraoxonase)
and its influence on the organoleptic characteris-
tics of the fillets.
Materials and methods
Essential oil from Aloysia triphylla
Leaves of A. triphylla (L’H
er.) Britton cultivated in
Frederico Westphalen, Rio Grande do Sul state,
Brazil, were collected in August 2013. A voucher
specimen (SMDB No. 11169) was deposited in the
herbarium of the Department of Biology, Universi-
dade Federal de Santa Maria.
Oil extraction from fresh leaves was performed
by steam distillation (120 min) using a Clevenger
apparatus according to the British Pharmacopoeia
(2007). The essential oil was stored at 4°Cin
amber glass bottles until biological testing and
chemical analysis by CG-MS according to Parodi
et al. (2014) were performed (Table 1).
Animals
Juveniles of Nile tilapia, Gift lineage, sex-reversed
male, were purchased from Piscicultura de Bebe-
douro, Petrolina, Brazil. The fish were housed for
15 days in continuously aerated 250-L masonry
tanks with a semi-static system. The fish were fed
Table 1 Chemical constituents of Aloysia triphylla
essential oil used in the present study
N RT Compound RI Calc RI Lit %
1 10.399 a-pinene 931 933 0.36
2 12.762 Hepten-2-one
<6-methyl-5>
990 986 1.51
3 14.224 Limonene 1027 1028 2.70
4 14.362 Eucalyptol 1031 1030 3.04
5 15.070 b-Z-ocimene 1049 1048 2.52
6 17.096 Linalool 1100 1100 1.47
7 18.547 NI 1139 2.23
8 18.943 NI 1150 0.67
9 19.535 NI 1166 2.07
10 19.940 Rosefuranepoxide 1177 1177 1.37
11 20.202 NI 1184 3.87
12 21.811 Linalyl formate 1229 1216 3.99
13 22.255 Neral 1242 1238 16.12
14 22.736 Z-geraniol 1256 1256 3.47
15 23.290 Geranial 1272 1270 28.97
16 26.447 Nerolacetate 1366 1365 0.35
17 26.785 b-cubebene 1376 1386 0.32
18 27.054 geranylacetate 1385 1381 1.59
19 28.186 b-caryophyllene 1420 1418 8.50
20 29.253 a-caryophyllene 1455 1454 1.71
21 30.150 a-curcumenene 1484 1485 3.79
22 30.582 Bicyclogermacrene 1498 1500 1.19
23 31.035 b-curcumenene 1513 1516 0.91
24 31.375 a-cadinene 1525 1524 0.80
25 32.531 NI 1564 0.76
26 32.967 Spathulenol 1579 1576 1.99
27 33.138 Caryophyllene oxide 1585 1583 1.83
28 34.760 s-cadinol 1643 1642 1.87
Total Identified 90.35
RT, retention time; RI Calc, Calculated retention index; RI Lit,
Literature retention index (NIST 2008; Adams 2009); NI, not
identified.
©2016 John Wiley & Sons Ltd, Aquaculture Research,48, 3383–33923384
Anaesthesia with Aloysia triphylla in Nile tilapia R R Teixeira et al. Aquaculture Research, 2017, 48, 3383–3392
twice a day at a ratio of 5.0% body mass with a
commercial diet (320 g kg
1
crude protein and
3500 kcal digestible energy, Purina Nutripeixe SI).
During the experiments, water quality parame-
ters were maintained for temperature (24.12°C),
pH (6.22), conductivity (0.78 mS cm
1
), total
ammonia (0.25 mg L
1
N-NH
3
) and dissolved
oxygen (7.88 mg L
1
). The individuals were fasted
for a period of 24 hours prior to the experiments.
The experiments were conducted in accordance
with the rules of the Ethical Committee of the Biol-
ogy Institute (UFBA) under registration No. 19/
2012.
Anaesthetic induction and recovery
Time to anaesthesia induction and recovery was
tested in juveniles (n=80) weighing 16.05
5.14 g and measuring 9.62 1.16 cm. The
procedure involved transferring juveniles to aqua-
ria containing 2 L of water and EOAT at concen-
trations of 10, 20, 30, 40, 80, 150, 300 or
450 lLL
1
(diluted 1:10 with ethanol 99.6%).
An ethanol group was transferred to aquaria that
contained only ethanol at a concentration that
was equivalent to the dilution used for 450 lLL
1
EOAT. Control fish were submitted to the same
handling process using water only. Eight juveniles
were used for each tested concentration, and each
juvenile was used only once. The induction time
to different stages of anaesthesia and recovery was
evaluated for up to 30 min. The juveniles were
transferred to an anaesthetic-free aquarium (20 L)
to measure the anaesthesia recovery time. Fish
were considered recovered when they presented
swimming and other behaviour similar to that
seen in the maintenance tanks (adapted from
Small 2003).
Handling and biometry
Juvenile fish (n=48) weighing 107.31
18.70 g and measuring 18.52 1.25 cm were
anaesthetized with 300 lLL
1
of EOAT first
diluted in ethanol 99.6% (1:10) (the lowest con-
centration to induce anaesthesia within 2–3 min
exposure, see results) in a 5-L aquarium (one fish
at a time). Afterwards, fish were handled for bio-
metric measurements and thus exposed to air for
one minute. Following biometry, blood was col-
lected from the caudal vein of eight juveniles (at
time zero). The remaining anaesthetized juveniles
were placed in two 250-L tanks. Blood samples
were collected from eight fish from the first tank
one hour after anaesthesia and from another
eight fish from the second tank four hours after
anaesthesia. The control group was subjected to
the same procedures as the test group, but with-
out the addition of EOAT (adapted from Cunha,
Barros et al. 2010; Cunha, Zeppenfeld et al.
2010; Hohlenwerger et al. 2016).
Biochemical determination of stress parameters
Blood samples were collected using heparinized
syringes (1 mL), transferred to 2-mL plastic tubes
and centrifuged at 6°C at 3000 g(15 min) to sep-
arate the plasma. The samples were stored under
constant refrigeration (18°C). The plasma sam-
ples were sent to the Laboratory of Clinical Bio-
chemistry at the Faculty of Pharmacy (UFBA) for
measurement of the cortisol, glucose, lactate and
paraoxonase levels.
The cortisol S kit was used for the determination
of cortisol in the plasma aliquots in the mini-
VIDAS
â
equipment. This is an automated test that
is used for the quantitative determination of corti-
sol in plasma using an enzyme-linked fluorescent
assay. The measurement values of the Vidas corti-
sol S kit range from 2 to 650 ng mL
1
. The ana-
lytical detection limit, defined as the smallest
concentration of serum cortisol which is signifi-
cantly different from zero concentration with a
probability of 95%, is 2 ng mL
1
. The repeatabil-
ity, inter-run reproducibility and the inter-lot
reproducibility all were calculated by using the
recommendations of NCCLS document EP5-A2.
The observed values of total precision, dependent
from serum concentration, range from 7.42 to
12.98 coefficient of variance (%). The coefficient of
variation for the fish ranged from 8.68% to
13.59%, and the detection limit of the assay was
2ngmL
1
.
Plasma glucose levels were determined enzymat-
ically using glucose oxidase (GOD)/glucose peroxi-
dase (POD). This is a colorimetric method used in
a fully automated BT 3000 instrument (500 tests/
hour; Wiener Lab, Rosario, Argentina). Lactate
analysis was performed using the same instrument
(Lactate, Wiener lab
â
).
Paraoxonase activity was assayed by measuring
the p-nitrophenol reaction product according to the
method described by Senti, Tomas, Fito, Weinbren-
ner, Covas, Sala, Masi
a and Marruga (2003). The
©2016 John Wiley & Sons Ltd, Aquaculture Research,48, 3383–3392 3385
Aquaculture Research, 2017, 48, 3383–3392 Anaesthesia with Aloysia triphylla in Nile tilapia R R Teixeira et al.
result was achieved by multiplying the mean absor-
bance variation by the factor (factor =total reac-
tion volume (TRV) (mL)/e405 9sample value (SV)
(mL) 9cuvette thickness (cm), in which e405–
1805 mL
1
cm
1
). Therefore, paraoxonase activ-
ity =factor 9Dabs/minute.
Sensory evaluation
After blood collection, the animals of the time zero
group were killed by decapitation and the fillets
were prepared. The taste and odour were com-
pared between the control group and fish exposed
to 300 lLL
1
EOAT (see the previous section
‘Handling and biometry’).
The sensory evaluation adapted from Costell
(2002) was used. The fillets were cooked in a
microwave oven (portions of 25 g for 1 min) and
evaluated by 32 untrained judges in a blinded
comparison. Samples were coded by random
numbers. The degree of taste and odour difference
from the control was measured using a seven-
point scale, in which 1 =substantially better
than control, 2 =moderately better than control,
3=slightly better than control, 4 =not different
from control, 5 =slightly worse than control,
6=moderately worse than control and 7 =sub-
stantially worse than control.
Statistical analyses
All data are expressed as the mean SEM. All
data were subjected to the Levene test to verify the
homogeneity of the variances. The normality of
the data was assessed by Shapiro–Wilk test. Evalu-
ation of the anaesthetic activity was performed by
regression analysis (concentration 9time of seda-
tion; concentration 9time of anaesthesia; concen-
tration 9time of recovery from anaesthesia) using
SigmaPlot (version 8.0, Systat Software, San Jose,
CA, USA). In the event of no significant regression,
Kruskal–Wallis analysis was performed followed by
multiple comparisons of mean ranks for all groups.
The plasma cortisol, lactate, glucose and paraox-
onase levels were analysed using a two-way ANOVA
(time X concentration) followed by Tukey post hoc
tests. The sensory analyses were performed using
a one-way ANOVA followed by Tukey post hoc tests.
Statistica software (version 7.0) was used for the
analyses, and significance was set at a critical level
of 95% (P<0.05).
Results
Chemical composition of essential oil of Aloysia
triphylla
Geranial (28.97%) and neral (16.12%) were the
major chemical constituents of the EOAT
(Table 1).
Anaesthetic induction and recovery
Nile tilapia juveniles exposed to EOAT recovered,
and no mortality was observed during the experi-
ments. The application of 4050 lLL
1
of ethanol
alone (the concentration equivalent to the dilution
used for the 450 lLL
1
EOAT treatment) did not
induce anaesthesia or sedation.
Essential oil of A. triphylla caused sedation at all
of the tested concentrations (10–450 lLL
1
). No
relationship was observed between the EOAT con-
centration and the duration of sedation in Nile
tilapia juveniles, and there was no significant dif-
ference between the EOAT concentrations. Fur-
thermore, for concentrations at which the fish
only reached sedation (10–40 lLL
1
of EOAT),
the recovery time was <45 s (Table 2).
Anaesthesia occurred at concentrations above
80 lLL
1
EOAT, with 300 and 450 lLL
1
showing the shortest time for anaesthetic induc-
tion (170 and 163 s, respectively), where
300 lLL
1
was the minimum effective concentra-
tion. The recovery time was between 149 and
197 s. Our results showed that higher concentra-
tions resulted in a shorter time being required for
anaesthesia and a longer time being required for
anaesthetic recovery (Table 2).
Stress parameters
Plasma cortisol levels were significantly higher in
the control group one hour after biometric mea-
surements and in the anesthetized fish at four
hours after biometric measurements (P<0.05).
Nile tilapias anaesthetized with 300 lLL
1
EOAT
had significantly lower plasma cortisol levels than
the control group one hour after biometric mea-
surements (P<0.05) (Fig. 1a).
Anaesthetized Nile tilapias had significantly
higher plasma glucose levels than the control
group one hour after handling, whereas glucose
levels did not differ between anaesthetized and
control fish. However, at zero and four hours
©2016 John Wiley & Sons Ltd, Aquaculture Research,48, 3383–33923386
Anaesthesia with Aloysia triphylla in Nile tilapia R R Teixeira et al. Aquaculture Research, 2017, 48, 3383–3392
after biometric measurements, anaesthetized fish
showed significantly higher glucose levels than
control fish (P<0.05) (Fig. 1b). The paraoxonase
activity was similar in all groups at all of the
time points (P<0.05) (Fig. 1c). At time zero,
anaesthetized individuals had significantly higher
Table 2 Time required for sedation, anaesthesia and recovery in Nile tilapia with the essential oil of Aloysia triphylla
(lLL
1
) (EOAT). The time taken to reach each stage is shown in seconds (s). Data are expressed as mean SEM
(n=8)
Concentration (lLL
1
) Sedation (s) Anaesthesia (s) Recovery (s)
10 45.42 12.11
a
–<45
20 36.00 9.26
a
–<45
30 55.33 4.51
a
–<45
40 36.50 9.69
a
–<45
80 41.00 7.46
a
511.86 102.63 149.67 15.66
150 44.00 4.45
a
328.00 52.68 159.25 24.45
300 39.75 5.23
a
170.37 21.15 186.14 15.45
450 30.50 2.71
a
163.00 9.19 197.50 15.84
Equations *y=512.79 0.90x;
r
2
=0.80
y=140.29 +0.13x;
r
2
=0.97
Different letters in the columns indicate significant differences between concentrations according to the Kruskal–Wallis test. y =time
for anaesthesia induction (s) and x =EOAT concentration (lLL
1
).
*No regression.
Figure 1 Effect of the essential oil of Aloysia triphylla (EOAT) (300 lLL
1
) on plasma cortisol (a), glucose (b),
paraoxonase (c) and lactate (d) in Nile tilapia after 0, 1 and 4 h of exposure to handling (1 min). Data are
expressed as mean SEM (n=8). Different capital letters indicate significant differences at the same time point
between the control group and anaesthetized fish. Different lower case letters indicate significant differences between
different time points with the same treatment (with or without anaesthetic) (P<0.05).
©2016 John Wiley & Sons Ltd, Aquaculture Research,48, 3383–3392 3387
Aquaculture Research, 2017, 48, 3383–3392 Anaesthesia with Aloysia triphylla in Nile tilapia R R Teixeira et al.
plasma lactate levels than the control group and
the same situation occurred in anaesthetized fish
at one and four hours after biometric measure-
ments (P<0.05) (Fig. 1d).
Sensory evaluation
The fish fillets prepared from fish anaesthetized
with 300 lLL
1
EOAT had sensory scores for
taste (3.70 0.30) and odour (4.20 0.35) sim-
ilar to the scores obtained for fillets made with the
control fish (4.09 0.32 and 4.38 0.37,
respectively) (P>0.05).
Discussion
Chemical composition of essential oil of Aloysia
triphylla
The functional properties of essential oils are often
assigned to their major constituents (Benovit,
Silva, Salbego, Loro, Mallmann, Baldisserotto,
Flores & Heinzmann 2015). Citral is a monoter-
pene formed by mixing isomers of geranial and
neral (Nunes, Veloso, Pereira & Andrade 2005),
which were the two most abundant components
of the EOAT in this study. Geranial and neral have
also been reported as being the most abundant
components of EOAT in other studies with anaes-
thesia of white shrimp and silver catfish, respec-
tively (Parodi et al. 2012; Daniel, Veeck, Klein,
Ferreira, Cunha, Parodi, Zeppenfeld, Schmidt,
Caron, Heinzmann, Baldisserotto & Emanuelli
2014), demonstrating their potential for use in
aquaculture.
These two isomers are also abundant in other
essential oils, such as those obtained from Cymbo-
pogon citratus (Bassol
e, Lamien Meda, Bayala,
Obame, Ilboudo & Franz 2011) (48.1% geranial
and 34.6% neral) and Lippia alba (Silva, Oliveira,
Costa, Bizzo & Oliveira 2006) (39.9–46.9% gera-
nial and 30.7–32.9% neral). Although the propor-
tions of geranial and neral found by other authors
describing the sedative and anaesthetic properties
of EOAT were different to those in the present
study, the EOAT used in this work was as effective
as that used in previous studies. The intensity of
its action cannot be directly compared, because it
depends not only on the ratio between these two
isomers, but may also be influenced by the minor
components of EOAT (Benovit et al. 2015). In
addition, its effectiveness may vary according to
the fish species (King, Hooper, Hillsgrove, Benton
& Berlinsky 2005).
Anaesthetic induction and recovery
The absence of anaesthesia caused by ethanol
alone (control group) has been reported in previ-
ous studies in gilthead seabream (Sparus aurata)
(Ortu~
no, Esteban & Meseguer 2002), silver catfish
(Cunha, Barros et al. 2010) and Nile tilapia
(Hohlenwerger et al. 2016).
The time taken to achieve sedation can vary
depending on the concentration of the anaesthetic,
but this did not occur in the present study. Nile
tilapia seems to stand for a few seconds on seda-
tion, regardless of the concentration tested. How-
ever, because high concentrations cause
anaesthesia in Nile tilapia, in situations of pro-
longed exposure to EOAT (>30 min), concentra-
tions above 80 lLL
1
EOAT should be avoided.
On the other hand, an increased concentration of
EOAT (20–800 lLL
1
) reduced the time taken for
sedation in silver catfish (Parodi et al. 2014). The
same relationship was observed by Hohlenwerger
et al. (2016) with an essential oil of L. alba (10–
500 lLL
1
) in Nile tilapia. In addition, EOAT
(40 lLL
1
) used during transport of silver catfish
improved animal welfare (Parodi et al. 2014; Zep-
penfeld et al. 2014).
Similar to our results, Parodi et al. (2014) found
that increased EOAT concentration (100–
800 lLL
1
) reduced the time to anaesthesia and
increased anaesthetic recovery time in silver cat-
fish. Essential oils are mixtures of lipophilic compo-
nents and have a high affinity for fatty tissue
(Zahl, Kiessling, Samuelsen & Olsen 2010), which
could explain their longer recovery times (Gressler
et al. 2014), because this affinity could slow elimi-
nation via the gills (Hunn & Allen 1974). Addi-
tionally, Delbon and Ranzani Paiva (2012) and
Hohlenwerger et al. (2016) using eugenol
(40–120 mg L
1
) and essential oil of L. alba (200–
500 lLL
1
), respectively, also found that increas-
ing concentrations reduce the time taken for
induction of anaesthesia in Nile tilapia.
An ideal anaesthetic should induce anaesthesia
in a period of 60–180 s and anaesthetic recovery
time should be <300 s (Marking & Meyer 1985)
and cause minimal hyperactivity to the animal
(Bagheri & Imanpoor 2011). Our results showed
that 300 and 450 lLL
1
EOAT are within this
range, with 300 lLL
1
the recommended
©2016 John Wiley & Sons Ltd, Aquaculture Research,48, 3383–33923388
Anaesthesia with Aloysia triphylla in Nile tilapia R R Teixeira et al. Aquaculture Research, 2017, 48, 3383–3392
concentration, because it is the minimum effective
concentration and would avoid essential oil
wastage and unnecessary expense.
Stress parameters
In previous studies, plasma cortisol levels in non-
stressed Nile tilapia ranged from 16.43 to
65.67 ng mL
1
(Volpato & Barreto 2001; Biswas,
Maita, Yoshizaki & Takeuchi 2004; Barreto & Vol-
pato 2006), similar to our results with EOAT. Cor-
tisol is widely used as both a long- and short-term
primary stress indicator (Maricchiolo & Genovese
2011). Plasma cortisol levels in Nile tilapia reach
a peak one hour after stress and may remain ele-
vated for up to four hours after handling (Barcel-
los, Nicolaiewsky, De Souza & Lulhier 1999).
Similar with the results of our study, EOAT in sil-
ver catfish (135 and 180 mg L
1
) (Gressler et al.
2014) and essential oil of L. alba in silver catfish
(300 mg L
1
; Cunha, Barros et al. 2010) and Nile
tilapia (500 lLL
1
) (Hohlenwerger et al. 2016)
reduced plasma cortisol levels after handling.
The reduction in cortisol level can be related to
blockage of sensory information transmission to
the hypothalamus, thereby inhibiting a series of
hormonal changes that characterize stress
(Iversen, Finstad, Mckinley & Eliassen 2003).
These physiological defence responses are stimu-
lated in a cascade system and are primarily related
to the release of cortisol and catecholamines,
which influence the energy supply mechanisms
required by the individual in response to a stressor
(Perry & Capaldo 2011).
The rise in glucose levels is not necessarily
related to elevated cortisol, although cortisol can
lead to hyperglycaemia due to glucose synthesis in
the liver (gluconeogenesis) (Wendelaar Bonga
1997; Perry & Capaldo 2011). The hypergly-
caemia may be associated with an increase in fish
activity (swimming) and/or hypoxia after of expo-
sure to anaesthesia (Hohlenwerger et al. 2016).
An increased plasma glucose level seems to be
common in anaesthetized fish. Deriggi Pisani,
Inoue and Moraes (2006), Sim~
oes and Gomes
(2009) and Hohlenwerger et al. (2016) verified
that anaesthesia with eugenol (20–80 mg L
1
),
menthol (250 mg L
1
) and essential oil of L. alba
(500 lLL
1
), respectively, also promoted hyper-
glycaemic effects in Nile tilapia. Additionally,
Sanches, Rodrigues, Nunes, Oliveira, Fantini and
Campos (2014) proposed that plasma glucose
levels in fish are higher at the start of anaesthesia
but tend to gradually return to normal levels,
regardless of the substance and concentration
used. This was verified in our study.
The higher levels of lactate immediately after
handling in the anaesthetized fish possibly reflected
the significantly higher levels of glucose one hour
after handling stress, because lactate can be con-
verted to glucose via gluconeogenesis (Silveira,
Logato & Pontes 2009). Additionally, the use of
anaesthetic can reduce the respiratory capacity of
the fish, resulting in elevated plasma lactate levels
via the anaerobic route (Iversen et al. 2003). This
may have occurred in this study after exposure to
EOAT. Similarly, increased plasma lactate levels
were reported in silver catfish anaesthetized with
essential oil of Hesperozygis ringens (30 and
50 lLL
1
) (Toni, Martos Sitcha, Ruiz Jarabo,
Mancera, Mart
ınez Rodr
ıguez, Pinheiro, Heinz-
mann & Baldisserotto 2015) and in tambaqui
(Colossoma macropomum) anaesthetized with euge-
nol (60 lLL
1
) (Inoue, Boijink, Ribeiro, Silva &
Affonso 2011).
Paraoxonase is important for the protection of
the organism against damage to the immune sys-
tem and oxidative stress (Correia & Perry 2010;
Ming, Xie, Xu, Ge, Liu & Ye 2012). However, we
did not verify protective mechanisms related to
plasma paraoxonase in our study.
Sensory evaluation
The choice of anaesthetic should take into account
its influence on fish taste and odour and consumer
acceptance (Ribas, Flos, Reig, Mackenzie, Barton &
Tort 2007). Daniel et al. (2014) demonstrated that
the use of EOAT (40 lLL
1
) in the transport of
silver catfish before slaughter extended the fresh-
ness and increased the life of the fillet during cold
storage, demonstrating the potential of EOAT in
maintaining the quality of the fillet. Our results
showed that EOAT did not influence taste or odour
in fillet. This was also observed for the essential oil
of L. alba (300 mg L
1
) in silver catfish (Cunha,
Barros et al. 2010) and Nile tilapia (Hohlenwerger
et al. 2016), but not for eugenol (50 lLL
1
)in
silver catfish (Cunha, Zeppenfeld et al. 2010).
Conclusions
In conclusion, EOAT showed anaesthetic proper-
ties, which contributes to fish welfare. Nile tilapia
©2016 John Wiley & Sons Ltd, Aquaculture Research,48, 3383–3392 3389
Aquaculture Research, 2017, 48, 3383–3392 Anaesthesia with Aloysia triphylla in Nile tilapia R R Teixeira et al.
can reach anaesthesia with EOAT in the
80–450 lLL
1
range without mortality. Addition-
ally, 300 lLL
1
EOAT had ideal induction time
(2–3 min) and was considered a promising anaes-
thetic because it reduces plasma cortisol levels and
does not affect the organoleptic characteristics of
the fillets.
Acknowledgments
This work was supported by CAPES (Coordenac
ß
~
ao
de Aperfeic
ßoamento de Pessoal de N
ıvel Superior,
Brazil) through a research grant awarded to the
first author. The authors also thank CODEVASF
for donating Nile tilapia exemplars.
References
Adams R.P. (2009) Identification of Essential oil Compo-
nents by gas Chromatography/Mass Spectrometry (4th
edn), 804 pp. Allured Publishing Corporation, Carol
Stream, IL, USA.
Bagheri T. & Imanpoor M.R. (2011) The efficacy, physi-
ology responses and hematology of Persian sturgeon
(Acipenser persicus) to clove oil as an anesthetic agent.
Turkish Journal of Fisheries and Aquatic Sciences 11,
477–483.
Baker M.R., Gobush K.S. & Vynne C.H. (2013) Review of
factors influencing stress hormones in fish and wildlife.
Journal for Nature Conservation 21, 309–318.
Barcellos L.J.G., Nicolaiewsky S., De Souza S.M.G. & Lul-
hier F. (1999) Plasmatic levels of cortisol in the
response to acute stress in Nile tilapia, Oreochromis
niloticus (L.), previously exposed to chronic stress.
Aquaculture Research 30, 437–444.
Barreto R.E. & Volpato G.L. (2006) Stress responses of
the fish Nile tilapia subjected to electroshock and social
stressors. Brazilian Journal of Medical and Biological
Research 39, 1605–1612.
Bassol
e I.H.N., Lamien Meda A., Bayala B., Obame L.C.,
Ilboudo A.J. & Franz C. (2011) Chemical composition
and antimicrobial activity of Cymbopogon citratus and
Cymbopogon giganteus essential oils alone and in combi-
nation. Phytomedicine 18, 1070–1074.
Benovit S.C., Silva L.L., Salbego J., Loro V.L., Mallmann
C.A., Baldisserotto B., Flores E.M.M. & Heinzmann
B.M. (2015) Anesthetic activity and bio-guided frac-
tionation of the essential oil of Aloysia gratissima in sil-
ver catfish Rhamdia quelen.Anais da Academia Brasileira
de Ci^
encias 87, 1675–1689.
Biswas A., Maita M., Yoshizaki G. & Takeuchi T. (2004)
Physiological responses in Nile tilapia exposed to differ-
ent photoperiod regimes. Journal of Fish Biology 65,
811–821.
British Pharmacopoeia Commission (2007) British Phar-
macopoeia (5th edn). The Stationery Office, London,
UK.
Carter K.M., Woodley C.M. & Brown R.S. (2011) A
review of tricaine methanesulfonate for anesthesia of
fish. Reviews Fish Biology Fisheries 21,51–59.
Correia J.D. & Perry I.D.S. (2010) Modulac
ß
~
ao diet
etica da
atividade da paraoxonase: revis~
ao de estudos em
humanos. Revista Hospital das Cl
ınicas de Porto Alegre
30, 271–278.
Costell E. (2002) A comparison of sensory methods in qual-
ity control. Food Quality and Preference 13,341–353.
Coyle S.D., Durborow R.M. & Tidwell J.H. (2004) Anaes-
thetics in Aquaculture, 6 pp. SRAC Publication 3900,
Stoneville, MS, USA.
Cunha M.A., Barros F.M.C., Garcia L.O., Veeck A.P.L.,
Heinzmann B.M., Loro V.L., Emanuelli T. & Baldis-
serotto B. (2010) Essential oil of Lippia alba: a new
anesthetic for silver catfish, Rhamdia quelen.Aquaculture
306, 403–406.
Cunha M.A., Zeppenfeld C.C., Garcia L.O., Loro V.L., Fon-
seca M.B., Emanuelli T., Veeck A.P.L., Copatti C.E. &
Baldisserotto B. (2010) Anesthesia of silver catfish
with eugenol: time of induction, cortisol response and
sensory analysis of fillet. Ci^
encia Rural 40, 2107–2114.
Daniel A.P., Veeck A.P.L., Klein B., Ferreira L.F., Cunha
M.A., Parodi T.V., Zeppenfeld C.C., Schmidt D., Caron
B.O., Heinzmann B.M., Baldisserotto B. & Emanuelli T.
(2014) Using the essential oil of Aloysia triphylla
(L’Her.) Britton to sedate silver catfish (Rhamdia quelen)
during transport improved the chemical and sensory
qualities of the fish during storage in ice. Journal of
Food Science 79, 1205–1211.
Delbon M.C. & Ranzani Paiva M.J.T. (2012) Eugenol em
juvenis de til
apia do Nilo: concentrac
ß
~
oes e adminis-
trac
ß
~
oes sucessivas. Boletim do Instituto de Pesca 38,
43–52.
Deriggi Pisani G.F., Inoue L.A. & Moraes G. (2006) Stress
responses to handling in Nile tilapia (Oreochromis niloti-
cus Linnaeus): assessment of eugenol as an alternative
anesthetic. Acta Scientiarum Biological Sciences 28,
269–274.
Gressler L.T., Riffel A.P.K., Parodi T.V., Saccol E.M.H.,
Koakoski G., Costa S.T., Pavanato M.A., Heinzmann
B.M., Car
on B., Schmidt D., Llesuy S.F., Bascellos L.J.G.
& Baldisserotto B. (2014) Silver catfish (Rhamdia que-
len) immersion anaesthesia with essential oil of Aloysia
triphylla (L’Herit) Britton or tricaine methanesulfonate:
effect on stress response and antioxidant status. Aqua-
culture Research 45, 1061–1072.
Hohlenwerger J.C., Copatti C.E., Sena A.C., Couto R.C.,
Baldisserotto B., Heinzmann B.M., Caron B.O. &
Schimdt D. (2016) Could the essential oil of Lippia alba
provide a readily available and cost-effective anaes-
thetic for Nile tilapia (Oreochromis niloticus)? Marine
and Freshwater Behaviour and Physiology 49, 119–126.
©2016 John Wiley & Sons Ltd, Aquaculture Research,48, 3383–33923390
Anaesthesia with Aloysia triphylla in Nile tilapia R R Teixeira et al. Aquaculture Research, 2017, 48, 3383–3392
Hoshiba M.A., Gonc
ßalves F.D. & Urbinati C. (2009) Phys-
iological stress responses of matrinx~
a(Brycon amazoni-
cus) after chasing. Acta Amazonica 39, 445–452.
Hunn J.B. & Allen J.L. (1974) Movement of drugs across
the gills of fishes. Annual Review of Pharmacology 1,
4755.
Inoue L.A.K.A., Boijink C.L., Ribeiro P.T., Silva A.M.D. &
Affonso E.G. (2011) Avaliac
ß
~
ao de respostas metab
oli-
cas do tambaqui exposto ao eugenol em banhos
anest
esicos. Acta Amazonica 41, 327–332.
Iversen M., Finstad B., Mckinley R.S. & Eliassen R.A.
(2003) The efficacy of metomidate, clove oil, Aqui-Sk
and Benzoak
â
as anaesthetics in Atlantic salmon
(Salmo salar L.) smolts, and their potential stress-redu-
cing capacity. Aquaculture 221, 549–566.
King W.V., Hooper B., Hillsgrove S., Benton C. & Berlin-
sky D.L. (2005) The use of clove oil, metomidate, tri-
caine methanesulphonate and 2-phenoxyethanol for
inducing anaesthesia and their effect on the cortisol
stress response in black sea bass (Centropristis striata
L.). Aquaculture Research 36, 1442–1449.
Maricchiolo G. & Genovese L. (2011) Some contributions
to knowledge of stress response in innovative species
with particular focus on the use of the anaesthetics.
The Open Marine Biology Journal 5,24–33.
Marking L.L. & Meyer F.P. (1985) Are better anesthetics
needed in fisheries? Fisheries 10,2–5.
Ming J., Xie J., Xu P., Ge X., Liu W. & Ye J. (2012)
Effects of emodin and vitamin C on growth perfor-
mance, biochemical parameters and two HSP70s
mRNA expression of Wuchang bream (Megalobrama
amblycephala Yih) under high temperature stress. Fish
Shellfish Immunology 32, 651–661.
Nist (2008) NIST/EPA/NIH Mass Spectral Library and
Search/Analysis Programs. J. Wiley and Sons, Hoboken,
NJ, USA.
Nunes F.M.N., Veloso M.C.C., Pereira P.A.P. & Andrade
J.B. (2005) Gas-phase ozonolysis of the monoter-
penoids (S)-(+)-carvone, (R)-()-carvone, ()-carveol,
geraniol and citral. Atmospheric Environment 39,
7715–7730.
Ortu~
no J., Esteban M.A. & Meseguer J. (2002) Effects of
four anaesthetics on the innate immune response of
gilthead seabream (Sparus aurata L.). Fish & Shellfish
Immunology 12,49–59.
Parodi T.V., Cunha M.A., Heldwein C.G., De Souza D.M.,
Martins
A.C., Garcia L.O., Junior W.W., Monserrat J.M.,
Schmidt D., Caron B.O., Heinzmann B. & Baldisserotto
B. (2012) The anesthetic efficacy of eugenol and the
essential oils of Lippia alba and Aloysia triphylla in
post-larvae and sub-adults of Litopenaeus vannamei
(Crustacea, Penaeidae). Comparative Biochemistry and
Physiology Part C: Toxicology & Pharmacology 155,
462–468.
Parodi T.V., Cunha M.A., Becker A.G., Zeppenfeld C.C.,
Martins D.I., Koakoski G., Barcellos L.G., Heinzmann
B.M. & Baldisserotto B. (2014) Anesthetic activity of
the essential oil of Aloysia triphylla and effectiveness in
reducing stress during transport of albino and gray
strains of silver catfish, Rhamdia quelen.Fish Physiology
and Biochemistry 40, 323–334.
Paulus D., Valmorbida R., Toffoli E., Nava G. & Paulus E.
(2013) Teor e composic
ß
~
ao qu
ımica do
oleo essencial e
crescimento vegetativo de Aloysia triphylla em difer-
entes espac
ßamentos e
epocas de colheita. Revista Ceres
60, 301–305.
Perry S.F. & Capaldo A. (2011) The autonomic nervous
system and chromaffin tissue: neuroendocrine regula-
tion of catecholamine secretion in non-mammalian
vertebrates. Autonomic Neuroscience: Basic and Clinical
165,54–66.
Ribas L., Flos R., Reig L., Mackenzie S., Barton B.A. &
Tort L. (2007) Comparison of methods for anaesthetiz-
ing Senegal sole (Solea senegalensis) before slaughter:
Stress responses and final product quality. Aquaculture
269, 250–258.
Sanches M.S.S., Rodrigues R.A., Nunes A.L., Oliveira
A.M.S., Fantini L.E. & Campos C.M. (2014) Effect of
menthol and eugenol on the physiological responses of
pacu Piaractus mesopotamicus.Semina Ci^
encias Agr
arias
35, 2799–2808.
Schwerz L., Caron B.O., Manfron P.A., Schmidt D. & Elli
E.F. (2015) Biomassa e teor de
oleo essencial em Aloy-
sia triphylla (l’h
erit) Britton submetida a diferentes
n
ıveis de reposic
ß
~
ao h
ıdrica e
a variac
ß
~
ao sazonal das
condic
ß
~
oes ambientais. Revista Brasileira de Plantas
Medicinais 17, 631–641.
Senti M., Tomas M., Fito M., Weinbrenner T., Covas M.I.,
Sala J., Masi
a R. & Marruga T.J. (2003) Antioxidant
paraoxonase 1 activity in the metabolic syndrome. Jour-
nal of Clinical Endocrinology e Metabolism 88, 422–5426.
Silva N.A., Oliveira F.F., Costa L.C.B., Bizzo H.R. & Oli-
veira R.A. (2006) Caracterizac
ß
~
ao qu
ımica do
oleo
essencial da erva cidreira (Lippia alba (Mill.) N. E. Br.)
cultivada em Ilh
eus na Bahia. Revista Brasileira de
Plantas Medicinais 8,52–55.
Silveira U.S., Logato P.V.R. & Pontes E.C. (2009)
Utilizac
ß
~
ao e metabolismo dos carboidratos em peixes.
Revista Eletr^
onica Nutritime 6, 817–836.
Sim~
oes L.N. & Gomes L.C. (2009) Efic
acia do mentol
como anest
esico para juvenis de til
apia-do-nilo (Ore-
ochromis niloticus). Arquivo Brasileiro de Medicina Veter-
in
aria e Zootecnia 61, 613–620.
Small B.C. (2003) Anesthetic efficacy of metomidate and
comparison of plasma cortisol responses to tricaine
methanesulfonate, quinaldine and clove oil anes-
thetized channel catfish Ictalurus punctatus.Aquaculture
218, 177–185.
Summerfelt R.C. & Smith L.S. (1990) Methods for fish
biology. In: Anesthesia, Surgery and Related Techniques
(ed. by C.B. Schreck & P.B. Moyle), pp. 213–272.
American Fisheries Society, Bethesda, MD, USA.
©2016 John Wiley & Sons Ltd, Aquaculture Research,48, 3383–3392 3391
Aquaculture Research, 2017, 48, 3383–3392 Anaesthesia with Aloysia triphylla in Nile tilapia R R Teixeira et al.
Tabatabaie S.J. & Nazari J. (2007) Influence of nutrient
concentrations and NaCl salinity on the growth, pho-
tosynthesis and essential oil content of peppermint and
lemon verbena. Turkish Journal of Agriculture and For-
estry 31, 245–253.
Toni C., Martos Sitcha J.A., Ruiz Jarabo I., Mancera J.M.,
Mart
ınez Rodr
ıguez G., Pinheiro C.G., Heinzmann B.M.
& Baldisserotto B. (2015) Stress response in silver cat-
fish (Rhamdia quelen) exposed to the essential oil of Hes-
perozygis ringens.Fish Physiology and Biochemistry 41,
129–138.
Valent~
ao P., Fernandes E., Carvalho F., Andrade P.B.,
Seabra R.M. & Bastos M.L. (2002) Studies on the
antioxidant activity of Lippia citriodora infusion: scav-
enging effect on superoxide radical, hydroxyl radical
and hypochlorous acid. Biological and Pharmaceutical
Bulletin 25, 1324–1327.
Volpato G.L. & Barreto R.E. (2001) Environmental blue
light prevents stress in the fish Nile tilapia. Brazilian Jour-
nal of Medical and Biological Research 34, 1041–1045.
Wendelaar Bonga S.E. (1997) The stress response in fish.
Physiological Reviews 77, 591–625.
Zahl I.H., Kiessling A., Samuelsen O.B. & Olsen R.E.
(2010) Anaesthesia induces stress in Atlantic salmon
(Salmo salar), Atlantic cod (Gadus morhua) and Atlantic
halibut (Hippoglossus hippoglossus). Fish Physiology and
Biochemistry 36, 719–730.
Zeppenfeld C.C., Toni C., Becker A.G., Miron D.S., Parodi
T.V., Heinzmann B.M., Barcellos L.J.G., Koakoski G.,
Rosa J.G.S., Loro V.L., Cunha M.A. & Baldisserotto B.
(2014) Physiological and biochemical responses of sil-
ver catfish, Rhamdia quelen, after transport in water
with essential oil of Aloysia triphylla (L’Herit) Britton.
Aquaculture 418–419, 101–107.
©2016 John Wiley & Sons Ltd, Aquaculture Research,48, 3383–33923392
Anaesthesia with Aloysia triphylla in Nile tilapia R R Teixeira et al. Aquaculture Research, 2017, 48, 3383–3392
