Aquaculture 456 (2016) 70–75

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Aquaculture

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Jambu, Spilanthes acmella as a novel anaesthetic for juvenile tambaqui,

Colossoma macropomum: Secondary stress responses during recovery
Luis André Luz Barbas a,c,⁎, Giovanna Rodrigues Stringhetta b, Luciano de Oliveira Garcia b,
Mario Roberto Chim Figueiredo b, Luís André Sampaio a
a
Laboratório de Piscicultura Estuarina e Marinha, Programa de Pós-graduação em Aquicultura, Universidade Federal do Rio Grande, Instituto de Oceanografia, Rio Grande, Brazil
b
Laboratório de Aquacultura Continental, Programa de Pós-graduação em Aquicultura, Universidade Federal do Rio Grande, Instituto de Oceanografia, Rio Grande, Brazil
c
Laboratório de Aquacultura de Espécies Tropicais, Instituto Federal de Educação Ciência e Tecnologia do Pará — IFPA Campus Castanhal, Pará, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this study was to evaluate the efficacy of the waxy extract of jambu flowers, Spilanthes acmella as an
Received 24 July 2015 anaesthetic for fish, using juvenile tambaqui Colossoma macropomum as a model. The times to induction and
Received in revised form 24 January 2016 recovery after short-term anaesthesia were evaluated by concentration-response trials and secondary stress
Accepted 29 January 2016
responses. Juveniles were placed in aquaria containing five different concentrations of jambu extract (5, 10, 15,
Available online 2 February 2016
20, 25 mg L−1) and the times of anaesthetic induction and recovery were determined. Sham control fish and
Keywords:
fish exposed to ethanol-added water were used as controls. The secondary stress responses of fish following an-
Sedation aesthesia with jambu extract (20 mg L−1) were investigated through an assessment of whole blood variables:
Brazil cress glucose, ions (Na+, K+, and Ca++), osmolality, haematocrit (Htc), haemoglobin (Hb), partial pressures of CO2
Plant extract (pCO2) and O2 (pO2), bicarbonate concentration (HCO− 3 ), and pH. Deep anaesthesia was observed at all concen-
Spilanthol trations tested in this study. The use of 20 mg L−1 of this extract is recommended for rapid induction (b 3 min)
Blood parameters and uneventful recovery (b5 min) from deep anaesthesia; while the concentration of 2 mg L−1 is sufficient to
promote sedation. Only transient changes in secondary stress responses were observed in tambaqui during re-
covery, with most parameters returning to initial values within 48 h post-anaesthesia. Therefore, the extract of
jambu flowers may be considered an efficient anaesthetic for tambaqui and other fish species.
Statement of relevance: This is the first study using extract of Jambu, Spilanthes acmella as an anaesthetic for
aquatic organisms.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction Alternative anaesthetics such as menthol and eugenol have been

previously tested on juvenile tambaqui (Façanha and Gomes, 2005;
Anaesthetics are important in aquaculture to reduce handling stress Roubach et al., 2005), the latter being largely used for other tropical
and mortality and have also been used for research and veterinary med- teleost species (Vidal et al., 2007, 2008; Inoue et al., 2011). Essential
icine purposes (Sneddon, 2012). In addition to the advantages of using oils of Lippia alba, Ocimum gratissimum and Aloysia triphylla have been
anaesthetics to mitigate stress and decrease mortality rates, recent stud- recently investigated for their anaesthetic properties and were present-
ies have raised awareness about the importance of using anaesthetics ed as new natural compounds for anaesthesia of aquatic organisms
for aquatic organisms from an ethical perspective. Since there is reason- (Cunha et al., 2010; Azambuja et al., 2011; Benovit et al., 2012; Parodi
able evidence that fish are capable of nociception or pain perception et al., 2012; Silva et al., 2012).
(Ashley et al., 2007; Roques et al., 2010), welfare and pain are important The genus Spilanthes is comprised of approximately 60 species wide-
aspects to be addressed. ly distributed over tropical and subtropical regions of the world, namely
Tambaqui C. macropomum is the most widely farmed native fish spe- in Africa, America and Asia (Chung et al., 2008; Tiwari et al., 2011).
cies in Brazil and in several other countries in South and Central America Spilanthes acmella var. oleracea is commonly known as jambu, toothache
(FAO, 2014). Therefore, this species has the potential to be selected as a plant and Brazil cress. This species has long been used in traditional
model to establish studies with anaesthetics for tropical species. cuisines and medicines of different civilizations (Hind and Biggs, 2003).
Previous studies have assessed S. acmella extract bioactivity as an an-
algesic and local anaesthetic in rats and mice (Ansari et al., 1988;
⁎ Corresponding author at: Laboratório de Piscicultura Estuarina e Marinha,
Universidade Federal do Rio Grande, Instituto de Oceanografia, CP 474, 96201-900, Rio
Chakraborty et al., 2002; Nomura et al., 2013). Ethanolic extract of the
Grande, Brazil. leaves was investigated and significant peripheral analgesic activity
E-mail address: [email protected] (L.A.L. Barbas). was observed in experimental animal models (Barman et al., 2009).

http://dx.doi.org/10.1016/j.aquaculture.2016.01.026
0044-8486/© 2016 Elsevier B.V. All rights reserved.
 L.A.L. Barbas et al. / Aquaculture 456 (2016) 70–75 71

Other studies report the extract also proved to possess anti-nociceptive ethanol before using in anaesthetic baths for fish. The stock solution of
activity against continuous inflammatory pain and anti-hyperalgesic ac- S. acmella extract was prepared by weighing and diluting the jambu
tivity, possibly by inhibiting prostaglandin synthesis (Ratnasooriya and flowers extract in commercial alcohol (96%) yielding a 2.24 g L−1
Pieris, 2005). The presence of N-alkylamides in jambu, namely solution which was stored in an amber glass bottle at 4 °C until its use.
“spilanthol” (N-isobutyl-2E,6Z,8E-decatrienamide) suggests that the
therapeutic effect observed is a result of its marked anaesthetic activity
2.4. Biological activity
(Nomura et al., 2013).
Frequently, the use of anaesthetics to reduce handling stress in fish is
2.4.1. Experiment 1: anaesthetic efficacy of extract of jambu, S. acmella
advantageous based on comparisons of plasma cortisol levels, glycemia,
Juvenile fish (46.6 ± 6.2 g; 14.6 ± 0.8 cm, total length) were
plasma lactate concentration, haematology, and osmolality between
transferred to aquaria containing 30 L of continuously aerated water.
anaesthetized and non-anaesthetized animals (Bressler and Ron,
Concentrations of the extract at 5, 10, 15, 20 and 25 mg L− 1 were
2004; Small, 2005; Crosby et al., 2006). Contradictorily, anaesthetics
used in this experiment. A sham control group was used and animals
themselves can also induce stress responses by potentializing catechol-
(n = 5) were transferred to aquaria with anaesthetic-free water and
amine release into the blood stream (Bressler and Ron, 2004; Zahl et al.,
observed for 30 min. A vehicle control added with the same volume of
2010; Weber, 2011). It has been reported that MS-222 caused a ‘detri-
ethanol to reach the concentration of 25 mg L− 1 of jambu extract in
mental physiological impact’ in silver catfish, Rhamdia quelen (Gressler
the water was also evaluated. In order to evaluate the cumulative time
et al., 2014).
required to reach the different stages of induction and recovery from
To date, there are no reports about sedative and/or anaesthetic effi-
anaesthesia a digital stopwatch was used. Groups of 10 juveniles were
cacy of jambu, S. acmella on fish. The aim of this study was to evaluate
used for each concentration tested and each juvenile was used only
the anaesthetic activity of jambu on juvenile tambaqui, through deter-
once, observed individually and considered a replicate. All animals
mination of time to induction and recovery by concentration-response
were starved for 24 h prior to the tests.
trials, and secondary stress responses after short-term anaesthesia.
Cumulative time to reach the different stages of anaesthesia and re-
covery were characterized according to Park et al. (2008) with modifi-
2. Materials and methods
cations as follows: agitation (A1), loss of equilibrium and erratic
swimming (A2) and absence of or minimum opercular beating with
2.1. Animals
loss of reaction to tail pinch stimulus (A3) were used as behavioural in-
dicators associated with anaesthesia induction; erratic swimming and
Tambaqui juveniles were purchased from a commercial fish farm in
recovery of equilibrium (R1), normal opercular beating and normal
Brazilian Amazon and transported to the laboratory where they were
swimming (R2) were used as markers of recovery from anaesthesia.
maintained in continuously aerated 250 L tanks, with controlled water
The maximum observation time was 30 min. After induction, juve-
parameters. Fish were acclimated for 15 days in two recirculation sys-
niles were transferred to tanks with anaesthetic-free water, and the
tems prior to the beginning of the experiments. Photoperiod was fixed
time elapsed for recovery was registered. Animals were considered to
at 12 L/12 D. Fish were fed twice a day at 2% of biomass with commercial
have recovered when they showed normal swimming behaviour.
feed (28% crude protein). Juveniles were fasted for a period of 24 h prior
After recovery, fish were grouped according to the anaesthetic concen-
to the experiments.
tration and transferred to continuously aerated 250 L tanks, where they
The experiments were approved by the Ethical and Animal Welfare
were observed for two weeks to check for mortalities.
Committee of the Federal University of Rio Grande — FURG.
In order to investigate the effectiveness of a sedation state (slight an-
aesthesia), a group of fish (n = 10) was exposed for 10 min to 2 mg L−1
2.2. Water quality
of jambu extract which corresponded to 10% of the anaesthetic concen-
tration (20 mg L−1) considered adequate to induce deep anaesthesia of
The parameters (mean ± S.D) were maintained as follows: dis-
juvenile tambaqui (see results).
solved oxygen (DO) (experiment 1: 6.45 ± 0.39 mg L−1; experiment
2: 6.82 ± 0.09 mg L−1) and temperature (experiment 1: 25.6 ±
0.7 °C; experiment 2: 25.7 ± 0.1 °C) were measured using an oxygen 2.4.2. Experiment 2: evaluation of secondary stress responses
metre (Yellow Springs Instruments, Yellow Springs, OH, USA); pH This experiment was conducted to verify stress response of fish in
(experiment 1: 7.1 ± 0.2; experiment 2: 7.05 ± 0.1) was determined recovery after short-term exposure (3 min) to S. acmella extract at the
with a Five Easy FE20, Switzerland. Total ammonia nitrogen (TAN) concentration of 20 mg L− 1. This concentration was used because it
(experiment 1: 0.80 ± 0.08 mg L−1 NH+ −
4 + NH3 − N; experiment 2: was the minimal concentration tested capable of inducing stage A3
−1 + −
0.90 ± 0.05 mg L NH4 + NH3 − N) was quantified according to within 3 min (see results in Table 1). Tambaqui juveniles (50.5 ±
Unesco (1983), and nitrite was determined according to 3.9 g; 15.0 ± 0.4 cm, total length) were assayed in five groups (n =
Bendschneider and Robinson (1952) (experiment 1: 0.03 ± 10 per sampling time post-anaesthesia). Instead of using the same spec-
0.01 mg L−1; experiment 2: 0.02 ± 0.01 mg L−1). Total alkalinity was imens for blood collection over time, groups of 10 animals were used for
evaluated by titration in accordance with Eaton et al. (2005) guidelines each sampling time in order to avoid cumulative stress due to handling.
(experiment 1: 41.7 ± 0.09; experiment 2: 52.6 ± 0.05 mg CaCO3 L−1, A sham control group (CT) and an ethanol control (EC) were used (n =
respectively). 10 in each control group). In the EC fish were exposed to water added
with ethanol at the same volume used to provide the concentration of
2.3. Plant materials 20 mg L−1 of jambu extract.
Fish were captured with a dip net and individually transferred to
Extract of the flowers of Spilanthes acmella Var. oleracea (L.) was ob- continuously aerated 30 L aquaria previously added with jambu extract
tained by means of fractionated supercritical fluid extraction methodol- at 20 mg L−1 where they were exposed to anaesthetic baths of 3 min.
ogy with the use of CO2 [SFE(CO2)] to remove less polar compounds Immediately following anaesthesia, all fish were handled for biometric
from the flowers of the plant. For details on the methodology of extrac- measurements and transferred to their respective anaesthetic-free
tion, global and spilanthol yields of the extract see Dias et al. (2012). 100 L recovery tanks. Similarly, CT and EC groups were transferred to
Vegetal extracts, in the form of a wax, such as those obtained from identical aquaria and were also maintained for 3 min in their respective
jambu flowers through [SFE(CO2)] extraction methodology are poorly anaesthetic-free water tanks, simulating the same handling procedures
diluted in water and therefore it is necessary to pre-dilute them in of anaesthetized fish.
72 L.A.L. Barbas et al. / Aquaculture 456 (2016) 70–75

Table 1
Anaesthesia induction and recovery times (in seconds ± SD) in juvenile Tambaqui, Colossoma macropomum exposed to five concentrations of jambu extract Spilanthes acmella.

Concentration (mg L−1) Induction (s) Recovery (s)

Stage A1 Stage A2 Stage A3 Stage R1 Stage R2

5 199 ± 39a 348 ± 72a 459 ± 107a 34 ± 17a 271 ± 118ab

10 104 ± 55b 181 ± 48b 269 ± 70b 47 ± 30a 281 ± 63ab
15 82 ± 40bc 120 ± 46c 216 ± 63bc 34 ± 27a 233 ± 90b
20 58 ± 29bc 103 ± 32c 173 ± 40c 37 ± 22a 300 ± 64ab
25 48 ± 21c 75 ± 17c 144 ± 26c 37 ± 28a 338 ± 37a
y = 820.92×-0.88 y = 1531.5×-0.925 y = 1416.4×-0.706 y = 0.4913x2–11.671× + 324.82
Equations -
r2 = 0.99 r2 = 0.99 r2 = 0.99 r2 = 0.74

Different superscripts in the same column indicate statistical difference among concentrations after ANOVA and Tukey test (n = 10, p b 0.05).

Secondary stress responses were followed for 72 h after fish were tested can be predicted by equations presented in Table 1. Clear de-
anaesthetized. Blood samples (300–500 μL) were taken from the caudal creases (r2 = 0.99, p b 0.05) in the induction time stages (A1, A2 and
vasculature with 1 mL syringes coated with heparin at 0 (immediately A3) occurred with increasing concentrations of jambu extract whereas
after recovery), 2, 24, 48, and 72 h. an increase (r2 = 0.74, p b 0.05) in the recovery time was observed
Whole blood was used to determine glucose, ionic concentration with increasing concentrations of the extract and were best predicted
(Na+, K+, and Ca++), haematocrit (Htc), haemoglobin (Hb), partial by power regression. On the other hand, no relation (p N 0.05) between
pressure of CO2 (pCO2), partial pressure of O2 (pO2), pH, and bicarbon- increasing concentrations of anaesthetic and time to show behaviour
ate concentration (HCO− 3 ) using a portable i-STAT® clinical analyser associated with stage R1 was present.
with CG8 + cartridges (Abbott laboratories, Chicago, IL, USA). The Sedation or superficial anaesthesia was observed when fish were ex-
values obtained for pCO2, pO2, pH and HCO− 3 were corrected for the posed for 10 min to 2 mg L−1 of jambu extract.
water temperature (25 °C) according to the manufacturer's instructions No mortalities were observed after the two-week observation.
and also as described by Hanley et al. (2010). The use of i-STAT® and Water in sham control group and water comprised solely of ethanol ad-
calculations for blood gases have been described for several fish species dition at the same volume of the highest concentration of jambu extract
(Jacobs et al., 1993; Pidetcha et al., 2000; Harrenstien et al., 2005; used (25 mg L−1) did not produce any anaesthetic effect on tambaqui
Kristensen et al., 2010). Blood osmolality was determined using a va- through 30 min observation.
pour-pressure osmometer (Vapro 5520; Wescor, Inc.; Logan, Utah,
USA).
3.2. Secondary stress responses
2.5. Statistical analysis
No differences were observed between controls (CT and EC) for the
secondary stress response in this study (p N 0.05), therefore only values
All data are presented as mean ± SD. To verify the homogeneity of
of CT were used for pair-wise comparisons among whole blood vari-
variances and normality, data were submitted to Levene and Kolmogo-
ables of animals in recovery. There was an increase in blood glucose in
rov–Smirnov tests, respectively. Statistical differences between controls
juvenile tambaqui following anaesthesia baths with jambu extract at
(CT and EC) were tested by Student's t-test (p b 0.05). The results ob-
0 h and 2 h and glycemia returned to CT levels at 72 h after a slight os-
tained for stages A1, R2 (experiment 1), pO2 and Na+ (experiment
cillation. Blood K+ and Ca++ concentration decreased after 2 h in recov-
2) were Ln transformed previously to statistical analysis. One-way
ery and returned to CT values at 24 h. Blood Na+ concentration
ANOVA and Tukey test were used for data of anaesthesia induction
decreased after 2 h not returning to CT levels after 72 h post-
and recovery, and also for osmolality. One-way ANOVA and Dunnet
anaesthesia. Osmolality was unchanged (p N 0.05) throughout the re-
post-hoc test were used for the other whole blood variables. Trends of
covery period when compared to CT group. Haematological values of
the anaesthesia and recovery stages were fitted using non-linear re-
Hct and Hb showed a consistent biphasic decrease between 2 h and
sponse models with measured times to reach stages being the response
48 h, with both returning to CT values at 72 h. Partial pressure of oxygen
variables and concentrations of extract of S. acmella the predictor vari-
(pO2) measured in CT did not show significant changes throughout re-
able. Pearson correlation was performed among blood variables. The
covery and pCO2 decreased between 2 and 24 h compared to CT. How-
minimum significance level was set at p b 0.05 in all cases.
ever, pCO2 returned to CT levels at 48 h. A transient decrease was
observed in HCO− 3 concentration in 0 h when compared to control.
3. Results
Blood pH increased (p b 0.05) after 2 h in recovery when compared to
CT and was negatively correlated to pCO2 (r = − 0.84, p b 0.001)
3.1. Short-term anaesthesia induction and recovery
(Table 2).
Tambaqui showed a shorter time to reach stage A1 at the concentra-
tion of 10 mg L−1 and above, compared to the lower concentration used. 4. Discussion
The same pattern was noticed for stage A2 in which increasing concen-
trations of the extract led to a faster emergence of this stage, being the Studies focusing on fish welfare and pain assessment have drawn
time at the concentration of 15 mg L−1 or above, significantly shorter much attention recently with the emphasis on improving fish welfare
to show loss of equilibrium compared to 5 or 10 mg L−1. All the tested (Animals Scientific Procedure Act, 1986; Ashley et al., 2007; EU
concentrations were effective in inducing anaesthesia (stage A3). Fish Directive, 2010; Weber, 2011; Readman et al., 2013). Concerns have
exposed to the lowest concentration (5 mg L−1) reached anaesthesia been raised that MS-222, which is the most frequently used anaesthetic
in 7.6 min (459 s) and the subsequent increasing concentrations tested for aquatic organisms worldwide, may cause an aversive reaction and
reduced the induction time, the fastest being 45 s at 25 mg L−1. Based irritation of tissues in fish following its exposure to the drug (Williams
on visual assessment, fish were acknowledged fully recovered after ex- et al., 2009); much incidental evidence exists as to the adverse reaction
posure to all concentrations tested (Table 1). seen in fish during initial stages of anaesthesia (APC, 2009). Wong et al.
The overall trends observed as a result of the relation between the (2014) exposed zebrafish Danio rerio to MS-222 and clove oil and less
time required for anaesthesia or recovery stages over the concentrations pronounced aversion was observed for the latter.
 L.A.L. Barbas et al. / Aquaculture 456 (2016) 70–75 73

Table 2
Glycemia, ionic concentration (K+, Ca++, Na+) and osmolality, haematological indicators (Ht and Hb), partial pressure of gases (pCO2 and pO2), bicarbonate concentration (HCO−
3 ) and pH
in whole blood of juvenile tambaqui, C. macropomum recovering from anaesthesia with extract of jambu, Spilanthes acmella at 20 mg L−1. Values are expressed as means ± SD.

Glucose K+ Ca++ Na+ Osmolality Ht (%) Hb pCO2 pO2 HCO−

3 pH
(mg dL−1) (mmol L−1) (mmol L−1) (mmol L−1) (mOsm kg−1) (g dL−1) (mm Hg) (mm Hg) (mmol L−1)

CT 63.30 ± 8.30 3.96 ± 0.39 1.47 ± 0.20 150.40 ± 1.35 294 ± 5 23 ± 3 7.8 ± 1.0 8.76 ± 0.95 7.00 ± 2.34 5.65 ± 0.33 7.42 ± 0.05

S. acmella
0h 74.10 ± 10.47* 3.87 ± 0.41 1.33 ± 0.27 149.60 ± 1.65 297 ± 7 22 ± 3 7.5 ± 1.0 8.62 ± 1.41 8.87 ± 2.63 4.71 ± 0.60* 7.35 ± 0.09
2h 73.20 ± 8.05* 2.89 ± 0.44* 1.20 ± 0.20* 145.30 ± 1.06* 284 ± 6 19 ± 3* 6.3 ± 0.9* 5.78 ± 0.73* 10.69 ± 5.53 5.54 ± 0.83 7.59 ± 0.07*
24 h 65.40 ± 9.22 3.80 ± 0.24 1.37 ± 0.18 147.70 ± 1.70* 292 ± 8 22 ± 3 7.6 ± 0.9 7.23 ± 0.64* 6.32 ± 4.67 5.87 ± 0.68 7.52 ± 0.05
48 h 47.40 ± 6.17* 4.12 ± 0.67 1.47 ± 0.19 147.10 ± 2.28* 285 ± 4 19 ± 3* 6.4 ± 1.2* 8.29 ± 1.39 6.93 ± 1.11 5.24 ± 0.43 7.41 ± 0.09
72 h 51.86 ± 10.30 3.77 ± 0.45 1.24 ± 0.25 145.80 ± 1.14* 290 ± 6 21 ± 3 7.0 ± 0.9 7.32 ± 0.95 7.47 ± 4.01 5.09 ± 0.54 7.45 ± 0.07

Values with an asterisk are significantly different from sham control fish (CT) (non-anaesthetized fish) after ANOVA and Dunnet test (n = 10, p b 0.05).

The stage of agitation was observed for all concentrations of jambu. control fish, resembling the results of this study despite the slight oscil-
Rapid flaring of the opercula, “coughing”, surfacing and frantic swim- lation observed before returning to CT levels at 72 h. On the other hand,
ming were present. However, time to the onset of agitation behaviour Roubach et al. (2005) reported no differences in glycemia levels of
was concentration dependent and 4-fold faster at the highest concen- tambaqui over increasing concentrations of eugenol (35–135 mg L−1)
tration (25 mg L−1) compared to the lowest concentration used. The ag- or exposure to benzocaine at 100 mg L− 1. It is important to notice
itation observed suggests transitory distress in juvenile tambaqui in this that these authors did not compare glucose levels with a sham control
study. Similar behaviour was also observed with tambaqui anaesthe- group.
tized with benzocaine (Gomes et al., 2001). Benzocaine anaesthesia at 200 mg L−1 also increased glycemia levels
All five concentrations tested promoted anaesthesia with minimum immediately after recovery in juvenile tambaqui when compared to
opercular beating and loss of reaction to tail pinch stimulus (A3) within lower concentrations (50–150 mg L−1) (Gomes et al., 2001). Blood glu-
the stipulated observation time limits. However, only at the concentra- cose concentrations increased in red pacu, Piaractus brachypomus ex-
tion of 20 mg L−1 of jambu extract, animals reached anaesthesia (A3) in posed to MS-222 or eugenol and increases were also reported even in
2.8 min (173 s), therefore complying with the recommended maximum fish exposed to adequate short-term anaesthetic baths (i.e., b 3 min)
induction time of 3 min (180 s) and recovery within 5 min (300 s) (Bell, (Sladky et al., 2001).
1987; Iwama and Ackerman, 1994; Ross and Ross, 2008). Based on Blood glucose concentrations increased rapidly after induction with
these evaluation criteria, 20 mg L−1 was regarded as the most appropri- jambu extract, even after short-term exposure. Despite of this incre-
ate anaesthetic concentration. Furthermore, it has been reported that ment in blood glucose levels compared to CT, glycemia levels observed
fish submitted to rapid inductions at high concentrations of anaesthetic in this study were all close to the concentration of 70 mg dL−1 which is
were more likely to recover uneventfully (Sladky et al., 2001). accepted as normal glycemia for this species under rearing conditions
Other compounds such as benzocaine, menthol and eugenol have (Gomes et al., 2003).
been tested for tambaqui with recommended concentrations of 100– Blood Na+ concentration decreased and it was the only variable that
150, 150 and 65 mg L−1 respectively (Gomes et al., 2001; Façanha and did not return to CT levels after 72 h in recovery. Ion losses following an-
Gomes, 2005; Roubach et al., 2005). Those were regarded as appropriate aesthesia in fish have been described for silver catfish (Rhamdia quelen)
concentrations to promote fast anaesthesia in less than 3 min and are (Gressler et al., 2014) and may occur due to initial hyperactivity when
higher effective concentrations compared to the ones used in the pres- fish are exposed to anaesthetic baths which in turn can lead to increased
ent study. Nevertheless, menthol and eugenol allowed recovery only blood flow through the gills with consequent ion losses and water in-
after 5 min, thereby exceeding the maximum time recommended for flux. Transitory osmoregulation disturbances are frequently reflected
recovery from anaesthesia in fish (Bell, 1987; Iwama and Ackerman, in the osmolality, which did not occur in this study. Non-severe electro-
1994; Ross and Ross, 2008). Although in those cases the same species lyte alterations in fish also could be explained by normal ion diffusion in
was used and water quality conditions were quite similar to the condi- the gills, because a flux of ions of small magnitude could occur as a result
tions verified herein, great care should always be used for comparisons of many physiologic or environmental manipulations (Stoskopf, 1993).
and extrapolation of results. Recent study has demonstrated that al- In the face of mild changes in the concentration of the ions K+ and
++
though taxonomic classification is not the main reason for the variations Ca and more consistently Na+ after 2 h, coupled with a presumable
in behaviour response observed during anaesthetic induction and re- transitory incapacity of controlling excessive water influx through the
covery, many other factors are implicated, such as fish size, anaesthetic gills, it may have been possible that this scenario contributed to the
concentration used and water quality parameters at the moment of in- onset of the oscillatory pattern observed in Hct and Hb after 2 h in re-
duction (Gomes et al., 2011; Bowker et al., 2015). covery. The exact mechanism of action contributing to the reduction
A sedation state is characterized by lethargy, partial loss of equilibri- of Hct and Hb content is undetermined. Pseudoanemia may be caused
um and reaction to tail pinch stimulus, and is recommended for non- by perturbations of fluid balance that lead to plasma expansion, thus
invasive procedures such as biometrics, gill scrapes, tagging, or trans- causing apparent reductions of haemoglobin, red cell count and
port (Sneddon, 2012). Menthol at 100 mg L− 1 has been regarded as haematocrit, i.e., haemodilution (Evans, 2009).
an ideal concentration for sedation of juvenile tambaqui (Façanha and Unexpectedly, fish did not show significant alterations in blood pO2
Gomes, 2005). However, the use of 2 mg L−1 of jambu extract which at any given sampling time. Environmental and/or physiological condi-
corresponded to a small fraction of the most suitable anaesthetic con- tions that lower ventilation, such as exposure to hyperoxia or in this
centration observed herein, was enough to induce sedation in tambaqui. case anaesthetic induction, typically elicit a respiratory acidosis owing
Plasma glucose plays an important role in fish metabolism and along to CO2 retention (Perry and Gilmour, 2006). Such a response was not
with cortisol is one of the most reliable stress responses in fish observed in tambaqui of this study. A respiratory alkalosis characterized
(Wedemeyer et al., 1990). Our findings are similar to those of Inoue by depressed arterial pCO2 and elevated arterial pH occurs when venti-
et al. (2011) who observed an increase in plasma glucose in juvenile lation volume is increased in response to aquatic hypoxia even at the ex-
tambaqui following anaesthesia baths with eugenol at concentrations pense of disturbances of acid–base balance (Gilmour, 2001). Along with
of 20 and 60 mg L−1. Furthermore, those authors verified that glycemia hypocapnia observed in this study, blood pH increased, which was con-
returned to normal concentrations after 24 h when compared to sham sistent with respiratory alkalosis during the first 24 h in recovery.
74 L.A.L. Barbas et al. / Aquaculture 456 (2016) 70–75

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fact that those variables were negatively correlated is consistent with following acute stress in threespot gourami Trichogaster trichopterus. J. Aquat. Anim.
Health 18, 58–63.
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Blood HCO− 3 levels are increased during compensation of acidosis heliantheae) in Taiwan. Bot. Stud. 49, 73–82.
and decreased during compensation of alkalosis (Perry and Gilmour, Cunha, M.A., Barros, F.M.C., Garcia, L.O., Veeck, A.P.L., Heinzmann, B.M., Loro, V.L.,
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for silver catfish, Rhamdia quelen. Aquaculture 306, 403–404.
after recovery from anaesthesia (0 h) preceded the alkalosis observed Dias, A.M.A., Santos, P., Seabra, I.J., Júnior, R.N.C., Braga, M.E.M., de Sousa, H.C., 2012.
at 2 h in recovery, however, HCO− 3 returned to CT levels at 2 h, which Spilanthol from Spilanthes acmella flowers, leaves and stems obtained by selective su-
suggests a minor interference on HCO− 3 blood levels after anaesthesia.
percritical carbon dioxide extraction. J. Supercrit. Fluids 61, 62–70.
Eaton, A.D., Clesceri, L.S., Rice, E.W., Greenberg, A.E., 2005. Standard Methods for the Ex-
Anaesthesia with jambu extract in this study proved to be efficient amination of Water and Wastewater. 21st ed. American Public Health Association,
and elicited temporary physiological changes in blood mostly between Springfield.
2 and 24 h in recovery. It has been suggested that the alleged Evans, G.O., 2009. Animal Hematotoxicology: A Practical Guide for Toxicologists and Bio-
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hypoventilation observed during anaesthesia rather than a direct effect tion of Animals Used for Scientific Purposes 22 September 2010. Available at: http://
of the anaesthetic (Rothwell et al., 2005). No mortalities were observed eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2010:276:0033:0079:EN:PDF
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L.A. Sampaio and L.O. Garcia are research fellows of Brazilian CNPq.
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during their graduate studies at FURG. We are thankful to Dr Mara concentrations in yellow perch (Perca flavescens), walleye pike (Sander vitreus), and
Braga from the Chemical Engineering Department at the University of koi (Cyprinus carpio). J. Am. Vet. Med. Assoc. 236, 1104–1108.
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