vers?o impressa ISSN
Cienc. Rural vol.40 no.10 Santa Maria out. 2010
Epub 01-Out-2010
http://dx.doi.org/10.-00154
Anesthesia
of silver catfish with eugenol: time of induction, cortisol response and sensory
analysis of fillet
Anestesia de
jundi&s com eugenol: tempo de indu&&o, resposta ao cortisol
e an&lise sensorial do fil&
Mauro Alves
da CunhaI; Carla Cristina ZeppenfeldI; Luciano de Oliveira
GarciaII; Vania Lucia LoroIII; Milene Braga da FonsecaIII;
Tatiana EmanuelliIV; Ana Paula de Lima VeeckIV; Carlos
Eduardo CopattiV; Bernardo BaldisserottoI,
IDepartamento
de Fisiologia e Farmacologia, Universidade Federal de Santa Maria (UFSM), ,
Santa Maria, RS, Brasil. E-mail:
IIInstituto de Oceanografia, Esta&&o Marinha de Aquacultura,
Universidade Federal do Rio Grande (FURG), Rio Grande, RS, Brasil
IIIDepartamento de Qu&mica, UFSM, Santa Maria, RS, Brasil
IVDepartamento de Tecnologia e Ci&ncia dos Alimentos, Universidade
Federal de Santa Maria (UFSM), Santa Maria, RS, Brasil
VDepartamento de Ci&ncias da Sa&de, Universidade de
Cruz Alta (UNICRUZ), Cruz Alta, RS, Brasil
The aim of this
study was to identify the time of anesthetic induction and recovery of silver
catfish (Rhamdia quelen) exposed to eugenol. It was also determined
the efficacy of the anesthetic as a stress reducing agent and performed a sensory
analysis of the fillets from fish exposed to this substance. The silver catfish
were exposed to air for 1min to carry out biometry, and blood was collected
at 0, 1 and 4 hours later. Eugenol can be used in the range of 20-50mg L-1
for anesthetic induction in silver catfish, and recovery time from anesthesia
was not affected by eugenol concentration. The control group showed significantly
higher cortisol levels 4 hours after biometry than at time zero. Fish anesthetized
with eugenol (50mg L-1) presented significantly lower plasma cortisol
levels than control fish at the same time. These data indicate that eugenol
inhibits the rise of cortisol in the blood. The sensory analysis test demonstrated
that eugenol modifies the flavor of the fillet and therefore is contra-indicated
for anesthetization of silver catfish that are intended for human consumption.
Key words:
clove oil, Rhamdia quelen, sedation, stress, taste.
O objetivo deste
estudo foi identificar o tempo de indu&&o e recupera&&o
anest&sica de jundi&s (Rhamdia quelen) expostos
ao eugenol, bem como a efic&cia desse anest&sico na inibi&&o
do estresse e realizar an&lise sensorial dos fil&s dos peixes
expostos a essa subst&ncia. Os jundi&s foram expostos ao ar por
um minuto para realiza&&o da biometria, e o sangue foi coletado
zero, uma e quatro horas depois. O eugenol pode ser usado na faixa de 20-50mg
L-1 para a indu&&o da anestesia em jundi&s,
e o tempo de recupera&&o da anestesia n&o foi afetado pela
concentra&&o do eugenol. O grupo de controle mostrou n&veis
significativamente mais elevados do cortisol quatro horas ap&s a biometria
que no tempo zero. Os peixes anestesiados com eugenol (50mg L-1)
apresentaram n&veis significativamente mais baixos do cortisol plasm&tico
do que peixes do grupo de controle do mesmo tempo. Esses dados indicam que o
eugenol inibe o aumento do cortisol no sangue. O teste sensorial demonstrou
que o eugenol modifica o sabor dos fil&s e consequentemente &
contra-indicado para a anestesia do jundi& quando o fil& for destinado
ao consumo humano.
Palavras-chave:
&leo de cravo, Rhamdia quelen, seda&&o, estresse,
palatabilidade.
INTRODUCTION
Currently, procedures
for managing and manipulating silver catfish (Rhamdia quelen)
do not involve the use of anesthetics. However, due to increased interest in
product quality, the use of sedatives is occasionally necessary to facilitate
management and to reduce fish stress during handling. Stress induces cortisol
secretion by the interrenal tissue, and this hormone causes various secondary
stress responses, including increased glucose and lactate plasma levels (ROTLLANT
et al., 2001; SKJERVOLD et al., 2001). Increased plasma levels of these parameters
is an indication of glycogen mobilization and is associated with poor quality
and rigidity of fish fillets (SKJERVOLD et al., ). High levels of
plasma cortisol can also induce a decrease in the immunologic capacity of salmon
(Oncorhynchus tshawytscha) (PICKERING & POTTINGER 1989) and
channel catfish (Ictalurus punctatus) (DAVIS et al., ).
Certain anesthetics, as metomidate (OLSEN et al., 1995), eugenol and isoeugenol
(Aqui-S) (IVERSEN et al., 2003) can reduce or obstruct hypothalamus-hypophysis-interrenal
axis activation, resulting in lower cortisol discharge (ROTLLANT et al., 2001;
SKJERVOLD et al., 2001).
Clove oil is an
anesthetic derived from distillation of plant parts from the Eugenia
genus (Eugenia caryophyllata and E. aromatica) (LEE
& SHIBAMOTO, 2001), and the active ingredient is eugenol (makes up to 90-95%
of the clove oil), which functions as a depressor of the Central Nervous System
(ANDERSON et al., 1997). Moreover, clove oil has been indicated as an alternative
to traditional anesthetics as metomidate, quinaldine, and tricaine methanesulphonate
because it is a natural oil and safe to use (SLADKY et al., 2001). The clove
oil concentrations necessary for anesthesia induction vary by species. In channel
catfish (Ictalurus punctatus), bluegill (Lepomis macrochirus)
(STEHLY & GINGERICH 1999), Atlantic salmon (Salmo salar) (IVERSEN et al.,
2003), rainbow trout (Oncorhynchus mikiss) (KEENE et al., 1998),
black pacu (Piaractus brachypomus) (SLADKY et al., 2001), Chinook
salmon (Oncorhynchus thsawytscha) (CHO & HEATH 2000) and largemouth
bass (Micropterus salmoides) (COOKE et al., 2004) clove oil is
effective at concentrations of 10-50mg L-1.
The aim of this
study was to determine the optimal eugenol concentration for silver catfish
anesthesia and to evaluate the time of anesthetic induction and recovery from
anesthesia. Furthermore, it was examined the effect of eugenol on plasma cortisol
levels of silver catfish exposed to handling stress and the sensory characteristics
of the resulting fillets.
MATERIAL AND
Juvenile silver
catfish were purchased from a fish culture and transported to the laboratory,
where they were maintained for two weeks in continuously aerated 250L tanks,
21&1&C, pH 6.6-7.0, dissolved oxygen levels 5.8-7.2mg L-1.
Juveniles were fasted for 24h prior to experiments.
Anesthesia induction
and recovery
Juvenile fish (2.14&0.01g
and 7.0&0.1cm) were transferred to aquaria containing 1L of water and
eugenol (Eugenol, Odontofarma&, Porto Alegre, Brazil) at concentrations
(in mg L-1) of 5, 10, 20, 30, 40, 50, 60 or 70, first diluted in
ethanol (1:20). Control experiments were performed using aquaria containing
ethanol alone at the same concentration as used to the dilution of the highest
eugenol concentration. To evaluate the time required for anesthesia induction,
20 juveniles were used for each concentration tested, and each juvenile was
used only once, according to SCHOETTGER & JULIN (1967). The maximum observation
time was 30min. After induction, juveniles were transferred to anesthetic-free
aquaria to measure anesthesia recovery time.
Measurements of
plasma cortisol
Juvenile fish (194.89&12.5g
and 26.0&0.6cm) were divided into two treatment groups: eugenol first
diluted in ethanol (1:20) (50mg L-1) (this concentration was used
because is the lower concentration to obtain anesthesia within a short period
of time, around 111s, see results) or control (without anesthetic). Anesthesia
was carried out in an aquarium containing 5L of water (1-2min), and biometry
(weight and length measurements - time of air exposure: 1min) was performed.
Following biometry, blood was collected from the caudal vein of 12 juveniles
(time 0). The remaining anesthetized juveniles were placed in two 250L tanks
and blood samples of 12 fish from one tank were collected 1h later and from
12 fish from another tank 4h after anesthesia. The control group was subjected
to the same procedures as the test group except by the exposure to eugenol,
and the fish were held while the biometric measurements were taken. Blood was
collected using a Hamilton syringe, transferred to 2mL plastic tubes and later
centrifuged 3000xg to separate plasma, which was kept under constant refrigeration.
Plasma cortisol level was measured using a commercially available solid phase
competitive chemoluminescent enzyme inmunoassay kit (Immulite 2000) (Diagnostic
Products Corporation, Los Angeles CA, USA). The specificity of the test was
evaluated by comparing the parallelism between the standard curve and serial
dilutions in PBS (pH 7.4) of the plasma samples. No differences were observed
between human and fish samples. The standard curve, constructed with human samples
ran parallel to that obtained using serial dilutions of silver catfish plasma.
A high positive correlation (r2=0.9725) was obtained between curves.
The coefficient of variation observed from fish ranged from 9 to 12%. Fish from
which blood was collected at time 0 were euthanized by severing the spinal cord
immediately after blood collection. Fillets were obtained from these fish, which
were later used for sensory analysis.
Sensory analysis
To determine whether
there was a difference in the taste and odor attributes between the fillets
from fish submitted to eugenol treatment (50mg L-1, 1-2min, see previous
section &Measurements of plasma cortisol&) and control fish (no eugenol)
the difference from a standard method described by COSTELL (2002) was used.
Fillets were cooked in a microwave oven (20g 1min-1) and evaluated by 26 untrained
judges. The degree of taste and odor difference from control was measured on
a seven-point scale, where 1= extremely 2=moderately be 3=slightly 4=not di
5=slightl 6=moderatel 7=extremely
worse than control. Samples were coded by random numbers and presentation of
the samples included a hidden control. Sensory scores were obtained for the
treated sample and for the control sample.
Statistical analysis
All data were submitted
to a Levene test to verify the homogeneity of variance. Variance was found to
be equivalent between different groups, so analysis of plasma cortisol levels
were carried out using two-way ANOVA, followed by Tukey post hoc test. Data
from sensory analysis were analyzed using the Mann-Whitney U test. The time
of anesthetic recovery data were analyzed using the Kruskal-Wallis and Mann-Whitney
tests. Comparisons of time to anesthesia induction to stage 4 with 50mg L-1
eugenol in silver catfish of two different sizes were analyzed using the Student
T test. STATISTICA (version 5.1) was used for analyses and significance was
set at a level of 95% (P&0.05).
Fish exposed to
5 and 10mg L-1 eugenol showed no evidence of anesthesia during the
30min evaluation period. In juveniles exposed to higher concentrations of eugenol,
increasing eugenol concentration proportionally decreased induction time for
2 to 4 stages of anesthesia (). No mortality was found during anesthesia induction and recovery between
20 and 50mg L-1 eugenol administration. However, at 60 and 70mg L-1,
some fish reached stage 5 (respiratory movements ceased) and consequently mortality
increased to 20% and 65%, respectively. Ethanol alone showed no anesthetic effect.
No significant
difference was found in the recovery time at the different concentrations of
eugenol tested (206.4-260.2s). Likewise, there was no significant difference
between the latency to anesthesia induction and recovery in juveniles of different
sizes induced to stage 4 with 50mg L-1 eugenol (time for anesthesia
induction to stage 4-2g: 206.4&22.5s and 177g: 243.2&19.4s).
The control group
showed significantly higher cortisol levels 4h after biometry than at time zero.
Silver catfish anesthetized with eugenol showed significantly lower plasma cortisol
levels than fish from the control group at the same time (P&0.05) ().
The median sensory
score (interquartile range) of the odor of fillets obtained from silver catfish
anesthetized with eugenol was 4 (3-5), like that from control fish 4 (3-5).
However, the median taste score (interquartile range) of fillets from fish anesthetized
with eugenol was significantly higher 6 (6-7) than that from control fish 4
(3-4) (P&0.05), indicating that the taste of filets from fish exposed to
eugenol was considered worse than that from control fish (slightly to moderately).
DISCUSSION
Anesthesia induction
and recovery
In this study,
eugenol concentrations above 20mg L-1 induced stage 4 of anesthesia
in silver catfish within 15min, while lower concentrations had no anesthetic
effect within 30min. A positive effect was determined by a rapid induction to
stage 4 of anesthesia and a lack of harm to the fish at a concentration of 50mg
L-1. At this concentration, stage 4 anesthesia was reached in 111s
and death among the tested animals did not occur. Therefore, this concentration
of eugenol was indicated for induction to stage 4 of anesthesia. The effect
of clove oil varies according to the species, but 20-50mg L-1 induced
stage 4 of anesthesia within 120-360s in juvenile Chinook salmon (CHO &
HEATH 2000), rainbow trout (KEENE et al., 1998), black pacu (SLADKY et al.,
2001), and Atlantic salmon (IVERSEN et al., 2003).
The size of fish
appears to be positively correlated with the latency to anesthesia induction
(OLSEN et al., 1995), but no significant difference in the time to anesthesia
induction to stage 4 with eugenol 50mg L-1 was found relative to
fish size for silver catfish in this study.
Evaluation of plasma
Clove oil at concentrations
higher than 20mg L-1 prevented plasma cortisol elevation above resting
levels in Atlantic salmon, but its mechanism of action is not known. However,
it is reasonable to presume that the drug disrupts transmission of sensory information
to the hypothalamus (IVERSEN et al., 2003). One of the main fish responses to
adverse situations is the production of catecholamines and corticosteroids,
which are responsible for physiological and biochemical changes and are usually
characterized as stress responses. Cortisol levels in non stressed fish vary
from 5 to 51ng mL-1 and after acute stress from 30 to 309ng mL-1
(DAVIDSON et al., 2000). The resting cortisol level in non stressed silver catfish
is 23.80&5.45ng mL-1, which is significantly lower than the
level of chronically stressed fish (55.23&11.44ng mL-1) (BARCELLOS
et al., 2006), and similar to the values obtained in the control fish (20-25ng
mL-1) in the first two measurements. The increase of plasma cortisol
values in control fish 4h after biometry was also observed by CUNHA et al. (2010)
in an experiment using the same handling methodology. Cortisol secretion is
dependent on the severity and value of the stressor applied (SUMPTER et al.,
1985). Rainbow trout exposed to air for 30s showed increased plasma cortisol
levels 30min later (SLOMAN et al., 2001). In chronically stressed silver catfish
the cortisol peak occurs 1h after exposure to a new stress factor (BARCELLOS
et al., 2006). In our study at time zero the levels of plasma cortisol in control
fish were already significantly higher than those anesthetized with eugenol,
and a further increase (compared to time zero) occurred 4h after air exposure
and handling. However, silver catfish anesthetized with eugenol presented significantly
lower plasma cortisol levels than control fish at all analyzed times, which
supports the hypothesis that this anesthetic prevents an increase of cortisol
in the plasma at the moment of handling and air exposure. A similar result was
obtained by CUNHA et al. (2010) using the essential oil of Lippia alba
as anesthetic.
Sensory analysis
The sensory analysis
is relevant in this study because it evaluates fish quality in the same way
that the consumer would perceive fish quality. Recent studies propose some method
of anesthesia for fish slaughter practice (SAVENIJE et al., 2002; LINES et al.,
2003; MATOS et al., 2010). AQUI-STM is a fish anesthetic/sedative
approved for use in several countries, and its active ingredient, isoeugenol,
is approved for human consumption in the U.S. when used as a food flavoring.
AQUI-STM has been developed as an anesthetic that may be used on
food-fish with no withdrawal period. Isoeugenol residues were detected in rainbow
trout fillets after exposure to AQUI-STM (MEINERTZ et al., 2006),
but this compound is not known to produce adverse taste responses. Recently,
it was demonstrated that exposure of Solea senegalensis to clove
oil can assure a high quality product for the market. Moreover, the authors
concluded that the results can also be applied to other species used in aquaculture
as a method to assure high quality fish for human consumption (RIBAS et al.,
2007). However, they did not evaluate the taste of their fish. In the present
study, sensory analysis demonstrated that anesthesia with clove oil adversely
affects the taste of silver catfish fillets. This change probably occurred due
to the strong flavoring capacity of clove oil, and suggests that some residues
of the oil remain in the fish muscle. The Food and Drug Administration (FDA)
does not approve clove oil as an anesthetic in fish for human consumption because
of safety concerns (US FDA 2007). Aqui-S (or isoeugenol) is also not approved
yet for use in fish (US FDA 2010) However, clove oil and eugenol have been widely
used as a flavoring and antimicrobial agent in the food industry (VELLUTI et
al., 2003; JECFA 2006). Although clove oil and many of its components are considered
to be &generally recognized as safe& (GRAS) substances for use as
food additives by the United States FDA (1978), there is some evidence of carcinogenicity
for eugenol and methyleugenol (US FDA 2007). The joined results suggest that
due to sensory concerns eugenol is contra-indicated for anesthesia at the moment
of slaughter if the fish is intended to be used for human consumption.
Eugenol is an effective
anesthetic for silver catfish and can be used at 20-50mg L-1 without
problems for the fish or the handlers, but care must be taken if the fish is
to be used for consumption after anesthesia because eugenol leaves an unpleasant
taste in the fillet.
ACKNOWLEDGEMENT
This research was
supported by Conselho Nacional de Desenvolvimento Cient&fico e Tecnol&gico
(CNPq). B. Baldisserotto received a CNPq research grant.
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Received 04.07.10
Approved em 08.07.10
Received from the author 09.01.10
Autor para correspond&ncia.
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