Comparison of Electroanesthesia with Chemical Anesthesia (MS222 and Clove Oil) in Rainbow Trout (Oncorhynchus mykiss) using Plasma Cortisol and Glucose Responses as Physiological Stress Indicators
This study investigates Alternating Current (AC) electroanesthesia of rainbow trout (Oncorhynchus mykiss) in comparison with MS222 and clove oil, using plasma cortisol and glucose concentrations as stress assessment indicators. A microcontroller-based apparatus was designed and constructed to allow a programmable voltage-time Pulse-Width Modulated (PWM) electrical wave application through 19x20 cm submersible electrodes for 91sec in a 33 cm long tank to induce loss of equilibrium and immobility with recovery after 52±27 sec. Recovery after 660±102 sec was observed in MS222-anesthetized fish (after induction for 720±72 sec) and a recovery time of 546±102 sec was observed in clove oil-anesthetized fish (after induction for 144±42 sec) both are significantly longer recovery times in comparison with electroanesthesia (p<0.001). Using direct enzyme-linked immunosorbant assay (ELISA) for cortisol and enzymatic colorimetric assay for glucose assessments at 0, 1, 6, 12 h after each anesthesia, the anesthetics indicated similar trend of cortisol responses during 12 h of investigation. The dilatory trend of glucose changes and response derived from anesthetics and electricity and its surge at 6 h after anesthesia (p<0.05) confirmed glucose as a second order indicator of stress responses. Electroanesthesia is a fast, economic, eco-friendly and safe anesthetic method provides desirable trout immobility for aquaculture activities.
to cite this article:
A. Sattari, S.S. Mirzargar, A. Abrishamifar, R. Lourakzadegan, A. Bahonar, H.E. Mousavi and A. Niasari, 2009. Comparison of Electroanesthesia with Chemical Anesthesia (MS222 and Clove Oil) in Rainbow Trout (Oncorhynchus mykiss) using Plasma Cortisol and Glucose Responses as Physiological Stress Indicators. Asian Journal of Animal and Veterinary Advances, 4: 306-313.
Fish anesthetics (physical/chemical) are valuable tools that help to reduce
fish struggling and physical damages due to stressful rearing and propagation
activities (e.g., handling, sorting, spawning, transportation, vaccination,
injection etc.). A number of parameters such as expense, availability, ease
of use and human safety, have to be considered prior to the choice of an anesthetic.
Appropriate induction and recovery times of the anesthesia and level of anesthetic-induced
stress responses also require consideration. Although, chemical anesthetics
like tricaine methane sulphonate (MS222 is the most-frequently used and only
FDA approved anesthetic for edible fish in U.S.) and clove oil (eugenol) and
derivatives (generally considered as safe by FDA; GRAS) are used to alleviate
negative consequences of stress responses (Summerfelt and
Lynwood, 1990) but there is some evidence of undesirable and stressful consequences
after chemical anesthetic application. For example MS222, Metoimdate and AQUI-S
reduce the contractile efficacy of ventricular myocardium of Chinook salmon
(Oncorhynchus tschawytsha) (Hill et al., 2002).
MS222 alters brain currents needed to extract an action potential of supramedullary/dorsal
neurons of Cunner (Tautogolabrus adspersus) (Arnolds
et al., 2002) . Also MS222 and eugenol are not considered to be completely
suppressive of stress-induced cortisol and glucose responses in rainbow trout
(Wagner et al., 2002). Palic
et al. (2006) believe that MS222 and eugenol do not prevent the cortisol
response of stress and MS222 is not capable of preventing the degranulation
of neutrophil primary granules as a stress indicator in fish. In toad Bufo
marinus an immense disturbance of hematological and cardiorespiratory parameters
accures after anesthesia with benzocain (Andersen and Wang,
2002). Kiessling et al. (2009) described
that both MS222 and isoeugenol caused a marked increase in plasma cortisol at
the end of exposure and believe that different anesthetics provoke physiological
stress on their own. Although, the eugenol derivatives are relatively less expensive
than MS222, but extraction of a standard pharmaceutical form from clove plant
needs laboratory facilities and investments which may not always be available.
The cost beneficial alternative which eliminates the problems of chemical residues,
withdrawal periods and ecological pollution and is assumed to supply short induction
and recovery time and immobility for aquaculture activities, is electroanesthesia
which is considered to have fewer effects on plasma and tissue electrolyte changes
in comparison with MS222 (Jenning and Looney, 1998).
The development of an apparatus to investigate fish electroanesthesia was defined as the first objective using evaluation of endocrine anesthetic-induced stress indicators (plasma cortisol and glucose) in comparison with current chemical anesthetics (MS-222 and clove oil), introduced as second objective of this study.
MATERIALS AND METHODS
Three-hundred rainbow trout (fasted for 24 h) weighing 60±15 g (Mean±SD),
were purchased two weeks before the experiment (Parvaresheh Ghezelala Jajrood
Co., Tehran, Iran) and transported to experimental tanks (stocking density =
3 kg m-3 ) at the Faculty of Veterinary Medicine, University of Tehran,
supplied with 15±2°C aerated flowing well water. Water quality parameters
(pH = 7.8, Electrical conductivity; EC = 870 μs cm-1, Dissolved
oxygen; DO = 8.3 mg L-1 and Hardness = 300 mg L-1 as CaCO3)
were recorded using a Multi-340i detector (WTW, Germany).
Feeding started 24 h after arrival with the same batch of food used at the original rearing farm (Chinneh Co., Iran). Experimental procedures started after 14 days of acclimation on December 2007 and final raw data were elicited until April 2008.
The experiment was performed for three treatments of anesthesia; electricity,
MS-222, clove oil and control group. The experiment was triply repeated for
each treatment and control (n = 48). The control group fish were transferred
(in nets) from the acclimation tank to a recovery tank located at the Ichthyology
Lab. Fish intended as treatment designated animals were transported (in nets)
to an anesthetic bath supplied with air stones. After treatment these fish were
placed in recovery tanks under similar conditions to the control group. Blood
samples were taken from all animals (treatment and control) through peduncle
vessels (Wagner et al., 2002) at 0, 1, 6 and
12 h after placement in recovery tanks.
A prototype of an alternating current (AC) electroanesthesia apparatus was
designed and constructed based on Chiba et al. (2006)
study and Ross et al. (2008). This unit was equipped
by microcontroller system to allow an automatic voltage-time adjustments of
the anesthesia steps instead of manual adjustments (Chiba
et al., 2006). In order to adjust and calibrate the apparatus, a
number of pilot experiments were performed on rainbow trout, common carp (Cyprinus
carpio) and gold fish (Carassius auratus) to select the optimal voltage
wave form and time of insult for the minimum vigorous movements of fish during
anesthesia. The sinusoidal PWM electrical waves were found to have the least
effect on behavior in rainbow trout allowing the authors to devise a pattern
of voltage-time application. Groups of 4 fish were transported to the anesthesia
tank (19x20x33 cm) and placed between two submersible stainless steel plate
electrodes (19x20 cm) which inserted in a distance 33 cm from each other. The
PWM voltage waves form was applied to electrodes for 91 sec with the defined
pattern of voltage-time variations.
The fish were immersed in 100 mg L-1 of Tricaine methane sulphonate
(Ross et al., 2008) (MS-222, Finquel®;
Argent TR2905, Redmond, WA, USA) and 100 mg L-1 of clove oil (Soltani
et al., 2002) (Zardband Co., Tehran, Iran) up to stage 3 of anesthesia
(surgical anesthesia) (Ross et al., 2008). Then
the fish were immediately transported to recovery tanks and time of recovery
Blood samples collected in 1.5 mL tubes containing Heparin (5000 IU) were
centrifuged for 12 min (1500 r min-1) at laboratory room temperature
to separate the plasma which was preserved at-18°C.
Although, radio immunoassay (RIA) is the most accepted method for quantitative
detection of cortisol in fish (Patino et al., 1987;
Small and Davis, 2007) the problems of handling radio
isotopes are immense due to short half-life and human health risk. Therefore,
in the present study we applied direct enzyme-linked immunosorbant assay (ELISA)
which has been validated by Sink et al. (2008)
for non salmonid species.
An ELISA kit based on the competitive link between cortisol and related monoclonal
antibody was purchased (RE 52061, Lot: 43K028-2, 43K107, IBL, Hamburg, Germany).
Since, the kit was fabricated for human samples it was necessary to validate
its use for fish samples prior to analysis. Validation was performed by filling
the first 8 wells of the ELISA plate with 20 μL of 0, 2.5, 5, 10, 20, 50,
100, 400 and 800 ng mL-1 of standard solution (Sink
et al., 2008). The following four wells were filled with 20 μL
of a plasma sample from a highly-stressed rainbow trout (kept out of water for
2 min), assumed to contain a high amount of cortisol diluted in 1/4, 1/3, 1/2,
1 proportions. The next eight wells contained 20 μL of undiluted plasma
from another 8 highly stressed fish. According to the kit instructions, the
parallelism and linearity in trend of optic absorbance of cortisol between serial-diluted
standard solutions of the kit and highly-stressed fish plasma, indicated the
kit reliability for use as quantitative assays of cortisol in rainbow trout.
Subsequent cortisol assay of treatments and control samples were analyzed according
to booklet instructions.
An enzymatic-colorimetric kit (1500017, Pars Azmoon Co., Karaj, Iran) was
applied for single-spot detection of glucose (Adetunji et
al., 2008). This method allows the oxygen released by glucose (catalyzed
by glucose oxidase) to react with phenol and 4-amino pyridine (catalyzed by
peroxidase) to produce Kinonimin which is photometric-detectable and its quantity
is indicative of glucose volume.
A student t-test was used to define level of differences of induction and
recovery derived from each method of anesthesia. Based on statistics used by
Wagner et al. (2002) the confidence level of
95% was used for analysis performed by SPSS software version 16 and the Kolmogorov-Smirnov
test and examination of box plots and histograms were used for preliminary assessment
of the normal distribution. Also, the Pearson correlation coefficient was applied
to investigate presence of correlation between cortisol and glucose changes.
Fish were anesthetized after 720±72 sec (Mean±SD, n = 30) of induction with MS222, after 144±42 sec of induction with clove oil (n = 30) and after 84±33 sec (n = 24) of induction of immobility using the apparatus. Recovery times for anesthetics were 660±102 sec for MS-222, 546±102 sec for clove oil and 52±27 sec for electroanesthesia (n = 13). Significant differences were observed between anesthetics in induction and recovery times (p<0.001, Table 1).
No significant differences of plasma cortisol concentrations were observed
among three anesthetic treatments and among anesthetic treatments and control
group at 0, 1, 6 and 12 h after anesthesia (Fig. 1). Plasma
cortisol concentrations of anesthetized and non anesthetized fish increased
at 1 h after anesthesia so that the highest concentration belonged to clove
oil and MS222, respectively however after 6 h of anesthesia cortisol concentrations
of all groups indicated a decrease that in clove oil, electroanesthetized and
control fish was to level lower than the time of anesthesia (0 h) (Fig.
||Duration of induction and recovery times (Mean±SD)
for electroanesthesia (n = 24, n = 13 respectively), clove oil and MS222
(n = 30)
|Values in each column with different letters are significantly
||Cortisol responses of rainbow trout (Mean±SE) to clove
oil, MS222 and electroanesthesia during 12 h after anesthesia. No significant
difference is observed among groups in trend of cortisol changes
||Glucose responses of rainbow trout (Mean±SE) to clove
oil, MS222 and electroanesthesia during 12 h after anesthesia. At 6 h plasma
glucose of control group is significantly different from other treatments
This decrement was observed for anesthetized groups until 12 h of anesthesia
while level of cortisol in control group started to increase after 6 h nevertheless
at 12 h was still lower than time of anesthesia (Fig. 1).
Plasma glucose concentrations of anesthetized groups were not different from each other at all sampling times and from control group at 0, 1 and 12 h after anesthesia while they were significantly higher than control group after 6h of anesthesia (p<0.05) (Fig. 2). A notable increment of plasma glucose concentrations of anesthetized fish from 0 to 6 h after anesthesia and its recurrence to the level higher than time of anesthesia (0 h) was observed afterwards. The recurrence was notable in electricity and clove oil anesthetized fish (Fig. 2).
During normal aquaculture activities some unavoidably stressful procedures
have to be performed. Therefore it has become necessary to introduce a number
of practical approaches in an attempt to alleviate the detrimental consequences
of stress in fish (Barton and Iwama, 1991). One such
method applied to reduce the risk of procedural stress is anesthesia, however
the application of anesthetics is known to be stressful in itself (Barton
and Barton, 1982; Wagner et al., 2002; Kiessling
et al., 2009). For some routinely performed activities e.g., sorting,
weighing, injections etc. anesthesia with shorter induction and recovery times
are more appropriate. This characteristic is achieved by electro anesthesia
in our study with the shorter induction (84 sec) and recovery (52 sec) times
in comparison with other treatments. The resulting stress responses of anesthesia
can be categorized as endocrine, metabolic and whole animal responses (Barton,
2002). It is of primary importance that reliable indicators are defined
in order to evaluate anesthetics in relation to their stress responses. Plasma
cortisol and glucose have been introduced as stress indicator in fish (Pickering,
1981; Pickering and Pottinger, 1989; Ortuno
et al., 2002) and a number of anesthetic-induced increases in plasma
cortisol have been observed in several species i.e. sea bream, red drum, rainbow
trout and channel catfish (Robertson et al., 1998;
Barton and Barton, 1982; Tort et
al., 2002; Small, 2003).
In Chinook and Atlantic salmons, MS222 and eugenol derivatives provoke similar
post-recovery cortisol and glucose responses. Cho and Heath
(2000) and Kiessling et al. (2009) concluded
that the level of nervous depression achieved by anesthetics does not necessarily
mitigate certain physiological stress responses.
In Channel catfish when MS222 was compared to metomidate by Small
(2003), it caused an eightfold higher concentration of plasma cortisol than
metomidate and when it was used at the required dose for anesthesia of Japanese
eel, MS222 raised the cortisol level more than 2-phenoxyethanol (Chiba
et al., 2006). This study showed that the anesthetic-induced cortisol
responses did not differ among anesthetics in comparison with control. The electro-anesthetized
trout showed no significant difference of cortisol concentration from chemical
anesthetics which is a similar observation to that found in electro-immobilized
Gold fish (Carassius auratus) in comparison with MS222-anesthetized fish
(Singley and Chavin, 1975). This similarity in stress
responses among electroanesthesia and two chemical anesthetics of our study
on one hand and fast induction (84 sec) and recovery time (52 sec) of electroanesthesia
on the other hand, are criteria of a suitable anesthetic (Son
et al., 2001) which was previously confirmed in stripped bass surgery
and electroanesthesia was considered as an alternative to MS222 that eliminates
risk of chemical overdose-caused death (Jenning and Looney,
1998). Increment of cortisol concentrations in treatment and control groups
at 1 h which can be caused by anesthesia, netting and handling and its decrement
from 1 to 6 h after anesthesia is in agreement with Wagner
et al. (2002) findings although this decrement in our anesthetized
fish continued to the level lower than time of anesthesia (0 h) in clove oil
and electro anesthetized fish until hour 12. The decrement to this level is
in contrast to findings of Park et al. (2008)
in clove oil anesthetized kelp grouper and is representative of species differences
in stress responses and suggests efficacy of electroanesthesia besides chemical
anesthetics to recover fish from stress caused plasma cortisol increment and
its probable long term negative consequenses.
Continuation of decreasing trend of cortisol responses in anesthetized fish
from hour 6 to 12 in this study is in contrast to increasing cortisol concentrations
in Aqui-s® and MS222 anesthetized trout brood stock and similar
to stress responses of CO2 anesthetized fish performed by Wagner
et al., (2002). This decreasing trend from 6 h after anesthesia approves
alleviating role of anesthetics and electroanesthesia against physiological
consequences of stressful handlings nettings and other activities. Increment
of cortisol in control group since 6 h after anesthesia in spite of its decrement
in anesthetized groups after 6h, implies that anesthesia speeded up recovery
from handling and netting stresses.
Lack of significant difference of cortisol response among electro anesthetized
fish and other treatments and control is in contrast with Barton
and Dwyer (1997) study in juvenile bull trout (Salvelinus confluentus)
suggesting different physiological responses in different species.
During 12 h of investigation, plasma glucose fluctuations of anesthetized fish
followed a similar trend and did not return to 0 h level. Although electro anesthesia
and MS222 induced faster increase of glucose concentration than other groups
during first 6 h, after this time glucose response of electrical and clove oil
anesthetized fish indicated a faster recurrence to normal level than MS222 anesthetized
fish. The trend in plasma glucose fluctuations and its magnitude 6 h after anesthesia
with electricity and two anesthetics which is in accordance with Wagner
et al. (2002) findings in Aqui-s® anesthetized trout
and observed correlation (p = 0.00007 and r = 0.27) between cortisol and glucose
concentrations in electro anesthetized and control group of this study could
be indicative of the influence of cortisol magnitude 1 h after anesthesia and
confirm the categorization of cortisol and glucose responses by Barton
(2002) as indicators of first and second phase of stress responses, respectively.
It is concluded that in comparison with current chemical anesthetics (MS222, clove oil), electroanesthesia with fast induction and rapid recovery times offers an inexpensive, safe and eco friendly anesthetic alternative which can be useful in aquaculture activities need a kind of brief immobility to do something. This method facilitates rearing activities for more fish per time unit while reducing residues in fish and the environment. The fish electro anesthesia apparatus allows adjustable anesthesia for different sizes and types of fish improving farm application and enhancing physiological studies. The anesthetics used in this study suggest no different impact on stress responses of plasma glucose and cortisol and no controlling effect on stress responses but precipitating influence on recovery from stress responses. In order to improve fish welfare, future neurophysiologic studies can provide advantageous information on level and duration of unconsciousness, gained by application of this apparatus.
This study was financially supported by University of Tehran (grant number:7505004/6/2). We are extremely grateful to professor Reza Omidbaigi for producing and supplying clove oil, Asghar Moghiseh who performed ELISA for cortisol measurements and Mr. Majidi and his staff at Tehran Nour pathobiology laboratory who performed plasma glucose measurements. The authors greatly appreciate the cooperation of Jajrood trout farming complex which supplied fish for the experiments. Vincent Hindle kindly accepted to do language editing of the manuscript.
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