Evaluation of electronarcosis and clove oil for short-term anesthesia in common carp, Cyprinus carpio L. 1758

[Research]

Evaluation of electronarcosis and clove oil for short-term anesthesia in common carp, Cyprinus carpio L. 1758

S.F. Monsef Rad¹*, S.Y. Paighambari¹, A. Haji Moradlou¹, F. Mashayekhi², R. Qorbani¹

1- Department of Fishery, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

2- Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran

* Corresponding author’s E-mail: monsefrad@gmail.com

ABSTRACT

Anesthesia is considered to be the best way to ensure animal welfare during handling etc. Clove oil and electronarcosis are among the most popular chemical and physical anesthetics used in fisheries and biology; however there is a little information available on the effect of anesthesia using direct electric current in fish. In the present study, the impacts of clove oil (30 mg.l^-1) and electro-anesthesia (constant direct current, 0.5 v.cm^-1) (DC) were assessed in common carp, Cyprinus carpio during narcotic stage in two experimental sets. Experiment 1 was conducted to quantitatively compare induction of anesthesia and recovery time. The biochemical and hematological effects of anesthetics were analyzed as experiment 2. The results revealed that induction and recovery times in fish anesthetized with electric current were significantly lower than clove oil treatment. Significant differences were observed in cortisol concentrations, red blood cells and mean corpuscular hemoglobin concentration between anesthetic methods. Our results suggest that constant direct current electronarcosis was more quickly than clove oil and associated with lower side effects in common carp.

INTRODUCTION

The use of anti-stress agents is a common practice in modern aquaculture. Such substances are utilized to induce sedation or anesthesia during normally stressful procedures such as handling, sorting, tagging, artificial reproduction procedures, and surgery (Velisek et al. 2011). Therefore, reducing these substances may lead to changes in metabolism and growth, immune function, and normal behavior (Barton 2002; Velisek et al. 2011). Furthermore, notably, all sedative and anesthetic procedures themselves induce the primary stress response and cause side effects which may not be considered desirable (Ross & Ross 2008).

Stress is defined as a natural reaction to a negative environmental stimulus which impairs the normal performance, physiology and activity of an animal (Barton 2002; Ross & Ross 2008). On the other hand, with respect to animal welfare, fish should be free from physiological discomfort, disease and functional impairment, fear and chronic stress during handling or surgical procedures (Renault et al. 2011). Proper use of anesthetics may attenuate the primary stress response and, in turn, minimize the occurrence of negative consequences after veterinary practices (Trushenski et al. 2012c).

Basically, there are two types of anesthesia techniques: chemical anesthesia, using plethora of molecules and protocols (Topic Popovic et al. 2012; Ghanawi et al. 2013; Witeska et al. 2014), and physical anesthesia, using electricity, gas pressure or temperature (Vandergoot et al. 2011).

Chemical anesthesia in fish may be achieved by different agents, mainly tricaine methane sulphonate (MS-222), benzocaine, quinaldine sulphate, methomidate and clove oil (Velisek et al. 2011). At present, clove oil is commonly used in aquaculture because of its low cost and rapid effect (Renault et al. 2011); however this chemical anesthetic and its components are not approved as an anesthetic for fish (FDA 2007). Clove oil is a natural product distilled from stems, leaves and flower buds of Eugenia caryophyllata, and its active ingredient, eugenol (4-allyl-2-methoxyphenol), makes up about 80% of the oil by weight (Ross & Ross 2008; Javahery et al. 2012).

An interesting alternative to chemical anesthesia is the use of electricity. The constant direct current of electricity may act as a nonchemical anesthetic by generating electronarcosis in fish. Electronarcosis occurs through the inhibition of medullary motor paths and thus spinal reflex. During electronarcosis, no cerebral message reaches the motor paths, the fish loses equilibrium, and muscle tone, but gill ventilation continues (Henyey et al. 2002). The fish remains in a state of narcosis with the body relaxed while submerged in water and exposed to direct current (DC) within the appropriate voltage gradient range. It is not possible to physiologically achieve the narcotizing effect with alternative current (AC) or pulsed direct current (PDC). The relatively low DC voltage gradient presumably translates to reduced stress and injury to the fish, and a safer working environment for the handler (Hudson et al. 2011).

The stages of anesthesia described by Summerfelt & Smith (1990) are widely referenced (Bowzer et al. 2012; Javahery et al. 2012; Trushenski et al. 2012c). Stage 1 anesthesia refers to light sedation which described by slight loss of reactivity to external stimuli and slightly decreased opercular rates whereas the stage 6 refers to cardiac arrest and asphyxia. According to Summerfelt & Smith (1990), total loss of muscle tone and equilibrium, slow and regular opercular movements, and a loss of spinal reflexes refers to stage 4 anesthesia. This level of anesthesia is consistent with stage 2 anesthesia as described by Mc Farland (1959).

The physiological effect of anesthesia and sedation using electrical current and clove oil have been evaluated in numerous studies (Cooke et al. 2004; Velisek et al. 2005; Altun et al. 2006; Renault et al. 2011; Trushenski et al. 2012a, b, c), but a direct comparison between anesthesia using clove oil and constant direct current of electricity is lacking in the literature.

The objectives of this study  were to compare the induction of anesthesia and recovery times  in common carp (Cyprinus carpio), as a model fish, anaesthetized with clove oil and low - voltage constant direct current of electricity, and to evaluate the effects of two types of anesthesia with regard to some biochemical and hematological parameters.

MATERIALS AND METHODS

Anesthetics

Clove oil (eugenol concentration 75%) obtained from the Giah Essensse Company (Giah Essessense, Gorgan, Iran). Eugenol is the major active ingredient of clove oil and poorly soluble in water; thus clove oil diluted 1:10 with 95% ethanol to yield a working stock solution of 100 mg.ml^-1 (each ml of clove oil contains ̴ 0.75 g of active ingredient) (Noga 2010).

The portable electronarcosis unit was constructed based on Hudson et al. (2011). Briefly, the unit consisted of a holding tank modified from a 70- L marine grade cooler, two electrodes which were installed facing each other 45cm apart at opposite ends of the holding tank, and a 0-30-V DC (Direct Current) power supply (DAZHENG PS- 305D).

Fish

One hundred and twenty juvenile carp (50.08 ± 1.75 g body weight and 15.03 ± 0.19 cm total body length) were obtained from a local fish farm and after transportation to the laboratory acclimatized for 3 weeks to the aquarium facilities at the University of Guilan. The fish were kept in 1000 liter fiberglass tank; the physiochemical parameters of the water during experiment were: NH₄⁺: 0.06 mg.l^-1; NO₂^-: 0.02 mg.l^-1; NO₃^-: 0.5 mg.l^-1; salinity: 2- 3 mg.l^-1; conductivity: 2.24 mS.cm^-1; pH 7.5. During the acclimatization fish were offered food at a rate sufficient to maintain growth. Water temperature (21.3 ± 1.1°C) was continuously monitored. Fish were starved 24 h before the experiment.

Experimental procedure

A preliminary experiment was conducted to achieve proper dosage according to the literature (Velisek et al. 2005; Hudson et al. 2011). Briefly, thirty five fish were randomly collected from the reference stock and divided to six groups; Five groups were exposed to clove oil concentration of 20, 30, 45, 65 & 90 mg.l^-1 and the other group, comprised 10 fish, subjected to the voltage gradient increasing gradually to the range of 0.25-0.56 V.cm^-1. In all cases, fish were ensured to be in the stage IV of anesthesia; the clinical sings were identified as total loss of equilibrium, responsiveness to handling with maintenance of a slow, steady, opercular ventilation rate (Bowzer et al. 2012). With regard to induction time, recovery time and post-sedation distress, 30 mg.l^-1 clove oil and 0.5 V.cm^-1 electricity were used for the principal investigation.

Experiment 1: induction and recovery times

Two groups of 15 fish were randomly collected from the reference stock and transferred to individual holding tanks one day before the experiment. Fish were placed into anesthesia chamber filled with aerated culture water either dosed with clove oil or equipped with the electro-anesthesia unit. Care was taken to monitor fish and determine induction time to attain stage IV of anesthesia. Fish were considered induced to stage IV when they no longer responded to the visual and tactile stimulus, but the opercular rate remained steady and slow. In the case of electro-anesthesia, a tremor was observed immediately following electrical exposure and fish were not responsive to the stimuli during this tremor. Thus, induction was considered complete after the tremor had ceased. After induction fish were weighed and measured to determine total length, and then transferred to an aerated recovery tank. In the tank, fish were monitored to determine recovery of normal equilibrium according to the literature (Bowzer et al. 2012; Trushenski et al. 2012c). The survival rate of recovered fish was controlled for 24 h.

Experiment 2: hematological and biochemical response

Eight groups of fish were used in this experiment. Each treatment group consisted of six fish transferred from the holding tank to an anesthesia chamber. Each anesthesia regime was conducted in triplicate. In each treatment group, one fish sampled immediately after all fish had reached stage IV anesthesia and the rest were returned to a holding tank for further sampling at 0.5, 1, 2, 6 & 24 h post-anesthesia (one fish per group per time point; no fish were sampled more than once). Two control groups out of eight experimental ones were also sampled every hour over the course of the experiment. Since no differences were found between the control fish, the results of these groups were pooled.

The entire capture and blood sampling was conducted within two minutes for each fish. Blood samples collected by caudal vessels puncture with a heparinized syringe and kept on ice (<4 h) until analysis. Subsamples of whole blood were used for determination of hematological parameters; remaining whole blood was centrifuged at 13000 rpm at 4°C for 10 min and resultant plasma was stored at -80°C for further analysis.

Hematocrit was measured using microhematocrit capillaries filled with blood, centrifuged at 3000 rpm for 5 min and expressed as percentage of total blood volume. Hemoglobin was determined with a spectrophotometer (UV- 2100, Unico, USA) at 540 nm absorbance using the cyanomethemoglobin method (Dacie & Lewis 1975). Red blood cell (RBC) and white blood cell (WBC) counts were conducted with Neubauer chamber using Dacie’s dilution fluid (3 g sodium citrate, 99 ml distilled water and 1 ml formalin). Differential leukocyte count was performed with blood smears stained with Giemsa solution. The smears were observed by light microscopy (Olympus, Tokyo, Japan). The RBC indices including mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) were calculated according to Pradhan et al. (2014).

Biochemical indices in plasma included cortisol, glucose and lactate. Plasma cortisol levels were measured by ELISA reader (ELx800, Bio Tek, USA), and glucose and lactate were colorimetrically assessed using an auto-analyzer (Technicon RA- 1000, USA).

Statistical analysis

In experiment 1, individual fish were considered as experimental unit (n = 15).  Data were first tested for normality (the Shapiro – Wilk test) and any abnormally distributed data were transformed logarithmically. Then, induction and recovery times were analyzed using one-way analysis of variance (ANOVA) to reveal significant differences in measured variables among the experimental groups.

Data from experiment 2 were analyzed by one-way, repeated measures ANOVA. For this experiment, replicate groups were considered experimental unit (n = 3). Although each anesthetic was used to triplicate groups, each composed of six fish, it is determined that groups, not individuals, should be served as experimental units. Thus, experimental units represent independent observations. Fish sampled at each time point represented repeated observation made on the same experimental unit.

Data are expressed as mean ± SEM. Statistical analyses were conducted with SPSS 17.0 for windows where significance level was set at 0.05.

RESULTS

Induction and recovery time

Induction and recovery times differed significantly between anesthetics (p<0.05, Table 1). Fish were anaesthetized within 110.47 ± 4.20 sec and 37.73 ± 4.58 sec using clove oil and electric current, respectively. The recovery pattern was to regain equilibrium, then tactile responsiveness in rapid succession. These stages were achieved rapidly in the constant DC when electricity was turned off; the mean times were 3.07 ± 0.41 sec and 4.40 ± 1.27 sec to regain equilibrium and tactile responsiveness, respectively. These figures were 99.14 ± 14.46 sec and 159.41 ± 18.02 sec respectively, for fish exposed to clove oil (p< 0.05).

Table 1. Duration of induction and recovery times for clove oil and electroanesthesia (mean ± SEM, n= 30).

Stress and plasma analyses

Plasma concentrations of cortisol varied significantly between treatments (Pillai’s trace = 1.00, F (4, 1) = 623.3, p = 0.03). Plasma cortisol levels were significantly elevated at 0.5 h, 2 h and again at 24 h post-anesthesia among fish anaesthetized with clove oil, whereas cortisol concentration of electro-anesthetized fish increased at 0.5 h and then stabilized and decreased during the sampling period.

In the clove oil treatment, the highest value of glucose and lactate were observed 1 h after treatment and returned to below resting levels within 24 h post-anesthesia; while the peak of these biochemical responses under electric current treatment were observed 0.5h after anesthesia and returned to the control value by 24 h. There were no significant difference between fish anaesthetized with clove oil and electro-anesthesia (glucose: Pillai’s trace = 0.900, F (4, 1) = 2.250, p = 0.459; lactate: Pillai’s trace = 0.954, F (4, 1) = 5.161, p = 0.317; Fig. 1). The plasma factors for fish in stress - free condition were reported for the reference population.

Fig. 1. Time course of cortisol (a), glucose (b) and lactate (c) in common carp after anesthesia to stage IV with clove oil and constant direct current of electricity. Date are presented as means± SEM. Different letters stand for significant differences within the same group (one-way repeated measures ANOVA: p<0.05).

Table 2. Hematological parameters in common carp anesthetized with clove oil and electricity constant direct current (mean ± SEM).

^a significant effect of anesthesia (repeated- measures ANOVA with Bonferroni correction): p< 0.05

^b significant effect of time (repeated- measures ANOVA with Bonferroni correction): p< 0.05

^c significant effect of anesthesia × time (repeated- measures ANOVA with Bonferroni correction): p< 0.05

Hematological responses

The levels of RBC and MCHC were significantly higher in fish anaesthetized with clove oil than with electro-anaesthetized fish (RBC: Pillai’s trace = 1.00, F (4, 1) = 9862.9, p = 0.008; MCHC: Pillai’s trace = 1.00, F (4, 1) = 22079, p = 0.005; Table 2). The somewhat similar pattern was observed for WBC values; whereas the changes were not statistically significant (Pillai’s trace = 0.594, F (4, 1) = 0.366,

p = 0.826; Tables 2 & 3). Hematocrit and hemoglobin also decreased during the course of sampling (Hct: Pillai’s trace = 0.594, F (4, 1) = 0.366, p = 0.826; Hb: Pillai’s trace = 0.958, F (4, 1) = 5.712, p = 0.303). During these two experiments only one mortality was observed after 24h - observation period and general behavior of the experimental population seemed normal.

Table 3. Differential leukocyte in common carp anesthetized with clove oil and constant direct electric current (mean ± SEM)*.

                 *p>0.05 (significant difference at p= 0.05).