Effects of Clove Oil as a Euthanasia Agent on Blood Collection Efficiency and Serum Cortisol Levels in Danio rerio

Abstract

Zebrafish are an important laboratory animal model for biomedical research and are increasingly being used for behavioral neuroscience. Tricaine methanesulfonate (MS222) is the standard agent used for euthanasia of zebrafish. However, recent studies of zebrafish behavior suggest that MS222 may be aversive, and clove oil might be a possible alternative. In this study, we compared the effects of MS222 or clove oil as a euthanasia agent in zebrafish on the volume of blood collected and on serum levels of cortisol. Greater amounts of serum could be collected and lower serum levels of cortisol were present in fish euthanized with clove oil compared with equipotent dose of MS222. Euthanasia with clove oil did not blunt the expected elevation of serum cortisol levels elicited by an acute premortem stress. According to our findings, clove oil is a fast-acting agent that minimizes the cortisol response to euthanasia in zebrafish and allows the collection of large volumes of blood postmortem. These results represent a significant refinement in euthanasia methods for zebrafish.

Despite the widespread popularity and applicability of the zebrafish as a biomedical research model, the most humane method of euthanasia remains to be defined, not only because our understanding of zebrafish pain and distress is a work-in-progress, but also because few studies have assessed the limited number of euthanasia methods currently available.^10,8 The standard method for killing zebrafish typically involves anesthetic overdose with tricaine methanesulfonate (MS222). Recent work with MS222 has shown that zebrafish find the substance aversive, according to behavioral tests.^9,16 If the aversion behavior represents minor distress, euthanasia by MS222 might be suboptimal for certain types of studies. Specifically, as zebrafish usage increases for neuroscience research,¹³ investigators may prefer to use agents that minimize any additional load on the hypothalamic–pituitary–adrenal axis, which coordinates responses to stress. If the previously noted aversion behavior^9,16 represents at least moderate distress, then switching from MS222 to a more humane agent for the euthanasia of zebrafish may be warranted.

Clove oil has been proposed to be an equally or more effective euthanasia agent than MS222.^3,4,16 Clove oil is a natural anesthetic that is derived from distilled flowers, stems, and leaves of the clove tree, Syzgium aromaticum. Since antiquity, clove oil has been used as a mild topical anesthetic for toothache, headache, and joint pain in humans.⁶ In fish, clove oil has been used successfully for immobilizing fish and suppressing sensory systems during handling, surgery, and invasive procedures. Although most of the reports regarding its effectiveness are anecdotal, details of clove oil's mechanism of action provide support for its use.⁶ Clove oil is highly lipophilic and is absorbed through the skin and gills rapidly. Clove oil enters the blood stream, crosses the blood–brain barrier, and induces death by respiratory inhibition and, ultimately, cardiac arrest.⁶ The 2013 AVMA Guidelines for the Euthanasia of Animals states that the advantages of clove oil include its wide availability, low cost, short induction time, and wide range of efficacy.⁷ Disadvantages include a lack of FDA approval for use as a euthanasia agent.⁷ The general recommendation of the AVMA guidelines is that clove oil is an acceptable agent for euthanasia in fish not intended for consumption.

One of the challenges with zebrafish as a model is that often collected blood samples have insufficient volume for analysis. Traditional methods of collection, involving microcapillary tubes and syringes, typically yield only 2.0 to 4.5 μL per fish. These volumes are often insufficient for biochemical and cytologic analyses.² An alternative method of blood collection by centrifugation allows significantly higher and more predictable yields of 5.5 to 11.0 μL.²

The purpose of our study was to compare differences between euthanasia with MS222 and clove oil. Because time to apparent death is affected by the dosage of agent used, we performed dose–response studies with both clove oil and MS222 to identify equipotent doses. As part of this dose-response study, we also determined the number of fish that recovered after ‘apparent death’ as defined by cessation of opercular movement and nonresponse to tactile stimulation. After euthanizing fish with equipotent doses of clove oil and MS222, we measured the amount of blood collected via centrifugation and assessed serum cortisol levels. We show that euthanasia with clove oil led to larger blood volume yields and lower serum cortisol levels compared with those after MS222. These results have important implications not only for maximizing sample recovery in zebrafish but also for improving fish welfare and minimizing the potential adverse effects of stress on research data.

Materials and Methods

Animals and housing.

Transgenic zebrafish, Danio rerio carrying either Tg(isl1-hsp70l:mRFP), Tg(UAS:GCaMPHS), or Tg(5xUAS:RFP) on an AB background, between 2 to 3 y of age were bred in-house and housed separately by their strains. These strains carry different genetic alterations that have no known phenotypic effect other than expression of fluorescent proteins in different tissues. WT zebrafish were purchased from Aquatica Biotech (Sun City Center, FL). Fish were maintained in recirculating 3-L tanks at 27 ± 2 °C on a 14:10-h light:dark cycle and fed commercial fish diet. Fish were reared according to published guidelines.^5,14 The protocol for the experimental use of fish was approved by the University of Missouri IACUC.

Dose–response experiments.

We used 3 doses of each of the 2 agents to determine an equipotent dose. For MS222 (Sigma–Aldrich, St Louis, MO), the doses were 1.7, 5.1, and 17 mM. For clove oil (Frontier Natural Products, Norway, IA), the doses were 0.01%, 0.03%, and 0.1%. After apparent death, as determined by cessation of opercular movement and nonresponse to tactile stimulation, fish were put into recovery tanks filled with tank water for 30 min; the numbers of fish that recovered and did not recover were recorded. Prism 6.0 (GraphPad Software, La Jolla, CA) was used to plot the dose–response curves of the agents. A nonlinear regression of the log drug concentration was compared with response (3 parameters) was fitted to each data set and used to interpolate the concentration of agent necessary to produce apparent death in 1 min (MS222, 7.8 mM; clove oil, was 0.1%).

Euthanasia and blood collection.

Euthanasia was performed by using modifications of previously described techniques.² Briefly, each fish was placed into an individual 50-mL conical tube containing either 30 mL of unbuffered (pH 2.7 to 3.7) 7.8 mM (approximately 2300 mg/L) MS222 or sterile water containing 0.1% (100 mg/L) of clove oil. The time of death was determined according to cessation of opercular movement and nonresponse to tactile stimulation. The fish were weighed, and then blood was collected by cutting off the tail a few millimeters cranial to the caudal fin. Fish were placed in a fenestrated microtube (0.6 mL) nested within a 1.5 mL microfuge tube for centrifugation at 400 × g for 5 min at room temperature. The 1.5-mL tubes containing the blood samples were centrifuged at 4 °C, 13,800 × g for 15 min. Supernatants were recovered and the serum volumes measured. Samples were stored at –80 °C until analysis.

Acute stressor.

As previously described,¹ fish were stressed by chasing them with a fish net for 2 min immediately prior to euthanasia.

Cortisol ELISA.

Serum cortisol concentrations were determined by using a cortisol ELISA kit (Salimetrics, Carlsbad, CA) according to the manufacturer's instructions. The sensitivity of the assay is less than 0.007 μg/dL, and cortisol concentrations were read on a plate reader (SpectraMax M3, Molecular Devices, Sunnyvale, CA).

Statistics.

Data were analyzed by using Prism (GraphPad Software) and SigmaPlot (Systat Software, San Jose, CA). Statistical significance was set at a P value of less than 0.05. Serum volumes were normalized to body weight and then analyzed by using 2-factor ANOVA, with sex and euthanasia method as factors. In addition, ANOVA was performed to determine differences in serum cortisol levels, with sex and euthanasia method as factors. The Student t test was used to compare euthanasia time between treatment groups (MS222 or clove oil) and to compare cortisol levels between stressed and nonstressed groups euthanized with clove oil. All ANOVA were analyzed by using the Student–Newman–Keuls posthoc test.

Results

Determination of equipotent dose for euthanasia.

Multiple doses of MS222 and clove oil were tested to obtain an equipotent dose (Figure 1). The equipotent dose for a euthanasia time of 1 min was interpolated from the nonlinear curves for the 3 doses of each agent. Accordingly, 7.8 mM MS222 or 0.1% clove oil is required to cause death of zebrafish within 1 min. The number of fish that recovered after apparent death (as defined by cessation of opercular movement and nonresponse to tactile stimulation) was assessed by placing treated fish in a recovery tank containing tank-system water. At the highest doses of each agent tested (n = 4 per group; MS222, 17 mM; clove oil, 0.1%), no fish recovered; therefore we selected an equipotent dose near these concentrations for use in the remaining experiments in this study.

Figure 1.

Time (min, mean ± SEM) from first exposure until apparent death, as measured by cessation of opercular movement and nonresponse to a tactile stimulus, required to euthanize wild-type zebrafish (n = 4/group) by clove oil and MS222. Doses for each agent shown below figure. Dotted line represents equipotent dose chosen for the remaining experiments in this study.

Serum cortisol.

Male and female WT zebrafish were used to test the effect of equipotent doses of MS222 and clove oil on serum cortisol levels. The time to death was measured to ensure equipotency (Figure 2 A). Serum cortisol concentrations were greater (P <0.05) in fish euthanized with MS222 compared with clove oil, as determined by 2-way ANOVA (Figure 2 B). Posthoc tests revealed a significant effect of sex in female (P = 0.02) but not male (P = 0.08) zebrafish.

Figure 2.

Time to death (s) and cortisol levels (μg/dL) at equipotent doses of MS222 and clove oil in WT zebrafish (n = 5 male and 5 female fish per group). (A) Equipotency was confirmed by measuring the time to death, which did not differ between groups. (B) Serum cortisol levels were significantly (*, P < 0.05) increased in zebrafish euthanized by using MS222. Data are given as mean ± SEM.

Serum recovery.

Three strains of zebrafish—WT, Tg(isl1-hsp70l:mRFP) and Tg(UAS:GCaMPHS)—were used to test the volumes of serum that could be collected (Figure 3). All groups showed increased serum recovery when clove oil was used as the euthanasia agent. Figure 3 A shows the serum recovery at equipotent doses of clove oil and MS222 in WT zebrafish. For the transgenic stains tested, serum was normalized to body weight, and more serum was collected from fish euthanized with clove oil compared with MS222 in Tg(isl1-hsp70l:mRFP) (Figure 3 B) and Tg(UAS:GCaMPHS) (Figure 3 C) zebrafish.

Figure 3.

Serum volumes (μL per 1 g body weight) after euthanasia by clove oil and MS222. (A) WT (n = 5/group). (B) Tg(isl1-hsp70l:mRFP) (n = 5/group). (C) Tg(UAS:GCaMPHS) (n = 6/group). Bars denote mean ± SEM; †, P < 0.01; ‡, P < 0.001.

Cortisol levels after acute stressor.

To test whether clove oil masked the ability to detect changes in serum cortisol levels produced in response to acute stress, fish were chased with a net for 2 min prior to euthanasia. Tg(5xUAS:RFP) zebrafish had significantly (P = 0.01) higher circulating cortisol levels after exposure to an acute stressor than did nonstressed controls (n = 6 per group; stressed, 5.2 ± 0.9 μg/dL; unstressed, 1.8 ± 0.7 μg/dL; P = 0.01).

Discussion

In zebrafish, MS222 is an accepted and widely used agent for euthanasia. Recent reports show that zebrafish find MS222 aversive in conditioned place-avoidance paradigms and exhibit signs of distress when exposed to MS222.^9,15,16 Studies involving clove oil suggest that it may be less aversive to zebrafish and provide a more humane alternative to MS222 as an agent for euthanasia.^4,16 Barriers to the use of clove oil include a lack of FDA approval and some safety concerns regarding carcinogenicity, and whether use of the agent renders fish unfit for human consumption.⁵

Indicators of an aversive or distressing stimulus in zebrafish include a behavioral tendency to avoid the stimulus, excessive movement of the opercula (hyperventilation) in response to the stimulus, and the degree to which the stimulus induces elevations in serum cortisol.^1,8,16 Here, we show that zebrafish euthanized with MS222 have higher serum cortisol levels than do those euthanized with an equipotent dose of clove oil. These results suggest that MS222 causes increased distress, corroborating previous studies indicating that MS222 is aversive or distressing.^9,15

Buffering MS222 with Tris or bicarbonate has no effect on the agent's efficacy.¹² However, some researchers have speculated that the acidity of an unbuffered solution of MS222 is a potential source of aversion. Previous work has shown that fish display signs of distress in the presence of MS222 regardless of whether it is buffered or not.¹⁵

To ensure that clove oil did not interfere with the ability to detect increases in serum cortisol levels in response to acute stress, we exposed a group of fish to an acute stressor and measured their serum cortisol concentrations. As seen in previous studies,¹ chasing a fish for 2 min prior to euthanasia was sufficient to increase cortisol levels. We measured increased serum cortisol in acutely stressed fish compared with controls after both groups were euthanized with clove oil. In light of this finding, clove oil is a suitable agent in studies evaluating aspects of the stress response because this agent does not interfere with the detection of increased cortisol levels.

Another benefit of clove oil over MS222 is the ability to recover an increased volume of serum after euthanasia. Blood collection in zebrafish is challenging due to their rapid blood clotting time and the small total blood volume in the fish. Traditional blood collection methods use microcapillary tubes and syringes, which can be tedious and result in small volumes of blood collected.² Clove oil mainly contains a substance called eugenol, which makes up between 75% to 95% of the oil. Eugenol strongly inhibits platelet aggregation induced by platelet-activating factor in rabbits.¹¹ This inhibition of platelet aggregation makes clove oil an effective anticoagulant and most likely explains the increased blood collection after euthanasia of zebrafish. This study is the first known to link clove oil as an agent for euthanasia to increased blood-volume collection in zebrafish.

According to the Guide,⁵ “The selection of the most appropriate analgesic or anesthetic shall reflect professional judgment as to which best meets clinical and humane requirements without compromising the scientific aspects of the research protocol.” Our results suggest that the use of clove oil as a replacement for MS222 is not only a more humane method that significantly decreases distress during euthanasia but also one that facilitates the collection of increased amounts of serum. Compared with MS222, clove oil may be a more appropriate euthanasia agent for studies in zebrafish where uncompromised measures of the stress response are important readouts for addressing scientific questions.