The effect of three anaesthetic protocols on the stress response in cane toads (Rhinella marina)

R E S E A R C H P A P E R

The effect of three anaesthetic protocols on the stress

response in cane toads (Rhinella marina)

Sandra E Hernandez, Conrad Sernia & Adrian J Bradley

School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia

Correspondence: Sandra E Hernandez, School of Biomedical Sciences, The University of Queensland, Brisbane, 4072 Queensland, Australia.

E-mail: [email protected]

Abstract

Objective Three anaesthetics (MS222, clove oil and

a mixture of ketamine/diazepam) were administered

to cane toads to determine their effect on the

hypothalamic-pituitary-adrenal (HPA) axis. Time to

induction and recovery and any adverse events

were also evaluated.

Study design Prospective randomized experimental

trial.

Animals Thirty adult male cane toads (Rhinella

marina) with body mass ranging between 130 and

250 g were captured from the field.

Methods Three groups of 10 toads were anaesthe-

tized with ketamine (200 mg kg)1) and diazepam

(0.2 mg kg)1) by intramuscular injection, MS222

(3 g L)1) or clove-oil (0.3 mL L)1) both by immer-

sion. Blood samples were collected to determine

plasma corticosterone concentrations. Induction

and recovery time were recorded in each treatment.

After full recovery animals were euthanized and a

complete post-mortem examination was performed.

Results Significant differences were found in the

activation of the HPA axis and in the times of

induction and recovery between treatments

(p < 0.001). Animals anaesthetized with clove-oil

had the highest levels of corticosterone in plasma

(42.5 ± 21.6 ng mL)1). No differences were found

between ketamine/diazepam (15.0 ± 13.3 ng mL)1)

and MS222 (22.0 ± 13.6 ng mL)1) groups. The

mean ± SD induction (minutes) and recovery

(hours) times respectively were; ketamine/diazepam

66.5 ± 11 and 8 ± 3, clove oil 39 ± 12 and 7.6 ± 3,

and MS222 42.5 ± 11 and 1.5 ± 0.5. Clove oil

exposure had 30% mortality. Death followed a

period of respiratory distress with changes consistent

with non-cardiogenic oedema observed at post-

mortem examination.

Conclusions and Clinical relevance Based on shorter

induction and recovery times and minimal

activation of HPA, MS222 is the anaesthetic of

choice in cane toads. If it is not possible to use

immersion methods of anaesthesia, ketamine/

diazepam can be used but induction and recovery

times are prolonged. Clove oil had unacceptable

mortality in this study and should be used with

extreme caution.

Keywords anaesthesia, cane toads, clove-oil, keta-

mine, MS222, stress.

Introduction

Many laboratory or field interventions require the

use of anaesthetics to reduce distress and pain in

animals. An ideal anaesthetic will provide anaes-

thesia (loss of consciousness and reduced reflex

response), analgesia and amnesia for the course of

the intervention. In the case of field anaesthesia it is

vital that induction and recovery times are short or

that the anaesthetic can be easily antagonized.

1

Veterinary Anaesthesia and Analgesia, 2012 doi:10.1111/j.1467-2995.2012.00753.x

Since it is difficult to assess pain and distress by

direct observation in amphibians, mediators of pain

or stress that are released commonly in vertebrates

may be used as markers. The level of activation of the

hypothalamic-pituitary-adrenal (HPA) axis is con-

sidered an acceptable method of assessing the

response of an organism to a stressor (Sapolsky et al.

2000). As part of the early stress response catechol-

amines (epinephrine and norepinephrine), followed

soon after by glucocorticoids (cortisol and corticoste-

rone) usually increase in concentration in the blood

(McEwen & Wingfield 2003). It is common to use

glucocorticoids as markers of stress, because the

plasma concentration of this class of adrenocortical

hormones increases in response to handling, surgical

intervention, or anaesthesia (Bentson et al. 2003). In

amphibians, plasma corticosterone concentration is

a good indicator of physiological or environmen-

tally induced stress since it can be related to the

stage of development (Krug et al. 1983; Wright

et al. 2003), seasonal changes (Mukherji 1968;

Piezzi & Burgos 1968), level of activity and energy

expenditure (Emerson & Hess 2001; Romero et al.

2004), and to anthropogenic stressors (Hopkins

et al. 1999). However, no studies have been per-

formed to determine the effect of general anaesthe-

sia on the HPA axis in amphibians.

Anaesthesia in amphibians can be achieved by

injection, inhalation, topical application, or immer-

sion (dissolving the agent in water and placing

the animal in the solution) (Wright 2001b,c). Top-

ical and immersion administration are the most

commonly used, with MS222 (Tricaine methanesulf-

onate [3-aminobenzoic acid ethyl ester methanesul-

fate]) as the preferred choice (Wright 2001b,c) and

having the American Veterinary Medicine Associa-

tion (AVMA) and Australian approval (Haskell et al.

2004; Gentz 2007). Clove oil has been mentioned as a

possible alternative anaesthetic in amphibians (Gentz

2007). Clove oil and its principal chemical component

eugenol (4-allyl-2-methoxyphenol) have been used

regularly in human dentistry because of their local

anaesthetic and antiseptic properties (Sticht & Smith

1971). Clove oil has been used for chemical restraint in

amphibians, where it is reported to have anaesthetic

and analgesic activity (Guenette et al. 2007; Mitchell

et al. 2009; Goulet et al. 2010). Apart from its

chemical properties, clove oil is also attractive because

of its low cost and wide availability (Keene et al. 1998;

Walsh & Pease 2002; Iversen et al. 2003).

Ketamine, an N-methyl-D-aspartate (NMDA)

antagonist that produces dissociative anaesthesia,

has been reported as an option in amphibians with

diverse results (Wright 2001c). Used alone it gives

poor muscle relaxation and therefore is normally

given in combination with benzodiazepines. Diaze-

pam is most commonly reported in amphibians

(Wright 2001b) and gives central muscle relaxation

and additional tranquilization. Previous reports

show that while a number of topical and injectable

preparations have been used as anaesthetics in

amphibians, there has not been a systematic study

of their comparative effectiveness which includes

physiological parameters of stress. Knowledge of the

extent to which the chosen chemical restraint is

exacerbating the stress response, and whether it is

provoking secondary effects, is extremely important

in order to assess the physiological impact of general

anaesthesia on an animal.

In this study the effect of three anaesthetic proto-

cols on the stress response in cane toads (Rhinella

marina) was assessed. Plasma corticosterone was

measured to evaluate the effects of anaesthesia on the

HPA. Time to induction and recovery of anaesthesia

were also assessed, as were any adverse reactions

to the anaesthetic agents. The overall objective of

the study was to form conclusions regarding the

relative suitability of the agents for anaesthesia of

cane toads and potentially other amphibians.

Material and methods

Animal subjects

All procedures were approved by The University of

Queensland Animal Ethics Committee (SBMS/437/

09/URG/GOVTMEX/HSF/CFOC).

A total of 30 male cane toads Rhinella marina

weighing more than 100 g (range 130–250 g),

were captured from The University of Queensland

Lakes, St Lucia, SE Queensland, Australia. Toads

were housed individually with a wet bed, a depth of

2 cm of water and a dry refuge area containing a

section of poly-vinyl-chloride pipe (120 · 300 mm).

An ambient temperature of 22–24 �C was main-

tained with a light cycle of 12 hours of dark:

12 hours of light. All animals were fasted for

24 hours before exposure to the anaesthetics.

The experimental protocol consisted of three

groups of ten toads. Toads were assigned randomly

to a group as they were captured. The first group

received a dose of 200 mg kg)1 of ketamine

(Ketamil; Troy laboratories Pty Ltd., Australia)

combined with 0.2 mg kg)1 of diazepam (Pamlin;

Three anaesthetic protocols in cane toads SE Hernandez et al.

� 2012 The Authors. Veterinary Anaesthesia and Analgesia2 � 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists

Parnel Pty Ltd., Australia), administered intramus-

cularly (IM.) in the hind leg (semimembranosus)

(Wright 2001b,c). They were placed in a dry

container to avoid drowning during the induction

period. The containers utilized to maintain and

induce anaesthesia were opaque plastic containers

of 4 L in capacity with lids perforated to facilitate

ventilation. The second group was immersed in

3 g L)1 of MS222 (SIGMA-Aldrich, Ethyl 3-amino-

benzoate methanesulfonate 98%, MO, pH7) (Wright

2001b,c). The third group was immersed in

0.3 mL clove oil L)1 water (Wright 2001b). Clove

oil is not soluble in water at low temperature

(<15 �C), therefore it was prepared as a stock of

3 mL of clove oil (Biotech pharmaceuticals Pty Ltd.,

Australia) in 10 mL of ethanol (ETOH 90%). The

quantity of ethanol used to solubilise clove oil in this

study was 100 times lower than concentrations

reported in the literature (Keene et al. 1998; Ross &

Ross 2008) therefore no secondary effect attributable

to ethanol was expected. In Bufo sp. the inguinal area

has the highest capacity for transcutaneus absorp-

tion (Ogushi et al. 2010), so the volume of water

used to induce the animals was enough to submerge

a third of their height. Furthermore maintaining

the animals in a low volume of water facilitated the

introduction of a sponge or a piece of fabric to

the container enabling the head to be kept clear of

the water once the righting reflex was abolished.

Surgical procedure and evaluation of anaesthesia

Time of induction of anaesthesia was defined as the

time from the administration of the anaesthetic to

the time the animal lost righting reflex, palpebral

reflexes and involuntary muscular movement.

Righting reflex was tested placing the animal in the

dorsal position and observing whether the animal

tried to return to the ventral position. Palpebral

reflex was tested by gently tapping the palpebral

area with the finger and observing eyelid move-

ment. Involuntary muscular movement was tested

via the withdrawal reflex, by grasping each limb

distally and extending it. Patellar junction and tar-

sal junctions were tapped with a light object to test

myotactic reflex (Wright 2001c; Wright et al.

2001). Reflexes were assessed every 5 minutes

during induction. When the appropriate level of

anaesthesia was achieved, nociceptive withdrawal

was tested by applying a hard pinch on the inter-

digital skin in the hind limbs, inguinal area and

ventral area using straight blunt tipped tweezers.

A two centimetre incision was then made in the

skin below the xiphoid process in the abdominal

midline to expose the abdominal muscles and

ventral vein. A catheter 24 gauge · 19 mm was

inserted into the vein (Introcan Certo, Braun,

Germany). The catheter was fixed by suturing the

connecting hub to the pectoral muscles and sutur-

ing the skin around it, leaving the end of the

catheter with the cap exposed for blood sampling.

Catheters were placed to facilitate blood sampling

and to allow humane euthanasia at the end of the

study period. Once the placement of the catheter

was completed a blood sample (500 lL) was

collected into heparin treated syringes (Wright

2001a). The collected blood was placed into Eppen-

dorf tubes and centrifuged at 1,300 g for 5 minutes

to separate plasma from blood cells. Plasma was

stored at )20 �C for later corticosterone analysis.

Following surgery and blood collection flunixin

meglumine 1 mg kg)1 IM (Finadyne; Schering-

Plough Pty Ltd., Australia) an analgesic and

non-steroidal anti-inflammatory, was administered

IM in the front leg (deltoid muscle) (Wright 2001b).

Animals were then rinsed with fresh water and

returned to their cages to recover. Reflexes in

animals were monitored every 5 minutes for 1 hour

to determine recovery time. Reflex testing was then

conducted every 30 minutes for the next 2 hours

and finally every hour until recovery. A full

recovery was defined by the appearance of the

righting reflex and a return of ambulatory behav-

iour defined as the animal performing purposeful

and coordinated movement. After full recovery, all

animals were euthanized with 100 mg kg)1 of

pentobarbital (Lethabarb; Virbac Pty Ltd., Australia)

administered intravenously (IV) through the

catheter (Wright 2001b,c). After euthanasia all

animals were subject to a full post mortem exam-

ination. Special attention was paid to the examina-

tion of the respiratory, cardiovascular, hepatic and

renal systems.

Plasma corticosterone analysis

Plasma samples (20 lL) were assayed for cortico-

sterone by radioimmunoassay (Millis et al. 1999).

The tracer used was tritiated corticosterone (Corti-

costerone [1,2,6,7-3H] 2.59 TBq mmol)1; Perkin

Elmer Lab, MA, USA). Radioactivity was counted in

a liquid scintillation spectrometer (Beckman LS

6000 TA). For this study we used a polyclonal

antibody against corticosterone developed by


� 2012 The Authors. Veterinary Anaesthesia and Analgesia� 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists 3

AbCam laboratories (Product Number: ab77798),

raised in sheep and based on corticosterone-

3-cmo-urease as immunogen. The cross-reactions

documented were: 0.67% to 11-dehydrocorticos-

terone, 1.5% to deoxycorticosterone, and <0.01% to

18-OH-DOC, cortisone, cortisol and aldosterone.

The plasma samples were measured in duplicate

and the corticosterone concentration was calculated

from a standard curve, as described by Dudley

(Dudley et al. 1985).

Statistical analysis

The D’Agostino-Pearson omnibus test demonstrated

that the variables had a normal distribution

(p > 0.05). ANOVA analysis was used to determine

differences between treatments, followed by a Tukey

test to compare group means. The null hypothesis

was rejected at p < 0.05. The data were expressed

as mean ± SD. All statistical analyses were con-

ducted using GraphPad Prism (GraphPad Software,

CA, USA, 2009).

Results

The induction time was significantly different

between the three treatment groups (p < 0.001,

Fig. 1a). Ketamine/diazepam had the longest time of

induction of anaesthesia and was significantly dif-

ferent to MS222 and clove oil (Fig. 1). Time of

recovery was similar between ketamine/diazepam

and clove oil) but markedly longer than for MS222

(p < 0.001, Fig. 1b).

The highest levels of corticosterone during anaes-

thesia were observed for clove oil and were signif-

icantly greater (p < 0.05) than the plasma

corticosterone concentrations found for ketamine/

diazepam or MS222 (Fig. 2).

During the recovery period three of the ten toads

exposed to clove oil died and two others presented

with respiratory distress. The toads with respiratory

distress were euthanized to achieve a humane end

point. It was observed that these five toads were

those with the highest body weight and that they

were in contact with the clove oil solution for the

longest period of time to achieve the required level

of anaesthesia. Physical examination of the animals

with respiratory distress revealed a cyanotic oral

mucosa and an increase of serous exudates from

nares and mouth. The major findings of the post-

mortem in these animals were cyanosis of the oral

mucosa, haemorrhage and oedema in lung paren-

chyma with an accumulation of liquid in the

pulmonary sac and upper airways. No change in

the normal morphology of heart, vascular system or

other systems was found. These findings are con-

sistent with non-cardiogenic pulmonary oedema as

the cause of death. No important changes were

found at post-mortem in the other animals used in

the study.

Discussion

While amphibians are common research subjects,

information on suitable anaesthetic agents is very

limited. In this study we examined three anaesthetic

agents using plasma adrenal steroid concentration

as an index of stress, in addition to using stan-

dard observational criteria. The corticosterone

concentration was markedly higher in the clove oil

group than the MS222 and ketamine/diazepam

groups. Previous studies using cane toads report

Clove oil Ketamine/diazepam MS2220

20

40

60

80

a

b

a

Tim

e of

indu

ctio

n (m

inut

es)


2

4

6

8

10

12a

a

bTim

e of

reco

very

(ho

urs)

(a)

(b)

Figure 1 Times (mean ± SD) to induction of (1a) and

recovery from (1b) anaesthesia in cane toads anaesthetised

with one of three anaesthetic protocols (n = 10 in each

group).Different letters indicate statistical differences

between treatments (p < 0.05). Clove oil (0.3 mL L)1)

and MS222 (3 g L)1) were administrated by Immersion,

and ketamine (200 mg kg)1) diazepam (0.2 mg kg)1)

given by IM injection.



concentrations of corticosterone in plasma ranging

between 5–20 ng mL)1 (Orchinik et al. 1988).

These concentration are similar to those found in

the group anaesthetized with ketamine/diazepam

and MS222 but are approximately half of those

found when clove oil was used to induce anaes-

thesia. An increase in glucocorticoid concentration

in response to anaesthesia is an observation com-

mon in fish (Sink et al. 2007), dog (Fox et al. 1998)

and horses (Taylor 1991) and this study confirms

that it also occurs in the cane toad and presumably

in amphibians in general. Our observations with

clove oil are mirrored by a study in rainbow trout by

Sink et al. (2007) that reported clove oil to be less

effective than MS222 in reducing stress-induced

cortisol elevation. In addition, there was evidence of

a greater mortality in field-handled fish following

clove oil anaesthesia. These authors suggested that

the stress and higher mortality were due to the

irritant nature of clove oil odour, and possibly to

poor anaesthetic properties which rendered the fish

immobile without loss of consciousness (Sink et al.

2007). Data from rat nerve-muscle preparations

show that eugenol, the main component of clove oil,

is effective in blocking neuromuscular transmission

at low concentrations, thereby supporting the view

that clove oil may be causing neuromuscular

paralysis without anaesthesia (Ingvast-Larsson

et al. 2003). Despite these reservations eugenol can

cross the blood-brain barrier and reduce brain

activity when it is administered intraperitoneally in

rats (Sell & Carlini 1976). Furthermore IV admin-

istration of eugenol produces a reversible, dose-

dependent anaesthesia in male rats (Guenette et al.

2006). Therefore the route of administration is a

significant factor to consider since topical adminis-

tration would lead to paralysis while requiring a

much longer time for sedation and anaesthesia.

Studies on fish have shown that, even though the

primary route of adsorption of anaesthetic dissolved

in water is via the gills, the skin also plays an

important role. Studies in fish reported by Ferreira

et al. (1984) and Gilderhus (1989) showed that the

highest concentrations of anaesthetic are needed as

the size of the animal and skin thickness increases.

A report in amphibia (Xenopus laevis) exposed to

clove oil showed that body size influences induction

and recovery times (Goulet et al. 2010, 2011).

Young and smaller animals require shorter induc-

tion times and have a shorter surgical anaesthesia

period, compared with bigger animals (Goulet et al.

2010, 2011). Consequently age, body mass, body

surface area in contact with the water, and thick-

ness of the skin can affect the uptake of anaesthetics

diluted in water. In this study the effect of life cycle

was minimized by using toads with body weights of

more than 100 g, which is considered adult stage

(Zug et al. 2001).

The induction and recovery times found in the

present study were longer than those previously

reported for induction with MS222 (15–30 minutes)

(Wright 2001b,c; Ross & Ross 2008; Torreilles et al.

2009) and clove oil (15–30 minutes) (Lafortune

et al. 2000; Guenette et al. 2007; Goulet et al.

2010). Previous studies using MS222 and clove oil

were performed in aquatic amphibians completely

submerged in the water containing the anaesthetic.

In this study we used a species that spends most of its

life out of the water and its skin is thicker compared

with aquatic species. Furthermore the animals in this

study were not totally submerged in the water

containing the anaesthetic to avoid animals drown-

ing during the induction period. Therefore the total

skin area in contact with the anaesthetic was reduced

and in consequence this increased the time of

induction. The recovery time for MS222 was similar

to previous reports (40 minutes) (Wright 2001c;

Mitchell et al. 2009), but for clove oil (2–3 hours)

(Lafortune et al. 2000; Guenette et al. 2007; Goulet

et al. 2010) it was longer. One probable explanation

is that as the animals have to stay for longer in

contact with the anaesthetic, the recovery times in

the animals increase too. Therefore time of exposure

to clove oil seems to be another factor that needs

careful consideration. Moreover, the mortality rate in

the animals exposed to clove oil was 30%, increasing

to 50% after our decision to euthanase toads in


20

40

60

80

a

bb

Cor

ticos

tero

ne (n

g m

L–1 p

lasm

a)

Figure 2 Plasma levels of corticosterone (means ± SD) in

cane toads during anaesthesia induced with one of three

anaesthetic protocols.Different letters indicate statistical

differences between treatments (p < 0.05).



respiratory distress, which may will have failed to

recover if they had been left to do so.

Our observations on adverse reactions to clove oil

are supported by findings in frogs (Xenopus laevis)

exposed to eugenol (Goulet et al. 2011). Histopathol-

ogy on the respiratory system showed that clove oil

provoked hyperplasia of the epithelium around alve-

oli in the lung sacs and hyaline membranes in the

lung parenchyma. There is a report of interstitial

pulmonary haemorrhage and oedema, after tracheal

instillation of clove oil in rats and hamsters (LaVoie

et al. 1986). In humans the use of clove cigarettes

has been linked to pulmonary oedema (LaVoie et al.

1986; JAMA 1988), and there is a case report of non-

cardiogenic pulmonary oedema following IV injec-

tion of clove oil (Kirsch et al. 1990). The mechanism

by which clove oil induces inflammatory responses is

not clear. In vitro studies suggest the redirection of

arachidonic acid into the inflammatory pathways

involving prostaglandins, thromboxane and leuko-

trienes may be involved (Rasheed et al. 1984).

It is worth noting that among all the groups

ketamine/diazepam had the longest period of induc-

tion of anaesthesia. This finding is similar to those

reported for other amphibian studies (30–60 min-

utes) (Wright 2001b,c), even though our dose was

higher. In the case of recovery, ketamine/diazepam

and clove oil have the longest periods of recovery, a

finding consistent with previous reports (18 hours)

(Wright 2001c; Mitchell et al. 2009).

In conclusion MS222 anaesthesia of cane toads is

superior to ketamine/diazepam or clove oil anaes-

thesia due to shorter induction and recovery times,

and minimal activation of the HPA axis. If it is not

practical to perform immersion anaesthesia, a

combination of ketamine and diazepam can be used

IM. Induction and recovery times are prolonged

when using this method, making it unsuitable for

field use. However it appears to be no more stressful

than MS222 anaesthesia. Clove oil administration

in cane toads caused adverse changes in the

respiratory system leading to an unacceptable high

mortality rate. Therefore clove oil should be used

with extreme caution until further investigations

into the mode of action, general toxicity, and routes

of administration have been performed.

Acknowledgements

The National Council of Science and Technology

(CONACYT), Mexico for providing scholarship

support to SEH.

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Received 26 July 2011; accepted 27 January 2012.



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The effect of three anaesthetic protocols on the stress response in cane toads (Rhinella marina)