Fish Physiology and Biochemistry 27: 19–28, 2002.

© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

19

The effects of fish anaesthetics (MS222, metomidate and AQUI-S) on heart

ventricle, the cardiac vagus and branchial vessels from Chinook salmon
(Oncorhynchus tshawytscha)

J.V. Hill, W. Davison and M.E. Forster∗

School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand; ∗ Author
for correspondence (E-mail: malcolm.forster @canterbury.ac.nz)

Accepted: September 30, 2003

Key words: anaesthetics, branchial arteries, heart, teleost, vagus nerve

Abstract
The acute effects of 3 fish anaesthetics (MS222, metomidate and AQUI-S) were investigated on 3 parts of the
cardiovascular system of Chinook salmon (Oncorhynchus tshawytscha). All 3 anaesthetics reduced the contractile
force of paced strips of ventricular myocardium. MS222 reduced the contractility by almost 75%, and was more
potent than metomidate and AQUI-S, which reduced the contractility by about 25%. MS222 blocked vagal nerve
transmission to the heart at the normally applied anaesthetic concentration (NAAC) for Chinook salmon, whereas
metomidate and AQUI-S required 100 times their NAACs to have the same effect. Using myography, MS222 and
AQUI-S caused a maximal 30–40% dilation of EBAs at 10% NAAC, whereas the equivalent effect with metomidate
was only seen at 100% NAAC. MS222 again caused the greatest dilation of the ABAs. AQUI-S dilated the ABAs
at up to 50% NAAC, but this was reversed so that there was no dilation at 100% NAAC. Metomidate did not affect
the ABAs. These data from in vitro and in situ experiments, which generally show inhibitory effects, are used to
suggest possible cardiovascular outcomes in anaesthetised Chinook salmon.

Abbreviations: ABA – afferent branchial artery; EBA – efferent branchial artery; NAAC – normally applied
anaesthetic concentration (MS222 100 ppm; AQUI-S 60 ppm, metomidate 7 or 10 ppm).

Introduction blood, especially through the venous system where

Impaired cardiac function during anaesthesia can have
detrimental consequences in fish. Reduced gill perfu-
sion will cause hypoxia which would eventually lead
to death. A reduced cardiac output will also increase
the recovery time as fish anaesthetics are removed
from the site of action in the blood and then de-
graded and/or excreted elsewhere. In dogfish (Squalus
acanthias) 95% of accumulated MS222 is excreted by
the gill and 5% is metabolised and excreted via the
kidney (Stenger and Maren 1974).
Cardiac output, as a product of heart rate and stroke
volume, is controlled to meet demand. Agents that af-
fect the heart directly or by interference with intrinsic
cardiovascular control could compromise cardiac out-
put. In addition, skeletal muscle contractions move
blood pressures are low (Satchell 1991). As anaesthet-
ised fish are often immobilised, greater emphasis is
placed on the heart for adequate perfusion.
Local anaesthetics affect nerves and muscles by
interfering with ion movements. Nerves become in-
capable of transmitting action potentials and all types
of muscle lose contractile ability (Roweland 1974;
Hall and Clarke 1991). In mammals, local anaesthet-
ics can cause the heart to reduce contractile force and
induce arrhythmias (Hall and Clarke 1991). Neural
control of the heart may also be impaired. Some
general anaesthetics such as halothane, enflurane, iso-
flurane and nitrous oxide are known to have a negative
inotropic effect on the heart in vitro but often no effect
in vivo, probably due to compensatory homeostatic
control (Stoelting and Miller 1989). MS222, a chem-
20
ical with local anaesthetic properties, is known to
affect the contractility of the heart of rainbow trout
(Oncorhynchus mykiss, Ryan et al. 1993). Similarly,
analogues of the active component of AQUI-S signi-
ficantly affect the cardiovascular systems of mammals
(Dundee and Clarke 1979).
Vagal outflow has a negative chronotropic effect on
the heart in many teleosts and elasmobranchs (Ran-
dall 1970; Satchell 1991; Olson 1998) but, in some
teleosts, there are also sympathetic fibres in the vagus
which can increase heart rate (Saito and Tenma 1976;
Donald and Campbell 1982; Taylor 1992). It has been
suggested that MS222 can block excitatory adrener-
gic vagal stimulation of the heart of parore (Girella
tricuspidata, Montgomery et al. 1986). In contrast,
a decrease in heart rate in rainbow trout after 30
minutes of 100 ppm MS222 anaesthesia was attributed
to increased cholinergic vagal tone and was reversed
by intraperitoneal and/or intrapericardial injections of
atropine (Lochowitz et al. 1974). Other authors sug-
gest tonic cardiovagal inhibition is depressed centrally
during MS222 anaesthesia (Houston et al. 1971).
The fish gill fulfils a number of vital functions
including ion regulation and respiration. The gills
receive all the cardiac output immediately after it
is ejected from the heart and this blood must be
oxygenated adequately before it enters the systemic
circulation. Fish gill haemodynamics are controlled
by hormonal, neural, local and physical parameters
(Wood 1974; Olson 2002) that can react to numer-
ous exogenous and endogenous changes in order to
maintain gill function. Changing gill haemodynamics
with exogenous compounds is potentially detrimental
to the fish. Anaesthetics, and local anaesthetics in par-
ticular, are known to affect vasoactivity in mammals
(Hall and Clarke 1991). Vasoconstriction or vasodila-
tion can alter the pattern of blood flow through gill
filaments and lamellae (Farrell et al. 1979). This may
in turn influence respiratory gas and ion exchanges
(O2 , CO2 /HCO− 3 ) and could lead to hypoxia and/or
hypercapnia.
The following work examines the direct chem-
ical effect of three commonly used fish anaesthetics:
MS222, metomidate and AQUI-S, on the contractile
ability of ventricular myocardium, the control the car-
diac vagus has over a spontaneously beating perfused
heart, and the vasoactivity of the branchial arteries.
Materials and methods
Chinook salmon from a local aquaculture facility were
transported to the University of Canterbury. They were
kept unfed in a large (1.5 m3 ) tank with flow through
artesian well water.
The NAAC for MS222 and AQUI-S were found
to be 100 ppm and 60 ppm, respectively. These con-
centrations anaesthetise most Chinook salmon within
5 min. Initial tests showed the equivalent NAAC of
metomidate to be 10 ppm, however, later work showed
this was over-anaesthetising fish during surgery so the
NAAC was reduced to 7 ppm. The paced ventricle
strip experiment uses the original 10 ppm concentra-
tion, while the cardiac vagus and myography exper-
iments use 7 ppm. It should also be noted that the
recommended dose of AQUI-S 17–25 ppm.
All experiments were approved by the University
of Canterbury animal ethics committee.
Paced ventricle strips
Chinook salmon (mass 78 ± 4 g, range 58.0–117.0 g,
n= 16) were killed with a blow to the head. The
heart was exposed, excised and placed in a 15 ◦ C
fresh water fish saline (Gesser et al. 1982) bubbled
with 0.5% CO2 , 19% O2 , 80.5% N2 . The ventricles
(mass 55 ± 4 mg, range 34 – 86 mg, n= 16) were
dissected free then cut in half in the median plane.
Two pieces of 5–0 silk were tied to each ventricle half,
one to a remnant piece of bulbus arteriosus and the
other to the apex. The preparation was placed in a
water jacketed tissue bath at 15 ◦ C containing gassed
saline. One piece of the silk was tied to the base of
the tissue bath and the other to the arm of an isomet-
ric force transducer (1 g, Ugo Basile, model 7003).
The transducer was attached to a Gould transducer
pre-amplifier and the signal sent to a chart recorder
(Yokogawa, LR4110). To induce the muscle to con-
tract silver wire electrodes were placed either side
of the ventricle and 3 to 5 mV pulses (5 ms dura-
tion, 12 pulses/minute) were applied with a stimulator
and voltage amplifier manufactured in the Biology
department. Stimulator output was verified using an
oscilloscope. Many authors add adrenaline to heart
strips to maintain contractility (Farrell 1998). This is
important in the performance of the intact heart, but
less so for isolated strips which are not moving fluid.
After a 15-min equilibration period, anaesthetic
was placed in the external Ringer’s in a concentrated
bolus which was mixed with the action of the bub-
 21

bling gas. The 3 anaesthetics were tested at 10%, 25%,

50% and 100% of their NAAC. Each concentration
was left for 15 min before applying the next higher
concentration. After exposure to the last anaesthetic
concentration ventricles were washed in situ with fresh
Ringer’s 3 times over 15 min, and the force of con-
tractions measured a final time. Controls were run in
parallel without the addition of anaesthetics.
Contractions during the final minute of each period
was used for analysis. A 2-way ANOVA and Tukey’s
HSD post-hoc test were used to compare the anaes-
thetics’ effects at different concentrations. P ≤ 0.05
was used to indicate a significant difference.

The cardiac branch of the vagus

Chinook salmon (mass 95 ± 6 g, range 44.2–196.3 g,

n = 16) were killed with a blow to the head and
then pithed. A mid ventral incision was made from
the vent to the isthmus and the heart exposed. The
left operculum was removed, the gills on the left side
severed, and the animal placed on its right side. The
posterior cardinal vein or hepatic vein was cannu-
lated and perfusate (Ringer’s solution) was introduced
immediately. Perfusate pressure was adjusted until
the heart was pumping spontaneously and the sinus
venosus was not over expanding. After opening the
pericardium the ventral aorta was cannulated and out-
put pressure arbitrarily set at 47 cmH2 O. Input and
output pressures were recorded with pressure trans-
ducers (Model 4-327, Bell and Howell Instruments
Division, Pasadena, California) and the data amplified
and recorded on a Devices MX4 recorder.
A 1 to 2 cm incision was made in the skin, along
the line where the edge of the operculum meets the
body, from a mid-lateral position to the top of the oper-
culum. When pulled free from the muscle this skin
formed a pocket at the base of which a branch of the
vagus could be found. The pocket was immediately
filled with Ringer’s to prevent desiccation and death
of the nerve. The volume of the pocket was estimated
at 200–400 µl. A platinum hook electrode, attached
to the variable voltage and pulse rate stimulator, was
placed under the nerve.
A range of voltages at 25 Hz was used to stim-
ulate the vagus. Changes in heart rate or pressures
could be observed from the output pressure chart. A
voltage range (2 V maximum) was chosen from a
voltage having no effect to a voltage producing a large
effect. Anaesthetic was then added to the well of sa-
line around the vagus branch. Over the next 5 min the
Figure 1. a-b. Graphs of proportional decrease in the contractile
force of the ventricle with increased anaesthetic concentration or
with time. The anaesthetics were MS222 (
), metomidate () and
AQUI-S (♦) and a control (
). (a) shows data as a percentage of
maximum contractile force and (b) shows data as a percentage of
the controls. Error bars are ±1 SEM. n = 8 for each data point.
C=significant difference from control at 15 min. ∗ = significant
difference from the control value at the equivalent time. + = MS222
data significantly different from metomidate and AQUI-S treated
ventricles at the equivalent time.
same range of voltages was applied to the vagus and
the effect on the heart rate and perfusate pressures ob-
served. The anaesthetic in the pocket was then washed
out thoroughly with Ringer’s for 5 min and the same
range of stimulation voltages was repeated. The 3 an-
aesthetics were tested at their NAAC. If no response
occurred the concentration was increased by 10 times
and the stimulations tried again. This was repeated up
to 100 times the original anaesthetic concentration.
The data were qualitative. An effect was defined as
the requirement for an increase in voltage needed to
elicit a particular change in heart rate or pressure that
was also followed by a decrease in voltage required
after washout. As the voltage required for particular
effects could increase spontaneously with time this
was not always easy to determine.
22
The branchial arteries
Chinook salmon (359 ± 145 g, range 78 – 1362 g, n =
9) were killed with a blow to the head and their blood
heparinized via the caudal vein using a heparinized
Ringer’s. The ABAs and EBAs were then dissected
out and placed in cooled Ringer’s until required. Two
mm lengths of vessel were mounted in myographs
(myograph model 400A, myo-interface model 410A,
J.P. Trading, Aarhus, Denmark) in Ringer’s bubbled
with 0.5% CO2 in air at 15 ◦ C. Access to a limited
number of fish with a large size range meant ini-
tial tensions had to be estimated, based on previous
experience (Forster et al. 1998). The vessels were
then left for 1 h to equilibrate. After equilibration
the anaesthetics were placed in the saline as a small,
concentrated bolus and were mixed thoroughly with a
Pasteur pipette. The 3 anaesthetics were tested at 10%,
25%, 50% and 100% of their NAAC. Each dose was
left for 15 min before the next highest dose was added.
Analysis was performed on the final minute of each
exposure period. The effect of anaesthetic type, an-
aesthetic concentration and vessel type on vasoactivity
were compared using 2-way repeated measures AN-
OVA and Tukey’s HSD post-hoc tests. In all cases
P ≤ 0.05 was used to indicate a significant differ-
ence. All error values are 1 standard error of the mean
(SEM).
Results
Paced ventricle strips
In every trial the ventricle strips contracted for the
duration of the experiment. However, there was some
intrinsic decrease in contractile force with time. The
control ventricle contractions were significantly lower
at 45, 60, 75 and 90 min than at 15 min (Figure 1a).
Anaesthetic effects relative to control values are also
shown (Figure 1b).
All 3 anaesthetics had a negative inotropic ef-
fect although their potencies differed. MS222 af-
fected ventricle strips at its lowest tested concentra-
tion, whereas metomidate and AQUI-S only affected
the strips at their highest concentrations (Figure 1).
However, the data suggest all the anaesthetics had a
dose dependent effect. MS222 had a greater effect
than metomidate and AQUI-S at all concentrations
(Figure 1).
After washing out the anaesthetics the contractile
forces of all treated ventricle strips returned to control
levels (Figure 1) which shows that the effects of all of
the anaesthetics were reversible.
The cardiac branch of the vagus
The initial heart rate of the preparations was 89 ±
3 bpm (n = 16) whereas the mean heart rate of instru-
mented live fish 3–10 times larger was 50.8 ± 3.1 bpm
(unpublished data). The preparations generally las-
ted for 3–6 treatments during which time the output
pressure pulses were regular. With time, heart rate
tended to decrease and become irregular, and pressure
changes became more variable. Upon stimulation of
the vagus a variety of chronotropic and inotropic re-
sponses were observed (Figure 2). The intensity of the
changes was always voltage dependent although the
exact nature of the changes and the voltage required
to cause changes varied between preparations. Evid-
ence for a sympathetic branch of the vagus in Chinook
salmon was also observed (Figure 2d).
The application of 100 ppm MS222 to the vagus
increased the potential required to affect the heart
(Figure 3). This effect was readily and quickly revers-
ible after anaesthetic washout. A limited number of
tests with 20 and 50 ppm MS222 showed similar res-
ults. Metomidate and AQUI-S could affect conduction
down the vagus at 100 times their effective anaes-
thetic concentrations (Figure 3). However, recovery
required a greater number of washes than with MS222
and was usually incomplete. The results showed that
MS222 affected neural transmission through the vagus
at concentrations equal to and lower than its anaes-
thetic concentration whereas metomidate and AQUI-S
did not.
The branchial arteries
Mean starting tensions for the EBAs and ABAs after
1 h equilibration were 2.34 ± 0.25 mN, n = 9) and
1.73 ± 0.16 mN (n = 8) respectively. The afferent
vessels tended to relax more than the efferent arteries,
and, as noted, initial tensions were influenced by the
very different sizes of the donor fish. However, the
tension of the control vessels did not change over the
duration of the experiment.
Both anaesthetic type and concentration affected
EBA and ABA vasoactivity. EBAs dilated in response
to all 3 anaesthetics at all doses except 10% metomid-
ate (Figure 4a). MS222 and AQUI-S treated EBAs be-
haved similarly, whereas metomidate was a less potent
dilator at lower concentrations. The pattern of ABA
vasoactivity was more complex (Figure 4b). MS222
 23

Figure 2. a–e. Changes in cardiac output pressure of in situ spontaneously beating Chinook salmon hearts following electrical stimulation of a
branch of the vagus nerve. (a) and (b) show typical responses to vagal stimulation using a range of voltages. Lower voltages generally caused
a slight decrease in mean pressure and a slight decrease or irregularity in the heart rate. Higher voltages caused similar effects but of a greater
magnitude. The greatest effect was a complete cessation of the heart beating (c). Less common responses included increasing the regularity and
force of contractions (d) and a mild decrease in systolic pressure (e).

was again a potent dilator. AQUI-S initially dilated

vessels but this trend reversed at higher concentra-
tions. Metomidate had no effect. Although overall the
relative magnitude of dilation was greater in the EBAs
than ABAs, this might have been influenced by the
lower initial tension on the ABAs.

Discussion

Paced ventricle strips

MS222, metomidate and AQUI-S all decreased
myocardial contractility with MS222 having a greater
effect than metomidate and AQUI-S. The effect of
MS222 on chinook salmon ventricle strips was very
similar to its effect on the ventricle strips from rainbow
trout (Ryan et al. 1993). At MS222 bath concentra-
tions of 25 ppm, 50 ppm and 100 ppm the decreases in
contractility in this study were 40%, 54%, and 75%
Figure 3. Figure 3. Graph of proportion of nerves inhibited by ex-
posure to different concentrations of the three anaesthetics. n=7–10
for each treatment.
compared to approximately 30%, 60% and 85% in
rainbow trout (data read from Figure 1 in Ryan et al.
1993). Perfused heart preparations from rainbow trout
(Ryan et al. 1993) and tench (Tinca tinca, Shelton
24
Figure 4. a–b. Graphs of proportional change in tension of Chinook
salmon efferent (graph a, n = 9) and afferent (graph b, n = 8)
branchial arteries in the presence of MS222 (
), metomidate ()
and AQUI-S (♦) and the controls (
). Error bars are ±1 SEM. ∗ =
significant difference from the control value at the equivalent time.
MS, Me, Aq = significant difference from MS222, metomidate,
AQUI-S treated branchial vessels at the equivalent time.
and Randall 1962) show qualitatively similar results
to MS222 exposure.
The negative inotropic effects of metomidate and
AQUI-S were quantitatively similar, and smaller than
the effect of MS222. There are no comparable fish
studies on these anaesthetics or any of their analogues.
The active component in AQUI-S is isoeugenol. In
mammals, compounds related to both AQUI-S (pro-
panidid, propinal, estil), and metomidate (etomidate,
propoxate) are short or ultra-short acting intraven-
ous anaesthetics or hypnotics (Hall and Clarke 1991).
Propinal was abandoned as an anaesthetic because it
caused, amongst other negative effects, severe circu-
latory problems (Dundee and Clarke 1979; Hall and
Clarke 1991) and propanidid has negative inotropic
effects on the hearts of dogs and guinea pigs (Rene-
man and Janssen 1977). In contrast, etomidate does
not affect myocardium from these species or isolated
cat papillary muscle (Reneman and Janssen 1977) and
it is often noted for its minimal effects on the cardi-
ovascular system (Gooding and Corssen 1976, 1977;
Dundee and Clarke 1979; Stoelting and Miller 1989;
Hall and Clarke 1991; Aitkenhead and Smith 1996).
However, etomidate has been shown to cause a de-
crease in stroke volume and contractility in the cat
heart using an isolated heart lung preparation (Fischer
and Marquort 1977). During metomidate anaesthesia
the cardiovascular system is remarkably stable (Hall
and Clarke 1991). This information suggests com-
pounds related to AQUI-S have more severe effects on
hearts than metomidate and its analogues, but this was
not the case in this study.
The greater effect of MS222 versus metomidate
and AQUI-S is not unexpected as local anaesthetics af-
fect myocardium, and bronchial and vascular smooth
muscle (Stoelting and Miller 1989; Hall and Clarke
1991). The negative inotropic effects of local anaes-
thetics in mammals are well documented as are the
serious consequences of injecting significant amounts
of local anaesthetic into the blood stream which then
affects the heart and vascular smooth muscle (Mc-
Donald and Wann 1978; Hall and Clarke 1991). In
contrast, intravenous anaesthetics and hypnotics are
delivered directly into the blood, often into individuals
that are unwell, and so must have minimal deleterious
effects on the cardiovascular system. Therefore, the
lesser effects of metomidate and AQUI-S, compared
to MS222, on the ventricle strips were probably to
be expected. However, most mammalian general an-
aesthetics (Black 1980; Hall and Clarke 1991) and
short acting intravenous narcotics such as propanidid,
methohexital, ketamine and althesin do possess some
undesirable cardiovascular side effects (Fischer and
Marquort 1977).
The muscle relaxing effect of all 3 anaesthetics
were reversible within 15 min of removing them from
the external medium. In vivo the process could be
prolonged by a compromised heart as removal of the
anaesthetic to sites of excretion and/or degradation is
probably perfusion limited.
Ryan et al. (1993) demonstrated a decrease in
power output of the isolated rainbow trout heart ex-
posed to MS222. Reduced cardiac contractility will
reduce the stroke volume ejected by the ventricle if the
afterload is unchanged (Forster 1989). In vivo, this po-
tential effect could be countered. Anaesthetic induced
vasodilation of vascular smooth muscle may cause
systemic hypotension lowering ventral aortic pressure.
This would delay closure of the semi-lunar valves

permitting a longer ejection period and allowing the
heart to maintain it’s cardiac output. Any decrease
in venous tone, reducing preload on the heart, will
also impair cardiac output (Farrell and Jones 1992).
The negative inotropic effects observed in the vent-
ricle could be countered with neural, hormonal and/or
direct myogenic homeostatic responses (Laurent et al.
1983) so that the net effect on the whole anim-
als may be negligible (Fischer and Marquort 1977).
However, anaesthetics may also block or attenuate
homeostatic responses. Examples include: local an-
aesthetics which block nerves, metomidate and its
analogues block a step in the production of cortisol
(Olsen et al. 1995), and halothane is thought to block
the carotid baroreceptor mediated increase in heart
rate in response to hypotension in humans (Stoelt-
ing and Miller 1989). The negative inotropic effects
of local and some general anaesthetics (halothane,
enflurane) have been implicated in a reduced aortic
blood pressure in mammals (Black 1980; Stoelting
and Miller 1989). The cause of death due to local an-
aesthetic systemic toxicity is cardiovascular collapse
caused by myocardial depression in conjunction with
vasodilation (Aitkenhead and Smith 1996).
Impaired cardiac performance may cause fish ad-
ditional problems during recovery. Handling prior to
anaesthesia and/or the anaesthetic may cause stress
that can induce activity, and both stress and activity
will increase the work rate required by the heart. The
reduced maximum work rate of an inotropically im-
paired heart in an aerobically challenged fish would
decrease the time to exhaustion and prolong recovery
from hypoxia and anaesthesia. As one of the main
goals of anaesthesia is to mitigate stress, a stress free
induction and an anaesthetic that does not cause a
stress reaction would be advantageous.
The cardiac branch of the vagus
MS222 increased the potential required to initiate
and/or maintain a signal down the vagus nerve. This
nerve can decrease, and in some species of fish, in-
crease heart rate (Saito and Tenma 1976; Taylor 1992).
MS222 affects other neural paths in fish including:
the lateral line nerve fibres in cod (Gadus gadus),
Tilapia leucosticta and Rutilus rutilus; the Ampullae
of Lorenzini of dogfish (Scyliorhinus canicula); and
the trigeminal nerve activity caused by physical stim-
ulation of the skin in Tilapia leucosticta and Rutilus
rutilus (Hensel et al. 1975; Späth and Schweickert
1977). Work on squid axons showed that MS222 re-
25
versibly increases the excitation threshold and blocks
nerve conduction (Frazier and Narahashi 1975).
In contrast to MS222, metomidate and AQUI-S
could only block the vagus nerve at extreme concen-
trations, unlikely to be reproduced in vivo. Action
potentials can be blocked by inert gases, general an-
aesthetics, barbiturates and alcohols (McDonald and
Wann 1978) although the concentrations required to
block action potentials may be greater than the effect-
ive anaesthetic concentration. Etomidate affects ion
movement in frog (Rana esculenta) myelinated nerve
fibres at 24 ppm (Benoit 1995) but this concentration is
at least 6 times higher than the concentration required
to cause anaesthesia in fish (Summerfelt and Smith
1990). Etomidate has no effect on the nerves of dog
and guinea pig hearts at the anaesthetic concentrations
generally used (Reneman and Janssen 1977).
Although MS222 can affect the vagus in vitro at
100 ppm, a typical anaesthetic bath concentration, for
it to have an effect in vivo this concentration must oc-
cur at the site of the nerve. Accumulation studies of
MS222 in fish suggests this does occur. Hunn (1970)
found that 8 species of fish all accumulated more than
100 ppm MS222 in their brain after 5 min exposure to
MS222. Similar high concentrations of MS222 were
found in the brain of Pagrus auratus (Ryan 1992).
However, rainbow trout showed a reduced heart rate
after 30 min of 100 ppm MS222 anaesthesia which
was elevated after intraperitoneal and/or intrapericar-
dial injections of atropine (Lochowitz et al. 1974).
This suggests MS222 was not blocking the vagus or
inhibiting production of the negative chronotropic to-
nus centrally. In contrast, it has been suggested that
there is a rapid depression of central autonomic control
centres including tonic cardiovagal inhibition (Hous-
ton et al. 1971). The sympathetic branch of the vagus,
which increases heart rate in Girella tricuspidata,
is blocked by MS222 and its analogue benzocaine
(Montgomery et al. 1986). Additionally, the phasic
response to sound stimuli of sound sensitive neurons
in the medulla of Tilapia leucosticta and Rutilus ru-
tilus ceased during MS222 anaesthesia and the afferent
output from the medulla to the lateral line was de-
creased by 70% (Späth and Schweickert 1977). These
conflicting results show more work is needed.
No accumulation studies using metomidate or
AQUI-S have been published on fish, so the rate of
accumulation and the maximum attainable concen-
trations for these chemicals are unknown. However,
the present study suggests very high concentrations of
both metomidate and AQUI-S are required in order for
26
peripheral nerve inhibition to occur. However, metom-
idate and analogues of AQUI-S used in mammals are
intravenous anaesthetics and as such their primary site
of action is assumed to be the central nervous system.
The vagus nerve does not show an ‘all or nothing’
response because it is a nerve bundle. This was evident
during the vagal stimulations as application of higher
potentials to an MS222 affected vagus could increase
the response of the heart. Theoretically, an exogenous
agent could have a graded inhibitory response depend-
ing on how many nerve fibres were affected. The
magnitude of the effect on a nerve would be determ-
ined by the sensitivity of different neurons within the
bundle (Roweland 1974; Stoelting and Miller 1989)
and also the penetration of the agent into the bundle.
The amount of penetration of a local anaesthetic is
affected by the agent (dissociation constant, lipophili-
city), the solution (pH, ionic concentration), and the
nerve bundle morphology (anatomy, perfusion, adja-
cent tissue) (Stoelting and Miller 1989). The inability
of AQUI-S and metomidate to affect the vagus nerve
could be controlled by these factors. Similarly, these
factors could modify how these anaesthetics affect
groups of muscle fibres.
Carp (Cyprinus carpio) under prolonged MS222
anaesthesia eventually showed atrioventricular disso-
ciation, which the authors attributed to hypoxia (Ser-
faty et al. 1959). In the present study this was noted
in in situ perfused hearts as they were beginning to
fail and die, although this was probably not due to
hypoxia. It has been noted that some local anaesthet-
ics can also cause atrioventricular conduction blocks
in human patients as well as arrhythmogenesis (Mc-
Caughey 1992). Ryan et al. (1993) found that 10 ppm
MS222 caused arrhythmia and complete arrest of the
whole heart of rainbow trout.
Electrocardiograms (ECGs) directly measure elec-
trical activity in the heart and have been used to
monitoring changes that occur during fish anaesthesia.
Few notable changes were observed (McFarland 1959;
Campbell and Davies 1963) and where changes did
occur (greater lag before ventricular re-polarisation)
the anaesthesia was prolonged (at least 30 min) and
the changes were likely to be due to hypoxia (Yamam-
itsu and Itazawa 1988; Fredricks et al. 1993). Local
anaesthetics have been implicated in widening of the
QRS complex in human patients (McCaughey 1992).
This effect was also noted in carp (Cyprinus carpio)
anaesthetised for 2 h with 800 ppm 2-phenoxyethanol
(Yamamitsu and Itazawa 1988) but, again, separation
of anaesthetic effects from the effects of hypoxia is
difficult.
The branchial arteries
The myography experiments showed that all 3 anaes-
thetics dilate EBAs. MS222 dilated the ABAs while
metomidate did not. The effect of AQUI-S was not
straightforward, as in 6 out of 9 of the EBAs and
7 out of 8 of the ABAs the tension at 100% NAAC
was greater than the tension at 50% NAAC. As lower
concentrations cause dilation, and the increase in ten-
sion occurs late in the experiment, this may be a time
dependent effect of AQUI-S.
MS222 and AQUI-S had a maximal effect on
EBAs at 10% NAAC. This shows these anaesthet-
ics are potent vasodilators. MS222 and AQUI-S may
show a dose dependent effect using a range of lower
concentrations. In contrast, metomidate had a dose
dependent response over the anaesthetic range tested
and a maximal response may not have been elicited.
Although the anaesthetics had a greater propor-
tional effect on the EBAs than on the ABAs, the EBAs
had a greater absolute starting tension. The control
of gill blood flow is complex (Nilsson and Sundin
1998) and it is not possible to attribute functional
significance to the relative changes in tension. The
ABAs contain blood at a higher pressure and are vis-
ibly thicker than the EBAs. The ABAs have a greater
proportion of relatively inert connective tissue com-
pared to vascular smooth muscle. MS222 and AQUI-S
also vasodilate isolated, perfused salmon tails (S.E.
Rothwell and M.E. Forster, unpublished data).
Potentially, a general vasodilation could have ma-
jor consequences. Systemic vasodilation would reduce
dorsal aortic pressure. A reduction in venous tone
would affect filling of the heart and cardiac output.
Anaesthesia almost always causes some stress and
activity, but systemic vasodilation could counteract
any stress or exercise induced vasoconstriction. A
branchial vasodilation will alter gill haemodynamics,
but could also protect cardiac output from a heart
which is inotropically compromised by an anaesthetic.
As anaesthesia affects respiration, hypoxia may occur
which could induce further cardiovascular changes.
Acknowledgements
We gratefully acknowledge the assistance of Alistair
Jerrett and the New Zealand Institute for Crop and

Food Research, Seafood Research Unit for financial
support and donations of AQUI-S; and Isaac’s Salmon
Farm, McLean’s Island, Christchurch, for donations
of salmon and support of our research. We thank Dr
Chris Glover for his experimental expertise.
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