Chapter 21

Anesthesia and Restraint of Laboratory Fish

Michael Stoskopf and Lysa Pam Posner

I.Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
II.Fish and Pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 520
III.Physical Restraint of Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 520
IV. Basic Principles and Application of Anesthesia in Fish . . . . . . . . . . . . . . . . . . 521
V. Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522
VI. Anesthetic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523
A. Immersion Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523
B. Injectable Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529
VII. Analgesic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531
A. Opioids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531
B. Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) . . . . . . . . . . . . . . . . . . 531
VIII. Nonchemical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531
A. Hypothermia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531
B. Electroanesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531
IX. Euthanasia in Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532
A. Suitable Methods for Euthanasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532
B. Unsuitable Methods for Euthanasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533

I. INTRODUCTION
Properly administered anesthesia and analgesia are particu-
larly important in the management of fish in a research setting.
Often, they are the only suitable approaches to obtaining reliable
and repeatable data. The issues of the perception of pain by fish,
or the level of pain that might be expected from a procedure are
not the only issues to consider, and may not be the overriding
factors in deciding to apply anesthesia to a fish. Proper appli-
cation of anesthesia to fish spares not only the subjects but also
the researchers from stresses and situations that can seriously
impact the precision and even accuracy of data from invasive
procedures. Induction of anesthesia should always be consid-
ered, even for relatively minor manipulations of fish, whenever
a quiet and compliant subject would improve the outcome of the
research. This is equally true for very small and very large fish
that present physical challenges to safe handling.
The technology of providing anesthesia to fish has taken
important steps forward in the past decade, though there remains
considerable opportunity for improving our understanding of
the optimal approaches to providing for the comfort of fish
being manipulated in research settings. One of the significant

ANESTHESIA AND ANALGESIA IN LABORATORY ANIMALS, 2ND EDITION Copyright © 2008, 1997 by Elsevier Inc.
All rights reserved.

519
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challenges lies in the extreme diversity of the Piscean taxon.
There are more distinct species of fish than of all other vertebrate
taxa combined (Official Lists and Indexes of Names, 1987), and
they are adapted to an amazing breadth of environmental con-
ditions through a wide range of significant adaptations. Many
of these adaptations could impact the response of a species to
anesthetic and analgesic agents. Even within the relatively nar-
row range of teleost species routinely used in research, there
are important differences in physiology that bear consideration
and beg for a deeper understanding of the interactions between
anesthetic agents and fish physiology. For example, some infor-
mation is available regarding the impact of water temperature
on the absorption of antibiotics in fish, but far less is docu-
mented about anesthetic agents. Freshwater species should be
expected to absorb and excrete agents differently from eury-
haline and saline species because of the radical differences in
their handling of ionic homeostasis, but these differences are not
well studied. Commonly used laboratory fish are generally very
hardy species, often adapted to low-flow and low-quality waters
in their native habitats. They respond well to routine anesthe-
sia procedures, but fishes from pelagic and fast flowing stream
habitats can be more challenging to anesthetize. A lot remains to
be learned to allow optimization of anesthesia protocols across
the physiological diversity of the fishes. However, the need for
more knowledge should not be used as a crutch to avoid the use
of anesthesia and analgesia in fish in research settings. It should
rather be seen as a key reason for insisting on the application of
anesthesia and analgesia at every opportunity where, with even
slight adjunct effort, we might learn more about the physiology
of fish responses to state-of-the-art techniques.
II. FISH AND PAIN
The International Association for the Study of Pain (IASP,
1994) defines pain as “an unpleasant sensory and emotional
experience associated with actual or potential tissue damage, or
described in terms of such damage.” It further states that “the
inability to communicate verbally does not negate the possibility
that an individual is experiencing pain and is in need of appro-
priate pain-relieving treatment” (IASP, 1994). According to this
definition, animals do not need to communicate to experience
pain, but some may argue that animals cannot have the emo-
tional experience of pain and thus do not feel pain, but rather
only react to nociception (the perception of a noxious stimuli).
Because fish lack the structure analogous to the mammalian
neocortex, it has been argued that they do not feel pain. This
reasoning is also used as a rationale for not providing analge-
sia following invasive procedures. Fish do have well-developed
avoidance behavior and physical reaction to noxious stimuli
(Sneddon et al., 2003), and perhaps more telling, it has been
shown that fish have nociceptors that respond to pressure, prick,
and heat. Stimulation of those nociceptors causes neuronal
MICHAEL STOSKOPF AND LYSA PAM POSNER
activation to the higher brain centers, the telencephalon, in both
goldfish and trout (Dunlop and Laming, 2005). Fish also have
an endogenous opioid system with both kappa and mu recep-
tors in the brain, suggestive of an evolved need for modulation
of neuronal responses to complex factors including nociception
(Brooks et al., 1994). All of these points, i.e., evidence of a
functional neurologic pathway for nociception, reactive com-
plex behavioral responses and learned avoidance behavior to
noxious stimuli, and endogenous chemical modulation analo-
gous to those used by mammals, suggest it is likely that fish do
perceive painful stimuli. It is therefore incumbent upon inves-
tigators working with fish to provide anesthesia and analgesia
for invasive and potentially painful procedures.
Evaluation of pain in fish is difficult and not well standard-
ized. There is no single physiologic or behavioral parameter
that is documented to be well correlated with pain in fish. As
with terrestrial animals, a combination or matrix of behaviors
and physiologic parameters needs to be evaluated. Fish in pain
have been shown to change positions in the water column, show
avoidance, display rocking behaviors, and have increased oper-
cular movements and decreased appetite (Harms et al., 2005;
Sneddon et al., 2003). Pain in mammals has been shown to
cause adverse physiologic changes, such as increased sympa-
thetic tone, increased cardiac demands, increased metabolic
rate, and delays in healing. Allowing fish to remain in pain,
besides being inhumane, should therefore be expected to affect
research parameters, and should be avoided whenever possible.
III. PHYSICAL RESTRAINT OF FISH
Physical restraint of fish in laboratory settings is usually
required for catching and transferring individuals or groups
of fish between holding systems to experimental conditions.
Though “netting” fish is considered a routine procedure, if
improperly done, it can cause significant harm to the fish and
to the quality of the research data obtained from the fish. These
deleterious effects can be minimized by using proper catching
techniques, and by appropriate selection of equipment. It should
also be understood that fish can be acclimated to catch and trans-
fer procedures using behavioral techniques very similar to those
employed for mammals. When frequent transfers are expected
in a laboratory, the effort to acclimate the fish to the routine is
an excellent investment that will repay the scientist many times
over in terms of saved time and improved data.
The use of mesh nets is not the optimal approach to catching
and transferring most small fish species. A better approach does
not employ nets at all, or makes use of solid net-like devices
called bag nets. Even very finely meshed nets sold commer-
cially in pet stores for handling small fish are made of relatively
stiff and abrasive material, which can damage the protective
mucous layer of the fish and cause very fine abrasions. The
impact of abrasive nets is easier to appreciate on larger species
21. ANESTHESIA AND RESTRAINT OF LABORATORY FISH
such as goldfish and carp. Handling these fish using nets sold
commercially for fishermen or aquaculture harvesting can cause
abrasions, which can often be seen with the unaided eye and may
take many days to heal. Swim-through nets made from very
soft mesh materials such as those used to manufacture women’s
pantyhose are most appropriately used for slower swimming
species like koi or carp. The device consists of a hoop, usually
on a handle, and a long open-ended tunnel for a bag. The fish is
encouraged to swim into the net, and then the handler grasps the
far end of the tunnel with one hand to close the net as the fish
swims calmly down the tunnel. To release the fish after transfer,
the handler merely releases the end of the tunnel and allows the
fish to swim out forward, avoiding the need to flip the net or
force the fish against the grain of even the fine mesh of the tun-
nel. These devices should be considered for use in larger fish,
where capture with a bag net or box trap would result in very
heavy larger volumes of water that are difficult to lift.
Bag nets and box traps should be used to capture smaller
fish in laboratory settings. If properly used, they avoid forcing
the fish to contact the surface of the capture device and are at
least as effective and efficient in catching fish as the traditional
small mesh nets sold in pet stores. Bag nets resemble traditional
nets, but rather than a mesh bag, a solid, usually clear plas-
tic bag is attached to the net frame and handle. Box traps are
similar in concept, but are solid-walled containers, usually with-
out a handle. The keys to effective techniques of catching fish
for transfer or examination are patience and slow anticipatory
placement of the capture device. Ideally, obstacles and hiding
places should be removed from the system the fish are being
caught from; however, in some cases, properly designed “furni-
ture” or enrichment materials can be used as in situ box traps to
facilitate catching species that like to hide from predators. The
bag net or box trap should be maneuvered to come from behind
the swimming fish. When the fish is within the perimeter of the
bag or box, then the device is tilted and brought to the surface,
trapping the swimming fish within a contained volume of water.
Some of the water can be decanted carefully if needed, but the
fish and water are then removed from the system and moved to
where the fish needs to be studied or relocated. These devices
can even be used for induction of anesthesia without further
handling of the fish, although care should be taken to carefully
rinse the devices of residual anesthetic agents before they are
used again to catch fish from holding systems.
Some experimental protocols require chronic restraint of fish.
These protocols may be very necessary for collecting sophisti-
cated physiologic data from a fish. However, these protocols
can be very challenging to implement in a humane manner. The
nature of the species of fish and the level of acclimation of the
individual fish to the situation play a large role in whether or
not this type of experiment can be performed humanely without
anesthesia. Species that tend to occupy crevasses and remain
more or less sessile most of their day are more appropriate for
these applications than schooling and more pelagic species. The
restraint devices for these species can be constructed to resemble
521
to some degree, safe harborages, and with the constant provision
of proper water quality and life support, fish can be humanely
restrained sufficiently to allow various types of noncontact or
minimal contact data collection for prolonged periods. Unfortu-
nately, many research questions requiring chronic preparations
focus on issues with fish unsuited to chronic restraint without
anesthesia. Particularly when invasive dissection or implants
are involved, these types of experiments should be conducted
under anesthesia, and in cases where surgical dissections are
extensive, these should not be survival procedures.
IV. BASIC PRINCIPLES AND APPLICATION OF
ANESTHESIA IN FISH
The basic principles of anesthesia in fish will be quite famil-
iar to most scientists with experience in proper anesthesia of
mammals. However, it may be a bit of a surprise that many
of the principles also apply to fish. If in doubt, the anesthetist
should assume that principles that apply to mammals do apply to
fish as well. Fish responses to anesthetic agents are affected by
their physiologic state at the time of induction. A quiet, undis-
turbed environment prior to the induction, and calm transfers to
the induction system help ensure a smoother and more efficient
induction. Withholding food for 24 hours prior to the induction
is not a hardship for most fish, even for very small tropical ones.
While regurgitation in fish does not hold the same degree of
potential for harm that is present for mammals, it can disrupt an
induction or a procedure, and is easily avoided with a preanes-
thesia fast. Fish should be anesthetized using measured doses of
the agent, and careful records of the amounts of drug and timing
of application should be taken to guide future procedures and
to serve as a reference when troubleshooting unexpected data
results in experiments. The most successful fish anesthesiolo-
gists carefully assess fish size and condition prior to induction
and select measured doses based on experience with many pro-
cedures with the species. With experience, they may be able to
anesthetize several fish essentially simultaneously, and be able
to observe, monitor, and record the events successfully. When
anesthetizing a species of fish that you have never had experi-
ence with, it is best to limit yourself to anesthetizing one fish
at a time, and it is even more important to carefully record all
observations and administrations in the anesthesia record. Fish
should be followed for at least 24 hours after a procedure before
concluding that there were no untoward effects of the anesthe-
sia. Selection of the induction agent will often depend on the
experiments planned, but there is no substitute for experience
with the agent and the species being anesthetized when it comes
to having excellent results.
Essentially all routes of administration that are used in mam-
malian anesthesia are technically available for fish, including
oral, intramuscular (IM), intravenous (IV), and intraperitoneal
(IP) injection. The route of administration most commonly
522 MICHAEL STOSKOPF AND LYSA PAM POSNER

used for fish anesthesia is immersion in a concentration of the
anesthetic agent. The technique is analogous to the use of gas
induction chambers for mammals. The agents are absorbed pri-
marily across the gills of the fish and exert their impact centrally.
Prior to placing an animal in an immersion induction solution,
the recovery water should be prepared and made readily avail-
able to immediately receive a fish with an overdose or untoward
reaction to the drug. Proper aeration to ensure adequate oxy-
genation should be maintained for both the induction and the
recovery water.
Injection sites for fish vary to some degree with the fish
species, but IM injections are most commonly given above the
lateral line, usually in the area under the dorsal fin. IP injections
are most commonly given with a needle angled forward and
toward midline, entering the fish on the body wall just up from
the ventral surface of the fish, between the pelvic fin and the
anus. Doses of injectable drugs should be administered based
on the recorded weight of the fish. Often, considering the value
in reducing stress on the fish prior to induction, this is done by
first estimating the weight of the fish and then actually weigh-
ing the anesthetized fish to develop a useful length-to-weight
conversion table.
Once anesthetized, fish should be handled gently and with
care to avoid damaging their protective mucous layer and thin
skin epithelium. It is important to avoid the contact of the fish
with dry surfaces, even dry human hands. Wet latex or nitrile
gloves are preferable to bare hands. All surfaces that the fish
directly contacts should ideally be moist and smooth. During
anesthesia, fish must be supplied with adequately oxygenated
water to breathe. In prolonged procedures or when fish are held
in very deep planes of anesthesia that reduce respiratory effort,
it may be necessary to artificially supply the needed volumes of
oxygenated water to maintain the physiology of the fish. Though
fish can survive their removal from water and exposure of their
gills to air, and some fish are quite adapted to this, there are
physiologic consequences even for lungfish. Care should be
taken to minimize the time that fish are out of water or their
gills are not exposed to water.
V. MONITORING
Monitoring fish during anesthesia is a science that needs fur-
ther development. In most cases, monitoring is still limited
to visual observation of movement and respiratory rate and
effort. Advances in the use of electrocardiographic and other
physiologic monitoring are being made in larger species, but
the challenge of monitoring small species remains relatively
unsolved. The lack of physiologic monitoring during routine
fish anesthesia contributes significantly to the many areas of
ignorance that persist relative to the impact of anesthetic agents
of fish physiology. Investigators with concerns about the poten-
tial impact of anesthesia on a physiologic system under study
should be strongly encouraged to run pilot studies monitoring
the systems they are concerned about in fish anesthetized with
agents with different properties.
It is possible to categorize the stages and planes of anesthesia
in fish through observation of basic swimming, respiratory, and
cardiovascular patterns (Table 21-1). Many of the key responses
of fish to anesthesia can be monitored with minimal or no tech-
nical assistance. Useful reflexes include the menace reflex and
the righting reflex. In fact, these might better be referred to
as responses rather than reflexes because of the complexity of
their execution. The menace reflex of fish cannot be assessed
by the closing of eyelids, of course, but fish will startle and try
to move their head when an object approaches their eye. It is
not necessary to touch the eye for this response. The response
can be extinguished by overuse, just as occurs in mammals.

TABLE 21-1
Stages of Anesthesia

Stage Plane Descriptor Behavior of fish

0 Normal Swimming actively; reactive to external stimuli; equilibrium normal; muscle tone normal
I 1 Light sedation Voluntary swimming continues; slight loss of reactivity to visual and tactile stimuli; respiratory rate normal;
equilibrium normal; muscle tone normal
I 2 Deep sedation Voluntary swimming stopped; total loss of reactivity to visual and tactile stimuli; slight decrease in respiratory
rate; equilibrium normal; muscle tone slightly decreased; still responds to positional changes
II 1 Light narcosis Excitement phase may precede in increasing respiratory rate; loss of
tone decreased; still responds to positional changes weakly
II 2 Deep narcosis Ceases to respond to positional chances; decrease in respiratory rate to approximately normal; total loss of
equilibrium; no efforts to right itself; muscle tone decreased; some reactivity to strong tactile and vibrational
stimuli; suitable for external sampling, fin biopsies, gill biopsies
III 1 Light anesthesia Total loss of muscle tone; Responds to deep pressure; further decrease in respiratory rate; suitable for minor
surgical procedures
III 2 Surgical anesthesia Total loss of reactivity; respiratory rate very low; heart rate slow
IV Medullary collapse Total loss of gill movement followed by cardiac arrest in several minutes

Source: Stoskopf (1993).

21. ANESTHESIA AND RESTRAINT OF LABORATORY FISH
The righting reflex is particularly useful for judging the induc-
tion of anesthesia by immersion. The fish is gently held in the
water and turned to dorsal recumbency. Fully awake fish are
difficult, if not impossible, to grasp. As induction proceeds, the
fish can be held but it struggles against the manipulation of turn-
ing it on its back. With further induction, there is a delay in the
time taken for the fish to coordinate its finning motions to right
itself. Next, the animal becomes unable to right itself but will
still make fin and body motions in an effort to do so. Finally, the
fish will quietly remain in dorsal recumbency making no effort
to change position, indicating a deep state of central nervous
system (CNS) depression.
The respiratory rate can also be monitored with no special
equipment. The rate and strength of opercular excursion can
be visualized and monitored with only a timepiece that reads
to the second. The accurate evaluation of the strength of oper-
cular excursion does require some experience, particularly in
observing the species of fish being anesthetized in a nonstress-
ful awake condition to understand normal baseline gilling rates
and excursion. Weak opercular motions will not be as effec-
tive at moving oxygenated water across the gill surfaces, as the
shallow and ineffective respiration in mammals fails to optimize
gas exchange in the lungs. During induction, gilling rates often
increase initially in response to the handling and initial expo-
sure to the chemicals, but as induction proceeds, gilling rates
generally fall to normal and, with many agents, then proceed to
subnormal rates and excursions.
The heart rate of fish is more challenging to monitor than the
respiratory effort without some supplementation to the senses.
Even for very large fish, it is challenging to visualize the move-
ment of the relatively rigid body wall in the region of the heart.
Detection of a pulse in fish is an art that has yet to be well
developed. For relatively large fish, the esophageal stethoscope
and modifications of the concept can be used to obtain good
information about heart rate and, to some extent, depending on
experience, the quality of heart contraction. The device is passed
through only a very short distance to lie within the esophagus
over the region of the heart. This is not yet an option in the small
fish more commonly used in research facilities because of the
lack of suitably sized instruments and the need for amplification.
Nor are Doppler and pulse oximetry devices available that are
suitably miniaturized to be used on fish as small as the gold fish
or even smaller. Doppler devices designed for dogs and cats can
be used effectively in moderately sized and larger fish. When
external electrical signals are not a component of the experimen-
tal activity, it is possible to monitor the electrocardiogram (ECG)
of fish. The conductivity of water makes it possible to achieve
this monitoring without placing electrodes on the fish; how-
ever, standard ECG machines developed for mammals are not
suitable for the task. Special preamplification of the electrical
signals is required to obtain readable ECGs of fish in this manner
(Altimiras and Larsen, 2000). Another very sophisticated way
to obtain heart rate of anesthetized fish uses ultrasonography.
Again, the aquatic medium makes it possible to image the heart
523
of fish without making direct contact with the fish. The heart
rate and the quality of contractility are assessed directly by view-
ing real-time images of the heart. Rather high-quality probes of
10–12 MHz are required to visualize the heart of small fish effec-
tively. Pulse oximetry, a means of monitoring oxygen saturation
of blood in mammals, has not been particularly useful in fish,
primarily because of the challenges of locating sensors in posi-
tions that reliably can detect pulse. Differences in hemoglobin
characteristics also make calibration of the saturation infor-
mation and interpretation challenging even when readings are
obtained. With further advances in monitoring equipment being
made, it is possible that devices suitable for more effective
monitoring of fish anesthesia will become available in the future.
VI. ANESTHETIC AGENTS
There are a great number of agents available that have
been used to render laboratory fish compliant for experimen-
tal manipulation. Researchers have generally had the advantage
of ready access to a wide range of chemicals, and because
research fish do not usually enter the human food supply, reg-
ulations designed to prevent chemical contamination of food
have not inhibited the creativity of experimentalists. Over the
years, a large number of chemicals and agents have been applied
to fish in the hopes of inducing “anesthesia,” with varying
degrees of success. However, only a few of these agents have
found widespread usage sufficient to allow even a basic under-
standing of their effects and optimal application. We will focus
our discussions primarily on those agents which are currently
actively used by fish anesthesiologists for routine anesthesia
of fish. Many fish “anesthetics” reported in published compi-
lations have been used only sporadically and usually without
the adjunct assessment of key physiologic impacts of the drug.
For the most part, those drugs are no longer employed in fish
anesthesia, although they do seem to be rediscovered on occa-
sion. We include those agents in a secondary category of drugs
that have been used but without recommendation for their use.
Finally, a number of agents are listed in many compilations of
fish anesthesia agents that either have little or no efficacy, have
serious untoward effects on the patient, or present distinct health
risks to humans in application. We mention these in a separate
section of agents not recommended for use in fish anesthesia.
A. Immersion Agents
Immersion agents are those that produce anesthesia when
the fish is immersed in water in which the anesthetic agent
is dissolved. Although the agent must be water soluble, some
agents must first be dissolved in organic solvent and then diluted
with water. Concentrations are generally calculated as parts
per million (ppm), which is equivalent to milligrams per liter
(mg/L). Other less frequently used units may be seen in the
524
literature, such as grams per cubic meter of water (g/m3 ) which
is used when drugs are being applied to very large volumes of
water. This is rarely, if ever, appropriate for anesthetizing fish
in a research setting. To develop and practice good immersion
anesthesia in fish, it is very important that measured doses be
administered. Measured volumes of known concentration stock
solutions of anesthetic should be added to measured or reason-
ably accurately estimated volumes of water for induction, and
maintenance additions should also be carefully measured. The
practice of addition of dry agent haphazardly to unmeasured
volumes of water to save time is not appropriate.
Ideally, immersion agents should provide rapid immobiliza-
tion and rapid recovery, and have a wide safety margin (i.e.,
therapeutic concentration should be much smaller than the lethal
concentration). When at all possible, a separate tank should
be dedicated for use with anesthetics. When using immersion
agents, water quality is particularly important. Water with simi-
lar salinity, hardness, pH, dissolved oxygen, and temperature to
which the fish are acclimated should be used. The ideal situation
would be to use water from the tank where the fish are normally
housed if that water is not seriously compromised. The anesthe-
sia tank should be aerated, and there needs to be a method for
continuous flow of water over the gills (i.e., manual movement
of fish, or active flow through a tube placed near the gills, to
ensure constant flow of water over the gills).
There are four stages of anesthesia for aquatic species, which
are similar to anesthetic stages used in other species (Thur-
mon, 1996) (Table 21-1). Lighter planes of anesthesia might
be acceptable when only restraint is needed. For fish undergo-
ing painful procedures or surgery, deeper planes are required.
The ideal anesthetic depth is a compromise achieving slow,
steady, and effective opercular movement without response to
stimulation.
The ability to rapidly recover fish from anesthesia is one of
the advantages of the proper use of immersion agents; how-
ever, the pharmacokinetics of the immersion agents should be
kept in mind when managing the immersion. A drug continues
to be absorbed by the fish so long as the fish is maintained in
a concentration of the drug in the water. This may be neces-
sary to maintain the anesthesia, but depending upon the agent
being used, the compartmentalization of drug within the fish can
result in depot formation that will prolong the recovery from the
anesthetic. To recover fish from immersion-induced anesthesia,
the fish is placed in well-aerated, high-quality, anesthetic-free
water. Manual movement of the fish through the water can be
used to force water though the mouth and over the gills, to
facilitate depuration of the anesthetic and delivery of oxygen
to the fish. Effective use of this technique requires some expe-
rience and an understanding of the anatomy of the fish. It is
quite challenging to achieve effective water movement over the
gills in this manner for smaller fish. Simply opening and clos-
ing the mouth under water in a manner mimicking the motions
of the awake fish can be just as effective in pumping water over
the gills, and poses less risk to the patient compared to moving
MICHAEL STOSKOPF AND LYSA PAM POSNER
the fish manually around the tank. For very small fish, this can
be accomplished using a small cotton-tipped swab or similar
soft device such as a microsurgical point to move the lower jaw
of the fish down and back up simulating normal respiration.
Experience is a very important component of administering
anesthesia and that is particularly true for immersion anesthesia
of fish. Generally, anesthesia of fish should be conducted on one
fish at a time, although with experience anesthesiologists can
administer immersion anesthesia to large numbers of fish at one
time. Even experienced fish anesthesiologists find it prudent to
perform careful trials with a small number of fish one at a time
when anesthetizing a new fish species they are not familiar with
or when using a new agent.
1. Best Practices Agents for Use in Fish
a. Tricaine methanesulfonate (3-aminobenzoic acid ethyl
ester methanesulfonate: MS-222, TMS, Tricaine,
Metacaine, Fiquel)
Tricaine is the only anesthetic approved by the United States
Food and Drug Administration (FDA) for fish intended as food
and is the most commonly used agent for the anesthesia of fish.
Chemistry. Tricaine is a white crystalline powder that has a
water solubility of 1.25 g/ml at 20◦ C. A stock solution of 10 g/L
can be stored in an airtight container at room temperature. The
solution should be stored in darkened containers, as sunlight
will cause the solution to turn brown. The addition of tricaine to
water at relevant doses will cause the water to become acidic. At
100 mg/L, the pH of the solution can be as low 5. It is therefore
recommended to buffer the solution with sodium bicarbonate
to a pH between 7.0–7.5. This is most readily accomplished by
supersaturating the premixed solution of MS-222 with sodium
bicarbonate. This can be accomplished for stock solutions. The
precipitate sometimes reported with the procedure is simply
undissolved sodium bicarbonate. Care should be taken not to
decant this residual when taking from the stock solution. More
important to the efficacy of the stock solution is the formation
of an oily substance on the surface of the solution. This occurs
when the order of solution is reversed and water is buffered
prior to adding the anesthetic agent to the stock solution. The
same condition occurs with excessive exposure to light, which
can also cause a browning of the solution. The appearance of an
oily substance on the surface of the stock solution, cloudiness of
the stock solution that will not settle down, or darkening of the
solution all indicate that the stock solution should be discarded
and new stock made. The shelf life of stock solutions can be
extended by refrigeration, protection from exposure to light,
and even freezing the solution. The most popular stock solution
used is made up to a concentration of 10 g/L, which allows easy
calculation for working in 100 mg/L dilutions.
Mechanism of action. Tricaine is a water-soluble chemical
structurally related to benzocaine. It is a local anesthetic that
21. ANESTHESIA AND RESTRAINT OF LABORATORY FISH
works by blocking the conduction of sodium channels. It is
reasonable to question if the lack of movement seen in fish anes-
thetized with tricaine is at least partially associated with blocked
conduction of muscle as opposed to true CNS depression.
There is evidence of impact on nervous tissues as tricaine does
decrease neural conduction in toadfish (Palmer and Mensinger,
2004). The structurally related drug, lidocaine, has also been
shown to be analgesic and to function as a CNS depressant when
given centrally in domestic mammals (Doherty and Frazier,
1998; Valverde et al., 2004). It is possible that tricaine may
act similarly in fish.
Anesthetic effects. Tricaine is rapidly absorbed via gill dif-
fusion, and provides a fairly rapid induction and recovery.
The early stages can show some excitatory effects similar to
those seen in terrestrial animals as they pass through an exci-
tatory state (Stage II anesthesia) before becoming immobile.
Once immobile, tricaine is able to completely abolish muscle
movement.
Tricaine has been shown to cause physiologic changes in
fish, including increased cortisol, glucose, hematocrit, and lac-
tate (Cho and Heath, 2000). High doses (100 mg/L) of tricaine
decrease dorsal aortic pressure in Chinook salmon (Hill and
Forster, 2004).
Dose. There is a wide range of doses recommended for anes-
thesia of fish with tricaine in the literature, and this can be
primarily attributed to the variety of species, fish size, and even
the density of fish in a group being induced. Its wide margin
of safety in most applications also plays a role in broadening
the range of doses found to be “successful” in the literature.
Tricaine is more potent in warm water and in soft water, and
thus doses should be adjusted accordingly. Lower doses should
be used for sedation or with physiologically compromised or
diseased fish.
Tricaine is routinely administered at a dose between 25
and 100 mg/L for anesthesia. Most commonly used laboratory
species of fish are induced quite suitably at 100 mg/L. Tricaine
is used at doses between 400 and 500 mg/L for euthanasia by
anesthetic overdose.
Withdrawal. Although tissue concentrations of tricaine are
demonstrated to be near zero within 24 hours after adminis-
tration in salmonids (Bowser, 2001), the kinetics of the drug
would be expected to vary with a wide range of factors including
water temperature and body composition of the fish. Therefore,
the US FDA requires a 21-day withdrawal for fish used for food
or release into the wild (FDA, 2005).
b. Quinaldine sulfate (2-methylquinoline: quinate)
Chemistry. Quinaldine sulfate is a crystalline powder with
a solubility of 1.04 g/L in water. Stock solutions should be
stored in dark containers and protected from sunlight. The
525
aqueous solution is acidic and should be buffered with sodium
bicarbonate in a manner similar to that described for tricaine.
Humans exposed to quinaldine have reported irritation to eyes
and airways (Bowser, 2001).
Mechanism of action. Quinaldine has been purported to work
through mechanisms similar to those postulated for tricaine
(DeTolla et al., 1995), but even less is known of the exact
mechanisms of action.
Anesthetic effects. Quinaldine has been used for fish anes-
thesia and is more commonly used for collection of fish in
tide pools and coral reefs than is tricaine. In higher concentra-
tions, it can cause a rapid induction. Recovery characteristics
are more variable than with tricaine, in part because of the rel-
atively uncontrolled dosing that occurs with the drug in some
applications. Quinaldine often does not completely stop muscle
movement, and many anesthetists find it more appropriate for
sedation than for induction of surgical anesthesia. Long-term
sedation/anesthesia has been associated with mortality (Yanar
and Kumlu, 2000). Quinaldine can be an irritant to gills and
cause corneal damage, particularly when administered at high
concentrations for rapid immobilization in captures or when
using unbuffered stock solutions administered directly onto the
fish rather than mixing the stock into the anesthesia bath water
(Stoskopf, 1993).
Dose. As would be expected, the dose of quinaldine varies
with fish species, fish size, water temperature, and pH. Quinal-
dine is more effective in alkaline water, and in water with a
pH < 5, quinaldine is not effective (Bowser, 2001). At compa-
rable doses, larger fish are more heavily sedated and recovery
is more prolonged in warmer water. Quinaldine is generally
administered at concentrations of 15–60 mg/L to induce anes-
thesia or profound sedation. The most frequently used induction
dose for small laboratory fish species is 50 mg/L. Species dif-
ferences in response to the drug can be marked. For example,
25 mg/L will routinely cause loss of equilibrium in less than
4 minutes for salmonids, but for tilapia reported induction doses
range between 50 and 1000 mg/L (DeTolla et al., 1995).
Quinaldine can be administered in combination with tricaine,
and it is felt this mixture produces a faster induction. The most
common ratio of administration is 10:1, tricaine:quinaldine
sulfate (Rodger, 1999).
c. Imidazole anesthetics (metomidate: marinil, methomidate;
etomidate: amidate, R7464)
These agents are listed in our grouping of the best prac-
tice agents primarily because they are used extensively as
so-called “stress-free anesthesia agents”. We actually do not feel
they represent best practice agents but they may have specific
uses in certain situations. Unfortunately, the stress-free aspects
526
attributed to these drugs are a complete misnomer. Metomi-
date and etomidate block the 11-beta-hydroxylation of cortisol
both in mammals and in fish. This seriously perturbs the com-
plex positive and negative feedback pathways for control of the
neuroendocrine cascade. The lack of an increase in circulating
cortisol in fish given these drugs has been misinterpreted as
being due to the drug-relieving stress. This is not the case. The
fish are experiencing the same stress, but are not able to respond
to it by producing cortisol. This misinterpretation has resulted in
the misguided use of the drugs at low doses to “manage stress” in
fish shipments and transports. These drugs at appropriate doses
do provide a degree of immobilization and an apparent lack of
reaction to noxious stimuli. However, it is important to empha-
size that while plasma cortisol increases that occur when using
other fish anesthetics or even in minimally handling fish do not
occur with these drugs, this is a false picture of their neuroen-
docrine profile, which is characterized by increased levels of
hormones produced by the pituitary attempting to overcome the
synthetic blockade of cortisol production. Researchers should
be cautious when interpreting stress response in fish exposed to
imidazole anesthetics.
Chemistry. Metomidate and etomidate are imidazole com-
pounds. Metomidate is a white powder soluble in water and
ethanol. Etomidate is marketed as an aqueous solution com-
mercially prepared in 35% propylene glycol.
Mechanism of action. Imidazole anesthetics are gamma-
aminobutyric acid (GABA) receptor agonists that produce
anesthesia and amnesia in mammals (Stoelting, 1999). They
are poor muscle relaxants and provide no analgesia. The effi-
cacy of imidazoles in fish is greater in more alkaline water, and
therefore doses should be decreased with increasing pH of the
water.
Anesthetic effects. Imidazole anesthetics have a wide safety
margin in fish and produce a rapid induction with a more
prolonged recovery than induction, and these rates are dose
dependent (Amend and Goven, 1982). Metomidate at high con-
centrations (6–10 mg/L) is reported to cause no change in heart
rate, cardiac output, dorsal aortic pressure, or stroke volume in
Chinook salmon (Hill and Forster, 2004), but little physiologic
data is published on the effects on commonly used research
fish species. The effects reported in salmon are similar to what
has been reported in humans and other mammals (Stoelting,
1999). As mentioned earlier, metomidate has been suggested
to block the stress response. Imidazole anesthetics (e.g., meto-
midate and etomidate) interrupt cortisol synthesis in mammals
by suppressing the enzyme 11-beta-hydroxylase, which is nec-
essary for cortisol production (Stoelting, 1999). Fish exposed
to metomidate can have dark skin discoloration after exposure.
Because cortisol inhibits the release of ACTH and ACTH stim-
ulates the melanocyte-stimulating hormone, it is hypothesized
MICHAEL STOSKOPF AND LYSA PAM POSNER
that the decrease in cortisol production that occurs with the use
of these drugs results in an increase in melanocyte-stimulating
hormone (Harms and Bakal, 1994). Normal color returns after
recovery from the anesthetic. Metomidate has been successfully
used as an immersion agent, but has also been used successfully
to anesthetize turbot and halibut when used intravenously and
orally (Hansen et al., 2003).
Doses. Metomidate: 0.5–10 mg/L; 3 mg/kg IV; 7 mg/kg, oral
(Hansen et al., 2003). Etomidate: 2–20 mg/L.
d. Eugenol [4-allyl-2-methoxy-phenol: clove oil, isoeugenol,
AQUI-S (2-methoxy-4-propenyl phenol)]
AQUI-S is a 50% eugenol solution that is approved for use in
fish in Australia, New Zealand, Chile, and Korea, where it has
no withdrawal time for fish destined for human consumption or
release to the wild. The active ingredient, eugenol, is classified
by the FDA as GRAS (generally regarded as safe), but the use
of the compound for anesthesia of food fish is not approved.
Chemistry. Eugenol is a pale yellow liquid from the tree Euge-
nia aromatica. It is poorly water-soluble and must be dissolved
in ethanol (1:9) before dilution into water. The proprietary
formulation of AQUI-S is sufficiently soluble to allow direct
addition to induction water.
Mechanism of action. The mechanism of action of eugenol
in fish is unknown, but it has been used extensively as a topi-
cal anesthetic in human dentistry, and its mechanism of action
may be similar to that of other local anesthetics (e.g., tricaine,
lidocaine).
Anesthetic effect. Eugenol can cause immobility in fish. In
Chinook salmon, a high dose (60 mg/L) of anesthesia with
eugenol is accompanied by a decrease in heart rate, cardiac
output, dorsal aortic pressure, and stroke volume (Hill and
Forster, 2004). Anesthesia with eugenol is reported to cause a
75% decrease in arterial oxygen (PaO2 ) in fish (Hill and Forster,
2004), and an increase in catecholamines, glucose, and hema-
tocrit (Cho and Heath, 2000; Hill and Forster, 2004; Sladky
et al., 2001). It is possible that what appear as depression to the
CNS are the effects of hypoxemia.
Dose. Eugenol: 25–60 mg/L; AQUI-S: 20–60 mg/L.
2. Infrequently Used Agents in Fish
a. Benzocaine (ethyl aminobenzoate: anesthesin, anesthone,
ethyl aminobenzoate, orthesin, parathesin)
Benzocaine is commonly used as a local anesthetic for
humans (e.g., cough drops, sunburn aid). It is structurally
21. ANESTHESIA AND RESTRAINT OF LABORATORY FISH
similar to tricaine, but lacks the sulfate moiety of tricaine, which
renders the latter to be highly soluble in water. Benzocaine has
been used to produce anesthesia and for euthanasia. There is
no particular benefit of this agent over the closely related and
FDA-approved tricaine. The investigators should be guided to
use tricaine unless there is a specific reason that drug would
interfere with the data being collected. The similarities between
the drugs would make this a very rare situation.
Chemistry. Benzocaine occurs in two forms: a crystalline salt
(benzocaine HCl) that is soluble in water at 0.4 g/L, and a
nonwater-soluble basic form that must be dissolved in ethyl
alcohol at a concentration of 0.2 g/ml before it is sufficiently
soluble in water to create a functional induction dose. The HCl
salt of benzocaine can be prepared as a stock solution of 100 g/L,
which should be stored in a dark container and protected from
light. As with tricaine, benzocaine should be buffered with
sodium bicarbonate to raise the pH to 7.0–7.5.
Mechanism of action. Benzocaine is a local anesthetic that
blocks sodium channel conduction.
Anesthetic effects. Benzocaine produces a fairly rapid induc-
tion and recovery from anesthesia. However, even at high doses,
fish may retain some locomotor activity. Respiration is rapidly
depressed. Benzocaine is highly fat soluble, so obese or gravid
females may have prolonged recoveries.
Dose. Wide dose range reflects different species, size, water
temperature, and water hardness. Anesthesia can be accom-
plished with concentrations between 25 and 200 mg/L. Euthana-
sia can be accomplished with 3–5 times the concentration
needed to produce anesthesia.
b. Lidocaine (xylocaine, lignocaine)
Lidocaine is generally not considered as suitable an immer-
sion agent as tricaine, although it is used commonly for local
injectable analgesia. Investigators seeking to use this drug for
general anesthesia of their fish should be directed to the use of
tricaine unless there is a specific demonstrated reason to believe
that tricaine would interfere with the quality or usefulness of the
data collected.
Chemistry. Lidocaine is available in two forms: a hydrochloric
salt that is water soluble and a non-water-soluble basic form
that must be dissolved in acetone or alcohol before diluting in
water.
Mechanism of action. Lidocaine is a local anesthetic that
blocks sodium channel conduction (see information on
tricaine).
527
Anesthetic effect. Lidocaine produces a quick induction but
long recovery and causes cardiovascular depression. Increased
efficacy and decreased toxicity are seen when lidocaine is
administered with sodium bicarbonate (Carrasco et al., 1984).
Dose. Lidocaine has been administered at 250–350 mg/L with
1 g/L of sodium bicarbonate.
c. Volatile anesthetics (halothane, isoflurane, sevoflurane)
Although these agents are well studied in mammals and do
work through mechanisms that would be expected to induce
analgesia and anesthesia in fish, the challenges of their proper
safe use and achieving appropriate induction times with cur-
rently available technology keep them from being placed in the
category of “best practice agents.”
Chemistry. Volatile anesthetics are lipophilic and do not
always mix well with water. They can be added to the water
tank or distributed into the water by spraying the solution using
a syringe with a 25 g needle. Alternatively, it can be bubbled
through the water with oxygen through an anesthetic vaporizer
(Stetter, 2001).
Mechanism of action. Volatile anesthetics produce anesthesia,
analgesia, and amnesia by activating the inhibitory neuro-
transmitter GABA receptors in the CNS. Activation of GABA
receptors causes postsynaptic hyperpolarization which prevents
ascending neural transmission. It is also likely that volatile
inhalants block the activation of the excitatory neurotransmitter
glutamate.
Anesthetic effect. Induction can be slow with the excitement
stages observed. It is likely that the slow induction is due to
the difficulty in reaching therapeutic concentrations in water
because the volatile agents are continually being vaporized and
lost to the environment. At therapeutic concentrations, immo-
bility can be reached and recovery is quick following removal
from anesthetic exposure (Stetter, 2001).
Dose. Isoflurane: 0.25–0.75 ml/L (Stetter, 2001).
Fish require similar inhalant concentrations as mammals to
be effective (similar minimum alveolar concentration, MAC)
(Steffey, 1996). However, dosing is imprecise as agents are
highly volatile and vaporize quickly. This causes significant
changes to the water concentration and can increase atmospheric
concentrations exposing laboratory personnel to the anesthetic.
The ability to scavenge vaporized liquid is required. Due
to imprecision of dose and the exposure to humans, and the
need to scavenge waste gas, this method of anesthesia is rarely
suitable for the laboratory setting without highly specialized
equipment.
528
3. Inappropriate Agents for Use in Fish
a. Propanidid (epontol, sombrevin)
This drug is an ultra-short-acting, nonbarbiturate, general
anesthetic that was used successfully in human medicine until
it was prohibited from sale in the 1980s following anaphylactic
reactions.
Chemistry. Propanidid is insoluble in water, and thus must first
be dissolved in alcohol to a 5% stock solution before adding to
water.
Mechanism of action. Propanidid is presumed to work as a
GABA receptor agonist, but its mechanism of action in fish is
unknown.
Anesthetic action. Propanidid produces short- and long-term
anesthesia. The data with those reports are insufficient to estab-
lish whether or not anesthesia and analgesia are achieved.
Application of the drug is associated with significant respira-
tory and metabolic acidosis, with minimal changes to blood
chemistry (Thorsteinsson, 2002).
Dose. Propanidid is administered at 1.5–3 ml/L of an unspec-
ified stock solution. There is no compelling reason to pursue
this compound as a fish anesthetic.
b. Phenoxyethanol [2-phenoxyethanol phenyl ether: phenyl
cellosolve, phenoxethol, 2-phenoxyethanol (2-PE)]
Due to a low therapeutic index, and the potential for cardio-
vascular and toxic effects, phenoxyethanol is a poor choice as
anesthetic.
Chemistry. Phenoxyethanol is an organic compound that has a
water solubility of 27 g/L at 20◦ C. It is a topical irritant, and care
should be taken to protect human skin and eyes from contact.
There is no reason to believe it is any less irritating to fish tissues,
and thus application should only occur by exposing fish to the
lowest dilution needed for effect.
Mechanism of action. Mechanism of action of phe-
noxyethanol is unknown in fish.
Anesthetic action. Phenoxyethanol causes rapid induction and
recovery; however, fish may maintain muscle movement. Car-
diovascular parameters are depressed, and there is potential
for liver, kidney, and corneal damage. Fish may show initial
hyperactivity when first exposed, which is different from the
excitement of Stage II anesthesia and most likely attributable to
the irritant properties of the chemical.
MICHAEL STOSKOPF AND LYSA PAM POSNER
Dose. Phenoxyethanol is rarely used in modern research, and
investigators should be directed to use other drugs such as
tricaine.
c. Carbon dioxide (CO2 )
CO2 has a long history of use for sedation/anesthesia of fish.
It is inexpensive and can be delivered as a gas bubbled from
compressed gas tanks via air stones, or generated by use of
bicarbonate-of-soda antacids, in field situations. Approximately
200 mg/L of it is needed to immobilize fish. However, the gen-
eration of CO2 simultaneously displaces oxygen and increases
the acidity of water. The combination would not be expected to
be physiologically benign for the fish. Very high concentrations
of CO2 are required for sedation/anesthesia in mammals, and it
is unlikely that CO2 produces analgesia. Therefore, CO2 should
not be considered as an appropriate agent for anesthesia of fish.
4. Not Recommended for Use in Fish
a. Diethyl ether
Diethyl ether has a long history in human and veterinary
medicine as an anesthetic. Although it produces reliable anes-
thesia in mammals, it is extremely flammable. Its vapors are
denser than air, and will accumulate if proper ventilation is not
present. Simple static electricity can ignite the ether vapors.
Diethyl ether should only be used inside a fume hood, and thus
is not recommended for use in fish anesthesia.
b. Chloral hydrate
Chloral hydrate is a sedative that has been used extensively in
veterinary medicine, particularly in equine patients. It produces
sedation and muscle relaxation but does not produce analgesia or
anesthesia, and therefore it is not recommended as an anesthetic
agent for fish.
c. Urethane
Urethane has previously been used in the laboratory setting to
anesthetize laboratory animals and fish. However, urethane is an
established animal carcinogen and has been classified as “rea-
sonably anticipated to be a human carcinogen” (Program, 1983),
and thus should be not used in the routine laboratory setting.
d. Halothane
Halothane is a volatile anesthetic that has been used exten-
sively in human and veterinary medicine, and can be used
successfully to anesthetized fish. In people, hepatitis has been
reported following exposure to halothane (Stoelting, 1999). As
discussed previously, anesthesia of fish produces a significant
amount of environmental exposure to the agent. Thus, halothane
21. ANESTHESIA AND RESTRAINT OF LABORATORY FISH
is not recommended for this purpose due to the risks associated
with human exposure.
e. Chlorobutanol
Chlorobutanol has been used successfully to anesthetize fish,
but has been reported to be toxic in small fish (Canadian Council
on Animal Care, 2005). It poses human health hazards as it
is irritating to skin and eyes. Inhalation of large quantities can
cause unconsciousness (Thorsteinsson, 2002). Since it provides
no benefit over other available agents, it is not recommended
for use.
B. Injectable Agents
Injectable agents can be used successfully in fish intramus-
cularly or intravenously. This method does require either fish
handling before anesthesia which can increase stress, or the abil-
ity to dart or pole-syringe fish. For the researcher who needs to
anesthetize a large number of fish or routinely anesthetize fish,
this method may be less advantageous than immersion agents.
1. Best Practices Agents
a. Ketamine (ketaject, ketaset)
Chemistry. Ketamine is a white crystalline powder. One gram
of ketamine is soluble in 5 ml water or 14 ml alcohol. Commer-
cially prepared ketamine is a water-soluble liquid with a pH of
3.5–5.5.
Mechanism of action. Ketamine is an N -methyl-d-aspartate
(NMDA) antagonist that produces dissociative anesthesia by
dissociating the thalamo-cortical and limbic systems. In mam-
mals, it produces anesthesia and provides analgesia. It has been
suggested that ketamine provides better somatic than visceral
analgesia, but this distinction is inaccurate. NMDA antago-
nists has been shown to interrupt pain transmission in the dorsal
horn of the spinal cord, mediating somatic, visceral, neuropathic
and orthopaedic pain, often at subanesthetic doses (Visser and
Schug, 2006).
Anesthetic effects. Administration of ketamine is most com-
monly accomplished with an IM injection, although the anes-
thetic effects are more reliable intravenously. As in terrestrial
mammals, following administration of ketamine, fish may
struggle or show excitement during the early stages of anes-
thesia. During deeper anesthesia, muscle relaxation may still be
poor when ketamine is used alone. Muscle relaxation may be
improved by the addition of an alpha-2 agonist (e.g., xylazine).
Ventilation is minimally affected with ketamine alone, but may
be decreased when used in combination with an alpha-2 agonist
in sturgeon (Fleming et al., 2003).
529
Dose. Ketamine can be administered at 30 mg/kg IV (anes-
thesia <3 min) or 10–80 mg/kg IM. The very wide range of IM
doses is indicative of the relatively sparse research on the effi-
cacy and safety of this agent. As with other animals, it does
appear to have a wide margin of safety, and there may be signif-
icant interspecies differences in susceptibility. Another factor
that affects the required dose is the level of excitement of fish
at the time of injection.
b. Alpha-2 agonists (e.g., xylazine, dexmedetomidine,
medetomidine)
Chemistry. Alpha-2 agonists are crystalline substances that
are water soluble.
Mechanism of action. Alpha-2 agonists bind and activate
alpha-2 adrenergic receptors, resulting in sedation and anal-
gesia. At least five alpha-2 receptors have been identified in
zebrafish (Ruuskanen et al., 2005).
Anesthetic effects. Zebrafish show a dose-dependent decrease
in locomotion following dexmedetomidine administration
(Ruuskanen et al., 2005). It is possible that activation of alpha-2
receptors might also provide analgesia as it does in mammalian
species.
Alpha-2 antagonists (e.g., yohimbine, atipamezole) are effec-
tive reversal agents (Williams et al., 2004).
Dose. Medetomidine 0.06–4.0 mg/kg IM (given with ketamine).
c. Propofol
Chemistry. Propofol is an alkyl-phenol commercially avail-
able as an emulsion with a pH of 7–8.5.
Mechanism of action. Propofol is a GABA receptor ago-
nist that produces anesthesia in mammals (Stoelting, 1999). It
provides good muscle relaxation but no analgesia.
Anesthetic effects. In sturgeon, propofol produced light anes-
thesia accompanied with respiratory depression and bradycardia
(Fleming et al., 2003).
Dose. 3.5–7.5 mg/kg IV (Fleming et al., 2003).
2. Infrequently Used Agents
a. Alphaxalone–alphadolone (saffan, althesin)
This agent is a steroid anesthetic not available in the United
States, but commonly used in Europe and Australia in dogs and
cats. The older formulation had a Cremophor base and could
cause histamine release with swelling, pruritus, and edema. The
530
newer formulation (Alphaxalone CD) is alphaxalone only and
does not contain the Cremophor base.
Chemistry. Saffan is a poorly water-soluble synthetic steroid.
Each milliliter contains 9 mg alfaxalone plus 3 mg alfadolone.
Mechanism of action. Steroid anesthetics activate the
inhibitory GABA receptor producing anesthesia and muscle
relaxation.
Anesthetic action. Alphaxalone–alphadolone can produce
sedation and anesthesia in a variety of fish. At higher doses,
the induction is fairly rapid (within 5 minutes) but recovery to
normal swimming can take up to 5 hours (Harvey et al., 1987).
Dose. 0.3–1.5 ml/kg IM.
b. Azaperone (suicalm, stressnill)
Azaperone is a butyrophenone neuroleptic sedative/hypnotic
that is related to the drug haloperidol used in people with psy-
chiatric disorders. Azaperone has been extensively used in pigs
to prevent fighting and stress.
Chemistry. The commercial form is available in an aqueous
solution at 40 mg/ml.
Mechanism of action. Butyrophenones are dopamine (D2)
receptor antagonists. They also have agonist effects at serotonin
receptors and block activation of the reticular activating system.
Anesthetic action. Azaperone does not produce anesthesia or
analgesia but can be used as an anxiolytic. Fish administered
with azaperone do not show stress behaviors when netted or
moved to a new environment (Latas, 1987).
Dose: 4 mg/kg directly onto gills.
c. Barbiturates (pentobarbital, thiopental)
The barbiturates are often reported as potential fish anesthet-
ics in reviews, but apparently have only very rarely been applied.
They have been used on occasions to euthanize fish. Little actual
experience with these drugs in fish is reported in the literature.
Chemistry. These drugs are all derived from barbituric acid.
They are commercially available in aqueous solutions with a
basic pH. They are a class II controlled drug in the United States.
Mechanism of action. Barbiturates are commonly used anes-
thetics in mammals. They cause anesthesia by activation
and enhancement of GABA receptors in the CNS producing
unconsciousness and amnesia.
MICHAEL STOSKOPF AND LYSA PAM POSNER
Anesthetic effects. It is possible to use barbiturates intra-
venously in larger fish, but the difficulty of restraining larger
fish for IV injections makes this technique less useful for routine
anesthesia in research fish.
Dose. Thiopental 10–30 mg/kg IM; Pentobarbital 30–72 mg/kg
IM.
d. Diazepam
Diazepam has been used as an oral as well as an injectable
agent to sedate individual fish for safer handling and at lower
doses to stimulate appetite. It has been used effectively as a
sedative in larger groups but does not induce general anesthesia
or even mobility at the doses that have been applied. Published
data on the effects of this drug on fish species commonly used
in research laboratories is not readily available.
Chemistry. Diazepam has a water solubility of 3 g/L.The com-
mercially available liquid is 5 mg/ml with a vehicle that is 40%
propylene glycol and 10% ethanol. It is a Schedule IV controlled
drug.
Mechanism of action. Diazepam is a benzodiazepine that
produces sedation by activating GABA receptors in the CNS.
Anesthetic effects. Diazepam produces muscle relaxation and
CNS depression, but does not produce anesthesia or provide
analgesia. This type of drug is best used in conjunction with
other anesthetics and/or analgesics.
Dose. 1–4 mg/kg IM.
e. Gallamine triethiodide (Flaxedil)
Gallamine is a competitive, nondepolarizing neuromuscular
blocker (NMB) that has been extensively used to immobilize
alligators and crocodiles as well as fish.
Chemistry. Gallamine is supplied as 2% solution of gallamine
triethiodide, which needs to be protected from light.
Mechanism of action. NMBs work by blocking the binding
of acetylcholine to its receptor at the neuromuscular junction
causing paralysis of skeletal muscle. Neuromuscular blockers
do not produce any CNS depression or provide any analgesia. It
is considered inhumane and unacceptable to administer NMB
without a CNS depressant to mammals and should be considered
just as unacceptable for single agent use in fish.
Anesthetic action. Although NMBs do not provide sedation or
analgesia, they do provide immobility and chemical restraint.
21. ANESTHESIA AND RESTRAINT OF LABORATORY FISH
The paralysis of skeletal muscle can affect the muscles used
for respiration. The NMB, succinylcholine, can cause complete
cessation of respiration and death when used in fish. Gallamine
does appear to preserve gilling when properly dosed. Its use
alone in a painful procedure cannot be recommended.
Dose. Gallamine: 1–3 mg/kg IM.
3. Regional Anesthesia (Lidocaine, Bupivacaine)
Local anesthetics prevent the cranial migration of pain per-
ception by blocking sodium channel conduction. Local anes-
thetics can be used in fish as adjunct to general anesthesia or in
place of it. If the fish is not anesthetized, the injection of local
anesthetic can be painful and the manual restrain needed can be
stressful.
Dose. Lidocaine 4–10 mg/kg.
VII. ANALGESIC AGENTS
A. Opioids
There is evidence for the presence of mu and kappa opioid
receptors in teleost fish (Alvarez et al., 2006; Darlison et al.,
1997). This implies an endogenous opioid system that might be
manipulated to provide analgesia for research fish.
1. Butorphanol
Butorphanol is a kappa opioid agonist that results in sedation
and analgesia in mammals. The use of butorphanol (0.4 mg/kg
IM) in koi carp (Cyprinus carpio) preoperatively prevents
behavioral changes (Harms et al., 2005), suggesting either
decreased pain or stress or both. Butorphanol is a Schedule
IV controlled drug.
2. Morphine
Morphine is a mu opioid agonist that results in analgesia and
sedation in mammals. Morphine (5 mg/kg) has been shown to
attenuate postsurgical behaviors and decrease operculum rate
in rainbow trout following a noxious stimulus (Sneddon, 2006;
Sneddon et al., 2003). Naloxone has been shown to block
responses seen with administration of morphine. Morphine is a
Schedule II controlled drug.
B. Nonsteroidal Anti-Inflammatory Drugs (NSAIDs)
NSAIDs are commonly used drugs in human and veterinary
medicine. Ketoprofen (2 mg/kg IM) did not prevent behavioral
changes following noxious stimuli in koi carp, but did attenuate
531
the changes to creatine kinase indicating a diminished muscle
injury (Harms et al., 2005).
VIII. NONCHEMICAL METHODS
A. Hypothermia
Arguments are frequently given for the use of hypothermia
as an immobilizing method and even as an anesthetic for fish.
The question is quite complex, and based primarily on extrap-
olation of data from other species including humans. Many of
the fisheries-based investigators inappropriately equate anes-
thesia with immobilization, and little, if any, work has been
published assessing the actual issues looking at this method
of hypothermic analgesia or anesthesia in fish (Hovda, 2000).
Earlier work evaluating the physiologic impacts of lethal and
sublethal hypothermia on fish suggests that extreme caution be
used in considering this approach. Research has shown that
expected electrolyte and fluid shifts in tissues occur in fish
exposed to acute and chronic temperature shifts, and that the
rate of application of hypothermia, the acclimation tempera-
ture of the fish being induced, as well as the temperature being
applied as the endpoint of hypothermia affect survival and a
wide range of physiologic and biochemical processes in fish
(Elliott, 1981; Houston et al., 1968; Mark et al., 2002). While
the ability of hypothermia to immobilize fish is clearly estab-
lished, there is insufficient evidence to suggest that the process
provides anesthesia or analgesia.
Hypothermia applications in humans are frequently cited as
a basis for the use of the method in fish, but it should be pointed
out that humans subjected to surgical hypothermia to reduce
the impacts of hypoxia in certain types of surgery are under
general anesthesia before the technique is applied. Humans
experiencing hypothermia resulting in frostbite while conscious
generally report severe pain followed by localized numbness
before hypoesthesia occurs (Berg et al., 1999). Rewarming of
tissue has also been described as painful. Further investigation
into the impact on neural processing and the detailed impacts of
the application of the technique is needed before hypothermia
can be an accepted means of providing anesthesia for fish used
in research.
B. Electroanesthesia
Our understanding of electroanesthesia remains rudimentary.
The method immobilizes fish rapidly and hence has been recom-
mended for minor procedures particularly where large numbers
of fish are involved (DeTolla, 1995) and very specialized equip-
ment has been developed for the application of electroanesthesia
in wild fish for large-scale management assessments (United
States Patent 5305711). Studies on salmonids have shown that
532
electroanesthesia is a good alternative to MS-222 for short-
duration (<1 minute) immobilization (Gunstrom and Bethers,
1985; Orsi and Short, 1987; Sterritt et al., 1994) and the use
of electroanesthesia offers quick recovery of immobilized fish
(Gunstrom and Bethers, 1985).
Research in the area of human pain relief and anesthesia
reflected a major interest in the potential for electroanesthesia
and analgesia in the late 1960s to early 1970s, and mechanis-
tic assessments of the techniques began at that time. However,
the evaluation of electroanesthesia in fish suffers from the same
problems seen in the evaluation of other anesthetic modalities
for fish. Sophisticated physiologic assessments of anesthetized
fish are fairly rare. The reliance on plasma cortisol concen-
trations as a measure of anesthetic quality is fraught with
challenges, and there is an important need to determine stan-
dardized multifaceted methods for assessing quality in fish.
Recent studies comparing electroanesthesia and chemical anes-
thesia in juvenile Japanese eel (Anguilla japonica) provide a
good example (Chiba et al., 2006). The researchers looked at the
impact on plasma cortisol concentrations of eels anesthetized
using one of the several protocols used in electroanesthesia and
electroshocking (100 V, 2A—AC or 240 V, 6A—AC applied for
30 seconds and 500 V, 5A—DC or 1,000 V, 5A—DC pulsed
for 30 seconds). These results were compared to the effects of
the chemical anesthetics 2-PE and tricaine methanesulfonate
(MS-222) on plasma cortisol concentrations. Plasma cortisol
concentrations similar to those observed in eels anesthetized
with 2-PE, and somewhat elevated over what might be expected
as baseline cortisol levels based on studies in other fish species,
were observed in eels anesthetized with the lowest voltage
method (100 V). These results on their own would have been
consistent with the findings of earlier studies in goldfish using
MS-222 anesthesia to compare with electroanesthesia (Singly
and Chavin, 1975). However, the investigators noted that the
plasma cortisol concentrations in eels exposed to low voltage
were significantly elevated over the levels found in eels anes-
thetized with the higher voltage AC or DC electroanesthesia
methods, possibly supporting a conclusion that higher voltages
provide better anesthesia. Equally perturbing were the results
of the MS-222 anesthetized eels, which showed the highest
plasma cortisol concentrations of all groups studied. This dis-
parity, however, was most likely due to a quirk in the sampling
protocols where blood samples were drawn as soon as the fish
were deemed anesthetized rather than at a set time point after ini-
tiation of induction with the chosen agent. As in other vertebrate
species, there is a time lag in the response of elevated circulat-
ing cortisol concentrations and this time lag varies among fish
species to some degree. The use of high voltage caused a much
more rapid induction, allowing plasma samples to be obtained
much earlier after initial exposure than when the 100-V method
was employed. Similarly, the induction time for MS-222 in the
protocol used in the study was much longer than the 2-PE induc-
tion time, likely explaining much higher cortisol concentrations
in MS-222 anesthetized eels. The investigators saw increases in
MICHAEL STOSKOPF AND LYSA PAM POSNER
plasma cortisol concentrations over time in eels anesthetized
with electroanesthesia supporting this supposition. However,
the same is true of eels anesthetized with chemical anesthe-
sia. Eels, in particular, require much higher levels of chemical
anesthesia for induction than most other teleost fish, and their
response to exposure to these agents should not be ignored
when comparing the efficacy of electroanesthesia, particularly
for extrapolation to other fish groups.
IX. EUTHANASIA OF FISH
The goal of euthanasia of fish is the same as in all other species
(AVMA Panel on Euthanasia, 2001). It should be performed
quickly, humanely, and with minimal stress to the animal. The
unique differences in fish anatomy, physiology, and metabolism
need to be addressed when designing a plan for euthanasia.
Euthanasia may be accomplished by drug overdose or physical
methods. Unfortunately, many unsuitable methods have been
proposed and used in fish species.
A. Suitable Methods for Euthanasia
1. Chemical Overdose
Fish should be placed in water with anesthetic agents suffi-
cient to quickly anesthetize and then euthanize the fish (AVMA
Panel on Euthanasia, 2001). The fish should be left in treated
water for 10 minutes after movement has stopped.
Tricaine: 400–500 mg/L.
Benzocaine: >250 mg/L.
Phenoxyethanol: 0.5–0.6 mg/L.
Pentobarbital: 60–100 mg/kg IV or IP.
a. CO2
CO2 has been used to sedate and euthanize fish. It has been
applied by bubbling the gas through water using airstones or
by chemical generation of CO2 in the water. CO2 euthanasia is
accepted with caveats as humane in mammals (AVMA Panel
on Euthanasia, 2001). Further research should be conducted
on this agent and its impacts on fish. The common product,
Alka-seltzer™, and other carbonating antacids when added to
water produce CO2 . At high levels, CO2 does produce CNS
depression, however, in a tank atmosphere; it is more likely that
the fish die from hypoxemia.
2. Physical Methods
a. Decapitation
Decapitation is an acceptable form of euthanasia, but because
fish are tolerant to hypoxia, decapitation should be followed by
pithing to quickly stop brain activity. Because of differences
21. ANESTHESIA AND RESTRAINT OF LABORATORY FISH
in anatomy and difficulty in restraint, decapitation and pithing
should be conducted by a person experienced in the techniques.
When possible, decapitation should be performed following
induction of anesthesia.
b. Cranial concussion
Cranial concussion is an acceptable method of euthana-
sia. Unfortunately, some fish recover consciousness, and thus
cranial concussion should be followed by decapitation, exsan-
guination, or pithing. The procedure should be conducted by a
person experienced in the proper application of the technique in
the species being euthanized. When possible, cranial concussion
should be performed following induction of anesthesia.
B. Unsuitable Methods for Euthanasia
1. Cooling
Although cooling ectotherms will decrease movement and
metabolism, there is no evidence that it provided anesthesia
or analgesia. Many fish species are tolerant to cold and freeze.
Euthanasia by cooling/freezing should be considered inhumane.
2. Asphyxia
Asphyxia is not recommended due to the extended period of
time required for the fish to lose consciousness. Furthermore,
certain fish are resistant to hypoxia, and therefore the time to
death can be quite prolonged.
3. Formalin Immersion
It is quite unfortunate that formalin immersion of live fish
has been cited as an appropriate method for euthanasia of fish
in a number of publications including guidelines from scientific
societies that should be more cautious in their recommenda-
tions. The technique cannot be considered humane and should
not be employed even when logistical challenges of field con-
ditions are involved. Fish immersed in 10% buffered formalin
immediately exhibit extreme distress behaviors, rapid gilling,
and efforts to escape the solution, as would be predicted con-
sidering the irritant properties of formalin. They survive in the
solution far longer than would be expected, in some cases for
many hours (Stoskopf, personal observation). This technique
should not be condoned.
REFERENCES
Altimiras, J., and Larsen, E. (2000). Non-invasive recording of heart rate and
ventilation rate in rainbow trout during rest and swimming. Fish go wireless!
J. Fish Biol. 57, 197–209.
Alvarez, F.A., Rodriguez-Martin, I., et al. (2006). New kappa opioid receptor
from zebrafish Danio rerio. Neurosci. Lett. 405(1–2), 94–99.
533
Amend, D.F., Goven, B.A., et al. (1982). Etomidate: Effective dosages for a
new fish anesthestic. Trans. Am. Fish. Soc. 111, 337–341.
AVMA Panel on Euthanasia. (2001). Report of AVMA Panel on Euthanasia.
JAVMA 218, 669–696.
Berg, A., Aas, P., et al. (1999). Lokale frostskader. Tidsskr. Nor. Laegeforen.
119(3), 382–385.
Bowser, P. (2001). Anesthetic option for fish. In “Recent Advances in Vet-
erinary Anesthesia and Analgesia: Companion Animals.” (R. Gleed and
J. Ludders, eds.). International Veterinary Information Service, Ithaca, NY;
http://www.IVIS.org.
Brooks, A.I., Standifer, K.M., et al. (1994). Opioid binding in giant toad and
goldfish brain. Receptor 4(1), 55–62.
Carrasco, S., Sumano, H., and Navohro-Fierro, R., (1984). The use of
lidocaine–sodium bicarbonate as an anesthetic in fish. Aquaculture 41,
161–163.
Canadian Council on Animal Care. (2005). Anesthetic and sedative
drugs dosages fishes. Canadian Council on Animal Care; http://www.
ccac.ca/en/CCAC_Programs/Guidelines_Policies/GUIDES/ENGLISH/V1_
93/APPEN/APPXIII.HTM.
Chiba, H., Hattori, T., et al. (2006). Comparison of the effects of chemical
anesthesia and electroanesthesia on plasma cortisol levels in the Japanese eel
Anguilla japonica. Fish. Sci. 72, 693–695.
Cho, G.K., and Heath, D.D. (2000). Comparison of tricaine methanesulphonate
(MS222) and clove oil anaesthesia effects on the physiology of juvenile chi-
nook salmon Oncorhynchus tshawytscha (Walbaum). Aquaculture Res. 31,
537–546.
Darlison, M.G., Greten, F.R., et al. (1997). Opioid receptors from a lower
vertebrate (Catostomus commersoni): Sequence, pharmacology, coupling
to a G-protein-gated inward-rectifying potassium channel (GIRK1), and
evolution. Proc. Natl. Acad. Sci. U.S.A. 94(15), 8214–8219.
DeTolla, L., Srinivas, S., et al. (1995). Guidelines for the care and use of fish
in research. ILAR J. 37(4). Available at: http://dels.nas.edu/ilar_n/ilarjournal/
37_4/37_4Guidelines.shtml.
Doherty, T.J., and Frazier, D.L. (1998). Effect of intravenous lidocaine on
halothane minimum alveolar concentration in ponies. Equine Vet. J. 30(4),
300–303.
Dunlop, R., and Laming, P. (2005). Mechanoreceptive and nociceptive
responses in the central nervous system of goldfish (Carassius auratus) and
trout (Oncorhynchus mykiss). J. Pain 6(9), 561–568.
Elliott, J. (1981). “Some Aspects of Thermal Stress on Freshwater Teleost.”
Academic Press, London.
FDA. (2005). Database of approved animal drug products. http://dil.vetmed.
vt.edu/Display/NadaBrowse.cfm?NadaString=200–226.
Fleming, G.J., Heard, D.J., et al. (2003). Evaluation of propofol and
medetomidine–ketamine for short-term immobilization of Gulf of Mexico
sturgeon (Acipenser oxyrinchus de soti). J. Zoo Wildl. Med. 34(2), 153–158.
Gunstrom, G., and Bethers, M. (1985). Electrical anesthesia for handling large
salmonids. Prog. Fish-Culturist 47, 67–69.
Hansen, M.K., Nymoen, U., et al. (2003). Pharmacokinetic and pharmaco-
dynamic properties of metomidate in turbot (Scophthalmus maximus) and
halibut (Hippoglossus hippoglossus). J. Vet. Pharmacol. Ther. 26(2), 95–103.
Harms, C.A., and Bakal, B. (1994). Techniques in fish anesthesia. Proceed-
ings of the American Association of Zoo Veterinarians and Association of
Reptilian and Amphibian Veterinarians.
Harms, C.A., Lewbart, G.A., et al. (2005). Behavioral and clinical pathology
changes in koi carp (Cyprinus carpio) subjected to anesthesia and surgery
with and without intra-operative analgesics. Comp. Med. 55(3), 221–226.
Harvey, B., Denny, C., Kaiser, S., and Young, J. (1987). Remote intramuscular
injection of immobilizing drugs in to fish using a laser-aimed underwater dart
gun. Proc. Int. Assoc. Aquatic Anim. Med. 18, 144–152.
Hill, J.V., and Forster, M.E. (2004). Cardiovascular responses of Chinook
salmon (Oncorhynchus tshawytscha) during rapid anaesthetic induction
and recovery. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 137(2),
167–77.
534
Houston, A.H., Reaves, R.S., et al. (1968). Environmental temperature and the
body fluid system of the fresh-water teleost. I. Ionic regulation in thermally
acclimated rainbow trout, Salmo gairdneri. Comp. Biochem. Physiol. 25(2),
563–581.
International Association for the Study of Pain. (1994). “Classification of
Chronic Pain.” IASP Press, Seattle, Washington, DC.
Latas, P. (1987). The use of azaperone in the Spiney dogfish Squalus acanthus.
Proc. Int. Assoc. Aquatic Anim. Med. 18, 157–165.
Mark, F.C., Bock, C., et al. (2002). Oxygen-limited thermal tolerance in Antarc-
tic fish investigated by MRI and (31)P-MRS. Am. J. Physiol. Regul. Integr.
Comp. Physiol. 283(5), R1254–R1262.
Official Lists and Indexes of Names and Works in Zoology. (1987). International
Commission on Zoological Nomenclature; http://www.iczn.org/Official_
Lists_Indexes_pdfs.htm
Orsi, J., and Short, J.W. (1987). Modifications in electrical anesthesia for
salmonids. Prog. Fish-Culturist 49, 144–146.
Palmer, L.M., and Mensinger, A.F. (2004). Effect of the anesthetic tricaine
(MS-222) on nerve activity in the anterior lateral line of the oyster toadfish,
Opsanus tau. J. Neurophysiol. 92(2), 1034–1041.
Program, N.T. (1983). Urethane. Report on Carcinogens. US Department of
Health and Human Services.
Rodger, H. (1999). “Fish.” British Small Animal Veterinary Association,
Cheltenham.
Ruuskanen, J.O., Peitsaro, N., et al. (2005). Expression and function of alpha-
adrenoceptors in zebrafish: Drug effects, mRNA and receptor distributions.
J. Neurochem. 94(6), 1559–1569.
Singly, J., and Chavin, W. (1975). Serum cortisol in normal goldfish (Carassius
auraturs L.). Comp. Biochem. Physiol. 50(A), 77–82.
Sladky, K.K., Swanson, C.R., et al. (2001). Comparative efficacy of tricaine
methanesulfonate and clove oil for use as anesthetics in red pacu (Piaractus
brachypomus). Am. J. Vet. Res. 62(3), 337–342.
MICHAEL STOSKOPF AND LYSA PAM POSNER
Sneddon, L.U. (2006). Raleigh, (personal communication with L.P. Posner);
e-mail.
Sneddon, L.U., Braithwaite, V.A., et al. (2003). Novel object test: Examining
nociception and fear in the rainbow trout. J. Pain 4(8), 431–440.
Steffey, G. (1996). “Lumb & Jones’ Veterinary Anesthesia.” Lea & Febiger
Book, Baltimore, MD.
Sterritt, D., Elliot, S., et al. (1994). Electrical anesthesia for immobilizing adult
coho salmon in freshwater. North Am. J. Fish. Manag. 14, 453–456.
Stetter, M. (2001). “Fish and Amphibian Anesthesia.” WB Saunders, Philadel-
phia, PA.
Stoskopf, M.K. (ed.). (1993). “Fish Medicine.” WB Saunders, Philadelphia,
PA.
Stoelting, R. (1999). “Pharmacolgy and Physiology in Anesthetic Practice.”
Lippincott-Raven, Philadelphia, PA.
Thorsteinsson, V. (2002). Tagging methods for stock assessment and research
in fisheries. Marine Research Institute, Reykjavik, Iceland, pp 108–111.
Thurmon, J. (1996). “Lumb & Jones’ Veterinary Anesthesia.” Lea & Febiger
Book, Baltimore, MD.
Valverde, A., Doherty, T.J., et al. (2004). Effect of lidocaine on the minimum
alveolar concentration of isoflurane in dogs. Vet. Anaesth. Analg. 31(4), 264–
271.
Visser, E., and Schug, S.A. (2006). The role of ketamine in pain management.
Biomed. Pharmacother. 60(7), 341–348.
Williams, T.D., Rollins, M., et al. (2004). Intramuscular anesthesia of bonito and
Pacific mackerel with ketamine and medetomidine and reversal of anesthesia
with atipamezole. J. Am. Vet. Med. Assoc. 225(3), 417–421.
Yanar, M., and Kumlu, M. (2000). The anesthetic effect of quinaldine sul-
phate and or diazepam on sea bass juveniles. Turk. J. Vet. Anim. Sci. 25,
185–189.