Download Use of Injectable Potassium Chloride for

Comparative Medicine
Copyright 2000
by the American Association for Laboratory Animal Science
Vol 50, No 5
October 2000
Use of Injectable Potassium Chloride for Euthanasia
of American Lobsters (Homarus americanus)
A. Battison,1, * R. MacMillan1 A. MacKenzie1 P. Rose,2 R. Cawthorn,1,3 B. Horney1
Abstract  Potassium chloride (KCl: 330 mg/ml) was assessed as an euthanasia agent in American lobsters (Homarus
americanus). Two groups of 10 lobsters (408.2 to 849.9 g) were maintained at 11.9 to 12.1C (‘warm’) and 1.5 to 2.5C
(‘cold’) to evaluate the possible effect of ambient temperature on response to KCl. Death was defined as time of
cardiac arrest, as viewed and measured by use of ultrasound. The KCl solution was injected (100 mg of KCl/100 g of
body weight) at the base of the second walking leg to flood the hemolymph sinus containing the ventral nerve cord
with potassium. Disruption of this ‘central nervous system’ was immediate, followed by cardiac arrest within 60 to
90 seconds. Group median ( SD) baseline heart rate was 42 14 ‘warm’ and 36 5 ‘cold’ beats per minute. Time until
cardiac arrest ranged from 35 to 90 (57 18) seconds in the ‘warm’ group and from 40 to 132 (53 34) seconds in the
‘cold’ group. There was no significant difference between group medians for either parameter. Histologic lesions
were limited to mild to moderate acute degeneration, characterized by cell swelling, loss of contraction bands, and
occasional mild cytoplasmic vacuolation of skeletal muscle at the injection site. Injectable KCl solution was an
effective, reliable method for euthanasia of H. americanus.
Crustaceans, and other invertebrates, are often considered to be
‘lower life forms’ despite the recognized presence of complex behavioral responses and interactions (1). Descriptions of husbandry of
decapod crustaceans, including the American lobster, Homarus
americanus, currently recommend anesthetic overdose or anesthesia followed by freezing, as acceptable methods of euthanasia (2).
However, recovery from ‘death’ after periods of 24 hours or more
can occur in Homarus americanus after anesthetic overdose using
isobutanol (3). Freezing alone is an unacceptable alternative, as
formation of ice crystals in tissues may cause pain or distress in a
conscious animal (4). Earlier investigators also examined use of
electrical shock (5) and mechanical disruption, referred to as ‘sticking,’ of the ‘brain’ or ventral nerve cord (6) as non-chemical means
of euthanasia in large decapods. Electric shock is painful and unacceptable as a sole method of euthanasia by current standards
(4). ‘Sticking’ was considered an acceptable technique for use in
crabs, Cancer pagurus L., if preservation of the integrity of the surrounding tissues was not a requirement (6). Due to differences in
anatomy or physiology, techniques suitable for use in crabs might
not be effective in other decapods. The objective of the study reported here was to evaluate use of potassium chloride (KCl) as an
injectable euthanasia agent in H. americanus.
Potassium is a major intracellular cation, with extracellular
concentrations being carefully regulated within a narrow range
(7). In vertebrates, increase of blood potassium concentration,
hyperkalemia, can cause cardiac arrest due to depolarization of
cardiac cells (7). Potassium chloride, via intravenous injection,
is not approved when used as a sole agent for euthanasia in
mammals (4). The animal will remain conscious and capable of
experiencing distress, and potentially pain, during the period
prior to and including cardiac arrest. Death is ultimately attributable to hypoxia subsequent to circulatory failure.
1
Department of Pathology and Microbiology, 2Veterinary Teaching Hospital, and
Atlantic Veterinary College Lobster Science Centre, Atlantic Veterinary College,
University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada.
*Corresponding author.
3
In the protocol described here, the KCl solution is injected into
the hemolymph sinus that contains the ventral nerve cord and
communicates with the area surrounding the supraesophageal
ganglion. These structures can be considered the invertebrate
equivalent of the ‘spinal cord’ and ‘brain’ (i.e., central nervous system [CNS]) of vertebrate species. Flooding this region with high
concentration of potassium causes near-immediate depolarization
of neurons—effectively inhibiting transmission of sensory or motor
signals. The lobster is functionally ‘unconscious’ immediately. Cardiac arrest generally follows within 40 to 90 seconds due to increased extracellular potassium concentrations.
Distinguishing a dead lobster from one that is either simply
weak or moribund can be difficult. Evaluation of reflexes is difficult in any weakened animal. A pulse (as an indication of
heart rate) is not readily detectable in crustaceans. In this study, a
combination of M-mode and 2-D real-time B-mode ultrasound imaging was used to monitor heart rate and establish the time of
death in the experimental animals. To evaluate the potential for
ambient holding temperature on outcome, lobsters were acclimatized at 1.8 to 2.1C, ‘cold’ treatment group, and 11.9 to
12.3C, ‘warm’ treatment group, before injection with KCl.
Materials and Methods
The experimental protocol was approved by the Animal Care
Committee of the Atlantic Veterinary College (AVC) in accordance with the Canadian Council on Animal Care guidelines.
Twenty wild-caught, intermoult lobsters were purchased from a
dryland pound (Clearwater Inc., Halifax, Nova Scotia, Canada), acclimatized, and maintained for four to eight months in the lobster
holding facility at the AVC Lobster Science Centre (Charlottetown,
Prince Edward Island, Canada). The primary tanks utilize a recirculating water system with multiple filters, 300 and 100 m
(nominal) and 20 m (absolute), in place. Salinity is maintained
at 30 2 ppt. Ammonia (0.0 to 0.3 ppm), nitrates (10 to 40
ppm), nitrites (0.0 to 0.6 ppm), pH (7.41 to 8.48), water tempera-
545
Vol 50, No 5
Comparative Medicine
October 2000
ture (1.5 to 2.5C), and dissolved oxygen (9.65 to 12.9 mg/L) concentrations are monitored on a continuous basis (CR7 Measurement and Control System, Campbell Scientific Inc., Logan, UT)
and kept within their specified limits.
Lobsters were kept in individual compartments, six compartments per tray, with eight trays per tank. Lobsters were kept on
a 14 /10-hour low (< 3 lux) light/dark cycle except during periods
of room access when low-level incandescent lighting was increased. Lobsters were not fed during the holding period.
Lobsters were evaluated for presence of infective agents before being separated into the two treatment groups and at the
end of the trial prior to KCl injection. Hemolymph samples (3
ml) were collected aseptically (3-ml syringe, 20-gauge needle)
from the ventral abdominal sinus between the first and second
pleopods (swimmerets). Duplicate phenylethanol (PEA) broth
culture tubes were inoculated with 0.5 ml of hemolymph to examine for the presence of Aerococcus viridans, the etiologic
agent of gaffkemia. Tubes were kept at 28C in an aerobic incubator and checked for positive reactions at 48 and 96 hours.
Positive-reacting tubes were subcultured on blood agar plates
at 28C, colonies were Gram-stained and tested, using Api-20
Strep strips (bioMérieux, St-Laurent, Québec, Canada) for further bacterial identification. Lobsters were examined for infection with the scuticociliate Anophryoides haemophila by placing
a drop of hemolymph on a glass slide and examining it microscopically for presence of the parasite. Ciliate culture medium
(8) also was inoculated with 0.5 ml of hemolymph and incubated
at 4C for one week. Cultures were examined for An.
haemophila, microscopically, at the end of the incubation period.
Eight of the 20 lobsters (5 males and 3 females, weighing
from 702.6 to 849.9 g; median weight, 738.7 g) had been acclimatized in the system for 4 months before the experimental
trial. Twelve lobsters (8 males and 4 females, weighing 408.2 to
570.4 g; median weight, 508.4 g) had been acclimatized for 8
months. Lobsters were allocated to two treatment groups designated ‘cold’ and ‘warm.’ Groups were balanced as much as possible with respect to weight, sex, and prior acclimatization
periods. Adhesive labels were used for lobster identification.
‘Cold’ lobsters were kept in the primary system as previously
described. ‘Warm’ lobsters were placed, using an identical tray storage system, into an insulated holding tank (185 L). Water was obtained from the reservoir of the main system and allowed to warm
gradually in the holding tank over 9 days to 12.1C. Oxygen was
supplied continuously by use of an air compressor (Thomas Industries Inc., Sheboygan, WI) with a 7.5 by 2.5-cm air stone. Mortality detection was performed daily. Both groups of lobsters were
kept for a total of 11 days before injection with the KCl solution.
Hemolymph was again collected to examine for A. viridans and
An. haemophila infections at the end of the acclimatization period.
The 330 mg of KCl/ml solution was prepared from reagent grade
KCl (Fisher Scientific, Nepean, Ontario, Canada) and sterile distilled water. The solution was cold-filtered (0.2-m filter) under
vacuum and placed in a 45C warm water bath for 30 minutes to
ensure complete dissolution. Measured osmolality by freezing
point depression (OsmetteTM, model 5004, Precision Systems
Inc., 16 Tech Circle, Natick, MA) was 8,800 mOsm/kg on dilution.
To detect communication of the hemolymph sinus at the base of
the walking leg to the sinus surrounding the ventral nerve cord
(sternal sinus) and supraesophageal ganglion, a dye study was performed. Twenty drops of green commercial food coloring (Club
546
House®, McCormick Canada Inc., London, Ontario, Canada) were
added to 3 ml of the same artificial sea water (Instant Ocean®,
Aquarium Systems, Mentor, OH) used in the recirculation system.
Lobsters are considered osmoconformers (9); consequently, the
concentrations of major electrolytes in the hemolymph are similar to the surrounding seawater. This was confirmed by comparing the reported electrolyte concentrations in the artificial
seawater with the AVC Diagnostic Laboratory’s reference range
for hemolymph electrolytes for lobsters maintained under these
conditions. The colored solution was injected at the base of the
second walking leg of a non-study lobster, obtained for this purpose only (Figure 1). The movement of the dyed solution was
readily observed through the thin cuticle as dye passed from the
thorax into the abdomen. The lobster was then immediately killed
by manually severing the ventral nerve cord cranial to the large
chelae. The lobster was dissected to locate the ventral nerve
cord and supraesophageal ganglion confirming communication
of the sinuses (Figure 2).
Prior to KCl injection, baseline heart rate was determined
ultrasonographically (HDI 3000, P7-4 transducer, Advanced Tech-
Figure 1. Ventral view of the site of needle placement at the base of
the second pereiopod (walking leg) in Homarus americanus.
Figure 2. Lateral view of the lobster. The lateral carapace has been
removed to expose the supraesophageal ganglion (G), which is connected to the ventral nerve cord via connectives seen here, emerging
from the caudal border of the ganglion.
Euthanasia of Lobsters
Figure 3. Simultaneous 2-D real-time B-mode and M-mode cardiac
imaging obtained prior to injection of potassium chloride in the American lobster (Homarus americanus) maintained at 11.9 to 12.3C (HDI
3000, P7-4 transducer, Advanced Technology Laboratories, Markham,
Ontario, Canada). The small upper image is a real-time image of the
heart in a short-axis plane, with the M-mode cursor (thin white vertical line) positioned for measurement of wall movement. The lower
image is an M-mode tracing, following the movement of the selected
portion of the cardiac wall through the cardiac cycle. In this image,
the upper white band is the ventral cardiac wall, the lower white
band is the dorsal cardiac wall, and the black band in between is the
cardiac lumen. The walls appear closer in systole and farther apart in
diastole. Cardiac function is normal. The calculated heart rate is 53
beats per minute.
nology Laboratories, Markham, Ontario, Canada). A small amount
of ultrasound gel was used to provide contact. The probe was
placed on the ventral surface of the tail cranial to the first set of
pleopods (swimmerets) and directed craniodorsad toward the
heart. Real-time 2-D B-mode imaging was used to identify the
heart. Simultaneous M-mode imaging was performed to determine
the heart rate in beats per minute (bpm) (Figure 3).
Lobsters were lightly restrained manually in recumbent position, and injected (3-ml syringe, 20-gauge needle) with the KCl solution (100 mg of KCl/100 g of lobster) at the base of the second
pereiopod (walking leg). Rotating the leg internally and extending
it caudad provided easy access to the area. The dose was administered as a bolus injection. Heart rate was monitored continually
after injection. Death was defined as the time at which effective
heart contractions ceased (Figure 4).
Samples of heart, hepatopancreas, antennal gland, midgut,
gill, gonad, abdominal muscle, and muscle tissue from the injection site were collected 3 to 4 hours after euthanasia and were
placed in chilled (4C) 1G4F (1 part glutaraldehyde: 4 parts formalin) fixative. Tissues were processed in routine manner and
stained with hematoxylin and eosin for histologic evaluation.
Statistical analysis was performed, using the Minitab® release 11.12 software package (Minitab Inc., State College, PA).
A P-value < 0.05 was considered significant.
Results
All lobsters were culture negative for A. viridans and An.
haemophila at both sampling periods. Water temperature in the
insulated tank increased an average of 1.2C/d for 8 days, then
Figure 4. Simultaneous 2-D real-time B-mode and M-mode cardiac
imaging obtained 35 seconds after injection of potassium chloride in
the American lobster (Homarus americanus) maintained at 11.9 to
12.3C (HDI 3000, P7-4 transducer). The upper image is a real-time
image of the heart in a short-axis plane, with the M-mode cursor (thin
vertical white line) positioned for measurement of wall movement.
The lower image is an M-mode tracing, following the movement of
the selected portion of the cardiac wall through the cardiac cycle. In
this image, the broad white band is the ventral and dorsal cardiac
walls in close contact, and therefore, the cardiac lumen is not visible.
The heart is in systole, and cardiac function has ceased.
stabilized between 11.9 and 12.1C for the remaining 3 days of
the acclimatization period. Water in the insulated tank had to
be completely exchanged, with prewarmed (12C) reservoir water, on day 10 due to high ammonia concentration (> 5 mg/L).
The ammonia concentration was 1 mg/L the following day. Lobster 12 of this group was found dead on day 5 and removed.
The dyed seawater solution rapidly passed from the injection
site at the base of the second walking leg to the ventral midline of
the thorax and on to the mid-ventral region of the tail (abdomen).
Dissection revealed the ventral nerve chain and supraesophageal
ganglion surrounded by green-tinged fluid and tissue.
Initial heart rate ranged from 19 to 60 bpm. There was a
wider range of heart rate in the ‘warm’ group, 19 to 60 bpm
(42 14 bpm: median SD), compared with that of the ‘cold’
group, 29 to 44 bpm (36 5 bpm). However, there was no significant difference between group median heart rates (MannWhitney test, P = 0.46). There was no significant correlation
between lobster weight and heart rate when lobsters were evaluated as one large group (Pearson’s correlation coefficient, 0.249) or
within treatment groups (Pearson’s correlation coefficients,
0.483 warm, 0.094 cold).
Lobster 8 moved during the injection and did not receive the full
dose of KCl. Administration of an additional 1.0 ml of KCl solution
was required to cause cardiac arrest. Results from this animal are
included in the summary table (Table 1), but were not included in
the statistical analysis. The time until death in the ‘cold’ group
ranged from 40 to 132 (53 34) seconds. Time until death in the
‘warm’ group ranged from 35 to 90 (57 18) seconds. There was no
significant difference in median time until death between the
treatment groups (Mann-Whitney test, P = 0.76).
Immediately after the bolus KCl injection, there was a brief
547
Vol 50, No 5
Comparative Medicine
October 2000
Table 1. Summary of physiologic parameters and responses to potassium
chloride injection in American lobsters (Homarus americanus)
by treatment group
Cold (2C)
Lobster Body
Heart Time to Lobster
No.
weight
rate
death
No.
1
2
3
4
5
6
7
8
9
10
Median
SD
(g)
743.5
749.6
854.8
774.8
480.3
468.9
500.0
548.1
567.0
437.3
557.5
152.3
(bpm)
42
44
32
36
40
34
29
36
30
42
36
5.3
(s)
47
40
132
45
53
53
120
240b
73
50
53
33.9
11
12
13
14
15
16
17
18
19
20
Warm (12)
Body
Heart
weight
rate
(g)
746.8
NAa
739.6
703.2
406.5
524.9
515.9
514.9
533.6
490.4
524.9
122.4
(bpm)
32
NAa
56
57
19
34
42
48
60
29
42
14.3
Time to
death
(s)
71
NAa
57
44
76
35
90
58
50
40
57
18.1
a
Lobster died on day 5; data not available
Lobster did not receive full dose of KCl; results not included in statistical
analysis
bpm = beats per minute; SD= standard deviation
b
period (approx. 15 seconds) of limb extension followed by flaccidity
of the limbs and tail. There also was little to no ‘pinch’/closure response to objects placed within the chelae of the walking legs at
that time. The cardiac response was immediate, but variable in all
lobsters. In some lobsters, the heart rate would visibly slow and the
force of contraction would gradually diminish and eventually stop.
In others, there was cessation of contraction although movement of
the ventral wall was still present for a short time.
Lesions attributable to KCl injection were limited to mild to
moderate, multifocal myofiber degeneration of skeletal muscle at
the injection site. This was characterized by cell swelling, loss of
contraction bands, and occasional mild cytoplasmic vacuolation.
Discussion
The literal translation for ‘euthanasia’ is from the Greek eu
(good) and thantos (death). As stated in the Canadian Council
on Animal Care (CCAC) Guide, euthanasia procedures should
be ‘humane.’ This is defined as “…be painless, minimize fear
and anxiety, be reliable, reproducible, irreversible, simple, safe,
and rapid.” (10). The method of euthanasia should also be safe
and esthetically acceptable for personnel carrying out the procedure. It is also stated that a euthanasia method should “…produce rapid unconsciousness and thus assurance of insensitivity
to pain; this must be followed by cardiac and respiratory arrest.”
Death can be due to either direct or indirect hypoxia, depression
of essential neuronal activity, or physical disruption of brain activity resulting in unconsciousness.
Various physical and chemical (inhalant and injectable agents)
methods are approved for euthanasia of vertebrates (mammals,
birds, fish, reptiles, and amphibians) in laboratory settings.
Guidelines are in place to ensure that these procedures are carried out as humanely as possible for these species. Local institutional committees, the Animal Care Committees in Canada and
the Institutional Animal Care and Use Committees (IACUC) in
the United States, are present to ensure that these guidelines
are followed. There are often few, if any, guidelines in place for
invertebrate species. Publications involving invertebrate research may include no reference to the method used to terminate research subjects at the conclusion of experimental trials.
A manual describing the care of decapod crustaceans, such as
H. americanus, recommends anesthesia followed by freezing, or
548
an overdose of a recognized anesthetic, such as isobutanol, as
acceptable methods of termination (2). Freezing as a sole means
of euthanasia is deemed unacceptable—formation of ice crystals
within tissues may be painful (4). Electric shock (5) and physical disruption of the ‘brain’ or ventral nerve cord, ‘pithing,’ have
also been investigated as possible alternatives (6). Although
both procedures can induce unconsciousness if performed correctly, it is recommended that they be used in conjunction with
another procedure, such as exsanguination or decapitation, or
administration of pharmacologic agents to ensure death of the
animal (4). Electric shock may induce cardiac fibrillation while
the animal is still conscious if performed incorrectly (4). Pithing
may be suitable as a sole means of euthanasia in some species
(e.g., frogs) where the CNS is more readily accessible (4). This is
not the case in H. americanus.
The CNS of H. americanus consists of a ventral nerve cord
composed of a series of 15 paired ganglia connected by interneurons. The ‘brain,’ the supraesophageal ganglion, is the most cranial ganglion, lying dorsal to the esophagus, behind and between
the eyes. There are three more paired ganglia in the cephalic region. The most caudal, the subesophageal ganglion, contains
cardioinhibitor and cardioaccelerator nerve bodies. There are
five paired ganglia in the thorax and six paired ganglia in the
abdomen. The ventral nerve cord is found dorsal to the ventral thoracic and ventral abdominal arteries in the thorax and abdomen,
respectively (11).
Potassium chloride derives its effectiveness as an injectable
euthanasia agent from the potassium component. The cells,
more specifically, the neurons of H. americanus are similar
physiologically to their mammalian counterparts (11). Cells rely
on an electrochemical gradient/transmembrane potential across
the plasma membrane for proper function and survival. The
normal potassium ion gradient, high concentrations inside the
cell and low concentrations outside of the cell, is the principal
determinant of this potential. Maintenance of this potential is
essential for cellular function and, in the case of neurons, for
transmission of nerve impulses. Injecting a highly concentrated
potassium solution into the hemolymph increases the extracellular potassium concentration. Potassium then rapidly enters
the cell down the ‘new’ concentration gradient. Cells become depolarized due to the influx of positive charges. Depolarization
causes opening of voltage-gated sodium channels and subsequent generation of an action potential. The cell is unable to
repolarize because of the excess amount of intracellular potassium.
Disruption of the nervous system ensues, ultimately resulting in
failure of vital systems (e.g., circulatory and respiratory) and
death of the animal. The base of the second walking leg was chosen as the injection site for this trial after the dye study confirmed that this would result in immediate flooding of the sinus
containing the ventral nerve cord and the area surrounding the
supraesophageal ganglion.
Heart rate, initiation of contraction, and contractility are controlled principally by the cardiac ganglion, which is located at
the inner dorsal wall of the heart (12). The heart also receives
inhibitory and acceleratory signals from the cardioinhibitory
and cardioacceleratory nerves, respectively, which originate in
or slightly caudal to the subesophageal ganglion (12).
Initial heart rate in the lobsters ranged from 19 to 60 bpm.
There was no association between heart rate and body weight
when lobsters were evaluated as a single group or by treatment
Euthanasia of Lobsters
groups. This agrees with previous observations where the heart
rate for 425- to 500-g lobsters, kept at 16 to 20C, ranged from 50 to
130 bpm, with no correlation between heart rate, body weight, or
‘blood pressure’ (13). Although the median heart rate between the
two treatment groups was not statistically different, it was interesting to note that there was a wider range in heart rate in the
‘warm’ (19 to 60 bpm.) than the ‘cold’ (29 to 44 bpm) group. Ambient temperature has direct and indirect effects on heart rate in
crustaceans. Increasing temperature, within normal environmental range, generally results in increased heart rate (12).
The response to the KCl injection was virtually immediate in
all of the lobsters. Rigid extension of the claws and walking legs
was followed (approx. 15 seconds) by flaccid relaxation and loss
of muscular tone. This is the expected response after depolarization of the motor neurons. Muscles initially contract, then become
flaccid once the motor nerves lose the ability to repolarize and generate further electrical signals. Because the KCl solution is injected directly into the sinus surrounding the ventral nerve cord
and reaches the ‘brain,’ it can be concluded that disruption of
the CNS, its ability to process and transmit sensory input, and
loss of any awareness would be almost immediate. This would
satisfy the requirement for ‘anesthesia’ prior to cardiac arrest
under the definition of humane methods of euthanasia.
Time until visible cardiac arrest varied among animals, but
generally occurred in under 60 seconds in 12 of the 18 lobsters
and under 90 seconds in 16 of the lobsters. Slowing of the heart
rate and irregularity of contractions was generally evident within
15 to 30 seconds. The ventral wall was often the last area of the
heart muscle to continue attempts at contraction. Cardiac arrest for periods of up to 20 minutes, followed by resumption of
cardiac activity, has been reported in H. americanus (14). However, this was under quiescent laboratory conditions. The lobsters of this study were being handled and lightly restrained in
a recumbent position. These cannot be considered ‘quiescent
conditions.’ Cardiac arrest in this study was considered to be
chemically induced—an irreversible result of KCl injection,
rather than a transient, voluntary, physiologic state.
Potassium chloride, given by the intravenous route, for euthanasia is not approved for use in experimental animals (10, 15)
unless, the animal has received prior sedation or anesthesia because the conscious animal may experience pain and/or be distressed during cardiac and respiratory arrest. Using this protocol,
the KCl is injected directly into the CNS. This is expected to result
in near instantaneous disruption of the sensory and motor components of the nervous system. The rapid and sustained extension of the limbs after KCl injection provides evidence of loss of
motor control. Concurrent loss of the ability to process sensory
input is assumed. In this study, cardiac arrest occurred in most
animals in < 90 seconds after KCl injection.
The dose required, approximately 1 g of KCl/ kg of lobster, to
kill the lobsters in this study appears quite high. There are few
published reports of use of KCl as a euthanasia agent. Thirty to
35 mg of KCl/kg is the lethal intravenous dose in rabbits (16). Slow
intravenous injection of isotonic KCl at a dosage of 119 mg/kg was
fatal in dogs (17). The higher, 10- to 30-fold dose required in H.
americanus may be a characteristic of its physiology, which
makes it more resistant to the effects of high extracellular potassium concentration.
In those earlier reports in mammals, KCl was administered
by the intravenous route. An intrasinus injection would be the
closest equivalent in H. americanus with its open circulatory
system. Initial attempts by the authors to inject a similar total
dose of a less concentrated solution, 149 mg of KCl/ml (Astra
Pharma Inc., Mississauga, Ontario, Canada) into the large ventral abdominal sinus were less successful. Lobsters remained
alert for up to 20 to 30 minutes (personal observation by ALB).
A dilutional effect was suspected. This was not an acceptable
response nor did it meet the CCAC requirements for approved
methods of euthanasia. Cardiac and peripheral neuromuscular
effects would have preceded depression of the CNS. Similarly,
immersing a lobster in a KCl ‘bath,’ relying on diffusion of the
potassium across the gill membrane, would be unacceptable.
Isobutanol has been suggested as method of euthanasia in large
decapods (2). Isobutanol presents appreciable risk to personnel
handling the animals due to its properties of rapid percutaneous
absorption and vaporization. This chemical can cause severe irritation of skin, eyes, and mucous membranes. It is also a CNS depressant. Protective clothing and gloves should be worn when working
with this agent. Rooms should be well-ventilated (18). The efficacy
of isobutanol as an euthanasia agent may be questionable. Gilgans
and Burns (3), in their study of isobutanol as an injectable anesthetic agent, reported that, among lobsters that received ‘lethal’
overdoses of the agent, 50% of those kept at 6 to 8.5C and 30% of
those kept at 15C eventually ‘recovered.’ Lobsters were considered
to be dead if they were ‘limp’ and failed to withdraw their eye when
the visual surface was touched after a period of 24 hours.
Mild to moderate, multifocal, acute myofiber degeneration,
limited to the injection site, was the only histologic lesion identified in the tissues (heart, gill, hepatopancreas, midgut, gonad,
antennal gland, and abdominal and injection site muscle) examined. This was likely due to the hypertonicity of the KCl solution. Injection of a similar volume of autologous hemolymph did
not cause tissue injury (ALB, personal observation). Potassium
chloride injection, in addition to providing a rapid method for
euthanasia, would be advantageous in studies that require histologic evaluation of tissues.
Excellent ultrasound images of the heart were obtained, using
the 7-4 phased array probe. It was simple to obtain images in the
larger lobsters. However, imaging was more difficult in the
smaller lobsters. Four hundred grams may represent the minimal
practical size limit for this particular probe. A transducer with a
smaller contact surface would likely be required if smaller lobsters were to be examined using this technique.
Injectable KCl solution was an effective, rapid, reliable, and
safe method for the euthanasia of H. americanus in experimental studies conducted at the Atlantic Veterinary College Lobster
Science Centre. Disruption of the CNS is rapid and is followed
by cardiac arrest in < 90 seconds in most lobsters tested. There
is minimal risk to staff performing the procedure. The estimated
cost of the procedure is 0.7¢/ml of final solution. This method provides an acceptable and inexpensive alternative to the currently
recommended methods of anesthetic overdose or anesthesia followed by freezing for euthanasia of H. americanus. The procedure
should be readily adaptable for use in other crustaceans.
References
1. Atema, J., and R. Voight. 1995. Behaviour and sensory biology,
p. 313–348. In J. R. Factor (ed.), Biology of the lobster Homarus
americanus. Academic Press, Inc., San Diego, CA.
549
Vol 50, No 5
Comparative Medicine
October 2000
2. Ingle, R. W. 1995. Restraining, anaesthetising and killing, p. 78–
79. In The UFAW handbook on the care and management of decapod crustaceans in captivity. The Universities Federation for Animal Welfare, Potters Bar, Hertsfordshire, England.
3. Gilgan, M. W., and B. G. Burns. 1976. The anaesthesia of the
lobster (Homarus americanus) by isobutanol injection. Can. J. Zool.
54:1231–1234.
4. Andrews, E. J., B. T. Bennett, J. D. Clark, et al. 1993. 1993
report of the AVMA panel on euthanasia. J. Am. Vet. Med. Assoc.
202:230–249.
5. Baker, J. R., and M. B. Dolan. 1975. Experiments on the humane killing of lobsters (Homarus vulgaris) and crabs (Cancer
pagurus). Part 2. The exposure of lobsters to electric shock before
boiling. Sci. Paps. Humane Educ. Cent. 2:1–24.
6. Baker, J. R. 1955. Experiments on the humane killing of crabs.
J. Marine Biol. Assoc. UK 34:15–24.
7. Peterson, L. N., and M. Levi. 1997. Disorders of potassium metabolism, p. 192–240. In R. W. Schrier (ed.), Renal and electrolyte
disorders 5th edition. Lippincott-Raven, Philadelphia, PA.
8. Messick, G. A., and E. B. Small. 1996. Mesanophrys
chesapeakensis n. sp., a histophagous ciliate in the blue crab,
Callinectes sapidus, and associated hisopathology. Invertebrate
Biol. 115:1–12.
9. McMahon, B. R. 1995. The physiology of gas exchange, circulation, ion regulation, and nitrogenous excretion: An integrative approach, p. 497–517. In J. R. Factor (ed.), Biology of the lobster
Homarus americanus. Academic Press, Inc., San Diego, CA.
550
10. Canadian Council on Animal Care. 1993. Euthanasia, p. 141–
153. In E. D. Olfert, B. M. Cross, and A.A. McWilliam (ed.), Guide
to the care and use of experimental animals: volume 1, 2nd edition. Bradda Printing Services Inc., Ottawa, Ontario, Canada.
11. Beltz, B. Neurobiology and neuroendocrinology, p. 267– 289. In
J. R. Factor (ed.), Biology of the lobster Homarus americanus. Academic Press, Inc., San Diego, CA.
12. Maynard, D. M. 1960. Circulation and heart function, p. 161–
226. In T. H. Waterman (ed.), The physiology of crustacea, volume
1. Academic Press, New York, NY.
13. Burger, J. W., and C. Smythe. 1953. The general form of circulation in the lobster Homarus. J. Cell Comp. Physiol. 42:369–383.
14. McMahon, B. R., and J. L. Wilkens. 1975. Respiratory and circulatory responses to hypoxia in the lobster Homarus americanus.
J. Exp. Biol. 62:637–655.
15. NIH publication No. 92–3415. Institutional Animal Care and Use
Committee Guidebook.
16. Bhatkhande, C. Y., and V. D. Joglekar. 1977. Fatal poisoning
by potassium in human and rabbit. Forensic Sci. 9:33–36.
17. Winkler, A. W., H. E. Hoff, and P. K. Smith. 1938. Electrocardiographic changes and concentration of potassium in serum following intravenous injection of potassium chloride. Am. J. Physiol.
124:478–483.
18. Sigma-Aldrich Canada Ltd. Material safety data sheet. 1999.
Sigma-Aldrich Canada Ltd. Oakville, Ontario, Canada.