Rollins 1998
Abstract
This project focused on the development of an anesthetic regimen in yellowfin tuna
(Thunnus albacares), bonito (Sarda chiliensis), and Pacific mackerel (Scomber
japonicus), while monitoring effects on blood chemistry and hematology. Anesthesia
was induced with a combination of ketamine and medetomidine, and compared to MS-
222 (3-amino benzoic acid ethyl ester). Ketamine and medetomidine were injected into
the red muscle of fish, while MS-222 was applied by immersing a fish in a drug and
seawater solution. Effects of anesthesia were monitored and blood samples were taken.
Blood was then analyzed for hematocrit, hemoglobin, enzymes, metabolites and ions.
Survivorship between the species varied, with yellowfin tuna at 0% survival and bonito
the highest at 50% survival. A trend relating an increase in time of the anesthetic
induction period to decreasing metabolic rate was noted. Additionally. time until
recovery from anesthesia was found to increase with the ratio of ketamine to
medetomidine in yellowfin tuna, but was found to decrease with the ratio of the two
drugs in bonito. Anesthesia was found to have no effect on average yellowfin tuna
hematocrit (mean level = 33.4 +2.2). However, a positive relationship between
yellowfin tuna hematocrit and medetomidine dose was noted (r2-0.83). A negative
relationship between bonito hematocrit and the ratio of ketamine to medetomidine was
also present. A significant increase in ions (calcium, chloride, potassium and sodium)
and some enzymes (aspartate aminotransferase and creatine phosphokinase) was noted in
yellowfin and was most likely due to the high levels of activity associated with
anesthesia. Further studies into this form of intramuscular anesthesia are needed, both to
better determine the feasibility of usage of the drugs in yellowfin tuna, and to ascertain an
ideal anesthetic dose in bonito and mackerel.
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Introduction
Research on wild and nondomestic vertebrates has contributed significantly in many
fields, from ecology to physiology and microbiology. However, this research can require
sedating animals, or applying a form of analgesia. A regimen of anesthesia is an ideal
tool in vertebrate research, as it often provides both sedation and analgesia. This project
focused on the development and utilization of an anesthetic regimen in scombrids:
yellowfin tuna (Thunnus albacares), bonito (Sarda chiliensis) and Pacific mackerel
(Scomber japonicus). A primary goal of this work was to determine the specific dosage
of an intramuscular anesthesia for the three species of fish, while monitoring the
physiological effects of anesthesia through blood chemistry and hematology.
Interest in anesthetization of scombrids came about due to their commercial and
scientific importance. As major targets of global fisheries, research into their behavioral
patterns and physiological characteristics is of utmost significance. The fast-moving tuna
is also an anomaly among fish, due to its partial endothermy and high metabolic rate
(Brill 1987, Carey et al. 1971). The unique physiology of tunas makes the study of
yellowfin tuna important from a variety of physiological and evolutionary aspects. Such
studies require capture, transport, and short procedures with the fish, some of which
might benefit from anesthesia. Currently, either non-anesthetized methods, such as low
stress sling capture or immersion anesthesia, are used for most procedures (Fletcher et al.,
in preparation). While lowering the water level of a tank, and then herding a fish into a
vinyl, water-filled sling reduces the stress level of the fish and minimizes any
physiological or hematological changes, this technique is only sufficient for simple and
painless procedures. Furthermore, anesthetization of a single fish with MS-222 requires
either stress-inducing netting of the fish or the time-consuming sling technique.
Development of an intramuscular anesthetic technique, which could be delivered via dart.
would increase ease of capture and transport of scombrids, while reducing levels of stress
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and pain experienced by the fish. Moreover, if ketamine/medetomidine anesthesia is
shown to not significantly affect hematological parameters of yellowfin tuna, the regimen
could be used as an alternative method of obtaining the blood samples necessary for
physiological studies. Anesthesia will also benefit research in the field. Immersion
anesthetics such as MS-222 cannot be used on individual fish within a noncaptive school
without the problems mentioned above. Development of an effective intramuscular agent
that can be applied via a dart methodology will allow more extensive investigation of
wild populations by enabling insertion of acoustic and archival tags. Likewise, the
possibility that blood sampling while under ketamine/medetomidine anesthesia does not
significantly bias blood chemistry means that hematological studies could also be
performed.
While the bonito (Sarda chiliensis) does not have the endothermic capacity of a
yellowfin tuna, lacking the extreme capillarization of red muscle that helps to generate
elevated temperature, they share the tuna’s high metabolic rate of the tuna (Freund,
personal communication). As a scombrid, the bonito is important in comparative studies,
serving as a predecessor to the tuna in the evolution of endothermy. Additionally, no
baseline blood parameters for the bonito currently exist. The Pacific mackerel, is also a
fast-moving scombrid, but lacks the endothermy and the high metabolism of the
yellowfin and bonito. This species is also significant from a comparative viewpoint, as
an example of a fish closely related to the yellowfin and other tunas, yet preceding the
evolution of either elevated metabolism or endothermy. While blood sampling was not
possible in the small individuals captured, the development of anesthesia in the species
would still serve the same benefit in the capture and transport required by the Tuna
Research and Conservation Center (TRCC) in Pacific Grove, CA. Moreover, by
comparing the process and behavioral characteristics of anesthesia of all three species, the
effects of metabolism and endothermy on anesthesia were able to be ascertained.
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Preliminary research at the TRCC on yellowfin tuna showed a combination of
ketamine hydrochloride and medetomidine could be effective. However, consistent
results were not obtained (Block, personal communication). This study was aimed at
ascertaining an optimal and repeatable level of anesthesia via a dose response study,
while monitoring blood chemistry. Additionally, any variations in anesthetic
effectiveness or behavior between the three species of fish were noted.
A secondary aim of this study was to determine the extent of the physiological
changes resulting from anesthesia. Studies by Fletcher et al. (in preparation) found that
variations in capture methodology resulted in differing blood parameters in yellowfin.
Specifically, the study found that low-stress level capture via herding into a vinyl sling
resulted in much lower hematocrit and hemoglobin than did high-stress capture with a
net. Furthermore, plasma levels of alkaline phosphatase (AP), alanine aminotransferase
(ALT), aspartate aminotransferase (AST), bilirubin, cholesterol, creatinine, phosphate
and potassium were found to be significantly lower in sling-captured yellowfin than in
those captured in nets.
Anesthesia has also been shown to cause hematological effects similar to net capture
in non-scombrid teleosts, such as increased glucose, hematocrit and hemoglobin in
different species (Braley and Anderson 1992, Thomas and Robertson 1991, Hseu et al.
1996). These changes are most likely due to activation of the B-adrenergic response by
the anesthetic (Houston et al. 1971). This project measured hematocrit, hemoglobin,
enzymes, metabolites and electrolytes, and compared the data to the sling-capture data
from Fletcher et al. (in preparation). This comparison was performed in order to
ascertain the extent of the stress response and following hematological changes resulting
from ketamine/medetomidine anesthesia.
Ketamine and medetomidine, the main drugs used in the current study, have been
used previously, both in nondomestic mammals and in rockfish, and have been found to
lead to an anesthesia of good clinical quality (Jalanka 1991 and Williams, personal
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communication). Medetomidine is an alpha-2-adrenoceptor agonist, inhibiting the
activity of adenylyl cyclase, thus reducing signal transduction in the central nervous
system (Jalanka 1991). It has strong sedative and analgesic properties, and can be
reversed with atipamezole, an alpha-adrenoceptor antagonist and competitive inhibitor of
medetomidine (Jalanka 1991). However, used in isolation, medetomidine is not
sufficient for general anesthesia (Jalanka 1991).
Ketamine is an anesthetic that causes selective, rather than general depression of the
CNS (Ryall 1979). Its benefits include rapid induction of and recovery from anesthesia,
as well as stimulation of heart rate and cardiac output (Ryall 1979). It is a
noncompetitive antagonist of glutamate (NMDA) receptors (Moghaddam et al. 1997).
Ketamine also has been shown to have minimal hematological effects in mammals (Hall
and Clarke 1983). However, ketamine does not provide sufficient analgesia to allow
intrathoracic or visceral surgical procedures (Sawyer 1982). It often increases muscle
tension, and also causes hallucinations in humans (Ryall 1979). In combination,
ketamine and medetomidine induce a general, short-acting, reversible anesthesia with
good sedative and analgesic qualities. This drug combination was chosen partially for its
success in previous trials, as well as the benefits of its characteristics.
Though intramuscular anesthesia is not currently in use at the TRCC, MS-222 (3¬
amino benzoic acid ethyl ester), a widely used immersion anesthetic, has been used when
an anesthetic is required. MS-222, or tricaine, is a water soluble anesthetic agent that is
absorbed through the gill surface of a fish. It has been thoroughly studied in several
teleost species, including rainbow trout (Gingerich and Drottar 1989, Kebus et al., 1992,
Iwama et al., 1989), Atlantic salmon (Hunn and Greer 1991), blacknose dace (MacAvoy
and Zaepfel 1997) and red drum (Thomas and Robertson 1991). MS-222 acts by
depressing central autonomic functions, and is known to have an asphyxiant effect
(Houston et al., 1971) One yellowfin tuna and one bonito were anesthetized using MS-
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222 and behavioral and physiological effects were monitored, so that MS-222 could be
compared and contrasted with the intramuscular anesthesia regimen.
Materials and Methods
Intramuscular Anesthesia
The initial dosage applied in the study was 4 mg/kg ketamine and 0.4 mg/kg
medetomidine, and was determined from the preliminary work performed by Williams
and Block (personal communication). Atipamezole was always applied in the same dose
as medetomidine. Commercial versions of ketamine hydrochloride (Ketasat - Fort Dodge
Laboratories, Fort Dodge, IA) and medetomidine (Domitor - Orion, Espoo, Finland) were
used, in concentrations of 100 mg/ml and 1 mg/ml in saline, respectively. An
experimental version of atipamezole (Wildlife Pharmaceuticals, Fort Collins, CO) was
used, in a 5 mg /ml saline solution. Applied doses of the drugs varied from the ideal dose
depending on the actual weight of the fish, as well as human error.
A partial double dose study was performed (Figure 1). Experimental protocol varied
for the three species. For the 5 yellowfin tuna, the water level in the 10 m diameter tank
was lowered, and 5 to 7 people entered the tank via ladder. The ladder was then
removed. A fish was selected and separated from the school, using a vinyl crowder. The
fish was herded into a vinyl sling, filled with water. The curved length of the fish was
measured, an approximate weight determined using a length vs. weight chart, and the
appropriate volume for the dosage determined. The ketamine/medetomidine combination
was injected into the well-vascularized red muscle. A ridge running laterally along the
fish was used as a guide to the red muscle, and drugs were injected directly ventral to this
ridge, approximately 5 to 10 mm posterior to the pectoral fin. The passive, integrated
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transponder microchip (or "pit tag") number of the fish was read, a plastic, colored floy
tag was inserted for visual identification and the fish was released from the sling.
The fish was allowed to swim freely as the anesthetic took effect, with personnel
positioned along the walls to prevent the fish from harming itself. Behavioral effects of
the anesthesia on the fish were notéd, and when the fish was no longer able to equilibrate,
orient and swim independently, it was replaced in the vinyl sling. Forced ventilation of
the fish was started, using a submersible pump attached to a hose, which was placed in
the mouth of the fish. Once a fish was determined by a lack of muscle tension and
motility to be anesthetized, a blood sample was taken, following the method used in
Fletcher et al. (In preparation) drawing blood from the bulbus arteriosus. The blood
sample was then placed in a vial for hematocrit and hemoglobin analysis, containing
heparin (an anticlotting agent), and a vial for plasma separation, containing EGTA (a
clotting agent). Samples were then placed on ice until analysis. If the length previously
obtained was in question, another measurement was taken at this time. Finally, the dose
of atipamezole was measured and injected into the red muscle.
The fish remained in the sling under ventilation until it regained a strong tailbeat and
showed signs of righting itself and attempting to swim. Upon release, personnel
repositioned themselves around the tank, to protect the fish from harm until it regained its
orientation and equilibrium. When the fish sufficiently recovered, all personnel left the
tank, all equipment was removed, and the water level was restored.
Portions of the procedure were recorded using a Sony Hi-8 video camera, and
observations of the fish’s condition were made throughout the anesthetic process. Äfter
humans left the tank, observations continued to be made approximately every 10 to 15
minutes for approximately 2 to 3 hours. Time between observations continued to
increase until the fish died or a week passed, at which point observations were ceased.
The 6 bonito and 8 mackerel anesthetized were caught with a baited, barbless hook
and line and immediately transferred to a vinyl, water-filled sling held at the edge of the 6
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m diameter tank. Weights were estimated based on the relative size of the fish in the
tank, and doses were measured as in yellowfin tuna. A pre-determined volume of drug
was injected into the red muscle, as guided by the location of the pectoral fin, and the fish
was floy tagged. The fish was released and induction of anesthesia was observed. When
the fish was determined to be sufficiently anesthetized, the fish was returned to the sling,
or netted and then placed in the sling. At this point ventilation was started as before, and
a curved length of the fish was taken. Additionally, 3 bonito were weighed, in order to
obtain more accurate dosage information. The bonito underwent the same blood
sampling procedure as in the tuna; however, mackerel were neither weighed nor was their
blood sampled, due to their small size. The predetermined dosage of atipamezole was
then injected into the red muscle, and the fish was kept in the sling until an attempt to
swim or escape was made. We then released the fish and observed from the side of the
tank. Observations followed the same procedure as in yellowfin, but video was not
recorded due to lack of assistance.
Immersion Anesthesia
One yellowfin tuna and one bonito were anesthetized using MS-222. These
experiments began with the preparation of the anesthetic bath. A 1 g/L solution of MS-
222 in seawater was used, with a total volume of 30 L. As tricaine causes acidification of
seawater, which could lead to undesirable physiological effects in the fish, the drug was
buffered with 2 parts sodium bicarbonate per one part MS-222. 30 g of MS-222 and 60 g
of sodium bicarbonate were measured and placed in a container holding 30 L of filtered
seawater. The solution was then bubbled with an airstone for one hour. 10 L of the
bubbled solution were placed in a large cooler and another similarly sized cooler was
filled with pure seawater.
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Fish used for in MS-222 experiments were either netted for yellowfin tuna, or caught
with a baited, barbless hook and line for bonito. The fish were placed in a vinyl sling
within the MS-222 containing cooler, and the remaining drug solution was poured into
the sling. Ventilation of the fish was begun as before. The fish were held in the
anesthetic solution for approximately 3 minutes, and were then removed for weighing and
measurement of curved length. The fish were then placed in a vinyl sling in the cooler of
pure seawater and ventilation was resumed. Blood was then sampled from the bulbus
arteriosus. Fish were floy tagged, and when significant movement began, the fish was
moved to the tank, but remained ventilated in the sling. Once the fish showed signs of
righting and attempting to swim, the fish was released to swim freely in the tank.
Observations were made as in the intramuscular procedure, but video was not recorded.
Blood Analysis
Blood samples were analyzed for hematocrit, placing the heparin treated sample in a
standard capillary tube and centrifuging for 5 minutes (5000 rpm), separating red blood
cells from white blood cells and blood plasma. The percent of the liquid volume made up
by red blood cells was measured, giving a hematocrit level. Hemoglobin was analyzed
using a standard preparation (Sigma, St. Louis, MO) and measuring absorbance versus
hemoglobin standards using a spectrophotometer at 540 nm. Serum samples were
prepared by centrifugation as well, spinning the blood/EGTA-containing vial for 7
minutes (3500 rpm). The separated serum was then analyzed for enzyme and metabolite
levels (alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase
(ALT), bilirubin, cholesterol, creatine phosphokinase, creatinine and glucose) as well as
electrolytes (calcium, chloride, phosphate, potassium and sodium) at the Department of
Comparative Medicine at Stanford University. Resulting data from all three
measurements were compared to data from Fletcher et al. (in preparation), and were
analyzed for statistical significance (two-tailed Student's t-test).
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Data Analysis
Behavioral data were compared to ketamine and medetomidine doses, as well as to
the ratio of the two drugs. Stages of anesthesia investigated in the data were:
1. Period of anesthetic induction: defined as time from injection of anesthesia to
observation of fish being taken into vinyl sling, as well as being either “out", "under" or
otherwise relaxed, with a minimum of movement.
2. Time until release: defined as time from atipamezole injection until release of the
fish from the sling.
3. Time until recovery: defined as time from atipamezole injection until fish
recovers its orientation and equilibrium skills, as observed by fish avoiding objects in its
path and not listing in any direction.
4. Percent survival: defined as number surviving at present; deaths include those
attributable directly to anesthesia as well as those due to handling.
All averages of anesthetic stages were analyzed as mean, + standard error. Means
were then compared using a two-tailed Student's t-test for two means. Averages of blood
chemistry values were determined as mean, + standard error. Means between the two
sampling methods were compared using a two-tailed Student's t-test for two means of
unequal sample sizes. Significance to p + 0.05 was determined by comparing a critical t-
value to a test statistic; If the test statistic was greater than the critical value, the means
were determined to be significantly different, while if it was less than the critical value,
the values were not significantly different.
Results
Intramuscular Anesthesia
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The data from individual anesthesia experiments show distinct differences between
the three species (Table 1). Average time to anesthesia induction is at a minimum in
yellowfin tuna, increases in bonito and is highest in mackerel (Figure 2A).
Measurements of time to release in yellowfin are not significantly different from that in
bonito. However, mackerel time to release is much less than the other two species,
falling to a highly significant minimum (Figure 2B). Though a trend of decreasing time
of recovery is apparent, with a maximum in yellowfin and a minimum in mackerel, it is
not significant (Figure 2C). The overall percent of anesthetized fish surviving was
relatively low in all species, with 0 % survival in yellowfin, followed by mackerel at
38%, and bonito with highest survivorship (50%) (Figure 2D).
Analysis of the various anesthetic stages in yellowfin showed no apparent
relationship between ketamine dose and time to anesthesia induction, release or percent
survival. However, a positive relationship was found between time to recovery and
ketamine dose (Figure 3A). No trend was evident in comparing medetomidine dose with
time to induction or time to release. Nevertheless, a pattern between time to recovery and
medetomidine dose was present, with recovery time decreasing as medetomidine dose
increases (Figure 3B). Similarly, while comparisons of induction time or release time
versus ketamine:medetomidine ratio showed no trend, a positive trend surfaced in
comparison of recovery time to the ratio of the two drugs (Figure 3C).
No trends between ketamine dose or medetomidine dose and any of the categories of
anesthesia data were apparent in bonito. However, clear trends are evident in all data
categories when compared to the ratio of ketamine and medetomidine. In all 3 stages of
anesthesia, time measurements decrease with an increase in the ratio (Figure 4A-C).
Furthermore, the percentage of bonito surviving decreases significantly as the
ketamine: medetomidine ratio increases (Figure 4D).
In mackerel, all comparisons of anesthetic data versus individual drug dose gave no
patterns or trends. Ratio of ketamine to medetomidine was not analyzed with respect to
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anesthesia data, as all anesthetized mackerel received the same 5:1 ratio of drugs (Table
All three species anesthetized with ketamine and medetomidine displayed the same
characteristic behavior patterns. Äfter injection of anesthesia, a fish would begin to
increase speed, and fold its pectoral fins against its sides. As the anesthesia took further
effect, the fish would lose its equilibrium and orientation ability. The fish then lost its
tailbeat and experienced a relaxation of muscle tone in the last stages of induction. Once
under anesthesia, the fish would undergo discoloration, which lasted anywhere from
several hours to more than a day. Immediately following release, the fish would not be
able to orient and equilibrate properly. These skills were regained within 10 to 15
minutes of release. A fish would then enter a hyperexcitable and agitated state,
characterized by abnormally high speed, pectoral fins folded against the body, and a
general failure to swim with the school. In yellowfin tuna, this state was especially
pronounced and the fish never recovered. The agitated state was less severe in bonito,
and while it often lasted for at least one day, some of the fish did recover. Mackerel
experienced the shortest period of agitation, often returning to a normal swimming
pattern within the span of a few hours.
Immersion Anesthesia
Comparisons of the single yellowfin anesthetized with MS-222 show little difference
in time to anesthesia induction; the value of 3.42 minutes (min) is well within the average
yellowfin tuna range (mean = 6.73 min; S.D. = 3.50 min; Table 1). Time to release of the
MS-222 treated yellowfin is greater than the range for intramuscularly anesthetized
yellowfin, with a value of 17.98 min vs. a mean of 6.46 min (S.D. of 6.54 min; Table 1).
However, MS-222 measurements of time to recovery give a value within the range of
intramuscular values (17.98 min vs. mean = 12.39 min, S.D. = 8.36 min; Table 1). The
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quality of anesthesia obtained and the recovery of the fish were both qualitatively
comparable to that for the ketamine and medetomidine combination.
The single bonito anesthetized with MS-222 had a faster anesthesia induction time
than the range of values for intramuscularly injected bonito, with a value of 3.43 minutes,
versus a mean of 15.91 min (S.D. of 10.36 min; Table 1). Additionally, the times to
release and recovery were much longer than the ranges for ketamine and medetomidine
anesthesia; release time for MS-222 was 34.23 min, as compared to a mean of 8.81 min
(S.D. of 3.71 min), while recovery time for MS-222 was 36.13 min, versus a mean for
intramuscular of 11.53 min, (S.D. of 3.65 min; Table 1). The quality of the fish’s
recovery was comparable to intramuscular anesthesia, but the quality of the actual
anesthesia was much heavier.
Blood Chemistry and Hematology
Data on blood chemistry and hematological parameters in individual anesthesia
experiments are summarized in Table 2. Comparison of hematocrit levels obtained via
anesthesia and those obtained from the low-stress sling technique (Fletcher et al., in
preparation) are shown in Figure 5, and show no significant difference between the two
methods. However, analysis of hematology versus drug dosage in yellowfin shows a
significant correlation between increasing hematocrit levels and an increase in the ratio of
ketamine to medetomidine (Figure 80). No significant correlations exist in hematology
of yellowfin tuna vs. ratio of the two drugs or ketamine dose (Figure 8A and B). In
comparing mean enzyme and metabolite values, anesthesia fish had significantly greater
values of AST, CPK and cholesterol than in the sling technique (Figure 6). Levels of
blood calcium, potassium and sodium were found to be significantly lower in sling
technique sampling than anesthesia sampling (Figure 7A-C). However, blood chloride
was significantly higher in the sling technique (Figure 7D). Hemoglobin levels were not
statistically analyzed due to small sample size. In bonito, a significant correlation was
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found only between decreasing hematocrit and an increasing ratio of ketamine to
medetomidine (Figure 9). All other correlations and parameters in bonito were not
analyzed due to small sample size.
Discussion
Intramuscular Anesthesia
Distinct differences in anesthesia induction times were found between the three
scombrid species. This correlates with the differences in metabolic rate. The yellowfin,
with the highest metabolic rate, would be expected to respond to ketamine and
medetomidine most rapidly of the three species. The mackerel, conversely, with the
slowest metabolic rate would be expected to have the longest anesthetic induction period.
Bonito, with a metabolic rate similar to the yellowfin, but less red muscle capillarization,
would be expected to have intermediate induction times. This anticipated pattern is
reflected in the minimum induction time found in yellowfin and maximum induction
period in mackerel. However, induction time in bonito was not significantly different
from that in the yellowfin tuna. This result may be due to the small sample size of the
experiments. On the other hand, it is more likely that it reflects the importance of
metabolic rate rather than muscle capillarization in anesthetic induction.
Despite the differences in the three species' induction time, the data on average time
to release seem to contradict a metabolic explanation. The mackerel have an significantly
shorter time to release as compared to both bonito and yellowfin. Time to release was
defined as the time from injection of atipamezole until the release of the fish from the
sling. At this point, the fish showed a regular tailbeat and signs of righting itself, and was
also attempting to swim. Subjectivity of this measurement could have contributed to the
high variance and the lack of significant difference between time to release of yellowfin
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tuna and bonito. The minimal times to release in mackerel could mean anesthetic
differences result from non-metabolic rate causes. However, the mackerel never fully
entered anesthesia, and were actively swimming when recaptured for anesthetic reversal.
This could be due to their low metabolic rate. The semi-conscious state of mackerel,
therefore, allowed for earlier release, meaning their short release time may be a result of
their low metabolism.
Though no significant differences were found between the time to recovery in any of
the three species, a nonsignificant trend was apparent, with a maximum time for
yellowfin tuna and mackerel at a minimum value. The subjectivity inherent in measuring
time to recovery, determined as time from atipamezole injection to the time when the fish
recovered orientation and equilibrium ability, may have led to the high variance and lack
of significance in these values. Additionally, the low sample size may also contribute to
the lack of a significant difference.
When comparing results in the three species, the fact that the dosages varied
somewhat between species and individual fish must be kept in mind. This difference in
dosage may confound any conclusions that can be drawn from apparent patterns.
However, the difference in species dosages was often due to increasing resistance to
anesthesia, as bonito and mackerel were often not sufficiently anesthetized at doses
causing rapid induction in yellowfin. The fact that the highest doses were required in the
low-metabolizing mackerel, while low doses were effective in the well-vascularized, high
metabolizing tuna may reflect effects of metabolism and muscle vascularization on
anesthesia.
While average anesthesia data from yellowfin tuna is explainable by physiological
characteristics, the lack of surviving yellowfin was surprising. Doses of anesthesia in
yellowfin may have been too high, leading to observed rapid induction times but harming
the fish. However, it is possible that the version of the drugs makes a difference. The
differences between isomers of ketamine hydrochloride or medetomidine in the
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commercial and experimental versions could have caused yellowfin tuna deaths.
Experimental versions of ketamine and medetomidine should be applied before viability
of anesthesia is determined. In addition, further research into ketamine/medetomidine
anesthesia in yellowfin and increased sample sizes may lead to clearer relationships
between drug dosages and anesthetic stages. Since developing an ideal anesthetic dose
would be the primary goal of any further trials, correlations like those investigated would
be highly useful, helping to determine which dose produces the best quality of anesthesia,
with the best recovery and rate of survival.
In contrast to the yellowfin tuna, anesthesia in bonito appears to be a useful research
tool. The encouraging survival rate of bonito, in combination with overall good quality
of anesthesia make the ketamine and medetomidine combination an option that should be
further investigated, in order to increase sample size and determine correlations between
anesthetic stages, percent survival and drug dosage.
Though the survival rate and quality of anesthesia in mackerel is not as promising as
in bonito, intramuscular anesthesia is still viable. Mackerel agitation level was much
higher than in bonito, which led to large amounts of rough handling. Most mackerel
made a recovery to near normal behavior before death, but some were found to have
severe abrasions from handling. Death, therefore, was most likely due to handling, and if
an alternate capturing and restraining procedure is devised, successful anesthesia will be
possible. By increasing sample size and lowering death due to handling, the relationship
between drugs and anesthetic effects and an ideal ketamine and medetomidine dose will
be determined.
Immersion Anesthesia
In comparing anesthesia with MS-222 versus anesthesia with ketamine and
medetomidine, it seems evident that the immersion agent is no better overall than the
intramuscular anesthetic. While MS-222 did induce anesthesia more rapidly in bonito,
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the combination of the increased release and recovery time outweighs any benefit. In
yellowfin, the two forms of anesthesia seem quite comparable in every measure, except
time to release, in which the ketamine/medetomidine combination is actually preferable,
due to its shorter time period. In summation, I feel that the added convenience of an
intramuscular anesthesia, in that it can be applied via dart in the wild or captivity, makes
it the ketamine/medetomidine regimen preferable to MS-222.
Blood Chemistry and Hematology
The usefulness of anesthesia is promising, as supported by the lack of a significant
difference between hematocrit levels in yellowfin tuna sampling methods. Increases in
hematocrit have been shown to be a result of elevated stress in several species of fish
(Soivio and Oikara, 1976; Wood et al., 1983; Wells et al., 1984; Railo et al., 1985).
Therefore, the absence of any difference between anesthesia and sling hematocrits
implies that anesthesia with ketamine and medetomidine does not cause a physiological
stress response. While it does appear that hematocrit of yellowfin may be affected by
medetomidine increases, the small sample size of this data correlation means that
additional research must be performed before any definite conclusions can be drawn.
The significant differences in chloride, sodium, potassium and calcium with
anesthesia may be attributable to increased stress in the fish. Stress could result in other
changes in ionic concentrations, such as the increase in calcium that occurred in this
study or the fluctuations in chloride and sodium levels (Fletcher et al., in preparation;
Eddy 1981). However, sodium levels would be expected to increase, not decrease as
occurred in this study (Eddy 1981). Additionally, a study by Wells et al. showed no
increase in sodium or chloride in highly stressed yellowfin tuna (1986). Therefore, the
changes in these ions are most likely a result of some other physiological response to
anesthesia, or are simply due to the small sample size. The increase in potassium with
anesthesia may be a result of stress-released catecholamines causing potassium increases
page 18
Rollins 1998
in the blood (Bourne and Cossins 1982). In addition, potassium levels have been shown
to increase greatly following increased motor activity (Wood et al., 1983; Soivio and
Oikara, 1976). This result correlates, therefore, with the increase in speed noted during
the first stages of anesthesia induction. The significant increase in cholesterol between
the two sampling techniques is most likely not due to stress, but instead perhaps due to
diet differences between the two groups of fish.
Aspartate aminotransferase (AST), an enzyme concentrated in the liver (Mommsen
1984), has been shown to increase in stressed, wild yellowfin tuna (Wells et al., 1986).
However, the mechanism of such a response is not known (Fletcher et al., in preparation).
This means that the increase in AST with ketamine/medetomidine anesthesia is possibly
due to stress, but may also result from an unknown cause. However, the increase in
creatine phosphokinase (CPK), an enzyme that increases with motor activity (McDonald
and Milligan, 1992), also reflects higher level of activity in the anesthetized yellowfin.
The highly significant relationship between bonito hematocrit levels and the ratio of
ketamine to medetomidine could show an increased stress level with an increased amount
of ketamine, relative to medetomidine. However, the small sample size in bonito, in
combination with a large range of error, makes the correlation between bonito hematocrit
and drug ratio somewhat uncertain. The true effect of anesthetic on bonito hematocrit
must be further investigated before any conclusions can be made.
Conclusions
In conclusion, though the viability of anesthesia in yellowfin tuna seems low, I feel
further research should still be performed. The amount of procedural error present in this
project, in addition to the small sample size of fish anesthetized, and the lack of
investigation into the experimental version makes a final decision on the usefulness of
ketamine and medetomidine in yellowfin premature. Furthermore, the lack of change in
blood parameters means it remains a good candidate for an alternate blood sampling
page 19
Rollins 1998
technique. On the other hand, while viability of the anesthetic regimen in bonito and
mackerel seems reasonable, it too needs to be further investigated. Discrepancies in
procedural technique need to be eliminated, and an alternate handling method for
mackerel must be devised. Additionally, with further studies, the correlation between
bonito blood parameters and anesthetic drugs can be better elucidated. This could
perhaps add to the promise of this technique in blood sampling, as a baseline bonito
blood chemistry could be determined. In all, the limited results of this project should not
be viewed as the failure of intramuscular anesthesia in yellowfin tuna, nor should they be
regarded as the success of ketamine and medetomidine in bonito or mackerel.
Acknowledgments
My thanks to Dr. Barbara Block and Dr. Tom Williams for their guidance, assistance
and patience. Thanks also to Dr. Heidi Dewar, Ellen Freund, Alexandra Nelson, Tamara
Jaron, Andy Seitz, Daniel Dau, Andre Boustany, Simon Fletcher and everyone else who
helped out on my project. Dr. Jim Watanabe, thank you for helping me with the statistics
on the project.
References
Bourne, P.K. and A.R. Cossins. On the instability of K+ influx in erythrocytes of the
rainbow trout, Salmo gairdneri, and the role of catecholamine hormones in
maintaining in vivo influx activity. Journal of Experimental Biology. 101: 93-
104, 1982.
Braley, H. and Anderson, T.A. Changes in blood metabolite concentrations in response
to repeated capture, anaesthesia and blood sampling in the golden perch,
page 20
Rollins 1998
Macquaria ambigua. Comparative Biochemistry and Physiology. 103A(3): 445-
450, 1992.
Brill, Richard. On the standard metabolic rates of tropical tunas, including the effects of
body size and acute temperature change. Eishery Bulletin. 85: 25-35, 1987.
Carey, F.G., Teal, J.M., Kanwisher, J.W. and K.D. Lawson. Warm-bodied fish.
American Zoologist. 11: 137-145, 1971.
Eddy, F.B. Effects of stress on osmotic and ionic regulation in fish. In Stress and Fish
(ed. A.D. Pickering), pp. 11-47. Academic Press, London, 1981.
Fletcher, S., E. Freund, T. Williams, H. Dewar, C. Farwell and B.A. Block. Blood
sampling in captive yellowfin tuna (Thunnus albacares). in prep, 1998.
Gingerich, W.H. and K.R. Drottar. Plasma catecholamine concentrations in rainbow
trout (Salmo gairdneri) at rest and after anesthesia and surgery. General and
Comparative Endocrinology. 73: 390-397, 1989.
Hall, L.W., and K.W. Clarke. Veterinary Anesthesia (eighth edition). Balliere Tindall,
London, 1983.
Houston, A.H., J.A. Madden, R.J. Woods, and H.M. Miles. Some physiological effects
of handling and tricaine methanesulfonate anesthetization upon brook trout,
Salvelinus fontinalus. Canadian Journal of Fisheries Research Board. 28: 625-
633, 1971.
page 21
Rollins 1998
Hseu, J.R., S.L. Yeh, Y.T. Chu and Y.Y. Ting. Effects of anesthesia with 2-
phenoxyethanol on the hematological parameters of four species of marine
teleosts. Journal of the Fisheries Society of Taiwan. 23(1): 43-48, 1996.
Hunn, J.B. and I.E. Greer. Influence of sampling on the blood chemistry of atlantic
salmon. The Progressive Fish-Culturist. 53: 184-187, 1991.
Iwama, G.K., J.C. McGeer, and M.P. Pawluk. The effects of five fish anesthetics on
acid-base balance, hematocrit, blood gases, cortisol, and adrenaline in rainbow
trout. Canadian Journal of Zoology. 67: 2065-2073, 1989.
Jalanka, H.H. Medetomidine, Medetomidine-Ketamine Combinations and
Atipamezole in Nondomestic Animals: A clinical, physiological and comparative
study. Academic Dissertation. College of Veterinary Medicine, Helsinki, Finland,
1991.
Lumb, W.V. and E.W. Jones. Veterinary Anesthesia, Second Edition. Lea and Febiger,
Philadelphia, 1984.
Kebus, M.J., M.T. Collins, M.S. Brownfield, C.H. Amundson, T.B. Kayes, and J.A.
Malison. measurement of resting and stress-elevated serum cortisol in rainbow
trout Oncorhynchous mykiss in experimental net-pens. Journal of the World
Aquaculture Society. 23(1): 83-88, 1992.
MacAvoy, S.E. and R.C. Zaepfel. Effects of tricaine methanesulfonate (MS-222) on
hematocrit: first field measurements on blacknose dace. Transactions of the
American Fisheries Society. 126: 500-503, 1997.
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McDonald, D.G. and C.L. Milligan. Chemical Properties of the Blood. In Fish
Physiology, Vol. 12B (ed. W.S. Hoar, D.J. Randall, and A.P. Farrell), pp. 55-133.
Academic Press, San Diego, 1992.
Moghaddam, B. B. Adams, A. Verma and D. Daly. Activation of glutamatergic
neurotransmission by ketamine: a novel step in the pathway from NMDA receptor
blockade to dopaminergic and cognitive disruptions associated with the prefrontal
cortex. The Journal of Neuroscience. 17(8): 2921-2927, 1997.
Mommsen, T.P. Metabolism of the fish gill. In FEish Physiology, Vol. 10B (ed. W.S.
Hoar, D.J. Randall, and A.P. Farrell), pp. 203-238. Academic Press, San Diego,
1992.
Railo, E., Nikinmaa, M., and Soivio, A. Effects of sampling on blood parameters in
rainbow trout, Salmo gairdneri: richardson. Journal of Fish Biology. 26: 725-
732, 1985.
Ryall, R.W. Mechanisms of Drug Action on the Nervous System. pp. 70. Cambridge
University Press, Cambridge, 1979.
Sawyer, D.C. The Practice of Small Animal Anesthesia. W.B. Saunders Co.,
Philadelphia, 1982.
Soivio, A. and A. Oikari. Haemotological effects of stress on a teleost, Esox lucius.
Journal of Fish Biology. 8: 397-411, 1976.
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Thomas, P. and L. Robertson. Plasma cortisol and glucose stress response of red drum
(Sciaenops ocellatus) to handling and shallow water stressors and anesthesia with
MS-222, quinaldine sulfate and metomidate. Aquaculture. 96: 69-86, 1991.
Wells, R.M.G., et al. Recovery from stress following capture and anesthesia of Antarctic
fish: hematology and blood chemistry. Journal of Fish Biology. 25: 567-576.
1984.
Wells, R.M.G., R.H. Mclntyre, A.K. Morgan and P.S. Davie. Physiological stress
responses in big gamefish after capture: observations on plasma chemistry and
blood factors. Comparative Biochemistry and Physiology. 84A: 565-571, 1986.
Wood, C.M., Turner, J.D., and M.S. Graham. Why do fish die after severe exercise?
Journal of Eish Biology. 22:189-201, 1983.
page 24
Rollins 1998
Figure Legends
Figure 1: The figure shows the design of the double dose study that was only partially
completed due to mortality. Medetomidine doses are along the top and ketamine doses
are along the side. (B) indicates that the dose was applied to a bonito; (Y) indicates a
dose was applied to a yellowfin tuna; (M) indicates a dose was applied to a mackerel.
Figure 2: The panel shows averages of induction period (A), time to release (B), time to
recovery (C), as well as survivorship (D) for yellowfin tuna, bonito and mackerel.
Standard error bars are included in the three graphs of time vs. species.
Figure 3: The panel shows the time until recovery in yellowfin tuna, as compared to
ketamine dose (A), medetomidine dose (B) and the ratio of ketamine to medetomidine
(C. Standard error bars are included only where the value given is an average. Time is
along the y-axis, while drug or drug ratio is along the x-axis.
Figure 4: The panel gives the average of time of induction, time until release, time until
recovery and survivorship versus the ratio of ketamine to medetomidine for bonito.
Where the average is based on a sample size of greater than one, standard error bars are
included. Time is along the y-axis, and the drug ratio is along the x-axis.
Figure 5: This figure shows the average hematocrit of yellowfin tuna, versus which
sampling method was used. The sampling methods are along the x-axis, and are
anesthesia with ketamine and medetomidine, or the low-stress sling technique.
Hematocrit is measured along the y-axis. Values are presented as means, with standard
error bars. Sample size for anesthesia is 5, and is 24 for the sling method.
page 25
Rollins 1998
Figure 6: This panel measures the amount of alanine aminotransferase (A), creatine
phosphokinase (B) and cholesterol (C) versus blood sampling method in yellowfin tuna.
Sampling methods are anesthesia (left) and sling technique (right), and run along the x¬
axis.
Figure 7: This panel shows the level of calcium (A), chloride (B), potassium (C) and
sodium (D) versus sampling method in yellowfin tuna. Sampling methods are anesthesia
(left) and sling technique (right) along the x-axis.
Figure 8: This panel gives the relationship between yellowfin tuna hematocrit, and either
ratio of ketamine to medetomidine (A), ketamine dose (B) or medetomidine dose (C).
Ratios or drug dosages run along the x-axis, while hematocrit is on the y-axis. For Figure
8A, the regression line equation is y = -1.04x + 39.45 and R2 = 0.64. For Figure 8B, the
equation is y = -2.28x + 40.22 and R2 = 0.28. In Figure 8C, y = 52.67X + 8.65 and R2 =
0.83.
Figure 9: This panel gives the relationship of bonito hematocrit and either ratio of
ketamine to medetomidine (A), ketamine dose (B) or medetomidine dose (C). Ratios or
drug doses are on the x-axis, while hematocrit is on the y-axis. In Figure 9A, the
regression line equation is y =-0.94x + 32.4 with an R2 = 1. For Figure 9B, y =-4.50x +
38.61, and R2 = 0.50. In Figure 9C, y =-21.21x + 37.85 and R2 =0.44.
page 26
Table 1. Summary of Anesthesia: Part I - Yellowfin Tuna
Time to
Ratio of
Ketamine
Anesthesia
Time to
Dose Medetomidine ketamine:
Release
Species and
Induction
weight (kg)'
(mgkg) Dose (mgkg) medetomidine
(min)
(min)
Yellowfin
7.5
4.8
(7.3)
0.38
10.8:1
Yellowfin
(10)*
0.42
3.1:1
Yellowfin
(9)*
0.44
17.9
9.1:1
2.7
Yellowfin
0.51
5:1 3.6 4.1
2.53
(5.9)
Yellowfin
10.6 1.5
(4.6)
3.02
0.604
5:1
1gMS¬
Yellowfin
MS-222
222/L
n/a 3.4 18
(6.1)
seawater n/a
= estimated weight
page 27
Time to
Recovery
(min)
18.9
na
20.2
6.6
3.9
Comments and time of
death (after anesthesia
injection)
good anesthesia, died
(Q 3 hours, 50 minutes
good anesthesia;
underwent
fright response and
never recovered;
euthanised + 59
minutes, 53 seconds
good anesthesia; died
by 21 hours
good anesthesia; died
(Q 25 hours, 46 minutes
good anesthesia; died
(Q 10 hours, 35 minutes
good anesthesia;
decent recovery;
euthanised for research
by 144 hours
Table 1. Summary of Anesthesia: Part II - Bonito
Time to
Ketamine
Ratio of
Anesthesia
Time to
Species and
Dose Medetomidine ketamine:
Release
Induction
weight (kg)t
(mgkg) Dose (mgkg) medetomidine
(min)
(min)
5.5
2.7
Bonito (2.0)
0.4
10:1
1.4 0.28
5:1
8.1
6.5
Bonito (3.2)
Bonito (2.8)
15.5 13.7
1.82 0.36 5:1
2
0.4
5:1
22.1
11
Bonito (2.6)
12.8 7.8
Bonito (1.7)
0.6
5:1
Bonito
0.8
5:1
33 9.7
(2.5)
1gMS¬
Bonito-
MS-222
222/L
n/a 3.4 34.2
seawater n/a
(2.6)
* = estimated weight
page 28
Time to
Comments and time of
Recovery death (after anesthesia
injection)
(min)
good anesthesia;
jumped out of tank;
died by 17 hours, 30
minutes
good anesthesia and
8.9
recovery; survived
good anesthesia; long
recovery due to injury;
14.3
survived
anesthesia not
induced; fell on floor;
died by 20 hours, 32
minutes
16.3
light anesthesia; died
7.8
by 17 hours, 12 minutes
light anesthesia; slow
recovery but survived
13.7
anesthesia too heavy;
died by 42 hours; very
abraded
36.1
Table 1. Summary of Anesthesia: Part III - Pacific Mackerel
Time to
Ketamine
Ratio of Anesthesia
Species and
Dose Medetomidine ketamine:
Induction
length (kg)
(mgkg) Dose (mgkg) medetomidine (min)
Mackerel
(0.75)
0.4
5:1
n/a
Mackerel
(0.75)*
unknown unknown n/a
na
Mackerel
6.55
1.31
5:1
(0.6)
21
Mackerel
3.58
0.72
23.2
(1.1)
Mackerel
0.8
(0.5)*
Mackerel
0.77
3.84
5:1
60.2
(1.3)
Mackerel
(0.5)
5:1
24.3
Mackerel
1.39
6.94
5:1 40.1
(0.7)
* = estimated weight
page 29
Time to
Release
(min)
n/a
n/a
1.3
Time to
Recovery
(min)
n/a
n/a
27.4
Comments and time of
death (after anesthesia
injection)
anesthesia never
induced or reversed;
survival unknown
injection mistake;
anesthesia not induced
or reversed; survival
unknown
light anesthesia; bad
recovery; euthanised by
2 hours, 42 minutes
light anesthesia; good
recovery but euthanised
(Q 50 hours, 5 minutes
light anesthesia; good
recovery; survived
light anesthesia; good
recovery; died by 5
hours; very abraded
light anesthesia; good
recovery; survived
light anesthesia; good
recovery; died by 55
hours, 47 minutes; very
abraded
Table 2. Summary of Blood Chemistry: Part I - Hematocrit and Hemoglobin
Ratio of Hematocrit
Ketamine
Medetomidine ketamine to (% red Hemoglobin
Species and
Dose
Weight (kg)
(mgkg) Dose (mgkg) medetomidine blood cells) (gdL blood)
Yellowfin
0.38
10.8 to 1 27.5 na
(7.3)
Yellowfin
(10)*
1.3
0.42 3.1 to 1 33,5 n/a
Yellowfin
0.44 9.1 to 1 29.5 11
Yellowfin
2.53
5to 1 37 15.58
0.51
(5.9)
Yellowfin
5 to 1 39.5 16.32
0.604
3.02
(4.6)
1 gMS¬
Yellowfin-
222
n/a
22
n/a
MS-222 (6.1) L seawater
n/a
23
n/a
10 to 1
Bonito (2.0)
0.28
5 to 1
n/a
32.5
Bonito (3.2)
15.53
5 to 1
36.5
0.36
Bonito (2.8)
1.82
11.28
25
5 to 1
Bonito (2.6)
9.63
20.5
0.6
5 to 1
Bonito (1.7)
Bonito (2.5)
9.23
0.8
5 to 1 24
1 gMS¬
Bonito-
222/
40
n/a
n/a
MS-222 (2.6) L seawater n/a
* = estimated weight
page 30
Table 2. Summary of Blood Chemistry: Part II - Enzymes and Metabolites
Species and
AST
Bilirubin Cholesterol CPK Creatinine Glucose
Weight (g)
QUL)
ALTQUD)
APGUA)(mgd)(mgd)
(UT)(mgd) (mgd)
Yellowfin
123
183 0.3 107
(7.3)
Yellowfin
(10)*
187 1111 0.2 92
Yellowfin
(9)*
137
884 0.2 78
Yellowfin
(5.9)
186 1244 0.2
121
Yellowfin
0
126 1306 25
108
(4.6)
Yellowfin-
90
297
888 0.3 129
MS-222 (6.1)
239
109 0.3
192
Bonito (2.8)
260
143 0.3
102
Bonito (1.7)
0.3 219
282
180
Bonito (2.5)
* = estimated weight
page 31
Table 2. Summary of Blood Chemistry: Part III - Electrolytes
Species and
Chloride
Phosphorus Potassium
Calcium
Sodium
Weight (g)
(mgd)
(meql)
(mgd)
(meqL)
(meqL)
Yellowfin
168
5.4
2.5
14.2
210
(7.3)
Yellowfin
(10)
168
210
14.2
5.4
2.5
Yellowfin
(9)*
14.6
197
165
5.2
3.4
Yellowfin
203
13.7
166
(5.9)
3.1
Yellowfin
166
2.8
(4.6)
14.6
203
Yellowfin -
MS-222 (6.1)
18.1
207
4.7
6.2
232
15.6
4.3
184
Bonito (2.8)
149
6.4
169
3.2
206
Bonito (1.7)
16.6
191
3
Bonito (2.5)
11.5
4.3
154
* = estimated weight
page 32
Figure 1- Double Dose Study Model
Medetomidine
0.8 mgkg
0.4 mgkg
0.1 mgkg
0.2 mgkg
medetomidine
medetomidine medetomidine medetomidine
1 mgkg ketamine
Ketamine
B
2 mgkg ketamine
4 mgkg ketamine
Y, B
B.M
8 mgkg ketamine
Y = applied to yellowfin tuna
B = applied to bonito
M = applied to mackerel
page 33
Figure 2

—
A. Induction
Bono
Species
C. Recover
Species
uades
B. Release
Velonin Tuna
Bonto
Species
D. Survivorship
Veloufn Tuna
Bonito
Species
Mackel
naen
page 34
Figure 3
A. Recovery vs. Ketamine Dose
Ketamine Dose (mg/kg)
B. Recovery vs. Medetomidine Dose

Medetomidine Dose (mg/kg)
C. Recovery vs. Ketamine to Medetomidine Ratio
0
9.1 11:1

3:1
Ketamine to Medetomidine Ratio
page 35
0.
Figur
A. Induction vs. Ketamine to Medetomidine Ratic
B. Release vs. Ketamine to Medetomidine Ratio


Ketamine to Medetomidine Ratio
Ketamine to Medetomidine Ratio
C. Recovery vs. Ketamine to Medetomidine Ratio D. Survivorship vs. Ketamine to Medetomidine Ratio

10.1
101
Ketamine to Medetomidine Ratio
Ketamine to Medetomidine Ratio
page 36
Figure 5
35 -
20
10
anesthesia
Sampling Method
page 37
sling
Figure 6
20
eingnetaes
Method of Sampling
Anonenes

Method of Sampling
sing method
Method of Sampling
page 38
c.

sing method
Sampling Method
anestese
sing method
Sampling Method
anstesa
sing method
Sampling Method
sing mehos

Sampling Method
Figui
page 39
Figure 8

30
30
2
Ketamine to Medetomidine Ratio (x:1)
Ketamine Dose (mgkg)
0.40 0.45 0.50 0.55 0.60 0.65
Medetomidine Dose (mg/kg)
page 40
Figure
A.

38
10 11
Ketamine to Medetomidine Ratio (x:1)
Ketamine Dose (mg/kg)
02 03 04 05 0.6 0.7 0.8 0.9
Medetomidine Dose (mg/kg)
page 41