Abstract
Blood samples are used in many organisms as an indication of health and physiological
state. In yellowfin tuna, blood analysis is complicated by stress responses to handling.
These responses include changes in serum osmolarity, blood metabolites, and release of
red blood cells from the spleen leading to increased hematocrit. Samples from stressed
fish therefore do not accurately represent the resting health and physiology of an
individual. This study presents a new method developed to minimize stress levels during
blood sampling. Blood is drawn from the bulbus arteriosis of captive yellowfin tuna while
the fish are still in the tank. Twenty-seven samples were taken from 18 individuals over
the course of seven weeks. Hematocrit, hemoglobin, red blood cell counts, ions, and
metabolites were measured and compared to values obtained from fish sampled using
other common techniques. Hematocrit and hemoglobin values were similar to those
measured in chronically cannulated tuna, and lower than for fish sampled using a common
practice of capture in a net (hematocrit 31.6% + 0.8 vs. 44.1% + 8.5 ; hemoglobin 11.1
g/dl +0.3 vs. 17.0 g/dl +2.0). Creatine phosphokinase, alkaline phosphatase, potassium,
and creatinine (all related to stress and/or increased muscle activity) significantly increased
in net-sampled fish. Therefore, fish sampled using a bulbus puncture appear to be less
stressed. Using this sampling technique, an experiment was run in which diet in one tank
was changed from a normal 1% fat diet to a high 8% fat diet. Blood samples were taken
before and after the 8% diet treatment. Serum free fatty acid concentrations doubled
(0.25 mmol/L +0.03 vs. 0.524 mmol/L + 0.03) in the 8% fat fed fish that were sampled.
The results of these experiments demonstrate an ability to measure changes in blood
chemistry using the new sampling techniques. This study will help determine what is
physiologically "normal" in yellowfin tuna blood.
Introduction
Blood chemistry and hematology are commonly used as an indicator of health and
physiological status. However, the accurate interpretation of blood parameters relies on
the assumption that sampling procedure does not affect resting values (Houston, 1990).
At the Tuna Research and Conservation Center (TRCC), we are interested in determining
baseline values for captive yellowfin tuna (Thunnus albacares) blood hematology and
chemistry without injuring or stressing the fish.
The effects of handling stress and muscle activity on fish blood chemistry and
hematology are well documented. In rainbow trout, handling stress causes increased
hematocrit and disruption of ionic balance leading to increased potassium and decreased
chloride levels in the blood (Railo, E. et al). The stress response involves, in particular, an
adrenaline mediated release of red blood cells from the spleen into circulation (McDonald
and Milligan, 1992). Yamamoto et al. (1980) showed an increase in red blood cells in
circulation as measured by increased hematocrit and decreased spleen volume in capture-
stressed yellowtail. In addition, Wells et al (1984) showed increased hematocrit and
hemoglobin and decreased pH in Antarctic cod subjected to net-capture stress. Likewise,
Devries (1981) demonstrated large increases in blood potassium levels and altered
erythrocyte ion exchange abilities in fish that have undergone net-capture stress.
A variety of methods are used to control a fish before blood sampling. Past samples
have been obtained at the TRCC by capturing a fish with a net and removing it from the
water before drawing blood. Observation has shown that this method involves a
considerable amount of struggling by the fish for a prolonged period of time before the
sample can be taken.
Use of cannulation to obtain repeat samples has been helpful in determining resting
blood measurements in fish (Houston, 1990). Hematocrit values are generally lower in
cannulated fish, which implies less-stress and therefore more realistic measurements
(Sovio, et al, 1975). Cannulation of yellowfin tuna has been successful and has shown
relatively low hematocrit and hemoglobin values (Korsmeyer, unpublished results)
The use of anesthesia in blood sampling is another common method. A blood sample
can be taken without physically restraining the fish. However, anesthesia is known to
induce many changes in blood chemistry, including activation of adrenaline stress response
as well as increased hematocrit and hemoglobin measures (Houston, 1971). Fish must
also be handled first in order to anesthetize, and anesthesia has been shown to cause a high
level of mortality in tuna (Heidi Dewar, personal comment)
The dynamics of stress-mediated changes in blood chemistry and hematological
measurements, combined with our interest in determining average tuna blood
characteristics, led us to ask the question: can we develop a repeatable blood sampling
method that involves minimal stress and damage to the fish? The success of a sampling
technique can be assessed by comparing results to those obtained using techniques known
to cause stress-induced or other chemical changes in the blood. A consistent, accurate
sampling technique would prove valuable both in determining base-line values for captive
vellowfin tuna blood and for monitoring the health of the fish.
Methods:
Tanks and Maintenance
All yellowfin tuna sampled were held at the TRCC in large holding tanks 30 ft. in
diameter and 6 ft. deep. Fish from two tanks were used for blood sampling (T2 and T3).
Tank temperatures were held constant at 22°C. Oxygen and pH levels in the water were
monitored daily.
Blood Sampling
We recognized two possible sampling sites, the caudal vessels in the tail region and the
bulbus arteriosis, which leads from the ventricle to the ventral aorta (Figure 1). Contact
with the tail region is observed to cause the fish to struggle, so sampling from the caudal
region would require forced physical restraint of the entire body. Also, because the caudal
vein and artery are close to each other, it would not be known if samples were of arterial
or venous blood. Sampling from the bulbus is risky because of its close proximity to the
ventricle (Figure 1). However, the bulbus is fairly elastic, which could prevent bleeding
after sampling. Careful dissection showed that a needle entering anterior to the bone
covering the bulbus would not hit the ventricle. An angle of about 45 ° toward posterior
was required to avoid hitting the structurally weaker ventral aorta. The bulbus puncture
was used to obtain all samples. Blood samples were taken inside the tank. Water levels
were lowered to about 1 meter and an average of 7 workers were in the tank throughout
the sampling process. Fish were constrained in a small area and caught with water filled
vinyl slings.
The first three samples were carried out using a forced restraint method. A fish was
caught in an open-ended vinyl sling, turned upside down, and placed on a modified v¬
board with as little handling as possible. A vinyl covered foam flap (25 cm wide) was
then placed over the body and held firmly. The fish remained calm when a strip of blanket
soaked in PolyAqua fish Protectant was placed over the eyes. The sample was then
taken from the bulbus and the fish was released.
Fish sampled using forced restraint showed significant signs of handling trauma. This
led to the development of the sling technique, in which handling of the fish was minimized.
A fish was caught in a closed-ended vinyl sling, which was held closed under-water to
allow the fish to calm down. In this way, the fish was protected by an envelope of water.
The sling was opened and the fish was rolled over. Fish that fought or would not calm
down within a few seconds were immediately released to prevent injury and to insure low-
stress samples. The fish was held lightly in place and a blood sample was taken. Sampling
took less than 30 seconds. The relative stress levels of each fish in terms of struggle and
physical response to handling were recorded.
Approximately 3ml of blood was drawn into a 5 ml heparinized syringe (needle size 18-
20 ga 1.5 in.). A small amount was put into a tube containing EDTA and immediately
placed on ice. The remainder was put into a wax bottom serum separator with clot
activator. Blood from the EDTA tube was used to measure hematocrit, hemoglobin
(Sigma Diagnostics total hemoglobin kit), and red blood cell counts (Becton-Dickinson
unopette microcollection system). Blood in the serum separator was allowed to clot for
one hour, then centrifuged for 7 minutes at 3500 rpm. Serum was frozen at -80 C for
analysis of free fatty acid concentrations (Wako NEFA kit, Biochemical Diagnostics) and
chemistry analysis (Stanford University, Department of Comparative Medicine,
Diagnostic Laboratory). See table 1 for blood chemistry parameters measured.
Over the course of 7 weeks, 27 samples were obtained from 18 different fish using the
sling technique, and blood analysis results were compared to values obtained from both
net sampling methods (TRCC data) and chronically cannulated fish (Korsmeyer,
unpublished) In three individuals, repeat samples were taken 6 days apart, and
hematology values were compared in each fish between sampling events.
Feeding Manipulation
In order to test the ability of the sling technique in measuring changes in blood chemistry
over changes in conditions, fish were sampled from an experimental tank (T2) in which the
diet was manipulated. For two weeks the fish were maintained on the standard TRCC low
fat diet (28 Kcal/Kg/day, 1% fat) of squid and smelt. The diet was then switched to a high
fat diet (28 Kcal/Kg/day, 8% fat) of sardines for four weeks. Blood samples were taken
from T2 fish at the end of each of the feeding periods. T3 fish were held to a constant diet
over all sampling periods (low fat, 40 Kcal/Kg/day). T3 fish were sampled on three
occasions at least one week apart.
Results:
Repeat Sampling
Of the twenty-seven samples using the sling technique there was one mortality, giving a
success rate of 95%. The mortality was associated with the forced restraint method.
Hematocrit (Figure 2), hemoglobin (Figure 3), and red blood cell count (Figure 4)
values are shown for the three repeat samples. There was no significant differences in any
of the measurements between sampling dates (paired Students t-test, pr.05). There was
no significant difference in hematocrit (p=. 11) or hemoglobin (p=. 6) over dietary
treatment (Figure 5, one-way ANOVA, p=05). There were no significant changes in any
other blood parameter between diets or tank, therefore values from 27 sling-samples were
pooled to determine measured ranges. A summary of blood analysis from all samples in
this study is compared to results from net sampling techniques (Table 1).
Stress Indices
Hemoglobin values of net-sampled fish were significantly higher than sling-sampled
fish (Student's t-test, p.05). Significant increases in net-samples were also seen in
alkaline phosphatase, total bilirubin, cholesterol, creatinine, phosphorous, potassium,
SGOT, and SGPT (Student's t-test, pS.05). Although not statistically significant, an
increase was seen in creatine phosphokinase (2000 %).
A further comparison between sling-samples, net-samples, and chronically cannulated
samples showed significant increases in both hematocrit (Figure 6) and hemoglobin
(Figure 7) in net-sampled fish (one way ANOVA, Tukey's HSD, pS.05). There was no
significant difference between values obtained from cannulation (hemoglobin -11.3 +7.
hematocrit =30.7 + 1.3) and sling-samples (Tukey's HSD, p«05).
Diet Manipulation
Figure 8 shows the results of the T2 diet manipulation experiment. Free fatty acid
concentrations in blood serum doubled (0.25 mmol/L + O.03 to 0.524 mmol/L +0.03) in
the high fat diet (Student's t-test, p5.0005). This was the only significant change
recorded in the blood over change in diet.
Discussion
Results from measures of blood chemistry and hematology in this study indicate that
bulbus puncture blood sampling can be successfully repeated and results in a lower stress
response than in other techniques. We are interested in determining if this technique can
provide accurate, repeatable data on yellowfin blood.
The hematology results from the three repeat samples show that we can be fairly
confident about generating consistent data. This is necessary if we wish to use the
information either to set base-line blood parameters or use the results to evaluate the
physiological status of an individual. In other words, if we see a change in blood
chemistry from one week to the next, we want to be sure this change is due to actual
alteration of physiological processes, and not a result of the sampling process itself
Pooled data in this study includes samples from fish in different tanks being fed
different diets. All hematology and chemistry measures were compared between tanks and
diet with no differences being found. Thus, the pooled data represent a good estimate for
what the average captive tuna blood should look like. Only free fatty acid concentrations
show an increase over the two diet treatments. This is consistent with previous
experiments. Reynolds et al (1994) showed an increase in serum free fatty acid
concentration in dogs on a high fat diet compared to a high carbohydrate diet. Because the
free fatty acid measurements were done under variable conditions, our results tell us very
little about the normal range of free fatty acid in tuna blood. However, it does add
confidence to the repeatability of the sampling method. The way in which the blood was
sampled does not significantly alter the ability to accurately measure changes in blood
chemistry.
A number of lines of evidence show that samples obtained using bulbus puncture
techniques represent a relatively low level of stress. The hematocrit and hemoglobin
values obtained in this study are nearly identical to those from chronically cannulated fish.
Because cannulated fish are allowed to recover from handling prior to sampling, it is
assumed that they are only minimally stressed. These results indicate that the samples we
get using the sling technique are representative of fairly non-stressed tuna.
Blood chemistry differences were found between sling-sampled and net-sampled fish.
Increases in potassium, creatine phosphokinase (CPK), alkaline phosphatase, and
creatinine are all likely to be related to sampling condition. CPK is a muscle enzyme that
increases rapidly with motor activity (McDonald and Milligan, 1992). Average measures
for CPK were 2000% higher in net-sampled fish. This result shows CPK as a possible
indicator of stress in captive yellowfin tuna. In one instance, an individual that struggled
for up to 30 seconds in the sling before sampling had a CPK level that was 900% higher
than the average sling sample (personal observation). Alkaline phosphatase and creatinine
are also enzymes used in muscle activity (McDonald and Milligan, 1992). It is apparent
that one of the first effects of stress response in these fish is to increase muscle activity.
This is probably related to the adrenaline mediated changes in which red blood cells are
mobilized to prepare for increased oxygen demand in the body. Serum potassium has
been shown to increase during stress due to leakage from muscle cells and lysing of
erythrocytes (Railo, 1985). A combination of stress and cell lysis probably occurred in
this study, as evidenced by visible red coloring of serum from some net sampled fish
(personal observation). The increases we observed in potassium, CPK, alkaline
phosphatase, and creatinine in net-sampled fish therefore correlate well with what we
know about the probable stress state at the time of sampling.
Increases in SGOT and SGPT are often indicative of liver dysfunction (McDonald and
Milligan, 1992). We have to take into account the health state of the fish chosen for net-
sampling. These fish were usually sick or lethargic, and were chosen for sampling before
being euthenized. It is likely that other physiological factors contributed to the results we
see. However, many of the changes are probably a result of extended stress and muscle
activity that occurs when netting a fish.
The observed increases in phosphorous and cholesterol in net-sampled fish do not seem
to be directly attributable to a stress response. This increase could either be an artifact of
variable handling or environmental conditions that were not controlled, or a secondary
effect of changes in blood chemistry and osmolarity. To explain this effect, there will need
to be more samples taken using both methods. Likewise, to better quantify all the changes
we have observed in this study, more research needs to be done on the comparative
aspects of sampling techniques.
Conclusion
We developed a new method of blood sampling in which blood is taken from the
bulbus arteriosis of captive yellowfin tuna. This method has proven to be consistently
repeatable. Furthermore, based on hematology and blood chemistry comparisons between
this method and other common sampling methods, it appears that samples analyzed in the
current study are representative of minimally stressed fish. Hemoglobin and hematocrit
values closely resemble those obtained using cannulation methods Therefore, we are
confident that this technique can be used to determine accurate parameters for healthy
tuna blood, as well as aid in the comparison between various physiological states.
Literature Cited
Devries, A.L., and Ellory, T.C. 1981. The effect of stress on ion transport in fish
erythrocytes. Journal of Physiology. 324:51pp
Houston, A.H., Madden, J.A., Woods, R.J., and Miles, H.M. 1971. Some physiological
effect of handling and tricain methanesulphonite anesthesiation upon brook trout,
Salvelinus fontinalus. Journal of Fisheries Research Board of Canada. 28:625-633.
Houston, A.H. (1990) Blood and circulation. In Methods for Fish Biology (ed. C.B.
Schreck and P.B. Moyle). pp. 273-334. Maryland: American Fisheries Society.
Korsmeyer, 1996. Unpublished results.
McDonald, D.G. and Milligan, C.L. (1992). Chemical properties of the blood. In Fish
Physiology Vol. 12B (ed. W.S. Hoar, D.J. Randall, and A.P. Farrell). pp. 55-133.
San Diego: Academic Press.
Railo, E., Nikinmaa, M., and Soivio, A. 1985. Effects of sampling on blood parameters
in rainbow trout, Salmo gairdner: Richardson. Journal of Fish Biology. 26: 725-
732.
Reynolds, A.J., Fuhrer, L., Dunlap, H., Finke, M.D., and Kallfelz, F.A. 1994. Lipid
metabolite responses to diet and training in sled dogs. Journal of Nutrition.
124:27545-2759s.
Sovio, A., Nyholm, K., and Westman, K. 1975. A technique for repeated sampling of the
blood of individual resting fish. Journal of Experimental Biology. 63:207-217.
Wells, RM.G., et al. (1984). Recovery from stress following capture and anesthesia of
Antarctic fish: hematology and blood chemistry. Journal of Fish Biology. 25:567-
576.
Yamamoto, K.I., Itazawa, Y., and Kobayashi, H. 1980. Supply of erythrocytes into the
circulating blood from the spleen of exercised fish. Comparative Biochemistry and
Physiology. 65A:5-11.
Table 1
Comparison of blood analysis between fish sampled using sling technique and fish that were
were netted or dying. Data for sling sampled fish represents baseline values obtained during
this study.
Sling Technique
Netted Fish
Hematology
Mean t-SE
Mean t/-SEn t-test % increase
31.6 +.8
Hematocrit (%)
44.1 + 11.0 5 N.S. (p=.2) 40
11.1 +.3
Hemoglobin (g/dl)
16.2+2.1 8 “ (p=.02) 46
2.25 +.06
RBC (E6/cu mm)
Chemistry
7.6 +5
43.5 + 8.6 8 “ (p=.002) 472
Alkaline Phophatase (IU/L)
8.3 +.8
Anion gap
Bilirubin - direct (mg/dl)
Bilirubin - indirect (mg/dl)
.02 +.008
175 + 04 8 * (p=.004) 775
02 +.008
Bilirubin - total (mg/dl)
2.7 +.2
3.4 + 4 8 N.S. (p=.3)
BUN (mg/dl)
13.6 + 9 8 N.S. (p=.5)
12.9 +2
Calcium (mg/di)
155.0 + 15,5 8 N.S.(p=.24)
77.1 +-1.3
Chloride (meq.L)
193.6 + 24.8 8 " (p=.025)
119.0 + 3.0
Cholesterol (mg/dl)
1653 + 1018 8 N.S. (p=.16)
CPK (IU/)
78.5 + 25.6
2000
49 + 06 8 " (p=.002)
19 +.03
Creatinine (mg/dl)
158
94.8 + 3.8
80.9 + 14.3 8 N.S. (p=.44)
Glucose (mg/dl)
5.0 +.3
10.6 + 1.4 8 " (p=.002)
Phosphorous (mg/dl)
112
1.96 +.07
Potassium (meg/L)
7.4 + 1.5 8 * (p=.007)
278
25 +.06
RBC (E6/cu mm)
189.5 + 58.4 8 " (p=.01)
2.4 + 5
7795
SGOT (AST) (U/)
11 +.08
16 + 5.9 8 " (p=.03) 144454
SGPT (ALT) (U/)
197.7 + 6
193 + 14.2 8 N.S. (p=.8)
Sodium (meg/L)
4.4 +6 8 N.S. (p=.4)
3.9 +.06
Total protein (gm/di)
1.6 +.15 8 N.S. (p=.1)
1.2 +.07
Albumin (gm/di)
2.8 + 5 8 N.S. (p=.6)
Globulin (gm/di)
2.6 +.09
Figure Legends
Figure 1. Blood sampling locations on the yellowfin tuna. In this study, blood was
sampled from the bulbus arteriosis.
Figure 2. Repeat hematocrit measurements in three individual fish separated by 6 days
There is no significant difference (paired Student's t-test, p-66)
Figure 3. Repeat hemoglobin measurements in three individual fish separated by 6 days
There is no significant difference (paired Student's t-test, p-.44).
Figure 4. Repeat red blood cell counts in three individual fish separated by 6 days. There
is no significant difference (paired Student's t-test, p- 88).
Figure 5. Hematocrit and hemoglobin values in different tank and diet treatments. There
are no significant differences (one-way ANOVA, hematocrit p= 11, hemoglobin p-.6).
Figure 6. A comparison of hematocrit values in sling-samples (current study), cannulated
samples (Korsmeyer, 1996), and net-samples (TRCC data). Net samples are significantly
higher than sling samples and cannulated samples (one-way ANOVA, Tukey's HSD,
p5.05), while there was no significant difference between sling samples and cannulated
samples.
Figure 7. A comparison of hemoglobin values in sling-samples (current study), cannulated
samples (Korsmeyer, 1996), and net-samples (TRCC data). Net samples are significantly
higher than sling samples and cannulated samples (one-way ANOVA, Tukey's HSD,
pS.05), while there was no significant difference between sling samples and cannulated
samples.
Figure 8. Changes in free fatty acid concentrations ([FFA)) in high fat vs low fat diet
Increase in [FFA] in high fat diet is significant (Student's t-test, p5.005).
Acknowledgments
Thanks to everyone at the TRCC who made this project possible. Especially to my
advisor Dr. Barbara Block for her patience, enthusiasm, and motivational skills; Heidi
Dewar for making this project fun and always having time; Dr. Tom for teaching me
about the finer points and keeping things loose, Chuck Farwell because there is no one
better at catching a tuna; Ellen Freund for her wisdom and wit; Doug Fudge for his
technical support and tuna knowledge, Denise Imai for all of her help and support; Beth
Condie and Victor Tubbesing for their assistance; and everyone else who joined in when
extra hands were needed.

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Figure 2
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Figure 3
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Figure 4
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Figure 5
Tank 3 samples

Tank 2 before fat diet
Tank 2 after fat diet
14
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Figure 6
Sling Technique YFT
Chronically cannulated YFT
(Korsmeyer, 1996)
Netted or Dying YFT
n=5
20
15
10
Figure 7
— Sling Technique YFT
Chronically cannulated YFT
(Korsmeyer, 1996)
Netted or Dying YFT
n=9
n=6
n=27
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Figure 8
T2 before fat diet
T2 after fat diet