the acute effects of a handling stressor on select oreochromis aureus plasma components nora e....
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The Acute Effects Of A Handling Stressor On Select
Oreochromis aureus Plasma Components
Nora E. Demers
Florida Gulf Coast University
Ft. Myers, FL
Does the fight or flight response enhance innate immunity?
Or perhaps more realistically:
Is it possible to obtain a resting fish?
Innate immune parameters
– Lysozyme- published information:
• Immuno-suppressive with exposure to toxins
• Immuno-enhancing with exposure to toxins
• Immuno-enhancing within minutes of stress
Any conservation of response among species?
Possible role of other plasma components?
• Transferrin? Transferrin?
– Control iron available to pathogens
• Cholesterol?Cholesterol?
– Gross indicator of organelle trafficking
Fish Acquisition and Acclimation
Purchase Oreochromis aureus from local aquaculture facility.
Relocate to Lee County Hyacinth Control Ponds.
Seine net capture and transfer 10 fish to 400L circular tank with flow-through well water.
Acclimate for 1-2 weeks.
Experimental DesignAnesthetize all fish
in tank with 2-PE.
Bleed all fish from caudal vessel with heparinized needle.
Revive 5 fish while confined in nets in freshwater tank.
Stress fishStress fish by 30 seconds
in air and 5 minutes in shallow bucket.
Bleed again (same fish).
Bleed “control” fish again – had been in tank containing anesthesia entire time.
Spin blood and collect plasma.
Data acquired from plasma sample
lysozyme activitylysozyme activity --using fresh plasma– (kinetic assay- microplate) HEWL digestion of
Micrococcus lysodeckticus measured by change in optical density
• Glucose- oxidase-based colorimetric test• Total Protein- biuret reaction cupric ion in
alkaline medium• Iron-ferric iron removed from transferrin
by acidic pH, reduced and bound by dye• Cholesterol- esterhydrolase, oxidase and
peroxidase, leuco dye detected
Automated Assays on Frozen Plasma- (Vitros product) (compliments of DSI laboratories)
Automated Ion Assays Potentiometric analytical element
(compliments of DSI laboratories)
• Sodium- methyl monensin • Chloride- layer to prevent interference
from bromide and uric acidAll proficiency limits are very low. Data
shown are “real” changes in values, not machine noise.
Glucose Data Average final levels.
Control: 121 mg/dL.
Stressed 86 mg/dL.Ave. increase:
Control: 43 mg/dL (28/30).
Stressed 7 mg/dL (20/30).
Initial to final.p <.0001 (control).Stressed n.s.Control to
stressed n.s.-100
-50
0
50
100
150
200
Ch
an
ge
in g
luco
se le
vel m
g/d
L control
stressed
Glucose data interpretation?
““Control” fish were stressed.Control” fish were stressed.Initial anesthesia took >10 min. and keeping
fish anesthetized was a stressor.
Stressed fishStressed fish either had response inhibited OR they used up the plasma glucose that was released.
There was a significant difference between control and stressed groups.
Total protein data Average final levels
Control: 2.95 g/L
Stressed: 2.89 g/L
Ave. decrease:Control: -.075 g/L
( 15/30 )
Stressed -.174 g/L (17/30)
Initial to final
p =.05 (stressed)
p =.02 (control)
Control to stressed p=
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
chan
ge in
tota
l pro
tein
g/L
control
stressed
Total protein interpretation?
Expected to see increase in TP level.
(if Ho was supported).
Why did both groups show decreases?
due to changes in plasma volumes?
(hypervolume of blood is used to explain increases in some parameters but these are decreases…).
Why was stressed decrease greater?
Iron dataAverage final levels.Control: 127.7 ug/dL.Stressed: 120.7 ug/dL.Ave. increase:Control: 21ug/dL
(15/27).Stressed 4ug/dL
(13/26).Initial to final p=.02
(control).Control to stressed
n.s.
-50
0
50
100
150
200
chan
ge in
iron
ug/
dL
control
stressed
Iron interpretation?
• There was an increase in total iron in all samples.
• This could indicate an increase in plasma transferrin level.
• Does the “lesser increase” in stressed fish indicate a suppressed response or a return from more elevated level? (How would you determine?
Cholesterol dataAverage final
levelsControl: 148.3
mmol/LStressed 142
mmol/LAve. decrease:Control: 11.5
mmol/L (21/30) Stressed 17.3
mmol/L (22/30)Initial to final
p=.005 (control)=.0002 (stressed)
-80
-60
-40
-20
0
20
40
60
Cha
nge
in C
hole
ster
ol m
mol
/L
control
stressed
Cholesterol literatureToxicology.Plasma cholesterol-.Clarias -carbaryl 96 hours and 15 d sub- lethal dose (1 2
or 4 mg/L) no change (Sharma 1999).• Decreased of 15% (40 mg/dL) at 24 hour sub-lethal
dose (15 ppm) of carbaryl or phorate (Jyothi 2001) attributed to liver dysfunction.
Liver cholesterol-.
Channa punctatus nickel exposure for 30d – increasing levels of cholesterol with higher concentrations attributed to hepatic dysfunction or blocking of cholesterol synthesis.
Cholesterol literature
Exercise.Trout decreased total protein and cholesterol by 4
mmol/L within 1 hr (strenuous) w or w/out endrin (Grant & Mehrle 1973) (but they measured only 14 mmol/L as total level).
Piaractus decreased plasma chol. after 6 hr. confinement (Krieger-Azzolini et al, 1989).
Experimental:Perrier (1972) showed adrenaline I.M. injections (15
hr later) increases plasma cholesterol.
Cholesterol interpretation?Why do decreases in cholesterol occur within minutes?
(hypervolume is used to explain increases)
My Ho is that this demonstrates exocytosis of granules
How can this be tested?
Mark granule contents?Mark granule contents?
Mark cholesterol itself (how)?Mark cholesterol itself (how)?
Which question would yield more valuable information?
Do these results add useful information to interpretation of toxicology data?
Lysozyme data
-3
-2
-1
0
1
2
3
4
5
6
chan
ge in
lyso
zym
e ac
tivity
ug/
mL
control
stresssed
Average final levels.Control: 11.57 ug/mL.Stressed 11.72 ug/mL.Ave. change:Control: -.126 ug/mL
(18/30).Stressed +.327 ug/mL
(17/30).* Fish with increase of
4.73 was blind in one eye.
Initial to final n.s.Control to stressed n.s.
Lysozyme interpretation?Stressor of all samples may have exasperated
interpretation of results- Saw decreases in 2/3 of fish in both groups. Perhaps window of enhancement had passed (for C3 increase at 10 min. return to resting at 20 minutes).
Changes in lysozyme activity with stress do not appear to be very reproducible in these Oreochromis aureus -only 66% responded similarly.
With variation in published data, my confidence in lysozyme as an indicator of stress is lessened every day.
Sodium data
• Control: average loss of 2 mmol/L• Stressed: average increase of 3 mmol/L
-30
-25
-20
-15
-10
-5
0
5
10
15
20
25
chan
ge in
sod
ium
leve
l mm
ol/L
control
stressed
Chloride data
• Control: average decrease of 2mmol/L
• Stressed: average increase of 5 mmol/L
-30
-20
-10
0
10
20
30
Cha
nge
in c
hlor
ide
leve
l mm
ol/L
control
stressed
Conclusions• Fish respond more quickly to handling stressors than much
literature documents.• A closer look at the immediate response may help elucidate
confounding variables that confuse other research results.• Cholesterol and total protein data look to be closely related.• The role of iron and cholesterol would be especially
interesting to elucidate.