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.