Ontogenetic and energetic

consequences of an immunological challenge in

Japanese Quail (Coturnix coturnix

japonica)

Imran Rafi Ahmed PunekarDr. John Olson

• Birds must devote energy to a wide variety of functions

– Because the bird must strike a balance between devoting energy to growth, foraging, thermoregulation, digestion, and homeostasis

So, Like any good budget-keeper…

• Derive an equation for total energetics:

Etme = Em (maintenance) + Eb (biochemical) + Esda (specific dynamic action) + Etr (thermoregulation) + Eact (activity) + Ep (production)

– Em + Eb = Ebmr

– Precocial species have higher Em, higher Etr, and higher Eact

• Precocial Birds– Such as Japanese Quail (Coturnix coturnix japonica)

hatch with a layer of down feathers, with open eyes, and are capable of locomotion

– Incubation period is typically 17days– Naturally found across much of Europe and Asia,

came to Japan as pets in the 11th century, became popular as poultry in the early 1900’s.

– Introduced to the United States around 1870– Clutch size: 5-12 eggs– Sexually mature after 40 days– Range from 100-180g at maturity, depending on sex– Optimally lay 200-300 eggs a year– Japanese Quail typically grow up in grassy fields, or

near riverbanks– Are able to perform some thermoregulation – Are able to move at birth in order to forage for food,

avoid predation, and find suitable habitats– Display significant growth and development during

the post-hatch period

•In precocial species, less Energy is available for growth

BASELINE MASS GROWTH DATA

0

20

40

60

80

100

120

140

160

180

200

0 10 20 30 40 50 60 70

Age (days)

wei

ght,

g

Simons & Pacifici, NC State Univ. http://www.cals.ncsu.edu:8050/course/zo501/Notes/Chapter%2018%202005.pdf#search='altricial%20species%20growth%20curve'

•

Simons & Pacifici, NC State Univ. http://www.cals.ncsu.edu:8050/course/zo501/Notes/Chapter%2018%202005.pdf#search='altricial%20species%20growth%20curve'

• What happens if…– We add/increase a new energy cost?– Specifically,

by eliciting an immune response which would possibly imbalance the equationand cause less energy to be devoted elsewhere

Little is known about the ontogeny of the immune system in birds, much less about the energetic costs of mounting an immune response

• The Lab is performing an extensive

experiment that is testing the hypotheses that

(1)younger precocial Japanese Quail (Cotunix coturnix japonica) would be less effective in mounting an immune response than older birds That is, that the immune system develops with age and immunocompetence increases as the bird gets older

(2) an immunological challenge will adversely affect maintenance, growth and development of birds by reallocating energy from that devoted to overall growth, maintenance of hematology, organ growth, and thermoregulation

Dependent Variables– Hematology

•Hematocrit Data•Blood Smears and Differential counts• Ig’s• Immunohistochemistry

– Growth•Gross morphology•Organ growth

– Metabolic:•Max VO2 during cold challenge•BMR

– Biochemical •Catabolic enzyme activity

• This study concentrates on

hematology and some growth factors

• Predictions:Immunologically challenged birds will

experience:- less growth and organ development, - altered concentrations of immune cells in the blood- exhibit different changes in hematology over time

…when compared to control birds

Controloil suspension

vehicleMS-bac

in oil suspension

8-day chick Adult

Post-injection samples

Baseline ontogenetic changesBaseline ontogenetic changes

d 1 d 4 d 7 d 10

Pre-injection(1 h before )

d 13 d 16 d 19 d 22

d 4 d 8 d 12 d 16 d 20 d 24 d 28 d 32 d 36 d 40 d 44 d 48 d 52 d 56

Controloil suspension

vehicleMS-bac

in oil suspension

Ontogenetic changes in effect of Ontogenetic changes in effect of immunological challengeimmunological challenge

Experimental Design

Ages

• Hematology

– Blood samples take from brachial vein before injection, 1d post injection, and every 3 days after that.

• Organ Growth – Birds were raised, receiving blood

sample battery above until 22d post injection after which they were sacrificed via cardiac puncture, dissected immediately, and organs harvested.

RESULTS•Morphological Data

Baseline Mass Growth Curve

BASELINE MASS GROWTH DATA

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20

40

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80

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120

140

160

180

200

0 10 20 30 40 50 60 70

Age (days)

wei

ght,

g

•Morphological DataJuvenile Experimental Group Growth Curve

Juvenile Birds

0

20

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60

80

100

120

140

160

0 5 10 15 20 25 30 35

days

•Morphological DataAdult Experimental Group Growth Curve Adult Experimental Birds

0

50

100

150

200

250

0 10 20 30 40 50 60 70

days

wei

ght (

g)

0 5 10 15 20 2520

30

40

50

60

0 5 10 15 20 2520

30

40

50

60

8d MS-bac

8d Control

Hem

atoc

rit,

%

Time post-injection, d

Hematocrit DataInjection itself

causes a profound decrease in Hemotcrit

percentages in both experimental and control birds.Also, immunized birds also had a

decrease in hematocrit values

between 7-10 days, which is

when the immune response to the

vaccine is strongest, while

control birds experienced

smooth changes in hematocrit

percentages

0 5 10 15 20 2520

30

40

50

60

0 5 10 15 20 2520

30

40

50

60

Adult MS-bac

Adult Control

Hem

atoc

rit,

%

Time post-injection, d

Hematocrit Data

Hematocrit values were much more variable in adult birds, but similar fluctuations were

seen between both groups.

In adults, we see a huge discrepancy

between what happens to

hematocrit levels after injection. In

adult control specimens, we see

the expected drop of similar magnitude to

the drop seen in juvenile birds, but in

the experimental group, hematocrit

levels actually increase in all birds

Blood Smears• Few differences noted in ratio

of white blood cells to red blood cells in smears stained with Giemsa in juvenile birds

02468

10

MS C8 d

(7d post)8 d

(22d post)Adult

(7d post)Adult

(22d post)

MS C MS C MS C

Pectoralis Index (mean ± 1sd)M

ass

inde

x, %

bod

y m

ass

Pectoralis index did not change with an immunological challenge. However, the size of

the pectoralis does increase with age.

ANOVA with post hoc comparisons using student t-test : F= 3.7554

p=0.0217 d.f.=7

0.00.10.20.30.40.50.60.7

MS C8 d

(7d post)8 d

(22d post)Adult

(7d post)Adult

(22d post)

MS C MS C MS C

Gastrocnemius Index (mean ± 1sd)M

ass

inde

x, %

bod

y m

ass

Gastrocnemius index did not change either with an immunological challenge nor with age.

ANOVA: F= 1.6507 p=0.2126 d.f.=7

Liver Index (mean ± 1sd)

0

1

2

3

4

MS C8 d

(7d post)8 d

(22d post)Adult

(7d post)Adult

(22d post)

MS C MS C MS CMas

s in

dex,

% b

ody

mas

s

Liver index did not change with an immunological challenge. However, the size of

the liver does increase in adults.

ANOVA with post hoc comparisons using student t-tests: F= 3.7558

p=0.0217 d.f.=7

Heart Index (mean ± 1sd)

0.00.20.40.60.81.01.2

MS C8 d

(7d post)8 d

(22d post)Adult

(7d post)Adult

(22d post)

MS C MS C MS C

Mas

s in

dex,

% b

ody

mas

s

Heart index did not change either in response to an immunological challenge

nor with age.

ANOVA: F= 0.5195 p=0.8037 d.f.=7

Pancreas Index (mean ± 1sd)

0.00.10.20.30.40.50.6

MS C8 d

(7d post)8 d

(22d post)Adult

(7d post)Adult

(22d post)

MS C MS C MS C

Mas

s in

dex,

% b

ody

mas

s

Pancreas index did not change with an immunological challenge. However, the size

of the pancreas does decrease with age.

ANOVA with post hoc comparisons using student t-tests: F= 4.5449

p=0.0108 d.f.=7

Spleen (mean ± 1sd)

0.000.020.040.060.080.10

MS C8 d

(7d post)8 d

(22d post)Adult

(7d post)Adult

(22d post)

MS C MS C MS CMas

s in

dex,

% b

ody

mas

s

The size of the spleen, an important lymphoid organ, did not change with an immunological challenge. However, the size does increase

with age between day 15 and day 30.

ANOVA with post hoc comparisons using student t-tests: F= 4.5449

p=0.0108 d.f.=7

Bursa fabricius (mean ± 1sd)

0.00

0.05

0.10

0.15

0.20

MS C8 d

(7d post)8 d

(22d post)Adult

(7d post)Adult

(22d post)

MS C MS C MS CMas

s in

dex,

% b

ody

mas

s

ANOVA with post hoc comparisons using student t-tests: F= 4.3942

p=0.0147 d.f.=7

The size of the Bursa Fabricius, another important lymphoid organ in birds, did not change with an immunological challenge. Like the thymus, however, the size does

decrease significantly with age.

CONCLUSIONS• From this we conclude that:

– Our hypothesis that young birds would exhibit less of an immune response than older birds was….

– Our hypothesis that the cost of immunological challenges would result in trade-offs in growth, organ development and maintenance of hematology was…

Ongoing and Further Research– Comparison between the ontogeny of the

immune system in a Precocial vs. Altricial species.

– A comparison between the ontogeny of a T-cell mediated response vs. the ontogeny of a T-cell independent immune response.

– An analysis of Substrate metabolism; determining what effect an immune stress has on substrate metabolism as well as on levels of endogenous stress hormones

– In this experiment, we look at the effects of immunization on cold-hardiness and thermoregulatory capability, however a look at the opposite relationship, ie. that of cold stress on immuncompetence would be particularly interesting and helpful in the understanding of the effects of environmental stresses that exist in the wild.

ACKNOWLEDGMENTSSpecial thanks to:Special thanks to:

The Olson Lab Crew:The Olson Lab Crew:• Dr. John OlsonDr. John Olson• Doug CorwinDoug Corwin

• Lauren DiMennaLauren DiMenna• James “Rory” TuckerJames “Rory” Tucker

ACKNOWLEDGMENTS

• Dr. Anil BamezaiDr. Anil Bamezai• Dr. Norman DollahonDr. Norman Dollahon

• Dr. Robert CurryDr. Robert Curry

And thanks to all the Faculty, staff, And thanks to all the Faculty, staff, and students at the Biology and students at the Biology

Department at Villanova who Department at Villanova who helped in any way make this helped in any way make this

possible.possible.