Scott WilliamsARI Wet Lab Manager

Former Brood Operations Manager / Genetics Research Clear Springs Foods

What causes disease in fish?

The Environmentcommercial trout culture

• Clear Springs Foods Brood Division– Two facilities

• Snake River / Research– Selection site– Brood propagation site– 5% of egg production

• Soda Springs– Egg production facility

Improving the hostselection goals

PrimarilyGrowth rate Indirectly for feed conversion rate

SecondarilyDisease resistance

Improving the Fish through Animal Breeding

Selecting the Parents of the Improved population

Using an Animal ModelGenetic merit based on:

Individual records Family records

Family and Within Family Selection for Size at 273 Days

• 100 to 110 full-sib families per generation• 10 families per generation from randomly bred line• Families selection:

– Families with poor early survival were discarded• Families with high mean weights at 273 days were

saved• Within the selected families:

– pit tag top 15 to 20% of individuals

Selection for GrowthSize at 273 Days

• 40 days Post Spawn, reduced to 600 individuals• 63 days PS, reduced to 100 individuals• 76 days PS, combined with another family in 100 gal

tank• 161 days PS, each family was given unique

clip/brand• 162 PS, moved to raceway• 273 PS, final measurement taken

Mean Weights at 328 Days PFodd year generation

0

200

400

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800

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1991 1993 1995 1997* 1999*

655701

908

1350

1524

497 459

552

10231088

selected control

Mean Weights at 273 Dayseven year generations

Increasing size at 273 days post-fertilization

294

342

461

643

216236

217

437

669

450

0

100

200

300

400

500

600

700

800

1992* 1994* 1996 1998 2000

wei

ght (

g)

CSF Control

Correlated traits questions:• How does selection for rapid growth

affect other traits?• Conformation?• Fillet fat levels?• Disease resistance?

Genetics questionsrelated to disease:

• Is the ability to survive an IHNV infection a heritable trait, i.e., is it a trait that can be affected by selection?

• How does IHNV survival correlate to rapid growth?

Relationship of growth to IHNV Susceptibility

IHNV Mortality and size at 273 in BY 1999

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Relationship of growth to IHNV Susceptibility

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IHNV Mortality and size in BY 2000

What is heritability?

A measure of the degree to which the variance in the distribution of a phenotype is due to genetic causes. Or simply, the degree to which genes control expression of a trait.

How heritable is the IHNV survival trait?

Estimate of the heritability is <0.83

This estimate confounds sources of genetic variation other than additive.

This appears to be a highly heritable trait though the actual value is lower than this estimate.

Heritabilities and Genetic Correlations Between Size and IHNV Susceptibility

size @ 63 d 0.399 0.716 -0.304challenge CPM 0.828 0.215size @ 273 d 0.405

size @ 63 d

challenge CPM

size @ 273 d

Fish with higher mean weights at the time of challenge, had greater CPM

Groups with higher ending weights had sibling with higher CPM rates

Tagged families from the 2000 year classfamilies tagged based 1° on IHNV resistance

0%

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500 550 600 650 700 750 800 850

Perc

enta

ge o

f all

fam

ilies

Mean family weights

all families

selected families

Are there other traits correlated to disease resistance that could be

more easily measured?

Parental Humoral Response

humoral response - The immune response involving the transformation of B cells into plasma cells that produce and secrete antibodies to a specific antigen.

Traits Correlated to Disease Resistance: Parental Humoral Response

• Parents of the selected fish were PIT tagged• Eight months prior to spawn

– fish were IP exposed to attenuated form of the IHN virus.

• Six months later months later– Blood samples were collected from broodstock.– Samples and tag ID were correlated– Samples were used to evaluate each fish’s ability

to produce anti-IHNV neutralizing antibodies• Humoral response

Do the parental humoral responses correlate to the offspring challenge

results?

Weak effect of maternal titer on offspring survival rates.

Lower titer in the female associated with higher mortality rate in the offspring.

Improved Measurement of Disease Resistance

At 63 days post spawn• Two challenges

– Coldwater disease challenge• Two replicates of 50 fish each• Subcutaneous injection of Flavobacterium

psychrophilum– IHNV challenge

• One replicate of 50 fish• water borne exposure to IHNV in a closed, oxygenated

system for 1 hour– 10,000 plaque forming units/ml– Volume of water that is 10 times the total weight of the fish

Improved Measurement of Disease Resistance

• Mortality were collected and recorded daily• Necropsies were performed on a fraction of

the mortality to verify that the challenge organism was the cause of death

• Total cumulative percent mortality (CPM) and mean days to death (MDD) were calculated for each replicate

Distribution of Cold Water Disease Mortality 2001

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ent o

f fam

ilies

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in s

trai

n

Flavobacterium psychrophilum mortality range

Flavobacterium psychrophilum CPM histogram for all strains

CSF selected

control

CSI

Arlee

Average CWD Mortality Rate by Strain

Strain Flavobacterium psychrophilum CPMCSF Selected 45.5%a

Control 38.6%a

CSI 62.57%b

Arlee 53.1%ab

Distribution of IHNV Mortality Rates in 2001

IHNV CPM histogram for all strains

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IHNV mortality range

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ent o

f fam

ilies

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in s

trai

n

CSF selectedcontrolCSIArlee

Average mortality rate by strain

StrainFlavobacterium

psychrophilum CPM IHNV CPMCSF Selected 45.5%a 56.8%a

Control line 38.6%a 75.7%b

CSI 62.57%b 79.1%b

Arlee 53.1%ab 52.3%a

Distribution of CPM rates for Selected line 2002

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Cummulative Percent Mortality

2002 Selected Groups' CPM during IHNV and Fp challenges

IHNV Fp

Relationship between mortality rates 2001

y = -0.0165x + 0.6036R2 = 0.0003

p=0.87

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V C

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Flavobacterium psychrophilum CPM

Relationship between Fp and IHNV mortality

Relationship between Mortality rates in 2001

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IHN

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orta

lity

Flavobacterium psychrophilum mortality

Relationship between Fp and IHNV mortality within the control and selected lines

selected

control

Relationship between mortality rates 2002

y = 0.334x + 0.536R² = 0.104

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Fp C

PM

IHNV CPM

Fp and IHNV mortality

Mortality rates within Control and Selected lines in 2002

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Fp C

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IHNV CPM

Relationship between Fp and IHNV CPM for control and selected lines

selected families

control families

Selection Program Modifications

• 1º Family selection on disease resistance– Entire families were culled based on the

mortality rates of replicates challenged• IHNV• Coldwater disease (CWD)

• 2º Within family size– Saving the largest individuals within the

families with the best survival rates

Family selection decision based on mortality index

• Index based on mortality rates of both of the pathogens

• Families with highest index scores were saved for grow-out phase of evaluation

• All other families terminated after growth evaluation at 273 days

Assessing disease resistance &within family selection for size

273 days of age• Weigh every member of all remaining families• Return on day 274

– Pit tag largest 15% within each of these families

Directions for growth and disease resistance selection

• Livability in the selected populations appeared to be decreasing relative to the unselected control line

• Common occurrence for lines selected primarily for improved growth, e.g., poultry

Directions for growth and disease resistance selection

• 1° selection based on a survival index– Index takes into account survival traits

• 2° selection will be based on growth rate• Results:

– Improved survival to IHNV– However, reduced rate of weight increases

Identification of DNA Markers Associated With IHNV Resistance in Rainbow Trout

Develop a rainbow trout linkage map Find molecular markers linked to loci

affecting resistance to infectious hematopoietic necrosis virus in rainbow trout

Develop a breeding plan to increase IHN resistance in rainbow trout using the genetic markers (Marker Assisted Selection)

Identification of DNA markers• Two strain of rainbows

– One anadromous and with elevated resistance to IHNV– The other a domestic strain with good growth and low

resistance to IHNV• Produce F1 cross• Back-crossing members of each family (F1) with rainbow

trout produced an BC1 generation• Challenge with IHNV• Collect mortality and survivors• Use both AFLP & microsatellite markers• Look for polymorphisms associated with survival or

mortality

Marker Assisted Selection for Trout Improvement

Growth Disease Resistance

selective breeding

large & resistant

small large susceptible resistant

largeresistant

Molecular toolsDNA

extraction

QTL analysis

Marker Assisted Selection• Theoretically MAS has the potential to

greatly accelerate genetic improvement livestock, plants and aquatic animals.

• Initial data from livestock have demonstrated that MAS can be more productive than traditional breeding, but some cases can be equally or less effective than selective breeding.

• Limited data from aquatic species.