Trout Aquaculture Research at the National Center for Cool and Cold Water Aquaculture

Caird Rexroad

USDA/ARS National Center for Cool and Cold Water

Aquaculture

Leetown, West Virginia

USDASecretary of Agriculture

Under Secretary for Research, Education, and Economics

Agricultural Research

Service

Economics Research

Service

NationalAgricultural Statistics

Service

Cooperative State Research Education &

Extension Service

Leetown

National Center for Cool and Cold Water Aquaculture

To support and enhance the nation’s cool and cold water aquaculture production through research and

technology transfer.

US Trout Industry

Rainbow trout are the most cultured coldwater fish in the US, 747 locations

~390 Commercial farms composed a ~$95 million industry in 2007

• Foodfish ~$80 Million• Most sales to processors and restaurants

• Stockers ~$5.84 Million• Fingerlings ~$1.7 Million• Eggs ~$7.5 Million

~433 State/Federal/Private locations with production aimed at conservation and restoration valued at an additional $102 million in 2007

Majority of production in California, Wisconsin, Michigan, North Carolina, Pennsylvania, and Idaho (53% of sales of food size fish)

Imported ~$83 million fresh and frozen trout products, ~$.5 million live in 2006• Compete also with Salmon, Shrimp, Catfish, and Tilapia

ERS Aquaculture Outlook, NASS Trout Production

US Rainbow Trout Industry• Large Operations

– Troutlodge, Sumner, WA• Egg producer• Large Breeding program – evaluating Hatch-out rates,

Feed Conversion, Growth, Survivability, Uniformity, Flesh quality, Flesh yield, and Disease-resistance

– ClearSpring Foods, Buhl, Idaho• Vertically Integrated – feeds, broodstock,

production, processing, sales• Large Breeding program

• Smaller Operations, USA– USTFA– NAA– State Associations

• Associated Industries– Feed Production– Aquatic Health and Diagnostics– Genetic Services– Aquaculture Systems– Federal/State Hatchery Systems

Customer/Stakeholder Workshop:Issues faced by the US Rainbow Trout Industry

Disease• Fp, IHNV, IPNV• Certifications

Production Traits• Feed efficiency• Growth• Stress tolerance (handling, crowding, low O2)

Consumer Traits• Flesh color• Fillet quality

Nutrition • Next generation of feedsChromosome Set Manipulation• Tetraploid/triploid production

NCCCWA’s role in supporting the US Rainbow Trout IndustryTwo major companies from this industry have large breeding programs

Several of the issues arising from the first NCCCWA Customer/Stakeholder meeting can be addressed through breeding

NCCCWA Team Objectives:• Develop and evaluating selective breeding strategies for trout,

developing improved germplasm in the process, complementing ongoing industry efforts.

• Conduct basic and applied research to understandenvironmental factors and biological mechanisms controlling traits of interest.

Transfer technologies and germplasm to industry

Scientific StaffingGENETICS and PHYSIOLOGY

♣ Research Physiologist – Dr. Greg Weber ♣ Research Geneticist – Dr. Timothy Leeds ♣ Growth Physiologist - Vacant

GENOMICS♣ Molecular Geneticist – Dr. Yniv Palti♣ Molecular Biologist – Dr. Caird Rexroad III♣ Molecular Biologist – Vacant♣ Computational Biologist – Dr. Roger Vallejo

FISH HEALTH♣ Molecular Immunologist – Dr. Greg Wiens♣ Research Pathologist – Dr. Tim Welch♣ Microbiologist – Dr. Jason Evenhuis

Scientific Support Staffing

Research Technicians (13 )Administrative Assistant (2) Information Technologist (1)Facilities Management (2)Water Systems Operator (1) Wet Lab Crew (5)

Improved fish for producers / consumers

Production Systems

Fish Health

Integrated applied breeding program

QuantitativeGenetics

Physiology

Molecular Genetics

Hypothesis: Sufficient genetic variation exists inNCCCWA broodstock to realize genetic improvementthrough selection.

Selective Breeding

Goal: Develop and transfer technologies and germplasm to the aquaculture industry

GermplasmResearch and Production

♣ Biology High Fecundity Cryopreserve Sperm Response to Photoperiod Manipulation

♣ Crosses Intraspecific crosses – within species Interspecific crosses – crosses between two species F1 Crosses – first generation, abundance of heterozygosity and uniformity Backcrosses – cross of F1 and parent Full Sib Crosses regular Half Sib Crosses one common parent

♣ Genetic Manipulation Transgenics insert DNA Clonal lines – doubled haploids Sex reversal Chromosome Set Manipulation

Genetic Improvement of Aquaculture Species

1. Define trait2. Identify variation in a trait directly due to variation in DNA sequence

(Heritability) Sequence variation can be in or around a gene and changes how the gene

functions3. Understanding of the basic genetics of the trait

(mode of inheritance)4. Develop technologies designed to exploit positive genetic variation5. Use those technologies to development of genetically improved strains

Quantitative Genetics Definitions♣ Quantitative Genetics - use of statistics to assign

breeding values to broodstock used in selectivebreeding programs for the development ofsuperior strains for aquaculture

♣ Phenotype – category or classification of a trait

♣ Heritability – the extent to which an animalsbreeding value can be predicted from itsphenotype, = VG/VP

♣ Germplasm – biological resource material

Quantitative GeneticsVP=VG +VE+VGxE …

♣ VP= variation in phenotype♣ VG=variation due to genetics (VA+VD

+VAxD…)♣ VE=variation due to environment (VN+VT+VPP…)♣ VGxE=variation due to gene x environment

interactions ♣ Continuous variation♣ Discontinuous variation♣ Use of specific crosses to determine heritability of a

trait and to determine breeding values

Mapping Traits - Heritability

High Heritability Low Heritability

Broodstock Population of 100 Families

Performance EvaluationGrowth performance (even year)Disease resistance (odd year)

Selection of Top 10% Families

Selection Approach

Physiological characterization of selection traits

BW

EB

V, g

ram

s

Age, months

2002 base population

h2 ≈ 0.50

Growth Improvement Line

85 g

216 g

1010 g

547 g

Bod

y w

eigh

t (g)

297

dph

200

300

400

500r = 0.372P = 0.020

TGC

149

- 29

7 dp

h

1.4

1.6

1.8

2.0

2.2

2.4r = 0.533 P < 0.001

A B

DC

Trait Characterization: Thermal Growth Coefficient and Future Growth Performance

Body weight (g) 459 dph Body weight (g) 459 dph(Lankford and Weber 2006)

800 1000 1200 1400 800 1000 1200 1400

Preliminary heritability estimate TGC 9-12 months: h2 = 0.32

Feed Intake

Fed labeled diet 73 full-sib families 30 fish per family 3 occasions

Intake of each fish quantified Beads counted

Diseases

Courtesy of Rich Holt

Bacterial Coldwater DiseaseF. psychrophilumEconomic impact

Enteric Red-Mouth DiseaseY. ruckeriVaccination modelRecently emerging

biotype II

Family-Based Selective Breeding for Disease Resistance

x

x

Cross 1

Cross 2

Cross 100

Select for

breeding

Disease-Free Stock/Rooms

Challenge and record days until death and total mortality (day 21)

Calculate Heritability and Breeding Values

PathogenContainment Room

Cross 1

Cross 2

Cross100

xxx

0

10

20

30

40

50

60

70

80

90

100

Full-Sib Crosses

2005

Surv

ival

(%)

• Silverstein, J.T., Vallejo, R., Palti, Y., Leeds, T.D., Rexroad, C.E. III, Welch, T.J., Wiens, G.D. and Ducrocq, V. 2008. Journal of Animal Science. Submitted

• Leeds, T.D., Silverstein, J.T., Vallejo, R.L., Palti, Y., Rexroad, C. E. III, Welch, T.J. and Wiens G.D. Journal of Animal Science. In Preparation

Large Variation in Resistance to F. psychrophilum challenge (2005 year-class).

Selectedfor breeding

0

10

20

30

40

50

60

70

80

90

100

Full-Sib Crosses

2007

2005Surv

ival

(%)

Selective breeding increased average survival (2007 year-class).

• Silverstein, J.T., Vallejo, R., Palti, Y., Leeds, T.D., Rexroad, C.E. III, Welch, T.J., Wiens, G.D. and Ducrocq, V. 2008. Journal of Animal Science. Submitted

• Leeds, T.D., Silverstein, J.T., Vallejo, R.L., Palti, Y., Rexroad, C. E. III, Welch, T.J. and Wiens G.D. Journal of Animal Science. In Preparation

Characterization of Resistant Fish

Are fish resistant throughout life cycle? Yes – 2 g, 10g and 800g evaluation

Are fish resistant to other Fpstrains? Yes –2 other strains tested

What is the mechanism of resistance? More resistant to BCWD= bigger spleen Differences in immune gene expression

Hadidi, S., Glenney, G., Welch, T.J., Silverstein, J., Wiens, G.D. 2008. The Journal of Immunology. 180:4156-65.

Susc. Res.

Genotype by Environment High intensity recirc environment vs.

traditional raceway Will fish grow as well as , or better than in

“traditional” system? Environmental effect

Will the fish that grow best in traditional system be the same fish that grow best in high intensity system? Genotype x environment effect

G x E : a real concern? Tolerance:

Water hardness Temperature Waste product concentration Fish density

Examine performance of families across different environments NCCCWA, CFFI, NCSU Flow through vs. Partial Re-use

Comparison of families in different environments

Silverstein and Summerfelt (unpublished)

Correlation of BW (g) at NCCCWA and FI

0.0

100.0

200.0

300.0

0.0 100.0 200.0 300.0 400.0 500.0

Weight (g) at FI (263d)

Wei

ght (

g) a

t NC

CC

WA

(2

42d)

r=0.72, p<0.001

Partial Re-use systems

Silverstein and Hinshaw (unpublished)

Correlation of BW at NCCCWA and NCSU

0

20

40

0 20 40 60

Weight (g) at NCSU (7/26/04)

Wei

ght (

g) a

t N

CC

CW

A (7

/24/

04)

Flow Through systems

Creation of Tetraploids

First tetraploid hatched at NCCCWA

Haploid

TetraploidTriploid

Chromosome Set Manipulation can

produce rainbow trout which are sterile

Triploid Production

• Tripliod Benefits – More energy to growth and not reproduction– Protect germplasm/breeding strategies

• Similar protocol to developing tetraploids– Low efficiency, not 100% (diploid

contamination)• Cross Tetraploids with Diploids

– High efficiency– Less defects

What are the genes?

GenomeTranscriptome

Proteome

Use of Molecular Genetics for the Improvement of Rainbow Trout for Aquaculture Production

Maintain genetic diversity thru selection

Evaluation of families in common garden, ability to identify parentage

Identify genes for selection which affect traits which are expensive or difficult to measure or require sacrificing fish

Introgression – introgression of haplotypes associated with a phenotype into a population

Multi-trait selection, especially where multiple traits can not be evaluated on individuals

Association with a breeding program including “commercially relevant” germplasm to facilitate use of that information - NCCCWA broodstock

GenotypingMicrosatellites

Genetic Markers

Rainbow trout• ~835 anonymous

• 181 from BAC clones– Physical map integration

• 334 from genes

• Therion DNA, Int., Saratoga Springs, New York

• Striped bass– n=498– Kent Sea Tech Corporation

• Collaborations– Spanish mackerel– Greater amberjack– Red drum– Pacific sardine– Cobia

Useful for determining identifying individuals, parentage, characterizing population structures (migration, inbreeding, strain identification), estimation of genetic variation, conservation, evolutionary studies) and genetic maps having the goal of identifying genes affecting traits

YRO

1041

YRO

1131

YRO

1561

YRO

2235

YRO

2308

YRO

3596

YRO

3752

YRO

3764

YRO

3766

YRO

3775

YRO

3861

YRO

3865

YRO

3891

YRO

3894

YRO

3906

YRO

3920

YRO

3948

YRO

3973

YRO

3980

YRO1131 0.4YRO1561 0.5 0.3YRO2235 0.2 0.3 0.2YRO2308 0.5 0.3 0.4 0.2YRO3596 0.3 0.3 0.3 0.2 0.2YRO3752 0.2 0.3 0.2 0.3 0.3 0.2YRO3764 0.2 0.3 0.2 0.3 0.3 0.3 0.4YRO3766 0.3 0.3 0.2 0.3 0.2 0.3 0.3 0.7YRO3775 0.2 0.3 0.2 0.4 0.2 0.3 0.3 0.4 0.4YRO3861 0.3 0.3 0.2 0.4 0.3 0.3 0.3 0.4 0.4 0.4YRO3865 0.3 0.3 0.2 0.3 0.3 0.3 0.3 0.3 0.3 0.4 0.3YRO3891 0.3 0.4 0.2 0.3 0.3 0.2 0.4 0.3 0.2 0.3 0.3 0.4YRO3894 0.3 0.3 0.3 0.2 0.3 0.2 0.3 0.3 0.2 0.3 0.3 0.3 0.3YRO3906 0.3 0.3 0.3 0.2 0.2 0.3 0.3 0.3 0.3 0.4 0.2 0.4 0.4 0.3YRO3920 0.2 0.3 0.3 0.3 0.3 0.2 0.3 0.3 0.3 0.3 0.3 0.4 0.3 0.3 0.4YRO3948 0.2 0.3 0.3 0.3 0.3 0.4 0.3 0.4 0.5 0.5 0.5 0.3 0.2 0.3 0.3 0.3YRO3973 0.2 0.2 0.3 0.3 0.3 0.2 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3YRO3980 0.2 0.4 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.4 0.3 0.4 0.3YRO4054 0.2 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.4 0.2 0.3 0.2 0.2 0.3 0.2 0.3 0.3

Relationship Matrix Between Individuals

Population Substructure•Using LD data, we were able to determine that we have an effective breeding size (Ne) of ~150 in 2005 and 2006

•Actually used ~320 fish ratio of .48 actually contribute unique genetic variation

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SHSH

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SHSH

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SHSH

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SHSH

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SHSH

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TLTL

_004

TLTL

_007

TLTL

_008

TLTL

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TLTL

_012

TLTL

_016

TLTL

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TLTL

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TLTL

_022

TLTL

_023

TLTL

_024

TLTL

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TLTL

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TLTL

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TLTL

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TLTL

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TLTL

_039

UW

UW

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UW

UW

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UW

UW

_005

UW

UW

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UW

UW

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UW

UW

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UW

UW

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SHH

C_0

01SH

HC

_007

SHH

C_0

08SH

HC

_009

SHTL

_004

TLSH

_004

TLU

W_0

06TL

UW

_007

TLU

W_0

09TL

UW

_012

TLU

W_0

13TL

UW

_016

TLU

W_0

17TL

UW

_018

TLU

W_0

19TL

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_021

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UW

HC

_003

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HC

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_001

SHSH

_002

SHSH

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W_0

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W_0

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UW

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• Palti et al., 2006. Evaluation of family growth response to fish meal and gluten-based diets in rainbow trout (Oncorhynchus mykiss). Aquaculture 255(1-4):548-556.

• Johnson et al., 2007. Development and evaluation of a new microsatellite multiplex system for parental allocation and management of rainbow trout broodstocks. Aquaculture 266:53-62.

• Pierce et al., 2008. Family growth response to fishmeal and plant-based diets shows genotype x diet interaction in rainbow trout (Oncorhynchus mykiss). Aquaculture 278:37-42.

Genetic and Diet Effects on Growth Rate and Reproduction in the Rainbow Trout Strains of Troutlodge

11

2

2 2

1

♀

Mapping Traits

Rainbow TroutComparative

MapOMM17520.0OMM159926.3OMM164535.6OMM133736.6OMM500542.7OMY14DIAS45.3OMM108547.1OMM3120_BAFF-249.5OMM522050.1OMM1570OMM108061.7OMM105368.8OMM143472.3OMM516476.2OMM170680.1OMM5024OMM1766bOMM5146

82.9

OMM532484.3OMM520488.8OMM1058OMM108391.2OMM127892.1OMM1631OMM5275OMM3081b_TAP1

94.6

OMM1391a101.8OMM5062a109.2OMM5225bOMM1263bOMM1624aOMM5067

113.5

OMM1138OMM5121115.4OMM1137116.1OMM5109120.4OMM1336OMM5142a122.2OMM1778b127.1OMM1198OMM1016B142.2

0

20

40

60

80

100

120C

ortis

ol (n

g/m

l)

BA

CA BBCB

CCAAAB

ACBC

Plasma cortisol concentrations following a 3 hour confinement stress

Families (Mean + SEM)

Trait Evaluation: Stress Response

(Weber and Silverstein 2007)

Body weight (g) 297 dph200 300 400 500

Post

-stre

ssor

Plas

ma

Corti

sol (

ng/m

l)

0

25

50

75

100

r = 0.466P = 0.026

B

(Lankford and Weber 2006)

Heritability: h2 > 0.40

Contribution of Major Genes to Post-stressor Plasma Cortisol Levels

Contribution to total variancepolygenic 10%major gene 68%

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0 500 1000 1500 2000 2500 3000 3500 4000 4500

Variance

Den

sity

polygenicmajor gene

Marginal posterior densities* for polygenic and major gene variance from the mixed inheritance model for plasma cortisol (Gibbs sampling: samples per chain=500,000; burn-in=250,000; thinning=5,000; chains= 7)

USDA ARSNational Center for

Cool and Cold Water AquacultureLeetown, West Virginia