Fish Nutrition ResearchFish Nutrition ResearchDifferences and similarities with livestock nutrition and what the f t h ld P t IIfuture holds. Part II.

Ronald W. Hardy, DirectorRonald W. Hardy, DirectorAquaculture Research InstituteUniversity of Idaho U i itUniversity of Idaho University

of Idaho

Current areas of emphasis in fish pnutrition research

• Replacing marine protein and oil• Effects of nutrition on food quality and

fish healthfish health• Developing micro-particulate feeds for

ll l fi f dismall larvae at first feeding• How can these areas be investigatedHow can these areas be investigated

using new genomic tools?

Replacing marine protein and oilReplacing marine protein and oil• Rapid growth of aquaculture over the past 15 years isRapid growth of aquaculture over the past 15 years is

due to:– Industry growth in developed species–expanded production of new species–Switch from extensive to intensive production of pond fish th h hi h f d i t i SE A i d Chithrough higher feed inputs in SE Asia and China

• Proportion of annual global production of fish meal and fish oil used by aquaculture has tripledfish oil used by aquaculture has tripled

• Production of fish meal and oil cannot increase except in Alaska by increased recovery of processing wastey y p g

• Only way to meet expected demand for protein and oil in aquafeeds is to find alternatives to fish meal and oil

World fish meal use in livestock andWorld fish meal use in livestock and fish feeds in 1994, 2000 & 2006

60

50

40Poultry

20

30 SwineAquacultureOther

10

20 Other

01994 20062000

World fish feed productionWorld fish feed production1995, 2000, and 2010 (predicted), , (p )

30 000 000

25,000,000

30,000,000

15 000 000

20,000,0001995

10,000,000

15,000,000 20002010

5,000,000

0Salmonids Shrimp Catfish Marine Cyprinids

Summary of demandy• Average annual production of fish meal will be

equal to demand in fish feed by:equal to demand in fish feed by:– 2015 at current incorporation levels in fish feeds

B t if l b l d ti f fi h l d d t El– But, if global production of fish meal decreases due to El Nino or wild stock collapses, demand may equal supply as soon as 2010

• More likely scenario– Global production is adequate until at least 2020Global production is adequate until at least 2020

if percentage use in fish feeds decreases– Fish meal will no longer be used as the mainFish meal will no longer be used as the main

protein source, but rather as a feed supplement• Palatability of plant-based feeds• Source of taurine, carnitine, and other compounds• Essential amino acid balance in plant-based feeds

Can carnivorous fish be converted to a vegetarian diet?

• Trout fed a fish meal-free diet grow about g7% slower and are 10% less efficient

• W t f l t fi h l f di t• We cannot formulate a fish meal-free diet without adding several limiting amino acids or adding rendered animal products

• Basing veggie trout feeds on plant proteinBasing veggie trout feeds on plant protein concentrate increases fiber (phytate) in diet, thereby contributing to pollutionthereby contributing to pollution

Similarities to past situation in ppoultry feeds

• In USA, poultry feeds are based on soybean meal dand corn

• In 1970s, however, poultry performance could not beIn 1970s, however, poultry performance could not be sustained without 3-5% fish meal in the formulation

• Research over decades showed that fish meal contained various micronutrients, mainly ultra-trace elements that were essential to chickenselements, that were essential to chickens

• When these were supplemented, fish meal could be ppeliminated from poultry feeds

Poultry feed analogy is not perfectPoultry feed analogy is not perfect

• Ultra-trace mineral aspect may not be relevant to aquatic animalsbe relevant to aquatic animals

• More likely the growth factors in y gfish meal are related to:

–Amino acid imbalances–Amino acid imbalances–Palatability

Bi ti d i fi h l d l t–Bioactive compounds in fish meal and plant proteins

Challenges in feed formulation-1Challenges in feed formulation 1

• Replacing fish meal with plant proteins• Replacing fish meal with plant proteins–Corn or wheat gluten, soybean meal, soy

t i t t l t i t tprotein concentrate, canola protein concentrate

• Emphasis is on maintaining digestible p g gprotein and limiting amino acid levels

• Fish meal contains bioactive compounds• Fish meal contains bioactive compounds–Gonads, nucleotides, others

• Oilseed meals contain bioactive compoundsh t t d th d– phytoestrogens and other compounds

Challenges in feed formulation-2Challenges in feed formulation 2

• Replacing fish oil with plant oils• Replacing fish oil with plant oils–Canola, soy, flaxseed and others

• Fatty acid profiles of fish reflect dietary fatty acid intakeacid intake

• Fish oil contains bioactive fatty acids–Long-chain omega-3s

• Plant oils contain relatively high amounts of• Plant oils contain relatively high amounts of linoleic acid (C18:2, n-6)

I t f ith 3–Interferes with omega-3s–Can lead to production of inflammatory factors

Nutritional pathologies caused by p g ychanging feed formulations

• Fin erosion

• Skeletal deformitiesPhosphorus deficiency but– Phosphorus deficiency, but

complicated etiology with ascorbic id th f t iblacid or other factor possibly

involved

• Enteritis in distal intestine– soybean meal and salmonids

Effect of diet on fin erosion ofEffect of diet on fin erosion of rainbow trout

Standard Experimentalfish-meal based feed krill meal based feed

Atlantic salmon with jaw deformity (Screamer)(Screamer)

“Screamer” at harvest

Skeletal deformities in Atlantic salmonS e eta de o t es t a t c sa o

NormalNormal rainbow trout

Atlantic salmon

Phosphorus deficient

screamer

deficient rainbow trouttrout

Skeletal deformitiesSkeletal deformities• Deformities in farmed Atlantic salmon

– Behind head (chicken head)

– Mid-dorsal area (humpy)

C d l ( t )– Caudal area (stumpy)

• Fish look normal at seawater transfer, but develop spinal deformities during grow-out and are downgraded at harvestg g g

• Examination of fry and fingerlings prior to seawater transfer shows abnormal vertebra

– Abnormalities not grossly apparent, but seen in X-ray imaging

• Cause is thought to be inadequate mineralization (low dietary P ith other factors in ol ed) at j enile stage follo ed b inj rwith other factors involved) at juvenile stage, followed by injury

during seawater transfer and size-grading

Skeletal deformities in Atlantic salmonS e eta de o t es t a t c sa o

Example of problems with plant t i b l d t itiproteins: soybean meal and enteritis

• Morphological abnormalities of intestinal villi• Morphological abnormalities of intestinal villi

• Appearance related to dietary soybean meal pp y ylevel and duration of feeding

• Can be induced in Atlantic salmon andCan be induced in Atlantic salmon and rainbow trout, but not in Atlantic cod

• Causative factors unknown but not present in soy protein concentrate

SBM-induced enteritis- Distal intestine histology

PROXIMAL SECTIONSTOMACH

PROXIMAL SECTIONSTOMACH

DISTAL DISTAL SECTIONPYLORIC

SECTIONSECTIONPYLORIC

SECTION

Soybean meal-induced enteritis after 24 weeksiControl vs. 40% SBM diet

Control diet: No SBM. Photo showing normal villi

40% SBM diet: Villi are swollen and those in thePhoto showing normal villi

of distal intestine (X 75)swollen and those in the center fused. Note numerous large apicalnumerous large apical vacuoles(X 75)

Summary of results in our laboratory

• Trout growth performance• higher on diets containing 20% soybean meal than 40% soybean meal

• Expression of immune factors• Tumor necrosis factor (TNF) expression elevated in fish fed ( ) p40% soybean meal

• No differences in IL-8 or CD-8 expression

• Soybean meal-induced enteritis– No evidence in any treatment after 12 weeks growth trialNo evidence in any treatment after 12 weeks growth trial

– No evidence in Control fish (0% SBM) after 24 weeks

Very low incidence in 20% SBM after 24 weeks– Very low incidence in 20% SBM after 24 weeks

– High incidence in 40% SBM after 24 weeks

Chapter 10: Nutritional Pathologyp gy

Note to Dr. Roberts:

Chapter will need to be expanded tobe expanded to address new pathological p gconditions caused by feed imbalances

Marine fish larval feedsMarine fish larval feeds• Four problems with microparticulate feeds• Four problems with microparticulate feeds

– Larval fish cannot swim to catch feedLarval fish often do not recognize feeds as food– Larval fish often do not recognize feeds as food

– Very small feed particles are susceptible to nutrient leaching

– Larval fish do not have fully developed digestive systems, so special forms of protein are required

Early fish larvaeEarly fish larvae

Copepod: live prey for larval fishCopepod: live prey for larval fish

Marine fish larval feedsMarine fish larval feeds• Larval fish cannot swim to catch feedLarval fish cannot swim to catch feed• Larval fish often do not recognize

feeds as foodfeeds as food

• One solution: add little glass balls to increase pellet buoyancy and reflect

t i l th f li ht th t thcertain wavelengths of light that the fish can detect

Making microparticulate feeds float and visible to larvaeby imbedding very small glass balls in the feedby imbedding very small glass balls in the feed

Halibut larvae with microparticulateHalibut larvae with microparticulate feed (containing glass balls) in gut

Marine fish larval feedsMarine fish larval feeds

• V ll f d ti l• Very small feed particles are susceptible to nutrient leachingp g

Regular feed Coated feed

Marine fish larval feedsMarine fish larval feedsLarval fish do not have fully developedLarval fish do not have fully developed

digestive systems, so special forms of protein are requiredp q

• Line of research: Look at gene expressionLine of research: Look at gene expression of digestive enzymes and transporter proteins in gut of developing larvaep g p g

– Pep1: peptide transporter expressed early in marine larvae

• Develop diets containing amino acids and peptides rather than intact proteinsp p p

Omega-3 fatty acid levels in farmed fish

Problem: How to maintain omega 3 fattyProblem: How to maintain omega-3 fatty acid levels in fillets when plant oils are used in the feed??used in the feed??

f• Line of research: Use diets that contain fish oil at the end of the grow-o t periodout period

• Genomic research: Look at expression levels of fatty acid desaturase enzymeslevels of fatty acid desaturase enzymes

Fillet eicosapentenoic acid (EPA) compositionue 400

500Canola to menhaden oilMenhaden to canola oil

dibl

e tis

su

300

400

100/

g ed

200 *

mg

EPA

1

100

* Denotes significant differences within dietary groups over time (One factor

m 0Initial 6 weeks

Denotes significant differences within dietary groups over time (One factor ANOVA, P<0.05).

Diet history: Previously fed oil source for 17 weeks then switched

Initial weight 807g. At the completion of the experiment fish had attained an average weight of 1118 g fish-1 with an SGR of 0.905 and an FCR of 1.17.

Fillet n3/n6 fatty acid ratio

4.0Canola to menhaden oil

y ac

ids

3.0

Canola to menhaden oilMenhaden to canola oil

*

3/n6

fatt

y

2.0

atio

of n

3

*1.0

Ra

0.0I i i l 6 kInitial 6 weeks

* Denotes significant differences within dietary groups over time (One factor ANOVA, P<0.05).

D-6 fatty acid desaturase expressionD 6 fatty acid desaturase expression

1 0E+09*

8.0E+08

1.0E+09Canola to menhaden oilMenhaden to canola oil

on

6.0E+08

Exp

ress

io

*4.0E+08

-MG

B E

0 0E+00

2.0E+08

d6FA

D-

0.0E+00Initial 6 weeks

Genomics in fish nutrition researchGenomics in fish nutrition research

• N t iti l i ( t i i )• Nutritional genomics (nutrigenomics) involves measuring expression of genes that

d t diff t di t f trespond to different dietary factors– Digestion, nutrient transport, metabolism, nutrient

partitioning, protein synthesis, protein turnover, and so on respond to nutritional inputs

Studying expression of regulatory genes in various– Studying expression of regulatory genes in various pathways will provide insight into physiological processes

• Nutrigenomics is relevant to growth, immune function reproduction and just aboutfunction, reproduction, and just about everything

Genomic toolsGenomic tools• Micro-arraysMicro-arrays

– Zebrafish array (genome fully sequenced)– GRASP chip for salmonids

• RT-PCR• Proteomics

Problem with existing microarraysHave to sort through up to 25,000 genes to connect the dots in

metabolic pathways of interest, plus not all genes of interest exist on available arrays

Strategy to utilize genomics and proteomics in fish nutritionproteomics in fish nutrition

• Conduct feeding studies• Conduct feeding studies

• Measure physiological responses and correlate these with changes in gene expression using existing micro-arrays

• Identify genes of interest, e.g., key regulatory genes

• Create groupings (panels) of key regulatory genes in• Create groupings (panels) of key regulatory genes in specific pathways of interest

• Incorporate panels into mini-arrays that allow us to design precise, targeted experiments to test specific hypotheseshypotheses

Mini-arrays solve the problem of y pgene expression overload

• Mini-arrays measure gene expression in key regulatory or rate-limiting enzymes that are up orregulatory or rate-limiting enzymes that are up or down regulated in specific metabolic pathways

• Additional panels being developed by others can be added to expand to stress, effects of pollutants, intestinal enzyme expression

• Mini arrays will simplify nutritional studies in fish• Mini-arrays will simplify nutritional studies in fish compared to using global microarrays

Food restriction in zebrafish(why we need mini-arrays)(why we need mini arrays)

Microarrays ALDOLASEFRUCTOSE-1,6-

BIPHOSPHATASEMicroarrays(gluconeogenesis)

LiverBraines PYRUVATEKINASELiverBrain

Gen

e

1041genes

5 genes

KINASE

MALATEgenesgenes MALATEDEHYDROGENASE

(glucogenesis)

In LiverSUCCINATEDEHYDROGENASE

Persistent organic pollutants (POPs)Persistent organic pollutants (POPs)

• PCBs, dioxin, etc. were present in fish oil and the residual oil in fish meal

• Wild fish from Baltic and North Sea are known to have much higher concentrations of POPs than products from the Pacific

• Industry now removes POPs by extracting residual oil from meal with isohexane, then treating with activated carbon (same for oil)

f• After treatment, meals and oils are below EU and EPA action limits

• Farmed salmon have lower POP levels than wild salmon• Farmed salmon have lower POP levels than wild salmon, especially those from Washington State

• All salmon have lower POP levels than English muffinsAll salmon have lower POP levels than English muffins

Farmed fish and contaminantsFarmed fish and contaminants

Farming fish is really the only hope to produce fish with reduced levels of POPss t educed e e s o O s

– Dietary inputs can be managed

– High percentage of diet is plant protein in contrast to diet of wild fish which cannot be

ll dcontrolled