The Potential of In Ovo Feeding and Early Nutrigenomic Programming
Peter R. Ferket N.C. State University
The Greek Philosopher's Genetic Theory “An individual’s traits are acquired from contact with the environment, and can be inherited by their offspring.”
You are Programmed to Succeed!
Poultry Breeding Based on Mendelian Genetics
Gene6cs assumes heritable traits are based DNA sequence
Mendel
85% of Improvement in Poultry Production Due to Genetics!
1956 2006 42 d, Live Wt, g 540 2805 6 wk Feed/Gain 2.35 1.70 Days to 1.8 kg +112 32
• >45 g weight/year • >1% increase/year (Havenstein et al., 2007)
Growth
Incuba\on Neonate Produc\ve Growth
Maintenance
In 1956, the incuba6on/neonate stage represented <25% of a broiler’s life
1956
Growth
Incuba\on Neonate Produc\ve Growth
Maintenance
But Now It is 50%!
Genetics changed the playing field…
…but expression of gene6c poten6al is what drives performance and profits
Nutrition has not kept pace with genetics
The time has come to
CLOSE THE GAP
Molecular Biology is Changing Our Understanding of Heritability
• Study of gene expression by up-‐regula\on of mRNA
• Study of Proteomics
• Study of Metabolonics
Epigenetics = “On-Genes”
Epigene6cs assumes heritable traits are based on gene expression
Lamarck
It’s the link between gene6cs and nutri6onal adapta6on!
Swarming Locusts
EpigeneticEvidence
• Swarming phenotype is environmentally influenced
• Trait is passed genera\on to genera\on.
Exposure to an adverse s\muli during cri\cal
periods of development can permanently
reprogram normal physiological responses.
Early Life Programming
can turn on “Thrifty” Genes
Diego V. Bohórquez©
Why the difference between these two genetically identical bees?
Diego V. Bohórquez©
The first meal makes the difference!
Synchronize in-‐ovo feeding when the embryo begins to imbibe amnio6c fluid
(“The Avian Embryo”, Romanoff 1960)
Op6mum IOF Window
• E17-‐E18 for Broilers • E23-‐24 for Turkeys
Volume of amnio6c fluid (ml)
Days 1 3 5 7 9 11 13 15 17 19 21
8
6
4
2
IOF
• Administration of highly digestible nutrients (CHO, Protein, Minerals, Vitamins, etc. ) into the amnion of late term embryos
• Benefits: – Advanced gut development – Increased glycogen reserves – Advanced muscle development – Increase growth performance – Better humeral and innate immunity – Improved chick or poult quality – Changes gene expression
Uni and Ferket, 2003. US Patent No. 6592878.
Jejunum Cross-Sections 20 Day-old Broiler Embryo
Control
In Ovo-‐Fed* * 1 ml. Saline containing 10% Maltose + 10% Sucrose + 5% Dextran
Control
IOF
Turkey Embryo Jejunum 25 Days of Incubation
Oliveira et al. (2008)
Turkey Embryo Jejunum At Hatch
Control
IOF
Oliveira et al. (2008)
Control In-ovo Fed
Bohorquez et al. (2009)
Control In-‐ovo Fed
IOF advances villus development and func6on
of hatchling poults BohÓrqez et al. (2009)
In Ovo Feeding Increases Liver Glycogen
1 ml of Primary IOF solution given at 17 E
18E 19E 20E Hatch
Days before hatch
0
4
8
12
16
20
Glycogen
conten
t (mg/g o
f wet tissue
)
Control In-‐Ovo Fed
A A
A
A
A A
BB
18E 19E 20E HatchDays before hatch
0
4
8
12
16
20
Glycogen
conten
t (mg/g o
f wet tissue
)
Control In-‐Ovo
A A
A
B
A
A
B
B
Cobb Ross
In Ovo Feeding Increases Liver Glycogen Content in Poults
mg
Gly
coge
n
b
b
a
a
NC STATE UNIVERSITY
Oliveira et al. (2008)
Day 3 Hatch
0 7E-‐8E 16E 21E
Embryonic myoblasts
Fetal myoblasts
Adult myoblasts-‐ Satellite cells
Day 8
in-ovo feeding
In ovo Feeding Timed to Boost 2nd and 3rd wave of satellite cell development
Skeletal Muscle Satellite Cells (Thymidine incorporation - CPM)
7 d of age 3 d of age Broiler Chicks 310 16,803 Non-‐injected 1,524 29,453 Commercial IOF 492% 175% ∆
Kornasio et al. (2011) PSJ
Control IOF
Hematoxylin & Eosin X40
Breast Muscle Fibers in 35d Broilers
Kornasio et al. (2011) PSJ
Effect of In Ovo Feeding on Body and Breast Muscle Weights (g) of Ross Broilers
Day 25 Day 10 Day of Hatch
In Ovo Control In Ovo Control In Ovo Control
997* 943 254* 243 47.0* 45.3 BW (g)
+ 5.7 + 4.2 +3.7 BW Diff. (%)
13.0* 12.0 12.3* 11.4 2.05* 1.93 Breast muscle % of BW
+8.3 +5.2 +6.2 Diff. (%) in Breast Muscle
• 1ml of Primary IOF solu6on given at 17 E. • Age of breeding flock:35 weeks
Uni et al. (2005)
Effect of IOF and Post-Hatch Feeding Time on Cobb (500)
Post-‐Hatch Fas6ng Time
In Ovo Treatment
2 d BW, g 35 d BW, g 0-‐35 d FCR
36 hr (SP) Control 38.7 1,995 1.63 36 hr (SP) IOF 42.0 2,209 1.69 6 hr (EF) Control 63.7 2,374 1.72 6 hr (EF) IOF 63.7 2,272 1.60 Source of Varia6on -‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐ (P-‐Value) -‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
Post-‐Hatch Fas\ng Time .0001 .0001 .974 In Ovo Treatment .0005 .185 .514 Feeding X IOF .0005 .001 .0485
(Kornasio et al., 2011)
Effect of IOF and Post-Hatch Feeding Time on Muscle
Development of Cobb (500)
(Kornasio et al., 2011)
Effect of IOF and Post-
Hatch Feeding Time on
Breast Muscle of Cobb (500)
(Kornasio et al., 2011)
• Administration of highly digestible nutrients (CHO, Protein, Minerals, Vitamins, etc. ) into the amnion of late term embryos
• Benefits: – Advanced gut development – Increased glycogen reserves – Advanced muscle development – Increase growth performance – Better humeral and innate immunity – Improved chick or poult quality – Changes gene expression
Uni and Ferket, 2003. US Patent No. 6592878.
Effect of IOF on Bursa of Fabricius Development
• IOF-treated chicks have larger bursa
• Bursa has more and larger follicles
• =more B cells ready to produce antibodies
∆
Comm.IOF
Non
Injected
+20.4%
0.177
0.147
Bursa to
Body weight
+18.2%
447
379
Follicle No. at
hatch
Uni et al. (2008)
Control In-ovo Fed
Bohorquez et al. (2009)
Control In-ovo Fed
Bohorquez et al. (2009)
Mucin gene expression (MUC 2) in Chicks
Smirnov et al. (2006)
Control versus IOF:
Effect of Mineral Enrichment by IOF on Skeletal Development of Broilers
Control IOF Enriched (at E 18)
Amount per embryo Mineral / Vitamin concentration in the IOF solution
1.071mg Phosphorus (0.18%)
0.974mg Organic Iron (Bioplex) 0.16%
0.591mg Organic Zinc (Bioplex) 0.10%
0.36mg Organic Manganese (Bioplex) 0.06%
0.36mg Organic Calcium (Bioplex) 0.06%
0.018mg Organic Copper (Bioplex) 0.003%
136.8IU Vitamin D3 0.001%
(Yair and Uni, 2011)
Levels and uptake of minerals from the yolk in control and in ovo mineral
enrichment
(Yair and Uni, 2011)
Effect of IOF of Organic Minerals on Tibia Mechanical Properties of Broilers
The enrichment resulted in improvement in the mechanical properties of tibiae.
(Yair and Uni, 2011)
Blue=down regulated
Red=up regulated
Method = Ward
HBZ
HOXA3
CTGF
PDK1
SOD1
MYBPC3
TOP2B
SM22
AZIN1
TLR4
HSPE1
HB alphaA
EDNRA
ANF / ANP
HBE
IGFBP1
EFNB2
Tenascin precursor
Signal transducer and activator of transcription 3
Beta-actinCytoskeletal beta actin
GAPDH
CHICK DNA
TURKEY DNA
fructose biphosphatase (F1,6 BPase)
transketolase
creatine kinase
aconitate hydratase
malate dehydrogenase
maltase-glucoamylase
glutamate dehydrogenase
glyceraldehyde 3-phosphate dehydrogenase
growth hormone receptorphosphopyruvate hydratase
glycogen synthase 1 (muscle)
isocitrate dehydrogenase (NADP+)
carnitine O-palmitoyltransferasegrowth hormone receptor (GHR)
succinate dehydrogenase
EF2
CHICK DNATURKEY DNAaminopeptidase Ey
Std
Lsm
ean
Day
25
Std
Lsm
ean
Day
26
Std
Lsm
ean
Day
28
Std
Lsm
ean
Trt n
o
Std
Lsm
ean
Trt y
e
Std
Lsm
ean
Trt*
Day
no
25
Std
Lsm
ean
Trt*
Day
no
26
Std
Lsm
ean
Trt*
Day
no
28
Std
Lsm
ean
Trt*
Day
ye
25
Std
Lsm
ean
Trt*
Day
ye
26
Std
Lsm
ean
Trt*
Day
ye
28
Dendrogram
Hierarchical Clustering of Standardized LSMeans
Genes with similar pattern were grouped in the same cluster/color
43/120 genes expressed differently over \me by IOF
Oliveira et al. (2008)
Hori zontal reference l ine drawn at -log10(p ) = 4.01Frame Si ze for Gra phs BelowMarker Size for Graph s Below
0
10
-log1
0(p-
value
) for
Diff
of T
rt =
(CO
N)-(I
OF)
HOXA3
PA
MYL4
PLB
HIF1AACTA2
IFN? HMOX1
MYL9
EGFr
IGFBP1TLR2 -2IL13
Prepro endothelin -1
LEREPO4 protein - simila r
IL-8
FasL
creatin e ki nase
maltase-g lucoamylasehexokinase 1
fructose b iph osphate al dolase B
glycogen synthase kin ase-3 beta
aminopep ti dase
tyrosinase
F_actin
CDK1
14_3_3
-0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5Diff of Trt = (CON)-(IOF)
Diff of Trt = (CON)- (IOF)
Diff of treatment: Control -‐ IOF
Fold difference: 4.0 -‐ Log
10 (P-‐value
) of D
iff
Bohorqez et al. (2009)
• EGFr • ILGF-BP1 • Thyroid receptors α1 & β1
Epithelial growth
• IL-8 • TLR2 • Fas-Ligand
Innate Immunity
• Maltase-glucoamylase • Glycogen synthase kinase • Aminopeptidase 1
CHO and Protein Metabolism
• F-actin capping protein • Myosin light-chain 4 & 8
Smooth Muscle Development
Bohorqez et al. (2009)
Gene Expression Patterns: The Powerful Tool of Transcriptomics Decoding nutri6onal control: Nutrigenomics
Bridging the gap between
genetics and nutrition
Early nutri6on can imprint desired metabolic traits.
Imprint for the desired trait when they are young
Nutritional Imprinting
What Production Traits Can Be Imprinted by Adaptive Conditioning of Gene Expression?
• Tolerance to immunological stress • Tolerance to environmental stress • Tolerance to oxida\ve stress • Energy u\liza\on and requirement • Mineral u\liza\on and requirement
It is not just what we feed, but also when we feed it!
The first 90 hours of the chicks life
determines a bird’s ability to use specific nutrients
Nutritional Conditioning
Heat map shows differential gene expression patterns in intestinal tissues from broilers at 21 days
Control Condi6oned Uncondi6oned
The conditioned birds have totally different physiological baseline
Effects of the Programmed Nutrition (PN) Strategy on Ca
and P Digestion in Broilers Diet Ca Diges6on (%) P Diges6on (%) Corn-‐soy control diet 23.9b 23.1c
PN supplements without condi\oning 28.6b 28.8b
Complete PN strategy 44.6a 39.7a
Alltech-‐UK Nutri\on Research Alliance (2011)
Enhanced Mineral Retention a Using PN Strategy Mineral Content in Breast Muscle
Mineral content Control Alltech PN diet Calcium (ppm) 41 44 Selenium (ppb) 120 332 Zinc (ppm) 3.04 3.03 Manganese (ppb) 129 157 Copper (ppb) 513 457 Iron (ppm) 3.31 3.58
Note: PN diets contain up to 50% less Zn, Mn, and Cu
(Data from Alltech-‐UK Nutri\on Research Alliance, 2011)
Improved Meat
Quality Item Standard
diet Programmed Nutri6on
Total an\oxidant capacity (µMol)
1.76 1.85
Color (red index) 9.12 9.77 Drip loss (%) 2.19 1.32 pH 6.03 6.15
Oxidative Stability: Freshness
Quant et al. (2011) Alltech-‐University of Kentucky Nutri8on Research Alliance, Lexington, KY
Rancidity
Programmed Nutri6on
Control
Breast Filet Cooking Characteristics
Cooking characteris6cs Diet Cooking loss, % Control 17.05 Programmed Nutri\on 15.27 SEM 0.92
Potential Benefits of Nutrigenomic Programming • Nutrient U\liza\on • Performance efficiency • Disease Resistance • Meat yield and quality • Reproduc\ve efficiency • Enteric ecosystem • Behavior
This is only the beginning!
It is the next Step Forward in Nutrition.