The Potential of In Ovo Feeding and

Early Nutrigenomic Programming

Peter R. Ferket
N.C. State University

The Greek
Philosopher's
Genetic Theory
An individuals traits are
acquired from contact with
the environment, and can
be inherited by their
ospring.

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
+112

1.70
32

Days to 1.8 kg

>45 g weight/year
>1% increase/year
(Havenstein et al., 2007)

In 1956, the incuba6on/neonate stage

represented <25% of a broilers life
Growth

Maintenance

1956

Incuba\on Neonate Produc\ve Growth

But Now
Growth

It is 50%!
Maintenance

Incuba\on Neonate Produc\ve Growth

Genetics
changed
the playing
field
but expression of gene6c poten6al is
what drives performance and prots

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

Lamarck

Epigene6cs assumes heritable

traits are based on gene expression

Its the link between gene6cs

and nutri6onal adapta6on!

Swarming
Locusts

Epigenetic
Evidence
Swarming phenotype is environmentally inuenced
Trait is passed genera\on to genera\on.

Early Life
Programming
can turn on
Thrifty Genes
Exposure to an adverse
s\muli during cri\cal
periods of development
can permanently
reprogram normal
physiological responses.

Why the difference between these

two genetically identical bees?

Diego V. Bohrquez

The first meal makes the

difference!

Diego V. Bohrquez

Synchronize in-ovo feeding when the

embryo begins to imbibe amnio6c uid
(The Avian Embryo, Romano 1960)

Volume of amnio6c
uid (ml)
Op6mum IOF Window

IOF

E17-E18 for Broilers

E23-24 for Turkeys

8
6
4
2

Days
1

11

13

15

17

19

21

 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

Turkey Embryo Jejunum

25 Days of Incubation
Control

IOF
Oliveira et al. (2008)

Turkey Embryo Jejunum At Hatch

Control

IOF
Oliveira et al. (2008)

Control

Bohorquez et al. (2009)

In-ovo Fed

Control

In-ovo Fed

IOF advances villus

development and func6on
of hatchling poults
Bohrqez et al. (2009)

In Ovo Feeding Increases Liver Glycogen

Ross

Cobb
20

16

12
B

Glycogen content (mg/g of wet tissue)

Glycogen content (mg/g of wet tissue)

20

16

12

B
A

18E

19E

20E

Hatch

Days before hatch

Control

In-Ovo

18E

19E

20E

Hatch

Days before hatch

Control

In-Ovo Fed

1 ml of Primary IOF solution given at 17 E

NC STATE UNIVERSITY

In Ovo Feeding Increases Liver

Glycogen Content in Poults
mg Glycogen

Oliveira et al. (2008)

In ovo Feeding Timed to Boost 2nd and

3rd wave of satellite cell development
Adult myoblasts-
Satellite cells
Fetal myoblasts
Embryonic
myoblasts

7E-8E

in-ovo
feeding

16E

21E
Hatch

Day 3

Day 8

Skeletal Muscle Satellite Cells

(Thymidine incorporation - CPM)
Broiler Chicks
Non-injected
Commercial IOF

Kornasio et al. (2011) PSJ

3 d of age
16,803
29,453
175%

7 d of age
310
1,524
492%

Breast Muscle Fibers in

35d Broilers
Control

IOF

Hematoxylin & Eosin X40

Kornasio et al. (2011) PSJ

Effect of In Ovo Feeding on Body and Breast

Muscle Weights (g) of Ross Broilers
Day of Hatch
BW (g)

Di. (%) in Breast

Muscle

Day 25

Control

In Ovo

Control

In Ovo

Control

In Ovo

45.3

47.0*

243

254*

943

997*

+3.7

BW Di. (%)
Breast muscle %
of BW

Day 10

1.93

2.05*

+ 4.2
11.4 12.3*

+6.2

+ 5.7
12.0

+5.2

13.0*
+8.3

1ml of Primary IOF solu6on given at 17 E.

Age of breeding ock:35 weeks

Uni et al. (2005)

Effect of IOF and Post-Hatch

Feeding Time on Cobb (500)
Post-Hatch
In Ovo
2 d BW, g 35 d BW, g 0-35 d FCR
Fas6ng Time Treatment
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 PostHatch 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

Non

Injected

Comm.
IOF

Bursa
to
Body
weight

0.147

0.177

+20.4%

Follicle
No.
at
hatch

379

447

+18.2%

Uni et al. (2008)

Control

Bohorquez et al. (2009)

In-ovo Fed

Control

Bohorquez et al. (2009)

In-ovo Fed

Mucin gene
expression
(MUC 2) in
Chicks

Control versus IOF:

Smirnov et al. (2006)

Eect of Mineral Enrichment by IOF on

Skeletal Development of Broilers
Control

Mineral / Vitamin
concentration in the IOF solution
Phosphorus (0.18%)

IOF Enriched (at E 18)

Amount per embryo

1.071mg

Organic Iron (Bioplex) 0.16%

0.974mg

Organic Zinc (Bioplex) 0.10%

0.591mg

Organic Manganese (Bioplex) 0.06%

0.36mg

Organic Calcium (Bioplex) 0.06%

0.36mg

Organic Copper (Bioplex) 0.003%

0.018mg

Vitamin D3 0.001%

136.8IU

(Yair and Uni, 2011)

Levels and uptake of minerals from

the yolk in control and in ovo mineral
enrichment

(Yair and Uni, 2011)

Eect 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)

Method = Ward

Dendrogram
HBZ
AZIN1
PDK1
TOP2B
EDNRA
TLR4
Signal transducer and activator of transcription 3
MYBPC3
SM22
IGFBP1
glyceraldehyde 3-phosphate dehydrogenase
glycogen synthase 1 (muscle)
HOXA3
TURKEY DNA
Tenascin precursor
CHICK DNA
CHICK DNA
TURKEY DNA
aminopeptidase Ey
ANF / ANP
creatine kinase
malate dehydrogenase
transketolase
maltase-glucoamylase
HB alphaA
HBE
growth hormone receptor (GHR)
carnitine O-palmitoyltransferase
GAPDH
growth hormone receptor
phosphopyruvate hydratase
CTGF
SOD1
glutamate dehydrogenase
isocitrate dehydrogenase (NADP+)
fructose biphosphatase (F1,6 BPase)
aconitate hydratase
EF2
HSPE1
succinate dehydrogenase
EFNB2
Beta-actin
Cytoskeletal beta actin

Blue=down regulated
Red=up regulated

StdLsmean Trt*Day no 28

StdLsmean Trt*Day ye 28

StdLsmean Day 28

StdLsmean Trt*Day ye 26

StdLsmean Trt ye

StdLsmean Trt*Day no 26

StdLsmean Trt no

StdLsmean Day 26

StdLsmean Trt*Day ye 25

StdLsmean Trt*Day no 25

StdLsmean Day 25

43/120 genes
expressed
dierently over
\me by IOF
Genes with similar pattern
were grouped in the same
cluster/color
Oliveira et al. (2008)

Hori zontal reference l ine drawn at -log10(p ) = 4.01

F rame Si ze for G ra phs Below
Marker Size for Graph s Below

Diff of Trt = ( CON)- (IOF)

EGF r
10

-log10(p-value) for
Diff of Trt = (CON)-(IOF)

- Log10 (P-value) of Di

tyrosinase

CD K1

maltase-g lucoamylase
hexokinase 1
fructose b iph osphate al dolase B

ACT A2

PA

IL-8
HIF1A
IGF BP1
Prepro endothelin -1
aminopep ti T
dase
LR2 -2
IL13
IFN? PLB
LEREPO4
protein
- simila r
F asL
glycogen
synthase kin ase-3 beta

F _actin
MYL4

creatin e ki nase
MYL9
14_3_3

HMOX1

HOXA3

Fold dierence: 4.0

-0.5

-0.4

Bohorqez et al. (2009)

-0.3

-0.2

-0.1

0.1

0.2

Di of treatment: Control - IOF

Diff of Trt = (CON)-(IOF)

0.3

0.4

0.5

Epithelial growth

EGFr
ILGF-BP1
Thyroid receptors 1 & 1

Innate Immunity

IL-8
TLR2
Fas-Ligand

CHO and Protein

Metabolism

Maltase-glucoamylase
Glycogen synthase kinase
Aminopeptidase 1

Smooth Muscle
Development

Bohorqez et al. (2009)

F-actin capping protein

Myosin light-chain 4 & 8

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.

Nutritional
Imprinting
Imprint for the
desired trait when
they are young

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!

Nutritional Conditioning
The first

90 hours of the chicks life

determines a birds ability

to use specic nutrients

Heat map shows differential gene

expression patterns in intestinal
tissues from broilers at 21 days

Control

Condi6oned

Uncondi6oned

The conditioned birds have totally dierent 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
Calcium (ppm)
Selenium (ppb)
Zinc (ppm)
Manganese (ppb)
Copper (ppb)
Iron (ppm)

Control
41
120
3.04
129
513
3.31

Alltech PN diet
44
332
3.03
157
457
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 (Mol)
Color (red index)

1.76

1.85

9.12

Drip loss (%)

2.19

pH

6.03

9.77
1.32
6.15

Rancidity

Oxidative Stability: Freshness

Control

Programmed
Nutri6on

Quant et al. (2011)

Alltech-University of Kentucky Nutri8on Research Alliance, Lexington, KY

Breast Filet Cooking

Characteristics
Cooking characteris6cs
Diet
Control
Programmed Nutri\on
SEM

Cooking loss, %
17.05
15.27
0.92

Potential Benefits of
Nutrigenomic Programming
Nutrient U\liza\on
Performance eciency
Disease Resistance
Meat yield and quality
Reproduc\ve eciency
Enteric ecosystem
Behavior

This is only the

beginning!
It is the next Step
Forward in Nutrition.