3 Embryonic Development and Fundamentals of Incubation

Royal Pas Reform
Embryonic development
and fundamentals
of incubation
Contents
• Introduction
• Embryonic development during egg formation
• Embryonic development during incubation
• Fundamentals of incubation
(1 out of 5 not discussed!)
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Introduction
Introduction
Reflection on topic of presentation
Incubator management and parameters to

achieve the physiological embryo needs in
all stages of the embryonic development
• Practical → day-to-day incubation management
• Theoretical → fundamental points of incubation
• Embryo → support during different stages

Introduction
Good hatchery results is your goal!
But what is good?
• High hatchability
• Good chick quality
• Satisfied customers
Introduction
Good hatchery results is your goal!
What do we need to get good results?
1. Good quality hatching eggs
2. Optimal incubation conditions
3. Good management (knowledge & experience)

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Development during egg
formation (partially also discussed during “egg
quality and storage”)
Embryonic development during egg formation
Good quality hatching egg!

External Internal Other aspects
Shell intact, smooth and clean Fertile Nutritional aspects
Uniform weight, not Embryo at optimum Free of diseases (Se, Mg,

too big or too small stage of development etc)
Oval shape, sharp end down “Fresh” and properly Maternal antibodies
stored
Heredity (sexing
errors!)
Ovary and oviduct
Van Nassauw, 1997 Unfertilized oocyte

Embryonic development during

egg formation
Ovulation: release
unfertilized oocyte in
the infundibulum
< 15 minutes + 24 hours
at
41°C
=
Fertilization: fusion 105.8 °F
of sperm and oocyte:
one-cell embryo
(sperm storage tubules)
Development in the oviduct
First cleavage divisions

after fertilization
Development in the oviduct
Development of the head-to-tail axis

during formation of the egg shell
Development
continues
Embryonic stage at oviposition

Stage IX-X: (Ref; Eyal-Giladi 1976)
Embryonic stage after cooling to

room temperature
Stage XII-XIII: (Ref; Eyal-Giladi 1976)
Ref: Gilbert , 2006

Adaptation period = first hours after laying

(= oviposition)
• Cuticle is drying
• Air cell is being formed
• CO2 diffuses out of the eggs → pH gradient
• Blastoderm continues to develop as long as internal
egg temperature is > 25 °C / 77 °F
CO2
Air cell
Fertile eggs; can these be stored?

Diapause:
• The blastoderm displays the unique ability
to undergo developmental arrest at low
temperatures in a process called
“embryonic diapause”
• Very functional for natural incubation
• Also very practical for poultry industry:
– Storage of hatching eggs
– Export of hatching eggs
• Embryo has to be in correct stage to be
able to continue normal development after
storage (“diapause developmental window”)
Diapause
• Temporary arrested development
• When egg content is below 22 °C / 71.7 ° F
• Storage resistant phase
• Optimal storage stages are XII-XIII.
– Slower of quicker cooling down rates are
influencing the embryonic stage
Good quality hatching egg!
The good quality hatching egg contains an

embryo that:
• Is in the optimum stage of development;
• Contains 30.000 - 60.000 embryonic cells
• Differentiation has already started (tail-to-head structure)
Photos: courtesy of dr. M. Boerjan

The unincubated fertile and infertile egg
Fertile Fertile Fertile, Infertile

too far developed
The unincubated fertile and infertile egg
Fertile Infertile
An egg is not just an egg!

• Blastodisc or embryo?
• 30% yolk / 60% albumen
• 10% shell
In the embryo we recognize of 2 different cell-types

1. The area pellucida forms embryonic tissues
2. The area opaca develops to extra-embryonic
tissues
P A
P A
Area opaca Area pellucida Area Area

opaca pellucida
Differentiation starts in the hen

• 1-cell embryo → multi-cell embryo with differentiated
cells (tissues/organs)
• Starts in oviduct (and continues during incubation)
• Importance of undisrupted egg formation (rotation) →
contact with nutriënts
Pre-incubation egg handling
Will these un-incubated eggs give good

results?
1 2 3 4
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Development during
incubation
Embryonic development of the chick
Phases in development
Single-stage incubation as well as multi-stage
incubation provide the climate conditions to support
each of the critical embryonic phases optimally
1 22 33
Phase Period
1 Differentiation In the hen; first week setter
2 Growth Second week setter
3 Maturation Last days setter + hatcher + house
1 22 33
Phase Period
1 Differentiation Different organs develop
2 Growth Exponential increase of metabolism
3 Maturation Maturaturation of physiological control circuits
1 22 33
Start of incubation
Embryonic cells migrate

(arrow) after embryonic
temperature reached
incubation temperature!!
Gilbert, 2006
Migration of cells
Claudio D. Stern (2004): Gastrulation, from cells to embryo

Start of incubation
At start incubation, (embryo) temperature increases
and development of the blastoderm continues!!
Beyond point of no return!

Formation of sub-embryonic fluid
1 (0-24 h) day 3 6th day
Redistribution of water from albumen to the area below embryo

Extra embryonic tissues

• Yolk sac membrane (area
vasculosa): nutrition
• Chorion-allantois
membrane: gas-exchange
• Amnion cavity: protection,
support of embryonic
movements, transfer maternal
proteins (immunoglobulin)
• Allantois: collection metabolic
waste (urates etc.) Ref: Wolpert : Principles of development 4th edition
Subembryonic fluid changes

Extra-embryonic tissues and compartments is essential

for optimum nutrition and growth of the embryo
Baggott, Glenn K. (2001) Development of extra-embryonic membranes and fluid compartments. In: Deeming, D.C. (ed.)
Perspectives in Fertilisation and Embryonic Development in Poultry. Lincolnshire
Embryo complete; rapid growth
10th day 13
dayth day
3
th day
616 th day
Exponential increase of metabolic heat production until plateau phase

Maturation of physiological control

systems
Embryo becomes responsive
to external signals (heat
and cold stress; CO2 etc.)
• Thermo-regulation
• Digestive tract
• Hatching muscle
10th day
• Mobilization
day 3 of glycogen
6th day
19-20th day
Hatching phase: maturation of

lungs
Royal Pas Reform
Fundamentals of incubation
Incubation management supports the different phases
of embryonic AND extra-embryonic tissue development
1 2 3
---differentiation-----------growth------------------maturation
2 3
5.
1. Temperature: embryo temperature
H
2. Turning: embryo growth Y
G
I
3. Ventilation: supply O2; release CO2/H2O; air flow
E
N
4. Humidity: egg weight loss E
!!!
2 3
Biology & technology in balance

Single-stage as well as multi-stage incubation should
provide the conditions to support each of the critical
embryonic phases optimally
2 3
Fundamentals of incubation: temperature
Temperature
• Embryo temperature
• Egg shell temperature (EST)
• Air temperature
= air close to sensor
• Incubator temperature
= set point
2 3
Egg shell temperature profile

Joseph (2006) and Hulet (2007)showed that for optimum
development the embryo (egg shell) temperature should
follow a natural pattern
Average egg shell temperature (°F)

Hulet, et al (2007)
Joseph, et al (2006)
Day of incubation
2 3
Egg shell temperature: 0 – 10 days

Egg shell temperature regimes from day 0 to day 10:
post-hatch performance (Joseph et al. ,2006)
Egg shell temperature Weight (g) Weight (g) FCR

0 – 10 days day 7 day 42 (corr. 2kg)
Low 98.0 °F 138 3014* 1.36
(36.7 °C)
Control 100.0 °F 144 3103 1.35
(37.9 °C)
* P<0.05;n=156 (13 cages of 12 chicks) Joseph, et al (2006): Poultry Science 85:932
2 3
Egg shell temperature: 16 – 21 days

Egg shell temperature regimes from day 16 to day 21:
post-hatch performance (Hulet, 2007)
Factors Low Control High

99.5 °F (37.5 °C) 101.5 °F (38.6 °C) 103.5°F (39.7 °C)
BW (g) 44 days 2213 2263* 2166*
FCR 21 days 1.56 1.55 1.60*
FCR 44 days (corr. 2kg) 1.82 1.75 1.80
Mort. (%) 21 days 2.2 2.6 4.0
Hulet (2007): Poultry Science 86:1017
* P<0.05;n=272 (8 pens of 34 chicks)
2 3
“EST” is leading parameter
101,5
Incubator (air)
temperature supports 101
the development from 100,5

a poikilotherm embryo
to a partial homeo- 100
therm embryo/chick 99,5
99
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
mean maximum mean minimum
2 3
… but how to measure EST?

• Different devices give different result!
• In running setter or not?
• Not on aircell!
• Where on tray/trolley? Look for coldest/warmest egg?
2 3
Different devices compared
2 3
… and how to interprete?
100 °F?
2 3
Fast increase of metabolic heat > day 12

Broiler embryos produce more heat than layer embryos
Janke, Boerjan, Tzschentke, 2004
2 3
2 methods to “manage” egg shell temperature

100 102
90
• Single-stage incubation
101
80
70 100
– Regular down-ward adjustment

60
99
50
98
of temperature set point

40
30 97
20
96
10
0 95
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
• Multi-stage incubation
– Fixed temperature set point
– Metabolic heat transfer from
“old hot” eggs to “new cold”
eggs
2 methods to “manage” egg shell temperature
Egg shell temperature MS
Egg shell temperature SS
Set point temperature MS
temperature program SS
Multistage versus single-stage

Hatchabilities in single-stage (SS) incubator
compared to multistage (MS)incubator
(data hatchery Honduras)
% Hatchability of first quality chicks

86 SS
85
M…
84
83
%
82
81
80
79
78
May June July

Month
Maturation of thermoregulation
Maturation phase starts when body temperature is
constant (plateau-phase)
pipping
Janke, Boerjan, Tzschentke, 2004
2 3
Maturation of thermoregulation
• The hatchling develops Temperature in
Preferred ambient temperature °C

the capability to control hatcher:
34.5 and 37.5 °C
its body temperature
• Hatcher climate defines

set points of body
temperature
• A typical example of
epigenetic adaptation Day at farm
Tzschentke et al, 2004
2 3
Fundamentals of incubation: temperature → relation to daily practice
Critical phases for temperature

Phase Points of attention in relation to temperature
1 Differentiation Avoid cold spots → hatch window
2 Growth Be aware of risk of overheating
3 Maturation Hatcher climate defines set point of body temperature
1 22 33
Consequences 1
• Uniform start of incubation
• Accurate and uniform temperature control
• Prevent cold spots from activated humidifiers
• Breed specific temperature set points
Tem perature setpoints
102,0
101,5
101,0
100,5
tem perature (F)
100,0
99,5
99,0
98,5
98,0
97,5
0 2 4 6 8 10 12 14 16 18 20
days
broiler white layer brown layer egg shell temperature

Consequences 2
• ! RH and air speed influence the relation incubator (=
air) temperature and egg shell temperature
• ! Different types of setter need different set points of air
temperature
2. Turning: embryo growth
4. Humidity: egg weight loss
2 3
Fundamentals of incubation: turning
Why turning?
• Prevents adhesion of embryo to inner
shell membrane
• Stimulates rate of sub-embryonic
fluid formation
• Stimulates rate of development of
area vasculosa and chorio-allantois
• Allows normal transfer of albumen
proteins into amniotic fluid
• Promotes growth of embryo
• Reduces incidence of malpositions
2 3
Turning and embryo growth

Turning during the first 7 days of incubation promotes
development in the setter
turned
Not
turned
Deeming et al, 1967
2 3
Turning and hatchability (& growth)

Critical period period: 3 – 7 days
Day turned Hatchability (%) Embryo mass at 16

days (g)
0 – 21 78.6 18.5
3–7 76.9 17.0
7 – 21 64.3 16.5
0–3 64.3 15.6
0 – 3 & 7 - 21 42.9 15.2
Ref. Deeming (2002) in: practical aspects of
commercial Incubation in Poultry.
2 3 Ratite Conference Books
Turning frequency & duration

Summary table: ’The earlier (8-10-12-14 days)
turning is stopped the greater the benefit of 96x
versus 24x turning/day, but only for old flocks’
Elibol and Brake - 2006 Poultry Science 85:1498
2 3
Turning duration
‘… egg turning required during incubation until day 12,
at least, and should not be stopped until after day 15’
Tona et al, 2005 Poultry Science 84:315
2 3
Turning duration
Long-term experiment at hatchery customer USA 2018
2 3
Fundamentals of incubation: turning → relation to daily practice
Turning
Angle:
• Optimum turning angle 45 °
• Range 40 – 45 ° (big eggs / air flow)
Frequency:
• General concencus 1 x/hour
• Higher frequency ‘might’ benefit
Duration:
• Until transfer
• Horizontal for better air flow? From embryo age 16.00 onwards
2 3
Fundamentals of incubation: turning → relation to daily practice
Turning
• Check (for sure during 1st week)
• Measure the angle, do not assume!
• Know the symptoms (open CAM; sticky chicks)
2 3
2. Turning: embryo growth
4. Humidity: egg weight loss
2 3
Fundamentals of incubation: ventilation & humidity
Function of ventilation?
• Creating air flow
(uniformity)
• Supplying fresh air
(= O2)
• Releasing ‘dirty’ air
(= CO2 and H2O)
• Cooling
Link with RH% for

optimal egg weight
loss 2 3
Ventilation for O2-supply

Increasing demand for O2
50
45 Ross 308
Ross 508
40
Oxygen consumption (ml*h-1)
Leghorn
35 Broiler (Tullett & Deeming 1982)
Broiler (Burton & Tullett 1983)
30 Blue North Holland (Romijn & Lokhorst 1960)
25
20
15
10
5
0
8 10 12 14 16 18 20 22
Incubation time (d) Janke, Boerjan, Tzschentke, 2004
2 3
Ventilation = air refreshment

O2= essential
CO2 + H2O = waste products from embryo metabolism
O2 CO2 H2O
l/hr per 1000 eggs
O2
CO2
Incubation day
After Romanoff A.L. 1960: The Avian Embryo
2 3
Non-ventilation
Does embryo need high CO2 at start?

• Why (not)?
• Other effects on non-ventilation during 1st period in
setter?
• So what to do with damper?
Modern broiler embryos have shown to be tolerant to higher

levels of CO2:
• Upto 1.4% due to non-ventilation during the first 10 days
• Upto 4% (experimental) between days 13-18
De Smit et al., 2006; Everaert 2009
2 3
Humidity balance in setter
Air Nozzles
Eggs Leaks Air
Washing
Coming in by: Going out by:

Ventilation = dehumidification
RH% in setter determines weight loss pattern
2 3
Ventilation = dehumidification
16 gr H20/kg
Inlet air
should be
‘dry’ enough
to allow egg
weight loss
Example: 2 3
30 °C / 75 %RH
De-humidifying
= 20 gr water/kg air a must for
weight loss
Relevance of egg weight loss

• Optimum (natural) egg weight loss → approx. 12%
• Optimum mineral balance in embryonic compartments
• Optimum chick quality
2 3
Fundamentals of incubation: ventilation & humiliatie → relation to daily practice
Monitoring egg weight loss

Weight loss at transfer of 28 individually weighed
14 setter trays with eggs from same batch
10% weight
12 loss 12,50
weight loss (%)
11% weight
10 loss
12,00
8 12% weight
loss % weight loss at transfer
6 13% weight
11,50
loss
4
6 8 10 12 14 16 18
11,00
incubation time 1 3 5 7 9 11 13 15 17 19 21 23 25 27
2 3
Fundamentals of incubation: ventilation & humidity → relation to daily practice
Variation in egg weight loss

Individual egg weight loss within 1 setter tray
Real data of individual eggs;

R&D Pas Reform (November 2017)
2 3
Fundamentals of incubation: ventilation & humidity → relation to daily practice
Variation in egg weight loss

Risk of average weight loss 10% vs 12%
No. of eggs
5 6 7 8 9 10 11 12 13 14 15 16 17
egg weight loss
average 10 % average 12Conceptual
% data
2 3
Fundamentals of incubation: ventilation & humiliatie → relation to daily practice
Ventilation & humidity combined
Consequences for management

• Control ventilation based on RH% and CO2
• Monitor climate in inlet plenum
• Avoid excessive ventilation
o No ventilation at start incubation for … days;
o Might lead to active humidifiers = cold spot
o Low CO2 set point at end incubation (≤0.4 %).
2 3
Bringing it all together

5.
H
2. Turning: embryo growth Y
G
I
E
N
4. Humidity: egg weight loss E
!!!
2 3
Bringing it all together (setter)
---differentiation-----------------------growth------------------maturation
Bringing it all together (hatcher)

Summary
Further reading?
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Royal Pas Reform

• Embryonic development during egg formation

• Embryonic development during incubation

Reflection on topic of presentation

Incubator management and parameters to

• Practical → day-to-day incubation management

• Theoretical → fundamental points of incubation

• Embryo → support during different stages

Good hatchery results is your goal!

But what is good?

• Good chick quality

Good hatchery results is your goal!

What do we need to get good results?

1. Good quality hatching eggs

2. Optimal incubation conditions

3. Good management (knowledge & experience)

Good quality hatching egg!

Uniform weight, not Embryo at optimum Free of diseases (Se, Mg,

Ovary and oviduct

Van Nassauw, 1997 Unfertilized oocyte

Embryonic development during

Development in the oviduct

First cleavage divisions

Development in the oviduct

Development of the head-to-tail axis

Embryonic stage at oviposition

Embryonic stage after cooling to

Ref: Gilbert , 2006

Adaptation period = first hours after laying

Fertile eggs; can these be stored?

Good quality hatching egg!

The good quality hatching egg contains an

Photos: courtesy of dr. M. Boerjan

The unincubated fertile and infertile egg

Fertile Fertile Fertile, Infertile

The unincubated fertile and infertile egg

An egg is not just an egg!

In the embryo we recognize of 2 different cell-types

Area opaca Area pellucida Area Area

Differentiation starts in the hen

Will these un-incubated eggs give good

Embryonic cells migrate

Claudio D. Stern (2004): Gastrulation, from cells to embryo

Beyond point of no return!

Formation of sub-embryonic fluid

1 (0-24 h) day 3 6th day

Redistribution of water from albumen to the area below embryo

Extra embryonic tissues

Subembryonic fluid changes

Extra-embryonic tissues and compartments is essential

Embryo complete; rapid growth

Exponential increase of metabolic heat production until plateau phase

Maturation of physiological control

Hatching phase: maturation of

Biology & technology in balance

• Egg shell temperature (EST)

Egg shell temperature profile

Average egg shell temperature (°F)

Egg shell temperature: 0 – 10 days

Egg shell temperature Weight (g) Weight (g) FCR

Egg shell temperature: 16 – 21 days

Factors Low Control High

“EST” is leading parameter

the development from 100,5