R.

Keith Bramwell, PhD

Department of Poultry Science
The University of Arkansas
Fayetteville, Arkansas
Carbon Dioxide and the
Developing Embryo
Todays embryos seem to be more
susceptible and less viable in regard
to incubation

Simply will not take as much

temperature variation or stress
Carbon Dioxide in the Body
Bioenergetics of the Developing Egg
The avian embryo is a primary force for the
absorption, storage, and elimination of chemical
energy (heat).
The living matter of the embryo becomes a
machine for the transformation of the potential
energy of the egg into the kinetic energy of the
newly formed chick.
This is accompanied by the exchange of
respiratory gases and by the production of heat.
Romanoff, 1967
Gaseous Exchange
Embryonic respiration is the most obvious sign of
the activity of chemical changes, involving the
intake of oxygen and the expelling off of carbon
dioxide.
The magnitude of the gaseous exchange is
related to the size and age of the embryo as well
as the number of live embryos in our incubators.

Romanoff, 1967
Oxygen Consumption by the Embryo
Consumption of O2 may be considered a
measure of embryonic life, it represents an
immediate dissipation of energy.

Romanoff, 1967
Carbon Dioxide Production by the Embryo
CO2 production by the whole egg is greater than
just the embryo alone.
Other sources of CO2 production include:
Egg albumen, evolved during initial stages of
incubation.
Egg shell, from the carbonates dissolving for use by
the developing embryo, especially at the later stages
of development.
Extra-embryonic membranes, produced in proportion
to the weight of the living tissue.
Romanoff, 1967
 Carbon Dioxide
Carbon Dioxide in the air is approximately 0.04%
(400 ppm).
CO2 in a nest is approximately ten times the
concentration in air (0.4%, 4,000 ppm).
Studies show that CO2 concentration must be
0.4% to 0.6% (4,000 6,000 ppm) for normal
development to occur.
Hatch is substantially reduced at levels above
1% (10,000 ppm).
 Carbon Dioxide
CO2 plays a vital role in the process of water
movement and in the formation of the sub-
embryonic fluid in the first week of incubation.
Higher CO2 levels in the latter stages of
incubation (?):
May improve hatch and chick quality.
Have been associated with earlier timing of
pipping and hatching.
 Carbon Dioxide
More research is required to determine the
influence of flock age, breed and egg size on
CO2 at the time of hatch.
This is a topic of interest to the poultry industry,
for Single-Stage machines can operate to control
CO2 levels.
CO2 in Early Incubation
CO2 and O2 exchange are fundamental for
embryo development during incubation
(Romanoff, 1941; Tullett, 1990).
Hypoxia or oxygen insufficiency appears to
decelerate embryonic growth, reduce hatchability
(1956, 1982) cause developmental abnormalities
and increase embryo mortality (1987, 2002,
2005).
Therefore, it was believed that high CO2
concentrations were detrimental.
Willemsen, et al, 2008
CO2 in Early Incubation
However, recent reports suggest higher levels of
CO2 can have beneficial and persistent effects on
embryonic development, early hatching, and
improved overall hatch (Bruggeman et al., 2007;
Tona et al., 2007)

Willemsen, et al, 2008

CO2 in Early Incubation
Studying the effects of CO2 in early stages of
incubation on ascites in broilers.
Non-ventilation for the first 10 days
CO2 was 0.05% (500 ppm) @ day 0 and
0.7% (7,000 ppm) @ day 10
CO2 never surpassed 0.1% (1,000 ppm) in the control
group

De Smit, et al, 2008

CO2 in Early Incubation
0.8
Non-
0.7 ventilated
CO2 concentration (%)

0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1
0 2 4 6 8 10
incubation duration (d)
CO2 in Early Incubation
Non-ventilated=
faster embryo development, body weight
reduced duration to internal and external pipping,
time to hatch, hatch
Non-ventilated=
aircell pCO2 increased, pO2 decreased
Higher pCO2 stimulates hatching (Visschedijk, 1968)
Increased performance of embryo and post-
hatch growth (De Smit, et al., 2006; Tona, et al.,
2007)
De Smit, et al, 2008
CO2 in Late Incubation
Carbon dioxide is important, but pCO2 levels higher
than 1% is often considered to be deleterious
(Owen, 1991).
Older studies disagree (1960s and 1970s) and state
that the high CO2 help the embryo develop a better
tolerance to carbon dioxide.
This study they achieved CO2 at 2% (20,000 ppm) by
day 11 then 4% (40,000 ppm) on day 12 and held
until day 18.
This study did not show any benefits from late
increase in CO2
Everaert, et al, 2007
De Smit, et al., 2006
What are ALL the Benefits to this type
of Incubation?
How long does it take to produce a broiler chick?
6-7 weeks? Or, 6-7 weeks +21 days of incubation?

How much effect does incubation play in broiler

performance?

What if we provide the embryo what it needs EACH

DAY of embryo development, what are ALL the
benefits?
Hatch - 10/26/2006 Hatchability
Flock Age CM/Multi-stage JW/Single-stage Diff
3004-1 51 84.88 87.77 2.89
3004-2 51 86.81 86.81 0.00
3084-1 53 84.88 85.84 0.96

3084-2 53 87.77 88.73 0.96

3084-3 53 84.88 86.81 1.93
3085-3 54 82.95 83.91 0.96
3005-3 55 80.05 82.95 2.90

Egg Set 62208 62208

Chicks 52597 53574 977

%Hatch 84.55 86.12 +1.57

Note: One flock removed due to improper temperature profile parameter
 Hatchery
24 P120 Platinum Incubators and 12 P40 Hatchers
16 Super J Space Saver Incubator and 16 PX Hatchers

Weekly Egg Set Capacity

Platinum @ 967,680 eggs
Multi Stage @ 483,840 eggs = 1,451,520 eggs/wk.
Chick Cost = 26 cents USD

Data Compilation Date: March 1 June 30, 2009

Data: This hatchery data is not total eggs set and hatched at this hatchery for
this period but from samples where identical flocks were set and hatched in
both the Jamesway Multi Stage and Single Stage Incubation Systems.

Reported Average Fertility for Sample SS and MS combined @ 92.34%

First week Average Field Mortality @ .85 - .92 % (.885%)
Platinum @ .74 - .80% (.77%)
Multi Stage @ .89 1.00% (.945%)
Jamesway P120 Platinum Single Stage verses Jamesway Multi Stage Hatchery
Results 3/1 6/30/09

Number Total Total Number % 2nd % 2nd Total % 1st

Set Number % Culls Culls Grade Grade Number Grade
Hatch Hatch Chicks Chicks Hatch 1st Chicks
Platinum 831,096 719,732 86.60 1,624 0.23 4,008 0.56 714,100 85.92
(.195) (.482)

Multi 770,588 647,369 84.01 1,679 0.26 4,350 0.67 641,340 83.23
Stage (.218) (.564)

Platinum +2.59 <-55> <-.03> <-342> <-.11> +2.69

Adv. (-.023) (-.082)

Hatch of Fertile: Platinum @ 93.78% - Multi Stage @ 90.98% = Platinum Adv. @ +2.80%
Chick Weight: Platinum @ 44.84 grams Multi Stage @ 43.63 grams = Platinum Adv. +1.21 g
 Platinum verses Multi Stage Grow Out Results 3/1 6/30/09
Note: Same Total Amount of (same flocks)Eggs Set in Both Platinum and Multi Stage for This Data

Platinum Adv.
Total Chicks Placed: Platinum @ 4,193,180 Multi Stage @ 3,962,541 + 230, 639 chicks
Total Birds Processed: Platinum @ 4,016,518 Multi Stage @ 3,732,787 + 283,731 Birds
Per Cent Livability: Platinum @ 95.79% - Multi Stage @ 94.20% +1.59%
Total % Field Mortality: Platinum @ 4.21% - Multi Stage @ 5.80% <-1.59>%
Age of Bird @ Processing: Platinum @ 45.8 days Multi Stage @ 45.4 days +.4 days (9.6 hrs)
Average Weight @ Processing: Platinum @ 6.05 lbs. Multi Stage @ 5.92 lbs +.13 lbs.
Average Daily Gain: Platinum @ 59.9 Grams Multi Stage @ 59.1 Grams +.8 grams
Pay to Grower per Bird: Platinum @ .2232 Multi Stage @ .2080 +.0152 cents
Feed Conversion Ton: Platinum @ 1.894 Multi Stage @ 1.951 <-.057> better
Feed Cost per Ton @ $314.28 ---------
Total LBS. Meat Processed: Platinum @ 24,317,151 Multi Stage @ 22,106,851 +2,210,300 lbs.

Total Production Cost per LBS: Platinum @ 36.17cent Multi Stage @ 37.21cent 1.04 cents/lb
 Hatch of Fertile Calculation
Platinum:
831,096 eggs set X .9234% fertility = 767,434 fertile eggs
719,732 total chicks/767,434 fertile eggs = 93.78% HOF
Multi Stage:
770,588 eggs set X .9234% fertility = 711,561 fertile eggs
647,369 total chicks/711,561 fertile eggs = 90.98% HOF

Total LBS. of Meat Processed: 46,424,002 lbs.

Platinum @ 52.38% - Multi Stage @ 47.62% = +4.76% Platinum
46,424,002 X .0104 = $482,809.62 savings
Total Production Cost (4 months) X 3 = $1,448,428.86 yearly saving
(22,106,851 X .0104 = $229,911.25 savings
Using Multi Stage Only (4months) X 3 = $689,733.75 yearly saving)
 Hatch of Set
Platinum vs Multistage
Hatch of Fertile
Cull Chicks
120

100 96.4 94.85

83.68 82.65
80
Percent

60

40

20
1.03 2.425
0
Platinum Multistage
Comparison
SS MS SS Better
1. Hatchability 83.68 82.65 +1.03 SS
2. HOF 96.40 94.85 +1.55 SS
3. Culls 1.03 2.43 +1.40 SS
 JW/SS CM
Early Mortality
2.06% 2.17%
Late Mortality
2.26% 3.50%
Pips
.62% 1.44%
HOF 94.32% 92.84%

Uniformity 80.84% 79.79%

 Average Chick Weights for
~ 5 Month Trial

CM/Multi-Stage JW/Single Stage Diff Per Chick

Average Average
Total Weight Weight Total Weight Weight Variance Weight
Grams Grams Grams Grams Grams Difference

537925 44.83 557200 46.43 19275 1.61

CO2 in Incubation
At the heart of animal production efficiency is
how efficient energy is produced and utilized at
the cellular level.
Virtually all of the energy produced by a cell
(bioenergetics) needed for cell function, tissue
development, animal health (immune system) and
growth occurs by oxidative phosphorylation (by
mitochondria) and glycolysis.

Bramwell & Bottje, 2012

CO2 in Incubation
Mitochondria are cellular organelles that have been
called the power house of the cell since they are
responsible for at least 90% of the energy produced in
the cell in the form of ATP.
Thus, growth performance of animals is dependent
to a great extent on the ability of mitochondria to
produce sufficient amounts of ATP.

Bramwell & Bottje, 2012

CO2 in Incubation
Mitochondria can undergo a process called
mitochondrial biogenesis to produce more
mitochondria within the cell when there is increased
demand for cellular energy.
The process of producing ATP within the
mitochondria as well as mitochondrial biogenesis is
dependent upon the appropriate synthesis and
assembly of a large number of proteins that are
orchestrated by genes found in the nucleus as well as a
discrete set of DNA found within the mitochondria
Bramwell & Bottje, 2012
CO2 in Incubation
A by-product of mitochondrial energy production is
the generation of oxygen radicals that can damage cell
structures (e.g. lipids in membranes, protein and
DNA).
Oxygen radical generation can be attenuated by
various proteins and lipids that uncouple the electron
transport chain.
With higher oxidative damage, metabolism
becomes less efficient and cells must expend more
energy in repair processes at the expense of production
(e.g. cell growth, tissue development, etc.).
Bramwell & Bottje, 2012
CO2 in Incubation
So a critical balance between energy production,
uncoupling, and antioxidant levels is needed for
optimal function of cells.
Based on the improvements in embryonic
development and broiler growth performance, we
hypothesize that non-ventilation in single stage
setters from day 1 to day 10 of incubation:
a) improves energy utilization and reduces
oxidative stress in embryos, and
b) that these changes persist throughout grow-out
in broilers.
Bramwell & Bottje, 2012
Carbon Dioxide in the Body
Table 2. Exp. 1: Hatch weights of chicks incubated under normal (Control) and Non-ventilation (13 days to reach target of
10,000 ppm) conditions.

Controls n Non-Ventilation n P Value

1
Hatch Wt 42.8 + 0.2 8 43.3 + 0.2 8 P = 0.065
Hatch Wt2 42.7 + 0.1 61 43.2 + 0.1 61 < 0.001
1
Hatch weights by tray (using groups of 15 chick averages)
2
Hatch weights by groups of 15 chicks

Table 3: Exp. 1: Hatchery Residue Breakout for unhatched eggs incubated under Control and Non-Ventilation (13 day) conditions.
Hatch Infertile Early Dead Late Pipped
Treatment % % % Dead % % Cull
Control 90.2 3.93 3.13 2.74 0.92 0.37
Non-
Ventilation 91.7 3.54 2.68* 2.04* 0.85 0.51
*Non-ventilation values differ from Control (P < 0.05)
What is Going on Here?
An organisms role is to survive!!!
Populations of people that live in extremely high
altitude conditions live in a situation of slight oxygen
stress.
Their physiological makeup allows them to survive in
those conditions, then when they move to a less
stressful environment they can excel!!
What is Going on Here?
So, we hypothesize that:
Embryo development is occurring in a slightly hypoxic
environment (hypercapnia)
This stimulates the embryo to increase the number of
mitochondria through mitochondrial biogenesis
They undergo mild oxidative stress with no-ventilation
Low levels of oxidative stress triggers certain genes to be
turned on which enhances growth of the embryo and
subsequent chick.
Managing Carbon Dioxide
Eggs from old flocks, and old eggs (>7 days)
seem to perform better when CO2 levels were
elevated early in incubation.
Eggs from young flocks, and fresh eggs did not
benefit as greatly from elevated CO2 levels early
in incubation.
Embryo growth is enhanced when CO2 levels are
elevated early in incubation.
What Do We Know?
Our long held beliefs of carbon dioxide in the
incubation process need to be altered.
We know from limited research, but also from
industry field reports that CO2 levels should be
elevated from what we have previously believed.
Why? We dont know, more research in
controlled environments is needed on this
subject.
Factors That Affect Hatchability
Hatchery Management
-Egg handling
-Embryodiagnosis
-Embryo mortality
Breeder Management
-Male management
-Feeding strategies
-Breed/strain differences
-Physiology
-Behavior
-Courtship & mating