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Oxidative stress protection of embryos

by In Ovo supplementation
(Rutz et al., 2002). The objective of this paper is to
explore the potential applications of enhancing the
antioxidant status of the chicks during those critical
the last days of incubation.
1 - Department of Poultry Science, College of
Agriculture and Life Sciences, North Carolina
State University, Raleigh, NC 27695-76082
2 - DVM., MSc., PhD Student
Federal University of Pelotas, RS, Brazil
PDSE/CAPES

rdmalhei@ncsu.edu

he use of in ovo technology is a very innovative

field in the research and industry application,
including novel means of vaccination and
embryonic nutrient supplementation. As
defined by the early work developed by Uni and
Ferket (2003), in ovo feeding is the administration
of exogenous nutrients and other agents into the
amnion of the late-term avian embryo, and it can
advance the development of the growing embryo
before and after hatch. Similarly, in ovo feeding
technology could be used to benefit important
physiological and biochemical stasis including
enhance oxidative protection, key adaptation
function to metabolic stress during the perinatal
period of rapid early development. Early perinatal
development is a critical period to establish the
expression of genetic potential that influence
economically important production traits later in
birds life (Ohta et al., 2001; Ipek et al., 2004; Uni
and Ferket, 2004; Foye et al., 2005, Uni et al., 2005,
Pedroso et al., 2006, Leito et al., 2008, Leandro et
al., 2010, Leito et al., 2010). The ability to improve
economically traits, such as average daily gain, feed
conversion, meat yield, and disease resistance will
lead to increased returns and efficiency for the
industry (Schall, 2008). Considering that oxidative
protection against reactive oxygen species (ROS)
originated during body physiological processes
starts in embryo early development, egg content can
be a key factor in the extension of ROS generation
causing deleterious effects in poultry metabolism

Atoms are most stable in the ground state

when every electron in the outermost shell has a
complimentary electron that spins in the opposite
direction. By definition a free radical is any atom
(e.g. oxygen, nitrogen) with at least one unpaired
electron in the outermost shell, and is capable of
independent existence (Karlsson, 1997). A free
radical is easily formed when a covalent bond
between entities is broken and one electron
remains with each newly formed atom (Karlsson,
1997). Most biologically-relevant free radicals are
derived from oxygen and nitrogen. Both these
elements are important for animal life, but in some
circumstances they can be converted into free
radical molecules (Surai et al., 2003). Free radicals
are highly reactive due to the presence of unpaired
electron(s). Any free radical involving oxygen can
be referred to as reactive oxygen species (ROS)
(Figure 1). Oxygen centered free radicals contain
two unpaired electrons in the outer shell. When
free radicals steal an electron from a surrounding
compound or molecule a new free radical is formed
in its place. Thus, a chain reaction of free-radical
formation continues and can occur over a thousand
of events (Goldfarb, 1999). About 2 to 5% of the
total oxygen intake during both rest and exercise
may form the highly damaging superoxide radical
via electron escape (Sjodin et al., 1990).

Ramn D. Malheiros, P.R. Ferket and F.M. Gonalves

Introduction

Oxidative distress

Lipoperoxidation
Polyunsaturated fatty acids (PUFAs) are
abundant in cellular membranes and in low-density
lipoproteins (LDL) (Dekkers et al., 1996). The PUFAs
allows cellular membranes fluidity given passage to
non hydro soluble substances. Free radicals prefer
to steal electrons from the lipid membranes of a cell
because of their proximity to pro-oxidant agents,
thus initiating a free radical attack on the cell known
as lipid peroxidation (Figure 2). Reactive oxygen
species target the carbon-carbon double bond of
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electron
from
the
hydrogen
associated
with the carbon at the
Reactive oxygen species (ROS):
double bond. In turn
=Superoxide
this leaves the carbon
=Hydrogen peroxide
with
an
unpaired
=Hydroxyl
electron and hence
becomes a free radical.
Sources
In an effort to stabilize
the
carbon-centered
Exogenous:
free
radical
molecular
Endogenous:
= Xenobiotic-initiated
=Mitochondrial respiration
rearrangement occurs.
=Bacterial killing by white blood cells
The newly arranged
=Metabolism of endogenous substrates
molecule is called a
by oxidases & cytochromes P450
conjugated diene (CD).
=Metal-catalyzed reactions
The CD then very easily
=Diabetes
reacts with oxygen to
= etc
form a proxy radical
which steals an electron
Effects
from
another
lipid
molecule in a process
Oxidative damage to
called propagation. This
Signal transduction:
celullar macromolecules:
process then continues
=Physiological and pathophysiological
=Lipid membranes, proteins,
in a chain reaction
effects
DNA, RNA, carbohydrates
(Halliwell and Chirico,
Figure 1 - Source of ROS and the general mechanisms by which oxidative stress can 1993), consisting in
alter cellular function (from Well et al., 2009).
a propagation of free
radicals and formation
of non-radical products
such as (ketones, ethers, alkanes, aldehydes, etc.).
polyunsaturated fatty acids. The double bond on
This intermediates and products can change with
the carbon weakens the carbon-hydrogen bond,
reaction conditions and time, representing a big
allowing for easy dissociation of the hydrogen by
threat to animal health status. The process continues
a free radical. A free radical will steal the single
indefinitely until no hydrogen source is
available or the chain is intercepted.

Ramn D. Malheiros, P.R. Ferket and F.M. Gonalves

Oxidative stress

Free radicals formation

Types of free radicals
There are numerous types of free
radicals that can be formed within the
animal. The most common ROS include:
the superoxide anion (O2-), hydroxyl
radical (OH), singlet oxygen (O), and
hydrogen peroxide (H2O2).

Figure 2 - Oxidative modification of lipids

Area: Chicken Breeder and Broiler Production August 07

Superoxide anions; are formed

when oxygen (O2) acquires an additional
electron, leaving the molecule with
only one unpaired electron. Superoxide
anions (O2-) are continuously being
formed within the mitochondria. The
rate of formation depends on the
amount of oxygen flowing through the

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mitochondria at any given time.

Hydroxyl radicals; are short-lived, but the most
damaging radicals, within the body. This type of free
radical can be formed from O2- and H2O2. Copper
or iron catalysts and H2O2 also produce OH. These
reactions are significant as the substrates are found
within the body and could easily interact (Halliwell
and Chirico, 1993).
Hydrogen peroxide; produced in vivo by many
reactions. Hydrogen peroxide is unique in that it
can be converted to the highly damaging hydroxyl
radical or be catalyzed and excreted harmlessly as
water. Glutathione peroxidase, a seleno-protein
enzyme is essential for the conversion of glutathione
to oxidized glutathione, during which H2O2 is
converted to water (Goldfarb, 1999). If H2O2 is not
converted into water, O is formed.

Factorsthataffectoxidative
stress during incubation
According of Surai (2007), there are many
factors that can interfere with the antioxidant status
of a chick, and many of these oxidative stress factors
act at different stages of development before hatch
(Figure 3).

Temperature, humidity,
carbon dioxide
fluctuations
Vaccination

Delay in collecting
chicks from incubator

The 19th day of embryonic development is

an important point when risk of lipid peroxidation
is very high for chickens, especially high meat yield
strains of broilers. At this stage of development, the
embryonic tissues are characterized by comparatively
high levels of polyunsaturated fatty acids (PUFA).
At the same time natural antioxidant reserves have
not reached a sufficient level for innate protection.
Moreover, internal piping occurs at this critical
stage and oxygen availability increases as pulmonary
respiration begins. Low antioxidant status in
combination with high temperatures, humidity, and
PUFAs increases the embryos susceptibility to lipid
peroxidation.
Hatching time is considered as an
environmental stress for the chick. At this
time, natural antioxidant (Vitamin C and Vitamin
E) concentrations have reached a maximum level.
However, the risk of peroxidation of the high
concentrations of unsaturated lipids in tissues
increases as the concentration of ascorbic acid
needed to reduce oxidized vitamin E decreases,
especially during high temperature and humidity
conditions during the time of hatch.

Day 19 of incubation

Hatching time

Disease challenger

Figure 3 - Factors that can interfere with the antioxidant status of a chick at the
end of incubation (Adapted from Surai, 2007).

Ramn D. Malheiros, P.R. Ferket and F.M. Gonalves

Singlet oxygen; is not a free radical, but can

be formed during radical reactions and also cause
further reactions. When oxygen is energetically
excited one of the electrons can jump to an empty
orbital, creating unpaired electrons (Karlsson,
1997). Singlet oxygen can then transfer the energy
to a new molecule and act as a catalyst for free
radical formation. The molecule can also interact
with other molecules leading to the formation of a
new free radical.

Temperature, humidity and carbon dioxide

concentration fluctuations during incubation
can affect embryonic development, oxidation and
phosphorylation in tissues, leading to the production
of free radicals. For example, high carbon dioxide
concentration during incubation have been shown
to jeopardize the livability of the embryo. Everaert
et al. (2007) demosnstrated that broiler embryos
when submitted to 4% CO2 pressure inside the
incubator between 10th to 18th days, were not able
to affect the relative growth of chicks until 7 days,
when compared with the control group, however,
corticosterone and thyroxine concentrations were
significantly higher in the CO2 incubated chicks on
7 days post hatch.

Delay in collecting
chicks from incubator
can
exacerbate
oxidative stress of the
hatching chick. Since
not all chicks are hatched
at the same time because
the
heterogeneous
nature of the eggs set
(i.e eggs from older
breeders hatch earlier
than those from young
flocks and chicks from
smaller eggs hatch earlier

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Ramn D. Malheiros, P.R. Ferket and F.M. Gonalves

than those from larger eggs), some chicks may be in

the incubator for 2 12 hours longer than others.
This wide hatch window puts additional pressure
on antioxidant defense capacity. Furthermore, any
delay in food and/ or water intake after hatching
usually negatively affects a number of performance
parameters and delay occurs in the maturation of
the enzymatic system that control metabolism,
free radical production and antioxidant protection
systems.
Disease challenger of the breeder hens or
progeny immediately after hatch significantly
affects oxidative stress tolerance in perinatal chicks.
Phagocytic immune cells themselves produce
free radicals in the process of killing internalized
pathogens. Without adequate antioxidant nutrient
reserves, cellular membranes and hence, important
organelles, can be damaged by the free radicals
thereby reducing the effectiveness of the immune
cell. In addition, Se is considered to have a specific
role in immune system regulation, which could be
independent on its antioxidant functions.
Vaccination is a substantial stress; and in some
cases using vitamin E as a vaccine adjuvant can help
improve vaccination efficiency. In ovo vaccination is
a relatively new concept in vaccine administration
and has replaced post-hatch injection of Mareks
disease vaccine in broiler chickens. On the other
hand, the challenge of the immunization begins
sooner in chicks life. Its important that the embryo
have a good reserve of antioxidants to respond
vaccination with high efficiency. Bhattacharyya
et al. (2007) showed improvement of immune
response in Turkeys with
in ovo supplementation of
carbohydrates.

Worlds Poultry Congress 5 - 9

August - 2012 Salvador - Bahia - Brazil

either impede embryonic nutrient utilization or

increase their demand to maintain metabolic stasis.
These adverse incubation events may be related
to management of the incubation, environment,
air quality, contamination, genetic factors, and
many other issues. In ovo administration of some
compounds that positively addresses oxidative stress
may alleviate the associated adverse effects on chick
growth and welfare. Some of these antioxidant
compounds could enhance or protect critical
physiological or biochemical functions leading to
improved health and welfare of the perinatal chick.

Vitamins
Vitamin E (VE) is the major fat-soluble
antioxidant, that quenches the chain reaction of lipid
peroxidation. The antioxidant system of the brain is
of great importance because of the development of
nutritional encephalomalacia, which occurs in young
chicks as results of VE deficiency (Dror and Bartov,
1982; Bhanja et al., 2007). The VE is well known
for its antioxidant properties and is often used as
the standard by which all other antioxidants are
compared. The VE is distributed in all compartments
in the egg, and the concentrations changes as the
incubation period progresses (Figure 4) (Noble et al.,
1993). The use of antioxidants, like VE has been used
to alleviate the adverse effects of the peroxidation
of lipids, and cholesterol, in domestic animals (Singh
et al., 2005). Surai et al. (1997, 1999) observed a
highly positive correlation between dietary VE
levels in the maternal hens diet and the levels of
VE in egg and subsequent in chick tissues. The VE
levels can be correlated with the immune response
of chicks. Gore and Qureshi (1997) observed a

Early antioxidant
feeding:
How
this can help?
After the fertile egg is laid,
it must contain all the nutrients
to nourish and protect the
developing embryo until the
chick hatches and begins
to consume and assimilate
sufficient nutrients to support
subsequent development and
growth. But some adverse
events during incubation can

Figure 4 - Alfa-Tocopherol concentrations in: (a) yolk contents; (b) yolk

sac membrane; and (c) liver of the chick embryo during incubation. Each
result is the mean SE of 6 observations (from Noble et al., 1993).

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positive correlation between VE administered in ovo

on immune system and immunoglobulin levels in
turkey poults and broiler chicks at 7 and 14 days
post-hatch. Moreover, Schaall (2008) demonstrated
that in ovo VE administration was able to change the
total lipid content of tissues at hatch time, especially
in the brain and plasma. Schaall (2008) concluded
that VE enhances the antioxidant status of hatched
chicks tissues and protects lipid membranes from
radical oxygen species.

Carotenoids
Many studies have already shown that dietary
carotenoids have a very participative role in the
antioxidant defense mechanisms of live organisms.
Carotenoids are lipid-soluble organic compounds
that pigment and protect the integument of many
organisms by absorbing light and UV radiation, and
many carotenoids provide antioxidant protection and
immunomodulation (Goodwin, 1986). In humans,
several authors suggest an important relationship
exist between carotenoids and the prevention of
cancer (Mathews-Roth, 1985; Krinsky, 1989; Ziegler,
1991). It has been suggested that carotenoids may
act as anti-carcinogenic agents by an antioxidant
mechanism independent of their conversion to
vitamin A (Peto et al., 1981). Most studies have
focused on the antioxidant properties of -carotene
and research has been largely conducted in
liposomes (Krinsky and Deneke, 1982), lipoproteins
(Jialal et al., 1991), and isolated membranes
(Palozza et al., 1992). Lawlor and OBrien (1995)
demonstrated the effects of -carotene on chicken
embryo fibroblast when challenged with different

L- Carnitine
L-Carnitine acts as an antioxidant that ultimately
results in a decrease in reactive oxygen species by
removing excessive levels of intracellular acetylCoA, which induces mitochondrial reactive oxygen
species production (Vicari and Calogero, 2001;
Agarwal and Said, 2004; Agarwal et al., 2005).
Free radicals responsible for lipid peroxidation
are scavenged by L-carnitine (Rebouche, 1992).
Other antioxidants, such as ascorbic acid, vitamin
E, and antioxidant enzymes, are protected against
potential peroxidation by L-carnitine (Kalaiselvi
and Panneerselvam, 1998). In addition to its role
as an antioxidant, L-carnitine transports long chain
fatty acids across mitochondrial membranes for
-oxidation of fatty acids. Under circumstances of
increased metabolism, when the demand for energy
escalates, L-carnitine availability could become
a limiting factor for -oxidation of fatty acids. In
such situations, exogenous supplementation of
L-carnitine could prove advantageous (Buyse et al.,
2001) and could in turn be used by the chick during
hatching. Zhai et al. (2008) concluded that the in
ovo injection of dosimetric dosages of L-carnitine in
the range of 0.05 to 10 mol into fertile Leghorn
eggs at 17 or 18 d of incubation did not affect
hatchability, yolk sac weight, or BW. The possibility
exists that dosages of L-carnitine greater than 10
mol could improve hatchability and hatchling
BW because there was no evidence of carnitine
toxicity with the dosimetry utilized in the current
study. Moreover, the response of broiler and turkey
hatching eggs to in ovo carnitine injections may
differ from Leghorn embryos. Differences in lipid
metabolic rate between egg-laying and meat-type
strains of poultry could influence response to in ovo
injection of L-carnitine (Sato et al., 2006).

Ramn D. Malheiros, P.R. Ferket and F.M. Gonalves

Vitamin C (ascorbic acid, AA) has been

demonstrated to improve immunoresponsiveness
and increase disease resistance in poultry by
optimizing the immune system (Pardue et al., 1985;
Rund, 1989). Ascorbic acid has also been shown to
have a sparing effect on vitamin E by acting as a redox
system reducing tocopheroxyl radicals back to their
reduced state tocopherol (Wilson, 1983). Ipek et al.
(2003) utilized in ovo injection during the later part
of incubation to study the effect of AA and glucose
on embryonic mortality, hatchability, chick quality,
and chick weight of broiler chickens embryos. These
researchers observed that in ovo injection of 3 mg
AA per egg resulted in a higher hatchability rate.
Zakaria and Al-Anezi (1996) observed the best
hatchability results for chickens when 3 mg/egg of
vitamin C were injected on day 15 of incubation.
Nowaczewski et al. (2011) demonstrated that in ovo
injection of 6 mg of vitamin C per egg on the 20th
day of incubation (8 days before hatch) improved
hatchability rate of ducklings.

concentration of -carotene and with or without

a presence of paraquat (Potent oxidative agent).
They concluded that -carotene was effective as an
antioxidant in low concentrations of (0.1M), but it
was an effective pro-oxidant at high concentrations
(10M). Koutsos et al. (2006) demonstrated that
chicks with a lack of carotenoid exposure, either
in ovo or post hatch, increases parameters of
systematic inflammation.

Glucose
According Bhattacharyya et al. (2007), the
maintenance of glucose homeostasis during
few days pre and post-hatch is among greatest
challenge of a chicks life. The primary source
of glucose need for hatching activities is the liver
and gluconeogenesis from protein of amnion and
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XXIV

muscle. Glycolysis, rather than fatty acid oxidation,

is needed during hatching to provide energy, as
oxygen supply is limited during the transition from
chorio-allantois to pulmonary respiration (Hoiby et
al., 1987). At day of hatch glycogen stores decrease
substantially, and remain low until the newly hatched
chick has the reserves access to oxygen, necessary to
mobilize and utilize body fat reserves and assimilate
external diet (Hazelwood, 2000). Just after hatch,
the chick undergoes rapid physical and functional
development of gastrointestinal tract for digestion
and assimilation of nutrients. However in most
commercial operations, the chicks get access to feed
only after several hours after hatching. This may
decrease hatchling weight and immune-competence
of chicks. These limitations in early energy status may
be alleviated by administering refined carbohydrates
or amino acids into the amnion or yolk sac (in ovo
feeding) at different days of incubation, thereby
improving chick livability and growth (Uni and
Ferket, 2004; Branja et al., 2004). It has already been
noted that glucose added to drinking water helps to
suppress gluconeogenic enzyme activity (Donaldson,
1995), which is necessary to sustain blood glucose
homeostasis. The digestive tract of the hatchlings
has limited ability for digestion and utilization of diet
rich in proteins and carbohydrates (Uni and Ferket,
2004). Hence, early access to highly digestible feed
is beneficial but the benefits are solely dependent
on the initiation of feed consumption unlike in ovo
feeding. It has been observed that in ovo feeding of
carbohydrates into the amnion increase hatchling
weight in broiler and turkeys (Ferket and Uni, 2002).

Regulatory peptides
Some regulatory peptides could have some antioxidant properties. Zheng et al. (2010) reported that
leptin has considerable role in anti-oxidant defense
of body by activating super oxide dismutase. Ghrelin
is another multifunctional regulatory peptide. After
identification of ghrelin in mammals by Kojima et
al. (1999) many relative studies were conducted
to better understand its metabolic functions.
Studies conducted during last decade revealed
various homeostatic functions for ghrelin, such
as the release of growth hormone (Hashizume et
al., 2005), osteogenesis (Fukushima et al., 2005;
Delhanty, 2008), hematopoiesis (Akarsu, et al.,
2007; Dixit et al., 2007), food intake and energy
balance and regulation (Nakazato et al., 2001;
Toshinai et al., 2003), endocrine/ paracrine roles in
pancreas (Stevanovi et al., 2007, Dezaki et al., 2008),
and currently antioxidative effects (Kheradmand
et al., 2008; Kheradmand et al., 2010). Peptide
structure of chicken ghrelin has 26 amino acids with
54% similarity to rat ghrelin (Kaiya et al., 2008).
Area: Chicken Breeder and Broiler Production August 07

Worlds Poultry Congress 5 - 9

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Ghrelin has been identified in albumen and yolk

of fertilized chicken egg (Yoshimura et al., 2009).
Malondialdehyde (MDA) (CH2(CHO)2) is a bio-marker
of oxidative stress in an organism and an increase of
MDA level demonstrates oxidative stress (Nielsena et
al., 1997). Anti-oxidative activity of ghrelin has been
reported in numerous studies (Kheradmand et al.,
2009; Kheradmand et al., 2010; seri et al., 2005).
seri et al. (2005) reported the anti-oxidative activity
of ghrelin and its ameliorative ability for alendronateinduced oxidative stress, as MDA comes back to the
control level after ghrelin infusion. The chick embryo
attempts to increase capacity of its antioxidant
defenses as incubation progresses towards hatch
(Stock et al., 1990). Although the anti-oxidative
effects of ghrelin has been studied in mammalian
models, its antioxidant function in birds is unclear.
Lotfi et al. (2012) described in ovo administration of
ghrelin on day 5 of incubation was more efficient in
reducing MDA than when administered on day 10
(4.10 and 4.60 nmol/mL in comparison with 9.53
and 9.50 nmol/mL, respectively). Apparently in ovo
ghrelin supplementation during early embryonic
development (i.e. day 5 of incubation) had an
antioxidant protective effect on the embryo as
indicated by a significant decrease in serum MDA
level of newly-hatched chicks. These researchers
concluded that in ovo administration of ghrelin on
day 5 and day 10 of incubation at the dosages of 50
and 100 ng/egg can have anti-oxidative protection
and decrease serum MDA level. Further studies with
measuring specific enzymes (superoxide dismutase,
glutathione peroxidase, etc) activity in blood or
tissues of newly-hatched chicks following in ovo
administration of ghrelin on different incubation
days are suggested for further clearness of
antioxidant properties.

Elements to use in ovo

with potential antioxidant
effects
Thiamin (Vitamin B1)
Thiamin (Vitamin B1) is a co-factor for
several enzymes catalyzing descarboxylation and
transkeletolation type reactions. Deficiency of
thiamin in eggs causes high mortality of embryos
just prior to hatching, and chicks that hatch exibit
symptoms of polyneuritis (Charles et al., 1972).
Deficiency of vitamin B6 (Pyridoxine) leads to early
embryonic death (Landauer, 1967) and decreases
IgM and IgG response to antibody challenger
(Blalock et al., 1984).

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Essential oils

Trace minerals
Individual trace minerals levels in the avian egg
are variable and the total amount deposited in the
egg depends on the mineral chemical form and the
amount fed to the hen (Naber, 1979; Stadelman and
Pratt, 1989). Changes in environmental conditions
during incubation can impact avian embryonic
trace mineral metabolism. Briefs periods of hypoxia
(reduced oxygen tension, 15% O2) or hyperoxia
(increased oxygen tension, 60% O2) during the late
stages of incubation will alter growth, weight of
the extra-embryonic membranes, and tissue trace
mineral homeostasis in the chick embryo (Richards et
al., 1991). Wilson et al. (1992) studied hepatic zinc,
copper, and iron levels in the chick embryo and the
levels of specific antioxidant enzymes that require
these metals as co-factors. They found a correlation
between catalase activity and its co-factor, iron, in
liver and reported a positive relationship between
hepatic zinc and oxygen consumption by the
embryo, as well as changes in brain levels of
superoxide dismutase (CuZn-SOD), a copper-and
zinc-containing enzyme. Marked differences in the
developmental profiles of tissue antioxidant enzyme
system were found for brain versus liver; however,
the relative abundance of metal cofactors did not
seem to influence the activity of most antioxidant
enzymes in these two tissues during development
(Richards, 1997).

Conclusion
In conclusion, oxidative stress is among the most
limiting threatening conditions that the avian embryo
must overcome as it develops towards hatch through
to the initiation of feed consumption. Although
the embryo is endowed with various antioxidant
compounds deposited by the hen to combat
oxidative damage, incubation distress can place a
significant drain on these limited antioxidants, and
consequently adversely affect critical physiological
and metabolic functions. The antioxidant status
of the embryonated egg is determined by what
the hen deposits, which is influenced by her diet,
nutritional and physiological state, and her own
antioxidant status. Because antioxidant status of
hatching eggs are variable and unpredictable, in ovo
supplementation of antioxidants by in ovo feeding
technology may prove to yield significant benefits.
Indeed, the few preliminary studies indicate the
potential benefits of in ovo feeding solutions
containing various antioxidant compounds,
including vitamin E, ascorbic acid, L-carnitine,
organic trace minerals, carotenoids, and antioxidant
peptides. However, more research is necessary to
better assess the practical application of antioxidants
by in ovo feeding on subsequent health and growth
potential of commercial broilers and turkeys.

Ramn D. Malheiros, P.R. Ferket and F.M. Gonalves

The essential oils (EOs) also called volatile or

ethereal oils, are aromatic oily liquids obtained
from plant material. EOs are complex mixtures of
secondary plant metabolites consisting of lowboiling-phenylpropenes and terpenes. Many sources
of antioxidant of plant origin and antioxidant
properties of many aromatic plants and spices have
been studied in recent years.. Among these, the
antioxidant properties of many aromatic plants and
spices have shown to be effective in retarding the
process of lipid peroxidation in oils and fatty acids.
Particularly rosemary, oregano and sage have been
extensively studied for their antioxidant activity
(Brenes and Roura, 2010). Cinammon have also been
demonstrated by Kamel (1999), to have a similar
antioxidant activity as the synthetic antioxidant BHT.
Due the oily characteristics of these products, the
use in ovo supplementation has some restrictions
mainly because lipossoluble characteristics of this
substances.

Selenium also can provide beneficial impact in

redox system, since this mineral act in glutathione
peroxidase activity, an important enzyme of body
oxidative system protection. Sefton and Edens
(2004) showed that hatch can be improved by
selenium supplementation in breeders diets, since
this mineral is transferred to the eggs after layer.
According to Surai (2006), selenium in the liver of
newly hatched bird is rapidly diluted in post hatch.
In ovo supplementation of selenium might be
represent a good alternative to prevent this depletion
in young broilers preventing possible injuries due to
oxidative peroxidation in body system.

References
AGARWAL, A., PRABAKARAN, S.A. and SAID, T.M. (2005)
Prevention of oxidative stress injury to sperm. Journal
of Andrology 26:654-660.
AGARWAL, A. and SAID, T.M. (2004) Carnitines and male
infertility. Reproductive BioMedicine Online 8:376384.
AKARSU, S., USTUNDAG, B., GURGOZE, M.K., SEN, Y.
and AYGUN, A.D. (2007) Plasma Ghrelin levels in
various stages of development of iron deficiency
Area: Chicken Breeder and Broiler Production August 07

XXIV
anemia. Journal of Pediatric Hematology Oncology
29:384-387.
ALESSIO, H.M. and BIASI, E.R. (1997) Physical activity
as a natural antioxidant booster and its effects on a
healthy lifestyle. Research Quarterly for Exercise &
Sport 68:292-302.
BHANJA, S.K., MANDAL., A.B. and GOSWAMI, T.K.
(2004) Effect of in ovo injection of amino acids on
growth, immune reponse, development of digestive
organs and carcass yield of broiler. Indian Journal of
Poultry Science 39:212-218.
BHANLA, S.K., MANDAL, A.B., AGARWAL, S.K.,
MAJUMDAR, S., and BHATTACHARYYA, A. (2007)
16th European Symposium on Poultry Nutrition,
Strasbourg, France, August, pp. 26-30.

Ramn D. Malheiros, P.R. Ferket and F.M. Gonalves

BHATTACHARYYA, A. MAJUMDAR, S., BHANJA, S.K.,

MANDAL, A.B., DASH, B.B. and AGARWAL, S.K.
(2007) Effects of in ovo injection of glucose on growth,
immunocompetence and development of digestive
organs in turkey poults. 16th European Symposium on
Poultry Nutrition, Strasbourg, France, pp. 26-30.
BLALOCK, T.L., HAXTON, J.P. and GARLICH, J.D. (1984)
Humoral immunity in chicks experiencing marginal
vitamin B6 deficiency. The Journal of Nutrition 114:
12-322.

August - 2012 Salvador - Bahia - Brazil

second half of incubation on embryonic development,

hatching parameters, and posthatch growth. Poultry
Science 86:1372-1379.
FERKET, P.R. and UNI, Z. (2002) Early enteric development
of turkeys. Proceedings of the 25th technical turkey
conference held at Shrigley Hall Hotel on 24-26 April,
pp. 59-64.
FOYE, O., FERKET, P. and UNI, Z. (2005) The effects of
in ovo feeding of protein and beta-methyl-beta
hidroxybutyrate (HMB) on nutrient digestion and
absorption in neonatal turkey poults. International
Poultry Scientific Forum, Atlanta, pp. 5.
FUKUSHIMA, N., HANADA, R., TERANISHI, H., FUKUE,
Y., TACHIBANA, Y., ISHIKAWA, H., TAKEDA, S.,
TAKEUCHI, Y., FUKUMOTO, S., KANAGAWA, K.,
NAGATA, K. and KOJIMA, M. (2005) Ghrelin direct
regulates bone formation. Journal of Bone and
Mineral Research 20:790-798.
GOLDFARB, A.H. (1999) Nutritional antioxidant as
therapeutic and preventive modalities in exerciseinduced muscle damage. Canadian Journal of Applied
Physiology 24:249-266.
GOODWIN, T.W. (1986) Metabolism, nutrition, and
function of carotenoids. Annual Review of Nutrition
6, 273-297.

BRENES, A. and ROURA, E. (2010) Essential oils in poultry

nutrition: Main effects and modes of action. Animal
Feed Science and Technology 158:1-14.

HALLIWELL, B. and CHIRICO, S. (1993) Lipid peroxidation:

Its mechanism, measurement, and significance. The
American Journal of Clinical Nutrition 57, 715S-724S.

BUYSE, J., JANSSENS, G.P. and DECUYPERE, E. (2001)

The effects of dietary l-carnitine supplementation
on the performance, organs weights and circulating
hormone and metabolite concentration of broiler
chickens reared under a normal or low temperature
schedule. British Poultry Science. 42:230-241.

HASHIZUME, T., HORIUCHIA, M., NONAKAA, S., KASUYA,

E., KOJIMA, M., HOSODA, H. and KANGAWA, K.
(2005) Effects of ghrelin on growth hormone secretion
in vivo in ruminants. Regulatory Peptides 126:61-65.

CHARLES, O.W., RONALD, D.A. and EDWARDS JR,

H.M. (1972) Thiamine deficiency identification and
treatment in commercial turley and Coturnix quail.
Poultry Science 51:419-423.

HAZELWOOD, R.L. (2000) Pancreas, in: WHITTOW, G.C.

(Ed) Sturkie Avian Physiology 5th, pp. 539-555 (San
Diego, Academic Press).
HOIBY, M., AULIE, A. and BJONEES, P.D. (1987) Anaerobic
metabolism in fowl embryos during normal incubation.
Comparative Biochemistry Physiology 86:91-94.

DEKKERS, J.C., VAN DOORNEN, L.J.P. and KEMPER, C.G.

(1996) The role of antioxidant vitamins and enzymes
in the prevention of exercise-induced muscle damage.
Sports Medicine 21, 213-238.

IPEK, A., SAHAN, U. and YILMAZ, B. (2004) The effect of

in ovo ascorbic acid and glucose injection in broiler
breeders eggs on hatchability and chick weight.
Archiv fr Geflgelkunde, 68:132-135.

DELHANTY, P. (2008) Ghrelin and bone. Proceedings of

10th European Congress of Endocrinology, Berlin, pp.
S7.3.

SERI, S.O., SQENER, G., YKSEL, M., CONTUK, G.,

ETINEL, ., GEDIK, N., and YEEN B.. (2005) Ghrelin
against alendronate-induced gastric damage in rats.
Journal of endocrinology 187:399-406.

DEZAKI, K., SONE, H. and YADA, T. (2008) Ghrelin is a

physiological regulator of insulin release in pancreatic
islets and glucose homeostasis. Pharmacology &
Therapeutics 118:239-249.
DIXIT V.D., YANG, H., SUN, Y., WEERARATNA, A.T.,
OUM, Y.H., SMITH, R.G. and TAUB, D.D. (2007)
Ghrelin promotes thymopoiesis during aging. The
Journal of Clinical Investigation 117:2778-2790.
DROR, Y. and BARTOV, I. (1982) Dietary factors affecting
experimental models of nutritional encephalomalacia.
Poultry Science 61:84-93.
EVERAERT, N., KAMERS, B., WITTERS, A., De SMIT, L.,
DEBONNE, M., DECUYPERE, E. and BRUGGEMAN, V.
(2007) Effect of four percent carbon dioxide during the

Worlds Poultry Congress 5 - 9

Area: Chicken Breeder and Broiler Production August 07

JIALAL, I., NORKUS, E.P., CRISTOL, L. and GRUNDY, S.M.

(1991) -Carotene inhibits the oxidative modification
of low-density lipoproteins. Biochimica et Biophisica
Acta 1086:134-138.
KAIYA, H., MIYAZATO, M., KANGAWA, K., PETER, R.E.
and UNNIAPPAN, S. (2008) Ghrelin a multifunctional
hormone in non-mammalian vertebrates. Comparative
Biochemistry and Physiology Part A: Molecular &
Integrative Physiology 149:109-128.
KALAISELVI, C.J. and PANNEERSELVAM, C. (1998) Effects
of L-carnitine on the status of lipid peroxidation and
antioxidants in aging rats. The Journal of Nutritional
Biochemistry 9:575-581.

XXIV

Worlds Poultry Congress 5 - 9

August - 2012 Salvador - Bahia - Brazil

KAMEL, C. (1999) Use of plants extracts in European pig

diets. Feed Compounder 19:23-27.

NABER, E.C. (1979) The effect of nutrition on the

composition of eggs. Poultry Science 58:518-528.

KARLSSON, J. (1997) Exercise, muscle metabolism and the

antioxidant defense. World Review of Nutrition and
Dietetics 82, 81-100.

NAKASATO, M., MURAKAMI, N., DATE, Y., KOJIMA, M.,

MATSUO, H., Kenji KANGAWA, K. and MATSUKURA,
S. (2001) Role of ghrelin in the central regulation of
feeding. Nature 409:194-198.

KHERADMAND, A., ALIREZAEI, M., ASADIAN, P. RAFIEI

ALAVI, E., JOORABI, S. (2009) Antioxidant enzyme
activity and MDA level in the rat testis following
chronic administration of ghrelin. Andrologia. 41:335340.
KHERADMAND, A., ALIREZAEI, M. and BIRJANDI, M.
(2010) Ghrelin promotes antioxidant enxyme activity
and reduces lipid peroxidation in the rat ovary.
Regulatory Peptides 162:384-389.
KINSKY, N.I. (1989) Carotenoids and cancer in animal
models. The Journal of Nutrition 119:123-126.
KOJIMA, M., HOSODA, H., DATE, Y., NAKAZATO, M.,
MATSUO, H., and KANGAWA, K. (1999) Gherlin is
a growth-hormone-releasing acylated peptide from
stomach. Nature 402:656-660.

KRINSKY N.I. and DENEKE, S.M. (1982) Interaction of

oxygen and oxy-radicals with carotenoids. Journal of
the National Cancer Institute 69:205-210.
LANDAUER, W. (1967) The hatchability of chicken eggs
as influenced by environmental and hereditary.
Monograph, Storrs University of Connecticut
Agricultural Experimental Station.
LAWLOR, S.M. and OBRIEN, N.M. (1995) Modulation
of oxidative stress by-carotene in chicken embryo
fibroblasts. British Journal of Nutrition, 73:841-850.
LEANDRO, N.S.M., OLIVEIRA, A.S.C, GONZALES, E,
CAF, M.B., STRIGHINI, J.H. and ANDRADE, M.A.
(2010) Probitico na rao ou inoculado em ovos
embrionados: Desempenho de pintos de corte
desafiados com Salmonella Enteritidis. Revista
Brasileira de Zootecnia 39:1509-1516.

NOBLE, R.C., COCCHI, M. and BATH, H.M. (1993)

-Tocopherol absortion and polyunsaturated fatty
acid metabolism in developing chick embryo. British
Poultry Science 34:815-818.
NOWACZEWSKI, S., KONTECKA, H. and KRYSTIANIAK, S.
(2012) Effect of in ovo injection of vitamin C during
incubation on hatchability of chickens and ducks.
Folia Biologica 60:93-97.
OHTA, Y., KIDD, M.P. and ISHIHASHI, T. (2001) Embryo
growth and aminoacid concentration profiles of
broiler breeder eggs, embryos and chicks after in
ovo administration of aminoacids. Poultry Science
80:1430-1436.
PALOZZA, P. and KRINSKY, N.I. (1992) -Carotene and Tocopherol are synergistic antioxidants. Archives of
Biochemistry and Biophysics 297:184-187.
PARDUE, S.L., THAXTON, J.P. and BRAKE, J. (1985) Role of
ascorbic acid in chicks exposed to high environmental
temperatures. Journal of Applied Physiology 58:15111516.
PEDROSO, A.A., CHAVES, L.S., LOPES, K.L.A.M.,
LEANDRO, N.S.M., CAF M.B. and STRIGHINI J.H.
(2006) Inoculao de nutrientes em ovos de matrizes
pesadas. Revista Brasileira de Zootecnia 35:20182026.
PETO, R. DOLL, R.J., BUCKLEY, J.D. and SPORN, M.B.
(1981) Can dietary -Catotene materially reduce
human cancer rates? Nature 290:201-208.
QURESHI, M.A. and GORE, A.B. (1997) Vitamin E exposure
modulates prostaglandin and thromboxane production
by avian cells of the mononuclear phagocytic system.
Immunopharmacol Immunotoxicol. 19:473-487.

LEITO, A.R., LEANDRO, N.S.M., STRINGHINI, H.J., CAF,

M.B. and ANDRADE, M.A. (2010) Inoculao de
maltose, sacarose ou glicose em ovos embrionados
de baixo peso. Acta Scientiarum. Animal Sciences 32,
93-100.

REBOUCHE, C.J. (1992) Carnitine function and

requeriments during the life cycle. FASEB Journal
6:3379-3386.

LEITO, R.A., LEANDRO, N.S.M., CAF, M.B., STRINGHINI

J.H., PEDROSO, A.A. and CHAVES, L.S. (2008)
Inoculao de glicose em ovos embrionados de frango
de corte: parmetros de incubao e desempenho
inicial. Cincia Animal Brasileira 9:847-855.

RICHARDS, M.P. (1991) Mineral metabolism in

the developing turkey embryo I. The effects of
developmental age and shell-less culture on trace
elements content of selected tissues. Comparative
Biochemistry and Physiology 100:1009-1016.

LOTFI, A., SHAHRYAR, H.A., EBRAHIMNESHAD, Y.

and SHAYEGH, J. (2012) Effects of in ovo ghrelin
administration on serum malondialdehyde level in
newly-hatched chickens. Asian Pacific Journal of
Tropical Biomedicine 2:47-49.

RICHARDS. M.P. (1997) Trace mineral metabolism in the

avian embryo. Poultry science 76:152-164.

MATHEWS-ROTH, M.M. (1985) Carotenoids and cancer

prevention - experimental and epidemiological
studies. Pure and Applied Chemistry 57:717-722.

Ramn D. Malheiros, P.R. Ferket and F.M. Gonalves

KOUTSOS, E.A., LOPES, J.C.G. and KLASING, K.C. (2006)

Carotenoids from in ovo or dietary sources blunt
systematic indices of the inflammatory response in
growing chicks (Gallus gallus domesticus). The Journal
of Nutrition, 136:1027-1031.

NIELSENA, F., MIKKELSEN B.B., NIELSEN, J.B.,

ANDERSEN, H.R. and GRANDJEAN, P. (1997) Plasma
malondialdehyde as biomarker for oxidative stress:
reference interval and effects of life-style factors.
Clinical Chemistry 43:1209-1214.

RUND, B. (1989) Vitamin C plays a role in immunity.

Poultry Digest. 48:44-55.
RUTZ, F., BERMUDEZ, V., PAN, E.A. and FISCHER, G.
(2002) Impacto da nutrio vitamnica sobre a
resposta imunolgica das aves. Simpsio Brasil Sul de
Area: Chicken Breeder and Broiler Production August 07

XXIV
Avicultura, Chapec, pp. 105-117.

WELL P.G., MCCALLUM, G.P., CHEN, C.S., HENDERSON,

J.T., LEE, C.J.J., PERSTIN, J., PRESTON, T.J., WILEY,
M.J. and WONG, A.W. (2009) Oxidative stress
in development origins of disease: Teratogenesis
neurodevelopmental deficits, and cancer. Toxicology
Sciences, 18:4-18.

SCHAAL, T.P. (2008) The effects of in ovo feeding of fatty

acids and aAntioxidants on broiler chicken hatchability
and chick tissue lipids. Honors Scholar, Oregon State
University.

WILSON, J.X., LUI, E.M.K. and DEL MAESTRO, R.F. (1992)

Developmental profiles of antioxidant enzymes and
trace metals in the chick embryo. Mechanisms of
Ageing and Development 65:51-64.

SJODIN, T., WESTING, Y.H. and APPLE, F.S. (1990)

Biochemical mechanisms for oxygen free radical
formation during exercise. Sports Medicine 10:236254.

WILSON, R.L. (1983) Free radicals protection: Why vitamin

E not vitamin C, b-carotene, or gluthathione? Biology
of vitamin E. Ciba Foundation Symposium 101:19-37.

STEVANOVIC, D., MILOSEVIC, V., STARCEVIC, V.P.

and SEVERS, W.B. (2007) The effect of centrally
administered ghrelin on pituitary ACTH cells and
circulating ACTH and corticosterone in rats. Life
Sciences 80:867-872.

Ramn D. Malheiros, P.R. Ferket and F.M. Gonalves

August - 2012 Salvador - Bahia - Brazil

SATO, M., TACHIBANA, T. and FURUSE, M. (2006) Heat

production and lipid metabolism in broiler and layer
chicken during embryonic development. Comparative
Biochemistry and Physiology Part A: Molecular &
Integrative Physiology 143:382-388.

STADELMAN, J.L. and PRATT, D.E. (1989) Factors

influencing composition of the hens eggs. Worlds
Poultry Science Journal 45:247-266.

STOCK, M.K., SILVERNAIL, K.K. and METCALF, J. (1990)

Prenatal oxidative stress: I. Malondialdehyde in hypoxic
and hyperoxic chick embryos. Free Radical Biology and
Medicine 8:313-318.
SURAI, P.F. (2002). Natural antioxidants in avian nutrition
and reproduction. Nottingham University Press,
Nottingham: 616 p.
SURAI, P.F. (2006) Selenium in nutrition and health,
Nottingham University Press, Nottingham: 974 p.
SURAI, P.F. (2007) Natural Antioxidant in poultry nutrition:
new developments. 16th European Symposium on
Poultry Nutrition, Strasbourg, pp. 26-30.
SURAI, P.F., KARADAS, F. and SPARKS, N.H. (2003) The
importance of antioxidants in poultry. Annual North
Carolina Poultry Nutrition Conference, p.38-56.
TOSHINAI, K., DATE, Y., MURAKAMI, N., SHIMADA, M.,
MONDAL, M.S., SHIMBARA, T., GUAN, J.L., WANG,
Q.P., FUNAHASHI, H., SAKURAI, T., SHIODA, S.,
MATSUKURA, S., KANGAWA, K. and NAKAZATO, M.
(2003) Ghrelin-induced food intake is mediated via
the orexin pathway. Endocrinology 144:1506-1512.
UNI, Z., FERKET P.R., TAKO, E. and KEDAR, O. (2005)
In ovo feeding improves energy status of late-term
chicken embryos. Poultry Science 84:764-770.
UNI, Z. and FERKET, P.R. (2004) Methods of early nutrition
and their potential. Worlds Poultry Science Journal
60:101-111.
UNI, Z. and FERKET. P.R. (2003) Enhancement of
development of oviparous species by in ovo feeding.
North Carolina State University and Yissum Research
Development Company Assignees, US Pat No
6,592,878.
VICARI, E. and CALOGERO, A.E. (2001) Effects of
treatment with carnitines in infertile patients with
prostate-vesiculo-epididymitis. Human Reproduction
16:2338-2342.

10

Worlds Poultry Congress 5 - 9

Area: Chicken Breeder and Broiler Production August 07

ZAKARIA, A.H. and AL-ANEZI, M.A. (1996) Effects of

ascorbic acid and coolong during egg incubation on
hatchability culling, mortality, and body weights of
broilers chickens. Poultry Science, 75:1204-1209.
ZHAI, W., NEUMAN, S., LATOUR, M.A. and HESTER, P.Y.
(2008) The effect of in ovo injection of L-carnitine
on hatchability of white leghorns. Poultry Science
87:569-572.
ZHENG, J., FANG, J., YIN, Y.J., WANG, X.C., REN, A.J.,
BAI, J., SUN, X.J., YUAN, W.J. and LIN, L. (2010) Leptin
protects cardiomyocytes from serum-deprivationinduced apoptosis by increasing anti-oxidant
defense. Clinical and Experimental Pharmacology and
Physiology 37:955-962.
ZIEGLER, R.G. (1991) Vegetables, fruits, and carotenoids
and the risk of cancer. American Journal of Clinical
Nutrition 53:251S-259S.