Broiler egg storage induces cell death and influences embryo quality.
ABSTRACT It is well known that egg storage reduces embryo performance, but the fundamental reasons for reduced embryo quality remain unclear. The objective of this study was to investigate possible cellular and molecular mechanisms that might reduce embryo quality after egg storage. Broiler hatching eggs were obtained from the Ross 308 broiler strain, divided into 2 groups, and stored (4 and 14 d) under the same temperature and humidity conditions. Samples of the eggs were used to assess embryo quality by determining daily embryo weight (wet and dry) from 4 to 21 d of incubation. To understand possible cellular and molecular mechanisms that might affect embryo quality, blastoderms (unincubated embryos) were isolated from a sample of eggs from each storage group, dissociated into single cells, and subjected to flow cytometry analysis to differentiate between viable, apoptotic, and necrotic cell populations. Quantitative real-time PCR analysis was used to compare the expression of selected apoptotic genes (Bcl-2 homologous antagonist/killer gene, Bcl-2-associated X gene, Bcl-2-related ovarian killer gene, B-cell lymphoma 2 gene, and B-cell lymphoma xL gene) in blastoderms and embryos (6 d old after incubation). Data were analyzed by the MIXED model procedure of SAS (SAS Institute, Cary, NC), with significance set at P ≤ 0.05. After covariance analysis of initial egg weights, the results showed decreased daily embryo weights (wet and dry), an indication of decreased embryo quality that could affect hatch quality. In addition, a decrease in blastodermal cell viability was associated with an increased percentage of apoptotic cell deaths (P < 0.0001). Expression of pro-apoptotic genes (Bcl-2 homologous antagonist/killer gene, Bcl-2-associated X gene, and Bcl-2-related ovarian killer gene) were upregulated at the blastodermal level as the storage duration increased, but all genes were downregulated after 6 d of incubation. This suggests that an increase in egg storage duration could activate mechanisms of apoptotic cell death at the blastodermal level, which may be one of the molecular mechanisms that leads to reduced daily embryonic weight during incubation. Experimental controls capable of reducing the cellular and molecular mechanisms of egg storage should be used to increase embryo quality.
- SourceAvailable from: Amir Akhlaghi[Show abstract] [Hide abstract]
ABSTRACT: A hypothesis was tested that providing buffer solutions or antioxidants during egg storage may help embryos in combating the harmful effect of longer holding periods. Hatching eggs were obtained from a breeder ﬂock (35 wk) and stored for 13 d before setting. In experiment 1, the eggs were injected (d 4) with bicarbonate buffer solution (BBS) or PBS. For experiment 2, l-carnitine (LC), vitamin E (VE), and vitamin C (VC) were injected (d 7) at 3 different doses. The egg internal quality characteristics were evaluated at 2-d intervals after injection and the remaining eggs were incubated for 21 d under standard conditions. At 21 d, hatchability was recorded and unhatched eggs were broken open to assess the fertility and stage of embryonic mortality. No differences were noted in albumen pH due to using buffer solutions or antioxidants except for a decreased pH at 2 d postinjection of the high dose of VC (75 mg). In ovo injection of BBS increased the albumen index and Haugh unit at d 6 postinjection; however, the response to PBS was not different from that in the control group. In ovo injection of antioxidants did not influence the albumen index, Haugh unit, and yolk index; however, the yolk percentage was partly affected. Irrespective of the dosage, hatchability was greatly decreased following in ovo injection of buffers or antioxidants (as low as 4.3 vs. 87.5% in control), with the highest mortality percentage recorded at early embryonic stages (d 0 to 6). Data suggested that, despite improvement in certain egg internal qualities, preincubational in ovo injection of BBS, PBS, LC, VE, or VC was associated with a profoundly decreased hatchability for which the underlying mechanism(s) remain(s) to be clarified.Poultry Science 11/2012; 91(11):2970-6. · 1.52 Impact Factor
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ABSTRACT: For evaluating the ability of selenium (Se) in counteracting the adverse effects of aflatoxin B1 (AFB1), two hundred 1-day-old male Avian broilers, divided into five groups, were fed with basal diet (control group), 0.3mg/kg AFB1 (AFB1 group), 0.3mg/kg AFB1+0.2mg/kg Se (+Se group I), 0.3mg/kg AFB1+0.4mg/kg Se (+Se group II) and 0.3mg/kg AFB1+0.6mg/kg Se (+Se group III), respectively. Compared with control group, the decreased relative weight of thymus and percentages of mature thymocytes, congestion in medulla and much debris in cortex of thymus, and the increased apoptotic thymocytes were observed in AFB1 group. However, supplied dietary sodium selenite could increase the relative weight of thymus and percentages of mature thymocytes, and alleviate histopathological lesions. Compared with AFB1 group, the percentages of apoptotic thymocytes detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method and flow cytometry method in three +Se groups were decreased, the expression of Caspase-3 and Bax, through quantitative real-time PCR and immunohistochemical method, in three +Se groups were decreased, while the expression of Bcl-2 was increased. The results indicate that sodium selenite supplied in the diet, through a mechanism of apoptosis regulation, may ameliorated AFB1-induced lesions of thymus and accordingly improve the impaired cellular immune function.Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 06/2013; · 2.99 Impact Factor
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ABSTRACT: The purpose of this study was to investigate the effects of dietary NiCl2 on antioxidant function, apoptosis, and the protein expression, mRNA expression and contents of the bcl-2, bax and caspase-3 in the cecal tonsil of broilers. 280 one-day-old avian broilers were divided into four groups and fed on a corn-soybean basal diet as control diet or the same basal diet supplemented with 300, 600 and 900 mg/kg of NiCl2 for 42 days. The activities of SOD, CAT and GSH-Px, and the ability to inhibit hydroxy radical, and GSH content were significantly decreased in all experimental groups. MDA content was significantly increased. The protein expression, mRNA expression and contents of bcl-2 were decreased, and bax and caspase-3 were increased in all experimental groups. The percentages of apoptotic lymphocytes were significantly increased. In conclusion, dietary NiCl2 in excess of 300 caused oxidative stress, and then induced decreased the protein expression, mRNA expression and the contents of bcl-2, and increased protein expression, mRNA expression and the contents of bax and caspase-3 proteins in the cecal tonsil. The local intestinal mucosal immunity could finally be impaired due to the oxidative stress and apoptosis in the cecal tonsil caused by NiCl2.Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 10/2013; · 2.99 Impact Factor
Cold storage of fertile hatching eggs is a common
practice in the poultry industry. In Canada, broiler
hatching eggs are usually stored for short periods (3
to 7 d) on farms, in hatcheries, or both before incuba-
tion. This is because most breeder farms are located
at significant distances from the hatchery. Other logis-
tics, such as limited incubator space and maximization
of incubator energy, compel farmers and hatcheries to
store the eggs for periods beyond 7 d. Previous stud-
ies have shown that storage durations longer than 7 d
significantly reduce hatchability (Fasenko et al., 2001)
and increase embryonic mortality (Walsh et al., 1995;
Fasenko et al., 2001). However, from a management
point of view, the practice of having a longer storage
period helps to manage the large number of eggs col-
lected effectively and therefore cannot be disregarded.
Nevertheless, the fundamental reasons for how egg
storage typically reduces embryo viability during incu-
bation, which may be translated into posthatch chick
quality, are not well understood. Previously, embryos
from broiler eggs cold stored until d 14 displayed a
reduced growth rate and poor chick quality compared
with eggs stored for 4 d (Fasenko et al., 2001). In oth-
er studies, the storage process affected the yolk and
albumen, perivitelline membrane, blastoderm quality,
gas exchange, and CO2 assessment of embryonic me-
tabolism (Fasenko, 1996, 2007; Bakst and Akuffo 1999;
Lapão et al., 1999; Fasenko et al., 2002, 2009). A more
recent study showed that cold storage activated blasto-
dermal cell deaths; apoptosis (programmed cell death)
Broiler egg storage induces cell death and influences embryo quality
J. A. Hamidu ,†1 Z. Uddin ,* M. Li ,* G. M. Fasenko ,† L. L. Guan ,* and D. R. Barreda *
* Department of Agricultural, Food and Nutritional Science, Edmonton, Alberta, T6G 2P5, Canada;
and † Department of Animal and Range Sciences, New Mexico State University, Las Cruces 88003
ABSTRACT It is well known that egg storage reduces
embryo performance, but the fundamental reasons for
reduced embryo quality remain unclear. The objective
of this study was to investigate possible cellular and mo-
lecular mechanisms that might reduce embryo quality
after egg storage. Broiler hatching eggs were obtained
from the Ross 308 broiler strain, divided into 2 groups,
and stored (4 and 14 d) under the same temperature
and humidity conditions. Samples of the eggs were used
to assess embryo quality by determining daily embryo
weight (wet and dry) from 4 to 21 d of incubation. To
understand possible cellular and molecular mechanisms
that might affect embryo quality, blastoderms (unin-
cubated embryos) were isolated from a sample of eggs
from each storage group, dissociated into single cells,
and subjected to flow cytometry analysis to differenti-
ate between viable, apoptotic, and necrotic cell popula-
tions. Quantitative real-time PCR analysis was used
to compare the expression of selected apoptotic genes
(Bcl-2 homologous antagonist/killer gene, Bcl-2-asso-
ciated X gene, Bcl-2-related ovarian killer gene, B-cell
lymphoma 2 gene, and B-cell lymphoma xL gene) in
blastoderms and embryos (6 d old after incubation).
Data were analyzed by the MIXED model procedure
of SAS (SAS Institute, Cary, NC), with significance
set at P ≤ 0.05. After covariance analysis of initial
egg weights, the results showed decreased daily embryo
weights (wet and dry), an indication of decreased em-
bryo quality that could affect hatch quality. In addi-
tion, a decrease in blastodermal cell viability was as-
sociated with an increased percentage of apoptotic cell
deaths (P < 0.0001). Expression of pro-apoptotic genes
(Bcl-2 homologous antagonist/killer gene, Bcl-2-associ-
ated X gene, and Bcl-2-related ovarian killer gene) were
upregulated at the blastodermal level as the storage
duration increased, but all genes were downregulated
after 6 d of incubation. This suggests that an increase
in egg storage duration could activate mechanisms of
apoptotic cell death at the blastodermal level, which
may be one of the molecular mechanisms that leads to
reduced daily embryonic weight during incubation. Ex-
perimental controls capable of reducing the cellular and
molecular mechanisms of egg storage should be used to
increase embryo quality.
Key words: egg storage , cell viability , apoptosis , gene expression , embryo quality
2011 Poultry Science 90 :1749–1757
MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY
Received January 13, 2011.
Accepted April 22, 2011.
1 Corresponding author: email@example.com
© 2011 Poultry Science Association Inc.
and necrosis (injurious cell death) were real, and these
were identified in layer flocks (Hamidu et al., 2010).
In chickens, a healthy embryo (blastoderm) contains
on average 60,000 embryonic (blastodermal) cells after
oviposition (Petitte et al., 1990; Etches et al., 1996).
However, it has been hypothesized that a minimum
number of healthy blastodermal cells may be required
to initiate normal embryonic development (Fasenko
et al., 1992). The authors further suggested that the
practice of long-term cold storage may decrease healthy
embryonic cell numbers below a critical threshold, af-
fecting normal embryo development and inducing ab-
normal development or an increase in embryo deaths
in eggs stored for 14 d (Fasenko et al., 1992). These
decreases in viable embryonic cell numbers may be
due to apoptotic or necrotic cell death (Bloom et al.,
1998; Bakst and Akuffo, 1999; Hamidu et al., 2010).
The objective of the current study was to understand
the effect of the duration of egg storage on embryonic
development by investigating embryo viability (weight)
during incubation, blastodermal cell viability, and the
expression of selected apoptosis genes under 2 storage
conditions, 4 vs.14 d. However, because broiler breed-
ers have undergone intensive selection compared with
layers, it was expected that different expression levels
of apoptosis and necrosis would be observed in broiler
blastoderms and in embryos from incubated eggs com-
pared with those observed previously with layer eggs
(Hamidu et al., 2010).
MATERIALS AND METHODS
All experimental procedures involving live embryos
older than 6 d of incubation were approved by the Fac-
ulty of Agricultural, Life and Environmental Sciences
Animal Policy and Welfare Committee at the Univer-
sity of Alberta (Edmonton, Alberta, Canada) according
to the guidelines outlined by the Canadian Council on
Animal Care (1993).
Daily Embryo Weight
A total of 540 Ross 308 broiler-breeder hatching eggs
were obtained from 33-wk-old flocks, placed on racks,
and stored for 4 and 14 d under light between 16 and
18°C and 70 to 80% RH. The hatchery light was ob-
tained from fluorescent bulbs, which did not contribute
any significant heat to the cooler. To arrive at storage
durations of 4 and 14 d, half of the eggs were first ob-
tained and stored for 10 d, after which the eggs stored
for 4 d were obtained. Both were then stored for an
additional 4 d before incubation. The hatchery condi-
tions met standard conditions of a typical hatchery in
Alberta. The egg collection and storage practices were
repeated when the flock age reached 37 wk. For each
trial, a total of 270 eggs per storage treatment were
incubated at 37.5°C and 56 to 57% RH, and in the last
3 d, the eggs were transferred into a hatcher with a
temperature of 36.9°C and 60% RH. Beginning at 4 d
and up to 21 d of incubation, 30 eggs (15 eggs/storage
treatment) were opened to determine daily embryonic
wet weight. The wet samples were dried in an oven
(Despatch V Series, model VRC2-26-IE, Despatch,
Minneapolis, MN) at 65°C for 4 d to obtain the DM
Cellular and Molecular Evaluation
of Apoptosis and Necrosis
Blastoderm Preparation. Three groups of 120 fresh-
ly laid fertile hatching eggs were also obtained from
Ross 308 experimental flocks, divided into 2 groups, and
stored as mentioned above. For each storage treatment,
whole blastoderms with an intact area pellucida and
area opaca were obtained and separated into individual
cells for analysis on a flow cytometer (BD FACScan,
Becton Dickinson and Co., San Jose, CA) as described
previously (Hamidu et al., 2010). To separate each blas-
toderm, it was aspirated twice at 10 and 20 min in a 50-
μL, prewarmed (37°C), commercially available 0.25%
trypsin:0.04% EDTA solution (Invitrogen Canada Inc.,
Burlington, Ontario, Canada) after incubation at 37°C.
A total of 40 blastoderms were isolated and pooled to-
gether per storage treatment, and an aliquot of 100
μL of cell suspension containing approximately 5.0 ×
105 cells was stained with annexin V-fluorescein iso-
thiocyanate (Annexin V) and propidium iodide (PI)
fluorescent dyes to differentiate between apoptotic and
necrotic cell populations (Molecular Probes Inc., Eu-
gene, OR). The cell suspensions were subjected to flow
cytometry data analysis (BD FACScan) to differentiate
between live or viable cells (Annexin V−/PI−), early
apoptotic cells (Annexin V+/PI), necrotic cells (An-
nexin V−/PI+), and late apoptotic-necrotic cells (An-
nexin V+/PI+). The data were confirmed by ImageS-
tream multispectral flow cytometry analysis (Amnis
Corporation, Seattle, WA) with a photo snapshot of
each cell passing through the flow chamber as reported
previously (Hamidu et al., 2010).
RNA Extraction and First-Strand cDNA Synthe-
sis. In a similar manner, hatching eggs obtained from
Ross 308 flocks were stored for 4 and 14 d, as outlined
previously for gene expression analysis of typical apop-
tosis genes. Forty eggs per storage treatment were used;
intact blastoderms were isolated by using ribonuclease-
free water, pooled together, and snap-frozen in liquid
nitrogen for further studies. An additional 36 eggs
from each storage treatment were incubated for 6 d at
37.5°C and 56 to 57% RH for gene expression analysis
of growing embryos. The 6 d of incubation was cho-
sen because, at this time point, practically all embry-
onic morphology was present and the RNA extracted
was a representative sample of the entire embryo body.
The embryos were harvested under sterile conditions
HAMIDU ET AL.
for RNA extraction. Total RNA was extracted from
both blastoderms and 6-d-old embryos using TRIzol
reagent (Invitrogen Canada Inc.) and Precellys lysing
kits (Cayman Chemical, Ann Arbor, MI). The mRNA
was then reverse transcribed into cDNA using super-
script II reverse transcriptase (Invitrogen Canada Inc.)
and Oligo-dT primers (Invitrogen Canada Inc.). The
cDNA was used as a template for quantitative real-time
qRT-PCR. Five known genes associated with apopto-
sis mechanisms were selected for gene expression analy-
sis: pro-apoptotic-associated genes [Bcl-2-associated X
gene (Bax), Bcl-2 homologous antagonist/killer gene
(Bak), Bcl-2-related ovarian killer gene (Bok)] and
anti-apoptotic-associated genes [B-cell lymphoma 2
gene (Bcl-2) and B-cell lymphoma xL gene (Bcl-xL)].
Primers used in this study were designed using Primer
express 3.0 software (Applied Biosystems, Streetsville,
Ontario, Canada; Table 1). Quantitative real-time PCR
was performed using SYBR Green chemistry with a
7500 Fast Real-Time PCR System (Applied Biosys-
tems). The reaction solution contained 5 μL of Fast
SYBR Green master mix, 2.5 μL of cDNA, and 2.5 μL
of the primer set (3.2 μM) for each sample, and the
reaction was performed using the following program:
95°C for 2 min, 40 cycles at 95°C for 15 s, and 60°C for
1 min, and a dissociation stage of 95°C for 15 s, 60°C for
1 min, 95°C for 15 s, and 60°C for 15 s. For each gene,
qRT-PCR was performed in triplicate in a 96-well plate
and a negative control was included. The PCR prod-
ucts were quantified according to the standard curves
produced by amplification of serial dilutions, and the
products were identified by their melting points. The
serial dilutions were used to determine PCR efficien-
cies, and any reaction with efficiency above 85% was
used for further analysis. Relative abundance of mRNA
(cDNA) was determined at the exponential phase as
the difference between the threshold cycle values mea-
sured between 14- and 4-d storage durations. Each gene
was normalized using the average threshold cycle values
of 3 housekeeping genes (hypoxanthine-guanine phos-
phoribosyltransferase, β-actin, and ubiquitin).
All data were analyzed using SAS software (SAS
Institute, 2002–2003) with the PROC MIXED proce-
dure at P ≤ 0.05. Where significant differences were
observed, least squares means were separated by the
PDIFF option of SAS. The initial egg weight was sig-
nificant between treatments; therefore, it was used in
a covariance model to analyze final egg weight after
storage, embryo weight, chick weight, pipping time, and
Egg and Embryo Weights
Initial egg weight before storage, used to determine
embryo viability, was higher in the 4- vs. 14-d storage
treatment (Table 2). Covariance analysis showed that
the final egg weight was also higher, indicating that
differences were due to the effect of egg storage rather
than the effect of egg weight (Table 2). Egg storage also
had a significant effect on daily embryonic weight (Ta-
ble 2). Daily wet and dry embryo weights were higher
in eggs stored for 4 d compared with those stored for
14 d, except at 18 d (wet weight) and at 4, 14, 18, and
20 d (dry weight; Table 3).
Table 1. Selected genes and primers used in this study
1Bak = Bcl-2 homologous antagonist/killer gene; Bax = Bcl-2-associated X gene; Bok = Bcl-2-related ovarian killer gene; Bcl-2 = B-cell lymphoma
2 gene; Bcl-xL = B-cell lymphoma xL gene; HPRT = hypoxanthine-guanine phosphoribosyltransferase; UB = ubiquitin.
2Gene sequences were obtained from the National Center for Biotechnology Information (Bethesda, MD) and Ensembl (http://useast.ensembl.org/
index.html) genome databases. F and R indicate the forward and reverse primers, respectively, in the 5′ to 3′ direction.
3Quantitative real-time PCR.
CELL DEATH IN STORED EGGS REDUCES EMBRYO VIABILITY
Cell Viability and Cell Death
Results from the current study showed that percent-
age of live cells was significantly higher in eggs stored
for 4 d (81.17 ± 2.15) compared with those stored for
14 d (68.18 ± 2.13; P < 0.0001; Figure 1).The percent-
age of early apoptotic cells was significantly higher in
the 14-d storage treatment (17.88 ± 1.87%) compared
with the 4-d storage treatment (4.32 ± 1.89%; P <
0.0001). Percentages of necrotic and late apoptotic-ne-
crotic cells were not significantly different between eggs
stored for 4 d (10.86 ± 0.76%; 3.38 ± 0.56%) and 14 d
(10.48 ± 0.75%; 3.38 ± 0.55%), respectively (Figure 1).
Gene Expression in Broiler Blastoderms
The expression of pro-apoptotic genes (Bak, Bax,
and Bok) increased by 20-, 3.4-, and 7-fold respectively,
in blastoderms of eggs stored for 14 d compared with
those stored for 4 d (Figure 2). The expression of the
anti-apoptotic gene Bcl-2 was the same between storage
treatments (1.19-fold), but the Bcl-xL gene, a homolog
of Bcl-2, was slightly downregulated (0.85-fold) in eggs
stored for 14 d compared with those stored for 4 d.
Gene Expression in Embryos
from Incubated Broiler Eggs
The expression level of all genes investigated was
downregulated in embryos at 6 d of incubation (Fig-
ure 2). Although the downward regulation of the anti-
apoptotic genes Bcl-2 and Bcl-xL was consistent with
the current research objectives, the expression of the
pro-apoptotic genes was expected to increase rather
Table 2. Effect of the duration of egg storage on egg weight and chick performance
before storage1 (g)
after storage2 (g)
a,bMeans in the same column with different superscripts differ significantly (P ≤ 0.05).
1Egg weight at the time of collection, before storage.
2Egg weight at the time of setting into incubator.
3Chick weight immediately after hatching.
4Time taken from setting eggs in incubator to external pipping.
5Time taken from setting eggs in incubator to the chick being hatched out.
6Standard error of means belonging to different treatments but within the specific egg or embryo parameter.
7P-value of columnar means.
Table 3. Effects of the duration egg of storage on wet and dry embryo weights
Wet embryo weight1 (g)Dry embryo weight2 (g)
4 d14 dSEM3
4 d14 dSEM4
a,bMeans in the same column with different superscripts differ significantly (P ≤ 0.05).
1Least squares means of wet embryo weight after breaking open 30 eggs (15 eggs/storage) daily.
2Least squares means of dry embryo weight after drying the samples in oven for 4 d at 65°C.
3Standard errors of the means belonging to different treatments within each day and wet embryo weight.
4Standard errors of the means belonging to different treatments within each day and dry embryo weight.
HAMIDU ET AL.
The embryonic weight results established in the cur-
rent study are consistent with previous results in which
prolonged storage reduced daily embryo weight (Chris-
tensen et al., 2002). Other studies have reported simi-
lar reduced embryo weights when the duration of egg
storage exceeded 8 d (Yalcin and Siegel, 2003). The
reduction in embryo weight may be due to a reduc-
tion in blastodermal cell numbers even before the eggs
were incubated (Foulkes, 1990; Hamidu et al., 2010).
A reduction in embryonic cell numbers could limit the
number of cells available to take up O2 for metabolic
activities, but this has not been fully investigated. Al-
though previous results have shown that this is pos-
sible, using O2 uptake or CO2 production values alone
may underestimate or overestimate metabolic activi-
ties during different days of incubation (Fasenko et al.,
2002; Fasenko, 2007).
The results for cellular apoptosis agree with the re-
sults from our previous study (Hamidu et al., 2010),
in which viable embryonic cell numbers also decreased
when the duration of egg storage increased from 4 to
14 d. In our previous study, we also observed that the
reduction in blastodermal cell numbers was primarily
due to an increased number of cell deaths from apop-
tosis and necrosis (Hamidu et al., 2010). Although the
kinds of cell deaths identified in our previous study
were the extremes of apoptosis (late apoptosis) and ne-
crosis (often associated with a release of inflammatory
responses, such as toxins), only the early stage of apop-
tosis was significant between treatments in our current
study. Other studies have found similar trends (Foul-
kes, 1990; Bloom et al., 1998; Bakst and Akuffo, 1999).
However, Bakst and Akuffo (1999) reported on turkey
blastoderms, which are quite different from those of
broiler breeders. Although Bloom et al. (1998) also
measured apoptosis in broiler blastoderms, this study
appeared to be somewhat subjective. In that study, the
authors used a single dye to characterize nuclear frag-
mentation in addition to reporting on DNA fragment
sizes larger than the characteristic 180 bp of fragment-
ed DNA usually used as evidence of apoptosis. It can
therefore be argued that the DNA fragments measured
may not have been due to apoptosis or apoptosis alone.
We can also speculate that the DNA fragmentation
identified in their study may have been due to DNA
damage during sample preparation. In our previous
and current studies, we decided to provide additional
proof or evidence of apoptosis by snapshots of apopto-
sis events after identification of the 2 different types of
cell deaths with 2 popular vital dyes (Hamidu et al.,
2010). Because of the arguments that surround PI mea-
surement of necrosis events, in our studies we added
a dye that was able to measure the degree of nuclear
fragmentation as a control, to properly assess necrotic
events (Rieger et al., 2010). The discrepancy resulting
in the different types of cell deaths between layer eggs
Figure 1. Analysis of live, apoptotic, and necrotic cells in broiler breeder blastodermal cell populations, and identification of genes important
in the induction of apoptosis in Ross 308 broiler breeder eggs stored for 4 and 14 d. A flow cytometry evaluation (ImageStream multispectral
flow cytometry analysis, Amnis Corporation, Seattle, WA) of live and dead cells based on the intensity of 2 staining fluorescent dyes, annexin
V-fluorescein isothiocyanate and propidium iodide (Molecular Probes Inc., Eugene, OR): live cells (Annexin V−/PI−), early apoptotic cells (An-
nexin V+/ PI), necrotic cells (Annexin V−/PI+), and late apoptotic-necrotic cells (Annexin V+/PI+). Percentage of live cells was higher in eggs
stored for 4 d than in those stored for 14 d (P < 0.05; n = 3). Percentage of early apoptotic cells was higher in eggs stored for 4 than in those
stored for 14 d (P < 0.0001). Percentages of necrotic cells and late apoptotic-necrotic cells were not different between egg storage treatments.
CELL DEATH IN STORED EGGS REDUCES EMBRYO VIABILITY
in the previous study (Hamidu et al., 2010) and the
broiler breeders in the current study could have been
due to different mechanisms or pathways of cell deaths.
In addition, we believe that the same genes character-
ized in our current study may not have been the same
set of genes causing cell death in layer strains. The fact
that we obtained a significant amount of necrosis in our
previous study meant that certain genes or agents, such
as tumor necrosis factors, which are more reason for
necrosis, may have been one of the key players in the
cell death of layer embryos. However, these are hypoth-
eses that need to be investigated. Also, because broiler
blastoderms had only a significant amount of early
apoptosis between the 2 storage treatments whereas
layer blastoderms had later apoptosis (a more acute cell
death than early apoptosis), it could mean that broiler
blastodermal cells may be more tolerant of cold storage
than layer blastoderm cells. All these reasons could be
due to strain differences.
In the current study, a negative control obtained from
freshly laid eggs was not included because we showed
in our previous study that blastoderms obtained from
freshly laid eggs did not perform as well as eggs from
d 4 and 14 (Hamidu et al., 2010). We showed that the
percentage of unexplainable necrotic cell deaths was ap-
proximately 73% in freshly laid eggs. Similarly, Bakst
and Akuffo (1999) reported that in turkey blastoderms,
approximately 30% of blastodermal cell deaths observed
within 48 h of egg storage could not be explained. All
these results appear to indicate why producers are not
interested in incubating freshly laid eggs until after a
few days of storage: apart from proper cellular divi-
sion, this also helps to create the air cell of the egg for
proper embryonic development and respiration. Based
on the results of our previous (Hamidu et al., 2010)
and current studies, we suggest that the reduction in
blastodermal cell numbers after egg storage for 4 to14 d
could possibly be the reason for decreased embryo qual-
ity and poor embryonic performance in eggs stored for
a longer duration (Mather and Laughlin, 1977; Fasenko
et al., 1992, 2001; Fasenko, 2007). It is interesting that
the amount of early apoptotic cell death was higher in
broiler breeders. Because apoptosis is a metabolically
active cell death mechanism, it will lead to a reduction
in energy in embryos that experience a higher amount
of apoptosis. Early cell apoptosis may not lead to im-
mediate death of the embryo; however, a decrease in
viable embryonic cell numbers, embryonic activities,
embryo quality, and even hatch quality may be immi-
nent. During the early stages of apoptosis, only small
Figure 2. Gene expression profiling of pro-apoptotic and anti-apoptotic genes. This figure shows, on a log scale, how different genes were
expressed after storage of broiler eggs for 4 and 14 d. Each bar indicates the difference between the 14- and 4-d egg storage treatments after each
treatment was normalized with housekeeping genes. Genes that were upregulated are shown with positive bars (up), whereas genes that were
downregulated are shown with negative bars (down). A positive bar means that the difference between the 14- and 4-d treatments was greater
than 1 (upregulation), whereas a negative bar means that the difference between the 14- and 4-d treatments was less than 1 (downregulation).
The results indicate that in blastoderms, all genes were upregulated, the B-cell lymphoma 2 gene (Bcl-2) did not change, and the B-cell lymphoma
xL gene (Bcl-xL) was downregulated slightly. In broiler embryos from eggs incubated for 6 d, all the genes were downregulated. Bak = Bcl-2
homologous antagonist/killer gene; Bax = Bcl-2-associated X gene; Bok = Bcl-2-related ovarian killer gene.
HAMIDU ET AL.
amounts of phosphatidylserine, a phospholipid-like cell
membrane protein that is normally located on the in-
ner side of the cell membrane, have translocated to the
cell outer membrane, a physiological signal indicating
that apoptosis has begun. However, the cell membrane
still remains intact, which otherwise would indicate a
late stage of apoptosis. These early apoptotic cells will
most likely appear to be viable cells under a light mi-
croscope; therefore, their immediate negative effects
may not be felt suddenly. Historically, apoptosis has a
healthy physiological function for the embryo (Kerr et
al., 1972); nevertheless, the approximately 18% of ear-
ly apoptosis observed in the currently study in broiler
eggs stored for 14 d may have an additional effect on
embryonic development that may need to be taken seri-
ously. Additionally, because early apoptotic cells will
eventually continue into late apoptosis, this mechanism
will lead to a reduction in the number of viable embry-
onic cells, which may be important for critical embry-
onic development during incubation.
Physiologically, a reduction in blastodermal cell num-
bers may also reduce the amount of O2 that each em-
bryo can take up for metabolic activities. A reduction
in embryonic metabolism may suggest that the embry-
os do not have enough cells to make effective use of the
available O2 to break down carbohydrate, fat, or pro-
tein molecules to release the needed energy for embry-
onic growth. This may have been the mechanism result-
ing in the reduced embryo weight. Because apoptosis is
a mild form of cell death, a recent study suggests that
the potential to control the event and its impact on em-
bryonic or cell development could be higher than with
necrosis (Fabbri et al., 2006). Normally, when apop-
tosis reaches the late stages, when cells have already
divided into apoptotic bodies, it is acceptable that the
process is irreversible. However, some studies suggest
that cells can recover, but only if they have not reached
the late stage of apoptosis, where they can be engulfed
by nearby phagocytic cells (Green and Beere, 2001).
Other studies have reported that for cells in which
caspases (enzymes that execute apoptosis) have been
activated, the cells still progress through a state of be-
ing mostly dead, a stage that physically resembles the
early phase of apoptosis but from which cells can fully
recover (Hoeppner et al., 2001; Reddien et al., 2001).
Because apoptosis, unlike necrosis, has a well-known
molecular basis, an understanding of this mechanism
may provide information that will help in the design
of future experiments to reduce some of the negative
effects of prolonged egg storage (McKenna et al., 1998;
Tavernarakis, 2007; Hitomi et al., 2008). It was for this
reason that we selected a few apoptosis genes to under-
stand the trends in their expression, which may guide
us in our future experiments to use molecular regula-
tion of cell death to control the processes that reduce
viable embryonic cell numbers and embryo quality.
In our current study, the occurrence of apoptosis in
broiler blastoderms appeared to be dependent on in-
creased expression of the pro-apoptotic genes Bak, Bax,
and Bok. In a previous study (Bloom et al., 1998), tran-
scripts of the 2 anti-apoptotic genes, Bcl-2 and Bcl-xL,
were detected in blastoderms. However, no study has
reported on the expression of pro-apoptotic genes in
chicken blastoderms to date. The expression levels of
either pro-apoptotic or anti-apoptotic genes are very
important events during apoptosis. It is normally ex-
pected that for apoptosis to proceed, environmental ef-
fects, such as temperature, cause the expression of the
pro-apoptotic genes to increase above that of the anti-
apoptotic genes (Yin et al., 1997; Ikeda et al., 1999;
Myers et al., 2008; Osborne et al., 2008). Other studies
have confirmed that higher expression of Bax in partic-
ular, compared with Bcl-2, resulted in the cells sending
a death command signal, but when Bcl-2 dominated,
cell death was inhibited at the expense of cell survival
(Korsmeyer, 1999; Suyama et al., 2001; Walsh et al.,
2008). At the embryonic level, after incubation for 6 d,
the expected expression levels of most of these genes
were not the same and therefore may require further
investigation. Although we would have expected that
in apoptotic cells, the pro-apoptotic genes would be
upregulated, the downregulation of all the genes con-
tradicts the known mechanisms initiated by apoptotic
cells; that means this unknown mechanism changes the
ratio in favor of the pro-apoptotic vs. the anti-apop-
totic genes. It appears speculative that apoptosis was
prevented to some extent when eggs were incubated
(incubation involves application of heat and O2 to eggs
to initiate embryo development), as indicated by the
gene expression results at the embryonic d-6 level; how-
ever, previous studies have shown that warming eggs
before incubation appears to improve embryo quality,
increase the number of viable embryonic cells, and im-
prove hatchability, which may have been due to the re-
stricted expression of the pro-apoptotic genes (Fasenko
et al., 2001; Reijrink et al., 2010). Similarly, Foulkes
(1990) reported that the reduction in blastodermal cell
numbers was less in egg stored at higher temperatures
compared with lower temperatures. The results of the
current study and from previous research may indicate
that serious research on the manipulation of storage
practices, such as the application of heat and perhaps
O2 treatments, may reduce the negative effects of egg
storage on embryo quality and hatchability.
Based on the data established from the current study,
a focus on studying gene expression as well as using
previous industrial activities, such as prestorage warm-
ing, may be some of the most effective ways to reduce
the negative effects of prolonged egg storage. Currently,
a gene called eukaryotic initiation factor 5A has been
identified in some higher animals and plants to mini-
mize apoptosis and improve survivability of the embryo
(Caraglia et al., 2003; Hopkins et al., 2008). An inten-
sive study of such a gene in poultry species may po-
tentially be used to limit the amount of apoptosis that
occurs during cold storage. Such investigations could be
extended to identify broiler breeder eggs that are resis-
tant to cold storage. Recently, it has been revealed that
CELL DEATH IN STORED EGGS REDUCES EMBRYO VIABILITY
some layer strains that may be resistant to the negative
effects of prolonged egg storage have been identified,
but this knowledge may require further testing to con-
firm this information (C. Robert, Lohmann Tierzucht
GmbH, Cuxhaven, Germany, personal communication).
The current information provides hope that there may
be broiler strains resistant to the negative effects of egg
storage, but additional studies are required to identify
In conclusion, our study shows that storing broiler
breeder eggs for a longer duration led to a reduced
embryonic quality during incubation, reduced viable
embryonic cell numbers, and increased early apoptosis
events, such as increased expression of pro-apoptotic
genes and reduced expression of anti-apoptotic genes
at the blastodermal cell level. Because apoptosis is a
genetically regulated event, it can be controlled. There-
fore, additional research is needed to focus on reducing
the induction of apoptosis when the duration of egg
storage is extended beyond the 7-d period. Appropri-
ately studying the genes that execute apoptosis and
prioritizing effective mechanisms that can control their
actions should be the next direction to increase em-
bryonic survival in the future. The current study has
potentially provided some insights into why egg storage
reduces hatchability and increases embryonic mortality.
We hope that the findings of this study can also lead
to the discovery of new broiler breeder strains that can
withstand the impact of apoptosis caused by cold stor-
age and can increase embryo survival during egg stor-
age and incubation.
We thank the Agriculture and Food Council (Nisku,
Alberta, Canada), Poultry Industry Council (Guelph,
Ontario, Canada), Alberta Livestock Industry Devel-
opment Fund (Edmonton, Alberta, Canada), Alberta
Agricultural Research Institute (Edmonton, Alberta,
Canada), the Poultry Research Centre (University of
Alberta, Edmonton, Alberta, Canada), and the Natural
Sciences and Engineering Research Council of Canada
(Ottawa, Ontario, Canada) for their financial support.
We are also grateful to Erin Christopher and Dorothy
Rutkowski, University of Alberta, for their assistance.
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