Comparative Biochemistry and Physiology, Part A 156 (2010) 373–379

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Comparative Biochemistry and Physiology, Part A

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a

Growth of the chicken embryo: Implications of egg size

Jacopo P. Mortola a,b,⁎, Khalid Al Awam a,b
a
Department of Physiology, McGill University, 3655 Sir William Osler Promenade, Montreal, Quebec, Canada H3G 1Y6
b
Neonatal Research Chair, King Saud University, Riyadh, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history: Among avian species with large differences in egg size, changes in eggshell conductance and incubation time
Received 28 January 2010 permit the water loss necessary for embryonic development. To what extent this happens for different-size
Received in revised form 3 March 2010 eggs within a species is much less known. Chicken eggs with fresh egg weight (Wegg) either large (L,∼66 g)
Accepted 4 March 2010
or small (S,∼51 g) were incubated at 38 °C and 60% humidity; their yolk and albumen scaled almost in
Available online 20 March 2010
proportion to Wegg. Eggshell gas conductance scaled to 0.77 of Wegg, as it occurs inter-specifically, while
Keywords:
external pipping and hatching occurred at similar times in S and L. Hence, L lost less water during incubation
Allometry than S, and embryos of L were over-hydrated and those of S were dehydrated. The absolute values of
Avian development embryo's weight, growth rate, oxygen consumption and the weight of the chorioallantoic membrane were
Egg constituents similar between S and L during the first half of incubation, and greater in L in the second half. Incubation in
Hypoxia oxygen consumption hypoxia reduced growth rate in both sets and maintained the difference in growth trajectories between
S and L. The energetic cost of growth and tissue maintenance did not differ significantly. It is concluded
that,10 + 9 among chicken eggs of different sizes, 1) the growth rate of the embryo relates to the size of its
egg, probably genetically and because of differences in water content, 2) eggshell conductance contributes,
but incubation time does not, to the requirements for water loss. Therefore, the egg water balance during
incubation may be the physiological constraint that limits the maximal variability in egg size compatible
with embryonic survival.
© 2010 Elsevier Inc. All rights reserved.

1. Introduction crested cormorant (Phalacrocorax auritus) from 40 to 60 g (Dzialowski

During incubation, avian eggs lose water in the form of water vapour
through the eggshell pores, a process crucial for the normal develop-
ment of the embryo (Rahn and Ar, 1974). Because surfaces do not scale
in proportion to volumes, small eggs risk dehydration and large-eggs
risk an excess of water retention (Paganelli et al., 1974). Avian species
lay eggs with enormous differences in size, and various mechanisms
have evolved to balance the geometrical constraints on water loss. By
comparison to small eggs, large eggs, usually laid by large species, have
values of eggshell pore area, daily water loss and eggshell conductance
higher than would be expected by geometric scaling (Drent, 1970; Ar
et al., 1974; Ar and Rahn, 1985), and longer incubation times (Rahn and
Ar, 1974). In fact, the appropriate combination of incubation time and
eggshell conductance is the main feature established through evolution
that permits embryonic development in eggs ranging in weight from
less than half a gram (some hummingbirds) to more than 1 kg (ostrich)
(Rahn et al., 1974; reviewed in Mortola, 2009).
Some variability in egg size occurs also within a species. For example,
the egg weight of the emu (Dromaius novaehollandiae) ranges from 400
to 700 g (Dzialowski and Sotherland, 2004) and that of the Double-
⁎ Corresponding author. Department of Physiology, McGill University, 3655 Sir
William Osler Promenade, Montreal, Quebec, Canada H3G 1Y6. Fax +1 514 398 7452.
E-mail address: jacopo.mortola@mcgill.ca (J.P. Mortola).
et al., 2009). In the common chicken (Gallus gallus) fertile eggs can be as
small as 40 g or as large as 75 g. Genetic differences among females, food
availability, seasonal variation, maternal size and age are factors
possibly contributing to egg size variability, and important differences
can occur within the same clutch (Lundberg and Vaisanen, 1979;
Rhymer, 1988; Suarez et al., 1997; Michel et al., 2003, for additional
references). In genetically selected pure breeds of domestic species with
unlimited food availability, seasonal variations and maternal age are the
main reasons for changes in egg size. The heavier eggs produce larger
chicks (e.g., Schifferli, 1973; Lundberg and Vaisanen, 1979; Batt and
Prince, 1979; Rhymer, 1988; Michel et al., 2003; Dzialowski and
Sotherland, 2004; Ipek and Dikmen, 2007; Dzialowski et al., 2009), but
how this comes about is not totally clear. If embryonic growth followed a
fixed, genetically determined trajectory not dependent on the size of its
egg, differences in hatchling's weight must reflect events occurring
toward the end of incubation. In fact, close to hatching, the yolk
assimilation and oxygen availability (Høiby et al., 1983; Tazawa et al.,
1992; Szdzuy et al., 2008) may limit body growth more in embryos of
small eggs than in those of large eggs, because yolk circulation and
eggshell gas conductance should be less in small-size eggs. In addition,
larger eggs have greater yolks, and the abdominal incorporation of the
yolk residue at hatching should boost the weight of the embryos of
larger eggs (Williams, 1994). Another, undemonstrated, possibility is
that embryonic growth rate bears some correlation with the size of its

1095-6433/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpa.2010.03.011
374 J.P. Mortola, K. Al Awam / Comparative Biochemistry and Physiology, Part A 156 (2010) 373–379

egg since the early phases of incubation. In this case, large eggs should
differ from small eggs in eggshell conductance and incubation time to
meet the embryonic requirements for water balance, as it is the case of
different-size eggs of different species.
The current study was performed on chicken eggs, first, to
establish the growth patterns of embryos of small and large eggs.
The results indicated that the growth trajectories differed around the
middle of incubation, whether in normoxia or hypoxia, as if small and
large eggs belonged to different species. Therefore, we asked what
mechanisms permitted eggs of different sizes to meet the require-
ments for embryonic water balance.
2. Materials and methods
Experiments were performed on eggs and embryos of the domestic
chicken (G. gallus), White Leghorn variety. They were all from a local
producer, and fed the same diet of standard chicken feed (88%)
complemented by other protein sources, vitamin and mineral supple-
ments. The average fresh egg weight (Wegg) of ∼2700 eggs was 59.4 ±
5.3 SD. Of these, about half fell in the range 55–63 g, considered the
common range. Hence, we have arbitrarily defined as small (S) or large
(L) eggs with fresh Wegg respectively b55 g or N63 g. For each of the
measurements performed, the average Wegg of L and S was, respec-
tively, ∼51 g and ∼66 g; hence, the two groups differed by ∼30%
(Table 1). To construct the allometric relationship between a given
parameter Y and Wegg (Y = a · Weggb), also eggs of the normal range
(Wegg N55 g and b63 g) were included. Egg surface area (in cm2) was
calculated for each egg from its fresh Wegg in g, as 4.835 · Wegg0.662
(Paganelli et al., 1974).
Some eggs were studied fresh. The large majority, though, was studied
at one of various days of incubation, most commonly at embryonic day E5,
E8, E11, E14, E17 or E19. Additional experimental days were included for
special cases (see Results). Details about the number of eggs used for each
measurement are given in Table 1 and in the figures.
loss (g day−1), which was converted to water loss (mL day−1) after
accounting for the density of water vapour (Ar et al., 1974). The
difference in partial pressure of water ΔP(H2O) between the egg and the
incubator was calculated from the corresponding T and relative
humidity. From these data, the water conductance of the egg, G(H2O),
mL· day−1 · torr-1, corresponded to the ratio between the daily water
loss and ΔP(H2O).
2.2. Weights and dimensions
Eggshell, yolk and albumen of freshly laid eggs were weighed on a
digital scale accurate to 10−2 g. The thickness of the eggshell (including
the outer membrane) was measured on fragments dissected from two
regions, the egg smaller circumference and the blunted end covering the
air cell. The fragment was placed vertically under a microscope and its
cross section was projected on a TV-monitor by a camera connected to a
microscope; the thickness was measured with a caliper and converted in
µm according to the magnification factor. Eggshell density was
measured on similarly dissected fragments. To this end, first, the
fragments were weighed both in air and in water, with a scale accurate
to 10−4 g (Sartorius, adapted with a specific gravity determination kit).
Then, density (weight/volume) was calculated from the weight in air
and the difference in weight between air and water, with correction for
temperature-dependence of water density.
On the day of the measurement the egg was weighed and the embryo
killed by exposure to CO2 and cold. The egg was cut open, the embryo and
the chorioallantoic membrane (CAM) were dissected free from adjacent
tissues, drained of blood or any fluid contents, lightly blotted and
weighed on a digital scale accurate to 10−4 g. The head of the embryo
was similarly weighed, as representative of those organs that change
rapidly during embryonic development (Azzam and Mortola, 2007).
2.3. Oxygen consumption (V̇O2)
̇ was monitored with an open-flow methodology (Frappell et al.,
VO 2

2.1. Incubation
After a storage time (at 15 °C) of no more than 6 days from laying, the
fresh Wegg was noted and at midday (E0) the eggs were placed in
incubators set at the temperature (T) of 38 °C and 60% relative humidity.
For those incubated in hypoxia, the desired oxygen concentration (15%
O2) was obtained by leaking a small stream of warmed and humidified
N2 into the incubator from a pressurized tank, under the control of a flow
1992) adapted to the avian embryo and hatchling (Menna and Mortola,
2002). The egg respirometer was submerged in a water bath that
maintained the inside T at 38 °C. A steady gas flow of 100 mL/min was
delivered continuously through the respirometer, and calibrated gas
analyzers (Sable Systems International Fox, Henderson, NV) monitored
the inflow and outflow O2 and CO2 concentrations, after the gas had
passed through a drying column. The output of the analyzers was dis-
played on a computer monitor during on-line acquisition. VO ̇ was
2

meter (Azzam et al., 2007; Szdzuy and Mortola, 2007). The O2
concentration of the incubator was analyzed continuously (Foxbox
fuel cell gas analyzer, Sable Systems Int., Las Vegas, NV) and displayed on
a computer monitor. Incubation T and relative humidity were monitored
by a data logger and a hygrometer placed inside the incubator; the
former collected the T-data every 10 min, while humidity was read daily.
The difference between the initial and final egg weights, divided by
derived from the flow rate and the inflow–outflow concentration differ-
ence. The values are presented in mL/day at STPD (standard tempera-
ture, pressure, and dry conditions).
Estimates of the energetic cost of tissue maintenance and of
tissue growth were obtained assuming the former proportional to
embryo's weight (Wembryo) and the latter to the rate of growth
(GR = ΔWembryo/Δday). In this way, VO ̇ = a · Wembryo+ b · GR, a
2

the number of days of incubation, represented the average daily weight and b being the respective proportionality coefficients (Vleck et al.,
1980a; Mortola and Cooney, 2008). From the data of VO ̇ , Wembryo and
2

GR over the period E5-to-E19, the equation was solved for a and b using
Table 1
the linear regressions VO ̇ /Wembryo = a + b · GR/Wembryo and VO ̇ /
Number and weight of Small and Large eggs for each of the variables studied. 2 2

GR = b + a · Wembryo/GR. In these equations, a represents the average

Variable measured Small eggs Large eggs cost (mL O2) for maintaining 1 g of embryonic tissue for 1 day, and b
N Initial weight, g N Initial weight, g represents the average cost (mL O2) of growing 1 g of tissue (Mortola
Internal pipping and hatchability 73 51.5 ± 0.4 31 65.7 ± 0.3 and Cooney, 2008).
Eggshell, yolk, and albumen weight 74 51.4 ± 0.4 43 66.4 ± 0.4
Eggshell thickness and density 28 52.0 ± 0.4 27 66.5 ± 0.5 2.4. Data analysis
CAM weight 78 50.9 ± 0.4 72 67.3 ± 0.3
Egg shell H2O conductance 215 51.9 ± 0.2 253 66.4 ± 0.1
Embryo's growth (normoxia) 316 52.1 ± 0.4 375 66.5 ± 0.3 Data are presented as means ± 1 SEM. For each measurement, the
Oxygen consumption 169 51.9 ± 0.2 135 66.6 ± 0.2 data of S and L were compared by Student's t-test. Exponents (b) and
Cost of growth and maintenance 158 51.9 ± 0.2 127 66.6 ± 0.2 intercept (a) of the equations relating a variable to Wegg (allometric
Embryo's growth (hypoxia) 112 52.1 ± 0.2 182 66.4 ± 0.2 relationship) were derived from the least-squares regression analysis
Values are means ± 1 SEM. of the log-transformed equation Y = a · Weggb. Critical values of the
 J.P. Mortola, K. Al Awam / Comparative Biochemistry and Physiology, Part A 156 (2010) 373–379 375

correlation coefficients r, differences between slopes or between two

sets of data were considered statistically significant at P b 0.05.

3. Results

The frequency of sterile eggs was about 10% for both S and L, about
double the rate of common-size eggs. The time of the star fracture of the
external pipping phase in S (20.1 days ± 0.1) and L (20.2 days ± 0.1) did
not differ significantly. Hatching occurred a few hours later in both
groups, with no significant difference in time.

3.1. Fresh egg constituents

L eggs had greater eggshell, yolk and albumen (Fig. 1). The allometric
equations, which included data from common-size eggs, were Fig. 2. Weight of the chorioallantoic membrane at various days of incubation, in small-
size (triangles) and large-size eggs (circles). Numbers in brackets (italics) indicate
 
0:615 2 number of small and large eggs studied. Symbols are group mean, bars 1SEM. *,
Eggshell W ðg Þ = 0:61⋅Weggðg Þ r = 0:44; N = 135 ð1Þ
significant difference between the two groups (P b 0.05).

 
1:104 2
Albumen Wðg Þ = 0:39⋅Weggðg Þ ; r = 0:90; N = 129 ð2Þ
difference reaching statistical significance at E17 (Fig. 2). After
 
0:938 2 normalization by Wegg, the CAM of L tended to be smaller (∼ 89%)
Yolk Wðg Þ = 0:35⋅Weggðg Þ ; r = 0:60; N = 132 ð3Þ
than in S, and significantly so at E8 and E10.

The scaling exponent of the eggshell close to 2/3 implies that the
relative W of the eggshell progressively decreases the bigger the Wegg.
Indeed, eggshell W/Wegg averaged 0.134 ± 0.001 in S and 0.121 ± 0.001
in L (P b 0.001). Albumen and yolk scaled, respectively, slightly above
(1.104) and slightly below (0.938) direct proportionality. Hence, even
though L had a greater absolute quantity of yolk, the yolk–Wegg ratio
and the yolk–albumen ratio (respectively, 0.26 ± 0.01 and 0.42 ± 0.02)
were significantly lower than in S (respectively, 0.28 ± 0.003, P b 0.05,
and 0.48 ± 0.02, P b 0.005).
Neither eggshell density nor thickness differed significantly between
the two groups of eggs; the former averaged 2.03 ±0.02 g/mL and 1.98±
0.02 g/mL, the latter 365 ±8 µm and 357±7 µm in S and L, respectively.
3.2. Chorioallantoic membrane
The chorioallantoic membrane (CAM) begins to form at day 4 of
incubation and continues to grow until ∼ E12, when its weight reaches
a plateau. This growth pattern was very similar in S and L until days
10–11; later, the CAM of L was slightly heavier than that of S, the
3.3. Water loss and eggshell conductance
During incubation, the egg weight loss averaged 0.27 ± 0.005 g/
day in L and 0.22 ± 0.004 g/day in S (P b 0.001). The allometric
relationship of the daily weight loss (mg/day), which included data
from these and normal-size eggs, was
0:770
Daily weight lossðmg = dayÞ = 10:4⋅Weggðg Þ ð4Þ
ðF0:09 SDEV; N = 692; Fig: 3Þ
This exponent was not significantly different from 0.62 (the
scaling exponent of egg surface area, Eq. 1) and significantly lower
than 1 (P b 0.02) (Fig. 3). Direct comparison between S and L
confirmed that the daily weight loss did not differ significantly
when normalized by egg surface area (3.4 ± 0.06 mg/day/cm2 in both
S and L), and was significantly lower in L when normalized by Wegg
(4.3 ± 0.08 and 4.0 ± 0.07 mg/day/g in S and L, respectively; P b 0.02).
Because all eggs were incubated under the same conditions of

Fig. 1. Allometric relations (double-log plot) of albumen, yolk and eggshell weight in
fresh eggs of different weight. Albumen weight (g) = 0.39 · Wegg (g)1.104, (r2 = 0.90,
N = 129); yolk weight (g) = 0.35 · Wegg (g)0.938, (r2 = 0.60, N = 132); eggshell weight
(g) = 0.61 · Wegg (g)0.615 (r2 = 0.44, N = 135). The thick lines are the best-fit linear
relationships through the data points; the thin lines represent the 95% confidence
intervals.
Fig. 3. Allometric relation of the daily water loss and initial fresh weight of the egg
(double-log representation). Daily water loss (mg/day) = 10.41 · Wegg (g)0.770
(± 0.093 SDEV; N = 692). The exponent statistically lower than 1 indicates that, the
larger the egg, the smaller the daily water loss in relation to egg size. The thick line is the
best-fit linear relationship through the individual data points; the thin lines represent
the 95% confidence intervals.
376 J.P. Mortola, K. Al Awam / Comparative Biochemistry and Physiology, Part A 156 (2010) 373–379

temperature and humidity, that is, with the same partial pressure
gradient of water vapour, the exponent of Eq. 4 was the same as that
of the water vapour conductance, GH2O:

0:770
GH2 OðmL = day = mmHgÞ = 0:64⋅WeggðgÞ ðF0:093 SDEV; N = 692Þ
ð5Þ

3.4. Embryo's growth

No statistical difference in the average values of embryonic W

(Wembryo) occurred until day E11, although from E5 the group
averages were consistently lower in S (Fig. 4, bottom, continuous
line). From E11 included until the end of incubation, the Wembryo of
S was statistically lower than that of L (Fig. 4, top). After normalization
by Wegg, L were statistically smaller than S since E8. In conclusion, L
embryos were growing at a faster pace than S for almost the whole
incubation, but not in proportion to the mass of their eggs. The
allometric relationships at some of these ages are presented in Fig. 5.
At end-incubation (E20), the exponent of the function representing
the combined weight of embryo and yolk (1.03) did not differ from 1;
without yolk, the exponent (0.61) was significantly less than 1. The
difference between the two exponents, 0.416, is significantly higher
than 0, meaning that the fraction of embryo's W represented by the

Fig. 5. Allometric relations of the embryo's weight at different days of incubation

(double-log representation). The lines refer to the best-fit linear functions: Day 8,
Y = 1.99 · Wegg0.024 (r2 = 0.022, N = 107); day 11, Y = 2.64 · Wegg0.103 (r2 = 0.007,
N = 189); day 14, Y = 1.71 · Wegg0.454 (r2 = 0.12, N = 360); day 17, Y = 4.28 · Wegg0.382
(r2 = 0.09, N = 195); day 20 without yolk, Y = 2.5 · Wegg0.614 (r2 = 0.20, N = 71); day
20 with yolk, and Y = 0.663 · Wegg1.027 (r2 = 0.79, N = 169).

yolk residue is greater in L than in S. In fact, the equation scaling the

yolk residue to Wegg was:

 
2:050 2
Yolk residueðgÞ = 0:003⋅WeggðgÞ r = 0:438; N = 46; Pb0:01
ð6Þ

with the exponent (2.050) significantly higher than 1 (P b 0.001).

The differences in embryonic growth between S and L were
matched by proportional differences in head growth; hence, the
relationship between the weight of the head and Wembryo was the
same for the two groups of eggs (Fig. 6).

3.5. Hypoxic incubation

Incubation in 15% O2 decreased the rate of growth in both S and L; the

Fig. 4. Top panel: body weight of the embryos in small-size (triangles) and large-size
eggs (circles), at various days of incubation. The open symbols, dashed lines, refer to
embryos incubated in hypoxia. *, statistically significant difference between Small and
Large. Symbols are group mean, bars 1SEM. When not visible, SEM was within symbol
size. Bottom panel: percent ratios between the embryos of Small and Large eggs. Short
dashed line refers to embryos growing in hypoxia. Pairs of numbers in round and
squared brackets indicate number of, respectively, Small-eggs and Large-eggs embryos
Wembryo of S was less than that of L at all ages tested, on average about
12% less, and the difference was significant at E14, E17 and E19 (Fig. 4).
As it was the case in normoxia, in hypoxia the Wembryo/Wegg ratio of S
exceeded that of L at all ages, on average by 12% (±3).
The blunting effect of hypoxia on embryo growth did not modify
the normal relationship between head and embryo weights; the data
points described a unique relationship, irrespective of the size of the
egg (Fig. 6).
3.6. Oxygen consumption
The VO ̇ of S and L did not differ statistically untill E12 (Fig. 7, top); after
2
that age, it was consistently lower in S, by about 13–15% (Fig. 7, bottom).
These differences were no longer present after VO ̇ normalization by
2

in normoxia (round) and hypoxia (squared). Wembryo (mL/day/g).

 J.P. Mortola, K. Al Awam / Comparative Biochemistry and Physiology, Part A 156 (2010) 373–379
Fig. 6. Relationship between the weight of the embryo and that of its head for small-size
(triangles) and large-size eggs (circles). The open symbols, dashed lines, refer to
embryos incubated in hypoxia. Numbers of embryos analyzed is the same as indicated
in Fig. 4, bottom. Symbols are group mean, bars 1SEM. When not visible, SEM was
within symbol size. Irrespective of egg size and of the degree of embryo growth, the
head-embryo relationship is described by a unique function.
̇ at day 17 was:
The allometric relationship for the 35 data points of VO
2
V̇O2 ðmL=dayÞ = 37:9⋅Weggðg Þ ðF0:08 SDEV; N = 35Þ
0:60
ð7Þ
Fig. 7. Top panel: Oxygen consumption (VO ̇ ) of the embryos in small-size (triangles)
2
and large-size eggs (circles), at various days of incubation. *, statistically significant
difference between Small and Large (P b 0.05). Symbols are group mean, bars 1SEM.
When not visible, SEM was within symbol size. Bottom panel: percent ratios of VO ̇ The corresponding equations for Large were V̇O2/Wembryo= 14.19 + 35.4· GR/Wembryo
2
between Small and Large eggs. Pairs of numbers in brackets indicate number of,
respectively, Small and Large eggs studied.
377
Even though numerically lower, this exponent did not differ
statistically from that of GH2O (Eq. 5).
̇ into the component related to
In order to partition the total VO 2
growth rate (b · GR) and that related to tissue maintenance (a · Wem-
bryo), the coefficient a and b were solved from V̇O2, Wembryo and
growth rate (GR = ΔWembryo/Δday). The equations (Fig. 8) did not
differ significantly between the two groups. Over the period E5-to-
E19, the average cost of growing 1 g of tissue was ∼40 mL O2 and that
of maintaining it for 1 day was ∼ 12 mL O2.
4. Discussion
4.1. Body growth
That larger eggs result in heavier hatchlings is the common finding
in several avian species (e.g., Schifferli, 1973; Lundberg and Vaisanen,
1979; Batt and Prince, 1979; Michel et al., 2003; Dzialowski and
Sotherland, 2004; Ipek and Dikmen, 2007; Dzialowski et al., 2008).
How this comes about is probably a combination of several factors.
Because larger eggs have a larger surface for O2 diffusion, the
limitation in O2 availability toward the end of incubation must be
more pronounced in embryos with smaller eggshell surface areas
(Høiby et al., 1983; Burton and Tullett, 1985; Tazawa et al., 1992). A
limitation in the ability to extract nutrients, owing to the smaller yolk
Fig. 8. Embryo's weight (W)-specific growth rate (GR) versus W-specific oxygen
consumption (V̇O2) (top panel) and GR-specific W versus GR-specific V̇O2 (bottom panel)
during incubation of small (triangles and continuous lines) and large eggs (circles and
dashed lines). The continuous lines are the best-fit linear regression through the data
points. Δg indicates the variation in W, expressed in g. In Small, V̇O2/Wembryo= 11.56 +
44.96 · GR/Wembryo (r = 0.98) and V̇O2/GR = 46.35 + 11.14 · Wembryo/GR (r = 0.93).
(r = 0.94) and V̇O2/GR = 36.04 + 14.01 · Wembryo/GR (r = 0.96). The equations did not
differ significantly between Small and Large.
378 J.P. Mortola, K. Al Awam / Comparative Biochemistry and Physiology, Part A 156 (2010) 373–379
circulation, is another factor that may limit growth in embryos of
small eggs, although never demonstrated. At end-incubation large
eggs have a disproportionately larger yolk residue than small eggs do
(Eq. 6), and its incorporation at hatching increases the hatchling's
birth W (Williams, 1994). Indeed, at day 20 the allometric curves of
the embryos with or without yolk differed substantially from each
other (Fig. 5), as shown in other species (Dzialowski and Sotherland,
2004). The current data indicated that the growth curves of S and L
were following different trajectories by day 11 (Fig. 4); in fact, it is
probable that the trajectories departed earlier, because the transversal
collection of the Wembryo data must have added variability to the
growth curves and blurred significant differences. Although bigger in
absolute terms, relative to Wegg the embryos of L were consistently
smaller than embryos of S. Eventually, at E20, the scaling exponent of
Wembryo was only 0.61.
̇ curves of S and L
Like the embryonic growth trajectories, also the VO
2
were taking separate routes approximately around the middle of
incubation. The difference in V̇O2 was no longer present upon
normalization by Wembryo, which implies that the higher VO ̇ in L
2
was strictly due to the bigger embryo's size and greater growth rate, and
was not contributed by extra-embryonic tissues. In fact, the CAM growth
was very similar between the two groups of eggs; therefore, its
participation to total egg VȮ was probably similar in S and L. In both
2
groups of eggs the cost of growing 1 g of tissue averaged about four
times that of maintaining it, as previously measured (Mortola and
Cooney, 2008), and was a similar amount in S and L, suggesting that the
body composition and the cost of metabolic activities were comparable.
The similarity in energetics should also imply that organ and tissue
composition was similar between the two groups. We do not have data
in this regard, but the uniqueness of the head-body relationship (Fig. 6)
and in W-specific VO ̇ suggests uniformity in the gross aspects of body
2
composition and organ development between S and L. Previously,
hematocrit, Wembryo-specific heart weight and heart cellular content
did not differ between E18 chicken embryos of 73-g eggs and those of
54-g eggs (Xu and Mortola, 1988). In conclusion, the growth of L
occurred at a faster pace, and required more O2, than that of S through-
out at least the second half of incubation, which covers some 75% of the
total body growth. However, the greater W of L was less than
proportional to their bigger egg; hence, the mass of their yolk residue
was disproportionately abundant, and its incorporation at hatching
contributed to the bigger W of the hatchling.
4.2. Hypoxic incubation
Sustained hypoxia is known to blunt embryo's growth and VO
(reviewed in Mortola, 2009). We wondered whether this limitation in
body growth was manifest preferentially in the embryos growing at the
faster pace. This was not so. In fact, the results indicated that the S–L
differences observed in normoxia were equally apparent in hypoxia
(Fig. 4, bottom). Body growth was blunted in both S and L with no major
effects on the body distribution, as evaluated by the head–body
relationship (Fig. 6). The fact that the S–L differences in embryonic
growth during hypoxia were essentially the same as in normoxia
suggests that differences in O2 diffusion (e.g., eggshell conductance) and
convection (e.g., CAM circulation) probably play a minor role in the
growth trajectories of S and L. Because the growth trajectories of S and L
tend to diverge quite early in incubation, it is possible that genetic
factors link egg size to embryo growth, as it is the case among species.
The blunting effects of hypoxia on embryonic growth, approximately
similar in S and L, are consistent with the effects of hypoxia in various
avian species (e.g., Vleck et al., 1980a; Vleck et al., 1980b; Snyder et al.,
1982; Bagley and Christensen, 1989; Léon-Velarde et al., 1997).
Visschedijk et al. (1985) reported that, during the last days of
incubation, egg-specific VO ̇ was the highest in eggs with medium
2
values of eggshell conductance (GH2O), and decreased at both lower
and higher GH2O; this suggested that optimal embryonic development
occurred over a narrow range of GH2O. Hence, another interpretation of
the different embryonic growth trajectories is that the differences in
GH2O between S and L contributed differences in water balance
between the two sets of eggs, increasing the water content of L and
lowering it in S.
4.3. Requirements for water balance
In the chicken eggs GH2O scaled to 0.77 (Eq. 5), which is almost
identical to the scaling pattern (0.78) emerged from interspecies
analysis of GH2O in eggs of 29 species spanning three orders of
magnitude (Ar et al., 1974). According to geometric similarity the
scaling exponent of the egg surface area and of its GH2O should be two/
thirds of Wegg, or 0.66. The fact that both inter- and intra-specifically
scaling exponents are higher than 0.66 indicates the importance of
increasing the total pore functional area with the increase in egg size,
probably through a combination of higher pore density and larger cross
section of the individual pores (Mortola, 2009).
Nevertheless, for water loss during incubation to be exactly
proportional to Wegg the scaling exponent of GH2O should be equal
to 1. It is possible that evolution has avoided a direct proportionality
between GH2O and Wegg because of gas exchange and structural
constraints; in large eggs a high number of pores with large diffusion
area would increase eggshell fragility, while the opposite situation in
small-size eggs would increase eggshell density. Hence, among avian
species, differences in incubation time (from only 11 days in the 0.5-g
eggs of some hummingbirds to more than 45 days in the 0.5-kg egg of
the emu and other large birds) are the main mechanism to achieve
adequate water loss (Rahn and Ar, 1974). As the result, the total relative
water loss during incubation (mL/Wegg), which is the product of the
relative daily water loss (mL/day/Wegg) and incubation time (day), is
approximately constant among species (Rahn et al., 1974).
The same combination does not apply to the chicken eggs of
different size. From Eq. 4 one can calculate that the total water loss
over the 20-day incubation in an average S egg (50-g) is 4.25 g, or 8.5%
of the original Wegg, and that of an average L egg (65-g) is 5.2 g, or
8.0% of the original Wegg. Hence, to compensate the fact that the
eggshell conductance is not directly proportional to egg W, the larger
eggs would need an incubation lasting 20 · (8.5/8), or 21.2 days. A
similar calculation indicates that the incubation of 70-g eggs would
need to last 2.1 days more than in 45-g eggs in order to lose the same
relative quantity of water. We found that the difference between L and
S was only 0.1 day, and insignificant. In a previous study, no significant
differences in the timing of hatching occurred between groups of eggs
̇ averaging either 71 or 55 g (Xu and Mortola, 1988). If these data in the
2
chicken were generalized, it should mean that the idea developed
through evolution of adjusting incubation time to eggshell conduc-
tance does not apply to the egg-size diversity within a species, with
the result that at hatching the relative water content of large-size eggs
exceeds that of small-size eggs. The magnitude of the overhydration
in the hatchlings of L, and of the dehydration in those of S, is difficult to
calculate because of the complexity of the water distribution among
the egg compartments (Ar, 1991). Nevertheless, it has been shown
that the hydration of the chick at hatch presents a positive correlation
with the size of its egg (Tullett and Burton, 1982).
4.4. Possible implications
The importance of the correct humidity for the survival at hatching
has long been known (Romanoff, 1972); however, the precise
definition of the tolerable boundaries is difficult. Some studies have
pointed out that large variations in water loss are still compatible with
hatching (Simkiss, 1980; Carey, 1986). In chickens, variations in the
relative humidity between 40 and 60% did not cause important
changes in the percentage of succesful hatching, while failures
increased once these limits were passed (Lundy, 1969). Large-scale
 J.P. Mortola, K. Al Awam / Comparative Biochemistry and Physiology, Part A 156 (2010) 373–379
studies, however, have shown that the optimal range of water loss
during incubation may be narrow, and that small variations from
optimality reduce hatchability (Meir and Ar, 1987; Ar, 1991; Tona
et al., 2001). Eggs can differ in size because of many reasons, from food
availability and seasonal rhythms to maternal size and age, and within
the same clutch for strategies on hatchling's survival (Lundberg and
Vaisanen, 1979; Williams, 1994; Maddox and Weatherhead, 2008).
Hence, it is not surprising that some variation in the egg water content
does not carry serious consequences on hatchability. Excessive devi-
ations from optimal size, however, would require appropriate changes
in incubation time. Because this does not happen, eggs too small or too
large relative to the mean are not compatible with the survival of the
hatchling.
It is very likely that hatching initiates once the PCO2 threshold is
reached (Mortola, 2009). The ratio between the embryonic CO2
production and the CO2-conductance of the eggshell determines the
partial pressure of CO2 (PCO2) in the aircell and arterialized blood. The
̇ at E17 was 0.60 (Eq. 7) and that of eggshell
scaling exponent of VO 2 Freeman, B.M. (Eds.), The Fertility and Hatchability of the Hen's Egg. Oliver and
conductance was 0.77 (Eq. 5). Because these two exponents did not
differ statistically, the PCO2 values should be similar in L and S, a
similarity that justifies the close timing of pipping and hatching.
Conversely, one could argue that the residual exponent (0.6–0.77 =
−0.17), even if not statistically significant, should not be overlooked,
because it may indicate a trend for larger eggs to have slightly lower
PCO2 than smaller eggs. This view would agree with blood gas data
sampled at day 18 in chicken embryos selected from eggs with large
differences in eggshell conductance (Tazawa et al., 1983). If indeed
PCO2 differed, albeit slighlty, between S and L, the fact that hatching
time was the same implies that the PCO2 threshold for hatching differs
between S and L, as can occur occasionally among different species
(Vleck et al., 1980b).
5. Conclusions
Eggs of different size produce different-size hatchlings. Events
toward the end of incubation, namely the incorporation of the yolk
residue, contribute to this difference. In addition, embryos of small
and large eggs follow different growth trajectories. These differences
could reflect a genetic link between embryonic growth rate and the
size of the egg, as well as some degree of overhydration and dehy-
dration in embryos of, respectively, large and small eggs. Appropriate
combinations of eggshell conductance and incubation time permit
enormous changes in egg size among bird species; however, in
different-size eggs of the same species, only conductance varies, and
by a limited amount. The result is that deviations in egg size from the
optimal mean carry the risks of overhidration in large eggs and of
dehidration in the small ones. Hence, in a given avian species, the
constraints of water balance may represent the evolutionary force
that determines the maximal range of egg size.
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