Effect of egg size on Emu (Dromaius novaehollandiae) egg composition and

hatchling phenotype

Edward M. Dzialowski1* and Paul R. Sotherland2

1 Department of Biology, University of North Texas, P.O. Box 305220, Denton, TX 76203

2 Department of Biology, Kalamazoo College, Kalamazoo, MI 49007

Running Title: Consequences of Emu Egg Size

Keywords: Emu, Dromaius novaehollandiae, eggs, development, maternal effect, life history, allometry, scaling

*Corresponding author email: edzial@unt.edu

Phone: 940-565-3631

Fax: 940-565-3821

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Abstract

Parental investment in eggs and, consequently, in offspring can profoundly influence

embryo and juvenile phenotypes, survival, and ultimately evolutionary fitness of an organism.

Avian eggs and embryos are excellent model systems to examine the influence of maternal

allocation of energy and nutrients, translated through egg size variation, on offspring

morphology, physiology, and survival. We used the natural range in Emu (Dromaius

novaehollandiae) egg size, from 400 g to greater than 700 g, to examine the influence of

maternal investment in eggs on the morphology and physiology of hatchlings. Female Emus

provisioned larger eggs with a greater absolute amount of energy, nutrients, and water in yolk

and albumen. This maternal investment was partitioned by embryos into embryogenesis, which

produced significant variation in hatchling size, and into post-hatching parental care in the form

of the reserve yolk, which was positively correlated with egg and hatchling size. Egg size also

influenced the physiology of developing Emu embryos, such that late-term embryonic metabolic

rate was positively correlated with egg size and embryos developing in larger eggs consumed

more yolk during development. Large eggs produced hatchlings that were both heavier (wet and

dry mass) and structurally larger (tibiotarsus and culmen lengths) than hatchlings emerging from

smaller eggs. As with many other precocial birds, larger hatchlings also contained more water,

which was reflected in a greater blood volume. However, blood osmolarity, hemoglobin content,

and hematocrit did not vary with hatchling mass. Thus, Emu maternal investment in offspring,

measured by egg size and composition, is significantly correlated with the morphology and

physiology as well as nutrient and energy reserves of the hatchlings and may, therefore,

significantly affect the success of these organisms during the first days of the juvenile stage.

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Introduction

Parental investment, particularly nutrients and energy allocated to eggs, can profoundly

influence development of embryos, phenotypes and survival of hatchlings, and, therefore,

evolutionary fitness of both offspring and parents. Parental investment in embryogenesis (PIE)

provides for the successful development of a zygote into a complete hatchling, and parental

investment in care of the hatchling (PIC) constitutes the energy and nutrients allocated to an egg

beyond those needed to produce a hatchling and used by the hatchling to support growth and

maintenance after emerging from the egg (Congdon 1989). As phenotypes of oviparous mothers

that affect phenotypes of their offspring, parental investment in offspring via eggs frequently has

significant, and evolutionarily meaningful, maternal effects (Bernardo 1996a, 1996b). Reaching

a full understanding of the magnitude of these maternal effects, and how they evolve, requires an

examination of intraspecific variation in parental investment in eggs along with an examination

of how embryos respond physiologically to the egg environments within which they develop.

The trajectory followed by an embryo from zygote to hatchling stages is influenced by an

interaction between genetic instructions in the nuclei of the embryo’s cells and conditions in the

environment surrounding those cells. Conceptually similar to evolutionary paths blazed by

populations of organisms through phenotypic space over several generations (Raup 1966),

developmental trajectories (Burggren 1999) of oviparous amniotes can change as a result of

biotic and abiotic factors encountered outside the eggshell and factors, initially maternal in

origin, found within the eggs. Phenotypes of these embryos, developing toward hatching and

toward metamorphosis into a more independent (e.g. self- feeding, thermoregulating, lung-

ventilating, and ambulatory) phase in their lives, are shaped, therefore, both by genetic and

environmental effects (Burggren 1999). Acquiring in-depth knowledge of the sensitivity of

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developmental trajectories to environmental perturbations, including maternal investment of

nutrients and energy in eggs, will improve our understanding of the genesis and importance of

maternal effects manifested in phenotypes of hatchlings.

Requiring only heat and oxygen from the environment and containing all nutrients and

water necessary to sustain developing embryos, avian eggs are attractive models for investigating

effects of parental (maternal) investment on developmental trajectories of embryos and

hatchlings. Variation in the composition of avian eggs among species is correlated with

functional maturity of hatchlings (Carey et al. 1980, Sotherland and Rahn 1987); quantity and

composition of parental investment varies significantly within species and is frequently

correlated with hatchling mass (Williams 1994). Thus, choosing a species, which produces eggs

of convenient size and variation, along the avian altricial-precocial continuum (Nice 1962) and

investigating how intraspecific variation in egg size and composition affects attributes of

hatchlings can provide useful insights into the importance of maternal effects in oviparous

amniotes.

In this study we examined consequences of natural variation in maternal investment –

egg size and egg composition – on Emu hatchling phenotypes. Emu eggs and hatchlings make

good experimental subjects for a study of parental investment because they are large (egg mass

approx. 600 g and hatchling mass approx. 400 g); macroembryony (Burggren 1999/2000) used in

this investigation facilitated measuring hatchling characteristics (e.g. blood volume) that are

otherwise difficult to quantify. In addition, intraspecific variation in egg mass from 400 g to

over 700 g provides a reasonably, but not unusually, wide range of egg size. Female Emus lay

between 5 and 20 eggs, typically incubated by the males, during the breeding season, and, after

emerging from their eggs, the precocial hatchlings forage for food under guidance from the

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males (Davies, 1975). Thus, Emus provide eggs and hatchlings, at one end of the altricial-

precocial continuum, with which variation in egg and hatchling characteristics can be

investigated. Results described here reveal that parental investment (i.e. provisioning eggs with

nutrients and water) by Emus significantly affects developmental trajectories of embryos and is

positively correlated with size and provisioning of hatchlings with water, which contributes to

blood volume, and nutrients typically consumed immediately after hatching.

Materials and Methods

Animals

Emu eggs were randomly collected within five days after oviposition at the Cross

Timbers Emu Ranch, Flower Mound, TX from November to March in 2000 and 2001. At the

time of egg collection, the female breeding population at Cross Timbers Emu Ranch was 45

female birds ranging in age from 3-7 years. Forty-nine Emu eggs were used to determine egg

composition, and 53 eggs were incubated to obtain measurements of hatchling components,

metabolic rates, and hematological parameters. Though we do not know the source of each egg,

it is likely that more than one egg from some females were used in this study. All protocols used

in this study were approved by the University of North Texas Animal Care and Use Committee.

Egg Components

Fresh egg mass was determined by drilling two small holes through the shell over the air

cell, filling the air cell with water, and then weighing the eggs on a Denver Instruments digital

balance. Short of weighing eggs immediately after oviposition, this is the most reliable method

of obtaining fresh egg mass (Ar and Rahn 1980, Rahn et al. 1985). Eggs were then separated

into shell, yolk, and albumen following the methods described in Finkler et al. (1998). The intact

yolk was weighed with the digital balance to determine yolk mass. Yolk, albumen, and shell

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were then dried to a constant mass in a drying oven at 60 oC. Shell mass was measured by

weighing the dry shell on the digital balance, and albumen wet mass was determined by

subtracting yolk wet mass and shell dry mass from the mass of the egg. Water contents of yolk

and albumen were determined by subtracting dry mass of each from the respective wet mass; the

sum of mass of water in the yolk and mass of water in the albumen yielded total water content of

each egg. Mass of egg solids was computed by adding yolk and albumen dry masses.

Egg Shell Conductance

We measured water vapor conductance of fresh eggs ranging in mass from 487 to 778 g

(n = 16). Eggs were initially weighed and then placed in individual desiccators. Each desiccator

contained an ample amount of drierite desiccant in the bottom of the desiccator to ensure that

water vapor pressure around each egg was 0 torr. The mass of each egg was measured daily for

five days. Each day, desiccator temperature and atmospheric pressure were recorded. Who le

eggshell water vapor conductance was then determined following the protocol of Ar et al.

(1974). Finally, initial egg mass was measured by drilling a hole through the shell above the air

cell end, filling the air cell with water, and then weighing the egg on a digital balance.

Incubation

Eggs were stored at 4 oC for no more than seven days before incubation. Eggs were

incubated in forced draft incubators with automatic rotation at Cross Timbers Emu Ranch until

approximately day 40 of incubation. They were then transferred to the University of North

Texas (Denton, TX, USA) where incubation continued until hatching in forced draft Emu

incubators (G.Q.F. Manufacturers). Eggs were incubated at 36.5 oC and at a relative humidity

(of approximately 30%) adjusted to yield an average fractional mass loss of about 12 % from the

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eggs. Prior to internal pipping, all eggs were transferred to a hatching incubator with a

temperature of 36.5 oC and a relative humidity between 35 to 40%.

Gas Exchange of Near-term Embryos

Metabolic rates of 15 eggs were measured on day 46 of incubation (i.e. after about 92%

of incubation) using a flow through system similar to the methods of Dzialowski et al. (2002).

Eggs were placed in individual PVC respirometers (approx. vol. 1 liter) and then into a constant

temperature chamber regulated at 37.5 oC. Air was pumped through the individual chambers and

flow was measured at the inflow side of the chambers using a calibrated Brooks flow meter.

Outflow O2 concentration from each respirometer was measured using a Beckman OM11 O2

analyzer. Inflow O2 concentration to the respirometers was determined from the outflow of an

empty respirometer. Metabolic rate (i.e. rate of oxygen consumption) was calculated using the

equation of Hill (1972). Metabolic rates were corrected to STPD and expressed in units of

ml O2 h-1 egg-1.

Air cell Po2 was measured in eight Emu eggs on day 46 of incubation. On day 40 of

incubation a 5 mm diameter hole was drilled in the air cell end of each egg using a drill press. A

square patch of 0.4 mm thick Thera-bandTM latex was glued over the hole using Duro Quick

GelTM and the egg was replaced into the incubator for 6 days. Using a 1 ml syringe and a 27-

gauge needle inserted through the latex, a 1 ml sample of gas was withdrawn from the air cell

and then promptly analyzed for Po2 using a Cameron Instruments BGM2000 blood gas meter.

Hatchling Morphology and Composition

All measurements of morphology and composition were made on hatchlings that were

less than one day old. Hatchlings were sacrificed by exposure to either halothane or iso-flurane,

and then weighed to obtain hatchling mass (yolk-free hatchling carcass mass plus residual yolk

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and yolk sac). The yolk sac was carefully dissected from each hatchling and weighed to measure

the quantity of residual yolk; yolk-free hatchling (carcass) mass was determined by subtracting

residual yolk mass from hatchling mass. Culmen length and right tibiotarsus length were

measured on each hatchling using digital calipers (Mitutoyo) as another means of quantifying

hatchling size. Heart, gizzard, and liver were dissected from the body, weighed separately, and

then dried separately to a constant mass in an oven at 60 oC. The yolk sac and what remained of

the carcass were also dried to a constant mass in a similar way. Water contents of the various

components were determined by subtracting dry mass of each from the respective wet mass.

Mass of yolk-free hatchling solids was computed by adding heart, gizzard, and liver dry masses

to the dry mass of the dissected carcass. We estimated the quantity of yolk consumed by an

embryo during incubation by subtracting the measured dry yolk sac mass from the calculated

mass of dry yolk that the egg from which a neonate hatched would have contained at the outset

of incubation, using initial egg mass and the equation for dry yolk mass in Figure 1.

Hematology and Blood Volume

To obtain blood for hemoglobin, hematocrit, and blood osmolarity, hatchlings were

anesthetized using halothane and blood was taken from the heart by direct cardiopuncture.

Hemoglobin was measured with a Radiometer OSM2 Hemoximeter. Hematocrit was measured

by centrifuging blood in heparinized capillary tubes. Osmolarity of the blood was measured

using a Wescor 5500 vapor pressure osmometer. Two measurements of each variable were

made and averaged for each blood sample.

Blood volumes were measured in 11 Emu hatchlings using the Evan's Blue dilution

technique (El-Sayed et al. 1995). Hatchlings were anesthetized with Iso-flurane and attached to

a ventilator that maintained an Iso-flurane concentration of 1% in the inspired air. Both the right

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and left jugular veins were exposed and non-occlusively canulated with tips of 26 gauge needles

attached to PE 50 tubing. The right jugular vein was used as the injection site for the Evan's

Blue solution, and the left jugular vein was used to withdraw subsequent blood samples.

Initially, 500 µl of blood was withdrawn into a heparinized syringe from the right jugular vein.

This was followed by an injection of 400 µl of an Evan's Blue solution (5 mg ml-1 dissolved in

0.9% heparinized saline) into the right jugular vein. The Evan's Blue injection was followed by

a 200 µl injection of heparinized saline to wash the tubing. Samples of blood were then taken

from the left jugular vein at 10, 15, and 20 min after the initial injection of Evan's Blue.

After each blood sample was collected, a known amount of blood from each sample was

added to an equal amount of hepranized saline and centrifuged for 15 min. All volumes were

gravimetrically determined using a Denver Instruments digital balance to increase measurement

accuracy. A 200 µl aliquot of the supernatant was added to 800 µl of hepranized saline and the

absorbance was measured at 610 nm using a Bausch amd Lomb Spectronic 88

spectrophotometer. A sub sample of plasma from the initial blood sample, taken before injection

of Evan's Blue, was used to create a blank for zeroing the spectrophotometer for each hatchling’s

measurement.

A standard curve (r2 = 0.93) relating maximal absorbance with blood volume of a known

quantity, was generated using blood from four additional hatchlings. Blood volumes were then

calculated according to the methods in El-Sayed et al. (1995).

Statistical Analyses

Linear regressions of parameters on egg mass and yolk-free hatchling mass were carried

out using SPSS 11.0. Additionally, log- log regressions were performed on data to determine if

component masses varied isometrically (slope of log- log regression, b = 1.0) or if component

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masses showed a positive (b > 1.0) or negative (b < 1.0) allometry with egg mass or hatchling

mass. The regressions were considered isometric if the 95% confidence interval of the slope of

the log-log regression included 1. A significance level of p < 0.05 was adopted for all

regressions. Linear regression equations are provided in the figure legends and log- log slopes

are provided in the text. All values are presented as mean ± S.D.

Results

Egg Composition

Mass of each egg component increased significantly as egg mass (586.17 ± 78.06 g; n =

49) increased from approximately 400 g to approximately 700 g (Figure 1). Albumen mass

(274.00 ± 40.75 g; n = 49) increased significantly (F1,47 = 309; p < 0.001) with egg mass as did

albumen dry mass (28.43 ± 5.21 g; n = 47; F1,45 = 135; p < 0.001). Yolk mass (237.66 ± 33.07 g;

n = 49) increased significantly (F1,47 = 208; p < 0.001) with egg mass as did yolk dry mass

(136.36 ± 21.93 g; n = 47; F1,45 = 146; p < 0.001). Like the other two major components of

fresh eggs, shell mass (75.12 ± 11.90 g; n = 49) increased significantly (F1,47 = 99; p < 0.001)

with egg mass.

The relative contribution of albumen and yolk to the eggs did not vary with initial egg

mass. The slopes (b) of the log- log regressions of log albumen mass (b = 1.04 ± 0.13; mean ±

95% C.I.; r2 = 0.87), log dry albumen mass (b = 1.22 ± 0.22; r2 = 0.75), log yolk mass (b = 0.93

± 0.14; r2 = 0.82), and log dry yolk mass (b = 1.05 ± 0.18; r2 = 0.76) against log initial egg mass

were not significantly different from 1. As a result, fraction of yolk in the contents (0.47 ± 0.03),

typically correlated with developmental maturity of hatchlings (Sotherland and Rahn 1987), did

not change (F1,47 = 0.49; p = 0.49) with egg mass.

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Water and solid content of eggs increased with egg mass (Figure 2), but the fraction of

water and solids did not vary significantly over the range of egg masses examined. Water in

eggs (343.02 ± 46.21 g; n = 45) increased significantly (F1,43 = 648; p < 0.001) with egg mass, as

did the solid content of eggs (163.97 ± 25.84 g; n = 45; F1,43 = 245; p < 0.001). Approximately

71% of water in eggs was found in the albumen (244.56 ± 36.70 g; n = 47), which was composed

of an invariant fraction of water (0.90 ± 0.01; F1,45 = 1.8; p = 0.19). Similarly, neither the

fraction of water in the yolk (0.42 ± 0.03; n = 47) nor the fraction of water in the eggs (0.68 ±

0.02; n = 45) changed significantly with egg mass. However, the total amount of water in the

yolk (99.93 ± 13.84 g; n = 47) increased significantly (F1,45 = 66; p < 0.001) with egg mass as did

the total amount of water in the albumen (F1,45 = 265; p < 0.001).

Gas Exchange of Near-term Embryos

Metabolic rate (107.8 ± 11.6 ml O2 h-1; n = 15) of pre-pip embryos, measured on day 46,

increased significantly with initial egg mass (F1,13 = 7.7; p = 0.016; VO2 = 0.11 Me + 33.9; r2 =

0.37) and mass of yolk-free hatchlings that emerged from the same eggs (F1,13 = 14; p = 0.003;

VO2 = 0.20 Mh + 48.8; r2 = 0.51) . In a separate set of eggs, eggshell water vapor conductance

(59.5 ± 10.2 mg torr-1 day-1; n = 14; GH2O = 0.09 Me + 4.5; r2 = 0.51) increased significantly

(F1,13 = 9.4; p = 0.01) with initial egg mass. In contrast, neither pre-pip air cell PO2 (112.4 ± 5.9

torr; n = 8) nor mass of water lost by diffusion from eggs during incubation (70.8 ± 17.8 g; n =

15), which comprised approximately 11% of initial egg mass, varied significantly with egg mass

(F1,6 = 2.4; p = 0.17 and F1,13 = 2.5; p = 0.14, respectively).

Hatchling Morphology and Composition

Hatchling mass (both yolk-free hatchling carcass and residual yolk) increased with egg

mass (Figure 3). Hatchling mass (405.81 ± 44.78 g; n = 53) increased significantly (F1,47 = 216;

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p < 0.001) with egg mass, as did yolk-free hatchling mass (302.27 ± 37.75 g; n = 53; F1,47 = 57; p

< 0.001) and residual yolk mass (103.53 ± 26.86 g; n = 53; F1,47 = 11.7; p = 0.001). Dry mass of

yolk-free hatchling (80.23 ± 12.15 g; n = 47) and dry mass of residual yolk (57.84 ± 14.63 g; n =

47) also increased significantly (F1,44 = 27; p < 0.001 and F1,47 = 16; p < 0.001 respectively) with

egg mass.

Large hatchlings were composed of more water and solids than small hatchlings (Figure

2), but the fraction of water in hatchlings remained unchanged regardless of hatchling size. Mass

of water in yolk-free hatchlings (223.64 ± 28.91 g; n = 46) increased significantly (F1,44 = 46; p <

0.001) with mass of yolk-free hatchlings, but the fraction of water in those hatchlings (0.74 ±

0.02; n = 46) did not vary significantly (F1,44 = 0.23; p = 0.88) with hatchling mass. Mass of

solids in hatchlings (i.e. dry mass of yolk-free hatchling) also increased significantly (F1,44 = 120;

p = < 0.0001) with hatchling mass.

Yolk consumed by developing embryos (i.e. the difference between the dry mass of the

yolk - estimated using measured initial egg mass and the equation for dry yolk mass provided in

Figure 1 - and measured mass of solids remaining in the yolk-sac; 88.8 ± 13.3; n = 30) increased

significantly (F1,29 = 21.8; p < 0.001) with initial egg mass (Fig. 4).

Linear dimensions of heavier hatchlings were greater than those of lighter hatchlings

(Fig. 5). Length of both the right tibiotarsus (70.26 ± 3.97 mm; n = 49; F1,45 = 21; p < 0.001) and

culmen (36.75 ± 1.89 mm; n = 49; F1,45 = 16; p < 0.001) increased significantly with yolk-free

hatchling mass.

Wet and dry masses of heart, liver, and gizzard all increased significantly with yolk-free

hatchling mass (Table 1).

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Hematology of Hatchlings

Blood volume (27.8 ± 7.0 ml; n = 11), which constituted approximately 9.2 % of the

yolk-free hatchling mass, increased significantly (F1,9 = 12; p = 0.007) with yolk-free hatchling

mass (Fig. 6). However, none of the other blood parameters measured – osmolarity (304.6 ±

15.6 mOsm/kg; n = 44), hematocrit (38.4 ± 4.1 %; n = 45), or hemoglobin concentration (12.9 ±

1.8 g%; n = 44) – varied significantly with yolk-free hatchling mass (Fig. 6).

Discussion

Consequences of parental investment (i.e. investment of energy and nutrients; Congdon

1989) in reproduction, frequently manifested as maternal effects, can be observed as significant

variation in the physiology, morphology, and life history of organisms (Bernardo 1996a, 1996b).

Female birds vary this reproductive investment by allocating different amounts of albumen and

yolk to the eggs they produce or by producing eggs of different size. Maternal investment in

Emu eggs, the third largest egg laid by extant birds, varied considerably (Fig. 1), with eggs

differing in mass by as much as 300 g, and this variation was reflected in concomitant variation

in hatchling size and composition.

Yolk and Albumen in Eggs

One measure of parental investment in bird eggs typically is expressed as the fraction of

yolk in the contents (FYC) of eggs. Emus in this study laid eggs containing nearly 50% yolk

(FYC = 0.47), which is within the range of yolk content for precocial birds but larger than that

predicted for other precocial eggs of the same mass. Sotherland and Rahn (1987) examined the

relationship between egg mass and energy content for a wide variety of birds and found that FYC

for precocial species ranges from 0.32 to 0.69. Based on the equation for yolk content in

precocial species (Sotherland and Rahn 1987), we predicted the FYC for an average sized Emu

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egg (512 g wet contents) to be 0.39, which is less than the FYC of Emu eggs measured in this

study. Thus, female Emus provision their eggs with relatively more yolk and less albumen than

would be predicted for a typical large precocial egg. If we compare Emu eggs with those of

closely related species (Sibley and Ahlquist 1990), Emu eggs tend to have a larger FYC than

either the Ostrich (Struthio camelus, 1.2 kg egg, FYC = 0.38, Romanoff and Romanoff 1949) or

Cassowary (Casuarius casuarius, 546 g egg, FYC = 0.42, Carey et al. 1980). This finding is not

surprising, however, because the incubation period of the Emu is longer than that of the ostrich,

suggesting that Emu embryos may require more energy than Ostrich embryos to complete

incubation. In contrast, Emu eggs have a lower FYC than the smaller Kiwi eggs (Apteryx

australis, 440 g egg, FYC = 0.61, Reid 1971, Calder et al. 1978), which has an incubation period

about 25 d longer than the Emu.

A different but related comparison among species entails examining the contribution of

albumen and, therefore, water (albumen in all bird eggs is about 90% water; Sotherland and

Rahn 1987) to avian egg contents. We suggest here that always focusing on yolk and FYC

diverts attention from albumen, the only other component of eggs, and its important

contributions to embryo development and hatchling phenotype. Fraction of albumen in the

contents (FAC = 1-FYC) of eggs is very high (about 80%) in altricial species and drops to less

than 50% in more precocial species. (Eggs of some reptiles contain as little as 10% albumen, but

these eggs can take up water during development; Tracy and Snell 1985). Emu egg contents are

about half albumen (FAC = 0.53), and at the middle of the range observed in ratites where FAC

varies between 0.4 (Kiwi) to 0.6 (Ostrich).

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Scaling of Egg Composition

Parental (maternal) investment in avian eggs varies both interspecifically and

intraspecifically in two ways. First, the absolute size of eggs and egg contents (solids, water, and

energy in albumen and yolk) can vary among and within species. Second, the relative

contribution of yolk, albumen, and shell to the mass of an egg can vary with egg size and with

maturity of neonate at hatching.

Intraspecifically, Emu eggs exhibit isometric scaling between egg size and all egg

components. Large eggs contained more yolk and albumen (Fig. 1) as well as water and solids

(Fig. 2) than small eggs, but yolk and albumen mass increased isometrically with egg size; the

slope of natural log regressions of these components on egg mass did not differ significantly

from 1. Thus, Emu eggs in this study followed the precocial pattern (Williams 1994) where eggs

of all sizes had the same relative amount of yolk and albumen.

A number of studies have examined intraspecific variation of egg composition and have

revealed patterns of how yolk and albumen content vary with egg size along the altricial-

precocial continuum (Sotherland et al. 1990, Williams 1994, Hill 1995, Carey 1996). For most

species of birds, the vast majority of which are altricial, variation in albumen mass accounts for

most of the variation in egg mass, but yolk contributes more to variation in egg mass as FYC

increases toward the precocial end of the altricial-precocial continuum (Sotherland et al. 1990).

Williams (1994) reviewed 22 studies that examined intraspecific variation in egg components

and found that only half of these studies revealed an isometric relationship between egg size and

either yolk or albumen content. Hill (1995) found that wet albumen mass and wet yolk mass

tended to scale isometrically with egg mass in precocial species, whereas in altricial species

albumen showed positive allometry (b > 1.0) and yolk increased with mass more slowly (b <

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1.0). Thus, it seems that altricial species tend to change egg size by increasing the amount of

albumen while keeping yolk content relatively constant, whereas precocial species tend to alter

egg size by increasing both yolk and albumen content with an increase in egg mass. Further

support for this pattern has been observed in precocial Wood Ducks (Aix sponsa, Kennamer et al.

1997) and Ruddy Ducks (Oxyura jamaicensisI, Pelayo and Clark 2002), which lay eggs having

yolk and albumen varying isometrically with egg mass, and in altricial Great Tit (Parus major)

eggs in which much of the variation in egg mass is attributable to variation in albumen mass

(Lessells et al. 2002).

Egg Size and Hatchling Size

A developing embryo may partition its available energy and nutrients, invested in the egg

by its mother, into growth and maintenance of the developing body or into residual yolk. Energy

and nutrients invested in an egg by a female that are used to make and maintain the embryo is

parental investment in embryogenesis (PIE), whereas energy and nutrients left as residual yolk

(or as fat deposits in the hatchling) is parental investment in care of the hatchling (PIC; Congdon

1989). Increased parental investment in larger Emu eggs (Fig. 1) yielded larger hatchlings (Fig.

3) containing more residual yolk (Fig. 3) even though embryos in the larger eggs had consumed

more yolk during incubation (Fig. 4). Increased hatchling size is attributable to increased total

water content (Fig. 2), increased dry mass (Fig. 2 and 4), and increased structural size as

measured by the tibiotarsus and culmen lengths (Fig. 5). Heart, liver, and gizzard masses were

also larger in hatchlings from large eggs (Table 1). Thus, increased parental investment by Emus

yielded larger hatchlings that received greater post-hatching care in the form of residual yolk,

indicating that egg size can be equated with egg quality in Emus.

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In his review of the literature, Williams (1994) found that larger bird eggs produce

heavier hatchlings, but not necessarily structurally larger hatchlings. However, many of the

studies of the relationship between mass and structural size in hatchlings examined only

hatchling (including residual yolk) mass and concluded that hatchlings from larger eggs were

heavier because they contained more residual yolk and not because they were structurally larger

(Williams 1994). Ankney (1980) found a significant positive relationship between egg size and

length of the tarsus and culmen of Lesser Snow Goose (Anser caerulescens) hatchlings, but the

relationship between hatchling size and linear dimensions was not reported. Larger eggs laid by

the Thick-Billed Murre produced heavier hatchlings, but this was due mainly to increased water

content or residual yolk and not due to increased linear dimensions (Birkhead and Nettleship

1982). In a number of alcid species, most of the variance in hatchling mass in relation to pipped

egg mass was attributed to differences in residual yolk rather than increased water content or dry

mass of hatchlings (Birkhead and Nettleship 1984). In the King Eider (Somateria spectabilis),

large eggs produced larger hatchlings with larger wet and dry breast and leg muscle mass than

the hatchlings produced from small eggs (Anderson and Alisauskas 2002). However, the relative

structural size of larger King Eider hatchlings was less than that of small hatchlings. In the

altricial Blackbird (Turdus merula), large eggs produced both heavier and larger hatchlings

(Magrath 1992).

Emu hatchlings had a large residual yolk, which was positively correlated with initial egg

mass and with yolk-free hatchling mass (Fig. 3). Female Emus provisioned eggs with enough

yolk to support development and maintenance of embryos and to provide sufficient residual yolk

to support activity and survival after hatching such that females producing larger eggs provided

greater parental care than females producing smaller eggs. Ruddy ducks (Oxyura jamaicensis)

18

exhibit a similar pattern, where hatchling mass and mass of residual yolk are positively

correlated with initial egg mass (Pelayo and Clark 2002).

Though we did not examine survivorship consequences of variation in residual yolk, it is

likely that the amount of residual yolk we measured would influence early growth and survival

of these hatchlings because adult Emus do not feed the young (Davies, 1975). Parental

investment in hatchlings via egg yolk can provide hatchlings from larger eggs with more residual

yolk (i.e. parental investment in care), which serves as a post-hatching source of nutrients and

energy that can affect survivorship, especially during times of nutritional stress. A number of

studies of precocial species (Kear, 1965, Ankney, 1980, Peach and Thomas 1986, Thomas et al.

1988, Slattery and Alisauskas 1995, Visser and Ricklefs 1995, Dawson and Clark 1996, Nager et

al. 2000, Anderson and Alisauskas 2001) have shown that an increase in yolk reserves,

correlated with increased egg size, results in increased hatchling survival under limited food

conditions. However, this generalization may not apply when eggs hatch asynchronously and

then are fed by their parents. Under these conditions, hatching order within clutches influences

survivorship of neonates to a greater extent than does egg mass (Stokland and Amundsen 1988,

Magrath 1992; Bollinger 1994).

In general, Emu hatchlings have more residual yolk, as a fraction of the whole hatchling,

than many of the other avian species studied (Vleck and Vleck 1996). Whereas precocial

hatchlings retain residual yolk of 0.15 to 0.18 of their wet mass and 0.28 of their dry mass (Carey

1996,; Vleck and Vleck 1996), Emu hatchlings in our study had residual yolk amounting to 0.25

of their wet mass and 0.42 of their dry mass. There is also sizeable residual yolk in the ostrich,

accounting for 0.29 of wet mass and 0.56 of dry mass (Gefen and Ar, 2001). The ostrich and

19

Emu are both ratites, suggesting that members of this clade have noticeably high parental

investment in hatchling care and residual yolk.

Water Relations and Hatchling Mass

Water loss from avian eggs during incubation and metabolic water production by the

embryos occur at rates that cause the hydration of egg contents at the end of incubation to be

similar to that at the beginning of incubation (Ar and Rahn 1980), and these coincident rates also

cause hatchlings and the eggs from which they emerge to have similar water contents

(Sotherland and Rahn 1987). Emu eggs contained on average 68% water, which comprised 74%

of the hatchlings they produced (Fig. 2). Both of these values are in close agreement with the

water content of precocial eggs and hatchlings (Sotherland and Rahn 1987). There tended to be

an isometric increase in the water content of both the egg and the yolk-free hatchling with an

increase in initial egg mass and yolk-free hatchling mass (Fig. 2); a similar relationship was

observed in the dry mass of eggs and yolk-free hatchling solids (Fig. 2). Japanese Quail

(Coturnix coturnix) hatchling water content scales isometrically with egg size, but the proportion

of water in Laughing Gull (Larus atricilla) chicks increases with a positive allometry such that

larger hatchlings are composed of more water (Ricklefs et al. 1978).

The quantity of water in avian eggs has a significant influence on the mass of developing

embryos and hatchlings. Variation in Emu hatchling mass is attributable in part to variation in

mass of water in yolk-free hatchlings (Fig. 2). Studies examining effects of water loss from eggs

during incubation have shown that differences in wet embryo mass tend to be correlated with

water content of the embryo and that eggs losing the most water tend to produce embryos with

the lowest mass (Davis et al. 1988, Tullett and Burton 1982). Removing albumen from Chicken

(Gallus gallus) eggs caused a reduction in hatchling size (Hill 1993, Finkler et al. 1998) and

20

resulted in hatchlings with a reduced yolk-free wet body mass (Finkler et al. 1998). Though

hatchlings emerging from eggs from which albumen had been removed were smaller (i.e. length

of tibiotarsus was shorter), much of the decrease in wet body mass was attributed to the presence

of less water in the smaller hatchlings. The dry yolk-free body mass of hatchlings from control

eggs was not different from that of hatchlings emerging from eggs from which albumen had been

removed (Finkler et al. 1998). Thus, water availability in eggs may be one of the main

determinates of yolk-free hatchling mass in precocial species. A similar relationship between

water content and hatchling mass has been observed in turtle eggs, where increased levels of

water in eggs result in increased hatchling and organ sizes (Packard et al. 1987, Packard 1999,

Packard et al. 2000, Packard and Packard 2001).

Finkler et al. (1998) postulated that some of the observed variation in body mass,

correlated with variation in water mass, might be accounted for by variation in extracellular

liquid volume, including blood volume. Blood volume of Emu hatchlings increased with

hatchling size (Fig. 6), but this increase in blood volume was not accompanied by variation in

other hematological parameters (Fig. 6). Thus, a portion of the additional water found in larger

Emu hatchlings appears in a larger volume of blood, suggesting that one potential benefit of

allocating additional albumen (i.e. water) to eggs might be enhanced performance of the

cardiovascular system (Fortney et al. 1983, Nadel 1985).

Metabolic Rate, Egg Shell Conductance, and Air Cell Po2

Maximum metabolic rates of bird embryos and the conductance of the eggshells in which

the embryos develop are typically matched in such a way that levels of respiratory gases in the

air cell vary over an amazingly narrow range at the end of incubation, regardless of egg size,

length of incubation, or degree of hatchling maturity (Rahn and Paganelli 1990). Metabolic rates

21

of developing Emu embryos, which reach a plateau about 8 days prior to hatching (Vleck et al.

1980), reported here agree with those reported previously by Beutel et al. (1983) and Vleck et al.

(1980) and were significantly correlated with initial egg mass and the mass of the yolk-free

hatchling. Larger eggs produced larger hatchlings, and, not surprisingly, near-term embryos

from larger eggs had greater overall metabolic rates than those from smaller eggs. Because

metabolic rate of Emu embryos and water vapor conductance of the eggs in which they

developed covaried with egg mass, Po2 in air cells of Emu eggs did not vary with egg mass and

were in close agreement with the values calculated by Vleck et al. (1980). Using Fick’s law of

diffusion and our measurements of shell gas conductance and metabolic rate, we calculated that

air cell Po2 should have averaged about 107torr, which is less than 5% different from the values

measured.

Consequences of Egg Size Variation

Emu egg size influenced the morphological and physiological phenotypes of the resulting

hatchlings. Using the regressions from the results and Figs. 1 through 4, we predicted a number

of parameters for a small egg (450 g) and a (44%) larger egg (650 g) of Emus to illustrate

consequences of variation in the size of emu eggs (Table 2). Most phenotypic characters

measured here were between 38 and 51% larger in hatchlings from the larger egg and tend to

scale in a similar fashion with egg size. Using the energy content of dry solids in eggs (29 kJ g-1,

Sotherland and Rahn 1987), one can predict that a female Emu would invest 3596 kJ in a 450 g

egg whereas a 650 g egg would contain 48% more energy (5336 kJ). If mass-specific costs of

producing eggs were the same for eggs of all sizes within a species, then a female ovipositing a

650 g egg would allocate nearly 50% more energy per egg than a female ovipositing a 450 g egg.

22

Large Emu eggs produced large hatchlings that also had more energy reserve at hatching

than hatchlings from small Emu eggs. Thus, in addition to having an advantage due to a larger

body size, hatchlings emerging from larger eggs also have an advantage because of increased

levels of energy reserve in the residual yolk. A hatchling from a large egg (650 g) has more

residual yolk at hatching, which, using the energy content of 32.15 kJ g-1 of dry residual yolk

(Gefen and Ar 2001), provides an additional 514 kJ of reserve energy when compared with the

hatchling from a 450 g egg.

Parameters that did not scale the same as all the others were near term-embryo metabolic

rate and air cell Po2. Larger eggs had higher metabolic rates, but metabolism did not increase to

the same extent with increases in egg mass as hatchling body mass, suggesting that embryos in

larger eggs may have responded more to internal hypoxia caused by limitations imposed by a

relatively low eggshell conductance.

Embryos in larger eggs received more parental investment in embryogenesis and parental

investment in care of the hatchling, which allowed them to consume more yolk solids and grow

larger during incubation but still have more yolk to support them as hatchlings. However,

embryos in small and large eggs consumed nearly the same proportion of yolk solids with which

their eggs were provisioned.

Our investigation revealed that female Emus vary parental investment in their offspring

through changes in the absolute amount of yolk and albumen in eggs. Greater parental

(maternal) investment in their offspring via eggs produced hatchlings that are heavier and

structurally larger (i.e. greater PIE), and that contain more residual yolk (i.e. greater PIC). Thus,

embryos in larger eggs incorporate more energy into their bodies during development in the egg

yet still have more energy available for use upon hatching. We do not know, however, if

23

embryos that “find” themselves in larger eggs, containing more resources and a larger gas

exchange surface, respond by growing more or if embryos that would grow more are put into

larger eggs. Further research is needed to elucidate more clearly how maternal phenotypes affect

developmental trajectories.

Acknowledgements

All Emu eggs used in this study were donated by the Cross Timbers Emu Ranch, Lewisville, TX.

We thank Warren Burggren, Anne Dueweke, and two anonymous reviewers who provided

valuable comments on earlier versions of the manuscript. This research was partially funded by

NSF operating grant IBN 98-96388 and TARP grant 99466 to Warren Burggren, a Faculty

Development Grant and a Joyce Research Fellowship from Kalamazoo College to Paul

Sotherland and the Betz Chair Endowment, Drexel University to James Spotila.

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Table 1 Hatchling component masses (g) and their allometric regressions on yolk-free hatchling

mass.

Component Mean Std Dev Slope Intercept F P r2 n

Heart Wet 2.14 0.58 0.009 -0.53 25.8 <0.001 0.34 53

Heart Dry 0.37 0.11 0.001 -0.08 16.8 <0.001 0.27 47

Liver Wet 8.23 1.71 0.026 0.43 24.7 <0.001 0.33 53

Liver Dry 3.35 0.70 0.011 -0.03 26.6 <0.001 0.37 47

Gizzard Wet 5.60 1.02 0.014 1.36 18.5 <0.001 0.27 53

Gizzard Dry 1.04 0.29 0.003 0.26 6.0 0.018 0.12 47

30

Table 2 Predicted egg and hatchling components from 450 g and 650 g Emu eggs.

Parameter Egg Mass (g) % increase

450 650 44

Wet yolk in egg (g) 184 260 41

Dry yolk in egg (g) 102 150 47

Wet albumen in egg (g) 209 307 47

Dry albumen in egg (g) 22 34 55

Water in egg (g) 270 384 42

Energy in egg (kJ) 3596 5336 48

Water-vapor conductance (mg d-1torr-1) 45 63 40

Air cell PO2 (torr) 108 115 6

MR near-term embryo (ml O2 h-1) 83 105 26

Wet hatchling (g) 298 426 43

Yolk-free hatchling (g) 227 317 40

Yolk-free hatchling solids (g) 64 88 38

Water in yolk-free hatchling (g) 163 229 40

Residual yolk dry (g) 39 55 41

Yolk solids consumed (g) 63 95 51

Percent yolk solids consumed 62 64 3

31

Figure Legends

Figure 1. Mass of Emu egg components increase with fresh egg mass (Me). Filled circles show

albumen mass (Ma = 0.49 Me – 11.1; r2 = 0.87), open circles show albumen dry mass

(Mad = 0.06 Me – 5.1; r2 = 0.75), filled squares show yolk mass (My = 0.48 Me + 13.4;

r2 = 0.82), open squares show yolk dry mass (Myd = 0.24 Me – 6.2; r2 = 0.76), and

triangles show shell mass (Ms = 0.13 Me + 1.5; r2 = 0.68).

Figure 2. Mass of water and solids in yolk-free hatchlings and eggs increase with yolk-free

hatchling carcass mass (Mc) and with fresh egg mass (Me). Filled inverted triangles

show carcass water content (Mcw = 0.7 Mc + 1.6; r2 = 0.95) and filled diamonds show

egg water content (Mew = 0.57 Me + 13.4; r2 = 0.94). Open inverted triangles show

carcass solids (Mcs = 0.0.27 Mc – 1.6; r2 = 0.73) and open diamonds show egg solids

(Mes = 0.3 Me - 11.6; r2 = 0.85).

Figure 3. Mass of Emu hatchling components increase with fresh egg mass (Me). Filled circles

show hatchling (yolk-free hatchling carcass + reserve yolk) mass (Mh = 0.64 Me +

10.2; r2 = 0.82), filled triangles show yolk-free hatchling carcass mass (Mc = 0.45 Me +

24.1; r2 = 0.55), open triangles show yolk-free hatchling carcass dry mass (Mcd = 0.12

Me + 7.4; r2 = 0.38), filled squares show residual yolk mass in hatchling (Mry = 0.19

Me – 13.9; r2 = 0.2), and open squares show residual yolk dry mass (Mryd = 0.11 Me –

13.0; r2 = 0.26).

Figure 4. Mass of dry yolk solids consumed (predicted initial egg yolk solids – measured yolk

sac solids) by embryos during development increase with fresh egg mass (Myc = 0.16

Me – 8.8; r2 = 0.44).

32

Figure 5. Linear dimensions of hatchling Emus increase with yolk-free hatchling carcass mass

(Mc). Squares show right tibiotarsus length (Lt = 0.04 Mc + 48.7; r2 = 0.32) and circles

show culmen length (Lc = 0.02 Mc + 27.8; r2 = 0.26).

Figure 6. Hematological parameters – blood volume, blood osmolarity, hematocrit,

and hemoglobin content – plotted as a function of yolk-free hatchling carcass mass

(Mc). (A) Hatchling blood volume (Vb = 0.09 Mc + 0.43; r2 = 0.57), (B) hematocrit

(Hct = 0.02 Mc + 29.2; r2 = 0.05), (C) hemoglobin (Hb = 0.006 Mc + 9.4; r2 = 0.03),

and (D) blood osmolarity (Osm = - 0.03 Mc + 325.2; r2 = 0.02).

33

Figure 1

Egg Mass (g)

350 400 450 500 550 600 650 700 7500

50

100

150

200

250

300

350

34

Figure 2

Yolk-free Hatchling Mass (g)

200 300 400 500 600 700

Mas

s (g

)

0

50

100

150

200

250

300

350

400

450

500

Egg Mass (g)

35

Figure 3

Egg Mass (g)350 400 450 500 550 600 650 700 750

Hat

chlin

g C

ompo

nent

Mas

s (g

)

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100

200

300

400

500

36

Figure 4

Egg Mass (g)450 500 550 600 650 700 750

Dry

Yol

k C

onsu

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(g)

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60

70

80

90

100

110

120

37

Figure 5

Yolk-free Hatchling Mass (g)200 230 260 290 320 350 380

Lin

ear D

imen

sion

(mm

)

30

40

50

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70

80

39

Figure 6 Yolk-free Hatchling Mass (g)200 240 280 320 360 400

Osm

olar

ity

(mm

ol k

g-1)

260

280

300

320

340

Yolk-free Hatchling Mass (g)200 240 280 320 360 400

Hem

atoc

rit (

%)

20

25

30

35

40

45

50

Yolk-free Hatchling Mass (g)200 240 280 320 360 400

Hem

oglo

bin

(g%

)

0

4

8

12

16

20

A.

C.

B.

Yolk-free Hatchling Mass (g)200 240 280 320 360 400

Blo

od V

olum

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l)

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40

D.

Review of Dzialowski and Sotherland, Effect of egg size on emu egg composition and hatchling phenotype. J. Exp. Biol. MS #5360E Overview This interesting study examines correlations between egg size and egg composition (variation in the quality of eggs) and between these characteristics and phenotypic traits of the resultant hatchlings (e.g., body and organ size, metabolic rate, yolk reserves) in a precocial bird that produces exceptionally large eggs (in absolute terms), and discusses possible influences such phenotypic variation may have on survivorship and ultimately the fitness of the offspring. The topic of study is certainly applicable beyond the study organism and will be useful both in comparative studies of maternal investment in birds and among oviparous amniotes in general. Thus, I believe the study is of sufficient interest to the audience and provides enough novel insight into the subject that it is suitable for publication in Journal of Experimental Biology. However, I also believe that the manuscript needs a significant (albeit not preventative) amount of revision before it will be ready for publication in the journal. Foremost, since the journal focuses on insights gleaned through experimentation, I would urge the authors to recast the introduction and discussion with a more direct experimental approach—specific a priori hypotheses should be presented in the introduction, and the discussion should focus on how the data support or refute these hypotheses. I have a number of suggestions below that I hope the authors will consider as they are revising this paper. Specific Comments Throughout 1. “Emu” and other bird names should be lower case, based on the format established in previous

papers in this journal (e.g., J. Exp. Biol. 2003 206: 2703-2710; J. Exp. Biol. 1993 175: 233-249).

2. The reasoning behind taking measurements of vapor conductance and air cell PO2 is never well established, and these measurements seem to add little to the overall picture. They could be removed to help shorten this otherwise lengthy MS without detracting from the study.

Abstract 1. p.2, line 8 – insert “the” before “yolk” 2. p.2, line 9-11 “This maternal investment was partitioned by embryos into embryogenesis,

which produced significant variation in hatchling size, and into post- hatching parental care in the form of the reserve yolk, which was positively correlated with egg and hatchling size” is an awkward sentence. I would suggest recasting it into something like this: “Variation in maternal investment was reflected in differences in hatching size and post-hatching yolk reserves among hatchlings”. Brevity is certainly a virtue in abstracts.

3. p.2, last 2 lines—“…energy reserves of the hatchlings and may, therefore, significantly affect the success…” Change to “…energy reserves of the hatchlings and may, in turn, influence the success…”. The authors need to be more cautious here—nothing presented in the data or cited in the study provides direct evidence of differential survival as a result of variation in egg composition within emus. The strength of the language therefore needs to be toned down somewhat lest you run the risk of pulling a “Blair n’ Bush”.

Introduction 1. p.3, second to last line: “…into a more independent (e.g. self-feeding, thermoregulating, lung

ventilating, and ambulatory) phase in their lives…”. Remove “lung ventilating”, since at this point in your thesis you are talking in a broad context not limited to air-breathing organisms.

2. p.4, 2nd paragraph, last sentence: “Thus, choosing a species, which produces eggs of convenient size and variation, along the avian altricial-precocial continuum (Nice 1962) and investigating how intraspecific variation in egg size and composition affects attributes of hatchlings can provide useful insights into the importance of maternal effects in oviparous amniotes”. The injection of the altricial-precocial continuum statement makes this phrase awkward, and really adds little. I would recommend removing it.

3. p.5, top paragraph: “Thus, Emus provide eggs and hatchlings, at one end of the altricial-precocial continuum, with which variation in egg and hatchling characteristics can be investigated”. Again, the A-P continuum point seems to come out of nowhere, since the importance of this topic was not really established earlier in the introduction.

4. An overall point on the introduction: I quote here a line from the instructions for reviewers for JEB: “The single most important criterion for publication in the journal is scientific excellence. In general, this means that a manuscript must pose and test a significant hypothesis that is relevant to basic issues of experimental biology”. In what I have read in the introduction and throughout the paper there is clear evidence that this study provides relevant insight into intraspecific variation in maternal investment by precocial birds. However, the specific hypothesis(es) being tested in this study is(are) not clearly defined in the introduction. What exactly did the authors expect to see in their investigation? What rationale do they have to support these expectations? These would help bring better focus to the study help the reader understand the rationale behind the study, the techniques used, etc.

Methods 1. p.5, “Egg components” paragraph, first two sentences: Just a minor comment—estimating

initial mass for an egg using this technique and then opening the egg for determination could cause some questions regarding wet yolk and albumen mass, as it assumes that all of the water lost by the egg came from the albumen, and that no exchange of water occurred between the yolk and albumen during this period. I think it would make little difference in the overall picture, but I wonder how much exchange of water could occur between the yolk and albumen in 5 days.

2. p.6, “Egg shell conductance” paragraph, second sentence: what was the approximate volume of the desiccators?

3. p.6-7, “Incubation” paragraph: What was the range of temperature variation in the incubators (36.5 +/- ?)? Also, is an RH of 30% analogous to that experienced by eggs incubated in the field?

4. p.7, “Gas Exchange”, first paragraph, 2nd sentence: Just curious—why was the temperature chamber for respirometry measurements set to 1C higher than the incubation temperatures?

5. p.7, “Gas Exchange”, first paragraph, last line: “egg-1” means g egg mass? 6. p.7, “Hatchling Morphology” paragraph, 2nd sentence (slight clarification): “Hatchlings were

euthanized by exposure to either halothane or iso-flurane, and then weighed to the nearest 0.1 g(?) to obtain hatchling mass.

6. p.8, “Hatchling Morphology” paragraph (minor clarification): “Culmen length and right tibiotarsus length were measured to the nearest 0.1 mm (?) on each hatchling…”

7. p.8, “Hematology”, 2nd paragraph, 2nd sentence: remove caps from “Iso-flurane”. 8. p.9, 2nd paragraph: “heparinized” is misspelled (2x). Also, strictly speaking, you cannot

measure volume directly using a balance. 9. p.9-10, “Statistical Analyses”, 2nd sentence: is it a log-log regression (base-10 logarithms) or a

ln-ln regression (natural logarithms)? You refer to natural logs on page 15, 2nd paragraph.

Results 1. p. 10, “Egg Composition”, first paragraph, first line: insert “The” before “Mass” at the

beginning of the paragraph. 2. p. 10, “Egg Composition”, first paragraph: I recommend moving the mean values to a table if

you really want to keep them—it will help the text flow more smoothly, and allow you to reword sentences 2 and 3, which are nearly identical to one another. Since you are investigating intraspecific variation in egg mass, composition, and hatchling phenotype, though, I’m not sure it’s necessary to include these mean values at all.

3. p.10, “Egg Composition”, second paragraph, last sentence: insert “the” before “fraction of yolk”

4. p.10, “Egg Composition”, both paragraphs: For clarity, you should specify “yolk wet mass” and “albumen wet mass” rather than “yolk mass” and “albumen mass”.

5. p.11, first paragraph, 7th line (clarification): “…nor the overall fraction of water…” 6. p.11, “Gas Exchange” paragraph, first sentence: “…and mass of yolk-free hatchlings that

emerged from the same eggs…”—reword to “…and with the yolk-free mass of the hatchlings when they emerged…”

7. p.11, “Gas Exchange” 2nd sentence: Perhaps replace “increased significantly” with “was positively correlated” to give some variety to the text.

8. p.11, “Gas Exchange” last sentence: “…nor mass of water lost...which comprised approximately…varied significantly with egg mass…” Was the analysis conducted using fractional water loss (%) or absolute water loss (g)? It is unclear which in this statement, especially since if one measure remains constant among eggs of different mass the other will vary with varying egg mass.

9. p.11, “Gas Exchange”: You present results for linear regressions. How did these different measurements scale (log-log) with variation in egg mass or hatchling mass?

10. p.12, 1st paragraph: Again, I recommend either removing these mean values altogether or tabulating them.

11. p.12, 2nd paragraph, first sentence: Insert “were” between “than” and “small hatchlings”. 11. p.12, first two paragraphs: it would be helpful if allometric scalings with egg mass and

hatchling mass were provided here. 13. p.13, “Hematology” paragraph—again, what was the scaling of blood volume with hatchling

mass? Discussion 1. p.14, 2nd paragraph, first sentence: Change “A different but related…” to “Another related…” 2. p.15, 2nd paragraph, line 4: as mentioned above, it is unclear whether your data were base-10

log transformed or natural log transformed. 3. p.15, last line: suggest replacing “…and yolk increased with mass more slowly…” with

“…and yolk showed negative allometry…” to provide more effective contrast with the earlier part of the sentence

4. p.16, first paragraph, 4th line: need to italicize the species name in Aix sponsa. 5. p.17, second paragraph, second sentence: This is a long meandering sentence. Try something

like this: “Thus, female emus that produced more massive eggs provisioned their eggs with more yolk that not only supported more growth in the embryos but provided more residual yolk to support metabolism after hatching”.

6. p.18, 2nd paragraph, 1st sentence: Change “…it is likely that the amount of residual yolk” to “…it is plausible that the amount of residual yolk…” The reason being is that there is no clear knowledge of what impact variation in residual yolk has on the survival of emu chicks. The citations provided discuss the importance of this residual yolk in precocial species under food-limited conditions, but are such conditions encountered by emu chicks frequently

enough that differential survival would result? And if so, why is there such extensive variation in the size of the eggs (if bigger is indeed better) if there is directional selection favoring large, yolky eggs?

7. p.21, first two lines: Move “reported here” to just after “developing emu embryos” 8. p.22, 1st paragraph, 1st sentence: Insert “did” between “than” and “hatchlings from small emu

eggs”. 9. p.22, 2nd paragraph: I disagree with the assessment of why metabolism did not increase to the

same extent with increased egg size as did hatchling mass. If hypoxia was really a factor, wouldn’t one expect to see lower PO2 in the air cell in larger eggs? The difference in MR may simply be a matter of differential scaling, which could be evaluated if the log-log regressions for metabolism and hatchling composition were provided.

Literature Cited p. 24—citation for Burggren (1999/2000) – August Krogh should be capitalized. Tables 1. p.29, Table 1 caption: I suggest using “Hatchling organ masses” rather than “Hatchling

component masses”. Figures 1. Figure 1 is missing a y-axis legend 2. Figure 2: Suggest “Content Mass (g)” for the y-axis legend instead of simply “Mass (g). 3. Figure 6: All panel letters are too close to the top, and the “D” for panel D lies on the top

frame of the panel. All panel letters should be moved down somewhat. 4. Figure 7: Instead of “Linear Dimension”, use “Length”.

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Parental investment, particularly nutrients and energyallocated to eggs, can profoundly influence the development ofembryos, the phenotypes and survival of hatchlings, and,therefore, evolutionary fitness of both offspring and parents.Parental investment in embryogenesis provides for thesuccessful development of a zygote into a complete hatchling,and parental investment in care of the hatchling constitutes theenergy and nutrients allocated to an egg beyond those neededto produce a hatchling and used by the hatchling to supportgrowth and maintenance after emerging from the egg(Congdon, 1989). As phenotypes of oviparous mothers thataffect phenotypes of their offspring, parental investment inoffspring via eggs frequently has significant, and evolutionarilymeaningful, maternal effects (Bernardo, 1996a,b). Reaching afull understanding of the magnitude of these maternal effects,and how they evolve, requires an examination of intraspecificvariation in parental investment in eggs along with anexamination of how embryos respond physiologically to theegg environments within which they develop.

The trajectory followed by an embryo from zygote tohatchling stages is influenced by an interaction between genetic

instructions in the nuclei of the embryo’s cells and conditionsin the environment surrounding those cells. Conceptuallysimilar to evolutionary paths blazed by populations oforganisms through phenotypic space over several generations(Raup, 1966), developmental trajectories (Burggren, 1999) ofoviparous amniotes can change as a result of biotic and abioticfactors encountered outside the eggshell and factors, initiallymaternal in origin, found within the eggs. Phenotypes of theseembryos, developing toward hatching and towardmetamorphosis into a more independent (e.g. self-feeding,thermoregulating and ambulatory) phase in their lives, areshaped both by genetic and environmental effects (Burggren,1999). Acquiring in-depth knowledge of the sensitivity ofdevelopmental trajectories to environmental perturbations,including maternal investment of nutrients and energy in eggs,will improve our understanding of the genesis and importanceof maternal effects manifested in phenotypes of hatchlings.

Requiring only heat and oxygen from the environment andcontaining all nutrients and water necessary to sustaindeveloping embryos, avian eggs are attractive models forinvestigating effects of maternal investment on phenotypes of

The Journal of Experimental Biology 207, 597-606Published by The Company of Biologists 2004doi:10.1242/jeb.00792

Parental investment in eggs and, consequently, inoffspring can profoundly influence the phenotype, survivaland ultimately evolutionary fitness of an organism. Avianeggs are excellent model systems to examine maternalallocation of energy translated through egg size variation.We used the natural range in emu Dromaiusnovaehollandiaeegg size, from 400·g to >700·g, to examinethe influence of maternal investment in eggs on themorphology and physiology of hatchlings. Female emusprovisioned larger eggs with a greater absolute amount ofenergy, nutrients and water in the yolk and albumen.Variation in maternal investment was reflected indifferences in hatchling size, which increased isometricallywith egg size. Egg size also influenced the physiology ofdeveloping emu embryos, such that late-term embryonicmetabolic rate was positively correlated with egg size andembryos developing in larger eggs consumed more yolk

during development. Large eggs produced hatchlings thatwere both heavier (yolk-free wet and dry mass) andstructurally larger (tibiotarsus and culmen lengths) thanhatchlings emerging from smaller eggs. As with manyother precocial birds, larger hatchlings also containedmore water, which was reflected in a greater bloodvolume. However, blood osmolality, hemoglobin contentand hematocrit did not vary with hatchling mass. Emumaternal investment in offspring, measured by egg sizeand composition, is significantly correlated with themorphology and physiology of hatchlings and, in turn,may influence the success of these organisms during thefirst days of the juvenile stage.

Key words: emu, Dromaius novaehollandiae, egg, development,maternal effect, life history, allometry, scaling.

Summary

Introduction

Maternal effects of egg size on emu Dromaius novaehollandiaeegg compositionand hatchling phenotype

Edward M. Dzialowski1,* and Paul R. Sotherland2

1Department of Biological Sciences, University of North Texas, PO Box 305220, Denton, TX 76203, USAand2Department of Biology, Kalamazoo College, Kalamazoo, MI 49007, USA

*Author for correspondence (e-mail: edzial@unt.edu)

Accepted 17 November 2003

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embryos and hatchlings. Variation in the composition of avianeggs among species is correlated with functional maturity ofhatchlings (Carey et al., 1980; Sotherland and Rahn, 1987).The quantity and composition of parental investment variessignificantly within species and is frequently correlated withhatchling mass (Williams, 1994). Investigating howintraspecific variation in egg size and composition affectshatchling attributes can provide useful insights into theimportance of maternal effects in oviparous amniotes.

In this study we examined consequences of natural variationin maternal investment – egg size and composition – on emuhatchling phenotypes. Emu eggs and hatchlings make goodexperimental subjects for a study of parental investment becausethey are large (egg mass approx. 600·g; hatchling mass approx.400·g), facilitating measurements of hatchling characteristics(e.g. blood volume) that are otherwise difficult to quantify. Inaddition, intraspecific variation in egg mass from 400·g to>700·g provides a reasonably, but not unusual, wide range ofegg size. Female emus lay between 5 and 20 eggs, typicallyincubated by the males during the breeding season. Afteremerging from their eggs the precocial hatchlings forage forfood under guidance from the males (Davies, 1975). Thus, likeother precocial birds (Williams, 1994; Hill, 1995), emus shouldproduce eggs having component masses that vary isometricallywith egg mass, as well as hatchlings, emerging from those eggs,that vary isometrically with egg mass. Therefore we tested thefollowing hypotheses: (1) maternal investment, in the form ofnutrients and water in eggs, is positively correlated with egg sizeand varies in such a way that the proportional composition ofeggs remains constant; (2) morphological and physiologicalphenotypes of hatchlings correlate positively with egg size suchthat proportional composition of hatchlings remains constantregardless of hatchling size; (3) maternal investment in eggsprovides for greater energy use in larger eggs duringdevelopment while provisioning hatchlings with similaramounts of residual yolk regardless of hatchling size.

Materials and methodsAnimals

Emu Dromaius novaehollandiae Latham eggs wererandomly collected within 5 days after oviposition at the CrossTimbers Emu Ranch, Flower Mound, TX, USA fromNovember 2000 to March 2001. At the time of egg collection,the female breeding population at Cross Timbers Emu Ranchwas 45 female birds ranging in age from 3–7 years. Forty-nineemu eggs were used to determine egg composition, and 53 eggswere incubated to obtain measurements of hatchlingphenotypes. Though we do not know the source of each egg,it is likely that more than one egg from some females was usedin this study. All protocols used in this study were approvedby the University of North Texas Animal Care and UseCommittee.

Egg components

Fresh egg mass was determined by drilling two small holes

through the shell over the air cell, filling the air cell withwater, and then weighing the eggs on a Denver Instruments(Denver, CO, USA) digital balance. Short of weighing eggsimmediately after oviposition, this is the most reliable methodof obtaining fresh egg mass (Ar and Rahn, 1980). Fresh eggswere then separated into shell, yolk and albumen followingthe methods described in Finkler et al. (1998). The intact yolkwas weighed with the balance to determine yolk mass. Yolk,albumen and shell were then dried to a constant mass in adrying oven at 60°C. Shell mass was measured by weighingthe dry shell on the balance, and albumen wet mass wasdetermined by subtracting yolk wet mass and shell dry massfrom the mass of the egg. Water contents of yolk and albumenwere determined by subtracting dry mass of each from therespective wet mass; the sum of water mass in the yolk andwater mass in the albumen yielded total water content of eachegg. Mass of egg solids was computed by adding yolk andalbumen dry masses.

Incubation

Eggs were stored at 4°C for no more than 7 days beforeincubation. Eggs were incubated in forced draft incubatorswith automatic rotation at Cross Timbers Emu Ranch untilapproximately day 40 of incubation. They were thentransferred to the University of North Texas, where incubationcontinued until hatching in forced draft emu incubators (GQFManufacturers, Savannah, GA, USA). Eggs were incubated at36.5±1°C and a relative humidity of approximately 30%,corresponding to the relative humidity experienced in the nest.Prior to internal pipping, all eggs were transferred to a hatchingincubator maintained at 36.5°C and a relative humidity of35–40%.

Gas exchange of near-term embryos

Metabolic rates (V̇O2) of 15 eggs were measured on day 46of incubation (i.e. 92% of incubation) using a flow-throughsystem similar to the methods of Dzialowski et al. (2002). Eggswere placed in individual PVC respirometers (approx. vol. 1·l)and then into a constant temperature chamber regulated at37.5°C. Air was pumped through the individual chambers andflow was measured at the inflow side of the chambers using acalibrated Brooks (Hatfield, PA, USA) flow meter. Outflow O2

concentration from each respirometer was measured using aBeckman OM11 O2 analyzer (Anaheim, CA, USA). Inflow O2concentration to the respirometers was determined from theoutflow of an empty respirometer. Metabolic rate (i.e. rate ofoxygen consumption) was calculated using the equation of Hill(1972), corrected to STPD and expressed in units of ml·O2·h–1.

Air cell PO∑ was measured in eight emu eggs on day 46 ofincubation. On day 40 of incubation a 5·mm diameter hole wasdrilled in the air-cell end of each egg using a drill press. Asquare patch of 0.4·mm thick Thera-band™ latex was gluedover the hole using Duro Quick Gel™ and the egg wasreplaced into the incubator for 6 days. Using a 1·ml syringeand a 27-gauge needle inserted through the latex, a 1·ml sampleof gas was withdrawn from the air cell and then promptly

E. M. Dzialowski and P. R. Sotherland

599Consequences of emu egg size

analyzed for PO∑ using a Cameron Instruments (Port Aransas,TX, USA) BGM2000 blood gas meter.

Eggshell conductance

We measured water vapor conductance (GH∑O) of fresh eggsof mass 487–778·g (N=16). Eggs were initially weighed andthen placed in individual desiccators (approx. vol. 6·l). Eachdesiccator contained an ample amount of Drierite™ desiccantin the bottom of the desiccator to ensure that water vaporpressure around each egg was near 0·kPa. The mass of eachegg, desiccator temperature and atmospheric pressure weremeasured daily for 5 days. Whole eggshell GH∑O wasdetermined following the protocol of Ar et al. (1974). Finally,initial egg mass was measured as above by filling the air cellwith water and then weighing the egg.

Hatchling morphology and composition

All measurements of morphology and composition weremade on hatchlings that were less than 1 day old. Hatchlingswere euthanized by exposure to either halothane or iso-flurane, and then weighed to the nearest 0.1·g to obtainhatchling mass (yolk-free hatchling mass plus residual yolkand yolk sac). The yolk sac was carefully dissected from eachhatchling and weighed to measure the quantity of residualyolk; yolk-free hatchling mass was determined by subtractingresidual yolk mass from hatchling mass. Culmen length andright tibiotarsus length were measured to the nearest 0.1·mmon each hatchling using digital calipers (Mitutoyo, Aurora, IL,USA) as a means of quantifying hatchling structural size.Heart, gizzard and liver were dissected from the body,weighed separately, and then dried to a constant mass in anoven at 60°C. The yolk sac and what remained of the hatchlingwere dried to a constant mass in a similar way. Water contentsof the various components were determined by subtracting drymass of each from the respective wet mass. Mass of yolk-freehatchling solids was computed by adding heart, gizzard andliver dry masses to the dry mass of the dissected carcass. Weestimated the quantity of yolk consumed by an embryo duringincubation by subtracting the measured dry yolk sac massfrom the calculated mass of dry yolk that the egg from whicha neonate hatched would have contained at the outset ofincubation, using initial egg mass and the equation for dryyolk mass in Fig.·1.

Hematology and blood volume

To obtain blood for hematological measurements, hatchlingswere anesthetized using halothane and blood was taken fromthe heart by direct cardiopuncture. Hemoglobin was measuredwith a Radiometer (Brønshøj, Denmark) OSM2 Hemoximeter.Hematocrit was measured by centrifuging blood in heparinizedcapillary tubes. Osmolality of the blood was measured using aWescor (Logan, UT, USA) 5500 vapor pressure osmometer.Two measurements of each variable were made and averagedfor each animal.

Blood volumes were measured in 11 hatchlings using theEvan’s Blue dilution technique (El-Sayed et al., 1995).

Hatchlings were anesthetized with iso-flurane and attached toa ventilator that maintained an iso-flurane concentration of1% in the inspired air. Both the right and left jugular veinswere exposed and non-occlusively canulated with tips of 26-gauge needles attached to PE50 tubing. The right jugular veinwas used as the injection site for the Evan’s Blue solution,and the left jugular vein was used to withdraw subsequentblood samples. Initially, 500·µl of blood was withdrawn intoa heparinized syringe from the right jugular vein. Thiswas followed by an injection of 400·µl of an Evan’s Bluesolution (5·mg·ml–1 dissolved in 0.9% heparinized saline)into the right jugular vein. The Evan’s Blue injection wasfollowed by a 200·µl injection of heparinized saline to washthe tubing. Samples of blood were then taken from the leftjugular vein at 10, 15 and 20·min after the initial injection ofEvan’s Blue.

After each blood sample was collected, a portion of bloodfrom the sample was added to an equal amount of heprainizedsaline and centrifuged for 15·min. All volumes weregravimetrically determined using a Denver Instruments digitalbalance to increase measurement accuracy. A 200·µl sample ofthe supernatant was added to 800·µl of heprainized saline andthe absorbance was measured at 610·nm using a Bauschand Lomb (Rochester, NY, USA) Spectronic 88spectrophotometer. A subsample of plasma from the initialblood sample, taken before injection of Evan’s Blue, was usedto create a blank for zeroing the spectrophotometer for eachhatchling’s measurement.

A standard curve (r2=0.93) relating absorbance to Evan’sBlue concentration was generated using plasma from fouradditional hatchlings. Blood volumes were calculated from themeasured Evan’s Blue concentrations according to themethods in El-Sayed et al. (1995).

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Fig.·1. Mass of emu egg components increase with fresh egg mass(Me). Filled circles, albumen mass (Ma=0.49Me–11.1; r2=0.87); opencircles, albumen dry mass (Mad=0.06Me–5.1; r2=0.75); filled squares,yolk mass (My=0.48Me+13.4; r2=0.82); open squares, yolk dry mass(Myd=0.24Me–6.2; r2=0.76); triangles, shell mass (Ms=0.13Me+1.5;r2=0.68).

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Statistical analyses

Linear regressions of parameters on egg mass and yolk-freehatchling mass were carried out using SPSS 11.0. Additionally,log–log regressions were performed on data to determine ifcomponent masses varied in simple linear proportion to bodymass (slope of log–log regression, b=1.0) or if componentmasses showed a positive (b>1.0) or negative (b<1.0)allometry with egg mass or hatchling mass. The regressionswere considered to vary in simple proportion to body mass ifthe 95% confidence interval of the slope of the log–logregression included 1. In order for the log–log relationship tohold true the intercept of the untransformed data must passthrough the origin. Prior to log–log transformation allrelationships were examined using non-linear power fits(y=y0+axb; Sigmaplot 8.02) to the untransformed data. The50% confidence interval for the intercept (y0) was used todetermine if it differed significantly from zero. Log–logregressions were carried out on data when the intercept wasnot significantly different from zero. A significance level ofP<0.05 was adopted for all regressions. Linear regressionequations are provided in the figure legends and whendetermined allometric slopes are provided in the text. Allvalues are presented as means ±S.D. except for the slopes ofthe log–log regressions, which are presented as the slope (b)±95% confidence interval.

ResultsEgg composition

The mass of each egg component increased significantly asegg mass (586.17±78.06·g; N=49) increased fromapproximately 400·g to 700·g (Fig.·1). Albumen wet mass(274.00±40.75·g; N=49) increased significantly (F1,47=309;P<0.001) with egg mass as did albumen dry mass(28.43±5.21·g; N=47; F1,45=135; P<0.001). Yolk wet mass(237.66±33.07·g; N=49) increased significantly (F1,47=208;P<0.001) with egg mass as did yolk dry mass (136.36±21.93·g;N=47; F1,45=146; P<0.001). Like the other two majorcomponents of fresh eggs, shell mass (75.12±11.90·g; N=49)increased significantly (F1,47=99; P<0.001) with egg mass.

The relative contribution of albumen and yolk to the eggsdid not vary with initial egg mass. Slopes (b) of the log–logregressions of log albumen wet mass (b=1.04±0.13; r2=0.87),log dry albumen mass (b=1.22±0.22; r2=0.75), log yolk wetmass (b=0.93±0.14; r2=0.82), and log dry yolk mass(b=1.05±0.18; r2=0.76) against log initial egg mass were notsignificantly different from 1. As a result, fraction of yolk inthe contents (0.47±0.03), typically correlated withdevelopmental maturity of hatchlings (Sotherland and Rahn1987), did not change (F1,47=0.49; P=0.49) with egg mass.

Water and solid content of eggs increased with egg mass(Fig.·2), but the fraction of water and solids did not varysignificantly over the range of egg masses examined. Waterin eggs (343.02±46.21·g; N=45) increased significantly(F1,43=648; P<0.001) with egg mass, as did the solid contentof eggs (163.97±25.84·g; N=45; F1,43=245; P<0.001).

Approximately 71% of water in eggs was found in thealbumen (244.56±36.70·g; N=47), which was composed of aninvariant fraction of water (0.90±0.01; F1,45=1.8; P=0.19).Similarly, neither the fraction of water in the yolk (0.42±0.03;N=47) nor the overall fraction of water in the eggs (0.68±0.02;N=45) changed significantly with egg mass. However, thetotal amount of water in the yolk (99.93±13.84·g; N=47)increased significantly (F1,45=66; P<0.001) with egg mass asdid the total amount of water in the albumen (F1,45=265;P<0.001).

Gas exchange of near-term embryos

Metabolic rate (107.8±11.6·ml·O2·h–1; N=15) of pre-pipembryos, measured on day 46, was positively correlated withinitial egg mass Me (F1,13=7.7; P=0.016; V̇O2=0.11Me+33.9;r2=0.37) and with the yolk-free mass of the hatchlings Mh

when they emerged from the same eggs (F1,13=14; P=0.003;V̇O2=0.20Mh+48.8; r2=0.51). Metabolic rate scaled with anegative allometry with egg mass (b=0.56±0.41) and yolk-freehatchling mass (b=0.50±0.25). In a separate set of eggs,eggshell water vapor conductance (447.4±76.7·mg·kPa–1

day–1; N=14; GH∑O=0.09Me+4.5; r2=0.51) increasedsignificantly (F1,13=9.4; P=0.01) with initial egg mass. Incontrast, pre-pip air cell PO∑ (15.0±0.8·kPa; N=8) did not varysignificantly with egg mass (F1,6=2.4; P=0.17) (not shown).

Hatchling morphology and composition

Hatchling mass (yolk-free hatchling plus residual yolk)increased with egg mass (Fig.·3). Hatchling mass(403.59±45.67·g; N=48) increased significantly (F1,46=214;P<0.001) with egg mass, as did yolk-free hatchling mass

E. M. Dzialowski and P. R. Sotherland

Yolk-freehatchling mass (g)

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Fig.·2. Mass of water and solids in yolk-free hatchlings and eggsincrease with yolk-free hatchling mass (Myfh) and with fresh eggmass (Me). Filled inverted triangles, yolk-free hatchling watercontent (Myfhw=0.7Myfh+2.2; r2=0.95); filled diamonds, egg watercontent (Mew=0.57Me+13.4; r2=0.94). Open inverted triangles, yolk-free hatchling solids (Myfhs=0.25Myfh+1.0; r2=0.76); open diamonds,egg solids (Mes=0.3Me–11.6; r2=0.85).

601Consequences of emu egg size

(303.47±37.06·g; N=48; F1,46=83.6; P<0.001) and residualyolk mass (100.13±23.96·g; N=48; F1,46=14.3; P=0.001). Drymass of yolk-free hatchling (80.76±11.73·g; N=45) and drymass of residual yolk (57.13±13.93·g; N=46) also increasedsignificantly (F1,43=32.8; P<0.001 and F1,44=17; P<0.001respectively) with egg mass. Hatchling mass increased insimple linear proportion to egg mass. The slopes (b) of thelog–log regressions of log yolk-free hatchling wet mass(b=0.96±0.20; r2=0.66) and log yolk-free hatchling dry mass(b=0.94±0.29; r2=0.47) against log initial egg mass were notsignificantly different from 1.

Large hatchlings were composed of more water and solidsthan were small hatchlings (Fig.·2), but the fraction of waterin hatchlings remained unchanged regardless of hatchlingsize. Mass of water in yolk-free hatchlings (225.20±27.2·g;N=44) increased significantly (F1,43=747.2; P<0.001) withmass of yolk-free hatchlings, but the fraction of water in thosehatchlings (0.74±0.02; N=44) did not vary significantly(F1,43=0.05; P=0.82) with hatchling mass. Mass of solids inhatchlings (i.e. dry mass of yolk-free hatchling) alsoincreased significantly (F1,43=120; P<0.001) with hatchlingmass.

Yolk consumed by developing embryos, i.e. differencebetween mass of the dry yolk (estimated using measuredinitial egg mass and the equation for dry yolk mass providedin Fig.·1) and measured mass of residual yolk remaining inthe yolk-sac (83.64±14.44; N=46) increased significantly(F1,44=48.4; P<0.001) with yolk-free hatchling mass(Fig.·4A). The combination of initial yolk mass increasingwith egg mass and yolk consumed increasing with yolk-freehatchling mass yielded a constant residual yolk mass across

all hatchling masses (mean dry residual yolk 57.8±14.6).However, the statistical residuals from regressions of yolk-free hatchling dry mass and residual yolk dry mass on eggmass revealed that, independent of initial egg mass, largerhatchlings had less residual yolk upon hatching than smallerhatchlings (Fig.·4B).

Linear dimensions of heavier hatchlings were greater thanthose of lighter hatchlings (Fig.·5). Length of both the righttibiotarsus (70.44±3.79·mm; N=48; F1,46=107.12; P<0.001)and culmen (36.78±1.90·mm; N=48; F1,46=9.6; P=0.003)increased significantly with yolk-free hatchling mass.

Wet and dry masses of heart, liver, and gizzard all increasedsignificantly with yolk-free hatchling mass (Table·1).

Hematology of hatchlings

Blood volume (27.8±7.0·ml; N=11), which constituted anessentially constant proportion (approximately 9.2%) of theyolk-free hatchling mass, increased significantly (F1,9=12;P=0.007) with yolk-free hatchling mass (Fig.·6). The increase

Egg mass (g)

350 400 450 500 550 600 650 700 750

Hat

chlin

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)

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Fig.·3. Mass of emu hatchling components increase with fresh eggmass (Me). Filled circles, hatchling (yolk-free hatchling + residualyolk) mass (Mh=0.64Me+9.6; r2=0.82); filled triangles, yolk-freehatchling mass (Myfh=0.46Me+20.5; r2=0.64); open triangles, yolk-free hatchling dry mass (Myfhd=0.12Me+6.7; r2=0.43); filled squares,residual yolk mass (Mry=0.18Me–10.9; r2=0.24); open squares,residual yolk dry mass (Mryd=0.11Me–12.0; r2=0.28).

Yolk-free hatchling mass (g)

200 230 260 290 320 350 38040

50

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–20 –15 –10 –5 5 10 15 200–30

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A

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idua

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idua

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ry y

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Fig.·4. (A) Mass of dry yolk solids consumed (predicted initial eggyolk solids – measured residual yolk solids) by embryos duringdevelopment increases with yolk-free hatchling mass(Myc=0.29Myfh–3.94; r2=0.52). (B) Mass of residual yolk solids rMdecreased as mass of yolk-free hatchling increased irrespective ofinitial egg mass (rMryd=–1.03rMyfhd+0.41; r2=0.58). Statisticalresiduals from the regression of the mass of residual yolk solids andthe mass of yolk-free hatchling on initial egg mass were obtainedfrom regression equations in Figs·1 and 3 and plotted against eachother, revealing a trade-off between retaining residual yolk andproducing a hatchling.

602

in blood volume was proportional to the increase in yolk-freehatchling mass (b=1.05±0.46; r2=0.69). However, none of theother blood parameters measured [osmolality(304.6±15.6·mOsm·kg–1; N=44), hematocrit (38.4±4.1%;N=45), or hemoglobin concentration (12.9±1.8·g%; N=44)]varied significantly with yolk-free hatchling mass (Fig.·6).

DiscussionConsequences of parental investment (i.e. investment of

energy and nutrients; Congdon, 1989) in reproduction,frequently manifested as maternal effects, can be observed assignificant variation in the physiology, morphology and lifehistory of organisms (Bernardo, 1996a,b). Female birds varyreproductive investment by allocating different amounts ofalbumen and yolk to the eggs they produce or by producingeggs of different size. Maternal investment in emu eggs, thethird largest egg laid by extant birds, varied considerably(Fig.·1), with eggs differing in mass by as much as 300·g. Thisvariation was reflected in concomitant variation in hatchlingsize and composition.

Yolk and albumen in eggs

One measure of parental investment in bird eggs typically isexpressed as the fraction of yolk in the contents (FYC) of eggs.Emus in this study laid eggs containing nearly 50% yolk(FYC=0.47), which is within the range of yolk content forprecocial birds but larger than that predicted for precocial eggsof the same mass. Sotherland and Rahn (1987) examined therelationship between egg mass and energy content for a widevariety of birds and found that FYC for precocial speciesranges from 0.32 to 0.69. Based on the equation for yolkcontent in precocial species (Sotherland and Rahn, 1987), wepredicted the FYC for an average sized emu egg (512·g wetcontents) to be 0.39, which is less than the FYC of emu eggsmeasured in this study. Thus, female emus provision their eggs

with relatively more yolk and less albumenthan would be predicted for a typical largeprecocial egg. If we compare emu eggswith those of closely related species, emueggs tend to have a larger FYC than eitherthe ostrich Struthio camelus(1.2·kg egg,FYC=0.38; Romanoff and Romanoff,1949) or cassowary Casuarius casuarius(546·g egg, FYC=0.42; Carey et al., 1980).This finding is not surprising, however,because the incubation period of the emuis longer than that of the ostrich,suggesting that emu embryos require moreenergy than ostrich embryos to completeincubation. In contrast, emu eggs have alower FYC than the smaller kiwi eggs(Apteryx australis; 440·g egg, FYC=0.61;Reid, 1971; Calder et al., 1978), which hasan incubation period about 25 days longerthan the emu.

Another related comparison amongspecies entails examining the contributionof albumen and, therefore, water (albumenin all bird eggs is about 90% water;Sotherland and Rahn, 1987) to avian eggcontents. We suggest here that alwaysfocusing on yolk and FYC divertsattention from albumen and its important

E. M. Dzialowski and P. R. Sotherland

Yolk-free hatchling mass (g)

200 230 260 290 320 350 380

Len

gth

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30

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Fig.·5. Linear dimensions of hatchling emus increase with yolk-freehatchling mass (Myfh). Squares, right tibiotarsus length(Lt=0.09Myfh+44.4; r2=0.70); circles, culmen length(Lc=0.02Myfh+30.3; r2=0.17).

400360320280240200

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ity (

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ol k

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Fig.·6. Blood volume, blood osmolality, hematocrit and hemoglobin content plotted as afunction of yolk-free hatchling mass (Myfh). (A) Hatchling blood volume(Vb=0.09Myfh+0.43; r2=0.57), (B) hematocrit (Hct=0.02Myfh+29.2; r2=0.05), (C)hemoglobin (Hb=0.006Myfh+9.4; r2=0.03) and (D) blood osmolality(Osm=–0.03Myfh+325.2; r2=0.02).

603Consequences of emu egg size

contributions to embryo development and hatchlingphenotype. The fraction of albumen in the contents(FAC=1–FYC) of eggs is very high (about 80%) in altricialspecies and drops to less than 50% in more precocial species.Emu egg contents are about half albumen (FAC=0.53), and atthe middle of the range observed in ratites, where FAC variesbetween 0.4 (kiwi) to 0.6 (ostrich).

Scaling of egg composition

Maternal investment in avian eggs varies bothinterspecifically and intraspecifically in two ways. First, theabsolute size of eggs and egg contents can vary among andwithin species. Second, the relative contribution of yolk,albumen and shell to the mass of an egg can vary with egg sizeand with maturity of neonate at hatching.

Intraspecifically, emu eggs exhibit isometric scalingbetween egg size and all egg components. Large eggscontained more yolk and albumen (Fig.·1) as well as water andsolids (Fig.·2) than small eggs, but yolk and albumen massincreased isometrically with egg size; the slope of log–logregressions of these components on egg mass did not differsignificantly from 1. Thus, emu eggs in this study followed theprecocial pattern (Williams, 1994) where eggs of all sizes hadthe same relative amount of yolk and albumen.

A number of studies have examined intraspecific variationof egg composition and have revealed patterns of how yolk andalbumen content vary with egg size along thealtricial–precocial continuum (Sotherland et al., 1990;Williams, 1994; Hill, 1995; Carey, 1996). For most species ofbirds, the vast majority of which are altricial, variation inalbumen mass accounts for most of the variation in egg mass,but yolk contributes more to variation in egg mass as FYCincreases toward the precocial end of the altricial–precocialcontinuum (Sotherland et al., 1990). Williams (1994) reviewed22 studies that had examined intraspecific variation in eggcomponents and found that only half of these studies revealedan isometric relationship between egg size and either yolk oralbumen content. Hill (1995) found that wet albumen mass andwet yolk mass tended to scale isometrically with egg mass inprecocial species, whereas in altricial species albumen showedpositive allometry (b>1.0) and yolk showed negative allometry(b<1.0). Thus, it seems that altricial species change egg size

by increasing the amount of albumen while keeping yolkcontent relatively constant, whereas precocial species tend toalter egg size by increasing both yolk and albumen contentwith an increase in egg mass. Further support for this patternhas been observed in precocial wood ducks Aix sponsa(Kennamer et al., 1997) and ruddy ducks Oxyura jamaicensis(Pelayo and Clark, 2002), which lay eggs having yolk andalbumen varying isometrically with egg mass, and in altricialgreat tit Parus majoreggs, in which much of the variation inegg mass is attributable to variation in albumen mass (Lessellset al., 2002).

Egg size and hatchling size

The developing emu embryo may partition the maternalinvestment of energy and nutrients into growth andmaintenance of the developing body or into residual yolk. Theenergy and nutrients invested in an egg by a female that theembryo uses for growth and maintenance are parentalinvestment in embryogenesis, whereas energy and nutrientsleft as residual yolk or hatchling fat deposits comprise parentalinvestment in care of the hatchling (Congdon, 1989). Increasedparental investment in larger emu eggs (Fig.·1) yielded largerhatchlings (Fig.·3) that tended to contain similar amounts ofresidual yolk as smaller hatchlings due to the fact that the largerhatchlings consumed more of their yolk during incubation(Fig.·4). Increased hatchling size is attributable to increasedtotal water content (Fig.·2), increased dry mass (Fig.·2), andincreased structural size as measured by the tibiotarsus andculmen lengths (Fig.·5). Heart, liver and gizzard masses werealso larger in hatchlings from large eggs (Table·1). Thus, theincreased maternal investment was used by the developingembryo for embryogenesis to yield a larger hatchling that hadthe same level of post-hatching care in the form of residualyolk, suggesting that egg size can be equated with egg qualityin emus.

In contrast with our findings here, a review of the literatureby Williams (1994) found that larger bird eggs produce heavierhatchlings, but not necessarily structurally larger hatchlings.However, many of the studies of the relationship between massand structural size in hatchlings examined only hatchling massincluding residual yolk and concluded that hatchlings fromlarger eggs were heavier because they contained more residual

Table·1. Hatchling organ masses (g) and their regressions on yolk-free hatchling mass

Component Mean S.D. Slope Intercept F P r2 N

HeartWet 2.16 0.56 0.008 –0.386 20.33 <0.001 0.29 52Dry 0.38 0.11 0.001 –0.04 12.6 <0.001 0.22 46

LiverWet 8.30 1.65 0.024 0.97 18.9 <0.001 0.28 52Dry 3.39 0.65 0.010 0.44 18.56 <0.001 0.30 46

GizzardWet 5.63 1.07 0.014 1.51 15.05 <0.001 0.23 52Dry 1.05 0.29 0.003 0.30 4.7 0.036 0.10 46

604

yolk and not because they were structurally larger (Williams,1994). Ankney (1980) found a significant positive relationshipbetween egg size and length of the tarsus and culmen of lessersnow goose Anser caerulescenshatchlings, but the relationshipbetween hatchling size and linear dimensions was not reported.Larger eggs laid by the thick-billed murre produced heavierhatchlings, but this was due mainly to increased water contentor residual yolk and not due to increased linear dimensions(Birkhead and Nettleship, 1982). In a number of alcid species,most of the variance in hatchling mass in relation to pippedegg mass was attributed to differences in residual yolk ratherthan increased water content or dry hatchling mass (Birkheadand Nettleship, 1984). In the king eider Somateria spectabilis,large eggs produced larger hatchlings with larger wet and drybreast and leg muscle mass than the hatchlings produced fromsmall eggs (Anderson and Alisauskas, 2002). However, therelative structural size of larger king eider hatchlings was lessthan that of small hatchlings. In the altricial blackbird Turdusmerula, large eggs produced both heavier and larger hatchlings(Magrath, 1992).

Emu hatchlings had a large residual yolk, which waspositively correlated with initial egg mass (Fig.·3) but not yolk-free hatching mass. Female emus provisioned eggs withenough yolk to support development and maintenance ofembryos and to provide sufficient residual yolk to supportactivity and survival after hatching. By factoring out the effectof egg mass on both residual yolk dry mass and yolk-freehatchling dry mass (Fig.·4B) we found that larger-than-averagehatchlings, at any egg mass, have less residual yolk thansmaller-than-average hatchlings. Therefore, there appears to bea trade-off for the developing embryo: produce more tissue andhatch with less residual yolk or hatch smaller with moreresidual yolk.

In general, emu hatchlings have more residual yolk, as afraction of the whole hatchling, than many of the other avianspecies studied (Vleck and Vleck, 1996). Whereas precocialhatchlings retain residual yolk of 0.15–0.18 of their wet massand 0.28 of their dry mass (Carey, 1996; Vleck and Vleck,1996), emu hatchlings in our study had residual yolkamounting to 0.25 of their wet mass and 0.42 of their dry mass.There is also sizeable residual yolk in the ostrich, accountingfor 0.29 of wet mass and 0.56 of dry mass (Gefen and Ar,2001). The ostrich and emu are both ratites, suggesting thatmembers of this clade have noticeably high parentalinvestment in hatchling care and residual yolk.

Though we did not examine survivorship consequences ofthe levels of residual yolk measured here, it is plausible thatthe large amount of residual yolk we measured would influenceearly growth and survival of these hatchlings because adultemus do not feed the young (Davies, 1975). Parentalinvestment in hatchlings via yolk can provide hatchlings withenergy used to grow and sufficient residual yolk (i.e. parentalinvestment in care), which serves as a post-hatching source ofnutrients and energy that can affect survivorship, especiallyduring times of nutritional stress. A number of studies ofprecocial species (Kear, 1965; Ankney, 1980; Peach and

Thomas, 1986; Thomas et al., 1988; Slattery and Alisauskas,1995; Visser and Ricklefs, 1995; Dawson and Clark, 1996;Nager et al., 2000; Anderson and Alisauskas, 2001) haveshown that an increase in residual yolk, correlated withincreased egg size, results in increased hatchling survival underlimited food conditions.

Water relations and hatchling mass

Water loss from avian eggs during incubation andmetabolic water production by the embryos occur at rates thatcause the hydration of egg contents at the end of incubationto be similar to that at the beginning of incubation (Ar andRahn, 1980); these coincident rates also cause hatchlings andthe eggs from which they emerge to have similar watercontents (Sotherland and Rahn, 1987). Emu eggs contained onaverage 68% water, which comprised 74% of the hatchlingsthey produced (Fig.·2). Both of these values are in closeagreement with the water content of precocial eggs andhatchlings (Sotherland and Rahn, 1987). There was anisometric increase in the water content of both the egg and theyolk-free hatchling with an increase in initial egg mass andyolk-free hatchling mass (Fig.·2); a similar relationship wasobserved in the dry mass of eggs and yolk-free hatchlingsolids (Fig.·2). Japanese quail Coturnix coturnix hatchlingwater content scales isometrically with egg size, but theproportion of water in laughing gull Larus atricilla chicksincreases with a positive allometry such that larger hatchlingsare composed of more water (Ricklefs et al., 1978).

The quantity of water in avian eggs, found mainly in thealbumen, has a significant influence on the mass of developingembryos and hatchlings. Variation in emu hatchling mass isattributable in part to variation in mass of water in yolk-freehatchlings (Fig.·2). Studies examining the effects of water lossfrom eggs during incubation have shown that differences inwet embryo mass tend to be correlated with water content ofthe embryo and that eggs losing the most water tend to produceembryos with the lowest mass (Davis et al., 1988; Tullett andBurton, 1982). Removing albumen from chicken Gallus galluseggs caused a reduction in hatchling size (Hill, 1993; Finkleret al., 1998) and resulted in hatchlings with a reduced yolk-free wet body mass (Finkler et al., 1998). Though hatchlingsemerging from eggs from which albumen had been removedwere smaller (i.e. length of the tibiotarsus was shorter), muchof the decrease in wet body mass was attributed to the presenceof less water in the smaller hatchlings. The dry yolk-free bodymass of hatchlings from control eggs was not different fromthat of hatchlings emerging from eggs from which albumenhad been removed (Finkler et al., 1998). Thus, wateravailability in eggs may be one of the main determinates ofyolk-free hatchling mass in precocial species. A similarrelationship between water content and hatchling mass hasbeen observed in turtle eggs, where increased levels of waterin eggs result in increased hatchling and organ sizes (Packard,1999; Packard et al., 1987, 2000; Packard and Packard, 2001).

Finkler et al. (1998) postulated that some of the observedvariation in body mass, correlated with variation in water mass,

E. M. Dzialowski and P. R. Sotherland

605Consequences of emu egg size

might be accounted for by variation in extracellular liquidvolume, including blood volume. Blood volume of emuhatchlings increased isometrically with hatchling size (Fig.·6),but this increase in blood volume was not accompanied byvariation in other hematological parameters (Fig.·6). Thus, aportion of the water found in larger emu hatchlings appears ina larger volume of blood

Metabolic rate, eggshell conductance, and air cell PO∑

Maximum metabolic rates of bird embryos and theireggshell conductance are typically matched in such a way thatlevels of respiratory gases in the air cell vary over an amazinglynarrow range at the end of incubation, regardless of egg size,length of incubation, or degree of hatchling maturity (Rahn andPaganelli, 1990). Metabolic rates of developing emu embryosreported here, which reach a plateau about 8 days prior tohatching (Vleck et al., 1980), agree with those reportedpreviously by Beutel et al. (1983) and Vleck et al. (1980), andwere significantly correlated with initial egg mass and yolk-free hatchling mass. Larger eggs produced larger hatchlings,and, not surprisingly, near-term embryos from larger eggs hadgreater overall metabolic rates than those from smaller eggs.Because metabolic rate of emu embryos and water vaporconductance of the eggs in which they developed covaried withegg mass, PO∑ in air cells of emu eggs did not vary with eggmass and were in close agreement with values calculated byVleck et al. (1980). Using Fick’s law of diffusion and ourmeasurements of shell gas conductance and metabolic rate, wecalculated that air cell PO∑ should have averaged about14.3 kPa, which is less than 5% different from the valuesmeasured.

Consequences of egg size variation

Emu egg size influenced the morphological andphysiological phenotypes of the resulting hatchlings. Tosummarize the consequences of emu egg size variation weused the regressions from the results and Figs·1–4 to predicta number of parameters for a small emu egg (450·g) and a44% larger emu egg (650·g; Table·2). In support of ourhypotheses, hatchling phenotypic characters measured herewere 38–51% larger in hatchlings from the larger egg andscaled proportionally with egg size. Using the energy contentof dry solids in eggs (29·kJ·g–1; Sotherland and Rahn, 1987),we predict that a female emu would invest 3596·kJ in a450·g egg, whereas a 650·g egg would contain 48% moreenergy (5336·kJ). If mass-specific costs of producing eggswere the same for eggs of all sizes within a species, then afemale ovipositing a 650·g egg would allocate nearly 50%more energy per egg than a female ovipositing a 450·g egg.We hypothesized that increased maternal investment inthe form of increased yolk would result in largerhatchlings. Embryos in larger eggs received more parentalinvestment in embryogenesis, which allowed them toconsume more yolk solids and grow larger during incubationbut have sufficient yolk reserves to support them ashatchlings (Table·2).

Parameters that did not scale isometrically with egg masswere near term-embryo metabolic rate and air cell PO∑

(Table·2). Larger eggs had higher metabolic rates, butmetabolism did not increase to the same extent with increasesin egg mass as with hatchling body mass, suggesting thatembryos in larger eggs may have responded more to limitationsimposed by a relatively low eggshell conductance.

Our investigation revealed that female emus vary parentalinvestment in their offspring through changes in the absoluteamount of yolk and albumen in eggs, while keeping theproportion of the two constant. Embryos in larger eggsdeveloped into hatchlings that were heavier and structurallylarger than embryos in smaller eggs (i.e. greater parentalinvestment in embryogenesis yielded larger hatchlings), buthatchlings from eggs of all sizes contained the same amountof residual yolk (i.e. emus invest similar parental care, viaeggs, in their hatchlings). We do not know, however, ifembryos that ‘find’ themselves in larger eggs, containing moreresources and a larger gas exchange surface, respond bygrowing more or if embryos that would normally grow moreare put into larger eggs. Further research is needed to elucidatemore clearly how maternal phenotypes affect developmentaltrajectories and ultimately fitness.

All emu eggs used in this study were donated by the CrossTimbers Emu Ranch, Flower Mound, TX, USA. We thankWarren Burggren, Anne Dueweke, Mike Finkler and fouranonymous reviewers who provided valuable comments onearlier versions of the manuscript. This research was partiallyfunded by NSF operating grant IBN 98-96388 and TARPgrant 99466 to Warren Burggren, a Faculty DevelopmentGrant and a Joyce Research Fellowship from KalamazooCollege to Paul Sotherland and the Betz Chair Endowment,Drexel University to James Spotila.

Table·2. Predicted egg and hatchling components from 450·gand 650·g emu eggs

Egg mass (g) % increase

Parameter 450 650 44

Wet yolk in egg (g) 184 260 41Dry yolk in egg (g) 102 150 47Wet albumen in egg (g) 209 307 47Dry albumen in egg (g) 22 34 55Water in egg (g) 270 384 42Energy in egg (kJ) 3596 5336 48Water-vapor conductance 45 63 40

(mg·day–1·Torr–1)Air cell PO∑ (kPa) 14.4 15.3 6MR near-term embryo 83 105 26

(ml·O2·h–1)Wet hatchling (g) 298 426 43Yolk-free hatchling wet (g) 227 317 40Yolk-free hatchling solids (g) 64 88 38Water in yolk-free hatchling (g) 163 229 40Yolk solids consumed (g) 63 95 51

606

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