Am J Physiol Regul Integr Comp Physiol 310: R766 –R775, 2016.
First published January 27, 2016; doi:10.1152/ajpregu.00274.2015.
Ventilation changes associated with hatching and maturation of an
endothermic phenotype in the Pekin duck, Anas platyrhynchos domestica
Tushar S. Sirsat and Edward M. Dzialowski
Developmental Integrative Biology Research Group, Department of Biological Science, University of North Texas,
Denton, Texas
Submitted 17 June 2015; accepted in final form 22 January 2016
endothermy; ventilation; tidal volume; minute ventilation; hypercapnia
ENDOTHERMY IS A HIGH-ENERGY
phenotype that requires an elevated capacity to deliver oxygen from the atmosphere to the
tissues and ultimately, the mitochondria of the cells. Avian
aerobic metabolism involves an oxygen cascade of 1) convective oxygen delivery by the lungs from the environment to the
interface with the circulatory system, 2) convective transport
by the circulatory system from the lungs to the tissues, and 3)
diffusion from the tissue capillaries to the mitochondria. Endotherms typically have resting and maximal aerobic metabolic
rates 8 times higher than ectotherms (3, 8). This elevated
metabolic rate requires endotherms to have a greater capacity
to deliver oxygen to the lungs and transport it in the blood to
the tissues than ectotherms. A recent meta-analysis suggests
Address for reprint requests and other correspondence: E. M. Dzialowski;
Dept. of Biological Sciences, Univ. of North Texas, 1155 Union Circle, no.
305220, Denton, TX 76203 (e-mail: [email protected]).
R766
the cardiovascular system may place the greatest limits on
maximal oxygen transport associated with aerobic metabolism
of adult vertebrates (11). Whether this holds true for developing life stages is unknown. It may be more likely that during
ontogeny, multiple levels of the oxygen cascade develop together, and no one level is limiting.
Avian embryos begin life with an ectothermic metabolic
phenotype and are incapable of regulating body temperature by
internal heat production (30), but precocial species, like the
Pekin duck (Anas platyrhynchos domestica), develop an endothermic metabolic phenotype rapidly upon hatching. Whittow
and Tazawa (30) proposed a model for endothermic development in precocial birds that describes phases experienced
during the transformation from ectothermy to endothermy.
Initially, developing embryos are in a limiting stage where
embryo metabolism is limited by oxygen conductance across
the eggshell (30). During the paranatal hatching period, the
embryo begins to breathe with its lungs as it first internally pips
through the inner membrane of the shell and then externally
pips the eggshell with its beak. Whittow and Tazawa (30)
proposed that during the external pipping stage, precocial
species are no longer oxygen limited because they are beginning to breathe with their lungs, but instead are power-limited
by immature metabolic capacity of the tissues, thus limiting
endothermy. Endothermic capacity is reached once tissues and
organ systems express a metabolic phenotype with the ability
to increase aerobic respiration and generate enough heat to
maintain homeothermy.
At the end of incubation, oxygen limitation occurs across the
eggshell because of a limited number of eggshell pores and
reliance on the vascular chorioallantoic membrane (CAM) for
oxygen uptake. During the hatching paranatal period, a transition exists from reliance on gas exchange at the CAM to
exchanges at the lungs as the embryo first internally and then
externally pips (13, 19). As the lungs increase their contribution to oxygen delivery, limits placed on oxygen uptake by
eggshell conductance should decrease. One might expect that
the ability to increase oxygen delivery by changing breathing
patterns in externally pipped paranates might allow for hypercapnic and endothermic ventilatory responses when faced with
high CO2 or cooling ambient temperatures.
The ventilator chemosensitivity to hypoxia and hypercapnia
differs between externally pipped paranates and hatchlings (16,
27). In chicken hatchlings, a strong hypoxic and hypercapnic
response of breathing frequency (ƒ), tidal volume (VT), and
pulmonary ventilation (V̇E) exists. Chemosensitivity in V̇E
appears to be lacking in externally pipped chicken paranates:
there is little change in response to either 10% hypoxia or 4%
hypercapnia (27). Externally pipped paranates are apparently
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Sirsat TS, Dzialowski EM. Ventilation changes associated with
hatching and maturation of an endothermic phenotype in the Pekin
duck, Anas platyrhynchos domestica. Am J Physiol Regul Integr
Comp Physiol 310: R766 –R775, 2016. First published January 27,
2016; doi:10.1152/ajpregu.00274.2015.—Precocial birds begin embryonic life with an ectothermic metabolic phenotype and rapidly
develop an endothermic phenotype after hatching. Switching to a
high-energy, endothermic phenotype requires high-functioning respiratory and cardiovascular systems to deliver sufficient environmental
oxygen to the tissues. We measured tidal volume (VT), breathing
frequency (ƒ), minute ventilation (V̇E), and whole-animal oxygen
consumption (V̇O2) in response to gradual cooling from 37.5°C
(externally pipped paranates, EP) or 35°C (hatchlings) to 20°C along
with response to hypercapnia during developmental transition from an
ectothermic, EP paranate to endothermic hatchling. To examine potential eggshell constraints on EP ventilation, we repeated these
experiments in artificially hatched early and late EP paranates. Hatchlings and artificially hatched late EP paranates were able to increase
V̇O2 significantly in response to cooling. EP paranates had high ƒ that
decreased with cooling, coupled with an unchanging low VT and did
not respond to hypercapnia. Hatchlings had significantly lower ƒ and
higher VT and V̇E that increased with cooling and hypercapnia. In
response to artificial hatching, all ventilation values quickly reached
those of hatchlings and responded to hypercapnia. The timing of
artificial hatching influenced the temperature response, with only
artificially hatched late EP animals, exhibiting the hatchling ventilation response to cooling. We suggest one potential constraint on
ventilatory responses of EP paranates is the rigid eggshell, limiting air
sac expansion during inhalation and constraining VT. Upon natural or
artificial hatching, the VT limitation is removed and the animal is able
to increase VT, V̇E, and thus V̇O2, and exhibit an endothermic
phenotype.
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VENTILATION AND THE DEVELOPMENT OF ENDOTHERMY
Oxygen (%)
21.5
animal
inflow
animal
inflow
21.0
20.5
20.0
100
200
300
400
500
Arbitrary Time (s)
Fig. 1. Representative trace of inflow and outflow oxygen measurements. The
trace shows oxygen percentage of inflow oxygen and respiratory chamber
outflow from one animal, switching every 120 s and subsampling from the
multiplexer at rate of ⬃100 ml/min.
MATERIALS AND METHODS
Rates of oxygen consumption (V̇O2, ml O2/min) were calculated using
the following equations derived from Withers (31):
Animals
Pekin duck (Anas platyrhynchos) eggs were obtained from Blanco
Industries (McKinney, TX) and incubated at 37.5°C and 60% relative
humidity (RH) in an incubator (model 1202; G.Q.F. Manufacturing,
Savannah, GA). Eggs were automatically turned every 4 h. Just prior
to hatching, internally pipped eggs were moved into a clear Lyon
incubator maintained at 37.5°C and 85% RH. Ventilation patterns and
whole animal oxygen consumption (V̇O2) were measured in external
pipped paranates and hatchlings within the first 24 h of hatching. All
protocols were approved by the University of North Texas Institutional Animal Care and Use Committee.
Oxygen Consumption and Animal Temperature
Oxygen consumption of externally pipped paranates and hatchling
neonates was measured by flow-through respirometry. Animals were
placed inside a respirometry chamber with volumes of either 500 ml
(externally pipped) or 1,000 ml (hatchlings). Respirometry chambers
were placed inside a programmable incubation chamber set to 37.5°C
for externally pipped paranates and 35°C for hatchlings. Animals were
allowed to acclimate for at least 60 min prior to any changes in their
environment. Oxygen and nitrogen were mixed with a microprocessor
control unit (controller model 0154, Brooks Instruments, Hatfield,
PA) and flow controllers (model 5850E; Brooks Instruments) to
achieve an inflow gas mixture between 20.9 and 21.3% oxygen with
a balance of nitrogen. The gas flow rate to the chambers was measured
with a calibrated FlowBar1 mass flow meter (Sable Systems International, Las Vegas, NV). A sample stream of the outflow gas was
pulled by an R1 flow controller (AEI Technologies, Pittsburgh, PA)
through a nafion tube (ADInstruments, Colorado Springs, CO) surrounded by Drierite, a column of Sodasorb, and another nafion tube
drier (for water and CO2 removal) before it was pulled through a
FC-1B O2 analyzer (Sable Systems International) to measure oxygen
levels. In some experiments, CO2 in the inflow and outflow gas was
also measured using a CA-10 CO2 analyzer (Sable Systems International). In this case, the gas stream was dried by a single nafion tube
drier. Data were recorded with LabChart 7 and a PowerLab 16SP
(ADInstruments). Sampling of multiple respirometer chambers outflow gas was automatically controlled by a custom-built four-channel
solenoid multiplexer controlled by LabChart 7. At most, three animals
were measured at any given time, and each chamber was sequentially
sampled for 120 to 150 s during the course of an experimental run. An
example trace shows that 120 s is sufficient for the oxygen signal to
stabilize in our system (Fig. 1). An additional solenoid allowed for
sampling of inflow O2 and CO2 levels between sampling of the animal
chambers, allowing inflow gas to be sampled at least every 7.5 min.
V̇O2 ⫽ V̇I ⫻
共FIO2 ⫺ FEO2兲 ⫺ FEO2共FECO2 ⫺ FICO2兲
V̇O2 ⫽ V̇I ⫻
1 ⫺ F EO 2
共 F IO 2 ⫺ F EO 2兲
1 ⫺ F EO 2
(1)
(2)
where V̇I is incurrent flow rate (ml/min), FIO2 and FICO2 are incurrent
O2 and CO2 fractions of dry gas, and FEO2 and FECO2 are excurrent O2
and CO2 fraction in dry gas. When CO2 was not recorded, but
scrubbed with Sodasorb, Eq. 2 was used.
During measurements of V̇O2, animal temperature was continuously recorded. Body surface temperature was measured in externally
pipped paranates just under the shell. The eggshell was cleaned with
70% isopropyl alcohol, and a small hole was made with an 18-gauge
needle or dental drill. The egg was candled prior to inserting the
thermocouple to ensure that no major chorioallantoic blood vessels
were damaged. A 36-gauge copper constantan thermocouple was
placed just under the shell on the animal’s skin and secured in place
with dental wax (Kerr, Czech Republic). Shell temperature during
gradual cooling of three infertile eggs warmed to 40.4°C prior to
cooling were also measured to show the cooling response of an egg
without CAM blood flow or internal heat production. Body temperature of hatchlings was measured in the cloaca. Thermocouples were
placed in the cloaca and held in place with a small plastic disk glued
to the feathers of the animal, as in Ricklefs and Williams (20).
Temperatures were measured using temperature pods and recorded
with a PowerLab 16SP and LabChart 7 (ADInstruments). Wet thermal
conductance (C; ml O2·h⫺1·g⫺1·°C⫺1) was calculated for some of the
hatchlings in experiment 4 using the equation: V̇O2/[(Tb ⫺ Ta)·mass],
using whole hatchling mass, including the yolk sac.
Pulmonary Ventilation
Tidal volumes (VT) were estimated during the cooling trials and
CO2 exposure using a modification of the barometric technique (6,
27). Changes in pressure associated with breathing were measured
with a spirometer (ADInstruments) connected in-line with each metabolic chamber (14). Volume calibration was conducted on each
metabolic chamber after each trial by injecting known volumes of air
(Vcal) into the system using a Hamilton syringe (Hamilton, Reno,
NV). The corresponding change in pressure (Pcal) was used to calibrate the system (K ⫽ Vcal/Pcal as in Ref. 27), where K is a calibration
constant. Respirometer chamber relative humidity was measured on
excurrent air with a relative humidity sensor (HIH 4021, Honeywell,
Minneapolis, MN) and was used to estimate water vapor pressure in
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unable to alter ventilation to a great extent in response to CO2
and O2.
During hatching and development of endothermy, the response of the maturing respiratory system to cold thermal
stress in the paranatal, externally pipped stage and hatchling
stages is unknown. Could one constraint on attaining an endothermic metabolic phenotype in externally pipped paranatal
embryos be due to a limit placed by the eggshell on their ability
to increase V̇E and oxygen delivery to the lungs? In this study,
we examined the response of the developing respiratory system
of externally pipped paranates and day 0 hatchlings to cold
thermal stress and hypercapnia. We then looked at ventilation
patterns in animals as externally pipped paranates in the egg
and then after artificial hatching to address the possible constraints placed on ventilation by the eggshell during the externally pipped stage.
R768
VENTILATION AND THE DEVELOPMENT OF ENDOTHERMY
the chamber. All data were recorded with a PowerLab 16SP and
LabChart 7 (ADInstruments). Breathing frequency (ƒ, breaths/min)
was determined from the pressure waves using the cycle measurements function in LabChart 7. Tidal volume (␮l/min BTPS) was
estimated from K, chamber and body temperature, water vapor pressure, and the measured pressure changes as in Szdzuy and Mortola
(27). Minute ventilation (V̇E, ml/min BTPS) was calculated as VT·ƒ.
Experimental Design
Statistics
Ventilation data, V̇O2, and animal temperature were analyzed
within and between each age group using two-way repeated-measures
ANOVA with air temperature or CO2 level as the repeated variable,
followed by a Sidak post hoc test. Values from experiment 3 were log
transformed prior to analysis due to unequal variability between
treatments. To examine differences between externally pipped and
day 0 hatchlings, we used a t-test comparing resting thermoneutral
values at 37.5°C for externally pipped paranates with those at 35°C
for day 0 hatchlings. Mean minimum wet thermal conductance was
determined at air temperatures below 30°C and examined with a t-test.
Statistical analysis was carried out with Prism 6 (GraphPad Software,
La Jolla, CA) and SigmaPlot 12.0 (Systat Software, San Jose, CA).
For all statistical tests, level of significance was set at P ⬍ 0.05. All
data are presented as means ⫾ SD.
RESULTS
Body mass of developing animals did not differ between
external pipping and after hatching (Table 1). There was no
difference between externally pipped paranatal and hatchling
whole body mass (P ⫽ 0.064), residual yolk sac mass (P ⫽
0.21), or yolk-free body mass (P ⫽ 0.05).
Externally Pipped Paranate and Hatchling Responses to
Cooling
An endothermic phenotype developed rapidly upon hatching
(Fig. 2). Hatchlings had a significantly higher V̇O2 in their
thermoneutral zone than did externally pipped paranates at the
same air temperatures (Table 1). During external pipping, V̇O2
remained constant during cooling from 37° to 20°C (Fig. 1A;
P ⫽ 0.97). Upon hatching, the hatchlings were able to increase
V̇O2 in response to gradual cooling. Oxygen consumption
Table 1. Resting values in externally pipped paranates and day 0 posthatching Pekin duck from experiment 1
Age
Tb,°C
Whole body mass, g
Residual yolk mass, g
Yolk-free body mass, g
f, breaths/min
VT, ␮l
VT, ml/kg
V̇E, ml/min
V̇E, ml·kg⫺1·min⫺1
V̇O2, ml O2/min
V̇E/V̇O2
Pekin Duck
Pekin Duck
P
EP (n ⫽ 7)
40.2 ⫾ 1.3
65.2 ⫾ 5.8
11.2 ⫾ 5.8
54.0 ⫾ 1.6
100.4 ⫾ 13.2
141.5 ⫾ 48.2
2.2 ⫾ 2.4
14.4 ⫾ 6.1
223.4 ⫾ 107.2
0.89 ⫾ 0.16
Hatch (n ⫽ 5)
39.6 ⫾ 0.4
59.0 ⫾ 4.0
7.4 ⫾ 2.5
51.6 ⫾ 2.2
33.2 ⫾ 3.8
758.0 ⫾ 160.8
12.9 ⫾ 3.6
25.1 ⫾ 5.6
425.2 ⫾ 121.0
1.22 ⫾ 0.1
21.4 ⫾ 4.6
0.366
0.064
0.211
0.050
⬍0.001
⬍0.001
⬍0.001
0.005
0.040
0.004
Muscovy Duck,
Hatchlinga
Chicken,
EPb
Chicken,
Hatchlinga
Wedge-Tailed
Shearwaterc
Wedge-Tailed
Shearwaterc
Hatch
40.1
50.3
EP
Hatch
40.0
40.8
EP
Hatch
33
46.6
150
4.5*
7.09
212*
0.47
35
47.4
310
8.8*
14.5
411*
0.61
23.8
46
565
11.3
25.1
499
1.26
20.8
37.3
72.7
76.7
2.1
6.0
160
0.4
63
330
8.1
20.2
496
0.79
25.3
Values are presented as means ⫾ SD. EP, externally pipped; Tb, body temperature. aData for Muscovy duck and chicken hatchlings from Mortola and
Toro-Velasquez (16). bEP chickens from Szdzuy and Mortola (27). cWedge-tailed Shearwater from Pettit and Whittow (18) and Zhang and Whittow (33). For
correct comparisons with published values, mass specific values are based on whole body mass including yolk. *Estimated using externally pipped and hatchling
body masses from Zhang and Whittow (33).
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Experiment 1. Ventilation and V̇O2 patterns were measured in
externally pipped paranates (n ⫽ 7), and day 0 hatchlings (n ⫽ 5;
between 0 and 24 h old) during gradual cooling. After the resting
period, temperature of the incubation chamber was gradually decreased at a rate of 9.2°C/h until an air temperature of 20°C was
reached. During these runs, flow through the chambers was high
enough to maintain chamber CO2 below 1.5%. A second set of early
externally pipped paranates was exposed to 40% O2 during cooling
(n ⫽ 8).
Experiment 2. To ensure the hatchling ventilation changes that we
observed in experiment 1 were not in response to elevated CO2,
ventilation patterns were estimated in response to 0, 1.5, and 4% CO2
in another set of day 0 hatchlings (n ⫽ 4). Animals were maintained
at 35°C for the entire CO2 exposure experiment. After the resting
period, gas flowing into the chamber was changed to 21% O2 and
1.5% CO2 balanced with nitrogen for 15 to 20 min. Inflow CO2 level
was then increased to 4% CO2 for an additional 15 to 20 min. Inflow
gas was mixed with the Brooks flow controllers, as previously
mentioned.
Experiment 3. To determine whether limits to ventilation chemosensitivity are influenced by the eggshell, we measured hypercapnic
ventilation responses in externally pipped paranates in the eggshell
and then after artificially removing them from the egg (artificially
hatched). Animals were maintained at 37.5°C for this experiment.
Ventilation patterns were measured in externally pipped paranates for
30 min. After recording the baseline ventilation response, the incurrent CO2 level was increased to 4%, and ventilation was recorded for
an additional 20 min. The externally pipped paranates were then
quickly removed from the metabolic chamber, helped out of the
eggshell, and dried with a hair drier (Conair, Stamford, CT). In
some cases, the CAM artery and vein were tied off at the belly of
the animal. The artificially hatched animals were placed back into
the metabolic chamber, and baseline ventilation was measured for
45– 60 min. This was followed by an increase in the incurrent CO2
level to 4% for 20 min. Shell and cloacal temperatures were measured
as noted above.
Experiment 4. In the final set of animals, we examined the cooling
response after helping animals that were at various stages of external
pipping out of the eggshell. Externally pipped paranates were removed from the eggshell and dried as in experiment 3, and then their
response to cooling was measured as in experiment 1. We separated
the externally pipped paranates into two age classes: animals that had
a small eggshell star fracture and those that had made larger holes in
the eggshell. We denote these two groups as early EP hatchlings and
late EP hatchlings.
R769
VENTILATION AND THE DEVELOPMENT OF ENDOTHERMY
B
3
Day 0
EP
2
1
40
35
30
Ta
Infertile Egg
25
20
0
20
C
30
20
40
Ta (°C)
D
40
Tidal Volume (µl)
120
100
80
60
40
20
0
1500
1000
500
0
20
30
40
20
Ta (°C)
30
Ta (°C)
E
Minute Ventilation
(ml min-1)
30
Ta (°C)
100
40
Fig. 2. Pekin duck whole animal oxygen
consumption (A), cloacal/eggshell temperature (B), ventilation rate (breaths/min) (C),
tidal volume (␮l/breath) (D), and minute ventilation (ml/min) (E) in response to cooling
during external pipping () and the first day
of post-hatch life (). Animals were cooled
at a rate of 9.2°C/h. Hatchling temperature
was measured in the cloaca, and externally
pipped embryo body surface temperature was
measured just under the shell. Open symbols
represent significant differences from the
value at the highest air temperature for the
age at P ⬍ 0.05. The dashed line in B
represents the line of thermal equality. Shell
temperature of cooling infertile eggs are included in B (gray line; n ⫽ 4). Horizontal
lines above a temperature indicate a significant difference between the externally pipped
and hatchling values at that temperature.
Data are presented as means ⫾ SD; n ⫽ 7
externally pipped paranates and five hatchlings, except for V̇O2 where n ⫽ 4 hatchlings.
75
50
25
0
20
30
40
Ta (°C)
began to increase significantly at 27°C and continued to increase in a linear fashion during the rest of the cooling
exposure (P ⬍ 0.001).
Surface body temperature of externally pipped paranates
was not significantly different from hatchling cloacal temperature at the highest air temperatures (Table 1). Upon cooling,
both externally pipped paranates and hatchlings showed significant decreases in surface and cloacal temperatures, respectively (P ⬍ 0.001). Surface body temperature of externally
pipped paranates began to decrease significantly around an air
temperature of 30°C (Fig. 2B). Hatchling cloacal temperature
did not drop significantly until 27°C. At the lowest air temperature, hatchlings had a significantly higher body temperature
(⬃36.7°C) than externally pipped paranates (⬃32.1°C).
Externally pipped paranates and hatchlings exhibited significantly different ventilation patterns (Table 1; Fig. 1, C–E).
Ventilation rate was significantly higher in externally pipped
paranates compared with hatchlings (Table 1; P ⬍ 0.001).
Upon cooling, externally pipped paranates’ ƒ decreased significantly (Fig. 2C; P ⬍ 0.001). In contrast, hatchling ƒ was
initially constant during the first portion of cooling in the
thermal neutral zone and then increased significantly at temperatures below 27°C (P ⬍ 0.001).
The estimated VT was significantly higher in hatchlings than
externally pipped paranates, while both were within the air
temperature of the hatchlings thermoneutral zone (Table 1;
P ⬍ 0.001). There was a significant effect of temperature on
estimated VT in the externally pipped paranate with VT being
higher at air temperatures below 24°C compared with VT at
37.5°C (Fig. 2D; P ⫽ 0.007). Hatchlings exhibited a significant
increase in VT only at the lowest air temperature (20°C; P ⫽
0.015).
The V̇E differed significantly between the two ages (Table 1;
Fig. 2E): V̇E was significantly higher in hatchlings compared
with externally pipped embryos. During cooling, V̇E did not
change in externally pipped paranates (Fig. 2E; P ⫽ 0.56).
Hatchling V̇E remained constant during the first portion of
cooling and then increased significantly in a linear fashion
(Fig. 2E; P ⬍ 0.001). Below an air temperature of 28°C,
hatchling V̇E was significantly higher than that of externally
pipped paranates.
Hatchling Response to Hypercapnia
Hatchlings significantly increased ƒ (P ⫽ 0.0007), VT (P ⫽
0.0038), and V̇E (P ⫽ 0.0002) in response to hypercapnic
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Ventilation Frequency
(breaths min-1)
Temperature (°C)
Oxygen Consumption
(ml O2 min-1)
A
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VENTILATION AND THE DEVELOPMENT OF ENDOTHERMY
conditions (4% CO2; Fig. 3). In response to increasing CO2
from 0% to 1.5%, ƒ, VT, and V̇E did not change. Increasing
CO2 from 1.5% to 4% produced a significant increase in ƒ, VT,
and V̇E.
Ventilatory Response to Hypercapnia after Artificial
Hatching
Artificial hatching during the externally pipped stage resulted in significant changes in ventilation without changes in
V̇O2 (Fig. 4). The V̇O2 did not differ between the externally
pipped paranate in the eggshell, and once it was artificially
removed from the shell (Fig. 4A; P ⫽ 0.53). During external
Ventilation Frequency
(breaths min-1)
80
60
40
20
0
0
1
B
2
3
4
5
4
5
CO2 (%)
Tidal Volume (µl)
1500
1000
500
0
0
1
2
3
CO2 (%)
C
The endothermic V̇O2 response to cooling depended upon
the length of time an animal had been in the externally pipped
stage (Fig. 5). Externally pipped paranates with initial eggshell
star fractures exhibited an ectothermic V̇O2 response to cooling, while those that had been in the externally pipped stage for
longer had an intermediate V̇O2 response to cooling. With
increased length of time in the externally pipped stage, there
was an increase in V̇O2 with cooling that was not maintained at
the coolest air temperatures. Early externally pipped paranates
were unable to increase V̇O2 during cooling when exposed to
40% O2 (Fig. 6).
An endothermic V̇O2 response in artificially hatched animals
depended upon the length of time the animal had been in the
externally pipped stage (P ⬍ 0.001 for V̇O2, ƒ, and V̇E; P ⫽
0.01 for VT, Fig. 7). Animals that were removed from the shell
with star fractures maintained V̇O2, ƒ, or V̇E and significantly
increased VT, in response to gradual cooling (Fig. 7). If
allowed to stay in the eggshell for longer before artificial
hatching, V̇O2, ƒ, VT, and V̇E all increased significantly with
cooling after the artificial hatching (Fig. 7). At air temperatures
below 30°C, cloacal temperatures were significantly higher in
the late externally pipped hatchlings compared with the
early externally pipped hatchlings (P ⬍ 0.001; Fig. 7B).
Mean minimum wet thermal conductance was lower in the
early externally pipped hatchlings (0.10 SD 0.02 ml
O2·h⫺1·g⫺1·°C⫺1) compared with the late externally pipped
hatchlings (0.15 SD 0.02 ml O2·h⫺1·g⫺1°·C⫺1) across the
lowest air temperatures (P ⬍ 0.001).
DISCUSSION
100
Minute Ventilation
(ml min-1)
Ventilatory and Metabolic Response to Cooling after
Artificial Hatching
Development of an Endothermic Metabolic Response
75
50
25
0
0
1
2
3
4
5
CO2 (%)
Fig. 3. Breathing pattern of day old hatchlings in response to exposure to
increased CO2. Ventilation rate (breaths/min) (A), tidal volume (␮l/breath) (B),
and minute ventilation (ml/min) (C) are shown. Open symbols represent
significant difference from the value at 0% CO2 at P ⬍ 0.05; n ⫽ 4. Data are
presented as means ⫾ SD.
Upon hatching, Pekin duck’s metabolic response to cooling
transitioned rapidly from an ectothermic phenotype to an
endothermic phenotype (Fig. 2A). The mean response of externally pipped paranates in the eggshell was to not change V̇O2
and heat production during to gradual cooling. This is a typical
response for a precocial embryo at this stage. Whittow and
Tazawa (30) proposed that at the end of incubation, the
embryo’s ability to increase V̇O2 becomes limited by the
oxygen conductance of the eggshell. During the paranatal
hatching period, the embryo begins to ventilate the lungs first
during internal pipping and then after the eggshell is broken at
external pipping. As the lungs become a more prominent site of
gas exchange (13, 19), Whittow and Tazawa (30) predicted that
embryos become power-limited as the limits of eggshell conductance are countered by increasing lung ventilation. Pekin
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A
pipping in the eggshell, there were no changes in ƒ, VT, and V̇E
in response to hypercapnia (Fig. 4, B–D). Upon artificial
hatching, there was a significant increase in normocapnic VT
and V̇E and significant decrease in ƒ compared with the same
animal as an externally pipped paranate in the eggshell. In
response to 4% CO2, the artificially hatched animals significantly increased V̇E. This was accomplished mainly by significantly increasing ƒ (Fig. 4B). The response in VT was variable,
increasing in some and remaining constant or slightly decreasing in others (Fig. 4C).
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VENTILATION AND THE DEVELOPMENT OF ENDOTHERMY
3
A
Oxygen Consumption
(ml O2 min-1)
Fully Cracked Shell
Oxygen Consumption
(ml O2 min-1)
1.5
1.0
0.5
0.0
Hatched
20
30
40
a
a
Fig. 5. Examples of individual oxygen consumption (ml/min) from externally
pipped paranates at various stages of hatching in response to gradual cooling.
100
75
50
c
25
b
0
0% CO2
4% CO2
0% CO2
EP
4% CO2
Hatched
C
1000
b
800
b
600
400
a
a
200
0
0% CO2
4% CO2
0% CO2
EP
D
4% CO2
Hatched
75
d
50
c
25
duck embryos exhibited a varying ability to increase V̇O2
during external pipping depending on the extent of pipping
(Fig. 5). Newly externally pipped paranates were unable to
increase V̇O2, while older externally pipped paranates were
able to increase V̇O2 to a limited extent during gradual cooling.
Internally and externally pipped chicken embryos were unable
to increase V̇O2 in response to cooling at 30°C for 1 h (26).
Even when the blunt end of the eggshell covering the air cell
was removed, chicken embryos were unable to increase V̇O2 to
a great extent (26).
Once the paranate has hatched, resting V̇O2 increased, and
the ability to increase V̇O2 upon cooling appeared rapidly. A
strong endothermic response upon hatching, as in the duck, has
been observed in other water birds (9, 24), while other species,
such as the chicken, may not show as strong an endothermic
response until a day or two after hatching (1, 26, 32). The rapid
appearance of an endothermic V̇O2 response to cooling upon
hatching led us to examine changes in ventilation patterns
associated with obtaining an endothermic phenotype.
Body temperature of all ages (externally pipped and hatchlings) decreased with the cooling exposure. As expected, surface eggshell temperature of externally pipped paranates experienced a greater drop than cloacal temperature of the hatchling
(Fig. 2B). The externally pipped eggshell cooled in a similar
fashion to the eggshell of infertile eggs. Tazawa et al. (29)
found that chicken eggs tended to cool at similar rates with and
without a developing embryo present. In hatchlings, while V̇O2
and thus, heat production, increased, cloacal temperature
dropped during the second half of the cooling bout. The
capacity to maintain body temperature increased as the externally pipped paranatal stage progressed. Hatchlings that were
helped out of the eggshell early in the externally pipped stage
had a greater drop in body temperature than those artificially
b
a
0
0% CO2
4% CO2
EP
0% CO2
4% CO2
Hatched
State
Fig. 4. Breathing pattern response to exposure to 0% and 4% CO2 at 37.5°C in
animals as externally pipped paranates and then after artificial hatching by
removing the animal from the eggshell. Whole animal oxygen consumption
(ml/min) (A), ventilation rate (breaths/min) (B), tidal volume (␮l/breath) (C),
and minute ventilation (ml/min) (D). Values with different letters are significantly different from each other at P ⬍ 0.05. Data points express means ⫾ SD;
n ⫽ 7. Gray lines indicate individual responses.
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Ventilation Frequency
(breaths min-1)
1
Ta (°C)
B
125
Tidal Volume (µl)
Star Fracture
2
0
EP
Minute Ventilation
(ml min-1)
3/4 Cracked Shell
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VENTILATION AND THE DEVELOPMENT OF ENDOTHERMY
Oxygen Consumption
(ml O2 min-1)
3
40% O2
2
1
0
20
30
40
Fig. 6. Oxygen consumption of early externally pipped paranates during
cooling in a 40% oxygen environment. Open symbols represent significant
differences from the value at the highest air temperature at P ⬍ 0.05. Data
presented as means ⫾ SD; n ⫽ 8.
hatched later in external pipping (Fig. 7B) but had a lower wet
thermal conductance (Fig. 7C). Lower wet thermal conductance during the first hours after hatching compared with older
hatchlings have been observed in both Eider duck (25) and
Breathing Frequency, Tidal Volume, and Minute Ventilation
Resting ventilation patterns undergo significant changes during hatching. Externally pipped paranates had a higher resting
B
2
1
0
20
30
Temperature (°C)
Late EP Hatchling
Early EP Hatchling
40
40
30
Ta
20
20
Ta (°C)
Tidal Volume (µl)
Ventilation Frequency
(breaths min-1)
1500
100
80
60
40
20
20
30
40
1000
500
0
20
Ta (°C)
100
75
50
25
20
30
30
Ta (°C)
E
0
40
D
120
0
30
Ta (°C)
C
Minute Ventilation
(ml min-1)
Fig. 7. Oxygen consumption, body temperature, and breathing pattern responses to cooling of animals after artificial hatching by removing the animal from the eggshell during
the externally pipped stage (EP). Externally
pipped paranates were helped out of the eggshell with either a small star fracture (early EP,
) or larger hole in the eggshell (late EP, ).
Whole animal oxygen consumption (ml/min)
(A), ventilation rate (breaths/min) (B), tidal
volume (␮l/breath) (C), and minute ventilation
(ml/min) (D). Open symbols represent significant difference from the value at 37.5°C
within an age group at P ⬍ 0.05. Horizontal
lines above a temperature denote significant
differences between the late EP and early EP
response at that temperature. Data points express means ⫾ SD; n ⫽ 5 early EP and 7 late
EP. Gray lines indicate individual responses.
Oxygen Consumption
(ml O2 min-1)
A
3
40
Ta (°C)
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40
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Ta (°C)
chicken (15) hatchlings. The difference observed in Eider duck
minimum wet thermal conductance was suggested to be due to
changes in temperature regulation of the periphery (25). From
external pipping through hatching, the capacity to maintain
body temperature may be limited by maturation of the insulation and the extent of aerobic and thermogenic capacity of the
tissues (i.e., power-limited).
In externally pipped paranates, we measured body surface
temperature just under the surface of the eggshell. Because
body surface temperature is closer to the surface of the egg, a
more rapid drop in externally pipped egg surface temperature
is expected than that of the hatchling core temperature. Heat
loss across the eggshell might be greater because of the lack of
insulation. Core body temperature of externally pipped
paranates most likely did not drop as much as the measured
subsurface egg temperature and is probably closer to that of
hatchlings. We did not measure core temperature of the externally pipped embryo in the eggshell and were not able to
estimate minimum wet thermal conductance at this stage.
VENTILATION AND THE DEVELOPMENT OF ENDOTHERMY
pipped levels in the eggshell (Fig. 4C). While we did not
measure lung volume, chicken lung volumes were not significantly different between externally pipped paranates and
day-old hatchlings (22).
Pulmonary Ventilation and Endothermy
In response to cooling, externally pipped paranates were on
average unable to increase V̇O2 and ventilation. There was a
significant decrease in ƒ during cooling that was compensated
by a small, but significant, increase in VT at the lowest
temperature. This allowed the animal to maintain V̇E at resting
levels, but not increase it. A similar change was observed in
externally pipped Japanese quail ƒ that decreased from 82 to 41
breaths/min when cooled from 38°C to 28°C for 40 min (17).
During external pipping in the egg, ƒ may be near the maximal
frequency and unable to increase further.
Thermoregulatory heat production in externally pipped
paranates may be limited at this stage by their inability to
increase V̇E when exposed to cold. As noted above, externally
pipped embryo resting VT is only 20% of the hatchling value
at rest. If VT of externally pipped embryos is limited by the
animal’s ability to expand the air sacs, then they should not be
able to exhibit an endothermic metabolic response to cooling.
Upon hatching, the constraint of the eggshell on VT is removed
and the hatchling responds to cooling by increasing V̇E. During
initial cooling in the thermal neutral zone, there is no increase
in ventilation in the duck. At around 28°C, the hatchling
increases V̇O2 and V̇E in a typical endothermic response.
During gradual cooling to 20°C, ƒ, VT, and V̇E continue to
increase, providing oxygen to the lungs to meet the increasing
thermoregulatory V̇O2 demand. This increase in V̇E occurs by
increasing both ƒ and VT during cooling. Hatchling ƒ at the
coldest temperature measured (20°C) had increased to that of
the cold externally pipped embryo. This was lower than the
maximal ƒ measured in the resting externally pipped embryo.
The low VT in externally pipped paranates may explain the
limited capacity of thermogenesis during this stage, but there
may also be limits on the metabolic machinery to generate heat
during the externally pipped stage. To test this, we artificially
hatched externally pipped paranates, either early or late in the
external pipping stage. During external pipping, the length of
time in this stage influenced an animal’s ability to increase V̇E
in response to cooling. Animals artificially hatched early in the
externally pipped stage were unable to increase ƒ, VT, V̇E, and
V̇O2 in response to cooling; instead, they only maintained
resting levels. When artificial hatching occurred later in the
externally pipped stage, the hatchlings were able to respond to
cooling with increases in ƒ, VT, V̇E, and V̇O2. Therefore, during
the externally pipped stage, there is maturation of the ventilatory response possibly due to maturation of the thermosensors
in the animal. Additionally, early externally pipped embryos
with only small star fractures exposed to 40% O2 were unable
to increase V̇O2 during cooling (Fig. 6). This is unlike the emu,
in which V̇O2 increased during cooling in a hyperoxic environment as early as the internally pipped stage (7). Our data
suggest that along with maturation of the ventilatory response,
there is a maturation of the cardiovascular system and metabolic capacity of the shivering muscles required before the
animal exhibits an endothermic phenotype.
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ƒ when compared with hatchlings (Fig. 2C), roughly three
times higher than hatchlings. Similar high ƒ of internally
pipped Pekin duck embryos, ranging from 86 to 239, have been
previously observed (23). The difference in ƒ between Pekin
duck externally pipped paranates and hatchlings was greater
than in other species. The difference in resting ƒ between
externally pipped paranates and hatchlings in chickens was
only a 1.2-fold (Table 1; Refs. 16 and 27). In wedge-tailed
shearwater, ƒ of externally pipped embryos and hatchlings was
not different (Table 1; Ref. 33). However, the Pekin duck
differs from other species in that externally pipped ƒ is high,
and upon hatching, ƒ decreases significantly.
Pekin duck hatchlings have a lower ƒ when compared with
other precocial hatchlings (Table 1). Pekin duck hatchling ƒ is
31, 27, and 47% lower than wedge-tailed shearwater, Muscovy
duck, and chicken hatchlings (16, 33). At rest, hatchlings
breathe at a rate of 33 breaths/min, which is roughly three
times the breathing frequency of adult Pekin ducks (2, 5, 12).
A similar difference between hatchling and adult ƒ is observed
in chicken and Muscovy ducks (10, 12, 16).
Resting estimated VT differed significantly between externally pipped paranates and hatchlings. Externally pipped
paranatal VT was less than 20% that of day-old hatchlings.
Similar differences in VT have been observed in externally
pipped paranates and hatchling chickens. Comparisons of similarly sized externally pipped paranates and hatchlings reveal a
large difference in VT. As with the duck, VT in externally
pipped chicken paranates is about 20% that of similar sized
hatchlings (16, 27). Similar VT differences between externally
pipped paranates and hatchlings are seen in developing chicken
and wedge-tailed shearwater (Table 1). The Pekin duck hatchling has a larger absolute VT compared with Muscovy ducks
and larger VT normalized to body mass than the Muscovy
duck, chicken, and wedge-tailed shearwater (Table 1).
At each stage of development and environmental condition,
an animal needs to ensure that minute ventilation provides
adequate oxygen supply at the lungs to support resting or active
V̇O2. Externally pipped paranate resting V̇E was only 57% that
of a hatchling, while V̇O2 was 73%. At this stage, the externally
pipped paranate is also relying on the CAM for a portion of
oxygen exchange. When compared with hatchlings, V̇E was
lower in externally pipped paranates due to the significantly
lower VT, even though ƒ was three times greater in the
externally pipped paranate. Similar differences in V̇E have
been observed between externally pipped and hatchling chickens (Table 1; Refs. 16 and 27) and wedge-tailed shearwater
(18). As with Pekin duck, differences in VT between the two
stages were the main factor driving the different V̇E in these
other species.
Differences in resting ƒ, VT, and V̇E between externally
pipped paranates and hatchlings appear to be due to limits
placed on VT by the eggshell. This is supported by the observation that artificially hatching externally pipped paranates
resulted in a rapid change in ƒ, VT, and V̇E to hatchling levels,
regardless of the age of the externally pipped paranate. Ventilation in birds involves filling of multiple air sacs within the
animal that require an expansion and increase in the volume of
the animal (21). The eggshell may hinder full air sac filling,
resulting in low VT that was compensated in externally pipped
paranates by an elevated ƒ. Tidal volume increased six-fold
upon artificial hatching compared with their externally
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VENTILATION AND THE DEVELOPMENT OF ENDOTHERMY
Development of Ventilatory Chemosensitivity
To determine whether lack of a ventilatory response during
external pipping is due to an insensitivity of ventilation, we
examined hatchling ventilatory chemosensitivity in response to
hypercapnia. In response to 1.5% CO2, there was a small,
insignificant increase in hatchling ƒ, VT, and V̇E. This change
in ventilation at 1.5% CO2 was less than the changes in
hatchling Muscovy ducks or chickens in response to 2% CO2
(16). A level of 1.5% CO2 was chosen here because that was
the maximum CO2 level measured in the outflow from our
respirometry chambers at the lowest temperatures. The
changes in ƒ, VT, and V̇E at 1.5% CO2 were all well below
changes observed in the thermoregulating hatchling experiencing the coldest test temperatures (Figs. 2 and 3). Thus, we can
rule out ventilatory changes caused by increased metabolic
chamber CO2 levels and instead suggest any change is a
thermoregulatory response to cooling.
Pekin duck hatchling ventilatory changes in response to 4%
CO2 were of similar magnitude to changes occurring during
cooling. Under both conditions, the significant increase in V̇E
was due to both increased ƒ and VT. The increase in V̇E from
0 to 4% CO2 was larger than those observed in Muscovy
ducks, but similar in magnitude to changes in the chicken
hatchling (16). Pekin duck hatchlings had a similar ventilation
rate at 4% CO2 as the Muscovy duck, but a larger change in
VT, thus producing a greater change in V̇E. The V̇E hypercapnic
response of the chicken hatchling had a larger contribution of
VT than in the Pekin duck (16, 28). This large contribution of
VT in the chicken compared with the Pekin duck may be due
to the higher resting ƒ in the chicken when compared with the
duck. Hatchling ventilation responses to hypercapnia are similar to those observed in the adult Pekin duck (5).
In contrast to hatchlings, externally pipped Pekin duck
paranates were unable to alter ventilation in response to hypercapnia of 4% CO2, but after undergoing artificial hatching,
they showed a functioning hypercapnic chemosensitivity (Fig.
4). The ƒ and VT of externally pipped paranates did not change
significantly when exposed to hypercapnia. However, they may
possess a chemosensitivity that can be observed in response to
4% CO2: externally pipped paranates with lower ƒ at 0% CO2
increased ƒ when exposed to 4% CO2, resulting in similar
values of 4% CO2 ƒ for all animals, which served to decrease
variability compared with values at 0% CO2 (Fig. 4B). Exter-
nally pipped chicken paranates VT increased only modestly in
response to hypercapnia (8% CO2) and hypoxia (5% O2),
increasing from 82 ␮l under control conditions to 147 ␮l (14)
without increasing ƒ. After artificial hatching of externally
pipped paranates, the animals exhibited a clear chemosensitivity to elevated CO2. This suggests that the externally pipped
paranatal hypercapnic VT response may also be limited by the
high ƒ and physical inability to expand the air sacs.
Perspectives and Significance
An endothermic phenotype develops rapidly in the hatchling
Pekin duck. Upon hatching, animals respond to cooling with a
significant increase in V̇O2 and heat production. The Pekin
duck hatchling has both a ventilatory chemosensitivity and a
ventilatory thermal sensitivity. In contrast, externally pipped
paranates have high ƒ coupled with low VT. Resting ventilation
rate may be at a maximum during the externally pipped stage
to compensate for the low VT. This low VT and inability of
externally pipped paranates to increase VT and V̇E in response
to either temperature or hypercapnia appears to be a constraint
of the eggshell, as they both increase if the animal is helped
from the eggshell. During the externally pipped stage,
paranates appear limited by O2 uptake across the CAM and at
the lung, but they may also be power-limited as suggested by
Whittow and Tazawa (30). At the end of in ovo development,
one might predict that the systems develop together so that
externally pipped paranates are limited by the respiratory and
cardiovascular systems and capacity for aerobic metabolism in
the muscles. Natural ontogeny of thermogenesis during external pipping and hatching include significant improvements in
ventilation immediately after removal of eggshell, enabling
paranates to establish endothermy.
ACKNOWLEDGMENTS
Paul Sotherland and Sarah Goy Sirsat provided valuable comments on
initial drafts of the manuscript. We also thank three anonymous referees whose
comments and suggestions greatly improved our article.
GRANTS
This study was funded by Grant IOS 1146758 from the National Science
Foundation to E. M. Dzialowski.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: T.S.S. and E.M.D. conception and design of research;
T.S.S. and E.M.D. performed experiments; T.S.S. and E.M.D. analyzed data;
T.S.S. and E.M.D. interpreted results of experiments; T.S.S. and E.M.D. edited
and revised manuscript; T.S.S. and E.M.D. approved final version of manuscript; E.M.D. prepared figures; E.M.D. drafted manuscript.
REFERENCES
1. Azzam MA, Szdzuy K, Mortola JP. Hypoxic incubation blunts the
development of thermogenesis in chicken embryos and hatchlings. Am J
Physiol Regul Integr Comp Physiol 292: R2373–R2379, 2007.
2. Bech C, Johansen K, Brent R, Nicol S. Ventilatory and circulatory
changes during cold exposure in the Pekin duck Anas platyrhynchos.
Respir Physiol 57: 103–112, 1984.
3. Bennett AF, Ruben JA. Endotherm and activity in vertebrates. Science
206: 649 –654, 1979.
4. Bucher TL, Bartholomew GA. The early ontogeny of ventilation and
homeothermy in an altricial bird, Agapornis roseicollis (Psittaciformes).
Respir Physiol 65: 197–212, 1986.
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00274.2015 • www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.6 on June 17, 2017
The ventilatory response to cooling in the hatchling Pekin
ducks was different from that found in adult Pekin ducks (2).
Bech et al. (2) found that when measured at 20°C, 0°C, and
⫺20°C, the adult Pekin ducks increase V̇O2 by increasing V̇E,
mainly through increasing VT. In contrast, we found that
during cooling, the hatchlings increased VT and, to a greater
extent, ƒ.
Hatchling ventilation patterns in response to cooling in
altricial species differ from the precocial Pekin duck. The
altricial rosy-faced lovebird does not show an endothermic
ventilation response until 7 days posthatch, which increases
through day 11 (4). On day 11, the nestling is able to increase
V̇O2 during 5°C cooling bouts. Increase in V̇O2 is associated
with an increase in V̇E due solely to an increase in ƒ. This
example contrasts with the duck hatchling that increased both
ƒ and VT.
VENTILATION AND THE DEVELOPMENT OF ENDOTHERMY
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
edited by Johansen K and Burggren WW. Copenhagen, Denmark: Munksgaard, 1985, p. 199 –211.
Ricklefs RE, Williams JB. Metabolic responses of shorebird chicks to
cold stress: hysteresis of cooling and warming phases. J Exp Biol 206:
2883–1893, 2003.
Scheid P, Slama H, Willmer H. Volume and ventilation of air sacs in
ducks studied by inert gas wash-out. Respir Physiol 21: 19 –36, 1974.
Seymour RS. Patterns of lung aeration in the perinatal period of domestic
fowl and Brush Turkey. In: Respiration and Metabolism of Embryonic
Vertebrates, edited by Seymour R. Dordrecht: The Netherlands: Dr. W.
Junk Publishers, 1984, p. 319 –332.
Spear HG, Dawes CM. The effects of cooling the egg on the respiratory
movements of the hatching duck (Anas platyrhynchos). Comp Biochem
Physiol A 74A: 861–867, 1983.
Steen JB, Gabrielsen GW. Thermogenesis in newly hatched Eider
(Somateria mollissima) and Long-tailed Duck (Clangula hyemalis) ducklings and Barnacle Goose (Branta leucopsis) goslings. Polar Res 4:
181–186, 1986.
Steen JB, Gabrielsen GW. The development of homeothermy in common eider ducklings (Somateria mollissima). Acta Physiol Scand 132:
557–561, 1988.
Szdzuy K, Fong LM, Mortola JP. Oxygenation and establishment of
thermogenesis in the avian embryo. Life Sci 82: 50 –58, 2008.
Szdzuy K, Mortola JP. Monitoring breathing in avian embryos and
hatchlings by the barometric technique. Respir Physiol Neurobiol 159:
241–244, 2007.
Szdzuy K, Mortola JP. Ventilatory chemosensitivity and thermogenesis
of the chicken hatchling after embryonic hypercapnia. Respir Physiol
Neurobiol 162: 55–62, 2008.
Tazawa H, Wakayama H, Turner JS, Paganelli CV. Metabolic compensation for gradual cooling in developing chick embryos. Comp
Biochem Physiol 89A: 125–129, 1988.
Whittow GC, Tazawa H. The early development of thermoregulation in
birds. Physiol Zool 64: 1371–1390, 1991.
Withers PC. Design, calibration, and calculation for flow-through respirometry systems. Aust J Zool 49: 445–461, 2001.
Yoneta H, Dzialowski EM, Burggren WW, Tazawa H. Endothermic
heart rate response in broiler and White Leghorn chicks (Gallus gallus
domesticus) during the first two days of post-hatch life. Comp Biochem
Physiol A 147: 529 –535, 2007.
Zhang Q, Whittow GC. Embryonic oxygen consumption and organ
growth in Wedge-tailed Shearwater. Growth Dev Age 56: 205–214, 1992.
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00274.2015 • www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.6 on June 17, 2017
5. Dodd GAA, Scott GR, Milsom WK. Ventilatory roll off during sustained
hypercapnia is gender specific in pekin ducks. Respir Physiol Neurobiol
156: 47–60, 2007.
6. Drorbaugh JE, Fenn WO. A barometric method for measuring ventilation in newborn infants. Pediatrics 16: 81–87, 1955.
7. Dzialowski EM, Burggren WW, Komoro T, Tazawa H. Development
of endothermic metabolic response in embryos and hatchlings of the emu
(Dromaius novaehollandiae). Respir Physiol Neurobiol 155: 286 –292,
2007.
8. Else PL, Hulbert AJ. Comparison of the “mammal machine” and the
“reptile machine”: energy production. Am J Physiol Regul Integr Comp
Physiol 240: R3–R9, 1981.
9. Eppley ZA. Development of thermoregulatory abilities Xantus’ murrelet
chickes Synthliboramphus hypoleucus. Physiol Zool 57: 307–317, 1984.
10. Gleeson M. Analysis of respiratory pattern during panting in fowl, Gallus
domesticus. J Exp Biol 116: 487–491, 1985.
11. Hillman SS, Hancock TV, Hedrick MS. A comparative meta-analysis of
maximal aerobic metabolism of vertebrates: implications for respiratory
and cardiovascular limits to gas exchange. J Comp Physiol B 183:
167–179, 2013.
12. Jones DR, Holeton GF. Cardiovascular and respiratory responses of
ducks to progressive hypocapnic hypoxia. J Exp Biol 56: 657–666, 1972.
13. Menna TM, Mortola JP. Metabolic control of pulmonary ventilation in
the developing chick embryo. Respir Physiol Neurobiol 130: 43–55, 2002.
14. Meena TM, Mortola JP. Ventilatory chemosensitivity in the chick
embryo. Respir Physiol Neurobiol 137: 69 –79, 2003.
15. Misson BH. The relationships between age, mass, body temperature and
metabolic rate in the neonatal fowl (Gallus domesticus). J Therm Biol 2:
107–110, 1977.
16. Mortola JP, Toro-Velasquez PA. Breathing pattern and ventilatory
chemosensitivity of the 1-day old Muscovy duck (Cairina moschata) in
relation to its metabolic demands. Comp Biochem Physiol A 167: 35–39,
2014.
17. Nair G, Baggott GK, Dawes CM. The effects of a lowered ambient
temperature on oxygen consumption and lung ventilation in the perinatal
quail (Coturnix C. japonica). Comp Biochem Physiol A 76A: 271–277,
1983.
18. Pettit TN, Whittow GC. The initiation of pulmonary respiration in a bird
embryo: tidal volume and frequency. Respir Physiol 48: 209 –218, 1982.
19. Rahn H, Matalon S, Sotherland PR. Circulatory changes and oxygen
delivery in the chick embryo prior to hatching. In: Cardiovascular Shunts,
R775