Acclimation to hypothermic incubation in developing chicken embryos (Gallus domesticus) : II. Hematology and blood O2 transport
Department of Biological Sciences, University of North Texas, Denton, TX 76203, USA
* Author for correspondence (e-mail: burggren{at}unt.edu)
Accepted 22 January 2004
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Summary |
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Key words: chicken embryo, Gallus domesticus, thermoregulation, hypothermic incubation, hemoglobinoxygen affinity, heterokairy
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Introduction |
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Maturity of both the respiratory and cardiovascular systems is crucial to ensure the efficient delivery of O2 to support metabolic heat production and regulate body temperature. In this study we examine the effects of hypothermic incubation on the metabolic and thermoregulatory physiology of chicken embryos. We examine hematology, blood O2 transport characteristics, and in vivo arterial and venous blood PO2 and pH of late-stage chicken embryos incubated in either normothermal conditions (38°C) or hypothermal conditions (35°C). Our goal was to determine if differences in blood parameters might underlie our finding that lower incubation temperature negatively affects the ability of embryos to respond metabolically to decreases in ambient temperature. Specifically, we hypothesized that chronic incubation at low ambient temperature (35°C) would modify both blood O2-carrying capacity and hemoglobinO2 binding affinity in the late-stage chicken embryo, and that these changes might result in a modified thermoregulatory phenotype.
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Materials and methods |
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Samples were incubated at 38.0°C (control), or 35.0°C (hypothermic)
and a relative humidity of 60%. To determine the gross effects of incubation
temperature on development, nine or more embryos incubated at each temperature
were staged for developmental maturity on days (D) 1314, 1516,
1718 and 1920 (Hamburger and
Hamilton, 1951). Hypothermic embryos have a slower rate of
development than embryos incubated in control conditions, so staging was
completed to determine the length of incubation required for the 35°C
embryos to reach developmental stages equivalent to those of the 38°C
embryos.
All subsequent experiments were conducted on embryos at the following stages: HH 4142, reached on days 1516 for 38°C and days 1718 for 35°C; HH 4344, reached on days 1718 for 38°C and days 1920 for 35°C.
Surgical protocol
Each egg was candled to locate a large chorioallantoic membrane (CAM)
vessel. The overlying shell and membranes were removed without disturbing the
underlying vasculature. The tip of a 30 gauge needle attached to a 1 ml
syringe was inserted into a CAM artery and/or vein against the direction of
blood flow. Dead space in the needle (approximately 10 µl) was filled with
heparinized saline (53 mg 100 ml1). A minimum of 200 µl
of blood was drawn for determination of P50, hemoglobin
concentraction and hematocrit, while 100 µl was drawn for measurement of
arterial and venous PO2 and pH or from separate
embryos for ATP and 2,3-BPG determinations. Caution was taken to avoid
contamination by the accidental uptake of fluid.
Hemoglobin and hematocrit determination
A 50 µl blood sub-sample was injected into an OSM2 Hemoximeter
(Radiometer, Copenhagen) for determination of the hemoglobin content of the
blood (g%). Hematocrit was measured by drawing 10 µl of blood into a
heparinized capillary tube, sealing one end, and centrifuging for 5 min in a
micro-hematocrit centrifuge. The hematocrit was determined as the volume of
packed red blood cells to the total volume of blood.
P50 determination
A 50 µl sub-sample of freshly drawn arterial blood was added to a
cuvette containing 4 ml of Hemox solution, 20 µl of Additive A, and 10
µl of Anti-Foaming agent, all solutions from TSC Scientific (New Hope, PA,
USA). The sample was placed in the measurement cuvette of a HEMOXTM
Analyzer (TSC Scientific), which continuously records blood O2
saturation while changing blood PO2, plotting
the results as a continuous rather than punctuated O2 equilibirum
curve. Blood placed in the measurement cuvette was allowed to thermally
equilibrate at either 35°C or 38°C, depending on the experimental
protocol. Measurements of percentage saturation and
PO2 were simultaneously recorded on a computer
using Chart software (ADInstruments, Colorado Springs, CO, USA). Humidified
air was gently bubbled through the sample until 100% O2 saturation
was obtained (approximately 7 min). The sample was then bubbled with
humidified nitrogen for 7 min, until O2 saturation of the blood
reached zero. The sample was then reoxygenated through equilibration with air
for an additional 7 min. Recorded saturations and
PO2 values were plotted to obtain a sigmoidal
O2 equilibrium curve. Two P50 measurements were
determined on each sample, with the replicates averaged to generate the
P50 for that sample.
Hill plots and `n' coefficient determination
The percentage saturation (S) of the blood corresponding to
PO2 values of 1.6 kPa, 2.5 kPa, 4.1 kPa, 6.6
kPa, 10.5 kPa and 16.6 kPa was determined for each O2 equilibrium
curve to characterize the shape of the O2-equilibrium curve for
creation of a Hill plot. Values were converted as follows:
x=logPO2,
y=log[S/(100S)], where S is
saturation. A linear Hill plot with logPO2 as
the independent variable and log[S/(100S)] as the
dependant variable was fitted with a regression line (Sigmaplot 2001). The
`n' coefficient, the slope of the regression line, summarizes the
shape of the curve.
Arterial and venous PO2 and pH
A 100 µl sample of CAM arterial or venous blood was injected into
thermostatted PO2 and pH electrodes
(Microelectrodes Inc., Bedford, NH, USA) connected to a blood gas analyzer
(Blood Gas Meter BMG 200, Cameron Instrument Company, Port Aransas, TX, USA)
for simultaneous measurements of PO2 and
pH.
Physiological (in vivo) O2-equilibrium curves
The O2-binding properties of embryonic chick hemoglobin are
highly influenced by pH (Bohr shift). To allow direct comparisons of blood
properties between incubation temperatures without the confounding effects of
differences in acidbase status, most previously published O2
equilibrium curves have been generated at 7.47, a pH characteristic of
late-stage embryos incubated at 38°C
(Tazawa, 1980). We elected to
measure the P50 of each blood sample at two pH values,
7.47, corresponding to previous measurements of arterial pH for embryos
incubated at 38°C, and at 7.57, the arterial pH for embryos incubated at
35°C (Tazawa, 1973
,
1980
). Bohr shifts were
calculated for each developmental stage and incubation temperature. Then,
using the calculated Bohr shifts, a series of stage- and treatment-specific
O2-equilibrium curves determined in vitro at pH values of
7.277.87 (in 0.1 pH unit increments), were constructed. Each in
vivo blood PO2 value collected from a
sample was then plotted on the O2-equilibrium curve most closely
corresponding to that sample's pH. The in vivo
O2-equilibrium curves used for placement were then removed to
reveal the in vivo O2-equilibrium curves at the mean
arterial and venous pH values.
ATP and 2,3-bisphosphoglycerate concentrations
A 100 µl sample of blood was added to an equal volume of trichloroacetic
acid (TCA) for ATP measurement and to 300 µl of TCA for
2,3-bisphosphoglycerate (2,3-BPG) measurement. Samples designated for ATP
measurement were stored on ice for a maximum of 2 h before measurement.
Samples for 2,3-BPG measurement were stored at 70°C for 4 weeks
before measurement, conditions that maintain stable amounts of organic
compounds. Concentrations of organic phosphates were determined with an
end-point spectrophotometry technique at a wavelength of 345 nm
(SigmaAldrich measurement kit; St Louis, MO, USA).
Statistical analyses
All data was tested for normality of distributions (ShapiroWilks
normality test) and equality of variances before specific statistical analyses
were performed. Data from within and between stages and at the two incubation
temperatures were tested for statistical significance with an ANOVA.
Significance between groups was determined with a
StudentNewmanKeuls (SNK) multiple range post hoc test.
All statistical tests were conducted using SAS software and decisions were
made with a 0.05 level of significance. Values are means ± 1
S.E.M.
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Results |
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At HH 4142, blood of both 35°C and 38°C embryos had relatively high P50 value, low O2 affinity, and was insensitive to changes in measurement temperature (Fig. 2). By HH 4344, the bloodO2 affinity of the 38°C embryos at 38°C had not changed significantly, but was now temperature sensitive, with P50 dropping from 6.0 kPa±0.4 at 38°C to 4.4 kPa±0.4 at 35°C (Fig. 2B, F=4.55, P<0.0001). In considerable contrast to the 38°C embryos, the blood of the 35°C embryos remained temperature insensitive at HH 4344, showing no change in O2 affinity measured at 38°C (Fig. 2B, SNK, P>0.1).
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Temperature- and stage-induced O2 affinity changes in 35°C and 38°C embryos were not accompanied by any significant change in the shape of the O2 equilibrium curve, summarized by the Hill coefficients (n). Values of n ranged from 2.42±0.056 to 2.64±0.059 for the HH 4142 embryos (Fig. 3A inset). Similarly, at HH 4344 there were no significant changes in the shape of the curves for either the 35°C or 38°C embryos, with Hill coefficients ranging from 2.10±0.19 to 3.10±0.11 (Fig. 3B inset).
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In vivo respiratory properties of arterial and venous CAM blood
Blood from the CAM artery, carrying relatively deoxygenated blood from the
tissue of the embryos to the CAM gas exchanger underlying the shell, had low
PO2 values, in the order of 3.74.2 kPa
(Table 1). 35°C embryos at
HH 4142 had a significantly lower arterial
PO2 than the 38°C embryos (SNK,
P<0.05), but between HH 4142 and 4344 the arterial
PO2 of the 35°C embryos remained unchanged,
while that of the 38°C embryos dropped significantly (SNK,
P<0.001).
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Venous CAM blood (draining the CAM gas exchanger) of HH 4344 embryos had similar PO2 values regardless of incubation temperature (between 8.1±0.3 kPa and 9.1±0.4 kPa for 35°C and 38°C, respectively). Only the 38°C embryos experienced a significant drop in venous PO2 as development continued (SNK, P=0.024). Within each incubation temperature and stage the venous PO2 was always significantly higher than that of the arterial blood (SNK, P<0.001).
There were no significant differences in the pH of arterial or venous blood between incubation temperatures or stages, and the pH of the arterial blood was not significantly different from the venous blood for any of the treatment groups (Table 1).
When plotted on O2-equilibrium curves representing in vivo arterial and venous pH conditions, the in vivo physiological range of PO2 and saturation experienced by the embryo can be determined for each group (Fig. 4). HH 4142 embryos maintained a 6080% O2 saturation in `arterialized' blood in both incubation temperature groups. With further development to HH 4344, oxygen saturation climbed slightly to 6590% in both groups. There was more variation in CAM arterial blood saturations. Minimum saturation values of about 20% were evident in 35°C embryos at HH 4344 compared with less than 10% in 38°C embryos at HH 4344.
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Organic phosphate modifiers of hemoglobinoxygen affinity
Blood [ATP] ranged from approximately 80120 µmol
dl1 (Fig.
5A), while blood [2-3-BPG] ranged from approximately 0.10.6
µmol dl1 (Fig.
5B). The 35°C embryos had significantly lower concentrations
of ATP at HH 4142 (F=4.20, P=0.012), but by HH
4344 there was no significant difference in [ATP] between incubation
temperatures. Concentrations of 2,3-BPG were not significantly different
between incubation temperatures at HH 4142. By HH 4344 the
38°C embryos experienced a significant decline in blood 2,3-BPG, giving
the 35°C embryos a significantly higher [2,3-BPG] at the latest stages
(F=6.99, P<0.001).
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In general, 35°C embryos maintained constant [ATP] and [2,3-BPG] between HH 4142 and 4344, while the concentrations of both organic phosphates tended to decrease between these same stages in the 38°C embryos (Fig. 5).
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Discussion |
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Hypoxic incubation can increase both hematocrit and [Hb] of embryonic
chicken blood (Dusseau and Hutchins,
1988; Dzialowski et al.,
2002
), suggesting that increasing the total O2-carrying
capacity of the blood may be an important acclimation response ensuring
adequate O2 delivery to tissues prior to hatching. It is
interesting to note that, in contrast to the 38°C embryos, there was no
similar significant increase in hematocrit and [Hb] values in 35°C embryos
with development from HH 4142 to HH 4344
(Fig. 1). Perhaps hypothermic
incubation precludes important acclimation responses that operate at
38°C.
The lower O2-carrying capacity of the 35°C embryos
corresponds to the significant increase in HbO2 affinity
between HH 4142 and 4344. The concurrence of these events
suggests that the 35°C embryos may be maximizing the loading of
O2 at the respiratory surface to compensate for restriction of
total O2-carrying capacity. Blood gas data supports this notion,
with the 35°C HH 4344 embryos creating CAM venous (`arterialized')
O2 saturations of close to 90%
(Fig. 4). A left-shifted curve
should allow the embryo to saturate blood more completely at
PO2 values in vivo similar to those
experienced by the 38°C embryos. HH 4142 embryos incubated at
35°C and all 38°C embryos maintained CAM venous saturations of less
than 80%, corresponding to the findings of other studies
(Misson and Freeman, 1972;
Tazawa and Mochizuki, 1978
;
Tazawa, 1980
).
Incubation temperature and HbO2 affinity
HemoglobinO2 binding affinity represents a compromise
between the conflicting needs of efficient loading of O2 at the
respiratory surface and adequate delivery of O2 to the
metabolically active tissues (Reeves,
1984). Balancing these requirements becomes more complicated in
the late stages of incubation in the chicken embryo. An egg incubating in
normal temperature and O2 conditions becomes increasingly hypoxic
in ovo as the restrictions of diffusive gas exchange across the egg
shell fail to meet the growing O2 demands of the rapidly developing
embryos (Wagensteen et al.,
1970
; Rahn et al.,
1974
; Ar et al.,
1980
; Tazawa,
1980
; Reeves,
1984
). As a consequence, air cell
PO2 declines from 18.6 kPa in early incubation
to approximately 13.3 kPa just prior to pipping
(Reeves, 1984
;
Burggren et al., 2000
). Venous
PO2 follows that same pattern, decreasing from
10.6 kPa at D12 of incubation to 8.1 kPa by D17
(Tazawa et al., 1971
).
Following an inverted pattern, blood PCO2
increases during development, resulting in a progressive decrease in blood pH
(Tazawa, 1973
).
Maintaining adequate O2 loading in the face of these
developmental respiratory transitions is assisted by HbO2
affinity transitions in the chicken embryo. Development is characterized by a
progressive left-shift in the O2 equilibrium curve, from a
P50 of 7.0 kPa at D8 to P50 values at
D18 down to 4.25.8 kPa (Tazawa,
1980) or even as low as 4.1 kPa
(Reeves, 1984
). In the present
study, chicken embryos incubated normothermically at 38°C generally
followed the expected pattern, with a decrease in P50 from
6.8 kPa at HH 4142 down to 6.0 kPa at HH 4344 (days 17 and 18 of
incubation), with P50 values at these later stages
slightly higher than values reported in the literature. Embryos incubated at
35°C followed the expected pattern more closely, reaching a
P50 of about 4.0 kPa by HH 4344.
Why hemoglobin(s) of HH 4142 embryos remain insensitive to a 3°C
change in measurement temperature is unclear. The sensitivity of whole blood
to changes in temperature depends on its hemoglobin types
(Baumann and Meuer, 1992). By
D8 of incubation the blood of the chicken embryo contains three types of
hemoglobins: a hatching hemoglobin (HbH), which plays only a minor role in
O2 transport, and two adult hemoglobins (HbA and HbD). In adult
chickens the ratio of HbA to HbD is approximately 3:1, but HbD plays a larger
role in the chicken embryo, existing at a ratio of 1:1.05 at D9 of incubation
(Baumann and Meuer, 1992
). The
ratio of HbA to HbD continues to change in the late stages of incubation, and
although the influence of each hemoglobin on the O2 binding
affinity of the blood is not yet fully understood, it is possible that the
ratio of HbA to HbD may have an important impact on the temperature
sensitivity and other characteristics of embryonic hemoglobin. Incubation
temperature may impact the rate at which the ratio of HbA to HbD increases and
in this fashion delay the onset of blood temperature sensitivity.
In addition to being altered by changes in embryo acidbase status,
temperature and Hb type, O2 affinity of embryonic chicken blood is,
of course, also affected by organic phosphate concentrations
(Baumann and Meuer, 1992). Up
until approximately D12 of incubation in the chicken, ATP is the primary
organic modifier of hemoglobinO2 affinity
(Misson and Freeman, 1972
;
Bartlett and Borgese, 1976
;
Baumann and Meuer, 1992
;
Hochachka and Somero, 2002
).
The increase in hemoglobinO2 affinity between D8 and D18 of
incubation corresponds to the period where metabolic rate reaches its maximum
pre-pipping plateau, and hypoxia begins to develop within the egg. At this
point the aerobic production of ATP is more difficult to achieve and there is
a fall in blood [ATP] (Bartlett and
Borgese, 1976
; Nikinmaa,
1990
). As [ATP] drops, anaerobic production of
2,3-bisphosphoglycerate (2,3-BPG) increases and this organic phosphate then
acts as the primary allosteric modifier of hemoglobin until after hatching,
when inositol polyphosphate (IPP) becomes the adult allosteric modifier
(Bartlett and Borgese, 1976
;
Nikinmaa, 1990
).
Contrary to our expectations, the significant increase in
hemoglobinO2 affinity between HH 4142 and 4344
in the 35°C embryos is not associated with any change in [ATP] or
[2,3-BPG]. This suggests that in hypothermic embryos organic phosphates played
little role in modifying hemoglobinO2 affinity, and that
incubation temperature and the nature of the hemoglobins present were probably
the primary reasons for the significant drop in P50
observed by HH 4344. The effect of temperature on organic phosphate
concentration in the chicken embryo has never been examined but, in general,
[ATP] and [2,3-BPG] from these experiments are similar to those of numerous
other studies on chicken embryos (Isaacks
and Harkness, 1975; Isaacks et
al., 1976
; Bartlett and
Borgese, 1976
; Baumann and
Meuer, 1992
) and other precocial embryos including turkeys,
pheasants, guinea fowl and ducks (Isaacks
et al., 1976
; Bartlett and
Borgese, 1976
).
Although incubation temperature had no apparent affect on organic phosphate
concentrations in late-stage chicken embryos, hypoxic incubation results in an
earlier switch from ATP to 2,3-BPG. Exposure to hyperoxia allows chicken
embryos to maintain high levels of ATP throughout incubation and delay the
developmental switch to 2,3-BPG (Ingermann
et al., 1983; Baumann et al.,
1986
). The arterial and venous pH and
PO2 values collected for embryos incubated at
35°C and 38°C were similar at each stage of development
(Table 1), indicating that a
reduction in incubation temperature does not increase the level of hypoxic
stress beyond what is normally experienced by the late-stage embryo. From
previous research, it is clear that O2 regulates both the
concentrations of organic phosphates and the timing of the switch from ATP to
2,3-BPG. It is not surprising then, that incubation temperature alone did not
result in obvious changes in the patterns of organic phosphate concentration
in the late-stage chicken embryo.
Oxygen transport and consequences for metabolism and thermoregulation
The adult chicken can increase pulmonary ventilation and cardiac output to
elevate O2 transport rates in support of increased
O2. In contrast,
O2 in the chicken embryo
is limited by diffusive gas exchange across the shell, especially in later
developmental stages when O2 demands are increasing
(Tazawa et al., 1992
).
Essential changes occur in both [Hb] and blood HbO2 affinity
as the embryo reaches these final, most aerobically demanding stages.
Interestingly, embryos incubated at 35°C and 38°C maintained
surprisingly similar rates of O2 consumption at equivalent stages
of development less than might be predicted with from typical
temperature effects on animal metabolism
(Black and Burggren, 2004
).
Yet, the mechanisms for achieving these similar O2 demands appear
quite different. Initially, HH 4142 embryos present themselves very
similarly at both incubation temperatures, with near identical basal metabolic
rates, hematocrits, total hemoglobin contents and temperature-insensitive
hemoglobins with low O2 affinities. However, by HH 4344
important differences emerge between 35°C and 38°C embryos. The
38°C embryos have developed significantly higher hematocrit and [Hb], and
so presumably have a greater potential O2-carrying capacity than
the 35°C embryos. The 35°C embryos, on the other hand, have
hemoglobins with higher O2-binding affinities and are able to more
completely saturate the blood at the respiratory gas exchanger surface. The
similar metabolic rates of these two different incubation groups indicate that
either set of compensatory changes can effectively support the O2
demands of embryos.
Although basal metabolic rates of embryos incubated at 35°C and
38°C are not significantly different
(Black and Burggren, 2004),
35°C embryos were less effective in responding metabolically to acute
decreases in ambient temperature in late incubation. We speculate that at HH
4344 the more temperature-sensitive hemoglobins of the 38°C embryos
might provide an advantage over the temperature-insensitive hemoglobins of
35°C embryos at the same developmental stage. When exposed to decreases in
ambient temperature, the hemoglobins of the 38°C embryos at HH 4344
could have a higher O2 affinity at the cooler respiratory surface
and a lower O2 affinity at the site of the warmer tissues,
maximizing O2 loading at the CAM. The presence of descending
temperature gradients from the core of the embryo to the surface of the shell
in the developing chicken egg is well established, making the
temperature-sensitive nature of the hemoglobin molecule in the 38°C
embryos an efficient strategy for O2 transportation
(Tazawa et al., 1988
;
Turner, 1990
). The 35°C
embryos at HH 4344 maintain a high O2-affinity regardless of
temperature, which apparently still enables them to achieve efficient loading
of O2 but might inhibit efficient delivery of O2 to the
embryo. As one might expect, the 35°C embryos experienced a significant
decline in
O2 with a much
smaller acute ambient temperature decrease than the 38°C embryos,
suggesting a less developed or otherwise compromised capacity for
thermoregulation through elevated aerobic metabolism.
Beyond HH 4142, 35°C embryos experienced retarded hematological
development and a delayed metabolic response to acute temperature changes
compared to embryos incubated in control conditions of 38°C. These data
providing the first supporting evidence in an endothermic species for
`heterokairy' a within-individual change in the timing of the onset of
regulatory mechanisms during development
(Spicer and Burggren, 2003).
Exposure to decreased ambient temperature represents an ecologically relevant
threat to the chicken hatchlings (Whittow
and Tazawa, 1991
), especially the 35°C embryos with their
retarded thermoregulatory abilities. A series of future experiments will
examine how the hematological and metabolic differences revealed in these
studies affect the ability of the hatchlings to cope with such thermal stress.
Of course, physiological temperature-dependent differences induced by
incubation temperature differences are not likely to arise in a naturally
incubated chicken embryo receiving conductive heat from the incubating hen.
However, even though the experimental induction of such changes is not
directly relevant to the ecology of natural incubation, the present study has
shown that using incubation temperature as a developmental variable is clearly
a very useful tool for examining the capability of developing physiological
and hematological systems to respond to environmental challenges in
ovo.
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Acknowledgments |
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