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First published online February 29, 2008
Journal of Experimental Biology 211, 883-889 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.014068
`Blood-doping' effects on hematocrit regulation and oxygen consumption in late-stage chicken embryos (Gallus gallus)
Department of Biological Sciences, University of North Texas, PO Box 305189, Denton, TX 76203, USA
* Author for correspondence (e-mail: burggren{at}unt.edu)
Accepted 21 January 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSResultsDISCUSSIONCONCLUSIONSReferences |
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12% at day 15. Hct decline was strictly
proportional to the extent of blood withdrawal. Incremental Ringer solution
injection over an 8 h period, transiently increasing blood volume up to 85%
over initial values, did not decrease Hct, indicating that injected Ringer
solution rapidly left the circulating blood compartment. Blood doping with
erythrocyte-enriched whole blood artificially elevated Hct from 27% to 38%,
but caused no significant change in routine O2 consumption
(0.35–0.39 ml O2 min–1
egg–1) at any point over the subsequent 6 h period in day
15–17 embryos. We conclude that Hct is not protected acutely in day 15
chicken embryos, with no evidence of erythrocyte sequestration or release.
Additionally, at day 15–17, Hct increases of 10% do not enhance
embryonic oxygen consumption, suggesting that blood oxygen carrying capacity
per se is not limiting to oxygen consumption.
Key words: hematocrit, oxygen consumption, blood volume, development, embryo
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSResultsDISCUSSIONCONCLUSIONSReferences |
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; Wangensteen et
al., 1970/71; Wangensteen et
al., 1974; Erasmus and Rahn,
1976; Ar et al.,
1980; Ar et al.,
1991; Paganelli,
1980; Tazawa et al.,
1981; Rahn and Paganelli,
1982; Wakayama and Tazawa,
1988; Tazawa and Whittow,
2000; Wagner-Amos and Seymour,
2002; Wagner-Amos and Seymour,
2003). Yet, even in the heavily diffusion-limited respiratory
systems of bird eggs, effective gas exchange ultimately depends upon all
components of the `oxygen cascade', including blood perfusion and the blood's
ability to transport O2 to the embryo's metabolically active
tissues. Blood O2 transporting capacity, in turn, depends upon the
number of red blood cells and the concentration and character of the avian
embryonic hemoglobins.
The fundamental role of the red blood cell in oxygen transport in all but
the youngest avian embryos [where direct diffusion suffices – see
Burggren (Burggren, 2005)] has
led to several investigations of erythropoiesis (see
Dieterlen-Lievre, 1997;
Dragon and Baumann, 2003;
Maina, 2004). Changes in
hematocrit (Hct) and hemoglobin concentration ([Hb]; and thus changes in blood
O2 transporting capacity) are inducible well before hatching in
bird embryos. Hypoxia (either through high altitude or experimental ambient
hypoxia) is a potent erythropoietic stimulus in chicken embryos, though the
response is not universal to all birds (see
Monge and Leon-Velarde, 1991;
Dragon and Baumann, 2003;
Baumann and Dragon, 2005;
Chan and Burggren, 2005).
Presumably, the enhanced O2 carrying capacity associated with
stimulated erythropoiesis assists the chicken embryo in maintaining normal
levels of blood O2 transport. In adult vertebrates, at least,
limitations in blood O2-carrying capacity can generally be
partially or fully compensated for by increases in cardiac output or tissue
oxygen extraction (for a review, see Calbet
et al., 2006). Avian embryos exhibit relatively complex
chemoreceptor reflexes by at least the last third of embryonic development
(see Burggren and Crossley,
2002; Crossley et al.,
2003a; Crossley et al.,
2003b; Khandoker et al.,
2003), and arterial hypoxia may additionally stimulate an
increased cardiac output and redistribution of blood flow within the tissues.
Either increased cardiac output or, in the longer term, enhanced
erythropoiesis raising Hct – or certainly both in concert – could
ameliorate the negative effects of hypoxia and maintain tissue
oxygenation.
To investigate the ability of the late chicken embryo to regulate Hct, we
have investigated whether experimental modifications in Hct through hemorrhage
and Ringer solution infusion are compensated for acutely in day 15–17
chicken embryos. To investigate the specific role of Hct in the normal
functioning of the oxygen cascade from environment to tissues, we increased
Hct through red blood cell infusions [blood `doping' or `boosting' (e.g.
Ekblom, 2000;
Schumacher and Ashenden,
2004)] along with concurrent O2 consumption
measurements to understand how alterations in blood carrying capacity affect
oxygen consumption.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSResultsDISCUSSIONCONCLUSIONSReferences |
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Venous cannulation for Hct manipulation
For withdrawal or infusion of blood or Ringer solution, a vein in the
chorioallantoic membrane (CAM) was cannulated with a method adapted from that
of Tazawa et al. (Tazawa et al.,
1980). Briefly, each egg was candled to find the largest CAM vein
at the egg's blunt end. The egg was then half-buried in a sand bath set at
38°C to maintain egg temperature throughout surgery. A piece of eggshell
(4 mm in diameter) above the selected vein was removed. The inner
eggshell membrane was carefully removed to reveal the underlying vein, which
was then non-occlusively cannulated in a downstream direction. The cannula
comprised a 30 gauge needle, bent at 90° approximately 2 mm from the tip,
which was glued into 100 mm of PE 10 tubing, which in turn was glued into 100
mm of PE 50 tubing. Prior to its insertion, the cannula was filled with
heparinized (100 units ml–1) Ringer solution (commercial
lactate Ringer USP; 130 mequiv l–1 Na+, 4 mequiv
l–1 K+, 110 mequiv l–1
Cl–, 28 mequiv l–1 lactate and 3 mequiv
l–1 Ca2+; osmolality 275 mosmol
kg–1 H2O). The cannula was secured in place in the
vessel with cyanoacrylate glue. The open end of the cannula was then closed
with a small stainless steel pin. The egg was returned to the incubator (at
38°C) immediately after cannula implantation.
Hct determination
Hct was determined on 25 µl of undiluted blood drawn into a Hamilton
syringe through the implanted cannula. Blood volume in day 15 embryos is
2.5 ml (see Tazawa and Whittow,
2000), so the volume of this blood sample represents 1% of
blood volume in day 15–17 chicken embryos. Sampled blood was transferred
to a capillary tube, which was then sealed and centrifuged for 5 min in a
mircocentrifuge (ACCU-STAT MP Readacrit; Pittsburgh, PA, USA) before Hct was
determined.
Protocols for Hct manipulation
In the first series of experiments on day 15 embryos, controlled blood
withdrawal was used to reduce Hct. Every 30 min, 125 µl of blood (5%
of control estimated blood volume) was withdrawn from the CAM vein cannula
into a Hamilton syringe. In the second set of experiments on different day 15
embryos, hypervolemia was induced in an attempt to reduce Hct. Every 30 min,
150 µl of heparinized Ringer solution was injected into the CAM vein. Then,
10 min after each Ringer solution injection, 25 µl of blood was withdrawn
for Hct determination, resulting in an acute net blood volume increase of 125
µl for each such injection cycle.
Artificial erythrocythemia
Artificial erythrocythemia (blood doping) was used to acutely increase Hct.
Within 1 h prior to experiments, approximately 1.5 ml of blood was collected
by chorioallantoic venipuncture into a heparinized syringe from each of 15,
2–3 day donor embryos, which were subsequently killed. Collected blood
was then pooled. Preliminary experiments revealed no obvious agglutination or
similar reactions within the pooled blood sample. Immediately after pooling,
donor blood was centrifuged to separate erythrocytes from plasma.
Approximately 700 µl of plasma was removed from this pooled sample, and the
erythrocytes were then re-suspended in the remaining plasma. The reconstituted
blood sample was then stirred in a vortex mixer for 20 s to ensure complete
mixing. This procedure yielded whole blood with a Hct of approximately
50–65%, which was approximately 20–30% higher than in controls.
The blood sample was visually observed for color change induced by aeration
during re-suspension, to ensure the blood could still be re-oxygenated. This
sample of high Hct blood was then used immediately in blood doping
experiments.
The cannula inserted into a CAM vein served as the site for injection of
400 µl of the high Hct blood sample into a recipient embryo. Injection of
donor blood into a recipient embryo was always well tolerated, with no obvious
in vivo clotting or impairment of the microcirculation, even after
multiple injections over several hours. Blood doping resulted in a net, acute
blood volume elevation of 300 µl (400 µl injection with 100 µl
withdrawal for Hct determination), which represents an increase of
12–14% for day 15–17 embryos.
Embryos were then sampled for Hct and subjected to oxygen consumption measurements, as described below.
Oxygen consumption measurements
Routine oxygen consumption was measured on individuals within sealed,
flow-through respirometers (volume, 296 ml). Air warmed to 38°C flowed at
70 ml min–1 through a port into the bottom of the
respirometer and out of a port at its top, ensuring continual replenishment of
the gas in the respirometer. The gas stream exiting the chamber passed
initially through soda lime (to remove CO2) and then through
Drierite (to remove H2O) before entering an eight-channel oxygen
analyzer (model FC-1B, Sable Systems Inc., Las Vegas, NV, USA). A second minor
stream of gas tapped off the inflow stream to the respirometer was scrubbed
for CO2 and water vapor and also sent to the analyzer for analysis
of the inflow O2 level. Gas flow through the respirometer was
controlled with a Sable Systems gas analyzer sub-sampler (version 2.0), and
was adjusted so that the O2 differential between in-flowing and
out-flowing gas was 0.4–0.6% throughout the experiment. Prior to
beginning the oxygen consumption
(
O2)
measurements, each respirometer containing an egg to be measured was
completely submerged for a minimum of 30 min in a water bath (Fisher ISOTEMP
1028P, Pittsburgh, PA, USA) thermostatically held at 38°C to ensure
thermal equilibrium.
O2 of each
egg was calculated by Sable Systems data analysis software after appropriate
entry of variables. Three separate respirometers were run concurrently, with
duplicate measurements made for each egg. All
O2 values were
calculated on a per egg basis.
The protocol for the
O2 measurement
was started by placing a completely intact, non-cannulated egg into a
ventilated respirometer and letting it thermally equilibrate for 30 min, after
which a baseline (pre-cannulation) level of
O2 was
determined. The egg was then removed from the respirometer and a CAM vein
cannulated as described above. The embryo was allowed to recover in an
incubator (38°C) for 1 h following cannulation before being returned to
its respirometer for the remainder of the experiment. After a second 30 min
period in the respirometer following cannulation, another
O2 measurement
was taken, immediately followed by the first Hct determinations. The embryo
was then blood doped, as described above, and the attendant increase in Hct
documented. O2
was determined every 30 min over a course of 6 h after blood doping, followed
by a third and final Hct determination.
Statistical analysis
All O2 and
Hct data for each stage were tested for normality and equality of variances.
Hct data for blood volume change was non-parametric, resulting in the use of a
Kruskal–Wallis one-way analysis of variance (ANOVA) on ranks to
determine statistical significance. Significance between different groups was
tested for using Dunn's method. A one-way ANOVA was utilized to determine
significance between control Hct data at each stage, followed by a
Holm–Sidak pairwise multiple comparison test. Hct values determined
during the experimental procedure described above were tested for significance
with either Student's paired t-test or a Mann–Whitney
ranked-sums test, depending on normality.
O2 data were
analyzed using a Kruskal–Wallis ANOVA on ranks to determine statistical
differences between each treatment group, followed by a two-way repeated
measures ANOVA to determine treatment and stage effects. SigmaStat version 3.0
(Systat Software, Inc., San Jose, CA, USA) was used to conduct all statistical
analyses. All statistical decisions were made using a 0.05 level of
significance. All averages are presented as means ± 1 s.e.m.
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Results |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSResultsDISCUSSIONCONCLUSIONSReferences |
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20% in Hct evident on days 15 and 16, and of 15% on day 17.
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40% (Dunn's method,
P<0.05).
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5% increase in blood volume) every 30 min
for approximately 6–12 h (Fig.
2B). Despite repeated injections of Ringer solution that in some
cases totaled up to more than double the estimated initial total embryo blood
volume, there was no significant change in Hct from control measurements
(Kruskal–Wallis, P>0.1).
Artificial erythrocythemia
Embryos at day 15, 16 and 17 all showed a significant 10–15% increase
in Hct immediately following injection of erythrocyte-enriched blood
(Kruskal–Wallis one-way ANOVA on ranks, P<0.001;
Fig. 3A). This erythrocythemia
persisted for at least 6 h following injection in all three populations. In
day 15 and 17 embryos, there was no significant change in Hct during the 6 h
post-injection period (one-way repeated measures ANOVA). In day 16 embryos,
however, there was a significant but small decrease in Hct back towards
control values (one-way repeated measures ANOVA, P<0.05).
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O2 and Hct levelO2 measured in
day 15–17 embryos before and after cannulation are presented in
Table 1, which also provides
egg mass and N values for data presented in
Fig. 3B. Pre-cannulation
O2 values in day
15–17 embryos ranged from 0.35 to 0.38 ml O2
min–1 egg–1, and were not significantly
different between developmental days (P>0.05). Vein cannulation
had no significant effect on
O2 of embryos at
day 15 and 17 (Table 1). In day
16 embryos, O2
decreased significantly (Student's paired t-test, P=0.01)
but only slightly to 0.31±0.01 ml O2 min–1
egg–1.
|
Large, artificially induced increases in Hct through blood doping caused no
significant difference in
O2 at any point
over the 6 h post-injection period in the three populations
(Kruskal–Wallis, P0.985, 0.328 and 0.946;
Fig. 3B). There were also no
significant interactions between stage and treatment (P>0.05
two-way ANOVA on ranks).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSResultsDISCUSSIONCONCLUSIONSReferences |
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).
Acutely, rapid changes in Hct can result from fluid fluxes between the
circulating blood volume and non-vascular compartments. Erythrocytes are also
sequestered and released by the spleen. In many vertebrates, splenic
contractions release stored erythrocytes, a catecholamine-mediated response
especially prone during exercise, during decreases in blood volume, or upon
exposure to toxins (Hughes et al.,
1984; Jensen,
1987; Yamamoto,
1987; Ojiri et al.,
2002; Stewart and McKenzie,
2002; Marques et al.,
2006; Shah,
2006).
Normal circulating Hct appears overall to be relatively well regulated in
the late chicken embryo, since even the normal rapid increase in Hct of >4%
in just 2 days appears to be a consistent feature across studies
(Fig. 4). Mean Hct values from
these studies increased from 28.0±0.7% (day 15) to 30.6±1.0%
(day 16) to 32.3±1.1% (day 17), with little variation between studies.
However, as evident in our present study, at each developmental stage there
are outliers with considerably higher or especially lower Hct
(Fig. 1). Presumably, in late
incubation the erythropoietic mechanisms evident in juvenile and adult birds
(Luger, 2003) begin to assert themselves. Indeed, environmental hypoxia begins
to trigger erythropoiesis between day 14 and 18 in chicken embryos
(Tazawa et al., 1988;
Camm et al., 2004). However,
the reflex arcs that control embryonic erythropoiesis, and ultimately regulate
Hct, remain enigmatic.
|
Erythrocyte sequestration and release as a mechanism for Hct regulation
presumably occurs in adult birds as it does in mammals, but has received
little attention in birds of any developmental stage. In the present study,
day 15 chicken embryos experiencing graded blood removal were unable to
maintain Hct at pre-intervention levels even transiently, with Hct falling
progressively with each blood withdrawal
(Fig. 2A). Tazawa
(Tazawa, 1982) similarly
observed a decline in Hct caused by four repetitive samplings in day 16
embryos. Based on these findings, day 15–16 chicken embryos apparently
do not release sequestered erythrocytes, at least not in sufficient numbers to
offset red blood cell loss from even the initial mild hemorrhage.
Blood volume regulation in avian embryos
Regulation of blood volume in adults has been extensively studied in most
vertebrate taxa (for a review, see Takei,
2000), and involves a highly integrated and very complex suite of
mechanisms embodied in the Guyton model of mammalian circulation
(Simanonok et al., 1994). As
is the case for Hct regulation, however, very little is known about the
ontogeny of blood volume regulation generally, let alone the underlying
mechanisms. In the present study, day 15 chicken embryos receiving repeated
injections of Ringer solution showed no decrease in Hct, which would be
evidence of lasting hemodilution, despite dramatic increases in blood plasma
volume. Increased capillary permeability or blood pressure (or both) would
facilitate rapid ultrafiltration of fluid out of the circulating blood
compartment. In this scenario, blood volume actually would not increase, and
Hct would be maintained at pre-injection levels. Interestingly, our
observations are in contrast to earlier observations of hypervolemic
hemodilution (i.e. decreased Hct) by Tazawa
(Tazawa, 1982) in day 16
embryos.
Blood pressure associated with volume loading has been measured in very
early chicken embryos (e.g. Wagman et al.,
1990; Yoshigi et al.,
1997), but not in more advanced embryonic stages closer to
hatching. Whether blood volume increases actually increase blood pressure in
avian embryos will depend in part on vascular compliance. Up until internal
piping in the bird embryo, blood circulates through a very large
chorioallantoic membrane lining the bird shell. The compliance of this
membrane, how it changes during development, and whether this unique
circulatory structure influences blood pressure and volume regulation requires
additional experimentation. Altimiras and Crossley
(Altimiras and Crossley, 2000)
reported that baroreflex function in chicken embryos progressively matures
beginning around day 18 of incubation, so the underpinnings of some form of
physiological regulation of blood volume could be in place in day 15–17
embryos. Testing this hypothesis will require determination of blood pressure
and direct measurement of blood volume during the course of graded blood
removal or repeated Ringer solution injections in late-incubation chicken
embryos.
To some extent, blood volume will be maintained by simple, passive
mechanisms that drive water across capillaries in response to changing osmotic
gradients. However, capillary permeability changes are also heavily implicated
in blood volume regulation, and have received considerable attention in early
chicken embryos (e.g. Cruz et al.,
1997; Defouw and Defouw,
2001). Capillary permeability decreases during development in the
chicken embryo, especially after day 10
(Ribatti et al., 1993). The
physiological implications of these permeability changes at the system level
in older embryos have yet be explored.
Artificial erythrocythemia and O2 consumption
Experimental adjustment of Hct (and thus of blood O2 capacity)
is a powerful way of manipulating potential systemic O2 transport.
Indeed, there is a general positive relationship between short-term blood
O2 capacity and
O2 in a wide
range of vertebrates (Tazawa et al.,
1971; Hillman et al.,
1985; Yahav et al.,
1997; Tan and Lim,
2001; Gaudard et al.,
2003), though the correlation is not inevitable (e.g.
Wood et al., 1979;
Cooper and Morris, 2004). In
chicken embryos, total
O2 is near its
zenith by day 15–17 (see Tazawa and
Whittow, 2000; Dzialowski et
al., 2002), which would also argue for optimization of elements
participating in O2 transport between environment and tissues.
However, in the present study, day 15–17 embryos showed no increase in
routine O2
despite a 10–15% artificially induced increase in Hct. It is possible
that Hct might have been influential in affecting oxygen consumption if the
embryo was consuming oxygen at a far higher rate than that evident in our
measurements. While typically there is a considerable difference between
routine and maximal oxygen consumption in birds and other vertebrates, it is
not clear to what extent routine oxygen consumption would increase in an
embryo in its egg under normal conditions, given the limited opportunities for
`exercise' or even for being visually or mechanically stimulated.
That routine
O2 was not
increased by elevated Hct in these intermediate-to late-stage chicken embryos
suggests that blood oxygen capacity, as a key element of the oxygen cascade
from environment to tissues, is not a limiting factor. In this scenario,
enhancement of blood oxygen capacity will not have nearly as large an effect
as, for example, increasing oxygen diffusion across the shell and into the
egg. The large diffusion barrier across the bird egg has been well documented
(e.g. Pettit and Whittow,
1982; Rahn et al.,
1987; Meir et al.,
1999; Monge et al.,
2000; Wagner-Amos and Seymour,
2002). Acute exposure to hyperoxia increases the
PO2 gradient and subsequently increases
O2 diffusion across the egg shell. As a result, hyperoxic exposure
also markedly increases
O2 in day
16–18 chicken embryos (Tazawa et
al., 1992). Collectively, these data suggest that oxygen diffusion
into the shell may be a larger limiting factor in late embryonic gas exchange
than blood O2 carrying capacity, which could be circumvented by
increases in tissue blood flow, for example.
No discussion of the effects of artificial erythrocythemia on oxygen
consumption is complete without considering the blood viscosity effects
accompanying increased Hct. Blood viscosity increases with increasing Hct in
reptiles, birds and mammals (see Barshtein
et al., 2007; Viscor et al.,
2003). With increasing viscosity comes the specter of reduced
blood flow, which could offset any potential advantage to blood O2
transport associated with elevated Hct. Indeed, sharply elevated viscosity and
the associated decrease in blood flow reduces the diffusive capacity of gas
exchange organs (Piiper and Scheid,
1992). In fact, blood viscosity increase is one of the reasons why
severe blood doping in human athletes is considered ineffective at best and
dangerous at worst (Spivak,
2001). In the present study, increased Hct had no effect on
routine O2,
which theoretically could be explained by an increase in blood viscosity
accompanying the acute experimentally induced increase in Hct. However, at
least in humans, Hct generally needs to increase to values of greater than
50% before negatively affecting tissue blood flow (see
Lowe et al., 2002;
El-Sayed et al., 2005). In our
experiments the maximum induced Hct was 40%
(Fig. 3A), which suggests that
viscosity effects were probably not limiting blood flow.
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CONCLUSIONS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSResultsDISCUSSIONCONCLUSIONSReferences |
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O2 was tightly
linked to blood oxygen capacity, these variations in Hct could well have
negative implications for individual embryos during the last critical stages
of development. Yet, the clear independence of routine
O2 from large
variations in Hct in days 15–17 (Fig.
3) indicates that either (1) late embryonic routine
O2 is not oxygen
limited despite large variations in blood oxygen capacity, or (2) adjustments
in tissue perfusion may compensate for disturbances to blood oxygen carrying
capacity. Future experiments will test these ideas. |
Acknowledgments |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSResultsDISCUSSIONCONCLUSIONSReferences |
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