Adjustments of gastric pH, motility and temperature during long-term preservation of stomach contents in free-ranging incubating king penguins
1 Centre National de la Recherche Scientifique UPR 9010, Centre d'Ecologie
et Physiologie Energétiques, 23 rue Becquerel, F-67087 Strasbourg Cedex
2, France
2 earth&Ocean Technologies, Hasseer Strasse 75, 24113 Kiel,
Germany
* Author for correspondence (e-mail: cecile.thouzeau{at}c-strasbourg.fr)
Accepted 6 May 2004
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Summary |
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Key words: gastric pH, gastric motility, gastric temperature, stomach content preservation, penguin, Aptenodytes patagonicus, incubation fast
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Introduction |
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Such a long-term storage of food in the stomach must imply substantial
modifications of the usual gastric digestive processes, since the digestive
function of the stomach has been switched off and replaced by a storage
function. In incubating king penguins, the well-conserved stomach content is
characterized by an unchanged appearance, unchanged mass and energetic values,
as well as a pH close to 4 at the beginning and the end of the incubation
shift (Gauthier-Clerc et al.,
2002; Thouzeau et al.,
2003
). These observations argue both for a modification of food
breakdown in the stomach and for a delayed transfer into the intestine. One
way to assess these adjustments is to study physiological parameters of
gastric functions such as pH, motility and temperature.
Few studies have investigated the plasticity of seabird digestive
physiology using both intra- (Wilson et
al., 1989; Peters,
1997a
) and interspecific
(Jackson, 1992
) comparisons.
Intraspecific studies have been made on inshore feeders that forage close to
their nest and therefore modulate their digestion for a comparatively shorter
period than king penguins that do so for up to 3 weeks. Wilson and colleagues
were the first to show that breeding African penguins Spheniscus
demersus exhibit a substantial delay in gastric emptying when on shore to
feed the brood (Wilson et al.,
1989
). Furthermore, a slowing of the digestive processes by
variation in gastric pH has been observed in the Magellanic penguin (S.
magellanicus) foraging at sea
(Peters, 1997a
). Lastly,
modification of stomach temperature, through adjustment of gastric motility,
has been proposed to reduce the rate of digestion in several foraging penguin
species (Peters, 1997a
,
2004
). Technical difficulties
in studying physiological digestive parameters in free-living seabirds
certainly explain why there have been so few studies on digestive modulation
in penguins. The particular situation of long-term stomach food storage of
male king penguins while on shore therefore provides a unique opportunity to
better assess physiological digestive plasticity in seabirds.
Studies on digestion generally require the animals to be held under
controlled conditions. However, in penguins, as with many other species,
stress resulting from human disturbance with or without handling has been
demonstrated to influence physiological parameters such as body temperature
(Regel and Pütz, 1997),
heart rate (Nimon et al.,
1996
) and plasma metabolites and hormones
(Ménard, 1998
). Thus
human presence has to be minimized. In the present study we used recently
developed miniature gastric probes (Peters,
1997a
,
2004
) to record stomach
temperature and gastric pH or gastric motility in free-living birds incubating
in their colony.
The purpose of our work was to test whether any adjustments occur in gastric pH, motility and temperature during food conservation in the stomach of free-ranging incubating male king penguins. To this end, we continuously monitored the changes in gastric motility, pH and temperature and compared birds incubating in the colony with non-breeders held in temporary captivity.
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Materials and methods |
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Experiment 1: Non-breeding birds as controls for digestion
Fifteen non-breeding adult king penguins Aptenodytes patagonicus
(J. F. Miller) were studied in 3 series of 46 individuals. Non-breeders
are birds not yet engaged with a mate for reproduction at the time of capture.
Because it is impossible to retrieve non-breeding birds in the colony on a
daily basis, non-breeders were penned together under natural climatic
conditions for the duration of each experimental series. Birds were fasted for
48 h, following which they were fed once daily for 3 days with a fish diet in
order to acclimate them to captivity and daily manipulation. The diet
consisted of two Austral Ocean species, the mackerel icefish
(Champsocephalus gunnari) and grey rockcod (Notothenia
squamifrons). Before each meal, the birds were weighed to check for any
changes in body mass and if necessary, the fish ration was adjusted (diet mass
range: 476940 g). Twenty-four hours after the last meal, which is a
long enough period to allow complete gastric evacuation in king penguins
(Jackson, 1992), each bird was
given a gastric pH/temperature probe
(Peters, 1997a
) or a gastric
motility/temperature probe (Peters,
2004
), but was not provided with fish. Probes were given orally
followed by a massage down the oesophagus until they passed through the
stomach entrance. The first 24 h of recorded data were considered to refer to
the fasting state, before birds where refed with a single meal. Three days
later, the birds were reweighed and the logger was removed (see below for
details). The colour of droppings was noted to determine the state of
digestion, knowing that in birds excreta are whitish (due to uric acid) during
feeding and greenish during fasting
(Handrich et al., 1993
). A
large proportion of the birds regurgitated the device before the end of the
experiment [57% (4 out of 7) and 50% (4 out of 8) for gastric pH/temperature
probes and motility/temperature probes, respectively]. Finally, for the
pH/temperature and motility/temperature probes, respectively, 3 and 4 birds
retained the device for at least 1 day following the single meal and thus data
obtained from these birds could be used for the analyses. These birds were
used as controls for gastric pH, motility and temperature evolution during
digestion.
Experiment 2: Breeding birds
The female penguin undertakes the first and third incubation shifts, while
the male takes the second and fourth shifts
(Weimerskirch et al., 1992).
For our study, males were marked with a plastic flipper band at the beginning
of the second incubation shift. At the beginning of the fourth incubation
shift, 15 males were equipped with a stomach probe (7 and 8 males with gastric
pH/temperature probes and motility/temperature probes, respectively). For
this, the bird's head was covered with a hood so as to minimize handling
stress. As a precaution, the egg in the broodpatch was replaced by a dummy egg
and was temporarily held in an incubator. The bird was taken away from the
colony, its incubating site being marked and preserved with a piece of wood.
After weighing, a stomach food sample was collected (see below for the
technique used) for chemical composition determination (water, lipid and
nitrogen content), and the bird was equipped with a gastric pH/temperature
probe or a gastric motility/temperature probe. After this procedure, the bird
was brought back to the same place in the colony and its egg restored.
The probes remained in the stomach for 78 days, i.e. until about the middle of the incubation fast. This duration meant that we could manipulate incubating birds without compromising their incubation success. After the 78 days, the bird was recaught and the probe was retrieved. A stomach food sample was collected (see below) to determine the degree of conservation.
Depending on the type of stomach probe, a specific magnetic or noose-like
retrieval tool was used for recovery
(Wilson et al., 1998;
Wilson and Kierspel, 1998
).
Importantly, all equipped birds continued incubating after the end of the
experiments.
Stomach pH/temperature probe
Stomach probes were used that allowed gastric pH and temperature to be
continuously monitored in free-ranging animals. The pH-probe (earth&Ocean
Technologies, Kiel, Germany) is a self-contained pH-meter incorporating a
glass microelectrode, a separate reference electrode with a pressure balancing
system and a data logger (for details see Peters,
1997a,b
).
The pH electrode was calibrated before and after each deployment, using
traceable NIST standard reference buffers (three calibration points: pH 1.68,
4.01 and 7.01, uncertainty ± 0.02 pH units) under
temperature-controlled conditions. Thus any drift could be corrected for and
respective measurement uncertainties could be calculated for each deployment
(see Peters, 1997a
) using the
program pHG (Jensen Software Systems, Laboe, Germany). Before deployment, the
temperature sensor was calibrated in a water bath against a reference
thermometer. The temperature range was 1742°C with a resolution of
<0.1°C (see Peters,
1997a
). Data were archived in the programmable data-logger with
the sampling interval set to 20 s.
Stomach motility/temperature probe
A device for monitoring both stomach motion and temperature in free-ranging
animals was used (earth&Ocean Technologies). The motility probe contains a
piezoelectric film sensor (Peters,
2004), which allows the detection of gastric motor activity due to
the electric charge generated by a dynamic strain (bending) exerted on the
sensor. This type of sensor is rather independent of positioning and of a
certain prebending of the sensor, rendering results from different devices
comparable (Peters, 2004
). The
temperature measurement was the same as for the gastric pH/temperature device.
Data were archived in a programmable data-logger at a sampling interval of 40
s. This sampling interval was chosen because we wanted to obtain an overall
description of the stomach motion rather than the detection of every single
contraction wave. Thus the recorded signals summed several contraction waves
(cf. Peters 2004
, for details
on probe performance). The motility is expressed in relative units of the full
range (0255) of the device (Peters,
2004
).
Food sample collection and chemical composition
Samples of stomach contents were collected using a non-invasive tube
sampling method (by sucking up material with a rubber tube, carefully
introduced from the bill into the stomach). In the majority of the cases, two
successive samplings were made in order to obtain food samples as
representative as possible of the whole stomach contents. The two samples,
each about 1030 ml, were pooled into a tube maintained on ice and
homogenized. Several aliquots were taken and frozen at 20°C until
laboratory analysis in France.
Before analysis of the chemical composition of the food samples, we checked
for the presence of bile pigments that reflect duodenogastric refluxes, i.e.
we noted whether food samples were yellow-green. Each sample was weighed,
freeze-dried and reweighed. Water content was determined by the difference
between fresh and dry masses. Dry samples were ground to a fine powder for
lipid and total protein analyses. Lipid content was determined gravimetrically
by a method adapted from Folch et al.
(1957) on 0.2 g of powder.
Nitrogen content was determined using the Kjeldhal method
(Hiller et al., 1948
) on two
100 mg aliquots of the initial dried powder and converted to protein by
multiplying by 6.25.
Statistical analyses
Comparisons among days in experiment 1 were made using ANOVA for repeated
measures (RM-ANOVA) followed by multiple comparison
(StudentNewmanKeuls test). In experiment 2, comparisons between
the onset and the end of the experiment, for body mass and chemical
composition of the food, were made using a paired t-test. Comparison
among days of the experiment for gastric temperature was made using RM-ANOVA)
followed by multiple comparison (StudentNewmanKeuls test).
Non-linear regression analysis was performed using the software Sigmaplot
(Jandel SPSS, Chicago, USA). Data are expressed as means ±
S.D.
Daytime and night-time were identified using day-length data for Crozet Archipelago for the experimental period (http://www.bdl.fr/cgi-bin/levcou.cgi).
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Results |
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Experiment 2: Breeding birds
Body mass changes
The body mass of incubating birds significantly decreased over the period
studied (paired t-test, t=1.471; P<0.001), on
average by 10.4±1.4%, corresponding to a specific daily body mass loss
of 15.1±2.3 g kg1 day1.
Gastric pH evolution during the incubation fast
Fig. 3A shows an individual
example (bird 5) of pH and gastric temperature recordings during the first
week of incubation. For three birds (2, 5 and, to a lesser extent, 4), pH
showed unexplained fluctuations at the beginning of the recording period, so
that we decided not to consider those hours concerned for further analyses
(see Fig. 4).
|
|
The changes in gastric pH appeared to be highly different among incubating birds during the logger deployment (Fig. 4). For birds 1, 2, 3, 4 and 5, mean hourly pH values rarely fell below ca. pH 4 throughout the days of the experiment, i.e. values higher than the daily minimum values (range: 2.4±0.82.8±0.6, Table 1), and even the daily mean values (range: 3.1±13.4±0.5, Table 1) observed in the control group (Fig. 4). Conversely, in the two other incubating birds, the pH either decreased and was maintained at values as low as the minimum daily values observed for control birds from day 4 of the fast (bird 6) or repeatedly decreased down to these values after rather large oscillations (bird 7).
Compared to the mean value calculated for the control birds, the mean pH over the whole study period for birds 1, 2, 3, 4 and 5 was significantly higher (P<0.05) than that of control birds, with a difference of about 80%. No significant differences were found between control birds and incubating birds 6 and 7 (Fig. 5A).
|
Gastric motility evolution during the incubation fast
Fig. 3B shows an individual
example of gastric motility and temperature recordings during the first week
of incubation. As for pH, gastric motility evolution appeared to be very
different among incubating birds (Fig.
6). Birds 9, 11 and 12 showed extremely low values throughout the
fast with 94.4±1.7% of the mean hourly values lower than 10 (relative
units), i.e. values clearly lower than the daily mean values observed in the
control group during the days after feeding (range:
13.6±3.928.2±8.1 relative units;
Table 1). On the other hand,
for bird 14 a high proportion of values (63.2%) was above 30 (relative units),
i.e. values higher than the daily mean values observed in the control group.
Intermediate profiles of motility were also found between these two extremes
(Fig. 6).
|
Compared to the mean value calculated for the control birds, the mean motility over the whole experimental period appeared to be significantly lower for birds 912 (P<0.05), while values were comparable for birds 8, 13 and 15, and higher for bird 14 (Fig. 5B).
Gastric temperature evolution during the incubation fast
The daily rhythmic fluctuations in stomach temperature were much less
pronounced during the incubation fast (Fig.
3) than in the control group (Figs
1,
2). Except during the first day
(38.1±0.4°C), daily temperature did not vary significantly
(P>0.05) over the course of the experimental period, with a mean
value of 37.9±0.3°C (N=14 birds).
Chemical composition of stomach contents during the incubation fast
The chemical composition of stomach contents exhibited high variability,
both between the onset and the end of the experimental period as reflected by
the S.D. values and data ranges, and among individuals
of the same fasting stage (Table
2).
|
On an individual basis, fresh lipid content was either elevated, constant or decreased between the onset and the end of the experiment, with amplitude ranging from 83.7% (bird 14) to +16.3% (bird 12). On average for the whole group of birds, however, fresh lipid content decreased significantly (26.8±34.0%; paired t-test, t=2.858; P<0.02). Dry lipid content decreased between the beginning and the end of the experiment in all but one bird, for which lipid content was slightly increased, with amplitude ranging from 65.8% (bird 14) to +4.8% (bird 6). On average for the 15 birds, dry lipid content significantly decreased (28.0±17.9%; paired t-test, t=5.221; P<0.001).
As was the case for lipids, fresh protein content was either elevated, constant or decreased between the onset and the end of the experiment, with amplitude ranging from 56.5% (bird 15) to +79.7% (bird 7). On average there was no significant variation in the fresh protein content of food (P>0.05). Dry protein content increased between the beginning and the end of the experiment in all but one bird, with an amplitude ranging from 0.6% (bird 15) to +46.6% (bird 2). On the average, for the whole group of 15 birds, dry protein content increased between the beginning and the end of the experiment (+15.1±11.4%; paired t-test, t=5.909; P<0.001).
Notably, there was a significant (P=0.002) positive correlation between the decrease in dry food lipid content and the mean motility during fasting (Fig. 7).
|
None of the food samples at the beginning of the experiment showed bile pigment coloration (Figs 4, 6). At the end of the experiment, 33% of the birds had stomach contents coloured by yellow-green bile pigments (birds 6, 7, 13, 14 and 15).
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Discussion |
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Gastric pH
Our study demonstrates that a high modulation of gastric pH occurs in male
king penguins at the end of the incubation fast. The maximum pH is similar to
that observed for Magellanic penguins, i.e. around pH 6
(Peters, 1997a). However,
there is a substantial difference among penguin species when comparing the
duration of such an adjustment. While the smaller penguin species can maintain
a high gastric pH for only a couple of hours or possibly up to a few days, we
observed that a high pH can be maintained during the whole period of logger
deployment in incubating male king penguins, i.e. for at least a week. In
order not to threaten the reproductive success and also to ensure logger
recovery, we did not extend our logger deployments until the female's return.
From our data, however, it seems reasonable to suggest that the observed pH
adjustment could be maintained during the whole 23 weeks of incubation
until hatching.
Importantly, our study shows that pH profiles differ among the birds. This
finding, in addition to a high variability in both chemical composition and
visual aspect of the food, most probably means that the birds conserve food to
a greater or a lesser degree. Thus, the pH for birds 6 and 7, that reached
values as low as those observed during digestion in control birds and was
associated with bile pigment coloration of the stored food at the end of the
experiment, presumably reflects a lower efficiency in their food conservation
during incubation. Why such variability exists remains to be elucidated.
Gauthier-Clerc et al. (2000)
have suggested that in breeding king penguins some kind of internal clock
could exist that would allow the male king penguins returning from the sea to
bring back food in their stomach only if their arrival falls within the
hatching schedule, i.e. if the probability that the bird will be on land at
the hatching date is high. Such an internal clock could also influence the
level of food conservation during incubation.
The pH variations observed most certainly reflect fundamental modulations
in the gastric digestive function affecting enzymatic processes. Since avian
gastric proteinases are adapted to acid conditions
(Duke, 1986), the pH values
found in the present study, usually higher than 4 and sometimes even as high
as 6, are unfavourable for gastric protein breakdown. Thus, food protein is
probably preserved not only in quantity
(Gauthier-Clerc et al., 2002
)
but also in quality. It is more difficult to judge the extent of food lipid
conservation. Indeed, regular refluxes from the duodenum into the stomach are
a peculiarity of avian digestion and it is therefore likely that stomach
contents are exposed to pancreatic and duodenal lipases
(Duke, 1997
). In the hypothesis
of passage of some pancreatic enzymes from the duodenum to the stomach
via refluxes, the pH of about 6 monitored in incubating penguins
would allow these lipases to be active. Thus, some hydrolysis of food lipids
could occur during storage, which would be consistent with the decrease in
total lipid content observed during the period of food storage
(Gauthier-Clerc et al., 2002
;
this study).
Even if the presence of food in the stomach results in itself in a short-
to medium-term increase in pH (Rhoades and
Duke, 1975), as we observed in the control experiment on the day
of feeding, it does not explain the observed longer-term adjustments. The
particular mechanisms responsible for these pH adjustments are not yet
identified, as complex interactions among neural, hormonal and paracrine
mechanisms are involved in the regulation of acid secretion in the stomach
(Fanning et al., 1982
).
Factors leading to an inhibition of gastric acid secretions can originate from
the animal's own physiology or from external stimuli. In fasting incubating
king penguins, it seems unlikely that the high pH found in some birds would be
linked to external factors, like ingesting food, since food quality seems to
be the same among individuals irrespective of whether they conserved their
stomach contents to a greater or a lesser extent. Furthermore, the positive
correlation between the mass of the stomach contents brought back from sea and
the probability of the bird to be on land at the time of hatching
(Gauthier-Clerc et al., 2000
)
would indicate that the storage process is probably initiated by the bird
itself and not by an external factor.
Gastric motility
Gastric motility was markedly reduced for most of the incubating birds
fasting with food in their stomach. In contrast, a small proportion of the
incubating birds exhibited a gastric motility as high as that of control birds
during digestion. For these incubating birds, a bile pigment coloration was
found in the food at the end of the study period and their high gastric
motility was associated with a large decrease in food lipid content compared
to other birds. Thus, motility was variable, a lower motility probably being
associated with a better conservation of stomach content. Such a correlation
between inhibition of gastric digestion and stasis of gastric motility has
been made in several amphibians (Fanning
et al., 1982; Taylor et al.,
1985
).
Motor function is a major factor in the control of gastric emptying, and
the conservation of the amount of food stored in the stomach, which was
previously shown during incubation
(Gauthier-Clerc et al., 2000),
was probably correlated with an inhibition of the bird's gastric motility. In
the present study, we did not determine the whole mass of the stomach
contents, because flushing the birds would have threatened their reproductive
success. However, the lower the mean stomach motility during the period of
logger deployment, the lower was the decrease in lipid content of the gastric
contents. Moreover, the stomach contents of most birds were in an almost
unchanged state of preservation at the end of incubation compared to the
beginning, still containing intact pieces of fish and squid (data not shown).
This supports the hypothesis of a rather constant mass of stored food that has
been efficiently preserved in the stomachs of our birds.
While the reduction of gastric motility can be important in penguins
incubating with food in their stomach (see
Fig. 6 for birds 11 and 12), it
was never total. In fasting birds, the amplitude of motility was reduced but
it still persisted, suggesting the influence of a `pacemaker'
(Duke and Evanson, 1976),
which would initiate and regulate the gastric motility
(Chaplin and Duke, 1988
). The
minimal gastric motility observed in incubating king penguins fasting with a
full stomach might then be due to this gastric pacemaker.
Gizzard contraction frequency has sometimes been used as an index of
overall gastrointestinal motility (Savory,
1987). In domestic fowl, gastric contraction is generally followed
by duodenal spikes, and a reduction in stomach activity induces an increase in
antiperistalsis (Roche and Ruckebusch,
1978
). These observations led the authors to suggest a permanent
balance between intestinal and gastric activities in this species. However,
their hypothesis may not be valid for king penguins, since an increase in
antiperistalsis would have resulted in biliary pigmentation in the stomach
content, which was not the case in most of our birds. This observation leads
us to suggest a decrease in intestinal motility along with gastric motility in
penguins, similar to reports in the gastric brooding frog
(Fanning et al., 1982
). Such a
mechanism would be advantageous for food conservation in the stomach because
an inhibition of duodenal motility would preclude the entry of pancreatic and
intestinal enzymes into the stomach.
In penguins, the stomach constitutes the main retention organ of the solid
component of ingested food. Its control is mainly under pyloric influence
since this sphincter seems to prevent the gizzard from discharging material
that has not been ground into small enough particles
(Ferrando et al., 1987;
Vergara et al., 1989
). Thus,
in penguins, it can be hypothesized that the mechanisms of gastric motility
inhibition act principally on the pylorus. The high pH observed could also
stimulate food retention in the stomach, in accordance with the delayed crop
emptying caused by omeprazole-induced inhibition of gastric secretion of HCl
in the chicken (Mabayo et al.,
1995
).
Temperature
We found no difference in the stomach temperature of incubating king
penguins whatever the extent of their gastric content preservation, excluding
temperature adjustments from those factors involved in the mechanisms of
stomach food preservation. No daynight stomach temperature cycle
occurred in incubating birds, in contrast to our observations for
non-incubating penguins. In the latter, the diel temperature pattern observed
was probably the result of movements and thus of muscular heat generation
(Aschoff, 1970). Whereas
incubating king penguins remain active by day as well as by night
(Challet et al., 1994
;
Le Maho et al., 1993
), they
spend most of their time resting in a motionless state during the course of
fasting (Challet et al., 1994
).
This might contribute to the constancy of stomach temperature, but more
importantly, this stability can be explained by the need to hold the
temperature of the egg at an adequate level for embryo development,
independent of the bird's digestive status. Indeed, the average stomach
temperature (38.0°C; present study, or 38.2°C;
Thouzeau et al., 2003
) is
identical to the brood patch temperature, which is maintained constant during
incubation (Handrich, 1989
). A
similar absence of a distinct daynight cycle in core temperature has
been observed in incubating blue petrels Halobaena caerulea
(Ancel et al., 1998
) and only
reappeared in case of egg desertion, probably reflecting daynight bird
movements. In some of our incubating birds, however, a positive correlation
was observed between stomach temperature and motility (data not shown), which
might correspond to a breeder being more active and engaged in territory
defense behaviour.
Conclusions and perspectives
The present study demonstrates the existence of substantial adjustments of
pH and gastric motility in a way that contributes to the inhibition of
digestive gastric processes in incubating male king penguins. Mechanisms
underlying these adjustments are probably complex, including a combination of
neuronal, humoral and/or hormonal factors. The body mass of our birds was
always above the critical value that could jeopardize their survival in a
prolonged fast (Cherel et al.,
1988). Therefore, body condition was not involved in the decision
of the bird to conserve its stomach contents to a greater or a lesser degree.
The fact that incubating male penguins fast with a full stomach only when the
probability of their being on land for hatching is high
(Gauthier-Clerc et al., 2000
)
leads us to suggest the involvement of reproductive hormonal factors in
gastric digestion inhibition during the last part of the incubation. It would
therefore be interesting to determine whether different mechanisms are
involved in the initiation of food storage during foraging at sea and the
maintenance of this storage while on shore.
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Acknowledgments |
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