Post-prandial blood flow to the gastrointestinal tract is not compromised during hypoxia in the sea bass Dicentrarchus labrax
1 Department of Zoology, University of Göteborg, Box 463, S-405 30
Göteborg, Sweden
2 CREMA-L'Houmeau, (CNRS-IFREMER), France
* Author for correspondence (e-mail: M.Axelsson{at}zool.gu.se)
Accepted 6 June 2002
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Summary |
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Key words: gastrointestinal blood flow, cardiac output, hypoxia, post-prandial, sea bass, Dicentrarchus labrax
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Introduction |
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In their normal habitat, fish have to adjust continuously to multiple stimuli such as changes in water temperature, salinity and oxygen levels; they also have to escape predators or forage and catch prey. If the cardiovascular system cannot fully adjust to these stimuli, compromises have to be made. This will become even more pronounced after feeding when a higher demand is put on the cardiovascular system.
It has been shown that marine coastal environments are exposed to
large-scale and long-lasting hypoxic episodes
(Diaz and Rosenberg, 1995).
Even when not lethal, these conditions are liable to force fish to prioritize
their energy demands to make them fit within a reduced metabolic scope
(Priede, 1985
). For example,
in the Atlantic cod Gadus morhua, it was observed that fed fish
voided their stomach contents when exposed to hypoxia, indicating that systems
other than the gastrointestinal tract were prioritised during hypoxia
(Claireaux et al., 2000
).
The aim of the present study was to test whether the sea bass Dicentrarchus labrax shows a post-prandial increase in gastrointestinal blood flow and, if so, whether this increase is compromised during exposure to hypoxia. Sea bass was chosen as a model to test our hypothesis as it is exposed to hypoxia in its natural littoral habitat and its feeding habits are well documented because of its commercial importance. The experimental protocol involved the measurement of cardiac output and gut blood flow during a stepwise hypoxic challenge before and after feeding.
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Materials and methods |
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Surgical procedures
Seven sea bass (body mass 356±24g, mean ± S.E.M.) were
individually anaesthetised in tricaine methane sulphonate (MS-222; 100 mg
l-1, Sigma) until breathing movements ceased, and then placed on
their left side on an operating table. The gills were continuously irrigated
with aerated sea water containing the same anaesthetic (50 mg l-1).
To measure cardiac output (), the
ventral aorta was exposed through an incision on the left side of the isthmus.
Depending on the size of the animal, the freed portion of the ventral aorta
was placed inside a 4S- or 2S-type Transonic ultrasound flow probe (resolution
0.8 and 0.1 ml min-1, respectively; absolute accuracy ±15%
for both probes) (Transonic System Inc., Ithaca, NY, USA). The opening of the
probe head was closed either by the metallic lock (for 4S probes) or by a
small piece of plastic glued to the probe head itself (for 2S probes). The
lead from the probe was secured to the skin with two silk sutures. To record
GI, a 1 cm long incision was made just
posterior to the left pectoral fin. After careful blunt dissection, the
coeliac and mesenteric arteries were placed in a 2S- or 4S-type Transonic
ultrasound flow probe. Care was taken not to damage the nerves running along
the vessels. The leads from the probe were externalised through the incision
and secured to the skin with two sutures. After surgery, the animals were
transferred to the experimental chamber and allowed to recover for 24-36h
before experiments commenced.
Anatomical study of the gastrointestinal vasculature
Two animals were used to obtain corrosion casts of the main arteries. The
animals were killed by MS-222 overdose (1 g l-1). The heart was
exposed through a midline incision, and a P120 catheter was secured in the
ventricle. Physiological saline (0.9% NaCl) containing heparin (200 i.u.
ml-1) was used to rinse the vascular beds until the fluid that
returned via the atrium was clear of red blood cells. Subsequently,
Mercox resin (Ladd Research Industries Inc.) was slowly injected and allowed
to polymerise overnight before the tissue was digested using 30% KOH solution.
The cast was rinsed with distilled water and photographs were taken and
digitised.
Gastrointestinal emptying time
A group of 18 sea bass (body mass 258±10 g, mean ± S.E.M.)
were used to study gastric emptying time (GET) in non-instrumented animals.
Fish were anaesthetized with MS-222 (100 mg l-1) until righting
reflexes disappeared and were then force-fed 2.9% of their body mass with blue
mussels Mytilus edulis. The food ratio was chosen from the largest
daily feeding rates observed in sea bass using ad libitum
demand-feeding strategies (Azzaydi et al.,
1998). The fish were placed randomly in three separate tanks
maintained in identical conditions.
Three groups of six fish each were sampled after 24, 36 and 48 h. Each animal was killed by MS-222 overdose (1 g l-1), and the stomach contents were carefully separated, blotted dry and weighed.
Experimental protocol: hypoxia exposure
After 24-36 h of post-operative recovery, the flow probes were connected to
a two-channel Transonic flow meter (Transonic Systems Inc., model T206). The
water oxygen concentration was continuously recorded using an Orbisphere 27141
oxygen meter and a nitrogen/air mixture was used to set each hypoxia level
using calibrated glass flow meters. The experimental protocol started by
recording and
GI for 10 min at water
PO2=20.6 kPa (normoxia) to obtain a stable
control period. The animal was then exposed to a stepwise decrease in water
PO2 (to 14.2, 9.0, 6.4, 5.1 and 3.9 kPa,
manually adjusted), each step being held for 5 min. After the last step, water
PO2 was returned to full saturation (20.6 kPa)
and the fish was allowed to recover for 1 h. No struggling was observed during
the hypoxia exposure.
The fish was then lightly anaesthetised in the experimental chamber until righting reflexes were lost, then force-fed 3% of its body mass with blue mussels M. edulis. The experimental chamber was flushed to eliminate the anaesthetic, and the animals were left to recover for 24 h before the stepwise hypoxia protocol was repeated. After the second hypoxic exposure, the animals were killed with MS-222 overdose (1 g l-1) in the experimental chamber, and the stomach contents were carefully separated, blotted dry and weighed.
Data acquisition, calculations and statistical analysis.
The flow signals from the Transonic flow meter were fed directly into a
Dell Latitude Xpi Laptop computer running Labview (National Instruments
version 5.1). The flow signals were sampled at 20 Hz. Heart rate (fH)
was obtained from the pulsatile flow signals. Stroke volume (Vs) was
calculated as /fH.
and
GI were normalized per animal body
mass. Data are presented in graphs as means ± S.E.M.
Wilcoxon's signed-ranks test for paired (two-tailed) and MannWhitney
U test for non-paired (two-tailed) samples were used to evaluate the
statistical significance. The level of statistical significance was set to
P<0.05. In cases of repeated tests, a modified Bonferroni
procedure was used (Holm,
1979).
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Results |
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after feeding increased
significantly by 15% (from 40.0±3.4 to 45.5±4.2 ml
min-1 kg-1, Fig.
2) and
GI by
71% (from 9.6±1.6 to 14.9±1.6 ml min-1
kg-1, Fig. 3) in
normoxic conditions. Before feeding,
GI constituted
24.0±3.2% of total cardiac output, and this increased significantly 24
h after feeding to 34.0±4.4% (Fig.
3).
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During hypoxia alone, decreased
significantly from 40.0±3.4 ml min-1 kg-1 in
normoxia (20.6 kPa) to 29.8±2.5 ml min-1 kg-1 at
PO2=3.9 kPa. fH decreased
significantly from 51.3±3.8 to 26.0±2.4 min-1, while
VS increased significantly from 0.80±0.08 to
1.18±0.11 ml kg-1 (Fig.
2). Similar qualitative changes occurred 24 h after feeding, but
post-feeding decreased to
24.7±1.7 ml min-1 kg-1, which was significantly
lower than pre-feeding values at this PO2
(Fig. 2).
Hypoxia alone decreased
GI significantly from
9.6±1.6 ml min-1 kg-1 in normoxia (20.6 kPa) to
3.7±1.1 ml min-1 kg-1 at
PO2=3.9 kPa
(Fig. 3); this corresponded to
a decrease in
GI/
from 24±3 to 13±4%. After feeding, gastrointestinal blood flow
decreased significantly from 14.9±1.6 ml min-1
kg-1 in normoxia (20.6 kPa) to 6.5±1.3 ml min-1
kg-1 at PO2=3.9 kPa
(Fig. 3) but there was no
change in
GI/
because of the concomitant greater drop in
.
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Discussion |
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The kinetics of digestion in fish has been studied in a variety of species,
Sea bass of 26 g body mass fed 2% of their body mass emptied the stomach in 39
h at 15°C (Santulli et al.,
1993). Assuming that the effect of body mass on gastric emptying
time (GET) is similar to that in other species, a GET of 23 h is predicted for
the sea bass used in the present study. Under similar conditions of body mass,
amount of food and temperature, GET was 16 h for European plaice
Pleuronectes platessa (Basimi and
Grove, 1985
) and 23 h for turbot Scophthalmus maximus and
dab Limanda limanda (Grove et
al., 1985
; Gwyther and Grove,
1981
).
GET in our non-instrumented fish was approximately 48 h, which is much
greater than the predicted 23 h; it was even longer in the instrumented fish,
in which the stomach contents after 24 h were still above 60%, indicating that
force-feeding, anaesthesia and surgery delay gastric emptying, as previously
reported in Atlantic cod (Dos Santos and
Jobling, 1988) and rainbow trout Oncorhyncus mykiss
(Olsson et al., 1999
). The aim
of the present study was not to determine the absolute gastric emptying time
but to compare pre-prandial versus post-prandial states and the
effects of hypoxia on gastrointestinal blood supply in these two different
states. In the instrumented animals, the high gastric content 24 h after
feeding was accompanied by an increased blood supply to the gut.
Relative splanchnic blood flow before feeding (24% of cardiac output) was
within the range reported in other species: 40% in Atlantic cod
(Axelsson and Fritsche, 1991),
36% in chinook salmon Oncorhynchus tshawytscha
(Thorarensen et al., 1993
),
35% in red Irish lord Hemilepidotus hemilepidotus
(Axelsson et al., 2000
), 25% in
white sturgeon Acipenser transmontanus
(Crocker et al., 2000
) and 16%
in sea raven (Axelsson et al.,
1989
). After feeding, splanchnic blood flow increased by
71±19% from 9.6±1.6 to 14.9±1.6 ml min-1
kg-1). This increase is similar to that reported in Atlantic cod
fed a similar food ratio (60%; Axelsson and
Fritsche, 1991
). Greater increases have been observed in sea raven
(coeliac blood flow more than doubled;
Axelsson et al., 1989
) and red
Irish lord (112% increase in coeliac flow 4 days after feeding;
Axelsson et al., 2000
)
force-fed greater ratios (7-9% and 10-15% of body mass, respectively).
Red Irish lord and sea raven are both benthic ambush feeders with a
relatively low level of activity compared with the pelagic highly active sea
bass. In pelagic species, the active muscles need to be supplied with oxygen,
and any increase in demand from other vascular circuits such as the
gastrointestinal tract must be met either by a redistribution of blood or by
an increased cardiac output. The post-prandial increase in gut blood flow of
5.3±0.9ml min-1 kg-1 is paralleled by an increase
in cardiac output of 5.4±2.1 ml min-1 kg-1. This
increase is largely due to increase in heart rate, as in red Irish lord
(Axelsson et al., 2000) and
Atlantic cod (Axelsson and Fritsche,
1991
). This match between the increase in gastrointestinal blood
flow and cardiac output is in contrast to the post-prandial increase in
splanchnic blood flow in mammals, which involves an additional redistribution
of blood from other tissues (Matheson et
al., 2000
; Vatner et al.,
1974
).
During mild hypoxia, is maintained
despite the reduction in fH. This is due to a compensatory
increase in VH, similar to what has been reported in
rainbow trout (Wood and Shelton,
1980
) and Atlantic cod
(Fritsche and Nilsson, 1989
).
At a PO2 of 3.9 kPa
decreases because of a significant
bradycardia without inotropic compensation and, at the same time,
GI decreases by a
relatively greater percentage (61% at 3.9 kPa compared with normoxia). In
Atlantic cod, coeliac blood flow decreased by more than 40% and mesenteric
flow by more than 60% during hypoxia
(Axelsson and Fritsche, 1991
).
Thus, these results provide ample evidence that hypoxia affects the splanchnic
blood flow in sea bass, similar to in other fish species, and that feeding
itself also induces an increase in splanchnic blood flow.
Interestingly, and contrary to our initial hypothesis, relative splanchnic
blood flow
(GI/
)
during hypoxia did not decrease after feeding. Indeed
GI/
decreased significantly before feeding but not after feeding
(Fig. 3), indicating that, even
when facing a hypoxic challenge, gut blood flow is maintained at the expense
of other systems. Salmonids are able to digest food and swim at the same time,
but this occurs at a cost, namely a 10-15% reduction in the maximum prolonged
swimming performance (Thorarensen,
1994
).
Similarly, regional blood flow must be readjusted when oxygen availability
is reduced. Atlantic cod are known to regulate food intake during hypoxia.
They exhibit a direct relationship between growth rate and oxygen
availability, in the absence of changes in food conversion efficiency. Of the
reduction in growth, 97% can be accounted for by reduced food consumption
(Chabot and Dutil, 1999).
Although behavioural regulation of food intake may occur in sea bass as well, our results suggest that once food has been eaten it is digested and absorbed, even at the expense of a reduced oxygen supply to other organs.
A simple explanation of our results could be that sea bass lack reflex
mechanisms to regulate GI. However, we
consider this highly unlikely, given that reflex control of the splanchnic
circulation does occur in conditions such as fright or exercise (M. A., J. A.
and G. C., unpublished data). During fright episodes, the reduction in
is independent of the reduction in
GI, the latter recovering more slowly.
This probably indicates an independent reflex vasoconstriction of the
splanchnic circulation, as has been shown in other species
(Farrell et al., 2001
).
An alternative explanation for the post-prandial maintenance of
GI/
during hypoxia could be as follows. Preprandial regulation of
GI (as occurs during hypoxia or
exercise) is largely dependent on reflex vasoconstriction of the gut
vasculature. Following feeding, splanchnic blood flow increases as response to
the release of local factors such as nitric oxide or adenosine
(Matheson et al., 2000
). Once
this response has started, reflex vasoconstriction may not be as effective in
decreasing
GI because local hyperaemia
outcompetes reflex regulation.
Although this hypothesis is indirectly supported by the present results,
further studies are required to investigate the control mechanisms involved.
In mammals, it has been shown that the initiation and maintenance of
post-prandial hyperaemia is complex and involves gastrointestinal activity,
tissue oxygenation levels, the enteric nervous system, gastrointestinal
peptides and paracrine substances as well as sympathetic neuronal activity
(Chou and Coatney, 1994;
Vanner and Surprenant,
1996
).
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Acknowledgments |
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