J. exp. Biol. (198a), 99, 255-*68
255
With 4 figures
mPrinted in Great Britain
DIFFERENCES BETWEEN DIRECTLY MEASURED AND
CALCULATED VALUES FOR CARDIAC OUTPUT IN THE
DOGFISH: A CRITICISM OF THE FICK METHOD
BY J. D. METCALFE AND P. J. BUTLER
Department of Zoology and Comparative Physiology\
University of Birmingham, P.O. Box 363, Birmingham
(Received 20 November 1981 - Accepted 3 March 1982)
SUMMARY
Cardiac output has been measured directly, and calculated by the Fick
method, during normoxia and hypoxia in six artificially perfused dogfish
(Scyliorhinus canicula) in an attempt to estimate the accuracy of this method
in fish. The construction and operation of a simple extra-corporeal cardiac
bypass pump is described. This pump closely mimics the flow pulse profiles
of the fish's own heart and allows complete control of both cardiac stroke
volume and systolic and diastolic periods.
During normoxia [Po% = 21 kPa) there was no significant difference
between directly measured and calculated values for cardiac output. However, some shunting of blood past the respiratory surface of the gills may
have been obscured by cutaneous oxygen uptake. In response to hypoxia
(POi = 8-6 kPa) there is either a decrease in the amount of blood being
shunted past the respiratory surface of the gills and/or an increase in cutaneous oxygen uptake such that the Fick calculated value for cardiac output
is on average 38% greater than the measured, value. It is proposed that the
increase in the levels of circulating catecholamines that is reported to occur
in response to hypoxia in this species may play an important role in the
observed response to hypoxia. The results are discussed in terms of their
implications for the calculation of cardiac output by the Fick principle in
fish.
INTRODUCTION
In studies of respiratory function and blood flow in fish, cardiac output is frequently
calculated by use of the Fick principle (Randall, Holeton & Stevens, 1967; Butler &
Taylor, 1975; Kiceniuk & Jones, 1977; Short, Taylor & Butler, 1979), where cardiac
output equals oxygen consumption (A^o,) divided by the difference between the oxygen
content of venous blood entering the gills, and the oxygen content of arterial blood
leaving the gills. However, the use of the Fick principle can only be an accurate
method for estimating the rate of blood flow from the heart if it is assumed that all
the oxygen consumed by the fish is taken up across the gills and that all cardiac output
flows across the gills and enters the systemic circulation via the dorsal aorta. This
method for calculating cardiac output in fish has been borrowed from similar studies
9-2
256
J. D. METCALFE AND P. J. BUTLER
conducted on mammals in which the analogous assumptions are valid, but in fish thg
situation is more complex.
^
The importance of cutaneous gas exchange in many amphibians (Whitford &
Hutchinson, 1965; Piiper & Gatz, 1974) and some reptiles (Belkin, 1968; Graham,
1974) has been appreciated for some time. However, in many studies of gas exchange
in fish, the contribution that cutaneous oxygen uptake may make to the total oxygen
consumption appears to be given little consideration. In eels at 11 °C, cutaneous
oxygen uptake is reported to account for 35 % of the total oxygen consumed, while
in trout the proportion is 13% (Kirsch & Nonnotte, 1977). In the antarctic ice fish
Chaenocephahis aceratus cutaneous oxygen uptake has been estimated to contribute
as much as 40% to the total oxygen consumption (Hemmingsen & Douglas, 1970).
In a recent study, cutaneous oxygen uptake in the dogfish Scyliorhinus canicula
was reported to contribute about 20% to the total oxygen consumption at 13 °C
(Nonnotte & Kirsch, 1978). Both cutaneous oxygen uptake and oxygen consumption
by the gill tissue (Johansen & Pettersson, 1981) will result in the total oxygen consumption being greater than the amount of oxygen being taken up by the gills and
transferred to the blood, and consequently the calculated value for cardiac output will
be larger than the actual cardiac output.
Recent studies of the gill vascular anatomy in many species of fish have revealed a
complex vascular architecture, with both 'respiratory' and 'non-respiratory' blood
pathways (Steen & Kruysse, 1964; Richards & Fromm, 1969; Vogel, Vogel &
Kremers, 1973; Laurent & Dunel, 1976; Vogel, Vogel & Pfautsch, 1976; Dunel &
Laurent, 1977, 1980; Cooke, 1980; Cooke & Campbell, 1980; Olson & Kent, 1980:
J. D. Metcalfe & P. J. Butler, in preparation) and in many species it appears that
some of the blood entering the gills from the heart may be diverted away from the
gas exchange surface of the gills and return directly to the heart via the venous
circulation of the gills and head. Any diversion of cardiac output within the gills in
this manner will cause the calculated value for cardiac output to be smaller than the
actual value.
In a recent study of the eel, Hughes et al. (1981) report that as much as 30% of the
total cardiac output is shunted away from the respiratory surface of the gills and is
not involved in gas exchange, this blood presumably returns directly to the heart via
the venous circulation. In addition, these authors report that adrenaline (2 fig kg-1)
reduced the proportion of cardiac output that is shunted away from the respiratory
surface to about 6 % indicating a greater blood flow to the gas exchange surface, and
also reducing the difference between the calculated and actual values for cardiac
output.
In the present study, the Fick method for the calculation of cardiac output in the
dogfish has been evaluated by comparing simultaneously calculated and directly
measured values. Hypoxia causes an increase in the levels of circulating adrenaline
and noradrenaline in Scyliorhinus canicula (Butler et al. 1978; Butler, Taylor &
Davison, 1979) and since these hormones may affect the proportion of cardiac output
which enters the systemic circulation, the comparison has been made both during
normoxia and hypoxia.
In the dogfish, the anatomical arrangement of the afferent branchial arteries with
Cardiac output in dogfish
257
.respect to the heart, render it impossible to measure total cardiac output directly
'with a cannulating electromagnetic flow measuring device, without entering the
pericardium. The latter procedure is undesirable since the integrity of the rigid
pericardium in elasmobranchs is reported to be essential for normal cardiac function
(Hanson, 1967). For these reasons, circulation has been maintained in the dogfish in
the present study by means of an extra-corporeal cardiac bypass which has been
developed for this purpose.
A number of cardio-respiratory studies in intact fish have employed an extra
corporeal cardiac bypass (Saunders & Sutterlin, 1971; Kent & Pierce, 1978; Opdyke,
Holcombe & Wild, 1979; Hughes et al. 1981). Any such system should mimic as
closely as possible the flow pulse profiles of the fish's own heart (cf. Bacon et al. 1976)
and should allow independent control of stroke volume, and systolic and diastolic
periods during operation as well as maintaining the blood at the experimental temperature. These criteria should be fulfilled with the minimum of cellular damage to
the blood. The construction of such an extra-corporeal cardiac bypass is described in
in the present report.
MATERIALS AND METHODS
Construction of an extra-corporeal cardiac bypass
The blood pump (Fig. 1) is a modification of the design by Daly, Ead & Scott
(1978). Pulsatile flow is induced by the sinusoidal compression of a portion of an
18 cm loop of silicon rubber tube (I.D. 6 mm, O.D. 9 nuns Portex Ltd.) by a triangular
compression plate which is moved, via a Perspex drive shaft, by an off-centre drive
cam mounted on the shaft of a high torque (3-5 lb in) electric motor (Klaxon Ltd.).
The power to the motor is supplied from the control unit of a commercial peristaltic
pump (Watson-Marlow, MHRE 22). Silicon rubber tube has a high coefficient of
restitution and good biocompatability (M. de B. Daly, personal communication) and
was used for these reasons. Commercial plate valves (Tudor Accessories Ltd.) at
both ends of the silicon rubber tube maintain a unidirectional flow. In each valve the
plate is loaded by a small stainless-steel spring which effectively prevents any back
flow due to its short response time in closing. The valves are retained in a Perspex
valve block which is mounted on a Perspex anvil such that the loop of silicon rubber
tube lies across the upper surface of the anvil which provides support during compression. The anvil, with the valve/tube assembly, is mounted within the Perspex water
bath of the pump on a 27 cm length of o BA stainless steel studding connected to a
handle mounted on the outside of the water bath. Rotation of this handle allows the
anvil and the valve/tube assembly to be moved along a track on the base of the water
bath and so the length of the silicon rubber tube under the compression plate can be
varied. In this way stroke volume of the pump may be altered independently of any
other variable during operation. The drive shaft is mounted on the side of the water
bath by two Perspex yokes which are pivoted at either end on brass/steel bearings.
A round Perspex cap is mounted on the top of the drive shaft on a 1 cm long o. BA
thread. Rotation of this cap allows alteration of the length of the drive shaft, and thus
the proportion of the drive cam which makes contact with the drive shaft can be
controlled. Wear on the cap of the drive shaft by rotation of the cam is reduced by a
J. D. METCALFE AND P. J. BUTLER
Fig. i. The e^tra-corporeal cardiac bypass pump: (A) Front view. (B) Top view of anvil and
valve block assembly, showing position of compression plate. (C) Side view. A, Anvil; BT,
bubble trap; C, cap; CP, compression plate; DC, drive cam; DS, drive shaft; H, handle; MS,
micro switch; M, motor; RB, roller bearing; ST, silicon rubber tube; S, stainless steel
studding; T, track; TC, tripping cam; V, valve; VB, valve block; WB, water bath; Y, yokes.
brass roller-bearing, mounted in a Perspex yoke, situated between the cam and the
cap, which is also mounted on the side of the water bath.
Systolic and diastolic periods are independently controlled by means of a microswitch which is activated by a tripping cam mounted on the shaft of the motor behind
the drive cam. The micro-switch and tripping cam are set so that the switch is open
during the down stroke of the drive shaft (systolic period) but closed at all other times
(diastolic period). In this way the power supply to the motor is switched between two
Cardiac output in dogfish
259
o turn potentiometers which control the motor speed. This allows the motor speed,
nd thus the period during systole and diastole, to be controlled independently of
each other during operation of the pump.
Control of the pump in this way allows stroke volume to be continuously variable
between o and about 4-0 ml. Systolic and diastolic periods are continuously variable
between 0-2 and 30 s giving an overall pulse frequency range of 10-150 min"1. The
values of these variables span well outside those values measured in intact dogfish
of the size range used in the present study (c.f. Short et al, 1979).
Blood flowing to and from the pump passes through small glass bubble traps (Fig. 1)
which contain a small, variable, volume of air (about 3-5 ml), which acts as a ' Windkessel'. On the inflow side of the pump, the 'Windkessel' effect of the bubble trap
reduces the violence of the diastolic filling which was found to be important in
maintaining adequate venous return to the pump. On the outflow side of the pump,
the ' Windkessel' effect of the bubble trap damps out the high-frequency oscillations
observed inflowpulses at very low frequencies (less than 15 min"1) which appear to be
due to valve 'flutter*. These damping chambers do not affect the overall pulse duration. The temperature of the bloodflowingthrough the pump is controlled by passing
water at the desired temperature through the water bath surrounding the valve/tube
assembly.
R
Preparation of animals
In the present study cardiac output has been simultaneously calculated via the
Fick principle, and measured directly, during normoxia and induced environmental
hypoxia in six lesser spotted dogfish (Scyliorhinus canicula) of either sex, the mass of
which ranged from 0-603 t o 0-897 kg. These were obtained from the Plymouth
Laboratories of the Marine Biological Association of the U.K., and transported in
oxygenated sea water to the aquaria in Birmingham where they were held in aerated,
recirculating sea water maintained at 15 ± 1 °C for at least two weeks prior to any
experiments. Fish were fed periodically on either whitebait or sprats obtained from a
local fishmonger. All experiments were performed at the above temperature, and
each fish was starved for 4 days prior to any experiment. Having been washed with
strongly heparinized (2000 units i.u. ml"1 sodium heparin, Weddel) elasmobranch
Ringer's solution (Capra & Satchell, 1977 a), the perfusion circuit (the pump and
associated cannulae) was primed with 30-40 ml of heparinized (20 units i.u. sodium
heparin ml"1, Weddel) dogfish blood obtained from a donor fish. This blood recirculated around the perfusion circuit prior to the cannulation of the experimental
fish.
The experimental fish was anaesthetized in sea water containing 0-04 g I"1 tricaine
methanesulfonate (MS 222, Sigma Chemical Co.) and placed ventral side up on an
operating table in a constant temperature room maintained at 15 °C. The gills were
irrigated with recirculating, filtered, aerated sea water containing anaesthetic (as
above). The caudal artery was exposed via a 15 mm longitudinal, ventral incision
just posterior to the anal fin, and cannulated with a 40 cm length of polythene tubing
(o.D. 1 -o mm, Portex) filled with heparinized elasmobranch Ringer's. This cannula
allowed the measurement of dorsal aortic blood pressure and cardiac pump rate, and
260
J. D. METCALFE AND P. J. BUTLER
the sampling of post branchial blood for the measurement of arterial blood oxygeM
contents and tensions. The caudal vein wa9 plugged at this point with a i cm lengtfl
of heat-sealed polythene tubing (as above). The wound was sutured closed and
sealed with a patch of household rubber glove attached with cyanoacrylate adhesive
(R.S. Components Ltd.) to prevent the loss of blood. Heparin (2000 units i.u. sodium
heparin kg"1, as above) was injected via this cannula into the fish's blood stream and
allowed to circulate for 5-10 min before continuing the surgical procedure.
The pericardium was exposed and opened via a 20 mm ventral incision. The
junction between the ventricle and the conus arteriosus was ligated to prevent blood
loss and the ventral aorta was cannulated via the conus arteriosus with a 100 cm length
of blood-filled PVC tubing (o.D. 2-8 mm, Portex) which was connected to the outflow
from the pump via one of the bubble traps. This bubble trap was fitted with a short
cannula to allow sampling of venous blood leaving the pump (Fig. 2). Care was taken
to prevent any air from entering the circulation during this part of the procedure.
Once this cannula had been tied into place, the sinus venosus was cannulated with a
similar length of PVC tubing which was connected to the inflow to the pump via the
second bubble trap. The cannula placed in the sinus venosus was tipped with a small
perforated cage (7 x 2-8 mm O.D., curved through 450) manufactured from ' Araldite',
the tip of which lay in the opening of one of the Cuverian ducts. This cage prevented
the collapse of the sinus venosus and allowed venous blood to be drawn into the
pump under negative pressure. During the cardiac bypass procedure, blood flow was
arrested for about 15-20 min. However, this did not appear adversely to affect the
fish. Once the cannulae had been tied into place, perfusion was commenced at a low
pulse rate (about 8 beats min"1) with a stroke volume of i-o ml kg" 1 (Short et al.
1979). The pericardio-peritoneal canal was plugged with a small glass plug (10x4 mm
diam.) to prevent any blood loss subsequently leaking into the peritoneum from the
pericardium. The wound was sutured closed and sealed as previously described.
The fish was placed in a Perspex respirometer (capacity 5 1) and restrained by a
clamp at the base of its tail. The cannulae passed through bungs sealed into the lid
of the respirometer. Once sealed, the respirometer was placed in a black Perspex
experimental tank containing about 40 1 filtered, aerated, recirculating sea water at
15 ± 1 °C which covered the respirometer. The experimental tank was covered with
polystyrene foam so that the fish received the minimum of visual disturbance. The
respirometer was supplied with sea water drawn from the experimental tank via a
pair of gas exchange columns placed in series, at a flow rate of approximately 0-75
1 min"1, measured with aflowmeter (Rotameter 1100 sea water: GEC-Elliot) (Fig. 2).
The paired gas exchange columns allowed accurate control of the oxygen tension of
the water flowing into the respirometer during hypoxia independently of the oxygen
tension of the water being drawn from the experimental tank.
The dorsal aortic cannula was connected to a pressure transducer (s.E. Labs:
S.E.M. 4-86) for the measurement of dorsal aortic blood pressure, and its output was
displayed on a four-channel rectilinear pen recorder (Ormed Ltd.). Blood flow from
the cardiac pump was measured with a cannulating electromagnetic flow probe
(Biotronex: 1-5 mm I.D.) placed in the ventral aortic cannula. The flow probe was
connected to a Biotronex pulsed logic flow meter (BL-610) and the output displayed
Cardiac output in dogfish
261
Au>=
Fig. 2. The experimental arrangement used in obtaining the physiological measurements
from dogfish artificially perfused with blood by the extra-corporeal cardiac bypass pump.
D, Dogfish; A, dorsal aortic cannula; Q, electromagnetic flow meter; E, experimental tank;
P, extra-corporeal pump; F, filter; G, gas exchange columns; Ptno.o,. inflow to respirometer;
P« 0i> outlet from respirometer; H> , direction of sea water flow; • , direction of blood
flow; R, respirometer; T, sea water flow meter; V, venous canula; W, water bath.
on the pen recorder (as above). The flow probe was calibrated with the fish's own
blood at the end of each experiment. Oxygen tensions of blood and water samples
were measured with a blood gas analyser (Radiometer PHM 71) having its oxygen
electrode housed in a glass cuvette maintained at the experimental temperature. The
oxygen content of blood samples was measured with a Lex-08-Con total oxygen
analyser (Lexington Instruments) which was calibrated with oxygen-saturated,
distilled water at o °C so as to be accurate over the range of low oxygen contents
encountered in blood samples (< 2-3 m mol I"1). Haematocrit was measured with a
microhaematocrit centrifuge (Hawksley) from venous blood samples.
Once the fish had been placed in the experimental tank, the cardiac pump rate was
gradually raised to about 30 beats min"1 (Short et al. 1979) over a period of about
S min. In a number of preliminary experiments it was found that adequate venous
return to the pump could only be maintained by increasing the blood volume of the
fish by about 25 ml kg"1. Consequently all fish received extra blood from a donor
fish in approximately this proportion.
Fish were allowed 5 h to recover from the anaesthetic in normoxic water (POf =
about 21 kPa). At the end of the period measurements of normoxic cardiac pump
rate and stroke volume were recorded over a 3 min period and directly measured
cardiac output (J^6 mea) was calculated as stroke volume x cardiac pump rate. Mean
dorsal aortic blood pressure was calculated as diastolic blood pressure-!-J (systolic-
262
J. D . METCALFE AND P. J. BUTLER
diastolic blood pressure). The oxygen tensions of water incurrent (PiDCiOt)
excurrent (Pex.o,) t 0 r n e respirometer, and respirometer water flow, were measured
and total oxygen consumption (MOt) calculated by the method described by Short
et al. (1979). Blood samples of between 0-5-07 ml were drawn into glass syringes
from the dorsal aorta (arterial blood) and from the bubble trap on the outflow side
of the cardiac pump (venous blood). The oxygen contents (C^ O j and Cv Oi) and oxygen
tensions (P o>0 , and Pr>0J of these samples were measured and cardiac output was
calculated via the Fick principle {X?b cal.) by the method described by Short et al.
(1979)The oxygen tension of the water incurrent to the respirometer was then reduced
to about 8-6 kPa over a period of 30 min by passing nitrogen at an appropriate rate
through the second of the two gas exchange columns. This oxygen tension was
maintained for the rest of the experimental period. As the water oxygen tension was
reduced, the reflex bradycardia observed in intact dogfish in response to environmental
hypoxia (Butler & Taylor, 1975; Short et al. 1979) was simulated with the cardiac
pump; the pulse rate was halved and the stroke volume doubled thus maintaining
cardiac output at the normoxic value. Tests showed that at the water flow rates used
(0-75 1 min"1) the oxygen tension across the respirometer equilibrated after about
20-30 min. Accordingly animals were allowed 1 h to adjust to the reduced oxygen
tension and the above variables were again measured and cardiac output was calculated
and measured directly.
In four experiments the oxygen consumption of any organism living upon the
skin of the fish was estimated. At the end of the experiment the fish was killed in situ
by injecting either sodium cyanide or sodium azide (2 mg kg-1) into the blood stream
of the fish. This was allowed to circulate for a few min and then the cardiac pump was
turned off. One h after death, total oxygen consumption in normoxic water was
measured as previously described. After such metabolic poisoning, there was no
detectable oxygen consumption. It appears that any organisms that live on the skin
of the dogfish make no measurable contribution to the calculated value for total
oxygen consumption.
In the present report, all mean values are expressed ± S.E.M. The difference between
means has been compared by using the paired Mest (Bailey, 1959). The term 'significant' refers to the 95 % level of confidence (P < 0-05), unless otherwise stated.
RESULTS
The operation of the extra-corporeal cardiac bypass as reported maintained the
viability of the fish for the entire experimental period in all cases, and in one preliminary experiment an individual fish remained alive for 21 h, after which time the
experiment was terminated. Fig. 3 shows that flow pulse profiles in artificially perfused fish were similar to those obtained from intact dogfish (Short, 1976) although
the falling phase of the flow pulse is somewhat longer in perfused fish. Though
some haemolysis of the blood might have been expected due to the operation of the
cardiac pump, none was apparent. In both haematocrit samples, and in blood which
had been allowed to settle overnight after the perfusion experiments, the plasma was
always clear with no obvious red colouration.
263
Cardiac output in dogfish
-
25
~
30r
Fig. 3. Comparison of pulse flow waves from an intact dogfish (a) (from Short, 1976) and from a
dogfish perfused with blood using the extra-corporeal cardiac bypass pump (6). The time
marker (t) indicates one second intervals.
Table 1. The mean ( ± s.E. of mean) values of the measured variables in unanaesthetised
dogfish artificially perfused with blood, at rest during normoxia and hypoxia
Normoxia
Mass (kg)
J V o , (kPa)
•Po.oj (tPa)
C o O , (mmol.l- 1 )
P,oj (kPa)
C^>, (mmol.l- 1 )
Pump rate (strokes min" 1 )
Mean dorsal aortic blood pressure (kPa)
Haematocrit (%)
Pt mea. (ml min" 1 kg"1)
l?i cal. (ml. min" 1 kg"1)
JVifo» Oanol min" 1 kg"1)
Pt cal./P'j mea.
N, Number of observations,
,
0-71910-041
20-781008
14-54 ±°-59
1-8710-89
3-6210-28
0-7110-23
29-3! 1'o
3-47 ±0-23
i5-Slo-8
29-i4±i-9°
26-7112-30
27-2810-94
0-9210-08
Hypoxia
—
8-55 l o - 4 i #
4-i9lo-53*
1-0310-09*
1-7710-13*
o-33 io-09*
i4-3l°'55 #
2-99lo-2t*
i43±°-8
25-8912-30
35-9Ol4-i2t
22-3911-91
I-381O-I9*
(6)
(6)
(6)
(6)
(6)
(6)
(6)
(6)
(6)
(6)
(6)
(6)
(6)
,
• Significant differences in response to hypoxia at the 95 % of confidence (P < 0-05). ,
f Significant differences between P^ cal and Pt mea. at the 90% level of confidence (P < o-i).
Values for cardiac output measured directly and calculated by the Fick technique
During Normoxia and Hypoxia
The mean ± S.E.M. of the mean of the variables measured in intact, artifically
perfused dogfish during both normoxia and hypoxia are presented in Table 1. The
individual and mean differences between Vb cal. and Vb mea. (presented as the
ratio between these two parameters) during normoxia and in response to hypoxia for
the 6 fish are illustrated in Fig. 4.
In the present study during normoxia, the values for arterial and venous blood
oxygen contents and tensions, and dorsal aortic blood pressure in artifically perfused
dogfish are similar to those reported for intact dogfish at 15 °C (Short et al. 1979),
264
J. D. METCALFE AND P. J. BUTLER
2-2
2-0
1-8
1-6-
8
1-210-8-
I
0-60-40-2-
Fig. 4. A graphical representation of the ratio between calculated cardiac output (Pb cal.) and
directly measured cardiac output (J^4 mea.) in six artificially perfused dogfish during normoxia (#) and in response to hypoxia (A). Mean values, ±8.B.M. are also shown.
although the present value for oxygen consumption is about 75% of the value reported
by these authors. This appears to confirm the visual observation that dogfish tolerate
artificial perfusion well, at least over the period of the present experiments (about 9 h).
In normoxic animals the calculated value for Vrb is on average 8% lower than the
directly measured value, however, this difference is not significant. Vb csX/Vb mea.
values during normoxia cover a broad span from 0-69 to 1 -24 (Fig. 4).
In response to hypoxia both aterial and venous blood oxygen contents and tensions
decreased significantly and this was accompanied by an 18% decrease in oxygen
consumption, however, this decrease was not significant. There was a small reduction
in directly measured Vb following adjustment of the cardiac pump during hypoxia,
however this change was not significant. The changes in the measured variables in
response to hypoxia (POt = 8-6 kPa) in the present study are similar to those reported
for this species by Short et al. (1979). in response to a similar reduction in environmental oxygen tension (POi = 102 kPa).
In contrast to normoxic fish, calculated t>rb is much (38%) higher than measured
Vb during hypoxia. This difference is significant at the 90% level (P < o-i). P 6 cal./
Vb mea. values during hypoxia ranged from o-88 to 2-09 (Fig. 4). Since blood flow
cannot be greater than that actually measured, this difference may indicate that a
Cardiac output in dogfish
265
large proportion of the total oxygen consumption is being taken up across the skin.
In response to hypoxia the I^6 cal./J^6 mea. ratio increased in all six fish, the mean
value increasing from 0-92 during normoxia to 1-38 in response to hypoxia, and this
increase is significant at the 95% level of confidence (P < 0-05). This indicates a
marked increase in observed cutaneous oxygen uptake during hypoxia (Fig. 4).
DISCUSSION
In the present study dogfish survived artificial perfusion well for periods of at least
9 h. Mean dorsal aortic blood pressure, and arterial and venous blood oxygen tensions
in perfused fish were similar to those previously reported for this species (Short et al.
1979). This would indicate that normal gas exchange is maintained in the present
study. Although a number of studies have reported artificial perfusion to be successful
in a number of fish species (see Introduction for references), few appear to have
succeeded in maintaining viability for as long as 9 h. This may be related to the
ability of the extra-corporeal cardiac bypass reported in the present study to mimic
closely the flow characteristics of the fish's own heart.
During normoxia there was no significant difference between Vh cal. and Vb mea.
which indicates that there was no shunting of blood past the respiratory surface of
the gills despite the fact that such a shunt, via the vascular network of the interbranchial septum, has been shown to be anatomically possible in S. canicula (J. D.
Metcalfe & P. J. Butler in preparation). However, the present study only reveals the
minimum value for the shunting of blood past the respiratory surface during normoxia. If, in normoxic fish, some part of the total oxygen consumed is being taken
up across the skin, then the actual proportion of cardiac output that is being shunted
past the respiratory surface of the gills will be larger than that estimated in the
present study since, by the method of calculating X?b by the Fick technique, cutaneous
oxygen uptake and the shunting of blood past the gas exchange surface of the gills
mutually obscure each other. If the estimate of 20% for cutaneous oxygen uptake in
this species made by Nonnotte & Kirsch (1978) is accurate, then the real proportion
of cardiac output that is shunted past the gas exchange surface may be as high as 28 %.
However, this value of 20% for cutaneous oxygen uptake made by Nonnotte &
Kirsch (1978) may be unusually high due to their experimental method which would
have destroyed the stable boundary layers of water next to the skin of the fish, causing
oxygen uptake to increase.
The fact that normoxic oxygen consumption in the present study is lower than that
previously reported for intact dogfish at 15 °C (Short et al. 1979), despite the fact
that the arterio-venous oxygen content difference is similar, would suggest that
cardiac output in the present study was lower than that measured by Short et al.
(1979). However, the measured value for Vb in the present study is similar to that
calculated by the Fick technique by these authors. This indicates that the actual blood
flow across the gas exchange surface of the gills in the present study may have been
lower than in the earlier study.
In response to hypoxia oxygen consumption decreased by 18%. Although this
reduction was not significant in the present study, similar but significant reductions
266
J. D. METCALFE AND P. J. BUTLER
in oxygen consumption have previously been reported in response to hypoxia both
in this species (Butler & Taylor, 1975; Taylor, Short & Butler, 1977; Short et al.
1979) and other elasmobranchs (Piiper, Baumgarten & Meyer, 1970) at environmental
temperatures above 7 °C. During hypoxia cutaneous oxygen uptake appears to
increase and accounts for almost 40 % of the total oxygen consumed. This dramatic
increase in observed cutaneous oxygen uptake during hypoxia may be the result of
either haemodynamic changes within the gills which reduce any blood shunt, thereby
revealing the actual value for cutaneous oxygen uptake, or of a real increase in cutaneous oxygen uptake, or probably some combination of these two processes.
How such changes in the regional distribution of branchial blood flow and oxygen
transfer across the skin are brought about in response to hypoxia are as yet unclear.
However, they may be related to the increase in the levels of circulating catecholamines adrenaline and noradrenaline which have previously been reported to occur
in response to hypoxia in S. canicula (Butler et al. 1978, 1979). These hormones have
repeatedly been reported to enhance blood flow across the respiratory surface of the
gills of both teleost and elasmobranch fish (Rankin & Maetz, 1971; Randall, Baumgarten & Malyuse, 1972; Bergman, Olson & Fromm, 1974; Girard & Payan, 1976;
Dunel & Laurent, 1977; Hughes et al. 1980). The possible role of these humoral
agents may be of particular importance in the dogfish since this species appears to
lack any direct neural control of its gill blood vessels (J. D. Metcalfe & P. J. Butler, in
preparation). Noradrenaline has also been reported to cause vasodilatation of the
skin blood vessels in elasmobranchs (Capra & Stachell, 19776) and this may serve
to enhance oxygen transfer across the skin during hypoxia.
The present study does not reveal the absolute values for either the proportion of
cardiac output that bypasses the gas exchange surface, or for the proportion of the
total oxygen consumption which is taken up across the skin in 5. canicula. However,
it does demonstrate that the calculation of any variable by the Fick technique which
assumes that all oxygen is taken up across the gills, and that all cardiac output reaches
the dorsal aorta, may not only be inaccurate, but may also not reveal the real changes
in these variables that occur in response to hypoxia.
Financial support was provided by the Science and Engineering Research Council.
REFERENCES
BACON, D . R., DALY, C. DE B., DALY, M. DE B. & SCOTT, R. W. (1976). Modified roller pump with
improved haemodynamic characteristics and temperature control of perfusate. Laboratory Practice
25, 464-466.
BAILEY, N. T. J. (1959). Statistical Method* in Biology. English Universities Press. London.
BELKIN, D . A. (1968). Aquatic respiration and underwater survival of fresh water turtle species.
Respir. Physiol. 4, 1-14.
BERGMAN, H. L., OLSEN, K. R. & FROMM, P. O. (1974). The effects of vasoactive agents on the
functional surface area of the isolated perfused gills of the rainbow trout. J. comp. Pkytiol. 94, 267-286.
BUTLER, P. J. & TAYLOR, E. W. (1975). The effects of progressive hypoxia on respiration in the dogfish
(ScyliorUnui canicula L.) at different seasonal temperatures. J. exp. Biol. 63, 117-130.
BUTLER, P. J., TAYLOR, E. W., CAPRA, M. & DAVISON, W. (1978). The effect of hypoxia on the levels
of circulating catecholamines in the dogfish Scyliorkimu canicula L. J. comp. Phytiol. B IV], 3as~33 o BUTLER, P. J., TAYLOR, E. W. & DAVISON, W. (1979). The effect of long term, moderate hypoxia on
Cardiac output in dogfish
267
acid-base balance, plasma catecholamine and anaerobic end products in the unrestrained dogfish
ScyliorMmu camcula, J. comp. Phytiol. B 133, 297-303.
CAPRA, M. F. & SATCHELL, G. H. (1977 a). Adrenergic and cholinergic responses of the isolated salineperfused heart of the elasmobranch fish Squalus tucklyei. Comp. biochem. Phytiol. 8, 56-65.
CAPRA, M. F. & SATCHELL, G. H. (19776). The differential haemodyanic response of the elaimobranch,
Squalua acanthias to the naturally occurring catecholamines adrenaline and noradrenaline. Comp.
Biochem. Phytiol. 58C, 41-48.
C00KE, I. R. C. (1980). Functional aspects of the morphology and vascular anatomy of the gills of the
endeavour dogfish, Centrophorus tcalpratut (McCulloch) (Elasmobranchii: Squalidae). Zoomorphologie
94, 167-183.
COOKK, I. R. C. & CAMPBELL, G. (1980). The vascular anatomy of the gills of the smooth toadfish,
Torqiagmer glaber (Teleostii: Tetraodontidae). Zoomorphologie94, 151-166.
DALY, M. DE B., EAD, H. W. & SCOTT, R. W. (1978). A small pulsatile perfuaion pump-blood equilibrator system. J. Phytiol., Lond. a8o, 6-8p.
DUNEL, S. & LAURENT, P. (1977). La vascukriaation branchiale chea l'Anguille: action de l'ac^tylcholine et de l'adrenaline BUT la repartition d'une resine polymerisable dans les differents compartiments vasculaires. C.r. hebd. Seanc. Acad. Sri. Parit, 284, 2011-3014.
DUNBL, S. & LAURENT, P. (1980). Functional organisation of the gill vasculature in different classes of
fish. In epithelial Transport in the Lower Vertebrates (ed. B. Lahlou), pp. 37-58. Cambridge: Cambridge University Press.
GIRARD, J. P. & PAYAN, P. (1976). Effect of epinephrine on vascular space of gills and head of rainbow
trout. Am. J. Phytiol. 330, 1555-1560.
GRAHAM, J. B. (1974). Aquatic respiration in the sea snake PelamU platurus. Retpir. Phytiol. a i , 1-7.
HANSON, D. (1067). Cardiovascular dynamics and aspects of gas exchange in Chondrichthyes, Ph.D.
Dissertation, University of Washington, Seattle, Washington (quoted by Satchel], G. H.) in Circulation in Fithet (1971), Cambridge University Press, Cambridge).
HEMMINGSEN, E. A. & DOUGLAS, E. L. (1970). Respiratory characteristics of the haemoglobin-free
fish Chaenocephalut aceratut. Comp. Biochem. Phytiol. 3a, 733-744.
HUGHES, G. M., PBYRAUD, C ,
PEYRAUD-WAITZENEGGER, M. & SOULIHR, P. (1981). Proportion
of
cardiac output concerned with gas exchange in the gills of the eel (A. onguxlla). J. Phytiol., Lond. 310,
61-teP.
JOHANSEN, K. & PETTERSSON, K. (1981). Gill O, consumption in a teleost fish (Gadus morhua). Retpir.
Physiol. 44, 277-384.
KENT, B. & PIERCE, E. C. (1978). Cardiovascular responses to changes in blood gases in dogfish
shark, Squalus acanthiat. Comp. Biochem. Phytiol. 60C, 37-44.
KICENIUK, J. W. & JONES, D. R. (1977). The oxygen transport system in trout (Salmo gatrdneri) during
sustained exercise. J. exp. Biol. 69, 247-260.
KIRSCH, R. & NONNOTTE, G. (1977). Cutaneous respiration in three fresh water teleosts. Resp. Physiol.
*9, 339-354.
LAURENT, P. & DUNEL, S. (1976). Functional organisation of the teleost gill. I; Blood pathways.
Acta Zool. 57, 189-209.
NONNOTTE, G. & KIRSCH, R. (1978). La respiration cutanee chez un selacian (Scyliorhinus canicula).
C.r. hebd. Acad. Sci. Seanc. Paris. 286, 1597-1599.
OLSON, K. R. & KENT, B. (1980). The microvasculature of the elasmobranch gill. Cell Tistue. 309,
49-63.
OPDYKE, D. F., HOLCOMBE, R. & WILD, D. W. (1979). Blood flow resistance in Squalus acanMas.
Comp. Biochem. Physiol. 6aA, 711-717.
PIIPER, J., BAUMOARTEN, D. & MEYER, M. (1970). Effects of hyporia upon respiration and circulation
in the dogfish (Scyliorhinus ttellaris). Comp. Biochem. Physiol. 36, 513-520.
PIIPER, J. & GATZ, R. N. (1974). Exploitation des mechanismes des echanges gazeux chex une salamandre ne possedant ni poumons, ni branchies. J. Physiol., Paris 69, 287 A.
RANDALL, D. J., HOLETON, G. F. & STEVENS, E. D. (1967). The exchange of oxygen and carbon-
dioxide across the gills of the rainbow trout. J. exp. Biol. 46, 339-348.
RANDALL, D. J., BAUMGARTEN, D. & MAYLUSE, M. (1972). The relationship between gas and ion
transfer across the gills of fish. Comp. Biochem. Physiol. 41 A, 629-637.
RANKIN, J. C. & MAETZ, J. (1971). A perfused teleostean gill preparation: vascular actions of neurohypophysial hormones and catecholamines. J. Endocr. 51, 621-635.
RICHARDS, B. D. & FROMM, P. O. (1969). Patterns of blood flow through filaments and lamellae of
isolated-perfused rainbow trout gills. Comp. Biochem. Physiol. 39, 1063-1070.
SAUNDERS, R. L. & SUTTERLIN, A. M. (1971). Cardiac and respiratory responses to hypoxia in the
sea raven, Hemitripterus americamu, and an investigation of possible control mechanisms. J. Fish. Res.
Bd. Can. 38, 491-503.
268
J. D. METCALFE AND P. J. BUTLER
SHORT, S. (1976). Some aspects of cardioregulation in the dogfish (Scyliorhinus canicula). Ph.D. thesis.
University of Birmingham, U.K.
SHORT, S., TAYLOR, E. W. & BUTLER, P. J. (1979). The effectiveness of oxygen transfer during normoxia and hypoxia in the dogfish (Scyliorhinut canicula) before and after cardiac vagotomy. J. comp.
Phyriol. B 13a, 289-295.
STEEN, J. B. & KRUYSSE, A. (1964). The respiratory function of the teleostean gills. Comp. Biochem.
Pkytiol. 13, 127-142.
TAYLOR, E. W., SHORT, S. & BUTLER, P. J. (1977). The role of the cardiac vagus in the response of the
dogfish Scyliorhimu canicula to hypoxia. J. exp. Biol. 70, 57-75.
VOGEL, W., VOGEL, V. & KREMERS, H. (1973). New aspects of the intrafilamental vascular system in the
gills of an euryhaline teleost Tilapia mostambica. Z. Zellforsch. 144, 573-583.
VOGEL, W., VOGEL, V. & PFAUTSCH, M. (1976). Arterio-venous anastomoses in rainbow trout gill
filaments. Cell Tissue Res. 167, 373-385.
WHITFORD, W. G. & HUTCHISON, V. H. (1965). Gas exchange in salamanders. Physiol. Zool. 38, 228-242