J. exp. Biol. (1978), 73, U9-I29
With 3 figures
Printed in Great Britain
THE DISTRIBUTION OF BLOOD FLOW IN THE GILLS OF
FISH: APPLICATION OF A NEW TECHNIQUE TO
RAINBOW TROUT (SALMO GAIRDNERI)
BY JOHN H. BOOTH
Department of Zoology, University of Toronto, Toronto,
Ontario, Canada M$S 1A1
(Received 28 June 1977)
SUMMARY
Injecting vitally stained blood cells into the ventral aorta of unrestrained
cannulated fish, and rapid freezing in liquid nitrogen, provided a method of
investigating blood flow patterns in the gills. Rainbow trout in air-saturated
water perfused an average of 58% of the secondary lamellae of the gills.
Perfusion of the filamental central compartment was insignificant indicating
that the effects of any non-respiratory shunting would be unimportant. If
the functional surface area of the gills is variable, it seems likely that this
would be accomplished through lamellar recruitment. There was no evidence
that pillar cell contraction affected lamellar perfusion. There was preferential
perfusion of lamellae near the base of the filaments, and of filaments near the
dorsal end of the gill arches.
INTRODUCTION
A large body of indirect evidence, based primarily on changes in the diffusion
capacity and vascular resistance of the gills, suggests that fish are capable of regulating
the functional capacity of the gills to exchange gases (Keys & Bateman, 1932; Ostlund
& Fange, 1962; Steen & Kruysse, 1964; Randall, Holeton & Stevens, 1967; Wood &
Randall, 1971; Rankin & Maetz, 1971; Motais & Isaia, 1972; Bergman, Olson &
Fromm, 1974; Wood, 1974, 1975). Such regulation might serve to reduce the costs
of osmoregulation during periods of rest, when the fish does not require the full gas
exchange capacity of the gills (Steen & Kruysse, 1964). While gas exchange depends on
several factors, including convection of blood and water, diffusion distance, and permeability, much recent interest has centred on the possibility that fish might be able to
alter the proportion of the total gill surface area which is participating in gas exchange.
Two models have been described to explain how such changes in the functional
surface area might be achieved. The first proposes a non-respiratory shunt permitting
blood to move from the afferent to the efferent filamental artery through a 'central
compartment' in the body of the filament, or through an anastomosis of the filament
arteries at the tip of the filament, instead of through the respiratory pathway across the
secondary lamellae (Steen & Kruysse, 1964; Richards & Fromm, 1969). Recent
anatomical studies (Gannon, Campbell & Randall, 1973; Morgan & Tovell, 1973;
Vogel, Vogel & Kremers, 1973; Fromm, 1974; Laurent & Dunel, 1976; Vogel, Vogel
120
J O H N H. BOOTH
& Pfautsch, 1976; B. J. Gannon, G. Campbell, and D. J. Randall, in preparation) and
a study employing radioactive microbeads 15 /i and 50 /i in diameter (Cameron, 1974)
have failed to demonstrate such a shunt pathway.
The initial report of a non-respiratory shunt (Steen & Kruysse, 1964) was based
primarily on studies of eels, which possess a considerable capacity for cutaneous gas
exchange (Berg & Steen, 1965). Cameron (1974) estimated that in rainbow trout even
a small admixture of venous blood from a non-respiratory shunt would considerably
reduce the P O j of the blood in the dorsal aorta. Direct observations of the dorsal aortic
P o > in the resting rainbow trout have shown that this is not the case (Holeton &
Randall, 19676; Randall et at. 1967; Davis, 1971; Holeton, 1971). A non-respiratory
shunt would not be of obvious advantage to a fish unless further gas exchange could
occur at some other location, such as the skin or an auxiliary breathing organ.
For most fish, the second model, involving the perfusion of varying numbers of
secondary lamellae (Steen & Kruysse, 1974; Randall, 1970; Hughes, 1972), would
seem to be a better strategy for changing the functional surface area of the gills. Such a
system avoids the reduction in dorsal aortic P Oa which is inherent in shunting.
Furthermore, it would result in decreased branchial resistance during periods of high
oxygen demand, reducing the work load on the heart. During periods of low demand,
progressive shutdown of lamellae would reduce the exposure of the blood to unfavourable osmotic conditions or toxic compounds in the water around the fish.
Lamellar recruitment could be regulated by contraction of the pillar cells in the
lamellar blood spaces (Hughes & Grimstone, 1965; Newstead, 1967) or by the
contraction of muscle cells in the afferent and efferent lamellar arterioles (Richards &
Fromm, 1969; Morgan & Tovell, 1973). Apparently contracted pillar cells have been
reported (Hughes & Wright, 1970; Morgan & Tovell, 1973; Hughes & ByczkowskaSmyk, 1974) but these have never been correlated with lamellar perfusion.
The only direct evidence favouring lamellar recruitment has been provided by
Davis (1972). He observed increased bloodflowin the secondary lamellae at the tips of
the gill filaments of live intact rainbow trout following injection of adrenaline. The
resolution of his technique was not fine enough to obtain observations at the level of
the individual secondary lamellae. Other attempts to determine the model which best
describes how the functional surface area is regulated in vivo have lacked validity since
the techniques have involved either isolated gill arches (Steen & Kruysse, 1964;
Richards & Fromm, 1969; Rankin & Maetz, 1971; Bergman et al. 1974) or caused
severe disturbances of the fish through anaesthesia, unnatural positions, or surgery
(Cameron, 1974; Wood, 1974, 1975).
In order to be able to distinguish between 'anatomical' and 'physiological' pathways in the gills, a method is required which will permit observations to be made of
the distribution of blood flow in live undisturbed fish. This report describes a technique in which blood cells marked with a fluorescent vital dye were injected into the
ventral aorta of rainbow trout. Rapid freezing of the gills in liquid nitrogen was used to
stop the blood flow before the effects of handling could be expressed. The stained
cells provided direct evidence of the distribution of blood flow at the moment of
freezing.
Blood flow in gills of rainbow trout
121
MATERIALS AND METHODS
Rainbow trout (Salmogairdneri) weighing between 200 g and 350 g (Franklin Trout
Farm Ltd, Mount Albert, Ontario) were kept indoors in 850 1 tanks of aerated,
dechlorinated water maintained at 10 °C. The fish had rather large amounts of body
fat so they were starved until much of this fat had been metabolized. They were then
fed a maintenance diet of commercial trout pellets.
For experimentation, the ventral aorta was cannulated after the method of Holeton
& Randall (1967 a) using a 23 gauge hypodermic needle attached to 40 cm of PE50
polyethylene tubing. The fish was then placed in an opaque chamber (10 1 capacity) in
recirculated, aerated water maintained at 5 °C, where it was allowed to recover.
After 24 h, a 0-5 ml sample of blood was withdrawn through the cannula and
replaced with heparinized Courtland saline (Wolf, 1963) to maintain blood volume.
The blood sample was added to 5 ml of Courtland saline containing 15 i.u./ml
heparin. This mixture was then combined with 5 ml of r i x IO~3M solution of a
fluorescent vital dye, acridine orange (Fisher Scientific), in unheparinized saline.
Heparin formed a precipitate with acridine orange, so the above sequence was adopted
to permit the heparin to react with the plasma proteins before the dye was added. The
blood-dye mixture was left to stand for 10 min and then centrifuged for 1-5 to 2 min at
about 7000 g. This was long enough to sediment the blood cells but left most of the
remaining precipitate in suspension. The small amount of precipitate which remained
with the cells occurred as minute particles (approximately 1-2 /i diameter), much
smaller than the blood cells. The supernatant was discarded and the stained blood
cells resuspended in 0-5 ml of heparinized Courtland saline. The stained cell suspension was then injected into the fish through the cannula at a rate of o-8 ml/min.
Preliminary measurements showed that the injection rate, amounting to no more than
15% of the cardiac output (Cameron & Davis, 1970), was sufficiently slow that no
changes occurred in the heart rate or dorsal aortic blood pressure. However, the
injection rate was sufficiently fast that freezing could be completed before the marked
cells could complete a circuit of the circulatory system (Fig. 1). This prevented any
confusion which might result if the circulation through some regions of the gills is shut
down for a relatively short period of time.
While the stained cells were still being injected, the fish was plunged into a container of either isopentane cooled to —155 °C, or liquid nitrogen, to stop the blood
flow through the gills. Temperature measurements obtained with a thermistor bead
implanted among the gill filaments revealed that the rate of cooling was so slow that
changes in the distribution of blood flow would not be prevented unless the freezing
medium could come into direct contact with the gills. Thus, a method of exposing the
gills to the coolant was required which did not interfere with the normal breathing
movements of the fish before freezing.
Several techniques were tried for opening the opercula at the moment of freezing.
The one which proved most reliable, and least disturbing to the fish, involved attaching small loops (about 1-5 cm diameter) of stainless steel suture wire to the opercula
while the fish was anaesthetized for cannulation. The loops were passed through two
small holes drilled in the operculum, and through two 5 mm diameter plastic buttons
Iplaced one on either side of the operculum to prevent the wire from cutting. When the
122
JOHN H. BOOTH
(a)
I
1
2
1 2
L
0
10
20
30
40
50
Time (s)
Fig. i. Time courses relevant to the freezing procedure, (a) Minimum time required for blood
to complete one circuit (Davis, 1970). (6) Maximum time required for injection of the stained
blood cells (i.e. all blood injected), (c) Actual period of injection of stained cells (injection stops
when blood flow stops), (d) 1, Maximum time required for placement of the retractor; 2, maximum time required for temperature of the gills to reach — 10 °C. (e) 1, Minimum placement
time for retractor; 2, minimum time for reduction of gill temperature to —10 °C. (/) Time
required for ventral aorta smooth muscle to reach 50 % of maximum contraction (Kirby &
Burnstock, 1969).
fish was about to be frozen, the two arms of a specially made steel retractor were
slipped into the loops. When the fish was dropped into the cooling medium, the arms
of the retractor would spring apart, retracting the opercula and exposing the gills.
Placement of the retractor lasted 1-2 s. Measurements obtained with a thermistor
implanted among the gill filaments showed that the temperature was reduced from
5 °C to —10 °C less than two seconds after the fish was lifted from the chamber
(Fig- i).
Cold isopentane, which was used for the first six fish, is frequently used to freeze
small pieces of tissue because it does not boil on contact with warm tissue, facilitating
the transfer of heat. This study required that only a few millimetres depth of tissue be
frozen quickly, and it was found that liquid nitrogen alone caused the same rate of
cooling of superficial tissues. Since there was no special advantage in using isopentane
as well, its use was discontinued in favour of liquid nitrogen for the last four fish.
The frozen gill arches were removed from one side of the fish, separated, and cut
into blocks of about 10 filaments each. While still frozen, 8 fi sections of the tissue
block were cut along the longitudinal axis of the filaments in the plane of the arch,
using a microtome mounted in a cryostat maintained at — 15 °C. The sections were
thawed and dried on microscope slides and examined without further treatment.
Examination was carried out using a Leitz Wetzlar fluorescence microscope equipped
with a BG12 3 mm transmission filter (transmission peak at 400 nm) and a K530
barrier filter which transmits all wavelengths greater than 530 nm.
Journal of Experimental Biology, Vol. 73
Fig. 2
B
Fig. 2. Sections of trout gill tissue containing stained blood cells, viewed with a fluorescence
microscope. (A) A section of part of one filament showing variations in perfusion between
adjacent lamellae. Calibration bar 25 ft. (B) A section through part of two filaments showing
variations in perfusion between adjacent filaments and perfusion of the central compartment
with stained blood cells. Calibration bar 100 /i. Symbols: s, side branch of filament support
rod; c, central compartment.
JOHN H. BOOTH
(Facing p. 123)
Blood flow in gills of rainbow trout
123
To estimate the degree of perfusion of the gills, the numbers of completely perfused, unperfused, and half-perfused (0-25-0-75 of the blood cells stained) secondary
lamellae were obtained for every fifth filament from each hemibranch. Comparison of
the perfusion of the gills showed that the proportion of lamellae perfused was essentially the same on both sides of thefish.Thus estimates were made for the arches on one
side only. The proportion of secondary lamellae in a hemibranch which were perfused
could be obtained from the equation
c A = x+yjz
proportion perfused
^ J—
where x = number of completely perfused lamellae,
y = number of half-perfused lamellae,
z = total of perfused, half-perfused, and unperfused lamellae.
To calculate the proportion of lamellae perfused in a fish, the three perfusion totals
were summed for all eight hemibranchs and the totals were applied to the above
equation.
RESULTS
The nuclei of blood cells stained with acridine orange, when examined under the
fluorescence microscope, appeared reddish-orange if a high concentration of dye was
used. The colour of the nuclei changed to orange, yellow, yellow-green, or green as the
concentration of dye was reduced. Mixing well-stained cells with unstained cells in
vitro resulted in all of the cells in the suspension becoming stained. The colour of
fluorescence, which was uniform between cells, depended on the original proportions
of stained and unstained cells. The dye treatment did not appear to change the size or
shape of the blood cells at the concentrations used, when compared with unstained
cells in vitro. Also, stained cells appeared to have compressed and bent in the secondary lamellae, in the same manner as unstained cells, suggesting that the dye had little
or no effect on the flexibility of the blood cells.
Microscope observations of sections of gill tissue during thawing and drying
revealed no movement of blood cells in the secondary lamellae or small blood vessels,
and very little movement of cells in the branchial arteries. There was no apparent
shrinkage of tissue sections during thawing and drying, as long as all of the section lay
flat on the slide. New sections of tissue were almost invisible in the absence of stain.
After a few hours, the tissue developed a faint auto-fluorescence, appearing greenishbrown with the filters used.
Observations of the afferent branchial and afferent filamental arteries, using the
fluorescence microscope, showed that all of the blood cells fluoresced reddish-orange,
orange or yellow. Blood cells in the efferent branchial and efferent filamental arteries
were usually yellow or green.
Observations of the gill sections using conventional light microscopy revealed the
presence of blood cells in every secondary lamella. Using fluorescence microscopy, it
was observed that, while some secondary lamellae contained only stained blood cells
fluorescing orange, yellow, or green, others contained few or no stained cells (Fig. 2).
Areas of reduced perfusion consisted of (a) single lamellae, (b) groups of lamellae on
n n e or both sides of a filament, (c) whole filaments, (d) groups of filaments (Figs. 2, 3).
JOHN H. BOOTH
124
Table i . The proportion of secondary lamellae perfused in the gills of rainbow trout in
air-saturated water at 5 °C. Values are calculated for each hemibranch (HB) andfor each
fish
Proportion of lamellae perfused
Arch
I
Arch
2
Arch 3
Arch 4
*
A
jrisn
no.
4
5
II
12
14
15
45
46
47
48
X
S.D.
HBi
0-778
0-685
0738
0-495
0603
0-560
HB2
0-935
0701
0-785
0-548
0645
0679
0512
0-482
0761
0-626
0-II2
0-771
0707
O-733
0739
0-099
0788
0-706
HBi
0-892
0-540
0-655
HB2
0-882
0-559
0632
0759
0-641
HBi
0-619
0-153
0-273
0-517
0699
0-443
°'339
0486
O'433
0-377
0-269
0-501
0758
0336
0-576
0767
0-178
0183
0463
0-333
0-371
0-612
O-353
0-561
0-185
0636
0-440
0436
HB2
0-732
0-231
0-254
0-725
0228
0-520
0-172
0829
0-780
0631
HB 1
0-510
0-263
WVinlo
fish
0282
0867
0903
0683
HB2
0681
0513
O'355
0-823
0-354
0635
0-483
o-881
0836
0717
0782
0503
0531
0608
0-540
0-537
O-4I5
0-617
0-720
0-565
0603
0199
0-624
0198
0-582
0-106
0-721
0-533
0-410
0-628
0-426
0-579
(a)
(b)
Fig. 3. Diagram of part of a gill arch showing typical distribution patterns of stained blood
cells (darkened regions) in the secondary lamellae of the gill filaments. Patterns (o) and (/)
were found less frequently than the others.
The proportion of lamellae perfused for each fish and for each individual hemibranch
is shown in Table 1.
Stained cells were observed anywhere within the length of a partially perfused
lamella, but were more frequently seen near the base or outer margin (Fig. 2). No
particular preference was observed for the marginal channel. While the thickness o!
Blood flow in gills of rainbow trout
125
the secondary lamellae occasionally varied, there was no apparent relationship of this
variable with the perfusion of the lamellae. The distribution of perfused lamellae was
quite variable, but certain basic patterns were observed. Generally, a group of lamellae
containing only stained cells at or near the base of the filament was separated by a
number of lamellae containing a few stained cells from a group of lamellae near the tip
of the filament in which no stained cells were visible (Fig. 3). More lamellae containing
stained cells were found in filaments near the dorsal portion of the arch compared
to the ventral end.
A central compartment was visible in most of the filaments examined. It appeared to
be at least partially divided into subcompartments by cartilaginous side branches of
the filament support rods (Fig. 2B). The width of the compartment varied between
filaments, even when sectioned at the same location on the filament. Neither the
presence of stained cellsfluorescingyellow-green or green in the central compartment
(Fig. 2B), nor the size of the compartment space, seemed to be related to the level of
perfusion of the secondary lamellae in the same filament. In some sections, yellowgreen or green fluorescing blood cells were also found in vessels running beside the
afferent filamental artery.
DISCUSSION
The techniques described in this paper incorporate two significant improvements
over previous methods used to study gill blood flow. The system of rapid freezing
permits observations to be made on fish which, until the moment of freezing are alive,
intact, unrestrained, and undisturbed. Of course, the most meaningful results will only
be obtained if the blood can be stopped before the distribution of blood flow can
change in response to the handling. It would be very difficult to determine the precise
moment at which blood flow ceases. However, the rapid cooling rate achieved, combined with the very thin tissue involved, make it reasonable to assume that blood flow
should stop before the vascular smooth muscle in the vessel walls can significantly
alter the flow pattern of blood in the gill filaments (Fig. 1). The second improvement
in methodology involves vitally staining the blood cells to obtain information on their
movement. Previous studies have used india ink (Steen & Kruysse, 1964; Richards &
Fromm, 1969) which behaves more like a plasma marker, or have used rigid microbeads (Cameron, 1974). Neither marker can be expected to accurately mimic the
physical properties and movements of the blood cells. Yet this information is important since it is the red blood cells which are the blood component of the greatest
respiratory significance. By using a fluorescent vital dye, it was possible to definitely
identify vessels which blood cells had entered prior to the moment of freezing.
Observations made on the stained cells in vitro and in situ gave no indication that the
stained cells behaved any differently than unstained cells.
The in vitro observation that a mixture of stained and unstained cells became
uniformly stained explains why all the cells in the afferent branchial artery were
stained, even though the injected cells represented only 15 % of the cardiac output.
Obviously the dye is not firmly bound, and will move out gf the cell during mixing in
the presence of a suitable concentration gradient. Similarly, if the flow through a
region of the gills is slowed, more dye would be lost by stained cells to the vessel
walls before freezing. The cells frozen in such a region would appear greener than
5
EXD
73
126
J O H N H. BOOTH
those in a region with a high rate of blood flow, where each blood cell would lose
less dye.
The number of stained cells found in the central compartment was trivial compared
to the number in the secondary lamellae. Also, there was no inverse relationship in the
perfusion of the two pathways which might be expected if the exposure of the blood to
the respiratory surface was reduced by non-respiratory shunting. Thus the considerable reduction observed in the proportion of the respiratory surface perfused with
blood is probably achieved by regulating the recruitment of lamellar blood flow.
Some ability to control blood flow at the level of the filamental arteries is suggested
by the observations of complete filaments which remain unperfused. But the distribution of stained cells within filaments indicates that control must also occur at the level
of the individual secondary lamellae. The apparent preference for perfusion of
lamellae near the base of the filaments and of filaments near the dorsal end of the arch
may be the result of attempts by the fish to regulate the local ventilation/perfusion
ratio (Wood, 1975).
There was no evidence of any relationship between the thickness of a secondary
lamella and the presence of stained blood cells in the lamellar channels. Thus it seems
unlikely that lamellar blood flow is mediated by contraction of the pillar cells (Hughes
& Grimstone, 1965; Newstead, 1967). This does not preclude variations in flow
patterns within single lamellae being affected by pillar cell contraction, but total
lamellarflowis probably controlled by muscle tissue in the lamellar arterioles (Richards
& Fromm, 1969; Morgan and Tovell, 1973).
The lack of any obstruction of the marginal channel by pillar cells of the secondary
lamellae (Hughes & Grimstone, 1965) and its apparently larger diameter compared to
the other lamellar channels (Skidmore & Tovell, 1972; Hughes & Byczkowska-Smyk,
1974) have often led to suggestions that it might represent a preferred pathway for
lamellar blood flow (Hughes & Grimstone, 1965; Newstead, 1967; Rankin & Maetz,
1971; Hughes & Byczkowska-Smyk, 1974; Laurent & Dunel, 1976). Support for this
hypothesis is reduced somewhat by the observations that, in trout gills, the marginal
channel is by no means the only unobstructed channel through the lamellae (Morgan &
Tovell, 1973), that it is only wider than the other channels at the afferent and efferent
ends (B. J. Gannon, G. Campbell and D. J. Randall, in preparation), and that, in
casting experiments, it is never exclusively perfused (Laurent & Dunel, 1976). Muir &
Brown (1971) observed a similar arrangement of the pillar cells into distinct rows in
the secondary lamellae of scombroid fishes. They believed that this arrangement
served to organize the blood flow within the lamellae. In the present study, the
observed preference for flow through the channels near the base or outer margin, but
not the marginal channel exclusively, was probably due to the reduced resistance to
flow offered by the channels in these parts of the lamellae, compared to the less
regular channels in the middle. Certainly there was no evidence that lamellar blood
flow was directed exclusively through the marginal channels by the contraction of the
pillar cells.
The present study has demonstrated that the necessary conditions exist for lamellar
recruitment (i.e. unperfused lamellae in resting fish). Since 58% of the lamellae were
perfused in resting fish, the maximum possible increase in the number of lamellae
perfused is about 70%. Butfivefoldincreases in the oxygen uptake have been observed
Blood flow in gills of rainbow trout
127
in moderately exercised fish (Stevens & Randall, 1967). Thus, if variations in functional surface area of the gills do occur, their contribution to changes in the gas
exchange capacity of the gills might prove to be rather small.
The direction and role of blood circulation in the central compartment remains
uncertain. Gannon et al. (1973), Laurent & Dunel (1976) and Vogel et al. (1976)
found small vessels connecting the efferent filamental artery with the central compartment in the gills of rainbow trout. Other vessels connect the central compartment
with blood vessels running beside and parallel to the afferent filamental artery (Gannon
et al. 1973; Laurent & Dunel, 1976). They proposed that blood flowed from the
efferent filamental artery, through the central compartment and back toward the heart
through the vessels beside the afferent filamental artery.
Vogel et a/. (1973) found a similar arrangement of blood vessels in the gill filaments
of Tilapia mossambica. They also found evidence of vessels connecting the afferent
filamental artery with the central compartment. However they concluded that these
vessels were too narrow and too few in number to represent a significant shunt pathway. Although such vessels have never been observed in rainbow trout, that does not
mean that they do not exist. The severity of treatment of a fish required by vascular
casting could result in blockage or closing of pathways which might be available in
unhandled fish. The technique used by Vogel et al. (1976) was inadequate to screen
the afferent lamellar arterioles which, as they suggest, might also have connections
with the central compartment.
In the present study the stained cells in the central compartment, as well as those
lying in the vessels lying beside the afferent filamental artery, were dull green or
slightly brighter yellow-green in colour. They resembled the blood cells in the
efferent filamental artery more than the bright orange cells in the afferent filamental
artery. While this evidence cannot be considered proof that blood in the central
compartment comes from the efferent filamental artery, the amount of dye lost by the
cells in the central compartment does indicate that these cells had sustained a longer
contact with unstained tissue than would be likely if they had entered the compartment through short vessels directly from the afferent filamental artery.
If the role of blood flow in the central compartment is nutritive, as proposed by
Gannon et al. (1973) and Morgan & Tovell (1973), it is difficult to understand the
complex structure of the compartment in rainbow trout. A possible explanation, which
could account for the striking variability of the dimensions of the compartment, is that
changes in hydrostatic pressure, combined with the distensible wall of the compartment (Vogel et al. 1973) could serve to vary the position of the secondary lamellae in
the respiratory water flow.
I would like to thank Dr G. F. Holeton for his constant advice and encouragement,
and Dr J. Machin for valuable criticisms of the manuscript. Financial support for this
study was provided by a National Research Council of Canada grant to G. F. Holeton.
Personal support for the author was provided by the Fisheries Research Board of
Canada, and the Ontario Graduate Scholarship programme.
5-2
128
JOHN H. BOOTH
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