AMEB. ZOOL., 13:475-489 (1973).
Respiratory Responses to Hypoxia in Fish
GEORGE M.
HUGHES
Research Unit for Comparative Animal Respiration, The University,
Woodland Road, Bristol BS8 1UG, Great Britain
SYNOPSIS: Hypoxia is discussed in the widest sense, i.e., as the conditions under
which cells suffer a lack of oxygen. From a consideration of the transfer of oxygen
from the water to the cellular sites as a series of resistances, it is suggested that hypoxia can result from an increase in resistance anywhere along the chain, and possible types of hypoxia in fish are denned from this point of view.
More detailed discussion is given in relation to (1) interference with gas transfer at the gill surface; (2) reduction in the blood O2 carrying capacity; (3) modifications in the cardiac and ventilatory frequencies and in the coupling between
these two rhythms, and (4) the time course of the hypoxic stimulus.
It is concluded that most o£ the increase in knowledge of hypoxia during the
past ten years can be fitted into known theoretical frameworks, but there is still a
great need for measurements of oxygen tension at all levels in the respiratory chain;
attention needs to be directed to both diurnal and seasonal variations and especially
to long-term changes in those environmental conditions which tend to result in hypoxia.
The need for oxygen is one of the most
pressing demands on the physiology of fish
and is aggravated by the physical properties of their respiratory medium. During
the past ten years our knowledge of processes involved in the transfer of the O2
molecules from the water to the sites of
usage at the cellular level has increased
tremendously not only for individual species but also with respect to the variety
of adaptations to particular environments.
It would be impossible to review adequately all of these advances in the time
available and inevitably I shall emphasize
a particular viewpoint. This will be related to the type of work carried out during this period at Bristol, but I believe it
illustrates the overall development of work
in this field. Some of the theoretical background was presented at a previous symposium (Hughes, 1964).
THE RESPIRATORY CHAIN
In hypoxia the O2 available is less than
under normal conditions (normoxia),
whereas in hyperoxia it is greater. Just as
the normal functioning of the respiratory
I wish to thank the Natural Environment Research Council for financial support. I should also
like to thank Mr. D. W. Williams and Mr. R.
Adeney for research assistance.
system adjusts to maintain a particular
level of O2 at the cellular level, so the
strict definitions of hypoxia and hyperoxia
refer to the conditions of the cells, and
more specifically hypoxia means a lack of
oxygen for some oxidizable substrate. The
concept of a chain for the transfer of oxygen from the environment to the cell provides a useful framework for discussions
of this kind. Figure 1 shows a diagram of
the so-called respiratory chain along which
oxygen molecules pass from the environment to these cellular sites. During this
transfer they meet a series of resistances.
Under normal conditions the whole system adjusts in such a way that these resistances are overcome and maintains the
correct level of functioning of the cellular
components. Any one of these resistances
can increase and give rise to hypoxia at
the cellular level. Information about these
resistances has increased in recent years,
particularly in relation to transfer across
the secondary lamellae from the water to
blood (Hills and Hughes, 1970; Jones et
al., 1970; Hughes and Hills, 1971). Quantitative measurements of P02 levels at different positions along the chain, and consequently the relative sizes of the resistances offered to that transfer, still need
further investigation. We are especially ignorant about the oxygen tensions at the
475
476
GEORGE M. HUGHES
EXTERNAL
WATER
RESPIRATORY
CAVITIES
INTERLAMELLAR
WATER
CONVECTION
PLASMA
BBC
Hb
CONVECTION
DIFFUSION
HEACI|ON
VENOUS
TISSUE
FLUID
ADMIXTURE
MITOCHONDRIA
s
EXTERNAL
ENVIRONMENT
GILLS
CIRCULATION
F
D E
CELLS
G
HI
A
B
C
FIG. 1. Diagram indicating changes in O2 tension
along ihe respiratory chain of fish from the external water (Pln) to the mitochondria (P m i t ). Some
of the main resistances are shown as is a qualitative indication of the relative drop across them
(e.g., dPn and APm) . At the gills a very significant resistance is due to convection of the water
between individual secondary lamellae. Diffusion
is the physical process responsible for transfer of
O2 across the erythrocytes and the gill and cell
membranes. Less is known about subdivision of the
resistances between capillary blood and mitochondria. The fall in O2 tension in the convection region which links the sites of O2 transfer at the gill
and at the tissues is due to the variable effects of
shunting and ventilation/perfusion inequalities.
Parts of the chain mainly affected by different
types of hypoxia (A-I) mentioned in the text are
indicated.
tissue and cellular levels of fish. The admirable study of Garey and Rahn (1970)
illustrates the value of studies using the
gas pocket technique, which has been used
in relatively few lower vertebrates (Hughes,
1966). Garey and Rahn (1970) also showed
the value of measurements of the O 2 tensions in natural waters in order to gain
information about the physiological range
of the external environment to which given
species are subjected. In recent studies on
some Indian air-breathing fishes (Hughes
and Singh, 1970; Singh and Hughes, 1971,
1973), I was surprised to find that there are
no really detailed studies comparable to
the classical work of Carter and Beadle
(1931) for the ponds in which Anabas,
Clarias, Saccobranchus, etc., spend their
lives. However, much is known of the
ecology of these waters, and there are indications that particular species exploit
local differences in the environment. In addition to diurnal fluctuations there are also
marked seasonal changes. For example, the
levels of O;> in Indian fresh waters rise at
the time of the monsoon rains, and recent
morphometric studies of the respiratory organs of air-breathing fishes have indicated
that the relative development of the waterand air-breathing organs may be correlated
with these environmental changes (Hughes
et a!., 1973). Marine waters are generally
477
HYPOXIA IN FISH
fully aerated, but there is good evidence
for regions of low O 2 content particularly
at great depths. Thus, although the gill
areas of fish from such depths are generally small (Hughes, 1972c), it would not be
surprising to find some species equipped
with larger gill surfaces and other adaptations which enabled them to survive swimming through regions of low O 2 .
T o summarize, then, oxygen must pass a
series of resistances, the sizes of which are
indicated by reductions in Po., (AP02) which
may be summed to give the total O 2 tension differential along a particular pathway as indicated below:
P i n - 1 mit —
APr +- APU -f
APm4- AP pl -f AP e
A P h b - f APad -f APt
APcm- +• APe
where P r -> APC represent each of the 10
regions shown in Figure 1 where there is a
drop in P 0 2 between the respiratory cavities and cytoplasm. Clearly the total O,
flow would be affected by changes in these
resistances. If any one of them increases
too much, it may not be possible for the
P 0 2 level at the mitochondria (P mit ) to be
maintained. A given percentage change in
resistance would clearly have most effect
when it influences one of the larger resistances. From this point of view the parts
of the chain high in resistance might be
regarded as the most susceptible for hypoxia. Each of the many pathways in parallel with one another may differ slightly
because of local differences in resistance,
for example distance for diffusion at the
gill membrane. Such differences could also
be quite substantial because of the admixture of blood of varying degrees of oxygenation depending upon their path through
the gill exchanger. It has become clear that
we must regard this and other types of
heterogeneity as an important feature of
the respiratory mechanisms of most vertebrates (Hughes, 19736).
The main purpose of this paper is to
emphasize the way in which hypoxia can
be considered in relation to the whole
chain as it may affect the O 2 supply at the
cellular level and we may recognize dif-
ferent types of hypoxia according to the
site at which these resistances are increased.
Another important parameter is that of
time, for the adjustments to hypoxic conditions sometimes incorporate quite complex control mechanisms having different
time constants so that some adjustments
can occur fairly rapidly whereas others occur over longer periods, e.g., acclimatization.
CLASSIFICATION
Without giving an exhaustive description
of the many different types of hypoxia, several examples will be chosen to illustrate
our present knowledge. Perhaps the recognition of other points in the respiratory
chain where hypoxia could occur might
lead to further investigations.
TYPE A. Hypoxia due to a reduction
in P 0 2 of the inspired water. This is the
experimental procedure usually thought of
when dealing with hypoxia in fish (see
subsequent section entitled Environmental
Hypoxia). T h e primary effect is on the
level of the driving force (Pjn), and not on
any specific resistance, and is liable to
modify O 2 tensions along the whole chain.
Such hypoxias might arise in a fish's normal environment, e.g., in pools of a restricted volume when the O 2 uptake of the
organism exceeds that diffusing into the
water. The presence of plants in most freshwater ponds adds a further complication
because of diurnal fluctuations in O 2 levels
resulting from photosynthesis (Fig. 2). In
most marine waters, the O 2 levels are fairly constant, but in the deeper parts of the
ocean there are regions where fish may
suddenly encounter water of very low O2
tension. Bottom living forms may also be
subjected to hypoxia because just below
the surface of the mud the O 2 tension may
drop quite sharply. Conditions of lowered
O, are also found in polluted waters. At
high altitudes the P o= in the gaseous phase
is low so the O 2 tensions of any waters in
equilibrium will be equally low even at
full saturation. Hence, it would be of interest to study the respiratory biology of
the fishes of Lake Titicaca, for example.
478
GEORGE M. HUGHES
FIG. 2. Diurnal changes in oxygen tensions of water and tissue in trout and carp during the month
of June. Ordinate mm Hg: abscissa 4 hr intervals;
shaded area represents night. (From Carey and
Rahn, 1970).
It is evident that the onset of hypoxia
will have varying time courses depending
on the general nature of the local conditions. In the laboratory some of these are
simulated by a closed respirometer, or by
lowering the O2 tension of water entering
a continuous-flow respirometer by bubbling
nitrogen or some air and N2 mixture
through the water. In spite of much research in this field (Fry, 1957; Butler and
Taylor, 1971), there is still need for refinement in the apparatus used for controlled
production of experimental hypoxias of
varying rates.
TYPE B. Hypoventilation of the gill
surfaces such as can be induced experimentally or perhaps be the result of a malformation or paralysis of the normal ventilatory apparatus.
TYPE C. Involves interference with the
diffusion exchange across the gill surfaces
such as can occur if the thickness of the
barrier is increased, for example, by the
effect of certain pollutants. These effects
may also extend to other parameters involved in the gas diffusing capacities of
the gill system as discussed below.
TYPE D. Concerns the relationship between the ventilation and the perfusion
sides of the gill exchanger; hypoxia can
result from extreme unevenness in this respect. Normally, these seem to be adjusted,
but experimentally can be interfered with.
There has been relatively little study of
this aspect of fish respiration which could
prove to be extremely important. In rays
it appears that there is no control over
the relative ventilation of different gill
pouches, but that there is some control of
the blood flow (Satchell et al., 1970; Cameron et al., 1971).
The next three types include hypoxias
due to malfunctions within the circulatory
system.
TYPE E. Can result from the excessive
shunting of venous blood, by-passing the
respiratory exchange surfaces. Such shunts
are present in fish gills, but their detailed
mechanisms are not yet understood. The
possible modification of such a pathway resulting from changes in the hormonal content of the blood (Rankin and Maetz,
1971) could be an example. Such changes
occur in response to osmotic stresses and
serve to reduce the dangers of excessive
salt or water exchanges and could lead to
hypoxia under certain conditions. Among
air-breathing fishes there are shunt pathways which by-pass the gills or the airbreathing organs; excessive flows of blood
in these pathways could result in hypoxia.
TYPE F. Hypoxia resulting from a reduction in the amount of functional hemoglobin within the circulatory system. Such
anemias can arise from carbon monoxide
poisoning (Anthony, 1961; Holeton, 1971a),
and the injection of phenylhydrazine (Cameron and Wohlschlag, 1969; Cameron and
Davis, 1970), and these have been used
experimentally. There is also the possibility of certain phenolic substances entering
the blood and producing hypoxia by their
hemolytic effect. Lack of available iron is a
further possibility among natural populations. Certain species of fish, such as the
icefish, have evolved in which the amount
of functioning hemoglobin is very markedly reduced and limits the responses of
479
HYPOXIA IN FISH
such fish to environmental hypoxia (Hemmingsen and Douglas, 1970; Holeton,
1972).
TYPE G. Caused by poor circulation in
different parts of the body. Clearly, if the
conditions for blood flow are inadequate,
the tissue will become hypoxic. Within
the external gas exchangers themselves this
possibility must also be considered. One
factor which has not been given much consideration in fish is the possibility of
changes in flexibility of the red blood cells,
which would clearly increase the effective
viscosity of the blood and could lead to
conditions of insufficient flow in both the
respiratory organs and the tissues. Erythrocytes are wider than the capillary diameters and generally "squeeze" through the
channels between pillar cells (Muir and
Brown, 1971). Reduction in flexibility and
also changes in volume associated with increases in blood CO2 levels (Ferguson and
Black, 1941) could be serious.
Interference with diffusion between the
capillaries and the cells is again a theoretical possibility which could be aggravated, for example, if the interstitial spaces
became enlarged.
TYPE H. Even within the cells hypoxia
can result from poisoning of the oxidative
enzyme systems, e.g., with cyanide poisoning. Presumably this is one of the results
of poisoning in some pollution situations,
and possibly other types of pollutants affect living organisms in this way. This is
sometimes referred to as "histotoxic hypoxia" and is known with respect to cobalt
poisoning in mammals (van Liew and
Chen, 1972).
TYPE I. Results from cells "over using"
Oj. and, hence, leading to a hypoxia.
These are some of the main sites at
which hypoxia can occur. It is clear that a
number of them can be particularly important in fishes, and these types will be
discussed in more detail.
tance to gas transfer. This resistance is
increased in three ways: (1) by interfering
with the convective processes between the
secondary lamellae; (2) by increasing the
water/blood distance, and (3) by reducing
the surface area across which transfer can
occur. It is not surprising, then, that trout
placed in waters containing suspendedsolid pollutants increase their coughing
frequency (Hughes, 1973a) which would reduce the rate of decrease in gas transfer,
but such responses cannot be effective indefinitely. Unfortunately, it has not yet
been possible to carry out experiments on
the O2 consumption and O2 diffusing capacities of fish from such waters. At Bristol, however, such experiments have been
carried out in relation to zinc.
Skidmore (1970) studied changes in the
physiology of rainbow trout following treatment with zinc sulphate in concentrations
of about 40 ppm Zn. Although somewhat
high relative to naturally occurring concentrations of zinc, this proved to be a convenient concentration for laboratory
studies of this kind. The possibility that
death was mainly due to interference with
the osmotic and ionic regulation of the fish
was first investigated, but later the idea
that this and other pollutants produce hypoxia within the fish due to interference
with O2 transfer across the gill surfaces
was supported. Evidence was obtained from
the decline in arterial P 02 (Fig. 3) similar
S - ijmchronjr
£ 80
i
Po, (mm. Hg)
POLLUTANTS AND GAS TRANSFER ACROSS
THE GILL MEMBRANES
Suspended-solid pollutants adhere to the
secondary lamellae and increase the resis-
SurvMI Um< (%)
FIG. 3. Oxygen tension in dorsal aortic blood of
four rainbow trout in zinc-free water and in zinc
sulphate solution (40 ppm) until immobilization
of the opercula at 100% survival time. (From Skidmore, 1970.)
480
GEORGE M. HUGHES
to that observed in fish subjected to hypoxia by lowering of the ambient O2 tension. Injection of zinc into the blood
stream did not in itself produce these effects. From the histological changes which
occurred in the gills during such treatment
(Skidmore and Tovell, 1972), it was clear
that there was an increase in the diffusion
distance between the water and blood. This
would reduce the diffusing capacity of the
gills (Hughes, 1972a) which would be further aggravated because there was also a
reduction in the effective surface area due
to withdrawal of the surface epithelial layers from the underlying tissue. From the
micrographs it is possible to estimate that
even in early stages this must amount to
at least a 25% reduction in area. This, together with a doubling of the water/blood
barrier thickness would reduce the diffusing capacity to almost one-third. A further
reduction would be due to interference
with the convective transfer of gas from
water flowing between the secondary
lamellae. As this provides a very significant
part of the overall resistance to gas transfer
(Hills and Hughes, 1970), this is by no
means a negligible factor. During the later
stages when the epithelium has withdrawn
almost completely from the crypt regions
between the secondary lamellae, gas transfer to the blood could only occur between
the water and marginal channels of secondary lamellae.
Further confirmation of our hypothesis
that death during conditions of pollution
can result from hypoxia was reported in
relation to zinc toxicity by Burton et al.
(1972), who found that under these conditions tissue hypoxia also occurred as deduced from measurements of the muscle
and liver lactic and pyruvic acid levels.
The effect of some other pollutants on
the respiratory chain could also be conveniently mentioned here. Larsson (1973)
summarized some of the effects of chlorinated hydrocarbons on fish blood. Studies
on eels subjected to fresh and sea water
containing 0.001 ppm PCP (pentochlorophenols) indicated an increase in blood
hematocrit, but in this case there was a
far greater disturbance at the cellular levels
of the respiratory chain which led to increased ventilatory movements. The primary effect seemed to be an uncoupling of
the oxidative phosphorylation which resulted in a hypermetabolic state due to increased utilization in tissue-energy reserve.
Other pollutants, e.g., PCB (polychlorinated biphenyls), effectively reduce the blood
hematocrit when tested on trout.
BLOOD TRANSPORT
Experimentally, hypoxia can be generated by reducing the effective hemoglobin
concentration and, hence, increasing the resistance to gas transfer provided by a blood
transport system. Experiments carried out
by Holeton (1972) have given interesting
information on many aspects of the gas
transfer and have contributed to our understanding of the physiology of the remarkable icefish which lack hemoglobin in
their blood. Holeton (1971a) confirmed the
survival of trout at low temperatures when
nearly all of their hemoglobin had been
combined with carbon monoxide as had
been shown for goldfish (Anthony, 1961).
The way in which the respiratory and cardiovascular systems of trout become adjusted in relation to this situation was to
be expected from earlier analyses of gas
exchange in terms of the O2-carrying capacities and volumes of water and blood
pumped across the gill exchanger (Hughes,
1964). The capacity rate ratio was emphasized by that analysis and consists of
the ratio between the products of the O2
capacities and volume flows of these two
liquids. Reduction in the O2 capacity of
the blood could be compensated by an increase in cardiac output, and this seems to
be one of the ways in which the COpoisoned trout and icefish adjust to the
reduction in O2 capacity. It is important
for the proper functioning of the countercurrent exchanger at the gills that this capacity rate ratio should be close to unity,
and this appeared to be the case (Fig. 4)
following experimental anemias induced
by the injection of phenylhydra/ine hydrochloride (Cameron and Davis, 1970).
HYPOXIA IN FISH
481
a superimposed response which Holeton
(19716) concluded must be initiated by
changes in the O2 tension of the environment or arterial blood. As with many adult
fish, the initial response to hypoxia tends
to counteract its effects, but the later responses encourage conservatory mechanisms
reducing O2 uptake. The adult bradycardia response to hypoxia is seen as a mechanism whereby the fish makes no attempt
to increase flow on the blood side of the
exchanger and instead concentrates on
5
10
13
20
25
30
mechanisms which enable it to maintain a
HEMAT0CR1T
high effectiveness of O2 transfer to the
FIC. 4. Rainbow trout. Changes in capacity rate
blood flowing through the gills.
ratio in fish
Evidence for mechanisms assisting this
general response of rainbow trout to hyQ 1B 0 2
poxia is found from many studies on the
relationship between the cardiac and veno£ different haematocrits (%) (CR ratio = 1.668
— 0.203 HCT) . Note relatively small deviation
tilatory frequencies. In spite of early work
from unity. (From Cameron and Davis, 1970.)
emphasizing the possibility of synchrony,
many recordings from trout in normoxia
HYPOXIAS AND BRADYCARDIA
indicated the possibility that these two
rhythms were not so closely coupled
It is of considerable interest to observe (Hughes, 1961). Further studies have inthe effects of hypoxia on developing trout, dicated that this coupling increases during
for whereas a marked bradycardia has gen- hypoxia, but it is only recently that
erally been found in adult teleost fish (Shel- methods have been developed for the quanton and Randall, 1962; Randall and Smith, titative evaluation of such a mechanism
1967; Hughes and Umezawa, 1968), a (Hughes, 19726). Analysis of data obtained
tachycardia was observed in the newly for trout during hypoxia (Fig. 6) clearly
hatched trout (Holeton, 19716). At very
low P02's (less than 5 mm Hg) there was,
however, a marked fall both in heart rate
and ventilatory frequency (Fig. 5). A further difference betwen larval and adult
trout was observed on return of O2-rich
water which resulted in the abolition of the
bradycardia within a few beats with adults,
whereas this occurred gradually over 10
. so
min with larval trout. The latter also
i
showed similar responses to both carbon
monoxide and environmental hypoxia,
whereas in adults they are quite different,
there being no bradycardia during carbon
monoxide poisoning (Holeton, 1971a).
It seems that the bradycardia response to
hypoxia does not occur until at least 8
days after hatching, and the suggestion was FIG. 5. Rainbow trout 8 days after hatching. Effect of hypoxia at 10 C on heart rate (solid line
made that the responses of newly hatched and
figures) and venlilatory frequency (dotted line,
trout to CO and hypoxia represent basic open figures) . Heavy line indicates Po2 o£ water.
responses to anoxemia. The adult brady- Individual fish are represented by a particular
cardia response would thus appear to be shape of symbol. (From Holeton, 19716.)
12S
e
100 2
V
E
7S
SO
|
482
GEORGE M. HUGHES
ticular resistances within the respiratory
chain and thus serve to maintain a given
flow of O2 in spite of a reduction in the
overall O2 tension differential.
ENVIRONMENTAL HYPOXIA
•".'•
50
100
ISO
0,TENSION(mmHg)
FIG. 6. Rainbow trout. Plot showing change in
per cent coupling between the cardiac and ventilatory rhythms during gradual and step-wise lowering of inspired P o for seven trout at 15 C. Data is
based on histograms (Knights, 1971) showing the
per cent of total number of ECG's recorded for all
fish at each P0.2 (about 2000-4000) in each of ten
equal phases during a ventilatory cycle between
positive peaks of the buccal pressure. Figures for
the coupling of an individual specimen are given
above the appropriate Po o s.
indicates an increase in the percentage
coupling between the two rhythms as the
level o£ O2 in the inspired water was
lowered. As with many experiments of this
kind, the grouping of data from many specimens tends to obscure the effects. The
results for individual specimens may be
very striking, and as shown in Figure 6,
extremely high couplings of more than
90% have been observed at low Po2'sThe effect of both bradycardia and increased cardioventilatory coupling in trout
strongly suggests improved conditions for
effective transfer of O2 from the water to
the blood by ensuring that the flows of
the two media are closely related and that
maximal time is available for this transfer.
In view of the high resistance of O2 transfer offered by the interlamellar water and
the O2 demands of the two pumps, this
would seem to be energetically desirable.
Hypoxia also results in increased blood
pressure (Holeton and Randall, 19676)
which, combined with the contractile
mechanism of the pillar cells (Bettex-Galland and Hughes, 1972), helps to maintain
the form of the secondary lamella sieve and
to reduce the resistance both to water flow
and to O2 transfer to the blood. Here,
then, are mechanisms which reduce par-
Many experiments have been carried out
in which the P 02 of the ambient water is
lowered and different respiratory and circulatory parameters monitored. The relationship between O2 consumption and environmental Po2 has been studied for many
species, and the distinction has been made
between regulating (independent) and conforming (dependent) respiration (Fry,
1957). The term "regulating" is somewhat
misleading in this context and perhaps it
would be better to distinguish between
"conformers" where the O2 consumption
follows closely that of the environmental
P 02 a n d "non-conformers" which at least
during the initial hypoxia have a constant
or even an increased O2 consumption until
a critical tension is reached (Fig. 7). That
the distinction cannot always be maintained even with respect to individual species has been emphasized by a number of
workers including Spitzer et al. (1969) who
showed that a given species may behave as
a conformer or non-conformer depending
on the acclimation temperature. The common response on the ventilatory side of the
exchanger for non-conforming fish is an increased ventilation of the gills at least to
120110-
x
l0
°
»
90-
=•
8070-
S
60-
I 50I 10-
I M20-'
o
10-
Ojygen tension,
% saturation
FIG. 7. Oxygen consumption of three species of
fresh-water fish during gradual hypoxic stress, catfish (A-A)< t r o u t ( C - C ) . and bluegill (Q—D) •
Polynomial lines were fitted to grouped data by
computer. (From Marvin and Heath, 1968.)
483
HYPOXIA IN FISH
INTEGRATED
EMG
30
RESPIRATORY FREQUENCY
FIG. 8. Callionymus lyra. Relationship between integrated electromyograms from the hyohoideus
and adductor mandibulae and ventilatory frequency during changes in P o below and above normal
levels. When the Po2 is restored to its normal level
following a period of breathing water containing
low O2, the EMG drops almost immediately, though
frequency continues at a low rate as shown by two
circled points, thus indicating a dual control
mechanism. (From Hughes and Ballintijn, 1968.)
a certain level. It may take the form of an
increase in frequency and stroke volume,
either of which may be the greater, or as
in Callionymus (Fig. 8), an increase in
stroke volume with a concomitant decrease
in ventilatory frequency (Hughes and Ballintijn, 1968). In all cases there is a net
increase in the volume of available O2
presented at the gill surfaces. Increases in
ventilation volume in such experiments
have been used to estimate the cost of the
pumping movements which would clearly
require a significant proportion of the animal's resting O2 consumption. The relationship between ventilation volume and
O2 uptake for a number of experiments
(Fig. 9) have been summarized by Shelton
(1970), and in relation to experiments on
trout, it is of interest that other trout data
(Hughes and Saunders, 1970) fit closely
with that of Holeton and Randall (1967a).
Marvin and Heath (1968) also found an
increase in V02 during gradual hypoxia
(Fig. 7). These data indicate that an increase in ventilation volume is associated
with an increase in O2 consumption. The
slope of this part of the curve has been
used by various authors to estimate the
O2 cost of breathing. Originally Hughes
and Shelton (1962) used the available data
of van Dam (1938) to estimate a value of
at least 20% of the resting O2 consumption. Schumann and Piiper (1966) derived
even higher figures using tench and more
recently lower estimates (10%) have been
made (Hughes and Saunders, 1970). It
would, appear, then, that during hypoxia
the increased pumping produces a consequent rise in O2 demand by the fish. Up to
a certain point this can be met, but when
the O2 levels in the water are reduced to
a certain extent perhaps the energy requirements become limiting. Consideration of
the corresponding demands of the cardiac
pump led me to estimate that they consume about one-half the amount of O2
used by the ventilatory pumps (Hughes,
1964), and this question has recently been
investigated in further detail: a number of
authors suggest that the requirement of
RAINBOW TROUT
1000
2000
3000
4000
Ventilotion volume (ml/mpn/kg)
FIG. 9. Relationship between O™ consumption and
ventilation volume under hypoxic conditions. (•)
trout recovered from exercising in aerated water
at 12 C (van Dam, 1938) . Lines are drawn to indicate relationships between ventilation and O2 consumption which would exist in fully aerated water
at this temperature for different utilizations and
van Dam's results are extrapolated to the 25% utilization line. (O) Eel during hypoxia at 18 C (van
Dam, 1938) . (Q) Trout (Holeton and Randall,
1967) in closed respirometer reduce their environmental OL, to 30 mm Hg at 15 C. (•) O2 consumption of fish in continuous respirometer during stepwise reduction of inlet P<,2 (after Hughes and
Saunders, 1970) . The line for the data of Holeton
and Randall (1967) is extended (dotted) to include these data for lower ventilation volumes.
(Modified from Shelton, 1970.)
484
GEORGE M. HUGHES
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PPG
Control
20 30 40 50 60 70 150
Oxygen partial pressure (mm Hg)
FIG. 11. O2 dissociation curves of blood of hypoxic
eels, horizontal and vertical bars represent mean
± S.E. Insert shows the red cell organic phosphates
of control and hypoxic eels. (After Wood and Johansen, 1972.)
OL
0
/
/
/ //
CO
0
Control
f
c
/ /
T. tinea.
— *~~~\
9
tent
the cardiac pump is relatively greater
(Heath, 1964; Cameron and Davis, 1970;
Jones, 1971). The use of hypoxic stimuli
in experiments of this kind has a major
disadvantage of disturbing the resting condition of the fish. A slightly different experimental approach has recently been
adopted by Dejours (1973) who used tench
that had been kept in hyperoxic conditions (Fig. 10). These fish also showed an
increased O2 consumption associated with
an increase in gill ventilation as the inspired PO2 was lowered, but in this case
never to levels which led to excitation of
the fish. All the data presently available
seem to support the view that approximately 10% of the resting O2 uptake of
10
0
FIG. IO.I Changes in respiratory parameters of tench
in continuous flow respirometers during reduction
in P o from hyperoxia (250 mm) to moderate hypoxia (80 mm). Notice the large increase in venlilatory flow (VE) below air saturation and the increase in metabolic rate (Mo ) associated with this.
The increase in ventilation volume is mainly due
to an increase in frequency (f) and slightly augmented stroke volume (V,). (From Dejours, 1973.)
chronic stages of responses, there are
changes in the properties of the hemoglobin such that there is both an increase
in O2 capacity and in affinity associated
with a reduction in the ATP content of
the red cells (Fig. 11). The affinity increase improves the loading potential for
O2 at the gills in severely hypoxic water
and reduces the detrimental effect a Bohr
fresh-water fish is used by the ventilatory
100
muscles during normal pumping in sta90tionary water.
80
The time course of the hypoxic stim» 70
ulus is clearly important and has now been
13 60
shown in a number of studies that monitor the bradycardia, responses of the ven- ^>^ 50tilatory system, or changes in blood oxySi 30
gen carrying capacities.
For the eel, Wood and Johansen (1972)
20
have recently shown a significant difference
10
in the responses of fish kept in normoxic
0 10 20 30 40 50 SO 70 60 90 100 110 120 130
water and those having suffered hypoxia
WATER OXYGEN TENSION urn Hg
for about one week. Usually hypoxia reFIG. 12. Effectiveness of O2 uptake by the blood in
sults in increased ventilation and an in- control and hypoxic eels, and in trout as a funccrease in utilization by the tissues ot O2 tion of acute changes in water P o . (From Wood
in the circulating blood. During the more and Johansen, 1973.)
HYPOXIA IN FISH
DOGFISH
FIG. 13. Scyliorhinus canicula. Changes in ventilatory and cardiac frequencies during hypoxia together with associated changes in activity o£ the
adductor manibulae and constrictor hyoideus muscles, as indicated by the height of their integrated
myograms (Hughes and Ballintijn, unpublished).
shift would have where the entire O2 transport takes place at the lower part of the
dissociation curve (Pa02 > 10-15 mn). Uptake of O2 in the gills of severely hypoxic
fish when compared with controls (Fig. 12)
showed an increase in the effectiveness of
O2 transfer to the blood, i.e., the ratio between the quantity of O2 transferred to the
maximum that could possibly be taken up
by the blood (Hughes, 1964). Thus, just
as hypoxia can result from changes in the
resistance to the gas transfer at all levels
of the chain, so the responses can be composed of immediate reflexes, responses of
longer time course, and finally the longterm changes illustrated by recent work
showing the effects on affinity of the hemoglobin.
HYPOXIA AND GILL VENTILATION IN
ELASMOBRANCHS
So far hypoxia has been discussed mainly in relation to teleost fish. Recent studies
on cartilaginous fishes have shown similar responses to hypoxia, but have also revealed some different responses which may
be related to differences in the basic lay-
485
out of the gills.
Studies on dogfish subjected to environmental hypoxia have shown that increased
ventilation volume is mainly due to
changes in stroke volume as indicated by
increased EMG's of the adductor mandibulae and constrictor hyoideus muscles
(Fig. 13) which are generally associated
with greater amplitudes of oro-branchial
pressure. The ECG recordings in these experiments also suggest changes in the
bradycardia response that are related to
the rate of P 02 change; this has also been
studied by other workers (Butler and Taylor, 1972). There appears to be considerable variation between individual specimens, and hence, it is often preferable to
pay more attention to these rather than to
base conclusions on lumped data alone.
This is illustrated by such plots in Figure
14 which might be taken to indicate that
there was no significant change in ventilatory frequency, whereas this seems to be
the case in at least three of the four individual plots shown.
The relationship between the cardiac
and ventilatory rhythms has probably been
investigated more extensively in the dogfish than in any other fish. Recently a convenient method of expressing these relationships using polar coordinates has been
developed (Hughes, 19726), Both this study
and others (Satchell, 1971; Taylor and Butler, 1971) support the view that over the
long term there is not always such a definite relationship between the two rhythms
as had been previously thought. The increase in coupling during hypoxia which
has sometimes been suggested is not so
marked, nor so well defined as that illustrated for the trout (Fig. 6) by comparables analyses. A polar diagram (Fig. 15)
for ten individual dogfish shows no clear
distinction between the phase angles or
the per cent coupling determined during
periods of normoxia or hypoxia. Looked
at in further detail, however, there is evidence for an increase in coupling in at
least eight of the ten cases (Table 1), but
more detailed analysis is required to decide whether or not this is statistically significant.
486
GEORGE M. HUGHES
HI
DOGFISH
22 2.0
8 1.5
LLJ
1.0
LU
rr
0
150
50
02TENSION(mm.Hg.)
FIG. 14. Scyliorhinus canicula. Effect of reduced
O2 tension of inspired water on ventilatory frequency and amplitude of oro-branchial pressure. The
first two graphs are for four individual specimens
and the two on the right show the mean (± S.E.)
of summed results of experiments for six speci-
mens. Note the variability in response of individual specimens; an increase in the frequency and
amplitude of the pressure recorded is generally
found at least during the early stages of hypoxia.
Clearly we may find as many variations
among elasmobranchs as are known for
teleosts. Rays are probably more adapted
to hypoxic conditions than are dogfish and,
certainly, sharks. The electric ray (Torpedo
marmorata), for example, often lies partly
buried at the sea bottom, and some experiments carried out recently showed that
even during prolonged hypoxia the fish remained relatively "unmoved" at P02's which
would have caused a dogfish to swim quite
violently. A significant increase both in
frequency and in amplitude of the ventilatory pressures was recorded during these
experiments (Fig. 16), but the cardiac
rhythm generally remained almost constant
and contrasts with the well-defined bradycardia of dogfish.
Thus, although evidence is strongly in
favor of the existence of counterflow at the
gill exchangers of both elasmobranchs and
teleost fish, there are likely to be differences
when the patterns of water and blood are
considered at the secondary lamellar level.
This has been indicated by differences in
the effect of hypoxia on arterial blood pressure (Piiper et al., 1970) and on the relationship between the cardiac and ventilatory pumps.
The development of a more quantitative
HVPOXIA IN FISH
487
/ \
FIG. 15. Scyliorhinus canicula. Polar diagram showing the per cent coupling and phase angle between
the cardiac and ventilatory cycles of ten specimens,
TORPEDO
g 1-5
150
100
50
16
30
50
75
FIG. 16. Torpedo marmorata. Effect of lowering
P o of the inspired water on the ventilatory (V)
and cardiac (H) frequencies together with changes
in amplitude of the pressure recorded from the
oro-branchial cavity.
subjected to gradual hypoxia. Data for normoxic
and hypoxic conditions are indicated by different
symbols.
expression for the coupling between these
two rhythms should be of value in this
respect, but ultimately we need to know
the capacity flow rates of the blood and
water at the exchange surfaces themselves.
So far nearly all measurements relate to
the total flows into and out of the gill system as a whole, and in further investigations we must attempt to make measurements at the unit level.
Thus, the goal of our efforts during the
next decade remains the same as was indicated by the theoretical analysis of ten
years ago. The mechanisms for gas exchange in fish gills, although simpler in
some ways than at the mammalian alveolus,
have been shown to be far more complex
than was first believed. The comparative
respiratory physiologist need not approach
488
GEORGE M.
TABLE 1. Percentage coupling between the cardiac
and ventilatory rhythms of the dogfish Scyliorhiiras canicula. The mean values and standard
errors for 11 specimens are given under both
normoxic and hypoxic conditions. The total number
(N) of recordings analysed is also shown. Couplings for individual fish are given and the larger
of the two is italicized.
Dogfish
Normoxia
XXIII
XXIV
29.78
6.16
35.9
5.62
8.93
16.35
XXV
XXVI
XXVII
XXIX
XXXI
XXXIII
XXXIV
XXXV
XXXVI
Hypoxia
6.83
39.5
6.72
6.85
81.S
54.65
17.63
9.4
3.1
11.67
5.1
9.67
7.7
5.59
16.18
Dogfish cardio-ventilatory coupling
All
Hypoxia
Normoxia
All normoxia
Mean (%)
(S.E.M.)
N
14.701
14.298
12.780
15.209
(1.710)
(2.238)
(2.272)
(2.676)
79
44
30
35
the despair o£ his mammalian counterpart,
however, for there remain myriads of fresh
species to investigate, each of which can
yield one more fragment of the grand
mosaic.
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