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Respiratory responses to progressive ambient hypoxia in the sturgeon, Acipenser baeri
Respiration Physiology, 91 (1993) 71-82 © 1993 Elsevier Science Publishers B.V. All fights reserved. 0034-5687/93/$05.00
71
RESP 01981
Respiratory responses to progressive ambient hypoxia in the sturgeon, Acipenser baeri G. Nonnotte a, V. Maxime b, J. P. Truchot a, P. Williot c and C. Peyraud b aLaboratoire de Neurobiologie et Physiologie Compardes, CNRS URA 1126 et Universit~ de Bordeaux, I, Arcachon, France, b Laboratoire de Physiologie Animale, CNR S URA 648 et Universitd de Bretagne Occidentale, Brest, France, and CCEMAGREF, Division Aquaculture et P~che, Gazinet, France (Accepted 12 August 1992) Abstract. Changes in respiratory and acid-base variables were studied in siberian sturgeon, Acipenser baeri, during progressive deep hypoxia followed by recovery under normoxic conditions. During hypoxia, both ventilatory frequency and amplitude increased and this sturgeon was able to maintain standard oxygen consumption down to a low critical level of ambient Po2 (Pwo2<40 mmHg). During the posthypoxic period, an 02 debt was repaid by an elevated oxygen consumption (nearly double control value at 1 h), indicating that a shift to anaerobic metabolism had occurred during exposure to severe hypoxia. Gradually increasing ambient hypoxia initially induced a respiratory alkalosis. Below the critical Pwo2 level and during normoxic recovery, a sudden flush of lactate into the blood was associated with a typical metabolic acidosis which was almost totally compensated 3.5 h after return to normoxia. Thus, as for most other fish, respiratory responses of the sturgeon to progressive hypoxia reveal a typical 02 regulatory behavior.
Acid base, hypoxia; Control of breathing, hypoxia; Fish, sturgeon (Acipenser baeri); Hypoxia, respiration, acid base
With respect to the relationship between oxygen consumption and ambient oxygen tension, aerobic organisms have been classically described as oxyconformers when their 02 consumption varies directly with ambient P02, or oxyregulators if 02 consumption is independent of this factor, at least within a certain range above a critical 02 pressure, Pc. Oxyregulation is made possible by a number of compensatory responses increasing the various 02 conductances in the gas exchange system and allowing maintenance of an unchanged tissue 02 supply, in spite of reduced ambient 02 availability (see Dejours, 1981). Oxyconformity throughout the whole P02 range, or failure of oxyregulation below the Pc, result from either the absence or the limitation of such mechanisms, so that oxygen supply can no longer match oxygen demand. In these cases, tissue energy expenditure may either be depressed or more or less maintained by shifting to anaerobic metabolism, at the price of a so-called 02 debt which must be repaid at return to normoxic conditions. Correspondence to: G. Nonnotte, Laboratoire de Neurobiologie et Physiologie Comparres, place du Dr Peyneau, 33120 Arcachon, France.
72 Oxyconformity has rarely been described unambiguously in vertebrates (see Prosser, 1973; Ultsch etal., 1981). One of the best documented examples is the sturgeon Acipenser transmontanus, in which both 02 consumption and gill water flow rate were reported to be reduced steadily with declining ambient oxygen tension (Burggren and Randall, 1978). Furthermore, the absence of any 02 debt repayment and of any decrease of blood pH indicated that a reduced total energy expenditure took place during hypoxic exposure, rather than a shift to anaerobic metabolism maintaining energy production. Although based on clear and apparently consistent findings and fitting well the ancientness of the Chondrostei among vertebrate groups, this notion of oxyconformity being a characteristic of sturgeons was not supported by a large body of earlier data from Russian workers (see Vinberg, 1956; Klyashtorin, 1981) and has also been challenged by a more recent investigation on the same species (Ruer et al., 1987). However, these criticisms were based only on measurements of 02 consumption as a function of 02 tension. Therefore, in order to settle this problem, we have investigated as completely as possible the respiratory and acid-base responses to declining oxygen tension in the Siberian sturgeon Acipenser baeri.
Materials and methods
Fish and water. Three-year-old sturgeon (Acipenser baeri), each weighing about 1.8 kg were obtained from the experimental hatchery of C E M A G R E F (St Seurin sur l'Isle, Gironde, France). Prior to experimentation, they were maintained for three weeks in a large outdoor circular tank supplied with well-aerated running tap water at seasonal temperature. They were fed once a day (1 ~o of live weight) with a commercial diet (Aqualim, France) until 48 h before the experiments. Fish were anaesthetized by immersion in 2-phenoxyethanol (1/200) in an air-saturated solution for approximatively 10 min. After weighing, they were transferred to an operating table where the gills were continuously irrigated with aerated water containing a maintenance dose of 2-phenoxyethanol (1/600). Each fish was placed in a moist tray and chronically fitted with a dorsal aortic cannula (PE50) implanted by blind puncture at the caudal level (Williot, unpublished). A polyethylene PE-190 catheter was then inserted through the cleithrum bone into the branchial cavity. The dorsal aortic cannula was flushed daily with a saline containing 10 I.U..ml -~ of heparin (lithium salt; Sigma) in a Ringer solution adapted for sturgeon. Following the operation, the animals were placed on a perforated Plexiglas platform and transferred into a cylindrical darkened respirometer (37 L) in which aerated tap water was circulated in an open circuit at a flow rate of 1 L.min -1 at 15 °C. They were allowed to recover there for 24-48 h. An immersed pump was placed into the respirometer to ensure homogenization of the water. The size of the respirometer was such that the fish remained at rest and hence could be regarded as being in a state of standard metabolism. During experimental periods, water was re-circulated by a centrifugal
73 pump through the respirometer at a constant flow rate (1 L.min-1) from a large thermostatted (15 °C) tank (120 L), in a closed circuit. The acid-base balance of this water was automatically regulated with a p H - P c o 2 stat (Dejours et al., 1978). As CO2-free air was bubbled constantly, this device intermittently injected small quantities of pure CO2 into the water through a solenoid valve set to open when water pH rose above a preset value. The titratable alkalinity of the water (TAw) was measured each day using a modified Gran titration procedure and adjusted to a constant value. This allowed water pH and carbon dioxide tension to be regulated at the same value in normoxic and hypoxic conditions (pH=8.050, TAw = 1.90 meq. L - l ; Pco2 = 0.75 mmHg). The components of the carbonate system were characterized from the TAw and pH measurements (Truchot and DuhamelJouve, 1980).
Experimental protocols and measurements. Fish (n = 7) were sequentially exposed to various stages of environmental hypoxia (Pwo2 = 60, 40 and 20 Torr) obtained by bubbling pure nitrogen in the thermostatted tank in which immersed marbles allowed quick 02 depletion (within 10-15 min). Blood sampling and simultaneous measurements of respiratory and acid-base variables at each stage of imposed hypoxia were performed after a 1-h period of steady state in the water circuit. During the return to normoxic conditions, the same variables were then measured at 10, 20, 30 min, 1, 2 and 3.5 h. The average time course of Pwo2 variations into the respirometer during progressive hypoxia and normoxic recovery is depicted in Fig. 1.
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74 Measurements and calculation of respiratory variables. Standard oxygen consumption (1rio2) was determined at steady state by measuring the 0 2 partial pressure difference between water flowing into (Po2 inlet) and out of (Po2 outlet) the respirometer. A peristaltic pump allowed alternate sampling of the inlet and outlet water which passed at a constant flow rate (5 ml.min-1) through a thermostatted Po -measuring cell (Radiometer E 5046). Oxygen consumption was calculated according to the relation: Mo: = (Po2inlet - P o : outlet)'Qw'~woJ(Body weight) where Qw is the constant water flow through the respirometer and atWo: is the O2 solubility in freshwater = 2.01 #mol.L-1.Torr -x at 15 °C. Frequency (VF) and amplitude (VA) of ventilatory movements were obtained from continuous recording of hydrostatic pressure changes in the branchial cavity, measured by connecting the catheter inserted through the cleithrum bone to a Honeywell 156PC pressure transducer. The maximal change of pressure observed during each breathing cycle was used as an estimation of ventilatory amplitude. An average value of this variable measured for each fish in steady state during a 10-min period before the exposure to hypoxia was used as an arbitrary unit to estimate ventilatory amplitude changes which occurred during hypoxia. Partial pressure of oxygen in arterial blood (Pao~) was determined on a 200 #1 blood sample using a thermostatted Po-measuring cell (Radiometer E 5046). After each measurement, the blood was returned to the fish. Measurements of blood acid-base characteristics and plasma ion concentrations. A blood sample (about 240 #1) was drawn from the arterial cannula into three heparinized glass capillaries. Blood pH was measured immediately from one glass capillary using a Radiometer microelectrode G 222A calibrated with Radiometer precision buffer solutions type S 1500 and S 1510 and connected to a Radiometer PHM72 pH meter. The blood partial pressure of carbon dioxide (Paco2) was determined by the Astrup method (Astrup, 1956) using the two other capillaries. The bicarbonate concentration in arterial blood was calculated by the Henderson-Hasselbalch equation, using a CO2 solubility coefficient and a operational pK' from Boutilier et al. (1985). The measurement of plasma ionic concentrations needed an additional arterial blood sample of about 200/A. Chloride concentration [C1- ] was measured with a chloride titrator Radiometer CMT 10. Sodium [Na ÷ ] and potassium [K + ] concentrations were obtained with a Beckman flame photometer. Lactate concentration ([Lact-]) was measured by an enzymatic method (Boehringer-Mannheim, Kit 139 084). Results
Hypoxia. When acclimated to the respirometer in normoxia, the sturgeon showed a spontaneously interrupted ventilatory pattem with ventilatory periods consisting of 6-8 breathing cycles lasting 10-15 sec, separated by apneic periods of about the same duration. Usually, a progressive decrease of the depth of ventilation occurred during the ventilatory periods, as illustrated by the typical sample of ventilation recording
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Fig. 2. Typical examples of ventilatory patterns recorded in normoxia (A) and in hypoxia (B) at Pwo2 = 60 mmHg.
shown in Fig. 2A. When Po2 into the respirometer (Pwo2) began to decrease, the periodic breathing pattern was quickly altered, with reduced duration of apneas and lengthening of the ventilatory periods, until complete disappearance of apneas (Fig. 2B). This effect was associated with a highly significant increase in overall as well as intraburst ventilatory frequency. The amplitude of the breathing movements also increased significantly (Fig. 3). During the gradually imposed hypoxia, the standard oxygen consumption did not significantly differ from normoxia until the stage of 40 mmHg Pwo2. Then, it markedly decreased, so that the value measured at 20 mmHg was about half of control (Fig. 4). Thus, the critical oxygen tension below which aerobic metabolism became dependent on ambient Po2 was lower than 40 mmHg and higher than 20 mmHg. Oxygen tension in arterial blood (Pao2) decreased progressively until 40 mmHg, then more abruptly below the critical oxygen tension (Fig. 5). Carbon dioxide tension in arterial blood (Paco:) initially decreased while pHa rose significantly (Fig. 6), in parallel with the increases of both ventilatory frequency and amplitude. Then, at the lowest Pwo~ value, pHa returned to a level not significantly different from control while plasma lactate increased sharply (Fig. 7). Plasma Na ÷ , K ÷ , and C1- concentrations remained unchanged throughout hypoxia exposure. When Pwo2 was returned to normoxia, the ventilatory frequency and amplitude of pressure changes in the branchial cavity remained at their highest level for about 30 min, then fell back progressively to control values. However, as shown in Fig. 3, the time course of these ventilatory changes showed an hysteresis compared to that observed during progressive hypoxia, with higher values at the same Pwo2 levels.
B a c k to normoxia.
76
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Fig. 3. Ventilatory frequency (V.F.) and amplitude (V.A.) during normoxia (Nx), progressive hypoxia ( 0 ) and return to normoxia (O). Stages of hypoxia (I, II, III) and time into normoxic recovery are indicated (see Fig. 1). Normoxic ventilatory frequency (Nx) is calculated as an overall frequency, i.e. in reference to total time including apneas. Stars indicate a significant difference (P< 0.01) compared to normoxic values.
Measurements of standard oxygen consumption could be performed only 1 h after the end of hypoxic exposure, because during the initial period of restoration of normoxia, the time course of variations of Pwo2 was too rapid to get a steady state of outlet Po2 during measurement time, and consequently reliable values of Mo2 could not be obtained. The mean value of IVlo2 observed at 1 h was about twice compared with control while Pwo2 was only a little lower than the normoxic level (Fig. 4). Then, 10Io2 slowly returned within 3.5 h to control levels. Arterial 02 partial pressure gradually increased toward pre-hypoxic levels in parallel with the progressive increase of Pwo2. However, as observed for ventilatory frequency and amplitude, there was a difference between pre- and post-hypoxic Pat2 at the same Pwo~, with lower values during recovery than during progressive hypoxia at the same Pwo~ (Fig. 5). During the initial 30 min of the post-hypoxic period, pHa fell sharply to its lowest value (7.68 + 0.06), then gradually increased toward the control pre-hypoxic level
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Fig. 4. Changes in standard oxygen consumption (iVlo2) resulting from progressive hypoxia (O) and return to normoxic conditions ( 0 ) . Symbols and labels as in Fig. 3. 80
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Fig. 5. Changes in 02 tension in arterial blood (Paoz) resulting from progressive hypoxia (O) and return to normoxic conditions (O). The dotted line is the line of equality between blood and water Po2 values. Symbols and labels as in Fig. 3.
(Figs. 6 and 7). During the first 30 min into normoxic recovery, Paco2 remained almost steady at the lowest level observed during the deepest hypoxic stage. This coincided with the maximal hyperventilatory response. Then, ventilation decreased whilst Paco 2 returned to its normoxic level. Sodium, potassium and chloride plasma concentrations did not change significantly from control values ( [ N a + ] = 119.2+ 1.2mEq.L-~; [ K + ] = 2.32 + 0.07 mEq. L - 1; [ C1 - ] = 103.6 + 2.3 mEq. L - 1) during all the hypoxic and posthypoxic periods. By contrast, plasma lactate concentration continued to rise at the beginning of the post-hypoxic period to reach a value about 8 times higher than control at 10 min. Later on, [Lact- ] steadily returned to the normoxic reference value, which was attained by 3.5 h (Fig. 7).
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pHa Fig. 6. Diagram [ H C O 3 + C O 2 ] vs pH illustrating acid-base changes in arterial blood resulting from progressive hypoxia and return to normoxic conditions. Filled symbols: normoxia (m) and hypoxia stages I (&), II (O) and III (V). Hollow symbols: back to normoxia, 10min (©), 20min (rl), 30min (A) 1 h (~), 2 h (~7) and 3.5 h (~).
Discussion The sturgeon: a typical oxyregulator. Our results clearly demonstrate that sturgeon is able to maintain a standard 02 uptake during progressive hypoxia down to a critical ambient Po2 of 20-40 mmHg. This is made possible thanks to a marked hyperventilation, as shown by increases in both frequency and amplitude of gill respiratory movements, which lead to alkalosis and hypocapnia. Below the Pc, anaerobic metabolism is initiated, as indicated by lactate release to the blood, metabolic acidosis and repayment of an 02 debt after return to normoxia. These findings are in striking contrast with those reported by Burggren and Randall (1978) for similarly-sized specimens of another sturgeon species, Acipenser transmontanus. These authors found a steady decrease not only of oxygen uptake but also of gill ventilatory flow rate during progressive hypoxia and concluded that 'sturgeon is unusual amongst vertebrates in that it becomes an oxygen conformer as soon as Po2 is reduced below ambient'. The absence of any 02 debt repayment also led them to conclude that no shift to anaerobic metabolism takes place but that 'sturgeon simply shuts down metabolism' during hypoxia. We suggest that such a complete disagreement in results and interpretation between the two equally consistent sets of data might be due to the techniques used to study ventilation. Namely, the very flexible tubing sewn on the retractile mouth of the fish by
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[DWo2 (mmHg) Fig. 7. Changes in arterial pH (pHa) and plasma lactate concentration resulting from progressivehypoxia (0) and return to normoxic conditions (©). Symbols and labels as in Fig. 3.
these authors for direct measurement of gill ventilatory flow may have restricted the ability of the sturgeon to achieve the ventilatory adjustments needed to compensate for the decrease of ambient Po2. In support to this interpretation, it must be emphasized that at the same temperature, the mean normoxic ventilatory frequency reported by Burggren and Randall for Acipenser transmontanus was about twice that we observed in A. baeri, and about the same as that we recorded at the maximal hyperventilatory response during hypoxia. Moreover, at the beginning of our experiments, we tried to measure the ventilatory flow rate directly with the same device as that set up by Burggren and Randall (1978). The few fish (n = 3) fitted in this manner never exhibited a periodic ventilation in normoxia, which should be considered as a typical breathing pattern in bottom-dwelling fish such as the sturgeon (see below). In addition, we observed that the increased depth of breathing induced by exposure to hypoxia tended to cause a collapse of the thin wall of the tube inserted around the buccal orifice. Such an effect could be suspected to add a resistance to flow in proportion of ventilatory efforts and thus to account for the decreased ventilation observed by Burggren and Randall in hypoxic sturgeon. This interpretation is also borne out by the Pao~ values they report, which appear markedly lower than ours at similar levels of ambient Po2-
80 We cannot, however, explain why they found n o 0 2 debt repayment after hypoxia. Furthermore, in absence of any reported lactate measurement, the metabolic status of their fish cannot be evaluated. Effects of gradual hypoxia. Various respiratory characteristics of the sturgeon fall into a range corresponding to that reported for poorly active, bottom-dwelling teleosts. When appropriately corrected for body size (0.67 for the mass exponent) and temperature (Q10=2) differences, standard normoxic 1(/lo2 of Acipenser baeri at 15 °C (29.09#mol.min-l'kg-1) was lower than that of A. transmontanus (42.06 ktmol'min-~'kg -1, Burggren and Randall, 1978), but ranged between values reported for active fish (for example, salmon: 44.08 #mol-min-1 .kg-l, Maxime et al., 1990) and sluggish fish (for example, eel: 15.67 #mol.min-l'kg -1, Le Moigne et al., 1986). This could probably be related to the active growth of young sturgeon in spite of their relatively sluggish behavior. Normoxic sturgeon exhibited a spontaneously interrupted ventilation in normoxia. Such a periodic breathing rhythm has been observed in many teleosts in resting conditions: tench Tinca tinca (Shelton and Randall, 1962); carp Cyprinus carpio (Peyraud and Serfaty, 1964; Peyraud-WaRzenegger, 1979); eel Anguilla australis (Forster, 1981; Hipkins and Smith, 1983) and can be regarded as a typical eupneic ventilation in bottom-dwelling species known for their reduced motor activity and their ability to withstand hypoxic conditions. That normoxic A. baeri exhibited this pattern indicates that our fish were in a satisfactory physiological state and not unduly disturbed by experimental conditions. Increased gill ventilation has been commonly reported in fish subjected to a lowering of ambient Po2. In active species such as trout (Holeton and Randall, 1967) and Atlantic salmon (Maxime et al., unpublished), the increase in ventilatory flow rate resuits from a much more important rise of stroke volume than of frequency of breathing movements. This pattern of response appears the more marked the more active the species. The sturgeon showing about an equal increase in both frequency and amplitude displayed a pattern of ventilatory adjustments rather similar to that observed in bottom-dwelling species such as carp (Peyraud, 1965; Lomholt and Johansen, 1979) and tench (Randall and Shelton, 1963). Hyperventilation of the gills in hypoxic sturgeon led to a typical respiratory alkalosis with a decreased blood Pco2 and an increased pH. This helped maintain tissue 02 supply by a left shift of the 02 ' dissociation curve that kept arterial saturation high. According to data reported by Burggren and Randall (1978) forAcipenser transmontanus as well as to unpublished results by Salin et al. in A. baeri, blood 02 affinity is relatively high in sturgeon, so that the mean Pao2 = 23 mmHg recorded at the second stage of imposed hypoxia (Pwo2 = 40 mmHg) should correspond to a Hbo: per cent saturation of about 65 Yo. This is probably a reason for a relatively low critical level of ambient Po: in Acipenser baeri. Our Pc value at 15 ° C, 20-40 mmHg, is lower than that reported for active fish such as trout (Holeton and Randall, 1967), but of the same order of magnitude as that found for Acipenser galdenstiidti (Klyashtorin, 1981) and for fish
81 commonly experiencing environmental hypoxia in natural conditions such as the goldfish (Fry and Hart, 1948). Several aspects of the changes of respiratory variables during normoxic recovery deserve comment. Despite a short exposure time to the deepest hypoxic stage below Pc (1 h), the sturgeon obviously repaid an 02 debt with an elevated Mo2 lasting about 3.5 h. This 02 debt repayment was closely correlated with both the clearance of blood lactate and the recovery of blood pH (Fig. 7). It is additionally worth noting that blood lactate and the associated metabolic acidosis peaked together not during hypoxia but 10-30 min into normoxic recovery, suggesting a delayed but simultaneous release of both lactate ions and metabolic acid generated anaerobically during the hypoxic exposure. This contrasts with the asynchronous release of lactate and metabolic acid found in most teleosts during recovery from exhausting exercise (see Truchot, 1987). This is also in keeping with the absence of any significant change of plasma ion concentrations during either progressive hypoxia or normoxic recovery, which suggests that the generation and subsequent dissipation of the metabolic acidosis mainly involve internal metabolic events but no or minimal ionic exchanges. At the same ambient Po2, the values of Pao2 measured during normoxic recovery were lower than those found during progressive hypoxia (Fig. 5). This probably resulted from the increased tissue 02 uptake which must have substantially reduced oxygenation of the venous blood. As shown by the recorded ventilatory frequencies and amplitudes, the gill water flow rate also exhibited an hysteresis during normoxic recovery, but with values higher than during gradual hypoxia, at the same ambient Po2- This enhanced hyperventilatory response might be explained by a stronger hypoxic drive due to lower arterial Po2 values, perhaps reinforced by a persistent acidosis which could disappear only after metabolic processing of released lactic acid. This relatively long-lasting, post-hypoxic hyperventilation also delayed the restoration of normoxic Paco2 values. In conclusion, our data indicate that Acipenser baeri behaves as a typical 0 2 regulator when subjected to gradual hypoxia. This strengthens the idea (Ultsch et aL, 1981) that the few cases of 02 conformity reported for lower vertebrates may result from experimental artefacts and should be reexamined.
B a c k to norrnoxia.
References Astrup, P. (1956). A simpleelectrometrictechniquefor the determinationof carbon dioxidetension in blood and plasma, total content of carbon dioxidein plasma and bicarbonatecontent in 'separated' plasma at a fixed dioxidetension (40 mmHg). Scand..I. Clin. Invest. 8: 33-43. Boutilier, R.G., G.K. Iwama, T.A. Heming and D.J. Randall (1985). The apparent pK of carbonic acid in rainbow trout blood plasma between 5 and 15 °C. Respir. Physiol. 61: 237-254. Burggren, W.W. and D.J. Randall (1978). Oxygenuptake and transport during hypoxic exposure in the sturgeon Acipenser transmontanus. Respir. Physiol. 34: 171-183. Dejours, P., J. Armand and J.P. Gendner (1978). Importancede la rrgulation de l'rquilibreacide-basede l'eau ambiante pour l'rtude des 6changes respiratoireset ioniques des animaux aquatiques. C.R. Acad. Sci. Paris 287: 1397-1399.
82 Dejours, P. (1981). Principles of Comparative Respiratory Physiology. Amsterdam, New York, Oxford: Elsevier, 265 pp. Forster, M.E. (1981). Oxygen consumption and apnea in the shortfin eel, Anguilla australis schmidtii. New Zeal. J. Mar. Freshw. Res. 15: 85-90. Fry, F.E.J. and J. S. Hart (1948). The relation of temperature to oxygen consumption in the goldfish. Biol. Bull. 94: 66-77. Hipkins, S.F. and D.G. Smith (1983). Cardiovascular events associated with spontaneous apnea in the Australian short finned eel (Anguilla australis). J. Exp. ZooL 227: 339-348. Holeton, G. F. and D.J. Randall (1967). The effect of hypoxia upon the partial pressure of gases in the blood and water afferent and efferent to the gills of rainbow trout. J. Exp. BioL 46: 317-327. Klyashtorin, L.B. (1981). The ability of sturgeons (Acipenseridae) to regulate gas exchange. J. Ichthyol. 21: 141-144. Le Moigne, J., P. Soulier, M. Peyraud-Wa~'tzenegger and C. Peyraud (1986). Cutaneous and gill 02 uptake in the European eel (Anguilla anguilla L.) in relation to ambient Po2, 10-400 Torr. Respir. Physiol. 66: 341-354. Lomholt, J. P. and K. Johansen (1979). Hypoxia acclimation in carp. How it affects 02 uptake, ventilation, and 02 extraction from water. Physiol. Zool. 52: 38-49. Maxime, V., M. Peyraud-Wartzenegger, G. Clalreaux and C. Peyraud (1990). Effect of rapid transfer from seawater to freshwater on respiratory variables, blood acid-base status and 02 affinity of hemoglobin in atlantic salmon (Salmo salar L.). J. Comp. Physiol. B 160:31-39. Peyraud, C. and A. Serfaty (1964). Le rythme respiratoire de la carpe (Cyprinus carpio L.) et ses relations avec le taux de l'oxyg~ne dissous dans le biotope. Hydrobiologia 23: 165-178. Peyraud, C. (1965). Recherches sur la rrgulation des mouvements respiratoires chez quelques t616ostrens: Analyse du r6flexe opto-respiratoire. Thrse de Doctorat d~s Sciences. Universit6 de Toulouse, 258 pp. Peyraud-Waitzenegger, M. (1979). Simultaneous modifications of ventilation and arterial Po2 by catecholamines in the eel, Anguilla anguilla L.: participation of alpha and beta effects. J. Comp. Physiol. B 129: 343-354. Prosser, C.L. (1973). Comparative Animal Physiology, 3rd Edn., Philadelphia: Saunders, 966 pp. Randall, D.J. and G. Shelton (1963). The effects of changes in environmental gas concentrations on the breathing and heart rate of a teleost fish. Comp. Biochem. Physiol. 9: 229-239. Ruer, F.M., J.J. Cech and S.I. Doroshov (1987). Routine metabolism of the white sturgeon, Acipenser transmontanus: effect of population density and hypoxia. Aquaculture 62: 45-52. Shelton, G. and D.J. Randall (1962). The relation between heart beat and respiration in teleost fish. Comp. Biochem. Physiol. 7: 237-250. Truchot, J.P. and A. Duhamel-Jouve (1980). Oxygen and carbon dioxide in the marine intertidal environment: diurnal and tidal changes in rockpools. Respir. Physiol. 39: 241-254. Truchot, J.P. (1987). Comparative Aspects of Extracellular Acid-Base Balance. Zoophysiology, Vol. 20. Berlin, Heidelberg: Springer Verlag, 262 pp. Ultsch, G.R., D.C. Jackson and R. Moalli (1981). Metabolic oxygen conformity among lower vertebrates: the toadfish revisited. J. Comp. Physiol. B 142: 439-443. Vinberg, G.G. (1956). Rate of metabolism and food requirements of fishes. Fisheries Research Board of Canada, Translation Series No. 194, 202 pp.
71
RESP 01981
Respiratory responses to progressive ambient hypoxia in the sturgeon, Acipenser baeri G. Nonnotte a, V. Maxime b, J. P. Truchot a, P. Williot c and C. Peyraud b aLaboratoire de Neurobiologie et Physiologie Compardes, CNRS URA 1126 et Universit~ de Bordeaux, I, Arcachon, France, b Laboratoire de Physiologie Animale, CNR S URA 648 et Universitd de Bretagne Occidentale, Brest, France, and CCEMAGREF, Division Aquaculture et P~che, Gazinet, France (Accepted 12 August 1992) Abstract. Changes in respiratory and acid-base variables were studied in siberian sturgeon, Acipenser baeri, during progressive deep hypoxia followed by recovery under normoxic conditions. During hypoxia, both ventilatory frequency and amplitude increased and this sturgeon was able to maintain standard oxygen consumption down to a low critical level of ambient Po2 (Pwo2<40 mmHg). During the posthypoxic period, an 02 debt was repaid by an elevated oxygen consumption (nearly double control value at 1 h), indicating that a shift to anaerobic metabolism had occurred during exposure to severe hypoxia. Gradually increasing ambient hypoxia initially induced a respiratory alkalosis. Below the critical Pwo2 level and during normoxic recovery, a sudden flush of lactate into the blood was associated with a typical metabolic acidosis which was almost totally compensated 3.5 h after return to normoxia. Thus, as for most other fish, respiratory responses of the sturgeon to progressive hypoxia reveal a typical 02 regulatory behavior.
Acid base, hypoxia; Control of breathing, hypoxia; Fish, sturgeon (Acipenser baeri); Hypoxia, respiration, acid base
With respect to the relationship between oxygen consumption and ambient oxygen tension, aerobic organisms have been classically described as oxyconformers when their 02 consumption varies directly with ambient P02, or oxyregulators if 02 consumption is independent of this factor, at least within a certain range above a critical 02 pressure, Pc. Oxyregulation is made possible by a number of compensatory responses increasing the various 02 conductances in the gas exchange system and allowing maintenance of an unchanged tissue 02 supply, in spite of reduced ambient 02 availability (see Dejours, 1981). Oxyconformity throughout the whole P02 range, or failure of oxyregulation below the Pc, result from either the absence or the limitation of such mechanisms, so that oxygen supply can no longer match oxygen demand. In these cases, tissue energy expenditure may either be depressed or more or less maintained by shifting to anaerobic metabolism, at the price of a so-called 02 debt which must be repaid at return to normoxic conditions. Correspondence to: G. Nonnotte, Laboratoire de Neurobiologie et Physiologie Comparres, place du Dr Peyneau, 33120 Arcachon, France.
72 Oxyconformity has rarely been described unambiguously in vertebrates (see Prosser, 1973; Ultsch etal., 1981). One of the best documented examples is the sturgeon Acipenser transmontanus, in which both 02 consumption and gill water flow rate were reported to be reduced steadily with declining ambient oxygen tension (Burggren and Randall, 1978). Furthermore, the absence of any 02 debt repayment and of any decrease of blood pH indicated that a reduced total energy expenditure took place during hypoxic exposure, rather than a shift to anaerobic metabolism maintaining energy production. Although based on clear and apparently consistent findings and fitting well the ancientness of the Chondrostei among vertebrate groups, this notion of oxyconformity being a characteristic of sturgeons was not supported by a large body of earlier data from Russian workers (see Vinberg, 1956; Klyashtorin, 1981) and has also been challenged by a more recent investigation on the same species (Ruer et al., 1987). However, these criticisms were based only on measurements of 02 consumption as a function of 02 tension. Therefore, in order to settle this problem, we have investigated as completely as possible the respiratory and acid-base responses to declining oxygen tension in the Siberian sturgeon Acipenser baeri.
Materials and methods
Fish and water. Three-year-old sturgeon (Acipenser baeri), each weighing about 1.8 kg were obtained from the experimental hatchery of C E M A G R E F (St Seurin sur l'Isle, Gironde, France). Prior to experimentation, they were maintained for three weeks in a large outdoor circular tank supplied with well-aerated running tap water at seasonal temperature. They were fed once a day (1 ~o of live weight) with a commercial diet (Aqualim, France) until 48 h before the experiments. Fish were anaesthetized by immersion in 2-phenoxyethanol (1/200) in an air-saturated solution for approximatively 10 min. After weighing, they were transferred to an operating table where the gills were continuously irrigated with aerated water containing a maintenance dose of 2-phenoxyethanol (1/600). Each fish was placed in a moist tray and chronically fitted with a dorsal aortic cannula (PE50) implanted by blind puncture at the caudal level (Williot, unpublished). A polyethylene PE-190 catheter was then inserted through the cleithrum bone into the branchial cavity. The dorsal aortic cannula was flushed daily with a saline containing 10 I.U..ml -~ of heparin (lithium salt; Sigma) in a Ringer solution adapted for sturgeon. Following the operation, the animals were placed on a perforated Plexiglas platform and transferred into a cylindrical darkened respirometer (37 L) in which aerated tap water was circulated in an open circuit at a flow rate of 1 L.min -1 at 15 °C. They were allowed to recover there for 24-48 h. An immersed pump was placed into the respirometer to ensure homogenization of the water. The size of the respirometer was such that the fish remained at rest and hence could be regarded as being in a state of standard metabolism. During experimental periods, water was re-circulated by a centrifugal
73 pump through the respirometer at a constant flow rate (1 L.min-1) from a large thermostatted (15 °C) tank (120 L), in a closed circuit. The acid-base balance of this water was automatically regulated with a p H - P c o 2 stat (Dejours et al., 1978). As CO2-free air was bubbled constantly, this device intermittently injected small quantities of pure CO2 into the water through a solenoid valve set to open when water pH rose above a preset value. The titratable alkalinity of the water (TAw) was measured each day using a modified Gran titration procedure and adjusted to a constant value. This allowed water pH and carbon dioxide tension to be regulated at the same value in normoxic and hypoxic conditions (pH=8.050, TAw = 1.90 meq. L - l ; Pco2 = 0.75 mmHg). The components of the carbonate system were characterized from the TAw and pH measurements (Truchot and DuhamelJouve, 1980).
Experimental protocols and measurements. Fish (n = 7) were sequentially exposed to various stages of environmental hypoxia (Pwo2 = 60, 40 and 20 Torr) obtained by bubbling pure nitrogen in the thermostatted tank in which immersed marbles allowed quick 02 depletion (within 10-15 min). Blood sampling and simultaneous measurements of respiratory and acid-base variables at each stage of imposed hypoxia were performed after a 1-h period of steady state in the water circuit. During the return to normoxic conditions, the same variables were then measured at 10, 20, 30 min, 1, 2 and 3.5 h. The average time course of Pwo2 variations into the respirometer during progressive hypoxia and normoxic recovery is depicted in Fig. 1.
160
Normoxia (Nx)
140
Back to normoxia
Hypoxic exposure
f 120
t
100
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60 40
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0
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I
1 hour
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TIME Fig. 1. Averagetime course of changes of Pwo: during experiments.Arrows indicate the periods at which recording and blood sampling were performed.
74 Measurements and calculation of respiratory variables. Standard oxygen consumption (1rio2) was determined at steady state by measuring the 0 2 partial pressure difference between water flowing into (Po2 inlet) and out of (Po2 outlet) the respirometer. A peristaltic pump allowed alternate sampling of the inlet and outlet water which passed at a constant flow rate (5 ml.min-1) through a thermostatted Po -measuring cell (Radiometer E 5046). Oxygen consumption was calculated according to the relation: Mo: = (Po2inlet - P o : outlet)'Qw'~woJ(Body weight) where Qw is the constant water flow through the respirometer and atWo: is the O2 solubility in freshwater = 2.01 #mol.L-1.Torr -x at 15 °C. Frequency (VF) and amplitude (VA) of ventilatory movements were obtained from continuous recording of hydrostatic pressure changes in the branchial cavity, measured by connecting the catheter inserted through the cleithrum bone to a Honeywell 156PC pressure transducer. The maximal change of pressure observed during each breathing cycle was used as an estimation of ventilatory amplitude. An average value of this variable measured for each fish in steady state during a 10-min period before the exposure to hypoxia was used as an arbitrary unit to estimate ventilatory amplitude changes which occurred during hypoxia. Partial pressure of oxygen in arterial blood (Pao~) was determined on a 200 #1 blood sample using a thermostatted Po-measuring cell (Radiometer E 5046). After each measurement, the blood was returned to the fish. Measurements of blood acid-base characteristics and plasma ion concentrations. A blood sample (about 240 #1) was drawn from the arterial cannula into three heparinized glass capillaries. Blood pH was measured immediately from one glass capillary using a Radiometer microelectrode G 222A calibrated with Radiometer precision buffer solutions type S 1500 and S 1510 and connected to a Radiometer PHM72 pH meter. The blood partial pressure of carbon dioxide (Paco2) was determined by the Astrup method (Astrup, 1956) using the two other capillaries. The bicarbonate concentration in arterial blood was calculated by the Henderson-Hasselbalch equation, using a CO2 solubility coefficient and a operational pK' from Boutilier et al. (1985). The measurement of plasma ionic concentrations needed an additional arterial blood sample of about 200/A. Chloride concentration [C1- ] was measured with a chloride titrator Radiometer CMT 10. Sodium [Na ÷ ] and potassium [K + ] concentrations were obtained with a Beckman flame photometer. Lactate concentration ([Lact-]) was measured by an enzymatic method (Boehringer-Mannheim, Kit 139 084). Results
Hypoxia. When acclimated to the respirometer in normoxia, the sturgeon showed a spontaneously interrupted ventilatory pattem with ventilatory periods consisting of 6-8 breathing cycles lasting 10-15 sec, separated by apneic periods of about the same duration. Usually, a progressive decrease of the depth of ventilation occurred during the ventilatory periods, as illustrated by the typical sample of ventilation recording
75
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.
.
.
.
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i
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Fig. 2. Typical examples of ventilatory patterns recorded in normoxia (A) and in hypoxia (B) at Pwo2 = 60 mmHg.
shown in Fig. 2A. When Po2 into the respirometer (Pwo2) began to decrease, the periodic breathing pattern was quickly altered, with reduced duration of apneas and lengthening of the ventilatory periods, until complete disappearance of apneas (Fig. 2B). This effect was associated with a highly significant increase in overall as well as intraburst ventilatory frequency. The amplitude of the breathing movements also increased significantly (Fig. 3). During the gradually imposed hypoxia, the standard oxygen consumption did not significantly differ from normoxia until the stage of 40 mmHg Pwo2. Then, it markedly decreased, so that the value measured at 20 mmHg was about half of control (Fig. 4). Thus, the critical oxygen tension below which aerobic metabolism became dependent on ambient Po2 was lower than 40 mmHg and higher than 20 mmHg. Oxygen tension in arterial blood (Pao2) decreased progressively until 40 mmHg, then more abruptly below the critical oxygen tension (Fig. 5). Carbon dioxide tension in arterial blood (Paco:) initially decreased while pHa rose significantly (Fig. 6), in parallel with the increases of both ventilatory frequency and amplitude. Then, at the lowest Pwo~ value, pHa returned to a level not significantly different from control while plasma lactate increased sharply (Fig. 7). Plasma Na ÷ , K ÷ , and C1- concentrations remained unchanged throughout hypoxia exposure. When Pwo2 was returned to normoxia, the ventilatory frequency and amplitude of pressure changes in the branchial cavity remained at their highest level for about 30 min, then fell back progressively to control values. However, as shown in Fig. 3, the time course of these ventilatory changes showed an hysteresis compared to that observed during progressive hypoxia, with higher values at the same Pwo2 levels.
B a c k to normoxia.
76
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(mmHg)
Fig. 3. Ventilatory frequency (V.F.) and amplitude (V.A.) during normoxia (Nx), progressive hypoxia ( 0 ) and return to normoxia (O). Stages of hypoxia (I, II, III) and time into normoxic recovery are indicated (see Fig. 1). Normoxic ventilatory frequency (Nx) is calculated as an overall frequency, i.e. in reference to total time including apneas. Stars indicate a significant difference (P< 0.01) compared to normoxic values.
Measurements of standard oxygen consumption could be performed only 1 h after the end of hypoxic exposure, because during the initial period of restoration of normoxia, the time course of variations of Pwo2 was too rapid to get a steady state of outlet Po2 during measurement time, and consequently reliable values of Mo2 could not be obtained. The mean value of IVlo2 observed at 1 h was about twice compared with control while Pwo2 was only a little lower than the normoxic level (Fig. 4). Then, 10Io2 slowly returned within 3.5 h to control levels. Arterial 02 partial pressure gradually increased toward pre-hypoxic levels in parallel with the progressive increase of Pwo2. However, as observed for ventilatory frequency and amplitude, there was a difference between pre- and post-hypoxic Pat2 at the same Pwo~, with lower values during recovery than during progressive hypoxia at the same Pwo~ (Fig. 5). During the initial 30 min of the post-hypoxic period, pHa fell sharply to its lowest value (7.68 + 0.06), then gradually increased toward the control pre-hypoxic level
77 50
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PWo2 (mmHg)
Fig. 4. Changes in standard oxygen consumption (iVlo2) resulting from progressive hypoxia (O) and return to normoxic conditions ( 0 ) . Symbols and labels as in Fig. 3. 80
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Fig. 5. Changes in 02 tension in arterial blood (Paoz) resulting from progressive hypoxia (O) and return to normoxic conditions (O). The dotted line is the line of equality between blood and water Po2 values. Symbols and labels as in Fig. 3.
(Figs. 6 and 7). During the first 30 min into normoxic recovery, Paco2 remained almost steady at the lowest level observed during the deepest hypoxic stage. This coincided with the maximal hyperventilatory response. Then, ventilation decreased whilst Paco 2 returned to its normoxic level. Sodium, potassium and chloride plasma concentrations did not change significantly from control values ( [ N a + ] = 119.2+ 1.2mEq.L-~; [ K + ] = 2.32 + 0.07 mEq. L - 1; [ C1 - ] = 103.6 + 2.3 mEq. L - 1) during all the hypoxic and posthypoxic periods. By contrast, plasma lactate concentration continued to rise at the beginning of the post-hypoxic period to reach a value about 8 times higher than control at 10 min. Later on, [Lact- ] steadily returned to the normoxic reference value, which was attained by 3.5 h (Fig. 7).
78 12
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pHa Fig. 6. Diagram [ H C O 3 + C O 2 ] vs pH illustrating acid-base changes in arterial blood resulting from progressive hypoxia and return to normoxic conditions. Filled symbols: normoxia (m) and hypoxia stages I (&), II (O) and III (V). Hollow symbols: back to normoxia, 10min (©), 20min (rl), 30min (A) 1 h (~), 2 h (~7) and 3.5 h (~).
Discussion The sturgeon: a typical oxyregulator. Our results clearly demonstrate that sturgeon is able to maintain a standard 02 uptake during progressive hypoxia down to a critical ambient Po2 of 20-40 mmHg. This is made possible thanks to a marked hyperventilation, as shown by increases in both frequency and amplitude of gill respiratory movements, which lead to alkalosis and hypocapnia. Below the Pc, anaerobic metabolism is initiated, as indicated by lactate release to the blood, metabolic acidosis and repayment of an 02 debt after return to normoxia. These findings are in striking contrast with those reported by Burggren and Randall (1978) for similarly-sized specimens of another sturgeon species, Acipenser transmontanus. These authors found a steady decrease not only of oxygen uptake but also of gill ventilatory flow rate during progressive hypoxia and concluded that 'sturgeon is unusual amongst vertebrates in that it becomes an oxygen conformer as soon as Po2 is reduced below ambient'. The absence of any 02 debt repayment also led them to conclude that no shift to anaerobic metabolism takes place but that 'sturgeon simply shuts down metabolism' during hypoxia. We suggest that such a complete disagreement in results and interpretation between the two equally consistent sets of data might be due to the techniques used to study ventilation. Namely, the very flexible tubing sewn on the retractile mouth of the fish by
79 10"
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70
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110
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150
[DWo2 (mmHg) Fig. 7. Changes in arterial pH (pHa) and plasma lactate concentration resulting from progressivehypoxia (0) and return to normoxic conditions (©). Symbols and labels as in Fig. 3.
these authors for direct measurement of gill ventilatory flow may have restricted the ability of the sturgeon to achieve the ventilatory adjustments needed to compensate for the decrease of ambient Po2. In support to this interpretation, it must be emphasized that at the same temperature, the mean normoxic ventilatory frequency reported by Burggren and Randall for Acipenser transmontanus was about twice that we observed in A. baeri, and about the same as that we recorded at the maximal hyperventilatory response during hypoxia. Moreover, at the beginning of our experiments, we tried to measure the ventilatory flow rate directly with the same device as that set up by Burggren and Randall (1978). The few fish (n = 3) fitted in this manner never exhibited a periodic ventilation in normoxia, which should be considered as a typical breathing pattern in bottom-dwelling fish such as the sturgeon (see below). In addition, we observed that the increased depth of breathing induced by exposure to hypoxia tended to cause a collapse of the thin wall of the tube inserted around the buccal orifice. Such an effect could be suspected to add a resistance to flow in proportion of ventilatory efforts and thus to account for the decreased ventilation observed by Burggren and Randall in hypoxic sturgeon. This interpretation is also borne out by the Pao~ values they report, which appear markedly lower than ours at similar levels of ambient Po2-
80 We cannot, however, explain why they found n o 0 2 debt repayment after hypoxia. Furthermore, in absence of any reported lactate measurement, the metabolic status of their fish cannot be evaluated. Effects of gradual hypoxia. Various respiratory characteristics of the sturgeon fall into a range corresponding to that reported for poorly active, bottom-dwelling teleosts. When appropriately corrected for body size (0.67 for the mass exponent) and temperature (Q10=2) differences, standard normoxic 1(/lo2 of Acipenser baeri at 15 °C (29.09#mol.min-l'kg-1) was lower than that of A. transmontanus (42.06 ktmol'min-~'kg -1, Burggren and Randall, 1978), but ranged between values reported for active fish (for example, salmon: 44.08 #mol-min-1 .kg-l, Maxime et al., 1990) and sluggish fish (for example, eel: 15.67 #mol.min-l'kg -1, Le Moigne et al., 1986). This could probably be related to the active growth of young sturgeon in spite of their relatively sluggish behavior. Normoxic sturgeon exhibited a spontaneously interrupted ventilation in normoxia. Such a periodic breathing rhythm has been observed in many teleosts in resting conditions: tench Tinca tinca (Shelton and Randall, 1962); carp Cyprinus carpio (Peyraud and Serfaty, 1964; Peyraud-WaRzenegger, 1979); eel Anguilla australis (Forster, 1981; Hipkins and Smith, 1983) and can be regarded as a typical eupneic ventilation in bottom-dwelling species known for their reduced motor activity and their ability to withstand hypoxic conditions. That normoxic A. baeri exhibited this pattern indicates that our fish were in a satisfactory physiological state and not unduly disturbed by experimental conditions. Increased gill ventilation has been commonly reported in fish subjected to a lowering of ambient Po2. In active species such as trout (Holeton and Randall, 1967) and Atlantic salmon (Maxime et al., unpublished), the increase in ventilatory flow rate resuits from a much more important rise of stroke volume than of frequency of breathing movements. This pattern of response appears the more marked the more active the species. The sturgeon showing about an equal increase in both frequency and amplitude displayed a pattern of ventilatory adjustments rather similar to that observed in bottom-dwelling species such as carp (Peyraud, 1965; Lomholt and Johansen, 1979) and tench (Randall and Shelton, 1963). Hyperventilation of the gills in hypoxic sturgeon led to a typical respiratory alkalosis with a decreased blood Pco2 and an increased pH. This helped maintain tissue 02 supply by a left shift of the 02 ' dissociation curve that kept arterial saturation high. According to data reported by Burggren and Randall (1978) forAcipenser transmontanus as well as to unpublished results by Salin et al. in A. baeri, blood 02 affinity is relatively high in sturgeon, so that the mean Pao2 = 23 mmHg recorded at the second stage of imposed hypoxia (Pwo2 = 40 mmHg) should correspond to a Hbo: per cent saturation of about 65 Yo. This is probably a reason for a relatively low critical level of ambient Po: in Acipenser baeri. Our Pc value at 15 ° C, 20-40 mmHg, is lower than that reported for active fish such as trout (Holeton and Randall, 1967), but of the same order of magnitude as that found for Acipenser galdenstiidti (Klyashtorin, 1981) and for fish
81 commonly experiencing environmental hypoxia in natural conditions such as the goldfish (Fry and Hart, 1948). Several aspects of the changes of respiratory variables during normoxic recovery deserve comment. Despite a short exposure time to the deepest hypoxic stage below Pc (1 h), the sturgeon obviously repaid an 02 debt with an elevated Mo2 lasting about 3.5 h. This 02 debt repayment was closely correlated with both the clearance of blood lactate and the recovery of blood pH (Fig. 7). It is additionally worth noting that blood lactate and the associated metabolic acidosis peaked together not during hypoxia but 10-30 min into normoxic recovery, suggesting a delayed but simultaneous release of both lactate ions and metabolic acid generated anaerobically during the hypoxic exposure. This contrasts with the asynchronous release of lactate and metabolic acid found in most teleosts during recovery from exhausting exercise (see Truchot, 1987). This is also in keeping with the absence of any significant change of plasma ion concentrations during either progressive hypoxia or normoxic recovery, which suggests that the generation and subsequent dissipation of the metabolic acidosis mainly involve internal metabolic events but no or minimal ionic exchanges. At the same ambient Po2, the values of Pao2 measured during normoxic recovery were lower than those found during progressive hypoxia (Fig. 5). This probably resulted from the increased tissue 02 uptake which must have substantially reduced oxygenation of the venous blood. As shown by the recorded ventilatory frequencies and amplitudes, the gill water flow rate also exhibited an hysteresis during normoxic recovery, but with values higher than during gradual hypoxia, at the same ambient Po2- This enhanced hyperventilatory response might be explained by a stronger hypoxic drive due to lower arterial Po2 values, perhaps reinforced by a persistent acidosis which could disappear only after metabolic processing of released lactic acid. This relatively long-lasting, post-hypoxic hyperventilation also delayed the restoration of normoxic Paco2 values. In conclusion, our data indicate that Acipenser baeri behaves as a typical 0 2 regulator when subjected to gradual hypoxia. This strengthens the idea (Ultsch et aL, 1981) that the few cases of 02 conformity reported for lower vertebrates may result from experimental artefacts and should be reexamined.
B a c k to norrnoxia.
References Astrup, P. (1956). A simpleelectrometrictechniquefor the determinationof carbon dioxidetension in blood and plasma, total content of carbon dioxidein plasma and bicarbonatecontent in 'separated' plasma at a fixed dioxidetension (40 mmHg). Scand..I. Clin. Invest. 8: 33-43. Boutilier, R.G., G.K. Iwama, T.A. Heming and D.J. Randall (1985). The apparent pK of carbonic acid in rainbow trout blood plasma between 5 and 15 °C. Respir. Physiol. 61: 237-254. Burggren, W.W. and D.J. Randall (1978). Oxygenuptake and transport during hypoxic exposure in the sturgeon Acipenser transmontanus. Respir. Physiol. 34: 171-183. Dejours, P., J. Armand and J.P. Gendner (1978). Importancede la rrgulation de l'rquilibreacide-basede l'eau ambiante pour l'rtude des 6changes respiratoireset ioniques des animaux aquatiques. C.R. Acad. Sci. Paris 287: 1397-1399.
82 Dejours, P. (1981). Principles of Comparative Respiratory Physiology. Amsterdam, New York, Oxford: Elsevier, 265 pp. Forster, M.E. (1981). Oxygen consumption and apnea in the shortfin eel, Anguilla australis schmidtii. New Zeal. J. Mar. Freshw. Res. 15: 85-90. Fry, F.E.J. and J. S. Hart (1948). The relation of temperature to oxygen consumption in the goldfish. Biol. Bull. 94: 66-77. Hipkins, S.F. and D.G. Smith (1983). Cardiovascular events associated with spontaneous apnea in the Australian short finned eel (Anguilla australis). J. Exp. ZooL 227: 339-348. Holeton, G. F. and D.J. Randall (1967). The effect of hypoxia upon the partial pressure of gases in the blood and water afferent and efferent to the gills of rainbow trout. J. Exp. BioL 46: 317-327. Klyashtorin, L.B. (1981). The ability of sturgeons (Acipenseridae) to regulate gas exchange. J. Ichthyol. 21: 141-144. Le Moigne, J., P. Soulier, M. Peyraud-Wa~'tzenegger and C. Peyraud (1986). Cutaneous and gill 02 uptake in the European eel (Anguilla anguilla L.) in relation to ambient Po2, 10-400 Torr. Respir. Physiol. 66: 341-354. Lomholt, J. P. and K. Johansen (1979). Hypoxia acclimation in carp. How it affects 02 uptake, ventilation, and 02 extraction from water. Physiol. Zool. 52: 38-49. Maxime, V., M. Peyraud-Wartzenegger, G. Clalreaux and C. Peyraud (1990). Effect of rapid transfer from seawater to freshwater on respiratory variables, blood acid-base status and 02 affinity of hemoglobin in atlantic salmon (Salmo salar L.). J. Comp. Physiol. B 160:31-39. Peyraud, C. and A. Serfaty (1964). Le rythme respiratoire de la carpe (Cyprinus carpio L.) et ses relations avec le taux de l'oxyg~ne dissous dans le biotope. Hydrobiologia 23: 165-178. Peyraud, C. (1965). Recherches sur la rrgulation des mouvements respiratoires chez quelques t616ostrens: Analyse du r6flexe opto-respiratoire. Thrse de Doctorat d~s Sciences. Universit6 de Toulouse, 258 pp. Peyraud-Waitzenegger, M. (1979). Simultaneous modifications of ventilation and arterial Po2 by catecholamines in the eel, Anguilla anguilla L.: participation of alpha and beta effects. J. Comp. Physiol. B 129: 343-354. Prosser, C.L. (1973). Comparative Animal Physiology, 3rd Edn., Philadelphia: Saunders, 966 pp. Randall, D.J. and G. Shelton (1963). The effects of changes in environmental gas concentrations on the breathing and heart rate of a teleost fish. Comp. Biochem. Physiol. 9: 229-239. Ruer, F.M., J.J. Cech and S.I. Doroshov (1987). Routine metabolism of the white sturgeon, Acipenser transmontanus: effect of population density and hypoxia. Aquaculture 62: 45-52. Shelton, G. and D.J. Randall (1962). The relation between heart beat and respiration in teleost fish. Comp. Biochem. Physiol. 7: 237-250. Truchot, J.P. and A. Duhamel-Jouve (1980). Oxygen and carbon dioxide in the marine intertidal environment: diurnal and tidal changes in rockpools. Respir. Physiol. 39: 241-254. Truchot, J.P. (1987). Comparative Aspects of Extracellular Acid-Base Balance. Zoophysiology, Vol. 20. Berlin, Heidelberg: Springer Verlag, 262 pp. Ultsch, G.R., D.C. Jackson and R. Moalli (1981). Metabolic oxygen conformity among lower vertebrates: the toadfish revisited. J. Comp. Physiol. B 142: 439-443. Vinberg, G.G. (1956). Rate of metabolism and food requirements of fishes. Fisheries Research Board of Canada, Translation Series No. 194, 202 pp.