J Comp Physiol B (2001) 171: 483±489 DOI 10.1007/s003600100198
O R I GI N A L P A P E R
Roberto Huet de Salvo Souza á Roseli Soncini Mogens Lesner Glass á Jose Roberto Sanches Francisco Tadeu Rantin
Ventilation, gill perfusion and blood gases in dourado, Salminus maxillosus Valenciennes (Teleostei, Characidae), exposed to graded hypoxia Accepted: 19 March 2001 / Published online: 25 April 2001 Ó Springer-Verlag 2001
Abstract The dourado, Salminus maxillosus, is an active and migratory teleost found in lotic waters of Southern Brazil. We have studied the relationships of gas transport in dourado to the speci®c ecophysiology of thisspecies. Measurements were performed of blood gases, O2 uptake, gill ventilation and perfusion at normoxia and various levels of hypoxia. Thus, the study aimed at a detailed assessment of the causes of O2 transport failure, using recent models for gas transport in vertebrates. Oxygen uptake was maintained down to a critical water partial O2 pressure of 42 mmHg, below which it markedly decreased. This could be explained based on ventilatory and cardiovascular responses: Ventilation increased suciently to match decreases of water O2 partial pressure during moderate hypoxia (partial pressure of O2>42 mmHg) but failed to meet O2 demands below this value. Likewise, the cardiovascular responses were insucient to maintain an adequate transport below moderatelevels of hypoxia. Thus, combined failure of ventilation and blood gas transport account for the abrupt decreases of O2 transport. The species proved highly vulnerable to hypoxia, which is consistent with the normally well-aerated habitat and the active mode of life of the species. Communicated by G. Heldmaier R.H. de Salvo Souza Center for Research on Tropical Fish/Brazilian Institute for Environmental Management (CEPTA/IBAMA), Pirassununga, SP, Brazil R. Soncini á M.L. Glass Department of Physiology, Faculty of Medicine of RibeiraÄo Preto/USP, RibeiraÄo Preto, SP, Brazil J.R. Sanches á F.T. Rantin (&) Department of Physiological Sciences, Federal University of SaÄo Carlos, 13565-905 SaÄo Carlos, SP, Brazil E-mail:
[email protected] Tel.: +55-16-2608314; Fax: +55-16-2608327
Keywords Ventilation á Gill perfusion á Blood gases á Oxygen uptake á Dourado, Salminus maxillosus Abbreviations EO2 oxygen extraction from the ventilatory current á fR ventilatory frequency á Hct hematocrit á ODC O2 dissociation curve á PcO2 critical O2 pressure á V_G gill ventilation á V_ O2 oxygen uptake á VT ventilatory tidal volume
Introduction Environmental oxygen is a limiting factor for aerobic metabolism in teleost ®sh (Blazka 1958; Hochachka 1980). For the ``oxygen transport cascade'' to function, the ®sh must detect and respond to reductions of ambient O2 levels, and this homeostasis involves ventilatory and cardiovascular responses (Randall 1982; Milsom and Brill 1986; Glass et al. 1990; Sundin et al. 1999, 2000). Within a certain range of water O2 levels, most teleost ®sh maintain an adequate oxygen uptake but with larger reductions of ambient O2 the ®sh will reach a critical O2 tension (PcO2), below which an adequate oxygen uptake can no longer be maintained (PoÈrtner and Grieshaber 1993; Thillart et al. 1994). Most teleost ®sh hyperventilate as a primary response to ambient hypoxia(Smith and Jones 1982; Jensen et al. 1993). Concomitant cardiovascular adjustments to hypoxia lead to a recruitment of secondary lamellae and elevations of their basal portion, which results in an increase of gill surface area for gas exchange (Booth 1979; Soivio and Tuurala 1981). Most teleosts develop bradycardia when exposed to decreased levels of water O2 partial pressure (PO2). This response depends on O2 receptors generally located on the ®rst gill arch (Sundin 1995). It has been suggested that bradycardia reduces energy expense and also prolongs ®lling time before systole which may increase
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O2 extraction by the myocardium. In addition, a slower blood ¯ow through the secondary lamellae may facilitate the O2 equilibrium between the inspired water and the blood (Holeton and Randall 1967; Farrell 1984; Fritsche and Nilsson 1989). Moreover, hypoxia activates a cardio-respiratory coordination (Glass et al. 1991; Rantin and Kalinin 1996). Adjustments of O2 transport during hypoxia also include an increase of haemoglobin-O2 anity of the blood, which is activated through an adrenergic mechanism (Nikinmaa 1990). Relatively few studies on teleost ®sh address the species-speci®c adaptations of respiratory physiology to habitat and mode of life (Rantin et al. 1992, 1993; Harder et al. 1999; Sundin et al. 2000). It has, however, become clear that very active species such as the rainbow trout, Onchorhyncus mykiss, and the pacu, maintain a high arterial PO2 (PaO2) and are characterized by a low Hb-O2 anity, which facilitates liberation of O2 at tissue level, and permits sustained activity (Cameron 1971; Gilmour and Perry 1994; Randall and Cameron 1973). This feature will, however, leave the species vulnerable to hypoxic conditions that may exclude adequate O2 release at tissue level. Conversely, hypoxia-tolerant bottom dwellers such as carp, Cyprinus carpio, and eel, Anguilla anguilla, are characterized by a low PaO2 combined with a high Hb-O2 anity, which promotes O2 loading from hypoxic environments (Albers et al. 1983; Glass et al. 1990). Conversely, such species are vulnerable to lack of O2 if high levels of activity are required. Most data on respiratory physiology of teleost ®sh have been obtained for species of temperate regions. In tropical and subtropical environments, high temperatures increase O2 demand which stresses the performance of the respiratory system. The ``dourado'', Salminus maxillosus, inhabits the main course of the Parana river basin of Southern Brazil (subtropical clime) and is a typical activepredatory and migratory species (Godoy 1975). These conditions require a high O2 demand. In this context our objective was to evaluate the responses of this active species, normally exposed to temperatures up to 25°C during the summer months. In particular, the cardiovascular and respiratory responses to hypoxia were addressed. The measurements involved gill ventilation, relative branchial blood ¯ow and blood gases along with the Hb-O2 dissociation curve (ODC). This combined approach serves to quantify and specify the hypoxia-dependent limitations to O2 transport of this species and to relate the ®ndings to its ecology. This approach was motivated by the lack of studies combining these measurements to study an active species. A similar approach has earlier been applied to a typical bottom dweller, the carp, in which various components of the O2 transport system were evaluated and quanti®ed by a number of studies (Itasawa and Takeda 1978; Glass et al. 1990).
Material and methods Experimental animals Specimens of S. maxillosus weighing between 2.7 kg and 4.0 kg were collected from the Mogi Guassu River, near Pirassununga, SP. The ®sh were maintained in holding facilities at the National Center for Research on Tropical Fish/Brazilian Institute for Environmental Management (CEPTA/IBAMA) for at least 1 week before transfer to the Laboratory of Zoophysiology and Comparative Biochemistry, Federal University of SaÄo Carlos, SP. Surgical procedures Before surgery, ®sh were anesthetized by immersion into a solution of benzocaine (1 g/10 l). Subsequently, the position of the animal was ®xed using a support, and catheters were implanted for sampling the inspired and expired water. Thus, the buccal chamber was cannulated (PE90) and the second catheter (PE50) was inserted through the opercular cleithrum (for details see Hughes et al. 1983). For blood sampling, a PE50 catheter was inserted into the dorsal aorta using pointed steel string as support. Subsequently, the support was withdrawn and the catheter was exteriorized through the side of the mouth and secured by means of sutures (Soivio et al. 1975; Claiborne and Heisler 1984). Using a similar procedure, a PE50 catheter was also used to cannulate the ventral aorta. Both catheters were ®lled with a heparinized saline and ¯ushed on a regular basis. Finally, to measure cardiac frequency (fR) a cu, containing a sonic crystal, was sutured around the ventral aorta between the bulbus and the gills. This cu was connected to a Doppler ¯owmeter (545C-4, Bioengineering, University of Iowa, Iowa City, USA) coupled to a Narco Narcotrace 40 physiograph (Narco BioSystems, Houston, Tx., USA) that was used to display fR and relative blood ¯ow through the ventral aorta. After surgery, the ®sh were transferred to a ¯ow-through respirometer (measurements of O2 uptakeand ventilatory variables) or, alternatively, to a restraining tube (measurements of cardiac variables and samplings of blood). Both types of chambers were left in a larger experimental aquarium, supplied with aerated water at 25°C after which a recovery period of 18 h was allowed prior to experimentation. Exposure to hypoxia Fish were sequentially exposed to graded hypoxia (PwO2=140, 100, 70, 50, 40, 30, and 20 mmHg). The desired O2 tensions in water were monitored by a computerized feed back system as described by Rantin and Kalinin (1996), and each exposure was maintained for at least 30 min. The PO2 of the ingoing water ¯ow (PwO2) was adjusted to normoxia or to the various levels of hypoxia. Normoxia was achieved by bubbling the water with air and hypoxic levels were obtained by addition of N2. Oxygen uptake Oxygen uptake V_ O2 mlO2 kg 1 h 1 was measured by ¯owthrough respirometry according to Rantin et al. (1992). The oxygen tensions of the ingoing (PinO2) and outgoing (PoutO2) water were continuously measured. This was achieved by siphoning water samples via polyethylene catheters to O2 electrodes (FAC 001- O2; FAC, SaÄo Carlos, SP, Brazil) that were housed within temperaturecontrolled cuvettes and connected to a FAC-204A O2 Analyzer. Oxygen uptake was calculated as V_ O2 VR a (PinO2±PoutO2)/ Wt, where VR represents the constant water ¯ow through the respirometer (l h±1), a denotes the solubility coecient for O2 in water (mlO2 l±1 mmHg±1) and Wt the body mass (kg).
485 Gill ventilation Gill ventilation V_ G mlH2 O kg 1 min 1 was measured according to the method of Hughes et al. (1983). The permanently implanted PE catheters allowed continuous measurement of inspired (PiO2) and expired (PeO2) water O2 tensions. V_ G was calculated according to Hughes and Saunders (1970): V_ G VR
PinO2 PoutO2 =
PiO2 PeO2 =Wt. Respiratory frequency Respiratory frequency (fR, breaths min±1) was measured from the buccal pressure variations. The buccal PE catheter was connected to a Narco P-1000B pressure transducer that was coupled to the universal coupler of a Narco Narcotrace 40 physiograph. Ventilatory tidal volume Tidal volume (VT ± ml H2O kg±1 breath±1) was calculated by dividing gill ventilation by the respiratory frequency V_ G =fR . Arterial and venous blood PO2 and hematocrit Blood samples (0.7 ml each) were withdrawn via the catheters implanted into the ventral and dorsal aorta and the O2 tensions were measured by means of thermostatted O2 electrodes connected to a FAC 204A O2 Analyzer. Hematocrit (Hct) was determined using the microcapillary method (centrifugation for 4 min at 12,000 rpm in a Fanen 207 N centrifuge; SaÄo Paulo, SP, Brazil). Cardiac frequency and relative blood ¯ow The fH and relative blood ¯ow ofthe ventral aorta were computed based on the output from the ultra-sonic crystal, that was coupled to a Doppler ¯ow ampli®er (Bioengineering, Model 545C-4, The University of Iowa, USA), connected to a Narco Narcotrace-40 physiograph. In vitro determination of the Hb-O2 dissociation curve The Hb-O2 dissociation curve was obtained for the saturations of 10%, 30%, 50%, 70%, and 90% according to the mixing method described by Haab et al. (1960). In short, blood samples from seven specimens were obtained by caudal puncture and immediately used for the construction of the dissociation curves. For each ®sh the sample was divided and transferred to two glass tonometers, one ¯ushed with 30% O2+2% CO2+N2 to saturate the blood, the other with 2% CO2+N2 to desaturate the blood. Known saturations were obtained by quantitative mixing of blood volumes from the two tonometers. After mixing the O2 tension of the sample was measured, using the O2 electrode system along with pH (Micronal pH-meter B374; Micronal, SaÄo Paulo, SP, Brazil) The Bohr shift was determined repeating the curve with CO2 set at 0.2%.
42 mmHg (PcO2), below which the uptake decreased markedly to approach zero (Fig. 1). Ventilation increased 3.9 times from a normoxic value of 940 ml H2O kg±1 min±1 to 3770 ml H2O kg±1 min±1 at 20 mmHg, corresponding to the lowest water PO2 (Fig. 2). The increase of V_ G was achieved by joint modulations of ventilatory stroke volume and respiratory frequency (VT increased 5-fold, while fR increased 1.3-fold; Fig. 2). fH was constant from normoxia down to 70 mmHg. Below this point, fH decreased signi®cantly in an almost linear fashion. In spite of an increase in stroke volume, relative cardiac output (=gill blood ¯ow) also decreased markedly with reductions of ambient PO2 (Fig. 3). PaO2 declined with each reduction of the inspired O2 level. The lowest water PO2 (20 mmHg), was associated with an 11-fold reduction of PaO2 relative to previous normoxic values. Meanwhile, the (PaO2±PvO2) gradient was 68 mmHg in normoxia decreasing to as little as 2 mmHg at 20 mmHg (Fig. 4). The whole-blood oxygen dissociation curves (25°C) after equilibration to dierent gas mixtures are shown in Fig. 5. The Hill coecient, nHill, was 1.32 at pH 8.22 and 1.64 at pH 7.73, while the Bohr factor was ±0.65. The values for P50 were: 10.5 mmHg at pH 8.22 and 21 mmHg at pH 7.73, the latter value being in the range of in vivo pH.
Discussion The study documents that the ``dourado'' has low tolerance of hypoxic environments. Table 1 compares this species to another high-performance species (trout) and a hypoxia-resistant bottom dweller (carp). These
Statistics The Wilcoxon test was applied to detect statistically signi®cant dierences in relation to normoxic values (P
