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A technique for the determination of the available oxygen in living carp (Cyprinus carpio) muscle
Camp. Biochem. Physiol.Vol. 78A, No. 4, pp. 675-679, 1984 0
Printedin Great Britain
0300-9629/84 $3.00 + 0.00 1984 PergamonPress Ltd
A TECHNIQUE FOR THE DETERMINATION OF THE AVAILABLE OXYGEN IN LIVING CARP (CYPRINUS CARPI@ MUSCLE* P. H. Centro Parana,
LUCCHIARI, EDITH FANTA FEOFILOFF, ANA T. BOSCARDIM
and M.
BACILA
de Biologia Marinha, Universidade Federal do Parana, Rua Jaime Balao 575, 80,000 Curitiba, Brasil and Instituto de Biologia Basica Medica e Agricola, Universidade Estadual Paulista, “Julio de Mesquita Filho”, Botucatu, Sb Paulo, Brasil (Received 27 October 1983)
Abstract-l. A polarographic method for the measurement of the available oxygen in the muscle of living carp by the use of a platinum microelectrode is proposed. 2. The oxygen and the reference electrodes were assembled in a single insertion piece which was implanted in the muscle of a living carp maintained in a special experimental chamber. 3. Curves for normal oxygen levels corresponding to air-saturated water, as well as to a carbogenesaturated water, were obtained. 4. The method can be considered adequate for the measurement of tissue oxygen in living fishes.
INTRODUCTION
After the early work of Davies and Brink (1942) who demonstrated that tissue PO2 can be measured in uivo by a polarographic technique, a number of methods for the measurement of the PO, in living tissues have been proposed (Berezin and Epstein, 1968; Biron and Dittmar, 1977; Cobbold, 1974; Connelly, 1957; Hahn, 1969; Hahn et al., 1975; Jamieson and Van den Brenk, 1965; Lemy and Hauquet, 1970; Lucchiari, 1978; Meyer et al., 1954; Monte et al., 1962; Smith and Hahn, 1969; Whalen et al., 1973, among others). The oxygen assayed by polarography is the oxygen available to the cells (Strauss et al., 1968). It depends on several factors: the offering of arterial oxygen, the microcirculation, and the oxygen consumed by the cells (Montgomery and Horwitz, 1950; Sinagowitz et al., 1977). Furthermore, the assay of tissue oxygen also depends on the coefficients of diffusion and of solubility of the oxygen in the medium, as well as on some geometric characteristics of the tissues (Vasli, 1963). In spite of the amount of information on the 02-CO, exchange and the PO* of the fish blood (Black, 1951; Fry, 1957; Krogh, 1941; Piiper and Baumgarten-Schuman, 1968; Randall, 1970b, among many others) tissue gas tensions are only indirectly calculated. The PO, in the mixed venous blood is taken as an indication of the possible gas tension in the tissues. Thus, as the PO, in the mixed venous blood of the carp is 3.2 mmHg (Garey, 1964) and the gas gradient between blood and tissue has not been measured in fishes, it is assumed that tissue gas tensions probably lie in the range of 1-15 mmHg (Randall, 1970b). For fishes, such a gas gradient between tissue and blood is of an unknown extent and might be different for different species as well as for each tissue (Berezin *With grants in aid from I-PSRM, Comisslo Interministerial para OS Recursos do Mar (CIRM) and Conselho National de Pesquisas. 675
and Epstein, 1968). It depends, among other factors, on the distance between the arterial capillaries and the tissue itself (Lemy and Hauquet, 1970). In order to better define the physiological properties of the available oxygen in fish tissue, a special set up has been developed consisting of a microelectrode for the polarographic determinations of O,, coupled with a system of simultaneous measurement of pH, salinity, temperature and oxygen content of the water in the test chamber. MATERIALS AND METHODS
For the measurement of the available
oxygen in the muscle of a living carp, a microelectrode, connected to a polarograph and a recorder, was implanted in an animal maintained in an appropriate container. 1. The electrode The oxygen sensor used was the platinum cathode with a silver-silver chloride reference electrode, both built with 0.12 mm dia wires. Both wires were welded to a thin flexible and insulated conductor cable and then perpendicularly fixed at a distance of 3%6mm from each other on a thin 4 x 7 mm plastic base. The weld was covered with selfpolymerizing acrylic, and the wires with a layer of ultra-fast Araldite. Immediately before the insertion, the tips of the silver and the platinum wires were cut out to obtain electrodes with the length of 3 and 6 mm respectively. Finallv, the electrode was immersed in a 0.1 M HCl solution-the silver wire being used as anode in order to allow the electrode position of a layer of AgCl over the free surface of the silver. 2. The circuit In order to measure the polarographic current, the circuit indicated in Fig. 1 was used. The gain of the amplifier was adjusted to ten, through P, and the zero through P4 with the amplifier input open. The output of 723 nA supplies a stabilized tension of 2V. The potentiometer P, makes it possible to adjust the polarization tension to the desired values, which may be read at the DPM with the switch in position 2. The potentiometer P, allows to calibrate the readings of the tensions of polarization at DPM.
P. H. LUCCHIARIet al.
616
Reference electrode
made in the skin which was separated from the underlying tissues, the muscle surface being exposed. The electrode was implanted perpendicularly to the dry muscular surface, covered immediately with the two skin flaps and fixed by means of two surgical stitches. Immediately after, the animal was placed in the experimental chamber. The carp very soon assumed a normal body position and the respiratory frequency was recovered to normal values after 2-5 min. The whole procedure had the mean duration of 12 min. In order to fix the electrode in the animal body, a thin layer of a superbonder glue was applied to its base, just before its implanting in the muscle. 6. The observations
Recorder
Fig. 1. Circuit used for the measurement of the polarographic current. R, = 5 kQ; RZ= 2 kD; R, = 1.5 kQ; R4 = 0.030 kQ; R, = 10 kQ; R, = 8.5 kQ; R, and Rs = 1 kR; R9=10kQ;P,=1kQ;P,=100kQP,=1k~;P,=10kQ C, = 0.1 PF and C, = 100pF. 3. The&h
chamber
The fish chamber (Fig. 2) was built from acrylic. It consists of a 2.0 1. test chamber, fixed in a water bath which can be maintained at a constant temperature. When the gas is bubbled into the test chamber it goes first through a glass serpentine fixed in the water bath. This allows us to maintain the gas in a state of thermal equilibrium with the water. The chamber cover is detachable and possesses holes through which the sensors and the conductor wires may be introduced into the test chamber. 4. The experimental
animal
Ten healthy carp (Cyprinus carpio L.) with an average length of 22 cm were obtained from a fish farm and maintained in the laboratory at 21 “C, receiving food pellets once a day. 5. The implant of the oxygen electrode The animal was held without anesthesia, the mouth and the gill surface being maintained wet through a continuous flow of well aerated water. To prepare the site for the insertion of the electrode, the scales were taken off latero-dorsally just in front of the dorsal fin (Fig. 3). With the aid of a bistoury, a 2 cm cut was
Gi
After an adaptation period of 16 hr, the connections with the measuring apparatus were established, the polarization voltage adjusted to 600 mV and the tests started. The polarographic current, corresponding to the amount of oxygen available in the muscles of the living carp, was recorded. At the same time, the frequency of the respiration movements was counted with a chronometer. The experimental temperature was maintained constant and the animal was fed normally once a day. I. The recuperation After the measurements, the electrode was pulled out from the muscle, the skin was sewed and the animal returned to the acclimation aquarium. Its respiration and activity were observed, showing a complete recovery. This result has been obtained at least in duplicate determinations with ten experimental carp. RESULTS A register of the polarographic current in the interior of the muscle was obtained by switching on the system. It corresponds to the amount of the available 0, in that tissue (Fig. 4). Such value is directly related to the concentration of the oxygen in the water which is maintained continuously saturated with atmospheric air. The respiratory frequency was also determined, showing constant values for the animals submitted to normal conditions in the chamber, as well as for the control animals in the aquarium: 60 opercular movements per min at 21°C. A constant level of tissue oxygen was found in such
normal conditions. The sensitivity of the method has been established by increasing the oxygen concentration in the medium. This has been carried out by bubbling carbo-
SW0
Fig. 2. Schematic view of the experimental chamber for the study of fish respiration with implanted oxygen electrode. CV+over; EC--experimental chamber; GD-gas diffuser; GI-gas input; OEl-oxygen electrode; OE2-oxygen electrode; SE-serpentine; TS-temperature sensor; WB-constant temperature water bath; WI--constant temperature water inflow; W-water outflow.
Measurement
of the available
oxygen
in living carp muscle
677
Carbogene
0
IO
20
30
40
50
60
Time (rmn) Fig. 4. Recording of the polarographic current corresponding to the available oxygen in the muscle of a living carp. Arrows indicate the number of respiratory movements per min at the times considered.
P. H. LUCCHIARIet al.
678
gene (O,-CO,, 954.9%) in the test chamber. Besides providing oxygen to the water experimental medium it also causes an increase in the frequency of the respiratory movements, due to the CO, it contains. As expected, a rising curve was obtained {Fig. 4), corresponding to a higher amount of available O2 in the muscle, until a new equilibrium was reached. Bubbling air again, as the animal continues with a higher respiratory frequency, the curve falls until a new equilibrium is established. The original level was totally reestablished after 2 hr. This result has been obtained, at least in duplicate deter~nations, with ten experimental carp. DISCUSSION
Fish respiratory metabolism has been the subject of many studies since the measurements of its concentrations in arterial and venous blood (Hughes, 1961; Rahn, 1967; Randall, 1970b; Piiper and BaumgartenSchuman, 1968; Saunders, 1961, 1962). However, no information is available on the level of tissue oxygen in living fishes, which represents the amount of oxygen available for the cells. Since the pioneering work of Davies and Brinks (1942), the implanting of microelectrodes in animal tissues has been used for the assay of available oxygen in dogs, rats, cats and apes; for physiological studies of the brain (Bicher and Knsely, 1970; Lucchiari, 1978), the kidney (Sinagowitz et al., 1977; Strauss et al., 1968; Vercesi, 1982), the heart (Cicogna, 1980; Mazzela ef al., 1975), the muscles (Vasli, 1963; Whalen, 1971) and the skin (Huch et al., 1979; Montgomery and Horwitz, 1950). Several techniques of mammalian circulatory physiology have been already adapted to fish studies. Thus, the conception of a microelectrode for polarographic measurements could very well be a new important tool in fish physiology studies. Of the relatively small number of fish species studied on research into the experimental physiology of circulation, the carp has been one of the most frequently used. Much of the available data in this important field has been obtained by a variety of techniques, under a variety of conditions. In some instances the fish were not intact and were either out of the water, restrained, or anesthetized. In other experiments, the records were obtained from intact) unanesthetized and relatively unrestrained fish (Randall, 197Oa). The conditions in our experiments followed the idea of working with an animal subjected to a situation as naturally physiological as possible. Thus, the test chamber used in this experiment (Fig. 2) is relatively unstressing for the animal, which under normal conditions displayed a normal breathing frequency, no loss of mucus secretion after the adaptation time and normal feeding, color and body position, if compared with the control animals maintained in the aquarium. The implant was carried out without anesthesia in order to avoid any interference with the measurements. The carp reacted very well to it and, as soon as it was placed into the test chamber, performed normally. After the removal of the electrode, the healing was quite fast and complete.
One of the main features of the microelectrode used here was the joining in one single insertion piece of the platinum and the reference electrodes (Fig. 3) unlike the ones commonly used, which need two implants, one for each electrode (Cicogna, 1980; Lucchiari, 1978; Vercesi, 1982). Furthermore, the electrode was glued on the muscle and stuck under the skin. Such procedure allowed the recording of neat curves, with an acceptable level of noise, without the loss of their shape (Fig. 4). This result is still more interesting considering that it has been obtained in living fish maintained free in the test chamber and not immobilized or anesthetized. According to the present method it was possible to stabilize the polarization voltage at a level of more or less 1 mV. The answer obtained was constant in 25 repetitions and in ten different animals. Thus we conclude that the method is rehable. Carbogene was used to increase the respiratory frequency (Olthof, 1934; Saunders, 1961). The rise in the curve after increasing the oxygen tension in the medium was not due to diffusion. With pure oxygen, as the animal decreases markedIy the frequency of its breathing movements, the level obtained was the same as the ones obtained for atmospheric air. The knowledge of tissue oxygen tension, if simultaneously measured with the blood flow and the gas transport from the external environment through the gills, can greatly facilitate the study of tissue oxygen supply, its utilization and diffusion from the blood into the tissues. The method proposed may be well used to give this kind of information. Also it may be adapted for other species of fishes, increasing the field of studies on physiology of respiration.
REFERENCES Berezin 1. P. and Enstein I. M. (1968) Measurement of tissue PO, by a cylindrical electrode at normal and high atmosoheric nressure. Biomed. Enc. N. Y. 2, 319-323. Biiher H: I. and Knsely M.-H. (1970) Brain tissue reoxygenation time demonstrated with a new ultramicro oxygen electrode. J. a&. Physiol. 28, 387-390. Biron R. and Dittmar A. (1977) High sensitivity device for continuous measurement of tissue O2 uptake. J. uppl. Physiol. 43, 370-374.
Black E. C. (1951) Respiration
in Fish. Publ. Ontario
Fisheries Res. Lab. 71, 91-111.
Cicogna A. C. (1980) TensHo de oxigenio intramiocardico e do sangue do seio coronario. Estudo comparative dnrante varia@es da relago oferta-consumo deoxigenio em coracao de Go. Thesis. Fat. Med. Botucatu, UNESP. Cobboid R. S. C. (1974) Transducers for Biomedical Measurements: Principles and Applications. John Wiley, New York. Connelly C. M. (1957) Methods for measuring tissue oxygen tension: theory and evaluation. The oxygen electrode. Fed. Proc. 16, 681-684.
Davies P. W. and Brink Jr. E. (1942) Microelectrodes for measuring oxygen tension in animal tissue. Rev. scient. Inst~m. 13, 524-533. Fry F. E. .I. (1957) The aquatic respiration of fish. In The Physiology of Fishes (Edited by Brown M. E.), pp. i-63. Academic Press, New York. Garey W. F. (1967) Gas exchange, cardiac output and blood pressure in free summing carp (Cyprimcs carpio). Ph.D. Dissertation. Univ. of New York, Buffalo, New York. Hahn C. E. W. (1969) The measurement of oxygen micro-
Measurement
of the available
cathode currents by means of a field-effect transistor operational amplifier system with digital display. J. scient. Instrum. (J. Phys. E) Ser. 2, 2, 48-50. Hahn C. E. W., Davies A. H. and Albery W. J. (1975) Electrochemical improvement of the performance of PO, electrodes. Respir. Physiol. 25, 109-135. Huch R., Huch A. and Rolfe P. (1979) Transcutaneous measurements PO, using electrochemical analysis. In Non-invasive Physiological Measurements (Edited by Rolfe R.), pp. 313-331. Academic Press, New York. Hughes G. M. (1961) How a fish extracts oxygen from water. New Scient. 11, 346-348. Jamieson D. and Van den Brenk H. A. S. (1965) Electrode size and tissue PO, measurement in rats exposed to air or high pressure oxygen. J. appl. Physiol. 20, 514-518. Krogh A. (1941) The Comparative Physiology of Respiratory Mechanisms. University of Pennsylvania Press, Philadelphia, Pennsylvania. Lemy M. and Hauguet M. (1970) Mesures polarographiques de la POz dam le sang et les tissus. Acta anaesth. belg. 21, 141-163. Lucchiari P. H. (1978) Circuit0 polarografico optimizado para determina@o de niveis de oxigenio intracerebral de rato. Thesis. Inst. Bas. de Biol. Med. e Aaric. Botucatu. UNESP. Mazzela H., Fiandra O., Peluffo C., Spera E. and Gin&s F. (1975) Human intramyocardial oxygen detected through chronic polarographic electrodes. Archs Inst. Cardiol. Mex. 45, 301-306. Meyer J. S., Fang H. C. and Brown D. D. (1954) Polarographic study of cerebral collateral circulation. Archs Neural. Psychiat. 72, 296312. Monte U. Del Boni, I. and Maschepa G. (1962) Tecnica per la misura polarografica della tensione di ossigeno in vivo. Ric. Sci. 2, 238-247. Montgomery H. and Horwitz 0. (1950) Oxygen tension of tissues by polarographic method-I. Introduction: Oxygen tension and blood flow of the skin of human extremities. J. clin. Invest. 29, 1120-1130. Olthof H. J. (1934) Die Kohlensaure als Atemreiz bei
C.&P. 78/4A-E
oxygen
in living carp muscle
679
Wassertieren insbesondere bei den Stisswasserfischen. Z. vergl. Physiol. 21, 534-562. Piiper J. and Baumgarten-Schuman D. (1968) Carriage of 0, and CO, by water and blood in gas exchange of the dogfish (Scyorhinus stellaris). Resp. Physiol. 5, 326-337. Rahn H. (1967) Gas transport from the external environment to the cell. Ciba. Found. Symp., Development of the Lung, pp. 3-23. Randall D. J. (1970a) The circulatory System. In Fish PhysioZogy (Edited by Hoar W. S. and Randall D. J.), pp. 133-172. Academic Press, New York. Randall D. J. (1970b) Gas exchange in fish. In Fish Physiology (Edited by Hoar W. S. and Randall D. J.), Vol. 4, pp. 523-592. Academic Press. New York. Saunders R. L. (1961) The irrigation of gill in fishes. I. Studies of the mechanism of branchial irrigation. Can. J. Zool. 39, 637-653. Saunders R. L. (1962) The irrigation of the gills of fishes. II. Efficiency of oxygen uptake in relation to respiratory flow, activity and concentration of oxygen and-carbon dioxide. Can. J. Zool. 40, 817-862. Sinagowitz E., Golson M. and Halbfab H. J. (1977) Local tissue PO, in kidney survey and transplantation. Adv. exp. Med. Biol. 94, 721-727. Smith A. C. and Hahn C. E. W. (1969) Electrodes for the measurement of oxygen and carbon dioxide tensions. Br. J. Anaesth. 41, 731-741. Strauss J., Beran A. V., Brown C. T. and Katurich N. (1968) Renal oxygenation under “normal” conditions. Am. J. Physiol. 215, 1482-1487. Vasli S. (1963) Post-ischemic polarography in human calf muscle: Acta chir. stand. (Suppl. 315), l-61. Vercesi L. A. P. (1982) A@o do manitol na isquemia renal no c80. Estudo Polarogrbfico. Thesis. Fat. Med. Botucatu, UNESP. Whalen W. J. (1971) Intracellular PO, in heart and skeletal muscle. Physiologist 14, 69-82. Whalen W. J., Nair P. and Granfield R. A. (1973) Measurement of oxygen tension in tissue with micro oxygen electrode. Microvasc. Res. 5, 254-262.
Printedin Great Britain
0300-9629/84 $3.00 + 0.00 1984 PergamonPress Ltd
A TECHNIQUE FOR THE DETERMINATION OF THE AVAILABLE OXYGEN IN LIVING CARP (CYPRINUS CARPI@ MUSCLE* P. H. Centro Parana,
LUCCHIARI, EDITH FANTA FEOFILOFF, ANA T. BOSCARDIM
and M.
BACILA
de Biologia Marinha, Universidade Federal do Parana, Rua Jaime Balao 575, 80,000 Curitiba, Brasil and Instituto de Biologia Basica Medica e Agricola, Universidade Estadual Paulista, “Julio de Mesquita Filho”, Botucatu, Sb Paulo, Brasil (Received 27 October 1983)
Abstract-l. A polarographic method for the measurement of the available oxygen in the muscle of living carp by the use of a platinum microelectrode is proposed. 2. The oxygen and the reference electrodes were assembled in a single insertion piece which was implanted in the muscle of a living carp maintained in a special experimental chamber. 3. Curves for normal oxygen levels corresponding to air-saturated water, as well as to a carbogenesaturated water, were obtained. 4. The method can be considered adequate for the measurement of tissue oxygen in living fishes.
INTRODUCTION
After the early work of Davies and Brink (1942) who demonstrated that tissue PO2 can be measured in uivo by a polarographic technique, a number of methods for the measurement of the PO, in living tissues have been proposed (Berezin and Epstein, 1968; Biron and Dittmar, 1977; Cobbold, 1974; Connelly, 1957; Hahn, 1969; Hahn et al., 1975; Jamieson and Van den Brenk, 1965; Lemy and Hauquet, 1970; Lucchiari, 1978; Meyer et al., 1954; Monte et al., 1962; Smith and Hahn, 1969; Whalen et al., 1973, among others). The oxygen assayed by polarography is the oxygen available to the cells (Strauss et al., 1968). It depends on several factors: the offering of arterial oxygen, the microcirculation, and the oxygen consumed by the cells (Montgomery and Horwitz, 1950; Sinagowitz et al., 1977). Furthermore, the assay of tissue oxygen also depends on the coefficients of diffusion and of solubility of the oxygen in the medium, as well as on some geometric characteristics of the tissues (Vasli, 1963). In spite of the amount of information on the 02-CO, exchange and the PO* of the fish blood (Black, 1951; Fry, 1957; Krogh, 1941; Piiper and Baumgarten-Schuman, 1968; Randall, 1970b, among many others) tissue gas tensions are only indirectly calculated. The PO, in the mixed venous blood is taken as an indication of the possible gas tension in the tissues. Thus, as the PO, in the mixed venous blood of the carp is 3.2 mmHg (Garey, 1964) and the gas gradient between blood and tissue has not been measured in fishes, it is assumed that tissue gas tensions probably lie in the range of 1-15 mmHg (Randall, 1970b). For fishes, such a gas gradient between tissue and blood is of an unknown extent and might be different for different species as well as for each tissue (Berezin *With grants in aid from I-PSRM, Comisslo Interministerial para OS Recursos do Mar (CIRM) and Conselho National de Pesquisas. 675
and Epstein, 1968). It depends, among other factors, on the distance between the arterial capillaries and the tissue itself (Lemy and Hauquet, 1970). In order to better define the physiological properties of the available oxygen in fish tissue, a special set up has been developed consisting of a microelectrode for the polarographic determinations of O,, coupled with a system of simultaneous measurement of pH, salinity, temperature and oxygen content of the water in the test chamber. MATERIALS AND METHODS
For the measurement of the available
oxygen in the muscle of a living carp, a microelectrode, connected to a polarograph and a recorder, was implanted in an animal maintained in an appropriate container. 1. The electrode The oxygen sensor used was the platinum cathode with a silver-silver chloride reference electrode, both built with 0.12 mm dia wires. Both wires were welded to a thin flexible and insulated conductor cable and then perpendicularly fixed at a distance of 3%6mm from each other on a thin 4 x 7 mm plastic base. The weld was covered with selfpolymerizing acrylic, and the wires with a layer of ultra-fast Araldite. Immediately before the insertion, the tips of the silver and the platinum wires were cut out to obtain electrodes with the length of 3 and 6 mm respectively. Finallv, the electrode was immersed in a 0.1 M HCl solution-the silver wire being used as anode in order to allow the electrode position of a layer of AgCl over the free surface of the silver. 2. The circuit In order to measure the polarographic current, the circuit indicated in Fig. 1 was used. The gain of the amplifier was adjusted to ten, through P, and the zero through P4 with the amplifier input open. The output of 723 nA supplies a stabilized tension of 2V. The potentiometer P, makes it possible to adjust the polarization tension to the desired values, which may be read at the DPM with the switch in position 2. The potentiometer P, allows to calibrate the readings of the tensions of polarization at DPM.
P. H. LUCCHIARIet al.
616
Reference electrode
made in the skin which was separated from the underlying tissues, the muscle surface being exposed. The electrode was implanted perpendicularly to the dry muscular surface, covered immediately with the two skin flaps and fixed by means of two surgical stitches. Immediately after, the animal was placed in the experimental chamber. The carp very soon assumed a normal body position and the respiratory frequency was recovered to normal values after 2-5 min. The whole procedure had the mean duration of 12 min. In order to fix the electrode in the animal body, a thin layer of a superbonder glue was applied to its base, just before its implanting in the muscle. 6. The observations
Recorder
Fig. 1. Circuit used for the measurement of the polarographic current. R, = 5 kQ; RZ= 2 kD; R, = 1.5 kQ; R4 = 0.030 kQ; R, = 10 kQ; R, = 8.5 kQ; R, and Rs = 1 kR; R9=10kQ;P,=1kQ;P,=100kQP,=1k~;P,=10kQ C, = 0.1 PF and C, = 100pF. 3. The&h
chamber
The fish chamber (Fig. 2) was built from acrylic. It consists of a 2.0 1. test chamber, fixed in a water bath which can be maintained at a constant temperature. When the gas is bubbled into the test chamber it goes first through a glass serpentine fixed in the water bath. This allows us to maintain the gas in a state of thermal equilibrium with the water. The chamber cover is detachable and possesses holes through which the sensors and the conductor wires may be introduced into the test chamber. 4. The experimental
animal
Ten healthy carp (Cyprinus carpio L.) with an average length of 22 cm were obtained from a fish farm and maintained in the laboratory at 21 “C, receiving food pellets once a day. 5. The implant of the oxygen electrode The animal was held without anesthesia, the mouth and the gill surface being maintained wet through a continuous flow of well aerated water. To prepare the site for the insertion of the electrode, the scales were taken off latero-dorsally just in front of the dorsal fin (Fig. 3). With the aid of a bistoury, a 2 cm cut was
Gi
After an adaptation period of 16 hr, the connections with the measuring apparatus were established, the polarization voltage adjusted to 600 mV and the tests started. The polarographic current, corresponding to the amount of oxygen available in the muscles of the living carp, was recorded. At the same time, the frequency of the respiration movements was counted with a chronometer. The experimental temperature was maintained constant and the animal was fed normally once a day. I. The recuperation After the measurements, the electrode was pulled out from the muscle, the skin was sewed and the animal returned to the acclimation aquarium. Its respiration and activity were observed, showing a complete recovery. This result has been obtained at least in duplicate determinations with ten experimental carp. RESULTS A register of the polarographic current in the interior of the muscle was obtained by switching on the system. It corresponds to the amount of the available 0, in that tissue (Fig. 4). Such value is directly related to the concentration of the oxygen in the water which is maintained continuously saturated with atmospheric air. The respiratory frequency was also determined, showing constant values for the animals submitted to normal conditions in the chamber, as well as for the control animals in the aquarium: 60 opercular movements per min at 21°C. A constant level of tissue oxygen was found in such
normal conditions. The sensitivity of the method has been established by increasing the oxygen concentration in the medium. This has been carried out by bubbling carbo-
SW0
Fig. 2. Schematic view of the experimental chamber for the study of fish respiration with implanted oxygen electrode. CV+over; EC--experimental chamber; GD-gas diffuser; GI-gas input; OEl-oxygen electrode; OE2-oxygen electrode; SE-serpentine; TS-temperature sensor; WB-constant temperature water bath; WI--constant temperature water inflow; W-water outflow.
Measurement
of the available
oxygen
in living carp muscle
677
Carbogene
0
IO
20
30
40
50
60
Time (rmn) Fig. 4. Recording of the polarographic current corresponding to the available oxygen in the muscle of a living carp. Arrows indicate the number of respiratory movements per min at the times considered.
P. H. LUCCHIARIet al.
678
gene (O,-CO,, 954.9%) in the test chamber. Besides providing oxygen to the water experimental medium it also causes an increase in the frequency of the respiratory movements, due to the CO, it contains. As expected, a rising curve was obtained {Fig. 4), corresponding to a higher amount of available O2 in the muscle, until a new equilibrium was reached. Bubbling air again, as the animal continues with a higher respiratory frequency, the curve falls until a new equilibrium is established. The original level was totally reestablished after 2 hr. This result has been obtained, at least in duplicate deter~nations, with ten experimental carp. DISCUSSION
Fish respiratory metabolism has been the subject of many studies since the measurements of its concentrations in arterial and venous blood (Hughes, 1961; Rahn, 1967; Randall, 1970b; Piiper and BaumgartenSchuman, 1968; Saunders, 1961, 1962). However, no information is available on the level of tissue oxygen in living fishes, which represents the amount of oxygen available for the cells. Since the pioneering work of Davies and Brinks (1942), the implanting of microelectrodes in animal tissues has been used for the assay of available oxygen in dogs, rats, cats and apes; for physiological studies of the brain (Bicher and Knsely, 1970; Lucchiari, 1978), the kidney (Sinagowitz et al., 1977; Strauss et al., 1968; Vercesi, 1982), the heart (Cicogna, 1980; Mazzela ef al., 1975), the muscles (Vasli, 1963; Whalen, 1971) and the skin (Huch et al., 1979; Montgomery and Horwitz, 1950). Several techniques of mammalian circulatory physiology have been already adapted to fish studies. Thus, the conception of a microelectrode for polarographic measurements could very well be a new important tool in fish physiology studies. Of the relatively small number of fish species studied on research into the experimental physiology of circulation, the carp has been one of the most frequently used. Much of the available data in this important field has been obtained by a variety of techniques, under a variety of conditions. In some instances the fish were not intact and were either out of the water, restrained, or anesthetized. In other experiments, the records were obtained from intact) unanesthetized and relatively unrestrained fish (Randall, 197Oa). The conditions in our experiments followed the idea of working with an animal subjected to a situation as naturally physiological as possible. Thus, the test chamber used in this experiment (Fig. 2) is relatively unstressing for the animal, which under normal conditions displayed a normal breathing frequency, no loss of mucus secretion after the adaptation time and normal feeding, color and body position, if compared with the control animals maintained in the aquarium. The implant was carried out without anesthesia in order to avoid any interference with the measurements. The carp reacted very well to it and, as soon as it was placed into the test chamber, performed normally. After the removal of the electrode, the healing was quite fast and complete.
One of the main features of the microelectrode used here was the joining in one single insertion piece of the platinum and the reference electrodes (Fig. 3) unlike the ones commonly used, which need two implants, one for each electrode (Cicogna, 1980; Lucchiari, 1978; Vercesi, 1982). Furthermore, the electrode was glued on the muscle and stuck under the skin. Such procedure allowed the recording of neat curves, with an acceptable level of noise, without the loss of their shape (Fig. 4). This result is still more interesting considering that it has been obtained in living fish maintained free in the test chamber and not immobilized or anesthetized. According to the present method it was possible to stabilize the polarization voltage at a level of more or less 1 mV. The answer obtained was constant in 25 repetitions and in ten different animals. Thus we conclude that the method is rehable. Carbogene was used to increase the respiratory frequency (Olthof, 1934; Saunders, 1961). The rise in the curve after increasing the oxygen tension in the medium was not due to diffusion. With pure oxygen, as the animal decreases markedIy the frequency of its breathing movements, the level obtained was the same as the ones obtained for atmospheric air. The knowledge of tissue oxygen tension, if simultaneously measured with the blood flow and the gas transport from the external environment through the gills, can greatly facilitate the study of tissue oxygen supply, its utilization and diffusion from the blood into the tissues. The method proposed may be well used to give this kind of information. Also it may be adapted for other species of fishes, increasing the field of studies on physiology of respiration.
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