- Email: [email protected]
The role of gills in the responses of Salmo gairdneri during moderate hypoxia
THE ROLE OF GILLS IN THE RESPONSES OF SALMO GA~~~~~~~ DURING MODERATE HYPOXIA ANTTI SOIVIO*, MIKKO NIKINMAA*, KEIJO NYHOLM~ and KAI WESTMANS *Division of Physiology, Department of Zoology, University of Helsinki, Arkadiankatu 7, SF-00100 Helsinki 10 tLaukaa Fish Culture Research Station, SF-41360 Valkola and JThe Finnish Came and Fisheries Research institute, Fisheries Division, P.O. Box 193, SF-00131 Helsinki 13, Finland
(Received 2 Febrmy
1981)
Abstract-l. Gill function of rainbow trout, cannulated permanently to both ventral and dorsal aortae, was studied during moderate, short term hypoxia (5.5 mg/l) at 9°C. 2. Hb concentrations and Hct values were significantly and MCHC slightly higher in the dorsal aorta. 3. PO, difference between the aortae decreased from 11 kPa in normoxia to 6 kPa in hypoxia. 4. Blood pH as well as protein and inorganic ion concentrations of the plasma were the same in both aortae and remained constant. 5. Lactate concentration in the ventral aorta exceeded that of the dorsal one in the prehypoxic fish, but decreased to same value during hypoxia.
The gill function of rainbow trout has mostly been studied with perfused head or gill preparations. Less work has been done with “in uiuo preparations”, permanently cannulated fish, in which the gills are in
their natural surroundings both externally and internally. The in vivo studies have so far mainly been aimed at solving the effects of hypoxia, exercise and intravenously added substances on the gas exchange through the gills and the circulation of the gills (Holeton & Randall, 1967; Randall et al., 1967; Booth, 1978, 1979a,b; Wood & Shelton, 1980a,b). Driedzic & Kiceniuk (1976) suggested that the gills of rainbow trout utilize lactate during exercise in viva. In addition the fish gills have important functions in osmoregulation. For example the osmotic balance in freshwater fish is partly regulated by the uptake of NaCl by the gills. In view of the several functions of the gills both in gas exchange and osmoregulation, and as it is known that in addition to the blood gas tensions, several other blood parameters change in hypoxia (e.g. Soivio et al., 1980), this study was conducted to find out the role of gills in the blood responses of rainbow trout to moderate hypoxia. This goal could be achieved by taking blood samples from undisturbed fish, permanently cannulated both to the ventral and the dorsal aorta.
MATERIALAND
METHODS
The experiments were carried out at Laukaa Fish Culture Research Station in November 1976. The experimental rainbow trout (S&o g~~r~~erj Richardson), from the stock of the Station, had been acclimated for a minimum of 4 wk to 90-100% 0, saturation, 9 + 0S”C temperature and rearing hall conditions with continuous light. In the experiments lake water was warmed up and used with a flow of 2 I’min-‘. kg-’ from a recirculating system, in which 1% of the total volume was replaced every minute. The pH of
the water was 6.9, the specific conductivity at 20-C 37 $S.cm- ’ and the total hardness 0.92 dH. Nine 4-yr-old rainbow trout (weight 910 k 4Og, length 42.5 f l.Ocm) (Mean + SEM) were anaesthetized with MS-222 (0.1 g. 1.. I) and first cannulated to the dorsal aorta (Soivio et al., 1975) and immediately thereafter via the bulbus arteriosus to the ventral aorta. The heart was exposed with a 20-25 mm long ventral incision. The pericardium was cut open and the tapered end of cannula (Clay Adams PE-50) thread about 20mm into the ventral aorta through a small hole, which was made with the tip of a hypodermic needle in the wall of the bulbus arteriosus. The cannula was anchored to the ventral body wall either by three bulbs made on the cannula by heating it (cf Soivio et af., 1972), or by a bulb on the cannula and a collar made from PE-160, when the gash was sutured with surgical steel. The cannula was flushed clear with non-heparini~ed saline, filled with saline containing IOIU of NH4+heparinate.ml- ‘, and stopped by melting the tip. The fish were allowed to recover for a week from the operation. For 48 hr before the experiments the fish were enclosed in individual restrainers (Soivio et al., 1975). Daily feeding (EWOS pelleted trout food) was stopped 48 hr before the experiment. The normoxic blood sample (O-sample) was taken from all the fish simultaneously through the ventral and the dorsal aortic cannulae. The samples (I ml) were collected into disposable tuberculine syringes without anticoagulants. The water was made hypoxic (5.5 mg 0,/l) by pumping it through a partial vacuum (Mount, 1961). During hypoxia the fish were simultaneously sampled for pre- and postbranchial blood at 20min, 1 hr and 3 hr. After 3 hr of hypoxia the fish were given normoxic water again and they were sampled after I, 3, 6, 24 and 48 hr of recovery, as described above. All the blood samples were analysed for pO1, pC02 and pH as described by Nikinmaa & Soivio (1979) and for haematocrit value (Hct), haemoglobin- (Hb), glucose- and lactate concentrations as described earlier by Soivio et al. (1975). The plasma protein concentration of each sample was determined by the Biuret reaction, the Cl- ‘-concentration with Radiometer CTM chloride titrator and the Na’-, K+-, Ca’+- and Mg’+-concentrations with an atomic absorption spectrophotometer according to Kristoffcrsson er al. (1972). 133
ANTTI SOIVIOrt ul
134
60 -
30
iI* * ** ** **** * * * 0
IO1
I
I
1
l/3
I
3
I
3
***
3(
I
3
6
I
3
6
*
**
**
**
0
l/3
I
3
I
I
!
0
l/3
I
3
1
6
**
24
* 24
I
4t3hr
* 46 hr
230 **
**
?
24
46 hr
Fig. 1. The means for haemoglobin concentration (Hb), haematocrit value (Hct) and mean corpuscular haemoglobin concentration (MCHC) of the rainbow trout in the blood from dorsal (0) and ventral (0) aorta prior to (0 hr), during (U) and after (-) hypoxia. The bars indicate + SEM. P-values were calculated with dependent t-tests for the differences between dorsal and ventral aorta1 blood CO = 0.1: * = 0.05: ** = 0.01; *** = 0.001).
RESULTS
Both the Hct values and the Hb concentration were (P< 0.05) higher in the dorsal than in the ventral aorta in each sample throughout the experiment (Fig. 1). The mean corpuscular haemoglobin concentration (MCHC) was slightly higher in the dorsal than in the ventral aorta, although this difference was statistically significant only at 1 hr of hypoxia and 1 hr of recovery. Also, the MCHC of dorsal aortic blood tended to increase (NS) in hypoxia. The difference in pOz between the aortae decreased from
significantly
11 kPa (80mmHg) in normoxia to about 6 kPa (35 mmHg) in hypoxia. The dorsal and ventral ~0~s were ca. 17 and 6 kPa respectively in normoxia and ca. 10 and 4 kPa in hypoxia. Immediately at the onset of the recovery period the normal pOz of the dorsal aorta was restored, while the pOz of the ventral aorta remained almost at the hypoxic level throughout the experimental recovery period. This leads to an increase in the ApO, (Fig. 2). In undisturbed normoxic rainbow trout the pC0, in the ventral aorta was 213 k 13 Pa (1.6 mmHg) and in the dorsal aorta about 26 Pa (0.2 mmHg) lower.
Gills and responses of trout during hypoxia
1
1
1
135
I
I
I
I I 24 40hr l/3 3 3 6 0 Fig. 2. The oxygen tension @O,) of dorsal (0) and ventral (0) aorta1 blood of rainbow trout prior to (0 hr), during (I) and after (---) hypoxia. For further details, see the legend of Fig. 1.
The difference between the aortae persisted throughout the hypoxic period and the 2-day recovery. Both the dorsal and the ventral aortic pC0, decreased during hypoxia, but returned to their normoxic levels within the first hour of recovery (Fig. 3). The pH value of the blood was the same in the preand postbranchial blood of normoxic, undisturbed rainbow trout prior to the hypoxic exposure. The pH of ventral aortic blood remained constant throughout the experiment. However, the pH of the dorsal aortic blood tended to increase (NS) during hypoxia, returning to the prehypoxic level at 1 hr of recovery (Fig. 3). The lactate concentration, 0.067 k 0.011 g/I, of the dorsal aortic blood in normoxic, prehypoxic rainbow trout was slightly lower (P < 0.1) than in the ventral aortic blood (Fig. 3). In hypoxia the lactate concentration in the ventral aortic blood decreased significantly (P < O.OOl),especially during the first hypoxic hour, and the dorso-ventral difference disappeared. During the recovery the lactate concentration remained significantly lower (P < 0.01) than before the hypoxic period with no apparent differences between the dorsal and the ventral blood. The glucose concentrations (Table 1) in the blood of pre- and postbranchial blood were the same before hypoxia. Throughout the experiment the blood glucose concentration tended to increase and remained on a slightly (30%) elevated level at the end of the experimental recovery (P < 0.05). The total protein and ion concentrations (K+, Na+ Ca*+, Mgzf and Cl-) prior to the hypoxic exposure were the same in the plasma of dorsal and ventral aortic blood (Table 1). No systematic, osmotically significant trends in these parameters could be observed during the experiment. However, the protein, K+- and Na+-concentrations were slightly increased at the beginning of hypoxia, while the Ca*+-, Mg’+- and Cl--concentrations were decreased. Also,
during the recovery period, especially between hours 1 and 3, small fluctuations took place in the ion and protein concentrations (Table 1). DISCUSSION
The values from the dorsal aortic blood for normoxie, pre-experimented rainbow trout of this study agree well with those obtained earlier from the same stock, when similar sampling technique and experimental conditions have been used (Soivio et al., 1975, 1977, 1980; Nikinmaa et al., 1980). The decreasing trend of Hb concentration and Hct value throughout the experiment were evidently due to subsequent sampling (Soivio et al., 1975), which seemed not to have any effect on the other parameters studied. The most notable difference between the aortae was that throughout the experiment the Hb concentrations and Hct values were significantly higher in the dorsal aortic than in the ventral aortic blood. This indicates a systemic loss of plasma in the gills, and is actually the first direct evidence of plasma skimming taking place in the gills of undisturbed normoxic or hypoxic rainbow trout. In another connection we have got evidence that either anaesthetized or stunned sea trout, sampled for blood with cardiac puncture had higher Hb concentration than trout sampled from ductus Cuvieri (Soivio et al., manuscript). Therefore, it seems that the plasma, lost in the gills, does not return to the systemic circulation, but goes straight back to the heart, probably via the anterior cardinal veins. The sites of plasma skimming in gills have been supposed to lie on the afferent side of the filamental circulation mainly because of the high intralamellar Hct values measured morphometrically (Hughes, 1980). Recently, Soivio & Tuurala (1981) have speculated the possibility of plasma skimming through the secondary lamellar wall. On the other
0
L
8
1
1
f
I
i
i/3
I
3
I
3
6
24
Fig. 3. pCO1, pH and lactate concentration prior to (0 hr), during (III) and after (
I
48hr
of dorsal (0) and ventral (0) aorta1 blood of rainbow trout -) hypoxia. For further details, see the legend of Fig. 1.
hand, part of the plasma skimming may take place on the efferent side of the secondary lamellae. The arterio-venous anastomoses (AVA), when compared with the size of the efferent filamental arteries (EFA) (cf Vogel er ul., 1976) have similarities to the vascular bed in the systemic circulation; the AVA’s resembling capillaries and the EFA’s the thoroughfare vessels. In systemic microcirculation the thoroughfare vessels are usually high-haematocrit shunts, the true capillaries getting large amounts of plasma (Johnson, 1971) and having low Hct values. Whatever the site is, the skimming of plasma (2-37;) from the circulation in gills facilitates the oxygen transport in the fish. If the skimming takes place after the secondary lamellae it has no negative effects on the blood viscosity inside the gills. However, the high Hct counts of the secondary lamellar capillaries (Soivio & Hughes 1978; Hughes et a!., 1978) indicate that separation of plasma
and erythrocytes takes place in the secondary lamellae. The changes in the erythrocytic size in the gills agree well with the results of in cifro incubations (Soivio et al., 1973), which show that the erythrocytes of Salmonidae are bigger in low than in high pOz. According to Black & Irving (1938) an increase in pC0, increased the erythrocytic size. In this study. however, the observed changes in pCOz in hypoxia did not change the crythrocytic volume. In recent studies (Soivio & Nikinmaa, 1981; Nikmmaa & Soivio, 1981) it has been shown that the erythrocytes in the dorsal and also in the ventral aorta are swollen in hypoxia at high temperatures (18C). The swelling of erythrocytes leads to an increase in the blood oxygen affinity, mostly by increasing the intraerythrocytic pH by decreasing the intracellular ATP and the Hb concentrations (Soivio & Nikin-
Gills and responses of trout during hypoxia Table
1. The means
State Co, (mgil) Time (hr)
10 0 D
(g/l) V
D
(g/l) V
K+ (rn-equiv/l)
D
V
Na+ (m-equiv/l)
D
V
Ca’+ m-equiv/l)
SEMs for blood glucose, plasma protein and during and after the experimental hypoxia
Prehypoxia
Glucose
Protein
and
D
V
Mg2 + (m-equiv/l)
D
V
Cl_ (m-equiv/l)
D
V
The number
137
l/3
0.669 0.052 (8) 0.667 0.043
0.647 0.059 (7) 0.671 0.059
Hypoxia 5.5 1 0.701 0.053 (7) 0.681 0.056
ion concentrations
3
1
3
0.706 0.096
0.705 0.060 (7) 0.685 0.057
0.694 0.039
(5) 0.699 0.091
29.6 2.4 (7) 32.0 2.2
32.4 1.1 (5) 33.3 3.3
31.4 3.2 (6) 30.4 3.1
29.2 4.7 (4) 29.0 4.6
33.1 2.4 (6) 30.7 ’ 3.0
2.43 0.10
2.49 0.16
2.41 0.12
2.18 0.18
2.25 0.14
(7) 2.41 0.13
(7) 2.63 0.19
(7) 2.31 0.14
(4) 2.45 0.42
2.58
(8) 149.4 1.9
155.7 7.1 (6) 150.8 2.3
151.9 1.9 (7) 148.0 3.2
5.17 0.40
4.93 0.29
(9) 5.22 0.41
(6) 0.646 0.034
Recovery 10 6 0.728 0.043 (6) 0.673 0.054
prior
to,
24
48
0.897 0.099 (7) 0.960 0.104
0.915 0.045 (5) 1.110 0.061
(6) 23.9 2.8
27.6 7.2 (2) 28.2 7.3
30.1 3.2 (5) 29.8 3.3
29.3 9.5 (2) 28.0 9.6
2.43 0.12
2.37 0.15
2.53 0.20
0.18
(7) 2.77 0.22
(6) 2.43 0.17
(6) 2.82 0.30
2.80 0.45 (3) 2.17 0.13
147.3 12.5 (4) 152.5 5.3
148.8 5.4 (6) 1so.2 3.8
150.3 2.8 (7) 157.0 2.9
151.0 4.0 (6) 154.2 2.8
147.7 2.3 (7) 148.3 2.4
144.8 4.9 (4) 148.5 3.7
5.56 0.65 (5) 5.60 0.59
5.52 0.62 (5) 5.44 0.61
5.80 0.41 (5) 5.84 0.40
5.17 0.43 (7) 5.20 0.44
5.23 0.80 (3) 5.27 0.88
5.17 0.49
(8) 5.10 0.44
5.02 0.58 (5) 4.86 0.54
5.13 0.37
1.51 0.07 (8) 1.54 0.06
1.49 0.0 (8) 1.49 0.06
1.47 0.09 (5) 1.42 0.06
1.62 0.07 (4) 1.63 0.09
1.62 0.09 (4) 1.60 0.07
1.65 0.08 (4) 1.67 0.05
1.60 0.05 (6) 1.62 0.06
1.75 0.05 (3) 1.74 0.05
1.58 0.09 (5) 1.58 0.09
139.3 1.3 (8) 139.0 0.7
138.2 1.3 (6) 136.8 1.1
138.6 1.5 (8) 138.6 1.2
139.5 1.5 (6) 137.5 0.7
137.2 1.5 (6) 137.8 0.6
139.4 1.1 (8) 137.6 1.3
138.2 1.7 (5) 139.4 1.9
139.0 1.5 (7) 138.3 1.4
140.0 2.4 (4) 138.3 2.1
(dorsal/ventral)
analyses
145.6 2.4
of paired
maa, 1981; Nikinmaa & Soivio, 1981). However, in this study a similar swelling of the erythrocytes in the dorsal aortic blood would not be expected as the oxygen saturation of water was ca. 50% (C&, 5.5 mg/l) and Soivio & Nikinmaa (1981) found that the swelling of the erythrocytes begins when the oxygen saturation of water decreases below 50% at 18°C (when the Co, of water was ca. 4.5 mg/l). The swelling of the erythrocytes seems to take place when the pOz of blood decreases near the P50 value. In this study the oxygen saturation of dorsal aortic blood was probably near 90x, when calculated from the data of Nikinmaa & Soivio (1979). The P,, value was evidently reached first in the tissue capillaries. The swelling of the erythrocytes at this point could ensure the O2 transport also to the venous end of the capillary bed and make the distribution of oxygen to different parts of the capillary bed more even. The accumulation of lactate in the blood of rain-
(6)
27.3 3.8
(6)
are given in parenthesis.
bow trout as a result to environmental hypoxia has been demonstrated by Holeton & Randall (1967) and Burton & Shephard (1971). According to Black et al. (1961) the blood lactate concentration in Salmonoid fish reaches a maximum approximately 3 hr after the exercise has ceased. This delay is evidently due to the slow rate of transfer of muscle lactate into the blood, which probably is partly caused by the impaired circulation after exercise (cf Dando, 1969). The decreasing trend of the lactate concentration during the experiment show clearly that during moderate experimental hypoxia the tissues have enough oxygen to prevent anaerobiosis. Actually the situation in this sense seems to be better than before the hypoxic experiment. This may be an indication of ionic, pH and/or circulatory changes taking place in tissues. Also it is possible that the decreasing ~0, of the capillaries and mixed venous blood in hypoxia favours O2 delivery from the red blood cells more
ANTTI Sotvio
13x
than it decreases the rate of diffusion of 02, the net result being an increase in the amount of oxygen available in the tissues during moderate hypoxia. Such changes could facilitate the oxygen transfer from gills to tissues. The prehypoxic decrease of blood lactate concentration in gills agrees well with the studies of Bilinski & Jonas (1972) and Driedzic & Kiceniuk (1976) which show that the gill tissue has an ability to utilize lactate as an energy source. However. the results reveal that the ~0, during the experimental hypoxia was too low to enable the lactic utilization by gills. The continuously decreasing trend of lactate c~~ncentr~~tion of ventral aorta1 blood indicates that the facilities for oxygen utilization in tissues surrounding the systemic circulation are relatively better than in gills during moderate hypoxia. The changes in the osmotically active parameters studied are negligible during the experiment. However, the slight, non-significant peaks after the onset of hypoxia and between I-24 hr of recovery agree in timing with the findings of Hunn (1969) on rainbow trout and Kirk (1974) on Ictalurus punctatus. Kirk (1974) demonstrated that the increase of plasma osmolarity took place 1 hr after a severe hypoxia. This osmoconcentration was closely connected to elevated blood lactate concentration and decreased blood pH value, changes which were not observed in this study. This again is an indication that the SOS,< O2 saturation of the environment is easily tolerable for rainbow trout at 9’C. All in all, decreasing the oxygen saturation of water to 50% at 9°C causes none of the changes in blood parameters usually connected with hypoxia in rainbow trout; haemoconccntration. changes in plasma pH value, blood lactate concentration. changes in erythrocytic volume (Soivio P( (I/.. 1980: Soivio & Nikinmaa, 1981). Therefore the most notable finding about the role of gills in modifying blood parameters was the clear change in blood Hb concentration taking place in gills, both in normoxia and in hypoxia. Acknowledgements-We wish to express our thanks to Mrs Hannele Virtanen, MSc. and Mrs Ida Luhtanen for valuable technical assistance. This study was partly supported by a grant from the National Research Council for Science (Suomen Akatemia, Luonn(~ntieteellinen toimikunta).
REFERENCES BILINSKI E. & JONAS R. E. E. (1972) Oxidation of lactate to carbon dioxide by rainbow trout (Salmo yairdnrri) tissues. J. Fish. Res. Ed Can. 29, 1467 1471. BLACK E. C. & IRVING L. (193X) The effect of hemolysis upon the affinity of fish biood for oxygen. f. c&i. conrp. Physiol. 12, 255-262. BLACK E. C., ROBERTSON A. C. & PAKK~K R. R. (1961) Some aspects of carbohydrate metabolism in fish. In Comparative Physiology of Carhohydrutr Metabolism in Heterothermic Atlimals (Edited by MARTIN A. W.), pp. 89-124. Univ. of Wash. Press, Seattle, BOOTH J. H. (1978) The distribution of blood flow in the gills of fish: application of a new technique to rainbow trout (Salmo gairdneri). J. exp. Biol. 73, 119-l 30. BOOTH J. H. (1979a) The effects of oxygen supply, epinephrine and acetylcholine on the distribution of blood Row in trout gills. J. cup. Biol. 83, 31.39.
t’f ~ii.
BWTH J. H. (1979b) Circulation
in trout gdls: the rclatmnship between branchial perfusion and the width of the lamellar blood space. CLIP. J. Zool. 57. 2183 218.5. BVRTON D. T. & SPEHAK A. M. 11971) A re-evaluation of the anaerobic endproducts of fresh-water fish exposed to environment~ii hypoxia. Titn2p. Bi~~(.~zf,~~z. P~~,~j~)~. 40A, 945-954. DANDO P. R. (1969) Lactate metabolism in fish. J. )?IL~IT. hioi. Ass. I’.K. 49, 209-223. DKIEDZI(, W. R. & KI~~~I~JK .J. W. (1976) Blood lactate levels in free-swimming rainbow trout (Salvo gctirdneri) before and after strenuous exercise resulting in fatigue. J. Fisll. Ru\. Rd Can. 33, I73 176. HOI.ETON G. F. & RANIXLI. D. J. (19671 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. rxp. Eiol. 46, 317-327. HUGHES G. M. (1980) Functional morphology of fish gills. In Epithr/ia/ Transport itr rhr Lower L’ertrbrates. Part 1: Morphological aspects (Edited by LAHLOU), pp. 15.-36. C.U.P., Malta. HL’C;HESG. M., TIXRALA H. & Sorvro A. (1978) Regional distribution of blood in the gills of rainbow trout m normoxia and hypoxia: a morphometric study with two fixatives. Ann. Zoo/. Fcwuci 15, 226-234. HUNN J. B. (1969) Chemical composition of rainbow trout urine following acute hypoxic stress. Trans. Am. Fish. Sot. 98, 20-22. JOHNSON: P. C. 11971) Red cell separation in the mesenteric capillary network. Am. .I. Physiol. 221, 99-104. KIRK W. L. (1974) The effects of hypoxia on certain blood and tissue electrolytes of channel catfish, Ictulurus punctuttrs (Rafinesque), Trans. Am. Fish. Sot. 103, 593-600. KRISTOFFERSSOIVR.. BROBERC;S. & OII
NIKINMAA M. & SOI~IO A. (1979) Oxygen dissociation curves and oxygen capacities of a freshwater fish, SuImo qrlirdneri. .4m~. Zool. Femki 16, 2 17-221. NIKIN,MAAM. &Sotvio A. (1981) Blood oxygen transport of hypoxic Srrfmo ~~~z~rd~7~ri at high temperature. Submitted to J. rup. Zool. NIK~NMAA M., TUURALA H. & SOIVIO A. (1980) Thermoacclimatory changes In blood oxygen binding properties and gill secondary lamellar structure of Sulmo guirdneri J. wmp.
Physiol.
140, X5-
260.
RANDALI. D. J.. HXETON G. F. & STEVENSE. D. (1967) The exchange of oxygen and carbon dioxide across the gills of rambow trout. J. rup. Birii. 46, 329.-348. SOIVIO A. & H~J(~HESG. M. (1978) Circulatory changes in the secondary lamellae of Salmo gairdneri gills in hypoxia and in anaesthesia. Ann. Zoo!. Fennici 15, 221-22s. SOI~IO A. & NIKIXMAA M. (1981) The swelling of erythrocytes in relation to the oxygen aR’mity of the blood of the rainbow trout. Sdrm ~~~~~~~~f?~,~j Richardson. In Strrcc rortl F.islr (Edited by PICKI~HIN~;A, D.). Academic Press, New York. In press. SOIVIO A. & TU~JKALA H. (1981) Structural and circulatory responses to hypoxia m the secondary lamellae of Sulmo gairdnrri gills at two temperatures. Submitted to J. camp. Physiol. Sowro A., WESTMAN K. & NYHOLM K. (1972) improved method of dorsal aorta catheterization: haematoio~ical effects followed for three weeks in rainbow trout (Sulmo gairdneri). Finn. Fish. Rus. I, 11-21. Sorvro A., NYHOLM K. & WESTMAN K. (1973) Notes on haematocrit determinations on rainbow trout, Salmo goirdnrri.
Aqutrculturr
2, 3 I 35.
Gills and responses of trout during hypoxia SOIVIOA., NYHOLMK. & WESTMANK. (1975) A technique for repeated sampling of the blood of individual resting fish. J. exp. Biol. 62, 207-217. SOIVIOA., NYHOLMK. & HUHTI M. (1977) Effects of anaesthesia with MS 222, neutralized MS 222 and benzocaine on the blood constituents of rainbow trout, Salmo gairdneri. J. Fish Biol. 10, 91-101. SOIVIOA., NIKINMAAM. & WESTMANK. (1980) The blood oxygen binding properties of hypoxic Salmo gairdneri. .I. camp. Physiol. 136, 83-87.
139
VOGEL W., VIJGEL V. & F%AUT~CHM. (1976) Arteriovenous anastomoses in rainbow trout gill filaments. Cell Tiss. Res. 167, 373-385. W~~ID C. M. & SHELTONG. (1980) Cardiovascular dynamics and adrenergic responses of the rainbow trout in ho.
J. exp. Biol. 87, 247-270.
WOOD C. M. & SHELTONG. (1980) The reflex control of heart rate and cardiac output in the rainbow trout: interactive influences of hypoxia, haemorrhage and systemic vasomotor tone. J. exp. Biol. 87, 271-284.
(Received 2 Febrmy
1981)
Abstract-l. Gill function of rainbow trout, cannulated permanently to both ventral and dorsal aortae, was studied during moderate, short term hypoxia (5.5 mg/l) at 9°C. 2. Hb concentrations and Hct values were significantly and MCHC slightly higher in the dorsal aorta. 3. PO, difference between the aortae decreased from 11 kPa in normoxia to 6 kPa in hypoxia. 4. Blood pH as well as protein and inorganic ion concentrations of the plasma were the same in both aortae and remained constant. 5. Lactate concentration in the ventral aorta exceeded that of the dorsal one in the prehypoxic fish, but decreased to same value during hypoxia.
The gill function of rainbow trout has mostly been studied with perfused head or gill preparations. Less work has been done with “in uiuo preparations”, permanently cannulated fish, in which the gills are in
their natural surroundings both externally and internally. The in vivo studies have so far mainly been aimed at solving the effects of hypoxia, exercise and intravenously added substances on the gas exchange through the gills and the circulation of the gills (Holeton & Randall, 1967; Randall et al., 1967; Booth, 1978, 1979a,b; Wood & Shelton, 1980a,b). Driedzic & Kiceniuk (1976) suggested that the gills of rainbow trout utilize lactate during exercise in viva. In addition the fish gills have important functions in osmoregulation. For example the osmotic balance in freshwater fish is partly regulated by the uptake of NaCl by the gills. In view of the several functions of the gills both in gas exchange and osmoregulation, and as it is known that in addition to the blood gas tensions, several other blood parameters change in hypoxia (e.g. Soivio et al., 1980), this study was conducted to find out the role of gills in the blood responses of rainbow trout to moderate hypoxia. This goal could be achieved by taking blood samples from undisturbed fish, permanently cannulated both to the ventral and the dorsal aorta.
MATERIALAND
METHODS
The experiments were carried out at Laukaa Fish Culture Research Station in November 1976. The experimental rainbow trout (S&o g~~r~~erj Richardson), from the stock of the Station, had been acclimated for a minimum of 4 wk to 90-100% 0, saturation, 9 + 0S”C temperature and rearing hall conditions with continuous light. In the experiments lake water was warmed up and used with a flow of 2 I’min-‘. kg-’ from a recirculating system, in which 1% of the total volume was replaced every minute. The pH of
the water was 6.9, the specific conductivity at 20-C 37 $S.cm- ’ and the total hardness 0.92 dH. Nine 4-yr-old rainbow trout (weight 910 k 4Og, length 42.5 f l.Ocm) (Mean + SEM) were anaesthetized with MS-222 (0.1 g. 1.. I) and first cannulated to the dorsal aorta (Soivio et al., 1975) and immediately thereafter via the bulbus arteriosus to the ventral aorta. The heart was exposed with a 20-25 mm long ventral incision. The pericardium was cut open and the tapered end of cannula (Clay Adams PE-50) thread about 20mm into the ventral aorta through a small hole, which was made with the tip of a hypodermic needle in the wall of the bulbus arteriosus. The cannula was anchored to the ventral body wall either by three bulbs made on the cannula by heating it (cf Soivio et af., 1972), or by a bulb on the cannula and a collar made from PE-160, when the gash was sutured with surgical steel. The cannula was flushed clear with non-heparini~ed saline, filled with saline containing IOIU of NH4+heparinate.ml- ‘, and stopped by melting the tip. The fish were allowed to recover for a week from the operation. For 48 hr before the experiments the fish were enclosed in individual restrainers (Soivio et al., 1975). Daily feeding (EWOS pelleted trout food) was stopped 48 hr before the experiment. The normoxic blood sample (O-sample) was taken from all the fish simultaneously through the ventral and the dorsal aortic cannulae. The samples (I ml) were collected into disposable tuberculine syringes without anticoagulants. The water was made hypoxic (5.5 mg 0,/l) by pumping it through a partial vacuum (Mount, 1961). During hypoxia the fish were simultaneously sampled for pre- and postbranchial blood at 20min, 1 hr and 3 hr. After 3 hr of hypoxia the fish were given normoxic water again and they were sampled after I, 3, 6, 24 and 48 hr of recovery, as described above. All the blood samples were analysed for pO1, pC02 and pH as described by Nikinmaa & Soivio (1979) and for haematocrit value (Hct), haemoglobin- (Hb), glucose- and lactate concentrations as described earlier by Soivio et al. (1975). The plasma protein concentration of each sample was determined by the Biuret reaction, the Cl- ‘-concentration with Radiometer CTM chloride titrator and the Na’-, K+-, Ca’+- and Mg’+-concentrations with an atomic absorption spectrophotometer according to Kristoffcrsson er al. (1972). 133
ANTTI SOIVIOrt ul
134
60 -
30
iI* * ** ** **** * * * 0
IO1
I
I
1
l/3
I
3
I
3
***
3(
I
3
6
I
3
6
*
**
**
**
0
l/3
I
3
I
I
!
0
l/3
I
3
1
6
**
24
* 24
I
4t3hr
* 46 hr
230 **
**
?
24
46 hr
Fig. 1. The means for haemoglobin concentration (Hb), haematocrit value (Hct) and mean corpuscular haemoglobin concentration (MCHC) of the rainbow trout in the blood from dorsal (0) and ventral (0) aorta prior to (0 hr), during (U) and after (-) hypoxia. The bars indicate + SEM. P-values were calculated with dependent t-tests for the differences between dorsal and ventral aorta1 blood CO = 0.1: * = 0.05: ** = 0.01; *** = 0.001).
RESULTS
Both the Hct values and the Hb concentration were (P< 0.05) higher in the dorsal than in the ventral aorta in each sample throughout the experiment (Fig. 1). The mean corpuscular haemoglobin concentration (MCHC) was slightly higher in the dorsal than in the ventral aorta, although this difference was statistically significant only at 1 hr of hypoxia and 1 hr of recovery. Also, the MCHC of dorsal aortic blood tended to increase (NS) in hypoxia. The difference in pOz between the aortae decreased from
significantly
11 kPa (80mmHg) in normoxia to about 6 kPa (35 mmHg) in hypoxia. The dorsal and ventral ~0~s were ca. 17 and 6 kPa respectively in normoxia and ca. 10 and 4 kPa in hypoxia. Immediately at the onset of the recovery period the normal pOz of the dorsal aorta was restored, while the pOz of the ventral aorta remained almost at the hypoxic level throughout the experimental recovery period. This leads to an increase in the ApO, (Fig. 2). In undisturbed normoxic rainbow trout the pC0, in the ventral aorta was 213 k 13 Pa (1.6 mmHg) and in the dorsal aorta about 26 Pa (0.2 mmHg) lower.
Gills and responses of trout during hypoxia
1
1
1
135
I
I
I
I I 24 40hr l/3 3 3 6 0 Fig. 2. The oxygen tension @O,) of dorsal (0) and ventral (0) aorta1 blood of rainbow trout prior to (0 hr), during (I) and after (---) hypoxia. For further details, see the legend of Fig. 1.
The difference between the aortae persisted throughout the hypoxic period and the 2-day recovery. Both the dorsal and the ventral aortic pC0, decreased during hypoxia, but returned to their normoxic levels within the first hour of recovery (Fig. 3). The pH value of the blood was the same in the preand postbranchial blood of normoxic, undisturbed rainbow trout prior to the hypoxic exposure. The pH of ventral aortic blood remained constant throughout the experiment. However, the pH of the dorsal aortic blood tended to increase (NS) during hypoxia, returning to the prehypoxic level at 1 hr of recovery (Fig. 3). The lactate concentration, 0.067 k 0.011 g/I, of the dorsal aortic blood in normoxic, prehypoxic rainbow trout was slightly lower (P < 0.1) than in the ventral aortic blood (Fig. 3). In hypoxia the lactate concentration in the ventral aortic blood decreased significantly (P < O.OOl),especially during the first hypoxic hour, and the dorso-ventral difference disappeared. During the recovery the lactate concentration remained significantly lower (P < 0.01) than before the hypoxic period with no apparent differences between the dorsal and the ventral blood. The glucose concentrations (Table 1) in the blood of pre- and postbranchial blood were the same before hypoxia. Throughout the experiment the blood glucose concentration tended to increase and remained on a slightly (30%) elevated level at the end of the experimental recovery (P < 0.05). The total protein and ion concentrations (K+, Na+ Ca*+, Mgzf and Cl-) prior to the hypoxic exposure were the same in the plasma of dorsal and ventral aortic blood (Table 1). No systematic, osmotically significant trends in these parameters could be observed during the experiment. However, the protein, K+- and Na+-concentrations were slightly increased at the beginning of hypoxia, while the Ca*+-, Mg’+- and Cl--concentrations were decreased. Also,
during the recovery period, especially between hours 1 and 3, small fluctuations took place in the ion and protein concentrations (Table 1). DISCUSSION
The values from the dorsal aortic blood for normoxie, pre-experimented rainbow trout of this study agree well with those obtained earlier from the same stock, when similar sampling technique and experimental conditions have been used (Soivio et al., 1975, 1977, 1980; Nikinmaa et al., 1980). The decreasing trend of Hb concentration and Hct value throughout the experiment were evidently due to subsequent sampling (Soivio et al., 1975), which seemed not to have any effect on the other parameters studied. The most notable difference between the aortae was that throughout the experiment the Hb concentrations and Hct values were significantly higher in the dorsal aortic than in the ventral aortic blood. This indicates a systemic loss of plasma in the gills, and is actually the first direct evidence of plasma skimming taking place in the gills of undisturbed normoxic or hypoxic rainbow trout. In another connection we have got evidence that either anaesthetized or stunned sea trout, sampled for blood with cardiac puncture had higher Hb concentration than trout sampled from ductus Cuvieri (Soivio et al., manuscript). Therefore, it seems that the plasma, lost in the gills, does not return to the systemic circulation, but goes straight back to the heart, probably via the anterior cardinal veins. The sites of plasma skimming in gills have been supposed to lie on the afferent side of the filamental circulation mainly because of the high intralamellar Hct values measured morphometrically (Hughes, 1980). Recently, Soivio & Tuurala (1981) have speculated the possibility of plasma skimming through the secondary lamellar wall. On the other
0
L
8
1
1
f
I
i
i/3
I
3
I
3
6
24
Fig. 3. pCO1, pH and lactate concentration prior to (0 hr), during (III) and after (
I
48hr
of dorsal (0) and ventral (0) aorta1 blood of rainbow trout -) hypoxia. For further details, see the legend of Fig. 1.
hand, part of the plasma skimming may take place on the efferent side of the secondary lamellae. The arterio-venous anastomoses (AVA), when compared with the size of the efferent filamental arteries (EFA) (cf Vogel er ul., 1976) have similarities to the vascular bed in the systemic circulation; the AVA’s resembling capillaries and the EFA’s the thoroughfare vessels. In systemic microcirculation the thoroughfare vessels are usually high-haematocrit shunts, the true capillaries getting large amounts of plasma (Johnson, 1971) and having low Hct values. Whatever the site is, the skimming of plasma (2-37;) from the circulation in gills facilitates the oxygen transport in the fish. If the skimming takes place after the secondary lamellae it has no negative effects on the blood viscosity inside the gills. However, the high Hct counts of the secondary lamellar capillaries (Soivio & Hughes 1978; Hughes et a!., 1978) indicate that separation of plasma
and erythrocytes takes place in the secondary lamellae. The changes in the erythrocytic size in the gills agree well with the results of in cifro incubations (Soivio et al., 1973), which show that the erythrocytes of Salmonidae are bigger in low than in high pOz. According to Black & Irving (1938) an increase in pC0, increased the erythrocytic size. In this study. however, the observed changes in pCOz in hypoxia did not change the crythrocytic volume. In recent studies (Soivio & Nikinmaa, 1981; Nikmmaa & Soivio, 1981) it has been shown that the erythrocytes in the dorsal and also in the ventral aorta are swollen in hypoxia at high temperatures (18C). The swelling of erythrocytes leads to an increase in the blood oxygen affinity, mostly by increasing the intraerythrocytic pH by decreasing the intracellular ATP and the Hb concentrations (Soivio & Nikin-
Gills and responses of trout during hypoxia Table
1. The means
State Co, (mgil) Time (hr)
10 0 D
(g/l) V
D
(g/l) V
K+ (rn-equiv/l)
D
V
Na+ (m-equiv/l)
D
V
Ca’+ m-equiv/l)
SEMs for blood glucose, plasma protein and during and after the experimental hypoxia
Prehypoxia
Glucose
Protein
and
D
V
Mg2 + (m-equiv/l)
D
V
Cl_ (m-equiv/l)
D
V
The number
137
l/3
0.669 0.052 (8) 0.667 0.043
0.647 0.059 (7) 0.671 0.059
Hypoxia 5.5 1 0.701 0.053 (7) 0.681 0.056
ion concentrations
3
1
3
0.706 0.096
0.705 0.060 (7) 0.685 0.057
0.694 0.039
(5) 0.699 0.091
29.6 2.4 (7) 32.0 2.2
32.4 1.1 (5) 33.3 3.3
31.4 3.2 (6) 30.4 3.1
29.2 4.7 (4) 29.0 4.6
33.1 2.4 (6) 30.7 ’ 3.0
2.43 0.10
2.49 0.16
2.41 0.12
2.18 0.18
2.25 0.14
(7) 2.41 0.13
(7) 2.63 0.19
(7) 2.31 0.14
(4) 2.45 0.42
2.58
(8) 149.4 1.9
155.7 7.1 (6) 150.8 2.3
151.9 1.9 (7) 148.0 3.2
5.17 0.40
4.93 0.29
(9) 5.22 0.41
(6) 0.646 0.034
Recovery 10 6 0.728 0.043 (6) 0.673 0.054
prior
to,
24
48
0.897 0.099 (7) 0.960 0.104
0.915 0.045 (5) 1.110 0.061
(6) 23.9 2.8
27.6 7.2 (2) 28.2 7.3
30.1 3.2 (5) 29.8 3.3
29.3 9.5 (2) 28.0 9.6
2.43 0.12
2.37 0.15
2.53 0.20
0.18
(7) 2.77 0.22
(6) 2.43 0.17
(6) 2.82 0.30
2.80 0.45 (3) 2.17 0.13
147.3 12.5 (4) 152.5 5.3
148.8 5.4 (6) 1so.2 3.8
150.3 2.8 (7) 157.0 2.9
151.0 4.0 (6) 154.2 2.8
147.7 2.3 (7) 148.3 2.4
144.8 4.9 (4) 148.5 3.7
5.56 0.65 (5) 5.60 0.59
5.52 0.62 (5) 5.44 0.61
5.80 0.41 (5) 5.84 0.40
5.17 0.43 (7) 5.20 0.44
5.23 0.80 (3) 5.27 0.88
5.17 0.49
(8) 5.10 0.44
5.02 0.58 (5) 4.86 0.54
5.13 0.37
1.51 0.07 (8) 1.54 0.06
1.49 0.0 (8) 1.49 0.06
1.47 0.09 (5) 1.42 0.06
1.62 0.07 (4) 1.63 0.09
1.62 0.09 (4) 1.60 0.07
1.65 0.08 (4) 1.67 0.05
1.60 0.05 (6) 1.62 0.06
1.75 0.05 (3) 1.74 0.05
1.58 0.09 (5) 1.58 0.09
139.3 1.3 (8) 139.0 0.7
138.2 1.3 (6) 136.8 1.1
138.6 1.5 (8) 138.6 1.2
139.5 1.5 (6) 137.5 0.7
137.2 1.5 (6) 137.8 0.6
139.4 1.1 (8) 137.6 1.3
138.2 1.7 (5) 139.4 1.9
139.0 1.5 (7) 138.3 1.4
140.0 2.4 (4) 138.3 2.1
(dorsal/ventral)
analyses
145.6 2.4
of paired
maa, 1981; Nikinmaa & Soivio, 1981). However, in this study a similar swelling of the erythrocytes in the dorsal aortic blood would not be expected as the oxygen saturation of water was ca. 50% (C&, 5.5 mg/l) and Soivio & Nikinmaa (1981) found that the swelling of the erythrocytes begins when the oxygen saturation of water decreases below 50% at 18°C (when the Co, of water was ca. 4.5 mg/l). The swelling of the erythrocytes seems to take place when the pOz of blood decreases near the P50 value. In this study the oxygen saturation of dorsal aortic blood was probably near 90x, when calculated from the data of Nikinmaa & Soivio (1979). The P,, value was evidently reached first in the tissue capillaries. The swelling of the erythrocytes at this point could ensure the O2 transport also to the venous end of the capillary bed and make the distribution of oxygen to different parts of the capillary bed more even. The accumulation of lactate in the blood of rain-
(6)
27.3 3.8
(6)
are given in parenthesis.
bow trout as a result to environmental hypoxia has been demonstrated by Holeton & Randall (1967) and Burton & Shephard (1971). According to Black et al. (1961) the blood lactate concentration in Salmonoid fish reaches a maximum approximately 3 hr after the exercise has ceased. This delay is evidently due to the slow rate of transfer of muscle lactate into the blood, which probably is partly caused by the impaired circulation after exercise (cf Dando, 1969). The decreasing trend of the lactate concentration during the experiment show clearly that during moderate experimental hypoxia the tissues have enough oxygen to prevent anaerobiosis. Actually the situation in this sense seems to be better than before the hypoxic experiment. This may be an indication of ionic, pH and/or circulatory changes taking place in tissues. Also it is possible that the decreasing ~0, of the capillaries and mixed venous blood in hypoxia favours O2 delivery from the red blood cells more
ANTTI Sotvio
13x
than it decreases the rate of diffusion of 02, the net result being an increase in the amount of oxygen available in the tissues during moderate hypoxia. Such changes could facilitate the oxygen transfer from gills to tissues. The prehypoxic decrease of blood lactate concentration in gills agrees well with the studies of Bilinski & Jonas (1972) and Driedzic & Kiceniuk (1976) which show that the gill tissue has an ability to utilize lactate as an energy source. However. the results reveal that the ~0, during the experimental hypoxia was too low to enable the lactic utilization by gills. The continuously decreasing trend of lactate c~~ncentr~~tion of ventral aorta1 blood indicates that the facilities for oxygen utilization in tissues surrounding the systemic circulation are relatively better than in gills during moderate hypoxia. The changes in the osmotically active parameters studied are negligible during the experiment. However, the slight, non-significant peaks after the onset of hypoxia and between I-24 hr of recovery agree in timing with the findings of Hunn (1969) on rainbow trout and Kirk (1974) on Ictalurus punctatus. Kirk (1974) demonstrated that the increase of plasma osmolarity took place 1 hr after a severe hypoxia. This osmoconcentration was closely connected to elevated blood lactate concentration and decreased blood pH value, changes which were not observed in this study. This again is an indication that the SOS,< O2 saturation of the environment is easily tolerable for rainbow trout at 9’C. All in all, decreasing the oxygen saturation of water to 50% at 9°C causes none of the changes in blood parameters usually connected with hypoxia in rainbow trout; haemoconccntration. changes in plasma pH value, blood lactate concentration. changes in erythrocytic volume (Soivio P( (I/.. 1980: Soivio & Nikinmaa, 1981). Therefore the most notable finding about the role of gills in modifying blood parameters was the clear change in blood Hb concentration taking place in gills, both in normoxia and in hypoxia. Acknowledgements-We wish to express our thanks to Mrs Hannele Virtanen, MSc. and Mrs Ida Luhtanen for valuable technical assistance. This study was partly supported by a grant from the National Research Council for Science (Suomen Akatemia, Luonn(~ntieteellinen toimikunta).
REFERENCES BILINSKI E. & JONAS R. E. E. (1972) Oxidation of lactate to carbon dioxide by rainbow trout (Salmo yairdnrri) tissues. J. Fish. Res. Ed Can. 29, 1467 1471. BLACK E. C. & IRVING L. (193X) The effect of hemolysis upon the affinity of fish biood for oxygen. f. c&i. conrp. Physiol. 12, 255-262. BLACK E. C., ROBERTSON A. C. & PAKK~K R. R. (1961) Some aspects of carbohydrate metabolism in fish. In Comparative Physiology of Carhohydrutr Metabolism in Heterothermic Atlimals (Edited by MARTIN A. W.), pp. 89-124. Univ. of Wash. Press, Seattle, BOOTH J. H. (1978) The distribution of blood flow in the gills of fish: application of a new technique to rainbow trout (Salmo gairdneri). J. exp. Biol. 73, 119-l 30. BOOTH J. H. (1979a) The effects of oxygen supply, epinephrine and acetylcholine on the distribution of blood Row in trout gills. J. cup. Biol. 83, 31.39.
t’f ~ii.
BWTH J. H. (1979b) Circulation
in trout gdls: the rclatmnship between branchial perfusion and the width of the lamellar blood space. CLIP. J. Zool. 57. 2183 218.5. BVRTON D. T. & SPEHAK A. M. 11971) A re-evaluation of the anaerobic endproducts of fresh-water fish exposed to environment~ii hypoxia. Titn2p. Bi~~(.~zf,~~z. P~~,~j~)~. 40A, 945-954. DANDO P. R. (1969) Lactate metabolism in fish. J. )?IL~IT. hioi. Ass. I’.K. 49, 209-223. DKIEDZI(, W. R. & KI~~~I~JK .J. W. (1976) Blood lactate levels in free-swimming rainbow trout (Salvo gctirdneri) before and after strenuous exercise resulting in fatigue. J. Fisll. Ru\. Rd Can. 33, I73 176. HOI.ETON G. F. & RANIXLI. D. J. (19671 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. rxp. Eiol. 46, 317-327. HUGHES G. M. (1980) Functional morphology of fish gills. In Epithr/ia/ Transport itr rhr Lower L’ertrbrates. Part 1: Morphological aspects (Edited by LAHLOU), pp. 15.-36. C.U.P., Malta. HL’C;HESG. M., TIXRALA H. & Sorvro A. (1978) Regional distribution of blood in the gills of rainbow trout m normoxia and hypoxia: a morphometric study with two fixatives. Ann. Zoo/. Fcwuci 15, 226-234. HUNN J. B. (1969) Chemical composition of rainbow trout urine following acute hypoxic stress. Trans. Am. Fish. Sot. 98, 20-22. JOHNSON: P. C. 11971) Red cell separation in the mesenteric capillary network. Am. .I. Physiol. 221, 99-104. KIRK W. L. (1974) The effects of hypoxia on certain blood and tissue electrolytes of channel catfish, Ictulurus punctuttrs (Rafinesque), Trans. Am. Fish. Sot. 103, 593-600. KRISTOFFERSSOIVR.. BROBERC;S. & OII
NIKINMAA M. & SOI~IO A. (1979) Oxygen dissociation curves and oxygen capacities of a freshwater fish, SuImo qrlirdneri. .4m~. Zool. Femki 16, 2 17-221. NIKIN,MAAM. &Sotvio A. (1981) Blood oxygen transport of hypoxic Srrfmo ~~~z~rd~7~ri at high temperature. Submitted to J. rup. Zool. NIK~NMAA M., TUURALA H. & SOIVIO A. (1980) Thermoacclimatory changes In blood oxygen binding properties and gill secondary lamellar structure of Sulmo guirdneri J. wmp.
Physiol.
140, X5-
260.
RANDALI. D. J.. HXETON G. F. & STEVENSE. D. (1967) The exchange of oxygen and carbon dioxide across the gills of rambow trout. J. rup. Birii. 46, 329.-348. SOIVIO A. & H~J(~HESG. M. (1978) Circulatory changes in the secondary lamellae of Salmo gairdneri gills in hypoxia and in anaesthesia. Ann. Zoo!. Fennici 15, 221-22s. SOI~IO A. & NIKIXMAA M. (1981) The swelling of erythrocytes in relation to the oxygen aR’mity of the blood of the rainbow trout. Sdrm ~~~~~~~~f?~,~j Richardson. In Strrcc rortl F.islr (Edited by PICKI~HIN~;A, D.). Academic Press, New York. In press. SOIVIO A. & TU~JKALA H. (1981) Structural and circulatory responses to hypoxia m the secondary lamellae of Sulmo gairdnrri gills at two temperatures. Submitted to J. camp. Physiol. Sowro A., WESTMAN K. & NYHOLM K. (1972) improved method of dorsal aorta catheterization: haematoio~ical effects followed for three weeks in rainbow trout (Sulmo gairdneri). Finn. Fish. Rus. I, 11-21. Sorvro A., NYHOLM K. & WESTMAN K. (1973) Notes on haematocrit determinations on rainbow trout, Salmo goirdnrri.
Aqutrculturr
2, 3 I 35.
Gills and responses of trout during hypoxia SOIVIOA., NYHOLMK. & WESTMANK. (1975) A technique for repeated sampling of the blood of individual resting fish. J. exp. Biol. 62, 207-217. SOIVIOA., NYHOLMK. & HUHTI M. (1977) Effects of anaesthesia with MS 222, neutralized MS 222 and benzocaine on the blood constituents of rainbow trout, Salmo gairdneri. J. Fish Biol. 10, 91-101. SOIVIOA., NIKINMAAM. & WESTMANK. (1980) The blood oxygen binding properties of hypoxic Salmo gairdneri. .I. camp. Physiol. 136, 83-87.
139
VOGEL W., VIJGEL V. & F%AUT~CHM. (1976) Arteriovenous anastomoses in rainbow trout gill filaments. Cell Tiss. Res. 167, 373-385. W~~ID C. M. & SHELTONG. (1980) Cardiovascular dynamics and adrenergic responses of the rainbow trout in ho.
J. exp. Biol. 87, 247-270.
WOOD C. M. & SHELTONG. (1980) The reflex control of heart rate and cardiac output in the rainbow trout: interactive influences of hypoxia, haemorrhage and systemic vasomotor tone. J. exp. Biol. 87, 271-284.