Respiratory, Ventilatory, and Cardiovascular Responses to Experimental Anaemia in the Starry Flounder, Platichthys Stellatus

The ability of teleost fish to survive in the real or functional absence of most of their haemoglobin has been known for many years (e.g. Nicloux, 1923; Schlicher, 1926; Anthony, 1961). A few studies have examined specific aspects of compensation (Cameron & Wohlschlag, 1968; Cameron & Davis, 1970; Wood & Randall, 1971; Jones, 1971a; Holeton, 1971 a,b, 1977; Haswell & Randall, 1978). In the most detailed of these investigations, Cameron & Davis (1970) showed that the rainbow trout (Salmo gairdneri) in freshwater maintained an approximately normal resting O₂ uptake, in the face of a 70%+ reduction in haematocrit, by pumping a much greater volume of the reduced O₂ capacity blood through the gills. This was accomplished almost entirely by elevation of cardiac stroke volume and facilitated by a decrease in peripheral vascular resistance. The trout thereby sustained normal O₂ uptake despite a greatly reduced difference between arterial and venous O₂ contents. Ventilation and blood O₂ tensions remained unchanged.

Recently we have analysed respiratory gas exchange under resting conditions in the starry flounder (Platichthys stellatus) in sea water (Wood, McMahon & McDonald, 1979). The flounder is a benthic, inactive fish whereas the trout is pelagic and active. Relative to S. gairdneri, P. stellatus exhibits low blood O₂ tensions, a low blood O₂ capacity, a high blood O₂ affinity, a low arterial-venous O₂ content difference, and an extremely high cardiac output. It appears that the flounder maintains a much higher cardiac output than the trout when at rest, thereby gaining cardiac efficiency but sacrificing the high O₂ transport capacity during exercise which can be achieved by the trout. The latter operates less efficiently at rest, but can call upon a considerable circulatory reserve during frequent exercise by raising cardiac output to more efficient levels. In view of these differences, especially in resting cardiac output, responses to haemoglobin deficiency might well be very different in the two species. Therefore in the present study we have examined the respiratory, ventilatory, and cardiovascular adaptations to experimental anaemia in P. stellatus.

Symbols employed for respiratory parameters follow the system of Dejours (1975) and are defined in the text when first employed.

Starry flounder (Platichthys stellatus Pallas) were collected (from January to April) and acclimated for at least 10 days to running sea water (salinity = 26–28 %, temperature = 7·5–10·5 °C) at Friday Harbor Laboratories, University of Washington, as described previously (Wood et al. 1977, 1979).

The first series of experiments concentrated on respiration, ventilation, and cardiovascular function during progressive experimental anaemia. The fish (N = 6; 425–1128 g) were fitted with arterial and venous catheters, inspired water catheters, ventilation collection masks, and opercular impedance leads while under MS-222 anaesthesia. The operative procedures have been described in detail previously (Wood et al. 1979) and in general follow the methods developed by Watters & Smith (1973). After surgery, the animals were placed in individual chambers (60 × 35 cm × 15 cm deep) which were filled to a depth of 6 cm with fine beach sand and shielded from the investigators. During the whole experimental period (1–3 weeks), the fish remained buried in the sand with only the eyes and mouth exposed, a behaviour which appears to duplicate natural resting conditions (Wood et al. 1979). After at least 72 h recovery, a set of control measurements was taken at the unbled haematocrit (Ht = 10·7–20·3 %). Blood was then withdrawn via the caudal vein catheter, centrifuged at 3000 g for 5 min to separate the erythrocytes, and the homologous plasma plus ‘buffy coat’ re-infused with sufficient saline (see below) to replace the missing red cell volume. The flounder was allowed to stabilize for at least 24 h at the new lower Ht, and then the set of measurements was taken again. The procedure was repeated 3–6 times so as to progressively lower the Ht from the original level to approximately 1% (‘severe anaemia’) in a series of steps over 4–14 days. At no time was more than 1 ml of blood/100 g removed from a fish, so multiple bleedings were necessary to achieve low Ht’s.

For four of the flounder, the separated red cells were immediately resuspended to the original Ht in Cortland saline (Wolf, 1963) adjusted to 160 m-equiv. 1⁻¹ sodium concentration by addition of NaCl, and heparinized at 100 i.u./ml. The cells were stored at 4 °C and resuspended in fresh saline daily. Haemolysis was negligible. After measurements had been recorded from the flounder at a Ht of about 1 %, the accumulated erythrocytes in saline from that animal were slowly re-infused into the caudal vein over a period of about 30 min. A partial or complete set of measurements was then taken 24 h after re-infusion.

In the above experiments the complete set of measurements comprised direct determinations of Ht, inspired water O₂ tension ⁠, mixed expired water O₂ tension ventilation volume ventilation rate (f_R) arterial O₂ tension venous O₂ tension arterial O₂ content venous O₂ content heart rate dorsal aortic blood pressure (BP_a), caudal venous blood pressure (BP_v), arterial pH (pH_a), and venous pH (pH_v). For the original control samples and at selected other Ht’s, total blood O₂ capacity and plasma O₂ capacity were also measured. The analytical techniques have been described in detail previously (Wood et al. 1977, 1979). At least three separate determinations of each parameter, at each Ht, were taken for each fish. From these direct measurements, the following derived parameters were calculated as in Wood et al. (1979): O₂ consumption ⁠, ventilatory stroke volume⁠, haemoglobinbound O₂ capacity per unit ⁠, arterial O₂ saturation (S_a, o₂), venous O₂ saturation (S_v, o₂), cardiac output ⁠, and cardiac stroke volume (V_s, _H). Systemic vascular resistance (R_S) was calculated as ⁠.

For one of the six fish, some pH measurements were lost due to pH electrode failure, so an additional flounder was fitted with only arterial and venous catheters and subjected to the sequential bleeding procedure. In this animal, only pH_a, pH_v, and Ht were measured. measurements during progressive anaemia were also recorded from three fish of the second experimental series (see below) which were fitted with only venous catheters. Data from these fish bearing only blood catheters were not significantly different from those taken from the six fish bearing ventilation masks, impedance leads, and blood and water catheters.

A second series of experiments examined the influence of severe anaemia on the responses to exhausting activity. All fish (286–850 g) in this series were fitted with venous catheters and allowed to recover for at least 72 h in darkened individual chambers (30 × 30 cm × 15 cm deep) filled to a depth of 6 cm with sand. The experimental fish (N = 4) were then progressively bled down to a Ht of about 1 % over a period of 4–6 days as described above. The control fish (N = 6) were not bled. Both groups were then subjected to exhausting activity by 10 min of manual chasing. Blood samples were drawn prior to exercise and at selected times up to 24 h postexercise; these samples were analysed for Ht, pH_v, and blood lactate levels. The exercise, blood sampling, and analytical procedures are described in detail by Wood et al. (1977).

A third experimental series assessed the natural variability of Ht in starry flounder in the wild. Fish (N = 97) over a wide weight range (95–1656 g) were examined. (The animals were collected by otter trawl from East Sound of Orcas Island, ashington State, during April and May, 1976.) The flounder were held for at least 10 days after capture to negate the haemoconcentration reported to occur after trawling (‘capture stress’: Fletcher, 1975). Fish with evidence of trawl damage were not used. Blood samples were obtained by quickly removing the fish from the water and blindly puncturing (i.e. without incision) either the haemal arch or ventricle with a no. 22 needle attached to a heparinized syringe. Occasional haemolysed samples were rejected.

The respiratory, ventilatory, and cardiovascular responses of individual fish to progressive experimental anaemia are shown in Figs. 1–9, while the control and severe anaemia (Ht about 1 %) data are summarized in Table 1. As each fish was used as its own control, paired statistical comparisons (two-tailed Student’s paired t-test) were employed. The data of Cameron & Davis (1970) on the rainbow trout are included for comparison in Table 1.

Overall, there was no significant change in resting during severe anaemia (Fig. 1, Table 1), although minor decreases did occur in several animals at very low Ht’s. However, below Ht s of approximately 5 %, this maintenance of occurred at the cost of a progressively increasing (Fig. 2 A). The rise in was accompanied in an inverse fashion by a fall in the extraction of O₂ from the ventilatory flow

As expected, both and declined in approximate proportion to the Ht (Fig. 4A, B, Table 1). However for there occurred a slight but definite deviation from linearity, declining somewhat more steeply than Ht (Fig. 4B). was relatively low over the range of control Ht’s, but rose dramatically in severe anaemia (Fig. 5 A, Table 1). Most of the increase occurred below Ht’s of about 5 %. On the other hand, steadily declined over the whole range of Ht’s examined (Fig. 5 B), thereby explaining to some extent the slight non-linearity of the versus Ht relationship.

This does not mean that these increases in were unimportant in blood O₂ transport. figures refer only to haemoglobin-bound O₂, while at low Ht’s, O₂ physically dissolved in the blood becomes increasingly important. Indeed, O₂ delivery to the tissues by physical solution rose from 8·7 ± 0·6 % of the total at control Ht’s to 72·3 ± 8·8% under severe anaemia (Table 1). If had not risen during severe anaemia, overall O₂ delivery would have dropped about one third from the control values. The fall in also made a slight contribution to the increased transport by physical solution. However, the real significance of the decline is that it represents an approximate doubling of the unloading of haemoglobin-bound O₂, based on the preceding saturation calculations.

Another important factor in maintaining O₂ delivery may have been blood pH. In all animals, there were highly significant decreases in both pH_a and pH_v during progressive anaemia (Fig. 6A, B; Table 1), the latter effect being slightly greater. The decreases were most rapid below a Ht of about 5 %. This has been seen more clearly in a similar experiment on a different batch of flounder (C. M. Wood, B. R. McMahon, and D. G. McDonald, unpublished results). In view of the Bohr shift known to occur in the blood of this species (Watters & Smith, 1973), the fall in pH_v probably contributed to the fall in ⁠, and therefore helped to maintain O₂ transport. On the other hand, the fall in pHa probably had little or no effect on because of the accompanying rise in ⁠.

Fig. 7 A shows that the combined effect of all these factors was to maintain constant down to a Ht of about 5%. Below this point, declined significantly (Fig. 7 A, Table 1), but far less markedly than if adjustments in and pH_v had not occurred. As resting remained essentially constant during severe anaemia (Fig. 1), varied inversely with ⁠, increasing significantly below a Ht of about 5 % (Fig. 7B, Table 1). The rise in was accomplished entirely through an elevation of V_{s, H} ; there was no significant change in f_H (Fig. 8 A, B, Table 1).

The increased was not accompanied by an increased BP_a. Indeed, below a Ht of about 5%, BP_a fell significantly in all fish (Fig. 9A, Table 1) and BP_v increased slightly (Table 1). Consequently, the blood pressure differential across the systemic vascular bed declined while blood flow increased, indicating a profound drop in systemic vascular resistance (R_s) at low Ht’s (Fig. 9B, Table 1). Calculations based on viscosity versus Ht data for trout blood (Cameron & Davis, 1970; Wood, 1974) suggest that the observed decrease in R_s was too large to result directly from the reduced viscosity of low Ht. A real systemic dilation must also have occurred.

Re-infusion of the homologous red blood cells was performed in four fish; the procedure proved fatal to one animal (erythrocytes stored up to 13 days) but was tolerated by the other three (storage = 4–11 days). Accounting for estimated losses of erythrocytes in blood sampling and handling, the Ht after re-infusion should have been about 70 % of the control value, but the actual figures 24 h after re-infusion were only 38 %, 40 %, and 42 % of the control Ht’s. The data from one fish are summarized in Table 2; the results from the other two were qualitatively similar but C_o values in vivo and in vitro after re-infusion were not measured. Because of the ‘loss’ of erythrocytes in the re-infusion procedure, in Table 2, the data after re-infusion (column 4) have been compared with data at both the original control Ht (column 1), and at the closest haematocrit before severe anaemia (moderate anaemia, column 2) as well as at the severe anaemia Ht (column 3). The results clearly show that all the pronounced changes observed under severe anaemia (increases in ⁠, and V_{s, H} decreases in and pH_v) could be largely reversed by the increase in Ht. Therefore these changes resulted specifically from alterations in the red blood cell concentration and not from any non-specific effect of the experimental procedure. (For unknown reasons, BP_v remained elevated after re-infusion in this one flounder, but did not in the other two fish.)

and ⁠, unlike and ⁠, did not return to the levels expected on the basis of the Ht value after re-infusion (compare column 4 with column 2 in Table 2). In vitro equilibrations showed that this discrepancy was due to a substantial reduction in with little or no change in 24 h after re-infusion (Table 2). This observation, together with the ‘loss’ of erythrocytes after re-infusion and the death of one fish, indicates some deterioration of the red cells during storage. Although haemolysis was negligible in the storage tubes, substantial haemolysis and ‘loss’ of erythrocytes probably occurred after the cells were returned to the flounder.

The second series of experiments was designed to assess the possible influence of severe anaemia on the acid-base responses to exhausting activity, responses which have previously been analysed in control P. stellatus (Wood et al. 1977). In this series, mean Ht was 16·1 ± 1·2 % (6) in the control group and 0·7 ± 0·1 % (4) in the anaemic group. In agreement with the preceding results (Fig. 6B, Table 1), initial pre-exercise pH_v was significantly lower in the anaemic fish (Fig. 10A), although there was no difference in mean venous lactate level (Fig. 10B). Ten minutes of manual chasing tompletely exhausted both groups, with no noticeable difference in swimming behaviour. Up to 4 h after exercise, changes in pH_v and venous lactate concentrations were very similar in the two groups (Fig. 10A, B). At 6 h, Lactate levels were slightly higher and pH_v significantly lower in the anaemic fish. By 24 h, Ph_v had returned to normal in both groups; lactate had similarly returned to resting levels in the control fish but remained slightly elevated (P = 0·05) in the anaemic flounder. Anaemia therefore seemed to have only a small influence on the response pattern late in the recovery period.

The frequency distribution of Ht’s occurring in 97 flounder collected from the wild is shown in Fig. 11. The mean Ht was 19 · 9%, but there was considerable variability (range 4·3–34·6 %). The data were not normally distributed but skewed strongly to the left with a mode (25·5 %) considerably higher than the mean (19·9 %). Only one Ht below 6 % was observed. There was no significant relationship between Ht and body weight. However, virtually all animals were parasitized by gill copepods, and casual observation indicated that Ht was negatively correlated with the degree of infestation.

Down to a Ht of about 5 %, the starry flounder was able to sustain resting with little or no disturbance of any parameter except ⁠. By gradually reducing as Ht fell (Fig. 5 B), the animal increased the relative unloading of O₂ from the blood (i.e. decreased ⁠). Consequently was maintained constant

(Fig. 7 A) in the face of the declining ⁠, and there was no need for compensatory manipulation of or other cardiorespiratory parameters. Below a Ht of about 5 %, declined, and a host of other changes were implemented in addition to the continuing decrease in ⁠. These included increases in (Fig. 7B) through elevation of (Fig. 8 B), decreases in BP_a (Fig. 9 A) and increases in BP_C accompanied by a decline in R_a (Fig. 9B), increases in (Fig. 5 A), decreases in pH_a and pH_v (Fig. 6A, B), increases in (Fig. 2A) through elevation of V_s,R (Fig. 3B) and slight depression of f_R (Fig. 3 A), and increases in (Fig. 2B).

The frequency distribution of Ht in flounder recently captured from the wild (Fig. 11) indicates that anaemia is of significant natural occurrence. The physiological data on the responses to experimental anaemia would seem to explain this frequency distribution. The ‘ normal ‘ Ht of healthy fish is probably in the region of the mode (i.e. above 24%), but large numbers of animals occur with Ht’s as low as 6%, giving the distribution a strong skew to the left (Fig. 11). These moderately anaemic flounder have probably lost erythrocytes through branchial (or other) parasitism, or through injury. Red cell replacement is obviously a very slow process for we observed no recovery of Ht in experimentally anaemic flounder kept for up to 7 weeks after bleeding. Moderately anaemic flounder can survive in the wild because compensation involves minimal disturbance of the cardio-respiratory system (decreases in only), at least in the resting state. However, only one fish (out of 97) was found in the wild with a Ht less than 6 %. This cutoff point corresponds with the inflection point (5 %) seen in the physiological data. Below a Ht of about 5 %, the animals must invoke major compensations in order to maintain resting ⁠. Some of these, especially the increases in and may have considerable metabolic cost (Jones, 1971b), yet there is no accom-anying rise in (Fig. 1). Thus a greater fraction of resting must be devoted to fuelling the branchial and cardiac muscles, and less will be available for other main- tenance functions. This condition can be tolerated by the fish in the laboratory where it simply lies buried in the sand the whole time. However in the wild, far greater demands are placed on the animal, and the condition presumably becomes unviable.

Despite the great differences in blood O₂ transport capacity between normal and severely anaemic flounder, the responses of the two groups to enforced activity were remarkably similar (Fig. 10). This finding supports our previous contention that O₂ transport is not the primary factor limiting exercise (Wood et al. 1977). Rather, exertion may be limited by the acute acidosis which quickly lowers blood pH to an unacceptable level. This rapid acidosis is almost entirely respiratory in origin (i.e. due to increase; Wood et al. 1977). The pH_v changes during this period (0·4 h after exercise) were very similar in the two groups (Fig. 10). As the non-bicarbonate buffer capacity (β) was reduced about 30% by severe anaemia (Wood et al. 1977), a larger pH_v, decline for the same rise might have been expected in the anaemic fish. That this did not occur may mean that the critical pH limiting further exercise was reached at a lower in the anaemic animals. Only late in recovery (6–24 h) were there significant differences between the two groups. The slower return of pH_v to pre-exercise values in the anaemic fish was associated with higher blood lactate levels at this time. The slow time course of lactic acid release into the bloodstream ensures that this metabolic acid makes a significant contribution to the pH_v depression only late in the recovery period (Wood et al. 1977). The eventually higher blood lactates in the anaemic group do indicate a greater reliance on anaerobic metabolism, but the differences were not great (Fig. 10). In any event, exercise metabolism in this sluggish species may normally be largely anaerobic. The accumulation of other anaero- bic end-products besides lactate is also possible (Hughes & Johnston, 1978).

Comparison of the responses of starry flounder to severe anaemia with those of the rainbow trout (Cameron & Davis, 1970) reveals both similarities and differences in the mechanisms of adaptation (Table 1). A number of derived parameters based on gas exchange equations have also been summarized in Table 3 to aid in this com- parison. A detailed analysis of the gas exchange strategies of the two species in the resting state has been presented earlier (Wood et al. 1979).

Like the flounder, the trout maintains normal resting during severe anaemia (Table 1), but the compensatory mechanisms appear much less complex. The anaemic trout simply elevates by increasing V_{s, H} and decreasing R_s, thereby maintaining constant despite a greatly reduced ⁠. No adjustments are made in ventilation, ⁠, or blood O₂ tensions (Table 1). There are therefore no changes in the percentage utilizations of O₂ from the water by the blood or from the blood by the tissues ⁠, in the percentage effectiveness of O₂ removal from the water or uptake by the blood ⁠, in the mean gradient or transfer factor for O₂ across the branchial epithelium, or in the convection requirement for water (Table 3). However this strategy does mean that the convection requirement for blood must increase while the ventilation/ perfusion ratio falls sharply in such a fashion as to hold the capacity-rateio of the two convective media almost constant at a value close to unity (Table 3).

This effectively keeps the exchange system at maximal efficiency in terms of O₂ transfer per unit total flow of blood and water (Shelton, 1970).

The severely anaemic flounder similarly elevates by increasing V_s,_H and decreasing R_s ; the latter presumably helps to minimize the increase in cardiac work. However the flounder also manipulates ventilation, and blood O₂ tensions (Table 1). Although the experimental anaemia was more pronounced in the flounder (Ht = 0 · 8 %) than in the trout (Ht = 3 · 8 %), the relative increase in was lower (two- versus three-fold). Indeed Cameron & Davis (1970) present data indicating that at a comparably low Ht, the anaemic trout increases seven- to ten-fold. This reflects a much lower resting in the control trout and therefore a much greater scope for raising ⁠. In fact, the anaemic trout becomes remarkably similar to the control flounder in terms of ⁠, V_s,H, (Table 1), and the and ratios (Table 3). Control flounder apparently operate at high and low in the resting state, thereby gaining cardiac efficiency but sacrificing high O₂ transport capacity during exercise or severe anaemia (Wood et al. 1979). In this light, it is perhaps remarkable that the anaemic flounder is able to increase ⁠, and as much as it does (Tables 1, 3).

Because of the flounder’s limited scope for raising ⁠, the anaemic animal has to lower and raise to maintain O₂ transport, thereby increasing both the unloading of O₂ from the haemoglobin at the tissues and the delivery of O₂ by physical solution (Table 1). The success of these manoeuvres is reflected in an approximate tripling of and maintenance of ⁠, during severe anaemia (Table 3). If necessary, the trout also could conceivably lower and thereby further augment its scope for O₂ delivery; there is evidence that this occurs during exercise (Kiceniuk & Jones, 1977). However, the option of raising is probably only available to benthic fish such as the flounder and tench (Eddy, 1974) which saturate their haemoglobin at low under control resting conditions. is already so high in the control trout that further increases seem unlikely.

The most surprising feature of the flounder’s response is the increase in ⁠, since the metabolic cost of ventilation is considerable in flatfish (Edwards, 1971). The elevations of and are about equal, so the ratio falls only slightly in contrast to the trout (Table 3). As the increase in is accompanied by a rise in in the classical manner (Fig. 2; Table 1), it seems to make little sense, at first glance, for the animal to pump more water across its gills yet derive the same total amount of O₂ from that flow - a virtual doubling of the ratio (Table 3). falls, and O₂ expenditure is presumably increased without raising O₂ uptake. An explanation for this anomaly may lie in the great importance of physical solution in blood O₂ transport during anaemia (Table 1), for which the ⁠, rise (Fig. 5A) is of critical importance. A scatter diagram of all simultaneously determined values of and shows a strong positive correlation (r = 0 · 65; P <0 ·001) between the two parameters (Fig. 12). The regression relationship is:

Both pH_a and pH_v declined quite markedly during anaemia in the flounder (Fig. 6; Table 1), a compensation which may have aided blood O₂ transport by the Bohr effect (see Results). Cameron & Davis (1970) did not report on blood pH in the anaemic trout, but Cameron (personal communication) has indicated that pH_a did not change with Ht in their study. More recently Haswell & Randall (1978) have confirmed that pH_a and remain constant during severe anaemia in Salmo gairdneri (Table 1). The acidosis observed in the flounder could be associated with the accumulation of acidic end products of anaerobic metabolism in the blood, with the reduced non-bicarbonate buffer capacity of low Ht blood (Wood et al. 1977), with a rise in blood levels, or with any combination of these and other factors. The phenomenon is currently under investigation, and preliminary results point to an elevation of blood levels during anaemia. Janssen & Randall (1975) have shown that a rise in acts as a direct ventilatory stimulus in fish. A rise in in the flounder but not in the trout (Haswell & Randall, 1978) would therefore explain the difference in the ventilatory responses of the two species to anaemia.

We wish to thank the Director, Dr A. O. D. Willows, and staff of Friday Harbor Laboratories, University of Washington, for their assistance and hospitality. Mr Gr Hewlett, of the Vancouver Public Aquarium, kindly supplied. ‘ Furanace’. Financial support was provided by grants from the National Research Council of Canada.