Gas Tensions in Tissues of Trout and Carp Exposed to Diurnal Changes in Oxygen Tension of the Water^*

When an artificial gas pocket is formed by injection of air into a tissue space the gases equilibrate with the tissue-blood environment and assume a state of constant composition which is maintained until all the gas is eventually resorbed. In man (Rahn, 1957) and in rats (Van Liew, 1968) evidence has been presented that the O₂ and CO₂ tensions of gas in such pockets are virtually the same as those of the venous blood draining these tissues provided that air or gases of lower O₂ concentrations are breathed (see also review by Piiper, 1965). Since the techniques required for sampling and analysis of the equilibrated gas are relatively simple, this method provides a convenient approach for the determination of gas tensions of the blood-tissue environment of unanaesthetized, unrestrained animals.

To interpret the tissue gas tensions in fish it is important to relate these to the normal gas tensions of arterial and mixed venous blood in unrestrained, free-swimming animals. These have recently become availablefortherainbowtrout(Stevens&Randall, 1967) and for the carp (Garey, 1967), the two species chosen for this study.

Experiments were performed on free-swimming fish at the New York State Fish Hatchery at Caledonia, N.Y. The waters which supply this station come from a slowly moving spring-fed stream with considerable photosynthetic activity which imposes a large diurnal fluctuation in the O₂ content of the water. During the experiments in the summer the O₂ tensions of the water rose as high as 230 torr in the late afternoon and fell to pre-dawn values as low as 50. Such large changes might be expected to influence the tissue O₂ tensions. For this reason tissue gas-tension analyses were performed over long enough periods to establish a positive correlation with the changes in water O₂ tension.

Two experiments were carried out during the summer and one in the winter on fish which were maintained in the raceways of the fish hatchery. The rainbow trout (Salmo gairdneri) were 2 years old, weighing about 500 g., while the carp (Cyprinus carpio) weighed between 2–3 kg. Water temperatures fluctuated a few degrees about a mean of 10 or 11° C.

Two days prior to our observations fish were taken from the raceway and air was injected into their coelomic cavities using a syringe and a no. 20 needle. The amount of air introduced depended upon the size of the fish and ranged from 20 to 40 ml. Previous studies had shown that after 24–36 hr. gas composition is constant, indicating a state of near-equilibrium between the O₂ and CO₂ partial pressures of the gas pocket and that of the surrounding tissue.

An O₂-electrode (Instrumentation Laboratory, Inc., or Radiometer) was utilized to measure the ambient water ⁠. Temperature equilibration between the water and electrode was ensured by the continuous circulation of water from the raceway through the electrode housing. Calibrations were checked before each sequence of measurements by setting the zero O₂ point with boiled water or nitrogen gas and the high O₂ point with air-saturated water obtained from a submerged flask in which air was constantly bubbled through a sintered disk. Thus calibration samples and the electrode were maintained at the ambient water temperature.

Water samples for analysis were drawn into 5 ml. glass syringes after the syringes had been carefully filled and emptied several times to avoid release of gas bubbles, particularly during the afternoon when the waters become supersaturated with O₂.

The oxygen content of carp and trout blood was established at various O₂ tensions with CO₂ tensions maintained at 2, 5 and 8 torr. The equilibrations were made on the Astrup microtonometer at io° C. and the gas and blood-gas contents were determined by the micro-Van Slyke technique.

The large diurnal fluctuations in water for given days in June 1964, July 1966 and February 1965 are shown in Figs. 1−3 and are clearly related to the day and night period. However, the degree of supersaturation and undersaturation of O₂ depends upon many other factors including the degree of cloudiness. The amplitude of the O₂ cycle observed in February is smaller even though the water temperature was only a few degrees lower.

Mean values of the composition of the equilibrated gas pockets for the trout and carp are given in Table 1 and their O₂ tensions are plotted in Figs. 1-3 with the simultaneously observed water O₂ values. It will be noted in Figs. 1 and 3 that for the trout there is a parallelism between the O₂ tensions of water and tissue although the latter appears to lag by approximately 4 hr. The O₂ tensions in the carp (Fig. 1) do not show any cyclic changes, and furthermore their values are distinctly less than those for the trout.

These were established at 10° C. in both species at CO₂ tensions of 2, 5 and 8 torr. Fig. 4 shows the dissociation curves for both species at the CO₂ tension closest to the CO₂ values that were observed in the gas pocket. The average gas-pocket values (see Table 2) are also indicated on the graph.

Diurnal changes in O₂ tension due to aquatic photosynthesis and respiration of natural waters are well appreciated, but the amplitude of the variations above and below the normal air saturation level depends upon many more factors than the degree of solar radiation. One might mention here only the physical effects of the changes in water temperature during the day and night which contribute in a minor but predictable manner to this fluctuation. For example, a rise from 10−15° C. decreases the O₂ solubility enough to increase the tension by about 10% provided there is no change in O₂ content. A similar decrease in O₂ tension would be expected during the night upon cooling. The hatchery personnel have observed that on frequent occasions the pre-dawn O₂ tensions during the summer months fall to such low values that asphyxia and distress symptoms are seen in the rainbow trout. Presumably these occur when the O₂ tensions fall below 50 torr.

The steady-state O₂ and CO₂ tensions found in gas pockets of mammals closely reflect the partial pressures of gases in the surrounding tissues and the venous blood draining these tissues (Rahn, 1957; Van Liew, 1968). Relatively little information is available from similar comparisons in cold-blooded vertebrates. Recently Torre (1967) established in intra-abdominal and subcutaneous gas pockets of frogs (Rana catesbiana) that at body temperatures from 5 to 25° C. the CO₂ tensions were consistently higher than the arterial blood values by 4-6 mm. Hg., while Bondi (1967) found a similar relationship between the C0₂ tensions of the intra-abdominal gas pocket and the mixed venous blood in the turtle.

For a comparison of our tissue gas tensions with those of arterial and venous blood we may rely upon the recent observations of Stevens & Randall (1967), Holeton & Randall (1967) on the rainbow trout, and those of Garey (1967) on the carp. These blood values were all obtained by implanted catheters in free-swimming animals and at temperatures comparable to those of our studies. Table 2 shows the average gas tensions in blood and their variance for the trout and carp when the water O₂ tensions were 134 and 108 torr, respectively. The trout values were obtained at temperatures between 4 and 8° C. and the carp values at 10° C. These blood values are compared with our tissue gas tensions and represent the mean value of the individual analyses when the water O₂ tensions were above 90 torr.

It will be noted that in both species the tissue O₂ tensions lie between the arterial and mixed venous value while the CO₂ tensions are higher than the mixed venous value. This is in contrast to the mammalian gas pocket where the simultaneous O₂ and CO₂ values deviate from the mixed venous or arterial values in such a manner that they reflect a gas exchange ratio of the pocket tissue between o-8 and i-o (Rahn, 1957). In such a case a pocket CO₂ tension higher than the mixed venous value is associated with a pocket O₂ tension appropriately lower than the mixed venous O₂. For the O₂ tensions of gas pockets in our fish we would have predicted, therefore, a CO₂ tension between arterial and mixed venous blood. A possible explanation for the high value which we observed is that the tissue is releasing lactic acid or other acid metabolites into the blood. Such a mechanism was suggested by Van Liew (1968) to explain his finding of exceptionally high in gas pockets in rats when was unusually low. In our fish the acid might be a normal metabolic product of the particular tissue around the gas pocket or it might be a localized tissue reaction to the presence of a foreign body, i.e. the gas pocket.

It is clear from Figs. 1 and 3 that the tissue O₂ tensions in the trout follow the diurnal fluctuations in water O₂ tension, but there is a lag of several hours between the gas-pocket and the water. This is best explained by a relatively small perfusion surrounding the large gas-pocket volume. Any change in the blood gas tension would be slow to be reflected in the pocket composition.

An analysis of the cyclic changes of the tissue O₂ tension in trout is presented in Fig. 5. Here we have indicated the predicted O₂ tensions of arterial and mixed venous blood which might be expected during the diurnal changes in water O₂ tensions as described in Fig. 1. The blood values during the changes in water O₂ were taken from the data recently presented by Holeton & Randall (1967). Using implanted catheters they determined the gas tensions of arterial and mixed venous blood of trout subjected to a continuously decreasing water O₂ tension. These values are here translated into the fluctuating water O₂ values of Fig. 1 and interpolated for water O₂ values above 160 torr (dotted lines). Upon this background we have re-plotted the tissue values of Fig. 1 or Table 1 except for the fact that we compensated for a 4 hr. lag in response time. This 4 hr. compensation, as shown in Fig. 4, provides an excellent fit by bringing out the parallelism of the tissue values with the mixed venous values. These might be expected to parallel the latter even more closely were it not for the relatively large ratio of gas volume to tissue perfusion which not only introduces the time lag but must also reduce the amplitude of the tissue O₂ cycle.

The lag time also explains the tissue differences depicted for the two observations made in the month of February (Fig. 2 and Table 1). At the time of sampling at 5 p.m. and the following noon the water O₂ tensions were identical. Yet the afternoon sample had an average of 38 and the noon sample a value of 26. If one considers an approximate 4 hr. lag, the former reflects the previous high water O₂ tensions while the latter still reflects the low water O₂ value of the previous night. On this basis we have interpolated the expected values (dotted line) between the two observations.

Holeton & Randall (1967) observed a large increase in ventilation volume with the decrease in water ⁠. Since the metabolic rate was not changed appreciably this would have lowered the arterial and mixed venous provided the water remained essentially zero. One would, therefore, predict that a similar increase in ventilation with the fall of the ambient O₂ tension would affect the CO₂ tensions in our gas pockets. That this actually happened can be seen from the values in Table 1. Although the changes are not very large the lowest tissue CO₂ values are associated with the lowest water O₂ values and are most probably due to the increased ventilation.

The data on the carp are based on observation during one 24 hr. period. The O₂ tensions are not only appreciably lower than in the trout, but also appear to remain constant in spite of the large fluctuations in the water. The most probable answer is to be found in the O₂ dissociation curve, which, when compared with that of the trout, is shifted far to the left (Fig. 4). At the normal arterial CO₂ tension (Table 2) the haemoglobin is essentially saturated at an O₂ tension of 20 torr, and Garey (1967) has shown that fluctuations in the water between 130 and 50 torr do not appear to alter the arterial One may conclude that the whole O₂ transport mechanism is little affected by changes in water O₂ tensions of the magnitude which we observed. In contrast, with trout, it is quite clear from the experiments of Holeton & Randall (1967) that the ventilation and the arterial respond quite promptly to any changes in water O₂ below the air-saturation level.

The difference in the tissue O₂ tension between the carp and trout is best appreciated when we plot their average values (Table 2) on their corresponding O₂ dissociation curves (Fig. 4). By doing this we assume that the gas-pocket tensions are in near equilibrium with those of the venous blood draining the pockets. By interpolation between the O₂ dissociation curves established for a of 5 and 8 torr the tissue points were plotted on a dissociation curve for the carp and a dissociation curve for the trout. This analysis suggests that the degree of O₂ unsaturation (c. 60-70%) of the venous blood leaving the tissue is similar in both species, but that the O₂ tension difference is the result of the large displacement between the two curves.