Increased RBC Count and Pulmonary Respiration in Cold-Adapted Frogs

Many thermal compensatory mechanisms are known which involve changes in the blood cell number and oxygen-carrying capacity of the blood (Prosser & Brown, 1961; Houston & De Wilde, 1969). For example, warm-adapted aquatic fish (Houston & De Wilde, 1969) and homeotherms which are acclimated to high altitude (Prosser & Brown, 1961) maintain polycythaemia to obviate the effects of oxygen debt in the tissues. Precht (1962) suggested that a hypoxic environment induces rapid red cell (RBC) production to increase the oxygen-carrying capacity of the blood. The correlation between RBC count and environmental changes seems, however, to be somewhat different in amphibians. Foxon (1964) noted that the RBC count increased in cold-adapted amphibians and considered it to be one of the compensatory mechanisms of cold adaptation. In addition the habitat seems to affect the RBC count in certain amphibians (Galten & Brooks, 1969; Leftwich & Burke, 1964; Roofe, 1961). The oxygen affinity of the blood also altered when frogs were adapted to different temperatures (Kirkberger, 1953). Although the haematology of various amphibians is known (Roofe, 1961; Leftwich & Burke, 1964; Galten & Brooks, 1969), the influence of seasonal and sex variations on RBC has not been considered in amphibians. The present study was, therefore, initiated to discover whether correlations exist between the RBC count, carbon dioxide content, percentage oxygen saturation, sex, season and cold-acclimation.

Rana cyanophlyctis (20–25 g) were caught in Bangalore during January, April and September 1971·50% of the animals caught during each month were acclimated to 12±1 °C in a refrigerator for 15–20 days. They were kept in groups of six in bread boxes (12 in × 6 in × 6 in) containing water at a depth of 2·5 in. The other frogs were kept at room temperature in laboratory aquaria containing water at a depth of 2·5 in. All of the frogs were fed with earthworms and sliced pond snail on alternate days. At all seasons 16 to 33% mortality occurred during the first 5 days of acclimation.

The blood was drawn from pithed frogs into an oxalated syringe and the RBC count was performed using a haemocytometer (Kolmer, Spaulding & Robinson, 1969). The haemoglobin content of the blood was determined by the cyanomethaemoglobin method (Oser, 1965, p. 1096). The oxygen saturation of the blood (drawn from the cutaneous vein and truncus arteriosus vessels) was determined spectrophotometrically (Oser, 1965). The oxygen and carbon dioxide contents of the blood were estimated manometrically. Blood for the determination of gases was collected using a lubricated (paraffin oil) and oxalated syringe (Oser, 1965). After expelling all the air from the syringe and the needle, the needle was inserted into the blood vessel. About 0·2–o·5 ml of blood was withdrawn. Immediately the needle was removed and a drop of mercury was drawn into the syringe to prevent the blood from coming in contact with the air. The blood was vigorously shaken, cooled in an ice-water mixture and then transferred to manometric flasks containing a layer of mineral oil. The dissolved gases were liberated by potassium ferricyanide-bisulphate reagent (Oser, 1965) taken in the side arm of manometric flasks. The volume of total gases was measured in the absence of fluid in the central well. The oxygen content was measured with filter paper strips soaked in 10% alkaline pyrogallol which were placed in the central well of the Warburg flasks at 25 °C. Similarly, 10% KOH was used to determine the carbon dioxide content in another flask. Oxygen content was calculated from the change in volume due to absorption of oxygen by 10% alkaline pyrogallol placed in the central well of the Warburg flask.

The extent of carbon dioxide loss via the lung or skin was studied by trapping ¹⁴CO₂ with Ba(OH)₂ (Fig. 1). 1 ml 1% ¹⁴C₆-glucose-saline (= 2μCi) was injected into the dorsal lymphatic cavity of the frog. The frog was allowed to move freely in the main vessel (which contained 200 ml water) and a stream of CO₂-free air was drawn through the apparatus for 1 h, the respired ¹⁴CO₂ being trapped with the Ba(OH)₂. The ¹⁴CO₂ escaping through the skin dissolved in the water. After the experiment this water was transferred to a stoppered flask through a delivery tube and 50 ml of cone. HC1 added through a thistle funnel, the liberated ¹⁴CO₂ being collected with Ba(OH)₂ solution. The precipitated isotopic carbonate was separated from the baryta solution by centrifugation, dried at 100 °C and the isotopic content counted in a Gas Flow Counter (Bunshane, Trombay Electronics). Since the respirometer (Fig. 1) had a relatively large surface area of contact between the water and gaseous phase, any exchange across the water surface would tend to complicate the calculation. However, as this factor applied uniformly to both normal and cold-acclimated frogs the absolute difference between the two values remained unaffected.

The ratio of ¹⁴CO₂ in the respired gas and liquid phase was used to estimate the degree of aerial or aquatic respiration in the frog on cold acclimation.

Studies on the thermal adaptation of terrestrial, semi-terrestrial and amphibious anurans (Brattstrom, 1971) have been well documented. Such studies have not been extended to the frog, Rana cyanophlyctis (Boulenger, 1890), which is thoroughly aquatic and frequently found in fresh-water ponds.

Considerable seasonal variation is found in the RBC count of Rana cyanophlyctis, the January count being higher than in September (Table 1). Females showed significantly (P = 0·001) higher counts than the males. Cold acclimation resulted in the elevation of counts in all seasons and in both the sexes. However, the extent of increase in RBC count associated with cold acclimation at a given season varies with the sex. For example, it is evident from Table 2 that the increase in count is directly proportional to the gradient of temperature acclimation (i.e. difference between environmental and acclimation temperature) in males.

To determine whether the increased RBC count induced by cold acclimation could affect the oxygen transport, the oxygen and carbon dioxide contents in truncus arteriosus and cutaneous vein were measured. The results of this experiment, which was performed on male frogs during April, are presented in Table 3. The increase in RBC count associated with cold acclimation is accompanied by an increase in haemoglobin content. Cold-acclimated frogs retained a higher oxygen saturation in the truncus arteriosus than in cutaneous vein (Table 3). The carbon dioxide content decreased in the truncus on cold acclimation. Consequently, the O₂/CO₂ ratios are higher in truncus than in cutaneous vein of the cold-acclimated frogs.

The data summarized in Table 4 show that cold-acclimated frogs release more ¹⁴CO₂ through respired air (i.e. through lungs) than frogs adapted to room temperature.

Structural and functional modifications of the respiratory system occur with increased terrestrialism. Such modifications have been observed in species of crabs which invade land habitats from wholly aquatic and intertidal zones (Gray, 1957). Ter-restrialization increases the effective gill area in these crabs. Vernberg (1956) reported that the metabolic rate of the intact crab and its gill tissue is increased in species adapted to a more terrestrial habitat. Metabolic adaptations (Augenfeld, 1967) and enzymic modifications (Vernberg & Vernberg, 1968) have also been observed in semiterrestrial animals inhabiting intertidal zones. Evans (1939) and Vernberg (1952) pointed out that aquatic salamanders have a higher metabolic rate than their terrestrial relatives.

The relationship between temperature and metabolic rate of anurans from different geographical ranges indicates respiratory adaptation (Tashian & Ray, 1957). These authors have also observed geographical differences in the relative oxygen uptake by pulmonary and cutaneous surfaces in temperate and tropical species of anurans.

The lungs account for greater oxygen uptake, and an increased contribution of the lungs to respiration occurs at lower temperatures in the anurans of the Northern Hemisphere (Hutchison, Whitford & Kohl, 1968). The results of the present study obviously support the above correlation.

The absence of sexual difference or higher RBC counts in males is reported by many workers (see Foxon, 1964). The present results agree with those of Galten & Brooks (1969), who found higher counts in females.

The increased count induced on cold acclimation is particularly interesting because warm-acclimated aquatic vertebrates are known to elevate the RBC count to obviate the effects of hypoxic environment by facilitating greater transport of oxygen (Houston & De Wilde, 1969). Increased ¹⁴CO₂ in the respired air and in the oxygen/ carbon dioxide ratios in the truncus also indicate that the frog has increased air breathing on cold acclimation. Although the percentage saturation of the cutaneous vein is less during cold acclimation, the actual oxygen content would seem to rise because of the increase in haemoglobin concentration. Modification of the distribution of blood flow during cold acclimation must be related to these changes, a more detailed investigation being required to elucidate this aspect of the work.

It is clear from these results that there is change in the mode of respiration of the frog associated with cold acclimation. Increased lung respiration may, therefore, be correlated with a higher RBC count in frogs following cold acclimation. A correlation between the RBC count and the habitat of many amphibians has already been proposed: terrestrial and arboreal amphibians, for example, showed high counts (Hall, 1966), while semi-terrestrial forms exhibited intermediate (Foxon, 1964) and aquatic ones low counts (Leftwich & Burke, 1964). In view of this correlation it could be postulated that the amphibians were pre-adapted to a terrestrial environment, having been already equipped with a higher count of their RBCs, owing to their exposure to freezing waters in geological ages. Hence this non-genetic respiratory adaptation to cold could have preceded and, thus, helped their invasion of terrestrial environments.

The authors record their sincere gratitude to the late Professor K. Pampapathi Rafl for extending experimental facilities and are grateful to the CSIR, New Delhi, for the award of a grant.