A Note on the Oxygen Requirements of Xenopus Laevis

While experimenting on the blood distribution in Xenopus (de Graaf, 1957) the desirability of obtaining some idea of the oxygen requirements of the animal became more and more apparent. Quite unexpectedly an ideal starting point was encountered in the form of an abnormal specimen which appeared to have no red blood pigment at all. The specimen was one of a regular supply kept in the Department of Zoology and had probably been kept in captivity for some months.

It had been injected with o-6 ml. 20% ethyl-urethane as an anaesthetic, and the abdominal cavity had been opened for experiment before anything untoward was noticed. The toad struggled in a normal fashion while the anaesthetic was being injected. External features appeared to be quite normal except for a slightly more bluish tinge to the skin than usual. After opening the abdomen it was noticed that the muscles had a bluish tinge, and the blood vessels were colourless apart from the usual chromatophores in their walls. The ventricle was whitish in colour, the atria pale and translucent, the main arterial arches also pale and somewhat translucent, and the spleen was white with a very slight yellowish tinge. The general anatomical structure was quite normal, although the spleen was somewhat smaller than usual. The specimen was a partially mature female.

About 1 ml. of blood was removed and oxalated. It was quite colourless to the eye. Fresh blood smears revealed no cellular elements apart from some bodies which had the appearance of spindle cells. Unfortunately, it has not been possible either to have a spectroscopic examination or an iron estimation performed on the blood sample.

The toad was preserved in 5 % formalin for some days and the spleen was subsequently removed, sectioned and stained. Comparison of the sections with those of a normal Xenopus spleen prepared in the same way revealed that there were no normal mature erythrocytes, although the other blood elements and the reticular structure of the spleen looked normal. Mr Jacobson of the Medical School kindly examined the preparation and found normoblasts present, although these did not show the cytoplasmic staining typical of mature erythrocytes.

Wolvekamp & Lodewijks (1934) noted that the number of erythrocytes in Rana tended to drop markedly when they were kept in captivity. They accounted for this by the greatly diminished activity and metabolism of the frog. No reports have been found, however, of the red blood count decreasing to as low a level as seen in this Xenopus specimen. The apparently normal behaviour of this animal prompted a brief investigation of oxygen supply in relation to survival.

In the first series of experiments an attempt was made to block the haemoglobin with carbon monoxide. Fifteen apparently normal toads were placed in a glass jar into which illuminating gas was tapped for 5 min. After a short period of 1 or 2 min. for air to circulate, some of the toads were removed and placed in separate containers free from gas. With the other animals the gas application was repeated a number of times, up to a total exposure of 30 min. Immediately after the experiment blood was removed from one of the specimens and tested for carbon monoxide content by the colorimetric pyrogallic-tannic acid method (Kolmer & Boerner, 1945). This showed that after a 30 min. exposure to gas 80-100% of the haemoglobin was blocked by carbon monoxide. Nevertheless, all the toads survived the 30 min. exposure, although it took them some hours to revive from the semi-comatose state produced by the treatment. Subsequent inspection during 4 days did not show any ill effects whatsoever, although the haemoglobin had by no means been completely regenerated by the end of that time. It appears safe to draw the conclusion that under certain circumstances Xenopus can withstand a nearly complete elimination of its haemoglobin. It is not suggested, however, that the toad will be able to manage without haemoglobin at periods of great activity, for example, the breeding season.

Another series of experiments was carried out to obtain some information on the significance of cutaneous respiration. A number of animals were placed in a container and prevented from reaching the surface of the water by wire gauze. Tap water of an average temperature of 15° C. was then kept flowing through the container at a slow rate. The oxygen content of the inflowing water was just under 5 ml./I. No food was given to the animals. It was found that the animals were still alive after a period of 4 weeks, when the experiment was discontinued. Apparently they did not suffer any obvious discomfort. In fact, they did not lie motionless all the time, but swam about occasionally and also became active when disturbed. These experiments indicate that in spite of the very small size of the arteriae cutaneae magnae (cross-sectional area about one-ninth of that of the pulmonary artery) cutaneous respiration is well able to support the animals under the prevailing conditions.

The occurrence, even as an abnormality, of a vertebrate without haemoglobin is almost unprecedented, except for the leptocephalus larvae of eels, and certain sluggish South Georgian fishes, which possess neither erythrocytes nor blood pigments (Ruud, 1954). The absence of haemoglobin in an otherwise apparently normal toad, therefore, opens interesting speculations regarding the significance of haemoglobin in poikilothermic animals. Barcroft (1928) has said that as one passes from warm-blooded to cold-blooded vertebrates, quite a number of factors tend to make the demand for oxygen more nearly commensurate with the supply which can be maintained by a blood devoid of pigment. Amongst these factors he notes that the demand for oxygen is less, and that the oxygen is more soluble in plasma at lower temperatures. Barcroft estimates that from the points of supply and demand of oxygen, a frog without haemoglobin would be some two hundred times better off than a mouse in the same circumstances. Although purely speculative, this paints rather a vivid picture. His opinion is well in agreement with the experiments of Nicloux (1923a, b), who reported that carps, pikes and eels survive for at least 4 hr., apparently without trouble, when the haemoglobin is blocked to the extent of some 90% by carbon monoxide. Gréhant (1887) stated that Rana survived for at least 3 days in a gas mixture containing 50 % carbon monoxide.

Wolvekamp (1932) and Wolvekamp & Lodewijks (1934) have determined the oxygen dissociation curves of the blood of Rana esculenta. Since the tension of carbon dioxide in the lungs varies between 7 and 14 mm. Hg, since the carbon dioxide tensions in the circulatory system and in the tissues are very probably not higher than 14 mm. Hg, (Campbell, 1924,1926), and since the oxygen tension in the tissues is not usually below 28 mm. Hg, it follows from these dissociation curves that the haemoglobin does not deliver a large quantity of oxygen to the tissues. Most of the oxygen is kept in store by the blood. The blood might act, to a considerable extent, as an emergency mechanism for times of greater activity and increased metabolism.

Rana hibernates for long periods at low temperatures under conditions where only cutaneous respiration is possible. It has been shown by Serfaty & Gueutal (1943) that R. esculenta is able to survive for 2-3 weeks when totally submerged in well-oxygenated water at a temperature as high as 14-15⁰ C. Charles (1931) performed experiments which indicate that the cutaneous respiration of Xenopus accounts for about one-quarter to one-third of the total absorption of oxygen in richly oxygenated surroundings. Similar estimations by Krogh (1904) show a greater cutaneous respiration (one-third to one-half) in Rana esculenta and R. fusca. The difference between the genera can probably be explained by the relative sizes of the arteriae cutaneae magnae, those of Rana being much larger.

The present experimental results agree well with the findings of other workers and support the contention that the oxygen requirements of Rana and Xenopus may fall to a very low level at certain periods, although it is not claimed that such conditions always prevail in nature. Nevertheless, the question must be faced as to whether the type of selective distribution of blood advocated by the ‘classical theory’, or the kind of distribution found in Xenopus (de Graaf, 1957) is really related to the oxygen needs of the tissues. One wonders if the starting point of all considerations on blood distribution in Amphibia, that is, the oxygen need of head and body, is not altogether wrong. The possibility that selective distribution serves some quite different purpose must be seriously considered. This ‘different purpose’, however, is completely unknown at the moment and future research will have to clarify the position.