THE E F F E C T O F ATHTROInTSXl AND IIPPERTHY- ROIDTSM ON THE OXYGEN ('ONSUMPTION

OF THE ADULT SALAMANDER

AI~FHED TAYTiOR Depart i twi t of Zoology, Oregoii Stule College, C'ortwllis

TI IKEE WGIJRER

The salamaiiclei- tliyi*oid gland has been studied extensively, especially from the standpoint of tlie role of this gland in development and Inetamorplrosis. Studies on adult animals are chiefly confined to thc effects of thyroid removal. Hpper- thyroidism in the adult salainan~lor has received little atten- tion. Likewise the problem of the cliangcs in metabolisni associated with the athyi-oid aiid Iiypci*thyroid states in mature aniphibiaiis has not been investigated to any extent. Work of this nature has, of course, been done by many investi- gators working with higher types of animals.

It would seem that data of this kind for the salamander would be of special value since the reaction of these animals to disturbaiice of thyroid function is niost serere. On tlie basis of expcriciicc gained in this laboratory with many hun- dreds of tliyl.oiclectornize(~ salainaiitlci+s covering a period of 5 years, it appears t lint tliyroidless Triturus torosus will die in less than a year when kept at room temperature. A4dams and associates ('32) reported similarly for Triturus viri- descens. They found that the longest periods of survival after thyroid removal were 182, 183, 204, 229 and 286 days. Mammals, including man, are not nearly so adversely affected.

'Aided by a grant from the General Research Council of the Oregon State System of Higher Eduratioii.

135

136 ALFRED TAYLOR

THYROTI) ANI) METABOTJISM I N TRT'I'CHUS

The present study W'RS undcrtakcn, accordingly, to obtain data on the metabolic clianges, as measured by oxygcii utili- zation, associated with tlic atliyroid and hyperthyroicl condi- tions in the salamaiidci.. As the investigation progressed nnexpectecl data wtts o1)taincd in connection with the Iiyper- thyroid state and its effect 011 moulting.

MATERIALS AN I ) METHODS

The salamt~ndci* Tt*itut-us toiwsus (Ratlilte) was iitilized in this investigation. Inclivitluals of al,out the same size and of tlic iiialc scs were sclcctcd. Liii.g;c iiurribcrs of these aiii- mals have been used in this laboratory during the last 5 gears. l l any individnal sdamaiidcrs have bcen lwpt througliout tliat period. The fact tliai they have remained hcalthy for sue11 a space of time iiitlicates the method of liandling and feeding was adequate.

Smtill aquaria wcre used. Each aquarium contained ciglit to ten tinimals. Tlic ;iqiinriti wei*c 1-aisccl up aboiit 2 inches on one end and enough watci. kept in them so that the sala- manders coulcl be either out of the water on the raised end or entirely submergcd at thc oppositc cncl. The watcr mas changed oncc t i day. The aiiimals were fed twice a week throughout the year. The dict consisted of earthworms alter- nated with ground becf. This treatment insui*cd a supply oP well fleshed animals wliich had been kept long enough to clis- close any defective indivi~li ids mliich might 1)cl present. Such aiiinials wcrc also quieter tliaii f iwlily caught stock during tests for oxygen utilizatioii. Fni*tlici*, a closer approach to normal metabolism was possible with such salamanclers tlian with the use of emaciated refrigerator kept stock. It is well known that thc metabolic rate drops in animals insufficiently nourished.

Feeding was discoittiiiued 2 weeks prior to making metabo- lism tests. Both anesthetized ancl unanesthctized animals were utilized in the tests for normal metaholism. It was found that Nembutal (Pentobarhital sodium) if injected into the

THYROID AND METABOLISM I N TEITUKCS 137

visceral cavity in just the right aniouiit would produce an anesthesia lasting 8 or 9 hours and cs1,ccially suitable for quieting the animals. In the present study 1 p. of Nembutal was added to 100 cc. of Ringer's solution and 1 cc. of this mixture used for a 20 gm. salamander.

Athyroidism was produced as previously described (Taylor,

The hyperthyroid state was induced very cffectively by homotransplanl s of thyroid tissue. Four thyroids were trans- ~)lanted to each experiniciital aiiinial wliicli, including their own pair, gave tlie aiiinials so ti-catctl six thyroids or three h i e s tlic nornial.

Animals for this operation were anesthetized as previously described. A small incision was made in the body wall in the region of tlic splecii aiicl the freshly removed ~vholc thyroids were iiiscrtctl through this openiiig niicl placed on the ventral surface of tl1c liver. A single suture closed the wound.

For oxygen mcasuremciit, the animals were placed in a wide mouth flask. This container was scaled by a co1.B through which was iiisci*tcd a thermometer, one a rm of a maiiometcr, mid a sniall glass liypodcrniic syringe. With these in position the stopper was pressed down and the seal made perfect by 1)rushing over with melted paraffin. The flask was immersed in water for tcmperatui-e control. Pressure was kept at the barometric level by the manometer which also scrvcd to iiidicate ~vlictlier or not tlic seal was airtight. Carbon dioxide was taken out by having in the flask with the animals a gauze coveiwl vial containing 10% potassium hy- droxide solution.

The animals wci-e sealctl in for 1 Iiour after which a sample of gas was drawn off arid analyzed for oxygen content by a moclificatioii of the mic~*o-metliod described hy Camphell ancl Taylor ('35). A hrief description of one determination will clarify the procedure.

The flask used in this trial had a net volume of 391 cc. Five normal anesthetived salamanders weighing 98.5 g-m. were placed in the container. The specific gravity of the normal

'36).

animals was 1,036. Tlie iiet capacity of tlie flask after tlic iiitrotluctioii of tlic salami-liiders was accordiiigly 289 cc. The specific gravity of normal salamanders under the conditions givcii does iiot vary appreciably from animal to aninial. In the instance of the experimental animals their volume was taken directly.

The apparatus was next immersed in tlie water bath and left uiicovered until the coiitciits hecanie stable a t 17°C. The cover was placed in position ancl sealed. At the end of 1 hour, thc plunger of tlie Iiypodermic syringe w a s raised a id a samplc of gas drawn up over tlie coiitained liquid. This sample was nnalyzecl for oxygen content. ('alculations 011 the (late ob- taiiied from gas aiialysis were made as follows :

Where S = the initial roluttlc~ of gas t:tkcti into tlir :il)liar:itus for analysis 1; = rolunie of ~ : I B a f t e r rr1tioral of oxygen

S-N = oiygc'n pi-eseitt at tiiiic of :Analysis

~ l i c i i 0.7904 = pi-oportioii of 0x4-gcii 1)rcseiit after the animals have satisficd tlicii- nc!eds but in tcrms of tlic net volunie of t l iv co1it;iiilci.. If this volume is iiitlicatcd as V, the forniula (O.3ONi - 'iN 0.~104) V equals the total oxygen coiisuiiipt ion u i i co r iwk l f o r tcnil)eixture aiid pi-essniac.

By the above method of figuring, tlic 98.5 gm. of salaiiiantler mas fouiitl to have lowei*ecl tlic oxyg~ii coiiteiit of 289 cc. 1.28% in 1 hour. Tlie barometer rend 756, the temperature was 17"C., a r i d the gas was satui*atetl with water vapor. Hciicc the final figures iiitlicatcd a coiisuniption of 31.5 cu.mm. of oxygen iwlurccl to staiitln i d contlitioiis, i)c>i- gixm of sala- mander, per hour.

This method lias bceii used Iiclre fo i - the past 2 yea1-s during which time liuiidi-eds of dctermiiiat ioiis ha\-e hceii madc aiid the proredurc found to bc sufficiently reliable and accurate. ,4 temperature of 17°C. was used because the animals tencled to be quieter in a cool atmosphere and a t the same time this temperature is not low enough to introduce another factor. Tests m~erc made at othcr temperatures up to 30°C. and the same general trends appeared.

THYROID AhTD METABOLISM IN TRITVRUS 139

RESULTS

Table 1 and figure 1 summarize the results obtained in the investigation of normal metabolism both for the anesthetized and the unanesthetized. For the unanesthetized the average of twenty-four determinations involving more than 100 male salamanders gave an oxygen utilization of 68.6 cu.mm. per gram per hour. The anesthetized group show an oxygen con- sumption of 35.1 cu.mm. per gram per hour. It will be noted, as would be expected, that the standard deviation is much less for the anesthetized animals.

I n the instance of the thyroidectomized animals, metabolism tests were made daily from the first to the sixteenth day after

TABLE 1

Oxygen consumption under various conditions in terms of oubic millimeters per gram of body weight per hour

CONDITION

Uiianesthetized normals Anesthetized normals Athyroid 1 to 16 days Athyroid 21 to 32 days 4 extra thyroids 8 to 24 days 4 extra thyroids 46 to 53 days

I

10

8 5

77.2 49.7 10.3 40.3 6.4 128.6 25.2

thyroid removal. Table 1 and figure 2 give the average, 77.2 cu.mm. of oxygen for these determinations. This figure is slightly higher but not significantly so than that for normal unanesthetized animals. Beginning with the twentieth day there was a drop in the oxygen utilization of this group. The average of ten determinations between the twenty-first and thirty-second days after thyroidectomy is 49.7 or about 72.5% of the normal rate.

The results of homotransplants of two pairs of thyroids into an otherwise normal animal were not anticipated. Moult- ing was inhibited. The normally clear orange ventral surface began to blacken and become grimy after a lapse of 2 days and these animals soon had the appearance typical of thyroidless

THF) .JOURNAt O F EXPERIMENTAL ZOOLOOY, VOL. 81, NO. 1

140 ALFRED TAYLOR

ANAE STHETIZED

salamanders. If the transplant continued to function death resulted suddenly in periods extending from a week or '2 to 3 months. Animals autopsied after a month or more with functional transplants revealed spleens only a fraction of the usual size and often almost white in color.

Investigation of the oxygen utilization of these animals revealed a surprising trend. As the data summarized in figure 3 and table 1 show, for the first 3 weeks after the trans- plants of thyroids had been made, these salamanders used much less than the normal amount of oxygen. During this period 40.3 cu.mm. of oxygen were utilized, an amount not much in excess of the requirements of anesthetized normals.

THYROID A B D METABOLISM IN TRITURUS 141

Beginning about 48 days after the reception of the extra thyroids the metabolic rate attained a high level. An average of 128.6 cu.mm. of oxygen per gram per hour was recorded for this group. This was almost twice the normal metabolic rate.

Fig. 2 Oxygen consumption over a period of time following thyroideetomg.

Individual differences were marked. Some salamanders termi- nated the period of low metabolism much earlier or later than the group depicted in figure 3. Some individuals died suddenly after a few weeks in the hyperthyroid state. In every case that survived, however, there was an initial period of ab- normally low metabolism followed by a period of very high metabolism. Histological examination of the transplanted

142 ALFRED TAYLOR

and own thyroids disclosed an appearance of nonactivity dur- ing the time the metabolic rate was low and an appearance of activity correlating with the rise in oxygen utilization.

Moulting was inhibited as long as the transplants were functional without regard to the rate of metabolism.

...........................................................

AGE OF THYROID IMPLANTS IN D A Y S

Fig. 3 Oxygen consumption over a period of time following homotransplants of two pairs of thyroids into an otherwise normal salamander.

DISCUSSION

So far as could be determined, there are no published figures on the oxygen utilization of either athyroid or hyperthyroid adult salamanders. Investigations of this nature on normal adult salamanders are limited. Wills ('36) in association

THYROID AND METABOLISM I N TRITURUS 143

with an investigation of sex and respiration in developing amphibia, gives the oxygen utilization fo r adult male Triturus torosus as 45.53 cu.mm. per gram of body weight per hour at a temperature of 20°C. These animals had been without food for 'about 2 months' and this probably accounts for the lower rate in comparison with the figures given here. Pomerat and Zarrow ('37) have published the results of a study on the respiration of the newt but since the weights of the animals are not given, comparison is impossible.

The lag in the metabolic response to thyroidectomy cor- relates with the delay in skin reaction to this condition. The skin begins to blacken and the metabolic rate to fall at about the same time, 3 weeks after thyroid removal. Adams and associates ('32) have noted this delay in skin reaction after thyroidectomy in the instance of Triturus viridescens. The characteristic blackening began the second week in thyroid- less animals kept a t 69°F. and was delayed to the third or fourth weeks in animals kept a t a temperature of 54°F. A previous study (Taylor, '37) has shown that a period of above normal rate of cell division was characteristic of the spleen of athyroid salamanders and began about 3 weeks after thyroidectomy. It is interesting to note that this increased mitotic activity commenced after the metabolic rate was below normal. Apparently the salamander has enough of the thyroid principle stored in the tissues to last during the period when the metabolic rate remains normal.

The effect of implanting thyroids into a normal salamander was most surprising. The extreme lowering of the metabolic level which this procedure initiates is difficult to explain. So unexpected were these results that the data were checked several times with different groups of animals. Whenever the implanting of the two pairs of thyroids was successful and the animal survived, a period of lowered metabolism fol- lowed without any exception being noted. Attempts to obtain these results by homotransplants of one pair of thyroids rather than two pairs were not successful.

144 ALFRED TAYLOR

It may be said that the introduction of so much foreign thyroid tissue into the body of the salamander might have a toxic effect aside from the production of excessive amounts of thyroid hormone. This is apparently not so since other tissues were transplanted without ill-eff ect including in one instance a homotransplant of the whole cerebral sub- stance of the brain into one animal. Further, homotrans- plants of thyroid tissue as a replacement procedure in the instance of thyroidless salamanders worked successfully serv- ing to bring back to the normal condition animals near death from athyroidism. These implants were made in exactly the same way as described for the transplanting of the extra thyroids.

The animal's own thyroid gland appeared to become non- functional as a result of the presence of the extra thyroids. It assumed the appearance of a resting stage in accord with the criteria given by Uhlenhuth ( '27). The implants also went into the resting condition. Later as the metabolism became higher the thyroid tissue assumed an appearance of activity. It is hoped that further investigation specifically concerned with the effects of homotransplants of endocrine gland tissue into otherwise normal animals will reveal something of the baqis for this peculiar phenomenon.

The effect of the hyperthyroid state on moulting was not expected. It was especially surprising, in view of the work of others, to find moulting inhibited even when the metabolic rate was much higher than normal. Workers who have in- vestigated the effect of thyroid feeding, thyroxin injection, or thyroid implants agree that such procedures tend to increase moulting above the normal condition. Adams and associates ( '29, '30, '32) have shown this to be true for the salamander, and Noble and Bradley ( '33) have reported likewise for the lizard. Horning and Torrey ( '23), Torrey and Horning ( '25), Zawadowsky ( '25), Giacomini ( '26), Krieenecky and Nevalon-

nyj ('27) and many others have reported that feeding thyroid substance in excessive amounts to fowls is associated with increased moulting. Comparable results have been obtained with mammals (Gudernatsch, '15 ; Simpson, '22).

THYROID AND METABOLISM I N TRITURUS 145

It appears probable that the effect on moulting reported here was due to the fact that a relatively large amount of thyroid tissue was present in the experimental animals. Chang ( '26) has noted that either too much or too little of the thyroid principle inhibits hair growth. Implanting one pair of thy- roids, in the present study, did not inhibit moulting.

SUMMARY

1. The metabolic rate as measured by oxygen utilization was investigated for normal (anesthetized and unanesthetized), athyroid, and hyperthyroid adult male Triturus torosus. All salamanders used were well fleshed laboratory animals fed throughout the year except that feeding was discontinued 2 weeks before beginning the metabolism tests. Oxygen utili- zation is given in terms of cubic millimeters of oxygen, per gram of body weight, per hour.

2. The oxygen consumption for unanesthetized normal sala- manders was 68.6 and for anesthetized normal animals, 35.1.

3. The oxygen consumption of athyroid animals was measured over a period of time. The average of a series of determinations taken daily from the first to the sixteenth days after thyroidectomy was 77.2. The same group in the period from the twenty-first to the thirty-second days had an average oxygen consumption of 49.7 or about 72% of the normal rate. It appeared that cessation of moulting and the fall in the metabo- lic rate occurred at about the same time.

4. Hyperthyroid animals were produced by implanting two pairs of thyroids into otherwise normal salamanders. From the eighth to the twenty-fourth days after the operation, the oxygen utilization was, on the average, 40.3 or about 59% of the normal rate. Between the forty-sixth and fifty-third days the oxygen consumption of these animals was 128.6 or 187% of the normal rate. There appeared to be a general trend for animals which had received four extra thyroids to first undergo a period of abnormally low metabolism followed by a period, ending in death, of very high metabolism.

5. Contrary to the results of others, moulting was inhibited in the hyperthyroid animals regardless of whether the metabo-

146 ALFRED TAYLOR

lic rate was high or low. These animals quickly assumed and retained the blackened appearance which has been considered characteristic of the athyroid condition.

LITERATURE CITED ADAMS, A. E., AND L. RICHARDS

viridescens. Anat. Rec., vol. 44, pp. 222-223. ADANS, A. E., L. RICHARDS AND A. KUDER

pituitary glands t o moulting in Triturus viridescens.

1929 The effect of thyroidectomy in Triturus

The relation of the thyroid and Science, vol. 72,

ADAMS, A. E., L. KUDER AND L. RICHARDS 1932 The endocrine glands and moult- ing in Triturus viridescens. J. Exp. Zool., vol 63, pp. 1-55.

CAMPBELL, J. A., AND H. J. TAYLOR 1935 A modification of Krogh’s micro- method of gas analysis.

CHANG, HSI CHUNG 1926 Specific. influence of the thyroid gland on hair growth. Am. J. Physiol., vol. 77, pp. 562-567.

GUDERNATSCH, J. F. 1915 Feeding experiments on rats. 111. Am. J. Physiol., vol. 36, pp. 370-379.

HORNING, B., AND H. B. TORREY 1923 Effect of thyroid feeding on the moulting of fowls. Anat. Recc., vol. 24, p. 399.

KkfdENECKP, J., AND M. NEVALONNYJ 1927 Weitere Versuche iiber den EinflusN der Schilddruse und der Thymus auf die Entwicklung des Gefiedera bei den Huhncrkucken. Arch. f . Entwmech., Bd. 112, S. 594-639

The relation of the thyroid and the hypophysis to the moulting process in the lizard, Hemidactplus brookii.

1937 Studies on the respiration of the newt. I. Description of the method and da ta on the normal and gonad- ectomized animal. J. Cell. and Comp. Physiol., vol. 9, pp. 397415.

SIMPSON, S. 1922 Integumentary changes in the sheep following thyroidectomy and administration of thyroxin. Am. J. Physiol., POI. 59, pp. 445-446.

TAYLOR, ALFRED 1936 Athyroidism in the salamander Triturus torosus, Rathke. J. Exp ZOO]., V O ~ . 73, pp. 153-181.

1937 The effect of athyroidism on the rate of cell division. J.

The effect of thyroid feeding on the Riol.

UHLENHUTH, E. 1927 Die Morphologie und Physiologie der Salamander-schild- druse. I. Histologisch-embryologische Untersuchungen des Sekretions- prozesses in den verschiedenen Lehensperioden der Schilddruse des Marmorsalamanders, Ambystoma opacum. Arch. f . Entwmech., Bd. 109,

WILLS, IEVIN A. 1936 The respiratory ra te of developing amphibia with special J. Exp. Zool., vol. 73, pp. 481-510.

ZAWADOWSKY, BORIS 1925 The effect of feeding fowls on thyroid gland,

1930

pp- 323-324.

J. Physiol., vol 84, pp. 219-222.

NOBLE, G. K., AND HELEN T. BRADLEY 1933

Biol. Bull., vol. 64, pp. 289-298. POMWAT, C. M., AND M X. ZARROW

EX^. ZOO]., VOI. 75, pp. 239-244. TORREY, H. B., AND B. HORNING 1925

moulting process and feather structure of the domestic fowl. Bull., VOI. 49, PD. 275-289.

S . 611-749.

reference t o sex differentiation.

Endocrin., vol. 9, pp. 125-136.