(Received 31 August 1938)

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1 358 J. Physiol. (I938) 94, I2.492:6I2*44 THE YOUNG CHICK AS TEST FOR THE THYROTROPIC HORMONE BY CUTHBERT LESLIE COPE From the Medical Unit, University College Hospital, London (Received 31 August 1938) EVER since the early work of Loeb and of Aron on the thyrotropic hormone of the anterior pituitary lobe, the young immature guinea-pig of about 180 g. body weight has been the accepted test animal for work on this hormone, and its use has continued in spite of the recognition that such guinea-pigs have certain definite disadvantages. Numerous workers, for instance, have called attention to the fact that the more central parts of the thyroid glands of such guinea-pigs frequently show a degree of spontaneous hyperplasia which may readily be mistaken for the effects of a thyrotropically active substance [del Castillo, 1932; McGinty & McCullough, 1936; Cope, 1938a]. Because of this a number of investigators have used hypophysectomized rats as more sensitive and reliable test animals for the thyrotropic hormone. The normal rat has naturally a rather hyperplastic thyroid and is relatively insensitive to this hormone. But after removal of the pituitary gland in such rats this thyroid activity subsides and the gland assumes a resting condition with very flat epithelium and intact colloid. Such hypophysectomized rats are good and sensitive test animals for the thyrotropic hormone [Anderson & Collip, 1934; Hertz & Oastler, 1936]. But the operation of hypophysectomy on rats is one which can only be satisfactorily performed after considerable practice, and for this reason such animals have not been available in all laboratories where their use was desirable. It is because of this fact that we feel that a recent suggestion of Smelser [1937] is of special value and well merits wider recognition and consideration. Smelser has found that young, so-called " day-old chicks " have thyroids which are particularly suitable as tests for the thyrotropic hormone, and advocates their use for work in this field. Such young chicks have for some months been in use in this laboratory, and from our

2 CHICK AND THYROTROPIC HORMONE 359 experience with them we feel fully justified in advocating their more extended use. The advantages which they present more than outweigh the disadvantages, and it is the purpose of the present paper to discuss these advantages, our experience in this respect fully confirming the claims of Smelser. THE ADVANTAGES OF CHICKS Perhaps the greatest advantage of young chicks in the laboratory is their relative cheapness compared with guinea-pigs. For fully half the year, through the winter and spring months, they are readily obtained from poultry farms. Cockerels are much cheaper than pullets and have been found entirely satisfactory. The cost of such "day-old" light cross cockerels averages about one-tenth or less of the cost of young guineapigs. Consequently it is possible by using these to construct a weight response curve for a given preparation of thyrotropic hormone, entailing the use of four or five groups of five chicks each, for an outlay equivalent to the price of about two guinea-pigs. The advantage of such birds in work involving the frequent assay of extracts for thyrotropic activity is thus very considerable. Indeed, where such activity is measured by thyroid weight increase, this cheapness is the main advantage over the guinea-pig. But when histological criteria rather than thyroid weight are being used, the value of the chick is still greater. Smelser claimed as one of the main advantages of chicks that the thyroid gland was of uniform histological appearance throughout, there being no tendency to develop that area of central hyperplasia so frequently seen in young guinea-pigs, and so difficult to assess when present. This we are able to confirm. In P1. I, fig. 1, is shown a typical crosssection of the thyroid gland of a chick 10 days old, which shows well this uniformity of structure throughout the gland. Occasionally a slight degree of hyperplasia involving the whole gland is encountered, but in our experience this occurs considerably less frequently than it does in young guinea-pigs. We have never found glands showing any appreciable difference in histological structure between centre and periphery. In its response by weight increase to the action of thyrotropic hormone injected subcutaneously, the chick thyroid is of the same order of sensitivity as that of the guinea-pig. But as its body weight is only about one-third or less of that of the usual test guinea-pig, and equivalent dosage is proportional to body weight, it is evident that a considerable economy of test material is achieved by using young chicks compared with the amount required for assay on the same number of guinea-pigs.

360 C. L. COPE Finally, chicks when quite young seem to need considerably less space than do guinea-pigs, an additional advantage in a crowded animal house.

3 360 C. L. COPE Finally, chicks when quite young seem to need considerably less space than do guinea-pigs, an additional advantage in a crowded animal house. Compared with the above advantages the drawbacks of young chicks are relatively unimportant. It is desirable that during their early days of life they be maintained at an equable warm temperature in a heated cage. But such heating is very easily and simply provided by suspending in each cage a carbon filament electric lamp hanging in an open-top tin. The sides of this tin become hot and must be insulated from the chicks by a covering of thin cardboard. If, now, the sides and top of the cage are covered with felt or sacking, the temperature within is easily controlled by varying the amount of such covering. By such simple means we have had no difficulty in maintaining the cage temperature at the desirable 900 F. for the first week, and lowering this by 50 F. in each succeeding week. The mortality of our chicks from all causes in the first 3 weeks of life has been about 2 %. It has not been found desirable to use chicks older than this. The relatively small size of the chick thyroid is perhaps the greatest deterrent to its more extended use, but we have found the difficulty of dealing with such small thyroids to be considerably less than might be anticipated. The small size of the gland certainly tends to make the removal of the thyroid for weighing and sectioning a more delicate procedure than in the guinea-pig, but relatively little practice is required to dissect it out cleanly and rapidly, and we have not found it necessary to use a dissecting microscope for this purpose. The anatomical relations of the chick thyroid differ considerably from those of the mammal, and a note on its identification and removal may be of value to those unacquainted with its anatomy. DISSECTION OF THE CHICK THYROID The chick, killed with chloroform, is fixed on its back on a board by loops round neck and legs. The downy feathers over the trachea and the upper part of the breast are moistened with water and an incision is made with fine scissors in the mid-line of the neck over the trachea. This incision is continued downwards well on to the sternum, passing to the left side of the crop. Fat and lymphoid tissue (thymus) is dissected away from the base of the neck on the left side above the clavicle, exposing a tough transparent membrane which closes the anterior opening of the thoracic cavity and forms the anterior wall of the clavicular air sac. This membrane is cut through with fine scissors to expose the thyroid gland

4 CHICK AND THYROTROPIC HORMONE which will be found lying immediately beneath it close to the carotid artery. It appears as a small pinkish spherical globule which assumes a yellowish translucency when its blood is expelled by slight pressure. This translucency serves to distinguish it from the fragments of lymphoid tissue with which it is often associated, and which are more opaque. Immediately caudal to the thyroid on each side can usually be seen a second smaller yellowish and discrete globule which is the parathyroid gland. An attempt to remove the left thyroid gland cleanly from its position on the carotid artery is very liable to lead to much bleeding which obscures the field of dissection, and it is far more satisfactory, therefore, to cut out rapidly a miscellaneous mass of tissue containing the thyroid gland and then to dissect the gland free from the adherent pieces of artery and lymphoid tissue. It is also wise to insert a small plug of cotton wool into the bleeding wound to prevent blood running across to the right side and obscuring the field on that side. To reach the gland on the right side, the crop is pulled from its loose adhesion to the clavicle, thereby exposing the tough membranous wall of the clavicular air sac. This is cut through and the right thyroid is found immediately beneath it close to the carotid artery. The gland and adjacent tissue is cut out quickly, usually with a piece of artery attached, and is dissected clean at leisure well away from the bleeding wound. Bleeding is usually more severe on the right side than the left, and if this is not done the right thyroid is easily obscured by blood from the damaged vessels. With a little practice the dissection of the gland free from non-thyroid tissue is considerably easier than might be expected from its small size. THE ACTION OF THYROTROPIC HORMONE The mean thyroid weight in a number of successive groups of young chicks is shown in the following table and indicates the degree of constancy which may be obtained: Mean thyroid Date No. of chicks Approx. age weight (mg.) Feb days 4.4 Mar days 4-6 May days 4.5 Feb weeks 5-4 Apr weeks 4.7 Smelser's chicks at 6 days old gave a mean thyroid weight of only 2-8 mg., which is considerably less than our own. The cause for this PH. XCIV

5 362 C. L. COPE difference we have not determined, but it may well be due to difference in the breed of chicks. The effect of subcutaneously injected thyrotropic hormone on the thyroid of the young chick is essentially similar to that in the guinea-pig. The gland increases considerably in size and vascularity. This weight response provides the most convenient objective method of assay of 14 mg s Dose hormone daily for 5 days 2-0 mg. Text-fig. 1. The weight response curve of the chick thyroid to thyrotropic hormone. (1 mg. is equivalent to 0-3 Parkes unit.) thyrotropic activity and is considered more fully below. The colloid loses much of its power to stain with eosin, develops a mottled structure, and is finally absorbed completely. The epithelium changes from flat to cubical or definitely columnar with a wide clear protoplasm, and the cell nuclei increase in size, becoming spherical and often showing numerous mitoses. These changes are indicated in P1. I, figs. 2 and 3. The extent of thyroid weight increase in response to thyrotropic hormone is shown

6 THE JOURNAL O PHYSIOLOGY, VOL. 94, No. 3 PLATE I Fig. 1. The normal chick thyroid. x 300. Fig. 2. Chick thyroid response to O l Parkes unit of thyrotropic hormone. x 300. Fig. 3. Response to 0-6 Parkes unit of thyrotropic hormone. x 300. To face p. 362

7 CHICK AND THYROTROPIC HORMONE 363 graphically in Text-fig. 1. In this figure each plotted point represents the mean thyroid weight of a group of five chicks which had been injected subcutaneously with the charted dose of hormone daily for 5 days, and had been killed on the sixth. In the guinea-pig about 3*3 mg. of this active extract were required daily to double the thyroid weight, whence the preparation contains about 0*3 Parkes unit of hormonal activity per mg. [Rowlands & Parkes, 1934]. From Text-fig. 1 it is seen that 1 mg. or 0 3 Parkes unit will produce a chick thyroid weight of 9*1 mg., that is it will rather more than double the thyroid weight in the chick. The chicks used in producing this curve averaged 53 g. in weight compared with the 190 g. of the guinea-pig group. It appears, therefore, that the dose of thyrotropic hormone per g. body weight necessary to double the weight of the thyroid gland is approximately the same in the chick as in the guinea-pig, but that by virtue of the difference in body weight only one-third of the quantity is required for the former that is necessary for the latter. The weight response curve may be used satisfactorily to assay preparations with thyrotropic activity. We have used it recently to assay a preparation obtained from human pituitary glands [Cope, 1938b]. This preparation had previously been found by assay on young guinea-pigs to contain 1 Parkes unit in each 12 mg. of powder. 5 mg. of this powder were injected daily for 5 days into each of six chicks. These were killed on the sixth day and gave a mean thyroid weight of 10.8 mg. From the curve this weight corresponds to 0(4 unit, giving the powder a potency of 1 unit per 12-5 mg., a figure which agrees very closely with the result of assay in the guinea-pig. Chicks can thus be used as easily as, and much more cheaply than, guinea-pigs for the assay of unknown thyrotropic extracts. That they may also be used for work on antithyrotropic sera is clear from the following experiment which shows that an antithyrotropic serum prepared in the rabbit is as active an inhibitor of thyrotropic activity in the chick as it is in the guinea-pig. Rabbit serum daily Thyrotropic, & 5 Mean hormone Antithyro- thyroid daily Normal tropic No. in weight Group mg. c.c. c.c. group mg

364 C. l. COPE From this table it is seen that a daily dose of 0-6 c.c. of an antithyrotropic serum prepared in rabbits was more than adequate completely to inhibit the action of 1-5 mg.

8 364 C. l. COPE From this table it is seen that a daily dose of 0-6 c.c. of an antithyrotropic serum prepared in rabbits was more than adequate completely to inhibit the action of 1-5 mg. or 045 Parkes unit daily of a potent thyrotropic extract. The histological changes in the above series of glands corresponded reasonably well with the changes in weight. The detection of thyrotropic activity by means of the histological changes in the thyroid gland involves a much greater subjective element than does its estimation by the weight increase of the glands. Because of this it is difficult to indicate the minimum dose that can be detected by histological means in the chick thyroid. A daily dose of 0.1 Parkes unit produces a readily detectable rise in mean thyroid weight of a group of five chicks, and this dose will produce well-marked and quite unequivocal hyperplasia in a majority of the glands of the group. But some glands may show relatively little change with this dose, and it is therefore felt that doses of thyrotropic hormone much smaller than this cannot with certainty be detected unless a large group of birds is used. SUMMARY Smelser's claim that chicks are convenient test animals for work on the thyrotropic hormone is fully confirmed. The main advantages of chicks are their much greater cheapness than guinea-pigs, and the greater constancy and simplicity of the histological picture in their thyroid glands during the first 2 weeks of life. The difficulty of dissecting out cleanly such small thyroid glands is much less than might be expected. Thyrotropic substances can readily be assayed by the weight increase of the chick thyroid, this weight being approximately doubled by onethird of a Parkes unit of hormone. The behaviour of antithyrotropic serum in inhibiting the action of the thyrotropic hormone may also be conveniently studied in the young chick. REFERENCES Anderson, E. M. & Collip, J. B. [1934]. J. Physiol. 82, 11. Cope, C. L. [1938a]. Quart. J. Med. 31, 151. Cope, C. L. [1938b]. Lancet, 1, 888. del Castillo, E. B. [1932]. C.R. Soc. Biol., Paris, 111, 461. Hertz, S. & Oastler, E. G. [1936]. Endocrinology, 20, 520. McGinty, D. A. & McCullough, N. B. [1936]. Proc. Soc. exp. Biol., N.Y., 35, 24. Rowlands, I. W. & Parkes, A. S. [1934]. Biochem. J. 28, Smelser, G. K. [1937]. Proc. Soc. exp. Biol., N.Y., 37, 388.

hypophysectomized rat. Marenzi & Gerschman [1934] studied six of the University and Royal Infirmary, Glasgow (Received 13 December 1937)

hypophysectomized rat. Marenzi & Gerschman [1934] studied six of the University and Royal Infirmary, Glasgow (Received 13 December 1937) 124 J. Physiol. (I938) 92, I24-130 6i2.492.5:6I2.I26 THE EFFECT OF HYPOPHYSECTOMY ON THE BLOOD CALCIUM AND PHOSPHORUS OF THE RAT BY A. B. ANDERSON AND E. G. OASTLER From the Biochemical Laboratory, Department