Nevertheless, the length of the infecund period varies greatly in. WITsCHI [1935] has shown that certain pituitary and allied preparations

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1 SOME EFFECTS OF THYROID AND GONADOTROPHIC PRE- PARATIONS IN THE FOWL. By ALAN W. GREENWOOD and J. S. S. BLYTH. From the Institute of Animal Genetics, University of Edinburgh. (Received for publication 13th October 1941.) WITsCHI [1935] has shown that certain pituitary and allied preparations can induce maturation and even full activity in the resting gonads of sparrows and finches. But while hormonal control appears to be involved in the non-breeding season, as it is in the cestrous cycle of mammals and the brooding cycle of birds, there is general agreement that the initiation and termination of the quiescent period are also related to certain external stimuli such as light variations. The researches of Bissonnette and Benoit indicate that these external agents require the intermediation of the anterior pituitary in order to exert their effect [reviewed by Marshall, 1936; Van Dyke, 1939, p. 58]. As a rule, the annual autumnal cessation of egg production in the fowl coincides with the onset of the moulting period, and it might be assumed that the two processes are mutually incompatible, or too expensive physiologically to be carried on together. These views are supported by evidence from an inbred line, maintained at this Institute, in which a number of females have occurred which laid for two consecutive years without a break; such birds either did not moult, or replaced only a small percentage of their feathers while laying at a reduced rate. Nevertheless, the length of the infecund period varies greatly in individuals, and often far exceeds the time required for the completion of feather replacement. In a group of 58 hens, drawn from hatches in three successive years, the length of the gap at the end of the first laying year was 84-8 days, with a standard deviation of days. The gap also lengthens with age: for the same 58 birds the mean difference between the dates of renewal of production in the second and third years is 2029 days (S.E., 3.89), and between the second and fourth years days (S.E., 4 03). Greenwood et al. [1940] showed that although individual birds appeared to have a genetically determined date on which production ended, second-year birds tended to cease laying slightly earlier than pullets. In the same communication it was also pointed out that the range of dates on which birds cease to 175

2 176 Greenwood and Blyth lay is very wide: it varied from July to December in the stock examined. If, therefore, external agents are basically responsible for the changeover from production to moulting, it is obvious that they do not act alone, but are dependent on the presence of certain suitable conditions within the organism itself. The present report deals with the results of some preliminary investigations of the effect of anterior pituitary extracts and allied preparations, and of thyroid, administered to hens in and about the moulting period. MATERIAL. The gonadotrophic preparations used were provided through the kindness of the Medical Research Council. They were Pregnant Mare's Serum Q2, 30 I.U. per mg. (P.M.S.); Chorionic Gonadotrophin U.P. 31, 100 I.U. per mg. (U.P.), and three extracts of anterior pituitary, viz. Sheep A.P. 117B, Horse A.P. 118B, and Pig A.P. 74D; the biological activity of the pituitary fractions is discussed by Chance et al. [1939]: "B" signifies the alkaline soluble portion of acetone desiccated tissue, and "D'" the fraction of "B" soluble in aqueous solution between ph 3 and ph 6. The thyroid used was the B.D.H. commercial product "Thyroideum." The hens were part of the normal breeding stock of Brown Leghorns maintained at the Institute, and were aged from 2 to 4 years. The gonadotrophic preparations were introduced in three ways: (1) injections of a 10 mg. per c.c. aqueous solution, (2) subcutaneous implants of pellets in which the excipient employed was glucose, and (3) pressure tablets. The thyroid was administered in gelatin capsules until they were no longer available, when it was fed as pills made up with a little wet mash. EXPERIMENTAL. Gonadotrophic Preparations.-For 15 days from 3rd September 1940 the five preparations were administered to a like number of pens of hens at the rate of 1 mg. per bird per day; each pen contained two age groups. The majority of females in this stock begin to moult in September and October, but, as mentioned in the introduction, the time at which individuals commence varies widely, and to control the tests by comparison with untreated animals would not be satisfactory. The best estimate of the date would be the average for previous moults, but as the number of these varies from one to three, according to age, the data have been taken from the last year only: averaged figures show that the group receiving P.M.S. ceased production ± S.D days after 1st September 1939; those receiving U.P., ± 34-62; Pig Pituitary, ± 19-18; Sheep Pituitary, 33*37 ± 20-02, and Horse Pituitary, 24*14 ± 8-47 days.

3 Effects of Thyroid and Gonadotrophic Preparations in the Fowl 177 Apart from the group getting Horse Pituitary, the moulting dates in each pen are widely scattered, and some would have been expected normally to fall within the period of the test. Actually 7 birds, distributed over 3 groups, had already stopped production, though 4 of them laid within a week of the beginning of the experiment (Table I.). Since the comb regresses during ovarian inactivity, comb size (length + height) was used to indicate the course of changes in this organ. The results are summarized in Table I. No reaction was detectable in the groups receiving U.P., Sheep and Horse pituitary extracts. TABLE I.-GONADOTROPHIC EXTRACTS TO LAYING HENS. 1 mg. daily for 15 days. No. Difference from original comb size at of 7 Days. 14 Days. 21 Days. 30 Days. birds. The mean No. of new birds moulting at Mean Mean Mean. Mean mm. mm. mm. mm. Days. Days. Days ' P.M.S. 7 *329 0* * t Pig ' ' Pituitary U.P Sheep * Pituitary Horse Pituitary * 7 days' treatment only. t Total. comb size of birds injected with Pig Pituitary rose slightly, then fell away normally after treatment ended, but neither egg production nor the number of birds entering the moult showed any abnormality. The only striking results came from the P.M.S. group: in it comb size increased noticeably during treatment; egg production ceased 1 to 5 days after injections began, except for one egg laid on the 15th day, and the hens passed immediately into a heavy moult. In half of this group the injections were not carried beyond the 7th day, and in these comb regression appears to have begun less abruptly. One bird was killed after 14 days' treatment and showed a normally active ovary with the three largest follicles degenerating. Although during treatment the hens continued to look as if in full lay, and possessed widely separated springy pelvic bones, none of them showed any inclination to nest after the 5th day. The conclusion that P.M.S. was the only substance which definitely influenced the course of reproductive activity at this dosage-level was VOL. XXXI., NO

4 178 Greenwood and Blyth further confirmed when all the moulting dates became available: the mean differences (with S.E.) between the dates in 1939 and 1940 in the various groups are as follows: P.M.S., ± 4-64 days; Pig Pituitary, -3 0 ± 3X52; U.P., 1P27 ± 5d18; Sheep Pituitary, ; and Horse Pituitary, 2-17 ± 4 09 days. Following the failure to maintain egg production beyond the normal moulting date, attention was turned to hens that had already ceased laying and the same substances administered, usually in heavier doses, in an attempt to bring them back to production. In an endeavour to obtain a prolonged subjection to the hormone, pellets made up with glucose, and compressed tablets of the pure substances, were implanted in the breast muscles in some cases. As no really constructive indications had come out of the original experiments this set took the form of "feelers " carried out on single individuals or small numbers of birds. Periodic measurements made it possible to graph the course of each bird's comb regression and regeneration, and breaks in the smoothness of the curve indicated the effectiveness of the preparations tested. The details are summarized in Table II., and where fluctuations in comb size were small, and might be due to errors in measurement, the results have been tabulated as "no definite effect." Here, as in the first tests, Horse and Sheep pituitary, and U.P. are without effect. P.M.S., even in higher doses, has less influence on comb behaviour than before; it may be capable of checking comb regression temporarily, but given alone is ineffective on fully regressed combs. When administered with thyroid there were indications of a renewal of comb growth. On the other hand, the indefinite action of Pig Pituitary in the earlier experiments is replaced by a marked positive effect when higher doses are used; irrespective of the state of the comb at the time of testing it usually responded to this extract by increasing in size. The increase, however, was always temporary, even under prolonged treatment, and though some of the birds appeared to return to a laying condition, with an enlarged comb and wide springy pelvic bones, they did not lay. The case of the bird autopsied suggests that maturation of the follicles, but no ovulation, was taking place. Thyroid Feeding.-The fact that the comb showed growth when thyroid feeding was combined with the P.M.S. injections opened the question of whether the former would be effective alone, and 5 birds were fed 0-2 g. daily for 4 days; the combs began to increase sharply during treatment; when treatment ceased the increase continued in 3 at a slower rate, and in 2 the combs regressed again. After 10 days, medication with the same daily dosage of thyroid was recommenced in these and 3 other birds; the combs increased in size rapidly and by the 12th day all except one looked and handled as if in production. Hormone administration was stopped in 4 individuals at this point;

5 Effects of Thyroid and Gonadotrophic Preparations in the Fowl 179 TABLE II. Given as No. Total of per birds 3. bird. Duration treatment. State of comb. * Effect on comb. P.M.S. Aqueous solution Glucose pellet Saline mush Pressure tablet mg ( 14 iplus { plus 1 g. 12 days g. thyroid 4 days thyroid in I. R. Min. Min. Min. in last R. Min. l 5 days f Min.{ None. Regression inhibited 5 days. No definite effect. None. 2 increased for 1 week, 2 no definite effect. Regression inhibited 1 week. No definite effect. None; tablets sloughed out in 9 days. Pig Aqueous solution R. Increase 5 days, then return Pituitary to rapid regression. 6 days' increase, then re R. gression. Killed 15th day: active ovary with full R Min. Min. Do. sized degenerate yolks. 6 days' increase, then regression. 3 plus 4 mg. oestrone in last. Do. (CEstrone ineffective.) (Pls 4 days Glucose pellet R. Regression inhibited 1 week. Pressure tablet Min. Increased 1 week; regressed.,,, R. & Min. None. Sheep Aqueous solution R. No definite effect. Pituitary Glucose pellet Min. None. Horse Aqueous solution R. None. Pituitary,,,, Min. No definite effect R. None. Glucose pellet R. None. Pressure tablet Min None; sloughed out in 9 days R. No definite effect Min. Do.,, I. Do. U.P. Aqueous solution R. None. * State of comb at beginning of test. I. = Increasing in size; R. = Regressing; Min. = Minimum, i.e. fully regressed comb. 1 laid the next day, and in the others the combs regressed. The 3 in which it was continued laid on the 16th, 19th, and 24th days, the first being one which had not had the previous 4 days' feeding; the combs levelled out a few days before they commenced to lay. Treatment was stopped immediately laying began, but as it seemed curious that

6 180 Greenwood and Blyth no deleterious effects resulted from its abrupt cessation 1 hen was given thyroid for a further 14 days; it also continued to lay normally. Though none of these hens had shown any indications that comb growth was being renewed before the test began on 1st January, some had already passed the end of their previous moult gap, and there was just a chance that they might have come into production without thyroid medication. A second series of 7 were therefore selected, which had still some time to go before reaching their previous year's date for onset of laying. All the combs again responded immediately to a daily dose of 0-2 g., and 4 birds laid between the 18th and 31st day of treatment, dates which were from 13 to 36 days earlier than in the previous year. But, except in the case of the hen which laid on the 18th day, a peculiar retardation in the rate of comb growth began about the 11th day. The only obvious variation which could be related to this phenomenon was that the gelatin capsules in which the thyroid had been fed became unobtainable and from this time onwards the powder was fed as a pill made up with a little wet mash. In two individuals the comb became stationary and one began to regress despite continued feeding. Another pair which were showing practically no growth were given the addition of Pig Pituitary for 4 days in diminishing doses-5, 3, 2, and 1 mg. Though this caused renewed comb growth in both, and in one it continued until laying commenced at 26 days, regression supervened immediately in the other; similar diminishing doses of P.M.S. a week later produced the same results in this refractory individual. In the remaining two birds the check was not so pronounced and the comb continued its upward trend until they laid. The impression gained from individual comb graphs was that growth in the thyroid-fed birds was accelerated beyond that in normals coming into production, and while the small numbers of the former, and the minor variations of treatment to which they were subjected, do not lend themselves to statistical examination, a rough comparison with untreated hens has been made. The steepness of the ascending comb curve varies from beginning to end, as in a normal growth curve, so that the larger the section considered the better, and though not all thyroid-fed birds were treated for quite this length of time, a three weeks' period has been chosen. The rest of the hens in the original 5 pens were used as controls, and since the smallest increases resulting from thyroid feeding were 13 mm., untreated birds from all pens, making at least this amount of growth during the period of each test, were used for comparison (Table III.). In addition, the mean comb change for each of the original pens (excluding thyroid-fed birds) is given for the period of the first test. While the mean increase in thyroid-fed birds of both series is considerably higher than that in their respective

7 Effects of Thyroid and Gonadotrophic Preparations in the Fowl 181 TABLE III.-AVERAGE COMB GROWTH FOR THREE WEEKS' PERIOD. No. of Increase birds. mm. S.E. Thyroid-fed birds, Series ± 5-02 Untreated birds showing an increase of 13 mm ± 2-04 or more. Original group receiving P.M.S ± 1-44,, Pig Pituitary ± 2-04,, U.P ,,, Sheep Pituitary ± 3-36,, Horse Pituitary ± 3-72 Thyroid-fed birds, Series ± 3-31 Untreated birds showing an increase of 13 mm ± 2-68 or more. controls, the difference is not statistically significant in either case. The impression that they had a higher comb-growth rate is therefore not substantiated, though it seems possible that a positive difference might emerge with larger populations in the compared groups. It can be noted, however, that although all the hens in Series 1 and 2 show an increase of 13 mm. or more, only 9 out of 61 controls did so during the period of the first experiment and 16 during the second. DIscUSSION. In considering the negative nature of the results with some of the gonadotrophic preparations the question of the adequacy of the dosage arises. Certainly U.P. appears to have proved inactive in all previous tests on birds [Allen, 1939, p. 1027], but Witschi and Keck [1935] found Hill's extract of Horse Pituitary very effective in stimulating the quiescent ovary of the sparrow. Unfortunately, as these authors measure their doses in rat units, this does not allow of comparison with ours. In another communication, however, Witschi [1935] illustrated sparrow ovaries stimulated to complete development by daily doses of 2 RU of hypophyseal extract or 20 RU of Pregnant Mare's Serum. In the present experiments P.M.S. was active in the laying but not the non-laying hen, while similar quantities of Horse Pituitary extract were ineffective under both conditions. The report of Chance et at. [1939] indicates that Horse Pituitary is more active gonadotrophically (as tested in the immature rat) than either those from sheep or pig, yet a consistent positive effect was obtained with the last preparation in our fowls. Though further experimentation with different dosages is necessary to examine the adequacy of the amounts administered, it appears as if a species difference in relative reactivity to the various substances is present.

8 182 Greenwood and Blyth The failure of ovulation in stimulated ovaries and its actual inhibition in the hen by gonadotrophins have already been noted by Bates et al. [1935] and Walker [1925] respectively. On the other hand, Witschi [1935] obtained ovulation in both sparrows and finches by injections of P.M.S. Our results in laying hens with P.M.S. and nonlaying birds with Pig Pituitary are in conformity with the former workers', and the alternatives arise that the opposing findings are due to species differences in reactivity, or that there is an optimum level of hormone capable of inducing ovulation, and lying within definite limits which were exceeded in all cases but Witschi's. Hartman's opinion [Allen, 1939, p. 630] that pre-ovulation cell differentiation is important for ovulation is in line with the latter suggestion. The further possibility of a qualitative difference in the substances used is beyond the scope of this paper. Earlier researches on the effect of thyroid on ovarian activity are scanty and contradictory: Evans and Simpson [1930] and van Horn [1933] reported increased gonad-stimulating potency in the hypophysis of the hyperthyroid rat female, but Smelser [1934] found no increased potency in those of either the hypo- or hyperthyroid rat. From matings following a 3-5-day treatment of the female rat with thyroid substance, Kraatz [1939] obtained increased litter sizes in April, October, and December, but a deleterious effect supervened in hot weather. In fowls, Crew and Huxley [1923] were unable to demonstrate any effect of thyroid feeding on egg production, but Crew [1925] reported an increase in the production of senile hens under similar treatment. Though ovulation was not obtained in all the thyroid-fed birds there is no doubt, from the behaviour of the comb, that this hormone had a definite stimulatory effect on the ovaries. The hens which failed to ovulate under continued treatment were all in the second series, where the change-over from administration in gelatin capsules to wet-mash pills was associated with a retardation in the rate of comb increase. While it has yet to be determined that this relation was not fortuitous, it may be that the gelatin possesses "augmentation" properties similar to those disclosed in other proteins, like casein and albumin, when added to gonadotrophins [Saunders and Cole, 1936]. A single bird, not belonging to the experimental series, proved refractory when fed thyroid in a wet-mash pill, but responded at once and laid in 10 days when the same daily dose was provided as a pill bound with a little jelly made from domestic gelatin. The other point of note in this part of the experiment was that the abrupt cessation of feeding, either at the time of laying, or in one bird a fortnight after, was without effect on the continuance of ovarian function. Since administration of thyroid hormone is usually considered to depress the activity of the animal's own thyroid gland, this suggests that it is acting here not so much as a direct stimulatory agent

9 Effects of Thyroid and Gonadotrophic Preparations in the Fowl 183 but rather as a releasing mechanism which allows the chain of events leading to ovulation to come into play. Nevertheless it has to be remembered that the initial stimulation was not enough, and that in birds which were not quite close to the point of production when treatment ceased the ovary returned to an inactive state. Thus it would have to be assumed that the hormone fed "carried" the factors producing ovarian activity until they were fully developed and became self-supporting. In the case of the only gonadotrophin which evoked a comparable effect-pig Pituitary extract-stimulation of follicular development occurred but stimulation to ovulation was absent. Presumably the action was a direct one on the ovary, and any effect on the bird's own pituitary would be of a depressant nature, so that even had complete ovarian function ensued it would have been sustained at best only for so long as treatment lasted. Birds brought into lay by thyroid feeding, however, continued to do so, from which it may be inferred that this treatment instituted a regeneration of the gonadstimulating hypophyseal activities which normally take place in the course of renewal of production. Benoit and Aron [1934], who found that thyroid stimulated the testes of young cocks and drakes, suggest that the different actions observed with various gonadotrophins may be due to differences in their thyreotrophic activity. In this connection it may be noted that Chance et al. [1939] found the Pig Pituitary the most active species thyreotrophically, which puts the assumption of a direct effect with this substance in question. With thyroid feeding there were no instances of birds remaining in an apparently laying condition without ovulation, but this does not entirely rule out the possibility that the thyroid gland was involved in the reaction to Pig Pituitary extract. As a basis for further investigation along these lines our conception of the general endocrine situation in regard to the non-breeding season in the fowl may now be briefly discussed. Though the present results point to an insufficiency of thyroid activity, at least in the later part of the moult gap, Giacomini [1924] and Zavadowsky [1925] have shown that a single large dose of thyroid substance will precipitate a moult in the fowl. Hill et al. [1934] found that moulting followed hypophysectomy, but that the colour and structure of the new feathers were indicative of a marked hypothyroid condition. Greenwood [1936] concluded that moult is an inherent characteristic of all fowls irrespective of the presence or absence of sex glands, but that a certain degree of sexual activity is instrumental in inhibiting the moult. Seasonal thyroid weights suggest that thyroid activity is greater during the productive than the non-productive part of the year [Galpin, 1938], and from her figures it appears that thyroid weight is at a maximum (i.e. its activity is lowest) in the non-laying bird. On the other hand, Greenwood and Burns [1940] have demonstrated that the practically

10 184 Greenwood and Blyth continuous moult in the castrated bird is most intense at a time when thyroid secretion is regarded as approaching its maximum in the intact bird Ṡome clue to the meaning of these paradoxical observations may be found if the possibility is considered that the conditions essential to the onset of the moult are not necessarily the same as those which obtain once it is under way. It may be, as the work of Giacomini and Zavadowsky suggest, that the impetus to plumage change is derived from a brief release of an increased thyroid secretion, or, what would have the same effect, the normal secretion may become "available" for this purpose through the cessation or slackening of the inhibiting action of the gonad. The first supposition might be tenable in regard to Hill and Parkes' experimental birds, or to hens, where sexual activity ceases abruptly in the autumn, but the slower moult of cocks and capons, combined with the continuation of sperm production at a reduced rate in the former [Greenwood, 1936], suggest that the second postulation is more probable. Such a switch-over in the demands made on the hormone from one part of the organism to another in no way disagrees with the deduction from the study of gland weights, and from the present experiments, that it is a season of low activity for the thyroid gland. Indeed, it could conceivably have resulted from the lowering of the hormone-level below that adequate for gonad function, though the more obvious explanation of the cessation of gonad activity would be a deficiency of gonadotrophins. In this connection it has further to be considered whether lowered pituitary function is concerned solely with its gonadotrophic activities, or whether its thyreotrophic action is also involved, and so responsible for the change in level of thyroid secretion. Whatever may be the exact relationship between gonad, pituitary, and thyroid such considerations suggest that in the fowl the thyreotrophic action of the pituitary has an important reciprocal counterpart in the influence of the thyroid on that gland, and through it on the functioning of the gonads. SUMMARY. 1. Extracts of Horse, Sheep, and Pig Pituitary, Pregnant Mare's Serum and Chorionic Gonadotrophin were administered to hens prior to and during the moulting period. Dried thyroid powder was fed during the non-productive phase. 2. Injections of P.M.S. to laying hens resulted in cessation of egg production within 5 days, and the onset of a heavy moult. The combs increased in size during treatment. Apart from a slight increase in comb size in the group receiving Pig Pituitary extract, no reaction to the other gonadotrophins could be detected at the dosage-level employed.

11 Effects of Thyroid and Gonadotrophic Preparations in the Fowl In non-laying hens P.M.S. had little effect; Pig Pituitary appeared to cause a temporary reactivation of the reproductive organs, but in no case did ovulation occur. The other gonadotrophins were again inactive. 4. Consistent symptoms of ovarian stimulation resulted from the administration of thyroid, and a number of birds resumed egg production following this treatment. Variations in the degree of response obtained appeared to be related to the media in which this hormone powder was fed. REFERENCES. ALLEN, E. (1939). Sex and Internal Secretions. Bai11iere, Tindall & Cox, London. BATES, R. W., LAHR, E. L., and RIDDLE, O. (1935). Amer. J. Physiol. 111, 361. BENOIT, J., and ARON, M. (1934). C.R. Soc. Biol. 116, 221. CHANCE, M. R. A., ROWLANDS, I. W., and YOUNG, F. G. (1939). J. Endocrinol. 1, 239. CREW, F. A. E. (1925). Proc. Roy. Soc. Edin. 45, 252. CREW, F. A. E., and HUXLEY, J. S. (1923). Vet. J. 79, 343. EVANS, J. M., and SIMPSON, M. E. (1930). Anat. Rec. 45, 215. GALPIN, N. (1938). Proc. Roy. Soc. Edin. 58, 98. GIACOMINI, E. (1924). Report Second World's Poultry Congress, 45. GREENWOOD, A. W. (1936). Proc. Sixth World's Poultry Congress, 1, 265. GREENWOOD, A. W., BLYTH, J. S. S., and GALPIN, N. (1940). J. Agric. Sc. 30, 202. GREENWOOD, A. W., and BURNS, M. (1940). Quart. J. exp. Physiol. 30, 163. HILL, R. T., CORKILL, A. B., and PARKES, A. S. (1934). Proc. Roy. Soc., B, 116, 208. KRAATZ, C. P. (1939). Proc. Soc. exp. Biol. N. Y. 40, 499. MARSHALL, F. H. A. (1936). Phil. Trans. Roy. Soc., B, 226, 423. SAUNDERS, F. J., and COLE, H. H. (1936). Proc. Soc. exp. Biol. N. Y. 33, 505. SMELSER, G. K. (1934). Anat. Rec. 60, Suppl. 13. VAN DYKE, H. B. (1939). The Physiology and Pharmacology of the Pituitary Body, Vol. II. University of Chicago Press. VAN HORN, W. M. (1933). Endocrinol. 17, 152. WALKER, A. T. (1925). Amer. J. Physiol. 74, 249. WITSCHI, E. (1935). Wil-son Bull. 47, 177. WITSCHI, E., and KECK, W. N. (1935). Proc. Soc. exp. Biol. N.Y. 32, 598. ZAVADOWSKY, B. (1925). Endocrinol. 9, 125.