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Studies on Sex Differentiation in Fowls

1925; The Company of Biologists; Volume: 2; Issue: 4 Linguagem: Inglês

10.1242/jeb.2.4.439

1477-9145

G. F. Finlay,

Comparative Animal Anatomy Studies

For this investigation the fowl was chosen because of its marked sex dimorphism and its ability to withstand operative interference. The work was commenced and carried to an advanced stage at the School of Agriculture at Cambridge; later the birds were transferred to the Animal Breeding Research Department, Edinburgh. I wish to thank Dr F. H. A. Marshall, Dr F. A. E. Crew, Professor R. C. Punnett, and Mr S. T. P. Strangeways, for helping me in various ways during the course of the experiments. The histological part of this study has been carried out by Mr A. W. Greenwood and is reported in a separate paper.The funds for this work were provided largely from grants from the Ministry of Agriculture administered through the Animal Nutrition Institute, Cambridge. The writer also received grants as a holder of the Walter and Eliza Hall Veterinary Research Fellowship of Sydney University.Morgan has suggested that in the male Sebright, which has feathers of the henny type, the inhibitor of cocky feathering is located in the luteal cells of the testis of this breed. The logical conclusion, if one were to accept this hypothesis, would be that there must be more than one male sex-hormone, or that this internal secretion of the testis is not of a fixed character. In order to test this point attempts were made to graft testis from males of the hen-feathered variety into males with the more common cocky plumage to see if the cocky plumage of the latter would be inhibited. Attempts were also made to graft testis from the cocky feathered birds into the castrated henny feathered males. Unfortunately the grafts did not take. This may have been due to the fact that the birds were fully grown with the testis in a state of active spermatogenesis, and hence unsuitable for grafting ; or possibly to the fact that the birds were of rather heterozygous constitution. This experiment is only mentioned here to show that it formed one of the series, and also because it indicates what are unfavourable conditions under which to attempt gonad transplantation.The guinea-fowl has feathers of the henny type in both sexes. Two male birds were castrated when just under 6 months old to see if these birds would develop feathers of the common cocky type, when the influence of the testis was removed. At the time of the operation the testes were about the size and shape of haricot beans and were removed without difficulty. On post-mortem examination the castration was shown to have been complete. The birds remained in good health and feather until they were killed about fourteen months later. During this period moulting seemed to be going on fairly continuously, for loose feathers were always to be seen lying about, and no radical moult, as in the other fowls, took place. No change in the character of the new feathers was apparent. This experiment, therefore, indicates that the henny feathering of the male guinea-fowls is funda-mentally different from that of the Sebright bantam and other hen-feathered varieties of the domestic fowl.Previous workers in this field have usually operated upon chicks during the actively growing period, hence it has been somewhat difficult to know just what the influence of the gonads on sexual differentiation during the early period of development has been.One of my earlier cases is worth describing because it has some bearing upon the theory of gonad differentiation. This bird was a male chick, exact age unknown, but was about 6 weeks to 2 months old. The sex was fairly evident at this stage. It was castrated on both sides, and the chopped ovary from a young female inserted into the abdominal cavity. This bird (fig. 1, Plate VI.) showed the influence of the graft plainly. As regards feathering it was henny except for slight sickles in the tail. Its comb was very like that of a hen. The spurs were typically masculine. In behaviour it had some of the characteristics of a hen, though it was reported to me that it was seen occasionally to tread other hens. It never crowed, but was in the habit of singing. It did not behave like an asexed type, though it was difficult to say that it was definitely feminine. It would be more correct to describe it as abnormally sexed.It was killed when 11 months old. There was no trace of testis, but a flat ovary about 1 inch by 12 inch was growing in the mesentery. This case indicated that the ovary stimu lated comb growth, but not to the same extent as the testis would have done if it had been left to function. The ovary (fig. 2, Plate VI.) is apparently normal, and this point is of some interest in the light of the ovarian transplantations which are described later.The castration and other experiments performed on other types at this stage are not described, because they did not give any important information on the questions involved.I came to the conclusion that much better tests could be made if the operations were done on chicks just after hatching, in order to control the interaction between gonad and soma during practically the whole of the growing period. In general the first third of the growing period had been neglected by previous operators. As a preliminary experiment ten chicks about 4 days old were operated on in various ways—chiefly castrations and transplantations of gonads. All of the ten chicks survived till maturity, and though the operations were mostly failures, they indicated the possibilities of a valuable technique. The failures consisted mainly in the regeneration of small pieces of testes left behind, which grew into nearly full sized testes. Several, however, were completely castrated on one side. One case merits further description.A Columbian Wyandotte female chick was operated on when 4 days old It was anæsthetised and its body opened through the last intercostal space of the left side, and four testes from two chicks of the same age were inserted through the intercostal opening. When about 5 months old it was noted that this bird had the typical body and plumage of a pullet, but that it possessed a coarse masculine comb. The numerous cockerels about the place paid little or no attention to this bird, while they favoured other pullets. When about 7 months old it had every appearance of being in laying condition. It died when 8 months old, due to the stoppage of the oviduct with an abnormally large egg. There were also several yolks higher up the oviduct.Examination revealed that in addition to the normal ovary a nodule of testis about 1.5 mm. in diameter was projecting into the abdominal cavity from the site of the operative wound. This was found to be swarming with actively motile sperm, and on section showed normal testis tissue.The partial success achieved with the young chicks in the foregoing series led me to conclude that it would be profitable to continue this plan of experiment under more critical conditions. In particular it seemed desirable to use pure-bred Brown Leghorns, which, because of their relative homozygosity, would give more accurate comparative results and also because this breed probably shows more marked sexual differentiation than any other breed in this country. Pure-bred typical Brown Leghorn pullets were mated with an exhibition type cockerel. Pullets were used in order to obtain a maximum number of eggs from each female within a limited time. This made it possible to hatch out fairly large families of full brothers and sisters. It was perhaps a mistake to use pullets, for chicks from pullets are generally considered by poultrymen to be rather weakly. Certainly many of the chicks hatched were cripples and could not be used. Trapnest records were kept so that the relationship of the offspring could be determined. There are indications that grafting operations are more successful when done between closely related individuals, so it was planned to graft between brother and sister as far as possible. Chicks from a second pen, in which a Brown Leghorn cockerel was mated to Silver Duckwing Leghorn pullets, were also used in a few cases.Chicks were successfully operated on as early as 2 days old. The age recorded in all cases has been reckoned from the twenty-first day of incubation. These chicks hatched either at the twentieth or the twenty-first day, and for the operation it was found to be more convenient to wait until they were 3 days old. During the first two days the yolk sac is large and easily ruptured. By the third day also the chicks are active, strong, and accustomed to the brooder.The incubation was so arranged that groups of full brothers and sisters would all hatch on a certain day. These would be all taken to the operating room together and the transplantation experiments performed as rapidly as possible, so as to prevent undue exposure of the gonads.All possible combinations of gonad with soma were made. The operations were performed without assistance, for it proved a simple though delicate and tedious procedure. The difficulty of anæsthetising as reported by Goodale and others was not experienced, though it was found advisable to have the subject thoroughly under before opening the abdominal cavity, as the inhaled ether and air escapes through the wound.The only instruments used were a very fine pointed pair of scissors, the smallest silver probe, a special pair of dissecting forceps with very fine ends turned almost at right angles, and suturing material. The most important item was the light; direct sunlight was found to be best, while artificial light was quite useless.The chick was placed in a block of wood about 6 inches by 3 inches by I inch which was hollowed out in the shape of the chick’s body, with grooves for the head, neck, and legs. The wing was pinned above the body and the down plucked from the costal region. Sterile instruments were used, but rigid antiseptic measures were not taken, the skin being merely damped with warm water for the purpose of pushing aside the remaining down. The abdominal cavity was opened with the scissors through the last intercostal space, the air sac was cut through, and the gonad exposed.In order to make provision for gonads which might be removed from a chick’s body, several watch-glasses containing Ringer’s solution were placed in covered Petrie dishes which contained a little water, and the whole kept warm in an incubator.The removal of the testis had to be done very slowly and carefully. If the forceps were placed above the testis and pulled, the organ would come away, but a tiny fragment would be left, while the site would be obscured by hæmorrhage, It was found best to start at one end, and gently free the testis from its attachment bit by bit. To remove the opposite testis, the chick had to be turned over and a similar operation performed from the opposite side. The wounds were closed with two or three of the finest silk sutures.The removal of the ovary when the chick is but 2 or 3 days old is a matter of considerable difficulty. The area of attachment of the ovary had to be carefully crushed with the fine forceps, and after waiting some time to allow coagulation, the ovarian tissue was carefully picked off. By working in direct sunlight one could see when all the ovarian tissue was removed. As the sex of the chick was not known until its body cavity was opened, it was first put in the block with the left side uppermost, then if it proved a female, the chick was already on the side convenient for ovariotomy.Any gonad tissue which was removed was placed in the warm Ringer’s solution. When it was desized to graft a gonad into the chick’s body, the site just anterior to the kidney was used. This is vascular, and there are strands of connective tissue there to help immobilise the graft. Another advantage is that the growing gonad can project into the abdominal cavity and take up a position quite close to the normal gonad site. The anterior extremity of the kidney-is in close proximity to the upper part of the last intercostal space.The chicks were placed close to the fire immediately after operation to recover from the exposure. A few hours later they seemed to show little or no ill effects from the operation. Subcutaneous emphysema, however, invariably followed, the chick sometimes attaining almost twice its normal size. This, however, did not seem to cause any inconvenience, for such chicks were quite active and healthy. The skin invariably healed by first intention. Chicks which survived the operation, that is those which were not killed through hæmorrhage during the operation, almost invariably survived till maturity.As it was desized to study the reactions of soma and gonad, in all eight experimental groups were formed. These were as follows:(1) Testes left; (2) castrated; (3) castrated and ovary engrafted; (4) testes left and ovary engrafted.(1) Ovariotomised and testis engrafted ; (2) ovariotornised; (3) ovary left; (4) ovary left and testis engrafted.During the growing period it seemed as if the experimental birds were conforming to these groups, and a specimen of each group was selected and its portrait painted. There were, how ever, some interesting exceptional cases which will be described in detail later. Though not strictly accurate the coloured plates show the eight typical classes. The birds with silver hackles and saddles are from the second pen in which a Brown Leghorn was mated with Silver Duckwing Leghorns. However, except for this silver coloration, these birds are of the same type and size as those from the first pen.Bird No. 4 (Plate I.) is the only one which is not a good specimen of its class. The fact that this bird is only partially henny in its plumage was thought to be due to the presence of but a small quantity of engrafted ovary. On post-mortem examination this was found to be the case. Other cases support the view that if a certain amount of ovarian tissue is present, even in the presence of testis, a nearer approach to complete hen-feathering will result.Some normal males were reared as controls. In addition in two cases the vas deferens was resected, and in one case castration was performed and testis grafted subcutaneously. In all these cases normal development of the male characters followed.No. 1 (Plate I.) can be regarded as a typical Brown Leghorn cock. It was operated on when a few days old and it was noted that it was a male. Its testes were left intact and it was stitched up and left as a control. The size of this bird in relation to that of the representatives of the other seven classes can be seen by reference to the coloured plates, for in all illustrations care was taken to depict the birds in their relative sizes. Attention is drawn to the general body shape — espedally the constricted pelvis, the stance, strong but blunt spurs, large upright comb, large wattles and ear lobes, and the compact black and gold male plumage.No. 9 (Plate III.) was castrated when 11 days old and three testes from half-brothers grafted subcutaneously on the left side and two testes, also from half-brothers, on the right side. This bird developed as a normal cock. The nodules of testes could be felt on the lift side. When a year old it was killed and examined. Subcutaneously, on the left side, there was a testis nodule about one quarter the size of a normal testis, and also a smaller nodule about the size of a pea. Motile sperm were observable in the larger graft There was no trace of any testicular tissue inside the body cavity. The vasa deferentia were well developed, but were of course empty. I judged from this case in comparison with many others that the subcutaneous position is not so favourable for testis growth as inside the body cavity. Vascularisation is not so easily established, and the graft is constantly subject to slight injury. However, as regards spermatogenesis, these subcutaneous grafts were similar to the others.No. 10 (Plate III.) when 11 days old was opened and the left vas deferens, close to its junction with the testis, seized with the forceps and ruptured. This bird was typically cocky, but was slightly smaller than most of the males. It was examined when a year old. The right testis was of course normal; the left also appeared quite normal, and though it was only about three-quarters the size of the right, this is quite within the normal limits of variation. _The right vas was full of milky seminal fluid. The left was not connected to the epididymis, and though otherwise well developed it appeared very different to the right vas owing to the absence of seminal fluid. From this and other cases it would appear that a testis can function normally-at least for some months-without a duct. The sperm collect in a compact mass in the lower end of the tubules, while the more fluid secretion is evidently reabsorbed.No. 11 (Plate III.) is given merely as illustration of another normal control cock. It was not operated on at all. It has the same characteristics as the foregoing birds.It is apparent that testis can grow, function, and produce full masculinity whether it be in the body cavity or placed subcutaneously. The latter position, however, does not appear to be as favourable for growth of this organ as the body cavity. Case No. 9 (Plate III.) suggests that about one-seventh of the normal amount of testicular tissue is sufficient for full development of the sexual characters.In this group I attempted to perform complete castration in every case. However, this is very difficult in young chicks, and one must expect that in some cases a fragment of testicular tissue will be left. Such cases, however, give valuable indications as to the amount of testis required for full masculine development; and, I think, show that the reaction between soma and gonad is a quantitative one.No. 2 (Plate I.) was completely castrated when 7 days old. In size it was much the same as the normal males, and, contrary to what one might expect, the skeleton was similar. The spurs, however, differed somewhat, in that they were longer and sharper. It is possible that this was partly due to the inactivity of the bird, for in normal males the spurs are worn blunt by fighting and scratching. The plumage was of the same general type as the foregoing, but the feathers were considerably longer and not so compact. This was particularly noticeable in the hackles, saddles, and tail feathers. The comb and wattles remained infantile. The stance was somewhat different, this bird not having the upright proud carriage. This perhaps is due to the fact that the capon has not the same weight of head furnishings and can therefore adopt a more horizontal posture without changing the centre of gravity; and to the fact that he has not the sex pride which impels the normal male to assume an upright commanding position.No. 12 (Plate III.) was operated on when 3 days old. Complete castration was attempted, but examination when 18 months old showed that a piece of testis a little over 1 cm. in diameter and weighing 2.7 gm. was present. The spurs were sharp like those of a typical capon. The comb and wattles were cocky but were somewhat smaller than those of the normal cocks. The plumage was very similar to that of a typical capon, though it may have been somewhat more compact. As regards behaviour, this bird crowed and would tread hens, but the fact that its spurs were not worn, and that it did not fight or exhibit marked sexual instincts, showed that it was incompletely sexed.No. 13 (Plate III.) was also castrated when 3 days old, but a small piece of testis, about half the size of the piece in No. 12, was found when it was killed 18 months later. The comb and wattles were considerably smaller than in the foregoing, but this bird also would crow and tread hens, though its sexual instincts were still more suppressed (Plate III.).No. 14 (Plate III) isanother example of a, omplete capon, the castration having been performed when the bird was 4 days old.The photographs of Nos. 12, 13, and 14. were taken when these birds were 50 to 51 weeks old; and as they were all in good condition, the photographs give a very good indication of the quantitative relation between amount of testis and comb size. Previous experience had led me to always expect that when less than one-eighth of the normal amount of testis tissue was present, there was a degree of development of the head furnishings in quantitative relation to the size of the testis nodule left. More than that amount caused normal masculine development. A comparison of the measurements of the head furnishings of these fowls is given in the table of measurements. As regards behaviour, these cases indicated that manifestations of masculinity also varied according to the amount of testis present.With this group of birds complete castration was attempted, and at the same time some ovarian tissue was engrafted. This proved to be a particularly interesting group, because the ovary grafts either developed as ovarian tissue or became transformed into testicular tissue. Furthermore the time relationship gave some indication of the period in development when the ovary could be changed by environment. The group also indicates to some extent the degree to which feminisation of the male can be effected.No. 3 (Plate I.), when nearly II months old, and in Plate IV. when 7 months old. It was castrated when 6 days old, and two ovaries from sisters engrafted, one being put in each side. This bird was of the same size as the other males, and it possessed spurs like those of normal cocks. Its skeleton was like that of a cock, the pelvis being of the constricted male type. However, the secondary sexual characters were henny. The plumage was of the colour of a hen, the breast being salmon instead of black, while the rest of the plumage was the sombre brown of the hen. The back feathers, however, showed slight masculine tips, while the tail was larger than in normal hens. As can be seen from the photograph at 7 months the head furnishings are very much the same as those of pullets of that age.As regards behaviour and mentality, it was difficult to draw exact conclusions. This bird had not that lack of pride which characterises asexed birds. On the other hand it did not make any sexual advances towards other fowls, nor did it receive them. I suggest that it would be fairly accurate to describe it as having the general mental characteristics of the male without the aggressiveness and desize to lead which develops with the male secondary sexual characters. But this and similar birds had not the timid, shy disposition of pullets. When attacked it would repel its opponent vigorously and without fuss. These birds were the most peaceful in the pens, but they would resist attack.When killed just after the painting was made, examination revealed a normal ovary on the left side, just anterior to the kidney. It appeared to be about half the usual size, and there were yolks in all stages of development, the largest being almost ready to escape. The germinal spot was present. Some yolks had evidently already escaped, judging from the presence of free fluid yolk in the abdominal cavity. On the right side was a small undeveloped ovary.On the left side, attached to the last intercostal muscles, was a testis nodule about 1.5 cm. in diameter. It may be assumed that this nodule had the effect of causing a slight tipping of the feathers, and of stimulating comb growth to a slight extent. It is interesting to note, however, that this testis nodule did not cause masculine comb development up to 7 months of age, as can be seen from the photograph on Plate IV., nor did it evoke the sexual desizes of the cock.No. 15 (Plate III.) was castrated when 2 days old and an ovary from a sister was engrafted into each side. This bird grew up as a vigorous cock. The hackles were rather shorter than in a typical cock; the back feathers and saddles intermediate; while the comb and wattles were fully masculine. In behaviour this bird was fully cocky; it crowed often and would tread hens normally.Post-mortem examination, when a year old, showed a small nodule of testis the size of a pea on the left testicular site. This was quite insufficient to cause masculine secondary sexual characters to develop to any noticeable degree ; and if this had been the only gonad tissue present, the bird would have been of capon type, with long loose feathers, small comb and wattles, sharp capon’s spurs, and with the temperament of the capon.The cause of its masculinity was the grafted ovaries. A large grafted gonad, almost the size of a normal testis, was found attached to the anterior part of the left kidney on the site selected for grafting. Near this, attached to the intercostal muscles, was a small amount of gonad tissue about the size of a pea, and on the corresponding part of the right side was a third small growth. Multiple growths on the left side could be expected as the ovarian tissue was usually in several pieces when inserted. These three grafts all showed the same structure, viz, that of testicular tubules in a fibrous stroma. It differed from normal testis in that the tubules were either immature or irregular atrophic tubules, and there was much dense fibrous connective tissue. Normal testis is soft to the touch; normal ovary is tabulated; these engrafted gonads were smooth and tough and markedly different from normal gonads.There can be no doubt that the secondary sexual characters were caused by the large engrafted gonad. The adult plumage seen in the photograph was developed when the bird was about 6 months old. It may have been that at that time there were some ovarian elements present to cause the slight inhibition of the cocky type plumage, for cystic follicles projected from the surface of the graft. The comb and wattles and sexual instincts, however, were fully masculine.No. 16 (Plate III.) was castrated when 4 days old, and on each side an ovary from a sister was engrafted. This bird had similar characters to the foregoing, as can be seen from the illustration. Post-mortem examination showed a regenerated testis, about one-third normal size, and on the right and left intercostal sites were small ova-testis like growths. The amount of normal testis present was sufficient to account for the masculinity of this bird. The grafted ovaries proved to be of the same type as those in No. 15, viz., testicular tubules in a dense fibrous stroma. There can be no doubt that when the adult plumage of this bird was growing the secretion from the grafted gonads had some inhibiting effect.No. 17 (Plate III.) was castrated when 4 days old and about three-quarters of an ovary of a sister put in the left kidney site. The photograph of this bird was taken when it was just under 6 months of age, and though the skeleton had attained full size, the secondary sexual characters were not fully developed. In general this bird resembled a capon with a small nodule of testis. Its comb was erect, though but half the normal masculine size for that age. The plumage did not show greater inhibition than could be expected for a cockerel of that age. This bird died when 43 weeks old, but apart from feather growth showed no important change from the photograph.Examination revealed that it had been completely castrated, and that the engrafted gonad was but 3 mm. in diameter. It is not safe to draw many conclusions from this case, but the bird is described, for the grafted gonad proved to be another case of ovary which contained tubules similar to those of immature testes. In ill-health gonads, especially testes, become greatly reduced in size. The evidence that the gonad secretion bears a quantitative relation to the secondary sexual characters is taken from birds in perfect health and under standard conditions.No. 18 (Plate IV.) is a better case but is very similar to the preceding. When 4 days old it was castrated. An ovary of a sister was taken and three-quarters of it grafted into the left side and one-quarter into the right side. This bird died when 31 weeks old. The photograph shows it when about 24 weeks old. In general this bird had cocky secondary sexual characters. It had a black breast, but the back, saddle, and tail feathers were slightly restricted. The comb and wattles were about three-quarters full size; but as this bird was not very strong, this could be expected even if it had normal testes.Examination showed that the larger piece of ovary, which had been grafted into the left kidney site, had grown and was about 1 cm. in diameter. On the right side there were three small gonad growths, which al1 together were some-what smaller than the left graft. These grafts contained testicular tubules of the same type as that previously described. There was no trace of any of the bird’s original testicular tissue. It should be mentioned that the testicular growths derived from ovarian grafts are of a very different type to that which grows from fragments of original testis. The former are tough tissues due to the large amount of fibrous tissue, while the latter are normal testis growths. In any case histologically it is not possible to confuse the two types of growths.There can be no doubt that the masculine secondary sexual characters of No. 18 were due to the grafts. The slight inhibition of fully masculine plumage may have been due to some ovarian elements which remained till the time of moult.No. 19 (Plate IV.) was completely castrated when 11 days old, and a half ovary from a sister engrafted into the left kidney site. This bird was quite healthy till about 5 months old, when it developed partial paralysis. The photograph shows it at 6 months of age. It died from roup when 10 months old.Its size was that of a cock. Its spurs were typically cocky. Its plumage, comb, and wattles were typically henriy. Its temperament was similar to that of No. 3, that is, it was a peaceful bird unless attacked, when it would reply with vigour even though crippled. I