The Effect of Testis Hormone on the Preservation of Sperm Life in the Vas Deferens of the Fowl

In birds, however, the testes are normally carried in the body cavity, the temperature of which, moreover, is several degrees higher than that of mammals. It is clear, therefore, that in birds the same heat regulating mechanism does not exist. Moreover, the male germ cells have relatively a much longer life in the female tract, at least in the fowl, since it is well known that little decrease occurs in fertility during the first week or 10 days after the removal of the cock from the breeding pen, but thereafter it falls rapidly although fertile eggs may be secured as long as 19-20 days later (Elford, 1916; Curtis & Lambert, 1929). The author has secured fertile eggs 18 and 21 days after a single artificial insemination, while Crew (1926) reports an extreme case where 32 days elapsed between removal of the male and laying of the last fertile egg. Obviously temperature does not have the same deleterious effect in birds either on the process of spermatogenesis or on the life of mature spermatozoa. Unfortunately, the question of whether the bird possesses other regulating mechanisms known to exist in mammals cannot be settled merely by recourse to existing evidence.

Moore (1928 a) showed that sperm in the isolated epididymis of the rat will survive for 30–40 days and in the guinea-pig for 60–70 days, providing one testis is left intact to add its secretions to the body fluids. When both testes are removed and the sperm in the epididymis denied egress by ligatures, the length of survival is reduced in the rat to 17 or 18 days and in the guinea-pig to 23 days. The presence of a cryptorchid testis, or a living engrafted testis fragment, whether possessing functional or degenerate germinal epithelium, will maintain the normal life of sperm in the isolated epididymis and Moore concluded that the testis hormone is responsible for the maintenance of sperm life. As a result of this and confirmatory evidence supplied by further studies (Moore, 1928 b; Moore & McGee, 1928) the “spermatozoon motility test” became a standard test for detecting the presence of testis hormone.

The fact that, in the fowl, sperm can survive and maintain their functional capabilities in the female tract for a period of 3 or 4 weeks makes highly problematical the question whether their life is in any way supported or prolonged beyond this age in the excretory ducts of the male by virtue of the presence of a functional testis. Accordingly, experimental methods were resorted to in order to answer the question.

Young mature cocks, 8–9 months old and weighing between 1800 and 2200 g., were the experimental animals. The majority of these were inbred Exhibition White Leghorns, although a few Black × White Leghorn crosses were used. All birds were healthy and vigorous and were bred and reared on the Central Experimental Farm, Ottawa. The experiments were performed in each of 3 years (1933, 1934 and 1935) during the months of February and March. In each of these years the birds were divided into two groups and wherever possible full and half-brothers were paired and placed in opposite groups. About half the birds in group I were unilaterally vaso-ligated; the distal end of the duct just previous to its entrance into the wall of the cloaca being tied off. The remainder of group I and all of those in group II were bilaterally vaso-ligated at the same distal point. Operations were carried out aseptically under ether anaesthesia. The ligations were accomplished through incisions in the ventral body wall just anterior to the caudal extremity of each pubic bone. The coiled vas, white and distended with sperm, is easily discernible lying closely attached to the back. It was carefully freed with the aid of a hook and sharp-pointed tweezers from the surrounding peritoneum. Great care was taken to separate the vas from the ureter, a smaller, more opaque straight tubule lying just medially and very close to the vas. When freed of its attachments for a distance of about 1 cm., it was lifted with the hook and a ligature of silk placed around it. The body wall and skin were sutured separately and the operation repeated on the opposite side. The birds were restricted to water and 2 days later the unilaterally ligated birds were unilaterally castrated, the testis on the ligated side being removed; the bilaterally ligated birds in group I were again subjected to bilateral ligation of the vas deferens, this time at the proximal ends; while the birds of group II were totally castrated. The procedures were essentially as follows :

The bird is restrained on its side by means of cords around the shanks and the base of wings, and anaesthetized. The feathers are plucked just in front of the upper thigh and an incision about $112$ in. long is made through the skin and body wall immediately behind the last rib. The mesentery enclosing the air sacs is ruptured, exposing the testicle which is then freed of its outer envelope with tweezers and fine scissors. The epididymis in the fowl is a very small organ broadly and intimately connected to the hilus of the testis, and lying in close and intimate association with the adrenal anteriorly and the kidney dorsally. According to Gray (1937) it consists of rete testis, ductuli efferentia and epididymal tubules proper. In addition, the author has often noticed in histological section small areas of testicular tissue containing only Sertoli cells within their tubules. It seems likely that these areas give rise to the testis nodules often found after a supposedly complete castration and are probably responsible for most of the “slips” resulting from gonadectomy in the fowl. The nature of the epididymis makes a successful testis ligation difficult. However, it has been found that a double or triple ply of silk placed around the testis attachment so as to enclose in the ligatured portion as much of the epididymis as possible, will if tightly tied prevent all but a mild haemorrhage. The testis is excised with fine curved scissors and the ligated stump heavily cauterized to destroy as much of the epididymis as possible, thus minimizing the chances of regeneration of any epididymal testis nodules. The body wall and skin are sutured separately. The operation is repeated on the other side in the case of total castrates.

The bird is similarly held and the skin and body wall similarly incised. The mesentery is ruptured exposing the testicle, epididymis and vas. The latter is carefully freed from the kidney, which it overlies, at a point about $12$ in. from the epididymis. It is doubly ligated and a piece about $18$ in. long excised between the ligatures. The wound is closed as above, and the operation repeated on the opposite side. The points of ligation are illustrated in Fig. 1.

Due principally to respiratory failure, resulting from the necessary rupture of the abdominal air sacs, especially where this had to be done on both sides as in cases of complete castration and bilateral proximal vaso-ligation, mortality was high, amounting to 40 %. In addition a few birds were lost following the operation due to extensive subcutaneous emphysema, similar to that described by Domm (1931) and due, as he describes, to the escape of air from the ruptured sacs, through the body wall incision, and its accumulation in the subcutaneous spaces. Mild cases of subcutaneous emphysema are common following castration in young cocks, but the birds quickly recover. In adult males, however, the accumulation of air may become so extensive as to separate completely the skin from the muscular tissue over most of the body. The air accumulations extend even into the neck and head region and seriously interfere with respiration. A total of 120 birds were operated on; after allowing for mortality, and discards due to incomplete castration and general debility following operation, sixty-two, comprising thirty-one in each group, recovered to provide pertinent data.

The surgical procedures outlined above provided one series (group I) in which the formed sperm were imprisoned in the vas, either on both sides in the bilaterally vas isolated birds, or on one side in those unilaterally castrated; in the former both testes remained intact and functional, while in the latter one testis remained undisturbed. The procedures provided in group II a comparable series of vas isolated castrates, in which periodic post-operative sperm examinations could be made and compared with the results of similar examinations in group I. If the internal secretions of the testis provide an environment conducive to a prolonged life of mature sperm, as is the case in mammals, then sperm in the isolated vas of group I birds should retain the capacity for movement, on suspension in physiological saline, for a longer period of time than those in group II.

At this point it should, perhaps, be mentioned that the epididymis in the domestic fowl is an extremely small organ and cannot, because of the restricted nature of its capacity, act as a storehouse for sperm. At the height of the breeding season its weight is only about 1 or 2 % of that of the testis which it serves and it contains at the most not more than one-thirtieth of the total contents of the excretory ducts in the sexually rested male. It is clear therefore, that it does not function as a storehouse for sperm as in the mammal. This function, to the restricted extent that it occurs in the fowl, is performed by the ductus deferens. Consequently, the latter duct has been isolated in this study rather than the epididymis as was the case in Moore’s work. Moreover, the location of the epididymis, especially its intimate connexion with the adrenal and the vascular supply to the testis, make epididymal isolation practically impossible.

It was at first thought that a method of rating the activity of the sperm which took into account the percentage of motile cells, as well as the type of motility (progressive, rotary, etc.) which they exhibited, could be worked out. After careful consideration, however, it was decided that the simple classification adopted by Moore (1928 a) was, on the whole, more serviceable. Thus, when all sperm show vigorous progressive movement, the rating is × × × ×; when a few sperm are immotile and the entire mass less vigorous in movement, motility is designated × × ×; similarly × × denotes weaker vitality, while × means that the great majority show no movement, only a very occasional sperm exhibiting weak vibratile contractions. When all sperm are immotile the designation is 0. This method of classification while rather a rough quantitative one has the great advantage of consistency; that is to say that the different ratings are quite distinct one from the other and there is very little danger of confusing adjacent classes as would be the case were a multiple classification used.

Representative males from the two groups were killed at intervals beginning on the third day after the final operation. The body cavity was opened and the reproductive tract examined closely to check the completeness of vas isolation. When this was verified the isolated vas was carefully removed from the carcass, cut into anterior and posterior portions, the semen from each portion squeezed out, separately placed in a drop of Ringer’s solution on a glass slide and immediately examined under the microscope. While occasionally slight differences were found in the motility exhibited by sperm from the two portions of the vas they agreed very closely, and the male was given the highest rating exhibited by sperm in either section. In the case of bilateral vas isolation he was given the highest rating observed in either vas. Careful examination was made of the epididymal stump in group II and a few cases showing testis nodules were discarded, in other cases where the existence of testis tissue was in doubt serial sections of that area were made but no additional cases were found. In totally castrated cocks, the comb begins to shrink rapidly and after 2 weeks is noticeably smaller and paler. Especially after 25 days the comb is a quite definite indication of the success of the operation. Although the “all or none” law of the testis is definitely untenable in view of later work (see especially Payne, 1936) those birds possessing even minute testis fragments tend to retain a noticeable degree of turgidity although showing some comb shrinkage. According to every criterion all group II males contributing to the results included in this paper were complete castrates.

It may be stated at the outset that no differences were observed in length of sperm life between those males in group I which were unilaterally castrated and those which were ligated only. One would not expect to find a difference unless the constant production of sperm without means of exit from the testis and epididymis, with its consequent abnormally high rate of cytolysis and liquefaction in situ, results in the formation of spermatotoxins which attack and remove the living sperm in the isolated vas of the ligated males before they disappear from the unilateral castrates. This was not expected in view of the work of White (1932) who studied sperm life in the isolated epididymis of the rat by ligating the vasa efferentia. However, the object of isolating sperm in the group I males by unilateral castration as well as proximal vaso-ligation was for the purpose of testing this point. Since no differences were found, all males in group I are in fact comparable.

The results of this examination are shown in Table I.

One of the first things revealed by this table is the considerable individual variability exhibited. Individual variability is a universal characteristic of biological material and the present data are not exceptions to the rule. The important thing to note is that sperm can survive in the ductus deferens of the cock for a period of i month and still retain sufficient energy to show weak movement. It is not suggested that sperm are normally held for such a period, even in the absence of mating. The author has at hand evidence suggesting that the sperm pass through the excretory ducts in a very few days. Neither is it suggested that this is the maximum possible duration of life in the male ducts. It is quite possible that, could the vas be occluded by some means which did not involve operative technique necessarily producing considerable trauma, the survival time might be lengthened. It is interesting to note, however, that the maximum survival time of 32 days corresponds exactly with the maximum number of days reported in the literature as elapsing between removal of the cock and the laying of the last fertile egg (Crew, 1926). The average life of sperm in the male, however, as shown in Table I is apparently about 26 days. Since it is well known that sperm lose their fertilizing ability before their motility ceases (Lillie, 1915; Wolf, 1921; Hammond & Asdell, 1926), the average number of days elapsing between the cessation of mating and of fertility—apparently about 14 days with very frequent maxima of 20 or 21 days as quoted previously—means that the average sperm must survive in the female tract for about the same maximum time. If the testicular secretions create an optimum environment and thus prolong sperm life, one would quite reasonably expect to see this time increased in the male. The fact that this does not occur is an indication that the testis hormone is not concerned here, as it is in mammals, with prolonging sperm life.

The results of the sperm examinations made in a similar way on the total castrates are shown in Table II.

Here, as in group I, movement persisted for a maximum of 32 days with an average of about 26 or 28 days. The important point to observe is that, in spite of the fact that this group underwent the more serious operation, that of complete castration, and therefore suffered more through trauma than did group I, they show, if anything, slightly greater persistence of sperm motility. This indicates that the trauma has had little or no effect on formed spermatozoa. Furthermore, the persistence of sperm life in group II occurs in the face of a very considerable shrinkage of the vas deferens of the castrates, especially after the lapse of a 2-week post-operative interval. This evidence, it appears, is sufficient to warrant the definite conclusion that, in the fowl, retention of the capacity for movement on the part of sperm ageing in the isolated vas deferens is not enhanced by reason of the existence in the organism of actively secreting testicular tissue. It should, of course, be understood that this refers to movement only when exposed to a normal physiological saline solution; it is now generally recognized that no movement on the part of the sperm occurs in the excretory ducts (Simeone, 1933; Gunn, 1936).

A comparison of the data provided by the experimental groups in this paper, as well as a comparison of these observations with previously published records on the duration of fertility in the female after mating is interrupted, shows quite definitely that additions to the body fluids of the secretions arising from an active testis do not prolong the life of mature sperm in the isolated excurrent ducts of the male fowl. Since the effect of the testis has been shown, in the first instance, to be negative, we are not confronted with the task of Moore (1928 a), namely, of assigning responsibility for a positive effect to a specific testicular function. While thus freed of the burden of further research into the specific nature of a response, one is, nevertheless, confronted with the disturbing fact that mammalian sperm is armoured against the onset of senescence by virtue of a continuous supply of testis hormone, while the vulnerability of fowl sperm remains unaltered. While not necessarily requiring a teleological explanation, a comparison of the modes of reproduction in the two classes provides quite adequate reasons for such a functional divergence.

In the first place mammals are characterized by definite heat periods or at least by more or less widely spaced mating periods. The sequence of oestrus or mating, pregnancy, parturition, and lactation which may in some species include oestrus, in others be followed by oestrus after a shorter or longer period of anoestrus, is characteristic of female Mammalia in general. The female fowl, however, is constantly receptive to the male throughout her egg cycle which has greatly lengthened as a result of domestication. Many hens lay more or less continuously, especially non-broody breeds, pausing only during the annual moult. In any case, even in the wild state each militantly successful cock takes unto himself a number of mates and thus, at any given time even providing some are broody, has several physiologically receptive females to serve. In contrast to the mammalian male, who serves but infrequently, even during the breeding season, the cock must be prepared to deliver an effective quantity of sperm several times per day. Penquite et al. (1930) have counted as many as twenty-eight copulations performed by a single male within $1012$ hours, while Nicolaides (1934) has found that a single mating is as effective in producing fertility as are several successive matings, which means that the male is delivering a fully effective quantity of sperm at each mating.

Thus, the cock must produce a relatively large number of sperm in a given period of time which, coupled with the fact that the excurrent ducts can accommodate only a small quantity of semen, means that his testis must be a highly efficient producer of germ cells and that ejaculated sperm must be relatively fresh and must have traversed the excretory ducts in rather quick time. Birds which mate in pairs, while not requiring a continuous production of sperm as in the fowl, require neither the sperm-storing facilities of the mammal, since they only mate just previous to the laying of each clutch of eggs for the proper fertilization of which only a relatively small quantity of semen is required.

The male mammal, on the other hand, may possess a much less efficient testis and still perform his required cohabitation effectively, especially if he be provided with facilities for accumulating quantities of formed spermatozoa during his more inactive periods. This reservoir of sperm could be drawn upon when occasion demanded the serving of several females within a limited period; it would thus act as a reserve supply during odd periods when sperm demand exceeded supply or when the male, so to speak, was in negative germ cell balance.

The acquirement of such a sperm-storage organ would necessitate the parallel acquisition of a sperm-preserving mechanism whereby sperm in the reservoir could be maintained in a functional state. The epididymis, of course, provides just such a reservoir and the testis hormone just such a necessary preserving mechanism.

While the testis-controlled preserving mechanism definitely prolongs the life of sperm in forms requiring and possessing a storehouse, spermatozoa are nevertheless definitely perishable. Their life cannot be prolonged indefinitely within the male tract (Young, 1931; Simeone & Young, 1931). As in the case of goods designed for human consumption they must be withdrawn from storage before disintegrative processes are initiated. Nature has provided an effective catalogue of the stored sperm and arranged for an ordered sequence of withdrawal whereby the older cells are liberated first. In the conversion of the amphibian mesonephros into the mammalian epididymis, this cataloguing and withdrawal sequence has been simply and efficiently provided for by the retention only of the main Wolffian duct. The continuous nature of this duct preserves the necessary ordered sequence of sperm transport and withdrawal. Had the side-branching mesonephric tubules been retained, they would have provided side pockets where sperm might stagnate. The capacity of the original Wolffian duct at its anterior end has been greatly enlarged by a series of collings and convolutions, until it has become known as the epididymis.

In birds, however, whose mating habits do not demand storage and preservation, the mesonephros has degenerated into a vestigial epididymis. The task of storing the mature germ cells has been taken over to the small degree that it is necessary by the vas deferens, which is narrowly spiralled throughout most of its length.

The exact manner in which the testicular secretions produce their sperm-preserving effect is not known. White (1932) has shown that the preserving action of the testis is dependent on the presence of the pituitary and believes that the effect is produced either directly by testis hormone or indirectly by the action of this hormone on the epididymal secretions.

Since the data presented in this paper show that the testis hormone does not preserve sperm life in the isolated vas of the cock, it would appear that its effect, in mammals, is not direct but by way of the epididymis. It is, of course, possible that the function of sperm preservation is a specific property of the epididymis and that, in fact, the epididymis of the fowl exerts such an influence. However, since the epididymis is but a vestigial organ, and since the sperm in any case are not stored there, it is plain that such a function plays no important part in the general economy of avian reproduction.

1. Mature sperm in the isolated ductus deferens of the fowl retain the capacity for movement in physiological saline for an average period of 26 or 28 days and a maximum of slightly more than 30 days, irrespective of the presence or absence of functional testicular tissue. Considering that sperm lose fertilizing ability before motility, this time accords closely with the reported survival of fertility in the hen after segregation from the cock.

2. While thus showing that testis hormone is not directly concerned with the maintenance of sperm life in the excurrent reproductive ducts of the fowl and suggesting that its positive effect in mammals is likewise indirect, it does not preclude the possibility that its action is expressed indirectly only through the epididymis.

3. The fact that the epididymis in the fowl is rather a vestigial organ and does not act as a storehouse for sperm makes it clear that in any case a physiologically controlled sperm-preserving mechanism plays no important role in the economy of reproduction in the fowl.

4. It is suggested that the nature of the demands made upon the supply of sperm in ducts of the males, as a consequence of the evolution of the sexual cycle in their respective females, satisfactorily accounts for the wide divergence in degree of epididymal development between Aves and Mammalia. Such demands, if in fact responsible for the divergence, have probably conferred advantages on well-equipped males and have most likely been produced through the agency of natural selection.