FF 871 G5 opy 1 A Study of Some Factors Influencing Fertility and Sterility in the Bull BY HERBERT L. GILMAN Reprinted from the Annual Report of the New York State Veterinary College 1921-22 . ow ae C +e a A Study of Some Factors Influencing Fertility and Sterility in the Bull A THESIS Presented to the Faculty of the Graduate School of Cornell University for the Degree of Doctor of Philosophy BY HERBERT LESTER GILMAN, D. V. M., M.S. yt Reprinted from the Annual Report of the New York State Veterinary College ALBANY J. B. LYON COMPANY, PRINTERS 1922 Peg ee me SI ee A STUDY OF SOME FACTORS INFLUENCING FERTILITY AND STERILITY IN THE BULL HERBERT L. GILMAN Veterinary Experiment Station, Cornell University Normal reproduction is the fundamental foundation upon which the entire cattle industry rests. For this reason, any factor capable of interfering with it is a detriment to the industry, and a matter of prime importance to the breeder and the veterinarian. With the relative increase in number and value of eattle, and the fact that the profession is depending more and more on this in- dustry for a livelihood, these problems are assuming greater im- portance. The part played by the bull has been emphasized entirely too little, with the result that, as in human medicine, many fail to appreciate the effects of sterility or lowered fertility in the male. The part played by the sire in the spread of genital infections, though discussed frequently, has received little sys- tematic investigation. The bull must be regarded as at least half the herd, not only from the standpoint of the characters he imprints upon his prog- eny, but because of his relation to the reproductive efficiency in the herd. It seems quite probable that he does disseminate during copulation, infection associated with the genital organs, with the result that the bull is a very important factor in a study of the subject. Too frequently, his ability to copulate in an apparently normal manner, is taken as a standard of fertility. Gross changes in his genitalia, or the absence of spermatozoa from the semen are given due consideration, while other more obseure abnor- malities are not looked for nor regarded in their proper light. Neither fertility nor sterility are always absolute, but the terms should be used relatively inasmuch as we may have all degrees of infertility or impotency. All too frequently we forget the many delicate and intricate mechanisms involved in the reproduetive process, with the result that many phases of the problem are neg- lected or disregarded. The genital organs work as a unit, each part of which must function in perfect accord with the others to the end that full fertility may result. The physiological factors involved in the formation of the semen are too little understood or at best, our knowledge regarding them is more or less hazy. The purposes of the present work have been: (1) to summarize the work so far done on the subject, (2) to review briefly the known facts throwing light on the anatomy and physiology of the r ’ male genital organs, (3) to carry out systematic studies upon the pathology and bacteriology of the genital tract of the bull, and (4) to ascertain if possible whether the bull is a disseminator of those infections which interfere with reproduction in the female. [3] ? 4 The work has been carried on for the most part from the point of view of a laboratory man cooperating with clinicians. No attempt is made in this paper to give detailed clinical data, methods for physical examinations, etc. There are included many state- ments and some data given in a preliminary article on the sub- ject. While the subject is broad in its scope, in fact too broad for great detail, it is hoped that a start has been made toward future and more detailed investigations. History References to, and investigations relating to, the part played by the bull in the process of reproduction in the herd, and in the spread of genital infections, have been limited largely to those phenomena caused by Bact. abortum. Bang (1) originally called attention to the possibility of the male transmitting the organism discovered by him, but he reached no definite conclusion on the subject. James Law (2) writing on contagious abortion in cows, early suspected this possibility when stating under “‘ casual infec- tions,’’ that —‘‘ In a case which came under the observation of the writer recently, a family cow, kept in a barn where no abortion had previously occurred, was taken for service to a bull in a herd where abortion was prevailing, and though she was only present at the latter place for a few minutes, she aborted in the sixth month.’’ Jansen, as quoted by Sand, reports the case of a cow from an aborting herd having been taken into a herd that had been previously quite free from the disease. Soon after her arrival she aborted, and later cow after cow of the original herd aborted. The owner kept the matter a secret, and sent his cow to a neigh- bor’s bull for service, with the result that for two years abortion prevailed among cows served by this bull. MecFadyean and Stock-- man (3) later, in experimental work, attempted but failed to in- feet cows by using a soiled bull for service. Hadley and Lothe (4) state: ‘‘A large number of stockmen hold that the bull is an important factor in the transmission of contagious abortion in herds. A smaller number believe that the bull merely acts as a passive carrier of the abortion disease and is not actively concerned in the transmission.’’ In a subsequent bulletin, Hadley (5) re- marks: ‘‘ The abortion organisms may enter the body ...... during sexual intercourse.’’ In an experiment carried on by the same author and co-workers, abortion-free virgin heifers were mated to abortion-infected bulls, infection being evidenced by positive, reactions to the complement fixation and agglutination tests. His results indicate, he believes, ‘‘ that the bull is not so important a factor in transmitting abortion as many believe.’’ The con- clusions are: ‘‘Bulls may become infected with abortion. bacilli. Bulls that reacted to the blood tests were incapable of dissemi- nating the abortion disease to the abortion-free heifers with which they were mated. Bulls appear to possess a sexual or individual immunity to abortion infection that renders them less susceptible 5 than cows and induces a milder form of the disease. The resist- ance appears to be due to certain anatomic and physiologic differ- ences in their sexual organs which make them less favorable places for the growth of the abortion germs than those of the opposite Sex” Buck, Creech, and Ladson (6) appled the agglutination test to 325 mature bulls, of which 288 were negative and 37 positive. Bacillus abortus was isolated from five animals, of which three showed marked lesions, two in the seminal vesicles, and one in the left testicle. They conclude: ‘‘ B. abortus may involve organs of the generative apparatus of bulls, producing chronic inflammatory changes. Of the generative organs, the seminal vesicles appear to furnish the most favorable site for the lodgement and propagation of abortion infection.”’ Schroeder and Cotton (7) cite the case of a bull which reacted to the abortion test and, on post mortem, Bact. abortum was isolated from an abscess of one epididymis. They state: ‘‘Our attempts to produce a similar case of infection artificially failed, and, in agreement with the difficulties many investigators have had to obtain incriminating evidence against bulls, we have thus far failed to infect bulls in any way that justifies the assumption that they are important factors in the dissemination of abortion disease.’’ Further, they conclude: ‘‘ Regarding the dissemination of abortion disease by bulls, we may say, however, that it would be foolhardy in the dim hght of our present knowledge to take liberties with reacting bulls, or bulls from infected herds, or promiscuously used bulls.”’ Cotton (8) failed to demonstrate the presence of abortion bacilli in the genital organs of the bull used to serve aborting cows, or in the testicles of two bull ealves, one of which had been fed and the other injected with the cultures of the abortion bacillus. He con- eludes that the bull does not harbor the organisms in the testicles. Carpenter (9) injected both streptococci and Bact. abortum into the scrotal sacs of young calves, and intravenously in others. In no ease was he able to recover the organisms from any part of the genital canal, except for a streptococcus in one instance. Rettger and White (10) were unable to obtain cvidence of the presence of Bact. abortum in three bulls slaughtered after repeated reactions to the complement fixation and agglutination tests. The three bulls had been under observation for three years, with no conclu- sive evidence to indicate that they were a source of danger to the herds in which they were a part. They believe that the bull trans- mits the infection as a passive carrier. Attempts at artificial inoculation by natural channels have failed, with the possible exception of McFadyean, Sheather, and Minett (11) who were able to infect the bull by the prepuce in two eases and by the mouth in one case. The results, however. are by no means conclusive. They conclude, nevertheless, that cattle of any age of either sex may be infected by natural channels with the bacillus of epizootic abortion. 6 Schroeder (12) carried out investigations to ascertain the fre- quency with which bulls react to abortion tests, and the frequency with which lesions chargeable to abortion bacilli occur in the re- productive organs of reacting bulls. Studies were also pursued which he states conclusively prove that bulls with infected repro- ductive organs may expel abortion bacilli with the seminal fluid. In the first two mentioned investigations 325 bulls from a Wash- ington abbatoir were tested, and slaughtered upon reaction. ‘* Ap- proximately ten per cent of the bulls reacted, and approxi- mately ten per cent of the reacting bulls showed lesions of ihe reproductive organs from which abortion bacilli were isolated.’’ The value of these studies, he emphasizes, hes not in “‘ that they give us a measure of the proportion of bulls that react positively to abortion tests or the proportion of reacting bulls that are car- riers of abortion bacilli,’’ but in ‘‘ the fact that they show that abortion bacillus disease of the bull’s reproductive organs is not a wholly unique affection which practically may be ignored, but an important condition that must be taken into account in our efforts to combat infectious abortion, since it has been proved to be asso- ciated with contamination of the seminal fluid.’’ Jn discussing the method by which infected bulls transmit the organisms to cattle, he believes that leakage of semen from the penis, or vaginas of cattle after service, contaminates the food which subsequently gains entrance to their digestive tracts. As the result of a series of experiments, he states: ‘‘...... the results fail to justify in the least degree the assumption that cows are infected with abortion bacilli via their vaginas or uteruses at the time of copulation, or that the bull, through copulation, is an agent in the spread of abortion disease.”’ The work so far alluded to, has been limited to infection with, and the transmission of, Bact. abortum and the lesions associated with such infection. The last mentioned author, however, states: ““A search for other specific causes of abortions, among cattle has not been neglected, and bureau investigators could relate at great length stories similar to those which investigators have told about microorganisms isolated from the products of abortions and the uteruses of cows that have aborted. Bacilli of various kinds, different types of microecoeci, and spirilla or vibrio have been found repeatedly ; but when their pathogenicity has been tested in aceord- ance with widely recognized and accepted and required bacterio- logical standards, not one shred of evidence has been obtained to prove them true etiological faetors of bovine abortions. What role such microorganisms may have as causes of the sequellae of in- fectious abortions, and of other, possibly, independent, abnormal processes in the reproductive organs, is far from clear and merits eareful study.’’ Hadley (5) mentions the fact that: ‘‘ Unques- tionably the male often becomes infected with the germs that produce the various secondary diseases in the female, which are properly classed under the more inclusive term ‘abortion dis- > i A 4 ease. Also, speaking of the rarity with which the bull acquires 7 abortion infection, he alludes to the fact that he may act as a ‘* mechanical carrier of various disease germs from an infected to a healthy cow.’’ Carpenter (9) working on the female genital tract, comes to the conclusion that, in all probability, the genital organs are normally free from bacteria. Barney (13) quoting Huet finds that bacteria may be present in the seminal vesicles of healthy animals (horses, cattle, pigs, and laboratory animals). This, he states, corresponds with the well recognized findings in other parts of the genito-urinary tract, not only in animals, but in man. He (Huet) has further found that in animals dying of acute septi- eemia, the specific organism (anthrax, pneumococcus) is to be found in the vesicular secretions. Furthermore it was definitely shown that an infection could be transmitted to the female during the act of copulation. Williams, W. L. (14), calls attention to the lack of vet- erinary literature relating to the pathology and _ bacteriology of the male genital tract, except as related tc infection with Bact. abortum. Infection with other types of bacteria is empha- sized, the clinical recognition of such, with the accompanying pathological changes, and of the numerous phenomena involved in the process of reproduction in the male. The semen and its essen- tial germinal elements are taken up with reference to the entire lack of study devoted to them, and some of the abnormal changes are described. Jn a later contribution (15), he takes up the part played by the bull in the dissemination of genital infections and states: ‘‘ Clinical studies now indicate with great clearness that the bull is an active spreader of that group of genital infections which cause sterility, abortion, and related phenomena.’’ Wilhams, W. W. (16) studied the semen with reference to steril- ity, emphasizing the importance of its examination in the diag- nosis, giving methods for collecting samples, staining of sperms, and some of the abnormalities encountered. The work is funda- mental, and should be of great practical importance to all interested in the problem. In a later paper (17), he brings out a more ex- tended discussion of the question. He concludes that the clinical examination is of vital importance, and that the efficiency of the semen depends not only upon its physical properties but upon the number of spermatozoa that are motile, the degree of motility, degree of obligospermia, and the percentage of imperfect spermato- zoa, either deformed or immature. Of forty bulls examined, he finds that twenty, or fifty per cent, showed lessened fertility, and others, aside from this, showed minor changes in the genital organs or semen. The same author subsequently takes up the sub- ject of reproduction from the viewpoint of both sexes. but empha- sizing infection in the male, and the frequeney with which lowered vitality of the germinal cells occurs. Hopper (18) states: ‘‘A diseased bull may manifest non-fertility or decreased potency in different ways — by repeated service to apparently normal females without conception, by a high abortion rate in females that have been apparently normal, by characteristic infections following the 8 use of any particular sire, or by abnormalities in the breeding tract noted by rectal or physical palpation.’’ The observations of Williams (19) in a pure bred dairy herd bring out quite clearly the relation of the bull to the dissemination of genital infections. The bulls in this particular herd were ab- normal in many respects, as demonstrated by pathological changes in their genital organs, bacterial invasion of the parts, abnormali- ties of the semen and spermatozoa, and the probable transmission of infection to the females. Several of the sires from this herd furnished much of the material for the early basic work of this investigation. Since then the tracts of other sires have been worked upon with quite similar or identical results. To summarize the work already done, most investigators have considered the bull as merely a mechanical carrier of Bact. abor- tum infection, though all are more or less suspicious of his ability to become an active spreader. Schroeder, however, states that the organisms are eliminated with the semen, but infection of the fe- male oceurs secondarily through the digestive tract by contamina- tion of the food with the semen. Other investigators bring out fundamental points demonstrating the importance of other organ- isms than the Bang bacillus and eall attention to the need of a more thorough study of the anatomy, physiology, and pathology of the male genital tract. Any study of the genital organs must of necessity rest funda- mentally upon a thorough knowledge of the anatomy and phy- siology of those parts. Too few of us have stopped to consider these questions thoroughly, with the result that our ideas on the problem are more or less vague. It is much easier to understand why abnormal spermatozoa occur so frequently, or changes take place in the semen with death or weakening of the germinal ele- ments, if we realize or stop to consider the highly differential pro- eess of spermatogenesis, and the various structures which eontrib- ute to the formation of the semen. We must come to realize that each part of the genital tract is essential to the normal functioning of the whole, and that the genital tract and reproduction are in turn dependent upon the proper functioning of the entire body. Walker (20) emphasizes the importance of a thorough knowledge of physiology in stating, ‘‘Although the subject of sterility has attracted the attention of the medical profession; and although much has been written on its causes and treatment, it cannot be elaimed that the practical results obtained up to the present time are satisfactory, or that when consulted for sterility, the medical man of today ean hold out to his patient much more hope of sue- eessful treatment than the medical man of fifty years ago. Our failure in this respect is in the main due to an ignorance of the physiology of reproduction.”’ To bring out some of these points, the anatomy of the tract will be reviewed briefly, together with the physiology of reproduction, and the various factors which should be considered in a study of the problem. ANATOMY AND PHYSIOLOGY Tn origin and early development the ovary and testis are ident1- eal. The gonad and mesonephros or primitive kidney are de- veloped from the urogenital fold. The gonad first forms as a medio-ventral thickening of the fold, which gradually expands until it becomes attached by a mere stalk. At first, the gland is made up merely of a superficial epithelial layer, and an inner epithelial mass, or epithelial nucleus. In the process of develop- ment, large primordial germ cells migrate from the entoderm of the future intestinal canal, and pass through the stalk to the gonad. In the ease of the male gonad, seminiferous tubules are very difficult to make out in embryos smaller than 24 millimeters. Then they suddenly differentiate out as solid cords of germ cells, while the connective tissue grows in around them. These connective tissue sheaths unite at the center of the organ to form the anlage of the mediastinum testis. The testicular tubules unite and con- verge toward the hilus, there to meet the anlage ot the rete. At the mesonephriec end of the testis, the rete first appears as a col- lection of cells, differentiating out from the inner epithelial mass of the gonad. These cells gradually grow out to meet the collect- ing portions of the mesonephrie tubules on the one hand, and the seminiferous tubules on the other. The rete is represented as eords of cells at first, which in forty millimeter embryos hollow out to form tubules. The mesonephros, or primitive kidney, early starts to degenerate eranio-caudally,— the tubules becoming separated into a cranial and caudal group. The collecting and secretory parts of the cranial group separate, the collecting tubules growing out to meet _ the rete with which they unite to form the efferent ductules of the epididymis. The caudal group of tubules is vestigial and becomes the paradidymis. The mesonephrie duct becomes the vas deferens, connecting as it does with the tubules of the epididymis, and emptying into the urethra at Miiller’s tubercle or, as it later be- comes, the colliculus seminalis. The seminal vesicles arise as hollow saccules from the dorsal wall of the mesonephriec duct just as it empties into the urethra. The prostate develops as an outgrowth of the dorsal urethra just posterior to Miiller’s tubercle. The bulbo-urethral glands appear as solid, paired, epi hens} outgrowths from the entoderm of the urogenital sinus. Miiller’s duct, at first a solid tube growing from the anterior part of the mesonephros, and ending at Miiller’s tubercle, becomes a hollow tube, and in the female forms the entire genital tract ex- cept for the gonad and the lower part of the vagina. In the male, the anterior part remains as the vestigial appendix testis, and the posterior part, as the vagina masculina. Ellenberger states, how- ever, that this embryonic structure is very seldom seen in the mature bull. 10 The Male Reproductive Organs include the penis and testes, together with the excretory passages which connect the testes with the urethral canal. These excretory ducts include the epididymis, vas deferens, and seminal vesicles. Posterior to their termination in the urethra, there are connected the ducts of the prostate gland and the bulbo-urethral or Cowper’s glands. Tresres: The testicles of the bull are relatively large. Varying with the size and age of the animal they measure from fourteen to seventeen centimeters in length, including the epididymis, and from six to eight centimeters in diameter. Each testicle is en- closed within a serous sac, the tunica vaginalis, whose visceral layer is very intimately fused with the underly- ing covering of the organ, the tunica albuginea. The tunica albuginea is quite thin and consists of connective tissue which is rich in elastie fibres. Muscular tissue is not present as it is in the case of many mammals. Inside the tunica, and closely attached to, though separated from, the parenchyma by a thin layer of connective tissue, is a layer of very lobse connective tissue, which because of its rich supply of blood vessels is termed the tunica vasculosa. The parenchyma is of a yellowish gray color, and of a rather soft consistency. It is made up of the semi- niferous tubules, rete, and the connective tissue stroma, the medi- astinum testis. On section, the mediastinum appears as the center or axis of the entire organ. It is star-shaped, and radiates connect- ive tissue septa out into the parenchyma to support and separate the tubules. Ellenberger states that the testis of the bull and all ruminants lacks a closed system of interlobular septa, because of the feeble development of the connective tissue. The principal blood vessels and rete tubules are found in this structure, the function of the latter being to connect the seminifer- ous tubules and the efferent tubules of the epididymis. The epithelium of the rete is quite irregular,— consisting in places of a single layer; in others, of two layers. At some points there are formed groups of several cells lying over one another, with swollen homogeneous basal cells, which sometimes form projections into the lumen. The interstitial tissue, besides conveying the blood vessels to the organ, contains many ‘‘ interstitial cells.’’ These cells are rela- tively sparse in the adult bull, and are comparatively delicate, slightly granular, often shuttle-shaped, with a rather small nucleus. Embryologically they are derived from a syncitium arising from the mesothelium of the genital ridge, differentiating out by growth of the eytoplasm. They contain large quantities of fat, and elaborate the internal secretion of the testis. This secretion gov- erns the development of the secondary sexual characters, and has a profound influence on the general body metabolism, and develop- ment of the skeleton. The interstitial cells appear early in embryonic life even before there is any differentiation of sex, and id their greater relative development in the fetus is indicative of a future male development. In very young embryos, the growth is very rapid, followed, however, by a period of atrophy, during which the seminiferous tubules undergo marked development. Pende (21) states: ‘‘ There seems to be an inverse relation be- tween the growth of the tubular and interstitial tissues, as one is hypoplastic when the other is in full activity.’’ From birth to the onset of sexual maturity, which may be ealled a period of rest for the testicle, the cells are few in number. With the accentuation of the secondary sexual characters, and the beginning of sexual life, these cells again increase in number and activity. The parenchyma of the testis consists for the most part of the seminiferous tubules, which, on aceount of the courses they take in the different regions, are divided into groups. The peripheral tubules are the much-contorted tubuli contorti. These anastomose to form the much shorter tubuli recti. These in turn anastomose frequently, uniting to form the rete testis. The rete proceeds through the mediastinum to form the efferent ductules which break through the tunica albuginea to form the greater part of the head of the epididymis. The tubuli contorti are the longer and more numerous of the tubules, for it is here that practically all the spermatozoa are produced. The straight tubules are relatively so short that they may be regarded more in the light of the begin- ning of the system of excretory ducts. The seminiferous tubules consist of a thin peripheral membrana propria upon which rests the seminal epithelium, which is made up of the essential semen forming cells, and the cells of Sertol. The spermatogenic cells may be divided into three groups, from within outward: the peripheral single layer of small euboidal spermato- gonia; one or two rows of large spermatocytes; and three to five rows of spheroidal spermatids. The cells of Sertoli are more or less of the syncitial type,— large in size and irregular in outline. They oceur at various intervals between the layers of spermatogenic cells, with their bases resting upon the membrana propria. Centrally they send out protoplasmic processes for variable distanees,— some even reaching the border of the innermost cell layers. SPERMATOGENESIS: In this process, the primary germinal cells, the spermatogonia, divide to form the primary spermatocytes. Maturation consists of two cell-divisions of the primary sperma- toeytes, and these in turn form four spermatids. During the pro- cess, the number of chromosomes is reduced to half the number characteristic of the species. The spermatids then become con- verted into mature spermatozoa. This mode of transformation may be seen in Plate I. In the process, the nucleus of the sperma- tid forms a large part of the head; the centrosome divides, part passing to the extremities of the neck. One centrosome becomes the anterior, and remains attached to the head, while the other divides to form the posterior centrosome. The latter is divided into the anterior part, and the posterior nodule or annular ring. 12 Besides this, the posterior centrosome becomes elongated to form the axial filament, and the cytoplasm forms the sheaths of the neck and tail. The spiral filament of the connecting piece is derived from the cytoplasmic mitochondria. At this time, a large part of the cellular cytoplasm is east off. Meanwhile, the spermatozoa sink their heads into the long protoplasmic processes of the Sertoli or ‘‘ nurse ”’ cells which furnish nutritive material for their com- plete development. Finally the adult cells are cast off into the jumen. The structure of the spermatozoa, and a discussion of the semen will be taken up later. ee Epipipyuis: The epididymis is divided into three parts: the head, body and tail. The head is made up principally of the lobules formed by the much-coiled efferent ductules proceeding from the rete. The ductules, about twelve in number in the bull, unite to form the body, whieh remains coiled and runs along the postero-medial part of the testicle to which it is more or less closely attached. To quote Ellenberger (28): ‘‘ The transition from the rete into the ductules is gradual, as the characteristic epithelium of the latter (ductules) begins in eavities without walls, and at first, gradually form a wall which is well marked out as a thin ring of interstitial tissue...... The epithelium of the ductules is in sharp contrast to the rete in that it has a single-layered ciliated columnar epithelium, in which here and there one finds round basal cells. The dark and light columnar eells alternate; the cilia are often cemented together, and form cone-shaped, homogeneous appearing protuberances. The secretory activity is quite clearly observable. In the light cells one finds secretory globules, aceumu- lating in rows, sometimes above, other times below, arches of cells. The secretory droplets pass from the cells into the lumen, and often lie in irregular layers on the epithelium; also the basal cells appear swollen and shoved out between the cylindrical eells.’’ At the lower extremity of the testicle, the tail is formed, which is globular in shape, and more or less loosely attached to the testicle. Here the ductules anastomose freely, gradually become less coiled, and end in the single excretory tube, the vas deferens. The epithe- lium at the tail part is more or less of the pseudo-stratified columnar, ciliated variety. Outside this is a membrana propria, a circular muscular layer, and a connective tissue coat. The secre- tory activity is very marked here and one finds much secretion in the lumen. Courrier (24), working on the bat, suggests that the glandular activity is conditioned by the secretion from the inter- stitial (endocrine) gland. The action of the secretion is to dilute the large mass of spermatozoa present, nourish them to some extent and also stimulate them to active motilitv. Stigler (25) states that the properties of the sperms are modified in the epididymis; the motility, the ability to resist heat, and other properties are augmented, at least in the case of the guinea pig, rat, and mouse. Some authors state that the sperms first become motile when in contact with the prostatic secretion, but I have repeatedly exam- 13 ined the contents of the tail of the epididymis of the bull, rabbit, and guinea pig, finding full motility in each case, though the dura- tion is not nearly as long as when the sperms are ejaculated in the semen. The VAs DEFERENS is quite narrow (2 mm.) and runs from the tail of the epididymis to the verumontanum, or colliculus semin- alis, where it empties into the urethra in common with the duct of the vesicle. At first it is lined by epithelium similar to that of the vas epididymis, but this changes over into a peculiar low stratified type. Ellenberger describes it as follows: ‘‘ The epithelium shows a very pronounced basal coat. The overlying cell zone shows more (at the most, three) rows lying over each other of elongated nuclei, while an outline of cell form is not ordinarily noticeable, so that it may be spoken of as a syncitium, and at the same time as a many layered epithelium.’’ The mucosa forms low, broad folds into the lumen. The tunica propria is a thin con- nective tissue layer. The submucosa consists of thin connective tissue. Three muscular coats are present: an inner thin longitud- inal layer, middle circular layer, and an outer longitudinal layer. All are more or less intimately blended, and are surrounded by the adventitia, made up of connective tissue, elastic fibres and scattered longitudinal muscle cells of the internal cremaster mus- cle. Near the dorsal surface of the bladder, the ducts come in close apposition, and for ten to twelve centimeters dilate to form the ampullae. Here the mucous membrane beomes much plicated, forming long folds which anastomose freely. The function of the vas is to convey the spermatozoa and secretions from the epididymis to the urethra. Disselhorst (26) believes the ampulla acts as a seminal reservoir and states that he has found spermatozoa stored up in the little pockets in the walls of this structure in animals during the rutting time. He suggests, further, that there is a relation between the state of development of the ampulla and the time occupied by copulation. When the organ is small or absent, as in dogs, cats, and boars, the coition is a slow process, but when the ampulla is large and well developed, as in horses and sheep, the coitus requires a relatively short time. Inasmuch as coitus is so rapid in the bull, and the ampulla is so well developed, it seems as though this function is very probable. The Seminau VESICLES are very compact glandular structures \yeng on either side of the median line, on the dorsal side of the ladder, and ventral to the rectum. In the mature bull they sbeasure ten to twelve centimeters in length, four centimeters in -width, and about two and one-half to three centimeters in thick- ness. The glands are distinctly lobulated, quite tortuous, and are often asymmetrical in size and shape. They converge posteriorly, to empty into the urethra at the colliculus seminalis with the ampulla, in a slightly oval slit in the mucosa. Microscopically, the gland is of the anastomosing tubular type, with very poorly 14 developed excretory ducts to the glandular cavities. Posteriorly one finds centrally a few sinus-lke narrow excretory passages, which open into the somewhat larger collecting and excretory duct. The epithelium is of the simple columnar type and produces a relatively large amount of secretion. The gland cavities are sur- rounded by a membrana propria, over which is a relatively thick layer of smooth muscle. Outside this is a connective tissue covering which sends trabeculae or septa in between the lobules. The seere- tion of the seminal vesicles is a tenacious albuminous fluid with a slightly yellowish tinge, all or part of which appears in the ejaculate in the form of swollen sago-like grains which are soon dissolved following ejaculation and the liquefaction of the semen. The proteid compounds belong to the group of histones. The secretion is liquid when warm and coagulates when cold. Some say that the filling of the vesicles serves to excite sexual feeling, but this is doubtful in view of the fact that in some animals the sexual desire exists before the vesicles are filled. Likewise, Stein- ach found that rats, whose seminal vesicles had been removed, still retained their desire for copulation. The function of the secretion is to furnish much of the volume to the semen, and in some way it has a distinct bearing on fertility, inasmuch as ex- tirpation of the organs in rats leads to lowered fertility. The vesicles of the bull are in no sense a store-house for spermatozoa, as is usually understood. Repeated examinations in a large num- ber of bulls have led to the finding of spermatozoa there only in very rare instances. That they serve as a resorption place for sperms that are not ejaculated is also very unlikely. Normally, one sees on smear of the vesicles, occasional cells, leucocytes, lecithin gran- ules, sago bodies, and rarely a, few degenerated spermatozoa. The CoLuicuLus SEMINALIS is a rounded or cone-shaped emi- nence in the posterior urethra. upon which the ducts of the sem- inal vesicles and vasa deferentia open. The ducts open separately at the hottom of two narrow slits, one on each side of the mound, there being no distinct ejaculatory duct as in man. The function of the colliculus or verumontanum is not definitely known. It is generally believed that the structure is made up of blood spaces which become engorged during erection, causing a blockage of the posterior urethra, which prevents regurgitation of the semen. Rytina (27), however, demonstrated that the structure is not com- posed of any unusual number of blood vessels or spaces, and that removal of the organ was not followed by regurgitation of the semen into the bladder during ejaculation. He believes, and quite logically, that its function is to afford a prominence upon which the duets may empty. The mixture of the thick gelatinous semen with the thin prostatic secretion must occur at the moment of ejaculation and must be perfectly homogeneous, otherwise large numbers of the organisms remain in the thick gelatinous portion of the fluid. The eminence serves this purpose in that the pros- tatie ducts which converge toward it, may eject their secretion 15 toward the eminence, producing an admixture more evenly and quickly. Prostate: The bull possesses what Ellenberger calls a diffuse prostate. That is, there is no distinct glandular body as in man. It is composed principally of a glandular sheath around the urethral wall. Just posterior to the neck of the bladder, and in front of the urethral muscle, there is formed a slight dorsal trans- verse elevation, extending downward on the sides. This is what might be termed the body. The greater part of the gland is ‘‘disseminate’’ in form, being a sheath of glandular tissue em- bedded in the urethral wall. Dorsally it is about ten to twelve millimeters thick, and ventrally about two millimeters. The gland is a branched tubular structure, the interlobular tissue of which contains much unstriped muscle. The lobules are lined by a columnar type of epithelium. The ducts, about thirty to forty in number, open into the urethra in two rows posterior to the colliculus. The secretion is a thin, slightly turbid fluid, of a faintly alkaline reaction. Its function is to dilute the semen, stimulate the motility of the spermatozoa and nourish them. Fish (28) believes that the activating property of the secretion is due to enzymes, because boiling deprives the fluid of its power to accelerate the motility of the spermatozoa. Serrlach and Pares, quoted by Marshall (29), working on dog's, have adduced evidence indicating that the prostate is an internal secretory gland which controls the testicular functions, and regulates the process ot ejaculation. It is stated that if the prostate is removed, sperma- tozoa are no longer produced in the testis, and that the secretory activity of the aecessory genital glands ceases. These changes, however, can be prevented by the administration of extracts of the prostate. The fact that the prostate elaborates a secretion having a definite relation to the testis, has been verified by other authors. CowPer’s Guanps (Bulbo-Urethral): These glands are paired, oval structures about one and one-half by two and one-half centi- meters in size, situated on either side of the dorsal pelvie part of the urethra close to the ischial arch. They are deeply embedded, with the bulbus urethae, in the bulbo-ecavernosus muscle, thereby heing inaccessible to palpation. In general, they are of a well! developed anastomosing tubular type. The connective tissue stroma is relatively thin, and thickens only in between the larger lobules, where one finds the larger collecting ducts. Each gland opens by a single duct. The epithelium is of the simple cuboidal type. Little is known of the function of its secretion, though Kingsbury (30) regards it in the light of a mating gland; that is, it lubricates the genital passages during coitus, as does its homologous structure in the female, Bartholin’s eland. Ellenberger describes the urethra, slit ventrally, as presenting the following picture: “‘The colliculus seminalis distinctly ap- 16 pears as a process or offshoot of the ecrista urethralis of the Tri- gonium Vesicae. At the summit, and at the bottom of the two slits, open laterally the ducts of the vesicles, and medially the ductus deferens . . . . From the caudal slope of the colliculus eo two distinct mucous membrane folds which run through the entire pelvic urethra, near together, somewhat diverging, and then coming together, so that they form an elongated, narrow oval. At their caudal union, the excretory ducts of the bulbo-urethral glands open side by side. At the point of departure of the folds from the colliculus, arises a niche-shaped opening, between both folds, and likewise lateral to each fold. In these niches open the ducts of the prostate. The openings of the disseminate prostate he in rows as in the horse, but form not less than six rows. There is One row medial to each fold, and two lateral. Miiller mentions only the medial rows. These rows extend to the opening of the ducts of Cowper’s glands. The stratum glandulare (disseminate prostate) is very easy to recognize with the naked eye.”’ SEMEN: The semen is the mixed product of the testicles, their excretory passages, and the accessory sexual glands, a fact which complicates its study considerably. The freshly ejaculated fluid is cloudy, tenacious, more or less coagulable, and is rich in albumen. It is weakly alkaline in reaction, and contains eighty to ninety per cent of water. Of the solid constituents, there is forty per cent of ash, of which three-fourths is calcium phosphate. Besides the spermatozoa, the semen frequently contains epithelial cells, leucocytes, concentric amyloid concretions, and lecithin bodies. When cold, the characteristic phosphoric acid salts are precipi- tated. The fluid content is the product of the tubules of the testicles, their excretory ducts, and the accessory sexual glands. The characteristic odor of the semen is supplied by a slimy nucleo- albumen ‘‘spermin’’ which forms the spermatic crystals, and is furnished by the prostatic secretion. During ejaculation the spermatozoa and secretions added by the testicle and epididymis are probably carried to the ampulla by peristaltic muscular action, in the earlier stages of the orgasm. At the height of the orgasm, the ampulla is emptied into the pos- terior urethra in common with the secretion of the contracting vesicles, here to be admixed with the thin prostatic secretion. The entire mixture is then propelled, and ejaculation produced by strong museular contractions of the entire urethra. As was stated before, the semen is the product of the testicles, the excretory ducts, and the accessory sexual glands. The testes furnish the essential germinal elements, the sperms, and some of the fluid eon- tent. Then is added the product of the epididymis, vas, and ampulla, which stimulates the spermatozoa to active motility, nourishes the organisms, and adds somewhat to the fluid bulk of the secretion. Stigler (25) states: ‘‘During its sojourn in the epididymis, the properties of the spermatozoa are modified; the ‘tility, ability 17 to resist heat, and other properties are augmented, at least in the case of the guinea pig, rat, and mouse.’’ To the secretion is then added the product of the vesicles, which contributes markedly to its fluid content, nourishes the sperms, and supplies the ferment which induces clotting of the semen when ejaculated. This is very important because the clot formed in the vagina protects the delicate spermatozoa from the hostile acid vaginal secretion. The prostate likewise adds bulk and nourishing substances, besides stimulating the spermatozoa to fuller and more lasting motility. The addition of the spermin is perhaps unimportant. The func: tion of the secretion of the Cowper’s glands, which is added at this time, is problematical. It does, however, have a diluting action on the semen. Perhaps its secretion is poured out prior to ejacula- tion so as to lubricate the canal and prepare the way for the semen. Fish (28) has demonstrated by means of darkfield illumination, the presence of numerous minute particles or ultraparticles in this fluid. Their character and significance are matters of conjecture, but it would seem as though they were not identical with the *‘chylomicrons’” or fat particles found in the blood by Gage. Per- haps further researches will reveal some intimate connection be- tween the number present in a field, and the relative poteney of the animal. Each portion of the tract furnishes some essential element to the mixed product which is so remarkably adapted both as a vehicle for the ejaculation of the spermatozoa, and as a fluid in which their motility is initiated and maintained. Any derange- ment of one part is fraught with danger to the existence of viable spermatozoa, and the continuation of full fertility on the part of the animal. The physiology of each contributing gland must be borne in mind at all times. Walker (31) investigated the fer- tility of the semen of the dog, taken from various parts of the tract. His results were: (1) semen from the testicle and head of the epididymis showed no motility, (2) semen from the tail of the epididymis showed some motility in the more fluid contents of the preparations, (3) semen from the vas deferens appeared about the same, (4) a mixture of epididymis semen and pros- tatie secretion showed active motility, and (5) likewise in a mix- ture of epididymis semen, though only in those places where the fluids had become well mixed. My observations, however, differ in one respect with regard to the bull, as I have found full motility of the spermatozoa from the epididymis, but it is not so lasting as when augmented by the addition of the prostatie fluid. Boettcher (32) concludes: ‘“‘that the secretion of the accessory male genital organs possesses a protective colloid, which (1) hinders the sper- matozoid action of the vaginal secretion, at least until the sperms have time to reach the interior of the uterus which is an alkaline reaction, (2) that it makes the ejaculate more voluminous, so that by cohabitation, a very good part of the vagina becomes moistened, and the spermatozoa become distributed over the greater part 2 18 of the vaginal mucosa. This distribution is rendered necessary because some of the fluid flows from the vagina following coitus. In this manner the opportunity is given for a part of the ejacu- late containing the spermatozoa to be brought easier to the ex- ternal os, and (3) it happens that because of its content of sodium chloride the life of the spermatozoa is stimulated and prolonged.’’ A fuller discussion of the essential physiology of the various parts of the tract on the semen content, and fertility, will be taken up later. The changes in biochemical content, reaction, and the re- sult of the addition of bacterial products will also be more fully discussed. Spermatozoa: The history of the discovery of spermatozoa is very interesting, and for that reason a brief outline will be given. ‘The ‘‘semen threads’’ were first observed in the year 1667, by Ham, a student of Leeuwenhoek at Leyden. The dis- covery was announced, confirmed by findings in the dog and rabbit, and discussed by the latter author under the title: Ob- servationes Anthonii L. de natis e semine genitali animaculis (Upon the formation of young from procreative material). The sperms were taken to be animals on account of their motility, and their significance remained questionable if not unknown. Spal- lanzi, quoted by Marshall (29), was the first to show that the filtered fluid was impotent, and that spermatozoa in the semen were essential to fertilization. Kolliker, in 1841, discovered that the sperms arise from the cells of the testis, and Barry in 1843, observed the conjugation of sperm and ovum in the rabbit. This led to a clear understanding of the function of these important germinal elements. The spermatozoa are the male procreative cells, and are char- acterized by the possession of a head containing the chromosomes necessary for fertilization, and a tail capable of propelling the organism on its way to meet the ovum. The length of the entire sperm, including the head, is seventy-five to eighty microns. The head is nine and five-tenths microns long, and five and five-tenths microns wide. It may be divided into two principal parts, the head and tail. The head, for the larger part, is made up of the nucleus, and may be differentiated by staining reactions into a darker staining posterior part, an anterior lighter part, and often a still lighter area between the two. On the anterior part is a sharpened edge, the acrosome, which serves to perforate the ovum. The whole is surrounded by a very definite limiting membrane which often becomes obscured under abnormal conditions. The tail may be divided into three parts: connecting piece, principal part, and terminal filament. The connecting piece, the essential motile apparatus, is the thickest and strongest part, and joins the tail proper to the head. It consists of the central axial filament, a spiral filament around this, and an outer mitochondrial covering. Anteriorly it is limited by the anterior portion of the posterior chromosome, and posteriorly by the annular chromosome. The 19 anterior chromosome is directly connected with the head, there appearing a light break here at the neck where separation fre- quently occurs. This neck serves as a joint for the motion. The axial filament, therefore, does not reach the head, but extends back from the anterior part of the posterior chromosome. The principal part consists merely of the axial filament, and a thin outer covering, while the end piece is quite thin and is made up solely of the uncovered axial filament. The finer struetures are seen only when special staining reactions are used, and then only when the sperms are obtained directly from the testicle. The function of the sperms is of course primarily that of fertilization. Numerous observers have, however, thought that they might have some other definite, though unknown, use. An editorial in the Journal of the American Medieal Associa- tion (33) raises several important questions regarding this ob- secure phenomenon. The fact that an enormous number of sperma- tozoa are produced, and only one, or at most, a few perform the function of fertilization, raises the question as to what becomes. of the remainder. It is stated: ‘‘Zoologists have found that in some of the invertebrates the spermatozoa invade the entire body of the female, and in some species they reach the ovum by pene- trating the euticle from outside and migrating to their goal. Studies on rodents show that the sperms invade the epithelium of the generative mucosa and underlying connective tissue. These tissues seemed to be stimulated to growth, suggesting that this. may influence the uterine mucosa in its preparation for receiving and embedding the egg, and in forming the decidua.’’ It has been shown that the sperms contain a specific protein capable of producing antibodies in the blood plasma, by citing the fact that rabbits develop a distinct Aberhalden reaction for testicular pro- teins shortly after cohabitation. One very important observation showed that by immunizing female rabbits with sperms they were rendered sterile for some time, although after a few months they again became capable of impregnation. The question raised is: rh if the spermatozoa invade the female tissues and cause the for mation of specific antibodies which are capable of prevent- ing fertilization, may not such a process participate in the prob- lem of sterility ?”? This very problem seems to be a factor in ex- plaining why some couples who are not fertile to each other subsequently are both fertile when they cohabit with other indi- viduals. Motility: After clinical observation of the motility of the sper- matozoa of the bull, I find that it differs little or none from the types as observed by Reynolds (34) in his work on human sperma- tozoa. His observations are so accurate and well deseribed that they will be given in his words. ‘‘All normal motions appear to be consecutive phases. Initial motion, i. e., motion as seen in fresh semen under favorable conditions, consists of a lashing of the after part of the tail from side to side which is so rapid 20 as to constitute vibration. It produces rapid forward motion in a practically straight line, the head, middle piece, and forward por- tion of the tail maintaining their position in the line of motion with practically no swaying from side to side. The action of the flagellum is so rapid that it is quite impossible to follow its indi- vidual movements. Spermatozoa swimming in this manner head against the current and usually cross the field of observation in about five seconds in the absence of currents or obstacles. This type of motion will be described throughout the paper as ‘pro- eressive vibratile’ motion. Progressive vibratile motion is nor- mally succeeded after a variable length of time by what I regard as the second phase of normal motion. “‘The second normal motion differs from the first not only in its character but in markedly reduced speed. The tail movement alters to a long stroke from side to side and almost the whole length of the tail partakes in the stroke. This is, moreover, accom- panied by swaying of the head and middle piece through an are which is. always considerable and may even equal ninety degrees. The general outline of the spermatozoa, from being practically straight with almost non-detectable sharp, quick, small are vibra- tion of the aftertail, has become an S in outline, with large, slow, plainly perceptible undulations traveling gradually backward throughout the length of the spermatozoon. Speed has been lost and direction seems to be more specifically determined by the surroundings. Individuals at this stage show a pronounced choice of direction and go up to objects in the medium, from which they later make off as though the movement were determined by tactile reaction to some extent. This type of motion has, therefore, been named ‘undulatory tactile’ in contradistinction to ‘progressive vibratile.’ . ‘The third type of normal motion sueceeds the second and con- sists in a tendeney on the part of the spermatozoon to push itself against or into any small masses of cells, or sometimes other materials, which it may find in the neighborhood, bunting itself into any small cove that ean be found, and maintaining a slight burrowing motion by a lashing tail movement of the vibratile type not unlike the movements of the caudal fin of a fish. The move- ment of the flagellum in this third type is unlike the seeond type in that it is vibratile rather than lashing, but is slower than the vibratile. motion of the first type and less limited to the after- part of the tail. These spermatozoa are apparently not caught in the debris or unable to move off. From time to time, they back out of such a cove and seek another mooring place. ‘*This ‘stationary bunting’ motion is less universal than the other two. Many individual spermatozoa fail to attain it. It seems probable that only the most vigorous individuals ever reach this stage. It has not been encountered in unmixed semen or in any artificial mediums. It has been observed only when the sper- matozoon is in the secretions of the female genital tract. It is 21 most frequent when the spermatozoon is in contact with a nest of epithelial cells ‘