Chapter II: Part 2

“The three types of normal motion are not only distinctive but are always consecutive, i. e., the second follows the first after a period which is apparently determined both by initial vitality and by the favorable or unfavorable character of the medium, while the third has been observed to occur only in individuals which have already developed the second. They apparently constitute a normal cycle.

“This cycle is open to the theoretical explanation that these types of motion are directly adapted to the function of the spermatozoon; thus, the progressive vibratile motion which is characteristic of the earliest period of its existence appears especially suitable for its prolonged journey through the cervix and uterus to the fundus and tube. This is supported by the fact that during this motion it always heads directly against any existing current, and that during this stage of its journey it must under natural conditions be continuously exposed to the faint outward ciliary currents of the mucous membrane of these passages.

“The undulatory tactile motion which succeeds the progressive vibratile would then be well adapted to the later stage in which the spermatozoon has reached the tube, where its success in conjugation is dependent on its finding the ovum rather than on further progress.

“The stationary bunting type of motion is that which would be demanded by the passage of the spermatozoon through the egg membrane which has been so often observed in the lower animals. This very plausible explanation is, however, necessarily theoretical and must always remain so, as the conditions which surround the specimen on the field of the microscope vary in so many respects from those in which it accomplishes conjugation in the course of nature; but the practical importance of the study of types of motion is not affected by their explanation.”

The duration of motility is a variable factor, dependent entirely upon the environment in which the spermatozoa are placed. Within the body they usually survive at least a week. One author describes a case in which he found living sperms in a woman who stated that coition had not been experienced for three and one-half weeks. It has been stated with regard to human sperms, that their motion should continue or be capable of being re-established for twelve hours. Cary (35) states, “first, that in their proper medium and at the body temperature the viability of the sperm cells may extend over a period of a few days; second, that their prolonged vitality is probably dependent upon the normal lime salts of the prostatic fluid; third, that the sustaining power of the seminal fluid is increased by its union with the normal secretion of the female genital tract.” After death of the male animal they retain their motility in the genital tract for twenty-four to forty-eight hours.

Wolf (36) worked on this problem in rabbits, and summarizes as follows: “The motility of rabbit spermatozoa can be preserved for at least nine days by placing the juice of the epididymis in the Tyrode solution which had been buffered and to which glucose had been added. The solution is adjusted to a pH of about 7.4. Oxygen is passed in and a suitable amount of sodium bicarbonate added. The preparation must be kept at a temperature near the freezing point of water.” Under ordinary conditions motility persists outside the body only a few hours after ejaculation, but if the semen is kept quite cool till the time of examination on a warm stage, motility should be capable of being restored in at least a fair percentage of the sperms for twenty-four to forty-eight hours. The cells are more sensitive to heat than to cold, and even to dilute acids more than to alkalies.

Henle (quoted by Ellenberger) states that a spermatozoon under favorable conditions travels at the rate of twenty-seven millimeters in seven and one-half minutes, which makes three and five-tenths millimeters per minute. This is about sixty times the entire length of the spermatozoon, and twenty-one centimeters in an hour. Forward motion is also more pronounced when the swim is against the current, such as is produced by the cilia of the oviduct. It has been demonstrated that feebly motile sperms become very actively motile when placed on the mucosa of a fresh Fallopian tube.

TECHNIQUE

The material used in the work came from abattoir animals, bull calves and adult bulls raised in the department herd, and sires upon which clinical observations had been made by various veterinarians in the field. Semen samples, many of which were sent in, were collected as often as possible after the method described by Williams (16). The genital organs were removed with as little chance of contamination as possible, and taken or sent to the laboratory where the examinations were made soon after arrival.

All cultures were made by searing the surface carefully, tearing out a small portion of the tissue with sterile forceps, and placing it upon the media. In most cases, however, where fluids were present, tubes were inoculated with the material which had been drawn off with a sterile pipette. As stated by Carpenter (9), in his work on the female genital tract, the organisms usually live in the depths of the tissue. The media used principally were glucose glycerin agar (glucose 1 per cent, glycerin 3 per cent); plain agar, both with a pH value of 7.4, and Loeffler’s blood serum. Small amounts of sterile blood serum or defibrinated blood were added to most of the agar slants to insure better growths of streptococci when present. All tubes, to which the serum had been added, were incubated for forty-eight hours before inoculation to insure absolute sterility.

After inoculation, the agar tubes were sealed with sealing wax to give a partial oxygen tension which was quite necessary in isolating the streptococci. The growth of other organisms was by no means hindered by the procedure, for one tube from each organ was often left unsealed. Incubation was at 37° C, and the routine method of examining the tubes was identical with the method of Carpenter (9).

Whenever possible, a sample of blood was obtained from the animal either before, or at the time of slaughter, for agglutination with _Bact. abortum_ antigen. Extracts from the seminal vesicles, testes, and epididymes were injected into the guinea pigs and examined at the end of four to six weeks for the presence of _Bact. abortum_.

Sections of all organs were fixed as soon as possible in either Zenker’s or Helly’s fluid. Hematoxylin and eosin were used as routine tissue stains. Eosin and methylene blue, and Mallory’s connective tissue stain were, however, frequently utilized for special staining reactions.

The motility of the spermatozoa is best observed about half an hour after ejaculation, when the thick tenacious clot has started to liquefy. A drop of the fluid is placed upon a warmed slide, preferably one with a slight depression in it, and observation made with high or low powered objectives. The semen may be examined whole, or diluted with physiological saline solution. In the latter case, the sperms have a greater opportunity for freedom of motion in the absence of the thick viscid coagulate. A small vial of saline solution may be carried in one’s pocket where it will be kept warm, and a drop of this placed upon the glass slide. If the clot of semen is merely touched to this drop on the slide, plenty of spermatozoa will be deposited for an examination. This method is very satisfactory for the observation of motility, but needless to say, the undiluted semen must be used for the determination of the number of sperms present. If necessary, the specimen may be covered with a cover glass and the oil immersion objective used. While a warmed slide is quite sufficient to enable one to detect the presence of motility, the field soon cools and the sperms gradually become less motile. If possible, it is best to use a small electrically heated stage warmer, which keeps the field at a constant body temperature, so that the duration of the motion may be observed for hours if warmed physiological saline solution is added as the fluid evaporates.

Stained preparations are best made with _thin_ smears on the glass slides. This is conveniently done by placing a drop of the semen on a slide and smearing it over the surface with the edge of another slide. A fairly thin and even field is thus obtained. A still better method is to first dilute the semen with physiological saline solution, so as to obtain fewer sperms in the field. After drying the preparation in air, fixation may be produced by drawing the slide through a gas flame several times, by immersion in equal parts of alcohol and ether, or even by the use of tissue fixers such as Helly’s or Zenker’s fluids. For ordinary staining, heat fixation is the quickest, and at the same time is quite satisfactory. After the slide is cooled, or washed, to remove the fixing solutions, it should be placed for a few minutes in a _freshly_ prepared solution (1 per cent) of chlorazene, as recommended by Williams, to remove the mucus and proteid material which otherwise blur the field. Other authors (38) have recommended diluting the semen with about twenty volumes of a 0.12 per cent sodium carbonate solution in 0.8 per cent sodium chloride. From this liquid the cells should be centrifuged for several minutes, removed with a pipette, and smeared on the slide. Following this, the slide should be thoroughly washed, preferably for ten minutes in running water, after which it is ready for staining. Numerous methods have been used for this procedure, but the sperms are more or less erratic in their reactions to the dyes, and one must be very careful to use the same method in all samples, in order to obtain uniform results. For quick staining, to bring out gross abnormalities of structure, and number of sperms present, one may use Gram’s stain, or a light stain with any of the aniline dyes, such as fuchsin. To bring out the finer structure, particularly of the head, more careful technic must be employed.

Carnett and others (38) recommend the following: “The method of staining by iron-hematoxylin, particularly when supplemented by a cytoplasm stain, has proved, on the whole, the most satisfactory, and possesses the additional advantage of being absolutely permanent, a quality that few anilines can boast. The method consisted of treating the fixed object—and here the fixing agent was heat—with a two per cent solution of iron-alum for from two to four hours. The excess of iron-alum was then _completely_ removed by pure water, and the object treated with a solution of hematoxylin (one per cent aqueous) for twelve hours or longer. The cells by this time were perfectly black. However, a 1 per cent solution of iron-alum removed the stain from the cytoplasm, leaving the chromatin of the head, the centrosome, and the axial filament a brilliant blue-black. Care must be taken that the preparation is not over-decolorized. After decolorization a saturated aqueous solution of eosin was added for from one to three minutes. This stained the protoplasmic envelope pink, and, unless the envelope is overstained, the view of the inner structures is not impaired in the least.”

Williams (17) recommends using two staining solutions, one of alcoholic eosin and fuchsin, the other a diluted methylene blue. The results obtained are, however, more or less erratic, due to the unstable character of the former stain, and the ease with which one may over or under stain. Many beautiful specimens may, nevertheless, be obtained by this method. I have frequently used a fairly quick method, though one not satisfactory in all cases, which consists of staining for five or six minutes in a saturated aqueous solution of fuchsin, washing in water, and counterstaining for a few seconds in a strong solution of methylene blue. A quite satisfactory method is to stain from two to five minutes in a saturated aqueous solution of methyl green, with the application of gentle heat. The heat may be applied by warming the slide over a gas flame as it steams, or by placing the jar containing the stain in a hot water bath. The slide is then washed thoroughly and counterstained for five minutes in a strong aqueous solution of eosin. This is a fairly reliable method, and many excellent preparations may be obtained by its use. The nucleus is stained green, the anterior part of the head and all of the tail pink. So far, I have found this a very reliable stain for routine work.

PATHOLOGY

In the genital tracts that I have studied, a complete pathological and bacteriological examination was made wherever possible, but in many of the abattoir animals, and certain others, gross and microscopical examinations only could be made. The genital organs of one hundred and ninety-six males have been examined, and the gross or microscopical changes, or both, determined. The abattoir animals were from a large slaughter house, and a small local plant.

Of the tracts, the pathology of which was studied, two were from aborted fetuses; seven from apparently normal young calves; four from mature fertile bulls; and sixteen from mature infertile or sterile sires. The remainder (167) of those examined were from abattoir animals. Besides these, three specimens of seminal vesicles, and seven of testes were studied.

The tracts of the aborted fetuses and veal calves were apparently normal, both on gross and microscopic examination of the vesicles and testes. On gross examination, the tracts of the mature fertile bulls were normal, except for the presence of many fine connective tissue tufts and strands upon the serous covering of the tails of the epididymes, and adjacent portion of the parietal layer of the tunica vaginalis. Microscopic sections of all parts were apparently normal. The more important pathological changes in the tracts of the sixteen sterile or infertile bulls are given in the appended chart. The tracts are numbered the same as in Group VI of the report of the bacteriological findings; that is, any particular number in either table refers to the same animal. References are made throughout the text to some cases which appear in this group of animals. Prostate and Cowper’s glands are not included in the chart as they were not examined in some, and were negative in the others. Fibrous tufts and strands were present on the covering of the epididymes in each animal.

The study of sections from the abattoir animals, as well as those from the sterile or infertile bulls, forms the basis for the following observations upon the pathology of the male genital tract. The tracts secured from the abattoir were studied for the most part on the basis of the organ rather than on that of the animal. For example, all sections of testes were placed in the same bottle of fixer, and the same plan followed for the other organs.

TESTES: The testes seldom presented gross alterations of structure except for abscess formation, which, according to Williams, occurs more frequently in the bull than in any of the other domesticated animals. He also states that arrest in development by which the organs remain soft, flaccid, and somewhat smaller than normal is not uncommon. One very interesting specimen, which typifies abscess formation, came from a bull with a history revealing that one testis had become much enlarged, hot, and painful. These symptoms developed very rapidly. Anorexia was well marked. Local applications were used for several weeks, but at the end of two months the condition was so little improved that unilateral castration was performed. The general condition of the animal soon improved, but after a year of service he was so uncertain compared to what he had been, that he was sent to the butcher. It was impossible to obtain the other testicle for study, though it undoubtedly was abnormal. The testicle removed was considerably enlarged, measuring twenty by ten and one-half centimeters. The tunica albuginea presented a thickness of six millimeters, and was made up of firm sclerotic tissue. The epididymis was not recognizable in the mass. Testicular tissue was almost entirely gone. The only remains, of what appeared to have been parenchyma, was an elongated irregular area at one side of the organ. This tissue consisted of a whitish opalescent material, speckled with varying sized abscesses. This organ is pictured in Fig. 3. The remainder of the organ consisted of a thick yellowish caseous mass. _Streptococcus viridans_ was recovered from the outer portion of the organ, and guinea pig inoculations failed to demonstrate _Bact. abortum_.

Microscopically, changes are quite common and varied in character. In the seminiferous tubules, the changes range from a slight desquamation of the germinal epithelium to atrophy and complete degeneration of the entire tubule, as was the case in the left testis of Bull 1. In the mild cases, spermatogenesis occurs apparently in a normal manner up to the spermatid stage, at which point many of the cells degenerate and slough off. These appear in the seminal fluid, associated with the few sperms that reach maturity. This sloughing and degeneration may be localized in a few of the tubules, or it may be widespread over the entire organ. Likewise, the changes may involve not only the more mature cells, but they may be so severe as to cause almost total degeneration and desquamation of the seminal epithelium, as in Fig. 15. These defects in spermatogenesis are of course evidenced in the semen by the presence of immature, or abnormal types of sperms. With cessation of spermatogenesis or degeneration of the epithelium of the entire gland, no sperms are formed. Not infrequently one finds numerous tubules, or even the entire testis in which the germinal epithelium is intact, but there is little or no evidence of mitosis, as in some tubules of Bull 6. The cells are several layers deep as in the normal condition, but they are not dividing. This condition is shown in Fig. 13.

In the more chronic forms, the tubules become atrophied, and frequently disappear entirely. The membrana propria may become thickened, due to excessive connective tissue formation, or infiltration with serum or exudate. On the other hand, a distinct atrophy may occur. The stroma of the organ not infrequently is thickened by inflammatory exudates, or by a noticeable increase in the connective tissue. In some testes, the connective tissue is so much increased that the tubules rapidly become atrophied, and disappear. In abscess formation, due to acute inflammations, the entire organ becomes enlarged, markedly hyperaemic, and infiltrated with leucocytes. Necrotic areas appear here and there in the parenchyma. The rete often shows a marked degeneration of the lining epithelium, and atrophy caused by increase of the interstitial connective tissue.

EPIDIDYMIS: This organ not infrequently presents gross abnormalities, and very often is pathological on microscopic examination. Acute, inflammation, with induration or abscess formation, is very common in the tail, but less so in the head and body. Possibly this is caused by the fact that the tail is the most pendant portion of the organ. In these cases, the tail is enlarged, soft, and quite hot and painful on physical examination. Enlargement, due to a connective tissue induration, occurs occasionally in all three parts, and the inflammation may produce adhesions to the adjacent serous membranes. Inflammation of both the parietal and visceral layers of the tunica vaginalis is very common. In those cases, the membrane usually is quite hyperaemic, and on the surface it presents many small reddened tufts of newly-formed connective tissue. In adult bulls it is exceptional not to find at least slight evidence of some previous inflammation. In all of the numerous bulls examined, both apparently normal and sterile, I have found but one in which some evidence of inflammation (either present or past) could not be found. Along with the fibrous tufts, are numerous fine strands of connective tissues passing from the covering of the tail of the epididymis to the adjacent portion of the parietal layer of the tunica. The strands often extend even to the upper part of the head.

Microscopically, inflammation of the part is shown by hyperaemia, loss of cilia of the lining cells, and exudation. In the more severe forms, the lining cells which furnish considerable secretion for the nourishment and stimulation of the sperms, become degenerated, and are exfoliated into the lumen, as in Fig. 21. This condition is very common in sterile bulls, and those of lowered fertility. In the chronic types, the interstitial connective tissue is increased in amount, leading to degeneration and atrophy of part or all of the tubules, as in the case of Bull 2. Infiltration with leucocytes, and necrosis, are the predominating lesions in the pyogenic types of inflammation.

DUCTUS DEFERENS: This tube seems to be peculiarly free from severe inflammatory processes, and when these appear they are limited to the mucosa. The cells of the lining membrane not infrequently show a mild type of degeneration and exfoliation, or in the more chronic forms, the entire membrane degenerates and disappears. In man, the duct occasionally becomes occluded, but so far I have failed to find this condition in the bull. Undoubtedly, when the occlusion does occur, it is near the origin of the duct at the tail of the epididymis.

SEMINAL VESICLES: The seminal vesicles and epididymis, especially the tail, seem to be the parts most subject to extensive pathological changes, and bacterial invasion. In most instances, diseased vesicles present gross manifestations recognizable on clinical examination, while on the other hand microscopic changes may be present in the absence of gross lesions. As diagnosed on physical examination, or even on post mortem examination of the tract, the various forms may be classified into:

1. _Acute Catarrhal Type_: In this form, the vesicles are usually enlarged, soft, and more or less reddened by hyperaemia. On physical examination of the animal, distinct flinching is produced when pressure is applied to the organ. Enlargement may even be absent in the early stages, and the diagnosis may be made from the extreme sensitiveness alone.

2. _Suppurative or Cystic Types_: In both of these types, the vesicles are usually enlarged, either uniformly, or, as is usually the case, in localized areas. The suppurative form may extend over the entire gland, forming one large encapsulated abscess, or on the other hand, it may take the form of variable sized abscesses with thick sclerotic, or thin fluctuating walls. Occasionally the abscesses rupture and discharge their contents into the rectum. Dr. Williams presented one case of this type. One vesicle was apparently normal, whereas the other was about five times larger than normal, and consisted of a dense outer capsule which was adherent to all surrounding parts. On dissection, it was found that the organ consisted of abscesses of various sizes, the larger one of which had ruptured some time previously into the rectum, leaving the distinct remains of an opening into that part. The cystic form may occur either with or without suppuration. One case came to my attention in which both vesicles were made up of abscesses of varying sizes as well as of a smaller number of cysts. Evidently the cysts were of the retention type, and were secondary to the pyogenic infection.

3. _Chronic or Sclerotic Type_: This form is characterized by a distinct firmness with or without marked enlargement. The condition may be accompanied by disease of the parenchymatous tissue or it may take the form of a chronic productive inflammation of the interstitial tissue. This inflammation may be simply a superficial thickening, or it may extend in between the lobules.

4. The _Peri-vesicular or “pan-inflammatory” Type_ usually is the result of severe inflammation of the vesicles, with probable rupture of some of the smaller cysts or abscesses upon the surface. The vesicles are, as a rule, considerably enlarged and buried in a dense mass of adhesions which involve neighboring structures. The vesicles cannot be palpated on physical examination, and it is only on careful post mortem dissection that they may be studied. This type, however, is quite rare,—two cases only having come to my attention. In both, the vesicles themselves were markedly affected.

Microscopically, changes in the vesicles are quite frequently encountered, even in the absence of gross manifestations. In the acute catarrhal forms, the mucosa and submucosa are hyperaemic. The lining cells show various forms of degeneration, and there are, as a rule, inflammatory exudates in the lumen. As the inflammation progresses, the lining cells degenerate further, and become cast off into the lumen of the glandular cavities, as in Plate VI. The normal clear mucous secretion becomes mixed with fibrin, leucocytes, and cellular debris. These changes may involve merely parts of the organ, or they may be quite extensive. With large sections, one may find the inflammation in all stages, from the mildest catarrhal type, to complete degeneration and exfoliation of the secretion-forming mucosal cells, and filling of the cavities with degenerated cells, leucocytes, and debris. Frequently the interstitial tissue is in no way affected, but at times it is thickened by oedematous exudates, leucocytes, and fibrin. The chronic interstitial form is characterized by a considerable increase of connective tissue,—producing marked atrophy, or even complete obliteration of the glandular cavities. Microscopically the suppurative form may be diffuse over the entire gland, or as stated previously, may be in the form of localized abscesses, with or without a thick connective tissue wall. The parenchyma in these cases is usually extensively degenerated and atrophied in those parts that have not undergone suppuration and necrosis. The cysts appear to be of the ordinary retention type, and may or may not be accompanied by extensive changes in the lining epithelium.

Both the abscess formation and cystic conditions are undoubtedly initiated by an obstructive inflammation of all or part of the excretory duct. This is, however, a protective mechanism, for where the duct is closed the bacteria and exudates are unable to reach the urethra and contaminate the semen.

PROSTATE AND COWPER’S GLANDS: These glands were more or less neglected in the early part of the work, but later were subjected to the same examination as other parts. Of the thirty-six of each type of gland examined, I failed to find one with any gross changes, but two prostates were found that presented a mild catarrhal inflammation of the mucosa. It is probable that Cowper’s glands, as well, occasionally undergo inflammatory changes.

SEMEN: The semen, made up as it is of mixed products of the testes and accessory sexual glands, is very often abnormal, as would be expected in view of the frequency with which changes occur in the glands contributing to its formation. The normal semen is remarkably adapted to its function of nourishment and stimulation of the spermatozoa, and their conveyance to the internal female genital organs. The spermatozoa are extremely sensitive to changes in their environment, with the result that any alteration of the physical or biochemical content of the seminal fluid may cause death of the sperms. With this in view, we must remember that disease of any of the contributing organs is a potential danger, and threatens the potency of the animal. Each or all of the glands may add bacteria, acid secretions, or inflammatory exudates. On the other hand, they may not function at all. In each case, however, the semen is altered.

Unfortunately it is impossible with present methods to obtain the fluid absolutely free from vaginal mucus, but with care it may be secured reasonably free from contamination by douching the prepuce of the bull and vagina of the cow before service. This method was used as often as possible in collecting the samples. The usual amount of semen obtained was from six to ten cubic centimeters.

With a hypersecretion of one or all of the glands, the semen becomes quite thin and watery, with a deficiency of solid matter, together with changes in reaction. On the other hand, hypofunction results in a secretion too viscid, which is equally unsuited to the requirements of the spermatozoa. The thin watery semen clots imperfectly or not at all, and clotting is essential in protecting the spermatozoa from the acid secretions of the vagina. Likewise, a medium too viscid is a distinct hindrance to motility. Changes in reaction are very frequently encountered. The sperms are very sensitive to dilute acids, so that with even a slight acidity motility may diminish or entirely cease. Purulent inflammatory exudates are occasionally mixed with the semen, and although the pus cells themselves have not been found to be destructive to the sperms, certain degeneration products in the exudate are very toxic, and inhibit or destroy the motility. So far, I have failed to find red corpuscles present. One very interesting sample of semen was quite thick, of a yellowish green color, and of a distinctly acid reaction. The secretion from the vesicles was later found to be of this same character, and was due to a _Ps. pyocyaneus_ infection. The vesicles were highly inflamed and degenerated. The spermatozoa were in this case markedly decreased in number, and devoid of motility.

The early precipitation of the “Boettcherchen” crystals seems to be intimately connected with sterile semen, or spermatozoa of lowered vitality. Likewise, a decrease in solid matter is often seen in a deficient secretion. In normal semen, the clot disappears after standing a time, and a thick sediment settles out. This sediment is decreased in amount as a rule in abnormal semen.

SPERMATOZOA: Spermatozoa, the essential germinal elements, are very frequently abnormal, changes in which may be manifested in many ways. We may divide the deviations into changes in structure, and changes in the motility which is so indicative of the intrinsic vitality of the sperm. Reynolds (34) describes two forms of abnormal motion. The first is “rotary swimming,” in which the sperms move forward progressively, and sometimes with fair rapidity, but in a spiral screwlike manner. He states that this type of swimming is very awkward, easy to recognize, and is usually of quite long duration. The other form termed “pendulum swimming,” he states, is less common than the rotary swimming and is usually confined to relatively fewer sperms in a given field. “In this the middle piece and upper tail seem to lose their flexibility and balance to a considerable degree, and the lower tail motion swings the forward part of the spermatozoon to and fro with a pendulum movement. This type of swimming yields very poor progress.”

One factor we must bear in mind in the study of the semen obtained from the vagina, is that the spermatozoa may be highly motile before ejaculation, but the admixture of hostile vaginal mucus may inhibit or destroy the motility. On the other hand, the conditions may be reversed. Cary (35), in one instance, found that the spermatozoa in a sample of semen collected from a condom, appeared to be of very low vitality, while when they were mixed with the vaginal secretions, an exaggerated activity was manifested. May we not have to contend with this factor in some herds in which there is a very distinct acid and toxic vaginal secretion from the products of cervicitis and vaginitis?

In a study of motility, we must consider not only the abnormal types which may be encountered but the percentage of motile cells, and the duration of the movement. In _necrospermia_ all the ejaculated cells are motionless or dead. In other specimens, varying percentages of the cells are without motion, and the others may be possessed of full and lasting motility. On the other hand, the motility in some cases is very active at first, but quickly subsides even under the best of conditions. The appearance in freshly ejaculated semen of numerous sperms that have a tendency to take on the “undulatory tactile” type of motion when they should be in a highly active state, is very indicative of lowered vitality. Many specimens present this very picture, whereas the very active progressive movement should, under proper conditions, survive for a considerable time before it gives way to the second, and slower type. The cells frequently early bunt into epithelial cells or clumps of immotile sperms, then back out and move around sluggishly, only to repeat the same performance till they stop moving entirely. I have seen one specimen in which the sperms all tended to clump. Whether this was the result of some agglutinative substance in the vaginal secretion is problematical. I have seen several specimens of semen in which practically all the sperms were motile when first examined, but the motion did not survive for any great length of time. Even a small percentage of motionless sperms or of those showing lowered vitality is a considerable factor in potency. Although millions of the germinal elements are ejaculated into the vagina, large numbers of them are destroyed or become motionless there, and a small number is left behind in the cervix and uterus; so that even though but a single sperm is required for fertilization, the chances of impregnation are diminished in proportion to the number of dead or defective sperms.

_Aspermia_: Absence of spermatozoa in the semen is rarely encountered, and is probably due either to total cessation of spermatogenesis, or to an obstruction at some point in the system of excretory ducts. I have seen but one case of this character. The semen of this bull was greatly increased in amount, and of a thin watery consistency. Due to lack of cooperation on the part of the owners, the tract could not be obtained for study. _Oligospermia_, or a diminution of the number of spermatozoa, is quite common, and is undoubtedly associated with defective spermatogenesis, either as a result of poor mitosis of the seminal epithelium, or degeneration of the elements before maturity. This condition may vary from the finding of only occasional dead sperms in the field, to but a slight decrease in the usual number of normal sperms observed.

Abnormalities in morphology may be classified into immature types, and deformities or imperfect development of the head and tail. Defective spermatogenesis occurs so frequently that it is not surprising to find spermatozoa in various stages of development cast into the excretory ducts. The various stages passed through in the development, from spermatogonia to adult sperm, are numerous, and it therefore is to be expected that we should see in abnormal semen many different immature forms. No classification of the various types can be made, but a clearer understanding of them can best be obtained by a review of the process of spermatogenesis.

Spermatocytes and spermatids are seen in the more severe types of defective spermatogenesis, and are relatively uncommon, while the more mature forms that result from the transformations of spermatid to adult cell are very often seen. Some of these intermediate types are large oval cells without distinct nuclei and as a rule with poorly developed tails. Cells with no tails or distinct nuclei, those with protoplasmic appendages to the head or tail, and various other types, are occasionally encountered. Most of these are motionless and incapable of producing impregnation. Others are active, but survive a comparatively short time. According to Cary, the production of the immature cells is an effort on the part of the testes to supply an abnormal demand, and their presence indicates that the fertility of the semen is impaired.

The deformities, which may be divided into cephalic and caudal groups, are also the product of defective spermatogenesis, or they represent a degenerative process induced possibly by abnormalities of the fluid environment. It is rather difficult, however, to distinguish between deformities and immature types. The two most common cephalic deformities are what might be called macro and microcephalic forms. In the former, the head is enlarged to a greater or less extent, it is usually defective in staining qualities, and its outline is indistinct, due to degeneration of the covering membrane. This type is seen in Fig. 32. Also the shape of the head is usually abnormal, being either quite rounded, long and narrow, or short and very broad. Cells with protoplasmic appendages, though they are more properly an immature type, occasionally give the head a greater volume. Microcephalic sperms vary from those slightly smaller than normal to those in which the head is represented by a slight knob. In some cells, the head is small and round, in others, short and stubby, while another type is normal in outline but diminutive in size. These forms likewise are, as a rule, deficient in staining qualities, and are undoubtedly degeneration forms, occurring either as the result of faulty development, or degeneration subsequent to their formation. Cary believes they are degeneration types because in the majority of cells the tail is apparently fully formed, and in the normal process of evolution the tail is the last part of the cell to be exhibited. Double headed forms are quite rare, but they nevertheless appear at times. Their significance is difficult to explain. Another very frequent deformity of the head is a marked constriction at the posterior part so that it is the shape of a pear or top as in Fig. 27. In some, the head is otherwise normal in size, while in others it is much elongated, as in Fig. 28, or considerably atrophied. A constriction at the middle of the head, as in Fig. 29, is not uncommon. Both defects are undoubtedly the result of nuclear deficiency, as the nuclear part of the head in these cases is much diminished in size, and stains very deeply or not at all. I have seen spermatozoa, the heads of which were like an inverted cone, with a bulging rounded base. Other heads are even somewhat contorted and bent on themselves, as shown to some extent in Fig. 26.

Under caudal deformities, the most frequent form encountered is a thickening of the connecting piece. This may occur as a uniform thickening, or as a bulging appendage. Rudimentary development of the tail, the presence of two poorly formed tails, and defective development of the connecting piece occur rather infrequently.

All these immature and defective types are, as a rule, motionless, and of course incapable of producing fertilization. Their presence indicates lowered fertility of the semen. Besides these deformities, there are sperms showing a curvature of the tail at an acute angle just posterior to the neck,—the so-called “wry neck.” Their significance is difficult to explain, but they occur frequently in semen fixed and stained by the same routine methods used on samples in which they are absent. They probably are not the result of the methods used in fixing and staining. Some think they are slightly immature types, or that the condition is produced by abnormal contractions of the tail. The majority of sperms, however, especially those from highly fertile bulls, do not show this type at all.

The most common changes in the spermatozoa, are those in which there is a separation of the head from the tail, and degeneration of the head as evidenced by reaction to stains. The separation of the head from the tail always occurs at the neck, and often is associated with degeneration or abnormalities of the head. The separation, in the majority of cases, indicates some lowering of vitality in the elements, although in many instances traumatism produced in making smears or collecting the samples is responsible. Various forms of abnormal staining of the head are very common. The cell membrane, which is normally distinct and sharp, becomes blurred in outline. Normally, the head takes a good differential stain, the anterior part staining lightly, and the posterior part somewhat deeper. The nucleus is distinct in outline and well defined. The lighter “inner body” stands out in well stained specimens. As the result of degeneration, the whole head may take the stain uniformly, either slightly or much deeper than normal, according to the degree of degeneration. The whole problem of staining, however, depends very much upon the methods used, and the care with which they are applied. When a good method is obtained, it should be adhered to, and used uniformly on all specimens. As a rule, however, a certain amount of practice will enable one to differentiate between the sharply outlined, clearly staining normal forms, and those that show abnormal reactions to the stains.

BACTERIOLOGY

A complete bacteriological study was made of the genital tracts of fourteen normal young veal calves (six to twelve weeks old), four mature fertile bulls, and sixteen mature bulls, either sterile or impotent to some degree. Together with these, the tracts of eleven aborted fetuses, seven calves dying from calf infections (scours or pneumonia), and sixteen bulls slaughtered at an abattoir were studied bacteriologically. Occasionally, studies were made of individual seminal vesicles or testes, when these parts alone were brought or sent in. The history of the abattoir animals was, of course, quite indefinite or entirely negative. On the killing floor, many tracts could be studied for pathological changes, but in the bacteriological work it was difficult to care for more than two tracts on each visit.

The results of the bacteriological examinations are given in the appended tables, in which the tracts are divided into six groups. The results in Group I. consisting of normal veal calves, indicate that the genital organs of young male calves are, under normal conditions, free from bacteria. Carpenter (9) obtained like results in examining the genital tracts of heifer calves. The cultures made from the seminal vesicles and testes of all these veal calves were, with two exceptions, negative. Both seminal vesicles of one tract and one of another yielded cultures of _Staphylococcus albus_.

Adult bulls of known fertility were naturally difficult to obtain, only the four animals in Group II being available for examination. Two of these (Nos. 1 and 2) were from the experimental herd kept by the department, and at all times had a good breeding history. The other two were good breeders, but were slaughtered because of poor pedigrees. Bull 1, raised in the department herd, had a severe attack of scours when a few weeks old, while the calfhood history of the other is not known, he having been purchased after reaching sexual maturity. The cultures from the genital organs of the former (Bull 1) were entirely negative, except those from the left epididymis and scrotal sac, which yielded growths of _Streptococcus viridans_. All the organs of the tract from this animal were normal, except for the fact that numerous strands of connective tissue extended from the serous covering of the tail of both epididymes to the adjacent part of the parietal layer of the tunica. The tract of the other failed to show any organisms. The only evidence of any abnormality was the presence of the same connective tissue strands on the tail of the epididymis, as in the first tract. The other two bulls gave negative cultures from all parts.

Of the sixteen bulls in Group III, slaughtered at abattoirs, and in which no history was available, eight failed to show the presence of any organisms in their genitalia. Of the others, the vesicles yielded cultures of _Staphylococcus albus_ nine times, and _streptococci_ four times. _Staphylococcus albus_ was recovered once from the prostate, and once from Cowper’s glands. The testes gave cultures of staphylococci in two cases, and _Bact. abortum_ in one. No observable anatomical changes accompanied the presence of the Bang bacillus in this case. The epididymes showed growths of staphylococci five times, and streptococci on three occasions. _Streptococci_ were isolated from the scrotal sacs of eight testes.

The results in Group IV (aborted fetuses) show that bacteria are often present in the seminal vesicles or testes of these animals. As a rule, however, the organisms are identical with those isolated from the blood or other parts of the animal. This is to be expected, however, for because of the feeble resistance of the fetus to any infection, the organisms circulating in the blood may be isolated, as a rule, from many different organs and tissues. All samples of blood set with _Bact. abortum_ antigen were negative, irrespective of whether or not the organism was recovered from the blood or other tissues. This is in accordance with the findings of Carpenter in the female fetus,—the resistance is so feeble that few or no antibodies are formed to combat any existing infection. _Bact. abortum_ was recovered in two cases from the vesicles, and in four cases from the testes, but in each instance the same organism was present in the blood or other tissues of the body.

The results from the tracts of the calves dying of calf infections are given in Group V, and show that five were negative. The other two showed _B. coli_, staphylococci, and streptococci, in the organs indicated by the chart.

In Group VI, the mature infertile or sterile bulls, there was a comparatively wide variation in the type of organisms encountered, but streptococci and micrococci were the most common invaders. In the order of the frequency of infection, the organs would be enumerated as follows: Vesicles, epididymis (usually the tail), scrotal sac, testes, prostate, and Cowper’s glands. The first three parts mentioned usually contained bacteria. A streptococcus was the usual invader of the scrotal sac, and very probably was the cause of the connective tissue tufts and strands so frequently seen. The vesicles and epididymes gave, in the order of the frequency of their occurrence, staphylococci, streptococci, _B. coli_, and _Ps. pyocyaneus_. The streptococci were usually of the viridans group, though a few were hemolytic, and two strains were indifferent to blood. The testes gave growths in only eleven instances,—staphylococci eight times, streptococci two times, and an unidentified rod once. The prostate yielded staphylococci twice and Cowper’s gland once.

As emphasized previously, the vesicles and tail of the epididymis are most subject to infection and degenerative changes. At the same time, they are intimately connected with the secretion of the semen. Once the epididymis becomes infected, there is nothing to prevent the organisms, together with inflammatory products, from being mixed with the semen and ejaculated during coitus. Also in the vesicles, unless the inflammation is so severe as to occlude the excretory duct, the organisms are mixed with the vesicular secretion, which is emptied into the urethra during ejaculation. Carried along with the bacteria, are, of course, toxic products, degenerated cells, and the otherwise altered secretion of the glands. One interesting case noted was that of a bull that had passed from a state of fertility to that of complete sterility during a period of two months. The semen was semi-fluid, greenish yellow in color, and contained a very few non-motile spermatozoa. Post mortem examination showed that the vesicles had undergone abscess formation and that they contained yellowish green material similar to that which had been discharged during copulation. _Streptococcus hemolyticus_ and _Ps. pyocyaneus_ were isolated from both vesicles, and from the semen. _Micrococcus albus_ was isolated in nearly all cases of vesiculitis and was often associated with _Streptococcus viridans_ or _hemolyticus_.

Bacteriological studies of the semen are, on the whole, more or less unsatisfactory, due to the present difficulty in obtaining samples free from any chance of contamination. In most of the abnormal bulls, bacteria of various types were isolated from the semen, most of which agreed culturally with those later isolated from the internal genital organs of the same tracts. The method of culturing consisted of douching the prepuce of the bull and vagina of the female with sterile saline solution before breeding. Samples of vaginal mucus were taken before service, and the flora compared to that after douching. This method of douching produced vaginal samples relatively free from bacteria, at least so much so that the post coital fluid demonstrated that many organisms must have been introduced from without. Whether or not they came in with the semen is problematical, but in all probability this was the method of introduction.

I have so far failed to obtain _Bact. abortum_ from the tract of an adult animal, either by direct culture or guinea pig inoculation, except from the testicle of one abattoir bull. The agglutination tests with _Bact. abortum_ antigen were all negative, except for two abattoir bulls. The results so far obtained would seem to indicate that, in accordance with the findings of other workers, the Bang organism seldom invades the male genital tract, or does not thrive there after its introduction. Schroeder (12) and others, have, however, on various occasions, recovered the organism from the bull, and the former author even states that it invades the vesicles and is eliminated with the semen.

DISCUSSION

A complete discussion of those factors which have a bearing on reproduction and fertility in an animal, includes not only a thorough study of the genital tract, but an appreciative consideration of various extrinsic factors. The effect of environment has long been known to have a marked influence upon breeding, particularly with reference to animals in domestication. Diet, though long relegated to a minor phase of the question, has, within recent years, come to be a matter of prime importance with regard to its bearing upon the entire body metabolism. The endocrine organs preside over and regulate the growth and functioning of the genital organs from the earliest embryonic stage to the cessation of sexual life. Any derangement in one results in functional or organic changes in the other. In a given mating, we must take into consideration such factors as impediments to coitus, as well as those numerous agencies in the female which may interfere with the union of sperm and ovum, or with the successful implantation of the fertilized egg in the uterus, and its growth and development there till normal parturition takes place. Successful reproduction depends upon the mating of sexually sound females to equally sound males. Considering the various factors which govern reproduction, sexual soundness must necessarily depend, to a large extent, upon a good general condition of the entire body.

ENVIRONMENT: The effect of environment on fertility in the bull is no doubt a minor factor. Cases in which changes in environment affect fertility probably occur, however, particularly when fear and other psychic disturbances play a part. Marshall (29) states: “It would seem probable that failure to breed among animals in a strange environment is due not, as has been suggested, to any toxic influence on the organs of generation, but to the same causes as those which restrict breeding in a state of nature to certain particular seasons, and that the sexual instinct can only be called into play in response to certain stimuli,—the existence of which depends to a large extent upon appropriate seasonal and climatic changes.”

DIET: Under this heading we may include not only the effect of deficient food, but also constitutional disorders, as a result of which the organs of generation and those glands guarding their function receive insufficient nourishment. It is a well known fact, and long has been, that animals fail to breed when they are in a run down condition or when they are fed a deficient diet. Cary (35), quoting Hagner, states that the virility of the spermatozoa is often in direct proportion to the general physical condition of the patient.

Reynolds (34) emphasizes the fact that it is an established principle among animal breeders that a high protein diet in both sexes is essential to full fertility. “Oligospermia with deficient vitality of the spermatozoa is not infrequently found from constitutional disorders. It can easily be demonstrated in animals that both low diet and conditions of life that produce a nervous excitable state are attended by oligospermia.” Animals that are closely confined, those that are over-fat (show animals), as well as those fed a deficient ration very frequently fail to breed, but exercise and change of diet soon overcome the impotency.

Dutscher, Hart, Steenbock, and other biological chemists have done extensive work to show the essential importance of vitamines and minerals in the diet. Their results indicate that animals cannot thrive and breed normally when fed a diet composed solely of the products of one plant. There must be variety, and there must be not only a correct nutritive ratio, but the mineral and vitamine content must be present as well. Cows fed on the products of one plant often failed to breed, and if conception occurred, it invariably resulted in a premature birth, or the birth of weak and poorly nourished calves. The work of these authors is fundamental, and brings out many important points. Is it not probable that the deficient diet results in weakened tissues which are easier prey to the invasion of bacteria?