A study of some factors influencing fertility and sterility in the bull — Edition Insights

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.

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.