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.