Part 3

Motility: After clinical observation of the motility of the spermatozoa of the bull, I find that it differs little or none from the types as observed by Reynolds (34) in his work on human spermatozoa. His observations are so accurate and well described 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 as to constitute vibration. It produces rapid forward motion in a practically straight line, the head, middle piece, and forward portion 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 individual 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 ‘progressive vibratile’ motion. Progressive vibratile motion is normally 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, accompanied by swaying of the head and middle piece through an arc 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 arc vibration of the after-tail, 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 succeeds the second and consists in a tendency 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 can 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 movement of the flagellum in this third type is unlike the second 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 afterpart 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 hunting’ 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 stair”. It has not been encountered in unmixed semen or in any artificial mediums. It has been observed only when the spermatozoon is in the secretions of the female genital tract. It is most frequent when the spermatozoon is in contact with a nest of epithelial cells....

“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.