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.