New Methods of Handling Semen

Investigations into the physiology of spermatozoa stand at the peak of the new research related to artificial breeding.

In the operation of breeding associations, it is usually necessary to ship semen some distance, and it was difficult to maintain it at a temperature of about 40° F. A simple method has been developed that is satisfactory for use within the radius in which most federated associations operate. The semen is sealed in a test tube with cork and paraffin, and labeled as to name and other identification of the sire, when the semen was drawn, its sperm count, rate of dilution, and any other pertinent information. Three thicknesses of heavy paper are placed around the test tube. The package is then wrapped, together with a container of ice, in an insulated paper blanket and placed in a corrugated cardboard box or carton. The box is securely sealed or tied and addressed, ready for shipment. If semen is to be in transit more than 12 hours, a second container of ice ( which is a rubber toy balloon or a pint can) is added to the package.

Some of our most important new knowledge about the subject comes from several research workers at Cornell University, New York—G. W. Salisbury, Irvine Elliott, N. L. Van Demark, E. L. Willett, J. A. Zelaya, E. Mercier, and others. One of the problems they tackled was the extent to which semen can be diluted, thereby increasing the number of females upon which one ejaculate can be used. They studied bull semen diluted at the rate of 1 part to 8, 12, 16, 24, and 50 parts of yolk-citrate diluter. The average numbers of spermatozoa contained in 1 cubic centimeter were 150, 104, 80, 54, and 26 millions for the respective dilution rates. Results obtained in the use of this semen on large numbers of animals indicated that there were no statistically significant differences in the fertility of the semen diluted at these five rates.

H. H. Habibullin found that successful insemination resulted from doses of 0.025 cc. of ram semen containing 80,000,000 spermatozoa. Seventy-five percent of the ewes became pregnant, compared to 78.2 percent in ewes receiving 0.05 cc. of semen containing 160,000,000 spermatozoa. Experiments with goats showed that doses of 0.05 and 0.025 cc. of semen stored for 6 hours before insemination were enough to insure a normal number of conceptions. (A cubic centimeter is about a thimbleful.)

Egg yolk is an important constituent of both egg-yolk-phosphate and egg-yolk-citrate diluters. Dennis T. Mayer and John F. Lasley, during research at the University of Missouri, isolated an active resistance factor from egg yolk that gives a water-clear solution in phosphate buffer and that has proved more effective than the original ego, yolk-buffer mixture in increasing the resistance of spermatozoa to adverse conditions. A. H. Frank, C. A. Smith, and A. Eichhorn, of the Bureau of Animal Industry, described promising results with a diluter manufactured from chick embryos for bull semen. Still other tests have been made by workers at Cornell University in which yolk citrate, incubated egg, and chick embryo were compared. In an experiment in which semen of better than average quality was used, no differences were observed in livability of the spermatozoa. When semen of somewhat poorer quality was used, a slight difference in favor of the chick-embryo diluting agent was noted, compared to the yolk-citrate diluter.

The addition of 58 to 116 milligrams of glucose per 100 cc. of bovine semen, diluted at the rate of one part of semen to four parts of yolk-citrate diluter and incubated for 1 hour at 46.5° C., or stored for 10 days at 5° C., results in increased livability, according to workers at Cornell.

Studies made by V. K. Milovanov to determine the possibility of storing semen under anaerobic conditions show that under these conditions—that is, the absence of oxygen—spermatozoa retained motility and fertilizing capacity for long periods. To preserve bull and ram spermatozoa outside the body, he recommended that inert gas, hydrogen or nitrogen, be blown through the undiluted semen, after which it should be sealed in small capsules and cooled.

How to evaluate semen accurately perplexes all who work with artificial insemination. Various methods are used. One way is to count cells to determine the concentration of sperms. Others are to score their motility, and to take into account the proportion of abnormal cells. Direct and indirect measures are made of the rate that the semen uses up oxygen.

Some associations use the methylene-blue reduction test routinely to test the quality of semen. In studies at Cornell, 116 ejaculates from 39 bulls were used ; significant relationships were reported between the reduction time in fresh samples and semen volume, sperm concentration, motility, and lactic acid content. For routine prediction of semen quality, the Cornell workers recommend the use of the test, plus counts of spermatozoa and estimates of motility on fresh samples.

Various workers have determined the proportion of abnormal spermatozoa in semen as one way to check on fertilizing capacity. It has been customary to observe 300 to 500 cells per sample. Salisbury and Mercier believe that it is just as reliable to observe 100 cells as to make two slides and examine 500 cells on each.

A staining method to differentiate between live and dead ram spermatozoa is described by John F. Lasley, G. T. Easley, and F..F. McKenzie, on the basis of work at Missouri. An opal-blue stain is used, and. the results indicate that dead or dying spermatozoa are stained, while active and potentially active sperm do not take the stain.

Three scientists at New Jersey, S. Margolin, J. W. Bartlett, and O. L. Lepard, studied the possibility of determining semen quality by observing its longevity. They diluted samples of semen with the egg-yolk-phosphate buffer. A portion was placed in a refrigerator at 45° F. and estimates of motility were made at 24-hour intervals until motion ceased. The remainder of each sample was used in insemination. The men observed significant positive correlations between longevity and the conception rate in six of the eight bulls they studied. In the other two, they found negative relations that they considered of no significance.

We have also learned much more about the equipment used for semen collection. L. V. Panyseva and T. M. Kozenko, of the Soviet Union, found that the artificial vagina for bulls, shortened to 15.8 inches, is best suited for medium-sized boars. The ordinary vagina for bulls can be used only for very large boars. They also found that pulsation of the artificial vagina when the semen was being drawn has no effect on the quantity and quality of semen.