M. E. Friedman

“ Allowing sperm to equilibrate with the glycerol. Allowing sperm to stand in the presence of glycerol is considered by some to be necessary in order that the glycerol penetrate the sperm heads before freezing. From the first successful attempts at freezing bull sperm came the practice of allowing 12 to 20 hours for this process of equilibration. A long equilibration time results in aging the sperm. Data from a number of sources indicate that a drop of approximately 5 percent in fertility in the field occurs with each 24 hours of aging in the test tube. ”

“ STORAGE TEMPERATURE
In freezing and storing bull sperm, an alcohol bath containing dry ice at a temperature of -79° C. has been used as a cooling agent. In many areas, the availability of dry ice is limited and the cost is rather high. Mechanical means are available for obtaining temperatures as low as, or lower than, -79° C. but for the most part they are expensive. If warmer temperatures were suitable for storing frozen semen, the ordinary deep-freeze, which operates at -15° to -25° C., might be used. ”

“ Sperm cells centrifuged three times and washed twice in 0.9 percent sodium chloride solution withstood freezing well when finally resuspended and frozen in yolk-citrate diluent. The percentage of survival in three samples subjected to this treatment was 60 percent. Thus it appears that the seminal plasma itself is not essential for ejaculated sperm to survive the rigors of freezing. This is not surprising, since it had already been found that epididymal sperm, which also are free of accessory gland secretions, can withstand freezing and thawing. ”

“ However, results obtained by other workers show improved fertility with at least 12 hours equilibration. Some units getting good fertility results with frozen semen also are allowing the semen to stand at 5° C. for 12 to 18 hours before freezing. After the semen has equilibrated with the glycerol, 1-milliliter portions of the mixture are placed in 1.2- to 2-milliliter vials or ampules which are then sealed. Ampuling can be done with an automatic syringe or pipette, provided a large gage needle is used. ”

“ At the 30-percent level, the sperm moved slowly and could be seen to [18] rotate as they moved forward. Some samples were checked after slowly bringing the diluent up to a level of 40 percent glycerol; the sperm seemed to be immobilized completely in this solution.
Glycerol levels for freezing semen. The British procedure called for the use of 10 percent glycerol in the final mixture of semen and extender prior to freezing. Yet, as shown in Table 6, in our laboratory 5 percent glycerol resulted in the survival of a higher percentage of sperm than did 10 or 15 percent. ”

“ A mean of 32 percent of the sperm in the samples that were plunged directly into an alcohol bath at -79° C. were motile after thawing. There were no statistically significant differences among the samples frozen at 1.0°, 2.0° or 4.0° C. per minute. All of the others had significantly lower survival rates. Thus, it is obvious that too slow a cooling rate and plunging the samples directly into a -79° C. bath from a temperature of +5° C. cause greater harm to the sperm than cooling at a rate between 1.0° and 4.0° C. per minute. ”

“ In 1897, Davenport [1] [B] found that human sperm would withstand freezing. For thirty to forty years after that, little [6] attention was paid to freezing as a possible means of preserving semen. An excellent review of the early attempts to freeze sperm has been assembled by Polge and Parkes. [2] These investigators also gave a good account of their work at the British National Institute of Medical Research in London, where in 1949 they demonstrated that glycerol would protect fowl sperm so that it would survive freezing. ”

“ Also, if the glycerol addition could be made soon after the dilution with egg yolk-citrate extender at room temperature, time would be gained in processing the semen for use. Since aging in vitro is known to reduce the fertilizing ability of sperm, every effort should be made to keep the processing time at a minimum. ”

“ Little difference in survival during freezing was noted between the three rates of addition. Using 3 equal additions resulted in slightly better results, but the advantage was not statistically significant. While little difference was evident from adding the glycerol in 3 portions as compared to 1, many still use 3 additions in the hope of obtaining a slightly better sperm survival. In fact, some have gone to a procedure of adding the glycerol dropwise with constant gentle agitation. This method has not been tested in this laboratory. ”

“ A few ampules can be kept together easily by placing them in a polyethylene freezer bag that has had many small holes cut in it to let the alcohol of the bath contact the ampules. The ampules must be completely covered by the alcohol to insure uniform cooling. ”

“ The average percentages of motile sperm found after freezing 10 semen samples at each of the citrate and yolk levels in this experiment are shown also in Fig. 3. Little difference in freezability was found between citrate percentages of 1.55 and 1.95. When the rate of sperm motility following freezing and thawing was considered along with the percent of motile sperm, a slight advantage was found with 16 percent yolk and a citrate concentration of 1.55 percent. ”

“ Except for the fact that the viscosity seemed to reduce the rate of sperm motility, this frozen diluent stored for 65 days compared favorably with freshly prepared diluent for freezing semen.
Other diluents. Without the protective action of egg yolk or milk, few bull sperm will survive freezing. Several diluents were compared on a limited scale for freezing bull sperm. The results of these trials are compiled in Table 5. In this trial the yolk-citrate extender served best in maintaining sperm motility during freezing. ”