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.Storage at temperatures from -23° to -79° C. In testing the effects of storage temperatures on the survival of frozen bull sperm (in a diluent containing 7 percent glycerol), 9 ejaculates were frozen and kept at -23°, -37°, -51°, -65°, and -79° C. The desired temperatures were maintained by dropping pieces of dry ice into ethyl alcohol baths as needed. Samples were thawed after 1 hour, 1 day, 3 days, and 5 days. After 1 hour, the samples maintained at the various temperatures exhibited approximately equal motility (Fig. 5).

At the end of 1 day, samples stored at -79° C. exhibited approximately the same motility as did similar samples stored for 1 hour. The samples stored at -65° C. had declined slightly in motility and those maintained at -51° C. had only one-third the motility which they had displayed at 1 hour. The samples at -23° and -37° C. exhibited practically no motility after 1 day in storage. After 5 days, only 3 of the 8 ejaculates stored at -51° C. showed motility upon thawing. Apparently detrimental changes take place more rapidly when the samples are stored at temperatures warmer than -65° C. The nature of these changes has not been determined. Reports from other laboratories indicate that storage temperatures much lower than -79° C. are just as satisfactory as -79° C.

No tests of the effects of storage at -79° C. for periods longer than 51 days have been conducted in this laboratory. Portions of 12 ejaculates were frozen and stored at -79° C. for various periods. One portion of each of these was examined on the second, ninth, 16th and 51st day of storage. The percent of motile sperm and rate of motility at each of these examinations were as follows:

The average prefreezing motility percentage for the above samples was 58, with an average rate of motility of 2.9. It is apparent from these results that the loss in motility was greatest due to the initial freezing, and after that the drop was most pronounced during the first 16 days of storage.

The British and the Australians have both reported the successful maintenance of fertility in frozen semen stored at -79° C. for over two years.[5]Use of higher glycerol levels and a -20° C. storage temperature. In 1953, a report from Arkansas suggested that warmer storage temperatures could be used if a high percentage of glycerol were included in the freezing mixture.[7] To test the effectiveness of various glycerol levels on protecting sperm stored at deep-freeze temperatures, glycerol levels of 3.5, 5.5, 7.5, and 9.5 percent were used with portions of 4 semen samples. Survival in the portions frozen and stored at -20° C. was poor compared with the portions reduced and held at -79° C. In a second experiment, 4 samples were subdivided and frozen with a final concentration of 7, 11, 15, and 19 percent glycerol in the semen-diluent mixture. In this trial, poor results were obtained at -20° C. except that glycerol at a level of 19 percent protected the sperm more effectively than at lower levels. Maximal survival at -79° C. was obtained at the 7-percent glycerol level. A final trial was run, using glycerol levels of 7, 11, 15, 19, 23, 27, and 31 percent. The percentages of motile sperm present after storage at -79° C. and -20° C. are shown in Table 14.

Table 14.—Effect of Glycerol Level and Storage Temperature
on Freezability of Semen

(Average of 8 ejaculates)

While survival was fair over a short period of time with 19 percent glycerol at -20° C., deterioration was rapid during storage. After 18 hours of storage, the samples at -20° C. (19 percent glycerol) contained only one half as many motile sperm as were still present in the samples at -79° C. (7 percent glycerol). After 42 hours of storage, the best samples at -20° C. contained only one-third the number of motile sperm still present in the samples stored at -79° C. These trials leave little doubt that under the present system of freezing and storing, storage at ordinary deep-freeze temperatures is far inferior to storage at dry-ice temperatures.