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.
THAWING
The importance of carefully controlled cooling and storage has been emphasized in the foregoing sections. The need for controlling thawing rates and the temperature of thawing was not clearly defined in the early work on freezing bull semen. The British used a thawing temperature of 40° C., which was satisfactory. If there is a need to hold the semen for a time after thawing, then a lower thawing temperature might be more desirable so that cooling again will not be necessary.
Comparison of thawing temperatures of 5° C. and 38° C. The effects of thawing at temperatures of 38° (body temperature) and 5° C. (refrigerator temperature) were investigated. The first trial involved thawing as rapidly as possible by dropping glass ampules of frozen semen into water baths at the two temperatures. The frozen semen samples contained glycerol levels of 4, 6, 8, and 10 percent. The mean percentages of motile sperm found after thawing thirteen diluted semen samples treated in this manner are shown in [Figure 6].
| Effect of glycerol percentage and thawing temperature on sperm | |
| motility after freezing and thawing | (Fig. 6) |
The 5° C. thawing temperature resulted in a higher percentage of sperm survival at all the glycerol levels than 38° C., with the difference in favor of 5° C. becoming greater as the glycerol level increased. The reason for the interaction between glycerol level and thawing temperature is not known. It may be that the presence of the higher levels of glycerol at 38° C. brought about harmful metabolic activity. The difference in survival of sperm in semen thawed at 5° C. and at 38° C. continued during storage at 5° C. ([Table 15]). It was also evident that the interaction between glycerol level and thawing temperature continued during storage ([Fig. 7]).