Effect of freezing rate on sperm survival. Reports by one group of British workers in early trials on freezing bull semen indicated that the rate of cooling in freezing should not exceed 2° C. per minute between +5° and -15° C., although below -15° C. the rate could be faster. Another group expressed the view that semen could be plunged into dry ice at -79° C. after it had been cooled to -15° C. To clarify this part of the freezing procedure, 11 samples of semen were subdivided and portions of each were frozen at rates of 0.25°, 0.5°, 1.0°, 2.0°, and 4.0° C. drop per minute between +5° and -20° C. and then twice these rates between -20° and -79° C. Vials of each ejaculate at +5° C. were also plunged directly into an alcohol bath at -79° C. The samples which were cooled at the rates of 0.25°, 0.5°, 1.0°, 2.0°, and 4.0° C. per minute had the following percentages of motile sperm after thawing: 30, 40, 46, 44, and 44. 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.

Some investigators have suggested that rapid cooling below -20° C. is not detrimental to frozen semen. This idea was tested in conjunction with other experiments. Twenty-five samples cooled slowly (2° C. per minute to -28° C., then 4° C. per minute to -79° C.) showed 62 percent sperm survival compared with only 45 percent when cooled rapidly below -28° C. (2° C. per minute to -28° C. then plunged into bath at -79° C.). Thus, rapid cooling was detrimental even after the critical temperature range of +5° C. to -20° C. had been passed.

Rate of cooling in plastic and in glass. Plastic vials do not conduct the cold as rapidly as glass ampules do. The temperature in both glass and plastic containers tends to lag behind the change in the bath in which they are immersed as is shown in Figure 4.

Temperatures in the immersion bath were recorded in a 2-milliliter glass ampule containing 1 milliliter diluted semen and in an 8-milliliter plastic vial containing 2.5 milliliters of diluted semen. A second plastic vial and glass ampule filled to capacity with diluted semen showed a cooling rate almost identical to that shown in Figure 4. It was obvious from the comparison that samples in the plastic vials cooled slower than those in glass and that the volume of semen (at least the small volumes used) in the vials had little effect on the rate of cooling. In another experiment, it was shown that the volume of diluted semen in the ampule to be frozen (0.2, 1.0 or 5.0 ml.) had little or no effect on the survival of the sperm.