(Average of 10 ejaculates)

Stage of
observation		Sperm motility (percent)
Glycerol
only		Glycerol
and
glucose		Glycerol
and
arabinose		Glycerol
and
xylose		Glycerol
and
rhamnose
Fresh diluted semen6363636363
After glycerolization5455545760
After 18 hours equilibration3943443946
After freezing to -79° C. and
immediate thawing		2834342924
After 4 days at -79° C.2326262527

[K] Glycerol level in the final frozen mixture was 7 percent. Sugars were added to a level of 1.25 percent.

Substitutes for glycerol. Since glycerol was so effective in protecting sperm during freezing, many have assumed that related compounds might be even better. Several compounds, some related to glycerol and some not, have been tried as substitutes for glycerol in the freezing procedure. They include ethylene glycol, propylene glycol, trimethylene glycol, mannitol, sorbitol, dextrans, and seminal-plasma proteins. None of these materials has been as effective as glycerol in protecting sperm during freezing. In fact, several of the materials proved to be injurious to sperm prior to attempts to freeze the samples. While the work in our laboratory with these substances as glycerol substitutes was by no means finally conclusive, because of the many possible interactions of experimental conditions, sufficient data were gathered to lead us to abandon further study until greater promise of success might be evident.

FREEZING RATE

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.