=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 deg. C. per minute
between +5 deg. and -15 deg. C., although below -15 deg. C. the rate could be
faster. Another group expressed the view that semen could be plunged
into dry ice at -79 deg. C. after it had been cooled to -15 deg. 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 deg., 0.5 deg., 1.0 deg., 2.0 deg.,
and 4.0 deg. C. drop per minute between +5 deg. and -20 deg. C. and then twice these
rates between -20 deg. and -79 deg. C. Vials of each ejaculate at +5 deg. C. were
also plunged directly into an alcohol bath at -79 deg. C. The samples which
were cooled at the rates of 0.25 deg., 0.5 deg., 1.0 deg., 2.0 deg., and 4.0 deg. 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 deg. C. were motile
after thawing. There were no statistically significant differences among
the samples frozen at 1.0 deg., 2.0 deg. or 4.0 deg. 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 deg. C.
bath from a temperature of +5 deg. C. cause greater harm to the sperm than
cooling at a rate between 1.0 deg. and 4.0 deg. C. per minute.
Some investigators have suggested that rapid cooling below -20 deg. C. is
not detrimental to frozen semen. This idea was tested in conjunction
with other experiments. Twenty-five samples cooled slowly (2 deg. C. per
minute to -28 deg. C., then 4 deg. C. per minute to -79 deg. C.) showed 62 percent
sperm survival compared with only 45 percent when cooled rapidly below
-28 deg. C. (2 deg. C. per minute to -28 deg. C. then plunged into bath at -79 deg. C.).
Thus, rapid cooling was detrimental even after the critical temperature
range of +5 deg. C. to -20 deg. C. had been passed.
[Illustration: Cooling rates of diluted semen samples in plastic vials
and in glass ampules (Fig. 4)]
=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.