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 deg. 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 deg. C. and 38 deg. C.= The effects of
thawing at temperatures of 38 deg. (body temperature) and 5 deg. 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.
[Illustration: Effect of glycerol percentage and thawing temperature on
sperm motility after freezing and thawing (Fig. 6)]
The 5 deg. C. thawing temperature resulted in a higher percentage of sperm
survival at all the glycerol levels than 38 deg. C., with the difference in
favor of 5 deg. 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 deg. C. brought about harmful metabolic activity.
The difference in survival of sperm in semen thawed at 5 deg. C. and at 38 deg.
C. continued during storage at 5 deg. C. (Table 15). It was also evident
that the interaction between glycerol level and thawing temperature
continued during storage (Fig. 7).
[Illustration: Effect of thawing temperature on sperm motility during
storage at 5 deg. C. following freezing and thawing (Fig. 7)]
Table 15.--Effect of Glycerol Level, Thawing Temperature, and Storage at
5 deg. C. After Thawing on Sperm Motility (Average of 13 ejaculates)
=====================================================================
Thawing Glycerol Sperm motility (percent)
temperature level -------------------------------------------
( deg. C.) (percent) Post- After storage at 5 deg. C.
thawing ---------------------- Average
1 day 3 days
---------------------------------------------------------------------
38 4 28.5 17.3 5.1 17.0
6 31.5 22.4 9.2 21.1
8 33.1 15.0 4.6 17.6
10 19.5 3.6 0.8 8.0
Average 28.2 14.6 4.9 12.2
5 4 29.2 21.7 19.8 23.9
6 37.7 33.8 23.5 31.7
8 41.5 33.1 17.3 30.6
10 33.1 18.5 6.0 19.2
Average 35.4 26.8 16.6 20.6
---------------------------------------------------------------------
It is obvious that motility falls off rapidly after the semen is thawed.
In a field trial in which the initial intent was to test the effect of
glycerol levels on fertility of frozen semen, the semen was thawed in
the morning and used during the same day. Survival of the sperm with 4
percent glycerol was so poor that only a few breedings were made with
these samples. Even at 7 and 10 percent, the fertility results were much
lower than with semen that had not been subjected to freezing. At that
time it was felt that thawing the samples and using them throughout the
day may have caused the low fertility results. Since then, a
large-scale experiment by Cornell University investigators, in
cooperation with the New York Artificial Breeders' Cooperative, has
shown definitely that thawing should be delayed until a few minutes
prior to breeding.[11] If the semen is used immediately, a thawing
temperature of either 5 deg. or 38 deg. C. appears to be suitable. However,
there is less danger of cold shock due to recooling if 5 deg. C is used.
=Thawing rate in plastic and in glass.= Glass ampules transmit cold or
heat more readily than plastic ones. The temperature rise is rapid in
both glass and plastic when samples are taken from the storage box at
-79 deg. C. and placed in water at 5 deg. C. However, complete thawing occurs
more rapidly in glass than in plastic ampules. The changes in
temperature that occurred when glass and plastic ampules were thawed in
a water bath at 5 deg. C. are shown in Figure 8. The initial temperature
rise for the first minute or two was about the same, then the rate of
warming in the plastic slowed and actual melting of the frozen sample
occurred a little over a minute later in the plastic than it did in the
glass. Both were thawed in less than four minutes.
[Illustration: Warming rates of diluted semen samples in plastic vials
and in glass ampules (Fig. 8)]