In freezing and storing bull sperm, an alcohol bath containing dry ice
at a temperature of -79 deg. C. has been used as a cooling agent. In many
areas, the availability of dry ice is limited and the cost is rather
high. Mechanical means are available for obtaining temperatures as low
as, or lower than, -79 deg. C. but for the most part they are expensive. If
warmer temperatures were suitable for storing frozen semen, the ordinary
deep-freeze, which operates at -15 deg. to -25 deg. C., might be used.
=Storage at temperatures from -23 deg. to -79 deg. C.= In testing the effects of
storage temperatures on the survival of frozen bull sperm (in a diluent
containing 7 percent glycerol), 9 ejaculates were frozen and kept at
-23 deg., -37 deg., -51 deg., -65 deg., and -79 deg. C. The desired temperatures were
maintained by dropping pieces of dry ice into ethyl alcohol baths as
needed. Samples were thawed after 1 hour, 1 day, 3 days, and 5 days.
After 1 hour, the samples maintained at the various temperatures
exhibited approximately equal motility (Fig. 5).
[Illustration: Effect of freezing and storing bull sperm at various
temperatures on the sperm motility at thawing (average of 9 ejaculates)
(Fig. 5)]
At the end of 1 day, samples stored at -79 deg. C. exhibited approximately
the same motility as did similar samples stored for 1 hour. The samples
stored at -65 deg. C. had declined slightly in motility and those maintained
at -51 deg. C. had only one-third the motility which they had displayed at 1
hour. The samples at -23 deg. and -37 deg. C. exhibited practically no motility
after 1 day in storage. After 5 days, only 3 of the 8 ejaculates stored
at -51 deg. C. showed motility upon thawing. Apparently detrimental changes
take place more rapidly when the samples are stored at temperatures
warmer than -65 deg. C. The nature of these changes has not been determined.
Reports from other laboratories indicate that storage temperatures much
lower than -79 deg. C. are just as satisfactory as -79 deg. C.
No tests of the effects of storage at -79 deg. C. for periods longer than 51
days have been conducted in this laboratory. Portions of 12 ejaculates
were frozen and stored at -79 deg. C. for various periods. One portion of
each of these was examined on the second, ninth, 16th and 51st day of
storage. The percent of motile sperm and rate of motility at each of
these examinations were as follows:
Day 2 9 16 51
Percent of motile sperm 49 46 40 38
Rate of motility 2.5 2.3 2.2 2.2
The average prefreezing motility percentage for the above samples was
58, with an average rate of motility of 2.9. It is apparent from these
results that the loss in motility was greatest due to the initial
freezing, and after that the drop was most pronounced during the first
16 days of storage.
The British and the Australians have both reported the successful
maintenance of fertility in frozen semen stored at -79 deg. C. for over two
years.[5]
=Use of higher glycerol levels and a -20 deg. C. storage temperature.= In
1953, a report from Arkansas suggested that warmer storage temperatures
could be used if a high percentage of glycerol were included in the
freezing mixture.[7] To test the effectiveness of various glycerol
levels on protecting sperm stored at deep-freeze temperatures, glycerol
levels of 3.5, 5.5, 7.5, and 9.5 percent were used with portions of 4
semen samples. Survival in the portions frozen and stored at -20 deg. C. was
poor compared with the portions reduced and held at -79 deg. C. In a second
experiment, 4 samples were subdivided and frozen with a final
concentration of 7, 11, 15, and 19 percent glycerol in the semen-diluent
mixture. In this trial, poor results were obtained at -20 deg. C. except
that glycerol at a level of 19 percent protected the sperm more
effectively than at lower levels. Maximal survival at -79 deg. C. was
obtained at the 7-percent glycerol level. A final trial was run, using
glycerol levels of 7, 11, 15, 19, 23, 27, and 31 percent. The
percentages of motile sperm present after storage at -79 deg. C. and -20 deg. C.
are shown in Table 14.
Table 14.--Effect of Glycerol Level and Storage Temperature on
Freezability of Semen
(Average of 8 ejaculates)
================================================================
Storage Glycerol Sperm motility after storage (percent)
temperature level --------------------------------------
( deg.C.) (percent) 18 hours 42 hours
----------------------------------------------------------------
-79 7 61 61
-20 7 2 1
11 3 1
15 14 10
19 30 22
23 29 19
27 25 18
31 21 12
----------------------------------------------------------------
While survival was fair over a short period of time with 19 percent
glycerol at -20 deg. C., deterioration was rapid during storage. After 18
hours of storage, the samples at -20 deg. C. (19 percent glycerol) contained
only one half as many motile sperm as were still present in the samples
at -79 deg. C. (7 percent glycerol). After 42 hours of storage, the best
samples at -20 deg. C. contained only one-third the number of motile sperm
still present in the samples stored at -79 deg. 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.