Both of the extenders that are widely used in routine storage of bull
semen at 5 deg. C. are used for freezing semen. These are the egg
yolk-sodium citrate and whole or skimmilk extenders. Most of the
research with extenders for freezing bull semen in this laboratory has
been done with the yolk-citrate diluents.
=Proportion of egg yolk in the final diluent.= Some early experiences
with a diluent consisting of one part yolk and one part 2.9 percent
sodium citrate dihydrate in distilled water showed poor sperm survival
following freezing. The final mixture with this diluent consisted of
about 45 percent yolk. In other attempts at adding glycerol in order to
freeze semen, the final proportion of yolk was diminished and better
sperm survival was obtained. Several experiments were carried out to
test the effect of varying levels of egg yolk.
In the first efforts to find the optimum level of egg yolk, the level
of yolk in the final frozen mixture was varied from about 6 to 46
percent. These levels were obtained by varying the proportion of yolk to
2.9 percent citrate solution in the original extending media and also in
the media added in glycerolating the samples.
Split portions of 20 semen samples were frozen in each of the extender
combinations indicated in Table 4. The mean percentages of motile sperm
found before and after freezing and thawing are shown also. The highest
percentages were found with extenders containing 23 and 24 percent yolk.
The highest percentage of yolk, resulting when a 1:1 (yolk to citrate)
extender was used for both extending and glycerolating, proved to be
most detrimental to sperm survival during freezing. The lowest
percentage of yolk used (6 percent) was not as effective in protecting
sperm during freezing as the intermediate levels tested (Fig. 2).
Table 4.--Effect of Egg-Yolk Level in Diluent on Freezability of Semen
(Average of 20 semen samples)
=========================================================================
Medium Yolk in Pre- Post- Sur-
---------------------------- final freezing thawing vival
Diluent Extending Glycerolating[C] mix- motility motility (per-
------------ ---------------- ture[D] (percent) (percent) cent)
yolk:citrate yolk:citrate (percent)
-------------------------------------------------------------------------
1 1:1 1:1 45.7 65 5 8
2 1:1 1:3 34.9 64 27 42
3 1:3 1:1 33.6 63 33 52
4 1:1 0:1 24.2 64 39 61
5 1:3 1:3 22.8 63 37 59
6 1:3 0:1 12.1 59 33 56
7 1:7 1:7 11.4 56 35 62
8 1:7 0:1 6.0 52 26 50
9 1:15 1:15 5.7 49 25 51
-------------------------------------------------------------------------
[C] This mixture included 14 percent glycerol.
[D] The average initial sperm concentration was 900 x 10^{6}/ml.
Sufficient extender was added to give 30 x 10^{6}/ml. at the first
extension. Thus the final concentration was 15 x 10^{6} sperm/ml.
after glycerolization.
Since rather large changes in the percentages of yolk were used in this
experiment, two further trials were conducted in which 16, 24, and 32
percent yolk in the final mixture were compared, with the final citrate
percentages held constant. In these tests, 16 and 24 percent yolk
maintained sperm better at all citrate levels tried than 32 percent
yolk. The 16 percent level was slightly better at most of the levels of
citrate tested (Fig. 3).
=Citrate level in the final diluent.= The early work of the British
indicated that a final citrate level near 2 percent in the diluent was
satisfactory for freezing bull sperm. Later, in a personal
communication, Polge of the British group suggested that a citrate level
of about 2.35 percent might be best with a final glycerol concentration
of 7 percent. Some of the first attempts in this laboratory at
establishing the optimum yolk-to-citrate ratios are shown in Fig. 3. In
these experiments, the optimum levels of citrate appeared to be lower
than anticipated from the British work. Thus a more complex experiment
was set up to test a wider range of citrate levels using 16 and 24
percent egg yolk in the final freezing mixture. The average percentages
of motile sperm found after freezing 10 semen samples at each of the
citrate and yolk levels in this experiment are shown also in Fig. 3.
Little difference in freezability was found between citrate percentages
of 1.55 and 1.95. When the rate of sperm motility following freezing and
thawing was considered along with the percent of motile sperm, a slight
advantage was found with 16 percent yolk and a citrate concentration of
1.55 percent.
[Illustration: Percent of motile sperm after freezing and thawing semen
in diluents containing various levels of egg yolk (Fig. 2)]
From the results of these experiments, and from several reports in the
literature,[5],[6],[7],[9],[10] it appears that a diluting medium
resulting in a final concentration of 16 to 25 percent yolk and 1.55 to
2.2 percent sodium citrate dihydrate is highly satisfactory for
freezing.
=Storing and freezing diluent.= In some instances it would be
advantageous to have prepared diluent on hand for use at any time. The
suitability of stored diluent was tested with a yolk-citrate (equal
parts yolk and citrate without antibiotics added) diluent prepared and
stored at 5 deg. C. for 0, 2, 5, 7, and 9 days. Seven semen samples were
diluted and frozen in these diluents. No difference was noted in the
survival of sperm that could be attributed to the age of the diluent.
[Illustration: Percent of motile sperm after freezing and thawing semen
in diluents containing various levels of egg yolk and various
percentages of sodium citrate (Fig. 3)]
In another trial, a similar diluent (1:1 yolk to citrate with 1000 units
of penicillin and 5000 units of streptomycin) was prepared and stored in
the freezer compartment of a refrigerator at -15 deg. C. Upon thawing, it
was whitish in color and more viscous than freshly prepared diluent.
Except for the fact that the viscosity seemed to reduce the rate of
sperm motility, this frozen diluent stored for 65 days compared
favorably with freshly prepared diluent for freezing semen.
=Other diluents.= Without the protective action of egg yolk or milk, few
bull sperm will survive freezing. Several diluents were compared on a
limited scale for freezing bull sperm. The results of these trials are
compiled in Table 5. In this trial the yolk-citrate extender served best
in maintaining sperm motility during freezing. Yolk-phosphate and
homogenized whole milk were slightly less protective and yolk-saline
seemed to furnish the least protection to sperm during freezing.
A number of investigations in other laboratories have now proven that
milk can be used as effectively as the yolk-citrate diluent for freezing
bull sperm.[6],[7]
Table 5.--Comparison of the Freezability of 4 Semen Samples in Different
Extenders
======================================================================
Dilution Pre- Post- Motility
Extender rate freezing thawing Survival after
(semen: motility motility (percent) storage[E]
extender) (percent) (percent) (percent)
----------------------------------------------------------------------
Yolk-citrate 1:1 60 49 82 46
1:10 53 45 85 36
Yolk-saline 1:1 57 29 51 28
1:10 60 31 52 24
Yolk-phosphate 1:1 55 33 64 25
1:10 60 43 72 25
Whole milk 1:1 60 40 67 35
1:10 60 35 58 16
----------------------------------------------------------------------
[E] Stored at 5 deg. C. for 7 hours after thawing.