When the British procedure for freezing bull semen was first tried in
this country, many of the refinements of the technique still had not
been defined. It was known that glycerol worked well in protecting sperm
during freezing. The effects of glycerol on sperm at 5 deg. C., the
appropriate levels to use in freezing, and the manner of adding it were
not well established. Therefore, a number of trials were conducted in an
attempt to establish the best procedures.
=Effect of glycerol on sperm survival at 5 deg. C.= Since early work
indicated the need for adding glycerol to diluted semen in order to
protect the sperm during freezing, it was considered important to
determine the levels of glycerol that sperm would tolerate at 5 deg. C. Ten
semen samples were extended 1:9 (semen to diluent) in a 1:1 yolk-citrate
diluent (yolk to 2.9 percent sodium citrate dihydrate). Each sample was
then split into 6 portions and an equal volume of citrate solution
containing glycerol was added slowly to each to bring the glycerol in
the final mixture to 0, 5, 10, 20, or 30 percent (by volume). These
samples were stored at 5 deg. C. and examined for motile sperm after 1, 3,
and 7 days. The effects of glycerol levels on the percentage of sperm
surviving and the rate (or speed) of their forward motion (0 = no
forward motion; 4 = extremely rapid progressive motility) are presented
in Table 8.
The percentage of motile sperm decreased slightly at the higher levels
of glycerol. The most noticeable effect of the increase in glycerol
level was the reduction in the rate of forward motion of the sperm. At
the 30-percent level, the sperm moved slowly and could be seen to
rotate as they moved forward. Some samples were checked after slowly
bringing the diluent up to a level of 40 percent glycerol; the sperm
seemed to be immobilized completely in this solution.
=Glycerol levels for freezing semen.= The British procedure called for
the use of 10 percent glycerol in the final mixture of semen and
extender prior to freezing. Yet, as shown in Table 6, in our laboratory
5 percent glycerol resulted in the survival of a higher percentage of
sperm than did 10 or 15 percent. In order to define more clearly the
optimum glycerol level, several ejaculates of semen were subsampled and
portions were frozen after the addition of yolk-citrate extender and
glycerol in varying quantities. From Table 9 it can be seen that
glycerol levels of 6 and 8 percent in the final mixture resulted in
maximum sperm survival during freezing. These results were confirmed in
tests on the survival of sperm at 5 deg. C. storage for 3 days following
freezing and thawing with varying glycerol levels (see Table 10).
The results shown in Tables 9 and 10 were confirmed also in later
experiments. Thirty-six samples were subjected to various levels of
glycerol and no significant difference in freezability was found between
6 and 8 percent. Based on these findings, a glycerol level of 7 percent
was adopted for use in all experiments described in this bulletin,
unless otherwise indicated. Results in a number of other laboratories
have agreed with our findings regarding the use of approximately 7
percent glycerol with the yolk-citrate diluent.[5],[6],[7],[9],[10] With
milk as the extender, 10 to 13 percent glycerol has been preferred by
some.[5],[6],[7]
Table 9.--Effect of Glycerol Level on Sperm Motility After Freezing to
-79 deg. C. and Thawing
==========================================================
Glycerol Number Pre- Post- Survival
level of freezing thawing (percent)
(percent) samples motility motility
(percent) (percent)
----------------------------------------------------------
2 10 53 2 4
4 19 55 29 53
6 19 55 34 62
8 19 55 35 64
10 19 55 24 44
12 10 53 13 25
----------------------------------------------------------
Table 10.--Effect of Glycerol Level and Storage at 5 deg. C. After Thawing
on Sperm Motility
(Average of 13 ejaculates)
================================================
Sperm motility (percent)
Glycerol ----------------------------------
level Post- After storage at 5 deg. C.
(percent) thawing -----------------------
1 day 3 days
------------------------------------------------
4 29 22 20
6 38 34 24
8 42 33 17
10 33 18 6
------------------------------------------------
Table 11.--Effects of Temperature, Rate of Addition of Glycerol, and
Equilibration Time on Sperm Motility
(Average of 12 ejaculates)
===================================================================
Temperature Post-thawing motility (percent)
during Equilibration ----------------------------------
addition time Glycerol additions
of glycerol (hours) ----------------------------------
( deg. C.) 5 3 1 Average
-------------------------------------------------------------------
4.5 2 48 48 45 47.4
6 49 51 47 48.8
18 46 47 46 46.3
Average 47.8 48.6 46.0 47.5
10.0 2 44 43 45 43.9
6 48 50 46 47.9
18 43 46 42 44.0
Average 45.0 46.5 44.3 45.3
15.5 2 41 38 38 39.1
6 42 45 43 43.6
18 42 43 42 42.5
Average 42.0 41.8 41.4 41.7
-------------------------------------------------------------------
=Rate, temperature, and method of adding glycerol.= Closely associated
with the question of how much glycerol should be added is that of how
the additions should be made. Originally it was believed that the
glycerol should be added in stages so that changes would occur
gradually. However, there would be a saving in time if the entire amount
could be added at once. Also, if the glycerol addition could be made
soon after the dilution with egg yolk-citrate extender at room
temperature, time would be gained in processing the semen for use. Since
aging _in vitro_ is known to reduce the fertilizing ability of sperm,
every effort should be made to keep the processing time at a minimum.
The results of an experiment involving these items, along with that of
how much time should be allowed after the additions before freezing
(equilibration time), are presented in Table 11. One can see that sperm
survived freezing better when the diluted semen was cooled to 4.5 deg. C.
before the glycerol was added. The survival at 10 deg. and 15.5 deg. C. was
reduced with each rise in temperature. Thus, it appears that cooling to
refrigerator temperature (4-5 deg. C.) before adding the glycerol should be
a part of the routine procedure.
A comparison of the results from adding the glycerol in 5, 3, and 1
equal portions is given also in Table 11. Little difference in survival
during freezing was noted between the three rates of addition. Using 3
equal additions resulted in slightly better results, but the advantage
was not statistically significant. While little difference was evident
from adding the glycerol in 3 portions as compared to 1, many still use
3 additions in the hope of obtaining a slightly better sperm survival.
In fact, some have gone to a procedure of adding the glycerol dropwise
with constant gentle agitation. This method has not been tested in this
laboratory.
=Allowing sperm to equilibrate with the glycerol.= Allowing sperm to
stand in the presence of glycerol is considered by some to be necessary
in order that the glycerol penetrate the sperm heads before freezing.
From the first successful attempts at freezing bull sperm came the
practice of allowing 12 to 20 hours for this process of equilibration. A
long equilibration time results in aging the sperm. Data from a number
of sources indicate that a drop of approximately 5 percent in fertility
in the field occurs with each 24 hours of aging in the test tube. Thus
it would seem desirable to reduce the equilibration time to a minimum
commensurate with good freezability in order to reduce the effects of
aging (at 5 deg. C.). Results of attempting to reduce equilibration time are
given in Table 11. At 4.5 deg. C., little variation in motility following
freezing and thawing was found after equilibration times of 2, 6, and 18
hours. At the higher temperatures of 10 deg. and 15.5 deg. C., the shortest
equilibration time--2 hours--was slightly more detrimental with the
differences significant at the 5-percent level at 15.5 deg. C. For all
temperatures combined, 6 hours was significantly better than 2 or 18
hours.
=Sugar additions and equilibration time.= Early in their experiences in
freezing semen, the Australian workers found a short equilibration
time--30 minutes--to be satisfactory if sugars were added to the
diluent.[5] This protective action of sugars during the equilibration
period was confirmed in our investigations. The results of one phase of
this study are shown in Table 12. From these data it can be seen that
the presence of glucose or rhamnose at a level of 1.25 percent improved
sperm survival during the period of equilibration. In another trial
these sugars and two others, arabinose and xylose, were tested for their
protective action in freezing semen. The percentages of surviving sperm
remaining after the various steps in the freezing procedure with and
without the presence of these sugars are shown in Table 13.
Table 12.--Effect of Adding Sugars to Yolk-Citrate Diluent on Sperm
Motility During Equilibration With Glycerol[J]
============================================================
Sperm motility (percent)
-------------------------------------
Stage when observed Glycerol Glycerol Glycerol
only and glucose and rhamnose
------------------------------------------------------------
Fresh diluted semen 56 56 56
After glycerolization 54 54 54
After equilibration
2 hours 51 53 53
6 hours 48 52 53
12 hours 46 50 51
18 hours 40 46 46
------------------------------------------------------------
[J] Glycerol level in the final frozen mixture was 7 percent. Sugars
were added to a level of 1.25 percent.
Three of the sugars--glucose, arabinose, and rhamnose--protected the
sperm during equilibration and freezing. Xylose was less effective, but
its addition resulted in slightly better sperm survival than glycerol
alone. It was found also that the methylene-blue reduction time
(metabolic test for semen quality) was faster in samples to which the
sugars had been added--after glycerolization, after equilibration, and
after freezing the samples. This is confirming evidence for the presence
of more living and actively metabolizing sperm in the portions to which
sugars had been added.
Table 13.--Effect of Adding Sugars to Yolk-Citrate Diluent on Sperm
Motility During the Freezing Procedures[K]
(Average of 10 ejaculates)
======================================================================
Sperm motility (percent)
-------------------------------------------------
Stage of Glycerol Glycerol Glycerol Glycerol Glycerol
observation only and and and and
glucose arabinose xylose rhamnose
----------------------------------------------------------------------
Fresh diluted semen 63 63 63 63 63
After glycerolization 54 55 54 57 60
After 18 hours
equilibration 39 43 44 39 46
After freezing to
-79 deg. C. and
immediate thawing 28 34 34 29 24
After 4 days at -79 deg. C. 23 26 26 25 27
-----------------------------------------------------------------------
[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.