One of the first considerations in freezing semen is that of deciding
which semen samples are to be frozen. Since preservation of the
semen--the maintenance of the potential motility and especially the
fertility of the sperm--is the primary aim, some attention should be
directed to the kind of semen sample that will withstand freezing. Do
the initial characteristics of the sample indicate whether the sperm
will withstand freezing? Does maturity of the sperm affect their
freezability?
=Predicting freezability.= Estimates of semen quality in the past have
been based in part on the numbers of sperm present in a fresh sample and
on the percentage and rate of motility shown by the sperm. These
characteristics were used to determine the relationship between the
original concentration of sperm (in the fresh, undiluted sample), the
percentage and rate of sperm motility in the diluted samples just prior
to freezing, and the percentage and rate of sperm motility following
freezing and thawing. From data collected before and after freezing and
thawing 54 ejaculates, it was found that there was not a significant
correlation between the number of sperm present in the original sample
and the percent of motile sperm present after freezing and thawing (r =
0.03). A highly significant correlation (r = 0.45) was found, however,
between the percentages before freezing and after thawing. While this
correlation coefficient was highly significant, its magnitude indicates
that only about one fifth of the variation in percentage of motile sperm
observed after freezing was accounted for by the motility of the sperm
prior to freezing.
=Freezability of first and second ejaculates.= In the early days of
artificial breeding in this country, it was commonly believed that a
second ejaculate collected a few minutes after the first resulted in a
larger ejaculate containing more sperm. With the development of the
procedure of stimulating sexual excitement by restraint prior to
collecting semen, this difference between first and second ejaculates
has been greatly reduced. Still it was noted that second ejaculates
frequently withstood freezing better than first ejaculates, even though
restraint and stimulation of the bull occurred prior to collection of
the first ejaculate. During the course of a number of experiments, it
was possible to compare the freezability of 2 ejaculates that were
collected a few minutes apart from the same bull. Two consecutive
ejaculates were obtained one or more times from 24 bulls so that a total
of 58 comparisons could be made. The mean prefreezing and post-thawing
percentages of motile sperm in first and second ejaculates are presented
in Table 1.
An analysis of variance showed that in this comparison the differences
between first and second ejaculates in sperm survival during freezing
were highly significant. A later comparison of 27 first and second
ejaculates from 26 bulls did not show as great a difference between
first and second ejaculates in their ability to withstand freezing
(Table 1).
Table 1.--Comparison of the Freezability of First and Second Ejaculates
Taken a Few Minutes Apart From the Same Bull
=====================================================================
Number Number Prefreezing Post-thawing
of of Ejaculate motility motility Survival
bulls ejaculates (percent) (percent) (percent)
---------------------------------------------------------------------
24 58 1st 60 39 65
2d 62 45 74
26 27 1st 60 36 60
2d 65 40 61
---------------------------------------------------------------------
=Freezability of several consecutive ejaculates.= The fact that second
ejaculates sometimes withstood freezing better than first ejaculates
suggested that the maturity of the sperm might be a factor affecting
freezability. An opportunity to check this idea came when 20
consecutive ejaculates were collected from each of 6 bulls within a
4-hour period. The sperm in samples collected in this manner might be
expected to be less mature with each additional collection.
The results obtained in freezing several consecutive ejaculates are
shown in Figure 1 as averages for the ejaculates from 6 bulls. In same
instances, there was an insufficient quantity of semen available to test
the freezability. (Procedure: Diluted to 30 x 10^{6} sperm per ml. with
1:1 yolk-citrate, then cooled and glycerolated with an equal volume of
14 percent glycerol (percent by volume) in 2.9 percent sodium citrate.
Final sperm concentration, 15 x 10^{6}. Equilibration time, 15 hours.
Freezing rate, 2 deg. C. per minute from +5 deg. to -19 deg. C. then 4 deg. C. per
minute from -19 deg. to -79 deg. C. Held frozen for 5 or more hours then thawed
in water at 5 deg. C. and checked for motility.)
[Illustration: Percent of motile sperm before and after freezing
consecutive ejaculates collected within a 4-hour period from each of 6
bulls (Fig. 1)]
In general, the motility before freezing improved slightly from the
first to the fourth to sixth ejaculate and then declined until about the
12th or 14th ejaculate, at which point the prefreezing motility seemed
to level off through the 20th ejaculate (Fig. 1). The percentage of
motile sperm found after freezing and thawing followed the same trend at
an average level 10 to 15 percent lower than the prefreezing level. As
is readily seen from the trend lines in Fig. 1, the difference between
the prefreezing motility and the post-thawing motility increased
gradually after about the fifth ejaculate. Although the absolute
difference did not increase greatly, the percentage of survival after
freezing dropped from 81 percent on the first 5 ejaculates to 26.5
percent on the last 5 (Table 2).
Table 2.--Comparison of the Freezability of 20 Consecutive Ejaculates
Collected Within a 4-Hour Period
(Weighted averages for 6 bulls)
===================================================================
Number Prefreezing Post-thawing
Ejaculate of motility motility Survival
ejaculates (percent) (percent) (percent)
-------------------------------------------------------------------
1st to 5th 29 53.3 43.2 81.0
6th to 10th 26 43.8 30.2 69.0
11th to 15th 23 28.6 14.5 50.7
16th to 20th 19 18.1 4.8 26.5
-------------------------------------------------------------------
=Freezability of epididymal sperm.= Since the freezability of bull semen
seemed to be better in second than in first ejaculates and some
improvement in freezability was evident through the first 4 to 6
ejaculates taken consecutively, the question of whether epididymal sperm
would withstand freezing seemed to be important. Although when 20
collections were made, the later ejaculates no doubt contained fewer
mature sperm, the lowered freezability could have been due to accessory
gland secretions rather than changes in the sperm themselves. Removing
sperm directly from the epididymis would eliminate any effect that the
accessory gland secretions could be exerting. Further, if epididymal
sperm could be frozen, obtaining and using semen from a bull shortly
after his death should be possible.