By finding how methods of handling affect the sperm cells, one can
sometimes improve the procedures to avoid harmful effects. Some attempts
have been made in this laboratory to determine the effects of the
freezing procedures on the metabolic activity of bull spermatozoa. These
investigations have been limited in scope, involving the measurement of
oxygen-consumption and estimates of sperm motility during and after
incubation at 37 deg. C. in a Warburg apparatus.
=Effect of glycerol additions on oxygen uptake of diluted semen at 37 deg.
C.= The effect of adding glycerol to diluted semen on oxygen consumption
of the sperm was tested in a Warburg apparatus, using semen diluted with
an extender consisting of one part egg yolk and one part 2.9 percent
sodium citrate dihydrate. The yolk-citrate extender was added to the
semen at a rate which brought the sperm concentration in 0.5 milliliter
to 200 million to 500 million. An exact count was used to calculate the
oxygen uptake per 10^{8} sperm per hour (ZO_{2}).
[Illustration: Effect of glycerol additions on oxygen consumption of
sperm at 37 deg. C. (Fig. 9)]
Glycerol in various percentages in 2.9 percent sodium citrate dihydrate
solution was placed in the sidearm of the Warburg flasks. The diluted
semen was held in the main compartment. After a 60-minute preliminary
run, in which the rate of oxygen uptake of the sperm in yolk-citrate
diluent was determined, the contents of the sidearm were tipped into the
main compartment. The resulting glycerol percentages after mixing the
sidearm and main compartment contents were 0, 4, 8, and 12 percent. Ten
samples of semen were subsampled and the oxygen uptake of each was
determined at all four levels of glycerol.
Oxygen uptake was increasingly stimulated during the first 20-minute
interval by each increase in the amount of glycerol added (Fig. 9).
After the first 20 minutes, the rate of oxygen utilization decreased at
the two higher levels of glycerol but persisted at 4 percent. The rate
of oxygen consumption for the first 20-minute period at the 4-percent
glycerol level was 130 percent that of the control to which only sodium
citrate had been added. At 8 and 12 percent the values for the period
were 144 and 192 percent, respectively, of the control rate.
[Illustration: Effect of glycerol and glycerol-plus-catalase additions
on sperm motility during incubation at 37 deg. C. (Fig. 10)]
With each increase in glycerol level, motility was reduced during the
incubation period. This is shown in Figure 10 along with the effect on
motility of adding catalase, which is discussed in the next section.
=Effect of glycerol-plus-catalase on oxygen uptake of diluted semen.=
Certain bacteria have been shown to break glycerol down, forming
hydrogen peroxide as follows:
Glycerol + oxygen --> lactic acid + hydrogen peroxide.
Hydrogen peroxide is known to be detrimental to sperm. The addition of
glycerol to diluted semen first increased oxygen uptake and then reduced
it. Since a reduction in sperm survival followed, some harmful action
must have taken place with the addition of glycerol at 37 deg. C. To test
whether this action could be due to the release of hydrogen peroxide as
occurs in certain bacteria, glycerol with catalase--the enzyme which
breaks down hydrogen peroxide--was added to a portion of 8 diluted semen
samples and the oxygen uptake was recorded. Comparison of the resulting
oxygen uptake with glycerol and with glycerol plus catalase is shown in
Figure 11.
[Illustration: Effect of additions of glycerol-plus-catalase on oxygen
consumption of sperm at 37 deg. C. (Fig. 11)]
Oxygen consumption was increased by the presence of added catalase at
all glycerol levels and in the control. Sperm survival during the 3-hour
period at 37 deg. C. also was improved by the presence of catalase (Fig.
10). However, the general trend in oxygen consumption produced by the
addition of glycerol was not changed greatly. The higher levels of
glycerol still stimulated oxygen uptake during the first 20-minute
period after the additions and then slowed the rate of oxygen
utilization. The rate of utilization was generally higher during the
test period in the presence of catalase than without added catalase. It
appeared that a part of the harmful effect of glycerol might be due to
the formation of hydrogen peroxide. Still, the detrimental effects of
the higher levels of glycerol were not completely removed.
Table 16.--Effect of Freezing Procedures on Oxygen Utilization of Bull
Sperm in Yolk-Citrate Extender
(Average of 5 ejaculates)
=====================================================================
Microliters of oxygen
Semen sample tested utilized per 10^{8} sperm
--------------------------
First hour Second hour
---------------------------------------------------------------------
Fresh diluted semen 10.3 8.1
Fresh diluted semen glycerol tipped in
at end of first hour 9.7[L] 12.9[L]
Aged 20 to 24 hours at 5 deg. C. 11.2 8.3
Aged 20 to 24 hours at 5 deg. C. glycerol
tipped in at end of first hour 11.8[L] 12.9[L]
After 20 hours equilibration with glycerol 11.7[L] 7.8[L]
After freezing and thawing 9.7 6.3
--------------------------------------------------------------------
[L] Average of 3 ejaculates.
=Effect of freezing procedures on oxygen utilization by sperm.= Limited
data have been obtained on the effects of some of the freezing
procedures on the oxygen utilization of bull sperm. The results obtained
in these experiments confirmed the earlier findings that tipping
glycerol directly into the diluted semen at 37 deg. C. caused an increase in
oxygen consumption (Table 16). All other steps in the freezing procedure
had little effect on oxygen consumption by the sperm. Except where
glycerol was added during the determination, the rate of oxygen
utilization was lower the second hour than during the first. The oxygen
uptake of semen that had been frozen and thawed seemed to drop faster
than that of unfrozen samples.
=Effect of freezing procedures on methylene-blue reduction time.= The
methylene-blue reduction test has been used as a means of measuring
semen quality and is dependent on the metabolic activity of the sperm.
The effects of various freezing procedures on the ability of samples to
decolorize methylene blue were determined with 10 semen samples. Sperm
numbers were standardized to 300 x 10^{6} cells per milliliter and the
time required for these cells to reduce a 1:40,000 solution of methylene
blue was determined on freshly diluted semen, after the addition of
glycerol, after equilibration, and after freezing and thawing. Portions
of each diluted sample were tested at these stages of the procedure with
glycerol alone added and with glycerol and various sugars added.
A marked increase in the time required for the sperm to reduce methylene
blue occurred when the glycerol was added (Table 17). This increase was
greatest in the portions with glycerol alone and with glycerol and
glucose. The time increase was less pronounced in the presence of the
three pentose sugars used. Following equilibration, the samples regained
the ability to reduce methylene blue at a rate only slightly slower than
when they were fresh. Freezing and storage of semen resulted in slower
reduction of the methylene blue than was shown after equilibration with
glycerol. Since freezing usually kills some of the sperm, a slowing of
the reduction time after freezing would be expected.
Table 17.--Effect of Freezing Procedures on the Methylene-Blue Reduction
Time of Bull Semen With and Without the Addition of Sugars[M]
(Average of 10 ejaculates)
======================================================================
Methylene-blue reduction time (minutes)
------------------------------------------------
Glycerol Glycerol Glycerol Glycerol Glycerol
only and and and and
glucose arabinose xylose rhamnose
----------------------------------------------------------------------
Fresh semen 5.2 5.2 5.2 5.2 5.2
After glycerolization 26.4 25.2 17.3 14.3 19.4
After 18 hours
equilibration 7.4 6.5 6.4 5.3 6.2
Thawed immediately
after freezing 11.5 10.5 9.4 9.0 9.4
Thawed 48 hours
after freezing 14.3 10.2 11.3 10.1 9.5
----------------------------------------------------------------------
[M] Glycerol level in the final frozen mixture was 7 percent. Sugars
were added to a level of 1.25 percent.