The first trials by the British at freezing bull semen were made with
samples containing many millions of sperm cells. In routine artificial
breeding, it is common to add extenders to semen so that one milliliter
of diluted semen may contain only 10 million living sperm cells. (This
number still insures optimal fertility.) Frequently the addition of 100
or more parts of the yolk extender to each part of the original semen
sample is possible without reducing the sperm numbers below 10 million
per milliliter. No one knew if this process of dilution would affect the
resistance of bull sperm to freezing. The effect of various rates of
dilution on the freezability of bull sperm was tested with 10 semen
samples. The results, presented in Table 6, show that the numbers of
sperm between 10 and 90 million per milliliter did not influence the
percentage of sperm that survived freezing.
In a later trial it was found that sperm survival was slightly better at
lower dilution rates than in the same samples frozen following dilution
to 15 million sperm per milliliter. However, field trials with frozen
semen carried out by others, using sperm numbers as low as 15 million
per milliliter of semen inseminated or even lower, have been highly
satisfactory.[11],[12]
During the early studies in the Illinois laboratory, the effects of
glycerol level were also tested.[13] These effects are discussed in the
section on glycerol additions beginning on page 17.
=Effect of further dilution and refreezing after the initial freezing.=
Under some circumstances it might be advantageous to freeze semen with
a high concentration of sperm cells and then extend it further after
thawing. With such a procedure less storage space is needed than when
dilution is carried to the maximum before freezing. Two experiments were
conducted to test the effects of dilution and storage at 5 deg. C. and
dilution and refreezing following an initial freezing of concentrated
samples.
Table 6.--Effect of Sperm Numbers and Glycerol Level in Final Mixture on
Freezability of Bull Sperm at -79 deg. C. (Average of 10 ejaculates)
================================================================
Post-thawing motility (percent)[F]
---------------------------------------------
Glycerol level Number of sperm (millions/ml.)
(percent) --------------------------------
90 30 10 Average
----------------------------------------------------------------
5 36.0 34.0 36.0 35.0
10 22.0 24.0 23.0 23.0
15 3.2 0.9 0.2 1.4
Average 20.3 19.8 19.9 20.0
----------------------------------------------------------------
[F] Mean initial motility of sperm before freezing was 55 percent.
Four semen samples were split and extended at rates of 1:1 (semen to
extender) and 1:10. These were frozen, then thawed and halved. One half
was further extended to a level of 15 million sperm per milliliter; the
sperm numbers in the other remained unchanged. Each of these halves was
split again, and one portion of each was stored at 5 deg. C. for 3 to 7
hours. The other two portions were refrozen.
Table 7.--Effect of Further Dilution and Refreezing on Sperm Motility
After the Initial Freezing of Bull Semen
========================================================================
Post-thawing motility
Dilution Pre- ---------------------------------------------------
of freezing After After storage[G] After refreezing[H]
semen motility first ------------------- --------------------
(semen: (percent) freezing No Diluted No Diluted
extender) further to 15 further to 15
dilution million/ml dilution million/ml
------------------------------------------------------------------------
First trial: 4 samples
1:1 60 49 46 34 31 6
1:10 53 45 36 30 25 5
Second trial: 7 samples
1:9 67 47 41 35 28 11
15 million/ml 67 30 32 .. 18 ..
------------------------------------------------------------------------
[G] Stored at 5 deg. C. for 3 to 7 hours after first thawing.
[H] Refrozen following first thawing.
Table 8.--Effect of Glycerol Level and Storage at 5 deg. C. on Motility of
Sperm in Yolk-Citrate Extender
====================================================================
Sperm motility
--------------------------------------------------------
Post- After storage at 5 deg. C.
Glycerol thawing ---------------------------- Average
level 1 day 3 days 7 days
(percent) --------- --------- --------- --------- ---------
per- rate per- rate per- rate per- rate per- rate
cent cent cent cent cent
--------------------------------------------------------------------
Control[I] 56 2.5 55 1.9 46 1.8 38 1.4 48 1.90
0 54 2.4 44 1.9 46 1.8 36 1.4 45 1.87
5 52 2.2 50 1.9 46 1.7 32 1.4 45 1.80
10 52 2.3 46 1.8 42 1.7 28 1.6 42 1.85
20 52 2.1 50 1.7 44 1.6 38 1.1 46 1.62
30 50 0.7 44 0.5 42 0.4 30 0.4 42 0.51
Average 53 2.03 47 1.62 44 1.50 34 1.22 .. ....
--------------------------------------------------------------------
[I] The control differed from the 0-glycerol treatment in that no
additional citrate or glycerol solution was added.
A similar trial was carried out with seven samples; one portion was
diluted 1:9; the other was extended at the outset to 15 million sperm
per milliliter. Results for both tests are summarized in Table 7.
From Table 7 it can be seen that refreezing following an initial
freezing further reduced the number of surviving sperm. The second
freezing was more detrimental to the portion of the samples extended to
15 million sperm per milliliter than to the portion that was refrozen at
a higher sperm concentration. The percentage of motile sperm remained
fairly high in the portions that were diluted to 15 million sperm and
stored at 5 deg. C. However, in all cases, survival was best in the samples
at the lower dilution levels.