Good results usually can be obtained in freezing bull semen if care is
taken in collecting, diluting and processing the semen. Occasionally the
semen from certain bulls will not withstand freezing well. The reason
for this is not understood at present. However, carefully following the
directions and suggestions given below will usually produce satisfactory
results with semen samples that are of good quality at the start.
Experience in the field has shown that fertility results with frozen
semen are usually slightly lower during the first few months than with
liquid semen stored at 5 deg. C. (41 deg. F.). Most units that have worked with
frozen semen over a period of a few months are able to improve and do
get fertility results as good as, or better than, obtained in their
liquid semen program.
=Collection of the semen.= In order to obtain the best possible semen
for freezing, care and cleanliness should be exercised in making the
collection. The artificial vagina, and the glassware used should be
clean and dry. The underline of the bull should also be clean and dry.
The bull should be restrained near the teaser cow for a minute or two
prior to collection in order to excite the flow of secretions prior to
ejaculation. Allowing the bull to mount the teaser once without serving
the artificial vagina is a good practice to use in properly stimulating
the bull before collection of the semen.
If the bull has not been used for three or four days, the collection of
a second ejaculate for freezing may be advisable. The second ejaculate
seems to withstand freezing better than the first in many instances. A
clean, dry artificial vagina should be used for each ejaculate
collected. Repeated collections in the same artificial vagina may result
in contamination of the semen with bacteria, lubricating jelly and
minute particles of dirt. The semen sample should be protected from
contamination and from sudden temperature drops (cold shock).
=Preparation of extender.= A suitable egg yolk-citrate extender for
freezing bull semen can be prepared by the following procedure. One part
egg yolk (free of egg white and the membrane surrounding the yolk) is
mixed with 4 parts 2.4 to 2.9 percent sodium citrate dihydrate solution.
The citrate is prepared with distilled water and then boiled or
autoclaved. The citrate solution should be cooled before it is mixed
with the egg yolk. After the egg and citrate are mixed, 1000 units of
penicillin and 1000 micrograms of streptomycin are added per milliliter
of extender. Sulfanilamide should not be added. This extender can be
prepared 12 to 24 hours before use if it is stored at refrigerator
temperature. The portion of the extender needed for the original
dilution of the semen should be warmed to room temperature before it is
mixed with the semen.
=Dilution after collection.= As soon as possible after collection, the
semen sample should be diluted with the extender. The extender must be
at the same temperature as the semen (room temperature) when the two are
mixed together. At this time the semen can be partially diluted (1 part
semen to 4 parts of extender) or diluted to a sperm concentration twice
the final desired concentration (later in adding the glycerol for
freezing, the semen is diluted further with an equal volume of glycerol
containing extender). The diluted semen is slowly cooled (1-1/2 to 2-1/2
hours) to 5 deg. C. (41 deg. F.). Some units using frozen semen now allow the
semen to stand at 5 deg. C. for 5 to 6 hours before glycerolization to allow
the antibiotics to be more effective against any vibrio fetus organisms
that may be present. This step is taken because it has been shown that
glycerol inhibits the effectiveness of the antibiotics.[6] After
cooling, semen can be further diluted to twice the desired sperm
concentration if that were not done at the start. (Caution: Be sure
semen and diluent are at the same temperature.)
=Adding the glycerol.= The glycerol solution is prepared by adding 14
volumes of glycerol (reagent grade) to 86 volumes of yolk-citrate
diluent (same as yolk-citrate used for original dilution). This solution
may be added dropwise with constant gentle mixing to the already diluted
semen, or one-third at a time at 10-minute intervals with gentle mixing
during each addition. Either method should take about 20 to 30 minutes.
The total volume of glycerol-yolk-citrate solution added should be equal
to the volume of the original diluted semen. In this way a concentration
of 7 percent glycerol is obtained in the final mixture that is to be
frozen. Care must be taken to keep the temperature at 5 deg. C. (41 deg. F.)
during the time the glycerol is being added. (A cold room is best for
maintaining a temperature of 5 deg. C., but with care the operation can be
carried out at room temperature by using pans of ice water and a
refrigerator.)
=Equilibration.= The results presented in this bulletin suggest that
little or no time need be allowed after the glycerol is added before
freezing. However, results obtained by other workers show improved
fertility with at least 12 hours equilibration. Some units getting good
fertility results with frozen semen also are allowing the semen to stand
at 5 deg. C. for 12 to 18 hours before freezing. After the semen has
equilibrated with the glycerol, 1-milliliter portions of the mixture are
placed in 1.2- to 2-milliliter vials or ampules which are then sealed.
Ampuling can be done with an automatic syringe or pipette, provided a
large gage needle is used. Also, it is important not to force the fluid
mixture rapidly through the syringe or the sperm may be injured.
=Freezing.= The vials or ampules of diluted semen are placed in a bath
of isopropyl alcohol which has been cooled to 5 deg. C. (41 deg. F.). This bath
can be a wide-mouth thermos bottle or an insulated container of almost
any sort with a large opening at the top. The size needed depends on the
number of ampules being frozen. Some sort of convenient tray for holding
the ampules in an orderly fashion and enabling the samples to be
completely submerged is desirable. A few ampules can be kept together
easily by placing them in a polyethylene freezer bag that has had many
small holes cut in it to let the alcohol of the bath contact the
ampules. The ampules must be completely covered by the alcohol to insure
uniform cooling.
The alcohol of the bath and the ampules of semen are cooled by adding
chipped or ground dry ice in sufficient amounts to lower the temperature
of the bath 2 deg. C. (3.6 deg. F.) per minute from +5 deg. to -20 deg. C. From -20 deg.
down to -79 deg. C., the rate of cooling can be doubled (4 deg. C. or 7.2 deg. F.).
Electrical equipment that regulates the cooling rate to the desired
temperatures is available commercially, but the cost may be too high for
some small operations. The samples should be held at -79 deg. C. (-110 deg. F.)
until they are thawed. This can be done by using an alcohol bath and dry
ice or by special mechanical refrigerating equipment. At no time prior
to thawing should the samples be exposed to warmer temperatures.
=Thawing.= The ampules of frozen semen can be thawed by removing them
from the dry ice storage box and dropping them into a water bath at 5 deg.
C. (41 deg. F.). Thawing temperatures up to body temperature, 38 deg. C. (100 deg.
F.), can be used but extreme care must then be taken not to pass the
semen through a cold inseminating tube; for this would subject the sperm
to cold shock. The semen should be used for breeding within a few
minutes after thawing.