For the 15 minutes freezing the 4% glycerol was incorporated in the extender. Soon after
extension the semen was ampuled and frozen in liquid nitrogen vapour. The maximum time from
extension to freezing was less than fifteen minutes. The semen was frozen from within a few
seconds to up to |5 mnutes after addition of the extender.

For the | hour freezing, the semen was extended to ; volume with nonglycerolated extend-
er and cooled over | hour time to +5° C. After | hour the semen was made up to its total vol-
ume with glycerolated extender to yield a final glycerol concentration of 4%. The semen was
ampuled and frozen within fifteen minutes. For the 2; hour freezing, the semen was extended
as in the |4 hour method but the glycerol portion was added after 2 hours of cooling. The
semen was ampuled and frozen within ; hour after glycerol extender addition.

DIRECT DILUTION METHOD

When the semen was of high enough concentration (more than 200 million sperm cells per
cc) this method was utilized. Semen filtered of all gel was extended in the extender at 35°C.
The glycerol was already incorporated in the extender to give a final concentration of 4%.
Semen was quickly ampuled, sealed and frozen in liquid nitrogen vapour within 15 minutes.
There was no cooling necessary and the semen was transferred from room temperature to liquid
nitrogen vapour directly.

The semen was diluted to yield approximately either 50 million sperm cells/cc or 200 mil-
lion sperm cells/cc in different experiments.

Frozen semen was thawed by immersing the ampules in water at 40° C.

The thawed semen was stored at +5° C for evaluation of any latent damage to the cells.

Fertility tests were made on 10 mares with frozen stored from a period of several weeks
to one year.

RESULTS AND DISCUSSION

The semen froze very well with Y% glycerol. There were some ejaculates that froze
better with 3% glycerol. Fifteen minutes of glycerol equilibration was sufficient for good
recovery. The ampules that were frozen with a few seconds exposure gave equal or better re-
covery as the |5 minutes exposure showing that glycerol could afford maximum cryo-protection
without entering the sperm cell.

Table | shows the summary of recovery mttern obtained with different glycerol concen-
trations, eq times, osmotic pressure and methods.

It was interesting to note that although the osmotic pressure of the stallion semen was
in the region of 290 - 310 milliosmoles, best recovery was obtained repeatedly with extender
at 330 milliosmoles. The optimum glycerol concentration was 4% and equilibration time of 15
minutes was sufficient for good recovery.

Semen that was frozen without seminal plasma (centrifuged process) showed the most pro-
gressive motility. When semen was frozen with high seminal plasma concentration (Tow dilu-
tion rates of 1:1) the freeze recovery was lowered significantly. A high semen concentration
could be maintained in the ampule with good recovery only with the centrifuged process as
the seminal plasma was nearly absent. In the dilution method high dilution rates were re-
quired (at least Y times) for good recovery as otherwise the seminal plasma in the extended
semen was exerting a depressing influence on the freeze recovery. The centrifugation method
can be used conveniently for storing semen in pellets or straws.

Effects of seminal plasma removal was dramatically shown by two stallions whose semen
when collected during winter showed zero percent motility. When the seminal plasma was re-
moved and the sperm resuspended in an extender it was activated to up to 90% motility. This
semen, however, did not stand the freeze stress when frozen. Probably this anabiosis was
due to an overbearing effect of the inactivating material in the epididymal or accessory
fluid secretions, or it could be due to an inadequate amount of activating material in the
seminal fluid during winter. These stallions ejaculated normal semen with normal patterns

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