Vie CONG. INTERN. REPROD. ANIM. INSEM. ARTIF., PARIS, 1968, VOL. II

Freezing of ram semen in Cassou straws.

R.W. SAINSBURY
A.l. Research Unit, Ballycoolin, Finglas, Co. Dublin, Ireland.

The long term preservation of ram semen has presented a problem to
workers in many parts of the world. Low temperature storage of ram
spermatozoa was first described by Emmens and Blackshaw in 1950. A
slow rate of freezing resulted in poor survival of sperm. Some revival
was obtained by rapid freezing using diluents containing sugars. The
best appeared to be 40% sucrose in 0.9% sodium chloride. Diluents
containing glycerol and arabinose also gave good results.

Ram semen revived well from -89°C if frozen in 3% sodium citrate
adjusted to PH 7.1 with 0.1 M. phosphate buffer and a final concentration
of 7.5 to 10% glycerol and 1.5 arabinose.

Following the 5th International Congress on Anima! Reproduction
and Artificial Insemination work was commenced on freezing ram semen
by Mr. M. Keen at the National University of Ireland under the direction
of Professor Gordon and myself. It was decided that the experiments
should be conducted using straws rather than ampoules. Mr. Keen did
considerable work on the effect of various diluents, equilibration times,
freezing and thawing rates on the recovery of ram sperm frozen at -79°C
in an alcohol bath.

The best restlts were obtained using Lactose-egg yolk-glycerol.
Equilibration times of not less than four hours and not more than eight.
Fast freezing rates coupled with fast thawing at 37°C gave the best recovery.
Differences in percentage of motile sperm post freeze were significant
(P.05) after 2 days and 10 days but not after one month.

Work was commenced at the Department A.l. Research Unit in 1966
with a view to establishing the freezability of ram semen and its keeping
properties in the frozen state.

The diluent used was the Nagase lactose-yolk-glycerol as used in
pellets for bull semen, A 0.5 cc straw as a container and four hours equi-
libration time.

The semen was collected from four rams (three Finland and one
Suffolk) by electro-ejaculation and diluted immediately in the diluent
at 30°C. The diluted semen was allowed to remain at room temperature
for 30 minutes and then placed in the refrigerator at + 4°C for two hours.
The straws were then filled in a cold chamber (#4°C) and transferred to
iced water for a further one and a half hour. Freezing was carried out in
the horizontal position on racks over nitrogen vapour as for bull semen.
The frozen semen was stored in liquid nitrogen in a Linde 35, Samples of
this semen were examined at varying periods over the next two years.
Three of the rams (two Finland and one Suffolk) showed little deterioration
over the two year period, the numbers of live sperm and a good wave
motion being maintained. The semen obtained from these rams was of

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