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

Glxcerolisation and Freezing Techniques with Bull Semen
W. K. Hunter
Milk Marketing Board
England

Since commencing with 0.5 c.c. straws in July 1966 the
diluent used has been skim milk prepared to the following
formulae :

a) Skim milk 87% b) Skim milk 79%
Egg Yolk 10% Egg Yolk 10%
Glycerol 3% Glycerol 11%
Fructose per 100 c.c. 1.25 grams Fructose per 10EE c oc e T LD
Antibiotics Antibiotics grams

Apart from experiments, dose rate has been standardised
with normal semen to 20,000,000 total spermatozoa, calculated
by colorimeter, which on thawing gives approximately 14,000,000
live sperm.

Primary dilution to 50% of final volume is made in diluent
a) at 300.C. The diluted semen is then cooled to 180.C. in 15
minutes when diluent b) is added to make 75% of final volume and
finally the diluted semen is cooled to 5°.C. in 15 minutes, when
the final 25% of diluent b) is added; making a final concent-
ration of 7% glycerol.

An equilibration time of 6/7 hours is now standard although

with semen from a small proportion of bulls the equilibration

time varies to a minimum of two hours. The majority of bulls,
however, function satisfactorily at 6/7 hours and the failure

rate both before and after freezing is less than when a 16/20

hour equilibration was standard. Equally it has been found

that a small proportion freeze better with a reduced glycerol
level.

All freezing is carried out horizontally in vapour, in
an MVE.9000 with the straws 4/5 cms. above the nitrogen level,
exposed to the vapour for seven minutes and then plunged into
liquid nitrogen.

Of greatest importance is the fact that critical compar-
isons can be made between diluents, glycerolising techniques,
equilibration times, bulls, breeds and individual ejaculates
with the advent of 0.5 c.c. straws of Cassou and the

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