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

PROTECTIVE EFFECTS OF SUGARS AGAINST FREEZING INJURY OF BULL
SPERMATOZOA

H. NAGASE, S, YAMASHITA* and S. IRIE*
National Institute of Animal Industry, Chiba-shi, Japan

* Saitama Zootech. Expt. Stat., Kohnanmura, Ohsatogun, Saitama
Prefecture

Protective effects of sugars on spermatozoan survival with
pellet freezing were demonstrated by Nagase (1) and Nagase et al
(2-4) in the bull, goat and stallion, Two trials were conducted
to elucidate the sugar protection effect on survival and
fertility of spermatozoa in the pellet and with other freezing
procedures.

MATERIALS and METHODS. To elucidate the sugar protection in
different freezing methods, Holsteln semen which was extended

1:2 with yolk (20%)-sugar extenders soon after collection,
precooled to 5°C and equilibrated for 10 hrs, was frozen in three
different methods. Samples were thawed at 40°C. And the post-
thaw motility was observed at samples, which was added the
thawing media after thaw. Sugar and concentrations tested were
7.5% pentose and hexose, 10% disaccharides and 18% raffinose,
Egg-yolk citrate extender containing 0.31M (final level) of
glycerol was used as a control. The freezing methods were slow
versus the pellet and rapid freezing (dropping into liquid
nitrogen). Volume of semen was 0,1 ml both in slow and in
pellet, and 0,013-0.015 ml in the rapid method used for freezing.
Freezing rates of the semen were 5-7°C and 25°C per minute in the
slow and the pellet, respectively.

In the rapid method, the semen was dropped on liquid nitrogen
where it floated for 20 seconds and then sank,

In the fertility trial, 0.2 ml of the semen was extended 1:2
with egg yolk-lactose (10%), -raffinose (18%) and -lactose
containing glycerol (5.25%---control) extender, precooled to 586
and equilibrated for 6 hrs.

All were frozen in pellet form. The pelleted semen was
preserved in crushed dry ice for 7<54 days and thawed by adding
two pellets to 0,6 ml of the thawing media (5) kept at 35°C just
before insemination.

RESULTS and DISCUSSION, Results comparing sugar protection by
different freezing methods are given in Fig, 1. Glycerol in 0.31M
in the control shows good protection in the slow freezing and
pellet method, but was low in the rapld freezing. In the contrast
to glycerol, all sugars indicated remarkably better protection in

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