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

STUDIES ON DEEP FREEZING OF HORSE SPERMATOZOA

Y. NISHIKAWA (Faculty of Agr. Kyoto Univ., Kyoto, Japan)
Y. WAIDE (Nat. Inst. of Anim. Indust., Chiba, Japan)
S. SHINOMIYA (Kushiro A.I. Center, Kushiro, Japan)

Establishment of a technique with frozen semen for wide and
practical application to horse reproduction will be of great
significance industrially as well as scientificelly. There are
about 20 research reports on deep freezing of horse spermatozoa,
but no report seems to have dealt with its actual application
to horse breeding on a practical basis. In our laboratory an
experiment on deep freezing of horse spermatozoa was started a
few years ago, and is still being continued. The present experi-
ment is composed of two sections.

A. EXAMINATIONS ON FACTCRS WHICH SEEM TO INFLUENCE SPERM

FREEZABILITY
Materials and methods. The semen vial used exclusively in this
experiment was one ml vinyl straw. Freezing below —750C was
performed in 30 to 40 minutes with dry ice in the early tests,
but in the later tests it was accomplished with the use of liquid
nitrogen vapor in several minutes.

Results and discussion. 1. Effect of the composition of semen
dilutor: The composition of the dilutor has marked effects on
sperm viability of frozen semen. Egg yolk citrate dilutor
commonly used for cattle semen is quite harmful for horse semen.
When diluted with a kind of egg yolk-glucose-buffered solution
for horse semen we devised, however, the viability of frozen
spermatozoa was very high for horse. DBesides, when seminal
plasma was removed by centrifuging, the result was much better
than with the whole semen. The concentration of egg yolk giving
best result is 2 to 8 %. This is much lower than for cattle semen.

2. Effect of addition of glycerol: The best result was
obtained when the final percentage of glycerol was 4 to 5 %. and
the glycerol equilibration time was 3 to 5 hours. These figures
are all lower than for cattle semen. Glycerol was the best pro-
tective solute against freezing and ethylene glycol, polyethylene
glycol, and propylene glycol were not suitable.

3. Effect of horse breed: There was no difference in sperm
freezability among Anglo-Norman cross-bred, Percheron cross-bred
and Korean ponies used in this experiment.

4, Individual variations of stallions: It is clear that sperm
freezability varies greatly according to individual stallions.
Spermatozoa of some stallions showed viability almost as high as
before freezing, the recovery rate being 80 to 100 %. Such high
freezability of horse spermatozoa was not expected before the
test. With frozen spermatozoa of cattle, it is common that sper-
matozoa lose their viability very quickly during motility tests

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