under the microscope. There was, however, no decrease in
motility after more than 30 minutes under the microscope.
This was also far from what we had expected.

5. Effect of season of semen collection: Freezability of
spermatozoa collected in the off season was slightly lower than
that in the breeding season in some stallions. This result is
in accordance with Rombe's report (1).

6. Effect of temperature and duration of storage: When
stored in dry ice, that is at =79 C, viability decreased by 5
to 15 % after 3 to 4 weeks. But when stored at -196°C in liquid
nitrogen there was hardly drop in viability even after one year.

B. CONCEPTION TESTS

Materials and methods. Three conception trials were performed,
first in 1964, second in 1966 and third in 1967. Preparation
of frozen semen and insemination for the conception test was
conducted as follows. The semen was diluted with the dilutor
at the rate of 10;3 after the gelatinous portion was removed.
The diluted semen was then poured into a 50 ml precipitation
tube, centrifuged for 10 minutes at 2,300 to 2,500 r.p.m.

After the supernatant was removed, 2 to 3 ml of the dilutor was
added to the concentrated spermatozoa. Then the secondary
dilution was performed by adding an equal volume of the dilutor
containing glycerol. The final concentration of glycerol was

4 %, and the glycerol equilibration time was 2 to 3 hours.
After glycerol equilibration, the semen was poured into one ml
vinyl straws and cooled to about -80°C in % to 5 minutes with
liquid nitrogen vapor (2). Thawing was carried out by immersing
directly into water at 4°C. The number of straws for one in-
semination is ordinarily three. The content is diluted with
the dilutor to 25 ml per mare. The number of spermatozoa in
one injection was 450 to 1,800 milliom.

Results and discussion. In the first test, frozen semen pre-
served for 91 and 311 days in the dry ice cabinet was injected
into separate mares. Both conceived, but I regret to say, both
aborted due to falling down and malnutrltlon at 154 and 246 days
of gestation, respectively. In the second test, six mares were
inseminated and three conceived after the first insemination
and reached parturition. In the third test, the 105 mares were
inseminated. The conception rate after the first insemination

(1) S. ROMBE, V.KOTJAGINA, & N. PILER, 1965, Konevodo konnyi
- Sport, 35 : 8. (ABA, 34 ;169)

(2) Y. NISHIKAWA et al., 1965, Jape. J. Frozen Semen Res. Soc.,
15 & 16 ; 23