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

Studies on deep freezing preservation of fowl semen.

Moriyuki WATANABE
Dept. of Animal Husbandry, Faculty of Fisheries and Animal Husbandry,
Hiroshima University, Fukuyama, Japan.

Since the possibility of long-term storage of fowl spermatozoa
at low temperature was reported by Shaffner et al. (1), numerous
investigations have been made by many authors (2) (12), but in no
experiment did they obtain as good results as with bull semen.
Therefore, there is still much study to be done on the freezing pre-
servation of fowl semen.

The present investigation was carried out to establish techniques
for the freezing of fowl spermatozoa. Various in vitro treatments of
fowl spermatozoa were studied in an effort to determine the results
obtained from different diluents, freezing and thawing methods,
glycerol concentration, temperatures and equilibration times. holding
temperatures after thawing, and the correlation between the resumption
of fowl spermatozoa motility and storage periods and fertility.

MATERIALS and METHODS

The five cocks used for the present experiments were 2 or 3-
year old 8ingle Comb White Leghorn. The semen samples were collected
from 7:00 to 7:30 a.m. every three or four days by the abdominal
massage method. The four diluents A, B, C and D (prepared for the
present study), Yamane's and Locke-Ringer solutions were used for
freezing. The composition, pH and depression of freezing point of
these diluents are shown in Table 1.

The collected semen was diluted four or eight times with the
various diluents mentioned above at 37°C, and the semen samples were
frozen and thawed using the four methods shown in Diagram 1, respecti-

ve Glycerol concentration differed from one experiment to another.

In Trials 1 and II, glycerol concentration ranged from 7 to 50 percent,
whereas in Trials IIT and IV, it ranged from 5 to 12 percent. The
temperatures for glycerol equilibration and preservation of semen
samples after thawing were 20°C, 10°C, and 5°C for the former trials,
and 20°C, 30°C, and 37°C for the latter, respectively. The time for
glycerol equilibration varied with the experiment. In Trials Fiand IT;
it ranged from 30 to 90 minutes, and in Trials III and IV from 60 to
180 minutes, and from 60 to 90 minutes. As to the correlation between
various freezing periods and the motility of semen after thawing,
these were examined using semen thawed after 1, 7, T30 0 90 siE50is
180, and 365-day storage. The fertility test was practised on semen
stored at -79°C for 30, 90, and 100 days.

RESULTS and DISCUSSION

Diluent A was the most effective of the six above-mentioned
diluents for preserving spermatozoa motility after freezing. The
motility of spermatozoa diluted with Locke-Ringer solution was fairly
high just after thawing, but the movement was not normal, the sper-
matozoa behaving wildly and then losing motility rapidly. Yamane's
solution did not seem to be a suitable diluent for spermatozoa
freezing. The reason is not yet clear, but it is supposed that

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