Vle CONG. INTERN. REPROD. ANIM. INSEM. ARTIF., PARIS, 1968, VOL. I

DNA CONTENT OF BULL SPERMATOZOA AFTER FREEZING AND THAWING
A HANADA and H.NAGASE
National Institute of Animal Industry, Chiba-shi, Japan

Besides the function of the sperm tail, or middle piece, atten-
tion must be paid to the sperm head, especially the nucleic acid (DNA
), when sperm fertility is considered in connection with it's charact-
eristics. From this point of view, we investigated the DNA content in
bull spermatozoa before and after freezing and during preservation in
the frozen state.

GENERAL MATERTALS AND METHODS. Semen were collected from two normal
Holstein bulls. All freezing of semen was done in pellet form (Nagase
and Niwa, 196L) after dilution three times with two kinds of extender,
and glycerol equilibration at 5°C for L hours. The details of experi-
ments are described under each experimental results. DNA content in
spermatozoa was determined by Feulgen-microspectrophotometry on smear-
ed semen preparations, expressed in arbitrary units (A.U.), which
were calculated from multiplying ODSéO with nuclear area in each sperm
, and compared only within samples on a same slide but not with
another slides.

RESULTS, 1. One semen sample was divided into two parts. One part was
left as it was, and the other

Table 1. Comparison of DNA content was diluted with 20% yolk-10%
in bull spermatozoa before and after lactose-glycerol (3.5% after
freezing of semen. (Each N=28) dilution). After glycerol equi-
e Fenlyegmois EAéU') sample was frozen for ten min-
it N TSR Y- S i s utes and thawed. These three
Raw 7.L9 0.218 samples were smeared at the
Diluted 153 0.27hL same time. As shown in table 1
Frozen 735 0.318 , constant values of DNA con-

tent were demonstrated through

Difference within three samples was these semen processing.

not significant in 5% level.

2. One semen sample was divided into four parts, and diluted
with 20% yolk-3% citrate media, and glycerol (7% after dilution) was
added only to two parts. Freezing was carried out on three parts and
these were held frozen for one hour. Two frozen samples were thawed
and stored at 5°C in like manner with unfrozen sample. The last fro-
zen sample was stored in dry ice as a control. After preservation for
3 and 11 days, highest values of DNA content were demont irated in the
frozen control sample (table 2). The effect of freezing and thawing
was not clear after preservation for 3 days. But after 11 days, fro-
zen and thawed semen showed lower level of DNA content than the un-
frozen sample regardless of glycerol level.

3. Two bull semen samples were diluted with 20% yolk-3% citrate-
glycerol (3% after dilution). After freezing in pellet form, semen
was stored in dry ice or in liquid nitrogen in plastic tubes with
caps. The difference in DNA content between samples stored in two
cryogens was not significantly different during preservation for 55

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