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

STABILITY OF THYMINE IN
SPERMATOZOAL DNA DURING STORAGE IN VITRO

H.H.KOEFOED-JOHNSEN, J,FULKA and V. KOPECNY

Sterility Research Institute, Copenhagen, Denmark,
Laboratory of Physiology and Genetics of Animals,
Czechoslovak Academy of Sciences, Libechov, CSSR

During storage of spermatozoa in vitro their ferti-
lizing ability gradually decreases. Loss of DNA has been
recently considered as one of the possible causes for
this decrease. SALISBURY et al., (8) were the first to ex
press this supposition after detecting a decrease of
Feulgen positive material in stored bull spermatozoa.The
applicationdcytospectrophotometry in UV light and the
investigation of spermatozoa from different parts of the
genital tract, altered to a certain extent, the explicit-
ness of the original hypothesis (2,3).

The aim of the present work was to obtain further
information about the possible influence of in vitro age-

ing of spermatozoa on their DNA. For this purpose semi-
quantitative autoradiography (ARG) of rabbit gpermatozoa
labelled in thymine with tritium was employed,

MATERIAL AND METHODS. Two male rabbits with normal semen
production and good fertility were each injected at two
day intervals with four decreasing doses of thymidine

SH (Amersham, spec.activity 3.000 uc/mM),in a total of
1.3 mc/kg body weight., Ejaculates were collected regu-
larly twice a week, and spermatozoa from the 42nd to the
56th day after the first injection were used for experi-
ments. Fresh ejaculates were diluted in Tris buffer with
egg yblk (6) and stored at 4°C and 37°C under the con-
trol of antibiotics. Smears for ARG were prepared from
fresh spermatozoa (O hrs),and from spermatozoa stored
for 24 and 72 hrs at 4°C and 240 hrs at 37°C., Sperma-
tozoa from the same ejaculates stored at 4°C for 0,12,24
48,60, and 72 hrs, respectively, were used for the con-
trol of fertilizing ability. The procedure of insemina-
tion and evaluation of ova 24 hrs after application of
semen was the same as described previously (4).

Before coating with liquid Ilford K 5 emulsion,
smears were Feulgen stained (hydrolysis in 1 N HC1 for
12 min, at 60°C). Two exposure times , 8 and 12 days,
were used for all preparations, Evaluation of autoradio-
grams was carried out by two persons using a binocular
microscope NfpK, Carl Zeiss, Jena,obj.90Hi,oculars 15 K
in green light, Each observer counted from each storage
interval grains overlying 500 individua] ,randomly chosen

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