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

A SPECTRAL ANALYSIS OF RAM SPERMATOZOAL DNA
AFTER COLD SHOCK OR DEEP-FREEZING

P. J. QUINN and I. G. WHITE

Department of Veterinary Physiology,
University of Sydney, Sydney, Australia.

There is at present no satisfactory explanation for the low
fertility of deep-frozen ram semen. The spermatozoa must, however,
not only reach the site of fertilization, but also possess a com—
plete and presumably native complement of genetic material in order
that the developing embryo should survive.

Preliminary experiments in our laboratory have revealed that a
degradation of DNA into dialysable fragments occurs slowly in senes-
cent ram semen but the process is accelerated if the semen has been
cold shocked or deep—frozen (1). In addition the DNase 1 activity
of ram seminal plasma is extremely high, in contrast to the bull,
and freezing ram semen directly to =79°C resulted in a decrease of
DNase activity in the plasma and a subsequent increase in an extract
of washed sonicated cells during incubation 2).

These studies have now been extended to an investigation of the
changes in the amount and configuration of DNA in ram spermatozoa
after cold shock and deep freezing. DNA was extracted from the
spermatozoa by a method (3) in which, after an initial wash and re-
moval of lipid material, the residue was treated with 2-mercapto-
ethanol for several hours at 0°C and the nucleic acids released by
tryptic digestion. After centrifuging, the DNA was precipitated
from the supernatant with n-propanol and redissolved in saline-
citrate. Extraction of the pellet with hot perchloric acid and
determination of deoxyribose (4) showed that less than 5% of the
total DNA remained. Proteins and peptides were removed by repeated
shaking with chloroform until no residue was observed at the inter-
face. The samples were then shaken twice with equal volumes of
water—-saturated phenol and finally twice with ether to remove any
remaining phenol. The deproteinized samples were precipitated with
n-propanol and the DNA redissolved in a convenient volume of 10 mM

(1) P. J. QUINN, I. G. WHITE & K. W. CLELAND, 1968. J. Reprod.
Fert. (In press).

(2) P. J. QUINN, 1968. J. Reprod. Fert. (In press).

(3) E. BORENFREUND, E. PITT & A. BENDICH, 1961. Nature, 191: 1375.

(4) K. W. GILES & A. MYERS, 1965. Nature, 206: 92. e

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