NaCl buffered to pH 7.0 with 1 mM sodium phosphate.

A spectrum (220 to 290 mpuat 5 mmintervals) of each sample
contained in 1 cm light path, stoppered quartz cuvettes at 25°C was
obtained with a Hitachi UV spectrophotometer fitted with a tempera-
ture controlled cuvette housing. The temperature of the DNA samples
was then increased slowly to 80°C and after 10 minutes equilibration
the spectrum was recorded again and the absorbance corrected for
thermal expansion. The samples were then recooled to 25°C and a
final spectrum in the range 240 to 280 mmat 10 muintervals re-
corded. The spectra of native and denatured DNA and the hyper-—
chromic spectra were all analysed using the three term spectral
parameters derived from bacterial DNA (5).

In the first experiment (Table 1) aliquots of ram semen held
for 10 minutes at 37°C were cold shocked by placing in a water bath
at 0°C or frozen directly to -79°C in finely crushed solid CO,.
After 10 minutes the samples were incubated with an untreated control
at 37°C for 1 hour. Analyses showed a decrease in the DNA content of
the spermatozoa after freezing but not after cold shock. There was
no denaturation of the DNA or alteration of the base sequences.

To determine the effect of more favourable methods of freezing
on the DNA of ram spermatozoa during incubation after thawing, the
procedure of Lopatko (6) was used. DNA was extracted after 3 hours
equilibration of spermatozoa with glycerol i.e. immediately before
freezing, and thawed after 6 months storage at -79°C. After light
centrifuging the spermatozoa were resuspended in calcium-free Krebs-
Ringer—-phosphate containing antibiotics and glucose and incubated at
37°C. DNA was extracted at O, 4 and 24 hours incubation and sub-
jected to a spectral analysis. The results (Table 2) showed no
change in the amount of configuration of the DNA during freezing or
storage at =79°C. During subsequent incubation, however, there was
a decrease in the DNA content of the spermatozoa and an increase in
the proportion of denatured DNA. No apparent alteration in the base
ratio or in the sequence of base pairs was detected in contrast to
stored bull spermatozoa (7). There was a decrease of about 257 in
the viability of the spermatozoa as judged by Congo red stain,
motility score and the percentage of motile cells during the first 4
hours incubation; after 24 hours few spermatozoa were motile.

(5) S. Z. HIRSCHMAN & G. FELSENFELD, 1966. J. Mol. Bio., lé: 347.

(6) M. I. LOPATKO, 1963. Artificial Insemination of Farm Animals.
Kharkow Book Publishers: p. 64.

(7) M. R, SEGINA & C. NORMAN, 1964. Proc. V Int. Congr. Reprod.,
4: 276,

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