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

fortunately there was in existence a little known literature which,
when brought together, proved that highly competent people before us
had shown from 20 to 30 years ago by classical genetic techniques
that aging of spermatozoa in one species,at least (Drosoghila melano-
gaster) did increase the incidence of occurrenceof sex-linked lethal
mutations, and that the frequency of such mutations was temperature
dependent (15).

A great deal of effort designed to determine the purine-pyrimidine
base ratios of the DNA isolated from bull spermatozoa yielded evidence
indicating that aging produced no measurable change in those ratios.
Berchtold in our laboratory in 1962-63 isolated the DNA from many
samples of fresh spermatozoa and many from aged spermatozoa. It was
all purified by several trypsin digestions and reprecipitations.
The mean temperature for the characteristic increase in absorbance
of ultraviolet light was 86.43 * 0.25°C in all instances (3, 4),
yielding on calculation a g@nine-cytosine mol percent of 41.8 % 0.6.
Also, Graves (unpublished), in his more precise isolation of the
adenine, thymine, cytosine, and guanine bases from DNA of bovine
spermatozoan heads, found no measurable change in base ratios during
storage.

However, we have shown a marked incorporation of radioactive
carbon from glycine (9), into the DNA isolated from spermatozoa
after incubation at 37°C for several hours and,also, radioautographs
of spermatozoa stored that short interval at 37°c (10) or for a year
at -196°C show marked uptake of the C'® from radioactive glucose or
fructose into the sperm heads (Graves, unpublished). From such stud-
ies we know that the DNA of mature spermatozoa is undergaing measur-
able turnover during storage at every temperature tested from that
of near body temperature to that of the lowest temperature presently
used. All attempts at stopping this turnover by use of metabolic
inhibitors have so far resulted in death of the spermatozoa.

Thus we have established to our own satisfaction a means by
which the integrity of the genetic information transfer system can
be degraded; by the fact of continuing metabolic turnover of the
DNA of the spermatozoon during its mature, functional lifetime.
Outside of the animal body the rate of these reactions is tempera-
ture dependent but the rate of decline in the integrity of the ge-
netic information transfer system of the spermatozoon during low-
temperature storage is more rapid than temperature coefficients of
some of the reactions providing energy for motility (5,16).

Nevertheless, we do not yet have an adequate monitoring system
for establishing for living spermatozoa that their capacity for fer-
tilization and contributing to normal embryogenesis has, in fact,
been altered except by the ultimate fertility test. Neither measure-
ments of density of Feulgen staining (1,2,8,23), staining with acri-
dine orange, and quantitative measurement of resulting fluorescence

     

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