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

DETERMINATION OF SYERM DENSITY
BY MEANS OF ULTRAVIOLET ABSORPTIOMETRY

Co VAN DUIJN Jr.

Biophysics Department, Research Institute for Animal
Husbandry 'SCHOONOCRD', Zeist, The Netherlands

Nephelometric and absorptiometric methods with visible
light are in regular use for determining sperm density
of bull semen. These methods cannot be applied to anim-
als whers the seminal plasma is highly turbid, as is
the cas2 in boar, cock and human., Therefore a method
has been developed for spermatozoa in turbid media,
based on the specific ultraviolet absorption of nucleic
acids at 260 nm, where proteins show only slight absorpte
ion. Recent investigations have shown that the DNA
content of spermatozoa determined by UV-microspectro-
photometry is constant per species and independent of
ageing and death of the cell (2-6).

For calibration a series of dilutions was mede from
each ejacglate. The diluent was isotonic sodium citrate
(4= -0.547C, pH_6.75 + 0.05 for boar, bull and human
semen; A= -0.65C, pH 6.60 + 0.05 for cock semen), with
an addition of 6 ml methanal solution of 36 w% per li-
ter for preservation and killing of the spermatozoa.

Errors resulting from aspecific UV-absorption and
scattering were eliminated by using a part of the dilut-
ion from which the suspended cells had been remcved by
centrifugation as the reference standard, set at the
100% transmission value. Sperm numbers were determined
accurately by repeated haemocytometer countings from
each dilutior in the range.

The relative amounts of aspecific scattering at each
concentration were determined by separately measuring
thg directly transmitted rays and those scattered at
90°, with the pure non-scattering diluent as_the 100%
transmission standard. Aspecific scattering by the
spermatozoa and the seminal plasma was found to be
negligible in the range of dilutions that would be
employed for ordinary measuring purposes.

Fig.1 gives the relationship between percentage
+ransmission relative to the standard, and sperm
density, the latter at a logarithmic scale. Measuring
accuracy is optimal in the region where the relation-
ship between transmission and log (concentratigg) is
approximately linear. Optimel dilution factors are so
chosen that the average expacted measuring value for

1025