Pf. Levay et al., EFFECTIVENESS OF VARIOUS SPERM PROCESSING METHODS IN REMOVING SEMINALPLASMA FROM INSEMINATION MEDIA, Human reproduction, 10(8), 1995, pp. 2056-2060
The effectiveness of the wash and swim-up (1 and 2 wash cycle), two la
yer Percoll gradient, SpermPrep and underlay sperm preparation methods
in removing seminal plasma from insemination suspensions was investig
ated. The number of wash cycles needed to rid sperm suspensions of sem
inal plasma (n = 15) was also determined. All sperm preparation method
s were compared to the control wash and swim-up method, performed usin
g Earle's buffered salt solution (EBSS), without serum supplementation
so as not to mask seminal plasma concentrations. Control processing c
omprised 1 ml semen subjected to two wash cycles in EBSS followed by a
swim-up period of 60 min under 5% CO2 in air. The Percoll method comp
rised 1 ml semen layered on a discontinuous 36 and 81% Percoll gradien
t (n = 14). In the SpermPrep method, 1 ml semen was run through a Sper
mPrep II column (n = 10), and underlay samples (n = 12) were processed
by two wash cycles, after which sperm pellets were resuspended in the
remaining medium, layered under 1.2 ml EBSS and allowed to swim up un
der 5% CO2 in air for 1 h. Seminal plasma and supernatant fractions ob
tained after each processing phase were stored at -20 degrees C and pr
otein concentrations were determined by spectrophotometry. The wash an
d swim-up method was the most effective in removing seminal plasma fro
m sperm suspensions, followed by the two-layer Percoll gradient, under
lay and finally the SpermPrep II processing methods.