EFFECTIVENESS OF VARIOUS SPERM PROCESSING METHODS IN REMOVING SEMINALPLASMA FROM INSEMINATION MEDIA

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.