Effectiveness of various sperm processing methods in removing seminal plasma from insemination media

The effectiveness of the wash and swim-up (1 and 2 wash cycle), two layer Percoll gradient, SpermPrep and underlay sperm preparation methods in removing seminal plasma from insemination suspensions was investigated. The number of wash cycles needed to rid sperm suspensions of seminal plasma (n = 15) was also determined. All sperm preparation methods were compared to the control wash and swim-up method, performed using Earle's buffered salt solution (EBSS), without serum supplementation so as not to mask seminal plasma concentrations. Control processing comprised 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 comprised 1 ml semen layered on a discontinuous 36 and 81% Percoll gradient (n = 14). In the SpermPrep method, 1 ml semen was run through a SpermPrep 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 under 5% CO2 in air for 1 h. Seminal plasma and supernatant fractions obtained after each processing phase were stored at -20 degrees C and protein concentrations were determined by spectrophotometry. The wash and swim-up method was the most effective in removing seminal plasma from sperm suspensions, followed by the two-layer Percoll gradient, underlay and finally the SpermPrep II processing methods.