Andrology: 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 preparationmethods in removing seminal plasma from insemination suspensionswas investigated. The number of wash cycles needed to rid spermsuspensions of seminal plasma (n = 15) was also determined.All sperm preparation methods were compared to the control washand swim-up method, performed using Earle's buffered salt solution(EBSS), without serum supplementation so as not to mask seminalplasma concentrations. Control processing comprised 1 ml semensubjected to two wash cycles in EBSS followed by a swim-up periodof 60 min under 5% CO₂ in air. The Percoll method comprised1 ml semen layered on a discontinuous 36 and 81% Percoll gradient(n = 14). In the SpermPrep method, 1 ml semen was run througha SpermPrep II column (n = 10), and underlay samples (n = 12)were processed by two wash cycles, after which sperm pelletswere resuspended in the remaining medium, layered under 1.2ml EBSS and allowed to swim up under 5% CO₂ in air for 1 h.Seminal plasma and supernatant fractions obtained after eachprocessing phase were stored at -20°C and protein concentrationswere determined by spectrophotometry. The wash and swim-up methodwas the most effective in removing seminal plasma from spermsuspensions, followed by the two-layer Percoll gradient, underlayand finally the SpermPrep II processing methods.