TRANSFER OF SPERM INTO A CHEMICALLY-DEFINED ENVIRONMENT BY CENTRIFUGATION THROUGH 12-PERCENT (WT VOL) ACCUDENZ((R))/

Centrifugation is commonly used to wash sperm; however, most washing t echniques do not put sperm in a chemically defined environment. Rather , washing by centrifugation, in effect, dilutes seminal plasma compone nts. A 0.5-mL volume of 30% (wt/vol) Accudenz(R) was layered beneath 5 mL of 12% (wt/vol) Accudenz(R) in a 15-mL polypropylene centrifuge tu be. semen from individual males (n = 10) was upon the 12% (wt/vol) Acc udenz(R). After centrifugation at 1,250 x g at 4 C for 25 min, washed sperm were present at the interface of the Accudenz(R) layers. Based u pon hemacytometer counts, sperm recovery was 83% (CV = 12%). Neither s perm viability nor morphology was affected by washing. Efficacy of the washing procedure was evaluated by using extracellular glucose, gluta mic acid, Ca+2, and protein as markers. Washing eliminated 99% of the glutamic acid and glucose associated with sperm. Likewise, washing rem oved 98.5% of the extracellular Ca+2 associated with sperm. As evidenc ed by total protein analysis and SDS-PAGE, washing removed 98% of solu ble seminal plasma proteins from sperm. In addition, washing did not a ffect sperm mobility or fertilizing ability. This procedure returns ex tended sperm to a physiological concentration in a chemically defined environment. By suspending washed sperm in distinct media, we induced differential sperm mobility. Therefore, this procedure is suitable for the study of the effect of specific substances upon sperm cell functi on.