Transfer of Sperm into a Chemically Defined Environment by Centrifugation Through 12% (wt/vol) Accudenz®

Abstract

Centrifugation is commonly used to wash sperm; however, most washing techniques do not put sperm in a chemically defined environment. Rather, washing by centrifugation, in effect, dilutes seminal plasma components. A 0.5-mL volume of 30% (wt/vol) Accudenz® was layered beneath 5 mL of 12% (wt/vol) Accudenz® in a 15-mL polypropylene centrifuge tube. Diluted semen from individual males (n = 10) was overlaid upon the 12% (wt/vol) Accudenz®. After centrifugation at 1,250 × g at 4 C for 25 min, washed sperm were present at the interface of the Accudenz® layers. Based upon hemacytometer counts, sperm recovery was 83% (CV = 12%). Neither sperm viability nor morphology was affected by washing. Efficacy of the washing procedure was evaluated by using extracellular glucose, glutamic acid, Ca+2, and protein as markers. Washing eliminated 99% of the glutamic acid and glucose associated with sperm. Likewise, washing removed 98.5% of the extracellular Ca+2 associated with sperm. As evidenced by total protein analysis and SDS-PAGE, washing removed 98% of soluble seminal plasma proteins from sperm. In addition, washing did not affect sperm mobility or fertilizing ability. This procedure returns extended 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 function.