Isolation of motile spermatozoa by density gradient centrifugation in Percoll®

Abstract

A procedure using centrifugation in density gradients composed of Percoll was developed for isolation of spermatozoa from mammalian semen. To evaluate the technique, rabbit, human, or bovine semen was layered over continuous Percoll gradients ranging in density from 1.02 to 1.13 gm/ml and centrifuged at 1,500g for 45 min. After centrifugation, the seminal plasma remained above the gradient, whereas the spermatozoa and seminal particles were distributed within the gradient according to their buoyant densities. Unlike most washing techniques, no sperm pellet was formed; instead, the spermatozoa were concentrated into a compact band above the most dense layer of Percoll. The spermatozoa recovered from the gradient were easily resuspended by gentle techniques. Thus, the mechanical stress to the spermatozoa was minimized. Osmotic stress to the spermatozoa was also negligible as the Percoll gradients were isotonic throughout. Spermatozoa obtained by this technique possessed motility equivalent to that of spermatozoa in the unfractionated semen. Sperm suspensions recovered from the gradients contained less than 5% of the nonspermatozoal particles present in the original samples of unfractionated semen. Soluble seminal components were also efficiently removed from the spermatozoa. Thirty billion bovine spermatozoa could be fractionated on a single gradient without loss of effectiveness. Recovery of spermatozoa from these preparative separations averaged 80%. These results demonstrated that Percoll was a superior medium for efficient density gradient isolation of motile spermatozoa free of contamination by other seminal constituents.

Deficiency in Outer Dense Fiber 1 Is a Marker and Potential Driver of Idiopathic Male Infertility

“…A consent form was obtained from all donors. The semen samples were produced by masturbation and processed within 1 h of ejaculation using Percoll fractionation as described previously (23). The purified spermatozoa were obtained from the lower (80% Percoll) fraction.…”

Visualization of pig sperm chromosomes by in-vitro penetration of zona-free hamster ova

“…The pH was maintained at 74 using 20 mM-Hepes (BDH Chemicals Ltd, Poole, Dorset, U. (Lessley & Garner, 1983) and centrifuged at 1500 g for 40 min. A fraction containing >25% motile spermatozoa was selected by microscopic examination, washed twice with Medium BWW by centrifugation at 300 g and diluted to give a final concentration of 25 106 spermatozoa/ml.…”

Differential contribution of leucocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligozoospermic patients and fertile donors

“…Sweden). This preparation was designated 100% Percoli (Lessley & Garner, 1983) and 50% Percoli was prepared by dilution with an equal volume of medium BWW (Biggers et al, 1971).…”