Ficoll gradient isolation of immature sperm of high purity and intactness from goat epididymis.

An improved Ficoll density gradient centrifugation method has been described for the isolation of goat caput-epididymal immature spermatozoa of a high purity and intactness. The method consists of layering freshly extracted sperm suspension on the top of a Ficoll gradient comprising 2.5 ml each of 2%, 4%, 6%, and 8% Ficoll-400 in a modified Ringer's solution and centrifugation at 300 g for 3 min in a swing bucket table centrifuge. Spermatozoa, free from fat globules and blood cells, sedimented at the bottom of the 8% Ficoll layer. The plasma membrane of the isolated cells showed a high degree of intactness (approximately 96%) as assessed by lactic dehydrogenase marker enzyme and ethidium bromide-fluorescence methods.