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.
Haldar
S
S
Indian Institute of Chemical Biology, Calcutta.
Dey
C S
CS
Majumder
G C
GC
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D004822:Epididymis / Q000166:cytology*; D006041:Goats; D008297:Male; D013094:Spermatozoa / Q000166:cytology*