Bovine Sperm Fractionation

Abstract
Procedures have been developed for selective degradation of bovine sperm and for isolation of segments. Such degradation of sperm can be achieved by (1) pronase digestion in sodium citrate followed by carbonate-bicarbonate buffer at pH 10.5, (2) pronase in CaCl2, and (3) nitrogen pressure and cavitation. Pressure cavitation and pronase digestion cleaved sperm at the capitulum-implantation fossa junction. In addition, the plasmalemma was removed; the remaining intact unit was composed of the mitochondrial sheath and the outer dense fibers. When the digestion process was used, the capitulum was separated from the outer dense fibers. Also, the microtubules of the axoneme were degraded by pronase used for 5–6 h. Following cell cleavage, centrifugation at 23,300 g with a sucrose gradient yielded pure head and mitochondria outer dense fiber fractions. These methods can be carried out on large quantities of sperm and allow selective degradation and fractionation, structural relationship studies, and chemical analyses of the separate cell segments and components.