Immunomagnetic selection of acrosome-reacted bovine spermatozoa using anti-acrosin antibodies

Acrosome-reacted sperm cells were selected from thawed, cryopreserved samples of bovine spermatozoa using rabbit antibodies to bovine acrosin and goat anti-rabbit immunoglobulin-coated magnetic beads (IgG-MAG) or protein-A magnetic beads (PA-MAG). The process was monitored by staining the spermatozoa with Fluoresbrite(R)-labelled beads attached to anti-rabbit immunoglobulins (IgG-FL), Fura Red-AM (FRED) and propidium iodide (PI). The thawed spermatozoa were diluted in Tyrode's salt solution and exposed to FRED, antibovine acrosin, IgG-FL and/or IgG-MAG or PA-MAG. The reversible inhibitor, benzamidine, was used to control proteolysis during selection, while ionophore, A23187, was used in control samples to induce the acrosome reaction. Flow cytometry revealed three cell populations; dead cells stained only with PI, and two acrosome-reacted populations stained with IgG-FL and either FRED or PI. The acrosome-reacted spermatozoa were selected by exposing the samples to a magnet (2 min). Protein-A magnetic beads, when coupled to antibodies, were useful for selecting a specific sperm population.