Flow Cytometric Evaluation of Acrosome Function/dysfunction in the Stallion

Flow Cytometric Evaluation of Acrosome Function/Dysfunction in the Stallion. (August 2006) Tegan S. Bosard, B.S., University of Alaska, Anchorage Co-Chairs of Advisory Committee: Dr. Charles C. Love Dr. Dickson D. Varner The objective of this study was to establish a rapid and efficient assay that would assess acrosomal status and function of the stallion acrosome. Ejaculates from fertile and subfertile stallions were extended to 25x10/mL and divided into aliquots (1mL) treated with no ionophore (control) or 10μM A23187 and incubated at 37oC for 0, 1, 2, and 3h. Following incubation, samples were fixed with 2% paraformaldehyde for 10 minutes at room temperature; then stored at 4°C in Dulbecco’s Phosphate-buffered saline (DPBS) for 0, 24, and 72 hours (i.e. post-fixation storage). After post-fixation storage samples were then permeabilized with 95% ethanol at -20oC for 10 minutes. Samples were resuspended in 20% fetal bovine serum in DPBS, labeled with fluorescein isothiocyanate for 10 minutes, and analyzed by flow cytometry. Post-fixation storage produced fewer (P<0.05) acrosome intact (AI) spermatozoa and a higher (P<0.05) fluorescence intensity than respective fresh samples. Regardless of incubation time or treatment, cool-stored samples averaged ~6% lower (P<0.001) AI spermatozoa than the corresponding fresh semen; however, cooled storage did not alter (P>0.2) the overall fluorescence