Flow cytometric evaluation of acrosome function/dysfunction in the stallion
Bosard, Tegan S.
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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 25x106/mL and divided into aliquots (1mL) treated with no ionophore (control) or 10?M A23187 and incubated at 37?C 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 -20?C 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 properties as compared to fresh semen (730?8.08 vs. 734?8.01 fluorescence intensity units, respectively). For fertile stallions, the percentage of AI spermatozoa was higher (p<0.01) in control samples than A23187 samples at incubation times 1, 2, and 3h (Control-59, 56, and 51% vs. A23187- 46, 29, and 23%, respectively), but not at Time 0. For subfertile stallions, the percentage of AI spermatozoa was not affected by ionophore treatment (P>0.05) or incubation period (P>0.05). The results suggest that post-fixation storage in DPBS for up to three days is still representative of the acrosomal competence of the original sample. In addition, spermatozoa stored for 24 hours in an Equitainer? exhibited a small (~6%) but significant decrease in the percentage AI spermatozoa. Storage conditions may therefore, affect acrosomal integrity and contribute to reduced fertility when cooled-semen is used. Subfertile stallions exhibited little response [<11% acrosome reacted (AR)] after 3h of A23187 exposure, while the fertile stallions demonstrated a substantial response (? 36% AR) as soon as 1h after ionophore exposure. This assay diagnosed acrosomal dysfunction in stallions with unexplained subfertility.