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 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 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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-1875 |
| Date | 02 June 2009 |
| Creators | Bosard, Tegan S. |
| Contributors | Love, Charles C., Varner, Dickson D. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Thesis, text |
| Format | electronic, application/pdf, born digital |
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