Improving the Efficacy of In Vitro Assays to Detect Physiologic Differences in Anti-Porcine Zona Pellucida Sera Recovered from Mares Injected with SpayVac™ Public Deposited

http://ir.library.oregonstate.edu/concern/undergraduate_thesis_or_projects/5q47rr035

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  • Immunocontraception targeting the zona pellucida shows promise as a safe, effective and economically feasible method for controlling feral horse populations that roam public rangelands throughout the United States. The efficacy of porcine zona pellucida (pZP) vaccines to generate high titers can be evaluated with an indirect immunofuorescence assay or a sperm binding assay using equine, bovine or porcine oocytes and sperm. The objective of this research was to refine protocols for each of these assays that would yield high resolution results when comparing oocytes treated with high and low titer anti-pZP sera against various sets of control oocytes. Dilutions of rabbit anti-hose IgG-fluorescein isothiocyanate were varied in an indirect immunofluorescence assay which quantified fluorescence emitted from the zona pellucida. A dilution of 1:32 produced greater fluorescence intensity from oocytes treated with anti-pZP serum that control oocytes. Fluorescence intensity of oocytes treated with the 1:32 dilution also varied significantly by the titer level, high or low, of anti-pZP serum used. A sperm binding inhibition assay was performed using a longer incubation time for sperm binding than had been previously documented. However, this modification did not produce any higher resolution than results reported in previous studies.
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