The Bureau of Land Management (BLM) maintains a growing number of feral horses on public rangelands. With population growth rates as high as 22% annually, herds are exceeding their carrying capacity and millions of dollars are spent maintaining captured horses in holding facilities awaiting adoption. To manage the feral horse population, the BLM is seeking a contraceptive that is safe, can be remotely delivered, requires only a single administration and is effective for several years. Contraceptive strategies have been developed for feral horses that include hormone implants, chemical intrauterine devices, and immunocontraception. Porcine zona pellucida (pZP) immunocontraceptive vaccines have shown great potential for providing safe, long-term contraception in feral horses. ImmunoVaccine Technologies (Halifax, Nova Scotia, Canada) has developed a liposome encapsulated pZP formulation known as SpayVac™ (SpayVac), which after a single-dose provides multi-year contraceptive efficacy. In a continued effort to optimize the acceptability and efficacy of SpayVac, ImmunoVaccine Technologies developed alternative adjuvant preparations using either killed Mycobacterium butyricum (Modified Freund's Adjuvant; MFA) or a proprietary non-Mycobacterium based adjuvant (IVT) that are proposed to have less of the undesirable side-effects associated with Mycobacterium tuberculosis. Therefore, the objective of this research was to evaluate SpayVac in different adjuvant formulations for efficacy of contraception as measured by pZP titers and estrous cyclicity in treated mares. Domestic mares (n=28) were randomly assigned to four treatments (7 mares per treatment): adjuvant alone or saline (Control) or SpayVac vaccines in one of three adjuvant preparations: IVT or MFA in either an aqueous (MFA aq) or non-aqueous (MFA non-aq) suspension. Pre-immune blood samples were collected from each mare and mares were injected in the neck with a single injection of the Control or SpayVac. Subsequent blood samples were collected at weekly intervals for 26 weeks. Sera were analyzed for pZP titers and progesterone using ELISA. At the conclusion of the study, ovaries were recovered by ovariectomy (16 mares) or at necropsy (12 mares) for histologic analysis and collection of morphometric data and oocytes. Titers for pZP were greater (P<0.05) in IVT and MFA mares compared to Control mares and for MFA compared to IVT mares. Mares vaccinated with MFA aq had greater (P<0.05) pZP titers at 2 weeks post-injection compared to mares injected with IVT or MFA non-aq and at 3 weeks post-injection compared to mares injected with IVT. MFA non-aq mares had greater (P<0.05) pZP titers at 6 weeks post-injection compared to IVT mares and, although not significantly different, titers in MFA non-aq mares remained greater during weeks 8, 10, 14, 18 and 22 compared to IVT and MFA aq mares. Mean serum progesterone concentrations were greater (P<0.05) in Control compared to MFA non-aq mares. Mean ovarian weights, oocyte diameters, zona pellucida thicknesses and the number of horse sperm bound to oocytes recovered from vaccinated mares were greater (P<0.05) in Control mares compared to IVT and MFA mares. As judged by pZP titers and serum progesterone, these results suggest that SpayVac suspended in the MFA non-aqueous formulation exerted the greatest contraceptive effects in treated mares. This preparation of SpayVac may meet the criteria cited by the BLM for their most desirable immunocontraceptive. / Graduation date: 2012