Immunocontraception has been used effectively to decrease birth rates in many different animal species, wild and domestic, with contraceptive vaccines targeting one of two proteins that are required for reproduction. Some vaccines use porcine zona pellucida protein (PZP) as an antigen (Kirkpatrick et al., 2009). Antibodies to PZP bind to the zona pellucida and block fertilization in immunized females. The second target protein is gonadotropin-releasing hormone (GnRH), a key hypothalamic hormone that regulates synthesis and secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary gland.

Immunization against GnRH has been increasingly used to manage wild populations. The National Wildlife Research Center (USDA-APHIS Wildlife Services) developed an immunocontraceptive named GonaCon, which consists of a GnRH peptide combined with an adjuvant to trigger an immune response (Miller et al., 2004). This generates neutralizing antibodies against GnRH, thereby inhibiting FSH and LH synthesis and secretion. In females this vaccine blocks ovulation and estrus, resulting in infertility (Miller et al., 2004). In male white-tailed deer, GonaCon vaccination leads to reduced testosterone levels, reduction of testes size, and inhibition of reproductive behavior (Killian et al., 2005). However, infertility is temporary in both males and females, lasting only as long as GnRH-antibody titers remain high enough to block GnRH activity (Miller et al., 2000, Pinkham et al., 2022). The efficacy of GonaCon in reducing reproduction has been evaluated in a wide variety of mammalian species, such as prairie dogs and fox squirrels, wild boar, feral cats, and elk (Krause et al., 2014, Massei et al., 2012, Powers et al., 2014, Vansandt et al., 2017, Yoder and Miller, 2010).

GonaCon has also been considered for use in feral horse herds. Feral horses on public lands exhibit a high population growth rate with about 20% increase per year (National Research Council, 2013) and cause ecological damage when they reach high densities (Eldridge et al., 2020), necessitating some form of population control. Public sentiment favors contraception as a horse management tool over lethal methods or capture and removal, yet the public often responds poorly to permanent sterilization of horses. Reversibility of the GonaCon vaccine provides an appealing option for management of feral horse populations.

Baker et al. (2018) began research on the long-term effectiveness of GonaCon-Equine in free-roaming feral horses in Theodore Roosevelt National Park (TRNP) in 2009, with 25 mares receiving a primary vaccination that fall and a secondary vaccination four years later, by hand-injection at a roundup. Due to the success of the treatment, the study was expanded. To determine optimal reimmunization interval, 40 additional mares in three treatment groups were treated by remote delivery via dart, with the last group receiving their secondary vaccination in 2016 (Baker et al., manuscript in prep). Collectively, the reimmunization treatments were divided into groups based on the interval between primary and secondary injections: 4-year intervals, 2-year intervals, 1-year intervals, and 6-month intervals. A control group was treated with a saline injection on the same schedule as the 4-year group. Foaling rates of these mares were monitored from 2009 through 2021. While the vaccine was shown to be effective in reducing foaling rates, the duration of infertility was variable; some mares had one or two years of infertility while others remained infertile for the duration of the study. Factors influencing individual mares’ responses to treatment are as yet unknown.

Individual variation in reproductive rates following immunocontraception could be due to many factors, both environmental and/or genetic. Some genetic components of fertility are known, but many aspects of reproduction are complex and not fully understood. In humans, genes have been identified for some female reproductive disorders/diseases as well as markers contributing to fertility traits such as reproductive lifespan and ovarian function (Gajbhiye et al., 2018). The genetics of fertility traits in dairy cattle has also been a subject of interest, with several candidate markers identified (Ma et al., 2019). However, genes that influence fertility in horses have yet to be identified (Laseca et al., 2021, Raudsepp, 2020).

Variation in efficacy of immunocontraceptives could also be due to variation in genes underlying immune responses. As with fertility, immune responses are complex and difficult to attribute to any single gene. Research into human disease-resistance has demonstrated that epigenomic changes in gene expression play a role beyond purely genetic factors (Yamamoto et al., 2021). In cattle, some quantitative trait loci have been associated with immune response to vaccination (Leach et al., 2012). However, no previous studies have identified genetic factors in response to any immunocontraceptive agent in horses or response to GonaCon in any other species.

Here, we use SNP genotypes to conduct a genome-wide association study (GWAS) in search of loci associated with variation in the long-term effectiveness of GonaCon-Equine vaccine. We hypothesize that genes involved with fertility and/or immune response will impact the duration of infertility following vaccination. This research will improve our understanding of the mechanism by which the vaccine works and inform future use of GonaCon-Equine in other feral herds. More broadly, findings from this study may also provide insight into why vaccine induced immunity persists or wanes over time, which is an outstanding question in immunology (Kennedy et al., 2020).