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Management of overabundant free-roaming horse (Equus ferus caballus) populations has become a divisive and challenging effort for natural resource managers worldwide. Controlling these populations requires land managers to balance socio-political issues, multiple use management concepts, and endearing affection of horses to some stakeholders against negative ecological alterations created by excessive densities of these animals. One potential solution to this dilemma is controlling the fertility of female horses. During 2013–2020, research was conducted to develop and test a prototype dart configuration system to deliver an effective dose of GonaCon-Equine to free-roaming horses. This remote dart delivery system was demonstrated to be safe and reliable in free-ranging horses with a 90% (71/79) success rate for first attempts and provided the opportunity to design an experiment to determine the long-term effectiveness, reversibility, and physiological side effects of different reimmunization schedules. Five experimental groups of mares were established consisting of a control (n = 25), and a 4-yr reimmunization interval (n = 25) of mares from a previous experiment that were treated with this same vaccine by hand injection in 2013. Additional booster intervals of horses were (2-yr (n = 11), 1-yr (n = 15), and 0.5-yr (n = 14) and vaccinated by remote dart delivery during 2013–2016. Weekly ground observations were utilized to estimate foaling proportions, foal survival, body condition, and injection site reactions related to treatment groups. All GonaCon-Equine booster intervals reduced (P ≤ 0.001) foaling proportions between treated and control mares for all years of the experiment. Effective contraception was also achieved when GonaCon-Equine was applied by remote dart delivery at post-primary intervals shorter than 4 years. Reversibility of GonaCon-Equine immunization occurred in all treatment groups and averaged 0.40 (26/65). The only detectable adverse side effect of vaccination was intramuscular swelling observed in 89% (115/129) of mares and occasional draining abscesses at the site of vaccine injection. However, regardless of method of vaccine delivery (hand vs dart), none of these mares displayed any evidence of lameness, altered gait, or abnormal range of movement during the 6 years of this study. These results support previous findings that effective contraception of free-roaming horses will require a primary vaccination followed by reimmunization but that timing from 6 months to 4 years result in similar success and together can achieve greater reduction in population growth rates.

Wildlife and humans are increasingly competing for resources worldwide, and a diverse, innovative, and effective set of management tools is needed. Controlling abundance of wildlife species that are simultaneously protected, abundant, competitive for resources, and in conflict with some stakeholders but beloved by others, is a daunting challenge. Free-ranging horses (Equus caballus) present such a conundrum and managers struggle for effective tools for regulating their abundance. Controlling reproduction of female horses presents a potential alternative. During 2009-2017, we determined the long-term effectiveness of GnRH vaccine (GonaCon-Equine) both as a single immunization and subsequent reimmunization on reproduction and side effects in free-ranging horses. At a scheduled management roundup in 2009, we randomly assigned 57 adult mares to either a GonaCon-Equine treatment group (n = 29) or a saline control group (n = 28). In a second roundup in 2013, we administered a booster vaccination to these same mares. We used annual ground observations to estimate foaling proportions, social behaviors, body condition, and injection site reactions. We found this vaccine to be safe for pregnant females and neonates, with no overt deleterious behavioral side effects during the breeding season. The proportion of treated mares that foaled following a single vaccination was lower than that for control mares for the second (P = 0.03) and third (P = 0.08) post-treatment foaling seasons but was similar (P = 0.67) to untreated mares for the fourth season, demonstrating reversibility of the primary vaccine treatment. After two vaccinations, however, the proportion of females giving birth was lower (P

MicroRNAs (miRNAs) are small, non-coding RNAs which are produced throughout the body. Individual tissues tend to have a specific expression profile and excrete many of these miRNAs into circulation. These circulating miRNAs may be diagnostically valuable biomarkers for assessing the presence of disease while minimizing invasive testing. In women, numerous circulating miRNAs have been identified which change significantly during pregnancy-related complications (e.g. chorioamnionitis, eclampsia, recurrent pregnancy loss); however, no prior work has been done in this area in the horse. To identify pregnancy-specific miRNAs, we collected serial whole blood samples in pregnant mares at 8, 9, 10 m of gestation and post-partum, as well as from non-pregnant (diestrous) mares. In total, we evaluated a panel of 178 miRNAs using qPCR, eventually identifying five miRNAs of interest. One miRNA (miR-374b) was differentially regulated through late gestation and four miRNAs (miR-454, miR-133b, miR-486-5p and miR-204b) were differentially regulated between the pregnant and non-pregnant samples. We were able to identify putative targets for the differentially regulated miRNAs using two separate target prediction programs, miRDB and Ingenuity Pathway Analysis. The targets for the miRNAs differentially regulated during pregnancy were predicted to be involved in signaling pathways such as the STAT3 pathway and PI3/AKT signaling pathway, as well as more endocrine-based pathways, including the GnRH, prolactin and insulin signaling pathways. In summary, this study provides novel information about the changes occurring in circulating miRNAs during normal pregnancy, as well as attempting to predict the biological effects induced by these miRNAs.

Sex steroid hormones regulate developmental programming in many tissues, including programming gene expression during prenatal development. While estradiol is known to regulate placentation, little is known about the role of testosterone and androgen signaling in placental development despite the fact that testosterone rises in maternal circulation during pregnancy and in placenta-induced pregnancy disorders. We investigated the role of testosterone in placental gene expression, and focused on androgen receptor (AR). Prenatal androgenization decreased global DNA methylation in gestational day 90 placentomes, and increased placental expression of AR as well as genes involved in epigenetic regulation, angiogenesis, and growth. As AR complexes with histone lysine demethylases (KDMs) to regulate AR target genes in human cancers, we also investigated if the same mechanism is present in the ovine placenta. AR co-immunoprecipitated with KDM1A and KDM4D in sheep placentomes, and AR-KDM1A complexes were recruited to a half-site for androgen response element (ARE) in the promoter region of VEGFA. Androgenized ewes also had increased cotyledonary VEGFA. Finally, in human first trimester placental samples KDM1A and KDM4D immunolocalized to the syncytiotrophoblast, with nuclear KDM1A and KDM4D immunostaining also present in the villous stroma. In conclusion, placental androgen signaling, possibly through AR-KDM complex recruitment to AREs, regulates placental VEGFA expression. AR and KDMs are also present in first trimester human placenta. Androgens appear to be an important regulator of trophoblast differentiation and placental development, and aberrant androgen signaling may contribute to the development of placental disorders.