Detailed Summary of University-led Research Projects for Improved Fertility Control Tools for Wild Horses 1a. Title: Evaluation of minimally invasive methods of contraception in wild horse and burro mares: tubal ligation and hysteroscopically-guided oviduct papilla laser ablation

In an effort to develop minimally invasive, low risk techniques for contraception and population control in female wild horses and burros, we propose evaluating two procedures, tubal ligation and hysteroscopically-guided laser ablation of the oviduct papilla in standing sedated females. For tubal ligation, we hypothesize that a flexible endoscope inserted through a small incision in the vaginal vault will allow visualization of each oviduct in pregnant and non-pregnant mares. Use of a diode laser or cautery instrument will allow effective fulguration followed by bloodless sectioning of the oviduct. This procedure should allow successful sterilization of up to 100% of female wild horses and burros gathered in any particular location as a single event. For the hysteroscopic procedure, we expect to endoscopically visualize each oviduct papilla in standing, sedated, non-pregnant mares. A diode laser will be used to to seal the opening between the oviduct and each uterine horn, thus preventing subsequent fertilization. The authors feel the proposed procedures will be acceptable to the public because they do not involve major surgery, are expected to have minimal complications while approaching 100% effectiveness, and when applied, are expected to result in a static to decreasing population level. Additionally, tubal ligation is a technique commonly performed in humans. Fulfilling our objective of developing an acceptable sterilization technique will benefit the public by controlling the population levels of wild horses and burros. In the face of scare feed, drought, or grazing pressures by other herbivores, having some control of the number of wild equids will result in healthier animals and grazing lands. 2a, project 1. Title: Tubo-ovarian ligation via colpotomy as a method for sterilization in mares 2b, project 1. Recipient: University of Kentucky 2c, project 1. Additional Information: As reviewed in the recent report from the National Research Council [1], female methods of fertility control in equids include surgical ovariectomy, immunocontraceptives directed against the zona pellucida (pZP) or against gonadotropin releasing hormone (GnRH), GnRH antagonists, steroid hormones and intrauterine devices. One important and widely applied technique in humans, tubal ligation, was not addressed in the NRC report. The premise of the current proposal is that techniques for tubo-ovarian ligation can be performed safely, economically and effectively for sterilization of female equids under field conditions. Access to the reproductive tract would be achieved in the standing animal using well-defined sedation / analgesia / local anesthesia techniques and a surgical approach through the vaginal wall (colpotomy). Ovariectomy of the mare via colpotomy has been used for many decades as a method for sterilization in domestic horses [2-4]. The technique provides access to the ovary in the standing mare via an incision in the cranial vagina and is routinely performed in the standing animal in domesticated horses. Ovaries are removed, and hemostasis is achieved via the use of an ecraseur which simultaneously cuts and crushes the ovarian pedicle [2-4]. Although effective, the procedure can be accompanied by a high rate of complications (approximately 4% in one study [5]) due primarily to excessive hemorrhage from the ovarian pedicle, and such complications were described in the NRC report [1] as severely limiting application of ovariectomy through a colpotomy approach in addressing the needs for controlling fertility in wild equids. We propose an alternative technique (tubo-ovarian ligation) to induce ovarian necrosis / degeneration secondary to ischemia along with ligation of the oviduct to provide an additional measure to ensure complete loss of fertility in treated mares. As an alternative approach to ovariectomy in mares, procedures have been described for application of a ligature (nylon zip tie) to the ovarian pedicle in mares via laparoscopy [3]. With this procedure, the ovarian pedicle is ligated via flank laparoscopy, and the ovary undergoes ischemic necrosis or atrophy secondary to a loss of blood supply. Recovery of mares subsequent to the procedure is rapid, and loss of ovarian function is complete [3]. Two other studies have examined the use of zip ties to achieve ligation of the ovarian pedicle for hemostasis with subsequent removal of the ovary by sharp dissection [6, 7]. In the study by Cokelaere et al. [7], standing laparoscopic application of a zip tie was used to achieve hemostasis of the ovarian pedicle with subsequent ovarian excision in 10 mares. Hemostasis was reported to be effective in this method, thereby establishing the use of these devices to effectively control ovarian blood flow. In the second study by Rabelo et al., [6], application of the zip tie was performed through a surgical laparotomy in 12 mares. On the left side, the ovary was excised after application of a zip tie to the ovarian pedicle. On the right side, the zip tie was placed on the ovarian pedicle without removal of the ovary. Mares were examined 15 to 45 days postoperatively for gross and histologic changes in the right ovary which had been ligated in situ with the zip tie. The authors reported evidence of atrophy and ischemia of the right ovary subsequent to ligation without evidence of adhesion or abscess formation in the 12 mares examined [6]. The risk of significant hemorrhage from the ovarian pedicle is eliminated using this approach, and we propose to develop a procedure that can be applied in the field in standing mares under sedation / local anesthesia to accomplish simultaneous ligation of the ovarian pedicle and ligation of the oviduct in mares to achieve sterilization via a colpotomy approach for tubo-ovarian ligation in mares. Nylon (polyamide) cable ties (zip ties or tie raps) have been used in a number of experimental and clinical surgical applications [3, 7-9]. Although not approved medical devices for the horse, nylon cable ties can be heat sterilized and are available in a range of lengths and strengths that lend them to the proposed application in this study. As noted earlier, nylon cable ties have been used to ligate the ovarian pedicle in mares for ovariectomy as well as to induce ischemia and atrophy of the ovary in situ [3, 7]. Although long-term follow up to evaluate the biocompatibility of these nylon zip ties was not reported in these studies, there was complete encapsulation of the ovarian stump and zip tie within three to four weeks after application [6]. Nylon zip ties have been used in other species for ligation and hemostasis during ovariohysterectomy without problems associated with the device over time as an implanted foreign body [8]. A number of studies describe laparoscopic ovariectomy in the standing mare under sedation / analgesia and local anesthesia [3, 4, 7, 10]. Field application of laparoscopic surgery in wild horses is impractical due to considerations of equipment cost, technical and surgical skill requirements, as well as the relatively long surgical time and difficulties in appropriate restraint for the procedure in wild equids. Thereby, laparoscopic application of a tubo-ovarian ligature is impractical in a field setting. The use of vaginal surgical approaches (natural orifice transluminal endoscopic surgery NOTES) for access to the ovary for ovariectomy have been described in the mare [11, 12] based upon the use of a flexible videoendoscope introduced through a vaginal incision to allow imaging of the ovary and a bipolar vessel sealing device for ovarian removal. Cited advantages to the transvaginal approach include the absence of a skin and body wall incision, rapid healing, less tissue dissection, fewer wound complications as well as an improved post-operative recovery [12]. Again, as with laparoscopic approaches to the ovary, transvaginal endoscopy has relatively large requirements for equipment, technical and surgical skill, surgical time and need for appropriate restraint which limit the application of this approach to the ovary in wild equids. We propose, however, a two-phase approach to refine the use of colpotomy for tubo-ovarian ligation. In the first year, a standard manual colpotomy approach will be used for ligature placement to demonstrate the feasibility of this procedure to induce ovarian atrophy / tubal ligation. During the second year of the study, we will evaluate a custom designed video-driven instrument which will allow introduction of the ligature through a small vagina port without the need of introducing the operator’s hand into the abdomen. This approach will provide the advantages of absence of body wall incision, rapid healing, reduced operative time, reduced wound complications, and reduced risk of hemorrhage from the ovarian pedicle to facilitate tuboovarian ligation in the mare under standing sedation / analgesia. Because a high proportion of wild horses gathered from BLM – managed lands are likely to be pregnant during the period of July – March when it is possible to work with these animals, the impact of pregnancy on the application of the proposed tubo-ovarian ligation via colpotomy is an important consideration. Ovariectomy or disruption of ovarian function will cause pregnancy loss during the first 70 days of pregnancy due to the loss of ovarian progesterone which is the major progestational support of early pregnancy [13]. In addition, it appears highly likely that the proposed procedure will not be useful in mares during late gestation due to the difficulty in performing a colpotomy in such mares as well as the limited access to the ovaries as the reproductive tract moves cranially and ventrally with increasing gestation. Ultimately, the application of any contraceptive / sterilization procedure in wild horses must reduce the current level of population growth present in many Herd Management Areas [1]. Although male-directed procedures have been proposed as a means to regulate population growth [1], it is not at all clear that such approaches will effectively limit population growth under field settings. Likewise, the most widely applied contraceptive modality in wild horse mares, porcine zona pellucida vaccination, has been used in more than 4,562 mares in 80 of 179 HMAs since 2004 without significant reductions in population increase (http://www.blm.gov/wo/st/en/prog/whbprogram/science_and_research/fertility_control.html). Although the reversible contraceptive effect of pZP immunization may have application under some management situations, a permanent sterilization as proposed here may ultimately be a more practical method to control population growth. Although we anticipate that the proposed tubo-ovarian ligation will be a quick, effective and economical method to sterilize mares, there are a number of areas that remain to be assessed relative to application in wild horses. First, complete ablation of ovarian function as proposed here will affect the behavior, social structure and herd makeup in bands of wild horses; however, the same is true for all of the proposed or existing methods for reproductive control in the mare [1]. In the absence of normal ovarian cyclicity, mares will continue to demonstrate sporadic estrous behavior likely due to steroid production by the adrenal gland [14]. Continued estrous behavior in these mares may play an important role in retaining the harem structure of the band [1]. Second, application of the technique will likely be limited to nonpregnant mares or mares during the first half of pregnancy. Third, operator and animal safety is a concern using this approach. The NAS report [1] precluded ovariectomy in wild horse mares due to concerns about hemorrhage and peritoneal infection. We believe that the procedure proposed here eliminates the risk associated with hemorrhage during ovariectomy and thereby problems with peritoneal infections as well. Operator safety and training will also be an important consideration. Although wild horse mares have been ovariectomized under field conditions via a colpotomy approach, restraint and sedation present challenges that are not encountered with domesticated mares. Ultimately, we estimate total procedure time using this technique to be less than 7 to 10 minutes. The further development of a videoendoscopic approach for application of the tubo-ovarian ligature should further reduce the time required for the procedure as well as reduce the risk to the operator by allowing a more remote approach to the ovaries. Further refinements in restraint systems and sedation / analgesia protocols are also likely to greatly enhance animal and operator safety with this procedure. Attempts to affect population control in wild mammals have presented a real and persistent challenge across a wide range of ecosystems and animal types. Reproduction remains a very robust process and efforts to date to manage populations of wild mammals have been only variably successful. The proposed technique of tubo-ovarian ligation is a simple, direct and permanent method to prevent reproduction in the mare. The proposed application would have direct impacts on both wild populations of horses as well as domestic horses where a rapid, safe and inexpensive method to eliminate reproductive behavior may be desirable for many mare owners. Goals and Objectives: The overall goal of this proposal is to develop methodology for the safe, economical and effective sterilization of mares via colpotomy (vaginal incision) to achieve: 1) ovarian necrosis / atrophy via application of a ligature to the ovarian pedicle and 2) simultaneous sterilization via tubal ligation (i.e., tubo-ovarian ligation). Objectives: 1. Determine the effectiveness of a custom-designed instrument for placement of a polyamide (nylon) cable tie (zip tie) around the ovarian pedicle and oviduct of mares via colpotomy for tubo-ovarian ligation. The procedure, conducted in the standing animal under sedation and local anesthesia, is expected to induce permanent sterilization of treated mares. 2. To assess post-operative complications of the procedure in mares and the effects on the health of mares. 3. To determine long-term effects on the reproductive tract, and the overall health of mares and the fertility of mares undergoing the procedure. 4. To assess the feasibility of these procedures in pregnant mares. References 1. National Research Council (2013): Using Science to Improve the BLM Wild Horse and Burro Program: A Way Forward. The National Academies Press. 2. Colbern,G.T. and Reagen,W.J. (1987) Ovariectomy by colpotomy in mares. Compend.Contin.Educ.Pract.Vet., 9, 1035-1041. 3. Yarbrough,T.B. (2009): Ovariectomy Techniques. In: Current Therapy in Equine Medicine, edited by N.E.Robinson, et al, pp. 781-784. Saunders, 4. Seabaugh,K.A. and Schumacher,J. (2014) Urogenital Surgery Performed with the Mare Standing. Veterinary Clinics of North America: Equine Practice, 5. Hooper,R.N., Taylor,T.S., Varner,D.D., and Blanchard,T.L. (1993) Effects of bilateral ovariectomy via colpotomy in mares: 23 Cases (1984-1990). J.Am.Vet.Med.Assoc., 203, 10431046. 6. Rabelo,R.E., Silva,L.A.F., SantGÇÖAna,F.J.F., Silva,M.A.M., Moura,M.I., Franco,L.G., and OLIVEIRA,C.R. (2008) Use of polyamide tie-rap for ovariectomy in standing mares. Acta Scientiae Veterinariae, 36, 119-125. 7. COKELAERE,S.M., MARTENS,A.M., and WIEMER,P.E.T.E. (2005) Laparoscopic Ovariectomy in Mares Using a Polyamide Tie Rap. Vet.Surgery, 34, 651-656. 8. Barros,B.J., Sanches,A.W.D., and Pachaly,J.R. (2009) The efficiency of nylon 6.6 (polyamide) cable ties as a method for massive ligatures of ovarian pedicles and uterine stubs in ovariohysterectomy of bitches (Canis familiaris). Arquivos de Ciencias Veterinaírias e Zoologia da UNIPAR, 12, 47-60. 9. Downs,C. and Rodgerson,D. (2011) The use of nylon cable ties to repair rib fractures in neonatal foals. The Canadian Veterinary Journal, 52, 307. 10. Aziz,D.M., Al-Badrany,M.S., and Taha,M.B. (2008) Laparoscopic ovariectomy in standing donkeys by using a new instrument. Animal Reproduction Science, 107, 107-114. 11. Pader,K., Lescun,T.B., and Freeman,L.J. (2011) Standing ovariectomy in mares using a transvaginal natural orifice transluminal endoscopic surgery approach. Vet.Surgery, 40, 987-997. 12. Pader,K., Freeman,L.J., Constable,P.D., Wu,C.C., Snyder,P.W., and Lescun,T.B. (2011) Comparison of transvaginal natural orifice transluminal endoscopic surgery and laparoscopy for elective bilateral ovariectomy in standing mares. Vet.Surgery, 40, 998-1008. 13. Holtan,D.W., Squires,E.L., Lapin,D.R., and Ginther,O.J. (1979) Effect of ovariectomy on pregnancy in mares. J.Reprod.Fertil., suppl 27, 457-463. 14. Hedberg,Y., Dalin,A.M., FORSBERG,M., Lundeheim,N., Sandh,G., Hoffmann,B., Ludwig,C., and Kindahl,H. (2007) Effect of ACTH (tetracosactide) on steroid hormone levels in the mare: Part B: Effect in ovariectomized mares (including estrous behavior). Animal Reproduction Science, 100, 92-106. 15. Ginther,O.J. (1986): Ultrasonic imaging and reproductive events in the mare. Equiservices, Cross Plains, WI. 16. Ginther,O.J. (2007): Ultrasonic imaging and animal reproduction: color-doppler ultrasonography, book 4. 17. Alford,C. and Hanson,R. (2010) Evaluation of a transvaginal laparoscopic natural orifice transluminal endoscopic surgery approach to the abdomen of mares. Vet.Surgery, 39, 873-878. 18. Bollwein,H., Weber,F., Kolberg,B., and Stolla,R. (2002) Uterine and ovarian blood flow during the estrous cycle in mares. Theriogenology, 57, 2129-2138. 19. Ginther,O.J. (1992): Reproductive Biology of the Mare: Basic and Applied Aspects. Equiservices, Cross Plains, WI. 20. Tate,L.P., Fogle,C.A., Bailey,C.S., Tate,K.B., and Davis,J.W. (2012) Laparoscopic-Assisted Colpotomy for Ovariectomy in the Mare. Vet.Surgery, 41, 625-628. 21. Goodin,J.T., Rodgerson,D.H., and Gomez,J.H. (2011) Standing Hand-Assisted Laparoscopic Ovariectomy in 65 Mares. Vet.Surgery, 40, 90-92. 22. Bechert,U., Bartell,J., Kutzler,M., Menino,A., Bildfell,R., Anderson,M., and Fraker,M. (2013) Effects of two porcine zona pellucida immunocontraceptive vaccines on ovarian activity in horses. Jour.Wild.Mgmt., 77, 1386-1400. 3a. Title: Functional assessment of ovariectomy (spaying) via colpotomy of wild mares as an acceptable method of contraception and wild horse population control 3b. Recipient: Oregon State University 3c. Additional Information Abstract: Department of Interior Secretary Ken Salazar recently put forth new proposals for the management of wild horses and burros, including new strategies aimed at balancing wild horse and burro population growth with public adoption demand. Proposed strategies include slowing population growth rates of wild horses and burros on public rangelands through the aggressive use of fertility control. It is our hypothesis that ovariectomy via vaginal colpotomy can be safely and effectively performed on wild mares that have been selected for non-breeding status. Those individuals could then be returned to the range to live out their natural lives without individually contributing to population growth. The proposed research effort is based on recent pilot studies that have suggested the potential for surgery-related health complications from ovariectomy in adult female horses is low (near 1%). When evaluating options for field techniques, spaying (ovariectomizing) mares as a population control method is not recommended unless it can be performed in a safe, practical, and effective manner. This project proposes to conduct a large scope investigation of the safety and practicality of spaying mares as a tool for wild horse population control. The results of this study will provide standardized, baseline outcomes for this surgical procedure which can be directly compared to other less invasive procedures being conducted and evaluated by the same research team. Purpose, Objectives, and Relevance A. Why the project is needed by the applicant: This project will demonstrate that ovariectomy in wild mares is a procedure which can be safely and efficiently applied in the field to permanently sterilize wild horses. Successfully performed, it should be 100% effective. The need for permanent sterilization has become evident as the reproductive rate in wild horses exceeds the ability of the BLM to effectively and humanely manage horses (provide adequate feed and water) while protecting the ecology of the range. As the number of horses in holding facilities continue to escalate, the need for effective, practical, and economical techniques to control the reproductive rate in the wild horse populations is necessary. B. Applicant’s objectives: Our objectives are to utilize a proven field technique for ovariectomy (spay) and apply it in a trial that will evaluate the feasibility of utilizing surgery as a management tool in controlling the growth rate of wild horse herds. Since this procedure has been used as an effective tool for wild horse management on Sheldon National Wildlife Refuge, we propose that the BLM can also utilize spaying of wild mares to address the overpopulation issue on public lands. The objective of this trial is to show that, despite the risks associated with this surgery, the complication rate is very low and indeed very acceptable and that horses undergoing this procedure do not suffer unnecessary discomfort from the procedure. C. How do the objectives support the applicant’s mission: Our objectives are to develop a practical and cost-effective technique for permanent sterilization of wild mares. This and other proposed techniques will support our mission of seeking ways to alleviate excessive wild horse population growth rate. We think that mare management holds the key to population control. A concise procedure that is 100% effective and permanent is not only humane to the horses but of great benefit to the public by helping preserve the range in a healthy condition so that wildlife and plant biodiversity is not threatened by equine over population. Multiple uses of public lands maximize benefits to animals and the public. 4a. Title: Re-immunization of Free-Ranging Horses with GonaCon Immunological Vaccine: Effects on Reproduction, Safety, and Population Performance 4b. Recipient: Colorado State University 4c. Additional Information: BACKGROUND 1. Re-immunization In many areas of the western United States, overabundant and rapidly expanding populations of feral horses (Equus caballus) pose a significant dilemma for natural resource managers. The Wild Free-Roaming Horses and Burros Act of 1971(P.L.92-195) provided protection for feral horses and burros (Equus asinus) on most federal lands and established guidance for their management as a wildland species (Wagner 1983). There is, however, widespread concern among state, federal, and private land management agencies that unregulated feral horse populations are severely altering native plant communities and limiting the abundance and diversity of habitat resources allocated for native wildlife and other domestic livestock species. Current population control methods such as utilizing periodic roundups and adoption or sale of excess animals, or maintaining excess feral horses in long-term holding facilities are expensive, resource intensive, and unsustainable. Clearly, more efficient, cost effective, and humane approaches to reducing feral horse densities on public lands are needed. Controlling the fertility of female horses offers a potential non-lethal alternative to conventional methods (National Research Council 2013). A promising immunological approach to contraception in feral horses involves immunization against the neuropeptide gonadotropin releasing hormone (GnRH). Scientists at the National Wildlife Research Center (NWRC) have conjugated synthetic GnRH peptides to a highly immunogenic carrier protein that, when combined with a potent adjuvant, stimulates the host’s immune system to produce antibodies that bind to endogenous GnRH. This, in turn, prevents synthesis and secretion of important downstream reproductive hormones necessary for reproduction. Animals generally return to fertility as antibodies concentrations decline (Powers et al. 2011). Multiple years of infertility have been achieved in captive and free-ranging wild ungulates with a single inoculation with the GnRH-based vaccine, known as GonaCon. This vaccines has been experimentally tested and found to provide multiple years of infertility after a single application in white-tailed deer (Odocoileus virginianus)( Miller et al. 2008, Gionfriddo et al. 2011a), bison (Bison bison)( Miller et al. 2004), elk (Cervus elaphus)( Killian et al. 2009, Powers et al. 2011, 2014), wild pig (Sus scrofa)( Massei et al. 2012), and feral horses (Killian et al. 2008, Gray et al. 2010, Baker et al. 2013). However, multiple years of infertility are only experienced in a fraction of vaccinated animals. In free-ranging elk, there was approximately a 90% treatment effect the first year after vaccination but that dropped to 50% by the second year and by the third year of the study, there was no measureable response (Powers et al. 2014). Similarly, during the first 3 years of our current investigation in feral horses at THRO, we observed a 25-35% decrease in foaling in treated versus control mares for the first and second years of the study but no effect by year three (Baker et al. 2013). Repeat vaccinations generally result in a more profound and longer-lasting antibody production due to the anamnestic response (Tizard 1982). Therefore, we expect longerlasting contraceptive effects in re-vaccinated mares. The single-injection GonaCon vaccine is unique in that the formulation initiates high antibody titers that remain elevated in some applications; however, to our knowledge, no research has been conducted to evaluate booster doses of this vaccine in any mammalian species. Booster immunizations using a variety of GnRH vaccines in domestic horses have been shown to improve contraceptive efficacy and to suppress behavioral and physiological estrus (Garza et al.1986, Elhay et al. 2007, Botha et al 2008). However, these GnRH vaccines differ from GonaCon in that they incorporate different protein carrier molecules and adjuvants, and are formulated for short duration (< 1 yr.) contraceptive effectiveness that is generally achieved by using a primary immunization followed 35 days later by a booster inoculation. While a single vaccination is often preferred from a management perspective, GonaCon vaccine may prove to be more effective if repeat vaccinations are delivered on a periodic basis. Efficacy data collected from 25 mares treated with single application of GonaCon in 2009, at Theodore Roosevelt National Park (THRO) revealed a moderate 2-year decline of approximately 30% in foaling rates, with all mares regaining fertility by three years post-primary vaccination treatment (Baker et al. 2013). Surprisingly, re-vaccination of these same mares in the fall 2013 (four years post-primary vaccination) has resulted to date, in complete infertility during the 2015 foaling season (the first season to expect a re-vaccination effect on fertility). Clearly, these results are both statistically and biologically significant, as well as encouraging from a fertility control perspective. If these results persist over time and these mares remain infertile, it would lend support to our hypothesis that re-vaccination with GonaCon, even four years post-primary vaccination produces a strong anamnestic response in horses that stimulates anti-GnRH antibodies and suppresses fertility. At present, however, it is premature to predict how many of these revaccinated mares failed to conceive during the 2014 breeding season and will not foal or regain fertility during 2015 and beyond. It is possible that the booster vaccination simply delayed the estrous cycle in these mares, which could result in foals being born later in the foaling season. While these findings are tentative and inconclusive, they suggest that repeat vaccinations are likely needed to achieve high efficacy of GonaCon vaccine in free-ranging horses and these effects have not been investigated or determined. Thus, our proposed research offers a unique opportunity to address this question at THRO and will have relevance, not only to feral horses, but also to other wild ungulates that have been treated with a single treatment of GonaCon vaccine. Our proposed research will begin to define the vaccination schedule needed to maintain infertility in free-ranging horses and whether or not long-term or permanent sterility is a possible outcome. We will investigate the safety and efficacy of a repeat vaccination under the hypothesis that this vaccine will be more efficacious and longer-lasting than the original primary immunization. 2. Remote Dart Delivery Fundamental to practical field application of GonaCon vaccine in free-ranging horses is a safe, reliable, and effective method of administering a single dose of the vaccine to freeranging horses by means of a syringe dart. Many contraceptive agents have been successfully applied via syringe dart or biodegradable implant to an assortment of wild ungulate species including white-tailed deer (Turner et al. 1992, Jacobsen et al. 1995, DeNicola et al. 1997), elk (Shideler et al. 2002, Baker et al. 2005), feral horses (Kirkpatrick et al.1990, Roelle and Ransom 2009), and elephants (Loxodonta Africana) (Delsink et al. 2002). However, to our knowledge, evaluation of remotely-delivered GonaCon vaccine is limited to one field investigation with white-tailed deer (DeNicola unpublished data). Although dart performance in this study was less than expected, it provided important basic information regarding optimum dart configuration and delivery ballistics. Using this preliminary data, technicians at Pneu-Dart, Inc. developed a prototype dart configuration for delivering this highly viscous vaccine formulation to free-ranging horses. We tested this GonaCon-specific dart delivery system with captive feral horses at the 2013 scheduled roundup at THRO. Eleven adult mares (2-4 years of age), that had not been previously vaccinated, were held in small paddocks and remotely darted in the biceps femoris muscle with 2 ml (2000 μg) of GonaCon vaccine. All darts were weighed (± 0.01g) before and after injection to determine the precise dose delivered. Darting distance varied from 10-15 m. Nine out of 11 darts delivered, on average, 95% of the GonaCon vaccine formulation. Two darts failed to discharge possibly due to low muzzle velocity. All darts appeared to dispense the vaccine deep into the muscle mass and none of the darts were observed to bounce without penetration, partially discharge, blow-out, or show evidence of subcutaneous delivery of the vaccine. The two horses in which the darts failed to discharge were subsequently re-treated and the second darts successfully delivered a full dose. With 85% of the 2015 foaling season complete, 7/11 (63%) of these mares have not foaled. In contrast, only 16% of the untreated mares have not foaled to date. A dependable dart delivery system for administering GonCon remotely to free-ranging horses is critical to the determination of an optimum re-vaccination schedule in our proposed study. If successful, this technology will potentially provide resource managers with an alternative strategy for managing this feral horse population. 3. Biological Side-Effects Evaluation of the biological side-effects of GonaCon vaccine treatments have been reported for numerous wild ungulate species including white-tailed deer (Curtis et al. 2008, Gionfriddo et al. 2011b), elk (Powers et al. 2011, 2012, 2014), bison (Miller et al. 2004) and feral horses (Baker et al. 2013). Results from these investigations generally conclude that GonaCon does not cause serious adverse effects on general health, body condition, existing pregnancy, neonatal health, major organ systems, or fertility of male and female offspring of females treated during pregnancy. Granulomatous intramuscular injection-site lesions, that occasionally break and drain as abscesses, are the only adverse effect of vaccination consistently reported in these studies. The formation of these injection site lesions may be necessary for stimulation of a strong immune response and infertility. GonaCon vaccine contains AdjuVac; a water-in-oil based adjuvant developed from a USDA approved Johnes disease vaccine called Myocopar TM (Fort Dodge Animal Health). AdjuVac contains killed Mycobacterium avium, which is needed to induce a rapid, strong, and sustained contraceptive response (Miller et al. 2008a, Perry et al. 2008). This combination of water inoil emulsion and killed mycobacteria results in a highly potent adjuvant that stimulates both humoral and cellular immunity (Warren et al. 1986). Vaccines, like GonaCon, that contain mycobacteria may induce strong immune responses because of the formation of a repository or depot at the injection site (Fukanoki et al. 2000). In response to the presence of the depot, a granuloma forms as the immune system attempts to isolate the foreign material. The continued existence of this depot, which initiates a chronic inflammatory response, likely provides a long-term source of antigen stimulation and persistent antibody production. We speculate that this is the mechanism by which a single vaccination can provide multiple years of infertility in a portion of the population in many species that have been studied. However, even with this prolonged antigenic stimulation, the immune response from a single vaccination does not consistently provide multiple years of infertility in all or even a high proportion of animals (Powers et al. 2014, Baker et al. 2013). In all studies, where post-mortem examinations were performed, prevalence of injection-site inflammation and granulomas were present but in some species, such as white-tailed deer and elk, they were not apparent antemortem (Curtis et al. 2008, Powers et al. 2011, Gionfriddo et al. 2011b). In contrast to these species, injection site reactions in feral horses, following GonaCon vaccination at THRO, are readily observable as subcutaneous swellings. In past studies at THRO (2009-2013), all injection site reactions appeared to be confined to the general gluteus muscle where the vaccine was first hand-injected. Reactions to the vaccine were first observed 30 days post-treatment in 17.2% (5/29) of mares and by the second breeding season, 79.3% (23/29) of treated females showed some evidence of inflammation or swelling at the injection site. Saline control mares displayed no evidence of injection site reactions. Swellings of various sizes (marble to baseball size) were most common, followed by nodules, and rarely a draining abscess. Most of these reactions were observable for three years post-treatment, then began to resolve and become less visible by year 4 (many that could not be visually observed were still manually palpable at the 2013 roundup). However, similar to other studies where injection site reactions have been evaluated, we did not observe any clinical evidence of lameness, impaired mobility, depression, or decreased health or fitness in any animal that was associated with GonaCon vaccine treatment. While results from the above investigations are generally consistent relative to the effects of GonaConinduced injection site reactions, they are also limited to the consequences of a single vaccination usually delivered by hand-injection. At the 2013 THRO round-up, GonaCon –treated mares were re-vaccinated, four years post-primary vaccination, with a booster dose on the opposite side in the biceps femoris muscle. This investigation is in progress but thus far, injection site reactions appear to be less apparent than those observed following the 2009 vaccination (Baker et al. unpublished). At this time, the cumulative effects of re-vaccination are unknown and the potential for more intense immune reactions with additional doses of this vaccine delivered by syringe dart is a consideration (Broderson 1989, Roelle and Ransom 2009). 4. Behavioral Side-Effects Behavioral side-effects of GonaCon vaccination in wild ungulates have not been extensively investigated (Gray et al. 2010, Baker et al. 2012, Ransom et al. 2014). Given the physiological mechanism of action, GonaCon vaccine has the potential to suppress fertility and diminish the reproductive behaviors typically associated with estrus. However, in GonaConvaccinated female elk (Powers et al. 2011) and free-ranging horses (Gray et al. 2010, Baker et al. 2012, Ransom et al. 2014) such behaviors were maintained throughout the first breeding season after immunization and were not different from untreated females. In a previous study at THRO during 2009-2010, daily activity patterns, social interactions, and reproductive behaviors were similar for GonaCon treated and control mares (Baker et al. 2012, Ransom et al. 2014). But, since GonaCon only prevented conception in 50% of treated mares (n = 28), behavioral observations were limited to only 14 infertile females. Thus, inferences to free-ranging feral horse populations are not definitive and deserve further investigation prior to use in management applications. In an attempt to further our understanding of the behavioral side-effects GonaCon vaccine, we conducted behavioral observations during the first breeding season following revaccination of these same mares at THRO in 2013. We measured the effects of this vaccine on sociosexual behavior, harem dynamics, and activity budgets of treated (n = 25) and control (n =25) horses. To date (July 2