Animals

We selected and acquired 39 nonpregnant, 1–2-yr-old female bison of both Brucella-seropositive (29) and Brucella-seronegative (10) status during IBMP removal operations near the northern boundary of Yellowstone National Park in 2011. Animals were captured at the National Park Service's Stephens Creek, Montana, USA (45°2′56.76828″N, –110°45′4.76568″W) bison facility, as they migrated out of Yellowstone National Park (YNP) in late winter and early spring of 2011. We also collected ten 2-yr-old and 3-yr-old Brucella-seronegative bulls from IBMP operations at Stephen's Creek (2012–14) and additionally (2012) purchased four Brucella-seronegative bulls from a commercial source. Bison were manually restrained in a chute, then blood was collected via jugular venipuncture using a 16-gauge needle and 30 mL syringe for chute-side brucellosis screening using a fluorescence polarization assay (FPA) and the standard card test (United States Department of Agriculture, Animal and Plant Health Inspection Service, 2003). Bison were individually identified with visual ear tags (Y-Tex, Cody, Wyoming, USA) and a radio frequency identification button tag (AllFlex USA, Dallas Fort Worth Airport, Texas, USA). Animals were transported to the USDA, Animal and Plant Health Inspection Service (APHIS) bison facilities in Corwin Springs, Montana, USA (45°6′49″, –110°47′248″). We randomly assigned animals to a control group not treated with GonaCon (Control; n=14) or a treatment group that received one dose of 3000 mg GonaCon as described below (Treatment; n=15). Each group was housed separately in an approximately 10 ha pasture. In addition, each pasture also contained five Brucella-seronegative cows that served as a comparison of fertility to brucellosis-seropositive animals during the first year, as well as disease transmission sentinels for an associated study (Nol et al. 2024). Brucella-seronegative cows did not receive GonaCon.

Bison bulls were housed separately from the cows until breeding season. Bulls (two per pen) were comingled with cows from August to October. Commercially sourced bulls were removed from the study after the first breeding season.

GonaCon production and treatment

GonaCon was prepared using GnRH-blue protein hemocyanin conjugate and adjuvant formed into an emulsion, for depot effect, as described previously (Miller et al. 2008), with some differences in emulsification. Specifically, GonaCon was emulsified by passing it once through a microfluidizer processor run at 6,000 psi with an H30Z interaction chamber (Microfluidics M110L, Microfluidics, Westwood, Massachusetts, USA). At initiation of the study (2012), Treatment animals were manually handled in a chute and injected intramuscularly (IM) 2.5 cm from the hip, bilaterally with GonaCon (total of 3000 mg GnRH-blue protein conjugate in 2 mL adjuvant; 1500 mg in 1 mL was administered in each hip) approximately 90 d before first exposure to bulls. GonaCon was administered only one time to each individual treatment animal. Control bison were not treated with GonaCon, nor were Brucella-seronegative animals in either group.

Animal handling and sample collection

Each January, for five reproductive seasons, we manually restrained bison cows in a chute, during which time we collected blood and determined pregnancy status by rectal palpation. Blood samples were collected from the jugular vein as described above, transferred to serum separator tubes (Vacutainer, Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA), and submitted to the Montana Veterinary Diagnostic Laboratory, Bozeman, Montana, USA (MVDL), for brucellosis testing as described (Nol et al. 2024). Animals determined to be pregnant immediately received a vaginal transmitter (Advanced Telemetry Systems Inc., Isanti, Minnesota, USA) and were monitored daily thereafter for birth events. Blood was also collected from cows either directly post-partum or midyear (nonpregnant animals). Blood was collected from bulls annually just prior to breeding for brucellosis testing. We evaluated all bulls for breeding soundness over the course of the study. Electroejaculation (Lane Manufacturing Inc., Denver, Colorado, USA) of bulls was performed using warmed collection cups or tubes to avoid cold shock to the sperm. We evaluated and scored bull semen samples for gross and individual motility using Society for Theriogenology (SFT) -published criteria (Koziol and Armstrong 2018).

For post-partum handling of cows and breeding soundness examinations for bulls, animals were chemically immobilized using etorphine (0.01 mg/ kg) or thiafentanil (0.015–0.02 mg/kg) and xylazine hydrochloride (0.05–0.07 mg/kg; Wildlife Pharmaceuticals, Windsor, Colorado, USA) delivered IM via remote injection (Pneudart, Williamsport, Pennsylvania, USA). Immobilizing drugs were antagonized with naltrexone (Wildlife Pharmaceuticals; 50 mg IM per mg thiafentanil given or 25 mg IM per mg etorphine given) and 300 mg IM tolazoline (Akorn Animal Health, Lake Forest, Illinois, USA; 300 mg, half IM, half subcutaneously).

Brucellosis testing

Serologic testing by standard card, buffered acidified plate antigen, complement fixation, and FPA as described in the Uniform methods & rules for brucellosis eradication (United States Department of Agriculture, Animal and Plant Health Inspection Service, 2003) were performed at the MVDL, both on animal acquisition (for confirmatory testing of chute-side results) and for the remainder of the study.

Anti-GnRH antibody testing

We measured anti-GnRH antibodies using ELISA as follows. Microtiter plates (Greiner Bio-One North American Inc., Monroe, North Carolina, USA) were coated using 50 mL of a solution containing 4 ng GnRH-BSA (bovine serum albumin) conjugate in carbonate-bicarbonate buffer. Plates were incubated at 4 C overnight, then washed three times with 300 mL of 0.01 M phosphate-buffered saline (PBS) plus 0.05% Tween 20 (pH 7.4; giving PBST) per well at room temperature. To block nonspecific binding, 200 mL of a solution consisting of 20% (v/v) SeaBlock (Thermo Fisher Scientific; Waltham, Massachusetts, USA) and 5% (v/v) Tween 20 in 0.01 M PBS was added to each well, and plates were incubated for 1 h at 25 C, followed by another three washes with PBST. Serum samples were run in duplicate at a dilution of 1:2,000 in 50 lL of 0.01 M PBS and incubated for 1 h at room temperature. Plates were then washed three times with PBST. Rabbit anti-bovine IgG (Sigma-Aldrich, Inc., St. Louis, Missouri, USA), diluted 1:5,000 in 0.01 M PBS (50 mL), was added to each well and incubated for 1 h at 25 C, followed by two washes with PBST. Secondary antibody, goat anti-rabbit immunoglobulin G-horseradish peroxidase (IgG-HRP) (Sigma-Aldrich), diluted 1:3,000 in 0.01 M PBS (50 uL), was added to each well and incubated for 1 h at 25 C, followed by two washes with PBST. Enzyme substrate (3,3′,5,5′-tetramethyl-benzidine dihydrochloride) in 0.05 M phosphate citrate buffer with 0.14% urea hydrogen peroxide was then added to each well. After 3 min, 50 lL of 2 M sulfuric acid was added to terminate the reaction. Absorbance of each well was measured at 450 nm on a plate reader (VarioSkan Flash, Thermo Fisher Scientific). Plate background was corrected by subtracting the mean absorbance of all PBS wells from all other well values. Antibody responses were reported as optical densities (ODs). A positive control sample was included on each plate. Linearity was determined from eight concentrations of positive serum ranging from 1:1,000 to 1:128,000. The R² value was 0.98. The dilution used (1:2,000) to measure antibody response was at the upper end of the working range. Positive and negative control samples were included on each plate. The interassay coefficient of variation for the positive controls was 12%.

Data analysis

We limited the statistical analysis of pregnancy data to year 1. We limited statistical analysis of OD data to baseline (pre-treatment), 8 mo, and 44 mo post-treatment. Both sets of data were truncated due to potential bias issues resulting from missing values, animal removals, and, specific to the ELISA, unexplained output irregularities in other time points for all animals regardless of treatment. For the remaining data we compared differences between the treatment groups in number of calves produced and OD values of anti-GnRH antibodies using the methods described below.

The number of pregnancies or calves produced, alive or dead, was the variable of interest in evaluating GonaCon in its ability to prevent pregnancy. We used a Fisher's exact test, given the small sample size, to compare the number of calves produced between Treatment and Control, in year 1 only as stated above. Comparisons of anti-GnRH OD between Treatment and Control were conducted using a repeated measure ANOVA for three measurement periods: baseline, 8 mo post-treatment, and 44 mo post-treatment. Pairwise tests with Bonferroni correction were run to detect any differences between the within-treatment time periods. Prior to conducting the repeated measures, data were checked for normality using a Shapiro-Wilk's test and QQ plots. Values were considered significant at P=0.05. All statistical tests were performed in R (R Core Team 2022).