Management of intraspecific aggression in two bull giraffes

Maintaining nonbreeding individuals in zoological collections may sometimes necessitate housing bachelor groups. In turn, intact cohabiting males may express increased intraspecific agonistic behaviors, and management intervention may be indicated. Where castration is deemed inappropriate (e.g., future breeding, or anesthesia and surgery ‐ related risk), the immune contraceptive gonadotrophin ‐ releasing hormone (GnRH) is increasingly used as an alternative. When intraspecific aggression (sparring) in two bull giraffes housed as a bachelor pair at Knowsley Safari, UK, escalated in frequency and intensity (despite management interventions), further mediation was warranted to moderate sparring behaviors. The Ex situ Program recommendation was for one giraffe, the (slightly) older, outwardly mature (darker, strong musth) individual, to be treated with the GnRH vaccine Improvac ® (Zoetis). To gauge the efficacy of vaccination, behavioral observations were conducted during each vaccination phase to identify changes in the frequency of sparring behaviors. In addition, fecal samples were collected by keepers and sent to Chester Zoo's Endocrine Diagnostic Laboratory for analysis to compare androgen levels between the pre ‐ and postvaccination phases. Testicular atrophy was investigated using both visual inspection and photographic images. The GnRH vaccine Improvac ® initially appeared to be associated with reduced aggressive behaviors in the two bull giraffes. Sparring behaviors decreased in frequency after each vaccination phase, although these did not significantly diminish until phase 4. Physiological markers were inconclusive as testosterone concentrations varied throughout the phases, although levels remained low after the fourth vaccination phase. Approximately 8 months following the initial vaccination with Improvac ® , the unvaccinated bull exhibited heightened aggression, resulting in physical aggression and injury to the vaccinated bull. As a result, both bulls are now on an Improvac ® vaccination schedule, which has enabled them to remain housed together as a bachelor pair.


| STATEMENT OF THE PROBLEM
The ex situ management of Giraffa camelopardalis has resulted in conservation success, with populations breeding well in zoos (Schwarzenberger et al., 2022).As ex situ populations are currently stable, maintaining nonbreeding individuals in zoological collections may sometimes necessitate housing bachelor groups.Currently, Knowsley Safari houses two male Rothschild's giraffes (G.camelopardalis ssp.rothschildi), both 9 years old with an 8-month age difference and similar in size.There are no females on site.The pair have been housed together since birth and managed as a bachelor pair since 2017.Sparring behaviors initially occurred at a frequency typical of subadult bull giraffes and were infrequent and gentle, and the social relationship remained stable.However, in 2021, sparring sessions began to occur more frequently, with lining up, leg lifting, and mounting behaviors being observed.In 2022, sparring increased substantially in frequency, intensity, and duration, becoming a daily occurrence with significant body blows resulting in swelling and hair loss (Figure 1).The sparring sessions appeared to be exacerbated further by external stimuli (loud noises, keeper talks, high density of visitors), and the giraffes were becoming less responsive to keepers and more focused on each other.
Intraspecific aggression in giraffes usually presents itself through necking and sparring.It is thought to have two main functions: practice for subadult bulls to gain fighting skills (Bercovitch & Berry, 2015) and to establish dominance hierarchy and social cohesion (Leuthold, 1979;Wolf et al., 2018).Sparring is a natural behavior in bull giraffes that maintains a hierarchy of size and age.
Increased aggression levels in giraffes, particularly in those belonging to bachelor groups, have been attributed to the establishment of these hierarchies (Wolf et al., 2018).When sparring, bull giraffes will position themselves side by side, often head-to-head or head-to-tail (lining-up), and proceed to swing their neck, striking their head and ossicones against the body of the sparring partner (Granweiler et al., 2021).
In response to increased aggression between the bulls at Knowsley Safari, the animal management team consulted with the park's veterinary surgeon and the European Association of Zoos and Aquariums (EAZA) Ex situ Program (EEP) for Rothschild's Giraffe.
They decided to administer Improvac ® (Zoetis) for gonadal suppression, aiming to reduce testosterone-driven aggression (sparring).This intervention targeted the more mature bull (exhibiting darker coloration and strong musth), who frequently initiated sparring despite less effective neck swinging and thrusting.To assess effectiveness, we observed social interactions and recorded all sparring behaviors during each vaccination phase.Additional markers for the reduction of testosterone included visual observations and photographs to assess testicular atrophy and the collection of fecal samples to measure fecal testosterone metabolites (FAMs).

| GENERAL HUSBANDRY AND MANAGEMENT INTERVENTIONS
The indoor house measured 14.4 m × 14.3 m and was split into multiple stalls with multiple bedded areas providing separate resting areas or communal areas based on the giraffes' preferences.The exterior enclosure consisted of a sand area directly in front of the house, followed by a larger grass area, a feeding platform used for visitor experiences, along with several trees, scratching brushes, browse stations, and enrichment devices, such as barrel and puzzle feeders, to slow feeding behavior and increase the complexity of feeding.Giraffes were given paddock access when daytime temperatures exceeded 5°C.and overnight access when night-time temperatures exceeded 12°C.Giraffes were kept together 24/7 but usually chose separate sleeping/resting areas overnight.The daily diet consisted of Waterhouse browser ruminant cubes and Dengie Alfa-A (molasses free), with ad libitum alfalfa and salt licks.Leaf browsing was offered (ad libitum) throughout the spring, summer, and fall, and winter browsing was maintained with frozen leaves and browse silage.

| ADMINISTRATION OF GNRH VACCINATION IMPROVAC ®
The EEP recommendation was for one giraffe, the slightly older male, to be treated with the GnRH Improvac ® vaccine.The decision was based on known risk factors, for example, giraffes may have longterm reduction of androgen levels after the termination of Improvac ® vaccinations (Schwarzenberger et al., 2022).The older bull was chosen because he outwardly exhibited signs of a mature bull giraffe (darker coat coloration, emitting a strong musth smell).However, both individuals had been observed to initiate sparring sessions and exhibited varying degrees of dominance toward one another.
Vaccinations were administered intramuscularly using a dart gun by the zoo's veterinarian.Training for hand vaccination was ongoing when the first vaccination was deemed necessary; therefore, hand injections were not immediately actioned.Per Improvac ® guidelines, booster one (1) was provided no earlier than 3 weeks after the initial primer dose, and subsequent boosters were given no more than 10 weeks between doses, as evidenced in Table 1.Dosage was increased from 3 to 5 mL in the fourth vaccination phase due to continued sparring behavior observed by keepers and animal managers.
The higher dose has previously been used in males for immunocontraception with positive results (Schwarzenberger et al., 2022) (Table 1).

| BEHAVIORAL MONITORING
The effectiveness of the GnRH vaccine was determined by recording the relative frequencies of selected sparring behaviors (Table 2) and examining changes in the frequency of these behaviors performed throughout the vaccination phases.Data were collected between 09:00 and 17:00 across each phase, using one-zero focal sampling (Lehner, 1992)

| FECAL COLLECTION AND TESTOSTERONE METABOLITES
Keepers collected fecal samples daily during the morning husbandry routine.Once a giraffe had defecated, three to four subsamples were placed into a ziploc bag.The bag was put into a freezer at −20°C within 1 h of collection.Eighty-seven samples were collected for the VB, and 68 samples were collected for unvaccinated bull (UB) between November 21, 2021 and November 26, 2022, with samples collected for VB before vaccination and across all four vaccination phases.Samples for UB were collected prevaccination to establish baseline levels but were only collected opportunistically during the vaccination phases as he was not vaccinated.All samples were sent to Chester Zoo's Wildlife Endocrinology Diagnostic Laboratory for analysis.

| Testicular atrophy
Keepers conducted visual monitoring throughout each vaccination phase, and photographs of the VBs' testicles were taken during phase 1 and after phase 4. Photos were taken by the same individual, from a similar distance, angle, and with the giraffe in a similar stance.

| Behavior
A Kruskal-Wallis test revealed a statistically significant difference in sparring behaviors across the vaccination phases, H (4) = 13.74,p = .008.
Pairwise comparisons using DMC were used to pinpoint significant differences across the vaccination phases (Table 3).Sparring frequency remained similar between the prevaccination phase and phase 1 (Figure 2).However, phase 1 exhibited a significantly higher mean frequency of sparring per 15-min observation (M = 5.92, SD = 6.83) compared to phase 4 (M = 0.14, SD = 0.53).While no other pairwise T A B L E 2 Ethogram of observed agonistic behaviors.

Sparring behaviors Description
Necking/rubbing One giraffe rubs its head or neck against a conspecific's body

Lining-up
The two giraffes stand parallel to each other; they may be head-to-head or head-to-tail

Sparring
The giraffe swings its neck and strikes its head against the body of the sparring partner; this may include striking with ossicones comparisons between phases revealed statistically significant differences, a general trend of decreasing sparring behavior was observed across the vaccination phases (Figure 2).

| Hormone assay
The Chester Zoo Wildlife Endocrinology Diagnostic Laboratory reported that the giraffe exhibited varying androgen (testosterone) concentrations throughout both collection periods (Figure 3).Both

| Testicular atrophy
We compared photographs taken in phase 1 and a photograph taken after the fourth vaccination phase (Figure 4).Improvac ® vaccination in a single bull giraffe appeared to reduce intraspecific aggression levels initially.Sparring behaviors initiated by the VB decreased in frequency after each vaccination phase.Comparatively, other mammals immunized with Improvac ® , specifically to reduce aggression, showed behavioral changes after the second and third vaccinations (Bishop et al., 2016;Brewster & Nevel, 2013;Lueders et al., 2014;Martinez-Giménez et al., 2021;Rydhmer et al., 2010).Our findings were similar, with a decrease in the frequency of sparring in phase 2 and phase 3 compared to phase 1.However, a statistically significant reduction did not occur until phase 4, where the mean incidences of sparring were less than one per 15-min observation.This aligns with the findings of Schwarzenberger et al. (2022), advising that male giraffes may require four or five vaccinations at 4-week intervals for contraceptive effect.
Monitoring testicular atrophy in male giraffes is another measure for determining Improvac ® vaccination success (Schwarzenberger et al., 2022).Following the fourth vaccination phase, photographs revealed a significant reduction in scrotal and testicular size.While keepers observed a change in appearance near the end of phase 3, a consensus was lacking to the extent of reduction, with varying responses.This was likely due to slight variations in the giraffe's stance (weight bearing) and the keeper's angle of view.We recommend standardization for monitoring testicular atrophy, ideally

| CONCLUSION
Our case study demonstrates that vaccination with Improvac ® was associated with a reduction in sparring behavior between the giraffes, but only after the fourth dose.This coincided with low FAM and the reduction in size of the scrotum and testes.However, it is important to note that pre-and postvaccination FAM concentrations showed similar overall levels between the bulls, although the timing of their peak levels differed.Furthermore, we found that 8 months after treating one of the two bull giraffes with Improvac ® , there was a shift in social dynamics, requiring further management intervention and subsequent vaccination of both bulls.At the time of writing, the bulls remain housed together (separated at night), and the authors report that modest improvements in the social dynamics have been observed, including less avoidance and occasional social feeding at the browse station with no observed sparring behavior.In retrospect, vaccinating both bulls simultaneously might have circumvented injury and agonistic behavior.However, concerns regarding the potential for long-term reductions in androgen levels following the cessation of Improvac ® treatments led us to exercise caution and initially vaccinate only one bull.

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I G U R E 1 Pictured are the injuries sustained (swelling and hair loss) following physical contact during sparring bouts.[Color figure can be viewed at wileyonlinelibrary.com]In response to a significant increase in sparring behavior observed in 2021, the zoo implemented several management interventions to reduce its intensity and frequency.These efforts aimed to minimize potential stressors and encourage natural behaviors.Specifically, the zoo closed the house to visitors to provide a quiet area with minimal disturbance.The daily pellet feed was offered in smaller, more frequent feeds, interspersed with nettles alongside additional and more frequent enrichment and browse.Keepers ensured the browse was provided in multiple areas within the house and paddock, enabling the giraffe to choose where to feed and to engage in feeding behavior for longer.Despite these comprehensive efforts, anecdotal observations indicated that the interventions were not successful in diminishing the aggressive interactions and sparring behavior.In response to this finding, the park took action in 2022 to address the ongoing issue by administering the GnRH vaccine Improvac ® .
Abbreviation: N/A, not applicable.
bulls showed similar concentrations, although peaks in testosterone occurred at different times across the phases.The VB exhibited peaks in testosterone on November 16, 2021 (1168.08 ng/g) and during the second vaccination phase on August 9, 2022 (854.63 ng/g).In late September, there was a peak in the UB concentration (1068.42ng/g), but it had returned to its previous level when sampled on October 1, 2022, with the VB concentration of 146.31 ng/g and UB 180.03 ng/g.

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I G U R E 2 Sparring (mean ± SD) per 15 min observation/phase prevaccination, observation period (November 22, 2021−November 30, 2021), phase 1, the observation period after the first vaccination with Improvac ® (June 23, 2022-July 20, 2022); phase 2, the observation period after the second vaccination (July 21, 2022-August 17, 2022); phase 3, the observation period after the third vaccination (August 20, 2022-September 22, 2022); and phase 4, the observation period after the fourth vaccination (September 27, 2022-October 31, 2022).Sparring frequency significantly reduced between phases 1 and 4 (**).F I G U R E 3 Hormonal assay results for vaccinated bull (Alex) and unvaccinated bull (Orbit) conducted by Chester Zoo's Wildlife Endocrinology Diagnostic Laboratory.Fecal androgen metabolite (FAM) concentrations were calculated per 0.5 g of feces.Variable FAM concentrations occurred at different times for each bull.Vaccinated bull peaked after vaccination phase 2 in early August, then the levels decreased and remained low.A blue triangle indicates the administration of Improvac ® vaccinations.[Color figure can be viewed at wileyonlinelibrary.com] using a crush, to ensure consistency in distance, angle, and height for direct comparison, particularly if used as a single measure of vaccination efficacy.As reported by Chester Zoo, fecal androgen metabolite (FAM) concentrations exhibited significant variation throughout the four vaccination phases.While both bulls demonstrated similar overall FAM concentrations, the timing of peak levels differed significantly between individuals.This individual variation in peak timing makes it challenging to definitively attribute the observed decrease in FAM levels postvaccination solely to the Improvac ® treatment.However, 2 weeks after the third dose and throughout the fourth vaccination phase, the VB FAM concentrations remained low, similar to findings by Schwarzenberger et al. (2022), which concluded that four doses at monthly intervals are sufficient to suppress androgen concentrations.The mechanism underpinning the unexpected testosterone increase after initial vaccination remains unclear.This paradoxical effect contrasts with the expected action of GnRH vaccines, which block pituitary stimulation and suppress sex hormone production.Unlike GnRH agonists, which temporarily stimulate the pituitary gland and cause an initial surge in gonadotropins and gonadal activity, vaccines should theoretically prevent such hormonal increases.Interestingly, Moresco et al. (2022) discussed gonadal stimulation in some female giraffes and noted that collections observed estrus-like behavior in females after Improvac ® vaccination.These findings warrant further investigation to understand this unexpected response.A side effect of the vaccination protocol's efficacy is that the pair's social dynamics changed.Approximately 8 months following the initial vaccination with Improvac ® , the UB exhibited heightened aggression and dominance behaviors toward the VB.The UB was observed vocalizing, resource guarding, and dominance posturing toward the VB and the keepers.These behaviors resulted in significant injury to the VB and subsequent avoidance behaviors.These behavioral changes, including heightened aggression, dominance displays, and possessive behavior (similar to mate guarding, e.g., increased proximity, defensive dominance display), toward the VB resulted in an animal management decision to separate the giraffes overnight.Doing so allowed the VB to rest and sleep undisturbed by the UB's continual attention.However, management intervention alone was ineffective in mitigating the UB's aggression, and in March 2023, the UB received the first monthly Improvac ® injection.

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I G U R E 4 (a) Vaccinated bull's testicles; photograph taken at the start of phase 1.(b) vaccinated bulls' testicles taken after the completion of vaccination phase 4, illustrating the reduced size of testes as well as shriveled scrotum.[Color figure can be viewed at wileyonlinelibrary.com]