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Title: Effects of Lupron and surgical castration on fecal androgen metabolite concentrations and 
intermale aggression in capybaras (Hydrochoerus hydrochaeris)  
 
Running Title: Castration and aggression in capybaras 
 
Authors: Jennifer H. Yu1, Janine Brown2, Nicole Boisseau2, Tony Barthel3, Suzan Murray1 
 
Author Affiliations: 
1 Global Health Program, Smithsonian’s National Zoo and Conservation Biology Institute, Washington 
DC, USA 
2 Center for Species Survival, Smithsonian Conservation Biology Institute, Front Royal, VA, USA  
3 Animal Care Sciences, Smithsonian’s National Zoo and Conservation Biology Institute, Washington 
DC, USA 
 
Keywords: noninvasive hormone monitoring; reproductive endocrinology; fecal androgen metabolites; 
gonadotropin-releasing hormone; testosterone; capybara 
 
Research Highlights: Chemical sterilization with leuprolide acetate resulted in down-regulation of sperm 
and testosterone production, but did not reduce intermale aggression among three capybara males. 
Similarly, subsequent surgical castration of two males was unsuccessful in managing agonistic interactions.   
 
 
 
 
 
 
Abstract:  
To curb agonistic interactions in a bachelor group of three male capybaras (Hydrochoerus 
hydrochaeris), a single dose of leuprolide acetate (Lupron®) was used in an attempt to chemically sterilize 
the males. Concurrently, fecal androgen metabolite (FAM) concentrations were quantified via enzyme 
immunoassay to monitor changes in testosterone production after injection of the gonadotropin-releasing 
hormone agonist. When Lupron proved ineffective in suppressing intraspecific aggression, surgical 
castration was performed on two males, with continued noninvasive endocrine monitoring. In all three 
capybaras, FAM concentrations increased initially as a result of the luteinizing hormone surge, but then 
decreased significantly following chemical sterilization. Surgical castration resulted in further, persistent 
declines in FAM concentrations in two males, while the third, intact male demonstrated a rise in FAM to 
pre-Lupron concentrations at 8.5 and 9.5 months post-administration. Despite successful suppression of 
sperm and testosterone production, intermale aggression continued, ultimately necessitating separation of 
the animals and transfer to other holding institutions. Under this set of conditions, a single Lupron dose was 
inadequate for suppressing intraspecific aggression in a group of three males with a pre-established history 
of aggression.  
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1. Introduction: 
 Capybaras (Hydrochoerus hydrochaeris) are a gregarious, semi-aquatic rodent species native to 
South America and commonly maintained in zoological collections. Given their value to commercial 
production in some regions, and their pest status in others, capybara social behavior has been extensively 
studied to inform population control and management (as reviewed in Herrera, Salas, Congdon, Corriale, 
and Tang-Martínez, 2011). In the wild, capybaras tend to form relatively stable, territorial groups with a 
sex ratio strongly biased toward females. Male social structure exhibits a rigid and strongly linear 
dominance hierarchy, with a single dominant male maintaining priority access to mating opportunities, 
food, wallowing sites, and other resources (Herrera and Macdonald, 1993; Herrera et al., 2011; Rosenfield 
and Pizzutto, 2019). Dominance is maintained through the expression of androgen-driven secondary sexual 
characteristics (e.g. enlarged nasal scent glands), display of marking behavior, defense of central positions 
among the herd, and aggressive interactions directed at subordinate or satellite males. Intermale agonistic 
interactions may range from brief, relatively harmless chases to severe bite wounds sustained by 
subordinates (Costa and Paula, 2006; Herrera, 1992; Herrera and Macdonald, 1993; Morreira, Macdonald, 
and Clarke, 1997; Rosenfield and Pizzutto, 2019; Rosenfield et al., 2019).  
Given the likelihood of aggressive interactions and potential for serious injury when housed in 
groups, it is generally not advised to house male capybaras together for welfare reasons (AZA Rodent, 
Insectivore and Lagomorph TAG, 2018). However, the maintenance of heavily female-biased capybara 
groups is unsustainable indefinitely, resulting in a surplus of males in the global zoological population that 
often must be housed together. Surgical castration has been a strategy to curb undesirable agonistic 
behaviors, but it precludes the male from future breeding, an unfavorable outcome for conservation 
management. Thus, chemical sterilization has been proposed as an alternative, temporary, and less invasive 
means to reduce intraspecific aggression in wildlife maintained under human care (Penfold, Patton, and 
Jimchle, 2004).  
 Among chemical sterilization agents, gonadotropin-releasing hormone (GnRH) agonists like 
deslorelin and leuprolide acetate are commonly used in veterinary medicine. GnRH agonists are a class of 
drugs that function by binding to GnRH receptors, leading to an initial “flare” or acute surge of luteinizing 
hormone (LH) and a consequent short-term rise in circulating testosterone (Padula, 2005). Subsequently, 
they desensitize the pituitary gland through GnRH receptor downregulation, which reduces the secretion of 
gonadotropins and ultimately androgen production. GnRH agonists may theoretically modulate behavior 
by reducing circulating levels of testosterone (Goericke-Pesch, 2017; Padula, 2005). However, the impact 
of chemical or surgical castration on undesirable behaviors can be highly variable. This study evaluated the 
effects of a single-dose injection of leuprolide acetate (a GnRH agonist) and subsequent castration on 
intermale aggression and excretory fecal androgen metabolite (FAM) concentration in adult male 
capybaras.  
 
2. Materials and Methods: 
2.1 Animal History, Husbandry, Housing, and Social Behavior:  
Three male capybaras were acquired in November 2003 and housed in the collection of the 
Smithsonian’s National Zoological Park in Washington, DC. At the time of introduction, two siblings were 
from the same litter and aged 1.5 years, while the third male was 1 year of age (Table 1). Following a 
routine and uneventful 30-day quarantine, the males were introduced and housed together as a trio in one 
indoor-outdoor enclosure for 2 years. The habitat was composed of an outdoor 6 m x 6 m area on exhibit 
and a 4 m x 5.8 m indoor area with crushed granite flooring split into two accessible stalls. All three 
capybaras had access at all times to one shared indoor pool, and weather-dependent access to a shared 
outdoor pool. The capybaras were fed twice daily with pelleted rodent feed, hay, and mixed greens and 
produce, and water was provided ad libitum.  
 The three males were housed together uneventfully for 2 years until the time of sexual maturity. 
Agonistic interactions among the three males began abruptly and were first observed in early February 
2005. A serious altercation ensued among the three males, resulting in severe skin lacerations in one 
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individual and a fractured incisor and minor wounds in the first aggressor. At this time, they were separated 
in adjacent enclosures during the healing process; however, visual and auditory contact remained intact.  
  
2.2. Chemical and Surgical Interventions:  
Due to its temporary effects, chemical sterilization was attempted in the initial effort to manage 
intermale aggression and reduce further risk of injury. Capybaras were anesthetized for physical 
examination and injection of leuprolide acetate (Lupron®, TAP Pharmaceuticals, North Chicago, IL) on 14 
or 15 February 2005. Animals were induced with a combination of ketamine (3.0–3.6 mg/kg) and 
medetomidine (0.03–0.035 mg/kg), then maintained under anesthesia with supplemental oxygen and 
isoflurane (0.5–1%) as needed delivered via facemask. A single dose of Lupron (3.0–3.5 mg, or 
approximately 0.08–0.09 mg/kg) was injected intramuscularly in the left thigh of each male. Additionally, 
testicular measurements were taken from each male (Table 1). Following Lupron injection and reversal 
with atipamezole (5.5–7.0 mg), the capybaras were initially housed individually to avoid agonistic 
interactions during the initial testosterone surge. Each individual was prescribed a short course of 
haloperidol at 1 mg/kg daily to assist with the reintroduction process beginning 3 March 2005 and 
discontinued 21 March 2005. Slow reintroduction efforts were initiated 7 March 2005 but were ultimately 
unsuccessful, with capybara 1 sustaining severe wounds and injuries inflicted by enclosure-mates that 
required immediate veterinary attention.  
Given the severity of the injuries, the decision was promptly made to surgically castrate capybaras 
1 and 2 and to house all three males individually. Capybaras 1 and 2 were surgically castrated on 24 March 
and 4 April 2005, respectively. Both animals were induced using a combination of ketamine (3.4–3.5 
mg/kg) and medetomidine (0.033–0.035 mg/kg) and maintained at a surgical plane under anesthesia using 
isoflurane at 1–2% delivered via facemask. Both animals were sterilized using a closed castration technique 
without complication. As the presumptive dominant male, capybara 3 was not surgically castrated and 
remained intact. Testicular tissue from capybaras 1 and 2 were submitted for histological analysis.  
   
2.3. Fecal Processing and FAM Analysis: 
 Voided fecal samples were collected opportunistically from each male for fecal androgen 
metabolite (FAM) analyses between February and mid-December 2005. Fecal samples were attributed to 
specific individuals with the use of food coloring and stored frozen until time of processing and analysis as 
described by Putnam et al. (2015). Briefly, samples were dried in a lyophilizer (VirTis Ultra 35XL, SP 
Scientific, Warminster, PA), powdered, sifted, and 0.20 ± 0.02 g was weighed into 16x125 mm glass tubes 
(Fisherbrand, Thermo Fisher, Pittsburgh, PA). Five ml of 90% ethanol:10% de-ionized water was added to 
each sample along with ~20,000 dpm 3H–testosterone tracer (NEN Radiochemicals, Perkin Elmer, Boston, 
MA) to determine procedural loss. For 20 minutes, samples were boiled in a 95°C water bath and 
maintained at a 5-ml volume with the addition of 100% ethanol as needed. Samples were then centrifuged 
at 500 x g for 20 minutes (Sorval RC 3C Plus, Kendro Laboratory Products, Newtown, CT), the supernatant 
recovered and 5 ml of 90% ethanol:10% de-ionized water added to the pellet, which was vortexed (pulse 
rate 1/second, speed 65; Glas-Col, Terre Haute, IN) for 30 seconds. Samples were re-centrifuged (15 
minutes, 500 x g) and the supernatants combined and dried down under forced air. One ml of 100% 
methanol was then added to dried sample extracts, evaporated to dryness, and reconstituted in 1 ml of 
preservative-free buffer (0.2 M NaH2PO4, #S8282; 0.2 M Na2HPO4, #S7907, Sigma Aldrich, St. Louis, 
MO; 0.15 M NaCl, #S271, Fisherbrand; pH 7.0). After vortexing for 15 seconds, samples were placed in 
an ultrasonic cleaner water bath (Cole Parmer Instrument Company, Vernon Hills, IL) for 15 minutes. 
Sample extracts were further diluted 1:50 – 1:250 in buffer as needed. All sample extracts and dilutions 
were stored in polypropylene tubes at -20 °C until analysis. Average fecal extraction efficiency was 76.0 ± 
0.9% based on recovery of 3H-testosterone added samples prior to extraction. 
A single-antibody enzyme immunoassay (EIA) utilizing a polyclonal antibody to testosterone 
(R156/157, C.J. Munro, University of California, Davis, CA) and testosterone-horseradish peroxidase 
ligand (lot: 2005/2006, SCBI, Front Royal, VA) (Putman et al., 2015) was used to measure FAM 
concentrations. Briefly, testosterone antibody in coating buffer (0.015M Na2CO3, #S2127; 0.035 M 
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NaHCO3, #S8875, Sigma Aldrich, St. Louis, MO; pH 9.6) was adsorbed onto a flat-bottomed, high-binding 
96-well microtiter plates (Nunc-Immuno, Thermo Fisher) and incubated for 8 hours at 4˚C. The plates were 
washed five times (0.05% Tween 20, #P1379, Sigma Aldrich; in 0.15 M NaCl solution) followed by the 
addition of 0.05 ml standard (0.47–12.00 ng/ml, Catalog #46923, Sigma Aldrich), internal control or diluted 
sample in duplicate, and then 0.05 ml horseradish peroxidase solution. Plates were washed four times after 
incubation for 2 hours at RT, and 0.1 ml ABTS solution (0.04 M ABTS diammonium salt, #0400, Amresco, 
Solon, OH; 0.5 M H2O2 #BP2633, Fisherbrand, in 0.05 M citric acid buffer, #C0759, Sigma Aldrich; pH 
4.0) was added to each well. All EIA plates were read on a microplate reader (MRX, Dynex Technologies, 
Chantilly, VA) at 405 nm (ref. 490 nm) when the optical density (OD) of the 0.00 ng/ml standard was ~1.00 
(range 0.90–1.10). The testosterone EIA was validated for capybara fecal extracts by demonstrating 
parallelism between standard curves and serially diluted fecal extracts, and recovery of testosterone 
standard added to fecal extracts. 
 
3. Results: 
3.1 Testicular Histology:  
  Gross and histologic findings of the testes at the time of castration in two capybaras showed signs 
of testicular atrophy and fertility suppression following Lupron injection. Reduced sperm production in the 
seminiferous tubules was evident in both testes from both individuals, with minimal to no sperm storage 
noted in the epididymides. Mild to moderate hyperplasia of the interstitial cells was also observed in both 
males. These findings suggest that the Lupron did significantly reduce the production of sperm in capybaras 
1 and 2.  
 
3.2 FAM Concentrations: 
Data are reported as ng/g dry feces. The intra-assay coefficients of variation (CV) between 
duplicates for all samples were <10%. Inter-assay CV for two internal controls (high and low concentration) 
were 3.52% and 6.78%, respectively (n = 13). 
FAM concentrations for each male following Lupron injection and surgical castration are depicted 
in Figure 1, with descriptive statistics summarized in Table 2. In all three males, chemical sterilization with 
Lupron resulted in an initial surge of FAM concentrations as expected. For capybaras 1 and 2, FAM 
concentrations peaked at 5375.6 ng/g and 2131.2 ng/g at 4 and 6 days post-injection, respectively. After the 
initial FAM surge, concentrations then averaged 1292.3 ng/g and 782.1 ng/g, respectively. For the intact 
capybara 3, FAM concentrations peaked at 3824.8 ng/g 1 day following Lupron injection, followed by a 
decline to a mean of 702.5 ng/g over the remainder of the study period.  
 Following surgical castration, capybaras 1 and 2 demonstrated a sustained reduction in FAM for 
the remainder of the study (Fig. 1). Post-castration FAM concentrations dropped to means of 231.6 ng/g 
and 244.3 ng/g, respectively (Table 2). Conversely, the intact capybara 3 demonstrated FAM peaks of 
1595.8 ng/g and 2131.2 ng/g at approximately 8.5 and 9.5 months post-Lupron, respectively. 
 
3.3 Final Outcomes: 
 Intermale aggression in capybaras was insufficiently reduced by chemical and surgical castration 
in this study. All three capybaras were maintained as individuals, in exhibits separated by steel pipe barriers 
which restricted physical contact, but allowed for visual, auditory, and olfactory stimuli. Despite this 
individual housing arrangement, physical barriers, and chemical followed by surgical castration, aggressive 
behavior among the males continued, characterized by posturing and aggressive encounters at the gates. 
Due to this persistent and dangerous aggressive behavior, capybara 3 was transferred to a different 
institution in April 2006, while the remaining males were housed individually until transfer to another 
holding institution in September 2009. 
 
4. Discussion: 
Noninvasive monitoring of reproductive status and condition through fecal steroid analysis is an 
effective and valuable tool for the reproductive management of exotic species. Testosterone metabolites 
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excreted in feces reflect pooled activity over several hours, and thus better reflect average concentrations 
than measures of circulating testosterone (Brown, Terio, and Graham, 1996; Schwarzenberger, 2007). In 
this study, assessing FAMs proved to be an effective means of monitoring reproductive condition in male 
capybaras. 
In all three males, the administration of Lupron caused an initial increase in FAM concentrations, 
followed by a gradual reduction. For capybaras 1 and 2, surgical castration largely resulted in persistently 
low but detectible concentrations for the remainder of the study. The residual excretion of FAM suggests 
an extra-gonadal source of androgens, possibly through production by the adrenal cortex or through 
peripheral conversion of adrostenedione (Middlebrook and Schoener, 2020). That could also account for 
the single surge in FAM in capybara one month after castration (1731.8 ng/g on 1 December 2005), or it 
may be due to sample contamination with urine. For capybara 3, which retained testes throughout the study, 
Lupron induced an initial surge and subsequent decrease similar to the other two individuals; however, this 
pattern appeared to be subject to greater fluctuation. At approximately 8.5 and 9.5 months post-injection, 
FAM concentrations of capybara 3 increased to 1595.8 ng/g and 2131.2 ng/g, respectively, comparable to 
levels prior to Lupron administration. These spikes may indicate the waning efficacy of Lupron consistent 
with a recrudescence period, and potentially a ramping up of reproductive activity in preparation for the 
breeding season.   
The male capybaras in this study demonstrated lower FAM concentrations following chemical 
sterilization with Lupron and subsequent surgical castration, but did not have sufficiently lowered levels of 
aggression, ultimately necessitating their separation. These results are comparable to earlier studies that 
demonstrated the immunocontraceptive GonaCon – an anti-gonadotropin-releasing hormone vaccine – 
effectively suppressed steroidogenesis and fertility when used in male capybaras, but secondary sexual 
characteristics and intermale aggression were preserved (Rosenfield and Pizzutto, 2019; Rosenfield et al., 
2019). Similarly, Lupron did not extinguish agonistic behaviors in male capybaras under the specific 
circumstances and conditions in our study.  
The efficacy of GnRH agonists in mitigating unwanted aggression in conspecifics has been 
explored in a variety of other wildlife species. Deslorelin appears to be efficacious in curtailing interspecific 
aggression, especially in carnivores, but this pattern does not hold true for all taxa (Bertschinger  et al., 
2001; DeCaluwe, Wielebnowski, Howard, Pelican, and Ottinger, 2016; Harley, Power, and Stack, 2018; 
Molter, Fontenot, and Terrell, 2015; Norton et al., 2000; Penfold et al., 2002; Raines and Fried, 2016; 
Rowland, 2011; Vinke, van Deijk, Houx, and Schoemaker, 2008). Lupron in particular had promising 
results in suppressing musth-associated behaviors in an Asian elephant bull (de Oliveira, West, Houck, and 
Leblanc, 2004), but had minimal to no effect on aggression in collared lemurs and rut-associated behaviors 
in red deer (Barrell, Schaafsma, Ridgway, Wellby, and Miller, 2009; Ferrie, Becker, Wheaton, Fontenot, 
and Bettinger, 2011). Likewise, the success of surgical castration in eliminating intraspecific aggression 
can be variable and unpredictable in both wildlife and domestic species (Demas, Moffatt, Drazen, and 
Nelson, 1999; Farhoody et al., 2018; Ferrie et al., 2011; Garde, Pérez, Vanderstichel, Dalla Villa, and 
Serpell, 2016; Takeshita, Huffman, Kinoshita, and Bercovitch, 2017).  
As studied extensively in domestic dogs, the expression of aggressive behaviors in males is not 
solely testosterone-driven and can be influenced by additional factors; thus, the effects of chemical or 
surgical sterilization may be challenging to predict (Farhoody et al., 2018; Garde et al., 2016; Neilson, 
Eckstein, and Hart, 1997). The apparent age-dependent expression of androgen-driven behaviors (including 
aggression directed towards conspecifics) suggests that testosterone may play a role in agonistic behaviors 
that become learned with age (as reviewed in Goericke-Pesch, 2017). These learned behaviors may then 
persist despite the removal of hormonal drivers. Capybara males have a low gonadosomatic index and high 
proportion of non-spermatogenic testicular tissue, suggesting heavy investment in androgen production 
(Moreira et al., 1997; Costa and Paula 2006; Paula and Walker, 2013). It has been speculated that this 
reproductive strategy emphasizes chemical signaling and behavioral maintenance of social status over 
sperm output  (Herrera and Macdonald, 1993; Moreira et al., 1997). Interestingly, the presumed dominant 
male (Capybara 3) did not achieve the highest FAM concentrations pre- or post-intervention, although there 
appear to be a lot of intrinsic variability. This could suggest that he was not actually dominant, or that other 
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factors not captured here determine dominance, but monitoring over a longer period pre-intervention might 
elucidate more consistent patterns.    
Unfortunately, behaviors were not formally quantified via an ethogram or other methods in this 
study, so it is possible that changes in aggression levels occurred but were not perceived. However, the 
priority objective was to minimize harm, and address ethical concerns given the severity of aggression and 
serious risk of injury to all capybaras. For subsequent or similar studies in these or other species, rigorous 
objective measurement of behaviors would be essential to determine the effects of these interventions on 
conspecific aggression. 
Under the specific set of circumstances in this study, a single Lupron injection did not sufficiently 
suppress aggression in a bachelor group of male capybaras with established social antagonism. However,  
this study alone does not allow for conclusive determination of the efficacy of Lupron. For future attempts 
to maintain bachelor groups of capybaras, strong consideration should be given to age and timing of 
chemical interventions. In this study, the three males had already reached sexual maturity and developed 
agonistic behaviors prior to veterinary intervention. It is possible that chemical or surgical castration of 
capybaras at a younger, pre-pubertal age to prevent rather than treat aggression may have more favorable 
effects on group dynamics. However, it should also be noted that age is often not a reliable predictor of the 
effects of castration on problem behaviors (Neilson et al., 1997). Additionally, with no published guidelines 
for Lupron dosing at the time that these interventions were undertaken, it is uncertain whether higher or 
repeated doses might have produced greater suppression of intermale aggression. The use of deslorelin 
implants or other GnRH agonists for the control of problem behaviors also should be explored under more 
ideal conditions before definitive conclusions are drawn on efficacy for the control of aggressive behaviors.  
 
5. Conclusion: 
While contraception via single-dose Lupron and surgical castration had observable effects on 
excretory FAM concentrations in a group of three male capybaras, they did not abolish the expression of 
secondary sexual characteristics or agonistic interactions in this group. Subsequent studies and attempts at 
housing males together should consider earlier pre-pubertal interventions prior to the onset of conspecific 
aggression, and the addition of in study design for objective measurement of behavioral effects. Due to the 
possibility of persistent intermale aggression and risk of serious injury, caution is advised when housing 
capybaras in bachelor groups.  
 
Acknowledgements: 
 The authors would like to thank the animal care and husbandry team at Smithsonian National 
Zoological Park’s Elephant Trails for participating in behavioral observations and collection of samples. 
Thanks also to the veterinary team, especially Drs. Carlos Sanchez, Sharon Deem, and Ellen Bronson, who 
served as primary clinicians. Further thanks are due to pathologist Dr. Tabitha Viner. Additional support 
for this project and manuscript preparation was provided by the Smithsonian Institution, the Morris Animal 
Foundation and Dennis and Connie Keller through a training partnership, and James and Jamie Coss. This 
content has not been reviewed or endorsed by the Morris Animal Foundation, and the views expressed 
herein do not necessarily reflect the views of the Foundation, its officers, directors, affiliates, or agents. 
 
Data Availability Statement 
The data that support the findings of this study are available from the corresponding author, JHY, 
upon reasonable request. 
 
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Table Legends: 
 
Table 1. Summary of adult male capybaras (Hydrochoerus hydrochaeris) treated with Lupron (n=3) and 
subsequently castrated (n=2). 
 
Table 2. Summary of fecal androgen metabolite (FAM) concentrations (ng/mg dried feces) for 
approximately 1 week before and 5-7 weeks after injection of Lupron in three male capybaras, followed by 
subsequent surgical castration in two of the males. 
 
Figure Legend: 
 
Figure 1. FAM concentrations as determined by enzyme immunoassay for capybara bachelor group, 
February through December 2005. Dates of Lupron injection are denoted with (*), while surgical castration 
dates for Capybaras 1 and 2 are indicated by (†).  
8