Aggression Control in a Bachelor Herd of Fringe-Eared Oryx (Oryx ...

Zoo Biology 20:375–388 (2001)

Aggression Control in a Bachelor Herd of Fringe-Eared Oryx (Oryx gazella callotis), with Melengestrol Acetate: Behavioral and Endocrine Observations M.L. Patton,1* A.M. White,1 R.R. Swaisgood,1 R.L. Sproul,1 G.A. Fetter,1 J. Kennedy,2 M.S. Edwards,1 R.G. Rieches,2 and V.A. Lance1 1

Center for Reproduction of Endangered Species, Zoological Society of San Diego, San Diego, California 2 San Diego Wild Animal Park, Zoological Society of San Diego, Escondido, California

Aggression control is becoming an important component in the management of animals in captivity, but rigorous quantification of aggressive behavior has heretofore been lacking. This study was done to assess the ability of melengestrol acetate (MGA) given with feed (1.54 mg/kg) to control aggression in a bachelor group of fringe-eared oryx (Oryx gazella callotis). Systematic behavioral observations were conducted and fecal androgen content was measured for 42 and 90 days, respectively, before treatment, and during the 42 days of treatment. There was a significant reduction in concentrations of fecal androgen from 153 ± 6.0 to 95 ± 4.5 ng/g (T66 = 7, P < 0.0001). This reduction in androgen excretion was apparent after the first week of treatment. There was measurable MGA excreted in the feces during treatment. Although treatment did not arrest all aggressive behaviors among animals, the decline in androgens and increase in MGA was accompanied by a significant reduction in several measures of agonistic behavior. Posturing, aggressive contact, pursuit, and submission occurred significantly less frequently after treatment, and there was also a reduction in fighting-intention movements. Thus, both ritualized and nonritualized aspects of aggression were affected. Reductions in hormones and aggressive behaviors coincided temporally, suggestive of a potential causal relationship. Consistent with this hypothesis is a strong positive correlation between fecal androgen and total aggressive acts. This effect was not the result of a single behavioral element but occurred across several categories of agonistic behavior. Zoo Biol 20:375–388, 2001. © 2001 Wiley-Liss, Inc. Key words: melengestrol acetate; aggression control; antelope

*Correspondence to: M.L. Patton, Center for Reproduction of Endangered Species, Zoological Society of San Diego, San Diego, California 92112. E-mail: [email protected] Received for publication June 4, 2001; Accepted August 6, 2001.

© 2001 Wiley-Liss, Inc.

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INTRODUCTION

In male ungulates, it has been suggested that there is a causal link between aggressive behavior and androgens [Bouissou, 1983]. A synthetic progestogen, melengestrol acetate (MGA), originally developed as an orally active contraceptive [Kirk et al., 1962], has been reported to decrease aggression in three ungulate species. Silastic implants containing MGA reduced intraspecific aggression in bachelor herds of scimitar-horned oryx, Oryx dammah [Blumer et al., 1992]. Male muntjacs (Muntiacus reevesi) fed MGA had significantly decreased sperm numbers, and anecdotal reports of behavior indicated reduced aggression [Stover et al., 1987]. Orally administered MGA reduced aggression during the early rut in farmed fallow bucks (Dama dama) [Wilson et al., 2000]. None of these studies, however, systematically quantified aggression before and after MGA administration, leaving the question of efficacy unresolved. The fringe-eared oryx (Oryx gazella callotis), an east African subspecies of the gemsbok (Oryx gazella), is a horselike antelope that lives in the harsh arid environments of east Africa. This species has no particular breeding season. Most frequently, territorial males breed females in their postpartum estrus. Herds of bachelor males are seldom found [Price, 1986; Wacher, 1988]. In captivity, however, it is sometimes necessary to hold males in all-male groups. Elevated aggression in this unnatural situation can lead to injuries and even death of conspecifics [Blumer et al., 1992], especially during periods of social disruption. Methods to control aggression have thus become an important element in the keeping of wild ungulates in captivity. The purpose of this study was to assess the use of a feed containing MGA for the control of aggression in a bachelor group of fringe-eared oryx. Systematic behavioral observations were conducted to evaluate aggressive behaviors, and fecal androgen and MGA were measured to monitor a physiological response. MATERIALS AND METHODS Animals and Treatment

Four adult male fringe-eared oryx (Oryx gazella callotis) were housed at the San Diego Wild Animal Park (SDWAP), San Pasqual, CA in an enclosed 1.14-acre area out of public view. They were together for more than 1 year before the onset of this study. The ages ranged from 1.7 to 7.3 years at the beginning of the study. The Arabian oryx (O. leucoryx), the Arabian counterpart to the fringe-eared oryx, is reported to reach puberty at 7 months of age [Ancrenaz et al., 1998], and we estimate a similar age for puberty in the fringe-eared oryx. Before and during the first 6 weeks of the study, each animal was fed an estimated 2,000 g high-fiber herbivore (1/2′) pellets (O.H. Kruse Grain & Milling, Ontario, CA), 887 g Sudan grass hay and 887 g coastal Bermuda grass hay daily based on the daily group diet. All animals had access to water, trace mineral, and plain salt blocks. Six weeks after the initiation of the study, the oryx diet remained the same except for the substitution of high-fiber herbivore pellets (11/64′) (Central Soya, Fort Wayne, IN) that contained melengestrol acetate (17-hydroxy-6-methyl-16-methylenepregna-4,6diene-3,20-dione acetate; MGA; The Upjohn Co., Kalamazoo, MI). Each kilogram of feed contained 1.54 mg MGA. Each animal weighed approximately 170 kg and was assumed to consume 90% of the daily pellet ration. It was thus calculated that

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each animal received 2.77 mg/day or 0.016 mg/kg body weight/day. Body mass was based on an average obtained from immobilization records from more than 25 males at the SDWAP. Fifty days after the medicated diet commenced, two adult male fringe-eared oryx (both: age 1.7 years), which had received the medicated diet for the previous 18 days, were added to the group. Seventy-one days after this introduction, the medicated feed was discontinued in all animals. Therefore, the original four animals received the drug for 122 days, and the two additional animals received the medicated diet for 88 days. Fecal Collection and Extraction

Fecal samples from the four oryx were collected approximately every other day from several fecal piles, pooled into 30-mL plastic sample cups (Starstedt Inc., Newton, NC), and stored at –20°C. Fecal samples therefore reflected a pooled average of the study group. Sample collection began 90 days before treatment and continued for 50 days during the treatment of 4 males. Fecal collection continued after the introduction of two additional males and continued for an additional 30 days after treatment. Samples were lyophilized for 48 hours in a Flexi-Dry microprocessor manifold lyophilizer (FTS Systems, Inc., Stone Ridge, NY) to reduce variability in water content. Pellets were pulverized using a mortar and pestle. A 0.1 g sample was placed into a 16 × 150-mm glass tube for extraction. Diethyl ether anhydrous (5 mL; Mallinckrodt, Paris, KY) was added to each tube, vortexed (2 minutes), and flash frozen in a methanol:dry ice bath. The supernatant was poured into 12 × 75-mm culture tubes and allowed to evaporate in a water bath (37°C). The dried ether extract was then resolubilized in 1 mL absolute ethanol. Immunoassays Androgens

Testosterone content was analyzed in the fecal extracts by radioimmunoassay (RIA) using an antibody produced against testosterone 19-carboxymethylether:bovine serum albumin at a working dilution of 1:12,000 and a final dilution of 1:84,000. This antibody was characterized to cross react 100% with testosterone, 18.75% with 5α-dihydrotestosterone, 3.00% with 5α-androstane-3α, 17β-diol, and 1% with 5-androstene-3β, 17β-diol. Other hormones tested were found to cross react less than 1.00%. These were androstenedione, 5α-androstane-3, 17-dione, 5β-androstane-3, 17-dione, androsterone, estradiol-17β, 11β-hydroxy-androstenedione, progesterone, dihydroepiandrosterone, corticosterone, desoxycorticosterone, estriol, and estrone (ICN, Costa Mesa, CA). Tritiated testosterone (10,000 cpm/0.1 mL, Dupont, NEN, Boston, MA) was used to compete against standard testosterone (7–1,000 pg, Sigma, St. Louis, MO). Ten microliters of ethanolic fecal extract was diluted 1:100 in 0.1 M phosphate-buffered saline pH 7.0 (PBS) and 250 µL of this diluent was assayed in duplicate. After an overnight incubation at 4°C, the competitive reaction was terminated by the addition of 0.25 mL of charcoal dextran solution (6.25 g charcoal:0.625 g dextran in 1.0 L PBS) to separate bound from free hormone. The charcoal treated samples were held for 30 minutes at 4°C and then were centrifuged at 1,500g at 4°C for 15 minutes. The supernatant was decanted into scintillation vials, and scintillation fluid (5 mL, Ultima Gold, Packard Instrument, Meriden, CT) was added and counted for 2 minutes in a Beckman liquid scintillation spectrometer (LS 6500).

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MGA

An enzyme immunoassay (EIA) kit for the quantitative analyses of MGA (RBiopharm GmbH, Darmstadt, Germany) was used to assay fecal extracts. The kit was developed for measuring MGA in tissue and was modified for assay of fecal extracts. After sample extraction (see above), 20 µL of the extract were evaporated, reconstituted in 250 µL methanol:distilled water (40:60), and 10 µL was taken to the assay. Absorbance was measured at 450 nm against an air blank using an Emax precision microplate reader (Molecular Devices, Sunnyvale, CA). High-Pressure Liquid Chromatography Androgens

Reverse-phase high-performance liquid chromatography (HPLC) (Ultra Sphere C-18 Column; Beckman, San Ramon, CA) was used to characterize the immunoreactive fecal androgen metabolites. Tritiated testosterone (2,800 cpm) was added to pooled extracts of samples both before and after treatment and was analyzed in separate HPLC runs. Samples were first evaporated and then reconstituted in 20 µL 100% methanol (Optima grade, Fisher, Fair Lawn, NJ). Androgen metabolites were separated using isocratic methanol and distilled water (30:14) with 0.2 M potassium phosphate buffer, pH 5.35. Fractions were collected at a rate of 1 mL/min for 40 minutes, evaporated, and reconstituted in 500 µL PBS buffer. An aliquot (100 µL) of each was taken and counted in the LS 6500 to assess the elution profile of the reference 3H testosterone. Cross-reactivity against the androgen antibody was tested in each fraction by RIA. MGA

Past research in which radioactive testosterone was administered to cross-bred steers, and radioactive MGA administered to heifers demonstrated, in each case, that most of the label is excreted in the feces [Gassner et al., 1960; Krzeminski et al., 1981]. Therefore, pooled samples from pretreated and treated MGA fecal extracts were analyzed by HPLC. The protocol for HPLC differed from the androgen protocol only in the ratio of methanol to distilled water (30:10). Fractions were collected at a rate of 1 mL/min for 30 minutes. Each fraction was evaporated and reconstituted in 250 µL of methanol: distilled water (40:60) and MGA content was quantified by EIA. Retention times of known standards were used to identify peaks in the eluate. Behavioral Data Collection

Systematic behavioral observations were made during the last 2 hours of daylight (a period of peak activity) during the months of September through December 1999. Individual animals were recognized by natural differences in horn shape or pelage markings. Data were collected on the four study animals for 6 weeks before and 6 weeks after the commencement of the treatment. Behavioral data were not collected after the introduction of the two males or after removal of the medicated diet. However, keeper observations were noted. This study used “behavior sampling” methods, in which a group of animals is continuously observed for all occurrences of particular behaviors [Martin and Bateson, 1986]. Bouts of agonistic behaviors (aggressive or defensive social interaction) were recorded each time they occurred. Behavioral bouts are separated by 5 seconds. The ethogram was adapted from Walther [1979] as follows: “Posture” included ritualized displays, such as erect stance and


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horn threats that serve to accentuate either the overall body size or the length of the animal’s horns. “Fighting Intention” movements included symbolic gestures where an attack movement is simulated with no contact accompanying the motion. “Pursue” included chasing or continued direct approach of another animal after it had already shown submission. “Contact” included all aggressive contact in which an animal’s horns struck another animal’s body. “Submission” was recorded when an animal performed a defensive head-low posture or fled from another animal. “Total Aggressive Acts” included behaviors exhibited in all of the above behavioral categories, excluding Submission. In addition to social interaction behaviors, urination, defecation, and defecation posture (an indicator of dominance) were also recorded. At the end of each 2-minute interval, each animal was recorded as “Resting,” “Feeding,” “Agonistic,” or “Active.” In addition, an animal was scored as “Social” if it was within two body lengths of another animal. Data Analyses

For statistical purposes, each daily fecal sample representing the entire study group was the unit of analysis. Because these samples are not independent, we limit inferences from these results only to the original four animals, not the larger population. To test for hormonal differences between treatments, paired t-tests were used. Given that it was expected that androgen levels would decrease with MGA treatment, a simple regression was used to determine the relation between fecal androgens and MGA values. Simple regressions were also used to determine the relationship between hormone levels and the number of days on treatment. Spearman rank correlations were used to determine the relationship between fecal androgens and agonistic behaviors. In scimitar-horned oryx (O. dammah), a lag time of approximately 4 days exists between changes in circulating progesterone and the appearance of fecal pregnanes [Shaw et al., 1995]. Assuming a similar time for testosterone excretion, hormone levels in fecal samples were paired with behavior samples collected 4 days prior. For social interactions, data for each actor–recipient dyad was averaged for each of the treatment phases, so that each actor–recipient dyad contributed only two observations to avoid data pooling [Machlis et al., 1985]. Wilcoxon signed-rank tests for related samples were used to test for treatment effects. All reported values were corrected for ties. RESULTS Fecal Extraction, RIA, and EIA

Serial dilution of fecal extract yielded displacement curves parallel to those obtained with the testosterone standard (r = 0.997). Extraction efficiency of added tritiated testosterone was 53.4 ± 2.1% (mean ± SD, n = 10). Assay sensitivity was 8.87 pg/tube (calculated as mean pg/tube at 90% B/BO, n = 6). Buffer blanks were below the assay sensitivity. Accuracy was determined as 94.97 ± 6.57 (mean ± SD) by recovery of seven known quantities of standard (7.8–500 pg) that were equivalent to the quantities used in the standard curve added to a pool of fecal extract. A diluted fecal sample from a study female was used for this pool that contained an immunoreactive content just above the sensitivity of the assay. Interassay coefficients of variation (% SD/mean, n = 6) were 14.06% based on duplicates of a rhinoceros fecal

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pool with an immunoreactive content that yielded a %B/BO >80%, and 9.67% immunoreactive content that yielded a %B/BO >40%. Intraassay variation estimates (10 replicates of the same pools in a single assay) were 7.8% for the high pool and 7.7% for the low pool. Results are presented as nanograms per gram (equal to nanograms per gram dry fecal weight). HPLC-separated fractions of fecal testosterone metabolites showed an immunoreactive peak at fraction 15 minutes (Fig. 1; Peak III) that coeluted with 3H-testosterone followed by dihydrotestosterone at fraction 18 minutes (Peak IV). The testosterone peak accounted for 19% of the total immunoreactivity, and the dihydrotestosterone accounted for 23% in the fecal extract. Four additional unidentified peaks that cross reacted with the antibody in the RIA were noted at fractions 7 minutes (Peak I), 11 minutes (Peak II), 25 minutes (Peak V), and 31 minutes (Peak VI). Serial dilution of fecal extract from animals treated with MGA yielded a displacement curve parallel to that obtained with the MGA standard (r = 0.9878). The interassay coefficients of variation (% SD/mean, n = 6) were 17.7% based on a high control and 3.7% based on a low control. HPLC retention times of melengestrol and MGA were 11.5 (Fig. 2; Peak II) and 16.6 minutes (Peak IV), respectively. Separated fractions from a sample derived from animals receiving MGA showed immunoreactive peaks corresponding to these retention times. The peaks representing MGA and melengestrol accounted for 44% and 28%, respectively, of the immunoreactivity in the fecal extract. Three unidentified minor peaks (fractions 7, 14, and 27) were also present. An HPLC profile from a sample obtained from animals before receiving MGA showed no immunoreactive peaks (Fig. 2.).

Fig. 1. Immunoreactive androgens after high-performance liquid chromatography (solid circles). Peak III coeluted with 3H-testosterone (open squares). Peak IV corresponds to the retention time of dihydrotestosterone. Peaks I, II, V, and VI are four unknown steroids that cross react with the testosterone antibody.


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Fig. 2. Immunoreactive melengestrol acetate metabolites after high-performance liquid chromatography. Open squares represent fecal assays during treatment and solid circles, assays pretreatment. Peaks II and IV correspond to retention times of melengestrol acetate and melengestrol, respectively. Peaks I, III, and V are three additional unidentified compounds which cross-react with the melengestrol acetate antibody.

Pre- vs. During-Treatment Comparisons

The original four animals demonstrated a decrease in androgen levels in fecal samples after treatment with MGA (t66 = 7.0, P = 0.0001; Fig. 3) paralleled by an increase in fecal MGA concentration (t66 = 15.0, P = 0.0001; Fig. 3). Although treatment did not arrest all aggressive behaviors between animals, the decline in testosterone was accompanied by a significant reduction in several measures of agonistic behavior (Table 1). Posturing, Contact, Pursue, and Submission occurred significantly less frequently after treatment, and there was also a nonsignificant reduction in Fighting Intention movements. Thus, both ritualized and nonritualized aspects of aggression were affected. Reductions in hormones and aggressive behaviors coincided temporally (Fig. 4). Consistent with this hypothesis is a strong positive correlation between androgens and total aggressive acts (z = 3.2, rho = 0.63, P = 0.001). This effect was not the result of a single behavioral element, but occurred across several categories of agonistic behavior. Pursue (rho = 0.48, z = 2.4, P = 0.02), Fighting Intention (rho = 0.52, z = 2.6, P = 0.008) and Submission (rho = 0.59, z = 3.0, P = 0.0037) all showed significant positive correlations with androgens, whereas positive correlations with Posture (rho = 0.26, z = 1.3, P = 0.19) and Contact (rho = 0.31, z = 1.6, P = 0.12) did not attain statistical significance. Limited sample size precluded statistical analysis of the effects of MGA on noninteractive behaviors. These behaviors were averaged for all study animals, and those that changed pre- to posttreatment by more than 25% are reported later herein.

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Fig. 3. Changes in fecal androgen (shaded columns) and melengestrol acetate (MGA) (open columns) metabolites associated with time before MGA was fed and during the time MGA was fed. Bars represent standard errors.

Behaviors that increased after MGA treatment included percent time Active (X ± SE = 8.4 ± 2.9% vs. 10.9 ± 1.2%) and percent time Feeding (7.0 ± 1.3% vs. 11.5 ± 2.8%). Percent time engaged in Agonistic behaviors was higher before (6.8 ± 2.7%) than after (2.1 ± 0.8%) treatment. Behaviors that did not appear to be affected by the hormonal treatment included urination, defecation, defecation posture, and percent time Social. Introduction of Two Males and Cessation of MGA Treatment

After the end of the aforementioned study, two additional males were added to the study group of four. Although no behavioral observations were made, analysis of androgen in pooled fecal samples demonstrates a significant decline in androgen levels after addition of the two males (t55 = 8.0, P = 0.0001) (see Discussion). SevTABLE 1. Treatment effects on behavioral measures of aggressiona Behavior

Pretreatment

Posttreatment

T+

N

P

Posturing Fighting Intention Contact Pursue Submission Total Aggression

0.10 ± 0.05 0.07 ± 0.04 0.08 ± 0.03 0.21 ± 0.11 1.01 ± 0.48 0.46 ± 0.21

0.04 ± 0.02 0.03 ± 0.01 0.02 ± 0.01 0.09 ± 0.05 0.50 ± 0.21 0.18 ± 0.08

35 19 44 21 46 64

8 7 9 6 10 11

0.008 0.234 0.004 0.016 0.032 0.002

Values presented as mean ± SE, as calculated from average values from actor–recipient dyads. P values are Wilcoxon signed-rank tests for related pairs: T+ is the test statistic, N is the sample size. Sample size variation is caused by exclusion of ties. a


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Fig. 4. Solid squares represent mean androgen concentrations (±SE) of all samples collected during a given week. Shaded bars represent mean aggressive acts (±SE), as calculated from daily averages of the total number of aggressive acts across all subjects.

enty-one days later, MGA was discontinued, which was associated with a significant elevation in androgen values (t45 = 8.0, P = 0.0001) and decrease in MGA values (t41 = 8.0, P = 0.0001, Fig. 5). It was also of interest to evaluate whether MGA continued to produce further reductions in androgens throughout treatment. Androgen levels decreased significantly with increasing number of days on treatment (R2 = 0.384, F = 34.3, P < 0.0001, Fig. 6A), suggesting that MGA effects are strengthened as treatment duration increases. In contrast, MGA in fecal extracts did not increase across treatment period (R2 = 0.001, F = 0.05, P = 0.83, Fig. 6b). In two separate experiments, estimates of the half-life of MGA were 1.2 and 1.9 days based on radioactivity in excreta samples from heifers fed labeled MGA (per communication, Dawn A. Merritt, Ph.D., Pharmacia & Upjohn Animal Health, Kalamazoo, MI, August 2000), suggesting that it does not build up in the system. However, some may remain in muscle and other tissue. Androgen levels were negatively correlated with fecal MGA values (F1.109 = 25.6, R2 = 0.19, P = 0.0001), suggesting there is not substantive build up of MGA in fringe-eared oryx. DISCUSSION

Our results demonstrate that male fringe-eared oryx fed MGA had reduced fecal androgens associated with a concomitant decline in all forms of agonistic behavior, including both ritualized and escalated aggression. Although other studies have suggested such an effect of MGA on ungulate species [Stover et al., 1987; Blumer et al., 1992; Wilson et al., 2000], this study is the first to quantitatively document the drug’s behavioral effects on aggression. In this light it is interesting to note that the

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Fig. 5. Changes in fecal androgen (shaded columns) and melengestrol acetate (MGA) (open columns) metabolites during the treatment with MGA and post MGA. Bars represent standard errors.

effects appear to mediate aggression at all levels, ranging from symbolic posturing to escalated and injurious contact, suggesting specific motivational effects, rather than a general effect on activity level. Also of interest is the observation that dominance rank remained unchanged by the treatment (unpublished data). This observation suggests that dominance-related behavior and aggressiveness are to some extent under independent control [cf. Dixson, 1980]. Although behavioral data are not available, testosterone levels rebound after removal of MGA-treated feed, indicating that the effects of the drug are temporary and reversible. If these results hold true in subsequent studies, MGA could become an effective tool for mediating aggression and injuries in zoological settings. We also show that MGA may be measured in feces using an EIA kit developed for the quantitative analysis of MGA in bovine perirenal fat and muscle meat. The MGA assay showed some cross-reactivity to unidentified antigens in fecal samples of untreated oryx. These cross-reacting fecal antigens could be steroid metabolites, but their identity remains unknown and levels were not significant enough to impair the observation of the drug in post-treated samples. The manufacturer of the MGA EIA kit indicates a cross-reactivity of 3.9% for 17α-acetoxyprogesterone and less than 0.01% for 17α-methylprogesterone. Limited data exist on the mechanism by which MGA may exert its effect on males. Progestogens act as antagonists to androgens [Knowl and Egberink-Alink, 1989], and bulls receiving supplemental progesterone exhibit disrupted spermatogenesis, indicating inhibited testosterone secretion [Matsuyama et al., 1967]. When MGA is fed to domestic bulls, surges of luteinizing hormone (LH) and testosterone are


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Fig. 6. A: Changes in fecal androgen levels associated with number of days on melengestrol acetate (MGA) treatment (R2 = 0.384, F = 34.3, P < 0.0001). B: Changes in fecal MGA levels associated with number of days on MGA treatment (R2 = 0.001, F= 0.05, P = 0.83).

abolished on the second day of treatment. Testes of these bulls respond normally to LH stimulation, indicating that MGA blockage of testosterone secretion was through inhibition of LH release and not directly on the testes [Haynes et al., 1977]. Decreased agonistic behaviors observed in the fringe-eared oryx were probably caused by the decrease in androgens. Several lines of evidence suggest, but do not demonstrate, that such a causal relationship between MGA, androgen, and aggression exists. First, a decline in androgen and agonistic behavior coincided temporally. Second, correlations demonstrate that these hormonal and behavioral factors covary. Third, the reduction in agonistic behavior is not merely a side effect of reduced activity, because activity levels actually increased slightly after administration of MGA. Bouissou [1983] likewise argued for a causal relationship in a review of literature between androgen levels and aggressive behavior in male ungulates. A related six-methyl progestin, medroxyprogesterone acetate (MPA) decreases deviant behaviors in male sex offenders [Money, 1970], possibly because of its ability to reduce circulating testosterone. Increased metabolic clearance rate, decreased circulating gonadotropins, and inhibited testicular steroidogenesis have been suggested as

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mechanisms for MPA-associated reduction of testosterone in rats and/or humans [Barbieri and Ryan, 1980]. MGA may exert its effect on aggression through similar pathways in ungulates. Reduced androgens may not be the sole hormonal pathway mediating the effects of MGA on aggression in the present study. For example, other studies have suggested alternative mechanisms. In primates, behavioral effects of MPA may be mediated by brain mechanisms regulating sexual motivation that are relatively independent of circulating androgen levels [Michael et al., 1991]. Corticosteroids have also been implicated in regulating aggression [Knowl and Egberik-Alink, 1989], and some studies indicate that MGA acts biologically first as a progestogen and secondarily as a glucocorticoid [Lauderdale, 1983]. For example, Holstein heifers fed MGA had reduced adrenal weights, plasma cortisol, and adrenal cortisol and corticosterone concentrations when compared to controls [Purchas et al., 1971]. Atrophy of the adrenal cortex and lowered levels of circulating corticosterone were also found in rats given extremely high doses of MGA to inhibit tumor growth [Padilla et al., 1990]. Finally, reduced aggression may be a consequence of a progestational anesthetic effect [Gyermek, 1967], although this is an inadequate explanation for our results given that activity levels did not decrease after treatment. It is not clear precisely what dosage of MGA is adequate to produce the results we found for fringe-eared oryx. Subjects in our study received a higher daily dose of MGA (2.77 mg) than the dose found to be effective in reproductive cycle inhibition of domestic cows (0.5 mg) [Zimbelman et al., 1970]. The concentration of MGA the oryx received was determined on the basis of previous studies in which effective contraception was achieved for a variety of species [Raphael et al., 1992; Patton et al., 2000]. A lower dose (0.1 mg/head/day) appeared to decrease aggression in farmed fallow bucks but no decrease in sperm production when compared to controls [Wilson et al., 2000]. As in fallow bucks, when muntjac (Muntiacus reevesi) were treated with MGA (0.02– 0.3 mg/day), casual observation of behavior indicated reduced aggression. However, decreased sperm production was noted [Stover et al., 1987]. It is not known whether spermatogenesis was affected in the MGA-treated fringe-eared oryx. Finally, it should be noted that MGA need not be used permanently in many situations to mediate potentially injurious levels of aggression. Periods of social disruption, especially when new animals are added to an existing group, represent special managerial challenges because of the elevated aggressive interactions associated with social change. Behavioral management, such as giving access to new members through a fence before introduction to the group, is sometimes insufficient to avoid injurious aggression, forcing managers to seek innovative solutions. In the past, zoo managers have encountered significant difficulties when trying to form new all-male groups of ungulates of several species, and social disruption has led to serious injury [Blumer et al., 1992]. These risks provided the initial impetus for our test of MGA efficacy for aggression reduction. Our first experiment documented a significant decline in aggression and androgens after MGA treatment. Although we do not have baseline quantitative data for social introductions in untreated animals, animal caregivers monitored the introduction of the two males to the existing group of four males and reported no serious aggression. This stands in contrast to previous introductions that resulted in escalated aggression. Thus, it is reasonable to assume that prior treatment of all six animals served to modulate aggressive levels such that typical escalation did not take place.


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We should also note that the introduction period was associated with a significant decline in androgens (among the six males) compared to androgen levels in the four males before introduction. We do not have a ready explanation for this result, but offer two possibilities: 1) the two males that were introduced had lower androgen levels, bringing down the group mean; or 2) the cumulative effects of MGA treatment continued to produce a further reduction in androgen levels. Casual observations of these two males suggest subordinate status, providing support for the first hypothesis. The second hypothesis, however, is supported by the fact that androgen levels continued to decrease progressively throughout the duration of the treatment. Regardless, our results suggest a promising role for MGA in mediating aggression in all male groups of ungulate species. We encourage more tests to evaluate this drug’s efficacy across other species and situations. ACKNOWLEDGMENTS

We thank the behavioral observers, in particular Antimone Rowley, Dana Peimann, Sonia Quiett, the animal keepers at the SDWAP for conscientious collection of fecal samples, Sam Baker for organizing distribution of the medicated diet, R-Biopharm GmbH, Darmstadt Germany for providing MGA enzyme immunoassay (EIA) kits, Dawn A. Merritt, Ph.D., Pharmacia & Upjohn Animal Health, Kalamazoo, MI, for supplying MGA standard, and Dr. Lee Hagey for help with HPLC. REFERENCES Ancrenaz M, Blanvillain C, Delhomme A, Greth A, Sempéré AJ. 1998. Temporal variations of LH and testosterone in Arabian oryx (Oryx leucoryx) from birth to adulthood. Gen Comp Endocrinol 111:283–9. Barbieri L, Ryan KJ. 1980. Direct effects of medroxy-progesterone acetate (MPA) and megestrol acetate (MGA) on rat testicular steroidogenesis. Acta Endocrinol 94:419–25. Blumer ES, Plotka ED, Foxworth WB. 1992. Hormonal implants to control aggression in bachelor herds of scimitar horned orxy (Oryx dammah): a progress report. In: Proceedings, Joint Conference American Association of Zoo Veterinarians and the American Association of Wildlife Veterinarians. Oakland, California. p 212–6. Bouissou MF. 1983. Androgens, aggressive behaviour and social relationships in higher mammals. Horm Res 18:43–61. Dixson AF. 1980. Androgens and aggressive behavior in primates: a review. Aggressive Behav 6:37–67. Gassner FX, Martin RP, Shimoda W, Algeo JW. 1960. Metabolism of radioactive steroid esters in the bovine male and female. Fertil Steril 11:49–73. Gyermek L. 1967. Pregnenolone: a highly potent, naturally occurring hypnotic-anesthetic agent. Proc Soc Exp Biol Med 125:1058–62. Haynes NB, Kiser TE, Hafs HD, Marks JD. 1977. Prostaglandin F2α overcomes blockade of episodic LH secretion with testosterone, melengestrol acetate or aspirin in bulls. Biol Reprod 17:723–8.

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