Successful acclimatization of mandrills (Mandrillus sphinx) translocated to Conkouati-Douli National Park, Republic of Congo, as measured by fecal glucocorticoid metabolites.

Translocation and reintroduction are common tools in conservation management and can be very successful. However, translocation can be stressful for the animals involved, and stress is implicated as a major cause of failure in release programs. Conservation managers should therefore seek to understand how the stages of translocation impact stress physiology in the animals involved. We quantified fecal glucocorticoid metabolites (fGCMs) as a noninvasive measure of response to potential stressors during a translocation of 15 mandrills (Mandrillus sphinx) into Conkouati-Douli National Park, Republic of Congo. The mandrills were initially housed in a sanctuary, transferred to a pre-release enclosure in the National Park and then released into the forest. We collected repeated fecal samples (n = 1101) from known individuals and quantified fGCMs using a previously validated enzyme immunoassay. Transfer from the sanctuary to the pre-release enclosure correlated with a significant 1.93-fold increase in fGCMs, suggesting that transfer was a stressor for the mandrills. fGCM values decreased over time in the pre-release enclosure, suggesting that the mandrills recovered from the transfer and acclimatized to the enclosure. Release to the forest was not linked to a significant increase in fGCMs over the final values in the enclosure. Following release, fGCMs continued to decrease, fell below sanctuary values after just over a month and were about half the sanctuary values after 1 year. Overall, our results suggest that the translocation, although initially presenting a physiological challenge to the animals, was not detrimental to the well-being of the animals over the timescale of the study and, in fact, may have been beneficial. Our findings show the value of non-invasive physiology in monitoring, evaluating and designing wildlife translocations and, ultimately, contributing to their success.

Outcomes from an anti-gonadotropin-releasing hormone immunotherapeutic trial in large flying foxes (Pteropus vampyrus).

OBJECTIVE: The anti-GnRH immunotherapeutic product Improvest was administered to intact male large flying foxes (Pteropus vampyrus) under managed care for androgen mitigation, leading to a decrease in agonistic behaviors, falls, and injuries from conspecific attention. ANIMALS: 12 males were included in this study. PROCEDURES: Eleven bats received subcutaneous (SC) Improvest interscapular, and 1 animal received Improvest SC in its leg. Assessments included clinical presentation, treatment, behavior, and urine and fecal glucocorticoid metabolites and testosterone (T5) concentrations. RESULTS: Eleven of the 12 bats developed reactions, which included facial edema, localized irritation, swelling of the head and neck, and pruritus with varying degrees of skin ulceration and subsequent necrosis. Three of the animals required extensive treatments, and the 1 animal who received the injection in its leg was unaffected. Posttreatment, fecal glucocorticoid metabolite and/or T5 values were at or below the nonbreeding season baseline for 3 successive breeding seasons, and there was a reduction in agonistic interactions, falls, and injuries. CLINICAL RELEVANCE: A behavioral characteristic of this species is to focus on areas of irritation that exacerbated the extent of the skin wounds. Some cases required medical, surgical, and behavioral intervention. Large flying foxes may be particularly sensitive to this immunotherapeutic when given subcutaneously in the interscapular region. Despite this reaction, the positive long-term effects on behavior and multiyear reduction of hormones suggest that the use of this immunotherapeutic warrants further investigation, although the results should be taken into consideration with other factors such as handling, treatments, chronicity of lesions.

Using behavior and genital swellings to monitor social dynamics and track reproductive cycling in zoo-housed lion-tailed macaques (Macaca silenus).

Though lion-tailed macaques are managed ex situ as an assurance population, little information is available on the relationship between behavior and reproductive physiology to ensure successful management and evaluate welfare. To facilitate social and reproductive management in a group of one male and three female lion-tailed macaques at Disney's Animal Kingdom®, we observed behaviors associated with dominance and reproduction, rated genital swellings, recorded menses, tracked ovarian cycles as determined by measurement of fecal immunoreactive progesterone (P4) and estradiol (E2), and monitored fecal cortisol (CORT). We analyzed the dominance hierarchy of females, compared hormone patterns of ovarian cycles with genital swellings and copulations relative to menses, and tested for correlations between hormones and behavior. The strength of the dominance hierarchy indicated moderate linearity, but relaxed after the dominant female received anxiolytic medications to reduce intraspecific aggression. Fecal P4 and E2 patterns revealed ovarian cycle lengths averaging 29.7 ± 0.8 days. The timing of single-day E2 peaks varied between and within females' cycles. However, on average, E2 peaks occurred 6-10 days after the first day of menses, with maximum genital swellings and copulations occurring slightly thereafter in uncontracepted females. Female-to-female aggression and sexual behaviors with the male were related to ovarian cycle phase, but correlations between behaviors and hormones varied between females. Initiated aggression was positively correlated with E2 in the dominant and mid-ranked females, while aggression received was positively correlated with P4 or negatively correlated with E2 in the mid-ranked and subordinate females. In one uncontracepted female, sexual behaviors with the male were negatively correlated to P4. CORT was positively correlated to aggression in the dominant and mid-ranked females and negatively correlated to grooming in the subordinate female. Results indicate observations of visual and behavioral cues can be used to track ovarian cycles and evaluate social dynamics and welfare in zoo-housed lion-tailed macaques.

Seasonality of reproduction in wild cotton-top tamarins (Saguinus oedipus) in Colombia.

Seasonal availability of resources can influence the timing and success of reproduction in primates. This study examines the annual pattern of conceptions, mid-term (13 weeks) and full-term (26 weeks) pregnancy losses, births, and infant survivorship of the Critically Endangered cotton-top tamarin (Saguinus oedipus) at Proyecto Tití's long-term field site in Santa Catalina, Colombia. Using 18 years of behavior, physical exam, and fecal steroid data (N = 51 females, 168 conceptions, 121 liters), fecal estrone conjugate (E1 C) and pregnanediol-3-glucuronide (PdG) or progesterone (P4) profiles were examined to investigate the relationships between female reproductive patterns and reproductive success with feeding and climatic data. Cotton-top tamarins demonstrated seasonal patterns of reproduction and pregnancy losses. We found positive correlations between peak conception and nectarivory, births and rainfall, and mid-lactation and frugivory. High body mass mothers reproducing during the peak season were more likely to have infants survive. Overall, these results suggest that tamarins use a "relaxed income-breeder" strategy. Cotton-top tamarins appear to maximize reproductive output at the time of ovulation, and can reduce their investment in the current reproductive effort at many stages. Changes in food availability due to habitat loss and climate change will require the Critically Endangered cotton-top tamarins to alter their reproductive strategies to survive.

Simulated heatwave and fishing stressors alter corticosteroid and energy balance in neonate blacktip reef sharks, Carcharhinus melanopterus.

The increasing frequency and duration of marine heatwaves attributed to climate change threatens coastal elasmobranchs and may exacerbate existing anthropogenic stressors. While the elasmobranch stress response has been well studied, the role of the unique corticosteroid-1α-hydroxycorticosterone (1α-OHB)-in energy balance is not understood. Therefore, 1α-OHB's utility as a stress biomarker in elasmobranch conservation physiology is equivocal. Here, we analyse the roles of corticosteroids, 1α-OHB and corticosterone, and metabolites, glucose and 3-hydroxybutyrate (3-HB), in response to stress in a protected tropical shark species, the blacktip reef shark (Carcharhinus melanopterus). Wild-caught neonates were exposed to ambient (27°C) or heatwave conditions (29°C) and subsequently a simulated fishing stressor (1 min air exposure). Blood samples were taken prior to temperature exposure, prior to air exposure, and 30 min, 1 h, 24 h, and 48 h post-air exposure at treatment temperatures. Plasma 1α-OHB was elevated for 48 h in 27°C-exposed sharks but declined over time in 29°C-exposed sharks. Plasma 1α-OHB was not correlated with either metabolite. Plasma glucose was higher and plasma 3-HB was lower in 29°C-exposed sharks. In a separate experiment, blood samples were collected from both neonate and adult sharks immediately following capture and again 5 min later, and analysed for corticosteroids and metabolites. Plasma 1α-OHB increased in neonates within 5 min, but neonates displayed lower plasma 1α-OHB and higher glucose concentrations than adults. We conclude that 1α-OHB does not serve as a classic glucocorticoid role in C. melanopterus under these stressors. Furthermore, we show for the first time, ontogenetic differences in plasma 1α-OHB. Ultimately, our findings provide insights into hormonal control of energy mobilization during stress in C. melanopterus, particularly during simulated heatwave conditions, which seem to alter both endocrine and energy mobilization. Further work is needed to determine the utility of 1α-OHB as a biomarker for the mobilization of energy during a stress event in elasmobranchs.

Energy and corticosteroid mobilization following an induced stress response in an elasmobranch fish, the North Pacific spiny dogfish (Squalus acanthias suckleyi).

The dominant corticosteroid in elasmobranchs, 1α-hydroxycorticosterone (1α-OHB), has a described role in mineral regulation but a presumptive role in energy balance. Energy demand in vertebrates following exposure to a stressor typically involves an immediate but transient release of glucocorticoids as a means of mobilizing available energy stores, usually in the form of glucose. Although a glucocorticoid role for 1α-OHB would be expected, direct glucocorticoid function of this steroid has yet to be reported in any elasmobranch. In addition, elasmobranchs also utilize the metabolite ß-hydroxybutyrate (ß-HB), which is thought to replace the role fatty acids play in most vertebrates as a predominant fuel source in extrahepatic tissues. To determine the mobilization of metabolites and corticosteroids during a stress event, North Pacific spiny dogfish, Squalus acanthias suckleyi, were cannulated and held in a darkened isolation box to recover (24-48 h) before being subjected to an acute air exposure or corticosterone injection. Dogfish were then serially blood sampled at nine timepoints over 48 h. Glucose, ß-HB, 1α-OHB, corticosterone, as well as lactate, pH, and osmolality were quantified in plasma samples. All measured variables increased in control and treatment groups within 48 h from the start of experimentation, and ß-HB and 1α-OHB remained elevated for the duration of the experiment. There was no linear correlation between glucose and 1α-OHB, but there was a weak (R2 = 0.230) although significant (p = 0.001), positive correlation between ß-HB and 1α-OHB. Interestingly, there were also significant correlations between increasing circulating glucose and corticosterone (R2 = 0.349; p < 0.001), and decreasing ß-HB and corticosterone concentrations (R2 = 0.180; p = 0.008). Our data suggest that following successive stressors of capture, surgery, and confinement, 1α-OHB was not correlated with circulating glucose, only weakly correlated with circulating ß-HB concentrations (R2 = 0.230; p = 0.001), and that corticosterone may also serve a role in energy mobilization in this species.

Biochemical and biological validations of a faecal glucocorticoid metabolite assay in mandrills (Mandrillus sphinx).

Stress is a major factor in determining success when releasing endangered species into the wild but is often overlooked. Mandrills (Mandrills sphinx) are vulnerable to extinction due to habitat loss and demand for bush meat and the pet trade. To help bolster in situ populations, rehabilitated rescued mandrills recently were released into a protected area in the Republic of Congo. The goal of this study was to validate the use of faecal glucocorticoid metabolite enzyme immunoassays (EIAs) in mandrills and test field-friendly faecal hormone extraction techniques that can subsequently be used to monitor the stress physiology and welfare of mandrills throughout the release process. Using faecal samples collected from ex situ mandrills, we tested cortisol, corticosterone, 11ß-hydroxyetiocholanolone (69a), and 11-oxoetiocholanolone EIAs. Absolute concentrations, hormone profiles following medical procedures or translocation, and high-performance liquid chromatography fraction immunoreactivity showed that the 69a assay was the best choice to monitor the stress response in this species. Samples with delayed extraction or drying times had 40-80% lower 69a concentrations than samples extracted immediately post-collection and frozen. The 69a EIA is an appropriate assay for monitoring welfare in this species in situ or ex situ, and results indicated that consistent extraction methods are important for accurate comparisons.

Ultrasonographic and hormonal characterization of reproductive health and disease in wild, semiwild, and aquarium-housed southern stingrays (Hypanus americanus).

OBJECTIVE: To characterize physical examination, plasma biochemical, and ultrasonographic findings in aquarium-housed, managed semiwild, and wild southern stingrays (Hypanus americanus) with and without reproductive disease. ANIMALS: Southern stingrays from aquarium (n = 48), lagoon (managed semiwild; 34), and wild (12) habitats. PROCEDURES: Limited, opportunistic prosections were performed of presumed anatomically normal wild southern stingrays and compared with findings for aquarium-housed stingrays with reproductive disease. Ultrasonographic video data from both groups were used to assign a score (1 to 5) indicating increasing severity of ovarian and uterine reproductive disease. Plasma total 17ß-estradiol, estrone, progesterone, and testosterone concentrations were measured with enzyme immunoassays validated for use in southern stingrays. RESULTS: Ultrasonographic ovarian scores were significantly correlated with uterine scores. No reproductive disease was detected in semiwild or wild stingrays, but 65% (31/48) of aquarium-housed stingrays had developing or advanced reproductive disease (ie, ultrasonographic ovarian or uterine score of 4 or 5). Significant correlations were identified between ovarian and uterine disease status and plasma concentrations of all steroid hormones except testosterone. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that ultrasonography and plasma hormone concentrations may be useful in the identification of reproductive disease and determination of disease severity in southern stingrays.

Seasonal dynamics of agonistic behavior and hormones in an ex situ all-male colony of large flying foxes.

Large flying foxes (Pteropus vampyrus) are a socially complex species. In situ colonies typically comprise thousands of individuals in small harems of one male to many females. In ex situ environments, all-male colonies are becoming more common due to a surplus of males in the population. There is limited information describing the hormonal and behavioral patterns of all-male colonies during the breeding season. We assessed seasonal changes in hormones and behavior in an all-male colony of 12 large flying foxes at Disney's Animal Kingdom® . We validated hormone assays using morning urine and fecal samples to assess seasonal changes in excreted immunoreactive testosterone and glucocorticoid metabolites. We collected behavior data using an all-occurrence method, recording agonistic behaviors related to territorial defense (hooking, biting, wing flexing, vocalizing, and wrestling), and sexual behavior (mounting and frontal grabbing). Results indicated that (i) we could reliably measure testosterone and glucocorticoid metabolites concentrations from fecal and urine samples collected from individual bats; (ii) there were distinct relationships between changes in levels of agonism and hormone concentrations throughout the year; and (iii) three agonistic behaviors (chasing, wrestling, and open-mouth threat) peaked prior to the increase in testosterone and glucocorticoid hormones measured during the breeding season. These three behaviors could potentially be used as early indicators to signal the onset of the breeding season and allow time to implement ex situ management changes to reduce the incidence of agonism between individuals.

Challenges, pitfalls and surprises: development and validation of a monoclonal antibody for enzyme immunoassay of the steroid 1α-hydroxycorticosterone in elasmobranch species.

Sharks and rays are popular species used in wildlife ecotourism and aquariums to educate the public on the behavior, ecology and conservation challenges of elasmobranchs. To understand long-term physiological health and welfare under varying social and husbandry conditions, we developed and validated an enzyme immunoassay (EIA) to measure stress/ionoregulatory hormones in managed and semi-free range southern rays (Hypanus americanus). Banked serum and interrenal samples from 27 female rays managed at Disney's The Seas with Nemo and Friends® and Castaway Cay were used to evaluate measurement of 1α-hydroxycorticosterone (1αOHB) relative to corticosterone (B). Although commercial EIAs are available for B, those tested exhibit only low relative cross-reactivity to 1αOHB (3-5%). To improve measurement of 1αOHB, we developed a monoclonal antibody using a synthesized 1αOHB-derivative for evaluation using high-performance liquid chromatography (HPLC) and EIA. Relative displacements of cross-reactant compounds showed that the antibody had good sensitivity for the target antigen 1αOHB, and low sensitivity to related steroids (desoxycorticosterone and B), but greater sensitivity to 11-dehydrocorticosterone. Tests of competitive vs. noncompetitive EIA formats, reagent titration, and incubation times of the antibody and conjugate were used to optimize sensitivity, repeatability and precision of measured 1αOHB in standards and samples (4 ng/ml, 90% binding). Tests of sample pre-treatment (pH adjustment) and extraction with varying solvent polarity were used to optimize measurement of 1αOHB in <1 ml (serum) or 1 g (interrenal) samples. HPLC analysis revealed the 1αOHB EIA to be superior for measurement of 1αOHB compared to use of a B EIA with or without HPLC fractioning. Results may prove useful for extrapolation to guide best practices for 1αOHB measurement in other elasmobranch species. Improved measurement of stress/ionoregulatory hormones in sharks and rays will be important for many aspects of collection, transport, medical treatment in aquaria and conservation management of these charismatic and ecologically important species.

Examining factors that may influence accurate measurement of testosterone in sea turtles.

Differences in reported testosterone concentrations in male sea turtle blood samples are common in the veterinary literature, but may be accounted for by differences in sample handling and processing prior to assay. Therefore, our study was performed to determine best practices for testosterone analysis in male sea turtles (Caretta caretta and Chelonia mydas). Blood samples were collected into 5 collection tube types, and assay validation and measured testosterone concentrations were compared across different sample storage (fresh, refrigerated 1 week, or frozen), extraction (unextracted or ether-extracted), and processing treatment (untreated, homogenized, or dissociation reagent) conditions. Ether-extracted and dissociation reagent-treated samples validated in all conditions tested and are recommended for use, as unextracted samples validated only if assayed fresh. Dissociation reagent treatment was simpler to perform than ether extraction and resulted in total testosterone concentrations ~2.7-3.5 times greater than free testosterone measured in ether-extracted samples. Sample homogenization did not affect measured testosterone concentrations, and could be used to increase volume in gelled samples. An annual seasonal testosterone increase was observed in both species when ether extraction or dissociation reagent treatment was used. Annual deslorelin implant treatments in a Chelonia mydas male resulted in suppression of seasonal testosterone following the fourth treatment. Seasonal testosterone patterns resumed following discontinuation of deslorelin. Comparison of in-house and commercially available enzyme immunoassay kits revealed similar patterns of seasonal testosterone increases and deslorelin-induced suppression. Our study highlights the importance of methodological validation and provides laboratorians with best practices for testosterone enzyme immunoassay in sea turtles.

Nutrition and health in amphibian husbandry.

Amphibian biology is intricate, and there are many inter-related factors that need to be understood before establishing successful Conservation Breeding Programs (CBPs). Nutritional needs of amphibians are highly integrated with disease and their husbandry needs, and the diversity of developmental stages, natural habitats, and feeding strategies result in many different recommendations for proper care and feeding. This review identifies several areas where there is substantial room for improvement in maintaining healthy ex situ amphibian populations specifically in the areas of obtaining and utilizing natural history data for both amphibians and their dietary items, achieving more appropriate environmental parameters, understanding stress and hormone production, and promoting better physical and population health. Using a scientific or research framework to answer questions about disease, nutrition, husbandry, genetics, and endocrinology of ex situ amphibians will improve specialists' understanding of the needs of these species. In general, there is a lack of baseline data and comparative information for most basic aspects of amphibian biology as well as standardized laboratory approaches. Instituting a formalized research approach in multiple scientific disciplines will be beneficial not only to the management of current ex situ populations, but also in moving forward with future conservation and reintroduction projects. This overview of gaps in knowledge concerning ex situ amphibian care should serve as a foundation for much needed future research in these areas.

Development of husbandry practices for the captive breeding of Key Largo woodrats (Neotoma floridana smalli).

The Key Largo woodrat is an endangered rodent endemic to the island of Key Largo in the Florida Keys. After several reports documented a steep decline in the population, the US Fish and Wildlife Service developed a recovery plan, including captive breeding and reintroduction. Captive breeding efforts were to be focused on providing animals for future reintroduction to protected areas on Key Largo. However, little was known about the husbandry needs or reproductive behavior of this elusive nocturnal species. In 2005, Disney's Animal Kingdom(®) received 11 animals and began to systematically investigate methods of breeding Key Largo woodrats. Since the program's inception, 30 pups have been born and successfully parent reared. In this report, we describe some of the husbandry techniques that have contributed to the success of the Key Largo woodrat captive breeding program at Disney's Animal Kingdom(®) . The results obtained may be of use to other facilities maintaining woodrats and other rodent species.

Chemical and surgical interventions to alleviate intraspecific aggression in male collared lemurs (Eulemur collaris).

To reduce male-male aggression in collared lemurs (Eulemur collaris) at Disney's Animal Kingdom, a clinical trial of leuprolide acetate (Depo-Lupron) injections through one breeding season (24 wk), followed by surgical castration after the breeding season was implemented. Daily behavioral observations to record occurrences of aggression were conducted before and during the injection period, as well as after castration. Fecal sample collection began 2 wk before the first injection and continued throughout the clinical trial to determine testosterone metabolite concentration. Samples were collected 3 times per week. Fecal collection and behavioral observation continued for 5 mo after castration. Fecal samples from 3 intact males from other institutions were obtained to compare testosterone values. While the lemurs were treated with Depo-Lupron, testosterone concentrations spiked immediately after injection, and then returned to pretreatment, nonbreeding-season levels after varying lengths of time. Analysis of the behavioral data indicated that aggression was not significantly reduced with Depo-Lupron treatment. However, after castration, contact aggression and wounding decreased to zero. Although the sample is small, this study presents the first documented data on the effects that chemical treatment with Depo-Lupron and surgical castration have upon hormone levels and aggressive behavior in eulemurs.

Behavioral predictors of copulation in captive Key Largo woodrats (Neotoma floridana smalli).

The development of a captive breeding program for the endangered Key Largo woodrat (Neotoma floridana smalli) presents special challenges due to aggressive behavior toward conspecifics, a low reproductive rate, and limited information on estrous cycles. In an effort to identify behavioral predictors of copulation, we observed 17 Key Largo woodrats prior to and during 267 male-female pairing events, 76 of which resulted in copulation. Predictors of copulation include male-female interactions at the door of the tube connecting their enclosures, raspy vocalizations, pre-mounting lordosis, and chasing. A binary logistic regression model based on pre-pairing behaviors correctly predicted the outcome of 78.7% of observed pairing events, and a similar model based on intra-pairing behaviors correctly predicted the outcome of 86.1% of observed pairing events. Behavior-based models may be useful in the management of captive breeding programs for this and other endangered species.

Urinary steroid hormone analysis of ovarian cycles and pregnancy in mandrills (Mandrillus sphinx) indicate that menses, copulatory behavior, sexual swellings and reproductive condition are associated with changing estrone conjugates (E(1)C) and pregnanediol-3-glucuronide (PdG).

The objective of this study was to determine if sexual swellings in mandrills (Mandrillus sphinx) are a reflection of reproductive endocrine state. Urine samples were assayed using an enzyme immunoassay measuring pregnanediol-3-glucuronide (PdG) and estrone conjugates (E(1)C). Hormone patterns of ovarian cycles, pregnancy and lactation were characterized and compared with sexual swellings and copulations relative to menses and peak E(1)C. Cycle lengths averaging 28.7 days and pregnancy length of 181 days determined by hormonal and sexual swelling measures were similar to those reported in other Old World primate species. First day of copulation was observed during rising E(1)C concentrations and preceded observations of peak swelling by 1-2 days. Observations of peak sexual swellings occurred at or on the day after peak E(1)C and decreased following the ovulatory increase in PdG. Observations of menses and sexual swellings are a useful method to track mandrill ovarian cycles and can assist zoos in determining the reproductive state of females in their collections. Zoo Biol 27:320-330, 2008. (c) 2008 Wiley-Liss, Inc.

Pup development and maternal behavior in captive Key Largo woodrats (Neotoma floridana smalli).

This study describes the first systematic observations of maternal behavior and pup development of captive Key Largo woodrats (Neotoma floridana smalli) during the first 30 days of life. Data were collected on six litters of pups born to four dams between December 2006 and July 2007. Gestations for the six litters averaged 38 days and all dams exhibited adequate maternal care postpartum. Key Largo woodrat maternal and pup behavior was generally consistent with behavior observed in other woodrat species. We observed greater pup independence from the dam and a marked change in social interactions between days 13-22. No sex differences in pup development or maternal care were observed. Activity budgets were consistent across dams and across days within the observation period. Although dams spent much of their time inactive with pups attached to their teats, the average percent of intervals with at least one pup observed attached decreased steadily during the 30-day observation period. Attachment of pups to the dams' teats did not interfere with dams' ability to forage. Feeding with pups attached and feeding following active detachment of pups were both common. Dams were observed to actively detach pups by performing a circular turning motion. This information has application for the future management of this endangered species in captivity and in the wild. Zoo Biol 27:394-405, 2008. (c) 2008 Wiley-Liss, Inc.

Suppression of ovulation in Nile Hippopotamus (Hippopotamus amphibious) using melengestrol acetate-treated feed or high dose Depo-Provera injection.

Analysis of fecal progestogen profiles during Depo-Provera injection (1,200 mg; DEPO, Pfizer Inc., New York, NY), melengestrol acetate (MGA) in feed (2 or 3 mg/head/day), and a combination treatment (DEPO+MGA) are presented for nine captive female Nile hippos housed at Disney's Animal Kingdom in Florida. All tested treatments reduced fecal progestogen elevations successfully to durations consistent with prevention of ovulation for a portion of the treatment period. Percentage of treatment months with suppression of luteal phases indicative of ovulation was maximal for high-dose MGA (91.7+/-13.9%) and DEPO+MGA (91.7+/-20.4%), followed by DEPO injection alone (69.2+/-13.9%) and low-dose MGA (57.6+/-33.2%). Both 1,200 mg DEPO and low-dose MGA (2.0 mg/day) treatments were insufficient to prevent an apparent seasonal breakthrough of ovarian activity from June-August 2002. Although luteal phases were observed, no females conceived during those months. Overall, in 133.5 treatment months with females housed with an adult male, one female conceived during the transition period between treatments. After cessation of contraceptive treatment, average latency to first normal ovarian cycle was 80.6+/-19.5 days (range = 22-179 days). Up to 12 months post-treatment, however, successive cycles were often irregular with evidence of short periods of anovulation and shortened luteal phases in all females monitored. In conclusion, high dose and combination treatments were most successful in preventing progestogen increases indicative of ovulation in hippos. Zoo Biol 26:259-274, 2007. (c) 2007 Wiley-Liss, Inc.