BACKGROUND

1. Monitoring behavior, stress and reproductive hormones in the black rhinoceros using non-invasive fecal assays Mark Kamhout, Department of Zoology, Michigan State University and Kathy Carlstead Ph. D, Honolulu Zoo BACKGROUND Black Rhinoceros (Diceros bicornis) are a critically endangered species with ~3,600 individuals left in the wild (IRF, 2005). There are only ~119 individual animals housed at institutions within the United States (AZA, 2005). Captive reproduction has been generally good but has been offset by an unusually high mortality rate (AZA, 2005). The excessively high mortality rate could be the result of nutritional deficiencies and/or the exposure to chronic stress (Dierenfeld, 2001). Past research has found that measuring corticoid levels from fecal matter may be a very useful, non-invasive tool for measuring stress (Brown et al, 2001). Enzyme Immunoassay (EIA) techniques can effectively determine cortisol levels found in the feces in a cost efficient, environmentally friendly manner. OBJECTIVE To determine possible sources of stress in captive black rhinos we monitored changes in behavior, corticoids and reproductive hormones for two black rhino pairs located at two zoological institutions over an extended period of time. These time periods encompass potentially stressful events such as invasive training (blood draws, IM injections, chute training, etc.) irregular activity around the rhinos (construction and maintenance work, extremely high visitor attendance) and the female’s estrous cycles. SETTINGS OF THE STUDY The Study at Potter Park Zoo (PPZ): PPZ is located in Lansing, Michigan. PPZ has 1.1 black rhinoceros (Jimma and Ebony aged 13 and 11 years at time of study) that had been housed together at an early age but were separated in an attempt to improve breeding. The pair had been introduced several times without any copulation occurring prior to the study period where they were then housed separately. Behavioral and hormonal data were collected on 1.1 animals over a six-month period (November 2003 through April 2004). Due to the lack of copulation the PPZ is switching males with the Cleveland Zoo to better facilitate breeding. The Study at Honolulu Zoo (HZ): HZ is located in Honolulu, Hawaii. HZ has 1.1 black rhinoceros (Corky and Satsuki, aged 14 and 6 years at time of study) that had been housed in close proximity, but separated, for 1.5 years prior to the study period (December 2000-2001). The study period encompasses the time prior to their first introduction (Jan, 2002). The pair is now housed together on a daily basis in the same outdoor yard but separated at night in their respective stalls. They have not reproduced due to inadequate male copulatory behavior, although the female is cycling and the pre-copulatory behavior is normal. 3) Changes in the locations of the rhinos affected their stress hormone levels. Males appear to be more affected by these changes (Figures 5 and 6). Figure 5 Figure 6 Honolulu Zoo Potter Park Zoo 4) On days when corticoid levels are high for the females at each zoo, males are rated by keepers as performing more flehmen and they have higher testosterone levels, and females have higher progesterone (P4) levels (indicating the non-estrus or luteal phase of the cycle) (Figures 7 and 8). Figure 7 Figure 8 DISCUSSION and CONCLUSIONS - Each rhino has a unique pattern of hormonal stress responses to specific events, although machine noise affects the majority of the animals. - The positive correlation between the female’s cortisol levels and the male’s testosterone levels for both pairs indicates that male sexual behavior and/or hormonal state induces stress responses in females. - Switching the locations of rhinos at PPZ from their regular yards increased corticoids. The corticoid levels of males at both zoos were lower when pairs were locked in their stalls with no yard access. - Females have higher corticoids when they are not in estrus and males are performing sexually-motivated behaviors such as flehmen at high frequencies. This indicates that male (pre-) sexual behavior around a non-receptive female may be a stressor for females in a captive setting. - Fecal collection and EIA were useful in monitoring reproductive hormones and corticoids non-invasively in this species. METHODS Observational Data: Behavioral observations were made on a daily basis by keepers using a standardized behavioral and environmental monitoring checksheet. Hormonal Data: Daily fecal samples from each rhino were collected each morning from night stalls during cleaning. EIA protocols were followed in the Brookfield Zoo Endocrinology lab to determine the levels of corticoids and reproductive hormones in each sample. The behavioral, environmental and hormonal data for each rhino pair were then compared and analyzed for statistical significance using correlations, ANOVA and T-tests. RESULTS There were four main results that became evident after analyzing the hormonal and behavioral data from both zoos: 1) Corticoid responses to events such as concerts (HZ only) and positive-reinforcement training were specific to individuals. However, 3 of 4 rhinos had significant corticoid responses to machine noise (Figures 1 and 2). Figure 1 Figure 2 2) In both pairs there were significant positive correlations over time between female cortisol and male testosterone (Figures 3 and 4). Honolulu Zoo Potter Park Zoo Figure 3 Figure 4 Acknowledgements: Special thanks to the Brookfield Zoo Endocrinology staff, the volunteers, staff and keepers at the Honolulu Zoo and Potter Park Zoo, Dr.Tara Meyers-Harrison, Gerry Brady, Astrid Bellem, Dr. Richard Snider, and Dr. Nadja Wielebnowksi, who helped make this project possible. References: American Zoo and Aquarium Association. www.aza.org. Brown J.L., Bellem, A.S., Fouraker, M.,Wildt, D.,E., Roth, T.,L. 2001. Comparative analysis of gonadal and adrenal activity in the black and white rhinoceros in North America by noninvasive endocrine monitoring. Zoo Biology: 463-486. Dierenfeld E.S. 1996. Nutrition. In: M. Fouraker and T.Wagener (Eds.), AZA Rhinoceros Husbandry Resource Manual, Fort Worth Zoological. Park, Fort Worth, Texas. International Rhino Foundation. www.irf.org