2019 Rhino Taxon Advisory Group Research Masterplan

RHINOCEROS RESEARCH MASTERPLAN 2019

Association of Zoos and Aquariums Rhinoceros Taxon Advisory Group Rhino Research Council

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Introduction Rhino Research Council The Rhino Research Council is comprised of advisors with extensive rhino expertise in one of the following: genetics, nutrition, health, reproduction, behavior & ecology, and management. Each advisor selects several co-advisors to assist them with their area of responsibility. The AZA Rhino TAG’s Research Council is charged with developing a five-year Masterplan that encompasses research priorities for rhinos and prioritizes those challenges deemed most important to address during the life of the Masterplan (typically five years).

Masterplan Goals The Rhino Research Council Masterplan document serves several purposes. First, because much of the information shared and discussed by the Rhino Research Council is from studies that have yet to be published, the Masterplan provides a summary of the most recent progress in rhino research and some of the newest theories about the challenges still facing rhinos. Second, the Masterplan is an excellent source of reference when the Rhino TAG is asked to endorse proposed research projects. Finally, the priorities in the Masterplan help guide the International Rhino Foundation’s targeted request for proposals.

Over the years, the philosophy guiding the Rhino Research Masterplan has shifted. When first formed, the Rhino Research Council focused only on research priorities for ex situ rhinos. The rationale for this focused approach was that the Council was a part of the Rhino TAG and therefore, its role should be to assist the TAG with ex situ rhino management issues. However, during the development of the 2004 Masterplan, there was much discussion about the integration of both ex situ and in situ rhino research priorities into the document. That year, studies on wild rhinos that could shed light on problems in the ex situ population were included. During the 2009 Masterplan meeting, we took a step further and embraced research priorities for wild rhinos as part of our responsibility realizing that what we were calling “wild” rhinos were really minimally managed rhino populations since all rhinos are now managed to some extent. Finally, in the 2014 Masterplan, an attempt was made to fully integrate the research needs for minimally managed and intensively managed rhino populations in Africa, Asia and our zoos. We attempted to do the same for the 2019 Masterplan. In contrast to 2014, when the top research priorities were in situ based, the 2019 votes were primarily for challenges impacting rhinos managed ex situ.

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2019 Rhino Research Council

Chair: Dr. Terri Roth, Cincinnati Zoo/CREW, OH TAG Chair: Adam Eyres, Fossil Rim Wildlife Center, TX

Health (Veterinary Medicine) Advisor: Dr. Michele Miller, Stellenbosch University, South Africa Advisor: Dr. Eric Miller, St. Louis Zoo, MO Co-Advisor: Dr. Benn Bryant, Taronga Conservation Society, Australia Co-Advisor: Dr. Robin Radcliffe, Cornell University, NY Co-Advisor: Dr. Beth Hammond, Lion Country Safari, FL

Nutrition Advisor: Dr. Katie Sullivan, Disney’s Animals, Science and Environment, FL Co-Advisor Dr. Marcus Clauss, University of Zurich, Switzerland Co-Advisor Dr. Ellen Dierenfeld, Ellen S. Dierenfeld LLC, MO Co-Advisor Kerrin Grant, The Wildlife Center, NM Co-Advisor Barbara Henry, Cincinnati Zoo, OH Co-Advisor Dr. Eduardo V. Valdes, Disney’s Animals, Science and Environment, FL

Genetics Advisor: Dr. Peter J. van Coeverden de Groot, Queens University, Canada Co-Advisor: Dr. Alfred Roca, University of Illinois, IL Co-Advisor: Dr. James Austin, University of Florida, FL Co-Advisor: Dr. Candace Scott, Queens University, Canada

Behavior and Ecology Advisor: Dr. Lara Metrione, SE Zoo Alliance for Reproduction & Conservation, FL Co-Advisor: Dr. Elizabeth Freeman, George Mason University, VA Co-Advisor: Dr. Rachel Santymire, Lincoln Park Zoo, IL

Reproduction Advisor: Dr. Monica Stoops, Cincinnati Zoo/CREW, OH Co-Advisor: Dr. Justine O’Brien, Taronga Zoo, Australia Co-Advisor: Dr. Linda Penfold, SE Zoo Alliance for Reproduction & Conservation, FL Co-Advisor: Dr. Parker Pennington, San Diego Zoo Global/ICR, CA Co-Advisor: Dr. Mandi Schook, Disney’s Animal Programs, FL Co-Advisor: Dr. Jessye Wojtusik, Cincinnati Zoo/CREW, OH

Management Advisor: Adam Eyres, Fossil Rim Wildlife Center, TX Co-Advisor: Lance Aubrey, San Diego Zoo Global, CA (Black rhino, GOH) Co-Advisor: Paul Reinhart, Cincinnati Zoo, OH (Sumatran rhino) Co-Advisor: Randy Rieches, San Diego Zoo Global, CA (White rhino, GOH) Co-Advisor: Lisa Smith, Buffalo Zoo, NY (Black rhino) Co-Advisor: Clarice Brewer, White Oak Conservation Center, FL

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Primary Research Priorities

I. Impact of and control over body condition/weight ➢ Reproductive dysfunction ➢ Foot/joint problems ➢ Phytoestrogen relationship ➢ Diet composition and variety versus nutrient composition impact ➢ Health/disease impacts ➢ Overall effect on well-being ➢ How to best monitor/measure and alter to improve animal well-being

II. Iron overload in browsing rhinos ➢ Epidemiological review – what really is the significance/prevalence of IOD? ➢ Best biomarkers for detecting, monitoring and assessing condition or treatment ➢ Organ iron accumulation versus organ damage ➢ Association with other health issues (is it primary or secondary?) ➢ Interaction with other micro-nutrients

III. Understand/address early and late stage reproductive dysfunction ➢ Impact of over-conditioning ➢ Cause of stillbirths/pregnancy loss ➢ Why so much cyclic dysfunction (silent estrus, acyclicity, anovulation)?

IV. Investigate behavioral and environmental factors that affect rhino well-being ➢ Ex situ health, body condition, reproduction, socialization, enriched environment ➢ In situ impact of factors like dehorning and traumatic injury recovery

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Introduction It is important to note that these four research priorities are not ranked in order of importance. They were simply the research topics receiving the four highest vote counts from RRC advisors and SSP Chairs. It is also worth noting that there is a lot of overlap among these priorities.

I. Impact of and control over body condition/weight As with most animals managed ex situ, rhinos typically receive more nutrient dense diets and get less exercise than they do in situ. Not surprisingly, these conditions lead to over- conditioning and obesity, which probably impact rhinos in the same way they do people and pets. Just how much obesity accounts for health, reproductive and well-being issues and/or what physiological pathways are involved are not known, so research in these areas would be very enlightening and could contribute to future management/husbandry recommendations.

II. Iron overload in browsing rhinos Research on the prevalence and significance of iron overload disorder (IOD) in browsing rhinos continues to be a priority. It is generally accepted that many browsing rhinos exhibit high levels of serum iron saturation and accumulate iron deposits in organ tissues (hemosiderosis) that are noted post-mortem. However, the relationship between iron loading and disease appears complex, as it is not yet known if hemosiderosis is directly related to iron overload disease or is occurring in response to other disease states (i.e., dental disease, metabolic disturbance) or vice versa. It is also unclear if the iron deposits are actually damaging the organ tissue since iron overload is rarely identified as the primary cause of death in black rhinos. Recent studies have brought to question some of the previous held beliefs about monitoring IOD progression in rhinos, and new biomarkers/approaches may be necessary for future studies. Further work is also needed in determining the bioavailability and impact of dietary iron on IOD progression.

III. Understand/address early and late stage reproductive dysfunction Although reproductive challenges in white and GOH rhinos continue to rise to the top of the research priority list, there has been significant progress in recent years. There is a better understanding of acyclicity in white rhinos and treatments for overcoming it. Additionally, a dietary change that reduces phytoestrogen intake has been associated with resumed reproductive success in some white rhinos. Furthermore, artificial insemination is proving to be a useful tool for some rhinos that are not breeding naturally. However, the incidence of anovulation in all rhino species is much higher than previously realized and the cause is unknown. Early embryonic loss and stillbirths continue to plague rhino reproductive success.

IV. Investigate behavioral and environmental factors that affect rhino well-being Animal welfare and well-being are of utmost importance to animal managers caring for individuals in zoos, on private ranches or in sanctuaries/national parks. Although rhino welfare issues differ among these environments, in all cases, the goal is to provide the best possible conditions for the rhinos. Ex situ, the challenge is to provide an environment that is as stimulating as that rhinos would experience in their native habitat during good times to the greatest extent possible. In situ, the challenges range from identifying factors that impact translocation success to understanding how to lessen the trauma and facilitate the recovery of rhinos either suffering from poaching injuries or orphaned as calves so that they can resume a productive, natural life with their counterparts in their native habitat. Identifying how to achieve these well-being goals requires evidence-based scientific study.

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Detailed Areas in Need of Research Within Each Discipline

Health (Submitted by the Veterinary Advisory Group)

Goal: AZA Research initiatives of member institutions ideally should support global conservation programs with a focus on preserving the species in their native habitats.

ASIAN RHINO HEALTH

1. Sumatran and Javan rhinos

1.1.Disease risk analyses 1.1.1. Translocations within and between range states, or from abroad present risks for introduction of disease with or to introduced rhinos, especially if animals have been held ex situ for some time. Research into diseases that present potential risk to rhinos, logistically appropriate diagnostic techniques for screening, and incorporation into protocols should be considered part of the risk analysis and translocation process. 1.1.2. Disease prevention in and around sanctuaries and protected areas – particularly the role of hemoparasites (trypanosomes) and tick-borne diseases (Babesia, Theileria and Anaplasma) and other transmissible infectious diseases that can be spread from domestic animals to sympatric rhinos. This should include development of effective quarantine and biosecurity protocols to prevent, detect, and treat these diseases. Vaccination for tetanus should also be evaluated for this species. This topic was recently highlighted by the IUCN Asian Rhino Specialist Group as a very important area of study given the small populations for both Sumatran and Javan rhinos that are located in areas often completely surrounded by humans and animal-based agriculture.

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1.2. Iron overload disorder in Asian browsing rhino species. There should be a thorough pathology review of all ex situ rhino deaths for Sumatrans to better understand historical extent and impact of confinement and stress in this syndrome. In addition, further research on specific biomarkers for diagnosis are needed.

1.3.Nutrition of Asian rhino browsers 1.3.1 Comprehensive study of sanctuary rhino browse selections that have supported rhino health and reproduction to be used as a template for other developing sanctuaries to follow. 1.3.2 Are there any problems with ex situ diets due to low browse selection and availability? 1.3.3 Studies on browse nutrient degradation related to varied collection, handling and storage protocols employed at zoos and sanctuaries.

1.4.Protocol development 1.4.1. Animal movements among institutions (successful case examples are available from work done in preparation for movement of two male rhinos from the U.S. back to Indonesia; i.e. government requirements, vaccination strategies, etc.) 1.4.2. Capture and translocation protocols in situ 1.4.3. General anesthesia protocols in-situ (based on available drugs). Currently available data within range countries should be compiled and reviewed to continue improving protocols. Research on the use of short and long-acting tranquilizers is also needed for Sumatran rhinos. 1.5. Establish normal biological and health parameters for ex situ and wild animals (some work from SRS recently published, but need more normal data from truly wild rhinos to better understand health of ex situ animals or those moved for future translocation programs) 2. Greater one-horned (GOH) rhino

2.1 Obesity/body condition index – Overconditioning in ex situ rhinos (all species) leads to a multitude of health problems including musculoskeletal, foot, and reproductive problems. This has particularly been associated with pododermatitis along with ex situ husbandry conditions in this species of rhino. Research examining methods of standardizing body condition scores for all rhino species, along with improved nutritional management should be a priority for ex situ health. Existing species-specific rhino body condition scoring systems should be used as starting points and continue to be revised.

2.2 Gastrointestinal and cardiovascular problems – may be underreported in this species. These were significant contributors to adult mortality in a recently reported review of necropsy reports. Research to investigate the presence of health issues and contributing factors (nutrition, management, stress, etc.) should be considered for this species.

2.3 Disease risk analyses 2.3.1 Translocations within and between range states, or from abroad present risks for introduction of disease with or to introduced rhinos, especially if animals have been held ex situ for some time. Research into diseases that present potential risk

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to rhinos, logistically appropriate diagnostic techniques for screening, and incorporation into protocols should be considered part of the risk analysis and translocation process. 2.3.2 Disease prevention in and around sanctuaries and protected areas – particularly the role of transmissible infectious diseases that can spread from domestic animals to sympatric rhinos including mycobacteriosis. A recent case of Mycobacterium orygis was discovered in a free-ranging GAOH in Nepal. Since GOH live in high burden TB countries, further research should also investigate human-animal diseases. Similar to the Sumatran and Javan rhinos, GAOH rhinos are located in areas often completely surrounded by humans and animal-based agriculture. In addition, vaccination for tetanus and possibly other diseases that may be associated with trauma during capture, translocation or in sanctuaries should be investigated.

AFRICAN RHINO HEALTH

1. African Black rhino - Many of the disease issues of black rhinos have been well studied and it is clear there are significant challenges to keeping browsing rhinos in captivity. Certainly, better strategies can and should be established to help alleviate these concerns and research will be required to address these issues (iron overload disorder, (IOD); idiopathic hemorrhagic vasculopathy syndrome, (IHVS); nutrition, husbandry, infectious disease, etc.).

1.1 Nutrition- is the inclusion of cereal based concentrates in rhino diets detrimental (all rhinos)?

1.2 Quantifying stress (especially chronic stress) in rhino - develop laboratory and behavioral markers. What is the health impact, for instance, on development of GI ulcers, increased iron stores, etc. in both ex situ and recently captured free-ranging rhinos (all rhinos)? New ideas include evaluation of neutrophil function using portable luminometer and the analyses of five markers in fecal samples that include thyroid hormones.

1.3 Iron overload disorder – Does iron overload cause disease in ex situ browser rhinos or not (i.e., what is the clinical significance)? Does it increase susceptibility to other diseases, such as infections? Determine what is the most useful marker (or suite of markers) for iron overload; e.g., is rhino specific ferritin an accurate marker? Research is needed to determine whether dietary and therapeutic interventions are effective or even necessary, and if other methods such as stress/management play a role. What are the clinical/laboratory indications for therapy? Is there a link between excessive body condition &/or chronic stress and the presence of chronic inflammatory mediators in ex situ rhino (as in humans and domestic horses)? Is it possible that there is a ‘rhino metabolic syndrome’ characterized by insulin resistance and the chronic elaboration of inflammatory mediators by adipocytes that contributes to excessive iron uptake (and other black rhino diseases)? Current research is underway to investigate the role of the GI microbiome in diseases of black rhino, particularly IOD. This is a high priority research area for the ex situ population.

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1.4 Oro-dental health – Factors in development of dental disease in black rhino (all rhinos?) and methods for treating and preventing routine dental issues need further research. In a recent survey, only 29% of black rhinos in North American zoos have ever had an oral examination under general anesthesia; all of these individuals had orodental disease ranging from enamel points to severe periodontal disease and tooth loss. Association of renal disease with orodental disease has been shown in other species. Therefore, more information is needed, not just for browsers, but all rhino species. What is the prevalence of periodontal (or general orodental) disease? More information is needed to describe orodental abnormalities and determine the pathogenesis. What is the association between browse feeding and dental health? Is the problem simply caused by high starch ex situ diets or abnormal tooth wear and periodontal trauma due to chewing/grinding inappropriate forage?

1.5 In addition to the causes of black rhino mortality, a superficial necrolytic dermatitis (SND)-like disease continues to plague ex situ black rhinos affecting both their appearance and welfare, though not necessarily causing mortality. In the past, data suggested that SND affected as many as 50% of ex situ black rhinos. In one study, the hypothesis that SND was caused by hypoaminoacidemia was refuted. Furthermore, there was no positive correlation between corticoid levels and animals with lesions vs. those without lesions. Animals without lesions exhibited higher mean corticoid levels over time than animals with lesions. Disease could be immune-mediated.

1.6 Renal disease – Necropsy reports on ex situ black rhinos in the U.S. have shown an increased number of cases of renal disease as a cause of death with secondary tissue mineralization. Does renal disease predispose to hypertension with secondary arterio/atherosclerosis and possibly other health issues such as intermittent epistaxis? Is there an age or dietary association with development of renal disease? Ante-mortem bloodwork and urinalysis has not typically been useful diagnostically. Research to investigate predisposing factors, including diet, and improved methods for early diagnosis and intervention may benefit the ex situ population. In addition, relationship to other health syndromes should be re-examined.

1.7 IHVS, idiopathic epistaxis – Cases are becoming rare, however, the death of a 4-year- old black rhino was attributed to IVHS in 2014. When they occur, there is no obvious etiology or consistently successful treatment. Idiopathic syndromes in black rhinos should continue to receive research attention, including the role of stress, nutrition, management, and infectious diseases.

1.8 Tuberculosis - Mycobacteriosis (TB) caused by Mycobacterium bovis has been diagnosed in both free-ranging black rhinos and individuals on private game reserves in South Africa. This has led to quarantine and restrictions on future translocations. Further research is needed to develop accurate antemortem diagnostic tests, investigate the epidemiology, perform risk assessments and create management and control plans.

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1.9 Management of poacher injured and orphaned rhinos in range countries - Recent development of treatment options for poaching injuries have been implemented in South Africa but additional research is needed to improve pain management, infection control, and accelerate wound healing. Development of prognostic indicators would be very useful to best utilize limited resources. In addition, research into orphan rhino care including diet/formula and management (level of human contact, use of surrogate dams) are needed to develop evidence-based protocols. Ex situ Black Rhino Mortality (primary cause of death) Note: does not represent co-morbidities

Australia 2000-2017 (n=11) North America 2008-2017 (n=28)

0% infectious disease 14% infectious disease – Clostridial 0% IHVS/hemolytic anemia 4% IHVS/hemolytic anemia 0% renal disease 28% renal disease 19% stillbirth/weak calf 11% stillbirth/weak calf/congenital anomalies 0% trauma 0% trauma 0% unknown 14% unknown 9% cardiovascular/vasculitis 4% cardiovascular/vasculitis 27% GI 7% GI – colonic rupture, splenic mass rupture 9% neoplasia (uterine adenocarcinoma) 7% neoplasia (uterine, thyroid adenocarcinoma) 9% liver disease 4% liver disease (iron overload disorder) 9% multisystemic disease 7% multisystemic disease 18% orodental disease 0% orodental disease

2. African white rhino 2.1 Idiopathic neurologic disease – Several white rhinos have exhibited episodic tremors/ataxia in Australia and sporadic cases have been observed in white rhinos in Kruger National Park. Survey of neurologic disease in white rhino would be worthwhile to determine if this is an underreported condition.

2.2 Neosporosis – This infection has resulted in abortion and acute death in white rhinos. An experimental ELISA is available. Research to determine seroprevalence may reveal if this is a widespread issue for reproductive or potentially systemic health problems.

2.3 Gastrointestinal health issues – Investigation into potential factors such as diet/nutrition, management, infectious diseases and stress to determine if morbidity/mortality may be prevented. Anecdotal reports of Clostridial enterotoxemia exist in white rhinos. Research into the predisposing factors, treatment and prevention by use of available vaccines is needed.

2.4 Pharmacokinetics/dynamics of commonly used antibiotics and analgesics (all rhinos) – Little scientific work has been done in rhinoceros species on therapeutic drugs, especially on use of non-steroidal anti-inflammatory drugs. Empirical use may be

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inadequate, or potentially cause adverse effects. With the advent of husbandry training and restraint devices, sample collection is now possible for these types of studies.

2.5 Management of poacher injured and orphaned rhinos in range countries - Similar to the black rhino, there is an urgent need for research into effective management of poaching injuries and orphan rhino care.

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Nutrition Compiled by the 2018 Rhino Research Council Nutrition Advisors

There have been about 36 peer-reviewed studies on rhino diets and nutrition related factors over the last 5 years. Overall, rhino nutrition investigations have been geared towards understanding dietary nutrients as they impact serum nutrients in relation to health, characterizing practical diet needs under human care, characterizing consumption in wild areas, and how nutrients and diet can impact wellness and ultimately welfare. The general consensus remains the same as in 2009 and 2014 - the highest priority nutritional studies would be those in association with potential causes of rhino disease (in particular iron overload disorder) and/or reproductive problems.

1. Iron values (Iron Overload Disorder - IOD) – epidemiological information on diets, iron parameters and health serum markers, and current health status would be useful to re-evaluate the population since it has been many years since the original data were generated and many dietary/management changes have been implemented.

What has been done: 14 publications

Current Need: 1.1 Identifying and improving diagnostics of IOD 1.2 Documenting diet ingredient impact on iron loading vs. inflammation (ie. alfalfa, forms of phosphorus) 1.3 What is the upper limit for total dietary iron concentrations vs. pelleted diet iron? (< 300 ppm Fe currently recommended) 1.4 Understanding of antioxidant dietary impacts on iron loading (supplemented antioxidant / anti-inflammatory in the form of vitamins, omega 3’s, etc.) 1.5 Potential treatment options – investigating safety and efficacy of natural chelators, as well as long term large volume phlebotomy and their impacts on health 1.6 Iron loading in relation to pregnancy – potentially an epidemiological review

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Research being conducted / in progress:

A. Water contributions to iron load across institutions – Michigan State University (M.S. study) B. Iron related genetic mutations in relation to adenosine –San Diego Zoo Institute for Conservation Research C. Role of Gut Microbiota in Health and Disease Sensitivity of the Black Rhinoceros- Smithsonian’s National Zoo, Center for Species Survival D. Black rhino specific ferritin assay being tested - University of Florida E. Examining oxidant stress in black rhino in relation to IOD potential measurements – University of Florida F. Labile plasma iron and miRNAs as potential diagnostic markers of IOD – Cincinnati Zoo’s Center for Conservation and Research of Endangered Wildlife

2. Diet studies – several ongoing and relating to potential health hazards and impacts of diet items / nutrient levels.

What has been done: 15 publications (including documentation of free-ranging animal nutrient intakes in GOH, Sumatran and Javan species)

Current needs: 2.1 Continue monitoring serum versus dietary vitamin E (primarily in black rhinos, but potentially in white rhinos as well due to obesity / reproduction concerns) 2.2 Establish serum vitamin E ranges in current population using appropriate commercial laboratories (black rhinos and across species) 2.3 Investigate interaction of diet / nutrient content (macro or micro) and validated inflammatory markers in each species 2.4 Establish more suitable minimum dietary nutrient concentrations for animals during maintenance, gestation and breeding. Are horse requirements adequate / appropriate as guidelines? 2.5 Develop science-based recommendations regarding the use of alfalfa for each rhino species. The energy, protein, calcium, and bioavailable iron content of alfalfa, as well as its overuse in diets, may be a factor in health including potential renal, iron and obesity issues. 2.6 Investigate the use of phytase, versus presence of phytic acid, in conjunction with phosphorus supplement, to combat hypophosphatemia. Ex situ black rhinos have demonstrated serum hypophosphatemia and hypercalcemia, likely in relation to diet. Currently high levels of NaPO4 supplementation are required to achieve low normal serum P values, but there is a paucity of data regarding phosphorus digestibility. 2.7 Investigate rhino microbiomes by utilizing fecal DNA / RNA analyses as a means of defining gut populations in rhinos on particular diets under human care versus wild diets. 2.8 Re-evaluate serum nutrient profiles in wild rhino species considering current methodologies to update historical “reference” values for vitamins, minerals, etc.

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Research being conducted / in progress: A. Vitamin E in serum and fecal samples on varied supplementation of Emcelle for white and black rhinoceros species – Disney’s Animal Kingdom B. Vitamin D in serum across seasons in black rhinos – Blank Park Zoo C. Gut microbiome analysis in white and black rhinos – San Diego Zoo Global, Smithsonian Center for Species Survival and Disney’s Animal Kingdom

3. Oro-dental issues – A research priority under rhino health since it can lead to oral lesions and loss of body condition, but its cause could be related to diet (nutritional and/or mechanical). There have been several cases of rhinos requiring repeated teeth floating procedures, and much discussion on dental concerns across species. Potential evaluation of sugar content and amount of enrichment items may also be warranted.

Current Need: 3.1 Investigate epidemiological correlation of dietary nutrients and dental disease 3.2 Impact of browse on dental health (quantity / type/ etc.) 3.3 Investigate use of alfalfa in relation to dental disease

What has been done: 1 publication

4. Fatty acids – Fatty acid ratios differ significantly between ex situ and wild populations of black rhinos. Types of fatty acids available from diet could be associated with changes in immune function and/or diseases such as SND.

Current Need: 4.1 What are the appropriate dietary ratios of n-3: n-6 fatty acid? Supplements are being used to increase linolenic acid, but what is the minimum amount to offer to provide benefits? Documentation of benefits would potentially lead to diet re-formulations. 4.2 Interaction between fatty acids / composition of diet and inflammation

5. Trace and macro minerals – Recent investigations are ongoing, including copper which is an important anti-oxidant that interacts with iron. The recent reported incidence of renal disease in the population calls calcium and phosphorus content of diets into question.

Current Need: 5.1 Appropriate dietary concentrations of zinc and copper, considering their interactions, and potential binding with phytate and other secondary compounds. Zinc and copper are also sometimes being supplemented for hoof health. 5.2 Investigate macro minerals in total diet in relation to renal health

Research being conducted: A. Copper versus iron absorption using new in vitro methodology using rhino fecals – University of Ghent

6. Body condition / body weight – Another research area that has also been noted under rhino health. Body condition scoring (BCS) systems have been established for African and Asian

14 2019 Rhino Taxon Advisory Group Research Masterplan rhino species and should be distributed/utilized by researchers and animal managers. These scoring systems are very subjective at each institution, so using standardized protocols should be helpful. Furthermore, efforts to control for bias by sending images of animals from different institutions to one person for evaluation was equally as difficult because of the variation in animal appearance based on photo angle/quality. Perhaps physiological values for leptin, glucose, insulin, etc., could contribute to a continuum of data for a more solid method of evaluating body condition, in addition to computerized assessments, body measurements matched with weights and/or characterizing body types in the process. Tracking weight in some context in relation to dietary nutrients consumed (fiber versus starch as energy sources in pelleted diets for example), while challenging, is important for understanding nutritional impacts on health. This is an important issue since body condition has been suggested as a factor involved in potential iron loading, disease, historically skewed sex ratio of calves and reproductive issues of F1 white rhinos.

What has been done: 2 publications

Current Need: 6.1 Determining markers of body condition beyond BCS, correlated with body weight and other health assessment 6.2 Investigating use and tracking of the variable nutrient impact of dietary enrichments on BC/BW across institutions

7. Influence of dietary phytoestrogens on reproductive success of white rhinos There is continued concern that phytoestrogens may be impacting white rhino reproductive success. Work has evaluated the estrogen activity in rhino feeds; alfalfa and soy are predictably high but Sudan grass also demonstrated high activity. West Coast Bermuda hay was found low in activity but it was high in Bermuda hay grown on the East Coast. Therefore, both types of hay and growing locations/condition likely affect phytoestrogen content. Initial diet changes which lowered phytoestrogens, but also may have lowered energy intake and impacted body condition, have been associated with two successful births at San Diego Zoo Global (unpublished) warranting further investigation.

What has been done: 2 publications

Need: 7.1 Test impact of low phytoestrogen diet and examine in relation to nutrient changes / body weight and body condition, as well as reproductive success.

Research being conducted/ in progress: A. Survey of white rhino holding institutions for diet changes versus reproductive success in recent years - SDZG

8. Science-based browse recommendations – There are often requests for browse lists. In 2014, seasonal browse analyses were encouraged to help institutions make informed decisions about browse choices. Studies exist on the diversity of browse chosen by wild rhinos as well as the nutritional components of the browse. However, the fact is that most zoos will feed what browse

15 2019 Rhino Taxon Advisory Group Research Masterplan they can get locally and with the least amount of cost/effort. Regional browse studies on nutritional value and palatability of local species are helpful as general guides and exist for some areas. Recent papers have illustrated diversity of browse intake in wild areas for Greater One Horned (GOH) and Javan rhinos as well. Developing a published browse database by region could be helpful, but not a priority. Institutions with black rhino are encouraged to develop browse farms to help provide better diets for their animals and these regional studies are a great reference when initiating a browse farm.

What has been done: 2 publications

9. Influence of diet on calf sex ratios – no research published in 5 years This issue was a priority in the past, but the skewed sex ratio appeared spurious hence this is not considered a current research need.

10. Neonatal growth, milk composition, and assisted-rearing This issue was a priority in the past, and while existing information should be compiled for easier access, there has been previous work published describing and documenting these processes under human care.

What has been done: 2 publications

Rhino Nutrition-related References 2013-2018 (see below for citations) Prepared September 2018 Topics: # of references General Nutrition/Diets 13 Iron Overload Disorder 14 Inflammatory Markers 2 Phytoestrogens 2 Body condition scoring 2 Tooth-wear / dental issues 1 Neonatal growth/assist-rearing/milk composition 2 Total 36

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Reproduction

RRC Reproductive Discipline Since the 2014 Masterplan, a total of 49 new peer reviewed manuscripts and conference abstracts have been published in the area of rhino reproduction. As the wealth of research continues to grow so have advancements in addressing previously established reproductive priorities for ex situ, minimally managed and wild rhino populations. For example, mate compatibility, courtship and aggression previously emerged as a high priority issue for Asian rhinos but has since been effectively addressed through rhino manager/keeper/veterinary education regarding acceptable behaviors and utilization of established ovarian biomarkers associated with successful breeding in these species. Some priority issues still remain but have shifted research focus from physiologic origin to that stemming from a management need. Ex situ African black rhino populations were once plagued by skewed natal sex ratio of excess male births compared to that of female calves. While there is no longer significant skew among any of our ex situ rhino populations having an even birth sex ratio means that 50% of calves are males. For species like the African white rhino which excels in large herds with one bull and several cows this presents a population management challenge. Using novel approaches such as establishing protocols for housing/exhibiting multi-male groups and/or integrating sex sorted sperm into assisted reproductive techniques (ART) appears warranted for addressing this issue.

Emerging priorities in the reproductive discipline have and continue to be heavily focused on the female side of the rhino breeding pair. Not many over-arching issues are associated with male rhino reproduction. However, an area that continues to surface is a means by which to empower African male rhinos in natural breeding circumstances. The innate female dominant social dynamic of these species represents a particular research/management challenge. Reliable and repeatable methods of collecting and cryopreserving rhino semen have been established with significant advances in utilizing the biomaterial to provide proof of concept and applicability to

17 2019 Rhino Taxon Advisory Group Research Masterplan population management. There is continued need to strategically bank sperm from individual males and establish cooperatively managed rhino genome resource banks among institutions. While not a top priority, developing chemical induction for semen collection could be especially useful in establishing advanced ART such as IVM/IVF. This methodology could also increase the ability to bank sperm from more individual males (ie, institutions that do not want to use general anesthesia or are not comfortable with administering best practice anesthetic drugs for given species).

The reproductive discipline and the scientists involved in developing and conducting the research needed to meet priority goals could not do so without the assistance and input from rhino management and animal care teams. Operant conditioning for different procedures and overall structured management is key for rhino reproductive research success. The substantial data collected to date has helped understand the normative reproductive characteristics among the different rhino species and identify early and late stage reproductive dysfunctions. However, determining how to prevent these dysfunctions and identifying methods to treat underlying causes are of primary concern moving forward. A multidisciplinary research approach will likely be most effective in addressing select issues like over-conditioning (i.e., impact on reproduction but extends to disciplines of health, nutrition, management and behavior).

Whereas, natural breeding is and continues to be the primary means of maintaining and growing our rhino populations, the need for ART should not be overlooked and remains important for those critically endangered species or doomed populations. Tremendous strides have been made in developing ART for some of our managed rhino species. However, compared to the closest domestic relative the horse, rhino ART is lagging. Therefore, a priority focus must be placed on the advancement of available ART at our disposal to meet the genetic and demographic needs of managed breeding programs for African and Asian rhinos. Optimizing ART for rhino species will facilitate reproductive goals for each population and ease genetic as well as general management. From a welfare perspective it is important to establish the types of reproductive assessments that should be done and at what life stages to ensure optimal breeding recommendations are met for each species.

The five reproductive priorities that emerged for 2019 Rhino Research Masterplan are as follows:

1) Understand and address early and late stage reproductive dysfunction in African and Asian rhinos

1.1 Impact of over-conditioning on reproductive dynamics 1.2 Stillbirth; while stillbirths have occurred in both African species it has predominately been an issue in GOH rhinos. With up-to-date stats, there may emerge changes and/or protocols for expectant females that minimize occurrence. 1.3 Silent estrus 1.4 Acyclicity 1.5 Anovulation 1.6 Early pregnancy loss

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2) Conduct studies to improve AI success across rhino species and develop the in vitro laboratory techniques necessary to save doomed populations/genetically valuable individuals.

2.1 AI timing 2.2 Application of lower sperm numbers (to help conserve genetic material and to aid in the use of sorted sperm in which lower sperm concentration is mandated) 2.3 In vitro maturation/fertilization (northern white and Sumatran rhinos)

3) Develop novel biomarkers of follicular development to enhance natural and assisted reproductive efforts in African and Asian rhinos.

3.1 Estrogen detection. With the exception of GOH rhinos, there has not been a reliable method of measuring estrogen and having it reflect physiologic state. 3.2 Impending ovulation (biomarkers such as behavior, specific hormone milieu and/or ultrasound) 3.3 Anovulation vs. ovulation

4) Determine best practices for reproductive management of male and female rhinos with respect to age, prior reproductive performance and genetic contribution

5) Develop ideal test paradigms for operant conditioning for reproductive procedures in African and Asian rhinos - An improved understanding of rhino behavior as it relates to training for specific reproductive procedures and how reproductive status may influence behavioral response. Framework of established training responses, timeframes for adaptation and to reach steady state behavior.

5.1 Males; hand injection of anesthetic drugs, manual semen collection 5.2 Females; blood collection, ultrasound, artificial insemination

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Genetics

The potential benefits of employing genetic tools for studying rhino conservation and population genetics are tremendous, but considerable research and development is still needed in many areas before direct, efficient, reliable application will be possible. Areas where conservation genetics could be most valuable include studies on: genetic structure among and diversity within surviving populations; dispersal; paternity; and censusing. The potential methodologies would include analyses of: mtDNA, microsatellites, genetic sexing, single-nucleotide polymorphism (SNPs), immunogenetics and the microbiome.

Over the next five years, there needs to be an emphasis on developing the appropriate methodologies:

1. Microsatellite analysis - Microsatellites are powerful if they can be amplified from fecal DNA and should be a priority. However, fecal samples in the tropics deteriorate very rapidly.

2. High throughput sequencing (HTS) – Since fecal DNA typing is difficult and genetic diversity in some rhinoceros species is low, next generation sequencing (NGS) may be of great value in providing the initial screening for polymorphisms needed to develop markers to examine rhino population genetics. These sequences are being used to assemble the genomes of all rhinoceros species.

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3. Standardization – different labs using various loci are reporting different results. There needs to be some consistency, or the techniques may wrongly be considered unreliable for answering conservation/population questions.

4. Shorter amplicons (75-150 bp) need to be targeted by genetic markers and made available for fecal DNA studies since degradation can occur rapidly in fecal material.

5. Fecal microbiome analysis may be useful in some cases as an alternative to analysis of an individual’s own genetic material. Microbial DNA is more plentiful and in better condition when excreted in the feces and each individual has their own microbial profile. This was the methodology employed to determine that the fecal samples from the Viet Nam Javan rhino were all from the same individual.

6. Immunogenetic variability and its relationship to disease resistance and mate choice would be interesting to investigate. If pre-testing for mate compatibility or interest could occur prior to animal transport, it would be very valuable to animal managers in zoos and managed reserves/ranches. It may also be a means of ensuring that immune system diversity is maintained in small populations.

7. Environmental DNA – eDNA is an emerging tool in genetic studies but may be more challenging under tropical conditions just as fecal DNA is more challenging in samples from tropical forests.

Ongoing Priority Genetic Studies

➢ Immunogenetic variability – analyses of the major histocompatibility complex and of innate immune system genes such as Toll-like receptors are being planned and once established will help significantly when analyzing populations for variation in genes that have been under selection and may provide greater fitness when their diversity is maintained. This is particularly important information for species that appear to have low neutral genetic variation, for example the GOH and white rhinoceros.

➢ Fecal DNA analyses – Some work has been done, but the method needs to be optimized so that individual genotypes from fecals can be used for census work and assessing and studying breeding strategies, dispersal, etc. in reserves and parks. The analyses are especially needed for the Javan and Sumatran rhinos since it is the only material available for analyzing populations and learning more about animal numbers, locations, sex, relatedness, etc. Progress has been made towards this goal, and newly developed markers have been used to determine population structure in Sumatran rhinos, while the use of these markers on fecal samples is being implemented in Indonesia for both species. A project to optimize non- invasive genotyping from fecals as a tool for estimating numbers and sex of white and black rhinos from the Kruger National Park has been initiated. Finally, a project optimizing fecal genotyping of D. b. bicornis for census of the Etosha population is being launched.

➢ Study of disease history and parasite load - A critical emerging arena to which genetics can contribute is disease risk assessment of different rhino populations. This is particularly

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relevant in light of the tragic loss of five ex situ Sumatran rhino in Malaysia. A particular avenue worth pursuing is PCR techniques that can determine presence and load of different pathogens in wild populations from tissue collections and fecals. A major study investigating these possibilities is proposed for the Kruger National Park for their C.s.simum and D.b.minor populations.

➢ Social aggregation, dispersal, variance in reproductive success in extant D.b.bicornis (Namibia) - Nearly 200 samples have been collected from D. b. bicornis in Etosha National Park and Damaraland D.b. bicornis. With > 500 animals this is the largest population of endemic black rhinos. The characterization of their genetic structure using 9 microsatellite loci has been completed. Although there are suggestions that these animals cluster when numbers are higher - which is counter to the popular notion that these animals are solitary - genetic analyses at 9 microsatellite loci do not support any significant structure in this population. This may be because so little time has passed since the numbers of this population started to increase and a dispersal /genetic structure equilibrium may not have been reached. Using more NGS-derived genetic markers, another look at this population 15 years hence would be instructive with respect to emerging genetic structure in this, the largest black rhino population.

➢ Individual genetic variation – Such information should be examined and compared to help guide breeding recommendations. Such genetic studies can verify assumptions made regarding founder animals. Genetic markers can also be used to verify pedigree-based analyses and identify genetically valuable individuals.

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Behavior & Ecology

Priority Issue: Investigating behavioral and environmental factors that affect rhino wellbeing

1. Developing and validating methods to assess wellbeing: Documentation and evaluation of animal wellbeing is now a requirement of all AZA zoos, and WAZA encourages considerations for wellbeing even among conservation activities executed in situ. Wellbeing involves the promotion of positive states within five broad domains relevant to an animal’s life, including nutrition, physical health, environment, behavior, and mental state. Within the context of environment, behavior, and mental state, metrics for gauging whether an animal is in a more positive state or a more negative state need to be developed, and the validity of their application to rhinos and their appropriate interpretation in the context of rhino biology and husbandry require investigation. Some research has already evaluated potential associations between behavior, zoo environment, and successful (or unsuccessful) reproduction. Evidence supporting a relationship between other endpoints of wellbeing (i.e., good (or poor) health or nutrition, a positive (or negative) mental state and specific behaviors, environments, or measurable physiological parameters associated with psychological states (e.g., inflammatory markers, acute phase proteins) needs to be built. One assessment tool, WelfareTrak (by Brookfield Zoo), has been tested for its informative ability regarding black rhino welfare.

2. Specific factors that might affect wellbeing that are a priority for research

2.1 Behavioral effects of dehorning – Dehorning rhinos is one option being used to curtail the poaching epidemic. While dehorning itself is not painful and glucocorticoid concentrations appear to return to pre-procedure values within a few days, the rhino’s response to the sudden absence of the horn and how the rhino adapts behaviorally to that physical change in the short- and long-term are not understood, especially related to

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overall wellbeing. Sparring behavior, territorial defense, breeding success, and predator defense are all key areas where the absence of a horn might alter wellbeing outcomes, either positively or negatively.

2.2 Behavioral response to intensive medical care following traumatic injury and the re- release process – Following traumatic injury, such as poaching attempts, some wild rhinos are left in a grisly condition that requires many months of intensive medical care and rehabilitation. During this time, the rhinos are exposed to stimuli they have never experienced before and are isolated from natural stimuli and choices to which they were accustomed. Although treatment is necessary for the rhino to survive and metrics of health are monitored closely to determine the progress of recovery, how the rhino responds behaviorally to this dramatic change in life conditions also should be monitored. Behaviors that might be associated with distress or depressed mental state v. recovery need to be described. Once rehabilitated and deemed healthy for release, the rhino’s behavioral response to the release process needs to be monitored and evaluated to determine its relevance to positive or negative states of wellbeing.

2.3 Behavioral response to management in bomas and during transport – During translocation/reintroduction or quarantine for shipment, rhinos are kept in bomas. Behavioral and physiological measures of wellbeing for rhinos in transition should be developed. A scoring system has recently been developed for white rhinos in bomas (Miller et al., 2016) and additional study would be useful, especially for the other species. Non-invasive measures of the stress response other than glucocorticoids would be valuable.

2.4 Ex situ meta-analyses across management practices, environments, etc. – The effects of various demographic (e.g., age-sex ratios), social (e.g., group size and density), and environmental characteristics of zoos (e.g., climate, enclosure characteristics) on behavior, especially features that differ from what the species would experience in range country and those that involve human interactions (e.g., enrichment, training and education/guest-relations programs), need to be assessed from a wellbeing perspective for rhino populations in managed care. Beyond population-level data, the meta-analysis approach should make it possible to track changes in an individual rhino’s indices of wellbeing as it moves from one facility to another, and to subsequently design controlled experiments based on observed trends.

Additional Behavior and Ecology Research Needs

1. Understanding and managing small populations in the face of habitat fragmentation, poaching, and climate change: In the shrinking habitat that exists for rhinos, whether a viable population can sustain itself there is a concern, especially as environmental conditions change over time and the poaching crisis continues.

1.1 Population viability and habitat suitability – This is a very active area of research for all rhino species; much has been learned, but many questions need additional study. Assessments in each subpopulation and habitat pocket are needed to determine which

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subpopulations are sources and which are sinks. New research in this area can take advantage of techniques developed in other fields, including dung DNA analysis and fecal hormone metabolite analysis for determination of pregnancy status, to improve survey methods. Advancements in the use of recently-developed technologies, including drones and on-animal accelerometers, might improve managers’ abilities to monitor rhino populations. How the source versus sink fate of a given subpopulation or how the long- term viability of a habitat pocket will affect the status of the larger metapopulation is crucial to understand. The reasons for subpopulation growth or decline over time and for changes in habitat suitability across seasons need to be better understood in order to manipulate the outcome. In addition, a deeper understanding of how reproduction and population growth are affected by population density and spatial distribution, limitations on sub-adult dispersal, social constraints such as mate choice and dominance, and environmental variability and climate shifts are needed. Key factors that promote adult and sub-adult survival to maximize fecundity also need to be identified.

1.2 Corridor habitat – Assessments to determine key positive and negative attributes that affect rhino dispersal between subpopulations are needed. Habitat and climate changes over time need to be modeled to determine where corridors will be needed in the future.

1.3 Habitat restoration and management – Habitat restoration and management can improve conservation outcomes, but the techniques themselves require further research. How to control and eliminate the invasive plant Mikania micrantha in GOH rhino habitat, and how to prevent or mitigate desertification and habitat water loss in Africa are of particular concern. The effects of fire and changes in megaherbivore densities, including the potential loss of rhino populations, on savanna ecosystems need additional study. Recent studies have looked at the potential effects of competition between rhinos and other megaherbivores, such as elephants and the Javan bull (banteng), on habitat quality, plant food availability, and habitat carrying capacity. Additional studies of inter-species interactions, especially in marginal and human-modified habitats, would be very useful.

2. Determining behavioral and ecological factors that result in successful translocation/reintroduction: While many of the challenges noted above for small populations apply to situations in which groups of rhinos or individuals are translocated/reintroduced, there are some additional considerations specific to this situation that require further study.

2.1 Meta-analyses of data on translocation attempts and outcomes – As a first step, accessibility to this type of data needs to be improved in such a way that the safety of the rhinos is not compromised. Meta-analysis could provide insights about how individual rhino characteristics and specific release area characteristics might influence the outcome of the attempt.

2.2 Dispersal – How much and where rhinos move immediately after release, and what influences that movement are important areas of study. Recent research looking at what types of information rhinos communicate to each other through olfaction and vocalization

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might provide new opportunities for manipulating and improving translocation and reintroduction.

2.3 Territoriality and breeding success – Factors that influence which bulls establish a territory and secure reproductive opportunities need to be better understood. This area of study might benefit from developing methods for small reserve managers to document social interactions.

2.4 Sub-adults – As more calves are orphaned by poaching, successful reintroduction techniques for sub-adults after hand-rearing are essential. Important areas of study include the impact of boma location, the effects of age upon release, predator and territorial bull avoidance, and social and environmental factors that influence early home range establishment.

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Management

Priority Issue: Determine management aspects that can enhance areas of rhino conservation within the ex situ facilities and potential benefits to wild rhinos.

1. Optimize/Standardize Body Condition Scoring - Whereas, this is a veterinary/nutrition focus, the caretakers of the animals should all be aware of the different scales and what the scores mean in relation to their charges. Management will be affected by low and high scores, especially in regard to breeding success. There are several systems—mainly based on a five-point scale, for rhinos.

2. Determine factors contributing to success or failure of male/male housing or bachelor groups - There are more and more opportunities to house two young males together in traditional zoo settings, and larger groups of males in larger landscape facilities. The International Rhino Keepers Association (IRKA) is interested in helping to do the research with respect to ages, size of exhibit, exhibit design/layout, factors that have been known to help, issues to avoid, etc. to determine if/when this can work and for how long we should expect successful groupings to remain in-tact.

3. Determine success/failure in pairing older animals with younger animals - There are many situations in which one of two companion animals has died and the SSP wants to pair a younger animal with that lone animal. The IRKA is also interested in looking into the success of those introductions over the past years since this will most likely need to continue into the future as pairs become lone animals. Ultimately, when the other older animal dies, it would then make sense to put another compatibly aged animal with the lone animal.

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4. Conditioning recommendations to help with other RRC needs. 4.1 Ultrasonography, phlebotomy, oral exams, etc. These behaviors have already been conditioned in many rhinos throughout the US and Europe. Perhaps the Rhino Research Council (in conjunction with the TAG and SSP Coordinators) should encourssage a more formalized training/conditioning program that is focused on the aspects that each of the RRC disciplines might require. A compilation of best practices could be useful. 4.2 Are there other training opportunities that don’t fall under ‘medical, scientific research’ headings? Blood collection will be valuable for the genetics discipline, as well as nutrition and reproduction. Ultrasound conditioning is obviously beneficial to the reproductive research. Collection of urine and saliva could be beneficial for various research projects, and of course, fecal collections which require no training. Scale training, especially for those with portable scales, could be important for BCS monitoring. 5. Can research be done in captivity that helps the wild populations? There has been some opportunity for collecting measurements on ex situ rhinos to help with collar fitting in the wild. Is there more opportunity for this type of work?

6. Management decisions that affect the potential to be a release candidate - This was something discussed in 2013, regarding the ex situ animals being an ‘assurance’ population for potential reintroduction. It is doubtful there will be large scale reintroductions in the near future, but could managers participate in determining what makes a ‘good’ candidate for reintroduction? This would go hand in hand with some of the needs described in the behavior and ecology section.

7. Collection management styles 7.1 Exhibit strengths and weaknesses for non-breeding animals 7.2 Exhibit strengths and weaknesses for breeding groups 7.3 Conditioning for moves—crate training, facility design, factors that improve moves 7.4 Determine stressors and levels of stress in animals before, during and after moves (in conjunction with medical and behavior disciplines)

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In situ Research Priorities

A request for input regarding in situ rhino research priorities was distributed to a few rhino field researchers/conservationists. The topics received that are not already represented elsewhere in the document are summarized below.

African Rhinos

1.) Objective determination of conservation value of different rhino populations - This issue becomes important to influence more appropriate metapopulation management of rhinos by highlighting the fact that the conservation value of rhino populations drops disproportionately as numbers decline, an issue that is really important for countries that are not making appropriate policy decisions on the biological management of rhinos (i.e., 25% of rhinos are on private land but that 25% does not equate to 25% of the conservation value of all rhinos in Africa). What is happening in terms of genetic exchange between small, privately-owned groups of rhinos in South Africa? What are the effects of any age/sex skewing, derivation of trend data on inbreeding coefficients to help provide guidelines for the IUCN/SSC African Rhino Specialist Group to example current classifications (“Key”, “Important”, to also include “Marginally viable,” “Non-viable”, etc.

2.) Rhino parentage analysis to determine effects of management practices – These data are needed to determine what husbandry practices (translocations, dehorning, etc.) may be impacting breeding dynamics.

3.) Exploration of alternative low-power radio frequency systems to track rhinos and to integrate anti-poaching data – There is the need for a software programming expert to work on a simple “platform” that integrates primary digital data from devices in the field (digital radios that transmit GPS positions, Sigfox devices sending

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locational information from tagged rhinos and from Sigfox-linked devices such as vehicle trackers, gunshot detectors, etc., so that this information can be displayed in a customized way on a computer screen in an ops room.

4.) Economic analysis of rhino conservation - What economic values do rhinos contribute to national economies? How much do rhinos serve as proxy indicators for ecosystem processes and other components of natural capital and what are the economic implications arising from this? How much does rhino conservation cost over and above the basic per area protection costs that need to be met for a typical spectrum of other wildlife species in protected areas (both private and state) in Africa? What implications does this have for international performance-based financial support for productive rhino breeding situations? What economic stimuli can be applied to achieve new range expansion options in South Africa, which depends upon small land units being induced to amalgamate into larger ones?

Asian Rhinos

1.) Desk study documenting information on Sumatran rhino captures and translocations in the 1980s. The ecology and sociobiology of Asian rhinos differs vastly from that of the African species. Only anecdotal data are available on Sumatran rhino captures from the 1980s. Two young females, Ratu and Rosa, wandered out of the forest and were successfully transferred to the Sumatran Rhino Sanctuary in 2005, and a capture attempt in 2016 failed. A desk study documenting information and lessons learned from the captures in the 1980s would be a useful first step to lay the groundwork for future efforts.

2.) Genetic studies of Javan rhinos - The only existing population of Javan rhinos in Ujung Kulon National park has fluctuated in size over the years, but has never been estimated to be over ~85 animals and currently stands at ~68 individuals. Because there is no other wild population of this rhino species, the infusion of genetic diversity is not possible. Regardless, there is considerable concern about the extent of inbreeding within this population and a desire to conduct a population genetic study to determine relatedness and a parentage analysis of the existing animals.

3.) Nutritional analysis of food plants most frequently fed to Sumatran rhinos at the Sumatran Rhino Sanctuary in Way Kambas National Park - There is a large collaborative effort underway to capture some of the last wild Sumatran rhinos for intensive management in breeding centers. Only one native breeding center in SE Asia has succeeded in producing calves, and therefore any new center should be built in its likeness and staff should adopt similar husbandry and management practices. Because browse availability will likely vary in different locations, it would be helpful to conduct a nutrient analysis of the current diets of the rhinos at the Sumatran rhino sanctuary so that nutrient value can be replicated at new facilities with different browse.

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Addendum

Recent (2013-2018) Relevant Rhino Research Papers

Published Literature on Rhino Nutrition

Bapodra P, Dierenfeld E, Wolfe BA. Evaluation of season‐related dietary changes on the serum profiles of fat‐soluble vitamins, mineral, fatty acids, and lipids in the captive greater one horned rhinoceros (Rhinoceros unicornis). Zoo Biol. 2014; 33: 314-319. Dutta DK, Bora PJ, Mahanta R, et al. Seasonal variations in food plant preferences of reintroduced Rhinos Rhinoceros unicornis (Mammalia: Perrissodactyla: Rhinocerotidae) in Manas National Park, Assam, India. J Threatened Taxa. 2016; 8: 9525-36. Edwards KL, Shultz S, Pilgrim M, et al. Irregular ovarian activity, body condition and behavioural differences are associated with reproductive success in female eastern black rhinoceros (Diceros bicornis michaeli). Gen Comp Endocrinol. 2015; 214: 186-94. Gimmel A, Hoby S, Deillon L, von Houwald F, Schweizer R, Kölln M, Ratert C, Liesegang A. Milk composition of Indian rhinoceros (Rhinoceros unicornis) and changes over lactation. Journal of Zoo and Wildlife Medicine. 2018;49(3):704-14. Hariyadi AR, Sajuthi D, Astuti DA, Alikodra HS, Maheshwari H. Analysis of nutrition quality and food digestibility in male Javan rhinoceros (Rhinoceros sondaicus) in Ujung Kulon National Park. Pachyderm. 2016 Jul 19(57):86-96. Heidegger EM, von Houwald F, Steck B, Clauss M. Body condition scoring system for greater one‐horned rhino (Rhinoceros unicornis): Development and application. Zoo Biology. 2016 Sep 1;35(5):432-43. Huntley NF, Naumann HD, Kenny AL, Kerley MS. Black rhinoceros (Diceros bicornis) and domestic horse (Equus caballus) hindgut microflora demonstrate similar fermentation responses to grape seed extract supplementation in vitro. J Anim Physiol Anim Nutr. 2016; 1- 15. Lavin SR, Sullivan KE, Wooley SC, Stone K, Russell S, Valdes EV. Near infrared reflectance spectroscopy (NIRS) analyses of nutrient composition and condensed tannin concentrations in Carolina willow (Salix caroliniana). Zoo Biology. 2015 Nov 1;34(6):576-82. Lavin SR, Sullivan KE, Wooley SC, Robinson R, Singh S, Stone K, Russell S, Valdes EV. Nutrient and plant secondary compound composition and iron‐binding capacity in leaves and green stems of commonly used plant browse (Carolina willow; Salix caroliniana) fed to zoo‐managed browsing herbivores. Zoo Biology. 2015 Nov 1;34(6):565-75. Mbatha KR, Bakare AG, Browse silage as potential feed for captive wild ungulates in southern Africa, Animal Nutrition Journal (2018), doi: 10.1016/j.aninu.2017.12.003.

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Miller M, Chavey PS, Hofmeyr J, et al. Evaluation of serum ferritin and serum iron in free- ranging black rhinoceros (Diceros bicornis) as a tool to understand factors affecting iron- overload disorder. J Zoo Wildl Med. 2016; 47: 820-6. Mukhlisi M, Atmoko T, Yassir I, Setiawan R, Kusuma AD. Abundance and nutrient content of some food plants in Sumatran rhino habitat in the forest of Kutai Barat, East Kalimantan, Indonesia. Pachyderm. 2017 Oct 2(58):77-87. Oltman W, Olds J, Makowski AJ, et al. Seasonal variation of 25-hydroxy-vitamin D in two captive Eastern black rhinoceros (Diceros bicornis michaeli). National Merial Veterinary Scholars Symposium, The Ohio State University. 2016. Roth TL, Reinhart PR, Kroll JL. Serum Ferritin Concentration is Not a Reliable Biomarker of Iron Overload Disorder Progression or Hemochromatosis in the Sumatran Rhinoceros (Dicerorhinus sumatrensis). Journal of Zoo and Wildlife Medicine. 2017;48(3):645-58. Salyer, Lorien, Iron Metabolism Genes in Browsing and Grazing Rhinoceroses: Implications For Iron Overload Disorder. 2017. Honors Thesis Projects. 54. Schook MW, Wildt DE, Raghanti MA, et al. Increased inflammation and decreased insulin sensitivity indicate metabolic disturbances in zoo-managed compared to free-ranging black rhinoceros (Diceros bicornis). Gen Comp Endocr. 2015; 217: 10-19. Sullivan KE, Lavin SR, Livingston SE, Knutson M, Valdes EV, Warren LK. Comparative digestibility of dry matter, protein, and fiber between the horse and black rhinoceros. Journal of Equine Veterinary Science. 2015 May 1;35(5):405. Sullivan KE, Valdes EV. Update on Rhinoceros Nutrition. In: Miller RE, Lamberski N, Calle P, editors. Miller-Fowler's Zoo and Wild Animal Medicine Current Therapy, Volume 9. Elsevier Health Sciences; 2018. 699- 706. Taylor LA, Müller DW, Schwitzer C, et al. Tooth wear in captive rhinoceroses (Diceros, Rhinoceros, Ceratotherium, Perissodactyla) differs from that of free-ranging conspecifics. Contributions to Zoology. 2014; 83. Thakur S, Upreti CR, Jha K. Nutrient analysis of grass species consumed by greater one-horned rhinoceros (Rhinoceros unicornis) in Chitwan national park, Nepal. International Journal of Applied Sciences and Biotechnology. 2014 Dec 24;2(4):402-8. Tubbs CW, Durrant BS, Milnes MR. Reconsidering the use of soy and alfalfa in southern white rhinoceros diets. Pachyderm. 2017 Oct 2(58):135-9. Tubbs CW, Moley LA, Ivy JA, et al. Estrogenicity of captive southern white rhinoceros diets and their association with fertility. Gen Comp Endocr. 2016; 238: 32-38. Watanabe M, Roth TL, Bauer SJ, Lane A, Romick-Rosendale LE. Feasibility Study of NMR based serum metabolomic profiling to animal health monitoring: a case study on iron storage disease in captive Sumatran rhinoceros (Dicerorhinus sumatrensis). PloS one. 2016;11(5):e0156318. Wojtusik J, Roth TL. Investigation of Factors Potentially Associated with Serum Ferritin Concentrations in the Black Rhinoceros (Diceros Bicornis) Using a Validated Rhinoceros- Specific Assay. Journal of Zoo and Wildlife Medicine. 2018;49(2):297-306.

Published Conference Abstracts on Rhino Nutrition Brooks M, Lee B, Pera J. Case Study: Winos for rhinos: feeding grape pomace to black rhinoceros (Diceros bicornis) as a method for mitigating iron storage disease. 2017. Proceedings of the Twelfth Conference on Zoo and Wildlife Nutrition, AZA Nutrition Advisory Group, Frisco, TX.

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Ellis KB, Whisnant S, Fellner V, Koutsos E, Ange-van Heugten. 2013. The interaction of diet and fecal cortisol in the Southern white rhinoceros (Ceratotherium simum simum). In Ward A, Coslik A, Mahan K, Treiber K, Reppert A, Maslanka M, Eds. Proceedings of the Tenth Conference on Zoo and Wildlife Nutrition, AZA Nutrition Advisory Group, Salt Lake City, UT. Koutsos E, Clauss M, Valdes E. Designing iron controlled diets for exotic hoofstock- variability in raw materials and manufacturing contributions to total dietary iron. Comparative Nutrition Society Symposium, Rio Grande, PR. 2016. Mimiko J, Stringer E, Parsons J. 2017. Case Study: Iron In Black Rhinoceros Diets: The Impact of Pasture. Proceedings of the Twelfth Conference on Zoo and Wildlife Nutrition, AZA Nutrition Advisory Group, Friscoe, TX. Murtaugh K, Power M, Ward A. Macronutrient composition of milk from an Asian rhinoceros. 2017. Proceedings of the Twelfth Conference on Zoo and Wildlife Nutrition, AZA Nutrition Advisory Group, Frisco, TX. Paglia D. Human Medical Experience Provides Paradigms Relevant To Captive Breeding of Endangered Wildlife: Rationale for Prevention and Therapy of Hemolytic and Iron Overload Propensities In Browser Rhinoceroses, Tapirs and Other Susceptible Species. American Association of Zoo Veterinarian Conference. 2017, Frisco, TX. Ricketts V, C Sauer, ES Dierenfeld and K Whitehouse-Tedd. 2018. Feed intake and dietary content of iron (Fe) copper (Cu), tannic acid and vitamin E of five captive black rhinoceros (Diceros bicornis) in a UK collection. Marwell Wildlife Nutrition Conference, Colden Common, Winchester, UK. Sullivan KE, Valdes EV, Livingston SE, et al. Use of a novel iron chelator (HBED) in black rhinoceros. In Bissell H, Brooks M Eds. Proceedings of the Eleventh Conference on Zoo and Wildlife Nutrition, AZA Nutrition Advisory Group, Portland, OR. 2015. Sullivan KE, Coffey R, Lavin SR, et al. Sequencing the Black Rhino L-ferritin Gene: How Accurate is our Testing? Proceedings of the Nutrition Advisory Group to the AZA bi- annual conference, Frisco, TX. 2017. Sullivan KE, Lavin SR, Livingston SE, Knutson M, Valdes EV, Warren L. Safety and efficacy of a novel iron chelator in equine as a model for black rhinoceros. Flat Rock, NC: Comparative Nutrition Society. 2014. Sullivan KE, Livingston S, Williams S, Mylniczenko N, Rodriguez C, Pye G, Valdes EV. 2016. Iron Overload Disorder in Browsing Rhinos 2016 Workshop: A Review of Current Goals and Practical Action Planning. Comparative Nutrition Society 11th Biennial Symposium. Rio Grande, Puerto Rico. Sullivan KE, Mylniczenko ND, Emerson JA, Hall NH, Fontenot D, De Voe R, Nolan E, Stacy N, Livingston SE, Lavin SR, Valdes EV, Pye GW. 2015. Case study: a hemolytic event in an iron overloaded black rhinoceros (Diceros bicornis) in association with cessation of chelation therapy. In Bissell H, Brooks M Eds. Proceedings of the Eleventh Conference on Zoo and Wildlife Nutrition, AZA Nutrition Advisory Group, Portland, OR. Sullivan KE, Ardente A, Livingston S, Williams S, Valdes EV. 2018. Impact of differing levels of oral supplementation of vitamin E for white (Ceratotherium simum simum) and black rhinoceros (Diceros bicornis) on serum levels and fecal losses. Comparative Nutrition Society 12th Biennial Symposium. Quebec City, Canada.

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Publications on Rhino Reproduction Compilation of published rhino 'reproductive' research since last Masterplan *highlighted= references abstract/conference proceedings

2018 Gomez, M. C., Cates, Y., Stansfield, D. B., Young, C., Klee, R., & Durrant, B. (2018). 187 Morphological Appearance and Expression of Spermatogonial Stem Cell Markers in White Rhinoceros Testicular Tissue. Reproduction, Fertility and Development, 30, 233-234. Hermes, R., Hildebrandt, T.B., Goritz, F. 2018. Cryopreservation in rhinoceros- setting a new benchmark for sperm cryosurvival. PLoS One 13(7):e0200154. doi: 10.1371/journal.pone.0200154 Hildebrandt, T.B., Hermes, R., Colleoni, S., Diecke, S., Holtze, S., Renfree, M.B., Stejakal, J., Hayashi, K., Drukker, M., Loi, P., Goritz, F, Lazzari, G., Galli, C. 2018. Embryos and embryonic stem cells from the white rhinoceros. Nat Commun 9(1):2589. doi: 10.1038/s41467-018-04959-2 Pennington, P.M., Durrant, B.S. (2018). Assisted reproductive technologies in captive rhinoceroses. Mammal Review https://doi.org/10.1111/mam.12138 Stoops, M.A., Winget, G.D., DeChant, C.J., Ball, R.L., Roth, T.L. (2018). Early fetal sexing in the rhinoceros by detection of male-specific genes in maternal serum. Molecular Reproduction & Development, 85 (3) 197-204. https://doi.org/10.1002/mrd.22953. Wojtusik, J., Stoops, M.A., Roth, T.L. (2018). Comparison of soy lecithin, coconut water, and coconut milk as animal protein-free alternatives to egg-yolk in extender for semen cryopreservation of the African black rhinoceros (Diceros bicornis) and Indian rhinoceros (Rhinoceros unicornis). Theriogenology, 121: 72-77. https://doi.org/10.1016/j.theriogenology.2018.07.042

2017 Bickley, S.M., Pollock, K.E., Stoops, M.A. (2017). Biological and behavioral reasons for unsuccessful breeding in captive female African black rhinos: olfactory stimulation as a method for improvement. In Proceedings: the 6th International Society of Wildlife Endocrinology, Orlando, FL, USA Aug 14-16. P75. Korody, M. L., Pivaroff, C., Nguyen, T. D., Peterson, S. E., Ryder, O. A., & Loring, J. F. (2017). Four new induced pluripotent stem cell lines produced from northern white rhinoceros with non-integrating reprogramming factors. bioRxiv, 202499. Kottwitz, J., Stoops, M., Reeves, J., Harmon, R., Wilborn, R., Edmonson, M., Boothe, D. (2017). The negative effects of analgesic and anesthetic drugs on sperm motility: implications for assisted breeding in captive rhino. In Proceedings: the 49th Annual American Association of Zoological Veterinarians Conference, Dallas, TX USA Sept 22-29. P107-108. Roth, T. L., Schook, M. W., & Stoops, M. A. (2017). Monitoring and controlling ovarian function in the rhinoceros. Theriogenology. DOI: 10.1016/j.theriogenology.2017.12.007 Schwarzenberger, F. (2017). Comparative evaluation of gestation in three rhinoceros species (Diceros bicornis; Ceratotherium simum, Rhinoceros unicornis). In Proceedings: International Society of Wildlife Endocrinology, Orlando, FL USA Aug 14-16. P69. Stoops, M.A., O'Brien, J.K., Niederlander, J., Metrione, L., Pootoolol, J., Delk, K., Niemuller, C., Hagen, D., Douglas, R.H., Proudfoot, J. (2017). Administration of biorelease progesterone and estradiol or GnRH to induce estrous cycles and breeding in anestrous

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African white rhinoceroses (Certatotherium simum simum). In Proceedings: International Society of Wildlife Endocrinology, Orlando, FL USA Aug 14-16. P111. Tubbs, C. W., Durrant, B. S., & Milnes, M. R. (2017). Reconsidering the use of soy and alfalfa in southern white rhinoceros diets. Pachyderm, 58, 135-139. Ververs, C., Hostens, M., van Zijll Langhout, M., Otto, M., Govaere, J., & Van Soom, A. (2017). 109 REPRODUCTIVE PERFORMANCE PARAMETERS IN A LARGE HERD OF CONFINED FREE-ROAMING WHITE RHINOCEROSES (CERATOTHERIUM SIMUM). Reproduction, Fertility and Development, 29(1), 163-163. Wojtusik, J., Stoops, M.A., Roth, T.L. (2017). Use of animal-protein free extenders as alternatives to standard egg-yolk based extender in semen cryopreservation of the black (Diceros bicornis) and Indian (Rhinoceros unicornis) rhino. In Proceedings: 11th International Conference on Behaviour, Physiology, and Genetics of Wildlife, Berlin, Germany Oct 4-7th. P135. 2016 Galli, C., Hermes, R., Goeritz, F., Colleoni, S., Diecke, S., Drukker, M., Hayashi, K., Holtze, S., Lazzari, G., Payne, J., Sos, E., Steiskal, J., Wiesner, M., Zainuddin, Z.A., Hildebrandt, T.B. 2016. First results of oocyte maturation and in-vitro-fertilisation (IVF) in Sumatran and northern white rhinoceroses. In: Proceedings 15th International Elephant & Rhino Conservation & Research Symposium, Singapore Zoo, Singapore Nov. 13-18. 51.

Hermes, R., Schwarzenberger, F., Göritz, F., Oh, S., Fernandes, T., Bernardino, R., ... & Saragusty, J. (2016). Ovarian down regulation by GnRF vaccination decreases reproductive tract tumour size in female white and greater one-horned rhinoceroses. PloS one, 11(7), e0157963. O'Brien, J.K., Stoops, M.A., Roth, T.L., Ball, R.L., Montano, G.A., Stenman, K.L., Posy, J.L., Saiers, R., Ramer, J.C., Love, C.C., Robeck T.R. (2016). Progress in sperm sorting and cryopreservation technologies for modifying population sex ratio and preserving genetic diversity in the rhinoceros and elephant. In: Proceedings 15th International Elephant & Rhino Conservation & Research Symposium, Singapore Zoo, Singapore Nov. 13-18. P48. Roth, T. L., Stoops, M. A., Robeck, T. R., & O’Brien, J. K. (2016). Factors impacting the success of post-mortem sperm rescue in the rhinoceros. Animal Reproduction Science, 167, 22-30. Roth, T. L., Stoops, M. A., Robeck, T. R., & O’Brien, J. K. (2016). 116 FACTORS IMPACTING THE SUCCESS OF POSTMORTEM SPERM RECOVERY AND CRYOPRESERVATION IN THE RHINOCEROS. Reproduction, Fertility and Development, 28, 188-188. Santymire, R.M., Misek, S., Gossett, J., Kamhout, M., Chatroop, E., Rafacz, M. (2016). Male behaviours signal the female's reproductive state in a pair of black rhinoceros housed at Lincoln Park Zoo. Journal of Zoo and Aquarium Research, 4, 30-37. Saragusty, J., Diecke, S., Drukker, M., Durrant, B., Friedrich, B-N. I., Galli, C., Goritz, F., Hayashi, K., Hermes, R., Holtze, S., Johnson, S., Lazzari, G., Loi, P., Loring, J.F., Okita, K., Renfree, M.B., Seet, S., Voracek, T., Stejskal, J., Ryder, O.A., Hildebrandt, T.B. (2016). Rewinding the process of mammalian extinction. Zoo Biology, 35, 280-292. Stoops, M. A., Campbell, M. K., DeChant, C. J., Hauser, J., Kottwitz, J., Pairan, R. D., Shaffstall, W., Volle, K., Roth, T. L. (2016). Enhancing captive Indian rhinoceros genetics

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via artificial insemination of cryopreserved sperm. Animal Reproduction Science, 172, 60- 75. Tubbs, C. W., Moley, L. A., Ivy, J. A., Metrione, L. C., LaClaire, S., Felton, R. G., Durrant, B.S., Milnes, M. R. (2016). Estrogenicity of captive southern white rhinoceros diets and their association with fertility. General and Comparative Endocrinology, 238, 32-38. von Houwald, F. (2016). Husbandry, management and breeding of the Greater one-horned rhinoceros Rhinoceros unicornis at Zoo Basel. International Zoo Yearbook, 50, 203-214. 2015 DeCourcy, K., Campbell, M., Hauser, J., Kottwitz, J., Levens, G., Pairan, R., Volle, K, Stoops, M.A. (2015). Using urinary hormone analysis to predict gender and assess fetal viability in the Indian rhinoceros (Rhinoceros unicornis). In: Proceedings Annual Conference of the American Association of Zoo Veterinarians. Portland, OR USA Sept25-Oct 2. P82-83. Edwards, K. L., Shultz, S., Pilgrim, M., & Walker, S. L. (2015). Irregular ovarian activity, body condition and behavioural differences are associated with reproductive success in female eastern black rhinoceros (Diceros bicornis michaeli). General and Comparative Endocrinology, 214, 186-194. Edwards, K. L., Shultz, S., Pilgrim, M., & Walker, S. L. (2015). Male reproductive success is correlated with testosterone in the eastern black rhinoceros (Diceros bicornis michaeli). General and Comparative Endocrinology, 213, 40-49. Edwards, K. L., Walker, S. L., Dunham, A. E., Pilgrim, M., Okita-Ouma, B., & Shultz, S. (2015). Low birth rates and reproductive skew limit the viability of Europe’s captive eastern black rhinoceros, Diceros bicornis michaeli. Biodiversity and Conservation, 24, 2831-2852. Gener, S., Schwarz, C., Grothmann, P., Bernhard, A., Eulenberger, K., Einspanier, A., & Gottschalk, J. (2015). Oestrus and pregnancy detection in the black rhinoceros (Diceros bicornis) by faecal hormone analysis. Reproduction in Domestic Animals, 50, 33-34. Moresco, A., Larsen, R.S., Boon, D., O'Brien, J.K., Stoops, M.A. (2015). Semen collection in black rhinoceros (Diceros bicornis) via urethral catheterization. In: Proceedings Annual Conference of the American Association of Zoo Veterinarians. Portland OR USA Sept25- Oct2. P170-171. O’Brien, J. K., Roth, T. L., Stoops, M. A., Ball, R. L., Steinman, K. J., Montano, G. A., Love, C.C., Robeck, T. R. (2015). Sperm sex-sorting and preservation for managing the sex ratio and genetic diversity of the southern white rhinoceros (Ceratotherium simum simum). Animal Reproduction Science, 152, 137-153. Van der Goot, A. C., Martin, G. B., Millar, R. P., Paris, M. C. J., & Ganswindt, A. (2015). Profiling patterns of fecal 20-oxopregnane concentrations during ovarian cycles in free- ranging southern white rhinoceros (Ceratotherium simum simum). Animal Reproduction Science, 161, 89-95. Ververs, C., van Zijl Langhout, M., Govaere, J., & Van Soom, A. (2015). Features of reproduction and assisted reproduction in the white (Ceratotherium simum) and black (Diceros bicornis) rhinoceros. Vlaams Diergeneeskundig Tijdschrift, 84, 175-187. 2014 Cain, B., Wandera, A.B., Shawcross, S.G., Edwin, H.W., Stevens-Wood, B., Kemp, S.J., Okita- Ouma, B., Watts, P.C. (2014). Sex-biased inbreeding effects on reproductive success and home range size of the critically endangered black rhinoceros. Conservation Biology, 28, 594-603.

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Capiro, J. M., Stoops, M. A., Freeman, E. W., Clawson, D., & Schook, M. W. (2014). Effects of management strategies on glucocorticoids and behavior in Indian rhinoceros (Rhinoceros unicornis): translocation and operant conditioning. Zoo Biology, 33, 131-143. Edwards, K. L., McArthur, H. M., Liddicoat, T., & Walker, S. L. (2014). A practical field extraction method for non-invasive monitoring of hormone activity in the black rhinoceros. Conservation Physiology, 2(1). Freeman, E.W., Meyer, J.M., Bird, J., Adendorff, J., Schulte, B.A., Santymire, R.M. (2014). Impacts of environmental pressures on the reproductive physiology of subpopulations of black rhinoceros (Diceros bicornis bicornis) in Addo Elphant National Park, South Africa. Conservation Physiology, 2 doi:10.1093/consphys/cot034. Hermes, R., Göritz, F., Saragusty, J., Stoops, M. A., & Hildebrandt, T. B. (2014). Reproductive tract tumours: the scourge of woman reproduction ails Indian rhinoceroses. PloS one, 9(3), e92595. Metrione, L., & Eyres, A. (2014). Rhino Husbandry Manual. Fort Worth (TX): International Rhino Foundation. Penfold, L.M., Powell, D., Traylor-Holzer, K., Asa, C.S. (2014). "Use it or lose it": characterization, implications, and mitigation of female infertility in captive wildlife. Zoo Biology, 33, 20-28. Schwwarz, C., Grothmann, P., Gottschalk, J., Eulenberger, K., Einspainer, A. (2014). Breeding management of black rhinos (Diceros bicornis michaeli) in Magdeburg zoo. Tierarztliche Praxis Ausgabe G Grosstiere/Nuztiere, 42, 150-155. Stoops, M. A., West, G. D., Roth, T. L., & Lung, N. P. (2014). Use of urinary biomarkers of ovarian function and altrenogest supplementation to enhance captive breeding success in the Indian rhinoceros (Rhinoceros unicornis). Zoo Biology, 33, 83-88. Tubbs, C., McDonough, C.E., Felton, R., Milnes, M.R. (2014). Advances in conservation endocrinology: the application of molecular approaches to the conservation of endangered species. General and Comparative Endocrinology, 203, 29-34. 2013 Benco, A., Campbell, M., Barthel, M., Pinto, C., MacKinnon, K., Stoops, M. (2013). Urinary hormone concentrations and pharmacokinetics/pharmacodynamics of haloperidol in a female Indian rhinoceros (Rhinoceros unicornis). In: Proceedings International Elephant and Rhino Conservation and Research Symposium. Pittsburgh, PA USA Aug 26-30. P11-12. Nau, M., Pairan, P., Pinto, C., Sims, R., MacKinnon, K., Stoops, M. (2013). Relationship of salivary hormone concentrations to urinary hormone excretion profiles in the Indian rhinoceros (Rhinoceros unicornis). In: Proceedings International Elephant and Rhino Conservation and Research Symposium. Pittsburgh, PA USA Aug 26-30. P9. Van der Goot, A., Martin, G., Metrione, L., Paris, M., Schook, M., Penfold, L. (2013). Attempt to control estrus and ovulation in white rhinoceroses using a synthetic progestagen and slow release GnRH analogue. In: Proceedings International Elephant and Rhino Conservation and Research Symposium. Pittsburgh, PA USA Aug26-30. P9. Roth, T.L., Reinhart, P.R., Romo, J.S., Candra, D., Suhaery, A., Stoops, M.A. (2013). Sexual maturation in the Sumatran rhinoceros. Zoo Biology, 32, 549-555. Van der Goot, A.C., Dalerum, F., Ganswindt, A., Martin, G.B., Millar, R.P., Paris, M.C. (2013). Faecal progestagen profiles in wild southern white rhinoceros (Ceratotherium simum simum). African Zoology, 48, 143-151.

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Publications on Rhino Behavior and Ecology

Adhikari, K. 2015. Ecology, demography, conservation and management of greater one horned rhinoceros (Rhinoceros unicornis) in Chitwan Natioal Park, Nepal. PhD Dissertation. Saurashtra University, Rajkot, India. Aldila, D., A.J. Hutchinson, M. Woolway, N. Owen-Smith, and E. Soewono. 2015. A mathematical model of black rhino translocation strategy. Journal of ssMathematical and Fundamental Sciences 47(1):104-115. Anderson, T.M., P.M. Ngoti, M.L. Nzunda, D.M. Griffith, J.D.M. Speed, F. Fossøy, E. Røskaft, and B.J. Graae. 2018. The burning question: does fire affect habitat selection and forage preference of black rhinos (Diceros bicornis) in East African savannas? Oryx 1-10 doi:10.1017/S0030605318000388. Badenhorst, M., M. Otto, A.C. van der Goot, and A. Ganswindt. 2016. Stress steroid levels and the short-term impact of routine dehorning in female southern white rhinoceros (Ceratotherium simum simum). African Zoology 51(4):211-215. Barman, R., B. Choudhury, N.V.K. Ashraf, and V. Menon. 2014. Rehabilitation of greater one- horned rhinoceros calves in Manas National Park, a World Heritage Site in India. Pachyderm 55:78-88. Bhattacharya, A., and K. Chakraborty. 2017. Why do Indian rhinos eat elephant grasses? International Journal of Trend in Research and Development 4(5):355-357. Cain, B., A.B. Wandera, S.G. Shawcross, W.E. Harris, B. Stevens-Wood, S.J. Kemp, and B. Okita-Ouma. 2014. Sex-biased inbreeding effects on reproductive success and home range size of the critically endangered black rhinoceros. Conservation Biology 28(2):594-603. Capiro, J.M., M.A. Stoops, E.W. Freeman, D. Clawson, and M.W. Schook. 2014. Effects of management strategies on glucocorticoids and behavior in Indian rhinoceros (Rhinoceros unicornis): translocation and operant conditioning. Zoo Biology 33(2):131-143. Christie, K.S., S.L. Gilbert, C.L. Brown, M. Hatfield, and L. Hanson. 2016. Unmanned aircraft systems in wildlife research: current and future applications of a transformative technology. Frontiers in Ecology and the Environment 14(5):241-251. Cinková, I., and R. Policht. 2014. Contact calls of the northern and southern white rhinoceros allow for individual and species identification. PLoS ONE 9(6):e98475. Cinková, I., and R. Policht. 2015. Discrimination of familiarity and sex from chemical cues in the dung by wild southern white rhinoceros. Animal Cognition 18(1):385-392. Clark, J.H. 2013. Habitat use analysis of a reintroduced black rhino (Diceros bicornis) population. Honors Thesis. Western Kentucky University, Bowling Green, USA. Cromsigt, J.P.G.M., and M. te Beest. 2014. Restoration of a megaherbivore: landscape-level impacts of white rhinoceros in Kruger National Park, South Africa. Journal of Ecology 102(3):566-575. D’Amen, M., N.E. Zimmermann, and P.B. Pearman. 2013. Conservation of phylogeographic lineages under climate change. Global Ecology Biogeography 22(1):93-104. Deka, R.J., and N.K. Sarma. 2015. Studies on feeding behavior and daily activities of Rhinoceros unicornis in natural and captive condition of Assam. Indian Journal of Animal Research 49(4):542-545. Di Minin, E., J. Laitila, F. Montesino-Pouzols, N. Leader-Williams, R. Slotow, P.S. Goodman, A.J. Conway, and A. Moilanen. 2015. Identification of policies for a sustainable legal trade

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in rhinoceros horn based on population projection and socioeconomic models. Conservation Biology 29(2):545-555. Donaldson, J.E., S. Archibald, N. Govender, D. Pollard, Z. Luhdo, and C.L. Parr. 2018. Ecological engineering through fire-herbivory feedbacks drives the formation of savanna grazing lawns. Journal of Applied Ecology 55(1):225-235. Dutta, D.K., and R. Mahanta. 2015. A study on the behavior and colonization of translocated greater one-horned rhinos Rhinoceros unicornis (Mammalia: Perissodactyla: Rhinocerotidae) during 90 days from their release at Manas National Park, Assam India. Journal of Threatened Taxa 7(2):6864-6877. Dutta, D.K., A. Sharma, R. Mahanta, and A. Swargowari. 2017. Behaviour of post released translocated greater one-horned rhinoceros (Rhinoceros unicornis) at Manas National Park, Assam, India. Pachyderm 58:58-66. Edwards, K.L. 2013. Investigating population performance and factors that influence reproductive success in the eastern black rhinoceros (Diceros bicornis michaeli). PhD Dissertation. University of Liverpool, Liverpool, United Kingdom. Eyres, A., J. Capiro, and J. Ivy. 2017. Population analysis and breeding and transfer plan southern white rhinoceros (Ceratotherium simum simum) AZA species survival plan yellow program. Silver Spring, MD: Association of Zoos and Aquariums Population Management Center. 43 p. Ferreira, S.M., C. Greaver, G.A. Knight, M.H. Knight, I.P.J. Smit, and D. Pienaar. 2015. Disruption of rhino demography by poachers may lead to population declines in Kruger National Park, South Africa. PLoS ONE 10(6):e0127783. Freeman, E.W., J.M. Meyer, J. Adendorff, B.A. Schulte, and R.M. Santymire. 2014. Scraping behavior of black rhinoceros is related to age and fecal gonadal metabolite concentrations. Journal of Mammalogy 95(2):340-348. Freeman, E.W., J.M. Meyer, J. Bird, J. Adendorff, B.A. Schulte, and R.M. Santymire. 2014. Impacts of environmental pressures on the reproductive physiology of subpopulations of black rhinoceros (Diceros bicornis bicornis) in Addo Elephant National Park, South Africa. Conservation Physiology 2(1):cot034. Gedir, J.V., P.R. Law, P. du Preez, and W.L. Linklater. 2018. Effects of age and sex ratios on offspring recruitment rates in translocated black rhinoceros. Conservation Biology 32(3):628-637. Greaver, C., S. Ferreira, and R. Slotow. 2014. Density-dependent regulation of the critically endangered black rhinoceros population in Ithala Game Reserve, South Africa. Austral Ecology 39(4):437-447. Gyöngyi, K., and M. Elmeros. 2017. Forage choice of the reintroduced black rhino and the availability of selected browse species at Majete Wildlife Reserve, Malawi. Pachyderm 58:40-50. Hariyadi, A.R. 2015. Assessment of Halimun-Salak National Park as a potential site for establishing the second population of Javan rhinoceros (Rhinoceros sondaicus). http://www.rhinoresourcecenter.com/pdf_files/146/1460994396.pdf Harjanto, E. 2017. On a territorial competition between Rhinoceros sondaicus and Bos javanicus at Ujung Kulon National Park. Communication in Biomathematical Sciences 1(1):46-53. Kafley, H., M.E. Gompper, M. Khadka, M. Sharma, R. Maharjan, and B.P. Thapaliya. 2015. Analysis of rhino (Rhinoceros unicornis) population viability in Nepal: impact assessment of antipoaching and translocation strategies. Zoology and Ecology 25(4):288-294.

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Kruger, M. 2017. Capture and boma stress responses in the white rhinoceros (Ceratotherium simum). PhD Dissertation. University of the Witwatersrand, Johannesburg, South Africa. Landman, M., and G.I. Kerley. 2014. Elephant both increase and decrease availability of browse resources for black rhinoceros. Biotropica 46(1):42-49. Landman, M., D.S. Schoeman, and G.I. Kerley. 2013. Shift in black rhinoceros diet in the presence of elephant: evidence for competition? PLoS ONE 8(7):e69771. Law, P.R., B. Fike, and P.C. Lent. 2013. Mortality and female fecundity in an expanding black rhinoceros (Diceros bicornis minor) population. European Journal of Wildlife Research 59(4):477-485. Law, P.R., B. Fike, and P.C. Lent. 2014. Birth sex in an expanding black rhinoceros (Diceros bicornis minor) population. Journal of Mammalogy 95(2):349-356. Law, P.R., B. Fike, P.C. Lent. 2015. Dynamics of an expanding black rhinoceros (Diceros bicornis minor) population. European Journal of Wildlife Research 61(4):601-609. Law, P.R., and W.L. Linklater. 2014. Black rhinoceros demography should be stage, not age, based. African Journal of Ecology 52(4):571-573. le Roux, S.P., J. Marias, R. Wolhuter, and T. Niesler. 2017. Animal-borne behavior classification for sheep (Dohne merino) and rhinoceros (Ceratotherium simum and Diceros bicornis). Animal Biotelemetry 5:25. Linklater, W.L., K. Mayer, and R.R. Swaisgood. 2013. Chemical signals of age, sex and identity in black rhinoceros. Animal Behaviour 85(3):671-677. Linn, S.N., M. Boeer, and M. Scheumann. 2018. First insights into the vocal repertoire of infant and juvenile southern white rhinoceros. PLoS ONE 13(3):e0192166. Marneweck, C., A. Jurgens, and A.M. Shrader. 2017. Dung odours signal sex, age, territorial and oestrous state in white rhinos. Proceedings of the Royal Society of London B 284(1846):20162376. Mays, H.L. Jr., C. Hung, P. Shaner, J. Denvir, M. Justice, S. Yang, T.L. Roth, D.A. Oehler, J. Fan, S. Rekulapally, and D.A. Primerano. 2018. Genomic analysis of demographic history and ecological niche modeling in the endangered Sumatran rhinoceros Dicerorhinus sumatrensis. Current Biology 28(1):70-76. Menargues, A., V. Urios, and R. Limiñana. 2013. Seasonal pattern of salivary cortisol secretion in the greater one-horned rhinos. Animal Welfare 22(4):467-472. Miller, M., M. Kruger, M. Kruger, F. Olea-Popelka, and P. Buss. 2016. A scoring system to improve decision making and outcomes in the adaptation of recently captured white rhinoceroses (Ceratotherium simum) to captivity. Journal of Wildlife Diseases 52(2):S78- S85. Mulero-Pázmány, M., R. Stolper, L.D. van Essen, J.J. Negro, and T. Sassen. 2014. Remotely piloted aircraft systems as a rhinoceros anti-poaching tool in Africa. PLoS ONE 9(1):e83873. Murphy, S.T., N. Subedi, S.R. Jnawali, B.R. Lamichhane, G.P. Upadhyay, R. Kock, and R. Amin. 2013. Invasive mikania in Chitwan National Park, Nepal: the threat to the greater one-horned rhinoceros Rhinoceros unicornis and factors driving the invasion. Oryx 47(3):361-368. Ngoti, P.M. 2017. The feeding ecology of eastern black rhinocoeroses (Diceros bicornis michaeli) in southern Serengeti National Park, Tanzania. Master’s Thesis. Norwegian University of Science and Technology, Trondheim, Norway.

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Odendaal-Holmes, K., J.P. Marshal, and F. Parrini. 2014. Disturbance and habitat factors in a small reserve: space use by establishing black rhinoceros (Diceros bicornis). South African Journal of Wildlife Research 44(2):148-160. Ojah, S., A. Saikia, and P. Sivakumar. 2015. Habitat suitability of Laokhowa Burhachapori wildlife sanctuary complex of Assam, India for Rhinoceros unicornis Linn. The Clarion 4(2):39-47. Olsson, S. 2015. A survey of the available browse for the black rhinoceros (Diceros bicornis Linnaeus, 1758) in a farmland area in the Kunene region, Namibia. Bachelor’s Thesis. Halmstad University, Halmstad, Sweden. Plotz, R.D. 2014. The interspecific relationships of black rhinoceros (Diceros bicornis) in Hluhluwe-iMfolozi Park. PhD Dissertation. Victoria University of Wellington, Wellington, New Zealand. Plotz, R.D., W.J. Grecian, G.I.H. Kerley, and W.L. Linklater. 2016. Standardising home range studies for improved management of the critically endangered black rhinoceros. PLoS ONE 11(3):e0150571. Pluháček, J., B.L. Steck, S.P. Sinha, and F. von Houwald. 2017. Interbirth intervals are associated with age of the mother, but not with infant mortality in Indian rhinoceros. Current Zoology 63(3):229-235. Prinsloo, D. 2017. Impact of African elephant feeding on white rhinoceros foraging opportunities. Master’s Thesis. Nelson Mandela University, Port Elizabeth, South Africa. Pusparini, W. 2014. Ecology and conservation of endangered species in Sumatra: smaller cats and the Sumatran rhinoceros (Dicerorhinus sumatrensis) as case studies. Master’s Thesis. University of Massachusetts, Amherst, USA. Pusparini, W., P.R. Sievert, T.K. Fuller, T.O. Randhir, and N. Andayani. 2015. Rhinos in the parks: an island-wide survey of the last wild population of the Sumatran rhinoceros. PLoS ONE 10(9):e0136643. Pusparini, W., and H.T. Wibisono. 2013. Landscape-level assessment of the distribution of the Sumatran rhinoceros in Bukit Barisan Selatan National Park, Sumatra. Pachyderm 53:59-65. Schwabe, F., T. Göttert, N. Starik, S.R. Levick, and U. Zeller. 2015. A study on the postrelease behavior and habitat preferences of black rhino (Diceros bicornis) reintroduced into a fenced reserve in Namibia. African Journal of Ecology 53(4):531-539. Soka, G.E., A.A. Rija, and A. Owino. 2014. Modeling black rhinoceros (Diceros bicornis) population performance in East Africa: the case of Lake Nakuru National Park, Kenya. Biodiversity & Endangered Species 2(3):1-6. Subedi, N., S.J. Jnawali, M. Dhakal, N.M.B. Pradhan, B.R. Lamichhane, S. Malia, R. Amin, and Y.V. Jhala. 2013. Population status, structure and distribution of the greater one-horned rhinoceros Rhinoceros unicornis in Nepal. Oryx 47(3):352-360. Subedi, N., B.R. Lamichhane, R. Amin, S.R. Jnawali, and Y.V. Jhala. 2017. Demography and viability of the largest population of greater one-horned rhinoceros in Nepal. Global Ecology and Conservation 12:241-252. Thapa, K., S. Nepal, G. Thapa, S.R. Bhatta, and E. Wikramanayake. 2013. Past, present and future conservation of the greater one-horned rhinoceros Rhinoceros unicornis in Nepal. Oryx 47(3):345-351. Thompson, S., T. Avent, and L.S. Doughty. 2016. Range analysis and terrain preference of adult southern white rhinoceros (Ceratotherium simum) in a South African private game reserve: insights into carrying capacity and future management. PLoS ONE 11(9):e0161724.

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Thuo, D.N., J.O. Junga, J.M. Kamau, J.O. Amimo, F.M. Kibegwa, and K.E. Githui. 2015. Population viability analysis of black rhinoceros (Diceros bicornis michaeli) in Lake Nakuru National Park, Kenya. Biodiversity & Endangered Species 3(1):1-5. Tripathi, A.K. 2013. Social and reproductive behavior of great Indian one-horned rhino, Rhinoceros unicornis in Dudhwa National Park, U.P., India. International Journal of Pharmacy and Life Sciences 4(11):3116-3121. Veldhuis, M.P., M.I. Gommers, H. Olff, and M.P. Berg. 2018. Spatial redistribution of nutrients by large herbivores and dung beetles in a savanna ecosystem. Journal of Ecology 106(1):422-433. Ververs, C., M. van Zijll Langhout, M. Hostens, M. Otto, J. Govaere, B. Durrant, and A. Van Soom. 2017. Reproductive performance parameters in a large population of game-ranched white rhinoceroses (Ceratotherium simum simum). PLoS ONE 12(12):e0187751. Whitham, J.C., and L. Miller. 2016. Using technology to monitor and improve zoo animal welfare. Animal Welfare 25(4):395-409. Yadav, P.K., K. Sarma, and S. Dookia. 2013. The review of biodiversity and conservation study in India using geospatial technology. International Journal of Remote Sensing and GIS 2(1):1-10.

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