Greater One Horned Rhinoceros

Research

Introduction and Top Research Priorities

Largely due to the rhino poaching crisis that far out-weighs any other challenge facing rhinos today, the top five priorities of the Rhino Research Council, listed below, are skewed towards in situ issues. It is important to note that these research priorities are not ranked in order of importance, so the first and only priority focused on the ex situ population is not considered more important than the others. The Rhino Research Council realizes that conservation of genetic diversity via cell/gamete rescue from remnant populations is really not a research issue and is more of an implementation challenge, but it was identified as an important action to emphasize at this time of crisis. Following this list of the highest research priorities, the chapter continues with important areas of ex situ research, including reproduction, health, nutrition, behavior/ecology, and genetics. For more detailed discussions of in situ research needs, the reader is referred to the Rhino Research Council’s 2014 Rhino Research Masterplan.

Investigate Major Factors Affecting Health and Reproduction Ex Situ

Epidemiology of browser rhino health issues—Health concerns and unusual syndromes continue to impact black rhinos in captivity, but the incidence of various conditions shifts over time, and patterns require constant monitoring and re-evaluation to track current trends and conditions. Otherwise, outdated problems and historic records continue to be referenced at the cost of realizing emerging trends and realities. One example is the significant increase in the number of black rhino deaths due to renal disease compared to five years ago and a decreased incidence of mortality due to hemolytic anemia compared to 20 years ago. It would be extremely useful to secure ongoing support for a point person whose priority is to evaluate the historical data and current situations to keep abreast of the problems and factors associated with those health issues.
Iron overload syndrome (significance, detection, treatment, prevention)—Evidence that hemosiderosis is occurring in browsing rhinos is solid, but there are still many questions surrounding the impact of this condition on rhino health, whether it is primary or secondary, why it is occurring, and what can be done about it. In the past decade, relatively little progress has been made. Although there has been some progress in developing oral iron chelators for humans, the efficacy and safety of such medications are unknown in rhinos, and they are cost prohibitive. Research on dietary tannins as iron chelators has produced inconclusive results. Recent studies on the impact of large volume, regular phlebotomies for reducing iron marker values has shown promise but is unlikely to be adopted as a long-term management strategy at many institutions. Research in any of the four areas—cause, detection, prevention and treatment—could be valuable in producing insight on how to manage/prevent this condition.
Obesity/body condition scoring—Overconditioning in captive rhinos can lead to a multitude of health problems, including musculoskeletal, foot, and reproductive problems. One example is the almost certain exacerbation of pododermatitis associated with captive husbandry conditions in the greater one-horned rhino. Research on standardizing body condition scores for all rhino species, along with improved nutritional management, should be a priority for captive health.
Sub-optimal reproduction (stillbirths, embryo loss, phytoestrogens, pathology)—Although reproductive success has improved in white and greater one-horned rhinos by changing management strategies, providing larger spaces and more complex social groups that include experienced and inexperienced individuals, and allowing mate choice options, there are a few reproductive challenges that remain unresolved and challenge our ability to develop self-sustaining populations. For example, < 50% of all captive white rhinos are reproductively successful, almost 50% of greater one-horned rhino calves are stillborn, and as our ability to evaluate larger numbers of female rhinos has improved, incidence of known embryo loss and reproductive pathology continue to increase.

Improve Rhino Identification and Monitoring

Optimize/standardize survey methodologies—Given the current poaching crisis, research that can improve our ability to monitor and survey wild populations is extremely important. Post-release monitoring is our primary method of determining the success of translocation, dehorning and many other conservation efforts. Data compiled from tracking individuals after translocation can be used to develop specific criteria that individual rhinos should meet (sex, age, reproductive status) prior to translocation in order to improve the success rate. Better methods for signal transduction and reception would be helpful for forest-dwelling animals. Creative methods of transmitter attachment are needed since horn transmitters are difficult to insert in de-horned animals, and radiocollars are a challenge given the neck morphology of rhinos. Also, lesions can occur with collars. Given the abundance of research on this topic in the military field, adaptation and application of this newer technology may be useful for rhinos.
Genetic census—Advances in genetic analyses of fecal samples, hair, environmental-DNA and microbiomes could be used to determine the genetic diversity among wild populations, including sex ratios, number of individuals and extent of inbreeding. Identify Most Important Factors in Translocation Success Translocations have proven extremely valuable in saving rhinos from poaching, re-populating parks/ranches, bolstering dwindling populations and establishing new populations. However, there is room for improvement, and given the importance of this action for long-term rhino survival, research is needed to enhance survival and reproductive success of every rhino moved. Studies might focus on the following example topics: white rhino boma maladaptation, mortality rates in different habitats, impacts of dehorning, impact on source populations, and identifying the type of individuals most likely to succeed.

Identify Most Important Factors in Translocation Success

Translocations have proven extremely valuable in saving rhinos from poaching, re-populating parks/ranches, bolstering dwindling populations and establishing new populations. However, there is room for improvement, and given the importance of this action for long-term rhino survival, research is needed to enhance survival and reproductive success of every rhino moved. Studies might focus on the following example topics: white rhino boma maladaptation, mortality rates in different habitats, impacts of dehorning, impact on source populations, and identifying the type of individuals most likely to succeed.

Determine Ecological Factors Impacting Rhino Populations

To choose the best locations for rhinos and provide guidance for those who want to improve their land in support of rhino populations, more information is required regarding specific ecological traits that impact rhino population survival and ability to thrive. Spatial ecology is especially important and is integrated with habitat quality, usage and preference.

Conserve Genetic Diversity via Cell/Gamete Rescue from Remnant Populations (small/sub-populations and highly endangered)

Given the poaching crisis in Africa and India and the rapid decline of Sumatran rhinos throughout their range, it seems prudent that every effort should be made to preserve cell lines and gametes from all genetically valuable rhinos/populations. The methodologies already exist but may need to be modified for the field conditions encountered in the African savannah and the tropical forests of Asia.

Areas in Need of Research Ex Situ Reproduction

Reproduction:

There are several options for minimizing aggression when trying to breed greater one-horned rhinos: 1) move aggressive animals to a very large enclosure where the females can get away from the males, and the rhinos will usually work through their differences with minimal injury; 2) pair experienced males with inexperienced females and inexperienced males with experienced females; 3) use urinary hormone monitoring and behavioral observations to ensure introductions coincide with the female’s estrus; or 4) conduct artificial insemination, which has now proven successful in this rhino species. If these approaches are utilized and successful, this challenge will be resolved and will be dropped from future masterplans. Stillborn calves, however, continue to be a big problem for greater one-horned rhinos, and stillbirth the cause associated with 30% of all greater one-horned rhino mortalities in North America and Europe. Although first thought to be primarily occurring with primiparous cows, it is not uncommon in multiparous cows as well. A study is underway to determine if fetal presentation or amniotic sac rupture during delivery are associated with live/dead outcome. Other important areas of reproduction research are as follows:

Early embryonic death (EED)—Repeated documentation of EED in all captive rhino species is puzzling. In the past 15 to 20 years, there have been at least six confirmed cases of EED in white rhinos at four different institutions, five confirmed cases of EED in greater one-horned rhinos at three different locations, and seven EEDs confirmed in two Sumatran rhinos at two locations. Few rhinos are monitored closely enough to diagnose EED, so considering the number of confirmed cases, it appears to be a common occurrence in the captive population. It has also been reported in at least one field study in wild black rhinos. However, in most field studies females breed only once or twice before conceiving and carrying pregnancies to term, so the high incidence of EED in captivity seems aberrant. In addition to EED, abortions later in gestation have also been reported. There are many potential causes of EED and abortion in rhinos, including uterine infection, uterine scarring from a previous dystocia, endometrial hyperplasia, reproductive pathology, hormonal insufficiency, dietary insufficiency or toxicity, social/behavioral influence of enclosure co-inhabitants or other health-related conditions of the female rhinos. It is quite possible EED is occurring in many females that are breeding repeatedly without producing calves. Further investigation into the prevalence and potential causes of EED and abortion is warranted.

Reproductive pathology—Reproductive tract pathology in female rhinos has been reported in all captive rhino species and prevalence can be as high as 50% in females over 15 years of age (white rhinos). It can range from small, inconsequential cysts that do not affect fertility to large, invasive tumors that jeopardize the life of the rhino. There is general agreement that these pathologies typically develop in rhinos that spend the majority of their lives in a non-pregnant state and could be related to repeated exposure to hormone fluctuations, a condition not natural in wild rhinos that spend much of their time pregnant. However, there are some younger rhinos that also develop severe pathology, and more recent research has revealed that a fairly substantial proportion of white and greater one-horned rhinos develop pathologies in their early teens. Understanding the etiology of this pathology and identifying hormone therapies that might prevent it would be very useful. Furthermore, criteria for determining when the degree and types of pathology observed during an exam can be tolerated without loss of fertility versus those that render a female infertile would be very useful to establish so that appropriate management recommendations could be made.

Measuring estrogen—Quantifying changes in estrogen levels that correlate with meaningful reproductive events has been difficult in several of the rhino species and continues to be challenging. A more accurate and meaningful way to assess estrogen concentrations could be useful for monitoring cyclicity, timing introductions and assessing other reproductive characteristics.

Early pregnancy marker—Pregnancy can be diagnosed in rhinos through progesterone monitoring and ultrasound. It would be useful for management and for learning more about EED if a pregnancy-specific marker could be identified in rhinos not trained for ultrasound, but this is not a high priority. The use of assisted reproduction has the potential of becoming a very useful tool for regional and metapopulation management of rhinos. Additionally, assisted reproduction may provide a means of overcoming physical and/or behavioral problems that currently prevent reproduction in specific individuals. Progress towards these goals has been impressive, and it is now time to look at priorities considered feasible to overcome during the next five years.

Semen collection—Semen collection by electroejaculation (EEJ) has become more reliable and has been successful in all four captive rhino species. However, there are differences in the quality of samples collected with greater one-horned rhinos being the most reliable in producing a high quality, concentrated sample. In black, white and Sumatran rhinos, samples often are more dilute and more likely to be contaminated with urine or red blood cells. Samples are often adequate for cryopreservation, artificial insemination (AI) or in vitro fertilization but often are not able to withstand more complex processing like sperm sorting. More research into the effects of different anesthetic protocols and attempts at penile catheterization may prove valuable. Chemical induction of ejaculation has been attempted but has not proven very effective yet.

In vitro fertilization (IVF)—To date, IVF success has been minimal (one IVF attempt produced one embryo in a black rhino and one IVM/IVF attempt produced one embryo in a black rhino) and more research is needed. Success has been minimal, in part, because of limited opportunities.

Sperm sorting—Since 2005, 25 EEJ attempts across three species (n=10 black, n=9 white, n=6 greater one-horned) have been conducted for sperm sorting. Inconsistent ejaculate quality is the greatest challenge to application of sorting technology and integration into AI programs for each species. Only 36% of collections were good enough for sorting. Greater one-horned rhino sperm has a unique problem: an interaction between the seminal plasma and egg yolk that prevents staining of X and Y populations. Most progress has been with white rhino where sorted, cryopreserved samples are now available for use in AI procedures.

Artificial insemination—Successful AI protocols using cryopreserved semen now exist for white rhinos (five term pregnancies) and greater one-horned rhinos (three term pregnancies). AI has not yet been successful in Sumatran or black rhinos. Additional research is needed in developing successful procedures for black rhinos and Sumatran rhinos, and research to improve efficiency/success rates in all rhinos is also needed to ensure the technology is integrated into the captive management plan. Some challenges include: breaking down intact hymens in females that have never mated, determining ovulatory versus anovulatory cycles in greater one-horned rhinos, and controlling the cycle for planned, timed AI to improve feasibility of use.

Estrous cycle manipulation—In Europe, significant progress has been made with white rhinos using synthetic progestin and human chorionic gonadotropin or gonadotropin releasing hormone. Similar methodologies are being tested in the U.S. An ovulation induction protocol has been established for Sumatran rhinos. Research is still needed on effective methods for controlling the cycle in greater one-horned rhinos so that specifically-timed AI procedures can be planned and implemented.

Gamete rescue—Protocols for rescuing sperm post-mortem are well established but protocols for oocyte rescue are not. A few attempts have been made with oocyte rescue, and one two-cell embryo was produced following maturation and IVF with frozen-thawed sperm. However, all protocols still need substantial research before they will be applicable for management purposes

Health Related Research Needs

Obesity/body condition index – Overconditioning in captive rhinos leads to a multitude of health problems, including musculoskeletal, foot, and reproductive problems. Research examining methods of standardizing body condition scores for all rhino species, along with improved nutritional management, should be a priority for captive health. Methods applied to domestic livestock, and more recently elephants, should be considered for use in rhinos. Also, is the inclusion of cereal-based concentrates in rhino diets detrimental?
Gastrointestinal and cardiovascular problems—These problems might 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.
Quantifying stress (especially chronic stress)—Develop laboratory and behavioral markers of stress. What is the health impact, for instance, on the development of gastrointestinal ulcers, increased iron stores, etc.? New ideas include evaluation of neutrophil function using a portable luminometer and the analyses of five markers in fecal samples including thyroid hormones.
Disease risk analyses—Translocations within and between range states or from abroad present risks for introduction of disease. Research into diseases that present potential risk to rhinos and into logistically-appropriate diagnostic techniques for screening and their incorporation into protocols should be considered part of the risk analysis and translocation process. • Pharmacokinetics/dynamics of commonly used antibiotics and analgesics—Little scientific work has been done in rhinoceros species on therapeutic drugs. Non-empirical use may lead to inadequate or potentially adverse effects. With the advent of husbandry training and restraint devices, sample collection is now possible for these types of studies.
Pharmacokinetics/dynamics of commonly used antibiotics and analgesics—Little scientific work has been done in rhinoceros species on therapeutic drugs. Non-empirical use may lead to inadequate or potentially adverse effects. With the advent of husbandry training and restraint devices, sample collection is now possible for these types of studies.

Nutrition

Body condition scoring systems have been established for the African rhino species, however preliminary trials have proven that these scoring systems are very subjective if utilized by staff at each institution. Furthermore, efforts to control for bias by sending images of animals from different institutions to one person for evaluation were equally as difficult because of the variation in animal appearance based on photo angle/quality. Although similar scoring systems should be developed for Asian rhino species, less subjective methods for all species are needed. Perhaps physiological values for leptin, glucose, insulin, etc., could provide a more solid method of evaluating body condition in addition to computerized assessments, body measurements matched with weights and/or characterizing body types. This is an important issue since body condition has now been suggested as a factor involved in iron storage problems, disease, skewed sex ratio of calves and reproductive failure of captive-born white rhinos. There are often requests for browse lists. Many papers exist on the 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 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. Results of such studies have been reported for East coast species and a similar study on West coast species (in particular acacia) is ongoing.

In addition, a study comparing sumac and willow was also recently reported. San Diego Zoo Global is studying the nutrient composition of elephant grass (Pennisetum purpureum) following one to six months of regrowth. Institutions with black or Sumatran 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.

Other important topics for nutritional research are as follows:

• Research needs related to reproduction include determining if there is an impact of diet on calf sex ratio, evaluation of new hand-rearing formulas for supplementation and complete rearing, determining milk production, and estimating energy requirements of lactation.

• Minimum dietary nutrient concentrations for rhinos need to be established for maintenance, gestation and breeding to determine if utilizing the horse guidelines are adequate. • Science-based recommendations need to be developed regarding the use of alfalfa for rhinos.

• The use of fecal DNA analyses as a means of defining wild rhino diets should be investigated.

• The cause of tooth overgrowth could be related to diet (nutritional and/or mechanical), and further investigation could be helpful. There have been several cases of rhinos requiring repeated teeth floating procedures. Additional browse could be helpful since silica is known to reduce tooth growth.

• Recent investigations of trace minerals are ongoing, especially with copper, which is an important antioxidant that could combat iron. One study has changed the diet to enhance copper. Appropriate dietary concentrations of zinc and copper need to be determined as both are being supplemented for both hoof health and to bind with iron.

Behavior and Ecology

Large-scale manipulative experiments to test hypotheses relating to social environment, stress, foraging/nutrition, movement patterns, etc. on reproduction, health and survival are encouraged. To achieve this goal, multiple large, naturalistic enclosures will need to be established and experimental groups assigned to different treatment conditions in a way that establishes multiple experimental replicates to address each question. Such a systematic approach, on a more limited number of variables of interest, will advance understanding of the factors governing whether captive (and wild) populations thrive or fail. Areas of ex situ research might include:

• Role of communication (olfactory, acoustic) in reproduction, maintaining social relationships, endocrinology (stress and reproduction), and overall social organization (consider experimental manipulations, such as with chemical signals and acoustic playbacks)

• Evaluate social processes (courtship, aggression) leading to successful copulation

• How social mechanisms lead to establishment of social outcomes, such as territoriality, dominance, group composition, etc., and how these change through time as new animals move in and out of the group

• How do changes in ecological parameters affect social behavior, endocrinology (stress and reproduction) and organization?

• Role of climatic variables (temperature/rainfall/photoperiod) on social organization and behavior

• Development of methods to reduce bodily injuries in male-male conflict and inter-sexual conflict that inhibits reproduction

• Behavioral, hormonal, and health perspectives regarding social stress

• Manipulate social variables such as density, group size, and age-sex ratios to determine outcomes for social behavior, organization, stress, health, reproduction, and survival

• Avoidance and attraction patterns

• Social density and composition effects

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 /connectivity of surviving populations, dispersal, paternity, and census. The potential methodologies would include analyses of mitochondrial DNA, microsatellites, genetic sexing, single nucleotide polymorphisms, immunogenetics and the microbiome. Over the next five years, there needs to be an emphasis on developing appropriate methodologies, including:

• Microsatellite analysis—Microsatellites are powerful if they can be amplified from DNA from feces and should be a priority.

• Next generation sequencing (NGS)—Since fecal DNA typing is difficult and genetic diversity in some rhinoceros species is low, NGS may be of great value in providing the initial screening for polymorphisms needed to develop markers to examine rhino population genetics.

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

• Shorter amplicons—Amplicons of 75-150 base pairs need to be targeted by genetic markers and made available for fecal DNA studies since degradation can occur rapidly in fecal material.

• Fecal microbiome analysis—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.

• Immunogenetic variability—Immunogenetic variability and its relationship to 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.

• Environmental DNA—eDNA is an emerging tool in genetic studies but may be more challenging under tropical conditions. Ongoing priority genetic studies that might include ex situ components include:

• Major Histocompatibility Complex (MHC) variability—MHC analyses are being planned and, once established, will help significantly when analyzing populations for variation in genes that are under selection.

• 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. 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. 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 relevant in light of the tragic loss of five captive Sumatran rhino in Malaysia. A particular avenue worth pursuing is polymerase chain reaction techniques that can determine presence and load of different pathogens 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.