Tannin-binding salivary proteins in three captive rhinoceros species

Tannin-binding salivary proteins (TBSP) are considered to be counter-defences acquired in the course of evolution by animals whose natural forage contains such tannins. As tannins mostly occur in browse material but not in grasses, it is assumed that grazers do not have a need for TBSP. Whereas it has been shown in several non-ungulate species that TBSP can be induced by dietary tannins, their presence or absence in ungulates has, so far, been shown to be a species-specific characteristic independent of dietary manipulations. We investigated saliva from three rhinoceros species from zoological gardens fed comparable, conventional zoo diets. As expected, saliva from white rhinoceroses (Ceratotherum simum, grazer) had lower tannin-binding capacities than that from black rhinoceroses (Diceros bicornis, browser). Surprisingly, however, Indian rhinoceroses (Rhinoceros unicornis), commonly regarded as grazers as well, displayed the highest tannin-binding capacities of the three species investigated. It is speculated that this discrepancy might be a result of an evolutionarily recent switch to a grass-dominated diet in Indian rhinoceroses, and that the black rhinoceros, which is closer related to the white rhinoceros than the Indian species, has evolved an inducible mechanism of TBSP production. In separate trials during which the tannin content of the diets of black rhinoceroses was increased by the addition of either tannic acid or quebracho, the tannin-binding capacity of black rhinoceros saliva was increased to levels within the same range as that of Indian rhinoceroses on the conventional diets. While induction trials in white and Indian rhinoceroses remain to be performed for a full understanding of salivary anti-tannin defence in rhinoceroses, these results are the first report of an induced salivary response to increased dietary tannin levels in an ungulate species.     

Ovarian superstimulation, transrectal ultrasound-guided oocyte recovery, and IVF in rhinoceros

Numerous reports on reproductive pathology in all rhinoceros species illustrate the abundance of female infertility in captive populations. In infertile rhinoceroses, oocyte collection and embryo production could represent the best remaining option for these animals to reproduce and to contribute to the genetic pool. We report here on superstimulation, repeated oocyte recovery, and attempted in vitro fertilization (IVF) in white and black rhinoceroses. Four anestrous rhinoceroses (two white, two black) with unknown follicular status were treated with gonadotropin-releasing hormone analogue, deslorelin acetate, for 6 to 7 d. Number and size of follicles in superstimulated females was significantly higher and larger compared with those in nonstimulated anestrous females (n = 9). Ovum pick-up was achieved by transrectal ultrasound-guided follicle aspiration. Up to 15 follicles were aspirated per ovary. During six ovum pick-ups, a total of 29 cumulus-oocyte complexes (COCs) were harvested with a range of 2 to 9 COCs per collection. No postsurgical complications were noted on the rhinoceros ovaries using this minimally invasive approach. Various in vitro maturation (IVM) and IVF protocols were tested on the collected COCs. Despite the low total number of COCs available for IVM and IVF in this study, we can report the first rhinoceros embryo ever produced in vitro. The production of a 4-cell embryo demonstrated the potential of transrectal ultrasound-guided oocyte recovery as a valuable tool for in vitro production of rhinoceros embryos from otherwise infertile females.     

Allozyme variation and differentiation in African and Indian rhinoceroses

We studied variation at 25 to 31 allozymic loci in African and Asian rhinoceroses. Four taxa in three genera were examined: African Ceratotherium simum simum (northern white rhinoceros), C. s. cottoni (southern white rhinoceros), Diceros bicornis (black rhinoceros), and Rhinoceros unicornis (Indian rhinoceros). Extremely small amounts of intraspecific variation were observed in sample sizes of 2 to 10 presumably unrelated individuals per taxon: P = .00-.10, H = 0.00-0.02. We examined demographic bottlenecks and sampling errors as possible reasons for the low levels of detectable variation. The very small intraspecific genetic distance (D = 0.005) between the two living white rhinoceros subspecies is far less than the distance that has been reported for other mammal subspecies. The mean D value of 0.32 +/- 0.11 between the two African genera was also less than expected given the divergence time of greater than 7 million years suggested by the fossil record. Rhinoceroses may be evolving more slowly at the structural gene loci than are some other mammal groups. The estimate of D = 1.05 +/- 0.24 for the African-Indian split supports this idea, as the lineage diverged at least 26 million years ago. Our results contribute to the currently available scientific information on which management decisions aimed toward saving endangered rhinoceroses should be based.     

One Small Step for Rhinos,One Giant Leap for Wildlife Management- Imaging Diagnosis of Bone Pathology in Distal Limb

Chronic foot disease poses a threat to the general health, represents a tremendous clinical challenge, and often is a reason for euthanasia in captive megaherbivores, among them the elephant and rhinoceros. Nevertheless, apart from the elephant, foot pathology is handled as being confined only to soft tissues whereas bone pathology is often overlooked. As a case in point, the authors selected the second largest mammal on land, the rhinoceros. We performed a computed tomographic (CT) study using the highest resolution available in veterinary world, followed by digital radiography of eight distal limbs from two white and one Indian rhinoceroses. Our study demonstrated that bone pathology in rhinoceroses’ foot is present and in large numbers, yet none of these were diagnosed ante mortem. Even when the animals were euthanized due to foot problems, the decision was based on soft tissue pathology rather than orthopedic reasons. Even more worrying is the fact that the largest number of osteopathologies was present in one of the white rhinoceroses that showed no discernable related clinical signs. This study describes for the first time the existence of bone pathology in white rhinoceros foot, in addition to the two previously described rhinoceros species - Indian and black rhinoceroses. Furthermore, the chronic foot disease reported for the Indian rhinoceros in our study was not restricted to soft tissue structures as was presumed ante mortem but included severe bone pathology. New evidence suggesting that osteopathology in rhinoceroses’ distal limb is more widespread than it was thought before could force us to rethink of radiographic diagnosis in captive megaherbivores as routine examination incorporated into their health management. The anticipated improvements in radiologic examinations in megaherbivores will increase the effectiveness of their management and husbandry and open the way for improved animal welfare and better wildlife conservation.     

The Microbial Community in the Feces of the White Rhinoceros (Ceratotherium simum) as Determined by Barcoded Pyrosequencing Analysis

As a non-ruminant herbivore, the white rhinoceros has the ability to utilize fibrous plant matter through microbial fermentation in the hindgut. So far, there has been no report using molecular techniques to study the gut microbiota of the white rhinoceros. We used barcoded pyrosequencing to characterize 105,651 sequences of 16S rRNA genes obtained from fecal samples from five white rhinoceroses. Results showed that Firmicutes and Bacteroidetes were the predominant phyla in the samples, which were comprised largely of unclassified bacteria. The microbiota of one animal treated with drug therapy differed from those in other healthy animals, and was dominated by Aerococcus -related bacteria. The core microbiota in the healthy rhinoceros were dominated by phyla Firmicutes and Bacteroidetes, represented by the Ruminococcaceae, Lachnospiraceae, Rikenellaceae and Prevotellaceae families. The present work provides a phylogenetic framework for understanding the complex microbial community of the rhinoceros; however, further studies are required to link the distinctive microbiota with their digestive role in the hindgut of the white rhinoceros.     

Fat soluble vitamins in blood and tissues of free-ranging and captive rhinoceros

Several disease syndromes in captive rhinoceroses have been linked to low vitamin status. Blood samples from captive and free-ranging black (Diceros bicornis) and white rhinoceros (Ceratotherium simum) and tissue samples of captive individuals from four rhinoceros species were analysed for vitamins A and E. Circulating vitamin A levels measured as retinol for free-ranging versus captive black and white rhinoceros were 0.04 (+/- 0.03 SD) vs. 0.08 (+/- 0.08) and 0.07 (+/- 0.04) vs. 0.06 (+/- 0.02) microgram/ml, respectively. Circulating vitamin E levels measured as alpha-tocopherol were 0.58 (+/- 0.30) vs. 0.84 (+/- 0.96) and 0.62 (+/- 0.48) vs. 0.77 (+/- 0.32) microgram/ml, respectively. In contrast to earlier findings, there was no significant difference in vitamin E concentration between captive and free-ranging black rhinoceros. When the samples of captive black rhinoceros were grouped into those taken before 1990 and after 1990, however, those collected before 1990 had significantly lower (P < 0.001) vitamin E levels (0.46 +/- 0.83 microgram/ml) and those collected in 1990 or later significantly higher (P < 0.001) vitamin E levels (1.03 +/- 1.04 micrograms/ml) than the captive population as a whole. This is probably due to increased dietary supplementation. There were significant differences in circulating vitamin concentrations in black rhinoceroses from different regions in the wild. Serum 25-hydroxy (OH) vitamin D3 averaged 55.7 ng/ml in free-ranging rhinoceroses; no carotenoids were detected in any blood samples. Captive black and white rhinoceroses appear to be adequately supplemented in vitamin A and E. Captive Indian rhinoceroses (Rhinoceros unicornis) had significantly lower vitamin A concentrations in blood (P < 0.001) and higher vitamin A concentrations in liver tissue samples (P < 0.001) than other rhinoceros species. Equine requirements are not recommended as a model for rhinoceros vitamin requirements.     

Characterization of microsatellite loci in the black rhinoceros (Diceros bicornis) and white rhinoceros (Ceratotherium simum): their use for cross-species amplification and differentiation between the two species

As the population sizes of the black and white rhinoceroses continues to decline, more efforts are needed in multiple areas to help with the conservation efforts. One area being explored is the use of genetic diversity information to aid conservation decisions. In this study, we designed 21 microsatellite primers for white and black rhinoceroses, 16 and 17 of which amplified bands in the white and black rhinoceros, respectively. Out of these primers all 16 were polymorphic in the white rhinoceros and 12 of the 17 were polymorphic in the black rhinoceros. The mean number of alleles was 3.31 and 2.12, the expected heterozygosities were 0.420 and 0.372, and the observed heterozygosities were 0.436 and 0.322 for the white and black rhinoceroses, respectively. Seven of the primers produced different allele sizes and variations that distinguished between black and white rhinoceroses. Further genetic analyses with larger wild population sample sizes and markers are recommended to obtain a better understanding of the genetic structure of the black and white rhinoceros populations in order to be useful in the conservation efforts of these critically endangered species. A. Kilbourn—In memoriam.     

Black rhinoceros (Diceros bicornis) populations in northwestern Namibia are apparently not infected with piroplasms

Babesiosis is a potentially fatal disease in black rhinoceroses. Blood specimens collected from black rhinoceroses from Etosha National Park (n = 29) and Damaraland (n = 22), Namibia, were subjected to polymerase chain reaction using Theileria and Babesia genus-specific primers and reverse line blot, with negative results. The animals were sparsely infested with ticks. In the absence of suitable prophylactic measures, na?ve rhinoceroses would be at risk if translocated to Babesia-endemic areas.     

White rhinoceros range size in the south-western Kruger National Park

White rhinoceros range size was ascertained telemetrically in the south-western Kruger National Park. The mean annual range size of territorial males was 9.86 km² compared to 22.83 km² for adult females. White rhinoceros females' summer wet season range was larger (21.44 km²) than the winter dry season range (11.64 km²). It is argued that abundant field-water during the wet season enables animals to range further from permanent water supplies and to utilize larger foraging areas. White rhinoceros have core areas in their individual ranges that usually are situated along riverbanks in the preferred grazing regions. These core areas also include some favourite resting spots on high-lying areas. White rhinoceros range sizes in the south-western Kruger National Park were similar to those of other reserves with comparable white rhinoceros densities. In the Umfolozi Game Reserve, which has a higher white rhinoceros density than the Kruger National Park, the individual ranges are much smaller.     

Elevated free tyrosine in rhinoceros erythrocytes

Red blood cells of African black rhinoceroses (Diceros bicornis) are highly sensitive to oxidant-induced hemolysis and they possess a number of enzymatic and biochemical features that differ radically from other mammals. Here we show concentrations of free tyrosine in rhinoceros red blood cells which can approach levels as high as 1 mM, 50-fold higher than in human red blood cells. Elevated levels of tyrosine are also observed in red blood cells of other members of the order Perissodactyla such as the horse and zebra. Captive black rhinoceroses have significantly lower levels of red blood cell tyrosine than black rhinoceroses in the wild. Tyrosine transport studies indicate that black rhinoceros red blood cells have lost the ability to transport tyrosine as efficiently as human red blood cells.     

Ciliated intestinal protozoa of black (Diceros bicornis michaeli) and white rhinoceroses (Ceratotherium simum simum) in Kenya

The aim of this study was to survey ciliated intestinal protozoa of the black rhinoceros ( Diceros bicornis michaeli ) and white rhinoceros ( Ceratotherium simum simum ) in Kenyan National Parks. Faecal samples from 28 rhinoceroses that were chemically immobilized for translocation were opportunistically collected. Presence of ciliates was assessed using faecal floatation and sedimentation techniques. The ciliates were identified using cellular morphological features. Ophryoscoleciidae, Cycloposthiidae and Blepharocrythiidae were the three ciliate families represented. Ophryoscoleciidae had nine genera, Cycloposthiidae six genera and Blepharocorythiidae 1 genus. The dominant ciliate genus in all the rhinoceroses that were sampled was entodinium . It was found that the nutrient composition of the diet influences the diversity and numbers of intestinal ciliates, which in turn regulates the nutrient available to the animal. This interplay of nutrient composition of diet, ciliate diversity and nutritional benefits to the host has been used as an index to assess the nutritional state of ruminants. Because of the occurrence of rumenal ciliates in the hindgut fermentative chamber of the rhinoceros, such an index can be used to guide the formulation of feed mixtures for rhinoceros in captivity and remote nutritional assessments of rhinoceros both in free-range and in captivity.     

Gamete rescue in the African black rhinoceros (Diceros bicornis)

Mortality rates are high among captive African black rhinoceroses (Diceros bicornis), due to increased susceptibility to disease. The ability to rescue genetic material from individuals that die unexpectedly represents a practical approach to assist ex situ conservation efforts. The objectives of the present study were to attempt postmortem oocyte recovery from ovaries of African black rhinoceroses (N = 6) and to test the efficacy of equine protocols for rhinoceros oocyte IVM and IVF using cryopreserved rhinoceros sperm. The interval from ovary removal to oocyte recovery was 25.3 ± 13.9 h (mean ± SD). Ovaries were transported at 4 °C or 22 °C and effects of temperature on postmortem oocyte competence was evaluated. Numbers of oocytes collected per female averaged 15.8 ± 6.9. In total, 95 oocytes were recovered. Of these, 85 were inseminated using homologous sperm and 10 were inseminated using heterologous sperm. Overall, substantial numbers of viable oocytes were retrieved from African black rhinoceros ovaries 1 to 2 days postmortem from ovaries stored at ambient temperature. A proportion of these oocytes matured and underwent penetration and fertilization by heterologous or homologous frozen-thawed rhinoceros sperm. The reproductive competence of postmortem oocytes was further demonstrated by development of a single two-cell embryo. Despite the need for further refinements, gamete rescue in the rhinoceros has promise for producing rhinoceros embryos, as well as testing sperm functions in vitro.     

The effect of long non-reproductive periods on the genital health in captive female white rhinoceroses (Ceratotherium simum simum, C.s. cottoni)

White rhinoceroses suffer from a low reproductive rate in captivity. Intensive efforts to propagate specifically the northern white rhinoceros have been very limited. The dismal outlook for this subspecies in the wild makes successful ex situ breeding programs paramount. In this context, this study examined 48 southern and 6 northern white rhinoceroses using ultrasound and faecal hormone analysis to elucidate causes for female reproductive failure and to determine whether long non-reproductive periods have a detrimental impact on genital health. Results showed that 76% of the nulliparous females had intact hymenal membrane indicating these females had never been bred, at an age when their wild counterparts have delivered multiple offspring. Fifty-six percent of the studied population had various reproductive pathology. Cystic endometrial hyperplasia; leiomyomas of the cervix, uterus and ovary, adenoma; para-ovarian cysts and hydromucometra represent the scope of lesions identified. The stages of the lesions in nulliparous females correlated with age (r = 0.4, P < 0.05). Due to the severity of the lesions, 28% of the study population was considered post-reproductive. Therefore, the reproductive life span in some individuals was 10-20 years shorter than expected. However, in parous females the incidence of pathological lesions was significantly lower (P < 0.0001). Seventy-eight percent females studied had erratic or absent luteal activity. The hormone data corresponded with two ultrasonographic levels of ovarian activity, active and inactive, occurring within an age range of 3-19 years and 15-38 years, respectively. This suggests the lack of ovarian activity by reproductive mid-life in non-reproducing females. The accuracy of the ultrasound findings was validated by necropsy in nine animals showing a strong positive correlation (r2 = 0.9, p < 0.001). Our data suggests that the development of reproductive pathology and ovarian inactivity in white rhinoceros is an age-related consequence of long non-reproductive periods. This asymmetric ageing process of the genital organs can be prevented with the achievement of at least one pregnancy.     

Cage restraints for rhinoceroses

Captive breeding programs for the rhinoceros can be enhanced by studying their reproductive physiology. To do so requires repetitive manipulations under physically controlled circumstances. To facilitate these procedures, zoos throughout the world have constructed restraint devices, or chutes. Chute designs are usually determined by the space in which they are to be situated and the budget available for their construction. In this study, eight chute designs at seven institutions were compared for efficiency in controlling rhinoceroses during ultrasonography, semen collection, and blood sampling. Procedures were conducted on 16 rhinoceroses of four species: the white rhinoceros (Ceratotherium simum simum), the black (Diceros bicornis), the greater one-horned Asian (Rhinoceros unicornis), and the Sumatran (Dicerorhinus sumatrensis). Chutes were evaluated for dimensions, fabricating materials, procedures to be attempted, and structural features such as shoulder restraints. Permanent, indoor, side-adjustable, pass-through chutes were determined to be the most efficient and convenient type because they saved time and reduced stress on the animals. Zoo Biol 17:343–359, 1998. © 1998 Wiley-Liss, Inc.     

Observations on rhinoceros tongue morphology

The rhinoceros tongue is distinguished among perissodactyl organs by its sagittally divided intermolar eminence crowned by a dextral and a sinistral cluster of circumvallate papillae. These structures were originally noted in the Indian rhinoceros by Owen (1852) but have since remained unmentioned for this, or for any other rhinoceros form. They are here described for specimens of the Indian, African White and African Black rhinoceroses. The structural and functional nature of the intermolar eminence is established and this entity is distinguished from an hitherto unrecognized topographical component of the tongue, designated the pars elliptica linguae. The lingual morphology of the three rhinoceros forms is summarised and certain intergeneric differences are noted.     

Tourists' Effects on Drivers of Working Asian Elephants

ABSTRACT

A growing wildlife tourist industry in Asia sometimes includes the use of elephants to transport tourists. Elephant drivers are responsible for the safe management of their elephants while loading, transporting, and unloading tourists for rides to view wildlife. This study sought to investigate the preferences and perceptions of elephant drivers regarding tourists when viewing rhinoceroses in the field. Drivers were interviewed with standardized questions with translator assistance and asked to show the distances from the rhinoceros at which both they and tourists preferred to view a rhinoceros. According to drivers, they preferred to be a longer distance from a rhinoceros than did tourists. Drivers described that some tourists wanted to get closer than was safe to rhinoceroses and other wildlife so as to take good photographs; some even wanted to be within touching distance. Drivers noted that tourists with long camera lenses did not seek or request to be as close to the rhinoceros as tourists with cameras with short lenses.     

Assisted reproductive technologies in captive rhinoceroses

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THE CAPTURE AND TRANSLOCATION OF THE BLACK RHINOCEROS

Black rhinoceroses were hunted on foot, from a vehicle, or from a helicopter throughout their range of habitats. Animals weighing 370–1,260 kg could be handled an average of 13 min after darting with a mixture of etorphine (1.9 μg/kg) and acepromazine (19 μg/kg). Cases of respiratory depression at this dosage were reversed soon after immobilisation by the intravenous injection of cyprenorphine (1.0 μg/kg). The captured animal was transported to camp, lashed on its side to a sledge, for periods of up to 515 min without mishap. A rhinoceros that was unloaded 180 min after darting rose to its feet and tried to push its way through the corner of the pen until antidote was administered at 215 min. This action was not observed in animals that received a total dose of 3.4 μg/kg of cyprenorphine before being unloaded 90–413 min after darting. The addition of hyoscine (at doses as low as 18 μg/kg) to etorphine and acepromazine improved the tractability of rhinoceroses that were only partially immobilised. However, sometimes at this dosage and always at doses above 35 μg/kg, hyoscine prolonged the ‘pushing’ phase for 175–410 min after darting despite the administration of cyprenorphine (4.0 μg/kg). There were no mortalities due to drug action in the series of 59 healthy rhinoceroses that were dart-immobilised, but there were two deaths due to mishandling and one caused by subsequent mismanagement.