Conservation genetics of the black rhinoceros (Diceros bicornis)

We analysed genetic variation in mitochondrial DNA (mtDNA) control region in the Diceros bicornis minor and D. b. michaelianimals at the Western Plains Zoo, which form part of an international ex-situ breeding program. Six of the nine D. b. minor animals were wild-caught from Zimbabwe during the 1990s, and our study revealed five distinct mtDNA haplotypes, and a haplotype diversity of 0.86 in the colony. Phylogenetic relationships between mtDNA haplotypes analyzed using the neighbor joining method reveal that for the small sample available, D. b. minor and D. b. michaeli are reciprocally monophyletic and represent separate ancestral lineages. Nucleotide divergence between the black and and white rhinoceros(Ceratotherium simum) was 14.0%, and nucleotide divergencebetween the D. b. minor and the D. b. michaeli subspecies was 2.6%. This suggests a divergence time for the two blackrhinoceros subspecies of between 0.93 MY and 1.3 MY.     

Predicting the habitat usage of African black rhinoceros (Diceros bicornis) using random forest models

Species distribution models are often used in ecology to ascertain relationships between environmental variables and species presence. Modelling to understand this relationship can be used to aid conservation management strategies. In this paper, we applied the random forest classification method to predict habitat used by black rhino for browsing. The random forest model was created using detailed habitat data collected from Ol Pejeta Conservancy in Kenya. Variables from plots where rhino had been present were compared to those not used by rhino. Independent data were used to test the predictive accuracy of the rules generated. The model performed well with the independent test data, correctly classifying 69% of the sampling plots where black rhino were present. Important habitat features that affected rhino presence were browse availability and density of vegetation, with Vachellia drepanolobium (formerly Acacia) and Euclea divinorum being important components. The analysis also highlighted areas of potential high browse pressure, which should be the focus of continued monitoring and management.     

Measurement of fecal steroids in the black rhinoceros (Diceros bicornis) using group-specific enzyme immunoassays for 20-oxo-pregnanes

The metabolism and excretion of progesterone in different animal species results in several fecal 5-reduced progesterone metabolites (pregnanes), which in recent studies were quantified using progesterone antibodies. To increase the accuracy of fecal 20-oxo-pregnane evaluations in the black rhinoceros, enzyme immunoassays (EIA) using antibodies against 5α-pregnane-3β-ol-20-one 3HS:BSA (5α-20-one EIA) and 5β-pregnane-3α-ol-20-one 3HS:BSA (5β-20-one EIA) were developed. The assays showed high crossreactivities with pregnanes containing a 20-oxo group and are referred to as group-specific; results of these assays were compared with an EIA using an antibody against 6HS-progesterone (4-ene-20-one EIA). Fecal samples of both subspecies of the black rhinoceros (Diceros bicornis michaeli, n = 5, and Diceros bicornis minor, n = 1) during pregnancy were collected 1–3 times/week. HPLC separation showed three major immunoreactive fecal 20-oxo-pregnane peaks; their elution profiles and different crossreactivities in the three EIAs provided strong evidence that these peaks are 5α-pregnane-3, 20-dione, 5α-pregnane-3α-ol-20-one, and 5α-pregnane-3β-ol-20-one. Pregnane values in the pregnant animals continuously increased between months 3–7 and were significantly (P < 0.01) elevated above the levels of nonpregnant animals (0.2 μg/g) by week 11. During months 6–13 concentrations in the 5α-20-one and in the 5β-20-one EIA (5–11 μg/g) were significantly (P < 0.01) higher than in the 4-ene-20-one EIA (1.5–3 μg/g). In conclusion, the immunoreactive fecal 20-oxo-pregnane metabolites in the black rhinoceros are determined more accurately with antibodies against pregnane-20-one-C3 conjugates, as compared with a progesterone antibody. © 1996 Wiley-Liss, Inc.     

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.     

Characteristics of black rhinoceros (Diceros bicornis) bedding sites

Black rhino numbers have decreased greatly since the early 1970s, primarily as a result of poaching. A recent strategy to protect rhinos in Kenya has been to establish fenced sanctuaries. This has increased the rhino population and that of other species, but problems have emerged because of limited dispersal and rising animal densities. Unfortunately, how rhino utilize habitat, especially areas called bedding sites, is not well understood. These areas provide shade and may be a critical component of rhino habitat. We measured habitat variables at bedding sites located in bedding plots and compared them with control plots at Sweetwaters Reserve, Kenya. Euclea divinorum was the most common tree in the bedding site comprising 64.3% of the vegetation. Elephant dung was significantly more likely to be found in bedding plots than in control plots which suggests that elephants and rhinos use overlapping habitats. Elephants may be causing damage to the tree species that are important for bedding sites. Resource competition between large herbivores in small reserves is likely to negatively affect the tree species. Black rhino habitat, particularly bedding sites, may be at risk and rhino numbers may decrease.     

Gross anatomy of the reproductive tract of female black (Diceros bicornis michaeli) and white rhinoceros (Ceratotherium simum simum)

Reproductive tracts were collected from three black rhinoceros and two white rhinoceros at necropsy. All females were nulliparous, except for one primiparous white rhinoceros. The animals ranged in age from 7 days to 28 years. All nulliparous animals had a constriction in the vaginal canal, which appeared to be a hymen. The primiparous female had only remnants of a hymen. The total length of the tract averaged 102 cm in three adult animals (≥21 years old). The distance from the vulva to the external cervical os averaged 40 cm in these animals. The endometrium of a 28-year-old nulliparous black rhinoceros and a 27-year-old nulliparous white rhinoceros exhibited signs of hyperplasia, whereas this condition was not present in the other animals. It is not known if this condition was related to the fertility of these animals. The cervix of the rhinoceros was firm, and the lumen followed a very tortuous path through eccentric rings of tissue. Visual examination of the ovaries revealed the presence of surface follicles on the ovaries in both species. The overall size of the reproductive tract, especially the vagina and uterine horns, and the extremely tortuous cervical lumen will present obstacles that must be overcome when developing artificial insemination and embryo transfer proceduers for use in rhinoceros.     

Genetic variation and population structure in remnant populations of black rhinoceros, Diceros bicornis, in Africa

Black rhinoceros (Diceros bicornis) are one of the most endangered mammal species in Africa, with a population decline of more than 96% by the end of the last century. Habitat destruction and encroachment has resulted in fragmentation of the remaining populations. To assist in conservation management, baseline information is provided here on relative genetic diversity and population differentiation among the four remaining recognized subspecies. Using microsatellite data from nine loci and 121 black rhinoceros individuals, and comparing the results with those of other African species affected in similar ways, Diceros bicornis michaeli retained the most genetic diversity (heterozygosity 0.675) compared with Diceros bicornis minor (0.459) and Diceros bicornis bicornis (0.505), suggesting that the duration of the known bottlenecks in these populations has only had a limited impact on diversity. Comparable and moderate degrees of population differentiation were found between D. b. minor, D. b. bicornis and D. b. michaeli. Results from the single sample available of the most endangered subspecies, Diceros bicornis longipes, showed the least diversity of all individuals examined. This information should assist conservation management decisions, especially those affecting population viability assessments and selection of individuals for translocations, and will also facilitate subspecies identification for ex situ individuals of uncertain origin.     

Pregnancy diagnosis in the black rhinoceros (Diceros bicornis) by salivary hormone analysis

Saliva samples collected from 12 black rhinoceroses (Diceros bicornis) were analyzed for 20 alpha-hydroxyprogesterone-like and total estrogen immunoreactivity. Five nonpregnant, two conception, and nine pregnancy intervals were monitored. HPLC co-chromatography of immunoreactive free steroid components in saliva indicated that one of three immunoreactive progestin components eluted with 20 alpha-hydroxypreg-4-en-3-one, whereas the single major estrogen peak eluted with estradiol. Direct radioimmunoassay measurements of ether extracts for these two reproductive hormones in saliva provided accurate and consistent profiles in which pregnancy was detected 13 months prior to parturition, and parturition was predicted by approximately 1 month. Measurement of 20 alpha-hydroxypreg-4-en-3-one was more useful in pregnancy diagnosis, and estradiol measurement was useful as an indicator of impending parturition. © 1996 Wiley-Liss, Inc.     

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.     

Density dependence and population dynamics of black rhinos (Diceros bicornis michaeli) in Kenya’s rhino sanctuaries

Density‐dependent feedback mechanisms provide insights into the population dynamics and interactions of large herbivores with their ecosystem. Sex ratio also has particularly important implications for growth rates of many large mammal populations through its influence on reproductive potential. Therefore, the interrelationships between density‐dependent factors, comprising density, sex ratio and underlying growth rates (r) were examined for the Eastern black rhino (Diceros bicornis michaeli) living in three rhino sanctuaries in Kenya using four population models. The exponential and logistic models gave similar results and the former were accepted because they better portrayed the actual situation on the ground. Sex ratios in all sanctuary populations were positively correlated with r but interpreted with realization of other factors also affecting r. We caution that the results of population models should be interpreted alongside ground‐truthed observations. We recommend that future translocation strategies should take into account sex and age structures of the donor population, while future studies of density dependence should take into account both biotic and abiotic factors.     

Evaluation of habitat use and ecological carrying capacity for the reintroduced Eastern black rhinoceros (Diceros bicornis michaeli) in Ruma National Park,Kenya

We carried out a postrelease evaluation to determine predictors of habitat use and carrying capacity for the black rhinoceros (Diceros bicornis michaeli), which are critical for monitoring how the Ruma National Park sub-population may contribute to Kenya's meta-population strategy. We determined whether level of elevation, rockiness, shade, distance to fence, roads, and human settlements predict habitat use, differences in habitat and diet preference between female and male black rhinoceros, and the ecological carrying capacity (CC) of black rhinoceros in the park. We used standard ecological methods to collect data on predictors of habitat use, habitat preference and to estimate CC. Results show, first, that none of the environmental and anthropogenic factors evaluated predicted habitat use by black rhinoceros in the park. Second, although there was no significant difference in habitat preference between the sexes (U = 16.50, p = 0.306), there was a 60% difference in Jaccard's dissimilarity in diet selection between the sexes. Third, the park can support 65 black rhinoceros. Altogether, the findings suggest that the park has potential to support other sub-populations in Kenya. We recommend that future similar studies should incorporate population viability analysis and a community-based approach to forecast the species health and extinction risk.     

Predicting savanna vegetation structure on the basis of plant available moisture (PAM) and plant available nutrients (PAN): a case study from Australia

The use of the plant available moisture (PAM)/plant available nutrients (PAN) concept to compare savanna structure was examined using data from twenty Australian sites. Above-ground biomass was regressed on various combinations of seventeen different estimates of PAM (plant available moisture) and two estimates of PAN (plant available nutrients). The ratios of actual transpirational loss from the subsoil to potential evapotranspiration (PET), and total annual rainfall to PET, were most highly correlated with total biomass. Grass biomass is poorly predicted by PAM on its own, and requires inclusion of woody leaf biomass in the regression. PAN had little effect on total biomass, although it is likely to be important for other, functional aspects of vegetation. The woody : grass ratio is best predicted by an index involving the ratio of subsoil : topsoil moisture. For biomass comparisons the use of a detailed water-balance model to estimate PAM is not warranted.     

Serum alpha-tocopherol, all-trans retinol, total lipids and cholesterol in the black rhinoceros (Diceros bicornis)

1. Mean concentration of serum alpha-tocopherol (Vitamin E) in 28 free-living black rhinoceroses sedated during translocation in Zimbabwe was 1.92 (SD, 0.43) mg/l. 2. Alpha-tocopherol was not detectable (less than 0.15 mg/l) in five captive black rhinoceroses held at London Zoo. 3. Circulating levels of all-trans retinol (Vitamin A) were not different between the two groups. 4. The low level of alpha-tocopherol in captive rhinoceroses suggests a risk of acute haemolytic anaemia.     

Serum and fecal steroid analysis of ovulation,pregnancy, and parturition in the black rhinoceros (Diceros bicornis)

Studies were conducted to determine: (1) if fecal hormone metabolite concentrations correlated with serum estrogen and progesterone concentrations, follicular activity and reproductive behavior in the black rhinoceros (Diceros bicornis) and (2) if threshold values of respective fecal metabolite concentrations correlated with pregnancy. Blood and fecal samples were collected, in conjunction with transrectal ultrasound and behavior observations, for an 18-month period from one black rhinoceros female. Subsequently, serial fecal samples were collected from 13 females in 10 zoos. Quantitative analysis of serum progesterone (P₄) and estradiol (E₂) was performed by radioimmunoassay (RIA): analysis of fecal estrogen metabolites (E) and fecal progesterone metabolites (P) were performed by enzyme immunoassay (EIA). Serum P₂ concentrations identified two luteal phase patterns and two nadirs which corresponded with behavioral estrus. Fecal E patterns indicated a sharp peak which corresponded with breeding. concentrations of fecal P illustrated identifiable nadirs and several peaks which corresponded to serum P₄ nadirs and luteal phases. Serum P₄ concentrations were not different between the luteal phase and pregnancy. Fecal P concentrations started to rise above luteal phase concentrations approximately 150 days postbreeding and remained elevated until immediately before parturition. Serum E₂ and fecal E concentrations rose and subsequently declined after parturition. In the fecal samples from seven pregnant females, fecal P concentrations were similarly elevated compared to six nonpregnant females. Results indicated that fecal steroid metabolites accurately reflected serum steroid hormone concentrations and that the measurement of P and E concentrations permitted the characterization of the estrous cycle, the diagnosis of pregnancy, and the onset of parturition. Zoo Biol 16:121–132, 1997. © 1997 Wiley-Liss, Inc.