Alkaline phosphatase as an indicator of true ejaculation in the rhinoceros

The objective was to determine if seminal alkaline phosphatase (ALP) can serve as an indicator of true ejaculation in the rhinoceros. Concentrations of ALP activity were determined in seminal fractions collected from African black rhinos (Diceros bicornis), an African white rhino (Ceratotherium simum), and an Indian rhino (Rhinoceros unicornis) during electroejaculation. In addition, seminal fractions collected during penile massage of a Sumatran rhino (Dicerorhinus sumatrensis) were assessed. Correlations between ALP activity and sperm concentration, fraction pH, and fraction osmolality were evaluated in the Indian rhino and black rhino. Concentrations of ALP activity in rhino ejaculate fractions ranged from < 5 to 11,780 U/L and were positively correlated (P < 0.05) with sperm concentration for both Indian rhino (r = 0.995) and black rhino (r = 0.697), but did not exhibit a strong correlation with either pH or osmolality (P > 0.05). Data were insufficient for establishing meaningful correlation coefficients in the Sumatran rhino and white rhino, but preliminary results were in accordance with findings in the Indian rhino and black rhino. We concluded that ALP was present in rhinoceros semen, likely originated from the epididymides and/or testes, and could serve as a useful tool for assessing the production of ejaculatory versus pre-ejaculatory fluid in the rhinoceros.     

Mineral concentrations in serum/plasma and liver tissue of captive and free‐ranging Rhinoceros species

Mineral implications in health issues of captive rhinos have received much attention lately. This study was undertaken to establish reference values for the mineral status of rhinos. Serum/plasma samples of free‐ranging black (Diceros bicornis) and white (Ceratotherium simum) rhinos and of captive black, white, Indian (Rhinoceros unicornis), and Sumatran (Dicerorhinus sumatrensis) rhinos, as well as liver tissue samples of captive black, white, and Indian rhinos were analyzed for mineral content. Circulating mineral levels of free‐ranging animals were subject to variation according to region. In free‐ranging animals, high molybdenum (Mo) values compared to horse normals were striking. Captive animals displayed even higher circulating Mo concentrations. The significance of iron (Fe) overload in captive specimens of the browsing rhinos (black and Sumatran) was confirmed. Hepatic Fe levels increased in blacks with age. Although this Fe overload is suspected to be linked with diets, the data indicate that this is not due solely to an excessive dietary Fe supply. Whereas the grazing species (white and Indian) had high liver copper (Cu) levels, the browsing species had low to marginal liver Cu concentrations. Liver concentrations of K, Mg, Co, and Mo increased with age in captive black rhinos. Additional findings include high circulating Se levels in all rhino species. Future research should be directed at investigating factors leading to high Fe levels, and at investigating Cu metabolism in captive rhinoceros species. Zoo Biol 24:51–72, 2005. © 2005 Wiley‐Liss, Inc.     

Effects of browse availability and quality on black rhino (Diceros bicornis michaeliGroves 1967) diet in Nairobi National Park,Kenya

Investigations were conducted to study the effects of browse availability and quality on black rhinoceros (Diceros bicornis michaeliGroves 1967) diet in Nairobi National Park, Kenya, between September 1994 and February 1995. Line intercept method was used to assess availability of browse. The ratios of browsed crowns to the total crown were applied in the estimate of browse use. Proximate analysis and extraction methods were used, respectively, to appraise browse nutritional quality and phytochemical status of twelve species. Thirty‐four plant species were identified as potential black rhino browse, whose availability differed significantly (P < 0.001). Lippia javanica (20.581 ± 3.101, n = 544) and Lannea cornuta (0.963 ± 0.529, n = 544) were the most and least available species, respectively. Only 32 species, however, were utilized, as L. cornuta and Rhynchosia hirta did not show any level of rhino feeding activity. Browse use was found to vary significantly (P < 0.001) during the study period. Grewia similis was most (20.564 ± 2.764, n = 544) and Commelina africana least (0.974 ± 0.974, n = 544) exploited, respectively. Differences in total alkaloids and phenols, crude fibre, crude protein and total ash contents among species investigated were highly significant (P < 0.001). Ether extract values were similar (P= 0.0933) across plant taxa. The study shows that the black rhino is a selective browser feeding on plants with low phenol and alkaloid contents and high fibre values. Of the nutritionally similar species, browsing was driven by availability. These findings suggest that a key habitat factor important in black rhinoceros conservation include diverse plant species with low levels of phenols and alkaloids.     

Declining and low fecal corticoids are associated with distress, not acclimation to stress, during the translocation of African rhinoceros

Concentrations of adrenal steroid metabolites in feces are routinely used to assess the welfare of animals that are the subject of conservation efforts. The assumption that low and declining corticoid concentrations indicate the absence of stress and acclimation, respectively, is often made without experimental support or wild-animal comparisons, although intrinsic control of adrenal steroids might occur even under ongoing stress and distress. We adopted the capture and 11-week captivity of 18 black ( Diceros bicornis : 11 males, seven females) and 52 white ( Ceratotherium simum : 22 males, 30 females) rhinoceros as an experimental test of the relationship between corticoid concentrations and stress (translocation) and measured for suppressed gonad function as an indicator of distress – the biological cost of cumulative stressors. Fecal samples collected from the rectum at capture and during captivity were stored frozen and their corticoid, and androgen (in males) or progestin (in females), concentrations determined by radioimmunoassay. Corticoid profiles followed the expected pattern of being two to five times pre-capture levels (ng g⁻¹: black rhino: female 24.5±3.7, male 23.9±2.2; white rhino: female 16.3±1.6, male 12.3±2.4) for up to 17 days after capture and declined with time in captivity. Black rhinoceros and male white rhinoceros corticoids declined below pre-capture values and were associated with suppressed levels of androgens and progestins with increased time in captivity. Declining corticoids could not be interpreted as acclimation or the absence of stressors, without also measuring for distress in African rhinoceros. White rhinoceros female corticoid values remained elevated, although their gonad steroid levels were also suppressed. We discuss our findings for the management of rhinoceros in the wild and captivity.     

Effects of sociosexual environment on serum testosterone in captive male African rhinoceros

The relationships between testosterone concentrations in male African rhinoceros and the presence of conspecific males and females were investigated. Serum testosterone concentrations were measured using enzyme-linked immunoassay (EIA) in 37 male black rhinoceros (Diceros bicornis) and 21 male white rhinoceros (Ceratotherium simum) housed at 37 institutions in the USA. Testosterone concentrations in both black (n = 37) and white (n = 21) rhinoceros males rose with increasing numbers of females present (P < 0.05). Average testosterone concentrations also rose with an increased number of conspecific males (n = 34) in black rhinoceros (P < 0.05). However, no specific pattern was found among male white rhinoceros housed with other males. We inferred that introduction of females to a male may play an important role in stimulating libido and spermatogenesis. The similar response of black rhinoceros and white rhinoceros to increased numbers of females suggested that, at least historically, herd structure for blacks may have been more similar to whites than previously realized, and should be investigated further.     

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.     

Not all ants are equal: obligate acacia ants provide different levels of protection against mega‐herbivores

In obligate ant–plant mutualisms, the asymmetric engagement of a single plant species with multiple ant species provides the opportunity for partners to vary in their behaviour. Variation in behaviour has implications for the interactions with third‐party species such as herbivores. This study assessed the effect of obligate ant‐mutualists (Crematogaster mimosae, Crematogaster nigriceps and Tetraponera penzigi) inhabiting the African ant‐acacia (Acacia drepanolobium) on three mega‐herbivore browsers: the Maasai giraffe (Giraffa camelopardalis tippelskirchi), the reticulated giraffe (Giraffa c. reticulata) and the black rhino (Diceros bicornis). Giraffes are abundant and wide‐ranging herbivores of the acacias, whereas black rhinos are localized and perennial herbivores of the acacias. Multiyear field studies comparing the ants’ aggressive behaviour and browsing by mega‐herbivores suggested differences between the tending abilities of the primary ant species inhabiting A. drepanolobium. Trees occupied by the aggressive ant species C. mimosae had significantly less browsing by giraffes and black rhino than trees occupied by other ant species. The results of this study provide evidence that ant‐mutualists on African acacias can serve as deterrents to mega‐herbivores and that different ant species vary in their tending abilities.     

Reproductive behaviour of captive Sumatran rhinoceros (Dicerorhinus sumatrensis)

The Sumatran rhinoceros (Dicerorhinus sumatrensis) is on the verge of extinction in Malaysia. At the Sumatran rhinoceros Conservation Centre in Sungai Dusun, the reproductive behaviour of two female and two male rhinoceroses were studied for 8-10 months during attempts to breed them in captivity. Due to the paucity of scientific information on the reproductive biology of the Sumatran rhinoceros, this study was conducted to obtain information on the reproductive behaviour of this species. The male rhino was introduced to a female rhino in the morning for 1-2 h daily in order to observe for behavioural oestrus. Observations were made on the signs of oestrus and mating behaviour. Oestrus was determined by receptivity towards the male and lasted about 24 h. Common signs of oestrus were an increase in frequency of urine spraying, tail raising or swinging, anogenital and other contacts. Although the males exhibited mounting, the inability of the male to achieve intromission was poor. The study demonstrated that the pattern of courtship and copulation of the captive Sumatran rhinos were comparable with those of other rhino species, reported previously by other scientists and flehmen reflex was also exhibited by the male Sumatran rhinos. In a captive breeding programme, it is recommended that only an oestral female is introduced into a male enclosure due to the male solitary behaviour and to avoid serious injuries inflicted onto the females.     

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.     

Semen cryopreservation in the Indian rhinoceros (Rhinoceros unicornis)

The objective was to identify an extender and cryoprotectant combination for Indian rhinoceros (Rhinoceros unicornis) sperm that yielded high post-thaw sperm quality. Male Indian rhinoceroses (n = 6; 7.5-34 yr old) were anesthetized and subjected to a regimented electroejaculation procedure (75-100 mAmps; 4-10 volts; 7-150 stimuli; total of 10 electroejaculation procedures). High quality semen fractions from each ejaculate were divided into four aliquots and a 2 x 2 factorial design used to compare the effect of two sperm extenders (standard equine [EQ] and skim milk-egg-yolk-sugar [SMEY]), and two cryoprotectants (glycerol and dimethylsulfoxide [DMSO]). Cyropreserved samples were thawed and assessed for motility, viability and acrosome integrity over time. Electroejaculate fractions processed for cryopreservation had high sperm concentration (516 × 10⁶/mL) and motility (79%). Post-thaw sperm characteristics were higher (P < 0.05) when semen was cryopreserved in EQ versus SMEY. Post-thaw motility of sperm cyropreserved in EQ averaged 50-55% compared to 22-37% in SMEY, with no significant differences in sperm characteristics of samples cyropreserved in glycerol and DMSO. In conclusion, sperm collected from Indian rhinoceroses via electroejaculation were cryopreserved using EQ extender with either glycerol or DMSO; post-thaw quality was adequate for use in assisted reproductive procedures.     

Contemporary and historical impacts of megaherbivores on the population structure of tree euphorbias in South African subtropical thicket

African elephant and black rhinoceros – both megaherbivores – impact negatively on the abundance of succulent plants, including tree succulents, in South Africa's subtropical thicket. We sampled 35 sites in subtropical thicket to assess historical and contemporary impacts of African elephant and black rhinoceros on the population structure of three species of succulent tree euphorbia. Population age structures were highly variable and showed no species-specific effects. Almost half the sites had growing populations dominated by young individuals. Sites having contemporary impacts had significantly fewer individuals in the 30–75 years age range, but this effect declined with increasing terrain slope. Eighty-one percent of sites assumed to have been impacted by megaherbivores historically had individuals that predated rhino and elephant extirpation in the region. This and other population age structure data indicate that megaherbivores and tree euphorbias coexisted historically on terrain of low relief. However, in areas not subject to contemporary impacts, euphorbia populations are likely to be much higher now than historically, owing to population relaxation after the extirpation of megaherbivores in the mid 1800s. Given the sensitivity of tree euphorbias to megaherbivore impacts, managers should consider monitoring the population structure of these species in order to identify thresholds of potential concern regarding megaherbivore impacts.     

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.     

Genetic variation of complete mitochondrial genome sequences of the Sumatran rhinoceros (<Emphasis Type="Italic">Dicerorhinus sumatrensis</Emphasis>)

The Sumatran rhinoceros (Dicerorhinus sumatrensis) is the smallest and one of the most endangered rhinoceros species, with less than 100 individuals estimated to live in the wild. It was originally divided into three subspecies but only two have survived, D. sumatrensis sumatrensis (Sumatran subspecies), and D. s. harrissoni (Bornean). Questions regarding whether populations of the Sumatran rhinoceros should be treated as different management units to preserve genetic diversity have been raised, particularly in light of its severe decline in the wild and low breeding success in captivity. This work aims to characterize genetic differentiation between Sumatran rhinoceros subspecies using complete mitochondrial genomes, in order to unravel their maternal evolutionary history and evaluate their status as separate management units. We identified three major phylogenetic groups with moderate genetic differentiation: two distinct haplogroups comprising individuals from both the Malay Peninsula and Sumatra, and a third group from Borneo. Estimates of divergence time indicate that the most recent common ancestor of the Sumatran rhinoceros occurred approximately 360,000 years ago. The three mitochondrial haplogroups showed a common divergence time about 80,000 years ago corresponding with a major biogeographic event in the Sundaland region. Patterns of mitochondrial genetic differentiation may suggest considering Sumatran rhinoceros subspecies as different conservation units. However, the management of subspecies as part of a metapopulation may appear as the last resource to save this species from extinction, imposing a conservation dilemma.     

A study on the postrelease behaviour and habitat preferences of black rhinos (Diceros bicornis) reintroduced into a fenced reserve in Namibia

Translocations and reintroductions are key elements for the population management of the critically endangered black rhino (Diceros bicornis, Linnaeus, 1758). In this study, we investigated the postrelease behaviour and habitat preferences of a black rhino starter group (n = 4) on the individual level. The animals were reintroduced to a fenced game reserve (87 km²) in North‐Central Namibia 1 year prior to our study. We used camera traps and very high frequency (VHF) radiotelemetry to examine the animals' temporal and spatial behaviour over a period of 4 months at transition between wet and dry seasons. Our results underline a peak in drinking activity and waterhole visits occurring between 7 p.m. and 8 p.m. We found a shift in intensity in drinking activity during the period of the study. Satellite‐based woody cover estimations only suggest positive correlations between the density of woody cover and favoured black rhino habitat types. Although the area seems suitable to facilitate breeding success of this starter group, it does not support a self‐sustaining population. However, black rhinos were already successfully reintroduced to several additional fenced reserves in this region. The selective opening of fences in the future could help to enable genetic exchange between currently isolated groups of rhinos.     

Feasibility of sex-sorting sperm from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis)

The objective of these studies was to investigate the practicality of flow cytometric sex-sorting for spermatozoa from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis). In Experiment 1, four semen extenders were tested regarding their suitability for liquid preservation of spermatozoa before sorting. Dilution in MES-HEPES-based semen extender followed by incubation generated best sperm quality parameters (motility, viability, and acrosome integrity). In Experiment 2, the effect of staining method (15 °C for 4 to 6 h during transport or 37 °C for 1 to 1.5 h) on sort efficiency and sperm quality was investigated. Staining at 15 °C during transport resulted in a higher percentage of sperm samples showing a resolution of X- and Y-chromosome-bearing populations (60%) compared with that for staining at 37 °C after transport (33%) and resulted in superior sperm integrity after staining (43.8 ± 11.3% vs. 19.6 ± 12.1%). Sort rate was 300 to 700 cells/sec and sort purity, determined for one sorted sample, was 94% for X-chromosome-bearing spermatozoa. In Experiment 3, the highly viscous component of rhinoceros seminal plasma, which complicates the process of sperm sorting, was examined by gel electrophoresis and mass spectrometry. Results suggested a 250-kDa glycoprotein (most likely originating from the bulbourethral gland) to be responsible for the characteristic viscosity of ejaculates. In Experiment 4, viscosity of seminal plasma, as measured by electron spin resonance spectroscopy, was significantly decreased after addition of α-amylase or collagenase (0.5 and 3 IU per 100 μL seminal plasma, respectively) by 28% and 21%, respectively, with no negative effect on sperm characteristics. The results of this study demonstrate for the first time that rhinoceros spermatozoa can be successfully sorted into high-purity X- and Y-chromosome-bearing populations. Furthermore, the successful liquefaction of viscous ejaculates provides the means to greatly improve sort-efficiency in this species.     

Post-coital sperm recovery and cryopreservation in the Sumatran rhinoceros (Dicerorhinus sumatrensis) and application to gamete rescue in the African black rhinoceros (Diceros bicornis)

Sumatran rhinoceros (Dicerorhinus sumatrensis) sperm samples were collected from a post-copulatory female and characterized to determine their potential for sperm preservation and future use in artificial insemination. Five samples of acceptable quality from one male were used to compare the effect of two cryoprotectants (glycerol and dimethyl sulfoxide (DMSO)) and two post-thaw protocols (untreated and glass wool column) on sperm quality. The percentage of motile spermatozoa, sperm motility index (0-100) and sperm morphology were evaluated subjectively, and viability and acrosomal status were assessed using fluorescent markers. Evaluations of frozen-thawed spermatozoa were performed over a 6 h incubation interval. Post-coital semen samples (n = 5; 104.0 +/- 9.1 ml; 2.5 +/- 0.8 x 10(9) total spermatozoa; mean +/- SEM) exhibited a sperm motility index of 56.7 +/- 3.3, and contained 40.2 +/- 6.3%, 72.0 +/- 3.2% and 79.8 +/- 6.5% normal, viable and acrosome-intact spermatozoa, respectively. Glycerol and DMSO were equally effective as cryoprotectants and, regardless of post-thaw protocol, samples retained greater than 80% of all pre-freeze characteristic values. Processing semen samples through glass wool yielded higher quality samples, but only half the total number of motile spermatozoa compared with untreated samples. High values for pre-freeze sperm characteristics were also maintained after cryopreservation of epididymal spermatozoa from one black rhinoceros (Diceros bicornis) using the same protocol. In summary, Sumatran rhinoceros spermatozoa of moderate quality can be collected from post-copulatory females. Rhinoceros sperm samples show only slight reductions in quality after cryopreservation and thawing and have potential for use in artificial insemination.     

An analysis of threats,strategies, and opportunities for African rhinoceros conservation

The complexity and magnitude of threats to black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros conservation in Africa have triggered global concerns and actions. In this study, we analyzed (i) threats to rhinoceros conservation including external shocks, (ii) historical rhinoceros conservation strategies in Zimbabwe and Africa, more broadly, and (iii) opportunities for enhanced rhinoceros conservation in Zimbabwe and Africa. A literature search from 1975 to 2020 was carried out using a predefined search protocol, involving a number of filters based on a set of keywords to balance search sensitivity with specificity. A total of 193 articles, which were most relevant to key themes on rhinoceros conservation, were used in this study. The common threats to rhinoceros conservation identified in this paper include poaching, habitat fragmentation and loss, international trade in illegal rhino products, and external shocks such as global financial recessions and pandemics. Cascading effects emanating from these threats include small and isolated populations, which are prone to genetic, demographic, and environmental uncertainties. Rhinoceros conservation strategies being implemented include education and awareness campaigns, better equipped and more antipoaching efforts, use of innovative systems and technologies, dehorning, and enhancing safety nets, and livelihoods of local communities. Opportunities for rhinoceros conservation vary across the spatial scale, and these include (a) a well‐coordinated stakeholder and community involvement, (b) strategic meta‐population management, (c) enhancing law enforcement initiatives through incorporating real‐time surveillance technologies and intruder detection sensor networks for crime detection, (d) scaling up demand reduction awareness campaigns, and (e) developing more certified wildlife crime and forensic laboratories, and information repository for international corporation.