Northern White Rhinoceros Research Articles

The fate of Uganda’s Northern white rhino translocated to Murchison Falls National Park in 1961 and 1964

This historical note records the demise of Uganda’s northern white rhino (Ceratotherium simum cottoni) population for the scientific record. Ceratotherium simum was distributed in Africa between the Mahgreb and the southern Cape, wherever there was suitable grazing (though not necessarily in all places at the same time), until after the appearance of Homo sapiens on the continent. By the eighteenth century this extensive range had contracted into two widely separated and taxonomically distinct populations, one Sudanic and the other southern African. The Sudanic population of northern white rhino (NWR) were scientifically recognised in 1907 as (Ceratotherium simum cottoni) taxonomically separate from C.s.simum. The NWR previously occurred in Uganda but is currently believed to be extinct in the country. Given low human densities and the inaccessibility of much of the Central African Republic and Southern Sudan, it is possible that a few undetected individuals still exist in remote places. For example, while extinct in the Democratic Republic of the Congo’s Garamba National Park, a few may possibly remain in the contiguous ‘domain de chasse’. Cette note historique enregistre la disparition de la population de rhinocéros blancs du Nord (Ceratotherium simum cottoni) en Ouganda aux fins de l’enregistrement scientifique. Ceratotherium simum a été distribué en Afrique entre le Mahgreb et la région du Cap, partout où il y avait un pâturage convenable (mais pas nécessairement partout en même temps), jusqu'à l'apparition de l'Homo sapiens sur le continent. Au dix-huitième siècle, cette vaste aire de répartition s'était réduite en deux populations largement séparées et taxonomiquement distinctes, l'une soudanienne et l'autre d’Afrique australe. La population soudanienne de rhinocéros blancs du Nord (RBN) a été reconnue scientifiquement en 1907 comme étant (Ceratotherium simum cottoni) distincte sur le plan taxinomique de C.s.simum. Auparavant, le RBN se trouvait en Ouganda, mais on pense actuellement qu'il est éteint dans le pays. Compte tenu de la faible densité humaine et de l'inaccessibilité d'une grande partie de la République Centrafricaine et du Sud-Soudan, il est possible que quelques individus non détectés existent encore dans des lieux isolés. Par exemple, même s’ils sont éteints dans le Parc National de Garamba en République Démocratique du Congo, quelques-uns peuvent rester dans le «domaine de chasse» contigu.

Online sentiment towards iconic species

Studies assessing online public sentiment towards biodiversity conservation are almost non-existent. The use of social media data and other online data sources is increasing in conservation science. We collected social media and online news data pertaining to rhinoceros, which are iconic species especially threatened by illegal wildlife trade, and assessed online sentiment towards these species using natural language processing methods. We also used an outlier detection technique to identify the most prominent conservation-related events imprinted into this data. We found that tragic events, such as the death of the last male northern white rhinoceros, Sudan, in March 2018, triggered the strongest reactions, which appeared to be concentrated in western countries, outside rhinoceros range states. We also found a strong temporal cross-correlation between social media data volume and online news volume in relation to tragic events, while other events only appeared in either social media or online news. Our results highlight that the public is concerned about biodiversity loss and this, in turn, can be used to increase pressure on decision makers to develop adequate conservation actions that can help reverse the biodiversity crisis. The proposed methods and analyses can be used to infer sentiment towards any biodiversity topic from digital media data, and to detect which events are perceived most important to the public.

25 Cryopreservation of horse testicular tissue as a model for rhinoceros

Cryopreservation of testicular tissue (TT) allows retention of valuable genetic material that can be used for conservation of endangered species, such as the northern white rhinoceros (NWR; Ceratotherium simum cottoni). Previously, we found that cryopreservation of NWR TT with a slow controlled cooling rate (CR) method induced morphological alterations in the seminiferous tubules (ST). However, the relative influence of CR, type of medium, and condition of TT from the aged NWR male on TT integrity was not clear. Due to the limited availability of rhinoceros TT, we used the horse as a model for optimization of TT cryopreservation. We evaluated the effect of (1) cryoprotectant solution [PBS (PBS +1.5M dimethyl sulfoxide) v. DMEM (DMEM/F12+10.0% fetal bovine serum+0.05M sucrose+1.5M dimethyl sulfoxide)] and (2) CR [CR1 (−2.0°C min−1 from 0°C to −4.0°C, −15°C min−1 to −12°C, and −0.3°C min−1 to −40°C in a programmable freezer) v. CR2 (same as CR1 but cooled to −8°C and held for 5min before cooling to −40°C) v. CR3 (−1.0°C min−1 from 0°C to −80°C in a CoolCell® freezing device; Corning, Corning, NY, USA)] on the structural integrity of ST from a 2-year-old horse (n=20 ST), cell viability, and expression of spermatogonial stem cells (SSC; GFRα1, and GRP125) and pluripotent markers (SSEA-4, SSEA-1, and OCT-4) in spermatogonial cells isolated from TT frozen with the above treatments (n=3). We found a positive interaction between CR and cryoprotectant solution on structural integrity of fixed and stained TT after freezing in PBS and CR2 that resulted in lower detachment of epithelium cells from the basement membrane (score±standard error of the mean; 0.50±0.1) than that of TT frozen in PBS and CR1 and CR3 (1.00±0.1 and 1.80±0.1, respectively; P<0.001) or in DMEM and CR1 (1.25±0.1), CR2 (1.35±0.1), and CR3 (1.40±0.1; P<0.01) and in lower incidence of basement membrane damage (0.75±0.1) than that of TT frozen in PBS and CR1 (1.17±0.07) and CR3 (1.16±0.07) or in DMEM and CR1 (1.10±0.1), CR2 (1.15±0.1), or CR3 (1.45±0.1; P<0.01). A lower rate of pyknosis was observed in TT frozen with PBS (1.15±0.06) than in TT frozen in DMEM (1.43±0.06; P<0.001). Overall, integrity of ST was improved when TT was frozen in PBS at CR2 having similar percentages of ST with intact epithelium (60%) and basement membrane (35%) as that of refrigerated TT (45 and 50%, respectively) but different from that of TT frozen with PBS at CR1 (10 and 15%, respectively; P<0.05). Flow cytometry analysis of spermatogonial cells revealed that the percentages of live cells from TT frozen in PBS (CR1: 61.5±7.4%; CR2: 59.7±4.8%; CR3: 51.5±4.1%) or DMEM (CR1: 66.2±6.0%; CR2: 59.8±6.0%; CR3: 58.9±6.9%), and expression of SSC and pluripotent markers was similar among all freezing treatments. However, the percentages of live cells from frozen-thawed TT were lower than those of cells isolated from refrigerated TT (80.6±2.2%; P<0.001). Overall, our results showed that (1) structural integrity of horse ST was better maintained when TT was frozen in PBS at CR2 and (2) SSC can be isolated from frozen-thawed TT with a similar relative frequency to that of refrigerated TT.

189 Isolation and purification of rhinoceros and horse spermatogonial stem cells

The northern white rhinoceros (NWR; Ceratotherium simum cottoni) is critically endangered. Testicular tissue (TT) of an NWR that died at 46 years was cryopreserved and stored. Spermatogonial stem cells (SSC) have been isolated from TT and, following transplantation or in vitro culture, differentiated into mature spermatozoa. These SSC have been phenotypically characterised by the expression of markers specific for SSC, and in vitro culture methods optimized to isolate, purify, and enrich populations of SSC from mixed germ cells. The reproductive age may affect the percent of SSC in TT, affecting recovery from older animals. We previously identified in TT of NWR the surface markers GFRa1 and GPR125, both of which were expressed at various stages of spermatogenesis. However, characterisation, isolation, and purification of rhinoceros SSC have not been studied. Because of the limited availability of rhinoceros TT, we used the horse as a model for rhinoceros (both species are in the same order: Perissodactyla). In this study, we (1) identified and compared the expression of markers specific for SSC (GFRa1, GPR125, PLZF) and pluripotent markers (SSEA-1, SSEA-4, OCT-4) in rhinoceros and horse mixed germ cells; (2) evaluated whether rhinoceros and horse SSC could be purified and enriched by sequential culture with collagen (2 days) and laminin (3 weeks); and (3) determined whether age may affect the percentages of SSC by comparing the expression of SSC and pluripotent markers in horse mixed germ cells at different reproductive stages: pubertal (PU=1-1.5 years) v. post-pubertal (PP=2-3 years) v. adult (AD=>5 years). Mixed germ cells were isolated from testes of one male of each of 3 rhinoceros species: NWR, southern white rhinoceros (SWR; Ceratotherium simum simum, 49 years), and greater one-horned rhinoceros (GOHR; Rhinoceros unicornis, 5 years), and horses at different reproductive stages (PU=2, PP=2, AD=7). The SWR and GOHR testes were refrigerated overnight, whereas NWR mixed germ cells were isolated from frozen-thawed TT. Flow cytometry analysis showed expression of SSC and pluripotent markers but not for transcription factor PLZF. The marker expression was similarly distributed between the 3 species of rhinoceros, with a larger portion of cells positive for SSEA-4 (mean%±s.e.m.: 6.3±1.1%) and smaller proportion for GDNFa1 (0.4±0.2%), GRP125 (0.1±0.1%), OCT-4 (0.4±0.2%), and SSEA-1 (0.4±0.2%; P<0.05). Similarly, horse expressed the markers GDNFa1 (0.9±0.3%), GRP125 (0.9±0.4%), OCT-4 (1.7±0.5%), and SSEA-1 (0.12±0.1%), but the abundance of SSEA-4 (1.8±0.8%) was less than that of rhinoceros (P<0.05). In rhinoceros, sequential culture enhanced the numbers of cells expressing all markers compared with that before culture, whereas in horses, the increase was observed only for cells expressing GDNFa1, GPR125, and SSEA-1. Reproductive stage did not affect the percentages of horse cells expressing germ cell and pluripotent markers. Overall, these results showed that rhinoceros SSC can be isolated from TT and expressed the same SSC markers as horses, and that differential culture enriched a population of SSC.

Embryos and embryonic stem cells from the white rhinoceros

The northern white rhinoceros (NWR, Ceratotherium simum cottoni) is the most endangered mammal in the world with only two females surviving. Here we adapt existing assisted reproduction techniques (ART) to fertilize Southern White Rhinoceros (SWR) oocytes with NWR spermatozoa. We show that rhinoceros oocytes can be repeatedly recovered from live SWR females by transrectal ovum pick-up, matured, fertilized by intracytoplasmic sperm injection and developed to the blastocyst stage in vitro. Next, we generate hybrid rhinoceros embryos in vitro using gametes of NWR and SWR. We also establish embryonic stem cell lines from the SWR blastocysts. Blastocysts are cryopreserved for later embryo transfer. Our results indicate that ART could be a viable strategy to rescue genes from the iconic, almost extinct, northern white rhinoceros and may also have broader impact if applied with similar success to other endangered large mammalian species.

Hybrid white-rhino embryos created in last-ditch effort to stop extinction

DNA of northern white rhino — of which only two remain — mixed with that of close subspecies in a bid towards growing population using surrogates. DNA of northern white rhino — of which only two remain — mixed with that of close subspecies in a bid towards growing population using surrogates.

Evaluating recovery potential of the northern white rhinoceros from cryopreserved somatic cells.

The critically endangered northern white rhinoceros is believed to be extinct in the wild, with the recent death of the last male leaving only two remaining individuals in captivity. Its extinction would appear inevitable, but the development of advanced cell and reproductive technologies such as cloning by nuclear transfer and the artificial production of gametes via stem cells differentiation offer a second chance for its survival. In this work, we analyzed genome-wide levels of genetic diversity, inbreeding, population history, and demography of the white rhinoceros sequenced from cryopreserved somatic cells, with the goal of informing how genetically valuable individuals could be used in future efforts toward the genetic rescue of the northern white rhinoceros. We present the first sequenced genomes of the northern white rhinoceros, which show relatively high levels of heterozygosity and an average genetic divergence of 0.1% compared with the southern subspecies. The two white rhinoceros subspecies appear to be closely related, with low genetic admixture and a divergent time <80,000 yr ago. Inbreeding, as measured by runs of homozygosity, appears slightly higher in the southern than the northern white rhinoceros. This work demonstrates the value of the northern white rhinoceros cryopreserved genetic material as a potential gene pool for saving this subspecies from extinction.

Is The Northern White Rhino About To Become Extinct: Can The Species Be Saved?

Is The Northern White Rhino About To Become Extinct: Can The Species Be Saved?

Can IVF save the northern white rhino from extinction?

As the last male northern white rhino is euthanased, leaving just two females - his daughter and granddaughter - what is the future for this subspecies? Sophie Ingledew explains.

Observations on Southern White Rhinoceros Ceratotherium simum simum Translocated to Uganda

Translocation is increasingly common in conservation but remains controversial due, in part, to limited information. Here, in a brief study, we consider the translocation of the southern white rhinoceros Ceratotherium simum simum to a fenced 64-km2 reserve in Uganda (a replacement for the locally extinct native northern subspecies, Ceratotherium simum cottoni). We examined the condition, behavior, and habitat use of two female-calf groups. Rhinos were least active during the early afternoon. Both groups spent more time feeding in Microchloa kunthii grasslands than expected given the small extent of such areas but spent most time in the more abundant woodlands and Hyparrhenia grasslands. The small ranges of the two groups, 11.8 and 9.0 km2 (100 h of observations over a 1-month period in 2011), and their apparent good health suggest satisfactory feeding conditions. Reassuringly, we identify no concerns but also note that our judgments are constrained by limited information and guidance. There is scope for novel observations, for example, our observations suggest that mothers lead calves more frequently in habitats with reduced visibility versus more open habitats. Every translocation is an experiment, but lessons will be lost without greater emphasis on transparency and assessments.

187 Morphological Appearance and Expression of Spermatogonial Stem Cell Markers in White Rhinoceros Testicular Tissue

Spermatogonial stem cells (SSC) have been isolated from testicular tissue (TT) of several mammalian species and differentiated into mature spermatozoa following transplantation or in vitro culture. The northern white rhinoceros (NWR; Ceratotherium simum cottoni) is critically endangered. Thus, frozen NWR TT, cryopreserved and stored at the San Diego Zoo’s Frozen Zoo®, potentially contain SSC that could be a source of spermatozoa. The method used for cryopreserving TT may affect the integrity and number of SSC. Therefore, identifying alterations in the seminiferous tubules (ST) of frozen-ndash;thawed-NWR TT will provide insight into the condition of the SSC. Therefore, our aims were to (1) determine the effect of freezing rhinoceros TT on the structure of epithelium, and (2) identify SSC (GFRα1, GPR125) and pluripotent (SSEA-4 and Oct-4) markers. Testicular tissue of an adult NWR and a stillborn southern white rhinoceros (SWR) were frozen by equilibration of TT for 30 min at 4.0°C in PBS and 1.5 M dimethyl sulfoxide (DMSO), cooled at 2.0°C/min to −4.0°C, 0.3°C/min to −40°C, and plunged into liquid nitrogen. Tissues were thawed at 37°C in a water bath and DMSO removed in a 4-step dilution. Tissue was then fixed, dehydrated, and paraffin embedded. For morphological evaluations, frozen-ndash;thawed tissue was sectioned and stained with hematoxylin and eosin (H&amp;E). The TT from both rhinoceros collected immediately after death (fresh) and stained with H&amp;E were used as a control for cryopreservation. Localization of SSC and pluripotent markers in ST of frozen-ndash;thawed TT was detected by immunohistochemistry. Morphologically, fresh-NWR TT was severely altered, displaying large epithelium gaps and partial (62.2%) or total detachment (37.7%) from, and slight damage (35.5%) to, the basement membrane. The number of pyknotic nuclei per ST was moderate (15.6 ± 7.2%). Many of these changes could have resulted from autolysis and handling before tissue preparation. In contrast, histological appearance of fresh-SWR was good, with 98.3% of the tubules intact, and a small proportion of pyknotic cells (0.8 ± 1.5%). Seminferous tubule (n = 30/male) length and width (μm; ± SEM) differed between NWR (635.2 ± 34.4 × 214.6 ± 10.8) and SWR (277.7 ± 13.8 × 73.2 ± 2.4; P &lt; 0.05). Damages after cryopreservation compared with fresh tissue comprised (1) epithelium detachment, NWR = 100% (P &lt; 0.0001), and SWR = 43.3% (P &lt; 0.001); (2) basement membrane alteration, only in NWR (93.0%; P &lt; 0.001); and (3) decreased length and width in the ST, NWR = 409.4 ± 18.1 × 173.4 ± 8.2 (P &lt; 0.05), and SWR = 195.2 ± 8.3 × 61.6 ± 2.8 (P &lt; 0.05), with loss of lumen in both males. Immunohistochemistry revealed that NWR expressed GFRα1 and GPR125 at various stages of spermatogenqaesis, whereas Oct-4 was detected in few cells. In contrast to NWR, Oct-4 expression in SWR was located at the basement membrane; SSEA-4 was not detected in either male. In conclusion, freezing-induced morphological alterations in rhinoceros ST and positive expression of markers for SSC and pluripotency suggest the presence of SSC. Further studies are required to evaluate the viability of rhinoceros SSC.

Species definitions and conservation: a review and case studies from African mammals

The nature of species, especially as applied to large mammals, is of major concern in conservation. Here, we briefly comment on recent thinking in alpha taxonomy, and assert that species are in essence evolutionary lineages, and that the most effective way of recognising them is by their diagnosability, i.e. the so-called Phylogenetic Species Concept. We further assert that the amount of genetic distance is not a relevant datum for distinguishing species, and that the ability to interbreed is not relevant. We consider a few case studies, especially that of the Northern White Rhinoceros Ceratotherium cottoni, and also species in Loxodonta, Giraffa and Oreotragus.

Comparison of whole mitochondrial genome sequences of northern and southern white rhinoceroses (Ceratotherium simum): the conservation consequences of species definitions

The probable extinction of the last confirmed population of northern white rhinoceros (Ceratotherium simum cottoni) in the world has ignited debate regarding its species status compared to the southern white rhinoceros (Ceratotherium simum simum). Previous studies, based on partial mitochondrial sequences, have reported conflicting results regarding the species status of the northern white rhinoceros. We use whole mitochondrial genomes obtained using Next Generation Sequencing of four northern white rhinoceros and three southern white rhinoceros using novel primers in three overlapping fragments. Phylogenetic relationships were constructed, using Maximum Likelihood, and recovered monophyletic clades for northern white rhinoceros and southern white rhinoceros. The divergence time between the two mitochondrial DNA lineages was estimated to be between 0.46 and 0.97 million years ago using Bayesian inferences. Since there are currently only three surviving northern white rhinoceros individuals these results put into sharp focus the ongoing debate regarding the methods of species definition, and in particular the consequences of such definitions in conservation management of endangered species and subspecies. We conclude that the designation of sub-species status is more applicable to northern and southern white rhinoceros.

Can modern technology prevent extinction?

Conservation Biology![Figure][1] The northern white rhinoceros is a critically endangered species. PHOTO: © DANITA DELIMONT CREATIVE/ALAMY STOCK PHOTO Rising temperatures, habitat loss, and increasing urbanization mean that many animal species are on the brink of extinction. However, Saragusty et al. suggest that advances in stem cell and assisted reproductive technologies might allow scientists to save severely endangered species and to bring extinct species back to life. Examining the case of the northern white rhinoceros, an instance in which only three nonreproducing individuals are known to exist, the authors outline the potential and caveats of applying current methodological advances to save this iconic species. Furthermore, the guidelines laid out for the northern white rhino are likely to be applicable to many other critically endangered species. Zoo Biol. 10.1002/zoo.21284 (2016). [1]: pending:yes

Rewinding the process of mammalian extinction.

With only three living individuals left on this planet, the northern white rhinoceros (Ceratotherium simum cottoni) could be considered doomed for extinction. It might still be possible, however, to rescue the (sub)species by combining novel stem cell and assisted reproductive technologies. To discuss the various practical options available to us, we convened a multidisciplinary meeting under the name "Conservation by Cellular Technologies." The outcome of this meeting and the proposed road map that, if successfully implemented, would ultimately lead to a self-sustaining population of an extremely endangered species are outlined here. The ideas discussed here, while centered on the northern white rhinoceros, are equally applicable, after proper adjustments, to other mammals on the brink of extinction. Through implementation of these ideas we hope to establish the foundation for reversal of some of the effects of what has been termed the sixth mass extinction event in the history of Earth, and the first anthropogenic one. Zoo Biol. 35:280-292, 2016. © 2016 The Authors. Zoo Biology published by Wiley Periodicals, Inc.

Introduction: Wildlife Crime

The poaching and the subsequent illegal trafficking of wildlife has become a booming local and global business (Alacs and Georges 2008) and is directly related to significant population losses for many species (Schneider 2012). In the last decade, countless media stories have been shared about the rise in poaching incidents particularly as they relate to the more photogenic animals, such as elephants, rhinos, and tigers. Many of these animals are critically endangered or have already gone extinct in the wild. For example, both the Western Black Rhino (Save the Rhino International 2015) and the Northern White Rhino are believed to be extinct in the wild (WWF 2015) as a direct consequence of rhino poaching for their horns. The poaching and illegal trafficking of animals and/or their by-products is not limited to only photogenic species. Pangolins, an insect-eating mammal that is similar to an armadillo, are not particularly attractive (Gill 2012) and do not receive much media attention, but are among the most poached mammals in the world for their meat and scales (Sutter 2014). The illegal wildlife trade is driven by a number of factors including the pet trade, accessories or luxury items, bush meat, and alternative medicines. As a result of the increased attention to ‘wildlife crime’, researchers from a variety of disciplines have begun to give this problem more attention. One of those disciplines is criminology. In fact, many criminology-related publications on the topic have been published within the last couple of years. For instance, a quick search of Criminal Justice Abstracts using Bwildlife conservation^ produced 236 articles or books published from 1937 to 2005, and 247 articles and books in the ten years spanning 2006 to 2015. Clearly, criminologists have become more interested in the sub-discipline of wildlife crime, also known as ‘conservation criminology’, and have made great progress in explaining why it happens, how the trade operates and offer solutions to reduce its frequency. The purpose of this special issue on BWildlife Crime^ is to create awareness of this global issue, increase dialogue with conservationists, and promote partnership research with park rangers and others on the ground. This special issue on wildlife crime covers a broad range of topics and includes conceptual and empirical papers. Eur J Crim Policy Res (2015) 21:299–302 DOI 10.1007/s10610-015-9290-x

Conservation genomics

A female northern white rhinoceros, Ceratotherium simum cottoni, one of only seven remaining individuals of a form of African rhinoceros on the verge of extinction. Image courtesy of San Diego Zoo Global.

Social and reproductive behaviour of critically endangered northern white rhinoceros in a zoological garden

Northern and southern white rhinos have poor reproduction in captivity and social interactions between them, especially increased agonistic behaviour, are believed to be one of the possible reasons. Northern white rhino is currently on the brink of extinction with less than ten animals surviving. We studied the social behaviour of northern white rhinos in zoological garden and investigated the effects of separation of the oldest, wild-born female from the herd on the social behaviour of other group members. After the separation, the numbers of agonistic and the play interactions between the animals significantly increased, no change was found in cohesive behaviour. Our results suggest that a composition of white rhino groups has a significant influence on social interactions between the animals and that better knowledge of proper composition of their groups in captivity in terms of age, sex and wild or zoo origin might improve animals’ well-being and also increase a chance for reproduction.

Induced pluripotent stem cells from highly endangered species

For some highly endangered species there are too few reproductively capable animals to maintain adequate genetic diversity, and extraordinary measures are necessary to prevent extinction. We report generation of induced pluripotent stem cells (iPSCs) from two endangered species: a primate, the drill, Mandrillus leucophaeus and the nearly extinct northern white rhinoceros, Ceratotherium simum cottoni. iPSCs may eventually facilitate reintroduction of genetic material into breeding populations.

AfRSG northern white rhino strategy—an alternative view

AfRSG northern white rhino strategy—an alternative view