Northern White Rhinoceros Research Articles

Mapping the follicle-specific regulation of extracellular vesicle-mediated microRNA transport in the southern white rhinoceros (Ceratotherium simum simum)†.

Efforts to implement effective assisted reproductive technologies (ARTs) for the conservation of the northern white rhinoceros (NWR; Ceratotherium simum cottoni) to prevent its forthcoming extinction, could be supported by research conducted on the closely related southern white rhinoceros (SWR; Ceratotherium simum simum). Within the follicle, extracellular vesicles (EVs) play a fundamental role in the bidirectional communication facilitating the crucial transport of regulatory molecules such as microRNAs (miRNAs) that control follicular growth and oocyte development. This study aimed to elucidate the dynamics of EV-miRNAs in stage-dependent follicular fluid (FF) during SWR ovarian antral follicle development. Three distinct follicular stages were identified based on diameter: Growing (G; 11-17mm), Dominant (D; 18-29mm), and Pre-ovulatory (P; 30-34mm). Isolated EVs from the aspirated FF of segmented follicle stages were used to identify EV-miRNAs previously known via subsequent annotation to all equine (Equus caballus; eca), bovine (Bos taurus; bta), and human (Homo sapiens; hsa) miRNAs. A total of 417 miRNAs were detected, with 231 being mutually expressed across all three stages, including eca-miR-148a and bta-miR-451 as the top highly expressed miRNAs. Distinct expression dynamics in miRNA abundance were observed across the three follicular stages, including 31 differentially expressed miRNAs that target various pathways related to follicular growth and development, with 13 miRNAs commonly appearing amidst two different comparisons. In conclusion, this pioneering study provides a comprehensive understanding of the stage-specific expression dynamics of FF EV-miRNAs in the SWR. These findings provide insights that may lead to novel approaches in enhancing ARTs to catalyze rhinoceros conservation efforts.

Genetic load and viability of a future restored northern white rhino population.

As biodiversity loss outpaces recovery, conservationists are increasingly turning to novel tools for preventing extinction, including cloning and invitro gametogenesis of biobanked cells. However, restoration of populations can be hindered by low genetic diversity and deleterious genetic load. The persistence of the northern white rhino (Ceratotherium simum cottoni) now depends on the cryopreserved cells of 12 individuals. These banked genomes have higher genetic diversity than southern white rhinos (C. s. simum), a sister subspecies that successfully recovered from a severe bottleneck, but the potential impact of genetic load is unknown. We estimated how demographic history has shaped genome-wide genetic load in nine northern and 13 southern white rhinos. The bottleneck left southern white rhinos with more fixed and homozygous deleterious alleles and longer runs of homozygosity, whereas northern white rhinos retained more deleterious alleles masked in heterozygosity. To gauge the impact of genetic load on the fitness of a northern white rhino population restored from biobanked cells, we simulated recovery using fitness of southern white rhinos as a benchmark for a viable population. Unlike traditional restoration, cell-derived founders can be reintroduced in subsequent generations to boost lost genetic diversity and relieve inbreeding. In simulations with repeated reintroduction of founders into a restored population, the fitness cost of genetic load remained lower than that borne by southern white rhinos. Without reintroductions, rapid growth of the restored population (>20-30% per generation) would be needed to maintain comparable fitness. Our results suggest that inbreeding depression from genetic load is not necessarily a barrier to recovery of the northern white rhino and demonstrate how restoration from biobanked cells relieves some constraints of conventional restoration from a limited founder pool. Established conservation methods that protect healthy populations will remain paramount, but emerging technologies hold promise to bolster these tools to combat the extinction crisis.

The impact of induced pluripotent stem cells in animal conservation.

It is widely acknowledged that we are currently facing a critical tipping point with regards to global extinction, with human activities driving us perilously close to the brink of a devastating sixth mass extinction. As a promising option for safeguarding endangered species, induced pluripotent stem cells (iPSCs) hold great potential to aid in the preservation of threatened animal populations. For endangered species, such as the northern white rhinoceros (Ceratotherium simum cottoni), supply of embryos is often limited. After the death of the last male in 2019, only two females remained in the world. IPSC technology offers novel approaches and techniques for obtaining pluripotent stem cells (PSCs) from rare and endangered animal species. Successful generation of iPSCs circumvents several bottlenecks that impede the development of PSCs, including the challenges associated with establishing embryonic stem cells, limited embryo sources and immune rejection following embryo transfer. To provide more opportunities and room for growth in our work on animal welfare, in this paper we will focus on the progress made with iPSC lines derived from endangered and extinct species, exploring their potential applications and limitations in animal welfare research.

The neonatal southern white rhinoceros ovary contains oogonia in germ cell nests

The northern white rhinoceros is functionally extinct with only two females left. Establishing methods to culture ovarian tissues, follicles, and oocytes to generate eggs will support conservation efforts using in vitro embryo production. To the best of our knowledge, this is the first description of the structure and molecular signature of any rhinoceros, more specifically, we describe the neonatal and adult southern white rhinoceros (Ceratotherium simum simum) ovary; the closest relation of the northern white rhinoceros. Interestingly, all ovaries contain follicles despite advanced age. Analysis of the neonate reveals a population of cells molecularly characterised as mitotically active, pluripotent with germ cell properties. These results indicate that unusually, the neonatal ovary still contains oogonia in germ cell nests at birth, providing an opportunity for fertility preservation. Therefore, utilising ovaries from stillborn and adult rhinoceros can provide cells for advanced assisted reproductive technologies and investigating the neonatal ovaries of other endangered species is crucial for conservation.

On the fundamental incompatibility between wildlife conservation and animal ethics

Wildlife conservation aims to protect the natural world, plant and animal species, and the habitats they form part of and rely on for survival. More particularly, it focuses on species that are considered important, be it from economic, ecological and other perspectives, and preventing harm to these species. While conservation activities, based on common conservation values such as species fitness and biodiversity, are no doubt beneficial to animals in general, there seems to be a fundamental disjoint between this approach and the mainstay of current ethical theories regarding animals, which focus on the treatment and welfare of individual entities. I will argue that the conflict between wildlife conservation and animal ethics arises both in the aims, as well as the application of conservation. In its aims, the focus on species as a whole often comes at a cost to individual animals, and in its application individual animals are often harmed as well. To demonstrate these two points, I will use the case study of the cloning and reintroduction of the currently extinct northern white rhino. I will then conclude that wildlife conservation, while aiming to benefit animals, is fundamentally at odds with any animal ethic that considers individual wellbeing as central.

In vitro fertilization program in white rhinoceros.

To save endangered rhinoceros species, assisted reproductive technologies are warranted. We here report in vitro blastocyst generation of the Near-Threatened Southern white rhinoceros and, for the first time, also of the technically Extinct Northern white rhinoceros. The Anthropocene is marked by a dramatic biodiversity decline, particularly affecting the family Rhinocerotidae. Three of five extant species are listed as Critically Endangered (Sumatran, Javan, black rhinoceros), one as Vulnerable (Indian rhinoceros), and only one white rhino (WR) subspecies, the Southern white rhinoceros (SWR), after more than a century of successful protection is currently classified as Near Threatened by the IUCN, while numbers again are declining. Conversely, in 2008, the SWR's northern counterpart and second WR subspecies, the Northern white rhinoceros (NWR), was declared extinct in the wild. Safeguarding these vanishing keystone species urgently requires new reproductive strategies. We here assess one such strategy, the novel in vitro fertilization program in SWR and - for the first-time NWR - regarding health effects, donor-related, and procedural factors. Over the past 8 years, we performed 65 procedures in 22 white rhinoceros females (20 SWR and 2 NWR) comprising hormonal ovarian stimulation, ovum pick-up (OPU), in vitro oocyte maturation, fertilization, embryo culture, and blastocyst cryopreservation, at an efficiency of 1.0 ± 1.3 blastocysts per OPU, generating 22 NWR, 19 SWR and 10 SWR/NWR hybrid blastocysts for the future generation of live offspring.

Robust induction of primordial germ cells of white rhinoceros on the brink of extinction

In vitro gametogenesis, the process of generating gametes from pluripotent cells in culture, is a powerful tool for improving our understanding of germ cell development and an alternative source of gametes. Here, we induced primordial germ cell-like cells (PGCLCs) from pluripotent stem cells of the northern white rhinoceros (NWR), a species for which only two females remain, and southern white rhinoceros (SWR), the closest species to the NWR. PGCLC differentiation from SWR embryonic stem cells is highly reliant on bone morphogenetic protein and WNT signals. Genetic analysis revealed that SRY-box transcription factor 17 (SOX17) is essential for SWR-PGCLC induction. Under the defined condition, NWR induced pluripotent stem cells differentiated into PGCLCs. We also identified cell surface markers, CD9 and Integrin subunit alpha 6 (ITGA6), that enabled us to isolate PGCLCs without genetic alteration in pluripotent stem cells. This study provides a first step toward the production of NWR gametes in culture and understanding of the basic mechanism of primordial germ cell specification in a large animal.

Application of decision tools to ethical analysis in biodiversity conservation.

Achieving ethically responsible decisions is crucial for the success of biodiversity conservation projects. We adapted the ethical matrix, decision tree, and Bateson's cube to assist in the ethical analysis of complex conservation scenarios by structuring these tools so that they can implement the different value dimensions (environmental, social, and animal welfare) involved in conservation ethics. We then applied them to a case study relative to the decision-making process regarding whether or not to continue collecting biomaterial on the oldest of the two remaining northern white rhinoceroses (Ceratotherium simum cottoni), a functionally extinct subspecies of the white rhinoceros. We used the ethical matrix to gather ethical pros and cons and as a starting point for a participatory approach to ethical decision-making. We used decision trees to compare the different options at stake on the basis of a set of ethical desiderata. We used Bateson's cube to establish a threshold of ethical acceptability and model the results of a simple survey. The application of these tools proved to be pivotal in structuring the decision-making process and in helping reach a shared, reasoned, and transparent decision on the best option from an ethical point of view among those available.

Engineering a Sustainable Future (Vol. 26, No. 07n08, Full Issue)

For the months of July and August 2022, APBN considers how technology, for better or for worse, allows us to engineer a future where we can grow crops in the ocean, save endangered animals, and possibly even bring extinct animals back to life. Here, we have Alex Teo, Managing Director and Vice President of Siemens Digital Industries Software, Southeast Asia, on how we can look towards the ocean as a viable alternative to traditional farming; Dr Sebastian Diecke and Steven Seet on the use of stem cell-associated techniques to rescue the northern white rhino from extinction; and Vanessa Quek on considering what de-extinction really means for us and other animals. Other highlights in this issue include highlights from a webinar hosted by Professor Michael S. Strano, Joint Principal Investigator at Disruptive & Sustainable Technologies for Agricultural Precision (DiSTAP), on the recent advancements in AgriTech to help meet the increasing demand for food, and an interview with Dr Mee-ran Kim, Professor in Obstetrics and Gynaecology at Korea Seoul St. Mary's Hospital, Chair of the APAC Consensus, on the gynaecological condition, endometriosis.

Ethical Analysis of the Application of Assisted Reproduction Technologies in Biodiversity Conservation and the Case of White Rhinoceros (Ceratotherium simum) Ovum Pick-Up Procedures.

Originally applied on domestic and lab animals, assisted reproduction technologies (ARTs) have also found application in conservation breeding programs, where they can make the genetic management of populations more efficient, and increase the number of individuals per generation. However, their application in wildlife conservation opens up new ethical scenarios that have not yet been fully explored. This study presents a frame for the ethical analysis of the application of ART procedures in conservation based on the Ethical Matrix (EM), and discusses a specific case study—ovum pick-up (OPU) procedures performed in the current conservation efforts for the northern white rhinoceros (Ceratotherium simum cottoni)—providing a template for the assessment of ART procedures in projects involving other endangered species.

Na\xefve-like pluripotency to pave the way for saving the northern white rhinoceros from extinction

The northern white rhinoceros (NWR) is probably the earth’s most endangered mammal. To rescue the functionally extinct species, we aim to employ induced pluripotent stem cells (iPSCs) to generate gametes and subsequently embryos in vitro. To elucidate the regulation of pluripotency and differentiation of NWR PSCs, we generated iPSCs from a deceased NWR female using episomal reprogramming, and observed surprising similarities to human PSCs. NWR iPSCs exhibit a broad differentiation potency into the three germ layers and trophoblast, and acquire a naïve-like state of pluripotency, which is pivotal to differentiate PSCs into primordial germ cells (PGCs). Naïve culturing conditions induced a similar expression profile of pluripotency related genes in NWR iPSCs and human ESCs. Furthermore, naïve-like NWR iPSCs displayed increased expression of naïve and PGC marker genes, and a higher integration propensity into developing mouse embryos. As the conversion process was aided by ectopic BCL2 expression, and we observed integration of reprogramming factors, the NWR iPSCs presented here are unsuitable for gamete production. However, the gained insights into the developmental potential of both primed and naïve-like NWR iPSCs are fundamental for in future PGC-specification in order to rescue the species from extinction using cryopreserved somatic cells.

Individuality, species-specific features, and female discrimination of male southern white rhinoceros courtship calls.

Male vocalizations associated with courtship can play a key role in mate selection. They may help females obtain information about males' quality and identity and/or may contain species-specific properties that help prevent interspecies breeding. Despite vocalizations being a prominent part of the courtship of white rhinos, the role that they play in white rhino breeding behaviour has not been extensively studied. Both southern (SWR) and critically endangered northern white rhino (NWR) males intensively vocalize during courtship with hic calls. We examined these calls and found that call properties differed between NWR and SWR males. In addition, we found that individual SWR males could be identified with a high degree of accuracy using their hic calls and that the signature information capacity in hic calls would allow females to individually recognize about 11 adult males living in or moving through their home-ranges, which may help with mate selection. Then, we conducted playback experiments with wild anoestrus SWR females. The females discriminated between the NWR and SWR hic calls and between the SWR hic and SWR pant calls. However, we only found differences in the latency of observed behaviours, not in their duration or in the intensity of females' reaction. This might suggest that females which are not in oestrus are not highly responsive to a male's motivation (i.e., seeking contact or mating), but are more interested in assessing his dominance status or familiarity. Ultimately, our results indicate that courtship hic calls encode information which might help females choose mating partners.

Why stem cells might save the northern white rhino

Why stem cells might save the northern white rhino

On the Horns of a Dilemma: Let the Northern White Rhino Vanish or Intervene?

ABSTRACT Two females, Nadine and Fatu, are the sole surviving Northern White Rhinos (NWR). The subspecies is functionally extinct. Hope for NWR now lies in emerging reproductive and genetic technologies, which could potentially produce NWR from induced pluripotent stem cells. What is the rationale for this project? This question raises almost every philosophical issue facing conservation science today. I argue that NWR recovery is hard to justify via many traditional paths (e.g., historical fidelity, ecosystem health, biodiversity), but if we shift focus to white rhinos in general or even mammals then clear benefits emerge.

An Ethical Assessment Tool (ETHAS) to Evaluate the Application of Assisted Reproductive Technologies in Mammals' Conservation: The Case of the Northern White Rhinoceros (Ceratotherium simum cottoni).

Simple SummaryApplying assisted reproductive technologies (ARTs) to the conservation of endangered species may be the only way to save them from extinction. However, ART application can raise relevant ethical issues and could benefit from a comprehensive ethical assessment. Unfortunately, there is a lack of attention to the topic in the scientific literature and, to our knowledge, there is no tool for the ethical assessment of ARTs in the context of conservation that has been described. In the present paper, we show the effects of applying a dedicated ethical self-assessment tool, the Ethical Assessment Tool (ETHAS), to ovum pick-up and in vitro fertilization procedures performed within the BioRescue project. The BioRescue project is an international enterprise using ARTs to save the northern white rhinoceros from extinction. The situation of the northern white rhinoceros is particularly critical as there are only two individuals of this subspecies still alive and they are both infertile females. The application of the ETHAS to the procedures contributed to the overall acceptability of the project and improved communication among the project’s partners. In turn, the tool itself was also refined through an iterative consultation process between experts (both ethicists and scientists) and stakeholders.Assisted reproductive technologies (ARTs) can make a difference in biodiversity conservation. Their application, however, can create risks and raise ethical issues that need addressing. Unfortunately, there is a lack of attention to the topic in the scientific literature and, to our knowledge, there is no tool for the ethical assessment of ARTs in the context of conservation that has been described. This paper reports the first applications of the Ethical Assessment Tool (ETHAS) to trans-rectal ovum pick-up (OPU) and in vitro fertilization (IVF) procedures used in a northern white rhinoceros (Ceratotherium simum cottoni) conservation project. The ETHAS consists of two checklists, the Ethical Evaluation Sheet and the Ethical Risk Assessment, and is specifically customized for each ART procedure. It provides an integrated, multilevel and standardized self-assessment of the procedure under scrutiny, generating an ethical acceptability ranking (totally, partially, not acceptable) and a risk rank (low, medium, high), and, hence, allows for implementing measures to address or manage issues beforehand. The application of the ETHAS to the procedures performed on the northern white rhinoceros was effective in ensuring a high standard of procedures, contributing to the acceptability and improved communication among the project’s partners. In turn, the tool itself was also refined through an iterative consultation process between experts and stakeholders.

Conservation Research in Times of COVID-19 – The Rescue of the Northern White Rhino

Abstract COVID-19 has changed the world at unprecedented pace. The measures imposed by governments across the globe for containing the pandemic have severely affected all facets of economy and society, including scientific progress. Сonservation research has not been exempt from these negative effects, which we here summarize for the BioRescue project, aiming at saving the northern white rhinoceros (Ceratotherium simum cottoni), an important Central African keystone species, of which only two female individuals are left. The development of advanced assisted reproduction and stem-cell technologies to achieve this goal involves experts across five continents. Maintaining international collaborations under conditions of national shut-down and travel restrictions poses major challenges. The associated ethical implications and consequences are particularly troublesome when it comes to research directed at protecting biological diversity – all the more in the light of increasing evidence that biodiversity and intact ecological habitats might limit the spread of novel pathogens.

Rewinding Extinction in the Northern White Rhinoceros: Genetically Diverse Induced Pluripotent Stem Cell Bank for Genetic Rescue.

Extinction rates are rising, and current conservation technologies may not be adequate for reducing species losses. Future conservation efforts may be aided by the generation of induced pluripotent stem cells (iPSCs) from highly endangered species. Generation of a set of iPSCs from multiple members of a species can capture some of the dwindling genetic diversity of a disappearing species. We generated iPSCs from fibroblasts cryopreserved in the Frozen Zoo®: nine genetically diverse individuals of the functionally extinct northern white rhinoceros (Ceratotherium simum cottoni) and two from the closely related southern white rhinoceros (Ceratotherium simum simum). We used a nonintegrating Sendai virus reprogramming method and developed analyses to confirm the cells' pluripotency and differentiation potential. This work is the first step of a long-term interdisciplinary plan to apply assisted reproduction techniques to the conservation of this highly endangered species. Advances in iPSC differentiation may enable generation of gametes in vitro from deceased and nonreproductive individuals that could be used to repopulate the species.

The Generation Of Blastocysts From Induced Pluripotent Stem Cells Taken From Northern White Rhino Skin Cells

The Generation Of Blastocysts From Induced Pluripotent Stem Cells Taken From Northern White Rhino Skin Cells

Exploring the limits of saving a subspecies: The ethics and social dynamics of restoring northern white rhinos (Ceratotherium simum cottoni)

AbstractThe northern white rhino (Ceratotherium simum cottoni) is functionally extinct with only two females left alive. However, cryopreserved material from a number of individuals represents the potential to produce additional individuals using advanced reproductive and genetic rescue technologies and perhaps eventually a population to return to their native range. If this could and were done, how should it be done responsibly and thoughtfully. What issues and questions of a technical, bioethical, and societal nature will it raise that need to be anticipated and addressed? Such issues are explored in this article by an interdisciplinary team assembled to provide context to the northern white rhino project of the San Diego Zoo Global.

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.