• 98 Efficacy of commercial equine semen freezing extenders for cryopreservation of southern white rhinoceros (Ceratotherium simum simum) sperm

      Young, Carly; Ravida, Nicole; Pennington, Parker M.; Durrant, Barbara S. (2019)
      Once nearly extinct in the wild, the southern white rhinoceros is currently listed as near threatened by IUCN. This status is likely to change as poaching continues to escalate....
    • Activation of southern white rhinoceros (Ceratotherium simum simum) estrogen receptors by phytoestrogens: Potential role in the reproductive failure of captive-born females?

      Tubbs, Christopher W.; Hartig, P.; Cardon, M.; Varga, Nicole; Milnes, Matthew R. (2012)
      The captive southern white rhinoceros (SWR; Ceratotherium simum simum) population serves as an important genetic reservoir critical to the conservation of this vulnerable species. Unfortunately, captive populations are declining due to the poor reproductive success of captive-born females....
    • Advances in conservation endocrinology: The application of molecular approaches to the conservation of endangered species

      Tubbs, Christopher W.; McDonough, C. E.; Felton, Rachel G.; Milnes, Matthew R. (2014)
      …In this review we examine various in vitro approaches we have used to compare estrogen receptor binding and activation by endogenous hormones and phytoestrogens in two species of rhinoceros; southern white and greater one-horned. We have found many of these techniques valuable and practical in species where access to research subjects and/or tissues is limited due to their conservation status....
    • Advancing laboratory-based zoo research to enhance captive breeding of southern white rhinoceros

      Tubbs, Christopher W.; Minteer, Ben A.; Maienschein, Jane; Collins, James P. (University of Chicago PressChicago, IL, 2018)
      The captive southern white rhinoceros (SWR) is not currently self-sustaining because of low fertility of captive-born females. The cause of this phenomenon is believed to be high dietary levels of phytoestrogens; endocrine disrupting chemicals (EDCs) produced by plants that can mimic the reproductive hormone estrogen....
    • Contrasting evolutionary history, anthropogenic declines and genetic contact in the northern and southern white rhinoceros (Ceratotherium simum)

      Moodley, Yoshan; Russo, Isa-Rita M.; Robovský, Jan; Dalton, Desiré L.; Kotzé, Antoinette; Smith, Steve; Stejskal, Jan; Ryder, Oliver A.; Hermes, Robert; Walzer, Chris; et al. (2018)
      The white rhinoceros (Ceratotherium simum) has a discontinuous African distribution, which is limited by the extent of sub-Saharan grasslands. The southern population (SWR) declined to its lowest number around the turn of the nineteenth century, but recovered to become the world's most numerous rhinoceros....
    • Epidemiology of tuberculosis in multi-host wildlife systems: Implications for black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros

      Dwyer, Rebecca A.; Witte, Carmel L.; Buss, Peter; Goosen, Wynand J.; Miller, Michele (2020)
      Cases of tuberculosis (TB) resulting from infection with Mycobacterium tuberculosis complex (MTBC) have been recorded in captive white (Ceratotherium simum) and black (Diceros bicornis) rhinoceros. More recently, cases have been documented in free-ranging populations of both species in bovine tuberculosis (bTB) endemic areas of South Africa. There is limited information on risk factors and transmission patterns for MTBC infections in African rhinoceros, however, extrapolation from literature on MTBC infections in other species and multi-host systems provides a foundation for understanding TB epidemiology in rhinoceros species. Current diagnostic tests include blood-based immunoassays but distinguishing between subclinical and active infections remains challenging due to the lack of diagnostic techniques. In other species, demographic risk factors for MTBC infection include sex and age, where males and adults are generally at higher risk than females and younger individuals. Limited available historical information reflects similar age- and sex-associated patterns for TB in captive black and white rhinoceros, with more reports of MTBC-associated disease in black rhinoceros than in white rhinoceros. The degree of MTBC exposure in susceptible wildlife depends on their level of interaction, either directly with other infected individuals or indirectly through MTBC contaminated environments, which is dependent on the presence and abundance of infected reservoir hosts and the amount of MTBC shed in their excreta. Captive African rhinoceros have shown evidence of MTBC shedding, and although infection levels are low in free-ranging rhinoceros, there is a risk for intraspecies transmission. Free-ranging rhinoceros in bTB endemic areas may be exposed to MTBC from other infected host species, such as the African buffalo (Syncerus caffer) and greater kudu (Tragelaphus strepsiceros), through shared environmental niches, and resource co-utilization. This review describes current knowledge and information gaps regarding the epidemiology of TB in African rhinoceros.
    • Estrogenicity of captive southern white rhinoceros diets and their association with fertility

      Tubbs, Christopher W.; Moley, Laura A.; Ivy, Jamie A.; Metrione, Lara C.; LaClaire, Sydney; Felton, Rachel G.; Durrant, Barbara S.; Milnes, Matthew R. (2016)
      …In this study, we investigate the role of dietary phytoestrogens in this reproductive phenomenon by characterizing activation of southern white rhinoceros (SWR) estrogen receptors (ESRs) 1 and 2 by diet items from nine North American institutions and comparing female SWR fertility to total diet estrogenicity. Of the diet items tested, alfalfa hay and soy and alfalfa-based commercial pellets were found to be the most potent activators of SWR ESRs.…
    • Evaluating recovery potential of the northern white rhinoceros from cryopreserved somatic cells

      Tunstall, Tate S.; Kock, Richard; Vahala, Jiri; Diekhans, Mark; Fiddes, Ian; Armstrong, Joel; Paten, Benedikt; Ryder, Oliver A.; Steiner, Cynthia C. (2018)
      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.
    • Exploring the limits of saving a subspecies: The ethics and social dynamics of restoring northern white rhinos (Ceratotherium simum cottoni)

      Ryder, Oliver A.; Friese, Carrie; Greely, Henry T.; Sandler, Ronald; Saragusty, Joseph; Durrant, Barbara S.; Redford, Kent H. (2020)
      The 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.
    • Gut microbiota and phytoestrogen-associated infertility in southern white rhinoceros

      Williams, Candace L.; Ybarra, Alexis R.; Meredith, Ashley N.; Durrant, Barbara S.; Tubbs, Christopher W. (2019)
      With recent poaching of southern white rhinoceros (SWR [Ceratotherium simum simum]) reaching record levels, the need for a robust assurance population is urgent. However, the global captive SWR population is not currently self-sustaining due to the reproductive failure of captive-born females. Dietary phytoestrogens have been proposed to play a role in this phenomenon, and recent work has demonstrated a negative relationship between diet estrogenicity and fertility of captive-born female SWR. To further examine this relationship, we compared gut microbial communities, fecal phytoestrogens, and fertility of SWR to those of another rhinoceros species—the greater one-horned rhinoceros (GOHR [Rhinoceros unicornis]), which consumes a similar diet but exhibits high levels of fertility in captivity. Using 16S rRNA amplicon sequencing and mass spectrometry, we identified a species-specific fecal microbiota and three dominant fecal phytoestrogen profiles. These profiles exhibited various levels of estrogenicity when tested in an in vitro estrogen receptor activation assay for both rhinoceros species, with profiles dominated by the microbial metabolite equol stimulating the highest levels of receptor activation. Finally, we found that SWR fertility varies significantly not only with respect to phytoestrogen profile, but also with respect to the abundance of several bacterial taxa and microbially derived phytoestrogen metabolites. Taken together, these data suggest that in addition to species differences in estrogen receptor sensitivity to phytoestrogens, reproductive outcomes may be driven by the gut microbiota’s transformation of dietary phytoestrogens in captive SWR females. IMPORTANCE Southern white rhinoceros (SWR) poaching has reached record levels, and captive infertility has rendered SWR assurance populations no longer self-sustaining. Previous work has identified dietary phytoestrogens as a likely cause of this problem. Here, we investigate the role of gut microbiota in this phenomenon by comparing two rhinoceros species to provide the first characterizations of gut microbiomes for any rhinoceros species. To our knowledge, our approach, combining parallel sequencing, mass spectrometry, and estrogen receptor activation assays, provides insight into the relationship between microbially mediated phytoestrogen metabolism and fertility that is novel for any vertebrate species. With this information, we plan to direct future work aimed at developing strategies to improve captive reproduction in the hope of alleviating their threat of extinction.
    • Induced pluripotent stem cells from highly endangered species

      Ben-Nun, Inbar Friedrich; Montague, Susanne C; Houck, Marlys L.; Tran, Ha T; Garitaonandia, Ibon; Leonardo, Trevor R; Wang, Yu-Chieh; Charter, Suellen J.; Laurent, Louise C; Ryder, Oliver A.; et al. (2011)
      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.
    • Molecular phylogeny and evolution of the Perissodactyla

      Steiner, Cynthia C.; Ryder, Oliver A. (2011)
      The evolution of perissodactyls (rhinoceroses, tapirs, and horses) has been well studied primarily because of their extensive fossil record. Nevertheless, controversy persists regarding relationships of some of the extant taxa, reflecting inconsistencies between molecular and morphological studies. Here we examine the phylogenetic relationships of 16 living perissodactyl species by concatenating two mitochondrial and nine nuclear genes, and we estimate their divergence times using a relaxed Bayesian molecular clock approach....
    • Ovulation induction in anovulatory southern white rhinoceros (Ceratotherium simum simum) without altrenogest

      Pennington, Parker M.; Marshall, Kira L.; Capiro, Jonnie M.; Felton, Rachel G.; Durrant, Barbara S. (2019)
      Lack of ovulation is common in captive southern white rhino females and contributes to poor reproductive success. We show that ovulation can be induced efficien
    • Pregnancies following long luteal phases in southern white rhinoceros (Ceratotherium simum simum)

      Pennington, Parker M.; Marshall, Kira L.; Capiro, Jonnie M.; Howard, Lauren L.; Durrant, Barbara S. (2020)
      All extant species in the Rhinocerotidae family are experiencing escalating threats in the wild, making self-sustaining captive populations essential genetic reservoirs for species survival. Assisted reproductive technologies (ARTs) will become increasingly important for achieving and maintaining ex situ population sustainability and genetic diversity. Previous reports have shown that a large proportion of captive southern white rhinoceros (SWR) females are irregularly cyclic or acyclic, and that cycling females display two different estrous cycle lengths of approximately 30 or 70 days. It has been suggested that the longer estrous cycle length is infertile or subfertile, as no term pregnancies have been observed following long cycles. Here we report the achievement of two pregnancies following long luteal phases, using ovulation induction and artificial insemination with either fresh or frozen-thawed semen. One female SWR conceived on the first insemination attempt and gave birth to a live offspring. A second female conceived twice in consecutive long cycles although the first embryo was resorbed by 33 days post-insemination. A pregnancy from this female's second insemination is ongoing with expected parturition in November 2019. Whether prolonged estrous cycles in SWR are subfertile or infertile in natural breeding situations remains unclear. However, our findings demonstrate that the application of ARTs following prolonged cycles can result the successful establishment of pregnancies in SWR. Therefore, with ARTs, female SWR otherwise considered nonreproductive due to long estrous cycles may still have the potential for representation and contribution to the ex situ population.
    • Reconsidering the use of soy and alfalfa in southern white rhinoceros diets

      Tubbs, Christopher W.; Durrant, Barbara S.; Milnes, Matthew R. (2017)
      The captive southern white rhinoceros (SWR) population is not currently self-sustaining due to the reproductive failure of captive-born females. Our research into this phenomenon points to chemicals produced by plants common to captive diets, such as soy and alfalfa, as possible causes...
    • Reproductive performance parameters in a large population of game-ranched white rhinoceroses (Ceratotherium simum simum)

      Ververs, Cyrillus; Langhout, Martine van Zijll; Hostens, Miel; Otto, Michelle; Govaere, Jan; Durrant, Barbara S.; Soom, Ann Van (2017)
      The population of free-roaming white rhinoceroses (Ceratotherium simum) is under serious threat. Captive breeding of this species is therefore becoming more important, but this is challenging and often not successful. Obtaining reproductive reference values is a crucial aspect of improving these breeding results. In this study performed between 2008 and 2016, reproductive performance was analysed in 1,354 animals kept in a 8000 hectares game-ranched environment. Descriptive statistics of this captive population showed an average annual herd growth (%) of 7 .0±0.1 (min -9 –max 15). Average calving rates were calculated as an annual calving rate of 20% and biennial calving rate of 37% adult females calving per year. Females had a median age of 83.2 months at first calving (IQR 72.9–110.7) and inter-calving intervals of 29.2 (IQR 24.6–34.8) months. Furthermore, translocations of animals did not interfere with reproductive success in terms of inter-calving periods or age at first calving. Multivariate models showed a clear seasonal calving pattern with a significant increase of the number of calvings during December–April when compared to April–December. Our results did not show any significant skewed progeny sex ratios. Weather observations showed no significant influence of rain or season on sex ratios of the calves.
    • Rewinding extinction in the northern white rhinoceros: Genetically diverse induced pluripotent stem cell bank for genetic rescue

      Korody, Marisa L.; Ford, Sarah M.; Nguyen, Thomas D.; Pivaroff, Cullen G.; Valiente-Alandi, Iñigo; Peterson, Suzanne E.; Ryder, Oliver A.; Loring, Jeanne F. (2021)
      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 non-integrating 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 non-reproductive individuals that could be used to repopulate the species.
    • Rewinding the process of mammalian extinction

      Saragusty, Joseph; Diecke, Sebastian; Drukker, Micha; Durrant, Barbara S.; Friedrich Ben-Nun, Inbar; Galli, Cesare; Göritz, Frank; Hayashi, Katsuhiko; Hermes, Robert; Holtze, Susanne; et al. (2016)
      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.
    • The Frozen Zoo

      Ryder, Oliver A. (2012)
      Wildlife gene banks provide a tool for studying species and monitoring conservation efforts
    • The plight of the northern white rhinoceros (Ceratotherium simum cottoni): is there still hope to prevent extinction?

      Ryder, Oliver A.; Hermes, R.; Goeritz, F.; Durrant, Barbara S.; Stejskal, J.; Hrudy, J.; Vahala, L.; Loring, Jeanne F.; Hildebrandt, T.B.; Szentiks, C.A.; et al. (Leibniz Institute for Zoo and Wildlife ResearchBerlin, 2015)