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.

Avantea Laboratory of Reproductive Technologies 26100 Cremona Italy

Avantea Laboratory of Reproductive Technologies 26100 Cremona Italy Fondazione Avantea 26100 Cremona Italy

Department of Stem Cell Biology and Medicine Graduate School of Medical Sciences Kyushu University Fukuoka 812 0054 Japan

Faculty of Veterinary Medicine University of Teramo Teramo Italy

Institute of Stem Cell Research and the Induced Pluripotent Stem Cell Core Facility Helmholtz Center Munich 85764 Neuherberg Germany

Leibniz Institute for Zoo and Wildlife Research D 10315 Berlin Germany

Leibniz Institute for Zoo and Wildlife Research D 10315 Berlin Germany Freie Universität Berlin D 14195 Berlin Germany

Max Delbrück Center Berlin Germany Berlin Institute of Health Berlin Germany

School of BioSciences The University of Melbourne Melbourne VIC 3010 Australia

ZOO Dvůr Králové Štefánikova 1029 544 01 Dvůr Králové nad Labem Czech Republic

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$a Hildebrandt, Thomas B $u Leibniz Institute for Zoo and Wildlife Research, D-10315, Berlin, Germany. HILDEBRAND@izw-berlin.de. Freie Universität Berlin, D-14195, Berlin, Germany. HILDEBRAND@izw-berlin.de.

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$a 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.

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$a Hermes, Robert $u Leibniz Institute for Zoo and Wildlife Research, D-10315, Berlin, Germany.

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$a Colleoni, Silvia $u Avantea, Laboratory of Reproductive Technologies, 26100, Cremona, Italy.

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$a Diecke, Sebastian $u Max Delbrück Center, Berlin, Germany. Berlin Institute of Health, Berlin, Germany.

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$a Holtze, Susanne $u Leibniz Institute for Zoo and Wildlife Research, D-10315, Berlin, Germany.

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$a Renfree, Marilyn B $u School of BioSciences, The University of Melbourne, Melbourne, VIC, 3010, Australia.

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$a Stejskal, Jan $u ZOO Dvůr Králové, Štefánikova 1029, 544 01, Dvůr Králové nad Labem, Czech Republic.

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$a Hayashi, Katsuhiko $u Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, 812-0054, Japan.

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$a Drukker, Micha $u Institute of Stem Cell Research and the Induced Pluripotent Stem Cell Core Facility, Helmholtz Center Munich, 85764, Neuherberg, Germany.

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$a Loi, Pasqualino $u Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy.

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$a Göritz, Frank $u Leibniz Institute for Zoo and Wildlife Research, D-10315, Berlin, Germany.

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$a Lazzari, Giovanna $u Avantea, Laboratory of Reproductive Technologies, 26100, Cremona, Italy. Fondazione Avantea, 26100, Cremona, Italy.

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$a Galli, Cesare $u Avantea, Laboratory of Reproductive Technologies, 26100, Cremona, Italy. cesaregalli@avantea.it. Fondazione Avantea, 26100, Cremona, Italy. cesaregalli@avantea.it.

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