Translational research for developing assisted reproduction technologies for endangered mammals (EUROVA – European Oocyte Biology Research Innovation Training Net)

The EUROVA consortium aims at developing new techniques and methods for in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF) and in vitro culture (IVC) of embryos. These may be used to advance new conservation tools for highly endangered mammals, such as the endangered rhinoceros family.

The EUROVA consortium is developing novel biotechnological approaches for wildlife conservation, with a particular focus on in vitro maturation of oocytes (IVM), in vitro fertilization (IVF), and in vitro culture of embryos (IVC). The overarching goal is to establish transferable methodologies that support the conservation of highly endangered mammalian species.

The research is based on the regular collection of rhinoceros oocytes from European zoos using an oocyte retrieval device developed and patented at Leibniz-IZW. These oocytes are analyzed with respect to their genetic, structural, and functional characteristics to improve our understanding of species-specific reproductive requirements.

Given the high value of each individual oocyte, non-invasive time-lapse imaging technologies are employed to continuously monitor oocyte maturation and embryo development while providing data on relevant biomarkers. Selected molecular analyses complement these investigations. The resulting knowledge is used to adapt and further develop established in vitro protocols from human, bovine, and mouse reproductive biology for application in endangered wildlife species.

Goals, innovation, impact, applicative outcome(s)

The long-term objective is the optimization of an artificial developmental environment that mimics maternal conditions (ivDOME) in order to support oocyte maturation and early embryonic development, ultimately leading to the production of reproductively healthy offspring.

The project will provide a scientific foundation for future conservation strategies based on advanced reproductive technologies. Although conservation applications are subject to regulatory frameworks that limit direct commercialization, the technologies and devices developed within the project have considerable innovation potential for related biomedical and biotechnological applications. Protectable innovations will be secured through patenting.

• Hildebrandt TB*, Holtze S*, Biasetti B, Bohner J, Cizmar D, Colleoni S, de Mori B, Diecke S, Galli C, Hayashi K, Kangethe F, Korody ML, Lekolool I, Ndereeh D, Kariuki L, Lazzari G, Mijele D, Mutisya S, Ngulu S, Omondi P, Seet S, Zywitza V, Stejskal J, Goeritz F. (2026, accepted for publication): De-extinction of the Northern White Rhinoceros (Ceratotherium simum cottoni). Journal of Reproduction and Development.
• Simone R, Čižmár D, Holtze S, Mulot B, Lamglait B, Knauf‐Witzens T, Weigold A, Hermes R, Hildebrandt TB (2024): Cryopreservation of okapi (Okapia johnstoni) oocytes following in vitro maturation. THERIOGENOLOGY WILD 4, 100088. doi:10.1016/j.therwi.2024.100088
• Subiran Adrados C, Ørnes Olesen H, Vernimmen Olesen S, Pors SE, Holtze S, Hildebrandt TB, Andersen CY, Kristensen SG (2024): Exploring the effect of PRP on vascularization and survival of follicles in human ovarian tissue transplanted to immunodeficient mice. Reproductive BioMedicine Online, S1472-6483(24)00463-2, https://doi.org/10.1016/j.rbmo.2024.104274.
• Hildebrandt TB and Holtze S (2024): Advanced assisted reproduction technologies in endangered mammalian species. Reprod Dom Anim. 2024; 59(Suppl. 3):e14700. https://doi.org/10.1111/rda.14700
• Appeltant R, Hermes R, Holtze S, Hildebrandt TB, Williams SA (2024): Follicle-like and other novel structures found in ovaries of aged white rhinoceroses and their potential impact on oocyte recovery rate. Theriogenology Wild online 4 June 2024, 100096, https://doi.org/10.1016/j.therwi.2024.100096.
  
• Hildebrandt TB*, Holtze S*, Colleoni S*, Hermes R, Stejskal J, Lekolool I, Ndeereh D, Omondi P, Kariuki L, Mijele D, Mutisya S, Ngulu S, Diecke S, Hayashi K, Lazzari G, de Mori B, Biasetti P, Quaggio A, Galli C, Goeritz F. (2023). In vitro fertilization program in white rhinoceros. Reproduction.166:383-399. doi:10.1530/REP-23-0087.
• Simone R, Čižmár D, Holtze S, Michel G, Sporbert A, Okolo C, Hildebrandt TB. (2023). In vitro production of naked mole-rats’ blastocysts from non-breeding females using in vitro maturation and intracytoplasmic sperm injection. Sci Rep 13, 22355 (2023). https://doi.org/10.1038/s41598-023-49661-6.
• Hildebrandt TB, Hermes R, Goeritz F, Appeltant R, Colleoni S, de Mori B, Diecke S, Drukker M, Galli C, Hayashi K, Lazzari G, Loi P, Payne J, Renfree M, Seet S, Stejskal J, Swegen A, Williams SA, Zainuddin ZZ, Holtze S. (2021). The ART of bringing extinction to a freeze - History and future of species conservation, exemplified by rhinos. Theriogenology. 169:76-88. doi: 10.1016/j.theriogenology.2021.04.006. Epub 2021 Apr 18. PMID: 33940218.
• Hayashi K, Cesare G, Diecke S, Hildebrandt TB (2021): Artificially produced gametes in mice, humans and other species. REPROD FERTIL DEV 33, 91–101. doi:10.1071/RD20265
• Hildebrandt TB*, Holtze S*, Biasetti P, Colleoni S, de Mori B, Diecke S, Göritz F, Hayashi K, Hayashi M, Hermes R, Kariuki L, Lazzari G, Mijele D, Mutisya S, Ndeereh D, Ngulu S, Seet S, Zwilling J, Zywitza V, Stejskal J, Galli C (2021): Conservation Research in Times of Covid-19 – the Rescue of the Northern White Rhino. J APPL ANIM ETHICS RES 1, 1-22. doi:10.1163/25889567-BJA10009
• Hildebrandt TB, Hermes R, Colleoni S, Diecke S, Holtze S, Renfree MB, Stejskal J, Hayashi K, Drukker M, Loi P, Göritz F, Lazzari G, Galli C (2018): Embryos and embryonic stem cells from the white rhinoceros. NAT COMMUN 9, 2589. doi:10.1038/s41467-018-04959-2.
• Saragusty J, Diecke S, Drukker M, Durrant B, Ben-Nun I, Galli C, Göritz F, Hayashi K, Hermes R, Holtze S, Johnson S, Lazzari G, Loi P, Loring JF, Okita K, Renfree MB, Seet S, Voracek T, Stejskal J, Ryder OA, Hildebrandt TB (2016): Rewinding the process of mammalian extinction. ZOO BIOL 35, 280-292. doi:10.1002/zoo.21284.