The ART of bringing extinction to a freeze – History and future of species conservation, exemplified by rhinos

Hildebrandt, Thomas B.; Hermes, Robert; Goeritz, Frank; Appeltant, Ruth; Colleoni, Silvia; Mori, Barbara de; Diecke, Sebastian; Drukker, Micha; Galli, Cesare; Hayashi, Kozaburo; Lazzari, Giovanna; Loi, Pasqualino; Payne, Junaidi; Renfree, Marilyn B.; Seet, S.; Stejskal, Jan; Swegen, Aleona; Williams, Suzannah A.; Zainuddin, Zainal Zahari; Holtze, Susanne

doi:10.1016/j.theriogenology.2021.04.006

Cited by 46 publications

(48 citation statements)

References 165 publications

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“…If left uncontrolled, excessive ROS levels can lead to cell degradation and DNA damage [ 58 ]. Further examination of these rhinoceros sperm cell characteristics is vital to enhance understanding of the molecular nature of rhinoceros spermatozoa during the freezing process, and of how these may influence the outcome of artificial insemination programs, for which the rhinoceros currently records a low success rate [ 3 , 4 ]. Nevertheless, the above results provide an important benchmark for the species.…”

Section: Discussionmentioning

confidence: 99%

“…Cryopreservation and biobanking of gametes, tissues and cell lines from endangered species has the potential to preserve gametes for the long term, maintain genetic diversity and help fight against the threat of extinction. For example, the successful collection of male gametes from the endangered Sumatran rhinoceros in terms of quantity and quality can play a key role in the de-extinction of the species in the future [ 4 ]. The current study has contributed towards this goal by demonstrating the ability of papain to ensure high quantities of viable rhinoceros spermatozoa are collected from ejaculates, even if fractions are deemed low sperm and viscous.…”

Section: Discussionmentioning

confidence: 99%

“…Current numbers of white, black, greater one-horned, Sumatran and Javan rhinoceros in situ of 10000, 3100, 2200, 30 and 18 adult individuals, respectively [ 1 ], emphasise the importance of captive breeding programs for the maintenance of genetic diversity and survival of these species [ 2 , 3 , 4 ]. Furthermore, it highlights the need for continued development of advanced assisted reproductive technologies (ARTs) to increase the likelihood of success.…”

Section: Introductionmentioning

confidence: 99%

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Increasing the Yield and Cryosurvival of Spermatozoa from Rhinoceros Ejaculates Using the Enzyme Papain

Rickard

Pool

Graaf

et al. 2022

Biology

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The preservation of rhinoceros semen is vital for captive breeding programs. While successful collection and cryopreservation of rhinoceros semen has been reported, the volume and quality of semen produced is often low due to the high viscosity associated with ejaculates collected via electroejaculation. Reducing semen viscosity would enable access to previously unusable spermatozoa from viscous fractions and could improve quality post-thaw. The enzyme papain successfully reduced the viscosity of camelid semen but has yet to be tested in wildlife species. This study assessed the influence of papain on the in vitro quality of rhinoceros spermatozoa during cryopreservation using advanced semen assessment. In experiment 1, the motility of spermatozoa from the viscous fraction of an ejaculate, either untreated or treated with papain and its inhibitor E-64 prior to cryopreservation, was assessed post-thaw. In experiment 2, spermatozoa from papain-treated viscous fractions were compared to spermatozoa frozen from untreated sperm-rich fractions pre-freeze, as well as after 0, 1.5 and 3 h of incubation post-thaw (37 °C). Papain significantly increased the quantity of spermatozoa collected from ejaculates, as well as the motility prior to freezing. Papain also improved the post-thaw motility, velocity, linearity and straightness of samples compared to sperm-rich samples, with no detriment to sperm viability, lipid membrane disorder, production of ROS or DNA integrity (p < 0.05). Results show the benefit of supplementing rhinoceros spermatozoa with papain prior to cryopreservation on sperm cryosurvival and demonstrates the potential of using papain to improve the success of cryopreservation protocols, not only for the rhinoceros, but also for other wildlife species.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Increasing the Yield and Cryosurvival of Spermatozoa from Rhinoceros Ejaculates Using the Enzyme Papain

Rickard

Pool

Graaf

et al. 2022

Biology

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“…GRBs and ARTs are not a new concept in conservation per se (Wildt et al 1986;Holt et al 2003), but the investment of research resources and development of technologies has heavily favoured avian and mammalian models. Even within those taxa, the focus has been on sperm cryopreservation paired with artificial insemination (Rodger and Clulow 2021) rather than more advanced stem and cell biology approaches, with some notable exceptions involving advanced technologies such as stem cell generation and nuclear transfer (Folch et al 2009;Hildebrandt et al 2021;Holt and Comizzoli 2021;Sandler et al 2021). For reptiles and amphibians, the development of technologies has lagged the other vertebrate taxa both in resources and time by a couple of decades (Browne et al 2011;Clulow et al 2012;Clulow and Clulow 2016;Della Togna et al 2020).…”

Section: Introductionmentioning

confidence: 99%

Common goals, different stages: the state of the ARTs for reptile and amphibian conservation

Clulow

Marcec-Greaves

et al. 2022

Reprod. Fertil. Dev.

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Amphibians and reptiles are highly threatened vertebrate taxa with large numbers of species threatened with extinction. With so many species at risk, conservation requires the efficient and cost-effective application of all the tools available so that as many species as possible are assisted. Biobanking of genetic material in genetic resource banks (GRBs) in combination with assisted reproductive technologies (ARTs) to retrieve live animals from stored materials are two powerful, complementary tools in the conservation toolbox for arresting and reversing biodiversity decline for both amphibians and reptiles. However, the degree of development of the ARTs and cryopreservation technologies differ markedly between these two groups. These differences are explained in part by different perceptions of the taxa, but also to differing reproductive anatomy and biology between the amphibians and reptiles. Artificial fertilisation with cryopreserved sperm is becoming a more widely developed and utilised technology for amphibians. However, in contrast, artificial insemination with production of live progeny has been reported in few reptiles, and while sperm have been successfully cryopreserved, there are still no reports of the production of live offspring generated from cryopreserved sperm. In both amphibians and reptiles, a focus on sperm cryopreservation and artificial fertilisation or artificial insemination has been at the expense of the development and application of more advanced technologies such as cryopreservation of the female germline and embryonic genome, or the use of sophisticated stem cell/primordial germ cell cryopreservation and transplantation approaches. This review accompanies the publication of ten papers on amphibians and twelve papers on reptiles reporting advances in ARTs and biobanking for the herpetological taxa.

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“…The successful cryopreservation of spermatozoa collected from wildlife species is vital for ARTs, in particular the establishment of biobanks to preserve genetic diversity across species [ 13 ]. Yet, depending on the intended application of frozen-thawed sperm, the minimal requirements for motility and viability post-thaw can differ greatly, depending on whether the sperm will be used for artificial insemination (AI), in vitro fertilization (IVF), or intracytoplasmatic sperm injection (ICSI).…”

Section: Introductionmentioning

confidence: 99%

Cryopreservation of Giraffe Epidydimal Spermatozoa Using Different Extenders and Cryoprotectants

Hermes

Lécu²,

Potier³

et al. 2022

Animals

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Giraffe numbers have plummeted over the last 30 years by 30–40%. Thus, their conservation status has been raised from least concern to vulnerable. Efforts to manage in situ and ex situ populations are increasing. Assisted reproduction techniques (ART) such as sperm cryopreservation could help preserve the genetic diversity of giraffe subspecies and, when used for artificial inseminations, enhance genetic exchange between isolated populations. However, to date, the post-thaw motility of recovered sperm has been low and inconsistent. In this study, epididymal sperm collected from the testes of giraffes (n = 7) was frozen in three different extenders, namely, BotuCrio, Steridyl, and test egg yolk (TEY), each supplemented with one of two different cryoprotectants (5% glycerol or a mix of 1% glycerol and 4% methylformamide) and frozen over liquid nitrogen vapor. Across all three extenders, sperm showed significantly better post-thaw results when frozen with a mix of glycerol and methylformamide compared with glycerol alone. Sperm frozen with TEY and a mix of glycerol and methylformamide achieved superior post-thaw total and progressive sperm motility of 57 ± 3% and 45 ± 3%, respectively. These results show the benefit of using alternative cryoprotectants for freezing giraffe spermatozoa and could aid in the application of ARTs for giraffe subspecies or the closely related endangered Okapi.

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The ART of bringing extinction to a freeze – History and future of species conservation, exemplified by rhinos

Cited by 46 publications

References 165 publications

Increasing the Yield and Cryosurvival of Spermatozoa from Rhinoceros Ejaculates Using the Enzyme Papain

Increasing the Yield and Cryosurvival of Spermatozoa from Rhinoceros Ejaculates Using the Enzyme Papain

Common goals, different stages: the state of the ARTs for reptile and amphibian conservation

Cryopreservation of Giraffe Epidydimal Spermatozoa Using Different Extenders and Cryoprotectants

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