The last survivors of the northern white rhinoceros (NWR, Ceratotherium simum cottoni), once widespread across Central and East Africa, are two non-reproductive females under continuous human protection. In order to prevent the extinction of this white rhinoceros subspecies, the BioRescue consortium founded in 2019 has developed an innovative reproductive biotechnology program. This holistic rescue strategy implements (i) advanced assisted reproductive technologies (aART) including in vitro fertilization (IVF) and embryo transfer, (ii) stem cell associated techniques (SCAT) for establishing in vitro gametogenesis (IVG), and (iii) a pangenetic rescue strategy (PRS) that uses the full spectrum of genetic diversity still available by incorporating DNA sequence information from the globally available NWR-museum specimens combined with gene editing to enrich the genetic diversity of the future living population. Key milestones of the BioRescue consortium are: 26 non-surgical oocyte collections in NWR followed by in-vitro oocyte maturation and embryo generation, first pregnancy in a southern white rhino (SWR) surrogate after heterologous embryo transfer, establishment of two SWR embryonic stem cell (ESC) lines, NWR induced pluripotent stem cell (iPSC) lines derived from somatic tissue, and robust primordial germ cell-like cell (PGCLC) induction as a first step towards IVG. Beyond biological and technical challenges, an essential part of the BioRescue project’s operational framework addresses the ethical dimension of this new approach in conservation, ensuring transparency, animal welfare, and societal accountability. This multidisciplinary strategy offers a replicable model in conservation science for rescuing critically endangered or practically extinct species, linking the most advanced reproductive technologies with ethical oversight to safeguard biodiversity.

In brief: This study identifies pregnanolone as a previously unrecognized circulating progestogen that emerges during the implantation period following embryonic diapause in roe deer. Abstract: Roe deer (Capreolus capreolus) are seasonal animals with fertilization taking place in summer (July/August). Thereafter, the embryo enters a 4- to 5-month period of embryonic diapause. This reproductive strategy, displaying a reduced embryonic developmental velocity at the blastocyst stage, leads to reactivation and implantation in late December-January and parturition in spring (May/June). The endocrine mechanisms governing reactivation, implantation and placentation remain poorly understood. Here, we applied ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry to profile circulating progestogens from reactivation through late gestation (December-April), and in placental and luteal tissue. Notably, the neurosteroid pregnanolone (3α,5β-tetrahydroprogesterone; 3α,5β-THP) appeared exclusively in pregnant does from late December/early January onwards, coinciding with the period of implantation. Pregnanolone was detected in placental but not luteal tissue, pointing to the placenta as a potential source. Our results indicate a previously unrecognized shift in progestogens from reactivation/early placentation onwards, suggesting that nonclassical progestogens may contribute to the endocrine regulation of implantation and pregnancy maintenance. Further investigation into the origin and functional roles of pregnanolone are needed to elucidate its contribution to pregnancy and reproductive success in roe deer.

Since assisted reproductive technologies (ART) are becoming increasingly important in wildlife conservation breeding programs, we need to discuss their implications to ensure their responsible use regarding the environment, the animals, and the people involved. In this article, we seek to contribute to the ongoing ethical and philosophical debate on ART in conservation by discussing the current attempt to save the northern white rhino (Ceratotherium simum cottoni, NWR) from extinction. Only two female NWRs are known to the world, both unable to carry a pregnancy, and the last chance to avoid sure extinction relies on pushing the boundaries of contemporary science through the use of advanced ART and stem cell-associated techniques. The attempt to save the NWR constitutes a valuable testbed for assessing the use of ART in conservation and an occasion for identifying possible critical issues. It touches upon several ethically relevant points—that we identify and organize in an Ethical Matrix—such as the need to guarantee animal welfare, and it provides the opportunity to discuss some significant questions related to conservation. For instance, how far is it legitimate to go in trying to save a taxon? Is using sophisticated technologies to remedy anthropogenic harm a part of the problem rather than the solution?

This case report presents a rare occurrence of an intrauterine single twin demise in a captive European roe deer (Capreolus capreolus), representing to the best of our knowledge the first documented instance of this phenomenon in a wildlife species. Ultrasound examination of the reproductive organs of a twin-pregnant doe revealed that one twin was smaller and showed an absent heartbeat compared to the other twin. The viable twin displayed normal development and birth, while the dead twin exhibited signs of intrauterine fetal death. This phenomenon of single twin demise in an ungulate species known for its ability to undergo embryonic diapause highlights the importance of understanding reproductive anomalies in wildlife. Such findings enhance our knowledge of reproductive biology and have implications for wildlife conservation and captive breeding practices.

The application of Assisted Reproductive Technologies (ART) in breeding programs to save wild animal species is a relatively new approach to conservation and their ethical implications seem still to be underestimated. However, the ethical evaluation is a decisive step for conservation projects employing ART to address crucial questions like the welfare and life of the animals involved, the survival of the targeted species, the quality and safety of the procedures and the very idea of biodiversity conservation we want to pursue. Balancing the potential benefits of ART with the risks involved requires careful ethical analysis. This paper highlights the increasing role of ART in species conservation and emphasizes the need to address their ethical implications. After discussing the lack of ethical discussion in ART application to wildlife, the contribution outlines an ethical frame to address this gap and introduces an ETHical ASsessment tool (ETHAS) for the ethical self-assessment of ART procedures in vertebrate conservation. The tool allows respondents to highlight ethical aspects to be improved and risks related to the procedure, from its design to each application. The ethical approach to the ART application, relatively novel to wildlife conservation, can contribute to assure the ethical acceptability of conservation programs while favoring discussion and communication among project partners and the integration of ethical aspects in conservation programs.

Genome Resources Banks (GRBs) represent vital repositories for the systematic collection, storage, and management of genetic material across various taxa, with a primary objective of safeguarding genetic diversity for research and practical applications. Alongside the development of assisted reproductive techniques (ART), GRBs have evolved into indispensable tools in conservation, offering opportunities for species preservation, mitigating inbreeding risks, and facilitating genetic management across fragmented populations. By preserving genetic information in a suspended state, GRBs serve as backups against population vulnerabilities, potentially aiding in the restoration of endangered species and extending their genetic lifespan. While evidence demonstrates the efficacy of GRBs, ethical considerations surrounding biobanking procedures for wildlife conservation remain largely unexplored. In this article, we will discuss possible ethical issues related to GRBs and the need to ethically monitor biobanking procedures in wildlife conservation. We will then propose a methodological tool, ETHAS, already in use for the ethical self-assessment of assisted reproduction techniques, to assess also biobanking procedures. ETHAS can make it possible to monitor a GRB from its design phase to its actual operation, helping to build biobanking procedures that meet high ethical standards.

Chronic kidney disease (CKD) is a prevalent disease among felids; yet its origin is still poorly understood, and the disease often remains asymptomatic for years, underscoring the need for early diagnosis. This study aimed to investigate the diagnostic value of urinalysis in accurately staging CKD, particularly as routine health checks in large felids often overlook its significance. In this research, ultrasound-guided cystocentesis (UGC) was performed on 50 captive nondomestic felids during routine veterinary health checks under general anesthesia. Urinalysis included microscopic examination of the sediment, measurement of urine specific gravity (USG) and protein to creatinine ratio (UPC). Additional serum kidney markers, such as creatinine and symmetric dimethylarginine, were compared with USG and UPC to assess their diagnostic value as urinary biomarkers. The results demonstrated proteinuria (UPC > 0.4) or borderline proteinuria (UPC 0.2-0.4) in 49% of the animals. Among these cases, 62% were of renal origin, and 38% were postrenal causes. USG was significantly higher in felids with borderline proteinuria compared to those with proteinuria. A moderate, but significant negative correlation between serum parameters and USG was observed, emphasizing the importance of assessing both diagnostic parameters during kidney evaluations. Additionally, felids with CKD have an increased risk of urinary tract infections, necessitating microscopic urinalysis and bacterial culture analysis. Abnormalities, including hematuria, pyuria, crystalluria, and bacteriuria, were found in approximately 38% of cases through microscopical examination of urine. No complications associated with UGC were observed and abnormal findings were detected in 60% of the cases. Based on these results, the authors recommend the inclusion of UGC and urinalysis as standard diagnostic tools in general health checks for nondomestic felids. This approach provides valuable insights into the early detection and staging of CKD, supporting early intervention and supportive medical care to prolong renal health in these animals.

Behavior and innervation suggest a high tactile sensitivity of elephant trunks. To clarify the tactile trunk periphery we studied whiskers with the following findings. Whisker density is high at the trunk tip and African savanna elephants have more trunk tip whiskers than Asian elephants. Adult elephants show striking lateralized whisker abrasion caused by lateralized trunk behavior. Elephant whiskers are thick and show little tapering. Whisker follicles are large, lack a ring sinus and their organization varies across the trunk. Follicles are innervated by ~90 axons from multiple nerves. Because elephants don’t whisk, trunk movements determine whisker contacts. Whisker-arrays on the ventral trunk-ridge contact objects balanced on the ventral trunk. Trunk whiskers differ from the mobile, thin and tapered facial whiskers that sample peri-rostrum space symmetrically in many mammals. We suggest their distinctive features—being thick, non-tapered, lateralized and arranged in specific high-density arrays—evolved along with the manipulative capacities of the trunk.

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.

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.

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 SWRPGCLC 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.

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.

Less than 80 Sumatran rhinos (SR, Dicerorhinus sumatrensis) are left on earth. Habitat loss and limited breeding possibilities are the greatest threats for the species and lead to a continuous population decline. To stop erosion of genetic diversity, reintroduction of genetic material is indispensable. However, as the propagation rate of captive breeding is far too low, innovative technologies have to be developed. Induced pluripotent stem cells (iPSCs) are a powerful tool to fight extinction. They give rise to each cell within the body including gametes, and provide a unique modality to preserve genetic material across time. Additionally, they enable studying species-specific developmental processes. Here, we generate iPSCs from the last male Malaysian SR Kertam, who died in 2019, and characterize them comprehensively. Differentiation in cells of the three germ layers and cerebral organoids demonstrate their high quality and great potential for supporting rescue of this critically endangered species.

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.

Etorphine is widely used in zoological medicine for the immobilization of large herbivores. All reported immobilization protocols for kulans use etorphine as the primary immobilizing agent. However, etorphine can trigger severe side effects and is highly toxic for humans, its availability is occasionally limited for use in wildlife medicine. Therefore, two different alpha-2 agonist-based protocols for the general anesthesia of kulans were investigated and compared with the standard etorphine immobilization. In total, 21 immobilizations were performed within the scope of routine husbandry management at the Serengeti-Park Hodenhagen. Kulans were darted using a ketamine–medetomidine–midazolam–butorphanol (KMMB) protocol ( n = 8, treatment group (TG) 1), a tiletamine–zolazepam–medetomidine–butorphanol (TZMB) protocol ( n = 7, treatment group (TG) 2), or an etorphine–acepromazine–detomidine–butorphanol (EADB) protocol ( n = 6, control group). Vital parameters included heart rate, respiratory rate, arterial blood pressure (invasive), end tidal CO 2 (etCO 2 ), electromyography and core body temperature, which were all assessed every 10 min. For blood gas analysis, arterial samples were collected 15, 30, 45 and 60 min after induction. Subjective measures of quality and efficacy included quality of induction, immobilization, and recovery. Time to recumbency was longer for TG 1 (9.00 ± 1.67 min) and TG 2 (10.43 ± 1.79 min) compared to the induction times in the control group (5.33 ± 1.93 min). Treatment group protocols resulted in excellent muscle relaxation, normoxemia and normocapnia. Lower pulse rates combined with systolic arterial hypertension were detected in the alpha-2 agonist-based protocols. However, only in TZMB-immobilized kulans, sustained severe systolic arterial hypertension was observed, with significantly higher values than in the TG 1 and the normotensive control group. At 60 min following induction, medetomidine and detomidine were antagonized with atipamezole IM (5 mg/mg medetomidine or 2 mg/mg detomidine), etorphine and butorphanol with naltrexone IV (2 mg/mg butorphanol or 50 mg/mg etorphine), and midazolam and zolazepam with flumazenil IV (0.3 mg per animal). All three combinations provided smooth and rapid recoveries. To conclude, the investigated treatment protocols (KMMB and TZMB) provided a safe and efficient general anesthesia in kulans with significantly better muscle relaxation, higher respiration rates and improved arterial oxygenation compared with the immobilizations of the control group. However, the control group (EADB) showed faster recoveries. Therefore, EADB is recommended for ultra-short immobilizations (e.g., microchipping and collaring), especially with free-ranging kulans where individual recovery is uncertain, whereas the investigated treatment protocols are recommended for prolonged medical procedures on captive kulans.

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.

Complex odontoma is a rare odontogenic lesion reported in rodents (order: Rodentia) and odd-toed ungulates (order: Perissodactyla), to name a few, and only in bovine animals of the order Artiodactyla. A 3-year-old female giraffe presented with a steadily proliferating, firm mass in the rostral mandible. With further expansion and ulceration of the mass, the general condition of the giraffe deteriorated and it was euthanized. Post-mortem examination revealed greyish–white tissue with an irregular arrangement of yellowish hard tissue arranged in thin plates and intermingled areas of greyish soft tissue. Histologically, irregular proliferated odontogenic epithelium and mesenchyme, dentin, cementum and empty spaces, suggestive of decalcified enamel, were present. These findings are consistent with a diagnosis of complex odontoma, which should be added to the differential diagnoses of oral tissue proliferations in giraffes. To our knowledge, this is the first description of a complex odontoma in a giraffe.

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.

The ongoing mass extinction of animal species at an unprecedented rate is largely caused by human activities. Progressive habitat destruction and fragmentation is resulting in accelerated loss of biodiversity on a global scale. Over decades, captive breeding programs of non-domestic species were characterized by efforts to optimize species-specific husbandry, to increase studbook-based animal exchange, and to improve enclosure designs. To counter the ongoing dramatic loss of biodiversity, new approaches are warranted. Recently, new ideas, particularly the application of assisted reproduction technologies (ART), have been incorporated into classical zoo breeding programs. These technologies include semen and oocyte collection, artificial insemination, and in-vitro embryo generation. More futuristic ideas of advanced ART (aART) implement recent advances in biotechnology and stem-cell related approaches such as cloning, inner cell mass transfer (ICM), and the stem-cell-associated techniques (SCAT) for the generation of gametes and ultimately embryos of highly endangered species, such as the northern white rhinoceros (Ceratotherium simum cottoni) of which only two female individuals are left. Both, ART and aART greatly depend on and benefit from the rapidly evolving cryopreservation techniques and biobanking not only of genetic, but also of viable cellular materials suitable for the generation of induced pluripotent stem cells (iPSC). The availability of cryopreserved materials bridges gaps in time and space, thereby optimizing the available genetic variability and enhancing the chance to restore viable populations.

Significance In mammals, embryo development can halt at the hatched blastocyst stage. Uniquely, proliferation of diapausing embryonic roe deer cells decelerates to a doubling time of 2 to 3 wk over a period of 4 mo. We highlight nutrient sensing as an important factor regulating embryonic developmental pace. The resumption of embryo development is characterized by an increase in uterine fluid mTORC1-activating amino acids, embryonic mTORC1 activity, and expression of metabolism and cell cycle genes. We propose selective mTORC1 inhibition via reduced estrogen signaling and high let-7 levels as mechanisms for slow cell cycle progression. We hypothesize that it is the lack of embryonic mTORC2 inhibition during embryonic diapause in the roe deer that enables the continuous decelerated rate of proliferation.