Application of decision tools to ethical analysis in biodiversity conservation.

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.

Que se logren decisiones éticamente responsables es crucial para el éxito de los proyectos de conservación de la biodiversidad. Adaptamos la matriz ética, el árbol de decisión y el cubo de Bateson para apoyar con el análisis ético de escenarios de conservación compleja mediante la estructuración de estas herramientas de tal manera que puedan ejecutar las diferentes dimensiones de valor (ambiental, social y bienestar animal) involucradas en la ética de la conservación. Después aplicamos las herramientas a un estudio de caso relacionado con el proceso de toma de decisiones respecto a si se debe seguir o no recolectando material biológico del rinoceronte blanco del norte (Ceratotherium simum cottoni) más viejo (una subespecie funcionalmente extinta) de los dos que existen. Usamos la matriz ética como un punto de partida para una estrategia participativa para la toma ética de decisiones y para recopilar los pros y contras éticos. Usamos el árbol de decisión para comparar las diferentes opciones en juego con base en un conjunto de deseos éticos. Usamos el cubo de Bateson para establecer un umbral de aceptación ética y modelar los resultados de una encuesta simple. La aplicación de estas herramientas demostró ser central en la estructuración del proceso de toma de decisiones y en el apoyo para lograr una decisión compartida, razonada y transparente sobre la mejor opción a partir de un punto de vista ético entre aquellos disponibles.

Sperm motility, kinematics, morphometry and morphology over two seasons in free-ranging African elephants (Loxodonta africana).

This study aimed to address the lack of information on quantitative semen and sperm characteristics of free-ranging African elephants. Nineteen ejaculates were collected from 12 elephant bulls by means of electroejaculation in spring (Season 1, end of dry season, n=7) and in autumn (Season 2, end of rainy season, n=12). While most elephant cows are in oestrus in the rainy season, it is not evident whether sperm quality also improves during this period. Semen samples were assessed using computer-aided sperm analysis (CASA), brightfield microscopy and transmission electron microscopy. Seasonal differences and individual variation in sperm quality of bulls were apparent, with ejaculates collected during Season 2 revealing higher percentages for total motility, progressive motility, rapid-swimming spermatozoa and kinematic parameters compared with Season 1 (P<0.05). Although normal sperm morphology percentage was similar over the two seasons, more sperm tail defects were found in Season 2 (P<0.05). The baseline reference data and multivariate sperm parameter associations reported in this study can be used to predict elephant bull sperm quality and potential to fertilise. It is clear that CASA can detect subtle differences in sperm quality of African elephant ejaculates and should be the approach for future investigations.

Effects of deslorelin acetate on plasma testosterone concentrations in captive yellow-bellied sliders (Trachemys scripta sp.).

In Europe, the yellow-bellied slider (Trachemys scripta sp.) is a non-native species in competition with native freshwater turtles. Research on contraception could be useful to control the captive population. Identifying a method of contraception in chelonians would potentially help to control aggression in other chelonian species. The objective of the current study was to evaluate the effects of a single 4.7-mg deslorelin acetate implant on plasma testosterone concentrations in yellow-bellied sliders (Trachemys scripta sp.). Eleven adult male yellow-bellied sliders were used for the study. Males from the treatment group (n = 6) received a 4.7-mg deslorelin acetate implant, whereas males from the control group (n = 5) did not receive any treatment. All individuals were housed under the same environmental conditions. Testosterone plasma concentrations of the control group and the treatment group were measured at six time points (T0-T6) between April and September. No difference between the control group and the deslorelin treatment group was observed at T0, T2, T3, T4, T5 or T6. However, mean plasma testosterone concentration was significantly higher in the treatment group than in the control group at T1. This suggests that treatment with a 4.7-mg deslorelin acetate implant has a transient stimulatory effect on the anterior pituitary in yellowbellied sliders without a negative feedback on testosterone production. Further studies with a higher dosage of deslorelin acetate are needed to draw conclusions on its contraceptive effect.

Paternal intergenerational epigenetic response to heat exposure in male Wild guinea pigs.

Epigenetic modifications, of which DNA methylation is the best studied one, can convey environmental information through generations via parental germ lines. Past studies have focused on the maternal transmission of epigenetic information to the offspring of isogenic mice and rats in response to external changes, whereas heterogeneous wild mammals as well as paternal epigenetic effects have been widely neglected. In most wild mammal species, males are the dispersing sex and have to cope with differing habitats and thermal changes. As temperature is a major environmental factor we investigated if genetically heterogeneous Wild guinea pig (Cavia aperea) males can adapt epigenetically to an increase in temperature and if that response will be transmitted to the next generation(s). Five adult male guinea pigs (F0) were exposed to an increased ambient temperature for 2 months, i.e. the duration of spermatogenesis. We studied the liver (as the main thermoregulatory organ) of F0 fathers and F1 sons, and testes of F1 sons for paternal transmission of epigenetic modifications across generation(s). Reduced representation bisulphite sequencing revealed shared differentially methylated regions in annotated areas between F0 livers before and after heat treatment, and their sons' livers and testes, which indicated a general response with ecological relevance. Thus, paternal exposure to a temporally limited increased ambient temperature led to an 'immediate' and 'heritable' epigenetic response that may even be transmitted to the F2 generation. In the context of globally rising temperatures epigenetic mechanisms may become increasingly relevant for the survival of species.

Rewinding the process of mammalian extinction.

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.

Sperm rescue in wild African elephants.

This study investigated ways of improving the usefulness of ~1700mL of poor-quality frozen semen collected from wild African elephant (Loxodonta africana) bulls. Ten semen samples from six bulls, frozen with 5% glycerol in Berliner cryomedium, with or without prior removal of the seminal plasma by centrifugation, were tested. All samples were subjected to the following density-gradient centrifugation treatments: no centrifugation (control), sham centrifugation, Percoll, OptiPrep, Isolate and PureSperm. Sample evaluation included motility, concentration, viability, acrosome integrity and normal morphology after thawing and after gradient centrifugation. Motility was also evaluated 3h after thawing. While all treatments were similar to the Control in acrosome integrity and normal morphology, significant differences were noted in concentration, viability and motility. Samples treated by Percoll showed the best motility, which was maintained unchanged over 3h of incubation (37°C). Correlations between manual and automated evaluations of concentration were high (cytometer; rho=0.92), but were lower for viability (cytometer; rho=0.57) and motility (computer-aided sperm analysis; rho=0.66). By performing density centrifugation, the quality of these sperm samples may be improved to a level suitable for artificial insemination in elephants. Although a sizeable proportion of cells are lost in the process, combining samples may still allow for multiple inseminations.

CRYOPRESERVATION OF ONAGER (EQUUS HEMIONUS ONAGER) EPIDIDYMAL SPERMATOZOA.

Genetic diversity is a primary component of adaptive evolution, and its loss or reduction can decrease the long-term survival probability of populations. Utilization of cryopreserved semen may be considered a perfect tool to improve genetic diversity, reduce inbreeding, and avoid animal translocation for breeding. The present study aimed at finding a reliable epididymal sperm freezing protocol for the critically endangered onager (Equus hemionus onager). Six testicles from three animals were processed postmortem. The effects of two transportation temperatures (22°C and 4°C; testicles submerged in saline), two cryopreservation techniques (conventional liquid nitrogen vapor freezing in straws and directional freezing in 8-ml HollowTubes(TM)), and two postthaw incubation temperatures (22°C and 37°C; evaluated after 0.5, 1, 2, and 3 hr) were tested in a 2×2×2 experimental design. Sperm samples were evaluated for motility, viability, acrosome integrity, and sperm morphology. The resulting optimal freezing protocol includes transportation of testicles at 4°C, cryopreservation by directional freezing, and, if needed, postthaw incubation at 22°C. With this combination of transportation temperature and cryopreservation technique, the authors obtained the following postthaw values normalized to prefreezing values: 60.3±8.8% motility, 60.7±13.3% viability, 75.3±9.5% acrosome integrity, and 94.7±2.9% normal morphology (excluding defects due to the epididymal origin of the sperm). After incubation at 22°C, motility values for the above combination were 40±5.7%, 30.3±5.2%, 28.3±4.4%, and 16.7±4.4% for 0.5, 1, 2, and 3 hr, respectively. In conclusion, with this protocol, good quality semen can be stored for future use in artificial inseminations when and where needed.

A simple method to identify ether lipids in spermatozoa samples by MALDI-TOF mass spectrometry.

Plasmalogens (alkenylacyl glycerophospholipids) are important lipid constituents of many tissues and cells (e.g., selected spermatozoa). Since the molecular weights of plasmalogens overlap with that of diacyl- or alkyl acyl lipids, sophisticated mass spectrometry (MS; including MS/MS) analysis is normally used for the unequivocal identification of plasmalogens. We will show here that a simple matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (without MS/MS capability) in combination with acidic hydrolysis and subsequent derivatization with 2,4-dinitrophenylhydrazine (DNPH) and/or digestion with phospholipase A2 (PLA2) is sufficient to determine the contributions of ether lipids in spermatozoa extracts. As neither diacyl nor alkylacyl lipids are sensitive to acids and do not react with DNPH, the comparison of the mass spectra before and after treatment with acids and/or DNPH addition readily provides unequivocal information about the plasmalogen content. Additionally, the released aldehydes are readily converted into the 2,4-dinitrophenylhydrazones and can be easily identified in the corresponding negative ion mass spectra. Finally, PLA2 digestion is very useful in confirming the presence of plasmalogens. The suggested method was validated by analyzing roe deer, bovine, boar, and domestic cat spermatozoa extracts and comparing the results with isolated phospholipids.

The functional anatomy of elephant trunk whiskers.

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.

Histological and endocrine characterisation of the annual luteal activity in Eurasian lynx (Lynx lynx).

Lynx presents a unique sexual cycle with persistent corpora lutea (CLs) and elevated serum progesterone (P4) throughout parturition and lactation. In other mammals, CLs normally disintegrate after parturition, therefore the aim of our study was to characterise the annual life cycle of lynx CLs. Ovaries from Eurasian lynxes were obtained from the National Veterinary Institute in Sweden, where tissues from killed lynx were stored at -20 °C. Ovaries from 66 animals were weighed; each corpus luteum was segmented for histology and hormone analysis. Ovary and CLs weights were constant throughout the year, peaking during pregnancy. In non-pregnant lynxes, the seasonal level of intraluteal steroids was steady for P4 (3.2±1.9 s.d. µg/g, n=53) and total oestrogens (18.3±15.5 s.d. ng/g, n=53). Within histology slides, structurally intact luteal cells were found throughout the year with the highest incidence in March/April; evidence of luteal regression was predominantly found in post-breeding season. Ovaries from pregnant animals contained two types of CLs. Group A was bigger in size with large luteal cells (P4, 72.3±65.4 s.d. µg/g; oestrogen, 454.0±52.4 s.d. ng/g). In contrast, group B were smaller, with greater luteal regression and lower steroid concentrations (P4, 8.3±2.9 s.d. µg/g; oestrogen, 31.5±20.4 s.d. ng/g). Our results suggest that structural luteolysis proceeds throughout the year and into next breeding cycle, resulting in two CLs types on the same ovary.

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

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.

Induced pluripotent stem cells and cerebral organoids from the critically endangered Sumatran rhinoceros.

Less than 80 Sumatran rhinos (SR, Dicerorhinus sumatrensis) are left on earth. Habitat loss and limited breeding possibilities are the greatest threats to the species and lead to a continuous population decline. To stop the 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 the rescue of this critically endangered species.

Naïve-like pluripotency to pave the way for saving the northern white rhinoceros from extinction.

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.

Ethical Analysis of the Application of Assisted Reproduction Technologies in Biodiversity Conservation and the Case of White Rhinoceros (Ceratotherium simum) Ovum Pick-Up Procedures.

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.

Complex Odontoma in a Young Captive Reticulated Giraffe (Giraffa camelopardalis reticulata).

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.

An Ethical Assessment Tool (ETHAS) to Evaluate the Application of Assisted Reproductive Technologies in Mammals' Conservation: The Case of the Northern White Rhinoceros (Ceratotherium simum cottoni).

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.

Ultrasonographically documented early pregnancy loss in an Asian elephant (Elephas maximus).

Early embryonic resorption or fetal loss is known to occur occasionally in captive elephants; however, this has mostly been reported anecdotally. The present study documents the case of a 24-year-old, multiparous Asian elephant cow that suffered embryonic death and resorption at around 18 weeks of gestation. From ovulation onwards, this female was sonographically examined 58 times. Blood was collected twice weekly for progestagen determination via enzyme immunoassay. On Day 42 after ovulation, a small quantity of fluid was detected in the uterine horn, which typically indicates the presence of a developing conceptus. Repeated inspections followed what appeared to be a normal pregnancy until Day 116. However, on Day 124, signs of embryonic life were absent. Progestagen concentrations started declining two weeks later, reaching baseline levels one month after embryonic death. Retrospectively, ultrasound examination revealed several abnormalities in the uterine horn. Besides an existing leiomyoma, multiple small cystic structures had formed in the endometrium at the implantation site and later in the placenta. These pathological findings were considered as possible contributors to the early pregnancy failure. PCR for endotheliotropic elephant herpes virus (EEHV) (which had occurred previously in the herd) as well as serology for other infectious organisms known to cause abortion in domestic animals did not yield any positive results. Although no definitive reason was found for this pregnancy to abort, this ultrasonographically and endocrinologically documented study of an early pregnancy loss provides important insights into the resorption process in Asian elephants.

Immobilization and intravenous anesthesia in a Sumatran rhinoceros (Dicerorhinus sumatrensis).

This paper reports in detail, for the first time, on two anesthetic procedures performed on a 15-yr-old, 530 kg, adult male Sumatran rhinoceros (Dicerorhinus sumatrensis). The anesthetic procedures were carried out in order to perform semen collection via electro-ejaculation, using well-established methods, and in order to examine and sample bilateral corneal opacities. Anesthesia for the first procedure was induced with a combination of 5 mg (0.0094 mg/kg) butorphanol tartrate and 5 mg (0.0094 mg/kg) detomidine hydrochloride administered intramuscularly. Subsequently, 0.74 mg (0.0014 mg/kg) etorphine and 3 mg (0.0057 mg/kg) acepromazine, with an additional 1.5 mg butorphanol (0.0028 mg/kg) and 1.5 mg (0.0028 mg/kg) detomidine, were administered intramuscularly. The second procedure was carried out using an intramuscular combination of butorphanol (0.019 mg/kg) and detomidine (0.019 mg/kg), followed by etorphine (0.0023 mg/kg) and acepromazine (0.009 mg/ kg). During the second procedure, the depth of anesthesia was managed with very small, supplemental intravenous doses of 50 mg ketamine (0.094 mg/kg). Sequential arterial blood gas analysis demonstrated respiratory acidosis with hypoxemia. Heart rate and respiratory rate ranged between 60-74 beats per minute (bpm), and 10-20 breaths per minute, respectively. Reversal after 100 min, with the intravenous administration of 150 mg (0.28 mg/kg) naltrexone and intravenous 20 mg (0.038 mg/kg) atipamezole, was uneventful and rapid, with the animal standing after 2 min. This combination provides satisfactory general anesthesia in this critically endangered species and will facilitate veterinary management of this species in captivity.

Parturition in white rhinoceros.

In captive rhinoceros stillbirth and perinatal death are recorded at a rate of 6-17% in the various species. At the same time there is a substantial lack of knowledge on rhinoceros parturition. Yet, predicting parameters for birth and progress of parturition are fundamental for the recognition of dystocia and perinatal problems. Therefore, we here intended to pay close attention to the Achilles heel of the 1.5-2.5 year reproduction cycle in rhinoceros, the parturition. For the prediction of parturition we recorded timelines for pre-birth udder development, genital swelling, milk production, behavioral unrest, and decrease of serum progesterone concentration and the gestational length in 19 white rhinoceros. First, second and third labour stage, foetal presentation and events in perinatal period were recorded to describe normal parturition and establish a guideline for better birth management in rhinoceros. Udder development and genital swelling were observed 3 and 2 weeks prior birth, respectively. Milk production was observed to start up to 3 weeks prior birth and increased significantly in the last week with most significant increase one day before parturition to 50.6 ± 45.4 mL (p < 0.006). Serum progesterone concentration started to decrease 7 days prior parturition and more significantly 48 h before parturition. While behavioral unrest and first stage labour was not observed reliably in all females the break of foetal waters and thus the start of second stage labour was unmistakably observed. Second stage labour, when foetal membranes had ruptured until the foetus was born, took 1:50 ± 0:20 h:min. Eighty-four percent of fetuses were born in anterior presentation (n = 16/19) and the final expulsion took <25 min suggesting that this is the normal presentation in white rhinoceros. In the less frequent posterior presentation final expulsion took up to 47 min. Overall, 95% of calves were born alive. Calves were standing and nursing in 0:55 ± 0:12 min and 3:32 ± 0:53 h:min, respectively. In 10.5% of births (n = 2/19) in anterior presentation perinatal complications occurred. Stillbirth occurred once (5.3% n = 1/19) when the foetus was born in posterior presentation. The recorded gestational length was 506 ± 2d. Delivering live offspring is of key importance to establish a new generation and secure long-term survival of a species. Various pre-birth changes, significant decrease of serum progesterone 48 h prior birth, different labour stages, foetal presentation and perinatal events described here add substantial knowledge on the understanding of normal rhinoceros parturition and may help diagnose dystocia and perinatal complications.

Insights in the regulation of trimetylamine N-oxide production using a comparative biomimetic approach suggest a metabolic switch in hibernating bears.

Experimental studies suggest involvement of trimethylamine N-oxide (TMAO) in the aetiology of cardiometabolic diseases and chronic kidney disease (CKD), in part via metabolism of ingested food. Using a comparative biomimetic approach, we have investigated circulating levels of the gut metabolites betaine, choline, and TMAO in human CKD, across animal species as well as during hibernation in two animal species. Betaine, choline, and TMAO levels were associated with renal function in humans and differed significantly across animal species. Free-ranging brown bears showed a distinct regulation pattern with an increase in betaine (422%) and choline (18%) levels during hibernation, but exhibited undetectable levels of TMAO. Free-ranging brown bears had higher betaine, lower choline, and undetectable TMAO levels compared to captive brown bears. Endogenously produced betaine may protect bears and garden dormice during the vulnerable hibernating period. Carnivorous eating habits are linked to TMAO levels in the animal kingdom. Captivity may alter the microbiota and cause a subsequent increase of TMAO production. Since free-ranging bears seems to turn on a metabolic switch that shunts choline to generate betaine instead of TMAO, characterisation and understanding of such an adaptive switch could hold clues for novel treatment options in burden of lifestyle diseases, such as CKD.