Ceratotherium Simum Simum Research Articles

Rival assessment by territorial southern white rhinoceros males via eavesdropping on the contact and courtship calls

Eavesdropping on vocal signals allows animals to gather information about conspecifics. For males, eavesdropping can be a low-risk method for determining the dominance status (e.g. territorial or subordinate) and motivation (e.g. mate seeking) of rivals. Adult southern white rhino males, Ceratotherium simum, are territorial and, in addition to other vocalizations, use contact and courtship calls when communicating with females. Although male territories are exclusive, the owners may tolerate up to three resident subordinate males. However, rival males sometimes intrude. Moreover, these intrusions may lead to rivals interacting with females. We investigated whether territorial males eavesdrop on vocal signals directed towards females by intruding males to determine their dominance status (territorial or subordinate) and motivation (contact calling an anoestrous female or approaching a female in oestrus during courtship). To do this, we first recorded and analysed contact and courtship calls of adult southern white rhino males and determined that these calls signal the males' dominance status and motivation. Playback trials revealed that territorial males differentiated between the calls of subordinate and other territorial males, showing a shorter latency to approach and longer searching behaviour (i.e. walking and running) after the playback of a subordinate's call. The reason for this response could be that the intruding subordinate male might be looking to challenge the territorial male for his territory. However, subordinates probably pose the lowest risk to the resident male in terms of fighting ability. In contrast, the reactions of the territorial males to the calls of other territorial males suggest that the presence and not the motivation of an intruding male was the most important factor influencing the male's reaction. We conclude that territorial males eavesdrop on the acoustic signals of trespassing males to gather information about these rivals, which can help them to better defend their territory.

Historical Population Declines Prompted Significant Genomic Erosion​ in the Northern and Southern White Rhinoceros (​ <i>Ceratotherium simum</i>)

Large vertebrates are extremely sensitive to anthropogenic pressures, and their populations are declining fast. The white rhinoceros (​Ceratotherium simum) is a paradigmatic case: this African megaherbivore suffered a remarkable population reduction in the last 150 years due to human activities. The two white rhino subspecies, the Northern (NWR) and the Southern white rhino (SWR), however, underwent opposite fates: the NWR vanished quickly after the onset of the decline, while the SWR recovered after a severe bottleneck. Such demographic events are predicted to have an erosive effect at the genomic level, linked to increased genetic drift and inbreeding. However, there is little empirical data available with which to reconstruct the subtleties of such processes. We therefore generated a whole-genome, temporal dataset consisting of 52 re-sequenced white rhino genomes, that represents both subspecies at two time windows throughout the past ~ 170 years. Our data not only reveals previously unknown population substructure within both subspecies, but allowed us to quantify the genomic erosion they underwent, with post-bottleneck white rhinos showing significantly lower levels of heterozygosity and higherinbreeding coefficients than pre-bottleneck individuals. Moreover, the effective population size (N​e​) has suffered a decrease of two and three orders of magnitude in the NWR and SWR, respectively. Our data provides empirical support for theoretical predictions about the genomic consequences ofshrinking populations, which is relevant for understanding the process of population extinction. Furthermore, our findings have the potential to inform tailored management approaches for the conservation of the remaining white rhinos.

MOLECULAR CHARACTERIZATION OF FECAL ESCHERICHIA COLI ISOLATED FROM ZOO ANIMALS.

Accredited zoos and animal parks play an important role in animal health research and conservation, providing important insights on matters of public health including zoonotic infectious diseases and antimicrobial resistance (AMR). The emergence and spread of AMR is a complex phenomenon that jeopardizes human and animal health and also threatens the long-term survival of endangered species. The presence of β-lactamases in clinical isolates is particularly significant as they can jeopardize the efficacy of critically important antimicrobials. Although the presence of β-lactamases and extended-spectrum β-lactamases (ESBLs) producing Enterobacteriaceae in zoo animals has been reported, data are not available for northern European countries. In addition, few data are available on phylogenetic grouping of Escherichia coli isolated from zoo animals that can provide additional information on the host-bacterium relationship and on the pathogenicity of isolates. This study aimed to characterize fecal E. coli isolated from 33 healthy zoo animals from 22 species in Ireland, using conventional and molecular microbiological methods. All E. coli isolates were ampicillin resistant, but combined resistance to amoxicillin and clavulanic acid was not detected. Three E. coli isolates sampled from one Amur tiger, one Bornean orangutan, and one Southern white rhino were multidrug resistant, and blaTEM was detected in E. coli recovered from the Amur tiger and the Bornean orangutan. Other β-lactamases, including ESBLs and AmpCs and plasmid-mediated mcr-1 and mcr-2, were not detected. Overall, E. coli isolates investigated were susceptible to the majority of the antimicrobials tested, and only two animals shed E. coli carrying β-lactamase-encoding genes. The majority of isolates belonged to phylogenetic group B1. The screening of the AMR phenotype and genotype of zoo animal E. coli provides useful data that is relevant to antimicrobial stewardship in the zoo veterinary services and relevant to the bank of knowledge needed for tackling AMR.

Assessment of the Acute Phase Response in Healthy and Injured Southern White Rhinoceros (Ceratotherium simum simum).

Acute phase reactants (APRs) have not been investigated in white rhinoceros (Ceratotherium simum). This study aimed to identify clinically useful APRs in this species. Reference intervals (RIs) were generated for albumin, fibrinogen, haptoglobin, iron and serum amyloid A (SAA) from 48 free-ranging animals, except for SAA (n = 23). APR concentrations between healthy animals and those with tissue injury (inflammation) (n = 30) were compared. Diagnostic performance was evaluated using receiver-operator characteristic (ROC) curve and logistic regression analyses. RIs were: albumin 18–31 g/L, fibrinogen 1.7–2.9 g/L, haptoglobin 1.0–4.3 g/L, iron 9.7–35.0 μmol/L, SAA <20 mg/L. Iron and albumin were lower and fibrinogen, haptoglobin and SAA higher in injured vs. healthy animals. Iron showed the best diagnostic accuracy followed by fibrinogen, albumin, haptoglobin and SAA. Iron ≤ 15.1 μmol/L and haptoglobin >4.7 g/L were significant predictors of inflammatory status and together correctly predicted the clinical status of 91% of cases. SAA > 20 mg/L had a specificity of 100%. In conclusion, albumin and iron are negative and fibrinogen, haptoglobin and SAA positive APRs in the white rhinoceros. The combination of iron and haptoglobin had an excellent diagnostic accuracy for detecting inflammation.

The future of private rhino ownership in South Africa

Sustained poaching over the past decade has led to significant loss of black (Diceros bicornis) and southern white (Ceratotherium simum simum) rhinoceroses across South Africa. Whereas much research has focussed on the heavily targeted state-owned populations, there is little understanding of the trends and challenges faced by rhino populations held in the private sector. Private rhino ownership has increased substantially across South Africa over the past three decades, with over 42% of the entire rhino population now in private ownership. Although total rhino numbers on private properties are still increasing, the number of properties owning rhinos is declining. This suggests a move away from traditional extensive properties to large, single-species breeding facilities, which are less valuable from a conservation perspective. The economic impact associated with increased poaching of rhinos over the past decade is the major challenge to private rhino ownership and may encourage disinvestment in rhinos. Some private rhino owners advocate for trade in rhino horn to generate the funds necessary for continued protection of their animals. However, other options to reduce disinvestment, such as local community-engagement projects, are likely to be more favourably received by the wider conservation industry.

Pregnancies following long luteal phases in southern white rhinoceros (Ceratotherium simum simum).

All extant species in the Rhinocerotidae family are experiencing escalating threats in the wild, making self‐sustaining captive populations essential genetic reservoirs for species survival. Assisted reproductive technologies (ARTs) will become increasingly important for achieving and maintaining ex situ population sustainability and genetic diversity. Previous reports have shown that a large proportion of captive southern white rhinoceros (SWR) females are irregularly cyclic or acyclic, and that cycling females display two different estrous cycle lengths of approximately 30 or 70 days. It has been suggested that the longer estrous cycle length is infertile or subfertile, as no term pregnancies have been observed following long cycles. Here we report the achievement of two pregnancies following long luteal phases, using ovulation induction and artificial insemination with either fresh or frozen‐thawed semen. One female SWR conceived on the first insemination attempt and gave birth to a live offspring. A second female conceived twice in consecutive long cycles although the first embryo was resorbed by 33 days post‐insemination. A pregnancy from this female's second insemination is ongoing with expected parturition in November 2019. Whether prolonged estrous cycles in SWR are subfertile or infertile in natural breeding situations remains unclear. However, our findings demonstrate that the application of ARTs following prolonged cycles can result the successful establishment of pregnancies in SWR. Therefore, with ARTs, female SWR otherwise considered nonreproductive due to long estrous cycles may still have the potential for representation and contribution to the ex situ population.

Behavioral and adrenocortical responses of captive white rhino adolescents to the introduction of a new calf

Changes in group composition are not exclusive to zoos and conservation breeding centers. The recent increase in poaching of African rhino species has accelerated the arrival of orphan calves at rehabilitation centers. Introducing new members into an existing group is often stressful for many mammal species. However, when young animals are involved, such responses may be reduced or absent. The goal of the present study was to evaluate the effect of introducing orphan calves on the stress responses of young orphan rhino from existing groups. The behavior and fecal glucocorticoid metabolite (fGCM) concentrations of eight orphan southern white rhinoceros (Ceratotherium simum simum) were assessed 1 month before and after the introduction of a new calf. From the 10 variables measured, only the response to humans and submissive behaviors showed significant changes. Stereotypies were not observed during the course of the study, and adrenocortical activity, monitored by means of fGCM concentrations, did not increase after the introductions, showing values within the range observed in free-ranging white rhino. However, strong individual differences were evident in most variables. Our results suggest that the introduction of white rhino calves into an existing group of young rhinos caused minimal stress in existing group members. Although these findings should be treated with caution when generalizing to other captive populations due to the small and heterogeneous sample, our findings may have management implications for rhino orphanages as well as zoos and breeding centers where non-breeding pairs are frequently maintained.

Using drones and sirens to elicit avoidance behaviour in white rhinoceros as an anti-poaching tactic.

Poaching fuelled by international trade in horn caused the deaths of over 1000 African rhinoceros (Ceratotherium simum and Diceros bicornis) per year between 2013 and 2017. Deterrents, which act to establish avoidance behaviours in animals, have the potential to aid anti-poaching efforts by moving at-risk rhinos away from areas of danger (e.g. near perimeter fences). To evaluate the efficacy of deterrents, we exposed a population of southern white rhinos (C. simum simum) to acoustic- (honeybee, siren, turtle dove), olfactory- (chilli, sunflower), and drone-based stimuli on a game reserve in South Africa. We exposed rhinos to each stimulus up to four times. Stimuli were considered effective deterrents if they repeatedly elicited avoidance behaviour (locomotion away from the deterrent). Rhinos travelled significantly further in response to the siren than to the honeybee or turtle dove stimulus, and to low-altitude drone flights than to higher altitude flights. We found the drone to be superior at manipulating rhino movement than the siren owing to its longer transmission range and capability of pursuit. By contrast, the scent stimuli were ineffective at inciting avoidance behaviour. Our findings indicate that deterrents are a prospective low-cost and in situ method to manage rhino movement in game reserves.

Gut Microbiota and Phytoestrogen-Associated Infertility in Southern White Rhinoceros.

With recent poaching of southern white rhinoceros (SWR [Ceratotherium simum simum]) reaching record levels, the need for a robust assurance population is urgent. However, the global captive SWR population is not currently self-sustaining due to the reproductive failure of captive-born females. Dietary phytoestrogens have been proposed to play a role in this phenomenon, and recent work has demonstrated a negative relationship between diet estrogenicity and fertility of captive-born female SWR. To further examine this relationship, we compared gut microbial communities, fecal phytoestrogens, and fertility of SWR to those of another rhinoceros species-the greater one-horned rhinoceros (GOHR [Rhinoceros unicornis]), which consumes a similar diet but exhibits high levels of fertility in captivity. Using 16S rRNA amplicon sequencing and mass spectrometry, we identified a species-specific fecal microbiota and three dominant fecal phytoestrogen profiles. These profiles exhibited various levels of estrogenicity when tested in an in vitro estrogen receptor activation assay for both rhinoceros species, with profiles dominated by the microbial metabolite equol stimulating the highest levels of receptor activation. Finally, we found that SWR fertility varies significantly not only with respect to phytoestrogen profile, but also with respect to the abundance of several bacterial taxa and microbially derived phytoestrogen metabolites. Taken together, these data suggest that in addition to species differences in estrogen receptor sensitivity to phytoestrogens, reproductive outcomes may be driven by the gut microbiota's transformation of dietary phytoestrogens in captive SWR females.IMPORTANCE Southern white rhinoceros (SWR) poaching has reached record levels, and captive infertility has rendered SWR assurance populations no longer self-sustaining. Previous work has identified dietary phytoestrogens as a likely cause of this problem. Here, we investigate the role of gut microbiota in this phenomenon by comparing two rhinoceros species to provide the first characterizations of gut microbiomes for any rhinoceros species. To our knowledge, our approach, combining parallel sequencing, mass spectrometry, and estrogen receptor activation assays, provides insight into the relationship between microbially mediated phytoestrogen metabolism and fertility that is novel for any vertebrate species. With this information, we plan to direct future work aimed at developing strategies to improve captive reproduction in the hope of alleviating their threat of extinction.

VAGINOSCOPIC IDENTIFICATION OF A VERTICAL VAGINAL SEPTUM IN ONE PRIMIPAROUS AND THREE NULLIPAROUS SOUTHERN WHITE RHINOCEROS (CERATOTHERIUM SIMUM SIMUM).

Vaginoscopy using a 10-mm, 30° forward viewing rigid endoscope was used to evaluate the caudal reproductive tract of four subadult southern white rhinoceros (Ceratotherium simum simum). A vertical vaginal septum was documented in all four animals, including a primiparous cow that gave birth to a stillborn calf 14 months before vaginoscopy. Vaginoscopy using a 57-cm-long, 10-mm, 30° forward viewing endoscope provides adequate visualization of the caudal reproductive track in the southern white rhinoceros, and a detailed description of the vertical vaginal septum is presented. Additionally, the presence of a vertical vaginal septum in a primiparous southern white rhinoceros suggests the presence of this anatomic structure cannot be used as a proxy of nulliparity for captive southern white rhinoceros.

Combining molecular and incomplete observational data to inform management of southern white rhinoceros (Ceratotherium simum simum)

Conservation efforts have preserved the southern white rhinoceros (SWR) in protected areas and have resulted in substantial overall growth in population size, but in small, fragmented populations in which inbreeding is an important risk. However, field observation of breeding often lacks sufficient accuracy to inform translocation strategies that are intended to increase genetic variation. The purpose of this study was to integrate microsatellite genotypes with an incomplete, field-observed pedigree to make inferences about mean kinship and basic demographic data that could be used to inform translocation programmes for SWR in a confined population in Botswana. Using this approach, we identified parents for 29 out of 45 offspring born in the reserve between 1993 and 2013 and detected eight non-breeding bulls with high mean kinship as candidates for translocation. The method also allowed inferences about demographic parameters that could influence the effectiveness of intervention strategies, such as age and timing of reproduction, and natal sex ratios. Importantly, the reproductive dominance of the bulls was not as skewed as expected after the original dominant bull was removed from the population, suggesting that closed populations can maintain multiple, simultaneously breeding males. The genetic data also confirmed that the accuracy of field-based parentage assignment was increased after implementation of an ear-notching programme. This study demonstrates the value of combining genetic information with ongoing surveillance to inform management of threatened populations, and of using mean kinship to inform metapopulation management by identifying candidates for translocation.

Species-specific drought impacts on black and white rhinoceroses.

Unrelenting poaching to feed the illegal trafficking of rhinoceros (rhino) horn remains the principle threat to the persistence of south-central black and southern white rhino that live in the Kruger National Park (Kruger), South Africa. Other global environmental change drivers, such as unpredictable climatic conditions, impose additional uncertainties on the management and persistence of these species. The drought experienced in Kruger over the 2015/2016 rainy season may have affected rhino population growth and thus added an additional population pressure to the poaching pressure already occurring. Under drought conditions, reduced grass biomass predicts increased natural deaths and a subsequent decrease in birth rate for the grazing white rhino. Such variance in natural death and birth rates for the browsing black rhino are not expected under these conditions. We evaluated these predictions using rhino population survey data from 2013 to 2017. Comparisons of natural deaths and birth rates between pre- (2013/2014 and 2014/15), during- (2015/2016) and post-drought (2016/2017) periods in Kruger showed increased natural mortality and decreased births for white rhino, but no significant changes for black rhino, supporting our predictions. As a result, despite reduced poaching rates, the total mortality rate of white rhino remains significantly higher than the birth rate. Decreased poaching, decreased natural deaths and no apparent drought effects in black rhino resulted in a lower total mortality rate than the estimated birth rate in 2017. Active biological management and traditional anti-poaching initiatives together therefore represent the most likely way to buffer the impacts of decreased population growth through climate change and wildlife crime on the persistence of rhinos.

Ovulation induction in anovulatory southern white rhinoceros (Ceratotherium simum simum) without altrenogest.

All species in the extant Rhinocerotidae family are experiencing increased threats in the wild, making captive populations essential genetic reservoirs for species survival. However, managed species face distinct challenges in captivity, resulting in populations that are not self-sustaining. Captive southern white rhinoceros (Ceratotherium simum simum) have low reproductive rates and presumed acyclicity is common among females. Although many females fail to ovulate, follicle growth may occur and ovulation can be hormonally induced. Female southern white rhino (n = 6), housed as a bachelorette group, were determined to be ovulatory (n = 1) or anovulatory (n= 5) by serial ultrasound and fecal progestagen analysis. When follicles reached pre-ovulatory size (~35mm), females (n = 4) were induced to ovulate in 11 trials with a GnRH analog (4.5mg, SucroMate™) via single intramuscular injection. Nine trials resulted in ovulation (81.8%), all between 36 and 48hours post-treatment. Ovulations were confirmed by progestagen elevation above baseline coincident with visualization of a corpus luteum (CL). Luteal phases were characterized as short (<50days) or long (≥50days). Between short and long cycles, only the number of days of progestagen above baseline was significantly different (P < 0.05), while days with visible luteal structures was not significant (P = 0.11). Both cycle types were observed following both spontaneous and induced ovulations. Furthermore, we showed that longer cycle lengths do not necessarily indicate early pregnancy loss as none of the females were bred or inseminated during the study. While anovulation is common in the southern white rhino captive population, ovulation induction can be achieved efficiently and predictably for use in conjunction with artificial insemination or to facilitate natural breeding. This information will lead to more efficient use of assisted reproductive technologies to overcome reproductive challenges in this species and to generate genetically healthy captive populations as a hedge against extinction.

98 Efficacy of commercial equine semen freezing extenders for cryopreservation of southern white rhinoceros (Ceratotherium simum simum) sperm

Once nearly extinct in the wild, the southern white rhinoceros is currently listed as near threatened by IUCN. This status is likely to change as poaching continues to escalate. To preserve the species’ current genetic diversity, cryopreserving and biobanking white rhinoceros sperm is imperative. The horse is the closest domestic relative of the rhinoceros and a useful model for the development of assisted reproductive technologies, including semen cryopreservation. Two equine semen cryopreservation protocols were compared to a common rhinoceros freezing method. Semen was collected from a single male on 3 occasions by electroejaculation. Initial semen parameters were 86% motility; speed 3.2 (scale 1-5); 89% plasma membrane integrity; and 95% intact acrosomes. Semen was extended 1:1 in INRA 96 (IMV Technologies, L’Aigle, France) before centrifugation at 400×g for 10min. Supernatant was removed and the sperm pellet was subjected to 1 of 2 treatments: resuspension in 500µL of either BotuCrio (Botupharma, Botucatu, Brazil) or Cryomax (ARS Inc., Chino, CA, USA), both containing a proprietary combination of glycerol and an amide as cryoprotectants. Following a 40-min cool at 4°C, extended semen was frozen in vials at a cooling rate of 30°C/min for 3min before LN submersion. Control semen was extended 1:1 in TEST-Y buffer without cryoprotectant and cooled for 2.5h before adding glycerol to a final concentration of 4%. Extended sperm (500µL) was frozen in vials at 12.5°C/min for 15min before LN submersion. Initial motility score (IMS;% motile×speed of progression2), plasma membrane integrity (IPL), and acrosome integrity (IAC) were recorded after extension. All vials were thawed at 37°C for 60s and the cryoprotectant was removed by centrifugation. Sperm pellets were resupended in M199+HEPES and sperm was evaluated for the characteristics described above at 37°C at 0, 30, and 60min (T0, T30, T60) post-thaw. All data are expressed as a percentage of initial (%IMS,%IPL, and%IAC) to account for the differences in sperm parameters between ejaculates. Cryopreservation protocol significantly affected%IMS at T0 (P=0.0131, Table 1). Although the differences were significant only at T0, sperm frozen in Botucrio or Cryomax tended to maintain a higher%IMS than the control freeze at all time points. However, sperm frozen in Cryomax lost a greater percentage of%IMS over time (67% from T0 to T60v. 44 and 46% for Botucrio and TEST-Y, respectively). Cryopreservation protocol did not affect%IAC or%IPL at any time point, but again Cryomax and Botucrio tended to be higher than TEST-Y. This study indicates that rhinoceros sperm may suffer less cryodamage in Botucrio or Cryomax frozen at 30°C/min than in the conventional TEST-Y frozen at 12.5°C/min. Table 1.Percent of initial motility score (IMS), plasma membrane integrity (IPL), and acrosome integrity (IAC) at 0, 30, and 60min post-thaw (T0, T30, and T60, respectively)

189 Isolation and purification of rhinoceros and horse spermatogonial stem cells

The northern white rhinoceros (NWR; Ceratotherium simum cottoni) is critically endangered. Testicular tissue (TT) of an NWR that died at 46 years was cryopreserved and stored. Spermatogonial stem cells (SSC) have been isolated from TT and, following transplantation or in vitro culture, differentiated into mature spermatozoa. These SSC have been phenotypically characterised by the expression of markers specific for SSC, and in vitro culture methods optimized to isolate, purify, and enrich populations of SSC from mixed germ cells. The reproductive age may affect the percent of SSC in TT, affecting recovery from older animals. We previously identified in TT of NWR the surface markers GFRa1 and GPR125, both of which were expressed at various stages of spermatogenesis. However, characterisation, isolation, and purification of rhinoceros SSC have not been studied. Because of the limited availability of rhinoceros TT, we used the horse as a model for rhinoceros (both species are in the same order: Perissodactyla). In this study, we (1) identified and compared the expression of markers specific for SSC (GFRa1, GPR125, PLZF) and pluripotent markers (SSEA-1, SSEA-4, OCT-4) in rhinoceros and horse mixed germ cells; (2) evaluated whether rhinoceros and horse SSC could be purified and enriched by sequential culture with collagen (2 days) and laminin (3 weeks); and (3) determined whether age may affect the percentages of SSC by comparing the expression of SSC and pluripotent markers in horse mixed germ cells at different reproductive stages: pubertal (PU=1-1.5 years) v. post-pubertal (PP=2-3 years) v. adult (AD=&amp;gt;5 years). Mixed germ cells were isolated from testes of one male of each of 3 rhinoceros species: NWR, southern white rhinoceros (SWR; Ceratotherium simum simum, 49 years), and greater one-horned rhinoceros (GOHR; Rhinoceros unicornis, 5 years), and horses at different reproductive stages (PU=2, PP=2, AD=7). The SWR and GOHR testes were refrigerated overnight, whereas NWR mixed germ cells were isolated from frozen-thawed TT. Flow cytometry analysis showed expression of SSC and pluripotent markers but not for transcription factor PLZF. The marker expression was similarly distributed between the 3 species of rhinoceros, with a larger portion of cells positive for SSEA-4 (mean%±s.e.m.: 6.3±1.1%) and smaller proportion for GDNFa1 (0.4±0.2%), GRP125 (0.1±0.1%), OCT-4 (0.4±0.2%), and SSEA-1 (0.4±0.2%; P&amp;lt;0.05). Similarly, horse expressed the markers GDNFa1 (0.9±0.3%), GRP125 (0.9±0.4%), OCT-4 (1.7±0.5%), and SSEA-1 (0.12±0.1%), but the abundance of SSEA-4 (1.8±0.8%) was less than that of rhinoceros (P&amp;lt;0.05). In rhinoceros, sequential culture enhanced the numbers of cells expressing all markers compared with that before culture, whereas in horses, the increase was observed only for cells expressing GDNFa1, GPR125, and SSEA-1. Reproductive stage did not affect the percentages of horse cells expressing germ cell and pluripotent markers. Overall, these results showed that rhinoceros SSC can be isolated from TT and expressed the same SSC markers as horses, and that differential culture enriched a population of SSC.

97 Ovulation induction in anovulatory southern white rhinoceros (Ceratotherium simum simum)

The extant Rhinocerotidae family is experiencing threats in the wild, making captive populations important genetic reservoirs for species survival. Because each species faces distinct challenges in captivity, populations are not self-sustaining. Therefore, assisted reproductive technologies (ART) such as AI will be necessary to maintain or increase captive genetic variation. Captive-born female white rhinoceros (Ceratotherium simum simum) have low reproductive rates and apparent acyclicity is a common issue. Although females fail to ovulate and progesterone remains at baseline levels, follicle growth may occur and ovulation can be induced with exogenous hormones. Female southern white rhinos (n=6) housed as a bachelorette group were determined to be ovulatory (n=1) or anovulatory (n=5) by serial ultrasound and fecal progesterone (P4; ng g−1) analysis. However, all anovulatory females grew follicles beyond preovulatory size, which then regressed. At the time of study, when follicles were preovulatory size (35.4±1.2 mm; mean±SD), anovulatory females (n=4) were induced to ovulate (n=11) with a gonadotropin releasing hormone (GnRH) agonist (4.5mg, SucroMate™; Bioniche Animal Health, Bogart, GA, USA) in a single intramuscular injection. Nine treatments resulted in ovulation (81.8%), all between 36 and 48h post-treatment, while 2 hemorrhagic anovulatory follicles (18.2%; HAF) formed, both in the same female. Ovulations were confirmed by disappearance of the follicle by 48h and P4 elevation above baseline was coincident with corpus luteum (CL) and HAF formation. All resulting luteal structures were included in analysis. Follicle growth was permitted to proceed without GnRH treatment between inductions (n=6); dominant follicles grew beyond preovulatory size (43.8±6.1mm) followed by regression and growth of another preovulatory follicle that was subjected to GnRH treatment. Data were analyzed by R studio (ver. 1.1.383). Luteal phases were characterized as “short” (&amp;lt;50 days) or “long” (&amp;gt;50 days). Only P4 above baseline (days) was significantly different (P&amp;lt;0.05) between long and short cycles. Other cycle parameters, such as CL visibility (days), time from ovulation to P4 above baseline (days), maximum P4 (ng g−1), and maximum luteal size (mm), were not significantly different between cycle types. Both cycle types were observed following both spontaneous (short, n=5; long, n=3) and induced ovulations (short, n=8; long, n=3). These data provide additional insight into the differences between long and short cycles and that GnRH is a reliable and effective method to overcome anovulation in SWR. We also prove that long cycle lengths are not necessarily indicative of early pregnancy loss, as has been previously suggested. Taken together, this information can enhance captive breeding efforts and the genetic diversity of the ex situ, SWR insurance population.

Contrasting evolutionary history, anthropogenic declines and genetic contact in the northern and southern white rhinoceros (Ceratotherium simum).

The white rhinoceros (Ceratotherium simum) has a discontinuous African distribution, which is limited by the extent of sub-Saharan grasslands. The southern population (SWR) declined to its lowest number around the turn of the nineteenth century, but recovered to become the world's most numerous rhinoceros. In contrast, the northern population (NWR) was common during much of the twentieth century, declining rapidly since the 1970s, and now only two post-reproductive individuals remain. Despite this species's conservation status, it lacks a genetic assessment of its demographic history. We therefore sampled 232 individuals from extant and museum sources and analysed ten microsatellite loci and the mtDNA control region. Both marker types reliably partitioned the species into SWR and NWR, with moderate nuclear genetic diversity and only three mtDNA haplotypes for the species, including historical samples. We detected ancient interglacial demographic declines in both populations. Both populations may also have been affected by recent declines associated with the colonial expansion for the SWR, and with the much earlier Bantu migrations for the NWR. Finally, we detected post-divergence secondary contact between NWR and SWR, possibly occurring as recently as the last glacial maximum. These results suggest the species was subjected to regular periods of fragmentation and low genetic diversity, which may have been replenished upon secondary contact during glacial periods. The species's current situation thus reflects prehistoric declines that were exacerbated by anthropogenic pressure associated with the rise of late Holocene technological advancement in Africa. Importantly, secondary contact suggests a potentially positive outcome for a hybrid rescue conservation strategy, although further genome-wide data are desirable to corroborate these results.

Serum protein electrophoresis in healthy and injured southern white rhinoceros (Ceratotherium simum simum).

Investigation of globulin fractions by serum protein electrophoresis (SPE) is the first step towards evaluation of the proteome in the southern white rhinoceros (Ceratotherium simum simum). Furthermore, identification of changes in globulins in animals with poaching and other injuries can guide discovery of potentially useful biomarkers of inflammation. The aim of this study was to develop reference intervals for agarose gel SPE in healthy white rhinoceros and to compare these serum protein electrophoresis results to those from animals with tissue trauma. Reference intervals for total serum protein and agarose gel electrophoretic albumin and globulin fractions were generated using serum samples from 49 healthy free-ranging adult white rhinoceros. A standardised gating system together with identification of specific proteins by mass spectrometry aided in fraction identification. Six globulin fractions were identified: α1a, α1b, α2, β1, β2 and γ. Reference intervals were generated for total serum protein (76–111 g/L), albumin (10–27 g/L) and globulin fractions (α1a: 1.6–3.2 g/L; α1b: 1.7–3.6 g/L; α2: 16.1–26.6 g/L; β1: 6.6–18.2 g/L; β2: 11.8–30.4 g/L; γ: 10.4–23.1 g/L; albumin: globulin ratio: 0.12–0.39). Results were compared to those from 30 animals with various degrees and chronicities of tissue trauma. Wounded animals had lower concentrations of total serum protein, albumin, total globulin, α and β1 globulins, lower percentages of α2 and β1 globulins, and higher percentages of β2 and γ globulins. These protein changes are similar to those seen in human patients with wounds rather than classic acute phase or chronic inflammatory responses.

Embryos and embryonic stem cells from the white rhinoceros

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.

First insights into the vocal repertoire of infant and juvenile Southern white rhinoceros.

Describing vocal repertoires represents an essential step towards gaining an overview about the complexity of acoustic communication in a given species. The analysis of infant vocalisations is essential for understanding the development and usage of species-specific vocalisations, but is often underrepresented, especially in species with long inter-birth intervals such as the white rhinoceros. Thus, this study aimed for the first time to characterise the infant and juvenile vocal repertoire of the Southern white rhinoceros and to relate these findings to the adult vocal repertoire. The behaviour of seven mother-reared white rhinoceros calves (two males, five females) and one hand-reared calf (male), ranging from one month to four years, was simultaneously audio and video-taped at three zoos. Normally reared infants and juveniles uttered four discriminable call types (Whine, Snort, Threat, and Pant) that were produced in different behavioural contexts. All call types were also uttered by the hand-reared calf. Call rates of Whines, but not of the other call types, decreased with age. These findings provide the first evidence that infant and juvenile rhinoceros utter specific call types in distinct contexts, even if they grow up with limited social interaction with conspecifics. By comparing our findings with the current literature on vocalisations of adult white rhinoceros and other solitary rhinoceros species, we discuss to which extent differences in the social lifestyle across species affect acoustic communication in mammals.