Early life experience and sex influence acoustic repertoire use in wild-born, but hand-reared, captive cheetahs (Acinonyx jubatus).

Early deprivation of adult influence is known to have long-lasting effects on social abilities, notably communication skills, as adults play a key role in guiding and regulating the behavior of youngsters, including acoustic repertoire use in species in which vocal production is not learned. Cheetahs grow up alongside their mother for 18 months, thus maternal influences on the development of social skills are likely to be crucial. Here, we investigated the impact of early maternal deprivation on vocal production and use in 12 wild-born cheetahs, rescued and subsequently hand-reared either at an early (less than 2 months) or a later stage of development. We could distinguish 16 sound types, produced mostly singly but sometimes in repeated or multitype sound sequences. The repertoire of these cheetahs did not differ fundamentally from that described in other studies on adult cheetahs, but statistical analyses revealed a concurrent effect of both early experience and sex on repertoire use. More specifically, early-reared males were characterized by a high proportion of Purr, Meow, and Stutter; early-reared females Mew, Growl, Hoot, Sneeze, and Hiss; late-reared males Meow, Mew, Growl, and Howl; and late-reared females mostly Meow. Our study demonstrates therefore the long-term effects of maternal deprivation on communication skills in a limited-vocal learner and its differential effect according to sex, in line with known social differences and potential differential maternal investment. More generally, it emphasizes the critical importance to consider the past history of the subjects (e.g., captive/wild-born, mother/hand-reared, early/late-mother-deprived, etc.) when studying social behavior, notably acoustic communication.

Effect of a Probiotic Mixture in Captive Cheetahs (Acinonyx Jubatus) with Gastrointestinal Symptoms-A Pilot Study.

Cheetahs (Acinonyx jubatus) are classified as "vulnerable" species due to the low numbers persisting in the wild. Gastrointestinal diseases are very common in this species when they are kept in captivity, in particular gastritis. Clinical signs are predominantly characterized by vomiting, diarrhea, weight loss and anorexia. In this study, we evaluated the efficacy of a multi-strain probiotic in two groups of cheetahs: Group A (n = 4)-rescued cheetahs housed at the Cheetah Conservation Fund (Otjiwarongo, Namibia); Group B (n = 9)-captive cheetahs housed in Italian zoos. Animals showed gastrointestinal signs of different severity, and were positive for Helicobacter spp., detected by PCR in stool samples. Two sachets of probiotic formulation were administered to all cheetahs once a day for 21 consecutive days. Clinical conditions (appetite loss, vomiting, stool consistency and Body Condition Score) before (T0) and after 21 days of probiotic administration (T1) were then compared using a simplified Feline Chronic Enteropathy Activity Index (FCEAI) score. A slight but not significant improvement in the scores was observed in Group A, which had mild intestinal symptoms, while a significant decrease in vomiting and stool consistency (**p < 0.01) scores was observed in Group B, which had more pronounced symptoms. Results suggest that high concentrations of live probiotics can be of help in managing gastrointestinal signs in cheetahs.

Categorising cheetah behaviour using tri-axial accelerometer data loggers: a comparison of model resolution and data logger performance.

BACKGROUND: Extinction is one of the greatest threats to the living world, endangering organisms globally, advancing conservation to the forefront of species research. To maximise the efficacy of conservation efforts, understanding the ecological, physiological, and behavioural requirements of vulnerable species is vital. Technological advances, particularly in remote sensing, enable researchers to continuously monitor movement and behaviours of multiple individuals simultaneously with minimal human intervention. Cheetahs, Acinonyx jubatus, constitute a "vulnerable" species for which only coarse behaviours have been elucidated. The aims of this study were to use animal-attached accelerometers to (1) determine fine-scale behaviours in cheetahs, (2) compare the performances of different devices in behaviour categorisation, and (3) provide a behavioural categorisation framework. METHODS: Two different accelerometer devices (CEFAS, frequency: 30 Hz, maximum capacity: ~ 2 g; GCDC, frequency: 50 Hz, maximum capacity: ~ 8 g) were mounted onto collars, fitted to five individual captive cheetahs. The cheetahs chased a lure around a track, during which time their behaviours were videoed. Accelerometer data were temporally aligned with corresponding video footage and labelled with one of 17 behaviours. Six separate random forest models were run (three per device type) to determine the categorisation accuracy for behaviours at a fine, medium, and coarse resolution. RESULTS: Fine- and medium-scale models had an overall categorisation accuracy of 83-86% and 84-88% respectively. Non-locomotory behaviours were best categorised on both loggers with GCDC outperforming CEFAS devices overall. On a coarse scale, both devices performed well when categorising activity (86.9% (CEFAS) vs. 89.3% (GCDC) accuracy) and inactivity (95.5% (CEFAS) vs. 95.0% (GCDC) accuracy). This study defined cheetah behaviour beyond three categories and accurately determined stalking behaviours by remote sensing. We also show that device specification and configuration may affect categorisation accuracy, so we recommend deploying several different loggers simultaneously on the same individual. CONCLUSION: The results of this study will be useful in determining wild cheetah behaviour. The methods used here allowed broad-scale (active/inactive) as well as fine-scale (e.g. stalking) behaviours to be categorised remotely. These findings and methodological approaches will be useful in monitoring the behaviour of wild cheetahs and other species of conservation interest.

Female Southern White Rhinoceros Can Select Mates to Avoid Inbreeding.

Current management models for many endangered species focus primarily on demographic recovery, often ignoring their intrinsic ecological requirements. Across the protected area network of southern Africa, most southern white rhinoceros are managed in populations of less than 50 individuals, experiencing restricted dispersal opportunities, and limited breeding male numbers due to their exclusive home range requirements. In the absence of information on the breeding structure of these populations, poor management decisions may require females to either forego a breeding opportunity or select to inbreed with close relatives. Here, we use a combination of social pedigree data together with genetic analyses to reconstruct the parentage of all 28 offspring produced in a 5-year period in a managed free-ranging southern white rhinoceros population. During this period, all breeding females (founders and first-generation daughters) had access to both a founder male (father to most of the daughters) and two recently introduced inexperienced males. We report that while founder females were more likely to breed with the founder male, their daughters, in contrast, were more likely to breed with the introduced males, thus avoiding inbreeding. However, we also found evidence of father-daughter inbreeding in this population, and contend that in the absence of choice, rather than forego a breeding opportunity, female white rhinoceros will inbreed with their fathers. We argue that to effectively conserve the southern white rhinoceros, managers need to understand the breeding structure of these small populations, particularly in terms of parentage and kinship.

Regional variation in the cheetah (Acinonyx jubatus) revisited: Morphology of wild and captive populations.

The cheetah (Acinonyx jubatus) is listed as a vulnerable species by the International union for the conservation of nature (IUCN), including two critically endangered subspecies, the Saharan cheetah, and the Iranian cheetah, so it is imperative that we understand variation in cheetah morphology to make good decisions regarding the conservation of this species. Here, we aim to determine whether northeastern African cheetahs have smaller body sizes than southern African cheetahs. This study also adds to our knowledge of cheetah morphology from two cheetah populations that do not yet have comprehensive published data: Kenya, and northeastern Africa, including captive individuals. We calculated means and standard deviations on cranial and body measurements of live or in few cases, freshly dead, cheetahs from the aforementioned populations, plus previously published data on Namibian and Botswanan cheetahs and compared them to one another using multivariate analysis of variance. Results show that northeastern African cheetahs have smaller body sizes than southern and eastern African populations. We also found that captive cheetahs retain the morphological characteristics of their ancestral population- captive cheetahs from southern Africa have similar body sizes to wild southern African cheetahs and larger body sizes than captives from northeastern Africa. Other analyses regarding cheetah growth agree with previous studies on Namibian and Botswanan cheetah populations rates. As such, this study can serve as a baseline for the care of captive cheetah populations to maintain healthy weights and body proportions.

Age-associated and deslorelin-induced declines in serum anti-Müllerian hormone concentrations in female cheetahs, Acinonyx jubatus.

Anti-Müllerian hormone (AMH) is widely used in human medicine to non-invasively estimate the size of the ovarian follicle reserve and to predict the ovarian response to gonadotropin stimulation in the context of assisted reproductive technologies (e.g., IVF). These applications of AMH testing have recently expanded to non-human mammals, with production animals, such as cows, goats and sheep being the primary focus of AMH research. However, few investigations have involved exotic species, and in particular carnivores. In this study, we measured AMH concentrations (0.078-3.078ng/mL) in archived serum samples that had been collected from 36 adult female cheetahs across their reproductive lifespan (2-15years of age). Similar to other mammals, AMH concentration in cheetahs declined with age, and its variability among females of the same age was considerable. The rates at which AMH declined over time in individual cheetahs were also highly variable. Five cheetahs had been contracepted with the long-acting GnRH agonist deslorelin for 6-18months prior to sample collection, and their AMH concentrations were relatively low compared to untreated females. In this first study of AMH in an exotic carnivore, the findings demonstrate that the age-associated decline in AMH is highly variable and that deslorelin appears to suppress AMH concentration in serum. Owing to the increased use of assisted reproductive technologies in ex situ populations of threatened and endangered species, such as cheetahs, the present study's findings will need to be taken into consideration if AMH is to be used successfully to optimize breeding management decisions in exotic species.

The global decline of cheetah Acinonyx jubatus and what it means for conservation.

Establishing and maintaining protected areas (PAs) are key tools for biodiversity conservation. However, this approach is insufficient for many species, particularly those that are wide-ranging and sparse. The cheetah Acinonyx jubatus exemplifies such a species and faces extreme challenges to its survival. Here, we show that the global population is estimated at ∼7,100 individuals and confined to 9% of its historical distributional range. However, the majority of current range (77%) occurs outside of PAs, where the species faces multiple threats. Scenario modeling shows that, where growth rates are suppressed outside PAs, extinction rates increase rapidly as the proportion of population protected declines. Sensitivity analysis shows that growth rates within PAs have to be high if they are to compensate for declines outside. Susceptibility of cheetah to rapid decline is evidenced by recent rapid contraction in range, supporting an uplisting of the International Union for the Conservation of Nature (IUCN) Red List threat assessment to endangered. Our results are applicable to other protection-reliant species, which may be subject to systematic underestimation of threat when there is insufficient information outside PAs. Ultimately, conserving many of these species necessitates a paradigm shift in conservation toward a holistic approach that incentivizes protection and promotes sustainable human-wildlife coexistence across large multiple-use landscapes.

Serum Amyloid A Protein Concentration in Blood is Influenced by Genetic Differences in the Cheetah (Acinonyx jubatus).

Systemic amyloid A (AA) amyloidosis is a major cause of morbidity and mortality among captive cheetahs. The self-aggregating AA protein responsible for this disease is a byproduct of serum amyloid A (SAA) protein degradation. Transcriptional induction of the SAA1 gene is dependent on both C/EBPß and NF-κB cis-acting elements within the promoter region. In cheetahs, 2 alleles exist for a single guanine nucleotide deletion in the putative NF-κB binding site. In this study, a novel genotyping assay was developed to screen for the alleles. The results show that the SAA1A (-97delG) allele is associated with decreased SAA protein concentrations in the serum of captive cheetahs (n = 58), suggesting genetic differences at this locus may be affecting AA amyloidosis prevalence. However, there was no significant difference in the frequency of the SAA1A (-97delG) allele between individuals confirmed AA amyloidosis positive versus AA amyloidosis negative at the time of necropsy (n = 48). Thus, even though there is evidence that having more copies of the SAA1A (-97delG) allele results in a potentially protective decrease in serum concentrations of SAA protein in captive cheetahs, genotype is not associated with this disease within the North American population. These results suggest that other factors are playing a more significant role in the pathogenesis of AA amyloidosis among captive cheetahs.

Genomic legacy of the African cheetah, Acinonyx jubatus.

BACKGROUND: Patterns of genetic and genomic variance are informative in inferring population history for human, model species and endangered populations. RESULTS: Here the genome sequence of wild-born African cheetahs reveals extreme genomic depletion in SNV incidence, SNV density, SNVs of coding genes, MHC class I and II genes, and mitochondrial DNA SNVs. Cheetah genomes are on average 95 % homozygous compared to the genomes of the outbred domestic cat (24.08 % homozygous), Virunga Mountain Gorilla (78.12 %), inbred Abyssinian cat (62.63 %), Tasmanian devil, domestic dog and other mammalian species. Demographic estimators impute two ancestral population bottlenecks: one >100,000 years ago coincident with cheetah migrations out of the Americas and into Eurasia and Africa, and a second 11,084-12,589 years ago in Africa coincident with late Pleistocene large mammal extinctions. MHC class I gene loss and dramatic reduction in functional diversity of MHC genes would explain why cheetahs ablate skin graft rejection among unrelated individuals. Significant excess of non-synonymous mutations in AKAP4 (p<0.02), a gene mediating spermatozoon development, indicates cheetah fixation of five function-damaging amino acid variants distinct from AKAP4 homologues of other Felidae or mammals; AKAP4 dysfunction may cause the cheetah's extremely high (>80 %) pleiomorphic sperm. CONCLUSIONS: The study provides an unprecedented genomic perspective for the rare cheetah, with potential relevance to the species' natural history, physiological adaptations and unique reproductive disposition.

TREATMENT OF CHRONIC HERPESVIRAL DERMATITIS IN A CAPTIVE CHEETAH (ACINONYX JUBATUS) IN NAMIBIA.

A 9-yr-old male cheetah (Acinonyx jubatus) housed at the Cheetah Conservation Fund in Namibia developed cutaneous lesions consisting of alopecia, erythema, ulceration, and crusting on the left fore and hind limbs. Histopathology of skin biopsies in conjunction with indirect fluorescent antibody and polymerase chain reaction testing confirmed a diagnosis of feline herpesvirus-1 dermatitis; microbial culture indicated secondary bacterial infection. Therapy included targeted systemic antimicrobial and antiviral treatment, topical medications, and repeated cryotherapy. Lesions exhibited varying degrees of clinical improvement but, overall, progressed in extent, size, and severity during the subsequent 2.5 yr of intense treatment. The cheetah was ultimately euthanized due to a guarded prognosis and concerns about poor quality of life. Potential factors initiating or contributing (or both) to the severity and nonhealing nature of the cutaneous lesions include chronic unidentified stress, altered immune system function, and other environmental influences.

Single-Incision Laparoscopic Sterilization of the Cheetah (Acinonyx jubatus).

OBJECTIVE: To describe laparoscopic ovariectomy and salpingectomy in the cheetah (Acinonyx jubatus) using single-incision laparoscopic surgery (SILS). STUDY DESIGN: Prospective cohort. ANIMALS: Female cheetahs (Acinonyx jubatus) (n = 21). METHODS: Cheetahs were randomly divided to receive either ovariectomy (n = 11) or salpingectomy (n = 10). The use and complications of a SILS port was evaluated in all of cheetahs. Surgery duration and insufflation volumes of carbon dioxide (CO2 ) were recorded and compared across procedures. RESULTS: Laparoscopic ovariectomy and salpingectomy were performed without complications using a SILS port. The poorly-developed mesosalpinx and ovarian bursa facilitated access to the uterine tube for salpingectomy in the cheetah. The median surgery duration for ovariectomy was 24 minutes (interquartile range 3) and for salpingectomy was 19.5 minutes (interquartile range 3) (P = .005). The median volume of CO2 used for ovariectomy was 11.25 L (interquartile range 3.08) and for salpingectomy was 4.90 L (interquartile range 2.52), (P = .001) CONCLUSIONS: Laparoscopic ovariectomy and salpingectomy can be performed in the cheetah using SILS without perioperative complications. Salpingectomy is faster than ovariectomy and requires less total CO2 for insufflation.

The tiger genome and comparative analysis with lion and snow leopard genomes.

Tigers and their close relatives (Panthera) are some of the world's most endangered species. Here we report the de novo assembly of an Amur tiger whole-genome sequence as well as the genomic sequences of a white Bengal tiger, African lion, white African lion and snow leopard. Through comparative genetic analyses of these genomes, we find genetic signatures that may reflect molecular adaptations consistent with the big cats' hypercarnivorous diet and muscle strength. We report a snow leopard-specific genetic determinant in EGLN1 (Met39>Lys39), which is likely to be associated with adaptation to high altitude. We also detect a TYR260G>A mutation likely responsible for the white lion coat colour. Tiger and cat genomes show similar repeat composition and an appreciably conserved synteny. Genomic data from the five big cats provide an invaluable resource for resolving easily identifiable phenotypes evident in very close, but distinct, species.

Contrasting historical and recent gene flow among African buffalo herds in the Caprivi Strip of Namibia.

Population genetic structure is often used to infer population connectivity, but genetic structure may largely reflect historical rather than recent processes. We contrasted genetic structure with recent gene-flow estimates among 6 herds of African buffalo (Syncerus caffer) in the Caprivi Strip, Namibia, using 134 individuals genotyped at 10 microsatellite loci. We tested whether historical and recent gene flows were influenced by distance, potential barriers (rivers), or landscape resistance (distance from water). We also tested at what scales individuals were more related than expected by chance. Genetic structure across the Caprivi Strip was weak, indicating that historically, gene flow was strong and was not affected by distance, barriers, or landscape resistance. Our analysis of simulated data suggested that genetic structure would be unlikely to reflect human disturbances in the last 10-20 generations (75-150 years) because of slow predicted rates of genetic drift, but recent gene-flow estimates would be affected. Recent gene-flow estimates were not consistently affected by rivers or distance to water but showed that isolation by distance appears to be developing. Average relatedness estimates among individuals exceeded random expectations only within herds. We conclude that historically, African buffalo moved freely throughout the Caprivi Strip, whereas recent gene flow has been more restricted. Our findings support efforts to maintain the connectivity of buffalo herds across this region and demonstrate the utility of contrasting genetic inferences from different time scales.

Specifying and sustaining pigmentation patterns in domestic and wild cats.

Color markings among felid species display both a remarkable diversity and a common underlying periodicity. A similar range of patterns in domestic cats suggests a conserved mechanism whose appearance can be altered by selection. We identified the gene responsible for tabby pattern variation in domestic cats as Transmembrane aminopeptidase Q (Taqpep), which encodes a membrane-bound metalloprotease. Analyzing 31 other felid species, we identified Taqpep as the cause of the rare king cheetah phenotype, in which spots coalesce into blotches and stripes. Histologic, genomic expression, and transgenic mouse studies indicate that paracrine expression of Endothelin3 (Edn3) coordinates localized color differences. We propose a two-stage model in which Taqpep helps to establish a periodic pre-pattern during skin development that is later implemented by differential expression of Edn3.

Diagnosis-based treatment of helminths in captive and wild cheetahs (Acinonyx jubatus).

This study was designed to identify endoparasites in captive cheetahs (Acinonyx jubatus) living in a seminatural captive environment in north-central Namibia. Results were used to assess the need for anthelmintic treatment and for the selection of an appropriate drug. The study assessed fecal parasite excretion qualitatively and quantitatively using a fecal flotation method during the winter of 2009. Four different species of parasites (two nematodes and two coccidias) were identified. Parasite excretion rates were found to be significantly lower than that of wild cheetahs living in the same area. Samples of the wild cheetahs were obtained at the time of anesthesia or were attributed to the wild individuals using genetic profiling. Captive cheetahs were dewormed with fenbendazole, whereas wild cheetahs were treated using ivermectin. Efficacy of these treatments was demonstrated at the end of the study.

Digital gene expression for non-model organisms.

Next-generation sequencing technologies offer new approaches for global measurements of gene expression but are mostly limited to organisms for which a high-quality assembled reference genome sequence is available. We present a method for gene expression profiling called EDGE, or EcoP15I-tagged Digital Gene Expression, based on ultra-high-throughput sequencing of 27-bp cDNA fragments that uniquely tag the corresponding gene, thereby allowing direct quantification of transcript abundance. We show that EDGE is capable of assaying for expression in >99% of genes in the genome and achieves saturation after 6-8 million reads. EDGE exhibits very little technical noise, reveals a large (10(6)) dynamic range of gene expression, and is particularly suited for quantification of transcript abundance in non-model organisms where a high-quality annotated genome is not available. In a direct comparison with RNA-seq, both methods provide similar assessments of relative transcript abundance, but EDGE does better at detecting gene expression differences for poorly expressed genes and does not exhibit transcript length bias. Applying EDGE to laboratory mice, we show that a loss-of-function mutation in the melanocortin 1 receptor (Mc1r), recognized as a Mendelian determinant of yellow hair color in many different mammals, also causes reduced expression of genes involved in the interferon response. To illustrate the application of EDGE to a non-model organism, we examine skin biopsy samples from a cheetah (Acinonyx jubatus) and identify genes likely to control differences in the color of spotted versus non-spotted regions.

A domestic cat X chromosome linkage map and the sex-linked orange locus: mapping of orange, multiple origins and epistasis over nonagouti.

A comprehensive genetic linkage map of the domestic cat X chromosome was generated with the goal of localizing the genomic position of the classic X-linked orange (O) locus. Microsatellite markers with an average spacing of 3 Mb were selected from sequence traces of the cat 1.9x whole genome sequence (WGS), including the pseudoautosomal region 1 (PAR1). Extreme variation in recombination rates (centimorgans per megabase) was observed along the X chromosome, ranging from a virtual absence of recombination events in a region estimated to be >30 Mb to recombination frequencies of 15.7 cM/Mb in a segment estimated to be <0.3 Mb. This detailed linkage map was applied to position the X-linked orange gene, placing this locus on the q arm of the X chromosome, as opposed to a previously reported location on the p arm. Fine mapping placed the locus between markers at positions 106 and 116.8 Mb in the current 1.9x-coverage sequence assembly of the cat genome. Haplotype analysis revealed potential recombination events that could reduce the size of the candidate region to 3.5 Mb and suggested multiple origins for the orange phenotype in the domestic cat. Furthermore, epistasis of orange over nonagouti was demonstrated at the genetic level.