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.

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.

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.

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.

Core profile of volatile organic compounds related to growth of Mycobacterium avium subspecies paratuberculosis - A comparative extract of three independent studies.

Analysis of volatile organic compounds (VOC) derived from bacterial metabolism during cultivation is considered an innovative approach to accelerate in vitro detection of slowly growing bacteria. This applies also to Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of paratuberculosis, a debilitating chronic enteritis of ruminants. Diagnostic application demands robust VOC profiles that are reproducible under variable culture conditions. In this study, the VOC patterns of pure bacterial cultures, derived from three independent in vitro studies performed previously, were comparatively analyzed. Different statistical analyses were linked to extract the VOC core profile of MAP and to prove its robustness, which is a prerequisite for further development towards diagnostic application. Despite methodical variability of bacterial cultivation and sample pre-extraction, a common profile of 28 VOCs indicating cultural growth of MAP was defined. The substances cover six chemical classes. Four of the substances decreased above MAP and 24 increased. Random forest classification was applied to rank the compounds relative to their importance and for classification of MAP versus control samples. Already the top-ranked compound alone achieved high discrimination (AUC 0.85), which was further increased utilizing all compounds of the VOC core profile of MAP (AUC 0.91). The discriminatory power of this tool for the characterization of natural diagnostic samples, in particular its diagnostic specificity for MAP, has to be confirmed in future studies.

Crowd monitoring in dairy cattle-real-time VOC profiling by direct mass spectrometry.

Volatile organic compounds (VOCs) emitted from breath, faeces or skin may reflect physiological and pathological processes in vivo. Our setup employs real-time proton-transfer-reaction time-of-flight mass spectrometry (PTR-TOF-MS) to explore VOC emissions of dairy cows in stable air under field conditions. Within one herd of 596 cows, seven groups (8-117 cows per group) were assessed. Groups differed in milk yield and health status (two contained cows with paratuberculosis, a chronic intestinal infection). Each group arrived one after another in the area of air measurement in front of the milking parlour. A customised PTR-TOF-MS system with a 6 m long and heated transfer line, was used for measuring VOCs continuously for 7 h, 1.5 m above the cows. Three consecutive time periods were investigated. Twenty-seven VOCs increased while the animals were gathering in the waiting area, and decreased when the animals entered the milking parlour. Linear correlations between the number of animals present and VOC concentrations were found for (C4H6)H+ and (C3H6O)H+. A relatively high concentration of acetone above the cows that had recently given birth to a calf might be related to increased fat turnover due to calving and different nutrition. Changes in VOC emissions were related to the presence of animals with paratuberculosis, to different average milk yields per group and to the time of the day (morning versus noon milking time). We found that VOC monitoring of stable air may provide additional immediate information on an animal's metabolic or health status and foster novel applications in the field of breath research.

Evaluation of needle trap micro-extraction and solid-phase micro-extraction: Obtaining comprehensive information on volatile emissions from in vitro cultures.

Volatile organic compounds (VOCs) emitted from in vitro cultures may reveal information on species and metabolism. Owing to low nmol L-1 concentration ranges, pre-concentration techniques are required for gas chromatography-mass spectrometry (GC-MS) based analyses. This study was intended to compare the efficiency of established micro-extraction techniques - solid-phase micro-extraction (SPME) and needle-trap micro-extraction (NTME) - for the analysis of complex VOC patterns. For SPME, a 75 µm Carboxen®/polydimethylsiloxane fiber was used. The NTME needle was packed with divinylbenzene, Carbopack X and Carboxen 1000. The headspace was sampled bi-directionally. Seventy-two VOCs were calibrated by reference standard mixtures in the range of 0.041-62.24 nmol L-1 by means of GC-MS. Both pre-concentration methods were applied to profile VOCs from cultures of Mycobacterium avium ssp. paratuberculosis. Limits of detection ranged from 0.004 to 3.93 nmol L-1 (median = 0.030 nmol L-1 ) for NTME and from 0.001 to 5.684 nmol L-1 (median = 0.043 nmol L-1 ) for SPME. NTME showed advantages in assessing polar compounds such as alcohols. SPME showed advantages in reproducibility but disadvantages in sensitivity for N-containing compounds. Micro-extraction techniques such as SPME and NTME are well suited for trace VOC profiling over cultures if the limitations of each technique is taken into account.

Continuous real-time breath analysis in ruminants: effect of eructation on exhaled VOC profiles.

BACKGROUND: The analysis of volatile organic compounds (VOCs) in breath allows non-invasive investigations of diseases. Animal studies are conducted as a model to perform research of VOCs and their relation to diseases. In large animal models ruminants were often used as experimental targets. The effect of their physiological eructation on VOC exhalation has not been examined yet and is the objective of this study. METHODS: Continuous breath profiles of two young cattle, four adult goats and four adult sheep were measured through a mask, covering mouth and nose, in real-time (200 ms) by means of proton transfer reaction time of flight mass spectrometry. Each animal was analysed twelve times for 3 consecutive minutes. RESULTS: Real-time monitoring yielded a distinction of different episodes in the breath profiles of ruminants. An algorithm to separate eructation episodes and alveolar breath was established. In the first exhalation after eructation at least 19 VOC concentrations increased (up to 36-fold) and went back to initial levels in subsequent exhalations in all investigated ruminants. Decay of concentrations was substance specific. In goats, less VOCs were affected by the eructation compared to cattle and sheep. Breath profiles without exclusion of eructation episodes showed higher variations and median values than profiles where eructation episodes were excluded. CONCLUSION: Real-time breath analysis of ruminants enables the discrimination and characterisation of alveolar breath and eructation episodes. This leads to a better understanding of variation in breath data and possible origins of VOCs: breath or digestion related. To avoid impairment of breath gas results and to gain further information on bacterial products from the rumen, eructation and alveolar breath data should be analysed separately.

Comparative analysis of volatile organic compounds for the classification and identification of mycobacterial species.

BACKGROUND: Species of Mycobacteriaceae cause serious zoonotic diseases in mammals, for example tuberculosis in humans, dogs, parrots, and elephants (caused by Mycobacterium tuberculosis) and in ruminants and humans (caused by M. bovis and M. caprae). Pulmonary diseases, lymphadenitis, skin diseases, and disseminated diseases can be caused by non-tuberculous mycobacteria (NTM). Diagnosis and differentiation among Mycobacterium species are currently done by culture isolation. The established diagnostic protocols comprise several steps that allow species identification. Detecting volatile organic compounds (VOCs) above bacterial cultures is a promising approach towards accelerating species identification via culture isolation. The aims of this project were to analyse VOCs in the headspace above 13 different species of mycobacteria, to define VOC profiles that are unique for each species, and to compile a set of substances that indicate the presence of growing mycobacteria in general. MATERIALS & METHODS: VOCs were measured in the headspace above 17 different mycobacterial strains, all cultivated on Herrold's Egg Yolk Medium and above pure media slants that served as controls. For pre-concentration of VOCs, needle-trap micro-extraction was employed. Samples were subsequently analysed using gas chromatography-mass spectrometry. All volatiles were identified and calibrated by analysing pure reference substances. RESULTS: More than 130 VOCs were detected in headspace above mycobacteria-inoculated and control slants. Results confirmed significant VOC emissions above all mycobacterial species that had grown well. Concentration changes were measurable in vials with visually assessed bacterial growth and vials without apparent growth. VOCs above mycobacterial cultures could be grouped into substances that were either higher or equally concentrated, lower or equally concentrated, or both as those above control slants. Hence, we were able to identify 17 substances as potential biomarkers of the presence of growing mycobacteria in general. CONCLUSIONS: This study revealed species-specific VOC profiles for eleven species of mycobacteria that showed visually apparent bacterial growth at the time point of analysis.

Strategies for the identification of disease-related patterns of volatile organic compounds: prediction of paratuberculosis in an animal model using random forests.

Modern statistical methods which were developed for pattern recognition are increasingly being used for data analysis in studies on emissions of volatile organic compounds (VOCs). With the detection of disease-related VOC profiles, novel non-invasive diagnostic tools could be developed for clinical applications. However, it is important to bear in mind that not all statistical methods are equally suitable for the investigation of VOC profiles. In particular, univariate methods are not able to discover VOC patterns as they consider each compound separately. The present study demonstrates this fact in practice. Using VOC samples from a controlled animal study on paratuberculosis, the random forest classification method was applied for pattern recognition and disease prediction. This strategy was compared with a prediction approach based on single compounds. Both methods were framed within a cross-validation procedure. A comparison of both strategies based on these VOC data reveals that random forests achieves higher sensitivities and specificities than predictions based on single compounds. Therefore, it will most likely be more fruitful to further investigate VOC patterns instead of single biomarkers for paratuberculosis. All methods used are thoroughly explained to aid the transfer to other data analyses.

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.

Effects of biological and methodological factors on volatile organic compound patterns during cultural growth of Mycobacterium avium ssp. paratuberculosis.

Mycobacterium avium ssp. paratuberculosis (MAP) causes chronic granulomatous enteritis in ruminants. Bacterial growth is still the diagnostic 'gold standard', but is very time consuming. MAP-specific volatile organic compounds (VOCs) above media could accelerate cultural diagnosis. The aim of this project was to assess the kinetics of a VOC profile linked to the growth of MAP in vitro. The following sources of variability were taken into account: five different culture media, three different MAP strains, inoculation with different bacterial counts, and different periods of incubation. Needle-trap microextraction was employed for pre-concentration of VOCs, and gas chromatography-mass spectrometry for subsequent analysis. All volatiles were identified and calibrated by analysing pure references at different concentration levels. More than 100 VOCs were measured in headspaces above MAP-inoculated and control slants. Results confirmed different VOC profiles above different culture media. Emissions could be assigned to either egg-containing media or synthetic ingredients. 43 VOCs were identified as potential biomarkers of MAP growth on Herrold's Egg Yolk Medium without significant differences between the tree MAP strains. Substances belonged to the classes of alcohols, aldehydes, esters, ketones, aliphatic and aromatic hydrocarbons. With increasing bacterial density the VOC concentrations above MAP expressed different patterns: the majority of substances increased (although a few decreased after reaching a peak), but nine VOCs clearly decreased. Data support the hypotheses that (i) bacteria emit different metabolites on different culture media; (ii) different MAP strains show uniform VOC patterns; and (iii) cultural diagnosis can be accelerated by taking specific VOC profiles into account.

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.