Twelve toll-like receptor (TLR) genes in the family Equidae - comparative genomics, selection and evolution.

Toll-like receptors (TLRs) represent an important part of the innate immune system. While human and murine TLRs have been intensively studied, little is known about TLRs in non-model species. The order Perissodactyla comprises a variety of free-living and domesticated species exposed to different pathogens in different habitats and is therefore suitable for analyzing the diversity and evolution of immunity-related genes. We analyzed TLR genes in the order Perissodactyla with a focus on the family Equidae. Twelve TLRs were identified by bioinformatic analyses of online genomic resources; their sequences were confirmed in equids by genomic DNA re-sequencing of a panel of nine species. The expression of TLR11 and TLR12 was confirmed in the domestic horse by cDNA sequencing. Phylogenetic reconstruction of the TLR gene family in Perissodactyla identified six sub-families. TLR4 clustered together with TLR5; the TLR1-6-10 subfamily showed a high degree of sequence identity. The average estimated evolutionary divergence of all twelve TLRs studied was 0.3% among the Equidae; the most divergent CDS were those of Equus caballus and Equus hemionus kulan (1.34%) in the TLR3, and Equus africanus somaliensis and Equus quagga antiquorum (2.1%) in the TLR1 protein. In each TLR gene, there were haplotypes shared between equid species, most extensively in TLR3 and TLR9 CDS, and TLR6 amino acid sequence. All twelve TLR genes were under strong negative overall selection. Signatures of diversifying selection in specific codon sites were detected in all TLRs except TLR8. Differences in the selection patterns between virus-sensing and non-viral TLRs were observed.

The first European woolly rhinoceros mitogenomes, retrieved from cave hyena coprolites, suggest long-term phylogeographic differentiation.

The woolly rhinoceros (Coelodonta antiquitatis) is an iconic species of the Eurasian Pleistocene megafauna, which was abundant in Eurasia in the Pleistocene until its demise beginning approximately 10 000 years ago. Despite the early recovery of several specimens from well-known European archaeological sites, including its type specimen (Blumenbach 1799), no genomes of European populations were available so far, and all available genomic data originated exclusively from Siberian populations. Using coprolites of cave hyenas (Crocuta crocuta spelea) recovered from Middle Palaeolithic layers of two caves in Germany (Bockstein-Loch and Hohlenstein-Stadel), we isolated and enriched predator and prey DNA to assemble the first European woolly rhinoceros mitogenomes, in addition to cave hyena mitogenomes. Both coprolite samples produced copious sequences assigned to C. crocuta (27% and 59% mitogenome coverage, respectively) and woolly rhinoceros (Coelodonta antiquitatis; 27% and 81% coverage, respectively). The sequences suggested considerable DNA degradation, which may limit the conclusions to be drawn; however, the mitogenomes of European woolly rhinoceros are genetically distinct from the Siberian woolly rhinoceros, and analyses of the more complete mitogenome suggest a split of the populations potentially coinciding with the earliest fossil records of woolly rhinoceros in Europe.

Comparative analysis of gestation in three rhinoceros species (Diceros bicornis; Ceratotherium simum; Rhinoceros unicornis).

This comparative analysis evaluated endocrine profiles and gestation length data of captive pregnant black rhinoceros (Diceros bicornis), white rhinoceros (Ceratotherium simum), and greater one-horned (GOH) rhinoceros (Rhinoceros unicornis). Hormone profiles were collected over three decades as part of pregnancy diagnoses. After the third month of gestation, the luteo-placental shift in progesterone production in pregnant rhinoceroses causes a significant increase in the concentration of faecal progesterone metabolites. We defined a laboratory-specific value of 1000 ng/g faeces as a threshold for incipient feto-placental progesterone production. Using this value allowed a comparison between species and revealed significant individual differences within a species. The mean ± SEM gestation days for reaching the 1000 ng/g faeces threshold were 89.5 ± 2.9 (range 56-138 days; n = 39) in black, 96.0 ± 2.6 (58-138; n = 39) in white, and 117.8 ± 5.3 (74-173; n = 19) in GOH rhinoceroses. For the calculations of gestation length, we complemented our results from three decades of reproductive monitoring with data from the literature, resulting in about 70 values for each species. Gestation length in the black, the white and the GOH rhinoceros was 460.6 ± 1.5 (range: 436 - 486), 503.8 ± 1.3 (range: 480 - 525) and 480.5 ± 1.1 (range: 453 - 505) days, respectively. Daylight length significantly affected gestation length, while the sex of offspring had no effect. On average, pregnancies with parturitions in spring and summer were one week shorter than those in autumn and winter. Although rhinoceroses are non-seasonal breeders, most parturitions in captivity occur in autumn and winter. We also analysed preconception endocrine profiles in the white rhinoceros. Conceptions in this species occurred after oestrous cycles of approximately 35 days (n = 18), 70 days (n = 3), 15 days (n = 1), after periods of ovarian inactivity (n = 5), and during a foal heat within one month after stillbirth parturition (n = 1). In conclusion, this study provides a comprehensive overview of gestational parameters in three rhinoceros species.

Ultra-performance convergence chromatography tandem mass spectrometry analysis of adrenal and gonadal steroid hormones in southern white rhinoceros(Ceratotherium simum simum) faeces and serum.

Steroid hormone analysis is routinely undertaken in the assessment of stress response and reproductive function in the management of both captive and free-ranging wildlife species. Faecal samples have become the preferred sample type for analysis as collection is non-invasive and easily assessable. These investigations are generally aimed at aiding successful translocations, enhanced survival outcomes in captivity and improvement of reproductive rate. Immunoassays are the most common approach in the analysis of hormones, particularly in the case of the southern white rhinoceros (Ceratotherium simum simum). Non-specificity, attributed to structural similarity of steroid metabolites impedes accurate evaluations which can be eliminated by chromatographic techniques which are more specific, selective and provide comprehensive analyses. This study developed and validated three methods using ultra-performance convergence chromatography tandem mass spectrometry for the assessment of classical androgens, progestogens and adrenal steroids, as well as the C11-oxy androgens and C11-oxy progestogens in serum and faeces from white rhinoceros. The limit of detection and quantification were determined for each steroid, parameters such as accuracy (<19.8 % RSD) and precision (<20.2 % RSD) were established with recovery, matrix effect, and process efficiency within acceptable limits. Subsequent analysis of serum and faecal samples from five white rhinoceros identified novel steroids for the first time in this species. In addition to the classical adrenal steroids, the following C11-oxy steroids were detected in faecal samples: 11α-hydroxydihydroprogesterone (168 ng/g), 11α-hydroxyprogesterone (125.9 ng/g), 11ß-hydroxyprogesterone (210.2 ng/g) and 11-ketoandrostenedione (3.3-19.6 ng/g) with 11-deoxycortisol being the major glucocorticoid (24.2-67.3 ng/g) together with 21-deoxycortisone (40.7 ng/g) and deoxycorticosterone (7.6-14.6 ng/g). In serum samples 11ß-hydroxyandrostenedione (0.35-2.34 ng/mL) and 11ß-hydroxytestosterone (0.18-1.62 ng/mL) were the predominant androgens with cortisol (5.8-20.5 ng/mL), the predominant glucocorticoid, while corticosterone, 18-hydroxycorticosterone and aldosterone were also detected. These methods can be applied independently to assess either androgens, progestogens, or adrenal steroid panels or in combination to assess the cohort of gonadal and adrenal steroids in faeces and/or serum, in southern white rhinoceros as well as other wildlife species. Analysis would enable the accurate assessment of reproductive health and stress responses while also distinguishing between stress and distress thus contributing to the conservation of wildlife species.

Non-invasive assessment of fecal glucocorticoid, progesterone, and androgen metabolites and microbiome in free-ranging southern white rhinoceros (Ceratotherium simum simum) in South Africa.

Increased poaching in northern South Africa has necessitated relocation of large numbers of southern white rhinoceros (Ceratotherium simum simum) to the Eastern Cape Province. The climate and grassland ecology of this province differ from that of northern South Africa which may impact the health of this species. This assessment of fecal steroid levels and microbiome in 10 free-ranging southern white rhinoceros in the Eastern Cape will provide insights into white rhinoceros physiology in this biome. Fecal steroid metabolites were analyzed using enzyme immunoassay (EIA) and ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). Fecal microbial composition was assessed via next generation sequencing. EIAs with antibodies raised against progesterone (P4; mouse monoclonal - CL425 clone), testosterone (T; rabbit polyclonal), corticosterone (B; sheep polyclonal) were utilized. Pregnant females had large quantities of fecal progesterone metabolites (FPMs) detected by CL425 EIA. Pregnant females also had native P4 and 11α-hydroxydihydroprogesterone (11αOHDHP4; 4-pregnen-11α-ol-3,20-dione) detected by UPC2-MS/MS but these concentrations were 1000-fold less than the concentrations of FPMs detected by the CL425 EIA. By contrast, non-pregnant females had FPM concentrations detected by CL425 EIA which were similar to native P4 and 11αOHDHP4 concentrations detected by UPC2-MS/MS. Mean fecal androgen metabolite (FAM) concentrations detected by the T EIA were similar between males and females. 11-ketoandrostenedione (11KA4) detected by UPC2-MS/MS was higher in females than males. However, there was no difference between males and females in the concentration of fecal glucocorticoid metabolites (FGMs) detected by the B EIA. Bacteroidia, followed by Clostridia, was the most abundant classes of fecal microbes. The unfiltered microbiome of females was more diverse than that of males. The core fecal microbiome of young rhinoceros had a higher observed species richness (Shannon diversity index, and Simpson diversity index) than that of old rhinoceros. In the alpha male, immobilization was associated with an increase in FGMs detected by 11-deoxycortisol (S) detected by UPC2-MS/MS coupled with decreased abundance of Spirochaetia. We detected substantially different FAM and FPM concentrations from those previously reported for both captive and wild white rhinoceros. Comparison of our UPC2-MS/MS and EIA results underscores the fact that most EIAs are highly cross reactive for many steroid metabolites. Our data also demonstrates a distinct effect of stress not only on FGMs but also on the fecal microbiome. This is the first non-invasive assessment of fecal steroid metabolites by UPC2-MS/MS and the fecal microbiome in wild white rhinoceros.

The steroid metabolome of pregnancy, insights into the maintenance of pregnancy and evolution of reproductive traits.

Modes of mammalian reproduction are diverse and not always conserved among related species. Progesterone is universally required to supports pregnancy but sites of synthesis and metabolic pathways vary widely. The steroid metabolome of mid-to late gestation was characterized, focusing on 5α-reduced pregnanes in species representing the Perissodactyla, Cetartiodactyla and Carnivora using mass spectrometry. Metabolomes and steroidogenic enzyme ortholog sequences were used in heirarchial analyses. Steroid metabolite profiles were similar within orders, whales within cetartiodactyls for instance, but with notable exceptions such as rhinoceros clustering with goats, and tapirs with pigs. Steroidogenic enzyme sequence clustering reflected expected evolutionary relationships but once again with exceptions. Human sequences (expected outgroups) clustered with perissodactyl CYP11A1, CYP17A1 and SRD5A1 gene orthologues, forming outgroups only for HSD17B1 and SRD5A2. Spotted hyena CYP19A1 clustered within the Perissodactyla, between rhinoceros and equid orthologues, whereas CYP17A1 clustered within the Carnivora. This variability highlights the random adoption of divergent physiological strategies as pregnancy evolved among genetically similar species.

Inflammatory and oxidative status in European captive black rhinoceroses: A link with Iron Overload Disorder?

Iron Overload Disorder (IOD) is a syndrome developed by captive browsing rhinoceroses like black rhinoceroses (Diceros bicornis), in which hemosiderosis develops in vital organs while free iron accumulates in the body, potentially predisposing to various secondary diseases. Captive grazing species like white rhinoceroses (Ceratotherium simum) do not seem to be affected. The authors hypothesized that inflammation and oxidative stress may be implicated in the pathogenesis of IOD in captive black rhinoceroses, making this syndrome a potential common denominator to various diseases described in captivity in this species. In this prospective study, 15 black (BR) and 29 white rhinoceroses (WR) originating from 22 European zoos were blood-sampled and compared for their iron status (serum iron), liver/muscle biochemical parameters (AST, GGT, cholesterol), inflammatory status (total proteins, protein electrophoresis) and oxidative stress markers (SOD, GPX, dROMs). Results showed higher serum iron and liver enzyme levels in black rhinoceroses (P < 0.01), as well as higher dROMs (P < 0.01) and a trend for higher GPX (P = 0.06) levels. The albumin/globulin ratio was lower in black rhinoceroses (P < 0.05) due to higher α2-globulin levels (P < 0.001). The present study suggests a higher inflammatory and oxidative profile in captive BR than in WR, possibly in relation to iron status. This could be either a consequence or a cause of iron accumulation. Further investigations are needed to assess the prognostic value of the inflammatory and oxidative markers in captive black rhinoceroses, particularly for evaluating the impact of reduced-iron and antioxidant-supplemented diets.

Anti-Müllerian hormone in managed African and Asian rhino species.

Serum collected across the lifespan of four managed rhino species: black (Diceros bicornis, n = 16), white (Ceratotherium simum simum, n = 19), greater one-horned (GOH, Rhinoceros unicornis, n = 11) and Sumatran (Dicerorhinus sumatrensis, n = 6) were validated and analyzed in an anti-Müllerian hormone (AMH) enzyme- linked immunoassay. Concentrations of AMH were examined over time, between sexes and throughout different reproductive states which included n = 3 female white rhinos immunocontracepted with porcine zona pellucida (pZP). Across species, males produced higher AMH concentrations compared to females. Among males, AMH concentrations varied by species aside from comparable values secreted between black and white rhinos. The GOH and Sumatran rhino secreted the highest and lowest male AMH concentrations, respectively. However, within each species, AMH concentrations were similar across male age categories. Preliminary insight into male AMH changes from birth to sexual maturity suggest its potential as a marker for onset of testicular maturation. Female black, GOH and Sumatran rhinos secreted comparable AMH concentrations which were higher than those in white rhino. Within each species, inter-individual variation in AMH secretion occurred among females of similar age. While AMH secretion did not differ across the ages sampled for female white (4->26 yr) and GOH (4-26 yr) rhinos, black and Sumatran rhinos >26 and <4 yr, respectively secreted lower AMH compared to conspecific females 7-26 yr of age. Two idiopathic infertility cases corresponded to low (outside species range) AMH values. The establishment of normative AMH concentrations in managed African and Asian rhinos provides an additional metric beyond traditional sex steroids to assess gonadal function. Further work is needed to determine if AMH can predict fertility potential in rhinos.

Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2 of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5-although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7-9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)10. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.

Rare gut microbiota associated with breeding success, hormone metabolites and ovarian cycle phase in the critically endangered eastern black rhino.

BACKGROUND: Host microbiomes play a role in hormone production and subsequent fertility in humans, but this is less well understood in non-model organisms. This is of particular relevance to species in zoo-based conservation breeding programmes, as relationships between host microbiome composition and reproductive output may allow for the development of microbial augmentation strategies to improve success. Here, we characterise faecal bacterial communities of breeding and non-breeding eastern black rhino (Diceros bicornis michaeli) using 16S rRNA gene amplicon sequencing and quantify progestagen and glucocorticoid metabolite concentrations through enzyme immunoassays to identify such relationships. RESULTS: We identified significant differences in black rhino gut microbiome composition according to ID, institution, breeding success and ovarian cycle phase. In particular, the gut microbiome during pregnancy and post-parturition was significantly altered. Around a third of bacterial genera showed more than ± 10% correlation with either progestagen and/or glucocorticoid concentration, and in general, microbial genera correlated with both hormones in the same direction. Through a combination of analyses, we identified four genera (Aerococcaceae, Atopostipes, Carnobacteriaceae and Solobacterium) that were significantly associated with breeding success, pregnancy and/or post-parturition, and higher faecal progestagen metabolite concentrations. These genera had a lower-than-average relative abundance in the gut microbiome. CONCLUSION: Our results indicate that many members of the gut microbiome of black rhino are associated with hormone production and breeding success, and some members of the rare microbiota appear to be particularly important. Although the directionality of the relationship is unclear, the variation in gut microbiome communities represents a potential biomarker of reproductive health. We identified four genera that were associated with multiple indicators of reproductive output; these could be candidate probiotics to improve the breeding success of black rhino in zoo-based conservation breeding programmes. Further work is required to understand the efficacy and feasibility of this, either directly through microbial augmentation (e.g. probiotics) or indirectly via dietary manipulation or prebiotics.

Pharmacokinetics of an intravenous and oral dose of enrofloxacin in white rhinoceros (Ceratotherium simum).

South Africa currently loses over 1000 white rhinoceros (Ceratotherium simum) each year to poaching incidents, and numbers of severely injured victims found alive have increased dramatically. However, little is known about the antimicrobial treatment of wounds in rhinoceros. This study explores the applicability of enrofloxacin for rhinoceros through the use of pharmacokinetic-pharmacodynamic modelling. The pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin were evaluated in five white rhinoceros after intravenous (i.v.) and after successive i.v. and oral administration of 12.5 mg/kg enrofloxacin. After i.v. administration, the half-life, area under the curve (AUCtot ), clearance and the volume of distribution were 12.41 ± 2.62 hr, 64.5 ± 14.44 µg ml-1  hr-1 , 0.19 ± 0.04 L h-1  kg-1 , and 2.09 ± 0.48 L/kg, respectively. Ciprofloxacin reached 26.42 ± 0.05% of the enrofloxacin plasma concentration. After combined i.v. and oral enrofloxacin administration oral bioavailability was 33.30 ± 38.33%. After i.v. enrofloxacin administration, the efficacy marker AUC24 : MIC exceeded the recommended ratio of 125 against bacteria with an MIC of 0.5 µg/mL. Subsequent intravenous and oral enrofloxacin administration resulted in a low Cmax: MIC ratio of 3.1. The results suggest that intravenous administration of injectable enrofloxacin could be a useful drug with bactericidal properties in rhinoceros. However, the maintenance of the drug plasma concentration at a bactericidal level through additional per os administration of 10% oral solution of enrofloxacin indicated for the use in chickens, turkeys and rabbits does not seem feasible.

A study of the pharmacokinetics and thromboxane inhibitory activity of a single intramuscular dose of carprofen as a means to establish its potential use as an analgesic drug in white rhinoceros.

The alleviation of pain and prevention of suffering are key aspects of animal welfare. Unfortunately, analgesic drugs are not available for all species. White rhinoceros (Ceratotherium simum), representing one of such species, which survive poaching attempts inflicted with severe facial injuries and gunshot wounds, nonetheless require analgesic support. To improve treatment conditions, this study explored the use of carprofen for the treatment of pain and inflammation in white rhinoceros. The pharmacokinetics of 1 mg/kg intramuscular carprofen was evaluated in six healthy white rhinoceros. The half-life of λz and mean residence time was 105.71 ± 15.67 and 155.01 ± 22.46 hr, respectively. The area under the curve and the maximum carprofen concentration were 904.61 ± 110.78 µg ml-1  hr-1 and 5.77 ± 0.63 µg/ml, respectively. Plasma TXB2 inhibition demonstrated anti-inflammatory properties and indicated that carprofen may be effective for a minimum of 48 hr in most animals. With its long half-life further indicating that a single dose could be effective for several days, we suggest that carprofen may be a useful drug for the treatment of white rhinoceros.

Postinduction butorphanol administration alters oxygen consumption to improve blood gases in etorphine-immobilized white rhinoceros.

OBJECTIVE: To investigate the effects of postinduction butorphanol administration in etorphine-immobilized white rhinoceros on respiration and blood gases. STUDY DESIGN: Randomized crossover study. ANIMALS: A group of six sub-adult male white rhinoceros. METHODS: Etorphine, or etorphine followed by butorphanol 12 minutes after recumbency, was administered intramuscularly [2.5 mg etorphine, 25 mg butorphanol (1000-1250 kg), or 3.0 mg etorphine, 30 mg butorphanol (1250-1500 kg)]. Sampling started at 10 minutes after initial recumbency, and was repeated at 5 minute intervals for 25 minutes. Arterial blood gases, limb muscle tremors, expired minute ventilation and respiratory frequency were measured at each sampling point. Calculated values included alveolar-arterial oxygen gradient [ [Formula: see text] ], expected respiratory minute volume (V˙e), tidal volume (Vt), oxygen consumption ( [Formula: see text] ) and carbon dioxide production ( [Formula: see text] ). RESULTS: Etorphine administration resulted in an initial median (range) hypoxaemia [arterial partial pressure of oxygen 25.0 (23.0-28.0) mmHg], hypercapnia [arterial partial pressure of carbon dioxide 76.2 (67.2-81.2) mmHg], increased [Formula: see text] [41.7 (36.6-45.1) mmHg, [Formula: see text] [11.1 (10.0-12.0) L minute-1] and muscle tremors. Butorphanol administration was followed by rapid, although moderate, improvements in arterial partial pressure of oxygen [48.5 (42.0-51.0) mmHg] and arterial partial pressure of carbon dioxide [62.8 (57.9-75.2) mmHg]. In rhinoceros administered butorphanol, [Formula: see text] [4.4 (3.6-5.1) L minute-1] and [Formula: see text] [4.2 (3.8-4.4) L minute-1] were lower than in those not administered butorphanol. Increased arterial oxygen tension was associated with lower oxygen consumption (p=0.002) which was positively associated with lower muscle tremor scores (p<0.0001). CONCLUSIONS AND CLINICAL RELEVANCE: Hypoxaemia and hypercapnia in etorphine-immobilized rhinoceros resulted from an increased [ [Formula: see text] ] and increased [Formula: see text] and [Formula: see text] associated with muscle tremors. Rather than being associated with changes in V˙e, it appears that improved blood gases following butorphanol administration were a consequence of decreased [Formula: see text] associated with reduced muscle tremoring.

Estrogenicity of captive southern white rhinoceros diets and their association with fertility.

The captive southern white rhinoceros (SWR) population is not currently self-sustaining, primarily due to poor or absent reproduction of captive-born (F1+) females. In this study, we investigate the role of dietary phytoestrogens in this reproductive phenomenon by characterizing activation of SWR estrogen receptors (ESRs) 1 and 2 by diet items from nine North American institutions and comparing female SWR fertility to total diet estrogenicity. Of the diet items tested, alfalfa hay and soy and alfalfa-based commercial pellets were found to be the most potent activators of SWR ESRs. In contrast, most grass hays tested were not estrogenic. The estrogenicity of total diets varied across the institutions surveyed and the degree of diet estrogenicity was positively associated with the percentage of the total diet comprised by pellets. Comparisons of fertility records of the institutions surveyed showed no significant relationship between diet estrogenicity and fertility for female SWR conceived or born in the wild (F0). However, for F1+ females, there was a significant negative relationship between institutional diet estrogenicity and fertility. Taken together, these data suggest that developmental exposure to phytoestrogens may be the cause of poor fertility in captive-born female SWR. Whether the low fertility of the current population of captive-born female SWR is permanent or can be reversed by removing phytoestrogens from the diet remains unclear. However, our findings suggest that in order for the SWR population to become self-sustaining, the development and feeding of low phytoestrogen diets should be strongly considered.

Feasibility Study of NMR Based Serum Metabolomic Profiling to Animal Health Monitoring: A Case Study on Iron Storage Disease in Captive Sumatran Rhinoceros (Dicerorhinus sumatrensis).

A variety of wildlife species maintained in captivity are susceptible to iron storage disease (ISD), or hemochromatosis, a disease resulting from the deposition of excess iron into insoluble iron clusters in soft tissue. Sumatran rhinoceros (Dicerorhinus sumatrensis) is one of the rhinoceros species that has evolutionarily adapted to a low-iron diet and is susceptible to iron overload. Hemosiderosis is reported at necropsy in many African black and Sumatran rhinoceroses but only a small number of animals reportedly die from hemochromatosis. The underlying cause and reasons for differences in susceptibility to hemochromatosis within the taxon remains unclear. Although serum ferritin concentrations have been useful in monitoring the progression of ISD in many species, there is some question regarding their value in diagnosing hemochromatosis in the Sumatran rhino. To investigate the metabolic changes during the development of hemochromatosis and possibly increase our understanding of its progression and individual susceptibility differences, the serum metabolome from a Sumatran rhinoceros was investigated by nuclear magnetic resonance (NMR)-based metabolomics. The study involved samples from female rhinoceros at the Cincinnati Zoo (n = 3), including two animals that died from liver failure caused by ISD, and the Sungai Dusun Rhinoceros Conservation Centre in Peninsular Malaysia (n = 4). Principal component analysis was performed to visually and statistically compare the metabolic profiles of the healthy animals. The results indicated that significant differences were present between the animals at the zoo and the animals in the conservation center. A comparison of the 43 serum metabolomes of three zoo rhinoceros showed two distinct groupings, healthy (n = 30) and unhealthy (n = 13). A total of eighteen altered metabolites were identified in healthy versus unhealthy samples. Results strongly suggest that NMR-based metabolomics is a valuable tool for animal health monitoring and may provide insight into the progression of this and other insidious diseases.

Increased inflammation and decreased insulin sensitivity indicate metabolic disturbances in zoo-managed compared to free-ranging black rhinoceros (Diceros bicornis).

Black rhinoceros (rhinos) living in zoos express a host of unusual disease syndromes that are associated with increased morbidity and mortality, including hemolytic anemia, rhabdomyolysis, hepatopathy and ulcerative skin disease, hypophosphatemia and iron overload. We hypothesized that iron overload is a consequence and indicator of disturbances related to inflammation and insulin/glucose metabolism. The objectives of this study were to: (1) generate the first baseline information on biomarkers of inflammation (tumor necrosis factor alpha [TNFα], serum amyloid A [SAA]), insulin sensitivity (insulin, glucose and proxy calculations of insulin sensitivity), phosphate and iron stores (ferritin) using banked serum from free-ranging black rhinos; and (2) then compare serum biomarkers between zoo-managed (n=86 individuals) and free-ranging (n=120) animals. Enzyme immunoassays were validated for serum and then biomarker levels analyzed using mixed models while controlling for sex, age and year of sample collection. Concentrations of TNFα, SAA, insulin and insulin-to glucose ratio were higher (P<0.05) in black rhinos managed in ex situ conditions compared to free-living counterparts. Findings indicate that the captive environment is contributing to increased inflammation and decreased insulin sensitivity in this endangered species.

Management strategies of iron accumulation in a captive population of black rhinoceroses (Diceros bicornis minor).

During routine health screens for black rhinoceroses (Diceros bicornis minor) in a captive setting, serum iron and ferritin were analyzed as well as total iron binding capacity and total iron saturation. Trends for ferritin and percent iron saturation showed steady increases since 2003 in four of four animals (three males; one female) with two animals (one male; one female) consistently showing higher elevations over conspecifics. The historical diet had been comprised of a commercial or in-house complete pelleted feed; several species of fresh browse, Bermuda grass, alfalfa and timothy hays, as well as enrichment and training items (apples, carrots, sweet potatoes, and a small amount of leafy greens and vegetables). In 2009, one of the three male rhinoceroses showed a threefold increase in ferritin and concurrently exhibited clinical signs of lethargy, decreased appetite, and disinterest in training. The lone female showed a twofold increase; she also became reproductively acyclic in the prior year. The male was immobilized for examination and phlebotomy. During the same time period, a new version of the complete pelleted feed, with a reduced amount of iron, was introduced. Subsequent to the diet change, the male's ferritin levels have consistently declined, and the female started cycling again. Even with these corrective steps to reduce iron levels, levels of iron saturation remained high, and ferritin levels were still above 1,500 ng/ml. Therapeutic phlebotomy was instituted via a rigorous training program that allowed phlebotomies over a 30-min time frame. This was possible because of a long-term training program for the animals, consistent training personnel, routine collection of samples on a monthly basis, and general comfort level of the animals in the restraint chute. The results of this integrated approach showed some significant improvements and an overall positive impact on the animals.

Fecal corticosterone concentrations and reproductive success in captive female southern white rhinoceros.

Prolonged or frequent secretion of adrenal glucocorticoids in response to aversive stimuli can negatively impact reproduction. Because female southern white rhinoceros (Ceratotherium simum simum) reproduce poorly in captivity, we compared fecal corticosterone metabolite concentrations among parous, nulliparous, and adolescent females and examined social and physical aspects of the captive environment that might be related to differences in corticosterone metabolite concentrations. Aggression, dominance, sexual and play interactions, social group size and composition, enclosure size, and other housing characteristics were assessed though behavioral observations and review of historical and institution records. Concentrations of metabolized corticosterone in fecal samples were analyzed by enzyme immunoassay. The proportion of nulliparous females did not differ (p>0.05) between subordinate and dominant animals, and subordinates did not have a higher mean fecal corticosterone concentration than dominants (p>0.05). Of the behaviors examined, only the frequency of sexual play behaviors differed (p<0.05) between dominants and subordinates. Average corticosterone concentrations differed (p<0.05) across housing institutions but were not consistently elevated (p>0.05) for females housed in most of the environmental conditions assessed. Housing with a female companion known from adolescence, however, tended to be associated (p=0.06) with a lower mean corticosterone concentration than that when housing with a female companion introduced during adulthood or no female companion. Wild-caught females had a higher (p<0.05) average corticosterone concentration than captive-born females. Average corticosterone concentration did not differ (p>0.05) between acyclic and cycling, or nulliparous and parous females.

Another one bites the dust: faecal silica levels in large herbivores correlate with high-crowned teeth.

The circumstances of the evolution of hypsodonty (= high-crowned teeth) are a bone of contention. Hypsodonty is usually linked to diet abrasiveness, either from siliceous phytoliths (monocotyledons) or from grit (dusty environments). However, any empirical quantitative approach testing the relation of ingested silica and hypsodonty is lacking. In this study, faecal silica content was quantified as acid detergent insoluble ash and used as proxy for silica ingested by large African herbivores of different digestive types, feeding strategies and hypsodonty levels. Separate sample sets were used for the dry (n = 15 species) and wet (n = 13 species) season. Average faecal silica contents were 17-46 g kg(-1) dry matter (DM) for browsing and 52-163 g kg(-1) DM for grazing herbivores. No difference was detected between the wet (97.5 ± 14.4 g kg(-1) DM) and dry season (93.5 ± 13.7 g kg(-1) DM) faecal silica. In a phylogenetically controlled analysis, a strong positive correlation (dry season r = 0.80, p < 0.0005; wet season r = 0.74, p < 0.005) was found between hypsodonty index and faecal silica levels. While surprisingly our results do not indicate major seasonal changes in silica ingested, the correlation of faecal silica and hypsodonty supports a scenario of a dominant role of abrasive silica in the evolution of high-crowned teeth.

Stable isotope ((13)C/(12)C and (15)N/(14)N) composition of the woolly rhinoceros Coelodonta antiquitatis horn suggests seasonal changes in the diet.

The extinct woolly rhinoceros Coelodonta antiquitatis is a prominent member of the Mammuthus-Coelodonta faunal complex, but its biology is poorly known, partly because very few specimens with well-preserved soft tissues have been discovered to date. However, the permafrost-preserved horns of the woolly rhinoceros are recording structures which contain isotopic records of the diet, environmental conditions and physiological status of the animal during most of its life. In this study we report the first data on the pattern of carbon ((13)C/(12)C) and nitrogen ((15)N/(14)N) isotopic composition along the nasal horn of woolly rhinoceros. We found systematic variations in δ(13)C and δ(15)N values associated with morphologically expressed transverse banding of the horn. The comparative analysis of isotopic variation in keratinous tissues of extant and extinct herbivores suggests that the oscillation in isotopic composition of the horn was induced by seasonal changes in the diet. Although the compiled evidence is in part contradictory, we suggest that more positive δ(13)C and δ(15)N values associated with dark-colored and less dense zones of the horn indicate a summer diet. More dense and light-colored zones of the horn have lower δ(13)C and δ(15)N values possibly indicating a larger proportion of woody and shrub vegetation in the winter diet. The validity of these conclusions has to be proven in further investigations, but our data underline the potential of isotopic analysis for studies on diet and habitat use by extinct members of Pleistocene fauna.