Rhinoceros horn mineral and metal concentrations vary by sample location, depth, and color.

Poaching is again driving rhinos to the brink of extinction due to the demand for rhino horn products consumed for cultural, medicinal, and social purposes. Paradoxically, the same horn for which rhinos are killed may contain valuable clues about the species' health. Analyses of horn composition could reveal such useful bioindicators while elucidating what people actually ingest when they consume horn derivatives. Our goals were to quantify minerals (including metals) in rhino horn and investigate sampling factors potentially impacting results. Horns (n = 22) obtained during necropsies of white (n = 3) and black (n = 13) zoo rhinos were sampled in several locations yielding 182 specimens for analysis. Initial data exposed environmental (soil) contamination in the horn's exterior layer, but also confirmed that deep (≥ 1 cm), contaminant-free samples contained measurable concentrations of numerous minerals (n = 18). Of the factors examined in deep samples, color-associated mineral differences were the most profound with dark samples higher in zinc, copper, lead, and barium (p < 0.05). Our data demonstrate that rhino horns contain both essential and potentially toxic minerals that could be relevant to rhino health status, but low concentrations make their human health benefits or risks unlikely following consumption.

Label-Free Quantification (LFQ) of Fecal Proteins for Potential Pregnancy Detection in Polar Bears.

Reliable pregnancy diagnostics would be beneficial for monitoring polar bear (Ursus maritimus) populations both in situ and ex situ, but currently there is no method of non-invasive pregnancy detection in this species. Recent reports in several carnivore species described the identification of fecal proteins that may serve as pregnancy biomarkers; however, repeatability has been limited. The objective of the current analysis was to utilize an unbiased, antibody-free, label-free method for the identification and quantification of fecal proteins to determine if differences associated with pregnancy are detectable in polar bears. Protein was extracted from fecal samples (n = 48) obtained from parturient (n = 6) and non-parturient (n = 6) profiles each at four timepoints: pre-breeding season, embryonic diapause, early placental pregnancy, and mid-placental pregnancy. Protein was prepared and analyzed on the Thermo Orbitrap Eclipse nanoLC-MS/MS system. A total of 312 proteins was identified and quantified; however, coefficients of variation (CV) were high for both abundance ratio variability (384.8 ± 61.0% SEM) and within group variability (86.8 ± 1.5%). Results of this study suggest that the inconsistencies in specific protein concentrations revealed previously by antibody-based assays may not be due to that methodology's limitations, but rather, are reflective of true variation that exists among samples.

Case Studies in Polar Bear (Ursus maritimus) Sperm Collection and Cryopreservation Techniques.

Assisted reproductive technologies can aid conservation efforts via support of ex situ population management and preservation of genetic material. Data from 38 sperm collection attempts from 17 polar bears (1-5 procedures/bear) were evaluated. Sample collections were attempted via electroejaculation (EEJ; n = 6), urethral catheterization (UC; n = 25), or sperm rescue (SR; n = 7) during the breeding season (Jan. 1-May 21; n = 27) and nonbreeding season (May 22-Dec. 31; n = 11). Sperm retrieval was successful in 1 EEJ (16.7%), 18 UC (72.0%) and 4 SR (57.1%) collections. Initial sperm motility and viability were 50.0% and 77.0% for EEJ, 64.3 ± 7.4% and 80.9 ± 3.8% for UC, and 56.7 ± 8.8% and 80.5 ± 0.5% for SR. UC and SR were more likely to be successful during the breeding season (84.2-100%) than the nonbreeding season (25.0-33.3%). Testicular tumors were observed in four males (57%) during SR. In total, 13 samples were cryopreserved (n = 1 EEJ, 9 UC, and 3 SR) with egg-yolk-based equine extender (EQ) or OptiXcell (OP). For both extenders, post-thaw motility and viability were reduced by 20-60% and 30-65%, respectively. Further efforts to optimize procedures are warranted, but this summary provides data useful for enhancing the success of polar bear sperm collection and cryopreservation.

Rhinoceros serum labile plasma iron and associated redox potential: interspecific variation, sex bias and iron overload disorder disconnect.

A consequence of the poaching crisis is that managed rhinoceros populations are increasingly important for species conservation. However, black rhinoceroses (BR; Diceros bicornis) and Sumatran rhinoceroses (SR; Dicerorhinus Sumatrensis) in human care often store excessive iron in organ tissues, a condition termed iron overload disorder (IOD). IOD research is impeded by the challenge of accurately monitoring body iron load in living rhinoceroses. The goals of this study were to (i) determine if labile plasma iron (LPI) is an accurate IOD biomarker and (ii) identify factors associated with iron-independent serum oxidative reduction potential (ORP). Serum (106 samples) from SRs (n = 8), BRs (n = 28), white rhinoceros (n = 24) and greater one-horned rhinoceros (GOH; n = 16) was analysed for LPI. Samples from all four species tested positive for LPI, and a higher proportion of GOH rhinoceros samples were LPI positive compared with those of the other three species (P < 0.05). In SRs, the only LPI-positive samples were those from individuals clinically ill with IOD, but samples from outwardly healthy individuals of the other three species were LPI positive. Serum ORP was lower in SRs compared with that in the other three species (P < 0.001), and iron chelation only reduced ORP in the GOH species (P < 0.01; ~5%). Serum ORP sex bias was revealed in three species with males exhibiting higher ORP than females (P < 0.001), the exception being the SR in which ORP was low for both sexes. ORP was not associated with age or serum iron concentrations (P ≥ 0.05), but was positively correlated with ferritin (P < 0.01). The disconnect between LPI and IOD was unanticipated, and LPI cannot be recommended as a biomarker of advanced rhino IOD. However, data provide valuable insight into the complex puzzle of rhinoceros IOD.

Rhinoceros Serum microRNAs: Identification, Characterization, and Evaluation of Potential Iron Overload Biomarkers.

Iron overload disorder (IOD) in critically endangered Sumatran (Dicerorhinus sumatrensis) and black (Diceros bicornis) rhinoceros is an over-accumulation of iron in organs which may exacerbate other diseases and indicate metabolic disturbances. IOD in rhinos is not well understood and diagnostics and therapeutics are limited in effectiveness. MicroRNAs (miRNAs) are small non-coding RNAs capable of altering protein synthesis. miRNA expression responds to physiological states and could serve as the basis for development of diagnostics and therapeutics. This study aimed to identify miRNAs differentially expressed among healthy rhinos and those afflicted with IOD or other diseases ("unhealthy"), and assess expression of select miRNAs to evaluate their potential as biomarkers of IOD. miRNAs in serum of black (n = 11 samples; five individuals) and Sumatran (n = 7 samples; four individuals) rhinos, representing individuals categorized as healthy (n = 9), unhealthy (n = 5), and afflicted by IOD (n = 3) were sequenced. In total, 715 miRNAs were identified, of which 160 were novel, 131 were specific to black rhinos, and 108 were specific to Sumatran rhinos. Additionally, 95 miRNAs were specific to healthy individuals, 31 specific to unhealthy, and 63 were specific to IOD individuals. Among healthy, unhealthy, and IOD states, 21 miRNAs were differentially expressed (P ≤ 0.01). Five known miRNAs (let-7g, miR-16b, miR-30e, miR-143, and miR-146a) were selected for further assessment via RT-qPCR in serum from black (n = 61 samples; seven individuals) and Sumatran (n = 38 samples; five individuals) rhinos. let-7g, miR-30e, and miR-143 all showed significant increased expression (P ≤ 0.05) during IOD (between 1 and 2 years prior to death) and late IOD (within 1 year of death) compared to healthy and unhealthy individuals. miR-16b expression increased (P ≤ 0.05) in late IOD, but was not different among IOD, healthy, and unhealthy states (P > 0.05). Expression of miR-146a increased in IOD and late IOD as compared to unhealthy samples (P ≤ 0.05) but was not different from the healthy state (P > 0.05). Selected serum miRNAs of black and Sumatran rhinos, in particular let-7g, miR-30e, and miR-143, could therefore provide a tool for advancing rhino IOD diagnostics that should be further investigated.

Reduced Gut Microbiome Diversity and Metabolome Differences in Rhinoceros Species at Risk for Iron Overload Disorder.

Iron overload disorder (IOD) affects many wildlife species cared for ex situ. Two of the four rhinoceros species in human care, Sumatran rhinoceros (Dicerorhinus sumatrensis) and black rhinoceros (Diceros bicornis), are susceptible, whereas the other two, white rhinoceros (Ceratotherium simum) and greater one-horned (GOH) rhinoceros (Rhinoceros unicornis), are relatively resistant to IOD. Complex interrelationships exist between mammalian hosts, their indigenous gut microbiota, metabolome, physical condition, and iron availability. The goal of this study was to gain insight into these relationships within the family Rhinocerotidae. Specific objectives were to (1) characterize the gut microbiome and metabolome of four rhinoceros species; (2) compare the microbiome and metabolome of IOD-susceptible and IOD-resistant rhinoceros species; and (3) identify variation in the microbiome and metabolome associated with compromised health or disease in IOD-susceptible rhinoceroses. Fecal samples were collected from 31 rhinoceroses (Sumatran rhinoceros, n = 3; black rhinoceros, n = 6; GOH rhinoceros, n = 9; white rhinoceros, n = 13) located at five facilities, and matched fecal aliquots were processed for microbiome and metabolome analyses using 16S rRNA gene sequencing and nuclear magnetic resonance spectroscopy, respectively. Despite the phylogenetic disparity and dissimilar zoo diets of the hosts, the structure of the fecal microbiota of the two IOD-susceptible rhinoceros species were more closely related to each other than to those of the two IOD-resistant species (Bray-Curtis dissimilarity; IOD-susceptible vs. IOD-resistant p-value < 0.001). In addition, IOD-susceptible rhinoceroses exhibited less microbial diversity than their IOD-resistant relatives (Shannon diversity; p-value < 0.001) which could have health implications. Of note, the black rhinoceros was distinct among the four rhinoceros species with the most divergent fecal metabolome; interestingly, it contained higher concentrations of short chain fatty acids. Neither age nor sex were associated with differences in microbial community composition (p = 0.253 and 0.488, respectively) or fecal metabolomic profile (p = 0.634 and 0.332, respectively). Differences in the distal gut microbiomes between IOD-resistant and IOD-susceptible rhinoceroses support hypotheses that gut microbes play a role in host iron acquisition, and further studies and experiments to test these hypotheses are warranted.

Animal protein-free OptiXcell and shortened equilibration periods can replace egg yolk-based extender and slow cooling for rhinoceros semen cryopreservation.

OptiXcell (OP) was tested as an animal protein-free alternative to an egg yolk-based extender for rhinoceros semen cryopreservation and shorter chilling/equilibration periods were evaluated. Semen was collected from three rhinoceros species: black (Diceros bicornis; n = 2), white (Ceratotherium simum; n = 2), and greater one-horned (GOH; Rhinoceros unicornis; n = 3). Controls were diluted with equine extender (EQ) or OP and equilibrated for 1 h. Treatments were diluted with extender and cooled for 15 min (fast: FEQ; FOP) or not cooled (immediate: IEQ; IOP), prior to cryopreservation. Motility decreased post-thaw (EQ: 50.7 ±â€¯5.2%; OP: 52.9 ±â€¯3.4%) from fresh (82.9 ±â€¯2.9%), was higher in OP than IOP (38.6 ±â€¯4.9%; P ≤ 0.05) and decreased over time (P ≤ 0.05). Post-thaw acrosomal integrity was lower in EQ, FEQ, and IEQ (56.9 ±â€¯0.7; 56.6 ±â€¯4.5; 54.9 ±â€¯2.9%) than OP, FOP, IOP (71.8 ±â€¯4.7; 71.9 ±â€¯3.8; 69.9 ±â€¯4.5%) and fresh (72.6 ±â€¯1.4%; P ≤ 0.05). Progression and viability were lower in EQ (2.8 ±â€¯0.2; 61.9 ±â€¯7.4%) and OP (3.1 ±â€¯0.2; 53.4 ±â€¯6.9%) than fresh (3.7 ±â€¯0.2; 87.2 ±â€¯1.3%), decreased over time (P ≤ 0.05) but not different among treatments (P > 0.05). Morphology did not differ between fresh (75.0 ±â€¯4.9% normal) and any treatment group (70.0-77.8%) or over time (P > 0.05). OptiXcell is comparable to egg yolk-based EQ when used for rhinoceros semen cryopreservation. Furthermore, chilling/equilibration can be reduced with little impact on sperm characteristics.

Comparison of soy lecithin, coconut water, and coconut milk as substitutes for egg-yolk in semen cryodiluent for black rhinoceros (Diceros bicornis) and Indian rhinoceros (Rhinoceros unicornis).

Semen cryopreservation for the black rhinoceros (Diceros bicornis) and Indian rhinoceros (Rhinoceros unicornis) relies on extenders containing egg-yolk (EY). Use of such media is not ideal as inter-batch composition varies and there is risk of pathogenic contamination. The goal of this study was to test animal protein-free extenders. Semen collected via electroejaculation from 10 rhinoceros (6 black, 4 Indian) was diluted with extender containing EY, 1% or 2% soy lecithin (1%SL; 2%SL), coconut water (CW), or coconut milk (CM), cryopreserved and evaluated for sperm motility, viability, morphology, progression, and acrosomal integrity at 0, 1, 3, 6 and 24 h post-thaw. Mean ±â€¯SD fresh ejaculate motility was 84.5 ±â€¯7.6%, progression: 3.6 ±â€¯0.6 (scale 0-5), viability: 83.4 ±â€¯7.1%, intact acrosomes: 71.3 ±â€¯6.9%, and morphologically normal: 78.8 ±â€¯13.6%. Motility and progression decreased in all groups post-thaw, were greatest in EY, and decreased over time (P ≤ 0.05). Motility and progression did not differ (P > 0.05) between 1%SL and 2%SL, but were lower (P ≤ 0.05) in CM and CW, and acrosomal integrity was higher (P ≤ 0.05) in EY, 1%SL and 2%SL than in CM and CW. Post-thaw viability was greatest in EY and 2%SL followed by 1%SL, then CM and CW (P ≤ 0.05). Morphology did not differ among treatments (P > 0.05). Morphology, acrosomal integrity, and viability were maintained over time (P > 0.05). Although some rhinoceros sperm survived cryopreservation in SL treatments, reduced post-thaw motility rendered all treatments inadequate substitutes for EY-based extenders.

INVESTIGATION OF FACTORS POTENTIALLY ASSOCIATED WITH SERUM FERRITIN CONCENTRATIONS IN THE BLACK RHINOCEROS ( DICEROS BICORNIS) USING A VALIDATED RHINOCEROS-SPECIFIC ASSAY.

Iron overload disorder (IOD) can lead to organ dysfunction and may exacerbate other diseases in the critically endangered black rhinoceros ( Diceros bicornis). It is important to develop methods for monitoring the progression of iron storage (hemosiderosis), diagnosing the disease, and evaluating treatments in this species. Traditionally, an equine enzyme immunoassay (EIA) was used to measure rhinoceros ferritin, a serum protein correlated to iron stores. The goal of this study was to validate a rhinoceros-specific assay and investigate factors potentially associated with ferritin concentrations in black rhinoceros. A ferritin EIA developed for Sumatran rhinoceros was validated for black rhinoceros via Western blot analysis of liver ferritin and confirmed parallelism of serum samples to the EIA standard curve and used to analyze serum samples ( n = 943) collected from 36 black rhinoceros (<1-33 yr) at 14 U.S. institutions. Mean (±SEM) serum ferritin concentration was 6,738 ± 518 ng/ml (range: 85-168,451 ng/ml). Concentrations differed among individuals with eastern black rhinoceros (7,444 ± 1,130 ng/ml) having a higher mean ferritin than southern black rhinoceros (6,317 ± 505 ng/ml; P < 0.05) and higher mean values in wild-born (11,110 ± 1,111 ng/ml) than captive-born individuals (3,487 ± 293 ng/ml; P < 0.05). Ferritin concentrations did not differ between young rhinoceros (<5 yr old; 2,163 ± 254 ng/ml) and adults (7,623 ± 610 ng/ml) and were not correlated with age ( r = 0.143) or time in captivity ( r = 0.146, wild born; r = 0.104, all animals). Ferritin concentration was not impacted by sex (female: 2,086 ± 190 ng/ml; male: 8,684 ± 717 ng/ml), date, month, or season of collection ( P > 0.05). Data indicate ferritin concentrations are variable and not necessarily associated with IOD; ferritin is not recommended for diagnosing or monitoring IOD in black rhinoceros.

Early fetal sexing in the rhinoceros by detection of male-specific genes in maternal serum.

Genetic sexing of animals with long gestation time benefits the management of captive populations. Here, X and Y chromosome-specific primers, based on equine gene sequencing data, were developed and tested on captive rhinoceroses (10 males, 20 females) representing four species (Diceros bicornis, Certaotherium simum simum, Rhinoceros unicornis, and Dicerorhinus sumatrensis). The Y chromosome-specific primer set targeted SRY (Sex-determining region Y), and amplified a 177-bp product following PCR of DNA extracted from males, but not females, of all species. A primer set based on the equine AMEL (Amelogenin) gene resulted in a 232-bp product following PCR of all rhinoceros species. These gene-specific primer sets were then evaluated for their ability to determine gender in cell-free DNA from rhinoceros serum. Modifications to the original extraction and PCR protocols were required to obtain sufficient DNA quantities from serum, and both DNA yield and PCR amplification were substantially reduced or absent following multiple freeze-thaw cycles of serum. When fresh serum from 14 pregnant rhinoceroses (ultimately bearing seven male and seven female calves), representing four species at different stages of gestation (Days 61-490), were probed in a PCR-based assay, an accuracy of 71% was achieved for male-specific gene detection of SRY, which improved to 100% by including a reamplification step into the protocol. Such early sex determination should be a valuable tool for current management practices as well as future assisted reproduction of rhinoceroses.

Genomic Analysis of Demographic History and Ecological Niche Modeling in the Endangered Sumatran Rhinoceros Dicerorhinus sumatrensis.

The vertebrate extinction rate over the past century is approximately 22-100 times greater than background extinction rates [1], and large mammals are particularly at risk [2, 3]. Quaternary megafaunal extinctions have been attributed to climate change [4], overexploitation [5], or a combination of the two [6]. Rhinoceroses (Family: Rhinocerotidae) have a rich fossil history replete with iconic examples of climate-induced extinctions [7], but current pressures threaten to eliminate this group entirely. The Sumatran rhinoceros (Dicerorhinus sumatrensis) is among the most imperiled mammals on earth. The 2011 population was estimated at ≤216 wild individuals [8], and currently the species is extirpated, or nearly so, throughout the majority of its former range [8-12]. Understanding demographic history is important in placing current population status into a broader ecological and evolutionary context. Analysis of the Sumatran rhinoceros genome reveals extreme changes in effective population size throughout the Pleistocene. Population expansion during the early to middle Pleistocene was followed by decline. Ecological niche modeling indicated that changing climate most likely played a role in the decline of the Sumatran rhinoceros, as less suitable habitat on an emergent Sundaland corridor isolated Sumatran rhinoceros populations. By the end of the Pleistocene, the Sundaland corridor was submerged, and populations were fragmented and consequently reduced to low Holocene levels from which they would never recover. Past events denuded the Sumatran rhinoceros of genetic diversity through population decline, fragmentation, or some combination of the two and most likely made the species even more susceptible to later exploitation and habitat loss. VIDEO ABSTRACT.

SERUM FERRITIN CONCENTRATION IS NOT A RELIABLE BIOMARKER OF IRON OVERLOAD DISORDER PROGRESSION OR HEMOCHROMATOSIS IN THE SUMATRAN RHINOCEROS (DICERORHINUS SUMATRENSIS).

The aim of this study was to determine if ferritin is a reliable biomarker of iron overload disorder (IOD) progression and hemochromatosis in the Sumatran rhinoceros (Dicerorhinus sumatrensis) by developing a species-specific ferritin assay and testing historically banked samples collected from rhinos that did and did not die of hemochromatosis. Ferritin extracted from Sumatran rhino liver tissue was used to generate antibodies for the Enzyme Immunoassay. Historically banked Sumatran rhino serum samples (n = 298) obtained from six rhinos in US zoos (n = 290); five rhinos at the Sumatran Rhino Conservation Centre in Sungai Dusun, Malaysia (n = 5); and two rhinos in Sabah, Malaysia (n = 3) were analyzed for ferritin concentrations. Across all US zoo samples, serum ferritin concentrations ranged from 348 to 7,071 ng/ml, with individual means ranging from 1,267 (n = 25) to 2,604 ng/ml (n = 36). The ferritin profiles were dynamic, and all rhinos exhibited spikes in ferritin above baseline during the sampling period. The rhino with the highest mean ferritin concentration did not die of hemochromatosis and exhibited only mild hemosiderosis postmortem. A reproductive female exhibited decreases and increases in serum ferritin concurrent with pregnant and nonpregnant states, respectively. Mean (±SD) serum ferritin concentration for Sumatran rhinos in Malaysia was high (4,904 ± 4,828 ng/ml) compared to that for US zoo rhinos (1,835 ± 495 ng/ml). However, those in Sabah had lower ferritin concentrations (1,025 ± 52.7 ng/ml) compared to those in Sungai Dusun (6,456 ± 4,941 ng/ml). In conclusion, Sumatran rhino serum ferritin concentrations are dynamic, and increases often are not associated with illness or hemochromatosis. Neither a specific pattern nor the individual's overall mean ferritin concentration can be used to accurately assess IOD progression or diagnose hemochromatosis in this rhino species.

A simple, field-friendly technique for cryopreserving semen from Asian elephants (Elephas maximus).

The specific objectives of the present study were to investigate the effects of manual seeding, differing freeze and thaw rates as well as storage for 24h at 4°C prior to cryopreservation on post-thaw sperm quality in Asian elephants. Extended semen was cooled in an equitainer to 4°C, frozen in liquid nitrogen vapour at various rates with and without manual seeding or in a dry shipper and thawed at 37, 50 and 75°C. There was a significant effect of freeze rate on post-thaw motility (P<0.0001) and acrosomal integrity (P<0.005). The faster freeze rates in the dry shipper and at 1cm or 2cm above liquid nitrogen consistently provided better cryopreservation than slower freezing rates. Thaw temperature had no effect on post-thaw semen quality but there was an interaction between freeze and thaw rates with higher thaw rates resulting in superior post-thaw semen quality in straws frozen at fast rates. Storage of samples prior to freezing had a detrimental effect on post-thaw semen quality. In summary, our results indicate cooling extended semen in an equitainer and cryopreserving it by placing straws directly in a dry shipper is a simple technique for effectively cryopreserving Asian elephant semen in the field or zoo.

Integrating trans-abdominal ultrasonography with fecal steroid metabolite monitoring to accurately diagnose pregnancy and predict the timing of parturition in the red panda (Ailurus fulgens styani).

Red pandas (Ailurus fulgens styani) exhibit a variable gestation length and may experience a pseudopregnancy indistinguishable from true pregnancy; therefore, it is not possible to deduce an individual's true pregnancy status and parturition date based on breeding dates or fecal progesterone excretion patterns alone. The goal of this study was to evaluate the use of transabdominal ultrasonography for pregnancy diagnosis in red pandas. Two to three females were monitored over 4 consecutive years, generating a total of seven profiles (four pregnancies, two pseudopregnancies, and one lost pregnancy). Fecal samples were collected and assayed for progesterone (P4) and estrogen conjugate (EC) to characterize patterns associated with breeding activity and parturition events. Animals were trained for voluntary transabdominal ultrasound and examinations were performed weekly. Breeding behaviors and fecal EC data suggest that the estrus cycle of this species is 11-12 days in length. Fecal steroid metabolite analyses also revealed that neither P4 nor EC concentrations were suitable indicators of pregnancy in this species; however, a secondary increase in P4 occurred 69-71 days prior to parturition in all pregnant females, presumably coinciding with embryo implantation. Using ultrasonography, embryos were detected as early as 62 days post-breeding/50 days pre-partum and serial measurements of uterine lumen diameter were documented throughout four pregnancies. Advances in reproductive diagnostics, such as the implementation of ultrasonography, may facilitate improved husbandry of pregnant females and allow for the accurate prediction of parturition.

Enhancing captive Indian rhinoceros genetics via artificial insemination of cryopreserved sperm.

The objective of this study was to design an artificial insemination (AI) protocol using cryopreserved spermatozoa to obtain pregnancies in captive Indian rhinoceroses (Rhinoceros unicornis). Four methods developed varied by timing and approach, as follows; Method 1: females (n=2) were inseminated pre- and post-ovulation under general anesthesia, Method 2: females (n=2) were inseminated pre-ovulation without anesthetic via endoscopy, Method 3: females (n=1) were inseminated pre-ovulation without anesthetic via manual insertion of an insemination catheter, Method 4: females (n=2) were inseminated same as Method 3 with the addition of standing sedation. Semen deposition site varied as a result of changes in AI technology and experience. All females conceived following intrauterine AI using three methods. Four pregnancies (n=3 females) produced via Method 3 and 4 resulted in term births (n=2 male calves, n=2 female calves) at 481.8±12.8days post-AI. Unfortunately, two early pregnancy losses were documented in a fourth female conceiving via Method 2. Pregnancy rates were 0%, 22%, 17%, and 50% for Method 1-4, respectively. Method 3 and 4 rates improved to 29% and 67%, respectively when accounting for AI's conducted only on ovulatory estrous cycles. Spermatozoa (n=5 males) were cryopreserved 0.3-9.3 y prior to successful AI procedures. The lowest dose of frozen-thawed sperm resulting in conception was 500×10(6) motile sperm. Mean time from AI to ovulation in conceptive and non-conceptive cycles was 26±11.8h and 66±80.7h, respectively.

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.

Ovulation induction and artificial insemination of a captive polar bear (Ursus maritimus) using fresh semen.

In 2008, polar bears were listed as a species threatened with extinction by the U.S. Endangered Species Act. Unfortunately, reproductive success has been poor despite breeding recommendations for almost every reproductively viable bear by the Species Survival Plan. Assisted reproductive technologies could complement breeding efforts by overcoming the challenges of behavioral incompatibilities and deficiencies, facilitating genetic management and increasing cub production. The goal of this study was to artificially inseminate a female polar bear after inducing ovarian activity and ovulation with exogenous hormones (equine chorionic gonadotropin and porcine luteinizing hormone). Fresh semen collected from an adult male via electroejaculation/urethral catheterization was used for the insemination. Fecal steroid monitoring indicated that the female ovulated following the exogenous hormone treatment. Progestin concentrations increased in late summer, at the time implantation was expected to occur; however, no cubs were produced. To the authors' knowledge, this is the first report of ovulation induction and artificial insemination in a polar bear.

Use of urinary biomarkers of ovarian function and altrenogest supplementation to enhance captive breeding success in the Indian rhinoceros (Rhinoceros unicornis).

Urinary hormone analysis was conducted on two adult female Indian rhinoceroses (Rhinoceros unicornis) that exhibited minimal or no estrual behaviors traditionally used to time breeding. Urine was collected throughout two consecutive estrous cycles to establish preliminary data on each individual's pattern and concentration of estrogen conjugates (EC) and progesterone metabolites (PdG) during follicular and luteal phases. Following preliminary endocrine analysis, urine samples were shipped on a frequent basis to verify when each female was off baseline in EC. Estrus and breeding dates were then predicted. Females were introduced to fresh male rhinoceros fecal samples daily throughout the follicular phase to potentially stimulate estrous behaviors. Despite successful assessment of follicular phase dynamics, females sometimes failed to exhibit estrus. Both females conceived following mating introductions that were timed using hormone analysis. Pregnancy was diagnosed either by endocrine analysis or rectal ultrasonography. Progestational support (altrenogest) occurred after pregnancy confirmation and varied for each female (21 and 66 days post-breeding). One female experienced early pregnancy loss and the other successfully completed a term pregnancy. These results demonstrate that a science based management strategy that relies on urinary biomarkers of ovarian function can facilitate naturally breeding captive Indian rhinoceroses.

Sexual maturation in the Sumatran rhinoceros (Dicerorhinus sumatrensis).

To help save the Sumatran rhino from extinction, the captive breeding program must capitalize on each rhino's reproductive lifespan. Doing so requires knowing when calves are sexually mature. The goal of this study was to monitor physiological changes associated with sexual maturation in two captive born calves (one male and one female) to determine the approximate age of maturity for both sexes of this species. Fecal testosterone metabolites were monitored in the male calf from 6 months to 7 years of age, and fecal pregnane metabolites were measured in the female calf from 6 months to 5.5 years of age. In addition, rectal ultrasonography was employed to monitor changes in ovarian activity from 2 to 5.5 years of age. The male calf's fecal testosterone concentrations reached levels comparable to those detected in samples from adult males when he was 6-6.5 years of age. The first pre-ovulatory sized follicle was observed on the ovaries of the female calf when she was 4.75 years old, but fecal pregnane metabolite concentrations only reached maximum mean concentrations and variability when she was 5-5.5 years of age. Results from this study indicate that male and female Sumatran rhino calves are sexually mature at 6-6.5 and 5-5.5 years of age, respectively.