Rhinoceros Unicornis Research Articles

Risks from disease caused by Mycobacterium orygis as a consequence of Greater one-horned Rhinoceros (Rhinoceros unicornis) translocation in Nepal.

The greater one-horned rhinoceros (Rhinoceros unicornis) is listed as vulnerable by the IUCN Red List. Mycobacterium orygis-associated disease was identified in a single greater one-horned rhino in Chitwan National Park in February 2015 prior to a planned translocation of five greater one-horned rhinoceros from Chitwan National Park to Bardia National Park for conservation purposes. This paper describes a qualitative disease risk analysis conducted retrospectively post-translocation for Mycobacterium orygis and this translocation, with the aim to improve the understanding of disease threats to the conservation of greater one-horned rhino. The disease risk analysis method used was devised by Sainsbury & Vaughan-Higgins (Conservation Biology, 26, 2017, 442) with modifications by Bobadilla Suarez et al (EcoHealth, 14, 2017, 1) and Rideout et al (EcoHealth, 14, 2017, 42) and included the use of a scenario tree and an analysis of uncertainty as recommended by Murray et al. (Handbook on import risk analysis for animals and animal products. Volume 1. Introduction and qualitative risk analysis, 2004), and the first time this combination of methods has been used to assess the risk from disease in a conservation translocation. The scenario tree and analysis of uncertainty increased the clarity and transparency of the analysis. Rideout et al.'s (EcoHealth, 14, 2017, 42) criteria were used to assess the source hazard and may be useful in comparative assessment of source hazards for future conservation translocations. The likelihood of release into the destination site of Mycobacterium orygis as a source hazard was estimated as of low risk, the risk of exposure of populations at the destination was of high risk and the likelihood of biological and environmental consequences was low. Overall, the risk from disease associated with Mycobacterium orygis as a result of this translocation was found to be low. Recommendations on disease risk management strategies could be improved with a better understanding of the epidemiology including the presence/absence of Mycobacterium orygis in greater one-horned rhino to develop effective disease risk management strategies.

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 ± 5.2%; OP: 52.9 ± 3.4%) from fresh (82.9 ± 2.9%), was higher in OP than IOP (38.6 ± 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 ± 0.7; 56.6 ± 4.5; 54.9 ± 2.9%) than OP, FOP, IOP (71.8 ± 4.7; 71.9 ± 3.8; 69.9 ± 4.5%) and fresh (72.6 ± 1.4%; P ≤ 0.05). Progression and viability were lower in EQ (2.8 ± 0.2; 61.9 ± 7.4%) and OP (3.1 ± 0.2; 53.4 ± 6.9%) than fresh (3.7 ± 0.2; 87.2 ± 1.3%), decreased over time (P ≤ 0.05) but not different among treatments (P > 0.05). Morphology did not differ between fresh (75.0 ± 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.

The Insulin Signaling Substrate Chico and the Ecdysone Response Element Broad Both Regulate Growth of the Head Horns in the Asian Rhinoceros Beetle, Trypoxylus dichotomus.

Males of the Asian rhinoceros beetle, Trypoxylus dichotomus, possess exaggerated head and thoracic horns that scale dramatically out of proportion to body size. While RNAi-mediated knockdowns of the insulin receptor suggest that the insulin signaling pathway regulates nutrition-dependent growth including exaggerated horns, the genes that regulate disproportionate growth have yet to be identified. We used RNAi-mediated knockdown of several genes to investigate their potential role in growth and scaling of the sexually dimorphic, exaggerated head horns of T. dichotomus. Knockdown of the insulin signaling substrate chico and the ecdysone response element broad caused significant decreases in head horn length, while having no or minimal effects on other structures such as elytra and tibiae. However, scaling of horns to body size was not affected by either knockdown. In addition, knockdown of phosphatase and tensin homolog, a negative regulator of the insulin signaling pathway, had no significant effects on any trait. Our results do not identify any candidate genes that may specifically mediate the allometric aspect of horn growth, but they do confirm the insulin signaling pathway as a mediator of conditional trait expression, and importantly implicate the ecdysone signaling pathway, possibly in conjunction with insulin signaling, as an additional mediator of horn growth.

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

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

Dental microwear textures and dietary preferences of extant rhinoceroses (Perissodactyla, Mammalia)

Rhinoceroses were conspicuous elements in Cenozoic ecosystems, and studying the ecological behavior of extant species might unravel the ecology of their fossil kin. Microwear as a short-term recorder may detect subtle variations in the diet. Dental microwear texture analysis (DMTA) is extensively used to infer paleodiets. Yet, regarding ungulates, most microwear studies have been conducted on artiodactyls, and more particularly on ruminants (i.e., foregut fermenters), which may not be good models for hindgut fermenters, such as rhinoceroses. Moreover, rhinoceroses display a specific enamel ultrastructure with vertical Hunter–Schreger bands and a peculiar mastication cycle likely to impact tooth response to wear. Here, we studied the DMTA of the five extant rhinoceros species (17 specimens of Ceratotherium simum, four of Dicerorhinus sumatrensis, 21 of Diceros bicornis, 14 of Rhinoceros sondaicus, and 5 of Rhinoceros unicornis) and built up the present dataset. In parallel, we also compiled a taxon-based dataset of consumed plants for each rhinoceros species. Accordingly, we propose to reclassify the Indian rhinoceros (Rhinoceros unicornis) from mixed-feeder to variable grazer. Significant discrepancies were found between grinding and shearing facets on molars and between species on a given facet. Plotting the percentage of anisotropic specimens against that of complex specimens for each species discriminated well the different diets on both facets. This unprecedented dataset on rhinoceros texture microwear confronted to detailed diets appears critical for future diet reconstruction of fossil rhinocerotoids.

Human-Rhino Conflict: Local People’s Adaptation to Impacts of Rhino

The greater one-horned rhinoceros, a protected species, significant to growing eco-tourism industry and conservation efforts in Nepal poses a number of challenges to the local people residing in the vicinity of the Chitwan National Park (CNP). The article is based on a study of local people’s perceptions on impacts of rhino and various coping and adaptation measures being adopted by them. The study also aims to explore ways to enhance adaptation by securing local people’s participation in rhino conservation. Review of secondary literature, surveys, Focus Group Discussions (FGDs), and Likert scale analysis were carried out for the purpose of the study. Five per cent of the households (50) in Bachauli Village Development Committee that hosts Bagmara Buffer Zone Community Forest User Group of the CNP were randomly selected. The study found that despite 80 per cent of the respondents incurred damage in some form from rhinos, 40 per cent of them exhibited a positive perception towards rhino conservation. Among several coping and adaptive measures to damages, a combination of electric/solar fencing and trench is found to be most effective. Applied research through collaborations between social scientists and ecologists can help to address the gaps in the understanding of different elements of human-wildlife conflict.

189 Isolation and purification of rhinoceros and horse spermatogonial stem cells

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

A SURVEY OF GASTROINTESTINAL PARASITES OF WILD AND ORPHAN GREATER ONE-HORNED RHINO ( RHINOCEROS UNICORNIS) IN KAZIRANGA NATIONAL PARK, ASSAM, INDIA.

Wild greater one-horned rhinos ( Rhinoceros unicornis), orphaned juveniles in human care, and orphaned calves from Kaziranga National Park, Assam, India were surveyed coprologically for gastrointestinal parasites. Parasite infections were present in 100% of wild rhino samples, 96% of orphaned juvenile samples, and 27% of orphaned calf samples. In wild rhino, observed parasite ova were primarily of trematodes Paramphistomum sp. (100%), followed by those of strongyle nematodes (94%) and the cestode Anoplocephala sp. (56%). Orphaned juvenile and calf samples were positive only for strongyles. Total fecal parasite egg counts were recorded in wild rhino (mean 64 eggs per gram [epg], range 0-270), orphan juveniles (mean 43 epg, range 0-145), and orphan calves (mean 2 epg, range 0-10). Results suggest that parasite infection in rhinos in this setting is common, though more extensive sampling would provide further information on epidemiology and potential impacts on individual health and population viability.

Sexual dimorphism and heightened conditional expression in a sexually selected weapon in the Asian rhinoceros beetle.

Among the most dramatic examples of sexual selection are the weapons used in battles between rival males over access to females. As with ornaments of female choice, the most "exaggerated" sexually selected weapons vary from male to male more widely than other body parts (hypervariability), and their growth tends to be more sensitive to nutritional state or physiological condition compared with growth of other body parts ("heightened" conditional expression). Here, we use RNAseq analysis to build on recent work exploring these mechanisms in the exaggerated weapons of beetles, by examining patterns of differential gene expression in exaggerated (head and thorax horns) and non-exaggerated (wings, genitalia) traits in the Asian rhinoceros beetle, Trypoxylus dichotomus. Our results suggest that sexually dimorphic expression of weaponry involves large-scale changes in gene expression, relative to other traits, while nutrition-driven changes in gene expression in these same weapons are less pronounced. However, although fewer genes overall were differentially expressed in high- vs. low-nutrition individuals, the number of differentially expressed genes varied predictably according to a trait's degree of condition dependence (head horn>thorax horn>wings>genitalia). Finally, we observed a high degree of similarity in direction of effects (vectors) for subsets of differentially expressed genes across both sexually dimorphic and nutritionally responsive growth. Our results are consistent with a common set of mechanisms governing sexual size dimorphism and condition dependence.

INSAR COHERENCE AND POLARIMETRIC PARAMETERS BASED CHARACTERIZATION OF FLOODED AREA – CASE STUDY OF A NATURAL WORLD HERITAGE SITE KAZIRANGA NATIONAL PARK

Abstract. Flood is a major threat to one of the UNESCO world heritage site of India-The Kaziranga National Park. Every year during the monsoon several hundreds of animals which include globally threatened species like single-horned Indian Rhinoceros of Kaziranga lose their lives due to the flood. The Synthetic Aperture Radar (SAR) can be used to monitoring the flood than the optical remote sensors because of their capability of all-weather and time-independent operability. The microwave L band is most suitable for the flood studies because of its higher penetration capability even through the vegetation. In this study, the advantages of SAR polarimetry and Interferometry of multi-temporal L band dual-pol data of ALOS PALSAR 2 were used to characterize the flooded area and also to monitor the flood extent. The H/ A/ Alpha decomposition gives a better characterization of the flooded area. The separability analysis is done with a different combination of decomposition parameters and the parameters having high-class separability between water and non-water areas are selected. Polarimetric classification using Random forest classifier is done on these selected decomposition parameters to classify the study into water and non-water areas. The classified images of different months before, during and after the flood time is used to quantitatively estimate the flood extent and for time series analysis. The Interferometric SAR coherence images along with the backscatter images are used to generate the RGB composites which also gives times series information on the flood impact.

Scanning transmission electron microscopic analysis of nitrogen generated by 3, 3'-diaminobenzidine-besed peroxidase reaction with resin ultrathin sections of rhinoceros parotid gland acinar cells.

A 3, 3'-diaminobenzidine (DAB)-based method was used to detect the localization of endogenous peroxidase activity in Indian rhinoceros (Rhinoceros unicornis) parotid gland acinar cells. The tissue had previously been resin-embedded in gelatin capsules for routine electron microscopic observations and thus pre-incubation for endogenous peroxidase analysis was not possible. We attempted to demonstrate the relationship between secretory granules (SGs) in resin ultrathin sections of Indian rhinoceros parotid gland acinar cells and endogenous peroxidase activity. A JEM 1400 Plus scanning transmission electron microscope (STEM) was used to conduct energy dispersive X-ray spectroscopy (EDS) analysis of the presence of nitrogen generated by the DAB reaction in bipartite structural SG consisting of a dense body (or core). The mapping patterns of nitrogen were restricted to the dense body. We observed nitrogen localized in the rough endoplasmic reticulum (ER), nuclear envelope (NE) and several components of the Golgi apparatus (G) of rhinoceros parotid gland acinar cells participating in the synthetic pathway of secretory proteins. Moreover, we established a nitrogen-detection method by EDS analysis of rhinoceros parotid gland. The reliability of the method was validated by comparison of the test group (peroxidase detection in ultrathin resin sections) and the control group (ordinary peroxidase detection in semi-thin sections following glutaraldehyde pre-fixation) of rat submandibular gland. The same mapping patterns of nitrogen were detected by DAB reaction in the SG, ER, NE and G in these two groups. Hence, EDS-STEM approaches for endogenous peroxidase post-incubation analysis will prove useful for advanced cytochemical analysis for the identification of any other resin sections.

Identification and mapping of risk areas of rhino poaching; a geospatial approach: A case study from eastern sector of Chitwan National Park, Nepal

Nepal has an appreciative history on conservation of rhino with remarkable proportions of Asian rhino population protected and managed within the protected areas. Poaching risk map promises early warning and a way to target preventive action, which can safeguard both human and ecosystem. This study was designed to identify and map risk areas of rhino (Rhinoceros unicornis) poaching within and around eastern sector of Chitwan National Park (CNP). A multi criterion GIS method was used to analyze and derive the risky zone. A binary logistic regression and expert consultation were done to finalize variables and risk rating, then weighted sum index model was performed in ArcGIS to derive risk zonation map. Presence/poaching and pseudo absence data were dependent variables and distance to guard post, settlement and road network from poaching events, land cover, slope and elevation were predictor variables for logistic regression model. Poaching events were observed to be spatially distributed around the park except in the south part. Among the seven predictable variables, five variables except terrain (slope and elevation) were statistically significant at 10% level of test (p&lt;0.1). The poaching risk map indicates that areas near to roads, far from the guard post, and densely populated area of grasslands are high risk zone areas for rhino poaching. The GIS based maps will be practical and strategically to wildlife managers in CNP to facilitate decision making on intervention programmes and how best to direct law enforcement patrol within and around the park. Banko JanakariA Journal of Forestry Information for NepalVol. 27, No. 2, 2017, Page: 12-20

MILK COMPOSITION OF INDIAN RHINOCEROS ( RHINOCEROS UNICORNIS) AND CHANGES OVER LACTATION.

The objective of this study was to determine the major nutrient composition of Indian rhinoceros milk ( Rhinoceros unicornis) over the first 13 mo of an 18-mo lactation period and to compare the results to those of previous studies on rhinoceros, African elephant ( Loxodonta africana), and horse milk ( Equus ferus caballus). The following parameters were measured: dry matter (DM), crude ash (ASH), crude protein (CP), ether extract (EE), nitrogen-free extract (NFE; calculated), lactose, calcium (Ca), phosphorus (P), magnesium (Mg), fatty acids (FAs), and gross energy (GE). DM, ASH, CP, and EE were determined with a proximate analysis, lactose with infrared spectroscopy and an enzymatic method, minerals with an autoanalyzer, FA with gas chromatography, and GE with bomb calorimetry. Milk samples were collected from two Indian rhinoceros cows from Zoo Basel. Rhino A gave birth to her third calf on 10 September 2012; three samples were collected and analyzed (colostrum, milk 1 wk and 2 wk postpartum). Rhino B gave birth to her eighth calf on 05 October 2013; samples were collected and 15 were chosen for the analyses (from colostrum to 13 mo postpartum). The composition of rhino B's colostrum was 13.8% DM (wet-weight basis), 4.8% ASH, 61.8% CP, 0.7% EE, 32.6% NFE, 26.7% lactose, 0.59% Ca, 0.54% P, 0.2% Mg (DM basis), and 20.3 MJ GE/kg DM. Rhino B's sample collected 13 mo postpartum averaged 8.0% DM (wet-weight basis), 3.6% ASH, 16.3% CP, 1.8% EE, 78.3% NFE, 84.7% lactose, 0.54% Ca, 0.48% P, 0.09% Mg (on DM basis), and 17.43 MJ GE/kg DM. The main FAs in rhino B's and rhino A's samples were C10 : 0, C12 : 0, C16 : 0, C18 : 1n9c, and C18 : 2n6c. Milk of the Indian rhinoceros is low in fat and protein but high in lactose, which is comparable to the milk composition of other rhinoceros species and horses, but not African elephants.

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 ± SD fresh ejaculate motility was 84.5 ± 7.6%, progression: 3.6 ± 0.6 (scale 0–5), viability: 83.4 ± 7.1%, intact acrosomes: 71.3 ± 6.9%, and morphologically normal: 78.8 ± 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.

&lt;b&gt;Habitat suitability and threat analysis of Greater One-horned Rhinoceros &lt;I&gt;Rhinoceros unicornis&lt;/I&gt; Linnaeus, 1758 (Mammalia: Perissodactyla: Rhinocerotidae) in Rautahat District, Nepal&lt;/b&gt;

The Greater One-horned Rhinoceros Rhinoceros unicornis has been listed as a Vulnerable species on IUCN Red List, Appendix I of CITES, and a protected animal under the National Parks and Wildlife Conservation Act 2029 B.S., 1973. In Nepal, it was found only in Chitwan, Bardia, Shuklaphanta and Parsa national parks, but it has recently been also reported from the forests of Rautahat. The main objectives of the study were to assess habitat suitability and threats for rhinoceros in Rautahat at an elevation range of approximately 300–1,000 m. Remote sensing data and geospatial modeling techniques were used to assess habitat suitability of rhinoceros. Vegetation assessment was carried out for tree, shrubs, and herbs of plot size 10m × 10m, 5m × 5m, 1m × 1m respectively for habitat suitability. Threat analysis was carried out using purposive sampling among local people and their perceptions were collected on the movement of rhinoceros and threats. The integration of nine explanatory variables showed that about 0.06%, 29.18%, 20.45%, and 50.31% of the study area was found to be most suitable, suitable, moderately suitable and unsuitable habitat respectively for rhinoceros. Out of 30 respondents, 37%, 23%, 20%, and 20% identified the main threat to rhinoceros to be unmanaged habitat, poaching, human-wildlife conflict and environmental factors, respectively. This study recommends parts of the Rautahat District to be extended as the habitat of rhinoceros and starting of immediate conservation initiatives in the area.

A Chronicle of Law Implementation in Environmental Conflicts: The Case of Kaziranga National Park in Assam (North-East India)

This text is based on an analysis of conflicts concerning Kaziranga National Park in Assam. Famous for the one-horned rhinoceros, this Park has become a political arena where tension has been mounting since its 2007 inclusion in a project aimed at protecting tigers. The physical injury and damage caused by wildlife to people and their property (land, animals, houses), the eviction of populations from within the Park’s perimeter, and poaching have led to conflicts that combine ecological, social, political, cultural and symbolic features, and which are being brought more and more often before the law courts. The aim of this paper is to show how these features are utilized by the different protagonists and how, since 2006, new decision-making bodies with extensive powers, environmental legislation, and nature-protection or population-defense organizations have contributed to defining the forms these conflicts have taken. Through the lens of environmental law in the making, it highlights some of the workings of India’s nature protection policy. The Kaziranga example provides an overview of the problems key measures or laws have created in the field of environmental protection, whether it is a question of them being applied too rigidly—as is the case of the Wildlife (Protection) Act 1972—, of them being bent—this is the case of Public Interest Litigations the original purpose of which has completely changed—, or of them not being applied at all—which is the case of the Forest Rights Act 2006 in Assam.

Seasonality, crop type and crop phenology influence crop damage by wildlife herbivores in Africa and Asia

Wildlife species damaging crops can cause substantial losses to farmers and at the same time create negative attitudes against wildlife and conservation efforts that may result in negative interactions against wildlife and lead to human-wildlife conflicts (HWCs). For the analysis of negative interactions between humans and terrestrial wildlife species, a globally applicable scheme for monitoring was developed and applied over 6 years in study areas of two Asian (Nepal and India) and two African (Zambia and Tanzania) countries. Factors influencing crop consumption by eight different groups of herbivores were monitored and analyzed using generalized linear models. Seasonality, crop availability, type and the phenological stage of the crop seem to play an important role in the crop damaging behavior of herbivores. Crop consumers such as elephants (Loxodonta africana and Elephas maximus), zebra (Equus quagga spp.) and boars/hogs (Sus scrofa, Potamocherus larvatus and Phacochoerus africanus) show preferences for harvested and/or maturing crops. Rhinos (Rhinoceros unicornis) and antelopes/deer (Taurotragus oryx, Aepyceros melampus, Boselaphus tragocamelus and Axis axis) damage the highest numbers of fields with crops at an intermediate growth stage. The findings of this study can inform management of HWCs in areas where people and wildlife coexist. Furthermore, this study demonstrates the benefits of standardized HWC assessments in order to compare data from different continents and between different species to be able to draw generalized conclusions for the management of HWC.

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.

The Fat-Dachsous signaling pathway regulates growth of horns in Trypoxylus dichotomus, but does not affect horn allometry

Males of the Asian rhinoceros beetle, Trypoxylus dichotomus, possess exaggerated head and thoracic horns that scale dramatically out of proportion to body size. While studies of insulin signaling suggest that this pathway regulates nutrition-dependent growth including exaggerated horns, what regulates disproportionate growth has yet to be identified. The Fat signaling pathway is a potential candidate for regulating disproportionate growth of sexually-selected traits, a hypothesis we advanced in a previous paper (Gotoh et al., 2015). To investigate the role of Fat signaling in the growth and scaling of the sexually dimorphic, condition-dependent traits of the in the Asian rhinoceros beetle T. dichotomus, we used RNA interference to knock down expression of fat and its co-receptor dachsous. Knockdown of fat, and to a lesser degree dachsous, caused shortening and widening of appendages, including the head and thoracic horns. However, scaling of horns to body size was not affected. Our results show that Fat signaling regulates horn growth in T. dichotomus as it does in appendage growth in other insects. However, we provide evidence that Fat signaling does not mediate the disproportionate, positive allometric growth of horns in T. dichotomus.