Semen cryopreservation in the Indian rhinoceros (Rhinoceros unicornis)

The objective was to identify an extender and cryoprotectant combination for Indian rhinoceros (Rhinoceros unicornis) sperm that yielded high post-thaw sperm quality. Male Indian rhinoceroses (n = 6; 7.5-34 yr old) were anesthetized and subjected to a regimented electroejaculation procedure (75-100 mAmps; 4-10 volts; 7-150 stimuli; total of 10 electroejaculation procedures). High quality semen fractions from each ejaculate were divided into four aliquots and a 2 x 2 factorial design used to compare the effect of two sperm extenders (standard equine [EQ] and skim milk-egg-yolk-sugar [SMEY]), and two cryoprotectants (glycerol and dimethylsulfoxide [DMSO]). Cyropreserved samples were thawed and assessed for motility, viability and acrosome integrity over time. Electroejaculate fractions processed for cryopreservation had high sperm concentration (516 × 10⁶/mL) and motility (79%). Post-thaw sperm characteristics were higher (P < 0.05) when semen was cryopreserved in EQ versus SMEY. Post-thaw motility of sperm cyropreserved in EQ averaged 50-55% compared to 22-37% in SMEY, with no significant differences in sperm characteristics of samples cyropreserved in glycerol and DMSO. In conclusion, sperm collected from Indian rhinoceroses via electroejaculation were cryopreserved using EQ extender with either glycerol or DMSO; post-thaw quality was adequate for use in assisted reproductive procedures.     

Methods of semen collection in an ambulatory greater one-horned rhinoceros (rhinoceros unicornis)

Illegal poaching and reduced habitats have led to the endangered status of rhinoceroses. Collection of semen for preservation and future artificial insemination would therefore increase the reproductive potential of the rhinoceros. Although various collection methods have been attemped with individual rhinos, comparison between methods on a single animal have not been reported. This report details the application of six semen collection techniques on an unsedated and unrestrained greater one-horned rhino. The methods included different types of penile and/or rectal stimulation. Artificial vaginas and an inflatible probe for electroejaculation were specially constructed for semen collection. Of the various methods employed, penile massage consistently resulted in sperm-poor seminal fluid, but when preceded by either rectal massage or electroejaculation, seminal fluid with high sperm concentration was obtained.     

An unrecorded specimen of the Javan rhinoceros (Rhinoceros sondaicus)

An hitherto unpuhlicized specimen of the Javan (Sondaic) rhinoceros in the Manchester Museum is briefly described.     

Molecular basis for ATP/2,3-bisphosphoglycerate control switch-over (poikilotherm/homeotherm) an intermediate amino-acid sequence in the hemoglobin of the great Indian rhinoceros (Rhinoceros unicornis, Perissodactyla)

The complete primary structure of the two hemoglobin components of the Great Indian Rhinoceros (Rhinoceros unicornis) is presented. The ratio for the two components B(alpha 2 beta I2): A(alpha 2 beta II2) is 6:4. Polypeptide subunits were separated by chromatography on CM-cellulose in a buffer containing 8M urea. The sequence was studied by degradation of the tryptic and hydrolytic cleavage products in a liquid phase sequencer. At position beta NA2 component B has Asp, whereas component A has Glu, an ATP-binding site in fish and reptilian hemoglobins. The other phosphate binding sites i.e. beta NA1 Val, beta EF6 Lys and beta H21 His are identical with 2,3-bisphosphoglycerate-(DPG)binding sites in mammalian hemoglobins, whereby rhinoceros hemoglobin resembles both ATP-sensitive poikilotherm hemoglobin and DPG-sensitive mammalian hemoglobin. The two components (beta I/beta II) additionally differ by exchange of Glu----Gly at position beta A3 and Gln----Lys at position beta GH3. The significance of these changes is discussed. Oxygenation properties of the two hemoglobins components and their dependence on ATP and DPG are given. The structure and function of Rhinoceros hemoglobin may give an insight into the evolution of the organic phosphate binding in vertebrate hemoglobins.     

Kidney of the great Indian rhino Rhinoceros unicornis, Linnaeus

The kidney of R. unicornis has almost 80 closely apposed lobes, all appearing peripherally. Every lobe, almost enclosed by a collagenous septum, resembles a deformed truncated cone. The pelvis proper is a small pouch which divides into a cephalic and a caudal urothelial-lined fibromuscular conduit. The terminal collecting ducts of every lobe open into a tubus maximus. This is lined by cuboidal cells and otherwise has no wall. There is no papilla. All lobes finally empty through the 18 primary infundibular orifices at the pelvic conduits. A primary fibromuscular infundibulum typically yields a secondary one supplying an adjacent lobe. Two or three lobes can use a common tubus maximus by "convergence" of their medullae. Tubus maximus, terminal collecting ducts and deep outer medulla are embraced by a fibromuscular calyx which is the peripheral extension of an infundibulum and is fused to the outer medulla. There is thus no vault between medulla and calyx. Large intralobar veins are fused to the outer wall of the calyx. The possible significance of this is discussed. The cortex is the only part of a lobe which has contact with infundibulum, pelvic conduits, or pelvis proper. The kidney has about 16 million glomeruli which form 5.8% of the adult's cortical mass. Many adult mammals, from mouse to rhinoceros, fit into the log10-log10 slope relating number of glomeruli per kidney to body-mass. Neonatal rhinos at term have mature glomeruli throughout the cortex. The small size of the glomeruli and the large number per field allow 16 million in an 118-gm kidney.     

Urinary steroid evaluations to monitor ovarian function in exotic ungulates: III. Estrone sulfate and pregnanediol-3-glucuronide excretion in the Indian rhinoceros (Rhinoceros unicornis)

A study was undertaken to identify urinary estrogen and progesterone metabolites in the female Indian rhinoceros (Rhinoceros unicornis). Measurements of these metabolites were then used to monitor ovarian function and establish normal levels and patterns of steroid excretion during the estrous cycle and pregnancy. Urine samples were analyzed for estrone sulfate and pregnanediol-3-glucuronide (PDG) by direct radioimmunoassays. Both hormones produced discrete profiles reflecting ovarian activity in nonconceptive cycles. The estrous cycle was observed to be 48 days (range 39–64) with a mean follicular phase of 14.8 days (range 13–19), followed by a mean luteal phase of 19 days (range 17–21). Of the single gestation monitored, PDG levels rose above luteal phase levels by the third month after breeding and remained elevated throughout gestation. The combined estrogen and progesterone metabolite profiles present a complete evaluation of ovarian steriod production in the mature female Indian rhinoceros.     

Genetic diversity, phylogeny and conservation of the Javan rhinoceros (Rhinoceros sondaicus)

With a total population of less than 60 individuals limited to two locations, the Javan rhinoceros is perhaps the most endangered large mammal on earth. Although species specific information is crucial to its conservation, its precarious status, habitat inaccessibility, and behavioral adaptations pose major obstacles to its study. Here we report on the first genetic analysis of the two extant populations, in Ujung Kulon, Indonesia, and Cat Tien, Vietnam, and discuss their conservation. As its critically endangered status precluded invasive sampling, we extracted DNA from dung, amplifying and sequencing segments of the mtDNA 12S rRNA gene and the non-coding D-loop. Divergence between Javan rhinos from Ujung Kulon and Cat Tien was similar to that between recognized subspecies of African rhinos, and exceeded that between Sumatran rhinos. The Ujung Kulon and Cat Tien populations represent separate Evolutionary Significant Units, advocating independent management. However, given the precariousness of the Cat Tien population, demographic considerations may override genetic issues in the short term. Genetic diversity of Javan rhinos was low and population expansion in the immediate future will be critical for its survival.     

The complete mitochondrial DNA sequence of the greater Indian rhinoceros, Rhinoceros unicornis, and the Phylogenetic relationship among Carnivora, Perissodactyla, and Artiodactyla (+ Cetacea)

The sequence (16,829 nt) of the complete mitochondrial genome of thegreater Indian rhinoceros, Rhinoceros unicornis, was determined. Like otherperissodactyls studied (horse and donkey) the rhinoceros demonstrateslength variation (heteroplasmy) associated with different numbers ofrepetitive motifs in the control region. The 16,829-nt variety of themolecule includes 36 identical control region motifs. The evolution ofindividual peptide-coding genes was examined by comparison with a distantlyrelated perissodactyl, the horse, and the relationships among the ordersCarnivora, Perissodactyla, and Artiodactyla (+ Cetacea) were examined onthe basis of concatenated sequences of 12 mitochondrial peptide-codinggenes. The phylogenetic analyses grouped Carnivora, Perissodactyla, andArtiodactyla (+ Cetacea) into a superordinal clade and within this clade asister group relationship was recognized between Carnivora andPerissodactyla to the exclusion of Artiodactyla (+ Cetacea) . On the basisof the molecular difference between the rhinoceros and the horse and byapplying as a reference to Artiodactyl/Cetacean divergence set at 60million years ago (MYA), the evolutionary divergence between the familiesRhinocerotidae and Equidae was dated to approximately 50 MYA.     

Behavioural ecology of the Sloth bear (Melursus ursinus)

The Sloth bear ( Melursus ursinus ), which is endemic to the Indian subcontinent and Sri Lanka, has declined in numbers in many areas in recent years probably as the result of over-killing and habitat alteration. In the 544 km² Royal Chitawan National Park in the Nepal terai, we estimated there were a minimum of 55 bears or a crude density of 0˙1/km². During peak concentrations in the lowlying riverine forest/tall grass habitat in March, the ecological density was 0˙5/km².
Cubs still with their mothers comprised 24 % of the 161 bears classified between December 1972 and November 1975. Females comprised 48% of the adults classified. Average size of 24 litters was 1˙6 (1–2). Seventy-two per cent of the observations were of lone individuals. Three (an adult female with two cubs), was the largest group of bears seen. The only extended social group observed was the female-young unit although newly independent siblings may remain together for a short time. Maternal relations, adult interactions, tree marking, ranging, feeding and anti-predatory behaviour are described.
Examination of 139 droppings showed that these bears fed on at least 17 different fruits (47% occurrence), flowers, grass, honey, and six different insects (52% occurrence). Fruits were an important food from March to June with individual species predominating throughout their fruiting season. Termites, including Odontotermes obesus , were in the diet for most of the year. The Sloth bear was not observed preying on mammals and according to other observers, only rarely feeds on carrion. The ecological basis for selected aspects of Sloth bear biology and behaviour is discussed.     

Behavioural ecology of the black mongoose (Galerella nigrata) in Namibia

The black mongoose (Galerella nigrata) occurs in northern Namibia, where it occupies habitats dominated by large boulders (petrophily). Because of its restricted distribution, virtually nothing has been documented about its natural history. To fill this data gap, six males were radio-tracked in the foothills of the Erongo Mountains, Namibia in 2003. Although largely solitary, some home ranges overlapped nearly 100%. Males sometimes formed hunting diads similar to other congeners. The animals were diurnal and denned alone, using multiple sites haphazardly from night to night. The diet included many petrophilic vertebrates. The taxonomic status of the black mongoose is controversial; in addition to morphological evidence that it is a full species, it appears to be a habitat specialist compared with other forms of Galerella in southern African.     

The epizootiology of the baculovirus of the coconut palm rhinoceros beetle (Oryctes rhinoceros) in Tonga

The incidence and distribution of the baculovirus of the coconut palm rhinoceros beetle was determined in Tongatapu, Tonga, 7 years after being first introduced. Surveys showed the virus to be spread throughout the beetle population, affecting 14.6% of breeding sites and over 84% of all adult beetles taken. Counts of damaged palms and of breeding site occupancy indicated that beetle numbers had remained at low levels. It appears that the virus has the potential for long-term control of beetle populations in these habitats.     

Age estimation of the White rhinoceros (Ceratotherium simum)

Age estimation criteria for the southern White rhinoceros ( Ceratotherium simum simum ) are presented both for free-ranging live animals and for cranial material. These are based on: (i) size appearance and horn development of live animals; (ii) stages of tooth eruption; (iii) tooth wear classes; (iv) attrition in height of the first molar tooth; (v) counts of cementum lines visible in tooth sections. Selected measurements are presented for live animals, skulls and horns.
For live animals, eight size classes are distinguished, seven of these covering immature animals up to ten years of age. Sixteen tooth wear classes are established, based on eruption and surface wear of maxillary dentition. Chronological ages were assigned from individually known animals followed in the field, and from skulls from animals for which exact records of age were available, or which could be assigned to an age category from appearance at death. Cementum line counts corresponded approximately with age in years, despite difficulties in interpreting lines. Some variability was observed, possibly related to nutritional conditions.
The maximum cementum line count obtained indicates a longevity of at least 40 years. Full body weight and socio-sexual maturity are attained by males between 10 and 15 years of age, while females first give birth between six and eight years of age. Sequences and times of tooth eruption are similar to those reported for the Black rhinoceros ( Diceros bicornis ).
Comparative cranial and body measurements are presented for the northern subspecies ( Ceratotherium simum cottoni ).     

Conservation genetics of the black rhinoceros (Diceros bicornis)

We analysed genetic variation in mitochondrial DNA (mtDNA) control region in the Diceros bicornis minor and D. b. michaelianimals at the Western Plains Zoo, which form part of an international ex-situ breeding program. Six of the nine D. b. minor animals were wild-caught from Zimbabwe during the 1990s, and our study revealed five distinct mtDNA haplotypes, and a haplotype diversity of 0.86 in the colony. Phylogenetic relationships between mtDNA haplotypes analyzed using the neighbor joining method reveal that for the small sample available, D. b. minor and D. b. michaeli are reciprocally monophyletic and represent separate ancestral lineages. Nucleotide divergence between the black and and white rhinoceros(Ceratotherium simum) was 14.0%, and nucleotide divergencebetween the D. b. minor and the D. b. michaeli subspecies was 2.6%. This suggests a divergence time for the two blackrhinoceros subspecies of between 0.93 MY and 1.3 MY.