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.     

Polymorphic microsatellite loci in the endangered Indian rhinoceros,Rhinoceros unicornis

Eleven novel polymorphic microsatellite loci are presented for the highly endangered Indian (or greater one horned) rhinoceros Rhinoceros unicornis (Mammalia: Rhinocerotidae). These will be used to analyse the genetic variability within and between the two remaining large populations of the Indian rhinoceros and to manage captive breeding.     

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.     

Energy and mineral nutrition and water intake in the captive Indian rhinoceros (Rhinoceros unicornis)

In the captive Indian rhinoceros (Rhinoceros unicornis), two disease complexes with a high incidence—chronic foot problems and uterine leiomyomas—may be linked to excess body weight (BW). In this study, intake and digestion trials were conducted (by means of 7‐day weigh‐backs, and 5‐day total fecal collections, respectively) with 11 Indian rhinoceroses at four zoological institutions in Europe and the United States to quantify energy and mineral nutrition on conventional or roughage‐only diets. Diets comprising a variety of forages (grass hay only, a combination of grass hay and grass silage, straw, or a mixture of grass and legume hay) were offered as the roughage source, along with various concentrates, produce, and supplements. Water intake was quantified, and urine samples were obtained opportunistically. The animals consumed 0.5–1.1% of their BW in dry matter (DM) daily, with calculated digestible energy (DE, in megajoules MJ) values ranging from 0.27 to 0.99 MJ DE/kg BW^0.75/day compared to an estimated requirement of 0.49–0.66 MJ DE/kg BW^0.75/day. Seven of 11 rhinos (64%) fed restricted levels of concentrate plus forage consumed DE in excess of this estimate. Even on roughage‐only diets, some individuals consumed energy well above the presumed metabolic requirements. Hence, restriction of both concentrates and roughage may be important for weight management in this species. Water intake ranged from 30 to 49 mL/kg BW daily (3.4–5.2 L/kg ingested DM), similar to values that have been reported for domestic equids. Excretion amounts and patterns also resembled those found in horses. Endogenous fecal losses measured for Ca, P, Cu, Fe, and Zn indicate that the maintenance requirements of these minerals should be met in Indian rhinoceroses by diets that meet recommendations for domestic horses. It is particularly important to evaluate dietary adequacy in mineral nutrition in this species in concert with the need for restricted energy intake, especially with regard to the hypothetical involvement of a low Zn supply in chronic foot problems. Zoo Biol 24:1–14, 2005. © 2005 Wiley‐Liss, Inc.     

Behavioural ecology of the Greater one-horned rhinoceros (Rhinoceros unicornis)

In the Chitawan Valley of Nepal there were estimated to be between 270 and 310 Greater one-horned or Indian rhinoceroses (Rhinoceros unicornis L.). Population densities reached 4.85 rhinos/km² in the favoured areas of high diversity of early successional vegetation types on the valley floor. The overall population composition was 32.3% adult females, 19.9% adult males, 21.2% sub-adults and 26.6% calves. Females first calved at a mean age of 7.1 years and the median intercalving interval was 2.8 years. Causes of death included poaching, tiger predation on calves and fighting among males. The population was increasing.
Rhinos fed from 183 species of plants belonging to 57 botanical families but grass (50 species) made up between 70 and 89% of their diet according to the season. Considerable seasonal variations in food availability resulted in movements of rhinos between vegetation types. Rhinos' ranges were smallest in the areas of greatest vegetational diversity.
Rhinos rarely formed groups. The most common type consisted of sub-adults–mainly males. Ten auditory displays were distinguished and visual displays, although less striking, included baring the lower incisor tusks. Scents were carried in the dung, the urine and the pedal scent glands. Squirt urination and foot-dragging displays were performed by breeding males only.
There was some degree of range exclusivity among breeding males but no true territoriality. Poor visibility and the relatively unpredictable distribution of resources in time and space have perhaps selected against a territorial mating system. Relationships between ecology and social organization are discussed with reference to other rhino species.
Threats to the continued survival of the Indian rhino include poaching, agricultural encroachment and erosion. In order to spread the risk of a catastrophe, reintroductions of rhinos to other protected areas are proposed.     

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.     

Phylogenetic relationships of the five extant Rhinoceros species (Rhinocerotidae, Perissodactyla) based on mitochondrial cytochrome b and 12S rRNA genes

A major question in rhinocerotid phylogenetics concerns the position of the Sumatran rhinoceros (Dicerorhinus sumatrensis) with regard to the other extant Asian (Rhinoceros unicornis and R. sondaicus) and African (Diceros bicornis and Ceratotherium simum) species. We have examined this particular question through the phylogenetic analysis of the complete sequences of the mitochondrial 12S rRNA and cytochrome b genes. Three additional perissodactyls (one tapir and two equids) plus several outgroup cetartiodactyls were included in the analysis. The analysis identified a basal rhinocerotid divergence between the African and the Asian species, with the Sumatran rhinoceros forming the sister group of the genus Rhinoceros. We estimate the Asian and African lineages to have diverged at about 26 million years before present.     

Predicted declines in suitable habitat for greater one‐horned rhinoceros (Rhinoceros unicornis) under future climate and land use change scenarios

Rapidly changing climate is likely to modify the spatial distribution of both flora and fauna. Land use change continues to alter the availability and quality of habitat and further intensifies the effects of climate change on wildlife species. We used an ensemble modeling approach to predict changes in habitat suitability for an iconic wildlife species, greater one‐horned rhinoceros due to the combined effects of climate and land use changes. We compiled an extensive database on current rhinoceros distribution and selected nine ecologically meaningful environmental variables for developing ensemble models of habitat suitability using ten different species distribution modeling algorithms in the BIOMOD2 R package; and we did this under current climatic conditions and then projected them onto two possible climate change scenarios (SSP1‐2.6 and SSP5‐8.5) and two different time frames (2050 and 2070). Out of ten algorithms, random forest performed the best, and five environmental variables—distance from grasslands, mean temperature of driest quarter, distance from wetlands, annual precipitation, and slope, contributed the most in the model. The ensemble model estimated the current suitable habitat of rhinoceros to be 2610 km², about 1.77% of the total area of Nepal. The future habitat suitability under the lowest and highest emission scenarios was estimated to be: (1) 2325 and 1904 km² in 2050; and (2) 2287 and 1686 km² in 2070, respectively. Our results suggest that over one‐third of the current rhinoceros habitat would become unsuitable within a period of 50 years, with the predicted declines being influenced to a greater degree by climatic changes than land use changes. We have recommended several measures to moderate these impacts, including relocation of the proposed Nijgad International Airport given that a considerable portion of potential rhinoceros habitat will be lost if the airport is constructed on the currently proposed site.     

Phylogenetic reconstructions in the genus Capra (Bovidae, Artiodactyla) based on the mitochondrial DNA analysis

Mitochondrial genome fragments were examined in all species of the genus Capra (Bovidae, Artiodactyla). Phylogenetic analysis was carried out using 59 cytochrome b gene sequences (392 bp), and 22 sequences of the mtDNA variable fragment (402 bp). In the control region, two unique deletions were revealed. One of the deletions was found only in Capra cilindricornis (17 bp), while another one grouped C. caucasica with C. aegagrus (1 bp). The group of Caucasian wild goats splits into two clades, and furthermore, the sequences of C. caucasica demonstrate remarkable similarity to the sequences of C. aegagrus, while C. cylindricus seems to have evolved independently for a long period of time. It was demonstrated that C. pyrenaica and C. ibex were extremely close to one another. Capra sibirica formed an outer group relative to the other species, and according to our data, was the most ancient species of the genus. On the contrary, genetic distance separating C. falconeri (the most independent species of the genus related to its morphology) from the other species is small.     

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.     

Phylogenetic relationship among Libellula, Ladona and Plathemis (Odonata: Libellulidae) based on DNA sequence of mitochondrial 16S rRNA gene

Abstract. The type genus for the dragonfly family Libellulidae is Libellula. At present, Libellula s.l. includes twenty-nine species, whose distribution is largely Nearctic. Whether two other libellulid taxa, Ladona and Plathemis, should be considered synonyms of Libellula, subgenera of Libellula, or separate genera, has been a subject of intermittent debate for over a century. Earlier proposals concerning Ladona and Plathemis were based on a limited number of morphological characters and lacked rigorous phylogenetic analyses. Therefore, we used the DNA sequence of a portion of the mitochondrial 16S rRNA gene and parsimony, maximum likelihood and neighbour-joining analyses to explore whether Ladona and Plathemis are monophyletic lineages distinct from Libellula. We obtained ≈ 415 bp of DNA sequence from twenty-three taxa including thirteen species of Libellula s.s., all three recognized species of Ladona, the two species of Plathemis and representatives of four other libellulid genera. Tetragoneuria williamsoni (Odonata: Corduliidae) was included as the outgroup. Parsimony analysis suggested that Ladona and Plathemis are monophyletic lineages distinct from Libellula s.s. with a sister group relationship between Libellula and Ladona. The monophyly of Ladona, Plathemis and Libellula was supported in > 90% of bootstrap replications and in trees five to ten steps longer than the most parsimonious trees. Relationships inferred from maximum likelihood and neighbour-joining analyses also supported the monophyly of Ladona and Plathemis. The four other libellulid genera included in the study formed a monophyletic clade distinct from Libellula, Ladona and Plathemis. Based on our analysis, we propose that Ladona and Plathemis be considered either genera or subgenera within Libellulidae.     

The complete mitochondrial DNA sequence of the harbor seal,Phoca vitulina

Summary The nucleotide sequence of the mitochondrial DNA (mtDNA) of the harbor seal, Phoca vitulina, was determined. The total length of the molecule was 16,826 bp. The organization of the coding regions of the molecule conforms with that of other mammals, but the control region is unusually long. A considerable portion of the control region is made up of short repeats with the motif GTACAC particularly frequent. The two rRNA genes and the 13 peptide-coding genes of the harbor seal, fin whale, cow, human, mouse, and rat were compared and the relationships between the different species assessed. At ordinal level the 12S rRNA gene and 7 out of the 13 peptide-coding genes yielded a congruent topological tree of the mtDNA relationship between the seal, cow, whale, human, and the rodents. In this tree the whale and the cow join first, and this clade is most closely related to the seal.Offprint requests to: Ú. Árnason     

Phylogenetic relationships among the Canary Island Steganacaridae (Acari, Oribatida) inferred from mitochondrial DNA sequence data

The mite genus Steganacarus is represented in the Canary Islands by three endemic species, one recently discovered species, and several morphotypes of uncertain taxonomic position. We used a fragment of the mitochondrial cytochrome oxidase I gene to reconstruct the phylogenetic relationships among representatives of the different taxa from the three central islands of the archipelago, Tenerife, La Gomera and Gran Canaria. Sequence data were analysed by both maximum parsimony and maximum likelihood methods. The inferred phylogenetic relationships do not correlate well with current morphological taxonomy but reveal four deeply divergent and geographically coherent lineages, one each on Gran Canaria and La Gomera and two on Tenerife. No pattern of molecular differentiation was observed among different morphotypes. Possible explanations for this incongruence are suggested in relation to the ecology and biogeography of the group. A recently discovered Steganacarus species from La Gomera, morphologically quite distinct from the other Canarian Steganacarus, is clearly identified as a taxon distantly related to all the other Canarian samples.     

Phylogenetic and genomic analysis of the complete mitochondrial DNA sequence of the spotted asparagus beetle Crioceris duodecimpunctata

We report the complete mitochondrial DNA sequence of the spotted asparagus beetle, Crioceris duodecimpunctata. The genome complement, gene order, and nucleotide composition of this beetle's mitochondrial genome were found to be typical of those reported for other insects. Unusual features of this genome include the substitution of UCU for GCU as the anticodon for tRNA(Ser), an unusual TpsiC loop for the tRNA(Ile) gene, and the identification of a putative ATT start codon for cox1. The utility of complete mitochondrial genome data for phylogenetic inference of the insect orders was tested, and compared to that of cox1 and combined mitochondrial ribosomal DNA sequences. Even though the number of insect orders represented by complete mitochondrial genomes is still limited, several well-established relationships are evident in the phylogenetic analysis of the complete sequences. Monophyly of the orders Diptera, Lepidoptera, and Coleoptera were consistently recovered. Monophyly of the Holometabola was also observed in some (though not all) analyses. The accumulation of complete mitochondrial sequences from a broader array of insect orders holds the promise of clarifying the early diversification of insects.     

The complete mitochondrial DNA sequence of the crustacean Artemia franciscana

The complete mitochondrial DNA (mtDNA) sequence of the brine shrimp Artemia franciscana has been determined. It extends the present knowledge of mitochondrial genomes to the crustacean class and supplies molecular markers for future comparative studies in this large branch of the arthropod phylum. Artemia mtDNA is 15,822 nucleotides long, and when compared with its Drosophila counterpart, it shows very few gene rearrangements, merely affecting two tRNAs placed 3 downstream of the ND 2 gene. In this position a stem-loop secondary structure with characteristics similar to the vertebrate mtDNA L-strand origin of replication is found. This suggests that, associated with tRNA changes, the diversification of the mitochondrial genome from an ancestor common to crustacea and insects could be explained by errors in the mtDNA replication process. Although the gene content is the same as in most animal mtDNAs, the sizes of the protein coding genes are in some cases considerably smaller. Artemia mtDNA uses the same genetic code as found in insects, ATN and GTG are used as initiation codons, and several genes end in incomplete T or TA codons.Correspondence to: R. Garesse     

The complete mitochondrial DNA sequence of the Atlantic salmon, Salmo salar.

The complete sequence of the Atlantic salmon (Salmo salar) mitochondrial genome has been determined. The entire sequence is 16665 base pairs (bp) in length, with a gene content (13 protein-coding, two ribosomal RNA [rRNA] and 22 transfer RNA [tRNA] genes) and order conforming to that observed in most other vertebrates. Base composition and codon usage have been detailed. Nucleotide and derived amino acid sequences of the 13 protein-coding genes from Atlantic salmon have been compared with their counterparts in rainbow trout. A putative structure for the origin of L-strand replication (O(L)) is proposed, and sequence features of the control region (D-loop) are described.