Characterization of new microsatellite markers derived from sequence databases for the emu (Dromaius novaehollandiae)

The emu (Dromaius novaehollandiae), a member of ratite family, is native to Australia and has been introduced to other countries worldwide. In this work, 10 polymorphic microsatellite loci were isolated and characterized for emu from public sequences. Polymorphism was surveyed in 22 individuals from two different populations kept in captivity. Between two and 11 alleles were found per locus, and the observed heterozygosity ranged from 0.05 to 0.85, in accordance with expectations. These markers will be useful as tools for detecting levels of genetic variation, reconstructing pedigrees (for quantitative genetic analysis) and identifying markers associated to fitness traits in emu populations.     

Development of a CAPS (cleaved amplified polymorphics sequence) assay for sex identification of the emu (Dromaius novaehollandiae)

Polymerase chain reaction (PCR) based procedures that have been used to identify the sex of most birds cannot be used in ratites. This paper describes the identification of a female (W‐chromosome) specific randomly amplified polymorphic (RAPD) 1.3 kb DNA fragment (ESEXW) in the emu (Dromaius novaehollandiae). Southern blot experiments and sequence analysis revealed that a related (96% similarity) sequence exists on the emu Z‐chromosome (ESEXZ). The sequences of ESEXW and ESEXZ have been used for the development of a two‐primer CAPS (cleaved amplified polymorphic sequence) assay for reliable sex identification of the emu.     

Distribution and characterization of microsatellites in the emu (Dromaius novaehollandiae) genome

This study generates data concerning the genome of a flightless species of bird, the emu. We examined and ultimately rejected the following hypotheses: (1) Microsatellites are randomly distributed throughout the emu genome. (2) The relative order of abundance of dinucleotides will be constant across genomes. (3) Interspersion distances for a given dinucleotide will be equal across vertebrate genomes. (4) In all genomes, a dinucleotide will be more frequent than any trinucleotide. (5) The percentage of single-copy DNA will remain the same in emus as in other volant birds. A cosmid library representing 4.48% of the emu genome was probed with 23 microsatellites. Hybridizations were scored on a scale of 0-3. The average insert size, approximately 40 kb, was used to determine frequency and interspersion. The cosmid library was probed with genomic DNA to determine the percent single copy. Co-occurrence frequencies and confidence intervals were compared to expected using chi-squared. The genome is estimated to contain a microsatellite repeat every 48 kb. Of 1632 clones probed for single-copy DNA, 643 displayed maximal hybridization, 220 displayed moderate hybridization, and 202 had minimal hybridization. After 3 days, 567 showed no hybridization.     

Pelvic limb musculature in the emu Dromaius novaehollandiae (Aves: Struthioniformes: Dromaiidae): Adaptations to high-speed running

Emus provide an excellent opportunity for studying sustained high-speed running by a bird. Their pelvic limb musculature is described in detail and morphological features characteristic of a cursorial lifestyle are identified. Several anatomical features of the pelvic limb reflect the emus' ability for sustained running at high speeds: (1) emus have a reduced number of toes and associated muscles, (2) emus are unique among birds in having a M. gastrocnemius, the most powerful muscle in the shank, that has four muscle bellies, not the usual three, and (3) contribution to total body mass of the pelvic limb muscles of emus is similar to that of the flight muscles of flying birds, whereas the pelvic limb muscles of flying birds constitute a much smaller proportion of total body mass. Generally, the pelvic limb musculature of emus resembles that of other ratites with the notable exception of M. gastrocnemius. The presence and arrangement of four muscle bellies may increase the effectiveness of M. gastrocnemius and other muscles during cursorial locomotion by moving the limb in a cranio-caudal rather than a latero-medial plane. J. Morphol. 238:23–37, 1998. © 1998 Wiley-Liss, Inc.     

Thermoregulation in a large bird,the emu (Dromaius novaehollandiae)

The emu is a large, flightless bird native to Australia. Its habitats range from the high snow country to the arid interior of the continent. Our experiments show that the emu maintains a constant body temperature within the ambient temperature range-5 to 45°C. The males regulate their body temperature about 0.5°C lower than the females. With falling ambient temperature the emu regulates its body temperature initially by reducing conductance and then by increasing heat production. At-5°C the cost of maintaining thermal balance is 2.6 times basal metabolic rate. By sitting down and reducing heat loss from the legs the cost of homeothermy at-5°C is reduced to 1.5 times basal metabolic rate. At high ambient temperatures the emu utilises cutaneous evaporative water loss in addition to panting. At 45°C evaporation is equal to 160% of heat production. Panting accounts for 70% of total evaporation at 45°C. The cost of utilising cutaneous evaporation for the other 30% appears to be an increase in dry conductance.Abbreviations A _r Effective radiating surface area - BMR basal metabolic rate - C _dry dry conductance - CEWL cutaneous evaporative water loss - EHL evaporative heat loss - EWL evaporative water loss - FECO₂ fractional concentration of CO₂ in excurrent air - FF_H2O water content of chamber excurrent air - FEO₂ fractional concentration of O₂ in chamber excurrent air - FICO₂ fractional concentration of CO₂ in incurrent air - FIO₂ fractional concentration of O₂ in chamber incurrent air - MHP metabolic heat production - MR metabolic rate - REWL respiratory evaporative water loss - RH relative humidity - RQ respiratory quotient ; - SA surface area - SEM standard error of the mean - SNK Student-Newman-Keuls multiple range test - STPD standard temperature and pressure dry - T _a ambient temperature(s) - T _b body temperature(s) - T _e surface temperature(s) - flow rate of air into the chamber - carbon dioxide production - oxygen consumption - vapour pressure of water     

Comparative morphology,morphometry and distribution pattern of the trigeminal nerve branches supplying the bill tip in the ostrich (Struthio camelus) and emu (Dromaius novaehollandiae)

The subdivisions of the trigeminal nerve (N. ophthalmicus R. medialis and N. intramandibularis) innervating the upper and lower bill tip, respectively, were well developed in both the ostrich and emu and displayed extensive branching. Transmission electron microscopy revealed that both nerves displayed features typical of a mixed, peripheral nerve. Nerve fibre size in the ostrich and emu was larger than that reported in domestic poultry. There were a significantly higher number of myelinated nerve fibres in the N. ophthalmicus R. medialis in the emu by comparison with the same nerve in the ostrich, or by comparison with the N. intramandibularis of either species. As myelinated nerve fibres supply Herbst corpuscles, and these structures have been demonstrated in this region in these two species, this may indicate that the upper bill of the emu is more sensitive to vibrational stimuli than the upper bill of the ostrich or the lower bill of both species. The large size of the nerve fibres, the high nerve fibre count and the particular distribution of the nerves in the bill tip support the existence of a well‐developed sensory area in this region of the ostrich and emu.     

Contact stress distributions on the femoral head of the emu (Dromaius novaehollandiae)

Osteonecrosis of the femoral head remains a challenging orthopaedic problem. The disease frequently progresses to femoral head collapse, leading to debilitating osteoarthritis in the affected hip(s). Since a major goal of pre-collapse interventions is to forestall the need for hip arthroplasty, it is important that any animal models used to develop or study such interventions also have a natural history of progression to femoral head collapse. The emu (Dromaius novaehollandiae), a large flightless bird native to Australia, consistently progresses to femoral head collapse when osteonecrosis is experimentally induced cryogenically. Full biomechanical characterization of the demands this animal places on its hip is an important consideration in future usage of this model. This study reports in vitro measurement of the contact stress distributions on the emu femoral head during stance phase of the gait cycle, using Fuji pressure-sensitive film. Applied hip loadings were based upon ground reaction forces and hip flexion angles recorded in vivo. The contact stress data showed reasonable homology with the human hip, both in terms of stress magnitude and sites of habitual loading on the femoral head.     

Hip joint contact force in the emu (Dromaius novaehollandiae) during normal level walking

The emu is a large, (bipedal) flightless bird that potentially can be used to study various orthopaedic disorders in which load protection of the experimental limb is a limitation of quadrupedal models. An anatomy-based analysis of normal emu walking gait was undertaken to determine hip contact forces for comparison with human data. Kinematic and kinetic data captured for two laboratory-habituated emus were used to drive the model. Muscle attachment data were obtained by dissection, and bony geometries were obtained by CT scan. Inverse dynamics calculations at all major lower-limb joints were used in conjunction with optimization of muscle forces to determine hip contact forces. Like human walking gait, emu ground reaction forces showed a bimodal distribution over the course of the stance phase. Two-bird averaged maximum hip contact force was approximately 5.5 times body weight, directed nominally axially along the femur. This value is only modestly larger than optimization-based hip contact forces reported in literature for humans. The interspecies similarity in hip contact forces makes the emu a biomechanically attractive animal in which to model loading-dependent human orthopaedic hip disorders.     

Persistence of Meckel's cartilage in sub-adult Struthio camelus and Dromaius novaehollandiae

This study describes the persistence of an embryonal structure through to sub-adulthood in the ostrich and emu. Mandibles from sub-adult ostrich and emu were subjected to special staining, light microscopy and dissected to reveal and describe Meckel's cartilage. Meckel's cartilage, composed of hyaline cartilage, was present within the neurovascular canal of both species. The persistence through to sub-adulthood of Meckel's cartilage in the ostrich and emu is a feature not previously reported in any other avian species. The proximal end of Meckel's cartilage was ossified in the region of the articular bone and the distal end was ossified in some specimens. Although this structure may ossify at a much later stage in life, the function in young and sub-adult birds may be to dampen shockwaves along the intramandibular nerve that result from the action of pecking. In the ostrich, the M. pseudotemporalis superficialis tendon inserted onto the supra-angular bone and Meckel's cartilage. In the emu, a small portion of the tendon was attached to the supra-angular bone and the main part to Meckel's cartilage. The persistence of Meckel's cartilage in adult lepidosaurs, crocodilians and ratites represents an unusual shared trait between the extant members of the above groups.     

Structural peculiarities associated with multiflagellate sperm in the emu, Dromaius novaehollandiae

Multiflagellate sperm represent a rare defect in mammals and also in the emu where an incidence of only 1% multiflagellate sperm was recorded in semen samples from 15 birds. Biflagellate sperm were the most frequent form of the defect observed, but 3- to 5-tailed sperm were also noted. An association was apparent between multiple tails and macrocephalic sperm, which accounted for 0.6% of multiflagellate sperm. Structural features of the defect were generally similar to those seen in mammals. The duplicated tails shared a single midpiece, which housed supernumerary centriolar complexes, each surrounded by a mitochondrial sheath. A single row of mitochondria separated adjacent centriolar complexes. Elements of the connecting piece (segmented columns, capitellum) appeared normal. The nuclear base appeared flat, staggered, or scalloped depending on the number and depth of additional implantation fossae. Multiflagellate emu sperm of normal head dimensions displayed a widened nuclear base (in the form of an attenuated peripheral nuclear extension) to accommodate the attachment of the additional centriolar complexes. Defective mammalian sperm do not show this modification of the nuclear base as the inherently wider sperm head is able to accommodate the supernumerary centrioles. Although often spiraled around each other, the duplicated principal pieces of the tail were generally separated and free. However, in some cells the proximal parts were collectively bound within the plasmalemma. Multiflagellate sperm appear to have a dual origin with some defective cells originating from incomplete cytokinesis and others as a result of abnormal centriolar duplication.     

Molecular characterization of goose- and chicken-type lysozymes in emu (Dromaius novaehollandiae): evidence for extremely low lysozyme levels in emu egg white

Lysozyme (LZ), a bacteriolytic enzyme, is found in the egg white of many avian eggs and plays an important role in host defense; however, LZ activity in emu (Dromaius novaehollandiae) egg white is exceptionally undetectable. We cloned and characterized emu goose-type LZ (LZG) and chicken-type LZ (LZC) genes. RT-PCR analysis revealed very low LZG gene expression levels and absence of LZC gene expression in the emu oviduct. Sequencing of full-length LZG and LZC cDNAs indicated that their amino acid sequences show high similarities to ostrich LZG and LZC, respectively, with conserved catalytic residues for enzymatic activities. Whereas recombinant emu LZG prepared using Pichia pastoris exhibited similar enzyme activity as ostrich LZG, recombinant emu LZC exhibited significantly higher lytic activity than chicken LZC. We concluded that emus have functional genes for both LZG and LZC like many other avians, and the LZG gene is expressed in oviduct probably as in other ratite, however, its expression levels in egg white were low to be detected.     

Response of spermatozoa from the emu (Dromaius novaehollandiae) to rapid cooling, hyperosmotic conditions and dimethylacetamide (DMA)

Three experiments conducted to improve the survival of emu sperm during cryopreservation aimed to: (1) minimize chilling injury during the cooling phase; (2) determine the osmotic effects of dimethylacetamide (DMA), sucrose and trehalose; and (3) investigate the timing and nature of cryoprotectant toxicity. We measured sperm membrane integrity, motility, morphology and egg membrane penetration. In Experiment 1, semen diluted 1:1 with a pre-cooled diluent (5°C) prevented chilling injury. In Experiment 2, semen was diluted with DMA, trehalose or sucrose (300-2400mOsm/L) in deionized water. Only added DMA decreased the percentage of morphologically normal sperm. The percentage of motile sperm was higher with DMA than with the sugars, but membrane intact sperm were comparable amongst all cryoprotectants. As for the osmotic effects, the percentage of membrane intact sperm decreased with 2400mOsm/L and sperm motility decreased with 1200-2400mOsm/L, but sperm morphology was similar at all osmolarities. In Experiment 3, sperm membrane integrity, motility and morphology were comparable at all DMA osmolarities between sperm equilibrated for 0 and 15min, and remained unchanged after removal of DMA. We conclude that: (a) loss of sperm function during the cooling phase can be avoided by using a diluent maintained at 5°C; (b) emu spermatozoa tolerate upto 1400mOsm/L; (c) DMA results in a permanent change in sperm morphology when it is dissolved in deionized water, but does not alter sperm membrane integrity and motility; and (d) equilibration time of sperm with DMA can be less than 10min.     

Recombination and nucleotide diversity in the sex chromosomal pseudoautosomal region of the emu, Dromaius novaehollandiae

Pseudoautosomal regions (PARs) shared by avian Z and W sex chromosomes are typically small homologous regions within which recombination still occurs and are hypothesized to share the properties of autosomes. We capitalized on the unusual structure of the sex chromosomes of emus, Dromaius novaehollandiae, which consist almost entirely of PAR shared by both sex chromosomes, to test this hypothesis. We compared recombination, linkage disequilibrium (LD), GC content, and nucleotide diversity between pseudoautosomal and autosomal loci derived from 11 emu bacterial artificial chromosome (BAC) clones that were mapped to chromosomes by fluorescent in situ hybridization. Nucleotide diversity (pi = 4N(e)mu) was not significantly lower in pseudoautosomal loci (14 loci, 1.9 +/- 2.4 x 10(-3)) than autosomal loci (8 loci, 4.2 +/- 6.1 x 10(-3)). By contrast, recombination per site within BAC-end sequences (rho = 4Nc) (pseudoautosomal, 3.9 +/- 6.9 x 10(-2); autosomal, 2.3 +/- 3.7 x 10(-2)) was higher and average LD (D') (pseudoautosomal, 4.2 +/- 0.2 x 10(-1); autosomal, 4.7 +/- 0.5 x 10(-1)) slightly lower in pseudoautosomal sequences. We also report evidence of deviation from a simple neutral model in the PAR and in autosomal loci, possibly caused by departures from demographic equilibrium, such as population growth. This study provides a snapshot of the population genetics of avian sex chromosomes at an early stage of differentiation.     

Unusual Gangliosidosis in Emu (Dromaius novaehollandiae)

Abstract: A previous study has demonstrated an unusual gangliosidosis in emu that is characterized by the accumulation of gangliosides in the brain tissues with GM3 and GM1 predominating. To provide insight into this unique disorder of emu gangliosidosis, the current study focused on analysis of neutral glycosphingolipids and gangliosides from brain and liver tissues of affected birds and healthy controls. We found not only that the total lipid-bound sialic acid content was increased three- and fourfold in the affected brain and liver, respectively, but also that the ganglioside pattern was rather complex as compared with the control. The absolute ganglioside sialic acid content was significantly increased in the diseased tissues, with the highest elevation levels of GM3 (14-fold) and GM1 (ninefold) in the affected brain. Relative increases in content of these monosialogangliosides were also significant. GM2 was only detected in the affected brain, but not in normal controls. The neutral glycosphingolipid fraction showed accumulation of many oligosylceramides, with six- and 5.5-fold increases in lactosylceramide levels for brain and liver, respectively. The level of myelin-associated galactosylceramide (GalCer) in the brain was decreased to only 41% of that in the healthy control, whereas no difference was found in liver tissues from both groups. Besides GalCer, the brain content of sulfatide (cerebroside-sulfate esters), another myelin-associated glycolipid, decreased to only 16% of the control. The loss of myelin-associated GalCer and sulfatide strongly suggests demyelination in the affected emu brain. Our overall data are consistent with the presence of a unique form of sphingolipidosis in the affected emus, perhaps with secondary demyelination, and suggest a metabolic disorder related to total sphingolipid activator deficiency.