Tesi sul tema "Marsupialia Reproduction"

Bibliography: leaves 262-298. This thesis investigated the structure and glycoconjugate composition of the zona pellucida (ZP) surrounding marsupial oocytes and the changes that occur during ovarian development, following ovulation, and following cortical granule exocytosis. The glycoconjugates of the oviduct epithelial lining of the brushtail possum around the time of ovulation were also examined to determine if there was any contribution of the oviductal secretions to the post-ovulatory ZP.

Orientador: Sergio Furtado dos Reis
Tese (doutorado): Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Gracilinanus microtarsus (Marsupialia: Didelphidae) é um pequeno marsupial de hábitos noturnos e arborícola que habita áreas de Mata Atlântica e Cerrado no Brasil. Os objetivos deste trabalho foram determinar a posição de G. microtarsus ao longo de duas dimensões ecológicas ? estratégia reprodutiva e dieta ? e definir níveis de agregação relevantes à dinâmica populacional da espécie. O estudo foi realizado no período de agosto de 2000 a fevereiro de 2003 em uma área de cerradão localizada no município de Américo Brasiliense, São Paulo. Os dados foram obtidos de indivíduos capturados em uma grade de captura de 3.600 m2. No total, foram capturados 91 indivíduos de G. microtarsus. Os resultados mostraram que os machos apresentam altas taxas de mortalidade após o início do período reprodutivo, indicando que G. microtarsus é melhor descrito como semélparo parcial. Usando as estimativas de sobrevivência e dados adicionais sobre a biologia de G. microtarsus, foi construído um modelo de dinâmica populacional estocástico. Os resultados das simulações desse modelo estocástico mostraram que as probabilidades de quasi-extinção e de extinção de G. microtarsus são sensíveis ao número de indivíduos que sobrevivem à queimadas no cerradão. Quanto à dieta, os resultados mostraram que G. microtarsus é principalmente insetívoro e que sua dieta é significativamente influenciada pelo sexo, estação e recurso alimentar. Além disso, a dieta varia entre indivíduos e a variação interindividual também é influenciada pelo sexo e estação
Abstract: The gracile mouse opossum Gracilinanus microtarsus (Marsupialia: Didelphidae) is a small marsupial with nocturnal and arboreal habits that lives in the Atlantic Rainforest and forested areas of the Cerrado in Brazil. The objectives of this study were to determine the placement of G. microtarsus along two ecological scales, namely, reproductive strategy and diet, as well as to define levels of aggregation relevant to the dynamics of this species. This study was conducted from August 2000 to February 2003 in an area of cerradão located in Américo Brasiliense, São Paulo. Data were collected from individuals captured in a 3,600 m2 trapping grid. A total of 91 individual G. microtarsus was captured. The results showed that males have high mortality rates after the beginning of the mating period, which suggests that G. microtarsus is best described as partially semelparous. Using survival estimates and additional data on the biology of G. microtarsus, it was constructed a stochastic population dynamic model. The results of the simulations showed that quasi-extinction and extinction probabilities of G. microtarsus are sensitive to the number of individuals that survive fires in the cerradão. As for the diet, the results showed that G. microtarsus is primarily insectivorous and that its diet is significantly affected by sex, season, and food resource. Furthermore, diet varies among individuals and the variation also is affected by sex and season
Doutorado
Ecologia
Doutor em Ecologia

Many Australian marsupial species require active population management to ensure their survival in the wild. Such management should be based on a sound understanding of species biology. This thesis explores how knowledge of reproduction and genetics can be applied to the management of three Australian marsupial species faced with contrasting management scenarios. The ??vulnerable?? greater bilby is the sole remaining desert bandicoot in Australia. They are a secretive, solitary species whose mating system is unclear. This research examined temporal changes in genetic diversity within two captive breeding programs utilising different management strategies. Using seven microsatellite loci, this study found the regular translocation of new individuals into the population maintained genetic diversity. Parentage analysis revealed the bilby to have a promiscious mating system. Sires and non-sires could not be distinguished by morphological traits. The tammar wallaby is a polygynous, solitary species that is threatened on mainland Australia, but overabundant on some offshore islands. The population genetics of tammars from the Abrolhos Islands in Western Australia were examined using nine autosomal and four Y-linked microsatellite loci, and mitochondrial DNA. There was a relationship between island size, population size and genetic diversity. The Abrolhos populations have significantly lower genetic diversity and are more inbred than mainland tammars and all sampled populations were significantly differentiated. The Abrolhos and mainland populations should be treated as separate Management Units. The eastern grey kangaroo is a gregarious, polygynous species that is often locally overabundant. To determine traits influencing male reproductive success, behavioural, morphological, physiological and genetic data were examined and showed dominance status, body size and testosterone concentrations were important factors. Sires were also significantly more heterozygous and genetically dissimilar to females, than non-sires. As body condition influences individual fitness, and management decisions; five body condition indices (BCI) calculated from morphological data were validated using serum biochemistry and haematology in two kangaroo populations with contrasting body condition. Blood parameters were found to be more reliable indicators of condition, questioning the credibility of BCIs currently used in management. These studies demonstrate the importance of reproductive and genetic data in assisting wildlife management, regardless of a species conservation status.

Plasma progesterone concentrations were used as a measure to determine the "ram effect" on nine cycling springbok ewes. Blood was collected biweekly, prior to and after ram introduction. Ewes were subjected to the ram for a period of forty two days. Blood was analysed for plasma progesterone by means of radioimmunoassays. No significant changes in the plasma progesterone concentrations were detected. Superficially, a reduction in the variation of the follicular phase of the ewes oestrous cycle was noted for before- to after ram introduction. However, possibly because of a too small sample size, no statistical significance was found. It is suggested that the "ram effect" on aseasonal cycling species may only have a synchronisation effect (reduction in the variation of time between the follicular phase of females) but more individuals should be used in future experiments to make any clear and definite conclusions. Furthermore other hormones such as e.g. luteinizing hormone, which may be less affected by translocation and handling stress, should also be used to determine the "ram effect" on aseasonally breeding wild ungulates.
Dissertation (MSc)--University of Pretoria, 1999.
Veterinary Wildlife Unit
MSc
Unrestricted

Die Dissertation beschäftigt sich mit den möglichen Gründen für die divergente evolutionäre Entwicklung von Beuteltieren und plazentalen Säugetieren nach der Kreide/Tertiär-Grenze. Eine Erklärung könnten ihre unterschiedlichen Reproduktionsstrategien sein. Während die Beuteltiere sehr embryonale Jungtiere nach einer kurzen Tragzeit gebären, bringen Plazentalier deutlich weiter entwickelte Jungtiere nach einer relativ langen Tragzeit zur Welt. Die Aufrechterhaltung eines stabilen Metabolismus und thermoregulatorische Fähigkeiten der Jungtiere bieten einen großen Vorteil für die Anpassungsfähigkeit an ungünstige Umweltbedingungen, wie sie für die K/T-Grenze vermutet werden. Aus diesem Grund untersucht diese Studie den strukturelle Entwicklungsgrad der Lunge und die metabolischen Fähigkeiten von neonaten Marsupialia und Plazentalia. Histologische, ultrastrukturelle und kalorimetrische Untersuchungen erfolgten in einer integrativen Studie. Basierend auf den Ergebnissen wurde eine Grundplanrekonstruktionen der Neonaten von Marsupialia und Plazentalia durchgeführt. Als Vertreter für nesthockende Plazentalia wurde der Goldhamster (Mesocricetus auratus), die Moschusspitzmaus (Suncus murinus) und das Belangeri Spitzhörnchen (Tupaia belangeri) untersucht. Das Wildmeerschweinchen (Cavia aperea) und die Kurzohr-Elefantenspitzmaus (Macroscelides proboscideus) repräsentieren die nestflüchtenden Plazentalia. Als Marsupialia wurden die Hausspitzmaus-Beutelratte (Monodelphis domestica) und das Tammar-Wallaby (Macropus eugenii) untersucht. Die Ergebnisse bestätigen die starken Unterschiede im Entwicklungsgrad der Neonaten und in der postnatalen Entwicklung zwischen Marsupialia und Plazentalia. Die neonatalen Lungen von M. auratus und S. murinus befinden sich im späten “terminal air sac”-Stadium und weisen viele kleine Atemkammern von 50-80 µm Durchmesser auf. Die Alveolenbildung erfolgt bei M. auratus und S. murinus bereits im Alter von zwei beziehungsweise vier Tagen. Bei T. belangeri, C. aperea und M. proboscideus sind Alveolen bereits zum Zeitpunkt der Geburt vorhanden. Im Gegensatz dazu, befinden sich die Lungen der neonaten Beuteltiere M. domestica und M. eugenii im frühen “terminal air sac”-Stadium mit wenigen großen Atemkammern von 300-400 µm im Durchmesser. Die postnatale Lungenentwicklung erfolgt sehr langsam und die Alveolenbildung startet mit 28 Tagen bei M. domestica und mit 65 Tagen bei M. eugenii. Die Metabolismusmessungen ergaben, daß Marsupialia mit einer niedrigen Metabolismusrate geboren werden und den Adultmetabolismus erst spät in der postnatalen Entwicklung erreichen. Einhergehend mit der weit entwickelten Lungenstruktur weisen die Plazentalia hohe Metabolismusraten zur Geburt auf und erreichen den Adultmetabolismus innerhalb der ersten Lebenswoche. Die Ergebnisse zeigen, dass Jungtiere der Plazentalia, im Vergleich zu jungen Marsupialia, eine höhere Widerstandskraft gegen Umweltschwankungen haben, was als ein evolutiver Vorteil der Reproduktionsstrategie der Plazentalia unter ungünstigen Klimabedingungen interpretiert werden kann.
This project deals with the possible reasons for the evolutionary differentiation between marsupial and placental mammals after the K/T-event. One explanation could be their different reproductive patterns. Marsupialia bear virtually embryonic young after a brief gestation period. In contrast, many eutherians bear anatomically advanced, highly precocious young after a relatively long gestation period. A stable metabolism and thermoregulatory abilities of the young are considered to offer a large adaptive advantage in a changing environment, how it is presumed for the K/T-boundary. Therefore this study determines the developmental stage and the respiratory efficiency of the lungs of marsupial and placental young. Histological, ultrastructural and calorimetric investigations were carried out in an integrated study and from the results morphotype reconstructions of the marsupial and placental neonates were carried out. As representatives for altricial Placentalia, the Golden hamster (Mesocricetus auratus), the Musk shrew (Suncus murinus), and the Belanger’s tree shrew (Tupaia belangeri) were examined. Furthermore the Guinea pig (Cavia aperea) and Short-eared elephant shrew (Macroscelides proboscideus) as typical precocial Placentalia were included. The Marsupialia were represented by the Grey short-tailed opossum (Monodelphis domestica) and the Tammar wallaby (Macropus eugenii). The results confirm clear differences in the developmental degree of the neonates and the postnatal development between marsupial and placental mammals. The newborn lungs of the altricially born placentals M. auratus and S. murinus are at the late terminal air sac stage with numerous small air sacs of 50 - 80 µm in diameter. Alveoli are formed shortly after birth at the age of 2 days in M. auratus and at the age of 4 days in S. murinus. In T. belangeri and in the precocially born C. aperea alveoli are already present at birth. In contrast, the lungs of the newborn marsupials M. domestica and M. eugenii are at the early terminal air sac stage with few large air sacs of 300 – 400 µm in diameter. The postnatal lung development proceeds very slowly in marsupials and alveoli are not present before the age of 28 days in M. domestica and 65 days in M. eugenii. The metabolic investigations demonstrate that Marsupialia have a low metabolism at birth and achieve the adult metabolism late in the postnatal development. All examined Placentalia showed the same pattern of oxygen consumption. Corresponding to their advanced differentiation of the lungs they also exhibit high metabolic abilities at birth and reach the adult metabolism during the first week of life. During this critical period placental young have a higher resistance against certain environmental stresses than marsupial young and this could mean an evolutionary advantage of the placental “reproductive strategy” under suboptimal climatic conditions.

Les resultats presentees dans cette etude sont basees a la fois sur des experiences de capture-recapture effectuees en guyane francaise, et sur l'utilisation de marqueurs genetiques (microsatellites et cytochrome b) chez deux especes de marsupiaux didelphides : didelphis marsupialis et philander opossum. Les didelphides representent une des composantes les plus importantes de la faune neotropicale. De plus, leur position systematique, au sein des mammiferes et egalement des marsupiaux, en fait des modeles d'etudes particulierement interessants car ils representent un modele original d'organisation mammalienne. Les domaines vitaux se chevauchent largement entre les individus, avec toutefois un recouvrement moindre entre femelles qu'entre males. Le renouvellement de la population est tres important, quasiment 100% en un an, pour les deux especes. Peu de jeunes marques dans les poches marsupiales sont recaptures une fois adultes sur la zone d'etude. Pour les deux especes il existe : une saisonnalite basee sur des disponibilites en nourriture et une strategie de reproduction de type r. Les marqueurs genetiques ont montre qu'il existait une reproduction de type polygyne chez p. Opossum. Il existe de plus, une importante tendance a la polyandrie chez p. Opossum et d. Marsupialis avec jusqu'a environ 30% et 50% de multiple paternite chez ces deux especes respectivement. Nos deux especes presentent une structuration de population avec une grande variabilite intra-population et une faible differentiation inter-population correlee a d'importants flux geniques. Les reconstructions phylogenetiques, effectuees tant sur les microsatellites que sur le cytochrome b, ont permis de mettre en evidence l'existence d'un clade guyanais et de souligner la particularite de la zone de cayenne. Cette region aurait pu etre isolee du continent au cours des transgressions marines passees, qui auraient separe les populations continentales et insulaires. Ces donnees montrent que ces deux especes, tres proches phylogenetiquement, presentent d'importantes similarites quant a leur organisation spatiale, leur strategie et mode de reproduction et leur genetique de population. Elles presentent egalement une des structures sociales les moins complexes parmi celles pouvant etre rencontrees chez les mammiferes.

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A evolução do dimorfismo sexual de forma e tamanho do crânio e mandíbula foi estudada em 31 táxons de marsupiais didelfídeos, a fim de compreender melhor o desenvolvimento desse caráter na família. Para elucidar fatores que poderiam estar condicionando esse dimorfismo nos Didelphidae, foi analisada também a evolução de outros dois elementos: alometrias entre tamanho e forma do crânio e da mandíbula; e bionomia das mesmas 31 espécies. Foi realizado um mapeamento de todos estes caracteres, qualitativos e quantitativos, sobre uma filogenia da família, reconstruindo seus estados ancestrais utilizando métodos de parcimônia. Foram feitas também correlações utilizando contrastes independentes dos dados de dimorfismo sexual para auxiliar a esclarecer os padrões evolutivos do dimorfismo sexual. Tais correlações foram significativas, indicando coevolução entre os tipos de dimorfismo em ambas as estruturas estudadas. Não houve tendência unidirecional de surgimento/desaparecimento e aumento/decréscimo do dimorfismo nos Didelphidae. As reconstruções também indicaram coevolução entre os dimorfismos. Poucas espécies são altamente dimórficas e a maioria apresenta dimorfismo sexual de forma. Os resultados de alometrias entre os sexos foram iguais para crânio e mandíbula, mas elas também não apresentam padrão geral em direção à igualdade ou diferenciação das mesmas entre os sexos. Ambos os estados estão uniformemente distribuídos na filogenia. O comportamento das alometrias no crânio e na mandíbula coevoluiu, mas o padrão de alometrias entre os sexos não é conservado dentro de Didelphidae e não coevoluiu com o dimorfismo sexual, indicando que não explica a evolução deste caráter. Outros fatores, não-alométricos, devem condicionar este caráter nessa família. No entanto, apesar de padrões aparecem dentro de alguns clados, não há também evidência de coevolução entre bionomia e dimorfismo sexual nos Didelphidae. Espécies asazonais são poucas e não dimórficas de tamanho, talvez pelo fato de se reproduzirem continuamente e sofrerem menores pressões seletivas. A semelparidade é rara dentro de Didelphidae, surgindo apenas em Monodelphini e seguindo daí caminhos evolutivos distintos. Espécies semélparas exibem maiores tamanhos de ninhada nos Didelphidae por se reproduzirem uma única vez. Acredita-se que a seleção sexual direcione o padrão de dimorfismo sexual onde os machos são maiores que as fêmeas em espécies semélparas de Didelphidae. Portanto, nem alometrias nem bionomia, a princípio, explicam nem condicionam a evolução do dimorfismo sexual nos didelfídeos. A inclusão de uma maior quantidade de dados reprodutivos de marsupiais didelfídeos seria ideal para testar a veracidade da ausência de coevolução entre estratégias bionômicas e dimorfismo sexual. Associar dados ecológicos ou de padrões de distribuição poderiam ajudar a compreender melhor a evolução das estratégias bionômicas e a sua importância sobre a evolução do dimorfismo sexual nos Didelphidae.
The evolution of size and shape sexual dimorphism of the skull and mandible was studied in 31 taxa of didelphid marsupials, in order to better understand the development of this character in the family. And to elucidate factors that could be conditioning this dimorphism in Didelphidae the evolution of two other elements was also analyzed: allometries between size and shape of the skull and mandible and bionomy of the same 31 species. A mapping of all these qualitative and quantitative characters was carried through on a phylogeny of the family, reconstructing its ancestral states using parsimony methods. Correlations using independent contrasts of the sexual dimorphism data had been made also to assist clarifying the evolutionary standards of the sexual dimorphism. Such correlations were significant indicating coevolution among types of dimorphism in both studied structures. There is no unidirectional trend of sprouting/disappearance and increase/decrease of the dimorphism in Didelphidae. The reconstructions had also indicated coevolution among dimorphisms. Few species are highly dimorphics and the majority of them present shape sexual dimorphism. The results of allometries between the sexes had been the same for skull and mandible, but they also did not present a general pattern directing to the equality or differentiation between the sexes. Both states are uniformly distributed in the phylogeny. The results of the allometries in the skull and mandible coevoluted but the results of allometries between the sexes was not manteined in Didelphidae and they did not coevolute with sexual dimorphism indicating that they do not explain the evolution of this character and that other non-allometric factors must condition this character in this family. Although patterns appear inside of some clades there was no evidence of coevolution between bionomy and sexual dimorphism in Didelphidae. There are few aseasonal species and they are non-sized dimorphics perhaps by reproducing continuously and suffering less selective pressures. The semelparity is rare in Didelphidae arising only in Monodelphini and following from there distinct evolutionary ways. Semelparous species display the largest offspring size by reproducing only once. It is known that sexual selection directs the patterns of sexual dimorphism in those species in which males are larger than females in semelparous taxa of Didelphidae. Therefore neither allometries nor bionomy at first explain the evolution of sexual dimorphism in didelphids. The inclusion of a larger amount of reproductive data for didelphid marsupials would be ideal to test the veracity of the absence of coevolution among bionomic strategies and sexual dimorphism. The association of ecological data or distributional patterns could help in better understanding the evolution of the bionomic strategies and its importance on the evolution of the sexual dimorphism in Didelphidae.

Thesis by publication -- 8 co-authored articles.
Thesis (PhD)--Macquarie University, Division of Environmental and Life Sciences, Department of Biological Sciences.
Includes bibliographical references.
Preface -- Management issues of urban common brushtail possums (Trichosurus vulpecula): a loved or hated neighbour -- Effects of deslorelin implants on reproduction in the common brushtail possum (Trichosurus vulpecula) -- Brushtail possums (Trichosurus vulpecula) in metropolotan Sydney: population biology and response to contraceptive implants -- Strategic survey for Toxoplasma gondii and Neospora caninum in the common brushtail possum (Trichosurus vulpecula) from urban Sydney, Australia -- Leptospirosis serology in the common brushtail possum (Trichosurus vulpecula) from urban Sydney, Australia -- Conclusions.
The common brushtail possum (Trichosurus vulpecula) is indeed a common inhabitant of many Australian citites, and one of the few marsupials that has adapted well to the urban environment. Their close proximity to people provides a great opportunity to experience native wildlife in the backyard, however, their utilization of house roofs, bold behaviour and appetite for garden plants often leads to conflict with householders. Population numbers are sufficiently high to require ongoing management to minimise negative impacts for humans and brushtail possums alike in a socially acceptable manner. The aim of this thesis was to identify current management issues and address the need for improved and novel management strategies. The potential of slow-release implants, containing the GnRH agonist deslorelin, as a contraceptive agent for brushtail possums was tested on a captive population. Males appeared resistant to treatment, but deslorelin was found to inhibit reproduction in female brushtail possums for at least one breeding season, making it a promising tool to control fertility in some wild populations. A further aim was to trial deslorelin implants on a wild urban population, to collect more information about the urban biology of this species and to point out issues which have previously not been addressed. Close proximity and interaction of urban brushtail possums with humans and their domestic animals can increase the risk of disease exposure and transmission and influence the health of wild populations. Serosurveys showed that animals were readily exposed to Leptospira spp. and Toxoplasma gondii. This thesis also provides the first data on brushtail possum dispersal in urban areas, knowledge which is highly relevant to the development of management strategies such as fertility control. The findings from this research broaden our knowledge about urban brushtail possums and should assist wildlife authorities in developing alternative or improved management procedures.
Mode of access: World Wide Web.
xxv, 287 p. ill., maps

Bibliography: leaves 262-298.
300 leaves : ill. (some col.) ; 30 cm.
This thesis investigated the structure and glycoconjugate composition of the zona pellucida (ZP) surrounding marsupial oocytes and the changes that occur during ovarian development, following ovulation, and following cortical granule exocytosis. The glycoconjugates of the oviduct epithelial lining of the brushtail possum around the time of ovulation were also examined to determine if there was any contribution of the oviductal secretions to the post-ovulatory ZP.
Thesis (Ph.D.)--University of Adelaide, Dept. of Anatomical Sciences, 2003?

Research Doctorate - Doctor of Philosophy (PhD)
The mechanisms and molecules mediating marsupial fertilisation and reproduction will only be elucidated with a detailed understanding of the molecular nature of marsupial gametes and their post-testicular maturation. This thesis presents the first pivotal steps in defining the antigenic nature and maturation of marsupial spermatozoa. Numerous monoclonal antibodies produced against tammar wallaby (Macropus eugenii) and brushtail possum (Trichosurus vulpecula) sperm antigens provided specific tools for characterising the cellular, biochemical and molecular nature of these gametes. Marsupial spermatozoa, like those of their eutherian counterparts, are comprised of a complex array of antigens. Many of these antigens were restricted to specific cellular regions and surface domains, whilst others were distributed widely in the cell. The epitopes recognised by the monoclonal antibodies also displayed differential characteristics. Some were species-specific, whilst others were shared only by other marsupial spermatozoa or a wide variety of species. Similarly some antibodies bound sperm-specific epitopes, whilst others were common to somatic tissues. Most sperm antigens arose in the marsupial testis, however others were added or modified during post-testicular maturation in the epididymis. The characterisation of sperm antigens with potential roles in sperm maturation, fertilisation events were facilitated by the WSA- 1 monoclonal antibody. This antibody recognised a species and tissue-specific epitope shared by an acrosomal matrix antigen and an epididymal maturation antigen on wallaby spermatozoa. The acrosomal antigen arose in the wallaby testis, persisting unaltered during epididymal maturation and shared some sequence homology with proacrosin. The proacrosin/acrosin zymogen mediates sperm binding to ZP2 and zona penetration in eutherian species. This antibody might therefore be used to investigate the importance of proacrosin in marsupial fertilisation. The WSA- 1 maturation antigen was added to the whole surface of wallaby spermatozoa during epididymal transit. Secretion by the epididymal epithelium commenced in the proximal head of the epididymis and persisted distally in the tract whereas the antigen first associated with the sperm surface in the proximal body of the epididymis. Cross-linking of the WSA-1 carbohydrate epitope on the sperm surface resulted in potent midpiece-midpiece agglutination of wallaby ejaculated spermatozoa. Investigations in eutherian species suggest that the WSA-1 maturation antigen may function in sperm storage or the acquisition of sperm motility and fertility in the wallaby epididymis. As such the WSA-1 antigen is likely to be involved capacitation and/or fertilisation events in the female tract and has great promise as an immunocontraceptive target antigen. Intracellular antigenic maturation accompanies the morphological maturation of marsupial spermatozoa during epididymal transit. The PSA-10 monoclonal antibody recognised midpiece fibre network antigen(s) that arose concomitantly with the epididymal development of this structure in both possum and wallaby spermatozoa. Thus the PSA-lO monoclonal antibody provides an important tool for following the development and fate of a cytoskeletal structure which is unique to marsupial spermatozoa. The PSA-10 antibody also recognised an antigen associated with the outer acrosomal membrane of possum spermatozoa. Initial detection of PSA-10 acrosomal immunoreactivity also accompanied major morphological folding and consolidation of the possum acrosome during epididymal transit. Evidence of the antigenic modification of the marsupial sperm nucleus during spermiogenesis was provided by the differential binding patterns of two monoclonal antibodies. The WSA-3 and PSA-1 monoclonal antibodies each bound sperm-specific nucleoproteins in the wallaby testis. The WSA - 3 antigen was first detected in stage 10 spermatids and accumulated in spermatids in stages of spermiogenesis characterised by considerable nuclear condensation and elongation. However the PSA- 1 nucleoprotein was first detected quite late in spermiogenesis on stage 13 spermatids. These marsupial sperm nucleoproteins may play a role in the protein transitions thought to mediate chromatin binding and condensation in the mammalian spermatid nucleus. The construction of a wallaby testis cDNA library as part of this investiagation provides an invaluable tool for the identification of of the nucleotide sequences encoding marsupial sperm proteins. Screening the cDNA library resulted in the cloning of a partial sequence for a marsupial heat shock protein. The highly conserved heat shock proteins have important roles in the cellular processing and cytoprotection of testicular polypeptides. The significant contributions to our fundamental knowledge of marsupial sperm antigens and their epididymal maturation in this study provide a strong foundation for future examinations of marsupial capacitation and fertilisation events. Some of the antigens identified may also have applications to the regulation of fertility in pest marsupial species.

Thesis (Ph.D.) - University of Adelaide, School of Earth and Environmental Sciences, Discipline of Ecology and Evolutionary Biology, 2008.
"March 2008" -- T.P. Includes author's previously published papers. Bibliography: pages 104-118. Also available in print form.

This thesis examines the reproductive biology of red-tailed phascogales, an obligate male semelparous dasyurid species, which is part of a captive breeding colony at Alice Springs Desert Park. The red-tailed phascogale belongs to a group of dasyurids that shows an unusual reproductive strategy amongst mammals, one which provides opportunity for understanding means by which individuals maximise their reproductive success and the role of sperm competition. The broad aim was to gain an understanding of the reproductive biology of red-tailed phascogales and explore means by which individuals can affect their reproductive success. Examination of the red-tailed phascogale reproductive biology showed that females mated with multiple males and were capable of storing sperm in their oviducts for at least a five day period. Captive female red-tailed phascogales showed greater plasticity in their breeding season than has been observed in Antechinus, which exhibits the same life history strategy. Females were observed to invest heavily into the production of young, producing almost twice as many ova (15.1 ± 1.9) as young they can raise and 76% of females filling six to eight of the eight available teats in a breeding attempt. A 63% male bias was observed in young attaching to the teats, which could be produced through differential attachment of the sexes to teats at birth. Of the 846 young born in the captive breeding colony, 68% were weaned, with weaning occurring between 90-110 days of age and a 53% female bias observed in young being weaned. By weaning, a litter of young weighed 380% of the mothers mass with male young tending to be heavier than females by weaning. No relationship was observed between maternal weight and either litter sex ratio or sex biased growth of young. A positive relationship between maternal body mass and body mass of offspring at weaning was observed, with the body mass of young at weaning correlated with its body mass at maturity. Multiple paternity was observed in more than half of the litters examined, with heavier males having increased siring success compared to lighter males. Genotyping showed that the effective population size for the captive colony was 1.9x that observed from the group managed studbook. Male reproduction was also not as tightly constrained as in Antechinus, with spermatogenic failure not occurring in captive populations until after mating had occurred, meaning males are not reliant on epididymal stores alone for successful breeding. Scrotal diameter showed a positive relationship with testis and epididymal mass across male life, although this relationship was not evident when analysis was restricted to the time of peak sperm production. Captive males showed the opposite pattern of testosterone fluctuations to that observed in wild animals, with lowest levels occurring during the mating period. Captive animals were able to survive up to five years in captivity, in contrast to the obligate semelparity observed in wild males. Although most captive females can survive to breed in a second year and females are known to breed in a second year in the wild, the reproductive strategy of females appears to be aimed at maximising the returns on their first breeding attempt. In males, the need to maximise the investment into the first breeding season is amplified through the complete absence of opportunity to breed again; either through post-mating mortality in the wild or spermatogenic failure in captivity. The results of this study have implications for captive breeding of red-tailed phascogales, with their reproductive biology; spermatogenic failure, restricted breeding season, teat number limiting the number of young raised, high lactational investment into young, sex biases, the need to maintain genetic diversity and biases in siring success; providing challenges for the maintenance of a captive population. The results of this study also provide comparative information that contributes to understanding the unusual life history strategy of Phascogale and Antechinus, and contributes to the growing body of knowledge about mating strategies in marsupials.
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Thesis (Ph.D.) - University of Adelaide, School of Earth and Environmental Sciences, 2008

This thesis examines the reproductive biology of red-tailed phascogales, an obligate male semelparous dasyurid species, which is part of a captive breeding colony at Alice Springs Desert Park. The red-tailed phascogale belongs to a group of dasyurids that shows an unusual reproductive strategy amongst mammals, one which provides opportunity for understanding means by which individuals maximise their reproductive success and the role of sperm competition. The broad aim was to gain an understanding of the reproductive biology of red-tailed phascogales and explore means by which individuals can affect their reproductive success. Examination of the red-tailed phascogale reproductive biology showed that females mated with multiple males and were capable of storing sperm in their oviducts for at least a five day period. Captive female red-tailed phascogales showed greater plasticity in their breeding season than has been observed in Antechinus, which exhibits the same life history strategy. Females were observed to invest heavily into the production of young, producing almost twice as many ova (15.1 ± 1.9) as young they can raise and 76% of females filling six to eight of the eight available teats in a breeding attempt. A 63% male bias was observed in young attaching to the teats, which could be produced through differential attachment of the sexes to teats at birth. Of the 846 young born in the captive breeding colony, 68% were weaned, with weaning occurring between 90-110 days of age and a 53% female bias observed in young being weaned. By weaning, a litter of young weighed 380% of the mothers mass with male young tending to be heavier than females by weaning. No relationship was observed between maternal weight and either litter sex ratio or sex biased growth of young. A positive relationship between maternal body mass and body mass of offspring at weaning was observed, with the body mass of young at weaning correlated with its body mass at maturity. Multiple paternity was observed in more than half of the litters examined, with heavier males having increased siring success compared to lighter males. Genotyping showed that the effective population size for the captive colony was 1.9x that observed from the group managed studbook. Male reproduction was also not as tightly constrained as in Antechinus, with spermatogenic failure not occurring in captive populations until after mating had occurred, meaning males are not reliant on epididymal stores alone for successful breeding. Scrotal diameter showed a positive relationship with testis and epididymal mass across male life, although this relationship was not evident when analysis was restricted to the time of peak sperm production. Captive males showed the opposite pattern of testosterone fluctuations to that observed in wild animals, with lowest levels occurring during the mating period. Captive animals were able to survive up to five years in captivity, in contrast to the obligate semelparity observed in wild males. Although most captive females can survive to breed in a second year and females are known to breed in a second year in the wild, the reproductive strategy of females appears to be aimed at maximising the returns on their first breeding attempt. In males, the need to maximise the investment into the first breeding season is amplified through the complete absence of opportunity to breed again; either through post-mating mortality in the wild or spermatogenic failure in captivity. The results of this study have implications for captive breeding of red-tailed phascogales, with their reproductive biology; spermatogenic failure, restricted breeding season, teat number limiting the number of young raised, high lactational investment into young, sex biases, the need to maintain genetic diversity and biases in siring success; providing challenges for the maintenance of a captive population. The results of this study also provide comparative information that contributes to understanding the unusual life history strategy of Phascogale and Antechinus, and contributes to the growing body of knowledge about mating strategies in marsupials.
Thesis (Ph.D.) - University of Adelaide, School of Earth and Environmental Sciences, 2008

Copies of author's previously published works, inserted.
Bibliography :p.137-167.
xi, 167, [54]p. leaves of plates : ill. (some col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Research performed on several aspects of the reproductive biology of the female, fat-tailed dunnarts (Sminthopsis crassicaudata). Investigates the origin of the shell membrane, the stage of embryonic development at which it is lost and implantation takes place, the morphological features of fetal-maternal cellular interactions at implantation and placentation.
Thesis (Ph.D.)--University of Adelaide, Dept. of Anatomy and Histology, 1995

Research Doctorate - Doctor of Philosophy (PhD )
In Australia almost 40% of the carnivorous marsupials, or dasyurids, are threatened. Assisted reproductive techniques (ART), especially genome resource banking, have the potential to contribute to the conservation of these species by reducing the loss of genetic diversity. This project aimed to advance the knowledge of ART in dasyurids by focusing on the long term preservation of male and female gametes and establishing protocols for the production of mature oocytes for use in future ART. These studies used the fat tailed dunnart (Sminthopsis crassicaudata) as a model dasyurid and replicated many of the findings on threatened dasyurids. Dasyurid spermatozoa had a relatively unstable acrosome which lacked acrosomal membrane disulphide stabilisation. There was no evidence that S. crassicaudata spermatozoa were susceptible to high concentrations of cryoprotectants, but spermatozoa frozen with up to 40% glycerol using a rapid freezing protocol were not viable. Nonetheless the morphology and acrosomal integrity of frozen spermatozoa was normal and there was no evidence of DNA damage. The lack of success with cryopreservation is likely to be an artifact of cold shock, which was observed in S. crassicaudata and had not previously been described in any other marsupial. This susceptibility to low temperature can be overcome by slow cooling spermatozoa to 0 ºC at 0.5 ºC minute -1 with up to 20% egg yolk, and it is likely that this finding will result in successful sperm cryopreservation in the near future. Freeze drying spermatozoa represents an additional strategy for long term sperm preservation and freeze dried S. crassicaudata spermatozoa had normal morphology and nuclear integrity. In this study preserved dasyurid spermatozoa were immotile and non-viable but had no nuclear damage, suggesting that fertilisation may be achieved with intracytoplasmic sperm injection (ICSI). As ICSI requires a large number of mature oocytes to be collected, a reliable timed ovarian stimulation protocol was established in S. crassicaudata. This protocol enabled the collection of up to 28 oocytes which were either mature, or able to be cultured to the first polar body stage within 48 hours. Despite the success of induced ovulation, methods for preservation of the female gamete are essential to genome resource banking. This study also described a protocol for the enzymatic dissociation of dasyurid ovarian tissue allowing collection of high quality individual preantral follicles. The oocytes inside these follicles were able to be vitrified without any loss of viability and short term in vitro culture of immature follicles repaired the small amount of vitrification-induced damage to the surrounding granulosa cells. This collection of studies describes progress in genome resource banking for spermatozoa and oocytes from dasyurids and the development of protocols allowing the collection of a large number of oocytes for use in fertilisation experiments. These advances provide a solid and comprehensive framework for continuing the study of dasyurid ART which is timely due to the urgent need for genome resource banking in several threatened dasyurid marsupials.

Research Doctorate - Doctor of Philosophy (PhD )
In Australia almost 40% of the carnivorous marsupials, or dasyurids, are threatened. Assisted reproductive techniques (ART), especially genome resource banking, have the potential to contribute to the conservation of these species by reducing the loss of genetic diversity. This project aimed to advance the knowledge of ART in dasyurids by focusing on the long term preservation of male and female gametes and establishing protocols for the production of mature oocytes for use in future ART. These studies used the fat tailed dunnart (Sminthopsis crassicaudata) as a model dasyurid and replicated many of the findings on threatened dasyurids. Dasyurid spermatozoa had a relatively unstable acrosome which lacked acrosomal membrane disulphide stabilisation. There was no evidence that S. crassicaudata spermatozoa were susceptible to high concentrations of cryoprotectants, but spermatozoa frozen with up to 40% glycerol using a rapid freezing protocol were not viable. Nonetheless the morphology and acrosomal integrity of frozen spermatozoa was normal and there was no evidence of DNA damage. The lack of success with cryopreservation is likely to be an artifact of cold shock, which was observed in S. crassicaudata and had not previously been described in any other marsupial. This susceptibility to low temperature can be overcome by slow cooling spermatozoa to 0 ºC at 0.5 ºC minute -1 with up to 20% egg yolk, and it is likely that this finding will result in successful sperm cryopreservation in the near future. Freeze drying spermatozoa represents an additional strategy for long term sperm preservation and freeze dried S. crassicaudata spermatozoa had normal morphology and nuclear integrity. In this study preserved dasyurid spermatozoa were immotile and non-viable but had no nuclear damage, suggesting that fertilisation may be achieved with intracytoplasmic sperm injection (ICSI). As ICSI requires a large number of mature oocytes to be collected, a reliable timed ovarian stimulation protocol was established in S. crassicaudata. This protocol enabled the collection of up to 28 oocytes which were either mature, or able to be cultured to the first polar body stage within 48 hours. Despite the success of induced ovulation, methods for preservation of the female gamete are essential to genome resource banking. This study also described a protocol for the enzymatic dissociation of dasyurid ovarian tissue allowing collection of high quality individual preantral follicles. The oocytes inside these follicles were able to be vitrified without any loss of viability and short term in vitro culture of immature follicles repaired the small amount of vitrification-induced damage to the surrounding granulosa cells. This collection of studies describes progress in genome resource banking for spermatozoa and oocytes from dasyurids and the development of protocols allowing the collection of a large number of oocytes for use in fertilisation experiments. These advances provide a solid and comprehensive framework for continuing the study of dasyurid ART which is timely due to the urgent need for genome resource banking in several threatened dasyurid marsupials.

Copies of author's previously published articles inserted.
Bibliography: leaves 118-142.
vi, 156, [26] leaves, [22] leaves of plates : ill. (some col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Thesis (Ph.D.)--University of Adelaide, Dept. of Anatomy and Histology and Dept. of Animal Genetics, 1997?

Copies of author's previously published articles inserted.
Bibliography: leaves 118-142.
vi, 156, [26] leaves, [22] leaves of plates : ill. (some col.) ; 30 cm.
Thesis (Ph.D.)--University of Adelaide, Dept. of Anatomy and Histology and Dept. of Animal Genetics, 1997?

Research Doctorate - Doctor of Philosophy (PhD)
Oestrous synchronisation technology has the capacity to advance genetic conservation outcomes for threatened marsupials by making use of selected spermatozoa in artificial insemination (AI). A technique capable of precise control of oestrus and ovulation in marsupials, of which most are spontaneous ovulators, remains the key limiting factor in developing practical AI programs. The major impediment is the corpus luteum (CL) which in marsupials becomes independent of hypothalamic-pituitary support after formation and persists in both pregnant and non-pregnant cycles. For this reason, eutherian synchronisation techniques that rely on targeting CL life have failed to induce luteolysis in marsupials. The aim of this thesis was to investigate the potential to circumvent the marsupial CL and synchronise oestrous by targeting the hypothalamic-pituitary gonadal (HPG) axis with a gonadotrophin-releasing hormone (GnRH) agonist. GnRH agonists are small molecules rapidly removed from the body and thus are traditionally delivered as multiple injections or in slow release implants not suitable for assisted breeding applications. In contrast, Lucrin® Depot (AbbVie), a GnRH agonist in microspheres effects a one-month suppression of pituitary function after a single injection. In this thesis, I detail the potential for Lucrin Depot to synchronise oestrous in two taxonomically distinct marsupials with alternative reproductive strategies, a dasyurid, the fat-tailed dunnart (Sminthopsis crassicaudata,) and a macropod, the tammar wallaby (Notamacropus eugenii). In the fat-tailed dunnart, a dose of 5 mg kg^–1 or 10 mg kg^–1 of Lucrin Depot, resulted in reproductive suppression for 4 to 8 weeks, a return to reproductive activity at 8 to 12 weeks, and a complete return to cycling at 16 weeks. Following Lucrin-induced suppression, female dunnarts were fertile and conceived as early as 8 weeks after receiving 5 mg kg^–1 but did not conceive until 14 or 15 weeks after receiving 10 mg kg^–1. In tammar wallabies that underwent removal of pouch young (Day 0 RPY), Lucrin Depot inhibited the growth of pre-ovulatory follicles (all follicles <2mm, Day 31 RPY). An effective dose, 1.25 mg kg^–1, delays oestrus until between Day 39-66 RPY if Lucrin-treated at the time of RPY, and between Day 43-71 RPY if Lucrin-treated on Day 10 RPY. The outcome of work in both the fat-tailed dunnart and tammar wallaby resolved that Lucrin Depot has the capacity to inhibit ovarian follicular activity, but alone does not synchronise oestrous to a degree needed for assisted breeding. In the final research chapter, I show Lucrin Depot can be combined with exogenous gonadotrophins for ovarian stimulation and synchronisation. Pre-treatment of tammar wallabies with 1.25 mg kg^-1 of Lucrin Depot on Day 0 RPY prior to single doses of 20IU of PMSG on Day 20 RPY and 500IU of hCG on Day 23 RPY, synchronised oestrus and copulation to within 1 day (Day 26±0.1 RPY, n= 5 of 6). At autopsy and follow-up ovarian histology, it was determined that 20IU of PMSG adequately stimulates the heathy growth of pre-ovulatory follicles (around 10+ follicles >3mm per ovary Day 31 RPY). However, both the control superovulation group, and the Lucrin Depot-superovulation group did not ovulate in response to a single 500IU injection of hCG. Together the data presented in this thesis confirm that Lucrin Depot can form the basis of an oestrous synchronisation strategy in marsupials, and with further work to resolve the optimum ovulation treatment, it can be expected to become a practical assisted breeding tool for the recovery of threatened marsupials.

The newborn marsupial is born at a highly altricial stage of development when compared to eutherian newborn mammals. Interestingly, the marsupial young are born into a non-sterile environment and lack a developed immune system which prevents them from mounting an adaptive immune response. Two reviews are presented in this thesis. The first review examined the complementary mechanisms which protect the developing marsupial young. Immune protection through forms of innate immunity and maternal contributions has been examined in marsupials. However, as there are many facets to immune protection, our understanding of the protection of the developing marsupial young is far from complete and much of the developing marsupial's innate immune system remains unexplored. Importantly, the review also recognised that the availability of marsupial genomes should be exploited to help direct further research on the protection of marsupial young. The second review examined the role of the pouch in marsupial reproduction. While it is clear that the pouch provides immune protection to those species that have a pouch, the pouch cannot provide immune protection to the young of those species without a pouch. Thus, instead of using the pouch to explain immune protection of marsupial young, the review examined the role of the pouch in supporting highly altricial young and promoting reproductive success in various environments where a pouch may confer significant advantages over free hanging young. The reviews set the direction for the experimental research presented in this thesis. The model species tammar wallaby (Macropus eugenii) was used to further examine innate immune genes. First, innate immune genes, mucin and lysozyme were identified or predicted in the tammar wallaby. The mucin and lysozyme genes were then physically mapped to conserved regions of the tammar wallaby genome which facilitated the confirmation of the identification of the genes. Second, the expression of immune genes was examined in the skin and lung of developing tammar wallabies considered immune-incompetent and immune-competent using transcriptome sequencing. There were 390 and 429 immune genes expressed in developing tammar wallaby skin and lung, respectively, suggesting that both tissues play a role in immune defense. Unexpectedly, mucin and lysozyme were not expressed in the skin and lung of wallabies considered to be immune-incompetent. Further, enrichment analysis of genes identified in Gene Ontology terms showed that specific immune genes, complement component 3, complement factor B and apolipoprotein A1, have increased expression in the skin and lung of wallabies considered immune-incompetent. The increased expression of the immune genes suggests that there may be specific innate immune system processes in play to compensate for the delayed development of the adaptive immune system in marsupial. The research presented in this thesis identified gaps in the research of immune protection of marsupial young and identified, mapped and examined the expression of innate immune genes in the developing tammar wallaby. Thus, we are closer to understanding the complexity of the developing marsupial immune system. The identification of immune genes and their expression, lays the foundation for further functional research in marsupial developmental immunology.