Orientador: Maria Jaqueline Mamprim / Resumo: O objetivo deste estudo foi descrever a anatomia da coluna vertebral de gambás da espécie Didelphis albiventris, a partir da maceração de espécimes para a obtenção das vértebras, e pela realização de radiografias e tomografia computadorizada, uma vez que não foram encontrados na literatura consultada, trabalhos relacionados à coluna vertebral de gambás-de-orelha-branca. Foram utilizados 35 exemplares independente de sexo e idade de D. albiventris, que vieram a óbito por motivos não associados a este trabalho. Todos os cadáveres foram levados ao Setor de Diagnóstico por Imagem para realização de radiografias dos segmentos vertebrais em projeções tangenciais (lateral direita e ventrodorsal). A partir da avaliação das radiografias, foram selecionados cinco animais sem alterações morfológicas ou secundárias a trauma para serem submetidos ao exame tomográfico e dez para maceração. As informações obtidas a partir dos conjuntos de vértebras maceradas, das radiografias e das tomografias, foram reunidas a fim de se descrever anatomorradiograficamente a coluna vertebral dos gambás-de-orelha-branca. Os resultados foram comparados com os descritos para outros didelfídeos, animais selvagens e domésticos. Os animais estudados apresentaram um padrão de sete vértebras cervicais, 13 torácicas, seis lombares, duas sacrais e entre 24 e 29 caudais, não diferindo significativamente do relatado para outros marsupiais. As informações contidas no trabalho servirão como fonte de consulta no atendimen... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to describe the anatomy of the vertebral column of the white-eared opossum (Didelphis albiventris), using gross vertebra obtained from maceration, and imaging modalities such as radiographs and computed tomography, since there were not found papers about it. A total of 35 D. albiventris specimens were used regardless of sex and age, which deaths were not associated with this study. All the animals were taken to the Diagnostic Imaging Department to perform radiographs of the vertebral segments in tangential projections (right lateral and ventrodorsal). From the evaluation of the radiographs, five animals without morphological abnormalities (abnormalities secondary to congenital or acquired diseases) were selected to be submitted to tomographic examination and ten animals for maceration. Information obtained from the macerated vertebrae, radiographs, and CT scans were collected in order to describe the anatomical anatomy of the spine of the white-eared opossums. The results were compared with those described for other didelphids, wild and domestic animals. The animals studied presented a pattern of seven cervical, 13 thoracic, six lumbar vertebrae, two sacral and 24 to 29 caudal vertebrae, not significantly different from what was reported for other marsupials. The information contained in the study will serve as a literature source to aid in the clinical care of marsupials since the casuist is increasing in wildlife referral centers and speciali... (Complete abstract click electronic access below) / Doutor

The aim of this study was to develop improved methods to detect viable Coxiella burnetii in wastes from livestock production. The impetus for this work arose because there is a significant risk of infection for humans attributed to contact with waste products from the livestock production industry. This situation is further compounded by the lack of suitable tools to detect viable C. burnetii in these wastes. In addition, effective disinfection strategies for livestock wastes are also required to reduce the risk of infection with C. burnetii for individuals that come into contact with these waste products. A quantitative real-time PCR system (qPCR) with high sensitivity and specificity was developed to detect the C. burnetii in environmental samples associated with domestic ruminants and native Australian marsupials. Different detection chemistries and procedures were evaluated based on their sensitivity, specificity and reproducibility. Overall it was found that the TaqMan PCR targeting the IS1111a locus provided the most sensitive and reproducible test. The Geneworks PowerSoil(tm) DNA isolation kit provided the best compromise between reproducibility and recovery of DNA from livestock wastes. When combined, the IS1111a TaqMan qPCR and Geneworks PowerSoil DNA Extraction Kit provided a test which was capable of detecting as few as two C. burnetii genome equivalents in 0.2g of soil or faeces. Coxiella burnetii has been shown to display extreme resistance to environmental exposure. Therefore, assessment of the viability of the organism in environmental matrices is more useful for risk assessment programs than detection of DNA alone. A quantitative reverse transcriptase PCR was developed that was able to detect viable C. burnetii cells in soil. The sensitivity of the assay was enhanced by heat-treating the soil samples prior to extraction of RNA. The factor most often associated with transfer of C. burnetii to humans is exposure to livestock or their waste. Therefore, decontamination of waste from livestock production industries is a key factor in preventing outbreaks of Q fever. A system was developed to determine the efficacy of various disinfectant treatments against the environmental pathogen C. burnetii. Treatments evaluated included sodium hypochlorite, ozone, ultraviolet light, peracetic acid (PAA), and Virkon S®. Sodium hypochlorite at a concentration of 0.1 mM reduced the infectivity of C. burnetii by over 92% while treatment with the same sodium hypochlorite concentration in wastewater showed significantly reduced efficacy. Despite this reduced potency, sodium hypochlorite is still useful for control of C. burnetii in the liquid waste of animal production. Commercially available ELISA and CFT assays exist for ruminants but there are no immunological tests available for detecting C. burnetii in marsupials even though Australian marsupials are known to be susceptible to C. burnetii. An indirect ELISA for detecting anti-Coxiella antibodies in kangaroos was developed. Paired serum and faecal samples were taken from 379 ruminants from Western Australia and the serum was tested with a commercially available ELISA and the complement fixation test while the faeces was tested using the qPCR developed during this study. Paired serum and faecal samples were taken from 343 kangaroos from WA and were tested with the antibody-ELISA developed during this study and by qPCR. A very low prevalence of anti-Coxiella antibodies was observed in the ruminants sampled and results from immunological tests correlated poorly with qPCR data. The development of an ELISA for use with kangaroo serum was problematic because of the lack of reference sera from animals known to be infected with C. burnetii. Despite this results from the ELISA developed suggested that the apparent seroprevalence in the WA animals surveyed was approximately 34%. Results from testing kangaroo faeces with the qPCR correlated poorly with the results from the antibody-ELISA. These data suggest that kangaroos may be a significant reservoir of C. burnetii in Western Australia and due to cohabitation of kangaroos and domestic ruminants, may provide a link between the wildlife and domestic cycles of C. burnetii.

This thesis describes a comprehensive study of the cellular responses of a number of endangered marsupial species with a principal focus on the tammar wallaby (Macropus eugenii) as a model macropod species. The development of in vitro experimental assays for the assessment of immune responses in this model species are described, which provided a set of benchmarks for comparisons with other members of the Macropodidae and with eutherian mammals. Once this data was collected and protocols were established, the study was extended to include investigations of the immune responses in opportunistic samples obtained from the Rufous Hare-wallaby (Lagorchestes hirsutus), the Long-footed potoroo ( Potorous longipes) and the more common, but nonetheless still vulnerable, Long-nosed potoroo (Potorous tridactylus) with a view to investigating their apparent susceptibility to infection with intracellular pathogens, particularly mycobacterial species. The findings from the application of these assays suggest that the cellular immune responses of these species are relatively complex and involve a level of sophistication that rivals their eutherian counterparts. Specifically peripheral blood and tissue leukocytes were morphologically similar to those of other mammals, with the exception of tammar wallaby monocytes that appeared to contain few lysosomal granules, and the basophils of the Rufous Hare-wallaby that contained very large atypical granules. The overall findings of this study suggest that the immune systems of macropod species possess most of the sophistication associated with that of eutherian mammals. Whilst some differences were apparent in cells and their products in the test species, no single factor common to all macropods was identified as a cause for immune dysfunction. It appears likely that as yet undefined factors related to their confinement rather than an inherent defect in their immunocapacity is responsible for the apparent disease susceptibility of these animals. / Doctor of Philosophy (PhD)

The primary objective of the studies described in this thesis was to improve the cryopreservation success of koala spermatozoa for the purpose of establishing a genome resource bank for this species. A defining feature of the studies in this thesis was the implementation of an organelle-specific approach to better understand the causes of koala sperm cryo-injury. The functional attributes of spermatozoa, such as mitochondrial function, plasma membrane fluidity, membrane lipid asymmetry and DNA integrity were assessed as an indication of cryo-injury. Sperm mitochondrial function and plasma membrane integrity were examined by cryomicroscopy using the fluorescent probes JC-1 and propidium iodide (PI) respectively in a dual staining technique. Cooling and re-warming koala spermatozoa were more detrimental to mitochondrial function than to plasma membrane integrity. Mitochondrial membrane potential (MMP) was suppressed by freezing and thawing treatments; after thawing, MMP declined significantly during rewarming (from 5ºC to 35ºC). The distribution of GM1 ganglioside was examined using fluorescent-labelled cholera toxin B. No significant redistribution of GM1 was observed after chilling or cryotreatment. The externalisation of phosphatidylserine (PS) was examined using fluorescent-labelled annexin V. There was no significant increase in translocation of PS after chilling or cryopreservation. These observations imply that cryotreatment had little effect on plasma membrane lipid asymmetry. Koala spermatozoa were incubated in a range of anisotonic media to investigate whether nuclear swelling was caused by osmotic flux during the cryopreservation process. Although the most hypotonic solution tested (64 mOsm/kg) induced the highest incidence of nuclear relaxation (mean ± SEM; 12 ± 3%), this was not as severe as that previously documented following cryopreservation. Chromatin relaxation is a phenomenon observed in koala spermatozoa, where the sperm nucleus expands due to the result of structural changes in the natural conformation of the sperm DNA/protamine complex. DNA fragmentation was not a primary cause of cryopreservation-induced sperm chromatin relaxation, although in situ nick translation of putative DNA breaks indicated that these increased as the sperm head became progressively more relaxed. Using a Sperm Chromatin Dispersion test (SCDt) specifically developed and validated for koala spermatozoa, a continuum of nuclear morphotypes was observed, ranging from no apparent DNA fragmentation to spermatozoa with highly dispersed and degraded chromatin. A double comet assay was also developed to investigate DNA fragmentation in the koala spermatozoa. Conducted under neutral followed by alkaline conditions, this assay was able to differentiate between single- (SSB) and double-stranded (DSB) DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa; the majority of the koala spermatozoa had nuclei with DNA abasic-like residues. The ubiquity of these residues suggested that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with “true” DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA, to nuclei that exhibited both SSB and DSB. Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of inter-molecular disulphide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulphide bonds within their chromatin, but nevertheless, have been successfully cryopreserved. To examine the hypothesis that the cryoprotectants used for fish sperm cryopreservation will confer a similar degree of protection on koala spermatozoa, various concentrations of five cryoprotectants (dimethyl sulphoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Dimethylacetamide at a concentration of 12.5% (v/v) was found to be comparable to glycerol in the successful cryopreservation of koala spermatozoa although high inter-male variability was observed. However, when the new protocol was subsequently validated for a larger population of captive koalas (n = 22), glycerol emerged the better cryoprotectant with respect to all sperm viability parameters assessed except for that of the incidence of chromatin relaxation, which was not affected by the cryoprotectant. Significant difference was also observed in the post-thaw survival of spermatozoa from different animals, which was independent of pre-freeze semen quality. Based on post-thaw semen viability parameters, the koalas could be divided into two distinct groups, where one group had significantly higher sperm viability compared to the other group, regardless of cryoprotectant used. Positive correlation between motility and MMP was observed before and after cryopreservation. However, cryopreservation significantly reduced the dependency between these variables (P < 0.001), suggesting that cryopreservation reduced the dependency between mitochondrial function and motility.

The primary objective of the studies described in this thesis was to improve the cryopreservation success of koala spermatozoa for the purpose of establishing a genome resource bank for this species. A defining feature of the studies in this thesis was the implementation of an organelle-specific approach to better understand the causes of koala sperm cryo-injury. The functional attributes of spermatozoa, such as mitochondrial function, plasma membrane fluidity, membrane lipid asymmetry and DNA integrity were assessed as an indication of cryo-injury. Sperm mitochondrial function and plasma membrane integrity were examined by cryomicroscopy using the fluorescent probes JC-1 and propidium iodide (PI) respectively in a dual staining technique. Cooling and re-warming koala spermatozoa were more detrimental to mitochondrial function than to plasma membrane integrity. Mitochondrial membrane potential (MMP) was suppressed by freezing and thawing treatments; after thawing, MMP declined significantly during rewarming (from 5ºC to 35ºC). The distribution of GM1 ganglioside was examined using fluorescent-labelled cholera toxin B. No significant redistribution of GM1 was observed after chilling or cryotreatment. The externalisation of phosphatidylserine (PS) was examined using fluorescent-labelled annexin V. There was no significant increase in translocation of PS after chilling or cryopreservation. These observations imply that cryotreatment had little effect on plasma membrane lipid asymmetry. Koala spermatozoa were incubated in a range of anisotonic media to investigate whether nuclear swelling was caused by osmotic flux during the cryopreservation process. Although the most hypotonic solution tested (64 mOsm/kg) induced the highest incidence of nuclear relaxation (mean ± SEM; 12 ± 3%), this was not as severe as that previously documented following cryopreservation. Chromatin relaxation is a phenomenon observed in koala spermatozoa, where the sperm nucleus expands due to the result of structural changes in the natural conformation of the sperm DNA/protamine complex. DNA fragmentation was not a primary cause of cryopreservation-induced sperm chromatin relaxation, although in situ nick translation of putative DNA breaks indicated that these increased as the sperm head became progressively more relaxed. Using a Sperm Chromatin Dispersion test (SCDt) specifically developed and validated for koala spermatozoa, a continuum of nuclear morphotypes was observed, ranging from no apparent DNA fragmentation to spermatozoa with highly dispersed and degraded chromatin. A double comet assay was also developed to investigate DNA fragmentation in the koala spermatozoa. Conducted under neutral followed by alkaline conditions, this assay was able to differentiate between single- (SSB) and double-stranded (DSB) DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa; the majority of the koala spermatozoa had nuclei with DNA abasic-like residues. The ubiquity of these residues suggested that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with “true” DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA, to nuclei that exhibited both SSB and DSB. Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of inter-molecular disulphide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulphide bonds within their chromatin, but nevertheless, have been successfully cryopreserved. To examine the hypothesis that the cryoprotectants used for fish sperm cryopreservation will confer a similar degree of protection on koala spermatozoa, various concentrations of five cryoprotectants (dimethyl sulphoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Dimethylacetamide at a concentration of 12.5% (v/v) was found to be comparable to glycerol in the successful cryopreservation of koala spermatozoa although high inter-male variability was observed. However, when the new protocol was subsequently validated for a larger population of captive koalas (n = 22), glycerol emerged the better cryoprotectant with respect to all sperm viability parameters assessed except for that of the incidence of chromatin relaxation, which was not affected by the cryoprotectant. Significant difference was also observed in the post-thaw survival of spermatozoa from different animals, which was independent of pre-freeze semen quality. Based on post-thaw semen viability parameters, the koalas could be divided into two distinct groups, where one group had significantly higher sperm viability compared to the other group, regardless of cryoprotectant used. Positive correlation between motility and MMP was observed before and after cryopreservation. However, cryopreservation significantly reduced the dependency between these variables (P < 0.001), suggesting that cryopreservation reduced the dependency between mitochondrial function and motility.

Leukocytes play a central role in protecting the body against infectious organisms and their research is essential for understanding the mechanisms of immunity. By studying leukocytes across a range of species, insights are provided into differing strategies employed to ensure resistance to disease. Surprisingly, the structure and function of marsupial leukocytes has received very limited study. Marsupials represent a major evolutionary pathway with distinct differences in reproduction and development from placental mammals. These differences in the life history of marsupials place unique challenges on the immune system, and differences in leukocyte structure and function could be reasonably expected. In this thesis, studies were undertaken to examine the cytochemical, ultrastructural and functional features of leukocytes from species of marsupials, belonging to the family Macropodidae (kangaroos and wallabies). The aim of these studies was to elucidate the characteristics of macropodid leukocytes and to compare and contrast these features with the known characteristics of other mammalian leukocytes. Leukocytes from two species of macropodid, the tammar wallaby (Macropus eugenii) and the western grey kangaroo (Macropus fuliginosis), formed the basis of this study with additional material provided from quokka (Setonix brachyurus), woylie (Bettongia pencillata) and red kangaroo (Macropus rufus). Staining characteristics of cells were examined following reaction with Sudan black B, peroxidase, chloroacetate esterase, naphthyl butyrate esterase, alkaline phosphatase and periodic acid-Schiff. Peroxidase and Sudan Black B reactions were similar to domestic animal species but chloroacetate esterase and naphthyl butyrate esterase were unreliable as markers for macropodid neutrophils and monocytes, respectively. Significant variation in staining for alkaline phosphatase was seen between species of macropodid. Tammar wallabies and quokka demonstrated strong neutrophil alkaline phosphatase activity whereas western grey kangaroos, red kangaroos and woylies contained no activity within their leukocytes. Peroxidase and alkaline phosphatase cytochemistry were also assessed at the ultrastructural level with transmission electron microscopy. This allowed the identification of distinct granule populations within macropodid neutrophils. Two subcellular compartments containing alkaline phosphatase activity were identified within tammar wallaby neutrophils. These were considered equivalent to secretory vesicles and a subpopulation of specific granules. Tubular vesicles containing alkaline phosphatase were also identified within the eosinophils of tammar wallabies. These structures were a novel finding having not been reported previously in the eosinophils of other animal species. In addition to cytochemistry, the general ultrastructure of leukocytes from tammar wallabies and western grey kangaroos were reported. Results were similar to previous reports for other marsupial species. The current body of knowledge was extended by the first detailed description of the ultrastructure of basophils in a marsupial. To assess functional aspects of macropdid neutrophils, flow cytometric assays were performed examining oxidative burst responses and phagocytosis. Reactive oxygen species were generated by neutrophils from tammar wallabies and western grey kangaroos in response to phorbol 12-myristate 13-acetate but not N-formyl-Met-Leu-Phe or opsonised bacteria. Phagocytosis of opsonised bacteria was also measured in neutrophils from tammar wallabies, which was poor in contrast to ovine neutrophils. However, flow cytometric studies were limited by sample preparation. Further optimisation of isolation methods for tammar wallaby leukocytes should be undertaken before dogmatic conclusions are drawn. Overall, the results of this thesis demonstrate that, in the areas examined, the general characteristics of leukocyte structure and function of mammals are present in macropodids. However differences were identified both within and outside of the macropodid group. These differences have important ramifications for the use of ‘model’ species in the study of leukocyte biology in marsupials. The results also provide potentially useful tools for the clinical diagnosis of haematological disease in macropodids and may be of interest to those studying comparative and evolutionary aspects of leukocyte structure and function.

Information on the reproductive biology and behavioural ecology of southern hairy-nosed wombats (Lasiorhinus latifrons) is limited. Field reproductive and behavioural studies have been hindered by the difficulties associated with the routine recapturing and direct observation of wombats in the wild. Additionally, due to their extremely poor breeding success within captivity and the intrinsic complications associated with the monitoring of nocturnal and semi-fossorial activity, little formal research has been conducted on captive-held individuals. The overarching objectives of this research were to gain a better understanding of (1) both male and female L. latifrons reproductive physiology/behaviour that will lead to improved captive breeding program outcomes and (2) captive L. latifrons activity rhythms and behavioural time budgets in order to identify the impacts of captivity on wombat behaviour and wellbeing. The primary experiments of this research were centred around the development of ‘non-invasive methodologies’ for the collection of biological samples (Chapter 2), direct observation of behaviour and activity (Chapter 3), assessment of male reproductive function (Chapter 4), detection of female oestrus and cyclicity (Chapter 5), characterisation of activity patterns (Chapter 6) and monitoring of stress (Chapter 7). The last two experiments also tested the efficacy of gentling (Chapter 7) and enrichment (Chapter 8) to improve the wellbeing of the captive wombats. Faecal steroid analysis is a non-invasive tool that allows the stress-free monitoring of steroid hormones and has been used on a wide range of animal species to examine their reproductive physiology and adrenal function. The usefulness of faecal hormone analysis, however, is directly related to the reliable collection and identification of individual faecal samples. In group-housed animals, the identification of faecal samples can be difficult and time consuming and is generally only accurate if a marker is incorporated into the animal’s diets. Hence, Chapter 2 examined the usefulness of non-toxic plastic glitter as a faecal marker in group-housed L. latifrons. Forty-two food treats were tested as vehicles for the oral delivery of glitter; of these, vehicle palatability (> 75% consumed) and consistency of intake (eaten > 80% of times offered) was high for six treats: (1) golden syrup with horse pellets, (2) golden syrup with weetbix, (3) pitted-dates, (4) honey with kangaroo pellets, (5) nutrigel with rolled-oats and (6) strawberry sauce with rolled-oats. Marker mean rate of passage was 2.9 ± 0.5 d, with maximal output occurring 4.2 ± 0.3 d after oral administration. A minimum marker dose of 1.6 g was necessary to achieve high labelling consistency (> 2 flecks of glitter were defaecated in > 90% of pellets) and this dosage was required every 3 days to maintain a steady and detectable state of marker output. Twelve glitter colours were tested and optimum labelling results were obtained with gold, metallic red, metallic green and metallic blue. Once established, this technique was then used to facilitate the long-term collection of faecal samples in order to characterize patterns of reproduction (Chapter 4), monitor ovarian events (Chapter 5) and to quantify stress-responses (Chapter 7) via faecal steroid analysis. The direct observation of wombats is difficult; individuals are not easily identified and the animals are often out-of-view (residing in burrows) or obscured (low-light conditions) during sampling. Published behavioural data available on L. latifrons is largely restricted to visual observations made during dawn and dusk only, whilst published activity data pertains to time spent in or out of the burrows rather than actual physical activity. Hence, Chapter 3 tested the effectiveness of two electronic monitoring systems to remotely observe wombat behaviour and physical activity. Digital video-surveillance proved to be an effective technique for the recording and monitoring of captive wombat behaviour. Animal visibility was good, behavioural events unambiguous and the system enabled the long-term, concurrent recording of behaviour with no direct human presence. Similarly, radio-telemetry proved to be an effective way of recording captive wombat physical activity. The system was reliable, removed observer error and enabled the continuous and concurrent recording of wombat activity. Once established, these two remote monitoring systems were then used to describe wombat behaviour elements and activity patterns (Chapters 3 and 6). After a year of continuous monitoring it was established that the wombats spent, on average, 69.9% sleeping, 8.8% lying resting, 5.2% feeding, 5.2% exploring, 4.3% stereotyping, 2.5% sitting-at-rest, 1.7% digging, 1.4% foraging, 0.4% being handled, 0.3% sun-basking, 0.2% grooming and 0.1% courtship/mating. Temporal patterns were bimodal for 8/12 of the wombat behaviours and unimodal for the remaining four. The mean proportion of total daily time spent active was 18.2 ± 1.8%. Daily activity patterns were characterized by a strong circadian cycle, with high nocturnal activity and low diurnal activity. Daily onset (18:19 h) and cessation (04:34 h) of activity was seasonally constant and strongly associated (P < 0.01) with sunrise/set, but not influenced by either temperature or humidity (P ≥ 0.09). At night there was an alternating rhythm of active and rest periods, with activity peaking at the beginning (19:00 h) and end (03:00 h) of each night. Activity was seasonal with annual changes in temperature, humidity and night-time length being the triggers of variation. Mean daily activity was greater during winter (19.7%) and spring (18.9%), than during summer (16.3%) and autumn (17.2%), with the degree of activity being largely governed by ambient temperature. Feeding, sleeping and stereotyping varied significantly with season. Feeding and stereotyping were negatively associated with ambient temperature and humidity, whilst being positively associated with night-time length; the inverse relationship was true for sleeping. Ambient temperature exerted its largest effect on time spent feeding; feeding times decreased by 3.1 min / 1ºC above 13ºC and compared to spring, feeding times were reduced by 41% during summer. There is, at present, very little data published on male and female wombat reproduction. The reticence of wombats to breed in captivity makes it difficult to study reproduction in captive animals and their cautious, nocturnal nature makes field studies challenging. Non-invasive techniques to monitor reproductive status/function will assist in improving the general knowledge of wombat reproduction and the development of new captive breeding management strategies, by allowing the easy monitoring of captive animals. Thus, the series of experiments in Chapters 4 and 5 explored the efficacy of a number of non-invasive methodologies to assess male reproductive function, monitor female cyclicity and predict the timing of oestrus. To address the pulsatile nature of testosterone, a GnRH agonist stimulation test was developed in the male. IM injection of 4 μg of buserelin (a GnRH agonist) resulted in an increase (P < 0.05) in plasma testosterone concentrations, with maximum secretion occurring at 90 min. Thereafter, plasma testosterone concentrations remained near maximum for 150 min. There was a strong, positive correlation (r = 0.73, P < 0.01), between pre-stimulation testosterone concentrations and the maximal concentrations achieved post-stimulation with post-stimulation concentrations varying between individuals (P < 0.01). These findings indicate that individual male wombats can show large fluctuations in plasma testosterone concentrations over time and that a GnRH agonist can be used to obtain a diagnostic index of the prevailing testosterone biosynthetic capacity of the testes. This technique was then used as part of a larger experiment (Chapter 4) to investigate seasonal changes in male wombat reproduction. To date, the effects of breeding season on captive male wombat fertility have yet to be examined and a better understanding of this phenomenon will pinpoint the most favourable times for mating. Seasonal changes in a series of male reproductive parameters were non-invasively examined over a 18 month period: (1) testosterone concentration, both plasmic and faecal, was monitored using enzyme-immunoassays (EIA), (2) testicular volume was measured manually using digital vernier callipers, (3) sperm production was evaluated by way of spermatorrhoea, whilst (4) prostate and bulbourethral gland cross-sectional areas were assessed by ultrasonography. Plasma testosterone secretion increased in early-winter, peaked late-winter and declined in early-spring (P < 0.01). No seasonal variation (P = 0.22) in faecal testosterone metabolite concentrations was apparent. Testicular volume showed no significant variation (P = 0.29) over the sampling period and spermatozoa were found in the urine throughout the year; these two observations suggest that the captive male wombats remain spermatogenically active year round. While there was no significant seasonal change (P = 0.20) in prostate size, bulbourethral gland size increased in late-autumn, peaked in mid-winter and declined in early-summer (P < 0.01). Ultimately, captive male reproductive function was influenced by seasonality, with a peak in plasma testosterone and bulbourethral gland size occurring in winter (Jun-Aug). In an attempt to characterize oestrus-specific behaviour and develop a reliable method of oestrus detection in L. latifrons, the reproductive physiology and behaviour of eight adult females was monitored for a period of 12 months (Chapter 5). The reproductive behaviour of both sexes (4♂: 8♀) was monitored using 24-h video surveillance, whilst female physical activity was remotely measured using radio-telemetry. A faecal sample was collected every three days, from each female to assess changes in faecal progesterone and oestradiol-17β metabolite concentrations. Each female also received an injection of 0.01, 0.1 or 0.2 mg/kg of oestradiol benzoate (OB) in one of two hormone trials. Video surveillance revealed that the courtship (n = 426) and mating (n = 46) ritual of L. latifrons consisted of 13 distinctive behaviours expressed over six obvious phases: investigation, attraction, chase, restraint, copulation and recovery. Reproductive behaviour was observed in five (2♂, 3♀) wombats, with female receptivity occurring at night and lasting for only 13-h. Faecal progesterone metabolite analysis proved to be a reliable method for mapping oestrous cycle activity, but was not useful for the prediction of oestrus. Six out of the eight female wombats displayed periods of elevated progesterone secretion. From these six individuals, 23 luteal phases, 12 follicular phases and 12 oestrous cycles were recorded, with a mean (± SE) length of 20.9 ± 1.1 d, 11.6 ± 0.6 d and 31.8 ± 1.1 d, respectively. In contrast, changes in the secretion of faecal oestradiol-17β metabolites provided little instructive information on oestrous cycle activity and were not associated with oestrus. Administration of OB resulted in a spike of oestradiol-17β metabolites in the faeces 3-4 d later, but was not dose dependent nor did it elicit oestrus-behaviour. Activity monitoring does not appear to be a useful method for detecting oestrus in L. latifrons, as changes were not associated with key events in the oestrous cycle. However, 24-h video surveillance proved to be a reliable method for oestrus detection in the captive L. latifrons. Threatening or aversive stimulation is experienced in wild and captive conditions alike and evokes similar physiological responses. If an animal, wild or captive, cannot cope with this stimulation it may experience stress. An uncontrollable source of stress for all captive animals lies in their interactions with their human caretakers. A high or persistent fear of people can be a source of psychological stress for animals in captivity. Positive handling is a potent method of reducing the specific fear of human beings through desensitisation. The response of animals to handling by humans has been extensively evaluated in domesticated species, but rarely assessed in wild animals. Hence, Chapter 7 examined the usefulness of a regular handling program to lower the behavioural fear and physiological stress responses of L. latifrons to human interaction. Adult L. latifrons (n = 12) were exposed to two different treatments in a replicated design: (1) daily handling: 15 min of tactile contact from a human handling 5 d/wk for 12 wk and (2) no-handling: no contact apart from that received during routine husbandry. The effect of handling was assessed using overt response, human approach, stressor and novel stimulus tests. Synthetic ACTH was used to validate a method for monitoring faecal cortisol in L. latifrons by EIA. IM injection of 250 μg of Synacthen resulted in a significant (P < 0.05) increase in plasma and faecal cortisol concentration 30 min and 2 d after administration, respectively. Handling positively affected the behavioural responses of the wombats to human approach and contact in two ways: (1) a significant reduction (P < 0.01) in the wombat’s mean flight distances to human approach and (2) a significant (P < 0.01) decline in the strength of the wombat’s behaviour-based fear reactions (i.e. fear scores) to human proximity and contact. Handling had no discernable effect on the wombat’s physiological stress responses to human contact or on their reactions to novelty. While faecal cortisol secretion increased in response to a stressor test involving human contact, it was not alleviated by regular handling (P = 0.84). Similarly, the wombat’s reactions to unfamiliar objects during novel stimulus testing were also unaffected (P = 0.17) by the handling treatment. Therefore, handling exerted its main effect on the behavioural responses of the wombats, representing fear responses to human handlers, rather than reducing their anxiety. The main difference between wild and captive environments lies in the differential ability of control, i.e. a free-living animal is able to control its amount of incoming stimulation whereas a captive animal has a limited capacity to alter the external stimulation to which it is exposed. Without natural behaviour outlets, captive animals have to rely on abnormal behaviour patterns to modify their expectations of the captive environment and exert some control over incoming stimulation. A ‘stereotypy’, defined as a repetitive movement pattern with no apparent goal or function, is a common abnormal behavioural pattern expressed by zoo animals. Stereotypies are of concern because of their association with poor welfare. A previous behavioural study revealed that common wombats (Vombatus ursinus) in captivity are very susceptible to the development of stereotypic behaviour. For that reason, the final experiment (Chapter 8) was designed to further examine stereotypic behaviour in wombats and tested whether environmental enrichment could be used to reduce its prevalence. Adult L. latifrons (n = 12) were subjected to two different treatments in a replicated design with 12 week periods: (1) Enrichment – animals received feed and olfactory enrichment, 5 d/wk and (2) No-enrichment - animals received the standard captive diet only. Wombat behaviour was remotely observed via 24-h video surveillance. Eight out of the 12 wombats displayed one of four stereotyped movements (straight-line pacing, boundary pacing, figure-8 pacing or wall-climbing), with time spent stereotyping ranging from 61-129 min/d (mean 87 ± 7 min/d); time devoted to stereotyping took up 4-9% of the daily budget. There was a significant increase in foraging (333%) and exploration (13%) in response to enrichment. Enrichment also encouraged the expression of a wider range of naturalistic foraging behaviours. Despite these positive effects, enrichment had no discernable effect on the time spent stereotyping (P = 0.87) or inactive (P = 0.13). Despite the fact that stereotypies and inactivity were not reduced by enrichment, animal welfare was still enhanced as there was a notable improvement in natural wombat-specific behavioural expression and diversity.

The primary objective of the studies described in this thesis was to improve the cryopreservation success of koala spermatozoa for the purpose of establishing a genome resource bank for this species. A defining feature of the studies in this thesis was the implementation of an organelle-specific approach to better understand the causes of koala sperm cryo-injury. The functional attributes of spermatozoa, such as mitochondrial function, plasma membrane fluidity, membrane lipid asymmetry and DNA integrity were assessed as an indication of cryo-injury. Sperm mitochondrial function and plasma membrane integrity were examined by cryomicroscopy using the fluorescent probes JC-1 and propidium iodide (PI) respectively in a dual staining technique. Cooling and re-warming koala spermatozoa were more detrimental to mitochondrial function than to plasma membrane integrity. Mitochondrial membrane potential (MMP) was suppressed by freezing and thawing treatments; after thawing, MMP declined significantly during rewarming (from 5ºC to 35ºC). The distribution of GM1 ganglioside was examined using fluorescent-labelled cholera toxin B. No significant redistribution of GM1 was observed after chilling or cryotreatment. The externalisation of phosphatidylserine (PS) was examined using fluorescent-labelled annexin V. There was no significant increase in translocation of PS after chilling or cryopreservation. These observations imply that cryotreatment had little effect on plasma membrane lipid asymmetry. Koala spermatozoa were incubated in a range of anisotonic media to investigate whether nuclear swelling was caused by osmotic flux during the cryopreservation process. Although the most hypotonic solution tested (64 mOsm/kg) induced the highest incidence of nuclear relaxation (mean ± SEM; 12 ± 3%), this was not as severe as that previously documented following cryopreservation. Chromatin relaxation is a phenomenon observed in koala spermatozoa, where the sperm nucleus expands due to the result of structural changes in the natural conformation of the sperm DNA/protamine complex. DNA fragmentation was not a primary cause of cryopreservation-induced sperm chromatin relaxation, although in situ nick translation of putative DNA breaks indicated that these increased as the sperm head became progressively more relaxed. Using a Sperm Chromatin Dispersion test (SCDt) specifically developed and validated for koala spermatozoa, a continuum of nuclear morphotypes was observed, ranging from no apparent DNA fragmentation to spermatozoa with highly dispersed and degraded chromatin. A double comet assay was also developed to investigate DNA fragmentation in the koala spermatozoa. Conducted under neutral followed by alkaline conditions, this assay was able to differentiate between single- (SSB) and double-stranded (DSB) DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa; the majority of the koala spermatozoa had nuclei with DNA abasic-like residues. The ubiquity of these residues suggested that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with “true” DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA, to nuclei that exhibited both SSB and DSB. Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of inter-molecular disulphide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulphide bonds within their chromatin, but nevertheless, have been successfully cryopreserved. To examine the hypothesis that the cryoprotectants used for fish sperm cryopreservation will confer a similar degree of protection on koala spermatozoa, various concentrations of five cryoprotectants (dimethyl sulphoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Dimethylacetamide at a concentration of 12.5% (v/v) was found to be comparable to glycerol in the successful cryopreservation of koala spermatozoa although high inter-male variability was observed. However, when the new protocol was subsequently validated for a larger population of captive koalas (n = 22), glycerol emerged the better cryoprotectant with respect to all sperm viability parameters assessed except for that of the incidence of chromatin relaxation, which was not affected by the cryoprotectant. Significant difference was also observed in the post-thaw survival of spermatozoa from different animals, which was independent of pre-freeze semen quality. Based on post-thaw semen viability parameters, the koalas could be divided into two distinct groups, where one group had significantly higher sperm viability compared to the other group, regardless of cryoprotectant used. Positive correlation between motility and MMP was observed before and after cryopreservation. However, cryopreservation significantly reduced the dependency between these variables (P < 0.001), suggesting that cryopreservation reduced the dependency between mitochondrial function and motility.

Marsupial moles (genus Notoryctes) are the most highly specialised burrowing marsupials. The specialisations of the appendicular musculo-skeletal system of the marsupial moles are extensive and widespread; the major alterations are concentrated in, but not restricted to, the forelimb. Many of the derived features of the muscular system appear to be adaptations for improving the mechanical advantage of the limbs for burrowing. A number of the specialisations of the muscular system of the marsupial moles are convergent with those previously documented in other fossorial mammals, including golden moles, rodents and armadillos. There are, however, a number of unique specialisations of the musculo-skeletal system of Notoryctes. The functional morphology of the locomotor apparatus of marsupial moles is interpreted on the basis of the descriptions of the anatomy of the skeletal and muscular systems. The burrowing technique of the marsupial moles is a modified form of the parasagittal digging method that is used by other fossorial mammals, such as golden moles, armadillos and some rodents including pocket gophers. Differences in the functional morphology of the hindlimb between marsupial moles and other fossorial mammals are a reflection of the fact that marsupial moles do not construct permanent open burrow systems, but instead constantly dig through loose soil, backfilling as they progress. The functional morphology of the tail is uniquely specialised in the marsupial moles to function as the fifth limb during the pentapedal burrowing locomotion of marsupial moles. The remains of Miocene fossil marsupial mole, while clearly pleisiomorphic with respect to the appendicular skeletal morphology of modern notoryctids, demonstrate a high degree of specialisation for digging. It is hypothesised that the Miocene marsupial mole was already substantially specialised for a fossorial lifestyle, and thus pre-adapted for a subterranean lifestyle developed in correlation with the desertification of the Australian continent. Phylogenetic affinities of marsupial moles within the Marsupialia have long been enigmatic. While specialisation of the musculo-skeletal system have been so widespread as to obscure almost any phylogenetically relevant patterns, there is some evidence to support an association between notoryctids and peramelid bandicoots. Interspecific differences between the two species of marsupial moles, Notoryctes typhlops and N. caurinus, are minor but do support the separation of the two species.

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Zoologia, 2016. / Submitted by Raquel Almeida (raquel.df13@gmail.com) on 2017-08-15T20:53:45Z No. of bitstreams: 1 2017_LeonardoFerreiraMachado_PARCIAL.pdf: 3742030 bytes, checksum: 2daea5b27775cab71d509626539de6e0 (MD5) / Approved for entry into archive by Raquel Viana (raquelviana@bce.unb.br) on 2017-09-05T21:28:57Z (GMT) No. of bitstreams: 1 2017_LeonardoFerreiraMachado_PARCIAL.pdf: 3742030 bytes, checksum: 2daea5b27775cab71d509626539de6e0 (MD5) / Made available in DSpace on 2017-09-05T21:28:57Z (GMT). No. of bitstreams: 1 2017_LeonardoFerreiraMachado_PARCIAL.pdf: 3742030 bytes, checksum: 2daea5b27775cab71d509626539de6e0 (MD5) Previous issue date: 2017-09-05 / Os domínios morfoclimáticos da Caatinga e do Cerrado possuem características únicas tanto em termos climáticos, quanto físicos e biológicos. Apresentam ambientes e fitofisionomias particulares que sofreram alterações históricas em seus limites de distribuição geográfica em consequência de mudanças climáticas, eventos geológicos e até mesmo devido a ocupação humana e efeitos de queimadas. Apesar deste passado dinâmico, a história evolutiva e a diversificação dos organismos com distribuição nestes ambientes são pouco exploradas em estudos modernos que utilizam sequências de DNA e métodos biogeográficos, filogeográficos e de demografia histórica. Esta condição é ainda mais evidente em relação aos roedores e marsupiais de pequeno porte, que apesar de serem os grupos de mamíferos com maior diversidade de espécies da América do Sul, são pouco representados em investigações sobre suas diversificações e relações históricas com o Cerrado e a Caatinga. É neste contexto que a presente tese procura contribuir. Foram utilizadas sequências de DNA e métodos filogenéticos e filogeográficos para investigar a relação entre a diversificação de espécies de pequenos mamíferos com a evolução de seus habitats inseridos em fitofisionomias do Cerrado e Caatinga. O primeiro capítulo explora hipóteses filogenéticas e biogeográficas no gênero Phyllomys e contém a descrição de uma nova espécie com distribuição em matas de galerias do Cerrado e áreas de transição com a Floresta Atlântica. Além disso, com base em análise de distribuição ancestral de Phyllomys e outros gêneros distribuídos na Amazônia, é proposta uma hipótese de que habitats apropriados para a ocorrência de ratos de espinho arborícolas se estendiam na atual região sul e central do Cerrado promovendo uma conexão entre Amazônia e Floresta Atlântica durante o Mioceno. O segundo capítulo investiga as relações filogeográficas entre populações de Gracilianus agilis da Caatinga e Cerrado. Propõe que as alterações demográficas sofridas por esta espécie tem relação com a evolução das Matas Secas da região e que G. agilis pode abrigar mais de uma espécie. Por último, o terceiro capítulo avalia se houve alterações demográficas ao longo do tempo em populações de Calomys tener e propõe que eventos de fogo e a ocupação humana na região do Cerrado são fatores que alteram a paisagem natural, criando habitats que possibilitaram o crescimento populacional desta espécie. / The morphoclimatic domains of Cerrado and Caatinga are unique in terms of biotic, climatic, and geological features. The phytophysiognomies of Cerrado and Caatinga suffered multiple changes in theirs geographic distribution during the last millions years as a consequence of climatic and geologic modifications, fire in natural environments, and pre-historic human settlements. However, the diversification and historical evolution of organisms distributed in these regions are poorly studied. This is much more clearly if one tries to find Cerrado and Caatinga biogeographical studies based in small mammals (rodents and marsupials) as models, and using modern phylogenetic and phylogeographic methods. In this thesis, I used DNA sequences and phylogenetic and phylogeographic methods to investigate small mammals evolution and theirs relationship with the Cerrado and Caatinga phytofisiognomies. The first chapter propose phylogenetic and biogeographic hypothesis for the genus Phyllomys. It contains a description of a new species of Phyllomys and a hypothesis of past link between Amazon and Atlantic forest of South America through where the central/southern Cerrado biome is today. The second chapter investigates phylogeographical relationships of Gracilinanus agilis populations distributed in the Cerrado and Caatinga. I propose that historical expansions and retractions of dry forests of Cerrado and Caatinga are drivers for the diversification of this species, and that populations under G. agilis may represent more than one species. The third chapter focus on historical demographic changes in populations of Calomys tener. The results indicates that burning in natural areas, as well as the pre-historic human settlements may have favored the population expansion of Calomys tener during the late Quaternary.