Phylogenetic Analysis of 18S rRNA and the Mitochondrial Genomes of the Wombat, <Emphasis Type="BoldItalic">Vombatus ursinus,</Emphasis> and the Spiny Anteater, <Emphasis Type="BoldItalic">Tachyglossus aculeatus:</Emphasis> Increased Support for the Marsupionta Hypothesis

The monotremes, the duck-billed platypus and the echidnas, are characterized by a number of unique morphological characteristics, which have led to the common belief that they represent the living survivors of an ancestral stock of mammals. Analysis of new data from the complete mitochondrial (mt) genomes of a second monotreme, the spiny anteater, and another marsupial, the wombat, yielded clear support for the Marsupionta hypothesis. According to this hypothesis marsupials are more closely related to monotremes than to eutherians, consistent with a basal split between eutherians and marsupials/monotremes among extant mammals. This finding was also supported by analysis of new sequences from a nuclear gene—18S rRNA. The mt genome of the wombat shares some unique features with previously described marsupial mtDNAs (tRNA rearrangement, a missing tRNA^Lys, and evidence for RNA editing of the tRNA^Asp). Molecular estimates of genetic divergence suggest that the divergence between the platypus and the spiny anteater took place ≈34 million years before present (MYBP), and that between South American and Australian marsupials ≈72 MYBP. Received: 28 October 2000 / Accepted: 23 March 2001     

Sperm membrane fatty acid composition in the Eastern grey kangaroo (Macropus giganteus), koala (Phascolarctos cinereus), and common wombat (Vombatus ursinus) and its relationship to cold shock injury and cryopreservation success

Marsupial spermatozoa tolerate cold shock well, but differ in cryopreservation tolerance. In an attempt to explain these phenomena, the fatty acid composition of the sperm membrane from caput and cauda epididymides of the Eastern grey kangaroo, koala, and common wombat was measured and membrane sterol levels were measured in cauda epididymidal spermatozoa. While species-related differences in the levels of linolenic acid (18:3, n-6) and arachidonic acid (20:4, n-6) were observed in caput epididymal spermatozoa, these differences failed to significantly alter the ratio of unsaturated/saturated membrane fatty acids. However in cauda epididymidal spermatozoa, the ratio of unsaturated/saturated membrane fatty acids in koala and kangaroo spermatozoa was approximately 7.6 and 5.2, respectively; substantially higher than any other mammalian species so far described. Koala spermatozoal membranes had a higher ratio of unsaturated/saturated membrane fatty acids than that of wombat spermatozoa (t = 3.81; df = 4; p < or = 0.02); however, there was no significant difference between wombat and kangaroo spermatozoa. The highest proportions of DHA (22:6, n-3), the predominant membrane fatty acid in cauda epididymidal spermatozoa, were found in wombat and koala spermatozoa. While species-related differences in membrane sterol levels (cholesterol and desmosterol) were observed in cauda epididymidal spermatozoa, marsupial membrane sterol levels are very low. Marsupial spermatozoal membrane analyses do not support the hypothesis that a high ratio of saturated/unsaturated membrane fatty acids and low membrane sterol levels predisposes spermatozoa to cold shock damage. Instead, cryogenic tolerance appears related to DHA levels.     

An investigation into the similarities and differences governing the cryopreservation success of koala (Phascolarctos cinereus: goldfuss) and common wombat (Vombatus ursinus: shaw) spermatozoa

The aim of this study was to determine the relative cryopreservation success of koala and wombat spermatozoa and to investigate reasons for their respective post-thaw survival by examining the sperm's response to a range of osmotic media and determining the presence and distribution of F-actin. An hypothesis was proposed that F-actin may be imparting a degree of structural inflexibility to the koala sperm plasma membrane; hence, exposure of spermatozoa to cytochalasin D (5 microM), a F-actin depolymerisation agent, should result in increased plasticisation of the membrane and greater tolerance of cell volume changes that typically occur during cryopreservation. In experiment 1, koala (n = 4) and wombat (n = 4) spermatozoa packaged in 0.25 mL straws were cryopreserved using two freezing rates (fast-3 cm above liquid N2 interface; slow-6 degrees C/min in a freezing chamber) and two glycerol concentrations (8 and 14% v/v) in a tris-citrate glucose buffer with 15% (v/v) egg yolk. Wombat spermatozoa showed better (P < 0.01) post-thaw survival (% motile, % intact plasma membranes, % decondensed sperm heads) than koala spermatozoa. When exposed to media of varying osmolality, koala spermatozoa were less tolerant (% intact plasma membrane) of hyper-osmotic conditions (920 and 1410 mOsmol/kg) than wombat spermatozoa. F-actin was localised using a monoclonal antibody but only found in the wombat sperm head. When koala and wombat spermatozoa were exposed to media of varying osmolality, cytochalasin D had no beneficial effect on sperm survival (% intact plasma membranes). This study has demonstrated that wombat spermatozoa are highly tolerant of cryopreservation when compared to koala sperm but that spermatozoa from both species show greatest post-thaw survival when frozen slowly in 14% glycerol. Koala sperm are also particularly susceptible to hyper-osmotic environments but lack of detectable F-actin in the koala spermatozoan suggests that poor cryopreservation success in this species is unlikely to be associated with F-actin induced plasma membrane inflexibility.     

Digestive tract morphology and digestion in the wombats (Marsupialia: Vombatidae)

Summary Wombats consume grasses and sedges which are often highly fibrous. The morphology of the digestive tract and the sequence of digestion were studied in two species of wombats from contrasting habitats: Vombatus ursinus from mesic habitats and Lasiorhinus latifrons from xeric regions. Studies were performed on wild wombats consuming their natural winter diets, and on captive wombats fed a high-fibre pelleted straw diet. Vombatus had a shorter digestive tract (9.2 vs 12.5 times body length) of greater capacity (wet contents 17.9 vs 13.7% body weight) than Lasiorhinus. The most capacious region of the digestive tract was the proximal colon (62–79% of contents). The proportional length and surface area of the proximal colon were greater in Vombatus, but those of the distal colon were greater in Lasiorhinus. These digestive morphologies may reflect adaptations for greater capacity and longer retention of digesta in Vombatus, but greater absorption and lower faecal water loss in Lasiorhinus. Apparent digestion along the digestive tract was estimated by reference to lignin. The proximal colon was the principal site of fibre and dry matter digestion, whereas nitrogen was mainly digested in the small intestine. Depot fats in captive wombats were highly unsaturated and reflected those in the diet. Therefore, lipids, proteins and soluble carbohydrates in the plant cell contents were digested and absorbed in the stomach and small intestine. Conversely, dietary fibre was probably retained and digested by microbial fermentation along the proximal colon.Abbreviations ADF acid detergent fibre - DM dry matter - NDF neutral detergent fibre - SD standard deviation     

Hindgut fermentation in the wombats: two marsupial grazers

Summary The wombats Vombatus ursinus and Lasiorhinus latifrons have a capacious proximal colon with only a vestigial caecum. The pattern of microbial fermentation in the hindgut of both species was studied in captive animals fed a pelleted straw diet and in wild wombats feeding on their natural winter diets. Digesta pH was low in the stomach but near neutrality along the hindgut, indicating effective absorption and/or buffering of the colonic contents. Initial proportions and production rates of short chain fatty acids in vitro reflected the fermentation of plant cell walls. Proportions of isobutyrate, isovalerate and n-valerate increased towards the distal colon indicating proteolysis and subsequent fermentation of amino acids. The low ammonia content of digesta fluid suggested that ammonia released from these amino acids was absorbed and utilized by the wombats and their gut microbes. Wild wombats had higher concentrations and production rates of short chain fatty acids than captive animals, which was consistent with the higher apparent digestibility of their natural diet. The energy from short chain fatty acids in captive animals was 30–33% of digestible intake. Energy intakes were low and similar to resting metabolic rates estimated for marsupials. Actual resting metabolic rates of the wombats are probably lower than these estimates, and the proportion of energy derived from fermentation substantially higher than the 53–61% estimated in wild wombats. The energy from fermentation clearly enables wombats to utilize diets high in fibre.Abbreviations DEI digestible energy intake - DM dry matter - NDF neutral detergent fibre - SCFA short chain fatty acids - SD standard deviation - SMR standard metabolic rate     

Secretion of electrolytes,protein and urea by the mandibular gland of the common wombat (Vombatus ursinus)

Saliva was collected from the mandibular glands of anaesthetized common wombats (Vombatus ursinus) to ascertain maximal flow rates, salivary compostion and possible adaptations, particularly PO₄ ^3- secretion, to assist digestion. After temporary catheterization of the main duct through its oral opening, salivary secretion was evoked at flow rates ranging from 0.02±0.002 (±SEM) ml·min^-1 (0.7±0.07 l·min^-1·kg body weight^-1) to 0.4±0.05 ml·min^-1(14±1.9 l·min^-1·kg body weight^-1) by ipsilateral intracarotid infusion of acetylcholine. The [Na⁺] (15±5.1 to 58±8.6 mmol·l^-1) and [HCO₃ ^-] (35±1.9 to 60±1.9 mmol·l^-1) were positively correlated with salivary flow rate. The [K⁺] (58±5.2 to 30±2.4 mmol·l^-1), [Ca²⁺] (10.4±1.67 to 4.1±0.44 mmol·l^-1), [Mg²⁺] (0.94±0.137 to 0.17±0.032 mmol·l^-1), [Cl^-] (71±9.2 to 45±6.0 mmol·l^-1), [urea] (9.3±0.79 to 5.1±0.54 mmol·l^-1), H⁺ activity (29±1.6 to 17±1.6 nEq·l^-1) and amylase activity (251±57.4 to 92±23.3 kat·l^-1) were negatively correlated with flow. Both concentration and osmolality fell with increasing flow at the lower end of the flow range but osmolality always increased again by maximal flow whereas the relation between protein and flow was not consistent at the higher levels of flow and stimulation. Salivary [PO₄ ³⁺] was not correlated with flow and at 3–14% of the plasma concentration was extremely low. Thus, in contrast to its nearest relative, the koala (Phascolarctos cinereus), the wombat secretes little PO₄ ³⁺ presumably because it does not need high levels of PO₄ ³⁺ in its saliva to facilitate microbial digestion of plant fibre.Abbreviations bw body weight - ww wet weight