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1 ive ch Ar on ly o -N er ng lo cu rre nt

2 Genetics In comparison to eutherian (ÒplacentalÓ) mammals, little is known about the genetics of marsupials. Along with Monodelphis domestica (the American opossum) and Sminthopsis crassicaudata (the Australian fat-tailed dunnart), tammar wallabies are the best studied species. Studies of the genetics of the tammar wallaby have added to our understanding of the relationships between different tammar populations, provided general information regarding the level of genetic variability in macropods, allowed for assessment of the natural origins of the expatriate population in New Zealand and provided data regarding the evolution of the mammalian genome. There is very limited genetic variation in particular genes which are involved in immune reactions (McKenzie and Cooper, 1994). These genes, known as the major histocompatibility complex (MHC) class II genes, are extremely variable in many eutherian species except those which have suffered a severe reduction in numbers. It was therefore supposed that all mammals would show high levels of genetic variability in MHC genes if they had not passed through a population ÒbottleneckÓ. One of the most striking differences between eutherians and marsupials is their mode of reproduction. Marsupials give birth to very immature young therefore the majority of offspring development occurs in the pouch. Genetic variability at MHC genes has been implicated as being important in successful eutherian reproduction. It has been suggested that the lack of variation at these loci in tammars could be related to the shorter gestation period of marsupials in comparison to eutherian mammals (McKenzie and Cooper, 1994). Husbandry There are a large number of captive breeding colonies of tammar wallabies in Australia. These are associated with various universities, CSIRO Divisions, zoos and private fauna parks. Tammars, being small macropods, can be kept in large groups in relatively small enclosures. However, they browse heavily on grass and consequently it is necessary to spell tammar yards periodically. Additionally, tammars, along with other macropod species will ringbark trees, therefore tree guards made of wire are necessary. Tammars are fed pelleted food similar to that fed to domestic ruminants from feed bins which are opened every second day. If no grass is available, their diet may be supplemented with lucerne hay. The natural habitat of tammar wallabies is dry sclerophyll forest, heathlands, coastal scrub, mallee and woodland thickets (Kennedy, 1992). It is important therefore to provide shelter of some kind, so hides made of branches and/or large hollow concrete tubing are placed in each yard. Tammar wallabies are of a size which makes them relatively easy to catch, at least for experienced handlers. To minimise stress, captures should be well planned and experienced personnel should be used. Catching should be as efficient as possible so that the chance of injury to the animal from running into fences or other animals in the yard is minimised. The standard catching procedure is to make animals run along a fence. They are then caught with a long handled landing net. Animals are removed from the net by the base of the tail and placed into a close-mesh hessian bag which must allow adequate air flow to avoid hyperthermia. It is important that the bag is as light-proof as possible because once restrained and in relative darkness the animal will usually become calm, allowing for easier examination. The most common reason for catching tammars in captive situations is to monitor their breeding. Examination of the pouch is carried out (whilst the animal is still in a hessian bag) by holding the animal by the base of the tail and placing it on its back. The back legs are freed from the bag to prevent injury and the pouch is exposed. The state of the pouch can be used as an indicator of the animalõs reproductive state. Usually the pouch is dirty, and is covered in a dark, tacky secretion. If a pouch is moist and clean it is likely that the female has been licking it in anticipation of an imminent birth. The presence of an elongated teat in an empty pouch indicates continued suckling of an at foot pouch young. Female tammars can suckle two pouch young at the one time, one new born and one at foot. Anaesthesia Tammars can be anaesthetized by various methods depending upon the procedure to be carried out (see Tribe and Middleton, 1988; Blyde, 1994). Appropriate anaesthetic agents include: Diazepam (Valium) Ñ a useful sedative for short procedures such as pouch checking and blood collection at a dose rate of 1 mg/kg intravenously; Zoletil Ñ a good immobilising drug providing smooth recovery and induction at a dose rate of 20 mg/kg intramuscularly; Ketamine / Xylazine mixture Ñ similar effect to Zoletil at dose rate 15 mg/kg Ketamine mixed with 5 mg/kg Xylazine, intramuscularly; and Isoflurane or Halothane Ñ both can be administered by mask to reach surgical anaesthesia following immobilisation by any of the above agents. Euthanasia can be performed by intravenous injection of an overdose of sodium pentobarbitone (Reilly, 1993). Diseases Macropods may be affected by a number of different diseases, some of which are common to particular species, others are common to all macropods and some are rarely seen ( see Spielman, 1995). Many diseases are avoidable if good husbandry techniques are employed. Provided that stocking rates are low, there is an adequate number of feeding stations, water troughs are cleaned regularly, faeces are removed from concrete enclosures and animals that do become ill are isolated, disease incidence can be minimal in captive macropod colonies. 2 ANZCCART News Vol 8 No 4 December 1995 Insert

3 Necrobacillosis, or lumpy jaw, is one of the most well described and characterised diseases affecting macropods and although there is more to learn about its aetiology and inciting agents, it is relatively well understood. It is associated with overcrowding, poor hygiene or poor diet, although it can occur in situations where none of these factors is apparent. The first signs are swellings around the jaw associated with necrotic, foetid and erosive lesions. Internal organs may also be involved. Once the disease is detectable, antibiotic therapy is probably universally unsuccessful without surgery. However, precursor conditions (such as gum disease) may be effectively treated with antibiotics. Animals suffering will usually show some improvement with treatment, but the disease is only slowed, not cured. Unless the circumstances are exceptional, the animal should be euthanased. Toxoplasmosis can occur in marsupials, which act as an intermediate host for the coccidian parasite, Toxoplasma gondii. Transmission is usually via ingestion of oocysts excreted in the faeces of domestic cats. Slight to severe neurological signs are manifested, including head tilt, ataxia, difficult respiration and sometimes sudden death. Treatment is only effective if given early and should include a combination of vitamin E / selenium, corticosteroids, diazapam (to reduce anxiety and produce sedation), sodium bicarbonate and intravenous fluids. Helminths are common in macropods and do not produce problems, except in times of stress. Good husbandry procedures provide the best means of protection (see Spielman, 1995). Zoonoses There are a few known diseases which can be transmitted from macropods to humans. There is a high carrier rate of salmonella species in young macropodids and even normal adults and these bacteria can cause sudden death, diarrhoea, lethargy, poor appetite and pneumonia in affected animals. Ringworm can be severe and is highly contagious to humans. It can cause oval to round areas of redness, scaliness or itchiness. However, most cases are mild and self-limited. Larger macropods can harbour the cystic stage of the tapeworm (Echinococcus granulosus), and are a possible source for spread to domestic animals and humans. Uninspected carcases should not be fed to dogs. Macropods can be infected by encephalomyocarditis virus, which is spread by rats and perhaps mice. It can cause death in animals and is potentially a serious zoonosis (Spielman, 1995). Table 1. Some useful data for the tammar wallaby Body weight 3-5 kg Body temperature 36.4 o C Basal metabolic rate 8.19W Intake of lucerne hay 29.4kg day -1 Min. urinary N 2 excretion Creatine excretion 60mg.kg day mg.kg day -1 Total body H 2 O content 60.8 (% body weight) Total water turnover 65.2ml.kg -0.8 day -1 Adapted from Hume (1982). Captive breeding and maintenance of the fat-tailed dunnart Clive Chesson and Rory Hope Department of Genetics, University of Adelaide South Australia 5005 Introduction The fat-tailed dunnart, Sminthopsis crassicaudata (Marsupialia Dasyuridae) is a mouse-sized nocturnal marsupial, widely but patchily distributed across southern and central Australia west of the Great Divide (Archer, 1981). Its habitat ranges from wet grasslands to dry stony deserts. The demise of dunnart populations in suburban areas such as the Adelaide plains has probably resulted from alterations to the natural habitat and the presence of feral predators. Animals from different geographic areas differ in external morphology and behaviour (Morton and Alexander, 1982; Hope and Godfrey, 1988). Extensive studies on the ecology of the species were carried out by Morton (1978). Martin (1965) recognised the potential of this polyoestrous marsupial as a small laboratory mammal suitable for biological research. The history of the breeding colony of dunnarts at the University of Adelaide has been well documented (Smith et al. 1978; Bennett et al. 1982; 1990). In 1995, responsibility for management of the colony passed from the Department of Genetics to the Animal Services Division of the University of Adelaide. Currently the colony comprises approximately 1000 animals. The features of the species described below are based on our experience with the colony in the Department of Genetics. Husbandry Animals can be housed in windowless rooms with artificial light and constant temperature ( C). One room can have the day length reduced to eight hours as a pre-treatment to stimulate males to breed. Other rooms, one a nursery and animal store, the other a breeding room, have a day length of 16 hours. Galvanised metal breeding cages (about 50 x 35 x 20 cm) incorporate a mesh top with a hinged flap at the front, a removable plate glass front and a separate metal base tray. Standard laboratory rat cages can be used for weaning and storage. A wellventilated plastic nest box containing shredded paper is supplied in each cage, and plastic mouse wheels were provided for breeding animals but not for nursing females. Clean dry autoclaved sand (important for animal grooming) was maintained in the cage trays. The natural diet comprises invertebrates including locusts, moths, cockroaches, centipedes and scorpions, and vertebrates such as small lizards and baby mice. In the laboratory, animals can be fed canned pet food, dry cat food and mealworms (Tenebrio molitor). Water and dry cat food should be supplied ad libitum. Dunnarts can be held by the tail with the body supported, or for a short period, by the scruff of the neck. Pouch and health checks should be carried out once per week. More frequent checks inhibit litter production. ANZCCART News Vol 8 No 4 December 1995 Insert 3

4 Disease Research with the dunnart Disease was not a major problem in the colony. Respiratory infections occasionally occurred but were preventable by good husbandry. The most common parasite Ornithonyssus bacoti was ready controlled using commercially available flea powders. Reproduction and growth The oestrous cycle length is approximately 31 days (Godfrey and Crowcroft, 1971) and the gestation period 13.5 days post-coitus (Bennett et al., 1990). Females can nurture only 10 young in their pouch. Often more than 10 young are born, the excess failing to attach to one of the ten nipples and dying. Females can be first mated at three months of age and males at seven months. The optimal ratio is four females per male in each cage. Litters are usually weaned 70 days after birth with underweight litters left an additional 7-14 days until animals reach at least 5.5 g body weight. A few exceptional males can sire as many as 45 litters. Unlike the situation in the wild, litters in the colony can be produced throughout the year. Some characteristics of postnatal growth and development are summarised below (Ewer, 1968; Godfrey and Crowcroft, 1971). Age (days) Growth stage 0 Birth weight = mg 28 Pouch bulging and young visible 37 Young protruding from pouch 40 Permanent attachment to nipples ceases Eyes open First leave nest, eat solid food 70 Weaned - body weight = 5-8 g 91 Females sexually mature (1st oestrus) 98 Weight 8-11 g 126 Weight g 210 Males sexually mature - first matings Haematological data Haematological parameters and blood cell indices of the species have been determined by Haynes and Skidmore (1991). Special techniques An inhalation anaesthetic was used (ethrane 4%) carried by a 1:1 mixture of oxygen and nitrous oxide at one litre per minute. The room used for anaesthesia was kept warm to prevent rapid loss of body heat by anaesthetised animals (Bennett et al., 1982). Blood ( ml) can be obtained from anaesthetised animals by puncturing the orbital sinus if skilled, or the jugular or recurrent tarsal vein, with an 25- gauge needle attached to a heparinised syringe. The species has considerable potential for biomedical and genetical research. Animals can be housed in a relatively small area and bred efficiently in captivity. The species is well characterised cytogenetically and has a small number of large distinct chromosomes (2n = 14) that can be identified on the basis of their size and shape without the need to apply specialised chromosome banding techniques. Cultured cell lines are available and chromosome suspensions prepared from such cells can be sorted to high levels of purity with a fluorescent activated cell sorter (Hope, 1993). A number of genetic markers have been described and some linkage data are available (Bennett et al., 1990; Hope et al., 1992; Hope, 1993). The small body size of the dunnart in comparison with other model marsupial species such as the tammar wallaby and the grey short-tailed opossum (Monodelphis domestica) gives the species an obvious advantage for comparative biomedical research. Because the young are born at a relatively early stage of development and are accessible in the pouch, marsupials such as S. crassicaudata provide excellent models for developmental studies (Hope, 1993). The dunnart has a number of attributes that make it one of the best marsupial models (and one of the best vertebrate models) for investigating the factors of critical relevance to reintroduction biology (Bennett et al., 1990). A major benefit of using the dunnart to investigate reintroduction biology is that the results obtained are likely to be of direct relevance to the re-establishment of populations of endangered species, including the sandhill dunnart (S. psammophila), the mulgara (Dasycercus cristicauda), the kowari (Dasyuroides byrneii), and other threatened dasyurids. The common brushtail possum Janine A. Duckworth and Lynne M. Meikle Manaaki Whenua - Landcare Research PO Box 69, Lincoln 8152, New Zealand Introduction The common brushtail possum, Trichosurus vulpecula (Marsupialia Phalangeridae), is the most widespread of the three species of brushtail possum in Australia. It is a nocturnal, predominantly arboreal mammal commonly found in wooded and forested areas throughout eastern, central and south-western Australia. The possum is well adapted to suburban life, frequently cohabiting with humans (Strahan, 1983). In the 1830s, common brushtail possums were first introduced to New Zealand to establish a fur trade. They have spread throughout that country and occur at much higher densities than in their native Australia. Their impact on New Zealand plants and animals and their role as the main wildlife reservoir of bovine tuberculosis infecting farmed cattle and deer make the possum New ZealandÕs most serious vertebrate pest (Cowan, 1990). 4 ANZCCART News Vol 8 No 4 December 1995 Insert

5 The common brushtail possum is one of the most extensively studied non-macropod marsupials. It is a hardy animal, and large enough to make it suitable for endocrine studies using repetitive blood sampling. As with other marsupials, many of the physiological and biochemical systems of the possum are extremely immature at birth. The possum neonate provides an accessible model for the study of early development of mammalian organs and their physiological and neurological function (Tyndale-Biscoe and Janssens, 1988). These factors, together with an intensification of research to develop technologies to reduce possum numbers in New Zealand, have increased the need for information on successful animal husbandry methods for possums. Landcare Research maintains a captive animal facility for basic biology and possum control research at Rangiora, Canterbury, New Zealand. Trapping and acclimatisation Wire or solid sided box traps ( 0.6 m x 0.3 m x 0.3 m), baited with an apple and a lure of cinnamon oil, icing sugar and flour mix, are suitable for the capture of possums from the wild. Trapping success is best on dry nights in winter and early spring when natural food is less plentiful. In warm climates traps must be checked early in the morning since trapped possums are susceptible to heat exposure and dehydration. Possums are easily transferred from the trap to a thick sack for initial examination and transport. Newly captured possums may be anaesthetised with a 1:2 mixture of oxygen and carbon dioxide (Jolly, 1993), and then weighed, sexed and checked for signs of infection, injury, or the presence of pouch young. At Landcare Research, possums are individually caged indoors for a six week acclimatisation period before assignment to outdoor pens or research trials. Acclimatising animals are checked daily, weighed each fortnight and subjected to minimum disturbance. Newly captured possums may experience some initial weight loss (Presidente, 1982; Buddle et al., 1992, Meikle, unpublished data), probably due to stress. Some animals may not adjust, despite specialised care. They develop diarrhoea, become anorexic and dehydrated, and require euthanasia. Stress-related mortality may also be a problem in overcrowded conditions, especially during the breeding season (Hope, 1971; Presidente, 1982). Housing Adults generally avoid contact with each other except during the mating period (Winter, 1976) or when nesting and feeding sites are limited (Fairweather et al., 1987). To date no standardised cage sizes have been specified for possums. At Landcare Research, animals are kept in individual wire cages (1 m x 0.4 m x 0.4 m) with mesh floors and a removable nest box (0.35 m x 0.2 m x 0.2 m). Cages are currently being enlarged (1 m x 0.4 m x 0.55 m). Individual possums have also been successfully housed in galvanised wire rabbit cages (0.5 m x 0.3 m x 0.55 m) (Dellow and Harris, 1984) and in stainless steel cages (0.76 m x 0.6 m x 0.6 m) (Fitzgerald et al., 1981). Housing should provide opportunity for seclusion, such as the use of solid sides, with a drape over the cage front. Trays under cages should be cleaned and waste food removed daily. Nest boxes should be cleaned at least once a fortnight, more often for some individuals. Animals can be successfully group-housed outdoors in wire mesh pens with grassed or concrete floors. The pen should be well constructed and verminand bird-proof. Possums have been successfully bred in an enclosure with an area of about 11 m 2 for two animals (Bergin, 1978), 16 m 2 for three to four animals (Jolly et al., 1995), or eight m 2 for up to seven animals (Curlewis et al., 1985). Group-housed animals may be individually identified by a small numbered metal ear tag. Although possums can maintain a body temperature of C over a range of ambient temperatures from 10 to 30 0 C (Tyndale-Biscoe, 1973), outdoor pens should include a rain- and wind- proof shelter. Sufficient nesting boxes or hessian nesting sacks should be provided for each individual and numerous branches and perches for climbing. This will reduce aggression. In warm areas, effective ventilation and insulation against excessive heat may be required. Animal handling and anaesthesia Possums can inflict painful wounds with their sharp claws and teeth and should be handled with care. A possum may be handled by grasping its tail. If it is confined to a sack, it is possible to physically examine an animal, including the pouch, without anaesthesia. Use of forceful restraint causes stress to animals and handlers and should be avoided. Confining animals in nest boxes or hessian sacks makes the handling, transport, and anaesthesia of possums easier. A wide range of anaesthetic agents is effective (Tribe and Middleton, 1988; Blyde, 1994). Short-term anaesthesia can be maintained using a 1:2 mixture of oxygen and carbon dioxide gases (Jolly, 1993). Longterm anaesthesia for surgical procedures can be induced with a mixture of halothane (or isoflurane) and oxygen in a closed container and maintained by a mask (NHMRC, 1990). Ketamine/xylazine as intramuscular injection, (dose rate 20 mg/kg ketamine, 2mg/kg xylazine; or ketamine alone, dose rate mg/kg) gives reasonable restraint as does Zoletil (dose rate mg/kg intramuscularly). For surgical anaesthesia this may be followed with halothane and oxygen by mask. Diazepam ( mg/kg intramuscularly) can be useful in facilitating handling. Expert advice should be sought before using anaesthetics. Nutrition Possums are hindgut fermenters and their digestive system is adapted to high-fibre diets (Wellard and Hume, 1981). The caecum and proximal colon are well developed and are the principal sites of microbial fermentation (Hume, 1982). In the wild, possums are ANZCCART News Vol 8 No 4 December 1995 Insert 5

6 predominantly herbivorous, feeding on the leaves, blossom, and fruit of a wide range of tree species. They also feed on grass, clover, and broadleaf weeds and are known to eat small birds, eggs and invertebrates (Kerle, 1984; Cowan, 1990). In captivity, possums thrive and breed when fed a wide variety of foodstuffs such as carrots, apples, other fruits and vegetables, maize, eucalyptus and other leafy vegetation, bread, and rabbit or stock feed pellets (Hope, 1971; Presidente, 1982; NHMRC, 1990). A varied diet should be provided. Maintenance requirements for individually caged animals have been estimated as 40 g digestible DM/kg W 0.75 (Fitzgerald et al. 1981). At Landcare Research possums in pens are offered about 200 g of grain-based pellets each day plus seasonal fruit and greens. They tend to be wasteful feeders. A 3.0 kg animal eats about g of pellets daily. Animals should have free access to clean water. In outdoor pens dried grain-based pellets are provided ad libitum from water-proof hoppers, and clean water supplied from a self-filling water trough. As possums are susceptible to calcium toxicity resulting in calcinosis (Jackson, 1983), food with a high calcium content should be avoided. Adult live weight of possums varies greatly ( g), largely as a reflection of genotypic origin and nutrition (Tyndale-Biscoe, 1973; Presidente, 1982; Cowan, 1990). Reproduction In the wild, births may occur in every month of the year, though in most populations they are highly seasonal. The main season for births is in autumn (April-June), with a smaller, more variable peak in spring (October- November). The incidence of spring breeding and the proportion of females breeding in both autumn and spring (double-breeders) is not strongly related to latitude but more to habitat and density (Kerle, 1984; Cowan, 1990). Possums are seasonally polyoestrous and monovular with an oestrous cycle of about 25 days and gestation length of days (Pilton and Sharman, 1962; Curlewis and Stone, 1986; Tyndale-Biscoe and Renfree, 1987). Oestrus and ovulation are usually suppressed throughout lactation. If the pouch young dies or is removed during the breeding season, oestrus often follows 8-12 days later. Repeated removal of pouch young will induce repeat breeding throughout the breeding period (Pilton and Sharman, 1962; Jolly et al., 1995). Age at first-breeding in females varies from one to three years (Green, 1984). Female possums possess a large forward-directed pouch with two teats. The singleborn young weighs about 0.2 g at birth (Tyndale-Biscoe and Renfree, 1987) and climbs unaided from the urogenital tract to the pouch. The growth and development of the young in the pouch has been described by Lyne and Verhargen (1957), Gilmore (1966), and Crawley (1973). The young remains permanently attached to the teat for the first days. At first, young gain weight slowly to reach about 100 g at days of age and then more rapidly to attain about one kg live-weight at days. Fur is evident about days, eyes open at days, and homeothermy develops about days. The young emerge from the pouch for gradually increasing intervals from about days and leave permanently between 150 and 240 days (Winter, 1976). Orphaned pouch young older than 90 days can be successfully handreared, with good hygiene practices, patience, and care in feeding (Presidente, 1982; Walraven, 1990). Male possums reach sexual maturity at one to two years (Gilmore, 1966). They produce sperm throughout the year, and although testis weight does not vary seasonally, the weights of the epididymis and particularly the prostate gland are heavier in the breeding season (Gilmore, 1969; Tyndale-Biscoe and Renfree, 1987). Diseases Health problems in captive possums are often a consequence of stress associated with their capture and adjustment to new surroundings. For animals in group housing, fighting during the breeding season, introduction of new animals, cold wet weather, and overcrowding are all sources of additional stress (Presidente, 1982, 1984). Salmonellosis, yersiniosis, and staphylococcal and streptococcal infections can cause problems in captive colonies (Hope, 1971; Presidente, 1984), possibly because of a stress-induced suppression of the immune response (Buddle et al., 1992). The range of parasites and diseases that have been reported in possums has been reviewed by Presidente (1984). Diseases of concern to people in contact with possums include leptospirosis, giardia, cryptosporidiosis, Q fever (Australia), and bovine tuberculosis (New Zealand). General experimental techniques Blood samples may be collected by intracardiac puncture (only from an anaesthetised animal) (Buddle et al., 1992), by venepuncture from the jugular vein or from the lateral or ventral tail vein of an anaesthetised animal. For reproductive studies, the stage of the oestrous cycle can be determined through the cytology and presence of sperm in daily vaginal smears (Pilton and Sharman, 1962) or urine samples (Duckworth, unpublished data). Euthanasia can be performed by intravenous or intra-peritoneal injection of sodium pentobarbitone (150 mg/kg) (Reilly, 1993). For information on haematology, see Dawson and Denny (1968), Presidente (1982) and Buddle et al., (1992). 6 ANZCCART News Vol 8 No 4 December 1995 Insert

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