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Home , Agile antechinus, Animal (book), Antechinus, Brown antechinus, Dasyuridae, Dasyuromorphia

Diet, Nutrition and Haematology of

Dasyurid

Hayley Stannard

B.Anim.Sc., B.Sc.(Hons.)

Submitted for the completion of a Doctor of Philosophy degree at the University of

Western

March 2012

1 Table of Contents

TABLE OF FIGURES ...... 5 TABLE OF TABLES ...... 6 ACKNOWLEDGEMENTS ...... 7 STATEMENT OF AUTHENTICATION ...... 10 PREFACE ...... 11 PUBLICATIONS ...... 12 CONFERENCE PRESENTATIONS ...... 14 ABSTRACT ...... 16 INTRODUCTION ...... 20 1.1 MARSUPIALS ...... 24 1.2 DASYURIDS ...... 25 1.2.1 Threats to Dasyurids ...... 26 1.2.2 Conservation and management of Dasyurids ...... 27 1.3 NUTRITION ...... 31 1.3.1 Digestive physiology of Dasyurids ...... 32 1.3.2 Diet of Dasyurids ...... 34 1.4 HAEMATOLOGY ...... 35 1.4.1 haematology ...... 37 1.4.2 Blood parameters and translocation ...... 39 1.5 STUDY ...... 40 1.5.1 (Antechinomys laniger) ...... 41 1.5.2 Red-tailed (Phascogale calura) ...... 43 1.5.3 Eastern (Dasyurus viverrinus) ...... 45 1.5.4 Spotted-tailed quoll (Dasyurus maculatus) ...... 47 1.5.5 Fat-tailed (Sminthopsis crassicaudata) ...... 49 1.5.6 Stripe-faced dunnart (Sminthopsis macroura) ...... 50 1.6 AIM...... 53 1.7 CHAPTER 1 REFERENCES ...... 55 THE DIET OF RED-TAILED IN A TRIAL TRANSLOCATION AT ALICE SPRINGS PARK, NORTHERN , ...... 66 2.1 CHAPTER OUTLINE AND AUTHORSHIP ...... 67 2.2 INTRODUCTION ...... 68 2.3 MATERIALS AND METHODS ...... 69 2.3.1 Study site ...... 69 2.3.2 and sample collection...... 70 2.3.3 Scat analysis...... 70 2.3.4 Light trap sampling and arthropod identification ...... 71 2.4 RESULTS ...... 73 2.4.1 Scat component analysis ...... 74 2.4.2 Comparison of seasonality ...... 75 2.4.3 Light trap sampling and arthropod identification ...... 78 2.4.4 Incidental observations ...... 80 2.5 DISCUSSION ...... 80 2.6 CHAPTER 2 REFERENCES ...... 86 THE RATE OF PASSAGE AND THE MORPHOLOGY AND HISTOLOGY OF THE GASTROINTESTINAL TRACT OF THE KULTARR (ANTECHINOMYS LANIGER) ...... 90

2 3.1 CHAPTER OUTLINE AND AUTHORSHIP ...... 91 (A) DESCRIPTION OF THE GASTROINTESTINAL TRACT AND ASSOCIATED ORGANS OF THE KULTARR (ANTECHINOMYS LANIGER) ...... 92 3.2 INTRODUCTION ...... 92 3.3 MATERIALS AND METHODS ...... 93 3.4 RESULTS AND DISCUSSION ...... 94 3.5 CHAPTER 3 REFERENCES (A) ...... 100 (B) RATE OF PASSAGE THROUGH THE KULTARR (ANTECHINOMYS LANIGER) DIGESTIVE TRACT ...... 104 3.6 INTRODUCTION ...... 104 3.7 METHODS ...... 105 3.7.1 Animals ...... 105 3.7.2 Rate of passage trial ...... 105 3.7.3 Digestibility trial ...... 107 3.8 RESULTS AND DISCUSSION ...... 108 3.9 CHAPTER 3 REFERENCES (B) ...... 112 DIGESTIBILITY OF CAPTIVE FEEDING REGIMES OF THE RED-TAILED PHASCOGALE (PHASCOGALE CALURA) AND KULTARR (ANTECHINOMYS LANIGER) ...... 116 4.1 CHAPTER OUTLINE AND AUTHORSHIP ...... 117 4.2 INTRODUCTION ...... 118 4.3 MATERIALS AND METHODS ...... 120 4.3.1 Animals ...... 120 4.3.2 Digestibility experiments ...... 120 4.3.3 Nutritional analysis ...... 121 4.4 RESULTS ...... 122 4.5 DISCUSSION ...... 131 4.6 CHAPTER 4 REFERENCES ...... 138 THE ROLE OF DIETARY COMPOSITION IN OPTIMUM NUTRITION FOR THE SMINTHOPSIS MACROURA AND S. CRASSICAUDATA ...... 143 5.1 CHAPTER OUTLINE AND AUTHORSHIP ...... 144 5.2 INTRODUCTION ...... 145 5.3 ANIMALS, MATERIALS AND METHODS ...... 147 5.3.1 Animals ...... 147 5.3.2 Digestibility trials: diets ...... 148 5.3.3 Digestibility trials: procedure ...... 148 5.3.4 Sample analysis ...... 149 5.3.5 Statistical analysis ...... 150 5.4 RESULTS ...... 150 5.4.1 Dietary analysis ...... 150 5.4.2 Body condition ...... 153 5.4.3 Digestibility trials ...... 156 5.5 DISCUSSION ...... 163 5.6 CHAPTER 5 REFERENCES ...... 170 DIGESTIBILITY OF TWO DIETS BY CAPTIVE EASTERN (DASYURUS VIVERRINUS) ...... 177 6.1 CHAPTER OUTLINE AND AUTHORSHIP ...... 178 6.2 INTRODUCTION ...... 179 6.3 METHODS ...... 181 6.4 RESULTS ...... 183 6.4.1 Diets ...... 183 6.4.2 Food consumption ...... 184

3 6.4.3 Digestibility ...... 184 6.5 DISCUSSION ...... 186 6.6 CHAPTER 6 REFERENCES ...... 195 FURTHER INVESTIGATION OF THE BLOOD CHARACTERISTICS OF AUSTRALIAN QUOLL (DASYURUS SPP.) SPECIES ...... 202 7.1 CHAPTER OUTLINE AND AUTHORSHIP ...... 203 7.2 INTRODUCTION ...... 204 7.3 METHODS ...... 206 7.3.1 Study animals ...... 206 7.3.2 Blood collection and analysis ...... 207 7.4 RESULTS ...... 209 7.5 DISCUSSION ...... 216 7.6 CHAPTER 7 REFERENCES ...... 224 GENERAL DISCUSSION AND FUTURE DIRECTIONS ...... 231 8.1 FUTURE DIRECTIONS ...... 239 8.2 CHAPTER 8 REFERENCES ...... 243

4 Table of Figures

Figure 1.1 of metatherian orders and Notoryctemorphia adapted from Cardillo et al. (2004) ...... 25 Figure 1.2 The digestive tract morphology of Dasyurid spp. (S) stomach, (I) intestine, (R) rectum...... 33 Figure 1.3 A kultarr (Antechinomys laniger) (Stannard, 2009) ...... 42 Figure 1.4 A red-tailed phascogale (Phascogale calura) (Stannard, 2008) ...... 44 Figure 1.5 An (Dasyurus viverrinus) (Stannard, 2007)...... 46 Figure 1.6 A spotted-tailed quoll (Dasyurus maculatus) (Lynch, 2012) ...... 48 Figure 1.7 A fat-tailed dunnart (Sminthopsis crassicaudata) (Stannard, 2011) ...... 50 Figure 1.8 A stripe-faced dunnart (Sminthopsis macroura) (Stannard, 2011) ...... 51 Figure 2.1 Multidimensional scaling of seasonal red-tailed phascogale scat components 77 Figure 3.1 Pyloric region of the kultarr stomach showing parietal cells...... 97 Figure 3.2 Brunner’s glands in the kultarr at the pylorus duodenum junction...... 97 Figure 5.1 Body mass (a) and tail width (b) of S. macroura over the course of the nutrition trials. During rest periods animals were fed food. All data are means +/- 1 standard deviation. [a] significant change in body mass/tail width from pre-trial value P<0.05; [b] significant change in body mass/tail width during the course of a feeding trial P<0.05...... 154 Figure 5.2 Body mass (a) and tail width (b) of S. crassicaudata over the course of the nutrition trials. During rest periods animals were fed cat food. All data are means +/- 1 standard deviation. [a] significant change in body mass/tail width from pre-trial value P<0.05; [b] significant change in body mass/tail width during the course of a feeding trial P<0.05...... 155 Figure 5.3 The gastrointestinal tract of S. macroura, where (S) stomach, (Ps) pyloric sphincter (I) intestine, (M) mesenteric tissue, (R) rectum, and (Sp) spleen...... 162 Figure 7.1 White blood cells of eastern and spotted-tailed quolls a) annular nuclei leukocyte and lymphocyte, b) neutrophil with an annular nuclei, c) eosinophil and lymphocyte, d) neutrophil with a segmented nucleus ...... 210

5 Table of Tables

Table 2.1 Percentage of occurrence of components from 95 red-tailed phascogale scat samples ...... 73 Table 2.2 Pairwise comparison showing the differences between summer and spring .... 78 Table 2.3 Arthropods collected at ASDP and possible prey items for red-tailed phascogales ...... 79 Table 3.1 Morphology of the gastrointestinal tract of the kultarr (Antechinomys laniger) ...... 95 Table 3.2 Transit time and mean retention time of kultarr (Antechinomys laniger) and other small dasyurids ...... 109 Table 3.3 Mean (± s.d.) intake and apparent digestibility of minced beef fed to during trial (n = 8) ...... 111 Table 4.1 Composition of food items (mean ± s.d.) ...... 124 Table 4.2 Body mass, intake and apparent digestibility (AD) of red-tailed phascogale diets (mean ± s.d.) ...... 127 Table 4.3 Body mass, intake and apparent digestibility (AD) of kultarr diets (n = 8; mean ± s.d.) ...... 129 Table 5.1 Composition of food items on a dry matter basis (n = 2) All data are means ± 1 standard deviation. *P<0.01 ...... 152 Table 5.2 Body mass, tail widths, food intake and apparent digestibility of the diets (AD) for S. macroura (n = 11). All data are means ± 1 standard deviation...... 157 Table 5.3 Body mass, tail widths, food intake and apparent digestibility of the diets (AD) for S. crassicaudata (n = 8). All data are means ± 1 standard deviation...... 159 Table 5.4 Gastrointestinal tract measurements of S. macroura and S.crassicaudata. Data are presented for as means ± 1 standard deviation, ID 1-8 are from the diet trials and 9-12 are from independent experiments...... 163 Table 6.1 Composition of food items (mean ± s.d.) ...... 183 Table 6.2 Apparent digestibility (AD) of two eastern quoll diets (n = 16; mean ± s.d.) 185 Table 6.3 Apparent digestibility of dry matter, gross , protein and lipids in dasyurids and other insectivorous-carnivorous ...... 187 Table 7.1 Range and mean values for blood chemistry and differential white cell counts of eastern quolls ...... 211 Table 7.2 Seasonal blood chemistry values for eastern quolls (mean ± s.d.) ...... 213 Table 7.3 Blood chemistry and differential WBC for spotted-tailed quolls ...... 215 Table 7.4 Comparison of Dasyurid blood chemistry and differential WBC ...... 218

6 Acknowledgements

Dr. Julie Old thank you for all your support and encouragement, I would not have been able to get through my PhD without you. Thank you for putting up with me for so long and for your patience and guidance throughout these past four years. I would like to thank

Dr. Penny Trevor-Jones for being my co-supervisor for a short period of time. Thank you, A/Prof. Lauren Young for coming onboard halfway through my candidature and for always being impressed with my progress especially when I was not.

To all the staff at Alice Springs Desert Park, I would especially like to thank Bruce,

Yokham, and Pat for assistance in obtaining arthropod samples. Wes Caton collected red- tailed phascogale scats and sent them to me for analysis. Thank you Wes for the knowledge you shared. It not only assisted my learning, but also contributed to maintaining our captive colonies at UWS.

The quolls used for my PhD were from captive colonies maintained at Australian

Ecosystems Foundation Inc. (AEFI) and Featherdale Wildlife Park (FWP). Thank you to all the staff at AEFI (Trevor, Annette, Ben and Angel) and FWP (Chad) for allowing me to use your animals and for your assistance along the way. Thank you, Michelle Bingley for your help with blood collection from quolls.

I would like to extend my sincerest gratitude to Dr Bronwyn McAllan, for accommodating me at the (USyd) and allowing me to incorporate your dunnarts in my study. Also, for providing editorial feedback on a manuscript prior to

7 submission, your help and time was greatly appreciated. To the postgrads at USyd: Pippa,

Isabel and Tasha, thank you for taking an interest in my PhD and assisting with feeding

the dunnarts.

Thank you to Mr Jack Wolfenden, Dr Ricky Spencer, Dr Adam Munn and Dr Marissa

Parrott for providing feedback on draft manuscripts. To the technical staff, especially

Mark Emanuel for your technical assistance and help accessing equipment, though difficult at times due to moving and renovations of labs; and to the many technicians who helped maintain the colonies of captive marsupials at UWS, thank you.

Fiona Loudon, thank you for teaching me how to do hair analysis. To Fiona and Alicia

Kasbarian, your company was greatly appreciated during overnight scat collections. I wish you both the best of luck in your PhD and future endeavours.

This research would not have been possible without a Postgraduate Research Award provided by UWS, a grant from the Australian Geographic Society and a F.G. Swain grant from the Hawkesbury Foundation. The financial grant provided by the Australian

Geographic Society was through the Quoll Funding project; thank you to Shannon Troy at the University of for sharing this funding with me.

My brother, Lee thank you for teaching me how to use Photoshop, and not being too annoyed at me for asking for help during late hours of the night even though you had

8 work in the morning. Thanks Mum for always listening to my stories about the animals and showing an interest in my research.

9 Statement of Authentication

This thesis contains no material which has been accepted for the award of any other

degree in any University or other tertiary institution and, to the best of my knowledge, contains no material previously published or written by another person, except where due

reference has been made in the text.

……………………………….

Hayley Stannard

23rd March 2012

10 Preface

This thesis is presented as a series of manuscripts which describes aspects of dasyurid biology. Much of this work has been published, accepted for publication or is currently in review. To assist with continuity throughout, an introductory literature review and a general conclusion are included at the beginning and end of the thesis. The inter- relationship between topics has resulted in some repetition in the introductions of some chapters. Submitted manuscripts are formatted according to the guidelines of the journal and references for each manuscript are located at the end of each chapter. All manuscripts are jointly authored, but in each case I am the first author. An additional manuscript can

be found at the end of the thesis document that came from work that was conducted

during my PhD.

11 Publications *Stannard, H.J. and Old, J.M. 2012 Description of the gastrointestinal tract and associated organs of the kultarr (Antechinomys laniger). Australian Mammalogy In Press

*Stannard, H.J. and Old, J.M. 2011. Digestibility of captive feeding regimes of the red- tailed phascogale (Phascogale calura) and kultarr (Antechinomys laniger). Australian

Journal of 59, 257-263.

DOI: 10.1071/ZO11069

*Stannard, H.J. and Old, J.M. 2011. Rate of passage through the kultarr (Antechinomys laniger) digestive tract. Australian Journal of Zoology 59, 273-276.

DOI: 10.1071/ZO11103

*Stannard H.J., Caton W. and Old J.M. 2010. The diet of red-tailed phascogales

(Phascogale calura) in a trial translocation at Alice Springs Desert Park, Northern

Territory, Australia. Journal of Zoology 280, 326-331.

DOI: 10.1111/j.1469-7998.2009.00658.x

Stannard, H.J. and Old, J.M. 2010 Observation of reproductive strategies of captive kultarrs (Antechinomys laniger). Australian Mammalogy 32, 179-182.

DOI: 10.1071/AM10011 0310-0049/10/020179

12 Stannard H.J., Wolfenden J. and Old J.M. 2010. Evaluating the capacity of constructed

wetlands in sustaining a captive population of (Ornithorhynchus anatinus).

Australasian Journal of Environmental Management 17, 27-34.

*Stannard, H.J., McAllan, B. and Old, J.M. The role of dietary composition in optimum nutrition for dunnarts Sminthopsis macroura and S. crassicaudata. Under review in

Laboratory Animals

*Stannard, H.J. and Old, J.M. Digestibility of two diets by captive eastern quolls

(Dasyurus viverrinus). Under review in Zoo Biology

*Stannard, H.J., Young, L.J. and Old, J.M. Further investigation of the blood parameters of Australian quoll (Dasyurus spp.) species. Under review in Veterinary Clinical

Pathology

*a chapter in this thesis

13 Conference Presentations

Stannard, H.J., Young L.J. and Old, J.M. ‘Haematology and blood chemistry of eastern and spotted-tailed quolls’ Molecular and Experimental Pathology Society of Australasia

Conference, Brisbane AUS, 30th Nov-2nd Dec 2011.

Stannard, H.J. and Old, J.M. ‘Digestibility trials of two captive Dasyurids: the kultarr

(Antechinomys laniger) and red-tailed phascogale (Phascogale calura).’ American

Society of Mammalogists and Australian Society Conference, Portland USA,

23rd-29th June 2011.

Stannard, H.J. ‘Nutrition of two Dasyurid species.’ UWS College of Health and Science

Postgraduate Research Forum, Kingswood AUS, 8th-10th June 2011.

Stannard, H.J. and Old, J.M. ‘Digestibility trials of two captive Dasyurids: the kultarr

(Antechinomys laniger) and red-tailed phascogale (Phascogale calura).’ Comparative

Nutrition Society Conference, Arizona USA, 6th-11th August 2010.

Stannard, H.J. and Old, J.M. ‘The nutrition of captive kultarrs (Antechinomys laniger).’

Australian Mammal Society Conference, Canberra AUS, 5th-9th July 2010.

Stannard, H.J. ‘Dasyurid Nutrition.’ UWS College of Health and Science Postgraduate

Research Forum, Werrington AUS, 7th-9th June 2010.

14 Stannard, H.J. and Old, J.M. ‘Translocated red-tailed phascogale diet’ Australasian

Wildlife Management Society Conference, Napier NZ, 29th Nov-2nd Dec 2009.

Stannard, H.J. ‘Dasyurid Biology: a progress report.’ UWS College of Health and Science

Postgraduate Research Forum, Werrington AUS, 2nd-4th June 2009.

Stannard, H.J., Trevor-Jones, P., Caton, W and Old, J.M. ‘The diet of red-tailed

phascogales (Phascogale calura) in a trial translocation at Alice Springs Desert Park,

Northern Territory, Australia.’ Australian Mammal Society Conference, Darwin AUS,

28th Sept-1st Oct 2008.

15 Abstract

This thesis examines aspects of the biology of six Dasyurid species, which are a family of insectivorous and/or carnivorous Australian marsupials. In particular diet, nutrition and haematology were studied. Diet and nutrition were studied to obtain information on diet choice by translocated animals and diet digestibility in captive animals. Haematology was studied in captivity as it is associated with clinical health and is influenced by nutrition.

The six species used for the study were part of captive colonies housed at the University of Western Sydney, the University of Sydney, Alice Springs Desert Park, Australian

Ecosystems Foundation Inc., and Featherdale Wildlife Park. The broad aim of the study was to aid current wildlife management practices and future conservation efforts (such as reintroduction and translocation programs) for these six species and other marsupial species in Australia.

Examination of the diet of a population of translocated red-tailed phascogales

(Phascogale calura) at Alice Springs Desert Park showed that they are primarily insectivorous with 92.6% of all scats containing arthropods. They are also opportunistic predators within the park, consuming (51.6%), small mammals (33.3%) and on occasion , and plant material (27.4%). A seasonal variation in diet was found between spring and summer, due to a larger portion of birds present in the diet in spring.

The red-tailed phascogales were able to exploit a number of prey types and it is therefore likely that they would survive a ‘hard’ translocation into the wild provided the site chosen has an adequate food supply.

16 Investigation of the gastrointestinal tract of the kultarr (Antechinomys laniger) shows that on gross examination it is simple with no differentiation between the small and large intestine, and lacked a caecum. Mean gross length of the kultarr digestive tract was 165.2

± 32.1 mm. Microscopically, the tissues appeared healthy with cell types similar to other mammals. Rate of passage through the kultarr digestive tract was rapid, measuring 1.6 ±

0.2 h and mean retention time 3.9 ± 1.2 h. The rapid transit time was consistent for an animal of equivalent body mass, dietary preference and gastrointestinal tract morphology.

Study of nutrition in red-tailed phascogales and kultarrs showed apparent digestibility values were above 81% for dry matter, energy, protein and lipids on a number of captive fed diets. No significant difference was found between phascogales and kultarrs when maintained on the same diet for apparent digestibility of dry matter, energy, protein and lipids. Maintenance energy requirements were determined to be 954 kJ kg0.75 d-1 for the red-tailed phascogale and 695 kJ kg0.75 d-1 for the kultarr. Digestion studies undertaken on stripe-faced dunnarts (Sminthopsis macroura) and fat-tailed dunnarts (Sminthopsis crassicaudata) showed that depending on the diet, the digestible energy intake of the stripe-faced dunnart ranged from 359 to 816 kJ kg -0.75 d-1 and digestible intake ranged from 542 to 990 kJ kg -0.75 d-1 for the fat-tailed dunnarts. Both dunnart species had varied absorption of minerals with higher values observed for Na, P and K compared to the other minerals studied. The morphology of the gastrointestinal tracts of both dunnart species were simple and consisted of a unilocular stomach and relatively uniform intestine, like that of the kultarr. The length of the intestine of the stripe-faced dunnart ranged from 84 to 129 mm and that of the fat-tailed dunnart 78 to 131 mm. Fat-tailed

17 dunnarts need to consume more nutrients per unit of body mass for maintenance in

captivity compared with stripe-faced dunnarts.

Studying digestibility in a larger Dasyurid species, the eastern quoll (Dasyurus viverrinus), showed they had high apparent digestibility values for dry matter, gross

energy, protein and lipids (>84%). There was a significant difference in apparent

digestibility of dry matter, gross energy and protein between the two diets provided:

mince and chicken necks.

Analysis of blood parameters in the eastern and spotted-tailed quoll provided new data

for blood chemistry and differential white cell values. For many of the parameters, blood

chemistry results were comparable to other marsupials and no significant differences

between genders were detected (P<0.05). Seasonal differences were determined for total

bilirubin, glucose, creatinine and sodium levels in the eastern quoll. Generally higher

levels for these parameters were observed in summer; however, in autumn (southern

hemisphere) sodium levels were significantly higher. Eastern quolls one year of age and

under had significantly (P<0.05) higher alkaline phosphatase values than older animals.

The values obtained in this study can be used to assess clinical health of quolls and will

assist with captive management and future reintroduction programs.

The results from this thesis have implications for captive management and future

conservation efforts for Dasyurids. The study has shown the diet choice of translocated

phascogales in a new environment, which has contributed to improving translocation

18 techniques used for this species. Nutritional experiments suggest that no single diet, if fed alone is appropriate for feeding captive dasyurids; and live diets provide behavioural enrichment, and enhance mental and physical stimulation. The ability of captive animals to catch live food also increases the likelihood of their survival post- release, if they are subject to translocation in the future. Energy requirements differ between species and do not necessarily relate to body mass but likely relate to physiological adaptations and ecology of the species. The data gained in this study has been incorporated into the daily management/husbandry practices for these species; it can also be used as a model for other Dasyurids. The data has also contributed to the paucity of biological data available on Dasyurids and will contribute to conservation of

Australian native species.

19 CHAPTER 1

Introduction

20 Biodiversity is the variety and richness of life on earth (Jeffries, 2006). It encompasses genetic, taxonomic and ecological diversity of all organisms, and ecosystems

(Sinclair et al., 2006). Human evolution and civilisation has caused many changes across the world, particularly to biodiversity. Natural habitats have become degraded due to encroachment by humans into natural landscapes, which has contributed to a reduction, and in some cases a complete loss of flora and species (Jeffries, 2006; Sinclair et al., 2006).

The ability to reverse and minimise human impacts on the natural landscape is an

important environmental and social issue. The act of conservation is the ability to reverse

or minimise the threatening impacts caused by humans to the natural environment. The

aim of conservation is to conserve the integrity of fauna species and the environments in

which they live (Jones et al., 2003). Flora and fauna are important to conserve as they are

part of natural environmental processes such as food webs, nutrient cycling, oxygen

supply and water cycling (Jeffries, 2006; Sinclair et al., 2006). Conservation practices

preserve flora and fauna for the purpose of diversity, tourism, provision of food and

resources for humans, life support and function (Burbidge and Eisenberg,

2006).

Wildlife management involves human actions to manipulate or monitor wildlife for the

purpose of conservation. The goals of wildlife management are to increase population

numbers, decrease population numbers, harvest species or leave alone and monitor

(Sinclair et al., 2006). Increasing wildlife numbers through translocation programs

21 (Seddon et al., 2007) have been implemented for the beaver (Castor fiber), maned sloth

(Bradypus torquatus), bilby ( lagotis) and a dasyurid, the (Parantechinus apicalis) to increase population numbers in the wild (Nolet and Baveco, 1996; Moro,

2003; Moseby and O'Donnell, 2003; Chiarello et al., 2004). Decreasing numbers has been used extensively for the European red (Vulpes vulpes) to reduce their numbers and thus reduce their on native species in Australia (Kinnear et al., 1988;

Thompson and Fleming, 1994; Kinnear et al., 2002). Conservation practices are crucial to deciding whether interventions should occur or whether monitoring is necessary.

Conservation practices implemented for wildlife encompass in-situ or ex-situ methods.

In-situ conservation generally involves the establishment of protected areas such as national parks, marine parks and reserves. These areas allow wildlife to sustain itself within the ecosystem with minimal human interference (Jeffries, 2006; Sinclair et al., 2006). Ex-situ conservation establishes populations and biodiversity banks (Jeffries, 2006). Captive breeding populations are used for the purpose of translocation or reintroduction with the aim of re-establishing species within their historical range in the wild. Zoological parks are wildlife biodiversity banks that are involved in conservation through reintroduction programs, for example: the American bison (Bison bison) and golden-lion tamarin (Leontopithecus rosalia) have successfully been reintroduced to the wild from zoo populations (Baker, 2007). Zoos are also the main platform for education about conservation of wildlife to the general public. In addition, zoos raise funds to support in-situ conservation projects and research on wildlife

(Cosgrove, 1987; Croke, 1997; Baker, 2007).

22 Research and gaining knowledge is required to determine appropriate conservation practices for flora and fauna. When researching fauna for conservation purposes a number of issues arise such as low population density, sparse distribution, behavioural ecology and the ability to capture and restrain (if needed). In captivity, low population numbers, adequate husbandry, zoonotic disease, health, reproductive output, in-breeding, behavioural changes and the influence of captivity on animals pose a number of challenges when using animals for research. It is important to study animals both in the wild and in captivity due to the limitations within these populations and to ensure there is a wealth of information to adequately manage fauna species to prevent further population declines and .

Half of the mammal extinctions across the world have occurred in Australia (Maxwell et al., 1996), with terrestrial mammal species within the critical weight range (0.35-5.5kg) accounting for the majority of these extinctions. Fauna that inhabit arid and semi-arid regions are particularly at risk (Kennedy, 1992). Humans arrived in Australia between

60,000 and 40,000 years ago, during which the reduction of native fauna populations began (Tyndale-Biscoe, 2005). During this period Australia was inhabited by 50 species of megafauna, weighing between 250 and 1000 kg (Tyndale-Biscoe, 2005). The of these mammals is believed to be linked to human invasion and climatic changes (Johnson and Wroe, 2003; Tyndale-Biscoe, 2005). for food, using sophisticated weapons and sedentary behaviour by Aboriginal people is thought to have contributed to the initial decline (Johnson and Wroe, 2003; Tyndale-Biscoe, 2005;

Burbidge and Eisenberg, 2006). Climatic changes in Australia 12,000 years ago is

23 believed to be linked with the further decline of native fauna species (Tyndale-Biscoe,

2005). After European settlement 200 years ago the rate of extinctions in Australia

increased (Tyndale-Biscoe, 2005).

1.1 Marsupials

Marsupials are a subclass of mammal that once occupied every continent across the

world (Tyndale-Biscoe, 2005). However, marsupials now only inhabit Australasia and

the Americas (Tyndale-Biscoe, 2005; Archer and Kirsch, 2006). Marsupials diverged

from eutherian mammals 147.7 million years ago (Bininda-Emonds et al., 2007).

Marsupials then began to diversify 82.5 million years ago, during the period

(Kemp, 2005; Bininda-Emonds et al., 2007). The evolutionary divergence between

Metatherians and Eutherians millions of years ago created a group of marsupials in

Australia that are well adapted to the varied habitats across the continent (Kemp, 2005).

Marsupials are distinguished from other mammals by their reproductive characteristics

particularly their reproductive tracts and strategies. They have a distinctive reproductive

tract that consists of two vaginae, two uteri and two oviducts (Tyndale-Biscoe, 2005).

Marsupial young are born at a early stage of development (Tyndale-Biscoe and Renfree,

1987). The young have well developed forefeet that are used to climb up to the teat after

birth (Van Dyck and Strahan, 2008). Teats of marsupials are generally enclosed in a

. Once the young are born they become attached to the teat for a period of time.

The milk provides sustenance and some immunological protection in the form of

immunoglobulins (or antibodies) (Tyndale-Biscoe and Renfree, 1987). The majority of

24 maternal investment of reproduction in marsupials is post-natal (Tyndale-Biscoe and

Renfree, 1987). Marsupials differ from Eutherians as they have a lower body temperature and lower metabolic rate (Tyndale-Biscoe, 2005). The marsupial subclass is separated into seven living orders: Didelphimorphia, , ,

Peramelemorphia, , Notoryctemorphia and Dasyuromorphia (Van Dyck and Strahan, 2008). The focus of this thesis is species in the family .

Myrmecobiidae Dasyuridae Dasyuromorphia Notoryctemorphia

Figure 1.1 Phylogenetic tree of metatherian orders Dasyuromorphia and

Notoryctemorphia adapted from Cardillo et al. (2004)

1.2 Dasyurids

The Dasyuromorphia consists of three families, 19 genera and 64 species (Kemp,

2005; Archer and Kirsch, 2006) (Figure 1.1). Marsupials of the Dasyuromorphia order

range from small to larger more carnivorous species. Their habitats range

from arid to (Jackson, 2003; Van Dyck and Strahan, 2008). The largest

animal in this order, the ( cynocephalus), is now extinct; eight

species are endangered and seven species are listed as vulnerable in Australia

(Department of Sustainability, Environment, Water, Population and Communities, 2009).

25 One of the differentiating features of the Dasyuromorphia order is their teeth. They have

four upper and three lower incisors and very sharp molars (Van Dyck and Strahan, 2008).

Within the Dasyuromorphia order is the Dasyuridae family, which appeared in the fossil

record five million years ago (Kemp, 2005). Members of this family range from the very

small, long-tailed , (Planigale ingrami; 4-5 g) to the ,

( harrisii; 8-10 kg) (Van Dyck and Strahan, 2008). Dasyurids have

unspecialised limbs, with the kultarr (Antechinomys laniger) being the only exception.

Generally, dasyurid forefeet have five clawed toes and the hind foot has four or five toes

depending on the species (Van Dyck and Strahan, 2008). Members of this family have

between two and 12 teats, situated on the abdomen enclosed in a small fold of skin or

pouch (Tyndale-Biscoe, 2005).

1.2.1 Threats to Dasyurids

The main cause of population decline, reduced abundance and extinction of marsupials in

Australia has been human settlement, introduction of exotic species and agriculture

(Tyndale-Biscoe, 2005). The introduction of the fox, cat (Felis catus) and the European

(Oryctolagus cuniculus) were particularly threatening to native species (Johnson

and Wroe, 2003). There is evidence to suggest that the (Canis lupus dingo) was the

main cause for the extinction of the thylacine and the Tasmanian devil, on mainland

Australia (Burbidge and Eisenberg, 2006). These two species of the Dasyuromorphia

order were once apex predators across the Australian continent.

26 Agricultural land practices such as the introduction of inedible plant species, land

clearing and over grazing reduces the and food available to native fauna (Tyndale-

Biscoe, 2005; Burbidge and Eisenberg, 2006). In addition, many Australian native

species, including dasyurids, were hunted as pests during the 19th century by farmers

(Burbidge and Eisenberg, 2006). Since European settlement in Australia, 125 plants and

animals have become extinct, these include 10 species of marsupial, which includes the

thylacine (IUCN, 2010). The greatest extinctions have occurred in arid zones and account

for 33% of mammal decline in Australia (Wilson et al., 2003; Burbidge and Eisenberg,

2006).

1.2.2 Conservation and management of Dasyurids

The International Union for the Conservation of Nature (IUCN) is an organisation that

aims to support the conservation of nature across the world through projects, scientific

research and support for government and non-government organisations (IUCN, 2010).

The IUCN has a program that assesses the of species and publishes

these findings through the IUCN Red List of (IUCN, 2010). The list

provides information as to the global threatened status of flora and fauna and their

relative risk of extinction (IUCN, 2010). The listing also provides a status for many

Australian marsupials and provides an extract of information on their geographic range, habitat, population trends, threats and conservation actions.

In Australia, national legislation has been implemented to protect threatened flora and fauna species in the form of the Environment Protection and Biodiversity Conservation

27 Act 1999 (Department of Environment and Water NSW, 2010). In addition, legislation is developed and implemented on a state-by-state basis in Australia.

In the state of threatened flora and fauna are protected by the

Threatened Species Act 1995 (Department of Environment Climate Change and Water

NSW, 2010). The objective of the legislation is to conserve threatened species and prevent extinction.

Marsupials are particularly important to conserve, as modern day marsupials are restricted to the Australasian and American regions (Jones et al., 2003; Burbidge and

Eisenberg, 2006). They are unique and evolved separately from Eutherian mammals

(Tyndale-Biscoe, 2005). Research is needed to further understand population ecology, physiology and biology of dasyurids in order to effectively manage these species,

particularly those that are threatened. Generally, dasyurid species are at high risk of

becoming threatened as they occur in low densities (Tyndale-Biscoe, 2005). For example,

one of spotted-tailed quoll (Dasyurus maculatus gracilis) is now restricted to

six fragmented populations in (Jones et al., 2003). The ability to implement

actions to oppose threatening processes affecting marsupials is an important part of

securing their future. These actions include translocation programs, protected areas,

research, development of policies and public education.

Reintroduction programs, action and recovery plans and legislation are used to protect

and conserve dasyurids and other native fauna species in Australia, in addition to the

provision of suitable habitat, research, public education and captive breeding.

28 Reintroduction programs require management of areas, the creation of private parks and

island breeding populations of fauna (Tyndale-Biscoe, 2005; Sinclair et al., 2006).

Generally captive populations are bred then released into the wild, suitable park land or

to an island. Reintroduction programs are important for the future of many species to

ensure viable numbers exist to maintain wild populations. These programs rely heavily

on the provision of suitable habitat, food and low numbers of predators. Exotic predators

are generally controlled using 1080 (), and the construction of predator-proof fences (Tyndale-Biscoe, 2005).

Biological and ecological knowledge of fauna species form the knowledge basis for

action and recovery plans. These plans are designed to adequately manage fauna species,

particularly threatened species. An action plan was written in 1992 for Australasian

marsupials and monotremes (Kennedy, 1992). It provided a review of the issues

surrounding threatened species in the Australasia region. The plan recommended 12

actions to be carried out in order to preserve Australian native fauna. These

recommendations included: development of species specific recovery plans, feral animal

control, research and education (Kennedy, 1992). The 1996 Australian Marsupial and

Monotreme Action Plan provides recovery outlines for Australian native marsupials and

monotremes (Maxwell et al., 1996). The outlines summarise recovery objectives and

management actions for individual species in Australia (Maxwell et al., 1996). Recovery

plans are species specific and provide research and management objectives for the

recovery of a particular threatened species. The plans are usually written and

implemented on a state-by-state basis for example, plans have been written by the NSW

29 National Parks and Wildlife Service for the kultarr, long-nosed (Potorous

longipes), mountain pygmy possum (Burramys parvus),

(Cercartetus concinnus) and brush-tailed rock (Petrogale penicillata) (Molsher,

2001; NSW National Parks and Wildlife Service, 2001a; NSW National Parks and

Wildlife Service, 2001b; NSW National Parks and Wildlife Service, 2002a; NSW

National Parks and Wildlife Service, 2002b). Recovery planning has had varied success

in Australia. Recovery efforts of the northern hairy-nosed (Lasiorhinus krefftii)

have developed DNA fingerprints for the wild population and improved monitoring

techniques (Horsup, 1996). For the mala (Lagorchestes hirsutus), ecological information

has increased, release techniques have been perfected and an understanding of the key

threatening processes have been developed (Johnson et al., 1996). The success of

recovery planning relies heavily on adequate funding, increasing knowledge of species

biology and ecology, a balanced input of ideas from team members and community

involvement.

A number of Australian marsupials have been translocated and reintroduced into the wild

or into semi-wild enclosures. These include the burrowing and brush-tailed

(Bettongia spp.), (Myrmecobius fasciatus), eastern barred (

gunni), mala and dibbler (Dufty et al., 1994; Friend and Thomas, 1994; Gibson et al.,

1994; Short and Turner, 2000; Moro, 2003; Priddel and Wheeler, 2004). The success of

these programs has relied heavily on a basic understanding of each species’ biology,

threatening processes and choosing suitable sites for introduction/release. Maximising

health of captive populations used for release programs ensures survival and optimum

30 reproduction whilst in captivity and aids their long-term survival post-release. When using captive populations for the purpose of breeding and release, a basic knowledge of species biology is essential. More information is known for some marsupials and not others. Much is known of the external factors threatening dasyurids such as predation

from exotic species, habitat degradation and human land-use. However, currently there is

insufficient information available on the general biology of dasyurids including

nutritional requirements and general health indicators such as haematology.

1.3 Nutrition

Nutrition is the nourishment of an animal through the ingestion, digestion and absorption

of nutrients from food. The process allows for nourishment of animals on a cellular level

to allow an animal’s body to function (Barboza et al., 2009). Nutrition encompasses an

array of topics including nutritional ecology, the interaction between an animal and its

environment and nutritional physiology, the interaction between food resources and the

use of those resources by an animal (Hume, 1999). The nutritional niche of an animal

determines whether it is a , herbivore or and whether it consumes a

specialist or generalist diet. Wildlife nutrition is a developing area of research, with the

majority of nutrition-related research in the past being conducted on domestic animals

rather than wildlife. Domestic animal nutrition has been well investigated to improve

management practices and quality of products for human consumption. Due to the

ecological and physiological differences between dasyurids and domestic animals,

domestic species are not suitable as comparative models for nutrition research. Therefore,

31 research focused on dasyurids is required to further understand their nutritional ecology and physiology to aid conservation strategies.

1.3.1 Digestive physiology of Dasyurids

Diets of contain a high water, protein and vitamin content. For example, provide 54-60% water, 15-21% protein, 17-26% fat and 3-5% ash (minerals) to their predators (Robbins, 1993; Dierenfeld, 1997; Barboza et al., 2009). The highly digestible materials include muscle and viscera, while bones, feathers, hair and are much less digestible. Diet composition affects an animal’s ability to digest and absorb the nutrients to be utilised in the body. The eastern quoll, Tasmanian devil and dusky (Antechinus swainsonii) have been found to have a relatively high apparent digestibility of foods with values of 78% and above for dry matter and energy (Cowan et al., 1974; Nagy et al., 1978; Green and Eberhard, 1979). Similarly high digestibility values have been found for Eutherian carnivorous mammals, such as the bobcat (Lynx rufus), river otter (Lontra canadensis), dog (Canis familiaris), mink

(Mustela vison) and blue fox (Alopex lagopus) (Powers et al., 1989; Vhile et al., 2005;

White et al., 2007).

Nutritional requirements vary depending on species, size, age, health and reproductive status (Dierenfeld, 1997). Adequate nutrition is essential for maintenance, growth, reproduction, digestive function and preventing disease (Allen and Oftedal, 1996).

Gaining nutritional sustenance from food begins with physical digestion which commences in the mouth with teeth (mastication). Dasyurids have specialised teeth

32 designed for piercing, shearing and grinding their food. Their digestive system is relatively short and simple with no caecum (Figure 1.2). The amount of time a meal

spends in the digestive tract influences the digestion and absorption of nutrients by an

animal (Barboza et al., 2009). Rate of passage is the time taken for a meal to first appear

in faeces, while mean retention time is the average time a meal takes to pass through the

digestive system (Robbins, 1993). Rate of passage of dasyurids is rapid, in comparison

with other mammals (Hume, 1999). For example, two Planigale spp. had rate of passage

times of 30-60min on a minced meat diet with an marker (Read, 1987). The

fat-tailed dunnart (Sminthopsis crassicaudata) has mean retention time of 53min, the

(Dasyuroides byrnei) 87min, (Dawson and Paiz, unpub. as cited in Hume et al.

2000), the 3-4h (Hume et al., 2000) and the eastern quoll (Dasyurus

viverrinus) up to 17h depending on season and diet (Moyle, unpub. as cited in Hume,

1999).

Figure 1.2 The digestive tract morphology of Dasyurid spp. (S) stomach, (I) intestine,

(R) rectum.

33 A lack of understanding of nutrition often leads to improper management of animals in

captivity. Poor nutrition leads to disease, mineral and vitamin deficiencies, poor physical

condition and poor reproductive output (Barnes, 1968; Robbins, 1993; Barboza et al.,

2009). Diseases such as metabolic bone disease, mineral deficiencies, growth defects and

are nutrition related diseases that have been observed in captive dasyurids

(Barnes, 1968; Potkay, 1977; Attwood and Woolley, 1980; Jackson, 2003). Thyroid

disease was observed in a colony of captive fat-tailed dunnarts due to low levels of iodine

provided in a new diet (Breckon and Hulse, 1972). The thyroid disease led to the death of

43% of the captive colony. Rickets is caused by a low calcium diet and has been observed in captivity in the American marsupial the woolly (Caluromys philander) (Barnes, 1968).

1.3.2 Diet of Dasyurids

Nutrition is delivered to an animal’s body through the food items consumed or the diet.

The term diet is used to describe the food items consumed ‘most’ by an animal, which can consist of plant or animal material or a mixture of both (Barboza et al., 2009). Faecal and stomach content analysis are used to determine the diet of wild animals. Studying diets of wild animals provides valuable insight into the choice of food items for captive diets. The diets of numerous dasyurid species have been studied in the wild and it has been found that small dasyurid species are mostly insectivorous and consume a range of arthropods including , , arachnids and (Hall, 1980; Kitchener,

1981; Fox and Archer, 1984; Woolnough and Carthew, 1996; Bencini et al., 2001;

Gilfillan, 2001; Lunney et al., 2001; Miller et al., 2003; Allison et al., 2006; Bos and

34 Carthew, 2007; Stannard et al., 2010). Medium sized dasyurids such as the

(Dasyucercus cristicauda) and (Dasyurus hallucatus) are also mostly

insectivorous and in addition consume small mammals and reptiles (Chen et al., 1998;

Pollock, 1999). Larger dasyurid species, such as the spotted-tailed quoll and Tasmanian

devil are more carnivorous than smaller species, and prey on medium-sized mammals

and scavenge on carrion (Belcher, 1995; Jones and Barmuta, 1998; Glen and Dickman,

2006; Belcher et al., 2007; Dawson et al., 2007). Thus, choice of prey type and size is

correlated to body mass of the predator, digestive physiology and handling time (Fisher

and Dickman, 1993; Rychlik and Jancewicz, 2002).

As highlighted earlier more research has focused on food items chosen by dasyurids in

the wild rather than nutritional composition of those food items. Increasing knowledge of

nutritional requirements reduces the possibility of deficiencies, disease and death in

captive animals caused by inadequate nutrition. Poor nutrition influences an animal’s

body condition, fat storage, reproduction and haematology and blood chemistry levels.

Haematology and blood chemistry are used as measurements of clinical animal health.

1.4 Haematology

Mammals have a closed circulatory system that consists of a heart, arteries and veins. The

heart is a pump that sends blood to the arteries, and veins are vessels that transport blood

throughout the body (Schalm et al., 1975; Moore et al., 2010). The blood system supplies

cells with water, oxygen, electrolytes, nutrients and hormones essential for function and

is involved in immune function (Schalm et al., 1975). Blood removes waste products

35 created from . Blood consists of 55% plasma and 45% cellular elements. The plasma is made up of water, salts and proteins, while the cellular elements are erythrocytes, leukocytes and platelets (Schalm et al., 1975; Clark, 2004).

Marsupial mature erythrocytes (red blood cells) lack a nucleus and constitute 30-50% of total blood volume as do other mammalian erythrocytes (Schalm et al., 1975; Clark,

2004; Moore et al., 2010). Important measures of red blood cells include packed cell

volume (PCV) or erythrocyte concentration. Low PCV levels are generally a sign of anaemia (Schalm et al., 1975). Mean corpuscular haemoglobin concentration is the number of haemoglobins on erythrocytes (Schalm et al., 1975).

Leukocytes (white blood cells) are nucleated cells involved in immune function and response to foreign bodies (Moore et al., 2010). There are five types of leukocytes: lymphocytes, neutrophils, monocytes, eosinophils and basophils. Lymphocytes are involved in the generation of antibodies and destroy cells infected with viruses.

Neutrophils are involved in the inflammation response and use phagocytosis to remove bacteria. Monocytes, like neutrophils are involved with inflammation, use phagocytosis and release cytokines (protein messenger cells). Neutrophils and monocytes are particularly important in protection against fungal infections (Brown, 2011). Basophils initiate an inflammation and allergic response; they release histamine and cytokines

(Moore et al., 2010; Karasuyama et al., 2011). Eosinophils detoxify tissues at the point of foreign body entry and protect the body from parasitic infections (Moore et al., 2010).

Total leukocyte counts are conducted to determine the concentration of cells in blood.

36 Increased leukocytes counts suggest a response to an infection (Schalm et al., 1975;

Moore et al., 2010). Reference values are developed as a standardised level for key erythrocyte and leukocyte haematology. Blood samples taken from animals can be compared to reference values to determine a change in homeostasis and diagnose an infection or abnormality if present. Physiological conditions such as, nutrition status, stress levels, age and sex can influence blood parameters (Spencer and Canfield, 1994;

Stirrat, 2003; Aroch et al., 2007; Reiss et al., 2008; Shanmugam et al., 2008).

Measurement of haematology values over a period of time can detect changes in animal health.

1.4.1 Marsupial haematology

In comparison to eutherian mammals marsupials have no or very low numbers of basophils. Marsupials also have alkali resistant haemoglobin and higher serum enzyme levels than eutherians (Parsons et al., 1971a; Parsons et al., 1971b). Reference ranges have been determined for a number of marsupials, for example, the northern hairy-nosed wombat, brush-tailed rock wallaby, Gilberts potoroo (Potorous gilbertii), tammar wallaby (Macropus eugenii), and (Perameles bougainville)

(McKenzie et al., 2002; Bennett et al., 2007; Barnes et al., 2008; Reiss et al., 2008;

Vaughan et al., 2009). These values have been determined using wild and/or captive animals, to determine haematology ranges for healthy animals.

Haematology has been investigated in a few dasyurid species. Studies on the (Antechinus stuartii) and red-tailed phascogale identified stress-related

37 reductions in haematocrit and haemoglobin levels prior to post-mating mortality (Cheal et

al., 1976; Bradley, 1990). Haematology reference values have been determined for the

eastern quoll, (Dasyurus geoffroii), fat-tailed and striped-faced dunnarts

(Smithopsis crassicaudata and S. macroura) (Melrose et al., 1987; Haynes and Skidmore,

1991; Svensson et al., 1998), as well as blood chemistry values for the Tasmanian devil

and spotted-tailed quoll (Parsons et al., 1970; Parsons and Guiler, 1972). Previous studies

of eastern quoll blood have determined ‘normal’ ranges for haematology of males and

females, and blood chemistry for two males (Parsons et al., 1971a; Parsons et al., 1971b;

Melrose et al., 1987). Studies of the spotted-tailed quoll determined ‘normal’ values for

blood chemistry of two males (Parsons and Guiler, 1972). Haematology values have not been investigated in the spotted-tailed quoll. These studies did not assess the influence of age, sex, season, reproductive status or lactation on haematology and blood chemistry values.

Haematology values can be used to assess changes in biological parameters such as age, reproductive status, habitat, season and nutrition. These biological parameters show

variability among species. In some cases age influences susceptibility to disease and

haematology concentrations and sex can influence erythrocyte concentration (Clark,

2004). Age-related changes to haematology levels have been observed in the

(Phascolarctos cinereus) and (Trichosurus vulpecula), with younger

animals generally having lower red cell values and higher reticulocyte numbers

(Presidente and Correa, 1981; Spencer and Canfield, 1994). Age-related changes to red

cells and reticulocytes are not a consistent finding across different marsupial species and

38 there is much variability in haematology values between species. Sexually dimorphic values have been found in dunnarts for neutrophil and lymphocyte concentration (Haynes and Skidmore, 1991). Seasonal decline in red cell volumes has been observed in quokkas

(Setonix brachyurus), which possibly relates to food availability during summer and autumn (Shield, 1971). In some dasyurid species haemoglobin and haematocrit values have also shown a decline due to season (Schmitt et al., 1989; Bradley, 1990; Agar and

McAllan, 1995).

1.4.2 Blood parameters and translocation

Investigation of haematology coupled with translocation has shown values such as erythrocyte, haematocrit and haemoglobin concentrations influence the longevity and post-release survival of translocated water vole (Arvicola terrestris) (Mathews et al.,

2006). In the greater rhea (Rhea americana) blood antibody values showed minimal disease resistance and/or exposure, that means the captive population was suitable to reintroduce to the wild as there would be minimal threat of disease to existing populations (Uhart et al., 2006). Stress related to capture has been observed in the rhea, with elevated levels of glucose and cholesterol (Uhart et al., 2006). Stress related to animal capture has also been noted for young orang-utans (Kilbourn et al., 2003). It is important to ensure animals used for translocation are in the best possible health/condition to ensure their survival once they have been translocated or reintroduced into the wild (Griffith et al., 1989; Miller et al., 1999; Mathews et al., 2006).

39 Many species of fauna are being used in reintroduction and translocation programs and

these programs often require a captive breeding population to produce enough animals

for release. It is important to ensure that captive animals are healthy and have optimum

reproductive output. Knowledge of nutritional requirements ensures animals are being

fed adequate diets that meet their daily requirements, which increase during the breeding

season. It is also significant to assess the health and condition of animals whilst in

captivity and prior to their release into the wild (Mathews et al., 2006). Comparing blood samples against reference values can determine the general health of animals whilst in captivity and prior to their release. Healthy animals that have been well managed whilst in captivity are more likely to survive post-release. An adequate management plan and suitable release site are also influencing factors on successful translocations (Letty et al.,

2007).

1.5 Study Species

Dasyurids are physiologically different from carnivorous eutherian animals. Dasyurids like all marsupials have a distinctive reproductive tract and reproductive strategies when compared to eutherian mammals. Some dasyurid species such as the antechinus and phascogale exhibit a unique reproductive strategy; the complete die-off of all males post- mating in the wild (Bradley, 1997). The unusual reproductive strategy has seen much research conducted to further understand their breeding biology and the processes behind semelparity, whilst less is known of other facets of their biology. Generally there is limited biological data available for many dasyurid species particularly those with a small body mass. Dasyurids can be difficult, expensive and time consuming to house in

40 captivity (Aslin, 1982; Selwood and Coulson, 2006; Selwood and Cui, 2006). They require adequate lighting, heat and a ‘meat’ diet. Dasyurids play an important ecological role in insect control, predation of small fauna and provide prey for larger species. The species chosen for this study were the kultarr, red-tailed phascogale, eastern quoll, spotted-tailed quoll, fat-tailed dunnart and stripe-faced dunnart. The chosen species are those that inhabit arid zones and smaller than the critical weight range (kultarr and dunnarts). The red-tailed phascogale has undergone server declines and is within the critical weight range. The eastern and spotted-tailed quolls have also undergone

significant declines. The species were chosen for this study as they are readily available

in captive management systems. Although dasyurids differ in morphology, body mass,

geographical distribution, reproductive strategies and ecology the chosen species are a

representative group for a number of dasyurids. It is most likely these dasyurids as well

as many other marsupials’ futures rely on human intervention.

1.5.1 Kultarr (Antechinomys laniger)

The kultarr was first described in 1866 and is a terrestrial mammal which inhabits arid

zones of south and central Australia (Krefft, 1866; Valente, 2008). It is a small dasyurid

marsupial which is brown and fawn to sandy in colour with a white belly (Figure 1.3)

(Valente, 2008). They have large ears and protruding eyes and a long tail with a brush- like tip. Their hind legs are elongated with four toes. On average males grow to between

80-100mm long and weigh 30g (Valente, 2008). Females are smaller, growing to 70-

95mm long and weigh 20g (Valente, 2008). Females are able to carry up to eight pouch

young depending on the number of teats present (Valente, 2008). The life span of kultarrs

41 in the wild is not known; although females can live up to 48-67 months in captivity

(Aslin, 1982; Stannard and Old, 2010).

Figure 1.3 A kultarr (Antechinomys laniger) (Stannard, 2009)

There are two sub-species of kultarr, A. l. laniger and A. l. spenceri, which are recognised by morphological differences, and inhabit different ranges across Australia (Lidicker and

Marlow, 1970). However, their characteristics are very similar and they are generally recognised as one species (Lidicker and Marlow, 1970). The kultarr is the only species within its ; however, they were once thought to be a member of the Sminthopsis genus (Krefft, 1866; Blacket et al., 1999). Reclassification of the kultarr based on genetics found it is in a sister to the Sminthopsis (Blacket et al., 1999).

Population declines have occurred, with the disappearance of the kultarr from , southern New South Wales, south-eastern and Sandringham station in

Queensland (Maxwell et al., 1996; NSW National Parks and Wildlife Service, 2002a). In

2008 the kultarr was listed as ‘least concern’ on the IUCN red list of threatened species

42 (Morris et al., 2008). It is most affected by habitat degradation, predation, flooding, fire and pesticides (NSW National Parks and Wildlife Service, 2002a). Habitat degradation is

caused by and grazing, which also influences abundance of (prey

items). Kultarr’s ecological role in the food web is to control insect populations. In turn

kultarrs are prey for larger animals such as feral , barn (Tyto alba) and possibly

(Smith and Medlin, 1982; NSW National Parks and Wildlife Service, 2002a).

The kultarr is a species which has had a very small amount of published data regarding

biological and ecological characteristics. Previous research has included studies on

maternal behaviour (Happold, 1972), reproduction (Woolley, 1984) and (Geiser,

1986). Whilst these studies have provided fundamental knowledge of kultarr biology,

there is still lack of biological and ecological information available, which can lead to

difficulties with captive maintenance. There are currently two captive breeding

populations, one at Alice Springs Desert Park (Alice Springs, ) and the

other at the University of Western Sydney (Richmond, New South Wales). These

populations provide vital resources for tourism, education and research.

1.5.2 Red-tailed phascogale (Phascogale calura)

There are three species of phascogale in Australia, the red-tailed and two brush-tailed

species (P. tapoatafa and P. pirata). These species are visually distinguished by their tails

and body mass. Brush-tailed phascogales are larger than red-tailed phascogales, reaching

up to 311g (Rhind et al., 2008; Soderquist and Rhind, 2008).

43 Red-tailed phascogales (Gould, 1844) are small arboreal marsupials that nest in trees.

They are a grey colour with a cream to white belly (Bradley et al., 2008). They have a long tail, the basal portion of the tail is a rusty red colour and the tip has long black hairs

(Figure 1.4). Red-tailed phascogales exhibit with males being larger than females (Bradley, 1997; Bradley et al., 2008). On average, males are 113mm long and weigh 60g, while females are 101mm long and weigh 43g (Bradley et al., 2008).

Females live for approximately three years while males only live for 11.5 months in the wild (Bradley, 1997). Semelparity is exhibited after breeding, where the entire male portion of the population dies (Bradley, 1987; Bradley, 1997; Foster et al., 2008). In captivity males can live up to five years, however they become sterile after one year of age (Bradley et al., 2008). Females can produce up to 8 young per year (Bradley et al.,

2008).

Figure 1.4 A red-tailed phascogale (Phascogale calura) (Stannard, 2008)

Red-tailed phascogales are only found in south-western , along the

Wheat belt (Bradley et al., 2008). They live in dense, tall dominated by

44 Casuarina, and Eucalypt species (Kitchener, 1981; Bradley et al., 2008). They were once found in northern Western Australia, Alice Springs, Barrow Creek,

MacDonnell Ranges and Tennant Creek in the Northern Territory, Mildura in Victoria and (Krefft, 1866; Smith and Medlin, 1982; Bradley, 1997). IUCN lists the red- tailed phascogale as ‘near threatened’ on the 2008 red list (Friend, 2008). Its range has been reduced since European settlement, through habitat loss, frequent fires and predation by cats and foxes (Maxwell et al., 1996; Bradley et al., 2008). Extensive land clearance has caused habitat fragmentation which has been a major factor of decline for red-tailed phascogales (Wilson et al., 2003).

A colony of red-tailed phascogales was established at Alice Springs Desert Park in 2001.

It is currently being used to re-establish wild colonies in South Australia and the Northern

Territory. In addition, animals are presently being released in current distribution ranges in Western Australia (W. Foster pers. comm. 2010). Successful reintroductions and translocations will ensure the future population stability of this species in the wild.

1.5.3 Eastern quoll (Dasyurus viverrinus)

There are four species of quoll in Australia: the eastern quoll, spotted-tailed quoll, western quoll and northern quoll. The eastern quoll is either black or fawn with white spots on the body and no spots on the tail (Figure 1.5). They have four toes on their hind foot, unlike other quolls which have five (Godsell, 1995). Like many other dasyurids they exhibit sexual dimorphism, on average males weigh between 850-2000g and females between 600-1100g (Bryant, 1988; Jones, 2008).

45

Figure 1.5 An eastern quoll (Dasyurus viverrinus) (Stannard, 2007)

Research into quoll biology includes lactation and milk composition, and the hormones

involved in these processes (Hinds and Merchant, 1986; Green et al., 1987; Messer et al.,

1987). Captive breeding and growth has also been well studied in the eastern quoll

(Fleay, 1935; Nelson and Smith, 1971; Merchant et al., 1984; Fletcher, 1985; Bryant,

1988). Breeding occurs in May and June, after gestation (16-23 days) up to six young are carried in the pouch until weaning after 102 days of age (Hill and O'Donoghue, 1913;

Fleay, 1935; Merchant et al., 1984). Eastern quolls live for approximately four to six

years in captivity (Bryant, 1988).

The eastern quoll only occurs in Tasmania, however it once occurred in New South

Wales, Victoria and South Australia, but is now considered extinct on

(Jones, 2008). The last sighting of an eastern quoll on mainland Australia was at

Vaucluse, New South Wales in 1963 (Godsell, 1995).

46

The eastern quoll is listed as ‘near threatened’ on the 2008 IUCN red list (McKnight,

2008). It has been negatively affected by European settlement due to agriculture, introduced predators and loss of habitat. Its range has been reduced by 50-90% with its extinction from the mainland (Maxwell et al., 1996). Quolls are often prey for larger animals such as owls, feral cats and dogs (Bryant, 1988). In Tasmania eastern quolls, particularly juveniles, are vulnerable to road mortality, as they will scavenge on other deceased animals along roadsides (Godsell, 1982; Jones, 2000).

1.5.4 Spotted-tailed quoll (Dasyurus maculatus)

Resembling the eastern quoll, the spotted-tailed quoll is larger and has a thick brown to reddish coat covered in spots with a long tail also covered in spots (Figure 1.6). The spotted-tailed quoll is the largest carnivorous marsupial on mainland Australia, since the extinction of the thylacine and Tasmanian devil (Mansergh, 1984; Belcher et al., 2008).

Males grow between 380-759mm and weigh up to 7kg and females 350-450mm long and weigh up to 4kg (Dickman and Read, 1992; Belcher et al., 2008). The spotted-tailed quoll inhabits rainforests, woodlands and areas of coastal Queensland, New South Wales and Victoria (Mansergh, 1984; Maxwell et al., 1996; Belcher et al., 2008). They are also distributed across Tasmania, and considered extinct in South Australia (Belcher et al.,

2008). Spotted-tailed quolls are polyoestrus and have six teats, however the average number of young produced per year is five (Fleay, 1940; Conway, 1988; Jones, 2008).

47

Figure 1.6 A spotted-tailed quoll (Dasyurus maculatus) (Lynch, 2012)

The spotted-tailed quoll diet has been well studied in wild animals, it has been found they

eat a variety of food items from insects to birds, small mammals and carrion (Belcher,

1995; Glen and Dickman, 2006; Belcher et al., 2007; Dawson et al., 2007). The most important food source is medium-sized mammals including foxes, , brushtail

possums, ringtail possums (Pseudocheirus peregrinus), and swamp (Wallabia

bicolor) among other mammalian, avian and reptilian species (Belcher, 1995; Dawson et al., 2007; Jarman et al., 2007). Due to the arboreal nature and size of the spotted-tailed quoll, it is able to catch and consume larger and more arboreal prey than the eastern quoll, reducing direct dietary competition in Tasmania. Due to habitat preferences there is

little overlap between the Tasmanian devil and quolls (Fisher and Dickman, 1993).

48 The spotted-tailed quoll population has undergone a significant geographical range reduction (Maxwell et al., 1996), with much of their current distribution relying heavily upon the abundance of prey items (Belcher and Darrant, 2006). Reduced prey abundance along with other pressures has created the threat of low . Parasites such as ticks, mites and fleas can infest spotted-tailed quolls and prolonged exposure to heavy infestations can lead to loss of condition (Vilcins et al., 2008). Subcutaneous atypical myobacteriosis has been found in captive spotted-tailed quolls and chronic infection leads to poor body condition and in some cases death (Raymond et al., 2000). Other pressures include loss of habitat, predation, competition and humans (Dickman and Read, 1992).

Poisoning and trapping used for exotic species causes illness and death of spotted-tailed

quolls (Mansergh, 1984). They are also targeted as pests by farmers, as they take poultry

during the night (Dickman and Read, 1992; Belcher et al., 2008). The fox and cat place a

strain on this species through competition for similar food items (Jones and Barmuta,

1998). These threats have contributed to geographical decline and the spotted-tailed quoll

being listed as ‘near threatened’ on the 2008 red list (Burnett and Dickman, 2008). Quolls

play an important ecological role in insect control, cleaning up dead animal carcasses and

also provide prey to large species. Further loss of this species will contribute to loss of

food for predators and possibly prolong the time for the decomposition of carcasses.

1.5.5 Fat-tailed dunnart (Sminthopsis crassicaudata)

There are approximately 20 species of dunnart found in Australia. The fat-tailed dunnart

on average weighs 15g and is distinguished by the fatness of its tail (Morton and

Dickman, 2008a). The tail stores fat reserves to be used later, during times of nutritional

49 stress (Figure 1.7). The fat-tailed dunnart inhabits southern and central regions of

Australia and occurs across all mainland states. Generally they are a solitary species that mostly prey on arthropods (Morton, 1978b; Morton and Dickman, 2008a). Fat-tailed dunnarts nest in during the day and will often share burrows in the non-breeding season (Morton, 1978b; Morton, 1978a; Morton and Dickman, 2008a). Fat-tailed dunnarts are seasonal breeders from July-February and are able to raise more than one litter per season (Ewer, 1968; Godfrey and Crowcroft, 1971; Morton, 1978b). Gestation lasts for 13 days and young are weaned at around 70 days (Godfrey and Crowcroft,

1971). One study has shown that on average the female’s litter sizes are 7.5 young. At the time of weaning only an average of 5.4 young survive; however, females are able to carry up to 10 young per litter (Morton, 1978b).

Figure 1.7 A fat-tailed dunnart (Sminthopsis crassicaudata) (Stannard, 2011)

1.5.6 Stripe-faced dunnart (Sminthopsis macroura)

The stripe-faced dunnart is characterised by a dark stripe of down the centre of its face between the ears and eyes (Figure 1.8). It inhabits central and northern regions of

50 Australia. On average it grows 85mm in length and weighs 20g (Morton and Dickman,

2008b). The stripe-faced dunnart preys on arthropods and small (Morton and

Dickman, 2008b). Stripe-faced dunnarts are seasonal breeders and breed from June to

February; sexual maturity is reached at 80-240 days (Woolley, 1990). Females have eight teats and therefore can carry a maximum of eight pouch young per litter. Young are born after 11-20 days gestation and the young become independent at 70 days (Woolley,

1990). Developmental biology has been well studied in stripe-faced dunnarts. Compared with other dasyurids more is known about their reproductive development and physiology, embryonic development and development of immune system (Selwood,

1987; Hickford et al., 2001; Kress et al., 2001; Old et al., 2004; Au et al., 2010).

Figure 1.8 A stripe-faced dunnart (Sminthopsis macroura) (Stannard, 2011)

The stripe-faced dunnart is not nationally listed in Australia as under threat, however in

the state of New South Wales it is listed as ‘vulnerable’ under the Threatened Species Act

1995. This is due to localised population declines (Frank and Soderquist, 2005). There

are a number of other dunnart species which are in decline and are nationally listed as

vulnerable or endangered such as the Butler’s dunnart (S. butleri), Boullanger Island

51 dunnart (S. griseoventer) (S. aitkeni), (S. psammophila) and Julia Creek dunnart (S. douglasi) (Department of Sustainability,

Environment, Water, Population and Communities, 2009). These dunnart species are threatened by introduced predators, fires, agricultural land use, human encroachment and low population densities (Robinson, 2008; Woolley, 2008b; Woolley, 2008a). The stripe- faced and fat-tailed dunnarts provide suitable models for other endangered dunnart species. Studying the biology and ecology of these two species will allow comparisons to be made to other dunnarts species. These two dunnarts are commonly held in zoos and wildlife parks for display and education purposes. There are also a few populations held at research institutions, such as the University of Sydney, Sydney, and La Trobe

University, Bundoora with the aim of gaining knowledge of their biology.

52 1.6 Aim

The work described in this thesis examines the health and management of captive

dasyurid species to improve current knowledge, animal management and husbandry

practices. The purpose of this research is to support wildlife managers with the captive

maintenance of dasyurids in addition, to supporting the use of captive dasyurids for reintroduction and translocation programs. These programs allow the restoration of wild populations in areas where the species once occurred and boost population numbers in current habitat ranges. At present the red-tailed phascogale is being reintroduced in the

Northern Territory, South Australia and Western Australia (W. Foster pers. comm. 2010).

There are also future intentions to use some of the other species studied in this thesis for

reintroduction programs in other parts of Australia.

This thesis is separated into eight chapters. Five of the following seven chapters are written as publications, and differ in format according to the style of the journal they

were submitted to. The final chapter is a general conclusion. The aims of the following

seven chapters are to:

Chapter 2 - Determine the diet of a translocated population of red-tailed phascogales

at Alice Springs Desert Park

Chapter 3 - Determine the rate of passage, and the histology and morphology of the

kultarr gastrointestinal tract

Chapter 4 - Determine the apparent digestibility of current captive feeding regimes

fed to the red-tailed phascogale and kultarr

53 Chapter 5 - Determine the apparent digestibility of a current captive diet,

commercially available diet, and live food items of the fat-tailed and stripe-faced

dunnarts. Determine morphology and dimensions of the gastrointestinal tract of both dunnart species

Chapter 6 - Determine the digestibility of two captive diet items of the eastern quoll

Chapter 7 – Provide further information on eastern and spotted-tailed quoll blood; determine the influence of age, sex and season on blood chemistry values of the eastern quoll

Chapter 8 – Discuss the results from previous chapters, draw conclusions and discuss future directions for related research

54 1.7 Chapter 1 references

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65 CHAPTER 2

The diet of red-tailed phascogales in a trial translocation at

Alice Springs Desert Park, Northern Territory, Australia

H. J. Stannard1, W. Caton2 & J. M. Old1

1 Native and Pest Animal Unit, School of Natural Sciences, University of Western Sydney, NSW, Australia

2 Department of Environment and Conservation, Wildlife Research Centre, Science Division, WA,

Australia

Journal of Zoology; 2010, vol. 280, pp. 326-331

DOI: 10.1111/j.1469-7998.2009.00658.x

66 2.1 Chapter outline and authorship

Chapter 2 was based on a study of a translocated population of red-tailed phascogales at

Alice Springs Desert Park, Alice Springs (ASDP). ASDP is a native wildlife park situated

5km outside of the Alice Springs town centre. The study was conducted to determine the food items chosen and food items available to the phascogales that were released into the

1306ha feral-proof area of the park. The phascogales were ‘soft released’ into the fenced park as opposed to ‘hard released’ directly into the wild. It was important to determine whether the animals were able to hunt for food and sustain themselves post-release. The research will contribute to further translocations (hard release) of red-tailed phascogales into former habitat ranges in other geographical locations.

The following manuscript is jointly authored, I am the primary author and I performed all laboratory analysis of scats, collection of at Alice Springs Desert Park, statistical analysis of data, and wrote the manuscript. Wes Caton provided the scat samples and critiqued the manuscript. Dr Julie Old supervised and negotiated the development of the work, provided editorial feedback on previous versions of the manuscript and acted as the corresponding author.

We would like to acknowledge and thank the editor and anonymous reviewers who provided feedback on the following manuscript prior to its acceptance by the journal.

This chapter is a published paper and should be cited as:

Stannard HJ, Caton W & Old JM. (2010) The diet of red-tailed phascogales in a trial translocation at Alice Springs Desert Park, Northern Territory, Australia. Journal of Zoology, vol. 280, pp. 326-331. DOI: 10.1111/j.1469-7998.2009.00658.x

67 2.2 Introduction

Red-tailed phascogales (Phascogale calura) are small (~50g) arboreal Dasyurids

(Bradley, Foster & Taggart, 2008). They are named after their distinctive red tail that has a black brush-like tip (Bradley et al., 2008). Males of the species undergo complete male mortality at the end of the breeding season (Bradley, 1987). The species is nocturnal and hunts for food during the night (Bradley et al., 2008). Red-tailed phascogales are listed as near threatened on the International Union for Conservation of Nature and Natural

Resources red list (Friend, 2008).

Red-tailed phascogales were once widespread throughout central Australia, including

Alice Springs and other areas of the Northern Territory, New South Wales, Victoria and

South Australia (Smith & Medlin, 1982; Bradley, 1997). The current distribution of the red-tailed phascogale is limited to a small area in south-western Western Australia vegetated with Eucalyptus wandoo and Casuarina huegeliana (Kitchener, 1981; Bradley et al., 2008). This is most likely due to European settlement and changes to their natural habitats (Smith & Medlin, 1982).

In Australia and overseas, translocation is a common tool in species conservation. Within

Australia, the bilby (Macrotis lagotis), dibbler (Parantechinus apicalis), brush-tailed bettong (Bettongia penicillata), bridled nailtail wallaby (Onychogalea fraenata) and the hare-wallabies (Lagorchestes hirsutus and L. fasciatus), have been translocated (Moseby

& O’Donnell, 2003; Moro, 2003; Priddel & Wheeler, 2004; Sigg, Goldizen & Pople,

2005; Hardman & Moro, 2006). Overseas, the European beaver (Castor fiber), maned

68 sloths (Bradypus torquatus) and black howler monkeys (Alouatta pigra) have also been translocated (Nolet & Baveco, 1996; Chiarello et al., 2004; Gavazzi et al., 2008). In

2001, Alice Springs Desert Park (ASDP) established a breeding colony and more recently developed a captive breeding facility with the aim of reintroducing the red-tailed phascogale into the Northern Territory and South Australia. The red-tailed phascogales were initially released into the ASDP in 2006. Radio-tracking and scat collection was used to monitor the animals over this time period. With the exception of a study undertaken in Western Australia of wild red-tailed phascogales (Kitchener, 1981) little is known about the diet of this species. This study describes the diet of the red-tailed phascogales translocated into the ASDP prior to hard release of phascogales into the nearby National Park and Warraweena Conservation Park, South Australia (Foster pers. comm.).

2.3 Materials and Methods

2.3.1 Study site

ASDP is a 1306ha wildlife park approximately 5km from Alice Springs (23o42’S

133o50’E). The local mean annual rainfall is 278.5mm with the mean maximum rainfall occurring in February and the mean minimum rainfall occurring in September. The mean minimum temperature is 4.0oC in July and the mean maximum temperature is 36.4oC in

January (Bureau of Meteorology 2008).

The study site consisted of a dominant upperstorey of mulga (Acacia aneura), witchetty bush (Acacia kampeana) and river red gums (Eucalyptus camadulensis).

69 Understorey vegetation consists of flowering shrubs such as pink everlasting (Schoenia cassiniana) and common everlasting (Chrysocephalum apiculatum).

2.3.2 Animals and sample collection

Twenty-five red-tailed phascogales (five males and 20 females) were released into ASDP in 2006. Phascogale scats were collected opportunistically from nesting and resting sites during July-October, 2006 and January-March, 2007 and stored at -20oC until required.

2.3.3 Scat analysis

Each scat was thawed, dried and weighed prior to analysis. Separation of the scat contents was conducted employing methods used by Jarman et al. (2007) whereby each scat was soaked in hot water overnight to loosen the scat, the contents gently mixed and then particles separated using a 500µm and a 250µm sieve. The content collected on each sieve was then examined under a dissecting microscope (x 10; Olympus, Ina, Japan) to identify scat components.

Arthropods were identified based on characteristic leg, head, wing or other body parts where possible. Hair was analysed microscopically for length, cuticle scale and medulla and compared to reference hair samples using the Hair Identification CD-ROM (Triggs &

Brunner 2002). Feathers were described as present or absent as no keys are currently available to identify birds based on feathers only. Plant material was identified using a

70 key developed by Moore (2005) for native plants of Central Australia. As phascogales are arboreal, the possibility of earthworms in the diet was not investigated.

Frequency of occurrence was determined by the presence or absence of a food type

(arthropod group, hair, scales, feathers and plant material) in the individual scats and expressing the number with the food type present as a percentage of all scats examined.

Seasonal variation of diet, based on the percentage of occurrence in all scats, for each season, was analysed using non-metric multidimensional scaling (MDS) and global one- way analysis of similarities (ANOSIM), based on Bray-Curtis dissimilarity matrix.

Following the global test, differences between individual seasons were determined by pairwise comparison. Similarity percentages (SIMPER) were then used to determine the significant differences between in diet components between seasons (Similar to methods used by Glen and Dickman, 2006).

2.3.4 Light trap sampling and arthropod identification

Arthropod sampling was conducted to determine the potential prey items available in the study site. Sampling took place during October 2008, which had an average temperature of 30˚C (Bureau of Meteorology 2008). Arthropods were sampled using three light traps

(Australian Entomological Supplies, Australia) similar to methods used by Doucette

(2007). The traps had a light globe to attract flying arthropods; once they had flown toward the light the fan drew them downward into a collection bag. The traps were suspended 2-3m off the ground and were set for 12h overnight to target nocturnal flying insects likely to be consumed by phascogales. Light traps are presumably bias toward

71 flying arthropods and it is likely phascogales are able to consume other non-flying arthropods. All arthropods caught were identified in the field, based on body part characteristics to Order or Family level where possible, using an insect field guide

(Zborowski & Storey, 2003).

72 2.4 Results

Collected scats (n = 95) weighed an average of 0.10g. Overall, the samples were dominated by arthropods (92.6%), feathers (51.6%) and mammalian hair (47.4%), but frequencies varied by season (Table 2.1).

Table 2.1 Percentage of occurrence of components from 95 red-tailed phascogale scat samples

Winter (%) Spring (%) Summer (%)

Arthropods 100 88.89 91.43

Plant material 29.17 30.56 22.86

Mammals

Nyctophilus geoffroyi 0 2.78 2.86

Mus musculus 4.17 8.33 2.86

Pseudomys desertor 4.17 0 2.86

Sminthopsis macroura 4.17 11.11 5.71

Phascogale calura 29.17 33.33 40

Birds 62.96 66.67 20

Other

Rocks 0 8.33 0

Bones 0 5.56 0

Reptile scales 0 8.33 8.57

73 2.4.1 Scat component analysis

Some arthropod components were identified to order based on whole legs (Insecta or

Araneae), pedipalps and chelicerae (Araneae), head (Hymenoptera), or whole and partial wings (Insecta). The majority of the components appeared to consist of partial remains of exoskeleton and could not be identified further. The majority of arthropods identified in scats were from the orders Araneae, Coleoptera, Blattodea, Diptera and Hymenoptera.

One whole (Hymenoptera) was identified in a sample and an ant head was identified in another sample. It is not known whether the ants were prey items, accidentally ingested or became attached to the scat after defecation.

In addition, as it was very rare for entire arthropods to be identified in the scats, it was extremely difficult to assess the numbers of each arthropod in each scat due to fragmentation of the arthropod remains.

Material other than arthropods found in scats included hair, feathers and plant material.

Hair was identified in 47.4% of the scat samples, of those samples, 33.3% were found to contain hair from mammalian species other than the red-tailed phascogale. These included desert mouse (Pseudomys desertor) at 4.44%, lesser long-eared bat (Nyctophilus geoffroyi) 4.44%, stripe-faced dunnart (Sminthopsis macroura) 15.56% and house mouse

(Mus musculus) 11.11%. Hair identified as red-tailed phascogale was presumed to be ingested during grooming.

74 Plant material was usually rare in samples and may have been ingested accidentally during capture of prey or nest building. Several samples contained pink flower petals identified as pink everlasting (Schoenia cassiniana), as this species grows in the park and flowers from winter to spring (Moore, 2005), it may be that the phascogales are using these flowers as a nectar source. Only one sample was found to contain exclusively plant material.

The other materials identified included bones. These bones were hollow and always associated with feathers and therefore presumed to be avian bones. Reptilian scales were found, but could not be identified to species because they were extremely fragmented.

Small rocks were found in samples which were presumably ingested incidentally whilst feeding on the ground or became attached to the scat after defecation.

Few samples were found to have only one prey type present (21.1%) and only one sample

(1.05%) was found to have no prey types present. No arthropod components reflected the presence of ticks, fleas or lice, potential parasites of the phascogales.

2.4.2 Comparison of seasonality

Overall, arthropods were the dominant scat component across the seasons. MDS of the results indicates the similarity scat components across the seasons, with a stress coefficient of 0.12 (Figure 2.1). A global one-way ANOSIM showed that the variation due to season was not significant (global R= 0.02, P=0.15); however, pairwise comparison showed a significant difference between spring and summer (P = 0.009).

75 SIMPER analyses showed that this variation was primarily due to the greater number of birds consumed in spring (Table 2.2).

76 1

Stress: 0.12

Spring

Summer

Winter

2 3 Figure 2.1 Multidimensional scaling of seasonal red-tailed phascogale scat components

4

77 Table 2.2 Pairwise comparison showing the differences between summer and spring

Scat component Spring mean Summer mean Mean Cum. %

number number dissimilarity

Feathers (birds) 0.68 0.28 12.90 25.80

Arthropods 0.87 0.95 4.73 69.77

Phascogale calura 0.32 0.41 9.65 45.10

Sminthopsis macroura 0.13 0.05 3.42 76.61

Mus musculus 0.06 0.05 1.72 91.04

Plant material 0.26 0.28 7.62 60.32

Schoenia cassiniana 0.06 0.13 2.55 87.59

Reptile scales 0.06 0.10 2.95 82.50

2.4.3 Light trap sampling and arthropod identification

Several Orders and Families were identified at the study site (Table 2.3). The majority of the arthropods identified were insects from the Lepidoptera (moths & butterflies)

Order. Arthropods from this order were identified based on size as a large variety of families from this order were represented. They were classified as small (<10mm), medium (<20mm) and large (>20mm). Small insects (<5mm) were collected but could not be identified to order.

78

Table 2.3 Arthropods collected at ASDP and possible prey items for red-tailed phascogales

Class Order Family Quantity

Arachnida - - 2

Insecta Orthoptera (grasshoppers & crickets) - 1

Hemiptera (bugs, hoppers & aphids) 8 -

Cydnidae 1

Flatidae 17

Blattodea (cockroaches) - 2

Mecoptera (Scorpian flies) Choristidae 1

Odonata (dragon/damsel flies) - 1

Hymenoptera (wasps and bees) Ichneumonidae 6

Diptera (true flies) Tabanidae 4

Culicidae 4

Lepidoptera (moths & butterflies) small <10mm >100

medium <20mm >100

large >20mm 15

Neuroptera (Lace wings) Nymphidae 1

Chrysopidae 150

Myrmeleontidae 10

Coleoptera (beetles) - >35

Passalidae 1

Elateridae 1

- Family unidentified

79

2.4.4 Incidental observations

On several occasions half-eaten remains of Stimpsons pythons (Antaresia stimsoni) and red-capped robins (Petroica goodenovii) were observed in nest boxes. In the case of the pythons only the head portions of the animals had been eaten.

2.5 Discussion

The results of this study show red-tailed phascogales are primarily insectivorous, a finding consistent with that of Kitchener (1981). They prey upon a range of insects and appear to have a high proportion of , beetles and cockroaches in their diet.

It appears that they are also opportunistic hunters of small mammalian, avian and reptilian species.

Arthropod components have posed identification problems in past dietary studies of the red-tailed phascogale, mulgara (Dasycercus cristicauda) swamp antechinus

(Antechinus minimus) and spotted-tailed quoll (Dasyurus maculatus) (Kitchener,

1981; Chen, Dickman & Thompson, 1998; Allison, Gibson & Aberton, 2006; Jarman et al., 2007). Frequency of occurrence in faecal samples was used as a measure to reduce the bias towards hard bodied arthropods and indigestible materials (Dickman

& Huang, 1988; Glen & Dickman, 2006). Frequency of occurrence was also used in this study due to the difficulty identifying the well masticated and digested pieces of arthropod. Further arthropod sampling would provide more information on potential prey items of the phascogales.

Red-tailed phascogales, although mostly insectivorous, consume desert mice

(Pseudomys desertor), stripe-faced dunnarts (Sminthopsis macroura) and house mice

80

(Mus musculus) as identified from hair samples. In addition, a large number of scats were found to contain phascogale hair suggesting they are ingesting their own hair whilst grooming. Another study revealed that wild phascogales prey upon house mice

(Mus musculus) and juvenile European rabbits (Oryctolagus cuniculus) (Kitchener,

1981). Similarly brush-tailed phascogales (Phascogale tapaotafa), a much larger phascogale species, preys upon dunnarts (Sminthopsis spp.) and brushtail possums

(Trichosurus vulpecula) (Scarff, Rhind & Bradley, 1998). As ground dwelling species such as dunnarts were identified in the scat samples it suggests that the phascogales at

ASDP are also foraging for food on the ground; furthermore it seems to target mammalian prey weighing ≤30g. It is also likely that phascogales are able to locate nests of mammalian species and prey upon young or juvenile animals.

Lesser long-eared bat (Nyctophilus geoffroyi) hair was also identified in 3.16% of scats. This particular species of bat nests in tree hollows and forages low to the ground (Menkhorst & Knight, 2004) creating a spatial overlap between these bats and the phascogales. Due to their small size (<11g) and choice of nest locations it could be assumed that phascogales are consuming the bats; however, the hair identified occurred in very low quantities within the samples. It is likely that phascogales are accidentally ingesting the hair when searching for food or nest materials. Bats

(Microchiroptera) previously have been identified in brush-tailed phascogale scat samples and were also believed to be incidentally ingested (Scarff et al., 1998).

Feathers could not be identified to species. However based on incidental observations of partially eaten red-capped robins, it is evident that the phascogales are consuming at least one avian species. Based on observations and the size of the phascogales it

81 seems likely they are preying on small avian species such as zebra finches

(Taeniopygia guttata), painted finches (Emblema pictum) and kingfishers

(Todiramphus pyrrhopygius) which also inhabit ASDP. It is also likely that phascogales may be preying on hatchlings in nests. Kitchener (1981) found that avian species constituted only a very small portion of wild phascogale diets. Perhaps land management practices favour the high consumption of birds at ASDP. These practices include irrigation, revegetation and predator-proof fencing, creating a suitable habitat which would attract and support a rich variety of avian species.

Although no reptile bones or whole limbs were found in the scat samples, incidental observations of half eaten pythons in nest boxes and scales identified in the scats suggest that phascogales may opportunistically forage on reptiles. However phascogales may also be killing these small reptiles for defensive purposes.

No ectoparasites were identified in the scat samples, suggesting that the animals were relatively free of parasites or were not ingesting them during grooming. Ectoparasites have previously been described in scats from greater stick-nest rats (Leporillus conditor), hopping-mice (Notomys alexis) and spotted-tailed quolls

(Dasyurus maculatus) (Copley, 1988; Old, Hill & Deane, 2007; Jarman et al., 2007).

The difference between spring and summer, although statistically significant, was not large, and might have been caused by a higher portion of birds being consumed in spring. The greater number of birds consumed in spring is most likely reflective of the increased availability of birds, particularly hatchlings and juveniles, due to the birds’ breeding season. It is also possible that phascogales are eating eggs with developed

82 juveniles inside. Further studies should be conducted to determine whether there is a temporal difference between the seasons.

Arthropod sampling was undertaken to further classify arthropod prey items, particularly as the majority of arthropods in the scat samples were broken down into unidentifiable pieces during the mastication and digestion processes. The three orders of arthropod identified in the highest quantity were Lepidoptera (moths & butterflies),

Neuroptera (Lace wings) and Coleoptera (beetles), similar to the findings from previous arthropod surveys at ASDP by Doucette (2007). Lepidoptera and Coleoptera in particular are favoured food items of wild phascogales (Kitchener, 1981).

Arthropod sampling suggests prey arthropod species are present at ASDP and provide an adequate food supply for the phascogales during the drier months of the year.

Further investigation of seasonal diversity and density of arthropods, particularly following rainfall, would lead to a greater understanding of the seasonal variation and availability of arthropods for the phascogales. Seasonal variation and availability of prey is generally reflected in changes in the diet as shown in spotted-tailed quolls

(Glen & Dickman, 2006). It would be ideal to conduct further scat analysis to coincide with seasonal arthropod sampling to ensure results correlate. In addition, other methods could be employed for arthropod sampling, such as pitfall traps, and bark and litter sampling to identify other species which phascogales may target.

Arthropod sampling took place during spring. Late winter and spring is the peak period of reproductive stress in female phascogales, as nutritional requirements are higher than at other times because of the need to supply nutrition to the pouch young.

83

During this time of reproductive stress, female phascogales are known to increase their food intake (Green, King & Bradley, 1989). The scat samples collected during spring had a high portion of feathers suggesting the phascogales are utilising prey with a higher nutritional content to meet the elevated requirements during this time of stress. Similar results have been recorded previously for the mulgara, which utilised during times of nutritional stress (Chen et al., 1998).

Phascogales breed in July and all males die due to post-mating stress and increased plasma glucocorticoid concentrations by the end of July (Bradley, 1987). In captivity, males become senescent and are no longer able to breed as they become infertile.

Within the translocated population it is unknown if all the males disappeared naturally from the population at the end of July. If this is the case, the samples collected in

August-October, 2006 would have consisted of female phascogale scats only. Also, as red-tailed phascogales leave the pouch for the first time at 44 days of age and are weaned at 100 days (Foster et al., 2006), samples collected in the January-March,

2007 period would include adult females and juvenile male and female phascogale scats. A higher number of scats were collected in summer in comparison to winter most likely due to selective elimination of males via the semelparous stress response.

This study provides the first insight into the diet of a reintroduced red-tailed phascogale population in Alice Springs. The results from this study are broadly similar to results found by Kitchener (1981) in Western Australia. The study of reintroduced red-tailed phascogale population has shown they are able to endure translocation and will exploit prey items other than arthropods (i.e. birds and mammals). However, phascogales’ continued survival, similar to other native

84 translocated species relies on adequate management of habitat, through elimination of exotic predators (Moseby & O’Donnell, 2003; Priddel & Wheeler, 2004), protection from land clearing and infrequent burning as suggested by Kitchener (1981). Prior to the translocation of phascogales into the wild it would be appropriate to assess the habitat dynamics including availability of prey (arthropods), shelter, predators, and competitor species to ensure suitable sites are chosen for their translocation. Further studies of spatial movement, home range and nesting sites would further contribute to the effective management of translocating this species.

85

2.6 Chapter 2 references

Allison, L.M., Gibson, L.A. & Aberton, J.G. (2006). Dietary strategy of the swamp

antechinus (Antechinus minimus maritimus) (Marsupialia: Dasyuridae) in

coastal and inland heathland habitats. Wildl. Res. 33, 67-76.

Bradley, A.J. (1987). Stress and mortality in the red-tailed phascogale, Phascogale

calura (Marsupialia: Dasyuridae). Gen. Comp. Endocrinol. 67, 85-100.

Bradley, A.J. (1997). Reproduction and life history in the red-tailed phascogale,

Phascogale calura (Marsupialia: Dasyuridae): the adaptive-stress senescence

hypothesis. J. Zool. (Lond.) 241, 739-755.

Bradley, A.J. Foster, W.K. & Taggart, D.A. (2008). Red-tailed phascogale. In The

Mammals of Australia. 3rd edn: 101-102. Van Dyck, S. and Strahan, R. (Eds.).

Sydney: New Holland.

Bureau of Meteorology (2008). Climate statistics for Australian locations.

http://www.bom.gov.au/climate/averages/tables/cw_015540.shtml Retrieved

24.9.08

Chen, X., Dickman, C.R. & Thompson, M.B. (1998). Diet of the mulgara, Dasycercus

cristicauda (Marsupialia: Dasyuridae), in the Simpson desert, central

Australia. Wildl. Res. 25, 233-242.

Chiarello, A.G., Chivers, D.J., Bassi, C., Maciel, M.A.F., Moreira, L.S. & Bazzalo,

M.

(2004). A translocation experiment for the conservation of maned sloths,

Bradypus torquatus (Xenarthra, Bradypodidae). Biol. Conserv. 118, 421-430.

Copley, P. (1988). The Stick-nest Rats of Australia. South Australia: Department of

Environment and Planning.

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Doucette, L.I. (2007). Behavioural ecology and thermal physiology of Australian

owlet-nightjars (Aegotheles cristatus). PhD thesis, University of New

England, Armidale.

Foster, W.K., Bradley, A.J., Caton, W. & Taggart, D.A. (2006). Comparison of

growth and development of the red-tailed phascogale (Phascogale calura) in

three captive colonies. Aust. J. Zool. 54, 343-352.

Friend, T. (2008). Phascogale calura. In: IUCN 2008. 2008 IUCN Red List of

Threatened Species. www.iucnredlist.org Retrieved 09.03.09

Gavazzi, A.J., Cornick, L.A., Markowitz, T.M. Green, D. & Markowitz, H. (2008).

Density, distribution and home range of the black howler monkey (Alouatta

pigra) at Lamanai, Belize. J. Mammal. 89, 1105-1112.

Glen, A.S. & Dickman, C.R. (2006). Diet of the spotted-tailed quoll (Dasyurus

maculatus) in eastern Australia: effects of season, sex and size. J. Zool.

(Lond.) 269, 241-248.

Green, B., King, D. & Bradley, A. (1989). Water and energy metabolism and

estimated food consumption rates of free-living Wambengers, Phascogale

calura (Marsupialia: Dasyuridae). Wildl. Res. 16, 501-507.

Hardman, B. & Moro, D. (2006). Importance of diurnal refugia to a hare-wallaby

reintroduction in Western Australia. Wildl. Res. 33, 355-359.

Jarman, P.J., Allen, L.R., Boschma, D.J. & Green, S.W. (2007). Scat contents of the

spotted-tailed quoll Dasyurus maculatus in the New England gorges, north-

eastern New South Wales. Aust. J. Zool. 55, 63-72.

Kitchener, D.J. (1981). Breeding, diet and habitat preferences of Phascogale calura

(Gould, 1844) (Marsupialia: Dasyuridae) in the southern Wheat Belt, Western

Australia. Rec. West. Aust. Mus. 9, 173-186.

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Menkhorst, P & Knight, F. (2004). A field guide to the mammals of Australia.

Melbourne: Oxford University Press.

Moseby, K.E. & O’Donnell, E.O. (2003). Reintroduction of the greater bilby,

Macrotis lagotis (Marsupialia: Thylacomyidae), to northern South Australia:

survival, ecology and notes on reintroduction protocols. Wildl. Res. 30, 15-27.

Moore, P. (2005). A guide to plants of inland Australia. Sydney: New Holland.

Moro, D. (2003). Translocation of captive-bred dibblers Parantechinus apicalis

(Marsupialia: Dasyuridae) to Escape Island, Western Australia. Biol. Conserv.

111, 305-315.

Nolet, B.A. & Baveco, J.M. (1996). Development and viability of a translocated

beaver (Castor fiber) population in the Neatherlands. Biol. Conserv. 75, 125-

137.

Old, J.M., Hill, N.J. & Deane, E.M. (2007). Isolation of the mite Myocoptes

musculinus Koch from the Spinifex (Notomys alexis). Lab.

Anim. 41, 292-295.

Priddel, D. & Wheeler, R. (2004). An experimental translocation of brush-tailed

bettongs (Bettongia penicillata) to western New South Wales. Wildl. Res. 31,

421-432.

Scarff, F.R., Rhind, S.G. & Bradley, J.S. (1998). Diet and foraging behaviour of

brush-tailed phascogales (Phascogale tapoatafa) in the jarrah forest of south-

western Australia. Wildl. Res. 25, 511-526

Sigg, D.P., Goldizen A.W. & Pople, A.R. (2005). The importance of mating system in

translocation programs: reproductive success of released male bridled nailtail

wallabies. Biol. Conserv. 123, 289-300.

88

Smith, M.J. & Medlin, G.C. (1982). Dasyurids of the northern Flinders Ranges before

pastoral development. In Carnivorous Marsupials: 365-372. Archer, M. (Ed.).

Sydney: NSW Zoological Society.

Triggs, B. & Brunner, H. (2002). Hair Id: An interactive tool for identifying

Australian mammalian hair (CD-ROM). Melbourne: CSIRO Publishing.

Zborowski, P. & Storey, R. (2003). A field guide to the insects of Australia, 2nd edn.

Sydney: Reed New Holland.

89

CHAPTER 3

The rate of passage and the morphology and histology of the

gastrointestinal tract of the kultarr (Antechinomys laniger)

90

3.1 Chapter outline and authorship

Chapter 3 is a descriptive chapter on the gastrointestinal function of the kultarr. The chapter is two accepted research notes (listed below). Rate of passage was determined and gastrointestinal morphology and histology described for the kultarr. Kultarr physiology research has been limited to a few studies and no published studies have focused on nutritional physiology. Determination of basic digestive tract physiology such as rate of passage, morphology and histology was required for the further studies on kultarr digestion and nutrition presented in this thesis. The study was conducted with the approval of the University of Western Sydney’s Animal Care and Ethics

Committee, A6666.

The two manuscripts are jointly authored, I am the primary author and I designed and conducted all feeding trials, collected all scats, conducted all laboratory analysis, measurements of the gastrointestinal tracts and interpreted the histological slides. I wrote both manuscripts and acted as the corresponding author. Dr Julie Old supervised the development of the work and provided feedback on earlier versions of each manuscript. The kultarrs used for the study were from a captive colony managed by Dr Julie Old, at the University of Western Sydney.

The following chapter is two published papers and should be respectively cited as:

Stannard HJ and Old JM. 2012. Description of the gastrointestinal tract and associated organs of the kultarr (Antechinomys laniger). Australian Mammalogy. In press. DOI: 10.1071/AM12003

Stannard HJ and Old JM. 2011. Rate of passage through the kultarr (Antechinomys laniger) digestive tract. Australian Journal of Zoology 59, 273-276. DOI: 10.1071/ZO11103

91

(A) Description of the gastrointestinal tract and associated organs of the kultarr

(Antechinomys laniger)

3.2 Introduction

Mammalian declines and extinctions have mostly occurred in arid zones of Australia

(Maxwell et al. 1996) with 33% of the mammalian fauna in these zones becoming locally extinct (Burbidge and McKenzie 1989). There is limited information on many aspects of biology available for small arid zone species. One example is the kultarr

(Antechinomys laniger) a small (20-30g) arid zone marsupial (Valente 2008). It is thought to be primarily insectivorous; however, no published studies have investigated their diet. They are the only species within the Dasyuridae family that has an external morphological adaptation, elongated hind limbs (Valente 2008). It is possible this limb adaptation and torpor are used to conserve energy in a landscape where temperatures and food availability fluctuate significantly (Baudinette et al.

1976; Geiser 1986).

Kultarrs are held in only a few institutions for the specific purpose of increasing our understanding of their biology and to aid conservation of the species. Little information is available on kultarr biology and studies are limited to those on maternal behaviour (Happold 1972), reproduction (Woolley 1984; Stannard and Old 2010) torpor (Geiser 1986) and locomotion (Baudinette et al. 1976; Marlow 1969). Whilst these studies have provided fundamental knowledge, there is still a lack of biological and ecological information available, which can lead to difficulties with captive maintenance.

92

Digestive tract morphology has been examined previously in the kultarr (Beddard

1908; Schultz 1967), but was limited to a description of the gross morphology. No studies have provided a detailed description of the gastrointestinal tract or its morphometrics. Previously, much research dasyurid gastrointestinal tracts has focused on immunology and therefore there is a lack of detailed microscopic descriptions of the digestive structures (Poskitt et al. 1984b; Old et al. 2003, 2004). Determination of the ‘normal’ appearance of the gastrointestinal tract will aid post-mortem investigations and future nutrition studies on this species in captivity. The aim of this study was to describe the macro and microscopic morphology of the gastrointestinal tract and the associated organs of the kultarr.

3.3 Materials and methods

Due to the limited number of kultarrs in captivity additional animals could not be euthanased for this study and therefore, specimens were obtained opportunistically from animals that died from old age. These animals were from a captive colony maintained at the University of Western Sydney, Hawkesbury Campus (Richmond,

NSW). Post-mortems were conducted on these animals and their deaths were unrelated to any gastrointestinal disease or infection, based on no obvious trauma or discoloration of the organs. In total, six adult kultarrs (four males and two females) were used for the description of gastrointestinal tract morphology. Measurements were taken of the total intestinal tract length (from the pyloric sphincter to the rectum) using Vernier callipers. Care was taken not to stretch the intestine during measurement. Measurements of body mass, body length from nose-tip to rump and the length of the stomach (from fundic to pyloric region) were also recorded.

93

Tissues for histological examination were obtained from one adult male kultarr. All organs were removed post mortem and stored in 10% neutral buffered formalin.

Tissue samples from the intestine, stomach, liver, gall bladder and pancreas were processed in graded ethanols and HistoChoice (Sigma-Aldrich, United States) and then embedded in paraffin wax. Sections (7µm thick) were cut for each tissue and stained with haematoxylin and eosin (Bancroft and Cook 1995). Slides were examined using an Olympus BX60 microscope (Olympus, Japan) with a Jenoptik

ProgRes C14 camera (Jenoptik, Germany) and Image Pro plus 6.2 software.

Photomicrographs were prepared using Adobe Photoshop.

3.4 Results and discussion

The gastrointestinal tract of the kultarr was simple, with a monolocular stomach. The stomach was situated on the left side of the abdominal cavity, closely associated with the liver and spleen. The liver had four distinct lobes separated by fissures. The intestine was smooth and uniform along the tract, with no morphologically distinct separation between the small and large intestine. The absence of a caecum made it difficult from external examination to determine the point at which the large intestine began macroscopically. The empty stomach (from fundic to pyloric region) ranged from 19.9 to 22.9mm and the intestine 121.5 to 218.6mm in length (Table 3.1). On average the ratio of intestinal length to body length was 1.9:1.

94

Table 3.1 Morphology of the gastrointestinal tract of the kultarr (Antechinomys laniger)

Animal No. Mean ± s.d.

1 2 3 4 5 6

Sex M F M M M F

Body mass (g) 28 22 29 25 22 25 24.1 ± 2.7

Body length (mm) 88.2 89.4 85.4 88.3 96.3 83.0 87.6 ± 4.7

Stomach (mm) 13.0 17.1 21.1 19.9 22.9 17.7 18.6 ± 3.5

Total intestine length (mm) 150.1 171.2 121.6 157.1 218.6 130.1 165.2 ± 32.1

The morphology of the gastrointestinal tract of the kultarr follows that of the general structure of other insectivorous marsupials (Hume 1999). The tract is simple and lacks definition between the small and large intestine. The kultarr lacks a caecum, which was also noted by Beddard (1908) and Schultz (1967), like other Dasyurids (Poskitt et al. 1984a; Hume 1999; Old et al. 2006). There is a correlation between the length of the gastrointestinal tract and body size in dasyurids, with larger species such as the

Julia Creek dunnart (Sminthopsis douglasi; 170-220mm) and

(Antechinus swainsonii; 600mm) having longer digestive tracts than the kultarr

(Poskitt et al. 1984a; Hume et al. 2000).

Morphometrics on dasyurid gastrointestinal tracts are limited. However, some data is available for the American grey short-tailed opossum (Monodelphis domestica) a

Didelphid marsupial. The opossum has a similar body mass to the kultarr (34.3g);

95 however, their digestive tract is much longer (intestine length 284mm) as it includes a caecum (Santori et al. 2004).

Histologically, the gastrointestinal tract tissues observed had no evidence of lesions, discolouration or abnormalities. The structure of the digestive tissues appeared similar to that of other mammals and in particular is almost identical to that described for

Robinson’s mouse opossum (Marmosa robinsoni; Barnes 1977).

The histological structure of the kultarr stomach resembles that of other mammals

(Rouk and Glass 1970; Hume et al. 2000; O’Hara et al. 2011). The stomach is comprised of simple columnar mucus-producing cells lining the gastric pits, similar to the insectivorous bats (Family: Vespertilionidae), northern brown bandicoot (Isoodon macrourus) and Julia Creek dunnart (Rouk and Glass 1970; Hume et al. 2000; O’Hara et al. 2011). Large round parietal cells and smaller granulated chief cells were scattered throughout the epithelium below the gastric pits. Parietal and chief cells become less abundant in the pyloric region of the kultarr (Figure 3.1); however, they are not completely absent in this region as they are in Caenolestid marsupials

(Richardson et al. 1987). As in other dasyurids and macropods, Brunner’s glands

(Figure 3.2) form a thick collar at the pylorus at the proximal end of the duodenum in the kultarr (Krause 1972; Hume et al. 2000; O’Hara et al. 2011). It is thought

Brunner’s glands provide protection from stomach acid and sharp-edged insect exoskeletons in the duodenum of marsupials (Hume 1999; Hume et al. 2000).

Brunner’s glands presumably also perform this function in the kultarr.

96

Figure 3.1 Pyloric region of the kultarr stomach showing parietal cells.

Figure 3.2 Brunner’s glands in the kultarr at the pylorus duodenum junction.

97

The liver was large, multi-lobed and similar to that described for other mammals. As with other mammals, including dasyurids, the liver consisted of a single lamina of hepatocytes arranged around central veins (Barnes 1977; Old et al. 2006). Kupffer cells were visible between hepatocytes and scattered throughout the liver tissue.

The pancreas had well-defined lobules divided by connective tissue. The serous acini, centroacinar cells and islets of Langerhans were visible. The pancreatic tissue was similar to that of other mammals and to that described for dasyurids generally

(Attwood and Woolley 1980), and also for the insectivorous (Tachyglossus aculeatus; Griffiths 1965). The pancreas was deemed healthy as there were no cysts or lesions observed, in contrast to kultarrs described previously that were affected by toxoplasmosis (Attwood and Woolley 1980).

The transverse section of the gall bladder showed it to be an empty, -shaped organ. Internally the gall bladder was highly folded and comprised of tall columnar epithelium proximal to a layer of basal membrane and connective tissue, similar to that described for the house mouse (Mus musculus; Hayward 1962).

The intestinal section examined was from the duodenal portion of the small intestine, and had three large macroscopic folds or plicae circulares. Microscopically the villi were short and lined with columnar epithelial cells and mucus-producing goblet cells as observed in other marsupial species (Barnes 1977; Old and Deane 2001; O’Hara et al. 2011). Smooth muscle external of the villi contained blood vessels, layers of longitudinal muscle and a layer of serosa lined the external surface of the intestine.

The intestinal glands were simple straight tubular. The distal portion of the kultarr

98 duodenum lacked Paneth cells, unlike Robinson’s mouse opossum (Barnes 1977). It is however possible Paneth cells were present further along the adult kultarr intestine.

The macro and microscopic gastrointestinal anatomy of the kultarr is similar to that of other dasyurids and marsupials studied thus far. The information provided in this study represents the ‘normal’ appearance of the gastrointestinal tract and will assist in future post-mortem investigations.

99

3.5 Chapter 3 references (A)

Attwood, H. D., and Woolley, P. A. (1980). Pancreatic pathology in dasyurid

marsupials. Journal of Wildlife Diseases 16, 245-250.

Bancroft, J. D., and Cook, H. C. (1995). ‘Manual of histological techniques and their

diagnostic application.’ (Churchill Livingstone: Edinburgh)

Barnes, R. D. (1977). The special anatomy of Marmosa robinsoni. In ‘The Biology of

Marsupials.’ (Ed D. Hunsaker II.) Pp. 387-414. (Academic Press: New York.)

Baudinette, R. V., Nagle, K. A., and Scott, R. A. D. (1976). Locomotory energetics in

a marsupial (Antechinomys laniger) and a (Notomys alexis). Cell and

Molecular Life Sciences 32, 583-585.

Beddard, F. E. (1908). On the anatomy of Antechinomys and some other marsupials,

with the special reference to the intestinal tract and mesenteries of these and other

mammals. Proceedings of the Zoological Society London 1908, 561–605.

Burbidge, A. A., and McKenzie, N. L. (1989). Patterns in the modern decline of

Western Australia's vertebrate fauna: causes and conservation implications.

Biological Conservation 50, 143-198.

Geiser, F. (1986). Thermoregulation and torpor in the Kultarr, Antechinomys laniger

(Marsupialia: Dasyuridae). Journal of Comparative Physiology B 156, 751-757.

Griffiths, M. (1965). Digestion, growth and nitrogen balance in an egg-laying

mammal, Tachyglossus aculeatus (Shaw). Comparative Biochemistry and

Physiology 14, 357-375.

Happold, M. (1972). Maternal and juvenile behaviour in the marsupial jerboa

Antechinomys spenceri (Dasyuridae). Australian Mammalogy 1, 27–37.

Hayward, A. F. (1962). Aspects of the fine structure of the gall bladder epithelium of

the mouse. Journal of Anatomy 96, 227-236.

100

Hume, I. D. (1999). ‘Marsupial Nutrition.’ (Cambridge University Press: Cambridge.)

Hume, I. D., Smith, C., and Woolley, P. A. (2000). Anatomy and physiology of the

gastrointestinal tract of the Julia Creek dunnart, Sminthopsis douglasi

(Marsupialia: Dasyuridae). Australian Journal of Zoology 48, 475-485.

Krause, W. J. (1972). The distribution of Brunner’s glands in 55 marsupial species

native to the Australian region. Acta Anatomica 82, 17-33.

Marlow, B. J. (1969). A comparison of two desert-living Australian mammals,

Antecinomys spenceri (Marsupialia: Dasyuridae) and Notomys cervinus

(Rodentia: Muridae). Journal of Zoology 157, 159-167.

Maxwell, S., Burbidge, A. A., and Morris, K. (1996). ‘The 1996 Action Plan for

Australian Marsupials and Monotremes.’ (Wildlife Australia: Canberra.)

O’Hara, P. J, Murray, P. J, and Klieve, A. V. (2011). Histology of the gastrointestinal

tract of the northern brown bandicoot, Isoodon macrourus (Marsupialia:

Peramelidae). Australian Mammalogy 33, 44-46.

Old, J. M., and Deane, E. M. (2001). Histology and immunohistochemistry of the gut-

associated lymphoid tissue of the eastern grey kangaroo, Macropus giganteus.

Journal of Anatomy 199, 657-662.

Old, J. M., Carman, R. L., Fry, G., and Deane, E. M. (2006). The immune tissues of

the endangered red-tailed phascogale (Phascogale calura). Journal of Anatomy

208, 381-387.

Old, J. M., Selwood, L., and Deane, E. M. (2003). A Histological investigation of the

lymphoid and immunohaematopoietic tissues of the adult stripe-faced dunnart

(Sminthopsis macroura). Cells Tissues Organs 173, 115-121.

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Old, J. M., Selwood, L., and Deane, E. M. (2004). The appearance and distribution of

mature T and B cells in the developing immune tissues of the stripe-faced dunnart

(Sminthopsis macroura). Journal of Anatomy 205, 25-33.

Poskitt, D. C., Barnett, J., Duffey, K., Kimpton, W.G., and Muller, H. K. (1984a). A

novel structure in the stomach and intestine of two species of Australian

marsupial mice. Journal of Comparative Pathology 94, 481-485.

Poskitt, D. C., Duffey, K., and Barnett, J. (1984b). The gut-associated lymphoid

system of two species of Australian marsupial mice, Antechinus swainsonii and

Antechinus stuartii. Distribution, frequency and structure of Peyer’s patches and

lymphoid follicles in the small and large intestine. Australian Journal of

Experimental Biology and Medical Science 62, 81-88.

Richardson, K. C., Bowden, T. A. J., and Myers, P. (1987). The cardiogastric gland

and alimentary tract of Caenolestid marsupials. Acta Zoologica (Stockholm) 68,

65-70.

Rouk, C. S., and Glass, B. P. (1970). Comparative gastric histology of five north and

central American bats. Journal of Mammalogy 51, 455-472.

Santori, R. T., Astua DeMoraes, D., and Cerqueira Silva, R. (2004). Comparative

gross morphology of the digestive tract in ten Didelphidae marsupial species.

Mammalia 68, 27-36.

Schultz, W. (1967). Der Magen-Darm-Kanal der Monotremen und Marsupialier. In

‘Handbuch der Zoologie’. (Eds J. G. Helmcke, D. Starck and H. Wermuth.) Pp.

1-177. (Walter de Gruyter: Berlin.)

Stannard, H. J, and Old, J. M. (2010). Observation of reproductive strategies of

captive kultarrs (Antechinomys laniger). Australian Mammalogy 32, 179-182.

102

Valente, A. (2008). Kultarr. In ‘The Mammals of Australia.’ 3rd edn. (Eds S. Van

Dyck and R. Strahan.) Pp. 122-124. (New Holland: Sydney.)

Woolley, P. A. (1984). Reproduction in Antechinomys laniger (‘spenceri’ form)

(Marsupialia: Dasyuridae): field and laboratory investigations. Australian

Wildlife Research 11, 481–489.

103

(B) Rate of passage through the kultarr (Antechinomys laniger) digestive tract

3.6 Introduction

The kultarr (Antechinomys laniger), a small dasyurid (20-30g) that inhabits arid zones of inland Australia (Valente 2008), has undergone substantial contraction in its geographical range and localised extinctions in western New South Wales (Riverina and the northwest) and Queensland (Sandringham Station) (Morris et al. 2008).

Research conducted on the physiology of kultarrs has included studies on locomotion

(Marlow 1969; Baudinette et al. 1976) and thermoregulation and torpor (Geiser

1986). To date there are no published studies of kultarr digestion and little is known of their nutrient requirements, and utilisation of energy. Anecdotal information suggests the kultarrs’ wild diet consists of invertebrates (Lidicker and Marlow 1970;

Valente 2008); however, in captivity kultarrs will eat minced meat, small rodents, commercial pet food and commercially available marsupial food (pers. obs.).

Rate of passage has been studied in a few dasyurids to date including Giles’ and narrow-nosed (Planigale gilesi and P.tenuirostris), the eastern quoll

(Dasyurus viverrinus), kowari (Dasycercus byrnie) and the Julia Creek dunnart

(Sminthopsis douglasi) (Read 1987; Hume 1999; Hume et al. 2000). Rate of passage has been determined for eutherian insectivorous species including the badger (Taxidea taxus), Cape rock elephant (Elephantulus edwardii) and American water shrews (Sorex palustris) (Harlow 1981; Woodall and Currie 1989; Gusztak et al.

2005). These studies have contributed to understanding the digestive physiology of small insectivores and have highlighted that rate of passage is strongly influenced by

104 dietary preference, body mass and species. For example, as body mass increases the rate of passage increases (Hume 1999).

The current study aimed to determine the rate of passage through the digestive tract of the kultarr. By increasing knowledge of digestive processes in kultarrs, it will increase information on small insectivorous species’ digestive function, and provide a comparative model, of which there is a paucity of information available for small digestion and nutrition.

3.7 Methods

3.7.1 Animals

The captive kultarrs used for this study were from a colony held at the University of

Western Sydney (UWS), Richmond NSW. The animals were maintained in individual wooden enclosures (60cm x 60cm x 60cm) with air vents and a glass sliding door.

Animals were held under natural photocycles and room temperature was maintained at 22 ± 3°C.Each animal was supplied with a wooden nest box (25cm2) lined with straw. Water was available ad libitum.

3.7.2 Rate of passage trial

Eight adult kultarrs (n=4 male and n=4 female) were fed a diet of lean minced beef, in gradually increasing quantities for a four week adjustment period. Minced meat was chosen as: 1) it has been used previously in passage studies on dasyurids (Read 1987;

Hume et al. 2000); 2) there is no published research to suggest kultarrs are solely insectivorous and in captivity they readily accept processed food such as minced meat

105 and commercial pet foods; 3) minced meats and commercial pet foods are commonly used in captive diets for small dasyurids (Woolley 1982; Ng et al. 1999; Selwood and

Cui 2006; Munn et al. 2010; Pollock et al. 2010). The kultarrs were fed varying levels of minced beef (1.5g, 3g, 4.75g and 6g) to adjust them to being fed solely minced meat. The remaining portion of their diet (total weight 6g) was insects including mealworms, crickets and cockroaches. Prior to feeding, the mince was frozen at -80°C for one week, to reduce the possibility of toxoplasmosis infection (Attwood and

Woolley 1970).

Following the adjustment period the kultarrs were given minced beef (3g) marked with mealworm (<1g). Mealworm cuticle was used as a marker as it is relatively indigestible and intact mealworms are also a part of the usual diet of the captive kultarrs at UWS. However, only solid matter is tracked through the digestive tract with mealworm cuticle, in contrast to the separation between solid and soluble material movement that is possible when cobalt and chromium are used as markers.

The mealworm track on solid matter through the digestive tract and does not track liquid and solid matter whereas cobalt and chromium does. Similarly, invertebrate cuticles have been used as markers in rate of passage studies on the Julia Creek dunnart (Hume et al. 2000), Giles’ planigale, narrow-nosed planigale (Read 1987), dusky antechinus (Cowan et al. 1974) and the echidna (Tachyglossus aculeatus)

(Griffiths 1965).

The mealworms were cooled in a freezer (-20ºC) for 10min to reduce their activity prior to inserting them into a ball of minced beef. Kultarrs consumed marked mince within 15min (pulse-dose) and the meal was replaced with an unmarked 3g mince

106 ball. Faeces were collected hourly for 12h post-dose, and then every 3h for the following 12h similar to methods used by Read (1987) and Hume et al. (2000).

Following collection, faeces were macerated manually in warm water and spread evenly across a Petri dish. Samples were then analysed under a stereomicroscope

(Olympus, Japan) for the presence of mealworm cuticle. Transit time was defined as the time at which the cuticle first appeared in the faeces post-dose. Total excretion time was defined as the cumulative time at which mealworm cuticle was no longer present in the faeces. Mean retention time was calculated using the following equation

(Robbins 1993):

M i is the number of particles found in the ith excretion

T i is the time (h) of the ith excretion after ingestion of the marker

3.7.3 Digestibility trial

Animals were fed 6g of minced beef daily, which is the normal quantity of food fed in captivity. Samples of food offered, refused food and faeces were collected daily, weighed (to ±0.1g) and stored at -20°C until analysed. Food samples, refused food and faeces were analysed for dry matter determined by freeze drying at -60°C for 24h

(Edwards Freeze dryer Modulyo EF4, IMA Edwards). Gross energy contents were determined by analysing the dried samples (in triplicate) in an Oxygen Bomb

Calorimeter (Parr 6200, Parr Instrument Company) using a benzoic acid standard

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(Cowan et al. 1974). Apparent digestibility was then calculated by subtracting the nutrient excreted from the nutrient intake and expressing it as a percentage of nutrient intake digested (Robbins 1993).

3.8 Results and Discussion

The kultarrs voided faeces regularly throughout the first 12h of collection following a pulse-dose of mealworm in beef mince, with faeces deposited every 1-3h on average.

The transit time (from consumption to first appearance) was 1.6 ± 0.5h. The total cumulative elimination time for cuticle was 7.3 ± 1.2h. The maximum time elapsed before the last appearance of cuticle in the faeces from one animal was 9h since consumption of marked minced beef. Mean retention time was determined to be 3.9 ±

1.2h.

Transit time is strongly influenced by body size, species and diet (Hume 1999), and therefore it was expected the kultarr would have a relatively rapid transit time, due to its small body mass and diet type. A rapid transit time is a common feature of small dasyurids, as observed in Planigale spp., Julia Creek dunnart and dusky antechinus

(Antechinus swainsonii; Cowan et al. 1974; Read 1987; Hume et al. 2000). Transit time of the kultarr was quicker than the antechinus and slower than the morphologically smaller Planigale spp. (Table 3.2).

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Table 3.2 Transit time and mean retention time of kultarr (Antechinomys laniger) and other small dasyurids

Species Body mass Transit time MRT Reference

Giles’ planigale 7-12g 0.5h 4h (total Read 1987

(Planigale gilesi) excretion)

Narrow-nosed planigale 5-7g 0.5h 4.5h (total Read 1987

(P.tenuirostris) excretion)

Kultarr 20-30g 1.6 ± 0.2h 3.9 ± 1.2h This study

(Antechinomys laniger)

Julia Creek dunnart 40-70g 1.3 ± 0.4h 3.3 ± 0.8h Hume et al.

(Sminthopsis douglasi) 2000

Dusky Antechinus <178g 3h Cowan et al.

(Antechinus swainsonii) 1974

The kultarr had a slower mean retention time compared with the Julia Creek dunnart,

(3.29h) maintained on the same diet of minced beef and mealworm (Hume et al.

2000). The slower mean retention time could be due to retention of mealworm cuticle in the stomach of the kultarr, however due to the limited data available for passage times of small dasyurids it is difficult to determine reasons for a slower mean retention time. Further research is required to understand the influence of body mass on mean retention times and food retention in the digestive tract in small dasyurids.

There is limited data available on comparative wildlife species to the kultarr, with passage times being studied in a small number of eutherian insectivores. Compared to

109 small eutherians the kultarr has a similar transit time: the mink (Mustela vison) has a mean time of 1h (Sibbald et al. 1962) and the Cape rock elephant shrew, 1.8h

(Woodall and Currie 1989). The shrew has a more complex digestive tract than the kultarr, which presumably explains why their passage times are slightly slower than that of the kultarr. Kultarrs have a shorter transit time than a similarly sized eutherian omnivore, the house mouse (Mus musculus); transit time 2.8h (Dawson 1972). The shorter passage time of the kultarr may be attributed to a simple GIT that lacks a caecum. Selective retention has been noted in species with a caecum, which lengthens transit time (Foley and Hume 1987; Moyle et al. 1995).

The minced beef diet provided to the kultarrs during the trial was highly digestible and may have contributed to the rapid transit time. The results found in the current study show the diet provided had an apparent dry matter digestibility of 77.7%, and apparent energy digestibility of 82.5% (Table 3.3). These results are similar to a study on eastern quolls (Dasyurus viverrinus) and Tasmanian devils (Sarcophilus harrisii), maintained on a diet of rat (Rattus spp.), which had an apparent dry matter digestibility of 79% and 80% (respectively) and apparent energy digestibility of 87% and 89%, respectively (Green and Eberhard 1979). The apparent dry matter digestibility of the dusky antechinus fed on a diet of house mouse was ~80% and energy digestibility ~87% (Cowan et al. 1974). The similar apparent digestibility results suggest the digestive efficiency of the kultarr is similar to that of other

Dasyurids.

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Table 3.3 Mean (± s.d.) intake and apparent digestibility of minced beef fed to kultarrs during trial (n = 8)

Nutrients Quantity

Dry Matter

Intake g d-1 2.3 ± 0.2

Faecal output g d-1 0.5 ± 0.2

Apparent digestibility % 77.7 ± 9.7

Gross energy

Intake kJ d-1 80.2 ± 6.3

Faecal output kJ d-1 12.3 ± 4.3

Apparent digestibility % 82.5 ± 7.3

The kultarr has quick passage times similar to other dasyurids, however slightly longer than the larger Julia Creek dunnart. The data provides a basis for future digestion studies and timelines of faecal collection for the kultarr in captivity. The acceptance of food items such as minced meat and small mammals indicates there is a need for further investigation into the diet of kultarrs in the wild. The data obtained in the present study has contributed to current knowledge of the digestive physiology of the kultarr.

111

3.9 Chapter 3 references (B)

Attwood H. D. and Woolley, P. (1970). Toxoplasmosis in dasyurid marsupials.

Pathology 2, 77–78.

Baudinette, R. V., Nagle, K. A., and Scott, R. A. D. (1976). Locomotory energetics in

a marsupial (Antechinomys laniger) and a rodent (Notomys alexis). Cellular and

Molecular Life Sciences 32, 583-585.

Cowan, I. Mc. T., O’Riordan, A. M., and Cowan, J. S. Mc. T. (1974). Energy

requirements of the Dasyurid marsupial mouse Antechinus swainsonii

(Waterhouse). Canadian Journal of Zoology 52, 269-275.

Dawson, N. J. (1972). Rate of passage of a non-absorbable marker through the

gastrointestinal tract of the mouse (Mus musculus). Comparative Biochemistry

and Physiology A 41, 877-881.

Foley, W. J., and Hume, I. D. (1987). Passage of digesta markers in two species of

arboreal folivorous marsupials: the greater glider (Patauroides volans) and the

brushtail possum (Trichosurus vulpecula). Physiological Zoology 60, 103-113.

Geiser, F. (1986). Thermoregulation and torpor in the Kultarr, Antechinomys laniger

(Marsupialia: Dasyuridae). Journal of Comparative Physiology B 156, 751-757.

Green, B., and Eberhard, I. (1979). Energy requirements and sodium and water

turnovers in two captive marsupial carnivores: the Tasmanian devil, Sarcophilus

harrisii, and the native cat, Dasyurus viverrinus. Australian Journal of Zoology

27, 1-8.

Griffiths, M. (1965). Digestion, growth and nitrogen balance in an egg-laying

mammal, Tachyglossus aculeatus (Shaw). Comparative Biochemistry and

Physiology 14, 357-375.

112

Gusztak, R. W., MacArthur, R. A., and Campbell, K. L. (2005). Bioenergetics and

thermal physiology of American water shrews (Sorex palustris). Journal of

Comparative Physiology B 175, 87-95.

Harlow, H. J. (1981). Effect of fasting on rate of passage and assimilation efficiency

in badgers. Journal of Mammalogy 62, 173-177.

Hume, I. D. (1999). ‘Marsupial Nutrition.’ (Cambridge University Press: Cambridge.)

Hume, I. D., Smith, C., and Woolley, P. A. (2000). Anatomy and physiology of the

gastrointestinal tract of the Julia Creek dunnart, Sminthopsis douglasi

(Marsupialia: Dasyuridae). Australian Journal of Zoology 48, 475-485.

Lidicker, W. Z., and Marlow, B. J. (1970). A review of the dasyurid marsupial genus

Antechinomys Krefft. Mammalia 34, 212-227.

Marlow, B. J. (1969). A comparison of two desert-living Australian mammals,

Antecinomys spenceri (Marsupialia: Dasyuridae) and Notomys cervinus (Rodentia:

Muridae). Journal of Zoology (London) 157, 159-167.

Morris, K., Woinarski, J., Ellis, M., Robinson, T., and Copley, P. (2008).

Antechinomys laniger In ‘IUCN 2009: IUCN Red List of Threatened Species.’

Version 2009.1. . Downloaded on 23 July 2009.

Moyle, D.I., Hume, I. D., and Hill, D. M. (1995). Digestive performance and selective

digesta retention in the long-nosed bandicoot, Perameles nasuta, a small

omnivorous marsupial. Journal of Comparative Physiology B 164, 552-560.

Munn, A. J., Kern, P., McAllan, B. M. (2010). Coping with chaos: unpredictable food

supplies intensify torpor use in an arid-zone marsupial, the fat-tailed dunnart

(Sminthopsis crassicaudata). Naturwissenschaften 97, 601–605.

Ng, K., Vozzo, R., Hope, P. J., Chapman, I. M., Morely, J. E., Horowitz, M., Wittert,

G. A. (1999). Effect of dietary macronutrients on food intake, body weight, and

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tail width in the marsupial S. crassicaudata. Physiology and Behavior 66, 131-

136.

Pollock, K., Booth, K., Wilson, R., Keeley, T., Grogan, K., Kennerley, P., Johnston,

S. D. (2010). Oestrus in the Julia Creek dunnart (Sminthopsis douglasi) is

associated with wheel running behaviour but not necessarily changes in body

weight, food consumption or pouch morphology. Animal Reproduction Science

117, 135-146.

Read, D. G. (1987). Rate of food passage in Planigale spp. (Marsupialia: Dasyuridae).

Australian Mammalogy 10, 27-28.

Robbins, C. T. (1993). ‘Wildlife Feeding and Nutrition.’ (Academic Press: New

York.)

Selwood, L., Cui, S. (2006). Establishing long-term colonies of marsupials to provide

models for studying developmental mechanisms and their application to fertility

control. Australian Journal of Zoology 54, 197-209.

Sibbald, I. R., Sinclair, G. G., Evans, E. V., and Smith, D. L. T. (1962). The rate of

passage of feed through the digestive tract of the mink. Canadian Journal of

Biochemistry and Physiology 40, 1391-1394.

Valente, A. (2008). Kultarr. In ‘The Mammals of Australia’. 3rd edn. (eds. S. Van

Dyck and R. Strahan.) pp. 122-124. (New Holland: Sydney.)

Woodall, P. F., and Currie, G. J. (1989). Food consumption, assimilation and rate of

food passage in the Cape rock elephant shrew, Elephantulus edwardii

(Macroscelidea: Macroscelidinae). Comparative Biochemistry and Physiology A

92, 75-79.

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Woolley, P. A. (1982). The laboratory maintenance of dasyurid marsupials. In

‘Management of Australian Mammals in Captivity’. (ed. D.D. Evans.) pp. 13-21.

(Zoological Board of Victoria: Melbourne.)

115

CHAPTER 4

Digestibility of captive feeding regimes of the red-tailed

phascogale (Phascogale calura) and kultarr (Antechinomys

laniger)

H. J. Stannard & J. M. Old

Native and Pest Animal Unit, School of Natural Sciences, University of Western Sydney, Hawkesbury

Campus, NSW, Australia

Australian Journal of Zoology; 2011, vol. 59, pp. 257-263

DOI: http://dx.doi.org/10.1071/ZO11069

116

4.1 Chapter outline and authorship

The focus of chapter 4 is the digestibility of captive diets fed to the red-tailed phascogale and the kultarr. The study used items currently fed to captive animals including insects and small rodents. Increasing the information on nutrition of these species aimed to improve captive management techniques and ensure animal health in captivity. Minimal information is available on dasyurid nutrition and just as little is available for small eutherian insectivores. Thus, this study adds to the current information available for small insectivorous species. The study was conducted with the approval of the UWS ACEC; approval number A7045.

The following manuscript is jointly authored; I am the primary and corresponding author. I conducted the digestibility trials and collected all the samples during the trials. I performed nutrient analysis of scats and food, calculation of nutritional content and statistical analysis of data, and wrote the manuscript. Mineral analysis was conducted by Waite Analytical Services, South Australia. Dr Julie Old supervised the development of the work and provided editorial feedback on previous versions of the manuscript. The animals used for this study were from captive colonies maintained at the University of Western Sydney, managed by Dr Julie Old.

We would like to acknowledge and thank the editor and anonymous reviewers who provided feedback on the following manuscript prior to its acceptance by the journal.

This chapter is a published paper and should be cited as:

Stannard HJ and Old JM. 2011. Digestibility of captive feeding regimes of the red- tailed phascogale (Phascogale calura) and kultarr (Antechinomys laniger). Australian Journal of Zoology 59, 257-263. DOI: 10.1071/ZO11069

117

4.2 Introduction

Dasyurids are a family of insectivorous/carnivorous marsupials that inhabit

Australasia (Van Dyck and Strahan 2008). Their simple digestive tract lacks a caecum, and together with their diet, facilitates rapid passage times through the gut

(Cowan et al. 1974; Read 1987; Hume 1999; Hume et al. 2000). Passage times influence digestibility, which is the availability and supply of nutrients to an animal from food (Barboza et al. 2009). Digestibility has been studied in a few dasyurid species such as the eastern quoll (Dasyurus viverrinus), Tasmanian devil (Sarcophilus harrisii) and dusky antechinus (Antechinus swainsonii; Cowan et al. 1974; Green and

Eberhard 1979). These studies reported high apparent digestibility for dry matter and energy for diets comprising mice (Mus musculus) and rats (Rattus rattus).

Whilst aspects of digestibility have been studied in a few species of dasyurid, mineral absorption and daily mineral requirements remain unknown as well as how their ability to process nutrients (such as energy, protein and lipids) compares with eutherian mammals on similar diets. Several studies have shown that insectivorous shrews (family: Soricidae and Macroscelididae) and bats (Plecotus auritus and Myotis daubentoni) have high digestive efficiencies (Hanski 1984; Woodall and Currie 1989;

Genoud and Vogel 1990; Webb et al. 1993). Maintenance energy requirements of shrews (Soricidae spp.) increase with increasing body mass and up to 300% during lactation (depending on litter size and species; Genoud and Vogel 1990). Studies of mineral absorption in small insectivores is lacking, whilst larger more carnivorous species have been studied, information on mineral absorption in carnivores is limited

(Powers et al. 1989; Childs-Stanford and Angel 2006).

118

The red-tailed phascogale (Phascogale calura) is primarily an insectivorous marsupial in the dasyurid family (Kitchener 1981; Stannard et al. 2010). They inhabit a small area of south-west Western Australia, having undergone a decline in geographical range due largely to human induced pressures (Bradley et al. 2008). Red-tailed phascogales are displayed in zoos, and used for captive breeding and translocation programs to aid reintroduction into former habitats, increasing their current range (W.

Foster pers. comm.).

The kultarr (Antechinomys laniger) is a small dasyurid marsupial, fawn in colour with a white belly (Valente 2008). The kultarr inhabits central regions of Australia; however population densities are believed to be low (Valente 2008). The arid areas kultarrs inhabit have temporal fluctuation of temperature and food availability

(influenced by rainfall; Fisher and Dickman 1993). The phascogale however, inhabits areas of forest with an average annual rainfall between 300 and 600 mm (Bradley et al. 2008). Owing to the higher rainfall in the phascogales’ natural habitat food availability would not fluctuate as dramatically as it would in the kultarrs’ habitat and physiologically it is possible the kultarr would have differing energy requirements in order to cope with fluctuating food availability in the arid environment. On a physiological aspect the kultarr also has specialised elongated hind limbs, and is the only dasyurid to have this specialised feature. It is possible the elongated hind limbs are adaptations that help this species conserve energy whilst moving across long distances (see Baudinette et al. 1976a, 1976b).

Currently, the red-tailed phascogale and kultarr are bred in captivity for translocation, education and research. In the long term an increased knowledge of phascogale and

119 kultarr nutrition will ensure their health, prevent disease and optimise reproductive success in the captive environment (Allen and Oftedal 1996). The aim of this study was to determine the apparent digestibility of current food items of the red-tailed phascogale and kultarr in captivity. This research will assist in understanding the requirements of maintaining dasyurid species in captivity and provides a model for other small dasyurids commonly held in captivity.

4.3 Materials and methods

4.3.1 Animals

Nine adult red-tailed phascogales (8 males and 1 female) and eight adult kultarrs (3 male and 5 female) from captive colonies held at the University of Western Sydney

(UWS), Richmond NSW, Australia, were used for this experiment. The animals were housed individually in wooden enclosures (60 x 60 x 60cm), with a paper substrate and a wooden nest box, under natural photocycles of spring and summer. Room temperature was maintained at 22 ± 3°C and water was available ad libitum. The study was undertaken with the approval of the UWS Animal Care and Ethics

Committee A7045 and animals held under a National Parks and Wildlife Service license S12302.

4.3.2 Digestibility experiments

During the digestibility experiments the animals were fed (ad lib.) one food item at

1400hrs daily for a total of 12 days. The first five days of the experiment were an adjustment period; during the last seven days all faecal matter, refused food and a sample of food was collected daily, weighed (±0.1g) and stored at -20°C until

120 analysis. Animals were weighed (±1g) at the start and end of the experiment. There was a four week rest period between each experiment to allow animals to return to their usual body mass, and during this time their diet consisted of a mix of the experimental diets. The experimental diets were comprised of adult crickets (Acheta domesticus), Australian wood cockroaches (Panesthia australis), mealworm larvae

(Tenebrio molitor), small rat (Rattus rattus; ~30g) and Wombaroo small carnivore food (Wombaroo Food Products). These diet items form part of the normal diets fed to the animals in captivity. Wombaroo is a manufactured diet for small carnivores.

The Wombaroo experiment was abandoned for the phascogales as they only consumed small quantities of the diet. The rats (rat A) fed to the red-tailed phascogales were from a feeding colony bred at UWS. The rats were euthanised with carbon dioxide and then frozen at -80°C to reduce the possibility of toxoplasmosis

(Attwood and Woolley 1970). The rats (rat B) fed to the kultarrs were purchased from

DoLittle Farm and frozen. Prior to feeding, the rats were thawed and cut into 1cm sections.

4.3.3 Nutritional analysis

Food samples, faecal matter and leftover food were oven dried at 105°C to a constant mass to determine dry matter mass, according to Cowan et al. (1974) and Moyle et al.

(1995). All samples were then ground to make a homogenous mixture for further analysis. Gross energy was determined by compressing dried samples into pellets

(~0.2g) for analysis in an Oxygen Bomb Calorimeter (Parr 6200) with a benzoic acid standard (Cowan et al. 1974). Nitrogen content was determined using the Kjeldahl method with a selenium catalyst and crude protein calculated as nitrogen x 6.25

(Willits et al. 1949; Jones 1991). Minerals (calcium, phosphorus, sodium, potassium

121 and iron) were analysed using radial inductively coupled plasma atomic emission spectrometry (ICPAES) at the Waite Analytical Services Laboratory. Total lipids were extracted with a chloroform/methanol (1:1 v/v) mixture, the aqueous layer containing lipids was removed, freeze dried and reweighed to calculate lipid content

(Folch et al. 1957).

Samples were analysed in duplicate for each analysis and a 5% precision value was observed. Apparent digestibility (AD) was calculated by subtracting the nutrient excreted from the nutrient intake and expressed as a percentage (AD = Nin-Nout/Nin x

100; Robbins 1993). To allow for comparison between species of differing body mass, intake data were scaled as body mass in kilograms to the power of 0.75 (M 0.75).

Scaling the data enables comparisons to be made with previous data for marsupials

(see Green and Eberhard 1979; Moyle et al. 1995; Hume 1999; Gibson and Hume

2000). Statistical comparisons for scaled intake and digestibility values for the phascogale and kultarrs on the same diet were compared using independent t-tests in

SPSS. Prior to analysis, percentage data was arcsine transformed to conform to normality.

4.4 Results

The nutritional composition of the diets is shown in Table 4.1. The diet had the lowest energy and lipid content and the highest protein content of the diets fed to the animals. Both rat diets had a similar nutritional composition. Generally mealworm larvae had the lowest mineral composition of the diets provided. The varied composition of the insect diets would relate to the diet consumed by the insects on the

122 days prior to analysis and results show nutrient proportions are similar to previous studies (Barker et al. 1998; Finke 2002).

123

Table 4.1 Composition of food items (mean ± s.d.)

Crickets Cockroaches Mealworm larvae Rat A Rat B Wombaroo

Acheta domesticus Panesthia australis Tenebrio molitor Rattus rattus Rattus rattus

Dry matter (%) 28.9 ± 1.0 33.9 ± 0.4 42.6 ± 0.5 28.4 ± 0.2 28.8 ± 0.1 54.1 ± 0.9

Gross energy (kJ/g) 15.8 ± 0.4 21.5 ± 0.6 24.5 ± 0.4 25.3 ± 0.1 24.9 ± 0 20.5 ± 0.6

Crude protein (%) 63.5 ± 0.9 59.1 ± 0.5 45.9 ± 0.8 52.4 ± 0.1 52.3 ± 1.3 38.7 ± 0.7

Lipids (%) 9.3 ± 0.1 22.3 ± 0.3 27.0 ± 1.6 38.1 ± 0.4 30.9 ± 0.8 13.3 ± 0

Calcium (mg/kg) 2000 2000 340 21000 20000 17800

Phosphorus (mg/kg) 11000 8000 6600 18100 18000 16000

Iron (mg/kg) 56 81 61 98 132 340

Sodium (mg/kg) 5200 4900 1480 5500 5100 9500

Potassium (mg/kg) 14400 11400 8300 10100 10400 11000

Magnesium (mg/kg) 1630 1400 1800 990 1210 1590

124

The red-tailed phascogales consumed between 18-32% of their body weight on a wet matter basis with the lowest consumption rate for the mealworm diet (18.2 ± 2.9%) and the highest for the cricket diet (31.8 ± 6.0%). The rat and mealworm larvae diets had the highest refusal rate with an average of 112.2 ± 11.3g and 31.4 ± 16.4g

(respectively) of food refused per week on a wet matter basis. The majority of the refused rat diet consisted of rat heads, hind quarters, skin and fur. Phascogales on average lost 12-13% of their body weight during the cricket and trials.

There was an average weight gain of 5.3% on the rat diet and weights remained consistent on the mealworm larvae trial.

The kultarrs consumed between 17-39% of their body mass in food per day on a wet matter basis with the lowest consumption rate for the mealworm diet (19.8 ± 2.0%) and highest consumption rate on the rat diet (38.8 ± 5.4%). The majority of the refused rat diet consisted of the same body parts as those refused by the phascogales, although the kultarrs appeared to have a preference for the brain which they consumed throughout the trial period. Kultarrs on average lost 10-11% of body weight on the

Wombaroo and mealworm larvae diets and gained 11% on the rat trial. Body weights remained relatively consistent during the other trials. In comparison, there was no significant difference (cricket t14 =2.131 P=0.05; cockroach t15 =1.423; P =0.175;

Mealworm t16 =-1.801; P =0.09; Rat t14 =1.002; P =0.33) between weight lost or gained (when scaled for body mass) between the kultarrs and phascogales whilst maintained on the same diet.

There was no significant difference of species on the scaled intake values for dry matter, energy, protein and lipids, with the exception of the cricket diet. The

125 phascogales had significantly higher scaled intake values for the cricket diet compared with the kultarr (dry matter t14 =-6.514; P<0.01, energy t14 =-4.372; P<0.05, protein t14 =-5.478; P<0.01, lipid t14 =-6.514; P<0.01). The range of digestible energy intake of the phascogale was 461-1033 kJ kg0.75 d-1 and the kultarr 617-883 kJ kg0.75 d-1.

Apparent digestibility values for the both the phascogale and kultarr were above 81% for dry matter, energy, protein and lipids (Table 4.2, 4.3). Both species showed large variations in mineral absorption values. There was no significant difference in apparent absorption of dry matter, energy, protein and lipid values between the species when provided the same diet, with the exception of apparent digestible energy on the cockroach diet, phascogales showed a significantly higher (t15 =-2.722;

P<0.05) value than the kultarrs.

The red-tailed phascogale showed the highest absorption values for calcium, magnesium, sodium, potassium and phosphorus on the cricket diet compared with the other diets (Table 4.2). Whilst on the cricket diet the kultarrs had higher absorption values for magnesium, sodium and potassium compared with the other diets (Table

4.3). Calcium absorption was low on the mealworm diet for both the phascogale (15.6

± 8.1%) and kultarr (8.3 ± 4.4%).

126

Table 4.2 Body mass, intake and apparent digestibility (AD) of red-tailed phascogale diets (mean ± s.d.)

Cricket Cockroach Mealworm Rat A

N = 8 n = 9 n = 9 n = 8

Body mass

Initial (g) 53.6 ± 7.9 57.6 ± 13.6 57.8 ± 10.2 57.2 ± 6.1

Final (g) 47.8 ± 9.2 51.1 ± 11 57.8 ± 11 60.9 ± 7.9

Dry matter

Intake (g d-1) 9.0 ± 0 6.8 ± 0.1 5.2 ± 1.2 2.9 ± 0.7

Intake (g kg 0.75 d-1) 89.7 ± 11.5 65 ± 9.1 44.2 ± 7.2 24 ± 5.6

AD (%) 90 ± 3 90 ± 1 91.4 ± 1.2 81.5 ± 5.2

Energy

Intake (kJ kg 0.75 d-1) 1225.8 ± 171.3 1280.8 ± 173.5 1019.8 ± 169.6 537.7 ± 130.7

AD of GE (%) 86.4 ± 4.6 91.6 ± 0.7 93.9 ± 0.9 88.7 ± 2.4

DE (kJ kg 0.75 d-1) 1032.7 ± 191.6 1162.3 ± 150.4 954.2 ± 164 461.2 ± 129.7

ADE (%) 83.9 ± 6.4 90.8 ± 0.9 93.5 ± 1.0 85 ± 5.6

Protein

127

Intake (g d-1) 5.6 ± 0.3 4 ± 0.1 2.4 ± 0.5 1.7 ± 0.3

Intake (kJ kg 0.75 d-1) 56.1 ± 8.4 38.4 ± 5.4 20.3 ± 3.3 13.9 ± 2.8

AD (%) 90.6 ± 2.9 88.8 ± 1.2 90.8 ± 1.5 83.1 ± 5.6

Lipids

Intake (mg d-1) 835.4 ± 4.3 1520.1 ± 22.4 1407.4 ± 312.6 1112.4 ± 253.6

Intake (g kg 0.75 d-1) 8.3 ± 1.1 14.5 ± 2.0 11.9 ± 1.9 9.1 ± 2.1

AD (%) 91.6 ± 3.0 97.1 ± 0.8 98.2 ± 0.3 96.7 ± 1.0

Fe AD (%) 43.7 ± 8.5 50.1 ± 7.5 33.4 ± 8.3 51.5 ± 5

Ca AD (%) 77.5 ± 7.3 75.7 ± 4.0 15.6 ± 8.1 50.4 ± 8.7

Mg AD (%) 80.1 ± 5.8 71.1 ± 3.7 41.5 ± 8.9 48.2 ± 5.7

Na AD (%) 90.2 ± 4.5 88.5 ± 2.3 88.1 ± 5.8 80.3 ± 7.3

K AD (%) 92.8 ± 3.7 90.3 ± 1.7 89.4 ± 3.8 79.9 ± 8.5

P AD (%) 91.9 ± 3.5 88.8 ± 1.3 85.1 ± 4.8 69.5 ± 5

128

Table 4.3 Body mass, intake and apparent digestibility (AD) of kultarr diets (n = 8; mean ± s.d.)

Crickets Cockroaches Mealworms Rat B Wombaroo

Body mass

Initial (g) 23.5 ± 4.2 25.3 ± 2.7 33.3 ± 5.2 28 ± 3.4 32.3 ± 2.5

Final (g) 23.3 ± 2.6 24.5 ± 3.7 30.4 ± 3.0 31.4 ± 3.0 29 ± 2.6

Dry matter

Intake (g d-1) 3.6 ± 0.0 2.7 ± 0.1 3.0 ± 0.0 3.2 ± 0.4 3.0 ± 0.2

Intake (g kg 0.75 d-1) 60.6 ± 5.3 44.8 ± 5.5 41 ± 3.1 43.8 ± 7 43.1 ± 4.9

AD (%) 89.0 ± 2.2 89.0 ± 2.3 91.8 ± 0.9 84.3 ± 3.4 91.0 ±9.8

Energy

Intake (kJ kg 0.75 d-1) 826.9 ± 93.0 865.4 ± 115.9 944.1 ± 76.0 973.7 ± 134.5 740.4 ± 120.9

AD of GE (%) 86.1 ± 2.8 89.6 ± 2.0 93.9 ± 0.6 89.5 ± 2.9 85.3 ± 4.8

DE intake (kJ kg 0.75 d-1) 695.3 ± 104.6 766.1 ± 115.6 882.7 ± 75.7 853 ± 100 616.5 ± 146.5

ADE (%) 83.8 ± 3.8 88.4 ± 2.5 93.5 ± 0.7 87.9 ± 3.7 82.4 ± 6.6

Protein

Intake (g d-1) 2.3 ± 0.0 1.6 ± 0.0 1.4 ± 0.0 1.7 ± 0.2 1.1 ± 0.1

Intake (g kg 0.75 d-1) 38.5 ± 3.4 27.3 ± 2.7 23.0 ± 2.0 28.3 ± 4.7 19.2 ± 2.5

129

AD (%) 90.8 ± 1.8 89.8 ± 2.6 91.2 ± 1.4 86.4 ± 3.7 86.0 ± 4.3

Lipids

Intake (mg d-1) 332.6 ± 2.7 616.8 ± 10.5 800.4 ± 2.8 1090 ± 123.4 398.9 ± 34.4

Intake (g kg 0.75 d-1) 5.6 ± 0.5 10.1 ± 1.2 11.1 ± 0.8 14.7 ± 2.1 5.7 ± 0.7

AD (%) 92.0 ± 3.9 97.2 ± 0.7 98.1 ± 0.8 94.9 ± 2.2 95.7 ± 0.8

Fe AD (%) 47.8 ± 5.5 52.6 ± 11.3 51.3 ± 6.7 59.5 ± 9.2 63.3 ± 16.3

Ca AD (%) 65.4 ± 8.6 75.7 ± 5.9 8.3 ± 4.4 29.0 ± 11.3 44.8 ± 18.2

Mg AD% 72.1 ± 5.2 71.0 ± 7.6 55.6 ± 9.0 53.4 ± 8.7 47.8 ± 18.2

Na AD% 90.0 ± 2.1 91.0 ± 2.5 89.8 ± 2.3 84.3 ± 3.6 88.8 ± 3.3

K AD% 95.8 ± 0.8 95.0 ± 1.6 93.3 ± 1.2 88.0 ± 2.8 91.8 ± 2.4

130

4.5 Discussion

The red-tailed phascogales and kultarrs consumed up to 39% of their body mass in food per day, this is similar to findings for the dusky antechinus (Antechinus swainsonii; 31%) and a small eutherian insectivorous shrew (Elephantulus edwardii;

22-38%; Cowan et al. 1974; Woodall and Currie 1989). Free ranging animals have been found to consume food at a rate almost double that of captive animals; for example free-ranging brown antechinus (Antechinus stuartii) consumed 60%, whilst captive animals consumed 37% of their body mass per day (Nagy et al. 1978). Free- living red-tailed phascogales consumed between 16-60% of their body mass in food per day (calculated from Green et al. 1989). Thus kultarrs and phascogales in this study consumed food (as a percentage of body mass) within the expected range of values for small insectivores on a daily basis (Cowan et al. 1974; Nagy et al. 1978;

Green et al. 1989; Woodall and Currie 1989).

The phascogales lost weight on two of the diets provided (cricket and cockroach); this could be a result of being restricted to one food type. Phascogales do however, fluctuate in weight throughout the year and typically refuse large amounts of food offered to them on a daily basis. Although the animals are housed indoors, seasonal cues from natural light cycles could also influence body weight. The body mass of the kultarrs remained relatively constant during most of the experiment suggesting their requirements were being met. They did lose an average of 11% of their body mass on the Wombaroo and mealworm trials, suggesting these diets were not meeting their requirements even though Wombaroo was fed in excess of the manufacturer’s recommended quantity per day and in excess of consumption. Wombaroo is made from protein and meal products (Wombaroo Food Products 2011). It is in powder

131 form with no indigestible components like skin, hair, bones or exoskeletons which likely contributed to the generally high digestibility values observed in the kultarrs.

The high apparent digestibility values of dry matter, energy, protein and lipids are attributed to the type of diets provided. The soft sections of the insects and muscle and organ tissue of the rat were consumed first and harder parts generally discarded.

These high values can be attributed to the phascogale and kultarr avoiding chitin in the insect exoskeletons and using their sharp teeth to cut the exoskeletons into very small pieces to enable access to the nutrients inside. Animals such as the numbat

(Myrmecobius fasciatus) that are unable to avoid chitinous exoskeletons have lower digestibility values (Cooper and Withers 2004) There was no significant difference between the digestibility of dry matter, protein or lipids between the two species whilst maintained on the same diet, suggesting both species have similar digestive efficiency of these nutrients. The digestibility values for dry matter, energy, crude protein and lipids observed in the phascogale and kultarr were similar to other dasyurids (Table 4.4; Cowan et al. 1974; Green and Eberhard 1979).

Limited information is available for diet digestibility in small insectivores. When compared to eutherian insectivores the kultarr and red-tailed phascogale show similar digestibility of nutrients (Table 4.4). Shrews do however, show lower digestibility values for diets high in chitin (Table 4.4; Hanski 1984; Woodall and Currie

1989).

132

Table 4.4 Apparent digestibility values of various diets by small insectivores

Species Diet Dry matter ADE (%) Protein AD Lipids AD Source

AD (%) (%) (%)

Kultarr Insects, rat, Wombaroo 84-92 82-94 86-91 92-98 This study

Antechinomys laniger

Red-tailed phascogale Insects, rat 82-91 84-94 83-91 92-98 This study

Phascogale calura

Dusky antechinus Mouse 80 - 74 92 Cowan et al. 1974

Antechinus swainsonii

Eastern quoll Rat 81 - - - Green and

Dasyurus viverrinus Eberhard 1979

Tasmanian devil Rat 79 - - - Green and

Sarcophilus harrisii Eberhard 1979

Shrews Cockroaches, ant pupae, 70-85 - - - Hanski 1984

Soricidae spp. sawfly cocoons

133

Beetles, 31-60 - - - Woodall and

Currie 1989

- No value available

134

The maintenance energy requirement (MER) in captivity for the phascogale is 954 ±

164 kJ kg0.75 d-1 and for the kultarr 695 ± 105 kJ kg0.75 d-1. Interestingly both species lost weight on diets that provided a higher digestible energy intake. It is possible factors such as age and/or seasonal cues influenced weight loss during the experiment.

In comparison to other dasyurid species, the Tasmanian devil and eastern quoll (545 kJ kg0.75 d-1), both the phascogale and kultarr have a higher MER (Green and

Eberhard 1979). The higher energy requirement of these smaller species is expected as generally MER is reduced with increased body size in marsupials (Hume 1999).

The dusky antechinus, a dasyurid with a body mass (49g) between that of the kultarr and phascogale has a MER of 933 kJ kg0.75 d-1 higher than that of the smaller kultarr and only slightly lower than the larger phascogale (Cowan et al. 1974).

Kultarrs had a lower MER than the phascogale, possibly reflecting their naturally arid distribution, characterised by variable food availability. The kultarr is a desert dwelling species and has adapted to survive in an environment where food availability is influenced by adverse weather conditions and climate (Stafford Smith and Morton

1990). Physiologically kultarrs use torpor to conserve energy in times of low temperature and food availability (Geiser 1986). Using torpor during the nutrition trials would have reduced the kultarrs’ energy requirements on a daily basis. Further work is required to determine how kultarrs have a low MER and the role of torpor may play in MER. The phascogales are a much more active species than the kultarrs and spend their time climbing branches and leaping around their enclosures, possibly accounting for the higher MER.

135 The (BMR) of the kultarr is 195 kJ kg0.75 d-1 (MacMillen and

Nelson 1969), and generally MER is 150-250% of the basal metabolic rate (Hume

1999). Using this calculation it would be expected the kultarr’s MER would be between 293-488 kJ kg0.75 d-1. In this study MER was determined to be around 356% of BMR which is higher than the predicted value. As only limited data is available for

BMR of dasyurids it is difficult to suggest why this value is higher than expected.

Field metabolic rate (FMR) of the red-tailed phascogale is 759-1378 kJ kg0.75 d-1 depending on season (Green et al. 1989). Generally the range of MER found in this study fall within the range of FMR of free-living animals. Similarities between MER and FMR suggest similar energy requirements for activity and maintenance in captivity versus the wild. When maintained on the rat diet, the phascogales MER was much lower than FMR, likely due to the low food intake on this diet and numerous indigestible components.

MER of the phascogale and kultarr are higher than those determined for small shrews

(body mass 9.5-16.2g) ranging from 252-371 kJ kg0.75 d-1. The African giant shrew

(Crocidura olivieri), slightly larger than the kultarr has a slightly higher MER of 721 kJ kg0.75 d-1 (Genoud and Vogel 1990). MER determined for the phascogale and kultarr is higher than that determined for omnivorous marsupials, the bilby (Macrotis lagotis) 629 kJ kg0.75 d-1, long-nosed bandicoot (Perameles nasuta) 511 kJ kg0.75 d-1 and long-nosed potoroo (Potorous tridactylus) 494 kJ kg0.75 d-1 (Wallis and Farrell

1992; Moyle et al. 1995; Gibson and Hume 2000), which is consistent with requirements determined for the other dasyurids mentioned previously.

136 The absorption of minerals was very varied in this study and differences were shown between species and diet type. Apparent digestibility of minerals reflects demand and availability in the diet (Barboza et al. 2009). Mineral requirements of dasyurids are largely unknown, although the sodium turnovers of Tasmanian devils and eastern quolls are relatively high (Green and Eberhard 1979). Further work is required to understand mineral requirements in dasyurids.

Presented in this paper is the most comprehensive data for nutrition (nutrient intake and digestibility) for any dasyurid. It has shown the red-tailed phascogale and kultarr can efficiently digest a range of foods, which is related to their simple digestive tract, type of food available and species adaptation to habitat. The results have provided further understanding of these animals’ requirements in captivity which will contribute to their maintenance and use of these species in captive breeding and translocation programs.

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budgets in free-living Antechinus stuartii (Marsupialia:Dasyuridae). Journal of

Mammalogy 59, 60-68.

Powers, J.G., Mautz W.W., and Pekins, P.J. (1989). Nutrient and energy assimilation

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140 Read, D.G. (1987). Rate of food passage in Planigale spp. (Marsupialia:

Dasyuridae).Australian Mammalogy 10, 27-28.

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Australia. Journal of Arid Environments 18, 255–278.

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(Phascogale calura) in a trial translocation at Alice Springs Desert Park,

Northern Territory, Australia. Journal of Zoology 280, 326-331.

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Holland: Sydney.)

Wallis, I.R., and Farrell, D.J. (1992). Energy metabolism in potoroine marsupials.

Journal of Comparative Physiology B 162, 478-487.

Webb, P.I., Speakman J.R., and Racey, P.A. (1993). Defecation, apparent absorption

efficiency, and the importance of water obtained in the food for water balance in

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daubentoni) bats. Journal of Zoology 230, 619-628.

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141 Woodall, P.F., and Currie, G.J. (1989). Food consumption, assimilation and rate of

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92, 75-79.

142 CHAPTER 5

The role of dietary composition in optimum nutrition for the

dunnarts Sminthopsis macroura and S. crassicaudata

H. J. Stannard1, B. M. McAllan2 & J. M. Old1

1Native and Pest Animal Unit, School of Natural Sciences, University of Western Sydney, NSW,

Australia

2Department of Physiology and Bosch Institute, University of Sydney, NSW, Australia

Under review in Laboratory Animals

143 5.1 Chapter outline and authorship

Chapter 5 focused on nutrition of two species of dunnart. The study determined the apparent digestibility of current captive diet items and live insects. In addition, the study provides morphology and dimensions of the gastrointestinal tract of both species. General morphology has been described previously in small dasyurids; however, no data were available for dimensions of the tracts. The study contributes further information on nutrition of dasyurids and is an extension of the work that was undertaken in Chapter 4. The study was approved by the University of Western

Sydney’s Animal Care and Ethics Committee A7982 and the University of Sydney’s

Animal Ethics Committee K22/11-2010/3/5444.

The following manuscript is jointly authored; I am the primary and corresponding author. I conducted the digestibility trials and collected all the samples during the trials. I performed nutrient analysis of scats and food, calculation of nutritional content and statistical analysis of data, and wrote the manuscript. Mineral analysis was conducted by Waite Analytical Services, South Australia. Dr Julie Old and Dr

Bronwyn McAllan supervised the development of the work and provided editorial feedback on previous versions of the manuscript. Dr Bronwyn McAllan supplied the dunnarts for the trials from her captive colony at the University of Sydney.

144 5.2 Introduction

Australia is a geomorphologically ancient continent, and it is well recognised that the soil is nutrient-impoverished.1 Despite this nutrient deficiency, the continent is home to a wide range of plants, vertebrates, and a significant diversity of invertebrates.1,2

The bulk of the world’s marsupials live in Australia, and there are 53 species of carnivorous marsupials, from the family Dasyuridae, and almost half of the

Dasyuridae species live in the arid zone.3 The stripe-faced dunnart (Sminthopsis macroura) is a small (20-27 g) insectivorous marsupial, which inhabits arid zones of central and northern Australia.4 In contrast, the fat-tailed dunnart (Sminthopsis crassicaudata) is a smaller (13-20 g) insectivorous marsupial, but inhabits more diverse habitats, including mesic environments in southern and central Australia.5,6

Both species prey on arthropods, small mammals and small reptiles. However, in captivity their diet mostly consists of cat formulations, dry dog food and other commercially available pet foods.6,7,8,9 Both species store fat in their tails, and are capable of using torpor, which is a temporary, controlled reduction in metabolic rate and associated drop in body temperature.10,11,12

Both species of dunnart are listed as ‘least concern’ on the IUCN red list of threatened species.13,14 The stripe-faced dunnart however, is listed as ‘vulnerable’ in New South

Wales under the Threatened Species Act 1995. The status of the stripe-faced dunnart is considered vulnerable due to localised population declines within the state of New

South Wales.15 There are a number of other dunnart species which are in decline and are nationally listed as vulnerable or endangered such as the Kangaroo Island dunnart

(S. aitkeni), Butler’s dunnart (S. butleri), and Julia Creek dunnart (S. douglasi).16,17,18

These dunnart species are threatened by introduced predators, fires, agricultural land

145 use, human encroachment and low population densities.16,17,18 The stripe-faced and fat-tailed dunnarts provide suitable models for other endangered dunnart species because of their overlap in body size, habitat use and dietary preferences (when known). The stripe-faced dunnart and fat-tailed dunnart are readily maintained in captive environments, and are frequently held for display, research and education purposes.

Nutrition has been studied more extensively in the fat-tailed dunnart than the stripe- faced dunnart. Previous research on the fat-tailed dunnart has shown food intake is influenced by opioid peptides, leptin, gender, photoperiod, and macronutrient composition of food.19,20,21,22 The role of food availability on torpor and activity rhythms, and nutrient uptake by embryos has been studied in the stripe-faced dunnart.9,23,24,25,26 To our knowledge no studies have previously looked at nutritional composition of diets in relation to nutritional requirements for maintenance of these species in captivity.

Uptake and digestion of nutrients is related to choice of diet, and gastrointestinal tract morphology and histology. Generally, dasyurids have a simple gastrointestinal tract that lacks a caecum.27 Gastrointestinal morphology has been described in larger dasyurids species such as spotted-tailed quoll (Dasyurus maculatus), eastern quoll (D. viverrinus), kowari (Dasyuroides byrnei), brush-tailed phascogale (Phascogale tapoatafa), kultarr (Antechinomys laniger) and fat-tailed

(Pseudantechinus macdonnellensis).28,29,30 Previously, descriptions of the dunnart gastrointestinal tract have been limited to gross morphology and gut-associated lymphoid tissues.31,32,33,34 Gross morphology and dimensions were described for the

146 largest species in the Sminthopsis genus, the Julia Creek dunnart.35 All these studies, however, lack detailed morphological and dietary measurements of the gastrointestinal tract.

Because of the paucity of knowledge about gut morphology and appropriate diet intake in dunnarts, we aimed to determine the apparent digestibility of a current captive diet, commercially available diet, and live food items that are similar to those available in the wild for both the fat-tailed and stripe-faced dunnarts. In addition, the study provides morphology and dimensions of the gastrointestinal tract of both species.

5.3 Animals, materials and methods

5.3.1 Animals

Eleven adult male stripe-faced dunnarts and eight adult male fat-tailed dunnarts were used for this study. Males were used because a previous study demonstrated that they robustly adapt physiologically and behaviourally to variable amounts of food.9 The animals were from a captive colony based at the University of Sydney, Sydney, NSW,

Australia. Each animal was housed individually in a plastic enclosure (20 x 45 x 25 cm) with a mesh lid. Animals were provided with a wood shaving substrate, cardboard nest box and a toy (plastic ladder or swing) for behavioural enrichment.

Animals were held under photocycles of 12L:12D and room temperature was maintained at 22 ± 5°C. Water was available ad libitum, and fresh food, in excess to daily requirements, was provided in the late afternoon, before the animal became active. This study was undertaken with the approval of the University of Western

147 Sydney’s Animal Care and Ethics Committee A7982 and the University of Sydney’s

Animal Ethics Committee K22/11-2010/3/5444.

5.3.2 Digestibility trials: diets

On day one animals were moved into the experiment room and allowed to adjust to the room for five days prior to starting the nutrition trials. The food items for each of the five trials were given in the order as follows: 1) cat formulation – lamb and kidney loaf style (Whiskas, Mars Petcare, VIC), 2) adult crickets (Acheta domesticus), 3)

Australian wood cockroaches (Panesthia australis), 4) mealworm larvae (Tenebrio molitor) and 5) Wombaroo small carnivore mix (Wombaroo Food Products, Glen

Osmond, SA). Wombaroo – marsupial formulation is a commercial diet for small carnivorous marsupials.36 A two-week rest period was provided between trials, and during this time animals were maintained on their usual cat formulation diet

(Whiskas, lamb and kidney loaf style). Each of these dietary items was chosen for the trials because they are commonly used in captive diets for dunnart species.

5.3.3 Digestibility trials: procedure

The digestibility trials investigated five dietary protocols; each trial was spaced by a two-week rest period, where animals were exposed to their usual cat formulation diet

(Whiskas, lamb and kidney loaf style). The order was chosen as usual food fed first, followed by insects, which were readily accepted, whilst Marsupial formulation was left to last, as some dasyurids, such as the captive red-tailed phascogale (Phascogale calura) have refused to eat this diet.30 During the digestibility trials the animals were fed one food type at 1500 hrs daily for a total of 10 days. The first five days of the

148 trial were an adjustment period from the previous dietary regime; and during the last five days all faecal matter, uneaten food and a sample of food was collected daily, weighed (±0.1 g) and stored at -20°C until analysis. During the trials animals were provided with a paper substrate which absorbed urine and allowed for the easy collection of faeces directly from the paper. Animals were weighed (±0.1 g) and tail width (mm) recorded at the start and end of each of the food trials. Tail width was taken at the base of the tail using vernier callipers. Tail width was recorded as an indicator of fat storage and general health of the animal.

Dunnarts were weighed twice during the two-week rest periods to determine if they had returned to their pre-trial body mass. On completion of the nutrition trials eight stripe-faced dunnarts and eight fat-tailed dunnarts were euthanased, organs were removed and gastrointestinal lengths measured (± 0.1 mm). On the day prior to euthanasia animals were fed cat formulation. As euthanasia of animals occurred before their usual feeding time, they were not fed on the day of euthanasia, and all stomachs were empty prior to measurement. Measurements of gastrointestinal tracts from dunnarts (four of each species) were also obtained opportunistically from animals euthanased for another independent experiment where the diet of the dunnarts was the same as their usual cat formulation diet, and therefore should have no impact on gastrointestinal dimensions. The euthanased individuals were healthy.

5.3.4 Sample analysis

Food samples, faecal matter and uneaten food were analysed using the same methods described in Stannard and Old37. Samples were analysed for dry matter, protein, lipids, and minerals Fe, Ca, P, Na, K and Mg. Mineral analysis was conducted by

149 Waite Analytical services, Glen Osmond, SA. Samples were analysed in duplicate for each analysis and a 5% precision value was observed. Apparent digestibility (AD) was calculated by subtracting the nutrient excreted from the nutrient intake, and

38 expressing it as a percentage of intake (AD = ((Nin-Nout)/(Nin)) x 100). Intake data were normalised to metabolic body mass; body mass as kg to the power of 0.75 (M -

0.75) to compare species of differing body mass. The scale of 0.75 has been used previously to scale energy intake data for marsupials.27,39,40,41

5.3.5 Statistical analysis

A one way-ANOVA and Tukey post hoc tests were used to compare nutrient composition of the five diets. A repeated measures ANOVA and Least Significant

Difference post hoc tests were used to examine changes in mass and tail widths within each species. One-way ANOVAs were used to compare apparent digestibility of each diet within each species. Percentage data were arcsine transformed for normality before analyses were performed. Unpaired Students t-tests were used to compare scaled intake and digestibility values between the stripe-faced and fat-tailed dunnarts on the same diet and at specific time-points.

5.4 Results

5.4.1 Dietary analysis

Composition of the diets varied considerably (Table 5.1); cat formulation had the lowest dry matter (F4, 5 = 1436.9; P=0.001) and highest energy (F4, 5 = 138.4; P=0.001) of the diets provided. The insect diets all had significantly higher crude protein than both the cat formulation and marsupial formulation diets (F4, 5 = 1003.9; P=0.001; Cat

150 formulation P<0.01 marsupial formulation P<0.01), and although lipid content varied between the insect diets, all had significantly less lipid content than the cat formulation (F4, 5= 2864.5; P=0.001). Mineral and salt ion content varied considerably between diets, although these were more consistent for the insect diets than for the commercial diets (Table 5.1; Ca F4, 5 = 25460.2; P=0.001; P F4, 5 = 28290.0; P=0.001;

Fe F4, 5 = 76744.0; P=0.001; Na F4, 5 = 7092.183; P=0.001; K F4, 5 = 3253.5; P=0.001).

Marsupial formulation has the highest levels of dry matter, Ca, P, Fe and Na. The crickets had the lowest energy and highest protein composition of the diets provided.

151 Table 5.1 Composition of food items on a dry matter basis (n = 2) All data are means ± 1 standard deviation. *P<0.01

% Dry Matter

Crickets Cockroaches Mealworm larvae Cat food Wombaroo

Acheta domesticus Panesthia australis Tenebrio molitor

Dry matter (%) 28.9 ± 1.0 33.9 ± 0.4 42.6 ± 0.5 14.4 ± 0.4* 54.1 ± 0.9

Gross energy (kJ/g) 15.8 ± 0.4 21.5 ± 0.6 24.5 ± 0.4 27.1 ± 0.6* 20.5 ± 0.6

Crude protein (%) 63.5 ± 0.9 59.1 ± 0.5 45.9 ± 0.8 38.7 ± 0.7* 37.7 ± 0.2*

Lipids (%) 9.3 ± 0.1 22.3 ± 0.3 20.0 ± 0.1 51.3 ± 1.0* 13.3 ± 0

Calcium (mg/kg) 2000 2000 340* 8200* 17800*

Phosphorus (mg/kg) 11000 8000* 6600* 12700 16000

Iron (mg/kg) 56 81 61 220* 340*

Sodium (mg/kg) 5200 4900 1480* 8400 9500

Potassium (mg/kg) 14400 11400 8300* 14300 11000

Magnesium (mg/kg) 1630 1400 1800 640* 1590

152 5.4.2 Body condition

There was no significant difference in body mass measurements for the stripe-faced dunnart between the pre-trial and start measurements for each trial (Figure1a). The body mass of the stripe-faced dunnart varied across the course of the feeding trials

(F16, 10 = 8.9; P<0.01; Figure 5.1a). Tail widths of the stripe faced dunnart also varied across the course of the feeding trials. There was a significant increase in tail width during the mealworm trial (P<0.01; Figure 5.1b).

The body mass of fat-tailed dunnarts varied across the course of the feeding trials

(Figure 5.2a) and increased significantly (F16, 8 = 6.5; P=0.003) on the mealworm trial

(P<0.05) and decreased significantly on the cat formulation trial (P<0.01). A significant (P<0.05) loss of tail width occurred during the cockroach trial. During the mealworm trial a significant gain (P<0.05) in tail width of the fat-tailed dunnarts occurred (Figure 5.2b).

153 (a)

40.00 b 35.00 a 30.00 a 25.00 20.00 15.00 Weight (g) Weight 10.00 5.00 0.00

Rest Rest Rest Rest Rest Rest Pre-trial Cricket startCricket end Cat food startCat food end Cockrock start Mealworm end Cockroach end Mealworm start WombarooWombaroo start end Trial period

(b) 9.00 b 8.00 7.00 6.00 5.00 4.00 3.00 Tail (mm) width 2.00 1.00 0.00

Rest Rest Rest Rest Rest Rest Pre-trial Cricket startCricket end Cat food startCat food end Cockrock start Mealworm end Cockroach end Mealworm start WombarooWombaroo start end Trial period

Figure 5.1 Body mass (a) and tail width (b) of S. macroura over the course of the nutrition trials. During rest periods animals were fed cat food. All data are means +/-

1 standard deviation. [a] significant change in body mass/tail width from pre-trial value P<0.05; [b] significant change in body mass/tail width during the course of a feeding trial P<0.05.

154

(a)

20.00 b a 15.00 b a

10.00 Weight (g) Weight 5.00

0.00

Rest Rest Rest Rest Rest Rest Pre-trial Cricket startCricket end Cat food startCat food end Cockrock start Mealworm end Cockroach end Mealworm start WombarooWombaroo start end Trial period

(b) 7.00 b 6.00 a b 5.00 a

4.00

3.00

Tail (mm) width 2.00

1.00

0.00

Rest Rest Rest Rest Rest Rest Pre-trial Cricket startCricket end Cat food startCat food end Cockrock start Mealworm end Cockroach end Mealworm start WombarooWombaroo start end Trial period

Figure 5.2 Body mass (a) and tail width (b) of S. crassicaudata over the course of the nutrition trials. During rest periods animals were fed cat food. All data are means +/-

1 standard deviation. [a] significant change in body mass/tail width from pre-trial value P<0.05; [b] significant change in body mass/tail width during the course of a feeding trial P<0.05.

155 5.4.3 Digestibility trials

The stripe-faced dunnarts consumed the equivalent of 17-50% of their body mass in wet matter per day and on a dry matter basis 9-15%, and % consumption differed significantly between diets, (F4, 11 = 212.9; P< 0.01). The stripe-faced dunnarts consumed a significantly greater percentage of food with respect to body mass when presented with the cat formulation diet (P<0.01). Fat-tailed dunnarts consumed the equivalent of 27-81% of their body mass in wet matter per day and 5-9% on a dry matter basis, and % consumption differed significantly between diets (F4, 8 = 116.5;

P<0.01), with a significantly lower percentage (P<0.01) on the marsupial formulation diet and significantly higher percentage on the cat formulation diet (P<0.01).

The apparent digestibility (AD) of all diets was high for the stripe-faced and fat-tailed dunnarts, who were able to easily digest the diets provided (Table 5.2, 5.3). There were significant differences in AD between diets for the stripe-faced dunnart, with the mealworm diet having significantly higher AD for dry matter (F4, 11 = 18.4; P<0.01), energy (F4, 11 = 13.2; P<0.01) and protein (F4, 11 = 13.6; P<0.01). The cat formulation diet had the highest AD for lipids (F4, 11 = 47.567; P<0.01) for the stripe-faced dunnart. For the fat-tailed dunnart the mealworm diet provided significantly higher

AD for dry matter (F4, 8 = 11.6; P<0.01), energy (F4, 8 = 13.3; P<0.01), protein (F4, 8 =

6.1; P<0.01) and lipids (F4, 8 = 14.2; P<0.01). The stripe-faced and fat-tailed dunnarts, when maintained on the same diet, have similar AD of nutrients. The stripe-faced dunnart however, has significantly higher AD of dry matter than the fat-tailed dunnart when on the marsupial formulation diet (F17, 19 = 6.6; P<0.05; t = 1.609), energy on the mealworm diet (F17, 19 = 5.8; P<0.05; t = 1.873), and lipid on the marsupial formulation diet (F17, 19 = 5.2; P<0.05; t = 1.273).

156 Table 5.2 Body mass, tail widths, food intake and apparent digestibility of the diets (AD) for S. macroura (n = 11). All data are means ± 1 standard deviation.

Crickets Cockroaches Mealworms Cat food Wombaroo

Body mass

Initial (g) 25.7 ± 4.3 26.4 ± 4.4 25.8 ± 4.1 25.7 ± 5.3 26.8 ± 4.7

Final (g) 25.8 ± 4.2 26.7 ± 4.7 30.6 ± 5.6 25.7 ± 4.3 25.5 ± 3.8

Tail width

Initial (mm) 5.9 ±1.1 6.4 ± 1 6.2 ± 1.1 5.9 ± 2.2 6.4 ± 1.2

Final (mm) 6.3 ± 0.8 6.6 ± 1 7 ± 1.3 6 ± 1.1 6.4 ± 1.4

Wet matter intake (g d-1) 7.7 ± 0.9 5.7 ± 0.8 6.8 ± 1.2 12.9 ± 0.2 3.4 ± 0.9

Dry matter

Intake (g d-1) 2.5 ± 0.3 1.8 ± 0.3 2.9 ± 0.5 1.9 ± 0 1.8 ± 0.5

(g kg 0.75 d-1) 39.4 ± 4.9 27.8 ± 5 39.7 ± 4.3 29.4 ± 3.3 28.3 ± 8.2

AD (%) 81.7 ± 0.8 79.4 ± 3.2 88.8 ± 0.8* 83.6 ± 3.3 76 ± 6

Energy

Intake (kJ kg 0.75 d-1) 482.6 ± 77.8 498.1 ± 91.6 895.4 ± 100 689.9 ± 84.1 480.6 ± 170.7

AD of GE (%) 77.2 ± 5 83.1 ± 2.4 91.9 ± 0.6 86.5 ± 2.5 83.3 ± 4.2

157 DE intake (kJ kg 0.75 d-1) 359.1 ± 80.8 396.3 ± 78.3 816.3 ± 95.3* 595.2 ± 87 392.7 ± 180.1

ADE (%) 73.5 ± 8.2 79.5 ± 3.4 91.1 ± 0.7* 86.1 ± 3.5 80.1 ± 8.9

Protein

Intake (g d-1) 1.4 ± 0.2 0.8 ± 0.1 1.2 ± 0.2 0.6 ± 0 0.6 ± 0.2

Intake (g kg 0.75 d-1) 21.4 ± 3.3 12.2 ± 2.6 16 ± 1.8 9.8 ± 1.3 8.8 ± 3.1

AD (%) 84.8 ± 3.9 73.1 ± 6.5 87.5 ± 1.5* 85.9 ± 4.1 83.5 ± 4.4

Lipids

Intake (mg d-1) 194.6 ± 27.3 382.4 ± 65.3 554.5 ± 99.6 917.2 ± 22.6 208.9 ± 68.1

Intake (g kg 0.75 d-1) 3.1 ± 0.5 5.9 ± 1.1 7.6 ± 0.9 14.5 ± 1.7 3.3 ± 1.1

AD (%) 83.2 ± 4.8 94.3 ± 1.9 95.5 ± 0.7 96.1 ± 1.4* 87.6 ± 3.9

Fe AD (%) -57.6 ± 18.8* -44.1 ± 26.5* -38.7 ± 24.4* 25.8 ± 15.7* 64.3 ± 9.1*

Ca AD (%) 20.6 ± 7 53.2 ± 11 -68.2 ± 7.1* 46.6 ± 13.2 26.5 ± 14.8

Mg AD (%) 54.1 ± 8.9 34.7 ± 15.3* 20.4 ± 15.5* 48.7 ± 12 45.5 ± 12.1

Na AD (%) 72.4 ± 4.7 76.6 ± 7.1 87.5 ± 3.5* 74.4 ± 4.1 89.4 ± 2.9*

K AD (%) 89.2 ± 2.3 86.3 ± 5* 90.2 ± 3 85 ± 4.3* 93.1 ± 1.8

P AD (%) 83.7 ± 3.3 83.8 ± 4.2 80.2 ± 5.2 84 ± 5.3 60.4 ± 8.4*

*P<0.01

158 Table 5.3 Body mass, tail widths, food intake and apparent digestibility of the diets (AD) for S. crassicaudata (n = 8). All data are means ± 1 standard deviation.

Crickets Cockroaches Mealworms Cat food Wombaroo

Body mass

Initial (g) 13.4 ± 0.8 14.9 ± 1.2 14.7 ± 1 14.3 ± 1.5 14.9 ± 1.3

Final (g) 14 ± 1.1 14.4 ± 1.4 16.7 ± 1.9 13.4 ± 0.8 15 ± 1.3

Tail width

Initial (mm) 4.2 ± 0.6 4.8 ± 0.7 4 ± 0.3 4.6 ± 0.5 4.6 ± 1

Final (mm) 4.5 ± 0.4 4.2 ± 0.7 4.9 ± 0.8 4.3 ± 0.5 4.6 ± 0.9

Wet matter intake (g d-1) 7.1 ± 0.8 5.5 ± 0.4 5.3 ± 0.7 10.9 ± 0.8 4.1 ± 0.7

Dry matter

Intake (g d-1) 2 ± 0.2 1.7 ± 0.3 2.3 ± 0.3 1.6 ± 0.1 2.9 ± 0.2

(g kg 0.75 d-1) 49.3 ± 4.4 41.6 ± 6.1 48.9 ± 6.3 39.9 ± 4.5 68.1 ± 5.3

AD (%) 78.8 ± 4 81.5 ± 2.2 88.4 ± 1.3* 82.6 ± 3 63.6 ± 18.5

Energy

Intake (kJ kg 0.75 d-1) 742.9 ± 84.3 757.9 ± 113.3 1095.6 ± 145 924.3 ± 80.9 1144.4 ± 137.4

159 AD of GE (%) 95.1 ± 4.5 84.6 ± 1.7 91.2 ± 1 85.5 ± 2.8 82 ± 6.8

DE intake (kJ kg 0.75 d-1) 542.4 ± 84.4 620 ± 98 989.9 ± 136.9 765.4 ± 52.3 894.2 ± 207.1

ADE (%) 72.8 ± 4.3 81.8 ± 2.4 90.3 ± 1.2* 83 ± 3.9 77.3 ± 10.4

Protein

Intake (g d-1) 1± 0.2 0.8 ± 0.2 0.9 ± 0.1 0.5 ± 0 0.9 ± 0.1

Intake (g kg 0.75 d-1) 25.3 ± 3.1 19.5 ± 3.2 19.2 ± 2.5 13.2 ± 1.6 20.1 ± 2.4

AD (%) 80.2 ± 3.4 78.8 ± 2.8 85.5 ± 2.2* 85.4 ± 2.9 78.3 ± 7.2

Lipids

Intake (mg d-1) 163.4 ± 19.1 360.9 ± 62.8 435.5 ± 63.2 763.4 ± 66.8 319.7 ± 48.6

Intake (g kg 0.75 d-1) 4.1 ± 0.5 8.7 ± 1.3 9.4 ± 1.3 19.4 ± 2.3 7.5 ± 1.1

AD (%) 88.5 ± 4.2 93.5 ± 1.6 95.8 ± 1.1* 94.8 ± 1.6 82.1 ± 9.8

Fe AD (%) -45.9 ± 25.2* 7.6 ± 10.9* -5.8 ± 18.6* 24.1 ± 7.4* 54.4 ± 13.7*

Ca AD (%) 29.8 ± 8.7 40.6 ± 9.8 -66.7 ± 24.8* 32.1 ±10.8 30.8 ±14

Mg AD (%) 59.4 ± 8.6 19.8 ± 18.8* 43.8 ± 6.6 39.6 ± 8.8 42 ±15.2

Na AD (%) 72.4 ± 3.2 77.6 ± 6.3 89.4 ± 2.6* 71.1 ± 4.8 81.2 ± 7.4

K AD (%) 88.2 ± 1.2 83.7 ± 5.1* 90.2 ± 2 86.2 ± 3.6 86.2 ± 6.3

P AD (%) 83.1 ± 2.2 80 ± 3.3 84.9 ± 3.9 78.9 ± 4.2 42.8 ± 26.6*

*P<0.01

160 The apparent absorption of minerals was variable for both species, however, there were high values observed for Na, P and K for all diets, compared with the absorption of Ca, Mg and Fe. Apparent absorption of Ca differed between diets for both species, with AD for Ca on the mealworm diet significantly different to the other groups, and was -68% and -67% for the stripe-faced (F4, 11 = 25.5; P<0.01; mealworm P<0.01) and fat-tailed dunnart (F4, 8 = 18.6; P<0.01; mealworm P<0.01), respectively.

Negative AD values were also obtained for Fe for the cricket and mealworm diets for the fat-tailed dunnart, and Fe for the three insect diets fed to the stripe-faced dunnarts.

Digestible energy (DE) intake for the stripe-faced dunnarts ranged from 359.1 ± 80.8 to 816.3 ± 95.3 kJ kg -0.75 d-1 and were significantly greater when fed mealworm and cat formulation, compared to the other insect diets and the marsupial formulation diet

(F4, 11 =33.5, P<0.01; cat formulation P<0.01 mealworm P<0.01). The DE of the mealworm diet was also significantly higher than the cat formulation diet for the stripe-faced dunnart (P<0.01). DE intake for the fat-tailed dunnarts ranged between

542.4 ± 84.4 to 989.9 ± 136.9 kJ kg -0.75 d-1 depending on diet, and were significantly higher for the mealworm and commercial diets compared to the other insect diets (F4, 8

= 13.2, P<0.01; mealworm P<0.01).

The gastrointestinal tract of the stripe-faced and fat-tailed dunnart were similar, both had a unilocular stomach (Figure 5.3). There was no external differentiation between the small and large intestine, which was fairly uniform along the tract (Figure 5.3).

There was no caecum present in either species. The liver had 3-4 lobes, and was a dark reddish colour. The liver was located close to the stomach and the gallbladder was underneath the liver. The Y-shaped spleen was located in the upper left portion of

161 the abdomen proximal to the stomach and was dark purple-red colour. When adjusted for body mass, there were no significant differences in the measurements of both stomach (t22, 24 = -0.652; P=0.22; t22, 24=-0.354; P=0.920) and intestine (t22, 24 = 0.212;

P=0.220) between the stripe-faced and fat-tailed dunnarts (Table 5.4). There was also no significant difference in intestine (stripe-faced dunnart t10, 12 =0.962; P=0.494; fat- tailed dunnart t10, 12 =2.163; P=0.143) and stomach lengths (stripe-faced dunnart t10, 12

=-0.121; P=0.879; t10, 12 =2.239; P=0.781; fat-tailed dunnart t10, 12 =0.763; P=0.672; t10, 12 =-1.844; P=0.131) between dunnarts euthanased at the end of this experiment compared to those euthanased for a previous experiment.

Figure 5.3 The gastrointestinal tract of S. macroura, where (S) stomach, (Ps) pyloric sphincter (I) intestine, (M) mesenteric tissue, (R) rectum, and (Sp) spleen.

162 Table 5.4 Gastrointestinal tract measurements of S. macroura and S.crassicaudata.

Data are presented for as means ± 1 standard deviation, ID 1-8 are from the diet trials and 9-12 are from independent experiments.

S. macroura S. crassicaudata

ID 1-8 9-12 1-8 9-12

Body mass (g) 25.4 ± 2.7 20.8 ± 3.3 14.8 ± 1.3 11.8 ± 1.9

Stomach length (mm)* 12.4 ± 1.5 10.2 ±1.7 11.7 ± 1.7 10.8± 2.2

Stomach width (mm)* 6.5 ± 0.8 6.6 ± 0.8 5.7 ± 0.9 7.2 ± 2.0

Total intestine length (mm) 102.2 ± 15 94 ± 11 108.5 ± 16.4 85.7± 7.3

* Measurement taken at widest point

5.5 Discussion

The study demonstrates the differences in response to dietary challenges in two species of dunnart. We found that body mass and tail width change with exposure to diets of different digestibility and lipid and protein content. The digestibility of the diets was generally high, as would be expected for carnivore diets. Minerals, however, were variably absorbed, depending on composition and availability of the mineral in the diet.

The stripe-faced dunnarts consumed up to 50% and fat-tailed dunnarts up to 81% of their body mass equivalent in food per day. The higher consumption rate was for the cat formulation diet for both species. Cat formulation has a high water composition, and likely accounts for the high percentage of food relative to body mass consumed.

Not including the cat formulation, the stripe-faced dunnart consumed 17-30% and fat- tailed dunnart 27-53% of food relative to their body mass, similar to that observed in

163 other dasyurids. Findings from other dasyurids in captivity for food consumption were

31% in dusky antechinus (Antechinus swainsonii), 37% in the brown antechinus (A. stuartii), and 17-39% in the red-tailed phascogale and the kultarr (Antechinomys laniger).37,42,43

Tail fat accounts for 25% of total body fat in fat-tailed dunnarts and it plays an important role in fat storage in this species.20 A significant (P<0.05) loss of tail width occurred in the fat-tailed dunnart during the cockroach trial; body mass loss however, was not significant between the start and end of cockroach trial (P=0.42). It appears the physiological response of the fat-tailed dunnarts was to use some of their stored fat during this trial, as cockroaches are lower in fat and higher in protein than their usual cat formulation diet. The loss in tail width also suggests fat-tailed dunnarts have a higher daily energy requirement than was offered in the cockroach diet. During the mealworm trial a significant gain (P<0.05) in tail width and gain (P<0.05) in body mass was observed in the fat-tailed dunnart. Similar results have been shown with fat- tailed dunnarts increasing tail width on high fat, low carbohydrate diets.22 The stripe- faced dunnart showed a significant (P<0.01) gain in tail width on the mealworm diet.

Mealworms have a moderately high fat and high dry matter content and it is likely the dunnarts were storing excess energy as fat in their tail during this trial to be used when physiologically challenged at a later time if required.44

Nutrient digestibility was influenced by diet type and species of dunnart ingesting the food. In comparison to other dasyurids, the stripe-faced and fat-tailed dunnarts have similar dry matter digestibility values for their diets. For example, the Tasmanian devil (Sarcophilus harrisii; 79%), eastern quoll (81%) and dusky antechinus (80%),

164 however, these dasyurids were fed either a “whole rat” (Rattus rattus) or “whole mouse” (Mus musculus) diet.39,42 Dry matter digestibility of the red-tailed phascogale is 82-91% and the kultarr 84-92% maintained on insects and small rats.37 ADE values obtained for the dunnarts were similar to those determined in other dasyurids:

Tasmanian devil (87%), eastern quoll (89%), red-tailed phascogale (84-94%), kultarr

(82-94%) and dusky antechinus (74%).37,39,42

The dunnarts have similar digestibility of dry matter and energy when compared to eutherian insectivores, for example shrews (Macroscelididae, Sorex and Neomys spp.) dry matter 70-85%; and bats (Plecotus auritus and Myotis daubentoni) dry matter

85%; energy 90%.45,46,47 Lower digestibility values for beetle diets, which are high in chitin (31-60%), have been observed in shrews.45,46 When compared to more carnivorous eutherians, the dunnarts are generally able to digest nutrients at similar levels.48,49,50,51

Digestible energy intake is equivalent to daily maintenance energy requirement

(MER) when an animal is maintaining body mass,27 and we found the MER of the stripe-faced dunnart is 359 kJ kg 0.75 d-1 and of the fat-tailed dunnart 542 kJ kg 0.75 d-1.

These MER were determined from the diet trial that had a zero weigh change. Energy requirements above 595 kJ kg 0.75 d-1 were determined for the cat food diet. The cat food diet provided relatively low levels of protein and high lipid and water composition possibly accounting for the weight loss on higher energy intakes in the fat-tailed dunnarts. In larger dasyurids a higher MER has been observed compared to dunnarts (Tasmanian devil and eastern quoll 545 kJ kg 0.75 d-1).39 The dunnarts showed lower energy requirements than do other small dasyurids (kultarr 695 kJ kg

165 0.75 d-1; red-tailed phascogale 954 kJ kg 0.75 d-1; dusky antechinus 933 kJ kg 0.75 d-

1).37,42 In marsupials, generally the MER increases with decreased body mass.27 As expected, the smaller fat-tailed dunnart has a higher MER than the stripe-faced dunnart. Compared to other small dasyurids however, the stripe-faced dunnart has much lower MER than could be expected. It is possible energy use is different in these species due to activity levels and the use of torpor.

MER of the stripe-faced dunnart (359 kJ kg 0.75 d-1) is similar to that found for smaller eutherian shrews (body mass 9.5-16.2 g) where MER ranges from 252-371 kJ kg0.75 d-

1 (Genoud and Vogel 1990). The African giant shrew (Crocidura olivieri), which is

0.75 -1 ∼30% larger (38 g) than the stripe-faced dunnart has a MER of 721 kJ kg d , approximately double that found for the stripe-faced dunnart.52 The stripe-faced dunnart MER is approximately 50% lower than found for eutherian counterparts. It is expected the eutherians would have a higher requirements, as this has been seen previously, with marsupial requirements being approximately 30% lower than their eutherian counterparts.39,53 In contrast, in the case of the fat-tailed dunnart, a much higher MER than similar sized eutherian counterparts was found in the present study.

Nutrient intake levels were generally higher when scaled for body mass for fat-tailed dunnarts compared to stripe-faced dunnarts when maintained on the same diet, and our results show that fat-tailed dunnarts have significantly higher requirements for dry matter, energy, protein and lipids. This is highlighted by MER with stripe-faced dunnarts having a 18-56% lower energy intake compared to fat-tailed dunnarts when maintained on the same diet. Therefore, fat-tailed dunnarts need to consume more

166 nutrients per unit of body mass for maintenance in captivity than do the stripe-faced dunnarts.

For both species, negative values were obtained for Fe apparent absorption percentage when animals were on the insect diets (except for fat-tailed dunnarts on cockroach diet), and also for Ca when on the mealworm diet. Ca and Fe were only available in small quantities in those food items. It could be that the dunnarts were not able to extract the minerals presented in this type of food, or the abrasive nature of the chitin caused endogenous losses from the gut. It is also possible endogenous losses of Fe was related to exposure to the previous diet, where Fe transporters were down- regulated due to significant amounts of absorbable iron present in their earlier diets, such that iron transport was not necessary, and the absorption was related to current diet, as has been noted in rats with Zn digestibility.54 Apparent absorption of Fe from the cat formulation and marsupial formulation diets showed positive values suggesting they were able to digest and absorb Fe from these diets. Another possibility is that iron is protein-bound in some non-absorbable way such that it cannot be absorbed readily from the insect diets. The negative values could represent normal shedding of cells from the gut, and thus presenting more Fe in the faeces than was in the food.

Ideally Ca and P should be in a ratio of between 1:1 and 2:1.55 The diets studied here have unbalanced Ca:P ratios, with the exception of the cat formulation and marsupial formulation diets. Mealworms for example, have a very low Ca composition and the

Ca:P ratio is 1:19. The low proportion of Ca in the insect diets suggests the diets are inadequate to meet the daily requirements of the dunnarts, which could lead to

167 nutrition disorders such as nutritional osteodystrophy. The low Ca levels observed are likely attributed to the diet fed to the insects prior to them being fed to the dunnarts

(carrots and bran). To ensure the dunnarts were receiving adequate Ca in their diet, the insects should be fed a higher Ca diet or be supplemented with a Ca powder prior to feeding them to the dunnarts.

The dunnarts have relatively short and simple digestive tracts. They have the smallest tract lengths of the small dasyurids studied, which is expected as they are the two smallest species studied. The Julia Creek dunnart intestinal length was 170-220 mm and the kultarr 122-219 mm.30,35 On average the ratio of body mass to intestine length is 1:4 for the stripe-faced dunnart and Julia Creek dunnart, and 1:7 for the fat-tailed dunnart and the kultarr. The short and simple digestive tract allows food to pass quickly through the tract and is ideal for handling an insect diet. Arthropods are generally high in protein and lipids, which are easily digested and absorbed by animals.55,56,57 In didelphid marsupials including Caluromys philander, aurita and Philander frenata, gastrointestinal tract morphology and length have been related to diet type.58 Didelphids show a large amount of dietary variation within the family, eating foods from insects and fruits to vertebrates. Didelphids that are generally frugivorous have longer gastrointestinal tracts, whilst the more insectivorous species have shorter, simpler digestive tracts (Santori et al. 2004).

Similarly, gastrointestinal tract morphology and length has been related to diet and phylogeny in African -rats (Bathyergidae spp.).59 It is likely diet has influenced gastrointestinal tract morphology and dimensions of the stripe-faced and fat-tailed dunnart, although we did not test long term outcomes of diets on morphology in the present study.

168

The biggest difference between the dunnarts gastrointestinal measurements in this study was the length of intestine. The smaller fat-tailed dunnart had a longer intestine length. When comparing this with diet digestibility, digestive tract morphology was impacted most by the marsupial formulation diet. The stripe-faced dunnarts had a lower dry matter intake and higher digestibility values (of DM, GE, protein and lipids) compared to the fat-tailed dunnart. Differences in digestibility values would likely be due to a larger stomach capacity and lower dry matter intake. The fat-tailed dunnarts ate more on a dry matter basis and even though they had a slightly longer intestine it did not compensate for the large volume of food travelling through the digestive tract.

From the animals’ responses (body mass and tail width changes, food intake and digestibility values) presented here it can be seen that no single diet used in this study is appropriate for feeding captive dunnarts if fed alone. For example, mealworms had a low Ca and high DE composition, which caused a large increase in body mass.

Maintaining dunnarts on this diet alone would lead to obesity and/or calcium and iron deficiency related illnesses. Ideally, a captive diet could provide a combination of the diets presented in this study to provide nutrients in a range of absorbable availabilities to adequately meet the nutrient requirements of captive dunnarts. Additionally, insect diets provide behavioural enrichment, particularly for captive animal mental and physical stimulation.

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176 CHAPTER 6

Digestibility of two diets by captive eastern quolls (Dasyurus

viverrinus)

H. J. Stannard & J. M. Old

Native and Pest Animal Unit, School of Natural Sciences, University of Western Sydney, NSW,

Australia

Under review in Animal Biology

177 6.1 Chapter outline and authorship

Chapter 6 determined the digestibility of two diets currently fed to a captive eastern quoll population. Nutrition is particularly important for eastern quolls as they are often affected by calcium deficiencies and low reproductive output in the captive environment. Understanding nutrition requirements of the eastern quoll in captivity will contribute to successful captive management and breeding programs. This in turn will assist with future plans to reintroduce this species on mainland Australia. The following study was approved by the University of Western Sydney’s Animal Care and Ethics Committee, A7753.

The following manuscript is jointly authored; I am the primary and corresponding author. I conducted the digestibility trials and collected all the samples during the trials. I performed laboratory analysis of scats and food, calculation of nutritional content and statistical analysis of data, and wrote the manuscript. Mineral analysis was conducted by Waite Analytical Services, South Australia. Dr Julie Old supervised the development of the work and provided editorial feedback on previous versions of the manuscript. The eastern quolls were from a captive colony maintained at

Australian Ecosystems Foundation Inc., Lithgow NSW.

178 6.2 Introduction

Mammals in Australia have been negatively affected since European settlement and activities including hunting, changed land practices (land clearing and agriculture), and the introduction of exotic predators and competitors (Short & Smith, 1994;

Maxwell, Burbidge & Morris, 1996; Johnson & Wroe, 2003; Tyndale-Biscoe, 2005;

Burbidge & Eisenberg, 2006; Johnson, 2006). Species most at risk of population decline and becoming endangered are small to medium-sized terrestrial species ranging from 100 to 5500g, also known as the ‘critical weight range’ (Johnson &

Isaac, 2009). With a larger number of species at risk, conservation tools are employed such as legislation, creation of protected areas, predator control, recovery programs, breeding captive populations and translocations to protect those most at risk (Short &

Smith, 1994; Sinclair et al., 2006). Translocation and reintroduction programs of threatened species are conservation tools used in Australia (Dufty et al., 1994; Friend

& Thomas, 1994; Gibson et al., 1994; Short & Turner, 2000, Moro, 2003; Priddel &

Wheeler, 2004) and worldwide (Nolet & Baveco, 1996; Chiarello et al., 2004). The success of these programs has relied heavily on a basic understanding of each species’ biology, threatening processes and choosing suitable sites for introduction/release.

Maximising health of the captive populations used in release programs ensures survival and optimum reproduction whilst in captivity and aids their long-term survival post-release.

The eastern quoll (Dasyurus viverrinus) is a small to medium, ‘critical weight range’

(700-2000g) Dasyurid (carnivorous marsupial). It once inhabited a number of states along the east and south coast of Australia including New South Wales (NSW),

Victoria, South Australia and Tasmania (Jones, 2008). The last sighting of an eastern

179 quoll on mainland Australia was at Vaucluse, NSW in 1963 (Godsell, 1995). The eastern quoll is now restricted to Tasmania due to , human encroachment into their habitat, and competition and predation from larger carnivores on mainland Australia (Bryant, 1988; Jones, 2008). Due to these threatening processes the eastern quoll is listed as ‘near threatened’ on the 2008 IUCN red list of threatened species (McKnight, 2008). Eastern quolls play an important ecological role in insect control and also provide prey to larger species (Jones et al., 2003). Conservation practices currently being undertaken to prevent further decline and possible extinction of the eastern quoll include captive breeding programs, education and in the future, translocation programs.

Eastern quolls have been widely held for display in zoos and wildlife parks. From these captive populations a significant amount of knowledge has been gathered on reproductive biology and growth of eastern quolls (Hill & O’Donoghue, 1913; Fleay,

1935; Weber, 1975; Merchant, Newgrain & Green, 1984; Fletcher, 1985; Bryant,

1988). Mating occurs in May-June, and after gestation (16-23 days) up to six young are carried in the pouch until weaning after 102 days of age (Fleay, 1935; Merchant et al., 1984). Research on nutrition; however, is lacking and captive diets often consist of commercial pet foods (Collins, 1973; Fletcher, 1985; Bryant, 1988; Jackson, 2003).

Often captive quolls are unable to carry a full complement of pouch young to weaning, with some loss of pouch young noted by Weber (1975), and it is possible nutrition plays a crucial role in pouch young survival. In the wild, eastern quoll diets mostly consist of invertebrates, small birds and mammals and plant material

(Blackhall, 1980; Godsell, 1982), similar to the diets of other Australian quoll species, the northern (Dasyurus hallucatus) and western quoll (Dasyurus geoffroii; Oakwood,

180 1997; Pollock, 1999; Glen et al., 2010). The spotted-tailed quoll (Dasyurus maculatus) consumes similar food items to the other quolls but also consumes medium-sized mammals (Belcher, 1995; Glen & Dickman, 2006; Belcher, Nelson &

Darrant, 2007). Abundance and availability of prey items often influence the suitability of habitat and survival of quolls within an area (Belcher & Darrant, 2006;

Glen & Dickman, 2011).

Nutritional-related diseases, such as nutritional osteodystrophy have been observed in eastern quolls, and are generally caused by insufficient levels of Ca in the diet

(Canfield & Cunningham, 1993; Jackson, 2003). Physical signs of the disease include paralysis of the hind limbs, growth defects, lameness, dragging limbs and abnormal bounding (Jackson, 2003). To prevent such diseases, it is essential to provide captive animals with a diet adequate to meet their nutritional requirements. Nutritional studies have only been conducted on the eastern quoll and are limited to Na and water turnover in free-living and captive animals (Green & Eberhard, 1979; Green &

Eberhard, 1983), energy requirements of suckling pouch young and lactating females

(Green, Merchant & Newgrain, 1997), and energy requirements for maintenance in captivity (Green & Eberhard, 1979). The present study aims to determine the digestibility of two diet items currently fed to a captive population of quolls in an effort to better understand nutritional requirements; information that is needed to successfully maintain captive breeding colonies on a long term basis.

6.3 Methods

The eight pairs of eastern quolls used for this study were from a captive colony housed at Australian Ecosystems Foundation Inc. (Lithgow, NSW Australia). Animals

181 were housed in male-female pairs as they are part of a breeding colony. The outdoor wire enclosures measured 5m x 3m x 4m with the floor of the enclosures composed of a dirt substrate covered with plants, logs and grass patches. Each enclosure had two elevated wooden nest boxes lined with straw. Water was available ad libitum.

The nutrition trials were approved under the University of Western Sydney’s animal care and ethics committee, A7753. During the digestibility trials the quolls were fed either kangaroo mince or chicken necks at 1500hrs daily for a total of 10 days. The first five days of the trial were an adjustment period; during the last five days all faecal matter, uneaten food and a sample of food were collected daily, weighed

(±0.1g) and stored at -20°C until analysis. The first trial was kangaroo mince followed by a four week rest period then the chicken neck trial was conducted. The diets were chosen as they form part of the ‘normal’ captive diet of this population along with: insects, day-old chicks and rats, which were fed in rotation.

Food samples, faecal matter and uneaten food from each enclosure were analysed for dry matter (DM), crude protein, gross energy (GE), lipids and minerals using the same methods as described by Stannard and Old (2011). Mineral analysis (Ca, P, Mg, Na,

K, S, Cu and Zn) was conducted by Waite Analytical Services (Glen Osmond, SA).

Apparent digestibility was calculated by subtracting the nutrient excreted from the nutrient intake and expressing as a percentage (AD = Nin-Nout/Nin x 100; Robbins,

1993). Independent t-tests were used to compare the digestibility values of the two dietary items and percentage data were arcsine conformed for normality prior to analysis in SPSS (PASW statistics 18).

182 6.4 Results

6.4.1 Diets

The two diets provided had similar gross energy compositions (Table 6.1). The kangaroo mince diet was significantly higher in protein (t2= 18.419; P<0.01), K (t10= -

18.629; P<0.01), S (t10= -14.574; P<0.01), Cu (t10= -34.023; P<0.01) and Zn (t10= -

10.367; P<0.01) compared with the chicken neck diet. Gross energy was similar in both diets (t2= -1.381; P=0.301).

Table 6.1 Composition of food items (mean ± s.d.)

Kangaroo mince Chicken necks

Dry matter (%) 22.9 ± 0.1 32.5 ± 0.6*

Gross energy (MJ/kg) 22.9 ± 0.2 23.3 ± 0.4 NS

Crude protein (%) 81.5 ± 0.4 53.7 ± 1**

Lipids (%) 10.6 ± 0.3 22.4 ± 0.3**

Ca (mg/kg) 335 47000**

P (mg/kg) 8300 28000**

Mg (mg/kg) 1030 1405**

Na (mg/kg) 3100 3200*

K (mg/kg) 13000 6500**

S (mg/kg) 8600 5371**

Cu (mg/kg) 7 1.9**

Zn (mg/kg) 130 98**

Significant difference*P<0.05, **P<0.01, NS = not significant

183 6.4.2 Food consumption

The quolls usually consumed all kangaroo mince provided; however, during the chicken neck diet bone pieces were often found uneaten. On average 10g of chicken neck bones were found uneaten. As the quolls were housed in outdoor wire enclosures they were able to opportunistically catch food including invertebrates, plant material

(grass), small reptiles and possibly small mammals and birds. This ability to catch small prey is a form of enrichment and an important welfare consideration for the quolls. Plant material and invertebrate exoskeletons were observed in the scats collected for analysis, however the material accounted for less than 1% of the total scat weight.

6.4.3 Digestibility

High apparent digestibility values for DM, GE, protein and lipids were observed on both diets (Table 6.2). There was a significant difference in apparent digestibility of dry matter (t14 = 2.465; P<0.05) and gross energy (t14 = 2.489; P<0.05) between the two diets. Protein digestibility was significantly higher (t14 = 5.470; P<0.01) when animals were on the kangaroo mince diet. There was no significant difference between lipid digestibility of the two diets (t14 = 1.469; P=0.164). Generally kangaroo mince had significantly higher mineral digestibility values compared to the chicken neck diet: Ca (t14 = 4.199; P<0.01), Na (t14 = 5.647; P<0.01), K (t14 = 5.420; P<0.01),

P (t14 = 8.438; P<0.01), Cu (t14 = 6.439; P<0.01) and S (t14 = 5.325; P<0.01). Whereas differences in Zn (t14 = 0.212; P=0.836) and Mg (t14 = 0.639; P=0.537) digestibility values were not significant between the two diets. Negative values were obtained from the quolls for Ca digestibility on the kangaroo mince diet and Cu on the chicken neck diet.

184

Table 6.2 Apparent digestibility (AD) of two eastern quoll diets (n = 16; mean ± s.d.)

Kangaroo mince Chicken necks

Dry matter

Intake (g d-1) 52.3 ± 1.3 49.7 ± 12.9

AD (%) 88.4 ± 4.0 83.5 ± 8.1*

Energy

Intake (MJ d-1) 1.2 ± 0 1.1 ± 0.3

AD of GE (%) 97.1 ± 0.8 95.8 ± 2.1*

Protein

Intake (g d-1) 42.6 ± 1.0 26.7 ±6.9

AD (%) 97.7 ± 0.8 94.9 ± 2.2**

Lipids

Intake (g d-1) 5.5 ± 0.1 11.2 ± 2.9

AD (%) 95.7 ± 1.7 94 ± 2.3NS

Ca AD (%) -6.0 ± 10.0 55 ± 13.5**

P AD% 95.6 ± 1.8 59 ± 15.7**

Mg AD% 41.7 ± 20.9 34.4 ± 17.7NS

Na AD% 95.9 ± 1.4 82.6 ± 8.2**

K AD% 93.8 ± 1.9 81.8 ± 6.1**

S AD% 95.0 ± 1.0 86 ± 4.6*

Cu AD% 37.6 ± 14.4 -18.1 ± 13.5*

Zn AD% 26.7 ± 14.4 28.1 ± 24.5NS

Significant difference*P<0.05, **P<0.01, NS = not significant

185 6.5 Discussion

The chicken neck and kangaroo mince diets fed to the quolls had high digestibility values for DM, GE, protein and lipid, as expected for carnivore diets. Mineral digestibility was varied depending on diet composition and availability. In some cases minerals appeared not to be absorbed, but excreted. The digestibility values obtained in the present study were consistent with previous values determined for the eastern quoll, DM 81-82% and apparent digestible energy of 89% when fed a rat diet (Rattus rattus; Green & Eberhard, 1979; Green et al., 1997).

Similarly high digestibility values for nutrients have been determined in other dasyurid species (Table 6.3). Digestibility of GE and protein by the quoll is higher in this study compared with other Dasyurids. Lipid apparent digestibility by the quolls was consistent with other smaller Dasyurids (Cowan, O'Riordan & Cowan, 1974;

Green & Eberhard, 1979; Stannard & Old, 2011; Stannard et al., unpub.).

Compared to similar sized eutherian carnivores maintained on natural food items, the eastern quoll had similar apparent digestibility values for DM (Table 6.3). Eutherians maintained on artificial diets can exhibit lower values that the quolls (the maned wolf

(Chrysocyon brachyurus; 65-66%; Childs-Sanford & Angel, 2006). Quolls had high digestibility of protein (95-98%) whilst eutherians show a varied digestibility of protein on natural and artificial diets (Table 6.3). The varied nature of the values determined for the eutherian animals is likely due to the composition of the diets.

186 Table 6.3 Apparent digestibility of dry matter, gross energy, protein and lipids in dasyurids and other insectivorous-carnivorous mammals

Species Diet DM (%) Energy (%) Protein (%) Lipids (%) Reference

Eastern quoll Chicken necks and kangaroo mince 84-88 96-97 95-98 94-98 1

Dasyurus vivverinus

Rat 81 88 2

Tasmanian devil Rat 79 87 2

Sarcophilus harissii

Dusky antechinus Mouse 80 3

Antechinus swainsonii

Red-tailed phascogale Insects and rat 82-91 86-94 83-91 92-98 4

Phascogale calura

Kultarr Insects, rat and small carnivore food 84-92 85-94 86-91 92-98 4

Antechinomys laniger

Stripe-faced dunnart Insects, cat food and small carnivore 76-89 77-92 73-88 83-96 5

Sminthopsis macroura food

Fat-tailed dunnart Insects, cat food and small carnivore 64-88 82-95 78-86 82-96 5

S. crassicaudata food

187 Sand cat Raw meat diet 84 90 92 6

Felis margaritas

Ocelot Commercial food, mouse, rabbit, rat, 67-80 82-89 85-91 96-99 7

Leopardus pardalis quail, chick

Weasel Bat, rabbit, mouse, starling 71-83 8

Mustela nivalis

Maned wolf Extruded meat meal and plant based 65-67 72-75 9

Chrysocyon brachyurus

Mink Extruded dog food 76 75 93 10

Mustela vison

1. This study; 2. Green & Eberhard, 1979; 3. Cowan et al., 1974; 4. Stannard & Old, 2011; 5. Stannard et al., unpub.; 6. Crissey et al.,, 1997; 7. Bennett et al., 2010; 8.

Moors, 1977; 9. Childs-Sanford & Angel, 2006; 10. Ahlstrøm &Skrede, 1998

188 Digestibility is influenced by diet type, nutritional content and indigestible components

(Barboza, Parker & Hume, 2009). The diets provided to the quolls had high nutritive values and low indigestible components, such as hair, skin, bones and feathers. Compared with the quoll, lower digestibility ranges have been determined for the bobcat (Felis rufus; DM 68-96%, energy 77-95%, protein 80-97% and lipids 67-98%) on a whole animal diet that included indigestible components such as hair, bones and skin (Powers,

Mautz & Pekins, 1989).

The present study provides the first values for mineral digestibility in the eastern quoll.

Mineral digestibility was influenced by diet item mineral composition. The kangaroo mince had a low Ca content and presumably accounted for the negative digestibility value obtained in the quolls on this diet. Similar findings have been determined for other

Dasyurids, dunnarts Sminthopsis spp., with negative digestibility values shown on a mealworm diet which has a low calcium content (Stannard, McAllan & Old unpub.).

Calcium and P should be fed in a ratio between 1:1 and 2:1 to all mammals to ensure calcium is not mobilised from bones (Barboza et al., 2009). Requirements for Ca and P would increase for female quolls during late lactation when energy demands are high to supply young with adequate sustenance (Green et al., 1997). The Ca:P ratio of the food items provided to the quolls were unbalanced, kangaroo mince was 1:25 and chicken neck 1:0.6 which could lead to deficiencies and possibly nutritional osteodystrophy if these diets were to be fed alone. The chicken neck diet was high in calcium, however its relative absorption was low compared to its composition. Calcium was in an indigestible

189 form in the chicken neck diet, as most of the Ca is held in the bone, and only a portion could be extracted for digestion by the quolls.

The quoll had a higher apparent absorption of P on the kangaroo mince diet compared to the chicken neck diet. The chicken neck diet had a higher P content than the kangaroo mince diet however most P, like Ca, is held in the bone (Barboza et al., 2009). The ability of the quolls to use their sharp molars to crush and expose bone would increase the surface area and therefore increase availability of Ca and P for absorption. However, despite this the quolls were unable to absorb as much P from the chicken neck diet compared to the kangaroo mince diet.

Magnesium composition in the chicken neck diet was higher than in the kangaroo mince diet. In contrast to dietary composition, the kangaroo mince diet had a higher digestibility value of Mg compared to the chicken neck diet. Generally Mg is readily available in carnivore diets as it is stored in bones (Barboza et al., 2009).

Sodium and K are used for growth, reproduction and energy production in the body

(Barboza et al., 2009). High values for Na and K absorption were determined for the quolls and similarly, high Na turnovers have been observed previously in the eastern quoll and Tasmanian devil (Green and Eberhard 1979). These values were consistent with studies in other small Dasyurids (Stannard & Old, 2011; Stannard et al., unpub.).

Dasyurids are very active, and this quoll population show daily diurnal activity in addition to the ‘normal’ nocturnal activity (pers. obs.) possibly accounting for high

190 requirements of Na and K. Natural diets items of the quoll, such as insects, are high in Na and K (Barker, Fitzpatrick & Dierenfeld, 1998; Finke, 2002) and dasyurids would have evolved to cope with this.

Sulphur content was higher in the kangaroo mince diet and apparent absorption values were also higher on the kangaroo mince diet compared with the chicken neck diet.

Sulphur requirements are closely associated with protein and energy requirements

(Barboza et al., 2009). Higher absorption values of protein and energy were observed on the kangaroo mince diet compared to the chicken neck diet accounting for the higher absorption of S on the kangaroo mince diet. Apparent absorption of S appears to be related to quantity of S present in the diet, and thus availability of this mineral for absorption is relative to composition in both these diets.

Copper and Zn are trace minerals, which are only required in small quantities (Barboza et al., 2009), and the results from this study were consistent with this observation. Quolls were able to absorb 38% of the Cu from kangaroo mince, whereas on the chicken neck diet they were unable to absorb Cu (-18%), however there was a high variation in Cu absorption values of this mineral among indivduals. It is possible the low concentration of Cu in the chicken neck diet, or endogenous losses account for the apparent loss of Cu whilst animals were on the chicken neck diet. Zinc is important in metabolism of nutrients and immune function, and deficiency can lead to infertility (Barboza et al.,

2009). Hence Zn is an important mineral to study in these captive animals to ensure successful reproduction in captivity. Zn apparent absorption by the quolls was relative to

191 concentration in the diet items provided. Levels were relatively low compared with other minerals as were absorption levels.

With limited studies on eutherians available for mineral digestion, it is difficult to comment on whether marsupial requirements differ from other carnivorous eutherian mammals. A lack of knowledge, and using diets suitable for domestic pets to feed marsupials (such as commercial cat food) could lead to nutritional-related disorders. In herbivorous marsupials it has been found that lower requirements for macro and trace minerals are required in their diet when compared with eutherian mammals. For example the quokka (Setonix brachyurus) has been shown to have lower requirements of Cu and

Co than sheep on Rottnest Island (Barker, 1961). Similarly, compared to sheep and horses, captive koalas (Phascolarctos cinereus) have lower requirements for P, Na, Se,

Zn and Cu (Ullrey, Robinson & Whetter, 1981). When maintained on a commercial pig

(Sus scrofa domestica) diet, the southern hairy-nosed wombat (Lasiorhinus latifrons) showed signs of Cu toxicity, suggesting a much lower requirement for Cu than the domestic pig (Barboza & Vanslow, 1990 as cited in Hume, 1999). These domestic species are much larger than the marsupials and may explain why food designed for them is not ideal for smaller marsupials. With the Australian continent being a mostly arid landscape, with nutrient depleted soils and variable rainfall (Morton et al., 2011), it is not unreasonable to assume marsupials have evolved to cope with this and have low mineral requirements compared to eutherians from other continents.

192 Eastern quolls are an , and it is a conservation priority to maximise reproductive output. Green et al., (1997) determined maintenance energy requirements were double during peak lactation in quolls and it is therefore likely other nutrients would need to be doubled for reproductively active females, particularly during peak lactation.

Increasing food would ensure animals were meeting their increased nutrient needs and ensure mothers were able to support the maximum number of young until weaning.

Increasing reproductive output is required for captive breeding programs to ensure suitable numbers are bred for use in translocation/reintroduction.

The quolls were able to opportunistically catch other food items. As these only accounted for a very small portion of the scats collected (<1% of scat weight), it is believed that these additional food items were unlikely to greatly influence the results obtained. It is possible the quolls were seeking these items to meet particular nutrient requirements or for mental and physical stimulation. The ability to find and catch their own food suggests these animals will be suitable for translocation.

More data is needed for both eutherian and marsupial carnivores to gain a greater understanding of eutherian and marsupial nutrition and how this relates to their species specific physiological needs. Successful captive breeding programs are becoming more urgent for endangered species. The information presented here has contributed to the current understanding and management of eastern quoll nutrition in captivity. The information obtained in this study can be used as a model for the other Australian quoll

193 species, and assist with increasing reproductive output in captivity, and therefore increase the number of animals available for translocation programs.

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201 CHAPTER 7

Further investigation of the blood characteristics of Australian

quoll (Dasyurus spp.) species

H. J. Stannard, L. J. Young & J. M. Old

Native and Pest Animal Unit, School of Natural Sciences, University of Western Sydney, Australia

Under review in Veterinary Clinical Pathology

202 7.1 Chapter outline and authorship

Chapter 7 focused on blood chemistry and white cell counts in two quoll species. The paper aimed to add to the current information available on blood parameters of the eastern and spotted-tailed quolls. The research will assist with health assessment in captive populations and reintroduced individuals in the future. In the wild the nutritional status of an animal can influence changes in blood chemistry levels and this study provided a preliminary set of data for animals that will be reintroduced in the future.

There were intentions to use kultarrs for this study (as there is no data available for them); however, their veins were too small to obtain enough blood volume for analysis.

Sample collection and analysis was approved by the University of Western Sydney’s

Animal Care and Ethics Committee, A6087.

The following manuscript is jointly authored; I am the primary and corresponding author.

I collected blood with the assistance of a qualified veterinarian and conducted the laboratory analysis of samples. I conducted the statistical analysis of data and wrote the manuscript. Dr Julie Old and A/Prof. Lauren Young supervised the development of the work and provided editorial feedback on previous versions of the manuscript. The animals used for the study were from captive colonies maintained at Australian

Ecosystems Foundation Inc., Lithgow NSW and Featherdale Wildlife Park, Doonside

NSW.

203 7.2 Introduction

Quolls are carnivorous marsupials that inhabit Papua and Australia, with four species inhabiting Australia: the eastern quoll (Dasyurus viverrinus), spotted-tailed quoll (D.maculatus), western quoll or chuditch (D.geoffroii) and northern quoll

(D.hallucatus).1 Eastern quolls are either black or fawn with white spots on the body and no spots on the tail. They weigh between 600-2000g and exhibit sexual dimorphism.2,3

The eastern quoll occupies forests, woodlands and open heaths in Tasmania.3 Its geographical range has been reduced by 50-90% since European settlement and it is now extinct on mainland Australia.4 Pressures from European settlement such as hunting, agriculture, predation and competition from exotic pest species have contributed to the reduced range and loss of this species.3,5

Resembling the eastern quoll, the spotted-tailed quoll is larger (up to 4kg) and has a thick brown to reddish coat and long tail covered in spots.6,7,8 The spotted-tailed quoll is the largest carnivorous marsupial on mainland Australia, since the extinction of the thylacine

(Thylacinus cynocephalus) and Tasmanian devil (Sarcophilus harrisii).6,8 It inhabits forests, rainforests and heathland along the eastern coast of Australia including

Tasmania.6,8 The spotted-tailed quoll has also undergone a significant geographical range reduction,4 with much of their current distribution relying heavily on the abundance of prey.9 Both the eastern and spotted-tailed quolls are listed as ‘near threatened’ on the

2008 International Union for the Conservation of Nature red list of threatened species.10,11

204 Studies of hematology and blood chemistry have shown there are differences between marsupial and eutherian mammals; albeit minor ones. Generally, marsupials have no or very low numbers of basophils and have higher serum enzyme levels than eutherians.12,13

Determining reference intervals for hematological values within each species cohort can be more informative than reference to generic hematological data at the class-level but this data can be difficult to obtain for threatened, native animals. Data for different populations within a species can provide a baseline comparison to detect changes in homeostasis and immune status, as in the case of an infection.14,15 For example, hematology analysis was used to determine the occurrence of anemia, lymphocytosis, neutrophilia and polycythemia in ill western quolls from a captive population.16

A number of biological factors influence hematology and blood chemistry levels such as age, sex, reproductive status, habitat, season and nutrition. In some cases age influences susceptibility to disease and hematology concentrations and gender can influence erythrocyte concentration.17 For example, differences between gender have been found in dunnarts (Sminthopsis spp.) for neutrophil and lymphocyte concentration.18 Hemoglobin and hematocrit values have also shown a decline due to season in some dasyurid species.19,20,21 It is essential to measure the influence of different physiological factors on hematology and blood chemistry of different species to understand what is ‘normal’ for a particular species.

Hematology reference values have been reported for a few dasyurid species, including the fat-tailed dunnart (Sminthopsis crassicaudata), stripe-faced dunnart (S. macroura),

205 western quoll, Tasmanian devil and eastern quoll.17,18,19, 20, 22,23,24,25 Previous studies have determined ranges for eastern quoll hematology (males and females), and blood chemistry for only two animals.12,13,24 Studies of the spotted-tailed quoll have determined ranges for blood chemistry of only two males.23 Whilst baseline values have been determined for blood chemistry in spotted-tailed quolls and hematology values for eastern quolls, the influence of season and age on these values have not been determined in either case. The influence of sex has been determined in eastern quoll hematology but not blood chemistry.24 Detailed descriptions of blood cell morphology have been conducted for all four Australian quoll species.17

With the limited blood chemistry data available for the eastern and spotted-tailed quolls and no hematology data available for the spotted-tailed quoll, this paper aims to provide a more detailed study of blood chemistry and differential white cell values for eastern and spotted-tailed quolls. In addition, changes influenced by biological factors: age, sex and season are presented.

7.3 Methods

7.3.1 Study animals

Materials and Methods

Study animals

Captive eastern quolls (n=35; 16 males and 19 females) at the Australian Ecosystems

Foundation Inc. (Lithgow, NSW Australia) and spotted-tailed quolls (n=3; 2 male and 1 female) at Featherdale Wildlife Park (Doonside, NSW Australia) were used for this

206 study. Both quoll species were housed in outdoor wire enclosures; an environment that provided a natural photoperiod and temperature fluctuations consistent with the local area. Mean seasonal temperatures at Lithgow during the collection period were summer

24.5˚C, autumn 18.5˚C, winter 7.5˚C, spring 12.3˚C, and at Doonside summer 29.4˚C, autumn 24.5˚C, winter 17.3˚C and spring 23.8˚C.26 The quolls were maintained on a diet of kangaroo mince, chicken necks, insects, day-old chicks and rats.

7.3.2 Blood collection and analysis

Sample collection and analysis was approved by the University of Western Sydney’s

Animal Care and Ethics Committee, A6087. Blood samples were collected in the early morning hours to reduce the possibility of heat-related stress. Each animal was removed from the nest box and placed into a hessian bag. The individual was restrained inside the bag with the tail exposed to allow access to the lateral caudal vein. Thirty-five eastern quolls were available for this study and sufficient blood volumes for blood chemistry analysis were obtained from 26 of these animals. Up to 150µL blood was taken from the vein using a 25-gauge winged infusion needle and syringe, as reported for collection from large dasyurids.17 Animals were conscious throughout the procedure. Once the blood sample was taken, the animals were returned to their enclosures, fed and monitored for signs of stress for the following hour. Eastern quoll blood samples were collected in

February, April and October over two years, to obtain seasonal values. As a cautionary measure to reduce inadvertent influence on breeding cycles, samples were not taken in winter as it is the breeding season for eastern quolls and blood sampling may have interfered with breeding behavior and contributed to animal stress during this time.

207 Spotted-tailed quoll samples were obtained opportunistically from two animals in March; one, nine-month-old male and one, three-year-old female and are included here as a comparison with the eastern quoll.

After collection, a 100µL aliquot of whole blood without anticoagulant was immediately transferred into an analysis plate and analyzed in the VetScan Chemical Analyzer

(Abaxis, California USA). Blood chemistry values were obtained for albumin (ALB), amylase (AMY), total bilirubin (TBIL), alkaline phosphatase (ALP), alanine transaminase (ALT), globulin, glucose (GLU), sodium, potassium, urea nitrogen (BUN), creatinine (CRE), calcium, phosphorus and total protein (TP) using a comprehensive rotor (Abaxis, California USA). The remainder (<50µL) of the blood was used to make a blood smear. Differential white blood cell (WBC) counts were conducted on blood smears stained with Diff Quik (Sigma-Aldrich, St Louis USA). Further hematological tests were not performed as only small sample volumes could be collected.

Statistical differences between seasons and ages were determined using one-way

ANOVA and differences between sexes were determined using unpaired t-tests in SPSS.

Reference intervals were determined using freeware program, Reference Value

Advisor.27 As a wildlife species was used for this study and sample sizes were small

(n<40), Box-Cox transformation, robust method with 90% confident intervals were used.

Due to the small sample size for spotted-tailed quolls, samples are presented as the raw data obtained for each analyte.

208 7.4 Results

Eastern quoll erythrocytes were anucleated biconcave discs ranging in diameter from 6-9

µm. Lymphocytes were proportionally the most abundant WBC and ranged from 10-19

µm in diameter. Lymphocyte nuclei were round and with darkly stained chromatin surrounded by a fine layer of cytoplasm (Figure 7.1a). Neutrophils generally had a 3-5 lobed nucleus and were 15-19 µm in diameter with fine granules in the cytoplasm. A number of neutrophils were ring-neutrophils, with an annular nuclei (Figure 7.1b).

Monocytes were large ranging from 15-20 µm in diameter and had an indented to “horse- shoe” shaped nucleus. Eosinophils had a 2-3 lobed nucleus with course granules that stained a reddish purple color and ranged from 16-20 µm in diameter (Figure 7.1c).

Basophils were not observed in the blood samples taken from either species.

Morphology of spotted-tailed quoll blood cells was similar to that of the eastern quoll; however, annular nuclei leukocytes were only occasionally observed. Lymphocytes measured 10-16 µm in diameter. Neutrophils had segmented nuclei, 3-6 lobes, with fine granules in cytoplasm and were 16-20 µm in diameter (Figure 7.1d). Monocyte nuclei were generally ‘horse-shoe’ shaped and 18-22 µm in diameter. Eosinophils were 16-18

µm diameter.

209

Figure 7.1 White blood cells of eastern and spotted-tailed quolls a) annular nuclei leukocyte and lymphocyte, b) neutrophil with an annular nuclei, c) eosinophil and lymphocyte, d) neutrophil with a segmented nucleus

Blood chemistry values obtained for the eastern quolls are presented as medians, reference intervals and lower and upper 90% confidence intervals in Table 7.1. There was no significant difference (P>0.05) between male and female eastern quolls for blood chemistry and differential WBC counts. Significantly higher values for: TBIL (F2, 25 =

7.128; P=0.004), CRE (F2, 25 = 3.333; P=0.054), GLU (F2, 25 = 5.644; P=0.010) were observed in summer compared with autumn. Sodium had a significantly higher value (F2,

25 = 3.262; P=0.05; Table 7.2) in autumn compared to spring. There was no significant difference in blood chemistry values between spring and summer (Table 7.2).

210 Table 7.1 Range and mean values for blood chemistry and differential white cell counts of eastern quolls

Reference Interval Median (Min-Max) 90% confidence interval 90% confidence interval

lower limit upper limit

Blood Chemistry n = 26

ALB (g/L) 11.5-44.8 28 (8-34) 5.4-17.9 38.9-48.5

ALP (U/L) 44.0-503.7 171 (55-529) 31.7-71.4 369.2-637.5

ALT (U/L) 19.6-169.3 44 (19-153) 18.7-21.8 108.5-250.8

AMY (U/L) 21.0-1216.5 518 (219-1261) 116.8-193.3 1035.8-1382.2

TBIL (µmol/L) 3.2-9.0 5 (4-8) 3.0-3.8 7.7-9.3

BUN (µmol/L) 6.5-26.5 19 (5.5-27.1) 5.2-11.0 24.5-28.2

Calcium (µmol/L) 1.5-3.5 2.4 (1.1-2.7) 1.0-1.8 3.1-3.7

Phosphorus (mmol/L) 0.7-3.9 2.1 (0.8-3.8) 0.4-1.1 3.4-4.3

CRE (µmol/L) 11.5-70.3 31 (18-61) 8.9-16.5 56.8-78.8

GLU (mmol/L) 2.3-9.5 5.5 (2.3-9.9) 1.2-3.1 8.3-10.6

Sodium (mmol/L) 112.5-181.0 146 (100-155) 102.7-126.8 164.5-192.0

Potassium (mmol/L) 2.1-8.2 5.2 (2.1-8.5) 0.8-3.2 7.3-9.0

TP (g/L) 13.2-67.8 57 (20-66) 19.0-41.7 69.2-80.6

211 Globulin (g/L) 16.2-43.1 25 (19-37) 14.4-19.5 36.3-47.7

Differential White Cell Count Mean ± s.d n = 35

Lymphocytes (%) 66.9 ± 14.5 66 (28-95)

Neutrophils (%) 22.0 ± 13.4 20 (3-63)

Ring-neutrophils (%) 7.1 ± 3.9 6.5 (0-17)

Monocytes (%) 3.0 ± 2.1 3 (0-8)

Eosinophils (%) 1.0 ± 1.7 0 (0-8)

Basophils (%) 0 0 (0)

212 Table 7.2 Seasonal blood chemistry values for eastern quolls (mean ± s.d.)

Summer Autumn Spring

n= 9 n = 12 n = 5

Blood chemistry

ALB (g/L) 27.5 ± 9.2 26 ± 5 20.8 ± 9.7

ALP (U/L) 248.4 ± 137 217.7 ± 115.9 141 ± 70.4

ALT (U/L) 64.8 ± 31.4 49.4 ± 37.9 60.2 ± 48.4

AMY (U/L) 588.1 ± 254 710.5 ± 320.2 447.4 ± 157*

TBIL (µmol/L) 6.6 ± 1.2 4.8 ± 0.75 5.6 ± 1.1**

BUN (mmol/L) 19.5 ± 5.9 16.8 ± 3.1 16.6 ± 5.4

Calcium (mmol/L) 2.4 ± 0.49 2.2 ± 0.32 2.1 ± 0.68

Phosphorus (mmol/L) 2.4 ± 0.75 2.3 ± 0.76 1.7 ± 0.71

CRE (µmol/L) 44.3 ± 12 29.8 ± 10.3 36 ± 17.1**

GLU (mmol/L) 7 ± 1.5 5 ± 1.3 4.8 ± 1.8**

Sodium (mmol/L) 139.4 ± 16.1 147.9 ± 3.6 128.4 ± 26.1

Potassium (mmol/L) 6.4 ± 1.2 4.8 ± 0.69 4.5 ± 2**

TP (g/L) 53.7 ± 13 53.3 ± 6.2 50.8 ± 17.7

Globulin (g/L) 24.8 ± 3.4 27.1 ± 5.5 31.5 ± 8.4

* P<0.05; ** P<0.01

213 Twelve of the eastern quolls were 1 year old, 10 of them were 2 years old and 4 of them 3 years old. A significant difference (F2, 25 = 5.911; P=0.008) was found between 1 and 2 year old (P<0.05), and 1 and 3 year old (P<0.05) eastern quolls for ALP. Older animals’

ALP levels were around half that of the one year old animals (ALP: 1yr 328 ± 131 U/L;

2yr 176 ± 72 U/L; 3yr 136 ± 65 U/L).

In comparison with the eastern quoll the spotted-tailed quolls generally showed similar blood chemistry levels. The spotted-tailed quolls had lower levels of ALP, ALT, AMY and globulin (Table 7.3). It should be noted that one male spotted-tailed quoll had much lower values for each analyte than the other two quolls but there was no obvious rationale to exclude it from this report. Blood was not clotted and the animal appeared to be in good health at the time of sampling.

214 Table 7.3 Blood chemistry and differential WBC for spotted-tailed quolls

Male 1 Male 2# Female

Blood Chemistry

ALB (g/L) 46 2 40

ALP (U/L) 115 15 65

ALT (U/L) 22 8 31

AMY (U/L) 148 16 202

TBIL (µmol/L) 9 6 7

BUN (mmol/L) 13.2 2 18.2

Calcium (mmol/L) 2.47 0.85 2.37

Phosphorus (mmol/L) 2.06 0.47 1.64

CRE (µmol/L) 94 43 93

GLU (mmol/L) 7.9 2.6 7.6

Sodium (mmol/L) 144 100 143

Potassium (mmol/L) 6.2 - 5.3

TP (g/L) 67 10 69

Globulin (g/L) 21 - 29

Differential White Cell Count

Lymphocytes (%) 65 67 46

Neutrophils (%) 25 26 45

Ring-neutrophil (%) 5 7 3

Monocytes (%) 4 0 5

Eosinophils (%) 1 0 1

Basophils (%) 0 0 0

# animal appeared healthy and blood was not clotted

215 7.5 Discussion

Although hematology and blood chemistry have been studied previously in eastern and spotted-tailed quolls, sample numbers were often small and only able to represent a single time point for these species.13,23 Melrose et al.24 however, provided a detailed study of hematology values for male (n= 40) and female (n=40) eastern quolls, which did not include juvenile or old animals. The present study included age classes and seasonal data that are not represented in previous studies of quoll blood chemistry. This study reported relationships between sexes, age and season and blood factors of a captive population. In addition, we reported that for eastern quolls, some serum enzymes were influenced by season and ALP was influenced by age.

Generally, the morphology of erythrocytes and leukocytes in the eastern and spotted- tailed quolls was consistent with previous descriptions for both species and similar to those of other dasyurid species.17,18,24 Lymphocytes followed by neutrophils were proportionally the most abundant WBC as found previously in quolls, other dasyurids

12,17,18,24 and some marsupial species.28,29,30 Eosinophils were rarely observed and basophils were not observed in either quoll blood sample, which is consistent with other dasyurids.12,13,18,22

This study also provides measurements of erythrocytes and leukocytes for the eastern and spotted-tailed quoll. Erythrocytes, neutrophils and eosinophils were larger in diameter, and eosinophils were more numerous in quolls than reported for two dunnart species. 18

Ring-neutrophils have also been reported in dunnarts.18

216 Mean blood chemistry values determined for the two quoll species were within the range observed for other dasyurids (see table 7.4). Mean values for ALP and AMY were higher and ALB lower when compared with the ranges for the Tasmanian devil.22 Values for the eastern and spotted-tailed quolls were similar to those determined in the western quoll.25

The blood chemistry values of the quolls in this study were generally similar to those reported for other marsupials28,31-35 and domestic eutherians.36 Results for TBIL were higher and TP lower in both quoll species, and CRE lower in the eastern quoll compared to general eutherian ranges. Previously GLU and BUN values were determined to be higher in marsupials than humans,13 however the results from quolls in this study show values for GLU and BUN were within the normal human range.37 Stress, dehydration and differences in nutritional status could explain differences between our GLU and BUN levels compared to previous values.

Previous findings of blood chemistry for two eastern quolls were consistent with the ranges observed in this study (see table 7.4)13. However, mean ALP values are higher than those previously determined, due to the inclusion of young animals in the present study. ALP values in eastern quolls showed large variations between individuals; similarly large variations in ALP have been observed previously in three captive murid species.38 Mean CRE values in the present study were lower than those previously determined by Parsons et al.13 possibly due to differences in dietary protein intake.

Spotted-tailed quoll blood chemistry values for ALP were higher and AMY and BUN levels were lower than those determined in a previous study.23 This again is most likely due to the inclusion of young animals in the present study.

217 Table 7.4 Comparison of Dasyurid blood chemistry and differential WBC

D. viverrinus D. maculatus D. geoffroii D. viverrinus D. maculatus S. harrisii

n=26 n=3 n =36 25 n=2 12,13 n=2 23 n=3 22

Blood Chemistry

ALB (g/L) 11.5-44.8 2-46 27.2-35.8 28 32.9-38.8 35

ALP (U/L) 44.0-503.7 15-115 102-1020 43-86 29-36 14-43

ALT (U/L) 19.6-169.3 8-31 12-100 - - -

AMY (U/L) 21.0-1216.5 16-202 - 654 214-260 53-73

TBIL (µmol/L) 3.2-9.0 6-9 0.2-6.9 9 - -

BUN (mmol/L) 6.5-26.5 2-18.2 14-23.9 21-28 31-50 19-37

Calcium (mmol/L) 1.5-3.5 0.85-2.47 2.14-2.71 1.1-1.2 1-1.1 0.85-1.3

Phosphorus (mmol/L) 0.7-3.9 0.47-2.06 - - 0.45-1.8 2.5-2.9

CRE (µmol/L) 11.5-70.3 43-94 33-75 88.4 - -

GLU (mmol/L) 2.3-9.5 2.6-7.9 2.6-9.7 5.2-5.6 4.9-13.7 5.7-14

Sodium (mmol/L) 112.5-181.0 100-144 - 132-137 133-144 135-142

Potassium (mmol/L) 2.1-8.2 5.3-6.2 - 3.7-7.6 4.6-4.7 4.6-5

TP (g/L) 13.2-67.8 10-69 44.8-74.2 61 54-60 65

218 Globulin (g/L) 16.2-43.1 21-29 13.8-71.2 33 - -

Differential White Cell Count

Lymphocytes (%) 29-97 46-67 33.5 35 - 20-27

Neutrophils (%) 2-71 25-45 56.7 55 - -

Ring-neutrophils (%) 0-9 3-7 - - - 0-4

Monocytes (%) 0-3 0-5 2.6 9 - -

Eosinophils (%) 0 0-1 6.1 1 - -

Basophils (%) 0 0 0 0 - -

- Value not available

219

Compared to each other the eastern and spotted-tailed quolls generally showed similar blood chemistry values. The eastern quolls exhibited higher levels of some blood chemistry levels including ALP, ALT, AMY and globulin. Variations in ALP content between species have been also observed in macropods.39 Higher levels of AMY in eastern quolls compared to spotted-tailed quolls have been shown previously23,24 and new data from our study now confirms these findings as standard for this species.

The sex of eastern quolls appeared to have no influence on the blood chemistry values obtained. Similarly, hematological values studied previously in eastern and western quolls showed no significant difference between sexes.24,25 Sex-related differences have been determined in dunnarts, with neutrophil: lymphocyte ratios being influenced by sex.18 Male antechinus (Antechinus stuartii) have been noted to have lower hemoglobin and hematocrit concentrations compared with females. These changes in antechinus however, are influenced by semelparity.40 Although differences in blood chemistry are observed in dasyurids that exhibit semelparity, this process has not been observed in eastern quolls. Thus, it is not surprising that we did not find a gender-related effect.

In eastern quolls, the mean CRE varied significantly across seasons. Reduced levels of

CRE have been associated with poor nutrition and consequently loss of muscle mass in dolphins (Tursiops truncates), harbour seals (Phoca vitulina) and Eurasian badgers

(Meles meles).41,42,43 In the eastern quolls the lowest CRE levels were observed in autumn; these reduced levels should not be related to nutrition as feeding was managed by humans. Food items were rotated daily, possibly accounting for change in CRE

220

between sampling periods. Increased CRE in spring and summer could also suggest a

temporary increase in muscle mass, which could relate to increased activity. Low BUN

levels have also been attributed to poor nutrition in the agile wallaby (Macropus agilis),29

however, BUN levels in the quolls were higher than in the wallaby and did not differ

significantly across the seasons, thus confirming no seasonal change in nutritional status

in the eastern quolls.

A seasonal change in GLU occurred in the eastern quolls with lower levels observed in

autumn. Seasonal changes have also been reported in brushtail possum GLU levels,

which are associated with stress and possibly water availability.44,45 In bobcats (Felis rufus) capture stress was believed to be the reason for elevated GLU levels.46 Changes in

GLU in eastern quolls could relate to hydration, although water is available ad libitum. It is more likely GLU levels relate to stress either from capture or other stress during that season. Stress could be related to changes in housing (changing animal pairs) or hormonal changes related to breeding.

Eastern quoll TBIL was significantly higher in summer compared to autumn. Opposing results were found in free-living mountain brushtail possums (Trichosurus caninus).35

TBIL levels were significantly lower in summer and autumn compared to winter and spring.35 TBIL is a measure related to liver function; low levels and/or change in TBIL levels suggest a current or recent change in liver function.36 The quolls did; however, appear healthy at the time of sampling. The range of values observed fell within the range observed for the western quoll.25

221

Juvenile eastern quolls had significantly higher levels of ALP than adult animals. ALP is involved in calcification, the rate of calcium deposition and bone growth.47 Higher ALP values in juveniles have been determined for mammals both in captivity and in the wild, including western quoll, brush-tailed rock wallaby, tammar wallaby, northern hairy-nosed wombat, Japanese macaques (Macaca fuscata), wolves (Canis lupus), coyotes (Canis latrans) and rock hyrax (Procavia capensis).25,31,32,33,47,48,49,50 As ALP is associated with growth it is reasonable to assume that eastern quolls under one year of age were still growing and therefore required larger quantities of ALP to facilitate bone and organ growth. Eastern quolls reach sexual maturity at one year of age3 and it could be expected growth slows or ceases at this time, hence the lower ALP levels in older quolls.

There may be a difference between data from captive mammals compared to animals in the wild. Melrose et al.24 found no significant difference between captive and wild quoll hematology values. Clark and Spencer51 however, found erythrocytic values were significantly different between wild and captive quokka populations. Gaughwin and

Judson52 found significantly different hematology and blood chemistry levels in captive and free-ranging southern hairy-nosed wombats (Lasiorhinus latifrons). Although the data in the present study is from a captive population, the animals were exposed to natural environmental conditions rather than controlled housing, so our study has identified trends that may be related to seasonal cues and climatic conditions of the local area. These values may provide useful comparisons for eastern quolls translocated into the surrounding area.

222

Both quoll species have become important to conserve due to significant geographical range contractions and the extinction of the eastern quoll on mainland Australia. The data presents changes in blood levels for the eastern quoll at different ages and seasons.

Spotted-tailed quoll blood chemistry requires further investigation to determine whether the data obtained here are typical for this species. As all animals appeared healthy and no hemoparasites were observed in blood smears, these results can be used to inform later studies of quolls to determine health status and changes in homeostasis. The data can now be used in combination with other indicators of health such as body condition, appetite and mobility to provide a valuable resource for maintenance of captive animals and in the monitoring of translocated animals post-release.

223

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CHAPTER 8

General discussion and future directions

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Dasyurids, like other mammals in Australia have suffered major declines due to human activities (such as agriculture, land clearing and introduced species) and these activities have contributed to mammal extinctions. The dasyurid species studied in this thesis have undergone population declines and in some cases are listed as endangered. It is important to conserve species for biodiversity and ‘normal’ ecosystem functioning. Numerous management techniques have been employed to ensure the future sustainability and biodiversity of mammal populations in Australia including legislation, reintroduction programs, and action and recovery plans. To ensure these management techniques are successful, knowledge of ecology, physiology and biology are required.

The research conducted for this thesis determined 1) diet of translocated phascogales, 2) nutrient digestibility, mineral absorption and energy requirements in five dasyurid species and 3) blood chemistry and white blood cell counts in two dasyurid species. Translocated red-tailed phascogales were found to be mostly insectivorous but they also consumed small vertebrates. Digestibility values were determined to be high which is associated with the types of diets consumed. Mineral absorption was varied depending on diet, species and physiological status. The gastrointestinal tracts studied in three species

(kultarr, stripe-faced dunnart and fat-tailed dunnart) were simple, similar to that reported for other dasyurids (Hume 1999). Blood samples taken from the two quoll species

(eastern and spotted-tailed quoll) found that there were differences between species, age and season.

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Translocating animals can be an expensive, time consuming process and increasing knowledge of species biology and ecology provides a basis for using particular species for translocation. My study has shown translocated red-tailed phascogales were able to tolerate translocation and exploit prey items other than arthropods (i.e. birds and mammals) at Alice Springs Desert Park (ASDP) in the first six months post-translocation.

These larger food items would be particularly important to reproductively active females during lactation when nutrient requirements are higher. My research was undertaken in conjunction with ASDP and information from my study has been shared to improve their understanding of responses of translocated phascogales to their new environment. This has contributed to improving their translocation techniques. With the information gained in this study and further research into spatial movement, home range and nesting sites of red-tailed phascogales, larger scale translocations could take place for this species and prove successful in reintroducing the red-tailed phascogale to parts of Australia where it once occurred.

Results from my study determined that in the captive setting the red-tailed phascogale has a higher energy requirement than the other small dasyurids studied in this thesis and they also have a higher requirement than similar sized insectivorous shrews (Genoud and

Vogel 1990). Therefore, in captivity red-tailed phascogales require high energy foods for maintenance. The main problem encountered in researching nutrition in captive phascogales is their preference for live food items. I found that in captivity they prefer live food and refuse items, such as minced meat and commercial small carnivore food

(Wombaroo), and only consume small amounts of mouse, rat and pinkie mice. Nutrition

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experiments with processed food items were attempted and abandoned due to low consumption rates and high refusal of food items. Nutrition experiments with phascogales therefore required mindful planning and interpretation of data. Optimising nutrition and feeding the preferred diet of the phascogales will ensure reproductive output is maximised. As red-tailed phascogale breeding season is very short, optimising conditions

is important for obtaining maximum numbers of offspring. If suitable numbers are

produced in captivity it can increase the success of translocations and therefore re-

establish populations of the red-tailed phascogale in parts of Australia where they once

occurred.

Compared to the phascogale, the kultarr has a long breeding season and low reproductive

output. Experiments on the kultarr were therefore organised around breeding so it did not

interfere with reproductive output. The kultarrs, unlike the phascogales, consumed all

food items provided to them, making them very suitable for nutrition trials. The kultarrs

were used for rate of passage trials and the phascogales could not be used as they refused

to eat minced meat.

The kultarr provides an interesting comparison with other dasyurids as it is the only

dasyurid to show a specialised morphological adaptation that differs from other family

members. The kultarr has elongated hind-limbs that may also be associated with

differences in physiological responses when compared with other dasyurids. Whether or

not this is significant is still unknown. Physiological differences within the dasyurid

family have been noted previously in quoll species. Physiology of each quoll species is

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influenced by differing environments; for example, some use torpor to cope with extreme

temperatures whilst other quolls do not (Cooper and Withers 2010).

Results I obtained from histological examination of the kultarr showed that internal gastrointestinal morphology is of similar structure and composition to other dasyurids

and marsupials. I also found transit times were consistent with the kultarrs body mass,

diet and gastrointestinal morphology. My nutrition experiments demonstrated that

maintenance energy requirements of the kultarr are low and lower than that of the

phascogale. It is likely that a low energy requirement is an adaptation to survive in an

arid environment where food availability is often restricted by adverse weather conditions

and climate. This species often uses torpor, which might contribute to the low energy requirements. Therefore, when maintaining kultarrs in captivity, they require relatively low food intakes compared to the phascogale. Their acceptance of food items means they are relatively easy to feed in captivity; however, the long breeding season and low reproductive output will hinder the success of captive population continuance. The information gathered in this research has been incorporated into captive management protocols for this species at UWS and ASDP. Ensuring nutritional requirements are met can contribute to the breeding success and contributes to the limited knowledge of kultarr biology.

Like the kultarr, the gastrointestinal tract of the fat-tailed and striped-faced dunnart is

simple. The digestive tract of these small dasyurids would have evolved to cope with the

insectivorous diet consumed by these animals in the wild. The nutrition experiments I

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conducted on the dunnarts found, per unit of body mass, that the smaller fat-tailed dunnarts have higher energy requirements in captivity. Therefore, the smaller dunnarts need to be fed more food per unit of body mass than the stripe-faced dunnarts. When maintained on processed foods, they often consumed their own body mass in food each day. The dunnarts readily accepted both live food and commercially available foods in captivity. Both dunnarts inhabit arid zones of Australia, therefore as with the kultarr, lower energy requirements are a characteristic of these species when compared with the phascogale. When maintained on high fat diets, dunnarts were able to store fat in their tail which could be used later in times of nutritional stress. These dunnart species have long been used as model species for studies on reproduction (Gardner et al. 1996; Selwood and Cui 2006) and on the role of food in lipid storage and activity patterns (Coleman et al. 1989; Hope et al. 1997a, 1997b; Kennedy et al. 1996; Hope et al. 1999; Ng et al. 1999;

Munn et al. 2010). They are also models for other endangered arid zone dunnart species.

Integrating the findings from my nutrition research into daily husbandry practices will improve nutrition in captivity that in turn can increase animal longevity and the continued use of these species as models in research.

The eastern quolls along with all other dasyurids studied in this thesis had high digestibility values for dry matter, gross energy and lipids. The colony I used for the nutrition experiments were a captive breeding colony and there are intentions to use this colony for reintroductions on mainland Australia. Working with captive colonies maintained by other organisations restricts timeframes and organisation of experiments.

My research on the quolls had to be conducted outside of the breeding season. It is a

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priority to increase reproductive output in eastern quolls to ensure enough animals are available for translocation. Often female quolls are unable to carry a full complement of pouch young all the way to weaning and nutrition may play an important role in pouch young survival. During reproduction and lactation food should be increased for females and increased up to three fold during peak lactation. The information gained in this study has enabled the information to be incorporated into the daily management/husbandry practices. This has included increasing food for females during breeding and further understanding of availability of nutrients in some of the diets provided to the captive colony.

From the animal responses (weight and tail width changes, food intake and digestibility values) presented in the nutrition studies it indicated that no single diet is appropriate for feeding captive dasyurids if fed alone. For example, mealworms have a low calcium and high digestible energy composition, which can cause obesity and/or calcium deficiency.

Maintaining any of the dasyurid species studied in this thesis on one diet type alone would lead to severe weight loss, obesity and/or nutrient deficiency related illnesses.

Ideally, in captivity a varied diet that provides a combination of a number of diets (i.e. cat

food, mealworms, and crickets) would provide nutrients in a range of absorbable

availabilities to adequately meet the nutrient requirements of the captive animals.

Additionally, live insect diets provide behavioural enrichment, and enhance mental and

physical stimulation for the animals. The ability of captive animals to catch live food also

increases the likelihood of their survival post-release.

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Nutrition influences haematology and blood chemistry levels in animals and these values

can be used to determine health and nutritional status of captive and translocated

populations. Haematology and blood chemistry data collected in this thesis has identified trends that relate to season and age of eastern quolls in the captive setting. My study provided further information to what is known for the eastern and spotted-tailed quolls.

Eastern quolls have very fine, deep veins which often led to only small blood samples

being obtained. Blood samples could only be analysed if a large enough sample was

collected quickly. In some cases blood clotted before it could be analysed and was

discarded, or sample volume was too small for blood chemistry analysis. However, in

total 26 samples were successfully analysed for blood chemistry which is the largest

sample size for eastern quoll blood chemistry to date. Due to difficulties accessing the

captive population of spotted-tailed quolls, a one off sample collection occurred and results were included to add to current knowledge and provide useful data for other researchers studying quoll blood. Originally kultarrs were going to be used in this experiment, however as they have very fine veins and it was not possible to warm the animals (to increase blood flow) prior to bleeding and I was unable to collect their blood.

The haematology and blood chemistry data gathered in this experiment can be used to assess the health of captive populations and can be used in the future to assess and compare with animals used in translocations.

All of the dasyurid species studied in this thesis as well as others are important to conserve due to significant geographical range contractions and the extinction of the eastern quoll on mainland Australia. Preservation of these species will contribute to

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conserving biodiversity in Australia and prevent further loss of native species.

Preservation of fauna species allows the natural intraspecifc and interspecific relationships to occur and ensures the ‘normal’ functioning of an ecosystem. Some of the

dasyurid species studied here have been or will be used for translocation in the future to

re-establish wild populations. The data presented here provides a basis for understanding

the nutritional requirements in captivity and animal responses to changes in their captive

diets. The haematology and blood chemistry levels obtained highlight the physiological

response to changes in season, nutrition and ageing in a captive eastern quoll population.

The data presented here has already had an impact on the management of captive

populations. It has informed wildlife managers of husbandry practices to maximise

captive health and has contributed to the continued effort of conserving Australian native

species.

8.1 Future directions

The results presented in this thesis have contributed to the basic understanding of biology

and physiology in a number of dasyurid species, providing the basis for further areas for

investigation into this poorly studied field. Observations were made of phascogales in

torpor on one occasion and further research into torpor use in red-tailed phascogales

would be helpful in understanding energy requirements and energy saving mechanisms in

this species. It would also be pertinent to understand how this relates to their environment

as reintroducing phascogales in arid zones may increase the use of this energy saving

response.

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Investigation into the nutrient requirements of male phascogales pre-mating and post- mating could determine the role nutrition plays in the survival of captive males post- mating. In the wild, male phascogales exhibit semelparity, which they do not exhibit in captivity (Foster et al. 2008). In captivity males become infertile but continue to live

(Foster et al. 2008). It is possible that access to energy rich foods after mating contributes to their survival in the captive setting.

Biological data for the kultarr is very limited, studies on locomotion (Ride 1965; Marlow

1969; Baudinette et al. 1976), maternal behaviour (Happold 1972), reproduction

(Woolley 1984; Stannard and Old 2010), torpor (Geiser 1986), and growth in captivity

(Caton 2007) have been undertaken. These have contributed to the fundamental biological data on this species however more research is needed. Further experiments on diet in the wild would be useful to understanding dietary preference, how they exploit prey items and how this is influenced by their environment. Experiments have not been undertaken to determine the diet of kultarrs in the wild and it is presumed they are insectivorous. However, in captivity my studies have shown they also consume small mammals. This needs to be clarified in future studies since the use of captive animals to gather information on wild populations needs to be properly informed by such correlation studies.

Further investigation of haematology and blood chemistry parameters for the spotted-

tailed quoll would provide a comprehensive data set for measuring health in these

species. Results from my research showed the spotted-tailed quoll amylase and alanine

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transaminase levels vary compared with other quoll species. Investigation of blood

parameters would indicate differences in physiological normalities such as ‘normal’

amylase levels. These species differences could be attributed to evolution to cope with differing environments. Studies of haematology and blood chemistry in wild eastern and

spotted-tailed populations would provide a data set for health assessment in wild animals.

A long term study would highlight differences in haematology and blood chemistry levels

for different life stages, reproductive states and nutritional status. Blood chemistry can be

used to understand nutritional fluctuation and availability of food within the quoll’s

habitat. The continued study of eastern quoll blood parameters once animals have been

released would enable the development of a baseline data set to measure health of

translocated animals. Continued surveillance of these parameters post-release would also

assist in measuring stress levels in quolls and their adjustment to the new environment.

Further studies of activity patterns and energy usage in these captive dasyurids would

increase understanding of captive nutrition requirements. Research into energy

requirements for reproducing/lactating females would contribute to understanding the

increased nutrient requirements of reproducing females.

Humans have dramatically altered the Australian landscape and farming practices in

particular has negatively impacted dasyurids due to habitat clearing and poisoning

potential food items. Research in habitats that dasyurids (particularly the red-tailed phascogale and eastern quoll) currently occupy to investigate diet, species interactions and availability of prey would assist in understanding the desired nutritional

241

characteristics of a particular species within a habitat. A recent study by Short et al.

(2011) has identified key habitat features for red-tailed phascogales such as dense canopy cover and presence of tree hollows. Studies such as that on habitat requirements combined with studies of diet availability within a habitat will ensure suitable sites are chosen for future translocations and reintroductions of the red-tailed phascogale.

With the research presented in this thesis biology and ecology of dasyurid species a greater understanding has emerged about how to appropriately manage and maintain dasyurids in captivity. The research has increased our fundamental knowledge of dasyurid physiology and has informed our understanding of how these animals have evolved to cope with their environment. Future research will contribute to the continued success of captive breeding, translocation and reintroduction programs.

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CSIRO PUBLISHING www.publish.csiro.au/journals/am Australian Mammalogy, 2010, 32, 179–182

Observation of reproductive strategies of captive kultarrs (Antechinomys laniger)

Hayley J. Stannard A,B and Julie M. Old A

ANative and Pest Animal Unit, School of Natural Sciences, University of Western Sydney, Locked Bag 1797, Penrith South DC, NSW 1797, Australia. BCorresponding author: Email: [email protected]

Abstract. Captive kultarrs (Antechinomys laniger) were bred from June to February. Their gestation period was longer than 12 days, as suggested by previous research. Pouches were observed to determine whether births occurred. Individuals were capable of producing offspring in their second and third year of life, which has been suspected previously, but never demonstrated in other published studies.

Additional keywords: dasyurid, embryonic diapause, gestation, sperm storage.

Introduction There has been limited success breeding the kultarr in The kultarr (Antechinomys laniger) is a terrestrial mammal captivity, with only one captive study producing offspring (Caton fi inhabiting arid areas of central Australia (Lidicker and Marlow 2007). Caton (2007) successfully bred two rst-year females. 1970; Valente 2008). A small dasyurid marsupial, it is a fawn to Another study utilised wild-caught females with pouch young to sandy-brown colour with a white belly (Valente 2008) and weighs investigate reproduction; captive matings were attempted but 17–30 g (males) and 14–29 g (females). They have large ears, proved unsuccessful (Woolley 1984). The limited success of protruding eyes and a long tail with a brush-like tip (Woolley breeding this species in captivity is most likely due to the solitary 1984; Valente 2008). The IUCN red list categorises this species as nature of the kultarr, that it is not well adapted to the captive ‘least concern’ (Morris et al. 2008). environment and there is a lack of detailed biological information Reproductive studies of the kultarr have determined that it is available. The kultarrs were bred to maintain a captive colony and polyoestrous andin captivity has a longbreeding seasonfrom July this research details the observations made during the 2009 to January (Woolley 1984). Both males and females reach sexual breeding season. maturity at 11.5 months of age and are thought to be able to breed in more than one season (Woolley 1984). Gestation is believed to Methods be 12 days; however, 17 days has been noted for one female The kultarrs described here are part of a captive population housed (Woolley 1984; Caton 2007). When the young are born, they at the Hawkesbury Campus, University of Western Sydney attach themselves to the teats for 30–48 days before being left in (UWS), Richmond, New South Wales. The animals were the nest while the mother forages (Valente 2008). In captivity, maintained individually in wooden enclosures. Animals were juveniles are weaned at 80–90 days, after which they become exposed to natural photocycles and room temperature was independent solitary animals (Happold 1972; Woolley 1984; maintained at 22 Æ 3C. Animals were fed a range of insects and Valente 2008). rat, and provided with fresh water ad libitum. Previously, in the Dasyurids are classified by their reproductive strategy – the 2007 breeding season, the kultarrs (5 males and 3 females) were kultarr is Strategy V (polyoestrus, ) (Lee et al. bred from June to January. The breeding roster involved placing a 1982). Other dasyurid species that exhibit Strategy I, for example, male and female together for two weeks and then separating them Antechinus spp., have been studied in much more detail and for two weeks over the six-month period. One female (#8) much more is known about their reproductive biology (Selwood produced five pouch young. The pouch young were first sighted 1980; Selwood and McCallum 1987; Woolley 1991; Shimmin 27 days after the removal of the mate. In 2008 the breeding roster et al. 2000; Kraaijeveld-Smit et al. 2002; Parrott et al. 2006; was attempted again from June until January; however, no Foster et al. 2008; Lane et al. 2008). Strategy I breeders are offspring were produced. monoestrus synchronised breeders, and all males die off after Eight (3 male and 5 female) adult kultarrs made up the their first breeding season (Lee et al. 1982). In captivity, breeding population at the start of the 2009 breeding season (Table 1). The rosters for these species can be synchronised to a very breeding program began in early winter, males and females were specific short timeframe with rotation of multiple males for each first introduced for 7–14 days, followed by a 21–28 day break female. before being paired again. Females A1 and B2 were paired with

Ó Australian Mammal Society 2010 10.1071/AM10011 0310-0049/10/020179 180 Australian Mammalogy H. J. Stannard and J. M. Old

Table 1. Kultarrs in the University of Western Sydney’s captive have also shown epithelial cells produced in urine and weight gain population at the beginning of the 2009 breeding season before the introduction of a male (Woolley 1984; Caton 2007). Age is measured in months from birth to the start of the 2009 breeding season After pairs were separated, the kultarrs were weighed and observed regularly (Table 2). Pouches of females were examined Kultarr Id. Sex Age (months) Previous offspring to detect whether they were still in oestrus (either recorded as A1 F 18 No pouch or no pouch) or if pouch young were present in the pouch. A3 F 31 No The same males and females were paired together for the first five A6 F 18 No matings, due to relatedness and a lack of males. Kultarrs were B2 F 31 Yes paired on the basis of the results from a mate-choice study 03 F 17 No conducted before the 2009 breeding season (M. Parrott et al., 06 M 31 Yes unpubl.). The results of that study showed a female’s perceived 02 M 17 No receptiveness to a particular male based on olfactory cues. In the 05 M 17 No present study, female kultarrs were paired and reproduced with their first-preference male. Male 05, Females 03 and A6 were paired with Male 06, and Female A3 was paired with Male 02. The breeding roster was repeated several times until the end of February 2010. A similar Results and discussion breeding roster was used by Caton (2007); however, the length of Female B2 had three pouch young present 44 days after the mating and rest time between pairings was 7 days shorter than the removal of the male from the November mating. A second female program used at UWS. (A6) had three pouch young present 25 days after the removal of Prior to the introduction of a mate, oestrus was detected by the male (Fig. 1). When the females’ pouches were checked a observations of changes to the pouch (size, colour and secretions) second time, there was an increase in both litters, B2 had four and and increased bodyweight as described by Hesterman et al. A6 had six pouch young. Kultarrs are ableto rear up to eight pouch (2008) for the spotted-tailed quoll (Dasyurus maculatus) and young, as that is the greatest number of teats recorded in a female Tasmanian devil (Sarcophilus harrissii). In other dasyurids, such pouch (Lidicker and Marlow 1970; Woolley 1984). The length of as the red-tailed phascogale (Phascogale calura), it is thought that time between removal of the male and parturition suggests that the the introduction of a male may initiate the onset of oestrus (Foster gestation period may be longer than previously thought. It is et al. 2008).In kultarrs it appears thatthis maynot be thecase, with possible that the female kultarrs were exhibiting either sperm signs of oestrus (changes to the pouch) occurring before the storage or delayed development of the embryo, or the effects of introduction of any males. Similarly, previous studies on kultarrs torpor may have lengthened the gestation period.

Table 2. Pairings and observations made during the 2009 breeding season On each day noted below in the table all animals were weighed and a visual inspection of health (tail fatness, coat condition) was made and recorded

Day Observations 0 Weight and pouch check 29 Males and females paired 43 Separated pairs 59 Males and females paired 66 Pairs separated 93 Males and females paired 113 Pairs separated 134 Males and females paired 157 Pairs separated 166 Males and females paired 172 Pairs separated. B2: pouch young  3 178 Males and females paired. B2: pouch young  4 – tails and ears brown in colour 187 A6: pouch young  3 191 B2: pouch young fixation period complete, eyes closed, ears opening 206 A6: pouch young  6 – ears and eyes not open, fixation period complete. B2: pouch young – no teeth, eyes and ears open 218 Males and females paired. B2: pouch young weighed and sexed, 2 males and 2 females 232 B2: one male pouch young found deceased, cause unknown 237 Pairs separated 246 A6: pouch young eating solid food (cockroaches) 257 Pouch check of A1, A3, 03 – no pouch young 264 Pouch check of A1, A3, 03 – no pouch young 274 Pouch check of A1, A3, 03 – no pouch young. A6: pouch young sexed, 4 males and 2 females Kultarr reproduction Australian Mammalogy 181

1986). It is therefore possible that pregnant kultarrs entered torpor, which may have lengthened gestation. Multiple paternity would be a suitable strategy for kultarrs in the wild as population densities are believed to be low and fluctuate seasonally (Valente 2008). Multiple paternity has been exhibited by other dasyurids such as the (Kraaijeveld-Smit et al. 2002), red-tailed phascogales (Foster et al. 2008) and spotted-tailed quolls (Glen et al. 2009) presumably to increase fitness and genetic diversity and produce offspring that will have a greater chance of survival. It is also displayed in eutherian red squirrels (Tamiasciurus hudsonicus) from North America; however, this is believed to be for different costs/benefits from those described above. Instead, it is thought that red squirrels allow multiple paternity to decrease the cost involved in evading interested mates (Lane et al. 2008). Whilst multiple paternities are exhibited in some dasyurids, particularly those with semelparity to increase genetic diversity, it may also be beneficial to wild kultarr populations. Previously, Caton (2007) successfully bred first-year females. In the present study, female B2 was three years old at the time of producing her 2009 litter, which was her second litter. She is believed to be the oldest female to reproduce in captivity. The male that sired her young was 1.5 years old. Female A6 was two years old at the time of producing her litter, which was her first litter. The male that sired her young was three years old and had sired a litter previously. These results show that both males and females are capable of reproducing in their second and third year, Fig. 1. Pouch young of female A6 at ~15 days of age. with 50% of the total population over one year of age producing offspring. Previous studies have shown that both male and female kultarrs may breed through the onset of oestrus and spermatozoa It is interesting to note that there was an increase in the litter production (Woolley 1984; Caton 2007); however, there are no size of both females between pouch checks. It may be that the published records of successful breeding (i.e. production of pouch young were hidden behind folds of pouch skin or their litter offspring) from animals after their first season. It was suggested mates during pouch checks; however, an increase in litter size has by Caton (2007) that ‘older’ females may not have reproduced been seen previously in brush-tailed phascogales (Phascogale due to age, change of environment (wild to captive setting) or tapoatafa) and numbats (Myrmecobius fasciatus) (Millis et al. were post-productive. It may also be possible that nutrition, 1999). Other dasyurid species, for example, the red-tailed change in nutrition (and diet) or mate choice may have been phascogale, eastern quoll (Dasyurus viverrinus) and Tasmanian factors. Three years of age is relatively old for a kultarr, with the devil, give birth to numerous pouch young. Not all the young can oldest captive female recorded as 48 months old (Aslin 1982). be accommodated in the pouch and the first eight, six and four However, a female kultarr has lived for 67 months in the UWS pups (respectively) that reach the teats are able to fuse and live captive population (pers. obs.). It is believed that the conditions (Bradley et al. 2008; Jones 2008a, 2008b). provided in captivity (e.g. adequate nutrition, space, Sperm storage has been noted in the red-tailed phascogale temperature, daylength and a suitable mate) were suitable for (Foster et al. 2008), kaluta (Dasykaluta rosamondae), brown these older animals to reproduce. antechinus (Antechinus stuartii) and agile antechinus (Antechinus This study has shown that gestation of the kultarr is longer than agilis) (Selwood and McCallum 1987; Woolley 1991; Shimmin 12 days, suggesting that sperm storage and/or a pause in et al. 2000). Delayed embryo development may be due to embryonic development is being utilised. It has also shown that environmental conditions, food resources or females waiting for captive-bred males and females are capable of producing another mate to increase sperm competition and possibly multiple offspring in their second and third year of life. Further paternity. Embryonic diapause has been documented in investigation is required into whether, and for how long, sperm macropods and honey possums (Tarsipes rostratus), with storage and embryonic development occur in the kultarr. environmental resource availability and photoperiod being controlling factors (Taggart et al. 2005; Oates et al. 2007). This Acknowledgements diapause has also been recorded in one dasyurid, the brown We thank the technical staff of the School of Natural Sciences for assistance in antechinus (Antechinus stuartii), in which two periods of maintaining the colony on a daily basis and Wes Caton for his helpful embryonic diapause were observed (Selwood 1980). Torpor has suggestions and expertise on kultarr breeding. Thank you to Lauren Young been observed in a pregnant stripe-faced dunnart (Sminthopsis and Marissa Parrott for providing feedback on a previous version of this macroura) (Geiser et al. 2005); similarly, kultarrs use torpor, manuscript. We acknowledge the School of Natural Sciences at the University which is influenced by food availability and temperature (Geiser of Western Sydney for colony support. Colony maintenance and reproduction 182 Australian Mammalogy H. J. Stannard and J. M. Old

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