Tulk et al. SpringerPlus (2016) 5:1479 DOI 10.1186/s40064‑016‑3111‑7 Open Access RESEARCH Haematology and serum biochemistry in captive Australian native murids: black‑footed tree‑rat (Mesembriomys gouldii) and greater stick‑nest rat (Leporillus conditor) Melissa L. Tulk1, Hayley J. Stannard2 and Julie M. Old1* *Correspondence: j.old@westernsydney.edu.au 1 School of Science and Health, Hawkesbury, Western Sydney University, Locked Bag 1797, Penrith, NSW 2751, Australia Full list of author information is available at the end of the article Abstract The black-footed tree-rat (Mesembriomys gouldii) and greater stick-nest rat (Leporillus conditor) are near threatened and vulnerable native Australian murids. There is a paucity of health and welfare knowledge for these species and native murids in general. In this paper we aimed to address this deiciency in knowledge by describing some key haematological and blood biochemistry parameters for these species. Haematology and blood biochemistry data were obtained from clinical histories of the two murid species held in captivity at Taronga Zoological Park, Mosman, Australia. The data were analysed to establish conidence intervals for each parameter available and leukocyte morphology described. White blood cell counts were higher in females than males. Both species also had high neutrophil:lymphocyte ratios (tree-rat ratios were almost even). Haematocrit was higher in male stick-nest rats than females. Diferential leukocyte counts and leukocyte morphology was consistent with previous descriptions in other murids and between individuals. Blood biochemistry values were unremarkable except for the high level of globulin in stick-nest rats. The values provided in the study will add to the knowledge of health data for murids in captivity and aid captive and natural management of Australian native murids. Keywords: Australian native rodent, Blood, Health, Leukocyte morphology Background he continuous decline of Australian mammals has occurred since European settlement (Burbidge and McKenzie 1989) and is inluenced by a combination of factors. he Muridae family, the only family of rodents found in Australia, is not exempt from this decline. hey currently account for up to 40 % of all Australian mammalian species (Breed and Ford 2007), with at least 57 currently recognised (Van Dyck and Strahan 2008). Animals with a larger body mass are able to maintain a suicient population density, decreasing the risk of extinction, leaving the smaller animals, such as murids (in the critical weight range of 25–500 g) (Johnson and Isaac 2009) with a higher rate of extinction risk (Burbidge and McKenzie 1989). he near threatened (IUCN 2014) black-footed tree-rat (Mesembriomys gouldii) is one of Australia’s largest rodents weighing 550–800 g (Breed and Ford 2007). he arboreal © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Tulk et al. SpringerPlus (2016) 5:1479 species is only found in three remote locations: Kimberly/mainland Northern Territory, north Queensland and Melville Island. Due to their solitary behaviour and large home ranges (>60 ha) (Breed and Ford 2007; Griiths et al. 2002) population numbers are dificult to determine but are estimated at 10,000–12,000 in each location. Residing in tree hollows, this species is vulnerable to ires, habitat loss, fragmentation and degradation. Predation is also a major threat and has contributed to the decline of this species with a 30–50 % population decline in the last decade (Woinarski et al. 2014). he greater stick-nest rat (Leporillus conditor) is a vulnerable species (IUCN 2014) previously found across much of the semi-arid and arid zone of Australia (Van Dyck and Strahan 2008). Currently, there is only one natural population located on Franklin Island in the Nuyts Archipelago, South Australia (Robinson 1975). he species has been bred in captivity and successfully re-introduced to a conservation reserve near Roxby Downs (South Australia), Reevesby Island (South Australia) (Arid Recovery 2007), St Peters Island (South Australia) and Salutation Island (Western Australia) (Morris 2000). Weighing 180–450 g, the ground dwelling stick-nest rat is vulnerable to predation by native and introduced predators. Due to constant threats to the long-term population survival of the two species, in situ conservation eforts alone are not suicient to ensure their survival. Longterm survival of these wild populations relies on maintaining captive populations, and maintaining the health of these animals is critical in the captive setting. It is also very often assumed that the health parameters of native murids are the same as domesticated laboratory murids despite the two groups of murids being distinctly separated taxonomically at the sub-Family level (Van Dyck and Strahan 2008). In addition, the causes and consequences of disease in native murid species are generally poorly understood, particularly for Australian species, and long-term data sets are required for health and disease studies. For example, there is generally a paucity of information regarding the haematology, serum biochemistry and leukocyte morphology for most Australian native murids, with only a few having previously been documented for captive specimens (Bradley et al. 1988; Kemper et al. 1987; Monamy 1995; Old et al. 2005, 2007). Haematology and serum biochemistry values for the black-footed tree-rat and greater stick-nest rat are needed as diferences in physiology and life history traits compared to other murids, means values for other species are not directly comparable. his study aimed to establish baseline conidence intervals for haematology, serum biochemistry and leukocyte morphology for these two species living in captivity. he information obtained may aid the conservation of black-footed tree-rats and greater stick-nest rats, and increase our knowledge of the biology of captive Australian native murids. Methods Study animals he animals used in this study were all from the captive colonies housed at Taronga Zoological Park, Mosman, NSW, Australia. hese animals were housed in species-speciic enclosures. Male tree-rats were held in a 19.7 m2 enclosure, and the females in a 3.1 m2 enclosure. All stick-nest rats were held in a 17.5 m2 enclosure. he enclosures Page 2 of 16 Tulk et al. SpringerPlus (2016) 5:1479 contained dirt/leaf litter or sand substrate with structural complexities provided by lora and browse. he animals were fed on a mixed diet of insects, vegetation, seeds, nuts, and fruit. All samples were obtained from the park’s medical reports from October 1995 to February 2015. Animals were categorized as ‘healthy’ or ‘unhealthy’ based on the clinical notes provided. Blood samples were taken as part of routine health checks, quarantine procedures or enquiries based on clinical symptoms of illness. Blood collection and analysis he animals were removed from their enclosure and taken to the park’s in-house wildlife hospital for blood collection and analysis. Animals were anesthetized with isolurane/O2 during blood collection though an induction chamber and maintained through a facemask. Extraction location of the blood samples varied between species and individuals from the saphenous, femoral and jugular vein/artery. White blood cell (WBC) counts (×109 cells/L) were determined using improved Neubaur counting chambers by Taronga pathology at time of blood collection, if not, WBC counts were manually determined at Western Sydney University using the original blood smears. The leukocyte, platelet, differential leukocyte count and leukocyte morphology were determined from Diff Quik (Thermo Fisher, Scoresby, Victoria) stained blood smears. Digital images of the leukocytes were obtained using a BX60 microscope (Olympus, Japan) with a ProgRes C14 camera (Jenoptik, Germany). Once extracted, blood biochemistry values were measured using a Relotron Instrument and IDEXX Vet Test (IDEXX Laboratories, Rydalmere, NSW), until late November 2010, when the REM systems VetScan2 and associated consumables (REM Systems, North Ryde, NSW) were used. Rotor plates were used to determine the values of 26 different blood parameters. he parameters included haematological parameters: estimated platelets (per/HOIF), haemoglobin (HGB) (g/L) and haematocrit (HCT) (%). In addition we analysed the serum biochemical parameters: Gamma-glutamyl transpeptidase (GGT) (U/L), lipase (U/L), chloride (mmol/L), total carbon dioxide (CO2) (mmol/L), creatine kinase (U/L), glucose (mmol/L), blood urea nitrogen (BUN) (mmol/L), creatinine (mmol/L), calcium (mmol/L), phosphorus (mmol/L), sodium (mmol/L), potassium (mmol/L), total protein (g/L), albumin (g/L), globulin (g/L), alanine transaminase (ALT) (U/L), total bilirubin (μmol/L), amylase (U/L), alkaline phosphatase (ALP) (U/L) and aspartate aminotransferase (AST) (U/L). Data analysis All values from ‘healthy’ individuals were analysed, including those samples obtained from the same individual, at diferent times. We conducted the analysis in this way because of the paucity of information available on Australian native murids generally. By utilising all samples available it provides a more accurate representation of the true values for each parameter, and hence provides more accurate conidence intervals, than if we only included one value randomly chosen for each individual. Using the Analysis Toolpak available on Microsoft Excel 2010, 90 % conidence intervals for each parameter were determined. Due to the large number of diferent conditions and diseases afecting Page 3 of 16 Tulk et al. SpringerPlus (2016) 5:1479 the murids’ classiied as clinically ‘unhealthy’, none of the parameters for these individuals were statistically analysed. We have however included the parameters of those individuals classiied as clinically ‘unhealthy’ in this paper, to provide an indication of those values that are more likely to suggest poor health. Results In this study, data from 37 captive individuals (some sampled multiple times) were included, from samples collected over two decades. he past two decades saw a large change in how blood was analysed and what animal healthcare professionals investigated when analysing blood. A number of parameters were only tested on one individual and were therefore excluded from the results, as it was not regarded as representative of the species. Each individual sample was classiied as ‘healthy’ or ‘unhealthy’, as determined by the accompanying clinical notes. Statistical comparisons were only made on ‘healthy’ animals. Animals that were ‘unhealthy’ varied in their conditions and were therefore not collectively analysed, and were included as individual samples rather than as part of a population sample. A total of 32 black-footed tree-rat samples consisting of 25 ‘healthy’ (18 female and 7 male) and seven ‘unhealthy’ (3 female and 4 male) samples, aged between 0.6 and 7.1 years old were used in this study. Greater stick-nest rat samples were obtained from 25 ‘healthy animals’ (18 female and 7 male), aged between 0.3 and 6.3 years old and one ‘unhealthy’ female aged 2.8 years (Table 1). Haematology Results for haematology of ‘healthy’ black-footed tree-rats and greater stick-nest rats are shown in Tables 2 and 3. Mean total WBCs were higher in female tree-rats compared to males. he majority of female tree-rats between the ages of 2.5–5.0 years had total WBCs counts over 18 × 109 cells/L (up to 52.4 × 109 cells/L); with younger females (<2.5 years) below 10.0 × 109 cells/L and all male tree-rats below 20.0 × 109 cells/L. Stick-nest rat mean total WBCs were higher in females compared to males. HCT mean levels were higher in male stick-nest rats compared to females. Tree-rats had similar mean percentages of neutrophils (44.1 %) and lymphocytes (48.5 %), while the stick-nest rats had a higher mean percentage of neutrophils (63.9 %) compared to lymphocytes (32.0 %). he range of N:L ratios in both species was large; tree-rats 0.1–6.1 (x̄ = 1.4) and stick-nest rats 0.3–11.5 (x̄ = 2.2). Morphology of blood cells Only neutrophils, lymphocytes, and monocytes were observed in the blood smears of both species. No eosinophils or basophils were observed in blood smears. he nucleus of the neutrophils of the tree-rat were hypersegmented and polymorphonuclear, containing between 3 and 4 large lobes, and ranging from 5 to 10 μm (Figs. 1, 2). Tree-rat lymphocytes measured 5–12 μm (Fig. 2). Tree-rat monocytes measured 5–11 μm in diameter and had reticular chromatin and a moderate level of basophilic cytoplasm with vacuoles (Fig. 2). he greater stick-nest rat neutrophils observed had a darkly stained nucleus containing minimal deinitive segmentation, with 3–4 large lobes, and varied in size between 9 and Page 4 of 16 Tulk et al. SpringerPlus (2016) 5:1479 Table 1 Haematology values for healthy black-footed tree-rats Parameter Mean ± SD 9 WBC count (×10 cells/L) Haemoglobin (g/L) Haematocrit 90 % CI lower limit 90 % CI upper limit Male mean ± SD Female mean ± SD 18.70 ± 13.87 (n = 22) 13.84 23.57 8.76 ± 4.68 (n = 7) 23.34 ± 14.37 (n = 15) 143.05 ± 22.88 (n = 21) 135.19 150.90 145.57 ± 19.79 (n = 7) 141.79 ± 23.46 (n = 15) 43.77 ± 5.78 (n = 22) 41.75 45.80 44.86 ± 6.91 (n = 7) 43.27 ± 5.36 (n = 14) Neutrophil (%) 44.11 ± 19.92 (n = 22) 37.13 51.09 45.70 ± 21.10 (n = 7) 43.37 ± 20.06 (n = 15) Lymphocyte (%) 48.54 ± 20.71 (n = 23) 41.43 55.64 43.43 ± 18.69 (n = 7) 50.77 ± 21.76 (n = 16) Monocyte (%) 4.08 ± 7.78 (n = 21) 3.08 5.08 2.99 ± 1.55 (n = 6) 4.52 ± 3.07 (n = 15) Eosinophil (%) 6.63 ± 5.55 (n = 19) 4.53 8.72 9.67 ± 7.13 (n = 6) 5.22 ± 4.28 (n = 13) N:L ratio 1.39 ± 1.34 (n = 22) 1.86 2.96 1.74 ± 2.09 (n = 7) 1.22 ± 0.92 (n = 15) 28.08 ± 24.68 (n = 16) 17.93 38.23 15.67 ± 10.89 (n = 6) 35.53 ± 28.02 (n = 10) Est. platelets (/HOIF) N:L neutrophil to lymphocyte ratio, CI conidence intervals Page 5 of 16 Tulk et al. SpringerPlus (2016) 5:1479 Table 2 Haematology values for healthy greater stick-nest rats Parameter Mean ± SD 9 WBC count (×10 cells/L) Haemoglobin (g/L) 11.25 ± 4.87 (n = 25) 111.78 ± 21.95 (n = 9) 90 % CI lower limit 90 % CI upper limit 9.65 12.85 99.74 123.81 Male mean ± SD 9.00 ± 4.28 (n = 11) 103.33 ± 21.87 (n = 6) Female mean ± SD 13.01 ± 4.56 (n = 14) 128.67 ± 7.85 (n = 3) Haematocrita 34.08 ± 5.07 (n = 25) 32.00 36.00 36.73 ± 3.79 (n = 11) 32 ± 4.97 (n = 14) Neutrophil (%) 63.89 ± 17.36 (n = 25) 58.18 69.60 57.01 ± 16.17 (n = 11) 69.29 ± 16.31 (n = 14) Lymphocyte (%) 32 ± 16.22 (n = 25) 26.66 37.33 37.29 ± 15.30 (n = 11) 27.83 ± 15.69 (n = 14) Monocyte (%) 4.49 ± 3.79 (n = 18) 3.02 5.96 5.66 ± 4.52 (n = 9) 3.33 ± 2.36 (n = 9) Eosinophil (%) 2.08 ± 1.52 (n = 13) 1.39 2.78 1.84 ± 1.49 (n = 6) 2.30 ± 1.52 (n = 7) N:L ratio 3.23 ± 3.00 (n = 25) 27.89 46.22 1.85 ± 0.86 (n = 11) 37.05 ± 24.29 (n = 19) 2.00 4.00 Est. platelets (/HOIF) 28.67 ± 10.48 (n = 6) 4.32 ± 3.58 (n = 14) 44.60 ± 30.03 (n = 10) N:L neutrophil to lymphocyte ratio, CI conidence interval Page 6 of 16 Parametera GGT (U/L) Mean ± SD 90 % CI lower limit 90 % CI upper limit Male mean ± SD Female mean ± SD 12.55 ± 3.46 (n = 2) 8.52 16.58 – 12.55 ± 3.46 (n = 2) Chloride (mmol/L) 106.00 ± 1.41 (n = 2) 104.36 107.64 – 106.00 ± 1.41 (n = 2) Total CO2 (mmol/L) 20.35 ± 10.96 (n = 2) 7.60 33.10 – 20.35 ± 10.96 (n = 2) 0.95 ± 0.21 (n = 2) 0.70 1.19 – 12.06 ± 7.32 (n = 20) 9.37 14.75 Cholesterol (mmol/L) Glucose (mmol/L) BUN (mmol/L) Creatinine (mmol/L) 6.62 ± 2.24 (n = 21) 5.82 7.42 46.28 ± 16.37 (n = 17) 39.77 52.79 12.45 ± 6.77 (n = 5) 7.03 ± 1.18 (n = 6) 50.00 ± 12.35 (n = 4) 0.95 ± 0.21 (n = 2) 11.93 ± 7.71 (n = 15) 6.45 ± 2.56 (n = 15) 45.14 ± 17.63 (n = 13) Calcium (mmol/L) 2.56 ± 0.18 (n = 9) 2.47 2.66 2.55 ± 0.20 (n = 3) 2.57 ± 0.18 (n = 6) Phosphate (mmol/L) 1.55 ± 0.45 (n = 11) 1.32 1.77 1.69 ± 0.42 (n = 4) 1.46 ± 0.47 (n = 7) Sodium (mmol/L) 139.93 144.07 4.06 ± 1.41 (n = 11) 3.36 4.76 Total protein (g/L) 56.33 ± 13.48 (n = 12) 49.93 62.73 51.20 ± 19.04 (n = 5) 60.00 ± 7.33 (n = 7) Albumin (g/L) 46.73 ± 11.76 (n = 11) 40.90 52.56 50.50 ± 7.94 (n = 4) 44.57 ± 13.56 (n = 7) Globulin (g/L) 11.73 ± 11.48 (n = 11) 6.03 17.42 5.00 ± 6.22 (n = 4) 15.57 ± 12.37 (n = 7) ALT (U/L) 60.03 ± 34.61 (n = 14) 44.81 75.24 61.48 ± 9.49 (n = 5) AST (U/L) 95.04 ± 58.06 (n = 10) 63.20 126.88 Potassium (mmol/L) Total bilirubin (μmol/L) Amylase (U/L) ALP (U/L) 142.00 ± 3.77 (n = 9) 4.61 ± 2.07 (n = 10) 1996.89 ± 2215.15 (n = 9) 82.69 ± 72.95 (n = 13) 3.53 5.68 780.71 3213.06 49.41 115.97 140.67 ± 2.31 (n = 3) 3.25 ± 2.21 (n = 4) 76.70 ± 29 (n = 3) 6.33 ± 2.31 (n = 3) 1037 ± 39.40 (n = 3) 54 ± 36.30 (n = 4) Tulk et al. SpringerPlus (2016) 5:1479 Table 3 Blood biochemistry values for healthy black-footed tree-rats 142.67 ± 4.37 (n = 6) 4.52 ± 0.44 (n = 7) 59.22 ± 43.58 (n = 9) 102.90 ± 71.41 (n = 7) 3.87 ± 1.58 (n = 7) 2476.83 ± 2653.75 (n = 6) 95.44 ± 83.03 (n = 9) CI conidence interval a Lipase (U/L) and Creatine kinase (U/L) were not included due to numerical irregularities Page 7 of 16 Tulk et al. SpringerPlus (2016) 5:1479 Fig. 1 A male black-footed tree-rat multi-lobed neutrophil (left) with two distinct lobes and a lymphocyte (right). Scale bar 20 μm Fig. 2 Two neutrophils (left and right) and a vacuolated monocyte (center) from a male black-footed tree-rat. Scale bar 20 μm 21 μm in diameter (Fig. 3). Occasional neutrophils with an annular nucleus were seen. Lymphocytes were 6–10 μm in diameter (Fig. 4). he monocytes measured 6–17 μm in diameter and the nucleus was indented giving it a horseshoe appearance (Fig. 5). Serum biochemistry Serum biochemistry values for the black-footed tree-rat (Table 4) and greater stick-nest rat (Table 5) were similar to previously reported murids for most parameters analysed. Stick-nest rats however had a high globulin concentration (Table 5) in comparison to other murids (Bradley et al. 1988; Kemper et al. 1987; Monamy 1995; Old et al. 2005, 2007; hrall et al. 2012). All other values are reported in Table 5. Unhealthy animals Individual murids were classiied as ‘healthy’ or ‘unhealthy’ based on the notes provided in their medical reports. Although no statistical comparisons were possible, we were Page 8 of 16 Tulk et al. SpringerPlus (2016) 5:1479 Fig. 3 Multi-lobed neutrophil from a male greater stick-nest rat. Scale bar 20 μm Fig. 4 A male greater stick-nest rat lymphocyte with very little cytoplasm visible. Scale bar 20 μm able to identify some observational diferences between animals classiied as ‘healthy’ and ‘unhealthy’. For example, one ‘unhealthy’ tree-rat with hypopyon noted in the medical report, had elevated glucose, creatinine, globulin and ALT, and low BUN and albumin concentrations compared to ‘healthy’ animals (Table 6). Another ‘unhealthy’ male tree-rat said to have lost weight had elevated levels of phosphate and potassium, and lower levels of globulin and ALP (Table 6). Both of these male tree-rats had a lower total WBC and lymphocyte count and elevated neutrophils compared to the ‘healthy’ tree-rats (Table 2). he animal with hypopyon also had an elevated N:L ratio. he ‘unhealthy’ stick-nest rat had lower levels of glucose, BUN, creatinine, phosphate, ALT, and ALP than that of the ‘healthy’ stick-nest rats. his stick-nest rat also had elevated platelets and high N:L ratio (Table 6). Page 9 of 16 Tulk et al. SpringerPlus (2016) 5:1479 Fig. 5 Monocyte from a male greater stick-nest rat with slightly indented nucleus to form a horseshoe shape. Scale bar 20 μm Discussion In comparison to other captive murids, the captive Australian native tree-rats and sticknest rats presented diferences in their leukocyte morphology, haematology and serum biochemistry. he haematology and serum biochemistry values were relatively consistent between individuals, despite the use of diferent analysis equipment and regardless of some diferences in collection methods between individuals. WBC counts were higher in females in both species. Both species also had high N:L ratios (tree-rat ratios were almost even). HCT was higher in male stick-nest rats than females. Diferential leukocyte counts and leukocyte morphology was consistent with previous descriptions in other murids and between individuals. Blood biochemistry values were unremarkable except for the high level of globulin in stick-nest rats when compared to previous murid research (Bradley et al. 1988; Kemper et al. 1987; Monamy 1995; Old et al. 2005, 2007; hrall et al. 2012). Healthy specimens of both species had elevated total WBC counts in comparison to the other murids (Bradley et al. 1988; Kemper et al. 1987; Monamy 1995; Old et al. 2005, 2007; hrall et al. 2012). Tree-rats had a mean WBC count that was almost double that reported previously for murids, while the stick-nest rats were within the expected range for murids, but at the higher end. Stick-nest rats had a higher WBC count when compared to other murids (Bradley et al. 1988; Kemper et al. 1987; Monamy 1995; Old et al. 2005, 2007; hrall et al. 2012) and had a small standard deviation, suggesting the values are likely to be a true indication of ‘healthy’ stick-nest rat WBC counts. he diferences in tree-rat mean WBC counts were diferent between the two sexes, females having higher counts. A larger sample size is needed to accurately determine species reference values (Table 2). Both species had neutrophilia, as animals were classiied as ‘healthy’ and did not show signs of inlammation, the cause of the condition can be assumed to be physiologic as a result of epinephrine or from stress (Harvey 2012). Neutrophils, usually make up 20–30 % (Provencher Bolliger and Everds 2012) of leukocytes. In tree-rats (44 %) and stick-nest rats (64 %) numbers of neutrophils were much higher than anticipated. Page 10 of 16 Tulk et al. SpringerPlus (2016) 5:1479 Table 4 Blood biochemistry values for healthy greater stick-nest rats Parameter Creatinine kinase (U/L) Glucose (mmol/L) BUN (mmol/L) Mean ± SD 123.00 ± 55.93 (n = 14) 90 % CI upper limit Male mean ± SD Female mean ± SD 123.00 ± 55.93 (n = 6) 271.00 ± 328.58 (n = 8) 92.60 322.54 6.36 ± 3.15 (n = 19) 5.63 9.47 6.36 ± 3.15 (n = 9) 8.62 ± 6.15 (n = 10) 14.77 ± 3.02 (n = 21) 12.00 16.00 14.77 ± 3.02 (n = 9) 13.47 ± 6.96 (n = 12) 39.57 52.60 44.45 ± 8.57 (n = 8) 47.18 ± 21.71 (n = 12) 1.91 2.88 2.14 ± 0.73 (n = 4) 2.57 ± 1.02 (n = 6) Creatinine (mmol/L) 46.09 ± 17.72 (n = 20) Phosphate (mmol/L) 2.14 ± 0.73 (n = 10) Potassium (mmol/L) 90 % CI lower limit 3.56 4.13 4.32 ± – (n = 1) 3.69 ± 0.24 (n = 3) Total protein (g/L) 59.20 ± 8.23 (n = 11) 51.62 61.29 59.20 ± 8.23 (n = 5) 54.17 ± 10.30 (n = 6) Albumin (g/L) 23.00 ± 5.69 (n = 11) 16.20 23.80 23.00 ± 5.69 (n = 5) 17.5 ± 8.18 (n = 6) Globulin (g/L) 30.20 ± 2.23 (n = 11) 29.56 37.34 30.20 ± 2.23 (n = 5) 36.17 ± 9.62 (n = 6) ALT (U/L) 55.74 ± 21.32 (n = 17) 46.53 64.39 55.74 ± 21.32 (n = 8) 55.21 ± 23.29 (n = 9) AST (U/L) 52.87 ± 29.44 (n = 15) 51.12 74.75 52.87 ± 29.44 (n = 7) 71.74 ± 22.96 (n = 8) – – 144.73 272.66 Total bilirubin (μmol/L) ALP (U/L) 4.32 ± – (n = 4) 8.55 ± – (n = 3) 208.69 ± 140.21 (n = 13) 8.55 ± – (n = 1) 8.55 ± – (n = 2) 227.50 ± 97.43 (n = 6) 192.57 ± 166.77 (n = 7) CI conidence interval Page 11 of 16 Tulk et al. SpringerPlus (2016) 5:1479 Table 5 Haematology values of unhealthy murids Species Tree-rat I Tree-rat II Tree-rat III Tree-rat IV Tree-rat V Tree-rat VI Tree-rat VII Stick-nest rat I Gender Female Female Male Male Male Male Male Female Age (years) 2.91 1.85 3.28 2.66 2.57 2.63 2.05 2.76 WBC count (×109 cells/L) 5.39 6.93 5.94 10.56 7.26 9.70 13.31 Haemoglobin (g/L) 150 128 158 148 Haematocrit 45 42 46 46 50 31 36 33 Neutrophil (%) 50 22.94 56.00 37.04 53.03 80.03 62.99 79.04 Lymphocyte (%) 33 72.01 33.00 43.94 35.04 14.05 20 16 Monocyte (%) 10 4.04 11.00 10.94 3.03 4.96 12.99 3.01 Eosinophil (%) 7 1.01 8.08 9 0.10 4.02 2.03 N:L ratio 1.52 0.32 0.84 1.51 5.70 3.15 4.94 35 30 35 15 35 Weight loss Weight loss Hypopyon Hypopyon Dyspnea Est. platelets (/HOIF) Illness 1.70 10 Anorexia, Cataracts Hyphema, Cataracts Cataracts 101 N:L neutrophil to lymphocyte ratio Page 12 of 16 Species Tree-rat I Tree-rat III Tree-rat IV Tree-rat V Tree-rat VII Tree-rat VIII Stick-nest rat I Gender Female Male Male Male Male Female Female Age (years) 2.91 3.28 2.66 2.57 2.05 2.89 2.76 Lipase (U/L) 20.00 Chloride (mmol/L) 5.51 Creatinine kinase (U/L) 134.00 Glucose (mmol/L) 9.30 7.20 9.04 13.44 24.19 28.80 4.54 BUN (mmol/L) 6.30 6.40 7.00 8.00 6.39 6.10 8.28 Creatinine (mmol/L) 57.00 51.00 57.02 55.96 73.99 39.00 32.97 Calcium (mmol/L) 2.77 2.76 9.84 2.35 Phosphate (mmol/L) 1.58 0.68 1.58 2.11 Sodium (mmol/L) 146.00 141.00 145.00 136.00 147.00 149.00 Potassium (mmol/L) 4.40 4.80 3.80 5.60 4.70 4.00 2.60 2.91 2.63 3.13 1.69 Total Protein R (g/L) 62.00 54.00 Total Protein (g/L) 66.00 57.00 52.00 52.00 64.00 67.00 54.00 Albumin (g/L) 42.00 53.00 52.00 41.00 24.00 51.00 24.00 Globulin (g/L) 24.00 3.00 11.00 42.00 16.00 30.00 ALT (U/L) 83.00 83.00 66.00 63.00 188.00 199.00 20.00 47.00 Total bilirubin (μmol/L) 5.00 6.00 6.00 4.99.00 5.00 4.99.00 Amylase (U/L) 1121.00 931.00 959.00 804.00 919.00 390.00 ALP (U/L) 60.00 62.00 66.00 22.00 104.00 80.00 99.00 Anorexia, Cataracts, Hyphema Cataracts Weight loss Weight loss Hypopyon Anorexia, Cataracts, Hyphema Dyspena AST (U/L) 47.00 218.00 Triglycerides (mmol/L) Illness Tulk et al. SpringerPlus (2016) 5:1479 Table 6 Blood biochemistry values of unhealthy murids 35.00 Page 13 of 16 Tulk et al. SpringerPlus (2016) 5:1479 Lymphocytes are usually the predominant leukocyte and can be as high as 70–80 % of the diferential WBC count in the laboratory mouse (Provencher Bolliger and Everds 2012), however in the black-footed tree-rat lymphocytes were just below 50 % and made up 32 % of all WBCs in the greater stick-nest rats. High neutrophil to lymphocyte ratios are useful indicators of poor health or stress (Old et al. 2005). On average both species had high N:L ratios, possibly a result of neutrophilia. A ifth of the stick-nest rats were skewed (6.6–11.5), while all other ratios were <4.0, which may account for the high mean ratio. Compared to other captive murids (Bradley et al. 1988; Kemper et al. 1987; Monamy 1995; Old et al. 2005, 2007; hrall et al. 2012), both species had very high N:L ratios, with tree-rats three times and stick-nest rats six times larger than previously reported murid N:L values. Both species were rarely handled or removed from their enclosure for any medical procedure. he stress of being handled prior to anaesthesia may explain the irregularities in the values as it can increase the number of neutrophils (Hedrich 2012). Anaesthesia, speciically isolurane, can have an efect on the percentage of neutrophils found in C3H mice, with 30 min exposures leading to a 15.4 % reduction in the number of circulating WBCs, and speciically a 26.9 % reduction in neutrophils up to 48 h after exposure (Colucci et al. 2013; Jacobsen et al. 2004). Exposure to 4 % isolurane, if administrated for a duration longer than 5 min may also have had an efect on erythrocytes parameters (Nahas and Provost 2002). he length of time the murids in this study were under anaesthesia is unknown. he morphological appearance of leukocytes in the two species was similar to that described previously for other murids including the brown rat (Rattus norvegicus) (hrall et al. 2012), plains rat (Pseudomys australis), spinifex hopping-mice (Notomys alexis) (Old et al. 2005) and the central rock-rat (Zyzomys pedunculatus) (Old et al. 2005). Neutrophils of both species in this study were larger in diameter when compared to the house mouse (Mus musculus) and brown rat (hrall et al. 2012). Lymphocyte size greatly luctuates from the size of erythrocytes to neutrophils (hrall et al. 2012). Both species’ lymphocytes did not exceed the size of neutrophils. Monocyte size and morphology were similar to that previously described for other murid species (Bradley et al. 1988; Kemper et al. 1987; Monamy 1995; Old et al. 2005, 2007). he low numbers of eosinophils and basophils was not unexpected. Eosinophil numbers are normally only elevated under certain conditions such as eosinophilia during an allergic response or in individuals with parasites (Harvey 2012). As the two species in this study were both from captive populations it is unlikely they would have had high parasite loads (due to regular treatment), and if allergic reactions were evident, would likely have been recorded in the clinical notes. In mammals, basophils are generally never found in high numbers and in some species can be absent (Latimer 2011). Globulin values include levels of enzymes, antibodies, and ibrous and contractile proteins. he stick-nest rat had a mean of 30.2 g/L globulin, 8.9–17.8 g/L above the current reported murid range (Bradley et al. 1988; Kemper et al. 1987; Monamy 1995; Old et al. 2005, 2007; hrall et al. 2012). he cause or efect of high globulin in rodents has not been investigated in detail. However in humans, high globulin can indicate chronic inlammation, an infectious disease, leukaemia, diseases of the liver or kidneys, or an autoimmune disease (Willard and Tvendten 2012). Stick-nest rats over the age of 4 years Page 14 of 16 Tulk et al. SpringerPlus (2016) 5:1479 did display a higher globulin level than their younger counterparts, presumably as they had been exposed to more pathogens than the younger animals. As the expected longevity of free-ranging stick-nest rats is 4 years (Jackson 2007), advanced age (or the wide range of ages of murids in this study) is a reasonable explanation for these high values. ALP is associated with measurements of skeletal growth and can be used as an indicator of age, with levels decreasing as the animal reaches adulthood (Calabuig et al. 2010). Tree-rat ALP was higher in older animals than younger animals and was not consistent with previous murid values (Bradley et al. 1988; Kemper et al. 1987; Monamy 1995; Old et al. 2005, 2007; hrall et al. 2012). Stick-nest rat ALP values were low in young individuals, peaked around 2.5 years, and dropped again when animals reached 4 years. In quolls (Stannard et al. 2013) and other murid species (Old et al. 2005) ALP levels varied greatly between individuals. High ALP has been seen as an efect of captivity in the black vulture (Aegypius monachus), as well as poor health (Villegas et al. 2002). Whilst higher ALP values have also been reported in healthy captive southern hairy-nosed wombats (Lasiorhinus latifrons) compared to wild wombats (Gaughwin and Judson 1980). A larger number of samples with a wider range of ages are needed to determine the reasoning behind the variability in the results and whether captive management is afecting the ALP values of these species. Conclusions Comparative fundamental descriptions of the morphology, relative numbers of leukocytes, and the serum biochemistry of two native Australian murids were provided in this paper to establish a baseline for presumably healthy individuals living in captivity. Our data indicated the values had some variation when comparing genders; however further data is required to determine how age inluences blood parameters in these species, speciically in WBC counts in both species as well as N:L ratios/percentages and HCT in stick-nest rats. Compared to other captive murids stick-nest rats had higher levels of globulin and requires further investigation. Nevertheless, the conidence intervals established provide a basis for monitoring the health status of captive individual black-footed tree-rats and greater sticknest rats and aid the long-term survival of these captive murid populations. he cause, consequence and impact of disease in native murid species remain poorly understood, and further long-term data sets are required to fully understand health and disease in these species, as well as blood samples from wild individuals. Authors’ contributions The study was part of an Honours project undertaken by MT at Western Sydney University (WSU) WSU. MT collated and analysed the data, and drafted the manuscript. HS and JO conceived of the study, participated in its design and coordination, data analysis and drafting of the manuscript. All authors participated in the design of the study. All authors read and approved the inal manuscript. Author details 1 School of Science and Health, Hawkesbury, Western Sydney University, Locked Bag 1797, Penrith, NSW 2751, Australia. 2 School of Life and Environmental Sciences, Charles Perkins Centre, University of Sydney, Sydney, NSW 2006, Australia. Acknowledgements We would like to acknowledge Paul Thompson (Taronga Zoological Park) for assistance in obtaining medical records and Casey Borthwick for helping in the preparation of the graphics used in this manuscript. Competing interests The authors declare that they have no competing interests. Ethics approval The project was approved by the WSU Animal Ethics Committee (ARA #A10997). Page 15 of 16 Tulk et al. SpringerPlus (2016) 5:1479 Funding Funding for the study was provided by the School of Science and Health, WSU. Received: 24 June 2016 Accepted: 19 August 2016 References Arid Recovery (2007) Greater Stick-nest Rat Fact Sheet. www.aridrecovery.org.au. Accessed 26 Aug 2016 Bradley AJ, Kemper CM, Kitchener DJ, Humphreys WF, How RA, Schmitt LH (1988) Population ecology and physiology of the common rock rat, Zyzomys argurus (rodentia: muridae) in tropical northwestern Australia. J Mammal 69:749–764. doi:10.2307/1381630 Breed B, Ford F (2007) Native mice and rats. Natural history, 1st edn. CSIRO, Melbourne, VIC, Australia Burbidge AA, McKenzie N (1989) Patterns in the modern decline of Western Australia’s vertebrate fauna: causes and conservation implications. Biol Conserv 50:143–198 Calabuig CP, Ferrer M, Muriel R, Tilgar V (2010) Plasma alkaline phosphatase as a sensitive indicator of age and skeletal development in wild coscoroba swans. Wildl Res 37:504–511. doi:10.1071/WR09160 Colucci DG, Puig NR, Hernandez PR (2013) Inluence of anaesthetic drugs on immune response: from inlammation to immunosuppression. OA Anaesth 1:21 Gaughwin M, Judson G (1980) Haematology and clinical chemistry of hairy-nosed wombats (Lasiorhinus latifrons). J Wildl Dis 16:275–280 Griiths AD, Koenig J, Carrol F, Price O (2002) Activity area and day-time tree use of the black-footed tree-rat Mesembriomys gouldii. Aust Mammal 23:181–183. doi:10.1071/AM01181 Harvey JW (2012) Veterinary hematology: a diagnostic guide and color atlas. Elsevier Health Sciences, St. Louis, Missouri, USA Hedrich HJ (2012) The laboratory mouse, 2nd edn. Academic Press, London, UK IUCN (2014) The IUCN red list of threatened species. http://www.iucnredlist.org/. Accessed 8 April 1025 Jackson S (2007) Australian mammals: biology and captive management, 1st edn. CSIRO, Collingwood, VIC, Australia Jacobsen KO, Villa V, Miner VL, Whitnall MH (2004) Efects of anesthesia and vehicle injection on circulating blood elements in C3H/HeN male mice. Contemp Top Lab Anim Sci 43:8–12 Johnson CN, Isaac JL (2009) Body mass and extinction risk in Australian marsupials: the ‘Critical Weight Range’ revisited. Austral Ecol 34:35–40. doi:10.1111/j.1442-9993.2008.01878.x Kemper CM, Kitchener DJ, Humphreys WF, How RA (1987) The demography and physiology of Melomys sp. (Rodentia: Muridae) in the Mitchell Plateau area, Kimberley, Western Australia. J Zool 212:553–562. doi:10.1111/j.1469-7998.1987.tb02923.x Latimer KS (2011) Duncan and Prasse’s veterinary laboratory medicine: clinical pathology. Wiley, New York Monamy V (1995) Ecophysiology of a wild-living population of the velvet-furred rat, Rattus lutreolus velutinus (Rodentia : Muridae), in Tasmania. Aust J Zool 43:583–600. doi:10.1071/ZO9950583 Morris KD (2000) The status and conservation of native rodents in Western Australia. Wildl Res 27:405–419. doi:10.1071/ WR97054 Nahas K, Provost JP (2002) Blood sampling in the rat: current practices and limitations. Comp Clin Pathol 11:14–37. doi:10.1007/s580-002-8080-5 Old JM, Connelly L, Francis J, Branch K, Fry G, Deane EM (2005) Haematology and serum biochemistry of three Australian desert murids, the plains rat (Pseudomys australis), the spinifex hopping-mouse (Notomys alexis) and the central rock-rat, (Zyzomys pedunculatus). Comp Clin Pathol 14:130–137. doi:10.1007/s00580-005-0586-y Old JM, Connelly L, Francis J, Gogler J (2007) Haematology and serum biochemistry of the Carpentarian rock-rat (Zyzomys palatalis). Comp Clin Pathol 16:249–252. doi:10.1007/s00580-007-0694-y Provencher Bolliger A, Everds N (2012) Haematology of the mouse. In: Hedrich HJ (ed) The laboratory mouse. Academic Press, pp 331–347. doi:10.1016/B978-0-12-382008-2.00014-3 Robinson A (1975) The stick-nest rat, Leporillus conditor, on Franklin Island, Nuyts Archipelago, South Australia. Aust Mammal 1:319–327 Stannard HJ, Young LJ, Old JM (2013) Further investigation of the blood characteristics of Australian quoll (Dasyurus spp.) species. Vet Clin Pathol 42:476–482. doi:10.1111/vcp.12094 Thrall MA, Weiser G, Allison RW, Campbell TW (2012) Veterinary hematology and clinical chemistry, 2nd edn. WileyBlackwell, New York Van Dyck S, Strahan R (2008) The mammals of Australia. New Holland Publishers, Sydney Villegas A, Sánchez J, Costillo E, Corbacho C (2002) Blood chemistry and haematocrit of the black vulture (Aegypius monachus). Comp Biochem Physiol A 132:489–497. doi:10.1016/S1095-6433(02)00097-1 Willard MD, Tvendten H (2012) Small animal clinical diagnosis by laboratory methods, 5th edn. Elsevier, North Holland Woinarski J, Burbidge A, Harrison P (2014) Action Plan for Australian Mammals 2012. CSIRO Publishing, Melbourne Page 16 of 16