Fatty-acid, amino-acid and mineral composition of two milk replacers for marsupials

Authors


Corresponding author: Robert H. Glew, PhD, Department of Surgery, MSC08-4770, School of Medicine, 1 University of New Mexico, Albuquerque, NM 87131-0001, USA. Tel: (1) 505-272-2362. Fax: (1) 505-344-0617. E-mail: rglew@salud.unm.edu

Abstract

Although milk replacers are widely used to feed orphaned and injured marsupial joeys in Australia, little is known about the nutrient composition of these foods. We analysed two milk substitutes, Di-Vetelact (DiV) and Wombaroo Kangaroo (Wom) [milk stage >0·7 (joeys having completed 70% of their pouch life)], for their content of fatty acids, amino acids, and minerals and trace elements. The protein contents of DiV and Wom were 25·0% and 17·3% of fresh weight, respectively, and, except for tryptophan, the amino-acid profile compared favourably with three high-quality proteins. The fatty-acid content of DiV and Wom was 9·65% and 17·6%, respectively. Relative to marsupial milk, linoleic acid (DiV, 18·3%; Wom, 9·32%) and α-linolenic acid (DiV, 2·97%; Wom, 3·94%) were well represented in both milk replacers, but docosahexaenoic acid was not detected. Comparable amounts of zinc, iron, potassium, calcium, magnesium and phosphorus were present in DiV and Wom, but the copper content of DiV was only 5% that of Wom and manganese was not detected in DiV. These data indicate that two popular marsupial milk replacers contain healthful amounts of many essential nutrients relative to marsupial milk but lack the docosahexaenoic acid that is critical for brain growth and development in mammals.

Introduction

Milk replacers are widely used in Australia for rescued marsupials, such as the wombat, kangaroo, bandicoot and the Endangered Tasmanian devil Sarcophilus harrisii (Stephens, 2008; IUCN, 2012). The Tasmanian devil is on the brink of extinction owing to an infectious cancer named devil facial tumour disease (DFTD) (Bode et al., 2009) for which there is currently no vaccine or therapy. The ability to hand-raise orphaned or pouch young that have been bred in captivity has become critical to efforts to preserve the species because it has already declined by more than 80% in the wild. Ensuring that milk replacers are nutritionally balanced is fundamental to best husbandry practices not only for the Tasmanian devil but also for marsupials in general.

Despite the obvious importance of these milk replacers or substitutes and the evidence that the maternal diet can influence the nutritional quality of the milk produced by marsupials, and the growth rate and development of their offspring (Munks et al., 1991; Merchant et al., 1996; Rose et al., 2003), the scientific literature and manufacturers' websites supply little in the way of quantitative information regarding the essential nutrients provided in the foods, including amino acids, fatty acids, and minerals and trace elements. For example, while it is of some use to know the amount of total fat and protein in a particular formulation, equally important is information about the fatty-acid and amino-acid composition of those macronutrients. To assess the nutritional quality of a dietary protein requires knowledge about the amounts and proportions of the essential amino acids. Similarly, it is not sufficient to know just the percentage of fat in a particular milk substitute; one would also like to know the proportions of the essential fatty acids linoleic acid and α-linolenic acid, the ratio of omega-6:omega-3 fatty acids (Simopoulos et al., 1999; Simopoulos, 2002), and whether the fat of the milk replacer provides sufficient amounts of the long-chain polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) upon which normal growth and development of the nervous system of mammals depends (Harris et al., 2009; Milte et al., 2012; Swanson et al., 2012).

While the literature offers little in the way of information regarding the nutrient composition of maternal milk of Tasmanian devils, numerous studies of other marsupials support several generalizations. First, the proportions of protein, carbohydrate and lipid in marsupial milk vary greatly throughout lactation (Nicholas et al., 1997; Trott et al., 2003): in general, carbohydrate declines for most species, while protein and lipid increase about twofold and eightfold, respectively. Except for the Brushtail possum Trichosurus vulpecula (Cowan, 1989) and Ringtail possum Pseudocheirus peregrinus (Munks et al., 1991), the protein : lipid ratio for marsupial milk remains in the 0·5–0·9 range throughout lactation (Lemon & Barker, 1967; Green et al., 1987; Crowley et al., 1988; Knockenberger, 1996; Merchant et al., 1996; Muths, 1996). Second, with regard to the fatty-acid composition of the triglycerides in marsupial milk, there is agreement between the data reported for the Red kangaroo Macropus rufus (Griffiths et al., 1972), Eastern quoll Dasyurus viverrinus (Green et al., 1987) and Long-nosed potoroo Potorous tridactylus (Crowley et al., 1988) that palmitic acid (16:0) and oleic acid (18:1n-9) account for about two-thirds of the fatty-acid total and that the omega-6 : omega-3 (i.e. linoleic acid : α-linolenic acid) ratio can vary widely between different marsupial species. For example, at 20 weeks of lactation the n-6:n-3 ratio for the milk fat of the Red kangaroo, Eastern quoll and Long-nosed potaroo are c. 2:1, 6:1 and 10:1, respectively. Furthermore, for the same marsupial species (e.g. Red kangaroo) the n-6:n-3 ratio ranges from 10:1 early in lactation to 2:1 in the final stage (Griffiths et al., 1972). Crowley et al. (1988) did not find arachidonic acid (20:4n-6) in Long-nosed potaroo milk, but the milk of the Red kangaroo (Griffiths et al., 1972) and Eastern quoll (Green et al., 1987) contains low but measureable proportions of arachidonic acid in the 0·1–1·6% range. None of these three studies of the fatty-acid composition of marsupial milk mentioned finding either EPA or DHA. Linoleic acid accounts for 4–15% of the fatty acids in marsupial milk and α-linolenic acid 1–2% of the fatty-acid total (Griffiths et al., 1972; Green et al., 1987; Crowley et al., 1988).

Lacking in the literature are comprehensive reports regarding the content in marsupial milk of nutritionally essential minerals and trace elements, such as copper, zinc, iron, magnesium and manganese. Equally lacking are reports of the amounts and relative proportions of all amino acids, free as well as those comprising proteins, in marsupial milk.

The main aim of this study was to determine the fatty-acid, amino-acid and mineral content of two commercially available milk replacers manufactured in Australia and widely used to hand-rear orphaned or injured marsupials endemic to that part of the world: Di-Vetelact (DiV) and Wombaroo Kangaroo (Wom). The information contained in this report should provide a basis for comparing the amounts of these essential nutrients in Wom and DiV versus the amounts of these nutritionally critical substances in the milk of the marsupials for whom they are intended. Wom is marketed solely for marsupials and DiV is marketed as a low-lactose supplement for both eutherians and marsupials.

Materials and Methods

Materials

Wom milk replacer >0·7 (intended for joeys that have completed 70% of their pouch life) and DiV in powder form were purchased from Wombaroo Food Products and Sharpe Laboratories Pty Ltd, respectively. A single batch of each milk replacer was analysed. Prior to sampling, each batch was mixed thoroughly with the aid of a stainless-steel mill in order to ensure effective homogenization.

Fatty-acid analysis

Total lipids in the two milk replacers were extracted according to a modification of the Folch method (Folch et al., 1957). Briefly, 0·25 g of sample was extracted with 20 ml of chloroform : methanol (2:1, v/v) at room temperature for 1 hour. The extracted lipids in the chloroform phase were separated from the aqueous phase by shaking and partitioning with 4 ml of 0·9% (w/v) NaCl. The chloroform layer was collected and evaporated under a stream of nitrogen gas and the lipids were then dissolved in 5 ml of chloroform.

To prepare fatty-acid methyl esters, 0·2 ml of sample was evaporated under a stream of nitrogen gas and then treated with 14% (w/v) methanolic boron trifluoride (BF3) for 20 minutes at 95°C (Morrison & Smith, 1964). The fatty-acid methyl esters were extracted into hexane, analysed and quantified using an Agilent 6890 gas chromatograph equipped with a flame-ionization detector and a fused-silica capillary column (Omegawax: 30 m × 0·32 mm internal diameter, film thickness 0·25 μm). Helium was used as the carrier gas. The injector was set at 205°C and the detector was at 235°C. The temperature of the oven was initially 140°C, and then raised to 205°C at 6°C minute–1 and held for 20 minutes. The fatty-acid peaks were identified by comparing their retention times with those of a standard mixture of fatty-acid methyl esters (RL-461). Quantification was carried out using the technique of internal standardization with triheptadecanoin. Samples were analysed in triplicate.

Amino-acid analysis

Approximately 0·4 g of the two milk replacers was extracted with ice-cold acetone to remove lipids that would interfere with amino-acid analysis. The powder was suspended in 2·0 ml of water to which 6 ml ice-cold acetone was added. The contents of the glass tube were shaken vigorously followed by centrifugation at 5000 g in a clinical centrifuge. The supernatant was removed with the aid of a Pasteur pipette and the extraction was repeated twice more. The protein residue was dried at room temperature. Then 20 mg of acetone-extracted Wom and DiV was hydrolyzed in 6 N HCl containing 0·1% (w/v) phenol at 110°C for 24 hours under vacuum, and the resultant amino acids were separated and quantified as described elsewhere (Glew et al., 2010) using a Hitachi L-8800 chromatographic system with a transgenomic column, pickering buffers and a customized gradient profile optimized for amino-acid resolution. For determination of methionine and cysteine, samples were oxidized with performic acid (Hirs, 1967) prior to acid hydrolysis. Tryptophan was determined as described elsewhere (Penke et al., 1974; Molnár-Perl & Pintér-Szákas, 1989). The reproducibility of the method was in the 0·6–3% range for the amino acids reported. The two formulas were analysed in triplicate.

Mineral analysis

The amounts of various minerals and trace elements in the marsupial milk replacers were determined by Zeeman atomic absorption spectroscopy. The fresh specimens (10 mg) were weighed, dissolved in 80% (w/v) HNO3 and analysed using a Hitachi Z-2300 atomic absorption spectrometer according to the method described by Martín et al. (2000). Digested samples were diluted onefold to 50-fold as required using 0·5% (v/v) HCl. With the aid of an auto-sampler, 20 μl aliquots of the diluted samples and 5 μl of 1·5% (v/v) HNO3 were injected into the graphite furnace. Analyses were performed in triplicate.

Statistical analysis

Data were analysed by t-tests using IBM SPSS Statistics software. Results are reported as mean plus or minus one standard deviation. Differences between means were considered significant at the P ≤ 0·05 levels.

Results

Fatty-acid composition

Fatty acids accounted for 17·6 (2·6)% and 9·65 (2·5)% of the fresh weight of Wom and DiV powder, and the difference between the two values was significant (P < 0·001). Monounsaturated fatty acids accounted for nearly 50% of the fatty-acid total in both Wom and DiV, and oleic acid (18:1n-9) was the major monounsaturated fatty acid (Table 1). The proportion of all saturated fatty acids was higher in Wom than DiV (41·0% vs 31·1%, P < 0·001). The only omega-3 fatty acid in either milk replacer was α-linolenic acid, but it represented only 2·4–4·0% of the fatty-acid total. There was about twice as much linoleic acid in DiV compared with Wom (9·3% vs 18·0%, P < 0·001). The omega-6 : omega-3 ratio was 2·36 for Wom and 7·41 for DiV.

Table 1. Fatty-acid composition (mass %) and content (mg g−1 dry weight) of Wombaroo (n-6:n-3 = 2·36) and Di-Vetelact (n-6:n-3 = 7·41) milk replacers: each value represents the mean ± SD of three independent experiments; NS. not significant. Significance P ≤ 0·05
SAMPLENORMALIZED (%)WEIGHT (mg g−1)
WOMBAROODI-VETELACTPWOMBAROODI-VETELACTP
C12:00·80 (0·16)0·47 (0·02)0·0221·41 (0·29)0·46 (0·13)0·007
C14:06·53 (0·23)3·64 (0·22)<0·00111·5 (2·06)3·55 (1·08)0·004
C14:10·35 (0·03)0·19 (0·01)0·0010·62 (0·11)0·18 (0·06)0·004
C15:00·77 (0·02)0·46 (0·02)<0·0011·35 (0·23)0·45 (0·12)0·003
C16:021·7 (0·48)16·8 (0·31)<0·00138·3 (6·35)16·3 (4·21)0·008
C16:1n-71·17 (0·04)0·68 (0·03)<0·0012·06 (0·37)0·65 (0·15)0·004
C18:010·1 (0·06)8·61 (0·31)0·00117·7 (2·64)8·29 (2·06)0·008
C18:1n-939·9 (0·72)44·5 (0·46)0·00170·1 (9·11)42·9 (10·7)0·028
C18:1n-74·79 (0·02)3·26 (0·03)<0·0018·43 (1·20)3·16 (0·82)0·003
C18:2n-69·32 (0·10)18·3 (0·39)<0·00116·4 (2·22)17·6 (4·63)NS
C18:3n-33·94 (0·05)2·47 (0·07)<0·0016·95 (1.09)2·39 (0·65)0·003
C20:00·29 (0·01)0·26 (0·02)NS0·51 (0·08)0·24 (0·06)0·012
C20:10·47 (0·03)0·34 (0·08)NS0·82 (0·06)0·31 (0·04)<0·001
C20:5n-3<0·05<0·05<0·10<0·10
C22:6n-3<0·05<0·05<0·10<0·10
total100100176 (26)96·5 (24·6)0·018

Amino-acid composition

Protein, estimated by summating the individual amino acids, accounted for 25·0% and 17·3%, respectively, of the fresh weight of Wom and DiV (Table 2). The protein content of the specimens was not determined by measuring nitrogen content. To obtain some measure of the quality of the protein in the two milk replacers, we compared the patterns of the essential amino acids in these commercial marsupial foods with three high-quality reference proteins: the World Health Organization (1985) standard pattern for human infants, cow's-milk protein and hen's-egg protein (Milt-Ward et al., 2004) (Table 3). For both Wom and DiV, the proportions of all of the essential amino acids, except tryptophan, either exceeded or fell within the range of one or more of the three reference proteins. The percentages of tryptophan in the protein fraction of Wom and DiV were 0·95% and 0·81%, respectively, and both values fell below the 1·4–1·7% range for the amino-acid profiles of the reference proteins (Table 3). Collectively, these results indicate that Wom and DiV both contain substantial amounts of high-quality protein, with the exception of tryptophan which is under-represented in both milk substitutes relative to the three reference proteins.

Table 2. Comparison of the amino-acid content (μg g−1 dry weight) of Wombaroo and Di-Vetelact milk replacers: the numbers in parentheses indicate one standard deviation. Significance P ≤ 0·05.
AMINO ACIDWOMBAROODI-VETELACTP
Aspartic acid24·4 (0·3)15·8 (0·2)<0·001
Threonine15·9 (0·2)10·1 (0·2)<0·001
Serine11·8 (0·2)8·54 (0·22)<0·001
Glutamic acid42·6 (1·6)32·0 (1·1)<0·001
Proline17·5 (0·4)14·1 (0·2)<0·001
Glycine4·12 (0·04)2·88 (0·04)<0·001
Alanine10·7 (0·2)6·78 (0·09)<0·001
Valine15·1 (0·1)10·7 (0·2)<0·001
Isoleucine15·2 (0·1)10·0 (0·2)<0·001
Leucine26·7 (0·2)18·0 (0·3)<0·001
Tyrosine9·32 (0·1)7·35 (0·13)<0·001
Phenylalanine9·84 (0·04)7·39 (0·12)<0·001
Histidine5·16 (0·05)4·00 (0·08)<0·001
Lysine21·1 (0·1)11·4 (0·2)<0·001
Arginine7·36 (0·07)5·53 (0·08)<0·001
Cysteine5·12 (0·06)2·72 (0·08)<0·001
Methionine5·57 (0·06)4·04 (0·06)<0·001
Tryptophan2·38 (0·02)1·40 (0·02)<0·001
total250 (4)173 (3)<0·001
Table 3. Comparison of the amino-acid composition of Wombaroo and Di-Vetelact milk replacers versus three reference proteins:adata derived from Table 2; bWHO. World Health Organization (1985); cMilt-Ward et al., 2004.
AMINO ACID% OF TOTAL
WOMBAROOaDI-VETELACTaWHObEGGcCOW'S MILKc
Threonine6·45·84·34·74·4
Valine6·06·25·56·66·4
Isoleucine6·15·84·65·44·7
Leucine10·710·49·38·69·5
Tyrosine plus phenylalanine7·78·57·29·310·2
Methionine plus cysteine4·33·94·25·73·3
Lysine8·46·66·67·07·8
Tryptophan0·950·811·71·71·4

Minerals and trace elements

Except for copper and potassium for which the contents did not differ significantly between the two formulas, Wom contained significantly more zinc, calcium, magnesium, manganese and phosphorus compared with DiV (Table 4). However, DiV contained twice as much iron as Wom. Chromium, selenium and molybdenum were below the limits of detection for these elements.

Table 4. Comparison of the mineral content (μg g−1 fresh weight) of Wombaroo and Di-Vetelact milk replacers: the numbers in parentheses indicate one standard deviation; NS. not significant. Significance P ≤ 0·05.
MINERALWOMBAROODI-VETELACTP
Copper150 (6)140 (7)NS
Zinc30 (2)10 (0·5)<0·001
Iron70 (2)140 (4)<0·001
Potassium5790 (850)4880 (920)NS
Chromium<3<3
Selenium<3<3
Calcium11,400 (1290)8700 (730)<0·001
Magnesium1550 (210)1120 (70)<0·013
Manganese20 (7)<3
Molybdenum<3<3
Phosphorus10,200 (1130)5730 (440)<0·001

Discussion

The need for new knowledge about the nutrient composition of commercial milk replacers used to feed hand-reared young marsupials in Australia is growing for at least two reasons. First, higher marsupial mortality and injury have resulted from increased urbanization, population growth and motorized vehicular traffic on the nation's roadways (Ramp et al., 2006; Rowden et al., 2008). In southern Tasmania over the last 3 years, a 30% increase has been noted in the number of injured animals rescued by the Bonorong Wildlife Sanctuary (Greg Irons, Director, Bonorong Wildlife Sanctuary, pers. obs). Fortunately, many of these injured animals or orphaned young are being rescued because of the availability of milk replacers such as DiV and Wom. Second, the impending extinction of the Tasmanian devil as a result of DFTD (Bode et al., 2009; Murchison et al., 2009) underscores the need for greater knowledge about how to care for and feed these animals in the wildlife sanctuaries devoted to preserving this unique and iconic animal. Many young Tasmanian devils have to be separated from mothers that are too diseased to feed their infants and, therefore, have to be hand-reared (Jones et al., 2007). These unfortunate realities have heightened awareness among concerned scientists and government officials of the need for more knowledge about the nutritional needs of marsupials, especially newborn and adolescent animals. The main aim of this study was to increase our understanding of the adequacy of milk replacers in current use as alternatives to maternal milk at rescue facilities by determining the content of certain essential macro- and micro-nutrients in marsupial formulas in Australia.

Unfortunately, the literature lacks peer-reviewed reports about the nutrient content of Wom or DiV against which we might compare our data. However, the website of the manufacturer of Wom provides information about the nutrient composition of the Wom formula recommended for ‘late’ lactation. In general, our nutrient values and those reported by the manufacturer for ‘late’ milk stage (>0·7: completed 70% of pouch life) Wom are in reasonable agreement. For example, we found the protein content of fresh Wom formula to be 25·0%, which agrees well with the manufacturer's reported protein value of 29·6%. The 17·6% fat content we found for the late stage (>0·7) milk replacer fell within the 13·6–28·0% range of fat in ‘early’ (< 0·4: completed 40% of pouch life) and ‘mid-stage’ (0·4–0·7: completed 40–70% of pouch life) Wom milk replacer; however, it was threefold lower than the fat content of 46·8% reported by the manufacturer. This discrepancy could be the result, in part, of the different methods used by us and the manufacturer to determine the nutrient compositions, or to variations between batches of milk replacer. To our knowledge, the manufacturer does not make information regarding the fatty-acid or amino-acid composition of Wom available to the public.

As for minerals and trace elements, according to our findings (Table 4) as well as information provided by the manufacturer, Wom contained 11 400 μg g–1 calcium, which agrees well with the manufacturer's claim of a calcium content of 14 000 μg g–1 for late stage (>0·7) Wom milk replacer. In addition, the values we report in Table 4 for zinc, potassium, magnesium and manganese all fall within a factor of two of the corresponding values provided by the manufacturer of Wom. The only large discrepancies between our mineral and trace-element data and that of Wombaroo Food Products were for copper and iron: we found the copper content of Wom to be 150 μg g–1 fresh weight (Table 4) which is about 20-fold greater than that reported by the manufacturer (8·0 μg g–1), and the iron content to be three-and-a-half-fold greater than the manufacturer's estimate.

As stated above, our ability to comment on how closely the nutrient data we provide herein for rescue formulas relate to the needs of young marsupials in general and the Tasmanian devil in particular is restricted by the fact that maternal-milk composition changes as lactation progresses and because of the paucity of published reports about the amino-acid and fatty-acid compositions of the maternal milk of Australian marsupials, in particular that of the Tasmanian devil. Furthermore, the fact that the composition of marsupial milk changes greatly during lactation (Merchant et al., 1996) makes it difficult to compare the content of most nutrients in Wom and DiV versus that of marsupial milk. Nevertheless, we can state with some degree of confidence that based on the relatively high protein content of DiV and Wom (Table 2), and the fact that the amino-acid profiles of the proteins in these two formulas compare favourably with that of egg protein (Table 4) (Milt-Ward et al., 2004), Wom and DiV both appear to be excellent sources of all of the essential amino acids except tryptophan.

As to the question of how well the fatty-acid composition of DiV and Wom relate to the needs of marsupials for nutritionally critical fatty acids, once again the dearth of published data on the fatty-acid profile and content of the milk lipids of marsupials limits our ability to articulate definitive assessments of this nature. Nevertheless, it seems reasonable to compare the fatty-acid compositions of the two rescue foods with that of the milk of three different marsupial species, namely Red kangaroo, Eastern quoll and Long-nosed potaroo. On the positive side, DiV and Wom contain relatively large amounts of the two fatty acids that are essential in humans and most other mammals, namely linoleic and α-linolenic acid. Marsupial milk contains 3–15% total fat (Griffiths et al., 1972; Green et al., 1987; Crowley et al., 1988) compared with 9·62% and 17·6% fat in DiV and Wom, respectively (Table 1). The proportion of linoleic acid in the two milk replacer foods was in the 9–19% range, which compares with 4–16% in marsupial milk fat. The proportion of α-linolenic acid was 2·47% in DiV and 3·94% in Wom; in marsupial milk this proportion is in the 0·1–2·8% range (Griffiths et al., 1972; Green et al., 1987; Crowley et al., 1988). Furthermore, the ratio of linoleic acid to α-linolenic acid is <8 for each of the rescue formulas (Table 1), which is desirable because there is a direct relationship between this ratio and risk of inflammation, at least in humans (Simopoulos et al., 1999; Simopoulos, 2002).

On the negative side of the fatty-acid picture, the conspicuous absence of the two long-chain omega-3 fatty acids EPA and DHA from Wom and DiV should be cause for concern because the normal growth and development of the central nervous system of humans and most other mammals are critically dependent on EPA and DHA (Milte et al., 2012; Swanson et al., 2012).

Although no nutritional complications associated with use of Wom or DiV have been reported in the scientific literature, milk replacers may still fall short of meeting the nutritional requirements of joeys. Nevertheless, our findings raise several concerns about the nutrient composition of Wom and DiV. The first concern has to do with the fact that although arachidonic acid and DHA are the major fatty acids in neural cell membranes in the brains of all mammals that have been studied in this regard (Sinclair, 1975; Crawford, Casperd & Sinclair, 1976; Crawford, Williams et al., 1976; Farkas et al., 2000; Innis, 2007), we did not find either of these nutritionally critical fatty acids in Wom or DiV. Heightening this concern are the findings of the three studies on the fatty-acid composition of the triglycerides in the milk of the Red kangaroo (Griffiths et al., 1972), Eastern quoll (Green et al., 1987) and Long-nosed potoroo (Crowley et al., 1988): although the maternal milk of two of these three marsupial species (Red kangaroo and Eastern quoll) contained arachidonic acid, none of the three species contained measureable DHA. Because metabolic conversion of the essential omega-3 fatty acid α-linolenic acid into DHA is thought to be inadequate to satisfy a mammal's DHA needs (Burdge & Calder, 2005), we are left with two questions: ‘how do joeys raised on Wom or DiV satisfy their DHA requirement?’ and ‘should arachidonic acid and DHA be added to these and other marsupial rescue formulas?’. In light of the fact that plants do not synthesize arachidonic acid or DHA, the apparent absence of these polyunsaturated fatty acids from Wom and DiV suggests that these two formulas are made from plant oils and not from a marine-fish oil or animal fat.

The second concern raised from the data in the present study has to do with the balance between the protein and lipid content of Wom and DiV. The protein : lipid ratios for Wom and DiV, calculated from the information in Tables 1, 2, respectively. These values are much higher than that which has been reported for the corresponding ratios for the milk of most marsupials; with the exception of the Brushtail possum and Ringtail possum where the protein : lipid ratio is close to unity, this ratio for other marsupials is in the 0·5–0·9 range (Lemon & Barker, 1967; Green et al., 1987; Crowley et al., 1988; Knockenberger, 1996; Merchant et al., 1996; Muths, 1996). This discrepancy between the protein : lipid ratios for Wom and DiV vis-à-vis the maternal milk of most marsupials raises two questions: ‘should the balance between protein and lipid in the two rescue formulas be adjusted so as to fall in line with what is found in maternal milk?’ and ‘are there any adverse consequences from having such a relatively high protein and low lipid content in rescue milks?’.

With regard to the limitations of the present study, the small number of publications pertaining to the fatty acids, amino acids, and mineral and trace elements in marsupial milk has hampered our ability to assess the nutritional significance of the data in the present study vis-à-vis the needs of young marsupials for these three classes of nutrients. We plan to correct this knowledge deficit by analysing these same nutrients in the milk and blood of a variety of Australian marsupials at different stages of growth and development during the first year of life. The Tasmanian devil will be at the centre of such future studies. Second, we analysed only a single batch of Wom and DiV, which raises concerns about the extent of batch-to-batch variations in the composition of these formulas. Third, in future studies of this kind, it will be important to include the results of proximate analyses so as to facilitate comparison of results between different laboratories.

Future studies should be directed at assessing the fatty-acid composition and trace-element content of the various commercially available formulas used as marsupial milk replacers, with the goal of determining the extent to which the rescue foods meet the needs of these pouched animals. It would also be helpful to know more about the ingredients used to make the milk replacers. The aforementioned future studies also depend strongly on the availability of much more information about the nutrient content of maternal milk of different marsupial species than presently exists in the peer-reviewed literature. This knowledge gap could be filled by comprehensive investigations of the nutrient content of maternal milk at all stages of lactation. Also needed are studies that assess the growth, development and health of joeys raised on particular replacement milks. These kinds of studies should begin with the Tasmanian devil because, ironically, its extinction is imminent and yet less is known about the nutritional aspects of the maternal milk of this Red-Listed Endangered species relative to most other marsupials.

In conclusion, the main finding of this study is that while two commercially available formulas used to feed rescued marsupials in Australia appear to contain useful quantities of many nutrients essential for the growth and general well-being of these invaluable animals, they likely provide inadequate amounts of arachidonic acid, EPA and DHA.

Products mentioned in the text

  • Agilent 6890: gas chromatograph, manufactured by Agilent Technologies, Santa Clara, CA 95051, USA.
  • Di-Vetelect: low-lactose animal supplement, manufactured by Sharpe Laboratories Pty Ltd, 12 Hope Street, Ermington, NSW 2115, Australia. See http://www.sharpelabs.com.au for more details.
  • Hitachi L-8800: amino-acid analyser, manufactured by Hitachi High-Technologies Corporation, Tokyo, Japan.
  • Hitachi Z-2300: atomic absorption spectrometer, manufactured by Hitachi High-Technologies Corporation, Tokyo, Japan.
  • IBM SPSS Statistics: statistical analysis program, manufactured by IBM Corporation, Armonk, NY 10504-1722, USA.
  • Omegawax: fused silica capillary columns, manufactured by Supelco, Bellefonte, PA 16823, USA.
  • RL-461: fatty acid methyl esters, manufactured by Nu-Chek-Prep, Inc., Elysian, MN 56028, USA.
  • Triheptadecanoin: internal standard, manufactured by Sigma, St. Louis, MO 63178, USA.
  • Wombaroo: kangaroo milk replacer, manufactured by Wombaroo Food Products, PO Box 151, Glen Osmond, South Australia 5064. See http://www.wombaroo.com.au/kangaroo.htm for more details.

Ancillary