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P U B L I S H I N G

Australian Journal of Zoology

Volume 45, 1997 CSIRO Australia 1997

A journal for the publication of the results of original scientific research in all branches of zoology, except the taxonomy of invertebrates

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Australian Journal of Zoology, 1997, 45, 6573

Milk Composition in the Grey-headed Flying-fox, Pteropus poliocephalus (Pteropodidae:Chiroptera)

Michael MesserA and Kerryn Parry-JonesB
A B

Department of Biochemistry, University of Sydney, NSW 2006, Australia. School of Biological Science, University of New South Wales, Kensington, NSW 2033, Australia.

Abstract Milk samples from 11 captive flying-foxes were collected at various times during lactation from 5 to 139 days post partum and analysed for protein, carbohydrate, total solids and ash. In addition, samples from 14 free-living animals, collected on a single occasion, were analysed. No significant changes in milk composition were observed during lactation in the captive bats except for a small increase in protein and a small decrease in carbohydrate concentration late in lactation. The milk from captive bats contained less protein and total solids than that from free-living animals (mean values: protein, 259 and 364%, repectively; total solids, 111 and 127%, repectively) but there was no significant difference with repect to the carbohydrate (613 and 644%, respectively). The fat content, estimated from the total solids by difference, was low (19 and 22%, respectively) in both captive and free-living animals. The results are compared with previously published values for milk composition in Chiroptera and are discussed in the context of nursing behaviour and diet in captive and free-living flying-foxes.

Introduction Although there is a considerable amount of information on the composition of the milk of a large variety of mammalian species (Jenness and Sloan 1970; Oftedal and Iverson 1995), relatively little is known concerning milk composition in bats (Kunz et al. 1995). For example, there are over 60 species of Chiroptera in Australia (Strahan 1992) but none has until now been studied with respect to the composition of its milk. The grey-headed flying-fox, Pteropus poliocephalus (Suborder Megachiroptera) is the largest of eight species of fruit-eating bats found in Australia. Lactation usually begins in October and continues for 34 months or even longer (Nelson 1965); the mothers generally have only a single young. The bats are regarded as pests by many orchardists and lactating flying-foxes are frequently shot (Parry-Jones 1996). Their orphaned young are sometimes rescued and have been hand-reared on a variety of milk formulae whose composition is mostly based on trial-and-error. Information on the composition of their milk should therefore be of assistance to hand-rearers as well as provide comparative data that may increase our understanding of growth and development in this species. In this study we collected milk samples from both captive and free-living animals. All samples were analysed for protein and carbohydrate. Samples from the free-living bats that were sufficiently large were also analysed for total solids, as were pooled samples from captive bats. Methods
Milk Collection A total of 67 milk samples, each at least 100 mL in volume, was obtained from 11 captive bats at various stages of lactation, from 5 to 139 days post partum, between early November 1995 and March 1996. Milking was usually done once every 2 weeks between 1200 and 1400 hours. The milk was manually expressed from one mammary gland and collected into microcapillary tubes while the mothers young was attached to the nipple of the other gland. The lactating bats, whose ages ranged from 2 to 9 years, were housed in a common enclosure measuring 18 21 73 m. Their diet consisted of chopped mixed fruit, supplied ad libitum, and a ration, averaging 10 g per animal per day, of a supplement (Wombaroo Food

10.1071/ZO96052

0004-959X/97/010065$05.00

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M. Messer and K. Parry-Jones

Products, Glen Osmond, SA) reportedly containing a minimum of 50% protein and 12% fat as well as minerals and vitamins. Milk samples from 14 free-living bats, caught with the aid of a harp trap (Tidemann and Loughland 1993), were obtained near Cabramatta, NSW, on 13 December 1995 between 0400 and 0600 hours. The animals were milked, usually from both mammary glands, within 2 h of capture. Since very few of the young were with their mothers their ages were unknown but if one assumes that most P. poliocephalus are born in October (Nelson 1965; Parry-Jones 1987) then most of the young would have been between 40 and 70 days of age. The milk samples were stored in 05-mL Eppendorf tubes for up to 4 months at 20C, prior to analysis. Analytical The milk samples were thawed, warmed to 37C and thoroughly mixed. For carbohydrate and protein analysis, duplicate 10-mL aliquots were diluted to 200 mL with 10 mM NaOH. (We noted that when water was used as the diluent, an insoluble precipitate tended to form, but this could be prevented by use of a slightly alkaline solution. Control experiments with standards showed that 10 mM NaOH did not affect the analysis of either carbohydrate or protein). Of the diluted milk, 10-m L aliquots were assayed for carbohydrate by a modification (Messer and Green 1979) of the phenolsulphuric-acid method, with lactose as a standard. Aliquots (10 mL) of the diluted milk were then assayed for protein by the dye-binding (Coomassie Blue G-250) method (Bradford 1976) with bovine serum albumin as a standard. Since the dyebinding method is dependent on the amino acid composition of the protein being analysed (Oftedal and Iverson 1995), it was calibrated by Kjeldahl analysis (total nitrogen determination) of three separate 200-mL aliquots of pooled milk obtained during early, mid and late lactation. The protein values obtained by the Kjeldahl method were 134 05% higher than those obtained by the dye-binding method; all our values were therefore adjusted accordingly, even though part of the difference would have been due to non-protein nitrogen, which was not estimated. Oftedal and Jenness (1988) found that non-protein nitrogen accounted for a fairly large proportion of the total nitrogen in the milk of equids and that the use of total nitrogen assays to calculate crude protein overestimated the protein content by 0203 g per 100 g of equid milk. It is possible, therefore, that our adjusted values for crude protein similarly overestimate the true protein content. Total solids were determined by measuring the mass change, to the nearest 001 mg, after freeze-drying to constant mass. Since most of the samples from the captive bats were too small for accurate determination of total solids by this method, the determinations were done only on duplicate 200-mL aliquots of pooled samples. Pooling was done by combining 50-mL aliquots from each of the individual milk samples. Those samples from the free-living bats that were at least 250 mL in volume (10 of 14) were also analysed for total solids, with duplicate 100-mL aliquots. The ash of pooled milk samples (each 400 mL) from captive and free-living bats was determined by incineration at 400C for 20 h. Milk fat was estimated as the difference between total solids and the sum of protein, carbohydrate and ash. Our estimates for fat are therefore subject to considerable error. If the values for protein were overestimated (see above), those for fat would be underestimates. Attempts were made to dermine the fat content by the creamatocrit method (Lucas et al. 1978) but it became apparent that the concentration of fat was too low to permit reliable results by this method. Statistical comparisons of the data for captive bats were made by a mixed model ANOVA (BMDP Statistical Software, University Press of California, 1992) which controlled for individual contributions of each bat and stage of lactation. Pairwise differences were determined by t-tests. Comparisons between the data for free-living bats and those for captive bats during the same stage of lactation were made by unpaired t-tests.

Results Results of protein and carbohydrate analyses of 67 milk samples collected from 11 captive flying-foxes at various times post partum are shown in Fig. 1. The data are summarised in Table 1, which lists the mean values for samples obtained from 5 to 50 days, from 51 to 99 days and from 100 to 139 days post partum; these time intervals are arbitrarily designated as early, mid and late lactation, respectively. There was no significant change in the protein or carbohydrate content up to 99 days post partum ( P > 005) but late in lactation there was a small but significant increase in the protein content (P < 0008) and a decrease in the carbohydrate content (P < 0001).

Milk Composition in Grey-Headed Flying-Foxes

67

10

(a)
8

Protein (%)

0 0 50 100 150

10

(b)
8

Carbohydrate (%)

0 0 50 100 150 Time post partum (days)

Fig. 1. Composition of milk of 11 captive grey-headed flying foxes with respect to (a) protein and (b) carbohydrate from 5 to 139 days post partum. Each of the symbols used for the data points is specific to a given animal.

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Table 1. Milk composition in captive and free-living Pteropus poliocephalus Fat, total solids and ash of the milk of captive animals were determined on pooled samples only. Fat was estimated as the difference between total solids and the sum of protein, carbohydrate and ash. The values in parentheses represent the number of individual samples analysed. Those within square brackets are the number of samples from which equal aliquots were pooled. All percentages are expressed as weight per volume; n.d., not determined. Values showing the same superscript are significantly different: a = P < 0008; b = P < 0025; c, d = P < 0001 Days post partum Protein (%) Carbohydrate Fat Total solids (%) (%) (%) (Mean s.d.) Captive 622b 028 202 (15) [15] 628c 037 174 (31) [31] 590b,c 042 190 (21) [21] 626 033 n.d. (13) Free-living 644 037 216 086 (14) (10) Ash (%) Energy (kJ mL1)

550 5199 100139 4070

252 054 (15) 242a 048 (31) 283a 057 (21) 227d 053 (13) 364d 064 (14)

112 [15] 108 [31] 111 [21] n.d.

046 [15] 040 [31] 046 [21] n.d.

241 [15] 229 [31] 239 [21] n.d.

4070 (est.)

127 075 (10)

062 [10]

274 030 (10)

Total solids and ash content of pooled samples obtained during early, mid and late lactation are also listed in Table 1, thus allowing estimation of the fat content (see Methods). The data indicate that the milk of captive flying foxes is relatively dilute in terms of dry matter and contains only about 2% fat. Comparison of milk samples from 14 free-living bats with 13 samples from captive bats at the same stage of lactation (4070 days post partum; Table 1) shows that their protein content is significantly greater ( P < 0001) but there is no significant difference with respect to carbohydrate. Of the 14 samples from the free-living bats, 10 were also analysed for total solids; the mean value obtained was higher than that for the milk of the captive bats but most of this difference appears to be due to the higher protein content. The mean value for fat (22%) was slightly higher than that for the milk from the captive bats but the results for milk fat were very variable, ranging from 073 to 35%. Discussion The only previously published values for milk composition in Megachiroptera are limited to a single sample from the epauletted fruit bat, Epomophorus wahlbergi (Quicke et al. 1984). By contrast, there have been several studies on milk composition in Microchiroptera, including an extensive recent investigation on three insectivorous species (Kunz et al. 1995) (Table 2). With two exceptions, the milk of Microchiroptera is more highly concentrated in terms of total solids and energy density, and lower in carbohydrate, than that of P. poliocephalus. The exceptions are the long-nosed bat, Leptonycterus sanborni, which is a pollen and nectar feeder (Huibregtse 1966) and the Jamaican fruit bat, Artibeus jamaicensis, which, like P. poliocephalus, forages primarily on fruit, blossoms and leaves (Parry-Jones and Augee 1991; Kunz and Diaz 1995). It seems likely, therefore, that milk composition in bats is related to the maternal diet (see also Kunz and Stern 1995). In other eutherian mammals, dilute milks, with a total solids content of less than 20%, are mostly confined to herbivorous or frugivorous species and are rarely found in carnivores or omnivores (Jenness and Sloan 1970; Oftedal and Iverson 1995).

Milk Composition in Grey-Headed Flying-Foxes

Table 2. Milk composition in Chiroptera Mean values based on the number of samples in parentheses: n.d., no data Total solids (%) Microchiroptera Tadarida brasiliensisB Myotis lucifugusB Myotis veliferB Myotis thysanodes Eptesicus fuscus Leptonycterus sanborni Artibeus jamaicensis Megachiroptera Epomophorus wahlbergi Pteropus poliocephalus Captive Free-living Protein (%) Carbohydrate (%) Fat (%) Ash (%) EnergyA (kJ mL1) Reference

(21) (3) (3) (1) (4) (21) (1)

365 271 324 405 n.d. 121 (1) 178 120 111 (3) 127 (10)

769 850 107 121 617 437 (1) 36 42 259 (67) 364 (14)

336 395 438 34 252 539 (5) 61 40 613 (67) 644 (14)

258 158 199 179 164 171C (1) 90 35 189C (3) 216C (10)

n.d. n.d. n.d. 16 n.d 063 n.d. n.d. 044 (3) 062 (1)

123 875 109 104 837 261 532 302 236 274

Kunz et al. (1995) Kunz et al. (1995) Kunz et al. (1995) Jenness and Sloan (1970) Kunz et al. (1983) Huibregtse (1966) Oftedal and TaftD Quicke et al. (1984) Present results

A All Energy densities were calculated with energy equivalents of 245, 165 and 381 kJ g1 for protein, carbohydrate and fat, respectively (Oftedal and Jenness 1988). For the sake of simplicity, it was assumed that all milk samples had a specific gravity of 100. B Peak lactation. C Estimated by difference (see Methods) D Unpublished data cited by Oftedal and Iverson (1995).

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Ben Shaul (1962) was perhaps the first to suggest a relationship between the composition of the milk of different species and their nursing behaviour; species (e.g. primates) whose young suckle frequently or on demand have dilute, low-fat milks whereas those whose young feed at widely spaced intervals (e.g. pinnipeds) have concentrated milks with a high fat content (Jenness and Sloan 1970; Kunz et al. 1995). P. poliocephalus give birth in spring and carry their young on foraging flights during the first three weeks (Nelson 1965). The young remain permanently attached to their mothers during this time and are able to suckle on demand. After three weeks of age, in free-living animals, the young are found attached during the day, but during the night the mothers leave them in the colony site. The mothers may return during the night to feed their young (Ratcliffe 1931), who are unable to forage for themselves until they are about 3 months old. In captive animals we observed a similar pattern with older young being attached to the nipple throughout the day but only permitted the occasional feed during the night. The young of P. poliocephalus can therefore be regarded as frequent feeders and the low total solids and fat content of the milk of this species is consistent with Ben Shauls proposal. It should be noted, however, that the sampling procedure used in the present study may not have been optimal. In many species there is an increase in the concentration of milk fat during the course of milk let-down (e.g. Atwood and Hartmann 1992) and therefore our samples may not have been entirely representative of the milk ingested by the young during a normal feed. In future studies it may be worth determining the effect of prior administration of oxytocin, since this would most probably increase the size of the samples. Further, we did not investigate whether separating the young from its mother for an extended period before milking would affect milk composition. Kunz et al. (1995), who studied this in T. brasiliensis, found that milk samples collected after 46 hours of separation contained about 41% more fat than those obtained immediately after the pre-dawn feeding period; protein and carbohydrate were unchanged. It is possible therefore, that our values for milk fat are underestimates and that the milk is more concentrated than would appear from Table 1. (An increase of 41% would have the effect of changing our values for fat from about 2% to almost 3%). Kunz et al. (1995) noted significant increases in the fat and total solids content during lactation in all three of the insectivorous species they studied, with the carbohydrate and protein values remaining constant. In the present investigation, changes in milk composition were small and appeared to be confined to protein and carbohydrate concentrations towards the end of lactation. Studies on other megachiropterans and/or other frugivorous species should reveal whether an increase in the milk fat and total solids content during lactation is found only in insectivorous species of bats. In the present study the milk of the free-living bats was very significantly higher in protein than that of the captive animals. This difference may conceivably be due to diurnal variations in milk composition since the milk from the free-living bats was obtained during the early morning whereas the captive animals were milked in the afternoon. However, differences in milk composition between free-living and captive animals have previously been noted in other species. In bears (Jenness et al. 1972) the milk carbohydrate concentration was found to be lower in free-living than in captive animals, whereas in ringtail possums (Munks et al. 1991) the milk fat was higher but the protein was lower. Munks et al. (1991) have noted that the diet of their captive animals contained more protein than that of the free-living ones and suggested that this may have contributed to higher milk protein concentrations. It is quite possible that the protein content of the diet of our lactating captive bats was less than optimal. Lactation imposes a great metabolic demand on small animals and has to be met by very significant increases in food consumption (Jenness 1974). Protein may be the limiting factor in the diet of frugivorous bats (Thomas 1984; Steller 1986) and although our animals received a protein supplement this was not provided ad libitum. The preferred foods of freeliving P. poliocephalus are the nectar and pollen from blossoms of the family Myrtaceae and of the genus Banksia, as well as native figs (e.g. Ficus rubiginosa) and other native fruits, rather than cultivated orchard fruits such as those supplied to our captive bats (Parry-Jones 1987;

Milk Composition in Grey-Headed Flying-Foxes

71

Parry-Jones and Augee 1991). In addition, their diet probably includes leaves and some insects (Parry-Jones and Augee 1992; Parry-Jones 1993). Kunz and Diaz (1995) believe that folivory may be quite common in species that feed largely on fruits that are low in protein while Funakoshi et al. (1993) consider that insects form a small but nutritionally important component of the diet of the warm-temperate fruit bat, Pteropus dasymallus dasymallus. Since pollen, leaves and insects are higher in protein content than orchard fruits (Rasweiler 1977; Hume 1982), our free-living bats may have ingested more protein than the captive ones, which could explain the difference in milk composition. Nevertheless, all the young of our captive animals appeared to be healthy and gained weight steadily, their mean weight at 20 weeks post partum being 531 g. Since there are no published values for growth rates in P. poliocephalus we were not able to determine whether our captive young grew at the same rate as free-living animals. Our results suggest that none of the commercial milk substitutes currently available is entirely suitable for the hand-rearing of orphaned flying-foxes (Table 3). All these substitutes contain more fat and some contain either more protein (e.g. Wombaroo and Biolac) or less protein (e.g. Nan 1 or 2) than the milk of P. poliocephalus. When one compares the composition of the milk of free-living P. poliocephalus with that of other species (Jenness and Sloan 1970) it appears that equid milk, which contains 12% fat, 1618% protein and 67% carbohydrate (Oftedal and Jenness 1988), is the most similar. A milk substitute designed for foals, supplemented with about 15% protein and 05% fat, might therefore be close to optimal for suckling grey-headed flying-foxes.

Table 3.

Comparison of composition of P. poliocephalus milk with that of some milk substitutes Protein (%) Carbohydrate (%) Fat (%) Energy (kJ L1)

P. poliocephalus milk Captive Free-living Nan 1A Nan 2A DiVetelactB WombarooC Biolac for puppiesD
A B

26 36 15 23 30 50 60

61 64 76 73 50 63 40

19 22 34 32 37 30 70

2360 2740 2729 2743 2800 3200 4797

Nestle Aust Ltd, GPO Box 4320, Sydney, NSW 2001. Sharpe Laboratories Pty Ltd, 3/12 Hope St, Ermington, NSW 2115. C Wombaroo Food Products, PO Box 151, Glen Osmond, SA 5064. D Biolac, 15 OShannassy St, Mount Pritchard, NSW 2170.

Acknowledgements We thank Dr Chris Tidemann for permitting us to milk the flying-foxes caught at his study site in Cabramatta and Julie Spence for helping with the project on the site. We also thank the Wambina Flying-fox Research Centre in Gosford for the use of their captive breeding colony. We are indebted to Keith Newgrain (CSIRO Division of Wildlife and Ecology, Canberra) for performing Kjeldahl (total N) estimations of three samples of milk and to Dr Thomas Kunz (Department of Biology, Boston University, USA) for reviewing the initial manuscript of this paper. We are most grateful to Dr Samir Samman (Human Nutrition Unit, University of Sydney) for his assistance with the statistical analyses.

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