Haematology and iron status of the egyptian fruit bat, rousettus aegyptiacus

Camp. Biochem. Physiol. Vol. 9OA, No. I, pp. 117-120, 1988

0300-9629/88 $3.00 + 0.00 0 1988 Pergamon Press plc

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HAEMATOLOGY AND IRON STATUS OF THE EGYPTIAN FRUIT BAT, ROUSETTUS AEGYPTIACUS J. VAN DER WESTHUYZEN Nutrition Unit, Department of Haematology, School of Pathology of the South African Institute for Medical Research and the University of the Witwatersrand, PO Box 1038, Johannesburg 2000, South Africa. Telephone: 01 I-725-051 1 (Received 7 September 1987)

Abstract-l. Haematological values and iron status of wild and captive fruit bats (Rousettus aegyptiucus) were determined. 2. Plasma iron concentrations were 175 pg/dl in wild males, and 286-316 pg/dl in captive bats. 3. Total splenic stores were small (around 100 pg) in relation to hepatic stores (3 mg) and total haem iron (2.6 mg). 4. Haemoglobin levels, red cell counts and haematocrits were unusually high and mean corpuscular volumes low. 5. Lactating wild bats showed no deficits in iron status or in haematological values. 6. It is concluded that the ascorbic acid content of fruit, together with the bats’ high food requirement, has ensured an ample iron supply in this vegetarian species.

INTRODUflION

The diet of man contains iron in two forms, namely haem iron and non-haem iron with different’ absorption mechanisms. Although haem iron is generally better absorbed, non-haem iron is the main source of iron in the diet (Bothwell et al., 1979). Non-haem iron is generally poorly bioavailable unless some enhancing factor such as ascorbic acid is present. The iron content of fruit is generally fairly low (‘to.5 mg Fe/100 g) (Paul and Southgate, 1978). Although fruit contains ascorbic acid, fruit is also a source of fibre, which inhibits iron absorption. It was therefore considered to be of interest to assess the iron status and haematological values of a vegetarian species, the Egyptian fruit bat, Roirsettus aegyptiacus (suborder Megachiroptera). This gregarious, cave dwelling species is the second largest fruit bat that occurs in the Southern African subregion (Smithers, 1983), and feeds on most pulpy wild fruits. Rousettus species have also been observed to feed on the pollen and nectar of various flowers (Jacobsen and du Plessis, 1976).

MATERIALS

AND METHODS

Animals

Two groups of wild bats were studied. Male Egyptian fruit bats Rousettus uegyptiacus (E. Geoffroy, 1810) were caught in November 1985 when returning to a cave in the Matlapitsi valley of the Drakensberg Mountains, northeastern Transvaal. This is a semi-tropical region and fruit, especially figs (Ficus spp.), is available the whole year round (Lombard 1961). Female fruit bats were caught during the litchi season (December 1986) in an orchard at Trichardsdal in the Lowveld, close to the Drakensberg, where these bats roosted in caves. These bats were all lactating. A group of bats caught in the litchi orchard was kept in captivity on a diet of peeled, sliced banana (Muss cav-

endishii) and diced paw-paw (Car&

papaya). Vitamin Btz levels were maintained by intramuscular injections of cyanocobalamin (0.5 pg/lOO g body weight fortnightly). Animals also received a multivitamin preparation (“Abidec”, Parke-Davis) weekly to supplement their intake of vitamin A, B, and D and thiamin, riboflavin and niacin (van der Westhuyzen, 1983). Experimental procedures Wild animals. Male cave bats (n = 10) were killed by exsanguination on the afternoon of the day of capture. Mean body weights were 142.4 + 7.3 g (mean + SD). The lactating female bats (n = 10) caught in the orchard, were killed during the afternoon following the night of capture. Mean body weights were 112.3 & 4.5 g, significantly lower than found in the group of male cave bats (P i 0.001). For both groups blood was withdrawn by cardiac puncture into heparinized tubes for the full blood counts and the estimation of plasma iron. Livers and spleens were surgically removed, weighed and placed in buffered formyl saline for measurement of tissue iron concentration. Cupfive animals. The results presented below suggested a sex difference in iron status and haematological indices among the wild bats. Consequently, plasma iron determinations and full blood counts were carried out on both male (n = 1I) and non-pregnant adult female bats (n = 27) held in captivity on the standard all-fruit diet for 3&32 weeks. Body weights of the male bats (136.5 + 8.6 g) were significantly higher than in the female group (129.3 + 9.8 g; P < 0.05). Laboratory methods

Full blood counts were carried out within one day on a Coulter FN electronic counter with Coulter “4c” calibration (Coulter Electronics Inc.). The attenuation was suitably adjusted to count the bat red blood cells which normally have a low MCV. Plasma iron concentrations were determined by the guanidine/FerroZine method (Roche). Non-haem iron concentrations of formalinized tissue samples were measured using the method of Torrance and Bothwell (1980). Plasma ferritin and transferrin concentrations could not 117

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118

VAN DER

be estimated by the standard immunoassay methods, as these bat proteins were inadequately bound by the specific antibodies, which were raised to human ferritin and transferrin, respectively. Slaristical analysis

Statistical evaluation was performed by Student’s f-test (two-tailed). The level of significance was chosen as P c 0.05. Data are expressed as means i SD.

RESULTS

for the wild bats are summarized in Table 1. Red cell counts were relatively high (12-14 x lO12/l), as were haemoglobin concentrations (over 17 g/dl). The mean haematocrit varied from 44% in cave male bats to 57% in the orchard females. Red blood cells were small, with mean corpuscular volumes (MCVs) of the order 3641 fl. Mean cell haemoglobin values (MCH) were therefore relatively low, in the range 12-l 5 pg. Mean cell haemoglobin concentrations (MC~C) varied from 3@-42g/dl. Surprisingly, the red ceil count, haematocrit and MCV were significantly higher and MCH and MCHC significantly lower, in the orchard females compared with the male cave group. Since the male and female wild bats were sampled in different locations (and I1 months apart), haematological values of captive male and female bats were also investigated (Table 2). The red cell count, haemoglobin and MCHC were significantly lower in the captive female compared to male bats. The MCV on the other hand, was significantly higher in the female group. These results are in contrast to the differences between the cave male bats and the lactating females caught in the orchard (see Discussion). The plasma iron and tissue iron concentrations in wild bats are summarized in Table 3. The orchard female bats, although lactating and having recently given birth to offspring, had significantly higher hepatic and splenic iron concentrations than the wild male bats. The mean splenic iron concentration in the female animals was double that found in the cave male animals. Total hepatic and splenic stores were similar in both groups, although values tended to be higher in the females. Plasma iron levels were substantial. The mean plasma iron level in male cave bats (175 pg/dl), was significantly lower than in captive male (316 pgg/dl, P < 0.001) and captive female groups (286 pg/dl, Haematological

parameters

P < 0.01). Table 1. Haematology of wild fruit bats (means rt SD) Cave males

Parameter White cell count

(x 109/1)

Red cell count (x IO”,@ Haemoglobin (g/dl) Haematocrit (%) Mean corpuscular volume (A) Mean cell haemoglobin (Pg) Mean cell haemoglobin concentration (g/dl)

(n = 10) --

Orchard females (Jr= 10) ~-..__

8.8+ 2.5 Il.95 2 0.94 17.1 i: 1.1 43.9 * 2.4

8.8 & 5.8 13.88 i 1.52’ 17.4 i: 2.0 57.0 * 6.2i

36.8 + I .4

41.1 It 1.7t

14.4 i_ 0.5

12.6 _+0.V

39.0 zt 1.6

30.6 * o.st

Significantly different from male group: *P < 0.01:

tP

< 0.001.

WFZTHUYZEN Table 2. Haematology and plasma iron levels in fruit bats in captivity (means t SD) Parameter __I_. ~ ..-. White cell count ( x 109/l) Red cell count (x 10’2/1) Haemoglobin (gjdl) Haematocrit (%) Mean corpuscular volume (fl) Mean cell haemoglobin (pg) Mean cell haemoglobin concentration (g/dlf Plasma iron (ng/dl)

Males (n = II)

Females (rt = 27)

12.1 k4.2

10.8 k 3.4

15.42 i 1.50 15.6 + 0.8 58.4 f 4.4

14.32 5 1.25’ 14.4 & 1.4t 56.4 ri: 5.1

38.0 + I .h

39.5 + 1.5’

IO.1 ri_O.6

10.0 IO.8

26.7 + I.2 316 i45

24.6 + 5.0’ 286 rt 82

Significantly diRerent from male

group: *P < 0.05;

tP

< 0.01.

Table 3. Plasma and tissue iron in wild fruit bats (means t SD) Parameter -____ __--__Plasma Fe (pgidlf Hepatic Fe (pgjg fresh wt) Splenic Fe trig/g fresh wt) Total haem Fe$ (mg) Hepatic Fe stores (mg) Splenic Fe stores (,ng)

-.

Cave males (n = IO) 175*94

Orchard females (n = IO) ND

473.5 i 155.6

752.1 i 342.1’

174.4k41.3 2.95 _i 0.29 2.84 + 0.99 87.1 i 17.6

361.8 * 140.4t 2.48 + 0.22t 3.19 f 1.69 1162~41.4

ND, not done. Significantly different from male group: *P < 0.05; tP < 0.001. $Calculated for each bat on the basis of a red cell volume of 35Sml/kg (van Tender, 1974) and on the assumption that haemoglobin contains 0.34% iron by weight (Underwood, 1971).

DISCUSSJON

Lactating female R~usefrus caught in the litchi orchard were selected as a group whose iron status was under stress through providing for the needs of the foetus and via lactation, for post natal development. It was therefore surprising that their haematological status was superior to that of male bats obtained from the cave. However, the two groups of wild bats represented different populations and were sampled at different times. The studies on the bats in captivity failed to show the same sex difference in haematological values; indeed, the male group showed higher red cell counts, higher haemoglobin levels and higher MCHCs than the non-pregnant, non-lactating females. This suggests that the observed differences between the male and lactating female bats in the wild are not physiological but determined by other factors, of which diet is most important. Dietary factors necessary for haemopoiesis include vitamin B,, and folate for DNA synthesis, amino acids for globin synthesis and iron for haem synthesis. Previous studies have shown that the fruit bat in the wild maintains satisfactory vitamin B,, and folate nutrition (van Tonder et al., 1975). The protein requirement of the fruit bat is unknown, but their all-fruit diet is certainly adequate to support rapid growth of the young. The results presented here suggest that the fruit bat Ro~se~?u~ in the wild obtains an ample dietary supply of iron. How is this achieved? The iron content of conventional fruit (with a few exceptions such as prunes and mulberries)

Haematology

is generally low (c 0.5 mg Fe/100 g) (Paul and Southgate, 1978). Although the iron content of wild fruits consumed by Rousettus is unknown, ascorbic acid, a major promotor of iron absorption, is commonly present. A recent study in man has shown that the ability of fruit juices to enhance iron absorption from a meal with low iron availability (rice), was correlated with their ascorbic acid contents (Ballot et al., 1987). None of the fruits in that study contained known inhibitors of iron absorption, such as phytate and condensed polyphenols, in amounts likely to be inhibiting. Moreover, fructose, which is present in fruit, also forms iron chelates (Bothwell et al., 1979) and therefore facilitates absorption. In addition to these facilitating factors, fruit bats have high food’requirements. In captivity, Rousettus consume 75% of their body weight per night (van der Westhuyzen, 1976). A high food intake increases the chances of absorbing sufficient iron from a diet of relatively low iron 1 bioavailability. The haematological values of Rousettui provide an interesting comparison with those of other species. Haemoglobin values of wild bats (17 g/dl) were higher than in most neotropical bats reported by Valdivieso and Tamsitt (1971). The haemoglobin concentration of iron-supplemented rats, 9 weeks old, was similar to that of the fruit bat (McCall et al., 1962; Williams et al., 1983, 1985). The red cell count in wild Rousettus (12-14 x 1012/1)is fairly high, similar to the little brown bat Myotis lucifugus (11 .O x lO’2/1,Worth 1932) but much higher than in the rat (7-8 x 1012/1)(McCall et al., 1962; Adelekan and Thurnham, 1986). In 4 species of neotropical bats, the red cell count varied from 3.9-7.8 x 1012/1 (Valdivieso and Tamsitt, 1971). Haematocrits in these species (29952.5%; average 43%) were similar to those of male cave bats (43.9%) and the rat (44.3%) (McCall et al., 1962). Haematocrits in the wild female and all the captive bats, (5658%) were however higher. White cell counts in Rousettus (8.8 x 109/1) were higher than reported in 4 species of neotropical bats (3.CL8.4, average 5.5 x 109/1) (Valdivieso and Tamsitt, 1971) and were even higher in captivity. The mean corpuscular volume (MCV) in Rousettus. around 40 fl, is substantially less than in the young rat fed a commercial diet (56.5 fl) (Williams et al., 1983). Interestingly, rats reared on an ironrestricted diet had MCVs of 43347 fl, viz microcytosis was present and the animals were anaemic (Hb of 67 g/d]) (Williams et al., 1983; Adelekan and Thurnham, 1986). The bats however, have considerable levels of all the haematinics and the low MCVs are physiological. For comparison, MCVs in 4 species of neotropical bats varied from 61-90 fl (calculated from data of Valdivieso and Tamsitt, 1971). Plasma iron concentrations in wild male bats averaged 175 kg/d1 (3 1.3 pmol/l), somewhat above values found in man, bovine adults (mean I45 pg/dl), ovine rams (152 pg/dl) and non laying hens (158 pgg/dl) (Underwood, 1971). Values reported in rats are either similar (Williams et aI., 1985) or somewhat higher (McCall et al., 1962; HSimllHinen and ‘Mlkinen, 1983; Williams et al., 1983; Adelekan and Thurnham, 1986) depending on their diet. As discussed earlier, plasma iron levels in the captive bat were higher than in their counterparts in the wild.

of fruit bat

119

In male Rousettus, hepatic iron concentrations (474 pg/g) exceeded splenic iron levels (174 pgg/g) by a factor of 2.7. In females the ratio was 2.1. In rats the reverse situation pertained (Williams et al., 1983; Hamllainen and Miikinen, 1983). Hepatic iron concentrations in young rats may, however, vary from only 3Opg/g (Williams et al., 1983) to at least 262pg/g (Adelekan and Thurnham, 1986), depending on the composition of the diet. Total splenic stores were small (around 100 pg) in relation to total hepatic stores (around 3 mg) and the calculated total for haem iron (around 2.6 mg). In relation to bodyweight, haem iron amounted to 2&22 mg/kg, somewhat lower than the 28.3 mg/kg found in iron replete growing rats (McCall et al., 1962). Total inorganic iron in liver and spleen in male bats amounted to 2.93 mg or 20.55 mg/kg bodyweight, about 5 times more than the rat on a body weight basis (Spray and Widdowson, 1950). These comparisons with the rat clearly demonstrate that the liver is the major storage site for iron in the fruit bat. In summary, facilitating factors such as the ascorbic acid and fructose content of fruit, together with the high food requirement of fruit bats, have ensured an ample iron supply in this vegetarian species. Acknowledgemenrs-Monica Simons is thanked forming the tissue iron assays. This work was supported in part by a grant South African Medical Research Council.

for perfrom

the

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