Unique Pits on the Erythrocytes of the Lesser Mouse-Deer, Tragulus Javanicus

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J. Anat. (1996) 189, pp. 211-213, with 3 figures Printed in Great Britain 211 Short Report Unique pits on the erythrocytes of the lesser mouse-deer, Tragulus javanicus

KATSUHIRO FUKUTA', HIROSHI KUDO2 AND SYED JALALUDIN3 1 National Institute of Animal Health, and I National Institute of Animal Industry, Ibaraki, Japan, and I Universiti Pertanian Malaysia, Selangor Darul Ehsan, Malaysia

(Accepted 13 March 1996)

ABSTRACT The lesser mouse-deer, Tragulus javanicus, is the smallest living ungulate and it possesses the smallest erythrocytes amongst mammalian species. We observed pits on its erythrocytes by scanning electron microscopy. They were 68-390 nm in diameter (135 + 65 nm) and appeared in 12.8 % of erythrocytes examined. They represent unique structures that have not previously been reported physiologically or pathologically.

capillaries. A small amount of the blood was spread INTRODUCTION on a microscope slide, fixed with methanol, stained The mouse-deer or chevrotain is a small mammal, with Giemsa, and examined by light microscopy. belonging to the order Artiodactyla and the suborder For scanning electron microscopy, blood was added Ruminantia. The lesser mouse-deer, Tragulus javani- to 2 different cold fixatives: 1 % glutaraldehyde in cus, is the smallest among living hoofed animals (Heck 0.1 M phosphate buffer (pH 7.2) and 2.5 % gluta- et al. 1968; Nowak, 1991), adults weighing about raldehyde in 0.1 M cacodylate buffer (pH 7.2). After 1.5 kg and rarely over 2 kg (Medway, 1978; Tweedie, fixation for 2 h, precipitated blood cells were sus- 1978). The erythrocytes of the lesser mouse-deer are pended in the same buffer. The blood cell suspension known to be the smallest amongst mammals (Altman was placed on a small glass slip (6 mm x 6 mm) coated & Dittmer, 1961; Hawkey, 1977). We examined the with poly-L-lysine, and left for 1 h for the blood cells blood cells of this species by scanning electron to adhere to the glass slip. The fixed blood cells on the microscopy and detected unique pits on the erythro- glass slip were postfixed in 1 % osmium tetroxide in cytes. 0.1 M phosphate buffer for 1 h, dehydrated in an ascending ethanol series and immersed in tertiary butanol. The specimens were dried using a freeze- MATERIALS AND METHODS drying device (JFD-300, JEOL), coated with gold at Blood from the lesser mouse-deer was collected from - 50 nm thickness using an ion sputtering device 5 males maintained at the Universiti Pertanian (JFC- 1500, JEOL) and examined by scanning electron Malaysia and 1 female at the National Institute of microscopy (JSM-6301F, JEOL). Animal Health, Japan. One male was captured in the wild and bred in the laboratory for over 2 y. The other lesser mouse-deer were laboratory bred. Except for 1 RESULTS young male aged 6 mo, the animals were mature at 1 y The haematocrit for the lesser mouse-deer was 5 mo to 3 y 7 mo in age. 50.2 + 6.7 % (range 44-61 %). On examination of the For the collection of blood samples a 26 gauge blood film by light microscopy, the erythrocytes were needle was inserted into the saphenous vein. Blood very small and varied in size. Most were round, but was collected in Pasteur pipettes and haematocrit elongated and triangular cells were also recognised.

Correspondence to Dr Katsuhiro Fukuta, National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305, Japan. 212 K. Fukuta, H. Kudo and S. Jalaludin

Fig. 1. Scanning electron micrograph of erythrocytes of the lesser mouse-deer. Small and variously shaped erythrocytes, such as biconcave discs (d), oval (o), spheroid (s), ellipsoid (e) and rod (r) forms are observed. Some of them have a pit (arrow) or rarely 2 pits (double arrows) centrally or at the edge of the cells.

Fig. 2. Erythrocytes with pits (arrows). Pits occur at the central part Fig. 3. High magnification ofmouse-deer erythrocytes. A biconcave of discs and at edge of rods. disc and a swollen disc display pits.

The erythrocytes were nonnucleated and their con- and rods. Discoid or biconcave disc erythrocytes were tents were homogeneously eosinophilic except for 1.65+0.23 pm in diameter and 0.83+0.15 pm in central pallor due to biconcavity. There was no sign of thickness. Statistically there was no difference in the blood parasites, such as Plasmodium, Piroplasma and dimensions of the erythrocytes between the 2 kinds of Babesia. fixative. The diameter of the discs in the present study On scanning electron microscopy, the erythrocytes was less than that obtained from dry films (2 or varied in shape, including discoid, biconcave discs, flat 2.3 gm) by Gulliver (1870) and Vidyadaran (1979), oval, spheroidal, ellipsoid, and rod, triangular and but slightly larger than those in tissue sections irregular shapes (Fig. 1). Discs or biconcave discs were (1.5 ptm; Duke, 1963). dominant amongst the various cell shapes, but it was In many erythrocytes, 1 or rarely 2 unique pits were difficult to classify the erythrocytes into each cell observed (Figs 1-3). The diameters of the pits were shape rigorously from their surface view on scanning 68-390 nm (135 +65 nm; mean+S.D.). Except for a electron microscopy. Moreover, there were inter- few shallow pits, most were deep as the floor was mediate shapes between discs and spheroids, and discs not seen. However, the deep pits did not seem to Unique pits on mouse-deer erythrocytes 213 perforate the cell. They appeared in all cell shapes these pits appear to be unique although their mentioned above and at any region on the cells, i.e. significance is uncertain. To investigate their nature, some pits were present at the central region of discs we intend to study newborn animals, haematopoiesis and rods and others at the edge (Figs 1, 2). Pits did not in bone marrow and other organs, and the molecular occur in all erythrocytes. By counting the pits in structure of erythrocyte membranes. scanning electron micrographs, 130 cells out of a total 1012 cells (12.8 %) had pits. Two pits per erythrocyte were found in 10 cells (7.7 % of the cells with pits). As ACKNOWLEDGEMENTS the cell count was carried out on the surface view by We thank the Department of Wildlife Protection and the scanning micrography, the actual number of cells National Park, Malaysia, for the permission and with pits would be more numerous if the opposite side license to rear the lesser mouse-deer in captivity. of the erythrocytes were also considered. The pits occurred in all examined animals. They were derived REFERENCES from different colonies and were clinically healthy, ALTMAN PL, DITrMER DS (1961) Blood and Other Body Fluids, but the incidence of pits differed between individuals. pp. 119-120. Bethesda, MD: Federation of American Societies for Experimental Biology. CASTOLDI GL, BEUTLER E (1988) Erythrocytes. In Atlas of Blood DISCUSSION Cells-Function and Pathology, 2nd edn (ed. Zucker-Franklin D, Greaves MF, Grossi CE, Marmont AM), pp. 49-80. Phila- Snyder & Weathers (1977) reported that the small delphia: Lea and Febiger. erythrocytes of the lesser mouse-deer were compen- DuKE KL (1963) Erythrocyte diameter in Tragulus javanicus, the chevrotain or mouse deer. Anatomical Record 147, 239-241. sated for functionally by an extremely high erythro- GULLIVER G (1870) On the size of the red corpuscles of the blood cyte count, and that their shape appeared to be of Moschus, Tragulus, Orycteropus, Ailurus, and some other unique with a spherical configuration. In the present mammalia, with historical notices. Proceedings of the Zoological Society of London, 92-99. study, spherical and ellipsoidal erythrocytes were HAWKEY C (1977) The haematology of exotic mammals. In observed, but discs and biconcave discs were domi- Comparative Clinical Haematology (ed. Archer RK, Jeffcott nant. LB), pp. 103-160. Oxford: Blackwell. The pits observed on the erythrocyte are unique: HECK L, HEINEMANN D, TBENIus E (1968) The cud chewers. In Grzimek's Animal Life Encyclopedia, vol. 13 Mammals IV (ed. similar structures have not been reported either Bannikow AG et al.), pp. 149-153. New York: Van Nostrand physiologically or pathologically. In erythrocytes, Reinhold. various abnormal shapes occur, such as echinocytes, MEDWAY L (1978) Mouse-deer or chevrotains, family Tragulidae. In The Wild Mammals of Malaya (Peninsular Malaysia) and stomatocytes, spherocytes, elliptocytes, drepnocytes Singapore, 2nd edn, pp. 106-107. Kuala Lumpur: Oxford (sickle cells), acanthocytes, and others, related to University Press. hereditary and pathological disorders, osmotic and NOWAK RM (1991) Chevrotains, or mouse deer. In Walker's Mammals ofthe World, vol. 2, 5th edn, pp. 1359-1362. Baltimore: pH alterations and exposure to chemicals such as Johns Hopkins University Press. phenothiazines and chlorpromazine (Costoldi & SNYDER GK, WEATHERS WW (1977) Hematology, viscosity, and Beutler, 1938; Wintrobe et al. 1981). However, respiratory functions of whole blood of the lesser mouse deer, structures similar to the pits on the lesser mouse-deer Tragulus javanicus. Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology 42, 673-678. erythrocytes have not previously been recognised. TWEEDEE MWF '(1978) Tragulidae, chevrotains or mouse-deer. In Stomatocytes have cup-shaped depressions of various Mammals of Malaysia, pp. 60-61. Petaling Jaya, Malaysia: sizes, but differ from the pits. Longman Malaysia. VIDYADARAN MK, HILMI M, SIRIMANE RA (1979) Haematological The lesser mouse-deer used in the present study studies on the Malaysian lesser mouse-deer (Tragulusjavanicus). were clinically healthy and no parasites were found on Pertanika 2, 101-104. inspection of blood films. Despite this, we cannot WINTROBE MM, LEE GR, BoGGs DR, BITHELL TC, FOERSTER J, exclude the ATHENS JW et al. (1981) Disorders of red cells. In Clinical possibility that the animals were affected Hematology, 8th edn, pp. 527-1042. Philadelphia: Lea and by an infective or hereditary disorder. Nevertheless, Febiger.