LM and SEM Studies on Tongue and Lingual Papillae in the Donkey (Equus asinus)

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1 LM and SEM Studies on Tongue and Lingual Papillae in the Donkey (Equus asinus) Hanna Jackowiak 1, Hassen Jerbi 2, Kinga Skieresz-Szewczyk 1 and Ewelina Prozorowska 1 1 Department of Histology and Embryology, Poznan University of Life Sciences, Wojska Polskiego71C, PL Poznań, Poland 2 Department of Anatomy, Veterinary School of Sidi Thabet, CP2020, Tunisia The macroscopic, LM and SEM observations were carried out on 27 tongues of the donkey. The three age groups were determined: year, 2-5 year, 6-10 year. The tongues of the donkeys in all studied age groups are elongated and consist of the spatula-like apex, narrow body, torus linguae and the root of tongue. It was found no sexual dimorphism of the morphological features of the donkey's tongue. The length of the tongue in studied age groups increases from 20 cm to 26 cm and the width of the tongue on torus area increases from 4.8 cm to 6.0 cm. The special-specific features, in comparison to the tongue of horses, is rare presence of the cartilage of dorsum of the tongue (cartilago dorsi linguae). The mucosa of the tongue forms filiform, fungiform, foliate and vallate papillae. The filiform papillae as only one type of mechanical papillae, cover the entire surface of the tongue up to the area of the vallate papillae. SEM studies reveal the presence of the two types of filiform papillae, differing in density and size of processes on the anterior part of tongue and on torus linguae. The fungiform papillae are scattered between the filiform papillae. Interesting observation in the donkey tongue is age related reduction of the number of fungiform papillae positioned on lateral surface of donkey's tongue, from 85 to 60. In each age group foliate papillae are located on the lateral surface of the posterior part of torus linguae and consist of 7-8 mucosal folds. Characteristic feature in donkey is variable number and shape of the vallate papillae. There are predominantly three vallate papillae located in one row behind the torus linguae. We also observed 2 to 5 vallate papillae in each age groups. The results are discussed in terms/aspect of feeding function, food manipulation in oral cavity and grooming behavior. Keywords: Functional Morphology; Tongue; Lingual papillae; Donkey; LM&SEM 1. Introduction The donkey is a member of the Equidae family along with horses and mules. Donkeys are domesticated asses, and they constitute their own species, Equus asinus. The genetic studies on donkey and wild ass from all over the world showed that wild asses in Northeast Africa are the ancestors of modern donkey [1]. So far the studies on morphology of organs of the oral cavity in Equidae were focused mainly on horses [2-7]. The donkey as a member of the Equidae family plays a significant economic importance in some areas of the world. Some morphological data about tongue were given by Abd-Elmaeim et al. [3]. Therefore, we aimed to prepare detailed macroscopic description of donkey s tongue and also scanning electron microscopic (SEM) and light microscopy (LM) observations of the distribution and microstructure of mechanical and gustatory lingual papillae on the surface of the tongue to look on species-specific features and functional adaptations. Such description could be important not only for veterinary practice but also be used in the discussion of the function of the lingual papillae according to the diet, mechanism of food intake (grazing and chewing) in herbivorous animals. 2. Material and methods The macroscopic observations were carried out on 27 tongues (16 female tongues and 11 male tongues) of the donkey collected from the slaughterhouse. The tongues after dissection were rinsed in saline and immersed in 4% neutralized formalin. The tongues were documented with a digital camera and divided into three age groups. Group 1: year, group 2: 2-5 year and group 3: 6-10 year. Tissue samples from the apex, body and torus linguae were collected. For light microscopy (LM) observation, samples were dehydrated in a series of ethanol (70% - 96%), and embedded in Paraplast. Paraplast blocks were cut µm slides stained with the Masson-Goldner trichrome [8]. The slides were examined using an Axioscope2plus light microscope (Zeiss, Germany). For scanning electron microscope (SEM) analysis the samples were dehydrated in a series of ethanol (70-100%) and acetone (96% - abs.), and subsequently dried at critical point using CO 2 (CPD300, Leica, Germany). All specimens were mounted on aluminums stubs covered with carbon tabs, sputtered with gold (Sputter Coater S150B, Edwards) and observed under the LEO 435 VP at an accelerating voltage of kv. Morphometric analysis were conducted by using a Multiscan system (ver. 10.2, CSS, Warsaw, Poland). 216

2 3. Results The tongue of the donkey is elongated and consists of the spatula-shape apex, narrow body, torus linguae and the root of the tongue (Fig. 1). The macroscopic observations reveal no sexual dimorphism of the morphological features of the tongue in donkey. The morphometric data reveals that the length of the tongue increases in age groups from 20.0 cm to 26.0 cm. The width of the apex increases from 3.8 cm to 4.6 cm and the width of the torus linguae increases from 4.8 m to 5.6 cm. (Graph 1). Four types of lingual papillae, i.e., fungiform papillae, filiform papillae, vallate papillae and foliate papillae are observed on the dorsal surface of the tongue (Figs. 4, 6, 7, 10, 13). Among 20 examined tongues only in 2 specimens cartilage of the dorsum of the tongue (cartilago dorsi linguae) is observed (Fig. 2). Fig. 1. Dorsal view on the tongue in the donkey. A-apex, B-body, T-torus linguae, R-root of the tongue. Scale bar =1 cm. Fig. 2. Cross section through the torus linguae. Arrow shows the cartilage of the dorsum of the tongue. Scale bar =1 cm. Graph 1. Length of the tongue and width of the apex and torus linguae in the donkey in the three age groups Filiform papillae The filiform papillae are the only one type of the mechanical papillae present on the entire surface of the tongue, from the lingual apex to the area of the vallate papillae. Two subtypes of filiform papillae differ in size of keratinized processes and density. On the anterior part of tongue i.e. the apex and the body of the tongue, the filiform papillae are sparsely arrange and their height is about µm (Figs. 3, 4). On the torus of the tongue the densely distributed filiform papillae create the dense brush and their height is µm (Figs. 5, 6). 217

3 Fig. 3. Cross section through the apex of the tongue of the donkey. Arrow shows the short keratinized processes of the filiform papillae. Ep-epithelium, Lp-lamina propria. LM, scale bar =100 µm. Fig. 4. Dorsal surface of the apex of the tongue of the donkey with keratinized processes of the filiform papillae (arrows). SEM, scale bar =100 µm. Fig. 5. Cross section through the torus linguae in donkey. Arrow shows elongated processes of the filiform papillae. Ep-epithelium, Lp-lamina propria. LM, scale bar =50µm. Fig. 6. Dorsal surface of the torus linguae of the donkey with densely distributed processes of the filiform papillae (arrow). SEM, scale bar =50 µm Fungiform papillae Fungiform papillae are evenly scattered between the filiform papillae on the entire dorsal surface of the apex and body of the tongue and are also observed on the lateral surface of the anterior part of the tongue (Fig. 7). These papillae, on the dorsal surface of the lingual apex, are slightly raised over the surface of mucosa, so that they are not well visible in macroscopical observations (Figs. 8, 9). The papillae on the lateral surface of the tongue are visible and interestingly the number of papillae decreases from 85 to 60 in studied age groups. Microscopic observations show that the fungiform papillae are covered with multilayered keratinized epithelium with long connective tissue papillae. The horny layer of epithelium is relative thin and is approximately 15.8 µm (Figs. 8). The average diameter of the fungiform papillae is µm. 218

4 Fig. 7. Lateral view on the lingual body (B) in the donkey. Dashed line marks the surface with the fungiform papillae. Scale bar =300 µm. Fig.8. Cross section through the fungiform papillae. Arrow points the keratinized layer of the epithelium. Ep-epithelium, Lp-lamina propria. LM, scale bar =100 µm. Fig. 9. Higher magnification of the surface of slightly protruded fungiform papillae (Fu) on surface of the body of the donkey's tongue. SEM, scale bar =100 µm Vallate papillae The vallate papillae are present on the posterior part of the torus linguae. In each age groups the number of vallate papillae vary between 2 and 5. Predominantly there are 3 vallate papillae located in one row, positioned perpendicular to the medial line of the tongue (Fig. 10). Characteristic feature of these papillae is variable shape, from rounded papillae, through the oval shape papillae to heart - shape papillae (Fig. 9). The diameter of the papillae varies between 1.7 mm and 3.5 mm. The LM and SEM observations show that the middle part of the vallate papillae i.e. the surface of the body of these papillae is concave (Fig. 11, 12). The body of each papilla is surrounded by wide furrow and flat outer wall. The height of multilayered, partly keratinized epithelium on the body of papillae, is about µm. In the thinner nonkeratinized multilayered epithelium of the lateral wall of the furrow, which height is about µm, are present numerous of the taste buds (Fig. 11). In the lamina propria below the furrows are located serous mucous glands (Fig. 11). 219

5 Fig. 10. Macroscopic pictures of the different shape of the vallate papillae in the donkey's tongue (V). Scale bar =1 mm. Fig. 11. Cross section through the vallate papillae. Arrowhead points the taste buds. Ep-epithelium, Lp-lamina propria, M-muscles, Sg-serous glands. LM, scale bar =1mm. Fig. 12. Higher magnification of the vallate papillae with the wide furrow of the papilla (arrowhead) and concave middle part of the papillae (arrow). SEM, scale bar =1 mm Foliate papillae Foliate papillae are located symmetrically on the lateral surface of the posterior part of the torus linguae near pallatoglossal arch (Fig. 13). Each papillae consist of about 7-8 leaves in each age groups of male and female donkey. Leaves are separated by deep furrows (Fig. 14, 15). Height of the leaves is approximately 1.2 mm and their wide is about 1.2 mm. The foliate papillae are covered with nonkeratinized epithelium, which height is about µm (Fig. 13). In the epithelium of the lateral wall of the leaves are present taste buds (Fig. 14). In the lamina propria below the vallate papillae numerous of the secretory units of the serous glands are present (Fig. 14). 220

6 Fig. 13. Lateral view on the posterior part of the torus linguae (T). Dashed line marks the surface with foliate papillae. Scale bar =4 mm. Fig. 14. Cross section through the foliate papillae (F). Arrows show grooves. Arrowhead points the taste buds. Ep-epithelium, Lp-lamina propria, Sg-serous glands. LM, scale ba r=0,5 mm. Fig. 15. Higher magnification of the foliate papillae (F). Arrow shows groove. SEM, scale bar =0,5 mm. 4. Discussion The macro - and microscopical studies on the tongue of donkey show the general similarity with a tongue in horses. In the age groups studied in the donkey we observe the elongation of 6 cm of the tongue, whereas its width extends of 2 cm. This shows that donkey s tongue is well constituted in postnatal period. The presence of spatula-like apex, elongated torus and muscular eminence called torus linguae is characteristic for all Equidae, studied so far [2-7]. It is important to mention that such eminence called intermolar prominence is typical for some Artiodactyls - ruminants, Perissodactyls - equines, but also is described in rodents [5,6,10,11-16]. The torus linguae or intermolar prominence is in contact with posterior keratinized part of hard palate and soft palate and also with teeth and play an important function during transport of food, mastication and formation of food bolus in all herbivorous species. The macro- and microscopic observations of the lingual papillae in donkey allow us to describe the distribution and microstructure of the mechanical papillae represented by filiform papillae and also three kinds of gustatory papillae. To describe a functional relationship between the feeding and the mucosal structure is important to know, that donkey is characterise with small nutrient requirements, hence its vegetable diet is varied (grass, hay, twigs and bark). Donkey is adapted to digest high fibre diets and utilize organic acids for energy [9]. The mechanical filiform papillae are visible on the surface of the lingual mucosa as single processes embedded deeply in the high epithelium. These mechanical papillae in donkey shows the similarity with horse and clear difference with ruminants, in which filiform papillae possess one main process and two lateral shorter processes [3-7,10,14-16]. According to the length of the well-keratinized processes of filiform papillae there are distinguished, similar to horse, two subtypes of filiform papillae. The short filiform papillae in the anterior part of the tongue in donkey play an important function in food intake. This papillae are responsible for the adhesion of the plants parts and water and also for the hair grooming. On the apex and anterior body of the tongue between these filiform papillae, the low fungiform papillae are dispersed. Our study shows that the gustatory papillae are partly covered with keratinized epithelium, what suggests the protection of the epithelium against to the hard particles of the food. The explanation of the age related 221

7 decrease of number of lateral fungiform papillae in postnatal period in donkey is probably an aging effect or phenomenon connected with destruction of papillae in contact of food. Both hypotheses could be study deeply in future. Observations of the long densely arranged filiform papillae, which form a dense comb on the surface of the torus, bring to the conclusion that this area is specialized in adhesion of masticated food, which is prepared to swallowing. The occurrence of numerous green food particles and hairs between these papillae on the macro - and microscopic photos is a sign of strong adhesion of masticated food. The long filiform papillae on torus occur in horse and hippo [5,6,16]. In hippo the papillae help in positioning of wet food. Contrary observations are delivered by studies of the ruminants torus, that is covered with short, well-keratinized conical papillae, which help grinding the solid food [10,14,15]. The present observations of the vallate papillae in donkey show diversity of the shape and size of vallate papillae. Our study reveals that the number of these papillae varied between 2 and 5, with predominantly number of 3 papillae. This results is contrary to the earlier studies on donkey s tongue where the authors observe only 2-3 vallate papillae, so that similar with horse [2,3]. As a comparison, the vallate papillae are absent in hippo and are distributed in rows along of intermolar prominence in ruminants [10,14-16]. Interesting, from functional point of view, is the presence of very thick multilayered epithelium of the concave surface of vallate papillae which has a special protective function. Such protective layer of the epithelium both, on the surface of the vallate papillae, as well as previously mentioned fungiform papillae, seems to be a species-specific feature of the donkey. The structure of third type of gustatory papillae i.e. foliate papillae of the donkey is typical to the other mammals, however the species-specific feature in donkey is constant number of foliae of papillae in male and female. References [1] Beja-Pereira A, England PR, Ferrand N, Jordan S, Bakhiet AO, Abdalla MA, Mashkour M, Jordana J, Taberlet P, Luikart G. African Origins of the Domestic Donkey. Science. 2004; 304:1781. [2] Nickel R, Schummer A, Seiferle E. Lehrbuch der Anatomieder Haustiere Aufl. 8. Berlin: Parey Buchverlag. [3] Abd-Elmaeim MMM, Zayed AE, Leiser R. Morphological characteristic of the tongue and ist papillae in the donkey (Equus asinus): a light and scanning electron microscopical study. Ann of Anat. 2002; 184: [4] Rezian M Absence of Hyaline Cartilage in the Tongue of Caspian miniature horse.anat Histol Embryol. 2006; 35: [5] Kobayashi K, Jackowiak H, Frackowiak H, Yoshimura K, Kumakura M. Comparative morphological study on the tongue and lingual papillae of horses (Perissodactyla) and selected ruminantia (Artiodactyla). Ital J Anat Embryol. 2005; 110: [6] de Paz Cabello P, Chamorro J, Sandoval M, Fernandez M. Comparative scanming electron microscopic study of the lingual papillae in two species of domestic mammals (Equus caballus and Bos primigenius). Acta Anat. 1988; 132: [7] Pfeiffer CJ, Levin M, Lopes MA. Ultrastructure of horses tongue: Further observations on the lingual integumentary architecture. Anat Histol Embryol. 2000; 29: [8] Romeis B. Mikroskopische Technik. 1989; Urban and Schwarzenberg, München. [9] Aganga AA, Letso M, Aganga AO. Feeding donkeys Livestock Res for Rural Dev; 12:1-7. [10] Shao B, Long R, Ding Y, Wang J, Ding L, Wang H. Morphological adaptations of yak (Bos grunniens) tongue to the foraging environment of the Qinghai-Tibetan plateau. J Anim Sci. 2010; 88: [11] Jackowiak H, Godynicki S. The distribution and structure of the lingual papillae on the tongue of the bank vole Clethrionomys glareolus. Folia Morph. 2005; 4: [12] Kobayashi K, Miyata K, Takahashi K, Iwasaki S. Three-dimensional architecture of the connective tissue papillae of the mouse tongue as viewed by scanning electron microscopy. Kaibogaku Zasshi. 1989; 64: [13] Kobayashi K. Three-dimensional architecture of the connective tissue core of the lingual papillae in the guinea pig. Anat Embryol. 1990; 182: [14] Emura S, Tamada A, Hayakawa D, Chen H, Shoumura S. Morphology of the dorsal lingual papillae in the Barbary sheep, Ammotragus lervia. Okajimas Folia Anat Jpn. 2000; 77: [15] Jackowiak H, Godynicki S, Skubis J, Łakomy P. Distribution of the mechanical and gustatory papillae in the roe deer, red deer and fallow deer (Cervidae). 2008; Abstracts of Congress EAVA, July, Budapest. p [16] Yoshimura K, Hama N, Shindo J, Kobayashi K, Kageyama I. Light and scanning electron microscopic study on the tongue and lingual papillae of the common hippopotamus, Hippopotamus amphibious amphibious. Anat Rec. 2009; 292: