Morphology of Lingual Papillae of Bear: Light Microscopic and SEM Study

Essam H. Ibrahim1,2,3, Attalla F. El-kott1,4, Ali Alshehri1, Mona Kilany2,5, Reza Yavari6, Salahud Din7* and Diaa Massoud8,9 1Biology Department, Faculty of Science, King Khalid University, P.O. Box 9004, Abha 61413, Saudi Arabia. 2Research Center for Advanced Materials Science (RCAMS), King Khalid University, P.O. Box 9004, Abha 61413, Saudi Arabia. 3Blood Products Quality Control and Research Department, National Organization for Research and Control of Biologicals, Cairo, Egypt. 4Zoology Department, Faculty of Science, Damanhour University, Damanhour, Egypt. 5Microbiology Department, National Organization for Drug Control and Research (NODCAR), Cairo, Egypt. 6Faculty of Veterinary Medicine, Razi University, Kermanshah, Iran. 7Department of Anatomy and Histology, University of Veterinary and Animal Sciences, Lahore. 8Biology Department, College of Science, Jouf University, P.O. Box 2014, Sakaka, AlJouf, Saudi Arabia 9Zoology Department, Faculty of Science, Fayoum University, Fayoum, Egypt. Article Information Received 13 August 2020 Revised 20 September 2020 Accepted 28 September 2020 Available online 14 January 2021


INTRODUCTION
T he tongue is a principle organ, has a direct functional role in the prehension, mastication, and swallowing of food and indirect effect in helping the animal to adapt to its environment (Darwish, 2012). The anatomical and morphological features of the tongue are determined by the feeding habits of a given animal species and habitat in which animal lives (McClung and Goldberg, 2000;Schwenk, 2000;Iwasaki, 2002;Darwish, 2012).
Lingual papillae, distributed on the dorsal surface of the tongue, are the most important features that can be O n l i n e

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inspection.
The purpose of the present study was to examine the macroscopic and microscopic morphology of the lingual papillae of the bear and compare the results with those previously reported in other species.

Sampling
The specimens of the present study were provided from two male individuals that were collected after dying due to shooting by hunters. The animals were found in a poor physical condition and referred to the Faculty of Veterinary Medicine, Razi University. The cooperation of Natural Resources Organization of the Kermanshah city is highly appreciated. Immediately after dying, the whole tongue was dissected and removed from the oral cavity and fixed in 10% neutral buffered formaldehyde.

Gross examination
For macroscopic observations, the tongues were examined using a stereomicroscope (UNICO, ZM191, USA) connected to a digital camera (Dinocapture V.2) and anatomical features on the dorsal surface of the tongues were photographed.

SEM examination
For scanning electron microscopy, the tongues were fixed in 2.5% glutaraldehyde for 48 h. The samples were then post-fixed with 1% osmium tetroxide solution. After dehydration in ascending concentrations of ethanol, the specimens were dried in a freeze dryer, mounted on the aluminum stubs and coated with gold. Finally, the specimens were examined under a scanning electron microscope (Quanta 450, FEI, USA) at accelerating voltages of 15 kV (Goodarzi, 2014).

Light microscopy
Following immediate dissection, the tongues were fixed in 10% neutral buffer formaldehyde for two weeks. Afterwards, the 1×1 cm specimens were removed from the apex, body and root and were processed and embedded in paraffin for light microscopic observations. The paraffinembedded blocks were sectioned at 5µm thickness and stained with Masson's trichrome method (Goodarzi et al., 2019).

RESULTS
In macroscopic observation, the filiform papillae covered the dorsal surface of the lingual apex and lingual body, while, fungiform papillae were scattered among them (Fig. 1a). The discoid-shaped fungiform papillae were more densely on the tip of the lingual apex. The filiform papillae were extended to the lateral margin of the lingual body (Fig. 2a). The filiform papillae were transformed into the conical papillae on the caudal part of the lingual body and lingual root (Fig. 3a). Totally, 14-16 of spheroid and oval vallate papillae at different sizes were present posteriorly on the root of the tongue (Fig.  4a). There were no foliate papillae on the lingual root. In SEM examination, the dorsal surface of the lingual apex was covered with numerous long and slender filiform papillae which their sharp tips directed caudally (Fig. 1b,  1c). The discoid fungiform papillae were scattered among the filiform papillae mainly on the lingual apex ( Fig. 1b). At higher magnification, some taste pores could be seen between the shingle-like flattened cells of a stratified squamous epithelium covered the surface (Fig. 1b). The lateral surface of the tongue was covered with caudallydirected filiform papillae (Fig. 2b). The conical papillae on the lingual body were represented by a flat pyramidal shape with several slender accessory processes (Fig. 3b). On the lingual root, the body of the vallate papillae was encircled by a wide continuous gustatory groove and a thick annular pad of the lingual mucosa. The dorsal surface of the vallate papillae was highly irregular which appears to be lobulated and epithelial lining revealed irregular microplicae (Fig. 4b, 4c).  Light microscopic evaluation showed that the tongue was covered by a keratinized stratified squamous epithelium. A dense connective tissue composed the lamina propria and thick masses of striated muscles constituted the bulk of the tongue (Fig. 1d). The lamina propria didn't penetrate completely into the filiform papillae of the lateral border (Fig. 2c). Some lingual gland were present in the lamina propria of the vallate papillae (Fig. 4d). Furthermore, numerous lingual salivary glands with seromucous appearance were intermingled with the lingual muscles in the lingual root (Fig. 4e).

DISCUSSION
The lingual prominence presents in many species such as rodents (Ciena et al., 2013;Kilinc et al., 2010) and ruminants (Zheng and Kobayashi, 2006), while it was not seen in carnivores and Pigs (Emura et al., 2006;Kumar and Bate, 2004). In general, lingual prominence is a characteristic feature of many herbivorous mammals which helps them to cellulose-rich materials against hard palate (Massoud and Abumandour, 2019). The tongue of the bear has no lingual prominence nor median sulcus.
Due to difference in feeding habits, diet, and handling of food inside the buccal cavity, the lingual papillae might be varied among species in their shape, size, number, orientation and distribution (Iwasaki, 1992;Iwasaki et al., 1996;Abumandour and El-Bakary, 2013;Emura et al., 2002).
In the present study, four types of lingual papillae were found on the lingual mucosa of the bear tongue. Two of them were mechanical type and represented by filiform and conical forms, while the other two represented the gustatory papillae which were fungiform and vallate papillae. Numerous types of the filiform papillae can be seen among mammalian species. Moreover, different shapes of filiform papillae may be present according to their location on the lingual mucosa (Yoshimura et al., 2009). Three types in rats and mice (Iwasaki et al., 1987a, b), and six types of filiform papillae in Egyptian fruit bat were reported previously (Abumandour and El-Bakary, 2013). Emura et al. (2001) reported smooth finger-like projections as filiform papillae on the margin of the lingual apex of newborn Asian black bear, while, they were budshaped on the lingual body. In the present study, the filiform papillae had a long and slender appearance with caudallydirected sharp tips. These observations were similar to those reported in the tiger (Emura et al., 2004), fishing cat (Emura et al., 2014), raccoon dog and fox (Emura et al., 2006). The shape and number of the papillae showed a marked change from the lingual apex toward the lingual body. This finding is consistent with the observations in the cat (Bushell et al., 1982), newborn panther (Emura et al., 2001), and lion (Emura et al., 2003). The lingual papillae on the mid-portion of the tongue of the bear were

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conical with several accessory processes, similar to those reported in the raccoon dog and fox (Emura et al., 2006). The fungiform papillae can be different in their shape and distribution among mammalian species. The shape of the fungiform papillae has a wide variety of rectangle in Egyptian fruit bat (Abumandour and El-Bakary, 2013), dome-shape in dog and fox (Emura et al., 2006), Mushroom-shape in the rat (Nasr et al., 2012;Kurtul and Atalgin, 2008) and elliptical or circular in Sorex caecutiens (Park and Lee, 2009). Two types of fungiform papillae including hemispherical and club-shaped papillae were reported in the newborn black Asian bear (Emura et al., 2001), tiger (Emura et al., 2004) and jaguar (Emura et al., 2013). The fungiform papillae of the present study had a discoid shape and the existence of taste pores on the apical periphery of the papillae suggest that they have a gustatory role as revealed in other species (Kilinc et al., 2010;Chamorro et al., 1986;Goodarzi, 2014).
The fungiform papillae in the guinea pig (Kobayashi, 1990), goat (Kurtul and Atalgin, 2008), Persian squirrel (Goodarzi, 2014) and maned sloth (Benetti et al., 2009) are reported to be concentrated on the lingual apex and also on both lateral borders. However, fungiform papillae in the common shrew are restricted to the lingual corpus (Jackowiak et al., 2004). Nasr (2012) reported that fungiform papillae of Erinaceous auritus are populated on the apex of the tongue and form clusters of two or three papillae. The fungiform papillae of the bear were scattered on the dorsum lingua with a more population on the tip of the lingual apex.
In this study, the bear showed 14-16 vallate papillae which were more than that was reported by Emura et al. (2001) in the newborn Asian black bear (7-8 papillae). The structure of the vallate papillae can be varied. The vallate papillae of the jaguar were surrounded by a discontinuous annular pad and a very shallow groove (Emura et al., 2013), while in the newborn Asian black bear (Emura et al., 2001) and equine (Chamorro et al., 1986) the vallate papillae were composed of a primary papilla which was divided into several secondary papillae by intermediate grooves.
In the tiger, the vallate papillae had an irregular surface with the openings of glandular ducts (Emura et al., 2004). Although observation of the this work such as the presence of lingual salivary glands in the connective tissue of the vallate papillae and opening of glandular ducts on the dorsal surface of this papillae indicated similarity between the tiger and bear, however, the structure of the vallate papillae in the bear was similar to that of the panther reported by Emura et al. (2001).