Comparative histological and Histochemical study of epiglottis cartilage in Deccani sheep and Bidri goat

Introduction

Socioeconomic growth and national economy of the country has been significantly backed by small ruminants sector. Small ruminants contribute about 10% GDP in the total value of livestock sector. Sheep and goat are the important source of food (milk & meat) and main source of income for small and marginal farmers. The heavy mortality in the small ruminants especially in goats has been reported due to respiratory tract disease. The larynx is a conducting part of respiratory system which protects the lower respiratory passage against inundation. Epiglottis cartilage being one of the important cartilage and unpaired one. The predominant breeds of sheep and goat in Bidar district are Deccani sheep and Bidri goat respectively and limited scientific work has been carried out, hence the present study was undertaken to study and to correlate any histological and histochemical variations in the Epiglottis cartilage of Deccani sheep and Bidri goat.

Materials and Methods

The materials for the study were collected from Six Deccani sheep and Six Bidri goat immediately after slaughter from local slaughter house. The collected Epiglottis cartilage samples were washed in normal saline and fixed in different fixatives like 10% Neutral buffered formalin, Zenker's fluid and Bouin's fluid. The samples were processed in isopropyl alcohol-xylene sequence and embedded in paraffin by routine method (Luna, 1968) [9] . Sections were cut at 5-6μm thickness and were utilized for histomorphological and histochemical studies. The Epiglottis cartilage tissue fixed in Neutral buffered formalin were pre-frozen for 1-2 hrs and embedded in freezing media; using cryostat 6-7 μm thickness sections were cut and utilised for lipid studies. The histomorphological study of Epiglottis cartilage was carried out by Ehrlich's Haematoxylin and Eosin stain (Luna, 1968) [9] , Van Geison's stain for collagen fibers (Bancroft et al., 2008) [3] , Gomori's method for reticular fibers (Luna, 1968) [9] , Verhoeff's method for elastic fibers (Bancroft et al., 2008) [3] and Toludine blue method for mast cells (Singh and Sulochana, 1996) [11] whereas histochemical study was carried out using PAS technique for mucopolysaccharides (Bancroft, et al., 2008) [3] , Alcian blue pH 2.5 for sulphated sulfomucins and sialomucins (Bancroft et al., 2008) [3] and Oil red O for lipids (Luna, 1968) [9] . The histometrical parameter of the thickness of the epithelium of Epiglottis cartilage in Deccani sheep and Bidri goat were measured by using image analyser software Motic Image Plus 2.0 and the available data was analyzed (Snedecor and Cohran, 1994) [13] .

Results and Discussion

Histomorphology: The epiglottis cartilage of Deccani sheep and Bidri goat consisted of lamina epithelia, lamina propria, submucosa and a cartilaginous layer (Lee, 1959 Trautmann and Fiebiger, 2002 Alsadi, 2006, Chunhui et al. 2009 and Sinha et al., 2012) [8,15,1,4,12] (Fig. 1, Fig 2). The epithelium on both the surface of epiglottis cartilage in Deccani sheep and Bidri goat was lined by stratified squamous epithelium with finger like projection extended in to the lamina propria of laryngeal epithelium and no projection in the pharyngeal epithelium observed (Kahwa and Purton, 1996 Alsadi, 2006 Sinha et al., 2012) [7,1,12] (Fig. 1, Fig 2). The taste buds were present on both the surface of epithelium of epiglottis cartilage in Deccani sheep and Bidri goat indicating the role of these areas in gustatory sensation. (Lee, 1959, Sweazey et al., 1994 Kahwa and Purton, 1996 Sinha et al., 2012) [8,14,7] (Fig. 12). The thickness of laryngeal epithelium in Deccani sheep was 251±20.53µm and Bidri goat was 384.5±47.56µm whereas, the thickness of pharyngeal epithelium in Deccani sheep was 71.96±2.74µm and in Bidri goat was 76.63±3.75µm. The laryngeal epithelium was found thicker than the pharyngeal epithelium in Deccani sheep and Bidri goat (Table 1). The lamina propria-submucosa layers in Deccani sheep were continuous, no distinct demarcation was observed. Whereas, in Bidri goat in between the lamina propria and submucosa consisted of muscularis mucosa which had smooth muscle fibers. This may be due to species variation. Lamina propria-submucosa consisted of loose connective tissue more of collagen fibers, few elastic and reticular fibers. Fibroblast, fibrocyte, plasma cells, macrophages, mast cells, blood vessels and nerve fibers were also noticed (Fig. 6, 7, 8, 10, 11). The aggregated lymphatic cells near the epithelium were also observed (Sinha et al., 2012) [12] (Fig. 12). The lamina propria-submucosa in Deccani sheep and Bidri goat consisted of glands and loose connective fibers. The glands were of tubulo-acinar types and sero-mucous in nature. Some acini and tubules were serous in nature and some were mucous. The serous demilunes were also observed (Fig. 9). The glands on laryngeal side were more compared to pharyngeal side. Glands were lobulated and lobules were separated from each other by connective tissue strands predominantly collagen fibers, reticular fibers, and elastic fibers (Lee, 1959 [8,1,12,2,5] (Fig. 6,7,8). The cartilage was elastic in nature and was distributed plate like structure and fat cells were observed in between the plates of cartilage in Deccani sheep and Bidri goat (Lee, 1959, Alsadi, 2006 Sinha et al. 2012) [8,1,12] (Fig. 1, 2 and11). The perichondrium covering the cartilage in Deccani sheep and Bidri goat consisted of two zones Viz., a layer consisting of elliptical lacunae with elongated chondroblast and the fibrous layer composed of mainly collagen fibers, elastic fibers and reticular fibers. The elastic fibers were predominantly present in the interterritorial matrix of the cartilage (Fig. 6). The lacunae were polyhedral shaped in the deeper part of the cartilage whereas, towards the periphery were in elliptical shaped. These lacunae were having single, two and multiple chondrocytes (cell nest) (Sinha et al., 2012) [12] .

Histochemistry:

The epiglottis cartilage in Deccani sheep showed a mild PAS positive reaction in all secreting units of the glands whereas, in Bidri goat showed strong PAS positive reaction in few secreting units of the glands indicating the presence of mucoplolysaccharides and variation in the secretory nature of the glands. Few secreting cells of the glands and the cartilage showed the Alcian Blue positive reaction at pH 2.5 indicating presence of sulfated sialomucins and sulfomucins (Majid, 1986). (Fig4, 5) Lipid droplets were observed in between the plates of the cartilage with Oil red O stain (Fig. 3). The cartilage showed metachromasia with Toluidine blue stain indicating presence of proteoglycans in the matrix (Fig. 4). Toluidine blue stain Х 40

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