Comparative morphological analysis of minor salivary glands in porcine species using histological and histochemical evaluation

Comparative morphological analysis of minor salivary glands in porcine species using histological and histochemical evaluation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Comparative morphological analysis of minor salivary glands in porcine species using histological and histochemical evaluation Prateek Rao, Amandeep Singh, Pawan Kumar, Simran Kaur This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7550163/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract The present work was undertaken to explore the anatomical distribution, histology, and histochemistry of minor salivary glands of pigs during their postnatal life. The heads of ten healthy adult pigs were procured from a local slaughter house immediately after decapitation. The fresh tissues were collected from minor salivary glands. Histologically, these glands were found in the propria-submucosa layer of the mucous membrane of the oral cavity. Depending on their location, the secretory acinar units were either mucous, serous, or mixed in nature. Serous demilunes were also observed at the periphery of the mucous secretory units. In addition to the plasma cells, myoepithelial cells were also found between the base of the secretory cells and the surrounding basal lamina. The duct system consists of intralobular and interlobular ducts. The intercalated ducts were characterized by the presence of small-sized lumens and lined with simple cuboidal epithelium. Striated ducts were lined with simple to stratified columnar epithelium having one to two cell layer thickness with characteristic longitudinal striations in the basal third. Large interlobular ducts were localized at the level of interlobular septae and were lined with stratified columnar epithelium and also showed the presence of goblet cells. Histochemical studies revealed the presence of weakly acidic sulfated mucosubstances, sialomucins, glycogen, mucopolysaccharides, mucin, and protein in the mucous secretory units. However, the serous acini and ducts showed a negative reaction to all these histochemical moieties. Micrometrical studies revealed that the acinar and luminal diameters of the mucous acini were larger than that of serous units. However, the diameter of the interlobular ducts was larger than that of the intercalated and striated ducts, which indicates that there was a significant difference both at the 1% and at the 5% levels. This study provided detailed insights into the anatomical and histological characteristics of minor salivary glands of pigs. Thus, understanding the anatomy, morphometry, and histology of the minor salivary glands is essential for a correct decision concerning medical and/or surgical intervention of such affections. This study will be helpful in the field of surgery and also aid in the diagnosis of pathological conditions of these glands, and therefore this study establishes a basis for future research on this model. Health sciences/Anatomy Biological sciences/Cell biology Health sciences/Diseases Health sciences/Medical research Biological sciences/Physiology Biological sciences/Zoology Anatomy Buccal Histology Labial Lingual Micrometry Palatine Porcine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Introduction Pigs' physical and nutritional health are the primary determinants of their production performance, which is also influenced by the effectiveness of their digestive systems. The first biological medium to come into contact with substances that are ingested as food is saliva. As a result, it acts as the initial oral defense strategy 1 . These secretions are crucial for lubricating the upper digestive tract, moistening food, and shielding the mucosa's lining cells from harm. It also secretes lysozymes to regulate the bacterial flora 2 . Lactoperoxidase and Immunoglobulin A in saliva serve as a salivary antimicrobial system 3 . The enzymes present in the saliva may help in preparing the food for further digestion in the stomach 4 . The salt content of the saliva helps to regulate electrolytes. The saliva also has an excretory role as some heavy metals and other organic and inorganic salts were eliminated in part through it 5 . In addition, dysfunction of salivary secretion (hyposalivation) causes xerostomia (dry mouth) and sequentially leads to severe dental caries as well as oral mucosal disorders 2 . Both major and small salivary glands secrete saliva, which is extremely vital. Saliva is a seromucous fluid secreted by a variety of glandular structures found in the salivary glands. The parotid, mandibular, and sublingual salivary glands are the three main salivary glands found in domestic animals. The lingual, labial, buccal, and palatine glands are located in the tongue, lips, cheek, and palate, respectively. These minor glands, which are found within the oral mucous membrane, were categorized as minor salivary glands 6 . This wide distribution of the minor salivary glands is advantageous for the protection of the oral cavity against pathogens 7 . The excretory ducts carried the secretions of these glands to the mouth cavity. Typically, there are two types of salivary secretions: mucous and serous. Whereas mucous discharge is more viscous and contains a higher proportion of organic proteinaceous matter, including neutral and acidic glycoproteins, serous secretion is thin and watery and comprises water, inorganic ions, and some proteinaceous material 8 . The literature revealed that most of the research work is confined to major salivary glands of buffalo 9 , sheep 10 , goat 1 and pig 11 , whereas research on minor salivary glands is very scanty in general and pig in particular. Hence, the present study has been planned in pig to explore the detailed structure and nature of the minor salivary glands, which will certainly be helpful to the histologists, physiologists and pathologists to understand the histoarchitectural organization of these glands. Materials and Methods Collection of the specimens The present study was conducted on minor salivary glands of ten healthy adult pigs of local mixed breed of either sex. The heads were procured from local slaughter house immediately after decapitation. The fresh tissues were collected from these glands and fixed in 10% neutral buffered formalin (NBF) (HT501128, Sigma-Aldrich, India) for 48-72 hours. For buccal glands, the tissues were collected from cranial, middle and caudal portions of dorsal and ventral rows. The tissues from the hard palate and soft palate were also collected for the study of palatine glands. For lingual glands, the tissues were collected from apex, body and root of the tongue. The tissues from the upper, lower and commissural portions of the lips were also collected for the study of labial glands. Tissue processing, sectioning and staining After fixation in 10% NBF for 48-72 hours at room temperature, the tissue samples were then processed for paraffin sectioning technique which includes various steps. The first step was dehydration of the fixed tissue in ascending grades of ethanol (459836, Sigma-Aldrich, India) i.e., 50%, 70%, 90% and three absolute grades of ethanol, keeping in each solution for one hour. After this step, clearing was done by passing the tissues through two sets of benzene solution (401765, Sigma-Aldrich, India), keeping in each solution for at least one hour. Then, infiltration was done to replace the clearing agent completely from the tissue with the paraffin wax of melting point 58-62oC (327212, Sigma-Aldrich, India). Cleared tissues were then passed through three sets of melted paraffin wax for one hour each, which were kept in an oven adjusting its temperature 1 or 2oC above the melting point of wax used. Then embedding of the tissues was done, in which, the melted paraffin wax of the same melting point as used for infiltration, was poured in the moulds having infiltrated tissues and then allowed to cool which results in hardening of the paraffin wax. Then, trimming of the blocks was done to expose the tissue surfaces to a level where a representative section could be cut. The paraffin sections of 5 µm thickness were cut on a microtome (RM2125 RTS, Leica Biosystems, Germany), mounted on microscope slides with DPX mounting media (06522, Sigma, India) and then stained with various histomorphological and histochemical stains 12-14 (Table). Photomicrography High-resolution photomicrographs of paraffin sections were taken by a light microscope equipped with a camera (DS-Fi3, Japan) having imaging software ViewNX. Statistical analysis The diameter of serous and mucous acini as well as intralobular and interlobular ducts of minor salivary glands was measured and the data was tabulated and statistically analyzed. The data was subjected to statistical analysis for determining correlation between these structures by using SPSS statistics software version 22. Results Histological and histochemical analysis of buccal glands The buccal glands of pig were observed in the cheek region and found arranged in two rows as dorsal and ventral groups. Histologically, the dorsal buccal glands of pig showed similar structure in rostral and caudal portions. The mucous membrane of the cheek was lined by stratified squamous non-keratinized epithelium. The lamina propria blends with the tunica submucosa without an intervening lamina muscularis, forming a propria submucosa. The dorsal buccal glands were located in the propria-submucosa layer and also scattered among the skeletal muscle bundles of the cheek (Fig. 1a). These were of compound tubuloacinar type being surrounded by a thin capsule having a mixed distribution of collagen, reticular and few elastic fibres. Blood vessels and nerve bundles were seen distributed with in the capsule. From the capsule, the interlobular septae arises which divided the parenchyma into lobes and lobules (Fig. 1b). The lobules showed the presence of secretory acinar units and the ducts. The secretory units were purely mucous in nature (Fig. 1c). The collagen fibres were observed surrounded the secretory acinar units, blood vessels and the muscle bundles (Fig. 1d). The mucous acini were comprised of pyramidal cells having distinct cell boundaries, located around a wide lumen and the flattened basophilic nuclei were situated towards the basement membrane of the cell. Their chromatin material was very dense which marked the appearance of nucleoli. Few mucous cells were noticed surrounded by long, spindle-shaped, flat cells known as myoepithelial cells, which were interposed between the base of the secretory cells and the surrounding basal lamina. Plasma cells were also found scattered in connective tissue present in between the secretory acinar units (Fig. 1e). The mucous acini measured 48.18±1.68 μm in diameter. The cytoplasm of these pyramidal cells was eosinophilic in nature and presented a vacuolated appearance due to washing of mucous secretions during the processing of tissues. The luminal diameter of the mucous acini was 11.88±1.32 μm. Blood vessels, nerves, adipocytes and interlobular ducts were distributed with in the interlobular septae. In addition to the collagen fibres, reticular fibres were seen around the capsule and also surrounding the ducts and blood vessels (Fig. 1f). Fine elastic fibres were observed in the blood vessels located in the interlobular septa (Fig. 2a). Large plexus of ganglionic cells and blood vessels were also noticed in the capsule from which nerve fibres traversed through the septa and ramified along the basement membrane of the acini and ducts (Fig. 2b). In addition to the secretory units, the ducts were also observed in the dorsal buccal gland, consisted of intralobular and interlobular ducts. Intralobular ducts were located within the lobules and were further subdivided into small and large intralobular ducts on the basis of their diameter and type of epithelium. The small intralobular ducts, known as intercalated ducts were characterized by the presence of small sized lumen and lined with simple cuboidal epithelium (Fig. 2c) and measured 15.18±0.80 μm in diameter. The intercalated ducts opened into large intralobular ducts, known as striated ducts, within the lobules. The striated ducts measured 50.16±1.23μm in diameter. These ducts were lined with simple to stratified columnar epithelium having one to two cell layer thickness with characteristic longitudinal striations in the basal third. The cytoplasm of the cells lining the striated ducts was eosinophilic, while the nuclei were basophilic and darkly stained (Fig. 2d). Myoepithelial cells were observed between the base of the cells and basement membrane of the intralobular ducts. The striated duct extended to the periphery of the lobule to open into interlobular ducts. Large interlobular ducts were localized at the level of interlobular septae and were lined with stratified columnar epithelium and measured 62.70±1.04 μm in diameter. The interlobular ducts also showed the presence of unicellular mucous glands known as goblet cells. The lumen of these ducts contains the varying amount of secretions which were eosinophilic in nature (Fig. 2e). The interlobular ducts drain the secretions into terminal excretory ducts where the epithelium changed to stratified squamous. These excretory ducts opened in the mucosal membrane of the cheek. The concentration of collagen fibres increased surrounding the intralobular and interlobular ducts (Fig. 2f). Histochemically, the mucous acinar cells showed a strong Alcian blue reaction in the secretory cells indicating the presence of weakly acidic sulfated mucosubstances, sialomucins and hyaluronic acid in the secretions. However, the ducts revealed negative reaction to all these histochemical moieties (Fig. 3a). The mucous secretory cells were intensely PAS positive by McManus’ method showing the presence of high concentration of glycogen in the secretions (Fig. 3b). The diastase treatment showed that PAS activity was reduced indicating the presence of glycogen in addition to other mucopolysaccharides. It showed a negative reaction with Best’s carmine confirming that mucopolysaccharides other than glycogen was present in the mucous cells. Combined PAS-AB method revealed intense positive reaction for acidic mucopolysaccharides in mucous cells and ducts were negative (Fig. 3c). Mucous acini showed strong positive reaction to mucin and the ducts were negative by Mayer mucicarmine method. However, the goblet cells of interlobular ducts showed positive reaction with this method (Fig. 3d). Colloidal iron method showed the moderate positive reaction for acidic mucopolysaccharides in mucous acini and ducts were negative (Fig. 3e). Weak protein activity was observed in mucous cells, however, ducts showed negative reaction by mercury bromphenol blue method (Fig. 3f). The histoarchitecture of ventral buccal glands of pig showed similar structure as of dorsal row of glands with the exception of serous type of secretory units. These glands were located in the propria-submucosa layer and also scattered among the skeletal muscle bundles of the cheek (Fig. 4a). The serous acini were comprised of pyramidal shaped cells, located around a narrow lumen and the basophilic spherical vesicular nuclei were situated towards the basal one third of the cell. The nucleoli showed distinct appearance due to their dense chromatin material. The cell boundaries were comparatively indistinct (Fig. 4b). The ventral buccal glands were of compound tubuloacinar type encapsulated by a thick dense capsule and was divided into lobules by means of thin connective tissue septa having distribution of reticular fibres (Fig. 4c) fibres in addition to fine blood capillaries, plasma cells and nerve fibres. The lobules showed the presence of serous secretory units and the ducts. Fine elastic fibres were also observed in the blood vessels located in the interlobular septa. The serous acini measured 23.76±1.23 μm in diameter and their luminal diameter was 4.62±0.80 μm. The cytoplasm of these pyramidal cells was basophilic in nature and presented numerous granules towards the apical part of cell. The duct system of ventral buccal glands was also similar to that of dorsal glands which consisted of intralobular and interlobular ducts. The small intralobular ducts, known as intercalated ducts were characterized by the presence of small sized lumen and lined with simple cuboidal epithelium and measured 8.58± 0.80 μm in diameter. The striated ducts were lined with simple to stratified columnar epithelium having one to two cell layer thickness with characteristic longitudinal striations in the basal third. These striated ducts measured 26.40±1.16 μm in diameter. The cytoplasm of the cells lining the striated ducts was eosinophilic, while the nuclei were basophilic and darkly stained. The striated duct extended to the periphery of the lobule to open into interlobular ducts. Large interlobular ducts were lined with stratified columnar epithelium and measured 41.58±0.80 μm in diameter. The interlobular ducts also showed the presence of goblet cells (Fig. 4d). The lumen of these ducts contains the varying amount of secretions which were eosinophilic in nature. The interlobular ducts drain the secretions into terminal excretory ducts where the epithelium changed to stratified squamous epithelium. Histochemically, the goblet cells of the interlobular ducts showed strong positive reaction to acidic mucopolysaccharides (Fig. 4e), neutral mucopolysaccharides (Fig. 4f), however the serous acini exhibited negative reaction to all histochemical staining techniques carried out during the present investigation indicating the absence of mucopolysaccharides, glycogen, mucin and protein. Histological and histochemical analysis of palatine glands For palatine glands, the tissues were collected from the ridged portion of the hard palate (rostral, middle and caudal portions) and also from oropharyngeal region of the soft palate. Histologically, no glands were found in the region of hard palate (Fig. 5a). The mucous membrane of the oropharyngeal region of the soft palate was lined by stratified squamous epithelium and the palatine glands were located in the propria-submucosa layer. These were of compound tubuloacinar type being surrounded by a thin capsule having a mixed distribution of collagen, reticular and few elastic fibres. In addition to the glands, lymphatic tissue was also seen in the propria-submucosa layer (Fig. 5b). Blood vessels and nerve bundles were seen distributed with in the capsule. From the capsule, the interlobular septae arises which divided the parenchyma into lobes and lobules. Blood vessels, nerves and interlobular ducts were distributed with in the septae. The lobules showed the presence of secretory units and the ducts. The secretory units were purely mucous in nature. The pyramidal cells of mucous acini were located around a wide lumen and the flattened basophilic nuclei were situated towards the basement membrane of the cell. They have distinct cell boundaries and their chromatin material was dense which marked the appearance of nucleoli. The cytoplasm of these pyramidal cells was eosinophilic in nature and presented a vacuolated appearance due to washing of mucous secretions during the processing of tissues (Fig. 5c). The mucous acini measured 38.94±1.23 μm in diameter and their luminal diameter was 9.24±0.66 μm. The myoepithelial cells were interposed between the base of the secretory acinar cells and the surrounding basal lamina. Plasma cells were also found scattered in connective tissue present in between the secretory acinar units. The collagen fibres were observed surrounded the blood vessels and acinar cells (Fig. 5d). Fine elastic fibres were also seen in the blood vessels located in the interlobular septa (Fig. 5e). In addition to the mucous acini, the ducts were also present in the palatine glands, consisted of intralobular and interlobular ducts. The intercalated ducts were lined by simple cuboidal epithelium (Fig. 5f) and measured 12.21±0.99 μm in diameter. The intercalated ducts opened into large striated ducts within the lobules, measuring 41.58±0.80 μm in diameter and were lined with simple to stratified columnar epithelium having two to three cell layer thickness. The cytoplasm of the cells lining the striated ducts was eosinophilic, while the nuclei were basophilic and darkly stained (Fig.5f). The striated duct extended to the periphery of the lobule to open into large interlobular ducts. The interlobular ducts were lined with stratified columnar epithelium (Fig. 6a) and these ducts measured 52.80±1.47 μm in diameter. The concentration of collagen fibres increased surrounding the intralobular and interlobular ducts. The interlobular ducts drain the secretions into terminal excretory ducts where the epithelium changed to stratified squamous. Histochemically, a strong alcian blue reaction was observed in the mucous secretory cells indicating the presence of weakly acidic sulfated mucosubstances, sialomucins and hyaluronic acid in the secretions. However, the ducts revealed negative reaction to all these histochemical moieties (Fig. 6b). The mucous secretory cells were intensely PAS positive by McManus’ method showing the presence of high concentration of glycogen in the secretions however, the ducts were showing negative reaction (Fig. 6c). Combined PAS-AB method revealed strong positive reaction for acidic and neutral mucopolysaccharides in mucous cells and ducts were negative (Fig. 6d). Mucous acini showed moderate positive reaction to mucin and the ducts were negative by Mayer mucicarmine method (Fig. 6e). Colloidal iron method showed the moderate to strong positive reaction for acidic mucopolysaccharides in mucous acini whereas, ducts revealed negative reaction (Fig. 6f). Histological and histochemical analysis of lingual glands The lingual glands of pig were collected from root, body and apex of the tongue. Histologically, the dorsal surface of tongue was lined by stratified squamous keratinized epithelium. The lingual glands present at the root of the tongue were of compound tubuloacinar type and were located in the propria-submucosa layer and also noticed among the skeletal muscle bundles (Fig. 7a). These glands were encapsulated by a thin layer of connective tissue made up of large amount of collagen fibres and sparse number of reticular fibres. Blood vessels and nerve bundles were found distributed with in the capsule. The interlobular connective tissue septae divided the parenchyma into lobes and lobules (Fig. 7b). The collagen fibres were also seen surrounded the blood vessels and the muscle bundles. The lobules showed the presence of secretory acinar units and the ducts. The secretory units in the tongue's root were made up of mucous acinar cells. The mucous acini were comprised of pyramidal cells having distinct cell boundaries, located around a wide lumen and the flattened basophilic nuclei were situated towards the basement membrane of the cell. Their chromatin material was very dense which marked the appearance of nucleoli. Occasionally, the crescent-shaped serous demilunes were also observed in the root of the tongue (Fig. 7c). Myoepithelial cells were seen between the base of the mucous secretory cells and the surrounding basal lamina. The duct system was comprised of intralobular and interlobular ducts. Intralobular ducts were further subdivided into small (intercalated) and large (striated) ducts on the basis of their diameter and type of epithelium. The intercalated ducts opened into striated ducts within the lobules. The striated ducts were lined with simple to stratified columnar epithelium having one to two cell layer thickness with characteristic longitudinal striations in the basal third (Fig. 7d). The cytoplasm of the cells lining the striated ducts was eosinophilic, while the nuclei were basophilic and darkly stained. The striated duct extended to the periphery of the lobule to open into interlobular ducts. Large interlobular ducts were localized at the level of interlobular septae and were lined with stratified columnar epithelium. Histochemically, the mucous acini showed a strong Alcian blue reaction indicating the presence of weakly acidic sulfated mucosubstances, sialomucins and hyaluronic acid in the secretions (Fig. 7e) however, the ducts revealed negative reaction to all these histochemical moieties (Fig. 7f). The mucous secretory cells were intensely PAS positive by McManus’ method showing the presence of high concentration of glycogen in the secretions (Fig. 8a). However, the ducts were negative and unicellular mucous glands showed intense PAS positive reaction which indicates the presence of large amount of glycogen (Fig. 8b). Combined PAS-AB method revealed strong positive reaction for acidic and neutral mucopolysaccharides in mucous cells and ducts were negative. However, goblet cells showed positive activity for neutral mucopolysaccharides (Fig. 8c). Mucous acini showed strong positive reaction to mucin and the ducts were negative by Mayer mucicarmine method (Fig. 8d). Colloidal iron method showed the moderate to strong positive reaction for acidic mucopolysaccharides in mucous acini (Fig. 8e). However, goblet cells of interlobular ducts showed positive reaction to this method (Fig. 8f). The lingual glands present at the body of tongue were of compound tubuloacinar type and located in the propria-submucosa layer. Lymphocytes and adipocytes were also found within the propria-submucosa in addition to the lingual glands (Fig. 9a). From the capsule, the connective tissue septae, made up of collagen and reticular fibres, arises which divided the parenchyma into lobes and lobules. The lobules showed the presence of secretory acinar units and the ducts. The secretory units were comprised of only mucous acini, having pyramidal cells with distinct cell boundaries, located around a wide lumen and the flattened basophilic nuclei were situated towards the basement membrane of the cell (Fig. 9b). The duct system was comprised of intralobular and interlobular ducts. The intercalated ducts opened into striated ducts within the lobules. The striated ducts were lined with simple to stratified columnar epithelium having one to two cell layer thickness (Fig. 9c). The cytoplasm of the cells lining the striated ducts was eosinophilic, while the nuclei were basophilic and darkly stained. The striated duct extended to the periphery of the lobule to open into interlobular ducts. Large interlobular ducts were localized at the level of interlobular septae and were lined with stratified columnar epithelium (Fig. 9d). Histochemically, a strong Alcian blue reaction was seen in the mucous acini, showed the presence of weakly acidic sulfated mucosubstances, sialomucins and hyaluronic acid in the secretions however, the ducts were showing negative reaction to all these histochemical moieties (Fig. 9e). McManus’ method revealed that the mucous secretory cells were intensely PAS positive indicating the presence of high concentration of glycogen in the secretions (Fig. 9f). However, the ducts showed negative reaction to this method. Combined PAS-AB method revealed strong positive reaction for acidic and neutral mucopolysaccharides in mucous cells and ducts were negative (Fig. 10a). Colloidal iron method showed the moderate to strong positive reaction for acidic mucopolysaccharides in mucous acini. A moderate positive reaction was seen in mucous acini indicating the presence of mucin and the ducts were negative by Mayer mucicarmine method (Fig. 10b). The circumvallate papillae were large, flattened structures completely surrounded by an epithelium-lined sulcus. The epithelium on the papillary side of the sulcus contains many taste buds. These taste buds were ellipsoid clusters of specialized epithelial cells embedded in the stratified squamous epithelium of the circumvallate papillae of the tongue. However, the glands that were present just beneath the circumvallate papillae were comprised of only serous secretory units (Fig. 10c). The serous acinar cells were pyramidal in shape with less distinct cell boundaries. The rounded nuclei of acinar cells with their deeply stained nucleoli were situated near to the basal third of the cells (Fig. 10d). These serous gustatory glands present ducts that opened into the sulcus at various levels. The duct system was comprised of intralobular and interlobular ducts. The intercalated ducts extended to the periphery of the lobule to open directly into large interlobular ducts (Fig. 10e). The interlobular ducts were localized at the level of interlobular septae and were lined with simple to stratified squamous epithelium and they opened into the sulcus at the base of the circumvallate papillae (Fig. 10f). The serous acinar cells showed negative reaction to all histochemical staining techniques carried out during the present investigation indicating the absence of mucopolysaccharides, glycogen, mucin and protein. The micrometrical studies revealed that the mucous acini measured 59.40±1.80 μm in diameter and their luminal diameter was 15.84±1.61 μm (Table 1). However, the serous acini measured 28.38±1.68 μm in diameter and their luminal diameter was 5.94±1.23 (Table 1). The intercalated ducts of the mucous acini measured 18.48±0.80 μm in diameter while that of serous acini was 10.23±0.80 μm. The striated ducts of the mucous acini measuring 61.38±1.41 μm in diameter. The interlobular ducts of mucous acini measured 74.58±1.32 μm in diameter while that of serous acini measured 48.18±0.80 μm in diameter (Tables 2 and 3). Histological and histochemical analysis of labial glands The labial glands of pig were distributed in the superior, inferior and commissural portions of the lips. The commissural labial glands were found distributed around the commissure of the lips on either side. The labial glands were placed under the skin rostral to the insertion of the zygomaticus muscle embedded in the orbicularis oris muscle. These glands extended between the superior labial artery and inferior labial vein along the angle of mouth. Histologically, the mucosa of the oral surface of lips is covered by stratified squamous non-keratinized epithelium. The superior labial glands were of compound tubuloacinar type and extending from the corner of the mouth to the length of the lips and situated in the propria-submucosa layer. The gland was surrounded by a thin layer of capsule having loose irregular connective tissue with varying distribution of collagen, reticular and elastic fibres. Blood vessels and nerve bundles were found distributed with in the capsule. The lobules showed the presence of secretory acinar units and ducts. The secretory units were predominantly mucous in nature with very few independent serous secretory units (Fig. 11a). Occasionally, in addition to the plasma cells, crescent-shaped serous cells known as serous demilunes, were also observed at periphery of the mucous secretory units (Fig. 11b). The interlobular connective tissue septae divided the parenchyma into lobes and lobules (Fig. 11c). The collagen fibres were also observed and surrounded blood vessels and ducts (Fig. 11d). Reticular fibres were also seen around the capsule and surrounding the acinar cells. Fine elastic fibres were observed in the blood vessels located in the interlobular septa (Fig. 11e). Mucous acini showed strong positive reaction to mucin whereas, serous acinar cells and ducts were negative reaction with Mayer mucicarmine method (Fig. 11f). The mucous acini measured 43.56±1.61 μm in diameter and their luminal diameter was 10.56±1.23 μm (Table 1). Myoepithelial cells were seen between the base of the mucous secretory cells and the surrounding basal lamina. The diameter of the serous acini measured 21.12±1.68 μm and their luminal diameter was 3.96±0.66 μm (Table 1). The histoarchitecture of labial glands of inferior lip as well as commissural part was similar to that of superior lip as described earlier. However, their population varies according to the regions as labial glands were most numerous in the superior lip as compared to that of inferior and commissural regions. The duct system of labial glands consisted of intralobular (Fig. 12a) and interlobular ducts (Fig. 12b). The intercalated ducts measured 13.20±1.04 μm in diameter, whereas diameter of striated ducts was 44.22±0.80 μm (Table 2). The large interlobular ducts measured 58.74±1.23 μm in diameter (Table 2). Goblet cells with bulging apical portion into the duct lumen were seen in the interlobular ducts (Fig. 12c). The lumen of these ducts contains the varying amount of secretions which were eosinophilic in nature. The concentration of collagen fibres increased surrounding the intralobular and interlobular ducts (Fig. 12d). The interlobular ducts drain the secretions into terminal excretory ducts where the epithelium changed to stratified squamous epithelium. Histochemically, a strong Alcian blue reaction was observed in the mucous secretory cells indicating the presence of weakly acidic sulfated mucosubstances, sialomucins and hyaluronic acid in the secretions. However, the serous units and ducts revealed negative reaction to all these histochemical moieties (Fig. 12e). The mucous secretory cells were intensely PAS positive by McManus’ method showing the presence of high concentration of glycogen in the secretions. The serous acinar cells and ducts were negative to glycogen (Fig. 12f). Combined PAS-AB method revealed strong positive reaction for acidic and neutral mucopolysaccharides in mucous cells. The serous acini and ducts showed negative reaction however, goblet cells showed mixed activity for neutral and acidic mucopolysaccharides with this stain (Fig. 13a). Colloidal iron method showed the moderate to strong positive reaction for acidic mucopolysaccharides in mucous acini however, the serous acini and ducts were showing negative reaction (Fig. 13b). However, the goblet cells of interlobular ducts showed positive reaction with Mayer mucicarmine method (Fig. 13c). Weak protein activity was observed in mucous cells, however, ducts showed negative reaction by mercury bromphenol blue method (Fig. 13d). Micrometry was conducted for the acinar and luminal diameters of minor salivary glands of pig and the data was obtained. Also, the micrometry of ducts of mucous as well as serous type of glands was conducted and the data was obtained. ANOVA has been carried out for the above-mentioned parameters and the significant as well as non-significant differences were obtained both at 1% and 5% levels. Discussion In the present study, the buccal glands were arranged in dorsal and ventral rows as reported in goat and sheep 15-16 . Contrary to the above findings, the buccal glands were arranged in dorsal, middle and ventral rows as reported in domestic ruminants 17-19 and horse 20 . The middle row which was found in cattle 21 and the Indian buffalo 22 was not observed in the present study. However, in horse the dorsal row was placed on the outer surface of the buccinator muscle near its upper border 23 . The buccal glands of goat were arranged in dorsal, middle and ventral rows 24 . The ducts of the dorsal and ventral buccal glands of bovines opened between papillae within the vestibule of the mouth 25 . The minor salivary glands appeared as clusters of serous and mucous aggregations in the propria submucosa between the intrinsic muscle bundles as reported in domestic animals 26 . These glands were seen outside the mucosa extending from the submucosa between the muscle fibers into the subcutis in domestic animals 17 . The buccal glands were of compound tubuloacinar type and contained mucous acini with serous demilunes as reported in human beings 27-30 . In the present investigation, the dorsal row of buccal glands were of mucous type. These findings were in parallel agreement with those reported in ruminants 17 and in Indian buffalo 22 . The parenchyma of dorsal buccal glands in buffalo was consisted of mainly mucous, a few serous and mixed type of acini with serous demilunes 31 . The dorsal buccal gland of sheep was of compound tubuloacinar type being surrounded by less distinct capsule. The gland was seromucous with predominance of mucous acini 32 . On the other hand, the middle buccal gland of sheep was consisted of mainly mucous with few serous types of acini 33 . However, the ventral buccal gland of sheep was comprised of seromucous secretory units with predominance of serous acini 34 . The buccal glands of dog were of tubuloacinar type and were located in the submucosal connective tissue 35 . The ventral row of buccal glands was of serous type in cattle and camel 36,37 which were in parallel agreement with those reported in the present study. The anterior part of the ventral row of buccal glands of Indian buffalo was purely mucous in nature whereas the posterior lobules were dorsally mixed and ventrally serous 22 . However, the ventral buccal gland of goat showed in anterior portion and upper part of the middle portion entirely the mucous acini while the lower part of the middle portion and the posterior portion the serous acini 38 . In goat, the dorsal and middle buccal glands were mainly mucous in nature with a very few serous and mixed acini, whereas ventral buccal gland was purely serous in nature 24 . In the present study, the mucous secretory units were lined by pyramidal cells resting on a basement membrane. However, the acinar cells were of irregularly cuboidal in form as reported in human beings 39 . Myoepithelial cells were observed between the base of the cells and basement membrane as reported in rats 40 . The present investigation showed that the mucous acini opened into the intercalated ducts which in turn joined to form the intralobular ducts. This is in contrary to the findings where the mucous secretory units opened directly into intralobular ducts which were striated in nature 40 . However, in the present study basal striations were seen in the striated ducts. The intralobular ducts of sheep were lined by flat cells which showed the presence of goblet cells 41 . The serous acini were lined by low columnar cells resting on a basement membrane though described the lining epithelium as cuboidal in the ruminants 42 . In the ventral buccal glands of sheep and goats, the serous acini had a wide lumen 17 , whereas in the present study the serous acini had a narrow lumen. Intercalated ducts that drained the acinar cells were observed in the present study while, in the camel that the serous acini like mucous acini opened directly into the striated ducts or into the excretory ducts 37 . In mammals, except in cattle, the presence of myoepithelial cells was noticed which were not extended beyond the intercalated ducts 43 . Similar findings were reported in the intralobular ducts of the rat submandibular gland 44 , while the present study revealed the presence of myoepithelial cells extending upto the intralobular ducts. Histochemically, the mucous acini showed PAS positive reaction indicating the presence of glycogen as described in the mucous secretory units of the salivary glands in mammals 45-50 . However, positive reaction was observed with AB indicating the presence of acidic mucopolysaccharides in mucous secretory cells. The present study also revealed the similar findings with these histochemical moieties. Presence of acidic mucopolysaccharides were also noticed in the mucous secretory units of the buccal glands of rats 51-52 . The mucous acini of dorsal row of buccal gland in buffalo contained weak amount of glycogen, acidic and neutral mucopolysaccharides 31 . In sheep, the mucous acinus of the dorsal buccal gland of sheep contained mucosubstances, glycogen, mucopolysaccharides and mucin 32 . However, the mucous acinus of middle buccal gland of sheep contained mucosubstances, glycogen, mucopolysaccharides, mucin and sudanophilic lipids while serous cells showed negative reaction 33 . The serous acini showed negative reaction to all the histochemical staining techniques carried out during the present investigation indicating the absence of mucopolysaccharides, glycogen, mucin and protein. However, in Japanese macaque, the presence of neutral, weak and strong acid mucopolysaccharides in the serous units of buccal glands 53 . While the presence of neutral, weak and acid mucopolyaccharides with varying intensities were observed in the serous acini of salivary glands in the humans 45 , horse 54 , buffalo 55 , dog 56 , opossum 57 and the Indian buffalo 58 . The present investigation revealed that the palatine glands were absent in the region of the hard palate. However, the smooth portion of the hard palate contained mucous glands in the propria submucosa as reported in the humans 59 and domestic animals 26 . However, some studies revealed the presence of seromucous glands in the human hard palate 30 . In camel, the palatine glands were located in the caudal part of the hard palate and in the entire length of the soft palate 60 . The palatine gland was most numerous at the apex of the soft palate. The palatine glands which were branched tubuloacinar type were observed with mucous secretory units in the submucosa scattered in between the muscle bundles on the oral side of the soft palate and the serous secretory units with a few scattered mucous acini in the propria submucosa on its nasal side as suggested in the domestic animals 17,26 though reported in humans the absence of any gland in the mucous membrane of the soft palate 61 . Some authors reported that the palatine glands were of mucous type in rats and mice 62 . However, they also claimed that they have observed the glands on the nasal side of the rat soft palate though they were not noticed in the rat soft palate 63 . The palatine glands of humans were histologically consisted of the gland tissue proper and supporting connective tissue frame work 30 . These glands were divided into lobules by connective tissue septa in which ducts and vessels were found. The soft palate in mesaticephalic breeds of dog was comprised of predominantly serous glands, and only few were mixed in nature with separate mucous and serous acini 64 . The rostral aspect of palatine glands of the Chukar partridge ( Alectoris chukar) was of simple branched tubuloacinar nature having both mucous and seromucous characteristics, while the caudal aspect included mucous type only with simple branched tubular glands 65 . The seromucoid glands were present in the rostral and caudal parts of the hard palate, as well as in the soft palate of domestic rabbits 66 . Histochemically, the mucous acini revealed the presence of neutral mucopolysaccharides in the current study. However, the presence of sulfated mucosubstances and minimal amount of non-sulfated mucosubstances was observed in the soft palate of the rat 63 . The periodic acid-Schiff (PAS) positive, alcianophilic, and metachromatic cells were present with a more homogeneous distribution pattern in the palatine glands of chicken 67 . Some studies revealed that the mucous glands were embedded in the submucosal layer of the soft palate in cat 68 . Histochemically, PAS-AB combined stain imparts magenta colour due to neutral secretion. These findings were in parallel agreement with those reported in this study. The lingual glands were of mixed type and were abundant under the circumvallate papillae as reported in the present study. The lingual glands were seen in the propria-submucosa and among its muscle bundles of the tongue except in the tip region however, they were observed in the humans 39 even in the tip region and these findings were in agreement with the above authors in man and domestic animals 26 . Serous glands were seen in the lamina propria in the vicinity of circumvallate papillae, while the mucous acini were placed deeper to the serous glands among muscle bundles as reported in the domestic animals 17 . Serous glands were of compound tubuloacinar type while the mucous glands were of simple branched tubuloacinar type though in the domestic animals 26 both serous and mucous glands were of simple branched tubuloacinar type. In the ox that the lingual glands of the root were of mixed type 17 . But in the present study mucous glands with occasional serous demilunes were observed in the root region. However, some studies reported the presence of mucous acini in the posterior lingual glands of spiny tailed lizard 69 and also reported in the mongoose the presence of serous, mucous and mixed glands in the posterior part of the tongue 70 . The serous and mucous glands were found in the lamina propria of the foliate and circumvallate papillae of the tongue in horse and dog and only serous acini in the pig 19 . The lingual glands in Japanese monkey consists of both serous and mucous secretory units 71 . The posterior lingual glands in the gray short-tailed opossums were of tubuloacinar type, consisting of predominantly mucous secretory cells and seromucous acinar units 72 . However, the lingual salivary glands of the Persian squirrel were confined to the body and root, which were composed of serous cells located anteriorly and mucosal and seromucosal cells placed posteriorly 73 . These findings were in partial agreement with the results of present investigation. Histochemically, the serous acini showed negative reaction for PAS except the basement membrane. However, in mongoose, the serous acinar cells and occasionally duct cells contained granules which gave reactions for neutral mucosubstances 70 . The mucous acini indicated the presence of neutral mucopolysaccharides and the absence of glycogen and acid mucopolysaccharides in Columba columba 74 . However, it was reported that the mucous cells and mixed cells contained acid and neutral mucins in the tongue of pagrosomus 53 . The secretion of the lingual glands contained neutral mucins, proteoglycans containing carboxylic acid, weak and strong sulphated groups, N-acetylated sialomucins, but lacked glycogen 75 . The secretory cells of the lingual salivary gland of the rock dove ( Patagioenas livia ) showed strongly positive reaction to Periodic Acid Schiff's stain 76 . The labial glands extended from the corner of the mouth to the length of the lips and located in the propria submucosa layer. The structure and arrangement of the labial glands were similar as reported in the ox 21 and ruminants 23 . The occurrence of labial glands was reported at the corner of the mouth in the sheep 41 . However, the labial glands of goat were grouped as commissural, superior and inferior glands 24 . In the present study, the labial glands were of mixed type as reported in the sheep 41 , humans 28 and ruminants 36 . Contrary to these findings, the labial glands were of mucous type a reported in the sheep, goat and carnivores 17 , however, in the camel, the labial glands were either serous or seromucous type 37 . The labial salivary glands of the one‐humped camel were of branched tubuloacinar type comprising of mucoserous acini capped by serous demilunes 69 . The labial glands of goat were mainly mucous in nature with a very few serous and mixed acini 24 . The labial glands of the camel were of compound tubuloacinar and mixed (predominantly mucous) type 77 . On the other hand, the labial salivary glands of dog were of compound tubuloacinar type having mixed and predominantly mucous acini with presence of serous demilunes 78 . The labial glands of mixed breed dog were of compound and mixed tubuloacinar nature having distribution of mucous acini, mucous acini with serous demilunes and isolated serous acini 79 . The labial glands of dog were of tubuloacinar type and were located in the submucosal connective tissue 35 . In the humans, the mucous secretory portions directly opened into the striated ducts contrary to the present findings in which the mucous units continued into the intercalated ducts and these ducts united and formed the striated ducts 39 . In the current study, histochemical studies revealed the presence of glycogen in the mucous cells by PAS staining. However, in sheep, the mucous cells secreted neutral mucosubstances, sialomucins and hyaluronidase resistant sulfomucins 80 . The serous component was highly evocative for PAS and colloidal iron techniques 15 . Some of the findings stated that the mixed labial glands of the camel were rich in both neutral and acidic mucopolysaccharides 77 . However, the labial glands of sheep revealed that the secretory mucous cells contained acidic and neutral mucopolysaccharides while serous cells showed negative reaction 80 . Conclusion It may be concluded that the minor salivary glands of pig were located in the propria-submucosa layer of mucous membrane of oral cavity. The lobules showed the presence of secretory acinar units and the ducts. Depending on their location, the secretory acinar units were either mucous, serous or mixed in nature. These glands were of compound tubuloacinar type being surrounded by a capsule having a mixed distribution of collagen, reticular and few elastic fibres. Serous demilunes were also observed at periphery of the mucous secretory units. In addition to the plasma cells, myoepithelial cells were also found between the base of the secretory cells and the surrounding basal lamina. In addition to the secretory acini, the ducts were also observed in the lobules which consists of intralobular and interlobular ducts. Histochemical studies revealed the presence of weakly acidic sulfated mucosubstances, sialomucins, glycogen, mucopolysaccharides, mucin and protein in the mucous secretory units. However, the serous acini and ducts showed negative reaction to all these histochemical moieties. Micrometrical studies revealed that the acinar and luminal diameters of the mucous acini were larger than that of serous units. However, the diameter of the interlobular ducts, especially of lingual glands, was larger than intercalated and striated ducts which indicates that there was a significant difference at both 1% and 5% levels. Thus, understanding the anatomy, morphometry and histology of these glands is essential for a correct decision concerning medical and/or surgical intervention of such affections. This study will be helpful in field of surgery and also aid in diagnosis of pathological conditions of these glands and therefore this study establishes a basis for future research on this model. Declarations Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Acknowledgements The authors would like to thank Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar for providing all type of necessary facilities to carry out the study. Author contributions Prateek Rao performed the experiment; Amandeep Singh conceived and designed, gathering information, drafting the manuscript, analysis and interpretation of data; Pawan Kumar supervising the study and Simran Kaur performed the statistical analysis of data. 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Histomorphological and histochemical stains employed on paraffin sections of minor salivary glands of pig. S.N o. Stain(s) (with Catalog number and supplier) Reference(s) Purpose 1. Haematoxylin and Eosin (230251, Sigma-Aldrich, India) Luna (1968) General histomorphology 2. Crossman’s Trichrome (HT15, Sigma-Aldrich, India) Crossman (1937) Collagen fibres 3. Alcian Blue (at pH 2.5) (05500, Sigma-Aldrich, India) Luna (1968) Acid mucopolysaccharides 4. Periodic Acid Schiff (PAS) (TTR009, Sigma-Aldrich, India) Luna (1968) Neutral mucopolysaccharides 5. PAS- Alcian Blue (05500, TTR009 Sigma-Aldrich, India) Luna (1968) Mucosubstances 6. Gomori’s stain (100380, Sigma-Aldrich, India) Luna (1968) Reticular fibers 7. Weigert’s stain (TTR003, Sigma-Aldrich, India) Luna (1968) Elastic fibers 8. Holme’s stain (R0283, Sigma-Aldrich, India) Luna (1968) Nerve cells and fibers 9. Mayer’s mucicarmine (41325, Sigma-Aldrich, India) Luna (1968) Mucin 10. Colloidal iron (85193, Sigma-Aldrich, India) Luna (1968) Acid mucopolysaccharides 11. Diastase digestion (09962, Sigma-Aldrich, India) Luna (1968) Glycogen 12. Mercury bromphenol blue (B0126, Sigma-Aldrich, India) Pearse (1968) Proteins Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 19 Dec, 2025 Reviews received at journal 17 Dec, 2025 Reviewers agreed at journal 17 Nov, 2025 Reviews received at journal 18 Oct, 2025 Reviews received at journal 13 Oct, 2025 Reviewers agreed at journal 18 Sep, 2025 Reviewers agreed at journal 16 Sep, 2025 Reviewers invited by journal 16 Sep, 2025 Editor assigned by journal 16 Sep, 2025 Editor invited by journal 15 Sep, 2025 Submission checks completed at journal 11 Sep, 2025 First submitted to journal 11 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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14:09:45","extension":"png","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":493762,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/2c655c3e718b045c8c3a60ba.png"},{"id":92185985,"identity":"ef88875b-1ada-4bb5-9a90-0a69c32b44e6","added_by":"auto","created_at":"2025-09-25 14:17:44","extension":"xml","order_by":41,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":179592,"visible":true,"origin":"","legend":"","description":"","filename":"d0a108525aa84411acf101ca29696b3d1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/3464ca281b0984af6ac5b5e9.xml"},{"id":92185480,"identity":"6ce037ad-68ac-4314-80de-f63ff95e6331","added_by":"auto","created_at":"2025-09-25 14:09:53","extension":"html","order_by":42,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":194418,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/2cff1ee0a1f77a1f524bec26.html"},{"id":92185431,"identity":"e86a65b4-3a5b-439c-b7d2-57f5d09f2f20","added_by":"auto","created_at":"2025-09-25 14:09:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3772527,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of dorsal buccal glands of pig showing: \u003cstrong\u003e(a) \u003c/strong\u003eDistribution of glands (G) in propria submucosa layer of the cheek.\u003cstrong\u003e \u003c/strong\u003eH\u0026amp;E. Scale bar = 40 μm; \u003cstrong\u003e(b)\u003c/strong\u003e Connective tissue septa (S) divided the parenchyma into irregular lobes. H\u0026amp;E. Scale bar = 100 μm; \u003cstrong\u003e(c)\u003c/strong\u003e Mucous acinar cells (M) of different lobes separated from septa (S). (H\u0026amp;E. Scale bar = 200 μm; \u003cstrong\u003e(d)\u003c/strong\u003e Distribution of collagen fibres (arrow) around the mucous acini, blood vessels and muscles. Crossman’ s trichrome. Scale bar = 40 μm; \u003cstrong\u003e(e)\u003c/strong\u003e Pyramidal shaped mucous secretory cells (M) arranged around a wide lumen. Plasma cells (arrow) were found scattered among the connective tissue. H\u0026amp;E. Scale bar = 400 μm; \u003cstrong\u003e(f)\u003c/strong\u003e Distribution of reticular fibres (arrow) around the ducts (D) and in the interlobular connective tissue septa (arrow) Gomori’s method. Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/f0fd948efc854b303c446f8f.png"},{"id":92185436,"identity":"59f3579f-2ca6-4c06-9193-226233a51f29","added_by":"auto","created_at":"2025-09-25 14:09:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3642725,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of dorsal buccal glands of pig showing: \u003cstrong\u003e(a) \u003c/strong\u003eDistribution of elastic fibres (arrow) in the blood vessels (Bv) of interlobular septa.\u003cstrong\u003e \u003c/strong\u003eWeigert’s method. Scale bar = 100 μm; \u003cstrong\u003e(b)\u003c/strong\u003ePresence of nerve fibres (N) in the interlobular septa. Holmes method. Scale bar = 40 μm; \u003cstrong\u003e(c)\u003c/strong\u003e Mucous secretory units (M) and intercalated ducts (Id) lined with simple cuboidal epithelium. H\u0026amp;E. Scale bar = 200 μm; \u003cstrong\u003e(d)\u003c/strong\u003e Striated ducts (Sd) lined with simple columnar epithelium with longitudinal striations. H\u0026amp;E. Scale bar = 400 μm; \u003cstrong\u003e(e)\u003c/strong\u003e Interlobular ducts (ILd) lined with stratified columnar epithelium and the acidophilic secretions were found in lumen. H\u0026amp;E. Scale bar = 400 μm; \u003cstrong\u003e(f)\u003c/strong\u003e Distribution of collagen fibres (arrow) around mucous acini (M) and ducts. Crossman’ s trichrome. Scale bar = 200 μm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/27d74851a9abc60f45ecdf40.png"},{"id":92185432,"identity":"939be0f2-0ab4-4ca7-8bbe-d26a21cfb7fb","added_by":"auto","created_at":"2025-09-25 14:09:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3737934,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of dorsal buccal glands of pig showing: \u003cstrong\u003e(a) \u003c/strong\u003eAlcianophilic reaction in mucous acini (M) indicating the presence of weakly acidic sulfated mucosubstances and sialomucins in the secretions.\u003cstrong\u003e \u003c/strong\u003eAlcian blue. Scale bar = 40 μm; \u003cstrong\u003e(b)\u003c/strong\u003e Intense PAS positive reaction in mucous acini (M) indicating the presence of high amount of glycogen in the secretions. McManus’ method. Scale bar = 40 μm; \u003cstrong\u003e(c)\u003c/strong\u003e Intense positive reaction for acidic mucopolysaccharides in mucous acinar cells (M). PAS-AB. Scale bar = 40 μm; \u003cstrong\u003e(d)\u003c/strong\u003e Strong positive reaction for mucin in mucous acini (M). Mayer’s mucicarmine. Scale bar = 200 μm; \u003cstrong\u003e(e)\u003c/strong\u003e Moderate positive reaction for acidic mucopolysaccharides in mucous acini (M). Colloidal iron. Scale bar = 100 μm; \u003cstrong\u003e(f)\u003c/strong\u003e Weak protein activity in mucous secretory cells (M). Mercury bromphenol blue. Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/c65014f951659643a9cb065a.png"},{"id":92185466,"identity":"91a44c4e-cd7b-4f71-8fb7-ef7377007118","added_by":"auto","created_at":"2025-09-25 14:09:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3842779,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of ventral buccal glands of pig showing: \u003cstrong\u003e(a) \u003c/strong\u003eDistribution of glands (G) in propria submucosa layer of the cheek.\u003cstrong\u003e \u003c/strong\u003eH\u0026amp;E. Scale bar = 40 μm; \u003cstrong\u003e(b)\u003c/strong\u003e Serous acinar units (S) having pyramidal shaped cells around a narrow lumen. H\u0026amp;E. Scale bar = 100 μm; \u003cstrong\u003e(c)\u003c/strong\u003e Distribution of reticular fibres (arrow) around the serous acini (S) and ducts (D). Gomori’s method. Scale bar = 200 μm; \u003cstrong\u003e(d)\u003c/strong\u003e Interlobular ducts having goblet cells (arrow) and lumen contains acidophilic secretions. H\u0026amp;E. Scale bar = 40 μm; \u003cstrong\u003e(e)\u003c/strong\u003eStrong alcianophilic reaction in goblet cells (arrow) of interlobular ducts and the serous acini (S) were negative. Alcian blue. Scale bar = 40 μm; \u003cstrong\u003e(f)\u003c/strong\u003e Strong PAS reaction in goblet cells (arrow) and the serous acini (S) were negative. McManus’ method. Scale bar = 40 μm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/00ab2db072366c076a19262a.png"},{"id":92185417,"identity":"b8b6e55b-19e6-4499-b0ac-3017ac3e922f","added_by":"auto","created_at":"2025-09-25 14:09:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3739722,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of palatine glands of pig showing: \u003cstrong\u003e(a) \u003c/strong\u003eAbsence of glands in the region of hard palate.\u003cstrong\u003e \u003c/strong\u003eH\u0026amp;E. Scale bar = 40 μm; \u003cstrong\u003e(b)\u003c/strong\u003e Distribution of glands (G) in propria submucosa layer of the oropharyngeal region of soft palate. H\u0026amp;E. Scale bar = 100 μm; \u003cstrong\u003e(c)\u003c/strong\u003eMucous secretory units (M) with distinct cell boundaries and peripheral flattened nuclei. H\u0026amp;E. Scale bar = 200 μm; \u003cstrong\u003e(d)\u003c/strong\u003e Distribution of collagen fibres (arrow) around the mucous acini (M). Crossman’ s trichrome. Scale bar = 200 μm; \u003cstrong\u003e(e)\u003c/strong\u003e Distribution of elastic fibres (arrow) in the blood vessels (Bv) of interlobular septa. Weigert’s method. Scale bar = 100 μm; \u003cstrong\u003e(f)\u003c/strong\u003eMucous acini (M), intercalated (Id) and striated ducts (Sd) within lobule. H\u0026amp;E. Scale bar = 200 μm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/329a232c611313df81b03830.png"},{"id":92185470,"identity":"b0cd3338-febc-4b1a-a893-85f71673e85d","added_by":"auto","created_at":"2025-09-25 14:09:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3571539,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of palatine glands of pig showing: \u003cstrong\u003e(a) \u003c/strong\u003eMucous secretory acini (M) and large interlobular duct (ILd) present in the interlobular connective tissue.\u003cstrong\u003e \u003c/strong\u003eH\u0026amp;E. Scale bar = 200 μm; \u003cstrong\u003e(b)\u003c/strong\u003eStrong positive alcianophilic reaction in mucous acini (M) indicating the presence of weakly acidic sulfated mucosubstances and sialomucins in the secretions. Alcian blue. Scale bar = 100 μm; \u003cstrong\u003e(c)\u003c/strong\u003eIntense PAS positive reaction in mucous acini (M) indicating the presence of high amount of glycogen in the secretions. McManus’ method. Scale bar = 100 μm; \u003cstrong\u003e(d)\u003c/strong\u003e Intense positive reaction for acidic mucopolysaccharides in mucous acini (M). PAS-AB. Scale bar = 100 μm; \u003cstrong\u003e(e)\u003c/strong\u003eModerate positive reaction for mucin in mucous acini (M). Mayer’s mucicarmine. Scale bar = 100 μm; \u003cstrong\u003e(f)\u003c/strong\u003e Moderate positive reaction for acidic mucopolysaccharides in mucous acini (M). Colloidal iron method. Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/58e7eeb1c2858de53fec62d2.png"},{"id":92185484,"identity":"42d67cb5-3cdb-4903-a758-2e5b2d92bf2e","added_by":"auto","created_at":"2025-09-25 14:09:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3691183,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of lingual glands of pig showing: \u003cstrong\u003e(a) \u003c/strong\u003eDistribution of glands (G) in propria submucosa layer of the root region of tongue.\u003cstrong\u003e \u003c/strong\u003eH\u0026amp;E. Scale bar = 40 μm; \u003cstrong\u003e(b)\u003c/strong\u003e Connective tissue septa (S) divided the parenchyma into irregular lobes. H\u0026amp;E. Scale bar = 100 μm; \u003cstrong\u003e(c)\u003c/strong\u003e Crescent-shaped serous demilunes (arrow) at the periphery of mucous acini (M). H\u0026amp;E. Scale bar = 200 μm; \u003cstrong\u003e(d)\u003c/strong\u003e Intercalated ducts (Id) and mucous acini (M) within the glandular lobules. H\u0026amp;E. Scale bar = 200 μm; \u003cstrong\u003e(e)\u003c/strong\u003eAlcianophilic reaction in mucous acini (M) indicating the presence of weakly acidic sulfated mucosubstances and sialomucins in the secretions. Alcian blue. Scale bar = 40 μm; \u003cstrong\u003e(f)\u003c/strong\u003e Strong Alcianophilic reaction in mucous acini (M) and the ducts (D) revealed negative reaction. Alcian blue. Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/11edc328702ccda9df018287.png"},{"id":92186005,"identity":"13ff72a6-e7c1-4f6c-b8ba-d58df333fc86","added_by":"auto","created_at":"2025-09-25 14:17:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3598571,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of lingual glands of pig showing: (a) Intense PAS positive reaction in mucous acini (M) indicating the presence of high amount of glycogen in the secretions. McManus’ method. Scale bar = 40 μm; \u003cstrong\u003e(b)\u003c/strong\u003e Intense PAS positive reaction in mucous acini (M) and goblet cells (arrows) of interlobular ducts. McManus’ method. Scale bar = 40 μm; \u003cstrong\u003e(c)\u003c/strong\u003e PAS-AB positive reaction in mucous acini (M) and goblet cells (arrows) of interlobular ducts. PAS-AB. Scale bar = 40 μm; \u003cstrong\u003e(d)\u003c/strong\u003e Strong positive reaction for mucin in mucous acini (M). Mayer’s mucicarmine. Scale bar = 200 μm; \u003cstrong\u003e(e)\u003c/strong\u003e Moderate positive reaction for acidic mucopolysaccharides in mucous acini (M). Colloidal iron. Scale bar = 40 μm; \u003cstrong\u003e(f)\u003c/strong\u003e Moderate positive reaction for acidic mucopolysaccharides in mucous acini (M) and goblet cells (arrow) of interlobular ducts. Colloidal iron. Scale bar = 40 μm.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/3d8ebb8da6407588993dd80e.png"},{"id":92185489,"identity":"c6aee848-3532-4154-84e6-9d4058913228","added_by":"auto","created_at":"2025-09-25 14:09:58","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3489404,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of lingual glands of pig showing: \u003cstrong\u003e(a) \u003c/strong\u003eDistribution of glands (G), lymphocytes (arrow) and adipocytes (A) in propria submucosa of tongue. H\u0026amp;E. Scale bar = 100 μm; \u003cstrong\u003e(b)\u003c/strong\u003eMucous secretory units (M) and adipocytes (A). H\u0026amp;E. Scale bar = 200 μm; \u003cstrong\u003e(c)\u003c/strong\u003e Mucous units (M) and small interlobular ducts (Id). H\u0026amp;E. Scale bar = 200 μm; \u003cstrong\u003e(d)\u003c/strong\u003eMucous units (M) and large interlobular ducts (Id). H\u0026amp;E. Scale bar = 200 μm; \u003cstrong\u003e(e)\u003c/strong\u003e Positive alcianophilic reaction in mucous (M) secretory units. Alcian blue. Scale bar = 100 μm; \u003cstrong\u003e(f)\u003c/strong\u003e PAS positive reaction in mucous acini (M). The lymphocytes (arrow) were also present in propria submucosa of the tongue. McManus’ method. Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/3b956c8fb05056c34ae43a36.png"},{"id":92185983,"identity":"5a164772-6508-4b48-9d6c-0bd779c5ff87","added_by":"auto","created_at":"2025-09-25 14:17:44","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":3874531,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of lingual glands of pig showing: \u003cstrong\u003e(a) \u003c/strong\u003ePositive PAS-AB reaction in mucous (M) secretory units.\u003cstrong\u003e \u003c/strong\u003ePAS-AB. Scale bar = 100 μm; \u003cstrong\u003e(b)\u003c/strong\u003e Moderate reaction for mucin in mucous secretory cells (M). Mayer’s mucicarmine. Scale bar = 100 μm; \u003cstrong\u003e(c)\u003c/strong\u003e Distribution of lingual glands (G) at the vicinity of vallate papillae (P). H\u0026amp;E. Scale bar = 40 μm; \u003cstrong\u003e(d)\u003c/strong\u003e Serous acinar cells (S) with rounded nuclei placed near to the basal third of the cells. H\u0026amp;E. Scale bar = 200 μm; \u003cstrong\u003e(e)\u003c/strong\u003eIntercalated ducts (arrow) lined with simple cuboidal epithelium. H\u0026amp;E. Scale bar = 100 μm; \u003cstrong\u003e(f)\u003c/strong\u003e Large excretory duct (arrow) lined with simple squamous epithelium. H\u0026amp;E.. Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/d05fd573d22461cfd504d5c3.png"},{"id":92185428,"identity":"62a81b86-8193-4b77-ba24-659a032273d2","added_by":"auto","created_at":"2025-09-25 14:09:45","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":3670326,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of labial glands of pig showing: \u003cstrong\u003e(a) \u003c/strong\u003eDistribution of large number of mucous acini (M) with very few independent serous units (S). H\u0026amp;E. Scale bar = 100 μm; \u003cstrong\u003e(b)\u003c/strong\u003eCrescent-shaped serous demilunes (arrow) at the periphery of mucous acini (M). H\u0026amp;E. Scale bar = 200 μm; \u003cstrong\u003e(c)\u003c/strong\u003e Interlobular connective tissue septae (S) divided the parenchyma into lobes. The central part showed the interlobular duct having goblet cells. H\u0026amp;E. Scale bar = 40 μm; \u003cstrong\u003e(d)\u003c/strong\u003e Distribution of collagen fibres (arrow) around the mucous acini (M) and interlobular duct (D). Crossman’ s trichrome. Scale bar = 100 μm; \u003cstrong\u003e(e)\u003c/strong\u003eDistribution of elastic fibres (arrow) in the blood vessels of interlobular septa. Weigert’s method. Scale bar = 100 μm; \u003cstrong\u003e(f)\u003c/strong\u003e Positive reaction for mucin in mucous acini (M) however, ducts (D) were negative. Mayer’s mucicarmine. Scale bar = 40 μm.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/86aea16a655cab85e63a06da.png"},{"id":92185463,"identity":"f8680866-420b-43d0-9845-864ea59109a0","added_by":"auto","created_at":"2025-09-25 14:09:47","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":3494803,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of labial glands of pig showing: \u003cstrong\u003e(a) \u003c/strong\u003eIntercalated (arrow) and striated (SD) ducts within the lobule. H\u0026amp;E. Scale bar = 100 μm; \u003cstrong\u003e(b)\u003c/strong\u003e Small interlobular ducts having goblet cells (arrow). H\u0026amp;E. Scale bar = 100 μm; \u003cstrong\u003e(c)\u003c/strong\u003e Large interlobular ducts having goblet cells (arrow) with secretions in lumen. H\u0026amp;E. Scale bar = 100 μm; \u003cstrong\u003e(d)\u003c/strong\u003e Distribution of collagen fibres (arrows) around the mucous acini (M), intra-and interlobular ducts (D). Crossman’ s trichrome. Scale bar = 40 μm; \u003cstrong\u003e(e)\u003c/strong\u003e Strong Alcianophilic reaction in mucous acini (M) however, serous acini (S) and ducts (arrow) were negative. Alcian blue. Scale bar = 40 μm; \u003cstrong\u003e(f)\u003c/strong\u003e Strong PAS reaction in mucous acini (M) however, serous acini (S) and ducts (D) were negative McManus’ method. Scale bar = 40 μm.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/3fa5a23456ebe3c84dee07f4.png"},{"id":92185419,"identity":"e28ba456-8d1f-499d-af2e-ff56b5e803f7","added_by":"auto","created_at":"2025-09-25 14:09:44","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":2338230,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs of labial glands of pig showing: \u003cstrong\u003e(a) \u003c/strong\u003eStrong PAS-AB reaction in mucous acini (M) while, serous acini (S) and ducts (D) were negative.\u003cstrong\u003e \u003c/strong\u003ePAS-AB method. Scale bar = 40 μm; \u003cstrong\u003e(b)\u003c/strong\u003ePositive reaction for acidic mucopolysaccharides in mucous acini (M) however, serous acini (S) and ducts (D) were negative. Colloidal iron method. Scale bar = 40 μm; \u003cstrong\u003e(c)\u003c/strong\u003e Positive reaction for mucin in mucous acini (M) and goblet cells (arrow) however, serous acini (S) were negative. Mayer’s mucicarmine. Scale bar = 40 μm; \u003cstrong\u003e(d)\u003c/strong\u003e Weak protein activity in mucous secretory cells (M). Mercury bromphenol blue. Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/1edf21e7bdf8685728962d6a.png"},{"id":92187256,"identity":"0debe959-e4f1-475d-a194-6053016b48b2","added_by":"auto","created_at":"2025-09-25 14:26:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":41471020,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7550163/v1/ee8433b0-8650-421c-9363-7a1a053463e2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative morphological analysis of minor salivary glands in porcine species using histological and histochemical evaluation","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePigs' physical and nutritional health are the primary determinants of their production performance, which is also influenced by the effectiveness of their digestive systems. The first biological medium to come into contact with substances that are ingested as food is saliva. As a result, it acts as the initial oral defense strategy\u003csup\u003e1\u003c/sup\u003e. These secretions are crucial for lubricating the upper digestive tract, moistening food, and shielding the mucosa's lining cells from harm. It also secretes lysozymes to regulate the bacterial flora\u003csup\u003e2\u003c/sup\u003e. Lactoperoxidase and Immunoglobulin A in saliva serve as a salivary antimicrobial system\u003csup\u003e3\u003c/sup\u003e. The enzymes present in the saliva may help in preparing the food for further digestion in the stomach\u003csup\u003e4\u003c/sup\u003e. The salt content of the saliva helps to regulate electrolytes. The saliva also has an excretory role as some heavy metals and other organic and inorganic salts were eliminated in part through it\u003csup\u003e5\u003c/sup\u003e. In addition, dysfunction of salivary secretion (hyposalivation) causes xerostomia (dry mouth) and sequentially leads to severe dental caries as well as oral mucosal disorders\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBoth major and small salivary glands secrete saliva, which is extremely vital. Saliva is a seromucous fluid secreted by a variety of glandular structures found in the salivary glands. The parotid, mandibular, and sublingual salivary glands are the three main salivary glands found in domestic animals. The lingual, labial, buccal, and palatine glands are located in the tongue, lips, cheek, and palate, respectively. These minor glands, which are found within the oral mucous membrane, were categorized as minor salivary glands\u003csup\u003e6\u003c/sup\u003e. This wide distribution of the minor salivary glands is advantageous for the protection of the oral cavity against pathogens\u003csup\u003e7\u003c/sup\u003e. The excretory ducts carried the secretions of these glands to the mouth cavity. Typically, there are two types of salivary secretions: mucous and serous. Whereas mucous discharge is more viscous and contains a higher proportion of organic proteinaceous matter, including neutral and acidic glycoproteins, serous secretion is thin and watery and comprises water, inorganic ions, and some proteinaceous material\u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe literature revealed that most of the research work is confined to major salivary glands of buffalo\u003csup\u003e9\u003c/sup\u003e, sheep\u003csup\u003e10\u003c/sup\u003e, goat\u003csup\u003e1\u003c/sup\u003e and pig\u003csup\u003e11\u003c/sup\u003e, whereas research on minor salivary glands is very scanty in general and pig in particular. Hence, the present study has been planned in pig to explore the detailed structure and nature of the minor salivary glands, which will certainly be helpful to the histologists, physiologists and pathologists to understand the histoarchitectural organization of these glands.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCollection of the specimens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was conducted on minor salivary glands of ten healthy adult pigs of local mixed breed of either sex. The heads were procured from local slaughter house immediately after decapitation. The fresh tissues were collected from these glands and fixed in 10% neutral buffered formalin (NBF) (HT501128, Sigma-Aldrich, India) for 48-72 hours. For buccal glands, the tissues were collected from cranial, middle and caudal portions of dorsal and ventral rows. The tissues from the hard palate and soft palate were also collected for the study of palatine glands. For lingual glands, the tissues were collected from apex, body and root of the tongue. The tissues from the upper, lower and commissural portions of the lips were also collected for the study of labial glands.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue processing, sectioning and staining\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter fixation in 10% NBF for 48-72 hours at room temperature, the tissue samples were then processed for paraffin sectioning technique which includes various steps. The first step was dehydration of the fixed tissue in ascending grades of ethanol (459836, Sigma-Aldrich, India) i.e., 50%, 70%, 90% and three absolute grades of ethanol, keeping in each solution for one hour. After this step, clearing was done by passing the tissues through two sets of benzene solution (401765, Sigma-Aldrich, India), keeping in each solution for at least one hour. Then, infiltration was done to replace the clearing agent completely from the tissue with the paraffin wax of melting point 58-62oC (327212, Sigma-Aldrich, India). Cleared tissues were then passed through three sets of melted paraffin wax for one hour each, which were kept in an oven adjusting its temperature 1 or 2oC above the melting point of wax used. Then embedding of the tissues was done, in which, the melted paraffin wax of the same melting point as used for infiltration, was poured in the moulds having infiltrated tissues and then allowed to cool which results in hardening of the paraffin wax. Then, trimming of the blocks was done to expose the tissue surfaces to a level where a representative section could be cut. The paraffin sections of 5 µm thickness were cut on a microtome (RM2125 RTS, Leica Biosystems, Germany), mounted on microscope slides with DPX mounting media (06522, Sigma, India) and then stained with various histomorphological and histochemical stains\u003csup\u003e12-14\u0026nbsp;\u003c/sup\u003e(Table).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhotomicrography\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh-resolution photomicrographs of paraffin sections were taken by a light microscope equipped with a camera (DS-Fi3, Japan) having imaging software ViewNX.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe diameter of serous and mucous acini as well as intralobular and interlobular ducts of minor salivary glands was measured and the data was tabulated and statistically analyzed. The data was subjected to statistical analysis for determining correlation between these structures by using SPSS statistics software version 22.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eHistological and histochemical analysis of buccal glands\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe buccal glands of pig were observed in the cheek region and found arranged in two rows as dorsal and ventral groups. Histologically, the dorsal buccal glands of pig showed similar structure in rostral and caudal portions. The mucous membrane of the cheek was lined by stratified squamous non-keratinized epithelium. The lamina propria blends with the tunica submucosa without an intervening lamina muscularis, forming a propria submucosa. The dorsal buccal glands were located in the propria-submucosa layer and also scattered among the skeletal muscle bundles of the cheek (Fig. 1a). These were of compound tubuloacinar type being surrounded by a thin capsule having a mixed distribution of collagen, reticular and few elastic fibres. Blood vessels and nerve bundles were seen distributed with in the capsule. From the capsule, the interlobular septae arises which divided the parenchyma into lobes and lobules (Fig. 1b). The lobules showed the presence of secretory acinar units and the ducts. The secretory units were purely mucous in nature (Fig. 1c). The collagen fibres were observed surrounded the secretory acinar units, blood vessels and the muscle bundles (Fig. 1d). The mucous acini were comprised of pyramidal cells having distinct cell boundaries, located around a wide lumen and the flattened basophilic nuclei were situated towards the basement membrane of the cell. Their chromatin material was very dense which marked the appearance of nucleoli. Few mucous cells were noticed surrounded by long, spindle-shaped, flat cells known as myoepithelial cells, which were interposed between the base of the secretory cells and the surrounding basal lamina. Plasma cells were also found scattered in connective tissue present in between the secretory acinar units (Fig. 1e). The mucous acini measured 48.18\u0026plusmn;1.68 \u0026mu;m in diameter. The cytoplasm of these pyramidal cells was eosinophilic in nature and presented a vacuolated appearance due to washing of mucous secretions during the processing of tissues. The luminal diameter of the mucous acini was 11.88\u0026plusmn;1.32 \u0026mu;m. Blood vessels, nerves, adipocytes and interlobular ducts were distributed with in the interlobular septae. In addition to the collagen fibres, reticular fibres were seen around the capsule and also surrounding the ducts and blood vessels (Fig. 1f). Fine elastic fibres were observed in the blood vessels located in the interlobular septa (Fig. 2a). Large plexus of ganglionic cells and blood vessels were also noticed in the capsule from which nerve fibres traversed through the septa and ramified along the basement membrane of the acini and ducts (Fig. 2b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition to the secretory units, the ducts were also observed in the dorsal buccal gland, consisted of intralobular and interlobular ducts. Intralobular ducts were located within the lobules and were further subdivided into small and large intralobular ducts on the basis of their diameter and type of epithelium. The small intralobular ducts, known as intercalated ducts were characterized by the presence of small sized lumen and lined with simple cuboidal epithelium (Fig. 2c) and measured 15.18\u0026plusmn;0.80 \u0026mu;m in diameter. The intercalated ducts opened into large intralobular ducts, known as striated ducts, within the lobules. The striated ducts measured 50.16\u0026plusmn;1.23\u0026mu;m in diameter. These ducts were lined with simple to stratified columnar epithelium having one to two cell layer thickness with characteristic longitudinal striations in the basal third. The cytoplasm of the cells lining the striated ducts was eosinophilic, while the nuclei were basophilic and darkly stained (Fig. 2d). Myoepithelial cells were observed between the base of the cells and basement membrane of the intralobular ducts. The striated duct extended to the periphery of the lobule to open into interlobular ducts. Large interlobular ducts were localized at the level of interlobular septae and were lined with stratified columnar epithelium and measured 62.70\u0026plusmn;1.04 \u0026mu;m in diameter. The interlobular ducts also showed the presence of unicellular mucous glands known as goblet cells. The lumen of these ducts contains the varying amount of secretions which were eosinophilic in nature (Fig. 2e). The interlobular ducts drain the secretions into terminal excretory ducts where the epithelium changed to stratified squamous. These excretory ducts opened in the mucosal membrane of the cheek. The concentration of collagen fibres increased surrounding the intralobular and interlobular ducts (Fig. 2f).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHistochemically, the mucous acinar cells showed a strong Alcian blue reaction in the secretory cells indicating the presence of weakly acidic sulfated mucosubstances, sialomucins and hyaluronic acid in the secretions. However, the ducts revealed negative reaction to all these histochemical moieties (Fig. 3a). The mucous secretory cells were intensely PAS positive by McManus\u0026rsquo; method showing the presence of high concentration of glycogen in the secretions (Fig. 3b). The diastase treatment showed that PAS activity was reduced indicating the presence of glycogen in addition to other mucopolysaccharides. It showed a negative reaction with Best\u0026rsquo;s carmine confirming that mucopolysaccharides other than glycogen was present in the mucous cells. Combined PAS-AB method revealed intense positive reaction for acidic mucopolysaccharides in mucous cells and ducts were negative (Fig. 3c). Mucous acini showed strong positive reaction to mucin and the ducts were negative by Mayer mucicarmine method. However, the goblet cells of interlobular ducts showed positive reaction with this method (Fig. 3d). Colloidal iron method showed the moderate positive reaction for acidic mucopolysaccharides in mucous acini and ducts were negative (Fig. 3e). Weak protein activity was observed in mucous cells, however, ducts showed negative reaction by mercury bromphenol blue method (Fig. 3f).\u003c/p\u003e\n\u003cp\u003eThe histoarchitecture of ventral buccal glands of pig showed similar structure as of dorsal row of glands with the exception of serous type of secretory units. These glands were located in the propria-submucosa layer and also scattered among the skeletal muscle bundles of the cheek (Fig. 4a). \u0026nbsp;The serous acini were comprised of pyramidal shaped cells, located around a narrow lumen and the basophilic spherical vesicular nuclei were situated towards the basal one third of the cell. The nucleoli showed distinct appearance due to their dense chromatin material. The cell boundaries were comparatively indistinct (Fig. 4b). The ventral buccal glands were of compound tubuloacinar type encapsulated by a thick dense capsule and was divided into lobules by means of thin connective tissue septa having distribution of reticular fibres (Fig. 4c) fibres in addition to fine blood capillaries, plasma cells and nerve fibres. The lobules showed the presence of serous secretory units and the ducts. Fine elastic fibres were also observed in the blood vessels located in the interlobular septa. The serous acini measured 23.76\u0026plusmn;1.23 \u0026mu;m in diameter and their luminal diameter was 4.62\u0026plusmn;0.80 \u0026mu;m. The cytoplasm of these pyramidal cells was basophilic in nature and presented numerous granules towards the apical part of cell.\u003c/p\u003e\n\u003cp\u003eThe duct system of ventral buccal glands was also similar to that of dorsal glands which consisted of intralobular and interlobular ducts. The small intralobular ducts, known as intercalated ducts were characterized by the presence of small sized lumen and lined with simple cuboidal epithelium and measured 8.58\u0026plusmn; 0.80 \u0026mu;m in diameter. The striated ducts were lined with simple to stratified columnar epithelium having one to two cell layer thickness with characteristic longitudinal striations in the basal third. These striated ducts measured 26.40\u0026plusmn;1.16 \u0026mu;m in diameter. The cytoplasm of the cells lining the striated ducts was eosinophilic, while the nuclei were basophilic and darkly stained. The striated duct extended to the periphery of the lobule to open into interlobular ducts. Large interlobular ducts were lined with stratified columnar epithelium and measured 41.58\u0026plusmn;0.80 \u0026mu;m in diameter. The interlobular ducts also showed the presence of goblet cells (Fig. 4d). The lumen of these ducts contains the varying amount of secretions which were eosinophilic in nature. The interlobular ducts drain the secretions into terminal excretory ducts where the epithelium changed to stratified squamous epithelium. Histochemically, the goblet cells of the interlobular ducts showed strong positive reaction to acidic mucopolysaccharides (Fig. 4e), neutral mucopolysaccharides (Fig. 4f), however the serous acini exhibited negative reaction to all histochemical staining techniques carried out during the present investigation indicating the absence of mucopolysaccharides, glycogen, mucin and protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological and histochemical analysis of palatine glands\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor palatine glands, the tissues were collected from the ridged portion of the hard palate (rostral, middle and caudal portions) and also from oropharyngeal region of the soft palate. Histologically, no glands were found in the region of hard palate (Fig. 5a). The mucous membrane of the oropharyngeal region of the soft palate was lined by stratified squamous epithelium and the palatine glands were located in the propria-submucosa layer. These were of compound tubuloacinar type being surrounded by a thin capsule having a mixed distribution of collagen, reticular and few elastic fibres. In addition to the glands, lymphatic tissue was also seen in the propria-submucosa layer (Fig. 5b). Blood vessels and nerve bundles were seen distributed with in the capsule. From the capsule, the interlobular septae arises which divided the parenchyma into lobes and lobules. Blood vessels, nerves and interlobular ducts were distributed with in the septae. The lobules showed the presence of secretory units and the ducts. The secretory units were purely mucous in nature. The pyramidal cells of mucous acini were located around a wide lumen and the flattened basophilic nuclei were situated towards the basement membrane of the cell. They have distinct cell boundaries and their chromatin material was dense which marked the appearance of nucleoli. The cytoplasm of these pyramidal cells was eosinophilic in nature and presented a vacuolated appearance due to washing of mucous secretions during the processing of tissues (Fig. 5c). The mucous acini measured 38.94\u0026plusmn;1.23 \u0026mu;m in diameter and their luminal diameter was 9.24\u0026plusmn;0.66 \u0026mu;m. The myoepithelial cells were interposed between the base of the secretory acinar cells and the surrounding basal lamina. Plasma cells were also found scattered in connective tissue present in between the secretory acinar units. The collagen fibres were observed surrounded the blood vessels and acinar cells (Fig. 5d). Fine elastic fibres were also seen in the blood vessels located in the interlobular septa (Fig. 5e). In addition to the mucous acini, the ducts were also present in the palatine glands, consisted of intralobular and interlobular ducts. The intercalated ducts were lined by simple cuboidal epithelium (Fig. 5f) and measured 12.21\u0026plusmn;0.99 \u0026mu;m in diameter. The intercalated ducts opened into large striated ducts within the lobules, measuring 41.58\u0026plusmn;0.80 \u0026mu;m in diameter and were lined with simple to stratified columnar epithelium having two to three cell layer thickness. The cytoplasm of the cells lining the striated ducts was eosinophilic, while the nuclei were basophilic and darkly stained (Fig.5f). The striated duct extended to the periphery of the lobule to open into large interlobular ducts. The interlobular ducts were lined with stratified columnar epithelium (Fig. 6a) and these ducts measured 52.80\u0026plusmn;1.47 \u0026mu;m in diameter. The concentration of collagen fibres increased surrounding the intralobular and interlobular ducts. The interlobular ducts drain the secretions into terminal excretory ducts where the epithelium changed to stratified squamous.\u003c/p\u003e\n\u003cp\u003eHistochemically, a strong alcian blue reaction was observed in the mucous secretory cells indicating the presence of weakly acidic sulfated mucosubstances, sialomucins and hyaluronic acid in the secretions. However, the ducts revealed negative reaction to all these histochemical moieties (Fig. 6b). The mucous secretory cells were intensely PAS positive by McManus\u0026rsquo; method showing the presence of high concentration of glycogen in the secretions however, the ducts were showing negative reaction (Fig. 6c). Combined PAS-AB method revealed strong positive reaction for acidic and neutral mucopolysaccharides in mucous cells and ducts were negative (Fig. 6d). Mucous acini showed moderate positive reaction to mucin and the ducts were negative by Mayer mucicarmine method (Fig. 6e). Colloidal iron method showed the moderate to strong positive reaction for acidic mucopolysaccharides in mucous acini whereas, ducts revealed negative reaction (Fig. 6f).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological and histochemical analysis of lingual glands\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe lingual glands of pig were collected from root, body and apex of the tongue. Histologically, the dorsal surface of tongue was lined by stratified squamous keratinized epithelium. The lingual glands present at the root of the tongue were of compound tubuloacinar type and were located in the propria-submucosa layer and also noticed among the skeletal muscle bundles (Fig. 7a). These glands were encapsulated by a thin layer of connective tissue made up of large amount of collagen fibres and sparse number of reticular fibres. Blood vessels and nerve bundles were found distributed with in the capsule. The interlobular connective tissue septae divided the parenchyma into lobes and lobules (Fig. 7b). The collagen fibres were also seen surrounded the blood vessels and the muscle bundles. The lobules showed the presence of secretory acinar units and the ducts. The secretory units in the tongue\u0026apos;s root were made up of mucous acinar cells. The mucous acini were comprised of pyramidal cells having distinct cell boundaries, located around a wide lumen and the flattened basophilic nuclei were situated towards the basement membrane of the cell. Their chromatin material was very dense which marked the appearance of nucleoli. Occasionally, the crescent-shaped serous demilunes were also observed in the root of the tongue (Fig. 7c). Myoepithelial cells were seen between the base of the mucous secretory cells and the surrounding basal lamina. The duct system was comprised of intralobular and interlobular ducts. Intralobular ducts were further subdivided into small (intercalated) and large (striated) ducts on the basis of their diameter and type of epithelium. The intercalated ducts opened into striated ducts within the lobules. The striated ducts were lined with simple to stratified columnar epithelium having one to two cell layer thickness with characteristic longitudinal striations in the basal third (Fig. 7d). The cytoplasm of the cells lining the striated ducts was eosinophilic, while the nuclei were basophilic and darkly stained. The striated duct extended to the periphery of the lobule to open into interlobular ducts. Large interlobular ducts were localized at the level of interlobular septae and were lined with stratified columnar epithelium. Histochemically, the mucous acini showed a strong Alcian blue reaction indicating the presence of weakly acidic sulfated mucosubstances, sialomucins and hyaluronic acid in the secretions (Fig. 7e) however, the ducts revealed negative reaction to all these histochemical moieties (Fig. 7f). The mucous secretory cells were intensely PAS positive by McManus\u0026rsquo; method showing the presence of high concentration of glycogen in the secretions (Fig. 8a). However, the ducts were negative and unicellular mucous glands showed intense PAS positive reaction which indicates the presence of large amount of glycogen (Fig. 8b). Combined PAS-AB method revealed strong positive reaction for acidic and neutral mucopolysaccharides in mucous cells and ducts were negative. However, goblet cells showed positive activity for neutral mucopolysaccharides (Fig. 8c). Mucous acini showed strong positive reaction to mucin and the ducts were negative by Mayer mucicarmine method (Fig. 8d). Colloidal iron method showed the moderate to strong positive reaction for acidic mucopolysaccharides in mucous acini (Fig. 8e). However, goblet cells of interlobular ducts showed positive reaction to this method (Fig. 8f).\u003c/p\u003e\n\u003cp\u003eThe lingual glands present at the body of tongue were of compound tubuloacinar type and located in the propria-submucosa layer. Lymphocytes and adipocytes were also found within the propria-submucosa in addition to the lingual glands (Fig. 9a). From the capsule, the connective tissue septae, made up of collagen and reticular fibres, arises which divided the parenchyma into lobes and lobules. The lobules showed the presence of secretory acinar units and the ducts. The secretory units were comprised of only mucous acini, having pyramidal cells with distinct cell boundaries, located around a wide lumen and the flattened basophilic nuclei were situated towards the basement membrane of the cell (Fig. 9b). The duct system was comprised of intralobular and interlobular ducts. The intercalated ducts opened into striated ducts within the lobules. The striated ducts were lined with simple to stratified columnar epithelium having one to two cell layer thickness (Fig. 9c). The cytoplasm of the cells lining the striated ducts was eosinophilic, while the nuclei were basophilic and darkly stained. The striated duct extended to the periphery of the lobule to open into interlobular ducts. Large interlobular ducts were localized at the level of interlobular septae and were lined with stratified columnar epithelium (Fig. 9d). Histochemically, a strong Alcian blue reaction was seen in the mucous acini, showed the presence of weakly acidic sulfated mucosubstances, sialomucins and hyaluronic acid in the secretions however, the ducts were showing negative reaction to all these histochemical moieties (Fig. 9e). McManus\u0026rsquo; method revealed that the mucous secretory cells were intensely PAS positive indicating the presence of high concentration of glycogen in the secretions (Fig. 9f). However, the ducts showed negative reaction to this method. Combined PAS-AB method revealed strong positive reaction for acidic and neutral mucopolysaccharides in mucous cells and ducts were negative (Fig. 10a). Colloidal iron method showed the moderate to strong positive reaction for acidic mucopolysaccharides in mucous acini. A moderate positive reaction was seen in mucous acini indicating the presence of mucin and the ducts were negative by Mayer mucicarmine method (Fig. 10b).\u003c/p\u003e\n\u003cp\u003eThe circumvallate papillae were large, flattened structures completely surrounded by an epithelium-lined sulcus. The epithelium on the papillary side of the sulcus contains many taste buds. These taste buds were ellipsoid clusters of specialized epithelial cells embedded in the stratified squamous epithelium of the circumvallate papillae of the tongue. However, the glands that were present just beneath the circumvallate papillae were comprised of only serous secretory units (Fig. 10c). The serous acinar cells were pyramidal in shape with less distinct cell boundaries. The rounded nuclei of acinar cells with their deeply stained nucleoli were situated near to the basal third of the cells (Fig. 10d). These serous gustatory glands present ducts that opened into the sulcus at various levels. The duct system was comprised of intralobular and interlobular ducts. The intercalated ducts extended to the periphery of the lobule to open directly into large interlobular ducts (Fig. 10e). The interlobular ducts were localized at the level of interlobular septae and were lined with simple to stratified squamous epithelium and they opened into the sulcus at the base of the circumvallate papillae (Fig. 10f). The serous acinar cells showed negative reaction to all histochemical staining techniques carried out during the present investigation indicating the absence of mucopolysaccharides, glycogen, mucin and protein.\u003c/p\u003e\n\u003cp\u003eThe micrometrical studies revealed that the mucous acini measured 59.40\u0026plusmn;1.80 \u0026mu;m in diameter and their luminal diameter was 15.84\u0026plusmn;1.61 \u0026mu;m (Table 1). However, the serous acini measured 28.38\u0026plusmn;1.68 \u0026mu;m in diameter and their luminal diameter was 5.94\u0026plusmn;1.23 (Table 1). The intercalated ducts of the mucous acini measured 18.48\u0026plusmn;0.80 \u0026mu;m in diameter while that of serous acini was 10.23\u0026plusmn;0.80 \u0026mu;m. The striated ducts of the mucous acini measuring 61.38\u0026plusmn;1.41 \u0026mu;m in diameter. The interlobular ducts of mucous acini measured 74.58\u0026plusmn;1.32 \u0026mu;m in diameter while that of serous acini measured 48.18\u0026plusmn;0.80 \u0026mu;m in diameter (Tables 2 and 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological and histochemical analysis of labial glands\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe labial glands of pig were distributed in the superior, inferior and commissural portions of the lips. The commissural labial glands were found distributed around the commissure of the lips on either side. The labial glands were placed under the skin rostral to the insertion of the zygomaticus muscle embedded in the orbicularis oris muscle. These glands extended between the superior labial artery and inferior labial vein along the angle of mouth. Histologically, the mucosa of the oral surface of lips is covered by stratified squamous non-keratinized epithelium. The superior labial glands were of compound tubuloacinar type and extending from the corner of the mouth to the length of the lips and situated in the propria-submucosa layer. The gland was surrounded by a thin layer of capsule having loose irregular connective tissue with varying distribution of collagen, reticular and elastic fibres. Blood vessels and nerve bundles were found distributed with in the capsule. The lobules showed the presence of secretory acinar units and ducts. The secretory units were predominantly mucous in nature with very few independent serous secretory units (Fig. 11a). Occasionally, in addition to the plasma cells, crescent-shaped serous cells known as serous demilunes, were also observed at periphery of the mucous secretory units (Fig. 11b). The interlobular connective tissue septae divided the parenchyma into lobes and lobules (Fig. 11c). The collagen fibres were also observed and surrounded blood vessels and ducts (Fig. 11d). Reticular fibres were also seen around the capsule and surrounding the acinar cells. Fine elastic fibres were observed in the blood vessels located in the interlobular septa (Fig. 11e). Mucous acini showed strong positive reaction to mucin whereas, serous acinar cells and ducts were negative reaction with Mayer mucicarmine method (Fig. 11f). The mucous acini measured 43.56\u0026plusmn;1.61 \u0026mu;m in diameter and their luminal diameter was 10.56\u0026plusmn;1.23 \u0026mu;m (Table 1). Myoepithelial cells were seen between the base of the mucous secretory cells and the surrounding basal lamina. The diameter of the serous acini measured 21.12\u0026plusmn;1.68 \u0026mu;m and their luminal diameter was 3.96\u0026plusmn;0.66 \u0026mu;m (Table 1). The histoarchitecture of labial glands of inferior lip as well as commissural part was similar to that of superior lip as described earlier. However, their population varies according to the regions as labial glands were most numerous in the superior lip as compared to that of inferior and commissural regions.\u003c/p\u003e\n\u003cp\u003eThe duct system of labial glands consisted of intralobular (Fig. 12a) and interlobular ducts (Fig. 12b). The intercalated ducts measured 13.20\u0026plusmn;1.04 \u0026mu;m in diameter, whereas diameter of striated ducts was 44.22\u0026plusmn;0.80 \u0026mu;m (Table 2). The large interlobular ducts measured 58.74\u0026plusmn;1.23 \u0026mu;m in diameter (Table 2). Goblet cells with bulging apical portion into the duct lumen were seen in the interlobular ducts (Fig. 12c). The lumen of these ducts contains the varying amount of secretions which were eosinophilic in nature. The concentration of collagen fibres increased surrounding the intralobular and interlobular ducts (Fig. 12d). The interlobular ducts drain the secretions into terminal excretory ducts where the epithelium changed to stratified squamous epithelium.\u003c/p\u003e\n\u003cp\u003eHistochemically, a strong Alcian blue reaction was observed in the mucous secretory cells indicating the presence of weakly acidic sulfated mucosubstances, sialomucins and hyaluronic acid in the secretions. However, the serous units and ducts revealed negative reaction to all these histochemical moieties (Fig. 12e). The mucous secretory cells were intensely PAS positive by McManus\u0026rsquo; method showing the presence of high concentration of glycogen in the secretions. The serous acinar cells and ducts were negative to glycogen (Fig. 12f). Combined PAS-AB method revealed strong positive reaction for acidic and neutral mucopolysaccharides in mucous cells. The serous acini and ducts showed negative reaction however, goblet cells showed mixed activity for neutral and acidic mucopolysaccharides with this stain (Fig. 13a). Colloidal iron method showed the moderate to strong positive reaction for acidic mucopolysaccharides in mucous acini however, the serous acini and ducts were showing negative reaction (Fig. 13b). However, the goblet cells of interlobular ducts showed positive reaction with Mayer mucicarmine method (Fig. 13c). Weak protein activity was observed in mucous cells, however, ducts showed negative reaction by mercury bromphenol blue method (Fig. 13d). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMicrometry was conducted for the acinar and luminal diameters of minor salivary glands of pig and the data was obtained. Also, the micrometry of ducts of mucous as well as serous type of glands was conducted and the data was obtained. ANOVA has been carried out for the above-mentioned parameters and the significant as well as non-significant differences were obtained both at 1% and 5% levels.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, the buccal glands were arranged in dorsal and ventral rows as reported in goat and sheep\u003csup\u003e15-16\u003c/sup\u003e. Contrary to the above findings, the buccal glands were arranged in dorsal, middle and ventral rows as reported in domestic ruminants\u003csup\u003e17-19\u003c/sup\u003e and horse\u003csup\u003e20\u003c/sup\u003e. The middle row which was found in cattle\u003csup\u003e21\u003c/sup\u003e and the Indian buffalo\u003csup\u003e22\u003c/sup\u003e was not observed in the present study. However, in horse the dorsal row was placed on the outer surface of the buccinator muscle near its upper border\u003csup\u003e23\u003c/sup\u003e. The buccal glands of goat were arranged in dorsal, middle and ventral rows\u003csup\u003e24\u003c/sup\u003e. The ducts of the dorsal and ventral buccal glands of bovines opened between papillae within the vestibule of the mouth\u003csup\u003e25\u003c/sup\u003e. The minor salivary glands appeared as clusters of serous and mucous aggregations in the propria submucosa between the intrinsic muscle bundles as reported in domestic animals\u003csup\u003e26\u003c/sup\u003e. These glands were seen outside the mucosa extending from the submucosa between the muscle fibers into the subcutis in domestic animals\u003csup\u003e17\u003c/sup\u003e. The buccal glands were of compound tubuloacinar type and contained mucous acini with serous demilunes as reported in human beings\u003csup\u003e27-30\u003c/sup\u003e. In the present investigation, the dorsal row of buccal glands were of mucous type. These findings were in parallel agreement with those reported in ruminants\u003csup\u003e17\u003c/sup\u003e and in Indian buffalo\u003csup\u003e22\u003c/sup\u003e. The parenchyma of dorsal buccal glands in buffalo was consisted of mainly mucous, a few serous and mixed type of acini with serous demilunes\u003csup\u003e31\u003c/sup\u003e. The dorsal buccal gland of sheep was of compound tubuloacinar type being surrounded by less distinct capsule. The gland was seromucous with predominance of mucous acini\u003csup\u003e32\u003c/sup\u003e. On the other hand, the middle buccal gland of sheep was consisted of mainly mucous with few serous types of acini\u003csup\u003e33\u003c/sup\u003e. However, the ventral buccal gland of sheep was comprised of seromucous secretory units with predominance of serous acini\u003csup\u003e34\u003c/sup\u003e. The buccal glands of dog were of tubuloacinar type and were located in the submucosal connective tissue\u003csup\u003e35\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe ventral row of buccal glands was of serous type in cattle and camel\u003csup\u003e36,37\u003c/sup\u003e which were in parallel agreement with those reported in the present study. The anterior part of the ventral row of buccal glands of Indian buffalo was purely mucous in nature whereas the posterior lobules were dorsally mixed and ventrally serous\u003csup\u003e22\u003c/sup\u003e. However, the ventral buccal gland of goat showed in anterior portion and upper part of the middle portion entirely the mucous acini while the lower part of the middle portion and the posterior portion the serous acini\u003csup\u003e38\u003c/sup\u003e. In goat, the dorsal and middle buccal glands were mainly mucous in nature with a very few serous and mixed acini, whereas ventral buccal gland was purely serous in nature\u003csup\u003e24\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the present study, the mucous secretory units were lined by pyramidal cells resting on a basement membrane. However, the acinar cells were of irregularly cuboidal in form as reported in human beings\u003csup\u003e39\u003c/sup\u003e. Myoepithelial cells were observed between the base of the cells and basement membrane as reported in rats\u003csup\u003e40\u003c/sup\u003e. The present investigation showed that the mucous acini opened into the intercalated ducts which in turn joined to form the intralobular ducts. This is in contrary to the findings where the mucous secretory units opened directly into intralobular ducts which were striated in nature\u003csup\u003e40\u003c/sup\u003e. However, in the present study basal striations were seen in the striated ducts. The intralobular ducts of sheep were lined by flat cells which showed the presence of goblet cells\u003csup\u003e41\u003c/sup\u003e. The serous acini were lined by low columnar cells resting on a basement membrane though described the lining epithelium as cuboidal in the ruminants\u003csup\u003e42\u003c/sup\u003e. In the ventral buccal glands of sheep and goats, the serous acini had a wide lumen\u003csup\u003e17\u003c/sup\u003e, whereas in the present study the serous acini had a narrow lumen. Intercalated ducts that drained the acinar cells were observed in the present study while, in the camel that the serous acini like mucous acini opened directly into the striated ducts or into the excretory ducts\u003csup\u003e37\u003c/sup\u003e. In mammals, except in cattle, the presence of myoepithelial cells was noticed which were not extended beyond the intercalated ducts\u003csup\u003e43\u003c/sup\u003e. Similar findings were reported in the intralobular ducts of the rat submandibular gland\u003csup\u003e44\u003c/sup\u003e, while the present study revealed the presence of myoepithelial cells extending upto the intralobular ducts.\u003c/p\u003e\n\u003cp\u003eHistochemically, the mucous acini showed PAS positive reaction indicating the presence of glycogen as described in the mucous secretory units of the salivary glands in mammals\u003csup\u003e45-50\u003c/sup\u003e. However, positive reaction was observed with AB indicating the presence of acidic mucopolysaccharides in mucous secretory cells. The present study also revealed the similar findings with these histochemical moieties. Presence of acidic mucopolysaccharides were also noticed in the mucous secretory units of the buccal glands of rats\u003csup\u003e51-52\u003c/sup\u003e. The mucous acini of dorsal row of buccal gland in buffalo contained weak amount of glycogen, acidic and neutral mucopolysaccharides\u003csup\u003e31\u003c/sup\u003e. In sheep, the mucous acinus of the dorsal buccal gland of sheep contained mucosubstances, glycogen, mucopolysaccharides and mucin\u003csup\u003e32\u003c/sup\u003e. However, the mucous acinus of middle buccal gland of sheep contained mucosubstances, glycogen, mucopolysaccharides, mucin and sudanophilic lipids while serous cells showed negative reaction\u003csup\u003e33\u003c/sup\u003e. The serous acini showed negative reaction to all the histochemical staining techniques carried out during the present investigation indicating the absence of mucopolysaccharides, glycogen, mucin and protein. However, in Japanese macaque, the presence of neutral, weak and strong acid mucopolysaccharides in the serous units of buccal glands\u003csup\u003e53\u003c/sup\u003e. While the presence of neutral, weak and acid mucopolyaccharides with varying intensities were observed in the serous acini of salivary glands in the humans\u003csup\u003e45\u003c/sup\u003e, horse\u003csup\u003e54\u003c/sup\u003e, buffalo\u003csup\u003e55\u003c/sup\u003e, dog\u003csup\u003e56\u003c/sup\u003e, opossum\u003csup\u003e57\u003c/sup\u003e and the Indian buffalo\u003csup\u003e58\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe present investigation revealed that the palatine glands were absent in the region of the hard palate. However, the smooth portion of the hard palate contained mucous glands in the propria submucosa as reported in the humans\u003csup\u003e59\u003c/sup\u003e and domestic animals\u003csup\u003e26\u003c/sup\u003e. However, some studies revealed the presence of seromucous glands in the human hard palate\u003csup\u003e30\u003c/sup\u003e. In camel, the palatine glands were located in the caudal part of the hard palate and in the entire length of the soft palate\u003csup\u003e60\u003c/sup\u003e. The palatine gland was most numerous at the apex of the soft palate. The palatine glands which were branched tubuloacinar type were observed with mucous secretory units in the submucosa scattered in between the muscle bundles on the oral side of the soft palate and the serous secretory units with a few scattered mucous acini in the propria submucosa on its nasal side as suggested in the domestic animals\u003csup\u003e17,26\u003c/sup\u003e though reported in humans the absence of any gland in the mucous membrane of the soft palate\u003csup\u003e61\u003c/sup\u003e. Some authors reported that the palatine glands were of mucous type in rats and mice\u003csup\u003e62\u003c/sup\u003e. However, they also claimed that they have observed the glands on the nasal side of the rat soft palate though they were not noticed in the rat soft palate\u003csup\u003e63\u003c/sup\u003e. The palatine glands of humans were histologically consisted of the gland tissue proper and supporting connective tissue frame work\u003csup\u003e30\u003c/sup\u003e. These glands were divided into lobules by connective tissue septa in which ducts and vessels were found. The soft palate in mesaticephalic breeds of dog was comprised of predominantly serous glands, and only few were mixed in nature with separate mucous and serous acini\u003csup\u003e64\u003c/sup\u003e. The rostral aspect of palatine glands of the Chukar partridge (\u003cem\u003eAlectoris chukar)\u0026nbsp;\u003c/em\u003ewas of simple branched tubuloacinar nature having both mucous and seromucous characteristics, while the caudal aspect included mucous type only with simple branched tubular glands\u003csup\u003e65\u003c/sup\u003e. The seromucoid glands were present in the rostral and caudal parts of the hard palate, as well as in the soft palate of domestic rabbits\u003csup\u003e66\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHistochemically, the mucous acini revealed the presence of neutral mucopolysaccharides in the current study. However, the presence of sulfated mucosubstances and minimal amount of non-sulfated mucosubstances was observed in the soft palate of the rat\u003csup\u003e63\u003c/sup\u003e. The periodic acid-Schiff (PAS) positive, alcianophilic, and metachromatic cells were present with a more homogeneous distribution pattern in the palatine glands of chicken\u003csup\u003e67\u003c/sup\u003e. Some studies revealed that the mucous glands were embedded in the submucosal layer of the soft palate in cat\u003csup\u003e68\u003c/sup\u003e. Histochemically, PAS-AB combined stain imparts magenta colour due to neutral secretion. These findings were in parallel agreement with those reported in this study.\u003c/p\u003e\n\u003cp\u003eThe lingual glands were of mixed type and were abundant under the circumvallate papillae as reported\u0026nbsp;in the present study. The lingual glands were seen in the propria-submucosa and among its muscle bundles of the tongue except in the tip region however, they were observed in the humans\u003csup\u003e39\u003c/sup\u003e even in the tip region and these findings were in agreement with the above authors in man and domestic animals\u003csup\u003e26\u003c/sup\u003e. Serous glands were seen in the lamina propria in the vicinity of circumvallate papillae, while the mucous acini were placed deeper to the serous glands among muscle bundles as reported in the domestic animals\u003csup\u003e17\u003c/sup\u003e. Serous glands were of compound tubuloacinar type while the mucous glands were of simple branched tubuloacinar type though in the domestic animals\u003csup\u003e26\u003c/sup\u003e both serous and mucous glands were of simple branched tubuloacinar type. In the ox that the lingual glands of the root were of mixed type\u003csup\u003e17\u003c/sup\u003e. But in the present study mucous glands with occasional serous demilunes were observed in the root region. However, some studies reported the presence of mucous acini in the posterior lingual glands of spiny tailed lizard\u003csup\u003e69\u003c/sup\u003e and also reported in the mongoose the presence of serous, mucous and mixed glands in the posterior part of the tongue\u003csup\u003e70\u003c/sup\u003e. The serous and mucous glands were found in the lamina propria of the foliate and circumvallate papillae of the tongue in horse and dog and only serous acini in the pig\u003csup\u003e19\u003c/sup\u003e. The lingual glands in Japanese monkey consists of both serous and mucous secretory units\u003csup\u003e71\u003c/sup\u003e. The posterior lingual glands in the gray short-tailed opossums were of tubuloacinar type, consisting of predominantly mucous secretory cells and seromucous acinar units\u003csup\u003e72\u003c/sup\u003e. However, the lingual salivary glands of the Persian squirrel were confined to the body and root, which were composed of serous cells located anteriorly and mucosal and seromucosal cells placed posteriorly\u003csup\u003e73\u003c/sup\u003e. These findings were in partial agreement with the results of present investigation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHistochemically, the serous acini showed negative reaction for PAS except the basement membrane. However, in mongoose, the serous acinar cells and occasionally duct cells contained granules which gave reactions for neutral mucosubstances\u003csup\u003e70\u003c/sup\u003e. The mucous acini indicated the presence of neutral mucopolysaccharides and the absence of glycogen and acid mucopolysaccharides in \u003cem\u003eColumba columba\u003c/em\u003e\u003csup\u003e74\u003c/sup\u003e. However, it was reported that the mucous cells and mixed cells contained acid and neutral mucins in the tongue of pagrosomus\u003csup\u003e53\u003c/sup\u003e. The secretion of the lingual glands contained neutral mucins, proteoglycans containing carboxylic acid, weak and strong sulphated groups, N-acetylated sialomucins, but lacked glycogen\u003csup\u003e75\u003c/sup\u003e. The secretory cells of the lingual salivary gland of the rock dove (\u003cem\u003ePatagioenas livia\u003c/em\u003e) showed strongly positive reaction to Periodic Acid Schiff\u0026apos;s stain\u003csup\u003e76\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe labial glands extended from the corner of the mouth to the length of the lips and located in the propria submucosa layer. The structure and arrangement of the labial glands were similar as reported in the ox\u003csup\u003e21\u003c/sup\u003e and ruminants\u003csup\u003e23\u003c/sup\u003e. The occurrence of labial glands was reported at the corner of the mouth in the sheep\u003csup\u003e41\u003c/sup\u003e. However, the labial glands of goat were grouped as commissural, superior and inferior glands\u003csup\u003e24\u003c/sup\u003e. In the present study, the labial glands were of mixed type as reported in the sheep\u003csup\u003e41\u003c/sup\u003e, humans\u003csup\u003e28\u003c/sup\u003e and ruminants\u003csup\u003e36\u003c/sup\u003e. Contrary to these findings, the labial glands were of mucous type a reported in the sheep, goat and carnivores\u003csup\u003e17\u003c/sup\u003e, however, in the camel, the labial glands were either serous or seromucous type\u003csup\u003e37\u003c/sup\u003e. The labial salivary glands of the one‐humped camel were of branched tubuloacinar type comprising of mucoserous acini capped by serous demilunes\u003csup\u003e69\u003c/sup\u003e. The labial glands of goat were mainly mucous in nature with a very few serous and mixed acini\u003csup\u003e24\u003c/sup\u003e. The labial glands of the camel were of compound tubuloacinar and mixed (predominantly mucous) type\u003csup\u003e77\u003c/sup\u003e. On the other hand, the labial salivary glands of dog were of compound tubuloacinar type having mixed and predominantly mucous acini with presence of serous demilunes\u003csup\u003e78\u003c/sup\u003e. The labial glands of mixed breed dog were of compound and mixed tubuloacinar nature having distribution of mucous acini, mucous acini with serous demilunes and isolated serous acini\u003csup\u003e79\u003c/sup\u003e. The labial glands of dog were of tubuloacinar type and were located in the submucosal connective tissue\u003csup\u003e35\u003c/sup\u003e. In the humans, the mucous secretory portions directly opened into the striated ducts contrary to the present findings in which the mucous units continued into the intercalated ducts and these ducts united and formed the striated ducts\u003csup\u003e39\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn the current study, histochemical studies revealed the presence of glycogen in the mucous cells\u0026nbsp;by PAS staining. However, in sheep, the mucous cells secreted neutral mucosubstances, sialomucins and hyaluronidase resistant sulfomucins\u003csup\u003e80\u003c/sup\u003e. The serous component was highly evocative for PAS and colloidal iron techniques\u003csup\u003e15\u003c/sup\u003e. Some of the findings stated that the mixed labial glands of the camel were rich in both neutral and acidic mucopolysaccharides\u003csup\u003e77\u003c/sup\u003e. However, the labial glands of sheep revealed that the secretory mucous cells contained acidic and neutral mucopolysaccharides while serous cells showed negative reaction\u003csup\u003e80\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIt may be concluded that the minor salivary glands of pig were located in the propria-submucosa layer of mucous membrane of oral cavity. The lobules showed the presence of secretory acinar units and the ducts. Depending on their location, the secretory acinar units were either mucous, serous or mixed in nature. These glands were of compound tubuloacinar type being surrounded by a capsule having a mixed distribution of collagen, reticular and few elastic fibres. Serous demilunes were also observed at periphery of the mucous secretory units. In addition to the plasma cells, myoepithelial cells were also found between the base of the secretory cells and the surrounding basal lamina. In addition to the secretory acini, the ducts were also observed in the lobules which consists of intralobular and interlobular ducts. Histochemical studies revealed the presence of weakly acidic sulfated mucosubstances, sialomucins, glycogen, mucopolysaccharides, mucin and protein in the mucous secretory units. However, the serous acini and ducts showed negative reaction to all these histochemical moieties. Micrometrical studies revealed that the acinar and luminal diameters of the mucous acini were larger than that of serous units. However, the diameter of the interlobular ducts, especially of lingual glands, was larger than intercalated and striated ducts which indicates that there was a significant difference at both 1% and 5% levels. Thus, understanding the anatomy, morphometry and histology of these glands is essential for a correct decision concerning medical and/or surgical intervention of such affections. This study will be helpful in field of surgery and also aid in diagnosis of pathological conditions of these glands and therefore this study establishes a basis for future research on this model.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar for providing all type of necessary facilities to carry out the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrateek Rao performed the experiment; Amandeep Singh conceived and designed, gathering information, drafting the manuscript, analysis and interpretation of data; Pawan Kumar supervising the study and Simran Kaur performed the statistical analysis of data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence and requests for materials should be addressed to A.S. or S.K.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eElewa, Y. 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Histochemistry of mucosubstances in the lingual glands of the mongoose. \u003cem\u003eActa Anat.\u003c/em\u003e \u003cstrong\u003e107\u003c/strong\u003e, 430-438.\u0026nbsp;(1980).\u003c/li\u003e\n \u003cli\u003eSuzuki, A. \u0026amp; T. Utsunomiya. A histological and histochemical study of posterior lingual gland in Japanese monkey (\u003cem\u003eMacaca fuscata\u003c/em\u003e). \u003cem\u003eInt. J. Oral Med. Sci.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 142-150.\u0026nbsp;(2010).\u003c/li\u003e\n \u003cli\u003eOkada, H., M. Suemitsu, T. Kanno, R. Tamamura, K. Kuyama, H. Murakami, T. Kato, \u0026amp; K. Suzuki. Morphological Features of the Posterior Lingual Glands in the Gray Short-Tailed Opossums (\u003cem\u003eMonodelphis domestica\u003c/em\u003e). \u003cem\u003eJ. Hard Tissue Biol\u003c/em\u003e. \u003cstrong\u003e22\u003c/strong\u003e, 489-492.\u0026nbsp;(2013).\u003c/li\u003e\n \u003cli\u003eJavad, S., T. Zahra \u0026amp; J. Faezeh. Anatomical and histological structure of the tongue and histochemical characteristics of the lingual salivary glands in the Persian squirrel. \u003cem\u003eAnat. Sci. Int.\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;93\u003c/strong\u003e, 58-68.\u0026nbsp;(2016).\u003c/li\u003e\n \u003cli\u003eNavas, P., Hidalgo \u0026amp; Buenoc. Histochemical analysis of the lingual glands in Columba Columba. \u003cem\u003eArch. Biol. Sci.\u003c/em\u003e \u003cstrong\u003e90\u003c/strong\u003e, 437-444.\u0026nbsp;(1980).\u003c/li\u003e\n \u003cli\u003eErdogan, S., H. Sagsoz, \u0026amp; M.E. Akbalik. Anatomical and histological structure of the tongue and histochemical characteristics of the lingual salivary glands in the Chukar partridge. \u003cem\u003eBr. Poult. Sci.\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e53\u003c/strong\u003e, 307-315.\u0026nbsp;(2012).\u003c/li\u003e\n \u003cli\u003eFatma, A. Al-Nefeiyl \u0026amp; Bedoor, A. Alahmary. Morphological, histological and histochemical studies of the lingual salivary glands of the rock dove, \u003cem\u003ePatagioenas livia\u003c/em\u003e (Columbidae). \u003cem\u003eInt. J. Curr. Res.\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 280-289.\u0026nbsp;(2015).\u003c/li\u003e\n \u003cli\u003eNabipour. Gross and histological study on the minor salivary glands of camel (\u003cem\u003eCamelus dromedarius\u003c/em\u003e). \u003cem\u003eJ. Camel Pract. Res.\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 123-129.\u0026nbsp;(2011).\u003c/li\u003e\n \u003cli\u003eGalvao, S. R., Queiroz, da Cunha D.N., R. J. De Carlo, A. P. B. Borges, L. D. A. Benjamin, C. C. Fonseca, L. Marcon, \u0026amp; C. E. R. Pereira. Morphological, morphometric and histochemical aspects of the labial salivary glands of dogs. \u003cem\u003eVet. Med.\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 3079-3086.\u0026nbsp;(2016).\u003c/li\u003e\n \u003cli\u003eMohamed, R. The labial and zygomatic salivary glands in mixed breed dogs in Trinidad: Anatomical location, histological features and histochemical characteristics. \u003cem\u003eWorld\u0026apos;s Vet. J.\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, 223-230.\u0026nbsp;(2020).\u003c/li\u003e\n \u003cli\u003eSingh A.D., R.K. Jain, P. Kumar, and K. Kapoor. Microscopic analysis of the labial salivary glands of the sheep (\u003cem\u003eOvis aries\u003c/em\u003e). \u003cem\u003eInt. J. Sci. Res\u003c/em\u003e. \u003cstrong\u003e3\u003c/strong\u003e, 536-538. (2014).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Histomorphological and histochemical stains employed on paraffin sections of \u0026nbsp;\u0026nbsp;minor salivary glands of pig.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"105%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eS.N\u003c/strong\u003e\u003cstrong\u003eo.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStain(s) (with Catalog\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003enumber and supplier)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;Reference(s)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; Purpose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHaematoxylin and Eosin\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;(230251, Sigma-Aldrich, India)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLuna (1968)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGeneral histomorphology\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCrossman’s Trichrome\u003c/p\u003e\n \u003cp\u003e(HT15, Sigma-Aldrich, India)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCrossman (1937)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCollagen fibres\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAlcian Blue\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;(at pH 2.5)\u003c/p\u003e\n \u003cp\u003e(05500, Sigma-Aldrich, India)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLuna (1968)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAcid mucopolysaccharides\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePeriodic Acid Schiff (PAS)\u003c/p\u003e\n \u003cp\u003e(TTR009, Sigma-Aldrich, India)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLuna (1968)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNeutral mucopolysaccharides\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePAS- Alcian Blue\u003c/p\u003e\n \u003cp\u003e(05500, TTR009 Sigma-Aldrich, India)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLuna (1968)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMucosubstances\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGomori’s stain\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(100380, Sigma-Aldrich, India)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLuna (1968)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eReticular fibers\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eWeigert’s stain\u003c/p\u003e\n \u003cp\u003e(TTR003, Sigma-Aldrich, India)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLuna (1968)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eElastic fibers\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHolme’s stain\u003c/p\u003e\n \u003cp\u003e(R0283, Sigma-Aldrich, India)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLuna (1968)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNerve cells and fibers\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e9.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMayer’s mucicarmine\u003c/p\u003e\n \u003cp\u003e(41325, Sigma-Aldrich, India)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLuna (1968)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMucin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e10.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eColloidal iron\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(85193, Sigma-Aldrich, India)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLuna (1968)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAcid mucopolysaccharides\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e11.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDiastase digestion\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(09962, Sigma-Aldrich, India)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLuna (1968)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGlycogen\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e12.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMercury bromphenol blue\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(B0126, Sigma-Aldrich, India)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePearse (1968)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eProteins\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Anatomy, Buccal, Histology, Labial, Lingual, Micrometry, Palatine, Porcine","lastPublishedDoi":"10.21203/rs.3.rs-7550163/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7550163/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The present work was undertaken to explore the anatomical distribution, histology, and histochemistry of minor salivary glands of pigs during their postnatal life. The heads of ten healthy adult pigs were procured from a local slaughter house immediately after decapitation. The fresh tissues were collected from minor salivary glands. Histologically, these glands were found in the propria-submucosa layer of the mucous membrane of the oral cavity. Depending on their location, the secretory acinar units were either mucous, serous, or mixed in nature. Serous demilunes were also observed at the periphery of the mucous secretory units. In addition to the plasma cells, myoepithelial cells were also found between the base of the secretory cells and the surrounding basal lamina. The duct system consists of intralobular and interlobular ducts. The intercalated ducts were characterized by the presence of small-sized lumens and lined with simple cuboidal epithelium. Striated ducts were lined with simple to stratified columnar epithelium having one to two cell layer thickness with characteristic longitudinal striations in the basal third. Large interlobular ducts were localized at the level of interlobular septae and were lined with stratified columnar epithelium and also showed the presence of goblet cells. Histochemical studies revealed the presence of weakly acidic sulfated mucosubstances, sialomucins, glycogen, mucopolysaccharides, mucin, and protein in the mucous secretory units. However, the serous acini and ducts showed a negative reaction to all these histochemical moieties. Micrometrical studies revealed that the acinar and luminal diameters of the mucous acini were larger than that of serous units. However, the diameter of the interlobular ducts was larger than that of the intercalated and striated ducts, which indicates that there was a significant difference both at the 1% and at the 5% levels. This study provided detailed insights into the anatomical and histological characteristics of minor salivary glands of pigs. Thus, understanding the anatomy, morphometry, and histology of the minor salivary glands is essential for a correct decision concerning medical and/or surgical intervention of such affections. This study will be helpful in the field of surgery and also aid in the diagnosis of pathological conditions of these glands, and therefore this study establishes a basis for future research on this model.","manuscriptTitle":"Comparative morphological analysis of minor salivary glands in porcine species using histological and histochemical evaluation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 14:08:35","doi":"10.21203/rs.3.rs-7550163/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-19T14:25:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-17T09:58:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"251426288400671225026534478458272295499","date":"2025-11-17T09:44:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-18T17:03:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-13T22:05:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178270371720999203205826248077214784479","date":"2025-09-18T09:22:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"202696491805270975058673198995611970108","date":"2025-09-17T03:21:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-16T06:38:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-16T06:20:46+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-15T14:44:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-11T15:18:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-11T15:10:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"14552091-b0fa-49fb-b90e-b362168a5216","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":54932232,"name":"Health sciences/Anatomy"},{"id":54932233,"name":"Biological sciences/Cell biology"},{"id":54932234,"name":"Health sciences/Diseases"},{"id":54932235,"name":"Health sciences/Medical research"},{"id":54932236,"name":"Biological sciences/Physiology"},{"id":54932237,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-03-13T04:53:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-25 14:08:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7550163","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7550163","identity":"rs-7550163","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}