Developmental Changes in Sublingual Salivary Gland of Indian Buffalo during Prenatal and Neonatal Life

The sublingual salivary gland contributes to the secretion of saliva which plays vital role in maintaining ruminants healthy by facilitating mastication and deglutition, helping in restoration of normal ruminal pH and microbial protein synthesis to be used as dietary proteins (Moghaddam et al., 2009). In general, the major salivary glands in herbivores are better developed than those of carnivores. The salivary glands may be classified on the basis of their secretions as serous, mucous or seromucous (mixed) glands. The distribution of these types varies from species to species (Konig and Liebich, 2009).
       
Saliva is secreted into oral cavity via a series of ducts in ductal system. The salivary glands also secrete IgA, potassium and sodium (Aspinall and Reilly, 2004). Dysfunction of salivary secretion (hyposalivation) causes xerostomia (dry mouth) and sequentially leads to severe dental caries as well as oral mucosal disorders (Featherstone, 2000). This precious role of sublingual salivary gland in digestion prompted to study the histogenesis which may be serving as a tool in future research on stem cell analysis of primordia of salivary gland.
       
Various studies have been done on this gland in buffalo (Venkatakrishnan and Mariappa, 1969; Singh and Singh, 2016), rat (Taga and Sesso, 2002; Uchida et al., 2013) and pangolin (Munyala et al., 2008), however, there is no detailed information about the histogenesis of sublingual salivary gland of buffalo during prenatal as well as neonatal life. Therefore, the present work was undertaken with an aim to highlight the ontogenetic events in histogenesis of sublingual salivary gland of foetal and neonatal buffalo.

This study was conducted after approval by the research committee and Institutional animal ethics committee.
 
Collection of the specimens
 
The present study was conducted on sublingual salivary gland of 36 buffalo foetuses, during different stages of prenatal development, as well as 6 neonates. Immediately after collection of samples from post-mortem hall, the foetuses were measured for their curved crown rump length (CVRL) in centimetres with a calibrated inelastic thread. The approximate age of foetuses was calculated by using the following formula in buffalo (Soliman, 1975).
  Where
Y= Age in day(s).
X= Curved crown rump length (CVRL) in cm(s).
       
Depending upon CVRL, foetuses were divided into three groups with a minimum of eight samples in each group: Group I (CVRL between 0-20 cm), group II (CVRL >20-40 cm) and group III (CVRL >40 cm). Group IV comprised of buffalo neonates and their age was estimated by dentition. Immediately after measuring CVRL, the foetuses as well as head of neonates were fixed in 10 per cent neutral buffered formalin (NBF) solution for 48-72 hours.
 
Processing, sectioning and staining of the specimens
 
After fixation in 10 per cent NBF, the tissue samples were processed for paraffin sectioning (5-6 µm thickness) and frozen sectioning (10-12 µm thickness) techniques. For paraffin sectioning, the dehydration of the fixed tissue was done in ascending grades of ethanol, keeping in each solution for one hour. It was done to remove water, as water was not miscible with paraffin. After this step, clearing was done by passing the tissues through two sets of benzene solution, 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= 58-62°C). 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 1-2°C above the melting point of wax used. Then embedding of the tissues were done, in which, the melted paraffin wax of the same melting point as used for infiltration, was pour 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 were then stained with various histomorphological and histochemical stains as mentioned in Table 1. Lipids and phospholipids were demonstrated on fresh cryostat sections. The cryo-sectioning was done with the help of cryo-microtome having temperature of -20°C to -23°C. In cryo-sectionig, fresh unfixed tissues of 10-12 µ thickness were obtained on clean glass slides, followed by mounting of slides with glycerol-jelly. These cryo-sections were then stained with various stains as mentioned in Table 1.


 
Statistical analysis
 
The diameter of mucous acini as well as serous acini, diameter of intercalated ducts, striated ducts and large excretory ducts of sublingual salivary gland of buffalo foetuses and neonates, was measured and the data was tabulated and statistically analysed as mentioned in Table 2.

The primordial anlage of monostomatic sublingual salivary gland was first noticed as a solid epithelial bud from oral epithelium at 39^th day of development. The epithelial bud was located just lateral to the mandibular gland and grew into underlying mesenchyme at 40th day of development. This concurred with the findings in domestic animals (Latshaw, 1987) and in human beings (Holsinger and Bui, 2007).
       
The polystomatic part of sublingual gland was formed from a series of 6-10 small independent epithelial invaginations from linguo-labial groove at 53^rd day of development. The glandular tissue was observed as a small mass without any lobulation in between tongue and mandible at 57^th day of development, which was in agreement with the reports in domestic animals (Latshaw, 1987; McGeady et al., 2006) and in human beings (Holsinger and Bui, 2007).
       
At 88^th day of development, most of the terminal buds were arranged in the form of clusters with undifferentiated epithelial cells. Lumen formation was observed first in the primary cords at this stage of gestation. The primary cords were surrounded with a layer of loose mesenchyme in the early stages of gland development.
       
Terminal buds and primary ducts were weakly positive for neutral mucopolysaccharides at this stage. Lumen formation was first evident in the terminal buds at 107^th day of development to form the terminal tubules (Fig 1). These terminal tubules were lined by double layered epithelium, which changed gradually to single layer (Fig 2). Condensation of embryonic mesenchyme was observed around the developing acini and ducts. At this stage, the intercalated and striated ducts were found and the lobulation of the gland also appeared first. A thin layer of collagen fibres and reticular fibres were seen around the lobes. The parenchyma of the gland was well developed. Similar findings were reported in prenatal period of the pig (Pospieszny et al., 2010) and mandibular salivary gland of prenatal buffalo (Singh and Singh, 2017).
 

 

       
The differentiation of stroma from embryonic mesenchyme started at 121^st day of development. The capsule formation around the gland was also initiated by the aggregation of mesenchymal tissue at this stage. The terminal tubules attained the structure of the acini. The lining epithelium of the primary ducts was two layered at 121^st day of development. There was a steep increase in the number of canalized ducts as age of the foetus increased. At this stage, interlobular ducts were also found.
       
The acinar cells were moderate positive for acidic mucopolysaccharides, however, intralobular ducts were devoid of these substances (Fig 3). Moderate to strong reaction for neutral mucopolysaccharides was observed in acinar cells and goblet cells (Fig 4).
 

 

       
Combined PAS-AB method revealed both acidic and neutral mucopolysaccharides in acinar cells whereas moderate Alcianophilic reaction was observed in connective tissue (Fig 5). Mucinous substances were found in acinar cells; however, these were not localized in ducts.
 

       
The acini were predominantly mucous in the age groups from 138^th day of development onwards. The cells of mucous acini were pyramidal in shape with spherical or flattened nuclei against the base (Fig 6 and 7). Flattened myoepithelial cells were evident between the acinar cells and basement membrane and were confined to the acini and intercalated ducts. Similar finding were reported in the sublingual gland of humans (Attie and Sciubba, 1981) and rat (Wolff et al., 2002) during late foetal period. Fine collagen fibres and reticular fibres (Fig 8) were also seen at this stage of gestation. Well-developed connective tissue septa was observed in the interlobular space around the acini and ducts from 150^th day of development onwards. The lining epithelium of the intercalated ducts was double layered cuboidal type at 150^th day of development and changed gradually to single layer at 155^th day. The striated ducts were lined by double layered epithelium with inner cuboidal and outer cuboidal or flattened cells. These findings were in fully agreement with the findings in mandibular salivary gland of prenatal buffalo (Singh and Singh, 2017).
 

 

 

       
The mucous cells were moderate to strong positive for sulphomucins while these were not localized in ducts (Fig 9). Moderate to strong reaction for acidic mucopolysaccharides (Fig 10) and neutral mucopolysaccharides (Fig 11) was seen in mucous acinar cells; however, ducts were devoid of these carbohydrates. Mucous acinar cells also showed moderate to strong reaction for mucinous substances (Fig 12). Combined PAS-AB method revealed both acidic and neutral mucopolysaccharides in goblet cells and mucous acinar cells while strong Alcianophilic reaction was observed in connective tissue (Fig 13). The phospholipids were localized in the cell membrane of secretory cells and large ducts of the gland (Fig 14).
 

 

 

 



 

 
       
The mean diameter of serous acini, mucous acini, intercalated duct, striated duct and large duct of sublingual gland of group II foetuses was 1.04±0.1 µm, 1.30±0.1 µm, 0.40±0.05 µm, 1.10±0.05 µm and 2.57±0.5 µm, respectively (Table 2).
       
The monostomatic and polystomatic parts of the gland were clearly distinguishable by connective tissue septae made up of collagen fibres at 170^th day of development (Fig 15). The monostomatic part of sublingual gland exhibited strong positive reaction for neutral mucopolysaccharides in mucous acinar cells, however, polystomatic part showed moderate positive reaction in secretory cells (Fig 16). Combined PAS-AB method revealed strong mixed reaction for acidic and neutral mucopolysaccharides in monostomatic part than polystomatic part (Fig 17). Mucous secretory acini of monostomatic part showed strong positive reaction for mucinous substances while moderate positive reaction was seen in polystomatic part (Fig 18).
 

 

 

 

       
Interlobular ducts lined with double layered epithelium surrounded by well-developed connective tissue were evident in the interlobular space at 185^th day of development (Fig 19). Infiltration of lymphocytes was also noticed with the advancement of age of foetus (Fig 20). The connective tissue showed distinct collagen fibres, reticular fibres as well as elastic fibres (Fig 21) from 194^th day onwards.
 

 

 

 
Mucous cells were strong positive for sulphomucins. Mucous acinar cells as well as goblet cells were strong to intense positive for acidic (Fig 22) as well as neutral mucopolysaccharides (Fig 23). Mucous cells also showed strong positive reaction to mucinous substances. Moderate positive protein activity was observed in interlobular septa and striated ducts. Fine sudanophilic lipid droplets were observed in the intralobular as well as interlobular connective tissue. Moderate amount of phospholipids were observed in the cell membrane of secretory cells and ducts. In group III foetuses, the mean diameter of serous acini, mucous acini, intercalated duct, striated duct and large excretory duct was 1.30±0.1 µm, 2.83±0.1 µm, 0.89±0.05 µm, 1.88±0.05 µm and 3.81±0.6 µm, respectively (Table 2).
 

 

       
In neonatal buffalo, the sublingual salivary gland was of compound tubulo-acinar type with a well-defined capsule. The gland was surrounded by a thick connective tissue capsule made of dense collagen fibres along with few elastic and reticular fibres, which was in agreement with the reports in yak (Sudhakar, 2006).
       
The mean diameter of serous acini, mucous acini, intercalated duct, striated duct and large excretory duct of day-old buffalo was 1.53+0.1 µm, 3.51+0.1 µm, 1.94+0.4 µm, 2.71+0.1 µm and 4.95+0.2 µm, respectively (Table 2).
       
The connective tissue traversed the gland to form septae and separated the glandular parenchyma into lobes and lobules. Large plexus of ganglion cells and blood vessels were also noticed in the capsule. The parenchyma of the sublingual gland comprised of mixed acini, predominantly mucous acini and few serous demilunes along with several orders of ducts distributed in the stroma (Fig 24). These findings were in total agreement as reported in buffalo calves (Venkatakrishnan and Mariappa, 1969) and in birds (Fujii and Tamura, 1966). The mucous acini composed of five to six pyramidal cells enclosing a narrow indistinct lumen. The nuclei were flattened and located in the basal part.
 

       
Stellate shaped myoepithelial cells were scattered around the basement membrane of mucous acini as well as the intercalated and striated ducts of the gland. These were dark basophilic in nature. The myoepithelial cells lining the duct epithelium were spindle shaped with few cytoplasmic processes. Similar finding were reported in chick (Shi and Gibson, 1977).
       
The duct system of the sublingual gland was comprised of intercalated duct, striated duct, interlobular duct and large excretory duct. The ducts of various orders predominated the secretory parenchyma in neonatal age.
       
The intercalated ducts, with defined morphology, were short with little cytoplasm delimiting a very small lumen. The striated ducts, with morphology close to that of the adult animal, were seen with a wide lumen and consisted of prismatic cells having characteristic longitudinal striations in the basal third and central spherical nuclei (Fig 25). The striated ducts were lined by simple columnar epithelium. 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.


       
Several interlobular ducts opened into large excretory duct. The larger ducts situated in the stromal tissue within the lobule and in between the lobes were lined by pseudostratified columnar epithelium and showed basal cells and also few goblet cells in between the columnar cells. These findings were in total agreement as reported in amphibian (Zylberberg, 1977).
       
In neonatal buffalo, mucous acinar cells were intense positive for acidic mucopolysaccharides (Fig 26) as well as neutral mucopolysaccharides (Fig 27); however, serous cells were devoid of these carbohydrates. A strong positive reaction for acidic mucopolysaccharides was observed in mucous acini by colloidal iron method; however, ducts were devoid of these carbohydrates (Fig 28). Mucous secretory acini showed intense positive reaction for mucinous substances (Fig 29), which was in agreement with the reports in domestic fowl (Arthitvong et al. 1999).
 

 

 

 

       
Fine granular sudanophilic lipids were observed in acinar cells. Fine lipid droplets were localized at luminal and basal positions in the epithelium of striated and large ducts. Strong positive activity was observed for phospholipids in acinar cells. Fat cells were more widely scattered in neonatal age group when compared to prenatal groups.
       
With advancement of age, the lobules were larger and showed a marked increase in the number of acinar cells and a reduction in intralobular connective tissue. Increase in the acinar cells completely filled the parenchyma.
       
In neonatal buffalo, the mean diameter of serous acini, mucous acini, intercalated duct, striated duct and large excretory duct was 2.55+0.1 µm, 4.01+0.2 µm, 3.17+0.1 µm, 4.76+0.3 µm and 7.83+0.1 µm, respectively (Table 2). The mean values of micrometrical parameters varied significantly between groups at p<0.05 and p<0.01 level.

On the basis of above observations, it may be concluded that the primordial anlage of monostomatic part of sublingual gland were seen first to that of polystomatic part. The primary ducts were first observed at 80^th day of development. The lobulation of the gland started at 107^th day, whereas the capsule formation was initiated at 121^st day of development. The duct system was also completed at 121^st day. At 121^st day of development, the terminal tubules attained the structure of the acini. The typical compound tubulo-acinar nature of the gland was first observed at 122^nd day. The sublingual gland showed predominantly mucous type of cells from 137^th day of development onwards. The myoepithelial cells were first appeared as flattened basal cells initially around the developing acinar cells at 138^th day of development. The two parts of the sublingual gland were clearly distinguishable by connective tissue at 170^th day of development of prenatal life. In neonatal age groups, the sublingual gland was of compound tubulo-acinar nature with a well-defined capsule. Localization of acidic as well as neutral mucopolysaccharides was observed in mucous cells and goblet cells of sublingual gland in prenatal and neonatal age groups. Phospholipids were observed in the cell membrane of secretory cells and ducts of sublingual gland.