Developmental morphological analyses on the preglottal salivary gland in Japanese quails (Coturnix japonica)

To understand the development of the mucous preglottal salivary gland in Coturnix japonica (Japanese quail), morphological and histochemical studies were performed on 20 healthy Japanese quail embryos (aging from 10th to 17th incubation days) and 25 healthy quail chicks (aging from 0th to 60th days). The primordia of preglottal salivary gland were observed as an epithelial bud at the early embryonic stage, which then elongated and differentiated into secretory units by the end of this stage. In Japanese quails, the preglottal salivary gland was a mucous polystomatic tubuloalveolar unpaired gland composed of two lateral portions and a middle one embedded into the submucosa of the lingual root. The gland openings accompanied taste pore (8.17 μm) of taste buds associated salivary glands type; some skeletal muscle fibers embedded among secretory lobules extended from muscle cricohyoideus at 14th day‐old quail chick. Also, both herbts corpuscles and secretory motor plexus could be detected among secretory lobules. Based on our investigations, the development of the preglottal salivary gland could clearly be distinguished in the embryonic stage into pre bud and bud stages at 10th day old, cord and branching stages ended by cavitation at 11th day old, canalization stage at 13th day old, lobulation and secretory stages by the 17th day old. The secretory materials showed different histochemical reactions ended with highly alcinophilic mucous indicated highly sialomucin (acidic) content. Myoepithelial cells could be demonstrated at a 17‐day old quail embryo and thereafter surrounded the secretory endpieces of the preglottal salivary gland.

which can facilitate feed moisten and lubrication (Bell & Freeman, 1971;Gargiulo, Lorvik, Ceccarelli, & Pedini, 1991;Nickel et al., 1977;Reece, 1996 ). In addition, the saliva had an antimicrobial effect particularly in the protection of the mucosal surface of the tongue against pathogenic micro-organisms. Furthermore, the saliva forms a sensation necessary hydrophilic environment on the tongue (Gargiulo et al., 1991).
Several investigations on the histology and histochemistry of the salivary glands of the birds have been made. However, the knowledge about the histogenesis and the mucins histochemistry of the salivary glands is not known during the pre-and posthatching period. The present investigation focused on the studying the histology and histochemistry of the preglottal salivary glands of the tongue in female and male Japanese quails during the pre-and posthatching period in addition to determine alcinophilic changes accompanied the secretion.

| Collection of specimens
This research was done in compliance with the ARRIVE guidelines (https://arriveguidelines.org). The Ethics Committee of Veterinary medicine, Assiut University, Egypt approved this study. The quails were obtained from a poultry farm, faculty of agriculture, Assiut University. A total of 45 healthy Japanese quails (Coturnixcoturnix japonica) were used starting from the sixth day prehatching till hatching day then at 7, 14, 30, and 60th day posthatching old. The quails were slaughtering according to local ethical board guidelines of the animal ethics committee of Veterinary medicine, Assiut University (20152016). After slaughtering, the heads were cut out and rinsed in running tap water and then by phosphate buffer saline to remove traces of blood after that both right and left commissures of the beak were incised. Finally, the oropharyngeal floor immersed in 10% neutral-buffered formalin for 24 hr. Three specimens from each age 7-and 14-days old quail chicks were fixed in 10% neutral buffered formalin and stained by alizarin red and alcian blue stains after maceration (KOH 1%) then photographed using stereomicroscope (LEICA S6D).

| Paraffine sections
For histological investigations three specimens of each age were used after proper fixation, the samples were kept in 10% formic acid/ formol saline for the process of decalcification then histological processing. The specimens are embedded in paraplast (Sigma Aldrich).
Serial 5-6 μm cross, longitudinal and frontal sections from the oropharyngeal floor was cut by a LEICA 2155rm automatic microtome. Some of these sections were routinely stained using: 1. Harris haematoxylin and eosin stain for general histological studies (Harris, 1900).
While the other sections were subjected to various histochemical techniques described below: 1. Periodic acid-Schiff (PAS) technique for detection of neutral mucopolysaccharides (Mc Manus, 1946).

| Semithin sections
Three specimens of 30 days old quail chicken from the preglottal part of the tongues were used for preparation of semithin sections (0.5 μm thickness) cut and stained with toluidine blue.
All stained techniques were adopted after (Bancroft & Gamble, 2002). The histological sections were examined by OLYM-PUS BX51 microscope and the photos were taken by OLYMPUS DP72 camera adapted into the microscope.

| Scanning electron microscope
For the scanning electron microscope (SEM) investigations of a 60-day old quail: Three samples from each age were used. The components of the floor of the oropharynx were washed several times in 0.1 M phosphate buffer at pH (7.2 ± 0.1). Post hatching samples rinsed with acetic acid 2%, then fixed in 4% glutaraldehyde solution for 24 hr.
Post fixation was made in 1% sodium tetroxide solution for 2 hr at 4 C. After that, the fixed samples were washed in 0.1 M phosphate buffer at pH = (7.2 ± 0.1), then dehydrated in ascending grades of ethanol followed by critical point-dried in liquid carbon dioxide. All specimens were mounted on aluminum stubs covered with carbon tabs, sputtered with gold. The prepared specimens were examined and photographed using JEOL SEM (JSM-5400) at an accelerating voltage of 15 kv in the electron microscope unit of Assiut University (Abou-Elhamd, Abd-Elkareem, & Zayed, 2018). The nomenclature used in the present study was coped with the (Nomina Anatomica Avium) as well as that was synonymized and homologized with names in previous and recent studies of the chicken and other avian species by different authors.

| RESULTS
The present study revealed that at 10 days prehatching the primordia of the preglottal salivary gland was observed as a thickening in the dorsal epithelium of tunica mucosa of the lingual root (epithelial placode; pre bud stage; Figure 1a). Then a solid bud-like cellular mass was formed (bud stage) (Figure 1a). By mitotic division, the epithelial bud was elongated and invading the underlining mesenchyme (submucosa) forming elongated solid epithelial cords (Cord stage) surrounded by concentric layers of mesenchymal cells (Figure 1b At 13 days prehatching, the epithelial cords of the salivary gland had undergone more branching and formation of the secretory endpieces. The epithelial cords and the secretory endpieces were surrounded by a concentric layer of mesenchymal cells (Figure 1f). In this age, the canalization began within the epithelial cords ( Figure 1f). The canalization process proceeded from the proximal part to the distal part where the secretory endpieces were located (canalization stage;  The cell size, shape, and nuclear arrangement were very homogeneous (cytodifferentiation; Figure 2d). At 14-, 30-, and 60-days old quails, the preglottal salivary gland was an unpaired gland located in the submucosa of the preglottal part of the tongue (radix linguae) extended longitudinally from the caudal process of the paraglossale (mid-length) to the apical end of the larynx. It consisted of two lateral portions, which were extended beyond the level of the caudal end of the glottis and one middle portion. The middle portion was ended at the level of the apical end of the glottis to be continued caudally as laryngeal salivary glands (Figures 3a-c and 5a). The gland had branched tubuloalveolar endpieces, whose cells were characterized by a light foamy cytoplasm and rounded nuclei.
The duct of the gland opened onto the dorsal surface of the preglottal part of the tongue with one or more than one opening. (Figures 3d,   4a,b, and 5b). The secretomotor plexus and herbst corpuscles were embedded between secretory lobules ( Figure 5d). The openings of salivary glands accompanied by pores of the taste buds associated salivary glands (Figures 4b and 5b,c). The preglottal salivary gland was supported by muscle cricohyoideus whose fibers could be demon-
Our results revealed that the pre-and posthatching development of the preglottal salivary gland is divided into the following stages: In salivary glands, branching morphogenesis and tubulogenesis is a multistep mechanism requiring coordinated cell proliferation,  to us (Gargiulo et al., 1991;Hodges, 1974;Menghi et al., 1993;Nalavade & Varute, 1977;Zaccone, 1977) do not define the preglottal or posterior lingual gland but they have only mentioned that the preglottal gland was unpaired as we did, also (Homberger & Meyers, 1989) do not agree with us. However (Liman et al., 2001) agree with us.
Again, in contrast to us, (Homberger & Meyers, 1989) do not declare the continuation of the preglottal gland as the laryngeal gland.
Also, Homberger and Meyers (1989)  taste pore's width is still constant that has been explained in the previous reports that have been described the size of taste pores as 5-10 μm (Gentle, 1971) or 6 μm (Saito, 1965) in width. Thus, these two openings are thought to correspond to the openings to the salivary glands and taste pores, respectively. Also, it is corresponding to (Kudo, Nishimura, & Tabata, 2008) who have assumed that the taste pore is 3-7 μm in width and is further observed in the apical surface of the taste bud. Moreover that (Li & Sugita, 2013) have found that the width of the opening of the salivary gland and taste bud in the large-billed Crow is more than 50 μm and less than 10 μm, respectively at the caudal lingual epithelium only. The present investigation indicates that the connective tissue capsule surrounding the entire gland as septa which envelopes each lobe, lobule, and acinus. (Calhoun, 1954) has reported the presence of muscle fibers in these septa of adult glands.
This in agreement with our present studies indicated that some muscle fibers inserted between lobular capsules of the preglottal gland and lymphoid tissues are present between septa also in older ages. However, (Mccallion & Aitken, 1953) do not find any muscle fibers or lymphoid tissues within the capsule.
Our results indicated that the gland was strong positive to PAS stain, while it was weak positive to AB stain at 0 day hatching old then start to be negative to PAS stain and strong to AB stain in 30-and 60-days posthatching old. The present results showed that the quails have well-developed lingual salivary glands even though it feeds on moist food. This finding agrees with the results of (Samar et al., 1995) who have described highly developed salivary glands in penguins.
Nevertheless, our results do not agree with the report of (Ziswiler & Farner, 1972) who found that the salivary glands of fish-eating birds are poorly developed. It was emphasized that all the salivary glands in the adult bird are of purely mucous type (Calhoun, 1954;Grossman, 1927;Schauder, 1923). It was reported that posterior salivary glands of the Common Myna are more strongly positive for neutral mucopolysaccharides than the anterior one whereas, both the glands are strongly positive for acid mucopolysaccharides (Kadhim, Hameed, & Abass, 2013). Similar results are reported in the red jungle fowl (Kadhim, Zuki, Babjee, Noordin, & Zamri-Saad, 2011) whereas, the lingual salivary glands of the little egret are considered free of neutral mucosubstance (Al-Mansour & Jarrar, 2004).
The nature of the neutral mucin of the lingual salivary glands may act as a lubricant for the food to facilitate swallowing. Besides, the mucin may also preserve hydration by providing a hydrophilic environment. In quail of all ages, the histochemical reactions showed that the cytoplasm of the secretory cells of the preglottal gland contained sialomucins, which were slightly variable according to the ages (Liman et al., 2001). Mucins have important roles in the maturation and maintenance of the ductal network in developing and mature human salivary glands. It also has many biological functions, such as protection of mucosal surfaces from adverse environmental influences, facilitation of glandular secretion, promotion and modulation of cell adhesion, and regulation of signaling (Teshima et al., 2011). In birds, saliva is produced by the minor salivary glands, which are mostly concentrated in the tongue. (Gabrielli & Tomassoni, 2014). In quails, the dorsum of the tongue coated with mucous fluid seems to be suitable for intaking various foods (Pourlis, 2014).
The present study revealed that myoepithelial cells could be demon- F I G U R E 1 2 Drawing diagram summarized the different pre-and posthatching developmental stages of the preglottal salivary gland. Note the myoepithelial cells (MC) They are associated with adenomeres of the most exocrine