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Title Page Developmental Morphological Developmental Morphological Analyses on the Preglottal Salivary Gland in Japanese Quails (Coturnix Japonica) Mahmoud Osman Khalifa Aswan University Mahmoud Abd-ElKareem ( [email protected] ) Assiut University Wafaa Gaber Assiut University TS Li Nagasaki University Abdelmohaimen M. Saleh Assiut University Research Article Keywords: Preglottal salivary gland, tubuloalveolar, myoepithelial cells, quails, sialomucin Posted Date: March 2nd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-255124/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Microscopy Research and Technique on August 3rd, 2021. See the published version at https://doi.org/10.1002/jemt.23892. Title Page Developmental Morphological Analyses on the Preglottal Salivary Gland in Japanese Quails (Coturnix Japonica) Mahmoud Osman Khalifa1,3, Mahmoud Abd-El Kareem2, Wafaa Gaber2, Li TS3 and Abdelmohaimen M. Saleh2 1Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Aswan University, Aswan, Egypt. 2Department of Anatomy, Histology, and Embryology, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt 3Department of Stem Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan Corresponding author: Mahmoud Abd-El Kareem* Department of Anatomy, Histology, and embryology, Faculty of Veterinary Medicine, Assiut University, Assiut 71526, Egypt [email protected] [email protected] Phone 00201094453945 1 Abstract 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 14thday-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. This mucous secretion 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. Keywords: Preglottal salivary gland, tubuloalveolar, myoepithelial cells, quails, sialomucin. 2 Introduction The salivary glands are characteristic features of the upper digestive tract of birds and these glands have been identified in numerous avian species 1 (Japanese quail); 2,3 (Avian); 4 (birds); 5 (chicken); 6 (The little egret); 7 (The White-tailed eagle); 8 (the black African ostrich) including domestic poultry 4,9–13. Salivary glands are well developed in birds, which consume a dry diet4. In some species, glandular tissue is absent while others display a seasonal change in gland size 2. The main functions of the salivary glands were to produce the saliva which can facilitate feed moisten and lubrication13–16 . 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 form a sensation necessary hydrophilic environment on the tongue 15. 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 post-hatching 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 post-hatching period in addition to determine alcinophilic changes accompanied the secretion. Materials and methods A-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 forty-five healthy Japanese quails (Coturnixcoturnix japonica) were used starting from the 6th-day pre-hatching till hatching day then at 7, 14, 30, and 60th day post-hatching 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 in10% neutral-buffered formalin for 24 h. Three specimens from each age 7- and 14-days old quail 3 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). B- 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 um cross, longitudinal and frontal sections from the oropharyngeal floor were 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 17. 2- Crossmon's triple technique 18 to study general structure. While the other sections were subjected to various histochemical techniques described below: 1- Periodic acid-Schiff (PAS) technique for detection of neutral mucopolysaccharides 19. 2- Alcian blue technique for demonstration of acidic mucopolysaccharides 20. 3- Combined Alcian blue-PAS technique 19. 4- Grimelius - Pascual's modified silver stain technique for demonstration of muscle fibers 21. C- Negative image analysis using CMEIAS color segmentation CMEIAS Color Segmentation is an Improved Computing Technology to Process Color Images for Quantitative Microbial Ecology Studies at Single-Cell Resolution 22. Negative image analysis was performed using CMEIAS color segmentation to assess the complex color micrographs that were obtained and give more details 23,24. D- 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 25. The histological sections were examined by OLYMPUS BX51 microscope and the photos were taken by OLYMPUS DP72 camera adapted into the microscope. 4 E- Scanning electron microscope (SEM): 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 0.1M phosphate buffer at pH (7.2±0.1). Post hatching samples rinsed with acetic acid 2%, then fixed in 4% glutaraldehyde solution for 24 hours. Post fixation was made in 1% sodium tetroxide solution for two hours 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 scanning electron microscopy (JSM-5400) at an accelerating voltage of 15kv in the electron microscope unit of Assiut University 26. 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 pre-hatching 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) (Fig. 1A). Then a solid bud-like cellular mass was formed (bud stage) (Fig. 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 (Figs. 1B & 1C). At 11 days pre-hatching, the initial epithelial cords of the gland had undergone branching and
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