Morphological studies on the prehatching development of the glandular stomach of Japanese quails using light, electron, and fluorescent microscopy

Gaber, Wafaa; Mostafa, Heba; Abdel-Rahman, Yousria A.; Abd El-Hafeez, Hanan H.

doi:10.1038/s41598-023-45355-1

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Published: 23 October 2023

Morphological studies on the prehatching development of the glandular stomach of Japanese quails using light, electron, and fluorescent microscopy

Scientific Reports volume 13, Article number: 18096 (2023) Cite this article

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Abstract

The development of the glandular stomach was studied using light, electron, and fluorescent microscopy. The research used 130 Japanese quail eggs from the second to the seventeenth days of incubation.The proventriculus could be distinguished on the3rd day. Its wall consisted of four tunics: tunica mucosa, very thin tunica submucosa, tunica muscularis, and outermost tunica serosa. Mucosal folds appeared on the 8th day. The luminal epithelium was pseudostratified columnar in type and transformed into simple columnar by the 10th day. The mucosal papillae emerged on the 11th day, spiraled on the 15th day, and had a distinct whorled look by the 17th day. Two types of proventricular glands were recognized: compound tubuloalveolar and simple tubular glands. Both types were situated within the tunica mucosa. On the 4th day, the compound glands emerged as evaginations of the lining epithelium. It began to branch on the 8th day and became well established by the 11th day. The simple glands appeared on the 11th day as localized down-growths of the luminal epithelium forming solid cords. On the 15th day, many of them showed complete canalization. On the 8th day, the muscular coat was differentiated into the lamina muscularis mucosae and tunica muscularis.

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Introduction

In developmental research, various animal models, including avian species, are currently employed. Among avian species, the chick embryo serves as a paradigm for developmental biologists. The Japanese quail (Coturnix japonica) was recently introduced as a popular model for embryological research¹. Several aspects account for the utility of this bird. First of all, it has become important to the economy as a species that can be farmed and makes tasty eggs and meat². Second, it is a great lab animal because it doesn’t need much care, is small (80–300 g), has a fast rate of reproduction (three to four babies a year), and is resistant to diseases^3,4,5,6. Its short incubation period and easy access to its egg makes it a great model for developmental biology. Its early maturity (it can reproduce at 6 weeks old) make it useful for studying ageing and disease^{7,8,9,10,11,12}.

The avian stomach is composed of two basic morphologically and physiologically distinct regions: a glandular portion, or proventriculus, and a muscular portion, or gizzard. Both parts are characterized by great morphological and functional variability, both between and within species. It is mostly influenced by accessibility, with the amount and kind of food changing over time^13,14. The proventriculus is a fusiform structure found in most birds between the esophagus and the gizzard. Its size varies in different kinds of birds, where it appears small in graminivorous birds and huge and distensible in carnivorous birds¹⁵. The proventriculus' major purpose is to produce hydrochloric acid and pepsinogen into the digestive compartments, which will churn the ingested materials via muscular mechanisms^13,14,16.

The quail stomach consists of two basic compartments having the same morphology and function as those of other birds¹⁷. Many text books^{18,19,20,21,22,23}, as well as numerous publications^{24,25,26,27,28,29,30,31}, have detailed the histomorphology and development of the glandular stomach of diverse avian species. The histomorphology and development of the avian digestive system has been described in a number of published works^{32,33,34,35,36,37,38,39,40,41,42}.

Except for Attia’s⁴³ and Soliman et al.³⁰ studies on the histogenesis of the stomach of pre-hatching quails, there has been little developmental research on the glandular stomach of quails. However, these researchers noted differences in some ages. Therefore, the purpose of this study is to investigate and provide detailed information about the gross, histological, and histochemical changes of the proventriculus at different stages of development during the pre-hatching period of Japanese quails, as well as the proventriculus’ fine structure, using a, using a light, electron (scanning and transmission), and fluorescent microscope.

Results

Two-days embryos

The primordium of the stomach could be observed as the most dilated part of the gut, dorsal, and cranial to the liver primordium and ventral to the level of the notochord. It appeared in the form of a spindle-shaped hollow organ with a strongly convex dorsal border and slightly concave ventral border. It continued cranially with the esophagus and caudally with the duodenum (Fig. 1A). Moreover, it was suspended by the dorsal mesentery and the dorsal section of the ventral mesentery extended to the liver primordium. The stomach was lined with a layer of thin endodermal epithelium (Fig. 1D) and a thick mesenchymal layer on the outside. (Fig. 1D).lining and an exterior thick mesenchymal layer. The epithelium appeared to be pseudostratified columnar, with vacuolated basophilic cytoplasm and a vesicular nucleus. The mesenchymal layer was thin cranially (prospective section of the glandular stomach) and thick caudally dorsally (prospective part of muscular stomach). This layer was made up of two types of cells: mesenchymal cells in the shape of stars and telocytes. The nucleus of the star-shaped cells was big and vesicular. Telocytes were spindly-shaped cells with a big oval nucleus and lengthy processes that connected to mesenchymal cells. The mesenchymal layer's outermost layer was darkly stained and consisted of stratified columnar epithelium. This layer represented the tunica serosa’s future mesothelium. Because the mesenchymal part of the wall was greater dorsally, the wall of the stomach primordium was thicker dorsally than ventrally. There were no materials in the lumen (Fig. 1B,C).

Three-day embryo

The stomach could be divided into two distinct parts: a small cranial portion (potential glandular stomach) and a large caudal portion (potential muscular stomach) (Fig. 2A). The pseudostratified columnar epithelium of the glandular stomach contained an ovoid vesicular nucleus. The various phases of mitotic divisions were visible (Fig. 2B). This epithelium was supported by a non-uniform basement membrane. Some mesenchymal cells formed clusters beneath the epithelium. The tunica serosa mesothelium consisted of a single layer of flattened or cuboidal cells with ovoid or rounded nuclei (Fig. 2C).

Four-day embryo

Evaginations of the lining epithelium marked the appearance of the compound proventricular glands' primordium. The epithelial cells showed shortening and were arranged radially at the base of these evaginations, beneath which the basement membrane evidently slightly protruded into the mesenchyme. Near the middle of the mesenchymal layer, the condensation of the mesenchyme was clearly observed wherein some mesenchymal cells differentiated into myoblast cells forming the prospective smooth muscle layer. Furthermore, the mesothelium became wholly flattened (Fig. 3A–C).

The glandular stomach’s long axis (proventriculus) extended craniocaudally from the median plane to the left side. It was placed just left of the median plane on the cranium, and it was connected medially to the right lobe of the liver, laterally to the left lung, dorsally to the aorta, and ventrally to the heart. Caudally, it was linked ventromedially to the liver, dorsally to the left mesonephros, and laterally to the left body wall. The proventriculus seemed flattened dorsoventrally in the transverse section. The lumen was slit-like and free of materials along its length (Fig. 3D,E).

Five-day embryo

The gland primordium increased in number, spreading throughout most of the mucosa. The special arrangement of the glandular epithelium became clearer and the gland primordium projected more deeply into the underlying mesenchyme. The mesenchymal cells consolidated and flattened under the epithelium of the gland rudiments. Vascular components were found within the mesenchymal layer. Mitotic patterns were found in both the glandular epithelium and the mesenchymal cells (Fig. 4A–D).The cranial part of the glandular stomach became separated laterally from the left body wall by the left lung and left lobe of the liver. Caudally, it was linked to the spleen as well as the liver, and it was linked dorsally to the well-developed mesonephros and ventrally to the liver, giving a deeper gastric impression. The dorsal mesentery extended obliquely from the roof of the body cavity ventrally and to the left to be attached to the dorsal aspect of the glandular stomach. It increased in length caudal wards and extended vertically and then transversely. Its caudal attachment with the proventriculus was interrupted by the spleen forming the gastrosplenic ligament (Fig. 4E,F).

Six-day embryo

The proventricular glands showed varying degrees of development. Some of them became more developed, protruding deeply into the underlying mesenchyme. They took the simple alveolar profile consisting of a spherical tip and a stalk representing the future primary duct. The gland epithelium was pseudostratified columnar in some regions and simple columnar in others. Most glandular epithelium’s apical section became elongated, and thin, and protruded into the lumen beyond the point of cell connections, giving the epithelium a serrated look. (Fig. 5A,B).

Scanning electron microscopy showed that the proventricular epithelial cells had a polygonal form on their surface. On the surface of the lumen, the apertures of the gland rudiments were seen as hollows of different sizes and shapes (Fig. 5C). Telocytes with several processes were observed under the epithelium (Fig. 5D).

Seven-day embryo

The glands elongated without branching. The elongation of the apical portion of the glandular epithelium became evident forming profuse finger-like projections bulged into the lumen and some of them sloughed off. These projections were observed on the apical portion of the luminal epithelium, but they were fewer and smaller. Semithin sections showed that granules stained with toluidine blue were at the end of the luminal and glandular epithelium as well as the projections. Moreover, pale oval cells with rounded nuclei were observed in between the luminal and glandular epithelium. Some of them possessed a narrow elongated cytoplasmic process pointing toward the luminal surface and a long axis perpendicular to thebasement membrane. Moreover, some cells showed mitotic figures. These cells were suggested to be the open-type endocrine cells with luminal contact (Figs. 6A,B and 7A–C).

The luminal epithelium was pseudostratified columnar in type, with rounded or oval nuclei having 1–3 nucleoli, as revealed by transmission electron microscopy pictures. Numerous electron-dense granules of various sizes and shapes were seen in the luminal and glandular epithelium, as well as the projections. Open-type endocrine cells were detected in the glandular epithelium. They showed an electron-lucent cytoplasm with supranuclear mitochondria, rough endoplasmic reticulum, and Golgi apparatus. Few pleomorphic electron-dense granules were observed in a basal location. The nucleus was rounded with a prominent nucleolus, distinct nuclear membrane, and marked nuclear pores (Fig. 7D,E).

The glandular stomach was positioned in the left dorsal quadrant of the body cavity. It became separated from the left body wall cranially (by the left lung) as well as from the left cranial thoracic air sac caudally (by the left abdominal air sac). Caudally, it was related dorsally to the gonad along with the mesonephros (Fig. 8A,B). Along its longitudinal axis, the glandular stomach was spindle-shaped in outline with its broad end located caudally and the narrow end located cranially. A slight constriction could be observed cranially demarcating it from the esophagus. Caudally, a slight indentation could be detected between the glandular stomach and the muscular stomach ventrally, whereas dorsally, they continued without any line of demarcation (Fig. 8C).

Eight-day embryo

The proventricular mucosal surface showed many small folds (plicae) with intervening depressions (sulci). The proventricular glands began to be branched. Most of the glandular epithelium became simple columnar in type, whereas the pseudostratified columnar epithelium was observed in small areas. The apical projections became increasingly apparent, and the lumina of the glands revealed a significant amount of cellular debris containing nuclei. Open-type endocrine cells were observed in between the luminal and glandular epithelium. Some of these cells had narrow elongated cytoplasmic process toward the basement membrane (Fig. 9A,B). Telocytes with long processes (telopodes) were observed in the mesenchyme around the glands, muscles, and blood vessels (Fig. 9C,D).

The muscular coat was differentiated into lamina muscularis mucosae and tunica muscularis. The lamina muscularis mucosae consisted of a thin discontinuous inner layer and continuous outer layer with few muscle fibers extending between the glands. The tunica muscularis had two layers as well: a thick inner circular layer and a thin discontinuous outer longitudinal layer. The tunica submucosa, which lay between the outer layer of muscularis mucosae and the inner layer of tunica muscularis, was highly thin. (Fig. 10A,B).

The scanning electron microscopic images confirmed the light microscopic results wherein most of the glandular epithelium became simple columnar in type with very prominent finger-like projections on its apical portion (Fig. 10C). A network of telocytes was observed under the luminal epithelium. The telocytes were connected with each other and with the surrounding structures. The telopodes consisted of alternating thin segments (podomeres) and dilations (podoms), giving it a beading appearance (Fig. 10D,E).

Nine-day embryo

Branching of the proventricular glands increased (Fig. 11A). Sloughing of the apical portion of the glandular epithelium became greater, and some of the epithelial cells lining the glands as well as the ducts transformed into low columnar cells (Fig. 11B). Ultra-structurally, the low columnar cells lining the glands showed some short microvillus-like structures on their apical border. The cytoplasm contained numerous mitochondria mainly at the apical portion of the cell and abundant rough endoplasmic reticulum concentrated at its basal portion. A large number of free ribosomes are distributed throughout the cytoplasm. Some cells' apical portions protruded into the lumen to be sloughed off. The centrally located nuclei were rounded or oval in shape with evenly distributed chromatin and distinct nucleolus. Moreover, the nuclear membrane was distinct with marked nuclear pores (Fig. 11C–F).

A slight indentation appeared between the glandular stomach and the muscular stomach dorsally, whereas the ventral indentation became deeper forming the isthmus (Fig. 12A).

Ten-day embryo

The primary duct of the proventricular gland is divided into two secondary ducts connecting it with the secretory units (Fig. 12B). The muscularis mucosae of the esophagus are divided into inner and outer layers at the esophago-proventricular junction, which continued with the inner and outer layers of the muscularis mucosae of the proventriculus. The outer layer was thick, whereas the inner layer appeared thin and discontinuous, enclosing the compound proventricular glands in between (Fig. 12C).

Eleven-day embryo

The mucosal folds became more developed and the adjacent folds approached each other and connected to surround the openings of the proventricular glands forming the mucosal papillae (Fig. 13A–D).

The primordium of the simple tubular glands could be detected as localized down-growths of the luminal epithelial cells into the lamina propria forming solid cords (Fig. 14A).

The proventricular glands established themselves as compound tubuloalveolar glands within the lamina propria, constituting the thickest portion of the proventricular wall. These glands consisted of numerous round or polymorphic lobules arranged in small groups and separated by septa of connective tissue. The septa were composed mainly of collagenous fibers. The secretory units connected to the secondary ducts by tertiary ducts, which were lined by simple cuboidal epithelium (Fig. 14B,C).

Cranially, the relations of the glandular stomach were similar to the previous stages, but caudally, it was separated from the mesonephros and gonad by the dorsally extended left abdominal air sac (Fig. 15A,B). The dorsal indentation between the glandular stomach and the muscular stomach became deeper and the isthmus became well-defined (Fig. 15C).

Twelve-day embryo

The compound tubuloalveolar glands became more branched and most of their lining epithelium was of low columnar type. The connective tissue septa separating the gland lobules became thinner (Figs. 16A,B).

The simple tubular glands increased in number and very few of them began to be canalized (Fig. 16C).

Thirteen-day embryo

Some simple tubular glands became more canalized (Fig. 16D). The inner and outer layers of the muscularis mucosae re-joined prior to the isthmus (Fig. 16E,F). The mucosal folds became longer, lined with simple columnar epithelium, and had a connective tissue core of lamina propria (Fig. 16G). Closed-type endocrine cells were observed in the luminal epithelium. These cells had no luminal contact. They were pale, with rounded nuclei, and possess short bipolar cytoplasmic processes (Fig. 16H). The lining epithelium of the compound glands’ secretory units was composed of low columnar cells with few cuboidal cells interspersed (Fig. 16I).

Fifteen-day embryo

The tip of the mucosal fold was lined with simple columnar epithelium, which transformed into simple cuboidal toward the base of the fold. In some areas, additional folds and sulci were arranged concentrically around the glandular duct opening, giving the mucosal papilla a spiral appearance (Fig. 17A–C).Many of the simple tubular glands showed complete canalization, lined with simple cuboidal epithelium and opened into the base of the sulci between the mucosal folds. They were located in the lamina propria, superficial to the discontinuous inner layer of the muscularis mucosae (Fig. 17D,E).

The secretory units of the compound glands became lined solely with simple cuboidal epithelium (oxyntico-peptic cells), and the apical portion of the cells appeared regular. The lumina of the secretory units were free from cellular debris (Fig. 17F).

Seventeen-day embryo

At this age, the four tunics constituting the wall of the proventriculus became well established as tunica mucosa, very thin tunica submucosa, tunica muscularis, and outer most tunica serosa. The two types of proventricular glands situated within the tunica mucosa.

The light and scanning electron microscopic results revealed that more folds were added to the concentric plical structure of the mucosal papilla, giving it a definite whorled appearance (Fig. 18A,B). The plicae became much higher than that of the previous age and the sulci became much deeper. The tip of the fold was lined with simple columnar epithelium, which decreased in height to be transformed into simple cuboidal toward the base of the fold. The folds’ connective tissue core was densely packed with smooth muscle fibers and telocytes (Fig. 18C,D). The lining epithelial cells' luminal surface was polygonal in form, with numerous short microvillus-like features. (Fig. 18E).

Most of the wall’s thickness is now made up of the branched compound proventricular glands. The gland lobules were close to each other and were only divided by a very thin layer of smooth muscle fiber-filled connective tissue that wrapped around the secretory units. (Fig. 19A,B). Moreover, at this age, some of the epithelial cells lining the secretory units made contact with the adjacent cells only at their basal portions, giving the lining epithelium a dentate appearance while the apical portion of other cells remained regular (Fig. 19C). The glandular epithelium contained closed-type endocrine cells. (Fig. 19D).

Telocytes with long telopodes showing a beaded appearance were observed between the muscle fibers (Fig. 19E,F).

Histochemical investigations

Neutral and acid mucopolysaccharides

The epithelium lining the lumen of the proventriculus as well as the gland primordium showed strong PAS and moderate Alcian blue positive reactions on the 6th and 7th day of incubation (Fig. 20A,B,E,F). On the 8th day, strong positive reactions for PAS and Alcian blue were observed in the luminal content, apical border of the luminal epithelium, and along the primary ducts of the glands, whereas the gland primordium showed weak reactions (Fig. 20C,G). In older embryos, from 9 to 17 days (Fig. 20D,H,I–P), the glandular epithelium showed negative PAS and Alcian blue reactions. Moreover, mucus strands showing positive PAS and Alcian blue reactions extended from the lumen of the proventriculus into the ducts of the glands. The simple tubular glands gave strong positive reactions for PAS and Alcian blue from the 12 and 13th day of incubation (Fig. 20I,M). On the 17th day, the secretory products filling the apical portions of the epithelial cells lining the mucosal folds as well as the intraluminal mucus coat were strongly stained with PAS and Alcian blue. These products decreased in amount toward the base of the folds. The ductal epithelium showed positive PAS and Alcian blue reactions, whereas the epithelium lining the secretory units of the compound proventricular glands was negative for both reactions (Fig. 20J,K,L,N,O,P).

Protein

The apical portion of the luminal and glandular epithelium as well as the muscular coat and the mesothelium was stained with mercury bromphenol blue (intense blue) which increased in strength from day 6 to day 17 of incubation (Fig. 21A–D).

Acridine orange

From day 6 to day 17 of incubation., acidic secretions in the apical area of the luminal and glandular epithelium, as well as secretory products filling the apical portions of epithelial cells lining the mucosal folds increased (Fig. 22A–H).

Enzymatic activity

The luminal epithelium and epithelium lining the gland rudiments gave weak positive reactions for alkaline and acid phosphatase and ATPase enzymes on the 5th day of incubation. These reactions increased as fetal age increased from the seventh day to the end of incubation including the luminal and glandular epithelium as well as the epithelium lining the ducts (Figs. 23, 24, 25).

Macroscopic investigations

The form of the glandular stomach did not alter much from the 7th day to the end of incubation. It had a spindle-shaped appearance, with a larger caudal end separated from the muscular stomach by an isthmus, and a little constriction separating it from the esophagus (Fig. 26).

The weight of the glandular stomach was about 0.005 gm at 7 days old embryo, and it continuously increased until the end of the incubation period, when it reached about 0.052 gm at 17 days old embryo. The glandular stomach formed around 21% of the weight of the stomach complex at 7 days old embryo, and this percentage decreased until it reached its lowest point at 17 days old embryo, when it was about 13.5% of the weight of the stomach complex (Table 1).

Table 1 Weight of the stomach complex, glandular stomach and the ratio between them.

Full size table

The length and width of the glandular stomach measured about 1.74 and 1.35 mm at 7 day old embryo respectively. Then the two parameters increased with the advancement of age until reached its maximum value on the 17th day being about 4.25 and 3.89 mm, respectively (Table 2).

Table 2 Length and width of the glandular stomach at different stages of development.

Full size table

Conclusion of main age changes

Figure 27 depicts the major morphological changes.

Discussion

Development of the chick embryo’s stomach complex began on the third day of incubation with the establishment of an expansion of the foregut directly anterior to the hepatic diverticula⁴⁴. However, in the present work, the primordium of the stomach in the quail could be identified on the 2nd day of incubation. Based on our findings and that of Attia⁴³ the stomach could be distinguished into two parts: a small cranial part (prospective glandular stomach) and a large caudal part (prospective muscular stomach) on the 3rd day of incubation. In the chick embryo, the proventriculus could be identified from the gizzard on the 6th day⁴⁵. However, on the tenth day of incubation, the two sections of the stomach in the helmeted guinea fowl could be distinguished from one another⁴⁶. The development and differentiation of the epithelium in the chick proventriculus were dependent on inductive signals from the underlying mesenchyme⁴⁷. In this respect, Yasugi⁴⁸ and Roberts⁴⁹ stated that, in birds, the epithelial-mesenchymal interaction was essential for normal gut development. Moreover, in the present study as well as that of Gallagher⁵⁰, epithelial-mesenchymal contact was probably enhanced in areas where the basement membrane became thin or discontinuous. The lining epithelium of the primitive quail proventriculus was pseudostratified and changed into the simple columnar type by the 10th day of incubation in the current investigation. Attia⁴³ as well as Soliman et al.³⁰ observed in the same bird that the epithelial transformation occurred on Day 9 and 12, respectively. Different sources of fertilized eggs and incubation methods were postulated by later authors as a possible explanation for such contradictory findings. The light and scanning electron microscopical results revealed that many small mucosal folds with intervening depressions were observed for the first time in an 8-day-old embryo. These folds increased in number and height with the advancement of age. This is consistent with the results obtained by Martínez et al.⁵¹ in a chick embryo at Day 14 as well as Wali et al.⁵² at Day 16. The projection of the mucosa into numerous folds leads to the increase in the surface area and consequently the amount of mucin secreted by their lining cells in order to give more protection against the harmful effects of the gastric juice and ingested materials²⁶.

Numerous papillae could be observed on the proventriculus’s mucosal surface on the 11th day. These papillae were formed by the connection of two folds after approaching each other around the opening of the proventricular glands. Similar papillae could be detected in the chick embryo on the 14th day⁵¹. With the advancement of age, some mucosal folds were concentrically arranged around the glandular duct openings, giving the mucosal papilla a definite whorled appearance on the 17th day. Stinson and Calhounin⁵³ and Dellman⁵⁴, as well as Salem et al.²⁵ described that, in the chick, the mucosal folds had a concentric arrangement. Salem & Abdel-Rahman²⁶ suggested that the concentric manner of arrangement of the mucosal folds around the openings of the proventricular glands probably gives better support and protection to the gland opening than the finger-like shapes. This is because the finger-like shapes could be damaged by the different ways this organ is stretched the concentric manner of arrangement of the mucosal folds around the openings of the prIn this work, the endocrine cells in the quail proventriculus were first seen on the 7th day of incubation between the luminal and glandular epithelium. However, Romanoff¹⁸ and Walter⁵⁵, stated that in chick embryo, these cells began to appear in the epithelium of the lumen at embryonic day 8. They also saw that the number of endocrine cells in the main lumen epithelium decreased over time. These cells’ functions were mostly taken over by cells that appeared in the epithelium of the proventriculus glands on the 12th and 16th days of incubation, respectively. Yamaguchi et al.⁵⁶ could observe in the quail proventriculus the embryonic endocrine cells for the first time on the 11th day of incubation in the superficial epithelium and proventricular glands.

According to the present study as well as that of Martínez et al.⁵¹, in the chick embryo, the open type of the endocrine cells was detected on day 7–12 or day 9–13 of incubation, respectively, after which their number decreased with the increased number of the closed type. Yamaguchi et al.⁵⁶ observed in the quail proventriculus that no “open type” cells could be observed in all developmental stages. Moreover, Aksoy & Cinar⁵⁷ observed in the chick embryo that both the gastrin and serotonin-IR cells were of the closed type in all stages studied.

In the present study, the proventricular glands had two types: superficial and deep (as previously reported by Langlois⁵⁸ and Zaher, et al.²⁸. The primordium of the compound deep proventricular glands in quail embryos appeared as evaginations of the lining epithelium on the 4th day of incubation. Similar results were observed in chick embryos around the 7th day of incubation⁵⁹. It was unclear whether the proventricular glands lie in the lamina propria or within the submucosa. In the present study as well as that of Alsanosy et al.³³on domestic fowls, Bezuidenhout, & Van Aswegen⁶⁰ on ostriches, Van Alten& Fennell⁶¹ and Romanoff¹⁸ on chicken embryos, and Liman et al.⁶² on 1-day-old hatched chicks, the deep tubuloalveolar glands were located in the lamina propria. On the other hand, the studies of on fowls considered the glands to be submucosal Bradley, and Grahame⁶³, Toner⁶⁴.

Many authors have reported the fact that some change occurred in the glandular epithelium of the presumptive secretory unit of the proventriculus. The glandular epithelium changed from pseudostratified columnar to cuboidal epithelium on the 15th day of incubation in the chick embryo^57,59,65 and in the helmeted guinea fowl⁴⁶. Similarly, Attia⁴³ stated that in the quail, the glands were still lined by stratified columnar epithelium and some glands were still undifferentiated and appeared as masses of stratified cells. However, Soliman, et al.³⁰ in the same bird observed that on the 12th day of development, the pseudostratified epithelium of the glands transformed into a simple columnar type at the location of oxyntico-peptic cells. However, none of these investigators stated what actually happens. In the present study, the glandular epithelium of the quail embryos showed gradual changes from pseudostratified columnar on day 6 to simple columnar on day 12 and then into the established type on day 15 which was the simple cuboidal epithelium. In accordance with Thomson⁶⁶, both light and electron microscopical observations of the proventricular glands of the studied bird revealed that the reorganization of the glandular epithelium occurred as follows: Firstly, by bulging of the apical portion of the superficial cells of the pseudostratified columnar epithelium into the lumen of the glands beyond the level of the terminal bars and their sloughing off, followed by the sloughing of the remaining part of the superficial cells. This was evidenced by the presence of cellular debris containing nuclei in the gland lumina. Secondly, by remolding of the basal cells from simple columnar to low columnar and finally to simple cuboidal.

Concerning the simple tubular glands, the present investigation revealed that their primordium could be detected on the 11th day as localized down-growths of the luminal epithelial cells into the lamina propria forming solid cords. These glands began to canalize on the 13th day. This was in accordance with that described in the chick embryo by Sjögren et al.⁶⁷ in the lower and upper parts of the glandular stomach on the 11th and 13th day of incubation, respectively, as well as Martínez et al.⁵¹ on the 14th day. However, the studies of Bezuidenhout & Aswegen⁶⁰ on ostriches, Ogunkoya & Cook⁶⁸ on three species of Australian passerines, Liman et al.⁶² on 1-day-old chicks, and Le Guen et al.⁶⁹ on gray-backed shrike stated that these glands formed by the invagination of the surface epithelial cells into the lamina propria. In earlier studies on domestic chickens, these glands were considered as artifacts of preparation⁶⁴.

Our findings indicate that in 10-day-old embryos, the muscularis mucosae of the esophagus diverged at the esophago-proventricular junction into the thin inner and thick outer layers, which continued with the corresponding ones of the proventriculus. Similar results were reported by Czarnecki⁷⁰ at 14 days of age and became more prominent on the 16th to 19th day. We also could observe the inner and outer layers of the muscularis mucosae rejoining prior to the isthmus, so we proposed that such arrangement of the muscularis mucosae probably plays apart in the emptying of the glands.

The histochemical observations revealed that the lining epithelium of the proventriculus showed strong PAS and moderate AB positive reactions on the 5th and 6th day of incubation, indicating its ability to produce both acidic and neutral mucin from these embryonic ages. With the advancement of age, these reactions became stronger in the lining epithelium as well as the primary ducts of the compound glands and the simple tubular glands from the 9th day till the day of hatching. These results were similar to that described by Hinsch⁷¹ in chick embryos. Ventura et al.⁶⁵ stated that only the surface epithelium was strongly positive mainly on themost advanced stages. However, Soliman et al.³⁰ mentioned that in quail embryos, the ductal epithelium of the proventricular glands showed no PAS and AB reactions from Day 12 to Day 17 of incubation.

The primordia of the compound glands showed strong PAS and moderate AB positive reactions on the 5th and 6th day of incubation; these reactions became weak on the 8th day and negative afterwards. This indicated that in the early stages of development, the glands could produce both acidic and neutral polysaccharides, whereas they originated from the luminal epithelium, keeping its same character. In the advanced ages, the compound glands became differentiated and hence could not produce neither acidic nor neutral polysaccharides. We agree on the importance of previous studies of Udoumoh et al. and Demirbağ et al.^72,73. As a result, this study found The production of neutral and acidic mucins in the stomach at embryonic day 14 may reflect early secretory roles of glands in the stomach of broiler chicken. They suggested that morphological evidence of rapid development of broiler stomach glands in late pre hatch periods, as well as maturation of the glands at hatch. These modifications could be required for early food ingestion, digestion, and a strong innate immune response quickly after hatching.

Acridine Orange is a cationic dye that stains proteins-containing membranous vesicles, including as secretory vesicles, membrane-bound acidic compartments, and acidic lysosomes. The metachromatic reaction of Acridine Orange is connected with the liberation of green and red fluorescence. Acridine Orange reacted with membrane-bound vesicles to produce an orange or red colour. Secretory vesicles and lysosomes are identified with Acridine Orange^74,75. Acidic secretions in the apical area of the luminal and glandular epithelium, as well as secretory products filling the apical portions of epithelial cells lining the mucosal folds, increased and were paraellel with an increase secretions of acidic mucin stained by alcian blue from the 6th to the 17th day of incubation.

The epithelium lining the ducts, as well as the proventricular mucosal epithelium and glands, showed positive ATPase, alkaline phosphatase, and acid phosphatase activity, similar to the findings of many previous studies^76,77,78. We hypothesized that elevated enzymes, which were linked to an increase in development ages, might be required for early meal ingestion, digesting, and absorption.

In conclusion, in a two-day embryo, the stomach primordium was seen to be the most dilated region of the gut, dorsal and cranial to the liver primordium and ventral to the level of the notochord. An inner, thin endodermal epithelial lining and an outer, thick mesenchymal layer comprised the stomach wall. The quail proventriculus wall consists of four tunics on the seventh day of embryogenesis: tunica mucosa, very thin tunica submucosa, tunica muscularis, and the outermost tunica serosa. On the seventh day embryo the wall of the quail proventriculus consists of four tunics; tunica mucosa, very thin tunica submucosa, tunica muscularis and outer most tunica serosa. There are two types of proventricular glands; compound tubuloalveolar glands and simple tubular glands whereas, both types are situated within the tunica mucosa. The compound tubuloalveolar glands develop on the 4th day of incubation. Beginning as an evagination in the pseudostratified columnar epithelium lining the lumen of the proventriculus, the gland rudiment, by repeated bifurcation of the evaginated portion, by sloughing of the superficial cells, and by remodeling of the residual basal cells from columnar to cuboidal, becomes a multilobular compound tubuloalveolar gland composed of numerous secretory units. The simple tubular glands develop on the 11th day as localized down-growths of the luminal epithelial cells into the lamina propria forming solid cords which later become canalized. Figure 27 displays the primary morphological changes and serves as a conclusion to the preceding section.

Materials and methods

Ethical approval

The Committee for the Use and Care of Animal Experiments and the Faculty of Veterinary Medicine in Assiut, Egypt, also gave their approval to the project. The number of the ethics license (No. 06/2023/0046). All methods were done according to the relevant guidelines and regulations.

ARRIVE guidelines

The research conformed to the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines⁷⁹.

The nomenclature used in the present study was adapted to the Nomina Anatomica Avium⁸⁰ as well as the available literature.

Sample collection and fixation

In this study, 130 healthy Japanese quail (Coturnix japonica) eggs were used. The fertile eggs came from the Poultry Production Department at Assiut University’s Faculty of Agriculture. The incubation took place in an automatic incubator where the temperature (37˚C) and humidity (60–65%) were managed. From the second day of development until the eggs hatched (Table 3). Scarification occurred on days 15–17, when the skin was incised from the recti of the beak to the thoracic inlet, enabling access to the body cavity. The samples were gathered in conformity with Egyptian animal legislation and the Institutional Ethical Committee of Veterinary Medicine at Assiut University.

Table 3 Age and number of the used embryos.

Full size table

The eggshells were carefully cracked apart at the broad end, and the embryos that appeared to be healthy were carefully removed from their shells. The embryos were extracted from the eggs and processed as a whole for gross morphology and light microscopy. For electron microscopy, the embryos were dissected then the glandular stomach was obtained, opened, washed in normal physiological saline and small pieces were taken.

For gross morphological studies

The quail embryos were fixed in 10% neutral buffered formalin then dissected for studying the length and width of the glandular stomach at different stages of development. The length of the glandular stomach (ab) was measured from the esophago-proventricular junction to the level of the beginning of the isthmus. The width (cd) was taken at the level of the midpoint of the length. After taking the images, these measurements were made using the Image J software (http://fiji.sc/Fiji, National Institutes of Health, USA).The weight of the stomach complex as well as the glandular stomach were recorded (The measurements are explained in the method Fig. 28). Stomach complex was described in Romanoff¹⁸.

For light macroscopically studies

Bouin’s fluid was used to preserve the embryos. After being dehydrated in a series of ethanol alcohol concentrations, the fixed specimens were cleared and embedded in paraffin wax (Table 4 displays the processing time for paraffin embedding). Transverse, sagittal, and frontal serial slices were cut at thicknesses of 3–5 µm using a Leica RM2125 microtome (Leica Microsystems, Wetzlar, Germany).For general histological studies, we used Harris haematoxyline and eosin stain; to demonstrate collagenous and muscle fibers, we used Mallory's trichrome stain; to identify such fibers, we used Crossmon’s trichrome stain; to detect acidic mucopolysaccharides, we used Alcian blue pH 2.5; and for neutral mucopolysaccharides, we used the PAS technique. Survan et al. described the staining procedures⁸¹. Mercury bromphenol blue stain for demonstration of protein⁸².

Table 4 The processing time of the samples in paraffin embedding techniques.

Full size table

Use acridine orange dye to colour paraffin-cutting sections

The method used is based on the work of Hoff et al.⁸³ and has been modified by⁸⁴.

For enzyme histochemistry

For frozen sections, we take half-cm3 samples of each age. Following formal calcium fixation, samples were immersed in OCT overnight at 4 °C in the refrigerator and then frozen at − 20 °C for cryosectioning⁸⁵ as directed by Suvarna et al.⁸¹.

The tissues were processed using the methods outlined by Suvarna et al.⁸¹ to identify the following enzymes: Acid phosphatase, Adenosine triphosphatase (ATPase), alkaline phosphatase. Leitz Dialux 20 microscope and Canon digital camera (Candison power shot A 95) were used to evaluate and take pictures of the produced sections.

For electron microscopy

Karnovesky’s solution (10 ml paraformaldehyde 25%, 10 ml gluteraldehyde 50%, 50 ml 0.1 M phosphate buffer PH 7.2, and 30 ml bidistilled water84) was used to fix small portions of the glandular stomach for 4–6 h at 4 °C. After washing the samples in a 0.1 M phosphate buffer, they were fixed in 1% osmium tetroxide for 2 h. The samples were then washed in the same buffer.

For semi thin section and transmission electron microscopy (TEM)

The fixed specimens were dehydrated in an increasing graded alcohol series before being imbedded in an Epon-Araldite combination, as described by Abd-Elhafeez et al.⁸⁶, and as stated by Suvarna et al.⁸¹. Karnovsky’s Fixative⁸⁷ was used to fix small sections of the specimens (2.0–3.0 mm length) at 4C overnight. The details of the process are explained in the Supplementary Materials. Semi-thin sections of 1 µm thickness were cut and stained with toluidine blue so that the specimens could be evaluated by light microscopy. The (600-800A°) ultrathin sections were stained with uranyl acetate and lead citrate⁸⁸, investigated with a JEOL-100 CX II transmission electron microscope, and then photographed.

For scanning electron microscopy

Critical-point drying with liquid Co₂ was used to dry the fixed tissues (0.5 × 0.5 cm²), after which they were dehydrated in ethanol and amyl acetate. They were sputter-coated with gold, placed on specimen stubs, and examined with a JEOL 5400 LV scanning electron microscope operating at 15 kV.

Digitally colored scanning and transmission

Photoshop (Adobe, CS6) was used to digitally colour the scanning and TEM images, as described by⁸⁹.

Data availability

On reasonable request, the corresponding author will provide the datasets used and/or analyzed during the current work.

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Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt
Wafaa Gaber, Heba Mostafa & Yousria A. Abdel-Rahman
Department of Cell and Tissues, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt
Hanan H. Abd El-Hafeez

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Wafaa Gaber
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Heba Mostafa
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Yousria A. Abdel-Rahman
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W.G., H.H.A.E, H.M.M., Y.A.A.-R. designed the research study contributed to the analysis and interpretation of data. The final version of the paper was written by H.H.A.E. and W.G., who grouped the photographs and produced the final version of the paper. The final version of the manuscript has been read and approved by all authors.

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Gaber, W., Mostafa, H., Abdel-Rahman, Y.A. et al. Morphological studies on the prehatching development of the glandular stomach of Japanese quails using light, electron, and fluorescent microscopy. Sci Rep 13, 18096 (2023). https://doi.org/10.1038/s41598-023-45355-1

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Received: 30 May 2022
Accepted: 18 October 2023
Published: 23 October 2023
DOI: https://doi.org/10.1038/s41598-023-45355-1

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Abstract

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Introduction

Results

Two-days embryos

Three-day embryo

Four-day embryo

Five-day embryo

Six-day embryo

Seven-day embryo

Eight-day embryo

Nine-day embryo

Ten-day embryo

Eleven-day embryo

Twelve-day embryo

Thirteen-day embryo

Fifteen-day embryo

Seventeen-day embryo

Histochemical investigations

Neutral and acid mucopolysaccharides

Protein

Acridine orange

Enzymatic activity

Macroscopic investigations

Conclusion of main age changes

Discussion

Materials and methods

Ethical approval

ARRIVE guidelines

Sample collection and fixation

For gross morphological studies

For light macroscopically studies

Use acridine orange dye to colour paraffin-cutting sections

For enzyme histochemistry

For electron microscopy

For semi thin section and transmission electron microscopy (TEM)

For scanning electron microscopy

Digitally colored scanning and transmission

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