Morphometrical and Histochemical study of glandular stomach (Proventriculus) in local domestic male ducks (Anas Platyrhynchos)

1- The study focused on the morphometrical and histochemical characteristics of proventriculus in domestic ducks.

2- Proventricular location, shape, and dimensions were studied.

3- Evaluation of the carbohydrate histochemistry of proventricular glands.

Introduction

The essential elements of the birds' alimentary tube are the oral cavity, esophagus (containing the crop in several types of birds), the proventricular and ventriculus), and intestinal segments. Anatomically, the gastric portion of domestic ducks is allocated as two main segments: the glandular part, known as gastric proventriculus (glandular stomach), which is linked to the esophagus cranially (1), and the muscular portion, known as the ventriculus (gizzard), that is divided by an isthmus or intermediary zone (2). The proventriculus varies in size according to the species variation of birds. It looked to be very small in the graminivorous, whereas found to be more significant in carnivorous (3). The length and breadth vary among regions and species (4). The wall of the proventriculus comprises the four general layers for the most digestive organs, which are tunica mucosa, submucosa, muscular layer, and serosa (5). The surface mucosa of glandular stomach was described by the presence of papilla with abundant tiny folds (plicae) ranging in its elevation and grouped in concentric manner round the solitary duct opening at the papilla's apex (6). Liman et al. (7) mention that the mucosa is markedly wrinkled, creating flat folds split by fissures or grooves where the tubular mucous glands opened in the bases of the furrows, elevation of Tmuscularis (papilla) contains the opening of leaving duct of submucosal proventricular glands (8). The stomach proventriculus mucosa liberating pepsinogen enzyme, hydrochloric acid, and mucin (7). The mucosal layer is lined with simple columnar cells. These cells extend to the interior of the furrows (9,10). The submucosa is studded by many tubular submucosal glands, ramified or complex types; only a primary simple cuboidal exocrine cell and a few stomachs argyrophil endocrine cells make up the glandular epithelium. (9). Most of the deep proventriculus wall thickness was taken up by glands that were conic or pear in form (11). T muscularis is somewhat adapted where the innermost layer is oriented longitudinally and the external circularly. The tunica serosa is constructed from connective tissue supported by mesothelium (8).

This work explores histological, morphometrical, and carbohydrate histochemical foundations in the proventriculus in adult domestic male ducks. It also aims to provide an adequate anatomical and histological database about the proventriculus of local male ducks to fulfill their necessities of food and energy.

Materials and methods

This study used six healthy adult male ducks (Anas Platyrhchos). The Birds were purchased from local sellers in Mosul province's animal market with body weights ranging between 2.5-3 Kg. After using chloroform inhalation, the birds were slaughtered, and the digestive tube was exposed (Figure 1). The proventriculus is harvested and divided equally into three segments (proximal, middle, and distal). The entire length of the proventriculus was measured using a measuring tape, as well as the length and width of each segment were measured with the aid of a vernier caliper.

Representative specimens of 6-8mm were taken as follows: esophageal proventriculus junction (proximal), proventriculus (middle), and proventricular gizzard junction (distal). All specimens were washed with buffered saline and then fixed immediately in standard neutral buffer formalin 10% for about 48 hours. They were dehydrated progressively by ethyl alcohol and cleared with xylene before embedding in wax of paraffin. Formerly, they were cut by rotatory microtome to obtain a histological section of 4-6 micrometer thickness and applied to the routine histological stain H&E for common inspection, Masson's Trichrome stain to recognize fibrous tissue, Toluidine blue, and Periodic acid Schiff reagents (PAS) for identification of mucins and glycosaminoglycan respectively (12-14).

The included micromorphometric measurements were included the tunica mucosa thickness as well as that of tunica submucosa, tunica muscularis, and tunica serosa of the studied areas measured in micrometer (unit/µm). The length or span of the folds in the studied areas /µm. Dimensions (length * width) of the compound gastric glands in the submucosal region and the simple tubular gastric glands in the mucosal region, and the duct diameter of the studied areas measured / µm.

All tissue sections we re-photographed, and the micromorphometric parameters were analyzed with the aid of an OMAX 8.0MP digital USB microscopic camera and sophisticated picture analysis software (OMAX Toup view 3.7), the camera was fitted to Microscope-Olympus-CX31, and the aid of stage micrometer did the calibration of objective lenses to the software.

Statistical analyses were carried out with the SigmaPlot 12.5 (Systat Software Inc 2016(software package. All numerical numbers were expressed as mean±SE. Then ANOVA with Duncan's test were assessed at P≤0.05. 

Figure 1: Photograph illustrates the tubular organs of the digestive tract of male ducks showing: 1esophagus, 2 proventriculus, 3 ventriculus (gizzard), 4 small intestines, and 5 duodenums.

Ethical approve

This work was conducted in the laboratory of anatomy, College of Veterinary Medicine, University of Mosul under reference number UM.VET.2021.011.

Results 

Anatomical approach

Gross anatomical results showed that the proventriculus in ducks was positioned cranial to esophagus and caudal to ventriculus in the left ventral region of the abdominal cavity (Figure 2). The liver's left lobe partly buried the organ. Conversely, well-allocated proventriculus from the esophagus and ventriculus were stated, probabilmente delineation of the esophageal-proventriculus junction.

The gross anatomical investigation of the stomach of local breed duck revealed that the stomach of the examined ducks looked to be a thin-walled part, noticeably composed of two segments which are the glandular one (proventriculus) and muscular one (ventriculus), these two segments separated by intermediate zone (Figure 2). The proventriculus, according to observations, of local duck was large, fusiform, or tubular with a mean length of 52.91±1.83 mm and meant width or diameter in three regions proximal, middle, distal 7.804±0.21, 12.522±1.18 and 10.56±0.19 mm, respectively (Figure 3).

Figure 2: Photograph illustrates a ventral side of duck bowl presenting: (H) Site of head, (E) Esophagus, (P) Proventriculus, (IS) Isthmus, (V) Ventriculus (gizzard), (C) Cloaca, (IN) intestine (PA) Pancreas, (Black stars) Testes.

Figure 3: Histogram showing the length and diameter of proventriculus in mail ducks.

Histological approach 

Histological investigations exhibited the three parts' proventricular wall constructed from the main tunicae of any classic tube-like structure: tunica mucosa, tunica submucosa, tunica muscularis, and serosa (Figure 4). The tunica mucosa varied in thickness among the three segments of the proventriculus, the highest thickness was recorded in the distal part 650.85±60.65 µm (Table1), and it consisted of three layers epithelium, lamina propria, and muscularis mucosa. The mucosa appeared thrown with folds (plicae proventricular) and sulcus at its luminal surface. The folds diverse in length. The highest lengths were recorded in proximal region 412.11±7.3 µm (Table 2). Some of the folds combined, and the lining epithelium was simple columnar with clear cytoplasm and intermingled mucus-secreting cells (goblet cells) may be present. The cells of the basal region of the folds were low and stained intensely more than those of the superficial region (Figure 5). Mucin granules were shown in supranuclear parts of columnar cells (Figure 6).

Figure 4: Photomicrograph of proventricular wall in duck presented (1) mucosa, (2) submucosa, (3) muscularis and (4) Serosa, H and E, (X100).

The proventricular glands at the base of these folds consist of both superficial and profound gastric glands. The superficial glands were dispersed in l. propria of mucosae, while profound glands existed in submucosa. The outer most superficial glands are short, tubular simple glands extended through the lamina propria (Figure 6). The glandular cells in the basal portions and body of the superficial gastric glands consisted of many pepsinogen granules (Figure 6). Muscularis mucosa, which was made up of circular smooth muscle fibers establishing a net appearance and neighboring the units of the large number of deep gastric glands (Figure 7). The glands contained numerous secretory tubules lined by one layer of cuboidal cells, those situated at a basal position to give a serrated or dentate appearance (Figure 10C). All of the branched tubular glands are divided by septa of connective tissue then each tubule connected to one duct opened into the central main duct, that opens into the proventricular luminal surface. The lobules of the glands were extended ovoid in the duck and were determined from each other by well-vascularized collagen fibers (Figure 7). Each glandular lobule consisted of tubuloalveolar units. Tunica submucosa was made up of a thin layer of loose connective tissue containing several fine blood capillaries and nerve endings. 

Submucosal inner glands are arranged into five longitudinal bulges or conical shapes. The lumen of the proventriculus was significantly reduced, being taken by its considerably thickened glandular walls. (Figure 6A). Tunica muscularis comprises an inner longitudinal and outer thick circular layer of smooth muscle fibers in the middle and distal parts (Figure 7). The anterior part contains inner and outer longitudinal and middle circular layers. The rich blood vessels and nerve plexus are found between such muscular layers. (Figure 8). The mean thickness of tunica muscularis varied in three different regions of the proventriculus. (Table 1). Tunica serosa consisted of connective tissue rich in blood vessels, nervous plexus, and adipose tissue, all covered by mesothelium. (Figures 7 and 8). The statistical analysis revealed that the average lengths of folds in the proximal part of the proventricular are high, while the average width or diameter of the proventricular glands was high in the middle part (Table 1).

Figure 5: Photomicrograph of proventriculus of local duck showed the folds lining epithelium was simple columnar(Black arrow) with clear cytoplasm cells, (Black stars) Goblet cells, Masson’s trichrome (X 400).

Table 1: The thickness of proventriculus in different regions of male duck

The similar letters among region in revealed no statistically significant difference at P≤0.05.

Table 2: The diameter of proventricular‎ glands, canals and lengths of folds of proventriculus in different regions of male duck

The similar letters among region in revealed no statistically significant difference at P≤0.05.

Figure 6: Microphotograph of crosswise section of proventriculus of duck (A and B) showing the(M) Mucosa, (SM) Submucosa, (EP) Epithelium, (LP) lamina propria, (SGG) Superficial gastric glands, (DGG) Deep gastric glands. H&E stain, A(X100), B(X100).

Figure 7: Microphotograpg of crosswise section for Proventriculus of duck. (M) Mucosa, (F) Folds, (Orang stars) Muscularis mucosa, (D) Main collecting ducts, (ST) Secretory tubules, (DGG) Deep gastric glands, (Black stars) Connective tissue, (MS) Muscular layers of Proventriculus, (S) Serosa and (Orang row) Blood vessels, Masson’s trichrome (X 40).

Figure 8: Photomicrograph of a transverse section from the anterior part of Proventriculus of male duck. (DGG) Deep gastric glands, (OCM) Outer circular layer of tunica muscularis in Proventriculus, (ICM) Inner circular longitudinal part of muscular tunica in Proventriculus, (LM) middle longitudinal part of muscular (MP) myenteric plexus between muscle bundle(G) ganglion plexus from large bundles of axons in tunica serosa. Masson’s trichrome (X 40).

Histochemical results

In the current study microscopic examination of the glandular stomach of duck shown strong +ve reaction of surface epithelial cells of mucosal folds to PAS indicating the presence of neutral mucins, moderate to AB ph 2.5 as the representing few amounts of acidic mucins. Lamina propria tissue between mucosal folds reacts moderately to PAS/AB pH2.5. The luminal surface of submucosal acini in the proventriculus of male duck reacts strongly with PAS and mild to moderate with AB pH2.5, signifying the presence of both acidic and neutral mucins. Lamina muscularis showed weak to moderate +ve reactions with PAS and vigorous with AB pH2.5. The tunica muscularis showed mild reaction toward PAS/AB pH 2.5 respectively (Table 3 and Figure 9). On the other hand, some structures of proventriculus showed a mild reaction against toluidine blue stain, indicating the presence of small quantities of glycosaminoglycan (Table 3 and Figure 10). Mast cells appeared in the connective tissue under the epithelium and showed a strong reaction when used the toluidine blue stain (Figure 10C).

Table 3: Response of proventriculus to PAS stain, AB pH2.5, and tunica blue stains at different regions

Very strong: +++ Strong: ++, Strong: +, Mild: ±, No reaction: -

Figure 9: Microphotograph of a crosswise section of the proventricular wall of male duck (A and B). showing (Blue rows) (E) Epithelium of mucosal folds and (ELG) Epithelium of luminal gland gave a positive reaction to PAS stain. (ST) Secretory tubules, (White stars) Connective tissue, and (MM) Muscularis mucosa were positive reactions to AB pH2.5. PAS/AB pH 2.5 stain in (A&B). A (X100), B (X400).

Figure 10: Microphotograph of a crosswise section of the proventricular wall in male duck. (A, B, and C) in A (Blue rows), Superficial gastric glands showed mild reaction against toluidine blue stain, B (Brown rows) caudal part glandular cells are put net to at their basal parts and presenting a dentate look. C (Black rows) Mast cells showed a strong reaction with toluidine blue, (E) Simple columnar epithelium. Toluidine blue stain in (A-C). A(X100), B (X100), C (X100).

Discussion 

The stomach in the local duck looked divided into cranial proventriculus and distal ventriculus, such mannerism was detected in different types of, as Saeed and Al-Nimma (15) in pigeon, Conversely, Abumandour (16) cited that stomach of Eurasian hobby comprises third additional pyloric part. Present work shows that the glandular proventriculus appeared to be a large, ovoid organ in ducks. Comparable discoveries were previously detected by Abumandour (16), who specified that the proventricular shape was ovoid or fusiform in different types of birds with different sizes. Contrariwise, the demarcation between proventriculus, esophagus, and ventriculus was not well-defined in the ducks of this study. The proventriculus of local breed ducks founded in the anterior aspect of ventral abdominal nook between the esophagus and gizzard. The liver covered a small part of the studied stomach. Similar findings on the location of this organ were cited by Abumandour (16) in Falco subbuteo, Tadjalli et al (17) in male adult Ostrich and Zaher et al (18) in common quail (Coturnix coturnix). 

The apparent long glandular stomach of local duck has a role in food storage like that cited in ratite birds (19). actually, the proventriculus is devoid of papillae, and its ducts open directly on the mucosal surface. The proventriculus showed the existence of four primary tunics of a classic tubular structures, including (Tunica mucosa, tunica submucosa, tunica muscularis, and tunica serosa). These tunicae were equally recognized in the proventriculus of several birds, such as the domestic pigeon (11), Japanese quail (20), and Coot bird (21). A recent study showed that the lining cells of proventricular mucosa was columnar studded with goblet cells, and this result came in agreement with Eman et al (22) when studying the proventriculus of common moorhen (gallinule chloropus). The fibers of the lamina propria spread inside the folds and possessed simple tubular mucous glands. Das et al (23) detected that the connective tissue of lamina propria in the stomach of Kadaknath fowl comprises nodular or diffuse lymphatic tissue. Thin, smooth muscle fibers of muscularis mucosa as bunches are scattered among the clusters of glands. Tunica Submucosa is occupied by abundant mucous compound glands, constructed of a layer of cuboidal cells and opened directly to the surface. Rossi et al (24) mentioned that the glands lobules in partridge Rhynchotus rufescens were separated by fibrous septa. These findings were consistent with our results in this study. Das et al (23) mentioned that the tunica muscularis in partridge is formed of an inner muscular layer and an outer one. In domestic ducks, the muscular was composed of an inner longitudinal and an outer thick circular layer of smooth muscle fibers in the middle and distal parts, while the anterior part contained an additional middle layer. The tunica serosa was constructed of loose connective tissue in which nerves, blood vessels, and adipose cells were observed, and a layer of the mesothelium covered such structures. These findings were similarly observed by Hussein et al (8) in studies of the stomach in the Iraqi falcon (Falcon perigord), while in moorhen Taher et al (25) and in partridge Rhynchotus rufescens Rossi et al (24) stated is composed of collagen fibers and a cuboidal cells layer. Histochemical, the surface of mucosal folds was intensely +ve to PAS, moderate to AB pH 2.5, indicating the presence of neutral mucins and few acidic mucins.

The fibrous tissue of L. propria, which intermingled among mucosal folds, presented a moderate reaction to PAS/AB pH2.5. The luminal surface of submucosal glands in the proventriculus of male duck reacts strongly with PAS and mild to moderate with AB pH2.5, indicating the presence of both neutral and acidic mucin, and these results support those of Taher et al (25) and AL-A´araji (26) in moorhen (Gallinula chloropus) and birds respectively and disagree with Udoumoh and Ikejiobi (27) stated that the complex tubular glands of the proventriculus of African pied crow displayed PAS adverse reactions. The muscularis mucosa and the blood vessel wall in the submucosa exhibited mild to moderate responses with PAS and strong with AB pH 2.5. Whereas the tunica muscularis showed mild reaction toward PAS/AB pH 2.5 respectively, Recently, Zaher et al. (18) recognized equally in the stomach of quail, rich neutral and acidic mucins in the gastric glands where they +ve reactions with PAS-AB stain. On the other hand, some structures of proventriculus showed mild reaction against toluidine blue stain, indicating the presence of small quantities of glycosaminoglycan. This is consistent with what was mentioned by Cook (28), Mahmud et al (29) and Khaleel et al (30) for the Gland Cells of the Stomach in different types of birds.

Conclusion

 The study concluded that the domestic duck's proventriculus contains two types of glands, the superficial and profound or deep gastric glands. The superficial glands are short, simple tube-shaped glands drawn-out through the lamina propria, while its inner or deep glands are arranged into four to five longitudinal bulges or conical shapes. Histochemical PAS staining positively reacted with epithelial of tunica mucosa and wall of proventricular glands.

Acknowledgement 

The authors thank the University of Mosul, College of Veterinary Medicine, for supporting this work.

Conflict of interest 

The authors declared that there is no conflict of interest.