Age-dependent microstructural and histochemical alterations in the pancreas of duck after hatching

Introduction

Ducks (family Anatidae) represent a diverse group of waterfowl with a cosmopolitan distribution, thriving in varied ecological niches from wetlands to agricultural landscapes worldwide (del Hoyo et al., 1992) [7] . The pancreas, a composite glandular organ strategically positioned in the limbs of the duodenum, is crucial to duck digestion, combining both exocrine and endocrine functions required for metabolic equilibrium (King & McLelland, 1984) [12] . The exocrine pancreas, made up of acinar cells and ductal systems, secretes a bicarbonate-rich pancreatic juice that neutralizes acidic chyme from the stomach entering the duodenum. This juice contains high concentrations of sodium bicarbonate (NaHCO₃) and digestive enzymes like amylase, lipase and proteases (Solomon, 2006; Rawles, 2015) [25,21] . Duck pancreases undergo many alterations after hatching. Acinar tissue expands rapidly, zymogen granules mature and several kinds of endocrine cells arise. These changes occur as the ducks' diets shift from yolk sac reserves to solid food. These ontogenetic changes are pivotal for establishing digestive efficiency, yet even subtle histological variations such as alterations in lobular architecture, ductal proliferation, or endocrine cell distribution can impair exocrine secretion or endocrine signaling, leading to growth stunting, maldigestion, or metabolic disorders (Nitsan et al., 1991) [20] . This paucity of knowledge hampers targeted interventions in duck health and production. Therefore, the present study aims to elucidate the microscopic architecture, histological organization and histochemical profile of the pancreatic lobes in post-hatched ducks, across key developmental stages.

Materials and Methods

The present investigation was conducted on pancreatic samples collected from forty ducks of both sexes, procured from a registered duck farm. The birds were divided into four age-based groups, with ten ducks in each group. Group I comprised newly hatched ducklings, Group II included ducks aged one month, Group III consisted of ducks aged three months and Group IV comprised ducks aged six months. Immediately after collection, the pancreas along with its respective lobes (dorsal, ventral and splenic) was carefully excised, washed thoroughly in normal saline to remove blood and debris and fixed in 10% buffered neutral formalin for histological examination. After adequate fixation, tissue samples from each lobe were processed routinely through paraffin embedding. Serial sections of appropriate thickness were cut and stained with Haematoxylin and Eosin (H&E) for general histoarchitecture. In addition, special staining techniques were employed to demonstrate specific tissue components, including Van Gieson's stain, Gomori's stain, Weigert's elastic stain (Luna, 1968) [13] , Maldonado's stain (Maldonado and Jose, 1967) [17] , Johnson's stain, Masson's trichrome stain (Bancroft and Stevens, 1996) [4] and Periodic Acid-Schiff (PAS) stain (Singh and Sulochana, 1997) [23] . Micrometric measurements were performed wherever necessary using an ocular micrometer.

Results

In the present investigation, the pancreas of ducks in all experimental groups exhibited a well-organized parenchyma composed of both exocrine and endocrine components (Fig 12). The pancreatic lobes were distinctly demarcated by delicate connective tissue septa, which subdivided the gland into numerous lobules of variable sizes. Within the parenchyma, the exocrine portion constituted the predominant component, occupying the major volume of the glandular tissue. In contrast, the endocrine portion was comparatively sparse and appeared in the form of small, clearly defined islets scattered irregularly among the exocrine acini (Fig 3). These islets were separated from surrounding acinar tissue by a thin connective tissue boundary and were more lightly stained than the surrounding exocrine tissue. Histological sections stained with Hematoxylin and Eosin revealed that the exocrine pancreas in all age groups consisted of serous tubuloacinar glands. The acini were predominantly spherical to ovoid in outline and closely packed with minimal interacinar space. Each acinus was lined by a single layer of tall columnar to pyramidal epithelial cells (Fig 4). The cytoplasm of these cells exhibited basophilia in the basal region and eosinophilia in the apical region, indicating the presence of abundant rough endoplasmic reticulum basally and zymogen granules apically. In the experiment it was found that the nuclei of acinar cells mainly found towards the basal portion of the cells and appears in the form of spherical to oval in shape (Fig 4). The nuclear chromatin appeared moderately condensed with a distinct nucleolus in many cells. These acinar cells formed the major cellular population within the pancreas of the experimental ducks. Distinct centroacinar cells were also observed within the lumen of the acini in all examined samples (Fig 5). These cells appeared as darkly stained oval to spherical cells projecting into the acinar lumen and represented the initial portion of the intercalated ducts. In all age groups, delicate strands of connective tissue were frequently observed surrounding individual acinar units and also extending between adjacent acini, thereby forming a supportive stromal framework. Special staining methods further clarified the nature of this connective tissue. Sections stained with Weigert's elastic stain, Van Gieson's stain and Masson's Trichrome stain demonstrated that the interacinar connective tissue strands and septa were composed predominantly of collagen fibers with only scanty elastic fibers (Fig 67). In the experiment of all pancreas of ducks, the collagen fibers were found to be dense and arranged regularly mainly towards the interlobular septa. On other hand the elastic fibers were found to be sparse and discontinuous. Overall, the pancreatic architecture remained constant throughout all experimental age groups, displaying typical avian pancreatic structure defined by a strong exocrine component, distributed endocrine islets, serous acinar layout and a collagen-rich supporting stroma.

Histochemistry

In the current study it was found that the Periodic Acid-Schiff (PAS) stained tissues showed mild to moderate reaction in pancreatic acini in all experimental duck that suggesting the existence of a small quantity of neutral mucopolysaccharides (Fig 8).

Discussion

In the current study it was found that parenchyma of all experimental ducks were composed of exocrine and endocrine tissue in which exocrine tissue was mostly composed of serous tubuloacinar arranged somewhat concentrically around the islets of Langerhans and considered as major portion of parenchyma. Similar observation was also made by Das et al. (2003) [8] in ducks, Mobini (2011) [18] in gooses, Faris (2012) [9] and Mobini (2013) [19] in pigeons, Hamodi et al. (2013) [10] in common gulls and guinea fowls, Basir and Abdi (2015) [5] in caspsian gulls, Al-Sharoot (2016) [3] in early-hatched gooses, Kadhim et al. (2010) [11] in red jungle fowls and Al-Agele and Mohammed (2012) [1] in golden eagles. In contrast Madhavi et al. (2000) [14] domestic duck (Anus boschas domesticlls) found tubuloalveolar acini around the islets. The nuclei of acinar cells were revealed to be spherical and oval in shape and were found close to the basal region of acinar cells darkly pigmented oval or spherical centroacinar cells were also observed in the lumen of acini. Similar results on the location and appearance of acinar cells may be found in adult ducks by Das et al. (2003) [8] , in domestic ducks by Madhavi et al. (2000) [14] and in chabro chickens by Yadav et al. (2018) [26] . Yadav et al. (2018) [26] in chabro chickens observed elongated or spherical tiny centroacinar cells, while Madhavi et al. (2000) [14] in domestic duck observed elongated centroacinar cells. In contrast centroacinar cell were absent in the result of Saadatfar et al. (2009) [22] in Mynah (Acridotheres tristis), Basir and Abdi (2015) [5] in Caspian Gull and Mahdi et al. (2023) [15] in Guinea fowl (Numida meleagris) Fine strands of connective tissue were frequently seen surrounding the acinar units and appeared between the acini which were mostly composed of collagen and scanty of elastic fibres in all experimental ducks. According to Beheiry et al. (2018) [6] in geese and Mobini (2013) [19] in pigeons, connective tissue fibers were found between interacinar gaps and septa separated the pancreas into lobes and lobules, which is consistent with the findings of the current study on connective tissue septa. Septa separated the gland into lobules and continued to spread across acinar cells, according to Agele and Mohammed (2012) [1] in Golden Eagle. Similar results were found in red jungle fowl by Khadim et al. (2010) [11] and in geese by Al-Sharoot (2016) [3] . According to Sivakumar et al. (2000) [24] , acinar units and smaller ducts are connected to small connective tissue strands. Mobini (2011) [18] reported that collagen and reticular fibres were found in both the interlobular and interacinar connective tissue of the goose pancreas, which was consistent with the results of the current study concerning the components of connective tissue septa. The exocrine parenchyma of pancrease showed a small quantity of neutral mucopolysaccharides which were in accordance with Al-Khakani et al. (2019) [2] in white-eared bulbuls and Mahmood et al. (2022) [16] in native ducks for pancreatic acini.

Conclusion

The exocrine portion made up the majority of the parenchyma under which discrete units of endocrine portion can be identified. Serous tubuloacinar glands with spherical to ovoid forms comprised the exocrine parts. It was made up of a single layer of pyramidal to columnar cells. Acinar cell nuclei were found to be oval and spherical in form. These cells were mostly located around the basal part of acinar cells in the experimental ducks' pancreas. The lumen of acini also included oval or round centroacinar cells with dark pigmentation. In between the acini and around the acinar units, fine strands of connective tissue were commonly seen. Periodic acid-Schiff (PAS) staining showed mild to moderate reaction in pancreatic acini in all experimental duck that suggesting the existence of a small quantity of neutral mucopolysaccharides.

Acknowledgement

AcknowledgmentThe authors are grateful to Sanghars Farm and Hatchery, Bihar for providing ducks for the research work. we also thanks to Department of Veterinary Physiology and Biochemistry for time to time support.

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