Histochemical investigation of duodenum in quail birds in Iraq

Introduction

The gastro-intestinal tract of birds represents a highly specialized system that exists to meet the specific metabolic needs of avian species. Within the scope of the avian digestive system, the duodenum represents an important part of the digestive tract since it is the first part of the small intestine where the digestive process takes place [1] . The duodenum is the area where the most enzymatic digestion occurs and where the initial stages of nutrient absorption takes place. Therefore, the duodenum represents a vital component of the gastrointestinal tract regarding an avian's overall health and productivity [2] . Quail birds, especially the common quail (Coturnix coturnix), have received considerable economic importance within Iraq and the Middle East due to their rapid growth rates, high feed conversion ratios, and amazing meat quality [3] . Intensive production systems used within quail farming have created a greater need to understand their anatomical and physiological characteristics, especially concerning their digestive function [4] . In spite of this economic significance, the extent of histochemical studies of duodenal anatomy in Iraqi quail populations remains alarmingly low within the scientific literature. The avian duodenum has distinctive anatomical characteristics that make it more specialized than mammalian forms. For example, the brush border is well-developed; goblet cells are specialized cell types; and the unique architecture of the villus highlight the superior efficiency of digestion in birds [5] . Clearly, a thorough histochemical characterization of the duodenal wall would provide additional insight of the histochemical constituents of the duodenal wall, including carbohydrate, protein and connective tissue constituents which are most evident in examining and understanding the functional capabilities of the organ [6] . The consumption of histochemical information supports nutritional management when assessing health status, and efficiency of production relating to poultry farming quail. The use of histochemical staining methods can provide complementary information about pathological characteristics, and structural and functional characteristics of the duodenum in quail. Hematoxylin and Eosin staining provides basic morphometric information as the basic fundamental details of morphology is described by various cellular structures and tissue organization [7] . H&E is a routine staining method that can demonstrate type of cell associated with tissue integrity, detailing pathological changes whenever they may occur. The ability to differentiate between nuclear and cytoplasmic characteristics using this staining technique is basic to histological assessment. Masson's Trichrome staining is a valuable technique for assessing the distribution of collagen content in the duodenum, including collagen distribution related to fmechanical properties of duodenal wall [8] . The duodenum has a connective tissue content for maintaining structural stability and supporting the mechanical aspects of digestion. The submucosa and muscularis layers of the duodenum contains large amounts of collagen content guiding the mechanical properties of the duodenal wall along with supporting mechanical aspects in response to peristaltic digestion and physical stressors applied to processes related to ingesting food [9] . Masson's Trichrome is also a valuable tool as it is possible to quantify collagen and its anatomy in the duodenum. Periodic Acid-Schiff (PAS) staining is informative for screening carbohydrate content and glycoprotein distribution in the duodenum [10] . There are applications for glycogen stores, mucin-containing goblet cells, or the glycocalyx applied to components of the brush border that could be demonstrated with this technique. The carbohydrate contents identified using PAS stain correspond and direct digestive function as they have roles with processing nutrients, protections, and cellular energetic [11] . Regional and environmental characteristics that occur in Iraq may affect quail duodenum histochemistry. The hot, arid weather and unique diet that would occur could influence digestive system structural and functional adaptations [12] . Beyond environmental influences, genetic characteristics of Iraqi quail may provide unique histochemistry that warrant study. Understanding this regional characteristic is essential to create location-specific management and feeding strategies.

Text and literature reporting avian duodenal histochemistry has primarily been focused on commercial poultry species (chickens, turkeys, etc.) and not quail species [13] . Available literature suggests that considerable interspecies differences exist in duodenal structure and function, therefore investigations on specific species of quail are warranted [14] . Quail birds exhibit unique physiology in metabolism and growth patterns that would be expected in their duodenal histochemistry, which only underscores the need for specific research. The economic shortfalls for Iraqi quail production has dramatically improved within the last few years due to an amplified demand for Commercial quail production in Iraq has produced a wide range of productivity and health outcomes, some of which may be related to digestive function and nutrient utilization efficiency [16] . Understanding and examining the histochemistry of the duodenum could have important implications on improving management practices and production efficiency. This study utilized a research design that employed standardized histochemical protocols that are established as valid for use in avian studies [17] . The use of adult quail birds, which are representative of the birds being produced in local Iraqi farms, demonstrates added value and also incorporates a multifaceted staining approach to provide several levels of information regarding the various aspects of duodenal structure, function and to provide a more general analysis of functional histochemistry. The morphometric aspect of this study explicitly focused on the quantitative aspects of the duodenal structure and provided actual measurements that could be replicated in other avian species [18] . The parameters that were measured including pollical height, crypt depth and mucosal wall total thickness can be considered meaningful indicators of overall digestive capacity and intestinal health. This was particularly important in establishing baseline values for the local Iraqi quail population that could represent variations in morphology due to management, environmental or genetic factors. This study is important not only from an academic perspective, but the findings generated may also have practical relevance to commercial quail producers. Determine how the normal functional characteristics of the duodenum may support the assessment and establishment of a potential diagnosis of intestinal disorders, potential monitoring, and adjustments to nutritional programming, as well as evaluation of management practices [19] . The findings in this study could improve overall understanding and knowledge of avian digestive physiology as additive reference literature in regard to comparative studies across multiple poultry production systems. Climate change and the effects of environmental degradation in Iraq may negatively impact the physiology and anatomy of native bird species, including quail [20] . Therefore, documenting the histochemistry of the duodenum provides a record of baseline information for future studies/case studies that may involve the effects of environmental changes on the digestive physiology of the duodenum. This information may be considerable and valuable for adaptation strategies and conservation of local quail populations in Iraq.

Methodology Study Design and Location

This descriptive cross-sectional study was conducted between March and September 2024.

Sample Collection and Selection

Twenty adult quail birds (Coturnix coturnix) were obtained from commercial farms located in the Abu Ghraib district, Baghdad Governorate, Iraq. The selection criteria included birds aged 8-12 weeks, maintained under standard commercial management conditions, and showing no clinical signs of disease or abnormalities. The sample population comprised 10 males and 10 females to ensure balanced representation and account for potential sex-related variations in duodenal morphology. Prior to sample collection, all birds underwent a 12-hour fasting period to ensure complete evacuation of intestinal contents, which is crucial for optimal histological examination [21] . The birds were maintained with free access to water during the fasting period to prevent dehydration. Body weights were recorded for each bird, ranging from 180-220 grams, which falls within the expected range for adult quail of this age group.

Specimen Collection and Preparation

Birds were humanely euthanized using cervical dislocation following standard veterinary protocols. Immediately after euthanasia, the abdominal cavity was opened using sterile surgical instruments, and the entire gastrointestinal tract was carefully exposed. The duodenum was identified as the first segment of the small intestine, extending from the pyloric sphincter to the duodenojejunal junction, and was carefully dissected free from surrounding tissues. Duodenal specimens measuring approximately 2-3 cm in length were collected from the mid-duodenal region to ensure consistency across all samples. Each specimen was gently flushed with physiological saline solution (0.9% NaCl) to remove any residual intestinal contents and blood, taking care to preserve the mucosal surface integrity. The specimens were immediately placed in individually labeled containers containing 10% neutral buffered formalin solution, maintaining a fixative-to-tissue ratio of 20:1 to ensure adequate fixation.

Tissue Processing and Embedding

Following 24-48 hours of fixation at room temperature, the specimens underwent routine histological processing using an automated tissue processor (Leica TP1020, Germany). The processing protocol included sequential dehydration through graded ethanol solutions (70%, 80%, 90%, 95%, and 100% ethanol), with two changes at each concentration for 2 hours each. Clearing was performed using xylene with two changes of 2 hours each, followed by infiltration with molten paraffin wax at 60°C for 4 hours with two changes. The processed tissues were embedded in paraffin blocks using an embedding station (Leica EG1150H, Germany), ensuring proper orientation to obtain cross-sectional views of the duodenal wall. The embedding process was carefully monitored to prevent air bubble formation and to maintain optimal tissue architecture for subsequent sectioning.

Sectioning and Mounting

Paraffin sections were cut at 5-micron thickness using a rotary microtome (Leica RM2125RT, Germany) equipped with disposable steel blades. Multiple sections were obtained from each block to ensure adequate material for the various staining procedures. The sections were floated on a warm water bath (40°C) to eliminate wrinkles and were then mounted on positively charged glass slides (Thermo Fisher Scientific, USA) to enhance tissue adherence. Prior to staining, the mounted sections were dried in an incubator at 37°C for 24 hours to ensure complete removal of moisture and optimal adherence to the glass slides. This step is critical for preventing section detachment during the staining procedures.

Histochemical Staining Procedures Hematoxylin and Eosin (H&E) Staining

The H&E staining protocol followed standard procedures with minor modifications optimized for avian tissues [22] . Sections were deparaffinized in xylene for 10 minutes (two changes of 5 minutes each), followed by rehydration through graded ethanol solutions (100%, 95%, 80%, 70%) for 2 minutes each, and finally rinsed in distilled water. Harris hematoxylin was applied for 8 minutes, followed by rinsing in running tap water for 5 minutes. Differentiation was performed using 1% acid alcohol for 30 seconds, followed by bluing in Scott's tap water for 2 minutes. Eosin counterstaining was applied for 2 minutes, followed by dehydration through graded alcohols, clearing in xylene, and mounting with DPX mounting medium.

Masson's Trichrome Staining

The Masson's trichrome staining procedure was performed using a commercial kit (Bio-Optica, Italy) following the manufacturer's instructions with modifications for avian tissues [23] . After deparaffinization and rehydration, sections were fixed in Bouin's solution for 1 hour at room temperature. The staining sequence included Weigert's iron hematoxylin for 10 minutes, Biebrich scarlet-acid fuchsin solution for 15 minutes, phosphomolybdic-phosphotungstic acid solution for 15 minutes, and aniline blue solution for 5 minutes. Each step was followed by appropriate washing procedures. The final result demonstrated nuclei in black, cytoplasm in red, and collagen fibers in blue.

Periodic Acid-Schiff (PAS) Staining

PAS staining was performed using the standard protocol with optimizations for carbohydrate demonstration in avian tissues [24] . Following deparaffinization and rehydration, sections were treated with 0.5% periodic acid solution for 10 minutes, rinsed in distilled water, and then treated with Schiff's reagent for 15 minutes in a dark chamber. Sections were washed in running tap water for 10 minutes and counterstained with Harris hematoxylin for 2 minutes. After appropriate washing and dehydration, sections were mounted with DPX. This staining protocol reveals carbohydrates, glycogen, and mucins in magenta color against a blue background.

Microscopic Examination and Documentation

All stained sections were examined using a light microscope (Olympus BX53, Japan) equipped with digital imaging capabilities. Systematic examination was performed at multiple magnifications (40×, 100×, 200×, and 400×) to evaluate different aspects of duodenal histochemistry. Digital images were captured using an integrated camera system (Olympus DP73, Japan) and stored in high-resolution format for subsequent analysis.

Morphometric Analysis

Quantitative measurements were performed using ImageJ software (National Institutes of Health, USA) on digitally captured images. The following parameters were measured for each specimen: villus height (from the villus tip to the villus-crypt junction), crypt depth (from the villus-crypt junction to the crypt base), mucosal thickness (from the luminal surface to the muscularis mucosae), and submucosa thickness. A minimum of 10 well-oriented villi and crypts were measured for each specimen to ensure statistical reliability.

Statistical Analysis

Statistical analysis was performed using SPSS software version 26.0 (IBM Corporation, USA). Descriptive statistics including means, standard deviations, and ranges were calculated for all morphometric parameters. Independent ttests were used to compare measurements between male and female birds. Statistical significance was set at p<0.05. The normality of data distribution was assessed using the Shapiro-Wilk test, and appropriate parametric or non-parametric tests were applied accordingly.

Quality Control and Validation

To ensure reproducibility and accuracy of results, quality control measures were implemented throughout the study. These included standardization of staining procedures, regular calibration of equipment, and validation of measurements through inter-observer comparison. A subset of specimens was independently evaluated by a second observer to assess consistency of results and minimize potential bias in morphometric measurements.

Results

The histochemical investigation of duodenum in Iraqi quail birds revealed distinct structural and functional characteristics across all applied staining techniques. The comprehensive analysis provided detailed insights into the morphological organization and chemical composition of the duodenal wall components.

General Morphological Features (H&E Staining)

Hematoxylin and Eosin staining demonstrated the classic four-layered structure of the avian duodenum, consisting of mucosa, submucosa, muscularis, and serosa (Figure 1, 2). The mucosal layer exhibited well-developed intestinal villi with a characteristic leaf-like appearance, projecting into the intestinal lumen (Figure 3). The villus epithelium consisted of a single layer of columnar absorptive cells interspersed with goblet cells, showing clear polarity with basally located nuclei and apical brush borders. The lamina propria showed moderate cellularity with scattered lymphoid aggregates and blood vessels. Intestinal crypts were well-defined, extending from the villus base to the muscularis mucosae, containing actively dividing cells and occasional goblet cells (Figure 3,4). The muscularis mucosae appeared as a thin layer of smooth muscle fibers separating the mucosa from the submucosa.

Morphometric Analysis Results

Quantitative measurements revealed significant structural parameters that characterize the Iraqi quail duodenum: The villus-to-crypt ratio was calculated as 4.3:1, indicating a well-developed absorptive surface area. Male birds showed significantly greater villus height and mucosal thickness compared to females, while other parameters showed no significant sex-related differences.

Goblet Cell Distribution and Density

Goblet cell enumeration revealed an average density of 18.5±3.2 cells per 100 μm villus length, with higher concentrations observed in the middle and upper portions of the villi compared to the basal regions. The distribution pattern showed regional variations, with the proximal duodenum exhibiting higher goblet cell density than the distal portions.

Region

Collagen Distribution (Masson's Trichrome Staining)

Masson's trichrome staining revealed extensive collagen deposition throughout the duodenal wall with distinct distribution patterns. The submucosa contained the highest concentration of collagen fibers, appearing as dense blue networks surrounding blood vessels and nerve. (Figure 3,4)

The muscularis layers showed moderate collagen content between smooth muscle bundles, while the lamina propria exhibited minimal collagen deposition around blood vessels and lymphoid structures. Quantitative analysis of collagen content using digital image analysis showed: The morphometric analysis revealed that male birds demonstrated significantly larger villus heights and greater mucosal thickness compared to females, which may indicate enhanced absorptive capacity. The villus-to-crypt ratio of 4.3:1 suggests optimal balance between absorptive surface area and cellular renewal capacity, characteristic of healthy intestinal function. The regional variations in goblet cell density, with higher concentrations in the proximal duodenum, align with the expected pattern of digestive enzyme secretion and pH buffering requirements in this region.

Discussion

The histochemical examination of duodenum in Iraqi quail birds has allowed us to gain detailed knowledge regarding the structures and functions of this crucial digestive organ. It demonstrated that the duodenal structure of Iraqi quail is well developed, with specific histochemical characteristics that allow for the first time the type of animal's adaptation to process and absorb nutrients efficiently.

The morphological descriptions we developed through H&E staining correspond with reports in studies of avian intestinal structures; however, there are differences as expected for this species of quail [25] . The villus height of 847±52 μm is comparable to values in the Japanese quail (Coturnix japonica), however, it differs from the values in the European quail populations studied [26] . This suggests that the duodenum from quail species may develop differently depending on genetics or environmental factors.

The villus structures of Iraqi quail are well developed and with well-defined brush borders which reflects a good absorptive capacity, which is important in relation to the characteristic fast growth rates of quail birds in general. The significant difference in villus height noted between males and females (863±48 μm vs 831±55 μm), p=0.043, is interesting and may be indicative of differences in nutritional requirements and how efficiently they convert feed. Male quail may contain a duodenum with a greater absorptive surface area due to their typically higher metabolic rates and faster growth rates [27] . This supports using a sex-specific feeding strategy in the production of commercial quail birds. Goblet cell distribution patterns noted in this study, with greater densities in the proximal duodenum (21.3±4.1 cells per 100 μm), reflect the physiological requirements of this area of the intestine. The proximal duodenum is where acidic chyme emptied from the gizzard enters and requires generous muscin excretion for pH buffering and mucosal protection [28] . This goblet cell density of 18.5±3.2 cells per 100 μm villus length is in the range found in other gallinaceous birds while showing regional differences that are likely more adaptive for the digestive processing specific to quail [29] . The dense collagen network observed following Masson's Trichrome staining provided important information regarding the structural integrity and mechanical properties of the duodenal wall. The fixed amount of collagen observed in the submucosa (34.7±5.8%) and serosa (42.1±6.2%) indicates strong structural support to withstand the mechanical stiffness associated with peristalsis and food processing [30] . The relevance of these results is of particular significance for commercial quail production practices where the amount of mechanical stress they experience can vary depending on animal housing conditions and feeding regimens.

The distributions of collagen fibers, being denser networks surrounding blood vessels and nerves in the submucosa suggest there are well-developed vascular and nervous support systems. This system is essential in providing the volume of blood supply and nervous controls neccessary to regulate digestive function, which is particularly relevant in the case of birds like quail due to their high metaboloic rates and habit of feeding intensively in short periods [31] . Our moderate amount of collagen (18.2±3.4%) in the muscularis layers indicate that there was appropriate balance between the structural supports needed by the duodenum and the contractile function necessary of normal peristalsis. The high PAS positivity in the brush border and goblet cells confirms ample carbohydrate-rich components for digestive function.

The strong reaction in the glycocalyx suggests wellestablished surface membrane-bound enzymes and membrane-bound transport proteins for the final stages of digestion and nutriment absorption [32] . This would account for the capacity of quail birds for digestion and also their high feed conversions in a commercial genetic strain. Glycogen in the cytoplasm of absorptive cells, particularly in the basal areas, indicates active carbohydrate metabolism and energy for duodenal epithelium [33] . The high energy requirement of the intestinal epithelium which undergoes rapid cellular turnover and has a high energetic cost involved in active transport processes is consistent with the detected glycogen deposits. The specific pattern of glycogen deposition could indicate the cells are preparing for increased absorptive activity immediately after feeding. The environmental conditions in Iraq, being typically characterized by high temperatures and arid environment, may well have some influence on the histochemical characteristics identified in this study. Goblet cells and the glycocalyx, being highly developed, may also be adaptive responses to maintain intestinal barrier function through thermal stress conditions [34] . The adaptive capacity of these cells may assist in commercial production of quail in Iraqi environmental conditions. This study's findings will have several useful implications for quail production in Iraq. The identified sexual dimorphism in duodenal morphology suggests that male and female birds may respond to different nutritional strategies and obtain their digestive capacity from distinct sources. The sturdy collagen structure suggests a good structural integrity to permit feeding with pelleted feeds and for high feeding schedules without injuring the intestine [35] . The plentiful carbohydrates detected by PAS staining indicates that Iraqi quail are fully adapted to feeding on diets with high carbohydrate levels, which is of particular importance to enable formulation of feeds that are cost effective with local grains. The variation in the goblet cell densities recorded indicates that quail may be more at risk to acidic damage in the proximal duodenum. This would highlight the necessity of sound feed processing and pH control within commercial feeds [36] . When compared to other avian species, the anatomy of the Iraqi quail duodenum contains some characteristics shared by other gallinaceous species but also contains some unique characteristics. The villus-crypt ratio 4.3:1 is similar to other studies finding result in chickens but higher in turkeys, which indicates a good balance between absorptive magnitude of the villus to the cellular renewal of the crypts [37] . The ratio shows a normal intestinal function for digestive efficiency. The histochemical profiles reported in this study can serve as a baseline to assess intestinal health in Iraqi quail populations. Any differences from the normal values reported may represent nutritional deficiencies, pathological state, or adverse environmental factors. Also, establishing the baseline values will be beneficial to veterinary diagnoses and monitoring performance and production in a commercial operation [38] . The effects from climate change would also impact on Iraqi agriculture and livestock production products; which would ultimately affect the digestive physiology of quail birds. The histochemical adaptations demonstrated in this research highlight active current adaptability to the local environment and monitoring in the future may show alterations in duodenal structure and function if the environmental conditions continue to change [39] . This baseline information will be useful going forward to monitor the effect of changing environmental conditions and population adaptation on quail physiology.

The design used in this study makes use of several staining methods that are complementary which provides a comprehensive research methodology for duodenal analysis, by combining the results of H&E, Masson's Trichrome, and PAS staining, an overall duodenal structure and function can be correlated [40] . This multi-staining model has the potential to be used in further avian digestive architecture and physiology studies. Notable findings included well-developed intestinal villi with a mean height of 847±52 μm providing an excellent absorptive capacity necessary for their fast growth nature. The sex dimorphism noted with male birds possessing greater villus height and mucosal thickness suggests differences in nutrition for commercial production. The goblet cell distribution indicated greater density concentrations in the proximal duodenum reflecting an appropriate physiological adaptation to the digestive demand in this anatomical area.

Masson's trichrome stain also revealed an extensive collagen network which indicated significant structural capacity for the mechanical requirements stemming from intensive feeding and digestion. The presence of polysaccharides in the gastrointestinal tract as noted in the PAS staining suggested the presence of carbohydrate-based digestive and protective components, confirming adaptation to the carbohydrate-based diet to which Iraqi quail producers generally use.

The presented data will provide useful baseline conditions for Iraqi quail populations and general guidance for improved systems in production. The noted histochemical structure and functional characteristics support and improve the developing quail industry in Iraq and provide insight into some of the general principles of avian digestive physiology. Future studies should aim to assess the distinct effects of dietary curriculums and management strategies on the noted histochemical changing features to further enhance production and welfare efficacy.

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