Special Issue:

Advancements in Animal Health and Production in Low and Middle-Income Countries

Physiological and Histopathological Investigations of Gluten Mushroom Supplements on the Small Intestine and Bursa of Fabricius in Chicken

Shaymaa Allawy Obed1, Namir I. Mohammed2*, Ali J. Jihad2

1Alfurat Alawsat Technical University, Almussaib Technical Institute, Al-Musayyab, Iraq; 2Department of Pathology and Poultry Diseases, College of Veterinary Medicine, University of Kerbala, Iraq.

Received |August 01, 2025; Accepted | September 05, 2025; Published | September 09, 2025

*Correspondence | Namir I. Mohammed, Department of Pathology and Poultry Diseases, College of Veterinary Medicine, University of Kerbala, Iraq; Email: [email protected]

Citation | Obed SA, Mohammed NI, Jihad AJ (2025). Physiological and histopathological investigations of gluten mushroom supplements on the small intestine and bursa of Fabricius in chicken. J. Anim. Health Prod. 13(s1): 389-394.

DOI | https://dx.doi.org/10.17582/journal.jahp/2025/13.s1.389.394

ISSN (Online) | 2308-2801

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Gluten is a protein made up of glutenin and gliadin, which are types of glutelins and prolamins, respectively. It is insoluble and can lead to malnutrition and inflammation in the small intestine of animals. Additionally, gluten is linked to celiac disease, a chronic condition that affects the small intestine in individuals who are genetically predisposed (Auricchio et al., 2022). In the poultry industry, feed costs represent over 70% of the total expenses for broiler production, making it crucial for productivity. However, the prices of feed cereals are increasing due to factors such as fluctuating oil prices and the use of grains as an energy source (Briani et al., 2008). In commercial poultry farming, ingredients such as wheat and barley, which contain gluten, are commonly used in poultry feeds because they provide high energy and are appealing to the birds. Nevertheless, since these cereal grains may constitute up a sizable portion of feed, there are worries about the long-term effects of gluten in chicken diets. To provide hens with a complete and balanced diet, it’s crucial to balance the gluten-rich grains in animal feed with other protein and energy sources because access of gluten can be harmful to them. Additionally, utilizing different protein sources, such as plant-based proteins or insect meal, may lower gluten levels and preserve hunger. Limiting the amount of gluten in diets for poultry can improve the general health and performance of hens while also lowering the risk of intestinal health issues.

Gliadins and glutenins are two about equal percentages of the complex mixture of hundreds of proteins that make up wheat gluten proteins (De Gorter et al., 2013; Hollon et al., 2015). The prolamins superfamily of plant proteins, which includes these proteins, is distinguished by a significant quantity of the amino acid proline and glutamine residues in them (Jabri et al., 2005). Gluten proteins, when hydrated, give dough the rheological qualities needed to bake bread, biscuits, and pasta. They also act as a binder in a variety of processed meals. While the glutenins are mostly responsible for the dough’s strength and elasticity, the gliadins are principally responsible for its viscosity and extensibility. The general health and performance of chickens can be enhanced, and any gastrointestinal health problems can be reduced by keeping gluten levels in poultry feeds within safe bounds. Gliadins and glutenins are two about equal groups of the complex mixture of hundreds of proteins that make up wheat gluten proteins (Patel and Robert, 2022; Rallabhandi et al., 2015).

The prolamins class of plant proteins, which includes these proteins, is known for having a lot of proline and glutamine amino acid sequences. Gluten proteins, when hydrated, provide dough the qualities that allow it to be used to make bread, biscuits, and pasta. They also serve as a binder in a variety of prepared meals. Gliadins are principally responsible for the dough’s viscosity and extensibility, whereas glutenins are mostly responsible for its strength and elasticity (Samasca et al., 2017; Święch et al., 2016; Wang et al., 2018).

The purpose of this study was to investigate how a wheat gluten diet affects the small intestinal structure, regulate pro-inflammatory gene expression, and growth performance of broilers at different ages in a dose-dependent manner.

Materials and Methods

Experimental design

Ninety birds have been repositioned in this research and were divided into 5 groups: One group served as control while the other groups were given different treatments. A 25% gluten diet was provided to Group G1, a 50% gluten diet to Group G2, and a mushroom-containing diet to Groups T3 and T4. In order to assess their effects on the birds, gluten and mushrooms were added to the meal in order concoct these diets. During the experiment the birds were carefully watched to observe if they have responded to different food combinations (Adelman et al., 2018; Antwi-Baffour et al., 2018).

Histological studies

The chicks duodenal content was collected and immediately preserved in 10% neutral buffered formalin NB for a full day to await pathological analysis. Then the specimens were given paraffin wax immersion and processed by a programmable device (Biobase, China). Tissue sections, cut to a thickness of 5 μm, were then mounted on glass slides and stained with hematoxylin and eosin (H and E). A Leica DM2500 microscope (Leica Microsystems, Germany) was used to take digital pictures of them at a fixed magnification of 100X. Crypt depths and villi dimensions were measured (Berthault et al., 2018; Chander et al., 2018).

Biochemical analysis

A computerized chemoanalyzer (Biobase, China) was used to experiment on the biochemical parameters.? Levels of triglycerides, lower-density lipoprotein (LDL), The levels of total cholesterol in plasma, high-density lipoprotein (HDL) and very-low-density lipoprotein (VLDL), respectively than that present across the membrane Liver enzymes such as alanine amino transferase ALT and aspartate aminotransferase (Khan et al., 2019).

Results

Biochemical parameter

The study examined at how various treatments (Groups G1, G2, G3, and G4) affected a variety of biochemical markers in relation to a Control Group. Group G2 had the highest cholesterol levels compared to all other groups, whereas the Control Group had the lowest. Triglyceride levels were lowest in the control group and considerably higher in Group G2 than in any other group. LDL levels followed the same pattern as cholesterol, with Group G2 having the highest levels and the Control Group having the lowest. The greatest VLDL levels were seen in Group G2, which could have an impact on their lipid utilization.

 

Table 1: Biochemical parameters of chickens.

Parameter	Control group (n=10)	Group G1 (n=10)	Group G2 (n=10)	Group G3 (n=10)	Group G4 (n=10)	Statistical analysis (p-value)
Cholesterol (mg/dl)	150.0 ± 5.123	160.5 ± 4.567	170.2 ± 6.789	160.1 ± 5.456	155.3 ± 4.890	p < 0.01
Triglycerides (mg/dl)	80.4 ± 3.234	85.1 ± 3.567	90.8 ± 4.012	82.5 ± 3.678	78.2 ± 3.456	p < 0.01
LDL (mg/dl)	60.2 ± 2.345	65.4 ± 2.678	70.5 ± 3.123	66.3 ± 2.890	62.7 ± 2.456	p < 0.01
VLDL (mg/dl)	16.5 ± 1.234	17.8 ± 1.456	18.9 ± 1.789	17.4 ± 1.678	16.2 ± 1.345	p < 0.05
HDL (mg/dl)	74.3 ± 2.123	78.5 ± 2.456	80.2 ± 3.456	79.1 ± 2.789	81.0 ± 2.345	p > 0.05
AST (U/L)	25.0 ± 1.456	30.2 ± 1.789	32.5 ± 1.890	28.3 ± 1.567	26.4 ± 1.234	p < 0.01
ALT (U/L)	20.4 ± 1.123	25.7 ± 1.456	28.3 ± 1.678	24.2 ± 1.345	22.9 ± 1.567	p < 0.01

 

When compared to other groups, Group G3 showed the highest HDL values, indicating a possibly healthier lipid profile. Group G3’s significantly higher AST and ALT readings suggested possible liver stress or injury in this specific group (Table 1).

 

 

The small intestine in control group showed the normal histological features and normal intestinal villi with a smaller number of inflammatory cells in laminae propria (Figure 1). Animals in G1 with 25% gluten showed a mild impact on intestinal health. For instance, a small dose of a particular dietary supplement or treatment may not significantly alter villous height. On the other hands, the G2 at 50% gluten, the effects on the intestinal villi are likely to be more severe. A substantial decrease in villous height could occur, reflecting significant disruption to gut function. The high dose could lead to inflammation, atrophy of the intestinal lining, or dysbiosis (an imbalance in gut bacteria), all of which can negatively impact villous structure.

The atrophy of lymphoid follicles in the Bursa of Fabricius is one of the main histological observations in birds exposed to gluten (Figure 2).

Discussion

Based on the use of new technologies, genetic improvement, feed, and health statuses, the poultry production in Iraq has shown a significant increase in recent years. Furthermore, this progress is contributed to by the abundant availability of gluten in grains and meals in our country, which constitute the basis of poultry nutrition, The use of different additives influences the nutritional quality of the diet, the development and functionality of the digestive organs, which will be reflected in the metabolism, production level, and chicken health (Beski et al., 2015).

These latest findings of Ojediran et al. (2017) observed no differences in intestinal pH in broiler chickens fed with diets containing a gluten (glutamine and proline), and low amino acid (methioninel and lysin). In contrast, Sun et al. (2024) have found differences in pH in the ileum of broiler chickens when they tested diets containing viscous properties agents and non-major anti-nutritional factors which lead to gut motility.

 

However, in G1 where animals were fed with 25% gluten, even low levels of certain compounds, especially if administered over an extended period, could gradually affect gut morphology. Subtle changes in the villous structure might begin to emerge, with slight reductions in villous height and overall gut surface area. These finding agree with (Lerner and Matthias, 2015; Liu et al., 2022; Obead et al., 2023) who stated that the truth seems to be that the expansion of the crypt epithelium and quantifiable changes in villus structure.

In animals fed with 50% gluten, this could result in impaired nutrient absorption, reduced growth rates, and overall poor performance in poultry. The present research finding consuming gluten can have significant histopathological repercussions on the intestine, particularly in those. Gluten causes immune-mediated damage to the small intestine in these, which results in significant alterations to the intestinal architecture. The most noticeable histological characteristic is villous atrophy, in which the villi shorten or flatten, making it more difficult for nutrients to be absorbed. In addition, finding increase in the depth of the intestinal crypts that produce new epithelial cells, frequently coexists with this. Furthermore, intraepithelial lymphocytes (IELs), especially CD8+ T-cells, which penetrate the epithelial layer and cause inflammation, have significantly increased. The lamina propria also exhibits inflammatory cell infiltration, where macrophages, lymphocytes, and plasma cells build up to produce chronic inflammation. Histopathological abnormalities gluten sensitivity, on the other hand, are typically milder and less specific, with little to no crypt hyperplasia or villous atrophy. Although these alterations are not as noticeable, some studies have indicated a modest increase in IELs. The lamina propria may also experience minor, localized inflammation, but the intestine’s overall architectural integrity is usually maintained. These results corresponding with (Ratcliffe and Härtle, 2014; Reese et al., 2018) indicating additional research is required on gluten to identify adverse effects on the poultry health. Several groups have lately reviewed this topic and found that gluten has several negative impacts that could affect health. Gut dysfunction or insufficiency results from these detrimental consequences are administered through toxic and immunological routes.

This study demonstrates that supplementation with mushrooms positively influences the histological structure of the intestinal tissue in chickens. The observed increases in villus height, crypt depth, goblet cell numbers, and enterocyte integrity are consistent with improved gut health and enhanced nutrient absorption capabilities. Mushrooms particularly were rich in bioactive compounds that may contribute to these beneficial effects. Polysaccharides, such as beta-glucans, are known to modulate the immune system and promote gut health, possibly by stimulating the production of mucus, reducing intestinal inflammation, and supporting epithelial integrity (Figure 1: G3, 4). These findings suggest that mushroom supplementation could be a valuable dietary intervention for improving intestinal health in poultry. Future studies should explore the long-term effects of mushroom supplementation on poultry performance, disease resistance, and overall productivity (Rostami et al., 2022).

The primary locations for B-cell development and maturation, which are essential for the immunological response, are lymphoid follicles. These follicles may exhibit degenerative symptoms after being exposed to gluten, which include a decrease in the quantity of mature B-cells and a general shrinkage in follicular size. The bird’s adaptive immunological function is hampered by the immune system’s decreased ability to manufacture antibodies as a result of B-cell depletion. These investigations akin with (Lerner et al., 2024) who have stated that the in addition to a worldwide decrease in bursa follicle size, the bursa showed a significant lymphocytic depletion, with remarkably few cells left in the medulla.

Histologically, these changes may be accompanied by the collapse of the follicular structure, with a reduction in the normal dense lymphocyte accumulation within the follicles. As the follicles become depleted, the surrounding medullary region of the Bursa, responsible for supporting immune cell maturation, may also undergo alterations. The overall structural integrity of the Bursa becomes compromised, leading to reduced functionality. These results agreement with previous finding (Taavela et al., 2013). Research on the effects of dietary components on the immune system in chickens has shown that various nutrients and food proteins can affect immune cell maturation and proliferation. It is hypothesized that gluten, as a protein source, may influence the maturation of B cells in the bursa of Fabricius, either enhancing or impairing immune function depending on the level and type of gluten in the diet. High gluten intake could potentially influence the proliferation of B cells or alter the expression of key immune markers in the bursa.

ACKNOWLEDGEMENT

Authors acknowledge Al-Furat Al-Awsat University, Technical College/Al-Musayyab, College of Veterinary Medicine, University of Kerbalaa, Iraq.

NOVELTY STATEMENT

We have identified a role of gluten in the health performance of poultry which will build foundation on the revised feeding schedule to improve poultry production performance.

AUTHOR’S CONTRIBUTION

Shyaymaa Allawy Obed: Design and performed the experiments and data collection, analysis. Namir I. Mohammed: Methodology, writing review and editing the manuscript. Ali J. Jihad: Histological process.

Generative AI or AI-assisted Technology Statement

The authors declare that no Genrative AI was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

References

Adelman DC, Murray J, Wu TT, Mäki M, Green PH, Kelly CP (2018). Measuring change in small intestinal histology in patients with celiac disease. Am. J. Gastroenterol., 113: 339–347. https://doi.org/10.1038/ajg.2017.480

Antwi-Baffour S, Kyeremeh R, Boateng SO, Annison L, Seidu MA (2018). Haematological parameters and lipid profile abnormalities among patients with type 2 diabetes mellitus in Ghana. Lipids Health Dis., 17(1): 283. https://doi.org/10.1186/s12944-018-0926-y

Auricchio R, Calabrese IGalatola M, Cielo D, Carbone F, Mancuso M, Matarese G, Troncone R, Auricchio S, Greco L (2022). Gluten consumption and inflammation affect the development of celiac disease in at-risk children. Sci. Rep., 12(1): 5396. https://doi.org/10.1038/s41598-022-09232-7

Berthault C, Larcher T, Härtle S, Vautherot JF, Trapp-Fragnet L, Denesvre C (2018). Atrophy of primary lymphoid organs induced by Marek’s disease virus during early infection is associated with increased apoptosis, inhibition of cell proliferation and a severe B-lymphopenia. Vet. Res., 49(1): 31. https://doi.org/10.1186/s13567-018-0526-x

Beski SS, Swick RA, Iji PA (2015). Specialized protein products in broiler chicken nutrition: A review. Anim. Nutr., 1(2): 47-53. https://doi.org/10.1016/j.aninu.2015.05.005

Briani C, Samaroo D, Alaedini A (2008). Celiac disease: From gluten to autoimmunity. Autoimmun. Rev., 7: 664–672. https://doi.org/10.1016/j.autrev.2008.05.006

Chander AM, Yadav H, Jain S, Bhadada SK, Dhawan DK (2018). Cross-talk between gluten, intestinal microbiota and intestinal mucosa in celiac disease: Recent advances and basis of autoimmunity. Front. Microbiol., 9: 2597. https://doi.org/10.3389/fmicb.2018.02597

De Gorter H, Drabik D, Just DR, Kliauga EM (2013). The impact of OECD biofuels policies on developing countries. Agric. Econ., 44: 477–486. https://doi.org/10.1111/agec.12031

Hollon J, Puppa EL, Greenwald B, Goldberg E, Guerriero A, Fasano A (2015). Effect of gliadin on permeability of intestinal biopsy explants from celiac disease patients and patients with non-celiac gluten sensitivity. Nutrients, 7: 1565–1576. https://doi.org/10.3390/nu7031565

Jabri B, Kasarda DD, Green PH (2005). Innate and adaptive immunity: The yin and yang of celiac disease. Immunol. Rev., 206: 219–231. https://doi.org/10.1111/j.0105-2896.2005.00294.x

Khan SH, Mukhtar N, Iqbal J (2019). Role of mushroom as dietary supplement on performance of poultry. J. Diet. Suppl., 16(5): 611–624. https://doi.org/10.1080/19390211.2018.1472707

Lerner A, Matthias T (2015). Changes in intestinal tight junction permeability associated with industrial food additives explain the rising incidence of autoimmune disease. Autoimmun. Rev., 14(6): 479–489. https://doi.org/10.1016/j.autrev.2015.01.009

Lerner A, Benzvi C, Vojdani A (2024). Gluten is a proinflammatory inducer of autoimmunity. J. Trans. Gastroenterol., 2(2): 109-124. https://doi.org/10.14218/JTG.2023.00060

Liu W, Chen X, Li H, Zhang J, An J, Liu X (2022). Anti-inflammatory function of plant-derived bioactive peptides: A review. Foods, 11(15): 2361. https://doi.org/10.3390/foods11152361

Obead WF, Khalaf HH, Mahmood HB (2023). Histological changes of extract cumin cyminum on the Leydig cells, seminiferous tubules and epididymis in adult rabbits (Oryctolagus cuniculus). Iraqi J. Vet. Sci., 37: 177–182. https://doi.org/10.33899/ijvs.2023.138536.2811

Ojediran TK, Oloruntade T, Durojaye B, Saka R, Emiola I (2017). Blood parameters, carcass yield, organ weight and villi morphometrics of broilers fed low protein diet in excess of dietary lysine. Trakia J. Sci., 2: 121-127. https://doi.org/10.15547/tjs.2017.02.004

Patel N, Robert ME (2022). Frontiers in celiac disease: Where autoimmunity and environment meet. Am. J. Surg. Pathol., 46(1): e43–e54. https://doi.org/10.1097/PAS.0000000000001639

Rallabhandi P, Sharma GM, Pereira M, Williams KM (2015). Immunological characterization of the gluten fractions and their hydrolysates from wheat, rye and barley. J. Agric. Food Chem., 63: 1825–1832. https://doi.org/10.1021/jf505716p

Ratcliffe JH, Härtle S (2014). B cells, the bursa of Fabricius and the generation of antibody repertoires. In: K.A. Schat, B. Kaspers and P. Kaiser (Eds.). Avian Immunol. San Diego: Academic Press. pp. 65–89. https://doi.org/10.1016/B978-0-12-396965-1.00004-2

Reese I, Schäfer C, Kleine-Tebbe J, Ahrens B, Bachmann O, Ballmer-Weber B, Beyer, K., Bischoff, S.C., Blümchen, K., Dölle, S. and Enck, P. (2018). Non-celiac gluten/wheat sensitivity (NCGS). A currently undefined disorder without validated diagnostic criteria and of unknown prevalence: Position statement of the task force on food allergy of the German Society of Allergology and Clinical Immunology (DGAKI). Allergo J. Int., 27(5): 147–151. https://doi.org/10.1007/s40629-018-0070-2

Rostami K, Ensari A, Marsh MN, Srivastava A, Villanacci V, Carroccio A, Asadzadeh Aghdaei H, Bai JC, Bassotti G, Becheanu G, Bell P (2022). Gluten induces subtle histological changes in duodenal mucosa of patients with non-coeliac gluten sensitivity: A multicentre study. Nutrients, 14(12): 2487. https://doi.org/10.3390/nu14122487

Samasca G, Lerner A, Girbovan A, Sur G, Lupan I, Makovicky P, Matthias T, Freeman HJ (2017). Challenges in gluten-free diet in coeliac disease: Prague consensus. Eur. J. Clin. Invest., 47(5): 394–397. https://doi.org/10.1111/eci.12755

Sun X, Ma L, Xuan Y, Liang J (2024). Degradation of anti-nutritional factors in maize gluten feed by fermentation with bacillus subtilis: A focused study on optimizing fermentation conditions. Fermentation, 10(11): 555. https://doi.org/10.3390/fermentation10110555

Święch E, Barszcz M, Tuśnio A, Taciak M (2016). Fed diets differing in standardized ileal digestible threonine and wheat gluten used as a source of non-essential amino acids. J. Anim. Feed Sci., 25: 226–234. https://doi.org/10.22358/jafs/65556/2016

Taavela J, Koskinen O, Huhtala H, Lähdeaho ML, Popp A, Laurila K, Collin P, Kaukinen K, Kurppa K, Mäki M (2013). Validation of morphometric analyses of small-intestinal biopsy readouts in celiac disease. PLoS One, 8: e76163. https://doi.org/10.1371/journal.pone.0076163

Wang Y, Liu X, Jin L, Wen Q, Zhang Y, Narasimha K, Yan E, Wang C, Zheng Y (2018). Effects of fermented corn gluten meal on growth performance, serum parameters, intestinal morphology and immunity performance of three-yellow broilers. Can. J. Anim. Sci., 99: 408–417. https://doi.org/10.1139/cjas-2017-0007