Performance, Serum Biochemical and Immunological Parameters, and Digestive Enzyme and Intestinal Barrier-Related Gene Expression of Broiler Chickens Fed Fermented Fava Bean By-Products as a Substitute for Conventional Feed

Improving the nutritional quality of unconventional feed ingredients such as fava bean by-products can enhance their utilization by broiler chickens. Hence, the quality of fermented fava bean by-products (FFB), in addition to growth, nutrient digestibility, digestive enzyme, and intestinal barrier-related gene expression, and serum biochemical and immunological parameters were evaluated in response to different levels of FFB. A total of 500 1-day-old broiler chicks (46.00 ± 0.388 g) were allocated to five groups with 10 replicates each (100 chicks per treatment). The first group was fed a corn–soybean diet (control diet), and the other four groups were fed a diet containing 5, 15, 25, and 35% FFB for 38 days. Birds fed 25% FFB exhibited maximum body weight gain (increase by 12.5%, compared with the control group) and the most improved feed conversion ratio. Additionally, birds fed FFB at 15, 25, and 35% showed improved dry matter and crude protein digestibility. Moreover, birds fed FFB at 25 and 35% exhibited a decrease in ileal pH and an increase in fiber digestibility (p < 0.05). Upregulation of digestive enzyme genes (AMY2A, PNLIP, and CCK) was observed in groups fed with FFB. The most prominent upregulation of genes encoding tight junction proteins (claudin-1, occludin, and junctional adhesion molecules) in the duodenum was observed in chicks fed 25 and 35% FFB (increase of 0.66-, 0.31-, and 1.06-fold and 0.74-, 0.44-, and 0.92-fold, respectively). Additionally, the highest expression level of enterocyte protective genes [glucagon-like peptide (GLP-2), mucin-2 (MUC-2), and fatty acid-binding protein (FABP-6)] was detected in duodenum of chicks fed high levels of FFB. Substitution of corn–soybean diet with FFB had an inhibitory effect on cecal pathogenic microbes (Escherichia coli and Clostridium perfringens) and increased beneficial microflora (Lactobacilli and Bifidobacterium), especially at high levels. Additionally, an increase was observed in IgM and lysozyme activity, with no effect on IgA in all groups fed FFB. All levels of FFB decreased cholesterol levels. Based on our results, we concluded that substitution of corn–soybean diet with FFB can improve the growth rate and nutrient digestibility of broiler chickens, enhance their intestinal barrier functions, and increase the number of beneficial microorganisms. Using FFB at 25% had a positive effect on the growth performance of broiler chickens, and it could be utilized in poultry farms.

Improving the nutritional quality of unconventional feed ingredients such as fava bean by-products can enhance their utilization by broiler chickens. Hence, the quality of fermented fava bean by-products (FFB), in addition to growth, nutrient digestibility, digestive enzyme, and intestinal barrier-related gene expression, and serum biochemical and immunological parameters were evaluated in response to different levels of FFB. A total of 500 1-day-old broiler chicks (46.00 ± 0.388 g) were allocated to five groups with 10 replicates each (100 chicks per treatment). The first group was fed a corn-soybean diet (control diet), and the other four groups were fed a diet containing 5, 15, 25, and 35% FFB for 38 days. Birds fed 25% FFB exhibited maximum body weight gain (increase by 12.5%, compared with the control group) and the most improved feed conversion ratio. Additionally, birds fed FFB at 15, 25, and 35% showed improved dry matter and crude protein digestibility. Moreover, birds fed FFB at 25 and 35% exhibited a decrease in ileal pH and an increase in fiber digestibility (p < 0.05). Upregulation of digestive enzyme genes (AMY2A, PNLIP, and CCK) was observed in groups fed with FFB. The most prominent upregulation of genes encoding tight junction proteins (claudin-1, occludin, and junctional adhesion molecules) in the duodenum was observed in chicks fed 25 and 35% FFB (increase of 0.66-, 0.31-, and 1.06-fold and 0.74-, 0.44-, and 0.92-fold, respectively). Additionally, the highest expression level of enterocyte protective genes [glucagon-like peptide (GLP-2), mucin-2 (MUC-2), and fatty acid-binding protein (FABP- 6)] was detected in duodenum of chicks fed high levels of FFB. Substitution of corn-soybean diet with FFB had an inhibitory effect on cecal pathogenic microbes (Escherichia coli and Clostridium perfringens) and increased beneficial microflora (Lactobacilli and Bifidobacterium), especially at high levels. Additionally, an increase was observed in IgM and lysozyme activity, with no effect on IgA in all groups fed FFB. All levels of FFB decreased cholesterol levels. Based on our results, we concluded that substitution of corn-soybean diet with FFB can improve the growth rate and nutrient digestibility of broiler chickens, enhance their intestinal barrier functions, and increase the number of beneficial microorganisms. Using FFB at 25% had a positive effect on the growth performance of broiler chickens, and it could be utilized in poultry farms.

INTRODUCTION
Increasing demand for conventional or basic feed ingredients such as corn and soybean meal (SBM) in the biofuel industry and poultry ration leads to increased costs, which encourages poultry nutritionists to search for low-cost unconventional feed alternatives and locally cultivated food crops, especially from high-protein legumes (1,2). Supplementation of poultry feed with native legumes and their by-products can offer additional protein, energy, and minerals that improve animal productivity (3,4). Fava beans are an alternative legume that can partially replace SBM as a protein source (5). Previously, feeding of broiler chickens on green beans processing by-products with enzymes significantly enhanced their performance up to 16% (6). Fava beans contain a high level of proteins (∼26%), carbohydrates (up to 77%), dietary fiber, niacin, folic acid, and vitamin C (7). Bean crops' by-products comprise stems, empty or partly filled pod with seeds, and leaves that vary in composition according to the ratio of each, with the highest crude protein content found in seeds and leaves (∼22-25%) (8). Regardless of its high nutritional value, utilization of fava beans and their by-products as an alternative to conventional feed ingredients in poultry is limited because of the presence of many anti-nutritional factors (ANFs) (9), such as vicine, convicine, tannins, protease inhibitors, oligosaccharides, and non-starch polysaccharides (NSPs). Additionally, they have a low content of sulfur-containing amino acids that reduce their nutritional value, has a negative effect on nutrient digestibility, promotes pathogen proliferation, and reduces overall animal performance (10,11). Various processing methods including soaking, boiling, germination, roasting, autoclaving, microwaving, micronization, and fermentation can be utilized to counteract negative effects of these ANFs (12). Solid-state fermentation with the aid of beneficial bacteria and fungi can utilize agricultural byproducts as substrates for NSP-degrading microorganisms and convert them to nutritive feed ingredients (13,14). Thus, using such an approach can improve the nutritional properties of original products (15), increase nutrient bioavailability (16), and remove undesirable components from legumes and other feed constituents (17). Moreover, microbial fermentation can enhance the proportion of digestible phosphorus, increase protein concentration and digestibility (18), and improve fiber digestibility (19). Moreover, this process helps enrich the raw material with vitamins and minerals and increase the effective release of methionine, lysine, threonine, and small peptides (20,21). Lactic acid bacteria (LAB) including Lactobacillus, Streptococcus, Pediococcus, and Leuconostoc are important because of their unique organoleptic properties (22). Previous studies have shown that fermented soybeans, soybean by-products, and rapeseed meal have a positive effect on broiler chicken performance (21,23). However, the influence of fermentation on the nutritional value of fava bean by-products lacks sufficient support, and there is a lack of information on the effect of fermented fava bean by-products (FFB) in broilers. Accordingly, the objective of this study was to elucidate the effects of substitution of corn-soybean diets with different levels (5,15,25, and 35%) of FFB on growth performance, nutrient digestibility, digestive and intestinal barrier gene expressions, cecal microbial population, and serum biochemical and immunological parameters in broiler chickens.

Preparation of FFB
Fungal and bacterial strains including Lactobacillus acidophilus (PTCC1643), Bacillus subtilis (PTCC1156), Lactobacillus plantarum (PTCC1058), and Aspergillus oryzae (PTCC5163) were used for fermentation. Each kilogram of fava bean byproducts (stem, leaf, and empty or poorly filled pod by-products) as fermentation substrate was inoculated and mixed with 1 L distilled water containing 10 6 spores/ml of A. oryzae and 10 8 CFU/ml of L. acidophilus, B. subtilis, and L. plantarum in fermentation tanks fitted with a one-way valve to allow leakage of produced gases and obstructed air from entry for 7 days. The fermented samples were then dried at 50 • C for 2 days. Dried samples were ground and mixed with other feed ingredients, and chemical analysis of fermented and unfermented fava beans was performed according to Latimer (24) ( Table 1).

Study Animals
Five hundred 1-day-old male Ross-308 broiler chicks were obtained from a local hatchery and were weighed on arrival (46.00 ± 0.388 g). Chicks were reared in a naturally ventilated open house with sawdust as litter. Lighting, room temperature, and relative humidity were monitored according to the recommendations of Ross-308 management (25). All animal

Experimental Design and Diets
Broiler chicks were randomly assigned to five groups (100 chicks per group), with 10 replicates each and 10 birds per replicate. The treatment groups received a basal corn-soybean diet (control) or a diet supplemented with 5, 15, 25, and 35% FFB. The experimental period was 38 days. All chicks were allowed free access to feed and water. All experimental diets were provided in mash and formulated according to the Ross Manual Guide (25), as presented in Table 2. Proximate analysis of different nutrients [dry matter (DM), crude protein (CP), crude fiber (CF), and ether extract (EE)] in feed ingredients and diets was performed according to the standard methods of the AOAC (26).

Growth Performance
Body weights (BWs) of chicks of each replicate were determined at 1, 23, and 38 days. The average feed intake (FI) per individual in each replicate was calculated as the difference between provided feed weight and remaining feed weight, which was then divided by the number of chicks in each replicate. At each time interval, BW gain (BWG) was estimated as the difference between final and initial BW. The feed conversion ratio (FCR) was calculated during the starter, grower, finisher, and overall phases (days  [FI (g/bird)/weight gain (g/bird)]. For apparent digestibility of nutrients, TiO 2 was used as an indigestible marker (3 g) and was added to each experimental diet. Chicken excreta were collected for 7 days, dried at 65 • C for 72 h, and dry matter, crude protein, ether extract, and crude fiber were analyzed according to the Association of Official Agricultural Chemists (26). The TiO 2 content in diet and excreta was calculated after acid digestion according to the method of Short et al. (27). The calculation was performed as follows: apparent nutrient digestibility = 100 -[100 × (indicator content (diet)/indicator content (feces) × nutrient content (feces)/nutrient content (diet)] (28).

Sample Collection
At the end of the experiment, chicks were killed by cervical dislocation (29), de-feathered, eviscerated, and weighed, after which dressing percentages were determined. Abdominal fat weight was determined and expressed as a percentage of the live BW. Blood samples (n = 10 per group) were collected from 10 randomly selected chicks of each replicate. Blood samples from the brachial vein were placed in dry sterilized tubes without anticoagulant and centrifuged at 3,000 rpm for 5 min for serum collection, for further clinico-biochemical analysis.

Clinico-Biochemical Analysis
The serum biochemical indices triglycerides (TAG), total cholesterol (TC), high density lipo-protein (HDL), low-density protein (LDL), alanine aminotransferase (ALT), and aspartate amino transferase (AST) were quantified using diagnostic kits (Spinreact, Santa Coloma, Spain). Serum lysozyme concentrations were measured according to the method of Lie et al. (30). The concentrations of immunoglobulins (IgA and IgM) were determined using ELISA kits for chickens (ABCAM Co. UK, cat. no. AB157692), according to the manufacturer's instructions.

RNA Extraction and Reverse-Transcription PCR
RNA was isolated from pancreatic and duodenal tissues (∼3 cm from the distal loop) using the QIAamp RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany). RNA concentration was measured using a NanoDrop TM 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) at an optical density of 260 nm.

Bacteriological Assay
At the end of the experimental period, the spread plate technique was used for counting selected microbes in cecal contents. Serial 10-fold dilution from 1 g of cecal content (n = 10 per group) was prepared in sterile saline.

Statistical Analysis
Data were analyzed using the general linear model (GLM) procedure of SPSS (SPSS Inc., Chicago, Illinois, USA) after confirming the homogeneity among experimental groups using Levene's test and normality using Shapiro-Wilk's test. The significant difference between the mean values was tested using Tukey's test, and the variation in the data was expressed as the standard error of the mean (SEM). The significance level was set at 0.05. Relative fold changes in the expression of target genes were calculated by the 2 − Ct method (33).

Chemical Analysis of Unfermented FFB and FFB
Crude protein and fat contents were significantly increased (p < 0.05) after fermentation of fava bean by-products; however, crude fiber, lignin, tannins, saponins, and cyanogenic glycosides significantly decreased (p < 0.05) in fermented fava beans compared to UFFB ( Table 1).

Growth Performance
Growth performance parameters of the broilers are presented in

Carcass Traits and Nutrient Digestibility
Data regarding the effect of FFB on dressing percentage and nutrient digestibility are shown in Table 5. The dressing percentage increased (p < 0.05), whereas the percentage of abdominal fat decreased (p < 0.05) in all experimental treatments. Substitution of the corn-soybean diet with 25 and 35% FFB significantly decreased the ileal pH. Nutrient digestibility of dry matter and crude protein was significantly (p < 0.05) increased in groups fed 15, 25, and 35% FFB, whereas the nutrient digestibility of crude fiber was increased (p < 0.05) in groups fed 25 and 35% FFB.

EXPRESSION OF INTESTINAL BARRIER, GUT PROTECTIVE, AND DIGESTIVE ENZYME GENES
mRNA expressions of genes encoding occludin, junctional adhesion molecules (JAM), and claudin were significantly upregulated (p < 0.05) in the duodenum with increasing levels of fermented FFB. The most prominent upregulation was observed in the 35% FFB group (increased by 0.74-, 0.44-, and 0.92-fold, respectively, vs. the control group) (Figure 1). Feeding broiler chickens with higher substitution levels of FFB (25% and 35%) significantly upregulated (p < 0.05) the expression of GLP-2 and FABP genes, compared with the control group. Moreover, the group fed 35% FFB showed the most significant level of MUC-2 (Figure 2). mRNA expression of the AMY2A gene was significantly upregulated (p < 0.05) in all groups fed FFB, whereas increasing the inclusion levels from FFB significantly upregulated PNLIP and CCK gene expressions (Figure 3). Table 6 shows that substitution of corn-soybean diet with different levels of FFB significantly (p < 0.05) increased the abundance of Bifidobacterium spp. and decreased (p < 0.05) Clostridium perfringens counts, compared with the controls. Lactobacillus spp. significantly (p < 0.05) increased groups fed 25% or 35% FFB diets. E. coli counts were significantly decreased (p < 0.05) in groups fed 15, 25, or 35% FFB.

Serum Biochemical and Immunological Parameters
The effects of FFB on liver enzymes, the lipid profile, and immunological parameters of broiler chickens are presented in Table 7. Compared to the control, different substitution levels of FFB had no effect on AST, ALT, uric acid, and creatinine (p > 0.05). Additionally, substitution of corn-soybean diet with different levels of FFB significantly decreased (p < 0.05) cholesterol and increased (p < 0.05) HDL. TAG and VLDL decreased (p < 0.05) in broilers fed diets substituted with 15,  , and Claudin-1 (CLDN-1; C) in the duodenum. FFB5% (basal diet substituted with 5% fermented fava beans by-products), FFB15% (basal diet substituted with 15% fermented fava beans by-products), FFB25% (basal diet substituted with 25% fermented fava beans by-products), FFB35% (basal diet substituted with 35% fermented fava beans by-products). a−c Means within the same column carrying different superscripts are significantly different at p < 0.05.

DISCUSSION
This study examined the application of microbial fermentation to an unconventional feed, fava bean by-products, and showed that it could be considered a successful tool to decrease ANFs and enhance their nutritive value. These beneficial effects may be related to the enrichment of feed with fermentation products such as probiotics and other bioactive functional ingredients. The benefit of using A. oryzae was the removal of oxygen from the fermentation media and achieving anaerobic conditions for L. acidophilus, B. subtilis, and L. plantarum growth and development. Once these bacteria were activated, the conditions for growth and proliferation of LAB were improved (34) with a subsequent reduction in feed pH that reduces viability and growth of pathogens. The pH of fermented FFB was 1.5-fold lower than that of unfermented fava beans. Previous studies have shown similar outcomes of decreased pH and increased LAB populations in fermented products (35,36). Additionally, in our study, FFB had higher concentrations of crude protein and fat and lower levels of crude fiber and ANFs such as lignin, tannins, saponins, and cyanogenic glycosides, compared to raw fava bean by-products. Ashayerizadeh et al. (37) reported that fermentation of rapeseed meal with L. acidophilus, B. subtilis, and Aspergillus    niger significantly reduced ANFs such as glucosinolates, phytic acid, total tannin, and phenolic compounds. Similarly, it was found that fermentation improved the nutritional quality of legumes by increasing crude protein and reducing crude fiber content (38,39). The increased protein content in FFB may arise from proteases produced from some microbial strains during fermentation, which decompose proteins to peptides and free amino acids in fermented products (40). Additionally, an increase in protein and fat content may be caused by a decrease in carbohydrate content during fermentation; subsequently, microorganisms can consume carbon and energy to produce microbial proteins (17). Moreover, higher crude protein content may also be derived from microbial protein synthesis accompanied by an increased microbial population at the time of fermentation (37,41). Moreover, a reduction in fiber content after fermentation was caused by production of fiber-degrading enzymes (42), reducing lignin and indigestible polyphenolic constituent levels (43) and decreasing NDF content (44). The decrease in ANFs in fava beans in the current study may be attributed to fungal and microbial enzymes degrading these compounds, which is in accordance with Soumeh et al. (45) who described similar benefits from microbial fermentation of SBM. Lactobacillus and B. subtilis have been described to produce phytase, xylanase, cellulase, and glucanase enzymes (46,47), which are responsible for degradation of non-beneficial components (48). Thus, subjecting legume feeds such as fava beans to fermentation may be a sound alternative to improve the nutritive value of legumes. Moreover, the results of growth performance parameters indicated that feeding fermented fava beans with improved nutritional value had a positive effect on BWG and FCR of broiler chickens, which allowed its application to formulated diets up to 35%. Furthermore, the maximum BWG and feed efficiency were observed in the group fed FFB at 25%. Overall improvement in broiler performance may be due to the improvement in nutritional quality of fava beans and nutrient apparent digestibility, activity of gut protective and digestive enzyme gene expressions, gut microbiology, and immunity of broiler chickens. The improvement effect of FFB on growth performance of broilers may be attributed to the lowering of ANFs in legumes after fermentation and increasing nutrient absorption and utilization (49). Similar studies have shown that microbial fermentation is an efficient process to eliminate ANFs and tannins in legume feed, thereby enhancing the nutritional quality and improving the performance of broilers (50). With fermentation, fava bean oligosaccharides and NSPs become more available for cecal microbes that facilitate their digestion (51). Substitution of the diet with 25 and 35% FFB significantly decreased ileal pH, because the fermentation process was associated with the production of organic acids and enhanced growth of LAB, leading to reduced gastric pH (34). Similarly, Drazbo et al. (44) reported that feeding turkeys fava beans led to lower ammonia levels in cecal digesta and lowered the pH of the intestinal digesta. Higher abundance of probiotic lactobacilli and bifidobacterial counts in the ileum was in accordance with the findings of Yamauchi and Suetsuna (52), who showed that fermented SBM (FSBM) increased the populations of yeasts, LAB, and Bacillus, which can improve the microecology balance and health of broiler gut and their growth performance. Additionally, reduced abundance of enteric pathogenic bacteria including C. perfringens and E. coli after feeding on higher levels of FFB may be related to the higher concentration of organic acids associated with the fermentation process, which enhanced LAB growth and proliferation, leading to reduced gastric pH, thereby inhibiting pathogen growth (34). Yin et al. (53) confirmed that FSBM feed increased the number of beneficial microbes and inhibited pathogen proliferation, and a more acidified environment in the intestine promotes proliferation of more desirable microbial taxa. Fermented feed inhibits growth of pathogenic bacteria and increases the population of desirable microbes by reducing the pH of the digestive tract (34,54). Fermentation increases the efficiency of feed and improves the growth rate by controlling the growth of non-pathogenic and pathogenic microbes in the intestinal tract of broilers (55).
Nutrient digestibility of dry matter and crude protein was increased in all groups fed different levels of FFB, except for the 5% FFB-fed group, whereas nutrient digestibility of crude fiber increased with increasing levels of FFB (25 and 35%). In accordance with our results, dietary supplementation with FSBM increased crude protein utilization in fodder and reduced the molecular size of peptides (56). Fermentation has also been shown to enhance the digestibility of different nutrients such as organic matter, nitrogen, amino acids, and fiber (57). Additionally, during L. acidophilus fermentation of fava bean byproducts, a significant amount of proteases, phytases, amylases, and β-glucanases can be generated and activated and improve the nutrient digestibility in animals (58). Jeong et al. (59) demonstrated that FSBM supplementation improved nutrient digestibility and productive performance of pigs. Moreover, fermentation with B. subtilis can improve the taste of feed, secrete digestive enzymes, stimulate digestion and absorption of nutrients (60), and produce bacitracin, polymyxin, nystatin, and gramicidin, which inhibit pathogen growth (61). Addition of FSBM to feed increased the activity of digestive enzymes (trypsin, lipase, and protease), improved the FCR and growth of weaned piglets (62), and increased the average daily gain (ADG) of finishing pigs (63). Similarly, using fermented feed, such as SBM, improved the morphological parameters of the intestine, increased nutrient absorption, and increased BWG in broilers (64). Additionally, Usayran et al. (65) found that broilers fed 30% tannin-free bean diets had better weight gain and FCRs than those fed a soybean control diet. Additionally, substituting SBM with fava beans had no adverse effect on the growth performance of guinea fowl broilers and broiler chickens (66,67). Farrell et al. (68) showed that fava beans can be added at up to 36% in broiler diets, where they partially replaced SBM without reducing bird performance. Moreover, Chachaj et al. (64) found that feeding turkeys 9 or 10% FSBM resulted in increased BWG, compared to the control group.
In line with the results of growth performance and nutrient digestibility, expression of digestive enzyme genes (amylase, lipase, and cholecystokinin) was upregulated after 38 days of feeding FFB. Moreover, with increasing levels of FFB, expression of these enzymes was more prominent. Similarly, activities of pancreatic enzymes in broilers were enhanced after feeding on FSBM (45). Microbial fermentation of cottonseed meal with the aid of B. subtilis has been shown to increase the activities of amylase and protease enzymes (69), which may result from B. subtilis, contributing to the production of protease and amylase enzymes. Moreover, increasing the consumption of carbohydrates can enhance mRNA expression levels of glucose transmitters, thereby increasing glucose absorption (70). Accordingly, feeding broiler chickens with higher levels of microbially fermented dried brewer grain enhanced pancreatic gene expression (amylase, protease, and lipase) and GLUT2 expression (71). Additionally, Lee et al. (72) showed that B. subtilis-based supplemented feed upregulated pancreatic lipase and carboxypeptidase genes in the gut.
Tight junctions (TJs), intercellular junctional complexes, consist of unique proteins including occludin, claudin, and JAM, which maintain epithelial cell integrity, allow nutrient transportation, and represent a barrier between the lumen and the host to inhibit bacterial invasion (73,74). TJ disruption may impair intestinal function, triggering gut leaking, which increases intestinal permeability and leads to systemic bacterial invasion, affecting animal health and growth performance (75,76). The MUC-2 gene is expressed by goblet cells, which form a mucus layer that prevents pathogen invasion, along with TJs (77). In the current study, maintaining the integrity of the intestinal barrier was evidenced by elevated TJPs and MUC-2 expression after feeding FFB. These results are in agreement with those of Lin and Lee (78), who reported that feeding Laetiporus sulphureusfermented products elevated zonula occludens-1, claudin-1, and mucin-2 expression. Fermented feeds were reported to enhance the intestinal barrier and immune function in poultry (79). Herein, improving the function of TJs may be attributed to the production of fermentation bioactive components such as oligosaccharides, isoflavones, and peptides, which protect intestinal cells and support their recovery (80). In addition to the presence of probiotic bacteria in fermented feed, TJ integrity and mucus secretion occurred in the gastrointestinal tract of broiler chicks. Moreover, dietary supplementation with B. subtilis and L. plantarum elevated mRNA expression of barrier functionrelated genes in broiler intestines (81,82). In the current study, substitution of corn-soybean diet with 5, 15, 25, and 35% FFB significantly decreased the abdominal fat percentage, while increasing the dressing percentage. Similarly, in previous reports, abdominal fat percentage was decreased by inclusion of 30% fava beans (65).
In the present study, substitution of corn-soybean control diet with fermented fava beans decreased the concentration of cholesterol and TAG, while increasing the concentration of HDL. Meanwhile, no considerable alterations were detected in AST, ALT, uric acid, and creatinine among different experimental groups. These results were consistent with those of Usayran et al. (65), who found that feeding on 30% tannin-free fava bean diets decreased the concentrations of cholesterol and TAG in broiler chicks, with no effect on AST, unlike the control diet. Additionally, Moschini et al. (83) found that AST, ALT, and plasma urea concentrations of birds fed 25 or 50% FFB were similar to those fed corn-SBM diets. High levels of HDL were found in the serum of turkeys fed 7 or 9% FSBM (64). Moreover, pigs fed FSBM diets had significantly lower creatinine concentrations than those fed control diets (63). The reduction of cholesterol and TAG levels after feeding on FFB may be attributed to the role of probiotic produced during fermentation in inhibiting 3-hydroxy-3-methylglutaryl CoA reductase enzyme incorporated in cholesterol biosynthesis (84).
In our study, the use of FFB in chicken diets significantly improved the immune response, which was represented by an increased concentration of IgM and lysozyme activity. Fermented feed can affect immune responses, which may be caused by the high content of LAB and bioactive peptides, as well as antioxidant compounds, compared to unfermented meals (85), and live microbes in fermented meals may act as probiotics and improve the humoral immune response of birds (36). In addition to bacteria in fermented feed producing lactic and acetic acid, which creates an acidic environment at pH 4, acidic molecules can penetrate cell membranes of bacteria and increase their acidity, which interferes with enzymatic processes and kills the bacteria (86). Our results were in line with those of Chachaj et al. (64), who reported that feeding turkeys FSBM at 7, 9, or 10% increased IgM levels, with no effect on IgA. Dietary supplementation with FSBM also plays an important role in relieving diarrhea and generating immune-related effectors, such as IgA and haptoglobin (87). In addition to Feng et al. (62), Fazhi et al. (88) showed that FSBM elevated the levels of serum IgA and IgM in broiler chickens and ducks. Additionally, FSM lowers the level of soy allergens (glycinin, β-conglycinin, and trypsin inhibitors) and reduces the risk of food hypersensitivity reactions (89).

CONCLUSION
Application of microbial fermentation, as novel processing technologies, for non-conventional feed resources such as fava bean by-products can enhance their nutritional value and utilization. Herein, feeding of broiler chickens on FFB can promote their growth performance by boosting digestive and intestinal barrier functions. These findings encourage the poultry feed industry to recommend FFB as an alternative nutritious unconventional feed ingredient, consequently minimizing the dependence on conventional feed sources and ensuring profitable broiler production.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Zagazig University, Egypt.

AUTHOR CONTRIBUTIONS
All authors shared in the study design, methodology, data collection and analysis, statistical analysis, and writing of the manuscript.