Fermented soybean meal exhibits probiotic properties when included in Japanese quail diet in replacement of soybean meal

Fermented soybean meal exhibits probiotic properties when included in Japanese quail diet in... ABSTRACT This study was conducted to investigate and compare the effect of dietary probiotic mixture (PM) and organic acid (OA) mixture with fermented soybean meal (FSBM) on performance, crop, and ceca microbiota, small intestine morphology, and serum lipid profile in Japanese quails. A total of 800 day-old Japanese quails was randomly allotted to 5 treatments with 8 replicate pens of 20 birds each, for 35 days. The experimental diets consisted of a control corn-soybean meal diet and 4 test diets: 1) control diet + 0.1% PM; 2) control diet + 0.2% OA mixture; 3) control diet + the combination of both PM and OA; and 4) an additives-free diet in which the soybean meal in the control diet was replaced with FSBM. The results indicated that in starter and the entire rearing periods, FSBM, PM, and PM+OA diets had significantly lower FCR compared to control or OA diets (P < 0.05). Birds in the FSBM group gained higher weight than control and OA birds (P < 0.05; 1 to 35 d). At d 21 and 35, birds fed the control diet showed significantly lower numbers of lactic acid bacteria in the crop, while coliforms were higher in the cecal content compared to the other diets (P < 0.05). At d 21, the villus height and villus height to crypt depth ratio in the duodenum and jejunum of birds fed PM, PM+OA, and FSBM diets were greater than in other treatments (P < 0.05). The serum concentrations of cholesterol and low-density lipoprotein cholesterol of birds fed PM, PM+OA, and FSBM diets were significantly lower than birds in control and AO groups (P < 0.05). The results obtained herein suggest that FSBM exhibits probiotic properties and, when used in substitution of SBM in Japanese quail diet, can improve growth performance, balance of desirable gastrointestinal microbiota in crop and ceca, small intestinal morphology, and serum lipid profile—likewise, a probiotic supplement. INTRODUCTION In-feed antibiotics have long been used in poultry feeds to improve feed efficiency and birds’ overall health status. However, bio-security threats for human and poultry health, arising from the antibiotic-resistant bacteria and the accumulation of antibiotic residues in poultry products, call for a worldwide removal of antibiotics, in particular at sub-therapeutic doses and as growth promoters from the poultry industry (Toghyani et al., 2010). In this regard, probiotics, prebiotics, organic acids (OA), and fermented feeds/ingredients, have been used as in-feed antibiotic alternatives to modulate poultry feed in order to improve animal health status and growth performance (Nava et al., 2009). The beneficial effects of probiotics and organic acids on performance indices and general health of different domesticated avian species are well documented (Levy et al., 2015; Seifi et al., 2017), although these effects have not always been consistent (Lee et al., 2010; Nosrati et al., 2017). Inclusion of fermented products in animal feed has been shown to induce advantageous effects on performance and gastrointestinal health by acting and exhibiting probiotic effects and could therefore be considered as an alternative for antibiotics growth promoters (Missotten et al., 2015). Fermented feed has a low pH, high concentration of lactic acid (>150 mM), and a high number of lactic acid bacteria (LAB) (approximately 109 CFU/mL of feed) (Ashayerizadeh et al., 2017). Engberg et al. (2009) showed that feeding fermented feed in laying hens increases LAB in the crop and decreases coliforms in the ileum. Furthermore, the OA content of fermented feed has been reported to modulate gastrointestinal microbiota balance through increasing acidity and lowering the pH (Niba et al., 2009). Besides the desired adverse effect on pathogenic bacteria, fermented feeds have some nutritional implications (Engberg et al., 2009). Microbial fermentation has been reported as an effective technique to eliminate or reduce anti-nutritional factors and improve nutritional value in plant-based protein meals (Ashayerizadeh et al., 2017; Jazi et al., 2017). Soybean meal (SBM) is the most commonly used plant protein source in poultry feed (Chiang et al., 2010). However, its application in poultry diets could often be limited due to some anti-nutritional factors, such as trypsin inhibitor (TI), oligosaccharides, phytic acid, and allergenic proteins, which interfere with digestion, absorption, and utilization of nutrients, especially for young chicks (Li et al., 2014; Feng et al., 2007a). Previous reports have indicated the reduction of TI and other anti-nutritional factors in SBM following fermentation (Wang et al., 2014; Sharawy et al., 2016). The hypothesis tested in the current study was that the antimicrobial compounds present in fermented SBM (FSBM) may act in a similar way to probiotics and OA in improving growth performance of Japanese quails through affecting gut microbiota and morphology. Furthermore, not many studies have tested FSBM against probiotic and/or OA in poultry, in particular Japanese quail. Therefore, in view of the dearth of data, the present study was conducted to investigate the chemical and biological characteristics of FSBM and to compare the efficacy of dietary probiotic and OA with FSBM on performance, gastrointestinal microbiota, morphometric analysis, and serum lipid profile in Japanese quails. MATERIALS AND METHODS Preparation of FSBM FSBM was prepared following the method used by Sun et al. (2013). Briefly, Lactobacillus plantarum (PTCC1058), Bacillus subtilis (PTCC1156) bacteria, and Aspergillus oryzae (PTCC5163) fungus were obtained from the Persian Type Culture Collection of Iranian Research Organization for Science and Technology. Each kg of SBM was inoculated and mixed with 1 L of distilled water containing approx. 105 CFU/mL of Lactobacillus plantarum and Bacillus subtilis and 106 spores of Aspergillus oryzae in a special tank (with a one-way valve to outflow gases produced and prevent air entry) for 7 d at 30°C. Ultimately, FSBM was dried for 2 d at 50°C. The dried samples were ground to pass through a 1.0 mm sieve and kept at room temperature until mixed in the diets. Chemical Analysis of SBM and FSBM Sub-samples from SBM and FSBM were obtained using a sampling probe, and a triple-rifled representative composite sample for each ingredient was used for the chemical analyses (conducted in triplicate). To determine the population of LAB, 1 g of SBM or FSBM was used to make serial 10-fold dilutions using buffered peptone water. Then, 0.1 mL of appropriate dilutions was spread on the plates containing modified de Man, Rogosa, and Sharpe agar. Plates were incubated in anaerobic conditions for 24 h at 37°C. After counting the number of colonies in each plate, the obtained number was multiplied by reversed dilution and reported as the number of colony forming unit (CFU) per 1 g sample. To determine the pH values, 20 g of each sample were transferred to a 250 mL beaker, and 200 mL of distilled water were added. The pH values were then measured by using a portable pH meter (pH Meter CG 804, Schott Gerate). The lactic acid concentration was determined by HPLC according to the method described by Marsili et al. (1983). Samples were analyzed for dry matter (DM), ash, crude protein (CP), ether extract (EE), and crude fiber (CF) according to AOAC (2005). Phytic acid was determined through the extraction of the samples with HCl and Na2SO4 and absorbance measured at 660 nm. TI activity in the samples was determined according to the method of Smith et al. (1980), and results are expressed as mg trypsin inhibited per g of dry sample. The contents of glycinin and β-conglycinin were estimated by the method of Wang et al. (2014). The amino acid (AA) compositions of SBM and FSBM were determined using an automated AA analyzer after hydrolyzing the samples with 6 M HCl at 110°C for 24 hours. Sulphur-containing AA were oxidized using performic acid before the acid hydrolysis. The changes of the chemical composition before and after the fermentation process are reported in Table 1. Table 1. Analyzed chemical composition of soybean meal pre- and post fermentation (% of dry matter basis). Soybean meal Item1 Pre-ferment Post ferment SEM P-value pH 5.92a 3.84b 0.28 0.007 Lactic acid (mmol/kg) 21.38b 172.78a 2.64 <0.001 LAB (Log10 CFU/g) 4.22b 10.64a 0.04 <0.001 DM 91.64 89.55 0.81 0.141 CP 44.03b 47.61a 0.63 0.011 EE 1.35 1.31 0.02 0.340 CF 7.31a 5.04b 0.19 0.001 Ash 6.26 6.34 0.24 0.83 Phytic acid (g/100 g) 0.63a 0.17b 0.01 <0.001 Trypsin inhibitor (mg/g) 2.85a 0.62b 0.25 0.003 β-conglycinin (mg/g) 59.62a 32.98b 1.07 <0.001 Glycinin (mg/g) 76.61a 28.26b 2.31 0.002 Indispensable amino acids Arginine 3.04 3.10 0.23 0.85 Histidine 1.15 1.19 0.04 0.57 Isoleucine 1.89 1.98 0.10 0.55 Leucine 3.16 3.26 0.21 0.19 Lysine 2.44 2.70 0.19 0.32 Methionine 0.57 0.58 0.03 0.79 Phenylalanine 2.12 2.20 0.15 0.73 Threonine 1.73 1.82 0.06 0.34 Valine 1.93 2.18 0.09 0.15 Dispensable amino acids Alanine 1.79 1.94 0.13 0.46 Asparagine 4.75 5.07 0.28 0.45 Cysteine 0.65 0.76 0.04 0.94 Glutamine 7.88 8.88 0.49 0.22 Glycine 1.74 1.90 0.04 0.07 Proline 2.14 2.24 0.13 0.62 Serine 2.10 2.32 0.08 0.13 Tyrosine 1.52 1.54 0.10 0.89 Soybean meal Item1 Pre-ferment Post ferment SEM P-value pH 5.92a 3.84b 0.28 0.007 Lactic acid (mmol/kg) 21.38b 172.78a 2.64 <0.001 LAB (Log10 CFU/g) 4.22b 10.64a 0.04 <0.001 DM 91.64 89.55 0.81 0.141 CP 44.03b 47.61a 0.63 0.011 EE 1.35 1.31 0.02 0.340 CF 7.31a 5.04b 0.19 0.001 Ash 6.26 6.34 0.24 0.83 Phytic acid (g/100 g) 0.63a 0.17b 0.01 <0.001 Trypsin inhibitor (mg/g) 2.85a 0.62b 0.25 0.003 β-conglycinin (mg/g) 59.62a 32.98b 1.07 <0.001 Glycinin (mg/g) 76.61a 28.26b 2.31 0.002 Indispensable amino acids Arginine 3.04 3.10 0.23 0.85 Histidine 1.15 1.19 0.04 0.57 Isoleucine 1.89 1.98 0.10 0.55 Leucine 3.16 3.26 0.21 0.19 Lysine 2.44 2.70 0.19 0.32 Methionine 0.57 0.58 0.03 0.79 Phenylalanine 2.12 2.20 0.15 0.73 Threonine 1.73 1.82 0.06 0.34 Valine 1.93 2.18 0.09 0.15 Dispensable amino acids Alanine 1.79 1.94 0.13 0.46 Asparagine 4.75 5.07 0.28 0.45 Cysteine 0.65 0.76 0.04 0.94 Glutamine 7.88 8.88 0.49 0.22 Glycine 1.74 1.90 0.04 0.07 Proline 2.14 2.24 0.13 0.62 Serine 2.10 2.32 0.08 0.13 Tyrosine 1.52 1.54 0.10 0.89 a,bMeans with different superscripts in each row are significantly different based on t test at P < 0.05. 1LAB = lactic acid bacteria; DM = dry matter; CP = crude protein; EE = ether extract; CF = crude fiber. View Large Table 1. Analyzed chemical composition of soybean meal pre- and post fermentation (% of dry matter basis). Soybean meal Item1 Pre-ferment Post ferment SEM P-value pH 5.92a 3.84b 0.28 0.007 Lactic acid (mmol/kg) 21.38b 172.78a 2.64 <0.001 LAB (Log10 CFU/g) 4.22b 10.64a 0.04 <0.001 DM 91.64 89.55 0.81 0.141 CP 44.03b 47.61a 0.63 0.011 EE 1.35 1.31 0.02 0.340 CF 7.31a 5.04b 0.19 0.001 Ash 6.26 6.34 0.24 0.83 Phytic acid (g/100 g) 0.63a 0.17b 0.01 <0.001 Trypsin inhibitor (mg/g) 2.85a 0.62b 0.25 0.003 β-conglycinin (mg/g) 59.62a 32.98b 1.07 <0.001 Glycinin (mg/g) 76.61a 28.26b 2.31 0.002 Indispensable amino acids Arginine 3.04 3.10 0.23 0.85 Histidine 1.15 1.19 0.04 0.57 Isoleucine 1.89 1.98 0.10 0.55 Leucine 3.16 3.26 0.21 0.19 Lysine 2.44 2.70 0.19 0.32 Methionine 0.57 0.58 0.03 0.79 Phenylalanine 2.12 2.20 0.15 0.73 Threonine 1.73 1.82 0.06 0.34 Valine 1.93 2.18 0.09 0.15 Dispensable amino acids Alanine 1.79 1.94 0.13 0.46 Asparagine 4.75 5.07 0.28 0.45 Cysteine 0.65 0.76 0.04 0.94 Glutamine 7.88 8.88 0.49 0.22 Glycine 1.74 1.90 0.04 0.07 Proline 2.14 2.24 0.13 0.62 Serine 2.10 2.32 0.08 0.13 Tyrosine 1.52 1.54 0.10 0.89 Soybean meal Item1 Pre-ferment Post ferment SEM P-value pH 5.92a 3.84b 0.28 0.007 Lactic acid (mmol/kg) 21.38b 172.78a 2.64 <0.001 LAB (Log10 CFU/g) 4.22b 10.64a 0.04 <0.001 DM 91.64 89.55 0.81 0.141 CP 44.03b 47.61a 0.63 0.011 EE 1.35 1.31 0.02 0.340 CF 7.31a 5.04b 0.19 0.001 Ash 6.26 6.34 0.24 0.83 Phytic acid (g/100 g) 0.63a 0.17b 0.01 <0.001 Trypsin inhibitor (mg/g) 2.85a 0.62b 0.25 0.003 β-conglycinin (mg/g) 59.62a 32.98b 1.07 <0.001 Glycinin (mg/g) 76.61a 28.26b 2.31 0.002 Indispensable amino acids Arginine 3.04 3.10 0.23 0.85 Histidine 1.15 1.19 0.04 0.57 Isoleucine 1.89 1.98 0.10 0.55 Leucine 3.16 3.26 0.21 0.19 Lysine 2.44 2.70 0.19 0.32 Methionine 0.57 0.58 0.03 0.79 Phenylalanine 2.12 2.20 0.15 0.73 Threonine 1.73 1.82 0.06 0.34 Valine 1.93 2.18 0.09 0.15 Dispensable amino acids Alanine 1.79 1.94 0.13 0.46 Asparagine 4.75 5.07 0.28 0.45 Cysteine 0.65 0.76 0.04 0.94 Glutamine 7.88 8.88 0.49 0.22 Glycine 1.74 1.90 0.04 0.07 Proline 2.14 2.24 0.13 0.62 Serine 2.10 2.32 0.08 0.13 Tyrosine 1.52 1.54 0.10 0.89 a,bMeans with different superscripts in each row are significantly different based on t test at P < 0.05. 1LAB = lactic acid bacteria; DM = dry matter; CP = crude protein; EE = ether extract; CF = crude fiber. View Large Experimental Birds and Diets All animal procedures were approved to be in compliance with the Institutional Animal Care and Use Committee of the Islamic Azad University, Isfahan (Khorasgan) Branch, Isfahan, Iran. A total of 800 day-old mixed-sex quails was purchased from a commercial hatchery, weighed, and randomly allocated into 5 treatment groups with 8 replicate pens of 20 birds based on a completely randomized design. The birds were housed in wire-floored pens (100 × 100 × 70 cm) in an environmentally controlled room with continuous light and were allowed ad libitum access to water and feed. The initial temperature of 37˚C was gradually reduced according to the age of the chicks until reaching 25˚C at the end of the 35-day experiment. Nutritional requirements of the Japanese quails were adopted from National Research Council (NRC, 1994) tables, and the basal diets were formulated accordingly (Table 2). The experimental diets consisted of a control corn-SBM diet and 4 test diets as follows: control + 0.1% probiotic mixture (PM, Protexin; Probiotics International Ltd., Somerset, UK), control + 0.2% OA mixture (Salkil; Anpario, UK), control + the combination of both PM and OA, and an additives-free diet in which the SBM in the control diet was replaced with FSBM. The PM used was a lyophilized mix comprising 2 × 109 CFU/g of 9 various microbial species, including: Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium bifidum, Streptococcus thermophilus, Enterococcus faecium, Aspergillus oryzae, and Candida pintolopesii. The OA mixture contained a combination of 7% ammonium propionate, 30% ammonium formate, 6% formic acid, and 57% material preservative. The inclusion rate of PM and OA was according to the manufacturer recommendations. Table 2. Composition and analysis of the experimental diets. Diet Ingredients (%) SBM based FSBM based Corn 49.10 54.71 Soybean meal (SBM) 41.89 - Fermented soybean meal (FSBM) - 37.74 Fish meal 3.0 3.0 Vegetable oil 3.54 2.04 CaCO3 0.85 0.83 Di-calcium phosphate 0.68 0.74 Sodium chloride 0.34 0.34 Vitamin premix1 0.30 0.30 Mineral premix2 0.30 0.30 Calculated composition3 Metabolizable energy (kcal/kg) 2950 2950 Crude protein (%) 24.41 24.41 Lysine (%) 1.32 1.32 Methionine (%) 0.35 0.35 Methionine + cysteine (%) 1.03 1.03 Calcium (%) 0.81 0.81 Available phosphorus (%) 0.40 0.40 Sodium (%) 0.16 0.16 Analyzed composition (%) Dry matter 89.1 88.29 Crude protein 25.07 25.54 Crude fiber 3.41 2.92 Ether extract 5.95 4.92 Calcium 0.91 0.87 Total phosphorus 0.58 0.56 Sodium 0.18 0.17 Chloride 0.23 0.21 Diet Ingredients (%) SBM based FSBM based Corn 49.10 54.71 Soybean meal (SBM) 41.89 - Fermented soybean meal (FSBM) - 37.74 Fish meal 3.0 3.0 Vegetable oil 3.54 2.04 CaCO3 0.85 0.83 Di-calcium phosphate 0.68 0.74 Sodium chloride 0.34 0.34 Vitamin premix1 0.30 0.30 Mineral premix2 0.30 0.30 Calculated composition3 Metabolizable energy (kcal/kg) 2950 2950 Crude protein (%) 24.41 24.41 Lysine (%) 1.32 1.32 Methionine (%) 0.35 0.35 Methionine + cysteine (%) 1.03 1.03 Calcium (%) 0.81 0.81 Available phosphorus (%) 0.40 0.40 Sodium (%) 0.16 0.16 Analyzed composition (%) Dry matter 89.1 88.29 Crude protein 25.07 25.54 Crude fiber 3.41 2.92 Ether extract 5.95 4.92 Calcium 0.91 0.87 Total phosphorus 0.58 0.56 Sodium 0.18 0.17 Chloride 0.23 0.21 1Supplied per kg of diet: 1.8 mg all-trans-retinyl acetate; 0.02 mg cholecalciferol; 8.3 mg alphatocopheryl acetate; 2.2 mg menadione; 2 mg pyridoxine HCl; 8 mg cyanocobalamin; 10 mg nicotine amid; 0.3 mg folic acid; 20 mg D-biotin; 160 mg choline chloride. 2Supplied per kg of diet: 32 mg Mn (MnSO4_H2O); 16 mg Fe (FeSO4_7H2O); 24 mg Zn (ZnO); 2 mg Cu (CuSO4_5H2O); 800 μg I (KI); 200 μg Co (CoSO4); 60 μg Se (NaSeO3). 3Crude protein, calcium, phosphorus, and amino acid contents are analyzed values. The metabolizable energy was calculated. View Large Table 2. Composition and analysis of the experimental diets. Diet Ingredients (%) SBM based FSBM based Corn 49.10 54.71 Soybean meal (SBM) 41.89 - Fermented soybean meal (FSBM) - 37.74 Fish meal 3.0 3.0 Vegetable oil 3.54 2.04 CaCO3 0.85 0.83 Di-calcium phosphate 0.68 0.74 Sodium chloride 0.34 0.34 Vitamin premix1 0.30 0.30 Mineral premix2 0.30 0.30 Calculated composition3 Metabolizable energy (kcal/kg) 2950 2950 Crude protein (%) 24.41 24.41 Lysine (%) 1.32 1.32 Methionine (%) 0.35 0.35 Methionine + cysteine (%) 1.03 1.03 Calcium (%) 0.81 0.81 Available phosphorus (%) 0.40 0.40 Sodium (%) 0.16 0.16 Analyzed composition (%) Dry matter 89.1 88.29 Crude protein 25.07 25.54 Crude fiber 3.41 2.92 Ether extract 5.95 4.92 Calcium 0.91 0.87 Total phosphorus 0.58 0.56 Sodium 0.18 0.17 Chloride 0.23 0.21 Diet Ingredients (%) SBM based FSBM based Corn 49.10 54.71 Soybean meal (SBM) 41.89 - Fermented soybean meal (FSBM) - 37.74 Fish meal 3.0 3.0 Vegetable oil 3.54 2.04 CaCO3 0.85 0.83 Di-calcium phosphate 0.68 0.74 Sodium chloride 0.34 0.34 Vitamin premix1 0.30 0.30 Mineral premix2 0.30 0.30 Calculated composition3 Metabolizable energy (kcal/kg) 2950 2950 Crude protein (%) 24.41 24.41 Lysine (%) 1.32 1.32 Methionine (%) 0.35 0.35 Methionine + cysteine (%) 1.03 1.03 Calcium (%) 0.81 0.81 Available phosphorus (%) 0.40 0.40 Sodium (%) 0.16 0.16 Analyzed composition (%) Dry matter 89.1 88.29 Crude protein 25.07 25.54 Crude fiber 3.41 2.92 Ether extract 5.95 4.92 Calcium 0.91 0.87 Total phosphorus 0.58 0.56 Sodium 0.18 0.17 Chloride 0.23 0.21 1Supplied per kg of diet: 1.8 mg all-trans-retinyl acetate; 0.02 mg cholecalciferol; 8.3 mg alphatocopheryl acetate; 2.2 mg menadione; 2 mg pyridoxine HCl; 8 mg cyanocobalamin; 10 mg nicotine amid; 0.3 mg folic acid; 20 mg D-biotin; 160 mg choline chloride. 2Supplied per kg of diet: 32 mg Mn (MnSO4_H2O); 16 mg Fe (FeSO4_7H2O); 24 mg Zn (ZnO); 2 mg Cu (CuSO4_5H2O); 800 μg I (KI); 200 μg Co (CoSO4); 60 μg Se (NaSeO3). 3Crude protein, calcium, phosphorus, and amino acid contents are analyzed values. The metabolizable energy was calculated. View Large Sampling and Measurements Growth performance parameters, including average feed intake (FI) and body weight gain (BWG), were measured at d 21 and 35 of the feeding trial, and feed conversion ratio (feed:gain, FCR) was calculated, and corrected for mortality within the same intervolves for each pen. At d 21 and 35, 2 birds/replicate were randomly selected from each treatment to examine pH, microbial population of the crop and ceca, and morphological characteristics of the small intestine. The birds were euthanized by cervical dislocation, and the digestive tract was carefully excised. Intestinal Morphological Analysis For intestinal morphometric analysis, birds on the designated evaluation d were euthanized, and duodenum, jejunum, and ileum samples were collected. A 1-cm segment of the midpoint of the duodenum, jejunum, and ileum from each bird was removed and fixed in 10% buffered formaldehyde for 48 hours. Each of these intestinal segments was embedded in paraffin, and a 5-μm section of each sample was placed on a glass slide and stained with hematoxylin and eosin for examination under a light microscope. All morphological parameters were measured using the ImageJ software package (http://rsb.info.nih.gov/ij/). Villus height (VH) was measured from the top of the villus to the top of the lamina propria. Crypt depth (CD) was measured from the base upwards to the region of transition between the crypt and villus. A total of 10 intact, well-oriented crypt-villus units was selected in duplicate from each tissue sample, and the averages of 20 values were obtained for each bird. Enumeration of Bacteria and pH Whole crop and ceca were aseptically removed and separated into sterile bags and homogenized. Samples were weighed, and 1:4 wt/vol dilutions were made with sterile 0.9% saline. Tenfold dilutions of each sample from each group were made in a sterile 96 well Bacti flat-bottom plate, and the diluted samples were plated on 3 different plates of medium to evaluate total number of LAB in modified de Man, Rogosa, and Sharpe agar; total coliforms in violet red bile agar; and total anaerobic bacteria (TAB) in plate count agar. For measuring the pH, about 1 gram of the crop and cecum contents of each bird was collected and transferred into 2 ml distilled water, then the pH values were measured using the portable pH meter. Serum Metabolites At d 21 and 35, 2 birds/pen were randomly selected, and blood samples were collected from the wing vein; the samples were centrifuged (at 2,000 × g for 5 min), and serum was separated and then stored at –20 ˚C until analyzed. Total cholesterol, triglycerides, and high-density lipoprotein cholesterol (HDL-C) values were measured using commercial laboratory kits (Pars Azmoon Kits; Pars Azmoon, Tehran, Iran). Very-low-density lipoprotein cholesterol (VLDL-C) values were calculated by dividing triglyceride values to unit 5 and low-density lipoprotein cholesterol (LDL-C) values by subtracting total values of HDL-C and VLDL-C from total cholesterol, according to the method described by Panda et al. (2006). Statistical Analysis Chemical composition, LAB numbers, and AA content of SBM pre- and post fermentation were compared using a t test. Data obtained during the feeding period of quail chicks were checked for normality and then analyzed based on a completely randomized design using the GLM procedures of SAS software (SAS, 2003). When a significant effect of treatment was detected, means were compared using Tukey's HSD test at P < 0.05 level of probability. RESULTS Chemical Composition of SBM and FSBM Chemical composition of the SBM pre- and post-fermentation process are presented in Table 1. The pH, CF, phytic acid, TI, β-conglycinin, and glycinin were significantly decreased and population of LAB and CP increased in SBM by the fermentation process (P < 0.05). The fermentation process had no significant effect on dry matter, ash, ether extract and amino acid content of SBM (P > 0.05). Growth Performance The results of the effects of experimental treatments on growth performance of Japanese quails are summarized in Table 3. BWG in birds fed FSBM diet was significantly higher than those fed the control and OA diets in starter and grower periods (P < 0.05). The BWG in the control treatment was significantly lower than FSBM and PM+OA in the grower period (P < 0.05). In the starter and entire rearing periods, birds fed FSBM, PM, or PM+OA diets had significantly lower FCR (P < 0.05). Also, over the grower period, FCR in FSBM and PM+OA treatments was significantly lower than the other treatments (P < 0.05). FI was not affected by dietary treatments (P > 0.05). Table 3. Effects of the experimental treatments on performance parameters of Japanese quails.1 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Starter (1–21 d) BWG 93.51b 96.23a,b 92.06b 97.55a,b 103.45a 2.36 0.031 FI 241.75 240.91 237.15 245.47 255.72 6.38 0.33 FCR 2.58a 2.50b 2.57a 2.51b 2.47b 0.01 0.002 Grower (21–35 d) BWG 78.36b 82.32a,b 81.88a,b 86.23a 87.80a 1.88 0.02 FI 286.25 299.89 297.43 307.34 308.45 7.28 0.25 FCR 3.65a 3.64a 3.63a 3.56b 3.51b 0.02 0.001 Overall (1–35 d) BWG 171.87b 178.56a,b 173.95b 183.79a,b 191.25a 4.10 0.02 FI 528.00 540.80 534.58 552.81 564.18 13.1 0.34 FCR 3.07a 3.03b 3.07a 3.01b 2.95c 0.01 <.001 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Starter (1–21 d) BWG 93.51b 96.23a,b 92.06b 97.55a,b 103.45a 2.36 0.031 FI 241.75 240.91 237.15 245.47 255.72 6.38 0.33 FCR 2.58a 2.50b 2.57a 2.51b 2.47b 0.01 0.002 Grower (21–35 d) BWG 78.36b 82.32a,b 81.88a,b 86.23a 87.80a 1.88 0.02 FI 286.25 299.89 297.43 307.34 308.45 7.28 0.25 FCR 3.65a 3.64a 3.63a 3.56b 3.51b 0.02 0.001 Overall (1–35 d) BWG 171.87b 178.56a,b 173.95b 183.79a,b 191.25a 4.10 0.02 FI 528.00 540.80 534.58 552.81 564.18 13.1 0.34 FCR 3.07a 3.03b 3.07a 3.01b 2.95c 0.01 <.001 a–cMeans with different superscripts in each row are significantly different (P < 0.05). 1Data represent means of 8 replicates of 20 quails per treatment. 2BWG = body weight gain; FI = feed intake; FCR = feed conversion ratio. 3CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large Table 3. Effects of the experimental treatments on performance parameters of Japanese quails.1 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Starter (1–21 d) BWG 93.51b 96.23a,b 92.06b 97.55a,b 103.45a 2.36 0.031 FI 241.75 240.91 237.15 245.47 255.72 6.38 0.33 FCR 2.58a 2.50b 2.57a 2.51b 2.47b 0.01 0.002 Grower (21–35 d) BWG 78.36b 82.32a,b 81.88a,b 86.23a 87.80a 1.88 0.02 FI 286.25 299.89 297.43 307.34 308.45 7.28 0.25 FCR 3.65a 3.64a 3.63a 3.56b 3.51b 0.02 0.001 Overall (1–35 d) BWG 171.87b 178.56a,b 173.95b 183.79a,b 191.25a 4.10 0.02 FI 528.00 540.80 534.58 552.81 564.18 13.1 0.34 FCR 3.07a 3.03b 3.07a 3.01b 2.95c 0.01 <.001 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Starter (1–21 d) BWG 93.51b 96.23a,b 92.06b 97.55a,b 103.45a 2.36 0.031 FI 241.75 240.91 237.15 245.47 255.72 6.38 0.33 FCR 2.58a 2.50b 2.57a 2.51b 2.47b 0.01 0.002 Grower (21–35 d) BWG 78.36b 82.32a,b 81.88a,b 86.23a 87.80a 1.88 0.02 FI 286.25 299.89 297.43 307.34 308.45 7.28 0.25 FCR 3.65a 3.64a 3.63a 3.56b 3.51b 0.02 0.001 Overall (1–35 d) BWG 171.87b 178.56a,b 173.95b 183.79a,b 191.25a 4.10 0.02 FI 528.00 540.80 534.58 552.81 564.18 13.1 0.34 FCR 3.07a 3.03b 3.07a 3.01b 2.95c 0.01 <.001 a–cMeans with different superscripts in each row are significantly different (P < 0.05). 1Data represent means of 8 replicates of 20 quails per treatment. 2BWG = body weight gain; FI = feed intake; FCR = feed conversion ratio. 3CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large Enumeration of Bacteria and pH The effect of experimental treatments on pH and microbial population in crop and cecal contents of Japanese quails are presented in Table 4. The PM, OA, PM+OA, and FSBM diets decreased pH of the crop and ceca at d 21 and 35 compared to the control diet (P < 0.05). Also, the LAB population was lower in the crop, while coliforms were higher in the ceca section of birds fed the control diet than in all the other treatments, for both ages tested (P < 0.05). At the end of starter and grower periods, the population of TAB in the crop and also ceca decreased significantly under the influence of PM, PM+OA, and FSBM diets compared to the other treatments (P < 0.05). Table 4. Effects of the experimental treatments on gastrointestinal microbiota composition (Log10 CFU/g) and pH value in Japanese quails.1 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Day 21 Crop pH 4.90a 4.77a,b 4.68b 4.64a 4.61b 0.05 0.012 LAB 7.32b 7.75a 7.52a,b 7.79a 7.86a 0.11 0.011 TAB 6.87a 6.36b 6.29b 6.12b 6.24b 0.13 0.005 Ceca pH 6.55a 6.43a,b 6.47a,b 6.35b 6.32b 0.04 0.020 Coliforms 5.65a 4.97b 5.50a 5.10b 4.91b 0.13 0.001 TAB 6.98a 6.32b 6.75a 6.31b 6.25b 0.12 0.001 Day 35 Crop pH 4.75a 4.47b 4.51b 4.42b 4.39b 0.03 0.005 LAB 7.81b 8.13a 7.98a,b 8.19a 8.25a 0.08 0.017 TAB 7.04a 6.49b 6.40b 6.32b 6.44b 0.12 0.003 Ceca pH 6.62a 6.43b 6.52a,b 6.39a 6.41b 0.04 0.011 Coliforms 6.07a 5.52b 5.91a 5.44b 5.58b 0.10 0.001 TAB 7.59a 7.17b 7.46a 7.01b 7.12b 0.09 0.001 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Day 21 Crop pH 4.90a 4.77a,b 4.68b 4.64a 4.61b 0.05 0.012 LAB 7.32b 7.75a 7.52a,b 7.79a 7.86a 0.11 0.011 TAB 6.87a 6.36b 6.29b 6.12b 6.24b 0.13 0.005 Ceca pH 6.55a 6.43a,b 6.47a,b 6.35b 6.32b 0.04 0.020 Coliforms 5.65a 4.97b 5.50a 5.10b 4.91b 0.13 0.001 TAB 6.98a 6.32b 6.75a 6.31b 6.25b 0.12 0.001 Day 35 Crop pH 4.75a 4.47b 4.51b 4.42b 4.39b 0.03 0.005 LAB 7.81b 8.13a 7.98a,b 8.19a 8.25a 0.08 0.017 TAB 7.04a 6.49b 6.40b 6.32b 6.44b 0.12 0.003 Ceca pH 6.62a 6.43b 6.52a,b 6.39a 6.41b 0.04 0.011 Coliforms 6.07a 5.52b 5.91a 5.44b 5.58b 0.10 0.001 TAB 7.59a 7.17b 7.46a 7.01b 7.12b 0.09 0.001 a,bMeans with different superscripts in each row are significantly different (P < 0.05). 1Data represent means of 8 observations per treatment. 2LAB = lactic acid bacteria; TAB = total anaerobic bacteria. 3CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large Table 4. Effects of the experimental treatments on gastrointestinal microbiota composition (Log10 CFU/g) and pH value in Japanese quails.1 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Day 21 Crop pH 4.90a 4.77a,b 4.68b 4.64a 4.61b 0.05 0.012 LAB 7.32b 7.75a 7.52a,b 7.79a 7.86a 0.11 0.011 TAB 6.87a 6.36b 6.29b 6.12b 6.24b 0.13 0.005 Ceca pH 6.55a 6.43a,b 6.47a,b 6.35b 6.32b 0.04 0.020 Coliforms 5.65a 4.97b 5.50a 5.10b 4.91b 0.13 0.001 TAB 6.98a 6.32b 6.75a 6.31b 6.25b 0.12 0.001 Day 35 Crop pH 4.75a 4.47b 4.51b 4.42b 4.39b 0.03 0.005 LAB 7.81b 8.13a 7.98a,b 8.19a 8.25a 0.08 0.017 TAB 7.04a 6.49b 6.40b 6.32b 6.44b 0.12 0.003 Ceca pH 6.62a 6.43b 6.52a,b 6.39a 6.41b 0.04 0.011 Coliforms 6.07a 5.52b 5.91a 5.44b 5.58b 0.10 0.001 TAB 7.59a 7.17b 7.46a 7.01b 7.12b 0.09 0.001 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Day 21 Crop pH 4.90a 4.77a,b 4.68b 4.64a 4.61b 0.05 0.012 LAB 7.32b 7.75a 7.52a,b 7.79a 7.86a 0.11 0.011 TAB 6.87a 6.36b 6.29b 6.12b 6.24b 0.13 0.005 Ceca pH 6.55a 6.43a,b 6.47a,b 6.35b 6.32b 0.04 0.020 Coliforms 5.65a 4.97b 5.50a 5.10b 4.91b 0.13 0.001 TAB 6.98a 6.32b 6.75a 6.31b 6.25b 0.12 0.001 Day 35 Crop pH 4.75a 4.47b 4.51b 4.42b 4.39b 0.03 0.005 LAB 7.81b 8.13a 7.98a,b 8.19a 8.25a 0.08 0.017 TAB 7.04a 6.49b 6.40b 6.32b 6.44b 0.12 0.003 Ceca pH 6.62a 6.43b 6.52a,b 6.39a 6.41b 0.04 0.011 Coliforms 6.07a 5.52b 5.91a 5.44b 5.58b 0.10 0.001 TAB 7.59a 7.17b 7.46a 7.01b 7.12b 0.09 0.001 a,bMeans with different superscripts in each row are significantly different (P < 0.05). 1Data represent means of 8 observations per treatment. 2LAB = lactic acid bacteria; TAB = total anaerobic bacteria. 3CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large Intestinal Morphological Analysis Table 5 summarizes the effects of the experimental treatments on intestinal morphometric analysis. At d 21, the VH and VH to CD ratio (VH:CD) in the duodenum and jejunum of the birds fed PM, PM+OA, and FSBM diets were greater than other experimental groups (P < 0.05). The use of feed additives or FSBM reduced the CD in the jejunum determined at the end of the starter period (P < 0.05). Also, at d 35, feeding the PM, PM+OA, and FSBM diets increased the VH and VH:CD ratio and decreased the CD in the jejunum (P < 0.05). Table 5. Effects of the experimental treatments on intestinal morphometric analysis (μm) in Japanese quails. Treatment1 Item CON PM OA PM+OA FSBM SEM P-value Day 21 Villus height (VH) Duodenum 765.29b 803.28a 761.13b 798.50a 810.51a 8.63 0.001 Jejunum 603.65b 675.87a 618.75b 663.26a 672.40a 8.11 <.001 Ileum 455.83 471.63 469.07 479.95 487.11 11.62 0.45 Crypt depth (CD) Duodenum 120.75 116.31 119.39 117.84 112.48 3.55 0.54 Jejunum 139.31a 127.54a,b 132.91a,b 121.36b 123.05b 4.05 0.03 Ileum 107.65 101.71 105.31 102.97 99.63 5.05 0.59 VH:CD Duodenum 6.34b 6.92a 6.40b 6.78a,b 7.23a 0.14 0.002 Jejunum 4.35b 5.30a 4.66b 5.47a 5.47a 0.10 <.001 Ileum 4.27 4.67 4.48 4.71 4.91 0.18 0.54 Day 35 Villus height (VH) Duodenum 890.25 918.22 895.04 902.68 932.54 14.99 0.28 Jejunum 637.07b 706.10a 649.19b 720.55a 731.66a 10.71 <.001 Ileum 520.85 547.03 522.24 534.20 542.71 15.38 0.67 Crypt depth (CD) Duodenum 169.04 163.21 165.73 168.96 157.80 5.20 0.53 Jejunum 142.12a 131.59a,b 142.44a 129.05a,b 126.37b 4.21 0.03 Ileum 128.76 123.01 127.22 126.64 121.51 7.17 0.94 VH:CD Duodenum 5.28 5.65 5.43 5.36 5.91 0.19 0.20 Jejunum 4.51b 5.37a 4.57b 5.59a 5.80a 0.13 <.001 Ileum 4.08 4.46 4.17 4.22 4.50 0.16 0.36 Treatment1 Item CON PM OA PM+OA FSBM SEM P-value Day 21 Villus height (VH) Duodenum 765.29b 803.28a 761.13b 798.50a 810.51a 8.63 0.001 Jejunum 603.65b 675.87a 618.75b 663.26a 672.40a 8.11 <.001 Ileum 455.83 471.63 469.07 479.95 487.11 11.62 0.45 Crypt depth (CD) Duodenum 120.75 116.31 119.39 117.84 112.48 3.55 0.54 Jejunum 139.31a 127.54a,b 132.91a,b 121.36b 123.05b 4.05 0.03 Ileum 107.65 101.71 105.31 102.97 99.63 5.05 0.59 VH:CD Duodenum 6.34b 6.92a 6.40b 6.78a,b 7.23a 0.14 0.002 Jejunum 4.35b 5.30a 4.66b 5.47a 5.47a 0.10 <.001 Ileum 4.27 4.67 4.48 4.71 4.91 0.18 0.54 Day 35 Villus height (VH) Duodenum 890.25 918.22 895.04 902.68 932.54 14.99 0.28 Jejunum 637.07b 706.10a 649.19b 720.55a 731.66a 10.71 <.001 Ileum 520.85 547.03 522.24 534.20 542.71 15.38 0.67 Crypt depth (CD) Duodenum 169.04 163.21 165.73 168.96 157.80 5.20 0.53 Jejunum 142.12a 131.59a,b 142.44a 129.05a,b 126.37b 4.21 0.03 Ileum 128.76 123.01 127.22 126.64 121.51 7.17 0.94 VH:CD Duodenum 5.28 5.65 5.43 5.36 5.91 0.19 0.20 Jejunum 4.51b 5.37a 4.57b 5.59a 5.80a 0.13 <.001 Ileum 4.08 4.46 4.17 4.22 4.50 0.16 0.36 a,bMeans with different superscripts in each row are significantly different (P < 0.05). 1CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large Table 5. Effects of the experimental treatments on intestinal morphometric analysis (μm) in Japanese quails. Treatment1 Item CON PM OA PM+OA FSBM SEM P-value Day 21 Villus height (VH) Duodenum 765.29b 803.28a 761.13b 798.50a 810.51a 8.63 0.001 Jejunum 603.65b 675.87a 618.75b 663.26a 672.40a 8.11 <.001 Ileum 455.83 471.63 469.07 479.95 487.11 11.62 0.45 Crypt depth (CD) Duodenum 120.75 116.31 119.39 117.84 112.48 3.55 0.54 Jejunum 139.31a 127.54a,b 132.91a,b 121.36b 123.05b 4.05 0.03 Ileum 107.65 101.71 105.31 102.97 99.63 5.05 0.59 VH:CD Duodenum 6.34b 6.92a 6.40b 6.78a,b 7.23a 0.14 0.002 Jejunum 4.35b 5.30a 4.66b 5.47a 5.47a 0.10 <.001 Ileum 4.27 4.67 4.48 4.71 4.91 0.18 0.54 Day 35 Villus height (VH) Duodenum 890.25 918.22 895.04 902.68 932.54 14.99 0.28 Jejunum 637.07b 706.10a 649.19b 720.55a 731.66a 10.71 <.001 Ileum 520.85 547.03 522.24 534.20 542.71 15.38 0.67 Crypt depth (CD) Duodenum 169.04 163.21 165.73 168.96 157.80 5.20 0.53 Jejunum 142.12a 131.59a,b 142.44a 129.05a,b 126.37b 4.21 0.03 Ileum 128.76 123.01 127.22 126.64 121.51 7.17 0.94 VH:CD Duodenum 5.28 5.65 5.43 5.36 5.91 0.19 0.20 Jejunum 4.51b 5.37a 4.57b 5.59a 5.80a 0.13 <.001 Ileum 4.08 4.46 4.17 4.22 4.50 0.16 0.36 Treatment1 Item CON PM OA PM+OA FSBM SEM P-value Day 21 Villus height (VH) Duodenum 765.29b 803.28a 761.13b 798.50a 810.51a 8.63 0.001 Jejunum 603.65b 675.87a 618.75b 663.26a 672.40a 8.11 <.001 Ileum 455.83 471.63 469.07 479.95 487.11 11.62 0.45 Crypt depth (CD) Duodenum 120.75 116.31 119.39 117.84 112.48 3.55 0.54 Jejunum 139.31a 127.54a,b 132.91a,b 121.36b 123.05b 4.05 0.03 Ileum 107.65 101.71 105.31 102.97 99.63 5.05 0.59 VH:CD Duodenum 6.34b 6.92a 6.40b 6.78a,b 7.23a 0.14 0.002 Jejunum 4.35b 5.30a 4.66b 5.47a 5.47a 0.10 <.001 Ileum 4.27 4.67 4.48 4.71 4.91 0.18 0.54 Day 35 Villus height (VH) Duodenum 890.25 918.22 895.04 902.68 932.54 14.99 0.28 Jejunum 637.07b 706.10a 649.19b 720.55a 731.66a 10.71 <.001 Ileum 520.85 547.03 522.24 534.20 542.71 15.38 0.67 Crypt depth (CD) Duodenum 169.04 163.21 165.73 168.96 157.80 5.20 0.53 Jejunum 142.12a 131.59a,b 142.44a 129.05a,b 126.37b 4.21 0.03 Ileum 128.76 123.01 127.22 126.64 121.51 7.17 0.94 VH:CD Duodenum 5.28 5.65 5.43 5.36 5.91 0.19 0.20 Jejunum 4.51b 5.37a 4.57b 5.59a 5.80a 0.13 <.001 Ileum 4.08 4.46 4.17 4.22 4.50 0.16 0.36 a,bMeans with different superscripts in each row are significantly different (P < 0.05). 1CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large Serum Metabolites The effect of experimental treatments on serum lipid profile of Japanese quails are shown in Table 6. The serum concentrations of cholesterol and LDL-C in birds fed PM, PM+OA, and FSBM diets were significantly lower than other treatments at d 21 (P < 0.05). The PM, PM+OA, and FSBM diet decreased serum concentrations of cholesterol, triglycerides, LDL-C, and VLDL-C compared to the control and OA diets at d 35 (P < 0.05). Table 6. Effects of the experimental treatments on serum lipid profile (mg/dL) in Japanese quails. Treatment2 Item1 CON PM OA PM+OA FSBM SEM P-value Day 21 Cholesterol 162.18a 144.81b 158.74a 140.17b 141.85b 3.12 <.001 Triglycerides 95.46 88.30 90.50 92.45 90.07 3.20 0.58 HDL-C 81.59 82.67 83.91 82.72 79.25 3.16 0.87 LDL-C 61.50a 44.48b 56.73a 38.95b 44.58b 3.14 <.001 VLDL-C 19.09 17.66 18.10 18.49 18.01 0.64 0.58 Day 35 Cholesterol 150.46a 136.20b 146.48a 131.60b 134.61b 3.58 0.001 Triglycerides 72.25a 53.84b 67.55a 52.31b 54.80b 3.29 <.001 HDL-C 85.40 89.60 82.18 88.75 86.01 2.99 0.43 LDL-C 50.60a 35.82b 50.79a 32.38b 37.63b 3.82 0.001 VLDL-C 14.45a 10.77b 13.51a 10.46b 10.96b 0.65 <.001 Treatment2 Item1 CON PM OA PM+OA FSBM SEM P-value Day 21 Cholesterol 162.18a 144.81b 158.74a 140.17b 141.85b 3.12 <.001 Triglycerides 95.46 88.30 90.50 92.45 90.07 3.20 0.58 HDL-C 81.59 82.67 83.91 82.72 79.25 3.16 0.87 LDL-C 61.50a 44.48b 56.73a 38.95b 44.58b 3.14 <.001 VLDL-C 19.09 17.66 18.10 18.49 18.01 0.64 0.58 Day 35 Cholesterol 150.46a 136.20b 146.48a 131.60b 134.61b 3.58 0.001 Triglycerides 72.25a 53.84b 67.55a 52.31b 54.80b 3.29 <.001 HDL-C 85.40 89.60 82.18 88.75 86.01 2.99 0.43 LDL-C 50.60a 35.82b 50.79a 32.38b 37.63b 3.82 0.001 VLDL-C 14.45a 10.77b 13.51a 10.46b 10.96b 0.65 <.001 a,bMeans with different superscripts in each row are significantly different (P < 0.05). 1HDL-C = high-density lipoprotein cholesterol; LDL-C = low-density lipoprotein cholesterol; VLDL-C = very-low-density lipoprotein cholesterol. 2CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large Table 6. Effects of the experimental treatments on serum lipid profile (mg/dL) in Japanese quails. Treatment2 Item1 CON PM OA PM+OA FSBM SEM P-value Day 21 Cholesterol 162.18a 144.81b 158.74a 140.17b 141.85b 3.12 <.001 Triglycerides 95.46 88.30 90.50 92.45 90.07 3.20 0.58 HDL-C 81.59 82.67 83.91 82.72 79.25 3.16 0.87 LDL-C 61.50a 44.48b 56.73a 38.95b 44.58b 3.14 <.001 VLDL-C 19.09 17.66 18.10 18.49 18.01 0.64 0.58 Day 35 Cholesterol 150.46a 136.20b 146.48a 131.60b 134.61b 3.58 0.001 Triglycerides 72.25a 53.84b 67.55a 52.31b 54.80b 3.29 <.001 HDL-C 85.40 89.60 82.18 88.75 86.01 2.99 0.43 LDL-C 50.60a 35.82b 50.79a 32.38b 37.63b 3.82 0.001 VLDL-C 14.45a 10.77b 13.51a 10.46b 10.96b 0.65 <.001 Treatment2 Item1 CON PM OA PM+OA FSBM SEM P-value Day 21 Cholesterol 162.18a 144.81b 158.74a 140.17b 141.85b 3.12 <.001 Triglycerides 95.46 88.30 90.50 92.45 90.07 3.20 0.58 HDL-C 81.59 82.67 83.91 82.72 79.25 3.16 0.87 LDL-C 61.50a 44.48b 56.73a 38.95b 44.58b 3.14 <.001 VLDL-C 19.09 17.66 18.10 18.49 18.01 0.64 0.58 Day 35 Cholesterol 150.46a 136.20b 146.48a 131.60b 134.61b 3.58 0.001 Triglycerides 72.25a 53.84b 67.55a 52.31b 54.80b 3.29 <.001 HDL-C 85.40 89.60 82.18 88.75 86.01 2.99 0.43 LDL-C 50.60a 35.82b 50.79a 32.38b 37.63b 3.82 0.001 VLDL-C 14.45a 10.77b 13.51a 10.46b 10.96b 0.65 <.001 a,bMeans with different superscripts in each row are significantly different (P < 0.05). 1HDL-C = high-density lipoprotein cholesterol; LDL-C = low-density lipoprotein cholesterol; VLDL-C = very-low-density lipoprotein cholesterol. 2CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large DISCUSSION In the present study, Lactobacillus plantarum, Bacillus subtilis, and Aspergillus oryzae were used for fermentation of SBM. Aspergillus oryzae fungus created an anaerobic environment for the growth of the facultative anaerobes Lactobacillus plantarum and Bacillus subtilis. The reduced pH value (subsequent to the production of OA by anaerobic bacteria) and simultaneously the increase in LAB population suggest that the essential and necessary conditions happened during the fermentation process (Ashayerizadeh et al., 2017). The extension of the fermentation process depends on the amount of lactic acid production. The lactic acid produced should be sufficient to lower the pH to 4 to 4.5, and remains stable during storage to prevent the growth of spoilage and pathogenic bacteria (Hasan, 2003). Similar to the fermentation conditions in our experiment, other studies also have shown that the use of Streptococcus thermophilus, Bacillus subtilis MA139, and Saccharomyces cerevisae or Aspergillus oryzae and Lactobacillus casei in the fermentation of SBM reduces pH and increases the concentration of lactic acid (Chen et al., 2010; Wang et al., 2014). Also, reduction of pH value and increased probiotic bacteria population in FSBM using Bacillus subtilis, Aspergillus niger, and Saccharomyces cerevisiae are reported (Wang et al., 2012). Microbial fermentation of SBM significantly reduced phytic acid, TI, β-conglycinin, glycinin, and CF, besides increasing CP concentration. Similar to our findings, in the studies of Sharawy et al. (2016) and Gao et al. (2013), decreased phytic acid and TI and increased CP were observed in FSBM compared to SBM. Other reports have shown that fermentation of SBM with Aspergillus oryzae (Chen et al., 2013a), Bacillus amyloliquefaciens vs. Lactobacillus spp. and Saccharomyces cerevisiae (Chi and Cho, 2016) reduces CF or oligosaccharides and increases nitrogen content. Decreased concentration of the allergenic protein (β-conglycinin and glycinin) in SBM following fermentation also has been reported (Li et al., 2014; Seo and Cho, 2016). Decreasing TI during fermentation is associated with structural change, precipitation, and inactivation of the TI binding site to trypsin (Chen et al., 2013a; Chi and Cho, 2016). The degrading activity of the enzymes, such as cellulases and phytase, which are produced by microorganisms, could be responsible for the reduction of CF and phytic acid (Chi and Cho, 2016; Ashayerizadeh et al., 2017). Li et al. (2014) reported that after fermentation, β-conglycinin and glycinin would be degraded to small-molecule peptides. The previous studies on the fermentation of SBM with fungi or bacteria suggest that the increase in CP content could be attributed to the simple protein constituents of microbial mass as well as microbial metabolism during fermentation (Kook et al., 2014; Jazi et al., 2017). In the current study, the addition of a mixture of OA was not effective in improving performance parameters of Japanese quails. The beneficial impact of OA mixture on performance traits of poultry has not always been consistent. Similar to our findings, there are reports indicating no or minimal effect of OA on weight gain and feed efficiency (Basmacioglu-Malayoglu et al., 2016; Nosrati et al., 2017), while in contrast, others have reported higher BW and improved FCR by supplementation of OA in Japanese quail (Ocak et al., 2009) and broiler chicks (Levy et al., 2015). This discrepancy in the literature could be due to the different inclusion levels of acid, the chemical form, and their pKa values (Khan and Iqbal, 2016). Birds fed the PM and PM+OA supplemented diets gained higher weight and had a lower FCR compared to birds in the control group. Similarly, other studies also have reported and observed better productive traits by dietary supplementation of probiotics in Japanese quails (Banisharif et al., 2016; Seifi et al., 2017). Probiotics, in general, improve the characteristics of the intestinal microbiota mainly through reducing the pH of the gastrointestinal tract and suppression of pathogenic bacteria (by production of volatile fatty acids and bacteriocins), inhibition of the colonization of the bacteria by competitive exclusion, and also stimulating the immune system (Patterson and Burkholder, 2003). Replacing SBM with FSBM in the diet improved growth performance indices of Japanese quails, and the birds fed the FSBM even out-performed their counterparts in PM groups in terms of FCR. FSBM could have exhibited probiotic properties due to its high population of lactic acid producing bacteria and acidification capacity, thus lowering gastrointestinal tract (GIT) pH, resulting in improved performance (Ashayerizadeh et al., 2017; Jazi et al., 2017). Furthermore, the reduced anti-nutritional factors in FSBM, such as TI, β-conglycinin, glycinin, and also phytic acid, compared to SBM should have prevented gut inflammation and improved nutrient bioavailability and digestibility (Feng et al., 2007a). In line with these results, other studies also have shown that partial inclusion of fermented rapeseed meal (Chiang et al., 2010) and soybean meal (Feng et al., 2007b) in broiler chicks diet can improve weight gain and feed efficiency. It has long been proven that microbial activity in the GIT has a significant impact on growth performance and general health of poultry (Niba et al., 2009). Results of this experiment indicated that PM supplementation and FSBM inclusion in Japanese quails diet can manipulate the gastrointestinal microbiota balance in favor of beneficial and more desirable bacteria, and also reduce pH throughout the GIT. However, diet supplementation with OA failed to induce any effect on the population of LAB, TBA, and coliforms in the crop or in the cecum. Apparently, OA—in particular, butyrate—could be quickly absorbed in a bird's foregut, such as the crop, and thus may not be as effective as other feed additives that can remain active and effective even in lower parts of the GIT (Van der Wielen, 2002). Similarly, Goodarzi Boroojeni et al. (2014) did not observe any marked effect of OA supplementation on population of Escherichia coli and Bifidobacterium Spp. in the crop and cecal content in broiler chicks. Micro-encapsulated OA could remain active in the entire digestive tract and thus have a higher efficacy than free OA in lowering bacteria proliferation in the GIT (Levy et al., 2015). It is well documented that supplementation of multi-strain probiotics of diet could fortify intestinal beneficial microorganisms (such as Lactobacillus) and suppress the growth of potentially pathogenic bacteria, such as Escherichia coli in poultry (Zhang and Kim, 2014; Nosrati et al., 2017). Probiotics can inhibit the gastrointestinal colonization of pathogenic bacteria by facilitating antibody production, competition on adherence site, competition for nutrients between microorganisms, and bactericidal effects (Zhang and Kim, 2014). From this perspective, one of the main factors determining the efficacy of probiotics is the ratio of LAB to pathogenic bacteria (Zhang and Kim, 2014). Likewise, fermented feedstuff such as SBM can increase LAB populations throughout the GIT by acidification of the foregut (especially the crop) and provide a favorable condition for the establishment and growth of beneficial bacteria such as the LAB (Niba et al., 2009). An increase in beneficial microbiota population leads to higher production of short-chain fatty acids and consequently decreased pH of GIT and also creation of a competitive exclusion phenomena, both of which help in forming a natural defense barrier against infection and pathogenic bacteria such as coliforms (Engberg et al., 2009; Ashayerizadeh et al., 2017). Higher LAB and lower fecal Escherichia coli counts in piglets have been observed by inclusion of FSBM (Yuan et al., 2017). Similarly, Engberg et al. (2009) also reported that feeding fermented feed in laying hens increases LAB in the crop and decreases coliforms in the ileum. The greater VH and CD and subsequently higher VH:CD ratio recorded at duodenal and jejunal segments of the small intestine for birds on PM and FSBM treatments imply better morphological development, which could to some extent account for the superior productive performance of these birds. Increased VH could result in a greater absorptive capability for available nutrients (Caspary, 1992), whereas low CD values indicate decreasing metabolic cost of the intestinal epithelium turnover (Floch and Seve, 2000), which may be reflected by the lower FCR observed in the current study. Apparently, the improved intestinal microbiota balance in favor of beneficial bacteria and increased amylase secretion in PM and FSBM groups can be responsible for improved morphological indices (Jin et al., 2000). Fermentation of SBM decreased the level of β-conglycinin and glycinin, which are known as potential antigenic and allergenic compounds for young monogastrics, causing villus atrophy and crypt hyperplasia in the small intestine (Wang et al., 2014). Consistent with our findings, previous studies on fermented protein meals have shown that inclusion of fermented rapeseed meal (Chiang et al., 2010) and SBM (Feng et al., 2007a) improves small intestine morphological parameters in broiler chicks. Various studies have shown that some Lactobacillus spp. could lower total plasma cholesterol and LDL-C (Anderson and Gilliland, 1999; Sanders, 2000; Kalavathy et al., 2010). These Lactobacillus spp. possess bile salt deconjugation ability and can hydrolyze bile salts, thus interfering with the reabsorption cycle of bile salts and increasing their fecal excretion. As cholesterol is the precursor of primary bile salts that are formed in the liver, a higher excretion of bile salts is associated with a higher excretion of cholesterol (Liong and Shah, 2005). Thus, the hypocholesteremic effects of PM and FSBM treatments observed in this study could be explained in light of a higher intestinal population of LAB in these treatments. Lactobacilli also have been reported to show hypolipidemic properties by inhibiting the activity of 3-hydroxy-3-methyl-glutaryl-CoA (Seifi et al., 2017), which could explain the lower serum concentration of triglycerides in PM and FSBM treatments observed at d 35. Similarly, other researchers also have reported lower serum cholesterol and triglycerides in Japanese quails (Seifi et al., 2017), broiler chicks (Kalavathy et al., 2010), and geese (Chen et al., 2013b) through diet supplementation with probiotic and/or fermented feed. CONCLUSION In summary, the results obtained in the current study indicate that microbial fermentation significantly reduces the content of some of the anti-nutritional factors present in SBM and also improves its nutritional value. In addition, FSBM exhibited the capability to perform effectively in the Japanese quail's GIT as a potential probiotic source. Diet supplementation with a PM or replacing SBM with FSBM in the feed can improve growth performance of quails, largely through improving gastrointestinal microbiota balance in favor of beneficial bacteria, and small intestine morphological parameters. No synergistic or additive effects seem to exist between the OA and PM used in this study. ACKNOWLEDGMENTS This project was funded and financially supported by Young Researchers and Elite Club, Islamic Azad University, Isfahan (Khorasgan) Branch, Isfahan, Iran. REFRERNCES Anderson J. W. , Gilliland S. E. . 1999 . Effect of fermented milk (yoghurt) containing Lactobacillus acidophilus L1 on serum cholesterol in hypercholesterolemic humans . J. Am. Coll. Nutr. 18 , 43 – 50 Google Scholar CrossRef Search ADS PubMed AOAC . 2005 . Association of Official Analytical Chemists. 2005 . 21th ed . Gaithersburg, M. D. : AOAC International . Ashayerizadeh A. , Dastar B. , Shams Shargh M. , Sadeghi Mahoonak A. R. , Zerehdaran S. . 2017 . Fermented rapeseed meal is effective in controlling Salmonella enterica serovar Typhimurium infection and improving growth performance in broiler chicks . Veterinary Microbiology . 201 : 93 – 102 . Google Scholar CrossRef Search ADS PubMed Banisharif M. , Kheiri F. , Jalali S. M. A. . 2016 . Hypericum perforatum and probiotic effects on performance, carcass characteristics and intestinal morphology in Japanese quails (Coturnix japonica) . J. Herb. Drugs. 7 : 83 – 88 . Basmacioglu-Malayoglu H. , Ozdemir P. , Bagriyanik H. A. . 2016 . Influence of an organic acid blend and essential oil blend, individually or in combination, on growth performance, carcass parameters, apparent digestibility, intestinal microflora and intestinal morphology of broilers . British Poultry Science . 57 : 227 – 234 . Google Scholar CrossRef Search ADS PubMed Boroojeni Goodarzi , Vahjen F. W. , Mader A. , Knorr F. , Ruhnke I. , Rohe I. , Hafeez A. , Villodre C. , Manner K. , Zentek J. . 2014 . The effects of different thermal treatments and organic acid levels in feed on microbial composition and activity in gastrointestinal tract of broilers . Poult. Sci . 93 : 1440 – 1452 . Google Scholar CrossRef Search ADS PubMed Caspary W. F. 1992 . Physiology and pathophysiology of intestinal absorption . Am. J. Clin. Nutr . 55 : 299S – 308S . Google Scholar CrossRef Search ADS PubMed Chen C. C. , Shih Y. C. , Chiou P. W. S. , Yu B. . 2010 . Evaluating nutritional quality of single stage- and two stage-fermented soybean meal . Asian Australas. J. Anim. Sci. 23 : 598 – 606 . Google Scholar CrossRef Search ADS Chen L. , Madl R. L. , Vadlani P. V. . 2013 . Nutritional enhancement of soy meal via aspergillus oryzae solid-state fermentation . Cereal Chemistry Journal. 90 : 529 – 534 . Google Scholar CrossRef Search ADS Chen W. , Zhu X. Z. , Wang J. P. , Wang Z. X. , Huang Y. Q. . 2013 . Effects of Bacillus subtilis var. natto and Saccharomyces cerevisiae fermented liquid feed on growth performance, relative organ weight, intestinal microflora, and organ antioxidant status in Landes geese . J. Anim. Sci . 91 : 978 – 985 . Google Scholar CrossRef Search ADS PubMed Chi C. H. , Cho S. J. . 2016 . Improvement of bioactivity of soybean meal by solid-state fermentation with Bacillus amyloliquefaciens versus Lactobacillus spp. and Saccharomyces cerevisiae . LWT - Food Science and Technology 68 : 619 – 625 . Google Scholar CrossRef Search ADS Chiang G. , Lu W. Q. , Piao X. S. , Hu J. K. , Gong L. M. , Thacker P. A. . 2010 . Effects of feeding solid-state fermented rapeseed meal on performance, nutrient digestibility, intestinal ecology and intestinal morphology of broiler chickens. Asian-Aust . J. Anim. Sci . 23 : 263 – 271 . Engberg R. M. , Hammershoj M. , Johansen N. F. , Abousekken M. S. , Steenfeldt S. , Jensen B. B. . 2009 . Fermented feed for laying hens: Effects on egg production, egg quality, plumage condition and composition and activity of the intestinal microflora . British Poultry Science . 50 : 228 – 239 . Google Scholar CrossRef Search ADS PubMed Feng J. , Liu X. , Xu Z. R. , Wang Y. Z. , Liu J. X. . 2007a . Effects of fermented soybean meal on digestive enzyme activities and intestinal morphology in broilers . Poultry Science . 86 : 1149 – 1154 . Google Scholar CrossRef Search ADS Feng J. , Liu X. , Xu Z. R. , Liu Y. Y. , Lu Y. P. . 2007b . Effects of Aspergillus oryzae 3.042 fermented soybean meal on growth performance and plasma biochemical parameters in broilers . Animal Feed Science and Technology . 134 : 235 – 242 . Google Scholar CrossRef Search ADS Floch N. L. , Seve B. . 2000 . Protein and amino acid metabolism in the intestine of the pig: From digestion to appearance in the portal vein . Prod. Anim . 13 : 303 – 314 . Gao Y. L. , Wang C. S. , Zhu Q. H. , Qian G. Y. . 2013 . Optimization of solid-state fermentation with Lactobacillus brevis and Aspergillus oryzae for trypsin inhibitor degradation in soybean meal . Journal of Integrative Agriculture . 12 : 869 – 876 . Google Scholar CrossRef Search ADS Hasan B. 2003 . Fermentation of fish silage using lactobacillus pentosus . J. Natur Indonesia . 6 : 11 – 15 . Jazi V. , Boldaji F. , Dastar B. , Hashemi S. R. , Ashayerizadeh A. . 2017 . Effects of fermented cottonseed meal on the growth performance, gastrointestinal microflora population and small intestinal morphology in broiler chickens . British Poultry Science . 58 : 402 – 408 . Google Scholar CrossRef Search ADS PubMed Jin L. Z. , Ho Y. W. , Abdullah N. , Jalaludin S. . 2000 . Digestive and bacterial enzyme activities in broilers fed diets supplemented with Lactobacillus cultures . Poultry Science . 79 : 886 – 891 . Google Scholar CrossRef Search ADS PubMed Kalavathy R. , Norhani A. , Michael C. V. L. W. , Chinna K. , Yin W. H. . 2010 . Bile salt deconjugation and cholesterol removal from media by Lactobacillus strains used as probiotics in chickens . J. Sci. Food Agric. 90 : 65 – 69 . Google Scholar CrossRef Search ADS PubMed Khan S. H. , Iqbal J. . 2016 . Recent advances in the role of organic acids in poultry nutrition . Journal of Applied Animal Research , 441 : 359 – 369 . Google Scholar CrossRef Search ADS Kook M. C. , Cho. S. C. , Hong Y. H. , Park H. . 2014 . Bacillus subtilis fermentation for enhancement of feed nutritive value of soybean meal . J. Appl. Biol. Chem. 57 : 183 – 188 . Google Scholar CrossRef Search ADS Lee K. W. , Lee S. H. , Lillehoj H. S. , Li G. X. , Jang S. I. , Babu U. S. , Park M. S. , Kim D. K. , Lillehoj E. P. , Neumann A. P. , Rehberger T. G. , Siragusa G. R. . 2010 . Effects of direct-fed microbials on growth performance, gut morphometry, and immune characteristics in broiler chickens . Poultry Science . 89 : 203 – 216 . Google Scholar CrossRef Search ADS PubMed Liong M. T. , Shah N. P. . 2005 . Bile salt deconjugation ability, bile salt hydrolase activity and cholesterol co-precipitation ability of lactobacilli strains . International Dairy Journal . 15 : 391 – 398 . Google Scholar CrossRef Search ADS Levy A. W. , Kessler J. W. , Fuller L. , Williams S. , Mathis G. F. , Lumpkins B. , Valdez F. . 2015 . Effect of feeding an encapsulated source of butyric acid (ButiPEARL) on the performance of male Cobb broilers reared to 42 d of age . Poult. Sci. 94 : 1864 – 1870 . Google Scholar CrossRef Search ADS PubMed Li C. Y. , Lu J. J. , Wu C. P. , Lien T. F. . 2014 . Effects of probiotics and bremelain fermented soybean meal replacing fish meal on growth performance, nutrient retention and carcass traits of broilers . Livestock Science . 163 : 94 – 101 . Google Scholar CrossRef Search ADS Marsili R. T. , Ostapenko H. , Simmons R. E. , Green D. E. . 1983 . High performance liquid chromatographic determination of organic acid . J. Food Prot . 46 : 52 – 57 . Google Scholar CrossRef Search ADS Missotten J. A. M. , Michiels J. , Degroote J. , De Smet S. . 2015 . Fermented liquid feed for pigs: An ancient technique for the future . J Animal Sci Biotechnol . 6 : 4 . Google Scholar CrossRef Search ADS Nava G. M , Attene-Ramos M. S , Gaskins H. R , Richards J. D . 2009 . Molecular analysis of microbial community structure in the chicken ileum following organic acid supplementation . Veterinary Microbiology . 137 : 345 – 353 . Google Scholar CrossRef Search ADS PubMed Niba A. T. , Beal J. D. , Kudi A. C. , Brooks P. H. . 2009 . Potential of bacterial fermentation as a biosafe method of improving feeds for pigs and poultry . Africa. J. Biotechnol . 8 : 1758 – 1767 . Nosrati M. , Javandel F. , Camacho L. M. , Khusro A. , Cipriano M. , Seidavi A. , Salem A. Z. M. . 2017 . The effects of antibiotic, probiotic, organic acid, vitamin C, and Echinacea purpurea extract on performance, carcass characteristics, blood chemistry, microbiota, and immunity of broiler chickens . J. Appl. Poult . Res . 26 : 295 – 306 . NRC . 1994 . Nutrient Requirements of Poultry . 9th rev. ed . Natl. Acad. Press , Washington, DC . Panda A. K. , Rao S. V. R. , Raju M. V. , Sharma S. R. . 2006 . Dietary supplementation of Lactobacillus sporogenes on performance and serum biochemico-lipid profile of broiler chickens . J. Poult. Sci. 43 : 235 – 240 . Google Scholar CrossRef Search ADS Patterson J. A. , Burkholder K. M. . 2003 . Application of prebiotics and probiotics in poultry production . Poultry Science . 82 : 627 – 631 . Google Scholar CrossRef Search ADS PubMed Ocak N. , Erener G. , Altop A. , Kop C. . 2009 . The effect of malic acid on performance and some digestive tract traits of Japanese quails . J. Poult. Sci. 46 : 25 – 29 . Google Scholar CrossRef Search ADS Sanders M. E. 2000 . Considerations for use of probiotic bacteria to modulate human health . J. Nutr . 130 : 384S – 390S . Google Scholar CrossRef Search ADS PubMed SAS Institute . 2003 . User's Guide: Statistics , Version 9.1 . SAS Institute, Inc. , Cary, NC, USA . Seifi K. , Karimi Torshizi M. A. , Rahimi S. , Kazemifard M. . 2017 . Efficiency of early, single-dose probiotic administration methods on performance, small intestinal morphology, blood biochemistry, and immune response of Japanese quail . Poult. Sci. 96 : 2151 – 2158 . Google Scholar CrossRef Search ADS PubMed Seo S. H. , Cho S. J. . 2016 . Changes in allergenic and antinutritional protein profiles of soybean meal during solid-state fermentation with Bacillus subtilis . LWT - Food Science and Technology . 70 : 208 – 212 . Google Scholar CrossRef Search ADS Sharawy Z. , Goda A. M. A. S. , Hassaan M. S. . 2016 . Partial or total replacement of fish meal by solid state fermented soybean meal with Saccharomyces cerevisiae in diets for Indian prawn shrimp, Fenneropenaeus indicus , Postlarvae . Animal Feed Science and Technology 212 : 90 – 99 . Google Scholar CrossRef Search ADS Smith C. , Van Megen W. , Twaalfhoven L. , Hitchcock C. . 1980 . The determination of trypsin inhibitor levels in foodstuffs . J. Sci. Food Agric. . 31 : 341 – 350 . Google Scholar CrossRef Search ADS PubMed Sun H. , Tang J. W. , Yao X. H. , Wu Y. F. , Wang X. , Feng J. . 2013 . Effects of dietary inclusion of fermented cottonseed meal on growth, cecal microbial population, small intestinal morphology, and digestive enzyme activity of broilers . Trop Anim Health Prod . 45 : 987 – 993 . Google Scholar CrossRef Search ADS PubMed Toghyani M. , Toghyani M. , Gheisari A. , Ghalamkari G. , Mohammadrezaei M. . 2010 . Growth performance, serum biochemistry and blood hematology of broiler chicks fed different levels of black seed (Nigella sativa) and peppermint (Mentha piperita) . Livestock Science . 129 : 173 – 178 . Google Scholar CrossRef Search ADS Van der Wielen P . 2002 . Dietary strategies to influence the gastrointestinal microflora of young animals and its potential to improve intestinal health . Pages 37–60 in Nutrition and Health of the Gastrointestinal Tract , Blok M. C , Vahl H. A. , de Lange L. , vande Braak A. E , Hemke G. , Hessing M. eds. Wageningen Academic Publishers , Wageningen, the Netherlands . Wang L. C. , Wen C. , Jiang Z. Y. , Zhou Y. M. . 2012 . Evaluation of the partial replacement of high-protein feedstuff with fermented soybean meal in broiler diets . The Journal of Applied Poultry Research . 21 : 849 – 855 . Google Scholar CrossRef Search ADS Wang Y. , Liu X. T. , Wang H. L. , Li D. F. , Piao X. S. , Lu W. Q. . 2014 . Optimization of processing conditions for solid-state fermented soybean meal and its effects on growth performance and nutrient digestibility of weanling pigs . Livestock Science . 170 : 91 – 99 . Google Scholar CrossRef Search ADS Yuan L. , Chang J. , Yin Q. , Lu M. , Di Y. , Wang P. , Wang Z. , Wang E. , Lu F. . 2017 . Fermented soybean meal improves the growth performance, nutrient digestibility, and microbial flora in piglets Animal Nutrition . 3 : 19 – 24 . Google Scholar CrossRef Search ADS Zhang Z. F. , Kim I. H. . 2014 . Effects of multistrain probiotics on growth performance, apparent ileal nutrient digestibility, blood characteristics, cecal microbial shedding, and excreta odor contents in broilers . Poult. Sci . 93 : 364 – 370 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. 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Fermented soybean meal exhibits probiotic properties when included in Japanese quail diet in replacement of soybean meal

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© 2018 Poultry Science Association Inc.
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0032-5791
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Abstract

ABSTRACT This study was conducted to investigate and compare the effect of dietary probiotic mixture (PM) and organic acid (OA) mixture with fermented soybean meal (FSBM) on performance, crop, and ceca microbiota, small intestine morphology, and serum lipid profile in Japanese quails. A total of 800 day-old Japanese quails was randomly allotted to 5 treatments with 8 replicate pens of 20 birds each, for 35 days. The experimental diets consisted of a control corn-soybean meal diet and 4 test diets: 1) control diet + 0.1% PM; 2) control diet + 0.2% OA mixture; 3) control diet + the combination of both PM and OA; and 4) an additives-free diet in which the soybean meal in the control diet was replaced with FSBM. The results indicated that in starter and the entire rearing periods, FSBM, PM, and PM+OA diets had significantly lower FCR compared to control or OA diets (P < 0.05). Birds in the FSBM group gained higher weight than control and OA birds (P < 0.05; 1 to 35 d). At d 21 and 35, birds fed the control diet showed significantly lower numbers of lactic acid bacteria in the crop, while coliforms were higher in the cecal content compared to the other diets (P < 0.05). At d 21, the villus height and villus height to crypt depth ratio in the duodenum and jejunum of birds fed PM, PM+OA, and FSBM diets were greater than in other treatments (P < 0.05). The serum concentrations of cholesterol and low-density lipoprotein cholesterol of birds fed PM, PM+OA, and FSBM diets were significantly lower than birds in control and AO groups (P < 0.05). The results obtained herein suggest that FSBM exhibits probiotic properties and, when used in substitution of SBM in Japanese quail diet, can improve growth performance, balance of desirable gastrointestinal microbiota in crop and ceca, small intestinal morphology, and serum lipid profile—likewise, a probiotic supplement. INTRODUCTION In-feed antibiotics have long been used in poultry feeds to improve feed efficiency and birds’ overall health status. However, bio-security threats for human and poultry health, arising from the antibiotic-resistant bacteria and the accumulation of antibiotic residues in poultry products, call for a worldwide removal of antibiotics, in particular at sub-therapeutic doses and as growth promoters from the poultry industry (Toghyani et al., 2010). In this regard, probiotics, prebiotics, organic acids (OA), and fermented feeds/ingredients, have been used as in-feed antibiotic alternatives to modulate poultry feed in order to improve animal health status and growth performance (Nava et al., 2009). The beneficial effects of probiotics and organic acids on performance indices and general health of different domesticated avian species are well documented (Levy et al., 2015; Seifi et al., 2017), although these effects have not always been consistent (Lee et al., 2010; Nosrati et al., 2017). Inclusion of fermented products in animal feed has been shown to induce advantageous effects on performance and gastrointestinal health by acting and exhibiting probiotic effects and could therefore be considered as an alternative for antibiotics growth promoters (Missotten et al., 2015). Fermented feed has a low pH, high concentration of lactic acid (>150 mM), and a high number of lactic acid bacteria (LAB) (approximately 109 CFU/mL of feed) (Ashayerizadeh et al., 2017). Engberg et al. (2009) showed that feeding fermented feed in laying hens increases LAB in the crop and decreases coliforms in the ileum. Furthermore, the OA content of fermented feed has been reported to modulate gastrointestinal microbiota balance through increasing acidity and lowering the pH (Niba et al., 2009). Besides the desired adverse effect on pathogenic bacteria, fermented feeds have some nutritional implications (Engberg et al., 2009). Microbial fermentation has been reported as an effective technique to eliminate or reduce anti-nutritional factors and improve nutritional value in plant-based protein meals (Ashayerizadeh et al., 2017; Jazi et al., 2017). Soybean meal (SBM) is the most commonly used plant protein source in poultry feed (Chiang et al., 2010). However, its application in poultry diets could often be limited due to some anti-nutritional factors, such as trypsin inhibitor (TI), oligosaccharides, phytic acid, and allergenic proteins, which interfere with digestion, absorption, and utilization of nutrients, especially for young chicks (Li et al., 2014; Feng et al., 2007a). Previous reports have indicated the reduction of TI and other anti-nutritional factors in SBM following fermentation (Wang et al., 2014; Sharawy et al., 2016). The hypothesis tested in the current study was that the antimicrobial compounds present in fermented SBM (FSBM) may act in a similar way to probiotics and OA in improving growth performance of Japanese quails through affecting gut microbiota and morphology. Furthermore, not many studies have tested FSBM against probiotic and/or OA in poultry, in particular Japanese quail. Therefore, in view of the dearth of data, the present study was conducted to investigate the chemical and biological characteristics of FSBM and to compare the efficacy of dietary probiotic and OA with FSBM on performance, gastrointestinal microbiota, morphometric analysis, and serum lipid profile in Japanese quails. MATERIALS AND METHODS Preparation of FSBM FSBM was prepared following the method used by Sun et al. (2013). Briefly, Lactobacillus plantarum (PTCC1058), Bacillus subtilis (PTCC1156) bacteria, and Aspergillus oryzae (PTCC5163) fungus were obtained from the Persian Type Culture Collection of Iranian Research Organization for Science and Technology. Each kg of SBM was inoculated and mixed with 1 L of distilled water containing approx. 105 CFU/mL of Lactobacillus plantarum and Bacillus subtilis and 106 spores of Aspergillus oryzae in a special tank (with a one-way valve to outflow gases produced and prevent air entry) for 7 d at 30°C. Ultimately, FSBM was dried for 2 d at 50°C. The dried samples were ground to pass through a 1.0 mm sieve and kept at room temperature until mixed in the diets. Chemical Analysis of SBM and FSBM Sub-samples from SBM and FSBM were obtained using a sampling probe, and a triple-rifled representative composite sample for each ingredient was used for the chemical analyses (conducted in triplicate). To determine the population of LAB, 1 g of SBM or FSBM was used to make serial 10-fold dilutions using buffered peptone water. Then, 0.1 mL of appropriate dilutions was spread on the plates containing modified de Man, Rogosa, and Sharpe agar. Plates were incubated in anaerobic conditions for 24 h at 37°C. After counting the number of colonies in each plate, the obtained number was multiplied by reversed dilution and reported as the number of colony forming unit (CFU) per 1 g sample. To determine the pH values, 20 g of each sample were transferred to a 250 mL beaker, and 200 mL of distilled water were added. The pH values were then measured by using a portable pH meter (pH Meter CG 804, Schott Gerate). The lactic acid concentration was determined by HPLC according to the method described by Marsili et al. (1983). Samples were analyzed for dry matter (DM), ash, crude protein (CP), ether extract (EE), and crude fiber (CF) according to AOAC (2005). Phytic acid was determined through the extraction of the samples with HCl and Na2SO4 and absorbance measured at 660 nm. TI activity in the samples was determined according to the method of Smith et al. (1980), and results are expressed as mg trypsin inhibited per g of dry sample. The contents of glycinin and β-conglycinin were estimated by the method of Wang et al. (2014). The amino acid (AA) compositions of SBM and FSBM were determined using an automated AA analyzer after hydrolyzing the samples with 6 M HCl at 110°C for 24 hours. Sulphur-containing AA were oxidized using performic acid before the acid hydrolysis. The changes of the chemical composition before and after the fermentation process are reported in Table 1. Table 1. Analyzed chemical composition of soybean meal pre- and post fermentation (% of dry matter basis). Soybean meal Item1 Pre-ferment Post ferment SEM P-value pH 5.92a 3.84b 0.28 0.007 Lactic acid (mmol/kg) 21.38b 172.78a 2.64 <0.001 LAB (Log10 CFU/g) 4.22b 10.64a 0.04 <0.001 DM 91.64 89.55 0.81 0.141 CP 44.03b 47.61a 0.63 0.011 EE 1.35 1.31 0.02 0.340 CF 7.31a 5.04b 0.19 0.001 Ash 6.26 6.34 0.24 0.83 Phytic acid (g/100 g) 0.63a 0.17b 0.01 <0.001 Trypsin inhibitor (mg/g) 2.85a 0.62b 0.25 0.003 β-conglycinin (mg/g) 59.62a 32.98b 1.07 <0.001 Glycinin (mg/g) 76.61a 28.26b 2.31 0.002 Indispensable amino acids Arginine 3.04 3.10 0.23 0.85 Histidine 1.15 1.19 0.04 0.57 Isoleucine 1.89 1.98 0.10 0.55 Leucine 3.16 3.26 0.21 0.19 Lysine 2.44 2.70 0.19 0.32 Methionine 0.57 0.58 0.03 0.79 Phenylalanine 2.12 2.20 0.15 0.73 Threonine 1.73 1.82 0.06 0.34 Valine 1.93 2.18 0.09 0.15 Dispensable amino acids Alanine 1.79 1.94 0.13 0.46 Asparagine 4.75 5.07 0.28 0.45 Cysteine 0.65 0.76 0.04 0.94 Glutamine 7.88 8.88 0.49 0.22 Glycine 1.74 1.90 0.04 0.07 Proline 2.14 2.24 0.13 0.62 Serine 2.10 2.32 0.08 0.13 Tyrosine 1.52 1.54 0.10 0.89 Soybean meal Item1 Pre-ferment Post ferment SEM P-value pH 5.92a 3.84b 0.28 0.007 Lactic acid (mmol/kg) 21.38b 172.78a 2.64 <0.001 LAB (Log10 CFU/g) 4.22b 10.64a 0.04 <0.001 DM 91.64 89.55 0.81 0.141 CP 44.03b 47.61a 0.63 0.011 EE 1.35 1.31 0.02 0.340 CF 7.31a 5.04b 0.19 0.001 Ash 6.26 6.34 0.24 0.83 Phytic acid (g/100 g) 0.63a 0.17b 0.01 <0.001 Trypsin inhibitor (mg/g) 2.85a 0.62b 0.25 0.003 β-conglycinin (mg/g) 59.62a 32.98b 1.07 <0.001 Glycinin (mg/g) 76.61a 28.26b 2.31 0.002 Indispensable amino acids Arginine 3.04 3.10 0.23 0.85 Histidine 1.15 1.19 0.04 0.57 Isoleucine 1.89 1.98 0.10 0.55 Leucine 3.16 3.26 0.21 0.19 Lysine 2.44 2.70 0.19 0.32 Methionine 0.57 0.58 0.03 0.79 Phenylalanine 2.12 2.20 0.15 0.73 Threonine 1.73 1.82 0.06 0.34 Valine 1.93 2.18 0.09 0.15 Dispensable amino acids Alanine 1.79 1.94 0.13 0.46 Asparagine 4.75 5.07 0.28 0.45 Cysteine 0.65 0.76 0.04 0.94 Glutamine 7.88 8.88 0.49 0.22 Glycine 1.74 1.90 0.04 0.07 Proline 2.14 2.24 0.13 0.62 Serine 2.10 2.32 0.08 0.13 Tyrosine 1.52 1.54 0.10 0.89 a,bMeans with different superscripts in each row are significantly different based on t test at P < 0.05. 1LAB = lactic acid bacteria; DM = dry matter; CP = crude protein; EE = ether extract; CF = crude fiber. View Large Table 1. Analyzed chemical composition of soybean meal pre- and post fermentation (% of dry matter basis). Soybean meal Item1 Pre-ferment Post ferment SEM P-value pH 5.92a 3.84b 0.28 0.007 Lactic acid (mmol/kg) 21.38b 172.78a 2.64 <0.001 LAB (Log10 CFU/g) 4.22b 10.64a 0.04 <0.001 DM 91.64 89.55 0.81 0.141 CP 44.03b 47.61a 0.63 0.011 EE 1.35 1.31 0.02 0.340 CF 7.31a 5.04b 0.19 0.001 Ash 6.26 6.34 0.24 0.83 Phytic acid (g/100 g) 0.63a 0.17b 0.01 <0.001 Trypsin inhibitor (mg/g) 2.85a 0.62b 0.25 0.003 β-conglycinin (mg/g) 59.62a 32.98b 1.07 <0.001 Glycinin (mg/g) 76.61a 28.26b 2.31 0.002 Indispensable amino acids Arginine 3.04 3.10 0.23 0.85 Histidine 1.15 1.19 0.04 0.57 Isoleucine 1.89 1.98 0.10 0.55 Leucine 3.16 3.26 0.21 0.19 Lysine 2.44 2.70 0.19 0.32 Methionine 0.57 0.58 0.03 0.79 Phenylalanine 2.12 2.20 0.15 0.73 Threonine 1.73 1.82 0.06 0.34 Valine 1.93 2.18 0.09 0.15 Dispensable amino acids Alanine 1.79 1.94 0.13 0.46 Asparagine 4.75 5.07 0.28 0.45 Cysteine 0.65 0.76 0.04 0.94 Glutamine 7.88 8.88 0.49 0.22 Glycine 1.74 1.90 0.04 0.07 Proline 2.14 2.24 0.13 0.62 Serine 2.10 2.32 0.08 0.13 Tyrosine 1.52 1.54 0.10 0.89 Soybean meal Item1 Pre-ferment Post ferment SEM P-value pH 5.92a 3.84b 0.28 0.007 Lactic acid (mmol/kg) 21.38b 172.78a 2.64 <0.001 LAB (Log10 CFU/g) 4.22b 10.64a 0.04 <0.001 DM 91.64 89.55 0.81 0.141 CP 44.03b 47.61a 0.63 0.011 EE 1.35 1.31 0.02 0.340 CF 7.31a 5.04b 0.19 0.001 Ash 6.26 6.34 0.24 0.83 Phytic acid (g/100 g) 0.63a 0.17b 0.01 <0.001 Trypsin inhibitor (mg/g) 2.85a 0.62b 0.25 0.003 β-conglycinin (mg/g) 59.62a 32.98b 1.07 <0.001 Glycinin (mg/g) 76.61a 28.26b 2.31 0.002 Indispensable amino acids Arginine 3.04 3.10 0.23 0.85 Histidine 1.15 1.19 0.04 0.57 Isoleucine 1.89 1.98 0.10 0.55 Leucine 3.16 3.26 0.21 0.19 Lysine 2.44 2.70 0.19 0.32 Methionine 0.57 0.58 0.03 0.79 Phenylalanine 2.12 2.20 0.15 0.73 Threonine 1.73 1.82 0.06 0.34 Valine 1.93 2.18 0.09 0.15 Dispensable amino acids Alanine 1.79 1.94 0.13 0.46 Asparagine 4.75 5.07 0.28 0.45 Cysteine 0.65 0.76 0.04 0.94 Glutamine 7.88 8.88 0.49 0.22 Glycine 1.74 1.90 0.04 0.07 Proline 2.14 2.24 0.13 0.62 Serine 2.10 2.32 0.08 0.13 Tyrosine 1.52 1.54 0.10 0.89 a,bMeans with different superscripts in each row are significantly different based on t test at P < 0.05. 1LAB = lactic acid bacteria; DM = dry matter; CP = crude protein; EE = ether extract; CF = crude fiber. View Large Experimental Birds and Diets All animal procedures were approved to be in compliance with the Institutional Animal Care and Use Committee of the Islamic Azad University, Isfahan (Khorasgan) Branch, Isfahan, Iran. A total of 800 day-old mixed-sex quails was purchased from a commercial hatchery, weighed, and randomly allocated into 5 treatment groups with 8 replicate pens of 20 birds based on a completely randomized design. The birds were housed in wire-floored pens (100 × 100 × 70 cm) in an environmentally controlled room with continuous light and were allowed ad libitum access to water and feed. The initial temperature of 37˚C was gradually reduced according to the age of the chicks until reaching 25˚C at the end of the 35-day experiment. Nutritional requirements of the Japanese quails were adopted from National Research Council (NRC, 1994) tables, and the basal diets were formulated accordingly (Table 2). The experimental diets consisted of a control corn-SBM diet and 4 test diets as follows: control + 0.1% probiotic mixture (PM, Protexin; Probiotics International Ltd., Somerset, UK), control + 0.2% OA mixture (Salkil; Anpario, UK), control + the combination of both PM and OA, and an additives-free diet in which the SBM in the control diet was replaced with FSBM. The PM used was a lyophilized mix comprising 2 × 109 CFU/g of 9 various microbial species, including: Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium bifidum, Streptococcus thermophilus, Enterococcus faecium, Aspergillus oryzae, and Candida pintolopesii. The OA mixture contained a combination of 7% ammonium propionate, 30% ammonium formate, 6% formic acid, and 57% material preservative. The inclusion rate of PM and OA was according to the manufacturer recommendations. Table 2. Composition and analysis of the experimental diets. Diet Ingredients (%) SBM based FSBM based Corn 49.10 54.71 Soybean meal (SBM) 41.89 - Fermented soybean meal (FSBM) - 37.74 Fish meal 3.0 3.0 Vegetable oil 3.54 2.04 CaCO3 0.85 0.83 Di-calcium phosphate 0.68 0.74 Sodium chloride 0.34 0.34 Vitamin premix1 0.30 0.30 Mineral premix2 0.30 0.30 Calculated composition3 Metabolizable energy (kcal/kg) 2950 2950 Crude protein (%) 24.41 24.41 Lysine (%) 1.32 1.32 Methionine (%) 0.35 0.35 Methionine + cysteine (%) 1.03 1.03 Calcium (%) 0.81 0.81 Available phosphorus (%) 0.40 0.40 Sodium (%) 0.16 0.16 Analyzed composition (%) Dry matter 89.1 88.29 Crude protein 25.07 25.54 Crude fiber 3.41 2.92 Ether extract 5.95 4.92 Calcium 0.91 0.87 Total phosphorus 0.58 0.56 Sodium 0.18 0.17 Chloride 0.23 0.21 Diet Ingredients (%) SBM based FSBM based Corn 49.10 54.71 Soybean meal (SBM) 41.89 - Fermented soybean meal (FSBM) - 37.74 Fish meal 3.0 3.0 Vegetable oil 3.54 2.04 CaCO3 0.85 0.83 Di-calcium phosphate 0.68 0.74 Sodium chloride 0.34 0.34 Vitamin premix1 0.30 0.30 Mineral premix2 0.30 0.30 Calculated composition3 Metabolizable energy (kcal/kg) 2950 2950 Crude protein (%) 24.41 24.41 Lysine (%) 1.32 1.32 Methionine (%) 0.35 0.35 Methionine + cysteine (%) 1.03 1.03 Calcium (%) 0.81 0.81 Available phosphorus (%) 0.40 0.40 Sodium (%) 0.16 0.16 Analyzed composition (%) Dry matter 89.1 88.29 Crude protein 25.07 25.54 Crude fiber 3.41 2.92 Ether extract 5.95 4.92 Calcium 0.91 0.87 Total phosphorus 0.58 0.56 Sodium 0.18 0.17 Chloride 0.23 0.21 1Supplied per kg of diet: 1.8 mg all-trans-retinyl acetate; 0.02 mg cholecalciferol; 8.3 mg alphatocopheryl acetate; 2.2 mg menadione; 2 mg pyridoxine HCl; 8 mg cyanocobalamin; 10 mg nicotine amid; 0.3 mg folic acid; 20 mg D-biotin; 160 mg choline chloride. 2Supplied per kg of diet: 32 mg Mn (MnSO4_H2O); 16 mg Fe (FeSO4_7H2O); 24 mg Zn (ZnO); 2 mg Cu (CuSO4_5H2O); 800 μg I (KI); 200 μg Co (CoSO4); 60 μg Se (NaSeO3). 3Crude protein, calcium, phosphorus, and amino acid contents are analyzed values. The metabolizable energy was calculated. View Large Table 2. Composition and analysis of the experimental diets. Diet Ingredients (%) SBM based FSBM based Corn 49.10 54.71 Soybean meal (SBM) 41.89 - Fermented soybean meal (FSBM) - 37.74 Fish meal 3.0 3.0 Vegetable oil 3.54 2.04 CaCO3 0.85 0.83 Di-calcium phosphate 0.68 0.74 Sodium chloride 0.34 0.34 Vitamin premix1 0.30 0.30 Mineral premix2 0.30 0.30 Calculated composition3 Metabolizable energy (kcal/kg) 2950 2950 Crude protein (%) 24.41 24.41 Lysine (%) 1.32 1.32 Methionine (%) 0.35 0.35 Methionine + cysteine (%) 1.03 1.03 Calcium (%) 0.81 0.81 Available phosphorus (%) 0.40 0.40 Sodium (%) 0.16 0.16 Analyzed composition (%) Dry matter 89.1 88.29 Crude protein 25.07 25.54 Crude fiber 3.41 2.92 Ether extract 5.95 4.92 Calcium 0.91 0.87 Total phosphorus 0.58 0.56 Sodium 0.18 0.17 Chloride 0.23 0.21 Diet Ingredients (%) SBM based FSBM based Corn 49.10 54.71 Soybean meal (SBM) 41.89 - Fermented soybean meal (FSBM) - 37.74 Fish meal 3.0 3.0 Vegetable oil 3.54 2.04 CaCO3 0.85 0.83 Di-calcium phosphate 0.68 0.74 Sodium chloride 0.34 0.34 Vitamin premix1 0.30 0.30 Mineral premix2 0.30 0.30 Calculated composition3 Metabolizable energy (kcal/kg) 2950 2950 Crude protein (%) 24.41 24.41 Lysine (%) 1.32 1.32 Methionine (%) 0.35 0.35 Methionine + cysteine (%) 1.03 1.03 Calcium (%) 0.81 0.81 Available phosphorus (%) 0.40 0.40 Sodium (%) 0.16 0.16 Analyzed composition (%) Dry matter 89.1 88.29 Crude protein 25.07 25.54 Crude fiber 3.41 2.92 Ether extract 5.95 4.92 Calcium 0.91 0.87 Total phosphorus 0.58 0.56 Sodium 0.18 0.17 Chloride 0.23 0.21 1Supplied per kg of diet: 1.8 mg all-trans-retinyl acetate; 0.02 mg cholecalciferol; 8.3 mg alphatocopheryl acetate; 2.2 mg menadione; 2 mg pyridoxine HCl; 8 mg cyanocobalamin; 10 mg nicotine amid; 0.3 mg folic acid; 20 mg D-biotin; 160 mg choline chloride. 2Supplied per kg of diet: 32 mg Mn (MnSO4_H2O); 16 mg Fe (FeSO4_7H2O); 24 mg Zn (ZnO); 2 mg Cu (CuSO4_5H2O); 800 μg I (KI); 200 μg Co (CoSO4); 60 μg Se (NaSeO3). 3Crude protein, calcium, phosphorus, and amino acid contents are analyzed values. The metabolizable energy was calculated. View Large Sampling and Measurements Growth performance parameters, including average feed intake (FI) and body weight gain (BWG), were measured at d 21 and 35 of the feeding trial, and feed conversion ratio (feed:gain, FCR) was calculated, and corrected for mortality within the same intervolves for each pen. At d 21 and 35, 2 birds/replicate were randomly selected from each treatment to examine pH, microbial population of the crop and ceca, and morphological characteristics of the small intestine. The birds were euthanized by cervical dislocation, and the digestive tract was carefully excised. Intestinal Morphological Analysis For intestinal morphometric analysis, birds on the designated evaluation d were euthanized, and duodenum, jejunum, and ileum samples were collected. A 1-cm segment of the midpoint of the duodenum, jejunum, and ileum from each bird was removed and fixed in 10% buffered formaldehyde for 48 hours. Each of these intestinal segments was embedded in paraffin, and a 5-μm section of each sample was placed on a glass slide and stained with hematoxylin and eosin for examination under a light microscope. All morphological parameters were measured using the ImageJ software package (http://rsb.info.nih.gov/ij/). Villus height (VH) was measured from the top of the villus to the top of the lamina propria. Crypt depth (CD) was measured from the base upwards to the region of transition between the crypt and villus. A total of 10 intact, well-oriented crypt-villus units was selected in duplicate from each tissue sample, and the averages of 20 values were obtained for each bird. Enumeration of Bacteria and pH Whole crop and ceca were aseptically removed and separated into sterile bags and homogenized. Samples were weighed, and 1:4 wt/vol dilutions were made with sterile 0.9% saline. Tenfold dilutions of each sample from each group were made in a sterile 96 well Bacti flat-bottom plate, and the diluted samples were plated on 3 different plates of medium to evaluate total number of LAB in modified de Man, Rogosa, and Sharpe agar; total coliforms in violet red bile agar; and total anaerobic bacteria (TAB) in plate count agar. For measuring the pH, about 1 gram of the crop and cecum contents of each bird was collected and transferred into 2 ml distilled water, then the pH values were measured using the portable pH meter. Serum Metabolites At d 21 and 35, 2 birds/pen were randomly selected, and blood samples were collected from the wing vein; the samples were centrifuged (at 2,000 × g for 5 min), and serum was separated and then stored at –20 ˚C until analyzed. Total cholesterol, triglycerides, and high-density lipoprotein cholesterol (HDL-C) values were measured using commercial laboratory kits (Pars Azmoon Kits; Pars Azmoon, Tehran, Iran). Very-low-density lipoprotein cholesterol (VLDL-C) values were calculated by dividing triglyceride values to unit 5 and low-density lipoprotein cholesterol (LDL-C) values by subtracting total values of HDL-C and VLDL-C from total cholesterol, according to the method described by Panda et al. (2006). Statistical Analysis Chemical composition, LAB numbers, and AA content of SBM pre- and post fermentation were compared using a t test. Data obtained during the feeding period of quail chicks were checked for normality and then analyzed based on a completely randomized design using the GLM procedures of SAS software (SAS, 2003). When a significant effect of treatment was detected, means were compared using Tukey's HSD test at P < 0.05 level of probability. RESULTS Chemical Composition of SBM and FSBM Chemical composition of the SBM pre- and post-fermentation process are presented in Table 1. The pH, CF, phytic acid, TI, β-conglycinin, and glycinin were significantly decreased and population of LAB and CP increased in SBM by the fermentation process (P < 0.05). The fermentation process had no significant effect on dry matter, ash, ether extract and amino acid content of SBM (P > 0.05). Growth Performance The results of the effects of experimental treatments on growth performance of Japanese quails are summarized in Table 3. BWG in birds fed FSBM diet was significantly higher than those fed the control and OA diets in starter and grower periods (P < 0.05). The BWG in the control treatment was significantly lower than FSBM and PM+OA in the grower period (P < 0.05). In the starter and entire rearing periods, birds fed FSBM, PM, or PM+OA diets had significantly lower FCR (P < 0.05). Also, over the grower period, FCR in FSBM and PM+OA treatments was significantly lower than the other treatments (P < 0.05). FI was not affected by dietary treatments (P > 0.05). Table 3. Effects of the experimental treatments on performance parameters of Japanese quails.1 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Starter (1–21 d) BWG 93.51b 96.23a,b 92.06b 97.55a,b 103.45a 2.36 0.031 FI 241.75 240.91 237.15 245.47 255.72 6.38 0.33 FCR 2.58a 2.50b 2.57a 2.51b 2.47b 0.01 0.002 Grower (21–35 d) BWG 78.36b 82.32a,b 81.88a,b 86.23a 87.80a 1.88 0.02 FI 286.25 299.89 297.43 307.34 308.45 7.28 0.25 FCR 3.65a 3.64a 3.63a 3.56b 3.51b 0.02 0.001 Overall (1–35 d) BWG 171.87b 178.56a,b 173.95b 183.79a,b 191.25a 4.10 0.02 FI 528.00 540.80 534.58 552.81 564.18 13.1 0.34 FCR 3.07a 3.03b 3.07a 3.01b 2.95c 0.01 <.001 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Starter (1–21 d) BWG 93.51b 96.23a,b 92.06b 97.55a,b 103.45a 2.36 0.031 FI 241.75 240.91 237.15 245.47 255.72 6.38 0.33 FCR 2.58a 2.50b 2.57a 2.51b 2.47b 0.01 0.002 Grower (21–35 d) BWG 78.36b 82.32a,b 81.88a,b 86.23a 87.80a 1.88 0.02 FI 286.25 299.89 297.43 307.34 308.45 7.28 0.25 FCR 3.65a 3.64a 3.63a 3.56b 3.51b 0.02 0.001 Overall (1–35 d) BWG 171.87b 178.56a,b 173.95b 183.79a,b 191.25a 4.10 0.02 FI 528.00 540.80 534.58 552.81 564.18 13.1 0.34 FCR 3.07a 3.03b 3.07a 3.01b 2.95c 0.01 <.001 a–cMeans with different superscripts in each row are significantly different (P < 0.05). 1Data represent means of 8 replicates of 20 quails per treatment. 2BWG = body weight gain; FI = feed intake; FCR = feed conversion ratio. 3CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large Table 3. Effects of the experimental treatments on performance parameters of Japanese quails.1 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Starter (1–21 d) BWG 93.51b 96.23a,b 92.06b 97.55a,b 103.45a 2.36 0.031 FI 241.75 240.91 237.15 245.47 255.72 6.38 0.33 FCR 2.58a 2.50b 2.57a 2.51b 2.47b 0.01 0.002 Grower (21–35 d) BWG 78.36b 82.32a,b 81.88a,b 86.23a 87.80a 1.88 0.02 FI 286.25 299.89 297.43 307.34 308.45 7.28 0.25 FCR 3.65a 3.64a 3.63a 3.56b 3.51b 0.02 0.001 Overall (1–35 d) BWG 171.87b 178.56a,b 173.95b 183.79a,b 191.25a 4.10 0.02 FI 528.00 540.80 534.58 552.81 564.18 13.1 0.34 FCR 3.07a 3.03b 3.07a 3.01b 2.95c 0.01 <.001 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Starter (1–21 d) BWG 93.51b 96.23a,b 92.06b 97.55a,b 103.45a 2.36 0.031 FI 241.75 240.91 237.15 245.47 255.72 6.38 0.33 FCR 2.58a 2.50b 2.57a 2.51b 2.47b 0.01 0.002 Grower (21–35 d) BWG 78.36b 82.32a,b 81.88a,b 86.23a 87.80a 1.88 0.02 FI 286.25 299.89 297.43 307.34 308.45 7.28 0.25 FCR 3.65a 3.64a 3.63a 3.56b 3.51b 0.02 0.001 Overall (1–35 d) BWG 171.87b 178.56a,b 173.95b 183.79a,b 191.25a 4.10 0.02 FI 528.00 540.80 534.58 552.81 564.18 13.1 0.34 FCR 3.07a 3.03b 3.07a 3.01b 2.95c 0.01 <.001 a–cMeans with different superscripts in each row are significantly different (P < 0.05). 1Data represent means of 8 replicates of 20 quails per treatment. 2BWG = body weight gain; FI = feed intake; FCR = feed conversion ratio. 3CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large Enumeration of Bacteria and pH The effect of experimental treatments on pH and microbial population in crop and cecal contents of Japanese quails are presented in Table 4. The PM, OA, PM+OA, and FSBM diets decreased pH of the crop and ceca at d 21 and 35 compared to the control diet (P < 0.05). Also, the LAB population was lower in the crop, while coliforms were higher in the ceca section of birds fed the control diet than in all the other treatments, for both ages tested (P < 0.05). At the end of starter and grower periods, the population of TAB in the crop and also ceca decreased significantly under the influence of PM, PM+OA, and FSBM diets compared to the other treatments (P < 0.05). Table 4. Effects of the experimental treatments on gastrointestinal microbiota composition (Log10 CFU/g) and pH value in Japanese quails.1 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Day 21 Crop pH 4.90a 4.77a,b 4.68b 4.64a 4.61b 0.05 0.012 LAB 7.32b 7.75a 7.52a,b 7.79a 7.86a 0.11 0.011 TAB 6.87a 6.36b 6.29b 6.12b 6.24b 0.13 0.005 Ceca pH 6.55a 6.43a,b 6.47a,b 6.35b 6.32b 0.04 0.020 Coliforms 5.65a 4.97b 5.50a 5.10b 4.91b 0.13 0.001 TAB 6.98a 6.32b 6.75a 6.31b 6.25b 0.12 0.001 Day 35 Crop pH 4.75a 4.47b 4.51b 4.42b 4.39b 0.03 0.005 LAB 7.81b 8.13a 7.98a,b 8.19a 8.25a 0.08 0.017 TAB 7.04a 6.49b 6.40b 6.32b 6.44b 0.12 0.003 Ceca pH 6.62a 6.43b 6.52a,b 6.39a 6.41b 0.04 0.011 Coliforms 6.07a 5.52b 5.91a 5.44b 5.58b 0.10 0.001 TAB 7.59a 7.17b 7.46a 7.01b 7.12b 0.09 0.001 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Day 21 Crop pH 4.90a 4.77a,b 4.68b 4.64a 4.61b 0.05 0.012 LAB 7.32b 7.75a 7.52a,b 7.79a 7.86a 0.11 0.011 TAB 6.87a 6.36b 6.29b 6.12b 6.24b 0.13 0.005 Ceca pH 6.55a 6.43a,b 6.47a,b 6.35b 6.32b 0.04 0.020 Coliforms 5.65a 4.97b 5.50a 5.10b 4.91b 0.13 0.001 TAB 6.98a 6.32b 6.75a 6.31b 6.25b 0.12 0.001 Day 35 Crop pH 4.75a 4.47b 4.51b 4.42b 4.39b 0.03 0.005 LAB 7.81b 8.13a 7.98a,b 8.19a 8.25a 0.08 0.017 TAB 7.04a 6.49b 6.40b 6.32b 6.44b 0.12 0.003 Ceca pH 6.62a 6.43b 6.52a,b 6.39a 6.41b 0.04 0.011 Coliforms 6.07a 5.52b 5.91a 5.44b 5.58b 0.10 0.001 TAB 7.59a 7.17b 7.46a 7.01b 7.12b 0.09 0.001 a,bMeans with different superscripts in each row are significantly different (P < 0.05). 1Data represent means of 8 observations per treatment. 2LAB = lactic acid bacteria; TAB = total anaerobic bacteria. 3CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large Table 4. Effects of the experimental treatments on gastrointestinal microbiota composition (Log10 CFU/g) and pH value in Japanese quails.1 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Day 21 Crop pH 4.90a 4.77a,b 4.68b 4.64a 4.61b 0.05 0.012 LAB 7.32b 7.75a 7.52a,b 7.79a 7.86a 0.11 0.011 TAB 6.87a 6.36b 6.29b 6.12b 6.24b 0.13 0.005 Ceca pH 6.55a 6.43a,b 6.47a,b 6.35b 6.32b 0.04 0.020 Coliforms 5.65a 4.97b 5.50a 5.10b 4.91b 0.13 0.001 TAB 6.98a 6.32b 6.75a 6.31b 6.25b 0.12 0.001 Day 35 Crop pH 4.75a 4.47b 4.51b 4.42b 4.39b 0.03 0.005 LAB 7.81b 8.13a 7.98a,b 8.19a 8.25a 0.08 0.017 TAB 7.04a 6.49b 6.40b 6.32b 6.44b 0.12 0.003 Ceca pH 6.62a 6.43b 6.52a,b 6.39a 6.41b 0.04 0.011 Coliforms 6.07a 5.52b 5.91a 5.44b 5.58b 0.10 0.001 TAB 7.59a 7.17b 7.46a 7.01b 7.12b 0.09 0.001 Treatment3 Item2 CON PM OA PM+OA FSBM SEM P-value Day 21 Crop pH 4.90a 4.77a,b 4.68b 4.64a 4.61b 0.05 0.012 LAB 7.32b 7.75a 7.52a,b 7.79a 7.86a 0.11 0.011 TAB 6.87a 6.36b 6.29b 6.12b 6.24b 0.13 0.005 Ceca pH 6.55a 6.43a,b 6.47a,b 6.35b 6.32b 0.04 0.020 Coliforms 5.65a 4.97b 5.50a 5.10b 4.91b 0.13 0.001 TAB 6.98a 6.32b 6.75a 6.31b 6.25b 0.12 0.001 Day 35 Crop pH 4.75a 4.47b 4.51b 4.42b 4.39b 0.03 0.005 LAB 7.81b 8.13a 7.98a,b 8.19a 8.25a 0.08 0.017 TAB 7.04a 6.49b 6.40b 6.32b 6.44b 0.12 0.003 Ceca pH 6.62a 6.43b 6.52a,b 6.39a 6.41b 0.04 0.011 Coliforms 6.07a 5.52b 5.91a 5.44b 5.58b 0.10 0.001 TAB 7.59a 7.17b 7.46a 7.01b 7.12b 0.09 0.001 a,bMeans with different superscripts in each row are significantly different (P < 0.05). 1Data represent means of 8 observations per treatment. 2LAB = lactic acid bacteria; TAB = total anaerobic bacteria. 3CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large Intestinal Morphological Analysis Table 5 summarizes the effects of the experimental treatments on intestinal morphometric analysis. At d 21, the VH and VH to CD ratio (VH:CD) in the duodenum and jejunum of the birds fed PM, PM+OA, and FSBM diets were greater than other experimental groups (P < 0.05). The use of feed additives or FSBM reduced the CD in the jejunum determined at the end of the starter period (P < 0.05). Also, at d 35, feeding the PM, PM+OA, and FSBM diets increased the VH and VH:CD ratio and decreased the CD in the jejunum (P < 0.05). Table 5. Effects of the experimental treatments on intestinal morphometric analysis (μm) in Japanese quails. Treatment1 Item CON PM OA PM+OA FSBM SEM P-value Day 21 Villus height (VH) Duodenum 765.29b 803.28a 761.13b 798.50a 810.51a 8.63 0.001 Jejunum 603.65b 675.87a 618.75b 663.26a 672.40a 8.11 <.001 Ileum 455.83 471.63 469.07 479.95 487.11 11.62 0.45 Crypt depth (CD) Duodenum 120.75 116.31 119.39 117.84 112.48 3.55 0.54 Jejunum 139.31a 127.54a,b 132.91a,b 121.36b 123.05b 4.05 0.03 Ileum 107.65 101.71 105.31 102.97 99.63 5.05 0.59 VH:CD Duodenum 6.34b 6.92a 6.40b 6.78a,b 7.23a 0.14 0.002 Jejunum 4.35b 5.30a 4.66b 5.47a 5.47a 0.10 <.001 Ileum 4.27 4.67 4.48 4.71 4.91 0.18 0.54 Day 35 Villus height (VH) Duodenum 890.25 918.22 895.04 902.68 932.54 14.99 0.28 Jejunum 637.07b 706.10a 649.19b 720.55a 731.66a 10.71 <.001 Ileum 520.85 547.03 522.24 534.20 542.71 15.38 0.67 Crypt depth (CD) Duodenum 169.04 163.21 165.73 168.96 157.80 5.20 0.53 Jejunum 142.12a 131.59a,b 142.44a 129.05a,b 126.37b 4.21 0.03 Ileum 128.76 123.01 127.22 126.64 121.51 7.17 0.94 VH:CD Duodenum 5.28 5.65 5.43 5.36 5.91 0.19 0.20 Jejunum 4.51b 5.37a 4.57b 5.59a 5.80a 0.13 <.001 Ileum 4.08 4.46 4.17 4.22 4.50 0.16 0.36 Treatment1 Item CON PM OA PM+OA FSBM SEM P-value Day 21 Villus height (VH) Duodenum 765.29b 803.28a 761.13b 798.50a 810.51a 8.63 0.001 Jejunum 603.65b 675.87a 618.75b 663.26a 672.40a 8.11 <.001 Ileum 455.83 471.63 469.07 479.95 487.11 11.62 0.45 Crypt depth (CD) Duodenum 120.75 116.31 119.39 117.84 112.48 3.55 0.54 Jejunum 139.31a 127.54a,b 132.91a,b 121.36b 123.05b 4.05 0.03 Ileum 107.65 101.71 105.31 102.97 99.63 5.05 0.59 VH:CD Duodenum 6.34b 6.92a 6.40b 6.78a,b 7.23a 0.14 0.002 Jejunum 4.35b 5.30a 4.66b 5.47a 5.47a 0.10 <.001 Ileum 4.27 4.67 4.48 4.71 4.91 0.18 0.54 Day 35 Villus height (VH) Duodenum 890.25 918.22 895.04 902.68 932.54 14.99 0.28 Jejunum 637.07b 706.10a 649.19b 720.55a 731.66a 10.71 <.001 Ileum 520.85 547.03 522.24 534.20 542.71 15.38 0.67 Crypt depth (CD) Duodenum 169.04 163.21 165.73 168.96 157.80 5.20 0.53 Jejunum 142.12a 131.59a,b 142.44a 129.05a,b 126.37b 4.21 0.03 Ileum 128.76 123.01 127.22 126.64 121.51 7.17 0.94 VH:CD Duodenum 5.28 5.65 5.43 5.36 5.91 0.19 0.20 Jejunum 4.51b 5.37a 4.57b 5.59a 5.80a 0.13 <.001 Ileum 4.08 4.46 4.17 4.22 4.50 0.16 0.36 a,bMeans with different superscripts in each row are significantly different (P < 0.05). 1CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large Table 5. Effects of the experimental treatments on intestinal morphometric analysis (μm) in Japanese quails. Treatment1 Item CON PM OA PM+OA FSBM SEM P-value Day 21 Villus height (VH) Duodenum 765.29b 803.28a 761.13b 798.50a 810.51a 8.63 0.001 Jejunum 603.65b 675.87a 618.75b 663.26a 672.40a 8.11 <.001 Ileum 455.83 471.63 469.07 479.95 487.11 11.62 0.45 Crypt depth (CD) Duodenum 120.75 116.31 119.39 117.84 112.48 3.55 0.54 Jejunum 139.31a 127.54a,b 132.91a,b 121.36b 123.05b 4.05 0.03 Ileum 107.65 101.71 105.31 102.97 99.63 5.05 0.59 VH:CD Duodenum 6.34b 6.92a 6.40b 6.78a,b 7.23a 0.14 0.002 Jejunum 4.35b 5.30a 4.66b 5.47a 5.47a 0.10 <.001 Ileum 4.27 4.67 4.48 4.71 4.91 0.18 0.54 Day 35 Villus height (VH) Duodenum 890.25 918.22 895.04 902.68 932.54 14.99 0.28 Jejunum 637.07b 706.10a 649.19b 720.55a 731.66a 10.71 <.001 Ileum 520.85 547.03 522.24 534.20 542.71 15.38 0.67 Crypt depth (CD) Duodenum 169.04 163.21 165.73 168.96 157.80 5.20 0.53 Jejunum 142.12a 131.59a,b 142.44a 129.05a,b 126.37b 4.21 0.03 Ileum 128.76 123.01 127.22 126.64 121.51 7.17 0.94 VH:CD Duodenum 5.28 5.65 5.43 5.36 5.91 0.19 0.20 Jejunum 4.51b 5.37a 4.57b 5.59a 5.80a 0.13 <.001 Ileum 4.08 4.46 4.17 4.22 4.50 0.16 0.36 Treatment1 Item CON PM OA PM+OA FSBM SEM P-value Day 21 Villus height (VH) Duodenum 765.29b 803.28a 761.13b 798.50a 810.51a 8.63 0.001 Jejunum 603.65b 675.87a 618.75b 663.26a 672.40a 8.11 <.001 Ileum 455.83 471.63 469.07 479.95 487.11 11.62 0.45 Crypt depth (CD) Duodenum 120.75 116.31 119.39 117.84 112.48 3.55 0.54 Jejunum 139.31a 127.54a,b 132.91a,b 121.36b 123.05b 4.05 0.03 Ileum 107.65 101.71 105.31 102.97 99.63 5.05 0.59 VH:CD Duodenum 6.34b 6.92a 6.40b 6.78a,b 7.23a 0.14 0.002 Jejunum 4.35b 5.30a 4.66b 5.47a 5.47a 0.10 <.001 Ileum 4.27 4.67 4.48 4.71 4.91 0.18 0.54 Day 35 Villus height (VH) Duodenum 890.25 918.22 895.04 902.68 932.54 14.99 0.28 Jejunum 637.07b 706.10a 649.19b 720.55a 731.66a 10.71 <.001 Ileum 520.85 547.03 522.24 534.20 542.71 15.38 0.67 Crypt depth (CD) Duodenum 169.04 163.21 165.73 168.96 157.80 5.20 0.53 Jejunum 142.12a 131.59a,b 142.44a 129.05a,b 126.37b 4.21 0.03 Ileum 128.76 123.01 127.22 126.64 121.51 7.17 0.94 VH:CD Duodenum 5.28 5.65 5.43 5.36 5.91 0.19 0.20 Jejunum 4.51b 5.37a 4.57b 5.59a 5.80a 0.13 <.001 Ileum 4.08 4.46 4.17 4.22 4.50 0.16 0.36 a,bMeans with different superscripts in each row are significantly different (P < 0.05). 1CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large Serum Metabolites The effect of experimental treatments on serum lipid profile of Japanese quails are shown in Table 6. The serum concentrations of cholesterol and LDL-C in birds fed PM, PM+OA, and FSBM diets were significantly lower than other treatments at d 21 (P < 0.05). The PM, PM+OA, and FSBM diet decreased serum concentrations of cholesterol, triglycerides, LDL-C, and VLDL-C compared to the control and OA diets at d 35 (P < 0.05). Table 6. Effects of the experimental treatments on serum lipid profile (mg/dL) in Japanese quails. Treatment2 Item1 CON PM OA PM+OA FSBM SEM P-value Day 21 Cholesterol 162.18a 144.81b 158.74a 140.17b 141.85b 3.12 <.001 Triglycerides 95.46 88.30 90.50 92.45 90.07 3.20 0.58 HDL-C 81.59 82.67 83.91 82.72 79.25 3.16 0.87 LDL-C 61.50a 44.48b 56.73a 38.95b 44.58b 3.14 <.001 VLDL-C 19.09 17.66 18.10 18.49 18.01 0.64 0.58 Day 35 Cholesterol 150.46a 136.20b 146.48a 131.60b 134.61b 3.58 0.001 Triglycerides 72.25a 53.84b 67.55a 52.31b 54.80b 3.29 <.001 HDL-C 85.40 89.60 82.18 88.75 86.01 2.99 0.43 LDL-C 50.60a 35.82b 50.79a 32.38b 37.63b 3.82 0.001 VLDL-C 14.45a 10.77b 13.51a 10.46b 10.96b 0.65 <.001 Treatment2 Item1 CON PM OA PM+OA FSBM SEM P-value Day 21 Cholesterol 162.18a 144.81b 158.74a 140.17b 141.85b 3.12 <.001 Triglycerides 95.46 88.30 90.50 92.45 90.07 3.20 0.58 HDL-C 81.59 82.67 83.91 82.72 79.25 3.16 0.87 LDL-C 61.50a 44.48b 56.73a 38.95b 44.58b 3.14 <.001 VLDL-C 19.09 17.66 18.10 18.49 18.01 0.64 0.58 Day 35 Cholesterol 150.46a 136.20b 146.48a 131.60b 134.61b 3.58 0.001 Triglycerides 72.25a 53.84b 67.55a 52.31b 54.80b 3.29 <.001 HDL-C 85.40 89.60 82.18 88.75 86.01 2.99 0.43 LDL-C 50.60a 35.82b 50.79a 32.38b 37.63b 3.82 0.001 VLDL-C 14.45a 10.77b 13.51a 10.46b 10.96b 0.65 <.001 a,bMeans with different superscripts in each row are significantly different (P < 0.05). 1HDL-C = high-density lipoprotein cholesterol; LDL-C = low-density lipoprotein cholesterol; VLDL-C = very-low-density lipoprotein cholesterol. 2CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large Table 6. Effects of the experimental treatments on serum lipid profile (mg/dL) in Japanese quails. Treatment2 Item1 CON PM OA PM+OA FSBM SEM P-value Day 21 Cholesterol 162.18a 144.81b 158.74a 140.17b 141.85b 3.12 <.001 Triglycerides 95.46 88.30 90.50 92.45 90.07 3.20 0.58 HDL-C 81.59 82.67 83.91 82.72 79.25 3.16 0.87 LDL-C 61.50a 44.48b 56.73a 38.95b 44.58b 3.14 <.001 VLDL-C 19.09 17.66 18.10 18.49 18.01 0.64 0.58 Day 35 Cholesterol 150.46a 136.20b 146.48a 131.60b 134.61b 3.58 0.001 Triglycerides 72.25a 53.84b 67.55a 52.31b 54.80b 3.29 <.001 HDL-C 85.40 89.60 82.18 88.75 86.01 2.99 0.43 LDL-C 50.60a 35.82b 50.79a 32.38b 37.63b 3.82 0.001 VLDL-C 14.45a 10.77b 13.51a 10.46b 10.96b 0.65 <.001 Treatment2 Item1 CON PM OA PM+OA FSBM SEM P-value Day 21 Cholesterol 162.18a 144.81b 158.74a 140.17b 141.85b 3.12 <.001 Triglycerides 95.46 88.30 90.50 92.45 90.07 3.20 0.58 HDL-C 81.59 82.67 83.91 82.72 79.25 3.16 0.87 LDL-C 61.50a 44.48b 56.73a 38.95b 44.58b 3.14 <.001 VLDL-C 19.09 17.66 18.10 18.49 18.01 0.64 0.58 Day 35 Cholesterol 150.46a 136.20b 146.48a 131.60b 134.61b 3.58 0.001 Triglycerides 72.25a 53.84b 67.55a 52.31b 54.80b 3.29 <.001 HDL-C 85.40 89.60 82.18 88.75 86.01 2.99 0.43 LDL-C 50.60a 35.82b 50.79a 32.38b 37.63b 3.82 0.001 VLDL-C 14.45a 10.77b 13.51a 10.46b 10.96b 0.65 <.001 a,bMeans with different superscripts in each row are significantly different (P < 0.05). 1HDL-C = high-density lipoprotein cholesterol; LDL-C = low-density lipoprotein cholesterol; VLDL-C = very-low-density lipoprotein cholesterol. 2CON = control; PM = probiotic mixture; OA = organic acids mixture; PM+OA = probiotic mixture plus organic acids mixture; FSBM = fermented soybean meal. View Large DISCUSSION In the present study, Lactobacillus plantarum, Bacillus subtilis, and Aspergillus oryzae were used for fermentation of SBM. Aspergillus oryzae fungus created an anaerobic environment for the growth of the facultative anaerobes Lactobacillus plantarum and Bacillus subtilis. The reduced pH value (subsequent to the production of OA by anaerobic bacteria) and simultaneously the increase in LAB population suggest that the essential and necessary conditions happened during the fermentation process (Ashayerizadeh et al., 2017). The extension of the fermentation process depends on the amount of lactic acid production. The lactic acid produced should be sufficient to lower the pH to 4 to 4.5, and remains stable during storage to prevent the growth of spoilage and pathogenic bacteria (Hasan, 2003). Similar to the fermentation conditions in our experiment, other studies also have shown that the use of Streptococcus thermophilus, Bacillus subtilis MA139, and Saccharomyces cerevisae or Aspergillus oryzae and Lactobacillus casei in the fermentation of SBM reduces pH and increases the concentration of lactic acid (Chen et al., 2010; Wang et al., 2014). Also, reduction of pH value and increased probiotic bacteria population in FSBM using Bacillus subtilis, Aspergillus niger, and Saccharomyces cerevisiae are reported (Wang et al., 2012). Microbial fermentation of SBM significantly reduced phytic acid, TI, β-conglycinin, glycinin, and CF, besides increasing CP concentration. Similar to our findings, in the studies of Sharawy et al. (2016) and Gao et al. (2013), decreased phytic acid and TI and increased CP were observed in FSBM compared to SBM. Other reports have shown that fermentation of SBM with Aspergillus oryzae (Chen et al., 2013a), Bacillus amyloliquefaciens vs. Lactobacillus spp. and Saccharomyces cerevisiae (Chi and Cho, 2016) reduces CF or oligosaccharides and increases nitrogen content. Decreased concentration of the allergenic protein (β-conglycinin and glycinin) in SBM following fermentation also has been reported (Li et al., 2014; Seo and Cho, 2016). Decreasing TI during fermentation is associated with structural change, precipitation, and inactivation of the TI binding site to trypsin (Chen et al., 2013a; Chi and Cho, 2016). The degrading activity of the enzymes, such as cellulases and phytase, which are produced by microorganisms, could be responsible for the reduction of CF and phytic acid (Chi and Cho, 2016; Ashayerizadeh et al., 2017). Li et al. (2014) reported that after fermentation, β-conglycinin and glycinin would be degraded to small-molecule peptides. The previous studies on the fermentation of SBM with fungi or bacteria suggest that the increase in CP content could be attributed to the simple protein constituents of microbial mass as well as microbial metabolism during fermentation (Kook et al., 2014; Jazi et al., 2017). In the current study, the addition of a mixture of OA was not effective in improving performance parameters of Japanese quails. The beneficial impact of OA mixture on performance traits of poultry has not always been consistent. Similar to our findings, there are reports indicating no or minimal effect of OA on weight gain and feed efficiency (Basmacioglu-Malayoglu et al., 2016; Nosrati et al., 2017), while in contrast, others have reported higher BW and improved FCR by supplementation of OA in Japanese quail (Ocak et al., 2009) and broiler chicks (Levy et al., 2015). This discrepancy in the literature could be due to the different inclusion levels of acid, the chemical form, and their pKa values (Khan and Iqbal, 2016). Birds fed the PM and PM+OA supplemented diets gained higher weight and had a lower FCR compared to birds in the control group. Similarly, other studies also have reported and observed better productive traits by dietary supplementation of probiotics in Japanese quails (Banisharif et al., 2016; Seifi et al., 2017). Probiotics, in general, improve the characteristics of the intestinal microbiota mainly through reducing the pH of the gastrointestinal tract and suppression of pathogenic bacteria (by production of volatile fatty acids and bacteriocins), inhibition of the colonization of the bacteria by competitive exclusion, and also stimulating the immune system (Patterson and Burkholder, 2003). Replacing SBM with FSBM in the diet improved growth performance indices of Japanese quails, and the birds fed the FSBM even out-performed their counterparts in PM groups in terms of FCR. FSBM could have exhibited probiotic properties due to its high population of lactic acid producing bacteria and acidification capacity, thus lowering gastrointestinal tract (GIT) pH, resulting in improved performance (Ashayerizadeh et al., 2017; Jazi et al., 2017). Furthermore, the reduced anti-nutritional factors in FSBM, such as TI, β-conglycinin, glycinin, and also phytic acid, compared to SBM should have prevented gut inflammation and improved nutrient bioavailability and digestibility (Feng et al., 2007a). In line with these results, other studies also have shown that partial inclusion of fermented rapeseed meal (Chiang et al., 2010) and soybean meal (Feng et al., 2007b) in broiler chicks diet can improve weight gain and feed efficiency. It has long been proven that microbial activity in the GIT has a significant impact on growth performance and general health of poultry (Niba et al., 2009). Results of this experiment indicated that PM supplementation and FSBM inclusion in Japanese quails diet can manipulate the gastrointestinal microbiota balance in favor of beneficial and more desirable bacteria, and also reduce pH throughout the GIT. However, diet supplementation with OA failed to induce any effect on the population of LAB, TBA, and coliforms in the crop or in the cecum. Apparently, OA—in particular, butyrate—could be quickly absorbed in a bird's foregut, such as the crop, and thus may not be as effective as other feed additives that can remain active and effective even in lower parts of the GIT (Van der Wielen, 2002). Similarly, Goodarzi Boroojeni et al. (2014) did not observe any marked effect of OA supplementation on population of Escherichia coli and Bifidobacterium Spp. in the crop and cecal content in broiler chicks. Micro-encapsulated OA could remain active in the entire digestive tract and thus have a higher efficacy than free OA in lowering bacteria proliferation in the GIT (Levy et al., 2015). It is well documented that supplementation of multi-strain probiotics of diet could fortify intestinal beneficial microorganisms (such as Lactobacillus) and suppress the growth of potentially pathogenic bacteria, such as Escherichia coli in poultry (Zhang and Kim, 2014; Nosrati et al., 2017). Probiotics can inhibit the gastrointestinal colonization of pathogenic bacteria by facilitating antibody production, competition on adherence site, competition for nutrients between microorganisms, and bactericidal effects (Zhang and Kim, 2014). From this perspective, one of the main factors determining the efficacy of probiotics is the ratio of LAB to pathogenic bacteria (Zhang and Kim, 2014). Likewise, fermented feedstuff such as SBM can increase LAB populations throughout the GIT by acidification of the foregut (especially the crop) and provide a favorable condition for the establishment and growth of beneficial bacteria such as the LAB (Niba et al., 2009). An increase in beneficial microbiota population leads to higher production of short-chain fatty acids and consequently decreased pH of GIT and also creation of a competitive exclusion phenomena, both of which help in forming a natural defense barrier against infection and pathogenic bacteria such as coliforms (Engberg et al., 2009; Ashayerizadeh et al., 2017). Higher LAB and lower fecal Escherichia coli counts in piglets have been observed by inclusion of FSBM (Yuan et al., 2017). Similarly, Engberg et al. (2009) also reported that feeding fermented feed in laying hens increases LAB in the crop and decreases coliforms in the ileum. The greater VH and CD and subsequently higher VH:CD ratio recorded at duodenal and jejunal segments of the small intestine for birds on PM and FSBM treatments imply better morphological development, which could to some extent account for the superior productive performance of these birds. Increased VH could result in a greater absorptive capability for available nutrients (Caspary, 1992), whereas low CD values indicate decreasing metabolic cost of the intestinal epithelium turnover (Floch and Seve, 2000), which may be reflected by the lower FCR observed in the current study. Apparently, the improved intestinal microbiota balance in favor of beneficial bacteria and increased amylase secretion in PM and FSBM groups can be responsible for improved morphological indices (Jin et al., 2000). Fermentation of SBM decreased the level of β-conglycinin and glycinin, which are known as potential antigenic and allergenic compounds for young monogastrics, causing villus atrophy and crypt hyperplasia in the small intestine (Wang et al., 2014). Consistent with our findings, previous studies on fermented protein meals have shown that inclusion of fermented rapeseed meal (Chiang et al., 2010) and SBM (Feng et al., 2007a) improves small intestine morphological parameters in broiler chicks. Various studies have shown that some Lactobacillus spp. could lower total plasma cholesterol and LDL-C (Anderson and Gilliland, 1999; Sanders, 2000; Kalavathy et al., 2010). These Lactobacillus spp. possess bile salt deconjugation ability and can hydrolyze bile salts, thus interfering with the reabsorption cycle of bile salts and increasing their fecal excretion. As cholesterol is the precursor of primary bile salts that are formed in the liver, a higher excretion of bile salts is associated with a higher excretion of cholesterol (Liong and Shah, 2005). Thus, the hypocholesteremic effects of PM and FSBM treatments observed in this study could be explained in light of a higher intestinal population of LAB in these treatments. Lactobacilli also have been reported to show hypolipidemic properties by inhibiting the activity of 3-hydroxy-3-methyl-glutaryl-CoA (Seifi et al., 2017), which could explain the lower serum concentration of triglycerides in PM and FSBM treatments observed at d 35. Similarly, other researchers also have reported lower serum cholesterol and triglycerides in Japanese quails (Seifi et al., 2017), broiler chicks (Kalavathy et al., 2010), and geese (Chen et al., 2013b) through diet supplementation with probiotic and/or fermented feed. CONCLUSION In summary, the results obtained in the current study indicate that microbial fermentation significantly reduces the content of some of the anti-nutritional factors present in SBM and also improves its nutritional value. In addition, FSBM exhibited the capability to perform effectively in the Japanese quail's GIT as a potential probiotic source. Diet supplementation with a PM or replacing SBM with FSBM in the feed can improve growth performance of quails, largely through improving gastrointestinal microbiota balance in favor of beneficial bacteria, and small intestine morphological parameters. No synergistic or additive effects seem to exist between the OA and PM used in this study. ACKNOWLEDGMENTS This project was funded and financially supported by Young Researchers and Elite Club, Islamic Azad University, Isfahan (Khorasgan) Branch, Isfahan, Iran. REFRERNCES Anderson J. W. , Gilliland S. E. . 1999 . Effect of fermented milk (yoghurt) containing Lactobacillus acidophilus L1 on serum cholesterol in hypercholesterolemic humans . J. Am. Coll. Nutr. 18 , 43 – 50 Google Scholar CrossRef Search ADS PubMed AOAC . 2005 . Association of Official Analytical Chemists. 2005 . 21th ed . Gaithersburg, M. D. : AOAC International . Ashayerizadeh A. , Dastar B. , Shams Shargh M. , Sadeghi Mahoonak A. R. , Zerehdaran S. . 2017 . Fermented rapeseed meal is effective in controlling Salmonella enterica serovar Typhimurium infection and improving growth performance in broiler chicks . Veterinary Microbiology . 201 : 93 – 102 . Google Scholar CrossRef Search ADS PubMed Banisharif M. , Kheiri F. , Jalali S. M. A. . 2016 . Hypericum perforatum and probiotic effects on performance, carcass characteristics and intestinal morphology in Japanese quails (Coturnix japonica) . J. Herb. Drugs. 7 : 83 – 88 . Basmacioglu-Malayoglu H. , Ozdemir P. , Bagriyanik H. A. . 2016 . Influence of an organic acid blend and essential oil blend, individually or in combination, on growth performance, carcass parameters, apparent digestibility, intestinal microflora and intestinal morphology of broilers . British Poultry Science . 57 : 227 – 234 . Google Scholar CrossRef Search ADS PubMed Boroojeni Goodarzi , Vahjen F. W. , Mader A. , Knorr F. , Ruhnke I. , Rohe I. , Hafeez A. , Villodre C. , Manner K. , Zentek J. . 2014 . The effects of different thermal treatments and organic acid levels in feed on microbial composition and activity in gastrointestinal tract of broilers . Poult. Sci . 93 : 1440 – 1452 . Google Scholar CrossRef Search ADS PubMed Caspary W. F. 1992 . Physiology and pathophysiology of intestinal absorption . Am. J. Clin. Nutr . 55 : 299S – 308S . Google Scholar CrossRef Search ADS PubMed Chen C. C. , Shih Y. C. , Chiou P. W. S. , Yu B. . 2010 . Evaluating nutritional quality of single stage- and two stage-fermented soybean meal . Asian Australas. J. Anim. Sci. 23 : 598 – 606 . Google Scholar CrossRef Search ADS Chen L. , Madl R. L. , Vadlani P. V. . 2013 . Nutritional enhancement of soy meal via aspergillus oryzae solid-state fermentation . Cereal Chemistry Journal. 90 : 529 – 534 . Google Scholar CrossRef Search ADS Chen W. , Zhu X. Z. , Wang J. P. , Wang Z. X. , Huang Y. Q. . 2013 . Effects of Bacillus subtilis var. natto and Saccharomyces cerevisiae fermented liquid feed on growth performance, relative organ weight, intestinal microflora, and organ antioxidant status in Landes geese . J. Anim. Sci . 91 : 978 – 985 . Google Scholar CrossRef Search ADS PubMed Chi C. H. , Cho S. J. . 2016 . Improvement of bioactivity of soybean meal by solid-state fermentation with Bacillus amyloliquefaciens versus Lactobacillus spp. and Saccharomyces cerevisiae . LWT - Food Science and Technology 68 : 619 – 625 . Google Scholar CrossRef Search ADS Chiang G. , Lu W. Q. , Piao X. S. , Hu J. K. , Gong L. M. , Thacker P. A. . 2010 . Effects of feeding solid-state fermented rapeseed meal on performance, nutrient digestibility, intestinal ecology and intestinal morphology of broiler chickens. Asian-Aust . J. Anim. Sci . 23 : 263 – 271 . Engberg R. M. , Hammershoj M. , Johansen N. F. , Abousekken M. S. , Steenfeldt S. , Jensen B. B. . 2009 . Fermented feed for laying hens: Effects on egg production, egg quality, plumage condition and composition and activity of the intestinal microflora . British Poultry Science . 50 : 228 – 239 . Google Scholar CrossRef Search ADS PubMed Feng J. , Liu X. , Xu Z. R. , Wang Y. Z. , Liu J. X. . 2007a . Effects of fermented soybean meal on digestive enzyme activities and intestinal morphology in broilers . Poultry Science . 86 : 1149 – 1154 . Google Scholar CrossRef Search ADS Feng J. , Liu X. , Xu Z. R. , Liu Y. Y. , Lu Y. P. . 2007b . Effects of Aspergillus oryzae 3.042 fermented soybean meal on growth performance and plasma biochemical parameters in broilers . Animal Feed Science and Technology . 134 : 235 – 242 . Google Scholar CrossRef Search ADS Floch N. L. , Seve B. . 2000 . Protein and amino acid metabolism in the intestine of the pig: From digestion to appearance in the portal vein . Prod. Anim . 13 : 303 – 314 . Gao Y. L. , Wang C. S. , Zhu Q. H. , Qian G. Y. . 2013 . Optimization of solid-state fermentation with Lactobacillus brevis and Aspergillus oryzae for trypsin inhibitor degradation in soybean meal . Journal of Integrative Agriculture . 12 : 869 – 876 . Google Scholar CrossRef Search ADS Hasan B. 2003 . Fermentation of fish silage using lactobacillus pentosus . J. Natur Indonesia . 6 : 11 – 15 . Jazi V. , Boldaji F. , Dastar B. , Hashemi S. R. , Ashayerizadeh A. . 2017 . Effects of fermented cottonseed meal on the growth performance, gastrointestinal microflora population and small intestinal morphology in broiler chickens . British Poultry Science . 58 : 402 – 408 . Google Scholar CrossRef Search ADS PubMed Jin L. Z. , Ho Y. W. , Abdullah N. , Jalaludin S. . 2000 . Digestive and bacterial enzyme activities in broilers fed diets supplemented with Lactobacillus cultures . Poultry Science . 79 : 886 – 891 . Google Scholar CrossRef Search ADS PubMed Kalavathy R. , Norhani A. , Michael C. V. L. W. , Chinna K. , Yin W. H. . 2010 . Bile salt deconjugation and cholesterol removal from media by Lactobacillus strains used as probiotics in chickens . J. Sci. Food Agric. 90 : 65 – 69 . Google Scholar CrossRef Search ADS PubMed Khan S. H. , Iqbal J. . 2016 . Recent advances in the role of organic acids in poultry nutrition . Journal of Applied Animal Research , 441 : 359 – 369 . Google Scholar CrossRef Search ADS Kook M. C. , Cho. S. C. , Hong Y. H. , Park H. . 2014 . Bacillus subtilis fermentation for enhancement of feed nutritive value of soybean meal . J. Appl. Biol. Chem. 57 : 183 – 188 . Google Scholar CrossRef Search ADS Lee K. W. , Lee S. H. , Lillehoj H. S. , Li G. X. , Jang S. I. , Babu U. S. , Park M. S. , Kim D. K. , Lillehoj E. P. , Neumann A. P. , Rehberger T. G. , Siragusa G. R. . 2010 . Effects of direct-fed microbials on growth performance, gut morphometry, and immune characteristics in broiler chickens . Poultry Science . 89 : 203 – 216 . Google Scholar CrossRef Search ADS PubMed Liong M. T. , Shah N. P. . 2005 . Bile salt deconjugation ability, bile salt hydrolase activity and cholesterol co-precipitation ability of lactobacilli strains . International Dairy Journal . 15 : 391 – 398 . Google Scholar CrossRef Search ADS Levy A. W. , Kessler J. W. , Fuller L. , Williams S. , Mathis G. F. , Lumpkins B. , Valdez F. . 2015 . Effect of feeding an encapsulated source of butyric acid (ButiPEARL) on the performance of male Cobb broilers reared to 42 d of age . Poult. Sci. 94 : 1864 – 1870 . Google Scholar CrossRef Search ADS PubMed Li C. Y. , Lu J. J. , Wu C. P. , Lien T. F. . 2014 . Effects of probiotics and bremelain fermented soybean meal replacing fish meal on growth performance, nutrient retention and carcass traits of broilers . Livestock Science . 163 : 94 – 101 . Google Scholar CrossRef Search ADS Marsili R. T. , Ostapenko H. , Simmons R. E. , Green D. E. . 1983 . High performance liquid chromatographic determination of organic acid . J. Food Prot . 46 : 52 – 57 . Google Scholar CrossRef Search ADS Missotten J. A. M. , Michiels J. , Degroote J. , De Smet S. . 2015 . Fermented liquid feed for pigs: An ancient technique for the future . J Animal Sci Biotechnol . 6 : 4 . Google Scholar CrossRef Search ADS Nava G. M , Attene-Ramos M. S , Gaskins H. R , Richards J. D . 2009 . Molecular analysis of microbial community structure in the chicken ileum following organic acid supplementation . Veterinary Microbiology . 137 : 345 – 353 . Google Scholar CrossRef Search ADS PubMed Niba A. T. , Beal J. D. , Kudi A. C. , Brooks P. H. . 2009 . Potential of bacterial fermentation as a biosafe method of improving feeds for pigs and poultry . Africa. J. Biotechnol . 8 : 1758 – 1767 . Nosrati M. , Javandel F. , Camacho L. M. , Khusro A. , Cipriano M. , Seidavi A. , Salem A. Z. M. . 2017 . The effects of antibiotic, probiotic, organic acid, vitamin C, and Echinacea purpurea extract on performance, carcass characteristics, blood chemistry, microbiota, and immunity of broiler chickens . J. Appl. Poult . Res . 26 : 295 – 306 . NRC . 1994 . Nutrient Requirements of Poultry . 9th rev. ed . Natl. Acad. Press , Washington, DC . Panda A. K. , Rao S. V. R. , Raju M. V. , Sharma S. R. . 2006 . Dietary supplementation of Lactobacillus sporogenes on performance and serum biochemico-lipid profile of broiler chickens . J. Poult. Sci. 43 : 235 – 240 . Google Scholar CrossRef Search ADS Patterson J. A. , Burkholder K. M. . 2003 . Application of prebiotics and probiotics in poultry production . Poultry Science . 82 : 627 – 631 . Google Scholar CrossRef Search ADS PubMed Ocak N. , Erener G. , Altop A. , Kop C. . 2009 . The effect of malic acid on performance and some digestive tract traits of Japanese quails . J. Poult. Sci. 46 : 25 – 29 . Google Scholar CrossRef Search ADS Sanders M. E. 2000 . Considerations for use of probiotic bacteria to modulate human health . J. Nutr . 130 : 384S – 390S . Google Scholar CrossRef Search ADS PubMed SAS Institute . 2003 . User's Guide: Statistics , Version 9.1 . SAS Institute, Inc. , Cary, NC, USA . Seifi K. , Karimi Torshizi M. A. , Rahimi S. , Kazemifard M. . 2017 . Efficiency of early, single-dose probiotic administration methods on performance, small intestinal morphology, blood biochemistry, and immune response of Japanese quail . Poult. Sci. 96 : 2151 – 2158 . Google Scholar CrossRef Search ADS PubMed Seo S. H. , Cho S. J. . 2016 . Changes in allergenic and antinutritional protein profiles of soybean meal during solid-state fermentation with Bacillus subtilis . LWT - Food Science and Technology . 70 : 208 – 212 . Google Scholar CrossRef Search ADS Sharawy Z. , Goda A. M. A. S. , Hassaan M. S. . 2016 . Partial or total replacement of fish meal by solid state fermented soybean meal with Saccharomyces cerevisiae in diets for Indian prawn shrimp, Fenneropenaeus indicus , Postlarvae . Animal Feed Science and Technology 212 : 90 – 99 . Google Scholar CrossRef Search ADS Smith C. , Van Megen W. , Twaalfhoven L. , Hitchcock C. . 1980 . The determination of trypsin inhibitor levels in foodstuffs . J. Sci. Food Agric. . 31 : 341 – 350 . Google Scholar CrossRef Search ADS PubMed Sun H. , Tang J. W. , Yao X. H. , Wu Y. F. , Wang X. , Feng J. . 2013 . Effects of dietary inclusion of fermented cottonseed meal on growth, cecal microbial population, small intestinal morphology, and digestive enzyme activity of broilers . Trop Anim Health Prod . 45 : 987 – 993 . Google Scholar CrossRef Search ADS PubMed Toghyani M. , Toghyani M. , Gheisari A. , Ghalamkari G. , Mohammadrezaei M. . 2010 . Growth performance, serum biochemistry and blood hematology of broiler chicks fed different levels of black seed (Nigella sativa) and peppermint (Mentha piperita) . Livestock Science . 129 : 173 – 178 . Google Scholar CrossRef Search ADS Van der Wielen P . 2002 . Dietary strategies to influence the gastrointestinal microflora of young animals and its potential to improve intestinal health . Pages 37–60 in Nutrition and Health of the Gastrointestinal Tract , Blok M. C , Vahl H. A. , de Lange L. , vande Braak A. E , Hemke G. , Hessing M. eds. Wageningen Academic Publishers , Wageningen, the Netherlands . Wang L. C. , Wen C. , Jiang Z. Y. , Zhou Y. M. . 2012 . Evaluation of the partial replacement of high-protein feedstuff with fermented soybean meal in broiler diets . The Journal of Applied Poultry Research . 21 : 849 – 855 . Google Scholar CrossRef Search ADS Wang Y. , Liu X. T. , Wang H. L. , Li D. F. , Piao X. S. , Lu W. Q. . 2014 . Optimization of processing conditions for solid-state fermented soybean meal and its effects on growth performance and nutrient digestibility of weanling pigs . Livestock Science . 170 : 91 – 99 . Google Scholar CrossRef Search ADS Yuan L. , Chang J. , Yin Q. , Lu M. , Di Y. , Wang P. , Wang Z. , Wang E. , Lu F. . 2017 . Fermented soybean meal improves the growth performance, nutrient digestibility, and microbial flora in piglets Animal Nutrition . 3 : 19 – 24 . Google Scholar CrossRef Search ADS Zhang Z. F. , Kim I. H. . 2014 . Effects of multistrain probiotics on growth performance, apparent ileal nutrient digestibility, blood characteristics, cecal microbial shedding, and excreta odor contents in broilers . Poult. Sci . 93 : 364 – 370 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. 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Poultry ScienceOxford University Press

Published: Mar 15, 2018

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