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. 2023 Mar 8;102(6):102629. doi: 10.1016/j.psj.2023.102629

Effects of fermented feed on growth performance, immune organ indices, serum biochemical parameters, cecal odorous compound production, and the microbiota community in broilers

Xin Zhu 1, Lijuan Tao 1, Haiying Liu 1, Guiqin Yang 1,1
PMCID: PMC10091030  PMID: 37004289

Abstract

The aim of this study was to explore the effects of dietary fermented feed addition on growth performance, immune organ indices, serum biochemical parameters, cecal odorous compound production, and the bacterial community in broilers. A total of 480 broiler chicks (1-day-old) were randomly assigned to 6 groups, including a basal diet (control group), a basal diet supplemented with 10, 15, 20, and 25% dried fermented feed, and 10% wet fermented feed. Each group contained 8 replicates of 10 chicks each. The results showed that fermentation increased (P < 0.05) the total acid level and the number of Lactobacillus, Yeast, and Bacillus. The 15% dried fermented feed group had an increased (P < 0.05) body weight (BW) than the control, while the 25% dried fermented feed group had the lowest (P < 0.05) BW on 42 d. Compared to the control group, the feed intake (FI) was increased (P < 0.05) in the 10, 15% dried and 10% wet fermented feed groups from 22 to 42 d and from 1 to 42 d. No significant difference (P > 0.05) was observed in feed conversion ratio (FCR) among all groups. Supplementation with fermented feed increased (P < 0.05) the bursa of Fabricius index but not (P > 0.05) the thymus and spleen indices. Compared with the control, the broilers fed fermented feed had increased (P < 0.05) serum total protein, albumin, globulin, IgA, IgG, IgM, lysozyme, complement 3, and complement 4 levels. The cecal concentrations of acetic acid, propionic acid, butyric acid, and lactic acid were increased and the pH values were decreased in the fermented feed groups (P < 0.05). Among the groups, the 15% dried fermented feed group showed the lowest concentrations of skatole and indole in the cecum (P < 0.05). The composition of the cecal microbiota was characterized, in which an increased abundance of Ruminococcaceae, Lactobacillaceae, and unclassified Clostridiales and a decreased abundance of Rikenellaceae, Lachnospiraceae, and Bacteroidaceae were found in the fermented feed groups. Taken together, dietary fermented feed supplementation can improve growth performance, immune organ development, and capacity and decrease cecal odorous compound production, which may be related to the regulation of microbial composition.

Key words: fermentation, immune, skatole, microbiota, broiler

INTRODUCTION

By 2050, the global population may reach over 9 billion, approximately 2 billion more than the current population, and this population growth will lead to an increased demand for animal protein (Goujon, 2019). To meet this demand, livestock and poultry operations will remain the mainstay for commercial production of animal protein, and the feeding scale will become larger. However, intensive animal production systems create a huge amount of fecal and odor substances. Odor emissions during animal production have become a major challenge for animal/human health and the environment, along with the continuously growing public complaints (Wang et al., 2021; Piccardo et al., 2022). To control livestock and poultry odor production, strict regulations have been enacted by governments, such as the livestock and poultry banned zone and the offensive odor control law.

Various odorous compounds have been identified in large-scale livestock and poultry farms, such as ammonia gas and volatile amines, sulfur-containing compounds, volatile fatty acids (VFAs), phenols, and indoles (Nie et al., 2020; Konkol et al., 2022). Among these components, skatole is a well-known typical N-heterocyclic aromatic compound with foul odor distributed in various environmental matrices, such as intestinal tract and feces slurry, sludge, and wastewater (Ma et al., 2021). In the animal gut, skatole is produced from the transformation of tryptophan via bacterial enzymes. Generally, skatole is relatively resistant to biodegradation and may persist in the environment. Due to its wide presence and lower odor detection threshold, skatole is usually regarded as the third main contributor, following ammonia and hydrogen sulfide, to odor emissions in livestock and poultry farms (Tesso et al., 2019). To lower odorous compound production in animal husbandry, limited methods have been adopted, including the modification of diets (Cho et al., 2015; Recharla et al., 2017; Khattak and Helmbrecht, 2019; Li et al., 2019) and the addition of oligosaccharides (Zhu et al., 2020; Liu et al., 2021), enzymes (O'Shea et al., 2014; Kim et al., 2022), organic acids (Øverland et al., 2008), microbes (Borowski et al., 2017; Greff et al., 2022), and absorbent materials (Schubert et al., 2021).

Fermented feed has recently attracted increasing attention for its improved nutritional characteristics, digestibility, palatability, and safety (Xu et al., 2019; Yang et al., 2022). Usually, manufacturing of fermented feed can be performed through the addition of natural or exogenous microorganisms into feed substrates under artificial control conditions (Yang et al., 2021). The fermentation process not only decomposes or transforms the antinutritional factors into nontoxic components, but also enhances the concentration of beneficial probiotics, enzymes and metabolites (Liu et al., 2023; Su et al., 2022). The application of fermented feed has been demonstrated to increase production performance, improve immune ability and modulate gastrointestinal ecology in broilers (Li et al., 2020). The intestinal microbiota plays important roles in nutrition, physiology, gut morphology, and host immune defense mechanisms. Many previous studies have demonstrated that the addition of fermented feed could be beneficial for maintaining health in poultry by lowering gut pH and stimulating the growth of lactobacilli, thus leading to higher lactic acid and short-chain fatty acid production. Changes in the composition of the gut microbiota may affect the immune response of chickens. Ao et al. (2011), Gao et al. (2009), and Tang et al. (2012) found that feeding fermented feed increased the weight of the bursa of Fabricius and spleen, serum IgM, IgG, complement 4, lysozyme content, and albumin:globulin ratio, while Choi et al. (2014) found decreased serum IgG content in broilers fed a fermented feed diet. Thus, the mechanism of induction and regulation of the immune response by dietary supplementation with fermented feed in broilers remains unclear. In addition, modulation of the gut microbial community may be an effective strategy for reducing odorous compound production (Zhu et al., 2020; Liu et al., 2021). However, to date, there is limited information concerning the effect and mechanism of fermented feed supplementation on odorous compound production in broilers. Our previous study demonstrated that fermented feed addition can enhance the average daily feed intake and average daily gain in broilers, contributing to growth performance and nutrient digestibility (Li et al., 2022). Herein, in the present study, we investigated the effects of dietary supplementation with fermented feed on immune function and odorous compound production and characterized the microbiota in the cecum of broilers. The results we obtained will be potentially useful for the application of fermented feed in improving health and controlling odor emissions and understanding the underlying mechanism in the livestock and poultry industries.

MATERIALS AND METHODS

Experimental Design and Bird Management

All experimental procedures were approved by the Animal Care and Use Committee of Shenyang Agricultural University (No. 202006046).

A total of 480 one-day-old Arbor Acre broilers with similar initial body weights (BWs) were randomly allocated into 6 groups with 8 replicates each and 10 chicks per replicate (5 males and 5 females). The chicks in each group were fed a basal diet supplemented with 0 (control), 10, 15, 20, 25% dried fermented feed (DFF) or 10% wet fermented feed (WFF). The diet components and nutrient levels are shown in Tables 1 and 2.

Table 1.

Ingredients and nutrient composition of diets in the starter period (1–21 d, air dry basis, %).

Content
Items Control 10% DFF 15% DFF 20% DFF 25% DFF 10% WFF
Ingredients
 Corn 56.50 50.20 47.05 43.90 40.74 50.20
 Soybean meal 25.55 22.70 21.28 19.85 18.43 22.70
 Corn gluten meal 4.60 4.09 3.83 3.57 3.32 4.09
 DDGS 3.00 2.67 2.50 2.33 2.16 2.67
 Soybean oil 1.00 1.00 1.00 1.00 1.00 1.00
 Extruded soybean powder 5.00 5.00 5.00 5.00 5.00 5.00
 Limestone 1.20 1.20 1.20 1.20 1.20 1.20
 Calcium hydrophosphate 1.80 1.80 1.80 1.80 1.80 1.80
 Sodium chloride 0.25 0.25 0.25 0.25 0.25 0.25
 Choline chloride 0.10 0.10 0.10 0.10 0.10 0.10
 Premix1 1.00 1.00 1.00 1.00 1.00 1.00
 Fermented feed 0.00 10.00 15.00 20.00 25.00 10.00
 Total 100.00 100.00 100.00 100.00 100.00 100.00
Nutrient content2
 Metabolizable energy (MJ/kg) 12.42 12.67 12.56 12.49 12.55 12.47
 Crude protein 20.03 20.22 20.77 20.67 20.79 20.31
 Ether extract 4.61 3.83 4.17 3.96 3.87 3.75
 Calcium 0.98 0.82 0.78 1.02 0.98 0.85
 Total phosphorus 0.66 0.67 0.69 0.62 0.63 0.68
 Available phosphorus 0.45 0.46 0.47 0.42 0.43 0.46
 Lysine 1.11 1.11 1.11 1.11 1.11 1.11
 Methionine 0.48 0.48 0.48 0.48 0.48 0.48
 Threonine 0.73 0.73 0.73 0.73 0.73 0.73
 Tryptophan 0.19 0.19 0.19 0.19 0.19 0.19
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1

The premix provided the following per kg of the diets: vitamin A, 18,000 IU; vitamin D3, 2,800 IU; vitamin E, 90 mg; vitamin K3, 7.2 mg; vitamin B1, 6.8 mg; vitamin B2, 27 mg; vitamin B6, 13.5 mg; vitamin B12, 0.1 mg; nicotinamide, 108 mg; calcium pantothenate, 45 mg; folic acid, 19.8 mg; biotin, 0.7 mg; Fe, 100 mg; Cu, 25 mg; Mn, 120 mg; Zn, 80 mg; I, 1 mg; Se, 0.15 mg.

2

Crude protein, ether extract, calcium, and total phosphorus were measured values, and the others were calculated values.

Table 2.

Ingredients and nutrient composition of diets in the grower period (22–42 d, air dry basis, %).

Content
Items Control 10% DFF 15% DFF 20% DFF 25% DFF 10% WFF
Ingredients
 Corn 55.40 49.04 45.86 42.68 39.50 49.04
 Soybean meal 22.20 19.65 18.38 17.10 15.83 19.65
 Corn gluten meal 3.00 2.66 2.48 2.31 2.14 2.65
 DDGS 3.00 2.66 2.48 2.31 2.14 2.65
 Wheat bran 3.50 3.10 2.90 2.70 2.50 3.10
 Soybean oil 1.00 1.00 1.00 1.00 1.00 1.00
 Extruded soybean powder 5.00 5.00 5.00 5.00 5.00 5.00
 Limestone 1.20 1.20 1.20 1.20 1.20 1.20
 Calcium hydrophosphate 1.80 1.80 1.80 1.80 1.80 1.80
 Sodium chloride 0.30 0.30 0.30 0.30 0.30 0.30
 Choline chloride 0.10 0.10 0.10 0.10 0.10 0.10
 Premix1 1.00 1.00 1.00 1.00 1.00 1.00
 Fermented feed 0.00 10.00 15.00 20.00 25.00 10.00
 Total 100.00 100.00 100.00 100.00 100.00 100.00
Nutrient content2
 Metabolizable energy (MJ/kg) 12.78 13.38 13.98 12.84 12.76 13.27
 Crude protein 18.77 18.94 18.57 18.90 18.78 18.36
 Ether extract 6.13 7.04 6.12 6.68 6.12 6.85
 Calcium 1.09 0.98 0.98 0.98 1.00 1.02
 Total phosphorus 0.62 0.73 0.63 0.68 0.73 0.74
 Available phosphorus 0.42 0.49 0.43 0.46 0.49 0.50
 Lysine 0.98 0.98 0.98 0.98 0.98 0.98
 Methionine 0.44 0.44 0.44 0.44 0.44 0.44
 Threonine 0.65 0.65 0.65 0.65 0.65 0.65
 Tryptophan 0.18 0.18 0.18 0.18 0.18 0.18
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1

The premix provided the following per kg of the diets: vitamin A, 18,000 IU; vitamin D3, 2,800 IU; vitamin E, 90 mg; vitamin K3, 7.2 mg; vitamin B1, 6.8 mg; vitamin B2, 27 mg; vitamin B6, 13.5 mg; vitamin B12, 0.1 mg; nicotinamide, 108 mg; calcium pantothenate, 45 mg; folic acid, 19.8 mg; biotin, 0.7 mg; Fe, 100 mg; Cu, 25 mg; Mn, 120 mg; Zn, 80 mg; I, 1 mg; Se, 0.15 mg.

2

Crude protein, ether extract, calcium, and total phosphorus were measured values, and the others were calculated values.

During a 6-wk experimental period, all chicks had ad libitum access to feed and water and were housed in a thermostatically controlled room. On D 1, the room temperature was 35°C and was gradually decreased at 0.5°C per d until it was decreased to 21°C, which was kept until the end of trial. The lighting was provided for 24 h each day. All chicks were vaccinated against Newcastle disease and infectious bronchitis via intranasal and intraocular administration on D 7, infectious bursal disease via oral drip on D 10, fowlpox by wing-web puncture on D 12 and Newcastle disease and infectious bursal disease by drinking water on D 24.

Fermented Feed Preparation

Lactobacillus plantarum (1.0 × 1011 CFU/g), Bacillus subtilis (1.0 × 1011 CFU/g), and Saccharomyces cerevisiae (2.0 × 1010 CFU/g) were selected and provided by Jiuzhou Biological Technology Co. Ltd. (Shenyang, China). The composition of the fermentation substrate was 63.02% corn, 28.51% soybean meal, 5.13% corn gluten meal, and 3.34% corn dried distillers grains soluble for a starter period (from 1 to 21 d) or 63.61 corn, 25.49% soybean meal, 3.44% corn gluten meal, 3.44% corn dried distillers grains soluble and 4.02% wheat bran for a grower period (from 22 to 42 d). A manufacturing process was briefly summarized as follows: The starter culture was inoculated into the fermentation substrate to be fermented (107 CFU/g feed), which was mixed and supplemented with sterile water to meet a 30% moisture content. The mixed wet substrate was transferred into breathing bags with a 1-way breathing valve (only exhaust but not intake) and fermented at 32°C for 5 d. After fermentation, a part of the fermented feed was dried at 35°C for 3 d, and the other part remained wet. Before feeding, the basal diet was mixed with DFF in different proportions (10, 15, 20, 25%) or 10% WFF, and the mixed diets were used as the experimental diets. Five grams of fermented sample was mixed with 50 mL of distilled water, shaken for 30 min, and the supernatant was used to measure the pH (PB-20, Sartorius, Gottingen, Germany). L. plantarrum were counted on MRS agar incubated at 36°C for 72 h under anaerobic conditions. The number of S. cerevisiae was measured by plate counting on PDA agar incubated at 28°C for 5 d under aerobic conditions, while that of B. subtilis was counted on MYP agar aerobically incubated at 30°C for 48 h. The viable colonies were enumerated in colony-forming units (CFUs) per gram of fermented sample.

Growth Performance

BW and feed intake (FI) were recorded at 1, 21, and 42 d of age, and feed conversion ratio (FCR, g feed/g gain) was calculated to evaluate the growth performance of broiler chickens.

Sample Collection

On D 42 of the trial, 1 chick from each replicate (4 males and 4 females in each group) was selected, weighed, and then sacrificed by bleeding from the jugular vein, after which the thymus, spleen, bursa of Fabricius, and ceca were dissected and removed. The cecal contents were collected and stored at −80°C for further analysis. Ten milliliters of blood were collected aseptically in aseptic capped tubes and centrifuged at 3,000 × g for 10 min at 4°C to obtain serum samples, which were then stored at −20°C for biochemistry parameter analysis.

Immune Organ Index

Weights of the thymus, spleen, and bursa of Fabricius were individually recorded. The index was expressed as follows:

Immune organ index = immune organ weight (g)/live body weight (kg).

Serum Biochemical Parameters

Serum biochemical parameters, including total protein, albumin, globulin, IgA, IgG, IgM, complement 3, complement 4, and lysozyme, were determined using commercial kits following the manufacturer's instructions (Jiangsu Meimian Industry Co., Ltd., Yancheng, China) and measured using a microplate reader (iMark, BIO-RAD, Hercules, CA).

Skatole, Indole, VFAs, and Lactic Acid Analysis

One gram of cecal contents was mixed with 2 mL of distilled water in a 5-mL centrifuge tube, and then homogenized by a tissue homogenizer for 20 s. After centrifuging at 3,000 × g for 10 min, 1 mL of supernatant liquid was collected and used to determine the concentrations of skatole, indole, VFAs and lactic acid by a high-performance liquid chromatography method as described previously (Zhu et al., 2020).

Microbiota Community Analysis

Total DNA of each cecal content sample was isolated using a commercial Mag-Bind DNA kit (Omega Bio-Tek, Waltham, MA). The quality of the extracted DNA was measured using electrophoresis on 2% agarose gels, and the purity was determined using a Qubit 3.0 fluorometer (Invitrogen, Carlsbad, CA). The forward primer 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and the reverse primer 806R (5′GGACTACHVGGGTWTCTAAT-3′) were used to amplify the V3 to V4 hypervariable region of the 16S rRNA gene. A polymerase chain reaction (PCR) amplification reaction system was prepared according to a commercial kit instructions (Yeasen Biotechnology, Shanghai, China). Program a thermal cycler as follows: 94°C for 3 min; 5 cycles of 94°C for 30 s and annealing temperature of 45°C for 20 s; 65°C for 30 s; 20 cycles of 94°C for 20 s, annealing temperature of 55°C for 20 s; 72°C for 30 s; and hold at 72°C for 5 min. PCR-amplified products were extracted by 2% agarose gel electrophoresis, purified with a Hieff NGS DNA Selection Beads kit (Yeasen Biotechnology, Shanghai, China) and quantified using a Qubit DNA assay kit (Invitrogen, Carlsbad, CA) following the manufacturer's protocols. The library was sequenced on an Illumina MiSeq platform (Sangon Biotech, Shanghai, China) and 250 bp paired-end reads were generated.

After sequencing, according to the unique barcodes, the raw paired-end reads were assigned to 1 sample read by discarding the primer connector sequences and were then processed to obtain qualified sequences by removing nonamplification and chimeric sequences using the Usearch and Uchime software packages (drive5.com). An Uclust algorithm was used to obtain operational sequences, and operational taxonomic units (OTUs) for species classification were clustered with 97% similarity. Species annotation and statistical analysis of the species composition for each sample at the phylum and genus levels were conducted using the Ribosomal Database Project classifier software and the Greengenes database.

Statistical Analysis

Data analysis was performed by using 1-way ANOVA with SPSS 22.0 software (SPSS Inc., Chicago, IL). Replicates were considered the experimental units. Duncan's multiple range tests were used to compare pooled SEMs, and a value of P < 0.05 was considered statistically significant.

RESULTS

Fermented Feed Characteristics

As shown in Figure 1, after fermentation, the level of total acid was increased (P < 0.05), while that of pH was decreased (P < 0.05) in both starter and grower wet fermented feed. Compared with the dried fermented feed, the number of Lactobacillus, Yeast, and Bacillus was much higher in both starter and grower wet fermented feed (P < 0.05).

Figure 1.

Figure 1

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Changes in the nutrient composition of feeds before and after fermentation. Fermentation substrates for starter (D 1–21) and grower (D 22–42) periods were fermented. The values were expressed as the means. Statistical differences were calculated by Duncan's multiple range tests. * P < 0.05.

Growth Performance

The effects of fermented feed on growth performance are presented in Table 3. Compared to the control group, dietary supplementation with 10 and 15% dried fermented feed did not affect (P > 0.05) BW on 21 d, while the 25% dried fermented feed group had a lower BW (P < 0.05). On 42 d, the chicks fed 15% dried fermented feed had an increased (P < 0.05) BW than the control group, while the 25% dried fermented feed group had the lowest (P < 0.05) BW among all groups. Supplementation with fermented feed had a significant (P < 0.05) effect on FI from 22 to 42 d and from 1 to 42 d in broilers. Chicks fed 10 and 15% dried or 10% wet fermented feed had an increased (P < 0.05) FI than those fed the control, 20 and 25% dried fermented feed. No significant differences (P > 0.05) were observed in FCR during the starter (1–21 d), grower (22–42 d), and whole periods (1–42 d). In addition, there were no significant differences (P > 0.05) in BW, FI and FCR between the 10% dried and 10% wet fermented feed groups.

Table 3.

Effects of fermented feed on growth performance of broiler chickens.

Item1 Groups
 SEM P value
Control 10% DFF 15% DFF 20% DFF 25% DFF 10% WFF
BW (g), 1 d 42.6 42.3 43.5 43.4 42.9 42.4 1.01 0.07
BW (g), 21 d 725.0a 728.1a 730.6a 709.0ab 696.9b 716.9ab 26.96 0.09
BW (g), 42 d 2080.0b 2125.8ab 2162.5a 2083.1b 2016.5c 2135.6ab 83.37 <0.01
FI (g), 1–21 d 909.9 915.0 921.2 910.9 921.2 946.8 35.94 0.34
FI (g), 22–42 d 2235.6b 2346.9a 24025a 2254.3b 2239.9b 2345.5a 98.79 <0.01
FI (g), 1–42 d 3110.5b 3261.9a 3323.7a 3112.1b 3137.4b 3294.9a 143.00 <0.01
FCR (g/g), 1–21 d 1.39 1.35 1.36 1.37 1.41 1.41 0.01 0.257
FCR (g/g), 22–42 d 1.70 1.68 1.68 1.64 1.70 1.66 0.01 0.452
FCR (g/g), 1–42 d 1.57 1.54 1.54 1.50 1.56 1.54 0.02 0.139
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Abbreviations: BW, body weight; DFF, dried fermented feed; FCR, feed conversion ratio; FI, feed intake; SEM, standard error of the mean; WFF, wet fermented feed.

a–c

Means within each row with different superscripts are statistically significantly different (P < 0.05).

1

Means were calculated using 8 replicates (10 birds/replicate) per treatment.

Immune Organ Index

Supplementation of diets with 15, 20, and 25% dried fermented feed significantly increased (P < 0.05) the bursa of Fabricius index, and the 20% dried fermented feed group showed the highest index. However, there were no significant differences (P > 0.05) in thymus and spleen indices among groups (Figure 2).

Figure 2.

Figure 2

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Effects of fermented feed on the immune organ index of broilers. The values were expressed as the means from 8 replicates per treatment. Statistical differences were calculated by Duncan's multiple range tests. *P < 0.05. Abbreviations: DFF, dried fermented feed; WFF, wet fermented feed.

Serum Biochemical Parameters

As shown in Figure 3, supplementing with fermented feed had significant effects (P < 0.05) on serum total protein, albumin, globulin, IgA, IgG, IgM, lysozyme, complement 3, and complement 4 levels in broilers. The levels of total protein and albumin in 15, 20, and 25% dried and 10% wet fermented feed groups were higher (P < 0.05) than that in the control and 10% dried fermented feed group. Similarly, the globulin levels of the 20 and 25% dried and 10% wet fermented feed groups were higher (P < 0.05) than other groups. The 20% dried fermented feed group showed the highest (P < 0.05) levels of total protein and globulin, while 15% dried fermented feed group showed highest (P < 0.05) level of albumin among all groups. Compared to the control, both dried (10, 15, 20, and 25%) and wet (10%) fermented feed groups had higher levels of IgA, IgG and IgM except that the 25% dried fermented group had a comparable (P > 0.05) IgG level with the control. In addition, the 20% dried fermented feed group showed the highest (P < 0.05) levels of IgG and IgM among all groups. The complement 3 level of the dried (10, 15, 20, and 25%) fermented feed groups was increased (P < 0.05) than that of the control and 10% wet fermented feed groups. Compared to the control, dietary supplementing with dried (10, 15, 20, and 25%) or wet (10%) fermented feed increased (P < 0.05) the levels of complement 4 and lysozyme. Among all groups, the 20% dried fermented feed group showed the highest (P < 0.05) level of complement 3 and the both 10 and 15% dried fermented feed groups showed the highest (P < 0.05) level of complement 4.

Figure 3.

Figure 3

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Effects of fermented feed on serum biochemical parameters of broilers. The values were expressed as the means from 8 replicates per treatment. Statistical differences were calculated by Duncan's multiple range tests. Means with different letters are significantly different (P < 0.05). Abbreviations: DFF, dried fermented feed; WFF, wet fermented feed.

Odor Compound Concentrations

Supplementation of diets with fermented feed had a significant (P < 0.05) effect on the concentrations of skatole and indole. Compare to the control, both dried (10, 15, 20, and 25%) and wet (10%) fermented feed groups showed a lower (P < 0.05) concentration of indole. However, only the 15 and 20% dried fermented feed groups showed a lower (P < 0.05) concentration of skatole. In addition, there were no significant differences (P > 0.05) in the concentrations of skatole and indole between the 10% dried and 10% wet fermented feed groups (Figure 4).

Figure 4.

Figure 4

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Effects of fermented feed on cecal skatole and indole levels of broilers. The values were expressed as the means from 8 replicates per treatment. Statistical differences were calculated by Duncan's multiple range tests. Means with different letters are significantly different (P < 0.05). Abbreviations: DFF, dried fermented feed; WFF, wet fermented feed.

As shown in Figure 5, the cecal pH and the concentrations of acetic acid, propionic acid, butyric acid, and lactic acid were affected (P < 0.05) by the dietary fermented feed supplementation. Compared with the control, the cecal pH values were decreased (P < 0.05) in both dried (10, 15, 20, and 25%) and wet (10%) fermented feed groups and the 20% dried fermented feed group had the lowest pH (P < 0.05). The cecal lactic acid concentration of the 20% dried fermented feed group was higher (P < 0.05) than that of the control and 20% dried fermented feed groups. Dietary supplement with both dried (10, 15, 20, and 25%) and wet (10%) fermented feed increased the acetic acid concentration of the cecum. The 15% dried and 10% wet fermented feed groups had a higher (P < 0.05) concentration of propionic acid than the control, 20% and 25% dried fermented feed groups. Supplementation of diets with dried (10, 15, 20, and 25%) fermented feed increased (P < 0.05) the concentration of butyric acid than the control and 10% wet fermented feed groups, and the 15% dried fermented feed groups had the highest butyric acid concentration (P < 0.05).

Figure 5.

Figure 5

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Effects of fermented feed on cecal pH and lactic acid and VFA levels in broilers. The values were expressed as the means from 8 replicates per treatment. Statistical differences were calculated by Duncan's multiple range tests. Means with different letters are significantly different (P < 0.05). Abbreviations: DFF, dried fermented feed; WFF, wet fermented feed.

Cecal Microbiota Characterization

The results from Figure 6A show that within the 6 groups (48 samples), 1,389 OTUs were shared among all groups, and 356, 438, 612, 432, 437, and 595 elements were unique in the control, 10, 15, 20, 25% dried and 10% wet fermented feed groups, respectively. However, there was no significant difference (P > 0.05) in the total number of OTUs among the groups. Moreover, we also found no significant differences (P > 0.05) in the ACE, Chao1, Shannon and Simpson indices among groups (Figure 6B), indicative of little effect of fermented feed on the richness and diversity of the microbiota. For the unweighted UniFrac PCoA principal component PC1, PC2, and PC3 explained 40.4, 12.4, and 9.1% of the between-sample variation, respectively. However, there was no clear separation by fermented feed supplementation from samples collected on D 42, except that plots from the 15% dried fermented feed group were more clustered than other groups (Figure 6C). The circus plot at the family level revealed that 14 top families with abundance >1% were present in all groups (Figure 6D). Compared to other groups, Ruminococcaceae, Lactobacillaceae, and unclassified Clostridiales were abundant and Rikenellaceae, Lachnospiraceae, and Bacteroidaceae were less abundant in the 15% dried fermented feed group (Figure 6E), suggesting that dietary supplementation with the fermented feed altered microbial composition of the cecum in broilers.

Figure 6.

Figure 6

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Effects of fermented feed on cecal microbiota of broilers. (A) Venn diagram depicts OTUs in all groups. (B) Alpha-diversity analysis in all groups. (C) PCoA beta-diversity analysis in all groups. (D) Circos plot visualization of the relationship and abundance of the microbial composition in all groups. (E) Analysis of microbial composition at the family level. The values were expressed as the means from 8 replicates per treatment. Statistical differences were calculated by Duncan's multiple range tests. Abbreviations: DFF, dried fermented feed; WFF, wet fermented feed. Groups 1 to 6 were the control, 10% DFF, 15% DFF, 20% DFF, 25% DFF, and 10% WFF groups, respectively.

DISCUSSION

Fermentation, which has always been used for processing and preserving food for thousands of years worldwide, has recently gained increasing attention from researchers as a method of adjusting the nutritive value of feed and output of livestock and poultry products (Hamza and Gunyar, 2022). The way in which beneficial microorganisms and/or enzymes are added to the diet by controlling incubation parameters to ensure correct management of fermented feed production is now preferred in the feed industry (Chen et al., 2009). Usually, during a fermentation process, a variety of antinutritional factors of animal feed are reduced, and nutritional quality and nutrient bioavailability are improved, along with increased beneficial metabolites, enzymes, and probiotics, such as Lactobacillus and Bacillus. Bacterial strains used to start fermentation could promote the degradation of complex carbohydrates, consisting of cellulose and hemicelluloses, into short chain organic acids, thus leading to a lower pH and creating an anaerobic microecology environment in the gut (Hui et al., 2021; Lv et al., 2022). The results from this study suggested that fermentation could indeed increase the total acid level and decrease the pH of diets, accompanied by enhancement of the number of beneficial bacteria, such as Lactobacillus and Bacillus. This probiotic activity of fermented feed could have the potential not only to improve production performance but also to modulate gastrointestinal ecology and metabolic behavior. Thus, it is reasonable that feeding fermented feed would also be an effective strategy to change gut microflora because of its unique characteristics of acidification of the gastrointestinal tract and establishment of beneficial bacterial growth conditions to enhance immune function and resist odor-producing bacterial growth and/or colonization.

Many previous studies have shown that feeding fermented feed had beneficial effects on growth performance in pigs (Xu et al., 2019), goose (Yan et al., 2019), and hens (Lv et al., 2022). In the current study, the findings indicated that supplementation with 10 and 15% fermented feed increased BW and FI in broilers during the whole feeding period (from 1 to 42 d). Similar results were also reported, in that improved growth performance of broilers fed with fermented feed was observed (Chen et al., 2009; Hamza and Gunyar, 2022). The positive effect of fermented feed on growth performance may be due to an improvement in the nutritive value and digestibility of feed components during fermentation. In addition, we also found that compared to the control group, broilers fed with 25% fermented feed had a compromised BW on 42 d, indicating that a higher inclusion of fermented feed in the diet led to a weak growth performance. Missotten et al. (2013) also found that BW and FI were decreased in broilers fed with fermented feed. One explanation may be that the low pH and high concentration of some metabolites (e.g., acetic acid, biogenic amines) in fermented feed impaired palatability and consequently decreased FI, leading to negative effects on growth performance.

Generally, modern broilers with great performance are highly susceptible to environmental stressors or external stimuli, and the development of immune function is connected with growth performance, morbidity and mortality. The immune organ index can always be used to reflect the levels of the broiler's immune response. In broilers, the thymus, spleen, and bursa of Fabricius are important immune organs. The bursa of Fabricius is an avian-specific immune organ, where B lymphocytes begin to develop and obtain diversity in the antibody repertoire. Generally, bursa of Fabricius was more easily influenced by the exogenous feed prior to other organs in broilers (Wu et al., 2020). Upon immunological suppression, the weight of the bursa of Fabricius is decreased. Ao et al. (2011) found that the relative weights of the bursa of Fabricius and spleen were increased by the dietary inclusion of fermented ingredients for broilers. Similarly, in this study, our results showed that supplementation with fermented feed had no effects on the weights of thymus and spleen, but a middle and high (15, 20, and 25%) inclusion of fermented feed increased the bursa of Fabricius index in broilers. Presumably, fermented feed may contain large quantities of beneficial bacteria (e.g., Lactobacillus and Bacillus) to stimulate the growth of immune organs. Rajput et al. (2013) demonstrated that feeding broilers with Saccharomyces and Bacillus contributed to the development of thymus and bursa of Fabricium. Another possibility was that fermentation media may contain prebiotic like compounds (e.g., oligosaccharides) which had an immune booster function. Sahail et al. (2013) found that supplementation of mannan oligosaccharide can restore the development of bursa of Fabricium in broilers exposed to an exogenous stressor that decreased the weight of viscera. In addition, fermentation can decrease the antinutritional factors of feed ingredients, thus reducing attack on the immune system of the host by these antinutritional factors. Antinutritional factors (e.g., trypsin inhibitor, phytic acid, etc.) have been demonstrated to inhibit nutrient digestion and absorption and have negative effects on immune organ development (Suprayogi et al., 2022; Zhang et al., 2022). Fermentation can increase the nutrition value of feed ingredients and FI, contributing to enhancement of nutrients supply, and then promoting organ development.

Albumin is the most abundant protein in serum and in extracellular fluids, and it has multiple roles, including regulation of colloid osmotic pressure, binding and transportation of various substances within the blood, antioxidant properties, nitric oxide modulation, and buffer capabilities (Vincent et al., 2014). Serum globulin is also a major component of serum total protein and is typically calculated as the difference between total protein and albumin and thus includes all nonalbumin proteins, including globulin, fibrinogen, C-reactive protein, interleukins, leukotrienes, and other regulatory and prothrombotic proteins (Shamji et al., 2012). In this study, our results demonstrated that dietary supplementation with higher levels (20 and 25%) of dried or 10% wet fermented feed can increase serum globulin levels, indicative of enhanced immune function. The reason of increased production of globulin after fermented feed supplementation may be because of the presence of beneficial bacteria, such as Lactobacillus and Bacillus, which predominant components of cell walls are peptidoglycan and lipoteichoic acid and are immunologically active to stimulate the production of cytokines (e.g., interleukins and C-reactive protein) in cells from animals (Zhu et al., 2020). Immunoglobulins, including IgA, IgG, and IgM, are produced from B cells upon activation and differentiation in lymph nodes in response to antigens or allergens and are an important component of host humoral immunity to resist attacks from pathogens and viruses (Shamji et al., 2012). Our findings in this study showed that serum IgA, IgG, and IgM levels were increased in broilers fed fermented feed diets. Similar results have been reported in the study of broilers, in that dietary supplementation with fermented feed increased serum immunoglobulin levels (Feng et al., 2007; Xu et al., 2011). One explanation for this finding may be that fermentation increased amino acid or small peptide production, thus leading to more substrate supply for immunoglobulin synthesis. Li et al. (2020) observed that fermentation increased the levels of small peptides (<600 Da) in the broiler diet. Wang et al. (2003) reported that adding 3 g/kg small peptides into the diet can increase the concentrations of serum immunoglobulin. Another explanation may be that fermented feed supplementation increased the supply of beneficial bacteria, such as Lactobacillus, which had been demonstrated to promote the synthesis of immunoglobulin in broilers (Wu et al., 2019). The complement system is one of the central protagonists of innate and acquired immunity, playing important roles in resisting pathogens, maintaining body homeostasis, and facilitating tissue repair (Santiesteban-Lores et al., 2021). Lysozyme performs highly important protective biological functions in which it destroys gram-positive bacteria by hydrolyzing the β-1,4-glycosidic bonds of peptidoglycans, breaking the murein layer and decreasing the mechanical strength of the bacterial cell wall, finally resulting in the death of the bacteria (Wu et al., 2019). In this study, our results showed that adding fermented feed to the diet can increase the levels of complement and lysozyme, thus improving the immune function of broilers.

In broilers, odorous compounds, including skatole, indole, and VFAs, are mainly produced in the cecum. VFAs, including acetic acid, propionic acid, and butyric acid, can be produced from the fermentation of dietary carbohydrates, while a portion of dietary protein and amino acids can also be fermented into VFAs and lactic acid, and the levels of VFAs and lactic acid can reflect the status of gut health and microbial activity. Intestinal epithelial cells can absorb and utilize VFAs as an energy source to accelerate the proliferation and maturation of enterocytes, thus contributing to gut health. Meanwhile, with an increase in the level of VFAs, gut pH decreases, which further stimulates gastrointestinal motility and promotes digestion and absorption of nutrients. Demeckova et al. (2002) found that adding fermented liquid feed to the diet increased the levels of acetic acid, propionic acid, and butyric acid in sow feces. Similarly, our study demonstrated that feeding fermented feed can increase the levels of VFAs in the cecum of broilers, leading to a lower pH. The explanation for this result may be that fermented feed supplementation can contribute to improving gut health and enhancing microbial activity.

In addition, in this study, we also found that the cecal odorants skatole and indole were decreased by dietary supplementation with fermented feed in broilers. One explanation for this finding may be that adding fermented feed into the diet improved the biological availability of nutrients for growth and decreased the supply of undigested fermentation substrates for odorous compound production. Another possibility may be that part of the large-molecular carbohydrates in the diet were fermented into small-molecular carbohydrates, including oligosaccharides, and enzymes secreted by microbes decreased digesta viscosity and increased digesta flow into the cecum, leading to increased VFA and lactic acid production and lowering pH. This will stimulate the growth of beneficial bacteria, such as Lactobacillus, to resist indole- and sktole-producing or metabolizing bacterial proliferation.

The intestinal microbial community plays an important role in the overall health and digestion in broilers. Ceca, pairs of elongated blind sacs, have the longest feed retention time and most abundant bacterial density and are extremely suitable for fermentation. Numerous bacteria in the cecum are capable of degrading nondigestible carbohydrates into short-chain fatty acids and proteins and peptides into odorous compounds such as NH3, H2S, indole, and its derivatives (e.g., skatole). On the basis of the characterization of microbial communities in the present study, we found that ACE, Chao1, Shannon, and Simpson indices were not significantly different among groups, suggesting that dietary supplementation with fermented feed has little effect on the richness and diversity of cecal microbiota in broilers. Similarly, Yan et al. (2019) also found no significant responses of Chao1, Shannon and Simpson values to increasing dietary fermented feed levels. Notably, compared to the control, dietary supplementation with 15% dried fermented feed increased the abundance of Ruminococcaceae, Lactobacillaceae, and unclassified Clostridiales and decreased that of Rikenellaceae, Lachnospiraceae, and Bacteroidaceae in the cecum. Ruminococcaceae, which contains endo-1,4-beta-xylanase and cellulase genes, can degrade diverse cellulose and hemicellulose components of plant material into short-chain fatty acids, such as acetic acid and butyric acid (Liu et al., 2021). In addition, lactic acid, produced from the fermentation of Lactobacillaceae, could be utilized by butyric acid-producers through a cross-feeding pattern, resulting in beneficial effects on gut health. A previous study found that Lactobacillus supplementation significantly increased the relative abundance of Ruminococcaceae in pigs (Yang et al., 2020). Clostridiales were previously shown to boost cellulose decomposition in collaboration with Ruminococcaceae, indicating that they played important roles in the fermentation of carbohydrates. Lachnospiraceae and Rikenellaceae were demonstrated to have the capacity to ferment fibers and produce butyric acid in the gut (Biddle et al., 2013). Bacteroidaceae represents one of the predominant anaerobic microbiota in the broiler cecum and belongs to propionate producers and has polysaccharide-degrading activity, which are considered beneficial microbes in the gut. However, some species of Bacteroidaceae harbor particular pathogenic potential in facilitating epithelial penetration of intestinal bacteria and producing enterotoxin to the lateral surface of enterocytes, thus leading to gut health damage (Liu et al., 2021). In addition, previous studies have demonstrated that Bacteroides from Bacteroidaceae played an important role in the process of deamination and decarboxylation of tryptophan to produce malodorous compounds (e.g., skatole) and the decreased cecal abundance of this bacteria was correlated with lower odorous compound production in broilers (Zhu et al., 2020). In this study, we found that the cecal levels of skatole and indole were lower in broilers fed fermented feed, presumably correlating with the decreased abundance of Bacteroidaceae, but more elaborate studies should be conducted to better understand their dynamic roles in odorous compound production.

In conclusion, the results from the present study indicate that adding fermented feed to broiler diets increases the BW, FI, bursa of Fabricius index, and elevates serum total protein, albumin, globulin, IgA, IgG, IgM, lysozyme, complement 3, and complement 4 levels. The supply of fermented feed also enhances the concentrations of acetic acid, propionic acid, butyric acid and lactic acid and decreases the pH of the cecum. Dietary supplementation with fermented feed can improve cecal fermentation, with Ruminococcaceae, Lactobacillaceae, and unclassified Clostridiales being more abundant and Rikenellaceae, Lachnospiraceae, and Bacteroidaceae being less abundant in fermented feed groups, thus helping minimize odorous compound skatole and indole production from the broiler cecum. On the basis of these observations, we can conclude that supplementing broiler diets with fermented feed can improve growth performance, immune function, and lower odorous compound production by modifying the microbiota in broilers.

ACKNOWLEDGMENTS

This study was supported by the National Natural Science Foundation of China (31772618).

DISCLOSURES

Guiqin Yang reports financial support was provided by the National Natural Science Foundation of China.

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