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. 2023 Oct 18;103(1):103210. doi: 10.1016/j.psj.2023.103210

Effects of Bacillus licheniformis on growth performance, immune and antioxidant functions, and intestinal microbiota of broilers

Songke Qin 1,1, Xiao Xiao 1,1, Zhenglie Dai 1,1, Guiling Zhao 1, Zhenchuan Cui 1, Yanping Wu 1, Caimei Yang 1,2
PMCID: PMC10684393  PMID: 37980737

Abstract

Bacillus licheniformis (BL) has been widely regarded as an important growth promoter in recent years. However, its usage in animal industry still needs more foundations. The aim of our study was to study the effects of BL on the growth performance, immunity, oxidative function and intestinal flora of broilers. A total of 760 one-day-old yellow-feathered broilers were randomly divided into 4 groups with 10 replicates per group and 19 broilers per replicate. The broilers in the control group (CON) were fed with basal diet. The treatment groups were supplemented with 250 mg/kg (BL250), 500 mg/kg (BL500) and 750 mg/kg (BL750) BL in the basal diet for 70 d. Results showed that BL groups significantly increased the body weight (BW) and average daily gain (ADG), decreased average daily feed intake (ADFI) and feed conversion ratio (FCR). In addition, the spleen and bursa indexes were higher in the BL groups than that in the CON group at d 70. BL supplementation also markedly increased the levels of immunoglobulins Y (IgY), IgA and anti-inflammatory interleukin 10 (IL-10), reduced the levels of proinflammatory IL-1β, tumor necrosis factor α (TNF-α) and IL-2 in the serum at 70 d in a concentration-dependent manner. Besides, BL addition significantly increased the levels of series antioxidant enzymes including total antioxidant capacity (T-AOC), glutathione peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT), and decreased the level of malondialdehyde (MDA) in the serum. Moreover, BL groups showed an obvious increase of isobutyric acid markedly and BL500 group significantly promoted the level of isovaleric acid in cecal contents of broilers. Finally, microbial analysis showed that BL supplementation presented visual separations of microbial composition and increased the relative abundance of p_Proteobacteria, g_Elusimicrobium, and g_Parasutterella comparing with the CON group. Together, this study inferred that dietary BL supplementation improved the growth performance, immune and antioxidant functions, changed the intestinal microflora structure and metabolites of yellow-feathered broilers, which laid a good basis for the application of probiotics in the future.

Key words: Bacillus licheniformis, yellow-feathered broiler, growth performance, immune function, intestinal microbiota

INTRODUCTION

In the past few decades, antibiotics have been widely used for their dual roles in promoting growth and treating various diseases. However, the prolonged abuse of antibiotics led to the rapid development of pathogen resistance and finally led to environmental pollution, which further did harm to human and animals (Tang et al., 2017). Thus, the prohibition of antibiotics has been implemented (Organization, 1999), and there was a dire need to find suitable growth promoters in animal production. In recent years, many alternative products, such as probiotics (Xu et al., 2021), antimicrobial peptides (Yi et al., 2017), plant extract (Abullais Saquib et al., 2021), acidifiers (Pearlin et al., 2020), plant essential oils (Montassier et al., 2021; Ayalew et al., 2022), have been highly favored due to their regulatory functions in growth performance, immune and antioxidant status, and microbial homeostasis in animal models (Roselli et al., 2005; Rossi et al., 2020; Luo et al., 2023). Among these substances, probiotics have been paid more and more attention for their safety and high efficacy in the animal field (Ningsih et al., 2023).

Bacillus species, 1 type of beneficial bacteria in the animal intestine, has been widely used for promoting growth performance and improving intestinal health under normal and pathogens challenge (Kan et al., 2021; Xu et al., 2021; Zhang et al., 2021; Liu et al., 2023). Moreover, in the last decades, advanced-progresses of Bacillus species have been achieved in broiler chickens (Manafi et al., 2018; Chang and Yu, 2022; Lena et al., 2022). Among which, Bacillus licheniformis (BL), characterized by high temperature and stress resistance, is one the most popular probiotics in animal industry. Previous studies have found that BL could produce various active substances, such as digestive enzymes, bacteriocin, and antibacterial peptides, to promote animal performance and stimulate the development of immune system (Giri et al., 2019; Devyatkin et al., 2021; Yang et al., 2021; Additives et al., 2023). For example, one previous study have shown that the combination of BL and Clostridium butyricum can improve the growth performance and reduce the incidence of diarrhea in weaned piglets (Zong et al., 2019). In addition, studies also reported that dietary supplementation with BL increased apparent digestibility of dry matter, organic matter, nitrogen and neutral detergent fiber, and further promoted growth performance (Deng et al., 2018). Moreover, dietary supplementation of BL increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX), decreased the concentration of ammonia nitrogen (NH3-N), and increased the content of microbial crude protein (MCP) in fattening lambs (Jia et al., 2018). More importantly, dietary BL or BL-fermented products supplementation changed the microbiota community and reshaped the microbial homeostasis, and further improved intestinal health in broilers (Chen and Yu, 2020; Han et al., 2023). However, probiotic strains differ regarding their unique properties, these differences even occurred when the strains belong to the same species. Therefore, whether BL HJ0135, isolated by our lab, can act as a growth promoter and regulator of gut health in broilers was unclear.

In this study, 3 dosages of BL HJ0135 were added into basal diet of yellow-feathered broilers, and further to investigate the effect of BL on growth performance, immune functions, antioxidant capacity, short-chain fatty acid production, and microbial structure. This research provided theoretical basis for probiotics applications in animal breeding.

MATERIALS AND METHODS

Ethic Approval

All experiments were performed in accordance with the Guidelines for the Use of Laboratory Animal Care in Zhejiang A & F University and approved by the Animal Ethics Committee of Zhejiang A & F University.

Bacteria Preparation

Bacillus licheniformis HJ0135 (BL) was originated from our lab. Frozen strain was cultured overnight in solid Luria-Bertani (LB) medium, and the single colony was picked into liquid LB broth at 37°C. Then the bacteria were collected by centrifugation and the concentration was calculated for latter experiments.

Experimental Design

A total of 760 one-day-old yellow-feathered broilers with an average body weight of 33.0 ± 0.30 g were selected. Broilers were randomly divided into 4 treatment groups, with 10 replicates per group and 19 chickens per replicate. The broilers in the control group (CON) were fed with the basal diet. The broilers in the treatment groups (BL: BL250, BL500, BL750) were fed with the basal diet supplemented with 250, 500 and 750 mg/kg BL (1 × 1010 cfu/g), respectively. The whole experiment lasted for 70 d. The basal diet was formulated to meet the nutritional requirements of broilers according to the Chicken Feeding Standard (NY/T33-2004), the composition and nutritional compositions of the basal diet were listed in Table 1.

Table 1.

Basic experimental diet composition and nutritional level (air-dried basis).

Ingredient, % Starter (1–35 d) Finisher (35–70 d)
Ingredients
 Corn 53.20 61.20
 Corn protein meal 2.00 3.50
 Soybean meal 25.10 14.10
 Extruded soybean 5.00 5.00
 DDGS 5.00 8.00
 Fermented soybean meal 2.50 0.00
 Wheat middlings 0.00 2.00
 Soybean oil 2.30 2.00
 Limestone 1.20 1.30
 CaHPO4 1.70 0.90
 Premix1 2.00 2.00
 Total 100.00 100.00
Nutrient levels
 ME2 (kcal/kg) 2972.62 3022.00
 CP 21.03 17.53
 Lys 1.26 0.95
 Met 0.55 0.44
 Met + Cys 0.90 0.76
 Thr 0.82 0.65
 Trp 0.22 0.16
 Ca 0.94 0.76
 Available P 0.73 0.29
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1

Supplied per kilogram of diet: vitamin A (retinyl acetate), 1,500 IU; cholecalciferol, 200 IU; vitamin E (DL-α-tocopheryl acetate), 10 IU; riboflavin, 3.5 mg; pantothenic acid, 10 mg; niacin, 30 mg; cobalamin, 10 µg; choline chloride, 1,000 mg; biotin, 0.15 mg; folic acid, 0.5 mg; thiamine, 1.5 mg; pyridoxine, 3.0 mg; Fe, 80 mg; Zn, 40 mg; Mn, 60 mg; I, 0.18 mg; Cu, 8 mg; Se, 0.15 mg; Lys, 3,000 mg; Met, 2,000 mg; Thr, 1,000 mg.

2

ME, apparent metabolizable energy.

Growth Performance Test

The body weight (BW) and feed consumption of broilers were recorded at 1, 35, and 70 d of age. The average daily feed intake (ADFI), average daily gain (ADG) and feed conversion rate (FCR) of broilers were calculated based on the recorded data.

Sample Collection

At the end of the experiment, 1 broiler from each replicate was weighed, and blood was taken from the carotid artery. Serum samples were collected by centrifugation at 6,000 × g for 6 min and stored at −80°C. The spleen, bursa, left thymus were weighed. The contents of cecum were collected for microbial analysis and short-chain fatty acids (SCFAs) measurement.

Immune Organ Index

The body weight and immune organ (spleen, bursa, and left thymus) of broilers were weighed, and the data were recorded and analyzed with Microsoft Excel 97-2003 software. Immune organ index was calculated as immune organ weight (g) /broiler body weight (kg).

Serum Biochemical Indicators

Serum biochemical parameters, such as levels of immunoglobulin A (IgA), IgM, IgY, interleukin-6 (IL-6), IL-18, IL-1β, IL-10, IL-2, tumor necrosis factor-α (TNF-α), total antioxidant capacity (T-AOC), GPX, SOD, catalase (CAT), and malondialdehyde (MDA) were measured according to the manufacturer's instructions (Angle Gene Biotechnology Co., Ltd., Nanjing, China).

SCFAs Measurement and Analysis

The quantification of cecal SCFAs was measured by gas chromatography referring to the method of Yang (Yang et al., 2020). Network gas chromatography (7890 B) and automatic liquid sampler (7693), equipped with 30 m × 0.25 mm × 0.25 mm DB-FFAP chromatographic column (Cat#122-3232, Agilent Technologies) and flame ionization detector G4513 A sampler (Agilent Technologies, Santa Clara, CA) were used in the process. Briefly, 0.5 g of cecal contents was dissolved in 1.5 mL of purified water. The supernatant was mixed with 25% phosphoric acid (m/v, 1:3) after high-speed centrifugation (12,000 × g, 4°C, 10 min). The mixture was placed overnight at 4°C and filtered into a special injection bottle for machine detection.

Gut Microbiota Analysis

The total genomic DNA of each sample microbial community was extracted according to the instructions of E.Z.N.A. soil DNA kit (Omega Bio-tek, Norcross, GA), and the quality of the extracted genomic DNA was detected by 1% agarose gel electrophoresis (Thermo Fisher Scientific Inc., Shanghai, China) to determine the DNA concentration and purity.

Using the DNA extracted above as a template, use the upstream primer 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and the downstream primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′) carrying the Barcode sequence to 16S rRNA gene V3 to V4 variable PCR amplification of the region. After mixing the PCR products of the same sample, the PCR products were recovered by 2% agarose gel, and purified using AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA). The recovered products were finally quantified with a Quantus Fluorometer (Promega Biotechnology Co., Ltd., Beijing, China). Use the NEXTFLEX Rapid DNA-Seq Kit to build a library of purified PCR products. Sequencing was performed on the Miseq PE300 platform of Illumina (Shanghai Meiji Biomedical Technology Co., Ltd., Shanghai, China). Fastp software was used for quality control of paired-end raw sequencing sequences, FLASH software was used for splicing.

Based on the default parameters, use the DADA2 plug-in in the Qiime2 process to denoise the optimized sequence after quality control splicing. Species taxonomic analysis of amplicon sequence variants (ASVs) was performed using the Naive Bayes classifier in Qiime2 based on the Silva database. The alpha diversity, indicated by Shannon and Chao1 indexes, and beta diversity, accessed by principal coordinate analysis (PCoA), were analyzed based on ASV table. Moreover, the relative abundance of species at different taxonomies was also obtained.

Statistical Analysis

Data were presented as the mean ± SEM and GraphPad Prism Software 8.0 (GraphPad Software Inc, Chicago, Illinois) was used for statistical analysis. One-way ANOVA was used to determine the significant difference among groups. A value of P > 0.05 was considered no significant difference, P < 0.05 was considered statistically significant.

RESULTS

Effects of BL on Growth Performance of Yellow-Feathered Broilers

As shown in Table 2, compared with the CON group, BL supplementation significantly increased the BW at d 70, ADG from d 35 to d 70 and d 1 to d 70 (P < 0.05). Moreover, ADFI and FCR from d 1 to d 35 and d 1 to d 70 were markedly decreased by BL addition (P < 0.05). No obvious changes of BW at d 1 and d 35, ADG from d 1 to d 35, ADFI and F/G from d 35 to d 70 was observed in our study (P > 0.05).

Table 2.

Effects of BL on growth performance of yellow-feathered broilers.

Items Treatments
 P value
CON BL250 BL500 BL750
BW, g/bird
 1 d 33.00 ± 0.30 32.20 ± 1.50 32.40 ± 0.40 33.60 ± 0.30 0.586
 35 d 555.30 ± 5.70 552.50 ± 5.40 566.40 ± 4.50 563.80 ± 3.50 0.154
 70 d 1515.50 ± 43.40b 1622.90 ± 12.20a 1616.00 ± 15.30a 1600.00 ± 14.70a 0.004
ADG, g/bird
 1–35 d 14.90 ± 0.20 14.80 ± 0.10 15.20 ± 0.10 15.00 ± 0.07 0.165
 35–70 d 27.60 ± 1.20b 30.70 ± 0.30a 30.30 ± 0.50a 31.00 ± 0.30a 0.007
 1–70 d 21.20 ± 0.60b 22.70 ± 0.20ab 22.60 ± 0.20ab 23.00 ± 0.20a 0.027
ADFI, g/bird
 1–35 d 30.60 ± 0.30a 24.90 ± 0.30c 25.90 ± 0.40b 26.10 ± 0.40b 0.010
 35–70 d 89.00 ± 3.60 96.00 ± 2.90 94.00 ± 2.60 94.00 ± 3.40 0.398
 1–70 d 91.90 ± 0.30a 88.80 ± 0.20b 85.20 ± 0.10b 77.50 ± 0.30b 0.076
FCR (g:g)
 1–35 d 2.10 ± 0.02a 1.70 ± 0.02b 1.70 ± 0.02b 1.70 ± 0.02b 0.001
 35–70 d 3.20 ± 0.05 3.10 ± 0.09 3.10 ± 0.06 3.00 ± 0.10 0.310
 1–70 d 2.80 ± 0.03a 2.60 ± 0.06b 2.50 ± 0.04b 2.50 ± 0.07b 0.022
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Data were presented as mean ± SEM, n = 8 in each group.

a,b

Different letter values in the same line indicate significant differences among groups (P < 0.05).

Abbreviations: ADFI, average daily feed intake; ADG, average daily gain; BL, Bacillus licheniformis; BL250, basal diet + 250 mg/kg BL; BL500, basal diet + 500 mg/kg BL; BL750, basal diet + 750 mg/kg BL; BW, body weight; CON, basal diet; FCR, feed conversion ratio.

Effect of BL on Immune Organ Index of Yellow-Feathered Broilers

As presented in Figure 1, compared with the CON group, BL treatment showed a tendency to increase the index of immune organs (thymus, spleen, and bursa) of broilers at d 35 (P > 0.05), while only BL250 group significantly increased the thymus index (P < 0.05). At 70 d, BL250 group significantly increased the spleen index of broilers (P < 0.05), BL750 group significantly increased the bursa index of broilers (P < 0.05), whereas there was no significant difference of thymus index between BL groups and the CON group (P > 0.05). In addition, the spleen index of BL250 group was significantly higher than that of BL500 group (P < 0.05), whereas there was no significant difference of thymus index and bursa index among BL250, BL500 and BL750 groups at d 70 (P > 0.05).

Figure 1.

Figure 1

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Effect of BL on immune organ indexes of yellow-feathered broilers. (A) Immune organ indexes at d 35 (n = 8); (B) Immune organ indexes at d 70 (n = 8). Different letter values indicate significant differences (P < 0.05). Abbreviations: BL, Bacillus licheniformis; BL250, basal diet + 250 mg/kg BL; BL500, basal diet + 500 mg/kg BL; BL750, basal diet + 750 mg/kg BL; CON, basal diet.

Effects of BL on Serum Immunoglobulin and Inflammatory Factors of Yellow-Feathered Broilers at 70 D

The levels of immunoglobulin in the serum were showed in Figure 2A. Compared with the CON group, BL supplementation all pronouncedly increased the level of IgA in 3 dosages (P < 0.05), while BL500 and BL750 groups significantly increased IgY levels (P < 0.05). Surprisingly, there was no significant difference of IgM between BL groups and the CON group (P > 0.05). In addition, there was no significant difference of immunoglobulins among 3 BL groups (P > 0.05). As for the levels of cytokines in the serum, results were shown in Figure 2B. Compared to the CON group, BL supplementation obviously reduced the levels of proinflammatory cytokines, including TNF-α, IL-2 and IL-1β (P < 0.05), and increased the level of anti-inflammatory IL-10 in a concentration-dependent manner (P < 0.05). Interestingly, only BL750 group exhibited reduced the level of IL-6 comparing to the CON group (P < 0.05), whereas no significant change of IL-18 was observed among 4 groups (P > 0.05). More importantly, supplemented with 750 mg/kg BL showed the best regulatory effect of cytokines in some extent among the 3 dosages.

Figure 2.

Figure 2

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Effects of BL on biochemical indexes in the serum of yellow-feathered broilers at 70 d. (A) Immunoglobulins (n = 8); (B) Cytokines (n = 8). Different letter values indicate significant differences (P < 0.05). Abbreviations: BL, Bacillus licheniformis; BL250, basal diet + 250 mg/kg BL; BL500, basal diet + 500 mg/kg BL; BL750, basal diet + 750 mg/kg BL; CON, basal diet; IgY/A/M, immunoglobulin Y/A/M; IL-2/6/1β/18/10, interleukin-2/6/1β/18/10; TNF-α, tumor necrosis factor-α.

Effects of BL on Antioxidant Functions of Yellow-Feathered Broilers at 70 D

The results were showed in Figure 3. Compared with the CON group, BL treatment significantly reduced the level of MDA, markedly improved the levels of T-AOC, GPX, and increased the activities of SOD and CAT in the serum (P < 0.05). In addition, the antioxidant indicators of BL750 group showed the largest change than that of BL250 and BL500 groups, suggesting that supplemented with 750 mg/kg BL showed the best regulatory effect of antioxidant capacity of yellow-feathered broilers among the 3 dosages.

Figure 3.

Figure 3

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Effects of BL on antioxidant functions of yellow-feathered broilers at 70 d (n = 8). Different letter values indicate significant differences (P < 0.05). Abbreviations: BL, Bacillus licheniformis; BL250, basal diet + 250 mg/kg BL; BL500, basal diet + 500 mg/kg BL; BL750, basal diet + 750 mg/kg BL; CAT, catalase; CON, basal diet; GPX, glutathione peroxidase; MDA, malondialdehyde; SOD, superoxide dismutase; T-AOC, total antioxidant capacity.

Effects of BL on SCFAs in Cecal Contents of Yellow-Feathered Broilers at 70 D

As shown in Figure 4, compared with CON group, BL groups all significantly increased the level of isobutyric acid in cecal contents (P < 0.05), while only BL500 group markedly increased the concentration of isovaleric acid (P < 0.05). In addition, BL treatments showed a tendency to increase the concentrations of acetic acid, propionic acid, valeric acid, and butyric acid, but there was no significant difference (P > 0.05). Moreover, there was no significant difference of SCFAs among 3 BL treatment groups (P > 0.05).

Figure 4.

Figure 4

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Effects of BL on SCFAs contents in cecal contents of yellow-feathered broilers at 70 d (n = 8). Different letter values indicate significant differences (P < 0.05). Abbreviations: BL, Bacillus licheniformis; BL250, basal diet + 250 mg/kg BL; BL500, basal diet + 500 mg/kg BL; BL750, basal diet + 750 mg/kg BL; CON, basal diet; SCFAs, short-chain fatty acids.

Effects of BL on Microflora in Cecal Contents of Yellow-Feathered Broilers at 70 D

Microbial analysis was based on ASVs table. Venn diagram was showed in Figure 5A, 418 ASVs were shared among the 4 groups, and 174, 187, 180 and 180 unique ASVs were present in the CON, BL250, BL500, and BL750 groups, respectively. Alpha diversity, indicated by Shannon and Chao1 indexes, was unchanged among 4 groups (Figure 5B). The PCoA plot was used for evaluating beta diversity and showed in Figure 5C. Results indicated that BL treatment partially changed the microbial composition, but with no significance (P > 0.05). To further investigate the effect of BL on microbial composition at different taxonomies, the dominant bacteria at phylum and genus levels were showed in Figure 5D and F. The top phylum was Firmicutes, followed by Bacteroidota and Verrucomicrobiota among 4 groups (Figure 5D), while only the relative proportions of Elusimicrobiota and Proteobacteria were significantly upregulated by BL supplementation comparing to the CON group (Figure 5E). Besides, Bacteroides, Prevotellaceae_UCG-001, unclassified_Lachnospiraceae, Rikenellaceae_RC-9_gut_group, and Faecalibacterium were the dominant bacteria of the TOP10 genera (Figure 5F). Analysis of differential species based on genus level was showed in Figure 5G, results indicated that the relative proportions of Elusimicrobium and Parasutterella were significantly upregulated by BL supplementation. More importantly, BL supplementation with 750 mg/kg showed the most obvious effect on microbial change.

Figure 5.

Figure 5

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Effects of BL on microflora in cecal contents of yellow-feathered broilers at 70 d. (A) Venn diagram (n = 8); (B) Alpha diversity as accessed by Shannon and Chao1 indexes (n = 8); (C) Beta diversity as accessed by Principal coordinate analysis (PCoA) (n = 8); (D) The relative abundance of Top 5 bacteria at phylum level (n = 8); (E) The relative abundance of differential bacteria at phylum level (n = 8); (F) The relative abundance of Top 5 bacteria at genus level (n = 8); (G) The relative abundance of differential bacteria at genus level (n = 8). Different letter values indicate significant differences (P < 0.05). Abbreviations: BL, Bacillus licheniformis; BL250, basal diet + 250 mg/kg BL; BL500, basal diet + 500 mg/kg BL; BL750, basal diet + 750 mg/kg BL; CON, basal diet.

DISCUSSION

Antibiotics, as growth promoters and agents for treating diseases, have been widely used for decades. However, a large amount of evidence have indicated that the long-term overuse of antibiotics as feed additives in livestock and poultry industry led to the rapid development of resistant bacteria and environmental pollution (Kuppusamy et al., 2018; Haque et al., 2020; Tian et al., 2021). Thus, searching for safety and high efficacy of alternatives seems to be so urgent. In this study, we demonstrated that 3 dosages of probiotic BL (250 mg/kg, 500 mg/kg, and 750 mg/kg) supplementation significantly improved growth performance, enhanced immune and antioxidant functions, increased the production of SCFAs and changed microbial structure.

A previous study demonstrated that BL (NO. 1.265) in drinking water (5.6 × 109 CFU/mL, 1.1 × 1010 CFU/mL) significantly increased growth performance as indicated by greater BW and ADG, and lower FCR of broilers (Liu et al., 2012). In another study, 3 g/kg of BL-fermented products supplementation also improved the BW and ADG of broilers (Chen and Yu, 2020). Furthermore, our previous study also showed that supplemented with 1.5 × 109 CFU/kg BL significantly improved the final BW, ADG and ADFI during the whole experiment, reduced the FCR of broilers from the 1- to 42-day period (Xu et al., 2021). In this study, we also observed that dietary BL supplementation significantly increased the final BW at d 70 and ADG during the whole experiment, reduced ADFI and FCR, which indicated that BL addition could take effect as a growth promoter in broilers.

Immune organ index reflects the immune function of the body to some extent. Previous study demonstrated that compared with the control group, the experimental probiotics (Lactobacillus fermentum, Bacillus subtilis, BL, Enterococcus faecalis) increased the thymus index, spleen index and bursal index of broilers (Zhao et al., 2022). Another study also showed that broilers fed with high doses of a mixture of BL AH-G202 and Bacillus subtilis AH-G201 (5 × 109 CFU/mL, for single strain) exhibited the greatest gain in spleen weight gain (Wang et al., 2022). In our study, low centration of BL (250 mg/kg) addition significantly increased the thymus index at d 35 and spleen index at d 70, while high concentration of BL (750 mg/kg) supplementation markedly promoted the bursa index. These results inferred that the same probiotic may show different effect on immune organs (thymus, spleen, bursa) at different dosage and stage.

Immunoglobulins are a group of globulins with antibody activity, which composed of the immune barriers to resist various viruses and bacteria (Criste et al., 2020). Robust release of cytokines including IL-1β, IL-18, TNF-α, etc., are usually accompanied by inflammatory response (Zhang et al., 2022; Zhou et al., 2023). Previous studies have shown dietary supplementation of Bacillus subtilis and BL can increase the levels of IgA and IgM levels in serum of broilers(He et al., 2019; Xu et al., 2021). Moreover, dietary Bacillus subtilis and BL (4 × 109 cfu/g) supplementation markedly increased the concentration of IgY in broiler chickens at 6 wk of age, while no obvious change of IgA and IgM was observed (Ebeid et al., 2021). Our study showed that supplemented with BL (250 mg/kg, 500 mg/kg, and 750 mg/kg) significantly improved the levels of IgA and IgY, with no impact on IgM. Moreover, compared to the control group, the mRNA expressions of IL-10 and IL-4 were elevated in all BL treatments (Wang et al., 2017). Previous research have found that children with intestinal inflammation after tumor radiotherapy which accepted the BL preparation treatment significantly reduced the levels of TNF-α, IL-1β, IL-6 in serum (Du et al., 2018). Another study also showed that upon inflammation induced by LPS, the combination of BL and Bifidobacterium downregulated the proinflammatory cytokines IL-1α, IL-6 induced by LPS stimulation (HS and Halami, 2021). In addition, supplemented with BL in weaned piglets was associated with a significant decrease in TNF-α and IL-1β levels in the ileal mucosa (Cao et al., 2019). In contrast, the gene expression of IL-8 and TNF-α were not changed by the BL HGA8B supplementation (Midhun et al., 2019). Our study showed that dietary BL could significantly reduce the levels of proinflammatory cytokines IL-1β, TNF-α, and IL-2, and increase the anti-inflammatory IL-10 in a concentration-dependent manner in serum of broilers. These indicators together inferred that BL supplementation can improve immune functions. However, the impact of BL in our study on different indexes was not completely identical with others, which may result from different environments and unique strains.

Oxidative stress refers to a state of imbalance between antioxidants and free radicals, which is dynamically regulated by the antioxidant enzymes, including GPX, SOD, and CAT (Ávila-Escalante et al., 2020; Sadasivam et al., 2022). Lipid peroxides (such as MDA) can damage cells and induce apoptosis by producing free radicals, and its cytotoxicity can also affect the activity of various enzymes and ATP synthesis (Anjum et al., 2022; Qi et al., 2022; He et al., 2023). Our study showed that dietary supplementation with BL significantly increased the levels of T-AOC and GPX, and promoted the activities of CAT and SOD in the serum of broilers. Moreover, BL supplementation in the basal diet also significantly reduced the level of MDA in serum. Previous studies have shown that dietary supplementation of BL H2 increased the levels of antioxidant enzymes, such as SOD, CAT, and T-AOC in the serum of broilers (Zhao et al., 2020). Another studies have shown that CAT expression is significantly higher in shrimp fed with added feed in the first 4 wk (Chen et al., 2020). In addition, other studies also showed that BL addition to the diet can significantly increase the levels of GPX, SOD, and CAT, and decrease the content of MDA in serum of broilers (Xu et al., 2021). Thus, our probiotic BL showed the similar effect on antioxidant capacity of broilers as others.

Fecal SCFAs represent dietary and bacterial fermentation in the intestinal ecosystem. As most probiotics have saccharolytic properties (Cardona et al., 2003), we also measured the effect of BL supplementation on SCFAs. Our results showed that dietary BL addition significantly increased the level of isobutyric acid in cecal contents of broilers, and the addition of 500 mg/kg BL to the diet could significantly increase the level of propionic acid and isovaleric acid in cecal contents of broilers. Previous studies have shown that dietary BL B26 supplementation can increase the concentrations of acetic acid, propionic acid, butyric acid and total SCFA in cecum (Musa et al., 2019). In addition, our previous study also showed that dietary BL and Bacillus subtilis could increase the concentration of butyric acid in cecum contents of broilers (Xu et al., 2021). However, another studies referred that dietary supplementation with BL can reduce the content of total SCFA in the intestinal contents of piglets (Collinder et al., 2003). SCFAs, as important microbial metabolites, whose levels might be regulated by the change of microbiota composition to a large extent.

Numerous studies have inferred that intestinal flora play an essential role in nutrition digestion and metabolism, maintaining intestinal barrier function and the development of the immune system (Fu et al., 2021; Sun et al., 2021). In our study, alpha and beta diversity of microflora in cecal contents were analyzed. Results showed that BL supplementation showed no impact on Shannon and Chao1 indexes, indicating no obvious change of alpha diversity. Moreover, the addition of BL showed a tendency to change microbial composition as accessed by PCoA plot. Similarly, another study also showed that no significant difference in ACE, Chao1, Simpson, and Shannon index was observed between BL and CON group (Kan et al., 2021). For further investigation, our results showed inferred that dietary BL supplementation significantly improved the relative abundance of Elusimicrobium and Parasutterella at genus level. One previous study has shown that Parasutterella may be associated with intestinal stress syndrome in mice (Chen et al., 2018), while another study indicated that Parasutterella played a potentially beneficial role in the mucosal secretion (Ju et al., 2019). Elusimicrobium is usually regarded as probiotic bacteria, which was reported to improve the dysbiosis of intestinal flora and restore intestinal barrier function in rats (Shi et al., 2017). Previous studies have shown that the relative abundance of probiotic Elusimicrobium and Parasutterella were pronouncedly increased in rats treated with Chinese herbs complex, which further improved intestinal health (Jin et al., 2022). Although dietary supplementation of BL can regulate microflora balance in cecal contents of Peking ducks and broilers (Kan et al., 2021; Li et al., 2022), the effect of BL on the increased Elusimicrobium and Parasutterella of broilers was not reported and the potential mechanism needs more work. In addition, the concrete function of Elusimicrobium and Parasutterella should be paid more attention in the future.

CONCLUSIONS

On the basis of these results, we can conclude that dietary supplementation with BL at 3 dosages significantly improved the growth performance of broilers by reducing ADFI and FCR, increased the immune organ indexes (especially spleen and bursa indexes), promoted the levels of immunoglobulins (IgY and IgA) and anti-inflammatory IL-10, and reduced the levels of proinflammatory cytokines including TNF-α, IL-2, and IL-1β in the serum of broilers. Moreover, BL supplementation significantly increased antioxidant functions, as indicated by decreased MDA and increased T-AOC, GPX, SOD, and CAT in the serum of broilers. Finally, only the levels of isobutyric acid and iscaleric acid in the cecal contents were increased in BL groups comparing to the CON group, and the microbial structure was changed by BL treatment. In detail, dietary BL supplementation significantly increased the relative abundance of p_Proteobacteria, g_Elusimicrobium, and g_Parasutterella. Considering all the indicators, BL supplementation with 750 mg/kg showed the best beneficial effect as a growth promoter and regulator of health in broilers.

Acknowledgments

ACKNOWLEDGMENTS

The authors extend their appreciation to the R & D plan of “Jianbing” and “Lingyan” in Zhejiang Province, Key R & D Program of Zhejiang Province, National Natural Science Foundation of China and Postdoctoral Science Foundation China for funding this research work.

Funding: The study was supported by the R & D plan of “Jianbing” and “Lingyan” in Zhejiang Province (2022C02043), Key R & D Program of Zhejiang Province (2021C02008), National Natural Science Foundation of China (32202687), and Postdoctoral Science Foundation of China (2023M733150).

DISCLOSURES

No potential conflict of interest was reported by the authors.

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