# Effects of DietaryBacillus velezensis LSG2-5 on Growth, Immunity, Antioxidant Capacity, and Disease Resistance of Amur minnow (Rhynchocypris lagowskii Dybowski)

Authors: Yurou Zhang; Mengnan Yu; Lili Lin; Jiajing Wang; Dongming Zhang; Qiuju Wang; Mahmoud M. Elsadek; Guiqin Wang; Qi Yao; Yuke Chen; Zhixin Guo
Journal: Aquaculture Nutrition
 (2022)

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## Abstract

The study was aimed at investigating the effects ofBacillus velezensis LSG2-5 isolated from host’s intestinal tract on growth performance, immune response, antioxidant capacity, and disease resistance of Amur minnow (Rhynchocypris lagowskii Dybowski). 535 fish were divided into 5 groups with 3 replicates per group. Diets containing different doses (0, 106, 107, 108, and 109 CFU/g, respectively) of B. velezensis LSG2-5 were used to feed fish (9.95±0.05g) for 56 days. Results showed that compared with control, weight gain rate and specific growth rate of fish were significantly increased by dietary 107-109 CFU/g B. velezensis (p<.05). While for the feed conversion ratio, all treatment groups were significantly decreased (p<.05). In serum, the contents of lysozyme and immunoglobulin M were significantly increased by dietary 106-109 CFU/g B. velezensis (p<.05); the activities of component 3, component 4, and alkaline phosphatase were significantly increased by dietary 108 CFU/g B. velezensis (p<.05). The activities of catalase, glutathione peroxidase, total antioxidant capacity, and superoxide dismutase in hepatopancreas were significantly increased by dietary 108-109 CFU/g B. velezensis (p<.05). The activities of catalase, glutathione, glutathione peroxidase, total antioxidant capacity, and superoxide dismutase in the intestinal tract of 108 CFU/g group were significantly increased (p<.05). However, malondialdehyde levels in the hepatopancreas and intestines were significantly decreased by dietary 107-108 CFU/g B. velezensis (p<.05). Dietary 107-109 CFU/g B. velezensis significantly upregulated catalase, glutathione peroxidase, and copper/zinc superoxide dismutase mRNA levels (p<.05). The levels of transforming growth factor β and interleukin-10 mRNA in the hepatopancreas of fish fed with B. velezensis of 108 CFU/g were significantly increased (p<.05), and these in the intestinal tract of 106-109 CFU/g groups were upregulated (p<.05). Feeding dietary 107-108 CFU/g B. velezensis significantly downregulated interleukin-8, interleukin-1β, and tumour necrosis factor α mRNA levels in hepatopancreas and intestines (p<.05). After 56 days of feeding, 225 fish (23.20±0.59g) were challenged with Aeromonas hydrophila (108 CFU/g), and survival rates were recorded for the next 14 days. Meanwhile, the serum immune parameters of fish were determined. Results showed that compared with control, the survival rates of the treatment groups were significantly increased (p<.05). Lysozyme, component 3, component 4, immunoglobulin M, acid phosphatase, and alkaline phosphatase levels in serum by dietary106-109 CFU/g B. velezensis showed significant promoting effects (p<.05). In conclusion, dietary 107-109 CFU/g B. velezensis boosts the growth, immunity, antioxidant capacity, and against A. hydrophila infection of Amur minnow. Regression analysis suggested that the optimal dose of B. velezensis LSG2-5 in the Amur minnow diet was 5.00×108CFU/g.


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## Body

## 1. Introduction

Probiotics have received attention as alternatives to antibiotics and other drugs in aquaculture [1]. Bacillus can regulate humoral immunity [2], mucosal immunity [3], or the expressions of immune-related genes [4] to enhance the immunity of fish. Studies have shown that both heterogeneous and indigenous Bacillus affect the growth, immunity, antioxidant capacity, and disease resistance of fish [5, 6]. Endogenous Bacillus has more excellent potential than exogenous Bacillus because of its ability to adapt to the same environment and avoid competitive rejection [7, 8].Bacillus velezensis is a new species that has been applied to aquaculture in recent years [9]. It mainly acts on the immune system and disease resistance of fish to inhibit the attack of pathogenic bacteria [10]. It is a potential candidate probiotic. A strain of B. velezensis isolated from healthy Macrobrachium nipponese has been proven to inhibit pathogenic bacteria in vitro and is not pathogenic [11]. The research on the prevention and control of aquatic animal diseases by B. velezensis is still limited to the isolation of superior microbial resources in specific habitats and the analysis of biological characteristics of superior strains. For large-scale applications, further research and systematic evaluation are needed. As a probiotic feed additive, B. velezensis comes from different sources, either in the gastrointestinal tract of the fish itself or in the environment where the fish live. It was found that B. velezensis from the intestinal tract of fish had probiotic effects on the immune system of channel catfish (Ictalurus punctatus) [12], Nile tilapia (Oreochromis niloticus) [13], crucian carp (Carassius auratus) [14, 15], hybrid grouper (Epinephelus lanceolatus♂ × E. fuscoguttatus♀) [16], and others, such as inhibiting pathogenic bacteria. Nonfish B. velezensis also had probiotic and disease-resistant effects on grass carp [10], rainbow trout (Oncorhynchus mykiss) [17], juvenile Atlantic salmon (Salmo salar L.) [18], and others. We speculate that B. velezensis still has probiotic effects on native fish in northern China. However, the adaptability of different doses of B. velezensis to different fish in vivo requires further research.The Amur minnow (Rhynchocypris lagowskii Dybowski) is an omnivorous cold-water fish with fresh and tender meat. It is one of the main economic fish promoted in Northeast China in recent years [19]. Countries have completely banned antibiotics worldwide [1]. Farming of Amur minnow has been facing diseases caused by pathogenic bacteria or environmental factors, which may cause economic losses. The current research on Amur minnow mainly focused on the nutritional requirements, including the effects of dietary carbohydrate sources [20], differential protein levels [21], β-conglycinin [22], glycinin [23], L-carnitine [24], and oxidized fish oil [25, 26]. In our laboratory, a strain LSG2-5 isolated from the intestinal tract of Amur minnow was identified as B. velezensis by 16S rDNA sequence analysis, and its probiotic evaluation and safety test were conducted [27]. To verify the positive effects of B. velezensis LSG2-5 on the host in vivo, Amur minnow was fed with different doses of dietary B. velezensis LSG2-5 in this research. The effects of B. velezensis LSG2-5 on growth performance, serum immunity, antioxidant enzymes, and resistance to Aeromonas hydrophila infection of Amur minnow were investigated, and the optimal dose of LSG2-5 was analyzed.


## 2. Materials and Methods

### 2.1. Probiotic Preparation

B. velezensis isolated and screened from the intestines of Amur minnow was cultured in the past. B. velezensis was placed into Luria-Bertani (LB) broth and cultured in a shaker incubator at 37°C for 16 h. B. velezensis was coated on LB agar plate and cultured at 37°C for 24 h. A single colony of B. velezensis was selected and placed into LB broth, divided into four groups, and cultured in new LB broth. The cells were collected and suspended in sterile phosphate-buffered saline (PBS). LB agar plate counting method was used to count, and the bacterial counts in the 4 groups were determined to be 106 CFU/mL, 107 CFU/mL, 108 CFU/mL, and 109 CFU/mL, respectively. Four groups of purified B. velezensis with different doses were prepared to feed immediately as needed.


### 2.2. Experimental Diets

The isonitrogenous and isolipidic basal diets were used in this study. The composition and nutritional composition of the basal diets are shown in Table1. On an air-dried basis, the crude protein content was 370.0 g/kg and the gross energy level was 168.0 MJ/kg. The ingredients were pulverized and passed through 250 μm sieves, weighted according to the proportion of the formula, and mixed evenly. An appropriate amount of water was added, and then fed pellets of 1.5 mm diameter were processed by a commercial feed extruder (9KLP-125, Yongfeng Machinery Equipment Co., LTD, Zhengzhou, China). During the production of diets, the temperature was controlled from 55°C to 65°C. The diets were naturally air-dried in the shade and stored at -20°C until used. Four different doses of B. velezensis (106 CFU/g, 107 CFU/g, 108 CFU/g, and 109 CFU/g) were added to the basal diet, and the control group was not added (0 CFU/g). Spraying bacterial solution was carried out in a sterile environment. B. velezensis was suspended in PBS and uniformly sprayed on the surface of the basal diets using a high-temperature sterilized 30 mL spray bottle. The surface of diets was covered entirely with bacterial suspension, at which time 15 mL of bacterial suspension was sprayed for every 100 g of diets. Before the experiment, the diets were dissolved to measure the living bacteria count. To ensure that the actual bacterial dose of each group in diets met the test requirements. All diets supplemented with B. velezensis were sprayed and dried at room temperature, sprayed daily, and stored at 4°C for short periods out of light.Table 1
Composition and proximate analysis of basal diets (g/kg air dry basis).
Ingredientsg/kgProximate compositiong/kgFish meal200.0Crude protein370.0Soybean meal330.0Crude lipid65.6Fish oil20.0Moisture45.9Corn oil20.0Ash77.5Wheat bran60.0Gross energy (MJ/kg)168.0Wheat flour100.0Nitrogen-free extract420.6Dextrin70.0Corn protein flour150.0Calcium dihydrogen phosphate30.0Compound premix 1%a10.0Lysine2.0Methionine5.0Choline chloride 50%3.0aCompound premix 1%: vitamins and mineral included. Vitamin A (IU/kg) 3600; vitamin D3 (IU/kg) 1200; vitamin E (mg/kg) 20, vitamin K3 (mg/kg) 5; vitamin B1 (mg/kg) 5; vitamin B2 (mg/kg) 7; vitamin B6 (mg/kg) 6; vitamin B12 (ug/kg) 20; calcium pantothenate (mg/kg) 20; nicotinic acid (mg/kg) 30; folic acid (mg/kg) 1.7; biotin (mg/kg) 0.05; VC phosphate (mg/kg) 171.4; inositol (mg/kg) 90; Mg (mg/kg) 150; Fe (mg/kg) 120; Zn (mg/kg) 60; Mn (mg/kg) 30; Cu (mg/kg) 4; Co (mg/kg) 0.5; Se (mg/kg) 0.1; I (mg/kg) 1.


### 2.3. Experimental Fish and Feeding Trial

Amur minnow (R. lagowski Dybowskii) was provided by Nian Nian You Yu Fishery (Jilin, China). The experiment was conducted at the Aquaculture Laboratory of Jilin Agricultural University (Changchun, China). The volume of the aquarium was 120 L, and the experimental units were cycled in natural light and dark (approx. 13 : 11 hours light: dark). The fish were fed the basal diets and domesticated for 14 days. A total of 535 fish (initial average weight: 9.95±0.05 g) were randomly assigned to 15 buckets and divided into 5 groups with 3 replicates per group. Fish were starved for 24 h before feeding experiment. The fish were artificially fed to apparent satiation twice a day (8 : 00 and 17 : 00) for 56 days during the experiment. The water temperature was (20±1)°C, the pH was 7.5±0.1, and the dissolved oxygen was >8.0 mg/L. A third of the water was exchanged every day. Clean the bottom of the tank every other day.


### 2.4. Sample Collection

After 56 days of rearing, the fish were fasted for 24 h. 6 fish were randomly selected from each replicate for analysis. All fish were anesthetized with eugenol solution (1 : 10,000). Then, the weight, length, viscera, and hepatopancreas of all fish were measured. Blood was collected from the tail vein for serum immunity analysis, stored at 4°C for 24 h, and centrifuged at3000×g for 15 min to separate the serum. The hepatopancreas and intestines (proximal intestine [PI], mid intestine [MI], and distal intestine [DI]) were isolated on ice for analysis of enzyme activities and gene expressions. Muscles were isolated on ice for the expressions of relative genes. All samples were immediately frozen in liquid nitrogen and stored at -80°C until analyzed.


### 2.5. Related Index Determination

#### 2.5.1. Growth Performance Measurement

Growth factors such as body weight gain rate (WGR), specific growth rate (SGR), feed conversion ratio (FCR), hepatosomatic index (HSI), viscerosomatic index (VSI), and condition factor (CF) were calculated as follows:(1)WGR%=100×FW−IW×IW−1,SGR%day=100×lnFW−lnIW×day−1,FCR=FIFW−IW,HSI%=100×hepatopancreasweightFW,VSI%=100×visceraweightFW,CF%=100×FWbodylength3.where FI is the feed intake, IW is the fish initial weight, and FW is the fish final weight.


#### 2.5.2. Analysis of Immune and Antioxidant Capacity

The levels of component 3 (C3), component 4 (C4), immunoglobulin M (IgM), lysozyme (LZM), acid phosphatase (ACP), and alkaline phosphatase (AKP) in serum were measured and analyzed following the kit’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels in hepatopancreas and intestines were measured and analyzed by the same methods.


#### 2.5.3. Analysis of Gene Expression

Total RNA was extracted from the hepatopancreas and intestinal tract using a TRIzol kit (Takara, Dalian, China). Fluorescence quantitative real-time PCR was used to detect the expression of genes related to inflammation and antioxidation. Follow the instructions of the One-Step TB Green PrimeScript RT-PCR Kit II(Takara, Dalian, China). Using 20 ɥL reaction system, 10 ɥL One-Step TB Green RT-PCR Buffer, 0.8 ɥL PrimeScript 1 Step Enzyme Mix, 0.8μL forward and 0.8 μL reverse primer, 2 μL of total RNA template, and 5.6 μL RNase free dH2O. Reverse transcriptional reaction conditions: 42°C for 5 min and 95°C for 10 s. Circulation conditions: 95°C for 5 s, 60°C for 30 s, and 40 times. Primers are shown in Table 2. The analysis was conducted according to the 2-∆∆CT methodology of a previous study [19].Table 2
Primer sequences of the gene.
GenesSequences (5′-3′)ReferenceGenBank numberβ-ActinForwardCGGTATCCATGAGACCACCTZhang et al. [24]AAB97964.1ReverseCTTCTGCATCCTGTCAGCAATGF-βForwardTTGGGACTTGTGCTCTATZhang et al. [24]EU099588ReverseAGTTCTGCTGGGATGTTTIL-10ForwardAA TCCCTTTGA TTTTGCCZhang et al. [24]AB110780ReverseGTGCCTTATCCTACAGTATGTGIL-8ForwardATGAGTCTTAGAGGTCTGGGTGZhang et al. [24]DQ453125ReverseACAGTGAGGGCTAGGAGGGIL-1βForwardCTGGAGCAATGCAATACAAAZhang et al. [24]AJ245635ReverseAGGTAGAGGTTGCTGTTGGAATNF-αForwardCCAGGCTTTCACTTCACGZhang et al. [24]FN543477ReverseGCCATAGGAATCGGAGTAGCATForwardACTACCAGTCAACTGCCCGTCWang et al. [28]NM_130912.1ReverseTTTAGCACCTGAGTGAAGAACGGSH-PXForwardACAGAGGGTGGGCGTTATWang et al. [28]AW232474ReverseTTCGGGCACCAGAGGACuZn-SODForwardGGCCAACCGATAGTGTTAGAWang et al. [28]Y12236ReverseCCAGCGTTGCCAGTTTTTAG


### 2.6. Challenge Test

A. hydrophila used was stored in the Aquaculture Laboratory of Jilin Agricultural University (Changchun, China), and its median lethal dose (LD50) was determined by the method described previously [29]. A. hydrophila was cultured in LB broth at 28°C for 16 h, and the cells were collected and suspended in sterile PBS. Amur minnow was challenged with 200 ɥL of serially diluted A. hydrophila suspensions for a final dose of 1.50×105, 1.50×106, 1.50×107, 1.50×108, and 1.50×109 CFU/mL. The cumulative mortality was calculated for 14 consecutive days.At the end of the feeding experiment, 225 fish (average weight:23.20±0.59 g) of B. velezensis treatment groups were each injected with 200 ɥL of PBS containing A. hydrophila, and the fish in the control group were each injected with 200 ɥL of PBS. Fishes were fed and observed for 14 days, and survival rates were recorded. At the end of the challenge experiment, the survival fish serum was collected according to the above methods for the analysis of immune indexes.


### 2.7. Data Analysis

All data were expressed asmeans±standard error of the mean (means±SEM). One-way ANOVA was used to analyze the difference between the treatment and control groups by SPSS22.0. Tukey’s test was used to determine the difference between the experimental groups (p<.05). The relationships between growth parameters and B. velezensis doses were analyzed by regression analysis.


## 2.1. Probiotic Preparation

B. velezensis isolated and screened from the intestines of Amur minnow was cultured in the past. B. velezensis was placed into Luria-Bertani (LB) broth and cultured in a shaker incubator at 37°C for 16 h. B. velezensis was coated on LB agar plate and cultured at 37°C for 24 h. A single colony of B. velezensis was selected and placed into LB broth, divided into four groups, and cultured in new LB broth. The cells were collected and suspended in sterile phosphate-buffered saline (PBS). LB agar plate counting method was used to count, and the bacterial counts in the 4 groups were determined to be 106 CFU/mL, 107 CFU/mL, 108 CFU/mL, and 109 CFU/mL, respectively. Four groups of purified B. velezensis with different doses were prepared to feed immediately as needed.


## 2.2. Experimental Diets

The isonitrogenous and isolipidic basal diets were used in this study. The composition and nutritional composition of the basal diets are shown in Table1. On an air-dried basis, the crude protein content was 370.0 g/kg and the gross energy level was 168.0 MJ/kg. The ingredients were pulverized and passed through 250 μm sieves, weighted according to the proportion of the formula, and mixed evenly. An appropriate amount of water was added, and then fed pellets of 1.5 mm diameter were processed by a commercial feed extruder (9KLP-125, Yongfeng Machinery Equipment Co., LTD, Zhengzhou, China). During the production of diets, the temperature was controlled from 55°C to 65°C. The diets were naturally air-dried in the shade and stored at -20°C until used. Four different doses of B. velezensis (106 CFU/g, 107 CFU/g, 108 CFU/g, and 109 CFU/g) were added to the basal diet, and the control group was not added (0 CFU/g). Spraying bacterial solution was carried out in a sterile environment. B. velezensis was suspended in PBS and uniformly sprayed on the surface of the basal diets using a high-temperature sterilized 30 mL spray bottle. The surface of diets was covered entirely with bacterial suspension, at which time 15 mL of bacterial suspension was sprayed for every 100 g of diets. Before the experiment, the diets were dissolved to measure the living bacteria count. To ensure that the actual bacterial dose of each group in diets met the test requirements. All diets supplemented with B. velezensis were sprayed and dried at room temperature, sprayed daily, and stored at 4°C for short periods out of light.Table 1
Composition and proximate analysis of basal diets (g/kg air dry basis).
Ingredientsg/kgProximate compositiong/kgFish meal200.0Crude protein370.0Soybean meal330.0Crude lipid65.6Fish oil20.0Moisture45.9Corn oil20.0Ash77.5Wheat bran60.0Gross energy (MJ/kg)168.0Wheat flour100.0Nitrogen-free extract420.6Dextrin70.0Corn protein flour150.0Calcium dihydrogen phosphate30.0Compound premix 1%a10.0Lysine2.0Methionine5.0Choline chloride 50%3.0aCompound premix 1%: vitamins and mineral included. Vitamin A (IU/kg) 3600; vitamin D3 (IU/kg) 1200; vitamin E (mg/kg) 20, vitamin K3 (mg/kg) 5; vitamin B1 (mg/kg) 5; vitamin B2 (mg/kg) 7; vitamin B6 (mg/kg) 6; vitamin B12 (ug/kg) 20; calcium pantothenate (mg/kg) 20; nicotinic acid (mg/kg) 30; folic acid (mg/kg) 1.7; biotin (mg/kg) 0.05; VC phosphate (mg/kg) 171.4; inositol (mg/kg) 90; Mg (mg/kg) 150; Fe (mg/kg) 120; Zn (mg/kg) 60; Mn (mg/kg) 30; Cu (mg/kg) 4; Co (mg/kg) 0.5; Se (mg/kg) 0.1; I (mg/kg) 1.


## 2.3. Experimental Fish and Feeding Trial

Amur minnow (R. lagowski Dybowskii) was provided by Nian Nian You Yu Fishery (Jilin, China). The experiment was conducted at the Aquaculture Laboratory of Jilin Agricultural University (Changchun, China). The volume of the aquarium was 120 L, and the experimental units were cycled in natural light and dark (approx. 13 : 11 hours light: dark). The fish were fed the basal diets and domesticated for 14 days. A total of 535 fish (initial average weight: 9.95±0.05 g) were randomly assigned to 15 buckets and divided into 5 groups with 3 replicates per group. Fish were starved for 24 h before feeding experiment. The fish were artificially fed to apparent satiation twice a day (8 : 00 and 17 : 00) for 56 days during the experiment. The water temperature was (20±1)°C, the pH was 7.5±0.1, and the dissolved oxygen was >8.0 mg/L. A third of the water was exchanged every day. Clean the bottom of the tank every other day.


## 2.4. Sample Collection

After 56 days of rearing, the fish were fasted for 24 h. 6 fish were randomly selected from each replicate for analysis. All fish were anesthetized with eugenol solution (1 : 10,000). Then, the weight, length, viscera, and hepatopancreas of all fish were measured. Blood was collected from the tail vein for serum immunity analysis, stored at 4°C for 24 h, and centrifuged at3000×g for 15 min to separate the serum. The hepatopancreas and intestines (proximal intestine [PI], mid intestine [MI], and distal intestine [DI]) were isolated on ice for analysis of enzyme activities and gene expressions. Muscles were isolated on ice for the expressions of relative genes. All samples were immediately frozen in liquid nitrogen and stored at -80°C until analyzed.


## 2.5. Related Index Determination

### 2.5.1. Growth Performance Measurement

Growth factors such as body weight gain rate (WGR), specific growth rate (SGR), feed conversion ratio (FCR), hepatosomatic index (HSI), viscerosomatic index (VSI), and condition factor (CF) were calculated as follows:(1)WGR%=100×FW−IW×IW−1,SGR%day=100×lnFW−lnIW×day−1,FCR=FIFW−IW,HSI%=100×hepatopancreasweightFW,VSI%=100×visceraweightFW,CF%=100×FWbodylength3.where FI is the feed intake, IW is the fish initial weight, and FW is the fish final weight.


### 2.5.2. Analysis of Immune and Antioxidant Capacity

The levels of component 3 (C3), component 4 (C4), immunoglobulin M (IgM), lysozyme (LZM), acid phosphatase (ACP), and alkaline phosphatase (AKP) in serum were measured and analyzed following the kit’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels in hepatopancreas and intestines were measured and analyzed by the same methods.


### 2.5.3. Analysis of Gene Expression

Total RNA was extracted from the hepatopancreas and intestinal tract using a TRIzol kit (Takara, Dalian, China). Fluorescence quantitative real-time PCR was used to detect the expression of genes related to inflammation and antioxidation. Follow the instructions of the One-Step TB Green PrimeScript RT-PCR Kit II(Takara, Dalian, China). Using 20 ɥL reaction system, 10 ɥL One-Step TB Green RT-PCR Buffer, 0.8 ɥL PrimeScript 1 Step Enzyme Mix, 0.8μL forward and 0.8 μL reverse primer, 2 μL of total RNA template, and 5.6 μL RNase free dH2O. Reverse transcriptional reaction conditions: 42°C for 5 min and 95°C for 10 s. Circulation conditions: 95°C for 5 s, 60°C for 30 s, and 40 times. Primers are shown in Table 2. The analysis was conducted according to the 2-∆∆CT methodology of a previous study [19].Table 2
Primer sequences of the gene.
GenesSequences (5′-3′)ReferenceGenBank numberβ-ActinForwardCGGTATCCATGAGACCACCTZhang et al. [24]AAB97964.1ReverseCTTCTGCATCCTGTCAGCAATGF-βForwardTTGGGACTTGTGCTCTATZhang et al. [24]EU099588ReverseAGTTCTGCTGGGATGTTTIL-10ForwardAA TCCCTTTGA TTTTGCCZhang et al. [24]AB110780ReverseGTGCCTTATCCTACAGTATGTGIL-8ForwardATGAGTCTTAGAGGTCTGGGTGZhang et al. [24]DQ453125ReverseACAGTGAGGGCTAGGAGGGIL-1βForwardCTGGAGCAATGCAATACAAAZhang et al. [24]AJ245635ReverseAGGTAGAGGTTGCTGTTGGAATNF-αForwardCCAGGCTTTCACTTCACGZhang et al. [24]FN543477ReverseGCCATAGGAATCGGAGTAGCATForwardACTACCAGTCAACTGCCCGTCWang et al. [28]NM_130912.1ReverseTTTAGCACCTGAGTGAAGAACGGSH-PXForwardACAGAGGGTGGGCGTTATWang et al. [28]AW232474ReverseTTCGGGCACCAGAGGACuZn-SODForwardGGCCAACCGATAGTGTTAGAWang et al. [28]Y12236ReverseCCAGCGTTGCCAGTTTTTAG


## 2.5.1. Growth Performance Measurement

Growth factors such as body weight gain rate (WGR), specific growth rate (SGR), feed conversion ratio (FCR), hepatosomatic index (HSI), viscerosomatic index (VSI), and condition factor (CF) were calculated as follows:(1)WGR%=100×FW−IW×IW−1,SGR%day=100×lnFW−lnIW×day−1,FCR=FIFW−IW,HSI%=100×hepatopancreasweightFW,VSI%=100×visceraweightFW,CF%=100×FWbodylength3.where FI is the feed intake, IW is the fish initial weight, and FW is the fish final weight.


## 2.5.2. Analysis of Immune and Antioxidant Capacity

The levels of component 3 (C3), component 4 (C4), immunoglobulin M (IgM), lysozyme (LZM), acid phosphatase (ACP), and alkaline phosphatase (AKP) in serum were measured and analyzed following the kit’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels in hepatopancreas and intestines were measured and analyzed by the same methods.


## 2.5.3. Analysis of Gene Expression

Total RNA was extracted from the hepatopancreas and intestinal tract using a TRIzol kit (Takara, Dalian, China). Fluorescence quantitative real-time PCR was used to detect the expression of genes related to inflammation and antioxidation. Follow the instructions of the One-Step TB Green PrimeScript RT-PCR Kit II(Takara, Dalian, China). Using 20 ɥL reaction system, 10 ɥL One-Step TB Green RT-PCR Buffer, 0.8 ɥL PrimeScript 1 Step Enzyme Mix, 0.8μL forward and 0.8 μL reverse primer, 2 μL of total RNA template, and 5.6 μL RNase free dH2O. Reverse transcriptional reaction conditions: 42°C for 5 min and 95°C for 10 s. Circulation conditions: 95°C for 5 s, 60°C for 30 s, and 40 times. Primers are shown in Table 2. The analysis was conducted according to the 2-∆∆CT methodology of a previous study [19].Table 2
Primer sequences of the gene.
GenesSequences (5′-3′)ReferenceGenBank numberβ-ActinForwardCGGTATCCATGAGACCACCTZhang et al. [24]AAB97964.1ReverseCTTCTGCATCCTGTCAGCAATGF-βForwardTTGGGACTTGTGCTCTATZhang et al. [24]EU099588ReverseAGTTCTGCTGGGATGTTTIL-10ForwardAA TCCCTTTGA TTTTGCCZhang et al. [24]AB110780ReverseGTGCCTTATCCTACAGTATGTGIL-8ForwardATGAGTCTTAGAGGTCTGGGTGZhang et al. [24]DQ453125ReverseACAGTGAGGGCTAGGAGGGIL-1βForwardCTGGAGCAATGCAATACAAAZhang et al. [24]AJ245635ReverseAGGTAGAGGTTGCTGTTGGAATNF-αForwardCCAGGCTTTCACTTCACGZhang et al. [24]FN543477ReverseGCCATAGGAATCGGAGTAGCATForwardACTACCAGTCAACTGCCCGTCWang et al. [28]NM_130912.1ReverseTTTAGCACCTGAGTGAAGAACGGSH-PXForwardACAGAGGGTGGGCGTTATWang et al. [28]AW232474ReverseTTCGGGCACCAGAGGACuZn-SODForwardGGCCAACCGATAGTGTTAGAWang et al. [28]Y12236ReverseCCAGCGTTGCCAGTTTTTAG


## 2.6. Challenge Test

A. hydrophila used was stored in the Aquaculture Laboratory of Jilin Agricultural University (Changchun, China), and its median lethal dose (LD50) was determined by the method described previously [29]. A. hydrophila was cultured in LB broth at 28°C for 16 h, and the cells were collected and suspended in sterile PBS. Amur minnow was challenged with 200 ɥL of serially diluted A. hydrophila suspensions for a final dose of 1.50×105, 1.50×106, 1.50×107, 1.50×108, and 1.50×109 CFU/mL. The cumulative mortality was calculated for 14 consecutive days.At the end of the feeding experiment, 225 fish (average weight:23.20±0.59 g) of B. velezensis treatment groups were each injected with 200 ɥL of PBS containing A. hydrophila, and the fish in the control group were each injected with 200 ɥL of PBS. Fishes were fed and observed for 14 days, and survival rates were recorded. At the end of the challenge experiment, the survival fish serum was collected according to the above methods for the analysis of immune indexes.


## 2.7. Data Analysis

All data were expressed asmeans±standard error of the mean (means±SEM). One-way ANOVA was used to analyze the difference between the treatment and control groups by SPSS22.0. Tukey’s test was used to determine the difference between the experimental groups (p<.05). The relationships between growth parameters and B. velezensis doses were analyzed by regression analysis.


## 3. Result

### 3.1. Growth Performance

The effects of dietaryB. velezensis LSG2-5 on fish growth performance are shown in Table 3. FW, WGR, and SGR of the fish fed with B. velezensis LSG2-5 (107-109 CFU/g) were significantly higher than those of the control (p<.05). FCR in experimental groups significantly decreased compared to the control (p<.05). Only the HSI of the 108 CFU/g group was significantly increased than the control group (p<.05). Compared with the control group, HSI in 107 and 109 CFU/g groups showed upward trends, but the differences were insignificant (p>.05). There were no significant differences in VSI and CF among all groups (p>.05).Table 3
Effects ofBacillus velezensis LSG2-5 on the growth performance of Amur minnow (Rhynchocypris lagowskii Dybowski).
IndexesAddition ofBacillus velezensis LSG2-5 (CFU/g)0106107108109IW (g)10.00±0.239.99±0.3610.00±0.5510.01±0.249.98±0.49FW (g)20.19±0.58a21.23±0.41a23.87±0.15b25.34±0.41b25.36±0.25bWGR (%)110.94±3.90a114.79±4.41a141.31±3.17b153.28±3.57b154.05±2.77bSGR (%/day)1.25±0.03a1.36±0.04a1.57±0.02b1.66±0.02b1.66±0.02bFCR1.42±0.05c1.28±0.04b1.04±0.02a0.97±0.03a0.98±0.02aHSI (%)3.04±0.03a3.07±0.03a3.14±0.04ab3.26±0.06b3.17±0.03abVSI (%)15.31±0.1615.52±0.0515.66±0.0715.49±0.1615.80±0.02CF (%)1.42±0.041.49±0.011.56±0.021.54±0.031.49±0.04Note: Values followed by different letters are significantly different (p<.05).According to the method described previously [30], different doses of B. velezensis LSG2-5 (X) in feed and the FW (Yw), WGR (Ys), and SGR (Yf) of Amur minnow were analyzed by parabolic regression analysis. The resulting equations are shown in Table 4. The maximums FW, WGR, and SGR of Amur minnow were obtained. The results combined with ANOVA analysis showed that the optimal dose of B. velezensis LSG2-5 was 5.00×108 CFU/g.Table 4
Effects ofBacillus velezensis LSG2-5 on the growth performance of Amur minnow (Rhynchocypris lagowskii Dybowski) (regression equation).
IndexesRegression equationXoptYmaxFWy=−4E−17x2+4E−08x+21.534,R2=0.70665.00×10831.53WGRy=−4E−16x2+4E−07x+117.09,R2=0.6715.00×108217.09SGRy=−3E−18x2+3E−09x+1.3795,R2=0.65215.00×1082.13Note: FW means fish final weight; WGR means weight gain rate; SGR means specific growth rate; Xopt means the optimal value of X; Ymax means the maximum value of Y.


### 3.2. Immunological Parameters

Table5 shows the serum immune parameters of fish fed with different doses of B. velezensis LSG2-5. The levels of LZM and IgM were significantly elevated in the four treatment groups relative to the control (p<.05). With the increase of B. velezensis LSG2-5 doses, the content of C3 and C4 showed a trend of first increasing and then decreasing. Among them, the content of C3 in the 108 and 109 CFU/g groups was remarkably increased than that in the control (p<.05), and only the content of C4 in the 108 CFU/g group was significantly higher than that in the control (p<.05). The activity of ACP in the 107 CFU/g group was significantly elevated than that in the control and other groups (p<.05). The activities of AKP in the 108 and 109 CFU/g groups were significantly higher relative to the control (p<.05).Table 5
Effects ofBacillus velezensis LSG2-5 on immunological parameters of Amur minnow (Rhynchocypris lagowskii Dybowski) in serum.
IndexesAddition ofBacillus velezensis LSG2-5 (CFU/g)0106107108109LZM (ɥg/ml)40.79±0.97a45.20±0.43b48.69±0.78b52.90±0.66c53.29±1.15cC3 (mg/L)30.59±0.71a32.54±0.63ab32.90±1.21ab36.38±0.42c34.25±0.16bcC4 (mg/L)51.02±1.08a53.91±0.83ab53.67±0.92ab57.20±0.90b54.73±0.39abIgM (mg/L)36.52±1.52a43.24±1.24b47.58±1.01b47.08±1.06b42.55±1.46bACP (U/L)55.68±1.89a58.11±1.13a65.01±1.52b59.20±1.12a58.33±0.88aAKP (U/L)85.22±1.79a86.54±1.11ab87.66±1.25ab93.68±1.18c91.61±1.20bcNote: Values followed by different letters are significantly different (p<.05).


### 3.3. Antioxidant Capacity

Table6 lists the antioxidant capacity in the hepatopancreas and intestines of Amur minnow fed with different doses of B. velezensis LSG2-5. In hepatopancreas, the activities of CAT in the 107-109 CFU/g groups were significantly higher than in the control (p<.05). The content of GSH was significantly increased in the 106 and 107 CFU/g groups compared with the control (p<.05). A significant increase in the activities of GSH-Px and T-AOC in the treatment groups was found compared with the control (p<.05). The activities of SOD in the 108 and 109 CFU/g groups were significantly increased than in the control (p<.05). A significant decrease of MDA levels in the 106-108 CFU/g groups was found compared to the control (p<.05).Table 6
Effects ofBacillus velezensis LSG2-5 on antioxidant capacity in hepatopancreas and intestines of Amur minnow (Rhynchocypris lagowskii Dybowski).
IndexesAddition ofBacillus velezensis LSG2-5 (CFU/g)0106107108109CAT (U/mgprot)H32.21±0.75a34.78±0.79a40.37±0.64b44.67±1.16c38.89±1.02bPI36.49±1.16a37.93±1.26ab38.15±1.17ab44.22±0.90c42.44±0.79bcMI35.12±1.53a38.47±0.35ab39.75±0.17b45.22±0.93c39.18±1.11abDI35.41±0.84a37.16±1.49a37.45±1.25a42.72±0.84b38.50±1.28abGSH (ɥmol/mgprot)H2.19±0.14a3.01±0.13b3.17±0.13b2.71±0.19ab2.69±0.11abPI2.26±0.14a2.87±0.09b2.83±0.11b2.65±0.08ab2.44±0.06abMI1.76±0.13a2.53±0.08b2.69±0.14b2.91±0.17b1.99±0.14aDI1.50±0.09a1.87±0.07ab1.96±0.14b2.52±0.07c2.63±0.07cGSH-Px (U/mgprot)H216.51±13.60a288.62±15.67b370.68±11.88c411.66±14.58c382.76±6.49cPI186.02±8.71a225.23±6.95abc244.78±11.20bc273.18±11.54c187.31±19.82abMI165.94±12.21a193.92±8.07ab200.58±17.71ab232.04±9.00b184.84±7.69abDI129.34±10.38a168.56±8.73b239.73±8.28c256.75±4.82c227.76±5.83cMDA (nmol/mgprot)H3.26±0.10c2.88±0.08b2.31±0.05a2.72±0.10b3.05±0.05bcPI3.25±0.15c2.94±0.11bc2.42±0.08a2.74±0.09ab2.93±0.07bcMI3.39±0.07c3.10±0.14bc2.89±0.04ab2.53±0.14a3.09±0.10bcDI3.28±0.08c2.76±0.07b2.26±0.10a2.45±0.08ab2.77±0.05bSOD (U/mgprot)H83.45±2.56a93.51±4.14ab95.02±2.55ab112.22±2.13c104.11±2.39bcPI79.60±2.03a85.03±2.12ab100.06±3.64c93.59±1.38bc88.80±3.26abMI76.23±0.91a82.62±2.25ab92.59±1.74c94.17±2.73c88.94±0.73bcDI66.00±2.56a67.88±1.98a70.62±4.41a86.52±3.55b88.51±1.07bT-AOC (U/mgprot)H2.54±0.05a3.52±0.16b3.84±0.11bc4.33±0.12c4.04±0.09cPI1.86±0.06a2.24±0.09b2.53±0.11bc2.88±0.07c2.79±0.05cMI1.58±0.07a2.44±0.08b2.49±0.15b2.78±0.09b2.84±0.10bDI1.35±0.04a1.67±0.13a1.82±0.06ab2.25±0.19bc2.53±0.13cNote: Values followed by different letters are significantly different (p<.05). H: hepatopancreas; PI: proximal intestines; MI: mid intestine; DI: distal intestine.In the proximal intestine, fish fed the 108 and 109 CFU/g diets demonstrated a significant increase in activity of CAT compared to the control (p<.05). The content of GSH in 106 and 107 CFU/g diets was significantly higher than the control (p<.05). The activity of GSH-Px in the 107 and 108 CFU/g groups was significantly higher than in the control (p<.05). Compared to the control group, a significant decrease of MDA levels in the 107 and 108 CFU/g groups was found (p<.05). The SOD levels were significantly elevated in the 107 and 108 CFU/g groups relative to the control (p<.05). However, T-AOC activity was significantly increased in all experimental groups (p<.05).In the mid intestine, CAT activity was significantly increased in the 107 and 108 CFU/g groups compared to the control (p<.05). The contents of GSH in the 106-108 CFU/g groups were significantly higher than the control (p<.05). Only the activity of GSH-Px in the 108 CFU/g group was significantly higher than the control (p<.05). A significant decrease of MDA levels in 107 and 108 CFU/g groups was found compared to the control (p<.05). The activity of SOD was significantly elevated in 107-109 CFU/g groups relative to the control (p<.05). T-AOC activity was significantly increased in the treatment groups (p<.05).In the distal intestine, the activity of CAT in the 108 CFU/g group was significantly higher than in the control (p<.05). Amur minnow fed 107-109 CFU/g diets demonstrated a significant increase in the content of GSH relative to the control (p<.05). Compared to the control group, the activity of GSH-Px in the four experimental groups was significantly increased (p<.05). The activities of SOD and T-AOC in the 108 and 109 CFU/g groups were significantly increased (p<.05), but the content of MDA in the four experimental groups was significantly decreased (p<.05).


### 3.4. Relative Gene Expression

#### 3.4.1. Relative mRNA Expression of Antioxidant-Related Genes

The effects ofB. velezensis LSG2-5 on related mRNA levels in hepatopancreas, intestines, and Amur minnow muscles are shown in Figures 1–3. The relative expressions of CAT mRNA in the hepatopancreas of fish treated with B. velezensis LSG2-5 were significantly increased compared with the control (p<.05, Figure 1). The relative expressions of CAT mRNA in the proximal, mid, and distal intestines of the 107-109 CFU/g groups were significantly higher than that of the control (p<.05, Figure 1). The relative expressions of CAT mRNA in the 106-108 CFU/g group muscle were significantly higher than that of the control (p<.05, Figure 1).Figure 1
Effects ofBacillus velezensis LSG2-5 on the relative expression of CAT mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 2
Effects ofBacillus velezensis LSG2-5 on the relative expression of GSH-PX mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 3
Effects ofBacillus velezensis LSG2-5 on the relative expression of CuZn-SOD mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Compared with the control group, Amur minnow fed 107-109 CFU/g B. velezensis demonstrated a significant increase in the relative expression of GSH-Px mRNA of the hepatopancreas and distal intestine (p<.05, Figure 2). The relative expressions of GSH-Px mRNA in the proximal and mid intestines of all B. velezensis LSG2-5 treatment groups were significantly increased (p<.05, Figure 2). The relative expressions of GSH-Px mRNA in the muscle of the 107 and 108 CFU/g groups were significantly increased (p<.05, Figure 2).The relative expressions of copper/zinc superoxide dismutase (CuZn-SOD) mRNA in hepatopancreas and proximal and mid intestines were significantly increased by feeding dietaryB. velezensis LSG2-5 (p<.05, Figure 3). In the distal intestine and muscle, the relative expressions of CuZn-SOD mRNA in the 107-109 CFU/g groups were significantly increased relative to the control (p<.05, Figure 3).


#### 3.4.2. Relative mRNA Expression of Genes Related to Inflammation

In this study, the relative expressions of mRNA of genes related to inflammation in the hepatopancreas, intestines, and muscles were also examined (Figures4–8). Compared to the control, adding 106-108 CFU/g of B. velezensis LSG2-5 to the diet significantly increased the expressions of transforming growth factor β (TGF-β) mRNA in hepatopancreas (p<.05, Figure 4); TGF-β mRNA expressions in mid intestines were significantly increased in the 107-109 CFU/g groups (p<.05, Figure 4). The expressions of TGF-β mRNA in proximal, distal intestines, and muscle of the four treatment groups were significantly higher than that of the control (p<.05, Figure 4).Figure 4
Effects ofBacillus velezensis LSG2-5 on the relative expression of TGF-β mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 5
Effects ofBacillus velezensis LSG2-5 on the relative expression of IL-10 mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 6
Effects ofBacillus velezensis LSG2-5 on the relative expression of IL-8 mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 7
Effects ofBacillus velezensis LSG2-5 on the relative expression of IL-1β mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 8
Effects ofBacillus velezensis LSG2-5 on the relative expression of TNF-α mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).The expression of interleukin-10 (IL-10) mRNA in hepatopancreas increased first and then decreased, which was significantly higher in the 108 CFU/g group than the control (p<.05, Figure 5). The expressions of IL-10 mRNA in proximal and distal intestines of the four experimental groups were significantly higher than that of the control (p<.05, Figure 5), and it in the mid intestine of the 108 and 109 CFU/g groups was significantly increased (p<.05, Figure 5). The expressions of IL-10 mRNA in the muscle of the 107 and 108 CFU/g groups were significantly higher than that in the control (p<.05, Figure 5). The expressions of IL-10 mRNA in the muscles of the 106 and 109 CFU/g groups also showed increasing trends but were not significant (p>.05, Figure 5).The expressions of interleukin-8 (IL-8), interleukin-1β (IL-1β), and tumor necrosis factor α (TNF-α) mRNA in hepatopancreas, intestines, and muscles showed opposite trends compared with the TGF-β and IL-10. The relative expressions of IL-8 mRNA in the hepatopancreas and mid intestine of the 107-109 CFU/g groups were significantly lower than that in the control group and 106 CFU/g group (p<.05, Figure 6). Compared to the control, the relative expressions of IL-8 mRNA in proximal intestines and muscles of the treatment groups were significantly decreased (p<.05, Figure 6). The relative expression of IL-8 mRNA in the distal intestine of the 108 CFU/g group was significantly decreased (p<.05, Figure 6), and there was no significant difference between 106, 107, and 109 CFU/g groups and the control group (p>.05, Figure 6).The expressions of IL-1β mRNA in the hepatopancreas and distal intestines of the 107-109 CFU/g groups were significantly lower than that of the control (p<.05, Figure 7). The expressions of IL-1β mRNA in proximal and mid intestines of the 108 and 109 CFU/g groups were significantly decreased compared to the control (p<.05, Figure 7). The IL-1β mRNA levels in the muscle of all the treatment groups were significantly decreased than that of the control (p<.05, Figure 7).Compared with the control, the expressions of TNF-α mRNA were significantly decreased in the hepatopancreas and proximal intestines of all the treatment groups (p<.05, Figure 8). The expressions of TNF-α mRNA were significantly decreased in mid intestines, distal intestines, and muscles of the 107-109 CFU/g groups (p<.05, Figure 8).


### 3.5. Effect ofA. hydrophila Challenge on Disease Resistance of Amur minnow (R. lagowskii Dybowski)

The mortality of challenged fish is shown in Figure9. The LD50 dose of Amur minnow injected with A. hydrophila at 14 d was 108 CFU/mL. The survival rate of Amur minnow challenged by A. hydrophila is shown in Figure 10. The survival rates of the B. velezensis LSG2-5 treatment groups were significantly increased compared to the control (p<.05). There was a significant increase in the 108 CFU/g group compared with the 106 CFU/g group (p<.05), and there was no significant difference between the 107-109 CFU/g groups (p>.05).Figure 9
Cumulative mortality of Amur minnow (Rhynchocypris lagowskii Dybowski) challenged with different dose of Aeromonas hydrophila by intraperitoneal injection for LD50 test.Figure 10
Cumulative survival rate following a 14-dayAeromonas hydrophila challenge. Different letters indicate significant difference (p<.05).Serum immunity of Amur minnow challenged byA. hydrophila is presented in Table 7. The levels of LZM, C3, IgM, ACP, and AKP in the B. velezensis LSG2-5 treatment groups were significantly higher than those in the control (p<.05). The content of C4 in the 107-109 CFU/g groups was significantly higher than that in the control (p<.05), and there was an increasing trend in the 106 CFU/g group but not significant (p>.05).Table 7
Effects ofAeromonas hydrophila challenge in serum immunity of Amur minnow (Rhynchocypris lagowskii Dybowski).
IndexesAddition ofBacillus velezensis LSG2-5 (CFU/g)0106107108109LZM (ɥg/ml)40.42±0.38a47.84±0.59b49.80±0.98b56.62±1.48c56.68±1.12cC3 (mg/L)32.91±0.91a38.03±0.14b41.55±0.77bc43.52±1.49c45.83±1.14cC4 (mg/L)54.27±0.64a56.07±0.66ab59.12±1.27bc63.72±0.34c60.12±0.65dIgM (mg/L)37.67±0.92a44.26±1.10b49.07±0.86b57.23±1.40c57.88±1.85cACP (U/L)51.30±0.40a53.96±0.78b66.34±1.33c69.29±0.59c66.54±0.58cAKP (U/L)98.46±0.91a104.96±1.20b108.98±0.95b123.52±1.89c118.79±1.12cNote: Values followed by different letters are significantly different (p<.05).


## 3.1. Growth Performance

The effects of dietaryB. velezensis LSG2-5 on fish growth performance are shown in Table 3. FW, WGR, and SGR of the fish fed with B. velezensis LSG2-5 (107-109 CFU/g) were significantly higher than those of the control (p<.05). FCR in experimental groups significantly decreased compared to the control (p<.05). Only the HSI of the 108 CFU/g group was significantly increased than the control group (p<.05). Compared with the control group, HSI in 107 and 109 CFU/g groups showed upward trends, but the differences were insignificant (p>.05). There were no significant differences in VSI and CF among all groups (p>.05).Table 3
Effects ofBacillus velezensis LSG2-5 on the growth performance of Amur minnow (Rhynchocypris lagowskii Dybowski).
IndexesAddition ofBacillus velezensis LSG2-5 (CFU/g)0106107108109IW (g)10.00±0.239.99±0.3610.00±0.5510.01±0.249.98±0.49FW (g)20.19±0.58a21.23±0.41a23.87±0.15b25.34±0.41b25.36±0.25bWGR (%)110.94±3.90a114.79±4.41a141.31±3.17b153.28±3.57b154.05±2.77bSGR (%/day)1.25±0.03a1.36±0.04a1.57±0.02b1.66±0.02b1.66±0.02bFCR1.42±0.05c1.28±0.04b1.04±0.02a0.97±0.03a0.98±0.02aHSI (%)3.04±0.03a3.07±0.03a3.14±0.04ab3.26±0.06b3.17±0.03abVSI (%)15.31±0.1615.52±0.0515.66±0.0715.49±0.1615.80±0.02CF (%)1.42±0.041.49±0.011.56±0.021.54±0.031.49±0.04Note: Values followed by different letters are significantly different (p<.05).According to the method described previously [30], different doses of B. velezensis LSG2-5 (X) in feed and the FW (Yw), WGR (Ys), and SGR (Yf) of Amur minnow were analyzed by parabolic regression analysis. The resulting equations are shown in Table 4. The maximums FW, WGR, and SGR of Amur minnow were obtained. The results combined with ANOVA analysis showed that the optimal dose of B. velezensis LSG2-5 was 5.00×108 CFU/g.Table 4
Effects ofBacillus velezensis LSG2-5 on the growth performance of Amur minnow (Rhynchocypris lagowskii Dybowski) (regression equation).
IndexesRegression equationXoptYmaxFWy=−4E−17x2+4E−08x+21.534,R2=0.70665.00×10831.53WGRy=−4E−16x2+4E−07x+117.09,R2=0.6715.00×108217.09SGRy=−3E−18x2+3E−09x+1.3795,R2=0.65215.00×1082.13Note: FW means fish final weight; WGR means weight gain rate; SGR means specific growth rate; Xopt means the optimal value of X; Ymax means the maximum value of Y.


## 3.2. Immunological Parameters

Table5 shows the serum immune parameters of fish fed with different doses of B. velezensis LSG2-5. The levels of LZM and IgM were significantly elevated in the four treatment groups relative to the control (p<.05). With the increase of B. velezensis LSG2-5 doses, the content of C3 and C4 showed a trend of first increasing and then decreasing. Among them, the content of C3 in the 108 and 109 CFU/g groups was remarkably increased than that in the control (p<.05), and only the content of C4 in the 108 CFU/g group was significantly higher than that in the control (p<.05). The activity of ACP in the 107 CFU/g group was significantly elevated than that in the control and other groups (p<.05). The activities of AKP in the 108 and 109 CFU/g groups were significantly higher relative to the control (p<.05).Table 5
Effects ofBacillus velezensis LSG2-5 on immunological parameters of Amur minnow (Rhynchocypris lagowskii Dybowski) in serum.
IndexesAddition ofBacillus velezensis LSG2-5 (CFU/g)0106107108109LZM (ɥg/ml)40.79±0.97a45.20±0.43b48.69±0.78b52.90±0.66c53.29±1.15cC3 (mg/L)30.59±0.71a32.54±0.63ab32.90±1.21ab36.38±0.42c34.25±0.16bcC4 (mg/L)51.02±1.08a53.91±0.83ab53.67±0.92ab57.20±0.90b54.73±0.39abIgM (mg/L)36.52±1.52a43.24±1.24b47.58±1.01b47.08±1.06b42.55±1.46bACP (U/L)55.68±1.89a58.11±1.13a65.01±1.52b59.20±1.12a58.33±0.88aAKP (U/L)85.22±1.79a86.54±1.11ab87.66±1.25ab93.68±1.18c91.61±1.20bcNote: Values followed by different letters are significantly different (p<.05).


## 3.3. Antioxidant Capacity

Table6 lists the antioxidant capacity in the hepatopancreas and intestines of Amur minnow fed with different doses of B. velezensis LSG2-5. In hepatopancreas, the activities of CAT in the 107-109 CFU/g groups were significantly higher than in the control (p<.05). The content of GSH was significantly increased in the 106 and 107 CFU/g groups compared with the control (p<.05). A significant increase in the activities of GSH-Px and T-AOC in the treatment groups was found compared with the control (p<.05). The activities of SOD in the 108 and 109 CFU/g groups were significantly increased than in the control (p<.05). A significant decrease of MDA levels in the 106-108 CFU/g groups was found compared to the control (p<.05).Table 6
Effects ofBacillus velezensis LSG2-5 on antioxidant capacity in hepatopancreas and intestines of Amur minnow (Rhynchocypris lagowskii Dybowski).
IndexesAddition ofBacillus velezensis LSG2-5 (CFU/g)0106107108109CAT (U/mgprot)H32.21±0.75a34.78±0.79a40.37±0.64b44.67±1.16c38.89±1.02bPI36.49±1.16a37.93±1.26ab38.15±1.17ab44.22±0.90c42.44±0.79bcMI35.12±1.53a38.47±0.35ab39.75±0.17b45.22±0.93c39.18±1.11abDI35.41±0.84a37.16±1.49a37.45±1.25a42.72±0.84b38.50±1.28abGSH (ɥmol/mgprot)H2.19±0.14a3.01±0.13b3.17±0.13b2.71±0.19ab2.69±0.11abPI2.26±0.14a2.87±0.09b2.83±0.11b2.65±0.08ab2.44±0.06abMI1.76±0.13a2.53±0.08b2.69±0.14b2.91±0.17b1.99±0.14aDI1.50±0.09a1.87±0.07ab1.96±0.14b2.52±0.07c2.63±0.07cGSH-Px (U/mgprot)H216.51±13.60a288.62±15.67b370.68±11.88c411.66±14.58c382.76±6.49cPI186.02±8.71a225.23±6.95abc244.78±11.20bc273.18±11.54c187.31±19.82abMI165.94±12.21a193.92±8.07ab200.58±17.71ab232.04±9.00b184.84±7.69abDI129.34±10.38a168.56±8.73b239.73±8.28c256.75±4.82c227.76±5.83cMDA (nmol/mgprot)H3.26±0.10c2.88±0.08b2.31±0.05a2.72±0.10b3.05±0.05bcPI3.25±0.15c2.94±0.11bc2.42±0.08a2.74±0.09ab2.93±0.07bcMI3.39±0.07c3.10±0.14bc2.89±0.04ab2.53±0.14a3.09±0.10bcDI3.28±0.08c2.76±0.07b2.26±0.10a2.45±0.08ab2.77±0.05bSOD (U/mgprot)H83.45±2.56a93.51±4.14ab95.02±2.55ab112.22±2.13c104.11±2.39bcPI79.60±2.03a85.03±2.12ab100.06±3.64c93.59±1.38bc88.80±3.26abMI76.23±0.91a82.62±2.25ab92.59±1.74c94.17±2.73c88.94±0.73bcDI66.00±2.56a67.88±1.98a70.62±4.41a86.52±3.55b88.51±1.07bT-AOC (U/mgprot)H2.54±0.05a3.52±0.16b3.84±0.11bc4.33±0.12c4.04±0.09cPI1.86±0.06a2.24±0.09b2.53±0.11bc2.88±0.07c2.79±0.05cMI1.58±0.07a2.44±0.08b2.49±0.15b2.78±0.09b2.84±0.10bDI1.35±0.04a1.67±0.13a1.82±0.06ab2.25±0.19bc2.53±0.13cNote: Values followed by different letters are significantly different (p<.05). H: hepatopancreas; PI: proximal intestines; MI: mid intestine; DI: distal intestine.In the proximal intestine, fish fed the 108 and 109 CFU/g diets demonstrated a significant increase in activity of CAT compared to the control (p<.05). The content of GSH in 106 and 107 CFU/g diets was significantly higher than the control (p<.05). The activity of GSH-Px in the 107 and 108 CFU/g groups was significantly higher than in the control (p<.05). Compared to the control group, a significant decrease of MDA levels in the 107 and 108 CFU/g groups was found (p<.05). The SOD levels were significantly elevated in the 107 and 108 CFU/g groups relative to the control (p<.05). However, T-AOC activity was significantly increased in all experimental groups (p<.05).In the mid intestine, CAT activity was significantly increased in the 107 and 108 CFU/g groups compared to the control (p<.05). The contents of GSH in the 106-108 CFU/g groups were significantly higher than the control (p<.05). Only the activity of GSH-Px in the 108 CFU/g group was significantly higher than the control (p<.05). A significant decrease of MDA levels in 107 and 108 CFU/g groups was found compared to the control (p<.05). The activity of SOD was significantly elevated in 107-109 CFU/g groups relative to the control (p<.05). T-AOC activity was significantly increased in the treatment groups (p<.05).In the distal intestine, the activity of CAT in the 108 CFU/g group was significantly higher than in the control (p<.05). Amur minnow fed 107-109 CFU/g diets demonstrated a significant increase in the content of GSH relative to the control (p<.05). Compared to the control group, the activity of GSH-Px in the four experimental groups was significantly increased (p<.05). The activities of SOD and T-AOC in the 108 and 109 CFU/g groups were significantly increased (p<.05), but the content of MDA in the four experimental groups was significantly decreased (p<.05).


## 3.4. Relative Gene Expression

### 3.4.1. Relative mRNA Expression of Antioxidant-Related Genes

The effects ofB. velezensis LSG2-5 on related mRNA levels in hepatopancreas, intestines, and Amur minnow muscles are shown in Figures 1–3. The relative expressions of CAT mRNA in the hepatopancreas of fish treated with B. velezensis LSG2-5 were significantly increased compared with the control (p<.05, Figure 1). The relative expressions of CAT mRNA in the proximal, mid, and distal intestines of the 107-109 CFU/g groups were significantly higher than that of the control (p<.05, Figure 1). The relative expressions of CAT mRNA in the 106-108 CFU/g group muscle were significantly higher than that of the control (p<.05, Figure 1).Figure 1
Effects ofBacillus velezensis LSG2-5 on the relative expression of CAT mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 2
Effects ofBacillus velezensis LSG2-5 on the relative expression of GSH-PX mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 3
Effects ofBacillus velezensis LSG2-5 on the relative expression of CuZn-SOD mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Compared with the control group, Amur minnow fed 107-109 CFU/g B. velezensis demonstrated a significant increase in the relative expression of GSH-Px mRNA of the hepatopancreas and distal intestine (p<.05, Figure 2). The relative expressions of GSH-Px mRNA in the proximal and mid intestines of all B. velezensis LSG2-5 treatment groups were significantly increased (p<.05, Figure 2). The relative expressions of GSH-Px mRNA in the muscle of the 107 and 108 CFU/g groups were significantly increased (p<.05, Figure 2).The relative expressions of copper/zinc superoxide dismutase (CuZn-SOD) mRNA in hepatopancreas and proximal and mid intestines were significantly increased by feeding dietaryB. velezensis LSG2-5 (p<.05, Figure 3). In the distal intestine and muscle, the relative expressions of CuZn-SOD mRNA in the 107-109 CFU/g groups were significantly increased relative to the control (p<.05, Figure 3).


### 3.4.2. Relative mRNA Expression of Genes Related to Inflammation

In this study, the relative expressions of mRNA of genes related to inflammation in the hepatopancreas, intestines, and muscles were also examined (Figures4–8). Compared to the control, adding 106-108 CFU/g of B. velezensis LSG2-5 to the diet significantly increased the expressions of transforming growth factor β (TGF-β) mRNA in hepatopancreas (p<.05, Figure 4); TGF-β mRNA expressions in mid intestines were significantly increased in the 107-109 CFU/g groups (p<.05, Figure 4). The expressions of TGF-β mRNA in proximal, distal intestines, and muscle of the four treatment groups were significantly higher than that of the control (p<.05, Figure 4).Figure 4
Effects ofBacillus velezensis LSG2-5 on the relative expression of TGF-β mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 5
Effects ofBacillus velezensis LSG2-5 on the relative expression of IL-10 mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 6
Effects ofBacillus velezensis LSG2-5 on the relative expression of IL-8 mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 7
Effects ofBacillus velezensis LSG2-5 on the relative expression of IL-1β mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 8
Effects ofBacillus velezensis LSG2-5 on the relative expression of TNF-α mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).The expression of interleukin-10 (IL-10) mRNA in hepatopancreas increased first and then decreased, which was significantly higher in the 108 CFU/g group than the control (p<.05, Figure 5). The expressions of IL-10 mRNA in proximal and distal intestines of the four experimental groups were significantly higher than that of the control (p<.05, Figure 5), and it in the mid intestine of the 108 and 109 CFU/g groups was significantly increased (p<.05, Figure 5). The expressions of IL-10 mRNA in the muscle of the 107 and 108 CFU/g groups were significantly higher than that in the control (p<.05, Figure 5). The expressions of IL-10 mRNA in the muscles of the 106 and 109 CFU/g groups also showed increasing trends but were not significant (p>.05, Figure 5).The expressions of interleukin-8 (IL-8), interleukin-1β (IL-1β), and tumor necrosis factor α (TNF-α) mRNA in hepatopancreas, intestines, and muscles showed opposite trends compared with the TGF-β and IL-10. The relative expressions of IL-8 mRNA in the hepatopancreas and mid intestine of the 107-109 CFU/g groups were significantly lower than that in the control group and 106 CFU/g group (p<.05, Figure 6). Compared to the control, the relative expressions of IL-8 mRNA in proximal intestines and muscles of the treatment groups were significantly decreased (p<.05, Figure 6). The relative expression of IL-8 mRNA in the distal intestine of the 108 CFU/g group was significantly decreased (p<.05, Figure 6), and there was no significant difference between 106, 107, and 109 CFU/g groups and the control group (p>.05, Figure 6).The expressions of IL-1β mRNA in the hepatopancreas and distal intestines of the 107-109 CFU/g groups were significantly lower than that of the control (p<.05, Figure 7). The expressions of IL-1β mRNA in proximal and mid intestines of the 108 and 109 CFU/g groups were significantly decreased compared to the control (p<.05, Figure 7). The IL-1β mRNA levels in the muscle of all the treatment groups were significantly decreased than that of the control (p<.05, Figure 7).Compared with the control, the expressions of TNF-α mRNA were significantly decreased in the hepatopancreas and proximal intestines of all the treatment groups (p<.05, Figure 8). The expressions of TNF-α mRNA were significantly decreased in mid intestines, distal intestines, and muscles of the 107-109 CFU/g groups (p<.05, Figure 8).


## 3.4.1. Relative mRNA Expression of Antioxidant-Related Genes

The effects ofB. velezensis LSG2-5 on related mRNA levels in hepatopancreas, intestines, and Amur minnow muscles are shown in Figures 1–3. The relative expressions of CAT mRNA in the hepatopancreas of fish treated with B. velezensis LSG2-5 were significantly increased compared with the control (p<.05, Figure 1). The relative expressions of CAT mRNA in the proximal, mid, and distal intestines of the 107-109 CFU/g groups were significantly higher than that of the control (p<.05, Figure 1). The relative expressions of CAT mRNA in the 106-108 CFU/g group muscle were significantly higher than that of the control (p<.05, Figure 1).Figure 1
Effects ofBacillus velezensis LSG2-5 on the relative expression of CAT mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 2
Effects ofBacillus velezensis LSG2-5 on the relative expression of GSH-PX mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 3
Effects ofBacillus velezensis LSG2-5 on the relative expression of CuZn-SOD mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Compared with the control group, Amur minnow fed 107-109 CFU/g B. velezensis demonstrated a significant increase in the relative expression of GSH-Px mRNA of the hepatopancreas and distal intestine (p<.05, Figure 2). The relative expressions of GSH-Px mRNA in the proximal and mid intestines of all B. velezensis LSG2-5 treatment groups were significantly increased (p<.05, Figure 2). The relative expressions of GSH-Px mRNA in the muscle of the 107 and 108 CFU/g groups were significantly increased (p<.05, Figure 2).The relative expressions of copper/zinc superoxide dismutase (CuZn-SOD) mRNA in hepatopancreas and proximal and mid intestines were significantly increased by feeding dietaryB. velezensis LSG2-5 (p<.05, Figure 3). In the distal intestine and muscle, the relative expressions of CuZn-SOD mRNA in the 107-109 CFU/g groups were significantly increased relative to the control (p<.05, Figure 3).


## 3.4.2. Relative mRNA Expression of Genes Related to Inflammation

In this study, the relative expressions of mRNA of genes related to inflammation in the hepatopancreas, intestines, and muscles were also examined (Figures4–8). Compared to the control, adding 106-108 CFU/g of B. velezensis LSG2-5 to the diet significantly increased the expressions of transforming growth factor β (TGF-β) mRNA in hepatopancreas (p<.05, Figure 4); TGF-β mRNA expressions in mid intestines were significantly increased in the 107-109 CFU/g groups (p<.05, Figure 4). The expressions of TGF-β mRNA in proximal, distal intestines, and muscle of the four treatment groups were significantly higher than that of the control (p<.05, Figure 4).Figure 4
Effects ofBacillus velezensis LSG2-5 on the relative expression of TGF-β mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 5
Effects ofBacillus velezensis LSG2-5 on the relative expression of IL-10 mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 6
Effects ofBacillus velezensis LSG2-5 on the relative expression of IL-8 mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 7
Effects ofBacillus velezensis LSG2-5 on the relative expression of IL-1β mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).Figure 8
Effects ofBacillus velezensis LSG2-5 on the relative expression of TNF-α mRNA in hepatopancreas, intestine, and muscle of Amur minnow (Rhynchocypris lagowskii Dybowski). Different letters indicate significant difference (p<.05).The expression of interleukin-10 (IL-10) mRNA in hepatopancreas increased first and then decreased, which was significantly higher in the 108 CFU/g group than the control (p<.05, Figure 5). The expressions of IL-10 mRNA in proximal and distal intestines of the four experimental groups were significantly higher than that of the control (p<.05, Figure 5), and it in the mid intestine of the 108 and 109 CFU/g groups was significantly increased (p<.05, Figure 5). The expressions of IL-10 mRNA in the muscle of the 107 and 108 CFU/g groups were significantly higher than that in the control (p<.05, Figure 5). The expressions of IL-10 mRNA in the muscles of the 106 and 109 CFU/g groups also showed increasing trends but were not significant (p>.05, Figure 5).The expressions of interleukin-8 (IL-8), interleukin-1β (IL-1β), and tumor necrosis factor α (TNF-α) mRNA in hepatopancreas, intestines, and muscles showed opposite trends compared with the TGF-β and IL-10. The relative expressions of IL-8 mRNA in the hepatopancreas and mid intestine of the 107-109 CFU/g groups were significantly lower than that in the control group and 106 CFU/g group (p<.05, Figure 6). Compared to the control, the relative expressions of IL-8 mRNA in proximal intestines and muscles of the treatment groups were significantly decreased (p<.05, Figure 6). The relative expression of IL-8 mRNA in the distal intestine of the 108 CFU/g group was significantly decreased (p<.05, Figure 6), and there was no significant difference between 106, 107, and 109 CFU/g groups and the control group (p>.05, Figure 6).The expressions of IL-1β mRNA in the hepatopancreas and distal intestines of the 107-109 CFU/g groups were significantly lower than that of the control (p<.05, Figure 7). The expressions of IL-1β mRNA in proximal and mid intestines of the 108 and 109 CFU/g groups were significantly decreased compared to the control (p<.05, Figure 7). The IL-1β mRNA levels in the muscle of all the treatment groups were significantly decreased than that of the control (p<.05, Figure 7).Compared with the control, the expressions of TNF-α mRNA were significantly decreased in the hepatopancreas and proximal intestines of all the treatment groups (p<.05, Figure 8). The expressions of TNF-α mRNA were significantly decreased in mid intestines, distal intestines, and muscles of the 107-109 CFU/g groups (p<.05, Figure 8).


## 3.5. Effect ofA. hydrophila Challenge on Disease Resistance of Amur minnow (R. lagowskii Dybowski)

The mortality of challenged fish is shown in Figure9. The LD50 dose of Amur minnow injected with A. hydrophila at 14 d was 108 CFU/mL. The survival rate of Amur minnow challenged by A. hydrophila is shown in Figure 10. The survival rates of the B. velezensis LSG2-5 treatment groups were significantly increased compared to the control (p<.05). There was a significant increase in the 108 CFU/g group compared with the 106 CFU/g group (p<.05), and there was no significant difference between the 107-109 CFU/g groups (p>.05).Figure 9
Cumulative mortality of Amur minnow (Rhynchocypris lagowskii Dybowski) challenged with different dose of Aeromonas hydrophila by intraperitoneal injection for LD50 test.Figure 10
Cumulative survival rate following a 14-dayAeromonas hydrophila challenge. Different letters indicate significant difference (p<.05).Serum immunity of Amur minnow challenged byA. hydrophila is presented in Table 7. The levels of LZM, C3, IgM, ACP, and AKP in the B. velezensis LSG2-5 treatment groups were significantly higher than those in the control (p<.05). The content of C4 in the 107-109 CFU/g groups was significantly higher than that in the control (p<.05), and there was an increasing trend in the 106 CFU/g group but not significant (p>.05).Table 7
Effects ofAeromonas hydrophila challenge in serum immunity of Amur minnow (Rhynchocypris lagowskii Dybowski).
IndexesAddition ofBacillus velezensis LSG2-5 (CFU/g)0106107108109LZM (ɥg/ml)40.42±0.38a47.84±0.59b49.80±0.98b56.62±1.48c56.68±1.12cC3 (mg/L)32.91±0.91a38.03±0.14b41.55±0.77bc43.52±1.49c45.83±1.14cC4 (mg/L)54.27±0.64a56.07±0.66ab59.12±1.27bc63.72±0.34c60.12±0.65dIgM (mg/L)37.67±0.92a44.26±1.10b49.07±0.86b57.23±1.40c57.88±1.85cACP (U/L)51.30±0.40a53.96±0.78b66.34±1.33c69.29±0.59c66.54±0.58cAKP (U/L)98.46±0.91a104.96±1.20b108.98±0.95b123.52±1.89c118.79±1.12cNote: Values followed by different letters are significantly different (p<.05).


## 4. Discussion

Probiotics such as yeast, lactic acid bacteria (LAB), andBacillus added to feed were widely used in aquaculture for growth promotion, immune enhancement, and disease prevention. Several studies have shown that probiotics positively affect fish, especially Bacillus [13, 31–35]. Due to its rich metabolites and wide distribution in nature, B. velezensis has become a new strain used in aquaculture in recent years [9]. The antimicrobial properties of probiotics were considered one of the criteria for application to aquatic animal feed [36]. Secondary metabolites produced by B. velezensis include bacilysin, difficidin, macrolactin, acetoin, and others [37]. Among them, bacilysin and difficidin wield intense antibacterial effects. In recent years, there have been more and more studies on the antibacterial and probiotic effects of B. velezensis in vitro or in vivo, including mandarin fish (Siniperca cheats) [38], common carp [39], hybrid snakehead [40], and grass carp [41]. However, there are few studies on the probiotic effects and resistance against pathogen infection of Amur minnow, which has hindered the development of Amur minnow culture. The microflora of fish intestines was complex and diverse and has not been fully exploited [42]. The probiotics isolated from the intestinal tract have proved to be more effective in improving the growth and immune function of the host. [38, 43–45]. Therefore, the purpose of this study was to verify the probiotic function and disease resistance of B. velezensis and to explore the requirements of host for different doses of B. velezensis. This is vital for the systematic evaluation of B. velezensis as a probiotic agent for different fish species.The growth performance indexes are big deals to evaluate the breeding effect of Amur minnow. This study showed that compared with the control group, the FW, WGR, and SGR of Amur minnow were significantly improved with the increase of the dose ofB. velezensis LSG2-5. Except for FCR, each growth index showed a positive correlation with the dose of B. velezensis LSG2-5. When the dose reached to 107 CFU/g, a significantly positive effect on the growth of Amur minnow was shown. It is found that diets supplemented with Bacillus in appropriate doses impact positively on growth performance of aquatic animals, such as rockfish (Sebastes schlegelii) [46], catfish (Ictalurus punctatus) [12], Pacific white shrimp (Litopenaeus vannamei) [47], and Epinephelus fuscoguttatus [48].According to Afrilasari & Meryandini [49], adding B. magaterium from the intestine of catfish (Clarias sp.) to the diet has beneficial effects on endogenous digestive enzymes and intestinal flora of catfish, thus improving the growth performance of catfish. Similarly, the addition of B. subtilis and B. licheniformis also optimized the performance of Asian sea bass (Lates calcarifer) [50]. The mechanism by which probiotics can promote the growth performance of fish is not precise. Reda & Selim [51] believe that probiotics can promote the growth of fish by providing nutrients for fish and affecting the digestive enzyme activity to improve the digestibility or feed utilization rate. The reason may be that probiotics can secrete exogenous enzymes to supplement the endogenous enzymes of the host to some extent. Or it could be that probiotics boost the production of endogenous enzymes in fish. Irianto & Austin [52] believe that Bacillus increases fish appetite by producing vitamins and organic acids. Bacillus degrades indigestible components in the intestine, improves the shape of the intestine, and promotes the absorption of nutrients to promote fish growth [52]. It is recommended that further investigation and research can be conducted to clarify the mechanism of action of Bacillus on the growth performance of fish.Existing studies have shown that dietaryBacillus can modulate immune function in fish because Bacillus can regulate different immunological parameters [53–55]. LZM is an important non-specific immune factor in the innate immune system of fish, which can hydrolyze the alkaline enzyme of mucopolysaccharides in pathogenic bacteria. It plays an essential role in resisting the invasion of pathogenic bacteria. Under the conditions of this study, fish fed B. velezensis LSG2-5 for 56 days could significantly increase the activity of LZM in serum. In the challenge test, LZM activity was also significantly increased in treatment groups. Suggesting that B. velezensis LSG2-5 can positively affect the nonspecific immune system of the host. The results are consistent with previous reports [18, 56]. C3 and C4 are essential components in the complement system of fish, which are the main bridges connecting specific and non-specific immune responses. In this study, feeding B. velezensis LSG2-5 (108 CFU/g) increased serum C3 and C4 levels in Amur minnow. When infected with A. hydrophila, the C3 and C4 levels in experimental groups were increased, indicating that B. velezensis LSG2-5 has a positive effect on the immune function of Amur minnow. Similar to our results, Bacillus also has a promoting effect on the content of C3 and C4 in the serum of grass carp (Ctenopharyngodon idellus) [57] and Nile tilapia (Oreochromis niloticus) [58].Interestingly, in the study of the effects ofB. pumilus and B. clausii on Epinephelus coioides, C3 and C4 levels in serum were significantly affected at 30 days but returned to normal levels at 60 days [59]. The inconsistent results may be related to the different strains of Bacillus [59]. IgM is an essential mediator in the specific immune response of fish, which plays a role in both cellular and humoral immunity. IgM level was increased in treatment groups fed with B. velezensis LSG2-5. In the challenge test, IgM levels were also significantly increased.Similarly, dietary supplementation withB. velezensis also increased serum IgM levels in Atlantic salmon (Salmo salar L.) [18] and Oreochromis niloticus [60]. Both ACP and AKP are critical components of fish lysosomal enzymes. In this experiment, B. velezensis LSG2-5 can increase the activities of ACP and AKP in the serum of Amur minnow at a suitable dose. In the challenge test, all test groups significantly increased ACP and AKP activities. Past reports have shown similar results on E. coioides [61], Tilapia (O. mossambicus) [62], and Psetta maxima [63] in diets supplemented with Bacillus. However, Zhao et al. [64] reported that B. cereus had no significant effect on the ACP activity of sea cucumber (Apostichopus japonicus), which may be due to the nature of B. cereus itself.The above results all indicate that when the dose ofB. velezensis LSG2-5 is appropriate, it can promote the immune parameters of Amur minnow. In addition, B. velezensis LSG2-5 also affected the expression of inflammation-related genes in Amur minnow. It has been reported that NF-κB signaling pathway is involved in developing classical inflammatory pathways. Activation of NF-κB signaling pathway leads to increased levels of IL-1β mRNA, TNF-α and IL-8 mRNA, and decreased levels of IL-10 and TGF-β mRNA. NF-κB signaling pathway plays a central role in inflammation and wound healing [65–67].In the present study,B. velezensis LSG2-5 at the appropriate dose could promote the expressions of TGF-β and IL-10 in hepatopancreas, intestines, and muscles. At the same time, inhibiting the expressions of IL-8, IL-1β, and TNF-α. These results suggest that B. velezensis LSG2-5 can inhibit the inflammatory response of fish by inhibiting the NF-κB signaling pathway. Similar results were found in the effect of B. subtilis [68] and B. licheniformis [69] on the expression of inflammatory genes in Labeo rohita. Inconsistent with our results, it was found in the influence of Bacillus, Enterococcus and Lactobacillus on tilapia (O. niloticus) [70] intestinal immunity that mixed probiotics showed a promoting effect on the expression of anti-inflammatory factors and proinflammatory factors. In the histological examination of the fish body, no evidence of inflammation was found. The author believes that the induction of pro-inflammatory factors improves the immune preparedness of the host. However, more research is needed to understand better the role of Bacillus in the activation of immune mechanisms in fish.Due to changes in the water environment or stimulating fish, the production and removal of reactive oxygen species in tissues or cells are unbalanced, resulting in the oxidative stress reaction of fish. Major antioxidant enzymes, including SOD, GSH, GSH-Px, and CAT, are the first line of defense in the antioxidant defense system of ROS produced by the body in the process of oxidative stress [18]. MDA reflects the peroxidation reaction of the body. Lipid peroxidation led by increased formation of MDA leads to the cross-linking of protein, nucleic acid molecules, and cytotoxicity [71]. Probiotics can act as antigens to stimulate the secretion of antioxidant enzymes, effectively removing excess free radicals and regulating the body’s free radical balance, thereby increasing antioxidant capacity [55, 72]. The activities of these enzymes can indicate the antioxidant status of fish. When the activities of SOD, GSH, GSH-Px, and CAT increase, it indicates that the antioxidant capacity of fish increases. A decrease in MDA usually means increased enzymatic antioxidant levels since MDA is direct evidence of a toxic process caused by free radicals [73]. In this study, SOD, GSH, GSH-Px, CAT, and T-AOC activities increased while MDA activities decreased.Similar trends have been shown in studies of the effects ofBacillus on Atlantic salmon (S. salar L.) [18] and common carp [55]. This study found that the diet supplemented with B. velezensis LSG2-5 significantly increased the mRNA expression levels of GSH-Px, CAT, and CuZn-SOD in hepatopancreas, intestines, and muscles of Amur minnow. These results were similar to those of the antioxidant enzymes mentioned above, which suggested that B. velezensis LSG2-5 could enhance the antioxidant capacity of Amur minnow. However, the primary mechanism between probiotics and antioxidant defense is not well understood and deserves further study.Bacterial infection is the main cause of disease in fish farming. Many reports have shown that the addition ofBacillus spp. to diets can improve the disease resistance of fish to A.hydrophila [74], S. agalactiae [6], V. harveyi [75], V. parahaemolyticus [76], A. salmonicida [18], A.veronii [15], Edwardsiella [77], and Streptococcus iniae [78]. The antibacterial activity of probiotic Bacillus is manifested in the active substance produced since it can have an antibacterial effect on various fish pathogenic microorganisms. Similarly, adding probiotics to the biofloc system can promote the resistance of red hybrid tilapia to S. agalactiae [79]. The study verified the probiotic effect of B. velezensis LSG2-5 on fish by injecting A. hydrophila into fish. The results showed that the survival rates were significantly improved in the treatment groups, and the treatment group showed a significant regulatory effect on serum immune response. In particular, LZM, C3, C4, IgM, ACP, and AKP were significantly regulated. These results indicated that B. velezensis LSG2-5 prevented pathogenic bacteria and improved the disease resistance of Amur minnow. However, further studies are needed to investigate the specific ways in which probiotics inhibit pathogenic bacteria in fish and whether probiotics have the same inhibitory effect on viral infection in fish.


## 5. Conclusion

Bacterial diseases frequently occur in fish farming, and probiotics are needed to prevent disease and replace antibiotics. The multifunctional immune feature ofB. velezensis LSG2-5 is confirmed in this study, especially for resistance to A. hydrophila. Meanwhile, dietary B. velezensis supplementation can improve the growth performance of Amur minnow. In conclusion, the supplementation of 107-109 CFU/g of B. velezensis LSG2-5 positively affected Amur minnow. Based on comprehensive considerations, we suggest that the optimal supplemental dose of B. velezensis LSG2-5 to Amur minnow is 5.00×108 CFU/g.



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*Source: 7199145-2022-02-24.xml*