Accepted Manuscript

Growth performance, innate immune responses and disease resistance of fingerling

blunt snout bream, Megalobrama amblycephala adapted to different berberine-dietary
feeding modes

Wei-Na Xu, Dan-Hong Chen, Qing-Qing Chen, Wen-Bin Liu

PII: S1050-4648(17)30445-X
DOI: 10.1016/j.fsi.2017.07.051
Reference: YFSIM 4733

To appear in: Fish and Shellfish Immunology

Received Date: 19 March 2017

Revised Date: 22 July 2017
Accepted Date: 24 July 2017

Please cite this article as: Xu W-N, Chen D-H, Chen Q-Q, Liu W-B, Growth performance, innate immune
responses and disease resistance of fingerling blunt snout bream, Megalobrama amblycephala adapted
to different berberine-dietary feeding modes, Fish and Shellfish Immunology (2017), doi: 10.1016/
j.fsi.2017.07.051.

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1 Growth performance, innate immune responses and disease resistance of fingerling blunt snout bream,

2 Megalobrama amblycephala adapted to different berberine-dietary feeding modes

3 Wei-Na Xu1,2, Dan-Hong Chen2, Qing-Qing Chen2, Wen-Bin Liu2*,

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4 Shanghai Key Laboratory for Veterinary and Biotechnology, School of Agriculture and Biology,

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5 Shanghai Jiao Tong University, No.800 Dongchuan Road, Shanghai 200240, People’s Republic of

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6 China

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7 Key Laboratory of Aquatic Nutrition and Feed Science of Jiangsu Province, College of Animal

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8 Science and Technology, Nanjing Agricultural University, No.1 Weigang Road, Nanjing 210095,

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9 People’s Republic of China
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11 Corresponding Authors at: College of Animal Science and Technology, Nanjing Agricultural
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12 University
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13 Tel (Fax): 86-025-84395382. E-mail: wbliu@njau.edu.cn (Wen-Bin Liu).

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23 Abstract：
：A 8-week feeding trial was conducted to evaluate the effect of different berberine-dietary

24 feeding modes on growth, non-specific immune responses and disease resistance of blunt snout bream,

25 Megalobrama amblycephala. Fish (average initial weight 4.70±0.02g) were fed two fat levels (5% and

26 10%) diets in three berberine-feeding modes (supplementing 50 mg/kg berberine continuously,

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27 two-week or four-week intervals) with four replicates, respectively. Then, fish were challenged by

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28 Aeromonas hydrophila and mortality was recorded for the next 96h after feeding trial. The results

29 showed that different feeding modes of berberine significantly influenced growth, innate immunity and

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30 antioxidant capability of fish. Fish fed normal diet with 50 mg/kg berberine at two-week interval mode

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31 reflected remarkably (P < 0.05) high weight gain (WG). Plasma TC and TG contents were significantly
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32 (P < 0.05) decreased. The lysozyme (LYZ) activities, complement component 3 (C3) and complement

33 component 4 (C4) concentrations were significantly (P < 0.05) increased. Fish not only exhibited
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34 relatively low hepatopancreas malondialdehyde (MDA) and lipid peroxide (LPO) contents, but also
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35 significantly (P < 0.05) improved superoxide dismutase (SOD) and catalase (CAT) activities. Fish
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36 mortality after challenged by Aeromonas hydrophila was decreased. Same results were also presented

37 in fish fed high-fat diet with 50mg/kg berberine at two-week, four-week intervals or continuous feeding
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38 modes. Based on fish healthy improvement and feeding cost saving, blunt snout bream fed normal diet
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39 with 50 mg/kg berberine at two-week interval or fed high-fat diet with berberine at two-week or
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40 four-week intervals were optimal feeding mode, respectively.

41 Key words: Blunt snout bream; Berberine; Feeding modes; Immunity; Disease resistance

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45 1. Introduction

46 In recent years, there has been a trend to increase dietary lipid levels in commercial fish feed

47 formulations to enhance protein sparing and to increase aquaculture productivity. However, high-fat

48 feeding results specifically in hepatic fat accumulation. In order to improving it, many methods have

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49 been tried by the fish nutritionist, which focus on the use of medicinal plant products as potential

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50 therapeutic measures for modulating the immune response[1, 2]. Some Chinese herbs have attracted

51 great interest as reverting hepatic lipid dysfunction and few side effects[3]. One of projects in our

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52 laboratory was to select Chinese herb and supplement into diets to improve lipid metabolism of fish.

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53 Berberine is the principal isoquinoline alkaloidal constituent of the stems and roots of various Berberis
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54 species, such as Hydrastis canadensis, Cortex phellodendri (Huangbai) and Rhizom/a coptidis

55 (Huanglian) [4]. It exerted a broad array of pharmacological activities, including antioxidant,

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56 anti-inflammatory and anti-hypertlipidemia[4-9]. Besides, it had been reported that berberine could
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57 reduce glucose blood levels and improve lipid deposition in diabetes [10-15]. Many in vitro and in vivo
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58 studies showed that berberine has potentially beneficial effects in the treatment of fatty liver and

59 obesity in human [12, 16, 17].

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60 In aquaculture, berberine as an effective component of herbal medicines was used for the prevention
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61 and treatment of fish diseases caused by some kinds of bacteria [18, 19]. Berberine could not only
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62 reduce cytochrome P4501A (CYP1A) mRNA expression in a dose-dependent manner but also directly

63 inhibit this enzyme competitively. And high berberine doses inhibit CYP3A through the

64 downregulation of its expression at both the mRNA and the protein level in Crucian carp[20]. Our

65 previous study showed (supplementing 50 mg/kg berberine continuously for 8-week) berberine could

66 attenuate oxidative stress, enhance the immunity, improve function of mitochondrial respiratory chain
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67 via increasing the complex activities, reduce the hepatocyte apoptosis level and hepatopancreas damage

68 of fish fed with the high fat [21-23]. Moreover, the results also showed that berberine should

69 up-regulated lipid related gens, peroxisome proliferator-activated receptors α (PPARα) and carnitine

70 palmitoyltransferase I (CPT IA) mRNA expression levels of fish fed with the high fat [22]. It maybe

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71 has influence on lipid metabolism of fish. However, berberine on fish lipid metabolism is still unknown

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72 and maybe it also can revert the metabolic syndrome in fish. The preliminary result showed that

73 berberine had a potential application as an immunostimulant in fish culture. Primarily because it can be

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74 easily obtained, is not expensive, act against a broad spectrum of pathogens, and can be given orally,

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75 which is the most convenient method of immunostimulation. However, some researchers had proved
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76 that continuous administration of immunostimulants had no beneficial effects on growth or/and

77 immunity, and even resulted in negative impacts[24, 25]. Same result was also found in our study of
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78 berberine [23]. Improvement effect of immunity was not showed in fish fed with normal diet (5% lipid
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79 level) supplied berberine, while slight oxidative stress of fish was happened during feeding trial. Why?
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80 Hence, scientific and reasonable application method was needed to study. Based on it, optimal feeding

81 mode or strategy of berberine was estimated in this study. Based on previous studies, a 8-week feeding
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82 trial was conducted to compare the effects of continuous and discontinuous administration of berberine
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83 on growth, innate immune responses and disease resistance to Aeromonas hydrophila in blunt snout
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84 bream fed with normal or high-fat diets respectively in this study. The data obtained here might prompt

85 some new insights into the application of herbal-additives.

86 2. Materials and methods

87 2.1. Berberine and experimental diets

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88 The composition of the basal diet and high-fat diet were shown in Table 1. Fish meal, soybean meal,

89 cottonseed meal and rapeseed meal served as protein sources. Soybean oil was used as lipid sources.

90 Wheat flour was adopted as carbohydrate sources. Berberine (HPLC≥98%) was bought from the spring

91 and autumn biotechnology company, Nanjing, China. Berberine (50mg/kg) were added into the basal

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92 diet and high-fat diet followed by mixing manually [23]. Dietary ingredients were ground into fine

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93 powder then thoroughly mixed, and then blended with an additional 100mL of water per kg of diet to

94 form a soft dough which was pelleted (without injected steam) using a Pillet Mill with a 2 mm diameter

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95 die. The experiment feed was dried at air temperature at 28 ℃ overnight and stored in sealed plastic

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96 bags at -4℃ until use. The proximate composition of the experimental diets was determined according
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97 to the standard AOAC methodology.

98 Table 1 here
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99
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100 2.2. Fish and experimental design

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101 Blunt snout bream were obtained from a local fish hatchery (Nanjing, China). Prior to the feeding trial,

102 fish were acclimated to experimental conditions for 2 weeks. During the acclimation period, fish were
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103 fed a control diet three times a day. And then, 960 healthy fish with an initial mean body weight of 4.70
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104 ± 0.02 g were randomly distributed into 32 cages which were anchored in an outdoor pond. The
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105 feeding mode and schedule followed in different treatments were presented in Table 2. Each treatment

106 has four replicates. 50mg/kg berberine was supplemented in diets [23]. Fish were fed three times daily

107 at 7:30,12:00 and 16:30 h, respectively, for 8 weeks. Fish were hand-fed to apparent satiation with

108 utmost care to minimize waste. Fish were held under natural photoperiod throughout the feeding trail.
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109 Water temperature ranged from 23 to 28 ℃, pH fluctuated between 6.5 and 7.6 and dissolved oxygen

110 was maintained approximately at 5.0 mg/L during the feeding trial.

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112 2.3. Sampling and analysis

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113 2.3.1. Sampling

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114 At the end of the feeding trial, fish were starved for 24 h prior to sampling. And then all individuals

115 were quickly removed from each cage and anesthetized in diluted MS-222 (tricaine methanesulfonate,

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116 Sigma, USA) at the concentration of 100 mg/L. Total number and weight of fish in each cage were

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117 determined to calculate the growth performance. Six fish were randomly removed from each cage and
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118 blood sample was collected by caudal vein puncture using heparinized syringes coated with lithium

119 heparin as anticoagulant. After centrifugation (3000 g for 10 min at 4℃), plasma was stored at -80℃
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120 for subsequent analysis. In addition, individual hepatopancreas was dissected over an ice bed and
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121 washed thoroughly with chilled saline (0.89 g NaCl L-1), dried quickly over a piece of filter paper and
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122 stored at -80℃ for the analysis of hepatopancreas oxidative stress state.

123 2.3.2. Growth performance

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124 The fish were weighed individually before (initial body weight, Wi) and after (final body weight, Wf)
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125 the 8-week feeding experiment. For each treatment, all fish were used to quantify the percent of weight
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126 gain (WG), specific growth rate (SGR), feed conversion ratio (FCR), viscera/body ratio (VBR),

127 hepatosomatic index (HIS), abdominal fat percentage (PAF). These parameters were calculated as

128 follows[26]:

129 Weight gain (WG) =100 × (Wf -Wi)/Wi

130 Specific growth rate (SGR, %/d) =100 × (Ln Wf-Ln Wi)/t
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131 Feed conversion ratio (FCR) = Feed consumed (g)/(Wf-Wi)

132 Viscera/body ratio (VBR, %) = 100 × viscera weight / body weight

133 Hepatosomatic index (HSI, %) = 100 × hepatopancreas weight/ body weight

134 where Wf was final body weight (g), Wi was initial body weight (g), t was experimental duration

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135 in days.

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136 2.3.3. Proximate composition analysis

137 The approximate ingredient of the trial diets was ensured based on the standard AOAC method.

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138 Moisture in diets was admeasured by oven drying at 105 ℃ till constant weight. Crude protein was

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139 determined by micro-Kjeldahl method and reduplicated with a factor 6.25 after acid digestion using an
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140 AutoKjeldahl System (1030- Auto-analyzer, Tecator, Hoganas, Sweden). And crude lipid was

141 determined by solvent extraction with a Soxtec System HT (Soxtec System HT6, Tecator, Hoganas,
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142 Sweden). Ash was measured by combustion at 550 ℃ for 4 h. Gross energy was measured by Bomb
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143 Calorimeter (Parr 1281, Parr Instrument Company, Moline, IL, USA).
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144 2.3.4 Measures of plasma biochemical parameters

145 Plasma total cholesterol (TC), triglyceride (TG), HDL cholesterol (HDL-c), and LDL cholesterol
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146 (LDL-c) levels were measured by colorimetric enzymatic methods using commercial kits (Jiancheng,
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147 Nanjing). Lysozyme activity was measured using the turbidimetric method given by Stolen with a little
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148 modification. Plasma acid phosphatase (ACP) activity was carried out based on a disodium phenyl

149 phosphate method. Complement component 3 (C3) and Complement component 4 (C4) levels were

150 determined according to the method described by a commercial kit (ref. no. A012) produced by Jian

151 Cheng Bioengineering Institute (Nanjing, China) [23].

152 2.3.5. Analysis of antioxidant status

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153 Hepatopancreas was homogenized in ten volumes (v/w) of chilled physiological saline in tissue

154 homogenizer and centrifuged at 3000 g at 4 ℃ for 10 min. The supernatant was used as enzyme source

155 for measuring enzymatic activities. All enzyme preparations were carried out on ice. Dilution of the

156 sample was done as and when required. Total superoxide dismutase (t-SOD) activity was measured at

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157 550 nm using an SOD detection kit (Nanjing Jian cheng Bioengineering Institute, China), and catalase

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158 (CAT) was determined following the methods described by Lu et al[21]. Lipid peroxide (LPO) was

159 measured following the method known as Quantitative colorimetric method. The malonaldehyde

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160 (MDA) concentration was examined by the thiobarbituric acid technique [27, 28]. The MDA

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161 concentration was expressed as n mol/mgprotein in the supernatant. Hepatopancreas protein
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162 concentration was determined using Lowry et al.’s method[29].

163 2.3.6. Detection of hepatocyte apoptosis

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164 The apoptotic hepatocytes were detected using the terminal deoxynucleotidyl transferase-mediated
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165 dUTP nick end labeling (TUNEL) method. TUNEL-positive cells were counted and expressed as the
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166 percentage of the total cell number[30].

167
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168 2.4. Challenge test

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169 A. hydrophila (A. hydrophila, BSK-10) was provided by Freshwater Fisheries Research Center,
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170 Chinese Academy of Fishery Sciences (Wuxi, Jiangsu Province, China) and was activated following

171 the methods described by Zhang et al [26] and Li et al [31]. A 96 h LD50 (Ah, BSK-10 does that killed

172 50% of the test fish) was determined before challenge test and the result showed that the LD50 was 107

173 CFU/mL. After the initial sampling, the fish were kept in prepared tanks for three days and then all

174 remaining 16 fish from each treatment were injected intraperitoneally with the A. hydrophila. Fish were
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175 carefully monitored and mortality was recorded twice daily for the next 96 h. Cumulative mortality rate

176 was determined using the following formula.

177 Cumulative mortality rate (%) = (N0- Nt)×100/N0

178 where Nt and N0 were the final and initial number of fish

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179

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180 2.5. Statistical analysis

181 Datum were received one-way analysis of variance (ANOVA) using the SPSS statistical package

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182 version 20.0 (SPSS Inc., Chicago Avenue, IL, USA). If significant (P < 0.05) discrepancy were

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183 discovered in factors, Duncan’s multiple range test was employed to rank the means. All data were
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184 presented as means ± S.E.M (standard error of the mean) of four replications.

185
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186 3. Result
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187 3.1. Growth performance

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188 Growth performances were shown in Table 3. No mortality was observed during the 8-week feeding

189 trial in all groups. Compared with basic control group (D1), WG of fish in D2 was significantly (P <
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190 0.05) increased. No significant (P > 0.05) differences were found in SGR, FCR, VBR and HIS of fish
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191 in all groups.

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192 Table 3 here

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194 3.2 Body composition

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195 As can be seen in Table 4, compared with basic control group (D1), the crude lipid content of fish in

196 D4 was significantly (P < 0.05) decreased. The crude protein, energy, ash and moisture contents were

197 no significant differences (P > 0.05) among all groups.

198 Table 4 here

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199

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200 3.3 Plasma biochemical parameters

201 As shown in Table 5, compared with basic control group (D1), plasm TG and TC contents of fish in D2

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202 and D4 were significantly (P < 0.05) decreased. TC content of fish in D5 was significantly (P < 0.05)

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203 increased. No significant differences (P > 0.05) were observed in HDL-C and LDL-C contents among
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204 all groups.

205 Table 5 here

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206
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207 3.4. Plasm Immune parameters

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208 Immune parameters of blunt snout bream were shown in Table 6. Compared with basic control group

209 (D1), plasma ACP, LYZ activities and C3 and C4 contents of fish in D2 were significantly (P < 0.05)
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210 improved but decreased in D5. Plasma ACP activities of fish in D6, D7 and D8 were significantly (P <
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211 0.05) increased. C4 contents of fish in D6 was significantly (P < 0.05) increased. Compared with D5,
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212 ACP, LYZ activities and C3 and C4 contents in D6, D7 and D8 were significantly (P < 0.05)

213 increased.

214 Table 6 here

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216 3.5. Hepatopancreas anti-oxidative and oxidative parameters

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217 Compared with basic control group (D1), hepatopancreas MDA content of fish in D2 decreased

218 significantly (P < 0.05), and increased significantly (P < 0.05) in D5. LPO contents of fish in D2 and

219 D7 decreased significantly (P < 0.05). CAT activities of fish in D2, D4, D6, D7 and D8 were improved

220 significantly (P < 0.05). SOD activities of fish in D2, D3, D4, D6, D7 and D8 showed improved

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221 significantly (P < 0.05), but decreased significantly (P < 0.05) in D5. Compared with D5, MDA

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222 contents of fish in D6, D7 and D8 were decreased significantly (P < 0.05), CAT and SOD activities of

223 fish in D6, D7 and D8 were increased significantly (P < 0.05).

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224 Table 7 here

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225
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226 3.6 Hepatocyte apoptosis

227 Hepatocyte apoptosis results of blunt snout bream subjected to different treatment were presented in
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228 Table 8 and Figure 1. The nucleus of normal cells was dyed with blue (As shown in figure the blue
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229 arrow), while apoptotic cells with brown (As shown in figure the brown arrow). Compared with basic
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230 control group (D1), the total numbers of apoptotic hepatocytes in D2, D3, D4, D6, D7 and D8 were

231 occurred no significant (P > 0.05) differences. The total number of apoptotic hepatocytes in D5 was
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232 increased significantly (P < 0.05). Compared with D5, the total numbers of apoptotic hepatocytes in
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233 D6, D7 and D8 were decreased significantly (P < 0.05).

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234 Table 8 and Figure 1 here

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236 3.7. Mortality after A. hydrophila challenge

237 The challenge test revealed that different feeding mode of berberine significantly affected bacterial

238 resistance in juvenile fish (Table 9). Compared with basic control group (D1), the mortality of fish in
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239 D3 and D4 was increased significantly (P < 0.05) after 24h challenge. The mortality of fish in D2 and

240 D8 was decreased significantly (P < 0.05) after 48h challenge. The lowest (P < 0.05) mortality of fish

241 was observed in D2 after 72h or 96h challenge. Compared with D5, the mortality of fish in D6 and D7

242 was decreased significantly (P < 0.05) after 72h or 96h challenge.

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243 Table 9 here

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244

245 4. Discussion

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246 Optimal feeding mode or strategy of dietary additive was the most important factor to success of any

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247 aquaculture operation. It might not only promote the best growth and feed efficiency of fish or shrimp,
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248 but also minimize feed wastage, thus improving the culture environment[32]. And it was particularly

249 appropriate for the juvenile fish because of their susceptibility to overfeeding and underfeeding which
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250 causes both increased incidences of disease and mortality[33]. In the present study, optimum
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251 berberine-supplement mode was investigated based on different dietary nutritional level. Supplement of
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252 berberine in normal diet at two-week interval could remarkably strengthen growth, innate immunity

253 and antioxidant capability of blunt snout bream. Fish reflected high growth performance but showed
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254 slight immunological enhancement of fish fed normal diet with continuous supplement of berberine or
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255 four-week interval during 8-week feeding trial. Growth, innate immunity and antioxidant capability of
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256 fish fed high-fat diet supplemented berberine were improved within all three mode (two-week,

257 four-week intervals or continuous). The present result showed long-term feeding of berberine could

258 increase oxidative stress and interval feeding of berberine might enhance the antioxidant capability of

259 juvenile blunt snout bream. It was supported by the fact that antioxidant enzymes (SOD and CAT)

260 were capable of scavenging reactive oxygen species and products of lipid peroxidation (MDA and
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261 LPO), thereby protecting cells from oxidative damage[34]. It was accord with the results in plasma

262 immune and anti-oxidative parameters. Due to improvement of immune and anti-oxidative abilities,

263 cumulative mortality of fish challenged by Aeromonas hydrophila were decreased at interval

264 application of berberine modes, but slight increased at continuous feeding mode. Related studies also

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265 proved that interval feed immunostimulants such as FOS and emodin could increase the antioxidant

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266 status of fish[26, 35, 36]. It hinted that berberine as a functional feed additive would be useful in

267 different feeding modes under different nutritional conditions in present study.

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268 The similar phenomenon was also observed by Matsuo who reported that long-term oral

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269 administration of peptide-glucans decreased the immune response of rainbow trout challenged by
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270 Vibrio anguillarum [46]. Other researches on feeding strategies of aqua-immunostimulants, such as

271 β-glucan and fructooligosaccharide had been reported [25, 26]. Bai et al [25]reported that shrimps fed
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272 with dietary β-glucan 2 days followed by the basal diet for 5 days showed the highest specific growth
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273 rate. Over the long-term period, no significant differences were observed in innate and specific immune
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274 parameters, survival, growth performances and conversion index in treated and control fish of fed diet

275 supplemented with Ergosan (0.5%) or Macrogard (0.1%)[37]. Long-term feeding of peptide-glucans
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276 decreased the immune response of catfish (Clarias gariepinus) [38]. Similarly, long-term dietary
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277 administration of glucans (35 d) did not influence the complement activity and lysozyme in turbot [39].
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278 Same results were showed by Chang et al [24]and Sun et al [40]. Due to quite limited relevant studies,

279 the mechanism underlying this process was still unknown. It was speculated oral application of

280 functional feed additive continuously or less all could reduce its efficacy. The negative effect might be

281 due to immunosuppression or immunity fatigue of fish. The effect of herb by oral delivery should be
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282 further investigated in relation to these aspects to evaluate its efficacy in different feeding mode or

283 strategy.

284 Additional, berberine could improve fish growth of fish fed high-fat diet and plasm lipid biochemical

285 parameters. The result was accord with the previous studies. High-fat diets led to excessive fat

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286 deposition, which may have a negative impact on fish such as poor growth, dysfunction of the

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287 mitochondria subsequently mediated oxidative stress, hepatocyte apoptosis and suppressive immune

288 [22, 41-45]. Berberine could attenuate oxidative stress and hepatocytes apoptosis in blunt snout bream

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289 fed high-fat diets. It might attribute to enhance the immunity of fish.

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290 Consistent with those results, an optimal feeding mode of berberine not only could contribute to the
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291 health of blunt snout bream and help to enhance disease resistance, but could decrease feeding cost.

292 Blunt snout bream fed normal diet with 50 mg/kg berberine at two-week interval feeding mode or fed
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293 high-fat diet with berberine at two-week or four-week intervals feeding modes were optimal feeding
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294 mode, respectively. Over feeding did not benefit the antioxidant ability and immune defense. It needed
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295 further investigation whether continuous supplement of berberine results in immunosuppression or

296 immunity fatigue.

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297 Acknowledgements
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298 This work was funded by National Nature Science Foundation of China (31472292) in
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299 collaboration with the National Technology System for Conventional Freshwater Fish Industries of

300 China (CARS-46-20).

301

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Figure Captions
Figure 1. Hepatocyte apoptosis of blunt snout bream subjected to different treatment. Hepatocyte

apoptosis of blunt snout bream. (D1) Normal diet without berberine (×200), normal cells,

amethyst (blue arrow), apoptotic cells, brown (black arrow), (D2) Normal diet +50 mg/kg

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berberine at two-week intervals feeding mode (×200), (D3) Normal diet +50 mg/kg berberine at

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four-week intervals feeding mode (×200), (D4) Normal diet +50 mg/kg berberine at continuous

feeding mode (×200), (D5) High-fat diet without berberine (×200), (D6) High-fat diet +50 mg/kg

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berberine at two-week intervals feeding mode (×200), (D7) High-fat diet +50 mg/kg berberine at

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four-week intervals feeding mode (×200), (D8) High-fat diet +50 mg/kg berberine at continuous
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feeding mode (×200).
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Table 1 Formulation and proximate composition of experimental diets

Ingredients Normal diet (%) High-fat diet (%)

Fish meal 5.0 5.0
Soybean meal 24.0 24.0
Rapeseed meal 15.0 15.0
Cottonseed meal 13.0 13.0
Wheat bran 15.0 15.0

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Wheat meal 16.7 16.7
Cellulose 5.0 0.0
Soybean oil 3.1 8.1

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Calciumbiphosphate 1.8 1.8
a
Premix 1.0 1.0

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Salt 0.4 0.4
Proximate composition (%)
Moisture 12.4 11.5

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Crude protein 30.6 30.4
Crude Lipid 5.0 9.7
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Energy (MJ kg-1) 17.2 18.7
a Premix supplied the following minerals (g kg_1 of diet) and vitamins (IU or mg kg_1 of diet): CuSO4·5H2O, 2.0 g; FeSO4·7H2O, 25 g; ZnSO4·7H2O, 22 g;

MnSO4·4H2O, 7 g; Na2SeO3, 0.04 g; KI, 0.026 g; CoCl2·6H2O, 0.1 g; Vitamin A, 900000IU; Vitamin D, 200000IU; Vitamin E, 4500 mg; Vitamin K3, 220 mg;
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Vitamin B1, 320 mg; Vitamin B2, 1090 mg; Niacin, 2800 mg; Vitamin B5, 2000 mg; Vitamin B6, 500 mg; Vitamin B12, 1.6 mg; Vitamin C, 5000 mg;

Pantothenate, 1000 mg; Folic acid, 165 mg; Choline, 60000 mg.
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Table 2 Berberine-feeding mode for juvenile blunt snout bream during the feeding trial.
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Berberine (50mg/kg) Normal diet High-fat diet

No-supplement D1 D5
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Supplement at two-week intervals feeding mode D2 D6

Supplement at four-week intervals feeding mode D3 D7
Supplement at continuous feeding mode D4 D8
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Table 3 Growth performance parameters of blunt snout bream subjected to different treatments

Treatment WG (%) SGR (%) FCR VBR HIS (%)

a
D1 496.87±25.27 3.15±0.09 1.59±0.06 9.15±0.32 1.41±0.13
D2 593.53±22.67b 3.43±0.04 1.37±0.02 9.22±0.08 1.46±0.05
D3 531.60±30.71ab 3.16±0.02 1.54±0.01 9.06±0.36 1.41±0.08
ab
D4 535.37±30.75 3.23±0.15 1.54±0.16 8.59±0.27 1.36±0.07
a
10.36±0.42 1.24±0.07

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D5 468.60±19.36 3.20±0.07 1.45±0.07
D6 497.94±26.24a 3.19±0.08 1.48±0.11 10.57±0.24 1.34±0.06
ab
D7 511.72±45.09 3.21±0.13 1.53±0.07 10.41±0.25 1.29±0.07

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ab
D8 498.96±21.07 3.08±0.05 1.58±0.07 9.81±0.22 1.27±0.08
Values were means S.E.M of 4 replicates. Means in the same line with different superscripts were
significantly different (P < 0.05).

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Table 4 Body composition of blunt snout bream subjected to different treatments

Treatment Crude protein (%) Crude lipid (%) Energy(KJ) Ash (%) Moisture (%)
a
D1 16.50±0.17 8.52±0.23 25.20±0.37 10.64±0.14 71.20±0.17
ab
D2 16.58±0.13 8.47±0.12 25.37±0.49 10.76±0.06 71.22±0.16
ab
D3 16.40±0.19 8.21±0.11 25.07±0.52 10.71±0.30 71.88±0.16
b
D4 16.24±0.08 7.85±0.20 25.44±0.44 10.33±0.54 71.94±0.25
a
D5 16.26±0.16 10.77±0.22 26.09±0.66 9.31±0.28 69.40±0.26

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a
D6 16.14±0.20 11.08±0.19 26.77±0.68 9.23±0.06 68.91±0.23
a
D7 16.16±0.21 11.01±0.17 26.70±0.60 9.71±0.58 68.98±0.46
a

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D8 16.47±0.27 10.16±0.64 26.75±0.50 8.35±0.60 69.19±0.15
Values were means S.E.M of 4 replicates. Means in the same line with different superscripts were
significantly different (P < 0.05).

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Table 5 Plasm biochemical parameters of blunt snout bream subjected to different treatments

Treatment TG(mmol/L) TC(mmol/L) HDL-C(mmol/L) LDL-C(mmol/L)

a a
D1 2.64±0.10 8.09±0.14 2.74±0.20 4.36±0.12
D2 2.22±0.10b 7.31±0.10b 3.07±0.13 4.79±0.15
D3 2.46±0.11ab 7.56±0.13ab 2.67±0.20 4.22±0.20
D4 2.09±0.13b 7.22±0.58b 2.53±0.22 4.35±0.48
D5 2.84±0.05a 9.46±0.36c 3.20±0.20 4.86±0.08

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D6 2.51±0.03a 7.44±0.30ab 2.79±0.25 4.66±0.13
D7 2.48±0.10a 8.02±0.23ab 2.75±0.15 4.74±0.05
D8 2.46±0.10a 7.95±0.27ab 2.53±0.27 4.55±0.13

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Values were means S.E.M of 4 replicates. Means in the same line with different superscripts were
significantly different (P < 0.05).

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Table 6 Plasm immunological parameters of blunt snout bream subjected to different treatments

ACP LYZ C3 C4
Treatment
(U/100ml) (µg/ml) (µg/ml) (µg/ml)
D1 80.59±5.19a 80.61±3.47a 329.74±11.97a 260.72±3.88a
D2 92.69±0.75b 93.41±1.95c 378.38±8.57b 343.16±5.94b
D3 87.79±4.47ab 83.08±2.08a 338.15±18.09ab 304.47±30.75ab
D4 80.06±3.20ac 83.25±2.17a 360.43±11.31ab 246.55±25.49a

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D5 72.82±1.64c 65.40±3.68b 286.28±6.01c 192.41±7.27c
D6 103.41±1.19b 80.29±6.39a 320.63±6.58a 302.99±20.96b
D7 106.58±3.46b 81.80±1.71a 311.11±5.34a 240.61±7.83a

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D8 111.58±4.95b 80.90±3.14a 319.14±9.38a 249.54±15.47ab
Values were means S.E.M of 4 replicates. Means in the same line with different superscripts were

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significantly different (P < 0.05).

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Table 7 Hepatopancreas anti-oxidative and oxidative biomarkers of blunt snout bream subjected to
different treatment

MDA LPO CAT SOD

Treatment
(nmol/mg) (nmol/mg) (U/mg protein) (U/mg protein)
D1 8.34±0.59a 3.85±0.43a 835.64±45.65ac 2583.18±177.12a
D2 6.42±0.37c 2.62±0.15b 939.55±31.81b 3395.98±254.84c
D3 7.67±0.57ac 3.31±0.40ab 987.18±74.81ab 3493.05±169.96c

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D4 7.35±0.42ac 3.56±0.26ab 1089.80±110.68b 2922.12±194.64b
D5 12.01±0.94b 3.86±0.32a 783.10±38.98c 2225.72±119.31b
D6 9.13±0.66a 2.82±0.13ab 1074.12±86.24b 3623.22±126.84c

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D7 9.49±0.46a 2.67±0.21b 1032.67±75.87b 3600.00±186.96c
D8 9.18±0.66a 3.55±0.39ab 1045.02±96.47b 3333.46±50.80c

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Values were means S.E.M of 4 replicates. Means in the same line with different superscripts were
significantly different (P < 0.05).

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Table 8 Hepatocyte apoptosis of blunt snout bream subjected to different treatment.

Treatment Apoptosis rate (%)

D1 8.44±0.65ab
D2 9.84±1.38a
D3 10.26±1.18a
D4 6.65±1.10b

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D5 23.37±1.61c
D6 7.22±1.00ab
D7 7.34±0.72ab

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D8 6.87±0.52b
Values were means S.E.M of 4 replicates. Means in the same line with different superscripts were
significantly different (P < 0.05).

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Table 9 The cumulative mortality of blunt snout bream subjected to different treatment after 96h
challenged by Aeromonas hydrophila

Treatment 24h 48h 72h 96h

D1 29.17±2.08a 51.17±2.08ac 79.17±2.08ab 79.17±2.08ab

D2 20.83±2.08a 35.42±2.08b 47.92±2.08c 47.92±2.08c
D3 35.42±2.08b 58.33±3.61ac 77.08±2.08ab 77.08±2.08ab

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D4 45.83±4.17b 52.08±2.08ac 74.58±2.08ab 74.58±2.08ab
D5 25.00±6.25a 58.33±4.16ac 83.33±4.17a 83.33±4.17a
D6 31.25±3.61ab 54.17±2.08ac 70.83±2.08b 70.83±2.08b

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D7 35.42±2.08ab 60.42±3.60a 72.91±2.08b 72.91±2.08b
D8 31.25±3.61ab 45.83±2.08c 77.08±2.08ab 77.08±2.08ab

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Values were means S.E.M of 4 replicates. Means in the same line with different superscripts were
significantly different (P < 0.05).

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Figures:

Figure1. Hepatocyte apoptosis of blunt snout bream subjected to different treatment

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Highlights
1. Different feeding modes of berberine significantly influenced growth, innate immunity
and antioxidant capability of fish.

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