Effects of dietary vitamin B1 on growth performance, intestinal digestion and absorption, intestinal microflora and immune response of juvenile golden pompano (Trachinotus ovatus)

Aquaculture 506 (2019) 75–83

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Effects of dietary vitamin B1 on growth performance, intestinal digestion and absorption, intestinal microflora and immune response of juvenile golden pompano (Trachinotus ovatus)

T

⁎

Pengwei Xuna,b, Heizhao Lina,c, Ruixuan Wanga, , Zhong Huanga,c, Chuanpeng Zhoua, Wei Yua,c, Qianqian Huanga,b, Lianjie Tana,b, Yun Wanga, Jun Wanga a Key Lab. of South China Sea Fishery Resources Exploitation &Utilization, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, People's Republic of China b College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201506, People's Republic of China c Shenzhen Base of South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shenzhen 518121, People's Republic of China

A R T I C LE I N FO

A B S T R A C T

Keywords: Golden pompano Vitamin B1 Growth performance Intestinal digestion and absorption Intestinal microflora Immune response

The study was conducted to investigate the effects of dietary vitamin B1 on growth performance, intestinal digestion and absorption, intestinal microflora, and the immune response of juvenile golden pompano. Six isonitrogenous and isolipidic diets were formulated, containing diet-1 (0 mg/kg), diet-2 (4.88 mg/kg), diet-3 (8.15 mg/kg), diet-4 (12.00 mg/kg), diet-5 (17.40 mg/kg) and diet-6 (20.40 mg/kg) of vitamin B1. The diets were fed to juvenile golden pompano (initial body weight:11.20 ± 0.15 g) for 8 weeks. The study indicated that dietary vitamin B1 significantly increased weight gain rate (WGR) (P < .05), specific growth rate (SGR) (P < .05), and feed efficiency ratio (FER) (P < .05) of golden pompano. Dietary vitamin B1 levels had a significant effect on serum total cholesterol (TC) (P < .05), glucose (GLU) (P < .05), C4 complement (C4) (P < .05) and lysozyme (LZM) (P < .05). A diet supplemented with 12.00 mg/kg vitamin B1 increased the activities of hepatic total antioxidant capacity catalase (CAT) (P < .05), superoxide dismutase (SOD) (P < .05), glutathione peroxidase (GPX) (P < .05), glutathione reductase (GR) (P < .05), transketolase (TK) (P < .05) and hepatic vitamin B1 accumulation (P < .05), decreasing the activities of hepatic malondialdehyde (MDA) (P < .05). A diet supplemented with 12.00 mg/kg vitamin B1 improved intestinal digestion and absorption by increasing the activities of intestinal chymotrypsin (P < .05), γ-glutamyl transferase (γ-GT) (P < .05), and creatine kinase (CK) (P < .05). Dietary vitamin B1 also increased the intestinal microvilli length and microvilli numbers to a certain degree. Dietary vitamin B1 increased the richness and the diversity of the microbial community (P < .05; Tukey test). Dietary vitamin B1 increased the number of Pseudomonas and restricted the number of Mycoplasma and E. shigella in intestine. A quadratic regression analysis on weight gain and hepatic vitamin B1 concentrations indicated that the optimum dietary vitamin B1 levels for the optimal growth of juvenile pompano were 12.94 and 12.61 mg/kg, respectively.

1. Introduction There are eight water soluble B-vitamins. It was once stated that water soluble vitamins were the most important medication needed for the basic health system, and they were listed among the World Health Organization essential medicines (Alizadeh and Reza, 2018). As a member of the B-vitamins, VitaminB1 (VB1), also known as thiamine,

performs important roles in animals. VB1 was discovered in 1926 (Carpenter, 2012). It occurs naturally in whole grains, meats, dairy products, nuts, and seeds (Kennedy, 2016). It can be readily dissolved in water and other polar solvents (Moshe and Rytwo, 2018). and becomes highly unstable when exposed to oxygen, light, temperature, and metal ions. Therefore, the activity of VB1 will be reduced when it is stored for a long time (Juveriya et al., 2016) VB1 is necessary for the

Abbreviations: VB1, VitaminB1; WG, weight gain rate; SGR, specific growth rate; FER, feed efficiency ratio; C C4, C4 complement; LZM, lysozyme; T-AOC, total antioxidant capacity; CAT, catalase; SOD, superoxide dismutase; GPX, glutathione peroxidase; GR, glutathione reductase; MDA, malondialdehyde; TP, total protein; TG, triglyceride; TC, total cholesterol; ACP, acid phosphatase ⁎ Corresponding author. E-mail address: [email protected] (R. Wang). https://doi.org/10.1016/j.aquaculture.2019.03.017 Received 3 January 2019; Received in revised form 9 March 2019; Accepted 9 March 2019 Available online 11 March 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.

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functioning of organisms (Dinicolantonio et al., 2018) and participates in energy metabolism as a cofactor for several enzymes (Lonsdale, 2006; Zastre et al., 2013; Lee et al., 2012; Witten and Aulrich, 2018; Maguire et al., 2018). After being absorbed in animals, it is converted to three phosphoester forms in various tissues and organs, including thiamine monophosphate (TMP), thiamine pyrophosphate (TPP), and thiamine triphosphate (Muroya et al., 2017; Lonsdale, 2015). Each form of VB1 is an important precursor that is involved in cellular energy metabolism (Jenčo et al., 2017). Thiamine, in particular thiamine pyrophosphate, is an essential cofactor for three key enzymes involved in intracellular glucose (GLU) metabolism: pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (α-KGDH), and transketolase (TK). TK has been shown to be the rate-limiting enzyme in the nonoxidative portion of the pentose phosphate pathway (Kochetov and Solovjeva, 2014). Most recent studies suggest that thiamine has a role in oxidative stress, protein processing, peroxisomal function, and gene expression (Gibson and Blass, 2007). VB1 deficiency could lead to decreased growth performance, decreased enzyme activity, and even death (Fattal-Valevski, 2011; Depeint et al., 2006). Reports in the literature indicate that thiamine deficiency in fish may cause decreased appetite, primarily impairing weight gain (El-Hindi and Amer, 1989). Thiamine deficient rainbow trout (Oncorhynchus mykiss) also have reduced TK activities (Masumoto et al., 1987). VB1 plays a critical role in GLU metabolism and its deficiency may result in the accumulation of anaerobic metabolites including lactate due to an imbalance between the caloric burden and the functioning of thiamine dependent enzymes (Maguire et al., 2018). VB1 is absorbed primarily in the proximal small intestine (Dudeja et al., 2001) and major sites of storage include striated muscle and liver (Gangolf et al., 2010). Diet and nutritional status are known to affect the immune response (Balfry and Higgs, 2001) and VB1 plays a role in the immune system. A number of studies using rats have also suggested that thiamin has antioxidative properties (Lukienko et al., 2000). VB1 is closely involved with hemin-dependent oxygenase, which affects the release of specific intercellular adhesion molecule (ICAM) proteins and in turn regulates disease resistance in fish (Dinicolantonio et al., 2018). The intestine can resist the invasion of microbial and intestinal microflora but is sensitive to a change in diet. It indicated that Lactobacillus counts increased gradually and highest population level of lactobacilli was obtained when the thiamine level was 0.79 mg/kg (Feng et al., 2011; Ringø et al., 2016). There has also been a study of the role of pantothenic acid, which can promote the growth of beneficial bacteria and inhibit the growth of harmful bacteria (Wen et al., 2010). The golden pompano (Trachinotus ovatus) belongs to the family Carangidae of the order Perciformes and is a warm-water fish species. In China, it is widely distributed in littoral areas, including the provinces of Guangdong, Fujian, and Hainan. Golden pompano has become an increasingly popular cultured marine fish species because of its fast growth, high flesh quality, wide salinity tolerance, and suitability for cage culture (Tan et al., 2016). The fish are fed formula feed primarily during the process of culture because formula feed can increase the nutrient value of diets and economic benefits. Golden pompano can acclimate formula feed well in aquaculture conditions because of their characteristics. Thus, it is important to understand the diet and nutritional status of golden pompano. In recent years, a few studies have been conducted regarding the VB1 requirement of fish. The purpose of this study was to quantify the VB1 requirement for golden pompano, based on data for growth performance, relational enzymatic activity, and intestinal microflora to provide a theoretical basis for feed preparation.

Table 1 Formulation and proximate analysis of the basal diet (on dry weight basis). Ingredients

Content (%)

Casein Soy protein concentrate Fishmeal Wheat flour Fish oil Soybean Lecithin Choline chloride Antioxidant Vitamin mixa, vitamin VB1-free Mineral mixb Monocalcium phosphate Micro-cellulose Attractant

20.0 18.0 16.0 23.0 8.0 4.0 2.0 1.0 2.0 1.0 1.0 3.0 1.0

Proximate composition (%) Moisture Crude protein Crude lipid Ash

8.20 42.71 12.31 6.67

a

Vitamin mix provided the following per kg of diet: VB2 45 mg, pantothenic acid 60 mg, VB12 0.1 mg, VK3 10 mg, inositol 800 mg, nicotinic acid 200 mg, folic acid 1.2 mg, biotin 32 mg, VD3 5 mg, VE 120 mg, VC 2.0 g, choline chloride 2.0 g, ethoxyquin 150 mg, avicel 14.52 mg; b mineral premix provided the following per kg of diet: NaF4 mg， KI 1.6 mg，CoCl2·6H2O (1%) 100 mg，CuSO4·5H2O 20 mg， FeSO4·H2O 160 mg，ZnSO4·H2O 100 mg，MnSO4·H2O 120 mg， MgSO4·7H2O 2.4 g，Ca(H2PO4)2·H2O 6.0 g，NaCl 200 mg，zelote power 30.90 g.

experimental diets were formulated according to different levels of VB1 (0, 4, 8, 12, 16 and 20 mg/kg diet). All ingredients were obtained from companies located in the People's Republic of China. Vitamin-free casein, soy protein concentrate, and fishmeal were used as dietary protein. Fish oil and soybean lecithin were used as a lipid source. The crude protein, crude lipid, and ash contents were 42.71%, 12.31%, and 6.67%, respectively. The formulation was considered to provide enough nutrition for the growth of golden pompano (Tan et al., 2016; Tan et al., 2018). The exact vitamin B1 content in the six diets was determined by a liquid chromatography method (GB/T 14700–2002). The value of the base diet (diet-1) was not detected because the content of VB1 in diet-1 was lower than the detection limit (1 mg/kg) of this method. In order to facilitate mapping, we use 0 instead of it. The VB1 values of diet-2, diet3, diet-4, diet-5 and diet-6 were 4.88, 8.15, 12.00, 17.40 and 20.40 mg/ kg, respectively. All the ingredients were crushed into powder, passed through a 60 mesh sieve, and thoroughly mixed with oil. Then, they were placed in a mincer and enough cold water was added to form a dough. The 2 and 2.5 mm diameter dough was wet-extruded by a pelletizer (F-26, South China University of Technology, Guangzhou, China) and air-dried in an air-conditioned room for two nights. All diets were placed into sealed bags and stored at −20 °C until use.

2.2. Experimental procedure Juvenile golden pompano were obtained from Shenzhen Long Qizhuang Industrial Development Co., Ltd. (China). Prior to the feeding trial, they were acclimated to laboratory conditions for 2 weeks in polythene cages and were fed commercial diets of pomfret (Guangdong Yuequn Marine Life Research and Development Co., Ltd.). They were fasted for 24 h at the beginning of the experiment. Juvenile gold pompanos were anesthetized with 100 mg/L eugenol (Shanghai Medical Instruments Co., Ltd., Shanghai, China). Twenty-five fish (initial body weight: 11.20 ± 0.15 g) were randomly stocked into 18 floating cages (1 × 1 × 1.5 m3; three cages per treatment). Each

2. Materials and methods 2.1. Experimental diets The formulations of the basic diet are given in Table 1. Six 76

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homogenizer in an ice bath. The homogenate was then centrifuged for 20 min at 3000 rpm/min. The supernatant was removed and was used to quantify hepatic TK, total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reductase (GR), and malondialdehyde (MDA) using an assay kit produced by Nanjing Jiancheng Bioengineering Institute and send to Guangdong Food Industry Institute (China) to quantify the hepatic VB1 concentration.

experimental diet was randomly assigned to cages in triplicate. The juvenile golden pompano were fed twice a day at 7:00 and 17:00 until apparent satiation on the basis of visual observation. The weight and number of dead fish and feeding quantity were recorded every day. Water temperature ranged from 28.0 to 32.8 °C, salinity from 14 to 18‰, and pH from 7.1 to 8.0. The dissolved oxygen content was > 6.0 mg/L. Ammonia nitrogen was lower than 0.05 mg/L. 2.3. Growth performance

2.7. Measurement of intestinal digestion and absorption Fish were fasted for 24 h before sampling and were anesthetized with 100 mg/L eugenol at the end of the feeding trial. The final weight and average body weight of all fish in each cage were determined. The following metrics were calculated:

Three fish were sampled randomly from each cage for obtaining three intestinal samples. The samples were weighed and placed in a centrifuge tube with sterilized physiological saline (0.86%, pH 7.4). Intestine samples were homogenized by a handheld homogenizer, in an ice bath. The homogenate was then centrifuged for 20 min at 3000 rpm and then the supernatant was removed. The supernatant was used to quantify intestinal chymotrypsin, γ-glutamyl transferase (γ-GT), amylase (AMS), creatine kinase (CK), and Na+ K+ ATPase using an assay kit produced by Nanjing Jiancheng Bioengineering Institute.

weight gain rate (WGR%) = [100 × (final body weight–initial body weight) /initial body weight];

specific growth rate (SGR%/day) = 100 × [Ln (final individual weight)–Ln (initial individual weight)]

2.8. Intestinal morphology and intestinal flora analysis

/number of days;

Three fish were sampled for obtaining three intestinal samples respectively from triplicate cages of gradient 1 (VB1 0 mg/kg), gradient 4 (VB1 12.00 mg/kg), gradient 6 (VB1 20.40 mg/kg). The intestines were dissected to obtain the foregut (1 cm after the stomach) (F-X1, F-X4 and F-X6), the midgut (M-X1, M-X4 and M-X6) and the hindgut (1 cm segment before the anus) (H-X1, H-X4 and H-X6) according to the method described by Tan et al. (2018) and three parts of the dissected intestine were fixed in 4% paraformaldehyde solution with 5 mL centrifuge tube, respectively. Intestinal samples fixed in 4% paraformaldehyde solution were sent to Google Biotechnology Ltd. to make segments. After fasted for 24 h, three fish were sampled for obtaining three intestinal samples respectively from triplicate cages of gradient 1, gradient 3 (VB1 8.15 mg/kg), gradient 4, gradient 6. Three replicates per treatment. The intestinal samples were sent to Guangzhou JiRui Gene Technology Co. Ltd. (China) for extraction of DNA and PCR amplification by Illumina MiSeq Sequencing platform. PCR was performed from V3~V4 variable regions of 16S rRNA to taxonomically identify the bacteria.

feed efficiency ratio (FER) = wet weight gain (g)/dry diet feed (g) condition factor (CFg/cm3) =100 × body weight (g)/[body length (cm)]3 hepatosomatic index (HSI%) = 100 × liver weight (g) /whole body weight (g) viscerosomatic index (VSI%) = 100 × viscera weight (g) /whole body weight (g).

survival rate (%) = 100 × (finial number of fish)/(initial number of fish). 2.4. Whole body composition At the end of the feeding trial, two fish were sampled randomly from each cage for the analysis of whole body composition. They were placed in an oven to measure moisture. Dried fish samples were ground into powder. Two gram of the powder was used for measuring ash in a muffle furnace (550 °C), 1 g was used for measuring crude lipids with a Soxhlet extraction method, and 0.2 g was used for measuring crude protein with a Kjeldahl method.

2.9. Data statistics and analysis Statistical analysis was performed using SPSS 21.0 (SPSS Inc., Michigan Avenue, Chicago, IL, USA) for Windows. The results were presented as the means ± SD (n = 3). Normality and homoscedasticity assumptions were confirmed prior to any statistical analysis. All evaluated variables were subjected to an analysis of variance (ANOVA) to determine whether VB1 levels significantly affected the observed response. Moreover, a follow-up trend analysis using orthogonal polynomial contrasts was performed to determine whether the significant effects were linear and/or quadratic. Quadratic regression model was performed by excel 2016 to estimate the optimum dietary VB1 levels based on weight gain rate and hepatic VB1 concentrations.

2.5. Serum biochemistry and immune response measurements Blood was collected using 2 mL heparinized syringes in a caudal venipuncture of five fish from each randomly sampled cage. Blood samples were collected into anticoagulation tubes. After collection, the blood samples were centrifuged (3000 rpm, 15 min, 4 °C) and the plasma was separated. The plasma samples were sent to Xinhai Hospital to quantify serum total protein (TP), triglyceride (TG), total cholesterol (TC), and GLU. The acid phosphatase (ACP), alkaline phosphatase (ALP), C4 complement (C4) and lysozyme (LZM) were quantified using an assay kit produced by Nanjing Jiancheng Bioengineering Institute (China).

3. Results 3.1. Effect of dietary VB1 levels on the growth performance of golden pompano

2.6. Hepatic antioxidative abilities and hepatic VB1 concentration measurements

The results shown in Table 2 demonstrated that the FBW, WGR and SGR of golden pompano fed VB1-supplemented diets were significantly higher than those fed the diet-1 group (P < .05). The highest FBW, WGR and SGR were recorded for the diet-4 group. FER of diet-2, diet-4, diet-5 and diet-6 was significantly higher than that of diet-1. There

Five fish were sampled randomly from each cage for obtaining five hepatic samples. The samples from each cage were weighed and placed in a centrifuge tube, to which sterilized physiological saline (0.86%, pH 7.4) was added. Hepatic samples were homogenized by a handheld 77

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Table 2 Effects of dietary vitamin B1 on growth performance in golden pompano (T. ovatus)§. Diets (VB1 mg/ kg)

IBW (g) FBW (g) WGR (%) SGR (%/d) FER CF (g/cm3) VSI (%) HSI (%) Survival (%)

Diet1

Diet2

Diet3

Diet4

Diet5

Diet6

ANOVA P

0

4.88

8.15

12.00

17.40

20.40

11.28 ± 0.01 39.63 ± 0.14a 251.36 ± 0.65a 2.24 ± 0.00a 0.48 ± 0.07a 3.31 ± 0.12 5.61 ± 0.03 0.91 ± 0.03 93.33 ± 0.06

11.14 ± 0.23 43.96 ± 0.56b 294.66 ± 13.36b 2.45 ± 0.06b 0.61 ± 0.06bc 3.29 ± 0.02 5.66 ± 0.09 0.89 ± 0.08 93.33 ± 0.06

11.25 ± 0.13 47.78 ± 0.38bc 324.86 ± 4.76bcd 2.58 ± 0.02bcd 0.59 ± 0.02ab 3.34 ± 0.02 5.73 ± 0.06 0.92 ± 0.08 89.33 ± 0.02

11.32 ± 0.12 50.17 ± 1.23c 343.39 ± 10.13d 2.66 ± 0.04d 0.75 ± 0.01d 3.28 ± 0.03 5.74 ± 0.18 0.93 ± 0.03 94.67 ± 0.06

11.07 ± 0.04 47.96 ± 1.94bc 333.36 ± 18.65cd 2.62 ± 0.08cd 0.63 ± 0.01bc 3.24 ± 0.12 5.57 ± 0.05 0.89 ± 0.06 88.00 ± 0.04

11.16 ± 0.16 45.32 ± 2.90b 303.27 ± 22.42bc 2.49 ± 0.10bc 0.72 ± 0.02cd 3. 22 ± 0.03 5.59 ± 0.13 0.87 ± 0.02 97.33 ± 0.02

Linear trend (P)

Quadratic trend (P)

.304 .000 .000 .000 .000 .413 .261 .748 .248

.284 .000 .000 .000 .000 .079 .442 .421 .695

.295 .000 .000 .000 .016 .775 .211 .742 .177

IBW: initial body weight; FBW: final body weight; WGR: weight gain rate; SGR: specific growth rate; FER: feed efficiency ratio; CF: condition factor; VSI: viscerosomatic index; HSI: hepatosomatic index. a,b,c,dMeans ( ± SD) values within the row unlike superscript letters were significantly different (P < .05). § Data represent the mean of triplicate groups. 380

y = -0.5498x2 + 14.224x + 246.87 R² = 0.9688 d

360

Weight gain rate/%

340

bcd

320

3.3. Effect of dietary VB1 levels on the serum biochemistry and immune response of golden pompano

cd bc

The effects of dietary VB1 levels on the serum biochemistry and immune response of golden pompano are shown in Table 4. The presence of VB1 in diets significantly decreased the TC (P < .05) and GLU (P < .05) of golden pompano. The TC and GLU levels in golden pompano fed the diet-4 were the lowest of all groups. There were no significant differences in the TG and TP of golden pompano. The C4 of golden pompano fed diet-3 was significantly higher than in fish fed on diet-1 (P < .05). The LZM activity of golden pompano fed diet-2 and diet-3 was significantly higher than in fish fed diet-1 (P < .05). There were no significant differences in the ACP and ALP of golden pompano.

b

300 280 260 a 240

x=12.94

220 200 0

5

10

15

Dietary vitamin B1 levels

20

25

mg/kg

Fig. 1. Relationship between the VB1 content in the diet and weight gain rate (QRA). Each point represents the mean ( ± SD) of three groups of fish (n = 3). Values with different superscript letters are significantly different (P < .05).

3.4. Effect of dietary VB1 levels on the hepatic antioxidative abilities and VB1 accumulation of golden pompano The effects of dietary VB1 levels on hepatic antioxidative abilities and VB1 accumulation of golden pompano are shown in Table 5. SOD and GPX levels in the liver of golden pompano fed diet-2, diet-3, and diet-4 were significantly higher than the group fed diet-1 (P < .05). Golden pompano fed diet-4 and diet-5 had significantly higher GR levels in their livers than the group fed diet-1 (P < .05). CAT in the liver of golden pompano fed diet-2 and diet-4 were significantly higher than the group fed diet-1 (P < .05). SOD, GPX, and GR in the liver of golden pompano first increased and then decreased as the VB1 content of the diets increased. There were no significant differences in T-AOC in all groups. The MDA of golden pompano fed VB1-spplement diets was significantly lower than the group fed on diet-1 (P < .05). TK activity in the liver of golden pompano fed diet-4 was significantly higher than in diet-1 group (P < .05). VB1 accumulation in the liver of golden pompano fed diet-3, diet-4, diet-5 and diet-6 were significantly higher than in diet-1 group (P < .05). Based on the hepatic VB1 concentrations, the optimal dietary VB1 requirement of golden pompano was estimated to be 12.61 mg/kg (Fig. 2).

were no significant differences in CF, VSI, HSI, and survival in all groups (P > .05). Based on the WGR, the optimal dietary VB1 requirement of golden pompano was estimated to be 12.94 mg/kg (Fig. 1).

3.2. Effect of dietary VB1 levels on the body composition of golden pompano The effects of dietary VB1 levels on the body composition of golden pompano are shown in Table 3. The crude protein content of whole fish fed diet-2, diet-3 and diet-4 was significantly higher than the diet-1 group (P < .05), with the highest value recorded in the diet-4 group. Fish fed diet-2, diet-3 and diet-4 had a significantly lower crude fat content than the diet-1 group (P < .05). There were no significant differences in moisture and ash in all groups.

Table 3 Carcass composition of golden pompano (T. ovatus) fed various levels of vitamin B1§. Diets (Vitamin B1 mg/ kg)

Moisture (%) Crude protein (%) Crude lipid (%) Ash (%) a,b,c §

Diet 1

Diet 2

Diet 3

Diet 4

Diet 5

Diet 6

0

4.88

8.15

12.00

17.40

20.40

69.43 56.19 30.46 14.36

± ± ± ±

2.00 0.44a 0.39a 0.82

69.77 60.02 25.98 14.35

± ± ± ±

2.10 0.65b 0.61bc 0.56

69.34 60.38 23.75 14.69

± ± ± ±

3.50 1.19b 1.40c 0.21

69.40 60.48 23.92 13.61

± ± ± ±

2.00 1.68b 2.51c 0.77

69.33 58.23 26.85 13.71

± ± ± ±

3.30 0.61ab 0.71abc 0.49

Means values within the row unlike superscript letters were significantly different (P < .05). Data represent the mean ( ± SD) of triplicate groups. 78

69.48 58.93 27.60 14.36

± ± ± ±

1.20 1.09ab 1.21ab 0.04

ANOVA P

Linear trend (P)

Quadratic trend (P)

1.000 .002 .000 .206

.934 .119 .099 .289

1.000 .000 .000 .446

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Table 4 Effects of dietary vitamin B1 on serum biochemical index in golden pompano (T. ovatus)§. Diets (Vitamin B1 mg/kg)

TG (mmol/L) TC (mmol/L) TP (g/L) GLU (mmol/L) ACP (U/L) ALP (U/L) C4(μg/ml) LZM (U/ml)

Diet 1

Diet 2

Diet 3

Diet 4

Diet 5

Diet 6

0

4.88

8.15

12.00

17.40

20.40

0.80 ± 0.09 4.24 ± 0.35a 24.73 ± 4.59 10.87 ± 0.49ab 5.25 ± 0.00 38.67 ± 6.66 37.40 ± 1.20a 21.14 ± 0.51a

0.76 ± 0.04 3.81 ± 0.06ab 23.50 ± 0.80 10.55 ± 0.54abc 5.38 ± 0.24 36.33 ± 4.04 44.50 ± 0.00ab 22.72 ± 0.55bc

0.85 ± 0.09 4.18 ± 0.21ab 25.13 ± 1.67 10.94 ± 0.00a 5.35 ± 0.05 38.00 ± 3.46 45.90 ± 7.10b 22.77 ± 0.67c

0.73 ± 0.03 3.62 ± 0.04b 23.23 ± 1.97 8.90 ± 1.02c 5.38 ± 0.14 33.67 ± 2.89 43.5 ± 0.90ab 22.33 ± 0.39abc

0.75 ± 0.08 3.82 ± 0.10ab 22.53 ± 0.64 9.23 ± 0.30bc 5.31 ± 0.05 32.67 ± 4.04 44.65 ± 0.25ab 21.12 ± 0.00a

0.67 ± 0.26 3.79 ± 0.34ab 23.17 ± 3.16 9.48 ± 0.82abc 5.24 ± 0.01 35.33 ± 5.03 39.25 ± 1.85ab 21.50 ± 0.20ab

ANOVA P

Linear trend (P)

Quadratic trend (P)

.638 .032 .794 .004 .551 .559 .029 .001

.224 .024 .324 .001 .660 .169 .630 .140

.521 .279 1.000 .443 .088 .560 .002 .001

TG, triglyceride; TC, total cholesterol; TP, total protein; GLU, glucose; ACP, acid phosphatase; ALP, alkaline phosphatase; C4, C4 complement; LZM, lysozyme. a,b,c Means values within the row unlike superscript letters were significantly different (P < .05). § Data represent the mean ( ± SD) of triplicate groups.

3.5. Effect of dietary VB1 levels on intestinal enzymatic activity, intestinal microflora, and intestinal morphology of golden pompano

mg/kg

4.5

c

3.5

Hepatic VB1 concentrations

The effects of dietary VB1 levels on the intestinal enzymatic activity of golden pompano are shown in Table 6. Chymotrypsin activity in the intestine of golden pompano fed diet-4 was significantly higher than that of fish fed diet-1 (P < .05). Fish fed diet-3 and diet-4 had a significantly higher CK activity in their intestines than that of fish fed diet1 (P < .05). The supplement of VB1 in diets significantly increased γGT activity in their intestines (P < .05). There were no significant differences in AMS and Na+ K+ ATPase in the intestines of fish among all groups. In this study, a total of 675,475 valid sequences and 43 Operational Taxonomic Units (OTUs) were obtained at the 97% similarity level from the 12 samples. ACE and Shannon index can evaluate the richness and diversity of the microbial community in the intestinal content, respectively. The completeness of sampling can be estimated with coverage profiles. The effects of dietary VB1 levels on the intestinal microflora of golden pompano are shown in Table 7. The coverage was 100% in all samples, which suggested almost all bacterial phylotypes present in the samples were identified. The richness estimated by the ACE indices and the diversity evaluated by Shannon indices of the microbial community were significantly different in diet-1, diet-3, diet-4 and diet-6 (P < .05; Tukey test). And the OTUs, ACE and Shannon in the intestine was the highest in diet-4, compared with other groups. The composition of intestinal microflora at the phylum level included mainly Proteobacteria, Firmicutes, and Tenericutes. They accounted for 99% of all microflora (Fig. 3). The composition of intestinal microflora at the genus level mainly included Exiguobacterium, Citrobacter, Acinetobacter, Pseudomonas, Mycoplasma, and Escherichia-Shigella. These species accounted for 94% of all microflora (Fig. 4). The effect of dietary VB1 levels on

c

c

4

b

3

ab

2.5

a

2 1.5 1

y = -0.0129x2 + 0.3254x + 1.7045 R² = 0.857

0.5

x=12.61

0 0

5

10

15

Dietary vitamin B1 levels

20

25

mg/kg

Fig. 2. Relationship between the VB1 content in the diet and the hepatic VB1 concentrations (QRA). Each point represents the mean ( ± SD) of three groups of fish (n = 3). Values with different superscript letters are significantly different (P < .05).

intestinal morphology of golden pompano was shown in Fig. 5. It was shown that the microvilli length and microvilli numbers of the foregut in golden pompanos fed diet-4 were higher than those in the diet-1 and diet-6 groups. 4. Discussion Growth performance can directly reflect the VB1 requirement of fish. An improvement in growth performance may be related to an improvement in the FER (Luo et al., 2014). The result of this study demonstrated that dietary VB1 levels had a significant positive effect on WGR and FER. The WGR and FER of golden pompano reached a

Table 5 Effects of dietary vitamin B1 on hepatic antioxidative abilities and hepatic VB1 accumulation in golden pompano (T. ovatus)§. Diets (Vitamin B1 mg/kg)

SOD activity (U/mg) CAT activity (U/mg) MDA (nmol/mg) GPX activity (U/g) GR activity (U/g) T-AOC (U/g) TK (U/g) VB1 accumulation (mg/ kg)

Diet 1

Diet 2

Diet 3

Diet 4

Diet 5

Diet 6

0

4.88

8.15

12.00

17.40

20.40

3.89 2.24 2.41 1.88 4.87 1.13 2.16 1.91

± ± ± ± ± ± ± ±

0.14a 0.08a 0.19a 0.28ab 0.19a 0.06 0.11a 0.02a

4.85 3.22 1.78 3.01 5.18 1.24 2.42 2.42

± ± ± ± ± ± ± ±

0.50c 0.00bc 0.10b 0.01c 0.01ab 0.02 0.25a 0.00ab

4.88 2.33 1.59 2.33 5.31 1.37 2.65 3.73

± ± ± ± ± ± ± ±

0.27c 0.25a 0.05b 0.00b 0.59ab 0.35 0.19ab 0.18c

4.70 3.81 1.54 2.27 7.52 1.45 3.16 3.81

± ± ± ± ± ± ± ±

0.27bc 0.50c 0.29b 0.33b 1.33c 0.09 0.19b 0.38c

4.16 2.58 1.48 1.67 6.75 1.37 2.69 3.65

± ± ± ± ± ± ± ±

0.00ab 0.25ab 0.37b 0.15a 0.71bc 0.04c 0.45ab 0.36c

4.28 2.42 1.56 1.56 6.48 1.22 2.44 2.78

± ± ± ± ± ± ± ±

0.00abc 0.12a 0.21b 0.00a 0.27abc 0.00 0.18a 0.10b

ANOVA P

Linear trend (P)

Quadratic trend (P)

.001 .000 .003 .000 .002 .166 .007 .000

.793 .750 .000 .000 .001 .222 .042 .000

.000 .000 .006 .000 .093 .019 .002 .000

SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde; GPX, glutathione peroxidase; GR, glutathione reductase; T-AOC, total antioxidant capacity; TK, transketolase. a,b,c,dMeans values within the row unlike superscript letters were significantly different (P < .05). § Data represent the mean ( ± SD) of triplicate groups. 79

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Table 6 Effects of dietary vitamin B1 on intestinal digestion and absorption abilities in golden pompano (T. ovatus)§. Diets (Vitamin B1 mg/ kg)

Diet 1

Diet 2

0 Chymotrypsin (U/mg) AMS (U/mg) γ-GT (nmol/mg) CK (U/g) Na+,K+ATPase (U/g)

Diet 3

4.88 a

1.92 ± 0.35 105.98 ± 1.86 93.47 ± 3.50a 0.23 ± 0.01a 0.36 ± 0.02

Diet 4

8.15 ab

2.76 ± 0.78 106.52 ± 12.12 145.30 ± 18.76b 0.30 ± 0.04ab 0.42 ± 0.06

12.00 ab

3.24 ± 0.49 123.01 ± 23.97 160.93 ± 9.99b 0.35 ± 0.02b 0.44 ± 0.10

AMS, amylase; γ-GT, γ-glutamyl transferase; CK, creatine kinase; § Data represent the mean ( ± SD) of triplicate groups.

Diet 5

a,b

17.40 b

3.74 ± 0.23 124.54 ± 9.61 166.96 ± 7.86b 0.35 ± 0.00b 0.44 ± 0.03

Diet Diet Diet Diet Diet Diet Diet Diet Diet Diet Diet Diet

Diet 3(8.15)

Diet 4(12.00)

Diet 6(20.40)

1a 1b 1c 3a 3b 3c 4a 4b 4c 6a 6b 6c

Linear trend (P)

Quadratic trend (P)

.039 .348 .000 .019 .072

.338 .470 .000 .955 .279

.017 .072 .000 .002 .052

20.40 ab

2.16 ± 1.17 114.49 ± 4.39 166.88 ± 3.05b 0.25 ± 0.06ab 0.57 ± 0.13

2.80 ± 0.04ab 109.98 ± 10.75 148.64 ± 16.71b 0.25 ± 0.09ab 0.36 ± 0.08

100%

OTUsa

ACEb

Shannon

Coverage

15 18 16 14 19 17 26 20 23 21 23 22

15.000 18.333 16.666 15.111 19.504 17.897 26.446 20.516 23.481 21.000 23.755 22.378

2.005 2.132 2.258 2.102 2.222 2.162 2.358 2.378 2.389 2.381 2.176 2.185

100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%

Exiguobacterium Citrobacter Acinetobacter Pseudomonas Mycoplasma Escherichia-Shigella Unclassified Bacillus Photobacterium Ochrobactrum Chryseobacterium Lactococcus Enterovibrio Sphingomonas Turicibacter Thermus Merismopedia Alkanindiges Vibrio Bradyrhizobium Stenotrophomonas Ruegeria Bacteroides Hydrogenophaga Ruminococcaceae_UCG-005

90%

80%

70%

Relative abundance

Diet 1(0)

ANOVA P

Means values within the row unlike superscript letters were significantly different (P < .05).

Table 7 OTU number, Alpha diversity and estimated sample coverage of intestinal microflora for 16S rRNA libraries in golden pompano (T. ovatus). Diets (Vitamin B1 mg/kg)

Diet 6

60%

50%

40%

Diet 1a-1c are three replicates of Diet 1. Diet 3a-3c are three replicates of Diet 3. Diet 4a-4c are three replicates of Diet 4. Diet 6a-6c are three replicates of Diet 6. a The operational taxonomic units (OTU) were defined at the 97% similarity level. b The richness estimators ACE, diversity indices Shannon and coverage percentage (coverage) were generated with Qiime program.

30%

20%

10% 100%

7.77

3.30

Diet 1 6.74

Relative abundance

80% 35.48

35.99

34.98

36.12

Diet 6

Proteobacteria

12.00 mg/kg VB1. Interestingly, the changing trend of TK activity was consistent with the accumulation of hepatic VB1. TK is a sensitive indicator of thiamin status and is involved in intracellular GLU metabolism, which plays an important role in the energy production of organisms. This suggests that moderate amounts of VB1 in diets can improve TK activity, enabling the liver to develop and absorb VB1 more effectively. Thiamine deficiency would compromise the enzyme and result in an altered metabolism of GLU. In the Gifu tilapia strain, the activity of TK in the liver significantly increased when VB1 in the diet was increased from 0.57 to 1.13 mg/kg (Ren et al., 2015). In rats, TK activity, with the exception of the brain, has been shown to decrease due to thiamine deficiency (Brin, 1962). It has also been reported that excess VB1 does not promote the TK activity and accumulation in the liver (Ren et al., 2015; Huang et al., 2007). The intestine plays an important role in nutrient digestion and absorption in fish. The intestinal absorption capacity provides the basis for fish to make full use of nutrients. Absorption is related to γ-GT activity, which is a key enzyme in the glutamate cycle. The enzyme is involved in peptide transport and provides raw materials for protein synthesis (Huang, 2009). Digestion is correlated with the activity of digestive enzymes, including chymotrypsin and α-amylase, because nutrients are digested by these enzymes (Lin and Xiao, 2006). CK catalyzes the reversible transfer of the high energy phosphate bond between creatine and adenosine triphosphate, and provides an energy source for muscle contraction and other life activities (Chen, 2016). In the present study, VB1 improved the activity of γ-GT, chymotrypsin, α-

Firmicutes

60% Tenericutes

50%

Cyanobacteria

40% 30%

Diet 4

Fig. 4. Bacterial composition of the different communities (% of relative read abundance of bacterial genus within each community).

90%

70%

Diet 3

4.24

Bacteroidetes

56.64

60.56

57.74

59.45

20%

Deinococcus-Thermus Unclassified

10% 0% Diet 1

Diet 3

Diet 4

Diet 6

Fig. 3. Bacterial composition of the different communities (% of relative read abundance of bacterial phyla within each community).

maximum at the same time when fish were fed a 12.00 mg/kg VB1 diet and they have a similar trend. The results of an investigation of Juvenile Jian carp (Huang et al., 2015) supported our study. It inferred that moderate amounts of VB1 in the diet can improve growth performance through an improvement in the FER. VB1 accumulation in tissues is often used as an index to evaluate the quantity required in aquatic animals (USA CONR, 2011). In the present study, compared to the diet-1, a VB1-supplemented diet significantly increased hepatic VB1 accumulation, with the accumulation of VB1 reaching a maximum when fish were fed the 12.00 mg/kg VB1 diet. It then decreased gradually when the VB1 concentration exceeded 80

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Fig. 5. Light microscopy images of the foregut, midgut, and hindgut morphology of golden pompano fed on diet-1 (F-X1, M-X1, and H-X1), diet-4 (F-X4, M-X4, and H-X4), and diet-6 (F-X6, M-X6, and H-X6) for 8 weeks. ml: microvilli length (mm); mn: microvilli numbers.

are regarded as pathogenic bacteria (Chaudhry et al., 2016; Bürki et al., 2016). In the present study, the proportion of Mycoplasma decreased gradually and then increased when the supplementation of VB1 was increased. The proportion of Mycoplasma in supplemented-VB1 diets was lower than that in the diet-1. This suggests that a moderate supplement of VB1 can restrict the number of Mycoplasma and improve the composition of intestinal microbiota, but the restriction gradually diminished when the supplement of VB1 exceeded 12.00 mg/kg. In our study, the dominant Proteobacteria included Citrobacter, Acinetobacter, Pseudomonas, and E. shigella. Pseudomonas were also shown to be the dominant bacteria in the intestine of juvenile flounder (Tanasomwang and Muroga, 1989). Pseudomonas have been regarded as candidate probiotics in grass carp and are an important biological control agent in aquaculture (Wu et al., 2012). Probiotics are widely used in aquaculture. They can help the host digest food, increase the nutritional value of food, prevent the colonization of pathogenic bacteria in the intestinal tract of the host, prevent the occurrence of disease, and purify water (Lazado et al., 2010). In the present study, the proportion of Pseudomonas initially increased and then decreased with the increasing VB1 content of the feed. However, some Pseudomonas are regarded as pathogenic bacteria. For example, P. putida can cause gill-rot disease in Anguilla anguilla (Fan, 2001). Further studies of the effects of Pseudomonas on the golden pompano intestine are required. Escherichia shigella is a Gram-negative facultative intracellular pathogen. It is the main pathogenic bacteria in the intestinal tract of the rhesus monkey (Hou and Chen, 1980). In the present study, the proportion of E. shigella among the total bacteria first increased, then decreased, and finally increased again with the increasing VB1 content of the feed. It is likely that at the beginning of the experiment, the VB1 supplement had less effect on E. shigella than the antagonism between Proteobacteria and Tenericutes. The effect of the VB1 supplement in restricting E. shigella was then gradually enhanced, but the greater antagonism between Proteobacteria and Tenericutes eventually caused the proportion of E. shigella among total bacteria to decrease. This restriction gradually diminished with the increasing amount of VB1 in the diet and the proportion of E. shigella among total bacteria increased. There has been only one published study of the effects of VB1 on the intestinal flora of fish, which demonstrated that VB1 can significantly increase the reproduction of Lactobacillus in the intestine of juvenile Jian carp (Huang, 2009). Intestine is a composite microbial ecosystem housing and it plays important roles in the nutrition and health of the host (Wang

amylase, and CK in the intestine as well as the crude protein content of golden pompano. This suggests that VB1 may enhance the digestion and absorption and promote protein synthesis in golden pompano by increasing the activities of γ-GT, chymotrypsin, α-amylase, and CK in the intestine, which may improve growth performance. There is only one report of the effects of thiamine on intestinal enzyme activity in fish. It was reported that VB1 can significantly increase intestinal chymotrypsin and amylase activity and the γ-GT activity of juvenile Jian carp (Huang, 2009) which is similar to the results of our study. A complete intestinal morphological structure is fundamental for maintaining normal intestinal function (Gao et al., 2013). In the intestine, shorter villi and deeper crypts are associated with the presence of toxins. Intestinal absorption capacity can be determined by fold height, and the increase of fold height and villus height can increase the surface area for nutrient absorption (Torrecillas et al., 2015). In the present study, VB1 increased the microvilli length and microvilli numbers of the foregut when fish were fed a 12.00 mg/kg (F-X4) VB1 diet (Fig. 5). This suggests that dietary VB1 promotes the early development of golden pompano intestines and enhances the absorption of nutrients in the gut. However, there was no difference in the microvilli length and numbers of the midgut and hindgut when fish were fed different diets. This may be because VB1 is absorbed primarily in the small intestine (Gangolf et al., 2010) and therefore when fish were fed a moderate VB1 diet the foregut was better developed than the midgut and hindgut. It is important to investigate the intestinal microbiota in farmed fish for health management in aquaculture. In the present study, compared to diet-1, the VB1 supplement of diet-4 increased the diversity and abundance of intestinal microflora. The best growth performance was obtained for diet-4. In terms of community composition, we found that the dominant gut bacteria of golden pompano belonged to three phyla: Proteobacteria, Firmicutes, and Tenericutes, which has also been reported for the turbot (Xing, 2013). Interestingly, the proportion of Firmicutes stabilized around 35%, with a contrasting trend for Proteobacteria and Tenericutes. This suggested that there may be a competitive relationship between Proteobacteria and Tenericutes. At the genus level, we found Mycoplasma was the dominant microbiota, which belong to the phylum Tenericutes. The Mycoplasma are the smallest self-replicating organisms with the smallest genomes (Dandekar et al., 2002). They have been previously detected in the intestine of the long-jawed mudsucker (Gillichthys mirabilis) (Bano et al., 2010). In many studies, the Mycoplasma

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reported that excess supplement in diets may consume more energy in metabolism, probably leading to toxic effects in the fish, with negative effects on growth (Tan et al., 2017). In the present study, enzyme activity was gradually decreased when the VB1 concentration in the diets exceed 12.00 mg/kg. This indicated the excess VB1 would have a negative effect on golden pompano to a certain degree. Golden pompano fed a VB1-deficient diet displayed poor growth, but had no obvious pathological symptoms. This result may have occurred because the feeding time was too short to exhaust the existing VB1 within golden pompano fed on diet-1. Channel catfish fed diet-1 for 20 weeks displayed VB1 deficiency symptoms (Murai and Andrews, 1978), which supported the results observed in our study.

et al., 2018). Intestinal microbiota can maintain normal gut function and it contains many enzymes, which can help the host to decompose the organic matter in food and enhance the digestion and absorption of nutrients (Ley et al., 2008; Li et al., 2015). In the present study, the underlying mechanism of Mycoplasma, Pseudomonas and Escherichia shigella is unknown, and further investigation is therefore required in next study. It has been reported that an improvement in disease resistance is positively related to the enhanced immune response of fish generated by nutrient (Feng et al., 2011). The health condition and nutritional metabolism of fish can be reflected by serum biochemistry parameters (Lin et al., 2015). In the present study, VB1 significantly decreased TC and GLU in the plasma of golden pompano fed the 12.00 mg/kg VB1 diet. Furthermore, the body fat of golden pompano significantly decreased when they were fed 12.00 mg/kg VB1 diets. This indicated that an appropriate VB1-supplement diet is likely to make blood and body fat decline and enhance disease resistance. Complement has an essential role in immune complex clearance, killing microbes, and antibody production (Holland and Lambris, 2002). Lysozymes are non-specific innate immunity molecules that play an important role in preventing the incursion of detrimental bacteria (Luo et al., 2007). In the present study, compared to the diet-1 group, the plasma C4 and LZM of golden pompano fed diet-3 were significantly increased, which agreed with previous results for juvenile Jian carp (Huang, 2009). These results suggest that VB1 supplements could improve the immune mechanisms of fish. Nutritional factors have an important effect on antioxidant defenses in fish and the antioxidant defenses and oxidative status of fish are influenced by dietary levels of vitamins (Martínez-Álvarez et al., 2005). Oxidative stress can produce free radicals (Slater, 1984) which could lead to liver disease if they are produced in the liver. In general, fish antioxidant systems consist of nonenzymatic compounds, glutathione (GSH), and antioxidant enzymes, including SOD, CAT, GR, and GPX. They play an important role in eliminating reactive oxygen species (ROS) as endogenous free radical scavengers (Chen et al., 2013; Wang et al., 2015). GSH is the major endogenous antioxidant scavenger that protects cells from oxidative stress (Sies, 1999). SOD can convert the highly reactive superoxide radical to H2O2, which reduces the concentration of ROS. CAT and GPX decompose H2O2 and protect tissues from highly reactive hydroxyl radicals (Williams and Burk, 1990). MDA indirectly reflects lipid peroxidation and the severity of free radical attacks on fish body cells. In the present study, VB1 can significantly increase the activity of SOD, CAT, GPX and GR and decrease the activity of MDA in the liver. These results suggest that VB1 may help liver tissue develop perfectly by increasing SOD, CAT, GPX and GR activity and decreasing MDA activity, so increasing the antioxidant ability of golden pompano. There were similar reports in the grouper Epinephelus coioides (Huang et al., 2007) and young grass carp (Wen et al., 2015). A recent study in rainbow trout (Oncorhynchus mykiss) found that antioxidant and antioxidant vitamins supplemented in diets controlled H2O2 production by immune cells (Thawonsuwan et al., 2010). A study in rats showed that thiamin supplementation prevented lipid peroxidation, glutathione depletion, and glyoxal-induced ROS formation, and caused a decrease in mitochondrial membrane potential in rat hepatocytes (Shangari et al., 2007). It has been reported that VB1 can directly interact with free radicals and hydroperoxides and continuously transmit (2H+ +2e−) on the pyrimidine ring NH2 group to free radicals; thus, eliminating the free radicals (Lukienko et al., 2000). The 12.00 mg/kg VB1 diet represented a boundary for the transition of enzyme activity in our study. When the VB1 supplement reached 12.00 mg/kg, the MDA activity reached a minimum, and the activity of almost all enzymes reached a maximum or second maximum in the liver or intestine. Therefore, the digestion, absorption, and the immune response of golden pompano were optimal when they were fed a 12.00 mg/kg VB1 supplement in their diet. However, there was no benefit for fish when the VB1 concentration was higher. A recent study

5. Conclusions The results of this study showed that diets supplemented with VB1 significantly promoted the growth performance and FER of golden pompano and also improved the intestinal digestion and absorption, intestine morphology, and composition of intestinal microbiota. Additionally, VB1 supplementation significantly increased the immune response and antioxidative ability of golden pompano. A quadratic regression analysis of weight gain rate and hepatic VB1 concentrations indicated that the optimum dietary VB1 levels for the optimal growth of juvenile pompano were 12.94 and 12.61 mg/kg, respectively. Acknowledgements The authors thank the participants who gave their time to this trial. The study was supported by Central Public-interest Scientific Institution Basal Research Fund, South China Sea Fisheries Research Institute, CAFS (2017ZD01); Modern agricultural biotechnology industry promoting and support projects (Shenzhen strategic emerging industry developmental special funds (biotechnology industry)) (SWCYL20150330010013); Natural Science Foundation of Guangdong Province, China (2017A030313112); the National Natural Science Foundation of China (31602175); Central Public-interest Scientific Institution Basal Research Fund, CAFS (2018HY-ZD0506); P. X. and H. L. designed the study. P. X. carried out most of experimental work and under the direction of R. W. wrote the manuscript. C. Z., Z. H., W. Y., Y. W., and J. W. assisted in the experimental design and manuscript revision; R. W. and H. L. approved the final version of the manuscript. P. X., Q. H., and L. T. carried out the rearing experiments and determination of sample. All authors have contributed to, seen and approved the final, submitted version of the manuscript. The authors declare that there are no conflicts of interest. References Alizadeh, T., Reza, M.A.M., 2018. An innovative method for synthesis of imprinted polymer nanomaterial holding thiamine (vitamin B1) selective sites and its application for thiamine determination in food samples. J. Chromatogr. B 1084, 166–174. Balfry, S.K., Higgs, D.A., 2001. Influence of dietary lipid composition on the immune system and disease resistance of finfish. In: Lim, C., Webster, C.D. (Eds.), Nutrition and Fish Health. the Haworth press Inc., New York, pp. 213–234. Bano, N., Derae, S.A., Bennett, W., et al., 2010. Dominance of Mycoplasma in the guts of the long-jawed Mudsucker, Gillichthys mirabilis, from five California salt marshes. Environ. Microbiol. 9, 2636–2641. Brin, M., 1962. Effects of thiamine deficiency and of oxythiamine on rat tissue transketolase. J. Nutr. 78, 179–183. Bürki, S., Spergser, J., Bodmer, M., et al., 2016. A dominant lineage of Mycoplasma bovis is associated with an increased number of severe mastitis cases in cattle. Vet. Microbiol. 196, 63–66. Carpenter, K.J., 2012. The discovery of thiamin. Ann. Nutr. Metab. 61, 219–223. Chaudhry, R., Ghosh, A., Chandolia, A., 2016. Pathogenesis of Mycoplasma pneumoniae: an update. Indian J. Med. Microbiol. 34, 7. Chen, X., 2016. Cloning, Expression and Functional Analysis of Creatine Kinase CK and Hepcidin Genes in Songjiang Perch. Shandong University, MD thesis. Chen, S., Zou, L., Li, L., et al., 2013. The protective effect of glycyrrhetinic acid on carbon tetrachloride-induced chronic liver fibrosis in mice via upregulation of Nrf2. PLoS One 8, e53662. Dandekar, T., Snel, B., Schmidt, S., et al., 2002. In: Razin, S., Herrmann, R. (Eds.),

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