Effects of dietary ginkgo biloba leaf extract on growth performance, plasma biochemical parameters, fish composition, immune responses, liver histology, and immune and apoptosis-related genes expression of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) fed high lipid diets

Accepted Manuscript Effects of dietary ginkgo biloba leaf extract on growth performance, plasma biochemical parameters, fish composition, immune responses, liver histology, and immune and apoptosis-related genes expression of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) fed high lipid diets Xiaohong Tan, Zhenzhu Sun, Qingying Liu, Huaqun Ye, Cuiyun Zou, Chaoxia Ye, Anli Wang, Heizhao Lin

PII:

S1050-4648(17)30628-9

DOI:

10.1016/j.fsi.2017.10.022

Reference:

YFSIM 4893

To appear in:

Fish and Shellfish Immunology

Received Date: 12 July 2017 Revised Date:

5 October 2017

Accepted Date: 10 October 2017

Please cite this article as: Tan X, Sun Z, Liu Q, Ye H, Zou C, Ye C, Wang A, Lin H, Effects of dietary ginkgo biloba leaf extract on growth performance, plasma biochemical parameters, fish composition, immune responses, liver histology, and immune and apoptosis-related genes expression of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) fed high lipid diets, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.10.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Effects of dietary ginkgo biloba leaf extract on growth performance, plasma

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biochemical parameters, fish composition, immune responses, liver histology,

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and immune and apoptosis-related genes expression of hybrid grouper

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(Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) fed high lipid diets

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Xiaohong Tana1, Zhenzhu Suna1, Qingying Liua, Huaqun Yea, Cuiyun, Zoua,

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Chaoxia Yea∗∗, Anli Wanga∗∗, Heizhao Linb, c∗∗

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Key Laboratory of Ecology and Environmental Science in Guangdong Higher

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Education, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture,

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School of Life Science, South China Normal University, Guangzhou 510631, PR

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China

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b

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Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese

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Academy of Fishery Sciences, Guangzhou 510300, PR China

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c

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of Fishery Sciences, Shenzhen 518116, PR China

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Shenzhen Base of South China Sea Fisheries Research Institute, Chinese Academy

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Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization,

These authors contributed equally to this work Corresponding author:

E-mail addresses: [email protected] (C.-X. Ye); E-mail address: [email protected] (A. Wang); E-mail address: [email protected] (H. Lin). 1

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Abstract For thousands of years, leaves from the Ginkgo biloba tree have been a common

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treatment in Chinese medicine. The present study was conducted to investigate the

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effects of dietary ginkgo biloba leaf extract (GBE) supplementation on growth

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performance, plasma biochemical parameters, fish composition, immune responses,

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liver histology, and immune and apoptosis-related genes expression of hybrid grouper

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(Epinephelus lanceolatus♂× Epinephelus fuscoguttatus♀) fed high lipid diets. A

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basal diet supplemented with GBE at 0, 0.50, 1.00, 2.00, 4.00 and 10.00 g kg-1 was

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fed to hybrid grouper for 8 weeks. The study indicated that dietary GBE did not

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improve growth performance and feed utilization but it reduced intraperitoneal fat rate.

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There were no significant differences in condition factor, viscerosomatic index,

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hepatosomatic index, spleen index, relative gut length, food intake, protein deposit

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rate and survival among all groups (P > 0.05). Dietary supplementation with

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0.50-4.00 g GBE kg-1 diets effectively increased plasma HDL content and decreased

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plasma GLU, LDL and TG content in fish. Furthermore, dietary GBE had a

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significant effect on moisture, crude protein and lipid in the liver, and protein in the

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whole body of fish (P < 0.05). Dietary supplementation with 0.50-1.00 g GBE kg-1

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diets effectively decreased occurrence rates of the hepatocyte swelling, hepatocyte

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vacuolization, and nuclei shifting to the cellular periphery cytoplasmic vacuolization,

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meanwhile hepatic antioxidant enzymes (SOD, CAT and T-AOC) activities

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significantly increased whereas MDA content significantly decreased in fish fed diets

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supplemented with GBE (P < 0.05). Moreover, dietary GBE up-regulated the

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expression of antioxidant genes (CAT, GPx and GR), immune-related genes (MHC2

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and TLR3) and anti-inflammatory cytokines (IL-10 and TGF-β1), while dietary

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supplementation with 0.50-4.00 g GBE kg-1 diets down-regulated apoptosis-related

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genes (p53, caspase-9, caspase-8 and caspase-3) expression in the head kidney of

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hybrid grouper. These results indicated that hybrid grouper fed diets supplemented

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with GBE did not improve growth performance and feed utilization but it had

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hypolipidemic effects, improved hepatic antioxidant status, maintained normal liver

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histology and preserved liver function, increased immune-related genes expression

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and decreased apoptosis-related genes expression in the head kidney of hybrid

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grouper.

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Keywords: hybrid grouper; ginkgo biloba leaf extract; growth performance; body

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composition;

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apoptosis-related genes

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biochemical

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plasma

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liver

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1. Introduction Hybrid grouper (Epinephelus lanceolatus♂ × E. fuscoguttatus♀) is a new species

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of grouper that was first produced in 2007 by Universiti Malaysia Sabah [1]. The

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female parent of this hybrid grouper, Epinephelus fuscoguttaus, commonly known as

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a brown marbled grouper or tiger grouper, is a slow growing but long-lived species

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with high disease resistance [2]. The male parent of this hybrid grouper, Epinephelus

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lanceolatus or giant grouper with a common name of queensland grouper, is popular

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breeding species for its rapid growth, reaching up to 3 kg in the first year [2]. Hybrid

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grouper obtains good properties of parents which has rapid growth and high disease

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resistance [2]. Hybrid grouper is euryhaline fish (suitable salinity: 10 to 20 psu,

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temperature: 26°C to 30°C) [1, 3], it has been widely cultured in China's southeast

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coast for their fast growth, popular taste, and high nutritional and economic value [4,

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5]. Previous study reported that suitable dietary protein and lipid requirement for

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hybrid grouper was estimated to be 45-53.5%, 7-13% of dry diets, respectively [6-8].

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Recently, high-lipid diets have increasingly been used for cost-effective farming in

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aquaculture, which is based upon the notion that dietary protein can be reduced when

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more energy is supplied in the form of dietary lipids [9, 10]. However, high-lipid diets

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often lead to ectopic lipid accumulation in the liver and abdominal adipose tissue

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tissues of farmed fish, which could induce fatty liver and metabolic disturbances [9].

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immune system of fish was suppressed , which increased the susceptibility of fish to

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infectious diseases, such events routinely occurred in aquaculture and caused

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substantial economic losses [11]. The farming of groupers also encountered

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increasing difficulties, especially with a host of infectious diseases including different

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viral, bacterial, and parasitic pathogens [12]. Recently, due to frequent occurring of

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food safety problems, consumers pay more attention to the quality and safety of food,

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and fish that is not fed with antibiotics and chemicals is more popular and thus use of

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chemicals and antibiotics in disease management is discouraged [12, 13]. Besides,

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frequent use of antibiotics and chemicals to control these diseases caused many other

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problems such as the spread of drug resistant pathogens, suppression of aquatic

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animal’s immune system, and environmental pollution [14]. Therefore, the researchers

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have hastened the search for the identification and development of safe dietary

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additives that can improve the physiological activity, liver health, and immune

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function of the farmed fish [15].

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Plant extracts have been reported to possess various activities like anti-stress,

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growth promotion, appetite stimulation, hepatoprotective effect, enhancement

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immunostimulation and anti-pathogen properties in fish aquaculture due to containing

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many active compounds such as alkaloids, terpenoids, tannins, saponins, glycosides,

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flavonoids, phenolics, steroids or essential oils, further they are often locally available,

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inexpensive, act against a broad spectrum of pathogens, easily biodegradable, and 5

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alternatives to traditional antibiotics, chemotherapies and vaccines. Ginkgo biloba,

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commonly known as ginkgo or gingko, is the oldest living fossil tree on the earth,

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more than 200 million years old and the only living species in the division

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Ginkgophyta, all others being extinct [17-19]. Ginkgo biloba has been used to calm

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wheezing, stop pain, and treat hypercholesterolemia, hypertension, coronary artery

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disease, angina pectoris, and cerebrovascular disease in traditional Chinese medicine

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for many years [20, 21]. Ginkgo biloba extract (GBE) mainly contains ginkgo flavone

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glycoside, terpenlactones and other special active components [19, 21], which has

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several beneficial effects including antioxidant functions [22], anti-inflammatory [19,

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23], hepatoprotective effects[24-26] and immunity regulation [27, 28]. Previous

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studies have shown that dietary supplementation with ginkgo biloba extract could

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improve slaughter performance, immune organ index and immunity in broilers [29,

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30]. Huang et al. also has reported dietary supplementation with ginkgo biloba extract

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could improve growth performance and intestinal mucosal morphology, decrease

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intestinal barrier permeability, protect intestinal barrier and enhance immune function

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in weaned piglets [28, 31]. These findings suggest that GBE has great potential as

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feed additives in aquaculture.

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To date, no study using GBE as a supplement in diet in groupers has been

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reported. Here, we studied the effect of GBE on growth performance, plasma

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biochemical parameters, fish composition, immune responses and liver histology of 6

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hybrid grouper (Epinephelus lanceolatus♂ × E. fuscoguttatus♀) fed high lipid diets.

125 2. Materials and methods

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2.1. Diet preparation

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The composition of the basal diet is given in Table 1. The basal diet (Diet 1) was

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used as control diet, and 0.50 (Diet 2), 1.00 (Diet 3), 2.00 (Diet 4), 4.00 (Diet 5) and

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10.00 (Diet 6) g kg-1 ginkgo biloba leaf extract (GBE with 24% ginkgo flavone

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glycoside and 6% terpenlactones, Shaanxi Ciyuan biotechnology Co., Ltd., China)

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were separately supplemented to formulate the five experimental diets. All the

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ingredients were ground into powder, sieved through 60-mesh. All diets were

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individually blended in a mixer and then homogenized after mixed oil was added. The

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water was included to achieve a proper pelleting consistency, and the mixture was

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further homogenized. The 4-mm diameter dough were wet-extruded by a pelletizer

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(F-26, South China University of Technology, Guangzhou, China) and air-dried to

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below 100 g kg −1 moisture of diet. After drying, all diets were sealed in bags and

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stored at −20 °C until used.

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2.2. Experimental fish and samples collection

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Experimental fish were obtained from Marine Fisheries Development Center of

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Guangdong Province (Huizhou, China), they were transported to the experimental

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condition in cylindrical tanks (500-L) with running water (the flow rate was 3 L/min). 7

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Prior to the feeding trial, the fish were fed with the basal diet (Diet 1) for 2 weeks to

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acclimate to the experimental diet and conditions. After fasting for 24 h, juvenile

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groupers of an average weight (121.76 ± 3.21 g) were anesthetized with

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Eugenol (Shanghai Medical Instruments Co., Ltd, Shanghai, China) and 20 fishes

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were randomly stocked into eighteen cylindrical tanks (500 L, three tanks per

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treatment). All rearing tanks were provided with continuous aeration and running

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water (the flow rate was 3 L/min). Each diet was randomly assigned to tanks in

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triplicate. Fish were fed two times daily at 8:00 and 16:00 until apparent satiation on

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the basis of visual observation. During the 8 weeks feeding trial, the number and

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weight of dead fish and feed consumption were recorded every day. During the

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feeding trial, water temperature ranged from 27.0 to 31.0 ℃, salinity from 30 to 33

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psu, pH from 7.8 to 8.25. Dissolved oxygen from 5.5 to 6.0 mg L-1 and total ammonia

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was lower than 0.2 mg L-1. The fish were reared and fed the diets, under natural

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daylight cycle.

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At the end of the feeding trial, fish were fasted for 24 h before sampling, then

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were euthanized with 100 mg· L−1 Eugenol (Shanghai Medical Instruments Co., Ltd,

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Shanghai, China). All fish populations and mean body weight in each tank were

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determined. Then three fish from each tank were sampled and stored at −20 °C for

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analysis of whole body composition. The blood from three fish randomly sampled

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from each tank was collected by caudal venipuncture using 2 ml heparinized syringes.

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Blood samples were collected into anticoagulation tubes in order to obtain plasma.

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plasma was separated and stored at –80 °C for analysis of biochemical indices.

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Viscera and liver of three fish sampled blood from each tank were collected and

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weighed and the ratios were expressed as a percentage of body weight, then muscle

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samples from the dorsal sides were removed and stored at –20 °C for further analysis

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of their composition. The three fish livers and head kidneys from each tank was

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excised, frozen in liquid nitrogen, and stored at –80 °C until analysis.

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2.3. Growth performance

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The parameters were calculated as per following formulae: Weight gain rate (WGR, %) =100 × (final body weight-initial body weight)/initial

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body weight;

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Specific growth rate (SGR, % day−1) = 100 × (Ln final body weight − Ln initial body

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weight) / number of days;

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Feed efficiency ratio (FER) = wet weight gain (g) / dry diet feed (g);

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Protein efficiency ratio (PER) = wet weight gain/protein intake;

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Protein deposition rate (PDR) = 100 × [(final body weight (g) × final mean

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whole-body protein (% dry basis) - initial body weight (g) × initial mean whole-body

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protein (% dry basis))]/ total protein intake (g);

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Condition factor (CF, g/cm3) =100 × (body weight, g)/(body length, cm)3；

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Viscerosomatic index (VSI, %) =100 × (viscera weight, g)/ (whole body weight, g) ;

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Spleen index (SI, %) =100 × (spleen weight, g)/ (whole body weight, g) ;

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Intraperitoneal fat rate (IFR, %) =100 × (intraperitoneal fat weight, g)/ (whole body

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weight, g) ;

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Relative gut length (RGL, %) =100× (gut length, cm)/ (body length, cm) ;

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Survival (%) = 100 × (final number of fish) / (initial number of fish).

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Plasma cholesterol (CHO), triglyceride (TG), glucose (GLU), total protein (TP),

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low density lipoprotein (LDL), high density lipoprotein (HDL) contents and alkaline

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phosphatase (AKP) activities were analyzed using a ROCHE-P800 automatic

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biochemical analyzer (Roche, Basel, Switzerland) by the colorimetric method

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previously used for fish blood biochemistry [32, 33].

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2.5. Fish composition

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Crude protein, crude lipid, moisture and ash in diets, liver, muscles and whole

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body were determined according to the established methods of AOAC (2005) [34]:

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moisture was determined by drying to constant weight at 105 °C; crude protein (N ×

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6.25) by Kjeldahl method using Kjeltec (FOSS 8400, Hoganos, Sweden) and after

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acid digestion; lipid was determined by petroleum ether (B.P. 30–60 °C for 3 h) using

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Soxtec™ 2055 (FOSS, Hoganos, Sweden). For ash content analysis, samples were 10

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placed in a muffle furnace (FO610C, Yamato Scientific Co., Ltd., Tokyo, Japan) at

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550 °C for 6 h.

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Three hepatic samples was weighed and added with ice-cold phosphate buffer

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(PBS: 0.064 mol/L, pH 7.4). Hepatic samples were homogenized by homogenizer in

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an ice bath. The homogenate was then centrifuged for 20 min at the speed of 3000

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r/min, remove supernatant, and the supernatant were used to quantify hepatic total

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antioxidative capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD)

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activities and malondialdehyde (MDA). The above-mentioned indices were measured

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by double antibody sandwich ELISA method using commercial kits (Shanghai

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Enzyme-linked Biotechnology Co., Ltd, Shanghai, China).

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2.7. Liver histological analysis

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For histological analysis, three liver samples per tank were fixed in 4 %

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paraformaldehyde solution for 48 h, then washed in 70% ethanol solution, finally

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transferred to a 70% ethanol solution for storage until processing into histological

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slides. Paraffin production process, images collection and samples measurement were

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determined according to the method described by Torrecillas et al. and Xu et al. [35,

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2.8. RNA isolation and gene expression analysis Three fish head kidney sampled from each tank were pooled and total RNA was

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extracted from the samples using 1 ml TRIzol reagent (Vazyme Biotech Co., Ltd,

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China) according to the manufacturer's instructions. The final RNA was eluted in an

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appropriate amount of 0.1% diethyl pyrocarbonate (DEPC) treated water

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(SigmaeAldrich, St. Louis, MO, USA). The quantity of isolated RNA was later

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determined by measuring their absorbance at 260 and 280 nm using a NanoDrop 2000

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spectrophotometer (NanoDrop Technologies, USA), and the quality of total RNA was

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detected by electrophoresis in 1.2% agarose gel. Single-stranded cDNA was

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synthesized from 1µg total RNA using PrimeScript RT reagent Kit With gDNA Eraser

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(Takara, Dalian, China) following the manufacturer’s instructions. The cDNA

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templates were then stored at -80 °C for later analysis.

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For quantitative real-time PCR, the specific primer pairs were designed as Table

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2 according to our transcriptome unigene (unpublished data). The β-actin gene was

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used as a housekeeping gene and it was amplified using β-actin-F and β-actin-R

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gene-specific primers. Real-time PCR was amplified in an ABI 7500 real-time PCR

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machine (Applied Biosystems, USA) using ChamQTM SYBR® qPCR Master Mix

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(Vazyme, Nanjing, China) following the manufacturer's recommendations. Before the

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RT-PCR experiments, the specificity and efficiency of the primes above were detected.

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The standard equation and correlation coefficient were determined by constructing a

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standard curve using a serial dilution of cDNA. The reaction mixtures were 20 µL,

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uM forward and reverse primers, and 7 µL dH2O. The real-time PCR conditions were

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as follows: 94 °C for 5 min, then 40 cycles at 95 °C for 15 s, 60 °C for 1 min. All

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samples were run in triplicate, and each assay was repeated three times. After

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finishing the program, the threshold cycle (Ct) values were obtained from each

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sample. Relative gene expression levels were evaluated using 2−∆∆CT method [37].

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256 2.9. Statistical analysis

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Results are presented with means ± S.D of three replicates. All data were

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subjected to one-way analysis of variance (ANOVA) followed by Duncan's multiple

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range tests. P-value of <0.05 was considered significant. All statistical analyses were

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performed using SPSS 16.0 (SPSS Inc., Michigan Avenue, Chicago, IL, USA) for

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Windows.

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3. Results

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3.1. Growth performance, feed utilization and body indices

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Growth performance, feed utilization, survival and body indices of hybrid

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grouper were fed the different experimental diets (Table 3 and Table 4). Dietary GBE

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levels did not significantly affect final body weight (FBW), specific growth rate

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(SGR), feed intake (FI), protein deposition rate (PDR), condition factor (CF),

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hepatosomatic index (HSI), relative gut length (RGL) and survival of fish after 8

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weeks of feeding (P ≥ 0.05). Fish fed Diet 6 had significantly lower weight gain (WG) 13

ACCEPTED MANUSCRIPT and feed efficiency ratio (FER) than those of fish fed Diet 1 (P < 0.05). There had no

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significant differences in WG and FER values of fish fed Diet 1, Diet 2, Diet 3, Diet 4

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and Diet 5 (P ≥ 0.05). The protein efficiency ratio (PER) and intraperitoneal fat rate

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(IFR) of fish fed Diet 5 and Diet 6 were significantly lower (P < 0.05) than those of

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fish fed Diet 1 (P < 0.05).

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3.2. Plasma biochemical indices

The contents of total protein (TP), cholesterol (CHO), triacylglycerol (TG),

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glucose (GLU), alkaline phosphatase (ALP), low density lipoprotein (LDL) and high

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density lipoprotein (HDL) in plasma were shown in Table 5. CHO of fish fed Diet 5

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and Diet 6 were significantly higher than that of fish fed Diet 1, Diet 2, Diet 3 and

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Diet 4 (P<0.05). TG and GLU of fish fed Diet 2, Diet 3 and Diet 4 was significantly

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lower than that of fish fed Diet 1 (P<0.05). TP of fish fed Diet 2 were significantly

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higher than that of fish fed Diet 1 (P<0.05). LDL of fish fed Diet 3, Diet 4 and Diet 5

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was significantly lower than that of fish fed Diet 1 (P<0.05). Dietary supplementation

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with GBE significantly improved ALP activity and HDL content of fish (P < 0.05).

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3.3. Whole body, muscle and liver composition The proximate compositions of whole body, muscle and liver of hybrid grouper

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fed the different experimental diets were shown in Table 6. Fish fed the Diet 2 and

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Diet 3 had significant lower crude protein contents of whole body, compared to that

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of the other groups. There was no significant differences in moisture and crude lipid

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contents of whole body among all treatments (P>0.05). The dietary administration of 14

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in muscle of hybrid grouper, compared to that of the control group (P>0.05). There

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was no significant difference in liver moisture among all treatments except for fish

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fed Diet 6 (P>0.05). Liver lipid content significantly decreased firstly and then

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significantly increased as dietary GBE levels increased (P<0.05). The lowest liver

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lipid content was observed in fish fed Diet 2, it was significantly lower than those of

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the other groups (P<0.05).

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The effects of GBE levels on hepatic oxidative status was shown in Table 7. The

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results showed dietary GBE levels had a significant effect on SOD, CAT activities,

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T-AOC and MDA content (P<0.05). The hepatic SOD activities of fish fed Diet 3 and

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Diet 4 were significantly higher than that of fish fed Diet 1 and Diet 2 (P<0.05).

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Dietary supplementation with GBE significantly increased hepatic CAT activities and

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T-AOC (P < 0.05), compared to that of the control group. The hepatic MDA content

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of fish fed Diet 3, Diet 4 and Diet 5 was significantly lower than that of fish fed Diet 1

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(P<0.05).

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3.5. Liver histology

A higher occurrence rates of the hepatocyte swelling, hepatocyte vacuolization,

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and nuclei shifting to the cellular periphery cytoplasmic vacuolization was observed

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in the hepatocytes of fish fed Diet 1, Diet 4, Diet 5 and Diet 6 (Fig. 1-1, Fig. 1-4, Fig.

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1-5 and Fig. 1-6), meanwhile the structure of liver lobule and liver sinusoid were not

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obvious in fish fed above diets. However, fish fed Diet 2 and Diet 3 showed regular 15

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size (Fig. 1-2 and Fig. 1-3). Hepatocyte swelling and hepatocyte vacuolization were

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slightly higher in fish fed higher GBE levels (Diet 4, Diet 5 and Diet 6) compared

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with those of fish fed Diet 1, although not significantly different with those found in

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fish fed Diet 1.

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3.6. Immune-related genes mRNA levels in the head kidney of hybrid grouper

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Expression profiles of immune-related genes were examined in the head kidney

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of fish at the end of the feeding trial (Figs. 2-5). Transcription levels of catalase

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(CAT), glutathione peroxidase (GPx), glutathione reductase (GR), manganese

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superoxide dismutase (MnSOD) and Kelch-like- ECH-associated protein 1 (Keap1) in

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fish were presented in Fig. 2. The mRNA levels of CAT in fish showed an increasing

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trend with the increasing dietary GBE levels. The mRNA levels of GPx were

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significantly up-regulated in the head kidney of fish fed Diet 2, compared to that of

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control group, whereas there were no significant differences in those of fish fed other

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diets. GR mRNA levels in fish were sharply increased with dietary GBE up to 1.00

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g/kg, then significantly depressed, and subsequently increased (P < 0.05). Keap1

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mRNA levels in fish were remarkably decreased with dietary GBE levels up to 0.50

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g/kg diet, and then increased with levels further increasing (P < 0.05). There were no

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significant differences in MnSOD mRNA levels among all groups (P>0.05).

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Transcription levels of interleukin 10 (IL-10), transforming growth factor β1

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(TGF-β1), interleukin 8 (IL-8), target of rapamycin (TOR) and IκB kinase α (IKKα)

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in the head kidney of fish were presented in Fig. 3. Dietary supplementation with

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GBE significantly increased IL-10, TGF-β1, IL-8 and TOR mRNA levels in fish (P < 16

ACCEPTED MANUSCRIPT 343

0.05), compared to those of the control group. IKKα mRNA levels in fish decreased

344

firstly with increasing dietary GBE levels up to 0.50 g kg-1, and thereafter increased

345

with higher levels of GBE, and remained nearly constant finally. The gene expression levels of major histocompatibility complex 2 (MHC2) and

347

toll-like receptor 3 (TLR3) in the head kidney of fish were presented in Fig. 4. Dietary

348

supplementation with GBE significantly increased MHC2 mRNA levels in fish (P <

349

0.05), compared to those of the control group. The mRNA levels of TLR3 were

350

significantly up-regulated in fish fed dietary supplementation with GBE (P < 0.05),

351

and no significant differences were found among 0-1.00 g /kg groups (P > 0.05).

SC

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Apoptosis-related genes mRNA levels in the head kidney of fish were presented

353

in Fig. 5. The mRNA levels of caspase-3 were significantly down-regulated in fish

354

fed dietary supplementation with GBE (P < 0.05). Caspase-8 and caspase-9 mRNA

355

levels in fish were remarkably decreased with dietary GBE levels up to 2.00 g/kg diet,

356

and then increased with levels further increasing (P < 0.05). The mRNA levels of p53

357

in fish showed a decreasing trend with dietary GBE levels up to 2.00 g/kg diet, and

358

then increased with levels further increasing, but no significant differences were

359

found among all groups (P > 0.05).

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361 362

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360 4. Discussion

Plant extracts or phytobiotics have received much attention as a potential

363

alternative to antibiotics to prevent disease in aquaculture. Plant extracts are known to

364

promote growth, increase appetite, enhance immune ability, as well as have

365

stress-reduction, sexual stimulation, hepatoprotective effects and anti-pathogenic

366

properties in fish [16, 24]. In previous studies, Yang et al. and Huang et al. reported 17

ACCEPTED MANUSCRIPT that dietary supplementation with GBE could significantly improve growth

368

performance in broilers [29] and weaned piglets [31], respectively. However, in the

369

present study, there were no significant differences in growth performance among all

370

groups except for fish fed Diet 6. While some of plant extracts and their mixture

371

enhanced growth performance, such as groupers (Epinephelus coioides) fed diets

372

containing aminarin [15], Indian major carp, (Labeo rohita) fed diets containing

373

Pedalium murex extracts [38] and golden pompano (Trachinotus ovatus) fed diets

374

containing dandelion extracts [39], others did not have growth promotion effects, such

375

as common carp (Cyprinus carpio) fed diets containing roasted coffee powder [40]

376

and pistachio (Pistacia vera) hull extract [41], and channel catfish (Ictalurus

377

punctatus) fed diets containing a phytogenic feed additive [42]. These inconsistent

378

results may be related to applied plant species, amounts of plant extract, difference in

379

extract solutions, plant extracts components and animal species [41]. In the present

380

study, diet supplemented with excess GBE (10.00 g kg-1) retarded growth and

381

decreased FER in hybrid grouper, similar to the results of common carp fed diets

382

supplemented with coffee bean [40] and rainbow trout fed diets supplemented with

383

persian oak fruit extract [43]. Excess GBE could lead to extra energy expenditure

384

toward metabolism, probably resulting in toxic effects and stress in the fish that had

385

adverse effects on growth and feed utilization. Further, ginkgolic acids in ginkgo

386

biloba leaf, which have been shown to possess allergenic as well as genotoxic and

387

cytotoxic properties [44], could cause adverse effect on health when fish fed excess

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18

ACCEPTED MANUSCRIPT 388

GBE. The blood parameters can reflect fish health condition and nutritional

390

metabolism which are useful for determining the health status of the fish in response

391

to dietary supplements [45, 46]. In the present study, dietary supplementation with

392

0.50-4.00 g GBE kg-1 diets increased plasma TP and ALP in fish. The increase in the

393

levels of plasma TP and ALP in fish is thought to be associated with a stronger innate

394

immunity response [39]. Similarly, dietary supplementation with soybean isoflavones

395

[47] and dandelion [39] increased levels of plasma TP and ALP. Previous study

396

showed that dietary GBE significantly increased serum HDL content and decreased

397

serum LDL and TG content in broilers [48]. Chen et al. and Zhang et al. also reported

398

that the administration of GBE significantly decreased TG, CHO, and LDL-C levels

399

and significantly increased HDL-C levels in rats with hyperlipidemia [49, 50].

400

Previous study showed that GBE supplemented-diet increased the contents of TP,

401

reduced the contents of GLU and TG in serum of crucian carp [51]. Similarly, in our

402

study, dietary supplementation with 0.50-4.00 g GBE kg-1 diets could effectively

403

increase plasma HDL content and decreased plasma GLU, LDL and TG content in

404

fish. This indicated that dietary adding GBE had hypolipidemic effect in fish.

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405

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389

Fish are susceptible to the effects of reactive oxygen and fish antioxidant

406

defenses which are dependent on feeding behavior and nutritional factor play an

407

important role in reactive oxygen scavenging [33]. Fish fed high lipid diets produced

408

excess reactive oxygen species (ROS) [52]. Reactive oxygen species (ROS) 19

ACCEPTED MANUSCRIPT scavenging ability has been correlated with the enzymatic and non-enzymatic

410

antioxidant defense systems [53]. SOD, GPx, GR and CAT are important anti-oxidant

411

enzymes and T-AOC directly reflected antioxidant capacity of fish, whereas MDA

412

levels indirectly reflected the severity of fish body cell from free radicals attack [33,

413

54]. GBE have been shown to exhibit antioxidant effects both in vitro and in vivo [17,

414

18, 55, 56]. Previous studies showed that GBE had strong scavenging activity to

415

hydroxyl radical, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide anion [17].

416

Bridi et al. reported that GBE could improve antioxidant abilities in rats [55].

417

Consistent with these studies, our study indicated that dietary supplementation with

418

GBE significantly increased activities of hepatic antioxidant enzymes (SOD, T-AOC,

419

CAT) while MDA content significantly decreased, compared to those of the control

420

group. The results demonstrated that GBE could improve hepatic antioxidative

421

abilities of hybrid grouper.

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409

To our knowledge, no study to date has investigated the effects of dietary GBE

423

on liver histology in fish. The liver is an important organ in metabolizing products

424

coming from the digestive tract, and histological changes are considered as a good

425

indicator in the evaluation of nutritional condition [57, 58]. As indicator of the

426

metabolic activity of hepatocytes, morphometric parameters are most often used:

427

hepatocytes number, hepatocytes surface area, hepatocytes nuclear area, and glycogen

428

and lipid content in the cytoplasm [59]. Previous study reported that dietary ginkgo

429

biloba leaf polysaccharide could significantly decrease hepatic fat accumulation,

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20

ACCEPTED MANUSCRIPT preserve liver function, and ameliorate changes in the lipid profile in rats fed high

431

lipid diets-induced nonalcoholic fatty liver disease [26]. In the current study, fish fed

432

Diet 2 and Diet 3 showed regular hepatocyte morphology with cytoplasmic lipid

433

vacuoles that did not alter hepatocyte size. However, a higher occurrence rates of the

434

hepatocyte swelling, hepatocyte vacuolization, and nuclei shifting to the cellular

435

periphery cytoplasmic vacuolization was observed in the hepatocytes of fish fed Diet

436

1, Diet 4, Diet 5 and Diet 6, meanwhile the structure of liver sinusoid were not

437

obvious in fish fed above diets. The increase of hepatocellular vacuolation observed

438

in fish fed Diet 1, Diet 4, Diet 5 and Diet 6 was caused by the accumulation of very

439

large lipid droplets, which reflect the higher lipid content in fish liver in comparison

440

to fish fed Diet 2. Previous studies have indicated that the size and volume of

441

hepatocytes may be a physiological response to lipid excess and energy storage [57].

442

These results indicated that dietary supplementation with 0.50-1.00 g GBE kg-1 diets

443

could maintain normal liver histology and preserve liver function when fish fed high

444

lipid diets.

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445

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The head kidney containing various immune cells is the main immunocompetent

446

organ in fish [53, 60]. Fish immunity has been correlated with normal structural and

447

function of the head kidney, which is also closely related to the antioxidant status in

448

fish [53]. Previous studies showed that antioxidant enzymes were stress- and

449

immune-response biomarkers to evaluate the fish health impact [61]. The results from

450

our study showed that dietary GBE significantly up-regulated the mRNA levels of 21

ACCEPTED MANUSCRIPT CAT, GPx and GR, whereas down -regulated the mRNA levels of Keap1 in the head

452

kidney of fish, compared with the control group. Previous study indicated that the

453

mRNA levels of antioxidant enzyme were negatively correlated with the Keap1

454

mRNA levels [62]. Therefore, dietary GBE up-regulation of antioxidant enzyme

455

mRNA levels might be partly related to the decreased Keap1 expression. Many

456

previous studies reported that increase of antioxidant genes expression could improve

457

fish antioxidant ability [62-64]. These results indicated that dietary GBE improved

458

antioxidant ability in head kidney of fish fed high lipid diets by increasing antioxidant

459

genes expression.

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In fish, the immune status was closely associated with the inflammation which

461

was initiated and regulated by inflammatory cytokines [64, 65]. IL-8 are important

462

pro-inflammatory cytokines, which can recruit and activate macrophages and

463

neutrophils to remove the cell debris and invaded microorganisms, and facilitate the

464

regrowth of injured tissues [15]. IL-10 and TGF-β1 are important anti-inflammatory

465

cytokines, which could counteract the production of pro-inflammatory cytokines and

466

limit inflammatory response [66]. Previous studies reported that GBE could suppress

467

inflammation in vitro and in vivo [21, 67-69]. Kotakadi et al. reported GBE had

468

anti-inflammatory properties and ameliorated colitis in mice by driving effector T cell

469

apoptosis [23]. Consistent with these studies, dietary supplementation with GBE

470

significantly up-regulated the expression of IL-10 and TGF-β1 in the head kidney of

471

fish. However, previous studies reported that plant immunostimulants up-regulated

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22

ACCEPTED MANUSCRIPT 472

pro-inflammatory cytokines and down-regulated anti-inflammatory cytokines in

473

Labeo rohita [66] and common carp [70]. The TOR pathway played an important role

474

in the inflammatory response in fish [65] and the inhibition of TOR signal pathway

475

could

476

anti-inflammatory cytokines [64]. In the present study, TOR mRNA levels were

477

significantly increased in fish fed diets supplemented with GBE. Further, the results

478

indicated that IL-10 mRNA and TGF-β1 levels were positively related to TOR mRNA

479

levels in the head kidney of fish. Similarly, previous studies reported that IL-10 and

480

TGF-β1 mRNA levels were positively related to TOR mRNA levels in grass carp [62,

481

64, 71]. These results implied that dietary GBE could suppress the inflammatory

482

response in head kidney of fish fed high lipid diets. However, the underlying

483

mechanism by which GBE influences the cytokine expression in fish is still unknown

484

and needs further study.

pro-inflammatory

cytokines

production

and

decrease

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increase

The head kidney is the primary immune organ in fish [53].The major

486

histocompatibility complex (MHC) molecules are cell surface glycoproteins which

487

play an important role in the vertebrate immune system, serving to present foreign

488

antigens to T cells [72]. Toll-like receptor 3 (TLR3), one member of TLR family

489

which play an essential role in the activation of innate immunity by recognizing their

490

cognate ligands, recognizes invading pathogens specifically double-stranded RNA

491

viruses [73]. Torrecillas et al. and Falco et al. reported that dietary supplementation

492

with concentrated mannan oligosaccharides and β-glucan significantly increased

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23

ACCEPTED MANUSCRIPT MHC2 and TLR3 mRNA levels in fish, respectively [74-76]. Similarly, our study

494

showed that MHC2 and TLR3 mRNA levels in the head kidney of hybrid grouper fed

495

dietary supplementation with GBE significantly increased. The increase of MHC2

496

mRNA levels found in fish fed GBE could be related to an increase of other

497

leucocytes populations or a potential stimulation of T cell population via antigen

498

presenting cells in order to initiate immune tolerance to the GBE itself, and the higher

499

level of TLR3 suggested a potential activation of innate immunity.

SC

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493

Previous studies demonstrated that rat fed high lipid diets not only caused

501

oxidative damage, but also initiated apoptosis [77, 78]. Fish fed high lipid diets could

502

produce excess reactive oxygen species (ROS) and initiate apoptosis. ROS can

503

regulate p53 which is a transcription factor that plays an important role in apoptosis

504

[79]. In the present study, dietary supplementation with 0.50-2.00 g GBE kg-1 diets

505

decreased the relative level of p53 mRNA in the head kidney of fish. Previous study

506

reported that caspase activity was a useful marker for determining stress-induced

507

apoptosis [80]. Apoptosis has two general pathways, one is the extrinsic death

508

receptor pathway which directly activates the initiator caspase-8 and the other is

509

intrinsic mitochondrial pathway which is initiated by the release of cytochrome c

510

activating downstream effector caspase-9 and caspase-3 [54, 80]. Previous study

511

reported GBE significantly attenuated mitochondrion-initiated apoptosis and

512

decreased the activity of caspase-3 in the cell [81]. Similarly, our study showed that

513

dietary supplementation with 0.50-4.00 g GBE kg-1 diets significantly decreased

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24

ACCEPTED MANUSCRIPT 514

caspase-9, caspase-8 and caspase-3 mRNA levels in head kidney of fish fed high lipid

515

diets. In conclusion, our study indicated that hybrid grouper fed high lipid diets

517

supplemented with GBE did not improve growth performance and feed utilization but

518

it had hypolipidemic effects and reduced intraperitoneal fat rate. Furthermore, dietary

519

supplementation with 0.50-1.00 g GBE kg-1 diets could maintain normal liver

520

histology and preserve liver function together with an increase in hepatic antioxidant

521

status via enhancing antioxidant enzyme activities. Moreover, dietary GBE

522

up-regulated the expression of antioxidant genes (CAT, GPx and GR),

523

immune-related genes (MHC2 and TLR3) and anti-inflammatory cytokines (IL-10

524

and TGF-β1), while dietary supplementation with 0.50-4.00 g GBE kg-1 diets

525

down-regulated apoptosis-related genes (p53, caspase-9, caspase-8 and caspase-3)

526

expression in the head kidney of hybrid grouper.

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529

Acknowledgement

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The research was supported by Special Scientific Research Funds for public

530

service industry (Agriculture) (201403011), the Special Scientific Research Funds for

531

Central Non-profit Institutes, Chinese Academy of Fishery Sciences Special Scientific

532

Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery

533

Sciences (2014ZD02), the Guangdong Provincial Oceanic Fisheries Science and

534

Technology Project (A201601C04), Guangdong Provincial Natural Science 25

ACCEPTED MANUSCRIPT 535

Foundation (2016A030313447), and the Innovation Project of Graduate School of

536

South China Normal University.

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ACCEPTED MANUSCRIPT Table 1 Composition and nutrient levels of experimental diets (g kg-1) Diet 1

Diet 2

Diet 3

Diet 4

Diet 5

Diet 6

Fish meal

450.0

450.0

450.0

450.0

450.0

450.0

Soybean meal

130.0

130.0

130.0

130.0

130.0

130.0

Flour

237.4

237.4

237.4

237.4

237.4

237.4

Beer yeast powder

50.0

50.0

50.0

50.0

50.0

50.0

Fish oil

50.0

50.0

50.0

50.0

50.0

50.0

Soybean oil

50.0

50.0

50.0

50.0

50.0

50.0

Lecithin

10.0

10.0

10.0

10.0

10.0

10.0

Vitamin premix1

2.00

2.00

2.00

2.00

2.00

2.00

Mineral premix2

5.00

5.00

5.00

5.00

5.00

5.00

Choline chloride (50% )

5.00

Antioxidant

0.10

Vitamin C

0.50

Monocalcium phosphate

10.0

Ginkgo biloba leaf extract3

0

Crude protein Crude lipid

AC C

Ash 1, 2

SC

M AN U 5.00

5.00

5.00

5.00

5.00

0.10

0.10

0.10

0.10

0.10

0.50

0.50

0.50

0.50

0.50

10.0

10.0

10.0

10.0

10.0

0.50

1.00

2.00

4.00

10.00

TE D

Moisture

5.12

5.73

5.61

5.65

5.10

5.10

46.63

46.78

46.58

46.24

46.65

46.23

14.81

14.67

14.72

14.72

14.75

14.73

10.36

10.31

10.33

10.40

10.40

10.42

EP

Nutrient levels (%)

RI PT

Ingredients

Mineral and vitamin premix provided by Guangzhou Feixite Aquatic Co., Ltd., China 1 Vitamin premix provides the following (mg kg-1 diet): vitamin A (500000 IU) 40 mg, vitamin B1 40 mg, vitamin B2 93.75 mg, vitamin B6 20 mg, vitamin B12 (1%) 45 mg, vitamin K3 (50%) 300 mg, inositol 400 mg, calcium pantothenate 250 mg, nicotinic acid 450 mg, folic acid 6 mg, biotin (2%) 10 mg, vitamin D3 (500000 IU) 15 mg, vitamin E (50%) 300 mg, unite bran 2990.25 mg 2 Mineral premix provides the following (mg kg-1 diet): Na2SeO3 (1%) 20 mg, CuSO4·5H2O (25%) 24 mg, FeSO4·H2O (30%) 266.65 mg, ZnSO4·H2O (34.50%) 100 mg, MnSO4·H2O (31.80%) 120 mg, Ca(IO3)2 (5%) 50 mg, CoSO4·7H2O (5%) 10 mg, zeolite power 4380.55 mg. 3 Shaanxi Ciyuan biotechnology Co., Ltd., China.

ACCEPTED MANUSCRIPT Table 2

Keap1 IL-8 IL-10 TGF-β1 IKKα TOR MHC-2 TLR3 Caspase-3

AC C

Caspase-8

SC

MnSOD

M AN U

GR

TE D

GPX

qPCR primers, forward/reverse (5’to3’) F: GCGTTTGGTTACTTTGAGGTGA R: GAGAAGCGGACAGCAATAGGT F: TACCCTACCAAGTCCTCCAACC R: AACAAACACCCGACACCCA F: CTTTCACTCCGATGTATCACGC R: GCTTTGGTAGCACCCATTTTG F: TACGAGAAGGAGAGCGGAAGA R: ATACCGAGGAGGGGGATGA F: CCAGAAGGAATGTGTGGCTAAA R: TGGTTGGTCATCGGGTTGTA F: AGTCATTGTCATCTCCATTGCG R: AAACTTCTTGGCCTGTCCTTTT F: TTCGACGAGCTCAAGAGTGAG R: TGCCGTTTAGAAGCCAGATACA F: AACATCCCGCTACCTCGCTT R: TCCGCTCATCCTCATTCCCT F: ACACCGACACAACGGCTCAT R: CCAGACGGCACAGTTTCACAG F: CCACTCTTTCTTTGCGGCTT R: GGGTTCTCGTCCCTCACTTG F: CCACCCGAACAAACAGACC R: TGATGCCCCCTCCAACACT F: TCTCCATTCCGTCACCTTCC R: TCATCCAGCCCGTTACTATCC F: CGCAAAGAGTAGCGACGGA R: CGATGCTGGGGAAATTCAGAC F: TGCTTCTTGTGTCGTGATGTTG R: GCGTCGGTCTCTTCTGGTTG F: TTTTCCTGGTTATGTTTCGTGG R: TTGCTTGTAGAGCCCTTTTGC F: GGCACCAAACAAACCAAAAAAC R: GTCAAGCAACTCCAGACCATCA F: TACGAGCTGCCTGACGGACA R: GGCTGTGATCTCCTTCTGC

EP

Primers CAT

RI PT

Sequences of primers used in this study.

Caspase-9 P53

β-Actin

CAT, catalase; GPx, glutathione peroxidase; GR, glutathione reductase; MnSOD, manganese superoxide dismutase; Keap1, Kelch-like-ECH-associated protein 1; IL-8, interleukin 8; IL-10, interleukin 10; TGF-β1, transforming growth factorβ1; IKKα,

ACCEPTED MANUSCRIPT IκB kinase α; TOR, target of rapamycin; MHC-2, major histocompatibility complex 2; TLR3, toll-like receptor 3.

Table 3 Effects of dietary ginkgo biloba leaf extract (GBE) on growth performance, feed utilization and body indices in hybrid grouper Diet 1

Diet 2

Diet 3

Diet 4

(GBE g kg-1 )

0

0.50

1.00

2.00

IBW (g)

124.17± 1.84 311.59± 22.69 150.81± 14.94b

123.68± 1.63 297.12± 19.88 140.34± 18.19ab

122.40± 3.74 289.08± 24.10 136.24± 19.75ab

125.71± 4.37 294.56± 18.37 134.16± 6.90ab

SGR (%/d )

1.64±0.11

1.56±0.14

1.53±0.15

FER (%)

0.99±0.03b

0.95±0.08b

FI (g/fish)

189.33± 15.02

PER

Diet 5

Diet 6

RI PT

Diets

10.00

123.80± 3.65 285.09± 21.05 130.08± 10.19ab

126.13± 1.30 279.72± 18.39 121.76± 14.11a

1.52±0.05

1.49±0.08

1.42±0.12

0.96±0.04b

0.95±0.04b

0.89±0.03ab

0.81±0.06a

182.51± 9.59

186.08± 4.10

178.21± 6.88

180.65± 13.70

188.60± 11.57

2.23±0.08c

2.26±0.00c

2.18±0.08bc

2.16±0.10bc

2.04±0.07ab

1.92±0.03a

PDR (%)

44.41±1.63

43.63±8.90

42.57±7.89

44.91±4.06

40.71±2.30

36.72±3.60

Survival (% )

98.33±2.89

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

Regression YWGR = 36.675x2 - 60.336x + 145.75

M AN U

WGR (%)

TE D

FBW (g)

SC

4.00

100.00±0.00

R² = 0.8863

P = 0.316

R² = 0.8924

P = 0.334

YFER = 0.0681x2 - 0.2358x + 0.9804

R² = 0.9678

P = 0.013

YFI = 37.626x2 - 35.947x + 187.11

R² = 0.6808

P = 0.79

YPER = 0.3154x2 - 0.6571x + 2.2598

R² = 0.9698

P = 0.005

R² = 0.8762

P = 0.49

2

AC C

EP

YSGR = 0.2644x2 - 0.442x + 1.6033

YPDR= -1.4464x - 6.162x + 44.254

Values are means ± SD of three replications. Means in the same raw with different superscripts are significantly different (P<0.05). IBW: initial body weight; FBW: final body weight; WGR: weight gain rate; SGR: specific growth rate; FER: feed efficiency ratio; FI: feed intake; PER: protein efficiency ratio; PDR: protein deposit rate.

ACCEPTED MANUSCRIPT Table 4 Effects of dietary ginkgo biloba leaf extract (GBE) on body indices in hybrid grouper Diets

Diet 2

Diet 3

Diet 4

Diet 5

Diet 6

0

0.50

1.00

2.00

4.00

10.00

CF ( g/cm3)

2.72±0.13

2.64±0.42

2.68±0.22

2.73±0.23

2.69±0.25

2.80±0.21

VSI (%)

8.58±0.82ab

9.11±0.93b

8.70±0.47ab

8.81±0.85ab

7.94±0.67a

7.92±0.80a

HSI (%)

2.32±0.45

2.60±0.40

2.45±0.54

2.34±0.51

2.38±0.45

2.32±0.54

SI (%)

0.25±0.05ab

0.27±0.09ab

0.21±0.04ab

0.30±0.07b

0.20±0.08a

0.19±0.05a

IFR (%)

2.02±0.28c

2.15±0.45c

1.74±0.31abc

1.86±0.17bc

1.57±0.33ab

1.41±0.22a

RGL (%)

136.44±12.72

146.48±8.36

142.26±10.63

140.45±15.08 141.40±20.77

g

M AN U

Regression

SC

(GBE kg-1 )

RI PT

Diet 1

YCF = 0.0962x2 + 0.013x + 2.6865 YVSI = 1.3624x2 - 2.4145x + 8.939 YIFR = 0.8858x2 - 1.5412x + 2.0629

141.51±22.90

R² = 0.6335

P = 0.763

R² = 0.6889

P = 0.063

R² = 0.8284

P = 0.006

TE D

Values are means ± SD of three replications. Means in the same raw with different superscripts are significantly different (P<0.05).

AC C

EP

CF: condition factor; VSI: viscerosomatic index; HSI: hepatosomatic index; SI: spleen index; IFR: intraperitoneal fat rate; RGL: relative gut length.

ACCEPTED MANUSCRIPT

Table 5 Effects of dietary ginkgo biloba leaf extract on plasma biochemical indices in hybrid grouper

Diet 1

Diet 2

Diet 3

Diet 4

( GBE g kg-1)

0

0.50

1.00

2.00

3.03±0.03a

2.98±0.10a

2.71±0.07a

2.93±0.25a

3.33±0.07d

1.90±0.23a

2.64±0.27bc

3.27±0.02c

0.89±0.03a

0.71±0.13a

42.25±2.76a

55.15±2.19b

125.67±

138.50±

3.06ab

17.68bc

0.57±0.04b

0.44±0.09ab

0.13±0.00a

0.17±0.00bc

TP/(g L-1) ALP/(U L-1) LDL/( mmol L-1) HDL/( mmol L-1) Regression

YCHO = -4.0857x2 + 5.6726x + 2.5928

10.00

4.77±0.53c

4.12±0.15b

2.50±0.23b

2.92±0.21bcd

3.08±0.23cd

0.93±0.18ab

1.67±0.21b

3.57±0.69c

SC

GLU/(mmol L-1)

Diet 6

4.00

M AN U

TG/(mmol L-1)

47.30±0.99ab

50.35±5.30ab

147.00±

159.50±

143.50±

110.00±

7.07cd

4.95d

4.95bcd

8.49a

0.35±0.01a

0.31±0.04a

0.33±0.03a

0.54±0.08b

0.18±0.01bc

0.19±0.02c

0.16±0.04abc

TE D

CHO/( mmol L-1)

Diet 5

RI PT

Diets

0.15±0.01ab

48.35±2.90ab

46.45±3.18a

P=0

YALP = -121.29x2 + 96.577x + 133.7

R² = 0.7967

P = 0.006

YLDL = 0.9974x2 - 0.949x + 0.4977

R² = 0.7799

P = 0.009

R² = 0.7249

P = 0.036

AC C

EP

R² = 0.6778

YHDL = -0.1834x2 + 0.2093x + 0.1391

Values are means ± SD (n=9) of three replications. Means in the same raw with different superscripts are significantly different (P<0.05). CHO: cholesterol; TG: triglyceride; GLU: glucose; TP: total protein; ALP: alkaline phosphatase; LDL: low density lipoprotein; HDL: high density lipoprotein.

ACCEPTED MANUSCRIPT

Table 6 Effects of dietary ginkgo biloba leaf extract on whole body, muscle and liver proximate composition in hybrid grouper Diet 1

Diet 2

Diet 3

Diet 4

Diet 5

Diet 6

( GBE g kg-1)

0

0.50

1.00

2.00

4.00

10.00

Whole body

RI PT

Diets

67.67±0.17

66.33±3.12

67.23±3.14

67.60±1.31

68.42±0.53

68.54±0.71

Crude protein

56.41±0.52b

54.41±0.22a

54.69±0.48a

57.58±0.51c

57.49±0.26c

57.32±0.25c

Crude lipid

23.55±1.03

23.34±0.64

23.15±0.26

22.16±0.34

21.01±2.31

21.52±0.10

14.91±0.43ab

14.85±0.23a

Moisture

75.13±0.61

75.53±0.74

Crude protein

79.76±1.37

78.35±1.98

Crude lipid

8.44±0.88

Ash

M AN U

Ash

SC

Moisture

15.00±0.27ab

15.59±0.04b

15.76±0.22b

15.58±0.54b

75.68±0.88

74.82±0.89

75.40±0.50

75.36±1.59

80.53±1.28

79.05±1.11

80.20±0.47

80.59±2.39

9.14±1.29

8.81±1.00

9.65±0.27

8.83±0.45

8.78±0.84

5.41±0.18

5.47±0.16

5.54±0.14

5.29±0.06

5.38±0.07

5.45±0.17

Moisture

58.12±2.30b

55.23±3.06ab

56.35±0.99b

54.58±2.95ab

54.49±0.61ab

51.34±1.18a

Crude protein

20.35±0.13e

18.41±0.11d

17.96±0.26c

18.33±0.03d

17.38±0.14b

16.91±0.04a

28.16±0.27c

29.31±0.12d

34.61±0.40e

40.48±0.37f

TE D

Muscle

Regression

AC C

Crude lipid

EP

Liver

24.12±0.45b

23.37±0.14a

Y moisture in whole body = -2.5372x2 + 4.1882x + 66.922

R² = 0.6194

P = 0.742

Y lipid in whole body = 7.2336x2 - 9.4938x + 23.761

R² = 0.9769

Y moisture in liver = 3.6002x2 - 9.1499x + 56.959

R² = 0.8449

P = 0.034

Y protein in liver = 5.3686x2 - 7.8385x + 19.423

R² = 0.7272

P=0

P = 0.229

ACCEPTED MANUSCRIPT Y lipid in liver = -17.78x2 + 34.829x + 23.442

R² = 0.9731

P=0

Values are means ± SD (n=9) of three replications. Means in the same raw with different superscripts are significantly different (P<0.05).

Diet 1

Diet 2

Diet 3

Diet 4

( GBE g kg-1)

0

0.50

1.00

2.00

SOD activity (U/g)

1034.7±12 9.6a

1145.4± 39.9a

1362.5± 18.6bc

CAT activity (U/g)

381.6±5.6a

425.2±6.8b

432.6±11.2b

MDA content(nmol/g)

120.9±5.7c

115.0±4.2bc

T-AOC (U/g)

134.0±2.4a

154.0±4.4cd

YMDA = 69.731x2 - 71.443x + 116.12

4.00

10.00

1375.7± 149.1c

1177.1± 72.0ab

1214.9± 41.0abc

428.9±6.7b

437.0±5.6b

437.0±7.8b

102.2±2.7ab

100.4±4.6a

103.9±8.7ab

113.8±3.0bc

158.5±4.9d

138.5±0.0ab

147.5±10.7bcd

144.5±4.7abc

TE D

YCAT = -120.04x2 + 152.37x + 403.56

Diet 6

M AN U

Regression

Diet 5

SC

Diets

RI PT

Table 7 Effect of dietary ginkgo biloba leaf extract on hepatic antioxidant abilities in hybrid grouper

R² = 0.5607

P=0

R² = 0.6279

P = 0.024

Values are means ± SD (n=9) of three replications. Means in the same raw with different superscripts are significantly different (P<0.05).

AC C

EP

SOD: superoxide dismutase; CAT: catalase; MDA: malondialdehyde; T-AOC: total antioxidant capacity

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

1

2

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

3

4

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

5

6

ACCEPTED MANUSCRIPT

RI PT

Figure 1. Liver sections of test fish fed diets supplementation with ginkgo biloba leaf extract levels (H&E staining 400×) 1. Liver from fish fed Diet 1 (0 gkg-1 GBLE); 2. Liver from fish fed Diet 2 (0.50 gkg-1 GBLE); 3. Liver from fish fed Diet 3 (1.00 gkg-1 GBLE); 4. Liver from fish fed Diet 4 (2.00 gkg-1 GBLE); 5. Liver from fish fed Diet 5 (4.00 gkg-1 GBLE); 6. Liver from fish fed Diet 6 (10.00 gkg-1 GBLE). “ ” nuclei shifted to the periphery of the hepatocytes; “ ” swelling cells; “ ” vacuolation.

b 2.5 2.0 1.5

a

d

a a a a a

a a a

cd bc abc ab a

SC

b a

c

bc

1.0

bc

a

a

a

a

a

a

abab aa

0.5 0.0 CAT

GPx 0

M AN U

Expression relative to β-actin

3.0

GR

0.5

1

2

Keap1

4

MnSOD

10

TE D

Figure 2. Relative expression of catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), Kelch-like- ECH-associated protein 1 (Keap1) and manganese superoxide dismutase (MnSOD) in the head kidney of hybrid grouper fed

EP

diets supplemented with graded levels of ginkgo biloba leaf extract for 8 weeks. 0, 0.5, 1, 2, 4, 10 represent the fish fed Diet 1, Diet 2, Diet 3, Diet 4, Diet 5 and Diet 6, respectively. Values are means, error bars indicate S.D. (n=9), and different letters

AC C

above a bar denote the significant difference between treatments (P < 0.05).

ACCEPTED MANUSCRIPT d

c 5.0

c 4.0 bcbc bc

3.0

bb b

cd d b b bc

ab

d

2.0 a

a

a

a

cd bc bc ab

1.0 0.0 IL-10

TGF-β1 0.5

1

2

TOR 4

10

ab

bcd cd d bc a

IKKα

SC

0

IL-8

RI PT

Expression relative to β-actin

6.0

Figure 3. Relative expression of interleukin 10 (IL-10), transforming growth factor β1

M AN U

(TGF-β1) and interleukin 8 (IL-8), target of rapamycin (TOR) and IκB kinase α (IKKα) in the head kidney of hybrid grouper fed diets supplemented with graded levels of ginkgo biloba leaf extract for 8 weeks. 0, 0.5, 1, 2, 4, 10 represent the fish fed Diet 1, Diet 2, Diet 3, Diet 4, Diet 5 and Diet 6, respectively. Values are means, error bars indicate S.D. (n=9), and different letters above a bar denote the significant

AC C

EP

TE D

difference between treatments (P < 0.05).

ACCEPTED MANUSCRIPT c

7.0 6.0

c

5.0

c

4.0

b b

b

3.0

b

b ab

2.0

ab a

a

1.0 0.0 MHC2 0.5

1

2

4

10

SC

0

TLR3

RI PT

Expression relative to β-actin

8.0

Figure 4. Relative expression of major histocompatibility complex 2 (MHC2) and

M AN U

toll-like receptor 3 (TLR3) in the head kidney of hybrid grouper fed diets supplemented with graded levels of ginkgo biloba leaf extracts for 8 weeks. 0, 0.5, 1, 2, 4, 10 represent the fish fed Diet 1, Diet 2, Diet 3, Diet 4, Diet 5 and Diet 6, respectively. Values are means, error bars indicate S.D. (n=9), and different letters

AC C

EP

TE D

above a bar denote the significant difference between treatments (P < 0.05).

ACCEPTED MANUSCRIPT

1.4 1.2

b

bc 0.8

c a

a a

a

ab ab abc

bc

ab

a

a a a

d

1.0

a

c

e

a

a

a

a

0.6 0.4 0.2 0.0 caspase-3

caspase-8 0.5

1

2

4

10

P53

SC

0

caspase-9

RI PT

Expression relative to β-actin

1.6

Figure 5. Relative expression of caspase-3, caspase-8, caspase-9 and p53 in the head

M AN U

kidney of hybrid grouper fed diets supplemented with graded levels of ginkgo biloba leaf extract for 8 weeks. 0, 0.5, 1, 2, 4, 10 represent the fish fed Diet 1, Diet 2, Diet 3, Diet 4, Diet 5 and Diet 6, respectively. Values are means, error bars indicate S.D. (n=9), and different letters above a bar denote the significant difference between

AC C

EP

TE D

treatments (P < 0.05).

ACCEPTED MANUSCRIPT Highlights： 1. Dietary supplementation with ginkgo biloba leaf extract (GBE) had hypolipidemic effects in hybrid grouper fed high lipid diets. 2. Dietary GBE improved hepatic antioxidant ability and maintain normal liver

RI PT

histology. 3. Dietary GBE increased immune-related genes expression and decrease

AC C

EP

TE D

M AN U

SC

apoptosis-related genes expression.