Effects of dietary hawthorn extracts supplementation on lipid metabolism, skin coloration and gut health of golden pompano (Trachinotus ovatus)

Journal Pre-proof Effects of dietary hawthorn extracts supplementation on lipid metabolism, skin coloration and gut health of golden pompano (Trachinotus ovatus)

Xiaohong Tan, Zhenzhu Sun, Meng Zhou, Cuiyun Zou, Hongyan Kou, Sarath Babu Vijayaraman, Yanhua Huang, Heizhao Lin, Li Lin PII:

S0044-8486(19)31791-0

DOI:

https://doi.org/10.1016/j.aquaculture.2020.734921

Reference:

AQUA 734921

To appear in:

aquaculture

Received date:

15 July 2019

Revised date:

3 January 2020

Accepted date:

3 January 2020

Please cite this article as: X. Tan, Z. Sun, M. Zhou, et al., Effects of dietary hawthorn extracts supplementation on lipid metabolism, skin coloration and gut health of golden pompano (Trachinotus ovatus), aquaculture (2019), https://doi.org/10.1016/ j.aquaculture.2020.734921

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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.

© 2019 Published by Elsevier.

Journal Pre-proof Effects of dietary hawthorn extracts supplementation on lipid metabolism, skin coloration and gut health of golden pompano (Trachinotus ovatus)

Xiaohong Tana, Zhenzhu Sunb, Meng Zhoua, Cuiyun Zoua, Hongyan Koua, Sarath Babu Vijayaramana, Yanhua Huanga , Heizhao Linc  , Li Lina   a

Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding,

oo

f

Guangdong Provincial Key Laboratory of Waterfo wl Healthy Breeding, College of Animal Sciences and Technology, Zhongkai University of Agriculture and

pr

Engineering, Guangzhou, Guangdong 510225, China b

e-

Institute of Modern Aquaculture Science and Engineering, Guangzhou Key

Pr

Laboratory of Subtropical Biodiversity and Biomonitoring, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, School of Life Science, South

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

rn

c

al

China Normal University, Guangzhou 510631, PR China

Jo u

Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, PR China



Corresponding author. Corresponding author.   Corresponding author. 

E-mail addresses: [email protected], [email protected], [email protected]. 1

Journal Pre-proof

Abstract Hawthorn fruit has long been used as a functional food in China due to its effects of increasing appetite, promoting digestion and protecting the gastrointestinal tract. The present study was conducted to assess the impact of dietary hawthorn extract (HTE) supplementation on plasma biochemical indices, proximate body composition,

oo

f

skin coloration, intestinal morphology and changes in mRNA expression of immune-related genes and tight junction proteins genes in the intestine of golden

pr

pompano. Basal diets supplemented with HTE at 0, 0.50, 1.00, 2.00, 4.00 and 10.00 g

e-

kg−1 were fed to golden pompano for eight weeks. The study showed dietary HTE

Pr

supplementation had hypolipidemic effects by reducing plasma cholesterol, triglyceride and low density lipoprotein content and improved skin coloration by

al

increasing yellowness values of the ventral skin color of golden pompano. Meanwhile,

rn

dietary supplementation of 0.50 and 1.00 g kg-1 HTE could improve gut morphology

Jo u

by increasing villus length, villus width and muscle thickness, and enhance barrier function by increasing tight junction protein genes mRNA levels. However, dietary 10.00 g kg-1 HTE supplementation not only caused damage to intestinal morphology and barrier function, but also induced oxidative stress and inflammatory response. Our study demonstrated that dietary 0.50 and 1.00 g kg-1 HTE supplementation was beneficial for skin coloration and intestinal health of golden pompano. Keywords : Golden pompano; Hawthorn extract; Skin coloration; Intestinal morphology; Tight junction protein genes 2

Journal Pre-proof

1. Introduction The ovate pompano Trachinotus ovatus, commonly known as golden pompano, is named for its golden color in the fins and abdomen, and is one of the commercially important mariculture species in the southern coast of China (Zhang et al., 2014). In natural waters, golden pompano is rich in color due to the intake of a large amount of

oo

f

carotenoid-rich algae and planktons. While, under artificial aquaculture conditions, the content of pigment sources such as carotenoids in the compound feed is lesser, and

pr

golden pompanos rapidly lose their natural color and turn white (Yang et al., 2017).

e-

The skin color of the golden pompano significantly affects the market price and

Pr

consumer acceptability of the species. The body color of fish is affected by many factors, such as background color, light environment, dietary lipid and carotenoids

rn

al

levels (Doolan et al., 2010; Doolan et al., 2010; Pham et al., 2014). Fish can synthesize melanin but cannot biosynthesize carotenoids de novo (Yi et al., 2014).

Jo u

Therefore, it is necessary for them to consume the corresponding pigment yielding substances to achieve and to maintain their normal body color. Despite considerable interest in this organism color, the application of dietary supplementation containing salt algae Dunaliella salina has been demonstrated in benefitting the yellow skin coloration of the back and the abdomen of golden pompano (Yang et al., 2017). Two dietary carotenoids, namely astaxanthin and lutein, a red to yellow pigment occurs naturally in certain algae, vegetables, and fruits, cause the pink or red color in salmon, trout, lobster, shrimp, and other seafood. Therefore, they are widely used as enhancing 3

Journal Pre-proof

agents to improve the colors that adorn the skin of the fish body (Yi et al., 2014). In prior studies, it was already established that the dietary supplementation of astaxanthin or xanthophylls improved the body coloration in Australian snapper Pagrus auratus (Doolan et al., 2010), olive flounder Paralichthys olivaceus (Pham et al., 2014) and large yellow croaker Larimichthys croceus (Yi et al., 2014).

oo

f

Golden pompanos are reported as susceptible to the intestinal tract disease during the feeding process that not only impair the gut functions but also reduces the

pr

digestibility and absorption of other valuable nutrients (Ahmadifar et al., 2019;

e-

Lauriano et al., 2016; Tan et al., 2018b; Van Doan et al., 2019; Zhao et al., 2014). The

Pr

excretion of partially assimilated or undigested food materials into the water body, resulting in the deterioration of water quality, which in turn challenges a threat or

rn

al

impairs fish health through various water sanitation-related diseases. Several studies have been reported, increased gut and growth responses of fish when supplementing

Jo u

with Phyto additives, such as European sea bass (Dicentrarchus labrax) supplemented with short-chain fructooligosaccharides and xylooligosaccharides (Guerreiro et al., 2018), tilapia (Oreochromis niloticus) fed diets supplemented with resveratrol, golden pompano (Trachinotus ovatus) supplemented with dandelion extracts (Tan et al., 2018) , rainbow trout (Oncorhynchus mykiss) supplemented with garlic (Etyemez Büyükdeveci et al., 2018), gilthead sea bream (Sparus aurata) supplemented with a mixture of carvacrol and thymol containing diets (Pérez-Sánchez et al., 2015). Crataegus monogyna commonly known as hawthorn fruit has long been used as 4

Journal Pre-proof

a functional food and is popular among people in China owing to antioxidant, anti- inflammatory, increase appetite, and protective properties on the gastrointestinal tract due to its a variety of active substances, such as flavonoids, phenolic acids, oligomeric proanthocyanidins, triterpene acids, and organic acids (Keser et al., 2014; Tadić et al., 2008). Dietary supplementation of aqueous hawthorn extracts increased

oo

f

the growth performance, nutrient digestibility and digestive enzyme activity, also improved intestinal microbiota and intestinal morphology in broilers chichens (Zhang,

pr

2009). Hawthorn leaf aqueous extract could improve intestinal digestive function by

e-

inhibiting the expression of 5-Hydroxytryptamine (5-HT) and 5-Hydroxytryptamine3

Pr

(5-HT3R) receptor in the intestinal mucosa of Irritable Bowel Syndrome model rats (Wu and He, 2011). Dietary fiber in freeze-dried hawthorn could not only increase the

rn

al

content of short-chain fatty acids such as acetic acid, propionic acid and butyric acid in the intestinal tract of mice, but also increase relative abundance of Bifidobacterium

Jo u

and Lactobacillus in colon and cecum, inhibit the proliferation of harmful microorganisms such as Enterobacter, Enterococcus and Clostridium perfringens (Yang et al., 2016). In our previous study, we observed that the golden pompano fish fed with hawthorn fruit aqueous extracts (HTE) containing diets increased in rates of growth, along with their antioxidant capacities and immunity against the pathogen (Tan et al., 2017a). With these above considerations, the present study was aimed to evaluate the potential effects of dietary hawthorn extracts supplementation in golden pompano 5

Journal Pre-proof

skin coloration, gut health, and plasma biochemical indices. Additionally, further experiments were conducted to test the most suitable hawthorn extracts inclusion levels in their feeds, which is necessary to benefit and not to cause intestinal impairment in golden pompano.

oo

f

2. Materials and methods 2.1. Diet preparation

pr

The basic diet used fishmeal, peanut meal and soybean meal as protein source,

e-

and fish oil and soybean oil as lipid source and its crude protein, crude fat and ash

Pr

content were (44.74±0.29)%, (10.61±0.10) % and (8.96 ±0.06)% respectively (Table 1). 0, 0.50, 1.00, 2.00, 4.00 and 10.00 g kg-1 hawthorn fruit aqueous extracts

rn

al

(abbreviated as HTE containing 40.0% flavonoids, Shaanxi Ciyuan biotechnology Co., Ltd., China) were separately added to the basal diets to make six experimental diets.

2017a).

Jo u

The preparation and processing of diets was the same as our previous study (Tan et al.,

2.2. Experimental fish and samples collection Healthy juvenile golden pompano fish was transported in polythene bags from Shenzhen Base of South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (Shenzhen, China), and acclimated to the local conditions with the basal diet for 2 weeks. After being fasted for 24 h, golden pompanos of average 6

Journal Pre-proof

weight (6.79 ± 0.08 g) were equally distributed in 18 floating cages (1 m × 1 m × 1.5 m ; three cages per group, n=25 in each cage). Each of the five treatment groups (0.50, 1.00, 2.00, 4.00, and 10.00 g kg-1 HTE supplemented diet) and control (0 g kg-1 ) groups were handfed two times (8:00 and 16:00) a day being close to apparent satiation without overfeeding based on visual observation.

f

During the 8 weeks of feeding trial, the fish were reared under natural daylight

oo

cycle, and water quality parameters were as follows: temperature = 27.0 ± 1.0 ℃, pH

pr

= 7.8 ±0.25, dissolved oxygen = 6.0 ± 0.66 mg L-1 , salinity = 20 to 23‰, and total

e-

ammonia-nitrogen = 0.05 ± 0.1 mg L-1 . After termination of experimental feeding, the

Pr

blood from each group (three fish) was randomly sampled through the caudal vasculature with heparinized tuberculin syringes and euthanized immediately with an

rn

al

overdose of Eugenol (100 mg·L−1 , Shanghai Medical Instruments Co., Ltd, Shanghai, China). The whole blood was centrifuged at 4 °C for 15 min at 8,000 × g, and plasma

Jo u

samples were aliquot in different plastic micro tubes and stored frozen at -80°C for biochemical analysis. The blood collected fish were dissected immediately, and the muscle tissue samples were collected for nutritional compos ition analysis and stored at –20 °C until analysis. The fish intestine was excised and divided into two segments (foreguts and remaining part); foreguts were placed in 4% paraformaldehyde for morphological analysis, and the rest part was placed in 2-mL centrifuge tubes, flash- frozen in liquid nitrogen and stored at -80˚C until being processed for gene expression analysis. All experimental protocols were conducted according to the 7

Journal Pre-proof

National legislation for fish welfare established by The Ministry of Science a nd Technology of the People's Republic of China. 2.3. Plasma biochemical analysis Biochemical indices were measured in the plasma between the gradient HTE fed groups to evaluate the best composition for nutritional support qualification. All the

oo

f

nutritional biochemical indices that including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, glucose (GLU) contents, plasma

cholesterol (LDL-C)

and high

density

lipoprotein

e-

lipoprotein

pr

cholesterol (CHO), triglyceride (TG), total protein (TP) content, low density

Pr

cholesterol (HDL-C) were evaluated by using a ROCHE-P800 automatic biochemical

rn

al

analyzer (Roche, Basel, Switzerland) following our previous study (Tan et al., 2017b).

2.4. Fish and diets proximate composition

Jo u

The proximate composition of the whole body and fillet of golden pompano obtained after 8 weeks of feeding trial were sampled and analyzed for crude protein, crude lipid, moisture, and ash contents according to our previous study (Sun et al., 2018).

2.5. Skin color measurement After 8-week feeding trial, skin color of 5 fish from each cage was measured separately. Before the measurement, the moisture on the surface of the fish body was 8

Journal Pre-proof

blotted with absorbent paper, and the skin color measurement was performed in ventral skin and dorsal (right back) skin at night by using a portable colorimeter (GEB-104 Pantone Color-Cue). The fish skin color was represented by L* for lightness, a* for redness or greenness, b* for yellowness or blueness according to the

oo

f

recommendation of the International Commission on Illumination (Yi et al., 2014).

2.6. Intestinal morphology analysis

pr

For intestinal morphology analysis, intestinal foregut (1 cm after the stomach) of

e-

golden pompano fixed with 4% paraformaldehyde solution were histologically

Pr

processed using paraffin technique and H&E (Haematoxylin and Eosin) staining system according to Anguiano et al. (Anguiano et al., 2013). The intestinal

rn

al

morphological information were obtained through histology, and the histometry was determined following the methods described in our previous studies (Tan et al., 2019b;

Jo u

Tan et al., 2018b).

2.7. RNA extraction and Quantitative real-time PCR analysis The mRNA expression in the intestinal tissues of golden pompano was performed by Quantitative real-time PCR (qRT-PCR) assay. Total RNA were isolated from three fish intestines of each treatment group with TRIzol ® reagent (Vazyme Biotech Co., Ltd, China), and the first-strand cDNA was synthesized by using PrimeScript RT reagent Kit With gDNA Eraser (Takara, Dalian, China), according to 9

Journal Pre-proof

the manufacturer’s instructions. The RNA quality integrity assessment and the primers for the golden pompano intestinal gene expression analysis were referred from our previous study (Tan et al., 2018b). The PCR reactions were carried out in ABI 7500 real-time PCR machine (Applied Biosystems, USA) using the ChamQTM SYBR® qPCR Master Mix kit (Vazyme, Nanjing, China). The methods for the qRT-PCR

oo

f

reactions and the evaluation of the relative gene expression levels were adopted from

pr

our previous studies (Tan et al., 2018a).

e-

2.8. Statistical analysis

Pr

The results were presented with means ± S.D. Firstly, the normal distribution (Kolmogorov-Smirnov test) and the homogeneity of the variance (Levene test) of all

rn

al

data were tested, and then the one-way analysis of variance (ANOVA) and Duncan's multiple range tests were performed by using SPSS 16.0 (SPSS Inc., Michigan

Jo u

Avenue, Chicago, IL, USA).

3. Results The effect of gradient HTEs dietary supplements on golden pompano fish survival and growth characteristics were displayed in our previous report (Tan et al., 2017a). 3.1. Effects of dietary hawthorn extract on plasma biochemical indices of golden pompano Plasma biochemical indices of golden pompano fed with different dietary HTE 10

Journal Pre-proof

levels after 56 days of feeding trial were presented in Table 2. Dietary supplementation of 2.00 g kg-1 HTE significantly decreased plasma cholesterol (CHO), triglyceride (TG) and glucose (GLU) (P < 0.05). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) contents of fish fed with 10.00 g kg-1 HTE diet were significantly (P < 0.05) higher than that of the control group, whereas

f

there was no significant difference observed between the other groups and the control

oo

group (P > 0.05). Dietary supplementation of 0.50 and 1.00 g kg-1 HTE significantly

pr

(P < 0.05) increased plasma high density lipoprotein (HDL) content, while dietary

e-

0.50, 2.00 and 4.00 g kg-1 HTE significantly decreased plasma low density lipoprotein

Pr

(LDL) content (P < 0.05), compared to that of control (0 g kg-1 HTE). However,

rn

in golden pompano.

al

dietary HTE had no significant (P > 0.05) effect on plasma total protein (TP) content

Jo u

3.2. Effects of dietary hawthorn extract on the proximate composition of the whole body and the muscle tissue of golden pompano At the end of the experiment, the proximate composition of the whole body and the muscle tissue of golden pompano were estimated and presented in Table 3. Supplementation of 0.50 and 1.00 g kg-1 HTE dietary groups significantly (P < 0.05) increased the whole-body crude protein contents, while the 10.00 g kg-1 HTE diet group showed higher crude lipid content in the whole body compared to the control group. Crude protein, crude lipid, and moisture contents in the muscle of fish after the 11

Journal Pre-proof

eight weeks of feeding trail were not significantly affected by various dietary HTE levels (P > 0.05).

3.3. Effects of dietary hawthorn extract on dorsal skin and ventral skin color of golden pompano

oo

f

The calorimetric outcomes of the fish skin (dorsal and ventral) coloration after the HTE supplementation feeding periods were presented in Table 4. Dietary HTE

pr

increased yellowness (b*) of ventral skin color and the values in fish fed with 0.50,

e-

2.00, and 4.00 g kg-1 HTE groups were significantly higher than that of the control

Pr

group (P < 0.05). No significant changes were recorded in the Light (L*), and redness (a*) values in both the dorsal and ventral skins, and neither in the yellowness (b*)

Jo u

inclusions (P > 0.05).

rn

al

value affected in the dorsal surface skin of golden pompano due to the dietary HTE

3.4. Effects of hawthorn extract on foregut morphology of golden pompano The results of the histological evaluation from the foregut of golden pompano fed over eight weeks with dissimilar supplementation of HTE diets are shown in Fig. 1 and detailed in Table 5. Fish foreguts displayed enhanced morphology with the expansion of mucous membrane and intestinal folds in proportion to the increased concentration from control to 1.00 g kg-1 HTE supplemented groups. While further increasing the HTE (2.0 to 4.0) denseness disturbed the foregut morphology with 12

Journal Pre-proof

loosely arranged intestinal margins, and the highest concentration completely distorted the golden pompano’s foregut when observed under a microscope. The histometric assessment between the various HTE treatment groups exposed dietary supplementation at 0.50 g kg-1 significantly increased in the length, width, and muscle thickness of the villus (P < 0.05). However, dietary 10 g kg-1 HTE supplementation

oo

f

significantly reduced villus length, when compared to control (P < 0.05). Decreasing level of villus numbers viz., 39.00±3.61, 35.67±2.08, 35.67±1.53, and 31.33±1.53

pr

were observed concerning the increasing 0, 0.50, 1.00, 2.00, g kg-1 HTE

e-

concentrations, then abruptly increased to 38.00±2.65 and 41.00±7.55 at 4.00, and

Pr

10.00 g kg-1 HTE concentrations, respectively.

pompano

rn

al

3.5. Effects of hawthorn extract on gene expression in the intestine of golden

Jo u

The mRNA expressions levels of antioxidant-related genes, inflammation-related genes, and tight junction protein genes in the intestine of fish were presented in Figs. 2–4. Glutathione peroxidase (GPx) mRNA level was decreased with increasing HTE level up to 1.00 g kg-1 diet, and increased thereafter, and achieved the highest expression level in fish fed diet supplemented with 4.00 g kg-1 HTE than the control group (P < 0.05). Dietary 0.50 g kg-1 HTE supplementation significantly reduced catalase (CAT) mRNA level, whereas rest of the supplemented groups showed an increasing trend, and dietary 2.00 and 10.00 g kg-1 HTE supplement group was 13

Journal Pre-proof

significantly higher than the control group (P < 0.05). Dietary HTE supplementation significantly decreased Kelch-like- ECH-associated protein 1 (Keap1) mRNA levels than the control groups (P < 0.05). The expression of glutathione reductase (GR) was not significantly affected by dietary HTE levels (P > 0.05) (Fig. 2). Dietary 10.00 g kg-1 HTE supplementation significantly increased interleukin 8

oo

f

(IL-8) and target of rapamycin (TOR) mRNA level (P < 0.05), but there was no significant difference in IL-8 and TOR expression between the other HTE

pr

supplemented groups and the control group (P > 0.05). Dietary HTE supplementation

e-

significantly increased interleukin 10 (IL-10) mRNA level except for fish fed 4.00 g

Pr

kg-1 HTE diet. Similarly, dietary HTE significantly increased transforming growth factor β1 (TGF-β1) mRNA level except for fish fed 0.50 g kg-1 HTE diet (Fig. 3).

al

Dietary HTE supplementation significantly decreased zonula occludens-1 (zo-1)

rn

mRNA level except for fish fed 0.50 g kg-1 HTE diet. Dietary 1.00 g kg-1 HTE

Jo u

supplementation significantly increased zonula occludens-3 (zo-3) mRNA level, whereas diets supplemented with 2.00, 4.00 and 10.00 g kg-1 HTE significantly reduced zo-3 mRNA level (P < 0.05). Compared with the control group, the mRNA expression level of occludin in 0.50 g kg-1 HTE-supplemented group was significantly up-regulated (P < 0.05), however those of other HTE-supplemented groups were down-regulated. Claudin3a mRNA level was decreased with increasing HTE level up to 1.00 g kg-1 diet, and increased thereafter, and claudin3a mRNA level was significantly higher in fish fed diet supplemented with 10.00 g kg-1 HTE than the 14

Journal Pre-proof

control group (P < 0.05).

4. Discussion Plant extracts are widely used as functional feed additives for aquatic animals, which can stimulate appetite, promote growth, resist stress, enhance immunity and

oo

f

improve intestinal health, possibly because they contain many active ingredients, such as polysaccharides, flavonoids, alkaloids, phenolic compounds and volatile oils

pr

(Awad and Awaad, 2017; Reverter et al., 2014; Ringø et al., 2018; Sun et al., 2018;

e-

Tan et al., 2019a; Tan et al., 2018a; Tan et al., 2017b). Our previous study indicated

Pr

that dietary 0.50 and 1.00 g kg-1 HTE supplementation significantly improved growth performance and immune capacity of golden pompano (Tan et al., 2017a). As far as

rn

al

we knew, this study was the first attempt to explore the effects of dietary HTE

pompano.

Jo u

supplementation on lipid metabolism, skin coloration and intestinal health in golden

In the present study, dietary HTE supplementation reduced CHO, TG, and LDL contents and increased HDL content. A previous study reported that the levels of CHO, TG, LDL, AST and ALT in the serum and liver significantly decreased and the serum HDL-C levels significantly increased in quails fed with high- fat diet and 1 mL of hawthorn leaf flavonoids in the doses of 0.02, 0.04 and 0.08 g kg-1 , respectively. (Ye et al., 2009). The levels of CHO and TG in blood significantly decreased in hyperlipoidemia mouse fed with 1.5 and 3.0 g kg-1 hawthorn flavonoids (Xie et al., 15

Journal Pre-proof

2009). Diets supplemented with 0.02 g kg-1 hawthorn leaf extract increased the abdominal fat rate of broilers fed for 42 days, whereas abdominal fat rate and serum TG and CHO contents were significantly decreased when the broilers were fed for 63 days (Li et al., 2009). Reduced CHO, TG and LDL contents and increased HDL level suggested that dietary hawthorn fruit extract supplementation might have

oo

f

hypolipidemic effects in golden pompano. In this study, dietary 10.00 g kg-1 HTE supplementation significantly increased plasma ALT and AST contents, which

pr

indicated that high doses of HTE supplementation might lead to liver dysfunction in

e-

golden pompano.

Pr

Previous studies have demonstrated that hawthorn leaf flavonoids cannot only reduce the blood lipids but also regulate the lipid deposition in animals (Li et al.,

rn

al

2009). In this study, dietary HTE supplementation increased crude protein and crude lipid contents in the whole body of fish, but reduced muscle lipid content, which

Jo u

suggested that HTE might regulate the distribution of fat and increase abdominal subcutaneous fat content in golden pompano. These results are in line with the study in chicken models, where dietary 0.2 g kg-1 hawthorn extract supplementation could reduce lipid deposition in broiler muscles (Zhang, 2009). When broilers were fed with diets supplemented with hawthorn leaf flavonoids for 28 days, the abdominal fat rate and subcutaneous fat rate of broilers were increased in the addition group. While the trend could be reversed by increasing the feeding cycle and the amount of hawthorn leaf flavonoids, which indicated that the regulation of hawthorn leaf flavonoids on 16

Journal Pre-proof

lipid deposition in broilers was periodic and dose-dependent (Li et al., 2009). Previous studies reported that increasing lipid deposition could increase pigmentation in fish (Christiansen and Wallace, 1988). In large yellow croaker Larimichthys croceus, a positive correlation was observed between skin yellowness and the whole-body lipid content as well as carotenoids content in fish (Yi et al., 2014). In the

f

present study, there were quadratic regression relationships between ventral skin

oo

yellowness and dietary HTE content (y = -0.1134x2 + 1.2351x + 1.6849, R² = 0.9222)

pr

as well as the whole-body lipid content (y = -0.5272x2 + 37.101x - 648.73, R² =

e-

0.4662). Dietary HTE might increase yellowness values of ventral skin color of

Pr

golden pompano by increasing lipid deposition in the whole body of fish. To our knowledge, this was the first report that HTE could be used as an additive in fish

rn

al

aquaculture to improve skin coloration of golden pompano. The integrity of the intestinal morphological structure is essential for maintaining

Jo u

the normal intestinal function (Gao et al., 2013). In aquatic animals, increasing muscle thickness, height or width of the villi are considered to increase the surface area of intestinal digestion and absorption, and the shortening of intestinal villi is re lated to the presence of toxins (Gao et al., 2013). In the present study, dietary 0.50 and 1.00 g kg-1 HTE supplementation increased villus length, villus width and muscle thickness in the foregut of golden pompano, whereas dietary 10.00 g kg-1 HTE supplementation impaired the foregut morphology. These results indicated that appropriate amount of HTE could promote intestinal villus growth, improve intestinal digestion and 17

Journal Pre-proof

absorption capacity, while high dose of HTE could damage the intestinal morphology of the golden pompano fish. Consistent with our study, dietary 0.2 g kg-1 hawthorn fruit extract increased growth performance and nutrient digestibility of broilers, which were associated with a significant increase in the height of duodenal villi, microvilli and jejunum microvilli (Zhang, 2009). Previous studies indicated that HTE could

f

improve intestinal morphology and health in animals, possibly because it improved

oo

intestinal microbiota. Zhang et al. reported that dietary supplementation of 0.2 g kg-1

pr

hawthorn fruit extract could significantly increase the average feed intake, average

e-

daily gain and feed conversion rate of broilers, and reduce the number of Escherichia

Pr

coli in ileum and cecum, and promote the proliferation of Lactobacillus in the jejunum, ileum and cecum (Zhang et al., 2009). Likewise, dietary fiber from the

rn

al

freeze-dried hawthorn increased the relative abundance of Bifidobacterium and Lactobacillus, also the content of acetic acid, propionic acid, and butyric acid in

Jo u

intestine of mice, and decreased the relative abundance of Enterobacter, Enterococcus and Clostridium perfringens (Yang et al., 2016). Hawthorn fruit contained many organic acids such as hawthorn acid, citric acid, ursolic acid and chlorogenic acid (Jin et al., 2006; Ren et al., 2007). Organic acids could not only improve intestinal morphology alone, but also promote the intestinal health by encouraging the reproduction of beneficial bacteria to produce large amounts of organic acids, such as acetic acid, propionic acid and butyric acid (Zhang et al., 2009). The changes of intestinal morphology and barrier function were closely related 18

Journal Pre-proof

to the changes of intestinal tight junction protein gene expression (Wu et al., 2015; Zou et al., 2016). Tight junction proteins such as zonula occludens, occludin and claudins acted as links between adjacent intestinal epithelial cells and played an important role in regulating intestinal permeability (Turner, 2009). Earlier studies indicated that increasing occludin and zonula occludens mRNA levels could improve

f

intestinal morphology and barrier function in fish (Turner, 2009). In the present study,

mRNA level respectively, whereas diets

pr

occludens-3 (zo-3) and occludin

oo

dietary 0.50 and 1.00 g kg-1 HTE supplementation significantly increased zonula

e-

supplemented with 2.00, 4.00 and 10.00 g kg-1 HTE significantly reduced zo-1 and

Pr

zo-3 mRNA level. Our study showed that low doses of HTE diets could improve intestinal barrier function and intestinal morphology, while high doses of HTE

rn

al

damaged intestinal barrier function and intestinal morphology. However, there was no report on the effect of HTE on intestinal tight junction in fish. The potential

study.

Jo u

mechanism of HTE on intestinal barrier function remained unclear and needed further

In fish, impaired intestinal mucosal barrier could lead to intestinal oxidative damage and inflammation (Burgos et al., 2018; Faggio et al., 2015; Fleming et al., 2010; Groschwitz and Hogan, 2009; Jiang et al., 2015). Previous studies indicated that the expression of antioxidant enzymes and inflammation-related genes is considered as a biomarker of stress and immune response to assess the health status of fish (Sagstad et al., 2010; Tan et al., 2018). Tadić et al. reported that 0.05, 0.10 and 0.20 g 19

Journal Pre-proof

kg-1 hawthorn extract had antioxidant, anti- inflammatory and gastroprotective effects (Tadić et al., 2008). In this study, dietary 0.50 g kg-1 HTE supplementation decreased GPx, CAT, GR and Keap1 mRNA level, while diets supplemented with 2.00, 4.00 and 10.00 g kg-1 HTE increased GPx, CAT and GR mRNA levels. Meanwhile, dietary 0.50 g kg-1 HTE supplementation had no significant effects on the expression of IL-8,

oo

f

TGF-β1 and TOR, whereas high doses of HTE diets significantly increased expression of IL-8, IL-10, TGF-β1 and TOR. These results showed that high doses of

pr

HTE containing diets might cause oxidative stress and inflammatory response in

e-

golden pompano. The findings in the present study suggested that the dietary 0.50 and

Pr

1.00 g kg-1 HTE supplementation could improve gut morphology by increasing villus length, villus width and muscle thickness in the foregut, and enhance intestinal barrier

al

function by increasing zonula occludens-3 (zo-3) and occludin mRNA level. However,

rn

high doses of dietary HTE (10.00 g kg-1 HTE) not only caused abnormal liver

Jo u

function, but also resulted in damage to intestinal morphology and barrier function which induced oxidative stress and inflammatory response. In conclusion, dietary HTE supplementation had hypolipidemic effects and improved skin coloration by increasing yellowness values of ventral skin color of golden pompano. Meanwhile, dietary 0.50 and 1.00 g kg-1 HTE supplementation could improve gut morphology by increasing villus length, villus width and muscle thickness, furthermore strengthen barrier function by increasing tight junction protein genes mRNA levels. Our study demonstrated that dietary 0.50 and 1.00 g kg-1 HTE 20

Journal Pre-proof

supplementation was beneficial for skin coloration and intestinal health of golden pompano and it can be used as a functional feed additive to promote the healthy development of fish aquaculture.

Acknowledgement

oo

f

The research was supported by Special Scientific Research Funds for public service industry (Agriculture) (201403011).

pr

References

e-

Ahmadifar E., Sheikhzadeh N., Roshanaei K., Dargahi N., Faggio C., 2019. Can

Pr

dietary ginger (Zingiber officinale) alter biochemical and immunological parameters and gene expression related to growth, immunity and antioxidant

rn

al

system in zebrafish (Danio rerio)? Aquaculture 507, 341-348. Anguiano, M., Pohlenz, C., Buentello, A., Gatlin, D.M., 2013a. The effects of

Jo u

prebiotics on the digestive enzymes and gut histomorphology of red drum (Sciaenops ocellatus) and hybrid striped bass (Morone chrysops × M. saxatilis). Brit. J. Nutr. 109, 623-629. Awad, E., Awaad, A., 2017. Role of medicinal plants on growth performance and immune status in fish. Fish Shellfish Immun. 67, 40-54. Burgos-Aceves M.A., Cohen A., Smith Y., Faggio C., 2018. MicroRNAs and their role on fish oxidative stress during xenobiotic environmental exposures. Ecotox. and Environ. Safe. 148, 995-1000. 21

Journal Pre-proof

Chen, L., Feng, L., Jiang, W., Jiang, J., Wu, P., Zhao, J., Kuang, S., Tang, L., Tang, W., Zhang, Y., Zhou, X., Liu, Y., 2015. Intestinal immune function, antioxidant status and tight junction proteins mRNA expression in young grass carp (Ctenopharyngodon idella) fed riboflavin deficient diet. Fish Shellfish Immun. 47, 470-484.

oo

f

Christiansen, J.S., Wallace, J.C., 1988. Deposition of canthaxanthin and muscle lipid in two size groups of Arctic charr, Salvelinus alpinus (L.). Aquaculture 69,

pr

69-78.

e-

Doolan, B.J., Booth, M.A., Jones, P.L., Allan, G.L., 2010. Effect of cage colour and

Pr

light environment on the skin colour of Australian snapper Pagrus auratus (Bloch & Schneider, 1801). Aquac. Res. 38, 1395-1403.

rn

al

Doolan, B.J., Allan, G.L., Booth, M.A., Jones, P.L., 2010. Effect of carotenoids and background colour on the skin pigmentation of Australian snapper Pagrus

Jo u

auratus (Bloch & Schneider, 1801). Aquac. Res. 39, 1423-1433. Etyemez Büyükdeveci, M., Balcázar, J.L., Demirkale, I., Dikel, S., 2018. Effects of garlic-supplemented diet on growth performance and intestinal microbiota of rainbow trout (Oncorhynchus mykiss). Aquaculture 486, 170-174. Faggio C., Fazio F., Marafioti S., Arfuso F., Piccione G., 2015. Oral administration of Gum Arabic: effects on haematological parameters and oxidative stress markers in Mugil cephalus. Iran. J. Fish Sci. 14, 60-72. Fleming, A., Jankowski, J., Goldsmith, P., 2010. In vivo analysis of gut function and 22

Journal Pre-proof

disease changes in a zebrafish larvae model of inflammatory bowel disease. Inflamm. Bowel Dis. 16, 1162-1172. Gao, Y., Han, F., Huang, X., Rong, Y., Yi, H., Wang, Y., 2013. Changes in gut microbial populations, intestinal morphology, expression of tight junction proteins, and cytokine production between two pig breeds after challenge with

oo

f

Escherichia coli K88: a comparative study. J. Anim. Sci. 91, 5614. Groschwitz, K.R., Hogan, S.P., 2009. Intestinal barrier function: molecular regulation

pr

and disease pathogenesis. J. Allergy Clin. Immun. 124, 3-20.

e-

Guerreiro, I., Serra, C.R., Oliva-Teles, A., Enes, P., 2018. Short communication: gut

Pr

microbiota of European sea bass (Dicentrarchus labrax) is modulated by

rn

279-288.

al

short-chain fructooligosaccharides and xylooligosaccharides. Aquacult. Int. 26,

Jiang, W., Tang, R., Liu, Y., Kuang, S., Jiang, J., Wu, P., Zhao, J., Zhang, Y., Tang,

Jo u

L., Tang, W., Zhou, X., Feng, L., 2015. Manganese deficiency or excess caused the depression of intestinal immunity, induction of inflammation and dysfunction of the intestinal physical barrier, as regulated by NF-κB, TOR and Nrf2 signalling, in grass carp (Ctenopharyngodon idella). Fish Shellfish Immun. 46, 406-416. Jin, G., Zhang, F., Xue, X., Xu, Q., Liang, X., 2006. Analysis of organic acids in hawthorn by reversed phase high performance liquid chromatography. Chinese J. Anal. Chem. 34, 987-990. 23

Journal Pre-proof

Keser, S., Celik, S., Turkoglu, S., Yilmaz, O., Turkoglu, I., 2014. The investigation of some bioactive compounds and antioxidant properties of hawthorn (Crataegus monogyna subsp. monogyna jacq.). J. Intercult. Ethnopharmacol. 3, 51. Lauriano E.R., Pergolizzi S., Capillo G., Kuciel M., Alesci A., Faggio C., 2016. Immunohistochemical characterization of Toll- like receptor 2 in gut epithelial

diet. Fish Shellfish Immun. 59, 250-255.

oo

f

cells and macrophages of goldfish Carassius auratus fed with a high-cholesterol

pr

Li, L., Zhu, X.L., Shu, G., Gao, P., Jiang, Q.Y., Chen, Y.C., 2009. Effect of hawthorn

Pr

Univ. Jiangxiensis 31, 610-615.

e-

leaf flavonoids on lipid metabolism in yellow-feathered broilers. Acta Agric.

Pérez-Sánchez, J., Benedito-Palos, L., Estensoro, I., Petropoulos, Y., Calduch-Giner,

rn

al

J.A., Browdy, C.L., Sitjà-Bobadilla, A., 2015. Effects of dietary NEXT ENHANCE®150 on growth performance and expression of immune and

Jo u

intestinal integrity related genes in gilthead sea bream (Sparus aurata L.). Fish Shellfish Immun. 44, 117-128. Pham, M.A., Byun, H.G., Kim, K.D., Lee, S.M., 2014. Effects of dietary carotenoid source and level on growth, skin pigmentation, antioxidant activity and chemical composition of juvenile olive flounder Paralichthys olivaceus. Aquaculture 431, 65-72. Ren, F., Wu, M., Gu, F., 2007. Determination of ursolic acid in hawthorn leaves by HPLC. Nat. Prod. Res. Dev. 19, 303-305. 24

Journal Pre-proof

Reverter, M., Bontemps, N., Lecchini, D., Banaigs, B., Sasal, P., 2014. Use of plant extracts in fish aquaculture as an alternative to chemotherapy: Current status and future perspectives. Aquaculture 433, 50-61. Ringø E., Faggio C., Chitmanat C., Doan H., Mai N.T., Jaturasitha S., Hoseinifar S.H., 2018. Effects of corncob derived xylooligosaccharide on innate immune growth performance in Nile tilapia

oo

f

response, disease resistance, and

(Oreochromis niloticus) fingerlings. Aquaculture 495, 786–793.

pr

Sagstad, A., Sanden, M., Haugland, A-C, H., Olsvik, P.A., G-I, H., 2010. Evaluation

e-

of stress- and immune-response biomarkers in Atlantic salmon, Salmo salar L.,

Pr

fed different levels of genetically modified maize (Bt maize), compared with its

rn

201-212.

al

near-isogenic parental line and a commercial suprex maize. J. Fish Dis. 30,

Sun, Z., Tan, X., Ye, H., Zou, C., Ye, C., Wang, A., 2018. Effects of dietary Panax

Jo u

notoginseng extract on growth performance, fish composition, immune responses, intestinal histology and immune related genes expression of hybrid grouper (Epinephelus lanceolatus ♂× Epinephelus fuscoguttatus ♀) fed high lipid diets. Fish Shellfish Immun. 73, 234-244. Sun, Z., Tan, X., Xu, M., Liu, Q., Ye, H., Zou, C., Ye, C., 2019. Liver transcriptome analysis and de novo annotation of the orange-spotted groupers (Epinephelus coioides) under cold stress. Comp. Biochem. Phys. D 29, 264-273. Sun, Z., Tan, X., Liu, Q., Ye, H., Zou, C., Xu, M., Zhang, Y., Ye, C., 2019. 25

Journal Pre-proof

Physiological, immune responses and liver lipid metabolism of orange-spotted grouper (Epinephelus coioides) under cold stress. Aquaculture 498, 545-555. Tadić, V.M., Dobrić, S., Marković, G.M., Ðorđević, S.M., Arsić, I.A., Menković, N.R.,

Stevic

́ ,

T.,

2008.

Anti- inflammatory,

gastroprotective,

free-radical-scavenging, and antimicrobial activities of hawthorn berries ethanol

oo

f

extract. J. Agr. Food Chem. 56, 7700-7709. Tan, X., Sun, Z., Ye, C., 2019a. Dietary Lycium barbarum extract administration

lanceolatus

♂× E.

fuscoguttatus♀) fed

high

lipid

diets.

e-

(Epinephelus

pr

improved growth, meat quality and lipid metabolism in hybrid grouper

Pr

Aquaculture 504, 190-198.

Tan, X., Sun, Z., Ye, C., Lin, H., 2019b. The effects of dietary Lycium barbarum

rn

al

extract on growth performance, liver health and immune related genes expression in hybrid grouper (Epinephelus lanceolatus♂ × E. fuscoguttatus♀)

Jo u

fed high lipid diets. Fish Shellfish Immun. 87, 847-852. Tan, X., Sun, Z., Huang, Z., Zhou, C., Lin, H., Tan, L., Xun, P., Huang, Q., 2017a. Effects of dietary hawthorn extract on growth performance, immune responses, growth- and immune-related genes expression of juvenile golden pompano (Trachinotus ovatus) and its susceptibility to Vibrio harveyi infection. Fish Shellfish Immun. 70, 656-664. Tan, X., Sun, Z., Liu, Q., Ye, H., Zou, C., Ye, C., Wang, A., Lin, H., 2018a. Effects of dietary ginkgo biloba leaf extract on growth performance, plasma biochemical 26

Journal Pre-proof

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 Shellfish

Immun. 72, 399-409. Tan, X., Sun, Z., Zhou, C., Huang, Z., Tan, L., Xun, P., Huang, Q., Lin, H., Ye, C.,

oo

f

Wang, A., 2018b. Effects of dietary dandelion extract on intestinal morphology, antioxidant status, immune function and physical barrier function of juvenile

pr

golden pompano Trachinotus ovatus. Fish Shellfish Immun. 73, 197-206.

e-

Tan, X., Sun, Z., Chen, S., Chen, S., Huang, Z., Zhou, C., Zou, C., Liu, Q., Ye, H.,

Pr

Lin, H., Ye, C., Wang, A., 2017b. Effects of dietary dandelion extracts on growth performance, body composition, plasma biochemical parameters,

rn

al

immune responses and disease resistance of juvenile golden pompano Trachinotus ovatus. Fish Shellfish Immun. 66, 198-206.

Jo u

Turner, J.R., 2009. Intestinal mucosal barrier function in health and disease. Nat. Rev. Immunol. 9, 799-809.

Van Doan H., Hoseinifar S.H., Ringo E., Esteban M.A., Dadar M., Dawood M.A.O., Faggio C., 2019. Host-associated probiotics: a key factor in sustainable aquaculture. Rev. Fis. Sci. Aquac. 1-27. Wu, L., He, Z., 2011. Effects of folium crataegi water extract on the expression of 5-ht and 5- ht3r in intestinal mucosa of IBS model rats. J. Mudanjiang Med. Univ. 32, 6-9. 27

Journal Pre-proof

Wu, Y., Jiang, Z., Zheng, C., Wang, L., Zhu, C., Yang, X., Wen, X., Ma, X., 2015. Effects of protein sources and levels in antibiotic- free diets on diarrhea, intestinal morphology, and expression of tight junctions in weaned piglets. Anim. Nutr. 1, 170-176. Xie, W., Sun, C., Liu, S., 2009. Effect of hawthorn flavanone on blood-fat and

China J. Chin. Mater. Med. 34, 224-229.

oo

f

expression of lipogenesis and lipolysis genes of hyperlipidemia model mouse.

pr

Yang, G., Zhou, Q., Dong, L., Hao, H., Liu, X., Zhao, W., 2016. Effect of increased

e-

dietary fiber in freeze-dried hawthorn on the intestinal health of mice. Mod. Food

Pr

Sci. Tech. 32, 20-25.

Yang, Y., Chen, X., Lin, H., Huang, X., Huang, Z., Zhou, C., 2017. Effects of dietary

rn

al

green alga Dunaliella salina levels on body pigmentation and growth of ovate pompano Trachinotus ovatus. Fisheries Sci., 336-340.

Jo u

Ye, X.Y., Xu, M.H., Li, X.F., Wang, Y.F., 2009. Effects of hawthorn leaf flavonoids on reducing blood lipids and preventing fatty liver in the quails. Fudan Univ. J. Med. Sci. 36, 142-148. Yi, X., Zhang, F., Xu, W., Li, J., Zhang, W., Mai, K., 2014. Effects of dietary lipid content on growth, body composition and pigmentation of large yellow croaker Larimichthys croceus. Aquaculture 434, 355-361. Yi, X., Xu, W., Zhou, H., Zhang, Y., Luo, Y., Zhang, W., Mai, K., 2014. Effects of dietary astaxanthin and xanthophylls on the growth and skin pigmentation of 28

Journal Pre-proof

large yellow croaker Larimichthys croceus. Aquaculture 433, 377-383. Zhang, M., 2009. The study of hawthorn extract in broilers. Henan Agricultural University, pp. 63. Zhang, M., Sun, Y., Fan, W., Wang, Z., 2009. Effects of hawthorn extract on growth performance and intestinal microflora of broilers. Acta Agr. Boreali-Occidentalis

oo

f

Sin. 18, 52-55. Zhang, Q., Yu, H., Tong, T., Tong, W., Dong, L., Xu, M., Wang, Z., 2014. Dietary

pr

supplementation of Bacillus subtilis and fructooligosaccharide enhance the

e-

growth, non-specific immunity of juvenile ovate pompano, Trachinotus ovatus

Pr

and its disease resistance against Vibrio vulnificus. Fish Shellfish Immun. 38, 7-14.

rn

al

Zhao, J., Bu, Y., Liu, Z., Shi, M., Xu, X., Song, X., 2014. A TNBS-induced enteritis model in grass carp. J. Fish. China 38, 559-568.

Jo u

Zou, Y., Xiang, Q., Wang, J., Peng, J., Wei, H., 2016. Orega no essential oil improves intestinal morphology and expression of tight junction proteins associated with modulation of selected intestinal bacteria and immune status in a pig model. Biomed Res. Int. 2016, 1-11.

29

oo

f

Journal Pre-proof

pr

Fig. 1. Effect of dietary supplementation with hawthorn extracts on foregut

e-

morphology in golden pompano. 0, 0.5, 1.0, 2.0, 4.0 and 10.0 represented the fish fed

Pr

diets supplemented with 0, 0.50, 1.00, 2.00, 4.00 and 10.00 g kg-1 hawthorn extracts,

al

respectively. Black arrows indicating gut impairments. Scale bar: 100 μm.

rn

2.00

Jo u

Expression relative to β-actin

2.50

d c

d

1.50

cd

bc

1.00

bc

bc

bc

b b a ba b

ab

b

b

a

c bc

a 0.50

d

a

a

ab

a

0.00 GPx

CAT 0

0.50

1.00

GR 2.00

4.00

Keap1 10.00

Fig. 2. Effect of dietary hawthorn extracts on relative expression of antioxidant capacity related genes in the intestine of golden pompano. 0, 0.50, 1.00, 2.00, 4.00, 10.00 represented the fish fed diets supplemented with 0, 0.50, 1.00, 2.00, 4.00 and 30

Journal Pre-proof 10.00 g kg-1 hawthorn extracts, respectively. Values were means (n=3), bars bearing the same letters were not significantly different (P > 0.05). GPx, glutathione peroxidase; CAT, catalase; GR, glutathione reductase; Keap1, Kelch-likeECH-associated protein 1.

3.00

e

c

2.50 cd

1.50 a

b

ab

a a a a

a

a a

c

ab

bc

ab ab a

pr

1.00

b

f

b

bc

oo

b

2.00

0.50

0.00 IL10 0

0.50

1.00

TGFβ1

2.00

4.00

Pr

IL8

e-

Expression relative to β-actin

d

TOR

10.00

al

Fig. 3. Effect of dietary hawthorn extracts on relative expression of interleukin 8

rn

(IL-8), interleukin 10 (IL-10), transforming growth factor β1 (TGF-β1) and target of

Jo u

rapamycin (TOR) in the intestine of golden pompano. 0, 0.50, 1.00, 2.00, 4.00, 10.00 represented the fish fed diets supplemented with 0, 0.50, 1.00, 2.00, 4.00 and 10.00 g kg-1 hawthorn extracts, respectively. Values were means (n=3), bars bearing the same letters were not significantly different (P > 0.05).

31

Journal Pre-proof

Expression relative to β-actin

2.50

c

2.00 c

d b

1.50

1.00

bc

b b

c

cd

bc ab

c b

ab

a a a

ab

a

a

0.50

a a a a

0.00

0

0.50

1.00

occludin 2.00

4.00

10.00

claudin3a

f

zo-3

oo

zo-1

pr

Fig. 4. Effect of dietary hawthorn extracts on relative expression of tight junction

e-

protein genes in the intestine of golden pompano. 0, 0.50, 1.00, 2.00, 4.00, 10.00

Pr

represented the fish fed diets supplemented with 0, 0.50, 1.00, 2.00, 4.00 and 10.00 g kg-1 hawthorn extracts, respectively. Values were means (n=3), bars bearing the same

Jo u

zonula occludens-3.

rn

al

letters were not significantly different (P > 0.05). Zo-1: zonula occludens-1, zo-3:

32

Journal Pre-proof Table 1 Composition and nutrient levels of experimental diets (g kg-1 ) Basal diet

Fish meal

300.00

Peanut meal

180.00

Soybean meal

120.00

Flour

254.40

Beer yeast powder

50.00

Fish oil

30.00

Soybean oil

30.00

Lecithin

10.00

oo

pr

e-

Vitamin premix1

Pr

5.00

Mineral premix2

Jo u

rn

Antioxidant

al

5.00

Choline chloride (50% )

Vitamin C

f

Ingredients

Monocalcium phosphate

5.00 0.10 0.50 10.00

Nutrient levels (%) Moisture

7.43

Crude protein

44.81

Crude lipid

10.66

Ash

8.92

1, 2

Mineral and vitamin premix provided by Guangzhou Fishtech Co., Ltd., China

33

Journal Pre-proof 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 K 3 (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): Na2 SeO3 (1%) 20 mg,

CuSO 4 ·5H2 O (25%) 24 mg, FeSO 4 ·H2 O (30%) 266.65 mg, ZnSO 4 ·H2 O (34.50%) 100 mg, MnSO 4 ·H2 O (31.80%) 120 mg, Ca(IO 3 )2 (5%) 50 mg, CoSO 4 ·7H2 O (5%) 10 mg,

oo

f

zeolite power 4380.55 mg.

Table 2 Effects of dietary hawthorn extract on plasma biochemical indices of golden

0

0.50

1.00

2.00

4.00

10.00

3.91±0.08c

2.88±0.08a

3.44±0.02abc

2.89±0.12a

3.60±0.27bc

3.11±0.39ab

2.66±0.13d

1.56±0.21ab

1.51±0.33a

1.96±0.04bc

2.27±0.03cd

1.64±0.00ab

3.50±0.71a

3.00±0.00a

2.50±0.71a

2.67±0.58a

3.50±0.71a

5.50±0.71b

19.00±8.49a

19.50±6.36a

19.00±8.49a

22.67±2.08a

28.00±1.41a

72.50±7.78b

13.11±1.29bcd 11.77±1.56bc

AST/( U L-1 ) GLU/(mmol L-1 ) TP/(g L-1 ) LDL/( mmol L-1 ) HDL/( mmol L-1 )

al

ALT/(U L-1 )

14.58±0.71d

rn

TG/(mmol L-1 )

13.62±1.58cd

10.88±0.23ab

9.14±0.16a

29.50 ±1.13

29.00 ±3.54

29.20 ±2.26

29.75 ±2.62

0.29±0.03b

0.22±0.02a

0.25±0.02ab

0.19±0.01a

0.21±0.01a

0.25±0.04ab

1.87±0.11a

2.18±0.04c

2.09±0.11bc

1.95±0.00ab

1.99±0.02ab

1.89±0.06a

Jo u

CHO/( mmol L )

Pr

-1

e-

Diets ( HTE g kg-1 )

pr

pompano (Trachinotus ovatus)

33.50 ±2.91

Values are means ± SD of three replications. Means in the same raw with different superscripts are significantly different (P<0.05). CHO: cholesterol; TG: triglyceride; ALT: alanine aminotransferase, AST: aspartate aminotransferase; GLU: glucose; TP: total protein; LDL: low density lipoprotein; HDL: high density lipoprotein.

34

27.20 ±0.71

Journal Pre-proof

0

0.50

1.00

2.00

51.10±1.09b

33.37±1.42a

33.38±1.45a

72.47±1.21

71.70±1.09

Crude protein Crude lipid

Jo u

Muscle Moisture

10.00

64.60±1.40

66.55±0.89

65.57±0.86

53.63±0.75c

48.87±0.51a

49.87±0.68ab

49.51±0.78ab

35.89±0.94ab

35.99±0.17ab

34.54±0.51ab

36.97±0.95b

72.01±0.84

72.60±1.32

72.73±0.73

72.64±0.79

Pr

48.56±1.94a

64.90±1.54

al

Crude lipid

64.79±0.08

rn

Crude protein

65.50±0.30

e-

Whole body Moisture

4.00

pr

Diets (HTE g kg-1 )

oo

composition of golden pompano (Trachinotus ovatus)

f

Table 3 Effects of dietary hawthorn extract on whole body and muscle proximate

71.79±1.89

70.08±2.74

71.48±1.11

71.14±2.65

71.23±0.89

71.13±1.17

22.41±1.64

22.07±1.10

21.35±0.38

21.19±0.98

20.78±0.51

20.29±0.50

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

35

Pr

e-

pr

oo

f

Journal Pre-proof

Table 4 Effects of dietary hawthorn extract on dorsal skin and ventral skin color of

0

0.50

1.00

2.00

4.00

10.00

rn

Diets (HTE g kg-1 )

al

golden pompano (Trachinotus ovatus)

Light (L*) Redness (a*) Yellowness (b*)

Jo u

Dorsal skin 58.83±3.05

59.09±4.34

58.33±2.15

58.52±3.86

60.30±4.37

57.69±6.74

0.82±0.54

1.53±0.62

1.22±0.69

1.40±0.48

0.97±0.38

1.71±0.91

-3.22±1.26

-2.79±1.08

-3.52±1.21

-3.21±1.37

-4.37±1.48

-2.78±1.07

92.73±4.44

91.06±4.24

93.40±3.25

94.03±3.00

92.52±4.83

91.92±4.14

-2.49±0.55

-2.00±0.62

-2.05±1.20

-2.43±0.40

-2.39±0.46

-2.27±0.54

1.31±0.81a

2.86±0.73b

2.63±1.29ab

3.72±1.95c

4.75±2.20c

2.71±1.02ab

Ventral skin Light (L*) Redness (a*) Yellowness (b*)

Values are means ± SD of three replications. Means in the same raw with different superscripts are significantly different (P<0.05). 36

Journal Pre-proof

Table 5 Effects of hawthorn extract on foregut morphology of golden pompano (Trachinotus ovatus)

0

0.50

1.00

2.00

4.00

10.00

Villus number

39.00±3.61b

35.67±2.08ab

35.67±1.53ab

31.33±1.53a

38.00±2.65ab

41.00±7.55b

Villus length (um)

1038.70±

1241.50±

1074.20

1197.90

1075.90

961.39

113.35b

131.96c

±141.84b

±133.40c

±135.68b

±129.94a

Villus width (um)

68.42±8.87a

85.94±7.06b

83.07±9.08b

85.52±8.36b

71.81±9.13a

71.77±8.77a

Muscle thickness

118.72±

146.96±

123.12±

122.18±

117.80±

114.03±

(um)

29.22a

19.38b

21.26a

18.72a

21.88a

19.68a

Jo u

rn

al

Pr

e-

pr

oo

f

Diets (HTE g kg-1 )

37

Journal Pre-proof Highlights 1. Dietary hawthorn extract (HTE) supplementation had hypolipidemic effects in golden pompano. 2. Dietary HTE supplementation improved skin coloration of golden pompano. 3. Dietary supplementation of 0.50 and 1.00 g kg-1 HTE could improve intestinal

Jo u

rn

al

Pr

e-

pr

oo

f

morphology and enhance its barrier function.

38

Journal Pre-proof Conflict of interest

We declare that we do not have any commercial or associative interest that

Jo u

rn

al

Pr

e-

pr

oo

f

represents a conflict of interest in connection with the work submitted

39