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Please cite this article as: Hong-Bo Lv, Food Chemistry, https://doi.org/10.1016/j.foodchem.2020.128479

Available online 28 October 20200308-8146/© 2020 Elsevier Ltd. All rights reserved.

The individual and combined effects of hypoxia and high-fat diet feeding on nutrient composition and flesh quality in Nile tilapia (Oreochromis niloticus)

Hong-Bo Lv, Ying-ying Ma, Chun-Ting Hu, Qiu-Yu Lin, Jun-jia-yu Yue, Li-Qiao Chen, Mei-Ling Zhang, Zhen-Yu Du *, Fang Qiao *

LANEH, School of Life Sciences, East China Normal University, Shanghai 200241, PR China

A R T I C L E I N F O

Keywords: Oreochromis niloticus Hypoxia High fat diet Nutritional value Flesh quality Growth

A B S T R A C T

Hypoxia and high-fat diet (HFD) feeding are two factors commonly existing in aquaculture. However, their in-dividual and combined effects on nutrient composition and flesh quality in fish have not been investigated. The present study evaluated the alterations of growth, nutrient composition and flesh quality in Nile tilapia (initially 7.0 ± 0.1 g and 5.6 ± 0.2 cm) fed with normal fat diet (5.95% fat) or HFD (11.8% fat) at two dissolved oxygen levels (1.1 ± 0.1 and 7.2 ± 0.1 mg/L) for 8 weeks. The results showed that hypoxia and HFD had similar effects in inducing lipid deposition, reducing flesh protein and amino acids content, pH values and water holding ability. Hypoxia had additional adverse effects in decreasing meat yield, flesh contents of n-3 PUFA and glycogen, increasing flesh fragmentation and causing liver damages. The combination of hypoxia and HFD significantly decreased feed intake, survival rate and muscle protein content, but didn’t affect flesh quality-related parameters.

1. Introduction

Fish is one of the primary protein sources for the human being, and currently, almost half of the global fish production are sourced from aquaculture (FAO, 2020). In the practical aquaculture, the high-density aquaculture pattern and formulated diets have been widely used (Benjamín, Claudia, Dias, Antonio & Luís, 2013; Liu, Liu, & Sun, 2017). However, a number of studies have shown that improper aquaculture pattern and nutrient-unbalanced diets would impair fish flesh quality, including the reduction of nutritional values and changes of texture parameters (Bjrnevik, Hansen, Roth, Foss, & Imsland, 2017; Refaey et al., 2018). Therefore, maintaining high fish flesh quality in the current aquaculture has been the main concern either for researchers or for popular consumers (Hosseini, Abedian-Kenari, Rezaei, Nazari, Feas, & Rabbani, 2010). It has been reported that fish flesh quality, including nutritional composition, chemical property (pH) and a series of physical parameters (color, water loss rate, hardness, firmness elasticity, etc.), are primarily affected by dietary components or environmental factors (Bjrnevik et al., 2017; Ostbye et al., 2018). However, detailed infor-mation about the effects of diets or environmental factors on fish flesh quality are still limited. Particularly, the effects of the combination of dietary components and environmental factors, which is very common

in practical aquaculture, in affecting fish flesh quality is rarely investigated.

Dissolved oxygen of water is one of the most important environ-mental factors in aquaculture. Hypoxia is a serious environmental stress, and occurs frequently in the high-density aquaculture (Refaey et al., 2018). It has been reported that different degrees of hypoxia seriously affect growth, survival, behavior and physiological activities of fish (Roman, Brandt, Houde, & Pierson, 2019). Hypoxia could also cause the activation of anaerobic respiration, decrease of glycogen content and increase of serum lactate content (Zhao, Cui, Liu, Sun, & Yang, 2020). Moreover, it was reported that hypoxia could change the amounts of whole-fish body composition in Nile Tilapia (Abdel-Tawwab et al, 2014). However, the effects of hypoxia on flesh quality, including muscle nutrient composition and flesh texture parameters, in economic fish have not been well studied.

Due to the limitation of traditional dietary protein sources, such as fish meal, high energy diets (high carbohydrate or high lipid diet) have been commonly applied in aquaculture to save dietary protein and cost (Jia, Cao, Du, He, & Yin, 2020). However, high fat diet (HFD) would also cause a series of physiological or chemical changes in fish muscle, including the increase of lipid (intramuscular fat) and decrease of water content, thus changing the tenderness, succulence and flavor of the

* Corresponding authors. E-mail addresses: zydu@bio.ecnu.edu.cn (Z.-Y. Du), fqiao@bio.ecnu.edu.cn (F. Qiao).

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier.com/locate/foodchem

https://doi.org/10.1016/j.foodchem.2020.128479 Received 11 May 2020; Received in revised form 21 October 2020; Accepted 22 October 2020

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flesh, as previously reported in triploid brown trout Salmo trutta (Regost, Arzel, Cardinal, Laroche, & Kaushik, 2001). In fact, because of the frequent occurrence of hypoxia and wide use of HFD in aquaculture, hypoxia and HFD usage could usually exist together in the practical aquaculture. However, to date, the interaction between HFD and hyp-oxia on flesh quality has not been investigated yet.

Tilapia is a kind of worldwide-farmed fish, and its fillets are also common food for human. Because tilapia has stronger stress resistance than most of other fish species, high-density culture and hypoxia commonly occur in the practical tilapia culture (Abdel-Tawwab et al, 2014). At the same time, the dietary fat content of tilapia feed has been increasing in recent years (Zhang et al., 2020). However, the effects of hypoxia and dietary fat content on the flesh quality of tilapia have not been intensively studied. Therefore, this study aims to explore the in-dividual and/or interactive effects of dietary fat level and water dis-solved oxygen on growth, nutrient composition and texture parameters of Nile Tilapia. The present study could provide overall information to understand the effects of hypoxia and HFD on the flesh quality of Nile Tilapia.

2. Materials and methods

2.1. Animal ethics approval

All experimental procedures and fish used in this study were gov-erned strictly under the Guidance of the Care and Usage of Laboratory Animals in China and approved by the Committee on Ethics of Animal Experiments of East China Normal University.

2.2. Fish and experimental design

Juvenile Nile tilapia (Oreochromis niloticus) were purchased from a local farm in Guangzhou, and acclimated in 6 tanks (440 L) for 2 weeks

before the experiment. During the acclimation, the fish were fed with a commercial diet (33% protein, 5% fat) twice daily to satiation, and the water dissolved oxygen was maintained at 7.17 ± 0.20 mg/L by air pumping. Afterwards, healthy fish with similar initial body weight (7.0 ± 0.14 g) and body length (5.6 ± 0.24 cm) were selected and distributed randomly into 12 fiberglass tanks (240L, 60 × 80 × 50 cm) in a closed cultivation system with 40 fish per tank and 3 tanks per group. The 4 treatment groups were set as: medium fat diet (MFD)-normoxia, MFD- hypoxia, HFD-normoxia and HFD-hypoxia. In the control group (nor-moxia), the dissolved oxygen in water was maintained at 7.22 ± 0.13 mg/L by air pumping. To create hypoxia condition, very tiny amounts of air was pumped into 6 tanks and the dissolved oxygen in the water was controlled at 1.14 ± 0.10 mg/L. The dissolved oxygen values were measured by using a dissolved oxygen analyzer (Hach hq30d, America) twice per day (8:30, 18:30). In the hypoxia or normoxia group, half fish (3 tanks) were fed with MFD (6% fat) and the rest (3 tanks) were fed with HFD (12% fat). During the acclimation and the experimental period, the water temperature was maintained at 28.0 ± 2.0 ◦C by using an automatic temperature controller and pH value was at 7.4 ± 0.2. The total ammonia nitrogen was <0.02 mg/L and the light was set at a 10-h/ 14-h light/dark cycle by using an automatic time controller (8:00–18:00 light/18:00–8:00 dark). The dietary feeding rate was set as 4% of total body weight of all fish in each tank, and the fish were fed twice daily. The total body weight of all fish in each tank were weighed every two

weeks and the daily feeding amount was changed accordingly. Fish wastes were siphoned every day along with 1/3 of the tank water, which was replaced by well-aerated dechlorinated tap water from a storage tank.

During the 8-week feeding trial, all diet residues were collected after 15 min of feeding, and then dried at 60 ◦C to calculate the feed intake. The weight of fish in each tank were weighed every 2 weeks and the feeding amounts were adjusted accordingly. The formulation and proximate composition of two experimental diets are shown in Supple-mental Table 1. The feed was pelleted to proper size (1.0 mm pellet diameter), and the pellets were dried in a cool and well-ventilated room for about 12 h and then kept at − 20 ◦C.

2.3. Sampling

At the end of the experiment, 9 fish from each group (3 fish/tank) were fasted overnight and sacrificed by anesthetizing with MS-222 (20 mg/L). Blood samples (1 mL) were collected with 1 mL heparinized syringes from anesthetized fish. Next, the serum was separated from blood cells by a centrifuge set at 10000 rpm for 10 min. Then, the upper serum (200 uL) was transferred into new cubes (2 mL), and maintained at − 80 ◦C until analysis. After the blood was withdrawn, the liver and mesenteric fat were collected and weighed for each sacrificed fish. Additionally, carcasses were weighted after cutting off the fins, tail and head. By careful dissection, dorsal muscles without subcutaneous adi-pose tissue were sampled using a sharp knife. All flesh muscle samples were divided into two parts, half were prepared for texture analysis, the other half were rapidly frozen in liquid nitrogen and stored in − 80 ◦C immediately for further analysis.

The feed intake, weight gain, survival rate, Feed conversion ratio (FCR), hepatosomatic index (HSI), mesenteric fat index (MFI), condition factor, carcass ratio and meat yield measurements were calculated by the following formulae:

Survival rate (%) = 100 × (Final fish number/Initial fish number)

Feed conversion ratio = Feed weight/(Final fish weight − Initial fish weight)

Hepatosomatic index(%) = 100 × (Hepatic weight/Body weight)

Mesenteric fat index (%) = 100 × (Mesenteric fat weight/Body weight)

Condition factor(%) = 100 ×(Fish wet weight/Total length3)

Carcass ratio(%) = 100 × [(Body weight − Head weight − Fin weight

− Visceral weight)/Body weight ]

Meat yield(%) = 100 × [(Body weight − Head weight − Fin weight

− Visceral weight − Bone weight)/Body weight ]

There are triplicate for final weight, weight gain, survival rate, FCR and FI assay, and 6 fish for condition factor, carcass ratio, HIS, and MFI measurement.

Feed intake (g/d/fish) = (Feed weight − Residue weight)/Number of days/Number of fishWeight gain(%)

= 100 × (Final fish weight − Initial fish weight/Initial fish weight)

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2.4. Biochemical indexes assay

The activities of glutamic propylic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), the content of serum glucose and tri-glycerides (TG), and the content of lactic acid and glycogen in tissue were measured by using commercial kits (Nanjing Jiancheng Bioengi-neering Institute, China). Six serum or tissue samples from each group were collected, and the values of these indexes were measured by using spectrophotometry method.

For serum glucose content assay, H2O2 was produced with the oxidation of glucose to gluconic acid catalyzed by glucose oxidase. Then peroxidase catalyzed the redox-coupled reaction of H2O2 with 4-amino-antipyrine and N-Ethyl-N-(3-sulfopropyl)-m-anisidine, producing a brilliant pruple color. The absorbance was determined at 505 nm. The serum glucose content was calculated as: (absorbance of serum – absorbance of blank)/(absorbance of standards – absorbance of blank) × concentration of standards.

For the tissue glycogen content measurement, the glycogen was determined by sulfuric acid-anthrone method. The absorbance was read at 620 nm in a microplate reader. The tissue glycogen content (mg/g tissue) was calculated as (absorbance of supernatant – absorbance of blank)/(absorbance of standard – absorbance of blank) × sample dilu-tion ratio × 0.01a × 10b/1.11c. “a” indicates the standard concentration, “b” indicates dilution multiple during the test, “c” indicates the coeffi-cient of converting measured glucose into real glycogen content.

The TG colorimetric assay used the enzymatic hydrolysis of TG by lipase to produce glycerol and free fatty acids. After the released glycerol was phosphorylated and oxidased, H2O2 was produced. Then peroxidase catalyzed the redox-coupled reaction of H2O2 with 4-aminoantipyrine and N-Ethyl-N-(3-sulfopropyl)-m-anisidine, producing a brilliant pru-ple color. The absorbance was determined at 510 nm. The TG content was calculated as (absorbance of serum – absorbance of blank)/ (absorbance of standards – absorbance of blank) × standard concentration.

For the lactate content assay, lactic dehydrogenase catalyzed lactate and NAD+ to produce pyruvic acid and H+. Then PMS was reduced by H+ and produced purple substance which has the absorbance of 530 nm. The lactic acid content was calculated as (absorbance of supernatant – absorbance of blank)/(absorbance of standard – absorbance of blank) ×standard concentration.

For the serum GPT activity assay, GPT catalyzed alanine and α-ketoglutarate to produce pyruvic acid and glutamic acid. Then 2, 4- dinitrophenylhydrazine reacted with pyruvic acid to produce phenyl-hydrazone, which showed red brown under alkaline condition at the absorbance of 510 nm. The GPT activity was calculated by the standard curve.

For the serum GOT activity assay, GOT catalyzed aspartic acid and α-ketoglutarate to produce glutamic acid and oxaloacetic acid, and oxaloacetic acid could turn into pyruvic acid by dehydroxylation. Then 2, 4-dinitrophenylhydrazine reacted with pyruvic acid to produce phe-nylhydrazone, which showed red brown under alkaline condition at the absorbance of 510 nm. The GOT activity was calculated by the standard curve.

All the procedures were carried out according to the commercial kits instruction. The absorbance was determined by using the microplate reader (Epoch, BioTek, USA).

2.5. Body composition analysis and muscle lipid separation

Six fish per group were selected randomly for determining the proximate composition of the whole fish body. The moisture content was determined by drying in the hot air oven at 105 ◦C for 15 h to constant weight. The total protein of whole fish and muscle were determined by a semi-automatic Kjeldahl System (FOSS, Sweden) after acid digestion. These parameters were assayed according to Association of Official Analytical Chemists (AOAC, 2005). The lipid content in the

whole body and muscle was determined after extraction by chloroform/ methanol (2/1, v/v) (Folch, Less, & Sloane Stanley, 1957). The lipid extracted from muscle was stored in − 20 ◦C for further analysis.

The separation of TG from muscle lipids was performed by thin layer chromatography (TLC) at room temperature as previously reported (Liu et al., 2019). TG of sample was separated by a solvent system containing isohexane /ether/acetic acid (80/25/1.5, v/v/v) for 160 min. After drying the TLC plates at 50 ◦C for 20 min, the plates were developed for 20 min in the iodine chambers. The TG in samples was identified and the content was calculated by comparing with the strip place and area of TG standard in the same TLC plate, respectively, in TLC scanner (KH-3000, KEZHE, Shanghai). Six muscle samples per group were used for TG content assay.

2.6. Fatty acid and amino acid analysis

Six muscle samples were used for fatty acid determination. The fatty acid compositions of muscle samples were determined according to the methods described by Liu with minor modifications (Liu et al., 2019). The dried lipid samples were added 0.5 mL methanolic KOH (2 mol/L) at 65 ◦C for 30 min. Two milligram of 14% BF3-CH3OH was added for FAMEs derivatization. The mixed liquid was heated at 75 ◦C for 30 min. After adding 1 mL of hexane (for HPLC, >95%) and 1 mL of ddH2O, the sample was centrifuged at 2000 rpm for 5 min. Afterwards, the upper layer, which dissolved fatty acids, was move to a new cube. The analysis was carried out using gas chromatography (Shimadzu 2010 Plus, Japan) equipped with a 100 m × 0.25 mm ID × 0.2 μm fused silica capillary column (RESTEK, USA). High purity nitrogen (99.99%) was used as the carrier gas with the flow rate of 7.8 mL/min. Temperature gradient rises from 150 ◦C to 200 ◦C at 4 ◦C/min and maintained at 240 ◦C for 30 min. The injector and detector temperatures were set at 250 and 260 ◦C, respectively. Fatty acids were identified by retention times compared with known standard mixtures (Supelco 37 Component FAME Mix in Dichloromethane, USA). By calculating the peak area ratio of each fatty acid (see the Supplemental Material 2 and 3), the result was presented as the relative percentages of each fatty acid (% total fatty acids), and the details of fatty acid composition were listed in Supplemental Table 2.

In the determination of amino acid composition, the muscles from 6 fish per group were collected and two fish were mixed as a pooled sample (a total of 3 pooled samples per group, n = 3). Twenty milligrams of dry samples were put into a glass tube with the addition of 2 mL of 6 N HCl, 0.1% (v/v) phenol and 2% (v/v) thiodiglycol, and the tubes were maintained at 110 ◦C for 24 h. After dried by pumping N2, 0.02 N HCl was added into the sample for dissolving the amino acid. The analysis was performed by amino acid automatic analyzer (Hitachi L-8900, Japan). By comparing the peak area of the standard, the contents of amino acids in muscle were presented as absolute concentration (mg/g muscle), and the details of amino acid composition were listed in Sup-plemental Table 3.

2.7. Measurement of flesh quality parameters

2.7.1. pH measurement The assessment of pH was performed according to a previous study

with minor modification (Wiklund, Finstad, Johansson, Aguiar, & Bechtel, 2008). Fresh muscle (1 g) was homogenized with deionized water (1/2, w/w). The pH of the homogenate was detected by pH Meter (PHSJ-3F, Leici, China) and used to represent the pH of fillet. Muscle samples of 6 fish per group were used for pH measurement (n = 6).

2.7.2. Centrifugal water loss and cooking loss Fresh muscle samples (1 g) were centrifuged in plastic pipe at

1000×g for 30 min at 4 ◦C. After drying the surface moisture using common qualitative filter paper, the centrifugal weight loss (%) was calculated as 100 × (the weight before centrifuge – the weight after centrifuge) /the weight before centrifuge (Sanchez-Alonso, Haji-Maleki,

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& Borderias, 2007). A high value of centrifugal weight loss indicates poor water holding capacity.

Cooking loss was detected by a similar method. Fresh muscle samples (1 g) were placed in centrifuge tubes, which have small holes to drain the drip. Then all tubes were cooked in a water bath at 70 ◦C for 15 min. After removing the drips, the samples were reweighed. Cooking loss was calculated by the following formulae:

Cooking loss (%) = 100 × (the weight before cooking – the weight after cooking) / the weight before cooking (Sanchez-Alonso et al., 2007).

Muscle samples of 6 fish per group were used for centrifugal water loss and cooking loss measurement (n = 6).

2.7.3. Total myoglobin measurement The total myoglobin of muscle samples was determined according to

a previous study (Yu et al., 2017). The white muscle (0.5 g) was coarsely minced and mixed with 1.5 mL of cold extracting medium (10 mM Tris–HCl, pH 8.0, containing 1 mM EDTA and 25 g/L Triton X-100). After complete homogenization for 90 s at 60 HZ by a homogenizer (Jinxin Tissuelyser-24, China), the mixture was centrifuged at 9600 × g for 10 min at 4 ◦C, and then the supernatant was filtered through a common qualitative filter paper. A preliminary assay was conducted to make accurate dilution curve before the formal assay. The results were presented as milligrams of total myoglobin per gram of muscle, calcu-lated from the millimolar extinction coefficient of the myoglobin in the filtrate at 576 nm using Micro Plate Spectrophotometer (BioTek Epoch, USA). Muscle samples of 6 fish per group were used for total myoglobin measurement (n = 6).

2.7.4. Hardness evaluation Fragmentation index was determined according to a published

method (Veiseth, Shackelford, Wheeler, & Koohmaraie, 2001). A flesh sample (1 g) was cut into small pieces and placed in a centrifuge tube with the addition of 1.5 mL of cold sucrose (0.24 M) and potassium chloride (0.02 M) solution. Then all samples were homogenized for 60 s at 60 Hz in a TissueLyser (Jingxin, China). The homogenate was then filtered through the sieve (250 μ pore size). The residue and sifter were blotted twice on an absorbent towel immediately and then weighed. Fragmentation index was calculated as follows: Fragmentation index (%) = 100 × (the weight of residue and sifter – the weight of sifter)/ initial sample weight. A low value of fragmentation index indicates low hardness. Muscle samples of 6 fish per group were used for hardness evaluation (n = 6).

2.8. Total RNA extraction, cDNA synthesis and quantitative real-time PCR (qRT-PCR) analysis

Total RNA was isolated by using a Tri Pure Reagent (Aidlab, China) according to the manufacturer’s protocol. RNA integrity was determined by 1% agarose gel electrophoresis. The quality and quantity of total RNA were tested by NanoDrop 2000 Spectrophotometer (Thermo Fisher, USA). Only RNA with an OD260/OD280 between 1.8 and 2.0 was used. The cDNAs were synthesized using a PrimerScriptTM RT reagent Kit with a gDNA Eraser (Perfect Real Time) (RR047A, Takara, Japan) ac-cording to the manufacturer’s protocol. Elongation factor 1 alpha (EF1α) and β-actin were used as double reference genes. Primer specificity was analyzed by melting curve analysis and product size determination by NCBI. RT-qPCR was performed in 96-well plates in Real-Time System (CFX Connect, Bio-Rad) with gene-specific primers (Supplemental Table 4). 2 μL cDNA was mixed with 10 μL 2 × Ultra SYBR Mixture (Aidlab, China), 2 μL forward and reverse (1:1) gene primers and 6 μL distilled water, and the total PCR reaction volume was 20 μL. The

Fig. 1. Influences of hypoxia and dietary lipid content on growth, survival and organ mass in Nile tilapia. (A) Final weight (g); (B) Weight gain (%); (C) Survival rate (%), CC means MFD – normoxia group (Control-Control), CL means MFD – hypoxia group (Control-Low oxygen), HC means HFD – normoxia group (HFD-Control), HL means HFD – hypoxia group (HFD-Low oxygen); (D) Feed Conversion Ratio (FCR); (E)Eight-week average feed intake (g/fish); (F) Condition factor (%); (G) Carcass ratio (%); (H) Hepatosomatic index (%); (I) Mesenteric fat index (%). Data are presented as mean ± SEM (n = 3 in A-E; n = 6 in F-I). # and ## indicates significant difference (p < 0.05) and very significant difference (p < 0.01), respectively, between different oxygen levels. The significant difference (p < 0.05) between the MFD and HFD groups is presented by *.

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procedure was 95 ◦C for the 30 s, 36cycles of 95 ◦C for 5 s and 60 ◦C for 20 s. Each qRT-PCR reaction was performed in duplicate. The expression levels were obtained from the data of the crossing points (Cp). The relative mRNA expressions were calculated by using the 2− ΔΔCt method. The RNA of 6 fish per group were isolated for qRT-PCR analysis (n = 6).

2.9. Statistical analysis

The data were expressed as mean ± SEM. The significant difference of each parameter between two given groups was tested using the in-dependent-samples t-test. In order to detect the possible interactions between the dietary lipid content and dissolve oxygen concentration in each parameter, the one-way ANOVA test was firstly used to detect the significant difference among all groups, and then the two-way ANOVA analysis was applied to calculate the individual or combined effects of dietary lipid content and dissolve oxygen concentration on the results. The statistical results of two-way ANOVA analysis were listed in Sup-plemental Table 5–9. The levels of statistical difference were set at P <0.05 (significant) and P < 0.01 (very significant). All data were carried out using the Statistical Package for the Social Sciences (SPSS) 23 soft-ware (IBM, Armonk, USA).

3. Results

3.1. Influences of hypoxia and dietary lipid content on growth, survival and organ mass

In both MFD and HFD groups, the fish in hypoxic condition showed significantly reduced final weight, weight gain, survival rate, feed intake and carcass ratio (Fig. 1A-C, E, G), while significantly increased hep-atosomatic index (HSI) (Fig. 1H). The increased mesenteric fat index (MFI) was only observed in the MFD-hypoxia group (Fig. 1I). And the hypoxia only decreased FCR in HFD group (Fig. 1D).

Compared to the MFD-fed fish, the HFD-fed fish showed the signif-icantly lower survival rate in both hypoxia and normoxia groups (Fig. 1C), and significantly reduced feed intake only in the hypoxia group (Fig. 1E). Moreover, the HFD-fed fish showed significantly increased final weight, weight gain, HSI and MFI only in normoxia status (Fig. 1A, B, H, I).

The two-way ANOVA test indicated that the interactions between dissolved oxygen and dietary lipid were seen in final survival rate, feed intake, HSI and MFI (Supplemental Table 5). This suggests that hypoxia could damage the growth and health of Nile tilapia, and reduced edible mass ratio in fish. Besides, the combination of HFD and hypoxia would result in higher death rate than the individual factor.

Fig. 2. Influences of hypoxia and di-etary lipid content on body composi-tion and serum biochemical indexes in Nile tilapia. (A) Whole body protein (% dry weight); (B) Whole body lipid (%); (C) Whole body moisture (%); (D) Hepatic glycogen (mg/g); (E) Hepatic TG (mmol/g); (F) Serum glucose (mmol/L); (G) Serum lactate (mmol/ L); (H) Serum TG (mmol/L); (I) Serum GPT (U/L); (J) Serum GOT (U/L). Data are presented as mean ± SEM (n = 6). # means significant difference in different oxygen levels (p < 0.05). * and ** indicates significant difference (p < 0.05) and very significant differ-ence (p < 0.01), respectively, between the MFD and HFD groups.

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3.2. Influences of hypoxia and dietary lipid content on body and liver composition

In the MFD groups, the hypoxia-treated fish showed less content of whole body protein and hepatic glycogen, but displayed higher content of whole body lipid and hepatic TG than those under normoxia state (Fig. 2A, B, D, E). However, hypoxia did not affect whole body moisture in MFD or HFD group (Fig. 2C).

In the normoxia groups, the HFD-fed fish showed lower body protein and moisture (Fig. 2A, C), but higher body lipid and hepatic TG (Fig. 2D, E) than the MFD-fed fish. Moreover, in the hypoxia state, the content of hepatic glycogen in the HFD group was higher than that in the MFD group (Fig. 2D).

The two-way ANOVA assay indicated that the interactions of dietary lipid content and dissolved oxygen were observed in the whole body protein, whole body lipid and hepatic TG (Supplemental Table 6). These results indicate that the hypoxia treatment in MFD feeding and the HFD

treatment in normoxia condition had the similar effects in reducing body protein and increasing lipid content in whole body and liver. Moreover, the dissolve oxygen and dietary lipid had interplaying effects on body and liver composition in Nile tilapia.

3.3. Influences of hypoxia and dietary lipid content on serum biochemical indicators

In the MFD groups, hypoxia induced lower serum glucose content and higher lactate than those in the normoxia state (Fig. 2F, G). Besides, in both MFD and HFD groups, hypoxia significantly enhanced serum GPT and GOT activities, which are the well-known liver damage markers (Fig. 2I, J).

As compared with the MFD group, HFD significantly increased serum glucose, lactate and TG contents in hypoxia and normoxia states, respectively (Fig. 2F-H).

The two-way ANOVA test indicated the interactions of dissolved

Fig. 3. Influences of hypoxia and dietary lipid content on flesh quality in Nile tilapia. (A) Total protein (%); (B) Total lipid (%); (C) Moisture (%); (D) Glycogen (mg/ g); (E) Lactate (mg/g); (F) TG (mg/g); (G) Meat yield (%); (H) Centrifugal water loss (%); (I) Cooking loss (%); (J) pH; (K) Fragmentation index (%); (L) Flesh myoglobin (mg/g). Data are presented as mean ± SEM (n = 6). # and ## indicates significant difference (p < 0.05) and very significant difference (p < 0.01), respectively, between different oxygen levels. * and ** indicates significant difference (p < 0.05) and very significant difference (p < 0.01), respectively, between the MFD and HFD groups.

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oxygen and dietary lipid only appeared in serum lactate (Supplemental Table 6). These results illustrate that dietary fat mainly affected serum metabolites in glucose and lipid metabolism, while hypoxia stress

mainly affected glucose metabolism-related metabolites and caused liver damages.

3.4. Influences of hypoxia and dietary lipid content on flesh biochemical composition

In the MFD groups, hypoxia significantly reduced the contents of total protein and moisture in flesh compared to the nomoxia state (Fig. 3A, C). Moreover, the fillets of MFD-hypoxia group showed higher contents of total lipid and TG than those in MFD-normoxia group (Fig. 3B, F).

At normoxia state, the HFD-fed fish showed higher glycogen and TG contents, while lower total protein and moisture than those in the MFD- fed fish (Fig. 3 A, C, D, F). On the contrary, at hypoxia status, HFD significantly increased flesh moisture content compared with MFD (Fig. 3C).

Two-way ANOVA analysis indicated that the significant interactions of two factors were observed on the flesh protein and moisture (Sup-plemental Table 7). All these data indicate that hypoxia caused loss of flesh protein and moisture, but increased flesh lipid content in the tilapia fed with normal diet. These similar results were also caused by the HFD feeding in normoxia status. However, in the HFD groups, the effects of hypoxia on flesh composition were not significant.

The composition of fatty acid in flesh is listed in Table 1 and Sup-plemental Table 2. Hypoxia had different effects on fatty acid content in MFD and HFD groups. In the MFD groups, hypoxia significantly reduced the percentage of Σ n-3 polyunsaturated fatty acids (n-3 PUFA), espe-cially DHA. However, in the HFD groups, there were no significant differences in fatty acid composition between hypoxia and normoxia states. Besides, HFD didn’t affect the fatty acid composition in normoxia state. However, under hypoxia condition, the fish in the HFD group showed significantly lower n-6 PUFA percentage and higher Σ n-3/Σ n-6 percentage than those in the MFD group. Two-way ANOVA analysis didn’t show significant interaction of two factors in flesh fatty acid composition (Supplemental Table 9). These results suggest hypoxia may have dietary lipid-related roles in affecting flesh fatty acids composition.

The composition of amino acid in flesh is listed in Table 1 and

Table 1 The fatty acid composition (%) and amino acid composition (mg/g) in the flesh of Nile tilapia.

Medium fat diet High fat diet Control Hypoxia Control Hypoxia

EPA 1.33 ± 0.25 2.16 ± 0.37 1.08 ± 0.11 2.32 ± 0.78 DHA 21.73 ± 2.69 13.51 ± 2.67# 16.06 ± 1.23 14.25 ± 1.43 SFA 24.27 ± 2.58 28.30 ± 2.13 23.67 ± 2.49 26.72 ± 0.50 MUFA 15.22 ± 0.71 9.81 ± 3.57 17.96 ± 1.00 16.14 ± 2.03 PUFA 60.51 ± 2.65 61.89 ± 2.15 58.37 ± 1.49 57.14 ± 1.83 Σ n-3

PUFA 24.89 ± 3.23 15.56 ± 2.93# 18.21 ± 1.14 18.67 ± 5.30

Σ n-6 PUFA

12.49 ± 0.63 13.99 ± 1.13 10.04 ± 0.85 10.64 ±1.91*

Σ n-3/Σ n- 6

1.99 ± 0.24 1.08 ± 0.24# 1.86 ± 0.21 1.85 ± 0.01*

DAA 71.30 ± 0.86 66.19 ±0.86##

66.48 ±0.319**

64.10 ±0.61#

FAA 78.87 ± 0.96 73.48 ±0.94##

73.57 ± 0.35** 71.23 ±0.69#

EAA 73.82 ± 0.93 68.58 ±0.63##

67.40 ± 0.32** 66.52 ± 0.72

SEAA 7.17 ± 0.26 6.71 ± 0.21 6.84 ± 0.09 6.95 ± 0.11 TAA 177.01 ±

2.11 164.61 ±1.73#

163.22 ±0.71**

159.41 ±1.59

EAA/TAA 0.42 ± 0.00 0.42 ± 0.00 0.41 ± 0.00** 0.42 ± 0.00#

EPA: eicosapentaenoic acid; DHA: docosahexenoic acid; Σ SFA: sum of saturated fatty acids; Σ MUFA: sum of monounsaturated fatty acids; Σ PUFA: sum of polyunsaturated fatty acids; Σ n-3: sum of n-3 polyunsaturated fatty acids; Σ n-6: sum of n-6 polyunsaturated fatty acids. For fatty acid composition, results were expressed as mean ± SEM (n = 6, wet matter). DAA: sum of delicious amino acids; FAA: sum of flavor amino acids; EAA: sum of essential amino acids; SEAA: sum of semiessential amino acids; TAA: sum of total amino acids. # or ##, p < 0.05 or p < 0.01 between normoxia and hypoxia within the same dietary lipid content; * or **, p < 0.05 or p < 0.01 between MFD and HFD within the same oxygen content. For amino acid composition, results were expressed as mean ± SEM (n = 3, dry matter).

Fig. 4. Influences of hypoxia and dietary lipid content on the expressions of the genes related to flesh quality in Nile tilapia. Data shows the expressions of the genes related to collagen synthesis (A-COLIA1, B-COLIA2), fat accumulation and breakdown (C-FAS, D-CPT1b), myogenic differentiation (E-MyoD1, F-MyoG) in muscle. Data are presented as mean ± SEM (n = 6). # and ## indicates significant difference (p < 0.05) and very significant difference (p < 0.01), respectively, between different oxygen levels. * and ** indicates significant difference (p < 0.05) and very significant difference (p < 0.01), respectively, between the MFD and HFD groups.

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Supplemental Table 3. Hypoxia significantly reduced the concentrations of delicious amino acids (DAA), flavor amino acids (FAA), essential amino acids (EAA) and total amino acids (TAA) in the MFD group, and also reduced the concentrations of DAA and FAA in the HFD group. As compared with the MFD-normoxia group, concentrations of DAA, FAA, EAA, and TAA were significantly decreased in the HFD-normoxia group. However, the dietary fat content didn’t significantly affect amino acid concentrations in hypoxia state. The interactive effects between dis-solved oxygen and dietary lipid content were found almost all above data except for DAA, FAA and semi-essential amino acid (SEAA) (Sup-plemental Table 9). These results indicate that hypoxia could reduce flavor-related amino acid composition in flesh regardless of dietary lipid content, as similar as the effects caused by the HFD feeding in normoxia state.

3.5. Influences of hypoxia and dietary lipid content on flesh quality

The results showed that, in the both MFD and HFD groups, the meat yield in the hypoxia state was lower than that in normoxia state (Fig. 3G). On the contrary, the fragmentation index in hypoxia was increased compared with normoxia state (Fig. 3K). Besides, in the MFD- fed fish, hypoxia exposure significantly decreased flesh pH, and increased centrifugal water loss and cooking loss, as compared with the normoxia state (Fig. 3H, I, J). However, there was no significant dif-ference in flesh myoglobin content between MFD and HFD groups, or between hypoxia and normoxia groups (Fig. 3L). In the normoxia status, the HFD group showed significantly higher centrifugal water loss, cooking loss and lower pH than MFD group (Fig. 3H, I, J). However, at hypoxia state, HFD caused significantly lower centrifugal water loss than MFD (Fig. 3H).

The two-way ANOVA analysis showed that the interactions between the two factors were found in the centrifugal water loss, cooking loss, pH and fragmentation index (Supplemental Table 7). The results suggest that hypoxia had adverse effects on the flesh quality, especially in the MFD group. Similarly, HFD also caused similar adverse effects as hyp-oxia, but only in the normoxia state.

3.6. Influences of hypoxia and dietary lipid content on the expressions of flesh quality-related genes

In the present study, mRNA expressions of the genes related to col-lagens synthesis (Colia 1, 2), lipid accumulation (FAS and CPT-1b) and muscle development (MyoD1 and MyoG) in muscle were determined (Fig. 4). Results showed that the hypoxia stress significantly down-regulated the Colia1, Colia2 expressions in both MFD and HFD groups (Fig. 4A, B), and decreased CPT1b expression in the MFD group (Fig. 4D). However, hypoxia significantly increased FAS expression in the MFD group (Fig. 4C). As shown in Fig. 4F, in both MFD and HFD groups, the mRNA expressions of MyoG were significantly higher in the hypoxia group than those in the nornoxia group.

At the normoxia state, the expressions of Colia1 and Colia2 were significantly lower in the HFD group than those in the MFD group (Fig. 4A, B). However, the expressions of FAS and MyoG were signifi-cantly increased in the HFD group, compared with the MFD group (Fig. 4 C, F). In addition, HFD didn’t affects these genes expressions in the hypoxia state.

The two-way ANOVA assay showed that the interactions between the two factors were observed in COLIA1 and COLIA2 (Supplemental Table 8). These results further indicate that hypoxia and HFD had similar effects in regulating the expressions of the genes related to flesh quality, and hypoxia tended to have stronger effects than the HFD treatment.

4. Discussion

4.1. Influences of hypoxia and dietary lipid content on growth and general physiological parameters

Water oxygen content is an important environmental factor for aquatic animals in regulating aerobic activities and metabolism involved in nutrition processing (Saravanan et al., 2013). For example, it was reported that hypoxia significantly reduced the weight gain by decreasing the food intake and food conversion efficiency in turbot ju-veniles (Person-Le, Lacut, Le Bayon, Le Roux, Pichavant, & Quemener, 2003). Similarly, the present study also indicated that the hypoxia- treated fish ate less feed and had lower weight gain than the normoxia group. Except reducing feed intake, hypoxia could also induce severe oxidative stress in fish liver in both MFD and HFD groups, and HFD would cause more severe hepatic steatosis than MFD (Yu, Zhang, Zhang, Wang, & Ji, 2019). In the present study, hypoxia significantly increased serum activities of GPT and GOT, which have been widely used as the marker of liver damage (Jia, Cao, Xu, Jeney, & Yin, 2012), in the both MFD and HFD groups, suggesting hypoxia also induced liver damage in Nile tilapia.

A large number of fish studies have indicated that the excessive di-etary fat content would induce severe fat accumulation and lipotoxicity, including increased oxidative stress and lowered immune functions, thus cause organ damages and low feed intake (Jia, Jing, Niu, & Huang, 2017). Similarly, in the present study, in the nomoxia state, HFD also induced lower survival rate and increased fat accumulation in organs of Nile tilapia, causing increased body weight. Moreover, at hypoxia state, HFD induced the lowest survival rate and feed intake as compared to the nomoxia state. This phenotype was also reported in a grass carp study, in which HFD induced more adverse hepatopancreas damage in hypoxia than in nomoxia state (Yu et al., 2019). Therefore, these data indicate that the combination of HFD and hypoxia would cause worse effects in growth and health than their individual effects in Nile tilapia.

4.2. Influences of hypoxia and dietary lipid content on nutrient deposition

The results of the present study showed that hypoxia and HFD also affected fish biochemical composition. Protein synthesis is one of the major energy consuming processes in the cellular energy costs (Pace & Manahan, 2006). Moreover, a previous fish study had indicated that hypoxia would reduce protein synthesis and stimulate breakdown of amino acids (Gracey, Troll, & Somero, 2001). As expected, the present study showed lower protein content in whole body and muscle in the MFD group, and this could be the output of lowered protein synthesis and increased protein breakdown. As the evidence, in the MFD group, hypoxia reduced amino acids contents in muscle, including DAA, TAA, EAA and TAA, verifying the less substrates for protein synthesis compared to the nomoxia state. Of note, in the HFD-fed fish, hypoxia didn’t reduce body and muscle protein. However, considering the HFD feeding itself had significantly reduced protein contents in fish body and muscle, as previously reported in other animals (Regost et al., 2001), the protein-lowing effect of hypoxia in the HFD-fed fish would be less sen-sitive as compared in the normal diet (MFD)-fed fish. Therefore, from the flesh quality aspects, HFD and hypoxia both reduced flesh protein content and could impair tastes by reducing flavor-related amino acids (DAA and FFA), but there were no combined effects of the both factors on flesh protein and amino acid-related parameters.

As is well known, the cellular oxidation of glucose is able to supply immediate energy for animals to respond to changing environmental conditions (Ishibashi, Ekawa, Hirata, & Kumai, 2002). When the oxygen is limited, glucose will be oxidized to pyruvate by activation of anaer-obic metabolism, which may cause lactic acid accumulation. Accord-ingly, in the present study, under hypoxia state, hepatic glycogen and serum glucose were decreased, accompanied with the increased serum lactate in the MFD group, indicating the anaerobic metabolism was

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induced. However, in the HFD group, the effects of hypoxia on body glycogen and lactate contents were not obvious, showing HFD could alleviate the effects of hypoxia in glycogen/glucose metabolism. In fact, in the present study, HFD significantly increased muscle glycogen and serum lactate in normoxia state, and also significantly increased hepatic glycogen and serum glucose in hypoxia state. Considering HFD feeding could provide excess lipid as the substrate for energy production, the energy sourced from carbohydrate would be reduced. This had been proved in our previous Nile tilapia studies, in which glycolysis was activated in the low-fat diet feeding, whereas the increased lipid catabolism would decrease glucose utilization (He et al., 2015). There-fore, in the HFD-fed fish, high glycogen and glucose content could be sourced by suppressed glycogen/glucose utilization. However, the HFD- caused different responses of glycogen deposition between liver and muscle in nomoxia and hypoxia states are still need further in-vestigations. At least from flesh quality aspects, hypoxia would elimi-nate the HFD-caused increase of glycogen deposition in muscle, which is an important sensory parameter for flesh taste.

In the present study, in the fish fed with normal diet (MFD), hypoxia significantly increased lipid or TG content in whole body, liver and muscle. It has been known that lipid oxidation reaction will consume more oxygen than the oxidation of protein and carbohydrate (Wu, Belardinelli, & Fraser, 2008). Previous studies also indicated that the hypoxia inducible factor (HIF) was involved in hypoxia-induced lipid accumulation via reducing CPT-1 expression-mediated fatty acid β-oxidation, but doesn’t influence fatty acid synthesis (Liu et al., 2014). Similarly, the present results also showed that hypoxia significantly decreased CPT-1b expression, while enhanced FAS expression, in muscle of the MFD group. These data verify that hypoxia could induce lipid accumulation in tissues through depressing cellular lipid oxidation. Except for hypoxia, numerous studies have showed that the long-term HFD feeding would also increase lipid deposition in fish (Regost et al., 2001). More intensive studies showed that the HFD feeding would decrease the phosphorylation degree of AMPKα, which is necessary to promote fatty acid oxidation by stimulating CPT1 (Yuan et al., 2018). In the present study, the significantly decreased CPT-1b expression was also found in the HFD-nomoxia group, and accordingly, the significantly increased total lipid or TG contents in fish body, liver and muscle were found in this group. However, although hypoxia and HFD feeding could individually elevate fat deposition in fish, the data showed that the combination of the both factors didn’t bring stronger effects in fat deposition, neither in organs nor in gene expressions. Considering the HFD-hypoxia-treated fish obtained the lowest feed intake among groups, the low energy intake should contribute to the loss of the combined effects of hypoxia and HFD on fat deposition.

In the present study, hypoxia significantly reduced n-3 PUFAs per-centage, especially DHA, only in the MFD-fed fish, while HFD didn’t change n-3 PUFAs composition in both nomoxia and hypoxia states. It has been reported that n-3 PUFAs, including DHA, is important in endogenous antioxidant system (Ishibashi et al., 2002), and intermedi-ate levels of n-3 PUFAs in Atlantic salmon diets gave the best protection against oxidative damage (Stbye, Kjr, Rr, Torstensen, & Ruyter, 2015). Therefore, fish is also supposed to utilize more n-3 PUFA to resist hyp-oxia stress. However, the HFD-hypoxia treatment induced high impair-ment towards fatty acid catabolism system, causing that the n-3 PUFAs could not be utilized efficiently. All these indicate that either HFD or hypoxia could increase fat accumulation in tissues, but the combination of the both factors would not strengthen the fat accumulation. Of note, hypoxia would do more harm to nutritional quality for fish fillets than the HFD feeding, through reducing the composition of n-3 PUFA, at least in the normal diet-fed fish.

4.3. Influences of hypoxia and dietary lipid content on flesh quality- related parameters

Meat (fillet) production, water holding ability, pH value,

fragmentation and flesh myoglobin are important flesh quality-related parameters for many cultured animals. In the present study, hypoxia significantly reduced meat yield, showing hypoxia would severely impair the economic value of farmed fish. According to the analysis and results above, all the lowered feed intake, reduced protein deposition and impaired physiological functions should contribute to this reduction of meat yield. Similarly, hypoxia also reduced the feed intake and growth of rainbow trout (Saravanan et al., 2013).

The flesh pH value of the MFD-fed fish was reduced under hypoxia state. As explained above, limited oxygen supply would induce anaer-obic metabolism, and cause lactic acid accumulation, which would directly lead to the lower pH. In addition, as shown above, the HFD feeding also disturbed glucose metabolism and increased lactate amount in serum and fillet, thus the decreased flesh pH value was also found in the HFD in this study. Some studies had demonstrated that the low flesh pH would induce the decline of water holding capacity in farmed cod (Bjrnevik et al., 2017). A cell study further indicated that the reduced pH decreased water holding capacity by clearing up the protein electric potential (Lambert, Nielsen, Andersen, & Ortenblad, 2001). Therefore, the present study indicates that either of hypoxia and HFD feeding could increase the lactate content, and decreased the pH value and water holding capacity in fillets of the fish. However, the combination of hypoxia and HFD would not strengthen these effects.

In flesh quality evaluation, the fragmentation index is an important parameter, which tightly correlates to the flesh hardness and chewiness (Tian, Han, Yu, Shi, & Wang, 2013). The present study clearly showed that hypoxia, but not HFD, significantly increased fragmentation index, indicating the muscular fibers in the hypoxia-treated fish were not easy to be broken. Collagen, along with fragmentation index, have positive effects on the hardness of flesh (Zhao et al., 2018). However, the mRNA expressions of COLIA 1 and COLIA 2, which are key genes in type I collagen synthesis in fish muscle (Zhao et al., 2018), were significantly down-regulated in the hypoxia groups. Previous studies indicated that the amino acids could stimulated the synthesis of collagen in Atlantic salmon (Ostbye et al., 2018). Considering the present study had shown that hypoxia significantly increased amino acids catabolism, this could be the important reason to lower muscular collagen synthesis. There-fore, the present study indicates that hypoxia would inhibit collagen synthesis, and the increased flesh hardness/fragmentation ability was not likely to be related to the collagen. Different from hypoxia, the HFD feeding didn’t change fragmentation index, but lower expressions of COLIA 1 and COLIA 2 were found in the HFD-nomoxia group than those in the MFD-nomoxia group. This suggests that HFD would also has adverse effects on collagen synthesis, and more severe impairment would probably appear in a longer HFD feeding trial.

MyoD and MyoG are important myogenic regulatory factors expressed in the skeletal muscle. In a previous study, a 7-day exercise induced the expression of MyoG in the muscle of Piaractus mesopotamicus (Rondinelle et al., 2019). The exercise could induce a low oxygen level in the muscle. Accordingly, the present study showed that although the flesh myoglobin content was comparable among groups, hypoxia significantly enhanced the MyoG expression in Nile tilapia, regardless of the MFD or HFD feeding. A study of Atlantic salmon indicated that the hardness of muscle had positive correlation with expression of MyoG (Ostbye et al., 2018). Therefore, the present study clearly indicates that hypoxia could increase the expression of MyoG, thus also increased the hardness of muscle in the both MFD group and HFD groups. Except hypoxia, HFD feeding also affected the expression of MyoG in fish muscle. A study of grass carp reported that the expression level of MyoG was higher in HFD-fed fish than in low fat diet-fed fish (Zhao et al., 2018). Differently, in this study, the HFD feeding significantly elevated the expression of MyoG in fish muscle in the normoxia state. However, the combination of hypoxia and HFD still didn’t strengthen the expression of MyoG, as compared with either of the individual treatment.

Taken together, hypoxia and HFD brought similar adverse effects on

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flesh quality-related parameters, including the reduced muscle water holding ability and pH values, down-regulated expressions of the collagen synthesis-related genes and the up-regulated MyoG expression. However, hypoxia had more severe effects to reduce meat yield and increase fragmentation than the HFD feeding, but the combination of hypoxia and HFD feeding would not result in more severe effects than their individual effects.

5. Conclusions

As two common factors which usually exist together in practical aquaculture, hypoxia and the HFD feeding had similar effects in reducing survival rate, flesh protein and delicious amino acids contents, flesh pH values and water holding ability, while increasing body lipid accumulation. However, hypoxia had additional adverse effects in reducing feed intake, meat yield and flesh n-3 PUFA content, and increasing flesh fragmentation and liver damage. Although the combi-nation of hypoxia and HFD feeding would not strengthen the adverse effects on flesh quality-related parameters, this combination signifi-cantly decreased feed intake, survival rate and muscle protein, but increased mesenteric fat accumulation (Fig. 5). Taken together, during the popular usage of HFD in aquaculture, the balance between the HFD- caused cost benefits and impairment on flesh quality should be carefully evaluated. More importantly, hypoxia during the HFD feeding should be seriously avoided.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence

the work reported in this paper.

Acknowledgement

This research was supported by National Key R & D Program of China (2018YFD0900400).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi. org/10.1016/j.foodchem.2020.128479.

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