# Effect of Dietary Cholesterol on Growth Performance, Cholesterol Deposition, and Lipid Metabolism in Adult Chinese Mitten Crab (Eriocheir sinensis)

Authors: Hui-xing Guo; Guang-zhen Jiang; Yong-jun Dai; Yang-yang Huang; Xi Wang; Kang Xiao; Mang-mang Wang; Kenneth Abasubong; Yun Xu; Ding-dong Zhang; Wen-bin Liu
Journal: Aquaculture Nutrition
 (2022)

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## Abstract

This research investigated the effects of dietary cholesterol (CHO) on growth, molting performance, CHO deposition, and lipid metabolism of Chinese mitten crab (Eriocheir sinensis). Six diets were formulated to contain 0% (control), 0.1%, 0.2%, 0.4%, 0.8%, and 1.6% CHO and were administered to E. sinensis43.35±0.05g, for 16 weeks. From our results, crabs fed with 0.2% and 0.4% CHO diet, the final body weight (FBW), and weight gain ratio (WGR) increased significantly and the feed conversion ratio (FCR) decreased significantly compared with that of the control group. Furthermore, the molting performance of E. sinensis was significantly improved in crabs fed 0.2% CHO. Meanwhile, 0.2% CHO tends to upregulate molt-promoting genes and downregulate the expression of molt-inhibiting genes. It indicated that CHO might improve the molting performance of crabs by regulating ecdysteroid signal pathway transduction. In addition, dietary CHO can significantly increase the content of CHO in the hepatopancreas, muscle, and hemolymph. Moreover, dietary CHO increases the content of triglyceride (TG) in the hepatopancreas and hemolymph. The gene-related lipid metabolism shows that CHO may increase the lipid content by promoting lipid synthesis and inhibiting lipolysis. According to the results of this study, the dietary CHO requirement of E. sinensis is 0.27% for growth requirement and 0.45% for tissue CHO deposition based on the broken-line model.


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## Body

## 1. Introduction

Cholesterol (CHO), as a kind of lipid, widely exists in various animal tissues [1], especially in the brain and nervous system. Plants generally contain limited or no CHO content [2]. The particularity structure of CHO makes it significant to all kinds of creatures. CHO is a key component of the cell membrane, which has great significance in maintaining cell membrane stability and fluidity [3]. CHO plays a significant role in lipid metabolism by synthesizing apolipoprotein [4]. In addition, CHO is a precursor for synthesizing a variety of active substances in organisms, including vitamin D and various steroid hormones [5]. Vertebrates can synthesize CHO with acetyl-CoA, usually without CHO deficiency [6, 7]. Due to the lack of some key enzymes in CHO synthesis, crustaceans cannot synthesize CHO. Therefore, crustaceans mainly obtain CHO through animal components in feed (e.g., fish meal and fish oil) [7, 8]. In recent years, with the replacement of animal components by plant components in the feed, the utilization of CHO is further limited [9]. Therefore, it is necessary to investigate the nutritional requirements of CHO in order to develop a more comprehensive formula feed.It has been reported that adding CHO in the feed ofScylla serrata is critical to maintain their survival [10]. The addition of CHO in the diet of Macrobrachium rosenbergii had no effects on the growth rate, but the addition of CHO enabled Macrobrachium rosenbergii to achieve optimal growth performance [11]. Meanwhile, as an important component of lipoprotein, CHO has a significant influence on lipid metabolism [12]. For instance, several studies have shown that adding CHO to rainbow trout (Oncorhynchus mykiss) feed can inhibit β-oxidation and increase the body’s lipid content [13]. In crustaceans, CHO is used to synthesize ecdysone, while the addition of 0.5% CHO in the diet of Procambarus clarkii was reported to promote molting [14, 15]. The lack of CHO in the diet can cause prolonged molting cycles in shrimp and crabs [16]. According to date from previous studies, the CHO requirement is 0.1%~0.2% for Penaeus japonicus, 0.14% for Litopenaeus vannamei, 0.5% for Homarus americanus, and 0.2% for Penaeus monodon [2, 17–19]. Previous studies have shown that there are great differences in CHO requirement of crustaceans at different developmental stages, but at present, the research on CHO requirement in Chinese mitten crab (Eriocheir sinensis) is focused on juvenile crabs [8, 20, 21], while the CHO requirement in adult Eriocheir sinensis has not been reported.In China,E. sinensis is a unique economic aquatic species, which plays a significant role in the Chinese aquaculture system in alleviating poverty and improving economic growth in many regions [22, 23]. In 2020, the output of E. sinensis has reached 775,000 tons, which has become an important part of people’s diet (Chinese fishery statistical yearbook, 2020). At present, juvenile E. sinensis can be fed with formula feed in the whole process, but for adult crab, the whole process of feeding formula feed is prone to problems such as unsuccessful molting and slow maturation. As an essential nutrient of crab, CHO plays an important role in growth and development. The deficiency of CHO in the feed may be one of the reasons for these problems [8, 24]. Given its great economic value, the research on the nutritional requirements of E. sinensis has been more in depth in recent years. However, there has been no report on the CHO requirement of adult E. sinensis. This study is aimed at evaluating the effects of dietary CHO on the growth, molting, CHO deposition, and lipid metabolism of adult E. sinensis. In addition, the optimum CHO content requirement in the diet will be assessed.


## 2. Materials and Methods

All experimental operations and feeding schemes in this study were approved by the Animal Care and Use Committee of Nanjing Agricultural University (Nanjing, China) (permit number: SYXK (Su) 2011–0036).


### 2.1. Diets

Six isonitrogenous and isolipid diets were formulated containing CHO 0% (control), 0.1%, 0.2%, 0.4%, 0.8%, and 1.6% (actually 0.06%, 0.14%, 0.23%, 0.44%, 0.82%, and 1.64%) at the cost of soybean oil. The fundamental diet formula of this experiment is shown in Table1.Table 1
Ingredient formulation and proximate composition (% dry matter) in this feeding trial.
Cholesterol supplementation (%)00.10.20.40.81.6Ingredients (%)Defatted fish meala18.5018.5018.5018.5018.5018.50Soybean meal15.0015.0015.0015.0015.0015.00Rapeseed meal2.502.502.502.502.502.50Cottonseed meal3.003.003.003.003.003.00Peanut meal28.5028.5028.5028.5028.5028.50α-Starch19.0019.0019.0019.0019.0019.00EPA oil: DHA oil (1 : 1)b1.201.201.201.201.201.20Soybean oil5.205.105.004.804.403.60Carboxymethyl cellulose1.001.001.001.001.001.00Ca(H2PO4)2·H2O2.202.202.202.202.202.20Cholesterol (purity 99%)c0.000.100.200.400.801.60Lecithin0.200.200.200.200.200.20Zeolite0.400.400.400.400.400.40Premixd1.001.001.001.001.001.00Mixturee2.302.302.302.302.302.30Total100.00100.00100.00100.00100.00100.00Proximate composition (%)Crude protein36.2136.1536.0136.3135.9636.24Crude lipid7.958.088.057.988.057.96Crude ash7.157.217.167.077.107.16aFishmeal had been skimmed from 0.38% to 0.11% cholesterol. bDHA oil and EPA oil (DHA content, 70% of oil; EPA content, 70% of oil) were purchased from Shanxi Pioneer Biotech Co. Ltd., Xian, Shanxi, China. cCholesterol (purity 99%) was purchased from Shanxi Pioneer Biotech Co. Ltd., Xian, Shanxi, China. dPremix supplied the following minerals (g/kg) and vitamins (IU or mg/kg): CuSO4·5H2O, 2 g; FeSO4·7H2O, 25 g; ZnSO4·7H2O, 22 g; MnSO4·4H2O, 7 g; Na2SeO3, 0.04 g; KI, 0.026 g; CoCl2·6H2O, 0.1 g; vitamin A, 900000 IU; vitamin D, 200000 IU; vitamin E, 4500 mg; vitamin K3, 220 mg; vitamin B1, 320 mg; vitamin B2, 1090 mg; vitamin B5, 2000 mg; vitamin B6, 500 mg; vitamin B12, 1.6 mg; vitamin C, 10000 mg; pantothenate, 1000 mg; folic acid, 165 mg; choline, 60000 mg; Biotin, 100 mg; and myoinositol, 15000 mg. eMixture includes the following ingredients (%): choline chloride 4.75%; antioxidants 1.72%; mildew-proof agent 2.35%; salt 22.06%; Lvkangyuan 59.30%; and biostimep 9.51%.All the experimental diets were made in laboratory conditions. First of all, all ingredients were processed through a 60 mm mesh; then, weigh all kinds of ingredients, and mix thoroughly with lipid sources. Thereafter, about 30% distilled water was continually added to the ingredient and was mixed manually to homogenize the mixture. Subsequently, the homogeneous mixture was processed through a 2.5 mm diameter die of a single-screw meat grinder extruder and later ventilated to dry at room temperature for 24 hours. After drying, the feed was ground into an appropriate size. Finally, all diets were stored at −20°C in a sealed plastic bag.


### 2.2. Experiment Crab and Feeding Trial

E. sinensis were purchased from a local farm in Pukou, Jiangsu province, China. A total of 240 healthy crabs 43.35±0.05g were randomly selected and divided into 6 groups, with four replicates (5 male crabs and 5 female crabs/replicate), and each replicate was fed in one cement pool, 24 cement pools in total 1.0×1.0×0.8m,L:W:H, with each cement pool having 12 pipes (20 cm long and 15 cm diameter) as shelters. Before the experiment, the crabs were fed for a week to adapt to the experimental condition. After the conditioning period, all crabs were hand fed with one of six experiment diets once daily (18 : 00) with 4%–6% body weight till obvious satiation for 16 weeks. During the experiment, clean up the residual feed and change 1/3 of the water every day, the number of death and molting crabs were recorded daily, the water temperature was at 22–28°C, dissolved oxygen was at 5.0–7.0 mg/L, pH was at 7.3–8.4, and ammonia was under 0.05 mg/L.


### 2.3. Sample Collection

Before sampling, all crabs were starved for 24 hours. Subsequently, all the crabs of each cement pool were quickly anesthetized on ice, then weighed, and counted. Four individuals from each cement pool were randomly selected to collect hemolymph with 1.0 mL syringes on the leg joints and mixed with precooled anticoagulant (EDTA, 15 mmol/L; citric acid, 30 mmol/L; citrate, 26 mmol/L; NaCl, 450 mmol/L; glucose, 100 mmol/L; andpH=7.2 1 : 1, then centrifuged at 4500 rpm, 4°C for 15 min, and stored at −20°C for subsequent analysis. Concurrently, the hepatopancreas and muscle samples were quickly collected, frozen in liquid nitrogen immediately, and then stored in the refrigerator at −20°C for gene expression and biochemical index analysis.


### 2.4. The Proximate Composition of Diet and Cholesterol Deposition

The total lipid, crude protein, moisture, and crude ash content in the diet was measured according to the standard method [25]. The specific procedures of determining crude protein, total lipid, crude ash, and moisture were the same as those in Cheng et al. [26]. The content of CHO in feed, hepatopancreas, and muscle samples were detected by high-performance liquid chromatography (HPLC). In summary, 1 gram of the sample was placed in a 50 mL centrifuge tube, and 15 mL ethanol absolute and 5 mL 60% KOH were added to it and then saponified in the water bath at 90°C for 1 hour. Subsequently, centrifuge at 5,000 g for 5 minutes; 1 mL supernatant was taken into 10 mL with ethanol absolute for HPLC detection (Agilent ZORBAX Eclipse Plus, column C18 5 μm 4.6×150mm. 100% methanol was used as the mobile phase with a flow rate at 1 mL/min.


### 2.5. The Biochemical Analysis of Hepatopancreas, Hemolymph, and Muscle

The triglyceride (TG) (cat. no. A110-1-1) and nonesterified fatty acid (NEFA) (cat. no. A042-2-1) contents in hemolymph, hepatopancreas, and muscle and total cholesterol (TC) (cat. no. A111-1-1), high-density lipoprotein cholesterol (HDL-C) (cat. no. A112-1-1), and low-density lipoprotein cholesterol (LDL-C) (cat. no. A113-1-1) contents in the hemolymph were measured. The abovementioned parameters were measured by the commercial kit (Jiancheng Bioengineering Co., Nanjing, China) according to the instructions.


### 2.6. Gene Expression

Total RNA was extracted from hepatopancreas using RNAiso Plus (TaKaRa, Dalian, China); then, the purity and concentration of RNA were measured by spectrophotometry on the basis of OD 260/280 using a NanoDrop ND-1000 UV Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Subsequently, 1μg RNA was reverse transcribed into cDNA immediately by HiScript®III RT SuperMix for qPCR (Vazyme, Nanjing, China) according to the manufacturer’s instructions and then stored at −20°C for real-time qPCR. After the reverse transcription, the expression level of mRNA was detected by real-time qPCR. The specific primer sequences of genes in this experiment are shown in Table 2. Briefly, the reaction system of RT-qPCR including 10 μL 2×ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), 2 μL cDNA template, and 7.2 μL DEPC-water and the primers of forward and reverse were 0.4 μL (10 μM). The PCR procedure included 95°C for 30 s and then at 95°C for 10 s and 60°C for 28 s for 40 cycles. The melting curve step was from 95°C to 60°C for 15 s and 60°C for 1 min; then, the temperature was raised to 95°C at a rate of 1.6°C per second for 15 s. At the end of the reaction, the specificity of the PCR product was determined by the melting curve. The β-actin gene was used as the internal reference and the target gene expression of crabs in the group with 0% CHO as the correct factor. The gene expression level was calculated by the 2−ΔΔCT [27] method.Table 2
Primer pair sequences and product size of the genes used for real-time PCR (qPCR).
GenePositionPrimer sequenceLengthProduct size (bp)Referencesrebp-1ForwardTCTTCACACCCTCTGGACGC20162[59]ReverseCCAAGGTTGTAATGGCACGC20cpt-1aForwardCATCTGGACACCCACCTCCA20183[22]ReverseATCTCCTCACCCGGCACTCT20fasForwardGTCCCTTCTTCTACGCCATCC21127[59]ReverseCGCTCTCCAGGTCAATCTTCAC22mttpForwardTAGGACAAGCAGGACTTTCCTCA24138[59]ReverseCCACATCCACAAACACATCAACA23ecrForwardCTCCCGGGTGCCATATTACC20212KF469223.1ReverseTGCTACACGGCACATTCACT20rxrForwardACCCTGTGCTAACCCTCTGA2074MK604180.1ReverseTGCTCACCACATCCTGCTTT20mihForwardTTTAGCTCCGTTCACGCCTT20102DQ341280.1ReverseTGGAGAACCCAGGAAAGCAC20β-ActinForwardTCGTGCGAGACATCAAGGAAA21178KM244725.1ReverseAGGAAGGAAGGCTGGAAGAGTG22srebp-1: sterol-regulatory element-binding protein 1; cpt-1a: carnitine palmitoyl transterase-1a; fas: fatty acid synthase; mttp: microsomal triglyceride transfer protein; ecr: ecdysteroid receptor; rxr: retinoid X receptor; mih: molt-inhibiting hormone.


### 2.7. Calculations

The related parameters were calculated as follows:(1)FBWg=totalfinalweighttotalcrabnumber,SR%=100×finalnumberofcrabsinitialnumberofcrabs,WGR%=100×finalbodyweight−initalbodyweightinitalbodyweight,FIg=totalfeedintakeofeachtanktotalnumberofcrabineachtank,FCR=dryfeedintakefinalbodyweight−initalbodyweight+deadcrabweightgain,MY%=totalmuscleweightfinalbodyweight,MF=totalmoltingtimesinitialnumberofcrabs+finalnumberofcrabs/2,where FBW is the final body weight, SR is the survival rate, WGR is the weight gain ratio, FI is the feed intake, FCR is the feed conversion ratio, MY is the meat yield, and MF is the molting frequency.


### 2.8. Statistical Analysis

The statistical analysis of the data in this experiment was implemented using the software SPSS23.0 (Chicago, IL, USA). The data were shown as means and pooled SEM (pooled standard error of means). Before analysis, test the normality and homogeneity of variance. Subsequently, a one-way analysis of variance was performed, and then, Duncan’s multiple comparison test to estimate the significant differencesP<0.05 among mean values was performed. Furthermore, orthogonal polynomial contrasts were used to determine whether the data effect was linear or quadratic.


## 2.1. Diets

Six isonitrogenous and isolipid diets were formulated containing CHO 0% (control), 0.1%, 0.2%, 0.4%, 0.8%, and 1.6% (actually 0.06%, 0.14%, 0.23%, 0.44%, 0.82%, and 1.64%) at the cost of soybean oil. The fundamental diet formula of this experiment is shown in Table1.Table 1
Ingredient formulation and proximate composition (% dry matter) in this feeding trial.
Cholesterol supplementation (%)00.10.20.40.81.6Ingredients (%)Defatted fish meala18.5018.5018.5018.5018.5018.50Soybean meal15.0015.0015.0015.0015.0015.00Rapeseed meal2.502.502.502.502.502.50Cottonseed meal3.003.003.003.003.003.00Peanut meal28.5028.5028.5028.5028.5028.50α-Starch19.0019.0019.0019.0019.0019.00EPA oil: DHA oil (1 : 1)b1.201.201.201.201.201.20Soybean oil5.205.105.004.804.403.60Carboxymethyl cellulose1.001.001.001.001.001.00Ca(H2PO4)2·H2O2.202.202.202.202.202.20Cholesterol (purity 99%)c0.000.100.200.400.801.60Lecithin0.200.200.200.200.200.20Zeolite0.400.400.400.400.400.40Premixd1.001.001.001.001.001.00Mixturee2.302.302.302.302.302.30Total100.00100.00100.00100.00100.00100.00Proximate composition (%)Crude protein36.2136.1536.0136.3135.9636.24Crude lipid7.958.088.057.988.057.96Crude ash7.157.217.167.077.107.16aFishmeal had been skimmed from 0.38% to 0.11% cholesterol. bDHA oil and EPA oil (DHA content, 70% of oil; EPA content, 70% of oil) were purchased from Shanxi Pioneer Biotech Co. Ltd., Xian, Shanxi, China. cCholesterol (purity 99%) was purchased from Shanxi Pioneer Biotech Co. Ltd., Xian, Shanxi, China. dPremix supplied the following minerals (g/kg) and vitamins (IU or mg/kg): CuSO4·5H2O, 2 g; FeSO4·7H2O, 25 g; ZnSO4·7H2O, 22 g; MnSO4·4H2O, 7 g; Na2SeO3, 0.04 g; KI, 0.026 g; CoCl2·6H2O, 0.1 g; vitamin A, 900000 IU; vitamin D, 200000 IU; vitamin E, 4500 mg; vitamin K3, 220 mg; vitamin B1, 320 mg; vitamin B2, 1090 mg; vitamin B5, 2000 mg; vitamin B6, 500 mg; vitamin B12, 1.6 mg; vitamin C, 10000 mg; pantothenate, 1000 mg; folic acid, 165 mg; choline, 60000 mg; Biotin, 100 mg; and myoinositol, 15000 mg. eMixture includes the following ingredients (%): choline chloride 4.75%; antioxidants 1.72%; mildew-proof agent 2.35%; salt 22.06%; Lvkangyuan 59.30%; and biostimep 9.51%.All the experimental diets were made in laboratory conditions. First of all, all ingredients were processed through a 60 mm mesh; then, weigh all kinds of ingredients, and mix thoroughly with lipid sources. Thereafter, about 30% distilled water was continually added to the ingredient and was mixed manually to homogenize the mixture. Subsequently, the homogeneous mixture was processed through a 2.5 mm diameter die of a single-screw meat grinder extruder and later ventilated to dry at room temperature for 24 hours. After drying, the feed was ground into an appropriate size. Finally, all diets were stored at −20°C in a sealed plastic bag.


## 2.2. Experiment Crab and Feeding Trial

E. sinensis were purchased from a local farm in Pukou, Jiangsu province, China. A total of 240 healthy crabs 43.35±0.05g were randomly selected and divided into 6 groups, with four replicates (5 male crabs and 5 female crabs/replicate), and each replicate was fed in one cement pool, 24 cement pools in total 1.0×1.0×0.8m,L:W:H, with each cement pool having 12 pipes (20 cm long and 15 cm diameter) as shelters. Before the experiment, the crabs were fed for a week to adapt to the experimental condition. After the conditioning period, all crabs were hand fed with one of six experiment diets once daily (18 : 00) with 4%–6% body weight till obvious satiation for 16 weeks. During the experiment, clean up the residual feed and change 1/3 of the water every day, the number of death and molting crabs were recorded daily, the water temperature was at 22–28°C, dissolved oxygen was at 5.0–7.0 mg/L, pH was at 7.3–8.4, and ammonia was under 0.05 mg/L.


## 2.3. Sample Collection

Before sampling, all crabs were starved for 24 hours. Subsequently, all the crabs of each cement pool were quickly anesthetized on ice, then weighed, and counted. Four individuals from each cement pool were randomly selected to collect hemolymph with 1.0 mL syringes on the leg joints and mixed with precooled anticoagulant (EDTA, 15 mmol/L; citric acid, 30 mmol/L; citrate, 26 mmol/L; NaCl, 450 mmol/L; glucose, 100 mmol/L; andpH=7.2 1 : 1, then centrifuged at 4500 rpm, 4°C for 15 min, and stored at −20°C for subsequent analysis. Concurrently, the hepatopancreas and muscle samples were quickly collected, frozen in liquid nitrogen immediately, and then stored in the refrigerator at −20°C for gene expression and biochemical index analysis.


## 2.4. The Proximate Composition of Diet and Cholesterol Deposition

The total lipid, crude protein, moisture, and crude ash content in the diet was measured according to the standard method [25]. The specific procedures of determining crude protein, total lipid, crude ash, and moisture were the same as those in Cheng et al. [26]. The content of CHO in feed, hepatopancreas, and muscle samples were detected by high-performance liquid chromatography (HPLC). In summary, 1 gram of the sample was placed in a 50 mL centrifuge tube, and 15 mL ethanol absolute and 5 mL 60% KOH were added to it and then saponified in the water bath at 90°C for 1 hour. Subsequently, centrifuge at 5,000 g for 5 minutes; 1 mL supernatant was taken into 10 mL with ethanol absolute for HPLC detection (Agilent ZORBAX Eclipse Plus, column C18 5 μm 4.6×150mm. 100% methanol was used as the mobile phase with a flow rate at 1 mL/min.


## 2.5. The Biochemical Analysis of Hepatopancreas, Hemolymph, and Muscle

The triglyceride (TG) (cat. no. A110-1-1) and nonesterified fatty acid (NEFA) (cat. no. A042-2-1) contents in hemolymph, hepatopancreas, and muscle and total cholesterol (TC) (cat. no. A111-1-1), high-density lipoprotein cholesterol (HDL-C) (cat. no. A112-1-1), and low-density lipoprotein cholesterol (LDL-C) (cat. no. A113-1-1) contents in the hemolymph were measured. The abovementioned parameters were measured by the commercial kit (Jiancheng Bioengineering Co., Nanjing, China) according to the instructions.


## 2.6. Gene Expression

Total RNA was extracted from hepatopancreas using RNAiso Plus (TaKaRa, Dalian, China); then, the purity and concentration of RNA were measured by spectrophotometry on the basis of OD 260/280 using a NanoDrop ND-1000 UV Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Subsequently, 1μg RNA was reverse transcribed into cDNA immediately by HiScript®III RT SuperMix for qPCR (Vazyme, Nanjing, China) according to the manufacturer’s instructions and then stored at −20°C for real-time qPCR. After the reverse transcription, the expression level of mRNA was detected by real-time qPCR. The specific primer sequences of genes in this experiment are shown in Table 2. Briefly, the reaction system of RT-qPCR including 10 μL 2×ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), 2 μL cDNA template, and 7.2 μL DEPC-water and the primers of forward and reverse were 0.4 μL (10 μM). The PCR procedure included 95°C for 30 s and then at 95°C for 10 s and 60°C for 28 s for 40 cycles. The melting curve step was from 95°C to 60°C for 15 s and 60°C for 1 min; then, the temperature was raised to 95°C at a rate of 1.6°C per second for 15 s. At the end of the reaction, the specificity of the PCR product was determined by the melting curve. The β-actin gene was used as the internal reference and the target gene expression of crabs in the group with 0% CHO as the correct factor. The gene expression level was calculated by the 2−ΔΔCT [27] method.Table 2
Primer pair sequences and product size of the genes used for real-time PCR (qPCR).
GenePositionPrimer sequenceLengthProduct size (bp)Referencesrebp-1ForwardTCTTCACACCCTCTGGACGC20162[59]ReverseCCAAGGTTGTAATGGCACGC20cpt-1aForwardCATCTGGACACCCACCTCCA20183[22]ReverseATCTCCTCACCCGGCACTCT20fasForwardGTCCCTTCTTCTACGCCATCC21127[59]ReverseCGCTCTCCAGGTCAATCTTCAC22mttpForwardTAGGACAAGCAGGACTTTCCTCA24138[59]ReverseCCACATCCACAAACACATCAACA23ecrForwardCTCCCGGGTGCCATATTACC20212KF469223.1ReverseTGCTACACGGCACATTCACT20rxrForwardACCCTGTGCTAACCCTCTGA2074MK604180.1ReverseTGCTCACCACATCCTGCTTT20mihForwardTTTAGCTCCGTTCACGCCTT20102DQ341280.1ReverseTGGAGAACCCAGGAAAGCAC20β-ActinForwardTCGTGCGAGACATCAAGGAAA21178KM244725.1ReverseAGGAAGGAAGGCTGGAAGAGTG22srebp-1: sterol-regulatory element-binding protein 1; cpt-1a: carnitine palmitoyl transterase-1a; fas: fatty acid synthase; mttp: microsomal triglyceride transfer protein; ecr: ecdysteroid receptor; rxr: retinoid X receptor; mih: molt-inhibiting hormone.


## 2.7. Calculations

The related parameters were calculated as follows:(1)FBWg=totalfinalweighttotalcrabnumber,SR%=100×finalnumberofcrabsinitialnumberofcrabs,WGR%=100×finalbodyweight−initalbodyweightinitalbodyweight,FIg=totalfeedintakeofeachtanktotalnumberofcrabineachtank,FCR=dryfeedintakefinalbodyweight−initalbodyweight+deadcrabweightgain,MY%=totalmuscleweightfinalbodyweight,MF=totalmoltingtimesinitialnumberofcrabs+finalnumberofcrabs/2,where FBW is the final body weight, SR is the survival rate, WGR is the weight gain ratio, FI is the feed intake, FCR is the feed conversion ratio, MY is the meat yield, and MF is the molting frequency.


## 2.8. Statistical Analysis

The statistical analysis of the data in this experiment was implemented using the software SPSS23.0 (Chicago, IL, USA). The data were shown as means and pooled SEM (pooled standard error of means). Before analysis, test the normality and homogeneity of variance. Subsequently, a one-way analysis of variance was performed, and then, Duncan’s multiple comparison test to estimate the significant differencesP<0.05 among mean values was performed. Furthermore, orthogonal polynomial contrasts were used to determine whether the data effect was linear or quadratic.


## 3. Results

### 3.1. Growth Performance

As shown in Table3, the FBW, SR, WGR, and FCR of crabs were markedly influenced by the supplementation level of CHO at quadratic P<0.05. Compared to those of the control group, CHO supplementation levels at 0.2% and 0.4%, FBW, SR, and WGR were significantly increased P<0.05 and FCR decreased significantly P<0.05. When the crabs were fed a 0.4% CHO diet, the FBW, WGR, and FCR reached the highest. However, the addition of CHO in the diet has no significant effects on MY and FI of crabs. In addition, through the broken-line model y=48.13x+99.89, R2=0.8885; y=−6.28x+114.55, R2=0.9036 (Figure 1) based on the relationship between the FBW and the dietary CHO levels, it was observed that the FBW of the crabs increased significantly P<0.05 with the addition of CHO from 0% to 0.4% and then decreased; when the CHO supplementation level was 0.27%, the crabs reached the highest FBW.Table 3
Effect of dietary cholesterol level on the growth performance ofE. sinensis.
Dietary CHO levelFBW (g)SR (%)WGR (%)MY (%)FI (g)FCR0102.40a57.50a140.10a20.24136.002.28a0.1102.70a65.00abc141.70a21.06135.902.26a0.2110.50bc75.00c161.30b19.78136.702.01bc0.4113.00c70.00bc163.30b20.15136.601.96c0.8104.80ab62.50ab144.60a19.32136.902.23ab1.6106.60abc62.50ab148.50ab19.37136.402.16abcSEM0.013.282.090.680.600.08P value0.040.030.040.600.850.04Regression analysisLinearAdj. R20.03−0.04−0.010.03−0.000.00P value0.200.730.330.150.340.31QuadraticAdj. R20.230.290.200.01−0.030.20P value0.030.010.040.330.500.04FBW: final body weight; SR: survival rate; WGR: weight gain ratio; MY: meat yield; FI: feed intake; FCR: feed conversion ratio; SEM: pooled standard error of:n=4; Adj. R2: adjusted R square. Values in the same column with different superscripts are significantly different at P<0.05.Figure 1
Broken-line relationship of the dietary CHO level to FBW forE. sinensis. Data appeared as means±SEMn=4. Values in the same curve with different superscripts are significantly different P<0.05.As shown in Figure2(b), the total molting times were increased significantly as the addition levels of CHO increased from 0% to 0.2% and then decreased. We observed that from the sixth week to the sixteenth week, the total molting times of crabs in the group with 0.2% CHO were always higher than those in other groups. As shown in Figure 2(a), the molting frequency (MF) was affected by dietary CHO supplementation, and it reached the highest when the CHO supplementation level is at 0.2%. Additionally, compared to the control group, no significant difference was observed among the groups with 0.1%, 0.4%, 0.8%, and 1.6% CHO.Figure 2
Effects of dietary CHO on (a) molting frequency (MF); (b) total molting times; (c) ecdysteroid receptor (ecr); (d) retinoid X receptor (rxr); and (e) molting-inhibiting hormone (mih). The values are the means±SEMn=4. Different letters indicate significant differences P<0.05.
(a)(b)(c)(d)(e)


### 3.2. The Molting-Related Gene Expression

The molting-related gene mRNA expression was markedly affectedP<0.05 by the dietary CHO supplementation as shown in Figures 2(c)–2(e). Compared with the group of CHO content at 0%, the mRNA expression of ecr (ecdysteroid receptor) and rxr (retinoid X receptor) was increased among the CHO addition groups and the significant differences were observed when the dietary CHO level is at 0.2% P<0.05 (Figures 2(c) and 2(d)). Compared to the control group, the mih (molt-inhibiting hormone) mRNA expression was decreased among the 0.1%, 0.2%, 0.4%, 0.8%, and 1.6% CHO groups. When the addition of CHO reached 0.8% and 1.6%, the expression of mih was significantly lower than that of the 0% CHO group P<0.05 (Figure 2(e)).


### 3.3. Analysis of Cholesterol Deposition

The content of CHO in hepatopancreas, muscle, and hemolymph of crab was significantly elevated with the increase of dietary CHO levelsP<0.05 (Figures 3(a) and 3(c)). And the CHO deposition in hepatopancreas was much higher than that in muscle and hemolymph. The broken-line model y=1.877x+1.144, R2=0.9392; y=0.3393x+1.837, R2=0.9854 (Figure 3(g)) of CHO deposition in hepatopancreas against the dietary CHO addition demonstrated that when the CHO content was lower than 0.45%, the CHO deposition efficiency in hepatopancreas was higher than that in the groups with 0.8% and 1.6% CHO. Meanwhile, the contents of HDL-C and LDL-C in hemolymph of crabs increased with the increase of the CHO level, and when the addition level exceeds 0.4%, significant differences were observed P<0.05 (Figures 3(d) and 3(e)). As shown in Figure 3(f), when the CHO level was less than 0.4%, the ratio of HDL-C/LDL-C could be significantly improved P<0.05 with the increase of CHO, while when the CHO content exceeded 0.4%, the ratio of HDL-C/LDL-C tends to decrease.Figure 3
Effects of dietary CHO on (a) the content of CHO in hepatopancreas; (b) the content of CHO in muscle; (c) the content of total CHO in hemolymph; (d) the content of high-density lipoprotein cholesterol in hemolymph (HDL-C); (e) the content of low-density lipoprotein cholesterol in hemolymph (LDL-C); and (f) the ratio of HDL-C/LDL-C in hemolymph. (g) Broken-line relationship of the dietary CHO level to the content of CHO deposition in hepatopancreas. The values are themeans±SEMn=4. Different letters indicate significant differences P<0.05.
(a)(b)(c)(d)(e)(f)(g)


### 3.4. Parameters of Lipid Metabolism

The content of TG in the hepatopancreas and hemolymph was significantly affected by the CHO addition levels when linear and quadratic. However, there is no significant difference in the content of TG in muscle (Table4). In addition, with the increase of CHO in the diet, the content of TG in hepatopancreas and hemolymph also increased significantly P<0.05, and when the amount of CHO was 1.6%, the content of TG reached the highest. Among the CHO addition groups, the contents of NEFA in the hepatopancreas, muscle, and hemolymph had no significant differences.Table 4
Effect of dietary CHO level on lipid metabolism related indices ofE. sinensis.
Dietary CHO levelHepatopancreasMuscleHemolymphTG (mmol/gprot)NEFA (mmol/gprot)TG (mmol/gprot)NEFA (mmol/gprot)TG (mmol/L)NEFA (mmol/L)00.45a0.400.170.120.73a0.780.10.50ab0.380.180.110.73a0.820.20.58bc0.390.180.110.79ab0.850.40.61bc0.400.180.100.86b0.840.80.70cd0.410.180.100.91b0.881.60.77d0.400.190.101.08c0.86SEM0.040.040.010.010.040.03P value0.000.900.930.500.000.47Regression analysisLinearAdj. R20.57−0.040.010.070.570.11P value0.000.680.250.120.000.07QuadraticAdj. R20.60−0.080.030.080.610.10P value0.000.850.230.160.000.13Effect of the dietary CHO level on lipid metabolism-related indices ofE. sinensis. SEM: pooled standard error of means n=4; Adj. R2: adjusted R square. Values in the same column with different superscripts are significantly different P<0.05. TG: triglyceride; NEFA: nonesterified fatty acid.


### 3.5. Related Gene mRNA Expression of Lipid Metabolism

The addition of CHO in the diet had a significant effect on the mRNA expression ofcpt-1a, srebp-1, fas, and mttp in hepatopancreas, and compared to the groups without CHO supplementation, the expression of cpt-1a was markedly decreased among the groups with CHO addition P<0.05 (Figures 4(a)–4(d)). On the contrary, the expression of mttp, srebp-1, and fas showed an upward trend after adding CHO. With the increase of the addition of CHO in diet, the expression of srebp-1 also increased significantly P<0.05. When the addition of CHO is below 0.2%, the expression of mttp does not change significantly. However, with the further addition of CHO, the expression of mttp increases significantly P<0.05. In addition, when the amount of CHO was less than 0.4%, the mRNA expression of fas tends to increase. In comparison, when the supplementation of CHO exceeds 0.4%, the expression of fas decreases significantly P<0.05.Figure 4
Effects of dietary CHO on (a) carnitine palmitoyl transterase-1a (cpt-1a); (b) fatty acid synthase (fas); (c) sterol-regulatory element binding protein 1 (srebp-1); and (d) microsomal triglyceride transfer protein (mttp). The values are the means±SEMn=4. Different letters indicate significant differences P<0.05.
(a)(b)(c)(d)


## 3.1. Growth Performance

As shown in Table3, the FBW, SR, WGR, and FCR of crabs were markedly influenced by the supplementation level of CHO at quadratic P<0.05. Compared to those of the control group, CHO supplementation levels at 0.2% and 0.4%, FBW, SR, and WGR were significantly increased P<0.05 and FCR decreased significantly P<0.05. When the crabs were fed a 0.4% CHO diet, the FBW, WGR, and FCR reached the highest. However, the addition of CHO in the diet has no significant effects on MY and FI of crabs. In addition, through the broken-line model y=48.13x+99.89, R2=0.8885; y=−6.28x+114.55, R2=0.9036 (Figure 1) based on the relationship between the FBW and the dietary CHO levels, it was observed that the FBW of the crabs increased significantly P<0.05 with the addition of CHO from 0% to 0.4% and then decreased; when the CHO supplementation level was 0.27%, the crabs reached the highest FBW.Table 3
Effect of dietary cholesterol level on the growth performance ofE. sinensis.
Dietary CHO levelFBW (g)SR (%)WGR (%)MY (%)FI (g)FCR0102.40a57.50a140.10a20.24136.002.28a0.1102.70a65.00abc141.70a21.06135.902.26a0.2110.50bc75.00c161.30b19.78136.702.01bc0.4113.00c70.00bc163.30b20.15136.601.96c0.8104.80ab62.50ab144.60a19.32136.902.23ab1.6106.60abc62.50ab148.50ab19.37136.402.16abcSEM0.013.282.090.680.600.08P value0.040.030.040.600.850.04Regression analysisLinearAdj. R20.03−0.04−0.010.03−0.000.00P value0.200.730.330.150.340.31QuadraticAdj. R20.230.290.200.01−0.030.20P value0.030.010.040.330.500.04FBW: final body weight; SR: survival rate; WGR: weight gain ratio; MY: meat yield; FI: feed intake; FCR: feed conversion ratio; SEM: pooled standard error of:n=4; Adj. R2: adjusted R square. Values in the same column with different superscripts are significantly different at P<0.05.Figure 1
Broken-line relationship of the dietary CHO level to FBW forE. sinensis. Data appeared as means±SEMn=4. Values in the same curve with different superscripts are significantly different P<0.05.As shown in Figure2(b), the total molting times were increased significantly as the addition levels of CHO increased from 0% to 0.2% and then decreased. We observed that from the sixth week to the sixteenth week, the total molting times of crabs in the group with 0.2% CHO were always higher than those in other groups. As shown in Figure 2(a), the molting frequency (MF) was affected by dietary CHO supplementation, and it reached the highest when the CHO supplementation level is at 0.2%. Additionally, compared to the control group, no significant difference was observed among the groups with 0.1%, 0.4%, 0.8%, and 1.6% CHO.Figure 2
Effects of dietary CHO on (a) molting frequency (MF); (b) total molting times; (c) ecdysteroid receptor (ecr); (d) retinoid X receptor (rxr); and (e) molting-inhibiting hormone (mih). The values are the means±SEMn=4. Different letters indicate significant differences P<0.05.
(a)(b)(c)(d)(e)


## 3.2. The Molting-Related Gene Expression

The molting-related gene mRNA expression was markedly affectedP<0.05 by the dietary CHO supplementation as shown in Figures 2(c)–2(e). Compared with the group of CHO content at 0%, the mRNA expression of ecr (ecdysteroid receptor) and rxr (retinoid X receptor) was increased among the CHO addition groups and the significant differences were observed when the dietary CHO level is at 0.2% P<0.05 (Figures 2(c) and 2(d)). Compared to the control group, the mih (molt-inhibiting hormone) mRNA expression was decreased among the 0.1%, 0.2%, 0.4%, 0.8%, and 1.6% CHO groups. When the addition of CHO reached 0.8% and 1.6%, the expression of mih was significantly lower than that of the 0% CHO group P<0.05 (Figure 2(e)).


## 3.3. Analysis of Cholesterol Deposition

The content of CHO in hepatopancreas, muscle, and hemolymph of crab was significantly elevated with the increase of dietary CHO levelsP<0.05 (Figures 3(a) and 3(c)). And the CHO deposition in hepatopancreas was much higher than that in muscle and hemolymph. The broken-line model y=1.877x+1.144, R2=0.9392; y=0.3393x+1.837, R2=0.9854 (Figure 3(g)) of CHO deposition in hepatopancreas against the dietary CHO addition demonstrated that when the CHO content was lower than 0.45%, the CHO deposition efficiency in hepatopancreas was higher than that in the groups with 0.8% and 1.6% CHO. Meanwhile, the contents of HDL-C and LDL-C in hemolymph of crabs increased with the increase of the CHO level, and when the addition level exceeds 0.4%, significant differences were observed P<0.05 (Figures 3(d) and 3(e)). As shown in Figure 3(f), when the CHO level was less than 0.4%, the ratio of HDL-C/LDL-C could be significantly improved P<0.05 with the increase of CHO, while when the CHO content exceeded 0.4%, the ratio of HDL-C/LDL-C tends to decrease.Figure 3
Effects of dietary CHO on (a) the content of CHO in hepatopancreas; (b) the content of CHO in muscle; (c) the content of total CHO in hemolymph; (d) the content of high-density lipoprotein cholesterol in hemolymph (HDL-C); (e) the content of low-density lipoprotein cholesterol in hemolymph (LDL-C); and (f) the ratio of HDL-C/LDL-C in hemolymph. (g) Broken-line relationship of the dietary CHO level to the content of CHO deposition in hepatopancreas. The values are themeans±SEMn=4. Different letters indicate significant differences P<0.05.
(a)(b)(c)(d)(e)(f)(g)


## 3.4. Parameters of Lipid Metabolism

The content of TG in the hepatopancreas and hemolymph was significantly affected by the CHO addition levels when linear and quadratic. However, there is no significant difference in the content of TG in muscle (Table4). In addition, with the increase of CHO in the diet, the content of TG in hepatopancreas and hemolymph also increased significantly P<0.05, and when the amount of CHO was 1.6%, the content of TG reached the highest. Among the CHO addition groups, the contents of NEFA in the hepatopancreas, muscle, and hemolymph had no significant differences.Table 4
Effect of dietary CHO level on lipid metabolism related indices ofE. sinensis.
Dietary CHO levelHepatopancreasMuscleHemolymphTG (mmol/gprot)NEFA (mmol/gprot)TG (mmol/gprot)NEFA (mmol/gprot)TG (mmol/L)NEFA (mmol/L)00.45a0.400.170.120.73a0.780.10.50ab0.380.180.110.73a0.820.20.58bc0.390.180.110.79ab0.850.40.61bc0.400.180.100.86b0.840.80.70cd0.410.180.100.91b0.881.60.77d0.400.190.101.08c0.86SEM0.040.040.010.010.040.03P value0.000.900.930.500.000.47Regression analysisLinearAdj. R20.57−0.040.010.070.570.11P value0.000.680.250.120.000.07QuadraticAdj. R20.60−0.080.030.080.610.10P value0.000.850.230.160.000.13Effect of the dietary CHO level on lipid metabolism-related indices ofE. sinensis. SEM: pooled standard error of means n=4; Adj. R2: adjusted R square. Values in the same column with different superscripts are significantly different P<0.05. TG: triglyceride; NEFA: nonesterified fatty acid.


## 3.5. Related Gene mRNA Expression of Lipid Metabolism

The addition of CHO in the diet had a significant effect on the mRNA expression ofcpt-1a, srebp-1, fas, and mttp in hepatopancreas, and compared to the groups without CHO supplementation, the expression of cpt-1a was markedly decreased among the groups with CHO addition P<0.05 (Figures 4(a)–4(d)). On the contrary, the expression of mttp, srebp-1, and fas showed an upward trend after adding CHO. With the increase of the addition of CHO in diet, the expression of srebp-1 also increased significantly P<0.05. When the addition of CHO is below 0.2%, the expression of mttp does not change significantly. However, with the further addition of CHO, the expression of mttp increases significantly P<0.05. In addition, when the amount of CHO was less than 0.4%, the mRNA expression of fas tends to increase. In comparison, when the supplementation of CHO exceeds 0.4%, the expression of fas decreases significantly P<0.05.Figure 4
Effects of dietary CHO on (a) carnitine palmitoyl transterase-1a (cpt-1a); (b) fatty acid synthase (fas); (c) sterol-regulatory element binding protein 1 (srebp-1); and (d) microsomal triglyceride transfer protein (mttp). The values are the means±SEMn=4. Different letters indicate significant differences P<0.05.
(a)(b)(c)(d)


## 4. Discussion

In this research, the result is that appropriate CHO in the diet could improve the SR, WGR, and FCR ofE. sinensis, which was consistent with the results of previous studies [1, 14, 28–30]. According to a previous report, CHO deficiency caused poor growth performance of Procambarus clarkia [14]. Similar results were found in this experiment, and the worst growth performance was observed when the crabs were fed the diet with 0% CHO. In addition, this study found that excessive CHO will also have adverse effects on the growth performance and previous studies have shown that dietary CHO may accumulate in the body and increase the metabolic burden on the body, resulting in poor growth performance [28, 31, 32].As an important nutrient, CHO is necessary for the growth of many aquatic animals. At present, the requirement of CHO for some aquatic animals has been reported, such as 0.51% forScylla serrata [32], 0.2% for Penaeus monodon [1], and 0.2%~0.5% for Litopenaeus vannamei [19]. However, the data on CHO requirement of E. sinensis are limited. In this experiment, according to the regression curve based on the FBW and dietary CHO levels, we inferred that the optimal CHO level of E. sinensis feed was 0.27%. This result is consistent with the finding in Penaeus monodon (0.2%; [1]), but lower than Scylla serrata (0.51%; [32]). The difference may be due to the differences in species, growth stages, and experimental conditions [18]. In addition, at present, the content of fish meal in commercial feed is about 30% and the CHO content of fish meal is about 0.4%; therefore, formula feed can only provide 0.12% CHO, which may not meet the needs.Molting is a necessary physiological process for the growth and development of crustaceans [8, 33]. In crustaceans, ecdysone and molt-inhibiting hormone (MIH) are two hormones that regulate the process of molting. Ecdysone is synthesized in the Y organ with CHO as a raw material, and then, ecdysone enters the nucleus to activate the heterodimer formed by the ecdysone receptor (EcR) and retinoid X receptor (RXR), so as to regulate the transduction of the ecdysone signal pathway [34–37]. Previous studies have reported that an appropriate amount of CHO can promote the activation of the ecdysone signal pathway and improve molting performance, while excessive CHO will inhibit the activation of the ecdysone signal pathway, resulting in the decline of the molting performance. Similar results were also obtained in this experiment, and 0.2% CHO could significantly improve the molting performance of the E. sinensis, while no obvious changes were observed in those supplemented with 0.4%, 0.8%, and 1.6%. [14, 38, 39]. In order to further investigate the effects of CHO on the ecdysone signal pathway, we measured the expression levels of molting-related genes in the ecdysone signal pathway. The results showed that the expression levels of ecr and rxr among the CHO-added group were higher than those in the control group and reached the highest in the 0.2% CHO group. This result is lower than the study of Tian et al. [14]. Tian et al. [14] reported that 0.5% CHO could significantly improve the expression level of ecr and rxr. This may be caused by species or different sampling times. The MIH can inhibit molting of crustaceans by inhibiting the secretion of ecdysone by the Y organ; after removing the eyestalk of Uca pugilator, its molting is advanced [40–42]. In addition, the secretion of MIH is negatively regulated by the molting hormone, so it is reasonable to add CHO and decrease the expression of mih in this experiment. [14, 43].In the present study, the content of CHO in the hepatopancreas, hemolymph, and muscle was positively correlated with the amount of CHO in the diet, which is similar to the results of previous studies onPenaeus japonicus [44]. As the most important organ for CHO storage and metabolism, the hepatopancreas is suitable for evaluating the deposition of CHO in tissues [8]. According to a previous study, when a nutrient in the environment will not be able to meet the demand of the animal growth, the animal will store this nutrient quickly to meet the needs of subsequent growth and development, and when the nutrient content is sufficient, the deposition efficiency will be greatly decreased [5, 45, 46]. This can also explain that the CHO content in the hepatopancreas increases rapidly when the content of dietary CHO is lower than 0.45% but increases slowly with further supplementation. Therefore, according to the results of CHO deposition in hepatopancreas, we concluded that the appropriate deposition amount of CHO in tissues of E. sinensis was 0.45%. The maturation of the E. sinensis can be divided into physical maturity and physiological maturity, and the nutrient requirements for these two physiological processes are different [47, 48]. At the stage of physical maturation, CHO is mainly used to maintain their basic growth requirement, and there is little demand for CHO [5, 47]. But when the crabs complete their pubertal molt, the gonad begins to enter a rapid development period and the demand for CHO also increases. In this process, the hepatopancreas must store sufficient CHO to meet the needs of physiological maturity [8, 10, 49]. As we all know, LDL-C and HDL-C are the transporters of CHO in the body. LDL-C is responsible for transporting CHO from the hepatopancreas to extrahepatic tissues, while HDL-C is responsible for transporting peripheral CHO back to the hepatopancreas [50, 51]. In this study, with the addition of CHO ranging from 0% to 1.6%, the contents of HDL-C and LDL-C in hemolymph markedly increased, similar to the results in Litopenaeus vannamei [52]. The ratio of HDL-C/LDL-C is an important indicator of CHO transport [52, 53]. According to our results, when the addition of CHO was from 0% to 0.4%, the ratio of HDL-C/LDL-C significantly improved, indicating that CHO was accumulated in the hepatopancreas, while when the addition of CHO was more than 0.4%, the ratio tends to decrease, which indicates that CHO was mainly transferred to peripheral tissue [5, 54, 55]. This further proves that when the content of CHO is lower than the requirement, it accumulates rapidly in the hepatopancreas, while when the demand is reached, it flows to the extrahepatic tissue.Lipid, as one of the most important energy substances, can provide enough energy for the development of the crabs. The TG and NEFA as the energy currency in the body can reflect the status of lipid metabolism to some extent [4, 56]. Liu et al. [36] reported that the level of CHO in the diet was positively correlated with the content of TG in hemolymph of Litopenaeus vannamei. Similarly, in this study, the content of TG also increased with CHO added. NEFA is decomposed by TG under the action of hormone-sensitive lipase. There is no significant difference in the content of NEFA in this experiment, which may be caused by the activity of hormone-sensitive lipase. However, the specific mechanism of cholesterol affecting the contents of TG and NEFA in crustaceans is still unclear ([52, 57]). This study speculated that it may be as follows: first of all, CHO can participate in the synthesis of lipoprotein, thus accelerating lipid transfer to promote lipid accumulation in the body; secondly, CHO may increase the lipid content of the body by promoting lipid synthesis and inhibiting lipid oxidation [5, 58]. Subsequently, we detected the expression of key genes involved in lipid synthesis and oxidation. The srebp-1 is an important transcription factor, which can activate the transcription of downstream lipid synthesis genes, such as fas, one of the key genes of fatty acid synthesis [59]. In this study, the expression of srebp-1 and fas increased significantly after crabs were fed the diet with CHO supplemented, which could be one of the reasons leading to the increase of TG; similar results have also been reported in other aquatic animals [52, 60, 61]. In addition, cpt-1a is one of the key genes in the process of β-oxidation, and its expression decreases significantly with the addition of CHO, indicating that the supplementation of CHO inhibits the β-oxidation of fatty acids [13]. The mttp is closely related to the assembly of VLDL. Furthermore, VLDL is mainly responsible for transporting TG from hepatopancreas to extrahepatic tissues. This can be interpreted as when there are excessive amounts of TG in the hepatopancreas, the expression of mttp will be upregulated to reduce the fat content in the hepatopancreas [59].


## 5. Conclusion

In conclusion, the results of this study show that adding 0.2% CHO to the diet can improve the molting performance ofE. sinensis. And an appropriate amount of CHO can promote the deposition of lipids in the hepatopancreas, which is beneficial to accumulating sufficient nutrients in the growth and development of E. sinensis. In addition, based on the regression analysis of the FBW, we found that the growth requirement of E. sinensis for CHO was 0.27%. Through the regression analysis of CHO deposition in the hepatopancreas, we concluded that the tissue CHO deposition of E. sinensis for CHO was 0.45%. Therefore, according to the results of the present investigation, we suggest that 0.27%–0.45% CHO should be added to the diet to promote the growth and development of E. sinensis.



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*Source: 2012958-2022-05-20.xml*