# Estimation of the Optimum Dietary Protein to Lipid Ratio in Juvenile Pengze Crucian Carp (Carassius auratus Var. Pengze)

Authors: Liyun Ding; Wenjing Chen; Huiyun Fu; Jun Xiao; Yilong Fu; Jingjing Ma
Journal: Aquaculture Nutrition
 (2022)

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## Abstract

An 8-week feeding experiment was conducted to evaluate the effect of dietary protein to lipid ratio on fish growth performance, digestive enzyme activity, body composition, serum biochemistry, immunity, and antioxidant capacity ofCarassius auratus var. Pengze juveniles. Nine experimental diets were formulated in a 3×3 factorial design to contain three protein levels (30%, 35%, and 40%) and three lipid levels (5%, 8%, and 11%), with protein-to-energy (P/E) ratios ranging 19.2–27.1 mg/kJ and named as Diet 1 (30/5), Diet 2 (30/8), Diet 3 (30/11), Diet 4 (35/5), Diet 5 (35/8), Diet 6 (35/11), Diet 7 (40/5), Diet 8 (40/8), and Diet 9 (40/11), respectively. Each diet was randomly assigned to triplicate groups of 20 fish per cylindrical fiberglass tank. The fish fed with Diet 4 had the highest specific growth rate (SGR) among the 9 groups. The whole-body lipid content increased significantly with increasing dietary lipid levels regardless of dietary protein content (P<0.05), whereas moisture content showed an opposite trend (P<0.05). The highest values of activities of serum lysozyme (LZM) and alkaline phosphatase (AKP) were detected in the fish fed Diet 4 (35/5), whereas malondialdehyde (MDA) was lowest in the fish fed Diet 4. In addition, serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) were the lowest, while high-density lipoprotein (HDL-C) and high-density lipoprotein to total cholesterol ratio (HDL-C/TC) were the highest in the Diet 4 group. The results indicate that a combination of 35.1% protein and 5.1% lipid with dietary CP/GE (mg/kJ) ratio of 24.0 is optimal for the culture of Carassius auratus var. Pengze juveniles.


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## Body

## 1. Introduction

Reducing the protein content in aquatic feeds is one of the strategies to reduce the feed cost and environmental pollution for sustainable aquaculture [1]. Protein is one of the most important nutrients in aquatic feeds. It is the main component of metabolic active substances in the body and a considerable factor affecting growth performance [2]. Generally, fish growth improves with increasing dietary protein up to the optimum level. However, excessive dietary protein content also increases the cost of feed production and affects profitability [3] as well as reduces the quality of culture water with higher ammonia levels [4, 5]. Considering this, much research has been conducted to decrease dietary protein incorporation, by increasing the lipid and/or carbohydrate content in fish diets [3]. Dietary lipid plays an important role in fish nutrition as a source of energy and essential fatty acids and could promote the absorption of fat-soluble vitamins. Within certain limits, increasing dietary lipid levels improves feed efficiency and growth performance [4–6]. Lipid and carbohydrate in fish diets also serve as energy sources for protein-sparing potential [7]. Therefore, the optimum ratio of dietary protein to lipid levels should be taken into consideration in formulated fish diets.Pengze crucian carp (Carassius auratus var. Pengze) is a kind of native omnivorous freshwater fish in China. This species was originally produced in several lakes of Pengze County (Jiangxi, China). It has been regarded as one of the most economically valuable species over the past decades in China because of its palatability, fast growth, better flavor, and high disease resistance [8]. Many studies on the molecular biology and reproductive biology of Pengze crucian carp have been conducted [9–11], but only few of them have reported on its nutriology. Therefore, the objective of our study was to evaluate the optimum protein to lipid ratio by analyzing the growth performance, digestive enzyme activity, body composition, blood lipid composition, antioxidant capacity, and immunity of juvenile Pengze crucian carp fed diets containing different protein and lipid levels.


## 2. Materials and Methods

### 2.1. Diet Preparation and Fish Culturing

A3×3 factorial design with three replicates was used. Nine practical diets were formulated to contain three protein levels (30, 35, and 40%) and three lipid levels (5, 8, and 11%) to produce protein-to-energy (P/E) ratios in the range of 19.2–27.1 mg/kJ. Fish meal and fermented soybean meal were used as the main protein sources and fish oil and soybean oil (1 : 1) as the main lipid sources. The diets were designated as Diet 1 (30/5), Diet 2 (30/8), Diet 3 (30/11), Diet 4 (35/5), Diet 5 (35/8), Diet 6 (35/11), Diet 7 (40/5), Diet 8 (40/8), and Diet 9 (40/11), respectively. Formulation and proximate composition of the diets are presented in Table 1.Table 1
Formulation and proximate composition of the experimental diets (g/100 g dry matter).
IngredientDiet groups (protein/lipid)30/530/830/1135/535/835/1140/540/840/11Fermented soybean meal1404040494949585858Fish meal1101010101010101010Corn starch1403734312825221916Fish oil1.534.51.534.51.534.5Soybean oil1.534.51.534.51.534.5Calcium biphosphate1.51.51.51.51.51.51.51.51.5Vitamin premix2111111111Mineral premix3222222222Choline chloride0.50.50.50.50.50.50.50.50.5Sodium alginate222222222Proximate compositionDry matter90.889.289.890.189.989.390.291.591.3Crude protein (CP)30.330.730.535.135.335.439.840.740.8Crude lipid4.77.811.15.1811.24.97.911.1Ash8.98.78.99.18.68.88.89.09.2Gross energy (GE, kJ/g)14.715.215.914.615.315.914.715.516.1CP:GE (mg/kJ)20.620.219.224.023.122.327.126.325.31Proximate composition as % dry-weight basis (fish meal (71.8% crude protein, 9.5% crude lipid); fermented soybean meal (54.2% crude protein, 2.7% crude lipid); corn starch (1.2% crude protein, 0% crude lipid)). 2Vitamin premix (per kg diet): thiamin, 15 mg; riboflavin, 25 mg; pyridoxine, 15 mg; cyanocobalamine, 0.2 mg; folic acid, 5 mg; calcium pantothenate, 50 mg; inositol, 500 mg; niacin, 100 mg; biotin (2%), 1.2 mg; ascorbic acid, 100 mg; vitamin A, 12000 IU; vitamin D3, 5000 IU; vitamin E, 55 mg; vitamin K3, 5 mg. 3Mineral premix (mg/kg diet): MgSO4·7H2O: 450; FeSO4·7H2O: 250; CuSO4·5H2O: 10; ZnSO4·7H2O: 108; MnSO4·4H2O: 40; KI: 1.5; NaCl: 600; KH2PO4: 1350; CoSO4·4H2O, 0.50.


### 2.2. Experimental Fish and Feeding Trial

Carassius auratus var. Pengze juveniles were obtained from Jiangxi Fisheries Research Institute, Nanchang. Prior to the experiment, the fish were stocked into experimental tanks to adapt to the new rearing conditions for 15 days. After acclimatization, a total of 540 fish with initial weight of 31.58±0.19g were randomly distributed into 27 cylindrical fiberglass tanks (Φ800mm×650mm), with 20 juveniles per tank. Each tank was then randomly assigned to one of three replicates of the nine experimental diets. The fish were cultured indoor with natural photoperiod. During the trial, the fish were fed to apparent satiation twice (09:00 and 17:00 h) daily for 56 days. The water temperature was 26±1.5°C, pH remained 7.53±0.12, dissolved oxygen>7mg/L, and ammonia nitrogen<0.1mg/L.


### 2.3. Sample Collection and Chemical Analysis

After the 56-day feeding trial, the total number and mean body weight of the fish in each tank were measured after 24 h starvation. Six fish per tank were randomly collected and stored (-20°C) for determination of proximate body composition. Another six fish from each tank were anesthetized with MS222 (100 mg/L); blood was collected from the caudal vein with a 1 mL syringe, left undisturbed for 4 h in the lab to clot (4°C), and centrifuged at4000g for 10 min at 4°C and immediately stored at -80°C until analysis. Samples of midintestine were dissected from six fish in each tank to determine digestive enzyme activity.Standard methods of the Association of Official Analytical Chemists [12] were followed for the analysis of crude protein, crude lipids, moisture, and crude ash contents in fish body and diets. Briefly, moisture was determined by drying the samples to a constant weight at 105°C. The crude protein contents (N×6.25) were determined using the Dumas combustion method with a protein analyzer (FP-528, Leco, USA). Ash contents were determined using a muffle furnace run at 550°C for 8 h. Crude lipid contents were determined via the ether extraction method using a Soxtec System HT (Soxtec System HT6, Tecator, Sweden). Gross energy content in diets was measured using a bomb calorimeter (Parr 1351; Parr Instrument Co., Moline, IL, USA).The levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in pooled serum were determined by an automatic Chemistry Analyzer (Hitachi 7600, Tokyo, Japan). Fish antioxidant capacity (total superoxide dismutase (TSOD, A001-3-2), catalase (CAT, A007-2-1), malondialdehyde (MDA, A003-1-2), and total antioxidant capacity (T-AOC, A015-1-2)), immunity (alkaline phosphatase (AKP, A059-2-2) and lysozyme (LZM, A050-1-1)), and intestinal digestive enzymes (trypsin (A080-2-2), lipase (LPS, A054-1-1), and amylase (AMS, C016-1-1)) and liver glycogen (LG, A043-1-1) were all determined using diagnostic reagent kits (Jiancheng Bioengineering Ltd., Nanjing, China) according to the instruction manuals of the manufacturer.


### 2.4. Statistical Analysis

All data were subjected to analysis of variance using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) for Windows. One-way ANOVA was used to determine whether there were significant differences between the treatments. Two-way ANOVA was used to determine the effect of dietary protein, dietary lipid, and their interaction. Differences among the means were tested by Tukey’s multiple-range test. The level of significance was chosen atP<0.05. The results are presented as means±SD.


## 2.1. Diet Preparation and Fish Culturing

A3×3 factorial design with three replicates was used. Nine practical diets were formulated to contain three protein levels (30, 35, and 40%) and three lipid levels (5, 8, and 11%) to produce protein-to-energy (P/E) ratios in the range of 19.2–27.1 mg/kJ. Fish meal and fermented soybean meal were used as the main protein sources and fish oil and soybean oil (1 : 1) as the main lipid sources. The diets were designated as Diet 1 (30/5), Diet 2 (30/8), Diet 3 (30/11), Diet 4 (35/5), Diet 5 (35/8), Diet 6 (35/11), Diet 7 (40/5), Diet 8 (40/8), and Diet 9 (40/11), respectively. Formulation and proximate composition of the diets are presented in Table 1.Table 1
Formulation and proximate composition of the experimental diets (g/100 g dry matter).
IngredientDiet groups (protein/lipid)30/530/830/1135/535/835/1140/540/840/11Fermented soybean meal1404040494949585858Fish meal1101010101010101010Corn starch1403734312825221916Fish oil1.534.51.534.51.534.5Soybean oil1.534.51.534.51.534.5Calcium biphosphate1.51.51.51.51.51.51.51.51.5Vitamin premix2111111111Mineral premix3222222222Choline chloride0.50.50.50.50.50.50.50.50.5Sodium alginate222222222Proximate compositionDry matter90.889.289.890.189.989.390.291.591.3Crude protein (CP)30.330.730.535.135.335.439.840.740.8Crude lipid4.77.811.15.1811.24.97.911.1Ash8.98.78.99.18.68.88.89.09.2Gross energy (GE, kJ/g)14.715.215.914.615.315.914.715.516.1CP:GE (mg/kJ)20.620.219.224.023.122.327.126.325.31Proximate composition as % dry-weight basis (fish meal (71.8% crude protein, 9.5% crude lipid); fermented soybean meal (54.2% crude protein, 2.7% crude lipid); corn starch (1.2% crude protein, 0% crude lipid)). 2Vitamin premix (per kg diet): thiamin, 15 mg; riboflavin, 25 mg; pyridoxine, 15 mg; cyanocobalamine, 0.2 mg; folic acid, 5 mg; calcium pantothenate, 50 mg; inositol, 500 mg; niacin, 100 mg; biotin (2%), 1.2 mg; ascorbic acid, 100 mg; vitamin A, 12000 IU; vitamin D3, 5000 IU; vitamin E, 55 mg; vitamin K3, 5 mg. 3Mineral premix (mg/kg diet): MgSO4·7H2O: 450; FeSO4·7H2O: 250; CuSO4·5H2O: 10; ZnSO4·7H2O: 108; MnSO4·4H2O: 40; KI: 1.5; NaCl: 600; KH2PO4: 1350; CoSO4·4H2O, 0.50.


## 2.2. Experimental Fish and Feeding Trial

Carassius auratus var. Pengze juveniles were obtained from Jiangxi Fisheries Research Institute, Nanchang. Prior to the experiment, the fish were stocked into experimental tanks to adapt to the new rearing conditions for 15 days. After acclimatization, a total of 540 fish with initial weight of 31.58±0.19g were randomly distributed into 27 cylindrical fiberglass tanks (Φ800mm×650mm), with 20 juveniles per tank. Each tank was then randomly assigned to one of three replicates of the nine experimental diets. The fish were cultured indoor with natural photoperiod. During the trial, the fish were fed to apparent satiation twice (09:00 and 17:00 h) daily for 56 days. The water temperature was 26±1.5°C, pH remained 7.53±0.12, dissolved oxygen>7mg/L, and ammonia nitrogen<0.1mg/L.


## 2.3. Sample Collection and Chemical Analysis

After the 56-day feeding trial, the total number and mean body weight of the fish in each tank were measured after 24 h starvation. Six fish per tank were randomly collected and stored (-20°C) for determination of proximate body composition. Another six fish from each tank were anesthetized with MS222 (100 mg/L); blood was collected from the caudal vein with a 1 mL syringe, left undisturbed for 4 h in the lab to clot (4°C), and centrifuged at4000g for 10 min at 4°C and immediately stored at -80°C until analysis. Samples of midintestine were dissected from six fish in each tank to determine digestive enzyme activity.Standard methods of the Association of Official Analytical Chemists [12] were followed for the analysis of crude protein, crude lipids, moisture, and crude ash contents in fish body and diets. Briefly, moisture was determined by drying the samples to a constant weight at 105°C. The crude protein contents (N×6.25) were determined using the Dumas combustion method with a protein analyzer (FP-528, Leco, USA). Ash contents were determined using a muffle furnace run at 550°C for 8 h. Crude lipid contents were determined via the ether extraction method using a Soxtec System HT (Soxtec System HT6, Tecator, Sweden). Gross energy content in diets was measured using a bomb calorimeter (Parr 1351; Parr Instrument Co., Moline, IL, USA).The levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in pooled serum were determined by an automatic Chemistry Analyzer (Hitachi 7600, Tokyo, Japan). Fish antioxidant capacity (total superoxide dismutase (TSOD, A001-3-2), catalase (CAT, A007-2-1), malondialdehyde (MDA, A003-1-2), and total antioxidant capacity (T-AOC, A015-1-2)), immunity (alkaline phosphatase (AKP, A059-2-2) and lysozyme (LZM, A050-1-1)), and intestinal digestive enzymes (trypsin (A080-2-2), lipase (LPS, A054-1-1), and amylase (AMS, C016-1-1)) and liver glycogen (LG, A043-1-1) were all determined using diagnostic reagent kits (Jiancheng Bioengineering Ltd., Nanjing, China) according to the instruction manuals of the manufacturer.


## 2.4. Statistical Analysis

All data were subjected to analysis of variance using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) for Windows. One-way ANOVA was used to determine whether there were significant differences between the treatments. Two-way ANOVA was used to determine the effect of dietary protein, dietary lipid, and their interaction. Differences among the means were tested by Tukey’s multiple-range test. The level of significance was chosen atP<0.05. The results are presented as means±SD.


## 3. Results

### 3.1. Effect of Dietary Protein and Lipid Levels on Fish Growth Performance and Feed Utilization

Dietary protein and lipid combinations significantly affected the WG, SGR, HSI, and Fulton’s condition factor (K) (P>0.05), but did not influence FCR and VSI (P<0.05) of Carassius auratus var. Pengze juveniles (Table 2). VSI and K were not significantly affected by dietary protein level, while WG, FCR, SGR, and HSI were not affected by dietary lipid level. Fish fed diet with 35% protein exhibited higher WG and SGR and lower FCR compared with those fed diets with 30% and 40% protein, whereas HSI was significantly depressed by dietary protein higher than 30%. VSI was elevated by dietary 8% and 11% lipid levels, while K of fish fed diets with 5% and 8% lipid levels was significantly lower than that of those fed diets with 11% lipid. Feed intake was lower in fish fed diets with 35% protein compared to fish fed diets with 30% and 40% protein, whereas feed intake was not influenced by dietary lipid levels. The fish fed diets with 35% protein showed lower FCR when compared with the groups fed 30% and 40% protein. According to one-way ANOVA, fish fed the diet with 35% protein and 5% lipid showed the highest WG and SGR, the lowest FCR, and lower VSI and HSI. Fish liver glycogen decreased from 36.16 mg/g to 25.09 mg/g among all dietary treatments and was significantly depressed by dietary protein levels at the same lipid level (P<0.05).Table 2
Growth response, feed utilization, and physical parameters of Pengze crucian carp (Carassius auratus var. Pengze) juveniles fed diets with different protein and lipid levels1.
Diet no. (protein/lipid)IBW2WG3 (%)Feed intake4 (g/100 g ABW5/d)FCR6SGR7VSI8 (%)HSI9 (%)K10Liver glycogenDiet 1 (30/5)31.58±0.1998.76±1.68a1.67±0.05b1.41±0.06b1.23±0.02a20.09±0.77ab3.26±0.34ab3.02±0.00a36.16±3.24bDiet 2 (30/8)31.53±0.15100.94±0.41ab1.59±0.03ab1.36±0.00ab1.25±0.00ab20.90±0.42ab4.02±0.12c3.21±0.05d35.72±3.38bDiet 3 (30/11)31.61±0.04101.35±0.78ab1.62±0.02ab1.34±0.02ab1.25±0.01ab20.71±0.42ab3.61±0.21bc3.22±0.02d34.55±3.48bDiet 4 (35/5)31.60±0.12105.01±1.63c1.63±0.0ab1.29±0.04a1.28±0.01c20.42±0.30ab2.99±0.06a3.19±0.02cd31.24±1.23abDiet 5 (35/8)31.69±0.34102.95±0.38bc1.59±0.02ab1.31±0.02a1.26±0.01bc21.79±0.38b3.16±0.51ab3.05±0.05ab30.59±3.52abDiet 6 (35/11)31.64±0.40101.99±1.28abc1.61±0.02ab1.29±0.03a1.26±0.01abc21.51±0.72ab3.26±0.08ab3.17±0.08cd29.70±1.43abDiet 7 (40/5)31.73±0.11100.60±0.72ab1.61±0.02ab1.35±0.02ab1.24±0.01ab19.43±2.19a3.04±0.39a3.11±0.05bc26.38±0.61aDiet 8 (40/8)31.74±0.07101.10±0.95ab1.56±0.06a1.34±0.03ab1.25±0.00ab20.99±0.57ab2.82±0.85a3.06±0.06ab25.86±2.06aDiet 9 (40/11)31.68±0.03101.43±1.24ab1.61±0.02ab1.34±0.03ab1.25±0.01ab21.67±2.05b3.03±0.03a3.16±0.00cd25.09±2.90aProtein level (%)30100.35±1.53u1.64±0.03v1.37±0.04v1.24±0.01u20.57±0.613.63±0.39v3.15±0.1035.48±0.83w35103.32±1.85v1.58±0.02u1.30±0.03u1.27±0.02v21.24±0.763.14±0.28u3.14±0.0830.51±0.77v40101.04±0.93u1.61±0.01uv1.34±0.03v1.25±0.00u20.70±1.822.96±0.23u3.11±0.0625.78±0.65uLipid level (%)5101.46±3.041.64±0.041.35±0.071.25±0.0319.98±1.25x3.10±0.293.11±0.75x31.26±4.898101.67±1.111.61±0.021.33±0.031.25±0.0121.23±0.58y3.33±0.603.11±0.09x30.72±4.9311101.59±1.251.59±0.031.32±0.031.25±0.0121.30±1.19y3.30±0.283.18±0.05y29.78±4.73Two-way ANOVA (P value)Protein0.0000.0030.0000.0000.4130.0000.1680.000Lipid0.9350.1020.2080.9230.0350.1360.0030.498Protein×lipid0.0100.6100.2440.0100.7650.0470.0001.0001Data are expressed as mean±SD (n=3). Values in the same column with different superscripts are significantly different (P<0.05). uvwSignificant (P<0.05) difference between protein levels within the same lipid level. xySignificant (P<0.05) difference between lipid levels within the same protein level. 2IBW: initial body weight (g). 3WG: weightgain%=100×finalbodyweightg–initialbodyweightg/initialbodyweightg. 4Feed intake g/100gABW/d=feedconsumptiong/ABWg×feedingdays. 5ABW: average body weight g=finalbodyweighg+initialbodyweightg/2. 6FCR: feedconversionratio=dryfeedfedg/weightgaing. 7SGR: specific growth rate %/day=100×lnfinalbodyweight−lninitialbodyweight/feedingdays. 8VSI: viscerosomatic index %=100×visceralweightg/wholebodyweightg. 9HSI: hepatosomatic index %=100×liverweightg/wholebodyweightg. 10K: K, Fulton’s condition factor g/cm3=100×finalbodyweightg/finalbodylength3cm3. 11LG: liver glycogen (mg/g).


### 3.2. Effect of Dietary Protein and Lipid Levels on Fish Whole-Body Proximate Composition

No interaction was found between dietary protein and lipid levels for fish whole-body moisture, crude protein, and crude ash contents except for crude lipids (Table3). Whole-body moisture, crude lipid, and crude ash contents were not significantly impacted by dietary protein levels, whereas crude protein and ash contents were not affected by dietary lipid levels to a significant extent (P>0.05). Whole-body crude protein content increased with increasing dietary protein levels and reached the highest in the 40% protein group. Whole-body moisture content decreased, whiles whole-body crude lipid content increased, with increasing dietary lipid level.Table 3
Body composition (wet weight basis) of Pengze crucian carp (Carassius auratus var. Pengze) juveniles fed diets with different protein and lipid levels1.
Diet no. (protein/lipid)MoistureCrude proteinCrude lipidAshDiet 1 (30/5)70.15±0.87c16.89±0.204.57±0.41abc4.78±0.06Diet 2 (30/8)67.99±0.52abc16.73±0.516.55±0.36ef4.88±0.30Diet 3 (30/11)67.91±1.19abc16.76±0.706.79±0.42f4.93±0.04Diet 4 (35/5)70.95±0.93c16.73±0.364.39±0.13ab4.85±0.04Diet 5 (35/8)68.96±0.75abc16.89±0.045.62±0.21de4.79±0.05Diet 6 (35/11)66.17±2.33ab17.61±0.055.48±0.27cd5.07±0.82Diet 7 (40/5)71.38±1.66c17.83±0.843.81±0.33a4.97±0.15Diet 8 (40/8)69.42±0.38bc17.98±0.485.28±0.04bcd4.83±0.05Diet 9 (40/11)65.47±2.19a18.13±1.056.07±0.64def5.03±0.10Protein level (%)3068.68±1.3516.80±0.45u5.97±1.114.86±0.173568.69±2.4517.08±0.44u5.16±0.614.90±0.434068.76±2.9517.98±0.72v5.05±1.064.94±0.13Lipid level (%)570.83±1.18z17.15±0.694.26±0.44x4.87±0.12868.79±0.80y17.20±0.685.82±0.61y4.84±0.161166.52±2.02x17.50±0.876.11±0.70y4.50±0.42Two-way ANOVA (P value)Protein0.9920.0010.0000.846Lipid0.0000.3970.0000.430Protein×lipid0.1400.6370.0370.9331Data are expressed as mean±SD (n=3). Values in the same column with different superscripts are significantly different (P<0.05). uvSignificant (P<0.05) difference between protein levels within the same lipid level. xyzSignificant (P<0.05) difference between lipid levels within the same protein level.


### 3.3. Effect of Dietary Protein and Lipid Levels on Fish Digestive Enzyme Activities

Significant interactive effects were found between dietary protein and lipid levels on fish intestinal trypsin and lipase activities (P<0.05), with no interaction detected in amylase activity (P>0.05) (Table 4). Trypsin activity was not significantly affected by dietary lipid level, but its activity increased with increasing dietary protein level, with no significant difference between the 35% and 40% protein groups. Amylase activity decreased with increasing dietary protein level. Lipase activity was the highest in fish fed diet with 35% protein and then decreased, with no significant difference detected between that of fish fed diets with 30% and 40% protein. Amylase activity was depressed by dietary lipid levels higher than 5%, and lipase activity was significantly improved by 11% dietary lipid.Table 4
Digestive enzyme activities of Pengze crucian carp (Carassius auratus var. Pengze) juveniles fed diets with different protein and lipid levels1.
Diet no. (protein/lipid)Trypsin activity (U/mg protein)Amylase activity (U/mg protein)Lipase activity (U/g protein)Diet 1 (30/5)3669.86±155.86a27.76±1.05c5.96±0.70aDiet 2 (30/8)3974.83±6.19ab24.42±1.03bc7.22±0.23bDiet 3 (30/11)4383.47±305.37bc21.49±0.83ab10.98±0.41eDiet 4 (35/5)4857.57±92.66c21.25±1.28ab9.23±0.14cDiet 5 (35/8)4532.42±299.31bc20.79±1.81ab10.48±0.44deDiet 6 (35/11)4786.46±118.86c18.39±4.15ab12.07±0.09fDiet 7 (40/5)4922.85±186.67c19.81±1.28ab8.69±0.13cDiet 8 (40/8)4847.20±166.25c19.40±1.49ab9.52±0.34cDiet 9 (40/11)4115.74±258.97ab15.88±3.52a8.78±0.11cProtein level (%)304009.4±354.33u24.56±2.85v8.05±2.30u354725.5±223.58v20.14±2.70u10.59±1.25v404628.6±462.95v18.36±2.75u9.00±0.44uLipid level (%)54483.4±624.5322.94±3.81y7.96±1.56x84451.5±419.1621.54±2.59xy9.07±1.48x114428.6±359.3118.59±3.68x10.61±1.47yTwo-way ANOVA (P value)Protein0.0000.0000.000Lipid0.8440.0010.000Protein×lipid0.0000.6200.0001Data are expressed as mean±SD (n=3). Values in the same column with different superscripts are significantly different (P<0.05). uvSignificant (P<0.05) difference between protein levels within the same lipid level. xySignificant (P<0.05) difference between lipid levels within the same protein level.


### 3.4. Effect of Dietary Protein and Lipid Levels on Fish Immunity and Antioxidant Capacity

Fish immunity and antioxidant capacity parameters determined in this study were significantly affected by dietary protein, dietary lipid, and their interactions (P<0.05), excluding CAT, which was not influenced by dietary protein levels (P>0.05) (Table 5). In the serum, both LZM and AKP were reduced by dietary lipids (8% and 11%) and dietary protein (40%). TSOD was elevated by dietary protein at 35% and dietary lipid levels higher than 5%. Liver TSOD and CAT were decreased by dietary lipid levels higher than 5%, while no significant differences in both parameters were found among fish from all the dietary protein groups. TAOC was highest in fish fed the diets with 35% protein, at 5% and 8% dietary lipid levels. MDA content generally reduced at 35% and 40% dietary protein levels and increased at 8% dietary lipid level. Fish fed Diet 4 had the lowest MDA content, the highest activities of the rest of the enzymes, and the highest TAOC content.Table 5
Immunity and antioxidant capacity of Pengze crucian carp (Carassius auratus var. Pengze) juveniles fed diets with different protein and lipid levels1.
Diet no. (protein/lipid)SerumLiverLZM (U/ml)TSOD (U/ml)AKP (king unit/L)TSOD (U/mgprot)CAT (U/mgprot)TAOC (mmol/gprot)MDA (nmol/mgprot)Diet 1 (30/5)372.10±17.26bc79.53±14.54a74.85±5.36abc1580.97±154.53cde16.81±0.67bcd0.22±0.03a17.40±0.63dDiet 2 (30/8)274.19±16.13a148.87±13.39abcd66.41±6.22abc1405.35±45.80bc17.73±2.04cd0.21±0.01a15.90±1.26bcDiet 3 (30/11)387.10±32.26c178.96±42.97cd77.85±10.98bc1637.03±24.43def13.07±2.11ab0.28±0.02b11.64±0.97bDiet 4 (35/5)397.10±22.26c188.99±10.89cd102.50±0.71d2034.76±34.42g23.87±2.01e0.33±0.02c5.99±0.10aDiet 5 (35/8)354.84±0.00abc222.93±49.84d78.51±4.73c1725.19±20.61ef14.99±0.29abc0.28±0.01bc16.00±1.97cdDiet 6 (35/11)290.32±64.52bc154.79±23.42abcd62.00±0.99a1186.96±72.27a11.50±0.12a0.20±0.02a10.80±0.50bDiet 7 (40/5)338.71±16.13abc86.95±25.57ab74.00±5.15abc1807.19±37.08f17.93±1.21cd0.23±0.01a13.26±2.51bcDiet 8 (40/8)290.32±32.26ab124.70±34.25abc63.39±3.36abc1483.76±5.18bcd10.93±1.58a0.21±0.01a13.32±0.20bcDiet 9 (40/11)290.32±32.26ab167.68±11.83bcd62.59±4.72ab1346.50±31.95ab20.18±2.23d0.22±0.00a11.54±0.57bProtein levels30344.46±56.74v135.79±50.09u7.30±0.86uv1541.1±132.6715.87±2.610.24±0.04uv14.98±2.73v35347.42±57.73v188.91±40.72v8.10±1.78v1649.0±373.8216.78±5.620.27±0.06v10.93±4.45u40306.45±34.21u126.44±41.42u6.67±0.67u1545.8±206.3216.34±4.440.22±0.01u12.71±4.56uvLipid levels5369.30±30.12y118.49±55.25x8.38±1.45y1807.6±212.65y19.53±3.50y0.26±0.0512.22±5.17xy8306.45±41.12x165.50±54.07y6.94±0.81x1538.1±146.56x14.55±3.24x0.24±0.0415.07±1.76y11322.58±62.47x167.14±27.27y6.75±0.98x1390.2±201.90x14.92±4.29x0.23±0.0411.33±0.73xTwo-way ANOVA (P value)Protein0.0200.0000.0000.0030.4820.0000.000Lipid0.0010.0030.0000.0000.0000.0060.000Protein×lipid0.0020.0040.0000.0000.0000.0000.0001Data are expressed as mean±SD (n=3). Values in the same column with different superscripts are significantly different (P<0.05). uvSignificant (P<0.05) difference between protein levels within the same lipid level. xySignificant (P<0.05) difference between lipid levels within the same protein level. LZM = lysozyme; TSOD = total superoxide dismutase; AKP = alkaline phosphatase.


### 3.5. Effect of Dietary Protein and Lipid Levels on Fish Blood Lipid Metabolism

Except TG, all the serum lipid parameters were significantly affected by dietary treatments (P<0.05) (Table 6). Interactions between dietary protein and lipid levels were detected for all indices but TG. Contents of TC and LDL-C were significantly improved by dietary lipid levels higher than 5%, irrespective of dietary protein contents. HDL-C and HDL-C/TC were all elevated by dietary protein at 35% and 40%, independent of dietary lipid levels. The HDL-C/LDL-C ratio was significantly enhanced by 35% dietary protein and 5% dietary lipid levels. Fish fed Diet 4 had the highest HDL-C, HDL-C/LDL-C, and HDL-C/TC, as well as the lowest TG, TC, and LDL-C.Table 6
Blood lipid composition of Pengze crucian carp (Carassius auratus var. Pengze) juveniles fed diets with different protein and lipid levels1.
Diet no. (protein/lipid)TG (mmol/L)TC (mmol/L)HDL-C (mmol/L)LDL-C (mmol/L)HDL-C/LDL-CHDL-C/TCDiet 1 (30/5)4.13±0.526.91±0.56ab1.96±0.22ab2.79±0.90ab0.73±0.15bc0.47±0.01abDiet 2 (30/8)4.41±0.688.31±0.62bc2.21±0.28abc4.24±0.78b0.52±0.03ab0.50±0.16abDiet 3 (30/11)4.28±0.449.64±0.17c1.55±0.08a4.30±0.39b0.36±0.05a0.36±0.02aDiet 4 (35/5)3.30±0.476.04±0.44a2.81±0.69c2.19±0.22a1.27±0.19d0.84±0.09deDiet 5 (35/8)3.31±0.788.44±0.28bc2.71±0.03bc4.18±0.09b0.65±0.02bc0.85±0.21eDiet 6 (35/11)4.12±0.088.48±0.29bc2.53±0.16bc4.00±0.09b0.63±0.02bc0.61±0.05bcDiet 7 (40/5)3.31±0.477.52±1.02ab2.34±0.06bc3.33±0.91ab0.74±0.18bc0.72±0.12cdeDiet 8 (40/8)4.27±0.217.54±0.65ab2.67±0.11bc3.65±0.67ab0.75±0.11c0.63±0.06bcDiet 9 (40/11)3.38±0.298.07±0.65bc2.29±0.06abc3.74±0.26ab0.61±0.03bc0.68±0.07cdProtein levels304.27±0.50v8.29±1.261.90±0.34u3.78±0.970.54±0.18u0.45±0.07u353.58±0.61u7.65±1.252.68±0.37v3.46±0.960.85±0.33w0.77±0.17v403.65±0.55u7.71±0.742.43±0.19v3.57±0.610.70±0.13v0.68±0.08vLipid levels53.58±0.596.82±0.90x2.37±0.522.77±0.81x0.91±0.31y0.68±0.1883.99±0.748.10±0.63y2.53±0.294.02±0.59y0.64±0.11x0.66±0.19113.93±0.498.73±0.80y2.12±0.454.01±0.34y0.54±0.13x0.55±0.15Two-way ANOVA (P value)Protein0.0130.0590.0000.5030.0000.000Lipid0.1830.0000.0150.0000.0000.000Protein×lipid0.0900.0070.4510.1460.0010.0201Data are expressed as mean±SD (n=3). Values in the same column with different superscripts are significantly different (P<0.05). uvSignificant (P<0.05) difference between protein levels within the same lipid level. xySignificant (P<0.05) difference between lipid levels within the same protein level. TG = triglyceride; TC = total cholesterol; HDL-C = high-density lipoprotein cholesterol; LDL-C = low-density lipoprotein cholesterol; HDL-C/LDL-C = HDL-C to LDL-C ratio; HDL-C/TC = HDL-C to TC ratio.


## 3.1. Effect of Dietary Protein and Lipid Levels on Fish Growth Performance and Feed Utilization

Dietary protein and lipid combinations significantly affected the WG, SGR, HSI, and Fulton’s condition factor (K) (P>0.05), but did not influence FCR and VSI (P<0.05) of Carassius auratus var. Pengze juveniles (Table 2). VSI and K were not significantly affected by dietary protein level, while WG, FCR, SGR, and HSI were not affected by dietary lipid level. Fish fed diet with 35% protein exhibited higher WG and SGR and lower FCR compared with those fed diets with 30% and 40% protein, whereas HSI was significantly depressed by dietary protein higher than 30%. VSI was elevated by dietary 8% and 11% lipid levels, while K of fish fed diets with 5% and 8% lipid levels was significantly lower than that of those fed diets with 11% lipid. Feed intake was lower in fish fed diets with 35% protein compared to fish fed diets with 30% and 40% protein, whereas feed intake was not influenced by dietary lipid levels. The fish fed diets with 35% protein showed lower FCR when compared with the groups fed 30% and 40% protein. According to one-way ANOVA, fish fed the diet with 35% protein and 5% lipid showed the highest WG and SGR, the lowest FCR, and lower VSI and HSI. Fish liver glycogen decreased from 36.16 mg/g to 25.09 mg/g among all dietary treatments and was significantly depressed by dietary protein levels at the same lipid level (P<0.05).Table 2
Growth response, feed utilization, and physical parameters of Pengze crucian carp (Carassius auratus var. Pengze) juveniles fed diets with different protein and lipid levels1.
Diet no. (protein/lipid)IBW2WG3 (%)Feed intake4 (g/100 g ABW5/d)FCR6SGR7VSI8 (%)HSI9 (%)K10Liver glycogenDiet 1 (30/5)31.58±0.1998.76±1.68a1.67±0.05b1.41±0.06b1.23±0.02a20.09±0.77ab3.26±0.34ab3.02±0.00a36.16±3.24bDiet 2 (30/8)31.53±0.15100.94±0.41ab1.59±0.03ab1.36±0.00ab1.25±0.00ab20.90±0.42ab4.02±0.12c3.21±0.05d35.72±3.38bDiet 3 (30/11)31.61±0.04101.35±0.78ab1.62±0.02ab1.34±0.02ab1.25±0.01ab20.71±0.42ab3.61±0.21bc3.22±0.02d34.55±3.48bDiet 4 (35/5)31.60±0.12105.01±1.63c1.63±0.0ab1.29±0.04a1.28±0.01c20.42±0.30ab2.99±0.06a3.19±0.02cd31.24±1.23abDiet 5 (35/8)31.69±0.34102.95±0.38bc1.59±0.02ab1.31±0.02a1.26±0.01bc21.79±0.38b3.16±0.51ab3.05±0.05ab30.59±3.52abDiet 6 (35/11)31.64±0.40101.99±1.28abc1.61±0.02ab1.29±0.03a1.26±0.01abc21.51±0.72ab3.26±0.08ab3.17±0.08cd29.70±1.43abDiet 7 (40/5)31.73±0.11100.60±0.72ab1.61±0.02ab1.35±0.02ab1.24±0.01ab19.43±2.19a3.04±0.39a3.11±0.05bc26.38±0.61aDiet 8 (40/8)31.74±0.07101.10±0.95ab1.56±0.06a1.34±0.03ab1.25±0.00ab20.99±0.57ab2.82±0.85a3.06±0.06ab25.86±2.06aDiet 9 (40/11)31.68±0.03101.43±1.24ab1.61±0.02ab1.34±0.03ab1.25±0.01ab21.67±2.05b3.03±0.03a3.16±0.00cd25.09±2.90aProtein level (%)30100.35±1.53u1.64±0.03v1.37±0.04v1.24±0.01u20.57±0.613.63±0.39v3.15±0.1035.48±0.83w35103.32±1.85v1.58±0.02u1.30±0.03u1.27±0.02v21.24±0.763.14±0.28u3.14±0.0830.51±0.77v40101.04±0.93u1.61±0.01uv1.34±0.03v1.25±0.00u20.70±1.822.96±0.23u3.11±0.0625.78±0.65uLipid level (%)5101.46±3.041.64±0.041.35±0.071.25±0.0319.98±1.25x3.10±0.293.11±0.75x31.26±4.898101.67±1.111.61±0.021.33±0.031.25±0.0121.23±0.58y3.33±0.603.11±0.09x30.72±4.9311101.59±1.251.59±0.031.32±0.031.25±0.0121.30±1.19y3.30±0.283.18±0.05y29.78±4.73Two-way ANOVA (P value)Protein0.0000.0030.0000.0000.4130.0000.1680.000Lipid0.9350.1020.2080.9230.0350.1360.0030.498Protein×lipid0.0100.6100.2440.0100.7650.0470.0001.0001Data are expressed as mean±SD (n=3). Values in the same column with different superscripts are significantly different (P<0.05). uvwSignificant (P<0.05) difference between protein levels within the same lipid level. xySignificant (P<0.05) difference between lipid levels within the same protein level. 2IBW: initial body weight (g). 3WG: weightgain%=100×finalbodyweightg–initialbodyweightg/initialbodyweightg. 4Feed intake g/100gABW/d=feedconsumptiong/ABWg×feedingdays. 5ABW: average body weight g=finalbodyweighg+initialbodyweightg/2. 6FCR: feedconversionratio=dryfeedfedg/weightgaing. 7SGR: specific growth rate %/day=100×lnfinalbodyweight−lninitialbodyweight/feedingdays. 8VSI: viscerosomatic index %=100×visceralweightg/wholebodyweightg. 9HSI: hepatosomatic index %=100×liverweightg/wholebodyweightg. 10K: K, Fulton’s condition factor g/cm3=100×finalbodyweightg/finalbodylength3cm3. 11LG: liver glycogen (mg/g).


## 3.2. Effect of Dietary Protein and Lipid Levels on Fish Whole-Body Proximate Composition

No interaction was found between dietary protein and lipid levels for fish whole-body moisture, crude protein, and crude ash contents except for crude lipids (Table3). Whole-body moisture, crude lipid, and crude ash contents were not significantly impacted by dietary protein levels, whereas crude protein and ash contents were not affected by dietary lipid levels to a significant extent (P>0.05). Whole-body crude protein content increased with increasing dietary protein levels and reached the highest in the 40% protein group. Whole-body moisture content decreased, whiles whole-body crude lipid content increased, with increasing dietary lipid level.Table 3
Body composition (wet weight basis) of Pengze crucian carp (Carassius auratus var. Pengze) juveniles fed diets with different protein and lipid levels1.
Diet no. (protein/lipid)MoistureCrude proteinCrude lipidAshDiet 1 (30/5)70.15±0.87c16.89±0.204.57±0.41abc4.78±0.06Diet 2 (30/8)67.99±0.52abc16.73±0.516.55±0.36ef4.88±0.30Diet 3 (30/11)67.91±1.19abc16.76±0.706.79±0.42f4.93±0.04Diet 4 (35/5)70.95±0.93c16.73±0.364.39±0.13ab4.85±0.04Diet 5 (35/8)68.96±0.75abc16.89±0.045.62±0.21de4.79±0.05Diet 6 (35/11)66.17±2.33ab17.61±0.055.48±0.27cd5.07±0.82Diet 7 (40/5)71.38±1.66c17.83±0.843.81±0.33a4.97±0.15Diet 8 (40/8)69.42±0.38bc17.98±0.485.28±0.04bcd4.83±0.05Diet 9 (40/11)65.47±2.19a18.13±1.056.07±0.64def5.03±0.10Protein level (%)3068.68±1.3516.80±0.45u5.97±1.114.86±0.173568.69±2.4517.08±0.44u5.16±0.614.90±0.434068.76±2.9517.98±0.72v5.05±1.064.94±0.13Lipid level (%)570.83±1.18z17.15±0.694.26±0.44x4.87±0.12868.79±0.80y17.20±0.685.82±0.61y4.84±0.161166.52±2.02x17.50±0.876.11±0.70y4.50±0.42Two-way ANOVA (P value)Protein0.9920.0010.0000.846Lipid0.0000.3970.0000.430Protein×lipid0.1400.6370.0370.9331Data are expressed as mean±SD (n=3). Values in the same column with different superscripts are significantly different (P<0.05). uvSignificant (P<0.05) difference between protein levels within the same lipid level. xyzSignificant (P<0.05) difference between lipid levels within the same protein level.


## 3.3. Effect of Dietary Protein and Lipid Levels on Fish Digestive Enzyme Activities

Significant interactive effects were found between dietary protein and lipid levels on fish intestinal trypsin and lipase activities (P<0.05), with no interaction detected in amylase activity (P>0.05) (Table 4). Trypsin activity was not significantly affected by dietary lipid level, but its activity increased with increasing dietary protein level, with no significant difference between the 35% and 40% protein groups. Amylase activity decreased with increasing dietary protein level. Lipase activity was the highest in fish fed diet with 35% protein and then decreased, with no significant difference detected between that of fish fed diets with 30% and 40% protein. Amylase activity was depressed by dietary lipid levels higher than 5%, and lipase activity was significantly improved by 11% dietary lipid.Table 4
Digestive enzyme activities of Pengze crucian carp (Carassius auratus var. Pengze) juveniles fed diets with different protein and lipid levels1.
Diet no. (protein/lipid)Trypsin activity (U/mg protein)Amylase activity (U/mg protein)Lipase activity (U/g protein)Diet 1 (30/5)3669.86±155.86a27.76±1.05c5.96±0.70aDiet 2 (30/8)3974.83±6.19ab24.42±1.03bc7.22±0.23bDiet 3 (30/11)4383.47±305.37bc21.49±0.83ab10.98±0.41eDiet 4 (35/5)4857.57±92.66c21.25±1.28ab9.23±0.14cDiet 5 (35/8)4532.42±299.31bc20.79±1.81ab10.48±0.44deDiet 6 (35/11)4786.46±118.86c18.39±4.15ab12.07±0.09fDiet 7 (40/5)4922.85±186.67c19.81±1.28ab8.69±0.13cDiet 8 (40/8)4847.20±166.25c19.40±1.49ab9.52±0.34cDiet 9 (40/11)4115.74±258.97ab15.88±3.52a8.78±0.11cProtein level (%)304009.4±354.33u24.56±2.85v8.05±2.30u354725.5±223.58v20.14±2.70u10.59±1.25v404628.6±462.95v18.36±2.75u9.00±0.44uLipid level (%)54483.4±624.5322.94±3.81y7.96±1.56x84451.5±419.1621.54±2.59xy9.07±1.48x114428.6±359.3118.59±3.68x10.61±1.47yTwo-way ANOVA (P value)Protein0.0000.0000.000Lipid0.8440.0010.000Protein×lipid0.0000.6200.0001Data are expressed as mean±SD (n=3). Values in the same column with different superscripts are significantly different (P<0.05). uvSignificant (P<0.05) difference between protein levels within the same lipid level. xySignificant (P<0.05) difference between lipid levels within the same protein level.


## 3.4. Effect of Dietary Protein and Lipid Levels on Fish Immunity and Antioxidant Capacity

Fish immunity and antioxidant capacity parameters determined in this study were significantly affected by dietary protein, dietary lipid, and their interactions (P<0.05), excluding CAT, which was not influenced by dietary protein levels (P>0.05) (Table 5). In the serum, both LZM and AKP were reduced by dietary lipids (8% and 11%) and dietary protein (40%). TSOD was elevated by dietary protein at 35% and dietary lipid levels higher than 5%. Liver TSOD and CAT were decreased by dietary lipid levels higher than 5%, while no significant differences in both parameters were found among fish from all the dietary protein groups. TAOC was highest in fish fed the diets with 35% protein, at 5% and 8% dietary lipid levels. MDA content generally reduced at 35% and 40% dietary protein levels and increased at 8% dietary lipid level. Fish fed Diet 4 had the lowest MDA content, the highest activities of the rest of the enzymes, and the highest TAOC content.Table 5
Immunity and antioxidant capacity of Pengze crucian carp (Carassius auratus var. Pengze) juveniles fed diets with different protein and lipid levels1.
Diet no. (protein/lipid)SerumLiverLZM (U/ml)TSOD (U/ml)AKP (king unit/L)TSOD (U/mgprot)CAT (U/mgprot)TAOC (mmol/gprot)MDA (nmol/mgprot)Diet 1 (30/5)372.10±17.26bc79.53±14.54a74.85±5.36abc1580.97±154.53cde16.81±0.67bcd0.22±0.03a17.40±0.63dDiet 2 (30/8)274.19±16.13a148.87±13.39abcd66.41±6.22abc1405.35±45.80bc17.73±2.04cd0.21±0.01a15.90±1.26bcDiet 3 (30/11)387.10±32.26c178.96±42.97cd77.85±10.98bc1637.03±24.43def13.07±2.11ab0.28±0.02b11.64±0.97bDiet 4 (35/5)397.10±22.26c188.99±10.89cd102.50±0.71d2034.76±34.42g23.87±2.01e0.33±0.02c5.99±0.10aDiet 5 (35/8)354.84±0.00abc222.93±49.84d78.51±4.73c1725.19±20.61ef14.99±0.29abc0.28±0.01bc16.00±1.97cdDiet 6 (35/11)290.32±64.52bc154.79±23.42abcd62.00±0.99a1186.96±72.27a11.50±0.12a0.20±0.02a10.80±0.50bDiet 7 (40/5)338.71±16.13abc86.95±25.57ab74.00±5.15abc1807.19±37.08f17.93±1.21cd0.23±0.01a13.26±2.51bcDiet 8 (40/8)290.32±32.26ab124.70±34.25abc63.39±3.36abc1483.76±5.18bcd10.93±1.58a0.21±0.01a13.32±0.20bcDiet 9 (40/11)290.32±32.26ab167.68±11.83bcd62.59±4.72ab1346.50±31.95ab20.18±2.23d0.22±0.00a11.54±0.57bProtein levels30344.46±56.74v135.79±50.09u7.30±0.86uv1541.1±132.6715.87±2.610.24±0.04uv14.98±2.73v35347.42±57.73v188.91±40.72v8.10±1.78v1649.0±373.8216.78±5.620.27±0.06v10.93±4.45u40306.45±34.21u126.44±41.42u6.67±0.67u1545.8±206.3216.34±4.440.22±0.01u12.71±4.56uvLipid levels5369.30±30.12y118.49±55.25x8.38±1.45y1807.6±212.65y19.53±3.50y0.26±0.0512.22±5.17xy8306.45±41.12x165.50±54.07y6.94±0.81x1538.1±146.56x14.55±3.24x0.24±0.0415.07±1.76y11322.58±62.47x167.14±27.27y6.75±0.98x1390.2±201.90x14.92±4.29x0.23±0.0411.33±0.73xTwo-way ANOVA (P value)Protein0.0200.0000.0000.0030.4820.0000.000Lipid0.0010.0030.0000.0000.0000.0060.000Protein×lipid0.0020.0040.0000.0000.0000.0000.0001Data are expressed as mean±SD (n=3). Values in the same column with different superscripts are significantly different (P<0.05). uvSignificant (P<0.05) difference between protein levels within the same lipid level. xySignificant (P<0.05) difference between lipid levels within the same protein level. LZM = lysozyme; TSOD = total superoxide dismutase; AKP = alkaline phosphatase.


## 3.5. Effect of Dietary Protein and Lipid Levels on Fish Blood Lipid Metabolism

Except TG, all the serum lipid parameters were significantly affected by dietary treatments (P<0.05) (Table 6). Interactions between dietary protein and lipid levels were detected for all indices but TG. Contents of TC and LDL-C were significantly improved by dietary lipid levels higher than 5%, irrespective of dietary protein contents. HDL-C and HDL-C/TC were all elevated by dietary protein at 35% and 40%, independent of dietary lipid levels. The HDL-C/LDL-C ratio was significantly enhanced by 35% dietary protein and 5% dietary lipid levels. Fish fed Diet 4 had the highest HDL-C, HDL-C/LDL-C, and HDL-C/TC, as well as the lowest TG, TC, and LDL-C.Table 6
Blood lipid composition of Pengze crucian carp (Carassius auratus var. Pengze) juveniles fed diets with different protein and lipid levels1.
Diet no. (protein/lipid)TG (mmol/L)TC (mmol/L)HDL-C (mmol/L)LDL-C (mmol/L)HDL-C/LDL-CHDL-C/TCDiet 1 (30/5)4.13±0.526.91±0.56ab1.96±0.22ab2.79±0.90ab0.73±0.15bc0.47±0.01abDiet 2 (30/8)4.41±0.688.31±0.62bc2.21±0.28abc4.24±0.78b0.52±0.03ab0.50±0.16abDiet 3 (30/11)4.28±0.449.64±0.17c1.55±0.08a4.30±0.39b0.36±0.05a0.36±0.02aDiet 4 (35/5)3.30±0.476.04±0.44a2.81±0.69c2.19±0.22a1.27±0.19d0.84±0.09deDiet 5 (35/8)3.31±0.788.44±0.28bc2.71±0.03bc4.18±0.09b0.65±0.02bc0.85±0.21eDiet 6 (35/11)4.12±0.088.48±0.29bc2.53±0.16bc4.00±0.09b0.63±0.02bc0.61±0.05bcDiet 7 (40/5)3.31±0.477.52±1.02ab2.34±0.06bc3.33±0.91ab0.74±0.18bc0.72±0.12cdeDiet 8 (40/8)4.27±0.217.54±0.65ab2.67±0.11bc3.65±0.67ab0.75±0.11c0.63±0.06bcDiet 9 (40/11)3.38±0.298.07±0.65bc2.29±0.06abc3.74±0.26ab0.61±0.03bc0.68±0.07cdProtein levels304.27±0.50v8.29±1.261.90±0.34u3.78±0.970.54±0.18u0.45±0.07u353.58±0.61u7.65±1.252.68±0.37v3.46±0.960.85±0.33w0.77±0.17v403.65±0.55u7.71±0.742.43±0.19v3.57±0.610.70±0.13v0.68±0.08vLipid levels53.58±0.596.82±0.90x2.37±0.522.77±0.81x0.91±0.31y0.68±0.1883.99±0.748.10±0.63y2.53±0.294.02±0.59y0.64±0.11x0.66±0.19113.93±0.498.73±0.80y2.12±0.454.01±0.34y0.54±0.13x0.55±0.15Two-way ANOVA (P value)Protein0.0130.0590.0000.5030.0000.000Lipid0.1830.0000.0150.0000.0000.000Protein×lipid0.0900.0070.4510.1460.0010.0201Data are expressed as mean±SD (n=3). Values in the same column with different superscripts are significantly different (P<0.05). uvSignificant (P<0.05) difference between protein levels within the same lipid level. xySignificant (P<0.05) difference between lipid levels within the same protein level. TG = triglyceride; TC = total cholesterol; HDL-C = high-density lipoprotein cholesterol; LDL-C = low-density lipoprotein cholesterol; HDL-C/LDL-C = HDL-C to LDL-C ratio; HDL-C/TC = HDL-C to TC ratio.


## 4. Discussion

### 4.1. Estimation of Optimum Dietary Protein and Lipid Levels

The purpose of this study was to estimate the optimum protein and lipid combination forCarassius auratus var. Pengze juveniles based on growth performance, feed utilization, body composition, and blood biochemistry. In the present study, the best SGR, WG, and FCR occurred in fish fed the diet containing 35.1% protein and 5.1% lipid, suggesting these levels are optimal for Pengze crucian carp. The lower growth rate as well as higher feed conversion ratio beyond this protein level can be attributed to the inability of the fish to effectively utilize the dietary protein above the optimum level [13]. Our results are comparable to previous research on common carp, in which the optimum protein and lipid levels were reported to be 34.61% and 5.58%, respectively [14]. Moreover, the determined P/E ratio (24.0 mg protein/kJ) in our study is within the estimated range of 19.45 to 26.89 mg protein/kJ [2] and is similar to that reported in hybrid grouper E. fuscoguttatus×E. lanceolatus (23.9 mg protein/kJ) [15]. However, similar to other fish species consuming various lipid diets, such as Sparus aurata [16], R. canadum [17], and S. rivulatus [6], no protein-sparing effect of dietary lipid was found in our study. This reveals that it is necessary to carefully investigate the appropriate dietary lipid level for all cultured species [18].


### 4.2. Effect of Dietary Protein and Lipid Levels on Fish Physical Parameters

Physical indices such as hepatosomatic index (HSI), viscerosomatic index (VSI), andK indicate the body condition of fish [13]. VSI is an important index that directly affects fish yield [17, 19]. In the present study, fish fed diets with higher lipid levels (8% and 11%) had higher VSI irrespective of dietary protein levels, indicating the increment of VSI mainly resulted from increased lipid retention in viscera. This result is similar to the findings in yellow drum Nibea albiflora (Richardson) [5]. HSI often serves as an important parameter for assessing the nutritional status and physiological conditions of fish [20], and high HSI was suggested to be correlated with worse health condition along with poor growth performance [21]. In the present study, HSI decreased dramatically with increasing dietary protein levels (y=0.067x+5.5883, R2=0.9334, y=HSI, x=dietaryproteinlevel), suggesting that the liver might not be the main organ for body lipid reserve in Carassius auratus var. Pengze. It is pertinent to note here that while formulating the diets in our study, dietary corn starch level was reduced with increasing dietary protein content. According to liver glycogen deposition data, we can deduce that the decline of HSI in our study is attributable to reduction in glycogen deposition caused by the decreasing corn starch inclusion in the diets, which is in agreement with previous studies [18, 22–24]. Interestingly, both the highest HSI and the inferior growth parameters occurred in the insufficient protein level (30%), which may lead to metabolic disorders [25] and shows that high HSI correlates with worse health conditions and poor growth performance [21]. In this trial, the highest K was recorded in fish fed diets with the highest lipid levels (11%) and the highest gross energy (15.9 and 16.1 kJ/g). This is in agreement with previous studies, in which high body lipid or visceral lipid occurred in fish fed high-energy (lipid) diets [16, 26–29]. Fish whole-body lipid content in our study also correlated positively with dietary lipid level. All the above results confirmed the view that K performs better as a predictor of energy concentration per g body weight [30].


### 4.3. Effect of Dietary Protein and Lipid Levels on Fish Whole-Body Proximate Composition

As well documented, lipid deposition in fish body is closely associated with dietary lipid levels [31]; increasing dietary lipid levels generally results in an increase in whole-body lipid and the decline of whole-body moisture [17, 32–36]. In the present study, with elevated dietary lipid level (x), whole-body lipid content (y1) increased and whole-body moisture content (y2) declined linearly (y1=0.3083x+2.93, R2=0.8642; y2=−0.7183x+74.46, R2=0.9991). Body protein content of the test fish tended to increase with the increase in the dietary protein level irrespective of dietary lipid content, which is consistent with the tendency of hybrid sunfish juveniles [33]. Whole-body lipid content was unaffected by dietary protein level in this study, suggesting that the juvenile Carassius auratus var. Pengze kept lipid content constant in the body when dietary protein level varied, which was also noted for juvenile Nibea diacanthus and U. cirrosa [37, 38].


### 4.4. Effect of Dietary Protein and Lipid Levels on Fish Digestive Enzyme Activities

Digestive enzymes play important roles in nutrient digestion, and their activities directly reflect the digestive capacity, nutritional status, and growth performance of aquatic animals [39–41]. Proteases can hydrolyze polypeptides into amino acids, which can be used to meet the growth and metabolism of fish [42]. In the present study, two-way ANOVA shows that trypsin activity was highest in fish fed diets with 35% protein and then plateaued indispensable of dietary lipid levels. From this aspect, we can explain why those fish fed the diet containing 35.1% protein and 5.1% lipid obtained the best growth performance among all dietary treatments. In agreement, researchers also detected that the proteases of Litopenaeus setiferus [43] and preadult red swamp crayfish [41] responded positively to dietary protein supplementation with the respective dietary energy of 13.5-16.1 kJ/g and 16.4 kJ/g. It is documented that lipase is an inducible enzyme influenced by dietary lipid levels [5, 44, 45]. This was shown in this study as intestinal lipase activity of the test fish positively correlated with dietary lipid level. Amylase activity has been shown to be adaptive to different nutrients in feed [42]. In this trial, amylase activity showed a decreasing trend with increasing dietary protein and lipid levels, which could also be as a result of the decreased corn starch levels with increased dietary protein and lipid content, as reported in red swamp crayfish (Procambarus clarkii) and Apostichopus japonicus [41, 46].


### 4.5. Effect of Dietary Protein and Lipid Levels on Fish Lipid Metabolism, Immunity, and Antioxidant Capacity

As reported, high-density lipoproteins (HDLs) are involved in the transport of cholesterol from plasma to the liver, acting as “cleaners” of body tissue cholesterol, and low-density lipoproteins (LDLs) play a role in the transport of cholesterol from liver to body tissues [47]; also, total cholesterol to high-density lipoprotein ratio (TC/HDL-C) is measured as an index for prediction of CHD risk [48]. In our study, fish fed with Diet 4 presented the highest HDL-C/LDL-C and relatively higher HDL-C/TC, suggesting that the diet with 35% protein and 5% lipid is good for fish health. In addition, all serum lipid metabolism parameters showed that the endogenous lipid transport was more active in fish fed diet with 35% protein and 5% lipid, and more cholesterol was transported from tissue cells back to the liver for metabolic transformation [49]. This may be another explanation for the best growth performance in fish fed Diet 4.The antioxidant system is vital for fish health and protects fish from oxidative stress [50, 51]. Inhibition of antioxidant enzymes like TSOD and CAT as well as reduction in TAOC could accelerate lipid peroxidation and lead to the formation of MDA in cells eventually. In the present study, the highest TSOD, CAT, and TAOC together with lowest MDA were found in the serum and liver of fish fed Diet 4, which shows that optimal dietary protein to lipid ratio could improve the antioxidant capacity of juvenile Pengze crucian carp by enhancing antioxidant enzymes. AKP and LZM are considered as reliable indices in the assessment of immune status [52, 53]. The highest activities of LZM and AKP were recorded in the serum of fish fed Diet 4, indicating that the combination of 35% protein and 5% lipid could improve fish immunity and thus enhance their ability of disease resistance.


## 4.1. Estimation of Optimum Dietary Protein and Lipid Levels

The purpose of this study was to estimate the optimum protein and lipid combination forCarassius auratus var. Pengze juveniles based on growth performance, feed utilization, body composition, and blood biochemistry. In the present study, the best SGR, WG, and FCR occurred in fish fed the diet containing 35.1% protein and 5.1% lipid, suggesting these levels are optimal for Pengze crucian carp. The lower growth rate as well as higher feed conversion ratio beyond this protein level can be attributed to the inability of the fish to effectively utilize the dietary protein above the optimum level [13]. Our results are comparable to previous research on common carp, in which the optimum protein and lipid levels were reported to be 34.61% and 5.58%, respectively [14]. Moreover, the determined P/E ratio (24.0 mg protein/kJ) in our study is within the estimated range of 19.45 to 26.89 mg protein/kJ [2] and is similar to that reported in hybrid grouper E. fuscoguttatus×E. lanceolatus (23.9 mg protein/kJ) [15]. However, similar to other fish species consuming various lipid diets, such as Sparus aurata [16], R. canadum [17], and S. rivulatus [6], no protein-sparing effect of dietary lipid was found in our study. This reveals that it is necessary to carefully investigate the appropriate dietary lipid level for all cultured species [18].


## 4.2. Effect of Dietary Protein and Lipid Levels on Fish Physical Parameters

Physical indices such as hepatosomatic index (HSI), viscerosomatic index (VSI), andK indicate the body condition of fish [13]. VSI is an important index that directly affects fish yield [17, 19]. In the present study, fish fed diets with higher lipid levels (8% and 11%) had higher VSI irrespective of dietary protein levels, indicating the increment of VSI mainly resulted from increased lipid retention in viscera. This result is similar to the findings in yellow drum Nibea albiflora (Richardson) [5]. HSI often serves as an important parameter for assessing the nutritional status and physiological conditions of fish [20], and high HSI was suggested to be correlated with worse health condition along with poor growth performance [21]. In the present study, HSI decreased dramatically with increasing dietary protein levels (y=0.067x+5.5883, R2=0.9334, y=HSI, x=dietaryproteinlevel), suggesting that the liver might not be the main organ for body lipid reserve in Carassius auratus var. Pengze. It is pertinent to note here that while formulating the diets in our study, dietary corn starch level was reduced with increasing dietary protein content. According to liver glycogen deposition data, we can deduce that the decline of HSI in our study is attributable to reduction in glycogen deposition caused by the decreasing corn starch inclusion in the diets, which is in agreement with previous studies [18, 22–24]. Interestingly, both the highest HSI and the inferior growth parameters occurred in the insufficient protein level (30%), which may lead to metabolic disorders [25] and shows that high HSI correlates with worse health conditions and poor growth performance [21]. In this trial, the highest K was recorded in fish fed diets with the highest lipid levels (11%) and the highest gross energy (15.9 and 16.1 kJ/g). This is in agreement with previous studies, in which high body lipid or visceral lipid occurred in fish fed high-energy (lipid) diets [16, 26–29]. Fish whole-body lipid content in our study also correlated positively with dietary lipid level. All the above results confirmed the view that K performs better as a predictor of energy concentration per g body weight [30].


## 4.3. Effect of Dietary Protein and Lipid Levels on Fish Whole-Body Proximate Composition

As well documented, lipid deposition in fish body is closely associated with dietary lipid levels [31]; increasing dietary lipid levels generally results in an increase in whole-body lipid and the decline of whole-body moisture [17, 32–36]. In the present study, with elevated dietary lipid level (x), whole-body lipid content (y1) increased and whole-body moisture content (y2) declined linearly (y1=0.3083x+2.93, R2=0.8642; y2=−0.7183x+74.46, R2=0.9991). Body protein content of the test fish tended to increase with the increase in the dietary protein level irrespective of dietary lipid content, which is consistent with the tendency of hybrid sunfish juveniles [33]. Whole-body lipid content was unaffected by dietary protein level in this study, suggesting that the juvenile Carassius auratus var. Pengze kept lipid content constant in the body when dietary protein level varied, which was also noted for juvenile Nibea diacanthus and U. cirrosa [37, 38].


## 4.4. Effect of Dietary Protein and Lipid Levels on Fish Digestive Enzyme Activities

Digestive enzymes play important roles in nutrient digestion, and their activities directly reflect the digestive capacity, nutritional status, and growth performance of aquatic animals [39–41]. Proteases can hydrolyze polypeptides into amino acids, which can be used to meet the growth and metabolism of fish [42]. In the present study, two-way ANOVA shows that trypsin activity was highest in fish fed diets with 35% protein and then plateaued indispensable of dietary lipid levels. From this aspect, we can explain why those fish fed the diet containing 35.1% protein and 5.1% lipid obtained the best growth performance among all dietary treatments. In agreement, researchers also detected that the proteases of Litopenaeus setiferus [43] and preadult red swamp crayfish [41] responded positively to dietary protein supplementation with the respective dietary energy of 13.5-16.1 kJ/g and 16.4 kJ/g. It is documented that lipase is an inducible enzyme influenced by dietary lipid levels [5, 44, 45]. This was shown in this study as intestinal lipase activity of the test fish positively correlated with dietary lipid level. Amylase activity has been shown to be adaptive to different nutrients in feed [42]. In this trial, amylase activity showed a decreasing trend with increasing dietary protein and lipid levels, which could also be as a result of the decreased corn starch levels with increased dietary protein and lipid content, as reported in red swamp crayfish (Procambarus clarkii) and Apostichopus japonicus [41, 46].


## 4.5. Effect of Dietary Protein and Lipid Levels on Fish Lipid Metabolism, Immunity, and Antioxidant Capacity

As reported, high-density lipoproteins (HDLs) are involved in the transport of cholesterol from plasma to the liver, acting as “cleaners” of body tissue cholesterol, and low-density lipoproteins (LDLs) play a role in the transport of cholesterol from liver to body tissues [47]; also, total cholesterol to high-density lipoprotein ratio (TC/HDL-C) is measured as an index for prediction of CHD risk [48]. In our study, fish fed with Diet 4 presented the highest HDL-C/LDL-C and relatively higher HDL-C/TC, suggesting that the diet with 35% protein and 5% lipid is good for fish health. In addition, all serum lipid metabolism parameters showed that the endogenous lipid transport was more active in fish fed diet with 35% protein and 5% lipid, and more cholesterol was transported from tissue cells back to the liver for metabolic transformation [49]. This may be another explanation for the best growth performance in fish fed Diet 4.The antioxidant system is vital for fish health and protects fish from oxidative stress [50, 51]. Inhibition of antioxidant enzymes like TSOD and CAT as well as reduction in TAOC could accelerate lipid peroxidation and lead to the formation of MDA in cells eventually. In the present study, the highest TSOD, CAT, and TAOC together with lowest MDA were found in the serum and liver of fish fed Diet 4, which shows that optimal dietary protein to lipid ratio could improve the antioxidant capacity of juvenile Pengze crucian carp by enhancing antioxidant enzymes. AKP and LZM are considered as reliable indices in the assessment of immune status [52, 53]. The highest activities of LZM and AKP were recorded in the serum of fish fed Diet 4, indicating that the combination of 35% protein and 5% lipid could improve fish immunity and thus enhance their ability of disease resistance.


## 5. Conclusion

Our results suggest that a diet containing 35.1% protein and 5.1% lipid with CP/GE (mg/kJ) ratio of 24.0 could be the optimum dietary protein and lipid levels based on WG and SGR as well as antioxidant and immunological parameters ofCarassius auratus var. Pengze.



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*Source: 2485134-2022-08-06.xml*