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common carp (Zhang & Sun 1994). These data ... Effect of dietary silkworm pupae meal on Jian carp H Ji et al. .... described by Mai, Zhang, Ai, Duan, Zhang, Li,.
Aquaculture Research, 2013, 1–13

doi:10.1111/are.12276

Effect of replacement of dietary fish meal with silkworm pupae meal on growth performance, body composition, intestinal protease activity and health status in juvenile Jian carp (Cyprinus carpio var. Jian) Hong Ji1,2,3, Jian-Lu Zhang1, Ji-Qin Huang1, Xiao-Fei Cheng1 & Chao Liu3 1

College of Animal Science and Technology, Northwest A&F University, Yangling, China Fisheries Research Institute, Northwest A&F University, Yangling, China

2 3

Ankang Fisheries Experimental and Demonstration Station of Northwest A&F University, Xianyang, China

Correspondence: H Ji, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China. E-mail: [email protected]

Abstract This study investigated the effect of replacement of graded dietary fish meal (FM) protein (0, 50%, 60%, 70% and 80%) with silkworm pupae meal (SP) in juvenile Jian carp. Triplicate groups comprising 18 fish (15.96  0.66 g) were fed one of five isonitrogenous and isocaloric diets (designated SP0, SP50, SP60, SP70 or SP80) for 8 weeks. The final body weight and specific growth rate of fish in the SP60, SP70 and SP80 groups were significantly lower than those for fish in the SP0 group (P < 0.05). The muscle protein content in the SP50 group was significantly higher than in the SP80 group (P < 0.05). With increasing FM replacement levels, the hepatic superoxide dismutase (SOD) activity decreased and the malondialdehyde (MDA) content increased among the groups, with the significant difference appeared in SP80 group. The gene expression level of heat shock protein 70 in the SP70 group was significantly higher than in the SP0 group (P < 0.05), while that in the SP80 group was significantly less than in the SP70 group (P < 0.05). Significantly decreased intestinal protease activity, increased serum ALT and AST activities, irregular-shaped hepatocytes and intestinal microvilli were found in the SP80 group. The study demonstrates that it is practical to replace 50% of the Jian carp dietary FM protein with SP, higher SP levels are not recommended and that oxidation status of the SP should be carefully assessed. © 2013 John Wiley & Sons Ltd

Keywords: silkworm pupae meal, fishmeal replacement, Cyprinus carpio var. Jian, HSP70

Introduction Most of the high dietary protein requirements of fish are best supplied by fishmeal (FM) because of its nutritional value and palatability (Hardy 1999). However, with the rapid development of the aquaculture industry, the long-term sustainability of fish farming could be at risk through overdependence on FM because of the emerging shortfall in FM production. Therefore, the partial or total replacement of FM in aquatic feeds with protein-rich animal or plant ingredients has become a focus of research (Refstie, Storebakken, Baeverfjord & Roem 2001; Watanabe 2002). In China, due to its resource characteristics and fierce price competition between aquatic feed companies in the market, the plant protein ingredients are predominant in the diets of non-carnivorous farmed fish and, animal protein ingredients are relatively minor components. However, researchers found that FM is indispensable in the diet of fish such as Black Sea turbot (Yigit, Erdem, Koshio, Erg€ un, T€ urker & Karaali 2006), Atlantic cod (Olsen, Hansen, Rosenlund, Hemre, Mayhew, Knudsen, Eroldo gan, Myklebust & Karlsen 2007) and also common carp (Hossain & Jauncey 1989); it can be substituted partially, but cannot be replaced completely. Therefore, the experimental

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Effect of dietary silkworm pupae meal on Jian carp H Ji et al.

design of this study was also FM partially replaced with SP. The use of plant protein in diets for aquaculture species has been very successful, but several problems have been identified including the presence of anti-nutritional factors (Francis, Makkar & Becker 2001) and unbalanced amino acid composition (Krogdahl, Bakke-McKellep & Baeverfjord 2003). Most animal protein resources are nutritionally more similar to FM and appear to be more suitable as a dietary replacement. Meals derived from poultry by-products, hydrolysed feathers, meat and bone meal, and blood meal have high protein contents that have been used in the aquatic feed industry (Zhu, Gong, Wang, Wu, Xue, Niu, Guo & Yu 2011). Silkworm pupae meal (SP), derived from reeled silk, is a suitable candidate as a FM replacement because of its high nutritional value and abundance in China. The production of SP in China accounts for approximately 80% of world production, with the annual capacity for dry pupae production being approximately 200 000 mt (Dong & Wu 2010). Dry pupae contains 50–70% crude protein and 24–33% crude lipid, and is a high quality insect protein source with a rich and balanced content of essential amino acids. Its nutritional value is comparable with that of FM, but the price is much lower. However, limiting factors in the use of SP include the presence of chitin (3%), which is difficult to digest, and the 27% lipid content rich in unsaturated fatty acids, oxidation of which can result in rancidity. Thus, the degree of SP substitution possibly appears to be limited. SP has been used as a dietary protein source for fish including Nile tilapia (Boscolo, Hayashi & Meurer 2001), Indian major carp (Begun, Chakraborthy, Zaher, Abdul & Gupta 1994) and common carp (Nandeesha, Gangadhara, Varghese & Keshavanath 2000), but the study results have been controversial. Begun et al. (1994) found significantly better food utilization by Indian major carp fingerlings when 50% of their dietary FM protein was replaced by silkworm pupae and clam meat protein, whereas Nandeesha et al. (2000) reported that the pupae could be used to completely replace FM, making up more than 50% of the diet of common carp. Jian carp (Cyprinus carpio var. Jian) is the first genetically modified new variety of common carp in China, and has a genetic stability >95%. Annual production of this variety exceeds

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100 9 104 tons, which is approximately 50% of the total annual production of cultured common carp in China (Zhou, Zhao, Jiang, Feng & Liu 2008). It contains a greater amount of protein, essential amino acids and taste amino acids (Lu & Bao 1994), and grows significantly better than common carp (Zhang & Sun 1994). These data indicate that Jian carp represent a huge sink for dietary inputs in China. The objective of this study was to determine the most appropriate level of SP in the diet of Jian carp by evaluating the effect of SP on growth performance, feed utilization, body composition, biological parameters, serum biochemical indices, activities of proteases and hepatic heat shock protein 70 (HSP70) gene expression. Such data will provide reference information for the development of a nutritionally complete formulated aquaculture feed for carp, and improve the utilization efficiency of SP. Materials and methods Experimental diets Rapeseed meal, Cottonseed meal, Soybean meal, Peruvian red FM (64.7% crude protein, provided by Huaqin Feed Factory, ShaanXi, China) and SP (52.3% crude protein, provided by AnKang Silk Factory, ShaanXi, China) and the other ingredients were used as dietary protein sources. Soybean oil was used as the main lipid source, and wheat meal (14.0% crude protein) was used mainly as the carbohydrate source. Based on these nutrients, five isonitrogenous (crude protein 36.8%) and isocaloric (gross energy 17 kJ g1) experimental diets, which replaced dietary FM protein with SP at 0, 50%, 60%, 70% or 80% levels (designated SP0, SP50, SP60, SP70 or SP80, respectively), were formulated to meet the protein and energy requirements of juvenile Jian carp (NRC, 1993; Zhou et al. 2008). Ingredients were grounded into fine powder through a 250-lm mesh. All the ingredients were thoroughly mixed with soybean oil, and water was added to produce stiff dough. The dough was dried for about 5 h in a cool and well-ventilated place pelleted. After drying, the diets were broken by-hand and sieved into proper pellet size (2.0 9 2.5 mm), and were stored at 20°C. The ingredients and approximate composition of the experimental diets are shown in Table 1. © 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–13

Effect of dietary silkworm pupae meal on Jian carp H Ji et al.

Aquaculture Research, 2013, 1–13 Table 1 Formulation (g kg1) and nutrient composition (%) of the experimental diets Group Component

SP0

Silkworm 0 pupae meal* Fish meal† 100 Meat and 50 bone meal Soybean meal 170 Full fat soybean 30 Rapeseed meal 220 Cottonseed meal 220 Wheat meal 127 Soybean oil 23 Dihydrogen 20 phosphate calcium Bentonite 20 Mixture‡ 20 Nutrient composition (%) Crude protein 36.76 (N% 9 6.25) Crude lipid 5.46 Crude ash 9.32 Energy (KJ/g) 16.63

SP50

SP60

SP70

SP80

57

68

79

90

50 50

40 50

30 50

20 50

170 30 220 220 128 15 20

170 30 220 220 129 13 20

170 30 220 220 130 11 20

170 30 220 220 131 9 20

20 20

20 20

20 20

20 20

36.79

36.78

36.78

36.77

5.55 8.86 16.77

5.53 8.77 16.78

5.49 8.68 16.80

5.46 8.59 16.81

*Supplied by AnKang silk factory (ShaanXi, China); silkworm pupae meal, crude protein, 52.3% dry matter, crude lipid, 27.8% dry matter. †Supplied by Huaqin Feed Factory (ShaanXi, China); Peruvian red fish meal, crude protein, 64.7% dry matter, crude lipid, 12.6% dry matter. ‡Contained 0.5% vitamin, 0.5% mineral, 1% limestone carrier. Ingredients included/kg: vitamin A 4000 IU, vitamin D3 800 IU, vitamin E 50 IU, vitamin B1 2.5 mg, vitamin B2 9 mg, vitamin B6 10 mg, vitamin C 250 mg, nicotinic acid 40 mg, pantothenic acid calcium 30 mg, biotin 100 lg, betaine 1000 mg, Fe 140 mg, Cu 2.5 mg, Zn 65 mg, Mn 19 mg, Mg 230 mg, Co 0.1 mg, I 0.25 mg, Se 0.2 mg.

Experimental procedure Juvenile Jian carp were obtained from the Ankang Fisheries Experimental and Demonstration Station of Northwest A&F University (Ankang, China). The fish were acclimated to the experimental conditions by stocking in a temperature-regulated experimental rearing system, and fed three times daily to satiation for 2 weeks using a commercial diet (Huaqin feed factory, Shaanxi, China). All rearing tanks were provided with continuous aeration and maintained under four fluorescent lights (08:00–20:00 hours, 12 h light, 600 lx in total; 20:00–08:00 hours, 12 h dark cycle) during the experiment period.

© 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–13

At the start of the experiment, the experimental fish were fasted for 24 h and then weighed. A total of 270 fish of similar size (initial weight 15.96  0.66 g) were randomly distributed among 15 circular tanks (130 L, r = 0.26 m, h = 0.62 m). Each diet was then randomly assigned to triplicate tanks. The fish were hand fed about 40 min for each feeding to apparent satiation three times daily (at 08:30, 12:30 and 16:30 hours) for 57 days. After each feeding finished about half an hour, we opened the drainage gate valve at the bottom of each tank, and filled fresh water after discharge about one third of the water. During the experimental period, the water temperature, dissolved O2, pH and ammonia content were maintained at 27.0  1.2°C, 7.80  0.54 mg L1, 7.5  0.29 and 0.10  0.02 mg L1 respectively. Sample collection and calculation At the end of the feeding trial, all of the fish were fasted for 24 h, then harvested and anesthetized with tricaine methanesulfonate (MS-222, 10 mg L1). The hepatopancreas, intestine and intraperitoneal fat were rapidly excised, weighed or measured (intestine length); hepatopancreas and intestines were frozen in liquid nitrogen (196°C) immediately and then stored at Ultralow temperature freezer (80°C) until analysis for HSP70 gene (two fish per tank), hepatic SOD activity and MDA content (two fish per tank) and intestinal protease activity (two fish per tank). Three fish per tank were randomly withdrawn for comparative analysis of fish muscle (dry matter, protein, lipid, ash) and frozen at 20°C. Data on the initial body weight (IBW), final body weight (FBW), feed intake (FI, g per fish) and proximate composition of the carcasses were used to calculate (formulae shown below) the specific growth rate (SGR), feed conversion rate (FCR), protein efficiency ratio (PER), hepatosomatic index (HSI), intraperitoneal fat body index (IFI) and condition factor (CF). The relative gut length (RGL) was also calculated. SGR ¼½ln (FBW)  ln (IBW)=feeding days 100 FCR ¼ wet weight gain (g)/FI (g) PER ¼ weight gain (g)/protein intake (g)

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Effect of dietary silkworm pupae meal on Jian carp H Ji et al.

HSI ¼½wet hepatopancreas weight (g)/ wet body weight (g)  100 IFI ¼½intraperitoneal fat weight (g)/ body weight (g)  100 CF ¼½body weight (g)/body length3 ðcmÞ 100 RGL ¼ ½intestinal length (cm)/ body length (cm)  100 Blood samples (6 fish/tank, 18 fish/dietary treatment) were removed from the caudal vein and centrifuged (966 9 g, 10 min) after incubate for 10 h at 4°C, and the recovered serum was immediately frozen and stored at 80°C prior to analysis of serum metabolites. All procedures were carried out according to national and institutional regulations on the care and use of experimental animals. Determination of diet composition and amino acid profile To assess the dry matter (105°C, 24 h; Hengkexue Co., Shanghai, China), crude protein (N 9 6.25) using the Kjeldahl method (Kjeltec 2300 Protein Analyzer; Hillerød, Denmark), crude lipid (ether extraction using the Soxhlet method. SZF-06;

Aquaculture Research, 2013, 1–13

Jinghong, Co., Shanghai, China) and ash (combusted at 550°C, SXL-1016; XinjiaCo., Shanghai, China) content of the diets, analyses of muscle samples were performed following standard laboratory procedures (Association of Official Analytical Chemists 1995). The amino acid composition (% fresh weight) of FM, SP, five experimental diets (1 sample per diets) and muscle samples (one fish per tank, three fish per group) was determined using the method described by Mai, Zhang, Ai, Duan, Zhang, Li, Wan and Liufu (2006). For amino acids (except for methionine and cystine), the whole fish tissue samples, feed ingredients or experimental diets were freeze-dried, and then hydrolyzed with 6 N HCl at 110°C for 24 h and the chromatographic separation and analysis of the amino acids was performed after orthophthaldehyde (OPA; Sigma, St. Louis, MO, USA) derivation using reverse-phase high performance liquid chromatography (HPLC, HP1100; Agilent, New Jersey, USA) that followed the modified procedure of Gardner and Miller (1980). Whereas for methionine and cystine, the samples were oxidized with performic acid at 10°C for 3 h to obtain methionine sulfone and cysteic acid, then freeze-dried twice with deionized water. The freeze-dried ingredients were analysed as the process of other amino acids. The results are shown in Table 2.

Table 2 Amino acid composition (% fresh weight) of the experimental fishmeal, silkworm pupa meal and diets Group Amino acids

FM

SPM

SP0

SP50

SP60

SP70

SP80

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine ∑AA*

4.93 2.33 2.09 7.03 4.40 3.32 3.45 1.01 2.65 1.62 2.25 4.26 1.68 3.09 4.31 1.77 3.00 53.19

4.45 1.84 1.81 4.99 3.82 2.04 2.13 1.05 2.31 1.65 1.65 3.01 2.49 2.23 2.87 1.34 2.27 42.05

2.91 1.29 1.52 5.79 3.39 1.74 1.54 0.93 1.64 0.59 1.14 2.33 0.44 1.87 1.80 0.80 2.24 31.99

2.84 1.23 1.47 5.69 3.32 1.69 1.45 0.91 1.67 0.62 1.17 2.30 0.52 1.85 1.76 0.81 2.34 31.64

2.82 1.22 1.46 5.59 3.39 1.74 1.51 0.85 1.63 0.51 1.16 2.26 0.54 1.80 1.79 0.78 2.28 31.34

2.82 1.21 1.45 5.56 3.40 1.74 1.51 0.85 1.65 0.57 1.13 2.27 0.51 1.77 1.78 0.79 2.30 31.32

2.75 1.19 1.45 5.41 3.65 1.72 1.48 0.86 1.63 0.59 1.14 2.22 0.49 1.75 1.72 0.75 2.22 31.03

*∑AA: sum of amino acids in the material and diets (%).

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© 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–13

Effect of dietary silkworm pupae meal on Jian carp H Ji et al.

Aquaculture Research, 2013, 1–13

Protease assay For each of two fish per tank, the complete intestinal tract was removed, squeezed out the intestinal contents and weighed, then homogenized using Hi-speed dispersator (XHF-D; SCIENIZâ, Ningbo, China) in 0.1 M pH 7.0 sodium phosphate buffer (1:9 w:v). The homogenate was centrifuged at 10 000 9 g for 15 min at 4°C (3–18 K; Sigmaâ, Germany). The supernatant (containing enzymes) was stored at 80°C prior to analysis. The acidic protease and alkaline protease activities were measured using the method of Liu, Wang, Xu and Xu (2008). One unit of specific activity was defined as the amount of enzyme needed to produce 1 lg tyrosine per min per mg soluble protein in the enzyme solution (U mg1 protein) at 37°C and pH 2.0 (acid protease) or pH 8.5 (alkaline protease). Antioxidant status The hepatic superoxide dismutase (SOD) activity and the malondialdehyde (MDA) content in the homogenate were measured using SOD and MDA assay kits (Jiancheng Biotech. Co., Nanjin, China) respectively. RNA extraction and real-time quantitative polymerase chain reaction (qPCR) The primers used in real-time PCR are showed in Table 3. RNA was extracted from the hepatopancreas by homogenization in TRNzol reagent (Tiangen, Beijing, China). To avoid genomic DNA amplification during real-time qPCR, the extracted RNA was treated with RNase-free DNase (TaKaRa, Dalian, China), and the RNA integrity was checked by electrophoresis on 2% agarose gels prior to reverse-transcription PCR. First-strand cDNA was synthesized from total RNA in pooled biological replicates using 200 U of SuperScript III RT (TaKaRa). Real-time qPCR was performed using a CFX 96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with SYBRâ Premix Ex TaqTM II (TaKaRa). The total volume of the PCR reaction was 20 lL, comprising 0.6 lL of each primer (10 lM), 1 lL of cDNA (101 dilution of the

first-strand synthesis product), 10 lL of 29 SYBRâ Premix Ex TaqTM II and 7.8 lL of sterile double distilled water. The PCR program comprised an initial 30 s denaturation at 95°C followed by 40 cycles of 95°C for 15 s and 57°C for 15 s. The primer sequences for b-actin and HSP70 were designed based on published Jian carp sequences. The forward and reverse primers are listed in Table 2. Following PCR, melting curve analyses were performed to confirm the presence of single products in these reactions. The relative quantification method described by Livak and Schmittgen (2001) was used to calculate the gene expression values. Histological examination of the intestine and hepatopancreas At the end of the feeding experiment, two fish per tank were randomly sampled and dissected for histological assessment of damage to the middle intestine and hepatopancreas. The middle intestine and hepatopancreas samples (about 2 9 2 mm in size) were removed and fixed in 2.5% glutaraldehyde (pH 7.2). The samples were subsequently processed according to standard histological procedures. The inner side of the intestine samples was examined using scanning electron microscopy (SM-6360LV; JEOL, Tokyo, Japan), and the hepatopancreas samples were examined using transmission electron microscope (JEOL-1230; JEOL). Images of samples using these techniques were obtained using digital photography. Statistics The results are presented as means  standard deviation. One-way analysis of variance was used to test the effect of diets. Tukey’s procedure was used for multiple comparisons. Differences were regarded as significant when P < 0.05. Results Growth performance During the feeding trial, no mortality was recorded and all fish accepted the experimental diets well.

Table 3 Primers used in real-time PCR Target gene

Genbank accession no.

Forward (5′–3′)

Reverse (5′–3′)

HSP70 b-actin

AY120894 M24113

TCAGTCTGCCCTTGTCATTGGTGA AGTTGAGTCGGCGTGAAGTGGTAA

TTTGAGCTGACAGGAATCCCACCT TCCACCTTCCAGCAGATGTGGATT

© 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–13

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Table 4 Growth performance and biological indices for the experimental fish* Group SP0 FBW (g)† SGR (%/d)† FI (g fish1)† FCR† PER† HSI (%)‡ IFI (%)‡ CF‡ RGL (%)‡

61.29 2.32 48.83 1.41 0.146 1.27 0.22 2.87 1.51

SP50         

a

2.37 0.09a 2.0a 0.13ab 0.01ab 0.07 0.02 0.39a 0.05

58.96 2.25 45.38 1.36 0.151 1.19 0.23 2.71 1.45

SP60         

ab

2.55 0.08ab 2.3ab 0.08b 0.01a 0.21 0.08 0.09ab 0.03

56.17 2.21 42.74 1.35 0.142 1.33 0.25 2.67 1.48

SP70         

b

1.53 0.05b 0.7b 0.05b 0.01ab 0.18 0.17 0.10b 0.10

56.04 2.16 43.04 1.44 0.132 1.21 0.23 2.58 1.48

SP80         

b

1.67 0.06b 0.7b 0.02ab 0.01bc 0.15 0.13 0.08b 0.06

53.85 2.10 42.35 1.46 0.126 1.26 0.22 2.64 1.47

        

4.49c 0.04c 0.8b 0.14a 0.01c 0.10 0.11 0.13b 0.07

FBW, final body weight; SGR, specific growth rate; FI, feed intake; FCR, feed conversion rate; PER, protein efficiency ratio; HSI, hepatosomatic index; IFI, intraperitoneal fat body index; CF, condition factor; RGL, relative gut length. *Values (mean  standard deviation of data for triplicate groups) with different superscripts in the same row are significantly different (P < 0.05) from each other. †with 18 fish in each group. ‡with 12 fish in each group.

The effects of each diet on growth performance and biological parameters are shown in Table 4. The results showed that for the SP60, SP70 and SP80 groups, the FBW, SGR and CF were significantly lower than for the SP0 group (P < 0.05), while there were no significant differences among the SP50, SP60 and SP70 groups. Voluntary FI decreased significantly with increasing dietary SP levels (P < 0.05). The FCR in groups SP50 and SP60 was lower than in the SP0 and SP70 groups (P > 0.05), and significantly lower than in the SP80 group (P < 0.05). The most efficient PER was found in the SP50 group (0.151), followed by groups SP0 (0.146), SP60 (0.142) and SP70 (0.132); the PER for group SP80 was significantly lower than for the other groups (P < 0.05). None of the diets affected HSI. A general increase in RGL was observed in group SP60, and significant increases in this parameter were found for groups SP70 and SP80 (P < 0.05).

values than the SP80 group (P < 0.05). The muscle ash content decreased with increasing SP in the diet, with the SP80 group having a significant lower level than the levels in the SP0 and SP50 groups (P < 0.05). No significant difference was found among the treatments for muscle and hepatopancreas moisture content; the total values remained relatively stable, but opposite trends of change were apparent. Amino acid composition The total amino acid content of muscle, and the content of individual amino acids, decreased with increasing dietary SP levels (Table 5). However, no significant differences were found except for proline in the SP60 group, which was lower than in the SP0 group, and cysteine in the SP50 group, which was significantly lower than in the SP0 group (P < 0.05). Serum biochemical indices

Muscle and hepatopancreas composition The muscle and hepatopancreas composition in fish fed each of the five diets are shown in Fig. 1. The muscle protein content peaked in the SP60 and SP70 groups, and was significantly higher in the SP50 group than in the SP80 group (P < 0.05). The muscle lipid content was inversely correlated with the muscle protein content, with groups SP50 and SP60 having significant lower

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The serum biochemical index values are shown in Table 6. No significant differences were observed among most treatments for serum alanine aminotransferase (AST) activity, and the content of triglyceride (TG), total protein (TP) and glucose (GLU). For aspartate aminotransferase (ALT) activity, there was a gradual increase with increasing dietary SP level, and the serum GLU content in the SP70 and SP80 groups was lower than in the © 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–13

Effect of dietary silkworm pupae meal on Jian carp H Ji et al.

Aquaculture Research, 2013, 1–13

a

42.0 40.0

SP0

38.0

SP50

36.0

SP60

ab

Value /%

34.0

ab

32.0

SP70

a

SP80

ab

30.0

b

b

28.0

ab ab b

26.0 24.0

ab

22.0

a ab ab bc

20.0 18.0

Muscle crude protein

Muscle crude lipid (×10) Muscle crude ash (×20)

Hepatic crude lipid

Figure 1 Proximate muscle and hepatic composition (%). Values are means (SD) of three replications containing three fish per replication. a, bMeans with different letters are significantly different (P < 0.05) from each other. Table 5 Muscle amino acid composition (% dry weight) of the experimental fish* Group Amino acids

SP0

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine ∑AA†

7.38 3.18 2.89 10.49 5.24 3.06 4.19 1.64 3.71 2.35 3.16 6.15 1.78 3.30 6.71 2.45 4.05 71.70

SP50                  

0.26 0.12 0.10 0.44 0.25a 0.04 0.12 0.10a 0.12 0.04 0.18 0.28 0.32 0.10 0.29 0.13 0.19 2.86

7.24 3.17 2.79 10.48 4.81 3.05 4.22 1.18 3.61 2.36 3.14 5.98 1.73 3.31 6.51 2.48 3.93 69.99

SP60                  

0.19 0.18 0.70 0.40 0.15ab 0.26 0.32 0.24b 0.17 0.20 0.38 0.20 0.38 0.26 0.41 0.22 0.10 5.81

7.14 3.06 2.77 10.39 4.56 2.99 4.13 1.34 3.60 2.36 3.16 5.81 1.63 3.25 6.43 2.34 3.88 68.83

SP70                  

0.47 0.16 0.15 0.52 0.09b 0.22 0.27 0.22ab 0.14 0.10 0.19 0.36 0.33 0.15 0.35 0.25 0.20 4.00

7.10 3.04 2.74 10.37 4.86 3.08 4.18 1.54 3.61 2.28 3.03 5.70 1.53 3.18 6.39 2.24 3.85 68.72

SP80                  

0.20 0.03 0.04 0.34 0.22ab 0.03 0.09 0.19a 0.04 0.02 0.09 0.14 0.07 0.13 0.10 0.24 0.02 4.32

7.01 3.02 2.73 10.37 4.79 2.99 4.11 1.59 3.58 2.28 3.00 5.78 1.51 3.13 6.42 2.43 3.83 68.58

                 

0.48 0.20 0.19 0.63 0.52ab 0.08 0.24 0.21ab 0.18 0.08 0.27 0.51 0.41 0.20 0.45 0.14 0.27 4.89

*Values are means (SD) of three replications, with three fish in each group. Values in the same row with different superscripts are significantly different (P < 0.05) from each other. †∑AA: sum of amino acids (%).

other treatments. The ALT activity of group SP80 was significantly higher than that of groups SP0, SP50 and SP60 (P < 0.05); the highest level of © 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–13

AST activity occurred in the SP80 group, and the HDL-c and LDL-c content of treatment groups SP0 and SP50 were significantly higher than those of

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Table 6 Serum biochemical indices of the experimental fish Group Serum indices 1

ALT (U mL ) AST (U mL1) TG (mmol L1) TP (g L1) Chol (mmol L1) HDL-c (mmol L1) LDL-c (mmol L1) GLU (mmol L1)

SP0 12.82 302.9 2.58 28.55 3.00 2.49 1.62 5.89

SP50        

a

2.52 118.7 0.21 1.17 0.14b 0.23b 0.09b 0.52

11.93 314.1 2.54 27.25 2.97 2.44 1.62 6.02

SP60        

a

3.15 109.2 0.50 0.70 0.31b 0.14b 0.09b 4.17

14.82 302.5 2.81 28.27 2.75 2.18 1.47 5.97

SP70        

a

1.12 106.4 0.16 1.86 0.15a 0.05a 0.07a 1.50

16.03 298.1 2.48 26.55 2.51 2.25 1.46 5.53

SP80        

ab

0.72 100.5 0.31 3.08 0.37a 0.01a 0.09a 2.20

23.87 379.6 2.62 27.52 2.72 2.25 1.45 4.69

       

7.50b 178.6 0.13 0.88 0.33a 0.24a 0.18a 2.28

ALT, alanine aminotransferase; AST, aspartate aminotransferase; TG, triglyceride; TP, total protein; Chol, cholesterol; HDL-c, highdensity lipoprotein; LDL-c, low-density lipoprotein; GLU, glucose. Values are means  SD of triplicate groups, with six fish in each group. Mean values with different superscripts in the same row are significantly different (P < 0.05) from each other.

Figure 2 Acid protease activity and alkaline protease activity in the entire intestine. Values are means (SD) of three replications containing three fish per replication. a,bMeans with different letters are significantly different (P < 0.05) from each other.

the other groups (P < 0.05). The serum cholesterol level was lower in fish fed the SP-based diets relative to the SP0 group, and was significantly lower in the SP60, SP70 and SP80 groups than in the SP50 and SP0 groups (P < 0.05). Protease activity Both the intestinal acid protease and alkaline protease activities initially increased, and then decreased with the increasing SP level (Fig. 2). However, there was no pronounced difference in acid protease activity among groups, but the alkaline protease activity of group SP50 was significantly higher than that of the SP80 group (P < 0.05). 8

Hepatic anti-oxidation indices The results of measurements of hepatic SOD activity and MDA content are shown in Fig. 3. SOD activity decreased with increasing dietary SP level, while the MDA content was inversely correlated with the SOD activity. The SOD activity of group SP80 was significantly lower than that of group SP50, and the MDA content of the SP80 group was significantly higher than that of the SP0 group (P < 0.05). Expression of HSP70 gene Based on the hepatic mRNA content, the HSP70 gene was up-regulated with increasing dietary SP content up to 70% replacement (Fig. 4). There © 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–13

Effect of dietary silkworm pupae meal on Jian carp H Ji et al.

Aquaculture Research, 2013, 1–13

Figure 3 Hepatic oxidation indices: superoxide dismutase (SOD) activity, malondialdehyde (MDA) content. Values are means (SD) of two replications containing three fish per replication. a,bMeans with different letters are significantly different (P < 0.05) from each other.

was no significant difference among the SP50, SP60 and SP70 groups, but the hepatic mRNA content of the SP70 group was significantly higher than in the SP0 group (P < 0.05). The HSP70 mRNA content of the SP80 group was lowest among all groups, and was significantly less than in the SP70 group (P < 0.05). Morphology of intestinal microvilli and hepatocytes

Discussion

The intestinal microvilli in the SP0 and SP50 groups were most regular in shape; those in the SP70 and SP80 groups were shorter in length, and in the SP60 group were associated with more debris (Fig. 5).

Expression of HSP70 mRNA

0.09

a

0.08 0.07 0.06 0.05

ab

ab

b

0.04 b

0.03 0.02 0.01 0.00

SP0

SP50

SP60

SP70

The hepatocytes in the SP0, SP50 and SP60 groups were polygonal in shape and had centrally located nuclei and clear cell boundaries. It was difficult to clearly distinguish the cell boundaries in the SP70 and SP80 groups, particularly the latter. However, in the SP80 group, the hepatocytes were of irregular shape, and apoptotic cells with small pyknotic nuclei were evident (Fig. 6).

SP80

Figure 4 Expression of hepatic HSP70 gene. Values are means (SD) of three replications containing two fish per replication. a,bMeans with different letters are significantly different (P < 0.05) from each other. © 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–13

In this study, we showed an inverse relationship between the growth performance of Jian carp and dietary SP levels. At an FM protein substitution level of 60% or higher, the SGR and FI were significantly lower, while the RGL was substantially higher. The finding that less than 60% of FM protein in Jian carp diets could be replaced with SP (6.8% SP in the diet) is consistent with the conclusions Nandeesha et al. (2000) and our laboratory (Ji, Cheng, Li, Zhang & Liu 2012). A reduction in diet palatability usually results in a decreased FI, which in turn can reduce growth (Arag~ ao et al. 2003). In our study, the FI of the SP-based diet groups was significantly lower than that of the control group. Considering low substitution level in this study had no significant effect on amino acids composition of each diet; thus, the retarded growth performance on these diets can be explained by the poor palatability of SP. On the other hand, it has been reported that dietary composition influences the organoleptic

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Effect of dietary silkworm pupae meal on Jian carp H Ji et al.

Aquaculture Research, 2013, 1–13

Figure 5 The surface structure of the intestinal microvilli. Scanning electron microscopy 9100; scale bar = 100 lm.

Figure 6 Hepatocyte construction. Transmission electron microscopy 98000; scale bar = 2 lm.

quality of fish (Grigorakis 2007). The results of this study revealed that 50% and 60% substitution of FM protein with SP resulted in the accretion of carp muscle protein content. This is consistent with the results of previous research. Nandeesha et al. (2000) reported that in common carp, the protein content increased as the percentage of the pupae content in the diet increased from 30% to 50%. We found that 50% substitution of FM protein with SP in the diet of mirror carp could improve whole-body protein deposition in our previous study (Ji et al. 2012). The inverse relationship found between the muscle protein and lipid contents we observed has been reported in several previous studies (De Silva, Gunasekera & Shim1991; Nandeesha et al. 2000). The utilization of dietary protein depends on the activities of digestive proteinases in the digestive organs (Natalia, Hashim, Ali & Chong 2004). Characterization and quantification of proteolytic enzyme activity may contribute to better understanding of the digestive physiology of fish. The results of this study show that the highest levels of intestinal protease activity and PER occurred in the SP50 group, and the lowest FCR levels occurred in the SP50 and SP60 groups, even though the FI of the SP50 group was lower than that of the SP0 group. This shows that the PER was positively correlated with protease activity, which is a similar result to that of Nandeesha et al. (2000), who reported that the activity of protease in both the intestines and hepatopancreas

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was higher in common carp fed diets based on 40% and 50% pupae. The heat shock protein (HSP) family is a group of cellular proteins present in most life forms (Lindquist 1986). Based on their molecular mass, HSPs are classified into several families including the HSP90s, HSP70s, HSP60s and low molecular mass HSPs (Cui, Liu, Luan, Li, Wu & Wang 2010; Zhang, Pang, Wu & Jian 2011). HSP70 is expressed at very low levels under normal conditions, but is induced rapidly in response to various stressors (Cellura, Toubiana, Parrinello & Roch 2006). Therefore, HSP70 plays an important role in marine crab exposed to environmental stressors and the occurrence of disease caused by feed factors (Zhang, Zhang, Zheng, Liu & Hu 2009); it has also been found to respond to diets of suboptimal composition (Martin, Vilhelmsson, Medale, Watt, Kaushik & Houlihan 2003; Hemre, Deng, Wilson & Berntssen 2004). Up-regulation of HSP genes in the hindgut of salmon fed with genetically modified maize (Sagstad, Sanden, Haugland, Hansen, Olsvik & Hemre 2007) has been reported. We found that with increasing dietary substitution of FM protein with less than 70% SP, there was enhanced hepatic HSP70 gene expression. This result differs from that of Hansen, Rosenlund, Karlsen, Olsvik and Hemre (2006), who reported no up- or down-regulation of the transcription of HSP70 in Atlantic cod fed a diet containing 16% soybean meal, 24% corn gluten meal, 16% soybean and 24% corn gluten meal compared © 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–13

Aquaculture Research, 2013, 1–13

with the FM control diet, which was interpreted as indicating no diet-induced stress response. This may be due to the different experimental conditions, such as difference in species, ages, feed factors, etc. In this study, we also found that the level of HSP70 gene expression in the SP80 group was significantly lower than in the control group (SP0); we speculate that the immune systems of fish in the SP80 group were too severely damaged to express the HSP70 gene normally. This group also showed the worst growth performance. In our study, high dietary SP levels significantly decreased hepatic SOD activity and increased MDA content, showing that the diets containing higher SP levels induced oxidative stress and lipid peroxidation, and return to damage the hepatopancreas. Moreover, the significantly increased serum ALT and higher AST activities, and markedly decreased intestinal protease activity in the SP80 group indirectly supported our hypothesis. As the primary site of food digestion, and nutrient uptake and transformation, the intestine and hepatopancreas are central to physiological functioning, and optimum utilization of dietary nutrients depends on their functional effectiveness (Caballero, Izquierdo, Kjørsvik, Montero, Socorro, Fern andez & Rosenlun 2003). As protein and lipid nutrients in particular can affect the construction of intestinal microvilli and hepatocytes, a number of histological studies have been reported of the impact of various dietary lipid sources and the substitution of proteins in fish including rainbow trout (Caballero, Obach, Rosenlund, Montero, Gisvold & Izquierdo 2002), gilthead sea bream (Caballero et al. 2003) and common carp (Ostaszewska, Dabrowski, Kamaszewski, Grochowski, Verri, Rzepkowska & Wolnicki 2010). Both free lysine and glycine, and the lysine deficiency in plant protein-based diets adversely affect fish liver and intestinal epithelium (Ostaszewska et al. 2010). The histological effects observed in this study may be partly explained by amino acid deficiencies in the diets used, based on assessment of the dietary amino acid profiles. Disturbed cellular homeostasis, and increased proliferation and apoptosis of the intestinal epithelium had also been observed in Atlantic salmon fed a soybean meal diet (Bakke-McKellep, Penn, Salas, Refstie, Sperstad, Landsverk, Ringo & Krogdahl 2007). Briefly, high levels of substitution of FM protein with SP in the Jian carp diet adversely affected the intestinal microvilli and hepatocytes of the fish. More© 2013 John Wiley & Sons Ltd, Aquaculture Research, 1–13

Effect of dietary silkworm pupae meal on Jian carp H Ji et al.

over, the higher activity of serum AST and ALT in the high SP-based diets confirms our speculation. Conclusion In general, when the substitution level was 60% or more in SP, the growth was significantly lower compared with the control group. Therefore, this study has shown that it is possible to replace 50% of the FM protein in Jian carp diets with SP without compromising growth performance and the health status of the fish.

Acknowledgments This research was supported by the New Agriculture Technology Demonstration and Extension System Development Program of the Ministry of Finance, People’s Republic of China. We are grateful for support from the Ankang Fisheries Experimental and Demonstration Station of Northwest A&F University.

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