Effects of fish protein hydrolysate on growth performance and humoral immune response in large yellow croaker (Pseudosciaena crocea R.)§

Effects of fish protein hydrolysate on growth performance and humoral immune response in large yellow croaker ( Pseudosciaena crocea R.) § § 1 2 first-author †E-mail:tangtotti@gmail.comwutx@tianbang.com †E-mail:tangtotti@gmail.comwutx@tianbang.com tangtotti@gmail.com 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:tangtotti@gmail.comwutx@tianbang.com †E-mail:tangtotti@gmail.comwutx@tianbang.com tangtotti@gmail.com 1 1 Department of Chemistry, Zhejiang University, Hangzhou 310027, China Department of Chemistry, Zhejiang University, Hangzhou 310027, China Key Laboratory for Molecular Design and Nutrition Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, China Key Laboratory for Molecular Design and Nutrition Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, China We investigated the effects of fish protein hydrolysate (FPH) on growth performance and humoral immune response of the large yellow croaker ( Pseudosciaena crocea R.). One thousand and two hundred large yellow croakers [initial average weight: (162.75±23.85) g] were divided into four groups and reared in floating sea cages (3 m×3 m×3 m). The animals were fed with 4 diets: basal diet only (control) or diets supplemented with 5%, 10% and 15% (w/w) FPH. The results show that dietary FPH levels significantly influenced the growth and immunity of the large yellow croaker. Compared with the control group, total weight gain (TWG) in all treatment groups, relative weight gain (RWG) and specific growth rate (SGR) in fish fed with diets supplemented with 10% and 15% FPH were significantly increased ( P < 0.05). Similar results were observed in immune parameters [lysozyme activity, serum complements, immunoglobulin M (IgM)]. Lysozyme activity, complement C4 and IgM were also significantly increased ( P < 0.05) in fish fed with diets supplemented with 10% and 15% FPH, while complement C3 level was significantly increased ( P < 0.05) in all treatment groups. In general, with the supplementation of FPH, particularly at dose of 10%, the growth performance and immunity of the large yellow croaker can be improved effectively. Fish protein hydrolysate (FPH) Growth performance Humoral immune response Large yellow croaker INTRODUCTION 1 As one kind of important nutrients, the utilization of protein in humans and animals is widely studied. Protein is digested into amino acids and peptides by digestive enzymes and then assimilated in the intestine. Absorption of peptides is considered as a major route of transport in the intestine of vertebrates, and small peptides are absorbed faster than free amino acids (Adibi, 1997 1997 ; Tesser et al., 2005 2005 ). In aquaculture, immunostimulants were considered as a promising tool to enhance the resistance of cultured fish to disease and stress (Bagni et al., 2000 2000 ). And some commercial feed supplements with immunostimulating capacities have already been applied (Cook et al., 2003 2003 ). Fish protein hydrolysate (FPH) has been used as an ingredient for diets of marine fish (Ouellet et al., 1997 1997 ; Aguila et al., 2007 2007 ). FPH contains antioxidative activity and some functional properties (Klompong et al., 2007 2007 ; Thiansilakul et al., 2007 2007 ; Liaset and Espe, 2008 2008 ). While some studies have investigated the effects of FPH on growth performance and nonspecific immunity in several fish species (Berge and Storebakken, 1996 1996 ; Bøgwald et al., 1996 1996 ; Carvalho et al., 1997 1997 ; Liang et al., 2006 2006 ), no similar studies have been done on the large yellow croaker. The large yellow croaker ( Pseudosciaena crocea R.) belongs to predatory fish species, and is one kind of economically important marine fish species in China. With high nutritive value and better flavor, it is popularly consumed by people in Zhejiang Province, China. Due to excessive harvest, the wild large yellow croaker has nearly been depleted prior to the 1980s, and been widely cultured in China since the success of artificial hatchery (Duan et al., 2001 2001 ; Mai et al., 2006 2006 ). The purpose of the present study was to evaluate the effects of dietary supplementation with FPH on growth performance and humoral immune response in the large yellow croaker. MATERIALS AND METHODS 1 Diets preparation 2 FPH was produced by hydrolyzing tissues of pollock ( Theragra chalcogramma ) with Flavourzyme and Alcalase. By the method of Adler-Nissen ( 1986 1986 ), degree of hydrolysis (DH) was calculated to be 45.5%. The water-soluble protein fraction was spray-dried and stored at 4 °C for further use. FPH contained 65.61% protein, 2.06% fat and 3.99% ashes (on dry matter basis). The experimental diets were formulated to be equal in protein, lipid and ash. White fishmeal, soybean meal and protein powder of blood corpuscles were used for sources of protein, and fish oil and α-starch were used for non-protein energy. After mixed homogeneously, the ingredients were extruded to be dry pellets (6 mm in diameter). The components and proximate analysis of the diets are presented in Table 1 1 . Note: Vitamin premix (mg/kg diet): Thiamin 25, riboflavin 45, pyridoxine HCl 20, vitamin B 12 0.1, vitamin K 3 10, inositol 800, pantothenic acid 60, niacin acid 200, folic acid 20, biotin 1120, retinol acetate 32, cholecalciferol 5, α-2-tocopherol 120, ascorbic acid 2000; Mineral premix (mg/kg diet): NaF 2, KI 0.8, CoCl 2 ·6H 2 O 50, CuSO 4 ·5H 2 O 10, FeSO 4 ·H 2 O 80, ZnSO 4 ·H 2 O 50, MnSO 4 ·H 2 O 60, MgSO 4 ·7H 2 O 1200, NaCl 100 2 Formulation and composition 4 Content (%, w/w) Diet 1 Diet 2 Diet 3 Diet 4 5 Ingredients  White fish meal 65.0 60.0 55.0 50.0  Soybean meal 4.5 4.5 4.5 4.5  α-starch 17.0 17.0 17.0 17.0  Protein of blood powder 1.8 1.8 1.8 1.8  Vitamin pre-mixture 1.0 1.0 1.0 1.0  Mineral pre-mixture 2.0 2.0 2.0 2.0  Cellulose 0.2 0.2 0.2 0.2  Fish oil 8.5 8.5 8.5 8.5  Fish protein hydrolysate 0 5.0 10.0 15.0 5 5 Proximate analysis (on dry matter basis)  Protein 41.36 41.78 42.05 42.24  Lipid 11.69 11.47 11.56 11.81  Fiber 2.45 2.38 2.64 2.57  Ash 11.14 11.53 11.28 11.15 Animals and rearing condition 2 Large yellow croakers weighing (162.75±23.85) g were purchased from net-cage culture field in Ningbo, Zhejiang Province, China. They were acclimated for 2 weeks before the trial started. Then 1200 healthy, energetic fish were put into 12 cages (3 m×3 m×3 m) and divided into 4 groups. Each group was repeated in triplicates and each cage was stocked with 100 fish. Fish were fed twice daily (5:00 a.m. and 17:30 p.m.) to apparent satiation. The trial lasted for 8 weeks [water temperature (26.1±2.2) °C; salinity 33‰ to 35‰; dissolved oxygen content 6.6 mg/L]. Growth performance 2 At the beginning and end of the trial, the animals were fasted for 24 h and their weights were determined. Growth performance was expressed as the total weight gain (TWG), relative weight gain (RWG) and specific growth rate (SGR). The calculation formulas were as follows: TWG (g)= W t − W i , RWG (%)=( W t − W i )×100/ W i , SGR (%)=(ln W t −ln W i )×100/ d , where W i and W t are the initial and final mean weights (g), respectively, and d represents the number of feeding days. Blood sampling 2 At the termination of the trial, nine fish per group (three fish randomly captured from each cage) were sampled. Blood was sampled from the caudal vein of the individual fish after anaesthetization. The whole blood was collected in a syringe, allowed to clot for 1 h in microtubes at room temperature and followed by 5 h at 4 °C, and then serum was harvested by centrifuging at 1500× g for 5 min at 4 °C. All serum samples were preserved at −20 °C prior to analysis. Detection of lysozyme 2 Lysozyme activity in the serum was measured with spectrophotometry based on lysis of freezer-dried particles of Micrococcus lysodeikticus (Sigma Chemical Co., USA) (Ellis, 1990 1990 ; Alcorn et al., 2002 2002 ). One unit of enzyme activity was defined as the amount of enzyme causing a decrease in absorbance of 0.001 per min per ml serum. Detection of total immunoglobulin M (IgM) 2 Total IgM was determined following the method of Siwicki and Anderson ( 1993 1993 ). The assay was based on the measurement of total protein contents in plasma using a micro protein determination method (C-690; Sigma) prior to and after precipitating down the IgM molecules employing a 12% (w/v) solution of polyethyleneglycol (Sigma). The difference in the protein contents was considered as the IgM content. Detection of serum complements (C3 and C4) 2 The method of immunoturbidimetry (Jiancheng Institute of Biotechnology, Nanjing, China) was adopted in the assay for serum complement level. C3 and C4 in serum samples were mixed with the antibody afforded by the kits, and then an antigen-antibody complex was produced. The optical density (OD) value was measured at 340 nm. Compared with the values of the standards from the kits, C3 and C4 contents were calculated in μg/ml. Statistical analysis 2 All data were analyzed by one-way analysis of variance (ANOVA) using the software of the SPSS 11.0 for Windows. When ANOVA identified differences among groups, multiple comparisons among means were made using Duncan’s new multiple-range test. The results are presented as mean± SD , and probabilities of P < 0.05 were considered significant. RESULTS 1 Growth performance 2 As shown in Table 2 2 , FPH supplemented to the diet could increase the TWG, RWG and SGR of large yellow croakers. Fish fed with Diet 1 (basal diet) showed the lowest TWG [(83.55±15.18) g] , RWG [(51.63±8.63)%] and SGR [(0.74±0.1)%] . The highest TWG [(101.9±11.89) g], RWG [(62.41±7.62)%] and SGR [(0.86±0.08)%] were recorded in the fish fed with Diet 3 containing 10% (w/w) FPH. Note: FPH: Fish protein hydrolysate; TWG: Total weight gain; RWG: Relative weight gain; SGR: Specific growth rate; Values (mean± SD ) in the same column sharing the same superscript letter are not significantly different ( P > 0.05) Group TWG (g) RWG (%) SGR (%) Diet 1 83.55±15.18 a 51.63±8.63 a 0.74±0.10 a Diet 2 91.05±5.61 b 55.84±5.41 ac 0.79±0.06 ac Diet 3 101.90±11.89 c 62.41±7.62 b 0.86±0.08 b Diet 4 93.24±13.40 b 58.06±9.00 bc 0.81±0.10 bc Lysozyme activity 2 The effects of FPH on the lysozyme activity of serum in large yellow croakers were shown in Fig. 1 1 . No significant difference was observed between fish fed with Diets 1 and 2. The lysozyme activities in fish fed with Diets 3 and 4 were significantly higher than those in fish fed with Diets 1 and 2 ( P < 0.05). Complement level 2 It was found that the levels of serum complements in large yellow croakers were significantly influenced by FPH. As shown in Fig. 2a 2a , C3 levels in Diets 2~4 were all significantly higher than that in Diet 1 ( P < 0.05). As shown in Fig. 2b 2b , C4 levels in Diets 3 and 4 were significantly higher than those in Diets 1 and 2 ( P < 0.05). Total serum IgM 2 FPH had a notable influence on total serum IgM level of large yellow croakers (Fig. 3 3 ). Total serum IgM levels were significantly higher in Diets 3 and 4 and peaked at (418±4) μg/ml for Diet 4, while there were no significant differences observed between Diets 1 and 2 treatments and between Diets 3 and 4 treatments. DISCUSSION AND CONCLUSION 1 Previous studies have shown that diets supplemented with FPH can improve growth performance of fish species (Berge and Storebakken, 1996 1996 ; Carvalho et al., 1997 1997 ; Refstie et al., 2004 2004 ). In contrast, other study reported that protein hydrolysate cannot serve as an efficient dietary component (Kolkovski and Tandler, 2000 2000 ). Positive results were observed in this study, growth performance of the large yellow croaker was improved by supplement with different doses of FPH to basal diets. Bitter flavor in FPH, caused by soluble peptides with a high content of amino acids with hydrophobic functional groups, may be controlled by the hydrolytic enzymes and separation methods (Refstie et al., 2004 2004 ). The major causes for positive effects of FPH on fish are high palatability stimulating digestive enzymes and channeling more biomass production (Cahu et al., 1999 1999 ; Oliva-Teles et al., 1999 1999 ; Aguila et al., 2007 2007 ). In seawater as well as in fresh water species, the best results were obtained by using a moderate level of hydrolysate, while high levels depressed fish development (Cahu et al., 1999 1999 ). In this study, growth performance of fish fed with Diet 3 was better than that of fish fed with Diet 4. It might have been caused by the bitterness of FPH, so moderate level of supplement is necessary. Lysozyme and complements are important indices of nonspecific immunity in fish (Murray et al., 2003 2003 ). Lysozyme is one of the major components in the immune defense system in both invertebrates and vertebrates (Song et al., 2006 2006 ). It is known to act as a nonspecific immune mediator against parasitic, bacterial and viral infections, and in response to infection its activity is found to increase in fish blood (Puangkaew et al., 2004 2004 ). In the present study, 10% and 15% (w/w) FPH in the diet could enhance the lysozyme activity of the large yellow croaker effectively. The results are in agreement with the previous study in sea bass (Liang et al., 2006 2006 ). Complement, an important component of the innate immune system, plays important roles in immune surveillance and clearance of invading pathogens. It has been demonstrated that fish complement can lyse foreign cells and opsonize foreign organisms for destruction by phagocytes (Gasque, 2004 2004 ). There are also indications that complement fragments participate in inflammatory reactions (Holland and Lambris, 2002 2002 ). The present results show that three doses of FPH in the diet could increase serum C3 level significantly. The complement C4 level in serum of fish fed with diets supplemented with 10% and 15% (w/w) FPH was observed to be significantly higher. Immunoglobulins are well recognized to provide protection in animals and humans against various diseases. IgM is a major component of the teleost humoral immune system (Watts et al., 2001 2001 ; Cuesta et al., 2004 2004 ). IgM is present in blood and other body fluids, and plays a role as an immune effector molecule (Ross et al., 1998 1998 ). Total serum IgM levels of fish fed with the assayed immunostimulant-supplemented diets were statistically higher than those of fish fed with a non-supplemented diet (Cuesta et al., 2004 2004 ). In the present study, compared with fish fed with basal diet, the large yellow croaker fed with diets supplemented with 10% and 15% FPH contained significantly higher levels of total serum IgM. It is reported that dietary protein can influence immune system of crustacea (Bachère, 2000 2000 ; Pascual et al., 2004 2004 ). The inclusion of FPH in diet may afford more sufficient digestible protein which could be impacted better into the immune system of fish. Furthermore, fish proteins are likely to contain a large amount of bioactive sequences and constitute a potential reservoir for potent immunomodulators (Duarte et al., 2006 2006 ). Biologically active peptides with immunostimulating and antibacterial properties can be produced during the hydrolyzing procedure (Bøgwald et al., 1996 1996 ; Kotzamanis et al., 2007 2007 ). We assume that FPH used in this study contains such bioactive peptides. In the current work, no significant effects were observed in large yellow croakers fed with Diet 2. It may be due to the deficient contents of digestible protein and bioactive peptides. Peptides with different molecular weight were mixed together in FPH. Further research is required to determine the components and their concentrations. And it is also necessary to evaluate the effects of size-fractionated FPH on large yellow croakers. From the results of this feeding trial, it is concluded that growth performance and immune parameters of the large yellow croaker can be improved by supplementing FPH to basal diets, and that 10% (w/w) FPH gives the best results. References SA Adibi . 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Isolation and partial characterisation of immunoglobulin from southern bluefin tuna Thunnus maccoyii Castelnau The papers for the Postdoctoral Fellows Association of Zhejiang Province, China 10.1631/jzus.B0820088 Project (No. 2006C12098) supported by the Science and Technology Department of Zhejiang Province, China 2008-03-18 2008-06-21 2022-01-05 Copyright © 2008, Journal of Zhejiang University Science 2008 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 09 09