Replacement of Dietary Fish Oil with Vegetable Oils Improves the Growth and Flesh Quality of Large Yellow Croaker(Larmichthys crocea)

DUAN Qingyuan,MAI Kangsen,SHENTU Jikang,AI QinghuiZHONG Huiying,JIANG Yujian,ZHANG LuZHANG Chunxiaoand GUO Sitong

1)Key Laboratory of Aquaculture Nutrition and Feed of Ministry of Agriculture of China, Ocean University of China, Qingdao 266003, P.R.China

2)Ningbo Academy of Ocean and Fishery, Ningbo 315012, P.R.China

3)College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035,P.R.China

? Ocean University of China,Science Press and Springer-Verlag Berlin Heidelberg 2014

1 Introduction

Fish oil rich in n-3 highly unsaturated fatty acids(HUFA)is an important lipid source of marine fish feeds,which primarily provides farmed fish with energy and essential fatty acids and increases the palatability of feeds.In recent years,there has been a shortage of fish oil due to the rapid development of aquatic feed industry.Vegetable oils are considered as an alternative lipid source of fish feeds because of their sustainable production,relatively low cost,and abundant polyunsaturated fatty acids(PUFA),such as linolenic acid(18:3n-3,LNA)and linoleic acid(18:2n-6,LA)(Regostet al.,2003b).Previous study indicated that replacement of 60% fish oil with palm oil in herring diets had no significant negative effect on fish growth(Watanabe,2002).Several studies showed that the addition of high level of vegetable oil into the diet had no significant effects on the sacrificing product qualities of Atlantic salmon,such as carotenoid content,muscle texture and liquid holding capacity(LHC)(Bellet al.,2001; R?r?et al.,2003).However,the lipid deposition,fatty acid profiles and other sensory qualities such as juiciness,rancid flavor and hardness were found significantly affected by dietary lipid sources and fatty acid compositions in rainbow trout(Boggioet al.,1985; Johanssonet al.,1991),Atlantic salmon(Hardyet al.,1987; Polvi and Ackman,1992; Waagb?et al.,1993),and coho salmon(Skonberget al.,1993).

Large yellow croaker(Larmichthys crocea)is an important marine fish species widely cultured in southeast China.During the past few years,intensive studies have been conducted on the nutrition of large yellow croaker(Aiet al.,2006,2007,2008,2011; Wanget al.,2010; Zuoet al.,2012).However,no study has been carried out to determine the effect of dietary fish oil replacement with vegetable oil on growth and flesh quality of this fish species.The replacement of fish oil with vegetable oil at an appropriate level can save substantial feed costs,further bringing enormous economic benefits.Thus,in this study we investigated the effect of dietary fish oil replacement with 50% or 100% vegetable oils(soybean oil or palm oil)on the growth performance and flesh quality of large yellow croaker.The fish at an initial weight of 245.29 g ±7.45 g were chosen for their large daily feed intake before reaching the market size.Results were used to determine whether anchovy oil(Menhaden oil)rich in n-3 HUFA could be partially or completely replaced by vegetable oil(soybean or palm oil)without significantly decreasing the growth performance and selected flesh qualities of cultivated large yellow croaker.

2 Materials and Methods

2.1 Experimental Diets

The practical basal diet(FO)was formulated to contain 45% crude protein and 11% crude lipid with fish meal as the main protein source and fish oil as the main lipid source(Duanet al.,2001).Other experimental diets were formulated by replacing the fish oil in FO with 50% soybean oil(SO50),100% soybean oil(SO100),and 100% palm oil(PO100),respectively(Table 1).The feed ingredients were ground into fine powder,passed through a 246 μm mesh,and then mixed with water(200 g kg-1)to make stiff dough.The dough was processed using an automatic pellet-producing machine.Oil mixture(6.4% of dry diet)in each diet was post-sprayed thoroughly on the produced pellets(5 mm × 5 mm).The feed were dried in a ventilated oven at 40℃ for about 12 h and then sealed in plastic bags for storage at ?20℃ before use.

2.2 Experimental Procedure

The feeding trial was conducted in 4 floating sea cages(3.0 m × 3.0 m × 3.0 m)at the Xihu Bay of Ningbo,Zhejiang Province,China.Artificially hatched large yellow croaker were obtained from a local commercial hatchery and fed with trash fish for one and a half years.Prior to experiment,the fish were fasted for 24 h,and then weighed after anesthetized with eugenol(1:10000)(Shanghai Reagent,China).Each cage was stocked with 250 fish individuals with an average weight of 245.29 g ± 7.45 g.The fish were fed to apparent satiation twice a day for 12 weeks.The daily feeding amount and dead fish number were recorded.Cages were cleaned every 20 d to ensure smooth water flow.During the experiment,water conditions were as follows:temperature 14.5–28.9℃,salinity 23–27,pH 8.0–8.3,and dissolved oxygen content about 7 mg L-1.

At the end of experiment,fish were fasted for 24 h and then weighed as described above.Thereafter,25 fish were randomly sampled each cage,frozen with ice in an incubator and suffocated to death,and then transported to laboratory for further analyses.Of these,15 fish werechosen for examination of the condition index(K),hepatosomatic index(HSI),viscerosomatic index(VSI),and body color.The liver and viscera(kidney,gonads and heart)were collected for determining the contents of moisture and crude lipid.Then,the same 15 fish were scaled and filleted for analyzing the flesh quality including the texture,proximate composition(moisture,cholesterol,crude protein,and crude lipid),acidity,LHC,and thiobarbituric acid reactive substance(TBARS).Fillets were obtained from pectoral fin to anus along the dorsal fin and divided into dorsal and ventral muscles.The positions where flesh quality properties were assayed are shown in Fig.1.

Table 1 Formulation,proximate composition and fatty acid composition of the experimental diets for large yellow croaker

Fig.1 Positions on the fillets where flesh quality properties were assayed.A,instrumental texture(n =15); B and C,proximal composition(n =5); and D,pH,TBARS,LHC(n =5).

2.3 Biochemical Analysis

Proximate compositions of fish diets,muscles,liver,and the rest viscera(kidney,gonads and heart)were analyzed according to Association of Official Analytical Chemists(AOAC)(1995).Moisture content was determined gravimetrically based on the weight difference in samples after oven-drying at 105℃ for 24 h.Crude protein content was determined with Kjeldahl method(N × 6.25),and crude lipid content was measured with Soxhlet extractor method.

The fatty acid profiles of feeds were determined using gas chromatography-mass spectrometry(GC-MS).Fish lipids were extracted from 2 g of freeze-dried flesh sample and then methylated according to the method of Folchet al.(1957).Fatty acid methyl esters were separated and quantified using GC-MS(Finnigan DSQ Trace GC-MS)with a column of CP-SIL8CB-MS(30 m × 0.25 mm × 0.25 μm).The column temperature was programmed to rise from 150℃ to 200℃ at 15℃ min-1and from 200℃ to 280℃ at 10℃ min-1.The temperatures of injector and detector were both set at 250℃.

Malonaldehyde(MDA)content(TBARS value)was determined with distillation method(Tarladgiset al.,1960).Fillet samples were stored under 4 storage conditions,i.e.,1 d at 4℃,7 d at 4℃,4 weeks at ?20℃ and 8 weeks at ?20℃,respectively,followed by homogenization with an electric blender.The absorbance of homogenized fillets was measured at 538 nm using spectrophotometry(Shimadzu corporation,Kyoto,Japan)with distilled H2O as the blank control.

Cholesterol content was determined according to the Chinese National Standard Method for food safety analysis(GB/T 5009-2003).The absorbance of cholesterol extract was measured at 560 nm using a spectrophotometer.

Texture parameters were measured using the Stable Micro Systems(TA.XT2,Surrey,England).Texture profile analysis(TPA)was performed using an 8 mm spherical probe with the compression rate set at 1 mm s-1and the descending distance at 60% of flesh thickness.Two measurements were made for each sample in dorsal muscle perpendicularly to the muscle fibers orientation.The highest fracturability obtained from the texture profile curve of each sample was used to calculate the hardness of fillets(Ginéset al.,2004).

LHC was determined by following the gravimetric method(G?mez-Guillénet al.,2000).Approximately 15 g of dorsal muscle was placed into a centrifuge tube along with 2 Gilson Pipetman pipet filters and centrifuged at 500 g and 10℃ for 10 min.The LHC was expressed as the percentage of water retained per 100 g water present in the muscle prior to centrifugation.The aqueous plus fatty fraction(AF)retained in the filter was dried to constant weight.Fat loss was calculated as the dry weight of AF∞100/initial weight of sample.All measurements were carried out in triplicate.

Flesh pH was determined using the electrode method.Briefly,10 g of flesh sample was mixed with 50 mL of distilled water and frequently shaken for 15 min,followed by pH measurement using a PHB-4 pH meter(Leizi Instrumental Factory,Shanghai,China).

The ventral aspect color was measured using a Chroma Meter CR-400(Minolta,Osaka,Japan).A three-dimensional color space(L,a* andb*)following the system of CIE(1976)was used to test the ventral epidermis color of the fish.

2.6 Data Analyses

The following variables were calculated:

whereSGRis specific growth rate,FCRis feed conversion ratio,CFis condition factor,VSIis viscerosomatic index,HISis hepatosomatic index,GFis gutted yield,Wt(g)is the final body weight,W0(g)is the initial body weight,t(d)is the feeding duration,S(g)is the fish flesh weight,V1(g)is the dry paper weight,V2is the wet paper weight,andV3(g)is the constant dry paper weight at 50℃.

All data were expressed as mean ± S.E.Differences among diet treatments were analyzed using One-way analysis of variance(ANOVA)and Tukey’s multiple comparison test(significant ifP<0.05).Statistical analyses were performed using Microsoft Excel and SPSS 11.5(SPSS,Inc.,Chicago,Illinois).

3 Results

3.1 Growth Performance and Biological Properties of Large Yellow Croaker

SGR of fish fed SO50 and PO100 was 0.67% d-1and 0.61% d-1,respectively,significantly higher than that of fish fed FO or SO100(0.49% d-1and 0.43% d-1,respect-tively)(P<0.05).In contrast,FCR of fish fed S050 and PO100 was 1.33 and 1.29,respectively,significantly lower than that of fish fed FO(1.62)or SO100(1.63)(P<0.05).Other biological properties of large yellow croaker showed slight but not significant variations among dietary treatments(P>0.05),which included condition factor(1.93%–2.29%),VSI(11.87%–13.34%),HSI(3.28%– 3.45%)and carcass yield(81.94%–83.38%)(Table 2).

Table 2 Growth performance and selected body parameters of large yellow croaker fed different experimental diets1

3.2 Proximate Composition of Dorsal and Ventral Muscles of Large Yellow Croaker

As shown in Table 3,partial or complete replacement of dietary fish oil with vegetable oil did not significantly affect the moisture content of muscle.However,complete replacement of dietary fish oil with soybean oil significantly increased the moisture content of viscera.Moisture content of viscera in fish fed SO100 was 63.50%,significantly higher than that in fish fed FO(60.86%),SO50(58.06%)and PO100(58.83%)(P<0.05).As for the liver,moisture content was the lowest in fish fed SO50(51.85%)(P<0.05),significantly lower than that in fish fed other dietary treatments(55.59%–58.14%).

Complete replacement of fish oil with vegetable oil significantly increased the lipid content of ventral muscle.Crude lipid content of ventral muscle was significantlyhigher in fish fed SO100(22.34%)or PO100(20.15%)in comparison with that in fish fed FO(19.25%)or SO50(18.60%)(P<0.05).Dietary soybean oil increased the lipid content of viscera and liver.Crude lipid content of viscera was significantly higher in fish fed SO100(21.75%)or SO50(21.19%)compared to that fed FO(15.86%)or PO100(18.07%)(P<0.05).Crude lipid content of liver was significantly lower in fish fed PO100(27.05%)than in those fed FO(33.24%),SO50(32.97%),or SO100(32.21%)(P<0.05)(Table 3).

Table 3 Proximal analysis of the dorsal and ventral muscles,viscera and liver of large yellow croaker fed different experimental diets1

Muscle cholesterol was significantly decreased after dietary fish oil was completely replaced with soybean oil.Total cholesterol content of dorsal muscle was significantly lower in fish fed SO100(84.03 mg(100 g)-1)than in those fed FO(88.25 mg(100 g)-1),SO50(87.56 mg(100 g)-1)and PO100(88.73 mg(100 g)-1)(P<0.05).In contrast,total cholesterol content of ventral muscle was significantly higher in fish fed SO100(54.24 mg(100 g)-1)than in those fed FO(41.55 mg(100 g)-1),SO50(38.16 mg(100 g)-1),or PO100(37.95 mg(100 g)-1)(P<0.05)(Table 3).

3.3 Texture,LHC,pH and Body Color of Fillets

The fish fed SO50 showed the highest hardness(279.32 g)and fracturability(198.44 g)in comparison with those fed other diets.However,no significant differences were observed in these two parameters among dietary treatments(P>0.05)(Table 4).

Table 4 Instrumental texture analysis,liquid holding capacity and pH in fillets of large yellow croaker fed different experimental diets1

Fish fed diets with FO and SO50 showed significantly higher liquid and water loss under the following two storage conditions.Liquid and water loss from fresh muscle(1 d at 4℃)were both significantly higher in fish fed FO(1.31%,1.18%)than in those fed SO50(1.22%,1.05%),SO100(1.14%,1.01%),or PO100(1.20%,1.07%)(P<0.05).After 7 d storage at 4℃,liquid and water loss from muscle were comparable in fish fed FO(1.27%,1.15%)or SO50(1.27%,1.14%),significantly higher than that in fish fed SO100(1.22%,1.09%)or PO100(1.21%,1.05%)(P<0.05).Fat loss from fresh muscle(1 d,4℃)was significantly higher in fish fed SO50(0.16%)than in fish fed FO(0.13%),SO100(0.14%),or PO100(0.13%)(P<0.05).No significant difference was observed in the pH of fish muscle regardless of the storage temperature at 4℃ or not(P>0.05)(Table 4).

As to colorimetric values in the ventral aspect,L value ranged from 74.24 to 75.26,a*from 2.58 to 4.30,b*from 23.28 to 26.39,Hue from ?1.10 to 0.61,Chroma from 23.78 to 26.57,?Efrom 29.40 to 31.97,but no signifycant differences were detected in these parameters among dietary treatments(P>0.05)(Table 5).

Table 5 Colorimetric values in the ventral muscle of large yellow croaker fed different experimental diets1

3.4 TBARS Value of Fillets

TBARS value of fillets in FO was the highest under any one of the four storage conditions.TBARS value of fillets in SO50 was relatively higher but this value was still lower than that in FO.TBARS value of fish fillets(1 day at 4℃)were significantly higher in fish fed FO(0.55 μmol kg-1)or SO50(0.45 μmol kg-1)than in those fed SO100(0.25 μmol kg-1)or PO100(0.36 μmol kg-1)(P<0.05).After 7 d storage at 4℃,TBARS value of fillets was significantly higher in fish fed FO(1.72 μmol kg-1)than in those fed the other diets,i.e.,SO50(1.22 μmol kg-1),SO100(0.97 μmol kg-1),or PO100(0.94 μmol kg-1)(P<0.05).After 4 weeks storage at –20℃,TBARS value of fillets was significantly higher in fish fed FO(1.21 μmol kg-1)or SO50(1.22 μmol kg-1)than in those fed SO100(1.11 μmol kg-1)or PO100(1.10 μmol kg-1).After 8 weeks storage at –20℃,TBARS values of fillets were significantly higher in fish fed FO(1.65 μmol kg-1)than in those fed SO50(1.39 μmol kg-1),SO100(1.43 μmol kg-1),or PO100(1.37 μmol kg-1)(P<0.05)(Table 6).

Table 6 TBARS values of fillets(μmol kg?1)under different storage conditions among dietary treatments?

4 Discussion

Results of the present study showed that partial or complete replacement of dietary fish oil with soybean oil or palm oil did not significantly reduce the growth performance of large yellow croaker.On the contrary,the fish growth performance was significantly improved after the dietary fish oil was replaced by 50% soybean oil or 100% palm oil.This was consistent with early findings on European sea bass and sharpsnout seabream(Mourente and Bell,2006; Richardet al.,2006; Piedecausaet al.,2007).However,Fountoulakiet al.(2009)have found that 69% dietary fish oil replacement with palm oil significantly decreased the growth performance of gilthead seabream(initial weight:100g).These inconsistent results could be attributed to different fish sizes,fish feeding histories,and levels of fish meal in the basal diet.Fish meal and fish oil are two main sources of n-3 HUFA,which play a critical role in maintaining normal growth,nonspecific immunity,and other physiological processes of fish such as reproduction and larvae development(Zuoet al.,2012).In the present study,nearly 70% fish meal was included in each diet,which potentially met n-3 HUFA requirement of large yellow croaker with the selected size(initial weight:245.29 g ± 7.45 g).Excessive n-3 HUFA has been found to retard fish growth performance,possibly due to the effect of oxidative stress(Sargentet al.,1993; ?stbyeet al.,2009).This might explain the relatively lower growth performance of fish fed FO compared to that of fish fed SO50 or PO100.The reduction of fish growth performance after complete replacement of dietary fish oil by soybean oil could be attributed to the high inclusion of dietary linoleic acid.High amounts of linoleic acid have been found to exert deleterious effects on the health of gilthead seabream by influencing the fatty acid composition of immune cells with high deposition of LA,altering the eicosanoids production,and even chronically increasing the basal expression of certain inflammation-related genes(Monteroet al.,2008,2010).On the other hand,palm oil has been verified as a good energy source for fish species(Turchini,2009).We suggest that compared to soybean oil,palm oil is a superior energy substance for yellow croaker as long as n-3 HUFA is guaranteed.There were no significant differences in the CF,VSI,HSI,or carcass yield of large yellow croaker among dietary treatments,consistent with previous findings in Atlantic salmon(initial weight:340 g)(Grisdale-Hellandet al.,2002).

In this study,crude lipid contents of fish ventral muscle and viscera were significantly increased after the replacement of dietary fish oil by vegetable oil.Similar results have been reported in Atlantic salmon by Ruyteret al.(2006).However,Lee and Cho(2009)found that the lipid level was reduced in fish fed with n-3 HUFA-deficient diets.Previously reported different effects of dietary fatty acids on the whole body and?or liver lipid stores of fish might be result from differences in the fish size and/or the duration of relevant feeding experiments(Turchiet al.,2009).In present study,no significant differences were observed in the crude protein content of fish dorsal or ventral muscles among dietary treatments(Table 3).Differently,Bellet al.(2001)found that complete replacement of dietary fish oil with rapeseed oil substantially increased the muscle protein content of Atlantic salmon.This inconsistence could be related to different diet formulations and/or compositions of the fish species.

Hardness,fracturability and liquid holding capacity(liquid loss and water loss)are important texture characteristics of fillets.In this study,instrumental texture analysis showed no significant differences in the hardness and fracturability of fillets among dietary treatments,de-spite slightly higher hardness scores of fish fed FO or SO50.This agrees well with previous findings in Atlantic salmon regarding the lack of significant effect of dietary fish oil replacement by vegetable oil on the texture of fish muscles(Waagb?et al.,1993; Koshioet al.,1994; Regostet al.,2003a).In contrast,the LHC of fillets in fish fed FO or SO50 was significantly lower than that in fish fed SO100 or PO100.This was inconsistent with the previous finding by R?r?et al.(2003),which showed that dietary oil sources had no significant influence on the LHC.The inconsistence could be attributed to different methods for determination of LHC(R?r?et al.,2003).However,it remains unclear how specific quality parameters were affected by the dietary fish oil replacement with vegetable oil,and future investigations are needed to elucidate associated mechanism(s).

MDA is a product of lipid peroxidation,which is commonly represented by the TBARS value.In this study,TBARS values of fillets of large yellow croaker fed the 4 diets were below 2 μmol kg-1,thus were considered ‘not rancid’ according to Keet al.(1984)(threshold 8 μmol kg-1).Under any of the 4 storage conditions(1 d at 4℃,7 d at 4℃,4 weeks at ?20℃,8 weeks at ?20℃),TBARS values of fillets were significantly decreased after the dietary fish oil was partially or completely replaced by vegetable oil.Correspondingly,relatively high rancid odor was detected in fillets of fish fed FO by sensory analysis,despite lack of significant differences between fish fed FO and the other diets.The relatively high TBARS value and rancid score of fillets in fish fed FO could be attributed to strong lipid peroxidation caused by high level of dietary n-3 HUFA(Sargentet al.,1993; ?stbyeet al.,2009).This indicated that fish oil replacement with vegetable oils decreased TBARS values and thus improved the final fillet quality to a large extent.On the other hand,results in this investigation verified that the growth retardation of fish fed FO diet was attributed to oxidative stress as reflected by the high TBARS values.

In conclusion,this study showed that dietary fish oil replacement with 50% soybean oil or 100% palm oil significantly enhanced the growth performance of large yellow croaker(initial weight:245.29 g ± 7.45 g)when about 60% fish meal was guaranteed in the diets.In addition,selected flesh quality properties(liquid holding capacity,TBARS value and overall flavor score)were improved after the dietary fish oil was replaced by vegetable oil.This indicates that fish oil replacement by vegetable oil decreases feed costs while improving the fillet quality to some extent.Further study is needed to investigate the molecular mechanism(s)of the dietary lipid source effects on fillet quality properties.

Acknowledgements

The study was supported by the National Key Technologies R&D Program for the 10thand 11thFive-year Plan of China(Grant No.:2001BA505B-06).We thank M.C.Cai for help in diet production,and W.T.Xu,T.G.Qian,H.Zhang for their assistance in the fish feeding and sampling.Thanks are also due to R.T.Zuo for his kind help in the revision of the manuscript.

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