Effects of dietary lysolecithin(LPC)on growth,apparent digestibility of nutrient and lipid metabolism in juvenile turbot Scophthalmus maximus L.

Boshn Li,Zheng Li,Yongzhi Sun,Shixin Wng,Bingshn Hung,Jiying Wng,∗

aShandong Provincial Key Laboratory of Restoration for Marine Ecology,Shandong Marine Resource and Environment Research Institute,Yantai 264006,China

bKemin Industries,(Zhuhai)Co.Ltd,Zhuhai 519040,China

Keywords:Turbot Lysolecithin(LPC)Growth performance Lipid metabolism

ABSTRACT An 8-week trial was conducted to investigate graded levels of dietary LPC on growth,apparent digestibility(ADs)of nutrients and lipid metabolism in juvenile turbot Scophthalmus maximus L.Five experimental diets were formulated which contain 11%crude fat with graded amounts of LPC(0,1000,2500,4000,5500 mg/kg diet).The fish fed diet with 11%crude lipid and 0 LPC was used as the negative control group(C-),meanwhile the fish fed diet with 12%crude fat and 0 LPC was used as the positive control group(C+).Each diet was fed to triplicate groups of turbot(initial body weight of 41 g)for 56d.Weight gain rate and specific growth rate of thefish fed LPC supplemented diets were significantly higher than the control groups,and the growth performance of the C-group was significantly lower than the others.The viscerosomatic index(VSI)was significantly decreased and the gall bladder somatic index(GSI)was significantly increased by dietary LPC.Crude lipid contents of muscle were elevated by dietary LPC.Apparent digestibility of energy was decreased by dietary LPC,but protein and lipid were not affected.Both total cholesterol(T-CHO)and high density lipoprotein cholesterol(HDL-C)were up-regulated,meanwhile neither alanine aminotransferase(ALT)nor total protein(TP)were down-regulated by dietary LPC.Enzymes involved in lipid metabolism(total lipase,hepatic lipase,lipoprotein lipase,fatty acids synthase,and lipase)were all elevated by dietary LPC.In conclusion,the lipid requirements of turbot were decreased by dietary LPC and the lipid utilization coefficient was enhanced.Based on SAS NLIN regression,dietary 870.37 mg/kg LPC was appropriate for turbot juvenile.

1.Introduction

Lipid supplies energy and acts as a structural component of cell membranes,and also may spare protein in diets for aquatic animals(Ghanawi,Roy,Allen Davis,&Patric Saoud,2011;Sargent,Henderson,&Tocher,1989).The lipids are poorly absorbed in an aqueous environment in the absence of an emulsifier.The limited capacity of young animals to secrete bile salts(one kinds of emulsifier)means there is inefficient utilization of dietary lipid(Román-Padilla,Rodriguez-Rúa,Ponce,Manchado,&Hachero-Cruzado,2017).Usually,choline chloride or bile salt is added to feeds to improve the growth performance of young fish(Gabaudan&Hardy,2000).Choline chloride can also improve the lipid metabolism and stress tolerance of juvenile giant grouper(Shinn-Ping Yeh et al.,2013).

Lysolecithin,also called Lysophosphatidylcholine(LPC),derives from lecithin by removal of its terminal fatty acid radical by phospholipase A or B.LPC is a natural metabolite of lecithin in the body and has strong surface activities.It can demyelinate nerves and destroy red blood cells(Robinson,1961).Due to its emulsification properties,it helps make the consistency of products smooth and easy to spread.LPC is an emulsifier which helps break down fats and forms micelles with fatty acids(Cristina Casals et al.,1984;Milada Dobiášová et al.,1975).The emulsifying capacity of LPC is 5-fold greater than lecithin(Zhang et al.,2007).LPC is widely used in the food and pharmaceutical industry and also in livestock and poultry feeds.The effect of LPC on the growth performance and fat utilization in fish has only been studied in the cruciancarp(Carassais auratus gibelio)(Li et al.,2010a,b)and hybrid tilapia(Oreochromis aureus♂×Oreochromis niloticus♀)(Li et al.,2010a,b).

Turbot(Scophthalmus maximus L.)is one of most important commercial culture species in Europe and Asia.Peng(2014)reported that growth,feed utilization and cardiovascular health of turbot would be enhanced by a suitable dietary lipid level(ranging from 9.38%to 15.73% diet)on the basisof50% dietary protein,buttherecommendation considered lipid and not digestible lipid in the feed.This led to research focused on the optimal lipid levels for turbot(Ma et al.,2014),and also a few studies directed at improving dietary fat utilization.In this context,the present study was conducted to evaluate whether LPC can decrease the dietary lipid requirements of turbot.

Table 1 Composition and proximate analysis of the experimental diets(g/kg,dry matter basis).

2.Materials and methods

2.1.Diets preparation

The basic feed formulation included 500g/kg protein and 110g/kg lipid,using white fish meal and soy meal concentrate as the source of protein and fish oil as the lipid source.LPC(purity 200g/kg,Kemin Industries Co.Ltd,Zhuhai,China)was added to the basic diet and the experimental diets formulated with 5 different LPC levels,0,1000,2500,4000,5500 mg/kg diet.The basic diet was used as the negative control group(C-);meanwhile a diet with 120 g/kg lipid and 0 LPC was given to the positive control group(C+)(Table 1).The procedures of diet preparation and storage have previously been described(Wang et al.,2017).All diets contained 0.03%yttrium trioxide(Y2O3)as an inert marker to determine the apparent digestibility(AD)of the nutrients and energy yield.

2.2.Fish and feeding regimes

Juvenile turbot were obtained from a commercial fish farm in Penglai,China.Prior to the beginning of the experiment,juvenile turbot was reared in a recirculating aquatic system for 2 weeks to acclimate to the experimental conditions.Fish were fed twice daily with a commercial feed to satiation during this period(CP 500mg/kg,CL 120mg/kg).

At the beginning of the trial,the fish were starved for 24 h and weighed.Fish of similar size(41.3±1.6g)were randomly distributed between 18,300 L green cylindrical fi berglass tanks and each tank was stocked with 30 juveniles.Each diet was randomly assigned to triplicate tanks.Fish were hand-fed to satiation twice a day(8:30 and 16:00).Thirty minutes after feeding the uneaten pelleted feed was collected from the drainage tube of the aquarium and the pellets counted.The feeding trial lasted for 8 weeks.During this period,the temperature ranged from 16.9 to 17.4°C,the salinity was held at 30,dissolved oxygen was approximately 6mg/L,and the total ammonia nitrogen was maintained at less than 0.1 mg/L.

2.3.Sampling collection and chemical analysis

During the last 2 weeks of the trial faeces were collected,washed in distilled water and then water removed by drying on filter paper.One sample of faeces per tank was analyzed and consisted of a pool of the faeces collected in the last 2 weeks of the experiment and had a minimum weight of 20g(wet weight).Ethoxyquin(ETQ:400mg/L,1mL 60/g wet faeces)was add to each sample which was stored at-20°C.

At the end of the trial,the fish were starved for 24h before sampling.The total number,body weight and length of each fish was measured after anesthetizing them with MS-222(100mg/kg,Jiangxi Jian Reagent,China).The values obtained were used to calculate survival rate(SR),weight gain rate(WGR),feed conversion ratio(FCR),protein efficiency ratio(PER),lipid efficiency ratio(LER),and condition factor(CF).Twenty fish per tank were randomly selected,and dorsal muscle,viscera,liver,intestine and the gall bladder were dissected out for analysis of proximate composition or calculation of organ indices.Blood was collected from the caudal vein of five fish from each tank using non-heparinized syringes and serum harvested after centrifugation at4000×gfor10minat4°C using a High-Speed Refrigerated Centrifuge(Hitachi Crg Series,Japan).All samples were stored at-20°C for further analyses.

Proximate composition analyses of feed ingredients,experimental diets,tissues and faeces samples were performed using established standard methods(AOAC,1990).Moisture content was determined by drying samples in an oven at 105°C overnight except for the faeces that were freeze dried.The crude protein content of samples was determined by measuring nitrogen(N×6.25)using the Kjeldahl method.The crude lipid content of samples was determined by ether extraction using Soxhlet,and the energy content of samples was determined using an adiabatic bomb calorimeter(PARR 6100,USA).

The diets and faeces samples were digested in concentrated nitric acid and analyzed by inductively coupled plasma-optical emission spectrometry(ICP-OES,Agilent 720,USA)to determine Y contents.Serum bio-chemical indices,such as alanine aminotransferase(ALT),total protein(TP),albumin(ALB),triglyceride(TG),total cholesterol(TCHO),high density lipoprotein cholesterol(HDL-C),and low density lipoprotein cholesterol(LDL-C)were analyzed using an automatic analyzer(Hitachi 7020,Japan).Total lipase(TL),hepatic lipase(HL),lipoprotein lipase(LPL),fatty acid synthetase(FAS),and lipase(LPS)in serum were analyzed using commercial kits(Shanghai Enzyme-linked Industrial Co.,Ltd.,Shanghai,China).The protein content of serum was determined using the Coomassie Brilliant Blue method using a commercial kit(Nanjing Jiancheng Bioengineering Institute,Nanjing,China).

2.4.Statistical analyses

Data from the trial was analyzed using One-Way analysis of variance(ANOVA)and the SPSS program Version for windows(SPSS Inc.,IL,USA).When significant differences were identified by ANOVA,multiple comparisons among means were made with a Duncan's multiple-range test with the significance cut-offset at P＜ 0.05.The results are presented as the means±SD(standard deviation).Statistical Analysis System 9.2(SAS institute,USA)NLIN regression was used to evaluate the optimal supplementation of LPC.

Table 2 Effect of dietary LPC on the growth performance and feed utilization of turbot.

3.Results

3.1.Growth and feed utilization

At the end of the trial,the SR of all groups was above 95%(Table 2).WGR and SGR were significantly increased by dietary LPC relative to the C+and C-groups.No significant differences in WGR and SGR were identified between the LPC supplemented groups.FCR presented the opposite trend,and D5500 group had a significantly lower FCR than the C+group,but did not differ from the D1000 and D2500 groups.The growth performance of the C-group was significantly lower than all other experimental groups.DFI of the C+group was significantly lower than all the other experimental groups.

SAS NILN analysis based on WGR showed that the optimal supplementation of LPC in turbot juvenile diet was 870.37mg/kg(Fig.1).

3.2.Figure indices

Dietary lipid and LPC significantly modified organ indices of turbot(Table 3).C-group had the highest viscerosomatic index(VSI)and intestinal somatic index(ISI),and the lowest gall bladder somatic index(GSI).VSI was significantly decreased and GSI was significantly increased in all groups fed diet supplemented with LPC.The ISI of the C+group was lower than all other experimental groups.

3.3.Proximate composition of tissues

The effects of dietary LPC on the proximate composition of tissues in turbot are presented in Table 4.There were no significant differences between experimental groups in the moisture and crude protein contents of muscle and liver.The crude lipid content of muscle and liver

were significantly increased by dietary LPC relative to the C-group.

3.4.Apparent digestibility of nutrients(ADs)

The effects of dietary LPC on the apparent digestibility(AD)coefficients of the feed are listed in Table 5.ADs of crude protein(CP)and crude lipid(CL)were relatively high,although there were no significant differences between the experimental groups.The energy digestibility of the C+group was lower than the C-,D1000 and D2500 groups,but was similar to the D4000 and D5500 groups.

3.5.Serum biochemical indices

The effects of dietary LPC on serum biochemical indices are presented in Table 6.Both ALT and TP were decreased with increasing dietary lipid levels.ALT and TG were decreased significantly by dietary LPC,but the TP contents increased and then decreased subsequently.TCHO,HDL-C,and LDL-C were decreased by dietary LPC levels,while TCHO and HDL-C of the C-group was significantly lower than the C+group.

3.6.Lipid metabolism enzymes

Dietary LPC increased lipid metabolism(Table 7).There were no differences in TL,HL,LPL,FAS,and LPS between the two control groups.Fish fed with LPC supplemented feeds have significantly increased enzyme activities relative to the C+and C-groups.

Fig.1.Relationship between weight gain rate(WGR)and dietary LPC levels based on the SAS NLIN regression analysis,where X represents dietary LPC levels of turbot juvenile.

Table 3 Effects of dietary LPC on VSI,HSI,ISI,GSI and CF indices in turbot.

Table 4 Effects of dietary LPC on the proximate composition of tissues in turbot(g/kg).

Table 5 Effects of dietary LPC on the apparent digestibility coefficients of nutrients in turbot.

Table 6 Effects of dietary LPC on the serum biochemical indices in turbot.

Table 7 Lipid metabolism enzyme in the serum from turbot fed LPC.

4.Discussion

4.1.Growth performances,feed utilization and organ indices

Many researchers have reported that the optimal dietary lipid levels for juvenile turbot range from 11.3 to 16.2%(Peng,2014;Regost et al.,2001).In the present study,the WGR of the C+group(CL 12%)was significantly higher than the C-group(CL 11%),and the growth performance was increased by 1000-5500 mg/kg feed of LPC.This phenomenon has previously been reported in tilapia(Li et al.,2010a,b).LER was significantly increased by dietary LPC suggesting it enhances theutilization oflipid and thelevelofLPC supplementation(1000-5500 mg/kg feed)was unimportant.Our results indicate that the utilization of lipids was improved by dietary LPC,which is more advantageous than just increasing lipid intake.There are few reports explaining how LPC brings about its effect.However,since LPC is a cellsignaling molecule,and acts as a ligand for a family of G-protein-coupled receptors(GPCRs),it may modify the cardiovascular,immune,and nervous systems,up-regulates vascular endothelial cell growth factors,and thorough its emulsifying capacity may significantly improve the fat digestion of turbot.

Why do researchers report differences in the lipid requirements of turbot?In fact,there are differences in the utilization of dietary lipid.That is to say,increasing lipid utilization coefficient can reduce lipid content in feed,reduce metabolic pressure,and increase the proportion of energy provided by lipid.Since lipid requirements can be affected by a range of different factors,it should be reevaluated taking into consideration the lipid utilization coefficient.

Liu(2012)have reported that the growth performance of turbot was not affected by dietary lecithin and soybean phospholipids.Growth performances of cobia Rachycentron canadum L.and orange-spotted grouper Epinephelus coioids(He,2013;Shi,2013)were increased by dietary lecithin.Meanwhile,Yun,Mai,Zhang,and Xu(2011)and Sun et al.(2014)reported that WGR and DFI were increased by dietary cholesterol and bile acid,respectively.These studies illustrate the different effect of these three phospholipids,and the importance of LPC as a key factor affecting growth.However,since Reynier,Lafont,Crotte,Sauve,and Gerolami(1985)have reported that cholesterol uptake is inhibited by lecithin but not by LPC,it may be this rather than the LPC causing the growth effect.

4.2.Approximately composition and apparent digestibility coefficients of nutrients

Lipid contents and deposition in the muscle and liver increases with increasing dietary lipid levels.In the broiler dietary LPC reduced liver lipid content(Yang,2008),but dietary LPC does not affect lipid in whole tilapia(Oreochromis aureus♂×Oreochromis niloticus♀)(Li et al.,2010a,b).We have been unable to find further reports relating body lipid content to dietary LPC,but dietary lipids increase the lipid contents of whole fish(Wang et al.,2016;Yi et al.,2014).In another word,adding LPC was equivalent to increasing the lipid content,or increasing the amount of effective lipid in the diet.

Dietary LPC had no effects on ADs of CP and EE,but significantly affected the apparent digestibility of energy.Zhang(2010)reported that LPC increases the performance of broilers due to the enhancement of lipid apparent digestibility.Furthermore,apparent metabolizable energy(AME)was also significantly increased in broilers by dietary LPC.However,in the present study of fish,the ADs of EE were high but dietary LPC did not have a significant effect and the AD of energy sources decreased with increased dietary LPC.Further studies will be required to investigate in more depth in fish the AD of feeds containing dietary LPC.

4.3.Lipid metabolism

LPC was found at the end of 19 century,and extensively studied of the mechanism in medicine since 1930's(Besterman et al.,1973;Robinson,1961;Rudolf Locher et al.,1992).LPC has different functions in the blood,chamber or cell.The most important physiological activities of LPC were haemolysis and regulating the release of adrenaline.These effects have little to do with lipid metabolism and the chemical structure of LPC is proposed to affect lipid metabolism.The 2 distinctly different hydrophilic and lipophilic regions in the molecule endow LPC with strong surface-active properties.The ability of LPC to emulsify lipids is about 5 times that of common phospholipids(Zhang,2007)and this may in part explain its effects in relation to lipid metabolism.

In this study,serum lipid indices and enzyme activities of lipid metabolism were significantly affected by dietary LPC.The serum concentration of TG,T-CHO,HDL-C and LDL-C were all decreased by dietary LPC.Gillett et al.(1975)reported that significantly decreased relative concentrations of LPC were found in blood platelets and erythrocytes as well as in plasma of patients suffering from chronic ischemic heart disease.Obviously,high serum lipids are closely related to this kind of disease,and serum lipids can be decreased by dietary LPC.In another word,dietary LPC increased the health of animal.TP,LPL,HL and TL are adipose decomposing enzymes,and mainly regulated by dietary lipid levels and lipid utilization coefficient(Qin et al.,2015;Sun et al.,2014).In the present study no differences in blood lipid decomposing enzyme activities were found between the C+and C-group,and this may be due to the relatively small differences in lipid content.Dietary LPC increased all the enzyme activities of lipid decomposition,and this no doubt contributed to the increase in the lipid utilization coefficient.FAS is the key enzyme in the pathway of lipid synthesis in vivo,which generated long chain fat acids by catalyzing acetyl CoA and malonyl CoA.FAS is regulated by the speed of transcription and the stability of FAS mRNA.Abundance of FAS mRNA is regulated by several factors,such as hormones,carbohydrates,proteins,fatty acids,minerals and vitamins.FAS mRNA transcription was down-regulate by fish oil and n-3 HUFA in the diet(Clarke et al.,1993;Ma et al.,2009).Dietary LPC reduced the fish oil content and the percentage of n-3 HUFA in the diet,and caused the increasing of FAS mRNA expression.In addition to its strong emulsifying ability,LPC is also one of lipids and it not only promotes lipid metabolism,but it can also replace part of the lipids in diets as illustrated in our study.The changes of serum lipid indices and the increase of lipid metabolism enzyme activities showed that LPC improved the efficiency of the lipid utilization coefficient and replaced part of the lipids.

5.Conclusion

LPC increased the growth performance of turbot by enhancing the lipid utilization coefficient efficiency.Adding between 1000 and 5500 mg/kg in the diets had the same effect and the higher concentrations with time may also have a negative effect on fish health.Based on the SAS NLIN analysis,dietary 870.37mg/kg LPC was optimal for turbot juveniles.

Acknowledgements

We all thank Kemin Industries(Zhuhai)Co.Ltd for supplying LPC and supporting many advices for the trial.The work was supported by key research and development Plan of Shandong Province(2016GSF115005),and Technology Development Plan Project of Shandong Province(2014GHY115006).