| Phospholipid is usually referred to as the polar lipid containing phosphorus,such as phosphatidylinositol(PI),phosphatidylethanolamine(PE),phosphatidylserine(PS)and phosphatidylcholine(PC).A large number of studies have shown that dietary phospholipid is an essential nutrient for various aquatic animals,which plays a very important role in their survival,growth,stress resistance,lipid metabolism and ovarian development.However,the important physiological functions of phospholipid have not been systematically studied in crustaceans.Therefore,the present study systematically investigated the effects of dietary phospholipid on growth,antioxidant capacity and lipid metabolism of juvenile Eriocheir sinensis as well as on vitellogenesis of adult crabs(female)using histological,nutritive and molecular biological methods.First of all,this study used three kinds of typical phospholipid sources(soy lecithin and egg yolk lecithin and krill oil)to explore the influences of different phospholipid sources on the juvenile and adult(female)E.sinensis with the aims of selecting suitable phospholipid sources as well as investigating the effects of different phospholipid sources on different growth stages of E.sinensis.Based on the common characteristics of lipid-lowering effects of three phospholipid sources at the juvenile stage of E.sinensis,further experiments were designed to explore whether dietary phospholipid could promote the utilization of high-lipid diet in juvenile E.sinensis.Meanwhile,phosphatidylcholine(PC)from soy source as the representative of phospholipid was selected to the lipid-lowering molecular mechanism of phospholipid.In addition,phospholipid remodeling reaction was also investigated to determine its role in the deposition of highly unsaturated fatty acids(HUFA)in the present study.The results of the present study not only provide valuable information for reasonable use of phospholipid in formula feed of E.sinensis,but also enrich the research content of phospholipid nutrition in crustacean.The main research results and conclusions of this paper are as follows:1.Influence of dietary phospholipid sources on growth performance,body composition,antioxidant capacity and lipid metabolism of juvenile E.sinensisThis experiment was conducted to investigate the effects of dietary phospholipid sources on growth performance,body composition,antioxidant capacity and lipid metabolism of juvenile E.sinensis.A total of seven isoproteic(35%)isolipid(8%)diets were designed to feed juvenile E.sinensis(0.26±0.01 g)for eight weeks,including one control diet(without phospholipid)and six experimental diet(1%or 3%krill oil,soy lecithin and egg yolk lecithin).The results showed that dietary phospholipid could significantly increase the final body weight(FBW),weight gain(WG)and specific growth rate(SGR)of juvenile E.sinensis,and 3%krill oil had the best growth-enhancing effect.Compared with phospholipid-devoid group,the activities of superoxide dismutase(SOD)and catalase(CAT)in hepatopancreas were significantly increased,while the content of malondialdehyde(MDA)was significantly decreased in phospholipid-added groups.In addition,the total lipid and triglyceride(TG)contents in the hepatopancreas and the total lipid of whole body were significantly decreased in phospholipid addition groups when compared with phospholipid-devoid group.Among all treatments,3%krill oil addition group showed the lowest total lipid and triglyceride(TG)contents in hepatopancreas.The fatty acid composition of hepatopancreas is related to that in the diets,particularly the C18:2n-6,C20:5n-3 and C22:6n-3 contents.Real-time quantitative PCR revealed that dietary phospholipid markedly down-regulated the gene expression related to lipid synthesis(sterol regulatory element-binding protein 1(srebp-1),fatty acid synthase(fas),?9 fatty acyl desaturase(?9 fad)and elongase of very long-chain fatty acids 6(elovl6))and oxidation(carnitine palmitoyl transterase-1a(cpt-1a),carnitine acetyltransferase(caat))as well as up-regulated the gene expression involved in lipid export(microsomal triglyceride transfer protein(mttp)).To conclude,the results above exhibits that the krill oil is a high-quality phospholipid source,and diets added with 3%krill oil can better promote growth,improve antioxidant capacity and facilitate lipid utilization for juvenile E.sinensis.2.Effects of dietary different phospholipid sources on antioxidant capacity,lipid metabolism and vitellogenesis of adult E.sinensis(female)Four isoproteic(40%)and isolipid(11.5%)experiment diets were formulated in the present study,including one control diet(without phospholipid)and six test diets(with 2.5%krill oil,2.5%egg yolk lecithin and 2.5%soy lecithin).The experimental period was 10 weeks,and the initial weight of adult E.sinensis(female)was 95.23±4.43 g.Dietary phospholipid increased the activities of glutathione peroxidase(GSH-Px),catalase(CAT)and superoxide dismutase(SOD)as well as decreased the content of malondialdehyde(MDA)in hepatopancreas.Among the three phospholipid sources,the krill oil exhibited the best antioxidant effect,which may be related to the fact that the krill oil contains a certain amount of astaxanthin and more n-3 highly unsaturated fatty acid(n-3 HUFA).The diets added with 2.5%krill oil,egg yolk lecithin and soy lecithin could promote the total lipid,polar lipid and neutral lipid accumulation in hepatopancreas and ovary.By contrast,the krill oil showed better the effects of promoting lipid deposition than the egg yolk lecithin and soy lecithin,probably because the krill was easier to up-regulate the expression of genes related to lipid absorption and synthesis in hepatopancreas and ovary,leading to higher lipid accumulation in hepatopancreas and ovary.Due to higher n-3 highly unsaturated fatty acid(n-3 HUFA)in krill oil,the krill oil significantly promoted n-3 highly unsaturated fatty acid(n-3HUFA)deposition in hepatopancreas,ovary and muscle,which not only could provide more energy for the ovarian development and essential fatty acids,but also greatly improve the nutritional value of edible parts(hepatopancreas,ovary and muscle).In addition,compared with the phospholipid-devoid group,the phospholipid-added group exhibited the higher the gonadosomatic index(GSI).The krill oil group showed a higher the gonadosomatic index(GSI)than the soy lecithin group,while the egg yolk lecithin group was in between.Combined with the results of 17?-estradiol(E2)and progesterone(PROG)contents in serum,vitellogenin(Vg)expression levels in ovary and hepatopancreas,vitellogenin receptor(Vgr)expression levels in ovary and cholesterol content in tissues,we presumed that dietary phospholipid could increase17?-estradiol(E2)and progesterone(PROG)contents by changing cholesterol to steroid hormones,which would enhance the vitellogenin(Vg)synthesis in ovary and hepatopancreas.The vitellogenin(Vg)bonding with steroids from hepatopancreas were transported via hemolymph,recognized by the vitellogenin receptor(Vgr)on oocyte,and absorbed by the oocyte and incorporated into yolk,finally promoting the vitellogenesis.Compared with the egg yolk lecithin and soy lecithin,the krill oil was easier to promote the conversion of cholesterol to steroid hormones(17?-estradiol(E2)and progesterone(PROG)),accelerating the synthesis of vitellogenin(Vg).Thus,the gonadosomatic index(GSI)in krill oil group was higher the egg yolk lecithin and soy lecithin groups.The above results indicate that the krill oil is an excellent phospholipid source,and it is recommended to add 2.5%krill oil in the diet of adult E.sinensis(female).3.Effects of phospholipids on growth performance,antioxidant capacity and lipid metabolism of juvenile E.sinensis fed high-fat dietOur previous study has shown that the diets added with soy lecithin,egg yolk lecithin or krill oil could significantly reduce total lipid content in hepatopancreas and whole body,promote the lipid utilization and improve the growth performance for juvenile E.sinensis.However,it is still unclear whether the dietary phospholipid can facilitate the utilization of a high-lipid diet in juvenile E.sinensis.Therefore,in the present study,soy lecithin commonly used in production as representative to investigate the effects of soy lecithin on the growth,antioxidant capacity and lipid metabolism of juvenile E.sinensis fed high-fat diet.Two lipid levels(13%and 8%)and two phospholipid levels(5%and 1%)were used to formulate four isoproteic(40%)experimental diets.The four dietary treatments were:LL-LP group(8%lipid and 1%phospholipid),LL-HP group(8%lipid and 5%phospholipid),HL-LP group(13%lipid and 1%phospholipid)and HL-HP group(13%lipid and 5%phospholipid).Compared with the HL-LP group,the specific growth rate(SGR),weight gain(WG)and final body weight(FBW)of juvenile crabs were significantly increased in the HL-HP group.Diets added with 5%soy lecithin effectively reversed the HL-LP diet-induced down-regulation on enzymatic-antioxidant activities and up-regulation of MDA content in this study,indicating that high dietary PL can reduce the lipid peroxidation induced by the high-lipid diet.In addition,5%soy lecithin in the diet significantly inhibited the increase of total lipid content in whole crab and hepatopancreas induced by high-lipid diet.Combined with the results of gene expression levels related to lipid metabolism in hepatopancreas and very low-density lipoprotein(VLDL)content in serum and hepatopancreas,we assume that dietary phospholipids inhibited excessive lipid accumulation in hepatopancreas by promoting lipid oxidation and exportation as well as inhibiting lipid absorption and synthesis.In conclusion,the diets added with 5%soy lecithin can improve growth performance and promote lipid utilization in juvenile E.sinensis fed a high-lipid diet by optimizing lipid metabolism.Besides,5%soy lecithin could also reduce lipid peroxidation induced by high-lipid diet and improve the health status of juvenile E.sinensis.4.Dietary phosphatidylcholine(PC)affects growth performance,antioxidant capacity and lipid metabolism of juvenile E.sinensisOur previous studies indicated that dietary soy lecithin,egg yolk lecithin or krill oil could reduce the lipid deposition of juvenile E.sinensis,but the lipid-lowering molecular mechanism was still unclear.The phosphatidylcholine(PC)is the major component of biological membranes with the function of growth promotion.Therefore,in the present study,phosphatidylcholine(PC)extracted from soya as the representative of phospholipid was used to explore the effects of different levels of phosphatidylcholine(PC)on growth performance,antioxidant capacity and lipid metabolism of juvenile E.sinensis.The specific aims were to evaluate the precise phospholipid requirement and explore the mechanism on lipid reduction in juvenile E.sinensis by feeding with highly purified phosphatidylcholine(PC).Six isonitrogenous(40%)and isolipidic(9%)diets were formulated to feed the juvenile E.sinensis(0.52±0.01 g)for 8 weeks.Six different levels of phosphatidylcholine(PC)were added in the diets:0%,1.5%,3%,4.5%,6%and 7.5%,respectively.The practical added levels of phosphatidylcholine(PC)in diets were 0%,1.36%,2.76%,4.34%,5.74%and 7.10%,respectively.The crabs in the phosphatidylcholine(PC)-added groups had a lower feed conversion ratio(FCR)and higher final body weight(FBW),weight gain(WG)and specific growth rate(SGR)than those in the phosphatidylcholine(PC)-devoid group.The dietary phosphatidylcholine(PC)supplement increased antioxidant capacity and reduced lipid peroxidation in the hepatopancreas of juvenile E.sinensis.The crabs fed the diet with phosphatidylcholine(PC)addition showed lower whole-body total lipid contents than those fed the diet without phosphatidylcholine(PC)addition.The diets supplemented with moderate phosphatidylcholine(PC)promoted whole-body protein deposition.The total lipid content of hepatopancreas was also markedly decreased in the phosphatidylcholine(PC)-added groups,while the total lipid content in muscle showed an opposite trend with the hepatopancreas.In addition,the appropriate dietary phosphatidylcholine(PC)facilitated the deposition of highly polyunsaturated fatty acids(especially C20:5n-3 and C22:6n-3)deposition in muscle and hepatopancreas.Dietary phospholipid significantly inhibited the gene expression related to lipid synthesis(fatty acid synthase(fas)and elongase of very long-chain fatty acids 6(elovl6))in hepatopancreas as well as up-regulated gene expression involved in lipid absorption(triacylglycerol lipase(tgl),lipoprotein lipase(lpl),fatty acid transport protein 6(fatp6)and fatty acid binding protein 9(fabp9)),oxidation(carnitine palmitoyl transterase 1a(cpt-1a),carnitine palmitoyl transterase 2(cpt-2),acyl-Co A oxidase(aco)and carnitine acetyltransferase(caat))and export(microsomal triglyceride transfer protein(mttp))in hepatopancreas,which indicated that the lipid-lowering mechanism of dietary phosphatidylcholine(PC)is mainly achieved by inhibiting the lipid synthesis and promoting the lipid oxidation and transport.In addition,based on the weight gain and specific growth rate,the broken-line regression analysis suggests that the optimum phosphatidylcholine(PC)requirement of juvenile E.sinensis should be in the range of2.89%?2.95%of the diet by weight.5.Effects of phosphatidylcholine(PC)deficiency and addition on growth performance,antioxidant capacity,fatty acid composition and phospholipid remodeling reaction of juvenile E.sinensis under different lipid sourcesOur previous studies showed that dietary phospholipid could promote the highly unsaturated fatty acids(HUFA)accumulation in muscle and hepatopancreas,suggesting that there may be a relationship between different lipid sources(different fatty acids)and phospholipid.Therefore,in the present study,phosphatidylcholine(PC)was used as the representative of phospholipid to study the effects of phosphatidylcholine(PC)deficiency and addition on growth performance,antioxidant capacity,fatty acid composition and phospholipid remodeling reaction of juvenile E.sinensis under different lipid sources.The specific objectives of this study were to(1)screen suitable lipid sources for juvenile E.sinensis;(2)investigate whether there was interaction between lipid source and phosphatidylcholine(PC),and the relationship between their interaction and phospholipid remodeling reaction.Two phosphatidylcholine(PC)levels and four different lipid sources were used to formulate eight isonitrogenous(35%)and isolipidic(9%)experimental diets,and fed to juvenile E.sinensis(4.19±0.01 g)for 8 weeks.Crabs fed diets added with 3%phosphatidylcholine(PC)showed dramatically higher the final body weight(FBW),weight gain(WG)and specific growth rate(SGR)than those fed diets without phosphatidylcholine(PC)under the same lipid source condition.Compared with the other lipid sources-added groups,the final body weight(FBW),weight gain(WG)and specific growth rate(SGR)were notably increased in the fish oil-added group at 3%phosphatidylcholine(PC)condition.There were significant effects of the dietary phosphatidylcholine(PC)levels and dietary lipid sources on the whole-body total lipid content.The total antioxidant capacity(T-AOC),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)activities in hepatopancreas were also significantly influenced by the dietary phosphatidylcholine(PC)levels and dietary lipid sources.The fatty acid composition of hepatopancreas and muscle tissue reflects the dietary fatty acid composition.The levels of C18:1n-9,C18:2n-6,C18:3n-3,C20:5n-3,C22:6n-3,monounsaturated fatty acids(MUFA),polyunsaturated fatty acids(PUFA),highly unsaturated fatty acids(HUFA)and the ratio of n-6/n-3 fatty acids in hepatopancreas and muscle were significantly affected by dietary lipid sources.Besides,the dietary lipid sources and the interaction between dietary lipid sources and phosphatidylcholine(PC)levels significantly influenced the C18:3n-3,C20:4n-6,C20:5n-3 and C22:6n-3levels in phosphatidylcholine(PC)of hepatopancreas.The 3%phosphatidylcholine(PC)-added group had markedly higher the phospholipase A2(PLA2)content in hepatopancreas than the phosphatidylcholine(PC)-devoid group at the same lipid source condition.Compared with the phosphatidylcholine(PC)-devoid group,the lysophosphatidylcholine acyltransferase(LPCAT)content in hepatopancreas was also significantly increased in the 3%phosphatidylcholine(PC)-added group when the dietary lipid source was fish oil and perilla oil.In conclusion,perilla oil(rich in C18:3n-3)and fish oil(rich in C20:5n-3,C22:6n-3 and other highly unsaturated fatty acids(HUFA))may more easily activate phospholipase A2(PLA2)and lysophosphatidylcholine acyltransferase(LPCAT)to accelerate the occurrence of phospholipid remodeling reaction,leading to fatty acids like C22:6n-3,C20:5n-3 and C18:3n-3 accumulation in hepatopancreas phosphatidylcholine(PC).In addition,at 3%phosphatidylcholine(PC)condition,dietary phosphatidylcholine(PC)may avoid the oxidation of fatty acids in fish oil and perilla oil by playing its antioxidant role as well as form physiologically important phospholipid with fatty acids in fish oil and perilla oil by phospholipid remodeling reaction,which make fish oil and perilla oil had better growth-promoting functions.To sum up,krill oil is the excellent phospholipid source for juvenile and female(adult stage)E.sinensis.Diets added with 5%soy lecithin could facilitate the utilization of high-fat feed for juvenile E.sinensis.Based on the weight gain(WG)and specific growth rate(SGR),the broken-line regression analysis suggests that the optimum phosphatidylcholine(PC)requirement of juvenile E.sinensis should be in the range of2.89%?2.95%of the diet by weight.The dietary phosphatidylcholine(PC)reduced lipid deposition in the hepatopancreas of E.sinensis mainly through inhibiting lipid synthesis as well as facilitating lipid oxidation and transport.In addition,perilla oil(rich in C18:3n-3)and fish oil(rich in C20:5n-3,C22:6n-3 and other highly unsaturated fatty acids(HUFA))may more easily activate phospholipase A2(PLA2)and lysophosphatidylcholine acyltransferase(LPCAT)to accelerate the occurrence of phospholipid remodeling reaction,leading to fatty acids such as C22:6n-3,C20:5n-3and C18:3n-3 accumulation in hepatopancreas phosphatidylcholine(PC). |
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