| Yellow River carp(C.carpio haematopterus)is a famous species of common carp in northcentral China due to its delicious flesh and profound traditional cultural connotation.However,the fillet of farmed Yellow River carp tends to be softer and less robust flavorful than that of wild ones,which has emerged long-term extraordinary low market price and consumer rejection.Recently,high-value aquatic products to meet market demand has become a central issue,thus it is urgent to find an efficient way to ensure predictable and firmer fillet of farmed fish.It is one of the most direct and effective strategies to improve the flesh quality of farmed fish through nutritional regulation.In this study,a 90-day rearing trial was conducted to investigate the effect of faba bean(Vicia faba L.)on growth performance and flesh quality of farmed Yellow River carp,and the regulatory mechanism of improving the flesh quality of Yellow River carp by nutritional strategy was preliminarily clarified.On the basis of the above regulation mechanism,a 8-week rearing trial was conducted to estimate the effect of dietary hydroxyproline(Hyp)supplementation on growth performance and flesh quality of Yellow River carp.The main research contents and results of this study are as follows:1.Effects of faba bean(Vicia faba L.)on antioxidant capacity,fillet texture characteristics and myofiber characteristics of Yellow River carp,C.carpio haematopterusIn this experiment,Yellow River carp with the same genetic and nutritional background(initial weight of 500 ± 20 g)were randomly assigned to 6 ponds.Carps were fed with commercial feed as the control group(CFG),and carps were fed with faba bean(knocked into small pieces after soaking in water for 12 hours)as the experimental group(FBG).A 90-day rearing trial was carried to evaluate muscle nutrient composition,antioxidant capacity,fillet texture characteristics,myofiber cell characteristics and intramuscular collagen content,the correlation between above indexes and fillet texture.The results showed that faba bean reduced the growth performance of Yellow River carp,changed the amino acid and fatty acid composition in muscle,especially,the contents of eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA)decreased significantly(P < 0.05).The contents of reduced glutathione(GSH)and hydrogen peroxide(H2O2)in white muscle and hepatopancreas of Yellow River carp in FBG increased significantly(P < 0.05).The antioxidant capacity decreased significantly(P < 0.05).In FBG,the myofiber diameter was smaller,the muscular collagen content and the space of myofiber increased(P < 0.05).the collagen content increased,and the myofiber spacing increased.The raw-and cooked-fillet texture in the FBG were better than those of the CFG.Therefore,this study found that there was a strong correlation between fillet texture and smaller myofiber diameter and higher content of intermuscular collagen content in FBG,while no significant correlation with myofiber density.In conclusion,the improvement of muscle texture characteristics and the decrease of muscle nutritional value of Yellow River carp in FBG were attributed to the significant increase of reactive oxygen species(ROS)level in muscle,which was caused by faba bean.This study could provide a solid practical basis for the study of nutritional regulation strategy and mechanism for the improvement of flesh quality of Yellow River carp.2.Determination and analysis of transcriptome of white muscle in Yellow River Carp(C.carpio haematopterus)and preparation of polyclonal antibodiesIn this study,we have carried out transcriptome sequencing analysis of white muscle and prepared polyclonal antibodies against key protein factors of the muscle development.The results showed that the transcriptome data could be used to screen the differentially expressed genes between the two experimental groups,and analyze the enrichment pathways of GO and KEGG.The genes expression of extracellular matrix involved in the collagen and the gene of lysyl oxidase showed significant down-regulated(P < 0.05).The genes involved in the structure of extracellular matrix and protease showed significant up-regulated(P < 0.05).Among the genes related to myofiber development,the expression of telethonin was significantly increased(P < 0.05),while the gene expression of proteasome-mediated ubiquitin-dependent protein catabolism was significantly down-regulated(P < 0.05).Go functional annotation showed that the differential genes were mainly concentrated in extracellular matrix structure,proteolysis,myofiber development,peptide crosslinking and calcium binding,etc.The results of KEGG enrichment showed that the differential genes enriched in amino acid and fatty acid metabolic pathway,and intracellular calcium homeostasis metabolic pathway were significantly down regulated in the muscle of experimental carp(P < 0.05);The differential genes enriched in metabolic pathways related to cytoplasmic matrix formation and carbohydrate utilization were significantly up-regulated in the muscle of experimental carp(P <0.05).In this study,rabbit derived polyclonal antibodies against the key molecule of myofiber development of Yellow River carp were successfully prepared.The antibody titer and tissue immunofluorescence test showed that the above antibodies had high stability and specificity,which could meet the detection and analysis of the expression level of related proteins in the process of muscle development,and provided a powerful tool for subsequent mechanism research.In summary,faba bean could affect extracellular matrix deposition,myofiber development,calcium ion binding,as well as amino acid,and lipid metabolism.Based on the transcriptome data of white muscle,this study have preliminarily discussed the regulation mechanism of improving the flesh texture characteristics of Yellow River carp fed with faba bean,which has important reference value for indepth study on the nutritional regulation mechanism of flesh quality improvement of Yellow River carp.3.Molecular regulation mechanism of promoting flesh texture of Yellow River carp(C.carpio haematopterus)based on strong oxidationBased on the aforementioned research results,this study discussed the mechanism of faba bean(Vicia faba L.)on improving the fillet texture of Yellow River carp.The genes related to intramuscular collagen metabolism,antioxidant system and the key genes of TGF-β/Smads signaling pathway were firstly studied.In order to explore the influence mechanism of faba bean on the fillet texture of Yellow River carp,this experiment detected the myogenic regulatory factors and the key genes of target of rapamycin(TOR)signaling pathway in the muscle development of Yellow River carp.The results showed that the expression of collagen synthesis related genes in the white muscle of carps in the experimental group(FBG)collagen 1 α1(col1 α1a),col1α1b,col1α2a,col1α2b were significantly lower than those of the control group(CFG)(P < 0.05).The genes expression of tgf-β,matrix metalloproteinases-2(mmp2)and tissue inhibitor of matrix metalloproteinases-2(timp2),and the protein expression of TGF-β were no significant difference(P > 0.05).The genes expression of glutathione reductase(gr),kelch like ECH associated protein-1(keap1)and heme oxidase 1(ho-1)in the muscle of FBG were significantly higher than those of CFG(P < 0.05).No significant difference in the genes expression of glutathione peroxidase 1(gpx1),superoxide dismutase(sod)and catalase(cat)between the two groups(P > 0.05).The genes expression of gpx1,gr and keap1 in hepatopancreas of FBG were significantly higher than those of CFG(P < 0.05),while the genes expression of sod and cat were significantly lower than those of CFG(P < 0.05),while the gene expression of ho-1 had no significant difference between the two groups(P > 0.05).The above results showed that the high level of reactive oxygen species(ROS)accumulated in the muscle of carps in FBG inhibited the synthesis of intramuscular collagen.The genes expression of paired box 7(pax7),myogenic differentiation antigen(myod),myogenic regulatory factor 4(mrf4),myogenin,myogenic factor 5(myf5),and myosin heavy chain(myhc)and myosin light chain(mylc)were significantly lower than those of CFG(P < 0.05),while the genes expression of telethonin and myostatin b(mstn b)were significantly higher than those of CFG(P < 0.05).The proteins expression of Pax7,Myod,Mrf4,Myogenin,Myhc and Mylc in white muscle of FBG were significantly lower than those of CFG(P < 0.05),this was consistent with the corresponding gene expression trend.Western blot showed that the proteins expression of Myod and Mrf4 in white muscle of FBG were significantly lower than that of CFG(P < 0.05).The gene expression of 4E binding protein 1(4ebp)in white muscle of FBG was significantly lower than that of CFG(P < 0.05).The above results showed that the satellite cells of of FBG were not activated and differentiated,and the normal growth and development of white myofibers were blocked.In conclusion,the increase of muscular ROS in Yellow River carp or the nutritional imbalance in faba bean affected the flesh texture characteristics by regulating the antioxidant capacity,collagen metabolism and myofiber structure.This study preliminarily elucidated the nutritional regulation mechanism of improving the flesh texture characteristics of Yellow River carp,which could provide an important theoretical basis for the precise nutritional regulation strategy for improving the flesh quality of farmed fish.4.Effects of dietary hydroxyproline supplementation on fillet texture,antioxidant capacity,myofiber structure and collagen content of Yellow River carp,C.carpio haematopterusThis study summarized and analyzed the existing achievements,an 8-week rearing trial was conducted,6 isoproteic and isolipidic practical diets were formulated with graded levels of Hyp(1.36,3.93,7.14,8.86,11.45 and 13.69 g/kg of dry matter).A total of 840 carps with an average initial weight of 17.90 ± 0.28 g were randomly allocated to 24 polypropylene tanks(400 L capacity filled to 300 L and 35 carps per tank).The results showed that different levels of dietary Hyp had no significant effect on the growth performance and morphological parameters(P > 0.05).With the increase of dietary Hyp level,the Gly content,22 : 6n-3,n-3 polyunsaturated fatty acids(n-3 PUFA)and intramuscular collagen concentrations showed a significant upward trend,while the proportion of Arg and Pro showed a downward trend.When dietary Hyp level was 11.45g/kg,muscular liquid holding capacity and p H reached the highest value.With the increase of dietary Hyp level,the activities of catalase(CAT)and total antioxidant capacity(T-AOC),and reduced glutathione(GSH)concentration in hepatopancreas showed a significant upward trend,and superoxide dismutase(SOD)activity and malondialdehyde(MDA)concentration had no significant difference among all treatments(P > 0.05).The trend of muscular CAT and T-AOC activity was the same as that of hepatopancreas,SOD activity showed a significant upward trend,MDA concentration showed a significant downward trend,and there was no significant difference in muscular GSH concentration among all treatments(P > 0.05).In addition to cohesiveness,the raw-fillet texture increased significantly with the dietary Hyp levels rising,and so did all of the cooked-fillet texture(P < 0.05).After cooking,the texture parameters of cooked fillet were less than those of raw-fillet except cohesiveness.Specifically,the maximum values of raw fillet were: firmness,chewiness and gumminess in dietary Hyp 11.45 g/kg group,springiness and shearing force in dietary Hyp 13.69g/kg group,respectively.As for cooked-fillet,the maximum values of firmness,chewiness,gumminess and springiness were observed in dietary Hyp 11.45 g/kg group,and the values of cohesiveness and shearing force were significantly increased with the increasing dietary Hyp up to8.86 g/kg(P < 0.05),and the two properties were not significantly influenced by dietary Hyp levels ranging from 8.86 to 13.69 g/kg(P > 0.05).The number of white myofibers with a small diameter(d ≤ 10 μm),the proportion of myofiber with minimum diameter and the density of myofiber were significantly higher in dietary Hyp 11.45 g/kg group than those of other groups(P < 0.05).The results of scanning electron microscope showed that the arrangement of myofibrils in raw fillet was more neater and denser than that in cooked fillet,and the connective tissue around the raw-fillet myofibrils showed a regular network structure,while that of cooked fillet obviously appeared agglutination.Among all treatments,there was no significant difference in the genes expression of paired box 7(pax7),myogenic differentiation antigen(myod),myogenic regulatory factor 4(mrf4),myogenin,myosin heavy chain(myhc)and myosin light chain(mylc)and the proteins expression of Pax7 and Myod(P > 0.05).However,the proteins expression of Mrf4 and Myhc increased significantly with the increase of dietary Hyp level(P < 0.05),the highest values appeared in dietary Hyp of 11.45 g/kg group.The protein expression of Myogenin firstly decreased and then increased with the increase of dietary Hyp level,and the lowest values appeared in dietary.The expression of collagen metabolism related genes collagen 1 α1(col1 α1a),col1α1b,col1α2a,col1α2b,small mother against decapitaplegic homologue 2(smad2)and smad4 were up-regulated with the increase of dietary Hyp level.In conclusion,an appropriate amount of dietary hydroxyproline supplementation could improve the nutritional composition and texture characteristics of Yellow River carp flesh and maintain its healthy growth and development.This study could provide an important reference for the application of the nutritional regulation mechanism of flesh quality improvement of farmed fish.In conclusion,faba bean can change the fillet texture characteristics by improving ROS levels in hepatopancreas and muscle of Yellow River carp,changing the structure of myofiber and the composition of cytoplasmic matrix,while hydroxyproline can improve the fillet quality by improving the antioxidant capacity of Yellow River carp,changing the structure of myofiber and the content of collagen.The application and promotion of nutrition strategy to improve the fillet quality of Yellow River carp need to be verified repeatedly in the rearing practice,evaluate the effect and optimize the feed formula in time. |
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