The Effects Of Stocking Density And Feeding Models On Muscle Quality And Metabolic Regulations Of Grass Carp Ctenopharyngodon Idellus

With the construction of ecological civilization,and the development of fishery transition in China,the aquaculture industry is experiencing the course of transition from the single yield-increasing model to quality and benefit-improving model.The improvement of the quality of farmed fish has attracted widespread public attention.In the process of fish-farming,the food sources,aquaculture environment,culture patterns and so on,are the major factors of affecting the formation of fish flesh quality.The intensive fish-farming model,based on the utilization of artificial formula feeds and high stocking density,contributes to a sustained increase in fish production.Therefore,it is an important mode for pond aquaculture in China.However,this farming model resulted in deterioration of water environment,and caused some negative influences on farmed fish,e.g.slowing down the growth,reducing immunity and disease resistance,as well as declining the flesh quality.Therefore,how to obtain high quality fish products has become an urgent challenge and the main direction of future aquaculture research.Grass carp(Ctenopharyngodon idellus)is one of the most commonly and important cultured fish species in China,its annual output exceeds 5 million tons,ranking the first in the production of freshwater aquaculture fish.To investigate the effects of different stocking densities and feeding diets on C.idellus flesh quality,and its related regulation mechanisms of metabolism,would help us to better understand the regulatory mechanisms inducing significantly differences in flesh quality.Furthermore,this work will provide theoretical foundations and suggestions for the establishment of quality and benefit-improving fish-farming model in the future.The doctoral dissertation includes two parts of research subjects,the one is related with farming C.idellus under different stocking densities;the other is feeding C.idellus with two different kinds of diets.The C.idellus used in the experiment on stocking density were originated from the same batch,with an initial average weight of 99.15±0.70g per tail.The fish were divided into three groups:low-stocking-density group(LSD,2.45kg/m~3),medium-stocking-density group(MSD,7.44 kg/m~3)and high-stocking-density group(HSD,14.47 kg/m~3),respectively.Four replicates were applied in each experimental group.All of the fish were reared for 78 days.The experiment on different feeding diets was conducted in aquaculture ponds(area:2666.4 m~2 per pond).The experimental fish were separated into natural grass-fed group(GF),and artificial feed-fed group(AF).Three replicate ponds were used in each experimental group.About 3000tails of C.idellus(initial average weight of 35 g per tail)were assigned to each pond(roughly 2666.4 m~2×depth 1.8 m per pond),which was co-housed with 550 tails of Hypophthalmichthys molitrix(average weight of 14 g per tail)and 350 tails of Aristichthys nobilis(average weight of 25 g per tail).During the 113-day rearing period,GF C.idellus were fed with 100 kg of Lolium perenne,Euphrasia pectinata and Sorghum sudanense for each pond per day,whereas 15 kg of artificial feed was supplied to each AG pond two times per day.At the end of the rearing experiment,the fish samples were collected for physiological and biochemical tests,as well as multiple-Omics detection.By the cross-comparative analysis,the effects of different stocking densities and different feeding diets on physiological and biochemical indexes,muscular characteristics and textural properties,as well as the expressions of muscle development-related genes were evaluated.Finally,to draw up the regulation and interaction networks of metabolisms,and to reveal the molecular mechanisms related to muscle growth and metabolisms of C.idellus.The main results of the research are as follows:1.The effects of stocking density on quality characteristics and transcriptional level of C.idellus muscleThe growth rate of C.idellus decreased with the increase in stocking density.The fish maintained at LSD exhibited the highest weight gain rate(WGR),the slowest growth rate was detected in HSD group(P<0.05).The levels of free amino acids(FAAs)and unsaturated fatty acids(UFAs)in MSD and HSD group fish muscles were higher than those in LSD samples.Compared with MSD and HSD groups,the saturated fatty acids(SFAs)with low carbon chains(namely,C12:0-C16:0)exhibited highest contents in LSD C.idellus muscles.Whereas C17:0-C22:0 SFAs were significantly elevated with the increase in stocking density.The transcriptomics profiling of muscle tissue revealed a total of 394 differential expressed genes(DEGs)among the 3 densities groups.Functional enrichment analysis of DEGs showed that the differences at transcription level among groups are mainly manifested in development process,muscle structure and contraction peculiarity,as well as metabolisms(including lipid,protein and carbohydrate)aspects.Meanwhile,the responses to stimulus and signal transduction were also significantly enriched by DEGs(P<0.05).Our findings suggested that both HSD and LSD could lead to muscle fat infiltration,anti-oxidative inhibition,and lower expressions of immune factors,thereby obtaining farmed fish with higher fat deposition and lower disease resistance.2.The effects of artificial feed and natural grass on muscular characteristics and metabolomics of C.idellus muscleIn measurement of serum biochemical indexes,the levels of lactate dehydrogenase,alkaline phosphatase,total cholesterol,high density cholesterol,and total protein,significantly elevated in grass-fed(GF)fish(P<0.05).Compared to the GF C.idellus,the markedly higher intramuscular fat was observed in artificial feed(AF)-fed fish(P<0.01).Texture profile analysis showed that adhesiveness,springiness,cohesiveness,gumminess and chewiness of AF group muscles were significantly higher(P<0.05),however,resilience and shear force were relatively lower,as compared with those in GF group.In addition,the expressions of MyoG and MyoD markedly increased in GF C.idellus muscles,whereas expressions of Myf-5,MRF4,igfs and MSTNs reduced in GF group.Moreover,the grass-feeding enhanced the expressions of CoL1a-1 and CoL1a-2.The experimental fish were divided into four test groups based on different sex and feeding diets-female artificial feed(FAF),female grass feed(FGF),male artificial feed(MAF)and male grass feed(MGF).The results of liquid chromatography-mass spectrometry(LC-MS)detection indicated that the AF C.idellus muscles had higher concentrations of docosapentaenoic acid(DPA),dihomo-gamma-linolenic acid(DGLA)and arachidonic acid(ARA).Whereas grass-feeding elevated n-3 UFAs in GF C.idellus muscles,such as eicosapentaenoic acid(EPA),alpha-linolenic acid(ALA)and gamma-linolenic acid(GLA).The docosahexaenoic acid(DHA)was significantly more abundant in MAF muscles relative to MGF(q-value<0.05),whereas no significance in the comparison of female samples.Metabolic pathway analysis revealed that the arachidonic acid metabolism and steroid hormone biosynthesis pathways were significantly enriched by significant discriminating metabolites(SDMs)between AF and GF groups(q-value<0.05).3.The effects of two different feeding diets on transcriptomics and proteomic of C.idellus muscleThe transcriptomics and proteomic detection were performed on GF and AF groups C.idellus muscles.The results reflected that AF C.idellus muscle displayed significantly higher fatty acid?-oxidation activity,characterized by increasing levels of fatty acyl-CoAs(FACs),fatty acid synthase(fasn),Acyl-CoA synthetase(acs)and carnitine palmitoyl-transferases(cpts),as well as fatty acylcarnitines.Additionally,the two different feeding diets also resulted in distinctive patterns of glucose uptake and metabolism between GF and AF groups.The expressions of gpia,fbps,pfkfb-4,aldo,tpi-1b,ga3pdh,eno3 and pkma,were significantly up-regulated in AF group;whereas the expressions of hk2,fbp2,pfkfb-1,pfkma,tpi-1a and pgks elevated in GF group muscles.In addition,the molecules(including genes and proteins)involved in JAK3-STAT,Ras-Raf and Wnt/?-catenin signaling differentially expressed between GF and AF groups.Further analysis of the enriched functions and interacted networks of DEGs and DEPs,revealed consistently alternations in muscle growth,motor function,nutrients metabolism,immune response and signaling between groups.The grass-feeding could strengthen the stress tolerance and antioxidant capacity of C.idellus,while relieve the insulin resistance and apoptosis by reducing expressions of insulin growth factors(igfs),leading to better nutrition and health in GF C.idellus.