Study On The Requirement And Bioavailability Of Dietary Copper In Juvenile Russian Sturgeon Acipenser Gueldenstaedtii

Copper(Cu)is a necessary nutrient element for all animals including fish,and Cu plays a role in the form of coenzyme.A series of physiological and biochemical processes such as organism redox reaction,iron ion metabolism,cell energy production,collagen synthesis and brain neurotransmitter production are related to Cu.Dietary Cu is the main source of fish to get Cu and excessive Cu can cause oxidative damage to fish.The content of Cu in the fish is regulated by the absorption and excretion.Cu absorption is main affected by the chemical form of Cu.Russian sturgeon Acipenser gueldenstaedtii is a widely cultivated aquatic species in the world.So far,copper nutrition research of Russian sturgeon is still lagging behind.Therefore,in order to explore the basic physiological role of Cu,and to determine the Cu requirement and the utilization in Russian sturgeon,this paper mainly studies the following four aspects:1)Effects of diet copper on growth,body biochemistry and antioxidant capacity of juvenile Russian sturgeon,2)Transcriptome analysis of the response of juvenile Russian sturgeon to dietary copper,3)A comparative study on the bioavailability of juvenile Russian sturgeon in different copper sources,4)Cloning,tissue distribution and expression analysis of copper transport protein in juvenile Acipenser gueldenstaedtii.The main results and conclusions were as follows:1.Effects of diet copper on growth,body biochemistry and antioxidant capacity of juvenile Russian sturgeonIn order to investigate the effects of Cu on the growth,body biochemistry and antioxidant capacity of Russian sturgeon and quantify the dietary Cu requirement of Russian sturgeon,copper sulfate(CuSO4·5H2O)was added into the basal semi-purified diet at levels 0,1,2,4,6,8 and 16 mg Cu/kg diet yielding the actual dietary values of 0.3,1.5,2.4,4.4,6.2,8.3 and 16.1 mg Cu/kg diet,respectively.Each diet was fed to sturgeon(11.84±0.07 g)in triplicate for 8 weeks.The Cu concentration in the rearing water was 0.8-1.2 ?g/L during the trial period.The weight gain rate(WGR)and feed efficiency were significantly higher in fish fed 2.4 to 8.3 mg Cu/kg than those fed 0.3,1.5 or 16.1 mg Cu/kg.The highest WGR was found in fish fed 6.2 mg Cu/kg.The whole-body and liver Cu concentrations were highest in fish fed 6.2 to 16.1 mg Cu/kg.Hepatic copper-zinc superoxide dismutase(Cu-Zn SOD)activities,total antioxidant capacity and serum ceruloplasmin activity were significantly higher and hepatic malondialdehyde were significantly higher lower in fish fed 6.2 and 8.3 mg Cu/kg diet than those in other treatments.No significant differences were in survival,hepatosomatic index,viscerosomatic index,condition factor,whole body moisture,crude protein,crude lipid and ash among all the treatments.Analysis of broken-line regression based on the WGR,whole-body Cu concentration,hepatic Cu-Zn SOD and serum ceruloplasmin activity of the fish fed different diets indicates that the optimal Cu concentration in the semi-purified diet for juvenile Russian sturgeon is 7-8 mg/kg.The results showed that Cu in the water could not meet the growth of Russian sturgeon.The addition of Cu in the diet was necessary,but the excessive Cu could lead to the decline of the growth performance and antioxidant capacity of Russian sturgeon.It was suggested that the dietary Cu of Russian sturgeon should be controlled at 7-8 mg/kg.2.Transcriptome analysis of the response of juvenile Acipenser gueldenstaedtii to dietary copperIn an effort to better understand the physiological role of Cu and to illuminate global gene expression patterns modulated by dietary Cu,we obtained the liver transcriptome information of Russian sturgeon fed diet supplemented Cu levels at 0,6 and 16 mg/kg(referred to as Cu deficiency,Cu optimum and Cu overdose group)by RNA sequencing(RNA-seq)technology.RNA-seq results showed that as many as 72270 unigenes with 1053 bp of average length and 1328 bp of unigene N50 were generated.Compared with Cu optimum group,a total of 1750 and 463 genes were significantly up-and downregulated in Cu deficiency group,and the numbers were 3228 and 1938 in Cu overdose group.GO terms with significant changes were 187 for biological process(BP),34 for cellular component(CC)and 118 for molecular function(MF)in Cu deficiency group,and the numbers were 225,29 and 140 in Cu overdose group.For KEGG analysis,30 pathway in Cu deficiency and 38 in Cu overdose significantly changed.Subsequent bioinformatics analysis revealed that the differentially expressed genes were mainly related to energy nutrient(carbohydrate and lipid)metabolism,immune system,and signal transduction,suggesting that these signaling pathways may be regulated by Cu content.The experimental results suggest that Cu affects the growth and physiological functions of Russian sturgeon mainly through energy and immune pathways.In the future,the study of fish Cu nutrient physiology can proceed from the significantly changed energy metabolism and immune related pathways.3.A comparative study on the bioavailability of juvenile Russian sturgeon in different copper sourcesThis study was conducted to evaluate the relative bioavailability(RBV)of copper sulfate(CuSO4),copper methionine(CuMet)and nano-copper oxide(CuONano)as diet additive in Russian sturgeon.The basal diet(control group)were supplemented Cu level at 2,4,6,8 and 16 mg/kg from Cu-Met or CuONano and 4,6 and 16 mg/kg from CuSO4.Sturgeon(9.82±0.08 g)were fed with these 14 diets for 8 weeks.Challenge test and apparent digestibility determination of Cu were carried out after the culture experiment for 1 weeks.Results showed that the sturgeon reached the same growth,tissue Cu deposition,antioxidant and immune capacity needed less diet Cu in the form of Cu-Met or CuONano than CuSO4.Fish fed diet Cu in the form of CuMet and CuONano exhibited higher AD and resistance to A.hydrophila.The highest weight gain rate(WGR),whole body Cu concentration,AD,copper-zinc superoxide dismutase(Cu-Zn SOD)activity,total antioxidant capacity,lysozyme and immunoglobulin M content were got at 6,4,and 4 mg/kg diet Cu supplemented from CuSO4,CuMet and CuONano respectively.There was no significant difference between these maximum values.Based on the WGR,hepatic Cu-Zn SOD activity,whole body Cu concentration the bioavailability of CuMet and CuONano is about 1.5-2.0 times that of CuSO4 and the optimal diet Cu requirements were 5-8 mg/kg depending on the form of diet Cu source.In summary,CuMet and CuONano have higher bioavailability than CuSO4,and can be effectively used by fish to reduce the amount of Cu supplemented in Russian sturgeon diet.This is an important reference for controlling the excretion of diet Cu in the aquaculture production and reducing the pollution of water environment.However,CuMet and CuONano had no additional growth promotion effect compared to CuSO4,higher absorption efficiency is the main reason leading to increased bioavailability.4.Cloning,tissue distribution and expression analysis of copper transport protein in juvenile Acipenser gueldenstaedtiiIn order to understand the absorption efficiency of different Cu sources in Russian sturgeon.Samples from sturgeon fed copper levels at 4,6 and 16 mg/kg in growth experiment of Cu utilization were selected to study the following three aspects at the molecular level:1)Full-length cloning of Cu transport protein Ctrl cDNA in Russian sturgeon;2)The mRNA expression of Ctrl,ATP7A and ATP7B in Russian tissues;3)Effects of different dietary Cu sources on the expression of Ctrl,ATP7A,ATP7B,CCS and SOD in Russian sturgeon.The results are as follows:1)Ctrl mRNA expression is higher in the gills,heart,liver and foregut.ATP7A mRNA expression is higher in the foregut,liver,spleen and gills.ATP7B mRNA expression is higher in the liver and gills.2)The Russian sturgeon Cu transport protein Ctrl,which encodes 202 amino acids,was cloned.The protein has three transmembrane regions and the presence of methionine-rich Cu binding sites was found at the N-terminus and C-terminus.3)The expression levels of Ctrl,ATP7A and ATP7B mRNA in CuMet and CuNano groups were higher than those in CuSO4 group,and the corresponding protein of Ctrl was higher in CuMet and CuNano groups when the dietary Cu levels at 4 and 6 mg/kg.When the dietary Cu content was 16 mg/kg,the protein expression of Ctrl was decreased.Cu-Zn SOD and CCS fluorescence quantitative results indicate that there may be no transcriptional regulation of Cu-Zn SOD expression.The above results suggest that the main diet Cu absorption site of Russian sturgeon is in the foregut,the liver is the central organ of Cu metabolism.The amino acid chelate or nano-form Cu in diet are more able to up-regulate Cu transport-related protein expression than copper sulfate,which effectively improved the absorption and retention rate of Cu.