Effect Of Riboflavin On Growth Performance And Lipid Metabolism In Pekin Ducks As Well As Its Regulatory Mechanism

Three experiments in vivo were conducted to investigate the effects of riboflavin on growth and lipid metabolism in Pekin ducks as well as its regulatory mechanism,while one experiment was conducted to investigate the effects of riboflavin on cell proliferation and mitochondrial function of HepG2 cells.Experiment 1 was conducted to determine riboflavin requirement of white Pekin ducks from 15 to 35 days of age.A total of 288 15-day-old ducks were allotted to 6 dietary treatments with 6 replicate pens of 8 birds per pen.These ducks were fed the experimental diets containing 1.38,2.38,3.38,4.38,5.38,and 6.38 mg of total riboflavin per kg respectively from 15 to 35 days of age.Compared to the ducks fed the basal diet(containing 1.3 8mg/kg riboflavin),riboflavin supplementation could increase average daily weight gain(ADG),average daily feed intake(ADFI),percentage yield of breast and abdominal fat,free riboflavin in plasma or liver,while decline feed/gain and liver index(P<0.05).According to the broken-line model,the riboflavin requirements of male white Pekin ducks from 15 to 35 days of age for growth performance and tissue riboflavin were 2.33?3.57 mg/kg.Experiment 2 was conducted to investigate the effects of riboflavin deficiency on growth and lipid metabolism of starter Pekin ducks as well as its underlying mechanism.360 one-day-old Pekin ducks were divided into three groups of 120 birds each,with 12 replicates and 10 birds per replicate.For 21 days,the ducks were fed ad libitum a control diet(CAL),a riboflavin-deficient diet(RD),or were pair-fed with the control diet to the mean daily intake of the RD group(CPF).Compared to the CPF and CAL groups,riboflavin deficiency decreased ADG,while increased feed/gain and mortality;decreased plasma and liver riboflavin content;increased plasma and liver triglyceride and total cholesterol content,liver total saturated fatty acids,fatty acids such as C6:0,C12:0,C16:0,C18:0,and liver index.Proteomics data showed that in RD 32 proteins were enhanced and 31 diminished(>1.5-fold)compared to CAL and CPF.GO analyis showed the differentially expressed proteins are mainly involved in fatty acid beta oxidation and the mitochondrial electron transport chain(ETC).Downregulation of the proteins in RD involved in fatty acid beta oxidation process(ACADS,ACADM,ACAD9,ETFDH)indicated fatty acid beta oxidation were impaired by RD,which may explain liver lipid accumulation.Downregulation of the proteins in RD involved in electron transport chain(ACAD9,NDUFS1,NDUFA8,and FXN)indicated electron transport chain were impaired by RD,which may explain growth depression in RD group.Experiment 3 was conducted to investigate the effects of maternal riboflavin on reproductive performance of maternal ducks and embryonic development of offspring.At 45 wk of age,a total of 80 female white Pekin ducks were randomly divided into two groups of 40 ducks each.The ducks received two experimental diets which supplemented the basal diet with 0 and 10 mg riboflavin/kg of diet for 8 weeks(riboflavin-deficient and control group).Maternal body weight,egg production,egg weight,egg fertility rate,and body weight of offspring did not differ in two groups.Plasma riboflavin from maternal ducks and egg yolk riboflavin content were decreased after 2-week riboflavin depletion in maternal ducks.The hatchability of eggs from the maternal ducks dropped dramatically after 2-week riboflavin depletion in maternal ducks,and dropped to approximately zero after 6-week riboflavin depletion.Comparisons of the relative abundance of proteins from the embryonic liver of maternal ducks fed the riboflavin-deficient diet with those fed the control diet showed that 67 proteins were up regulated and 120 down regulated.KEGG pathway showed that the differentially expressed proteins were enriched in TCA cycle,fatty acid beta oxidation,and electron transport chain processes.Maternal riboflavin deficiency downregulated proteins of embryonic liver involved in TCA cycle(DLD,SDHB,IDH1,SDHA,and ACO1),fatty acid beta oxidation(ETFDH,CPT1A,ACSL1,ACADS,ACAT1,ACSL5,DECR1,and ETFA),and electron transport chain(NDUFA9,NDUFS1,NDUFV1,NDUFA10,and ACAD9),which indicated these processes were impaired,leading to insufficient ATP production and virtually death of embryos.Experiment 4 was conducted to investigate the effects of riboflavin on cell proliferation and mitochondrial function of HepG2 cells,including 3 tests in vitro.Test 1 was conducted to investigate the effects of different riboflavin depletion period on mitochondrial function of HepG2 cells.HepG2 cells were cultured in riboflavin-deficient(RD)and riboflavin-sufficient(RS)medium for 2,4,6,8,10,and 12 days.Mitochondrial function of cells were measured at end of the experiment by Seahorse.Compared to RS group,maximal respiration and spare capacity were declined after 2-day riboflavin-depletion in RD group,and declined further with time,which indicates mitochondrial function was impaired.Test 2 was conducted to investigate the effects of different riboflavin concentrations on cell proliferation and mitochondrial function of HepG2 cells.HepG2 cells were cultured in mediums with 8 supplemental riboflavin concentrations(0,0.5,5,10,20,40,100,and 1064nmol/L)for 8 days.At 8 days,cell number,maximal respiration,and spare capacity of HepG2 cells were lower in the group with no riboflavin supplementation than the groups supplemented with riboflavin.These parameters increase as riboflavin increase,reached a plateau when riboflavin concentration increase to 10,20,and 20 nmol/L.Test 3 was performed to investigate whether riboflavin repletion can store cell growth and mitochondrial function of riboflavin-deficient cells.After deplete riboflavin for 8 days,HepG2 cells were cultured in mediums supplemented with 4 riboflavin concentrations(0,0.5,5,and 1064nmol/L)for 4 days.Compared to the cells without riboflavin supplementation,basal respiration,maximal respiration,and spare capacity were increased when supplemented with 5 nmol/L riboflavin;cell number,basal respiration,maximal respiration,and spare capacity were increased when supplemented with 1064 nmol/L riboflavin.These results showed that mitochondrial function of HepG2 cells was impaired after 2-day riboflavin-depeltion,while cell proliferation was decreased after 8-day riboflavin-depletion.Supplemented with 20nmol/L riboflavin in the medium is sufficient for mitochondrial function and cell proliferation of HepG2 cells.Mitochondrial function of riboflavin-deficient HepG2 cells can restore completely when replete riboflavin in mediums supplemented 1064 nmol/L riboflavin for 4 days,and cell proliferate rate was increased as well.In conclusion,riboflavin deficiency depressed embryonic development and growth of starter Pekin ducks,resulted in liver lipid accumulation.Liver proteome showed that riboflavin deficiency downregulated the proteins involved in fatty acid beta oxidation process(ACADS,ACAD9,and ETFDH),electron transport chain process(ACAD9 and NDUFS1),and TCA cycle(DLD),depressed lipolysis process,leading to liver lipid accumulation;blocked energy generation,resulted in growth depression.Furthermore,riboflavin deficiency impaired mitochondrial function in HepG2 cells,which can be restored by riboflavin supplementation,confirmed the data in vivo.