Study On Effects Of Replacing Fish Meal With Protein Hydrolysates On Groth,Antioxidation And Immune In Fish

Fish meal has been used as a major protein source in the aquafeed,which is high in protein,amino acid,unsaturated fatty acid and other micronutrients.During the past two decades,with the rapid development of aquaculture industry,the conflict between an increasing demand for fish meal and shortage of fish meal has become increasingly obvious.Therefore,it is crucial to reduce or substitute totally the use of fish meal in fish diet by replacing it with alternative protein sources,especially some protein sources that may be more sustainable and less expensive.Recently,it was demonstrated that protein hydrolysates(PH)include various bioactive peptides with different molecular weight,which peptides possessed various functional characteristics,such as antioxidant activities,enhancing immunity,antitumor,growth promotion,lipid reduction.Up to now,information on the application of PH in aquaculture focused on growth,body composition and biochemical parameter.However,there has been few studies on the antioxidation and immunoenhancement of bioactive peptides from PH in aquaculture animals.Therefore,the objective of the present study was to assess effects of replacing fish meal with protein hydrolysates on growth,antioxidative capacity and immune system in cyprinid fish.This study consisted of seven experiments as follows:1 Effects of replacing fish meal with cottonseed meal protein hydrolysate on growth and intestinal function of Megalobrama amblycephalaThis study evaluated effects of fish meal replacement by cottonseed meal protein hydrolysate(CPH)on growth and intestinal function of Megalobrama amblycephala.Fish(38.66 ± 0.08 g)were divided into five groups and fed with five isonitrogenous(320 g/kg crude protein)and isocaloric(17.8 MJ/kg gross energy)diets replacing fishmeal by CPH 0%(CPH0),1%(CPH1),3%(CPH3),5%(CPH5)and 7%(CPH7).Final weight and weight gain of fish fed CPH5 and CPH7 diets were lower than that of fish fed control diet(P<0.05).No significant(P>0.05)differences were found in FCR,CFI,HIS and VSI of fish among all groups.Protease and amylase,Na+,K+-ATPase,CK and γ-GT activities initially increased with supplementation levels increased up to 3%,then decreased significantly as further increasing supplementation levels.Microvillus length in anterior and mid intestine of fish fed CPH3 diets significantly higher than that of fish fed the control diet(P<0.05).The GH,GHR and IGF-I mRNA levels of fish fed CPH1 and CPH3 diets were significantly higher than that of fish fed the control diet(P<0.05).Dietary 5%and 7%CPH significantly increased the TNF-α,IL-6 and IKK-α mRNA levels(P<0.05),as well as decreased the IκB-α mRNA levels(P<0.05).Therefore,replacing fish meal with 3%CPH could strengthen digestive and absorptive function with exerting no negative effects on the growth performance of blunt snout bream.2 Replacing fish meal with cottonseed meal protein hydrolysate affects amino acid metabolism of Megalobrama amblycephalaThis study was conducted to evaluate effects of replacing fish meal with cottonseed meal protein hydrolysate(CPH)on amino acid metabolism of Megalobrama amblycephala.The experimental design is the same as the part 1.Dietary 3%CPH had no effect on plasma aspartate aminotransferase(GOT),alanine aminotransferase(GPT),succinate dehydrogenase(SDH)and xanthine oxidase(XOD)contents and liver SDH and XOD(P>0.05),but increased significantly hepatic GOT and GPT contents(P<0.05).The plasma threonine(Thr),valine(Val),lysine(Lys),histidine(His),total essential amino acids,total amino acids contents and thermal-unit growth coefficient(TGC)of fish fed CPH5 and CPH7 diets significantly decreased(P<0.05)compared with the control diet.Meanwhile,threonine(Thr),valine(Val),isoleucine(Ile)and leucine(Leu)contents in muscle significantly decreased(P<0.05)when fish fed CPH7 diet.Diets with 5%and 7%CPH significantly decreased target of rapamycin(TOR)and S6 kinase-polypeptide 1(S6K1)mRNA expressions levels in liver and gut,but increased eukaryotic translation initiation factor 4E-binding protein 2(4E-BP2)mRNA expression levels(P<0.05),as well as increased hepatic AMP-activated protein kinase α1(AMPKa-1),AMP-activated protein kinase α2(AMPKa-2)and sirtuin-1(SIRT-1)mRNA expressions levels and AMP-activated protein kinase α(t-AMPKα)protein contents.Overall,replacing fish meal with 5%and 7%CPH decreased the growth performance and amino acid catabolism,as well as inhibitedATP consumption via activating AMPK/SIRT1 pathway and inhibiting TOR signaling pathway.3 Effects of replacing fish meal with cottonseed meal protein hydrolysate on antioxidant capability and immunity of Megalobrama amblycephalaThis study aimed to assess effects of fish meal replacement by cottonseed meal protein hydrolysate(CPH)on antioxidant capability and immunity and its resistance to Aeromonas hydrophila.The experimental design is the same as the part 1.At the end of feeding trial,experimental fish per replicate were injected by Aeromonas hydrophila.Initially,after serial activation steps,LC50 was determined by intraperitoneal injection of 10 fish with graded concentrations(1×105 CFU/mL,1×106 CFU/mL,1×107 CFU/mL,1×108 CFU/mL and 1×109 CFU/mL)of A.hydrophila in advance,obtaining the 48 hours LC50 of 1×108 CFU/mL.After feeding experiment,20 fish from each group were injected intraperitoneally with 1mL/100g body weight of A.hydrophila using medical syringes.The mortality rate was recorded at 0h,6 h,12h,24h,48h and 96h after challenge.The results suggested that:Total superoxide dismutase(t-SOD)and catalase(CAT)contents of fish fed CPH1 and CPH3 diets were significantly(P<0.05)higher than that of fish fed the control diet.However,malondialdehyde(MDA)contents of fish fed CPH5 and CPH7 diets were significantly(P<0.05)higher than that of fish fed the control diet.Total protein,globulin,albumin,lysozyme and ACP contents of fish fed CPH3 diet showed the highest activity,which were significantly higher than that of fish in all the other groups(P<0.05).C3 and C4 level of fish fed CPH3 diet were higher than that of fish fed the control group(P<0.05).The relative expression levels of the immune-related genes(Leap-1and Leap-2)in liver and spleen of fish fed CPH1 and CPH3 diets were significantly(P<0.05)increased except for Leap-2 in liver of fish fed CPH1 diet.The lowest mortality rate was observed in fish fed CPH3 diet.Therefore,the diets supplemented with 1%and 3%CPH could enhance anti-oxygenic ability,and elevate immunity.4 Effects of replacing fish meal with yeast hydrolysate on growth performance and intestinal morphology of Cyprinus carpio var.JianA 10-week feeding trial was carried out to investigate effects of dietary fish meal replacement by yeast hydrolysate(YH)on growth performance and intestinal morphology of juvenile Jian carp(Cyprinus carpio var.Jian).Five isonitrogenous(340 g/kg crude protein)and isoenergetic(17.5 MJ/kg gross energy)diets replacing fish meal by YH 0%(YH0),1%(YH1),3%(YH3),5%(YH5)and 7%(YH7)were formulated.Replacement levels of YH significantly affected the growth of the fish with the highest values of weight gain(WG)occurred in fish fed YH3 diet.Furthermore,intestinal microvillus length of juvenile Jian carp fed YH3 diets were significantly higher than that of fish fed the control diet.The relative expression levels of intestinal inflammation-related genes(ALP,IL-1βand TNF-α)in YH7 were significantly higher than that in the control group(P<0.05).In proximal intestine,the expression levels of inflammation-related genes in YH3 and YH5 were lower compared with the control group,but exhibited no significant difference(P>0.05).However,in mid intestine,the expression levels of these genes in YH3 and YH5 were significantly lower compared to the control group(P<0.05).Therefore,replacing fish meal with YH at 3%could improve growth and intestinal healthy.5 Effects of replacing fish meal with yeast hydrolysate on antioxidation and immune system of Cyprinus carpio var.JianThis study aimed to evaluate the effects of fish meal(FM)replacement by yeast hydrolysate(YH)on liver antioxidation and immune system of juvenile Jian carp(Cyprinus carpio var.Jian).The experimental design is the same as the part 4.At the end of feeding trial,20 fish from each group were injected intraperitoneally with lmL/100g body weight of Aeromonas hydrophila using medical syringes.The concentration of Aeromonas hydrophila was 1×107 CFU/mL.Fish were sampled after 48h.The results shown that liver total superoxide dismutase(t-SOD),catalase(CAT),glutathione peroxidase(GPX)and glutathione(GSH)activities of fish fed YH1 and YH3 diets were significantly higher(P<0.05)compared to the control group(YH0).Pre-stressed plasma lysozyme activity,total protein and albumin contents and complement component 3(C3)and complement component 4(C4)levels of fish fed YH3 diet were significantly higher than those of fish fed the control diet.Besides,before stress,the expression levels of the inflammation-related genes(ALP,IL-1β and TNF-α)in the hepatopancreas and spleen of fish fed YH1 and YH7 diets were significantly higher than that of fish fed the control diet(P<0.05).However,after stress,the expression levels of these genes in YH diets showed no differences with those of fish fed the control diet(P>0.05).In the hepatopancreas and spleen,there was a trend that the expression levels of the complement-related genes initially increased and then decreased except for mbl-2 and masp,with the maximum values recorded in the fish fed YH3 diet.Besides,in these tissues,the expression levels of mbl-2 and masp in fish fed YH1,YH5 and YH7 diets were significantly lower than that in fish fed the control diet(P<0.05).In summary,the substitution of FM by 3%YH showed the optimal growth performance,activated complement system via ACP and CCP and improved the protection against A.hydrophila.6 Effects of cottonseed meal protein hydrolysate and yeast hydrolysate on antioxidant capability and immunity in hepatocytes of Megalobrama amblycephalaThis study was conducted to evaluate effects of cottonseed meal protein hydrolysate(CPH)and yeast hydrolysate(YH)on antioxidant capability and immunity in hepatocytes of Megalobrama amblycephala.The liver was carefully removed from the disinfected fish,and transferred into a sterile petri dish.Then,the liver was rinsed with 5 volumes of sterile PBS supplemented with streptomycin(100 μg/mL)and penicillin(100 IU/mL).The tissue was aseptically cut into 1mm3 pieces using the scalpel and scissors.After that,the liver was digested with the sterile trypsin at 37℃ for 40min.The hepatocytes were filtered through a cell strainer(70 μm)and collected in sterilized conical tubes.The isolated hepatocytes were centrifuged at 1000 r/min for 5 min,then washed twice using fresh DMEM/F12.Finally,the hepatocytes were diluted with DMEM/F12 medium to 105 cells/mL.3×105 primary hepatocytes were added into each well of 6-well plates and cultured for 48 h.The cells were divided into two groups with four replicates each group.The cells in control group were incubated with medium.The cells in the other group were incubated with CPH/YH.The CPH/YH were used at the concentration of 1g/L.After 24 hours,the culture media and primary hepatocytes were collected,respectively,for subsequent analysis.The results indicated that:CPH and YH had no effects on GOT,GTP and LDH activities,but increased significantly SOD and CAT activities,decreased MDA content in conditioned medium of hepatocytes(P<0.05).However,GOT and GTP contents of hepatocytes treated with CPH and YH were significantly higher than that in the control group(P<0.05).Besides,CPH and YH significantly up-regulated SOD,CAT,Leap 1 and Leap 2 mRNA levels(P<0.05),and down-regulated the expression of NOX2,Keap1,Nrf2 and Bach1 mRNA(P<0.05).Therefore,CPH enhanced hepatic cells metabolism,as well as improved immunity and antioxidant capacities of the primary hepatocytes of blunt snout bream via depression of Nrf2-Keap1 pathway.7 Discovery of antioxidation and innate immune of peptides from cottonseed meal protein hydrolysateThis study was designed based on that part 6 and aimed to explore a peptide with antioxidant activity and immunoenhancement from cottonseed meal protein hydrolysate(CPH)using cells experiment in vitro.Six different peptides(P1～P6)were selected and synthesized by Sangon Biotech(Shanghai)Co.,Ltd.(Shanghai,China).3×105 cells were seeded into each well of 6-well plates and cultured for 48 h.The cells were divided into six groups for testing six different peptides,and every group was assigned to three groups:control group(C),trial group(T)and amino acid group(A),four replicates each group.The cells in control group were incubated with medium.The cells in the trial group were incubated with peptide.The cells in the amino acid group were incubated with amino acids,the types and content of these amino acids were determined by the peptide sequence.After 24 hours,the culture media and primary hepatocytes were collected for subsequent analysis.The results suggested that:P1 and P2 significantly decreased GOT and GTP activities(P<0.05)in the conditioned medium.P4 had no effects on GOT and GTP activities(P>0.05),but increased SOD and CAT activities in the conditioned medium(P<0.05).No difference in MDA content among all groups were observed except for that in the trial group with P4.P2 and P6 had no effects on GOT activity in the hepatic cells,but P4 increased GOT and GTP activities(P<0.05),up-regulated SOD,CAT,Leap1 and Leap2 mRNA(P<0.05),down-regulated the expression of NOX2 and Nrf2 mRNA(P<0.05).Therefore,P4 is a bioactive peptide with antioxidation and immune-enhancement.