| 1.Effects of hydrolysable tannin on feeding performance,nutrition digestion and antioxidant ability of obscure puffer(Takifugu fasciatus)The possible roles of tannin on the obscure puffer(Takifugu fasciatus;initial weight 39.84 ± 3.09 g)were evaluated.Total 720 fish were fed with four isonitrogenous and isoenergetic semi-purified diets for eight weeks: control diet(OT0)with hydrolysable tannin-free protein sources(containing 43% fish meal)and three experimental diets supplemented with 0.25%,0.75%,and 1.25% hydrolysable tannin(called OT1,OT2,and OT3,respectively).In experimental diets,crude protein content was balanced by casein when fish meal level decreased,and the ratio of casein to tannin was nearly equal to that of protein content to tannin content in rapeseed meal(16.8:1).The results showed that the weight gain rate were not influenced,while feed coefficient ratio decreased significantly(P<0.05)with increasing tannin level;the muscle crude fat of OT2 was significantly higher than OT0 and OT1,and the amino acid level of OT0 was significantly higher than other groups(P<0.05);for liver antioxidant index,the activity of superoxide dismutase(SOD)and the content of malonaldehyde(MDA)in OT3 was significantly higher than other groups(P<0.05),total antioxidative capacity(T-AOC)in OT0 was significantly higher than other groups(P<0.05);for serum antioxidant index,the activity of SOD showed no significantly difference,while the content of MDA in OT0 was significantly lower than other groups(P<0.05),and the activity of catalase(CAT)decreased with increasing tannin level;for serum biochemical indices,the activity of GPT increased,and then decreased at OT2,paralleling the result of GPT in liver,the content of total protein(TP)in OT2 and OT3 were significantly lower than other groups(P<0.05);the activity of amylase in intestine increased and then decreased in OT3,the activity of protease increased with increasing tannin level in both stomach and intestine(P<0.05),the activity of lipase decreased in stomach(P<0.05);the expression of T2R1 m RNA in OT3 was significantly higher than OT0 both in tongue and intestine(P<0.01);the expression of HSP70 m RNA in liver in OT0 was significantly higher than OT3(P<0.01).Furthermore,the preference decreased with increasing tannin level before feeding test(P<0.05),while after 8-week domestication,the fish preferred to diet containing 0.75% tannin in all groups(P<0.05).In conclusion,feeding obscure puffer with diet containing no more than 1.25% tannin would not decrease growth and decrease feed coefficient ratio,but when tannin was higher than 1.25%,the digestion and metabolism of dietary protein were influenced,and the liver health and antioxidant ability were injured.In addition,domesticating obscure puffer with hydrolysable tannin-containing diet could enhance the feeding preference to the diet containing 0.75% hydrolysable tannin and increase the receptivity to bitter.2.Intestinal health and antioxidant status in obscure puffer(Takifugufasciatus)fed with rapeseed mealIn order to evaluate the effect of dietary rapeseed meal(RM)on obscure puffer,four isonitrogenous and isoenergetic diets were formulated with 0,10%,20%,and 30% RM respectively(OR0,OR30,OR50 and OR70,these diets contained similar level of tannin with the diets used in chapter one).After 56 d feeding trial,the weight gain rate of OR0 and OR10 were significantly(P<0.05)higher than OR30,the feeding conversion ratio showed opposite trend(P<0.05).In liver,the malonaldehyde content and catalase activity of OR20 and OR30 were higher than OR0 and lowest in OR10,superoxide dismutase activity was significantly(P<0.05)lower in OR0 and OR10 than other groups,total antioxidative capacity and glutathione increased with RM level,while glutathione peroxidase activity decreased(P<0.05).In serum,the activities of catalase and superoxide dismutase and the content of total protein and albumin decreased with RM level(P<0.05),while the malonaldehyde content and glutamic-pyruvic transaminase activity increased(P<0.05),the total protein in OR0 and OR30 were significantly higher than other groups(P<0.05),glutamic-pyruvic transaminase and glutamic oxalacetic transaminase activities in OR20 and OR30 were higher than other groups(P<0.05).In intestine,malonaldehyde content increased,while superoxide dismutase increased and then decreased(P<0.05).The catalase activity in OR0 and OR10 were higher than other groups(P<0.05).In hindgut,the height of the intestine folds in OR0 was higher than OR10 and R30(P<0.05),and the histomorphology was injured in OR30.The relative expression of TGF ?1 and IL-1 in intestine upregulated with RM level(P<0.05),the expression of IL-8 in OR20 was significantly higher than OR10,and lowest in OR0 and OR30(P<0.05),while HSP70 in liver decreased(P<0.05).The relative expression of intestinal TNF? in OR30 was higher than other groups(P<0.05).These results demonstrated that 20% or more dietary RM induced oxidative stress,damaged the hindgut structure and resulted in intestinal inflammation in obscure puffer.3.A GC-MS-based metabolomic assessment of obscure puffer in response to dietary rapeseed mealIn order to study the metabolomics responses of rapeseed meal on obscure puffer,the fish in chapter two were used to detect more parameters.The results showed that the muscle protein content in OR30 was significantly lower than other groups(P<0.05),the muscle lipid content were significantly decreased with RM level(P<0.05),while ash was opposite(P<0.05).The percentage of muscle essential amino acid in OR0 was significantly higher than other groups(P<0.05).In liver,the GPT activity decreased,GOT activity of OR0 was significantly higher than other groups(P<0.05),and MDA content decreased and then increased in OR20(P<0.05).In serum,the total protein,albumin and HDL-C in OR0 and OR10 were significantly higher than OR20 and OR30(P<0.05).The UN content in OR30 was significantly higher than OR0(P<0.05),and lowest in R10 and R20.Triglyceride and cholesterol increased significantly with RM(P<0.05).The LDL-C content in OR30 was significantly higher than OR20,OR20 was higher than OR0 and lowest in OR10(P<0.05).The intestinal activities of amylase,protease and lipase decreased significantly(P<0.05).In stomach,the amylase activity of OR20 was significantly higher than OR10 and OR30 and lowest in OR0(P<0.05),the lipase activity decreased(P<0.05),but the protease activity was opposite(P<0.05).The relative expression of GDH1 in intestine significantly upregulated with RM(P<0.05).In liver,the relative expression of FAS in OR20 was significantly higher than OR0 and lowest in OR10 and OR30(P<0.05).The relative expression of LPL in OR10 was lower than OR0,OR30 was significantly higher than OR20,and OR20 was higher than OR10(P<0.05).The relative expression of CTP1? in OR10 was significantly(P<0.05)higher than OR30,and lowest in OR0 and OR20.The expression of intestinal GDH1 upregulated with RM level(P<0.05).In metabolomics assay,29 differential metabolites(SDMs)were identified between OR0 and OR10,22 metabolites were down-regulated and 7 metabolites were up-regulated in the OR10 compared to the OR0.57 SDMs were identified between OR0 and OR30,41 metabolites were down-regulated and 16 metabolites were up-regulated in the OR30 compared to the OR0.31 SDMs were identified between OR10 and OR30,26 metabolites were down-regulated and 5 metabolites were up-regulated in the OR10 compared to the OR30.Part of these SDMs are related with lipid and protein metabolism.These indicated that 20% dietary rapeseed meal suppressed the absorption and biosynthesis of proteins,and the absorption of lipid was adversely influenced,these may be related with the change of metabolism pathways: TCA cycle,Cysteine and methionine metabolism,Tyrosine metabolism,Nicotinate and nicotinamide metabolism,Valine,leucine and isoleucine biosynthesis,Glycine,serine and threonine metabolism,Alanine,aspartate and glutamate metabolism.4.Hepatopancreas and intestinal health and antioxidant status in grass carp(Ctenopharyngodon idellus)fed diets contained hydrolysable tanninIn order to evaluate the effect of dietary rapeseed meal(RM)and hydrolysable tannin on the health of grass carp,eight isonitrogenous and isoenergetic diets were formulated,four of them were semi-purified diets with 0,0.75%,1.25%,and 1.75% tannin respectively(GT0,GT1,GT2 and GT3),and the other four were practical diets containing 0,30,50,and 70% RM respectively(GR0,GR30,GR50 and GR70),the tannin contents in practical diets were similar to semi-purified diets.After 56 d feeding trial,in semi-purified groups,no significant difference was observed in survival rate and weight gain rate(P>0.05),while the feeding conversion ratio and feed rate of GT2 were significantly higher than GT0(P<0.05).In hepatopancreas,the MDA content increased significantly and decreased in GT3(P<0.05),SOD activity was significantly lower in GT0 and GT1 than other groups(P<0.05),CAT activity in GT30 was significantly higher than GT0(P<0.05),the T-AOC of GT1 was higher than GT0 and decreased in GT2 and GT3(P<0.05).In serum,the SOD activity increased and then decreased in GT3(P<0.05),the GOT activity showed an inverse relationship with SOD(P<0.05),and the GPT activity of GT2 was significantly higher than GT1(P<0.05).The lysozyme activity in head kidney,MDA content and SOD activity in intestine significantly increased with tannin level(P<0.05),while the GPx activity and the GSH content in both intestine and hepatopancreas showed an inverse relationship(P<0.05).In hindgut,the height of intestine folds significantly decreased with tannin levels,and the histomorphology was injured.The relative expression of IL-8m RNA and Nrf2 m RNA in intestine of GT0 was significantly lower than other groups(P<0.05),IL-10 m RNA upregulated with tannin(P<0.05).The relative expression of Keap1 in GT2 was significantly higher than GT1 and lowest in GT0(P<0.05).In practical groups,no significant difference was observed in survival rate and weight gain rate(P>0.05),while the feeding conversion ratio in GR70 was significantly higher than other groups(P<0.05).In hepatopancreas,the content of MDA in GR50 was significantly(P<0.05)higher than other groups,the activity of SOD in GR70 was significantly(P<0.05)higher than other groups,the activity of CAT of GR50 and GR70 were higher than R0 and R30,the activity of T-AOC of R50 was higher than R30 and lowest R0 and R70(P<0.05).The content of GSH showed an inverse relationship with dietary RM(P<0.05).The activity of GPx increased and then decreased in R50 and R70(P<0.05).In serum,the activity of SOD increased with dietary RM(P<0.05),while the activity of T-AOC decreased significantly(P<0.05),the activity of GPT in GR70 was significantly lower than other groups(P<0.05),the activity of GOT in GR70 was significantly higher than GR30,and lowest in GR0.In intestine,the MDA content and the SOD activity increased significantly(P<0.05),while the GPx activity and the GSH content decreased(P<0.05).In head kidney,the activity of lysozyme in GR50 and GR70 were significantly(P<0.05)higher than GR0 and GR30,and the activity of ACP decreased(P<0.05).In the hindgut,the height of the intestine folds of GR50 group was higher than GR0(P<0.05),and the histomorphology was injured.In the intestine,the relative expression of IL-8m RNA in GR70 was significantly higher than other groups(P < 0.05),and IL-10 m RNA upregulated as dietary RM increased(P < 0.05).The relative expression of Nrf2 m RNA in R0 was significantly higher(P < 0.05)than GR30 and GR50,while Keap1 m RNA in R30 was significantly(P < 0.05)higher than GR50,GR50 was higher than GR70 and lowest in GR0.This study demonstrated that 50% dietary RM and 0.75% tannin induced oxidative stress,injured antioxidant ability,damaged the hindgut structure and resulted in intestinal inflammation in grass carp.5.Hydrolysable tannin supplementation alters digestibility and utilization of dietary protein,lipid,and carbohydrate in grass carp(Ctenopharyngodon idellus)In order to study the effect of dietary hydrolysable tannin on nutritional metabolism in grass carp,the fish of the semi-purified-diet groups in chapter four were used to detect more parameters.The results showed that the muscle protein content of GT0 and GT3 were significantly higher than GT2 and lowest in GT1(P<0.05),while lipid content of GT0 was significantly higher than other groups(P<0.05).Muscle and hepatic glycogen in GT0 were significantly lower than other groups(P<0.05).The percentage of muscleuscle saturated fatty acids in GT3 were significantly higher than GT0,and lowest in GT1 and GT2(P<0.05),the monounsaturated GT3 was significantly higher than other groups,while the poly-unsaturated fatty acids in GT1 and GT2 were higher than GT0 and lowest in GT3(P<0.05).Significant increases were obtained in trypsin and amylase activities as tannin levels increased(P<0.05),the lipase activity of GT0 and GT1 was significantly higher than GT2 and GT3(P<0.05).Activities of hepatic GPT decreased with increasing tannin level(P<0.05),the activity of GOT in GT2 and GT3 were significantly lower than other groups(P<0.05).The TP in serum of GT2 was significantly higher than GT0 and GT1 and lowest in GT3(P<0.05),whereas globulin of GT2 was significantly higher than GT0 and GT3 and lowest in GT1(P<0.05),while ALB of GT1 was significantly higher than other groups(P<0.05).Urea nitrogen of T0 was significantly higher than other groups(P<0.05),triglyceride and total cholesterol significantly increased with tannin level and decreased in GT3(P<0.05),significant decreases were obtained in LDL-C in GT3(P<0.05).m RNA expression of intestinal TOR was significantly upregulated as dietary tannin increased(P<0.05).In hepatopancreas,the expression of glucokinase in GT1 was significantly higher than GT2,GT2 was higher than GT0,and lowest GT3(P<0.05),pyruvate kinase in GT2 was significantly higher than GT0 and GT1 and lowest GT3(P<0.05).The expression of lipoprotein lipase was upregulated as tannin levels and then downregulated in GT3(P<0.05),and that of fatty acid synthase downregulated as tannin levels(P<0.05).In conclusion,grass carp could tolerate 1.75% dietary tannin without influencing growth.However,1.25% tannin impaired digestion and metabolism of protein,decreased the deposition of lipid and promoted utilization of carbohydrate.6.Rapeseed meal alters digestibility and utilization of dietary nutrition in grass carp(Ctenopharyngodon idellus)In order to study the effect of dietary RM on nutritional metabolism in grass carp,the fish of the practical-diet groups in chapter four were used to detect more parameters.The results showed that the muscle protein and lipid content decreased(P<0.05),while ash and muscle glycogen significantly increased(P<0.05).The percentage of muscle saturated fatty acids in GR50 were significantly lower than GR70 and GR30,and highest in GR0(P<0.05),the monounsaturated GT0 was significantly lower than other groups,while the poly-unsaturated fatty acids in GR50 was higher than GR0,GR0 was higher than GR70 and GR30(P>0.05).In intestine,the activity of trypsin and amylase increased significantly with RM level(P<0.05),the activity of amylase in GR50 was significantly higher than GR70,and lowest in GR0 and GR30(P<0.05),while lipase activity decreased(P<0.05).In hepatopancreas,the activities of GOT decreased,GPT decreased and increased in GR70(P<0.05),and hepatic glycogen in GR0 was lower than other groups(P<0.05).In serum,the content of total protein in GR50 was significantly higher than other groups(P<0.05),the album in GR50 and GR70 were significantly lower than other groups(P<0.05),while globulin showed opposite result(P<0.05).The urea nitrogen content decreased with RM level(P<0.05),the content of triglyceride decreased and increased in GR70(P<0.05),total cholesterol increased in GR30 and then decreased(P<0.05).The LDL-C content of GR30 was significantly higher than GR50,and lowest in GR0 and GR70,while HDL-C in GR70 was significantly lower than other groups(P<0.05).The relative expression of intestinal TORm RNA in GR0 was significantly higher than GR70,lowest in GR30 and GR50(P<0.05).In hepatopancreas,the expression of GK in GR50 was significantly higher than GR0 and GR30,and lowest in GR70(P<0.05),the expression of PK in GR30 was significantly higher than other groups(P<0.05),the expression of LPL upregulated with RM level and downregulated in GR70(P<0.05),the expression of FAS in GR0 and GR50 were significantly higher than GR70 and GR30(P<0.05).In conclusion,30% RM may improve the utilization of carbohydrate.But 30% and more RM in diet would injure the metabolism of protein,while 50% RM would promote the utilization of lipid and reduce lipid deposition.7.Different metabolomics responses and intestinal microbiota of grass carp(Ctenopharyngodon idellus)to dietary tannin and rapeseed mealIn order to study the metabolomics responses of rapeseed meal on grass carp and the role of inclusive tannin playing,grass carp were fed four diets for a 56-day trial.Two were practical-diets: GR0 diet contained 10% fishmeal without rapeseed meal,GR50 diet contained 50% rapeseed meal without fishmeal;the other two were semi-purified diets without(GT0)or with 1.25%(GT2)of supplemental hydrolysable tannin.The tannin in the GR50 diet was close to GT2.Weight gain rate of GR50 was significantly lower than GR0(P<0.05),while feed conversion ratio in GT2 was significantly higher than GT0(P<0.05).The muscle lipid and protein content were significantly lower in GR50 and GT2 compare to the other treatments respectively,while glycogen was opposite(P<0.05).The muscle PUFAs percentage in GT2 was significantly higher than GT0(P<0.05),while SFAs in GR50 were lower than GR0(P<0.05).In intestine,the activities of trypsin and ?-amylase were significantly higher in GT2 and GR50 groups compared with the other treatments(P<0.05),while lipase showed opposite trend (P<0.05).The hepatic activities of alanine aminotransferase and aspartate aminotransferase were lower in GT2 and GR50 than the other treatments(P<0.05),but the hepatic glycogen and malonaldehyde were higher(P<0.05).The total serum protein,globulin and triglyceride were significantly higher in GT2 and GR50(P<0.05).In metabolomics assay,29 differential metabolites were identified between GT0 and GT2,23 metabolites were downregulated and 6 metabolites were upregulated in GT2 compare to GT0.92 differential metabolites were identified between GR0 and GR50,31 metabolites were downregulated and 61 metabolites were upregulated in GR50 compare to GR0.High-throughput sequencing showed that Proteobacteria,Firmicutes,and Actinobacteria were the dominant bacterial phyla among groups.The intestinal microbial diversity was higher in GR50 and GT2.Redundancy analysis showed that tannin,rapeseed meal,and intestinal trypsin activity were positively or negatively correlated with the relative abundance of several different intestinal microbiota at phylum and/or genus levels.The results indicated that 1.25% tannin could not be the main reason for the poor growth of grass carp of the GR50 group;however,the protein metabolism was disturbed,while the lipid utilization and carbohydrate metabolism might be improved.The abnormal protein and lipid metabolism maybe partly caused by tannin in rapeseed meal,and the tannins contained in rapeseed meal seemed to bring more complex effects than tannin additive.These may be related with the change of metabolism pathways: beta-Alanine metabolism,Cysteine and methionine metabolism,Glycine,serine and threonine metabolism,Glutathione metabolism,Arginine and proline metabolism,Valine,leucine and isoleucine biosynthesis,Biosynthesis of unsaturated fatty acids.Furthermore,tannin and rapeseed meal supplementations modulated the intestinal microbiota,and may sequentially regulate the intestinal function by fermenting dietary nutrition,producing digestive enzymes,and modulating probiotics. |
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