| The blunt snout bream(Megalobrama amblycephala)is one of the peculiar economic freshwater fishes in China.The aquaculture industry is under increasing pressure to include higher levels of carbohydrates in diets,as this an effective way to minimize the costs of the diet.However,high-carbohydrate diets can produce negative impacts on growth and induce liver damage.As an effective methyl donor in animal organism,betaine supplemented in diet can improve growth performance,body composition,immunity,texture of flesh,gut microbial community,etc.Therefore,it is important to understand the betaine metabolism and the impacts of betaine on animals.A few studies have examined the effects of betaine supplementation on the gut microbial community composition and metabolism in M.amblycephala.Results suggested that high-carbohydrate diet decreased the content of hepatic betaine in M.amblycephala,but the mechanisms behind this decrease remain unknown.In this context,firstly,via a thorough study of the changes in growth performance,liver health status,metabolites in plasma,and expression of relative genes on juvenile M.amblycephala,we explored the mechanisms behind the high-carbohydrate diet-driven decrease in the content of hepatic betaine,and impacts on the growth of fish.Secondly,via a thorough exploration of impacts on the gut microbial community composition,TMA formation,TMAO metabolism and bile acids metabolism in high-carbohydrate and betaine diets,we explored the effects of betaine supplementation on TMAO metabolism and the development of NAFLD.The main results are:1.The specific mechanism via which a high-carbohydrate diet reduces the liver betaine contents in M.amblycephalaFour feeding trials[control(CD),high-carbohydrate(HCD),16-week betaine supplementation(LBD or HCB,1%of betaine),and 4-week betaine supplementation(SBD or HC4B)]were designed to explore the changes in growth performance,liver health status,metabolites in plasma and expression of relative metabolic enzyme genes in juvenile M.amblycephala.Initial body weight of fish is 5.47±0.085g in this trail.Results indicate:HCD significantly decreased the final body weight(BW~F),weight gain(WG)and specific growth rate(SGR),and significantly increased feed conversion ratio(FCR),hepatosomatic index(HSI)and crude lipid in the whole-body(p<0.05).HCB increased the BW~F,WG and SGR(p<0.05),decreased the HSI and crude lipid in the muscle(p<0.05).The results of liver histology indicated that exhibited elevated lipid accumulation in the HCD group,and decreased in the LBD group.The metabolites in plasma and relative gene expression levels revealed that:in the HCD group,compared with the CD group,S-adenosylmethionine(SAM)was increased significantly(p<0.05);whereas betaine,methionine,S-adenosylhomocysteine(SAH)and carnitine were decreased significantly(p<0.05).In the LBD group,compared with the HCD group,homocysteine,betaine,methionine,glycine,SAM,SAH and carnitine were increased significantly(p<0.05).In the SBD group,homocysteine,methionine,dimethylglycine,glycine,SAM,and carnitine were significantly lower compared with the LBD group(p<0.05).In the HCD group,compared with the CD group,the expression of gk,pfk1,serc,psph and cbs was increased significantly(p<0.001);whereas bhmt,metk and ahcy were not significantly different(p>0.05).In the LBD group,compared with the HCD group,the expression of bhmt,metk and ahcy was increased significantly(p<0.05),whereas that of gk,pfk1,serc,psph and cbs was decreased significantly(p<0.001).In the SBD group,compared with the LBD group,the expression of gk,serc,psph,bhmt and ahcy was increased significantly(p<0.05).These results indicate that the reduced content of hepatic betaine in response to a high-carbohydrate diet was caused by the increased amount of homocysteine metabolized from glucose(including glycolysis,serine metabolism,cystathionine metabolism and homocysteine remethylation),which in turn resulted in increased metabolic betaine consumption.On one hand,betaine addition improved the growth indices and liver condition,and lowered the lipid content in the whole-body and muscle.On the other hand,betaine supplementation enhanced the betaine metabolism,homocysteine metabolism,methionine methylation,transmethylation and homocysteine cycle.Short-term supplementation of 1%betaine to the high-carbohydrate diet-fed fish produced intermediate effects,but some parameters indicate that this fish species goes through a period of adjustment to a diet with supplemented betaine,during which its metabolic homeostais is somewhat disrupted.2.The effects of dietary carbohydrates and betaine on the gut microbial community composition,TMAO and bile acids metabolismAfter the feeding trial described above,we tested the diversity of gut microbial community and the abundance of bacterial taxa that may be associated with dietary carbohydrates and betaine.We also studied the metabolites in plasma and expression of related genes,to explore the impacts of dietary carbohydrates and betaine on the metabolism and health of this fish.Microbial community analysis results after HaeIII digestion indicated that the richness and stability index H′significantly decreased in the HCD group(p<0.05;compared to the CD group),and significantly increased in the HCB and HC4B groups(p<0.05;compared to the HCD group).MspI digestion results indicated that only H′significantly decreased in the HCD group(p<0.05).In the cluster analysis,HCB and HC4B samples were grouped in a separate subcluster at the T-RF level both in HaeIII and MspI datasets.The HCD group exhibited a highly significant increase in the abundance of Lactobacillus sp.(p<0.05),and a highly significant decrease in the abundance of Clostridium sp.and the expression of cutc gene(p<0.05).The HCB group exhibited a highly significant increase in expression of grdh(p<0.05),but a significant decrease in the abundance of Lactobacillus sp.and the expression of cnta(p<0.05),compared with the HCD group.Metabolites in plasma analysis indicated that both betaine and TMA levels were significantly decreased in the HCD group(p<0.05).This effect(decrease)was slightly less pronounced in the HC4B group,and much less pronounced in the HCB group.TMAO levels were also significantly reduced in these two groups(p<0.05).In the HCD group,the levels of Glu and Lys were significantly lower in comparison to the CD group(p<0.05).In comparison to the HCD group,the levels of 7 out of 12 studied amino acids(Ala,Gln,Glu,Ile,Leu,Pro,and Val)were significantly decreased in the HCB group(p<0.05);whereas 5(Gln,Ile,Leu,Trp and Val)were significantly decreased in the HC4B group.Expression levels of related genes showed that:compared to the CD group,the expression of cyp7a1,cyp27a1,oapt1 and fxr were highly significantly upregulated in the HCD groups(p<0.05),whereas the expression of mrp2 and tgr5 was decreased significantly(p<0.05).Compared to the HCD group,the expression of fmo3,cyp7a1,cyp27a1,bsep and tgr5was highly significantly increased in the HCB groups(p<0.05),whereas the expression of oatp1 was decreased significantly(p<0.05).The all the results showed:(i)high carbohydrates(starch)produced negative effects on the diversity of gut microbiota community and decreased the anaerobic choline metabolism and TMA levels,but betaine improved both parameters;(ii)betaine increased the fmo3 expression,but decreased the circulating concentrations of TMAO;(iii)high carbohydrates impaired the bile acids metabolism and induced NAFLD,whereas betaine supplementation reversed that trend;(iv)betaine may have reversed the negative effects of high dietary carbohydrates by decreasing their utilization.In conclusion,high-carbohydrate diet reduced the content of hepatic betaine,and produced negative impacts on the growth performance,liver condition,gut microbial community composition and bile acids metabolism,whereas dietary supplementation of betaine improved these parameters.All this indicates that a proper level of betaine in diets is good for health culture in M.amblycephala. |
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