| Fish meal has been widely used in aquaculture industry,however,its application is limited due to the lack of resources and rising prices.Soybean meal is one of the most important protein sources in aquatic animal feed.Studies on the substitution of soybean meal for fish meal have focused on the inhibition of fish growth,digestion and absorption by soybean meal at high dose,and intestinal inflammation.The mechanism of soybean meal on fish intestinal health has not been fully clarified,and the effect of soybean meal on fish intestinal energy metabolism has not been reported.Therefore,the application of soybean meal in aquatic feed is still limited.Alpha-ketoglutaric acid（AKG）has been partially studied in fish,which has the function of alleviating intestinal injury.However,its effect mechanism on fish intestinal health has not been clarified,and as an intermediate substance in the tricarboxylic acid cycle and a precursor substance of glutamine,The effect on intestinal energy metabolism of fish has not been reported.Songpu Mirror carp was used as the research object.The control group（FM）was fed a30.8% fish meal diet,and the test group 1 was fed a soybean meal group（SM,40% soybean meal）.Trial group 2 was AKG group（40% soybean meal +1% AKG）,trial group 3（Metf）and trial group4（Com）were supplemented with 300 mg/kg Metformin（Metf）and 0.2 mg/kg AMPK inhibitor compound-C（Com）on the basis of trial group 1,respectively.Test group 5（Metf+AKG）and test group 6（Com+AKG）were supplemented with 300 mg/kg Metf and 0.2 mg/kg Com on the basis of test group 2,respectively.The experiment lasted for 59 days.Based on the intestinal energy metabolism of carp and metabonomic analysis,this paper explored the effect mechanism of soybean meal on intestinal health and the mechanism of α-ketoglutaric acid alleviating intestinal injury of carp.The combination of the two will provide theoretical value for the application of soybean meal and α-ketoglutaric acid in aquatic feed.The results showed:1.Compared with FM group,weight gain rate（WGR）,specific growth rate（SGR）and hepato-somatic index（HSI）in experimental groups were significantly decreased（P<0.05）,the feed conversion rate（FCR）was significantly increased（P<0.05）,and the condition factor（K）in AKG group was significantly increased（P<0.05）.Crude protein content in all experimental groups had no significant difference（P>0.05）,while water content in AKG group was significantly increased（P<0.05）.2.The intestinal villi of proximal intestine（PI）in FM group and AKG group were compact and orderly,the intestinal mucosal epithelial integrity of PI was damaged in SM group,and the intestinal villi of PI in Metf group were compact and orderly.Compared with FM group,crypt depth（CD）of middle intestine（MI）and mucosal thickness（MT）of distal intestine（DI）in SM group were significantly decreased（P<0.05）.Compared with SM group,MT of DI and CD of PI in AKG group were significantly increased（P<0.05）.The MT of PI and MI in Metf group was significantly higher than that in SM group（P<0.05）,and the CD of PI in Metf+AKG group was significantly higher than that in SM group（P<0.05）.3.In the detection of intestinal gene expression,compared with FM group,AMPK-α,TOR and ACC of PI were significantly decreased in SM group（P<0.05）;TNF-α,IL-1β and TGFβ2 in PI,IL-1β,TGFβ1 in MI and TGFβ1 in DI were significantly decreased（P<0.05）,and TNF-α in DI was significantly increased（P<0.05）.Caspase9 in PI,Caspase8 in MI and Caspase9 in DI were significantly decreased（P<0.05）.Claudin3 c,Claudin7,Claudin11 and Occlaudin in PI and Claudin7 and Occlaudin in MI were significantly decreased（P<0.05）.Claudin3 c,Claudin11 and Occlaudin in the DI were significantly increased（P<0.05）.Compared with SM group,TOR of PI and MI in group AKG was significantly increased（P<0.05）.TNF-α in MI was significantly decreased（P<0.05）,and TGFβ1 in PI was significantly increased（P<0.05）.Claudin7 and Claudin11 in PI were significantly increased（P<0.05）,Claudin11 in MI（P<0.05）and Claudin11 and Occlaudin in DI were significantly decreased（P<0.05）.4.There were no significant differences in ATP,ADP and AMP contents in PI and MI among experimental groups（P>0.05）.Compared with FM group,ATP and ADP levels in DI of SM group were significantly increased（P<0.05）,but energy charge levels in PI and MI of SM group had a trend of decrease（P>0.05）.There were no significant differences in ATP,ADP and AMP contents and energy charge levels between AKG and SM groups（P>0.05）.5.1332 differential metabolites were found by intestinal mucosal metabonomics analysis in total.Enrichment analysis of metabolic pathways showed that the different metabolic pathways in SM group and FM group for 7 d included sphinolipid metabolism,necroptosis,apoptosis,glycerophospholipid metabolism,cholesterol metabolism and so on.The different metabolic pathways of SM group and FM group for 59 d included protein digestion and absorption,aminoacyl t RNA synthesis,sphinolipid metabolism,ABC transporters,m TOR signaling pathway,Fox O signaling pathway,necroptosis,cholesterol metabolism and so on.The metabolic pathways of the Metf group and FM group on 7 d were as follows: protein digestion and absorption,aminoyl t RNA synthesis,sphinolipid signaling pathway,necroptosis,and arginine synthesis and so on.The different metabolic pathways of Metf group and FM group on 59 d included ABC transporters,m TOR signaling pathway,Fox O signaling pathway,protein digestion and absorption,necroptosis,arginine and purine metabolism,sphinolipid signaling pathway,etc.The metabolic pathways of the Metf group and SM group on 7 d were as follows: apoptosis,necroptosis,D-glutamine and D-glutamate metabolism,sphingolipid signaling pathway,tricarboxylic acid cycle,thyroid hormone synthesis.The different metabolic pathways of Metf group and SM group for 59 d included protein digestion and absorption,amino sugar and nucleoside sugar metabolism,ABC transporters,aminoyl t RNA synthesis,apoptosis and necroptosis.The metabolic pathways of Com group and FM group on 7 d were as follows: protein digestion and absorption,aminoacyl t RNA synthesis,ABC transporters,sphingolipid signaling pathway,necroptosis,apoptosis,and arginine synthesis and so on.The differences in metabolic pathways between Com group and FM group for59 d included m TOR signaling pathway,Fox O signaling pathway,ABC transporters,sphingolipid signaling pathway,necroptosis,arginine and purine metabolism and so on.The metabolic pathways of Com group and SM group on 7 d were Fox O signaling pathway,AMPK signaling pathway,purine metabolism,and oxidative phosphorylation and so on.The different metabolic pathways of Com group and SM group on 59 d included ABC transporters,apoptosis,necroptosis,D-glutamine and D-glutamate metabolism,sphingolipid signaling pathway and so on.Conclusion:1.Soybean meal reduced the growth performance of carp,had the highest damage degree to distal intestinal mucosa,reduced the energy metabolism level of proximal and middle intestine,increased mitochondrial fission and aggravated oxidative damage by inhibiting AMPK pathway and Fox O signaling pathway;by reducing the utilization of amino acids in intestinal tract,soybean meal reduced the content of arginine,which inhibited TOR signaling pathway and reduced the tight junction protein expression in proximal and middle intestine.At the same time,inhibition of ACC expression in proximal and middle intestine resulted in increased lipid catabolism.Soybean meal mainly causes distal intestinal inflammation.2.AKG can improve the expression of tight junction protein by activating TOR and ACC signaling pathways,inhibit endogenous cell apoptosis,repair intestinal inflammation caused by soybean meal,promote intestinal development,improve intestinal digestive enzyme activities and intestinal health. |
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