Effects Of Feeding Broad Bean On Liver Fat Accumulation,Lipid Metabolism And Muscle Quality In Grass Carp(Ctenopharyngodon Idellus)

At present,the culture of crisp grass carp is the pillar industry of Zhongshan City in Guangdong Province.It is deeply loved by consumers because of its tight and crisp meat.However,in the feeding process,the muscles of the crisp grass carp tend to be semi-brittle,brittle and swollen;and the internal organs are prone to severe congestion during artificial pull nets or fish high-speed cruises;long-term feeding of the crispy meat of the broad bean,the liver will accumulate fat.It has been pointed out that excessive accumulation of fat in the liver will not only reduce the growth rate,immunity and disease resistance of the fish,but also reduce quality of the muscles and affect the taste.Therefore,these pathological phenomena have made a serious impact on the development of the crisp grass carp industry.So far,most research has focused on the comparative analysis of nutrient composition,texture characteristics and microstructure of the muscles of the crisp grass carp,but little research on the physiological and pathological changes of the crisp grass carp.In this paper,the effects of feeding broad bean?4 months?on the physiology and biochemistry of grass carp?broad bean group?were discussed from three aspects:blood physiology and biochemistry,liver lipid accumulation and lipid metabolism,and muscle physical and chemical properties.The results were compared with those of grass carp?control group?fed with common feed in order to provide basic research data for healthy culture of crispy grass carp.The following experiments were carried out as follows:1.Effects of feeding broad bean on blood physiological and biochemical characteristics of grass carpIn order to investigate the effects of feeding broad bean on the physiological and biochemical characteristics of grass carp,this paper compared the blood lipids,serum enzymes,blood routine and blood antioxidant capacity between the control group and the broad bean group.The results showed that compared with the control group,the triglyceride?TG?,total cholesterol?TC?and low density lipoprotein cholesterol?LDL-C?in the serum of the broad bean group were higher?P<0.05?,while the activities of alkaline phosphatase?AKP?,acid phosphatase?ACP?,lysozyme?LSZ?and the concentration of complement C3 were low?P<0.01?.White blood cells?WBC?,red blood cells?RBC?and platelets?PLT?in the blood of broad bean group were significantly lower than the control group?P<0.05?.Glucose-6-phosphate dehydrogenase?G6PD?,reduced coenzyme II/oxidized coenzyme II?NADPH/NADP+?,catalase?CAT?,reduced glutathione/oxidized glutathione?GSH/GSSH?and superoxide dismutase?SOD?in the serum of broad bean group were significantly lower than the control group?P<0.05?.Hydrogen peroxide(H₂O₂)and malondialdehyde?MDA?in the serum of broad bean group were significantly higher than the control group?P<0.05?.The above results indicate that long-term feeding of broad bean can induce the increase of blood lipids in grass carp,inhibit the activity of non-specific serum enzyme,reduce the blood antioxidant capacity of grass carp and induce the production of reactive oxygen species ROS?H₂O₂?.2.Effects of feeding broad bean on liver fat accumulation and fat metabolism in grass carpIn order to explore the effects of long-term feeding of broad bean on liver fat accumulation and lipid metabolism in grass carp.Pathological feature,Lipid accumulation in the liver and changes in cell structure were observed by H&E staining,oil red O staining,and transmission electron microscopy.Then,we expressed genes related to the de novo synthesis pathway of fatty acids,including Acc?acetyl-CoA carboxylase?and Fas?fatty acid synthase?;we expressed mitochondrial electron transport chain subunit-associated proteins,including Mt-Nd1?NADH-ubiquinone oxidoreductase chain 1?,UQCRC1?cytochrome b-C1 complex subunit 1?,Mt-Co1?cytochrome?C oxidase subunit 1)and Mt-Co2?cytochrome C oxidase subunit 2?;we expressed autophagy-related genes and their proteins,including Ulk1?Uncoordinated51-Like Kinase 1?,Bnip3?Bcl-2 Nine-kilodalton Interacting Protein 3?,Park2?Parkin RBR E3 ubiquitin protein ligase?,Beclin1,Ctsl?cathepsin-L?,PGC1-??peroxisome proliferator-activated receptor gamma coactivator 1-alpha?and LC3B?microtubule-associated protein light chain 3B?.In addition,we detected the metabolites of the liver by ultra performance liquid chromatography-mass spectrometry?UPLC-MS?,and analyzed the differential metabolites between the control group and the broad bean group in order to investigate the effects of broad beanon liver lipid metabolism and its possible mechanisms;from the liver H&E staining and oil red O staining of the broad bean group,liver fat accumulation was observed,which was characterized by steatosis;the ultrastructure showed that the cell structure of the liver of the broad bean group was disordered,the morphological structure of the mitochondria and endoplasmic reticulum in the cells was abnormal,and the number was significantly reduced.In addition,the expression of Acc and Fas genes in the liver of the broad bean group was up-regulated relative to the control group?P<0.01?,indicating that the fatty acid de novo synthesis pathway?DNL?was up-regulated;the expressions of mitochondrial electron-transporting respiratory chain subunit proteins UQCRC1,Mt-Co1 and Mt-Co2 in hepatocytes of broad bean group were down-regulated?P<0.01?,indicating mitochondrial dysfunction;the expressions of autophagy genes Ulk1,Bnip 3 and Park2 in the liver of broad bean group were up-regulated?P<0.01?,the protein expression of PGC1-?was down-regulated?P<0.01?,and the expression of LC3B protein was up-regulated?P<0.05?,indicating an increase in autophagy in the liver,especially mitochondrial autophagy;UPLC-MS technology was used to analyze 111 differential metabolites,including 9 up-regulated fatty acids and their derivatives,which is consistent with the result of liver oil red O staining.The results showed that long-term feeding of broad bean resulted in liver steatosis of grass carp,which may be due to up-regulation of fatty acid de novo synthesis,decreased mitochondrial number,mitochondrial dysfunction and increased mitochondrial autophagy in hepatocytes.These factors led to increased production and consumption of fatty acids in the liver directly,resulting in fat accumulation in the liver and fatty degeneration.3.Effects of feeding broad bean on muscle quality of grass carpIn order to explore the effects of long-term feeding of broad bean on grass carp muscle,this paper analyzed the basic nutrients,collagen content,pH,water holding capacity,fiber structure,texture characteristics and shearing force of the back,abdomen and tail muscles of the broad bean group.The results showed that compared with the three parts of the control grass carp,the three parts of the broad bean group had lower water and crude fat content,while the ash and crude protein were higher.The crude protein and collagen content in the back muscle of the broad bean group were the highest;and there was no significant difference in the pH of the three parts of the broad bean group and the control grass carp?P>0.05?.The water holding capacity of the three parts of the broad bean group was significantly lower than that of the common grass carp.Compared with different parts of the same species of fish,the two groups of fish showed higher water holding capacity in the abdomen.Compared with the control group,the three parts of the broad bean group had small muscle fiber diameter and high fiber density,and the hardness,chewiness,springiness,adhesiveness and shearing force of the three parts were high.What's more,the broad bean group has a high chewiness and springiness on the back.The results indicated that long-term feeding of broad beans can improve muscle nutrition,reduce muscle fiber diameter,and improve muscle texture characteristics.At the same time,broad bean had different effects on the physical and chemical properties of muscles in different parts of grass carp.