Evaluation And Mechanism Of Lipid Accumulation In The Hepatopancreas Of Grass Carp(Ctenopharyngodon Idella)

Grass carp(Ctenopharyngodon idella) is a kind of freshwater fish with the largest annual output in China. However, the fatty liver due to high density cultivation and commercial feed has been the threat to the health of the grass carp as well as the farmers’ benefits. Feed factors that may induce fatty liver of grass carp have been widely researched, but some basic problems, including the evaluation standard of fatty liver, effective indicators and its mechanism all need to be studied. It will be benefit to the diagnosis, prevention and treatment of fatty liver to establish the evaluation standard and research the mechanism of fatty liver in grass carp. In this study, we first constructed the evaluation standard of fatty liver in grass carp based on the hepatopancreas lipid content and other indirect evaluation indicators. Meanwhile, H-NMR, electron microscope and fluorescent quantitative PCR were used to explore the mechanism of fatty liver in grass carp. The main results were as follows:1. Establishment of fatty liver assessment index system for grass carpTwo hundred and forty five adult grass carp (average mass 1317.60±331.26 g) was used in this study. Body indicators, serum biochemical indices and hepatopancreas lipid were detected by physical detection, automatic biochemical analyzer and Soxhlet methods, respectively. Normal ranges of detected indicators from grass carp whose hepatopancreas lipid was below 5% were determimed by 68.26% confidence interval if they showed significant normal distribution. The direct evaluation standard of grass carp fatty liver was established mainly based on the hepatopancreas lipid content. When the mean of a certain indicator from grass carp with hepatopancreas lipid higher than 5 per cent was higher than the upper limit of normal range, then they would be selected out as indirect evaluation indicators of fatty liver. Results showed that when the hepatopancreas lipid of grass carp was 3.02% to 4.60%,4.60% to 7.56%,7.56% to 12.74% and higher than 12.74%, then the grass carp could be diagnosed to normal, mild, moderate and severe fatty liver, respectively. CF、VSI、HSI、MFI、ALT、AST、TBIL、DBIL、ALP、CHOL、LDL and GLU were indirect evaluation indicators of fatty liver due to their significantly higer means than the upper limits of normal ranges. Moreover, fatty liver grass carp was more obesity and showed much more hepatopancreas lipid and severe damaged liver function than non-fatty liver grass carp.2. Comparison of energy conservation and antioxidant capacity between normal and fatty liver grass carp.ATP, ADP and AMP in the hepatopancreas of non-fatty liver and fatty liver grass carp were detected by high pressure liquid chromatograph to partly explore the biochemical mechanism of fatty liver. Hepatopancreas ultrastructures as well as the anti-oxidant capacity of serum from normal and fatty liver grass carp were also detected by electron microscopic and conventional chemical methods, respectively. Results indicated that both ATP and EC value decreased with the severity of fatty liver. Though, significant differences in ATP, ADP, AMP and EC between the moderate and severe fatty liver groups were not founded (P>0.05), significant differences in ATP, ADP, AMP and EC between other groups were founded (P<0.05). Total adenylate in hepatopancreas of different degree of fatty liver grass carp was similar (P>0.05). With the deterioration of fatty liver, more and larger lipid vacuole appeared in the hepatopancreas tissues and the mitochondrion changed to be less and shrinkage, which suggested that the function of mitochondrion in the hepatopancreas was gradually damaged. The activity of SOD, GSH-Px, CAT and T-AOC in the serum decreased, but MDA and LPO increased with the deterioration of fatty liver of grass carp. In conclusion, the serum antioxidant capacity gradually reduced with the deterioration of fatty liver, too.3. Metabonomics analysis of hepatopancreas and serum in mormal and severe fatty liver grass carpTo explore the biochemical mechanism of grass carp fatty liver from the whole level, metabonomics analysis of hepatopancreas and serum in mormal grass carp with hepatopancreas lipid 3% to 4% and severe fatty liver grass carp with hepatopancreas lipid 14%-16% were carried out by H-NMR. Results indicated that PC A model could well separate normal and severe fatty liver grass carp. Fifty-eight and forty-seven metabolites were detected from the hepatopancreas and serum of grass carp, respectively. Glucose, oxypurinol, taurine, lactate, creatine, glutamic acid, alanine, sn-glycerol-3-phosphorylcholine, glycine, phenol, hypoxanthine, acetic acid, lysine, leucine and valine were main hepatopancreas metabolites distinguishing normal grass carp from the severe fatty liver grass carp. However, only glucose and oxypurinol in the hepatopancreas of severe fatty liver grass carp were higher than those in the normal ones. Lactic acid, glucose, creatine,citric acid, leucine, valine, proline, pyruvic acid, guanosine, isoleucine, glutamate, sn-3-of glycerol phosphocholine, alanine, lysine and glycine were main serum metabolites differing normal grass carp from severe fatty liver ones. Nevertherless, only glucose, citric acid, glutamic acid, proline, sn-glycerol-3-choline phosphate, arginine and glycine in severe fatty liver grass carp were higher than those in the healthy ones. Compared with the normal group, the energy metabolic pathways, especially the glycolysis and aerobic oxidation of glucose pathways in the hepatopancreas of severe fatty liver grass carp were obviously inhibited.4. Comparison of mRNA expression for genes associated with energy metabolism in hepatopancreas of normal and fatty liver grass carpTo explore the molecular mechanism of fatty liver, the mRNA expression levels of mitochondrion function genes (ND5, COI, UCP2 and cytb), lipid catabolism genes (LPL, APO- 14kDa and HSL) as well as lipid synthesis genes (ACACA and FAS) in the hepatopancreas of normal and fatty liver grass carp were analyzed by quantitative PCR. Results showed that the mRNA expression levels of UCP2, LPL, ACACA and FAS increased with the deterioration of fatty liver degree. However, the mRNA expression levels of ND5, COI, cytb, APO-14kDa and HSL decreased with the development of grass carp fatty liver. In conclusion, the aerobic oxidation pathway and lipid catabolic pathways were gradually inhibited, but the lipid synthetic pathway was gradually activated with the increase of lipid accumulation in the hepatopancreas of grass carp.