| In this experiment,carnivorous golden pompano,Epinephelus fuscoguttatus ? × E.lanceolatus ? grouper,and herbivorous grass carp were used as the experimental research objects.The GLUT2 gene of golden pompano was cloned by RACE-PCR technology,and three kinds of fish were analyzed.At the same time,through the comparison of breeding experiments,1)Effects of supplementary enzymes in high carbohydrate diet on growth,carbohydrate metabolism,digestive enzymes and GLUT2 gene expression of juvenile golden pompano;2)Effects of dietary carbohydrate to lipid ratios and exogenous enzyme supplementation on growth performance,digestive enzyme and hepatic carbohydrate metabolic enzyme activities of hybrid grouper;3)Effect exogenous enzyme supplementation in different carbohydrate levels on growth performance,digestive enzyme and hepatic carbohydrate metabolic enzyme activities of grass carp.Analyze its regulation mechanism in the carbohydrate metabolism to explore the difference of GLUT2 expression in different diet fish.The research results are as follows:1.Molecular cloning and sequence analysis of GLUT2 of golden pompanoThe gene for glucose transporter 2(Trachnotus ovatus,To GLUT2)was cloned and identified by RACE technology.To GLUT2 full-length c DNA consists of 2280 bp and 32 bp 5'-UTR,1638 bp open reading frame(ORF)and 610 bp 3'-UTR,encoding a 546 amino acid polypeptide(Gen Bank accession number: MK649830).The calculated molecular weight and p I of To GLUT2 are 53.1 k Da and 8.78,respectively.The molecular weight of To GLUT2 is similar to other species,and amino acid sequence analysis shows that it is a member of Class I of the GLUT family.Tissue distribution analysis showed that the expression of To GLUT2 was highest in the liver,followed by the intestine and brain.The values detected in muscle,heart and kidney are relatively low.Phylogenetic analysis showed that To GLUT2 had the highest similarity to sea bass GLUT2.There are QLS motif and GL motif in the amino acid sequence of pomfret pomfret,which is crucial for glucose transport.TOGLUT2 contains the STSIF motif of the seventh cell outer loop and the PGPIPW motif of the tenth helix,which belong to the GLUT class I transporter-specific motif.However,we found that the QLS motif of the seventh helix changed in different animals,including grass carp,mouse,mountain quail,zebrafish,and Homo sapiens,and other carnivorous fish were QLS motifs.Based on the above results,we speculate that this is one of the causes of carnivorous fish intolerance to carbohydrate.2.Effects of supplementary enzymes in high carbohydrate diet on growth,carbohydrate metabolism,digestive enzymes and GLUT2 gene expression of juvenile golden pompanoGolden Pompano were randomly divided into 5 groups with 3 replicates in each group.The fish were fed isoenergetic and isolipidic experimental diets(high carbohydrate group H,amylase group A,glucoamylase group G,pullulanase group P,complex enzyme group C).Amylase,glucoamylase,pullulanase and complex enzyme(the composition of the complex enzyme is the other three enzyme preparations added in the ratio of 1: 1: 1).The results showed that the weight gain rate WGR and specific growth rate SGR of the compound enzyme group(C)were significantly higher than other groups(P <0.05).The rate-limiting enzymes 6-phosphate fructokinase(PKF),glucokinase(GK)and pyruvate kinase(PK)are significantly lower in the complex enzyme group(C)than the high glucose group(H)(P <0.05).The key enzyme of gluconeogenesis phosphoenolpyruvate kinase(PEPCK)was increased in complex enzyme group(C),and significantly higher than the high glucose group(H)(P <0.05).The trypsin was significantly higher in the complex enzyme group(C)than the high glucose group(H)(P<0.05).The amylase(A)was significantly higher in the high glucose group(H)than the composite enzyme group(C)(P<0.05).Lipase is significantly higher in the amylase group(A)than the high glucose group(P <0.05).The expression of GLUT2 gene was significantly higher in glucoamylase group(G)than other groups(P<0.05).The liver glycogen content was significantly higher of the amylase group(A)and pullulanase group(P)than other groups(P <0.05).The muscle glycogen content are significantly higher of the high glucose group(H)and the glucoamylase group(G)than the other groups(P <0.05).Therefore,we can conclude that in the context of high carbohydrate nutrition,the supplement of compound enzyme preparations in the feed can significantly improve the growth performance of golden pompano;the supplement of enzyme preparations in the feed can effect the carbohydrate metabolism of golden pompano.The expression level of GLUT2 gene is the highest with glucoamylase added to the feed,and the ability to transport carbohydrate to the blood is the strongest,which improves the rate of carbohydrate utilization.3.Effects of dietary carbohydrate to lipid ratios and exogenous enzyme supplementation on growth performance,digestive enzyme and hepatic carbohydrate metabolic enzyme activities of hybrid grouper,Epinephelus fuscoguttatus,?×Epinephelus lanceolatus,?Grouper were randomly divided into 6 groups with 3 replicates in each group.The experimental formula uses fish meal and casein as protein sources,fish oil and phospholipid oil as fat sources,and corn starch as carbohydrate sources,with protein levels of approximately 43%.The composition of the enzyme preparation is that the other three enzymes(amylase,glucoamylase,pullulanase)are added at a ratio of 1: 1: 1,and formulated into six kinds of isoenergetic feeds(0.91,1.92,3.91,0.91 ENZ,1.92 ENZ,3.91ENZ).The results showed that the weight gain rate WGR and specific growth rate SGR of grouper were significantly affected by the ratio of carbohydrate-to-lipid and enzyme preparation(P<0.05).The medium and high carbohydrate-to-lipid ratio and its enzyme-added group were significantly higher(P <0.05)in the pyruvate kinase(PK)(P <0.05),and the enzyme-added group was significantly higher than that without enzyme group(P<0.05).High carbohydrate-to-lipid levels were significantly higher than those of 6-phosphate fructose kinase(PKF)(P <0.05).Glucokinase(GK)was significantly higher in the high carbohydrate-to-lipid ratio group than the middle and low(P<0.05),and the enzyme-added group was significantly lower than the enzyme-free group(P <0.05).The content of phosphoenolpyruvate kinase(PEPCK)was significantly higher(P <0.05)in the high carbohydrate-to-lipid group,and the enzyme-added group was significantly lower than that the enzyme-free group(P <0.05).The content of liver glycogen and muscle glycogen was significantly higher(P <0.05)in the high carbohydrate-to-lipid group,and the content of liver glycogen and muscle glycogen was significantly lower in the enzyme-added group than the enzyme-free group(P <0.05).The trypsin,amylase and lipase were significantly higher in high carbohydrate-to-lipid levels(P<0.05).The GLUT2 gene expression was significantly lowest in liver tissue of the high carbohydrate-to-lipid ratio group(P<0.05);the GLUT2 gene expression was significantly highest in the intestinal tissue of high carbohydrate-to-lipid ratio group(P <0.05).Therefore,we can conclude that the addition of carbohydrase can promote the growth of hybrid grouper and improve feed utilization in the high carbohydrate-to-lipid ratio nutritional background.And can increase the activity of enzymes related to carbohydrate metabolism in hybrid grouper,as well as the expression of the intestinal GLUT2 gene,and promote the rate of carbohydrate utilization.Adding carbohydrase can increase the digestive enzyme activity of hybrid grouper and enhance its digestive capacity in a high carbohydrate-to-lipid ratio nutritional background.4 Effect exogenous enzyme supplementation in different carbohydrate levels on growth performance,digestive enzyme and hepatic carbohydrate metabolic enzyme activities of grass carp,Ctenopharyngodon idellusThe grass carp were randomly divided into 6 groups with 3 replicates in each group.The experimental formula uses fish meal,soybean meal and soybean protein concentrate as the protein source,and wheat starch as the carbohydrate source,in which the protein level is about 28% and the lipid level is 5%.The composition of the enzyme preparation is that the other three enzymes(amylase,glucoamylase,pullulanase)are added in the ratio of 1: 1: 1,and formulated into six kinds of isoenergetic and isolipidic(34,42,50,34 ENZ,42ENZ,50ENZ).The experimental results show that the weight gain rate WGR and specific growth rate SGR of grass carp are not affected by carbohydrate level and enzyme preparation factors(P> 0.05).Those pyruvate kinase(PK),6-phosphate fructose kinase(PKF),and glucokinase(GK)contents were significantly higher in the high carbohydrate group(P <0.05).The enzyme-added group was significantly higher than without(P<0.05).The content of phosphoenolpyruvate kinase(PEPCK)was significantly higher in the high carbohydrate group(P <0.05),and the enzyme-added group was significantly higher than without(P <0.05).The content of liver glycogen and muscle glycogen is significantly higher in the high carbohydrate group(P <0.05).The liver glycogen and muscle glycogen were significantly lower in the enzyme-added group than without(P<0.05).Those trypsin,amylase and lipase were significantly higher in the high carbohydrate group(P<0.05);the content of trypsin and amylase was significantly higher in the enzyme-added group than without(P<0.05).Therefore,we found that adding carbohydrase can increase the activity of grass carp carbohydrate metabolism-related enzymes,promote carbohydrate utilization,can increase the activity of grass carp digestive enzymes,and enhance its digestive ability under the high carbohydrate level nutrition background.However,its inefficient circulation consumes a lot of energy,so it does not play a good role in growth performance. |
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