| Study 1:Effects of dietary carbohydrate sources on the growth performance,postprandial serum glucose,hepatic glucose metabolism and insulin pathway of largemouth bass（Micropterus salmoides）Four isoproteic（crude protein 52%）and isolipidic（crude lipid 10%）diets were formulated to contain 10%cassava starch,potato starch,pea starch and dextrin,respectively.Thereinto,fishmeal,shrimp meal,fermented soybean meal and corn gluten meal were used as the major protein source,rapeseed oil and soybean phospholipid were used as the main lipid source,using zeolite powder as a filler.Test fish,fasted for24h,with similar size（average initial weight 36.3±0.3g）were distributed into 12fiberglass tanks（1000L）at a stocking density of 35 individuals per tank.Each diet was randomly assigned to triplicate tanks,and the fish were fed to apparent satiation twice daily（8:00 and 17:00）for 11 weeks.During the feeding trial,the water temperature was27±1°C,and pH 7.2±0.2.All tanks were provided with a continuous flow of sand-filtered freshwater(2.0 L min-1)and aeration（dissolved oxygen,≥6 mg/L）.The tanks were maintained under a natural photoperiod.The research was conducted to investigate the effects of dietary starch sources on growth performance,postprandial serum glucose,hapatic glucose metabolism and insulin signaling pathway of largemouth bass and use these parameters to appraise suitable carbohydrate source in diet for largemouth bass.The results of this research are presented as follows:1.Effects of carbohydrate sources on growth performance,body composition and utilization of feed nutrients of test fishGrouth performance The research indicated that the final weight and specific growth rate of the test fish in the pea starch group were significantly higher than the other three starch groups（P<0.05）;there was no significant difference in condition factor and hepatosmatic index in four groups,and yet the viscerosomatic index in the dextrin group was significantly higher than that in the potato starch group（P<0.05）.Body composition of the test fish The result show that there was no significant difference in moisture and ash content of test fish whole body composition among the four groups（P>0.05）;however,the content of crude protein was higher in the group fed the pea starch added diet than in the dextrin group,and the content of crude lipid was lower（P<0.05）;the content of liver glycogen in the cassava starch group was significantly higher than that in the pea starch group（P<0.05）,but the dietary starch sources had no significant effect on the content of muscle glycogen in the test fish（P>0.05）.Feed utilization There was no significant difference in the feeding rate of the test fish fed among the four groups（P>0.05）,the feed efficiency of the test fish fed with the pea starch diet was significantly higher than that in the dextrin group（P<0.05）,meanwhile,the protein efficiency of the dextrin group was significantly lower than the other groups;moreover,the feed starch sources also had a significant effect on the protein deposition rate and lipid deposition rate（P<0.05）,the lipid deposition rate of the test fish fed with the pea starch group was significantly lower than that in the cassava starch group and the potato starch group（P<0.05）,while the protein deposition rate in the dextrin group was significantly lower than that in the other groups（P<0.05）.2.The effect of carbohydrate sources on the postprandial serum glucose,expression of genes related to glucose metabolism and insulin pathway in test fish.Postprandial serum glucose The result indicated that dietary starch sources had significant effects on serum glucose levels in the different time periods after meal time and the test fish was more tolerant to pea starch than other starch sources in accordance with the result of postprandial serum glucose content（P<0.05）.The serum glucose level of test fish fed with cassava starch had a tendency to gradually decrease to the basic level after 24h,while the other starch resourse had recovered after 12 h,meanwhile,the starch resourse had no significant effect on the content of postprandial serum glucose at 0,1,6 and 24h timepoint（P<0.05）,however,the serum glucose level of the fish in the pea starch group was significantly lower than that in the-cassava starch group at 3 h after meals,and the blood glucose level of the fish in the cassava starch group at 12 h after meals was significantly higher than that in the pea starch group and dextrin group（P<0.05）.Gene expression The pea starch led to a significantly increased expression of glucokinase（GK）and 6-phosphofructokinase isozyme（PFKL）（P<0.05）,while dietary starch sources had no significant influence on the expression of phosphoenolpyruvate carboxykinase（PEPCK）and fructose-1,6-bisphosphatase-1（FBP1）（P>0.05）;meanwhile,the expression of insulin receptor（IR）,insulin receptor substrate 1（IRS1）and phosphoinositide 3-kinase（PI3K）was significantly up-regulated in fish fed the diet with pea starch than others（P<0.05）,indicating the activation role of pea starch on insulin pathway to some extent.3.ConclusionThe research indicated that the pea starch compared with three other carbohydrate sources,significantly increased the growth performance and feed utilization of largemouth bass,and regulated the serum glucose levels,gene expression of glycolysis related enzyme in pea starch group is significantly higher than other three groups,meanwhile,pea starch had no inhibitory effect on gene expression of insulin pathway,therefore,pea starch was most suitable carbohydrate source used for largemouth bass.Study 2:Effects of cassava starch hydrolyzed by pullulanase and its supplementation level on the growth performance,postprandial serum glucose,hapatic glycogen,hapatic glucose metabolism and insulin pathway of largemouth bass（Micropterus salmoides）Six isoproteic（Crude protein 51%）and isolipidic（Crude lipid 11%）diets were formulated to contain 4%、8%and 12%cassava starch（CS4,CS8 and CS12）or cassava starch hydrolyzed by pullulanase（CS4,CS8 and CS12）,respectively.Thereinto,fishmeal,shrimp meal,fermented soybean meal and corn gluten meal were used as major protein source,soybean oil and soybean phospholipid were used as the main lipid source,using zeolite powder as a filler.Test fish,fasted for 24h,with similar size（average initial weight 13.08±0.02g）were distributed into 12 fiberglass tanks（1000L）at a stocking density of 40 individuals per tank.Each diet was randomly assigned to triplicate tanks,and the test fish were fed to apparent satiation twice daily（8:00 and17:00）for 11 weeks.During the feeding trial,the water temperature was 27±1°C,and pH was 7.2±0.2.All tanks were provided with a continuous flow of sand-filtered freshwater(2.0 L min-1)and aeration（dissolved oxygen≥6 mg/L）.The tanks were maintained under a natural photoperiod.The research was conducted to investigate the effects of cassava starch hydrolyzed by pullulanase and its supplementation level on the growth performance,postprandial serum glucose,hapatic glycogen,hapatic glucose metabolism and insulin pathway of largemouth bass and use these parameters to appraise suitable starch source and starch level in diet for largemouth bass.The research results are as follows:1.Effects of cassava starch hydrolyzed by pullulanase and its supplementation level on growth,body composition and utilization of nutrients in feed of test fish.Growth performance At the 4%addition level,the final weight and specific growth rate of the enzymatic treatment group were significantly higher than those of the cassava starch group（P<0.05）,while in the enzyme treated starch group,both the final weight and specific growth rate of the test fish showed a decreasing trend with the increase of the starch addition level（P<0.05）.Body composition Hepatosmatic index and viscerosomatic index of CS12 group were significantly higher than those of other groups（P<0.05）,while there was no significant difference among the three levels of enzymatic treatment group（P>0.05）.There was no significant difference among the six diets on the whole fish body composition of the test fish（P>0.05）.Different starch processing and adding level has a significant influence on liver composition of the test fish liver（P<0.05）,the liver protein content of the three added levels of enzyme treatment was significantly higher than the corresponding level of cassava starch group（P<0.05）,while the liver crude fat content was opposite,and with the increase of the added level,the liver protein content decreased,the liver crude fat content increased（P<0.05）.In addition,the glycogen content of the cassava starch group was significantly higher than that of the enzyme treatment group（P<0.05）,and the hepatic glycogen content of CS8 and CS12 group was significantly higher than that of CS4 group（P<0.05）,and the liver glycogen content of EH8 and EH12 group was significantly higher than that of EH4 group（P<0.05）.Feed utilization The cassava starch hydrolyzed by pullulanase and its supplementation level had significant effects on the fish feeding rate,feed efficiency,protein efficiency,protein retention rate and starch apparent digestibility（P<0.05）,but had no significant effects on lipid retention rate,protein and lipid apparent digestibility（P>0.05）.The feeding rate in the nzymatic treatment group was significantly higher than that in the cassava starch group at the same level（P<0.05）.The fish feeding rate in the cassava starch group decreased with the increase of starch addition level.The feeding rate in the enzymatic treatment group at the level of 4%and 8%was significantly higher than that in the group at the level of 12%（P<0.05）.The fish feed efficiency and protein efficiency of the CS12 group were significantly higher than those of the CS4 group（P<0.05）,and were significantly higher than those of the three levels of enzyme treated starch group（P<0.05）,while the feed efficiency and protein efficiency were not significantly different among the three levels of the enzymatic treatment group（P>0.05）.The apparent digestibility of starch in cassava starch group was significantly higher than that in enzymatic treatment group with the same addition level（P<0.05）,but there was no significant difference in the apparent digestibility of starch between the three addition levels in cassava starch group（P>0.05）.2.Effects of cassava starch hydrolyzed by pullulanase and its supplementation level on postprandial serum glucose,hapatic glycogen content,glucose metabolism and insulin pathway of test fishPostprandial serum glucose The content of postprandial serum glucose of test fish in six group decreased from 0h to 1h after feeding,then in addition to CS8group and EH4 group,the other four groups had an increasing tendency after 4h and reached the highest at the 12h（P<0.05）,the content of postprandial serum glucose of test fish in CS12 group was significantly higher than EH12 group at 24h time point（P<0.05）.The serum glucose level of enzyme treatment group was lower than that of cassava starch group,and the glycemic index was lower（P<0.05）.Postprandial hepatic glycogen The content of hepatic glycogen accumulation reached the highest in CS12 group at 0h time point,and was significantly higher than that in other groups at 1h time point（P<0.05）.The content of glycogen accumulation in CS12 group at 0 h was significantly higher than that in CS4 group and CS8 group（P<0.05）,and the content of glycogen accumulation in enzymatic treatment group was significantly lower than that in cassava starch group（P<0.05）.There was no significant difference among the three addition levels in enzymatic treatment group（P>0.05）.In enzymatic treatment group,the content of glycogen accumulation in EH8group at 4 time points,8 time points and 12 time points were significantly higher than EH4 group（P<0.05）,but the difference of hepatic glycogen accumulation between EH8group and EH12 group was not significant（P>0.05）.At the 24h time point,the content of hepatic glycogen in CS12 group was significantly higher than that in EH4 group and EH8 group（P<0.05）.Gene expression Cassava starch hydrolyzed by pullulanase and its supplementation level had a significant effect on the gene expression of key enzymes in glycolysis of the test fish（P<0.05）.The EH8 and EH12 groups had significant up-regulation of GK,and were significantly higher than CS4 group（P<0.05）.There was no significant difference in GK expression among CS8,CS12 and EH4 groups（P>0.05）.Among cassava starch groups,the gene expression of PK（Pyruvate Kinase,PK）in CS8group was significantly higher than that of CS4 group（P<0.05）,but there was no significant difference in CS12 group（P>0.05）.There was no significant difference in the expression of PK gene in three levels of enzymatic hydrolysis of cassava starch;the expression of G6PC（glucose-6-phosphatase,G6PC）in CS4 group was significantly higher than that in CS12 group（P<0.05）,and the other groups were not significantly different（P>0.05）.There was no significant difference in the expression of PFKL gene between starch treatment and addition（P>0.05）,and there was no significant difference in the expression of FBP1 and PEPCK genes in gluconeogenesis（P>0.05）.The expression of PI3K gene in CS4 and CS8 groups was significantly higher than that in CS12 group（P<0.05）;there was no significant difference in PI3K expression among the three levels of enzymatic treatment group（P>0.05）;the gene expression of protein kinase B（AKT）in CS8 group was significantly higher than that of EH4 group（P<0.05）,and there was no significant difference among other groups（P>0.05）;the gene expression of IR in CS8 group was the highest,which was significantly higher than the other five groups（P<0.05）;the gene expression of IRS1 in CS8 group and EH8 group was significantly higher than that in CS12 group（P<0.05）,and the difference between other groups was not significant（P>0.05）;starch treatment and level showed no significant difference in insulin receptor substrate 2（IRS2）gene（P>0.05）.3.ConclusionThe research showed that the cassava starch hydrolyzed by pullulanase is beneficial to promote the growth of largemouth bass,stabilize the postprandial blood glucose and reduce the accumulation of glycogen,which is a suitable starch source for largemouth bass.At the same time,the starch addition level of 4%（digestible starch5.68%）is more favorable for the growth health and more stable blood glucose of the bigmouth bass,which is more suitable as the starch addition level of the bigmouth bass in feed formulation. |
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