| An 8-week feeding trial was conducted to evaluate dietary leucine requirements of grouper(Epinephelus coioides),6 groups of diets containing 1.99%,2.79%,3.59%,4.39%,5.19% and 5.99% leucine contents were fed to grouper.The growth performance,whole body composition and biochemical indices were observed to investigate the optimum amount of Leucine in grouper feed.Besides the effects of different Leucine levels on the histomorphology of the liver and intestines and the genes related to the mTOR signaling pathway in the muscles,liver and intestines were investigated.Results of the present studies are presented as follows:1.The survival rate of grouper in each group was above 95% across the feeding period.All the parameters of growth performance such as weight gain rate,specific growth rate,feed intake,hepatopancreas somatic indices,and condition factor showed a tendency to increase first and then to decrease with the increase in dietary Leucine level.All of the above parameters reached a peak at the D3.59 group.The feed intake in group D1.99 was significantly lower than that in other groups(P<0.05);condition factor in D3.59 and D4.39 groups were significantly higher than other groups(P<0.05),and there was no significant difference in other indexes between dietary treatments(P>0.05).The feed conversion rate increased with the increase in Leucine level,but the lowest value for D4.39 group was not different from other groups(P>0.05).The whole body crude protein and crude fat contents increased first and then decreased with the increase in Leucine levels.The maximum value of crude protein for the D3.59 group was found higher than that of D1.99 and D5.99 groups(P<0.01),and the value in D4.39 group was higher than that in D5.99 group(P<0.05).There was no significant difference in between the other groups(P>0.05),the maximum value of crude fat for D4.39 group was higher than that for groups D1.99 and D5.99(P<0.05),but there was no difference between other groups(P>0.05).The crude ash and moisture contents of whole body decreased first and then increased with the increase in dietary Leucine levels,the lowest value occurred for D3.59 group.The content of crude ash in D3.59 group was significantly lower than that in D1.99 group(P<0.05),but there was no significant difference between the other groups(P>0.05).The moisture content of D3.59 group was lower than that of D5.19 and D5.99 group(P<0.05),and there was no significant difference between other groups(P>0.05).The activities of liver GPT,GOT,BCKDK and serum SOD increased and then decreased with the increase in dietary Leucine levels,and the activity ofGOT in D3.59 group was higher than that in D1.99,D5.19,and D5.99 groups(P<0.05).The activity of BCKDK in D5.19 was higher than that of D1.99,D2.79 and D5.99(P<0.01),and the values for D3.59,D4.39 were higher than that in D1.99(P<0.05),but there was no difference between the other groups(P>0.05).The contents of liver TG,T-CHO and the activities of serum GPT and GOT decreased first and then increased with the increase in dietary Leucine levels.The contents of TG and T-CHO in the liver were the lowest in D3.59 group,and were lower than that in D5.19 and D5.99 groups(P<0.05).The activities of serum GPT and GOT were lowest in D4.39 group,and the GPT activity in D1.99 group was higher than that in other groups(P<0.05).The optimum Leucine levels for the fish species were 4.26% or 4.48%(on fed basis),and 8.88%or 9.33%(on dietary protein level)respectively based on the regression analysis between weight gain rate or feed conversion rate and dietary Leucine level.2.With the increase of Leucine levels in diet,the height and number of mucosal folds and muscle thickness of the anterior intestine,mid-intestine and posterior intestine increased first and then decreased,the highest in the group of D3.59 or D4.39.The number of mucosal folds of the anterior intestinal in D2.79 and D3.59 were significantly higher than that of the other groups(P<0.05);The number of mucosal folds of mid-intestine in group D3.59 was significantly higher than that in group D5.19(P<0.05);The height mucosal folds of posterior in group D5.99 was significantly lower than that in other groups(P<0.05);The muscle thickness of the posterior intestinal in group D1.99,D2.79 and D3.59 was significantly higher than that in group D5.99(P<0.05).According to analyzing intestinal structure,the density of goblet cells in group D1.99 was smaller than that in other groups,and striated border is not neat;Group D5.99 had sparse mucosa fold,narrow lamina propria and irregular striated border;the striated border of the mid-intestinal D5.19 group was scattered and irregular.The mucosa folds of D1.99 and D5.99 groups were sparse in the posterior intestine.In group D1.99,the size of liver cells were not uniform,there are few the hepatic cell cords that was short,the cells were vacuolated,in D5.19 and D5.99 group,hepatocytes were expand,hepatic cell cords were short and less,and there are more fat droplets or vacuoles between hepatocytes.No abnormality was found in other groups.Therefore,the abnormal changes in the structure of the intestine and liver occurred for D1.99,D5.19 and D5.99,and the morphology of the intestinal and liver in D3.59 and D4.39 groups are better than those in other groups.3.The expression levels of m LST8,RagC/D,Raptor,and m TOR in the intestine,the SLC7A5,and Tel2 in the liver,and the Ragulator in the muscle,liver and intestine increased first and then decreased with the dietary increase in Leucine levels,and reached a significant peak(P<0.05)in D3.59 or D4.39.The intestinal expression level of mLST8 in D4.39 was the highestamong groups,and was higher than that in D1.99 and D2.79 group(P<0.05).The intestinal expression levels of RagC/D in D3.59 and D4.39 groups were higher than that of the other groups(P<0.05);The intestinal expression of Raptor in D2.79 and D3.59 groups was higher than that in other groups.The expression of intestinal TOR in the D4.39 group was higher than that in D1.99 group(P<0.05).The expression of liver SLC7A5 in D3.59 was higher than that in other groups(P<0.01),and the expression of liver Tel2 in D2.79 group was higher than that in D5.99group(P<0.05).The expression of muscle Ragulator in D4.39 group was higher than that of D1.99 and D2.79 group(P<0.05);the expression of Ragulator in the liver in D4.39 group was higher than that in other groups;the expression of intestinal Ragulator in D3.59 group was higher than in the D5.19 and D5.99 groups.The expression of V-ATPase in the intestine and SLC7A5 in the muscles decreased first and then increased with the increase in dietary Leucine levels(P<0.05),and the lowest values reached a lowest point for D4.39 group.The expression of muscle SLC7A5 in D4.39 group was lower than that in group D1.99(P<0.01)and in D5.99group(P<0.05).The expression of V-ATPase in the intestine in D1.99 group was higher than in other groups(P<0.05).The expression of S6 K in muscle showed a decreasing tendency(P<0.05)with the dietary increase in Leucine levels,and D1.99 was higher than that in other groups(P<0.05).It can be seen that the Leucine levels of diets have a more significant effect on the upstream gene expression of the mTOR signaling pathway protein synthesis pathway,but has less influence on the downstream gene expression. |
