| Feeding trials were conducted to investigate the effects of dietary arginine levels on the growth performence,body composition,feed conversation,intestine morphology,b0,+AT gene and related immune gene expression of orange-spotted grouper（Epinephelus coioides）at two different growth stages.fish for 5-10g and 80-100g of the feeding experiments were cultured 8 and 10 weeks.The brief summarys are as follows.1.In order to explore the absorption mechanism of arginine in orange-spotted grouper,the full length of b0,+AT gene（SLC7A9）were obtained using rapid amplification technology of cDNA ends.And expression of b0,+AT in different tissues was tested by Real-time quantification PCR.The results showed that the total length of b0,+AT gene was1808 bp,including 1509 bp of the open reading frame（ORF）which encoding 502 amino acids,the 5’-UTR of 80 bp and the 3’-UTR of 219 bp.The predicted molecular weight was55233.6,and the isoelectric point was 8.38.Multiple sequence alignment showed that grouper b0,+AT gene and the other species b0,+AT gene have a highly conservative.protein sequence of b0,+AT by SMART softwareshowed that it contained the typical domain of the12-putative-transmembrane domains.In addition,real-time QPCR indicated that b0,+AT could be expressed in all of the detected tissues,including heart,liver,spleen,kidney,brain,foregut,midgut,hindgut and muscle,respectively.The highest expression were found in hindgut and the second in kidney.2.Seven isonitrogenous and isolipidic commercial diets with arginine contents of2.13%,2.42%,2.71%,2.95%,3.20%,3.48%and 3.74%（dry matter）were formulated to investigate the effects of dietary arginine on growth performance and related physiological indexes of orange-spotted grouper,Epinephelus coioides at two growth stages.Triplicates of 30 fish（innitial body weight of 7.52±0.02 g）or 20 fish（innitial body weight of80.11±0.03 g）were randomly assigned to each treatment in separate tanks.The fish were fed the experimental diets for 8 and 10 weeks,respectively.The different levels of dietary arginine had no significant effect on the fish survival rate at two different growth stages（P>0.05）.However,the weight gain rate and specific growth rate with increasing dietary arginine levels increased first,and then decreased at two different growth stages.WGR and SGR of juvenile grouper fed with 2.95%arginine were significantly higher than those of fish in other groups（P<0.05）.WGR and SGR of subadult grouper fed with2.71%arginine were significantly higher than those of the fish fed with 2.13%and 3.48%arginine dietary（P<0.05）.The feed coefficient of juvenile grouper fed with 2.95%arginine was significantly lower than those of the fish fed 2.13%,2.42%and 2.71%arginine dietary（P<0.05）.FCR of subadult grouper was significantly higher in the 2.13%arginine group than in other groups（P<0.05）,with the exception of 3.48%arginine group（P>0.05）.With the increasing dietary arginine,the protein efficiency ratio was increased and then decreased in two different growth stages.PER of juvenile grouper fed with 2.95%arginine dietary was significantly higher than those of the fish fed with 2.13%,2.42%and 3.48%arginine dietary（P<0.05）.PER of subadult grouper fed with 2.71%arginine dietary significantly higher than those of the fish fed with 2.13%and 3.48%arginine dietary（P<0.05）.GLU of juvenile grouper fed with 2.13%and 2.42%arginine dietary were significantly higher than in other groups（P<0.05）.GLU of subadult grouper fed with3.48%arginine dietary was significantly higher than in other groups（P<0.05）.FH of foregut was increased and then decreased in two different growth stages with the increasing dietary arginine.FH of juvenile grouper was significantly higher in the 2.71%and 2.95%arginine group than in other groups（P<0.05）.FH of subadult grouper fed with2.71%arginine dietary was significantly higher than in other groups（P<0.05）,with the exception of 2.95%arginine group（P>0.05）.The fish fed 2.42%arginine group diet exhibited the highest TOR mRNA expression level in juvenile grouper muscle.In subadult,higher TOR gene expressions were obtained in fish fed with 3.48%arginine dietary than in fish fed with 2.13%、2.42%and 2.71%arginine dietary.A quadratic regression analysis of the specific growth rate against dietary arginine indicated that the optimum arginine level for the maximum growth of the juvenile grouper is 3.06%,corresponding to 6.07%of dietary protein on a dry weight basis.Broken-line model analysis of specific growth rate against dietary arginine levels indicated that the optimum requirement of arginine for maximum growth of subadult grouper was 2.72%,corresponding to 5.40%of dietary protein on a dry weight basis.3.Take samples from the experiment 2 to investigate the effects of dietary arginine on serum hormones level,enzyme activities in liver and muscle and b0,+AT gene expressions in two growth stages of orange-spotted grouper.With the increasing dietary arginine,serum insulin（INS）level was increased and then decreased in two different growth stages.INS of juvenile grouper fed with 2.95%arginine dietary was significantly higher than in the other groups（P<0.05）,with the exception of 3.20%and 3.48%arginine groups（P>0.05）.While INS of subadult grouper fed with 3.20%arginine group significantly higher than in the other groups（P<0.05）,with the exception of 3.48%arginine group（P>0.05）.GH of subadult grouper significantly increased with dietary arginine levels up to2.95%diet,and decreased thereafter.The arginase of juvenile grouper liver fed with 3.48%and 3.74%arginine dietary was significantly higher than those of the fish fed with 2.13%,2.42%and 3.48%arginine dietary（P<0.05）.The arginase of subadult grouper liver fed with 2.13%arginine dietary was significantly lower than in the latter 4 groups（P<0.05）.In juvenile grouper,gene expression of b0,+AT in hindgut and kidney significantly increased with dietary arginine levels up to 2.95%and 2.71%diet respectively,and decreased thereafter.Fish fed with 2.42%arginine dietary exhibited the highest b0,+AT mRNA expression level in subadult grouper hindgut.The b0,+AT mRNA expression level in subadult grouper kidney of the fish fed 2.95%arginine group than in other groups（P<0.05）,with the exception of 3.20%arginine group（P>0.05）.4.This experiment investigated the effects of dietary arginine on serum immune enzyme activities and mRNA expressions of related immune parameters in two growth stages of orange-spotted grouper.With the increasing dietary arginine,the serum total nitric oxide synthase（T-NOS）activity was increased and then decreased in two different growth stages.The T-NOS activity of juvenile grouper fed with 2.95%arginine group was significantly higher than in other groups（P<0.05）.The T-NOS activity of subadult grouper fed with 2.71%and 3.20%arginine dietary was significantly higher than those of the fish fed with 2.13%arginine dietary（P<0.05）.Gene expression of TLR22 in intestine significantly increased with dietary arginine levels up to 2.71%and 2.42%diet respectively in two growth stages,and decreased thereafter.Gene expression of MHC-II in kidney significantly increased with dietary arginine levels up to 3.48%and 2.42%diet respectively in two growth stages,and decreased thereafter.Fish fed with 2.13%arginine dietary exhibited the highest hepcidin mRNA expression level in subadult grouper liver.The hepcidin mRNA expression level in subadult grouper kidney of the fish fed with2.95%arginine dietary was significantly higher than in other groups（P<0.05）. |
