| Six experiments were carried out in this paper. Firstly, we studied the regularity of juvenile Jian carp body composition and the TOR gene expression in different organ and tissue at different weight stage. Based on the results of first study,we studied the effect of dietary threonine levels on growth performance, deposit ration of nutrients,activities of GOT and GPT in serum, hepatopancreas and muscle,development of digestive organ,activities of digestive enzyme, intestinal brush border enzyme activities, TOR gene expression in muscle and intestine, immue fuction of juvenile Jian carp;then we studied the effect of Thr concentration on cell proliferation and differentiation of Jian carp's IECs with cell culture research model;studied the effect of Thr concentration on protein synthesis ability of Jian carp's IEC,and the effect on TORgene expression of Jian carp's IEC.The content and results are as follows:1 Study on body composition and TOR gene expression of variable weight juvenile Jian carp which weight between 18.0-74.7gA total of 750 Jian carp (initial mean weight,18.0±0.2g) were randomly distributed into five groups of each three replicates, feeding the same diet, for the 0,14th,28th,42nd and 56th day sampling, to explore the effect of various weights on body composition of protein,lipid,ash and water, activities of GOT and GPT in serum,muscle and hepatopancreas, TOR gene expression in muscle, hepatopancreas,head kidney,hind kidney,spleen,heart and gill. The result shows:moisture content significantly decreased (P＜0.01) while fat content significantly increased(P<0.01)with the increasing of weight of juvenile Jian carp which weight between 18.0-74.7g; and the protein content increased firstly, after weight was 31.4g the protein content keep a stable level, while ash content decreased significantly (P<0.05), when the weight was 40.1g, the ash content keep a stable level (P>0.05)。The weight of juvenile Jian carp shows highly significant effect (P<0.01) on blood ammonia content, activities of hepatopancreas GPT and GOT. Blood ammonia content, activities of hepatopancreas GPT and GOT showed a highly line regression with weight respectively (Y1= 1083.781+16.457X,R2=0.964; Y2=1249.597 +16.190X, R2=0.947; Y3=2198.725+29.567X, R2=0.957,P<0.01).Activities of GPT and GOT in muscle significantily advanced with the increasing of weight of Jian carp (P<0.01); but activity of serum GOT significantily increased first after the weight reached 40.1 g significantly decreased(P<0.01), activity of serum GPT keep a stable value until weight reached 74.7g activity of GPT significantly increased(P<0.01); TOR gene expression of Jian carp's weight between 18.0 to 74.7g in head kidney, hind kidney, spleen and gill significantly increased with the increasing of weight (Y1=0.112+0.03X,R2= 0.776, P<0.05; Y2=0.036+0.06X, R2=0.862, P<0.05; Y3=0.255+0.002X, R2=0.770, P=0.05; Y4= 0.132+0.002X, R2=0.774, P<0.05);TOR gene expression in muscle increased with the increasing of weight and showed significantily direct correlation with body protein content and muscle protein content (r1=+0.890; r2=+0.848, P<0.05);while TOR gene expression of hepatopancreas significantily decreased with increasing of Jian carp's weight (Y=0.486-0.02X, R2=0.854,P<0.05), TOR gene expression of heart keep a stsble value until the weight reached 40.1g significantly decreased (P<0.05)。The result of study shows:juvenile Jian carp which weight between 18.0-74.7g, body protein content and fat content increased with the increasing of weight while moisture content and ash content decreased; the amino acid metabolism of hepatopancreas and muscle enhanced with increasing of weight; muscle,spleen, hepatopancreas, head kidney, hind kidney, gill and heart of juvenile Jian carp showed TOR gene expression, TOR gene expression of muscle,spleen, gill, head kidney, hind kidney increased with the weight increasing while hepatopancreas's and heart's deceased.2 Effects of dietary threonine on growth performance, body component, activities of GOT and GPT of serum, hepatopancreas and muscleA total of 1050 Jian carp (13.60±0.03g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Thr (0.70,0.95, 1.25,1.55,1.85,2.15,2.45%,) for 60 days to investigate the effects of Thr on growth performance, body deposition and the GPT and GOT activities in serum, hepatopancreas and muscle. Results showed:Threonine improved highly siginificantly juvenile Jian carp's specific growth rate and feed efficiency (P<0.01) and specific growth rate, feed efficiency and content of threonine, respectively,showed highly significantly (P<0.01) or significantly(P<0.05)quadratic regression(Y1=-0.3627x2+1.1765x+1.3705, R2=0.927; Y2=-5.8180x2+19.7028x+51.4993, R2=0.790), when threonine content was 1.62%and 1.69% respectively,specific growth rate and feed efficiency reached optimum value; Thr can significantly improve the deposition of protein and fat deposition rate of juvenile Jian carp (P<0.01), but the ash deposition rate of had no significant effect (P> 0.05), protein deposition rate and Thr content showed significant quadratic regression relationship (Y=-4.8344x2+15.2266x+21.3823, R2=0.820, P<0.05, X:Thr%), when the Thr content was 1.57%, the protein deposition rate achieved the best value; Thr can significantly increase activities of GOT and GPT in juvenile Jian carp's hepatopancreas and muscle (P<0.05), activity of GOT and GPT in hepatopancreas between Thr content showed significant or highly significant quadratic regression relationship (Yi=-1632.7x2+5584.86x-578.64, R2=0.787, P<0.05; Y2=Y=-586.98x2+1575.15x+2101.41, R2=0.961,P<0.01;Y3=-126.06x2+421.100x+703.604,R2=0.948,P<0.01;Y4=-174.89x2+56 4.995x+590.875,R2=0.813,P<0.05,X:Thr%), When Thr contents were 1.71,1.34,1.67 and 1.62%, GOT and GPT activity in hepatopancreas and muscle of juvenile Jian carp reached its maximum; Thr content showed significant effect to blood ammonia concentration (P<0.01), and the ammonia concentration and dietary Thr content showed a significant quadratic regression relationship(Y=1349.88x2-4183.5x+4392.23, R2=0.908, P＜0.01); dietary Thr content significantly effected TOR gene expression of juvenile Jian carp in muscle (P＜0.05), when dietary Thr reached 0.91%, TOR gene expression in muscle significantly decreased(P<0.05)and TOR gene expression significantly increased when Thr content reached 1.57%, but it had no significant difference with 0.70% group, with increasing of Thr content,TOR gene expression in muscle showed a increasing tendency, but had no significant difference (P<0.05)。Correlation analysis showed highly direct correlation between specific growth rate and feed intake, feed efficiency, protein deposition rate, Fatty deposition rate(r1=+0.976; r2=+0.883; r3=+0.923; r4=+0.932, P<0.01)。Presentation of results:Thr improved juvenile Jian carp's growth, and improment of growth had a direct correlation of increasing of feed intake, feed efficiency, protein and fatty deposition rate; Thr evalulated amino acid metabolism of juvenile Jian carp's muscle and hepatopancreas, and it significantly effected TOR gene expression in muscle; the optimum dietary Thr requirement for optimal specific growth rate of juvenile Jian carp(13.6-55.6g) was determined to be 1.62%or 5.07g/100gCP。3 Effect of dietary threonine on growth and development of digestive organ, function of digest and absorb ability, TOR gene expression of intestineA total of 1050 Jian carp (13.60±0.03g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Thr (0.70,0.95, 1.25,1.55,1.85,2.15,2.45%,) for 60 days to investigate the effects of Thr on growth and development of digestive organ, function of digest and absorb ability, TOR gene expression of intestine. Results showed:Thr can significantly increased juvenile Jian carp's hepatopancreas weight and protein content (P<0.01), significantly increased activities of hepatopancreas trypsin, chymotrypsin, lipase (P<0.01), hepatopancreas chymotrypsin activity, lipase activity and Thr content respectively showed significantly (P<0.05) or highly significantly (P<0.01) quadratic regression relationship (Yi=-4.0220x2+13.8680x-5.3260, R2=0.896, Y2=-189.38x2+624.789x+158.988, R2=0.915, X:Thr%), When Thr content reached 1.72 and 1.65%, chymotrypsin and lipase activity of hepotopancreas reached the highest value; Thr can significantly increase juvenile Jian carp's intestine index, intestinal length index, intestinal protein content and folds height in proximal,middle and distal intestine (P<0.01), significantly increased intestinal trypsin, chymotrypsin, lipase and amylase activity (P<0.01), intestinal pancreatic protease,lipase activity and Thr content showed a significantly (P<0.05) quadratic regression relationship(Y1=-0.7157x2+2.3674x+1.0750, R2=0.895, Y2=-140.57x2+454.669x+ 16.3032, R2=0.879, X:Thr%), When Thr content reached 1.65 and 1.62% respectively, intestinal trypsin and lipase activity reached the highest value; Thr significantly increased intestinal creatine kinase activity and Na+-K+ATP enzyme, alkaline phosphatase, y-glutamyltranspeptidase of proximal,middle and distal intestine (P<0.01), and between activities of these enzyme and content of Thr showed significant or highly significant quadratic regression relationship (Y1=-25.088x2+81.4732x-20.459, R2=0.894, P＜0.05; Y2=-106.30x2+363.988x-68.602; R2=0.945, P＜0.01; Y3=-127.01x2+430.508x-92.817; R2=0.943, P＜0.01; Y4=-70.337x2+226.023x-31.678, R2=0.938, P<0.01; Y5=-11.769x2+37.5870x-6.4615, R2=0.868,P＜0.05; Y6=-5.2309x2+16.5667x+1.5345;R2 =0.924,P＜0.01;Y7=-1.0622x2+3.5065x+0.9637,R2=0.883,P＜0.05;Y8=-22.440x2+71.1129x-9.7408,R2=0.971,P<0.01;Y9=-22.757x2+74.3064x+8.8138,R2=0.917,P<0.01;Y10=-25.413 x2+81.2129x+15.3137,R2=0.910,P<0.01,X:Thr%), when content of Thr reached 1.62. 1.71.1.69.1.61.1.60.1.58.1.65.1.58.1.63.1.60% respectively, Intestinal creatine kinase, proximal,middle and distal intestinal Na+-K+ATPase, alkaline phosphatase and y-glutamyl transferase enzyme activity reached the maximum value. Thr content significantly affect the number of intestinal Bacterium acidi lactici, E. coli and Aeromonas of juvenile Jian carp (P＜0.01),when Thr content reached 1.57%, number of intestinal Bacterium acidi lactici reached a peak, continue to add Thr, number of intestinal Bacterium acidi lactici showed a descend tendency, while the number chang of E. coli and Aeromonas showed a opposite tendency; Thr significantly affected intestinal TOR gene expression of juvenile Jian carp, intestinal protein content and middle intestinal TOR gene expression showed significant negative correlation (r=-0.917, P<0.01)。The results demonstrated that Thr improved Jian carp hepatopancreas growth and development, increased hepatopancreas's ability of secreting digestive enzyme; Thr improved Jian carp intestinal growth and development, increased intestinal ability of digesting protein.fatty and starch; improved Jian carp's absorb ability, at the same time Thr optimized intestinal microflora of juvenile Jian carp, increased the number of beneficia bacterium, decreased number of harmful bacterium; Thr content significantly affected intestinal TOR gene expression of Jian carp; the optimum dietary Thr requirement for optimal activities of digestive enzyme and intestinal brush border enzyme of juvenile Jian carp(13.6-55.6g) was determined to be 1.58～1.65%, which close to 1.62% determined by specific growth rate.4 Effects of threonine on intestinal enterocyte cell multiplication and differentiation of Jian carpUsing single-factor experiment design in order to sdudy the effects of threonine on intestinal enterocyte cell multiplication and differentiation of Jian carp by in vitro cell culture model.Experimental Design 6 treatment, with 4 replicates for each treatment. Threonine were added for each treatment concentration:0,135,170,205,240, and 275mg/ L. The results showed that:threonine levels significantly affected the MTT OD values of IEC of carp (P<0.05), with Thr concentration increasing, MTT OD of carp IEC increased significantly (P<0.05), when the Thr concentration reached 205mg/L, MTT OD value reached the max, when increased threonine concentration continually, MTT OD values decreased significantly (P<0.05); Thr significantly effected on alkaline phosphatase, Na+-K+ATP enzyme activity and cell protein content of carp IEC (P<0.05), when the Thr concentration reached 205mg/L, alkaline phosphatase, Na+-K+ATP enzyme activity reaches its maximum, continued to increase threonine concentration, both of these two enzyme activity decreased significantly (P<0.05),while the protein content increased with the increasing of threonine concentration;Thr significantly reduced the consumption of protein in culture medium (P<0.05), when Thr concentration reached 170mg/L, the protein consumption was the lowest; Thr significantly reduced the activity of lactate dehydrogenase in culture medium (P<0.05), when Thr concentration reached 205 mg/L, the lactate dehydrogenase activity in culture medium reached minimum; Thr significant effected GOT and GPT activity of carp IEC, significantly affected the ammonia concentration in the culture medium (P<0.05), when the Thr concentration reached 205 mg/L, GOT activity of carp's IEC was significantly higher than the rest of the treatments, while the GPT activity was first increased significantly (P<0.05), when Thr concentration reached 170mg/L then decreased significantly (P<0.05), when Thr concentration reached 135mg/L, ammonia concentration in the culture medium decreased significantly (P＜0.05), when the Thr concentration reached 205mg/L, the ammonia concentration reachede the lowest value.Research results indicate that:Thr promoted the proliferation and differentiation of carp IEC, promoted amino acid metabolism, thus ensuring the normal structure and function of IEC.5 Effect of threonine on protein synthesis and TOR gene expression of carp intestinal epithelial cells3 small pilot study was carried out in this study. the First one seted two treatment to study the effects of threonine on protein synthesis of carp IEC, with 10 replicates for each treatment, incubation time was 240min. threonine concentration of two treatment were: Omg/L and 205mg/L Thr; the second small study investigated the effects of TOR gene expression of carp IECs, seted two treatment respectively, Omg/L and 205mg/L Thr,6 replicates for each treatment, incubation time was 30min. The third study examined the time effect of TOR gene expression of carp IECs by threonine with 205mg/L, seted up five treatment 15min,30min, 100min,170min and 240min), at each time point seted 6 replicaation. The results showed that:Thr significantly increased protein synthesis rate of carp intestinal epithelial cells (P<0.05), significantly increased TOR gene espression of Jian Carp's IEC (P<0.05), increasment rate reached 55%; with the time extension, Thr significantly increased TOR gene expression of Jian carp IECs (P<0.05), when the culture time reached 30 min, TOR gene expression of Jian carp IECs significantly increased (P<0.05),when the culture time between 30min to 100min,TOR gene expression remained a stable level, when the cultue time reached 170 min, TOR gene expression increased significantly (P<0.05), then keep a stable level again. The results suggested that threonine increased the protein synthesis of Jian carp's IECs, the improvement of protein synthesis concerned with the increase of TOR gene expression by threonine,the effects of threonine on TOR gene expression of carp's IECs were significantly affected by culture time,when the culture time reached 170min, TOR gene expression of carp's IECs reached a stable value.In summary:TOR gene expression in muscle, spleen, head kidney, kidney and gill of juvenile jian carp weighted 20 to 80g increased with weight gain, while the hepatopancreas and heart decreased; Thr could promote growth performence, enhance body protein and body fat content, protein content concerned with TOR gene expression in muscle; Thr could promote the growth and development of juvenile Jian carp's digestive organs, improve its capacity of the secretion of digestive enzymes and improve intestinal absorptive capacity, intestinal protein content concerned with the TOR gene expression of intestine.Threonine could promote proliferation and differentiation of juvenile Jian carp's IECs, elevate the ability of protein synthesis and TOR gene expression of Jian carp's IECs; Threonine could improve nonspecific immunity and specific immunity ability, and the improvement of nonspecific immunity ability was responsed to its improvement of identify, sticking, bacteriostasis and bacteriolysis to pathogenic bacterium. According our study recult, the requirement of juvenile Jian carp(20-60g) was determined to be 1.62% or 5.07g/100gCP。6 Effect of Thr on immune function of juvenlile Jian carpThis study was carried out with juvenile Jian carp to investigate the effect of dietary threonine on the disease resistance and immune response. purified diets with seven levels (threonine content:0.70,0.95,1.25,1.55,1.85,2.15,2.45%) were fed to Jian carp (initial weight13.60±0.03g g, mean±SD). After 60 days feeding trial, the challenge experiment with Aeromonas hydrophila (AH) was conducted to determine the impact of the graded threonine levels on the disease resistance of Jian carp. Recults showes:Survival after vaccination was direct correlation with the increasing threonine levels(P＜0.01). when content reached 1.57% Survival after vaccination was 100%; threonine had a positive effect on the weight of head kidney and spleen (P＜0.05), significantly increased Blood counts of red cell and white cell(P<0.01); threonine also increased the Phagocytic activity of leukocyte significantly (P＜0.01), Phagocytic activity of leukocyte and threonine content showed a significantly quadratic regression relationship (Y=-26.408x2+88.0857x-21.261, R2=0.969, P＜0.01),when content of threonine reached 1.57%, Phagocytic activity of leukocyte reached the max; Serum immunoglobulin M (IgM) level and agglutination antibody titer to A. hydrophila were significantly increased (P＜0.05) in juvenile Jian carp fed the diets with the increasing of dietary threonine levels,when dietary content reached 1.57%, IgM level reached max, agglutination antibody titer to A. hydrophila and content of threonine showed a significantly quadratic regression relationship (Y=-54.651x2+179.867x-99.372, R2=0.969,P<0.05), when content of threonine reached 1.65%, agglutination antibody titer to A. hydrophila reached the max; thronine also significantly affected content of serum complement 3 and complement 4, lectin potency, activity of lysozyme (LA) and acid phosphatase (ACP) and total iron-binding capacity (TIBC) (P＜0.01), serum complement 3 and complement 4, acid phosphatase, total iron-binding capacity and content of thronine showed significantly quadratic regression relationship (Y1=-105.55x2+352.445x-127.43, R2=0.800; Y2=-14.066x2+46.1787x-12.768,R2=0.805;Y3=-186.01x2+594.124x-168.16,R2=0.883; Y4=-31.992x2+99.9297x+22.3843, R2=0.793, P<0.05, X:Thr%). Correlation analysis showed that there are highly significantly direct correlation between Survival after vaccination and weight of head kidney.spleen, Phagocytic activity of leukocyte, erum complement 3, IgM level (r1=+0.936, r2=+0.981, r3=+0.9841,r4=+0.932, r5=+0.934, P<0.01). These results suggested that threonine could enhance disease resistance of juvenile Jian carp,which response to the improvement of immune organ growth and development, nonspecific immunity and specific immunity ability, the optimum dietary Thr requirement for optimal immune function of juvenile Jian carp(20-60g) was determined to be 1.56-1.69%。...
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