Dietary manganese requirement of juvenile grass carp (Ctenopharyngodon idella Val.) based on growth and tissue manganese concentration

A growth trial was conducted to estimate the optimum concentration of dietary Manganese (Mn) for grass carp (Ctenopharyngodon idella). Triplicate groups of grass carp (3.97 ± 0.05 g) were fed diets containing graded levels (4.0, 8.9, 13.8, 18.7, 23.6 and 33.3 mg kg^?1) of Mn for 8 weeks. Weight gain, specific growth rate and feed efficiency were linearly increased up to the 18.7 mg kg^?1 dietary Mn and then levelled off beyond this level. For body composition, lipid contents in whole body, muscle and liver decreased significantly with increasing dietary Mn level. Grass carp fed with dietary Mn levels higher than 19.7 mg kg^?1 significantly decreased condition factor. Whole body, vertebrae and scales mineralization were all affected significantly by dietary Mn levels. Mn contents in whole body, vertebrae and scales were linearly increased up to the 18.7 mg kg^?1 dietary Mn and then levelled off beyond this level. Contrarily, Ca and P contents seem to be inversely related to dietary Mn. However, dietary Mn levels had no significant effect on body Fe contents. Broken‐line analysis indicated that 20.6 mg kg^?1 dietary Mn was required for maximal tissue Mn storage, as well as satisfied for the optimal growth of juvenile grass carp.     

Dietary potassium requirement of juvenile grass carp (Ctenopharyngodon idella Val.) based on growth and tissue potassium content

A growth trial was conducted to estimate the optimum concentration of dietary potassium (K) for grass carp (Ctenopharyngodon idella). Triplicate groups of grass carp (3.96 ± 0.06 g) were fed diets containing graded levels (0.87, 2.90, 5.37, 7.54, 9.87 and 12.4 g kg^?1) of K for 8 weeks. Final body weight, weight gain and feed efficiency and gill Na⁺‐K⁺ ATPase activity were highest in fish fed with 9.87 g kg^?1 dietary K and lowest in fish fed the basal diet (P < 0.05). The K contents in whole body and muscle were linearly increased up to the 9.87 g kg^?1 dietary K and then levelled off beyond this level, whereas in scales and vertebrae up to the 7.54 g kg^?1 dietary K (P < 0.05). However, dietary K levels had no significant effect on ash, Ca, P and Mg contents in whole body, scales, vertebrae or muscle. Analysis using polynomial regression of weight gain and gill Na⁺‐K⁺ ATPase activity and using the broken‐line regression of whole body K concentrations indicated that the adequate dietary K concentration for grass carp is about 9.45–9.99 g kg^?1 diet.     

Dietary available phosphorus requirement of juvenile grass carp (Ctenopharyngodon idella)

A growth trial was conducted to estimate the optimum concentration of dietary available phosphorus (P) for grass carp (Ctenopharyngodon idella). Triplicate groups of grass carp (5.59 ± 0.02 g) were fed diets containing graded levels (2.36, 4.27, 6.31, 8.36, 10.4 and 14.8 g kg^?1) of available P for 8 weeks. Grass carp fed with the P‐supplemented diets had significantly higher specific growth rate, weight gain, protein efficiency ratio and feed efficiency than fish fed with the basal diet. In whole‐body composition, protein content increased, while lipid content decreased with the increase in P level in diet (P < 0.05). Fish fed with the P‐supplemented diets had significantly higher whole body, vertebrae and scales mineralization (P < 0.05), but Ca/P ratios were not influenced. The blood chemistry analysis showed that dietary available P had distinct effects on P, Ca and Mg contents, as well as on the contents of triacylglycerol and total cholesterol. Broken‐line analysis indicated that 8.49 g kg^?1 dietary available P was required for maximal tissue storage and mineralization as well as optimal growth.     

Effect of dietary lipid level on growth, feed utilization and body composition by juvenile grass carp (Ctenopharyngodon idella)

A study was undertaken to determine the effect of dietary lipid level on growth, feed efficiency and body chemical composition of juvenile grass carp. Seven isonitrogenous diets (400 g kg^?1 crude protein) containing seven dietary lipid level (0, 20, 40, 60, 80, 100 and 120 g kg^?1 dry matter) were fed to triplicate groups of 40 fish with initial weight 6.52 g, for 70 days. No obvious and assured essential fatty acid deficiency symptom appeared in fish fed the lipid‐free diet. Excess dietary lipid level (100 and 120 g kg^?1) resulted in decreased feed intake. The best growth performance and feed utilization was observed in fish fed 20–40 g kg^?1 dietary lipid. The fish fed a lipid‐free diet had the lowest protein efficiency and protein retention. Growth performance and feed utilization increased with the increasing dietary lipid levels up to 40 g kg^?1 dietary lipid. Higher dietary level (above 40 g kg^?1) made growth performance and feed utilization decrease and no protein sparing effect was observed. Lipid retention decreased as dietary lipid level increased. Mesenteric fat index (MFI) increased, hepatosomatic index (HSI) decreased with dietary lipid level. The increased MFI and simultaneous decrease lipid retention can be explained by differences in growth. The effect of dietary lipid levels on the chemical composition of tissues was significant only for whole body and muscle. The excess lipid content of liver in all groups was regarded as a slight symptom of fatty liver, which was partly identified by microscopic structural study and lower plasma lipid indexes, comparing to the initial plasma data. In conclusion, grass carp is a fish with low energy requirement and excess dietary lipid level should be avoided.     

投饲率对草鱼生长、体组成和能量收支的影响

为了建立草鱼(Ctenopharyngodon idella)的精准投喂模型管理系统,研究了不同投喂水平下草鱼的能量收支。实验在水温(27.5±2.0)℃条件下,选用初始体重(71.03±1.13)g的草鱼,共设5个不同投饲水平(饥饿、1%鱼体重(1%BW)、2%BW、4%BW和饱食),研究摄食水平对草鱼生长、鱼体组分和能量收支的影响。结果显示:随投喂水平的增加,草鱼鱼体水分减少,粗蛋白含量、粗脂肪含量和能量增加,灰分含量先减少后增加。特定生长率和热积温生长系数均随投喂水平的增加先增加再降低,在5%鱼体重时达到最大,且高于饱食投喂组。饵料转化率(FE)在投喂水平为体重2%组最高,且显著高于其他各组。干物质和能量表观消化率随投喂水平的增加而显著增加,蛋白质表观消化率在投喂水平为体重2%组显著高于其他组。生长能比例随投喂水平的增加而显著增加,而在体重2%组到饱食组间均无显著性差异;代谢能与生长能相反。在最大投喂水平下的能量收支方程为:100 C=21.72 F+4.25 U+45.85R+28.18 G或100 A=61.94R+38.06G。结果表明,5%鱼体重的投喂量为草鱼此阶段的最佳投喂水平。     

Dietary zinc requirement of juvenile grass carp (Ctenopharyngodon idella) based on growth and mineralization

A growth trial was conducted to estimate the optimum requirement of dietary zinc (Zn) for grass carp (Ctenopharyngodon idella). Triplicate groups of grass carp (3.97 ± 0.05 g) were fed diets containing graded levels (13, 25, 34, 53, 89 and 135 mg kg^?1) of Zn for 8 weeks. Grass carp fed with dietary Zn levels higher than 34 mg kg^?1 significantly increased final body weight, weight gain and specific growth rate (P < 0.05). For body composition, fish fed with dietary Zn levels higher than 53 mg kg^?1 significantly decreased the moisture contents but increased the lipid contents of whole body and liver. Whole body, scales, vertebrae and liver mineralization were all affected significantly (P < 0.05) by dietary Zn levels. Zn contents in whole body, scales, vertebrae and plasma were linearly increased up to the 53 mg kg^?1 dietary Zn and then remained stable beyond this level. Grass carp fed with dietary Zn levels higher than 53 mg kg^?1 significantly increased triacyglyceride and total cholesterol contents and plasma alkaline phosphatase activity in plasma (P < 0.05). Broken‐line analysis indicated that 55.1 mg kg^?1 dietary Zn was required for maximal tissue storage and mineralization as well as optimal growth of grass carp.     

Effect of dietary carbohydrate‐to‐lipid ratios on growth performance,body composition,nutrient utilization and hepatic enzymes activities of herbivorous grass carp (Ctenopharyngodon idella)

Six isonitrogenous (390 g kg^?1) and isoenergetic (16.2 kJ g^?1) diets with varying carbohydrate : lipid (CHO : L) ratios (202.5–1.74), were fed to triplicate groups of 25 fish in indoor recirculation system. Over 8‐week‐growth trial, best weight gain (WG), specific growth rate, feed conversion ratio, protein efficiency ratio and protein production value (P < 0.05) were observed in fish‐fed diets with CHO : L ratio of 7.5. Fish fed either the lowest (1.7) or highest (202.5) CHO : L ratio tended to produce lower (P < 0.05) growth and feed conversion efficiencies. The values of viscerosomatic index, hepatosomatic index and intraperitoneal fat ratio increased as dietary CHO : L ratios decreased. There were no significant differences in whole body and liver crude protein among dietary treatments. Whole body and liver lipid increased as CHO : L ratios decreased. Plasma cholesterol and triacylglyceride levels increased linearly as dietary CHO : L ratios decreased. Activities of glucokinase and pyruvate kinase were stimulated by elevated levels of dietary carbohydrate; however, activities of lipase (LPS) and alkaline phosphatase were stimulated by elevated levels of dietary lipid. Based on a second‐order polynomial regression analysis of WG against dietary carbohydrate and lipid levels, 275 g kg^?1 of carbohydrate and 59 g kg^?1 of lipid, corresponding to a CHO : L ratio of 4.7, in a diet holding 390 g kg^?1 of crude protein and 16.3 kJ g^?1 of gross energy, proved to be optimal for grass carp. These results indicated that utilization of dietary lipid and carbohydrate was moderate in grass carp, but the fish were a little more capable of utilizing lipid compared with carbohydrate.     

Effects of dietary non‐protein energy source levels on growth performance,body composition and lipid metabolism in herbivorous grass carp (Ctenopharyngodon idella Val.)

A study was conducted to determine the effects of dietary non‐protein energy sources on growth, tissue lipid accumulation and lipid metabolism‐related genes expression of grass carp. Triplicate groups of fish were fed for 9 weeks on four isonitrogenous (300 g kg^?1) experimental diets with four levels of non‐protein energy (6.52 kJ g^?1 control diet, 5.32 kJ g^?1 high‐CEL diet, 8.46 kJ g^?1 high‐CHO diet and 8.53 kJ g^?1 high‐LIP diet respectively). Increasing dietary non‐protein energy source levels did not improve the growth, and the high‐CEL diet reduced the growth of grass carp. The high‐CHO diet tended to induce high hepatosomatic index, with high fat and glycogen content of liver. However, the high‐LIP diet caused the high mesenteric fat index, but did not increase liver fat. The mRNA abundance and activities of hepatic lipogenic enzymes were significantly increased in the high‐CHO diet group, whereas the opposite tendencies were observed in the high‐LIP diet group. Peroxisome proliferator‐actived receptor‐α (PPARα) in liver and PPARγ in mesenteric adipose tissue were up‐regulated in the high‐CEL diet group. Lipoprotein lipase (LPL) gene expression was significantly increased both in liver and mesenteric adipose tissue of fish fed the high‐LIP diet, while the LPL gene expression was up‐regulated in liver but down‐regulated in mesenteric adipose tissue of fish fed the high‐CEL diet. These findings suggest that an increase in dietary non‐protein energy sources alters the genes expression of lipid metabolism and increased lipid deposition.     

Dietary magnesium requirement and effects on growth and tissue magnesium content of juvenile grass carp (Ctenopharyngodon idella)

A growth trial was conducted to estimate the optimum concentration of dietary magnesium (Mg) for grass carp (Ctenopharyngodon idella). Triplicate groups of grass carp (5.56 ± 0.02 g) were fed diets containing graded levels (187, 331, 473, 637, 779 and 937 mg kg^?1) of Mg for 8 weeks. Weight gain, specific growth rate and feed efficiency were linearly increased up to 637 mg kg^?1 dietary Mg and then levelled off beyond this level. For body composition, dietary Mg levels higher than 473 mg kg^?1 significantly decreased the moisture content but increased the lipid content of whole body, muscle and liver. Dietary Mg levels higher than 473 mg kg^?1 significantly decreased the ash contents of vertebrae, scales and muscle. Mg contents in whole body, vertebrae, scales and plasma were increased up to 637 mg kg^?1 dietary Mg and then levelled off beyond this level. However, Ca and P contents seem to be inversely related to dietary Mg. Dietary Mg levels higher than 473 mg kg^?1 significantly decreased Zn and Fe contents in whole body and vertebrae. Broken‐line analysis indicated that 687 mg kg^?1 dietary Mg was required for maximal tissue Mg storage, as well as satisfied for the optimal growth.     

饲料中添加生物素对草鱼幼鱼生长、体成分和血清生化指标的影响

在纯化饲料中分别添加生物素0、0.05、0.10、0.20、0.40、0.80、1.60 mg/kg投喂初始质量为(5.92±0.25)g的草鱼(Ctenopharyngodon idellus)幼鱼8周,研究了不同生物素添加量对草鱼幼鱼生长性能、饲料系数、机体营养成分、血清生化指标的影响。试验结果显示:与对照组相比,添加生物素提高了草鱼幼鱼的增重率、特定生长率,降低了饲料系数。添加量为0.40 mg/kg时草鱼幼鱼的特定生长率和增重率最大,饲料系数最低,并与对照组存在显著差异(P<0.05);添加不同水平生物素对草鱼幼鱼全鱼水分、粗蛋白、粗脂肪含量无显著影响,但添加量为0.40 mg/kg时粗蛋白含量最大。0.10 mg/kg组和0.20 mg/kg组的全鱼灰分含量显著高于对照组(P<0.05);添加生物素对血清总蛋白(TP)、血糖(GLU)和总胆固醇(TC)无显著影响,但显著提高了血清甘油三酯(TG)含量,各添加组TG含量均显著高于对照组(P<0.05),1.60 mg/kg添加组的高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C)含量显著高于对照组(P<0.05)。综合本试验结果,草鱼幼鱼饲料中生物素适宜添加量为0.40 mg/kg。     

镁对草鱼生长、形体、肝功能和糖代谢的影响

在以酪蛋白和明胶为蛋白源的纯化日粮(镁含量76.9 mg/kg)中添加不同水平的镁(0、150、300、600、1200和2400 mg/kg),研究镁对体重为(10.68±0.14)g的草鱼(Ctenopharyngodon idella)生长、形体、肝功能和糖代谢的影响,时间为10周.结果显示:日粮中适当添加镁显著...     

Dietary calcium requirement and effects on growth and tissue calcium content of juvenile grass carp (Ctenopharyngodon idella)

A growth trial was conducted to estimate the optimum concentration of dietary calcium (Ca) for grass carp (Ctenopharyngodon idella). Triplicate groups of grass carp (4.52 ± 0.02 g) were fed diets containing graded levels (2.75, 4.51, 6.24, 7.99, 9.66 and 11.5 g kg^?1) of Ca for 8 weeks. Weight gain, feed efficiency and protein efficiency ratio were linearly increased up to the 7.99 g kg^?1 dietary Ca and then maintained stable beyond this level (P < 0.05). Dietary Ca levels higher than 7.99 g kg^?1 significantly increased the ash contents of whole body, vertebrae and scales. Ca contents in whole body, vertebrae and scales were linearly increased up to the 7.99 g kg^?1 dietary Ca and then maintained stable beyond this level (P < 0.05). In contrast, dietary Ca levels higher than 9.66 g kg^?1 significantly decreased Mg contents in whole body, vertebrae and scales. Dietary Ca levels higher than 7.99 g kg^?1 significantly increased plasma alkaline phosphatase activity. However, plasma Ca, P and Mg contents were not significantly affected by dietary Ca supplements (P > 0.05). Polynomial regression analysis indicated that 10.4 g kg^?1 dietary Ca was required for maximal tissue storage and mineralization as well as optimal growth.     

草鱼C1qC基因的克隆及表达分析

为了研究C1qC基因在草鱼(Ctenopharyngodon idella)免疫过程中所起的作用,利用RT-PCR和RACE方法克隆获得了C1qC基因cDNA全长序列,经序列分析表明,所克隆的C1qC cDNA全长为916 bp,包括开放阅读框(open reading frame,ORF)735 bp,5′端非编码区(untranslated region,UTR)89 bp和3′端非编码区(UTR)92 bp。735 bp的ORF共编码244个氨基酸,相对分子量为26 162.5 U。同源性分析表明,草鱼与斑马鱼(Danio rerio)的相似度最高,达到71%。经草鱼呼肠孤病毒(grass carp reovirus,GCRV)诱导后,草鱼C1qC基因在鳃、皮肤、肌肉、肝、中肾、心脏、头肾等组织中的mRNA表达水平均显著上调。在草鱼胚胎发育的各个阶段都能检测到C1qC mRNA的表达,说明该基因可能在草鱼胚胎和鱼苗的免疫反应和早期发育中起重要作用。本研究将为今后在草鱼免疫功能方面深入研究C1qC基因提供基础资料。     

长江草鱼群体MHC Class ⅡB的多态性和进化分析

克隆及测序草鱼(Ctenopharyngodon idella)长江3个群体的主要组织相容性复合体(MHC)Class II B基因编码β1和β2区的第2和第3个外显子及两个外显子之间的内含子,分析了草鱼MHC的进化模式和种群遗传结构。结果显示:实验共定义了34个等位基因,每条序列包括长为130~136 bp的第2个外显子,长为218 bp的第3个外显子以及长81~371 bp的内含子。序列分析揭示,第2个外显子有106个核苷酸变异位点(78%)和40个氨基酸变异位点(88%),而第3个外显子有100个核苷酸变异位点(45%)和41个氨基酸变异位点(56%),β1变异要大于β2区。用β1和β2区序列分别构建的邻接(NJ)系统树均显示5个具有高支持率的谱系,结合序列变异特点和内含子长度,推测草鱼至少存在5个MHC Class II座位。分别计算β1的肽结合位点(PBR)、非肽结合位点及β2的非同义替换率(dN)和同义替换率(dS),PBR的dN/dS为2.03(P<0.05),非肽结合位点和β2则小于1,表明草鱼MHC受到歧化选择作用。根据等位基因在群体中的分布频率作分子方差分析(AMOVA),得出FST为0.37%,提示长江草鱼MHC没有遗传分化。     

饲料中添加谷胱甘肽对草鱼生长、生理指标和抗病力的影响

选用初始体质量约8.50 g的草鱼(Ctenopharyngodon idella),在56 d的饲养期中分别投喂添加5种不同剂量谷胱甘肽(GSH)(添加量分别为0 mg/kg、100 mg/kg、200 mg/kg、300 mg/kg和400 mg/kg)的试验饲料,观察GSH对草鱼生长、生理指标和抗病力的影响。结果表明,饲料中添加GSH能够提高草鱼特定生长率、存活率和饲料效率。其中,300 mg/kg GSH组草鱼的特定生长率和400 mg/kg GSH组草鱼的存活率显著高于对照组;添加GSH各组草鱼的饲料效率均显著高于对照组,当添加量为200 mg/kg时草鱼饲料效率达到最高。与对照组相比,饲料中添加GSH的各组草鱼肝胰指数明显升高,其中200 mg/kg组达到显著水平。饲料中添加GSH能够提高血清IGF-1水平,其中300 mg/kg和400 mg/kg组显著高于对照组。与对照组相比,各实验组草鱼血液白细胞数目有不同程度升高,其中300 mg/kg和400 mg/kg组均达到显著水平。饲料中添加GSH可以提高草鱼对嗜水气单胞菌的抵抗能力,其中200 mg/kg GSH组草鱼攻毒后存活率达到最高。以特定生长率为判定指标,GSH在草鱼饲料中的适宜添加量为350 mg/kg。     

Dietary magnesium requirements of juvenile grass carp,Ctenopharyngodon idella

To determine dietary magnesium (Mg) requirements of juvenile grass carp, Ctenopharyngodon idella, magnesium sulphate was added to the basal diet at 0, 150, 300, 600, 1200, 2400 mg Mg kg⁻¹ diet. Each diet was fed to three replicate groups of juvenile grass carp (initial weight: 7.69 ± 0.13 g) in a closed, recirculating rearing system for 76 days. No mortality or nutritional deficiency signs were observed except the growth depression in fish fed the Mg‐deficient diet. Growth performance and activities of serum superoxide dismutase (SOD), glutathione peroxidase (GPx) and lysozyme (LSZ) were highest (P <0.05) in fish fed the diet supplemented with 600 mg Mg kg⁻¹. The serum malondialdehyde (MDA) content was higher (P <0.05) in fish fed the diets supplemented with 0 and 150 mg Mg kg⁻¹ than that in fish fed the diets with ≥300 mg Mg kg⁻¹. Mg concentrations both in whole‐body and vertebrae increased with the increase in dietary Mg level up to 300 mg kg⁻¹, whereupon the response reached a plateau. Analysis by second‐order polynomial regression of weight gain, by broken‐line regression of vertebrae Mg concentration and by linear regression of whole‐body Mg retention of fish indicated that the adequate dietary Mg concentration for juvenile grass carp was 713.5, 627.7 and 469.8 mg kg⁻¹ diet, respectively.     

草鱼HSP70基因cDNA部分序列克隆及其组织表达差异

根据鲤热休克蛋白70(Heat shock protein,HSP70)序列(AY120894)设计并合成一对引物,以草鱼(Cteno-pharyngodon idella)肝胰脏组织总RNA为模板,RT-PCR扩增获得草鱼HSP70基因cDNA部分序列,并进行了组织表达差异性研究。结果显示:所获为序列为480 bp,获得GeneBank登陆号为FJ483832。序列测序结果显示,HSP70扩增序列与鲤、斑马鱼、鲋的同源性为:93%、91%、93%。另外,所获序列HSP70在草鱼脂肪、肌肉、肠、脑、粘液、性腺、鳔、肝胰脏、心脏、脾脏、鳃、鳍12个组织的表达存在差异,HSP70在草鱼这12个组织中均检测到表达,其中在鳍中表达最高,极显著高于其他组织(P<0.01);在鳔中表达次之,且与脑、心脏、性腺中表达差异不显著;在粘液中表达最低。     

Molecular cloning of fatty acid synthase from grass carp (Ctenopharyngodon idella) and the regulation of its expression by dietary fat level

The fatty acid synthase (FAS) gene was cloned from liver of grass carp (Ctenopharyngodon idella) by degenerate oligonucleotide primed PCR. The obtained cDNA fragment was 683 bp, which encoded 227 amino acids. Then, grass carps with initial body weight of (134.89 ± 12.12) g were fed diet supplemented with 0, 20, 40 g kg^?1 fat to investigate the impacts of dietary fat levels on growth, liver FAS enzyme activity and mRNA expression. After 8 weeks feeding, final body weights of the three groups were 344.11, 347.23 and 373.02 g. Compared with control group (0 g kg^?1), growth rate of 40 g kg^?1 fat group was increased by 14.03%, and feed conversion rate decreased by 11.32% (P < 0.05), liver FAS enzyme activity of 20, 40 g kg^?1 fat groups were reduced by 33%, 64% (P < 0.05), and FAS mRNA expression level reduced by 18%, 74% (P < 0.05), respectively. Results above showed that 40 g kg^?1 fat addition can significantly improve growth performance of grass carp. Liver FAS activity and mRNA expression tended to be inhibited by the increasing dietary fat level. Fat containing high levels of polyunsaturated fatty acids had strongly inhibitory effects on liver FAS activity and gene expression.     

Use of probiotics Bacillus coagulans,Rhodopseudomonas palustris and Lactobacillus acidophilus as growth promoters in grass carp (Ctenopharyngodon idella) fingerlings

The present research evaluated the effects of three probiotics on the growth performance and intestinal digestive enzyme activity in fingerlings of grass carp, Ctenopharyngodon idella. Three treatments (T‐1, T‐2 and T‐3) were fed with diets containing different viable bacteria with a final concentration 10⁶ CFU g^?1 feed (Bacillus coagulans, Rhodopseudomonas palustris and Lactobacillus acidophilus, respectively) and the control was fed with basal diet without probiotics. All the diets supplemented with probiotics resulted in better (P < 0.05) final weight, daily weight gain and relative gain rate than control but no significant differences were observed among the treatment groups. The highest protease activity was observed in T‐1. However, T‐2 and T‐3 showed no difference (P > 0.05) in protease and cellulase activities compared with the control. The protease activity was higher in the foreintestine than in the hindintestine (P < 0.05), and the reverse was observed in cellulase activity. As for amylase activity, there was no difference between foreintestine and hindintestine. In conclusion, the three selected probiotics increased the growth performance of grass carp fingerlings. Furthermore, different digestive enzyme activity was observed in different intestine segment.