Effects of diet on amylase expression in the mosquitofish

Diets high in various carbohydrates were fed to mosquitofish,Gambusia affinis holbrooki, to determine the effects on amylase expression. Both amylase activity and amount of amylase protein were used as measures of amylase expression. Fish were fed for 21 days in one experiment, seven days in a second experiment and 24 h in a third. The first experiment compared responses of fish fed on a high-starch diet relative to a control diet. The second and third experiments compared responses on four diets relative to the control diet: maltose, starch, glucose, and glucose + cyclic adenosine monophosphate (cAMP). In the first two experiments whole visceral extracts were used. In the third experiment, gut and hepatopancreatic extracts were examined separately. Diet had a significant affect on the amount of amylase in all three experiments but affected amylase activity only in the 24 h experiment. Generally, glucose decreased amylase expression while maltose or cAMP + glucose increased it. Length of feeding period and tissue type also had significant effects on amylase expression.Paper IV on the amylase gene-enzyme system in fishes.     

不同生长阶段宝石鲈消化酶活性的初步研究

测定了出膜后30 d、70 d、120 d至成鱼4种规格,体重分别为(2.29±0.49)g、(23.16±4.23)g、(88.06±6.73)g、(270.26±9.17)g的宝石鲈(Scorturm barcoo)胃、肝胰脏、前肠、中肠、后肠的淀粉酶、胃蛋白酶和脂肪酶的活性。结果表明,随着宝石鲈的生长,淀粉酶在各生长阶段活力均很小,肝胰脏、前肠和后肠中的淀粉酶活力表现出逐渐增大的趋势;胃中的胃蛋白酶活性表现为逐渐增高,至规格3后胃蛋白酶活性趋于稳定;肠道是脂肪消化的主要场所,其中前肠脂肪酶变化趋势表现为先上升后下降,在前肠脂肪酶下降后中肠脂肪酶升高,起到了继续消化脂肪的作用。     

pH值对黄鳍鲷主要消化酶活性的影响

研究了pH值对黄鳍鲷肝、胃、肠3个部分主要消化酶(蛋白酶、脂肪酶和淀粉酶)比活的影响。结果显示,黄鳍鲷肝脏、胃和肠道蛋白酶最适pH值分别为:7.0、2.8和7.4,脂肪酶最适pH值分别为:7.2、7.6和7.6,淀粉酶最适pH值分别为4.8、5.2和6.8。胃蛋白酶的最适pH在酸性范围,肝和肠的最适pH值都在中性偏碱性范围。淀粉酶的最适pH值都在酸性范围,脂肪酶的最适pH都在碱性范围。     

团头鲂肠道肝胰脏消化酶的初步研究

本文采用动力学方法测定了团头鲂肝胰脏淀粉酶和肠道蛋白酶、淀粉酶的活性分布及pH值对各消化酶活性的影响,结果表明:食糜中的蛋白酶、淀粉酶活性沿消化道从前至后逐渐减弱,而肠黏膜中各消化酶活性则基本上是由前到后逐渐增加。在pH值6.0～9.0范围内,肠道蛋白酶活性在pH为8.6时为最大,在pH值6.2～8.6范围内,肝胰脏和肠道淀粉酶活性在pH值7.8时为最大。     

Digestive enzyme response to supplemental ingredients in practical diets for juvenile freshwater crayfish Cherax quadricarinatus

A feeding trial was conducted to determine digestive enzyme activity in Cherax quadricarinatus in response to diet composition. Eight experimental diets, each containing one of squid, red crab, sardine (600 and 650 g kg^?1 crude protein), sorghum, wheat meal, soy paste and the reference diet were evaluated over 30 days in terms of amylase, lipase, and protease activities in the hepatopancreas. The diet containing wheat meal produced the highest amylase activity. Lipase activity was not significantly different among treatments. Protease activity was higher in animals fed fish meal (60 g kg^?1 protein), soy paste and reference diet. Results suggest that C. quadricarinatus can modify hepatopancreatic enzyme levels in response to different compounds in the diet and can digest a broad variety of ingredients, particularly starch.     

黄鳍鲷主要消化酶活性在消化道不同部位的比较研究

研究了黄鳍鲷（Sparus latus Houttuyn）主要消化酶（蛋白酶、淀粉酶、脂肪酶）在消化道不同部位（肝胰脏、胃、前肠、中肠、后肠）的分布情况。结果表明，蛋白酶、淀粉酶、脂肪酶比活最高的部位分别是胃、肝胰脏和前肠，酶活性明显高于其他部分（P〈0．01）。从肠道消化酶的比活大小来看，蛋白酶比活是前肠、中肠、后肠依次降低，淀粉酶比活是中肠〉前肠〉后肠，脂肪酶比活是前肠〉后肠〉中肠。     

兴凯湖四种野生鲌消化酶活性比较研究

本文分析兴凯湖定点同日随机采集的翘嘴鲌Culter alburnus、蒙古鲌C.mongolicus、兴凯鲌C.dabryi和红鳍原鲌Cultrichthys erythropterus肝胰脏和肠道消化酶的活性,比较2龄鱼和≥3龄鱼两实验组样本的特点和差异。结果表明:四种鲌肠道蛋白酶和淀粉酶活性显著高于肝胰脏;蒙古鲌肝胰脏脂肪酶活性高于肠道,其他三种鲌类肠道脂肪酶活性高于肝胰脏(P0.05)。2龄和≥3龄鱼蒙古鲌肝胰脏蛋白酶活性最高,翘嘴鲌和兴凯鲌分别次之(P0.05);2龄鱼兴凯鲌、蒙古鲌、翘嘴鲌肠道蛋白酶活性较高,≥3龄鱼蒙古鲌和红鳍原鲌显著高于兴凯鲌、翘嘴鲌(P0.05)。2龄组和≥3龄鱼翘嘴鲌肠道脂肪酶活性和肝胰脏、肠道淀粉酶活性均较高,≥3龄蒙古鲌和兴凯鲌肝胰脏脂肪酶活性显著高于翘嘴鲌和红鳍原鲌(P0.05)。本研究结果对科学保护与利用相关鱼类有参考价值。     

温度和pH值对斜带髭鲷蛋白酶、淀粉酶活性的影响

研究了温度和pH值对斜带髭鲷Hapalogenys nitens蛋白酶、淀粉酶活力的影响。结果表明,斜带髭鲷胃内pH值范围为4.9～5.4;肝胰脏pH值为5.8～6.2;肠道pH值为6.5～6.9。蛋白酶活性随温度的上升而增加,在40℃达最大,40℃以上酶活性随温度的上升而下降。在不同消化器官中,蛋白酶活性大小顺序为：胃〉前肠〉幽门盲囊〉后肠〉肝胰脏。在15～50℃范围内,斜带髭鲷消化道不同部位淀粉酶活性的最适温度均为35℃,淀粉酶活性由高到低顺序为：肝胰脏〉幽门盲囊〉前肠〉后肠〉胃。在pH值为2.2～7.6范围内,胃蛋白酶活性的最适pH值为2.8;在pH值为4.8～8.0范围内,其他消化器官蛋白酶的最适pH值均为7.2;在最适pH值下,各消化器官中的蛋白酶活性由高到低顺序为：前肠〉幽门盲囊〉后肠〉肝胰脏。在pH值为4.8～8.0范围内,胃淀粉酶活性的最适pH值为6.0,肠、肝胰脏与幽门盲囊淀粉酶的最适pH值均为6.8,活性由高到低顺序为：肝胰脏〉幽门盲囊〉前肠〉后肠〉胃。在最适温度和pH值下,蛋白酶活性由高到低顺序为：胃〉前肠〉幽门盲囊〉后肠〉肝胰脏;淀粉酶活性由高到低顺序为：肝胰脏〉幽门盲囊〉前肠〉后肠〉胃。     

Effects of dietary Bacillus and medicinal herbs on the growth, digestive enzyme activity, and serum biochemical parameters of the shrimp Litopenaeus vannamei

White shrimp (Litopenaeus vannamei) was fed six different formulations of medicinal herbs (MH) and Bacillus in a feeding trial conducted for 56 days. The survival, growth, digestive enzyme activity, and serum biochemical parameters were assessed. A basal diet without MH and Bacillus was served as the control. At the end of feeding trial, survival ranged from 76.19% to 84.76% with no significant differences (P > 0.05) among all groups. Growth measured as final weight, specific growth rate (SGR), protein efficiency ratio (PER), protein productive value (PPV), and feed efficiency ratio (FER) were improved with increased dietary MH and Bacillus. Shrimp fed 0.2% MH and 0.3% Bacillus (Diet 23) were found to have the best growth performance. For shrimp fed Diet 23 final weight and SGR were significantly (P < 0.05) higher than for controls. The digestive enzyme activity of protease and amylase in hepatopancreas and intestines were significantly (P < 0.05) enhanced by the feed supplements, however amylase of the hepatopancreas was not affected. Serum protein and glucose content were enhanced by the supplement of MH and Bacillus. Serum cholesterol in shrimp of high Bacillus level groups was significantly (P < 0.05) higher than in the control group. However, no significant difference (P > 0.05) in serum Triacylglycerols between supplemented groups and control was found. Results revealed that the combined MH and Bacillus in diet could enhance growth because it can improve digestive enzyme activity and digestive metabolism.     

不同年龄黑尾近红鲌消化酶活性比较

用生物化学方法测定了一龄和二龄黑尾近红鲌(Ancherythroculter nigrocauda)肝胰脏及肠道蛋白酶、淀粉酶和脂肪酶的活性,并对两个年龄组个体的上述消化酶活性分别进行了比较。实验测得一龄鱼蛋白酶活性大小为前肠>中肠>后肠>肝胰脏,二龄鱼蛋白酶活性大小为中肠>前肠>后肠>肝胰脏;两个年龄组淀粉酶活性变化趋势相同,为肝胰脏>中肠>后肠>前肠;两个年龄组消化道中脂肪酶活性变化趋势也基本相同,为后肠>中肠>前肠>肝胰脏。总体来看,一龄鱼消化酶活性大于二龄鱼,蛋白酶和淀粉酶活性差异显著,而脂肪酶活性差异不显著。结果表明:在二龄鱼人工配合饲料中,要适当减少蛋白质和碳水化合物的比例。     

Ontogeny of protease, amylase and lipase in the alimentary tract of hybrid Juvenile tilapia (Oreochromis niloticus × Oreochromis aureus)

Juvenile tilapias (Oreochromis niloticus × O. aureus), with average body weights of groups I, II and III of 55.14, 122.82, and 225.68 g respectively were used to study changes in the activity of protease, amylase, and lipase in different organs and sections of the alimentary tract. Additionally, tract containing digesta, which is closer to physiological conditions, may help reveal the mechanisms of whole chemical digestion process. Another group of tilapias (mean body weight of 111.81 ± 0.14 g) was used to analyze changes in activity of protease, lipase, and amylase in response to varying pH and temperatures. The activity of enzymes in all digestive organs increased when body weight increased from 55 to 122 g, and the activity further increased when the weights increased from 122 to 225 g. The rates of increase in digestive enzyme activity were significantly greater than the rates of growth of the fish. When the activity was compared in different sections of the tract, a similar regularity was found for the three enzymes in all fish. The highest amounts of activity were seen in the foregut and the lowest in the rectum, with the middle amount of activity in the hindgut. The pH ranges for the maximum activity of tilapia protease in the stomach and intestine were 1.6–3.3 and 9.5–10.5 respectively and the optimal temperature in these organs was 55°C. The pH ranges for the maximum amylase activity in the intestine and hepatopancreas were 6–7 and 7.5 respectively, and high amylase activity in these organs was found at 25–35°C. The activity of lipase in the intestine remained unchanged at pH 6.0–9.0 and with temperatures between 25 and 35°C. These results suggest that the ability of tilapia to utilize feed varies at different growth stages and remains relatively stable with changes in environmental pH and temperature, which should be taken into account when designing feed formulas for tilapia.     

The effect of replacement of fish meal by yeast extract on the digestibility,growth and muscle composition of the shrimp Litopenaeus vannamei

Feeding trials were conducted to determine the dietary level of yeast extract (YE) for replacing dietary fish meal for evaluating whether yeast extract was superior to intact yeast as an alternative protein source for shrimp Litopenaeus vannamei. The basal diet (control, D0, containing 25% fish meal), was compared with five isonitrogenous and isoenergetic experimental diets [replacing 15% (D15), 30% (D30), 45% (D45), 60% (D60) or 100% (D100) of the fish meal in the basal diet with IYE]. The digestibility, growth and muscle composition of the shrimp were measured. The results showed that all replacement treatments displayed higher apparent digestibility of crude protein than did the control. The trypsinase activity in shrimp hepatopancreas increased significantly, whereas lipase activity decreased as the amount of dietary YE increased. The shrimp treated with D30 diet displayed the highest amylase activity in hepatopancreas. There was no significant difference in the weight gain (WG) and survival of shrimp between the control and the YE replacement treatments. Feed conversion ratio (FCR) increased as the dietary YE increased, and the FCRs of the D60 and the D100 treatments were significantly higher than that of the control (P < 0.05). The growth performance among the treatments was closely related to the similarity of the essential amino acids in the diets. There was no significant difference in muscle composition of the shrimp between control and other treatments. In conclusion, up to approximately 45% of the fish meal in shrimp diet can be replaced by yeast extract in the presence of supplemental fish oil, phosphorus and calcium.     

饲料蛋白质水平和加工工艺对克氏原螯虾生长性能、消化力和抗氧化性能的影响

为研究不同蛋白质水平和加工工艺饲料对克氏原螯虾(Procambarus clarkii)生长性能、消化力和抗氧化能力的影响,本研究采用4个蛋白质水平(28%、30%、32%和36%)和2种加工工艺即膨化饲料(EF)和硬颗粒饲料(PF)的4×2双因素实验设计,进行为期13周的养殖实验。结果显示,蛋白质水平和加工工艺对克氏原螯虾增重率和肝体比有显著的交互作用(P<0.05)。主效应结果显示,28%蛋白质可显著提高增重率和特定生长率,EF组的末重和增重率显著高于PF组(P<0.05)。蛋白质水平和加工工艺对肝胰脏的胰蛋白酶和淀粉酶活性有交互作用(P<0.05)。主效应结果显示,肝胰脏的胰蛋白酶、淀粉酶和肠道胰蛋白酶、淀粉酶均在28%蛋白质水平时最高,EF组肝胰脏胰蛋白酶、淀粉酶和肠道胰蛋白酶活力高于PF组(P<0.05)。蛋白质水平和加工工艺对肝胰脏的丙二醛和碱性磷酸酶活性有显著的交互作用(P<0.05)。主效应结果显示,肝胰脏碱性磷酸酶活性以及血清的碱性磷酸酶和酸性磷酸酶均在28%蛋白质水平时最低。PF组肝胰脏丙二醛含量显著低于EF组(P<0.05),碱性磷酸酶活性显著低于EF组(P<0.05)。4种模型(Logistic、Gompertz、Von Bertalanffy和Brody)均能很好地模拟克氏原螯虾体重和体长的生长曲线,拟合度均大于0.97,其中,以Logistic模型拟合克氏原螯虾体重和体长最优,且幂函数可以反映体重和体长之间的关系。综上所述,28%蛋白质水平和膨化加工工艺使克氏原螯虾具有最优的生长性能和消化力,28%蛋白质水平的硬颗粒饲料可提高克氏原螯虾的抗氧化能力。     

乳化剂对异育银鲫消化酶及血清指标的影响

选取8000尾异育银鲫,随机分为2个处理(对照组和试验组),每处理2重复,每重复2000尾,分别饲喂含乳化剂0%和0.05%的基础日粮,试验期为62 d。试验结果表明,饲养31 d后,添加乳化剂使异育银鲫的肝胰脏胰蛋白酶的活性显著提高(P<0.01),肠道胰蛋白酶活性也表现出升高的趋势(P=0.08);血清中总蛋白、球蛋白、葡萄糖和甘油三酯的含量也显著提高(P<0.01)。饲养62 d后,添加乳化剂使异育银鲫肠道淀粉酶活性显著高于对照组(P<0.05);血清中葡萄糖和甘油三酯的含量显著高于对照组(P<0.01)。试验结果表明,饲料中添加乳化剂能够提高异育银鲫的肠道和肝胰脏消化酶的活性,促进脂肪代谢和蛋白质沉积。     

Bacterial Change in the Gut and Hepatopancreas of Tiger Shrimp Reared in Bioaugmented System

The study was conducted in nine 150‐L capacity glass aquaria for 192 h to determine the changes in the microflora in the gut and hepatopancreas of tiger shrimp, Penaeus monodon, after bioaugmenting (water treatment and probiotics). Results showed that luminous bacterial counts were significantly lower in bioaugmented than in nonbioaugmented systems (range 0–5.9 × 10³ cfu/g and 0–3.2 × 10³ cfu/g in gut and hepatopancreas, respectively). Biochemical tests of isolates showed that the bioaugmented systems were dominated by Gram‐positive Bacillus and Streptococcus and the Gram‐negative Vibrio dominated the control. In water‐treated and probiotics‐fed bioaugmented system, the change in bacterial dominance to Bacillus became evident 2 h in the gut and 24 h in the hepatopancreas. Bacterial dominance shifted to Vibrio species after 120 h. Shrimp in the control were observed to become lethargic after 72 h as Vibrio species significantly increased in number. Dissolved oxygen and unionized ammonia levels were significantly lower in bioaugmented system compared with the control.     

Changes in lipid class and fatty acid composition of adult male Litopenaeus vannamei (Boone) in response to culture temperature and food deprivation

The effects of culture temperature and food deprivation on lipid class and fatty acid composition of adult male Litopenaeus vannamei (Boone) were investigated. Shrimp were maintained in recirculating seawater systems at temperatures of 26 and 32°C and fed 75% dry commercial feed and 25% fresh‐frozen squid for 42 days. Additionally, groups of fed and non‐fed shrimp were maintained at 26°C for 17 days. In shrimp fed at either 26 or 32°C, polar lipids were the main constituents of total identified lipid classes in muscle tissue (66–71%), while neutral lipids were more abundant in hepatopancreas (82–88%). Higher levels of triglycerides were observed in lipids of shrimp hepatopancreas kept at 32°C, but no other lipid class was affected by temperature. A significantly higher proportion of 22:6n‐3 was consistent in muscle and hepatopancreas polar and neutral lipids of shrimp maintained at 26°C. In response to food deprivation, the amount of polar lipids, but not neutral lipids, was reduced by approximately 28% in muscle tissue, whereas all lipid reserves were almost depleted in the hepatopancreas. The variable consumption of some individual fatty acids was observed in polar and neutral lipids of both tissues.     

Effect of substitution of dietary fish meal by soybean meal on different sizes of gibel carp (Carassius auratus gibelio): digestive enzyme gene expressions and activities,and intestinal and hepatic histology

A 7‐week growth trial was conducted to evaluate the effects of dietary soybean meal (SBM) on digestive enzyme activity of intestinal mucosa, mRNA levels of digestive enzymes in hepatopancreas, and the mid‐intestinal and hepatopancreas histology of gibel carp CAS III (Carassius auratus gibelio). Four different growth phases of gibel carp (initial body weight: fry, 0.8 g; juvenile, 5.0 g; 1‐year‐old, 62.7 g; and broodstock, 135.6 g) were tested. Seven isonitrogenous and iso‐energetic diets were formulated to contain different SBM replacement levels (0%, 20%, 40%, 60%, 80% and 100% of dietary fish meal protein), and another diet (SBMAA) contained all SBM protein and supplied crystalline amino acids. The results showed that the activities of mid‐intestine trypsin, α‐amylase and gamma glutamyl transpeptidase reduced with increased dietary SBM, while the chymotrypsin activity increased first and then decreased. The ultrastructures of intestinal epithelial cells and hepatopancreas cells in fry and broodstock fish were distinctly affected by 200 g kg^‐1 dietary SBM. Supplementation of dietary amino acid to the highest replacement groups was not sufficient to improve digestive and absorptive capacities and growth performance. Gibel carp may be adapted to dietary SBM through increase in gene expression of hepatopancreas digestive enzymes and has potential to utilize proceeded SBM as feedstuffs.     

Effects of dietary protein levels on the growth performance, digestive capacity and amino acid metabolism of juvenile Jian carp (Cyprinus carpio var. Jian)

This experiment was conducted to evaluate the effects of protein levels on the growth performance, digestive capacity and amino acid metabolism of juvenile Jian carp. Brown fish meal was used as the sole protein source in the present study. Six isoenergetic experimental diets containing 14.4 MJ kg^?1 of digestible energy and 220–495 g crude protein kg^?1 diets were fed to triplicate groups of 50 fish with a mean initial weight of 16.67 ± 0.01 g for 45 days. Per cent weight gain (PWG) and feed efficiency ratio (FER) improved with an increase in the dietary protein levels up to 330 g kg^?1 diet. The condition factor, relative gut length, intestinal folds height, hepatopancreas and intestine protein content improved with an increase in the protein levels up to 330–385 g kg^?1 diet. Trypsin, creatinkinase, Na⁺, K⁺‐ATPase and alkaline phosphatase activities generally followed the same tendency as that of growth parameters. Amylase and γ‐glutamyl transpeptidase (γ‐GT) activities were negatively correlated with increasing protein levels from 220 to 330 g kg^?1 diet, and no differences were found thereafter. Lipase activity was unaffected by protein levels. Lactobacillus amount was increased with protein levels up to 275 g kg^?1 diet, while Aeromonas amount followed the opposite pattern. Escherichia coli amount was not influenced by dietary protein levels. Glutamate–oxaloacetate transaminase (GOT) activities in the hepatopancreas and plasma ammonia concentration (PAC) were not influenced by protein levels between 220 and 275 g kg^?1 diet, but significantly increased with increasing protein levels from 275 to 440 g kg^?1 diet, and remained similar thereafter. Glutamate–pyruvate transaminase (GPT) activities significantly increased with protein levels >275 g kg^?1 diet. Based on the broken‐line model, the dietary protein requirement for PWG of Jian carp (16.7–55.0 g) was estimated to be 341 g kg^?1 diet with a digestible energy of 14.4 MJ kg^?1 diet.     

鲢、鳙蛋白酶、淀粉酶的研究

运用酶动力学方法研究了鲢、鳙的蛋白酶和淀粉酶的活性分布。鲢、鳙的蛋白酶活性在肝胰脏中最强,脾脏中也较强,而在胆汁中未测出。该酶活性在肠粘膜的第Ⅱ段最大,第Ⅰ段向第Ⅱ段递增,第Ⅱ段向肠后端递减,但第Ⅴ段的该酶活性比前段要高些。鲢的淀粉酶活性分布规律与上述情况相似,但鳙的淀粉酶活性在肠粘膜各段中由前向后呈递减趋势,并以第Ⅰ段为最大。在相同的器官组织中,鳙蛋白酶活性高于鲢,但差异不显著（Ｐ＞０．０５）,而淀粉酶活性则相反。这两种鱼的蛋白酶活性适宜ｐＨ为８．０～８．５,淀粉酶活性在ｐＨ７．５时最适。在鲢的肠内容物中发现有纤维素酶活性,但在肠粘膜中未检出该酶。     

Effects of lysine and methionine supplementation on growth,body composition and digestive function of grass carp (Ctenopharyngodon idella) fed plant protein diets using high‐level canola meal

A 76‐day feeding trial was carried out to evaluate the effects of Lysine and Methionine supplementation on growth and digestive capacity of grass carp (Ctenopharyngodon idella) fed plant protein diets using high‐level canola meal (CM). Fish with initial average weight 103.9 ± 0.6 g were fed three extruded diets. Fish meal (FM) diet was formulated as the normal control with 40 g kg^?1 FM and 300 g kg^?1 CM; CM diet was prepared by replacing all FM with CM (total 340 g kg^?1) without Lys or Met supplementation; CM supplement (CMS) diet was similar to CM diet but was supplemented with essential amino acids (EAA) to ensure the levels of Lys and Met similar to those in the FM diet. Feed intake, feed efficiency and specific growth rate of the grass carp fed CMS and FM diets were similar (P > 0.05), but higher than those of the grass carp fed CM diet (P < 0.05). The hepatosomatic index, relative gut length, intestosomatic index and intestinal folds height were significantly improved in fish fed FM and CMS diets as compared to CM diet (P < 0.05). Lower activities of trypsin, lipase and amylase in hepatopancreas were observed in fish fed CM diet (P < 0.05). Three hundred and forty gram per kilogram CM without Lys or Met supplementation significantly decreased trypsin, lipase and amylase mRNA levels in hepatopancreas (P < 0.05). These results indicated that the high supply of CM (340 g kg^?1) in plant protein (200 g kg^?1 soybean meal and 100 g kg^?1 cottonseed meal) diets decreased digestive ability through decreasing digestive enzyme activities and enzyme gene's expressions of grass carp, and these side effects can be reversed by supplementing Lys and Met. Therefore, CM could be high level used in a plant protein blend‐based extruded diet for grass carp as long as EAA were supplemented.