色氨酸对幼建鲤消化吸收能力和疾病抵抗能力及其组织器官中TOR表达影响研究
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摘要
本研究通过体内、体外实验相结合的方法,探索了幼建鲤消化酶活力及肠道刷状缘相关酶活力变化规律,在此研究基础上研究了色氨酸对幼建鲤生产性能,营养物质沉积效率,氨基酸代谢,消化吸收能力、免疫力以及蛋白质合成信号调控分子TOR基因表达的影响；运用鱼的原代细胞研究模型,研究了色氨酸对幼建鲤肠细胞增殖分化、蛋白质合成以及TOR基因表达的影响。主要的研究内容和结果如下：
1幼建鲤消化酶和肠道刷状缘相关酶活力变化规律研究
选择体重为18.0±0.2g的健康建鲤750尾,设5个阶段,每个阶段3个重复,每个重复50尾鱼,均饲喂相同饲粮,分别于0、14、28、42和56天采样,探索不同体重建鲤消化酶活力及肠道刷状缘相关酶活力的变化规律。结果发现：随幼建鲤体重(18.0-74.7g)增加,肝胰脏和肠道蛋白含量、消化酶活力呈极显著(P<0.01)或显著(P<0.05)升高。当建鲤体重为74.7g时,肝胰脏和肠道蛋白含量均显著高于其余各体重组(P<0.05),肝胰脏胰蛋白酶和糜蛋白酶活力、肝胰脏和肠道淀粉酶活力均极显著高于其它各体重组(P<0.01)；当体重分别达到58.6,40.1,58.6和58.6g时,肝胰脏脂肪酶活力、肠道胰蛋白酶、糜蛋白酶和脂肪酶活力分别进入平台值(P>0.05)。随幼建鲤体重增加,其肠道刷状缘相关酶活力也呈极显著(P<0.01)或显著(P<0.05)升高。当幼建鲤体重为74.7g时,后肠Na+,K+-ATPase活力,前肠、中肠和后肠的碱性磷酸酶(AKP)、前肠γ-谷胱甘肽转氨酶(y-GT)活力均极显著高于其它各体重组(P<0.01),中肠y-GT显著高于其余各体重组(P<0.05)；当幼建鲤体重达到58.6g时,全肠肌酸激酶(CK)活力、前肠和中肠Na+,K+-ATPase和后肠γ-GT活力均进入稳定值(P>0.05)。研究结果表明：20-80g的幼建鲤随体重增加,胰腺分泌胰蛋白酶、糜蛋白酶、脂肪酶和淀粉酶的能力增加,其分泌能力的增加与胰腺的发育逐步完善有关；肠道吸收能力随幼建鲤体重增加逐步提高,其吸收能力的提高与肠道的发育逐步完善有关。
2色氨酸(Trp)对幼建鲤生长、体成分沉积以及血清、肝胰脏和肌肉中GPT和GOT活力的影响
选择体重为7.73±0.03g的健康建鲤1050尾,分成七个处理,每个处理设3个重复,每个重复50尾,分别饲喂Trp水平为0.11%、0.17%、0.25%、0.38%、0.49%、0.60%和0.69%的纯合饲粮60d,探索色氨酸对幼建鲤生长性能、体成分沉积以及血清、肝胰脏和肌肉中谷草转氨酶和谷丙转氨酶活力的影响。结果表明：色氨酸对幼建鲤生长性能、体成分沉积和肝胰脏、肌肉、血清的GOT、GPT活力以及血浆氨含量均有极显著(P<0.01)或显著(P<0.05)影响,并呈开口向上或开口向下的抛物线模型,其顶点多为0.38%的色氨酸水平。当色氨酸水平从0.11%升高到0.38%时,特异性增长率(SGR)、采食量、体蛋白含量、蛋白质沉积率、蛋白质效率、体脂肪含量、脂肪沉积率、灰分沉积率、肝胰脏和肌肉的GOT和GPT活力均极显著(P<0.01)或显著(P<0.05)升高,到0.38%时最高,随后均极显著(P<0.01)或显著(P<0.05)降低。当色氨酸水平从0.11%升高到0.38%时,饵料系数、体水分含量、体灰分含量、血氨含量、血清GOT和GPT活力均极显著(P<0.01)或显著(P<0.05)降低,到0.38%时最低,随后均极显著(P<0.01)或显著(P<0.05)升高。进一步相关分析发现增重与蛋白质沉积率、脂肪沉积率、灰分沉积率和采食量呈现极显著(P<0.01)或显著(P<0.05)的正相关关系；饵料系数与增重、蛋白质沉积率和脂肪沉积率呈现极显著(P<0.01)的负相关。结果说明：Trp能促进幼建鲤的生长,提高体蛋白和脂肪的沉积能力,增强肝胰脏和肌肉中的氨基酸代谢强度；以生长速度为标识确定的10-40g的幼建鲤Trp需要量为0.35%或1.09/100g蛋白质。
3 Trp对幼建鲤肝胰脏和肠道生长发育、消化酶活力和肠道刷状缘相关酶活力的影响
选择体重为7.73±0.03g的健康建鲤1050尾,分成七个处理,每个处理设3个重复,每个重复50尾,分别饲喂Trp水平为0.11%、0.17%、0.25%、0.38%、0.49%、0.60%、0.69%的纯合饲料60天。结果表明：当色氨酸水平从0.11%升高到0.38%时,幼建鲤肝胰脏重量、肝体指数、肝胰脏蛋白含量、肠重、肠长、肠道蛋白含量、肠体指数和肠长指数以及前、中、后肠的皱襞高度、肝胰脏和肠道胰蛋白酶、脂肪酶和淀粉酶活力,全肠肌酸激酶活力,前、中、后肠的碱性磷酸酶(AKP)、Na+,K+-ATP酶和γ-GT酶活力均极显著(P<0.01)或显著(P<0.05)升高,到0.38%时最高,随后极显著(P<0.01)或显著(P<0.05)下降。肠道糜蛋白酶活力在0.25%组最高(P<0.05)。色氨酸对肝胰脏糜蛋白酶活力无显著影响(P>0.05)。进一步相关分析发现蛋白沉积率与肠胰蛋白酶、肝胰脏胰蛋白酶、肝胰GOT、肌肉GOT、肝胰GPT和肌肉GPT呈现极显著(P<0.01)或显著(P<0.05)的正相关关系；脂肪沉积率与肝胰脏脂肪酶呈现显著(P<0.05)的正线性相关；饵料系数与肝胰脏胰蛋白酶和脂肪酶、肠道胰蛋白酶和糜蛋白酶、前肠和后肠Na+,K+-ATP、前中后肠AKP和γ-GT、CK活力呈现极显著(P<0.01)或显著(P<0.05)的线性负相关。本试验结果表明：Trp可以提高幼建鲤消化吸收能力。消化吸收能力的提高与Trp促进肝胰脏和肠道生长发育,提高蛋白酶、脂肪酶和淀粉酶分泌能力,增加肠道吸收面积和肠道上皮吸收能力有关。
4 Trp对幼建鲤肠道菌群、特异性免疫和非特异性免疫能力的影响
选择体重为7.73±0.03g的健康建鲤1050尾,分成七个处理,每个处理设3个重复,每个重复50尾,分别饲喂Trp水平为0.11%、0.17%、0.25%、0.38%、0.49%、0.60%、0.69%的纯合饲料,进行为期60天的生长实验,测定肠道菌群、红白细胞数量和免疫器官重量。生长实验结束后用嗜水气单胞菌攻毒17天,考察色氨酸对幼建鲤疾病抵抗力和免疫功能的影响。结果表明：当色氨酸水平从0.11%升高到0.25%,肠道乳酸杆菌极显著(P<0.01)或显著(P<0.05)提高,0.25%组和0.38%组差异不显著(P>0.05),随后极显著(P<0.01)或显著(P<0.05)降低；肠道大肠杆菌呈现刚好相反的模型。色氨酸对鲤鱼肠道嗜水气单胞菌无显著影响(P>0.05)。色氨酸极显著(P<0.01)或显著(P<0.05)提高了头肾、后肾、脾脏重量和相应的指数,当色氨酸水平达到0.38%时达到最大,随着色氨酸水平的再升高,头肾、后肾、脾脏重量和相应的指数极显著(P<0.01)或显著(P<0.05)降低。当色氨酸水平从0.11%升高到0.38%时,红细胞和白细胞数量均极显著(P<0.01)或显著(P<0.05)升高,到0.38%时最高,随后均极显著(P<0.01)或显著(P<0.05)降低。当色氨酸水平从0.11%升高到0.25%时,攻毒后成活率极显著升高(P<0.01),0.25%-0.49%处于平台值(P>0.05),随后均显著(P<0.05)降低。当色氨酸水平从0.11%升高到0.49%时,血清IgM含量极显著(P<0.01)或显著(P<0.05)升高,随后极显著降低(P<0.01)。血清抗嗜水气单胞菌效价随着色氨酸水平升高到0.38%而增加,随后逐渐下降(P<0.05)。当色氨酸水平从0.11%升高到0.38%时,白细胞吞噬率、血清补体C3、C4、溶菌酶活力、酸性磷酸酶活力和总铁结合力均极显著(P<0.01)或显著(P<0.05)升高,随后均极显著(P<0.01)或显著(P<0.05)降低,但对血清凝集素效价无显著影响(P>0.05)。相关分析发现,攻毒后成活率与免疫器官重量、抗体效价、白细胞吞噬率、ACP、C3、C4和总铁结合力呈现极显著(P<0.01)或显著(P<0.05)的线性相关；白细胞吞噬率与红白细胞数量和补体C3、C4含量呈现极显著(P<0.01)或显著(P<0.05)的正线性相关；红白细胞数量与免疫器官重量呈现极显著(P<0.01)或显著(P<0.05)的线性相关关系。本试验结果表明：Trp能提高幼建鲤的疾病抵抗能力以及改善其肠道微生态平衡。疾病抵抗能力的提高与Trp提高特异性免疫和非特异性免疫能力有关。非特异性免疫能力的提高与Trp提高其对病原菌的粘附调理、抑菌、溶菌能力有关。
5 Trp对幼建鲤前、中、后肠和肌肉中TOR mRNA含量的影响
选择体重为7.73±0.03g的健康建鲤1050尾,分成七个处理,每个处理设3个重复,每个重复50尾,分别饲喂Trp水平为0.11%、0.17%、0.25%、0.38%、0.49%、0.60%和0.69%的纯合饲料60天,研究Trp对幼建鲤肠道和肌肉TOR mRNA表达量的影响。结果发现：鲤鱼前肠和中肠的TOR mRNA表达量随色氨酸水平升高而降低,随后逐渐升高,当色氨酸水平为0.49%和0.38%时,TOR mRNA表达量分别为最低(P<0.05)。当色氨酸水平从0.11%到0.17%时,后肠TORmRNA表达量显著降低(P<0.05),色氨酸水平为0.17%和0.25%时,其mRNA表达量最低,随后逐渐上升,到最高水平(0.69%)时最大(P<0.05)。肌肉TORmRNA的表达量在色氨酸中间水平(0.38%和0.49%)最低(P<0.05),色氨酸低水平(0.11%)和高水平(0.60%和0.69%)表达量最高(P<0.05)。综合结果说明：日粮Trp缺乏或过量均提高幼建鲤前、中、后肠和肌肉中的TOR基因表达,但Trp在适宜水平时降低,其影响机制有待进一步研究。
6 Trp对幼建鲤肠上皮细胞增殖分化的影响
试验分为6个处理,每个处理设4个重复,选择体重为80g左右的健康鲤鱼进行肠道上皮细胞接种培养后分别添加色氨酸浓度为0、16、36、66、96和126mg/L的DMEM培养液继续培养96h,收集培养液和细胞,对相关指标进行分析测试。结果表明：随着色氨酸浓度上升到36mg/L-66mg/L时,观察发现贴壁生长细胞数量明显增多,并形成大的细胞集落和成片的单层细胞,随后细胞数量有所下降。色氨酸水平从0到36mg/L时,鲤鱼肠上皮细胞的MTTOD值显著上升(P<0.05),36mg/L和66mg/L差异不显著(P>0.05),随后逐渐下降(P<0.05)。鲤鱼肠上皮细胞蛋白含量、谷草转氨酶活力、谷丙转氨酶活力、碱性磷酸酶活力、Na+,K+-ATP活力随色氨酸水平升高而显著升高(P<0.05),当色氨酸水平达到36,36,66,36和16mg/L时分别达到最大,随后逐渐降低(P<0.05)。培养液氨含量和乳酸脱氢酶活力随色氨酸水平升高而显著降低(P<0.05),当色氨酸水平达到96和66mg/L时分别达到最低,随后逐渐降升高(P<0.05)。结果表明：色氨酸促进了鲤鱼肠道上皮细胞的增殖和分化,保证了功能正常。
7 Trp对幼建鲤肠细胞蛋白合成和TOR基因表达的影响
本研究进行了3小试验。(1)试验设2个处理组(0mg/L和36mg/L Trp),每个处理8个重复,培养时间为240min,研究Trp对肠细胞蛋白质合成的影响。(2)试验设5个处理组,每个处理6个重复,分别于15min,30min,100min,170min和240min采样,研究36mg/L Trp对肠细胞TOR基因表达影响的时间效应。(3)试验设2个处理组(0mg/L和36mg/L Trp),每个处理8个重复,培养时间100min,研究36mg/L Trp对肠细胞TOR基因表达的影响。结果表明：(1)36mg/L色氨酸处理组细胞蛋白质合成率显著高于未添加色氨酸的对照组(0mg/LTrp)(P<0.05)。(2)30min时,TORmRNA的表达量显著低于15min,并达到最低(P<0.05),随后逐渐升高,在170min和240min时,达到最高(P<0.05)。(3)36mg/L组TOR mRNA表达量显著高于未添加色氨酸的对照组(0mg/LTrp)(P<0.05)。结果说明：Trp可促进鲤鱼肠细胞蛋白质合成,同时影响了蛋白质合成信号调控分子TOR的表达。
综上所述：20-80g的幼建鲤随体重增加,胰腺分泌胰蛋白酶、糜蛋白酶、脂肪酶和淀粉酶的能力增加,其分泌能力的增加与胰腺的发育逐步完善有关；肠道吸收能力随幼建鲤体重增加逐步提高,其吸收能力的提高与肠道的发育逐步完善有关。Trp能促进幼建鲤的生长,提高了体蛋白和脂肪的沉积能力；Trp通过促进肝胰脏和肠道生长发育,提高蛋白酶、脂肪酶和淀粉酶分泌能力,增加肠道吸收面积和肠道上皮吸收能力有关；Trp影响肌肉和肠道蛋白质合成信号调控分子TOR mRNA的表达量,缺乏和过量表达量都升高；Trp促进了鲤鱼肠道细胞生长发育,保证了功能正常,促进鲤鱼肠细胞蛋白质合成,但蛋白质合成信号调控分子TOR的表达降低。Trp提高了幼建鲤的疾病抵抗能力。疾病抵抗能力的提高与Trp提高特异性免疫、非特异性免疫能力以及改善了其肠道微生态平衡有关。非特异性免疫能力的提高与Trp提高其对病原菌的粘附调理、抑菌、溶菌能力有关。以生长速度为标识确定的10-40g的幼建鲤Trp需要量为0.35%或1.09/100g蛋白质。
The first study was conducted to evaluate the effects of various weights on the digestive enzyme and intestinal brush border enzyme activities in Jian carp. Based on the first study, other studies of the effects of Trp on production performance, nutrient deposition, amino acid metabolism, digestion and absorption capacity, immunity and protein synthesis regulatory factor TOR expression in Jian carp were carried out. Using fish primary cell culture, we investigated the effects of Trp on the proliferation and differentiation, protein synthesis, and TOR expression of intestinal cell of Jian carp. The main researches and results are as follows:
1. The effects of various weights on digestive enzyme activity and intestinal brush border enzyme activities in Jian carp
A 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 day,28th day, 42nd day and 56th day sampling, to explore the effects of various weights on digestive enzyme and intestinal brush border enzyme activities in Jian carp. The results indicated that:The hepatopancreas and intestinal protein content, digestive enzyme activity was great significantly (P<0.01) or significantly (P<0.05) increased with the increasing levels of body weights from 18.0g to 74.7g. When Jian carp weighed 74.7g, the hepatopancreas and intestinal protein content were significantly higher than the other treatments (P<0.05), hepatopancreas trypsin and chymotrypsin activities, hepatopancreas and intestinal amylase activities were great significantly higher than the other treatments (P<0.01). When the body weights levels up to 58.6,40.1,58.6 and 58.6g, the hepatopancreas lipase activity, intestinal trypsin, chymotrypsin and lipase activities were all significantly increased, respectively (P<0.05), and remained nearly constant thereafter (P>0.05). The effects of the body weights of juvenile Jian carp on the intestinal brush border enzyme activity also showed great significantly (P<0.01) or significantly (P<0.05) increased. When Jian carp weighed to 74.7g, the distal intestinal Na+, K+-ATPase activity, proximal, mid and distal intestinal alkaline phosphatase (AKP), and the proximal y-glutathione transaminase (y-GT) activities were great significantly higher than other groups (P<0.01), the y-GT activity in mid intestine was significantly higher than the other groups (P<0.05). The intestinal creatine kinase (CK) activity, proximal and mid intestinal Na+, K+-ATPase and distal intestinal y-GT activities were all increased with the increasing weights levels up to 58.6g (P<0.05), and no differences were found with further increase of zinc levels (P>0.05). The results indicated that following the body weight of Jian carp from 20 g to 80 g, the increased secretary capacity of hepatopancreas to secret trypsin, chymotrypsin, lipase and amylase, was due to the growth and development of the hepatopancreas; the improvement of intestinal absorption capacity was due to the growth and the development of the intestine.
2. Effects of Trp on the growth performance, body composition and the GPT and GOT activities in serum, hepatopancreas and muscle of the juvenile Jian carp
A total of 1050 Jian carp (7.73±0.03g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Trp (0.11%,0.17%, 0.25%,0.38%,0.49%,0.60% and 0.69%) for 60 days to investigate the effects of Trp on growth performance, body deposition and the GPT and GOT activities in serum, hepatopancreas and muscle. The results showed that the growth performance, body deposition, the GPT and GOT activities in serum, hepatopancreas and muscle and plasma ammonia levels were great significantly (P<0.01) or significantly (P<0.05) affected by Trp. When the Trp levels increased from 0.11% to 0.38%, the specific growth rate (SGR), feed intake, body protein content, protein production value (PPV), protein efficiency ratio, body lipid content, lipid production value (LPV), ash production value (APV), hepatopancreas and the activities of GOT and GPT in muscle were great significantly (P<0.01) or significantly (P<0.05) increased, the maximum value was on 0.38% Trp, and with further increase of Trp levels, were great significantly (P<0.01) or significantly (P<0.05) decreased. When the Trp levels increased from 0.11% to 0.38%, the FCR, body water content, body ash content, ammonia content, serum GOT and GPT activities were great significantly (P<0.01) or significantly (P<0.05) decreased, and the results of 0.38% Trp were the minimum, and with further increase of Trp levels, were great significantly (P<0.01) or significantly (P<0.05) increased. Further correlation analysis showed that there were great significantly (P<0.01) or significantly (P<0.05) positive correlation between the percent weight gain and the protein, lipid and ash production value, and feed intake; between the FCR and the weight gain, protein and lipid production value, there showed great significant negative linear correlation (P<0.01). Results indicated that Trp could promote the growth of juvenile Jian carp, improve body protein and fat deposition ability, and enhance the amino acid metabolism of the hepatopancreas and muscle. Based on the percent weight gain, the requirement of Trp in juvenile Jian carp (7.73-32.67g) was 0.35% or 1.09/100g protein
3 Effects of Trp on the growth and development of fish digestive organs, digestive enzyme and intestinal brush border enzyme activities of the juvenile Jian carp
A total of 1050 Jian carp (7.73±0.03 g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Trp (0.11%, 0.17%,0.25%,0.38%,0.49%,0.60%, and 0.69%) for 60 days. The results showed that, when the Trp levels increased from 0.11% to 0.38%, the hepatopancreas weight, hepatosomatic index, hepatopancreas protein content, intestinal weight, intestinal length, intestinal protein content, intestosomatic index and intestinal length index and proximal, mid and distal intestinal folds height, hepatopancreas and intestinal trypsin, lipase and amylase activities, fintestinal creatine kinase activity, proximal, mid and distal intestinal alkaline phosphatase (AKP), Na+, K+-ATPase enzyme and y-GT enzyme activities were great significantly (P<0.01) or significantly (P<0.05) increased, the highest value was on 0.38% Trp, and thereafter, were great significantly (P<0.01) or significantly (P<0.05) decreased. Intestinal chymotrypsin activity was the highest at levels of 0.25% Trp (P<0.05). There was no effect on hepatopancreas chymotrypsin activity (P>0.05). Further correlation analysis showed that there were great significantly (P<0.01) or significantly (P<0.05) positive correlation between the PPV and the intestinal trypsin, hepatopancreas trypsin, and the activities of GOT and GPT in hepatopancreas and muscle. Meanwhile, there were significant positive linear correlation between the LPV and the hepatopancreas lipase (P<0.05). Finally, between the FCR and the hepatopancreas trypsin and lipase, intestinal trypsin and chymotrypsin, proximal and distal intestinal Na+, K+-ATPase, proximal, mid and distal intestinal AKP and y-GT, CK activities, it was showed great significantly (P<0.01) or significantly (P<0.05) linear negative correlation. These results indicated that Trp could improve digestion and absorption capacity of Jian carp. The improvement of digestion and absorption capacity of fish by Trp were due to the enhanced growth and development of hepatopancreas and intestinal, improving the protease, lipase and amylase secretion ability, increasing intestinal absorption areas and the intestinal absorption ability.
4. Effects of Trp on the intestinal microflora, disease resistance, specific immunity and non-specific immunity in the juvenile Jian carp
A total of 1050 Jian carp (7.73±0.03 g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Trp (0.11%,0.17%, 0.25%,0.38%,0.49%,0.60%, and 0.69%) for 60 days to measure intestinal microflora, red and white blood cell counts and immune organ weights. After the growth experiment, fish was injected with Aeromonas hydrophila for 17 days to examine the effects of Trp on the disease resistance and immune function of Jian carp. The results showed that, followed the Trp levels increased from 0.11% to 0.25%, intestinal lactic acid bacteria was significantly{P<0.01) or significantly (P<0.05) increased, there was no significant difference(P> 0.05) between 0.25% group and 0.38% group, and thereafter, it was great significantly (P<0.01) or significantly (P<0.05) decreased. Intestinal E. coli showed the opposite model. There was no significant effect on intestinal Aeromonas hydrophila (P>0.05). Trp was great significantly (P<0.01) or significantly (P<0.05) increased head kidney, behind kidney, spleen weights and the indexes, when the Trp level of 0.38%, these values reached the maximum, and thereafter, the head kidney, behind kidney, spleen weights and the indexes was great significantly (P<0.01) or significantly (P<0.05) decreased. When the Trp levels increased from 0.11% to 0.38%, the red blood cells and white blood cell counts were great significantly (P<0.01) or significantly (P<0.05) increased,, when the Trp level of 0.38%, these values reached the maximum, and then were significantly (P<0.01) or significantly (P<0.05) decreased. When the Trp levels increased from 0.11% to 0.25%, the survival rate of post-infection was great significantly increased (P<0.01),0.25%-0.49% was on the plateaued (P>0.05), and then decreased significantly (P<0.05). When the Trp levels increased from 0.11% to 0.49%, the serum IgM was great significantly (P<0.01) or significantly (P<0.05) increased, and then great significantly decreased (P<0.01). Serum anti-Aeromonas hydrophila titre was increased with the increase Trp levels up to 0.38%, and then decreased gradually (P<0.05). When the Trp levels increased from 0.11% to 0.38%, the phagocytic activity (PA) of leucocytes, serum complement C3, C4, lysozyme activity and acid phosphatase enzyme activity, and total iron-binding capacity were great significantly (P<0.01) or significant (P<0.05) increased, and then were great significantly (P<0.01) or significantly (P<0.05) reduced, however, the serum haemagglutination titre had no significant effect (P>0.05). Correlation analysis found that between the survival rate of carp after injection and the immune organ weights, antibody titer, leukocyte phagocytosis rate, ACP, C3, C4, and total iron binding capacity, it showed great significantly (P<0.01) or significantly (P<0.05) linear positive correlation. The relationship between the phagocytic activity (PA) of leucocytes and the red and white cell counts, and complement C3, C4 levels was great significantly (P<0.01) or significantly (P<0.05) positive linear correlation; the red and white cell counts and the immune organ weights showed great significantly (P<0.01) or significantly (P<0.05) linear correlation. These results indicated that Trp could enhance disease resistance and improve intestinal micro-ecological balance in Jian carp. The enhanced resist disease was related to the improvement of the specific immune and non-specific immunity in Jian carp. The improvement of non-specific immunity was related to the enhanced the ability of chemotaxis, adherence, regulation, sterilization and bacteriostasis by Trp in fish.
5. Effects of Trp on intestinal and muscle TOR mRNA expression in the juvenile Jian carp
A total of 1050 Jian carp (7.73±0.03 g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Trp (0.11%,0.17%, 0.25%,0.38%,0.49%,0.60%, and 0.69%) for 60 days to study the effects of Trp on interstinal and muscle TOR mRNA expression. The results showed that the expression of proximal and mid intestinal TOR mRNA was decreased with the increase of tryptophan levels, when the Trp levels reached 0.49% and 0.38%, the values were the minimum, respectively, and then increased gradually (P<0.05). When the Trp levels increased from 0.11% to 0.17%, the expression of the distal intestinal TOR mRNA was also significantly decreased (P<0.05), when Trp levels of 0.17% and 0.25%, the mRNA expression was the lowest, and followed by a gradual increase to the highest with the further increase Trp levels (P<0.05). The mRNA expression of muscle TOR was the lowest in the levels of Trp on 0.38% and 0.49%(P<0.05), low levels (0.11%) and high levels (0.60% and 0.69%) of Trp got the highest TOR mRNA expression (P<0.05). These results indicated that the dietary Trp deficiency or excessive improved fish proximal, mid and distal intestinal and muscle TOR mRNA expression.
6. Effects of Trp on the proliferation and differentiation of Jian carp intestinal epithelial cell (IEC)
The present study explored the effects of Trp on the proliferation and differentiation of carp intestinal epithelial cell (IEC). IEC coming from 80g carp were treated with Trp (0, 16,36,66,96, and 126mg/L Trp) with 4 replicates of each treatment for 96h. The results showed that with the increase Trp levels up to 36mg/L-66mg/L, the IEC counts were increased significantly, and formed a large colony and into the monolayer film, and with the further increase Trp levels, the cell counts were decreased. When the Trp levels increased from 0 to 36mg/L, the MTTOD of IEC was significantly increased (P<0.05), 36mg/L and 66mg/L Trp had no significant difference effect on MTT OD (P> 0.05), and declined thereafter (P<0.05). The protein content, GOT, GOT, AKP, Na+, K+-ATPase activities of IEC were elevated with increase Trp levels up to 36,36,66,36 and 16mg/L, respectively (P<0.05), and then decreased gradually thereafter (P<0.05). The ammonia content and lactate dehydrogenase activity of the culture medium were decreased with elevated Trp levels up to 96 and 66mg/L (P<0.05), and then decreased gradually thereafter (P<0.05). These results indicated that Trp could promote the proliferation and differentiation, and ensure the function of IEC.
7. Effect of Trp on the protein synthesis and TOR mRNA expression of IEC in the juvenile Jian carp
This study was carried out three small trials. (1) IEC were treated with two levels of Trp (0mg/L and 36mg/L Trp) with 8 replicates of each treatment for 240min to research the effects of Trp on the protein synthesis of IEC. (2) IEC were treated with 5 levels of time (15min,30min, 100min,170min and 240min) with 6 replicates of each treatment to study the effects of time on the TOR mRNA expression of IEC using 36mg/L Trp DMEM. (3) IEC were treated with two levels of Trp (0mg/L and 36mg/L Trp) with 8 replicates of each treatment for 100min to research the effects of Trp on TOR expression of IEC. The results showed that the protein synthesis rate for 36mg/L Trp group was significantly higher than the control group which without added Trp (Omg/LTrp) (P<0.05). The second trail found that the TOR mRNA expression level on 30min was significantly lower than 15min (P<0.05), and after 30min, it followed by a gradual increase up to the highest on the 170min and 240min (P<0.05). The third trail found that the TOR mRNA expression for 36mg/L Trp group was significantly higher than the control group which without added Trp (Omg/L Trp) (P<0.05). These results indicated that Trp could promote protein synthesis and enhance the TOR mRNA expression of IEC.
In summary, following the body weight of Jian carp from 20 g to 80 g, the increased secretary capacity of hepatopancreas to secret trypsin, chymotrypsin, lipase and amylase, was due to the growth and development of the hepatopancreas; the improvement of intestinal absorption capacity was due to the growth and the development of the intestine. Trp could promote the growth of juvenile Jian carp, improve body protein and fat deposition ability, and enhance the amino acid metabolism of the hepatopancreas and muscle. Trp could improve digestion and absorption capacity of Jian carp. The improvement of digestion and absorption capacity of fish by Trp were due to the enhanced growth and development of hepatopancreas and intestinal, improving the protease, lipase and amylase secretion ability, increasing intestinal absorption areas and the intestinal absorption ability. The dietary Trp deficiency or excessive improved the fish proximal, mid and distal intestinal and muscle TOR mRNA expression. Trp could promote the proliferation and differentiation, and ensure the function of IEC. Trp could promote protein synthesis and enhance the TOR mRNA expression of IEC. Trp could enhance disease resistance and improve intestinal micro-ecological balance in Jian carp. The enhanced resist disease was related to the improvement of the specific immune and non-specific immunity in Jian carp. The improvement of non-specific immunity was related to the enhanced the ability of chemotaxis, adherence, regulation, sterilization and bacteriostasis by Trp in fish. Based on the percent weight gain, the requirement of Trp in juvenile Jian carp (7.73-32.67g) was 0.35% or 1.09/100g protein.
1幼建鲤消化酶和肠道刷状缘相关酶活力变化规律研究
选择体重为18.0±0.2g的健康建鲤750尾,设5个阶段,每个阶段3个重复,每个重复50尾鱼,均饲喂相同饲粮,分别于0、14、28、42和56天采样,探索不同体重建鲤消化酶活力及肠道刷状缘相关酶活力的变化规律。结果发现：随幼建鲤体重(18.0-74.7g)增加,肝胰脏和肠道蛋白含量、消化酶活力呈极显著(P<0.01)或显著(P<0.05)升高。当建鲤体重为74.7g时,肝胰脏和肠道蛋白含量均显著高于其余各体重组(P<0.05),肝胰脏胰蛋白酶和糜蛋白酶活力、肝胰脏和肠道淀粉酶活力均极显著高于其它各体重组(P<0.01)；当体重分别达到58.6,40.1,58.6和58.6g时,肝胰脏脂肪酶活力、肠道胰蛋白酶、糜蛋白酶和脂肪酶活力分别进入平台值(P>0.05)。随幼建鲤体重增加,其肠道刷状缘相关酶活力也呈极显著(P<0.01)或显著(P<0.05)升高。当幼建鲤体重为74.7g时,后肠Na+,K+-ATPase活力,前肠、中肠和后肠的碱性磷酸酶(AKP)、前肠γ-谷胱甘肽转氨酶(y-GT)活力均极显著高于其它各体重组(P<0.01),中肠y-GT显著高于其余各体重组(P<0.05)；当幼建鲤体重达到58.6g时,全肠肌酸激酶(CK)活力、前肠和中肠Na+,K+-ATPase和后肠γ-GT活力均进入稳定值(P>0.05)。研究结果表明：20-80g的幼建鲤随体重增加,胰腺分泌胰蛋白酶、糜蛋白酶、脂肪酶和淀粉酶的能力增加,其分泌能力的增加与胰腺的发育逐步完善有关；肠道吸收能力随幼建鲤体重增加逐步提高,其吸收能力的提高与肠道的发育逐步完善有关。
2色氨酸(Trp)对幼建鲤生长、体成分沉积以及血清、肝胰脏和肌肉中GPT和GOT活力的影响
选择体重为7.73±0.03g的健康建鲤1050尾,分成七个处理,每个处理设3个重复,每个重复50尾,分别饲喂Trp水平为0.11%、0.17%、0.25%、0.38%、0.49%、0.60%和0.69%的纯合饲粮60d,探索色氨酸对幼建鲤生长性能、体成分沉积以及血清、肝胰脏和肌肉中谷草转氨酶和谷丙转氨酶活力的影响。结果表明：色氨酸对幼建鲤生长性能、体成分沉积和肝胰脏、肌肉、血清的GOT、GPT活力以及血浆氨含量均有极显著(P<0.01)或显著(P<0.05)影响,并呈开口向上或开口向下的抛物线模型,其顶点多为0.38%的色氨酸水平。当色氨酸水平从0.11%升高到0.38%时,特异性增长率(SGR)、采食量、体蛋白含量、蛋白质沉积率、蛋白质效率、体脂肪含量、脂肪沉积率、灰分沉积率、肝胰脏和肌肉的GOT和GPT活力均极显著(P<0.01)或显著(P<0.05)升高,到0.38%时最高,随后均极显著(P<0.01)或显著(P<0.05)降低。当色氨酸水平从0.11%升高到0.38%时,饵料系数、体水分含量、体灰分含量、血氨含量、血清GOT和GPT活力均极显著(P<0.01)或显著(P<0.05)降低,到0.38%时最低,随后均极显著(P<0.01)或显著(P<0.05)升高。进一步相关分析发现增重与蛋白质沉积率、脂肪沉积率、灰分沉积率和采食量呈现极显著(P<0.01)或显著(P<0.05)的正相关关系；饵料系数与增重、蛋白质沉积率和脂肪沉积率呈现极显著(P<0.01)的负相关。结果说明：Trp能促进幼建鲤的生长,提高体蛋白和脂肪的沉积能力,增强肝胰脏和肌肉中的氨基酸代谢强度；以生长速度为标识确定的10-40g的幼建鲤Trp需要量为0.35%或1.09/100g蛋白质。
3 Trp对幼建鲤肝胰脏和肠道生长发育、消化酶活力和肠道刷状缘相关酶活力的影响
选择体重为7.73±0.03g的健康建鲤1050尾,分成七个处理,每个处理设3个重复,每个重复50尾,分别饲喂Trp水平为0.11%、0.17%、0.25%、0.38%、0.49%、0.60%、0.69%的纯合饲料60天。结果表明：当色氨酸水平从0.11%升高到0.38%时,幼建鲤肝胰脏重量、肝体指数、肝胰脏蛋白含量、肠重、肠长、肠道蛋白含量、肠体指数和肠长指数以及前、中、后肠的皱襞高度、肝胰脏和肠道胰蛋白酶、脂肪酶和淀粉酶活力,全肠肌酸激酶活力,前、中、后肠的碱性磷酸酶(AKP)、Na+,K+-ATP酶和γ-GT酶活力均极显著(P<0.01)或显著(P<0.05)升高,到0.38%时最高,随后极显著(P<0.01)或显著(P<0.05)下降。肠道糜蛋白酶活力在0.25%组最高(P<0.05)。色氨酸对肝胰脏糜蛋白酶活力无显著影响(P>0.05)。进一步相关分析发现蛋白沉积率与肠胰蛋白酶、肝胰脏胰蛋白酶、肝胰GOT、肌肉GOT、肝胰GPT和肌肉GPT呈现极显著(P<0.01)或显著(P<0.05)的正相关关系；脂肪沉积率与肝胰脏脂肪酶呈现显著(P<0.05)的正线性相关；饵料系数与肝胰脏胰蛋白酶和脂肪酶、肠道胰蛋白酶和糜蛋白酶、前肠和后肠Na+,K+-ATP、前中后肠AKP和γ-GT、CK活力呈现极显著(P<0.01)或显著(P<0.05)的线性负相关。本试验结果表明：Trp可以提高幼建鲤消化吸收能力。消化吸收能力的提高与Trp促进肝胰脏和肠道生长发育,提高蛋白酶、脂肪酶和淀粉酶分泌能力,增加肠道吸收面积和肠道上皮吸收能力有关。
4 Trp对幼建鲤肠道菌群、特异性免疫和非特异性免疫能力的影响
选择体重为7.73±0.03g的健康建鲤1050尾,分成七个处理,每个处理设3个重复,每个重复50尾,分别饲喂Trp水平为0.11%、0.17%、0.25%、0.38%、0.49%、0.60%、0.69%的纯合饲料,进行为期60天的生长实验,测定肠道菌群、红白细胞数量和免疫器官重量。生长实验结束后用嗜水气单胞菌攻毒17天,考察色氨酸对幼建鲤疾病抵抗力和免疫功能的影响。结果表明：当色氨酸水平从0.11%升高到0.25%,肠道乳酸杆菌极显著(P<0.01)或显著(P<0.05)提高,0.25%组和0.38%组差异不显著(P>0.05),随后极显著(P<0.01)或显著(P<0.05)降低；肠道大肠杆菌呈现刚好相反的模型。色氨酸对鲤鱼肠道嗜水气单胞菌无显著影响(P>0.05)。色氨酸极显著(P<0.01)或显著(P<0.05)提高了头肾、后肾、脾脏重量和相应的指数,当色氨酸水平达到0.38%时达到最大,随着色氨酸水平的再升高,头肾、后肾、脾脏重量和相应的指数极显著(P<0.01)或显著(P<0.05)降低。当色氨酸水平从0.11%升高到0.38%时,红细胞和白细胞数量均极显著(P<0.01)或显著(P<0.05)升高,到0.38%时最高,随后均极显著(P<0.01)或显著(P<0.05)降低。当色氨酸水平从0.11%升高到0.25%时,攻毒后成活率极显著升高(P<0.01),0.25%-0.49%处于平台值(P>0.05),随后均显著(P<0.05)降低。当色氨酸水平从0.11%升高到0.49%时,血清IgM含量极显著(P<0.01)或显著(P<0.05)升高,随后极显著降低(P<0.01)。血清抗嗜水气单胞菌效价随着色氨酸水平升高到0.38%而增加,随后逐渐下降(P<0.05)。当色氨酸水平从0.11%升高到0.38%时,白细胞吞噬率、血清补体C3、C4、溶菌酶活力、酸性磷酸酶活力和总铁结合力均极显著(P<0.01)或显著(P<0.05)升高,随后均极显著(P<0.01)或显著(P<0.05)降低,但对血清凝集素效价无显著影响(P>0.05)。相关分析发现,攻毒后成活率与免疫器官重量、抗体效价、白细胞吞噬率、ACP、C3、C4和总铁结合力呈现极显著(P<0.01)或显著(P<0.05)的线性相关；白细胞吞噬率与红白细胞数量和补体C3、C4含量呈现极显著(P<0.01)或显著(P<0.05)的正线性相关；红白细胞数量与免疫器官重量呈现极显著(P<0.01)或显著(P<0.05)的线性相关关系。本试验结果表明：Trp能提高幼建鲤的疾病抵抗能力以及改善其肠道微生态平衡。疾病抵抗能力的提高与Trp提高特异性免疫和非特异性免疫能力有关。非特异性免疫能力的提高与Trp提高其对病原菌的粘附调理、抑菌、溶菌能力有关。
5 Trp对幼建鲤前、中、后肠和肌肉中TOR mRNA含量的影响
选择体重为7.73±0.03g的健康建鲤1050尾,分成七个处理,每个处理设3个重复,每个重复50尾,分别饲喂Trp水平为0.11%、0.17%、0.25%、0.38%、0.49%、0.60%和0.69%的纯合饲料60天,研究Trp对幼建鲤肠道和肌肉TOR mRNA表达量的影响。结果发现：鲤鱼前肠和中肠的TOR mRNA表达量随色氨酸水平升高而降低,随后逐渐升高,当色氨酸水平为0.49%和0.38%时,TOR mRNA表达量分别为最低(P<0.05)。当色氨酸水平从0.11%到0.17%时,后肠TORmRNA表达量显著降低(P<0.05),色氨酸水平为0.17%和0.25%时,其mRNA表达量最低,随后逐渐上升,到最高水平(0.69%)时最大(P<0.05)。肌肉TORmRNA的表达量在色氨酸中间水平(0.38%和0.49%)最低(P<0.05),色氨酸低水平(0.11%)和高水平(0.60%和0.69%)表达量最高(P<0.05)。综合结果说明：日粮Trp缺乏或过量均提高幼建鲤前、中、后肠和肌肉中的TOR基因表达,但Trp在适宜水平时降低,其影响机制有待进一步研究。
6 Trp对幼建鲤肠上皮细胞增殖分化的影响
试验分为6个处理,每个处理设4个重复,选择体重为80g左右的健康鲤鱼进行肠道上皮细胞接种培养后分别添加色氨酸浓度为0、16、36、66、96和126mg/L的DMEM培养液继续培养96h,收集培养液和细胞,对相关指标进行分析测试。结果表明：随着色氨酸浓度上升到36mg/L-66mg/L时,观察发现贴壁生长细胞数量明显增多,并形成大的细胞集落和成片的单层细胞,随后细胞数量有所下降。色氨酸水平从0到36mg/L时,鲤鱼肠上皮细胞的MTTOD值显著上升(P<0.05),36mg/L和66mg/L差异不显著(P>0.05),随后逐渐下降(P<0.05)。鲤鱼肠上皮细胞蛋白含量、谷草转氨酶活力、谷丙转氨酶活力、碱性磷酸酶活力、Na+,K+-ATP活力随色氨酸水平升高而显著升高(P<0.05),当色氨酸水平达到36,36,66,36和16mg/L时分别达到最大,随后逐渐降低(P<0.05)。培养液氨含量和乳酸脱氢酶活力随色氨酸水平升高而显著降低(P<0.05),当色氨酸水平达到96和66mg/L时分别达到最低,随后逐渐降升高(P<0.05)。结果表明：色氨酸促进了鲤鱼肠道上皮细胞的增殖和分化,保证了功能正常。
7 Trp对幼建鲤肠细胞蛋白合成和TOR基因表达的影响
本研究进行了3小试验。(1)试验设2个处理组(0mg/L和36mg/L Trp),每个处理8个重复,培养时间为240min,研究Trp对肠细胞蛋白质合成的影响。(2)试验设5个处理组,每个处理6个重复,分别于15min,30min,100min,170min和240min采样,研究36mg/L Trp对肠细胞TOR基因表达影响的时间效应。(3)试验设2个处理组(0mg/L和36mg/L Trp),每个处理8个重复,培养时间100min,研究36mg/L Trp对肠细胞TOR基因表达的影响。结果表明：(1)36mg/L色氨酸处理组细胞蛋白质合成率显著高于未添加色氨酸的对照组(0mg/LTrp)(P<0.05)。(2)30min时,TORmRNA的表达量显著低于15min,并达到最低(P<0.05),随后逐渐升高,在170min和240min时,达到最高(P<0.05)。(3)36mg/L组TOR mRNA表达量显著高于未添加色氨酸的对照组(0mg/LTrp)(P<0.05)。结果说明：Trp可促进鲤鱼肠细胞蛋白质合成,同时影响了蛋白质合成信号调控分子TOR的表达。
综上所述：20-80g的幼建鲤随体重增加,胰腺分泌胰蛋白酶、糜蛋白酶、脂肪酶和淀粉酶的能力增加,其分泌能力的增加与胰腺的发育逐步完善有关；肠道吸收能力随幼建鲤体重增加逐步提高,其吸收能力的提高与肠道的发育逐步完善有关。Trp能促进幼建鲤的生长,提高了体蛋白和脂肪的沉积能力；Trp通过促进肝胰脏和肠道生长发育,提高蛋白酶、脂肪酶和淀粉酶分泌能力,增加肠道吸收面积和肠道上皮吸收能力有关；Trp影响肌肉和肠道蛋白质合成信号调控分子TOR mRNA的表达量,缺乏和过量表达量都升高；Trp促进了鲤鱼肠道细胞生长发育,保证了功能正常,促进鲤鱼肠细胞蛋白质合成,但蛋白质合成信号调控分子TOR的表达降低。Trp提高了幼建鲤的疾病抵抗能力。疾病抵抗能力的提高与Trp提高特异性免疫、非特异性免疫能力以及改善了其肠道微生态平衡有关。非特异性免疫能力的提高与Trp提高其对病原菌的粘附调理、抑菌、溶菌能力有关。以生长速度为标识确定的10-40g的幼建鲤Trp需要量为0.35%或1.09/100g蛋白质。
The first study was conducted to evaluate the effects of various weights on the digestive enzyme and intestinal brush border enzyme activities in Jian carp. Based on the first study, other studies of the effects of Trp on production performance, nutrient deposition, amino acid metabolism, digestion and absorption capacity, immunity and protein synthesis regulatory factor TOR expression in Jian carp were carried out. Using fish primary cell culture, we investigated the effects of Trp on the proliferation and differentiation, protein synthesis, and TOR expression of intestinal cell of Jian carp. The main researches and results are as follows:
1. The effects of various weights on digestive enzyme activity and intestinal brush border enzyme activities in Jian carp
A 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 day,28th day, 42nd day and 56th day sampling, to explore the effects of various weights on digestive enzyme and intestinal brush border enzyme activities in Jian carp. The results indicated that:The hepatopancreas and intestinal protein content, digestive enzyme activity was great significantly (P<0.01) or significantly (P<0.05) increased with the increasing levels of body weights from 18.0g to 74.7g. When Jian carp weighed 74.7g, the hepatopancreas and intestinal protein content were significantly higher than the other treatments (P<0.05), hepatopancreas trypsin and chymotrypsin activities, hepatopancreas and intestinal amylase activities were great significantly higher than the other treatments (P<0.01). When the body weights levels up to 58.6,40.1,58.6 and 58.6g, the hepatopancreas lipase activity, intestinal trypsin, chymotrypsin and lipase activities were all significantly increased, respectively (P<0.05), and remained nearly constant thereafter (P>0.05). The effects of the body weights of juvenile Jian carp on the intestinal brush border enzyme activity also showed great significantly (P<0.01) or significantly (P<0.05) increased. When Jian carp weighed to 74.7g, the distal intestinal Na+, K+-ATPase activity, proximal, mid and distal intestinal alkaline phosphatase (AKP), and the proximal y-glutathione transaminase (y-GT) activities were great significantly higher than other groups (P<0.01), the y-GT activity in mid intestine was significantly higher than the other groups (P<0.05). The intestinal creatine kinase (CK) activity, proximal and mid intestinal Na+, K+-ATPase and distal intestinal y-GT activities were all increased with the increasing weights levels up to 58.6g (P<0.05), and no differences were found with further increase of zinc levels (P>0.05). The results indicated that following the body weight of Jian carp from 20 g to 80 g, the increased secretary capacity of hepatopancreas to secret trypsin, chymotrypsin, lipase and amylase, was due to the growth and development of the hepatopancreas; the improvement of intestinal absorption capacity was due to the growth and the development of the intestine.
2. Effects of Trp on the growth performance, body composition and the GPT and GOT activities in serum, hepatopancreas and muscle of the juvenile Jian carp
A total of 1050 Jian carp (7.73±0.03g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Trp (0.11%,0.17%, 0.25%,0.38%,0.49%,0.60% and 0.69%) for 60 days to investigate the effects of Trp on growth performance, body deposition and the GPT and GOT activities in serum, hepatopancreas and muscle. The results showed that the growth performance, body deposition, the GPT and GOT activities in serum, hepatopancreas and muscle and plasma ammonia levels were great significantly (P<0.01) or significantly (P<0.05) affected by Trp. When the Trp levels increased from 0.11% to 0.38%, the specific growth rate (SGR), feed intake, body protein content, protein production value (PPV), protein efficiency ratio, body lipid content, lipid production value (LPV), ash production value (APV), hepatopancreas and the activities of GOT and GPT in muscle were great significantly (P<0.01) or significantly (P<0.05) increased, the maximum value was on 0.38% Trp, and with further increase of Trp levels, were great significantly (P<0.01) or significantly (P<0.05) decreased. When the Trp levels increased from 0.11% to 0.38%, the FCR, body water content, body ash content, ammonia content, serum GOT and GPT activities were great significantly (P<0.01) or significantly (P<0.05) decreased, and the results of 0.38% Trp were the minimum, and with further increase of Trp levels, were great significantly (P<0.01) or significantly (P<0.05) increased. Further correlation analysis showed that there were great significantly (P<0.01) or significantly (P<0.05) positive correlation between the percent weight gain and the protein, lipid and ash production value, and feed intake; between the FCR and the weight gain, protein and lipid production value, there showed great significant negative linear correlation (P<0.01). Results indicated that Trp could promote the growth of juvenile Jian carp, improve body protein and fat deposition ability, and enhance the amino acid metabolism of the hepatopancreas and muscle. Based on the percent weight gain, the requirement of Trp in juvenile Jian carp (7.73-32.67g) was 0.35% or 1.09/100g protein
3 Effects of Trp on the growth and development of fish digestive organs, digestive enzyme and intestinal brush border enzyme activities of the juvenile Jian carp
A total of 1050 Jian carp (7.73±0.03 g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Trp (0.11%, 0.17%,0.25%,0.38%,0.49%,0.60%, and 0.69%) for 60 days. The results showed that, when the Trp levels increased from 0.11% to 0.38%, the hepatopancreas weight, hepatosomatic index, hepatopancreas protein content, intestinal weight, intestinal length, intestinal protein content, intestosomatic index and intestinal length index and proximal, mid and distal intestinal folds height, hepatopancreas and intestinal trypsin, lipase and amylase activities, fintestinal creatine kinase activity, proximal, mid and distal intestinal alkaline phosphatase (AKP), Na+, K+-ATPase enzyme and y-GT enzyme activities were great significantly (P<0.01) or significantly (P<0.05) increased, the highest value was on 0.38% Trp, and thereafter, were great significantly (P<0.01) or significantly (P<0.05) decreased. Intestinal chymotrypsin activity was the highest at levels of 0.25% Trp (P<0.05). There was no effect on hepatopancreas chymotrypsin activity (P>0.05). Further correlation analysis showed that there were great significantly (P<0.01) or significantly (P<0.05) positive correlation between the PPV and the intestinal trypsin, hepatopancreas trypsin, and the activities of GOT and GPT in hepatopancreas and muscle. Meanwhile, there were significant positive linear correlation between the LPV and the hepatopancreas lipase (P<0.05). Finally, between the FCR and the hepatopancreas trypsin and lipase, intestinal trypsin and chymotrypsin, proximal and distal intestinal Na+, K+-ATPase, proximal, mid and distal intestinal AKP and y-GT, CK activities, it was showed great significantly (P<0.01) or significantly (P<0.05) linear negative correlation. These results indicated that Trp could improve digestion and absorption capacity of Jian carp. The improvement of digestion and absorption capacity of fish by Trp were due to the enhanced growth and development of hepatopancreas and intestinal, improving the protease, lipase and amylase secretion ability, increasing intestinal absorption areas and the intestinal absorption ability.
4. Effects of Trp on the intestinal microflora, disease resistance, specific immunity and non-specific immunity in the juvenile Jian carp
A total of 1050 Jian carp (7.73±0.03 g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Trp (0.11%,0.17%, 0.25%,0.38%,0.49%,0.60%, and 0.69%) for 60 days to measure intestinal microflora, red and white blood cell counts and immune organ weights. After the growth experiment, fish was injected with Aeromonas hydrophila for 17 days to examine the effects of Trp on the disease resistance and immune function of Jian carp. The results showed that, followed the Trp levels increased from 0.11% to 0.25%, intestinal lactic acid bacteria was significantly{P<0.01) or significantly (P<0.05) increased, there was no significant difference(P> 0.05) between 0.25% group and 0.38% group, and thereafter, it was great significantly (P<0.01) or significantly (P<0.05) decreased. Intestinal E. coli showed the opposite model. There was no significant effect on intestinal Aeromonas hydrophila (P>0.05). Trp was great significantly (P<0.01) or significantly (P<0.05) increased head kidney, behind kidney, spleen weights and the indexes, when the Trp level of 0.38%, these values reached the maximum, and thereafter, the head kidney, behind kidney, spleen weights and the indexes was great significantly (P<0.01) or significantly (P<0.05) decreased. When the Trp levels increased from 0.11% to 0.38%, the red blood cells and white blood cell counts were great significantly (P<0.01) or significantly (P<0.05) increased,, when the Trp level of 0.38%, these values reached the maximum, and then were significantly (P<0.01) or significantly (P<0.05) decreased. When the Trp levels increased from 0.11% to 0.25%, the survival rate of post-infection was great significantly increased (P<0.01),0.25%-0.49% was on the plateaued (P>0.05), and then decreased significantly (P<0.05). When the Trp levels increased from 0.11% to 0.49%, the serum IgM was great significantly (P<0.01) or significantly (P<0.05) increased, and then great significantly decreased (P<0.01). Serum anti-Aeromonas hydrophila titre was increased with the increase Trp levels up to 0.38%, and then decreased gradually (P<0.05). When the Trp levels increased from 0.11% to 0.38%, the phagocytic activity (PA) of leucocytes, serum complement C3, C4, lysozyme activity and acid phosphatase enzyme activity, and total iron-binding capacity were great significantly (P<0.01) or significant (P<0.05) increased, and then were great significantly (P<0.01) or significantly (P<0.05) reduced, however, the serum haemagglutination titre had no significant effect (P>0.05). Correlation analysis found that between the survival rate of carp after injection and the immune organ weights, antibody titer, leukocyte phagocytosis rate, ACP, C3, C4, and total iron binding capacity, it showed great significantly (P<0.01) or significantly (P<0.05) linear positive correlation. The relationship between the phagocytic activity (PA) of leucocytes and the red and white cell counts, and complement C3, C4 levels was great significantly (P<0.01) or significantly (P<0.05) positive linear correlation; the red and white cell counts and the immune organ weights showed great significantly (P<0.01) or significantly (P<0.05) linear correlation. These results indicated that Trp could enhance disease resistance and improve intestinal micro-ecological balance in Jian carp. The enhanced resist disease was related to the improvement of the specific immune and non-specific immunity in Jian carp. The improvement of non-specific immunity was related to the enhanced the ability of chemotaxis, adherence, regulation, sterilization and bacteriostasis by Trp in fish.
5. Effects of Trp on intestinal and muscle TOR mRNA expression in the juvenile Jian carp
A total of 1050 Jian carp (7.73±0.03 g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Trp (0.11%,0.17%, 0.25%,0.38%,0.49%,0.60%, and 0.69%) for 60 days to study the effects of Trp on interstinal and muscle TOR mRNA expression. The results showed that the expression of proximal and mid intestinal TOR mRNA was decreased with the increase of tryptophan levels, when the Trp levels reached 0.49% and 0.38%, the values were the minimum, respectively, and then increased gradually (P<0.05). When the Trp levels increased from 0.11% to 0.17%, the expression of the distal intestinal TOR mRNA was also significantly decreased (P<0.05), when Trp levels of 0.17% and 0.25%, the mRNA expression was the lowest, and followed by a gradual increase to the highest with the further increase Trp levels (P<0.05). The mRNA expression of muscle TOR was the lowest in the levels of Trp on 0.38% and 0.49%(P<0.05), low levels (0.11%) and high levels (0.60% and 0.69%) of Trp got the highest TOR mRNA expression (P<0.05). These results indicated that the dietary Trp deficiency or excessive improved fish proximal, mid and distal intestinal and muscle TOR mRNA expression.
6. Effects of Trp on the proliferation and differentiation of Jian carp intestinal epithelial cell (IEC)
The present study explored the effects of Trp on the proliferation and differentiation of carp intestinal epithelial cell (IEC). IEC coming from 80g carp were treated with Trp (0, 16,36,66,96, and 126mg/L Trp) with 4 replicates of each treatment for 96h. The results showed that with the increase Trp levels up to 36mg/L-66mg/L, the IEC counts were increased significantly, and formed a large colony and into the monolayer film, and with the further increase Trp levels, the cell counts were decreased. When the Trp levels increased from 0 to 36mg/L, the MTTOD of IEC was significantly increased (P<0.05), 36mg/L and 66mg/L Trp had no significant difference effect on MTT OD (P> 0.05), and declined thereafter (P<0.05). The protein content, GOT, GOT, AKP, Na+, K+-ATPase activities of IEC were elevated with increase Trp levels up to 36,36,66,36 and 16mg/L, respectively (P<0.05), and then decreased gradually thereafter (P<0.05). The ammonia content and lactate dehydrogenase activity of the culture medium were decreased with elevated Trp levels up to 96 and 66mg/L (P<0.05), and then decreased gradually thereafter (P<0.05). These results indicated that Trp could promote the proliferation and differentiation, and ensure the function of IEC.
7. Effect of Trp on the protein synthesis and TOR mRNA expression of IEC in the juvenile Jian carp
This study was carried out three small trials. (1) IEC were treated with two levels of Trp (0mg/L and 36mg/L Trp) with 8 replicates of each treatment for 240min to research the effects of Trp on the protein synthesis of IEC. (2) IEC were treated with 5 levels of time (15min,30min, 100min,170min and 240min) with 6 replicates of each treatment to study the effects of time on the TOR mRNA expression of IEC using 36mg/L Trp DMEM. (3) IEC were treated with two levels of Trp (0mg/L and 36mg/L Trp) with 8 replicates of each treatment for 100min to research the effects of Trp on TOR expression of IEC. The results showed that the protein synthesis rate for 36mg/L Trp group was significantly higher than the control group which without added Trp (Omg/LTrp) (P<0.05). The second trail found that the TOR mRNA expression level on 30min was significantly lower than 15min (P<0.05), and after 30min, it followed by a gradual increase up to the highest on the 170min and 240min (P<0.05). The third trail found that the TOR mRNA expression for 36mg/L Trp group was significantly higher than the control group which without added Trp (Omg/L Trp) (P<0.05). These results indicated that Trp could promote protein synthesis and enhance the TOR mRNA expression of IEC.
In summary, following the body weight of Jian carp from 20 g to 80 g, the increased secretary capacity of hepatopancreas to secret trypsin, chymotrypsin, lipase and amylase, was due to the growth and development of the hepatopancreas; the improvement of intestinal absorption capacity was due to the growth and the development of the intestine. Trp could promote the growth of juvenile Jian carp, improve body protein and fat deposition ability, and enhance the amino acid metabolism of the hepatopancreas and muscle. Trp could improve digestion and absorption capacity of Jian carp. The improvement of digestion and absorption capacity of fish by Trp were due to the enhanced growth and development of hepatopancreas and intestinal, improving the protease, lipase and amylase secretion ability, increasing intestinal absorption areas and the intestinal absorption ability. The dietary Trp deficiency or excessive improved the fish proximal, mid and distal intestinal and muscle TOR mRNA expression. Trp could promote the proliferation and differentiation, and ensure the function of IEC. Trp could promote protein synthesis and enhance the TOR mRNA expression of IEC. Trp could enhance disease resistance and improve intestinal micro-ecological balance in Jian carp. The enhanced resist disease was related to the improvement of the specific immune and non-specific immunity in Jian carp. The improvement of non-specific immunity was related to the enhanced the ability of chemotaxis, adherence, regulation, sterilization and bacteriostasis by Trp in fish. Based on the percent weight gain, the requirement of Trp in juvenile Jian carp (7.73-32.67g) was 0.35% or 1.09/100g protein.
引文
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