【作者】 周凡；

【导师】 邵庆均；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2011， 博士

【摘要】 本论文研究了饲料中分别添加不同水平的赖氨酸和精氨酸对黑鲷(A canthopagrus schlegelii)幼鱼生长、饲料利用效率、体组成、相关酶的活性及血清生化指标的影响,并由此得出黑鲷幼鱼对饲料中赖氨酸及精氨酸的需要量；并通过研究精氨酸对组织中生长相关主要基因表达来初步探讨饲料必需氨基酸调控黑鲷生长的主要机理。本论文还对饲料添加不平衡的精氨酸/赖氨酸对黑鲷幼鱼的影响,两种氨基酸在黑鲷体内的拮抗作用及其可能的机理进行探索。本论文的主要试验设计及研究结果如下。(1)室内流水养殖系统饲养黑鲷幼鱼(初重为9.13±0.09 g)8周来确定其对L-赖氨酸的需求量。设计6种等氮等能试验饲料,饲料必需氨基酸组成(除赖氨酸外)参照黑鲷鱼体38%蛋白的氨基酸模式,赖氨酸以0.40%的添加梯度从2.08%逐步上升到4.05%。试验结果表明：当饲料中的赖氨酸含量从2.08%上升至3.25%,黑鲷的增重率(WG)和特定生长率(SGR)随着饲料赖氨酸水平升高而升高(P<0.05),当饲料中赖氨酸含量为3.25%时,达到最大值(P<0.05)。饲料赖氨酸添加对饲料效率(FER)和蛋白质效率(PER)也有促进作用。赖氨酸对粗蛋白和总能的表观消化率,以及消化器官中的蛋白酶和脂肪酶活性有显著影响(P<0.05)。全鱼和背肌蛋白含量随饲料赖氨酸水平增加而增加,脂肪含量则呈现下降趋势；各处理组之间的灰分和水分含量没有显著性差异(P<0.05)。在体必需氨基酸(EAAs)沉积方面,背肌中的Val和His,肝脏的Val、Phe、Met和Thr的含量不受饲料赖氨酸水平的影响(P>0.05)；其余的各种必需氨基酸含量均为先上升后保持稳定的趋势。黑鲷幼鱼血清的甘油三酯、葡萄糖含量以及过氧化氢酶和超氧化物歧化酶活性均没有显著变化(P>0.05)。血清总蛋白(TP)含量随着饲料赖氨酸添加水平的增加而显著增加。总胆固醇(T-CHO)含量则与TP含量的变化趋势相反。血清谷草转氨酶(AST)和谷丙转氨酶(ALT)活性随着饲料赖氨酸含量的增加大致表现出下降的趋势。随着饲料赖氨酸含量增加,血清游离赖氨酸浓度先显著上升,在Diet 4组到达最高值。使用二次曲线回归模型,根据试验黑鲷幼鱼的SGR同饲料中赖氨酸水平的相关性得出其赖氨酸需要量为3.32%,占饲料蛋白的8.64%。(2)室内流水养殖系统饲养黑鲷幼鱼(初重为10.51±0.15 g)8周来确定其对L-精氨酸的需求量。设计6种等氮等能试验饲料,精氨酸以0.30%的添加梯度从1.85%逐步上升到3.46%。试验结果显示：六组试验鱼的存活率没有显著性差异(P>0.05)。SGR先随着饲料精氨酸的含量增加而显著上升(P<0.05),到2.51%精氨酸饲料组后,继续增加饲料精氨酸含量并不会进一步提高SGR(P>0.05)。Diet 1组的FER显著低于其它各组(P<0.05)；FER最高值(91.34%)出现在Diet 5组,与Diet 3,Diet 4和Diet 6组没有显著差异。PER和PPV的变化趋势类似,均随着饲料精氨酸水平先显著上升,随后保持稳定。饲料粗脂肪的表观消化率不受饲料精氨酸添加水平的影响,干物质,粗蛋白和总能的表观消化率均有显著变化。饲料精氨酸水平对黑鲷幼鱼全鱼和背肌的灰分和水分含量没有明显影响(P>0.05)。组织中蛋白含量随着饲料精氨酸的添加水平增加而呈上升趋势,而脂肪含量则呈现下降趋势。除Thr之外,全鱼体中的EAAs含量基本都随着饲料精氨酸的含量增加而显著上升,并在精氨酸添加水平较高的饲料处理组间保持稳定；全鱼精氨酸含量在Diet 1组显著低于其它各组(P<0.05),此后全鱼精氨酸含量显著递增,在Diet 5和Diet 6组时,其含量则显著高于之前四组(P<0.05)。肝脏Arginase活性在前三个饲料处理组呈上升趋势(P<0.05),此后继续增加饲料精氨酸含量,不再显著提高精氨酸酶活性(P>0.05)。Diet 1组的鸟氨酸脱羧酶(ODC)活性显著低于Diet 5和Diet 6组(P<0.05)。黑鲷幼鱼血清中除T-CHO之外,其它指标均受到饲料精氨酸水平的显著影响。基于折线回归模型模拟黑鲷幼鱼SGR与饲料精氨酸水平的关系可知,黑鲷幼鱼最适精氨酸需求量为2.79%(占饲料),相当于占饲料蛋白的7.74%。基于一元二次回归模型模拟黑鲷幼鱼PER与饲料精氨酸水平的关系可知：当PER达到最高值时,饲料精氨酸含量为3.09%,相当于占饲料蛋白含量的8.13%。(3)配制添加外源精氨酸和赖氨酸的含量分别为0.9%和1.3%(占饲料干重)的基础对照组饲料,使得两种必需氨基酸含量符合之前两个试验所得出的黑鲷最适需求量。在Diet 2和Diet 3组饲料中,赖氨酸水平高于对照组赖氨酸含量的20%,而精氨酸水平则分别高于和低于对照组精氨酸含量的20%；在Diet 4和Diet 5组饲料中,赖氨酸水平低于对照组赖氨酸含量的20%,而精氨酸水平则同样是分别高于和低于对照组精氨酸含量的20%；在Diet 6、Diet 7和Diet 8的饲料中,精氨酸含量与对照组设计成相同水平,并以相比与对照组赖氨酸含量20%的比例,逐渐提高这三组饲料的赖氨酸含量。各组饲料通过调整甘氨酸和天冬氨酸的含量保持等能等氮。8组饲料精氨酸/赖氨酸水平实测值分别为每100克饲料含2.83/3.25、3.46/4.06、2.27/4.08、3.51/2.70、2.31/2.74、2.87/4.03、2.91/4.68和2.85/5.24克。随机投喂初重为10.03±0.13 g的黑鲷幼鱼(每组三个重复),研究饲料不平衡精氨酸/赖氨酸添加对黑鲷的影响及探索是否存在拮抗作用。试验结果表明,饲料精氨酸/赖氨酸不平衡会严重影响黑鲷幼鱼的生长性能及饲料利用等,相关指标均在不同程度上低于饲喂对照组的试验鱼。根据生长表现、饲料利用效率、饲料赖氨酸与精氨酸的表观消化率、鱼体赖氨酸与精氨酸沉积量、肝脏精氨酸酶活性等指标综合判断,黑鲷存在精氨酸-赖氨酸的拮抗作用：当赖氨酸过量时,增加饲料中精氨酸含量可以一定程度缓解这种拮抗作用。(4)本试验以黑鲷肝脏和背肌组织总RNA为模板,oligo(dT)18为反转录引物,合成cDNA第一条链,以该cDNA为模板进行PCR反应。应用NCBI上已有的鱼类基因设计引物,分别扩增黑鲷IGF-Ⅰ、IGF-IR和Arginase基因片段,其序列片段长度分别为890bp、570bp和430 bp。根据BLAST比对分析结果表明,上述三种基因均与其它鱼类的相对应基因具有较高的同源性和相似度,可以认为是分别属于各自的功能基因家族。(5)取试验二(精氨酸需求试验)中的肝脏和背肌样品分析目的基因相对表达量。结果发现：饲喂最低精氨酸水平饲料(Diet 1)的黑鲷肝脏IGF-I相对表达量显著低于其它各组(P<0.05),随着精氨酸含量的增加,肝脏IGF-I的表达水平呈显著上升趋势,当添加的精氨酸含量超过3.25%之后,趋于稳定(P>0.05)。当投喂的饲料精氨酸水平超过2.88%时,背肌IGF-I表达量没有显著差异(P>0.05),但均显著高于对照组(P<0.05)。肝脏GHR mRNA水平随着饲料精氨酸水平的增加呈现先上升后稳定的趋势。背肌IGF-IRmRNA的最低表达量出现在饲喂Diet 1组饲料的试验鱼中(P<0.05);而最高相对表达量则出现在Diet 4组,显著高于精氨酸添加量小于3.25%的试验组(P<0.05),但与精氨酸添加量大于3.25%的试验组之间没有明显区别(P>0.05)。其它四个试验组之间的数据没有显著性差异(P>0.05)。饲料精氨酸水平对黑鲷肝脏精氨酸酶基因表达水平也有显著影响。在Diet 5和Diet 6试验组检测出相对较高的Arginase相对表达量,而表达量相对较低值则在Diet 1和Diet 2试验组出现(P<0.05)取试验三(精氨酸/赖氨酸不平衡试验)中的肝脏样品分析目的基因相对表达量。结果发现：当饲料精氨酸和赖氨酸添加量为适宜值时(Diet 1),肝脏的IGF-I mRNA相对表达量显著较高。在过量赖氨酸的饲料中增加精氨酸的水平(Diet 2),肝脏IGF-I的表达水平也显著高于其它各组(P<0.05),但与Diet 1组差异不显著(P<0.05)。饲料精氨酸和赖氨酸都不足量时(Diet 5),肝脏IGF-I表达量明显减少；而当精氨酸含量一定时,饲料中过量添加赖氨酸,也会使得肝脏IGF-I呈现下降的趋势。同样的,相比于对照组(Diet 1),饲料中过量的赖氨酸添加量明显抑制了肝脏Arginase基因的表达(Diet 7和Diet 8)；而降低饲料中的精氨酸含量,也会抑制Arginase基因的表达(Diet 3和Diet 5)。该基因的表达水平在饲喂Diet 2、Diet 4和Diet 6组饲料的黑鲷幼鱼之间没有显著差异,相比于对照组,差异也不显著(P>0.05)。以上结果表明,精氨酸对黑鲷的促生长机理与其对生长轴激素的转录水平上的调控有关；饲料中精氨酸添加水平可以一定程度促进精氨酸酶基因的表达,过量赖氨酸则对其起抑制作用。根据本研究的试验结果,得出以下结论。1、黑鲷幼鱼饲料需要添加适宜的赖氨酸和精氨酸来促进生长。2、本研究得到黑鲷幼鱼饲料中最适赖氨酸和精氨酸需求量(以生长表现评判)分别为：3.32%(占饲料比例),相当于8.64%(占饲料蛋白比例)；以及2.79%(占饲料)相当于占饲料蛋白的7.74%。3、根据生长表现、饲料利用效率和氨基酸代谢等多项指标综合判断,黑鲷存在精氨酸-赖氨酸的拮抗作用。4、饲料精氨酸可以影响生长作用轴相关基因表达。5、饲料精氨酸影响黑鲷体内精氨酸酶的基因表达,同时,该基因也受到饲料赖氨酸水平的调控作用。更多还原

【Abstract】 The effects of dietary L-lysine and L-arginine levels on growth performance, feed utilization, body compositions, enzymes activity and serum biochemical parameters of black sea bream Acanthopagrus schlegelii juvenile were studied in the present research. The effects of dietary arginine on growth relating genes in tissues of black sea bream were studied to discuss the mechanism of dietary EAA controlling growth of fish. In this research, the effect of dietary imbalanced arginine/lysine was estimated and the potential mechanism of antagonism between these two EAAs in black sea bream was also discussed. The main contents and results in the current research present as follows.Trial 1. An 8-week feeding experiment was conducted to determine the quantitative L-lysine requirement of juvenile black sea bream (initial mean weight:9.13±0.09 g, mean±SD) in eighteen 300-L indoors flow-through circular fibreglass tanks by feeding diets containing six levels of L-lysine ranging from 2.08% to 4.05% dry diet at about 0.40% increments. The isonitrogenous and isoenergetic diets were formulated to simulate the amino acid profile of 38% whole fish body protein except lysine. Each diet was assigned to triplicate groups of 20 fish in a completely randomized design. The results showed that weight gain (WG) and specific growth rate (SGR) increased with increasing levels of dietary lysine up to 3.25% (P<0.05) and both showed a declining tendency thereafter.Dietary increasing lysine level have promoting effect on feed efficiency ratio (FER) and protein efficiency ratio (PER). The apparent digestibility coefficients (ADCs) of crude protein and gross energy, the protease and lipase activies were also influenced by dietary lysine level (P<0.05). The whole body crude protein and crude lipid contents were significantly affected (P<0.05) by dietary lysine level, while moisture and ash showed no significant differences. While for EAAs compositions, except for Val and His contents in dorsal muscle, Val, Phe, Met and Thr contents in liver were independent with dietary treatments (P>0.05),, other EAAs contents were significantly affected by dietary lysine level. The highest free lysine leve in serum was obtained in Diet 4 group. The activity of glutamic-oxalacetic transaminase (AST) and the glutamic-pyruvic transaminase (ALT) in serum decreased significantly when lysine level increasing (P<0.05), however, no significant difference (P>0.05) were found in contents of triacylglycerol and glucose nor the activities of catalyse and superoxide dismutase.Analysis of dose (lysine level)-response (SGR) with second order polynomial regression suggested a requirement of juvenile black sea bream to be 3.32% dry diet or 8.64% dietary protein.Trial 2. An 8-week feeding trial was conducted to determine the dietary arginine requirement of juvenile black sea bream in eighteen 350-L indoors flow-through circular fibreglass tanks. Six isonitrogenous and isoenergetic diets were formulated to contain graded levels of L-arginine (1.85,2.23,2.51,2.86,3.20 and 3.46% dry diet) from dietary ingredients and crystalline arginine. Each diet was randomly assigned to triplicate groups of 25 juvenile fish (10.51±0.15 g). Results showed that no significant difference in survival in experimental fish (P>0.05). SGR increased with increasing dietary arginine levels up to 2.51% and remained nearly the same thereafter. PER and protein productive value (PPV) showed increase tendency and then level off. FER was the poorest in Diet 1(P<0.05), the highest value of FER (91.32%) was observed in Diet 5, but no statistical difference was found when comparing with the Diet 3, Diet 4 or Diet 6 (P>0.05). ADCs of dry matter, crude protein and gross energy significantly improved up to 2.86% arginine diet and decreased at different extent thereafter, however, ADCs of crude lipid were unaffected. Fish fed 1.85% arginine diet had significant lower protein content in whole body and dorsal muscle than those fed diets supplemented with or more than 2.86% arginine. Lipid content decreased and lower value occurred at 3.46% dietary arginine (P<0.05). There were no significant differences in ash or moisture contents among dietary treatment (P>0.05). The dietary EAAs composition in whole body of black sea bream was significantly influenced by dietary arginine with exception of Thr. Arginine retention increased with dietary arginine level, then declined slightly at 3.46%arginine diet. Dietary increasing arginine level elevate arginase activity in liver up to 2.51% arginine diet (P<0.05), and the enzyme maintained at stable level thereafter (P>0.05). Almost all serum biochemical parameters were significantly affected by dietary arginine level except for cholesterol level.Broken-line regression based on SGR and second order polynomial regression based on PER indicated optimum dietary arginine requirement for juvenile black sea bream were 2.79 and 3.09% diet, corresponding to 7.74 and 8.13% of dietary protein, respectively.Trial 3. An 8-week feeding trial was conducted to determine the effects of imbalanced arginine/lysine levels in the diets of black sea bream juvenile. Eight isonitrogenous diets were formulated using a combination of intact protein and crystalline amino acids to contain different arginine/lysine levels (2.83/3.25,3.46/4.06,2.27/4.09,3.51/2.70,2.31/2.74,2.87/4.03, 2.91/4.68 or 2.85/5.24,% dry diet). The control diet contained 2.83% and 3.25% of arginine and lysine, respectively, which is known to satisfy the requirements of this fish. Each diet was fed to triplicate groups of 25 fish (10.0±0.1g).The results showed that dietary imbalanced levels of arginine and lysine had serious influences on growth performance and feed utilization of black sea bream, these corresponding indicators exhibited decreasing tendency in different extent. Based on growth performance, feed utilization efficiency, ADCs of dietary lysine and arginine, lysine and arginine retention ratio in body as well as liver arginase activity obtained in the present study suggest that arginine/lysine antagonism may exist in black sea bream. Adding arginine level to diets which containing superfluous lysine could alleviate this antagonism in this marine fish species.Trial 4. In the present study, total RNA extracted from black sea bream liver and dorsal muscle were reverse transcribed as described for TaqMan assays. Cloning primers were designed based on conserved regions of the fish genes’sequences from NCBI website to amplify IGF-I, IGF-IR and Arginase genes of black sea bream, and the sequence length were 890 bp,570bp and 430 bp, respectively. Blast analysis showed that the high similarity among black sea bream and other fish species, which indicated that these three sequences correspond to their corresponding gene family.Trial 5. The liver and dorsal muscle samples obtained from the arginine requirement study were used for genes expression analyzing. The results showed that the lowest level of IGF-I gene expression in liver was found in fish fed diet without arginine supplement (Diet 1) (P<0.05). IGF-I mRNA expression level increased with increasing arginine level to 3.25% (P<0.05), and then level off (P>0.05). The IGF-I mRNA expression in dorsal muscle presented a similar variation tendency with it was in liver. The relative lower and higher IGF-IR mRNA expression in dorsal muscle were observed in fish fed Diet 1 and Diet 4 (P<0.05), respectively. GHR gene mRNA expression tended to be enhanced by dietary arginine level, however, no significant differeces were found among the later four arginine level diets (P>0.05). Higher arginase gene expression levels in liver were detected in experimental fish fed Diet 5 and Diet 6, while lower values were seen in Diet 1 and Diet 2 (P<0.05).The liver samples obtained from the arginine/lysine ratios study were also used for genes expression analyzing. The results showed that fish fed diet with appropriate level of arginine and lysine simultaneously could inducing relative higher IGF-I mRNA expression in liver (P<0.05). Also, adding arginine into diet with excessive lysine had comparable gene expression level with control group. Black sea bream fed diet with inadequate lysine and arginine (Diet 5), or fish fed diets with constant arginine level but increasing lysine content, hepatic IGF-I mRNA expression decline significantly (P>0.05). Similarly, when comparing with the control group, dietary superfluous lysine content (Diet 7 and Diet 8) depressed hepatic Arginase gene mRNA expression significantly. In Diet 3 and Diet 5, which containing insufficient arginine level, Arginase mRNA expression levels also showed down regulation. These was no significant difference on regulation effect by dietary treatment on hepatic Arginase gene mRNA level among the control diet, Diet 2, Diet 4 and Diet 6 group (P>0.05).Results of this trial indicated that the effect of growth-promoted of dietary arginine was by regulated growth axis genes transcriptional control in black sea bream. Elevations in liver arginase gene mRNA expression have been observed under the dietary arginine level, and lysine level had influence on this gene expression.The following conclusions could be drew based on the present research.1, Lysine and arginine are necessary for black sea bream juvenile in diet for growth.2, Based on the growth performance of black sea bream juvenile, the optimal lysine and arginine levels in diet are 3.32% dry diet, corresponding to 8.64% dietary protein; and 2.79% diet, corresponding to 7.74% of dietary protein, respectively.3, Judged by growth performance, feed utilization efficiency and metabolism of amino acid, antagonism between arginine and lysine exists in black sea bream.4, The growth of black sea bream could be influenced by dietary arginine level.5, Dietary arginine level influencing the arginase gene expression in black sea bream. Meanwhile, lysine level also could affect this gene expression.更多还原