【作者】 谷珉；

【导师】 麦康森；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2013， 博士

【摘要】 本研究以大西洋鲑(Salm)、大菱鲆(Scophthalmus maximus L)为实验对象,探讨植物蛋白源中抗营养因子对肉食性鱼健康的作用机制。同时以凡纳滨对虾(Litopenaeus vannanei)、大菱鲆为实验对象,探讨在含有植物蛋白源的饲料中补充不同形式的必需氨基酸对其生长和饲料利用率的影响。为提高鱼虾对植物蛋白源的利用提供理论基础。本论文共包括以下内容：1.大豆皂甙对大西洋鲑(Salmo salar,L)生长、消化酶和肠道健康的剂量效应本实验研究目的是探讨大豆皂甙对大西洋鲑(Salmo salar, L)生长、消化酶和肠道健康的剂量效应。配制两种基础饲料：鱼粉基础饲料(FM)和植物蛋白基础饲料(PM)。每种基础饲料中添加0、2、4、6和10g kg-1的大豆皂甙,共制备10种饲料。实验用大西洋鲑初始体重为442±33g,放养在20个玻璃钢桶中,每两个桶投喂一种实验饲料。养殖周期为10周。养殖结束后检测实验饲料对实验鱼体体重以及肠道健康的影响,包括：食糜胰蛋白酶活性、刷状缘膜酶活性以及肠道形态结构。植物蛋白饲料组的生长显著低于鱼粉饲料组(P<0.05)。大豆皂甙对鱼粉组大西洋鲑生长的影响表现出先上升后下降的变化(R2=0.953,P<0.001)。随着大豆皂甙添加剂量的提高,对大西洋鲑的造成如下负面影响：1)后肠刷状缘膜酶活性的下降；2)后肠胰蛋白酶活性的上升；3)上皮细胞的过量增殖。当大豆皂甙的添加剂量高于6g kg-1时,在两种基础饲料中都引起了大西洋鲑后肠炎症的发生。2.植物蛋白源和大豆皂甙对大西洋鲑(Salmo salar, L)肠道与肝脏脂肪积累、脂蛋白代谢和甾醇代谢的影响植物蛋白的使用造成了鱼类脂肪代谢的改变。本研究的目的是进一步探讨饲料中添加植物蛋白源以及大豆皂甙对大西洋鲑肠道和肝脏脂肪代谢的影响。配制两种基础饲料：鱼粉基础饲料(FM)和植物蛋白基础饲料(PM)。每种基础饲料中添加10gkg-1的大豆皂甙,共制备4种实验用饲料。实验用大西洋鲑初始体重为442±24g,放养在8个玻璃钢桶中,每两个桶投喂一种实验饲料。养殖周期为10周。实验结果表明植物蛋白的添加引起了大西洋鲑生长的下降、幽门盲囊和肝脏内脂肪积累的增多以及血液胆固醇水平的下降(P<0.05)。植物蛋白的添加还引起了肠道和肝脏内脂肪吸收、脂蛋白合成(载脂蛋白、脂肪酸转运载体、微粒体三酰转移蛋白、胆碱激酶、胆碱磷酸胞苷酰基转移酶A)和胆固醇合成(3羟基3甲基戊二酰辅酶A还原酶)过程中关键基因及其转录调控因子(甾醇调节因子结合蛋白2和过氧化物酶体增殖物激活受体)基因的上调。大豆皂甙的添加引起了大西洋鲑体内胆固醇和胆酸盐的下降。分子数据表明大豆皂甙引起了肝脏内胆酸盐生物合成限速蛋白酶基因(胆固醇7-羟化酶)、肝脏X受体蛋白基因以及胆酸盐转运蛋白基因的下调。基础饲料和大豆皂甙对于血清胆固醇水平具有显著的交互作用(P<0.05)。总之,本研究表明植物蛋白源的添加造成了大西洋鲑脂蛋白合成能力和胆固醇合成能力的提高。这可能是由于脂肪运输和胆固醇代谢的损伤所引起的。大豆皂甙具有降低胆固醇和胆酸盐含量的作用。分子数据表明大豆皂甙引起了大西洋鲑肝脏胆酸盐合成和分泌能力的下降。3.大豆皂甙、大豆异黄酮和植物甾醇对早期生长阶段大西洋鲑(Salmo salar, L)幼鱼的影响本研究通过14周的养殖实验探讨豆粕中常见的抗营养因子对早期生长阶段大西洋鲑的影响。检测指标包括：生长、消化酶活力、肠道和肝脏组织学以及骨骼发育情况。以鱼粉组饲料作为参照组(FM),在参照组中添加2gkg-1的大豆皂甙(SAP)、1.5gkg-1大豆异黄酮(IFL)、0.3g kg-1的植物甾醇(PHS)以及这三者的混合物(MIX),共制备5种等氮等能的饲料。每种饲料作为一个处理投喂给初次摄食的大西洋鲑仔鱼(初始体重约为0.18g),每个处理设三个重复。相比鱼粉组饲料,大豆皂甙的添加引起了实验鱼生长的提高,但是大豆异黄酮引起了生长的显著下降(P<0.05)。IFL组大西洋鲑前肠的麦芽糖酶活性显著低于FM组,胰蛋白酶活力显著高于鱼粉组(P<0.05)。肠道胆酸盐含量、亮氨酸氨基肽酶和淀粉酶活性在各处理组之间没有显著差异(P>0.05)。肠道组织形态学在各处理组之间没有出现显著差异(P>0.05)。IFL组肝脏细胞显著缩小,糖原含量显著减少。饲料处理具有提高鱼体骨骼发育畸形率的趋势(P<0.10)。PHS组和IFL组表现出最高的和次高的畸形率,分别为5.1%和4.3%。总之,本研究的结果表明大豆异黄酮对早期生长阶段大西洋鲑幼鱼的生长、肠道功能、肝脏组织和骨骼发育产生了负面影响。4.β-伴大豆球蛋白和大豆球蛋白对大菱鲆(Scophthalmus maximus L)生长、消化酶和免疫反应的影响本实验的研究目的是探讨β-伴大豆球蛋白和大豆球蛋白对大菱鲆(Scophthalmus maximus L)生长、消化酶和免疫反应的影响。以粗蛋白含量为48%和粗脂肪含量为12%的基础饲料作为对照组。在基础饲料中添加60gkg-1的p-伴大豆球蛋白(7S)、60g kg-1热处理的p-伴大豆球蛋白(H7S)、60gkg-1的大豆球蛋白(11S)和60g kg-1热处理的大豆球蛋白(HllS)。每种饲料作为一个处理投喂给初始体重约为6.80g的大菱鲆,每个处理设三个重复。7S处理组的大菱鲆特定生长率显著低于对照组,而饲料系数则显著高于对照组(P<0.05)。另外,7S的添加显著降低了大菱鲆肠道不同部位(幽门盲囊、中肠和后肠)消化酶活性,包括淀粉酶、脂肪酶和麦芽糖酶活性(P<0.05)。另外,7S处理组的大菱鲆后肠的酸性磷酸酶活性和溶菌酶活性显著低于其它处理组(P<0.05)。综上所述,β-伴大豆球蛋白(7S)的添加引起了大菱鲆生长、饲料利用率、消化酶活性的下降,并引起了后肠的免疫学反应。而热处理组后的7S以及大豆球蛋白并没有对大菱鲆产生上述负面影响。在本研究中,p-伴大豆球蛋白的免疫原性可能与加热处理过程有关。5.晶体蛋氨酸和蛋氨酸寡肽对于凡纳滨对虾(Litopenaeus vannanei)生长和饲料利用率的影响本研究探讨了晶体蛋氨酸(CMet)和蛋氨酸寡肽(OMet)对凡纳滨对虾(Litopenaeus vannanei)生长和饲料利用的影响。以凡纳滨对虾商业配方饲料作为对照组。以豆粕和花生粕的混合物作为替代蛋白源替代30%和60%的鱼粉。为平衡蛋氨酸水平,分别在30%的替代组中添加0.1%的晶体蛋氨酸(SPP30-CMet)或0.1%的蛋氨酸寡肽(SPP30-OMet),分在60%的替代组中添加0.2%的晶体蛋氨酸(SPP60-CMet)或0.2%的蛋氨酸寡肽(SPP60-OMet)。蛋氨酸来源显著影响了凡纳滨对虾的生长、体组成成分和肝体比(HSI)(P<0.05)。 SPP60-CMet处理组对虾的生长和虾体粗蛋白含量显著低于对照组,而肝体比显著高于对照组(P<0.05)。添加蛋氨酸寡肽的处理组与对照组之间没有显著差异(P>0.05)。SPP30-OMet处理组对虾的饲料效率和蛋白质效率显著高于SPP30-CMet处理组(P<0.05)。本研究表明相比晶体蛋氨酸,蛋氨酸寡肽能够更好的促进凡纳滨对虾对植物蛋白的利用。6.晶体蛋氨酸和蛋氨酸寡肽对于大菱鲆(Scophthalmus maximus L)餐后血清游离氨基酸水平、生长和饲料利用率的影响本研究通过8周的养殖实验探讨晶体蛋氨酸(CMet)(?)蛋氨酸寡肽(OMet)对大菱鲆(Scophthalmus maximus L)餐后血清氨基酸水平的影响(实验I)以及在高植物蛋白含量饲料中对大菱鲆生长和饲料利用率的影响(实验II)。在实验I中,配制蛋氨酸缺乏饲料并作为基础饲料,蛋氨酸含量为1.0%。为平衡饲料中的蛋氨酸,在基础饲料中添加0.5%的晶体蛋氨酸或蛋氨酸寡肽制备两种饲料,分别命名为CMet-0.5和OMet-0.5。每种饲料投喂给平均体重为35.8g的大菱鲆幼鱼,投喂周期为1周。在投喂后3h、6h、9h和12h取血清样品检测血液中的游离氨基酸含量。实验结果表明OMet-0.5处理组的大菱鲆血液蛋氨酸水平与大部分检测的氨基酸具有类似的餐后变化形式。而CMet-0.5处理组的大菱鲆血液蛋氨酸相比其它检测的氨基酸具有更早的吸收高峰值。在实验II中,除了CMet-0.5和OMet-0.5饲料外,在基础饲料中添加0.25%和1.0%的晶体蛋氨酸或蛋氨酸寡肽配制其它4种等氮等脂的饲料。将6种实验饲料投喂给初始体重为6.8g的大菱鲆幼鱼,投喂周期为8周。实验结果表明,蛋氨酸寡肽相比晶体蛋氨酸能够更好的促进大菱鲆的生长和饲料利用率。高浓度的蛋氨酸对大菱鲆的生长起到了负面的影响。更多还原

【Abstract】 The present studies were conducted to do basic research on how to improve plant meal utilization by fish and shrimp. Atlantic salmon (Salmo salar) and turbot (Scophthalmus maximus L) were used as experimental subjects to investigate the effects of anti-nutrients in plant meal on the health of these two carnivorous fish species. Studies were also conducted to investigate the effects of supplementation of different forms of essential amino acids on growth and feed utilization in white shrimp (Litopenaeus vannanei) and turbot fed with high levels of plant meal. The studies are summarized as follows:1. Dietary saponin dose response in Atlantic salmon (Salmo salar):. Effects on growth, digestive enzyme, and gut health.The goal of the current work was to examine the dose response of purified soya-saponins on growth, digestive physiology and gut health in Atlantic salmon (Salmo salar, L). Two basal diets were formulated:a fish meal based diet (FM) and a plant meal based diet (PM). Each basal diet was produced without or with soya-saponin supplementation (2,4,6or10g kg-1). Each diet was fed to duplicate tanks of Atlantic salmon (initial weight442±33g; mean±SD). The feeding trial lasted10weeks. At termination fish were sampled to analyze growth and intestinal responses, including chyme trypsin activity, tissue brush border enzyme activity and histology. Fish fed the PM based diets had lower final weights compared to fish fed the FM based diets. Saponins significantly influenced the growth performance of FM fed fish, showing higher growth at low (2-4g kg-1) saponin levels but lower growth at high (6-1Og kg-1) levels (R2=0.953,.P＜0.001). Increasing dietary soya-saponin concentration caused:1) Decreased enterocyte brush border enzyme activity in the distal intestine;2) Increased trypsin activity in digesta from the distal intestine;3) Increased enterocyte proliferation in distal intestine. Inflammation was observed in the distal intestine associated with high levels (6-10g kg-1) of dietary soya-saponin, regardless of the basal diet formulation.2. Effects of dietary plant meal and soya-saponin supplementation on intestinal and hepatic lipid droplet accumulation, lipoprotein and sterol metabolism in Atlantic salmon (Salmo salar L.).Altered lipid metabolism has been shown in fish fed with plant protein sources. This study aimed at gaining further insight into how intestinal and hepatic lipid absorption and metabolism are modulated by plant meal and soya-saponin inclusion in salmon feed. Post-smolt Atlantic salmon (initial weight442±24g; mean±SD) were fed one of four diets based on fish meal or plant meal, with or without1.0%soya-saponin for10weeks. Plant meal inclusion resulted in decreased growth performance, excessive accumulation of lipid droplets in pyloric caeca and liver and reduced plasma levels of cholesterol. Intestinal and hepatic gene expression profiling revealed up-regulation of genes involved in lipid absorption and lipoprotein synthesis (apolipoproteins, fatty acid transporters, microsomal triglyceride transfer protein, acyl-coA cholesterol acyltransferase, choline kinase, choline-phosphate cytidylyltransferase A), cholesterol synthesis (3-hydroxy-3-methylglutaryl-coenzyme A reductase) and associated transcription factors (sterol regulatory element binding protein2and peroxisome proliferator activated receptor gamma). Soya-saponin inclusion resulted in reduced body pools of cholesterol and bile salts. Hepatic gene expression of the rate-limiting enzyme in bile acid biosynthesis (cyp7a1) as well as the transcription factor liver X receptor and the bile acid transporter (abcb11) were down-regulated by soya-saponin inclusion. Significant interaction was observed between plant meal and soya-saponin inclusion in plasma cholesterol level. In conclusion, gene expression profiling suggested that the capacity for lipoprotein assembly and cholesterol synthesis were up-regulated by plant meal exposure, probably as a compensatory mechanism for excessive lipid accumulation and reduced plasma cholesterol levels. Soya-saponin inclusion had hypocholesterolemic effects on Atlantic salmon, accompanied by decreased bile salt metabolism.3. Effects of diet supplementation of soya-saponins, isoflavones and phytosterols on Atlantic salmon(Salmo salar, L) at early growth stage. A14-week trial was conducted to investigate the effects of antinutritional factors (ANFs) commonly present in soybean ingredients, singly and in combination, on Atlantic salmon (Salmo salar L.). Fry performance, including growth, digestive enzyme activities, intestinal and liver histology, and skeletogenesis were evaluated. The experimental diets consisted of a reference control fish meal diet (FM), and four diets based on the FM diet supplemented with2g kg-1soya-saponins (SAP),1.5g kg-1isoflavones (IFL),0.3g kg-1phytosterols (PHS) or a mixture of these (MIX). Each diet was randomly allocated to triplicate groups of fish fry (approximately0.18g) from start-feeding. Fish fed the SAP diet showed significantly higher growth performance than those fed FM, while the IFL treatment significantly decreased final body weight and specific growth rate (P<0.05). For digestive enzyme activities, fish fed the IFL diet had significantly lower maltase activity and higher trypsin activity in proximal intestine than fish fed the FM diet (P<0.05). No significant difference among the treatment groups was observed either in intestinal bile acids concentration or in activities of leucine aminopeptidase or amylase. Nor was intestinal histomorphology affected. Histological differences were observed in the liver of fish fed the IFL diet, characterized by reduced size of the hepatocytes due mainly to reduction in glycogen content. Dietary treatments tended to change the incidence of skeletal deformities in salmon fry (P＜0.10). Fish fed the PHS and IFL diets showed the highest frequencies (5.1and4.3%, respectively) of deformities among the five treatments, In conclusion, the results indicate that purified isoflavones may negatively affect growth performance, intestinal function, liver metabolism and bone formation of salmon fry.4. Effects of β-conglycinin and glycinin on growth, digestive enzymes and immune responses in juvenile turbot (Scophthalmus maximus L).The study was conducted to investigate the effects of β-conglycinin and glycinin on growth, digestive enzymes and immune responses in turbot (Scophthalmus maximus L). The basal diet was formulated to contain48%crude protein and12%crude lipid, and was used as the control. The other four diets were supplemented with6.0%β-conglycinin (diet7S),6.0%heat treated β-conglycinin (diet H7S),6.0% glycinin (diet11S) and6.0%heat treated glycinin (diet H11S), respectively. Each diet was randomly allocated to triplicate groups of fish (initial average weight of6.80g) for4weeks. Fish fed7S diet showed significantly lower specific growth rate (SGR) and higher feed conversion rate (FCR) than the others (P<0.05). Furthermore, it had reduced digestive enzyme activities (amylase activity, lipase activity, maltase activity and alkaline phosphatase activity) in different gastrointestinal (GI) sections (pyloric caeca, mid-intestine and distal intestine). Additionally, fish fed7S diet showed significantly higher acid phosphatase activity and lysozyme activity in distal intestine compared with fish fed the other diets (P<0.05). In conclusion, P-conglycinin (7S) significantly decreased the growth, feeding efficiency, digestive enzyme activities and induced the immunological responses in distal intestine of turbot. These changes were not observed in the fish fed heat treated7S diet. Glycinin (11S) had no significant influence on the health of turbot. Immunogenicity of β-conglycinin (7S) in turbot may be relative to its heat processing.5. Effects of dietary crystalline methionine or oligo-methionine on growth performance and feed utilization of white shrimp(Litopenaeus vannanei) fed high plant protein diets.An eight-week feeding experiment was conducted to investigate the effects of dietary crystalline methionine (CMet) or oligo-methionine (OMet) on growth performance and feed utilization of white shrimp, Litopenaeus vannanei. A practical diet was used as control diet. The other four isonitrogenous and isolipid diets replacing30%and60%fish meal by a mixture of soybean meal and peanut meal (SPP) were formulated. To balance the methionine content,0.1%CMet (SPP30-CMet) or0.1%OMet (SPP30-OMet) was added in30%fish meal replacing diets and0.2%CMet (SPP60-CMet) or0.2%OMet (SPP60-OMet) was added in60%fish meal replacing diets. Methionine source significantly affected growth performance, body compositions and hepatosomatic indices (HSI) of white shrimps (P＜0.05). Shrimps in SPP60-CMet treatment showed significantly lower weight gain, crude protein content in whole body and significantly higher HSI than those in control (P<0.05). However, no significant difference in these indices was observed between control and OMet supplemented treatments (P>0.05), Shrimps in SPP30-OMet treatment showed significantly higher feed efficiency ratio and protein efficiency ratio than those in SPP30-CMet treatment (P＜0.05). The present study indicated that compared with the CMet, dietary OMet resulted in better growth and feed efficiency of L. vannanei fed high plant protein diets.6. Effects of dietary crystalline methionine or oligo-methionine on postprandial plasma free amino acid level, growth performance and feed utilization of turbot (Scophthalmus maximus L) fed high plant protein diets.Two experiments were conducted to investigate the effects of dietary crystalline methionine (CMet) or oligo-methionine (OMet) on postprandial plasma free amino acid level (experimental I), growth performance and feed utilization of turbot (Scophthalmus maximus L) fed high plant protein diets (experimental Ⅱ). In experimental I, a methionine deficient diet (1.0%methionine) was formulated and used as basal diet. To balance the methionine content,0.5%crystalline methionine (CMet-0.5) or0.5%oligo-methionine (OMet-0.5) was added. Each diet was fed to triplicate groups of turbot (approximately35.8g) for a week. Plasma samples were collected3h,6h,9h and12h postprandially to analyze free amino acid concentration. The plasma methionine level of fish fed the OMet-0.5diet showed similar postprandial change pattern with most of the other assayed amino acids. However, in CMet-0.5treatment, the time observed for peak level of plasma methionine was earlier than that of any other assayed amino acids. In experimental II, exclusive of two diets used in Experimental I, another four isonitrogenous and isoenergetic diets with0.25%and1.0%of crystalline methionine (named as CMet-0.25and CMet-1.0) or oligo-methionine (named as OMet-0.25and OMet-1.0) were prepared. Each diet was fed to triplicate groups of turbot (approximately6.8g) for8weeks. The results showed that oligo-methionine could improve the growth performance and feed utilization of turbot compared with crystalline methionine. High supplementation of methionine showed negative effect on the growth performance of turbot.更多还原