【作者】 马晶晶；

【导师】 许梓荣； 邵庆均；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2008， 博士

【摘要】 本文研究了饲料中添加n-3高不饱和脂肪酸对黑鲷幼鱼生长、体组成及血浆生化指标的影响,并由此得出黑鲷幼鱼达到最佳增重时对饲料中n-3HUFA%及DHA/EPA的需要量,进而通过研究组织中主要生酯酶(FAS)及主要脂解酶(HSL)的活性及基因表达来初步探讨饲料n-3]HUFA调控黑鲷脂肪代谢的主要机理。研究内容包括：(1)饲料中添加不同水平n-3HUFA对黑鲷幼鱼生长及体组成的影响；(2)饲料中DHA/EPA比值对黑鲷幼鱼生长及体组成的影响；(3)黑鲷FAS及HSL基因克隆及序列分析；(4)饲料n-3HUFA含量及DHA/EPA比值对黑鲷幼鱼脂肪代谢相关酶活性及基因表达的影响。(1)选取平均体重为(8.08±0.09)g、健康、体质均匀的黑鲷幼鱼360尾,随机放养入18个水泥池中搭建的网箱中。通过用鱼油和玉米油调和脂肪酸含量,将试验饲料配制成n-3HUFA含量分别为0.79,0.83,0.85,0.88,0.92,0.94%,DHA/EPA比值固定(2.8／1)的试验饲料,随机分配给六组,每组三个重复。每天投饵时间为8：00和16：00,以眼观饱食(投饵率3-5%)为准,试验条件同上。八周饲养试验结束后,对黑鲷幼鱼生长性能、饲料效率,蛋白质效率等指标进行测定,并分析体组织常规成分组成及肝脏、肌肉和腹腔脂肪组织脂肪酸组成及血浆生化指标的变化。试验结果表明,黑鲷脂肪细胞直径呈减小趋势,n-3HUFA水平最高组显著小于试验组3。肌肉脂肪含量受n-3HUFA的显著影响,且在第四组时达到最低；黑鲷幼鱼全鱼组织常规营养成分含量在各组之间差异不显著。肝脏、肌肉及腹腔脂肪组织中∑SFA和16：0均随饲料n-3HUFA水平的增加呈下降趋势,∑n-3HUFA呈显著上升趋势。各组织中DHA/EPA比值不受饲料脂肪酸的影响。黑鲷幼鱼血浆甘油三酯和胆固醇含量随饲料中n-3HUFA的添加有下降趋势。以增重率为参考指标,在本试验条件下,黑鲷幼鱼[(8.08±0.09)g]获得最佳增重时对饲料中n-3 HUFA的需要量为0.87%DM,即8.7 g/kg饲料干物质。(2)选取平均体重为(11.01±0.02)g的黑鲷幼鱼420尾,在与试验一相同的条件下研究n-3HUFA含量一定,DHA/EPA比值不同(1.98,2.20,2.46,2.88,＼3.15,3.57,4.04)的试验饲料对黑鲷幼鱼生长性能、饲料效率、蛋白质效率,体组织营养组成及血浆生化指标的影响。试验结果表明,与DHA/EPA最低组相比,黑鲷幼鱼腹脂率(IPF ratio)有明显减少,并在试验组3时达到最低。脂肪细胞直径随饲料DHA/EPA比值的变化趋势不明显,但在试验组5,6时显著低于其它各组。试验结束后对试验鱼体组织营养成分进行分析。结果显示,肌肉脂肪含量在饲料DHA/EPA>2.46时有显著降低,降幅约为8.59%。全鱼组织脂肪沉积量在饲料DHA/EPA>3.15时显著减少,最大降幅可达28.3%。各处理组背肌和腹腔脂肪组织n-3HUFA含量不受饲料的影响,但DHA/EPA比值随饲料水平的增加而增加。以增重率(WGR)为参考指标,二次曲线分析(y=-9.9707x2+62.696x+10.309,R2=0.9258；x,饲料DHA/EPA比值,y,WGR)表明,本试验条件下,黑鲷幼鱼(11.01±0.02 g)获得最佳增重时饲料中DHA/EPA比值为3.15。(3)本研究克隆了黑鲷FAS和HSL基因片段,其片段长度分别为464 bp和664 bp,分别编码150和221个氨基酸。BLAST比对分析结果表明,黑鲷FAS基因片段同斑马鱼(Danio rerio)同源性为76%。黑鲷HSL基因片段同斑马鱼同源性为69%。本试验对黑鲷FAS及HSL基因片段的克隆主要为以下试验中黑鲷幼鱼FAS及HSL基因表达的荧光定量分析作准备。(4)本试验主要通过分析组织中脂肪代谢主要酶的活性及基因表达水平,探讨饲料中不同n-3HUFA及DHA/EPA比值对黑鲷幼鱼脂代谢影响的机理。对酶活的测定结果显示,肝脏组织FAS活性较高,而腹腔脂肪(IPF)组织几乎不能检测到该酶,说明FAS在黑鲷幼鱼中的表达具有组织特异性,以肝脏为脂肪合成主要器官。FAS活性在试验组1-4之间无显著差异,当饲料n-3HUFA含量在0.92%以上时开始有显著下降。腹腔脂肪组织中HSL活性随饲料n-3HUFA的添加有增加趋势,当饲料n-3HUFA含量达到最高时(0.94%),HSL活性增加到将近一倍。饲喂不同DHA/EPA比值饲料的试验鱼肝脏组织中FAS活性不受饲料1-4的影响,随着饲料DHA/EPA比值的进一步增加呈显著下降趋势。脂肪组织HSL活性随饲料DHA/EPA比值的增加有上升趋势,但当饲料DHA/EPA大于2.88时进入平台期,保持在0.79左右。荧光定量分析与酶活测定结果相似,同样表现为FAS表达水平在试验组4以后开始下降,而HSL随饲料n-3HUFA或DHA/EPA升高而上升。以上研究结果表明,饲料n-3HUFA及DHA/EPA对黑鲷幼鱼脂肪代谢影响主要是对脂肪酸合成酶(FAS)和激素敏感脂肪酶(HSL)活性及基因表达进行同步调控的结果。更多还原

【Abstract】 Four experiments were conducted to investigate the effect of dietary n-3 highly unsaturated fatty acids (HUFA, mainly EPA and DHA), on growth performance, body composition and plasma biochemistry in juvenile black sea bream, Sparus macrocephalus. The aim of this study was to deduce the optimum requirement of n-3 HUFA and DHA to EPA ratio for the best growth performance of juvenile black sea bream, Sparus macrocephalus, and then to explore the main mechanism of n-3 HUFA regulation on lipid metabolism in black sea bream. The present study include the following:(1) Effect of supplementation of dietary n-3 HUFA on growth and body composition of black sea bream juveniles, Sparus macrocephalus; (2) Effect of dietary DHA to EPA ratio on growth performance, body compositon and fatty acid profile of juvenile black sea bream, Sparus macrocephalus;(3) Cloning of fatty acid synthse (FAS) and hormone sensitive lipase (HSL) in black sea bream, Sparus macrocephalus;(4) Effect of dietary n-3 level and DHA to EPA ratio on lipogenic and lypolytic enzymes activities and relative gene expression in black sea bream, Sparus macrocephalus.(1) Three hundred and sixty black sea bream juveniles (average weight, 8.08±0.09) were randomly allocated into 18 net cages built in a big concrete pond. Six diets with a constant DHA to EPA ratio (2.8/1) were formulated to contain different levels (8.48,8.89,9.08,9.43,9.88,10.12%) of n-3 HUFA, by supplementing corn oil and fish oil to modulate the fatty aid content. All diets were assigned to six grous of fish with triplicate. Fish were fed twice every day (8:00 and 16:00) to apparent satiation. The experiment conditions were mentioned above. At the end of the feeding trial, growth performance, feed efficiency (FE) and protein efficiency ratio (PER) of black sea bream were calculated, and plasma biochemisty parameters, the proximate composition as well as fatty acid profiles in liver, dorsal muscle and white adipose tissue (WAT) were analysized. At the end of the experiment, hepotasomatic index (HSI) and intraperitoneal fat (IPF) ratio decreased with the increase of dietary n-3 HUFA and the values in group 5 and 6 were significantly lower than that in other four groups (P<0.05). Adipocyte diameter in IPF was decreased by dietary n-3 HUFA and the significance occurred between group 3 and 6. Proximate composition analysis showed that lipid content in muscle was affected by dietary n-3 HUFA significantly and reached the bottom at the level of 0.88%. No significance was found in whole body proximate composition of juvenile black sea bream. Concerning the fatty acid composition,ΣSFA and the main constituent 16:00 were negatively correlated with dietary n-3 HUFA; in contrast,Σn-3 HUFA content was significantly increased among all the treatment. DHA to EPA ratios in liver, dorsal muscle and WAT were unchanged with the supplementation of dietary n-3 HUFA. Plasma triglycerides (TG) and total cholesterol (T-CHO) were all significanlty decreased by dietary treatment. Quadratic analysis based on weight gain rate (WGR) indicated that dietary n-3 HUFA requirement for black sea bream was 0.87% DM, that is 8.7 g/kg in the diet.(2) Black sea bream juveniles (11.01±0.02 g, mean weight) were fed diets containing different DHA/EPA ratios (from 1.98 to 4.04) with constant dietary n-3 HUFA level (about 1.0%) for 8 weeks. Four hundred and twenty fish were allocated into seven groups with triplicate and were fed different experimental diets. Fish feeding and the experimental condition were all just the same with experiment 1. Growth performance, FE and PER were calculated, while tissue proximate composition, fatty acid composition and plasma biochemical parameters were all analyzed at the end of the feeding trial. IPF ratio in other treatments was significantly lower than that in group 1, and fish in group 3 showed the lowest fat deposit in viscera. Diameter of adipocyte in IPF was significantly reduced compared to the other groups. Proximate composition analysis showed that lipid content in muscle was decreased by 8.59% at large, and lipid deposit in whole body was decreased by 28.3%. Fatty acid profile analysis indicated that n-3 HUFA content in liver, dorsal muscle and WAT was independ of dietary DHA to EPA ratios, while DHA/EPA ratio in these tissues were positively correlated with dietary DHA/EPA ratio. Based on weigt gain rate (WGR), quadratic analysis (y=-9.9707x2+62.696x+10.309, R2=0.9258; x, DHA/EPA ratio; y, WGR) showed that at the present experiment condition, the DHA/EPA requiremet for juvenile black sea bream was 3.15.(3) FAS gene of 464 bp and HSL of 665 bp were cloned in this experiment. Blast analysis showed that the high similarity (76% and 69%) between black sea bream and zebrafish is a strong indication that these two sequences correspond to a FAS or HSL gene. Clone of partial FAS and HSL gene in the experiment made preparations for the next real-time PCR determination of FAS and HSL gene expression in the following experiment.(4) Postulated mechanism of fatty acid regulation in lipid metabolism in black sea bream was discussed according to the study of the effect of dietary n-3 HUFA and DHA to EPA ratio on FAS or HSL gene expression and enzyme activities. Determination of FAS and HSL activities showed that FAS activity was higher in liver than that in adipose tissue, which means the tissue specificity of FAS in black sea bream and liver was considered to be the main organ of fatty acid synthesis. In both experiment, FAS activity kept constant in group 1 to 4, and then decreased significantly with further increment of dietary n-3 HUFA or DHA/EPA ratio. HSL activity increased significantly with the elevation of dietary n-3 HUFA level or dietary DHA/EPA ratio, but reached a plateau when the ratio was higher than 2.88. Real-time PCR determination showed that FAS gene expression changed following the similar trend with FAS activity in liver; while liver HSL gene expression, just as in adipose tissue, was positively correlated with dietary n-3 HUFA level or DHA/EPA ratio. In summary, we can deduce that n-3 highly unsaturated fatty acid regulation in lipid metabolism was realized by affecting the two pathways, that is, lipogenesis and lipolysis at the same time.更多还原