【作者】 王爱民；

【导师】 王恬； 徐跑；

【作者基本信息】 南京农业大学 ， 水产养殖， 2011， 博士

【摘要】 吉富罗非鱼是遗传性状改良后的尼罗罗非鱼(Genetic Improvement of Farmed Tilapia Strain of Nile tilapia, Oreochromis niloticus),现已成为我国一个新的重要养殖品种。由于饲料营养失衡,如长期投喂低蛋白、高脂肪、高糖类和缺乏维生素的饵料,造成罗非鱼肝损害,生产存在规格小、品质差等问题。脂肪是鱼类重要的营养素,它的性质决定了其独特的生物学功能及其他营养素难以替代的作用。为探讨吉富罗非鱼对饲料脂肪适宜需求量及饲料脂肪水平对脂肪代谢及其关键酶的调节机制,本文开展了饲料中不同脂肪水平对吉富罗非鱼生长、体脂沉积及脂肪酸组成的影响研究,首次克隆了吉富罗非鱼脂肪酸合成酶(fatty acid synthetase, FAS)、脂蛋白脂酶(lipoprotein lipase, LPL) cDNA序列,并开展了饲料脂肪水平及再投喂对吉富罗非鱼脂肪酸合成酶、脂蛋白脂酶活性及其基因表达水平、血液脂肪代谢生化指标的调节研究。1饲料脂肪水平对吉富罗非鱼生长、营养物质消化、肌肉成分及血液生化指标的影响为探讨吉富罗非鱼对脂肪的适宜需求,将630尾(2.63±0.16 g)吉富罗非鱼随机分成6组,每组设置3个重复,每个重复35尾,其中第1组为对照组,投喂基础日粮(未添加鱼油,含脂肪1.73%),另外5组为试验组,饲喂在基础日粮中分别添加2%、4%、6%、8%、15%的鱼油含不同脂肪水平(3.71%、5.69%、7.67%、9.64%和16.55%)的等氮饲料,饲养90 d,试验结束时,测定增重率、饲料系数、营养物质表观消化率,禁食48 h,每个水族箱随机取3尾鱼,测定肌肉常规营养成分、肠道消化酶活性及血液常规生化指标。结果显示,随着饲料脂肪水平的提高,增重率和特定生长率呈现先升后降的趋势,蛋白质效率显著提高(P<0.05),而饲料系数显著下降(P<0.05)；增重率与饲料脂肪水平的二次多项式回归分析显示,吉富罗非鱼获得最高增长所需饲料的最佳脂肪水平为9.34%；饲料脂肪水平对粗蛋白表观消化率和饲料干物质表观消化率无显著影响(P>0.05),增加饲料脂肪水平显著提高了粗脂肪和磷的表观消化率(P<0.05)；随着饲料脂肪水平的升高,罗非鱼肌肉脂肪含量上升,变化范围为9.96%～17.27%,水分、粗蛋白、粗灰分及磷含量均呈下降趋势；吉富罗非鱼胃和肠道中蛋白酶活性没有显著变化(P>0.05),前肠和中肠的脂肪酶活性显著下降(P<0.05),前肠中的淀粉酶活性显著下降(P<0.05)；未添加鱼油的1.73%组血液中白蛋白和白球比均显著高于其他组(P<0.05),随着饲料脂肪水平的提高,胆固醇及碱性磷酸酶显著上升(P<0.05)；饲料脂肪水平对血糖有显著影响(P<0.05),对甘油三酯浓度、谷丙转氨酶和谷草转氨酶的活性无显著影响(P>0.05)。结果表明,饲料中一定含量的脂肪水平可以促进吉富罗非鱼生长,提高吉富罗非鱼对脂肪和磷的表观消化率,但脂肪水平过高会对鱼体增重及血液生化指标产生负作用,因此,在生产上吉富罗非鱼幼鱼对饲料中脂肪的适宜需求量为7.67%-9.34%。2饲料脂肪水平对吉富罗非鱼体脂沉积及脂肪酸组成影响饲养方案同1,饲养90 d,试验结束时,禁食48 h后,每一水族箱随机取3尾鱼的进行解剖,测定吉富罗非鱼的部分形体指标、肌肉肝脏及腹腔脂肪组织的脂肪沉积及脂肪酸组成。试验结果显示：5.69%、7.67%及9.64%脂肪组吉富罗非鱼肥满度较高；1.73%和16.55%脂肪组的肝体指数显著高于其他试验组(P<0.05)；除了1.73%脂肪组,其他各组中饲料脂肪水平越高,鱼体的脏体指数越高。7.67%脂肪组吉富罗非鱼肌肉脂肪含量显著高于3.71%组(P<0.05),同时显著低于16.55%组(P<0.05),但是其肝脏中脂肪含量与其他各组差异均不显著(P>0.05)；饲料脂肪水平越高,鱼体的脂肪含量越高,同时不饱和脂肪酸占总脂肪酸中的比例越高。结果表明,饲料脂肪水平影响吉富罗非鱼的部分形体指标,尤其对肝脏形态的影响较为明显。饲料中过多的脂肪容易在肌肉和肝脏组织中沉积,同时鱼体的脂肪含量和脂肪酸组成能够反映饲料的脂肪水平和脂肪酸组成。3吉富罗非鱼FAS、LPL基因的克隆及序列分析采用RT-PCR和cDNA末端快速扩增法(rapid amplification of cDNA ends, RACE)克隆了吉富罗非鱼FAS基因部分序列(GenBank:GU433188)及LPL基因的cDNA全长序列(GenBank: GU433189).克隆得到的吉富罗非鱼FAS基因的部分cDNA序列长557 bp,编码185个氨基酸,序列分析表明吉富罗非鱼脂肪酸合成酶与其他物种的同源性为62%～82%。吉富罗非鱼LPL基因的cDNA全长2298 bp,编码515个氨基酸,序列分析表明吉富罗非鱼脂蛋白脂酶与其他物种的同源性为57.3%～87.9%。一些重要的功能位点如脂肪结合域(lipid binding domain, LID)等在进化的过程中较为保守。同源建模分析显示LPL具有典型的脂肪酶家族结构。4饲料脂肪水平和再投喂对吉富罗非鱼脂肪酸合成酶活性及表达的影响为研究饲料脂肪水平对吉富罗非鱼FAS活性和表达的影响,选择平均体重为(2.63±0.16)g的健康的315尾吉富罗非鱼幼鱼,随机分成3组,每组3个重复,投喂3组不同脂肪水平(饲料中脂肪含量分别为3.71%、7.67%和16.55%)的等氮低脂肪组、中脂肪组、高脂肪组饲料,以低脂肪组为对照组,饲养90 d,试验结束时,禁食48 h,从每一水族箱随机取3尾鱼取其肝脏及肌肉,再投喂后6、12、24、48 h时再从每个水族箱(每个平行组)取3尾鱼取肝脏样品,测定吉富罗非鱼肝脏中FAS的生物活性,使用荧光实时定量PCR分别测定饲喂3.71%组、7.67%组和16.55%组饲料的吉富罗非鱼肝脏和肌肉中FAS mRNA的表达丰度以及再投喂后6、12、24、48 h肝脏中FAS mRNA的表达丰度。结果显示：饲料脂肪水平对肝脏中FAS活性无显著影响(P>0.05)：肝脏中FAS mRNA的表达丰度显著高于肌肉(P<0.05)；肝脏和肌肉中FAS mRNA的表达丰度随着饲料中脂肪水平增加而显著下降(P<0.05)；再次投喂后6～48 h,各个组的肝脏中FAS mRNA表达丰度显著下降(P<0.05)。结果说明,高脂肪饲料对肝脏中FAS活性分泌无诱导作用,吉富罗非鱼肝脏中FAS mRNA的表达丰度高于肌肉,高脂肪饲料能够抑制FAS mRNA表达,脂肪水平越高抑制作用越显著,再投饲后6～48 h,FAS基因表达受到抑制。5饲料脂肪水平和再投喂对吉富罗非鱼脂蛋白脂酶活性及表达的影响饲养方案同4,饲养90 d,试验结束时,禁食48 h,每一水族箱随机取3尾鱼的肝脏及肌肉,再投喂后6、12、24、48 h分别从每个水族箱(每个平行组)取3尾鱼取肝脏样品,使用实时荧光定量PCR分别测定了饲喂3.71%组、7.67%组和16.55%组饲料的吉富罗非鱼肝脏和肌肉中LPL mRNA的表达丰度以及禁食48小时再投喂后6、12、24、48 h肝脏中LPL mRNA的表达丰度。结果显示：LPL mRNA在吉富罗非鱼肝脏和肌肉中均有表达,但肝脏中LPL mRNA表达丰度显著高于肌肉中LPL mRNA表达丰度；随着饲料脂肪水平的升高,肝脏中LPL mRNA表达水平出现升高的趋势,16.55%高脂肪水平组LPL mRNA表达最高,显著高于3.71%低脂肪水平组(P<0.05)；禁食(饥饿)48h后,吉富罗非鱼肝脏LPL mRNA表达水平最高,再投喂后12 h显著下降,到48 h又逐渐回到饥饿时表达水平。研究表明：高脂肪显著促进了吉富罗非鱼肝脏LPL活性的分泌；LPL mRNA在吉富罗非鱼肝脏和肌肉中表达具有组织特异性,且肝脏是吉富罗非鱼LPL合成和表达的主要组织器官之一；高脂肪诱导了吉富罗非鱼肝脏LPL基因表达,同时吉富罗非鱼肝脏LPL基因表达受到饲养状态(饱食、饥饿)的调控。6饲料脂肪水平和再投喂对吉富罗非鱼血脂、血糖的影响为研究高脂肪饲料和再投喂对吉富罗非鱼摄食后血液脂肪代谢的影响,选择平均体重为(2.63±0.16)g的健康的315尾吉富罗非鱼幼鱼,随机分成3组,每组3个重复,投喂3组不同脂肪水平(饲料中脂肪含量分别为3.71%、7.67%和16.55%)的等氮低脂肪组、中脂肪组、高脂肪组,以低脂肪组为对照组,饲养90 d,试验结束后,禁食24 h,取样测定血液中脂肪代谢相关指标,再禁食24 h投喂,摄食3.71%组、7.67%组和16.55%组饲料后0、6、12、24、48 h每个水族箱随机取样3尾取样测定试验鱼血清中甘油三酯、胆固醇及血糖。结果显示：高脂肪饲料使吉富罗非鱼血清甘油三酯、胆固醇、碱性磷酸酶、谷丙转氨酶显著升高(P<0.05),血糖显著下降(P<0.05)；吉富罗非鱼摄食不同脂肪水平饲料后,48 h内,其血液甘油三酯、胆固醇和血糖均呈现先上升后下降的趋势,在摄食后同一时间点,摄食高脂肪试验鱼血液中甘油三酯和胆固醇显著高于低脂肪组(P<0.05),而血糖无显著变化,但有下降的趋势(P>0.05)。结果说明：高脂肪(16.55%)诱导吉富罗非鱼肝脏受到一定损害或病变。摄食后48 h内,吉富罗非鱼摄食低、中脂肪水平饲料后血液甘油三酯、胆固醇、血糖呈现单峰波形图变化规律,其中三组甘油三酯含量达到最高时间点均为摄食后第12 h；中低脂肪组胆固醇峰值为摄食后6 h,高脂肪为12 h；高中脂肪组血糖峰值为摄食后第6 h、低脂肪组为摄食后第12 h。高脂肪诱导血液甘油三酯合成代谢,但没有改变其变化基本规律,使胆固醇峰值时间比正常组滞后6 h,有抑制吉富罗非鱼血糖的浓度变化,不利于血糖的代谢趋势。更多还原

【Abstract】 GIFT is the genetically Improved Farmed Tilapia Strain of nile tilapia(Oreochromis niloticus), which have became an important aquaculture species. Unbalance nutrition in diet, i.e. low protein, high fat, high carbohydrate, and vitamins lacking, lead to the liver damage, smaller body and poor quality of fish. Lipid is an important nutrient for fish and it is difficult to be replaced by other nutrients because of its unique biological function. In order to explore the optimal lipid demand and the regulation mechanism of different lipid levels on fat metabolism and key enzyme in lipid metabolism for GIFT tilapia, the optimal lipid demand of GIFT tilapia, and the effects of different lipid levels on fat deposition, fatty acid composition were studied; the GIFT tilapia fatty acid synthase (FAS), lipoprotein lipase (LPL) cDNA were first obtained; the effects of lipid levels and refeeding on the activity, expression of FAS and LPS and blood biochemical parameters were studied.1 Effects of dietary lipid levels on growth, muscle composition, feed apparent digestibility, muscle composition and blood biochemical parameters of GIFT strain of nile tilapia(Oreochromis niloticus)In order to determine the optimal levels of the lipid of GIFT Strain of Nile tilapia (Oreochromis niloticus),630 GIFTs (average weight 2.63±0.16 g) were divided into six groups randomly, with one control group that fed with basal diet (1.73%lipid) and five experimental groups fed with different lipid level diet (3.71%,5.69%,7.67%,9.64%and 16.55%) by supplementing with 2%,4%,6%,8%and 15%fish oil as lipid source, respectively. Every group was triplicate. After rearing for 90 d, the fish was fasted for 48 h, then three fishes were randomly selected from each group, and the growth, feed conversion ratio, muscle composition, digestive enzyme activities, nutrient apparent digestibility and blood biochemical parameters were detected. The results showed that, along with dietary lipid level raising, weight gain rate and specific growth rate had a trend of going up first then falling down, protein efficiency ratio improved (P<0.05) and feed conversion ratio reduced (P<0.05). Lipid content of muscle increased with increasing dietary lipid levels by the range of 2.29%to 4.27%, protein, ash and phosphorus contents had a decreasing trend. There was no significantly change of the protease activities of stomach and intestine between each treatments (p<0.05), however the lipase activities of foregut and midgut, and amylase activities of foregut were significantly decreased with increasing of dietary lipid levels (P<0.05). Second-order regression analysis of weight gain rate on dietary lipid level indicated that the optimal dietary lipid for maximal growth of GIFT was about 9.34%. Crude protein and dry-matter digestibility had no significant effect by dietary lipid levels (P>0.05). Crude lipid and phosphorus digestibility increased significantly with dietary lipid level increasing (P<0.05). Albumin and albumin/globulin ratio in 1.73%group (control group) was significantly higher than other groups (P<0.05). Along with dietary lipid level increasing, cholesterol and alkaline phosphatase of fish serum raised significantly (P<0.05). Dietary lipid level had significantly influence on blood glucose (P<0.05), and had no significantly influence on the content of triglyceride, activities of glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase (P>0.05). Those results indicated that a certain content of lipid in diet could promote the growth and improve the apparent digestibility of lipid and phosphorus, but excessive amount of lipid might be negative for fish growth and blood biochemical parameters. So, the optimal added levels of lipid in GIFT diet was 7.67%-9.34%.2 Effects of dietary lipid levels on fat deposition and fatty acid composition of GIFT strain of nile tilapia (Oreochromis niloticus)The conceptual design of raising was the same as that of the One. Raised for 90 d, the fish was fasted for 48 h, then three fishes were randomly selected from each group, and the effects of dietary lipid levels on partial physique indices, the fat deposition and fatty acid composition in muscle, liver and celiac adipose tissue of GIFT were studied. The results showed the condition factor in 5.69%,7.67%and 9.64%group was higher than other groups; the hepatosomatic index of 1.73%and 16.55%group were significantly higher than that of other groups (P<0.05). Except 1.73%group, the index of viscera-body in other groups were increased as increasing lipid level in diet. The fat content of muscle in 7.67%group were significantly higher than that of 3.71%group (p<0.05) and lower than that of 16.55%group (P<0.05). The higher of dietary lipid level, the fat deposition in fish body and the proportion of unsaturated fatty acids in total fatty acids were higher, accordingly. Those results different dietary lipid levels could affect the partial fish physique indices, especially on the hepatosomatic index. Excessive lipid in diet made lipid easier to be accumulated in muscle and liver, and the fat contents and fatty acid composition of fish could reflect the lipid level and fatty acid composition in diet.3 Molecular Cloning and sequence analysis of FAS and LPL from GIFT strain of nile tilapia(Oreochromis niloticus)The FAS and LPL cDNA sequences were cloned from GIFT (Oreochromis niloticus) by using RT-PCR and RACE method. The obtained partial FAS cDNA was 557 bp in length encoding 185-aa (GenBank accession no. GU433188). Sequence analysis showed that GIFT FAS shared 62%-82%identities with other species FAS. The full length of GIFT LPL cDNA was 2298 bp in length encoding 515 amino acids (GenBank accession no. GU433189). Sequence analysis showed that GIFT LPL shared highly identities with other vertebrates LPLs, ranging from 57.3%to 87.9%. The residues and motifs needed for LPL function was also conserved in GIFT. The predicted protein structure of GIFT LPL showed that it possessed the typical structure of lipase.4 Effects of dietary lipid levels and refeeding on the activity and expression of FAS in GIFT strain of nile tilapia(Oreochromis niloticus)To study the FAS activity and gene expression affected by different dietary lipid levels, 315 healthy fish (average weight 2.63±0.16 g) was divided into three groups and fed with different lipid levels (3.71%,7.67%and 16.55%). The low lipid level group (3.71%) was set as control group. After feeding for 90 d, the fish were fasted for 48 hours and then the activity and gene expression level of FAS in liver and muscle were studied; Furthermore, using realtime PCR analysis the FAS mRNA expression level in liver was examined at 6,12, 24,48 h after refeeding with different lipid level diet. The results showed that there was no significantly effect of dietary lipid level on FAS activity in liver (P>0.05); the expression level of FAS mRNA in liver was significantly higher than that in muscle (P<0.05). Meanwhile, the expression levels of FAS mRNA in liver and muscle were significantly decreased with rising of dietary lipid level (p<0.05). After refeeding for 6 to 48 h, the expression level of FAS mRNA in liver was significantly decreased in each group (P<0.05). Those results indicated that the higher dietary lipid level could not induce the secretion of FAS in liver; the expression level of FAS mRNA in liver was significantly higher than that of in muscle. The higher dietary lipid level could inhibit the expression of FAS mRNA, and the tendency is the higher of lipid level the more inhibition. The expression of FAS was also inhibited after refeeding.5 Effects of dietary lipid levels and refeeding on the activity and expression of LPL in GIFT strain of nile tilapia (Oreochromis niloticus)The conceptual design of raising was the same as that of the Four. Raised for 90 d, the fish were fasted for 48 hours and then the activity and gene expression level of FAS in liver and muscle were studied; Furthermore, using realtime PCR analysis the FAS mRNA expression level in liver was examined at 6,12,24,48 h after refeeding with different lipid level diet. The results showed that GIFT LPL also was detected in adult liver and muscle which was in accordance with other piscine LPL. However, the expression level in liver was significantly higher than that in the muscle (P<0.05). GIFT LPL expression was increased with the increasing of lipid level in diet, with the highest in 16.55%group and significantly higher than that of 3.71%group (P<0.05). Furthermore, expression level of GIFT LPL in liver was increased after fasting for 48 h, and decreased after refeeding for 12 h and then back to the level at beginning of fasting. Those results indicated that the higher dietary lipid level could induce the secretion of LPL in liver; the expression of GIFT LPL was in a tissue specific pattern and the GIFT liver was the main organ for secretion and expression of LPL. The higher lipid in diet induced the expression of LPL in liver and this condition was regulated by the feeding status (satiation or starvation).6 Effects of dietary lipid levels and refeeding on lipid and sugar in the blood of GIFT strain of nile tilapia(Oreochromis niloticus)To explore further study of the effects of dietary lipid level on the blood lipometabolism parameters, the blood was randomly collected from three fish in 3.71%, 7.67%and 16.55%group after fasting for 24 h and refeeding for 0,6,12,24,48 h, and the blood indices related to lipometabolism, i.e. blood glucose(GLU), triglyceride (TG), cholesterol (CHO), were detected. The results showed that the higher lipid in diet could significantly increase the TG, CHO, alkaline phosphatase (ALP) and glutamic-pyruvic transaminase (GPT) (P<0.05), and decrease blood sugar (P<0.05); After refeeding for 48 h, the TG, CHO and GLU were first increased and then decreased. At the same time-points after refeeding, the TG and CHO of the high-lipid group were significantly higher than that of the low-lipid group (P<0.05), the GLU had a decreasing trend but no significant change was observed (P>0.05). Those results indicated that the high-lipid level (16.55%) might be harmful to liver. After refeeding for 48h, the TG, CHO, GLU appeared to be a singlet curve and reached to the highest at 12 h,6 h and 12 h after feeding, respectively. The same case was also observed in high-lipid group and middle-lipid group. The high-lipid could reduce the metabolism of TG and make time of CHO lag to 12 h after feeding.更多还原