【作者】 郭小泽；

【导师】 梁旭方；

【作者基本信息】 华中农业大学 ， 水生生物学， 2013， 博士

【摘要】 草鱼(Ctenopharyngodon idellus)是世界上产量第二大的鱼类,是我国主要的淡水养殖鱼类。草鱼是一种典型的草食性鱼类,在天然水域摄食水草,但在人工养殖中摄食配合饲料。然而,在养殖中由于营养不平衡,如长期投喂低蛋白高能量的人工饲料,结果导致草鱼体内脂肪大量蓄积的现象非常普遍,严重制约了草鱼养殖业的健康发展。为探讨草鱼脂肪蓄积及代谢调控机制,本文开展了饲料中不同非蛋白能量源对草鱼生长、体脂肪蓄积、血清生化指标及肝脏组织的影响。进而通过研究组织中脂肪代谢关键酶的基因表达和酶活来探讨饲料中不同非蛋白能量源对草鱼脂肪蓄积的主要机理,同时,研究在高糖、高脂饲料中添加氯贝丁酯对降低草鱼体脂肪含量、血清生化指标及脂肪代谢关键基因表达的影响。1.饲料中不同非蛋白能量源对草鱼生长、体组成及脂肪蓄积的影响为探讨饲料中不同非蛋白能量源对草鱼脂肪蓄积的影响,将360尾(40.10士0.15g)草鱼分为4组,每组3个重复,每个重复30尾鱼,分别投喂4种不同非蛋白能量源(对照组6.52kJ g-1、高纤维组5.32kJ g-1、高糖组8.46kJ g-1和高脂组8.54kJ g-1)的等氮(300gkg-1)饲料,进行为期9周的养殖实验。养殖实验结束,禁食24h,测定草鱼生长指标、饲料效率、体组成和形态指标,并统计草鱼各组织中脂肪含量。结果显示,与对照组相比,增加饲料中的非蛋白能量源(高糖组和高脂组)并没有显著提升草鱼的生长性能(p>0.05)；而降低饲料中的非蛋白能量源(高纤维组)却显著降低了草鱼生长性能(p<0.05)。在饲料效率和蛋白质效率方面,含有高非蛋白能量的高糖组和高脂组与对照物显著性差异(p>0.05),而非蛋白能量水平较低的高纤维组较对照组有显著性的降低(p<0.05)。随着饲料中非蛋白能量源水平的增加,高糖组和高脂组草鱼的体脂肪含量与对照组相比有显著的升高(p<0.05),尤其是高脂组草鱼体脂肪含量最高；与之相反,较低水平非蛋白能量的高纤维组的草鱼脂肪显著低于对照组(P<0.05)。鱼体水分含量变化与脂肪含量呈相反趋势,粗蛋白含量无显著变化(P>0.05)。肌肉中脂肪含量与全鱼脂肪含量的趋势一致。增加饲料中的糖类水平显著增加草鱼的肝体比(HSI)和肝脏脂肪含量(p<0.05),而饲料中高水平的脂肪则显著增加草鱼的肠脂比(MFI)(P<0.05)。而在产肉率(FI)方面各组无显著性的差异(P>0.05)。通过计算得出草鱼肌肉、肝脏和肠系膜脂肪组织中脂肪蓄积比率,其中肌肉中脂肪含量占全鱼脂肪总量的11.46%-14.54%,肝脏中脂肪含量的比率为2.35%-4.85%,肠系膜中脂肪含量占全鱼脂肪含量的比例范围是22.93%-28.61%。结果表明,增加饲料中非蛋白能量源不仅不能提高草鱼的生长性能,反而会增加鱼体脂肪的蓄积。2.饲料中不同非蛋白能量源对草鱼血清生化指标和肝脏组织的影响在与实验一相同的条件下,检测饲料中不同非蛋白能量源对草鱼血清生化指标和肝脏组织的影响。养殖实验结束后禁食24小时,每组随机取6条鱼,尾静脉采血,肝脏采用石蜡包埋和H-E染色(苏木精-伊红)法,在显微镜下观察肝脏组织形态并拍照。结果表明,随着饲料中非蛋白能量源含量的升高,高糖组和高脂组的血液中的TG和CHO浓度显著高于对照组和高纤维组(P<0.05),而高纤维组相对于对照组有下降的趋势但不显著(P>0.05)。高糖组的ALT、 AST和ALP这三种酶活性显著高于对照组和纤维组(P<0.05),而高脂组与对照组没有显著性的差异(P>0.05)。高糖组和高脂组草鱼血液中的TP和BUN的浓度与对照组没有显著性差异(P>0.05),而高纤维组显著性低于对照组(P<0.05)。饲料中不同水平的非蛋白能量源对草鱼血液中的TBIL和DB含量没有显著的影响(P>0.05)。高纤维组的GLU浓度显著高于对照组和高糖组(P<0.05)。在肝脏组织学方面,高糖组的肝脏细胞严重肿大变形,细胞中充满脂肪滴,细胞核挤压至一侧,甚至出现细胞核萎缩或消失,呈现透明的空泡化。高脂组的肝细胞有稍许的肿大,细胞浆内有大小不一的脂肪滴出现,部分出现细胞核偏移和空泡化。高纤维组的肝细胞排列致密整齐,肝细胞索明显,细胞核位于细胞中央,细胞大小正常,没有脂肪滴。因此,饲料中高糖组对草鱼肝脏组织的损伤最严重。3.饲料中不同非蛋白能量源对草鱼脂肪代谢相关基因表达和酶活的影响本实验主要通过研究草鱼肝脏组织和脂肪组织中脂肪代谢主要酶的基因表达和酶活性,探讨饲料中不同非蛋白能量源对草鱼体脂肪蓄积的影响机理。结果表明,高糖组草鱼肝脏组织中脂肪生成相关酶活性(脂肪酸合成酶FAS,乙酰辅酶A羧化酶ACC和葡萄糖-6-磷酸脱氢酶G6PD)较对照组有显著性的升高(p<0.05),而高脂组和高纤维组则有降低。在基因表达方面,高糖组草鱼肝脏组织和脂肪组织中FAS和ACC的mRNA水平显著上调(p<0.05),尤其是肝脏组织中FAS基因相对表达量较对照组上调了3倍多。高纤维组草鱼肝脏中PPARa基因mRNA水平与对照组相比有显著升高(p<0.05),脂蛋白脂酶(LPL)基因相对表达在高纤维组和高脂组草鱼肝脏中较对照组有显著升高(P<0.05),而在脂肪组织中则是高糖组和高脂组均显著升高,高纤维组则显著下降P<0.05)。上述结果表明,虽然高糖和高脂饲料均能增加草鱼体脂肪的蓄积,但是两者增加脂肪蓄积的方式不同。4.高糖、高脂饲料中添加氯贝丁酯对草鱼体组成、血清生化指标及脂肪代谢的影响本实验是在高糖和高脂饲料中添加1.25g kgq的氯贝丁酯,探讨其对草鱼降脂作用。实验鱼是实验一结束后高糖组和高脂组的草鱼(205±12g),各分为两组,一组投喂无氯贝丁酯饲料,一组投喂添加氯贝丁酯饲料,投喂率为4%鱼体重／天,草鱼每天所摄取的氯贝丁酯的量为50mg/kg体重,养殖实验共进行4周。养殖实验结束后,禁食24h,检测体重、鱼体组成、血脂含量及肝脏组织中脂肪代谢基因PPARα、 CPTI、LPL的表达。结果表明,在高糖和高脂饲料中添加氯贝丁酯对草鱼生长性状无显著变化,而显著降低了草鱼VSI和MFI(P<0.05),尤其是高脂组添加氯贝丁酯的草鱼MFI降低程度较大。在体组成方面,高糖和高脂饲料中添加氯贝丁酯较未添加组显著降低了全鱼、肌肉和肝脏中的脂肪含量(P<0.05)。在血脂方面,氯贝丁酯也显著降低了草鱼血液中甘油三酯、总胆固醇、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇(P<0.05)。高糖和高脂饲料中添加氯贝丁酯使草鱼肝脏中PPARa和LPL基因mRNA水平显著上调(P<0.05),而CPTI基因mRNA水平各组无显著差异(P>0.05)。上述结果表明,在高糖和高脂饲料中添加氯贝丁酯对草鱼有降脂作用。5.投喂青草、蚕豆和配合饲料对草鱼生长、肉质及脂肪蓄积的影响本实验通过投喂青草、蚕豆和配合饲料3种不同的饲料来探讨其对草鱼生长、肉质及体脂肪蓄积的影响。300尾草鱼(均重107+1.76g)分成3组,每组3个重复,每个重复30尾鱼,每天投喂2次,养殖16周。实验结果表明,摄食配合饲料组的草鱼增重率和特定生长率显著高于蚕豆组和青草组(p<0.05),蚕豆组草鱼生长性能最低。在体形特征方面,青草组草鱼VSI、HSI和MFI显著低于其它两组(P<0.05)。相反,蚕豆组草鱼的MFI显著高于配合饲料组和青草组(p<0.05)。在肌肉和肝脏组成组分方面,蚕豆组的草鱼肌肉粗蛋白和粗脂肪显著高于其它两组(P<0.05),青草组草鱼肌肉脂肪含量最低(p<0.05)。蚕豆组草鱼肝脏中脂肪含量显著高于其它两组(P<0.05),青草组最低。蚕豆组草鱼的肌肉质构参数(硬度、弹性、粘聚性、胶着性、咀嚼性和恢复性)是最高的。在血液生化指标方面,蚕豆组草鱼血清中胆固醇、谷草转氨酶和碱性磷酸酶含量都高于配合饲料组和青草组,青草组草鱼血清中谷丙转氨酶含量最低。在三组草鱼肝脏组织切片中可看到配合饲料组肝脏细胞排列混乱,细胞变形,细胞质中有脂滴；蚕豆组草鱼肝细胞明显肿大且充满脂滴,细胞核发生偏移,部分细胞核萎缩甚至消失；而青草组草鱼肝细胞排列整齐,细胞核清晰,细胞大小正常。上述结果表明,摄食蚕豆提高了草鱼肌肉的质构,但同时显著增加了草鱼体内脂肪的蓄积,造成肝脏一定程度的损伤。更多还原

【Abstract】 Production of grass carp(Ctenopharyngodon idellus) is the second large fish species of the world, and constitutes the largest aquaculture industry of finfish in China. As a typical herbivorous finfish, grass carp can eat artificial diets in aquaculture as well as aquatic weeds in natural environment. However, there is a great common phenomenon that the grass carp deposit extensive of fat in fish body when the fish fed commercial diets containing low protein and high lipid and/or carbohydrate levels. In order to explore the fat accumulation and lipid metabolic regulation mechanisms of grass carp, we studied the effects of different dietary non-protein energy source level and dietary supplemention of clofibrate on the growth performance, body composition, lipid deposition site, serum biochemical indices, liver histology and genes expression and enzymes activities involved in lipid metabolism of grass carp, and we also analyzed the fat deposite, physiological and biochemical changes in grass carp fed with vegetable food, broad bean and formulated diet.1. The effects of dietary non-protein energy source levels on growth performance, body composition and fat accumulation in grass carp (Ctenopharyngodon idellus)A study was conducted to analyze the effect dietary non-protein energy source leves on fat accumulation of grass carp.360grass carp (average weight40.10±0.15g) were divided into four groups randomly, each group was triplicate. Four isonitrogenous (300g kg-1)practical diets were formulated to contain four non-protein energy levels (control diet6.52kJ g-1. high-CEL diet5.32kJ g-1、high-CHO diet8.46kJ g-1and high-LIP diet8.54kJ g-1). After rearing for9weeks, the fish were fasted for24h, then six fish were randomly selected from each group, and the growth performance, feed conversion ratio, whole body and tissue composition, biometric parameters and fat deposition were detected. The results show that, compared with the control group, fish fed the high-CHO diet and the high-LIP diet had no significant difference in the growth performance and feed conversion ratio (P>0.05). In contrast, the high-CEL diet containing lower non-protein energy level led to significant lower WG, SGR and PER (P<0.05). The whole body lipid and muscle lipid were significantly increased in fish fed he high-CHO diet and the high-LIP diet (P<0.05), and the high-LIP diet group showed the highest values among the all groups. The opposite appearances were found in the moisture of whole body and muscle. The high-CHO diet group had the highest liver lipid in all groups. The lowest lipid content of whole body, muscle and liver were found in the high-CEL diet group(P<0.05). The high-CHO diet group had the highest HSI among the all groups (P<0.05). The high-LIP diet group had the significantly highest VSI and MFI in the all group (P<0.05). Proportion of fat in muscle (11.46%-14.54%), liver (2.35%-4.85%) and mesentery (22.93%-28.61%) out of the total body fat did not change significantly among treatments. Those results indicated that increasing dietary non-protein energy source levels did not improve the growth, but can increase the fat deposite in fish body.2. The effects of dietary non-protein energy source levels on serum biochemical indices and histology of liver in grass carp {Ctenopharyngodon idellus)The conceptual design of rearing was the same as that of the test One. After the rearing for9weeks, the fish was fasted for24h, then three fish were randomly selected from each group, and the effects of dietary different non-protein energy source levels on serum biochemical indices and histology of liver were detected. The results showed that the triglyceride (TG) and cholesterol (CHO) of the fish fed the high-LIP diet and high-CHO diet were significantly higher than those of the contol group and the high-CELgroup (P<0.05). However, the fish fed the high-CEL diet had the lowest TG and CHO in all the groups. The serum activities of aspartate amninotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) of the fish fed the high-CHO diet group were significantly higer than those of the control group and the high-CEL group (P<0.05), and there was no significantly different between the high-LIP group and the control group (P>0.05). There was no significantly different of total protein (TP) and blood urine nitrogen (BUN)between the high-CHO diet group, the high-LIP diet group and the control group (P>0.05), but the high-CEL group was markedly lower than that of the control group (P<0.05). The high-CEL group had the highest level blood glucose (GLU) among the all groups (P<0.05). As the aspect of liver histology, the hepatocytes of the fish fed the high-CHO diet were swelling by extreme lipid infiltration, and there were extreme lipid droplets in the cytoplasm, and the nucleus were dislocation, loss of cytoplasm staining affinity. However, the hepatocytes of the fish fed the high-CELdiet were arranged tidy, the nucleus is located in the central of cell, and the size of cell is normal, no fat droplets in cytoplasm. Therefore, the fish fed the high-CHO diet led the poor liver status among the all groups.3. The effects of dietary non-protein energy source levels on the genes expression and enzymes activities involved in lipid metabolism in grass carp (Ctenopharyngodon idellus)This study was to detect the effects of dietary non-protein energy source levels on the genes expression and enzymes activities involved in lipid metabolism of liver and mesenteric adipose tissue of grass carp. The results showed that the activities of fatty acid synthetase (FAS), acetylcoenzyme-A carboxylase (ACC) and glucose-6-phosphate dehydrogenase (G6PD) were significantly elevated in the fish fed the high-CHO diet (P<0.05). Compared with the control diet group, there was a decreasing trend of the FAS, ACC and G6PD activities in the fish fed high-LIP diet and high-CEL diet. There was no significant differnce of malic enzyme (ME) activity among all the groups (P>0.05). The mRNA expressions of FAS and ACC in liver were significantly higher of the high-CHO diet group than those of other group (P<0.05). In the mRNA levels of FAS and ACC there appeared a decreased trend in the high-CEL and high-LIP diet group compared with the control group. The expression of PPARa was significantly higher in the high-CEL diet group than that of the other group (P<0.05). Meanwhie, the LPL mRNA abundant was significantly increased in the fish fed high-CEL diet and high-LIP diet (P<0.05). In the mesenteric adipose tissue, the expression of FAS and ACC had similar tendencies as those in the liver. The LPL gene expression was significantly down-regulated in the high-CEL diet group, but up-regulated in the high-CHO and high-LIP diet group (P<0.05). Those results indicated that although the high dietary carbohydrate and lipid both can induce the lipid deposition in fish, the metabolic mechanism is markedly different.4. The effects of dietary supplemention clofibrate on the body composition, surem biochemical indices and lipid metabolism in grass carp (Ctenopharyngodon idellus)This study was to detect the hypolipidaemic effect of clofibrate in the grass carp fed the high-CHO diet and high-LIP diet. The grass carp was the end of test one fish (average weight205±12g), they were then treated with clofibrate (50mg/kg body weight) in the same high-CHO diet and high-LIP diet for4weeks. Afert rearing4weeks, the fish were fasted for24h, and then body weight, body composition, plasma lipid parameters and lipid metabolism-related genes in liver were measured. The results showed that there were no significant differences in the growth performance of the fish treated with clofibrate (P>0.05). However, there were markedly reduced the VSI and MFI of fish treated with clofibrate (P<0.05), especially the fish fed the high-LIP diet containing clofibrate. Clofibrate treatment decreased the lipid content of whole body, muscle and liver (P<0.05). The plasma contents of TAG, cholesterol, LDL-C and HDL-C were all significantly lowered after clofibrate treatment (P<0.05). The mRNA of PPARα and LPL were significanlty up-regulated after the clofibrate treatment (P<0.05), but the mRNA of CPT I was no change between the group (P>0.05). These data suggest that clofibrate has the hypolipidaemic effect in the grass carp fed the high-carbohydrate diet and high-fat diet.5. The effects on growth, body composition,biochemical and fat deposition in grass carp fed with vegetable food, broad bean and formulated dietA17-week feeding trial was conducted to evaluate the effects on growth, muscle quality and serum biochemical profile of grass carp (Ctenopharyngodon idellus) fed with the formulated diet (FD), broad bean (BB) or lettuce (L)(Lactuca sativa L.). The FD (Huamei6300) was purchased from a feed company (Conti Huamei Feed Co., Ltd.), BB and L(Lactuca sativa L.) were collected from the farmer’s market (Panyu, Guangdong province). The results showed that the weight gain of fish fed with the FD were significantly higher than the fish fed with L or BB. Fish fed with L had significantly lower lipid content in visceral and muscle than the fish fed with FD or BB. The hardness, springiness, choesiveness, gumminess, chewiness, resiliness of muscle in fish fed with the broad bean were all higher than that of the other two groups. Fish fed with the BB showed a significantly higher ALP activity and CHO content in serum than that with the L. The liver cell was big and loose when fish fed with BB or FD compared with the liver section of grass carp fed with L. The study proved that BB diet could increase the quality of texture, but with a slow growth ratio; FD could bring about a good growth ratio; and L or BB was not a proper feed when each was used alone for grass carp diet.更多还原