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脂肪水平对循环海水养殖道氏虹鳟的效应和机制研究
张静
学位类型硕士
导师李勇研究员
2017-05-17
学位授予单位中国科学院大学
学位授予地点北京
学位专业生物工程
关键词饲粮脂肪水平 道氏虹鳟 生长 消化吸收 抗氧化 脂肪代谢 封闭循环水养殖 基因表达
摘要虹鳟(Oncorhynchus mykiss),是目前世界范围内养殖最为广泛的冷水性经济鱼类之一,属三文鱼的一种,目前我国仍需大量进口,市场前景广阔。虹鳟养殖模式主要有流水、网箱以及循环水,前两种模式下营养与饲料的研究已较为成熟,而在工业化循环水养殖模式下其营养需求特点的变化尚未探明,且在该模式下存在一些特有的脂肪代谢问题。本试验研究,首次探寻了饲粮脂肪水平对工业化循环海水养殖优良虹鳟品系——道氏虹鳟生长性能、相关生理代谢及脂肪代谢关键酶基因表达的效应,为该模式养殖虹鳟的脂肪营养调控、腹脏脂肪沉积消减、及其生理代谢和分子营养机制探寻等,提供重要理论依据和实践参考。
本试验研究综合运用动物营养学、生理学、生物化学、分子生物学、生态营养学等理论和方法,利用营养调控和机制查寻手段,探究如何解决工业化循环水养殖虹鳟腹脏脂肪沉积过多问题。动物试验在工业化封闭循环海水养殖模式下进行。采用单因素试验设计,即试验饲粮在蛋白质、蛋白原料等营养素和营养源基本一致条件下,由4个脂肪水平组成——12%、15%、18%、21%(以L12、L15、L18、L21组表示),共形成4个试验处理组,每处理3重复,每重复25尾鱼。试验饲粮统一加工为缓沉性膨化颗粒饲料。试验鱼为初始体重(333.25±20.71)g的道氏虹鳟,试验期77 d。试验研究最终获得如下结果:
1. 生长性能结果显示,低脂肪组(L12)试验鱼的增重率显著优于中高脂肪组(L18、L21)29.12%-34.35%(P<0.05);而低中脂肪组(L12、L15)的饲料系数显著低于中高脂肪组12.95%-20.55%(P<0.05)。中高脂肪组显著增加腹脂率和脏脂率5.34%-9.34%(P<0.05),确实是导致虹鳟腹脂和脏脂沉积过多的主要营养因素,而低中脂肪组未发生此现象。可知,在饲粮蛋白质水平为45%时,饲粮低中脂肪水平更适合试验鱼的生长和饲料利用,并具减少腹脏脂肪沉积和促进体型正常的效应,循环水养殖道氏虹鳟的饲粮脂肪水平需求量不超过12%。
2.消化吸收和水质测定结果表明,低中脂肪组(L12、L15)主要通过显著提高小肠脂肪酶及AKP活力(P<0.05),改善试验鱼脂肪消化和其它营养物质吸收,可谓饲粮低脂肪水平(L12)生长性能较高的消化生理机制。低脂肪组脂肪酶活力显著高于其他三组5.98%-8.82%,低中脂肪组AKP活力显著高于中高脂肪组6.23%-11.25%(P<0.05)。各组水体氨氮相对浓度随饲粮脂肪水平升高而显著降低:低脂肪组相对氨氮浓度最高(0.127 mg/L/kg),显著高于其他组18.44%-24.91%(P<0.05);亚硝态氮(0.004 mg/L/kg)显著高于中高脂肪组95.04%-140.05%(P<0.05);然而,日常监测中低脂肪组水氨氮绝对浓度为0.34-0.48 mg/L,保持在合理范围内(小于0.5 mg/L)。饲粮低脂肪水平试验鱼较高的采食量和生长速度是其氮排泄增加的主要原因,也与饲粮高脂肪水平提高氮利用率有一定关系。
3.肝脏和血浆脂肪生理代谢测析表明,1肝脏脂肪酸合成酶(FAS)活力随饲粮脂肪水平升高而显著降低(P<0.05),而脂肪分解酶类LPL、HL、L-CPT1活力变化特征与之相反。其中,低脂肪组(L12)组FAS活力显著高于中高脂肪组(L18、L21)7.5%、8.7%(P<0.05),与L15组差异不显著(P>0.05);L-CPT1活力显著低于其他组13.08%-16.35%(P<0.05)。同时,低中脂肪组(L12、L15)LPL、HL活力分别显著低于高脂肪组(L21)26.81%-31.16%、18.92%-21.62%(P<0.05)。说明饲粮低中脂肪水平试验鱼主要通过增加肝脏脂肪合成和降低分解的自我调控方式,提高脂肪利用率,从而不仅保证鱼体脂肪营养需要,而且间接促进了生长。2初步显示,饲粮脂肪水平影响试验鱼血浆胆固醇(CHO)转运及肝脏相关脂质合成。L12、L15和L18组均通过协调血浆LDL-C及HDL-C含量,使得HDL-C/LDL-C显著高于L21组51.16-65.56%(P<0.05),从而促进外周CHO回肝代谢;L12组血液总胆固醇(TC)和甘油三酯(TG)显著高于后三组9.69%-15.72%、37.06%-59.55%(P<0.05)。即饲粮低脂肪水平通过增加CHO和TG的内源性合成,补充机体外源性摄取的不足;而饲粮高脂肪水平减缓了CHO向肝内转运,增加其在外周组织的积累。
4.肝脏抗氧化因子测析表明,不同饲粮脂肪水平影响试验鱼肝脏抗氧化能力的作用途径或机制存在差异。高脂肪组(L21)通过SOD和GSH两种酶联合清除活性氧自由基,分别显著高于低中脂肪组4.16%-10.64%和30.89%-43.50%(P<0.05)。低脂肪组(L12)则主要依靠CAT提高抗氧化能力和降低MDA含量,其CAT活力显著或极显著高于中高脂肪组(L18、L21)8.50%-19.39%(P<0.05,P<0.01),并通过SOD和CAT相互配合确保细胞膜氧化损伤最小,从而保证试鱼健康,为保持较高生长性能提供了内在条件。本试验条件下,饲料脂肪水平对试验鱼血浆免疫能力(总蛋白、球蛋白以及溶菌酶)无显著影响(P>0.05)。
5. 脂肪代谢酶基因表达测析表明,1FAS mRNA基因表达量,在肌肉中总体随饲粮脂肪水平升高而逐渐降低,低脂肪组(L12)显著高于高脂肪组(L21)66.82%;在肝脏中,低脂肪组显著高于其他组48.29%-55.15%(P<0.05)。LPL mRNA表达量,在肝脏中与饲粮脂肪水平呈正相关,高脂肪组显著或极显著高于其他组36.72%-113.59%(P<0.05或P<0.01),肌肉中也有类似趋势。该结果与脂肪代谢酶活力变化特征(肝脏FAS活力随饲粮脂肪水平升高而显著降低,LPL与之相反)具有同步相似性。2从测析结果中新发现:肝脏与肌肉对饲粮脂肪水平干预的分子生物学应答呈现组织差异性特征。即随饲粮脂肪水平变化,肌肉FAS基因表达比肝脏更明显且规律性更强,而肝脏LPL基因表达变化较肌肉明显,这一特征应该是由三文鱼肌肉脂肪沉积较强的特殊性所致。
6. 本研究的初步新发现是:1饲粮脂肪水平影响虹鳟血液胆固醇(CHO)转运:低中脂肪水平HDL-C/LDL-C比值较高,促进外周CHO回肝代谢,从而降低CHO在外周的沉积;而高脂肪水平比值最低,显著减缓其向肝内转运。2肝脏与肌肉对饲粮脂肪水平干预的分子生物学应答呈现组织差异性特征。即随饲料脂肪水平变化,肌肉FAS基因表达比肝脏更明显且规律性更强,而肝脏LPL基因表达变化较肌肉更明显。此可谓三文鱼肌肉脂肪分子营养调控的特征或机理显现。
总之,本试验研究表明,饲粮脂肪水平对工业化循环水养殖道氏虹鳟的生长性能、腹脏脂肪沉积、脂肪生理代谢、相关基因表达等,产生了显著或极显著的影响。研究初步揭示,试验鱼采食低中脂肪水平(L12、L15)饲粮后,主要通过调控肝脏和肌肉中脂肪关键酶基因表达,从而提高肝脏和肌肉中FAS和降低LPL的酶活性,加之降低CPT1、HL等脂肪分解酶类活性、协同脂肪转运蛋白等其他生理效应,总体上促进了体内脂肪代谢,最终达到既满足机体脂肪营养需求、又降低腹脏脂肪沉积、促进生长的目的。同时通过增强肠脂肪酶、AKP酶活力,促进脂肪等营养物质消化吸收;并凭借SOD和CAT组合效应增强机体抗氧化能力,保证了低中脂肪组试验鱼的营养供给和免疫抗病。研究初步证实,饲粮脂肪水平较高(18%-21%),是导致试验虹鳟腹脂和脏脂沉积过多的主要营养因素;而较低饲粮脂肪水平水平(12%-15%),不会发生此现象。研究初步确定,在工业化循环水养殖条件下,当饲粮蛋白质水平为45%时,330-600 g左右道氏虹鳟脂肪需求量不超过12%。该研究结果与目前国内外虹鳟养殖饲粮普遍采用较高脂肪水平现象形成鲜明对比,从营养调控手段和生理代谢及基因表达等机制方面基本解决了工业化封闭循环水养殖虹鳟腹脏脂肪沉积过多问题,也为不同养殖模式专用营养饲料研发的必要性找到了具体答案。
其他摘要Rainbow trout (Oncorhynchus mykiss), is a cold-water fish belonging to salmon species, which is considered as one of the most widely farmed fishes all over the world. In China, there is a vast market prospect on trout farming to satisfy the population demands due to the shortage of high value aquatic products. In general, the major culture models of rainbow trout are: flowing water culture, net cage culture and recirculating aquaculture system (RAS). Although, the first two models were intensively investigated, the nutrient requirements in RAS is not clear until now. To make the matter worse, there are some lipid metabolism problems of rainbow trout cultured in this model. Therefore, the present study aimed to exploring the effects of dietary lipid level on growth, physiological metabolism and gene expression of some key enzymes of Donaldson rainbow trout strain, in order to provide both theoretical and fundamental bases as well as the practical references of nutrition regulation, reduction in abdominal fat deposition, mechanism on physiological metabolic and molecular nutrition of rainbow trout in RAS.
Furthermore, the combination of animal physiology, biochemistry, molecular biology and eco-nutriology will help to better understand the effects of dietary lipid levels on growth, digestion and absorption enzymes, antioxidant ability, plasma immunity, fat metabolism and related genes expression of rainbow trout (Oncorhynchus mykiss) in RAS. For these reasons, four isonitrogenous diets containing 45% crude protein with increasing dietary lipid (L) levels of 12%, 15%, 18% and 21% (L12, L15, L18, L21) were fed to satiety to 25 fishes (333.25±20.71 g) for 77 days in triplicate groups. However, the following overview outlines the most important results and indications that included in this thesis as:
1. The growth and body indices results showed that the weight gain rate (WGR) in low-fat group (L12) was higher than that in middle and high fat groups (L18, L21) by 29.12% - 34.35% respectively (P<0.05), while the feed conversion ratio (FCR) in both low and middle groups (L12, L15) were lower than the other groups (L18, L21) by 12.95% - 20.55% respectively (P<0.05). Meanwhile, the abdominal fat (AFR) as well as the visceral fat rates (VFR) in group L18 and L21 were increased by 5.34% and 9.34% respectively, when compared with L12 group (P<0.05). These results are clearly indicating that the lipid levels were the main factor affecting the fat deposition in visceral mass, while there was no such phenomenon happened on group L12. In conclusion, when dietary protein level is 45%, diets with low or moderate fat content are more suitable for the growth and feed utilization of trial fish, which also has effects on decreasing fat deposition around visceral organs and coming into being normal shape, and lipid requirement of rainbow trout cultured in RAS is no more than 12%.
2. The results of lipase activities in group L12 were higher than other groups by 5.98% and 8.82% respectively (P<0.05). Also alkaline phosphatase (AKP) activities in groups L12 and L15 were higher than other two groups by 6.23% and 11.25% respectively (P<0.05). Ammonia nitrogen (NH4+-N) and nitrate nitrogen (NO2-N) concentrations were decreased with increasing lipid levels in the diet. Relative ammonia nitrogen concentrations in L12 were higher (0.127 mg/L/kg) than others by 18.44%, 23.30% and 24.91% respectively. Similarly, the relative nitrate nitrogen (0.004 mg/L/kg) in this group (L12) was higher than other groups by 140.05%, 95.04% and 110.51% respectively. However, ammonia nitrogen absolute concentration was between 0.34-0.48 mg/L in daily detection, which was within the acceptable limits (<0.05 mg/L), so there was no bad influence to the trial fish. The results clearly showed that the feed intake and growth rate in L12 group were higher than other groups, and therefore the elimination of nitrogen was more efficient than other groups. The diets with higher lipid levels improving the nitrogen utilization was another reason. Overall, The digestion, absorption and water quality results also showed that the L12 and L15 groups had better digestion and absorption of fat and other nutrition that both lipase and AKP activities were significantly increased (P<0.05), which was the digestive physiological characteristics or mechanism that ensured these two groups growing better.
3. The results of fat physiological metabolism in plasma and liver showed that, a) the activities of fatty acid synthetase (FAS) in liver were decreased with increasing the fat content in the diet, in contrary with the activities of lipolytic enzymes (LPL, HL, L-CPT1) that has been reversely changed . However, FAS activity of group L12 was significantly higher than groups of L18 and L21 with 7.5% and 8.7% respectively (P<0.05), while L-CPT1 activity of this group (L12) was lower than others by 16.35%, 13.08% and 15.93% respectively (P<0.05). Meanwhile, activities of both LPL and HL of L12 and L15 groups were lower than L21 by 31.16% and 26.81%, 21.62% and 18.92% (P<0.05) respectively, indicating that fishes fed on L12 and L15 diets have positively affected the fat utilization rate by increasing fat synthesis and decreasing lipolysis that satisfies the requirements for lipid in rainbow trout and promotes their growth consequently, and b) the dietary lipid levels have affected the transport of cholesterol (CHO) and the synthesis of some lipid. Overall, L12, L15 and L18 groups achieved higher lipoprotein-cholesterol HDL-C/LDL-C ratio than L21 with about 65.56%, 65.56% and 51.16% respectively (P<0.05) by elevating the content of high density lipoprotein-cholesterol (HDL-C) and decreasing the content of low density lipoprotein-cholesterol (LDL-C), thus the process of CHO entering liver was promoted. Meanwhile, group L12 made up the shortage of exogenous uptake by means of increasing endogenous formation of CHO and triglyceride (TG). Total cholesterol and TG contents in group L12 were significantly higher than other groups with 9.69% - 15.72%, and 37.06% - 59.55% respectively (P<0.05). In contrast, the HDL-C/LDL-C ratio in group L21 was the lowest (P<0.05), which slowed CHO transshipment to liver significantly, increasing the accumulation of CHO in peripheral tissues.
4. So far, the antioxidant mechanism was significantly differed among the groups. High-fat group (L21) mostly made use of superoxide dismutase (SOD) and glutathione (GSH) to increase antioxidant ability. While, SOD and GSH contents in group L21 were significantly higher (P<0.05) than groups L12 and L15 by 4.16% and 10.64%, 30.89% and 43.50% respectively. Low-fat group (L12) mainly depended on CAT to increase antioxidant ability and decrease MDA content, the CAT content in L12 was significantly higher (P<0.05) than L18 and L21 with 8.50% and 19.39% respectively. Furthermore, this group ensured the minimum oxidative damage to the cell membrane, thus supplied internal condition for better growth performance. In this trial, there was no significant effect of dietary lipid level (P>0.05) on immunologic indices in plasma (containing total protein, globin and lysozyme).
5. According to the gene expression results, FAS mRNA expression in muscle was decreasing significantly (P<0.05) with the increase of dietary lipid level. And low-fat group (L12) was significantly higher (P<0.05) than L21 group with 66.82%, while group L12 was significantly higher than others by 55.15%, 48.29% and 55.15% in liver, respectively. On the contrary, LPL genes expression in liver was positively correlated with fat content in the diet. High-fat group (L21) was significantly higher (P<0.05 and P<0.01) than low-fat groups (L12, L15, and L18) by 113.59%, 36.72% and 44.33%. There was the same trend in muscle. The result was similar to the change of fat metabolism enzyme activities in the liver. For the first time, the present results highlighted that the degree of molecular biological response to dietary lipid level was different between liver and muscle, which has not been reported before. Presented as FAS expression quantity changed faster in muscle than in liver, while LPL genes expression was opposite, which related to the importance of fat deposition in the muscle of trout.
6. Preliminary finding are as follow: (1) Fat levels affected the transport of plasma cholesterol (CHO) of rainbow trout, diet with low fat content could promote peripheral CHO to transport into liver for hepatic metabolism, while diet with high fat level significantly slowed the transshipment of CHO to liver. (2) FAS gene expression changed faster in muscle than in liver with increasing fat level in diet, while LPL gene expression in liver was more sensitive than muscle. Fundamentally speaking, the degree of molecular biological response to fat nutrition was different comparing liver with muscle.
In conclusion, this study showed that dietary lipid level had a significant effect on growth performance, abdominal fat deposition, fat metabolism and related genes expression of rainbow trout (Oncorhynchus mykiss) in the recirculating aquaculture system (RAS). On the one hand, the groups with low and middle fat contents improve the activities of FAS and reduce the activities of LPL by regulating gene expression of key enzymes in liver and muscle, and transport fat into liver effectively to promote fat metabolism, which satisfies the need on fatty acid, and reduces the abdominal fat deposition. On the other hand, these two groups enhanced antioxidant capacity by SOD and CAT to keep the healthy growth of the studied fish species.
The results of this trial showed that the Donaldsons rainbow trout strain (330-600 g) could achieve healthy growth without excessive fat supply, not more than 12%, in RAS with a dietary protein level of 45%, which is different with the results of previous studies that the diets of rainbow trout are always with higher fat content (15%-20%). The finding deals with the trouble of excessive fat deposition on rainbow trout by means of nutrition regulation, physiological metabolism and gene expression, and supplies the preliminary answer to the need for developing the special nutritional feed for different culture models.
学科领域水产养殖,水产动物营养与饲料科学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136580
专题实验海洋生物学重点实验室
作者单位中国科学院海洋研究所
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张静. 脂肪水平对循环海水养殖道氏虹鳟的效应和机制研究[D]. 北京. 中国科学院大学,2017.
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