1. 苦草粉对草鱼幼鱼生长性能与生理生化性能的影响 本研究评价了苦草粉(Vallisneria natans)在草鱼饲料中的应用效果。以不含苦草 粉的基础饲料（VN0 组）为对照，分别用 10%（VN1 组）、20%（VN2 组）、30% （VN3 组）的苦草粉替代基础组饲料中的次粉和米糠，配制出 4 种实验饲料，另 设置一组只投喂新鲜苦草的青饲料组（VN 组）。选用初始体重（18.85±0.20）g 的草鱼幼鱼在室内水泥池网箱进行为期 8 周的养殖实验。结果表明，添加苦草粉 不影响草鱼的存活率和饲料系数，30%苦草组增重率显著高于 20%苦草组。随着苦 草粉添加量的增加，内脏指数及肝胰脏指数显著降低；前肠淀粉酶活力显著增强， 中、后肠淀粉酶活力显著降低；对照组前、中、后肠蛋白酶活力依次增强，随着 苦草粉含量的增加，前肠蛋白酶活力显著增强，中肠蛋白酶活力表现出先增再降 再增的变化，VN1 组显著高于其他组，后肠蛋白酶活力呈现显著降低的趋势；肝 胰脏 SOD 活力显著提高，MDA 含量先降后升，VN3 组最高。血清总蛋白含量有 上升的趋势，VN3 组显著高于其他组；血清白蛋白含量呈现先增后降的趋势，VN1 组最高；血清 ALT 活力先增后降；与对照组相比血清 AST 活力显著降低，各苦草 粉组之间没有显著性差异。添加苦草粉显著降低了饲料表观消化率，但在一定程 度上增强了草鱼对嗜水气单胞菌的抗感染能力。VN 组出现负增长现象，内脏指数 及肝胰脏指数、血清白蛋白、球蛋白及 ALT 都显著低于其他组；肠道各段淀粉酶 活力显著高于各实用饲料组；中肠蛋白酶活力显著高于对照组，后肠蛋白酶活力 显著低于对照组。以上结果表明，饲料中添加 10%～30%苦草粉对草鱼生长没有影 响，但且有利于鱼体健康，可以作为草鱼饲料原料进行资源化的利用；苦草粉的 使用效果明显优于新鲜苦草。 2. 苦草粉对草鱼幼鱼肌肉营养成分、胶原蛋白含量及胶原蛋白基因表 达量的影响 为了探究苦草对草鱼幼鱼肌肉营养成分、胶原蛋白含量及胶原蛋白基因的表 达的影响，以不含苦草粉的基础饲料（VN0组）为对照，分别用10%（VN1组）、 20%（VN2组）、30%（VN3组）的苦草粉替代基础组饲料中的次粉和米糠，配制上海海洋大学硕士学位报告 II 出4种实验饲料，另设置一组只投喂新鲜苦草的青饲料组（VN组）。选用初始体 重（18.85±0.20）g的草鱼幼鱼在室内水泥池网箱进行为期8周的养殖实验。结果显 示，随着苦草粉添加量的增加，草鱼肌肉粗蛋白含量显著提高，粗脂肪和肌肉胶 原蛋白含量都显著降低；肌肉各种脂肪酸含量也是显著降低的，而n-3/n-6显著提 高；肌肉氨基酸总量上显著提高，苦草粉组鲜味氨基酸含量要显著高于对照组；2 种胶原蛋白基因的相对表达量都有下降的趋势。新鲜苦草组在肌肉粗脂肪含量上 显著低于其他各组，胶原蛋白含量显著高于其他组，而胶原蛋白基因的表达量要 显著低于其他组，n3/n6却显著高于其他各组，氨基酸的含量要显著低于其他组。 以上结果表明，饲料中添加30%以内的苦草粉可以提高肌肉的品质和营养价值，同 时苦草添加组草鱼肌肉可能更符合人体健康需求；30%添加量苦草粉的使用效果是 最好的。 3. 苦草对草鱼幼鱼肠道菌群的影响 本研究是在第一章和第二章的研究基础上对实验鱼进行的更深入的研究，为 了探究沉水植物苦草对草鱼肠道菌群的影响，本研究分别取基础饲料组(VN0)、 30%苦草粉添加组(VN3)、新鲜苦草组(VN)草鱼幼鱼肠道内容物样本进行16S rRNA 基因测序。结果显示，草鱼肠道优势菌门主要为厚壁菌门(Firmicutes)、变形菌门 (Proteobacteria)、放线菌门(Actinobacteria)；VN3组群落多样性和群落丰度都显著 高于其他组，VN组显著低于其他组(P＜0.05)；在门水平上，VN3组厚壁菌门、芽 单胞菌门(Gemmatimonadetes)的相对丰度显著高于VN0组(P＜0.05)；VN组厚壁菌 门的相对丰度也显著高于VN0组(P＜0.05)；在属水平上，VN3在微小杆菌属 (Exiguobacterium)和芽孢杆菌属(Bacillus)的相对丰度显著高于VN0组(P＜0.05)；VN 组在链球菌属(Streptococcus)、丹毒梭菌属(Erysipelatoclostridium)的相对丰度显著 高 于 VN0 组 (P ＜ 0.05) ； VN 在 微 小 杆 菌 属 (Exiguobacterium) 和 丹 毒 梭 菌 属 (Erysipelatoclostridium)属相对丰度都显著高于VN3组(P＜0.05)。结果表明，不同的 饲料可以影响草鱼肠道菌群结构；添加30%苦草粉可以提高草鱼肠道内容物菌群多 样性及丰度，促进肠道分解利用纤维素的能力，有利于鱼体健康；只投喂新鲜苦 草降低了肠道菌群多样性和丰度，不利于草鱼肠道健康。 4. 苦草对草鱼幼鱼肠道代谢组学的研究 本研究是利用GC/MS非靶向代谢组学技术对第一章所诉的VN0，VN3，VN组 草鱼中肠前段代谢组学进行更深入的探讨，并对所得结果进行两两分组比较。结 果显示，VN0、VN3两组共有差异代谢物114种，其中氨基酸有21种，糖类有8种， 核苷类有7种；VN0、VN3组共有差异代谢物88种，其中氨基酸14种，核苷类有11上海海洋大学硕士学位报告 III 种，糖类有10种，脂肪酸有4种；VN3、VN组共有差异代谢物58种，含量最多的差 异代谢物分别有核苷类7种，糖类4种，脂肪酸4种，有机酸3种，能量代谢产物3种。 此外，通过 KEGG pathway mapper 功能分析对差异代谢通路进行展示，苦草对草 鱼代谢通路也有显著影响，VN0、VN3组共有15个具有显著性差异的通路图，主要 包含氨酰生物合成，缬氨酸、亮氨酸和异亮氨酸的生物合成，甘氨酸、丝氨酸和 苏氨酸代谢，嘌呤代谢，精氨酸和脯氨酸代谢，缬氨酸、亮氨酸和异亮氨酸降解， 色氨酸代谢等通路。VN0、VN组有12个具有显著性差异的通路图，主要包括氨酰 生物合成，氨酰生物合成，缬氨酸，甘氨酸、丝氨酸和苏氨酸代谢，亮氨酸和异 亮氨酸，丙酸的代谢，苯丙氨酸的代谢等代谢通路。VN3、VN组有6条具有显著性 差异的代谢通路以嘌呤代谢，抗坏血酸和醛酸代谢，谷胱甘肽代谢，半乳糖代谢 等通路为主。以上结果表明，不同的饲料对草鱼代谢物、差异代谢物富集的代谢 通路有很大的影响。 5. 实用饲料搭配新鲜苦草对草鱼幼鱼生长性能、组织生化指 标及肌肉营养成分的影响 在第一章和第二章研究基础上，为了探究利用苦草的不同养殖模式对草鱼草 鱼幼鱼生长性能，组织生化指标，肌肉营养成分以肌肉胶原蛋白基因的影响。以 不含苦草粉的基础饲料（PA组）为对照，上午喂基础饲料下午喂新鲜苦草(PAB组) 为实验组，选用初均重为23.47g的草鱼在室内水泥池网箱进行为期8周的养殖实验。 结果显示，PAB组草鱼增重率和肝胰脏指数要低于对照组；肝胰脏中PAB的SOD、 MDA、ALT都要高于PA组，CAT和AST要低于PA组；PAB组血清总蛋白含量高于 PA组，其他指标都要低于PA组；PAB组肌肉的粗蛋白和粗脂肪含量都要低于对照 组，PAB组的氨基酸总量、非必需氨基酸总量和鲜味氨基酸总量都要高于PA组， PAB在饱和脂肪酸总量、单不饱和脂肪酸总量、多不饱和脂肪酸总量n-3系不饱和 脂肪酸总量和n-6系不饱和脂肪酸总量都低于PA组，而n-3/n-6高于PA组，胶原蛋白 基因表达量差异不显著。以上结果表明，只投喂实用饲料时，草鱼生长迅速，但 肌肉鲜味及营养价值偏低；实用饲料和新鲜苦草交替使用时，草鱼生长速度偏慢， 但整体肌肉品质更好，鱼体抗病力可能更强。 关键词，苦草，草鱼，生长性能，肌肉品质，胶原蛋白，肠道菌群，差异代 谢物上海海洋大学硕士学位报告 IV Feasibility study on the use of Vallisneria natans as feed material for grass carp, Ctenopharyngodon idellus ABSTRACT 1. Effects of adding Vallisneria natans meal to diet on growth performance, physiological and biochemical properties of juvenile grass carp (Ctenopharyngodon idellus) In this study, the effect of Vallisneria natans meal used as feed ingredient in the diet of grass carp (Ctenopharyngodon idellus) on growth, physiological and biochemical properties was evaluated. Four isonitrogenous and isoenergic diets were formulated by replacing the secondary powder and rice bran with 0 (VN0, control), 10% (VN1), 20% (VN2) and 30% (VN3) V. natans meal. In addition, the group that fed fish only with fresh V. natans was designed. Juvenile grass carp of initial weight (18.85 ±0.20) g were selected to be cultured for 8 weeks in indoor cement tank cages. The results showed that the addition of V. natans meal did not affect the survival rate and feed coefficient of grass carp , the weight gain rate of VN3 group was significantly higher than VN2 group. With the increase of the amount of V. natans meal, the visceral index and hepatopancreas index decreased significantly, the activity of amylase in the foregut was significantly increased, and that in the middle and rear intestines was significantly decreased. The activity of amylase in the front, middle and rear intestines in the VN group was significantly higher than that in the other groups. In the control group, foregut, middle and rear intestines protease activity in turn strengthened, and with the increase of the content of V. natans meal the activity of foregut protease was significantly increased, and midgut proteinase activity increased first, then decreased and increased again, the highest value was observed in the group VN1, and the protease activity in the posterior intestine showed a significant decrease trend. The SOD activity of hepatopancreas was significantly increased, while MDA content decreased first and then increased, and that in the group VN3 was the highest. The content of serum total上海海洋大学硕士学位报告 V protein increased significantly, the serum albumin content increased first and then decreased, and the highest value was found in group VN1. Compared with the control group, serum ALT activity was increased and then recovered to the control level, and serum AST activity significantly decreased in V. natans meal groups, and there was no significant difference among the V. natans meal groups. The addition of V.natans meal resulted in significantly reduced apparent digestibility of the diets and enhanced resistance ability of grass carp to Aeromonas hydrophila to some extent. The group VN showed negative growth and depressed tvisceral index, hepatopancreas index, serum albumin, globulin and ALT; The activity of amylase in the intestinal segments was significantly higher than that in all practical formulated diets; The activity of midgut protease in VN group was significantly higher than that of control group, and that of hindgut protease was significantly lower than that of control group. The above results showed that the addition of 10% ~ 30% V.natans meal in the diet had no effect on the growth of grass carp, but was beneficial to the health of the fish body. It indicated that V.natans meal used as the raw material of grass carp feed for resource utilization would be much better than fresh V. natans. 2. Effects of Vallisneria natans meal on muscle nutrient composition, collagen content and collagen gene expression of juvenile grass carp In order to explore the effects of V. natans meal on muscle nutrient composition, collagen content and collagen gene expression of juvenile grass carp. Four isonitrogenous and isoenergic diets were formulated by replacing the secondary powder and rice bran with 0 (VN0, control), 10% (VN1), 20% (VN2) and 30% (VN3) V. natans meal. In addition, the group that fed fish only with fresh V. natans was designed. Juvenile grass carp of initial weight (18.85 ±0.20) g were selected to be cultured for 8 weeks in indoor cement tank cages. The results showed that: With the increase of the content of V. natans meal, the content of grass carp muscle crude protein increased significantly, while the content of crude fat and muscle collagen decreased significantly. The contents of various fatty acids in muscle were also significantly reduced, while the contents of n-3/n-6 were significantly increased. The total amino acid content of muscle was significantly increased, and the umami amino acid content of the V. natans meal groups was significantly higher than that of the control group. The relative expression levels of both collagen genes showed a trend of decline.The content of crude fat in上海海洋大学硕士学位报告 VI muscle of fresh V. natans group was significantly lower than that of other groups, the content of collagen was significantly higher than that of other groups, and the expression level of collagen gene was significantly lower than that of other groups, but n3/n6 was significantly higher than that of other groups, and the content of amino acid was significantly lower than that of other groups. The above results showed that the addition of V. natans meal within 30% in the feed could improve the muscle quality and nutritional value, and at the same time, the fish muscle of the V. natans addition groups might be beneficial to the health of human.The best results were obtained with 30% added V. natans meal. 3. Effects of Vallisneria natans on intestinal flora of juvenile grass carp In order to explore the effect of submerged plant bitter grass on intestinal flora of grass carps. In this study, 16S rRNA gene sequencing was performed on the intestinal tract contents of juvenile grass carps from the basic feed group (VN0), 30% V. natans meal supplemental group (VN3) and fresh V. natans group (VN). The dominant phyla of grass carp intestinal was mainly Firmicutes, Proteobacteria and Actinobacteria. Community diversity and community abundance in the VN3 group were significantly higher than those in other groups, while those in the VN group were significantly lower than those in other groups (P < 0.05). At the phylum level, the relative abundance of Firmicutes and Gemmatimonadetes in VN3 group was significantly higher than that in VN0 group (P < 0.05)and the relative abundance of Firmicutes in VN group was also significantly higher than that in VN0 group (P < 0.05). At the genus level, the relative abundance of VN3 in Exiguobacterium and Bacillus was significantly higher than that in the VN0 group (P < 0.05).The relative abundance of VN group in Streptococcus and Erysipelatoclostridium was significantly higher than that in VN0 group (P < 0.05).The relative abundance of VN in the genera Exiguobacterium and Erysipelatoclostridium was significantly higher than that in the VN3 group (P < 0.05). The above results showed that different feeds could affect the structure of intestinal flora of grass carps, and the addition of V. natans meal could improve the diversity and abundance of intestinal flora of grass carps, promote the ability of the gut to disintegrate and utilize cellulose, and contribute to the health of fish. Feeding fresh V. natans not only reduced the diversity but abundance of intestinal flora, which was not conducive to intestinal health.上海海洋大学硕士学位报告 VII 4. Study on the metabolism for juvenile grass carp intestinal tract under different feed composition In this study, GC/MS non-targeted metabolomics was used to further explore the metabolomics of VN0(control group), VN3(30% V. natans group) and VN(fresh V. natans group) mentioned in chapter 1, and the obtained results were compared in two groups. The results showed that there were 114 different metabolites in group VN0 and VN3, including 21 amino acids, 8 sugars and 7 nucleosides.There were 88 different metabolites in group VN0 and VN, including 14 amino acids, 11 nucleosides, 10 sugars and 4 fatty acids.There were a total of 58 different metabolites in group VN3 and VN, among which 7 were nucleosides, 4 were sugars, 4 were fatty acids, 3 were organic acids, and 3 were energy metabolites. In addition, KEGG pathway mapper function analysis was used to show differences in metabolic pathways. V. natans have a significant effect on grass carp metabolic pathways, VN0 and VN3 group,and there was a total of 15 significant difference of path diagrams between VN0 and VN3 group, mainly containing ammonia acyl biosynthesis, valine, leucine and isoleucine biosynthesis, glycine, serine and threonine metabolism, purine metabolism, arginine and proline metabolism, valine, leucine and isoleucine degradation, tryptophan metabolism pathways. Between VN0 and VN group, there were 12 pathways with significant differences, including aminoacyl biosynthesis, aminoacyl biosynthesis, valine, glycine, serine and threonine metabolism, leucine and isoleucine, propionic acid metabolism, phenylalanine metabolism and other metabolic pathways. Compared VN3 group to VN group, there were 6 metabolic pathways with significant differences, mainly including purine metabolism, ascorbic acid and uronic acid metabolism, glutathione metabolism and galactose metabolism. The above results showed that different feeds had a great influence on the metabolites and the metabolic pathways enriched by different metabolites of grass carps. 5. Effects of practical feed with fresh Vallisneria natans on growth performance, tissue biochemical indexes and muscle nutrients of juvenile grass carp On the basis of the research in chapter 1 and chapter 2, in order to explore the上海海洋大学硕士学位报告 VIII effects of different cultivation modes of V. natans on growth performance, tissue biochemical indexes, muscle nutrients and muscle collagen gene of juvenile grass carps. The basic feed (PA group) without V. natans meal was used as the control group, and the combination of the basic feed fed in the morning and fresh V. natans fed in the afternoon(PAB group) were acted as the experimental group. Grass carp with the initial average weight of 23.47g were selected for the 8-week cultivation experiment in the indoor cement tank cage. The results showed that the weight gain rate and hepatopancreas index of grass carps in PAB group were lower than those in control group. The SOD, MDA and ALT levels of PAB in hepatopancreas were higher than those in PA group, and the levels of CAT and AST were lower than those in PA group. The total serum protein content in the PAB group was higher than that in the PA group, while other indexes were lower than those in the PA group. The content of crude protein and crude fat in muscle in the PAB group was lower than that in the control group. Total amino acids, non-essential amino acids and umami amino acids in PAB group were all higher than those in PA group. The total amount of saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, and n-6 unsaturated fatty acids of PAB were all lower than those of the PA group, while the n-3/n-6 was higher than that of the PA group. There was no significant difference in collagen gene expression. The above results showed that grass carps grew rapidly when only fed with practical feed, but their muscle flavor and nutritional value were lower. When the practical feed and fresh V. natans were used alternately, the growth speed of grass carp was relatively slower, but the muscle quality was better, and the disease resistance of the fish might be stronger. Keywords: Vallisneria natans, Ctenopharyngodon idellus, growth performance, muscle quality, collagen, intestinal flora, differential metabolites上海海洋大学硕士学位报告 IX 目 录 引 言.....1 沉水植物资源化利用的主要方式......1 1 治理富营养化水体..1 2 加工为肥料.....1 3 直接或间接用作燃料...... 2 4 药用价值2 5 用作饵料与饲料原料...... 2 6 其他资源化利用方式...... 3 研究展望及本研究目的和意义. 3 第一章 苦草粉对草鱼幼鱼生长性能与生理生化性能的影响....5 1 材料与方法...5 1.1 实验设计与实验饲料.. 5 1.2 实验用鱼与饲养管理.. 6 1.3 样品采集与指标测定. 7 1.4 攻毒实验.....8 1.5 数据处理与分析..8 2.结果......8 2.1 饲料中的苦草粉含量对草鱼生长性能与形体指标的影响.....8 2.2 饲料中的苦草粉含量对草鱼消化酶活力和表观消化率的影响......9 2.3 饲料中的苦草粉含量对草鱼生化指标的影响.....11 2.4 饲料中的苦草粉含量对草鱼抗感染能力的影响.12 3 讨论.....12 3.1 苦草粉作为饲料原料对草鱼生长性能的影响.....12 3.2 苦草粉作为饲料原料对草鱼肠道部分消化酶及饲料表观消化吸收率的影响.......13 3.3 苦草粉作为饲料原料对草鱼组织生化指标的影响......14 4 结论.....14 第二章 苦草粉对草鱼幼鱼肌肉营养成分及胶原蛋白基因表达量的影响15 1 材料与方法.16 1.1 实验设计与实验饲料16 1.2 实验用鱼与饲养管理16 1.3 样品采集与指标测定16 1.4 数据处理与统计分析........ 17 2 实验结果......17 2.1 草鱼幼鱼肌肉成分.... 17 2.2 草鱼幼鱼肌肉脂肪酸18 2.3 草鱼幼鱼肌肉氨基酸20 2.4 肌肉胶原蛋白基因相对表达量..21 3 讨论....21 3.1 苦草对草鱼幼鱼肌肉营养成分及胶原蛋白基因相对表达量的影响....21 3.2 苦草对草鱼幼鱼肌肉脂肪酸的影响..22 3.3 苦草对草鱼幼鱼肌肉氨基酸的影响..23上海海洋大学硕士学位报告 X 4 结论.....23 第三章 苦草对草鱼幼鱼肠道菌群的影响.......24 1.材料与方法..24 1.1 实验设计与实验饲料24 1.2 样本采集...24 1.3 实验方法...25 1.4 数据处理...25 2.结果....25 2.1 物种注释与评估........ 25 2.2 物种组成分析....28 3.讨论....33 4.结论....36 第四章 基于代谢组学研究苦草对草鱼幼鱼肠道代谢的影响..37 1 样品采集.....37 2 实验方法......37 3 样本分析与数据处理....37 3.1 分析流程...37 3.2 数据处理...38 4 结果.....38 4.1 多元统计分析结果.... 38 样本的对比分析.......39 4.2 差异代谢物........40 4.3 差异代谢物的代谢通路分析......48 5 讨论....52 6 结论.....54 第五章 实用饲料搭配新鲜苦草对草鱼幼鱼生长性能、组织生化指标及肌肉营养成分的影响55 1 材料与方法.55 1.1 实验饲料...55 1.2 实验用鱼与饲养管理55 1.3 样品采集与指标测定56 1.4 数据统计分析...56 2 结果....57 2.1 两种喂养模式下草鱼生长性能与形体指标57 2.2 草鱼消化酶活力的比较.....57 2.3 两种模式下草鱼生化指标的比较.......58 2.4 肌肉营养成分比较... 58 3 讨论....61 4 结论....62 全文总结........63