团头鲂锌的营养需求及锌对其精巢发育影响的研究

发布时间：2018-05-30 22:43

本文选题：团头鲂 + 锌需要量　；参考：《华中农业大学》2016年博士论文

【摘要】：锌(Zn)是维持鱼类正常生长、发育所必需的微量元素,它不仅参与机体的各种代谢而且还在骨骼发育、生殖、免疫、生物膜稳定和基因表达等生理机能中担负重要角色。饲料中Zn缺乏会导致鱼类生长缓慢、食欲减退、死亡率增高和骨骼受损,并影响免疫功能。Zn供给过量会增加饲料成本,影响钙、镁和铁等元素的吸收利用,同时引起养殖水体中Zn含量增加。这不仅会影响养殖鱼类的摄食,而且对鱼类的存活构成重大威胁。因此准确掌握饲料中Zn元素的配比是保障养殖鱼类正常生长和减少养殖过程中Zn排放的关键措施之一。团头鲂(Megalobrama amblycephala)是我国重要的养殖鱼类之一,其鱼种阶段(50~122g)对Zn的需要量为184.85 mg/kg。20世纪末,朱雅珠等采用L16(45)正交法研究了团头鲂幼鱼(4.9~18.6g)对Zn的需要量为20 mg/kg。鉴于已有的2篇报道中团头鲂对Zn的需要量差异较大,因此本论文研究了锌(Zn)对幼鱼和成鱼阶段的团头鲂(Megalobrama amblycephala)生长性能、饲料利用、抗氧化指标、基础生化指标、营养成分和精巢发育等的影响,以期确定幼鱼和成鱼阶段的团头鲂对饲料Zn的需要量;应用RNA-seq检测分析了团头鲂幼鱼(用含不同Zn含量的饲料饲喂10 w)的精巢转录情况,以期了解Zn调控精巢发育的分子机制;应用核磁共振技术(1H-NMR)检测分析了团头鲂幼鱼(用含不同Zn含量的饲料饲喂12 w)血清代谢物变化,以期发现团头鲂的Zn敏感生物标志物。具体研究结果如下:1.团头鲂幼鱼对饲料Zn的需要量以酪蛋白和明胶为蛋白源,七水硫酸Zn(Zn SO_4·7H_2O)为Zn源,分别配制成7种Zn含量(7.4、20.3、32.1、51.0、84.4、169.7和332.4 mg/kg)的半纯化饲料,投喂初始体质量为(3.6±0.1)g团头鲂12 w,考察Zn对团头鲂幼鱼生长性能、血清生化指标和抗氧化功能的影响,确定团头鲂幼鱼对饲料Zn的需要量。结果表明,随着饲料Zn含量增加,团头鲂增重率、特定生长率和全鱼Zn含量呈先增加后稳定的趋势;全鱼水分含量显著降低(P0.05),粗蛋白含量显著增加(P0.05)。饲料Zn含量对团头鲂饲料系数无显著影响(P0.05)。饲料中添加Zn显著影响血清总蛋白、尿素氮、高密度脂蛋白胆固醇、总胆固醇以及甘油三酯含量,而对血清白蛋白含量和碱性磷酸酶活性无显著影响。随着饲料中Zn含量的增加,团头鲂肝丙二醛含量显著降低(P0.05),而肝过氧化氢酶和超氧化歧化酶活性在各处理间均无显著差异(P0.05)。折线回归分析表明:团头鲂幼鱼(3.6~26.7 g)获得最佳生长时对饲料Zn需要量为32.6 mg/kg,获得最大鱼体Zn含量时Zn的需要量为47.6 mg/kg。通过延长养殖时间,48 w时7.4 mg/kg组约有20%的实验鱼出现脊柱弯曲、鳃盖外翻、臀鳍和尾鳍不完整症状中的一种或多种症状。2.团头鲂成鱼对饲料Zn的需要量为考察团头鲂成鱼对饲料中Zn的需要量,以七水硫酸Zn(Zn SO_4·7H_2O)为Zn源,配制Zn含量分别为7.8(对照组),32.7,50.3,87.2,165.4和328.5 mg/kg的6组等氮等脂饲料,分别饲喂初始体重为(128.60±0.74)g的团头鲂,每组设3个重复,每个重复放养15尾鱼,养殖12 w。结果显示,随着饲料Zn含量增加,团头鲂的增重率和全鱼Zn含量先显著升高,在Zn含量分别达到50.3 mg/kg和87.2 mg/kg后趋于稳定。饲料中添加Zn降低了饲料系数,但各添加组间并无显著差异。饲料中添加Zn对全鱼粗蛋白含量无显著影响(P0.05),但显著降低了全鱼水分含量,提高了全鱼灰分含量(P0.05)。饲料中添加Zn显著影响肝脏丙二醛含量、超氧化物歧化酶、过氧化氢酶、总抗氧化酶和谷胱甘肽过氧化物酶的活性,对血清葡萄糖、高密度脂蛋白胆固醇、总胆固醇含量无显著影响(P0.05),但显著提高碱性磷酸酶活性并降低甘油三脂含量(P0.05)。肝脏组织石蜡切片显示7.8 mg/kg和328.5 mg/kg组肝细胞出现核偏移,肝细胞的细胞质减少,出现了空泡现象。以增重率和全鱼Zn含量为评价指标,根据折线回归分析得出,以Zn SO_4·7H_2O为Zn源时,团头鲂成鱼对饲料中Zn的需要量分别为52.1和86.2 mg/kg。Zn含量不足时会对团头鲂成鱼的生长产生负面影响,但添加量过高(328.5 mg/kg)会引起肝脏损伤,降低抗氧化功能。3.饲料Zn对团头鲂成鱼精巢发育的影响本研究观察了团头鲂成鱼对锌需要量养殖实验结束时实验鱼的精巢切片及精子活力。精巢组织石蜡切片显示,各组精巢中均有发育成熟的精子,7.8和32.7 mg/kg组染色偏淡,其余四组颜色较深。7.8 mg/kg组的间质组织不发达,50.3、87.2、165.4和328.5 mg/kg四组间质组织较发达,其中分布的间质细胞核仁明显,核大而且圆。团头鲂成鱼精子运动参数方面,Zn含量对精子运动的摆动性和精子运动的前向性影响不显著,但显著影响精子平均曲线运动速度、平均直线运动速度、精子的平均路径速度、精子头侧摆幅度、精子平均鞭打频率、精子运动的直线性和精子平均移动角度等参数(P0.05)。随着饲料Zn含量的升高、团头鲂成鱼精子精子平均曲线运动速度、平均直线运动速度、精子的平均路径速度、精子头侧摆幅度、精子运动的直线性和精子平均移动角度先显著升高后下降(P0.05)精子平均鞭打频率则随着Zn含量升高呈下降趋势,50.3、87.2、165.4和328.5 mg/kg组显著低于对照组(P0.05)。以上结果表明饲料中Zn含量不足时会延缓团头鲂成鱼精巢发育,但添加量过高(328.5 mg/kg)会引起精子活力下降。4.饲料锌对团头鲂幼鱼精巢发育影响的转录组学分析本研究通过用3种Zn含量(8.5、30.9、328.1 mg/kg,分别记为ZN_L、ZN_M和ZN_H)的饲料饲养初始体质量为1.7g的团头鲂幼鱼10 w,应用RNA-seq检测分析了三组实验鱼精巢的转录情况,以期了解Zn调控团头鲂幼鱼精巢发育的分子机制。结果表明:三个转录本共组装出67497 Unigenes个,其中35999个Unigenes在Nr、Swiss-Prot、KEGG和COG/KOG四大数据库中被成功注释。组装出来的Unigenes与斑马鱼参考基因组的匹配率最高达39.71%(26798个Unigenes)。在三个处理组之间一共有39963个差异表达基因。其中34122个基因在ZN_M和ZN_L中差异表达(20200个上调基因和13922个下调基因);22875个基因在ZN_M和ZN_H组中差异表达(19763个上调基因和3112个下调基因);以及有30315个基因在ZN_H和ZN_L组中差异表达(18294个上调基因和12021个下调基因)。差异表达基因参与的生物学过程类别主要包括单组织过程、发育过程、生长、免疫系统过程和生物学过程调控等。差异基因所在的30条KEGG信号通路发生了显著性表达变化。信号通路主要包括神经活性的配体-受体相互作用、氧化磷酸化、核糖体生物合成、RNA聚合酶、核苷酸切除修复、DNA复制、细胞粘附分子、补体系统、肠道免疫Ig A生成网络、造血细胞系等。以上结果显示Zn可以诱导精巢发育相关基因的表达,对团头鲂性腺细胞的增殖具有正调控作用,能够促进性腺的发育。本研究发现的生物学过程和信号通路,可用于解释饲料Zn对团头鲂雄性生殖系统发育调控的分子机制。5.基于1H NMR技术的团头鲂幼鱼血清代谢组学分析以1H NMR代谢组技术测试比较了用Zn含量为7.4、32.1和332.4 mg/kg饲料喂养12 w团头鲂的血清代谢物,对血清中的51种代谢物进行了定性和定量,其中氨基酸及其衍生物22种,糖类3种、维生素5种和其它组分2种,经PCA-LS和VIP分析,差异代谢物依次为脯氨酸、乳酸、葡萄糖、丙氨酸、赖氨酸、谷氨酰胺、肌酸、羟脯氨酸、牛磺酸、丝氨酸、τ-甲基组氨酸、精氨酸、缬氨酸、蛋氨酸和甘露糖。其中脯氨酸指标反映Zn缺乏和过量都会对团头鲂产生营养性应激,乳酸则反映了肝功能状况,葡萄糖反映了团头鲂糖代谢状况。以上结果显示:脯氨酸、乳酸和葡萄糖可以作为反映团头鲂Zn营养状态的潜在生物标志物,饲料中Zn缺乏和过量会抑制团头鲂的氨基酸代谢和糖代谢。
[Abstract]:Zinc (Zn) is a necessary trace element to maintain the normal growth and development of fish. It not only takes part in the metabolism of the body, but also plays an important role in the physiological functions of bone development, reproduction, immunity, biofilm stability and gene expression. The deficiency of Zn in feed causes the slow growth of the fish, the loss of appetite, the increase of mortality and the damage of bone, The excessive supply of immune function.Zn will increase the feed cost, affect the absorption and utilization of calcium, magnesium and iron, and increase the content of Zn in the aquaculture water. This will not only affect the feeding of the cultured fish, but also pose a great threat to the survival of the fish. Therefore, the accurate mastery of the ratio of Zn elements in the feed is to guarantee the normal of the cultured fish. One of the key measures for the growth and reduction of Zn emission during the breeding process. Megalobrama amblycephala (Megalobrama amblycephala) is one of the most important fish in our country. The requirement of the fish seed stage (50~122g) for Zn is at the end of the 184.85 mg/kg.20 century, and the L16 (45) orthogonal method has been used to study the demand for Zn (4.9~18.6g) to Zn (20 mg/kg.). In this paper, the effects of zinc (Zn) on the growth performance, feed utilization, antioxidant index, basic biochemical indexes, nutritional components and spermary development in young and adult bream (Megalobrama amblycephala) were studied in order to determine the stage of juvenile and adult fish, in view of the large difference in the need for Zn in the 2 reports. The required amount of Zn in bream bream was analyzed by RNA-seq test. The transcriptional condition of the young blunt snout bream larvae (with 10 W feed with different Zn content) was used to understand the molecular mechanism of Zn regulating the development of the spermary, and the NMR (1H-NMR) assay was used to analyze the sera generation of the young blunt snout bream (with a feed of different Zn content in the feed of 12 W). In order to find the Zn sensitive biomarkers of blunt snout bream, the specific results are as follows: 1. the needs of young blunt snout bream young fish with casein and gelatin as the protein source, seven water sulphate Zn (Zn SO_4. 7H_2O) as Zn source, respectively, prepared into 7 kinds of Zn content (7.4,20.3,32.1,51.0,84.4169.7 and 332.4 mg/kg) semi purified feed, feeding early The mass of the beginning body was (3.6 + 0.1) g blunt snout Bram bream (12 W), and the effect of Zn on the growth performance, serum biochemical indexes and antioxidant function of Bram blunt snout bream was investigated to determine the requirement for the feed Zn in the young blunt snout bream. The results showed that with the increase of Zn content in the feed, the weight gain rate of the bream was increased, the specific growth rate and the total fish Zn content increased first and then the stable trend; the whole fish was the whole fish. The content of water content was significantly decreased (P0.05) and the content of crude protein increased significantly (P0.05). The feed Zn content had no significant effect on the feed coefficient of bream (P0.05). The addition of Zn in feed significantly affected the content of serum total protein, urea nitrogen, high density lipoprotein cholesterol, total cholesterol and glycerol three ester, while the content of serum albumin and alkaline phosphatase activity With the increase of Zn content in the feed, the content of malondialdehyde in the liver of bream was significantly decreased (P0.05), but there was no significant difference between the liver catalase and the activity of superoxide dismutase (P0.05). The fold regression analysis showed that the optimum growth of the young blunt snout bream (3.6~26.7 G) was 32.6 mg/kg for the feed Zn. When the maximum fish body Zn content is Zn, the requirement is 47.6 mg/kg. by prolonging the culture time. At 48 W, there are about 20% of the experimental fish in the 7.4 mg/kg group. With seven water sulfuric acid Zn (Zn SO_4. 7H_2O) as Zn source, Zn content was 7.8 (control group), 32.7,50.3,87.2165.4 and 328.5 mg/kg of 6 groups of equal fat feed were fed, respectively feeding the initial weight of (128.60 + 0.74) g bream, each set 3 repeats, each repeat 15 fish, 12 W. results showed, as the feed Zn content increased, reunion head. The weight gain rate and the total Zn content of the whole fish increased significantly first, while the Zn content reached 50.3 mg/kg and 87.2 mg/kg respectively. Adding Zn in the feed reduced the feed coefficient, but there was no significant difference between the added groups. The addition of Zn had no significant effect on the total fish crude protein content (P0.05), but significantly reduced the total fish water content and improved the whole fish content. The content of malondialdehyde in liver, superoxide dismutase, catalase, total antioxidant enzyme and glutathione peroxidase in the liver were significantly affected by the content of fish ash content (P0.05), and the activity of superoxide dismutase, catalase, total antioxidant enzyme and glutathione peroxidase had no significant effect on serum glucose, high density lipoprotein cholesterol and total cholesterol (P0.05), but the activity of alkaline phosphatase was significantly increased and decreased. The three fat content of low glycerin (P0.05). The liver tissue paraffin section showed that the hepatocytes in the 7.8 mg/kg and 328.5 mg/kg groups had nuclear migration, the cytoplasm of the hepatocytes decreased, and the vacuoles appeared. The weight gain rate and the total fish Zn content were the evaluation indexes. According to the regression analysis, Zn SO_4. 7H_2O was the source of Zn. When the required amount of 52.1 and 86.2 mg/kg.Zn, respectively, would have a negative effect on the growth of the adult blunt snout bream, but the excessive addition (328.5 mg/kg) could cause liver damage and reduce the effect of the antioxidant function.3. feed Zn on the development of the spermary of Bram bream. The semen section and sperm motility of the spermatozoon showed that the spermatozoa were mature in the spermary, the 7.8 and 32.7 mg/kg groups were dyed light, the other four groups were less developed in the.7.8 mg/kg group, and the interstitial tissues of the 50.3,87.2165.4 and 328.5 mg/kg four groups were more developed, among which the interstitial cells of interstitial cells were distributed. In terms of sperm motility parameters, Zn content has no significant effect on the swinging of sperm movement and the forward nature of sperm motility, but it has a significant effect on the average velocity of motility, the average speed of linear motion, the average speed of sperm, the amplitude of the sperm head side, the average whiplash frequency of sperm, and the motility of sperm. With the increase of feed Zn content, the average curve velocity of sperm sperm and sperm motility, the average velocity of sperm, the range of sperm head side pendulum, the linearity of sperm movement and the average movement angle of spermatozoon first increased significantly and then decreased (P0.05) sperm (P0.05) with the increase of feed content in the feed. The average whipping frequency decreased with the increase of Zn content, and the 50.3,87.2165.4 and 328.5 mg/kg groups were significantly lower than the control group (P0.05). The above results showed that the lack of Zn content in the feed would delay the development of the spermatozoa, but the excessive addition (328.5 mg/kg) would cause the spermatozoon vitality to decrease.4. feed zinc on the spermatozoon development of the young blunt snout bream. The transcriptional analysis was used to feed the 10 W of the young blunt snout bream by using 3 kinds of Zn content (8.5,30.9328.1 mg/kg, ZN_L, ZN_M and ZN_H, respectively, ZN_L, ZN_M and ZN_H). The transcriptional conditions of the three groups of experimental fish sperms were analyzed by RNA-seq detection, and the molecular mechanism of the Zn regulation of the spermary development of the young blunt snout bream was solved. The results showed that three transcripts were assembled to 67497 Unigenes, of which 35999 Unigenes were successfully annotated in the large databases of Nr, Swiss-Prot, KEGG and COG/KOG. The matching rate of the assembled Unigenes and zebrafish reference genome was up to 39.71% (26798 Unigenes). There were 39963 differentially expressed genes between the three processing groups. 34122 genes were expressed differently in ZN_M and ZN_L (20200 up-regulated and 13922 down-regulated genes); 22875 genes were expressed differently in ZN_M and ZN_H groups (19763 up-regulated and 3112 down-regulated genes), and 30315 genes were expressed differently in ZN_H and ZN_L groups (18294 up-regulated and 12021 down-regulated genes). Differential expression The biological process category of gene participation mainly includes the single tissue process, development process, growth, immune system process and biological process regulation. The 30 KEGG signaling pathways of differential genes have significant expression changes. Signal pathways mainly include ligand receptor interaction of neuroactive, oxidative phosphorylation and ribosome. Synthesis, RNA polymerase, nucleotide excision repair, DNA replication, cell adhesion molecule, complement system, intestinal immune Ig A generation network, hematopoietic cell line, etc. the above results show that Zn can induce the expression of the related genes of the spermary development, which can regulate the proliferation of the gonadal gland cells of the blunt snout bream, and can promote the development of the gonadal gland. This study found that Biological processes and signaling pathways can be used to explain the molecular mechanism of feed Zn on the development and regulation of male reproductive system of blunt snout brsnk..5. based on 1H NMR technology, the serum metabolomics of the young blunt snout bream bream was compared with the serum metabolites of Zn content for 7.4,32.1 and 332.4 mg/kg feed, and the serum metabolites of 12 W Group bream were compared with the 1H NMR metabolic group. 51 kinds of metabolites in the Qing Dynasty were qualitative and quantitative, including 22 amino acids and their derivatives, 3 saccharides, 5 vitamins and 2 other components. By PCA-LS and VIP analysis, the differential metabolites were proline, lactic acid, glucose, alanine, lysine, glutamine, creatine, hydroxyproline, taurine, serine, tau histidine, spermine Acid, valine, methionine, and mannose. The proline index reflects the nutritional stress of the blunt snout bream, while the lactic acid reflects the state of liver function. Glucose reflects the metabolic status of bream. The above results show that proline, lactic acid and grape sugar can be used as a potential source of Zn nutritional status of Bram blunt snout bream. Zn deficiency and excess in feed will inhibit amino acid metabolism and sugar metabolism in blunt snout bream.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S917.4

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