本文選題：團(tuán)頭魴 + 鋅需要量　； 參考：《華中農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：鋅(Zn)是維持魚類正常生長(zhǎng)、發(fā)育所必需的微量元素,它不僅參與機(jī)體的各種代謝而且還在骨骼發(fā)育、生殖、免疫、生物膜穩(wěn)定和基因表達(dá)等生理機(jī)能中擔(dān)負(fù)重要角色。飼料中Zn缺乏會(huì)導(dǎo)致魚類生長(zhǎng)緩慢、食欲減退、死亡率增高和骨骼受損,并影響免疫功能。Zn供給過量會(huì)增加飼料成本,影響鈣、鎂和鐵等元素的吸收利用,同時(shí)引起養(yǎng)殖水體中Zn含量增加。這不僅會(huì)影響?zhàn)B殖魚類的攝食,而且對(duì)魚類的存活構(gòu)成重大威脅。因此準(zhǔn)確掌握飼料中Zn元素的配比是保障養(yǎng)殖魚類正常生長(zhǎng)和減少養(yǎng)殖過程中Zn排放的關(guān)鍵措施之一。團(tuán)頭魴(Megalobrama amblycephala)是我國(guó)重要的養(yǎng)殖魚類之一,其魚種階段(50~122g)對(duì)Zn的需要量為184.85 mg/kg。20世紀(jì)末,朱雅珠等采用L16(45)正交法研究了團(tuán)頭魴幼魚(4.9~18.6g)對(duì)Zn的需要量為20 mg/kg。鑒于已有的2篇報(bào)道中團(tuán)頭魴對(duì)Zn的需要量差異較大,因此本論文研究了鋅(Zn)對(duì)幼魚和成魚階段的團(tuán)頭魴(Megalobrama amblycephala)生長(zhǎng)性能、飼料利用、抗氧化指標(biāo)、基礎(chǔ)生化指標(biāo)、營(yíng)養(yǎng)成分和精巢發(fā)育等的影響,以期確定幼魚和成魚階段的團(tuán)頭魴對(duì)飼料Zn的需要量;應(yīng)用RNA-seq檢測(cè)分析了團(tuán)頭魴幼魚(用含不同Zn含量的飼料飼喂10 w)的精巢轉(zhuǎn)錄情況,以期了解Zn調(diào)控精巢發(fā)育的分子機(jī)制;應(yīng)用核磁共振技術(shù)(1H-NMR)檢測(cè)分析了團(tuán)頭魴幼魚(用含不同Zn含量的飼料飼喂12 w)血清代謝物變化,以期發(fā)現(xiàn)團(tuán)頭魴的Zn敏感生物標(biāo)志物。具體研究結(jié)果如下:1.團(tuán)頭魴幼魚對(duì)飼料Zn的需要量以酪蛋白和明膠為蛋白源,七水硫酸Zn(Zn SO_4·7H_2O)為Zn源,分別配制成7種Zn含量(7.4、20.3、32.1、51.0、84.4、169.7和332.4 mg/kg)的半純化飼料,投喂初始體質(zhì)量為(3.6±0.1)g團(tuán)頭魴12 w,考察Zn對(duì)團(tuán)頭魴幼魚生長(zhǎng)性能、血清生化指標(biāo)和抗氧化功能的影響,確定團(tuán)頭魴幼魚對(duì)飼料Zn的需要量。結(jié)果表明,隨著飼料Zn含量增加,團(tuán)頭魴增重率、特定生長(zhǎng)率和全魚Zn含量呈先增加后穩(wěn)定的趨勢(shì);全魚水分含量顯著降低(P0.05),粗蛋白含量顯著增加(P0.05)。飼料Zn含量對(duì)團(tuán)頭魴飼料系數(shù)無顯著影響(P0.05)。飼料中添加Zn顯著影響血清總蛋白、尿素氮、高密度脂蛋白膽固醇、總膽固醇以及甘油三酯含量,而對(duì)血清白蛋白含量和堿性磷酸酶活性無顯著影響。隨著飼料中Zn含量的增加,團(tuán)頭魴肝丙二醛含量顯著降低(P0.05),而肝過氧化氫酶和超氧化歧化酶活性在各處理間均無顯著差異(P0.05)。折線回歸分析表明:團(tuán)頭魴幼魚(3.6~26.7 g)獲得最佳生長(zhǎng)時(shí)對(duì)飼料Zn需要量為32.6 mg/kg,獲得最大魚體Zn含量時(shí)Zn的需要量為47.6 mg/kg。通過延長(zhǎng)養(yǎng)殖時(shí)間,48 w時(shí)7.4 mg/kg組約有20%的實(shí)驗(yàn)魚出現(xiàn)脊柱彎曲、鰓蓋外翻、臀鰭和尾鰭不完整癥狀中的一種或多種癥狀。2.團(tuán)頭魴成魚對(duì)飼料Zn的需要量為考察團(tuán)頭魴成魚對(duì)飼料中Zn的需要量,以七水硫酸Zn(Zn SO_4·7H_2O)為Zn源,配制Zn含量分別為7.8(對(duì)照組),32.7,50.3,87.2,165.4和328.5 mg/kg的6組等氮等脂飼料,分別飼喂初始體重為(128.60±0.74)g的團(tuán)頭魴,每組設(shè)3個(gè)重復(fù),每個(gè)重復(fù)放養(yǎng)15尾魚,養(yǎng)殖12 w。結(jié)果顯示,隨著飼料Zn含量增加,團(tuán)頭魴的增重率和全魚Zn含量先顯著升高,在Zn含量分別達(dá)到50.3 mg/kg和87.2 mg/kg后趨于穩(wěn)定。飼料中添加Zn降低了飼料系數(shù),但各添加組間并無顯著差異。飼料中添加Zn對(duì)全魚粗蛋白含量無顯著影響(P0.05),但顯著降低了全魚水分含量,提高了全魚灰分含量(P0.05)。飼料中添加Zn顯著影響肝臟丙二醛含量、超氧化物歧化酶、過氧化氫酶、總抗氧化酶和谷胱甘肽過氧化物酶的活性,對(duì)血清葡萄糖、高密度脂蛋白膽固醇、總膽固醇含量無顯著影響(P0.05),但顯著提高堿性磷酸酶活性并降低甘油三脂含量(P0.05)。肝臟組織石蠟切片顯示7.8 mg/kg和328.5 mg/kg組肝細(xì)胞出現(xiàn)核偏移,肝細(xì)胞的細(xì)胞質(zhì)減少,出現(xiàn)了空泡現(xiàn)象。以增重率和全魚Zn含量為評(píng)價(jià)指標(biāo),根據(jù)折線回歸分析得出,以Zn SO_4·7H_2O為Zn源時(shí),團(tuán)頭魴成魚對(duì)飼料中Zn的需要量分別為52.1和86.2 mg/kg。Zn含量不足時(shí)會(huì)對(duì)團(tuán)頭魴成魚的生長(zhǎng)產(chǎn)生負(fù)面影響,但添加量過高(328.5 mg/kg)會(huì)引起肝臟損傷,降低抗氧化功能。3.飼料Zn對(duì)團(tuán)頭魴成魚精巢發(fā)育的影響本研究觀察了團(tuán)頭魴成魚對(duì)鋅需要量養(yǎng)殖實(shí)驗(yàn)結(jié)束時(shí)實(shí)驗(yàn)魚的精巢切片及精子活力。精巢組織石蠟切片顯示,各組精巢中均有發(fā)育成熟的精子,7.8和32.7 mg/kg組染色偏淡,其余四組顏色較深。7.8 mg/kg組的間質(zhì)組織不發(fā)達(dá),50.3、87.2、165.4和328.5 mg/kg四組間質(zhì)組織較發(fā)達(dá),其中分布的間質(zhì)細(xì)胞核仁明顯,核大而且圓。團(tuán)頭魴成魚精子運(yùn)動(dòng)參數(shù)方面,Zn含量對(duì)精子運(yùn)動(dòng)的擺動(dòng)性和精子運(yùn)動(dòng)的前向性影響不顯著,但顯著影響精子平均曲線運(yùn)動(dòng)速度、平均直線運(yùn)動(dòng)速度、精子的平均路徑速度、精子頭側(cè)擺幅度、精子平均鞭打頻率、精子運(yùn)動(dòng)的直線性和精子平均移動(dòng)角度等參數(shù)(P0.05)。隨著飼料Zn含量的升高、團(tuán)頭魴成魚精子精子平均曲線運(yùn)動(dòng)速度、平均直線運(yùn)動(dòng)速度、精子的平均路徑速度、精子頭側(cè)擺幅度、精子運(yùn)動(dòng)的直線性和精子平均移動(dòng)角度先顯著升高后下降(P0.05)精子平均鞭打頻率則隨著Zn含量升高呈下降趨勢(shì),50.3、87.2、165.4和328.5 mg/kg組顯著低于對(duì)照組(P0.05)。以上結(jié)果表明飼料中Zn含量不足時(shí)會(huì)延緩團(tuán)頭魴成魚精巢發(fā)育,但添加量過高(328.5 mg/kg)會(huì)引起精子活力下降。4.飼料鋅對(duì)團(tuán)頭魴幼魚精巢發(fā)育影響的轉(zhuǎn)錄組學(xué)分析本研究通過用3種Zn含量(8.5、30.9、328.1 mg/kg,分別記為ZN_L、ZN_M和ZN_H)的飼料飼養(yǎng)初始體質(zhì)量為1.7g的團(tuán)頭魴幼魚10 w,應(yīng)用RNA-seq檢測(cè)分析了三組實(shí)驗(yàn)魚精巢的轉(zhuǎn)錄情況,以期了解Zn調(diào)控團(tuán)頭魴幼魚精巢發(fā)育的分子機(jī)制。結(jié)果表明:三個(gè)轉(zhuǎn)錄本共組裝出67497 Unigenes個(gè),其中35999個(gè)Unigenes在Nr、Swiss-Prot、KEGG和COG/KOG四大數(shù)據(jù)庫中被成功注釋。組裝出來的Unigenes與斑馬魚參考基因組的匹配率最高達(dá)39.71%(26798個(gè)Unigenes)。在三個(gè)處理組之間一共有39963個(gè)差異表達(dá)基因。其中34122個(gè)基因在ZN_M和ZN_L中差異表達(dá)(20200個(gè)上調(diào)基因和13922個(gè)下調(diào)基因);22875個(gè)基因在ZN_M和ZN_H組中差異表達(dá)(19763個(gè)上調(diào)基因和3112個(gè)下調(diào)基因);以及有30315個(gè)基因在ZN_H和ZN_L組中差異表達(dá)(18294個(gè)上調(diào)基因和12021個(gè)下調(diào)基因)。差異表達(dá)基因參與的生物學(xué)過程類別主要包括單組織過程、發(fā)育過程、生長(zhǎng)、免疫系統(tǒng)過程和生物學(xué)過程調(diào)控等。差異基因所在的30條KEGG信號(hào)通路發(fā)生了顯著性表達(dá)變化。信號(hào)通路主要包括神經(jīng)活性的配體-受體相互作用、氧化磷酸化、核糖體生物合成、RNA聚合酶、核苷酸切除修復(fù)、DNA復(fù)制、細(xì)胞粘附分子、補(bǔ)體系統(tǒng)、腸道免疫Ig A生成網(wǎng)絡(luò)、造血細(xì)胞系等。以上結(jié)果顯示Zn可以誘導(dǎo)精巢發(fā)育相關(guān)基因的表達(dá),對(duì)團(tuán)頭魴性腺細(xì)胞的增殖具有正調(diào)控作用,能夠促進(jìn)性腺的發(fā)育。本研究發(fā)現(xiàn)的生物學(xué)過程和信號(hào)通路,可用于解釋飼料Zn對(duì)團(tuán)頭魴雄性生殖系統(tǒng)發(fā)育調(diào)控的分子機(jī)制。5.基于1H NMR技術(shù)的團(tuán)頭魴幼魚血清代謝組學(xué)分析以1H NMR代謝組技術(shù)測(cè)試比較了用Zn含量為7.4、32.1和332.4 mg/kg飼料喂養(yǎng)12 w團(tuán)頭魴的血清代謝物,對(duì)血清中的51種代謝物進(jìn)行了定性和定量,其中氨基酸及其衍生物22種,糖類3種、維生素5種和其它組分2種,經(jīng)PCA-LS和VIP分析,差異代謝物依次為脯氨酸、乳酸、葡萄糖、丙氨酸、賴氨酸、谷氨酰胺、肌酸、羥脯氨酸、�；撬�、絲氨酸、τ-甲基組氨酸、精氨酸、纈氨酸、蛋氨酸和甘露糖。其中脯氨酸指標(biāo)反映Zn缺乏和過量都會(huì)對(duì)團(tuán)頭魴產(chǎn)生營(yíng)養(yǎng)性應(yīng)激,乳酸則反映了肝功能狀況,葡萄糖反映了團(tuán)頭魴糖代謝狀況。以上結(jié)果顯示:脯氨酸、乳酸和葡萄糖可以作為反映團(tuán)頭魴Zn營(yíng)養(yǎng)狀態(tài)的潛在生物標(biāo)志物,飼料中Zn缺乏和過量會(huì)抑制團(tuán)頭魴的氨基酸代謝和糖代謝。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S917.4

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