普安银鲫胚胎发育中脂质代谢酶基因的表达及葡萄糖的影响

采用荧光定量PCR技术检测了普安银鲫胚胎发育中乙酰辅酶A羧化酶α、脂肪酸合成酶、脂蛋白脂酶和肝脂酶基因表达的特点及葡萄糖浸泡的影响,以探讨乙酰辅酶A羧化酶α、脂肪酸合成酶、脂蛋白脂酶和肝脂酶基因在普安银鲫胚胎发育中的表达及mRNA表达水平及葡萄糖对这4种基因mRNA表达量的影响。试验结果显示,乙酰辅酶A羧化酶α、脂肪酸合成酶、脂蛋白脂酶和肝脂酶基因在普安银鲫的成熟卵期均有表达,且随胚胎发育而上升。在15g/L葡萄糖处理组中,乙酰辅酶A羧化酶α、脂肪酸合成酶、脂蛋白脂酶和肝脂酶从原肠期至出膜前期mRNA表达量升高。在原肠期、晶体出现期和出膜前期,葡萄糖能显著上调脂肪酸合成酶和乙酰辅酶A羧化酶αmRNA的表达(P0.05)。在原肠期,葡萄糖能稍上调脂蛋白脂酶和肝脂酶mRNA的表达,但差异不显著(P0.05)。在晶体出现期和出膜前期,葡萄糖能显著上调脂蛋白脂酶和肝脂酶mRNA的表达(P0.05)。研究表明,乙酰辅酶A羧化酶α、脂肪酸合成酶、脂蛋白脂酶和肝脂酶属母源性酶类,其合子基因在原肠期已经开始表达。适宜水平的葡萄糖促进脂肪酸合成酶和乙酰辅酶A羧化酶αmRNA的表达有利于脂肪酸合成酶和乙酰辅酶A羧化酶α的积累,为仔鱼出膜做准备;促进脂蛋白脂酶和肝脂酶mRNA表达有利于脂质的分解供能。     

草鱼ghrelin基因的分子克隆与组织分布及其摄食调控作用分析

ghrelin是一种在脊椎动物摄食调节过程中起重要作用的脑肠肽,具有明显的摄食促进作用。实验利用同源克隆技术获得了草鱼ghrelin基因的cDNA序列和DNA序列,其中cDNA序列全长506 bp,包括90 bp的5′端非编码区(5′-untranslated region,5′UTR),312 bp的开放阅读框(open reading frame,ORF),以及104 bp的3′端非编码区(3′-untranslated region,3′UTR)。开放阅读框编码的103个氨基酸的ghrelin前体肽,经剪切加工后形成含有19个氨基酸的成熟肽。氨基酸序列分析结果显示,草鱼ghrelin与硬骨鱼类ghrelin相似度最高,而与其他脊椎动物相似度较低,同时草鱼ghrelin成熟肽N端的"活性中心"(active core)为鲤科鱼类中常见的GTSF形式。与大多数硬骨鱼类的ghrelin基因结构相同,草鱼ghrelin基因也包括4个外显子和3个内含子。荧光定量PCR检测到ghrelin mRNA大量分布于草鱼的前肠和脾,脑、肾、肝、肌肉、皮和鳔等组织也有ghrelin mRNA分布。草鱼脑和肠中的ghrelin表达水平在摄食后下降,随着饥饿时间的延长表达水平逐步升高,最后维持在较高水平,表明ghrelin作为摄食启动信号对草鱼的摄食活动起到了促进作用。     

银鲫繁殖中存在的问题及其对策

银鲫在天然水域产卵时，往往因水温低且变幅大等原因，产卵率和孵化率低，如有目的地控制亲鱼的性腺发育，提高催产和孵化的水温，催产时采用高剂量同时注射多种催情药物，并在人工繁殖中掌握好每个技术环节，可以使其产卵率，受精率，孵化率大幅度提高。     

黑鲫、金鲫、银鲫与白鲫的主要鉴别特征

鲫属是亚欧特产的淡水鲤科鱼类。我国记载很早，如《易经》已有“井谷射鲋”句。鲋是鲫在我国的古名，自古人民即很喜爱鲫，爱吃爱养。如1950年初在海南省辉县发掘出战国时期(公元前403—221年)的古墓中，常有鲫鱼被作为祭品，附近淇县在太行山麓产的“淇鲫”(淇县鲫鱼)古己著名．     

雄性金鲫与雌性银鲫交配产生的4倍体银鲫仔鱼及其染色体

在我国的银鲫中，除2体倍外，还有3倍体和4倍体。这一事实，小林等人第一次作了报告。之后，由于观察受精过程中的精核和卵核的行为，以及用其它种或其它亚种的精子，使卵受精，而产生的仔鱼，并观察这些仔鱼的形态，明确了8倍体银鲫的繁殖，是由雌核发育(gynogenesis)而来。     

温度与孔雀石绿浓度对银鲫胚胎发育的影响

温度与银鲫的胚胎发育有着密切的关系：温度升高，胚胎发育速度加快；银鲫胚胎发育的适宜温度为１６℃－３２℃．最适温度为１８℃—３０℃，临界温度为１５℃和３３℃。孔雀石绿对银鲫的胚胎发育有一定的毒害作用．其安全浓度与处理时间有关．     

葡萄糖、维生素C对普安银鲫早期发育的影响

为探究葡萄糖和维生素C（VC）对普安银鲫（Carassius auratus gibelio）早期发育的影响,采用静水溶液浸泡法研究了葡萄糖和VC溶液浸泡中普安银鲫仔鱼出膜时间、孵化率、成活率及卵黄囊仔鱼生长特点。结果显示,葡萄糖溶液质量浓度低于15 g·L^-1,仔鱼出膜时间随着葡萄糖质量浓度的增加而缩短,孵化率、成活率、体质量和全长则升高;葡萄糖溶液质量浓度高于15 g·L^-1对普安银鲫的早期发育表现出抑制作用。VC溶液质量浓度低于30mg·L^-1时,仔鱼出膜时间随着ρ（VC）的增加而降低,孵化率、成活率、体质量和全长则升高。ρ（VC）高于30mg·L^-1对普安银鲫的早期发育表现出抑制作用。研究表明适宜浓度的葡萄糖和VC能促进普安银鲫的早期发育。     

缩骨鲫MSTN基因的克隆与组织表达分析

采用同源克隆、生物信息学方法及荧光定量PCR(Real-time PCR)对缩骨鲫(Carassius auratus sogu var.)肌肉生长抑制素(myostatin,MSTN)基因进行克隆,分析和组织表达状况研究。结果显示:缩骨鲫基因组MSTN基因全长4 737 bp,含有两个内含子和三个外显子;编码区长度为1 128 bp,编码375个氨基酸,包括信号肽(1aa~23aa),TGF-β前肽保守区域(34aa~256aa),TGF-β功能区(281aa~375aa),保守的RXXR蛋白酶酶切位点以及C-端活性区域9个保守的半胱氨酸残基,这与其他物种MSTN相似。此外,缩骨鲫与鲫鱼(Carassius auratus)的MSTN氨基酸序列同源性最高,达到96.1%。以β-actin为内参基因,荧光定量PCR分析表明,MSTN在肌肉中表达量最高;脑、眼、心脏中次之;在肝胰脏、卵巢、肠和肾脏中微量表达。     

分级对银鲫生长和个体大小差异的影响

在室内水槽进行12周的饲养实验以检验分级对银鲫生长和鱼群内个体大小差异的影响。用隔网将实验水槽分为两部分,每个水槽内放养10尾大鱼（体重8.0~9.0 g）和10尾小鱼（体重6.5~7.5 g）,分级时将个体大小相近的10尾鱼分布在隔网同一侧。实验中,3组鱼分别隔1周、2周或4周分级1次,1组鱼不进行分级作为对照。鱼摄食率和生长率随分级间隔时间缩短而降低。实验结束时,隔1周分级1次的鱼体重低于对照组,隔4周分级1次的鱼体重与对照组无显著差异。实验结束时被分级的鱼群中大鱼体重的变异系数高于小鱼,表明大鱼对分级操作更敏感。本实验结果表明,分级未能明显提高银鲫生长速度和降低鱼群内个体大小的差异程度。     

The effects of dietary selenium on growth performances,oxidative stress and tissue selenium concentration of gibel carp (Carassius auratus gibelio)

A 100‐day growth trial was executed to determine the dietary selenium (Se) requirement of juvenile gibel carp (Carassius auratus gibelio). Selenomethionine was supplemented to casein‐gelatin diets at 0, 0.1, 0.25, 0.5, 1.0, 2.5 and 5 mg Se kg^?1, respectively. Each of these seven semi‐purified diets containing 0.34, 0.47, 0.66, 0.82, 1.23, 2.77 and 5.13 mg Se kg^?1 was fed to triplicate groups of gibel carp (2.74 ± 0.02 g) in a flow‐through system. No behaviour abnormalities and no mortality were found in fish exposed to dietary Se concentrations. With the increasing dietary Se supplemented concentrations, weight gain of fish remarkably increased at the levels of ≤1 mg Se kg^?1 diet and then showed no significant difference above 1 mg Se kg^?1 levels. Although growth performances (weight gain, hepatosomatic index, condition factor and survival) were not impaired in gibel carp fed at above the levels of 2.5 mg Se kg^?1, indicators of oxidative stress were changed significantly. Serum superoxide dismutase (SOD) activities significantly declined, hepatic glutathione peroxidase (GPx) activities significantly increased and the tissue Se concentrations significantly raised at the highest supplemented level of 5 mg Se kg^?1. A clear linear relationship between Se‐depended GPx activities and hepatic Se concentrations was observed. The present results indicated that the dietary Se requirement for gibel carp is 1.18 mg Se kg^?1 diet based on weight gain, GPx activities and tissue accumulation.     

异育银鲫病原温和气单胞菌表型及分子鉴定与溶血素基因检测

2008年10月江苏盐城大丰精养异育银鲫（Carassius auratus gibelio) 发生严重疾病,从病鱼肝脏、血液及腹水中分离到优势生长的细菌。对分离做纯培养的4株菌(JY081016-1至JY081016-4)进行形态特征、理化特性等表型生物学性状检验;测定了菌株（JY081016-1）的16S rRNA和gyrB基因序列,分析了16S rRNA和gyrB两种基因序列的同源性,并构建了系统发生树。结果表明分离菌对供试健康异育银鲫有强致病性,菌株（JY081016-1）所扩增的16S rRNA基因序列长度为1448bp（GenBank 登录号：GQ232759）,所扩增的gyrB基因序列长度为1177bp（GenBank 登录号：GQ232760）,其16S rRNA和gyrB基因序列与GenBank数据库中维氏气单胞菌和维氏气单胞菌温和生物型的16S rRNA和gyrB基因序列相似性均在97%以上;根据分离菌的表型及分子特征,判定分离鉴定的4株菌为温和气单胞菌（Aeromonas sobria）。胞外酶及溶血活性检测表明分离菌均能产生蛋白酶、卵磷脂酶、脂酶,在含7%家兔脱纤血液营养琼脂培养基上呈β型溶血;设计的特异性引物可扩增出溶血素基因。     

Partial and total replacement of fishmeal with poultry by-product meal in diets for gibel carp, Carassius auratus gibelio Bloch

Triplicate groups of gibel carp Carassius auratus gibelio Bloch (initial body weight: 4.89 g) were fed for 8 weeks at 24.8–30.8°C with nine isonitrogenous and isoenergetic diets. The control diet (F1) used white fishmeal (FM) as the sole protein source. In the other eight diets (F2–F9), 40.5–100% of FM protein was substituted by poultry by‐product meal (PBM) at 8.5% increments. The specific growth rate (SGR), feed efficiency ratio, protein efficiency ratio, protein retention efficiency and energy retention rate for fish fed PBM diets (F2–F9) were all higher, but not always significantly, than those for fish fed F1. All apparent digestibility coefficients for fish fed PBM diets were lower than those for fish fed F1. Fish fed F1 had a significantly higher hepatosomatic index value than fish fed PBM diets (P<0.05). No significant (P>0.05) effect of diet was found in whole‐body moisture and fat content. Whole‐body protein and energy content for fish fed PBM diets were slightly higher than that for fish fed F1. The optimal replacement level of FM by PBM was estimated by second‐order polynomial regression to be 66.5% in protein.     

方正银鲫微卫星序列的筛选及PCR扩增产物的初步检测

采用小片段克隆法构建了方正银鲫[（Carassius auratus gibelio（Block）]部分基因组文库，并采用非影印的方法将转化子转移到硝酸纤维素滤膜上。用人工合成、并经放射性同位素[γ-^32P]ATP标记的（CA）15探针对构建的基因组文库进行筛选，获得137个阳性克隆，经测序得到微卫星序列130个，其中完美型、非完美型、混合型分别占64．3％、16．9％和18.8％。应用引物设计软件Primer Prmmer5．0进行引物设计，得到引物103对。合成30对引物，随机选取其中5对进行PCR扩增。结果表明，这5对引物均可以稳定扩增，并且观测杂合度均较高。     

生物絮团技术对异育银鲫生长性能和抗性的影响

为研究生物絮团技术(Biofloc Technology,BFT)在沿海滩涂鱼类养殖中的应用效果,本实验以滩涂主要养殖种类——异育银鲫(Carassius auratus gibelio)为研究对象,按照BFT养殖模式(BFT组,不换水,只补存蒸发掉或取样部分的水分)和一般养殖模式(对照组,每日换水1次,每次换水1/4~1/3)分别饲养,测定各处理组异育银鲫的生长指标、消化酶活性和免疫相关酶活性,应用实时荧光定量PCR法定量分析热休克蛋白HSP70的相对表达,人工感染试验对比分析BFT养殖模式组和一般养殖模式组异育银鲫生长性能和抗性的变化。结果显示:(1)BFT组异育银鲫增重率、特定生长率和存活率均高于对照组(P0.01),肥满度、脏体比和肝体比与对照组间无显著差异(P0.05);(2)BFT组异育银鲫肠道中淀粉酶、脂肪酶和胃蛋白酶活性显著高于对照组(P0.05),分别提高了53.10%、28.10%和17.99%;(3)BFT组异育银鲫体表黏液中超氧化物歧化酶活性、血清中总抗氧化能力和溶菌酶活性高于对照组(P0.01);(4)BFT组脾、肾、肝和鳃中热休克蛋白HSP70的表达量分别上调了1.29倍、1.34倍、1.87倍和1.68倍;(5)嗜水气单胞菌(Aeromonas hydrophilia)人工感染试验证实,BFT组异育银鲫抗细菌感染能力显著增强。研究表明,BFT养殖模式适于异育银鲫养殖,可促进鱼体生长,增强其应激能力和抗病力。     

4种环境源性胁迫对异育银鲫血浆生化指标的影响

以异育银鲫(Carassius auratus gibelio)幼苗[(136.70±7.98)g]为实验对象,监测4种环境源性胁迫(高pH 9.2、低溶氧2 mg·L~(-1)、高亚氮2 mg·L~(-1)和高氨氮4 mg·L~(-1))下第3、7、10、15天血浆内皮质醇(COR)、葡萄糖(GLU)、I型干扰素(IFN)、白细胞介素-2(IL-2)、超氧化物歧化酶(SOD)、免疫球蛋白M(Ig M)、补体3(C3)以及丙二醛(MDA)的变化规律。结果显示:COR经高pH、高亚氮和高氨氮胁迫时显著升高,而SOD显著降低,Ig M经高亚氮和高氨氮胁迫时亦显著升高,表明COR、SOD和Ig M可能是广源性的胁迫状态指示指标,COR和Ig M的升高以及SOD活力的降低指示异育银鲫可能处于被胁迫状态。GLU、IFN和C3只经高氨氮胁迫时显著升高,且响应幅度剧烈,表明GLU、IFN和C3可能是灵敏且狭源性的应激指示指标,高水平指示机体可能处于氨氮应激状态。IFN和MDA经高亚氮和高氨氮胁迫时,表现出不同的响应趋势:氨氮胁迫时IFN和MDA显著升高,但亚氮胁迫时则显著降低,表明IFN和MDA响应模式可能具有特异性,不适合指示综合应激状态。     

Effect of partial replacement of dietary monocalcium phosphate with neutral phytase on growth performance and phosphorus digestibility in gibel carp,Carassius auratus gibelio (Bloch)

A feeding trial was conducted to study the effect of partial replacement of dietary monocalcium phosphate (MCP) with neutral phytase on growth performance and phosphorus digestibility in gibel carp, Carassius auratus gibelio (Bloch). Control diet was prepared with 2% MCP but without phytase (P0). Other three experimental diets were prepared by replacement of MCP by 25%, 50% and 75% respectively in comparison with control with supplementation of neutral phytase at 500 U kg^?1 diet in each and designated as P25, P50 and P75 respectively. Gibel carp (initial body weight of 30.22 ± 1.98 g) were reared in twelve 300‐L cylindrical fibreglass tanks provided with filtered flow‐through tap water at 26–28°C. After 8‐week experiment, gibel carp fed with P50 had no obvious differences from the control group on weight gain (WG), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency rate (PER) and survival rate. Phytase supplementation did not affect body compositions or muscle compositions. Crude protein and phosphorus (P) contents in the faeces of fish fed with the phytase‐supplemented diets were significantly lower than those of the control group. The apparent digestibility coefficients (ADCs) of crude protein and P in gibel carp were increased when fish fed with the diets in which MCP was replaced by neutral phytase. This study suggested that partial replacement of dietary MCP at 50% with neutral phytase was considered as a recommended dietary supplemental level and increased dietary P and protein availability.     

Response of gibel carp (Carassius auratus gibelio) to increasing levels of dietary lysine in zero fish meal diets

To investigate the response of gibel carp to dietary lysine levels in zero fish meal (FM) diets, 8 experimental diets were designed. Among them, diet 1 and diet 2 with 150 g/kg FM, methionine, lysine and threonine were supplemented in diet 2. Diet 3 was without FM but matched the amino acid profile of diet 2 except for lysine. Diets 4–8 were based on diet 3, with graded levels of lysine. After 8‐week feeding trail, final body weight, weight gain (WG), specific growth rate, feed conversion ratio (FCR) and feed intake were significantly improved by diets 1–2 and 6–8 compared with diet 3. Plasma glucose, triglyceride, immunoglobulin G and aspartate aminotransferase levels in diets 4–8 were higher than those in diet 1. Apparent digestibility coefficients (ADCs) of protein, energy, methionine, lysine, valine, histidine and phenylalanine in diet 1 were significantly lower than those in diet 6. The highest ADC of isoleucine was found in diet 3. TOR, IGF‐1, JAK3, STAT4, STAT6 and PepT2 mRNA levels were significantly increased with increasing dietary lysine up to a certain level in zero FM diet and lower than diets 1–2. Based on WG and FCR, the optimal lysine requirement of juvenile gibel carp was determined to be 24.4 g/kg and 24.2 g/kg (65.9 g/kg and 65.4 g/kg of dietary protein) in zero FM diet, respectively, using quadratic regression analysis.     

Effect of biofloc technology on water quality and feed utilization in the cultivation of gibel carp (Carassius auratus gibelio var. CAS III)

This study was designed to use sucrose as carbon source to investigate the effect of biofloc technology on water quality and feed utilization in the cultivation of gibel carp (Carassius auratus gibelio var. CAS III). Three isolipidic and isoenergetic diets were formulated containing graded levels of dietary protein (257.4, 300.7 and 353.4 g/kg). The control group (P34) was fed with 353.4 g/kg protein diet without biofloc. Three biofloc treatments (P24 + B, P29 + B and P34 + B) were fed with the three diets with daily sucrose supplementation and continuous air inflation. The results found that the sucrose supplementation could generate biofloc and increased biofloc volume (BFV, p < 0.05). The concentration of total ammonia nitrogen (TAN), nitrate nitrogen (NO₃‐N) and the total phosphorus (TP) of all tanks decreased in biofloc groups, and the total nitrogen (TN) of P24 + B treatment tanks was significantly lower than that of the control group (p < 0.05). The growth of P24 + B and P29 + B group was similar with that of control group (p > 0.05). Feed conversion ratio (FCR) of P29 + B was lower than P24 + B and P34 + B (p < 0.05). Protein retention efficiency (PRE) of P24 + B group was significantly higher than other treatments (p < 0.05). Phosphorus retention efficiency (PhRE) of P24 + B group was the highest among all treatments. Compared with the control group, P29 + B group had an improved total antioxidant capacity (T‐AOC), superoxide dismutase (SOD) activity and contained the lowest malondialdehyde level (MDA). The present results indicated that low‐protein diet is more suitable for gibel carp in zero‐water exchange biofloc system compared with high‐protein diet.