尼罗罗非鱼♀×萨罗罗非鱼♂杂交后代的遗传分离分析

应用微卫星标记对尼罗罗非鱼(Oreochromis niloticus)♀×萨罗罗非鱼(Sarotherodon melanotheron)♂杂交后代(F1、F2)的遗传分离情况进行了初步研究.在86对微卫星引物中,筛选出21对在尼罗罗非鱼和萨罗罗非鱼中存在差异等位基因的特异性引物,共检测出59个等位基因,包含32个尼罗罗非鱼和24个萨罗罗非鱼特异等位基因.F1观测杂合度(Ho=0.998)大于 F2(Ho=0.739), F2有效等位基因数(Ne=2.48)、平均多态信息含量(PIC=0.497)与 F1的(Ne=2.45、PIC=0.489)相似.F1中所有位点均表现为尼罗罗非鱼与萨罗罗非鱼杂合基因型.F2中位点等位基因分离情况各异,通过卡方检验,有18个位点符合孟德尔遗传(P>0.05),2个位点GM145、UNH003表现为偏离孟德尔遗传(P<0.05),1个位点GM276不分离.在18个孟德尔遗传位点中, F2中尼罗罗非鱼特异等位基因频率为48.3%,萨罗罗非鱼特异等位基因频率为47.7%;基因型出现F1型、尼罗罗非鱼型、萨罗罗非鱼型的比例分别为58.2%、19.9%、21.9%.结果表明, F2较好地继承了F1的遗传杂合性,但也存在一定程度的分离.     

尼罗罗非鱼与萨罗罗非鱼正反交鱼自繁后代F2耐盐性、生长性能及亲本对杂种优势贡献力的评估

为选育耐盐和生长兼优的罗非鱼,以原始亲本尼罗罗非鱼(Oreochromis niloticus)和萨罗罗非鱼(Sarotherodon melanotheron)为对照,正交鱼(尼罗罗非鱼♀×萨罗罗非鱼↑)自繁第二代(F2)和反交鱼(萨罗罗非鱼♀×尼罗罗非鱼↑)自繁第二代(F2)为试验对象,观察和比较他们在盐度为0、15、20及25时的耐盐性和生长性能,并估算杂种优势及亲本的贡献力.结果如下:(1)盐度20～25是正、反交鱼的适宜生长盐度,他们在这一盐度范围里的日均增重率为尼罗罗非鱼在盐度0下的75%左右、萨罗罗非鱼在盐度25下的4倍左右,表明杂交后代的生长速度比较接近尼罗罗非鱼,远优于萨罗罗非鱼.在盐度25下,正交鱼比反交鱼生长快7%左右.(2)在盐度15与20下,生长速度和体重变异系数的杂种优势较明显,未发现不同盐度会导致正反交鱼成活率有显著变化.(3)不同亲本鱼类的强势性状,如尼罗罗非鱼的快速生长、萨罗罗非鱼的高耐盐性,对杂交后代的不同性状在不同盐度中有不同的贡献力.运用F1≤a1P1 a2P2所作的估算表明,对杂交后代在15～25盐度下的生长率,尼罗罗非鱼的杂种优势贡献力比萨罗罗非鱼的大3～4倍;对杂交后代在25盐度下的耐盐性,尼罗罗非鱼的贡献力几乎为零,而萨罗罗非鱼的贡献力几乎为100%.     

耐盐罗非鱼育种回交效应评估

对罗非鱼2种回交子代、2种正反交子代(回交鱼的母本)、2种原始种(回交鱼的祖父、母本),共6种遗传型的生长特性、耐盐特性及遗传特征进行了研究.结果表明:(1)在盐度15、20及25时,2种回交子代的日均增重率皆比母本提高了30%～45%,差异显著(P=0.023).(2)SSR分析发现,2种回交子代接受尼罗罗非鱼(Oreochromis niloticus)的遗传特征带分别为87.5%和79.2%,接受萨罗罗非鱼(Sarotherodon melanotheron)的分别为35.2%和41.2%;而2种正反杂交子代接受尼罗罗非鱼的分别为66.7%和41.7%,接受萨罗罗非鱼的分别为47.1%和88.2%.(3)回交子代与原始亲本尼罗罗非鱼的遗传距离较近,同正反交子代相比,回交子代生长性能有所进步,耐盐性能基本保持,扩增的特异性条带数目较少,表明遗传多样性走低和基因型纯合性走强的趋势.(4)两种回交子代间有一定差异,萨尼♀[萨罗罗非鱼♀×尼罗罗非鱼(♀)(F2)]×尼罗罗非鱼(♂)生长快5%,有效等位基因数、平均杂合度、多态信息含量等遗传指标较高,是优选的对象.     

4种遗传型罗非鱼的耐盐慢性驯化表现

在实验室小水体中,对尼罗罗非鱼(Oreochromis niloticus,新吉富)、萨罗罗非鱼(Sarothrodon melanotheron)以及它们的杂交F1和F2("吉丽"罗非鱼)进行盐度梯度实验,观察其耐盐能力。采用4(遗传型)×4(水平或重复)随机区组设计。对照组全程维持淡水养殖;实验组起始盐度为0,盐度每天提高8直至实验鱼100%死亡。在水温保持(27.2±1.3)℃、正常投饵情况下,萨罗罗非鱼、尼罗罗非鱼、杂交F1及F2的50%死亡盐度平均值(MLS)分别为125.78±1.66、54.22±2.51、77.33±1.89和73.73±1.32;萨罗罗非鱼的累计存活率(CS)和MLS值均显著高于尼罗罗非鱼、杂交F1和F2(P<0.05);杂交F1和F2的CS值及MLS值均显著高于尼罗(P<0.05);F2和F1之间CS值和MLS值差异不显著(P>0.05)。     

罗非鱼杂交F1代与亲本的遗传关系及其杂种优势的利用

用ＲＡＰＤ 技术研究奥利亚罗非鱼和尼罗罗非鱼杂交子代和亲本的遗传关系及其在杂种优势中的应用。在所用的１６ 个随机引物中,有１１ 个引物在罗非鱼亲代和杂交子代间呈现多态。遗传相似性指数和遗传距离的计算结果表明,正交子代（ 尼罗罗非鱼♀×奥利亚罗非鱼♂） 在遗传关系上界于亲代之间,不表现出明显的倾向性;而反交子代（ 奥利亚罗非鱼♀×尼罗罗非鱼♂） 却与母本奥利亚罗非鱼极其相似。说明正、反交子代与两亲本遗传相似性有明显的不同,表明遗传性状介于两亲本之间的杂交子代更有可能形成显著的杂种优势。同时发现,扩增片段ＯＰＡ０７１９００ 、ＯＰＡ０７９６０ 、ＯＰＺ１４７２０ 和ＯＰＺ１４６００ 可以作为鉴别尼罗罗非鱼、奥利亚罗非鱼和正交子代的遗传标记。     

新引进萨罗罗非鱼与其它8种罗非鱼耐盐差异

以萨罗罗非鱼、尼罗罗非鱼、以色列红罗非鱼和奥利亚罗非鱼自交和双列杂交后代共9种罗非鱼为实验材料,通过耐盐致死实验、半致死盐度实验和慢性耐盐驯化实验,对萨罗罗非鱼与其他几种罗非鱼耐盐生态特点的差异进行了比较分析,结果表明,(1)萨罗罗非鱼MST为(9.24±1.353)h,ST50为(6.20±1.235)h,MLS-96理论值为30.2,观察值为31.2,远高于另外8种罗非鱼。(2)根据耐盐能力,9种罗非鱼可分为4类:第一类是萨罗罗非鱼,耐盐极强,各项耐盐指标都远高于其他罗非鱼;第二类是以色列红罗非鱼和尼罗罗非鱼♀×奥利亚罗非鱼♂,耐盐强,离差值为正值;第三类是奥利亚罗非鱼、尼罗罗非鱼♀×以色列红罗非鱼♂、以色列红罗非鱼♀×尼罗罗非鱼♂、尼罗罗非鱼♀×奥利亚罗非鱼♂和奥利亚罗非鱼♀×尼罗罗非鱼♂,耐盐较强,离差值为负值;第四类是尼罗罗非鱼,耐盐较弱。(3)当进行慢性耐盐驯化时,萨罗罗非鱼在5d内,死亡率由0达到30%,以后缓慢增长,14～15d有个死亡率的高增长期,然后即进入缓慢增长期;第11天,盐度32.8时,死亡率达到50%,第39天,盐度为102时全部死亡。死亡率与驯化时间之间的线性回归关系为LnY=3...     

慢性盐度胁迫对吉丽罗非鱼(尼罗罗非鱼♀×萨罗罗非鱼♂)及其两亲本耐盐性的影响

为深入研究养殖新品种吉丽罗非鱼[尼罗罗非鱼(♀)×萨罗罗非鱼(♂)]的耐盐性能,对吉丽罗非鱼及其两个亲本尼罗罗非鱼和萨罗罗非鱼进行了慢性盐度胁迫实验,分析了3种罗非鱼耐盐性能的差异,建立了盐度胁迫过程中死亡率与致死盐度及时间的回归模型,结果显示:(1)3种罗非鱼的耐盐能力差异显著,吉丽罗非鱼的耐盐性能接近于萨罗罗非鱼,远高于尼罗罗非鱼,耐盐胁迫时3种罗非鱼的平均致死盐度分别为57.9、66.7和18.5.(2)盐度胁迫实验中尼罗罗非鱼个体间死亡时间差异最大,萨罗罗非鱼个体之间差异最小,吉丽罗非鱼介于其间;吉丽罗非鱼和萨罗罗非鱼死亡时间都有极显著的正态负偏移,离群值和极值较多,尼罗罗非鱼有较显著正态正偏移,只有离群值,没有极值.(3)3种罗非鱼盐度胁迫实验中死亡率与死亡时间及盐度之间的回归关系更适合一元回归,吉丽罗非鱼盐度胁迫实验中死亡率(y)与死亡时间(t)的回归模型为增长模型Y=e (-7.694+0.031t)(R2=0.979),死亡率(Y)与盐度(s)的回归模型为二次模型Y =0.542-0.037s +0.001s2(R2=0.950).     

尼罗罗非鱼（ ♀） × 萨罗罗非鱼（ ♂） F1家系亲权关系微卫星分析

利用尼罗罗非鱼（Oreochromis niloticus）第2代遗传连锁图谱标记,对3组不同尼罗罗非鱼（♀）×萨罗罗非鱼（Sarotherodon melanotheron）（♂）杂交F1家系内亲权关系进行分析。结果显示,86个微卫星位点中共筛选出20个在尼罗罗非鱼、萨罗罗非鱼中存在差异的扩增位点,含13个种间特异性和7个共享带差异位点。尼萨杂交F1中,平均等位基因2.90,平均多态信息含量0.439,位点多态性较高。3个尼萨杂交F1家系组间遗传距离0.362~0.504,组内个体间遗传距离0.245~0.316,组内遗传距离明显小于组间。利用3个种间特异位点组合,可对3个不同家系组父、母本个体进行鉴别。通过对各组亲本与子代位点基因型分析,家系A、B和C组分别使用4、8和12个特异位点组合进行亲权鉴定,累积排除概率分别为99.99%、99.99%、99.91%,家系A、B组分别含3个半同胞家系,家系C组含2对非同胞或4个半同胞家系。     

不同盐度下尼罗罗非鱼、萨罗罗非鱼和以色列红罗非鱼幼鱼生长、成活率及肥满系数的差异

用1m×1m×1m的实验网箱,在盐度分别为0、10、20及30的水体中进行尼罗罗非鱼(Oreochromisniloticus)、萨罗罗非鱼(Sarotherodonmelanotheron)和以色列红罗非鱼(Israeliredtilapia)幼鱼的养殖实验。结果表明:(1)盐度、鱼的种类及盐度-鱼类的交互作用都对3种罗非鱼的生长及体重变异系数有显著影响(P<0 05);3种罗非鱼的成活率和肥满系数只受鱼的种类影响(P<0 05);盐度、盐度-鱼类的交互作用对其都无显著影响。(2)生长和盐度之间的回归关系在尼罗罗非鱼和以色列红罗非鱼较显著,在萨罗罗非鱼显著性较差。(3)尼罗罗非鱼和以色列红罗非鱼的生长随着盐度的降低而加快,萨罗罗非鱼的生长随着盐度的降低而减慢。在3种鱼中,盐度在6 9以下时,尼罗罗非鱼生长最快;盐度7 4～28 7时,以色列红罗非鱼生长最快;盐度高于29 0时,萨罗罗非鱼生长最快。(4)在实验的4种盐度下,萨罗罗非鱼的成活率都高于以色列红罗非鱼和尼罗罗非鱼,而除了在淡水中外,以色列红罗非鱼的成活率又都高于尼罗罗非鱼;萨罗罗非鱼的肥满系数都显著高于尼罗罗非鱼和以色列红罗非鱼,而以色列红罗非鱼的肥满系数和尼罗罗非鱼的没有显著差异。     

尼罗罗非鱼(♀)×萨罗罗非鱼(♂)杂交F_2与F_3群体遗传特征的微卫星分析

利用罗非鱼第二代遗传连锁图谱中微卫星标记对尼罗罗非鱼(Oreochromis niloticus)(♀)×萨罗罗非鱼(Sarotherodon melanotheron)(♂)杂交后代F_2、F_3群体遗传特征进行了初步比较分析。结果显示,28对引物中有22对引物能有效扩增,未观察到杂交后代F_2、F_3中有多倍体个体现象。筛选出7个在尼萨罗非鱼杂交后代F_2、F_3群体中存在差异的位点。杂交F_2群体平均等位基因(Na)为2.71,平均多态信息含量(PIC)为0.466,平均观测杂合度(Ho)为0.632;杂交F_3群体平均Na为2.14,平均PIC为0.370,平均Ho为0.432,杂交F_3遗传杂合性较杂交F_2降低。F_2自繁F_3过程中,F_3群体4个位点的等位基因与F_2完全相同,3个位点的等位基因少于F_2,且F_3群体在2个位点出现完全纯合,杂交F_3群体位点等位基因呈现纯合趋势。研究结果为尼萨罗非鱼杂交世代遗传变异与杂交利用积累基础资料。     

Detection of tilapia lake virus (TiLV) infection by PCR in farmed and wild Nile tilapia (Oreochromis niloticus) from Lake Victoria

Tilapia lake virus disease (TiLVD) has emerged to be an important viral disease of farmed Nile tilapia (Oreochromis niloticus) having the potential to impede expansion of aquaculture production. There is a need for rapid diagnostic tools to identify infected fish to limit the spread in individual farms. We report the first detection of TiLV infection by PCR in farmed and wild Nile tilapia from Lake Victoria. There was no difference in prevalence between farmed and wild fish samples (p = .65), and of the 442 samples examined from 191 fish, 28 were positive for TiLV by PCR. In terms of tissue distribution, the head kidney (7.69%, N = 65) and spleen (10.99%, N = 191), samples had the highest prevalence (p < .0028) followed by heart samples (3.45%, N = 29). Conversely, the prevalence was low in the liver (0.71%, N = 140) and absent in brain samples (0.0%, N = 17), which have previously been shown to be target organs during acute infections. Phylogenetic analysis showed homology between our sequences and those from recent outbreaks in Israel and Thailand. Given that these findings were based on nucleic acid detection by PCR, future studies should seek to isolate the virus from fish in Lake Victoria and show its ability to cause disease and virulence in susceptible fish.     

Tol2-mediated transgenesis in tilapia (Oreochromis niloticus)

The Nile tilapia (Oreochromis niloticus) is an important species in aquaculture and an excellent model system for laboratory studies. Functional genetic analysis using this species has been difficult because existing methods for producing transgenics are inefficient. Here we show that the Tol2 transposon system can be used to create transgenic tilapia with high efficiency. We constructed a line that is transgenic for GFP under control of a Xenopus elongation factor 1α (EF1α) promoter. The germline transmission rate of the Tol2 construct to the first generation was about 30%, which is much higher than conventional methods. GFP expression was strong and ubiquitous in the embryos. Application of the Tol2 system for constructing transgenics in tilapia and related species will promote research in many areas, but will be especially useful for studies of evolutionary developmental biology in the cichlid fishes of East Africa.     

硫酸新霉素在吉富罗非鱼体内的药代动力学及休药期

在实验水温(28±2)℃条件下,按25 mg·kg-1 的剂量对吉富罗非鱼(Genetically improved farmed tilapia,GIFT)单次口灌给药后,采用UPLC-MS/MS法测定吉富罗非鱼组织中的药物水平,研究硫酸新霉素在吉富罗非鱼体内的药代动力学及消除规律.结果表明,血药浓度时间数据符合一级吸收二室开放模型,药物在血浆中达峰时间Tmax、血药浓度高峰Cmax和消除半衰期T1/2β分别为1.299 h、16.138 μg·mL-1 和25.776 4 h.药物消除速度由快到慢依次为:肌肉、肝脏、肾脏,消除半衰期T1/2β分别为31.802 h、34.917 h、45.175 h.选取吉富罗非鱼可食性肌肉组织作为残留检测靶组织,参考中华人民共和国第235号公告中对禽肌肉MRL规定,以0.5 mg·kg-1为残留限量,建议休药期不低于6 d.     

Water-soluble vitamin requirements of tilapia: ascorbic acid (vitamin C) requirement of Nile tilapia, Oreochromis niloticus (L.)

Abstract. The quantitative dietary ascorbic acid requirements of fingerling Nile tilapia, Oreochromis niloticus L., were assessed by feeding seven diets containing graded supplements of this vitamin (0, 50, 75, 100, 125, 300 and 400mg/100g dry diet) to triplicate groups of fish for 12 weeks. Fish fed the diet devoid of ascorbic acid exhibited significantly ( P < 0·01) poorer growth and higher condition factor. Food conversion and protein efficiency ratios were improved with increasing dietary ascorbic acid level up to 125mg/100g of dry diet. Protein utilization, haematocrit, haemoglobin level and tissue ascorbate concentrations were depressed in fish fed the ascorbic-acid-free diet. Tissue ascorbate concentrations, especially for liver, were highly correlated with dietary ascorbic acid level. Fish fed the ascorbic-acid-free diet exhibited deficiency signs including erratic and convulsive swimming, anorexia, lethargy, caudal fin erosion, skin haemorrhages and mortality. Based on the nutritional and pathological parameters investigated, the recommended dietary inclusion level is 125mg/100g dry diet, which is equivalent to a net requirement (after processing and storage) of 42mg/100g diet.     

吉富罗非鱼成鱼胆碱的最适需要量

选用初始体质量为(220.00±8.34) g的吉富罗非鱼(Oreochromis niloticus)360尾, 随机分成6组,每组3重复(每重复20尾), 于1 m×1 m×1.5 m池塘网箱中饲养。分别饲喂胆碱含量为97.80(对照组)、375.04、565.74、974.27、      1 409.81、1 824.35 mg/kg的半纯化饲料10周, 研究胆碱对吉富罗非鱼成鱼生长、饲料利用、鱼体营养组成、胆碱蓄积量及部分血液生化指标的影响。结果显示, 经过10周的饲喂, 饲料中添加胆碱可显著提高鱼体增重率、特定生长率和饲料效率(P<0.05); 降低肝脂肪含量(P<0.05), 提高肌肉脂肪含量(P<0.05); 显著升高肝胆碱蓄积量(P<0.05); 胆碱添加组血清甘油三酯(TG)和总胆固醇(T-CHO)显著高于对照组(P<0.05), 并随饲料胆碱含量增加呈现升高的趋势; 肝甘油三酯(TG)和总胆固醇(T-CHO)随着胆碱含量的增加而显著降低(P<0.05); 血清谷草转氨酶(ALT)、谷丙转氨酶(AST)和碱性磷酸酶(ALP)均随着饲料胆碱含量的增大而显著降低(P<0.05)。结果表明, 饲料中添加适量的胆碱可以改善吉富罗非鱼成鱼的生长性能, 提高饲料利用效率, 降低肝脂肪含量, 促进肝脂肪转运; 对特定生长率进行回归分析, 得出吉富罗非鱼成鱼对饲料中胆碱的最低需要量为506.43 mg/kg, 而对肝胆碱蓄积量回归分析得出的需要量为981.38 mg/kg。

     

Dietary conjugated linoleic acid (CLA) for finishing Nile tilapia

A 8‐week feeding trial was conducted to evaluate the nutritional value of dietary conjugated linoleic acid (CLA) for finishing Nile tilapia evaluating its effects on growth performance, whole body and filet compositions, fatty acid composition, plasma lipid parameters, filet texture, liquid loss and holding capacity. Triplicate groups of Nile tilapia were hand‐fed until apparent satiation on diets in which CLA oil was supplemented at 0 (control), 5 or 10 g kg^?1. No differences were observed on weight gain, feed intake, feed/gain ratio, protein efficiency ratio, hepatosomatic index, visceral fat, filet yield and survival between treatments. Fish‐fed CLA diets had increased whole body crude protein and reduced filet lipid content. The total plasma cholesterol and triacylglycerol decreased with CLA supplementation. Neither 5 nor 10 g kg^?1 dietary CLA improved liquid‐holding capacity and filet texture when compared with fish fed the control diet. Fish‐fed dietary CLA showed lower whole body and filet C18:3(n‐6), C18:3(n‐3) and C20:2(n‐6) levels and higher levels of C18:0. CLA deposition in filet and whole body (16 mg–235 mg g^?1 of tissue) are higher than presented in natural sources of CLA, proving to be a great product to increase CLA consumption by humans.     

Growth performance and feed utilization of Nile tilapia Oreochromis niloticus (Linnaeus, 1758) and tilapia galilae Sarotherodon galilaeus (Linnaeus, 1758) fingerlings fed plant protein-based diets

This study was designed to determine the effect of complete substitution of fish meal (FM) by three plant protein sources including extruded soybean meal (SBM), extruded full‐fat soybean (FFSB) and corn gluten meal (CGM) on growth and feed utilization of Nile tilapia Oreochromis niloticus and tilapia galilae Sarothrodon galilaeus. Four isonitrogenous of crude protein (ca. 28.0%) and isocaloric (ca. 19 MJ kg⁻¹) experimental diets were formulated. The control diet (diet 1) was prepared with FM as the main protein sources. Diets 2–4, each FM control diet, were completely substituted with SBM (diet 2), FFSB (diet 3) and CGM (diet 4). l ‐lysine and dl ‐methionine were added to plant protein diets to cover the nutritional requirements of tilapia. Each treatment was allocated to three net pens and fed for 17 weeks. Nile tilapia fed the control diet showed significantly higher (P≤0.05) values for final body weight (FBW), feed intake (FI), weight gain (WG) and specific growth rate (SGR), whereas fish fed the diet with CGM achieved the lowest values. Tilapia galilae fed SBM diet recorded the highest (P≤0.05) values for growth performance. Better feed conversion ratio (FCR) for both Oreochromis niloticus and Sarothrodon galilaeus was observed when fish were fed SBM diet, whereas the worse FCR was recorded for FFSB diet. Feed utilization parameters including protein productive value (PPV), fat retention (FR) and energy retention (ER) showed significant differences (P≤0.05) for both the species fed different dietary protein sources. The present results suggest that, for Nile tilapia, both SBM and FFSB supplemented with dl ‐methionine and l ‐lysine can completely replace dietary FM. Meanwhile, S. galilaeus fed SBM diet exhibited comparable growth and feed utilization with those fish fed a fish‐meal‐based diet.     

吉富罗非鱼生长过程中氮收支变化的研究

为充实有关鱼类研究数据,提供实际生产理论依据,研究了吉富品系尼罗罗非鱼（Oreochromis niloticus）从鱼种（18 g）生长至成鱼（180 g）过程中氮收支变化。试验为期73 d,每日饱食投喂并收集鱼粪,溶解氧质量浓度为8.0-6.0 mg·L-1,pH为 7.5-6.5,水温为24-30 ℃。在鱼均质量达到50 g、100 g和180 g时测定并计算当前生长阶段氮收支。结果表明,生长氮比例在养殖初期最高（64%）,养殖中期最低（47%）;粪氮比例在养殖中期最高（9%）,养殖初期和末期分别为5%和4%;排泄氮比例随鱼的生长而逐渐增加。此外,试验期间水中总氮增加速度在养殖中期减慢,养殖末期再次加快。