饲料中不同脂肪酸对草鱼组织脂质含量和脂肪酸构成的影响

实验选用初始作重约1．4g的草鱼，在60天的饲养期中分别投喂1种不含脂肪的饲料和8种含3％不同脂质的饲料，观察了饲料中添加油酸（18：1n-9）、亚油酸（18：2n－6）、亚麻酸（18：3n－3）和n－3系列高度不饱和脂肪酸（n－3HUFA；20：5n－3＋22：6n－3）对草鱼肝胰脏、肌肉、头部和去肝胰脏内脏脂质含量和脂肪酸构成的影响。实验结果表明，与摄食单纯含油酸饲料的草鱼相比，含1％18：2n－6和1％18：3n－3或1％18：2n－6和0．5％n－3HUFA饲料组草鱼的肝胰脏脂质含量显著降低，但内脏脂质含量升高。向饲料中添加18：2n－6、18：3n－3和n－3HUFA能明显影响草鱼肝胰脏、肌肉和头部脂质多不饱和脂肪酸的相对含量；组织中n－6系列和n－3系列高度不饱和脂肪酸含量分别与饲料中添加18：2n－6和18：3n－3有相关关系，表明草鱼具有生物转化18：2n－6和18：3n－3为高度不饱和脂肪酸的能力。     

不同蛋白质源对草鱼和花鲈肉质影响的比较研究

本试验旨在比较不同蛋白质源对草鱼(Ctenopharyngodon idellus)及花鲈(Lateolabrax japonicas)体成分、肌肉游离氨基酸组成及鱼片质构特性的影响。设计草鱼和花鲈饲料各3种,分别等氮等能。3种草鱼试验饲料分别为以鱼粉为唯一蛋白质源的CI-FM组(对照组),以混合植物蛋白质(豆粕和大豆浓缩蛋白)替代80%鱼粉的CI-PPB80组及以混合植物蛋白质替代100%鱼粉的CI-PPB100组。3种花鲈试验饲料分别为以鱼粉为唯一蛋白质源的LJ-FM组(对照组),以混合植物蛋白质(棉籽浓缩蛋白和大豆浓缩蛋白)替代50%鱼粉的LJ-PPB50组和以混合植物蛋白质替代100%鱼粉的LJ-PPB100组。草鱼初始体重为(153.40±0.30)g,随机分为3组,每组3个重复,每个重复20尾鱼;花鲈初始体重为(12.97±0.03)g,随机分为3组,每组4个重复,每个重复25尾鱼。饲喂8周后,检测2种试验鱼的体成分、肌肉游离氨基酸含量及鱼片质构特性、剪切力、滴水损失及胶原蛋白含量。结果表明:与CI-FM组相比,CI-PPB100组草鱼肌肉粗脂肪含量显著降低(P0.05),粗灰分含量显著升高(P0.05);而花鲈肌肉粗脂肪含量则随替代水平的升高呈现先上升后下降的趋势,表现为LJ-PPB50组显著高于LJ-PPB100组(P0.05)。草鱼肌肉总游离氨基酸及生物胺前体含量各组间无显著差异(P0.05);随着替代水平的升高,花鲈肌肉总游离氨基酸及呈味氨基酸含量显著降低(P0.05),生物胺前体含量显著升高(P0.05)。2种试验鱼肌肉中存在各自的特异性成分。2种试验鱼生、熟鱼片的质构特性差异较大。草鱼的CI-FM组和花鲈的LJ-FM组生鱼片的硬度、黏性、咀嚼力、回弹力及剪切力均分别显著高于同鱼种的其他2组(P0.05)。对于熟鱼片,草鱼的CI-PPB80组内聚力显著高于其他2组(P0.05),其他各项指标各组间无显著差异(P0.05);花鲈的LJ-PPB100组硬度及黏附性显著高于LJ-FM组(P0.05),其他各项指标各组间无显著差异(P0.05)。根据结果,混合植物蛋白质替代鱼粉后降低了草鱼和花鲈鱼体脂肪的蓄积,对草鱼肌肉总游离氨基酸含量不造成影响,使花鲈肌肉呈味氨基酸含量下降,必需氨基酸含量减少,生物胺前体含量升高,货架期缩短;高植物蛋白质源饲料使得草鱼和花鲈的肉质均出现明显下降,而高鱼粉饲料可以保证2种试验鱼有较高的肉质。     

The megalocytivirus-induced protein CsMig1 enhances Cynoglossus semilaevis resistance against viral infection

Half-smooth tongue sole (Cynoglossus semilaevis) is an important economic fish species cultured in northern China. In this study, we identified and analyzed the expression and function of a megalocytivirus-induced gene, CsMig1, from tongue sole. The deduced amino acid sequence of CsMig1 is composed of 507 residues and contains no conserved domains. Blast analysis identified no close homologues of CsMig1. CsMig1 shares moderate sequence similarities in the N-terminal region with the Gig1 (i.e., grass carp hemorrhagic virus-induced gene) homologues of several teleost species. Quantitative real time RT-PCR analysis showed that constitutive CsMig1 expression occurred, in increasing order, in heart, spleen, muscle, kidney, liver, gill, and gut. Experimental infection with the viral pathogen megalocytivirus upregulated CsMig1 expression in kidney, spleen, and liver in time-dependent manners. Treatment of head kidney lymphocytes with the culture supernatant of megalocytivirus-stimulated cells significantly enhanced CsMig1 expression. When head kidney lymphocytes were transfected with the plasmid that constitutively expresses CsMig1, the cells exhibited significantly increased ability to resist megalocytivirus infection. Taken together, these results indicate that CsMig1 is a virus- and, possibly, interferon-induced novel immune factor that functions in the antiviral immunity of tongue sole.     

Molecular cloning of type I collagen cDNA and nutritional regulation of type I collagen mRNA expression in grass carp

Grass carp (Ctenopharyngodon idellus) are important Chinese freshwater fish, and in China, the faba bean has been used as the sole food source for grass carp to transform them into crisp grass carp. Because of this, crisp grass carp has become an economically important fish because of its increased muscle hardness. To study the nutritional regulation of type I collagen in faba bean‐fed grass carp, we isolated type I collagen alpha 2 (COL1A2) on the basis of our isolation of COL1A1. The COL1A2 cDNA was found to be 4899 bp in length and included a 4059‐bp coding sequence (CDS) and encoded a polypeptide of 1352 AA. The protein peptide molecular weight was 127.39 kD, and the theoretical isoelectric point was 9.37. The COL1A2 protein possessed five α‐helixes, eight β‐sheets, 16 regions of triple helical repeats, 21 low‐complexity regions, 10 function domains and two zinc‐binding sites; however, no calcium‐binding sites were observed. The mRNA expression of COL1A1 and COL1A2 was assessed in eight tissues (muscle, hepatopancreas, intestine, gills, skin, fin, kidney and spleen) from grass carp and crisp grass carp by semi‐quantitative RT‐PCR. Expression of COL1A1 in the muscle, intestines and skin of crisp grass carp was higher than that in grass carp, and expression of COL1A2 in the muscle, gills, fin and skin of crisp grass carp was higher than that in grass carp. In the muscle of crisp grass carp, expression of COL1A1 and COL1A2 was higher than that in grass carp, which was further confirmed by real‐time PCR, and collagen content also was enhanced. These results demonstrated that type I collagen was closely related to the increased muscle hardness of faba bean‐fed grass carp.     

Identification and characterization of Japanese flounder, Paralichthys olivaceus interferon-stimulated gene 15 (Jf-ISG15)

Previously, using cDNA microarray analysis, we demonstrated that an EST clone of Japanese flounder (Paralichthys olivaceus) with homology to mammalian interferon-stimulated gene 15 (ISG15) was strongly induced by treatment with DNA vaccine encoding the glycoprotein gene of Hirame rhabdovirus (HIRRV). In this study, we conducted molecular cloning and expression analysis of the Japanese flounder ISG15 (Jf-ISG15). Jf-ISG15 encoded two exons. The first exon was non-coding, while the second exon encoded a protein of 158 amino acids. The coded protein has two tandem ubiquitin-like domains with a carboxyl-terminus conjugation motif “LRLRGG”. Phylogenetic analysis revealed an evolutionary relationship among Jf-ISG15, mammalian and fish ISG15 orthologues. The interferon-stimulated response element (ISRE) sites were conserved among DNA sequences of Jf-ISG15 and mammalian ISG15 promoter regions. An RT-PCR analysis of healthy tissues showed that Jf-ISG15 mRNA was notably strongly expressed in gills, PBLs and spleen. Expression of Jf-ISG15 was strongly induced by poly-I:C treatment in head-kidney cells, peripheral blood leukocytes (PBLs) and spleen cells, and by HIRRV infection in kidney of juvenile fish suggesting that Jf-ISG15 plays a role in fish antiviral response.     

Molecular cloning and characterisation of a homologue of the alpha inhibitor of NF-kappaB in the griffon vulture (Gyps fulvus)

NF-kappaB has been found to play roles in many different compartments of the immune system during differentiation of immune cells and development of lymphoid organs and during immune activation. The activity of NF-kappaB is primarily regulated by a family of structurally related proteins known as the IkappaB proteins. Herein, we report the molecular cloning and characterisation of a griffon vulture (Gyps fulvus) orthologue of the alpha inhibitor of NF-kappaB (IkappaBalpha). The full-length cDNA consists of 1553 bp with an ORF encoding a 313 amino acids protein (GenBank accession number EU161944). The putative G. fulvus IkappaBalpha protein (Gf-IkappaBalpha) possesses the characteristic organization of the mammalian IkappaBalpha proteins. Gf-IkappaBalpha contains an N-terminal signalling receiver domain, a central ankyrin repeat domain, required for its interaction with NF-kappaB, and a putative PEST-like sequence in the C-terminus. Quantitative real-time RT-PCR (qRT-PCR) analysis indicated that Gf-IkappaBalpha mRNA levels were higher in vulture heart, lung, artery and PBMC cells than in small and large intenstine and kidney. The predicted amino acid sequence of Gf-IkappaBalpha was 73% identical to human IkappaBalpha, and 91% identical to chicken IkappaBalpha. These results confirm the existence of the NF-kappaB signalling pathway in vulture and suggest a similar functional interaction between IkappaBalpha and NF-kappaB. Based on the results and the homology to the vertebrate NF-kappaB cascade, these studies help to highlight a potentially important regulatory pathway for the study of the related functions in vulture immune system.     

草鱼幼鱼对饲料中核黄素的需要量

配制核黄素含量分别为0.54、2.32、4.08、5.78、9.28和19.35 mg/kg的6种纯化饲料,投喂初始均重为(11.21±0.16)g的草鱼幼鱼8周,通过研究核黄素对其生长性能、肝胰脏中D-氨基酸氧化酶(D-AAO)与肠道中消化酶活力及体成分的影响,以确定草鱼幼鱼对饲料中核黄素的需要量。每种饲料设3个重复,每个重复放养20尾鱼。结果表明:0.54和2.32 mg/kg组草鱼幼鱼的成活率显著低于其他各组(P0.05);随着饲料中核黄素含量的增加,草鱼幼鱼的增重率、特定生长率、饲料效率、肝胰脏中D-AAO及肠道中消化酶活力均先升高后趋于稳定,当饲料中核黄素含量为5.78 mg/kg时,以上指标均达到最大值;5.78 mg/kg组的肝体比显著高于0.54 mg/kg组(P0.05),但饲料中核黄素含量对脏体比、肥满度及全鱼的水分、粗蛋白质、粗脂肪和粗灰分含量无显著影响(P0.05)。折线模型回归分析表明,草鱼幼鱼获得最佳生长时对饲料中核黄素的需要量为5.54 mg/kg;肝胰脏中D-AAO活力达到最佳时对饲料中核黄素的需要量为5.99 mg/kg。     

五倍子预防草鱼细菌性烂鳃病效果的研究

五倍子能预防鱼类烂鳃病,但作用剂量不明确。为探明五倍子的推荐使用剂量,该研究从体内、体外两方面考查了不同剂量五倍子对病原菌抑制效果和饲料中添加不同剂量五倍子对草鱼烂鳃病预防效果的影响。将五倍子煎液以40、60、80、100 mg/kg剂量加入Shieh琼脂选择培养基,涂布柱状黄杆菌,培养72 h并计数,计算抑菌率;选择健康的草鱼幼鱼240尾,分为4个组,对照组饲喂基础饲料,3个试验组分别添加1%、2%、4%五倍子进行投喂,用柱状黄杆菌悬液向鱼缸连续泼洒3 d攻毒,饲养56 d后测定血清、肠、肝胰脏溶菌酶活力及血清补体C3、IgM含量。结果表明,80~100 mg/kg的五倍子抑菌效果明显,100 mg/kg能完全抑菌;2%五倍子剂量组肝胰脏溶菌酶活力显著（P<0.05）高于对照组,因此,五倍子在草鱼饲料中推荐添加剂量为2%。     

猪Toll样受体9基因的克隆、序列分析及其结构预测

本研究应用反转录-聚合酶链式反应(RT-PCR)扩增技术,从猪脾脏淋巴细胞中,克隆了猪Toll样受体9基因(pTLR9).基因序列分析表明,克隆的pTLR9基因ORF为3 093 bp,编码1 030个氨基酸,含18.5%的亮氨酸,含有24个氨基酸的信号肤序列,属于Ⅰ型跨膜受体,具有富含亮氨酸的重复序列(LRR)和Toll/IL-1R同源区结构域;与GenBank上登载的pTLR9参考序列(AY859728)的同源性为99.3%,与牛、马、羊和人的同源性较高,与家鼠、褐鼠的次之,TLR9的演化关系与亲缘关系密切.     

草鱼幼鱼对缬氨酸需要量的研究

试验选用体重(9.5±0.3)g的草鱼幼鱼,随机分成6个组,每组3个重复,每个重复20尾;以花生仁粕、酪蛋白及明胶为蛋白质源,分别饲喂缬氨酸水平为7.3～22.3 g/kg的6组等氮半精制饲料(粗蛋白质含量为32%),经70 d的生长试验研究其日粮缬氨酸的最适需求量.试验结果表明:日粮缬氨酸水平对草幼鱼的增重率、特定生长率、蛋白质效率、肌肉RNA/DNA比率、鱼体常规成分组成、消化酶活性、肝胰脏谷草-谷丙转氨酶和谷氨酸脱氢酶活性以及血液学指标都有显著的影响(P<0.05).随日粮缬氨酸水平的增加,增重率,特定生长率、蛋白质效率及肌肉RNA/DNA比率均呈先升后降趋势,均在16.3 g/kg组达到最大值(P<0.05).以增重率、特定生长率、肌肉RNA/DNA比率为基础对饲料缬氨酸水平进行二次回归分析,确定草鱼幼鱼日粮(32%粗蛋白质)缬氨酸适宜需要量分别为15.6、15.1及16.0 g/kg(日粮基础)或48.8、47.2及50.0 g/kg(日粮蛋白质基础).     

Growth,digestive and absorptive capacity and antioxidant status in intestine and hepatopancreas of sub-adult grass carp Ctenopharyngodonidella fed graded levels of dietary threonine

Background:This study was carried out to investigate effects of threonine levels on growth,digestive and absorptive capacity and antioxidant status in intestine and hepatopancreas of sub-adult grass carp(Ctenopharyngodonidella).Results:Weight gain,specific growth rate,feed intake and feed efficiency were significantly improved by dietary threonine(P0.05).Intestinal activities of trypsin,chymotrypsin,alpha-amylase,lipase,alkaline phosphatase,y-glutamyl transpeptidase and creatine kinase took the similar trends.Contents of malondialdehyde and protein carbonyl in intestine and hepatopancreas were significantly decreased by dietary optimal threonine supplementation(P0.05).Anti-superoxide anion capacity,anti-hydroxyl radical capacity,glutathione content and activities of superoxide dismutase,catalase and glutathione-S-transferase in intestine and hepatopancreas were enhanced by dietary threonine(P 0.05).Conclusions:Dietary threonine could improve growth,enhance digestive and absorptive capacity and antioxidant status in intestine and hepatopancreas of sub-adult grass carp.The dietary threonine requirement of sub-adult grass carp(441.9-1,013.4 g) based on weight gain was 11.6 g/kg diet or 41.5 g/kg of dietary protein by quadratic regression analysis.     

草鱼呼肠病毒研究进展

由草鱼呼肠病毒引起的草鱼出血病是一种高度传染性与致死性的病毒性疾病,该病的频繁暴发给我国草鱼养殖业及淡水养殖业造成了巨大的经济损失。论文就近年来对草鱼呼肠病毒在基因组序列及基因分型、流行病学、先天性免疫、检测方法、免疫防控等方面的研究进行综述,以期为草鱼呼肠病毒的研究提供借鉴和参考,为草鱼出血病预防和控制提供帮助。     

草鱼→鲤的细胞核移植

用草鱼的头肾组织细胞核、草鱼的头肾细胞核与鲤鱼成熟去核卵之杂种胚胎细胞核、草鱼的囊胚细胞核,分别与鲤鱼的去核卵细胞质配合进行核移植操作.结果,体细胞核移植,在亚科间的组合——草鱼核 鲤质中获得原肠期胚胎,得率为1%;体细胞继代核移植,在草鱼核 鲤质中获得胚孔封闭期胚胎,得率为5%;胚胎细胞核移植,在草鱼核 鲤质中获得眼球色素出现期胚胎,得率为2%.草鱼→鲤核-质杂种胚胎的胎盘形状、大小与鲤鱼的相似,而大于草鱼;胚盘细胞数量也多于草鱼胚胎同期胚盘,而类似于鲤鱼同期胚盘.     

鸭RIG-1基因的PCR扩增及其序列分析

维甲酸诱导基因1(RIG-1)是细胞质中侦测病原相关分子模式的识别受体。论文旨在扩增北京鸭RIG-1编码基因,并对其进行序列分析。通过PCR方法,从北京鸭脾脏中扩增得到RIG-1基因cDNA,并使用LaserGene分子生物学分析软件进行序列分析。结果发现北京鸭的RIG-1基因cDNA开放阅读框全长2 802bp,编码933个氨基酸。结构预测发现鸭RIG-1结构与哺乳动物类似,N端有两个串联CARD区,中间为DExD/H解旋酶区,C端为抑制区,各功能区起重要作用的氨基酸较为保守。同源性及进化树分析发现鸭RIG-1与鹅和斑马鱼的同源性最高分别为93.7%、78.4%,与哺乳动物同源性高于50%,与其他鱼类的同源性低于50%。成功扩增出北京鸭RIG-1基因cDNA编码序列,为鸭RIG-1的深入研究奠定了基础。     

Characterization of the immunoglobulin A heavy chain gene of the Atlantic bottlenose dolphin (Tursiops truncatus)

Immunoglobulin constant region heavy chain genes of the dolphin (Tursiops truncatus) have been described for IgM and IgG but not for IgA. Here, the heavy chain sequence of dolphin IgA has been cloned and sequenced as cDNA. RT-PCR amplification from blood peripheral lymphocytes was carried out using degenerate primers and a single sequence was detected. The inferred heavy chain structure shows conserved features typical of mammalian IgA heavy chains, including three constant (C) regions, a hinge region between constant region domain 1 (C1) and constant region domain 2 (C2), and conserved residues for interaction with the Fc alpha R1 and N-glycosylation sites. Comparisons of the deduced amino acid sequences of the IgA heavy chain for the dolphin and the evolutionarily related artiodactyl species showed high similarity. In cattle and sheep, as in dolphins, a single IgA subclass has been identified. Southern blot analysis as well as genomic PCR confirmed the presence of multiple IGHA sequences suggesting that IGHA pseudogenes may be present in the dolphin genome.