Apelin and apj in common carp (Cyprinus carpio L.): Molecular characterization,tissue expression and responses to periprandial changes and fasting–refeeding

Apelin and its receptor apj have a variety of biological functions and are involved in appetite and energy regulation. However, the comparative studies of apelin/apj system in fish are still absent. In this study, the open reading frames of apelin and apja and apjb were obtained. Apelin and apj mRNAs were widely distributed in all tissues detected, with the highest level in the brain, followed by the spleen, kidney and foregut. The mRNA expression of apelin and apjs increased rapidly at mealtime and significantly decreased after 30 min of feeding. In addition, there were obvious circadian rhythms (higher at nighttime and lower at daytime) in the expression of apelin and apja and apjb. Furthermore, fasting elevated the mRNA levels of apelin and apjs in the hypothalamus, foregut and hepatopancreas. When the 8‐day fasted fish were refed, apelin and apjs mRNA expression significantly decreased. However, there were differences in the response to fasting and refeeding between apja and apjb. The roles of apelin and its receptor apjs in fish are poorly understood. Herein, we employed molecular and physiological studies to characterize their structures, distribution and mRNA expression responses to periprandial changes and fasting–refeeding in Cyprinus carpio L. for the first time.     

中草药对鲤不同组织溶菌酶活性的影响

中草药具有增强机体免疫、抗氧化和抗疲劳等作用。在水温23℃下,给饲养在水族箱中平均体质量85.88±2.45g的鲤（Cyprinus carpio L）投喂添加了复方中草药AB（由甘草等组成）、CD（由茵陈、板蓝根等组成）的配合饲料和一个对照组,连续饲喂28d后,停食一天,采集实验鱼血清和组织（肝胰脏、脾、鳃）样本,测定溶菌酶含量。结果显示,29d时,CD组血清及各组织溶菌酶含量均与对照组有明显差异（P〈0.05）,AB组除肝胰脏组织外,血清和其余组织中的溶菌本科含量也和对照有显著性差异（P〈0.05）,但两种中草药之间溶菌本科含量除肝胰脏组织外无显著差异（P〉0.05）。提示这两种复方中药均有不同程度促进鱼类免疫力的作用。     

中草药对鲤不同组织溶菌酶活性的影响

中草药具有增强机体免疫、抗氧化和抗疲劳等作用。在水温23℃下,给饲养在水族箱中平均体质量85.88±2.45g的鲤（Cyprinus carpio L）投喂添加了复方中草药AB（由甘草等组成）、CD（由茵陈、板蓝根等组成）的配合饲料和一个对照组,连续饲喂28d后,停食一天,采集实验鱼血清和组织（肝胰脏、脾、鳃）样本,测...     

阿克曼菌对鲤糖代谢的调控作用研究

[目的]本试验旨在探究巴氏灭活的阿克曼菌（Pasteurized Akkermansia muciniphila，P-Akk）调控鲤（Cyprinus carpio L.）糖代谢的分子机制。[方法]本研究通过不同糖水平（20%、30%、40%、50%葡萄糖）试验，探究鲤（10.5±1 g）4周和8周时胰高血糖素样肽-1（glucagon-like peptide-1，GLP-1）分泌及阿克曼菌（Akkermansia muciniphila，Akk）定植的时空变化；在不同糖水平试验基础上，设置40%葡萄糖和三个不同浓度P-Akk（108 cfu/g P-Akk、109 cfu/g P-Akk、1010 cfu/g P-Akk）组，探究添加P-Akk 4周后对鲤（16.38±0.39 g）糖代谢及GLP-1的调控；通过P-Akk孵育鲤原代肝、肠细胞试验，共同探究不同糖水平下鲤肠道Akk、GLP-1对糖代谢的调控机制。[结果]结果显示：（1）随着饲料中葡萄糖含量的升高，鲤血清中GLP-1b含量显著降低（P < 0.05），4周时高糖组鲤肠道GLP-1a、GLP-1b以及肠道内容物中Akk丰度均显著降低（P < 0.05），但8周时无显著性差异（P > 0.05）。（2）P-Akk处理后，鲤血清中葡萄糖、GLP-1含量显著减少（P < 0.05）；中肠绒毛高度、肌层厚度显著增加，GLP-1含量减少，短链脂肪酸含量增加，muc2 mRNA表达水平升高（P < 0.05）；此外，P-Akk上调肝脏中ampk、pi3k、akt及糖酵解基因gk、pfk mRNA表达量，下调糖异生基因pepck mRNA表达量（P < 0.05）。（3）原代肝、肠细胞孵育结果显示，P-Akk处理肠细胞后GLP-1含量显著减少（P < 0.05），而gpr40的mRNA表达量升高；此外肝细胞中糖酵解基因gk、pfk mRNA表达量显著升高。[结论]综上，外源添加P-Akk可以缓解高糖饲料引起的血糖升高，维持葡萄糖稳态。[意义]该研究结果可为Akk作为益生菌在水产饲料中的应用提供理论基础。     

鲤免疫应答相关基因的克隆与鉴定

为研究鲤(Cyprinus carpio L.)白细胞免疫应答相关的分子机理,以体外培养的鲤外周血白细胞为实验材料,用荧光标记的mRNA差异显示(FluoroDDRT-PCR)技术,研究丝裂原(50μg/mL LPS、50μg/mL PHA和50μg/mL ConA)在刺激白细胞4、12和24 h内诱导白细胞免疫应答相关基因的mRNA表达差异,共获得92个差异片段,其中87个片段有再扩增产物,再扩增率为94.6%;将差异片段克隆,经PCR鉴定,获得81个阳性克隆,鉴定率为93.1%;差异片段序列同源性功能分析结果表明,本研究共获得3个免疫应答相关的cDNA克隆,它们分别编码鲤的蛋白酶体激活因子PA28α亚基、翻译延伸因子(EF-1α)和基质金属蛋白酶13(Mmp13)部分序列,为进一步研究这些差异表达基因在鱼类免疫中的作用机制奠定了基础。     

显微介导的远缘基因渐渗技术在鲤育种中的应用

以中国明对虾(Fenneropenaeus chinensis)为外源DNA供体,运用显微介导的远缘杂交技术,将中国明对虾总DNA直接导人鲤(Cyprinus carpio L.)受精卵内,创建鲤外源DNA导人系,并利用AFLP分子标记技术和肌肉营养成分分析,对显微介导中国明对虾基因鲤进行外源DNA检测与蛋白质和氨基酸含量进行测定,结果表明:(1)在30对AFLP引物中,有8对所扩增带,其显微介导中国明对虾基因鲤亲本和子代中都有和中国明对虾基因相同而对照鲤没有的带.说明受体鱼中,肯定有一段序列与目的基因序列一致,研究证明了亲缘关系甚远的异源DNA片段的超远缘杂交的可行性.(2)导人中国明对虾总DNA片段的显微介导中国明对虾基因鲤的蛋白质含量分别为18.37%,18.47%,17.99%,均高于对照组(17.06%);而氨基酸的总量分别为17.16%,17.56%,16.92%,也高于对照组((16.13%),其中天门冬氨酸、谷氨酸、甘氨酸、丙氨酸等4种鲜味氨基酸含量明显超过对照组,经数理统计分析,均有显著差异(P<0.05).说明直接导人中国明对虾总DNA对显微介导中国明对虾基因鲤的营养成分和氨基酸含量均产生影响,这可对鲤的品质改进,丰富鲤的遗传基础,为显微介导的超远缘杂交技术的应用提供思路.     

The effect of dietary bamboo charcoal supplementation on growth and serum biochemical parameters of juvenile common carp (Cyprinus carpio L.)

This study investigated the effect of dietary bamboo charcoal (BC) supplementation on the growth performance and health status of juvenile common carp (Cyprinus carpio L.). A 63‐day feeding trial was carried out wherein the carp were reared on diets containing increasing levels of BC (0%, 0.5%, 1%, 2% and 4%). Treatment with BC did not produce any obvious effect (p > .05) on the growth performance of the juvenile common carp and on the overall muscle fatty acid composition. However, significant improvements (p < .05) were reported on serum indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), triglycerides (TG), total cholesterol (TC), high density lipoprotein (HDL) and glucose (GLU) with the 4% inclusion level producing the most beneficial effects without adversely affecting some of the indices that were not improved at this level. Improvements were further observed in the intestinal villus length and goblet cells numbers at 4% supplementation. Although some evidence of potential toxicity exists, the overall findings of this study suggest that supplementation of common carp diets with 4% BC has a potential to enhance serum biochemical indices, intestinal functions and fatty acid content in common carp. Furthermore, while no positive effects were observed on the growth performance and total muscle fatty acid composition, no adverse effects were reported on these parameters. Therefore, BC has the potential to be used as a feed additive in the diets of juvenile common carp to improve health status and intestinal function.     

盐度对鲤能量收支的影响

以水蚯蚓为饵料，在温度为２７℃，盐度为淡水、３、５、７、９的条件下对幼鲤（初始体重为２．１９－３．３１ｇ）进行了摄食－生长实验，每一盐度下设４个摄食水平（饥饿－饱足），测定了最大摄食率、吸收率、特定生长率、转化效率和能量收支。结果表明：盐度对最大摄食率、特定生长率（ＳＧＲ）和转化效率有显著影响；盐度对排出废物所占比例（Ｅ／Ｃ）影响不显著，对代谢能所占比例（Ｒ／Ｃ）和生长能所占比例（Ｇ／Ｃ）有显著影     

利用OneMap软件构建鲤遗传连锁图谱

首次使用R环境中的OneMap软件包,以荷包红鲤抗寒品系(♂)和云南大头鲤(♀)为祖父母本所培育的110个F2个体为作图群体,以荧光扩增片段长度多态性(fluorescent amplification fragment length polymorphism,fAFLP)为主要分子标记,采用远交全同胞家系模型构建鲤的遗传连锁图谱。结果显示,110个F2个体中共产生1513个清晰的fAFLP标记,其中多态性标记911个;另开发多态性的EST标记12个,最后总计923个标记用于构建遗传连锁图谱;采用OneMap软件包构建的遗传图谱含有238个fAFLP标记和8个EST标记分布在50个连锁群上,总图距为2876.64cM,标记间平均间距为14.68cM,图谱覆盖率为66.56%。     

鲤嗜水气单胞菌感染症及其病原生物学特性

对一起养殖鲤(Cypr inus carp io )发生的病害进行了发病情况、临床特征、病理变化等方面的检验。以3尾病(死)鲤进行病变组织中的细菌检查及细菌分离, 对分离菌进行了形态特征、理化特性等较系统的表观分类学指征鉴定; 同时选择代表菌株进行了16S rRNA 基因的分子鉴定, 测定了16S rRNA 基因序列、分析了相关细菌相应序列的同源性、构建了系统发生树。结果表明, 分离菌为气单胞菌属(Aeromonas K luyv er and Van N iel 1936) 的嗜水气单[A.hydrophila (Chester 1901) Stan ier 1943] , 代表菌株( HC060718- 1株) 16S rRNA基因序列长度( 不包括引物结合区)为1 454 bp( GenBank登录号: EF669478)。择代表菌株做对健康鲤的人工感染试验, 表明了分离鉴定的嗜水气单胞菌在被检鲤病例的相应原发病原学意义及较强的致病作用。药敏试验结果显示, 对供试37种抗菌药物中的头孢噻肟等23种药物高度敏感, 对氨苄青霉素等5种药物敏感, 对青霉素G等9种药物耐药。     

鲤肌肉肌原纤维结合型丝氨酸蛋白酶的分子克隆

肌原纤维结合型丝氨酸蛋白酶(myofibril-bound serine proteinase,MBSP)是最近发现的一种蛋白酶。该酶参与肌原纤维蛋白的降解及鱼糜制品弹性的下降。但是,对该酶一级结构的研究,迄今为止,未有报道。本文根据已测定的鲤MBSP N-末端氨基酸序列以及丝氨酸蛋白酶活性中心保守序列设计兼并引物,结合RT-PCR技术实现了MBSP基因片段的扩增。再根据克隆到的MBSP片段序列设计基因特异引物,用于MBSP基因的5′和3′末端快速扩增。综合以上结果,鲤MBSP的全长被确定。序列分析表明,MBSP cDNA含有一732 bp的开放阅读框,编码243个氨基酸残基,其中信号肽长度为21个氨基酸残基。组成丝氨酸蛋白酶活性中心的氨基酸残基(His61,Asp107和Ser197)在MBSP中保守存在。成熟MBSP含有222个氨基酸残基,分子量为24.5 ku,比其天然蛋白的分子量30 ku略小。成熟MBSP的等电点为10.43。鲤MBSP与鲫MBSP,猪胰蛋白酶,牛胰蛋白酶,美洲鲽胰蛋白酶的同源性分别为80.6%,55.8%,55.3%和53.9%。而与仓鼠肌肉中具有胰凝乳蛋白酶性质的蛋白酶的同源性为39.2%。MBSP有高含量的赖氨酸残基(11.93%),此特性可能与该酶的肌原纤维结合特性有关。     

利用SSR﹑EST-SSR、SNP标记对鲤食物转化率、体厚、体质量3种性状的分析

利用Windows Map Manager2.0的标记回归法对鲤(Cyprinus carpio L.)F2群体进行单标记定位分析.用91个SSR标记、33个EST标记、364个SNP标记对鲤进行全基因组扫描,结果得到与食物转化率、体厚、体质量3个性状显著相关(P＜0.05)的标记,分别为27、35、27个,其中与食物转化率相关的标记SNP0041、SNP0044,其相关性达到极显著水平(P＜0.001),贡献率分别达到15%、16%.这些标记可作为分子标记辅助育种的参考标记.将得到的与食物转化率显著相关的EST座位HLJEl4、HLJE253和与体质量显著相关的HLJE253座位在NCBI上进行BLAST比对,结果显示HLJE253与斑马鱼上编码ORF2结构蛋白的基因相关,相关程度达61%.将得到的与3种性状相关的SNP标记在NCBI上进行序列查找,通过Blast比对找到相关的基因,并对可能的功能作了注释.     

镜鲤眼径及眼间距性状显著相关的微卫星标记筛选

利用镜鲤(Cyprinus carpio L.)全同胞家系68个个体,分析其眼径(ED)和眼间距(EC)与164个细菌人工染色体文库(bacterial artificial chromosome, BAC)末端序列来源的微卫星标记的相关性.结果164个微卫星标记共检测到401个等位基因,463种基因型,利用GLM模型对每个标记与对应的性状做回归分析, Permutation 10000次检验结果显示,共22个微卫星标记分别与眼径和眼间距具有显著相关性(P<0.05),其中CAFS1526等6个标记仅与眼径具有显著相关性(P<0.05), CAFS0882等11个标记仅与眼间距显著相关(P<0.05),而CAFS1298等5个标记与两个性状均具有显著相关性(P<0.05), CAFS1298和 CAFS1423与2个性状的相关性均达到极显著水平(P<0.01).用SPSS13.0的Duncan’s多重比较找到了每个标记与性状显著相关的基因型,进一步将与眼径和眼间距显著相关的标记整合到遗传连锁图谱上,其中9个微卫星标记与眼径或眼间距的 QTL 定位结果一致,其可解释表型变异率在13.3%~20.9%.     

中草药对鲤血清和免疫器官溶菌酶活性的影响

用添加刺五加、蒲公英、枸杞子、金银花和大蒜等单方中草药的配合饲料饲喂鲤(Cyprinus carpio L.),在开始饲喂后的第15d和第30d测定其血清和免疫器官(头肾、中肾、脾)的溶菌酶活性。结果显示,15d时,仅脾溶菌酶活性刺五加组和对照组有显著差异。30d时,血清溶菌酶活性蒲公英组、金银花组和大蒜组与对照组差异显著,头肾溶菌酶活性金银花组和对照组差异显著,中肾溶菌酶活性金银花组和大蒜组与对照组差异显著,脾溶菌酶活性所有试验组和对照组相比均有显著差异。     

彭泽鲫(♀)与黑龙江野鲤(♂)的杂交利用研究

以彭泽鲫为母本、黑龙江野鲤为父本进行杂交，将杂交一代(F1)的生长速度、抗病力、群体产量、经济效益、同工酶、生物学性状等与双亲进行对比试验。结果表明，杂交一代(F1)具有明显的优势，其生物学性状及同工酶谱与母本彭泽鲫相似。     

正常鲤外周血白细胞cDNA文库的构建

随着水产养殖业的发展,与鱼病防治紧密相关的鱼类免疫机制的研究越来越显示出其重要的理论与应用价值。鱼类的免疫系统虽不及哺乳动物发达,但其外周血白细胞已具有类似T、B淋巴细胞的分类,是机体体液免疫和细胞免疫的重要组成部分[1,2],但目前人们对鱼类免疫应答调控的机制     

Anaesthetic properties of Propiscin (Etomidaat) and 2-phenoxyethanol in the common carp (Cyprinus carpio L.), neural and behavioural measures

The objective of the study was to evaluate the anaesthetic effects of Propiscin (2% etomidate) and 2-phenoxyethanol on common carp ( Cyprinus carpio L.) by observing neural and behavioural measures. When exposed to the anaesthetic agent, the carp lost equilibrium after approximately 90 s of exposure. They did not breathe and the controls did not respond to tail pinch or prick or 6 V at the skin of the upper jaw. A shift to lower frequencies on the electroencephalogram (EEG) and computing fast Fourier transformation was observed when exposed to water containing Propiscin or 2-phenoxyethanol. Heart rate was reduced after placement in the water containing an anaesthetic agent. It may be concluded from our results that common carp were immobilized and sedated, when exposed to water containing 2 mL L⁻¹ Propiscin and immobilized and anaesthetized in water containing 0.5 mL L⁻¹ 2-phenoxyethanol. In addition, the use of EEG provides for a sound assessment of exposure of carp to these anaesthetics.     

Artificial spawning of carp (Cyprinus carpio L.): differences between females of Polish strain 6 and Hungarian strain W treated with carp pituitary homogenate, Ovopel or Dagin

Investigations of the effects of controlled reproduction were conducted on carp females of Polish strain 6 and Hungarian strain W after ovulation stimulation with carp pituitary homogenate (CPH) (0.3 mg+2.7 mg kg^?1 body weight), Ovopel (1/5+1 pellet kg^?1 body weight) or Dagin (1 dose kg^?1 body weight). In both strains, the highest percentage of spawning females was found after the Ovopel treatment. After treatments with CPH and Dagin this percentage did not differ in strain 6; however, after Dagin, it was higher in fish of strain W. After CPH or Ovopel stimulation the females of line 6 yielded eggs of higher weight compared with line W. After the Dagin treatment the weight of eggs obtained from females, both strains was similar but much lower than after the two remaining agents. The quality of eggs expressed both by the percentage of fertilization and that of living embryos after 24 and 36 h incubation was similar in the two strains only in the group treated with Ovopel. After CPH application eggs of better quality were obtained from strain 6 compared with strain W; however, after the treatment with Ovopel, eggs of much better quality were noted in strain W. In general, the best effects of reproduction were recorded in the case of Ovopel and the poorest in that of Dagin. Females of strain 6 yielded eggs of a higher weight but the percentages of spawning females were higher in strain W after each of the applied spawning agents. A new approach to this problem consisted in investigating the interaction between ovulation stimulators and origin of females of fish treated with Dagin.     

Growth testing of two breeds of common carp, Cyprinus carpio L. (Hungarian mirror and Třeboň scaly carp), in ponds with low and high stocking density

The growth of two breeds of common carp, Cyprinus carpio L., was tested in ponds under the climatic conditions of South Bohemia. T?eboň scaly carp (TR) and Hungarian mirror carp (M2) were kept in both low and high stocking densities during the second growing season and then stocked together for communal testing during the third growing season. Before the communal testing, the mean initial weights of fish from low‐ and high‐density stocks differed significantly (374.1 vs. 227.7 g for the TR breed and 766.7 vs. 317.3 g for the M2 breed respectively, P<0.01). After communal testing, mean weights of fish from low‐ and high‐density stocks gained 761.8 vs. 543.8 g for the TR breed and 1339.7 vs. 706.7 g for the M2 breed respectively. These observed weights were also significantly different (P<0.01). However, the test of corrected weight gain, i.e. gain not related to the initial weight of fish, revealed insignificant differences (P>0.01) between the weight gains after correction, i.e. the effect of different initial weights was successfully eliminated. These results seem to confirm the applicability of this method for the assessment of growth of purebred common carp under the climatic conditions of Central European fish farms.