Comparative effect of Freund's adjuvant and Aloe vera L. gel on the expression of TNF‐α and IL‐1β in vaccinated Cyprinus carpio L. with Aeromonas hydrophila C. bacterin

The comparative effects of Freund's and Aloe vera gel as adjuvants on the expression of IL‐1β and TNF‐α genes were studied in vaccinated common carp (Cyprinus carpio) with Aeromonas hydrophila bacterin. Fishes were intraperitoneally immunized with A. hydrophila bacterin in combination with Aloe vera gel or Freund's and also without any adjuvant. At day 28 after immunization, all groups were challenged by lethal dose of A. hydrophila (10⁷ cells/fish). Changes in the expression of IL‐1β and TNF‐α genes were evaluated in anterior kidney before challenge and 12, 24, 72 and 7 days postchallenge using quantitative real‐time PCR. Higher expression levels of both genes were observed in all vaccinated groups compared with non‐immunized group. Fishes which received Aloe vera gel showed higher expression of IL‐1β and TNF‐α in relation to animals which vaccinated with or without Freund's adjuvant. We concluded that Aloe vera gel in compared with Freund's adjuvant had a more stimulatory effect on the expression of immune‐related genes in vaccinated common carp and it can use as a novel adjuvant in aquaculture.     

Influence of dietary linoleic acid (18:2n‐6) and α‐linolenic acid (18:3n‐3) ratio on fatty acid composition of different tissues in freshwater fish Songpu mirror carp,Cyprinus Carpio

Six isonitrogenous and isoenergetic purified diets were formulated to feed Songpu mirror carp for 60 days. The control diet (CD) was only supplemented with soybean oil. The other five experimental diets contained soybean oil, linseed oil and lard oil blended at various inclusion levels to attain different linoleic acid (LA)/α‐linolenic acid (LNA) ratios (0.53, 1.04, 2.09, 3.95, 6.82) with a constant total C₁₈ polyunsaturated fatty acids (PUFA; LA+LNA, 2% dry weight) content. The fatty acid (FA) profiles of hepatopancreas, dorsal muscle, intestine, intraperitoneal fat (IPF), spleen and kidney reflected those of the diets, but with some differences. The spleen showed the lowest correlation with diet compared with other tissues, followed by the hepatopancreas (P < 0.05). The intestine and IPF showed relatively higher correlation. On the other hand, the control group had the lowest tissue‐diet correlation, followed by the LA/LNA0.53 group (P < 0.05), whereas the LA/LNA2.09 showed the highest. The LA/LNA ratios in the tissues were up‐regulated in the LA/LNA0.53, 1.04 groups and down‐regulated in the LA/LNA3.95, 6.82 groups. This was due to when LA (or LNA) was highly added in diet, the decrease in this FA was huge in tissue. The contents of saturated fatty acids and monounsaturated fatty acids increased in the control group, but seemed not influenced by dietary LA/LNA ratios. These results demonstrated that the FA deposition was tissue‐specific, and also influenced by the dietary FA composition in the experimental fish. Finally, we suggest that 2.09 is the optimal LA/LNA ratio (2% C₁₈ PUFA) of Songpu mirror carp for fillet FA composition.     

鲤鱼诱导型一氧化氮合成酶全长cDNA克隆及其在外周血白细胞中的差异表达分析

试验旨在获得鲤鱼诱导型一氧化氮合成酶(inducible nitric oxide synthase,iNOS)cDNA全长序列,并以此为基础探讨在丝裂原刺激下外周血白细胞中iNOS的表达变化。以从鲤鱼正常外周血白细胞cDNA文库中获得的iNOS的EST序列为基础,采用基因文库筛选和cDNA5'末端快速扩增技术(5'-RACE)相结合的方法,成功扩增出鲤鱼iNOScDNA全长序列,然后进行鲤鱼外周血白细胞原代培养,分成对照组和试验组,其中试验组分别为脂多糖(LPS,1.0μg/mL)刺激4、12h,刀豆蛋白A(ConA,1.0μg/mL)刺激4、24h,对照组为相同培养时间无丝裂原刺激的外周血白细胞,根据得到的iNOScDNA全长序列和鲤鱼β-actin序列分别设计特异性引物,应用实时荧光定量PCR方法检测各组外周血白细胞中iNOS在mRNA水平上的表达情况并进行分析。序列分析结果显示,最后获得的cDNA片段共3704bp,包含74bp的5'端非编码区,246bp的3'端非编码区,一个3384bp的完整的开放阅读框(ORF),共编码1127个氨基酸。序列同源性分析结果显示,该序列与鲫鱼iNOS基因同源性高达100%;实时荧光定量PCR结果显示,经LPS、ConA刺激的试验组外周血白细胞中iNOS表达量均升高,且短时间(4h)刺激的表达量高于长时间(LPS12h;ConA24h)刺激的表达量。综上所述,在LPS、ConA刺激过程中,iNOS在外周血白细胞中表达量均有上调,结果提示存在炎症反应的动态变化。     

丙氨酰-谷氨酰胺和γ-氨基丁酸对建鲤生长、饲料利用及体成分的影响

【目的】探讨丙氨酰-谷氨酰胺(Ala-Gln)和γ-氨基丁酸(GABA)对建鲤Cyprinus carpio var.jian生长、饲料利用及肌肉营养成分的影响。【方法】在建鲤配合饲料中添加不同比例Ala-Gln和GABA,配成7种等氮(质量分数为32%粗蛋白质)、等能(17.0 MJ·kg-1)饲料,以添加Ala-Gln和GABA对建鲤生长最适添加量(6.0 g·kg-1AlaGln、90 mg·kg-1GABA)的添加组添加比例为100%,在此基础上等比例缩减和增加,Ala-Gln和GABA的添加梯度分别为0(0 g·kg-1Ala-Gln、0 mg·kg-1GABA)、20%(1.2 g·kg-1Ala-Gln、18 mg·kg-1GABA)、40%(2.4 g·kg-1Ala-Gln、36 mg·kg-1GABA)、60%(3.6 g·kg-1Ala-Gln、54 mg·kg-1GABA)、80%(4.8 g·kg-1Ala-Gln、72mg·kg-1GABA)、100%(6.0 g·kg-1Ala-Gln、90 mg·kg-1GABA)、120%(7.2 g·kg-1Ala-Gln、108 mg·kg-1GABA),共7组。饲喂初始体质量为45.59 g建鲤8周。【结果】与对照组相比,饲料中添加Ala-Gln和GABA的添加组,建鲤平均增质量率和特定生长率均显著提高(P0.05);其中60%添加组(3.6 g·kg-1Ala-Gln、54 mg·kg-1GABA)的饲料效率、蛋白质效率及肌肉中粗蛋白质含量均显著高于不添加的对照组(P0.05),其他各组之间差异不显著(P0.05)。【结论】饲料中添加Ala-Gln和GABA在一定程度上促进了建鲤幼鱼生长、饲料利用和肌肉蛋白质的合成,其中以3.6 g·kg-1Ala-Gln、54 mg·kg-1GABA组的效果最佳。     

黄酒陈酿用陶缸的透氧模型建立

为获得黄酒陈酿用陶缸的透氧特性,采用3因子二次通用旋转组合设计,研究陶缸厚度(X_1)、环境温度(X_2)和陶缸内起始氧气含量(X_3)对陶缸氧气透气率影响,并建立了陶缸氧气透气模型。结果表明,3个因素对陶缸氧气透气性的影响大小依次为陶缸厚度(X_1)缸内起始氧气含量(X_3)环境温度(X_2),其中陶缸厚度和缸内氧气含量的影响达到极显著水平(P0.01),而环境温度的影响不显著(P0.05)。陶缸的厚度(X_1)和缸内起始氧气含量(X_3)、环境温度(X_2)和缸内起始氧气含量(X_3)的交互作用达到显著水平(0.01P0.05)。本研究能为黄酒不锈钢大罐存贮期间的加氧陈酿技术提供参数支持。     

基于转录组测序的兴国红鲤微卫星标记筛选

采用Illumina高通量测序技术,对兴国红鲤(Cyprinus carpio var.singuonensis)垂体和性腺等组织进行转录组测序分析,筛选微卫星标记并分析其组成及特征。结果显示:共获得13 652个微卫星标记,对所得位点进行分类,单核苷酸重复类型占47.86%,二、三、四、五、六核苷酸重复类型所占比例分别为34.43%、16.32%、1.25%、0.12%以及0.02%。随机选取30个SSR位点进行PCR验证,有20对可以扩增出清晰稳定的条带。在24个兴国红鲤个体中对上述位点进行多态性分析,结果表明,具有多态性的微卫星引物为9对。不同位点得到的等位基因范围为2~4,平均等位基因数为3.111 1±0.993 8,观测杂合度(Ho)、期望杂合度(He)以及多态信息含量(PIC)平均值分别为0.597 2±0.233 2、0.539 7±0.178 0和0.467 2±0.172 1。9个微卫星位点中,有4个显著偏离哈代-温伯格平衡(Hardy-Weinberg equilibrium,HWE)(P0.05)。Illumina高通量测序提供了一种直观、高效开发微卫星标记的方法,所选兴国红鲤群体遗传多样性维持较好。     

Effects of dietary vitamin K levels on growth performance,enzyme activities and antioxidant status in the hepatopancreas and intestine of juvenile Jian carp (Cyprinus carpio var. Jian)

A total of 1400 juvenile Jian carp (Cyprinus carpio var. Jian) (8.93 ± 0.03 g) were fed diets containing graded levels of vitamin K at 0.027 (basal diet), 1.52, 3.02, 4.51, 6.02 and 7.52 mg kg^?1 diet for 60 days to investigate the effects of vitamin K on growth, enzyme activities and antioxidant capacity in the hepatopancreas and intestine. Percentage weight gain (PWG), feed intake and feed efficiency of fish were improved by vitamin K. Activities of trypsin, chymotrypsin, amylase and lipase in the intestine and hepatopancreas and Na⁺, K⁺‐ATPase, creatine kinase, alkaline phosphatase and gamma‐glutamyl transpeptidase in the intestine were increased by vitamin K. Malondialdehyde and protein carbonyl contents in the hepatopancreas and intestine were decreased with vitamin K supplements. Certain level of vitamin K increased antihydroxyl radical, antisuperoxide anion, superoxide dismutase, catalase, glutathione‐S‐transferase, glutathione peroxidase and glutathione reductase activities and glutathione contents in the hepatopancreas and intestine. Intestinal Lactobacillus, E. coli and Aeromonas were changed with vitamin K supplements. Together, these results indicate that vitamin K improved fish growth, digestive and absorptive ability, and anti‐oxidant capacity. The dietary vitamin K requirement of juvenile Jian carp (8.93–73.7 g) based on PWG was 3.13 mg kg^?1 diet.     

瓯江彩鲤酪氨酸酶(TYR)基因的选择压力分析

酪氨酸酶是生物体黑色素合成的关键限速酶,黑色斑纹是瓯江彩鲤(Cyprinus carpio var. color) 体色的主要特点之一。以瓯江彩鲤的5种体色选育系F₅为材料,探讨酪氨酸酶基因5个外显子及其在不同体色间的变异特性和承受选择压力的敏感性。结果发现:酪氨酸酶基因外显子1和外显子5的核苷酸变异率最高,选择性位点最多,易承受到选择压力;5个外显子的非同义替换率(d_N) 与同义替换率(d_S) 的比值(ω值)均小于1(0.10~0.67),表明它们均受到净化选择压力;不同体色瓯江彩鲤的酪氨酸酶基因也均受到净化选择压力(ω=0.12~0.23)。应用PAML软件中的M2a和M8两种模型检测显示:无黑斑体色瓯江彩鲤所受正向选择压力位点数高于有黑斑体色,但不存在显著差异(P>0.05);酪氨酸酶基因中的部分氨基酸位点较易受正向选择压力。研究结果表明:瓯江彩鲤酪氨酸酶基因较保守,主要受净化选择作用,当前的人工选育未对酪氨酸酶基因造成显著的选择压力。     

池塘养殖锦鲤体表溃疡病的组织病理学观察

为临床诊断和防治锦鲤Cyprinus carpio koi皮肤溃疡病,采用病理解剖方法对患病锦鲤进行临床观察和组织病理学分析,试验用患病锦鲤取自上海市朝阳锦鲤养殖场发病池塘,确定病鱼10尾(编号1~10),取每条病鱼的组织进行病理解剖、石蜡切片、H-E染色。结果表明:患病锦鲤体表溃疡病处的病灶组织出现严重淤血、炎性细胞浸润并聚集、肌束溶解和坏死等典型病理表现;体表溃疡病是由致病菌局部感染所致,并未全身性感染,10尾病料的脾组织均表现出血管扩张、管壁增厚、血管周围的实质组织凝固变性,伴有红细胞坏死和凝固变性区域内部分组织坏死等较为一致的病理特征,可以诊断为血液中有毒物质引起的脾组织病变,导致脾功能低下;肝胰组织和肾组织较脾组织病变相对较轻;肠、心、鳃、头肾组织等基本正常。研究表明,本病例全部样品均有不同程度的脾凝固变性、肝组织淤血、肾组织间质充血及其组织中血管扩张、管壁增厚的病理变化,这与体表溃疡病变无直接关联。     

饲料中添加怀山药与怀菊花对黄河鲤幼鱼生长及血液理化指标的影响

在黄河鲤Cyprinus carpio基础饲料中加入质量分数为2％的怀山药与怀菊花,在室外水泥池中饲养42d后,测定其生长性能及血液理化指标,研究怀山药、怀菊花对黄河鲤生长及血液生理生化指标的影响。结果表明：怀菊花能显著提高黄河鲤的增重率、特定生长率（P〈0．05）,分别比对照组提高11．68％、5,60％;怀山药对黄河鲤的生长效果无显著影响（P〉0．05）。怀菊花组黄河鲤血液中红细胞（RBC）数量（P〈0．05）显著提高,红细胞平均体积（MCV）极显著提高,血清白蛋白（ALB）含量显著高于对照组（P〈0．05）;怀山药组黄河鲤血清中球蛋白（GLB）含量显著高于对照组（P〈0．05）。