CCl4对建鲤肝细胞DNA的毒性作用及对CYP3A基因表达的影响

四氯化碳(CCl₄)作为一种经典的肝脏毒物,被广泛应用于哺乳动物的肝损伤模型构建及保肝药物筛选。低、中、高浓度的CCl₄橄榄油溶液(6.25%、12.50%和15.00%,0.01 mL/g)腹腔注射建鲤72 h后,采用单细胞凝胶电泳和肝组织病理切片技术,测定血清中肝损伤生化酶来考察CCl₄对肝细胞DNA的毒性作用;同时采用实时荧光定量PCR法测定肝组织中CYP3A表达量的变化。结果显示,6.25%CCl₄作用建鲤72 h后肝组织切片检查没有发生明显的改变,对血清中酶学指标无显著影响,CYP3A的mRNA表达量与空白对照组相比也没有显著变化,而彗星实验结果显示该浓度的CCl₄作用肝细胞后,尾长、TDNA、尾矩及Olive 尾矩等DNA损伤指标与对照组相比,显著增大;随着CCl₄作用浓度的增加,肝细胞肿胀、广泛空泡变性,出现核固缩和核溶解等组织学的变化,12.50%和15.00% CCl₄均能显著引起血清中谷丙转氨酶(GPT)和谷草转氨酶(GOT)水平升高,15.00% CCl₄能显著促进乳酸脱氢酶(LDH)和丙二醛(MDA)大量生成;彗星实验中各项损伤指标也随着染毒剂量增加而极显著增大;同时,12.50%和15.00% CCl₄组的CYP3A mRNA表达量显著降低。结果表明,CCl₄对建鲤肝细胞DNA具有毒性作用,在实验的浓度范围内,CCl₄ 能抑制CYP3A mRNA表达;随着CCl₄作用浓度的增大,血清酶、病理切片和彗星实验结果表现出剂量效应,并有一定的一致性,彗星实验表现出更高的灵敏性。     

Aerobic Bacterial Flora of Common Carp (Cyprinus carpio L.) Cultured in Earthen Ponds in Saudi Arabia

ABSTRACT

Quantitative and qualitative estimation of bacterial flora present in pond water, sediments, gills, and intestine of healthy common carp Cyprinus carpio cultured in Saudi Arabia were performed and identified to species level where possible. Mean total viable bacterial counts in pond water ranged from 1.2?±?2.9?×?10⁴ to 2.5?±?3.5?×?10⁵ cfu/mL; in sediments, 9.3?±?2.1?×?10⁷ to 2.7?±?3.5?×?10⁹ cfu/g; in gills filaments, 4.3?±?2.9?×?10⁶ to 1.6?±?3.9?×?10⁷ cfu/g; and in intestine, 8.7?±?4.1?×?10⁹ to 5.4?±?3.2?×?10¹⁰ cfu/g. Gram-negative rod-shaped bacteria dominated (76%) the populations. In total, 12 bacterial genera and 15 species were identified. Pond water and sediment bacteria had the reflection on bacterial composition of gills and intestine of carp. Intestinal bacteria showed more diversification in contrast to gill bacteria. Aeromonas hydrophila, Shewanella putrefaciens, Vibrio cholerae, Corynebacterium urealyticum, Vibrio vulnificus, Vibrio sp., and Staphylococcus sp. were the common bacteria in all the populations. In pond water and carp intestine, A. hydrophila, S. putrefaciens, V. cholerae, and C. urealyticum were the most dominant bacteria (prevalence ≥ 10%) where pond sediments and the carp gills experienced with more one dominant bacterium V. vulnificus. Only the A. hydrophila covered one fourth (25%) of the total bacterial populations.     

建鲤生长性状QTL区间及其与镜鲤同源性比对

以建鲤(Cyprinus carpio var. jian)F₁群体的94尾个体为材料, 利用254个微卫星标记构建了建鲤的遗传图谱, 并对体长、体高和体厚性状进行了QTL定位, 共检测到17个生长相关性状QTL, 分布在10个连锁群上, 解释表型变异为10.8%~35.2%。以相同的分子标记, 构建建鲤与镜鲤(Cyprinus carpio L.)的比较图谱, 分析了建鲤和镜鲤群体生长性状QTL的同源性关系。结果表明, 建鲤与镜鲤图谱具有广泛的共线性或同线性关系, 建鲤每个连锁群在镜鲤图谱中均找到了对应的同源连锁群, 其中建鲤10个生长性状QTL与镜鲤13个相同性状QTL具有同源性, 同源比例高达58.8%。同时发现, 建鲤QTL置信区间相对较大, 与之同源的镜鲤QTL置信区间较小, 通常位于建鲤QTL子区间内, 定位结果更精细。此外, 建鲤与镜鲤连锁群上都存在QTL的富集区域, 而这些富集区域常常存在于同源连锁群的共线性区域, 可作为标记辅助选择的重点区域。鲤通用QTL的发掘, 为分子标记辅助育种和更进一步的精细定位打下基础。

     

不同糖脂比对建鲤幼鱼生长、体组成、消化及糖酵解能力的影响

本试验研究了饲料中不同糖脂比对建鲤幼鱼生长、体组成、消化及糖酵解的影响。试验共配制6组等氮等能的半纯合饲料,对应糖脂比分别为2.3,3.0,4.0,5.6,7.7和12.1。将鱼饱食投喂8周,每日投喂3次。试验结果表明,当饲料糖脂比从2.3升高至7.7时,增重率、特定生长率、蛋白质效率和蛋白保留率均显著升高(P < 0.05);而当糖脂比进一步升高时,其均呈下降趋势,但差异不显著(P > 0.05);饲料系数的变化趋势与其相反。建鲤幼鱼的脏体比以及全鱼、胴体和肝脏脂肪含量随着饲料糖脂比的降低均显著升高(P < 0.05),而全鱼、胴体和肝脏的蛋白质含量无显著差异(P > 0.05)。肠道淀粉酶活性随着糖脂比的升高显著升高(P < 0.05),而脂肪酶的变化趋势则相反(P < 0.05)。此外,血糖和胰岛素水平、肝糖原含量及肝脏葡萄糖激酶和丙酮酸激酶活性随饲料糖脂比的升高均显著升高(P < 0.05),血液中总胆固醇和甘油三酯含量则随着糖脂比的降低显著升高(P < 0.05)。根据二次回归模型得出,在等氮等能的饲料条件下,建鲤幼鱼最适宜的糖水平和脂肪水平分别为38.21%和4.69%,对应的糖脂比为8.14。     

松浦镜鲤越冬期的形态、组织结构及生化组成变化

利用电子标记跟踪同期孵化、同条件养殖的松浦镜鲤(Cyprinus carpio var.songpu mirror)在越冬前后的生长过程,比较实验鱼在125 d(越冬前)、170 d(结冰初期)及325 d(越冬后)的形态、组织结构及生化组成变化,并探讨越冬后鱼体主要供能物质含量与越冬体重损失的关系。结果显示,鲤325 d的体重(BW)、肥满度(CF)、脏体指数(VSI)、肝体指数(HSI)和肠体指数(ISI)均显著低于125 d(P0.05);与125 d相比,325 d时的肝和前肠组织结构变化明显,主要表现在肝细胞体积变小,细胞界限模糊,糖原和脂滴丢失。前肠直径、绒毛高度和肌层厚度均显著下降(P0.05),柱状上皮杯状细胞减少,纹状缘萎缩;鲤内脏脂肪和肝糖原含量在越冬期各测定时段均显著下降(P0.05),肌肉和全鱼蛋白质含量仅在冰下阶段(170~325 d)下降明显(P0.05)。越冬后肌肉蛋白质含量与体重损失率呈显著的负相关(P0.05),但内脏脂肪含量与越冬体重损失率无相关(P0.05)。以上结果表明,越冬导致鲤体重相关的形体指标显著变化;越冬长期饥饿引起了肝和前肠组织退化;越冬期间鲤贮能物质的消耗存在阶段性,在结冰前阶段(125~170 d),能量来源以肝糖原和脂肪为主。在冰下阶段(170~325 d),肝糖原、脂肪和蛋白质共同参与能量代谢。越冬后肌肉蛋白质含量可作为衡量越冬期体重损失程度的参考指标。     

镜鲤多家系体长和体质量QTL定位分析

为了克服单个家系数量性状位点(QTL)检测效率低、假阳性高等缺点,实验利用250对微卫星(SSR)标记对镜鲤8个全同胞家系的522尾子代进行基因组扫描,采用半同胞家系的分析策略对镜鲤体长(SL)和体质量(BW)性状进行QTL分析。结果显示,基于父系的QTL分析,共检测到4个QTL区间,其中,3个体长的QTL中,1个为95%基因组水平(genome-wide)显著性,位于LG24,可解释表型变异率为20.3%;其余2个均为95%染色体水平(chromosome-wide)显著性,分别位于LG6和LG30,可解释表型变异率分别为11.9%和11.6%。1个体质量的QTL达到99%基因组水平,位于LG24,可解释表型变异率达到38.3%,且与体长QTL区间重叠。基于母系的QTL分析,共检测到8个QTL区间,其中,5个体长的QTL中,1个为99%染色体水平,位于LG8,可解释表型变异率为16.6%;其余4个均为95%染色体水平,分别位于LG24、LG30、LG31和LG45,可解释表型变异率为9.6%~14.2%,且位于LG24和LG30上的QTL为父母本共有;3个体质量的QTL均与体长QTL区间重叠,1个为95%染色体水平,位于LG24,其余2个均为99%染色体水平,位于LG30和LG45,可解释表型变异率分别为14.1%和13.6%。进一步分析发现,位于LG24上的体长和体质量QTL区间重叠且均为父母本共有,体质量的3个QTL均与体长QTL存在重叠区域且呈现成簇分布的特点。本研究结果不仅可以为鲤分子育种提供更可靠的标记,而且为家系和品种间QTL变异规律的探索提供基础数据。     

Dietary microbial levan ameliorates stress and augments immunity in Cyprinus carpio fry (Linnaeus, 1758) exposed to sublethal toxicity of fipronil

A 45‐day feeding trial was conducted to study the stress ameliorating and immunomodulatory role of microbial levan in Cyprinus carpio fry exposed to sublethal dose (1/10th LC₅₀) of fipronil [(±)‐5‐amino‐1‐(2,6‐dichloro‐α,α,α‐trifluoro‐p‐tolyl)‐4‐trifluoromethylsulfinylpyrazole‐3‐carbonitrile]. Two hundred and twenty‐five fry were randomly distributed in five treatments in triplicates. Four purified diets were prepared with graded levels of microbial levan. Five different treatment groups were levan control L₀P₀ (basalfeed + 0% levan without exposure to pesticide); pesticide control L₀P₁ (basalfeed + 0% levan with exposure to pesticide); L_0.25P₁ (basalfeed + 0.25% levan with exposure to pesticide); L_0.50P₁ (basalfeed + 0.50% levan with exposure to pesticide) and L_0.75P₁ (basalfeed + 0.75% levan with exposure to pesticide). Lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and fructose‐1,6‐diphosphatase (FDPase) activites were significantly (P < 0.05) increased, whereas alkaline phosphatase (ALP), adenosine triphosphatase (ATPase) and acetyl choline esterase (AchE) activities were significantly (P < 0.05) reduced in higher levan‐fed groups. RBC, haemoglobin and WBC counts were significantly (P < 0.05) increased in the levan‐fed groups. Similar trends were also observed for the total serum protein, globulin, NBT and lysozyme activities. Blood glucose and serum cortisol exhibited a third order polynomial relationship with increasing level of dietary levan. Overall result showed stress ameliorating, immunostimulating and protective role of microbial levan against fipronil‐induced stress in C. carpio fry at 0.75% level of dietary levan supplementation.     

茵陈汤对饥饿鲤体质量、生物学性状、体成分及血清生化指标变化的影响

试验主要评估茵陈汤对饥饿鲤物质代谢及健康的作用,为鲤的健康高效养殖以及饲料开发提供依据。将210尾鲤(平均体质量19.90 g±2.65 g)以每箱35尾随机投放于6个室内网箱,分为对照组和茵陈汤组,饲以分别添加0%、0.9%茵陈汤的试验饲料56 d,然后每箱随机选取8尾体格健壮的鲤进行为期30 d的饥饿试验。饥饿试验前后进行鱼体生物学性状、体成分、血清指标的检测。结果表明在饲养试验中,鲤的生长性能、生物学性状、肌肉中水分、粗蛋白、粗脂肪含量、肝胰脏常规成分两组之间均无显著差异(P0.05),肌肉中粗灰分含量、血清ALT水平茵陈汤组显著低于对照组(P0.05),其他血清生化指标无显著差异(P0.05);饥饿后茵陈汤组的减量率显著低于对照组(P0.05),肥满度、肝胰脏水分含量显著高于对照组(P0.05),肌肉和肝胰脏粗蛋白、粗脂肪、粗灰分含量两组之间均无显著差异(P0.05);两阶段间,茵陈汤组鲤饥饿后脾脏指数显著高于饲喂后(P0.05),肥满度显著低于饲喂后(P0.05),其他生物学指标两组均分别显著低于饲喂后(P0.05)。肌肉中水分、粗蛋白、粗灰分含量饥饿后两组均分别显著高于饲喂后(P0.05),粗脂肪含量显著低于饲喂后(P0.05),茵陈汤组肝胰脏变化趋势与肌肉相符。饥饿后血清HDL、LDL、CHOL水平显著高于饲喂后(P0.05),其他血清指标两组均分别显著低于饲喂后(P0.05)。从饥饿前后鲤体质量、体成分及血清生化指标的变化,说明茵陈汤对鲤体内物质和能量代谢具有一定的影响,可以促进脂肪的利用,并减缓机体对营养物质的快速消耗,有利于体质量的维持。同时,茵陈汤有助于鲤的健康养殖,对鲤的肝胰脏具有一定保护作用。     

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.