复方中草药对镜鲤(Cyprinus carpio L.)血清转氨酶及红细胞抗氧化酶活性的影响

在基础日粮中添加2%由不同比例的刺五加、枸杞子、金银花和黄芪配伍的复方中草药饲喂镜鲤（Cyprinus carpio L）.,于第14天、第28天及第42天检测红细胞的过氧化氢酶（CAT）、超氧化物岐化酶（SOD）、血清的谷草转氨酶（GOT）以及谷丙转氨酶（GPT）活性,以研究复方中草药对镜鲤的毒副作用。在28及42d时,复方Ⅰ组红细胞CAT活性与对照组差异显著（P〈0.05）;28d时,各复方组红细胞SOD活性均与对照组差异极显著（P〈0.01）;28d时,复方Ⅱ组血清GOT活性及复方Ⅲ组血清GPT活性与对照组差异极显著（P〈0.05）;42d时,各复方组血清GPT活性与对照组差异极显著（P〈0.01）。结果表明,试验复方中药添加到镜鲤的饲料中,可以增强其血清转氨酶及红细胞抗氧化酶活性,其中复方Ⅰ效果最佳,建议用药时间为28d。

不同温度下不同蛋白水平对镜鲤(Cyprinus carpio L.)非特异性免疫的影响

选用德国镜鲤630尾(200±20)g,在18、22和26℃3个温度环境下用30%、33%、36%、39%、42%的五个不同的蛋白水平的饲料投喂,分别测定肝脏、脾脏中的超氧化物歧化酶(SOD)、丙二醛(MDA)、溶菌酶(LYZ)、血清中的碱性磷酸酶(ALP)和补体C3、C4等免疫指标。结果表明,18℃环境下,42%蛋白水平组肝脏SOD活性显著高于其他组(P<0.05);肝脏MDA含量随饲料蛋白水平的增加而降低;30%蛋白水平组脾脏溶菌酶的活性显著高于39%蛋白水平组(P<0.05)。22℃环境下,42%蛋白水平组肝脏SOD活性显著高于其他蛋白水平组(P<0.05);42%蛋白水平组肝脏MDA含量显著低于其他蛋白水平组(P<0.05);42%蛋白水平组血清C3显著高于30%蛋白水平组(P<0.05)。26℃环境下,肝脏SOD活性随饲料蛋白水平的增加而增加,但差异不显著(P>0.05);42%蛋白水平组肝脏MDA含量显著低于其他组(P<0.05)。结果显示,本试验条件下,在18、22和26℃温度下蛋白水平升高可在一定程度上提高鲤鱼的非特异性免疫,主要表现在抗氧化性方面,而对其他的非特异性免疫指标无显著的影响。

鲤鱼(Cyprinus carpio L.)体重和体长QTL的定位

利用265个AFLP标记、127个微卫星分子标记、37个EST-SSR标记和16个RAPD标记(共445个标记)对大头鲤/荷包红鲤抗寒品系的F2代雌核发育群体44个个体进行基因型检测,构建鲤鱼(Cyprinus carpio L.)遗传连锁图谱;利用软件WinQTLCart2.5采用复合区间作图法对体重和体长两个性状进行了QTL定位分析。结果共检测到与两个与体重性状相关的QTL,分别定位到LG24连锁群(qBWh-24-1)和LG20连锁群(qBWh-20-1)上,可解释的表型变异分别为7.98%和20.05%;检测到两个与体长性状相关的QTL,分别位于LG2连锁群(qFS-2-1)和LG20连锁群(qFS-20-1),可解释的表型变异分别为5.69%和12.69%,4个QTL的加性效应均为负值。

镉污染对鲤鱼(Cyprinus carpio Linn.)同工酶酶谱的影响

应用聚丙烯酰胺凝胶电泳和酶活性测定技术，比较和分析了饲养于不同浓度镉液中的鲤鱼在镉含量达到积累－释放平衡后，其肌肉和肝脏组织的酯酶同工酶、碱性磷酸酶同工酶及过氧化物酶同工酶酶谱。结果显示，镉的残留量使鲤鱼肌肉和肝脏组织酯酶同工酶和碱性磷酸酶同工酶谱带减少，活性减弱；过氧化物酶同工酶谱带增多，活性增强。

Effect of Metal Ions on Protease Activities in the Intestines and Hepatopancreas of Red-white Ornamental Carp (Cyprinus carpio L)

[Objective] The aim of this study was to study effects of metal ions on the protease activities in digestive tissues and gland of red-white ornamental carp（Cyprinus carpio L）.[Method] Effects of four kinds of metal ions （K＋,Na＋,Mg2＋ and Ca2＋） on protease activities in hepatopancreas,foregut,midgut,hindgut of red-white ornamental carp were studied by enzyme analysis method.[Result] Effects of four kinds of metal ions on protease activities of red-white ornamental carp were different in the range of experimental concentration from 25 mmol/L to 150 mmol/L.K＋ could promote protease activities in hepatopancreas and hindgut at different levels.Especially,K＋ had the promoting effect at low-concentration level,but the inhibitory effect at high-concentration level in midgut and the inhibitory effect in foregut.Na＋ had the promoting effect on protease activities in hepatopancreas,foregut and hindgut at different levels,but the inhibitory effect in midgut.Mg2＋ and Ca2＋ had the inhibitory effect on protease activities in intestinal and hepatopancreas at different levels.[Conclusion] This study provides basic data and theoretical foundation for researches on the digestive physiology of red-white ornamental carp or the development and optimization of compound feed.

Ontogeny of Immune Related Organs during Early Development of Carp (Cyprinus Carpio L.)

In this paper, ontogeny of immune-related organs during theearly development of carp was studied by histochemical technique-lcineBlue staining under acid conditions and PAS reaction(AB-PAS staining). Thekidney appeared one day before hatching, spleen, liver and pancreas emergedon the same day of hatching, and thymus was not found until the third day afterhatching. Ontogeny of these immune related organs of the carp in thisresearch is earlier than that reported by Botham.

Protective Effect of Bacillus subtilis Peptidoglycan (PG) on β-conglycinin-induced Intestinal Epithelial Cells Damage of Juvenile Carp (Cyprinus carpio)

[Objective] The paper was to investigate the protective effects of different concentrations of Bacillus subtilis peptidoglycan(PG) on β-conglycinin-induced inflammatory injury in intestinal epithelial cells of juvenile carp(Cyprinus carpio).[Method] In 24-cell microplates, the intestinal epithelial cells(IECs) of juvenile carp were primarily cultured for 72 h at 26°C and 6% CO2, and then the IECs were randomly divided into6 groups with 4 replicates per group. One of the six groups was set as negative control group, and the other groups were all supplemented with 1.0 mg/mL β-conglycinin in culture medium to establish inflammatory injury. At 24 h post induction, the culture media were changed into B. subtilis PG culture media with the concentrations of 0(positive control group), 0.15, 0.30, 0.45 and 0.60 mg/mL, respectively. The samples were collected to measure the antioxidant and anti-inflammatory indices at 12, 24 and 36 h post culture.[Result]β-conglycinin exposure significantly decreased the activity of ASA, AHR, SOD, CAT, GPx, and increased the PC content and the mRNA expression of inflammatory cytokines(IL-1β, IL-8, TNF-α1,IL-10 and TGF-β). At 12, 24 and 36 h post PG treatment, the activities of ASA, AHR, SOD, CAT, GPx and the content of PC in cells decreased in a dose-dependent manner;the mRNA levels of IL-1β, IL-8 and TNF-α1 were down-regulated and those of IL-10 and TGF-β were up-regulated.[Conclusion] Different concentrations of B. subtilis PG could protect IECs oxidative damage induced by β-conglycinin and improve the antioxidant capacity of IECs. High concentration of PG could improve the anti-inflammatory ability of IECs by inhibiting inflammatory factors and promoting the gene expression of anti-inflammatory cytokines.

Preliminarily study on the maximum handling size, prey size and species selectivity of growth hormone transgenic and non-transgenic common carp Cyprinus carpio when foraging on gastropods

The present study preliminarily examined the differences in maximum handling size, prey size and species selectivity of growth hormone transgenic and non-transgenic common carp Cyprinus carpio when foraging on four gastropods species( Bellamya aeruginosa, Radix auricularia, Parafossarulus sinensis and Alocinma longicornis) under laboratory conditions. In the maximum handling size trial, five fish from each age group(1-year-old and 2-year-old) and each genotype(transgenic and non-transgenic) of common carp were individually allowed to feed on B. aeruginosa with wide shell height range. The results showed that maximum handling size increased linearly with fish length, and there was no significant difference in maximum handling size between the two genotypes. In the size selection trial, three pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on three size groups of B. aeruginosa. The results show that the two genotypes of C. carpio favored the small-sized group over the large-sized group. In the species selection trial, three pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on thin-shelled B. aeruginosa and thick-shelled R. auricularia, and five pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on two gastropods species( P. sinensis and A. longicornis) with similar size and shell strength. The results showed that both genotypes preferred thin-shelled Radix auricularia rather than thick-shelled B. aeruginosa, but there were no significant difference in selectivity between the two genotypes when fed on P. sinensis and A. longicornis. The present study indicates that transgenic and non-transgenic C. carpio show similar selectivity of predation on the size-and species-limited gastropods. While this information may be useful for assessing the environmental risk of transgenic carp, it does not necessarily demonstrate that transgenic common carp might have lesser environmental impacts than non-transgenic carp.

Effect of Certain Toxicants on Gonadotropin-Induced Ovarian Non-Esterified Cholesterol Depletion and Steroidogenic Enzyme Stimulation of the Common Carp Cyprinus carpio in vitro

Isolated ovarian tissues from the common carp, Cyprinus carpio were incubated in vitro to obtain a discrete effect of four common toxicants of industrial origin, namely phenol, sulfide, mercuric chloride and cadmium chloride, on gonadotropin-induced alteration of nonesterified and esterified cholesterol and steroidogenic enzymes, △5-3β-HSD and 17β-HSD activity. Stage II ovarian tissue containing 30-40% mature oocytes were shown to be most responsive to gonadotropins in depleting only nonesterified cholesterol moiety and stimulating the activity of both. Safe doses of above mentioned toxicants when added separately to stage II ovarian tissue with oLH (1 μg/incubation) gonadotropin-induced depletion of nonesterified cholesterol and gonadotropin-induced stimulation of the activity of both enzymes was significantly inhibited. Esterified cholesterol remained almost unaltered. Findings clearly indicate the impairment of gonadotropin induced fish ovarian steroidogenesis by the four toxicants separately.

油酸诱导建鲤(Cyprinus carpio var. Jian)肝细胞脂肪变性模型的建立

通过对油酸诱导时间及有效作用浓度的研究,建立油酸致建鲤肝细胞脂肪变性模型。本研究以胰蛋白酶消化法获得的原代肝细胞作为实验材料,加入不同浓度油酸(0、0.05、0.1、0.2、0.4、0.8 mmol/L)与肝细胞共培养24–48 h,诱导肝细胞脂肪变性,分别收集不同时段的肝细胞及上清液,采用试剂盒测定肝细胞中甘油三酯(TG)、总胆固醇(TC)的含量,同时测定上清培养液中谷丙转氨酶(GPT)、谷草转氨酶(GOT)、γ-谷氨酰转肽酶(γ-GT)、乳酸脱氢酶(LDH)和超氧化物歧化酶(SOD)的活性,MTT法测定肝细胞的存活情况,确定油酸最佳诱导浓度,油红O染色法观察细胞内脂肪滴的形成情况。研究结果显示,0.4 mmol/L油酸与肝细胞共培养48 h,可以显著提高肝细胞内TG、TC含量(P﹤0.05或P﹤0.01),极显著提高上清培养液中GOT、GPT、LDH、γ-GT的活性,显著降低上清培养液中SOD的活性,但与24 h相比有升高趋势,0.4 mmol/L油酸与肝细胞共培养24 h,光镜下可见细胞内有脂肪滴形成,以48 h最明显。综上所述,油酸浓度0.4 mmol/L、诱导时间48 h成功构建了肝细胞脂肪变性模型,为研究鱼类脂肪肝类疾病奠定了基础。

鲤(Cyprinus carpio)Rh糖蛋白家族基因的克隆与组织mRNA表达

为了研究鱼类氨代谢机制,克隆了鲤(Cyprinus carpio)Rh糖蛋白家族的Rhag、Rhbg、Rhcg1基因的cDNA全序列,与人类Rh基因的氨基酸序列比对结果显示,鲤Rh糖蛋白保守性较高。系统分析表明,鲤3个Rh基因与斑马鱼(Danio rerio)Rh基因的亲缘关系较近。Rhag、Rhbg、Rhcg1基因在鲤鳃组织中表达较高,而在其它组织表达的相对量极低。比较3个基因在鳃组织的表达,Rhbg的表达量极显著高于其它2个基因。这一结果预示Rhag、Rhbg、Rhcg1与鲤的氨代谢密切相关,尤其是Rhbg基因,可能是鲤氨代谢的重要因子。

Effect of Mercuric Chloride and Cadmium Chloride on Gonadal Function and Its Regulation in SexuallyMature Common Carp Cyprinus carpio

Gonadal function in fish, CJprinus carpio was significantly affected by sublethal doses of mercuric chloride (HgCl2 ) and cadmium chloride (CdCl2 ) in chronic (45 days ) exposure.Parameters investigated were nonesterified (NE) and esterified (E) cholestcrol of ovary,liver and serum and ovarian 3β-Hydroxysteroid and 17β-Hydroxysteroid dehyrogenaseenzyme activity and serum and pituitary gonadotropin (GtH) levels. Both the pollutantswere able to reduce the hypothalamic extract (HE) or gonadotropin releasing hormone(GnRH) induced pituitary GtH release in vitro. Short term (96h) exposure of the fish tothe pollutants had no significant effect on the gonadal function. ln addition to thedeleterious effect of pollutants on the gonadal steroidogenesis and pituitary gonadotropin release, using [4-14C] cholesterol as a tracer it was found that for 45 days expeure, HgCl2'had an adverse effect on the transport of cholesterol from circulation to ovary.

鲤鱼(cyprinuscarpio)体细胞核移植的初步研究

以黑龙江野鲤的吻端和尾鳍组织为材料进行细胞体外培养，所得原代细胞进行传代，并以传至4～10代的尾鳍继代培养细胞作为供体，以同种鲤及鲫成熟具核未受精卵作为受体，采用显微注射方法进行移核，最终获得了发育至原肠胚期的胚胎．

The Use of Cyanobacteria Arthrospira platensis and Cladoceran Daphnia magna as Complementary Protein and Lipid Sources in Transitional Diet for Common Carp (Cyprinus carpio L.) Nursery

This study was conducted to evaluate the use of cyanobacteria Arthrospira platensis and cladoceran Daphnia magna biomass as complementary protein and lipid sources in diet supplementation for common carp (Cyprinus carpio L.) nursery. Three experimental diets containing A. platensis and D. magna meal at different concentrations were compared to the commercial (control) diet. Each experimental diet (ED) was set to contain D. magna and A. platensis meal at a specific combination: 2% and 5%, 2% and 7%, and 4% and 5% for ED1, ED2 and ED3, respectively. The protein and lipid content of the experimental diets ranged from 43.20% to 44.60% dry weight (DW) and 10.64% to 13.42% DW, respectively;while the protein and lipid content of the control diet were 43.00% DW and 6.72% DW, respectively. After 20 days of feeding period, ED3 group obtained the highest final body weight (BW) (58.18 ± 35.24 mg), total biomass (1936 ± 1625 mg), food conversion rate (1.34 ± 0.04), and specific growth rate (12.86 ± 0.03% BW day–1) among all treatment groups (P > 0.05);while ED1 group obtained the highest survival (75.5% ± 7.47%) among all treatment groups (P > 0.05). The total bacterial count and total pathogenic Aeromonas sp. in the culture water of the treatment groups (ranged from 2.00 to 2.65 × 105 CFU mL–1 and 3.50 to 4.12 × 103 CFU mL–1, respectively) were lower compared to the water of the control group fed (3.73 × 105 and 4.70 × 103 CFU mL–1, respectively). No significant differences in physicochemical water quality parameters were observed among treatments (P > 0.05). The current study suggests that the combination of A. platensis and D. magna biomass can be used as complementary protein and lipid sources in diet supplementation for common carp larvae and can result in a comparable fish growth, survival and feed utilization in common carp culture at the nursery phase.

鲤鱼(Crprinus carpio)体细胞系的建立及其生物学特性分析(简报)

动物细胞的培养技术是1907年哈里逊[1]在淋巴块中对蛙的神经板培养成功开始的,其后近一个世纪以来,陆续成功地培养了哺乳动物、昆虫等各种动物细胞,并广泛用于生物科学的各个分支.鱼类的细胞培养的系统研究和建系实践大约起始于60年代,被公认的真骨鱼类的第一个永久性的细胞系--虹鳟性腺细胞系(RTG-2)是由Wolf[2]建立的.

镜鲤体质量、体长、体高和体厚的QTL定位分析

利用SLAF-seq高通量测序技术,对107尾6月龄的镜鲤(Cyprinus carpio)F1群体的DNA样本进行基因分型,采用区间作图法定位分析镜鲤体质量、体长、体高和体厚4个生长相关性状的QTL。共检测出4个生长性状显著相关的29个QTL区间,分别定位到11个连锁群上(LG3、LG5、LG6、LG7、LG12、LG16、LG30、LG38、LG44、LG46和LG47),LOD值介于2.50~4.09之间,解释表型变异介于9.0%~24.2%之间。解释表型变异最大的为QTL6_5,分别解释体质量和体长性状表型变异的24.2%和24.1%。LG5、LG6和LG38的连锁群最多,均为6个QTL区间;其次是LG30和LG12,分别为3个和2个,其余6个连锁群(LG3、LG7、LG16、LG44、LG46和LG47)均含有1个QTL区间。本研究还发现17个QTLs在不同生长性状间共享,其余12个QTLs仅与一个性状相关。QTL区间与鲤全基因组物理图谱比对发现,在12个QTL区间存在生长相关候选基因。本研究结果可用于镜鲤生长性状分子标记辅助选择,以提高育种效率。

Fish Production, Water Quality and Bacteriological Parameters of Koi Carp Ponds Under Live-food and Manure Based Management Regimes

To test the effectiveness of introducing live zooplankton against direct manuring in ornamental fish ponds upon their survival and production, larvae of koi carp, Cyprinus carpio L., were cultured for 11 weeks in earthen ponds maintained according to four management regimes： （1） live zooplankton fed to carp larvae （LF）; （2） direct fertilization with poultry manure （PM）; （3） direct fertilization with cowdung （CD）; and （4） a control treatment （C）. There were three replicates for each treatment. The growth of heterotrophic bacteria and pathogenic microorganisms like Aeromonas sp. and Pseudomonas sp. were also examined in response to pond management. Values of dissolved oxygen were significantly higher （P〈0.05） in the water of LF ponds, compared to other treatments, while the PM and CD treatments recorded were significantly higher （P〈0.05） values of PO4 - P, NH4 - N, NO3 - N, NO2 - N, specific conductivity, alkalinity, and BOD, compared to the LF and C treatments. The percentages of organic carbon and total nitrogen in the bottom sediments were higher in the PM and CD treatments compared to LF （P〈0.05）. Average counts of heterotrophic bacteria in the water of PM and CD ponds were significantly higher than other treatments （P〈0.05）. The development of Aeromonas sp. and Pseudomonas sp. were significantly higher （P〈0.05） in the PM and CD treatments. Weight gain of koi carp stocked in LF was significantly higher （P〈0.05） than that of fish in the other treatments. There was a significant difference in the survival rate of koi carp among the treatments ranging from 67.21% in C to 90.11% in LF. The results suggest that raising koi carp larvae in ponds and feeding them exogenously with zooplankton would support high rates of survival and production through maintenance of better water quality and greater abundance of zooplankton in the system. Significantly lower abundance of Aeromonas sp. and Pseudomonas sp. in the LF treatment considerably lowered any possibility of occurrence of bacterial disease.

苯并芘对鲤鱼胆囊的毒性效应及响应机制

为探讨苯并芘对鲤鱼胆囊的毒理机制,以鲤鱼(Cyprinus carpio)为试验材料,采用15 d慢性毒性试验(苯并芘质量浓度分别为0、0.025和0.25 mg/L),通过检测鲤鱼胆囊中苯并芘质量分数、抗氧化参数、转录应答及免疫基因表达。结果显示:胆囊组织中的苯并芘质量分数积累随着苯并芘胁迫质量浓度显著升高,侧面反映了胆囊在解毒和排毒过程中扮演着重要角色;B[a]P暴露后胆囊抗氧化酶活性(超氧化物歧化酶、过氧化氢酶及谷胱甘肽过氧化物酶)和丙二醛浓度均升高,表明机体可通过激活抗氧化酶系统以应对氧化应激;低质量浓度B[a]P暴露会引起鲤胆囊组织的免疫应答,激活Notch受体信号通路,进而引起细胞凋亡并消除自身组织中过多或异常的细胞;而高质量浓度B[a]P暴露会抑制RIG-I和Notch受体信号通路,引起胆囊免疫稳态失衡,细胞凋亡受到抑制,从而延长异常细胞的存活时间,并进一步激活免疫细胞,导致自身组织的损伤。本研究初步揭示了鲤胆囊细胞应对苯并芘暴露的可能自我保护机制,实现对水环境中多环芳烃污染的早期预警及生态风险评估提供理论基础。

Cloning and expression analysis in mature individuals of two chicken type-Ⅱ GnRH (cGnRH-Ⅱ) genes in common carp (Cyprinus carpio)

Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide fam-ily, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II pre-cursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplica-tion hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the ex-pression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.