三种生态环境下尼罗罗非鱼免疫因子的比较

随机选取海水池塘(盐度28)和淡水池塘养殖及水库天然增殖的3种不同生态环境下、体质量为200~500g的尼罗罗非鱼为研究材料,采用生物化学方法测定3种生态环境尼罗罗非鱼血清中碱性磷酸酶、酸性磷酸酶、过氧化物酶、谷胱甘肽还原酶的活性和蛋白质量浓度。结果显示,3种生态环境中罗非鱼血清中碱性磷酸酶和酸性磷酸酶活力差异不显著(P0.05),其余各组间罗非鱼血清中过氧化物酶和谷胱甘肽还原酶的活性及血清蛋白质量浓度差异显著(P0.05),酶活力和蛋白质量浓度依次为:水库海水池塘淡水池塘。试验结果表明,水库天然增殖的尼罗罗非鱼具有较高的磷酸酶活性。     

高温胁迫对仿刺参抗氧化酶等指标的影响

在15、18、21、24、27、30℃水温下,分别制备仿刺参福建连江群体和辽宁大连群体的体腔和内脏团提取液,测定超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶、乳酸脱氢酶、碱性磷酸酶活力及丙二醛含量。试验结果显示,仿刺参连江群体内脏团提取液超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶和乳酸脱氢酶活力最大时的水温比大连群体分别提高了3~9℃;高温(24、27、30℃)胁迫下,连江群体体腔和内脏团提取液超氧化物歧化酶、过氧化氢酶、乳酸脱氢酶活力均极显著高于大连群体(P0.01),其内脏团提取液谷胱甘肽过氧化物酶活力亦极显著高于大连群体(P0.01),其体腔液谷胱甘肽过氧化物酶活力在24℃和30℃时极显著高于大连群体(P0.01);仿刺参连江群体和大连群体体腔液碱性磷酸酶活性均在15℃时最大,内脏团提取液的碱性磷酸酶活性则均在18℃时最大;连江群体仿刺参体腔和内脏团提取液丙二醛含量在18~30℃下均显著低于大连群体(P0.05,P0.01)。研究表明,福建连江夏季高温驯化的仿刺参子二代群体,在高温胁迫试验中,其体腔和内脏团提取液中超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶、乳酸脱氢酶、碱性磷酸酶活力显著高于大连群体,丙二醛含量则显著低于大连群体,这些酶耐热性的提高,有利于仿刺参机体清除因高温胁迫产生多余的自由基,减少机体因高温胁迫造成的损害。     

不同罗非鱼品系感染无乳链球菌后对血液和肝胰腺生化指标的影响

为探究无乳链球菌(Streptococcus agalactiae)感染后对罗非鱼的血液和肝胰腺生化指标的影响,本研究以吉富罗非鱼(Oreochromis niloticus)抗病选育系F5代、奥尼罗非鱼(Oreochromis aureus × O. niloticus)、吉富罗非鱼“百桂”品系为对象,通过人工腹腔注射感染无乳链球菌,检测感染后不同时期肝胰腺和血液中的酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、总抗氧化能力(T-AOC)和溶菌酶(LZM)活性的变化。结果显示,奥尼罗非鱼、吉富罗非鱼抗病选育系F5代和吉富罗非鱼“百桂”品系的平均死亡率分别为55.4%、60.5%和78.6%,表明奥尼罗非鱼抗感染能力最强;3个品系感染无乳链球菌后,肝胰腺组织中的ACP、AKP、SOD和T-AOC酶活力均呈先升高后下降的趋势,其中,在感染后24 h,奥尼罗非鱼的AKP和CAT酶活力显著高于吉富罗非鱼抗病选育系F5代和吉富罗非鱼“百桂”品系;而感染后24 h,血清中ACP、AKP、LZM、CAT和T-AOC酶均显著升高,而SOD酶则下降,其中,奥尼罗非鱼的ACP、AKP、CAT和T-AOC酶活性明显高于吉富罗非鱼抗病选育系F5代和吉富罗非鱼”百桂”品系。综合比较6种酶在感染后不同时期的活性变化及3个品系的感染存活率,筛选出AKP和CAT作为评估罗非鱼抗无乳链球菌感染能力强弱的指标。     

金属蛋白酶对奥尼罗非鱼生长、消化率及非特异性免疫功能的影响

在基础饲料中分别添加0、0.03%、0.05%、0.10%的金属蛋白酶,饲喂奥尼罗非鱼56d,测定奥尼罗非鱼的生长、消化率及血清溶菌酶活力和超氧化物歧化酶活力(SOD)。结果表明,添加0.10%的金属蛋白酶显著提高了奥尼罗非鱼的增重率(P<0.05),降低了饵料系数(P<0.05);添加0.10%的金属蛋白酶显著提高了奥尼罗非鱼对蛋白质的表观消化率(P<0.05),添加0.05%和0.10%的金属蛋白酶均显著提高奥尼罗非鱼对干物质的表观消化率(P<0.05);在血清酶活力方面,添加0.10%的金属蛋白酶组SOD活力比对照组高(P<0.05)。因此,饲料中添加金属蛋白酶能显著促进罗非鱼生长和营养物质的利用,提高罗非鱼血清SOD活力。     

饲料中添加三聚氰胺对吉富罗非鱼肝脏酶活性的影响

在水温23~28℃下,将体质量80~100g的吉富罗非鱼饲养在模拟养殖系统中,投喂10g/kg的三聚氰胺饲料10d,然后再喂养不含三聚氰胺的饲料10d,考察了不同时间下吉富罗非鱼肝脏中过氧化氢酶、超氧化物歧化酶、7-乙氧基—异酚噁唑酮—脱乙基酶和谷胱甘肽-S-转移酶活性的变化。结果显示,在饲料暴露的第1d,显著诱导了吉富罗非鱼肝脏中过氧化氢酶、超氧化物歧化酶、7-乙氧基—异酚噁唑酮—脱乙基酶和谷胱甘肽-S-转移酶的活性,诱导率分别为104.2%、15.6%、27.7%和75.8%。随着暴露时间的延长,4种酶的活性均有所下降,在暴露第6d、10d、10d和第6d开始被抑制,抑制率分别为52.2%、17.4%、5.9%和9.0%。在停止投喂含三聚氰胺的饲料后,再次诱导过氧化氢酶、7-乙氧基—异酚噁唑酮—脱乙基酶和谷胱甘肽-S-转移酶的活性,但超氧化物歧化酶的活性依然受抑制。试验结束时4种酶的活性均恢复到暴露初期的水平,与对照组差异不显著。在饲料三聚氰胺暴露的10d中,吉富罗非鱼肝脏中过氧化氢酶、超氧化物歧化酶、7-乙氧基—异酚噁唑酮—脱乙基酶和谷胱甘肽-S-转移酶活性的活性均呈现出"诱导—抑制"的趋势,在停止暴露10d后,上升到正常水平。饲料中的三聚氰胺扰乱了吉富罗非鱼肝脏酶系的正常功能,停止暴露后可逐步恢复正常。     

提取方法对淡水鱼油提取率及品质的影响

以白鲢(Hypophthalmichthys molitrix)内脏为原料,采用复合蛋白酶(Protemax)水解法提取鱼油,研究酶解条件和提取方法对鱼油提取率、品质及脂肪酸组成的影响,以获取高品质的鱼油制品.结果表明:提取方法和条件对鱼油的提取率、酸价及过氧化值(POV)均有明显影响;采用复合蛋白酶水解法提取鱼油时,其适宜的条件为酶解起始pH 7.5,酶解温度50 ℃,酶用量2 000 U/g粗蛋白,酶解时间3 h,鱼油的提取率达到85.67%,鱼油品质较好.复合蛋白酶水解法和碱性蛋白酶(Alcalase)水解法的提取率高于稀碱水解法,且复合蛋白酶水解法得到的n-3系列多不饱和脂肪酸含量更高.     

辛硫磷对岩原鲤肝脏抗氧化防御系统的胁迫与生物响应

利用不同浓度的辛硫磷进行10d暴露试验,测定了辛硫磷对岩原鲤（Procypris rabaudi）的急性毒性效应和亚慢性毒性条件下,鱼体内过氧化物酶（CAT）、超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、总胆碱酯酶（TChE）的活性和一氧化氮酶（NOS）、丙二醛（MDA）含量的变化,以期初步分析辛硫磷...     

罗非鱼肠蛋白酶的分离纯化及其性质

以罗非鱼肠为原料,采用超声波辅助提取技术、电泳技术和层析技术等对罗非鱼肠道蛋白酶进行研究,结果表明:将鱼肠匀浆经超声波提取的粗酶液,用30%～70%的硫酸铵盐进行盐析、HitrapTM Q FF阴离子交换柱纯化及Sephadex G-100凝胶柱分离纯化,得到罗非鱼肠蛋白酶纯品,其比活为335U/mg,得率为32.8%;SDS-PAGE电泳为单一蛋白酶带,分子量为28ku。该酶最适pH为8.0～8.5,在pH7.0～9.0的条件下稳定;最适温度为37～42℃,热稳定性好;该酶的Km值和Vmax值分别为0.605g/L和9.407μg/min。金属离子Ag+、Pb2+对蛋白酶有完全抑制作用,Na+、K+对该酶无抑制作用。丝氨酸蛋白酶抑制剂能完全抑制该酶活性,胃蛋白酶抑制剂和脲素对该酶有一定抑制作用,EDTA没有明显抑制作用,DTT能激活该酶活性,该酶为丝氨酸蛋白酶。     

罗非鱼肝脏中超氧化物歧化酶的提取、纯化与分析

以奥尼罗非鱼肝脏为原料,研究加热法提取超氧化物歧化酶(SOD)的工艺条件,丙酮沉淀回收酶法和HitrapTMQ FF阴离子柱纯化Cu,Zn-SOD,并对其酶学性质进行分析。结果表明加热法提取SOD时,在缓冲液中加入10mmol.L-1CuCl2,调节pH为5.6,65℃进行加热处理,可以提高SOD粗酶的稳定性和得率,其得率为57%,粗酶比活为(2580±6)U.mg-1。对酶的酶学性质研究表明纯化后SOD比活为(4895±2)U.mg-1,SDS-PAGE电泳为单一蛋白酶带,分子量为36ku,最大紫外吸收波长为265nm。该酶在pH6.0~9.0具有较好的稳定性,在75℃以下稳定,具有较好的耐热性。氰化钾、脲素、β-巯基乙醇、H2O2、对Cu,Zn-SOD具有明显的抑制作用;EDTA浓度小于3mmol.L-1时,对Cu,Zn-SOD活性有明显增强作用,EDTA浓度大于3mmol.L-1时,对Cu,Zn-SOD活性有抑制作用;DTT的修饰使Cu,Zn-SOD活性显著增加。为进一步研究和应用罗非鱼肝脏SOD提供了基础参数。     

光合细菌对罗非鱼鱼苗养殖水质及抗病力的影响

向罗非鱼鱼苗的养殖水体中投入光合细菌,研究不同浓度光合细菌对罗非鱼鱼苗养殖水体水质和鱼苗免疫力、成活率的影响.结果表明,水体中引入光合细菌浓度为4.5×102～4.5×106 cfu/mL时,各试验组的氨氮和亚硝酸盐氮含量都显著低于对照组(P<0.05);当浓度为4.5×104 cfu/mL时,罗非鱼鱼苗的抗病力增强,表现在鱼苗体内的组织过氧化物酶(POD)、碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)、溶菌酶(LSZ)和抗菌活力都显著高于对照组(P<0.05),罗非鱼鱼苗的存活率也提高了6.67%.适宜浓度的光合细菌,能有效改善鱼苗养殖水体的水质,提高鱼苗免疫力和成活率.     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.