三倍体雌性虹鳟性腺发育阶段细胞色素相关基因的表达

Cyp19a1b、Cyp11a1、Cyp11b2、Cyp17a1、Hsd3b1等细胞色素相关基因能够调节硬骨鱼类性类固醇的合成,对性腺发育和性别决定产生影响。本研究以全雌三倍体虹鳟(Oncorhynchus mykiss)为研究对象,正常雌性二倍体虹鳟为对照,选取31~68 dpf(days post fertilization)时间段的虹鳟仔鱼脑组织,采用q RT-PCR和酶联免疫的方法研究以上几种基因的表达状况和脑芳香化酶的活性变化,以期探明导致三倍体雌性虹鳟性腺发育异常的关键原因。q RTPCR结果显示,二倍体中Cyp19a1b在30~50 dpf时表达量上调并且维持在较稳定水平,但50~56 dpf时表达量逐渐下调,之后56~68 dpf表达量持续上调;三倍体中Cyp19a1b表达量在30~35 dpf开始上调,35~47 dpf逐渐下调,47~55 dpf开始第二次上调,之后维持在较稳定水平直至68 dpf,但三倍体Cyp19a1b的表达量显著(P0.05)低于同期二倍体的。二倍体Cyp11a1表达量在34 dpf出现峰值,三倍体Cyp11a1在38 dpf时出现峰值。二倍体Hsd3b1表达量在33~42 dpf时维持在较高水平,在38 dpf时出现高峰;三倍体Hsd3b1表达量在47~59 dpf时较高,在49 dpf出现高峰。二倍体中Cyp11b2在37 dpf出现峰值,之后开始下调;三倍体在40 dpf出现峰值,之后逐渐下调,但三倍体Cyp11b2表达量显著低于同期二倍体。二倍体Cyp17a1的表达量在35~46 dpf时逐渐上升,在45 dpf时达到高峰之后直至69 dpf逐渐下降,并且维持在较为平稳的水平上;但是在相同的实验条件下未检测到同一时期三倍体Cyp17a1的表达量。酶联免疫结果显示,在40 dpf时二者的脑芳香化酶活性到达高峰,但在40~60 dpf时期,二倍体虹鳟脑芳香化酶活性显著(P0.05)高于三倍体虹鳟,尤其在45~50 dpf时,该酶活性分别较三倍体的高1.15倍和1.12倍。以上结果表明三倍体虹鳟早期性腺发育迟缓的原因之一是Cyp19a1b、Cyp11a1、Cyp11b2、Cyp17a1、Hsd3b1等基因的表达晚于二倍体,且表达量低于二倍体,造成雌二醇不能正常合成,最终导致性腺发育迟缓。     

Embryonic expression of UCP2 in rainbow trout (Oncorhynchus mykiss)

Uncoupling proteins are mitochondrial anions transporters that dissociate respiration from ATP synthesis through proton leaks. Uncoupling protein 2 reportedly plays a role in several physiological processes such as energy partitioning, nutrition, and fatty acid metabolism. The mRNA expression of rainbow trout UCP2 genes (UCP2A and UCP2B) was monitored during embryogenesis and early larval development. Both genes were recruited early and displayed similar steadily decreasing patterns from fertilization until hatching. The expression of UCP2A and UCP2B appeared significantly differentiated after hatching and during the yolk sac absorption, with UCP2A displaying higher expression. We suggest that UCP2 expression profiles in the rainbow trout embryo could be associated with the utilization of lipids as a source of energy during development.     

Edwardsiellosis in farmed rainbow trout (Oncorhynchus mykiss)

Edwardsiella tarda was isolated from naturally infected rainbow trout (Oncorhynchus mykiss) in raceway culture on a commercial fish farm where the fish were kept at a density of 110 kg m^?3 and at a water temperature of 14°C and with a photoperiod of 13 h light:11 h dark. The clinical signs of diseased fish (150 ± 20 mm standard length) were anorexia and lethargy. The most striking lesions in the fish were in the liver. There were hyperaemia and haemorrhages; on histopathological examination, the liver displayed inflammatory infiltrate in portal area, focal necrosis, dilatation of blood sinus and activation of sinusoidal cells. Infection experiments, performed 2 years after isolation of the original culture of E. tarda, were carried out under laboratory conditions at water temperatures of 15, 18 or 24°C. All experimental fish (common carp, Prussian carp, tench), intraperitoneally injected with 8 × 10⁶ cells, demonstrated a total resistance to E. tarda.     

Metabolism of exogenous histamine in rainbow trout (Oncorhynchus mykiss)

Information on the metabolism of exogenous histamine in fish is of much concern regarding the effect of dietary histamine on fish. Histamine catabolic enzymes, diamine oxidase and histamine N-methyl transferase were measured in the tissues of rainbow trout. Diamine oxidase was detected in the stomach, pylorus caeca and intestine. Histamine N-methyl transferase was detected only in the liver.A change in the contents of histamine and its metabolites was observed in the tissues of rainbow trout after oral administration of histamine. A large amount of imidazole acetic acid was observed in the serum, kidney, liver and muscle. On the other hand, 1-methyl histamine was detected only in the liver. Histamine and its metabolites, imidazole acetic acid and 1-methyl histamine were metabolized and diminished within 48 hr in all tissues.These results showed that histamine was metabolized by two metabolic routes in rainbow trout. One is the main pathway producing imidazole acetic acid by intestinal diamine oxidase and the other is the complementary pathway producing 1-methyl histamine by liver N-methyl-transferase.     

Identification of a novel gill-specific calpain from rainbow trout (Oncorhynchus mykiss)

Calpains are calcium-dependent neutral proteases responsible for many cellular functions. The two forms of calpain ubiquitously expressed in mammalian tissues are known as μ-calpain and m-calpain. We report here the identification of a novel calpain that is similar to but distinct from the μ- and m-calpains in rainbow trout. The cDNA of the novel gene is 2623 bp in length with a single open reading frame. The predicted protein (676 amino acids) contains the conserved calpain characteristic domains that include: domain I (pro peptide), II (cysteine catalytic site), III (electrostatic switch), and IV (calmodulin-like) with five Ca²⁺-binding EF hands. Northern blot and RT-PCR analyses demonstrated that the novel calpain gene is predominantly expressed in rainbow trout gills. Comparison of the novel protein with the ubiquitously expressed calpains and several mammalian tissue-specific calpains revealed that the novel calpain is an orthologue of the mammalian digestive tract specific calpain (calpain 9).     

Nutritional regulation of somatostatin expression in rainbow trout, Oncorhynchus mykiss

Previously we characterized three DNAs from the endocrine pancreas (Brockmann body) of rainbow trout (Oncorhynchus mykiss) that encode for distinct preprosomatostatin (PPSS) molecules: one containing somatostatin-14 and its C-terminus (PPSS I) and two containing [Tyr⁷, Gly¹⁰]-somatostatin-14 at their C-termini (PPSS II′ and PPSS II″). In the present study, the regulation of PPSS expression was studied in rainbow trout placed on varying nutritional regimes (fed continuously, fasted, fasted then refed). Fish that were fasted for one week displayed reduced growth compared to their fed counterparts, but no alteration in pancreatic PPSS expression was noted between the two groups. Fish fasted for 4 and 6 weeks also were growth retarded and displayed increased levels of PPSS I mRNA and PPSS II″ mRNA compared to fed animals; PPSS II′ mRNA levels were not affected by food deprivation. Refeeding fish for two weeks following 4 weeks of food deprivation restored growth and reduced PPSS I and PPSS II″ mRNA expression to levels similar to those displayed by continuously fed fish. Changes in PPSS expression were correlated with increases in plasma SS. These results indicate that nutritional state modulates differential expression of PPSSs. This revised version was published online in July 2006 with corrections to the Cover Date.     

冷藏和冰藏条件下虹鳟鱼鱼片品质变化研究

为了科学地了解虹鳟鱼(Oncorhynchus mykiss)鱼片贮藏过程中的品质变化规律,以建立高效的品质控制技术,研究了虹鳟鱼鱼片在冷藏(3±1)℃和冰藏(0±1)℃条件下挥发性盐基氮(TVB-N)、色泽、腺苷三磷酸(ATP)关联物、K值及菌落总数(TAC)等指标的变化,评价不同温度对虹鳟鱼片品质的影响。结果显示:虹鳟鱼片TVB-N的增长速度在冷藏条件下显著高于冰藏条件,冷藏至第9天为20.72 mg/100g,冰藏至第15天为25.76 mg/100g;冷藏至第6天TAC为7.40 lg cfu/g,冰藏至第12天TAC为8.27 lg cfu/g;与冰藏相比,冷藏条件下虹鳟鱼片K值较高;肌苷酸(IMP)含量分别在冷藏8 h(8.36μmol/g)和冰藏24 h(8.70μmol/g)达到最大值,即冷藏和冰藏虹鳟鱼片的最佳食用时间分别是宰杀后的8 h和24 h。结合各项指标变化,冷藏和冰藏虹鳟鱼片的货架期分别为6 d和12 d。     

Nitrogen waste from rainbow trout (Oncorhynchus mykiss) with particular focus on urea

Particulate and dissolved nitrogen (N) waste components are removed in recirculating aquaculture systems (RAS) using different cleaning technologies, and to dimension and optimize their removal efficiency requires that the expected daily load of the different waste forms can be estimated. Using a laboratory, mass-balance approach, the current study examined the effects of commercially applied feeding levels on the loading of different N waste forms, including daily fluctuations in dissolved total nitrogen (TN), total ammonia nitrogen (TAN), urea-N, and non-characterized, dissolved N deriving from juvenile rainbow trout (Oncorhynchus mykiss). In addition, the study examined whether there was a removal of urea-N across a moving bed biofilter operated as end-of-pipe under commercial conditions. The laboratory, mass-balance study showed that there were no effects of feeding levels (1.3, 1.5 or 1.7% of the biomass per day ) on the excretion of dissolved N components, which constituted the majority of total N waste (>81.6% on average). The excretion of urea-N and non-characterized, dissolved N components constituted 12–13% and 9–11%, respectively of dissolved TN. The excretion of urea-N was largely constant and independent of the daily feeding practice, whereas that of non-characterized N appeared to reflect the daily feeding activity, following the trends in TN and TAN. The time limited feeding regime applied in the laboratory study resulted in a pulse in the excretion of TAN that a biofilter may be unable to fully level out, potentially resulting in unnoticed, critical water quality conditions in intensive RAS during certain times of the day. Particulate N waste constituted a minor fraction of total N waste (<18.4% on average), and the actual loading depended on the digestibility of dietary protein/nitrogen. Results from the commercially operated, nitrifying biofilter showed that urea-N was removed at a rate of 0.014 g N m² day⁻¹. Compared to the removal of TAN (0.208 g N m² day⁻¹), the moving bed biofilter was 1.07 times more active in removing dissolved N than immediately expected when only considering TAN.     

The effectiveness of adenoviral vectors to deliver and express genes in rainbow trout,Oncorhynchus mykiss (Walbaum)

The efficacy of adenoviral vectors for gene delivery into fish cells, both in vitro and in vivo, was evaluated. Vectors utilized were of human adenovirus serotype 5 (Ad), which are commonly used in human clinical trials, but have not been assessed for gene delivery to fish. Because nothing is known about Ad receptors in fish, both an Ad (Ad5Luc1) with natural tropism for the coxsackie and adenovirus receptor (CAR), as well as an infectivity enhanced Ad (Ad5LucRGD) were included within this study. Gene expression was detected in cell lines using either vector. The levels seen with Ad5LucRGD were much higher than for Ad5Luc1 in most lines except CHSE-214. Transduction of CHSE-214 cells with Ad5Luc1 could be blocked with an excess of a competitive inhibitor, suggesting that these cells possess a CAR homologue thatmediates attachment of Ad, similar to that seen in mammalian cells. In vivo gene delivery was attempted by several methods, with significant expression seen only via intramuscular injection, although infection efficiency was low. Thus it was observed that several teleost cell lines are capable of being infected and one cell line expressed a human serotype adenoviral receptor homologue that aids in Ad infection. Additionally, in vivo studies indicated that muscle tissue of rainbow trout could be infected with Ad vectors, suggesting an alternative gene delivery strategy for this animal.     

Androgen metabolism in the skin of the rainbow trout (Oncorhynchus mykiss)

Pieces of skin of male and female rainbow trout (Oncorhynchus mykiss) were incubated with testosterone and 11-ketotestosterone as substrates. In immature fish the conversion rate was low. In non-spawning adult males 11-ketotestosterone was reduced to 5α-11KDHT (up to 5.2%). In the fish in spawning condition the 5α-reduction rate was only about 1 to 2%. In the same specimens incubated with testosterone a high 11β-hydroxylase activity (23.8-25% in the male and 13% in the female skin) was found. Similar sex specific differences were observed for the occurence of 5α-reduced metabolites (about 20% in the male and 13% in the female tissue).

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Résumé Des fragments de peau de truites arc en ciel (Oncorhynchus mykiss, males ou femelles, ont été incubés avec de la testostérone ou de la 11-cétotestostérone, utilisées comme substrats. Chez les poissons immatures, les taux de conversion restent faibles. Chez les males adultes ne donnant pas de sperme, la 11-cétotestostérone est réduite en 5α-androstane-17β-ol-3,11-dione (jusqu'à 5.2%). Chezles poissons en conditions de fraie, le taux de 5α-réduction est seulement de l'ordre de 1 à 2%. Pour ce derniers individus, les incubations de peau en présence de testostérone montrent l'existence d'une forte activité 11β-hydroxylase (23.8-25% pour le male, et 13% pour la femelle). Des différences liées au sexe sont observées de la même manière dans la production de métabolites 5α-réduits (environ 20% avec le tissu male et 13% avect le tissu femelle).

     

虹鳟生长性状的随机回归分析

生长和抗逆是水产动物遗传育种工作中最重要的农艺性状,虹鳟的生长性状关乎虹鳟规模化养殖的生产经济效益,为了从遗传上精细解析虹鳟的生长性状,我们从渤海、丹麦、挪威、唐纳森和加利福尼亚5个虹鳟(Oncorhynchus mykiss)种系间的双列杂交开始,进行了连续4代的继代选育。本研究测量了第4代总共4368个实验个体在516日龄、608日龄、668日龄、883日龄和1036日龄5个时间点的生长数据。采用随机回归测定日模型,对虹鳟生长性状进行了动态遗传分析。根据贝叶斯信息准则,确定3阶勒让德多项式为拟合体重和体长的加性遗传效应和永久环境效应变化的最优子模型。利用双变量随机回归模型同时分析体重和体长两个生长性状。它们的遗传力在400～1000日龄之间呈现递减趋势,分别从0.288下降到0.164和从0.469下降到0.186,并且在该生长区间内体长的遗传力始终高于体重的遗传力。无论体重还是体长性状,在不同日龄之间的遗传相关都随着生长间隔的增大而降低,但是两个性状在生长初期和后期之间的遗传相关较高(遗传相关系数0.75以上),尤其是体重(遗传相关系数0.85以上),该研究结果为虹鳟早期的遗传选育提供了理论支撑。两个性状之间在相同日龄之间的遗传相关均在0.75以上,在不同日龄之间的遗传相关随着生长间隔的增大由0.83下降到0.63。以上的研究结论为虹鳟生长性状(主要是体长和体重)的遗传选育提供了理论基础,同时也为虹鳟的体长和体重两个性状的联合选育提供了精确的遗传分析结果,由于两性状在前期有较高的遗传相关,因此建议在虹鳟生长前期(400日龄)进行联合选择。