诺氟沙星在西伯利亚鲟体内蓄积规律及其对组织的病理损伤

为了确定诺氟沙星在鲟养殖中的科学使用方法,对不同剂量、不同给药次数情况下诺氟沙星在西伯利亚鲟(Acipenser baerii)体内蓄积规律及毒性进行了研究。分别以0、30 mg/kg、50 mg/kg、100 mg/kg的剂量对西伯利亚鲟连续口灌诺氟沙星3~5 d,每天1次,并于停药后24 h及240 h采集实验鱼血、肝、肾及软骨组织,对4种组织中药物蓄积量进行测定,并对肝、肾和软骨组织进行切片观察。实验结果表明:随着给药剂量增加、给药次数增多,实验鱼4种组织中诺氟沙星浓度逐渐升高,浓度由高到低依次为为:肾、软骨、肝、血清,且血清中药物浓度远远低于另外3种组织。相同给药剂量、不同给药次数时血清中药物浓度无显著差异;而肝、肾和软骨中均差异显著,停药240 h后,显著性消失。切片结果表明,50 mg/kg剂量连续给药5 d后,实验鲟肝、肾组织开始结构损伤变化,100 mg/kg剂量连续给药3~5 d后,损伤情况持续加重,停药240 h后,肝组织损伤情况有逐渐恢复趋势,而肾组织未见明显恢复趋势;而100 mg/kg连续给药3~5 d后,软骨组织出现软骨膜内层细胞减少;停药240 h后有恢复趋势。研究结果表明,诺氟沙星在鲟中使用剂量应小于50 mg/kg,连续给药次数低于5次,以避免对鲟肝组织造成损伤,引起肾不可逆损伤及影响软骨细胞发育和再生,对软骨组织造成潜在影响。     

Osmoregulation in juvenile Siberian sturgeon (Acipenser baerii)

The osmoregulation capabilities of juvenile Siberian sturgeon exposed to three experimental osmolalities (22, 250 and 387 mOsmol kg⁻¹) were studied over a 45-day period. Growth performance, haematological parameters, ion concentrations, gill and spiral valve Na⁺-K⁺-ATPase activities, as well as gill and spiral valve histology, were measured. At the end of the period, the plasma osmolality of fish kept in 250 and 387 mOsmol kg⁻¹ was higher than that of fish kept in 22 mOsmol kg⁻¹. Similar trends were observed in electrolyte concentrations. Spiral valve and gill Na⁺-K⁺-ATPase activity varied with exposure time and environmental salinity. Shortly after being transferred to hyperosmotic media, spiral valve Na⁺-K⁺-ATPase activity fell, while gill Na⁺-K⁺-ATPase activity remained constant. At the end of the experiment, gill Na⁺-K⁺-ATPase activities in fish kept in isosmotic and hyperosmotic media had increased in comparison to those of the control fish. Moreover, spiral valve Na⁺-K⁺-ATPase activities recovered and were similar to those recorded in fish kept in hyposmotic environments. Although some of the morpho-physiological mechanisms were operational in juvenile Siberian sturgeon in their adaptation to hyperosmotic media, fish cannot be considered hyperosmotic regulators as they were unable to maintain their plasma osmolality and electrolyte equilibrium in salinities higher than 250 mOsmol kg⁻¹. This suggests that the culture of juvenile Siberian sturgeon in brackish environments is unlikely to be successful. However, our data indicate that in natural environments, juvenile Siberian sturgeon in migratory populations (Ob and Lena Rivers) would be able to migrate successfully into estuarine brackish grounds with a salinity of up to 9%. This revised version was published online in July 2006 with corrections to the Cover Date.     

人工养殖西伯利亚鲟精子超低温冷冻保存研究

研究了人工养殖西伯利亚鲟精子的生物学特征及超低温冷冻保存方法。西伯利亚鲟的产精量为113.67±39.86 ml,精子密度为(6.49±3.10)×108/ml,精子活力为(85.4±9.5)%,精子寿命为353±23 s。精子密度与精子快速运动时间、精子寿命之间均存在线性相关,用方程分别表示为:y=1.0384x+1.5089(R2=0.7325);y=2.9069x+74.289(R2=0.6967)。结果表明精子密度可作为一项精子质量评价的标准。通过比较西伯利亚鲟精子在不同稀释液、不同抗冻剂和抗冻剂浓度、降温速率、解冻温度下的保存效果,结果表明:配方2作为稀释液,18%甲醇作为抗冻剂,二步法超低温(-196℃)冷冻保存精子,40℃水浴解冻取得最好的冻后活力,解冻后活力为(51.8±5.8)%。西伯利亚鲟授精的最佳精卵比为106∶1。在此精卵比下用冻精授精分别得到了(72.3±3)%的受精率和(52.9±4.1)%的孵化率,其中受精率与鲜精没有显著性差异,孵化率与鲜精有显著差异(P<0.05)。     

西伯利亚鲟消化道形态学和组织学的初步研究

对人工养殖2龄西伯利亚鲟消化道的形态解剖学和显微组织学结构进行了观察。研究结果表明,西伯利亚鲟的消化道从前至后分为口咽腔、食道、胃、幽门盲囊、十二指肠、瓣肠和直肠,食道与胃的分界不明显,形成一个"食道-胃过渡区",胃分为贲门部、胃体部和幽门部。组织学研究显示:西伯利亚鲟的消化道由内向外一般分为粘膜、粘膜下层、肌层和浆膜四层。其中食道粘膜上皮为复层扁平上皮,含杯状细胞和粘液细胞。"食道-胃过渡区"已有腺体存在。胃上皮细胞为单层柱状上皮细胞,无杯状细胞,胃的粘膜表面有许多上皮凹陷形成的胃小凹,幽门部的胃小凹较贲门部和胃体部深;贲门部和胃体部中含有大量的胃腺,幽门部无胃腺。幽门盲囊的管腔内被向内深入的肌层分割成许多完全或不完全的囊腔;粘膜褶皱和褶皱上的绒毛纵横交错,形成网状。肠上皮均为单层柱状上皮,上皮细胞间分布有大量的杯状细胞;粘膜褶皱的高度和数量、杯状细胞的数量从前至后递减:十二指肠粘膜褶皱细而高,上有许多绒毛;瓣肠粘膜和粘膜下层向管腔内突出并卷曲,形成螺旋瓣;直肠的粘膜褶皱矮且宽。     

Optimization of sperm irradiation protocol for induced gynogenesis in Siberian sturgeon,Acipenser baerii

The great diversity of optimal UV irradiation doses are used for DNA inactivation in fish sperm forcing authors to repeat optimization of irradiation treatment every time. Analysis of sperm UV irradiation protocol for induction of gynogenesis showed the importance of sperm UV light absorption estimations. The UV absorption investigation in Siberian sturgeon sperm showed average extinction coefficient 7.69 × 10^?8 ± 1.04 × 10^?8 cm². It is resulted in high heterogeneity of UV irradiation of undiluted sperm samples. Therefore, it is strongly suggested to specify doses only with defined concentration of spermatozoa; otherwise, the difference in absorbance level between samples can bring a significant error to optimal UV dose estimation. This was confirmed by UV-irradiated sperm motility investigation. Results of motility investigation of UV-irradiated sperm revealed high sensitivity of Siberian sturgeon spermatozoa motion mechanisms to UV irradiation, with complete loss of motility after homogeneous UV irradiation at doses above 2,000 J/m². Partial gynogenesis was conducted using diluted and undiluted sperm. Ploidy level of hatched larvae was estimated by flow cytometry. Percentage of haploid hatched larvae revealed sperm DNA inactivation efficiency. The highest percentage of haploid putative gynogenotes 19.67 ± 4.19 % was obtained at UV irradiation dose 200 J/m² with sperm diluted to 1:4.     

Impact of starvation on digestive enzymes activities and plasma metabolites in Siberian sturgeon (Acipenser baerii,Brandt, 1869)

To increase the current knowledge about the relationship between nutritional status and the digestive capacity of Siberian sturgeon (Acipenser baerii), we addressed the effect of starvation‐refeeding and macronutrient composition on growth parameters and key digestive enzyme activities in A. baerii. Acipenser baerii juveniles were fed four different diets for 3 weeks, then starved for 2 weeks and allowed to refed for 5 weeks with the same diets. Another group of fish were fed 10 weeks with the corresponding diets. Among 10‐week fed fish, high‐protein diets promoted higher body weight values, while the lowest specific growth rate was observed in fish fed a low‐protein, medium‐carbohydrate, high‐lipid diet (p < .05). At the end of the experiment, in fish refed for 5 weeks following a feeding‐starvation cycle and in 10‐week fed animals, the higher levels of blood glucose, triglycerides and cholesterol were found in fish fed low‐protein diets (p < .05). In all treatments, 2 weeks of starvation decreased α‐amylase activity in the intestine (p < .05), while 4 days of refeeding increased lipase (p > .05) and α‐amylase activity in the intestine as well as pepsin in the stomach (p < .05). Our findings suggest that A. baerii maintains a high capacity to digest proteins and lipids after 2 weeks of starvation and that α‐amylase can be used as an indicator of the nutritional status in fish submitted to starvation‐refeeding cycles. Indeed, refeeding with high‐protein and CHO:L ratio diets after starvation could improve the growth rate of A. baerii in culture.     

西伯利亚鲟Sox9基因cDNA全长克隆、序列分析及其表达检测

为了研究Sox9基因在西伯利亚鲟侧线神经发育过程当中所起的调控作用,本研究利用RT-PCR和RACE方法获得了西伯利亚鲟Sox9基因cDNA全长序列,经序列分析表明,所克隆Sox9cDNA全长为1409bp,包括开放阅读框(open reading frame,ORF)1083bp,5′端非翻译区(untranslate dregion,UTR)19bp和3′端非翻译区(untranslate dregion,UTR)307bp。1083bp的ORF共编码360个氨基酸,相对分子质量为41221.7U。序列分析表明,其与施氏鲟的亲缘关系较近,其次是斑马鱼。经实时荧光定量PCR技术对西伯利亚鲟Sox9基因在各组织以及在胚胎发育各时期中的相对表达量检测发现,Sox9在西伯利亚鲟中的表达具明显的组织差异性和时间差异性。其中,在肝,肾,肌肉,胰中的相对表达量较低。然而,大脑组织中的表达量与以上4个组织相比,处于极高水平,达到胰脏中相对表达量8.9倍,达到表达量最低点肾脏中的近21倍。在胚胎发育的不同阶段,Sox9均有表达。在原肠期、神经胚期及视泡形成期,Sox9基因的表达量均较高,特别是在原肠期其表达量达到西伯利亚鲟...     

Expression of dmrt1 and sox9 during gonadal development in the Siberian sturgeon (Acipenser baerii)

The sex differentiation period of the Siberian sturgeon was investigated through expression profiling of two testicular markers (dmrt1 and sox9). At the molecular level, a clear sexual dimorphism of dmrt1 and sox9 was observed in 3-year-old fish with immature gonads, in which males showed higher expression of these genes. Among 16-month-old sturgeons cultured in Uruguay, gonad morphology analyses showed one group of fish with undifferentiated gonads and a second group which had started their histological differentiation into ovaries or testes. dmrt1 showed a significantly higher expression in testes of recently differentiated fish, but this was not the case for sox9. In undifferentiated fish, we observed two clearly different groups in terms of expression: one group of fish over-expressing male markers (dmrt1, sox9) and another group of fish showing very low expression of these genes. This suggests that fish undergoing male differentiation can be identified by their profiles of gene expression before they undergo morphological differentiation.     

西伯利亚鲟鱼池塘养殖试验

2004年8月-2005年2月,笔者在石家庄井陉县进行了西伯利亚鲟鱼池塘养殖,成活率达90％以上,现将养殖情况总结如下。     

Dietary zinc requirement of Siberian sturgeon (Acipenser baerii,Brandt 1869) juveniles,based on the growth performance and blood parameters

International Aquatic Research - This study investigated the effect of dietary Zn levels on growth performance, feed utilization, and hematological parameters of juvenile Siberian sturgeon...     

超低温冷冻对西伯利亚鲟精子形态结构损伤的观察

为改进西伯利亚鲟精子超低温冷冻保存技术,探讨精子损伤机制,应用扫描和透射电子显微镜,观察了西伯利亚鲟鲜精与冷冻后精子的形态结构.结果显示,经过冷冻保存后精子的形态发生了很大变化.精子顶体长、精子头中部宽、头中部宽与前部宽比值及中段宽与鲜精相比显著增加(P<0.05);中段长度、后外侧延伸物长度比鲜精显著变短(P<0.05).精子经过冷冻后有30.5%的精子在形态、结构上受到不同程度损伤,受损精子显微结构表现为顶体后外侧延伸物与核糅合,顶体内容物丢失;核膜囊泡化、核膜断裂,核内出现空泡;线粒体内嵴弥散,线粒体脱落;鞭毛外膜松弛与鞭毛脱离等.部分受损伤精子出现顶体丢失,中段脱落,鞭毛自中段基部断裂的现象.精子损伤主要集中在膜系统,中心粒等微管系统基本完好.     

运动功能相关的中华鲟和西伯利亚鲟幼鱼侧视形态比较研究

通过选取11个侧视形态特征度量进行主成分分析(principal component analysis,PCA),同时设置26个标记点进行相对扭曲分析(relative warp analysis,RWA),比较了中华鲟(Acipenser sinensis)和西伯利亚鲟(A.baerii)幼鱼在侧视形态上的差异以及相对扭曲分析和传统多元分析在鲟鱼形态研究中的优劣。结合前期对它们的有氧游泳能力比较结果(西伯利亚鲟比中华鲟强25%),分析了鲟鱼形态的水动力功能。主成分分析比较表明,中华鲟头长、头高、背鳍前基点之后的躯干高度、尾柄长、背鳍前缘长度均显著大于西伯利亚鲟(P<0.05),而尾鳍上叶显著短于西伯利亚鲟(P<0.05)。相对扭曲分析计算样本的几何信息并可视化统计结果,表明中华鲟的吻厚、吻长、头高、头长、躯干后半段高、背鳍前缘长显著大于西伯利亚鲟,而尾柄长和尾鳍上下叶长显著小于西伯利亚鲟(P<0.001)。上述形态特征直接影响着两种鲟鱼的游泳能力。     

Outbreaks of Streptococcosis associated with Streptococcus iniae in Siberian sturgeon (Acipenser baerii) in China

Streptococcus iniae has emerged as an important fish pathogen over the past few decades causing high losses in aquaculture farms all over the world. At least 27 species of fish have been documented to be infected by S. iniae, including cultured and wild populations. In August and October 2013, a serious infectious disease characterized by body ulcer, internal organs haemorrhages and nodules showing on epicardium occurred on the Acipenser baerii farms in Ya'an country, China. Histological examination revealed a multisystemic, necrotising inflammatory response that was particularly marked in liver, kidney, heart and brain. Mass mortality (>40%) was observed in infected fish and two Gram‐positive cocci (Ab130920 and Ab131025) were obtained from kidneys and livers of diseased fish. Experimental infections with these two isolates resulted in marked symptoms in the sturgeons similar to those observed in natural outbreaks, and the LD₅₀ values of the two isolates were 5.1 × 10⁵ and 6.4 × 10⁵ cfu per fish respectively. The two microorganisms were identified as S. iniae through physiological and biochemical tests, 16S rRNA and lctO gene sequence analysis. Both two isolates showed a similar antibiotic susceptibility, which were sensitive to ampicillin, amoxicillin, cefazolin, amikacin, deoxycycline, florfenicol, azithromycin, ciprofloxacin, vancomycin and resistant to streptomycin, gentamicin, kanamycin, norfloxacin and sinomin (SMZ/TMP). Multiplex PCR assay for virulence genes showed both isolates possessed six main virulence genes: simA, scpI, pdi, pgm, cpsD and sagA genes. These results indicated that S. iniae could act as a pathogen of farmed A. baerii. This is the first report of S. iniae infection associated with mass mortality in A. baerii.     

Long-term culture of a cell population from Siberian sturgeon (Acipenser baerii) head kidney

In vitro cultures of native fish cell lines are of great importance, both for basic research and applied science. In particular, there is strong demand for long-term growable cell lines from breeding fish, like sturgeon. Here, we describe the culture of cells from Siberian sturgeon (Acipenser baerii) head kidney. The cells have so far been cultured over a period of 12 months (24 passages). Cytochemical and immunocytochemical examination suggests that, in vitro, the cells exhibit markers that are indicative for different cell types. In particular, fat storing cells (adipocytes) were observed, and the expression of cytokeratins and glial fibrilar acidic protein (GFAP) can be concluded on the basis of immuncytochemical analysis. The observation of different morphologies additionally underlines the heterogeneity of the cell population and matches the typical behaviour of in vitro cultures of stem/progenitor cells. Different applications can be imagined.     

西伯利亚鲟CXCR7基因cDNA全长的克隆及其表达分析

采用RT-PCR和cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE),首次克隆了西伯利亚鲟(Acipenser baerii)的CXCR7a和CXCR7b基因的全长cDNA。CXCR7a的cDNA全长为2 325 bp(登录号为:JQ034508),包括207 bp的5'端非翻译区(UTR)、可编码362个氨基酸的1 086 bp的开放阅读框(ORF)及包括poly(A)尾巴在内的大小为1 032 bp的3'端非翻译区(UTR);CXCR7b的cDNA全长为2 423 bp(登录号为:JQ034509),包括209 bp的5'端非翻译区(UTR)、可编码370个氨基酸的1 110 bp的开放阅读框(ORF)及包括poly(A)尾巴在内的大小为1 104 bp的3'端非翻译区(UTR),并对它们编码的蛋白质序列的分子特征进行了分析。氨基酸序列相似性(similarity)分析表明,CXCR7a与人(Homo sapiens)、小鼠(Mus musculus)、非洲爪蟾(Xenopus laevis)、斑马鱼(Danio rerio)的CXCR7相似性分别为82.6%、81.4%、80.9%、81.0%;CXCR7b与人、小鼠、非洲爪蟾、斑马鱼的CXCR7相似性分别为82.9%、82.3%、80.8%、82.5%;西伯利亚鲟CXCR7a与其CXCR7b的相似性最高为96.5%。系统进化树分析显示,西伯利亚鲟与斑马鱼聚为一簇,两栖类聚为一簇,其它高等脊椎动物再进行聚类。对CXCR7a和CXCR7b在各发育时期胚胎及仔鱼的表达状况分别进行定性分析表明,CXCR7a在除卵裂期、囊胚期的胚胎外,其在原肠胚中期至出膜1 d的仔鱼的各时期中均具有表达,CXCR7b在卵裂期至出膜1 d的仔鱼的各时期中均具有表达。另外,从基因组DNA水平上表明CXCR7a和CXCR7b来自不同的基因拷贝。这些结果为进一步研究CXCR7在西伯利亚鲟侧线系统发育中的作用提供了新的基础资料。     

Compensatory growth,plasma hormones and metabolites in juvenile Siberian sturgeon (Acipenser baerii,Brandt 1869) subjected to fasting and re‐feeding

A 7‐week study was conducted to investigate the effect of different fasting and re‐feeding regimes on compensatory growth and some physiological parameters of juvenile Siberian sturgeon (Acipenser baerii). Fish (46.5 ± 0.5 g) were fed on a diet (containing 450 g/kg crude protein and 20 MJ/kg digestible energy) according to four feeding regimes in triplicate including: control group (C, fed everyday), W1 (2 weeks of feeding followed by 1 week of fasting and 4 weeks of re‐feeding), W2 (1 week of feeding followed by 2 weeks of fasting and 4 weeks of re‐feeding) and W3 (3 weeks of fasting followed by 4 weeks of re‐feeding). The fasted groups including W1 (119.6 ± 2.1 g), W2 (118.0 ± 1.7 g) and W3 (108.5 ± 4.8) significantly lost their weights during fasting phase and did not attain the final weight of the C (137.3 ± 1.7 g) after re‐feeding phase. The re‐feeding phase increased the specific growth rate in the fasted groups compared to the C (p < .05). After the fasting phase, concentrations of T₃, T₄, glucose, total protein and triglyceride in plasma of fasted groups were decreased, but levels of total cholesterol, aspartate aminotransferase, alkaline phosphatase and lactate dehydrogenase increased compared to the C. After re‐feeding phase, except for glucose level, all mentioned metabolites were restored in the plasma of W1 group, but total protein level and aspartate aminotransferase concentrations in plasma were not restored in W2 and W3 groups. Overall, our finding demonstrated 4 weeks of re‐feeding was too short to induce full compensatory growth in A. baerii juveniles.     

Inheritance of microsatellite loci in polyploid Siberian sturgeon (Acipenser baeri Brandt) based on uniparental haploids

Haploid gynogenesis was induced in Siberian sturgeon (Acipenser baeri Brandt) using UV‐irradiated bester (hybrid of Huso huso L. and Acipenser ruthenus L.) sperm. The inactivated heterologous sperm was used successfully to produce gynogenetic haploids of Siberian sturgeon. All haploid progeny were screened using microsatellite DNA analysis and uniparental transmission in gynogenetic haploids was confirmed. The objective of this study was to obtain haploids of Siberian sturgeon and describe the inheritance of microsatellite loci in haploid embryos of this species. This initial work, based on inheritance in haploid Siberian sturgeon, suggests that three studied microsatellite loci segregate disomically, three tetrasomically and one octasomically.     

施氏鲟、西伯利亚鲟、达氏鳇及其杂交种种质鉴定方法的建立及其应用

以施氏鲟（Huso dauricus）为研究材料,分别从线粒体基因组及核基因组两个层面进行物种分子鉴定方法研究。在线粒体基因组层面,对3种鲟及未知鲟种类共计119个样品的D-Loop区进行测序,通过同源序列比对,构建NJ进化树、计算群体间遗传距离,以鉴定其中30尾未知种类。在核基因组层面,利用15对微卫星标记扩增3种鲟DNA,筛选出特异性标记Ls19和SX226。Ls19在施氏鲟中扩增出特异条带126 bp、130 bp,在西伯利亚鲟中扩增出特异条带139 bp、143 bp,在达氏鳇中扩增出特异条带124 bp、127 bp;SX226在施氏鲟中扩增出特异条带185 bp,在西伯利亚鲟中扩增出特异条带260 bp、273 bp、283 bp,在达氏鳇中扩增出特异条带180 bp、182 bp。通过特异条带对未知鲟进行鉴定,结果显示：30尾未知鲟种类中,有西伯利亚鲟17尾,施氏鲟1尾,达氏鳇1尾,达氏鳇×施氏鲟2尾,施氏鲟×达氏鳇1尾,施氏鲟×西伯利亚鲟8尾。结果表明,特异性微卫星引物Ls19和SX226可以应用于施氏鲟、西伯利亚鲟、达氏鳇的纯种及杂交种分子水平种质鉴定。     

Effects of olive pomace on growth performance,digestibility, body composition and fatty acid profile in yearling Siberian sturgeon,Acipenser baerii (Brandt 1896)

Replacement of olive pomace (OP) with wheat flour in diet was studied in diet of yearling Siberian sturgeon (Acipenser baerii). Fish (165 ± 6.8 g) were randomly allocated to 15 fibreglass tanks (15 fish/tank, in triplicate). Fish were assigned to one of five dietary treatments at 19.1 ± 1.5°C for 8 weeks: control diet (crude protein and crude lipid at 514.6 and 188 g/kg_, respectively) and four experimental diets with 20, 50, 75 and 100 g/kg OP to replace wheat flour in the experimental diets. No significant difference was observed in the final weight, feed conversion ratio, specific growth rate, hepatosomatic index, viscera‐somatic index and survival rate among the treatments (p > 0.05). No change was seen in digestibility of protein and lipid in diets containing 20 g/kg and 50 g/kg OP compared to control group, while these values decreased with increasing in OP above 50 g/kg. Digestibility of dry matter and gross energy among the treatments demonstrated no significant difference (p > 0.05). Polyunsaturated fatty acids, especially C22:6n3 (DHA), in the muscle of the fish fed 75 g/kg and 100 g/kg OP in diets were significantly higher than control group (p < 0.05). These data show that OP could be recommended as a substitute for wheat flour in diet of S. sturgeon but its utilization for other species warranted future works.