一种虹彩病毒感染大菱鲆的病理学研究

对我国虹彩病毒感染的大菱鲆Scophthalmus maximus进行的组织病理和超微病理学研究发现,该病典型的病理学特点是在病鱼的脾脏、肾脏、肠、肝脏、鳃、心脏和皮肤等器官组织内出现嗜碱性的肿大细胞。病毒感染导致患病大菱鲆多个器官组织发生了不同程度的病理变化,其中以脾脏组织的病理变化最为显著,表现为造血组织的严重坏死。此外,肾脏造血组织发生坏死、肠固有膜和黏膜下层出血和水肿、肝细胞水样变性、心肌局灶性坏死以及皮肤真皮层出血并伴有水肿和炎性渗出也是该病常见的组织病理学变化。超微病理研究表明,肿大细胞内有虹彩病毒粒子存在。病毒分布于受感染细胞的胞质、组织间隙以及血管腔内。受感染细胞出现线粒体和内质网等细胞器肿胀、崩解等细胞病理变化。研究认为,病毒感染造成皮下组织血管损伤出血,是虹彩病毒感染的大菱鲆发生＂红体病＂的原因所在。虹彩病毒感染所致的机体严重贫血是患病大菱鲆死亡的主要原因,而主要器官组织的病变使得病鱼器官功能衰竭则可加速鱼的死亡。     

过饱和溶氧对大菱鲆生长及消化酶的影响

探讨工程化养殖中过饱和溶氧对大菱鲆生长的影响,通过充入液态氧与空气,使试验水体的溶氧分别达到过饱和溶氧水平(12±1)mg/L和(14±1)mg/L,正常溶氧水平(7.0±0.5)mg/L.结果显示,过饱和溶氧组的大菱鲆生长速度及饵料利用率明显高于正常溶氧组(p<0.05),而在过饱和溶氧条件下与正常溶氧条件下大菱鲆的丰满度无显著差异(P>0.05).对大菱鲆胃、肠道消化酶、脂肪酶与淀粉酶的分析发现,大菱鲆胃蛋白酶活性在过饱和溶氧条件下较正常溶氧有明显提高(p<0.05),而脂肪酶与淀粉酶的酶活力未得到显著性提高(p>0.05).     

The importance of manual white blood cell differential counts and platelet estimates in elephant hematology: blood film review is essential

Unique features of elephant hematology are known challenges in analytical methodology like two types of monocytes typical for members of the Order Afrotheria and platelet counts of the comparatively small elephant platelet. To investigate WBC differential and platelet data generated by an impedance-based hematology analyzer without availability of validated species-specific software for recognition of elephant WBCs and platelets, compared to manual blood film review. Blood samples preserved in ethylenediaminetetraacetic acid (EDTA) of 50 elephants (n = 35 Elephas maximus and n = 15 Loxodonta africana) were used. A Mann-Whitney test for independent samples was used to compare parameters between methods and agreement was tested using Bland-Altman bias plots. All hematological variables, including absolute numbers of heterophils, lymphocytes, monocytes, eosinophils, basophils, and platelets, were significantly different (p < 0.0001) between both methods of analysis, and there was no agreement using Bland-Altman bias plots. Manual review consistently produced higher heterophil and monocyte counts as well as platelet estimates, while the automated analyzer produced higher lymphocyte, eosinophil, and basophil counts. The hematology analyzer did not properly differentiate elephant lymphocytes and monocytes, and did not accurately count elephant platelets. These findings emphasize the importance of manual blood film review as part of elephant complete blood counts in both clinical and research settings and as a basis for the development of hematological reference intervals.     

大菱鲆AFLP分析体系的建立

本研究构建了大菱鲆扩增片段长度多态性(AFLP)分析体系,对DNA提取、双酶切反应、连接反应、预扩增反应、选择性扩增反应和银染等步骤进行了分析。结果表明,用EcoRI/MseI双酶切、核心序列加3个选择性碱基的选择性扩增引物、1∶6(w∶w)的EcoRI端与MseI端选择性扩增引物比和20μl选择性扩增体积,可得到十分稳定的结果。所得产物经电泳和银染后可获得条带清晰、背景干扰小的图像。该体系的构建为AFLP技术在大菱鲆相关研究中的应用奠定了基础。     

Accuracy of pairwise methods in the reconstruction of family relationships, using molecular information from turbot (Scophthalmus maximus)

Many estimators and algorithms have been developed to infer the genealogical relationships from molecular marker data when there is no pedigree information. Most pairwise methods provide estimates in a continuous range that needs to be converted into genealogical relationships (namely full-sibs, half-sibs and unrelated) if there is a previous knowledge of the population structure. Transformations are usually based on arbitrary thresholds, but the possibility of correcting the coancestry estimates via explicit pedigree reconstruction methods has been suggested. Using molecular data for ten highly polymorphic microsatellite loci on a population of turbot (Scophthalmus maximus) with a known genealogy, the behaviour of four pairwise marker-based coancestry estimators and the molecular coancestry has been evaluated. The population consisted on 138 families with 4 full-sib individuals each and one family with 8 full-sib individuals (up to 15 half-sib families were also present due to the sharing of parents between some of the full-sibs families). Our results suggested that transforming the pairwise estimators and the molecular coancestry to family relationships through the establishment of thresholds performs slightly better than the explicit pedigree reconstruction method, when accuracy is measured in a pairwise basis. However, if joint relationships between more than two individuals were tested at a time, the threshold methods led to a high percentage of incongruous triads of full-sib individuals, with Mendelian incompatibilities appearing in congruous full-sib families (more than 70% and 60% in our study, respectively). The explicit pedigree reconstruction approach, due to its nature, is free from such problems. Therefore, the pedigree reconstruction approach seems to be a valuable tool to provide a congruent and compatible pedigree when the pairwise marker-based coancestry matrices or the molecular coancestry need to be transformed.     

盐度和温度对大菱鲆幼鱼抗氧化酶活性的影响

采用两因素交叉分组的方法,研究了温度和盐度对大菱鲆Scophthalmus maximus幼鱼行为活动、摄食、存活情况以及体表黏液、血液、鳃、肝脏中抗氧化酶活力的影响.结果表明:水温(17、20、23、25、28℃)和盐度(5、10、20、30、40)对大菱鲆幼鱼摄食、死亡率有显著影响(P＜0.05),在水温为17、20℃与盐度为20、30的组合条件下,大菱鲆幼鱼的摄食积极性以及存活率较高,显著高于其他组(P＜0.05);温度和盐度对大菱鲆幼鱼SOD、CAT与GP-X活力有显著影响(P＜0.05);当温度一定,盐度变化的条件下,肝脏、鳃、血清和黏液中的各种酶活力在正常海水盐度(30)时活力较低,并随盐度由30起降低或升高而均出现升高的趋势;当盐度一定,温度变化的条件下,随着水温的升高,各组织中的酶活力变化规律不显著;通过分析温度与盐度交互作用对各组织不同酶活的变化以及生命表征现象,得出了最适生存阈值,即大菱鲆幼鱼的适宜生活水温为17～20℃,适宜生活盐度为20～30.     

用饲料酵母替代鱼粉对大菱鲆幼鱼生长及免疫机能的影响

分别用质量分数为0、15%、30%、45%、60%的饲料酵母替代饲料中0、17%、34%、51%、68%的鱼粉,配制成5种等氮、等能的试验饲料,分别记为Ⅰ（对照组）、Ⅱ、Ⅲ、Ⅳ和Ⅴ组。将195尾大菱鲆Scophthatmus maximus（9.02 g±0.15 g）随机分为5组,每组设3个重复,每个重复组放养13尾鱼。将试验鱼饲养于设有循环流水系统的水族箱（60 cm×40 cm×50 cm）中,用5种饲料投喂,每天投喂3次,投饲率为鱼体质量的3%～4%,以生长及免疫指标来评价饲料酵母替代鱼粉对大菱鲆幼鱼生长的影响。经过56 d的饲养,结果表明：Ⅱ组的增重率（275.30%）和特定生长率（1.91%/d）最高,但与对照组（235.66%、1.82%/d）无显著差异（P〉0.05）;Ⅱ组的饲料系数（1.34）最低,但与对照组（1.53）无显著差异（P〉0.05）;Ⅱ组（97.44%）和Ⅳ组（94.87%）的成活率均显著高于对照组（84.62%）（P〈0.05）,而Ⅴ组的成活率（74.36%）则显著低于对照组（P〈0.05）;鱼体的粗蛋白含量随着饲料酵母替代比例的增加而逐渐降低（P〈0.05）,粗灰分则逐渐升高（P〈0.05）;溶菌酶、超氧化物歧化酶和血浆中蛋白的含量随着替代比例的增加而减少,且当饲料酵母替代比例超过51%时,与对照组差异显著（P〈0.05）。综合考虑大菱鲆的生长指标、形体指标、鱼体成分和免疫机能等因素,要获得最佳的养殖效益,大菱鲆幼鱼饲料中用饲料酵母替代鱼粉的比例以17%为宜。     

大菱鲆抗菌肽基因酵母表达载体的构建及表达

将大菱鲆（Scophthalmus maxinus）hepcidin基因成熟肽序列经过部分改造后采用PCR方法克隆到毕赤酵母胞内表达载体pGAPZB中,构建了重组胞内表达载体pGAPZB—tbhep6his。用BlnI线性化重组质粒后,电击转化毕赤酵母SMD1168。经过Zeocin筛选和PCR扩增转化子基因组筛选,得到了数株基因工程酵母菌。通过SDS—PAGE和Western blot鉴定,发现目的蛋白与预期蛋白的分子量相吻合,并能与特异抗体antihis特异性结合,表明大菱鲆抗菌肽hepcidin基因在酵母菌中进行了表达。大菱鲆抗菌肽酵母表达载体的构建和表达为进一步研究该抗菌肽的功能并开发新型鱼类饵料奠定了理论基础。     

周期性饥饿再投喂对大菱鲆幼鱼生长、生化组成和能量收支的影响

在水温为17～19℃条件下,分别以鲜杂鱼(x)和配合饲料(p)为饵料,采用一直投喂(对照组)和周期性饥饿1 d再投喂6 d(S1F6)、饥饿2 d再投喂5 d(S2F5)3种投喂方式,研究投喂不同饵料时周期性饥饿再投喂对大菱鲆Scophthalmus maximus幼鱼(体质量为7.80 g±0.01 g)生长、生化组成和能量收支的影响,试验共进行42 d。结果表明:投喂配合饲料时,S1F6p和S2F5p组均具有完全补偿生长现象;投喂鲜杂鱼时,S1F6x组具完全补偿生长现象,S2F5x具部分补偿生长现象;投喂配合饲料和鲜杂鱼时,周期性饥饿再投喂处理的大菱鲆幼鱼均通过饲料转化效率和摄食率共同提高来实现其补偿生长,且各试验组的鱼体成分和对照组均无显著性差异(P>0.05);与对照组相比,试验处理组幼鱼的生长能比例均有所增加,而代谢能比例均有所减少;无论是处理组还是对照组,鲜杂鱼的投喂效果均优于配合饲料。     

胆汁酸对大菱鲆幼鱼生长、脂肪代谢酶及血清生化的影响

在基础饲料中分别添加0、0.03%、0.06%、0.09%及0.12%的胆汁酸,配制5组等氮等能的实验饲料(D1、D2、D3、D4、D5),投喂体质量为26.5 g的大菱鲆(Scophthalmus maximus)幼鱼56 d,研究胆汁酸对其生长、体组成、脂肪代谢酶活力及血清生化的影响。结果显示:(1)实验鱼的增重率、特定生长率、蛋白质效率及脂肪效率呈先升后平稳的趋势,均在D3组达到最高值。(2)胆汁酸显著降低了全鱼粗脂肪及粗灰分含量,提高了粗蛋白含量(P0.05);降低了肌肉粗脂肪含量,提高了粗蛋白及粗灰分含量(P0.05)。(3)脂肪酶及脂蛋白脂酶活力呈先升后平稳的趋势;肝脂酶活力显著升高(P0.05)。(4)胆汁酸降低了血清谷丙转氨酶及谷草转氨酶活力,提高了碱性磷酸酶活力(P0.05);总蛋白及白蛋白的含量随胆汁酸含量的增加而升高(P0.05);甘油三酯、总胆固醇及高低密度脂蛋白胆固醇含量随胆汁酸含量的升高而下降(P0.05)。(5)鳗弧菌感染48 h后,D3、D4及D5组的累积死亡率显著低于D1组。以增重率为评价指标,经折线回归分析,体质量26.5 g的大菱鲆幼鱼饲料中胆汁酸的适宜添加量为0.09%。