人工养殖圆口铜鱼头肾组织学特征及操作胁迫对其影响

采用石蜡切片和组织涂片技术,对人工养殖圆口铜鱼(Coreius guichenoti)在正常和操作胁迫下头肾的组织、细胞形态结构及细胞数目进行研究,并探讨了其在操作胁迫后0.1、4、24 h 的变化特征.石蜡切片观察发现,圆口铜鱼头肾实质中无肾单位,主要由淋巴组织、造血组织、血管、肾间组织及黑色素巨噬细胞组成.各组织及细胞结构形态正常,分布规则.在操作胁迫后0.1、4 h 时,头肾中黑色素巨噬细胞数量较正常组显著增多,形成黑色素巨噬细胞中心,同时肾间组织增生,肾间细胞面积、核面积和核直径均有不同程度增大;在操作胁迫后24 h 时,黑色素巨噬细胞数量和肾间细胞面积、核面积、核直径均减小,并均低于正常组.组织涂片观察发现,正常圆口铜鱼头肾中主要有6类细胞:红细胞、淋巴细胞、单核细胞、嗜中性粒细胞、嗜碱性粒细胞、血栓细胞.应激后不规则红细胞增多,红细胞、嗜中性粒细胞数量和嗜碱性粒细胞持续减少;淋巴细胞和单核细胞数量在应激后0.1 h短暂增多,随后持续降低;血栓细胞数量持续增加.研究表明,圆口铜鱼头肾组织和细胞在应激后较短时间(0.1、4 h)内发生显著变化,部分组织的生物功能在24 h 时逐渐恢复正常.本研究可为圆口铜鱼及其他硬骨鱼类头肾的组织学研究、养殖与驯化过程中的健康监测等方面提供参考依据.     

重组鲮IGF-I对鲮GH表达的影响

使用半定量RT-PCR方法检测了鲮各组织中GH mRNA的表达,并分析重组鲮IGF-Ⅰ处理后鲮GH表达变化情况。结果表明：鲮的GH mRNA表达具有明显的组织特异性,在鲮的肝、肾、脾、肌肉、肠、鳃、性腺、心脏和皮肤中都未检测到GH mRNA表达,只在鲮脑中检测到GH mRNA表达;经重组鲮IGF-Ⅰ处理后,鲮脑中GH mRNA稍有升高,但在肝和肌肉中仍未检测到GH mRNA表达;重组鲮IGF-Ⅰ处理前后,鲮血清中GH浓度变化不明显。     

重组鲮GH对鲮IGF-Ⅰ表达的影响

使用半定量RT-PCR方法,研究了鲮（Cirrhinus molitorella）各组织中IGF-ⅠmRNA组织表达,并分析重组鲮GH处理后鲮IGF-Ⅰ表达变化情况。结果表明,鲮IGF-ⅠmRNA在肝组织中表达最高,其次是肾和脑,另外在肠、鳃、脾、性腺和心脏组织中也有表达,而在肌肉、皮肤中没有检测到鲮IGF-ⅠmRNA的表达;按120μg·g^-1鱼体重的剂量经腹腔注射重组鲮GH,观察12h后鲮IGF-Ⅰ表达变化情况,发现经重组鲮GH处理后,鲮肝组织IGF-ⅠmRNA表达水平显著升高,脑组织IGF-ⅠmRNA表达水平也稍有升高,而在肌肉中仍未检测到IGF-ⅠmRNA的表达;重组鲮GH处理前后,对照组中鲮血清中IGF-Ⅰ的含量为145.59±21.84ng·mL^-1,试验组中鲮血清中IGF-Ⅰ的含量为247.71±2.83ng·mL^-1,经t检验,P=0.043〈0.05,表明重组鲮GH处理前后鲮血清中IGF-Ⅰ的含量存在显著性差异。     

重组鲮IGF—I对鲮GH表达的影响

使用半定量 RT-PCR 方法检测了鲮各组织中 GH mRNA 的表达,并分析重组鲮 IGF-Ⅰ处理后鲮 GH 表达变化情况。结果表明:鲮的 GH mRNA 表达具有明显的组织特异性,在鲮的肝、肾、脾、肌肉、肠、鳃、性腺、心脏和皮肤中都未检测到 GH mRNA 表达,只在鲮脑中检测到 GH mRNA 表达;经重组鲮 IGF-Ⅰ处理后,鲮脑中GH mRNA 稍有升高,但在肝和肌肉中仍未检测到 GH mRNA 表达;重组鲮 IGF-Ⅰ处理前后,鲮血清中 GH 浓度变化不明显。     

外源生长激素注射对尼罗罗非鱼GH-IGFs生长轴通路因子及MyoD基因表达的影响

为探究生长激素(GH)对鱼类肌肉生长影响的分子机理,应用外源GH注射尼罗罗非鱼(Oreochromis niloticus)幼鱼,采用荧光定量PCR技术检测注射1~7周后GH试验组和对照组鱼的肝脏、肌肉中GH-IGFs生长轴通路因子(GHR1,GHR2,IGF-1,IGF-2)和肌肉中成肌分化因子基因(MyoD1,MyoD2)的mRNA表达变化。结果表明,外源GH注射后,肝脏、肌肉中GHR1 mRNA的表达水平均显著上调,GHR2 mRNA的表达水平均显著下调;肝脏、肌肉中IGF-1 mRNA、IGF-2 mRNA的表达水平均显著上调;肌肉中MyoD1 mRNA、MyoD2 mRNA的表达水平均显著上调(P0.05)。试验证实,肝脏和肌肉均为GH的效应器官,可能的作用途径为GH-GHR1-IGFs-MyoDs。     

运输胁迫对银鲳血清皮质醇、血糖、组织中糖元及乳酸含量的影响

研究分析了运输胁迫2、4、8和12 h后银鲳血清皮质醇、血糖、组织中糖元及乳酸含量的变化。选取平均体质量为(10.05±0.23) g的银鲳幼鱼为试验对象,共设3组运输密度,分别为4、8和16 g/L。每个时间段的运输结束后,将取样后剩余的鱼转入1 m³水体的圆形玻璃钢桶中观察24 h,以统计各自的累计死亡率。研究结果显示,16 g/L密度组在运输12 h后成活率为91.25%,其余均为100%;运输胁迫导致皮质醇含量显著升高(P<0.05),运输4 h后,血清皮质醇含量均达到最高值,12 h后皮质醇含量虽较运输4 h后有所降低,但仍显著高于试验初的水平,且此时16 g/L密度组的皮质醇含量均显著高于4和8 g/L密度组(P<0.05);血糖、血清与肌肉中乳酸含量在运输胁迫后均显著升高(P<0.05);肝脏与肌肉中糖元含量在运输胁迫后均显著降低(P<0.05),但在整个运输胁迫过程中,肝脏组织中糖元含量的降低程度明显高于肌肉组织。统计分析得出,较高的运输密度会加重银鲳的应激性反应,并推测在小水体开放式运输的情况下,规格为10 g左右的银鲳幼鱼,其运输密度不宜超过16 g/L。此外,在运输胁迫下,银鲳机体的能量供给主要来自肝脏糖元的动员。     

银鲳人工育苗技术研究

2005~2006年,在舟山海域银鲳繁殖季节(4~5月),随流刺网和张网捕鱼船出海,捕捞性腺成熟的银鲳进行人工授精,把胚胎运回岸上在孵化桶中继续孵化,得到初孵仔鱼并移入水泥池中进行培育。在水温19.0~24.0℃、盐度25.0~28.0的人工育苗条件下培育苗种。饵料为轮虫、卤虫无节幼体和配合饲料,孵化后3 d仔鱼开始开口摄食。银鲳初孵仔鱼~12日龄为仔鱼期,13~40日龄为稚鱼期,41日龄起转为幼鱼期。经50 d培育,获得20~27 mm叉长的银鲳幼鱼。育苗成活率为5%~14%。     

Establishment and characterization of a fin tissue cell line derived from silver pomfret,Pampus argenteus

A cell line (PaF) derived from the fin tissue of silver pomfret (Pampus argenteus) was established and characterized in this study. The cell line has been subcultured for more than 50 times in Dulbecco's modified Eagle's medium (DMEM) containing 15% foetal bovine serum (FBS) since the initial primary culture. PaF cells grew well at temperatures from 24°C to 28°C in DMEM supplemented with 15% FBS. Partial amplification and sequence analysis of the cytochrome B gene indicated that PaF originated from silver pomfret. Cytogenetic analysis demonstrated that the modal chromosome number was 48. A significant cytopathic effect was observed in PaF cells during viral haemorrhagic septicaemia virus (VHSV) infection, and the VHSV replication was confirmed by qRT‐PCR and viral titre assays. In contrast, PaF cells were resistant to red‐spotted grouper nervous necrosis virus infection. Moreover, PaF cells could respond to VHSV and lipopolysaccharide treatments, as indicated by the expression of immune‐related genes, TLR5 and TLR9. In conclusion, the establishment of PaF cell line will provide an appropriate in vitro tool for the study of mechanisms of pathogen–silver pomfret interaction.     

饲料组成对银鲳幼鱼生长率及肌肉氨基酸、脂肪酸组成的影响

研究分析了饲料组成对银鲳(Pampus argenteus)幼鱼特定生长率(SGR)及肌肉氨基酸与脂肪酸组成的影响。实验共设4组不同的饲料组成,依次为饲料A(鱼肉糜)、饲料B(鱼肉糜+配合饲料)、饲料C(鱼肉糜+配合饲料+蛏子肉糜)和饲料D(鱼肉糜+配合饲料+蛏子肉糜+桡足类)。实验用银鲳幼鱼的平均体重为4.80±0.11 g,每组饲料设3重复,实验周期为9周。研究结果显示,不同饲料组成可显著影响银鲳的特定生长率,饲料A组的特定生长率最低,并显著低于其它各饲料组;饲料D组的特定生长率最高,且显著高于饲料B、C组的特定生长率(P<0.05);但饲料B、C组间银鲳特定生长率无显著性差异(P>0.05)。银鲳肌肉氨基酸与脂肪酸的分析结果显示,4组饲料组成对银鲳肌肉氨基酸的组成及含量并无显著性影响(P>0.05),但可显著影响肌肉脂肪酸的组成及含量(P<0.05)。饲料D组的多不饱和脂肪酸(PUFA)、高度不饱和脂肪酸(HUFA)及n-3HUFA含量均分别显著高于饲料A、B和C组(P<0.05),饲料B和C组的PUFA、HUFA及n-3HUFA均分别显著高于饲料A组(P<0.05),但饲料B、C组间并无显著性差异(P>0.05)。综合分析得出,丰富的饲料组成以及饲料中较高的HUFA含量均有利于银鲳幼鱼的生长。     

舟山渔场银鲳人工授精及孵化

2005-2008年,在舟山海域银鲳(Pampus argenteus)繁殖季节(4-5月),随流刺网或张网捕鱼船出海,捕捞性腺成熟的银鲳,进行人工授精和孵化.人工授精试验采用干法、湿法和半干法,比较受精率和孵化率.通过4年连续测量银鲳成熟卵的卵径、油球径等形态指标,对卵子成熟度进行判别,在水温16.0～18.5 ℃、盐度28.0±0.5的条件下进行孵化.结果表明,以干法受精后间隔3～5 min水洗和半干法受精的方法受精率最高,受精率达18.50%～33.50%,最高达40%;孵化率达到43.83%～51.00%,最高达66%.银鲳成熟卵子的卵径为(1.368±0.082)mm,油球径为(0.550±0.031)mm,油球1个.对影响海捕银鲳亲体受精率和孵化率的环境条件也进行了讨论.     

养殖银鲳卵巢发育的组织学观察

每月定时取人工养殖子代银鲳(Pampus argenteus)样本,采用常规石蜡切片技术对样本卵巢发育情况进行周年观察。期间共采集到雌性银鲳样本92尾,叉长范围为113.1~185.7 mm,体质量33.6~187.5 g。按照各个时相形态特征和卵巢周年变化情况,养殖银鲳卵子发生分为6个时相,卵巢发育分为6个时期。研究表明,养殖条件下的银鲳群体与野生群体卵巢发育的组织学结构无明显差异,且繁殖季节基本一致,但养殖群体卵巢成熟系数明显低于野生群体。推测养殖过程中温度、光照和营养等因素对银鲳性腺发育和成熟具有较大的影响。本研究旨在为银鲳人工繁育研究提供基础科学支持。     

不同饲料对银鲳幼鱼增重率、肝脏脂酶及抗氧化酶活性的影响

利用营养学与生物化学的方法,研究分析了不同饲料对银鲳幼鱼增重率、饲料系数、肝脏脂酶及抗氧化酶活性的影响。试验共设4组不同饲料,依次为饲料1(新鲜鱼肉糜)、饲料2(新鲜鱼肉糜+饲料)、饲料3(新鲜鱼肉糜+饲料+蛏子肉糜)和饲料4(新鲜鱼肉糜+饲料+蛏子肉糜+桡足类)。试验用银鲳幼鱼的平均体重为(4.80±0.11)g,每组饲料设3重复,试验周期为9周。研究结果显示,不同饲料可显著影响银鲳的增重率,各饲料组中以饲料1组的增重率最低,并显著低于其它各饲料组;饲料4组的增重率最高,并显著高于饲料2、3组的增重率(P<0.05)。饲料系数以饲料1组最高,且显著高于其它3组饲料组;饲料2、3、4组饲料系数间并无显著性差异(P>0.05)。饲料1组肝脂酶与总脂酶活性均显著低于其它3组饲料组(P<0.05),但饲料2、3、4组间肝脏脂蛋白脂酶、肝脂酶和总脂酶活性均未有显著性差异(P>0.05)。各组间肝脏超氧化物歧化酶(SOD)与谷胱甘肽过氧化物酶(GSH-PX)活性均未有显著性差异(P>0.05),但均以饲料1组最低、饲料4组最高;饲料1、2、3组间肝脏过氧化氢酶(CAT)活性未有显著性差异(P>0.05),但饲料4组CAT活性显著高于饲料1组的CAT活性(P<0.05)。分析结果表明,单纯利用鱼肉糜作为饲料不利于银鲳的生长;饲料中添加桡足类不但有助于促进银鲳的生长,也可提高其抗氧化能力。     

Correlation between gut microbial diversity and flatulence of silver pomfret (Pampus argenteus) during ontogenesis

Gut microorganisms play an important role in host health and have susceptibility to diseases, undergoing a distinct transformation at early stages of development. This study aimed to determine the regulation of gut microorganism development from fertilized eggs to 150 DAH (days after hatching) and the differences between flatulent and healthy silver pomfret (Pampus argenteus). High‐throughput sequencing was used to analyze the diversity and relative abundance of microorganisms. PICRUSt was used to predict functional alterations of dominant microorganisms. The results showed that from all 42 samples, a total of 3,746,895 reads were obtained, representing 6,081 operational taxonomic units (OTUs). Top 3 phyla were Proteobacteria, Firmicutes and Bacteroidetes. From 0 to 32 DAH, the relative abundance of Proteobacteria continuously increased and peaked at 94.99%. After that, the abundance of Proteobacteria decreased, but Bacteroidetes and Firmicutes increased instead. Alpha diversity is the lowest at 32 DAH, the difference is significant compared with others, p < .05. Comparing flatulent fish with healthy fish, the former has more specific OTUs, higher diversity and abundance. There were four genera Ethanoligenens, Methanobacterium, Caproiciproducens and Leptolinea that only obtained from the flatulent samples, but almost none in the healthy samples. All of these four genera are directly or indirectly involved in gas metabolism. This study enriched the basic research of silver pomfret, for the first time, focused on flatulent individuals which would provide a theoretical basis for the subsequent breeding.     

Scuticociliatosis-associated mortalities and histopathology of natural infection in cultured silver pomfret (Pampus argenteus Euphrasen) in Kuwait

Sub-adults of silver pomfret (Pampus argenteus), produced and reared in the mariculture experimentation facilities of Mariculture and Fisheries Department of Kuwait Institute for Scientific Research (KISR), suffered 55–70% mortalities due to scuticociliatosis by Uronema sp. The parasitic infestation was noticed during April–May 2005. Affected fish showed varying levels of tissue damage including severe epidermal and dermal necrotic lesions. The disease occurred with the raise in water temperature from 20 to 22 °C during April. Loss of scales, appearance of bleached spots that coalesced to form brownish patches, haemorrhagic and severe dermal necrotic lesions were the major clinico-pathological manifestations. The parasite was found abundantly in the blood, peritoneal fluid and the cerebrospinal fluid, the skin and the gill wet mounts. Extensive fouling, necrotic degeneration of the gill epithelium and deep dermal necrosis resulted in mortality of the infected fish. The parasite was noticed in the lumen of the collecting ducts of the kidney and the alimentary canal. The parasite was also seen distributed extensively in the entire brain causing widespread nerve necrosis. Earliest separation of the clinically normal fish from the affected fish resulted in significantly higher survival. The investigations are of significant importance in view of the mariculture potentials of the fish especially after the success of captive spawning and larval rearing achieved by the KISR.     

渤海银鲳的营养级及摄食习性

根据2017年9—11月在渤海进行的3次定点底拖网调查采集的216个样品[包括120个银鲳(Pampus argenteus)样品, 51个饵料生物样品和45个比较研究鱼类样品],采用碳、氮稳定同位素技术测定了样品的稳定同位素比值。对银鲳各饵料种类的贡献比率进行了计算,探讨其摄食习性、营养级、生态位宽度和生态位重叠随叉长变化的特征。同时也通过对比研究验证了采用稳定同位素技术研究银鲳摄食生态的准确性。结果表明:(1)渤海银鲳的δ~(13)C值范围为-19.50‰～-14.85‰,平均值为-17.18‰,与叉长相关性不显著(r=-0.03,P=0.71);δ~(15)N值范围为11.55‰～14.77‰,平均值为13.30‰,与叉长相关性极显著,但相关强度是弱相关(r=0.26,P0.01)。(2)基于R语言的贝叶斯稳定同位素混合模型(SIAR)计算各饵料贡献率,表明渤海银鲳为杂食性,其主要饵料为底层虾类,其贡献率为11.52%～57.49%,平均贡献率为35.03%;其次是中国毛虾(Acetes chinensis),其贡献率为13.52%～42.82%,平均贡献率为27.87%。其他饵料的贡献率从高到低依次为日本枪乌贼(Loligo japonica)、浮游植物和浮游动物,其贡献率范围分别为6.41%～42.57%、0～21.33%和0～16.21%;平均贡献率分别为23.29%、8.08%和5.77%。(3)随叉长增加,银鲳的食物组成存在一定变化。生态位宽幅计算结果表明,渤海银鲳的营养生态位宽度随叉长增大存在先升高后下降的特征, 90～109 mm叉长组的生态位宽幅达到最大;但各叉长组营养生态位相似,生态位存在较多的重叠。(4)渤海银鲳的营养级范围为2.84～3.79,平均营养级为3.35;营养级随着叉长增大有升高的趋势。(5)MDS图分析得出银鲳与赤鼻棱鳀(Thryssa kammalensis)的δ~(13)C和δ~(15)N值相似性最高。本研究通过碳氮稳定同位素技术对银鲳的营养级和摄食习性有了较全面的了解,对构建渤海全程食物网,进而研究其动态变化具有重要的意义。