5种胃肠激素样内分泌细胞在斜带石斑鱼稚鱼期消化道中的定位

5-Hydroxytryptamine， 5-HT）、生长抑素（）、P物质（     

[07]5种胃肠激素样内分泌细胞在斜带石斑鱼稚鱼期消化道中的定位

应用免疫细胞化学SABC方法,用5-羟色胺(5-Hydroxytryptamine,5-HT)、生长抑素(Sonmtostatin,Som)、P物质(substance P,SP)、胃泌素(Gastrin,GAS)和神经肽Y(Neuropeptide,NPY)5种特异性哺乳类胃肠激素抗血清对斜带石斑鱼(Epinephelus coioides)稚鱼期的消化道进行免疫细胞化学定位.结果显示,5-HT-和Som-样免疫活性细胞定位在斜带石斑鱼稚鱼的胃各部,包括胃贲门部、胃体和胃幽门部;SP-、GAS-样免疫活性细胞则在胃和肠均存在,其中SP-样细胞定位在胃体部和胃幽门部以及整个肠道,GAS-样细胞则从胃幽门部至肠道各段均存在;NPY-样免疫活性细胞仅在肠道中被检测到.根据这5种胃肠激素样内分泌细胞的形态、染色强度及其分布,可判断其在稚鱼期已经发育成熟,已像成鱼胃肠激素内分泌细胞一样,调节消化道的运动和消化液的分泌.本研究结果提示,斜带石斑鱼稚鱼死亡的原因可能与其胃肠激素样内分泌细胞的功能没有直接关系.     

斜带石斑鱼(♀)×鞍带石斑鱼(♂)杂交子代幼鱼消化道粘液细胞和胃泌素细胞的研究

利用组织化学(AB-PAS)和免疫组织化学(SABC)的方法分别对斜带石斑鱼(Epinephelus coioides♀)×鞍带石斑鱼(E.lanceolatus♂)杂交子代(青龙斑)幼鱼消化道的粘液细胞和胃泌素(gastrin,Gas)分泌细胞的分布进行系统的研究。青龙斑幼鱼食道中有Ⅰ、Ⅱ、Ⅲ和Ⅳ型粘液细胞,含中性粘多糖和酸性粘多糖。贲门胃粘液细胞有Ⅰ、Ⅱ、Ⅲ种类型,胃体部有Ⅰ、Ⅲ、Ⅳ型粘液细胞,在胃腺的周围含有较多的Ⅰ和Ⅳ型粘液细胞,而幽门胃中只有Ⅰ型粘液细胞,只含有中性粘多糖。幽门盲囊和肠道都含有中性粘多糖和酸性粘多糖,幽门盲囊以Ⅱ型粘液细胞最多,少量的Ⅲ型粘液细胞,前肠、中肠和后肠均有4种类型的粘液细胞。肠道粘液细胞数量为中肠>后肠>前肠。利用免疫学的方法研究青龙斑幼鱼消化道Gas细胞的分布,表明幼鱼的整个肠道和幽门盲囊均有Gas细胞的存在,食道和胃中未发现Gas免疫阳性细胞。     

闽南地区斜带石斑鱼(Epinephelus coioides)神经坏死病毒的基因型与分子进化研究

本研究以闽南地区具有典型神经坏死病症状的斜带石斑鱼为材料,采用RT-PCR法对其进行病毒检测,对检测到的阳性序列进行双向测序和构建系统发育树,对病毒颗粒进行分离纯化,并通过分子进化模型分析病毒衣壳蛋白基因所受的选择压力。结果显示,采集到的6个石斑鱼样品均呈NNV阳性,系统树分析发现6个样品的PCR扩增片段均为RGNNV基因型序列,表明闽南地区感染石斑鱼的神经坏死病毒主要为RGNNV基因型病毒;通过PEG法对病毒进行分离提纯,获得直径为25~28 nm、呈二十面立体对称结构的无囊膜病毒颗粒;分子进化分析显示NNV外壳蛋白基因经历了纯化选择,表明病毒在进化过程中没有出现遗传变异并以相对恒定保守的速率进化。     

斜带石斑鱼消化系统胚后发育的组织学研究

利用形态学和连续组织切片技术，对出膜后1-60d的斜带石斑鱼各期仔鱼、稚鱼和幼鱼的消化系统进行了光镜观察，描述了其消化器官发育过程和组织学特性。研究表明，实验水温为22.0-27.8℃时，孵化后第4天，上下颌形成，卵黄囊被吸收，消化系统明显分化成食道、胃、肠、直肠以及肝脏、胆囊和胰脏等，鱼体由内源性营养转向外源性摄食营养，表明其消化系统的形态变化与食性的变化相适应。此后随着鱼体的生长，其消化系统从功能和结构上逐步完善成熟。胰脏在出膜后第4天出现，是和肝脏相互分开的一个独立的器官，但是发育到第35-60天，位于肠道后部的胰脏组织内出现许多大空泡。     

一株斜带石斑鱼（Epinephelus coioides）病原菌的分离与鉴定

2011年7月,福建省晋江某养殖场养殖的斜带石斑鱼( Epinephelus coioides )发病,病鱼体重3~10 g,症状为鱼体色变深,拒食,离群缓慢游动,背鳍下方局部鳞片脱落、表皮溃烂,肝脏点状发红。2011年7月从濒死病鱼肝脏、肾脏中分离到2株细菌,分别命名为201107-I与201107-II,经生理、生化指标和16S rRNA 基因鉴定,这两株均为哈维氏弧菌( Vibrio harveyi )。毒性实验发现这两株菌都能引起斜带石斑鱼表皮溃烂症,其中201107-II毒性较强,被用于后续感染实验。通过肌肉注射、腹腔注射与创伤浸浴3种方式进行回归感染实验,得到3种方式对石斑鱼的半致死量LD50分别为4．3×104、2．9×105和5．4×106 cfu/g鱼体重。从人工感染的石斑鱼肾脏中再次分离到该菌,经生理、生化指标和16 S rRNA 基因鉴定,为同株菌。药敏实验结果表明,该菌株对红霉素、大观霉素、氯霉素等16种抗生素高度敏感,对呋喃唑酮、克拉霉素、环丙沙星等9种抗生素中度敏感,对苯唑西林、青霉素G、万古霉素等13种抗生素不敏感。本研究对石斑鱼病害防治与健康养殖具有一定的指导意义。     

长鳍篮子鱼消化道内分泌细胞的免疫组织化学鉴别和定位

应用4种兔抗胃肠激素抗体和SABC免疫组织化学方法,对长鳍篮子鱼(Siganus canaliculatus)消化道中的内分泌细胞进行鉴别和定位。结果表明:5-羟色胺(5-HT)细胞在消化道各段均有分布,食道和胃中分布数量多,在幽门盲囊和肠道分布数量少;生长抑素(Som)细胞集中位于食道和胃部,数量多,幽门盲囊和肠道中未检出;神经肽Y(NPY)细胞在肠道有少量分布,其余各段均未检出;胃泌素(Gas)细胞在幽门盲囊和肠道有少量分布,食道和胃中均未检出。4种内分泌细胞的数量分布和形态特征存在一定的差异,并对其可能的生理作用进行了讨论。     

牙鲆消化道组织学观察及内分泌细胞分布

牙鲆(Paralichthys olivaceus)体质量约0.5 kg,活体取材,通过免疫组织化学染色和H.E染色,在光学显微镜下观察牙鲆消化道组织结构。结果表明,牙鲆消化道和高等脊椎动物类似,分为口咽腔、食道、胃、胃盲囊和肠。肠也可分为前肠、中肠和后肠,三者之间没有明显的界限。胃和前肠是消化吸收主要的部位,胃盲囊和前肠结构和功能非常类似。Serotonnin细胞和P Substance细胞在牙鲆胃前部的黏膜中零星分布,在胃体中较多见,平均每104μm2胃体黏膜上皮约各有2.2和2.0个阳性细胞。而Gastrin细胞分布在胃盲囊和前肠中,平均每104μm2黏膜上皮分别有0.98个和0.71个阳性细胞。可见,牙鲆和哺乳动物消化道一样都分布有肽能神经元。这些结果将为神经内分泌系统的研究提供资料。     

10种免疫相关基因在斜带石斑鱼组织中的表达分析

采用相对实时荧光定量PCR(Quantitative Real-time PCR,qRT-PCR)技术,以β-肌动蛋白(β-actin)的表达量作为内参,对健康养殖斜带石斑鱼(Epinephelus coioides)不同组织(头肾、心脏、肝脏、脾脏、鳃、肌肉、鳍、眼、肠道)中的10种免疫相关基因:免疫球蛋白(IgM)、CD4、MHCIIa、TCRβ、CD8a、MHCIa、ISP16、Mx、TNFR14、HSP90的mRNA表达量进行了研究。结果显示,10种免疫相关基因在斜带石斑鱼的10种组织中均有表达。其中体液免疫相关基因[免疫球蛋白(IgM)、CD4和MHCIIa]在鳃中的表达量最高,在其他组织部位的表达量相对较少。细胞免疫相关基因(TCRβ、CD8a和MHCIa)也是在鳃中的表达量最高,在眼、肌肉、肝和肠的部位表达量相对较少。ISP16在鳃和肝中的表达量较高,在肌肉和肠中的表达量较低。Mx在鳃中的表达量较高,在肌肉和肠的表达量较低。TNFR14在眼的表达量较高,在肠的表达量较低。HSP90在鳃和肝中的表达量相对较高,在心和肌肉的表达量较低。     

Apparent digestibility of selected ingredients in diets for juvenile grouper, Epinephelus coioides (Hamilton)

Apparent digestibility coefficients (ADCs) for dry matter (ADC_dm) and crude protein (ADC_cp) of selected feed ingredients were determined in vivo for grouper using passive faeces collection (Guelph System). A reference diet (RF) and test diets (consisted of 70% RF and 30% test ingredient) with 1% Cr₂O₃ as an inert indicator were used. An RF contained 45% protein, 10% fat and 15.7 kJ g^?1 metabolizable energy. Three isonitrogenous and isocaloric diets, each contained a test ingredient (white fish meal, white cowpea meal and ipil‐ipil leaf meal), were used in a growth study based on ADC_cp of feed ingredients. An RF without Cr₂O₃ was a control. The ADC values of experimental diets were also determined. In grouper, the ADC_dm of white cowpea meal, defatted soybean meal, wheat flour and shrimp meal (74–76%) were significantly lower than that of squid meal (99%), but comparable with those of the fish meals (84–89%). No significant difference was observed between the ADC_dm of ipil‐ipil leaf meal, rice bran and wheat flour (56–73%). The ADC_cp of white cowpea meal and defatted soybean meal were similar to those of the fish meals, squid meal and shrimp meal (94–99%). The ADC_cp of wheat flour was comparable with that of ipil‐ipil leaf meal (79–83%). Rice bran had the lowest ADC_cp value of 43%. Based on specific growth rate (SGR), the growth of fish fed white cowpea meal‐based diets was similar to that of the control fish (3.2–3.3% day^?1). Also, no significant difference was observed between the ADC_dm (68–72%) and ADC_cp (88–91%) of white cowpea meal‐based diet and the control diet. The results suggest that ADC values can be used as indicators to determine the nutritional value of feed ingredients. White cowpea meal can be incorporated as a protein source in practical diet for grouper at 20.5% of the diet with no adverse effect on growth.     

Growth and survival of the grouper Epinephelus coioides (Hamilton) at different loading rates in tanks

The effect of increasing fish loading (decreasing water exchange rate, while holding fish stocking density constant) on growth, survival and feed conversion ratio was determined for two size groups of the orange‐spotted grouper Epinephelus coioides (Hamilton). Fingerlings (124.3–145.8 g initial body weight) and subadults (990.6–1147.1 g initial body weight) were reared in 1‐m³ circular tanks for 14 weeks. For the fingerlings experiment, three loading rates of 0.5, 2 and 6 kg L^?1 min^?1 were used, while in the experiment with subadults, the loading rates were 1, 3 and 6 kg L^?1 min^?1. The mean growth rate of fingerlings ranged from 1.60 to 2.14 g fish^?1 day^?1, and survival was high (95–100%); however, there were no significant differences (P > 0.05) as a result of the different loading rates. Similar results were obtained in the subadults experiment, in which the growth rate (3.10–4.90 g fish^?1 day^?1) and survival (86.7–100%) were not significantly (P > 0.05) affected by the different loading rates. In both experiments, the feed conversion ratios were also not affected significantly by the loading rates. These results show that water exchange in aerated, flowthrough tanks can be reduced to as low as 6 kg L^?1 min^?1 without adversely affecting growth, survival and feed conversion of fingerling and subadult groupers.     

石斑鱼人工育苗技术研究

通过对点带石斑鱼人工育苗生产试验,从饵料准备、环境条件控制、管理措施等方面进行了探索。共培育出规格5～8cm的石斑鱼苗60多万尾。     

Application of logistic regression analysis to predict cannibalism in orange‐spotted grouper Epinephelus coioides (Hamilton) fry

Cannibalism is frequently observed in larviculture of orange‐spotted grouper Epinephelus coioides. Previously, based on measurements of morphometric characters, a linear equation of total length (TL) of prey to cannibals was proposed: TL_prey = 0.80 TL_cannibal – 1.50. To verify the reliability of the equation, experiments were performed with pairs of fish with different TLs. Cannibalism occurred only when the cannibal‐prey size ratios were equal to or larger than that predicted by the equation. To predict the probability of cannibalism among the grouper of known TLs, a logistic regression model was proposed. The logistic regression model is: The logistic regression model showed the following: when either TL_prey or TL_cannibal is constant, the probability of cannibalism increases with increase in the cannibal‐prey size ratios; if given a constant cannibal‐prey size ratio, probability of cannibalism is lower in early stages than in later stages. The prediction contrasts with that calculated from the linear equation where the minimum cannibal‐prey size ratios decrease with size of the cannibal. However, the prediction matches observed rearing experiences: cannibals prefer smaller prey to larger ones and the cannibalism rate declines as fry age.     

Alternative vegetable lipid sources in diets for grouper, Epinephelus coioides (Hamilton): effects on growth, and muscle and liver fatty acid composition

The aim of this study was to investigate the effects of different oils on growth performance and lipid metabolism of the grouper, Epinephelus coioides. Five experimental fish meal‐based isonitrogenous and isolipidic diets were formulated containing either 5.5%‐added fish oil (FO), soybean oil (SBO), corn oil (CO), sunflower oil (SFO) or peanut oil (PO). Each diet was fed to triplicate groups of 20 fish (initial body weight 13.2±0.02 g) grown in seawater at 28.0–30.5 °C for 8 weeks. Fish were fed twice a day to visual satiety. No significant differences in the survival, weight gain, specific growth rate, feed conversion ratio, protein efficiency ratio or hepatosomatic index were found between fish fed the FO or vegetable oils (VO) diets. Dietary lipid sources did not affect whole‐body composition among grouper fed the various diets. Muscle of fish fed the FO diet had significantly higher levels of 14:0, 16:0, 16:1n‐7, 20:5n‐3[eicosapentaenoic acid (EPA)] and docosahexaenoic acid (DHA)+EPA (except for PO fed fish) compared with those of fish fed VO diets. However, the levels of 18:1n‐9, 18:2n‐6 and DHA/EPA ratios in the muscle of fish fed FO diet were significantly lower than those of fish fed the VO diets. The liver of fish fed the FO diet had significantly higher levels of 18:0, 20:5n‐3, 22:6n‐3, n‐3 highly unsaturated fatty acids and DHA+EPA than those of fish fed the VO diets, whereas increases in 18:1n‐9, 18:2n‐6 and mono‐unsaturated fatty acid levels were observed in the liver of fish fed the VO diets.     

点带石斑鱼人工育苗技术的初步研究

通过对受精卵孵化及初孵仔鱼的饵料选择，仔鱼阶段海水比重，温度的调节等实验，研究了点带石斑鱼人工育苗技术。结果表明：采用牡蛎受精卵或担轮幼虫为初孵仔鱼的饵料是可行的。育苗的成活率与温度、比重、水质等因素关系密切。