4种多糖对皱纹盘鲍血细胞活性氧和血清凝集活力的影响

水温为(20.0±3)℃,盐度为30.0±1.5下,将灵芝多糖1、灵芝多糖2、脂多糖和酵母多糖分别配制成0、1.0、5.0、10、50、100、200μg·mL-1质量浓度,采用鲁米诺化学发光法(CL)和凝聚法检测其对皱纹盘鲍(HaliotisdiscushannaiIno)血细胞产生活性氧和血清凝集活力的影响。这些多糖均能增强鲍血细胞产生活性氧的能力和吞噬能力,其增强效果由强至弱依次为:灵芝多糖2>灵芝多糖1>脂多糖>酵母多糖;同种多糖中,急性组增强鲍血细胞活性氧产生和促进血清凝集活力的能力强于孵育组。本研究还比较了浸泡和注射灵芝多糖2(50μg·mL-1)和脂多糖(10μg·mL-1)对皱纹盘鲍血细胞活性氧产生和血清凝集活力的影响。结果表明,注射的效果优于浸泡。     

中国对虾血细胞吞噬活动中超氧阴离子（O↓（2）↑（-））的产生

用Zymosan A对中国对虾（Penaeus chinensis）血细胞进行体外刺激,用NBT还原法测定血细胞吞噬活动中产生O2^-的过程。结果表明,中国对虾血细胞在经刺激诱导的吞噬活动中有明显的呼吸爆发和O2^-的产生;不同浓度的Zymosan A对血细胞产生O2^-的影响不同。Zymosan A在0.25-1.0mg/ml对随质量浓度的增加,产生O2^-的量也增加,但当Zymosan A质量浓度继续增加到1.5和2.0mg/mL时,产生O2^-的量反而下降。用同一浓度的Zymosan A对不同密度血细胞进行刺激产生O2^-的强度也不同,随着密度的增加,血细胞产生O2^-的量增加。SOD、NEM和碘代乙酰胺（Iodoacetamide）对血细胞吞噬过程中O2^-的产生有抑制作用;Hg^2 、Cr^3 、Pb^2 、Cu^2 和Zn^2 5种重金属离子也能抑制血细胞产生O2^-;不同个体的中国对虾血细胞产生O2^-的能力不同。NBT还原法测定中国对虾血细胞吞噬时产生的O2^-可以作为检测对虾免疫状态的指标。     

不同类群九孔鲍免疫防御机能的比较

对不同类群的九孔鲍细胞与体液免疫机能进行比较,结果表明,不同类群的九孔鲍免疫机能具有显著的差异性,复壮鲍(养殖10个月,个体大小为51.8～59.2mm)细胞与体液免疫机能都明显高于退化鲍(养殖20个月,个体大小为37.4～43.1mm)(P<0.01),与野生九孔鲍(个体大小为67.7～76.7mm)相比,复壮鲍细胞免疫机能略高于野生鲍,体液免疫机能则明显低于野生鲍。九孔鲍血细胞在吞噬活动中具有呼吸爆发产生活性氧的功能与特点,活性氧的产生与九孔鲍自身生理、环境温度以及异物数量等因素有显著的相关性(P<0.01)。环境温度由25℃下降至18℃,九孔鲍的细胞与体液免疫机能都有明显的下降。退化鲍在25℃和18℃条件下血淋巴液的抗菌活力水平差异性不明显。酚氧化酶存在于九孔鲍血淋巴液中,但其活性水平不高,在各类九孔鲍之间的差异性也不明显,不能确认为九孔鲍的主要体液免疫因子。     

九孔鲍血细胞吞噬能力的研究

在体外研究九孔鲍(Haliotis diversicolor supertexta)血细胞的吞噬能力,初步了解九孔鲍血细胞的吞噬功能。结果表明,在室温20~22℃下,不同反应时间(30、60、90min)对血细胞吞噬作用的影响不显著;不同吞噬物比例(酵母聚糖与血细胞数量之比为2、5、10)对血细胞吞噬作用有显著影响,吞噬率和吞噬指数随着吞噬物比例的增大而上升。测定不同温度(10、20、30℃)下血细胞对酵母聚糖的吞噬作用,20℃时血细胞的吞噬率、吞噬指数最高;10℃与30℃时的吞噬率、吞噬指数均较低,但与20℃的相比没有显著差异。电镜观察证实酵母聚糖颗粒位于血细胞的吞噬体内。在室温20~22℃、反应时间60min的条件下,测定血细胞对金黄色葡萄球菌(10个菌/每个血细胞)的吞噬作用(吞噬率为23.7%、吞噬指数为0.51)。在硝基四氮唑蓝(NBT)还原试验中,血细胞在酵母聚糖的刺激下,发生呼吸暴发产生了超氧阴离子。     

温度胁迫对皱纹盘鲍生理和生化活动的影响

为探讨温度胁迫下皱纹盘鲍(Haliotis discus hannai Ino)的响应机制,采用室内控温实验,通过设置4个温度梯度(5℃、10℃、20℃和25℃),设计温度骤变处理组(皱纹盘鲍从15℃暂养温度直接转移至各实验温度)和温度缓变处理组(0.5℃/12 h),分析温度剧烈变化和温度缓慢变化对皱纹盘鲍耗氧率和排氨率的影响及其差异性;并对高温和低温处理下皱纹盘鲍消化腺中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、酸性磷酸酶(ACP)和溶菌酶(LSZ)活性的变化情况以及不同组织(血细胞和肌肉组织)中Cu/Zn–SOD基因的表达状况进行了研究。结果表明,皱纹盘鲍耗氧率和排氨率随海水温度的升高而增加,20℃达到最高值;25℃骤变处理组与缓变处理组耗氧率和排氨率间存在极显著差异(P0.01)。5℃和10℃骤变处理组皱纹盘鲍氧氮比(O/N)同缓变处理组间存在极显著差异(P0.01)。在高温胁迫后的3 h,消化腺中SOD、CAT和LSZ活性达到最高,而ACP活性在胁迫6 h后达到最高(P0.01);低温胁迫显著降低皱纹盘鲍LSZ的活性,于胁迫9 h后达到最低(P0.01)。不同温度胁迫下,皱纹盘鲍血细胞和肌肉组织中Cu/Zn–SOD基因的相对表达量均表现上调,与对照组存在显著差异性(P0.05)。本研究表明,温度胁迫能显著影响皱纹盘鲍的生理和生化活动,这将有助于探讨皱纹盘鲍夏季高死亡率的原因,为皱纹盘鲍健康养殖提供一定的理论依据。     

三倍体皱纹盘鲍活体倍性检测

以皱纹盘鲍的血细胞为材料，采用植物血球凝集素(PHA)体外浸泡法，运用正交实验设计，探讨PHA处理时间、PHA浓度、抽血时间对细胞分裂频度的影响。根据正交实验直观分析结果，得出用血细胞直接制备染色体标本进行三倍体皱纹盘鲍活体倍性检测的最优组合：PHA处理时间18h、PHA浓度0．04％，抽血时间22：00-24：00，3因素的主次顺序：PHA处理时间→PHA浓度→抽血时间。并可获得大量图象清晰、长度适中、分散良好的中期分裂相，是皱纹盘鲍活体倍性检测的最佳方法。同时，通过流式细胞仪测定皱纹盘鲍血细胞DNA相对含量进行三倍体皱纹盘鲍活体倍性检测，该方法制样简单，测试速度快。     

皱纹盘鲍与绿鲍杂交种生长比较

为探明皱纹盘鲍(Haliotis discus hannai)与绿鲍(Haliotis fulgens)种间杂交的受精、孵化和苗种发育规律,对皱纹盘鲍和绿鲍进行种间杂交,检测了不同组合受精率和孵化率,并以同期培育的皱纹盘鲍与绿鲍种间杂交F1(DF)、自繁F1(DD、FF)及回交F1(DD×DF、DD×FD)为研究对象。在相同养殖条件下,本文对皱纹盘鲍与绿鲍杂交、回交后代的生长、存活差异及相应的优势进行研究,为进一步探讨皱纹盘鲍与绿鲍的杂交效果、准确评价杂种优势提供理论依据。     

饵料对不同规格皱纹盘鲍能量收支的影响

对3种不同规格的皱纹盘鲍(41.40±2.05、54.22±2.66、63.17±2.52mm)分别进行不同饵料搭配的投喂,并对各生理活动的能量代谢进行了测量与计算。实验结果表明,搭配投喂的实验组中皱纹盘鲍能够摄取更多的有机物作为能量代谢的基质。扣除代谢能、排泄能和排粪能后,搭配投喂能提供给皱纹盘鲍的生长能显著高于对照组(P<0.05),尤其是孔石莼与裙带菜搭配投喂组以及裙带菜与海带搭配投喂组,其获得的生长能的比例在3种规格组中均处在很高的水平,是鲍的筏式养殖中值得推广使用的投喂方法。     

皱纹盘鲍营养生理学与疾病学研究进展

鲍类具有很高的营养价值,是一种非常重要的经济贝类,国内外对鲍类的研究主要集中在对皱纹盘鲍及其他一些种的摄食生理、营养需要及疾病防治等方面.本文主要从对皱纹盘鲍在不同生长阶段摄食、生长、发育等方面的研究及其在外界环境因素影响下的生理状况,皱纹盘鲍的营养需要与人工配合饲料的配制,以及皱纹盘鲍的主要细菌性、病毒性、寄生性病害的研究与防治等三个方面进行综述.     

皱纹盘鲍营养生理学与疾病研究进展

鲍类具有很高的营养价值，是一种非常重要的经济贝类，国内外对鲍类的研究主要集中在对皱纹盘鲍及其他一些种的摄食生理、营养需要及疾病防治等方面。本文主要从对皱纹盘鲍在不同生长阶段摄食、生长、发育等方面的研究及其在外界环境因素影响下的生理状况，皱纹盘鲍的营养需要与人工配合饲料的配制，以及皱纹盘鲍的主要细菌性、病毒性、寄生性病害的研究与防治等三个方面进行综述。     

橄榄蛏蚌血细胞形态及吞噬能力的初步研究

对橄榄蛏蚌(Solenaia oleivora)血细胞的形态特征和吞噬作用进行了初步研究。基于瑞氏染色后血细胞的形态、颜色、大小等特征,可将橄榄蛏蚌的血细胞分为大颗粒细胞、小颗粒细胞、透明细胞、类淋巴细胞共4种类型。透明细胞的直径最大,小颗粒细胞次之,类淋巴细胞最小;透明细胞的胞核直径最大,小颗粒细胞次之,大颗粒细胞最小。类淋巴细胞的核质比最大(0.67),其他3种细胞的核质比为0.37～0.48。4种血细胞所占比例以小颗粒细胞最高(43.6%),透明细胞次之(36.6%),类淋巴细胞最低(7.0%)。橄榄蛏蚌平均血细胞浓度为(4.21±1.51)×105个/mL,个体的血细胞浓度与体重(或壳长)不相关(P>0.05),不同体重组(或壳长组)的平均血细胞浓度无显著差异(P>0.05)。在37℃体外吞噬试验中,橄榄蛏蚌血细胞对金黄色葡萄球菌的吞噬比例为(43.6±7.0)%,吞噬指数为(0.743±0.145)。4种血细胞中,小颗粒细胞的吞噬比例最大(0.288±0.061),类淋巴细胞不具吞噬能力。     

Studies on the effects of LPS, ß‐glucan and metabolic inhibitors on the respiratory burst and gene expression in Atlantic salmon macrophages

Reactive oxygen species (ROS) production in macrophage‐like cells is induced as an antimicrobial defence against invading pathogens. In this study, we have explored how different stimuli and metabolic inhibitors affect the level of respiratory burst in Atlantic salmon (Salmo salar L.) head kidney macrophage‐like cells. Cells stimulated in vitro by bacterial lipopolysaccharide (LPS) and ß‐glucan showed increased production of ROS compared to unstimulated cells. Both stimulation and costimulation by curdlan (ß‐glucan) induced a higher production of ROS compared to stimulation and costimulation by LPS. Metabolic inhibitors co‐incubated with the stimulants did not, in most cases, perturb the level of ROS generation in the salmon macrophage‐like cells. The NAD⁺ content as well as the NAD⁺/NADH ratio increased in curdlan and LPS + curdlan‐stimulated cells compared to control cells, which indicated increased metabolic activity in the stimulated cells. Supporting these findings, gene analysis using real‐time quantitative PCR showed that arginase‐1 and IL‐1ß genes were highly expressed in the stimulated cells.     

The feasibility of using primary shrimp hemocyte culture to screen herbal immunostimulants

An analysis of the immune activity of herbal extract in vivo and in vitro assays was performed to explore the feasibility of using primary shrimp hemocyte culture to screen herbal immunostimulants. In vitro experiment was conducted by adding herbal extract into the medium of primary cultured hemocytes and incubating for 24h. In vivo experiment was carried out by feeding shrimp with diets incorporated with herbal extract for 21 days. Challenge test can be used to directly reflect comprehensive immune capacity of organisms. Four immune indexes, total hemocyte counts (THC and lysozyme, phenoloxidase (PO), and phagocytic activity were examined both in vitro and in vivo. The results showed that effects of herbal extract on THC or lysozyme activity were different between in vitro and in vivo. On the contrary, effects of herbal extract on PO activity and phagocytic activity in vitro and in vivo showed a strong correlation. Herbal extract, which enhanced PO activity and phagocytic activity in vitro, had ultimate immune-promoting function in vivo. Therefore, it was feasible to use primary hemocyte culture to screen herbal immunostimulants by determining the effects of herbal immunostimulants on PO and phagocytic activities.     

皱纹盘鲍血细胞的亚显微结构及分类研究

通过对皱纹盘鲍血细胞的超微结构的观察，将其血细胞分成5种类型：大颗粒细胞、小颗粒细胞、特殊颗粒细胞、透明细胞和淋巴样细胞。根据胞核的形态和胞质中细胞器的结构特征，研究认为大、小颗粒细胞是同一类细胞的不同发育阶段，而特殊细胞则是大、小颗粒细胞的原始细胞；透明细胞和淋巴样细胞是两类完全分化了的细胞。     

圆背角无齿蚌损伤后血细胞的吞噬活性

模拟人工育珠手术对圆背角无齿蚌外套膜进行务后，研究了其血细胞在０～２５ｄ对荧光极毛杆菌的吞噬活性的变化。结果发现，圆背角无齿蚌４种血细胞中有吞噬能力的主要是颗粒细胞和无颗粒细胞。手术蚌血细胞的吞噬活性从手术后１０天起有了显著升高，在第２０天时达到最大，而后开始下降；血清对手术蚌和对照蚌血细胞的吞噬都具有调理作用。     

Interaction of the fish pathogen Aeromonas hydrophila with tilapia, Oreochromis aureus (Steindachner), phagocytes

Abstract. Interaction of Aeromonas hydrophila and tilapia, Oreochromis aureus (Steindachner), phagocytes was studied in vitro. All virulent and avirulent strains of A. hydrophila tested could multiply in non-activated and Freund's complete adjuvant activated phagocytes. Activated phagocytes increased the uptake of bacteria into cells, and the rates of intracellular replication for these bacteria were faster than in non-activated phagocytes. Among the A. hydrophila strains examined, virulent strain PPD134/91 replicated at the fastest rate inside phagocytic cells and produced cytopathic effect in the phagocytes in the shortest incubation time. Opsonized avirulent A. hydrophila were sensitive to phagocyte-mediated killing or unable to grow in phagocytes. Serum components and phagocytes may together prevent the growth of avirulent A. hydrophila in fish. The release of extracellular oxygen radicals during phagocytosis was examined using chemiluminescence assay (CL). Virulent strains induced CL responses but avirulent strains did not. This suggests that the virulent strains interacted with the phagocytes somewhat differently from the avirulent strains.     

温度、盐度和pH对仿刺参体腔细胞活性氧产生的影响

无脊椎动物主要依靠天然免疫进行自身防御,而活性氧在保护宿主免受病原侵害方面发挥重要作用。本文研究了环境因子温度,盐度和pH对仿刺参体腔细胞吞噬过程中活性氧产生的影响。不同环境条件处理暂养7d的仿刺参,于不同时间点抽取体腔液,然后用鲁米诺化学发光法检测仿刺参体腔细胞体外吞噬酵母细胞时活性氧的产生。试验结果表明,与对照组相比(17℃),6℃和26℃水温中的仿刺参分别从第1d和第15d极大地增加了活性氧的产生。盐度25和盐度35两组仿刺参体腔细胞比对照组吞噬过程产生更多的活性氧,而盐度16和40两组第1d时活性氧的产生亦显著增强。较低pH同样极大增强了吞噬作用中活性氧的产生。研究结果表明,温度、盐度和pH均可影响仿刺参体腔细胞吞噬过程中活性氧的产生。该试验为进一步研究仿刺参在适应不同环境时的生理生化过程及无脊椎动物免疫机制奠定了基础。     

Enhancement of resistance to bacterial infection in rainbow trout, Oncorhynchus mykiss (Walbaum), by oral administration of bovine lactoferrin

Abstract Bovine lactoferrin was evaluated for its ability to enhance resistance of rainbow trout, Oncorhynchus mykiss (Walbaum), to infection with Vibrio anguillarum. Oral administration of lactoferrin (100 mg kg⁻¹) to fish daily for 3 days prior to an intraperitoneal challenge with V. anguillarum resulted in increased survival rates, and enhanced resistance against Streptococcus sp., although to a lesser extent. In lactoferrin-treated fish, an increase in phagocytic and chemiluminescent (CL) activities of pronephros cells against V. anguillarum was observed. The phagocytic and CL activities of cells against Streptococcus sp. were also significantly increased. However, no in vitro bactericidal activities of lactoferrin against V. anguillarum or Streptococcus sp. were observed. This suggests that the lactoferrin enhanced the resistance of the rainbow trout against bacterial infection through the activation of phagocytes.     

Time to recover the upright posture in juvenile abalones (Haliotis discus discus Reeve, H. gigantea Gmelin and H. madaka Habe)

The adoption of an upright posture in abalones is essential to enable them to use their foot, and hence to be able to move and seek shelter, as well as to avoid exposure of soft parts and possible predation. In mass restocking programs for abalone, juveniles are released by divers near the seabed, but without control over their posture when they reach the bottom. Thus, the time to recover the upright posture is an important consideration in abalone restocking programs as the quicker they assume this posture the higher the likelihood of survival. This study reports significant differences in the speed of recovering the upright posture between juveniles of the abalones Haliotis discus discus, H. gigantea and H. madaka and between tests conducted under stagnant and flowing water conditions. Longer times were required for recovery in all species in stagnant than flowing water. On average, juveniles of H. discus discus (17.16 and 10.43 s) and H. gigantea (22.54 and 11.89 s) recovered faster than those of H. madaka (161.13 and 49.02 s) under stagnant and flowing water conditions respectively. These results suggest that different species require different levels of care and that water flow or current at the time of release may affect post‐release survival.