The determination of lipase and phospholipase activities in gut contents of turbot (Scophthalmus maximus) by fluorescence-based assays

To study the potential of fluorescence based assays in the study of lipid digestion in fish, acyl esters of 4-methylumbelliferone and 1-acyl-2-[6 (7 nitro-1,3 benzoxadiazol-4-yl)amino]caproyl labeled phosphatidylcholine compounds (NBD-PC) were used as substrates for the assay of neutral lipase and phospholipase, respectively, in the gut contents of turbot. 4-Methylumbelliferyl hepatanoate (4-MUH) was hydrolysed at a higher rate than the butyrate or oleate esters whilst the hexanoic (C6) ester of NBD-PC was a more convenient substrate for the phospholipase assay than the dodecanoic (C12) ester. Neutral lipase activity was almost 10% higher when 50 mm potassium phosphate buffer pH 7.8 was used instead of 0.01 m citrate/sodium phosphate buffer pH 7.2. Both assays were very sensitive: neutral lipase and phospholipase activities were detectable at a minimum protein concentration in the digesta of 0.04 and 1.25 mg/ml, respectively. When the variations in lipolytic activities with gut segment and with size of fish were examined neutral lipase activity was always found to be higher in the hindgut and rectum segments than in the foregut. Although phospholipase activity was also found to be highest in the hindgut of the largest fish examined (av. wt. 182.3g), in fish of average weight 8g fish the activity was similar in all three segments. In the digesta from the whole gut of smaller fish (av. wt. 0.2, 0.6 and 1.43g) neutral lipase and phospholipase activities increased with increasing body mass when expressed as per ml of digesta. It is concluded that fluorescence-based assays are applicable to the study of lipid digestion in fish of different size.     

刺参肠组织蛋白质双向电泳体系的建立及优化

以刺参肠组织为材料,通过蛋白质提取方法、上样前处理方法、上样量以及聚焦条件的优化,建立了刺参肠组织蛋白质双向电泳技术体系.试验结果表明,采用TCA-丙酮沉淀法制备刺参肠组织蛋白质,能够得到高纯度的蛋白质提取液.刺参样品上样前不经过处理,等电聚焦电压1 000V,电流7mA,聚焦时间3h,平衡时间30min,最佳上样量25μg,pH 4～6.5、8.5cm的IPG胶条,银染能获得较好的双向电泳图谱.通过蛋白质双向电泳体系的优化,获得了刺参肠组织的蛋白质表达图谱,为进一步研究刺参差异表达蛋白的筛选及蛋白质组学研究提供技术保障     

单过硫酸氢钾粉对5种水产常见致病菌的体内外抑菌作用

研究了单过硫酸氢钾粉对5种水产常见致病菌的体内、外抑菌作用,测定了体外最小抑菌浓度(MIC)、最小杀菌浓度(MBC)及以保护率为指标的体内抑菌效果。结果表明,体外抑菌实验中,单过硫酸氢钾粉对爱德华氏菌Edwardsiella、温和气单胞菌Aeromonas sobria、嗜水气单胞菌Aeromonas hydrophila、迟钝爱德华氏菌Edwardsiella tard、荧光假单胞菌Psudomonas fluorescens均有明显的抑制作用,其对5种菌的MIC分别为1、0.25、0.125、0.25、0.5μg/ml;MBC与MIC相同。而体内抑菌实验中,随着单过硫酸氢钾粉浓度的增加,由5种致病菌引起鲫鱼的死亡率逐步降低。统计分析发现,使用0.25μg/ml单过硫酸氢钾粉药浴鲫鱼,即可对由5种致病菌引起的鲫鱼病害有治疗的作用。     

高锰酸钾氧化池塘藻毒素的研究

研究了高锰酸钾KMnO4 对池塘微囊藻毒素(MC - LR) 的去除效果, 探讨反应影响因素(高锰酸钾质量浓度、温度、pH 值、反应时间)对去除率的影响。结果表明, 高锰酸钾能有效地去除水中的微囊藻毒素。高锰酸钾对MC- LR的去除率与高锰酸钾投加的质量浓度、温度、反应时间成正相关, 其中高锰酸钾质量浓度对去除率的影响最大; 其次为温度和pH, 当温度为35e 时, 去除率可达96. 65%, 当pH 为3时, 去除率也达到了95. 1%; 反应时间对去除率的影响不太显著。正交试验的结果也进一步证明了这一点。     

Characteristics of divalent minerals extracted from liver of Japanese common squid Todarodes pacificus under various experimental conditions

In relation to the problem of how to utilize the waste from squid processing, we determined the proximate and mineral compositions of liver of the Japanese common squid Todarodes pacificus, and characterized the behavior of divalent minerals, focusing in particular on cadmium removal. The squid liver contained fat (44.0 g/100 g dry matter), protein (13.5 g/100 g dry matter), and ash (2.11 g/100 g dry matter). It also contained the macrominerals sodium, potassium, magnesium, and calcium, as well as the trace minerals iron, zinc, cadmium, and copper. Low- and high-pH treatments (pH 2 and 12) removed the cadmium entirely, but the trace minerals zinc, iron, and copper, as well as soluble protein, which are all important for proper functioning of the human organism, were also removed in large quantities. High percentages of soluble minerals and soluble protein were found in the low molecular weight (MW < 10,000) fraction. The solubility of zinc and copper were strongly related to the solubility of cadmium, but there was no relationship between the solubilities of magnesium and cadmium. On the other hand, while treatment with either 0.9 % NaCl or 100 mM phosphate buffer (pH 7.4) also removed the cadmium completely, it left some useful compounds (trace minerals and soluble protein) in the solid part, so these treatments may represent appropriate methods for removing cadmium from squid processing waste.     

牙鲆淋巴囊肿病毒纯化及不同来源病毒免疫特性比较

比较了3种不同的牙鲆淋巴囊肿病毒纯化方法,优化后的方法如下：剥离囊肿表面薄膜,收集内容物,匀浆后再用超声波细胞破碎仪破碎,反复冻融,650×g、1800×g差速离心,30%（W/W）蔗糖垫底超速离心（78500×g）浓缩病毒,最后蔗糖密度梯度超速离心（78500×g）纯化病毒。电镜观察发现,出现在47%～52%蔗糖密度区域的病毒带含有多量、纯净和结构一致的病毒粒子。此外,利用制备的兔抗血清对不同地区的病毒进行了免疫特性分析,Western blotting检测显示来自威海、青岛及秦皇岛3个地区的淋巴囊肿病毒反应结果是一致的,均有3条蛋白带发生反应,其分子量分别为125、66和55kDa。     

斑点酶联免疫吸附试验检测猪瘟血清抗体最佳条件的研究

以硝酸纤维膜为固相载体,以猪瘟病毒为抗原,确定了Dot-PPA-ELISA检测猪瘟抗体的最佳反应条件。试验表明,猪瘟兔化弱毒经仔猪睾丸原代细胞培养纯化,以0.05mol/LpH9.5CBS缓冲液稀释至浓度为0.98～1.15mg/mL制备诊检抗原膜片;采用4%蛋清蛋白0.05mol/LpH9.5CBS封闭;检测反应稀释液应用0.01MPPBST(含0.1%PEG、0.05Tween-20),酶结合物工作浓度以1∶30,底物选取4-氯1-萘酚等为检测猪瘟血清抗体的最佳反应条件。重复试验证明了其检测稳定性好。交叉试验、阻断试验证明了其特异性强。     

Gut pH as a limiting factor for digestive proteolysis in cultured juveniles of the gilthead sea bream (Sparus aurata)

After the development of the gastric function in juvenile fish, dietary proteins enter a two-phase digestive process comprising an acidic gastric phase followed by an alkaline intestinal phase. However, the main gastric protease, pepsin, is strictly dependent on the existence of a low-enough environmental pH. In 20-g gilthead sea bream, Sparus aurata, the mean minimal gastric pH is close to 4.5, while the mean pH in the duodenal portion of the intestine was nearly fixed at 6.5. The mean maximal gastric content of HCl was approximately 20 microEq for a low-buffering diet. Gastric proteases were more severely affected than intestinal proteases when assayed at actual sub-optimal pH values, 4.5 and 6.5, respectively. When the gastric proteases of juvenile fish were pre-incubated with a citric acid buffer at pH 6.0, the activity at pH 4.5 was very low, whereas when they were pre-incubated with the same buffer at pH 3.0, the activity at pH 4.5 was significantly increased; this fact suggests a deficient activation of zymogens during the gastric digestion and points to a potential approach to improve protein digestion in juvenile gilthead sea bream.     

Acidification of the stomach contents of weaned piglets--effect of feed quantity and composition

The pH in the stomach chyme (5-8 hours past offering the diet) of reared piglets was strongly correlated to the ingested feed quantity, a complete acidification of the stomach contents occurred in restrictively fed piglets (15 g diet/kg BW) only. Feeding ad libitum resulted in comparable low pH values in the fundus and pylorus region only, whereas in the cardiac region high pH values (5-6.5) were observed. Formic acid as feed additive (5 ml/kg diet) did not influence the pH in stomach contents significantly, on the other hand the ingestion of a diet with high CaCO3 content (59 g Ca/kg DM) prevented the acidification of the stomach chyme totally. The variation of pH-values in the different areas within the stomach was caused mainly by the variation of hydrochloric acid concentrations, the influence of lactic acid concentration was negligible.     

Evaluation of Sperm-Activating Solutions in Atlantic Salmon Salmo salar Fertilization Tests

The use of saline solutions was tested to determine their efficacy as replacements for ovarian fluid as sperm activators and to eliminate variability encountered with the use of Ovarian fluid. We tested fertilization rate of semen from eight males on Atlantic salmon Salmo salar eggs after five sperm-activating solutions and a non-activating saline were substituted for ovarian fluid. We used solutions shown acceptable for use with other salmonid species. The six solutions tested were a non sperm-activating phosphate-buffered saline (PBS, 7.2 g/L NaCl, 1.48 g/L Na₂HPO₄, 0.43 g/L K H₂PO₄), a Tris buffer (6.99 g/L NaCl, 3.63 g/L Tris and 2.42 g/L glycine), a Borax buffer (12.2 g boric acid/L in solution 1, 76 g disodium tetraborate/L combined 100:118, then 1 L combined with 3.7 L water and 18 g NaCl), and three solutions of 9.25 g/L (125 mM) NaCl buffered to pH 6.0, 7.5, and 8.9. The latter five solutions activated sperm immediately on contact, while PBS required additional water to activate sperm. The PBS solution was the least effective (mean percent eyed eggs 37.6%) for egg fertilization. The mean percent eyed eggs for the other five saline solutions (range 78% to 86%) were not significantly different. Sperm from one male provided significantly lower egg fertilization (39.6%) when compared with the other seven males (67.2–87.4% egg fertilization). Percent egg fertilization was not related to number of live sperm cells per egg. Our results show that osmotically-balanced sperm-activation solutions, even those with a pH range from 6.0 to 8.9 provide adequate media for fertilization of Atlantic salmon eggs. Fertilization in a deactivation saline and water reactivation of sperm resulted in low egg fertilization.     

pH胁迫对刺参存活、生长及抗氧化酶活性的影响

本研究比较分析了在对照组(pH为8.4)、低pH胁迫组(pH为6.8、7.0、7.2、7.4、7.6和7.8)和高pH胁迫组(pH为8.6、8.8、9.0、9.2、9.4和9.6)的养殖水环境下,胁迫36 d对刺参(Apostichopus japonicus Selenka)存活、生长及抗氧化酶活性的影响。结果显示,不同pH对刺参存活、生长及抗氧化酶活性有显著影响。在pH 7.4~9.0范围内,刺参存活率为100%,随着pH胁迫强度和胁迫时间的增加,刺参存活率逐渐降低,当pH为6.8、7.0和9.6时,自第3天起,刺参处于过度应激状态,继而有死亡个体出现,至30 d时刺参死亡率为100%。不同pH显著影响刺参生长,刺参特定生长率随pH胁迫程度增加呈下降趋势,当pH为9.0时,刺参出现负增长。各pH胁迫组刺参超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性均高于对照组,且随pH胁迫程度的增加呈先升高后下降的变化趋势。在低pH组中,以pH=7.4时刺参SOD和CAT活性最高,分别达到(74.92±2.24)U/ml和(14.99±2.38)U/ml,显著高于对照组;而在高pH组中,SOD和CAT活性分别以pH 8.8和9.0时最高,分别达到(72.90±1.10)U/ml和(15.68±0.89)U/ml,显著高于对照组。结果表明,pH在7.4~9.0范围内是刺参存活与生长的适宜水环境,过高或过低均会引起刺参不同程度的应激反应,进而导致刺参的死亡。     

Application of Computer‐assisted Sperm Analysis in Selecting the Suitable Solution for Common Carp,Cyprinus carpio L., Sperm Motility

The common carp, Cyprinus carpio L., sperm motility parameters were analyzed by using computer‐assisted sperm analysis system. The percentage of motile sperm (MOT, %), progressively motile sperm (PRG, %), curvilinear velocity (VCL, µm/sec), average path velocity (VAP, µm/sec), the wobbling index (WOB, %), movement linearity (LIN, %), beat cross frequency (BCF, Hz), and amplitude of lateral head displacement (ALH, µm) were determined. Five activation solutions (As) were used to activate sperm movement. As 1 solution: 68 mM NaCl, 50 mM urea, 0.5% bovine serum albumin (BSA), pH: 7.7, 181 mOsm/kg; As 2 buffer: 100 mM NaCl, 10 mM Tris, 0.5% BSA, pH: 9.0, 199 mOsm/kg; As 3 solution: 86 mM NaCl, 0.5% BSA, pH: 7.4, 167 mOsm/kg; As 4 buffer: 5 mM KCl, 45 mM NaCl, 30 mM Tris, 0.5%, pH: 8.0, 160 mOsm/kg; and As 5 solution: distilled water with the addition of 0.5% BSA, pH: 7.3, <3 mOsm/kg. Among five tested solutions, a buffer with a pH of 9.0 and osmolality of approximately 200 mOsm/kg (As 2) was the most suitable. After its activation, a significant increase in MOT and ALH values was observed, which can be of importance to the effectiveness of egg fertilization .     

草鱼鳃组织蛋白双向电泳技术的建立及优化

为建立并优化草鱼鳃蛋白质组双向电泳的技术体系,实验将草鱼鳃经裂解处理后,通过固相p H梯度胶条进行一向等电聚焦和SDS聚丙烯酰胺凝胶垂直电泳进行蛋白分离,对不同的裂解液配方、蛋白纯化方法、IPG胶条的分离范围、上样量和染色方法等进行了探索和优化,并应用lmage Master 2D Platinum 7.0软件对电泳图谱进行了分析。结果表明:采用裂解液中添加DTT来取代Tris和TBP、选择分离范围为pH4-7的IPG胶条和150μg的主动水化上样,电泳结束后对凝胶进行硝酸银染色,获得了覆盖范围广、低背景、高分辨率、适合差异蛋白分析的双向电泳参考图谱。     

Characteristics of minerals extracted from the mid-gut gland of Japanese scallop Patinopecten yessoensis at various pH values

This experiment was carried out to determine the proximate composition of the mid-gut gland (MGG) of the Japanese scallop Patinopecten yessoensis to evaluate the characteristics of divalent minerals and water-soluble protein at various pHs, and to examine Cd-binding protein at different molecular weights. MGG of scallop contained protein, fat and ash of 28.9 g/100 g dry matter, 44.6 g/100 g dry matter and 6.78 g/100 dry matter, respectively. MGG also contained the macrominerals sodium, potassium, magnesium and calcium, and the trace minerals iron, zinc, cadmium and copper. The solubility of divalent minerals and water-soluble protein was high in both acidic and alkaline conditions, except that magnesium was not affected by acidity. The solubility of copper and iron had a positive correlation with water-soluble protein at all pH values, whereas cadmium had a strong correlation at alkaline pH. Low-molecular-weight water-soluble protein (fraction III, 437.5 < MW < 1,355) bound cadmium strongly in acidic, neutral and alkaline conditions. However, in acidic conditions cadmium had the strongest binding to protein.     

Dietary potassium requirement of juvenile grass shrimp Penaeus monodon

ABSTRACT: A feeding trial was conducted to estimate the minimal dietary potassium (K) requirement for juvenile grass shrimp Penaeus monodon . Purified diets with seven levels (0, 0.3, 0.6, 0.9, 1.2, 1.5, 2.0 g/100 g) of supplemental potassium were fed to P. monodon (mean initial weight 0.75 ± 0.01 g) for 8 weeks. Each diet was fed to three replicate groups of shrimp. The rearing water contained 360 mg/L potassium. Weight gain and protein efficiency ratio (PER) of shrimp improved as dietary potassium supplementation level increased up to 1.5 g K/100 g diet and thereafter declined significantly ( P < 0.05). Feed efficiency (FE) of shrimp also showed a similar trend. However, whole body K concentration of the shrimp kept an increasing trend up to 2.0 g K/100 g diet. Analysis of the weight gain (percent) and PER of the shrimp by polynomial regression indicate that the minimal dietary potassium requirement in growing P. monodon is approximately 1.2 g/100 g.     

采用化学氧化法降解贝类养殖水中扑草净残留的研究

为去除贝类养殖过程中残留的农药扑草净,研究了不同处理(正常、超声波震荡、紫外线照射和不同pH)条件下高锰酸钾、过氧化氢和次氯酸钠对扑草净的降解效果.结果显示,次氯酸钠对扑草净的降解率最高,其降解效果不受紫外线照射、超声波震荡以及水体pH变化的影响.高锰酸钾和过氧化氢对扑草净的降解率较低,超声波震荡和紫外线照射辅助处理对...     

pH值对草鱼白细胞吞噬力影响的研究

用不同pH值控制的磷酸缓冲液为条件,测定pH值对草鱼血液吞噬活力的直接影响,其结果表明:在偏酸性和中性环境中(pH 值6 .0 ,6 .5 ,7.0 ) ,吞噬细胞活性受到抑制;相反,在偏碱性环境(pH 值7.5 ,8.0 )却使其吞噬细胞活性增强,与前者相比,差异显著。     

Development of a rapid immunochromatographic strip test for the detection of Vibrio parahaemolyticus toxin B that cause acute hepatopancreatic necrosis disease

Here, two monoclonal antibodies (MAbs) specific to different epitopes on ToxB, a toxin produced by Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis disease (VP_AHPND), were employed to develop a rapid strip test. One MAb was conjugated to colloidal gold to bind to ToxB at the application pad, and another MAb was used to capture colloidal gold MAb–protein complexes at the test line (T) on the nitrocellulose strip. To validate test performance, a downstream control line (C) of goat anti-mouse immunoglobulin G antibody was used to capture the free colloidal gold conjugate MAb. The sample in the application buffer could be applied directly to the application well, and the test result was obtained within 15 min. The sensitivity of the kit is approximately 6.25 µg/ml of toxin, which was equivalent to the toxin produced by approximately 10⁷ cfu/ml of bacteria. This kit is convenient and easy to use since it can be used to identify VP_AHPND directly using a single colony of bacteria grown on agar culture plates. Because of its high specificity and simplicity, as well as not being reliant on sophisticated equipment or specialized skills, this strip test could be used by farmers for surveillance for ToxB-producing bacteria.     

呋喃西林及其代谢物氨基脲的伏安检测方法

以石墨烯基电极为工作电极,研究了呋喃西林(NF)和氨基脲(SEM)的伏安检测方法。循环伏安(CV)扫速0.10 V/s时,在醋酸-醋酸钠缓冲溶液(pH 6.0)中NF发生不可逆氧化还原,过程受吸附控制。缓冲溶液种类、pH值、富集时间等优化情况下,NF的还原峰电流(ipc)与其浓度在20.0-80.0 nmol/L范围内呈线性关系,检测限为12.0 nmol/L(S/N=3)。CV扫速为0.10 V/s时,在pH4.1的醋酸-醋酸钠缓冲溶液中SEM也发生吸附控制的不可逆氧化还原。优化条件下,SEM的氧化峰电流(ipa)与其浓度在1.3-39.9μmol/L范围内呈线性关系,检测限为0.2μmol/L。优化条件下,NF和SEM可以顺序测定,检测限分别为71.0 nmol/L和0.3μmol/L,平均回收率分别为87%和90%。应用于测定淡水渔业水样中二者残留量,得到了较满意的结果。     

功能性电极快速伏安检测孔雀石绿

用阴离子表面活性剂十二烷基硫酸钠(SDS)修饰导电炭黑糊电极,制备了对孔雀石绿(MG)具有高灵敏度的功能传感器。实验对MG在该功能电极上的电化学行为进行了研究,并对影响测定的各种条件(制备糊电极所用导电材料种类;功能试剂修饰量;支持电解质的离子强度、pH值、种类;富集电位与时间等)进行了优化。在优化条件下,MG的氧化峰电流(ipa)与其浓度在5～200μg/L范围内呈线性关系,检测限为1.5μg/L(S/N=3)。该方法具有较高的灵敏度、良好的重现性和稳定性。应用于测定经过简单过滤预处理后的淡水渔业水样,平均回收率为90%。