养殖用水中孔雀石绿和结晶紫及其代谢物的测定

建立了用高效液相色谱紫外、荧光检测器同时检测养殖用水中的孔雀石绿、结晶紫及其代谢物隐色孔雀石绿和隐色结晶紫的分析方法。孔雀石绿和结晶紫用紫外检测器588 nm波长检测;隐色孔雀石绿和隐色结晶紫用荧光检测器检测,激发波长:265 nm,发射波长:360 nm。孔雀石绿和结晶紫的线性范围5.0~2500μg/L;隐色孔雀石绿和隐色结晶紫的线性范围1.0~1 000μg/L,四种物质的检出限均为0.5μg/L,回收率均在70%以上,RSD15%,此方法也可用于地下饮用水中孔雀石绿、结晶紫、隐色孔雀石绿和隐色结晶紫的检测。     

同位素内标法测定水产品中孔雀石绿、结晶紫及其代谢产物

建立了水产品中孔雀石绿、结晶紫及代谢物无色孔雀石绿、无色结晶紫的液相色谱—串联质谱测定方法。以氘代孔雀石绿、氘代结晶紫、氘代无色孔雀石绿、氘代无色结晶紫分别为孔雀石绿、结晶紫、无色孔雀石绿、无色结晶紫的同位素内标进行定量,4种化合物的质量浓度为0.5～10.0μg/L时线性关系良好(r2≥0.9992);在0.5、0.75、1.0μg/kg 3个质量浓度水平添加,平均回收率为101.4%～114.6%,相对标准偏差均小于7.0%。     

超高效液相色谱-串联质谱快速测定水产品中孔雀石绿、结晶紫及其代谢物残留量

建立了超高效液相色谱-串联质谱（UPLC-MS/MS）测定水产品中孔雀石绿（MG）、结晶紫（CV）及其代谢物隐色孔雀石绿（LMG）和隐色结晶紫（LCV）残留量的快速、准确检测方法。该法在SN/T1768-2006快速检测方法的基础上,采用空白样品添加不同质量浓度标准溶液的方式绘制校准曲线,并以氘代孔雀石绿（D5-MG）和氘代隐色孔雀石绿（D6-LMG）为内标进行检测,从而大大提高了定量的准确性。结果表明,MG、CV、LMG和LCV4种待测物在0～10.0ng·mL^-1范围内线性关系良好（R〉0.99）,方法检出限均为0.1μg·kg^-1。在加标量为0.5和1.0μg.kg-1水平下4种待测物平均回收率在92.1%～111.0%之间,测定结果的相对标准偏差为2.2%～9.5%。此外,该法在FAPAS国际能力验证中得以证实,改进后的方法快速且准确,能满足水产品出口检测要求。     

水产品中孔雀石绿、结晶紫药物残留检测方法的优化

对《水产品中孔雀石绿和结晶紫的残留量的测定高效液相色谱荧光检测法》(GB/T 20361—2006)中提取、浓缩等前处理过程进行了优化。结果表明，硼氢化钾的体积为4 mL、二氯甲烷为25 mL，且样品浓缩至近干时孔雀石绿、结晶紫回收率均较高，孔雀石绿回收率达91.0%以上，结晶紫回收率在72.5%以上。指出，优化后的方法回收率高，分离效果好，可用于水产品中孔雀石绿、结晶紫药物残留检测。     

QuEChERS-UPLC-MS/MS法测定水产品中孔雀石绿和酰胺醇类抗生素残留量

建立了超高效液相色谱质谱联用法同时测定水产品肌肉组织中孔雀石绿和酰胺醇类抗生素残留的方法。样品经QuEChERS方法提取净化后,采用液相色谱-串联质谱测定,内标法定量。孔雀石绿(malachite green,MG)、隐色孔雀石绿(leucomalachite green,LMG)、氯霉素(chloramphenicol,CAP)和氟甲砜霉素(florfenicol,FF)在0.5～50.0 ng/mL质量浓度范围内线性良好(r≥0.999 4)。甲砜霉素(thiamphenicol,TAP)在2.0～100.0 ng/mL质量浓度范围内线性良好(r≥0.999 6)。MG、LMG、CAP和FF的检出限为0.1μg/kg,定量限为0.5μg/kg;TAP的检出限为0.4μg/kg,定量限为2.0μg/kg。MG、LMG、CAP和FF在0.5、2.0和10.0μg/kg加标水平时,加标回收率为72.0%～102.6%,精密度小于6.4%;TAP在2.0、10.0和50.0μg/kg加标水平时,其加标回收率为76.5%～95.4%,精密度小于5.1%。本方法适用于检测水产品中孔雀石绿类和酰胺醇类抗生素的残留,可为提升水产品质量安全水平提供技术支持。[中国渔业质量与标准,2020,10(1):60-67]     

液相色谱-串联质谱法同时测定水产品中阿维菌素、伊维菌素及四种三苯甲烷类残留

建立了液相色谱-串联质谱法(HPLC-MS)同时测定水产品中阿维菌素(AVM)、伊维菌素(IVM)及四种三苯甲烷类(孔雀石绿MG、结晶紫CV、隐性孔雀石绿LMG和隐性结晶紫LCV)残留的分析方法。样品中加入内标物,经乙腈提取,中性氧化铝固相萃取柱净化,净化后溶液用HPLC-MS分析,采用试剂线性曲线,AVM和IVM采用外标法,三苯甲烷类采用内标法定量。AVM和IVM在2.0～80μg/kg浓度内具有良好的线性关系;四种三苯甲烷类在0.25μg/kg～10μg/kg浓度内具有良好的线性关系。最低检出限分别为4.0μg/kg和0.5μg/kg,加标回收率在89.8%～113%之间,相对标准偏差(n=6)在1.4%～10%之间。     

超高效液相色谱-串联质谱法测定鱼肉中孔雀石绿及其代谢物的残留量

对GB/T 19857—2005的方法进行优化,建立了鱼肉中孔雀石绿和隐色孔雀石绿残留量的超高效液相色谱-串联质谱分析方法。样品用乙腈提取,中性氧化铝小柱净化后,采用Zorbax Eclipse Plus-C18色谱柱进行分离,以乙腈-5 mmol/L乙酸铵缓冲溶液为流动相,以电喷雾正离子化模式(Electrospray ionization,ESI+)和多反应监测(Multiple reaction monitoring,MRM)扫描模式进行测定,同位素内标法定量。结果表明:孔雀石绿和隐色孔雀石绿含量在0.5~20.0 ng/m L的范围内线性关系良好(r0.999),方法检出限均为0.05μg/kg。在罗非鱼(Oreochromis mossambicus)、草鱼(Ctenopharyngodon idellus)、大菱鲆(Scophthalmus maximus)、鳜鱼(Siniperca chuatsi)、金鲳鱼(Trachinotus ovatus)、乌鳢(Ophiocephalus argus Cantor)6种鱼肉中添加水平为0.5、1.0、2.0μg/kg时,目标物的平均回收率为81.3%~103.3%,测定结果的相对标准偏差均小于10%(n=6)。该方法定量准确、分析时间短、试剂用量少,适用于鱼肉中孔雀石绿及其代谢物残留量的定量检测。     

水产品中孔雀石绿检测方法的优化

对水产品中孔雀石绿及隐色孔雀石绿的超高效液相色谱-串联质谱检测方法进行了优化。样品依靠缓冲盐体系将目标物质离子化后用酸性氧化铝除脂,利用乙腈提取,浓缩后过中性氧化铝小柱净化,35℃旋转蒸发近干,用1.00 m L 5 mmol/L乙酸铵0.1%甲酸-甲醇溶液(1∶1,V/V)定容,经0.22μm滤膜过滤后上机检测,采用同位素内标法定量。方法优化后孔雀石绿和隐色孔雀石绿的线性范围为0.5~20.0 ng/m L,相关系数均为0.999 9,检出限为0.500μg/kg,平均回收率分别为83.4%~101%和86.1%~101%,相对标准偏差(RSD,n=5)分别为3.44%~7.76%和2.28%~8.74%。该方法灵敏度高,准确性好,与现有国家标准相比,本方法减少了有机溶剂使用量、简化了前处理步骤,并且稳定了目标物质回收率。通过验证,该方法适用于水产品中孔雀石绿及隐色孔雀石绿残留的检测。     

鲈鱼组织中阿维菌素、伊维菌素残留的高效液相色谱荧光检测法研究

采用高效液相色谱分离,荧光检测器检测,建立鲈鱼组织中阿维菌素和伊维菌素残留的检测方法.样品用乙腈提取,碱性氧化铝SPE柱净化,N-甲基咪唑和三氟乙酸酐的乙腈溶液为衍生化试剂,常温衍生化15 rain.荧光检测器激发波长为365 nm,发射波长为475 nm.结果表明,阿维菌素和伊维菌素衍生化产物的浓度与峰面积在0.5～200 ng/ml范围内有很好的线性关系.添加水平为1～10 μg/kg,阿维菌素和伊维菌素的平均回收率在80.6%～88.0%和78.8%～82.8%之间,相对标准偏差均小于10%.阿维菌素的检出限为0.1/μg/kg,伊维菌素为0.2 μg/kg.该方法简便、灵敏、可靠,可用于鲈鱼组织中阿维菌素和伊维菌素残留的检测.     

应用ELISA试剂盒检测鱼肉中孔雀石绿和结晶紫

开发了一种用于检测鱼肉中孔雀石绿(MG)和结晶紫的间接竞争酶联免疫(ELISA)检测试剂盒,并对试剂盒性能进行了验证.结果表明,MG浓度在0.2～5.4ng/mL范围内有较好线性关系,50％竞争抑制率为0.50ng/mL,试剂盒检测鱼肉中MG的最低检测限为0.17ng/g.鱼肉中分别添加0.2、0.4、0.8ng/g的MG标准品,回收率均在72.4％以上,批内变异系数小于12.4％,批间变异系数小于12.9％.本方法灵敏度高,重复性好,检测时间短,可同时检测鱼肉中孔雀石绿、结晶紫、隐性孔雀石绿、隐性结晶紫等四种药物的残留总量.     

Association between the interannual variation in the oceanic environment and catch rates of bigeye tuna (Thunnus obesus) in the Atlantic Ocean

The environmental processes associated with variability in the catch rates of bigeye tuna in the Atlantic Ocean are largely unexplored. This study used generalized additive models (GAMs) fitted to Taiwanese longline fishery data from 1990 to 2009 and investigated the association between environmental variables and catch rates to identify the processes influencing bigeye tuna distribution in the Atlantic Ocean. The present findings reveal that the year (temporal factor), latitude and longitude (spatial factors), and major regular longline target species of albacore catches are significant for the standardization of bigeye tuna catch rates in the Atlantic Ocean. The standardized catch rates and distribution of bigeye tuna were found to be related to environmental and climatic variation. The model selection processes showed that the selected GAMs explained 70% of the cumulative deviance in the entire Atlantic Ocean. Regarding environmental factors, the depth of the 20 degree isotherm (D20) substantially contributed to the explained deviance; other important factors were sea surface temperature (SST) and sea surface height deviation (SSHD). The potential fishing grounds were observed with SSTs of 22–28°C, a D20 shallower than 150 m and negative SSHDs in the Atlantic Ocean. The higher predicted catch rates were increased in the positive northern tropical Atlantic and negative North Atlantic Oscillation events with a higher SST and shallow D20, suggesting that climatic oscillations affect the population abundance and distribution of bigeye tuna.     

Growth performance,digestive enzyme activity and immune response of Japanese sea bass,Lateolabrax japonicus fed with fructooligosaccharide

In this experiment, a feeding trial was performed to determine the effects of fructooligosaccharide (FOS) on growth performance, digestive enzyme activity and immune response of Japanese sea bass, Lateolabrax japonicus juveniles (initial weight 38.3 ± 0.5 g), and the fish were examined following feeding with six levels of FOS (0, 0.5, 1, 2, 4 and 6 g/kg) for 28 days. Significant enhancement of weight gain (WG) and specific growth rate (SGR) was found in fish fed 1 g/kg FOS incorporated diets (p < .05), while the feed conversion ratio (FCR) in the 1, 2 g/kg FOS groups reduced significantly compared with the control (p < .05). Besides, the crude lipid in the 4, 6 g/kg FOS groups increased significantly compared with the control (p < .05). On the other hand, the erepsin and lipase activities significantly elevated in intestine of fish fed 2 g/kg FOS (p < .05) and the lysozyme activity in serum of fish fed 2 g/kg FOS were significantly higher than that in the control (p < .05). Moreover, the alkaline phosphatase activities in serum of fish fed 0.5, 1, 2 g/kg FOS were significantly higher than in control (p < .05). Regression analysis showed that the relationships between dietary FOS levels and either SGR, FCR, erepsin or lysozyme activities were best expressed by regression equations, and the optimal inclusion levels are 1.37, 1.80, 3.06, 3.11, 1.93 and 1.80 g/kg for SGR, FCR, erepsin, lipase, lysozyme and total superoxide dismutase activities, respectively. Overall, this study revealed that FOS incorporated diets could beneficial for L. japonicus culture in terms of increasing the growth, digestion and immune activities. Under the present experimental condition, the optimal supplementary level of FOS in the diet of L. japonicus is 1–3 g/kg.     

Protein and amino acid composition from the mantle of juvenile O ctopus vulgaris exposed to prolonged starvation

Protein and amino acid composition of the mantle of juvenile O ctopus vulgaris (Cuvier, 1797) during fasting for 27 days were determined. Average protein content of octopus mantle was of 711.19 ± 46.80 g kg^?1 DW, and it decreased with increasing fasting days. The non‐essential amino acids content was higher (486.18 ± 11.08 g kg^?1 protein) than essential amino acids (425.82 ± 9.15 g kg^?1 protein) at the start of the experiment (unstarved animals). The results suggest that the amino acid profile of the mantle where the most abundant amino acids are Arg, His, Lys, Gly, Leu and Pro could indicate a prolonged fasting condition (>20 days) or poor nutrition of O . vulgaris. This study supports the idea of using mantle for metabolic needs of starved O . vulgaris suggesting that the degradation pathway of amino acids to pyruvate and tricarboxylic acid cycle intermediates was favoured contrary to the degradation pathway of ketogenic amino acids. Special considerations should be taken concerning Thr, Ile, Ser, Ala, Asx (Asp, Asn), Glx (Glu, Gln) (because of their fast intake) and Lys and His (due to their stable contents) during a prolonged period of fasting.     

Involvement of gonadal steroids in final oocyte maturation of white perch (Morone americana) and white bass (M. chrysops): in vivo and in vitro studies

Plasma estradiol-17 (E₂), testosterone (T), 17,20-dihydroxy-4-pregnen-3-one (DHP) and 17,20,21-tri-hydroxy-4-pregnen-3-one (20-S) levels were measured by radioimmunoassay (RIA) in white perch (Morone americana) and white bass (M. chrysops) that were induced to undergo final oocyte maturation (FOM) with human chorionic gonadotropin (hCG). Plasma DHP levels increased in females of both species in association with oocyte germinal vesicle migration (GVM) and germinal vesicle breakdown (GVBD) and decreased thereafter. Plasma 20-S levels also increased with oocyte GVM in white bass, but were several-fold lower than DHP levels. Circulating E₂ and T levels were greatest during GVM and GVBD in both species and decreased to low levels during oocyte hydration and ovulation. Follicles from white perch and white bass which received a priming injection of hCG in vivo, produced both DHP and 20-S in vitro after exposure to hCG and their oocytes underwent GVBD. Ovarian incubates from unprimed fish of either species produced only E₂ and T and their oocytes did not complete GVBD. Oocytes from unprimed bass, but not perch, matured when follicles were exposed to hCG in vitro. Both trilostane and cycloheximide blocked in vitro production of DHP and 20-S and oocyte GVBD by white perch follices. DHP and 20-S were equipotent inducers of FOM in the GVBD bioassay. None of several other structurally-related steroids tested were effective within a physiological range of concentrations. These results indicate a role for DHP and 20-S in the control of FOM in white perch and white bass.     

Cardiac,ventilatory and metabolic responses of two ecologically dissimilar species of fish to waterborne cyanide

Changes in heart rate, ventilatory activity and oxygen consumption were determined in trout (Salmo gairdneri) and brown bullhead catfish (Ictalurus nebulosus) during exposure to a steadily increasing concentration of waterborne cyanide selected to produce death in 8–9 hours for each species. The lethal cyanide concentration for the bullheads was an order of magnitude higher than for trout. Trout developed an immediate and gradually increasing bradycardia throughout the exposure period. Cyanide produced tachycardia in the bullhead followed by a gradual onset of bradycardia as the concentration of cyanide was raised. Pericardial injection of atropine (a muscarinic cholinergic antagonist) indicated that bradycardia in the trout was due initially to increased vagal tone but later due to the direct effect of cyanide on the heart. Hyperventilation in the trout persisted throughout the exposure period, although the rate and amplitude fluctuated and was variable between individual fish. During the last hour of exposure (highest cyanide concentration), ventilation was characterized by rapid, shallow breaths followed by a sudden respiratory arrest. The bullheads exhibited hyperventilation during the first 3 hours of exposure followed by a gradual, linear drop in ventilation rate and amplitude until death occurred. Cardiac and ventilatory responses in both species were attributed to stimulation of central and peripheral chemoreceptors by cyanide. Evidence is presented which suggests the initial response in the bullheads was due, at least in part, to gustatory stimulation by the cyanide. Oxygen consumption of the trout remained above pre-exposure levels for the majority of the test period. Oxygen consumption in the bullhead paralleled the changes in heart and ventilatory rates. Whole-body lactate levels of fingerlings of both species during cyanide exposure were measured to estimate the extent of anaerobiosis. Whole-body lactate levels were much greater in the bullheads than the trout, indicating a higher capacity for anaerobiosis, possibly due to a greater fuel supply. Overall, the trout responded to cyanide in a manner similar to that produced by environmental hypoxia whereas the bullheads experienced a gustatory stimulus which masked the hypoxia-like response.     

美人鱼发光杆菌对凡纳滨对虾非特异性 免疫功能及抗病力的影响

在基础饲料中分别添加0.1%和1%美人鱼发光杆菌灭活菌、0.1%美人鱼发光杆菌活菌配制成3种免疫实验饲料,以基础饲料为空白对照组饲料,每组设3个平行样。对个体质量为(4.83±0.36)g的凡纳滨对虾进行为期20 d的饲养实验,分别在0、5、10、15和20d进行取样,以血清中的酚氧化酶(PO)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)和溶菌酶(UL)活性为免疫指标,探讨了美人鱼发光杆菌作为免疫制剂对凡纳滨对虾非特异性免疫效应的影响;在投喂免疫饲料后的第22天,按0.004 2 kg/kg体重的剂量,直接投喂对虾白斑综合征病毒(WSSV)病料,并记录累积死亡率。结果表明,美人鱼发光杆菌免疫实验组对凡纳滨对虾血清中PO、ACP、AKP、UL和SOD活性影响明显高于对照组,并且在饲料中添加美人鱼发光杆菌后,明显提高了对虾抵御WSSV感染的能力。其中0.1%美人鱼发光杆菌活菌实验组的抗病毒感染能力最强,WSSV感染14d内累计死亡率为63.3%±5.8%;而对照组为96.7%±3.3%。研究表明,美人鱼发光杆菌添加在对虾饲料中能提高凡纳滨对虾非特异性免疫水平,增强抵抗疾病的能力,将其作为对虾免疫增强剂具有良好的应用前景。     

An overview of stress physiology of fish transport: changes in water quality as a function of transport duration

This study brings an integrated analysis about the relationship between water deterioration and its physiological consequences in live fish transport. The analysis was focused on the transport water and its deterioration, and physiological challenges imposed on the fish. Usual commercial handling procedures employed to mitigate fish stress during transport were discussed. Future topics of research for the establishment of safer fish transport protocols were proposed. Transport was classified into short (≤8 h) or long transport (>8 h). The main issue in short transports should be the prevention of water pH reduction, while in long transports it is the increase in ammonia. Plasma cortisol is the most employed marker for stress and is acutely elevated upon short episodes of transport, but remains elevated even in long‐transport events. Plasma glucose is perhaps a better marker for handling stress. Plasma lactate, pH, osmolality CO₂ and ions should be more often evaluated. Plasma Na⁺ and Cl^‐ are very useful markers of acidosis, due to their respective exchange for H⁺ and , for acid–base regulation. The establishment of species‐specific transport protocols should be preceded by such combined analyses of water and physiological parameters.     

Are hatchery‐reared abalone naïve of predators? Comparing the behaviours of wild and hatchery‐reared northern abalone,Haliotis kamtschatkana (Jonas, 1845)

Abalone populations have declined worldwide, generating interest in enhancement using hatchery‐reared individuals. In many cases, such restoration efforts have met with limited success due to high predator‐induced mortality rates. Furthermore, the mortality rates of outplanted hatchery abalone are often considerably higher than for wild individuals. This study uses northern abalone (Haliotis kamtschatkana) as a case study to determine whether hatchery‐reared abalone behave differently than their wild counterparts. In the field, outplanted hatchery‐reared abalone were significantly less responsive than wild abalone, in terms of number of abalone responding and intensity of response, to nearby movement and to physical contact with an inert probe. Also, when encountering a cue to which all abalone responded (a seastar predator), hatchery‐reared individuals remained subdued. Anti‐predator behavioural deficits in hatchery‐reared abalone were more pronounced in 4‐year‐old individuals than in 1‐year‐old individuals, suggesting an influence of either age or amount of time spent in the hatchery environment. These behavioural differences are expected to increase the vulnerability of hatchery‐reared abalone to predators, and are likely a major cause of their elevated predator‐induced mortality when outplanted.     

Effects of cadmium on the kinetics of calcium uptake in developing tilapia larvae, Oreochromis mossambicus

The toxic effects of Cd²⁺ on Ca²⁺ influx kinetics in developing tilapia (Oreochromis mossambicus) larvae were evaluated. Addition of 20 µg l^-1 of Cd²⁺ to the environment of 0 and 3 day-old larvae competitively inhibited the Ca²⁺ uptake within 4h resulting in a great increase in K_m values for Ca²⁺ influx (19.3 and 17.4 fold, respectively) as compared with their respective controls. Consequently, the actual Ca²⁺ influx of larvae in solutions of 0.2 mM Ca²⁺ are suppressed by 32–45%. Also, 3 day-old larvae were more sensitive to internally accumulated Cd²⁺ than 0 day-old larvae. Although the Ca²⁺ influx in 0 and 3 day-old larvae may be restored to the levels of their respective controls with 24h of being transferred to a 20 µg l^-1 Cd²⁺ solution, total body Ca²⁺ content was significantly reduced in 3 day-old larvae. Increased Ca²⁺ uptake efficiency ensures sufficient Ca²⁺ for normal growth. However, rapid increase in Ca²⁺ influx after hatching also leads to higher Cd²⁺ uptake. Exposure to Cd²⁺ will lead to a drop in body Ca²⁺ content resulting in retardation of larval growth. Therefore, we conclude that if Ca²⁺ uptake is interfered with at this critical stage of development, larvae will not be able to maintain normal levels of body Ca²⁺ and will show signs of Cd²⁺ poisoning.     

Migratory dynamics of stream-spawning longnose gar (Lepisosteus osseus)

Abstract– Literature evidence suggests that lake-dwelling longnose gar (Lepisosteus osseus) enter tributary streams to spawn, Until the present study, the dynamics of this breeding migration had never been investigated quantitatively. During the summers of 1991 and 1992, longnose gar were captured as they entered Weaubleau Creek, Missouri, a tributary of Harry S. Truman Reservoir. The in-stream spawning migration began in early April and ended in late May, and was positively correlated with stream flow and water level, and negatively correlated with water temperature. In-stream residence times ranged from 15 to 94 days, with males exhibiting longer residence times than females. Once in-stream, longnose gar travelled as far as 10 km upstream and occupied certain pools at greater relative frequencies. Although the reason for this preferential utilization is not completely understood, it may relate to pool depth and riffle proximity. Longnose gar disperse from the spawning stream great distances, with gar captured in Weaubleau Creek being recaptured up to 48 km away. This information should provide fisheries biologists the means to consider the reproductive ecology of this species in their conservation and management decisions.