恩诺沙星及其代谢产物在中华绒螯蟹血淋巴中的比较药代动力学

研究了不同水温(16℃、25℃)、不同给药剂量(10 mg/kg、20 mg/kg)和不同给药方式(肌注、口灌)等条件下,恩诺沙星及其代谢产物环丙沙星在中华绒螯蟹体内的药代动力学,比较了不同条件下药物在蟹血淋巴中的吸收、分布和代谢的差异。结果表明,恩诺沙星在蟹血淋巴中的药-时数据符合开放式二室模型。16℃时以10 mg/kg剂量肌注给药后,恩诺沙星在蟹血淋巴的主要药代动力学参数为:AUC96.818 mg/(L.h),Cmax6.54μg/mL,t1/2α0.851 h,t1/2β95.415 h;25℃时以10 mg/kg剂量肌注给药后,恩诺沙星的主要药代动力学参数为:AUC168.457 mg/(L.h),Cmax7.12μg/mL,t1/2α0.58h,t1/2β88.833 h;25℃时以20 mg/kg剂量肌注给药后,恩诺沙星的主要药代动力学参数为:AUC155.612 mg/(L.h),Cmax11.045μg/mL,t1/2α5.239h,t1/2β88.378 h;25℃时以10 mg/kg剂量口灌给药后,恩诺沙星的主要药代动力学参数为:AUC86.525 mg/(L.h),Cmax3.469μg/mL,t1/2α8.071h,t1/2β61.842 h。不同条件下,恩诺沙星在蟹血淋巴中的主要药代动力学参数差异较大。恩诺沙星的活性代谢产物环丙沙星在各种给药条件下的蟹血淋巴中均能检出,但含量均处于较低值,且药-时数据不能用房室模型拟合,表明恩诺沙星在蟹体内只有极少部分代谢为环丙沙星。     

Acrosome reaction of Chinese mitten-handed crab Eriocheir sinensis (Crustacea: Decapoda) spermatozoa: Promoted by long-term cryopreservation

The effects of three media, two temperatures, and fourteen durations of cryopreservation from 0 h to 450 d on in vitro acrosome reaction (AR) of spermatozoa in Chinese mitten-handed crab Eriocheir sinensis (Crustacea: Decapoda) were investigated. The spermatozoa of good quality were obtained from spermatophores by a glass homogenizer in an ice-bath and centrifugation at 4 °C. At 0 h, 2 h, 1 d, 3 d, 15 d, 30 d, 60 d, 90 d, 120 d, 150 d, 180 d, 270 d, 360 d, and 450 d of cryo-storage in Ca²⁺-free artificial seawater, 10% (v/v) glycerol, and 5% (v/v) dimethylsulfoxide at − 80 °C and in liquid nitrogen (− 196 °C), the changes in spermatozoal morphology, the time of beginning AR, and the time of maximum percentage of AR were observed. The relationships of the changes on AR presented with the different media, temperatures, and durations of cryopreservation were speculated. In this study, the cryopreserved spermatozoa all underwent AR in less than 1 h of settlement under room temperature while the percentage of AR in the control was only about 4.9%. Meanwhile, cryopreservation shortened both the time of beginning AR (from 30.11 min of the uncryopreserved spermatozoa to 0 min of cryopreserved spermatozoa on the 30th day) and the time of maximum percentage (from 59.88 min of the uncryopreserved spermatozoa to 0 min of cryopreserved spermatozoa on the 60th day). Whereas the effect of media on sperm cell AR was negligible (P > 0.05), the treatments of spermatozoa with short- and long-term cryopreservation resulted in extremely significant differences in the time of beginning AR as well as in the time of maximum percentage of AR (P < 0.01). The present data indicate that cryopreservation for long or short periods can promote the AR of sperm cells in E. sinensis and physiologically affect the ability to capacitate. It may be that the mechanism of AR in this study is the direct promotion of membrane fusion of the acrosomal cap, or destruction of the proteins inhibiting AR and activation of the proteins promoting AR, by cryopreservation. In addition, the results also show that cryopreservation can protect the spermatozoa because AR can occur in almost all sperm cells cryopreserved for less than 15 d.     

Study on the pharmacokinetics of enrofloxacin in the Chinese mitten-handed crab, Eriocheir sinensis, after different administration regimes

A pharmacokinetic study on enrofloxacin (ENR) following different administration regimes (i.e. intramuscular, oral and bath administrations) was carried out for the Chinese mitten-handed crab ( Eriocheir sinensis ) at 17 °C water temperature. Muscle and hepatopancreas samples were collected at each sampling time of 1 and 10 min and 0.5, 1, 2, 8, 16, 24, 48, 72 and 168 h for intramuscular administration and 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72 and 168 h for oral and bath administration. The levels of ENR and ciprofloxacin (CIP) were simultaneously determined ( n =5) by high-performance liquid chromatography. The results showed that similar absorption and elimination patterns of ENR level in muscle and hepatopancreas were observed for oral administration and bath treatment; however, the trend in ENR level changes in muscle and hepatopancreas after intramuscular administration was quite different from oral and bath treatments. Meanwhile, the level of ENR in hepatopancreas was much higher than that in muscle for the same administration regimes. The concentration–time data were analysed with the Practical Pharmacokinetics Program (3P97), and the kinetic profiles of ENR in the hepatopancreas and muscle conformed to two-compartment models. The absorption and elimination of ENR in hepatopancreas for bath treatment were faster than those for oral and intramuscular administrations. However, the pattern was quite different in muscle. The fastest absorption and elimination of ENR in muscle were seen for intramuscular administration.     

恩诺沙星及其代谢产物环丙沙星在牙鲆体内代谢消除规律

以80 mg/kg鱼体重对牙鲆单次口灌给药恩诺沙星,给药后在不同的时间点取样,用高效液相色谱荧光检测器检测,研究恩诺沙星及其主要代谢产物环丙沙星在牙鲆体内的代谢消除规律。研究表明,停药后0.25 h,肌肉中恩诺沙星残留量最低。各组织的消除半衰期依次为腮肝脏血液肾脏肌肉,其中肌肉中恩诺沙星消除半衰期最低为67.759 h,消除最快,停药后12 d检测不到恩诺沙星。停药后0.25 h,在牙鲆血液、肝脏、肾脏中均有环丙沙星残留,残留量依次为肝脏肾脏血液,肌肉和鳃中未检出环丙沙星。停药后22 d在血液、肝脏和肾脏3个组织中仍然能够检测出恩诺沙星,但是停药7 d后这3个组织中均检测不出环丙沙星。结果显示,恩诺沙星在牙鲆体内代谢速度较慢,而且只是在一段时间内有脱乙基代谢为环丙沙星的反应发生,但并不是在恩诺沙星消除的全过程都发生,而且代谢物环丙沙星在牙鲆体内的消除速度要比恩诺沙星快。     

中华绒螯蟹血淋巴抗菌活性的初步研究

主要研究了中华绒螯蟹血清和血细胞破碎上清液(HLS)对大肠杆菌、嗜水气单胞菌、蜡状芽胞杆菌和金黄色葡萄球菌的抗菌活性、不同浓度HLS对抗菌活性的影响以及血清中抗菌活性的诱导作用。结果显示,只有HLS对大肠杆菌、嗜水气单胞菌表现出显著的抗菌活性,而血清对4种菌、HLS对蜡状芽胞杆菌和金黄色葡萄球菌均未表现出抗菌活性;随着HLS浓度的降低,大肠杆菌的细菌存活指数(SI)迅速上升,抗菌活性迅速下降,HLS浓度高低直接影响抗菌活性的强弱;通过注射灭活大肠杆菌,血清在1~24h内表现出抗菌活性的诱导作用,在6h时抗菌活性最强。     

HPLC法测定牙鲆肌肉中恩诺沙星及其代谢产物环丙沙星

利用高效液相色谱-荧光检测器分析了牙鲆肌肉中恩诺沙星及其代谢产物环丙沙星的含量。样品前处理方法为:以磷酸盐缓冲液作为提取液,样品经均质、离心后,用正己烷除去脂肪,再用ODS(C18)固相萃取柱净化,洗脱液蒸发浓缩后,乙腈溶解残渣。其色谱条件为:色谱柱XDB-C18柱(250mm×4.6mm);流动相0.01mol/L四丁基溴化铵(pH3.0)∶乙腈=94∶6(v/v);流速1.5ml/min;进样量10μl;柱温40℃;荧光检测器激发波长280nm、发射波长450nm。结果表明,在牙鲆肌肉中添加50、100和200μg/kg3个水平的恩诺沙星和环丙沙星混合物标准后,其回收率均大于80%,相对标准偏差小于10%。恩诺沙星与环丙沙星检出限均为10μg/kg。     

中华绒螯蟹不同可食部位对恩诺沙星的富集效应研究

为了探究广谱抗菌药恩诺沙星暴露对中华绒螯蟹(Eriocheir sinensis)各可食部位(蟹腿肉、蟹身肉、性腺和肝胰腺)的影响,实验设置了空白对照组和三个恩诺沙星浓度试验组(空白对照、50、500和5000μg/L)来进行研究.结果表明:恩诺沙星在各部位的富集系数与水环境中恩诺沙星的浓度呈负相关关系,在蟹身肉中的富...     

pH胁迫对中华绒螯蟹5种非特异性免疫指标的影响

测定了不同pH(4.5,6.0,7.5,9.0,10.5)胁迫1、4、7、10d后中华绒螯蟹(Eriocheir sinensis)血细胞呼吸爆发(RP)、血细胞破碎物上清液(HLS)酚氧化酶(PO)、血清溶菌酶(LSZ)、酸性磷酸酶(ACP)及碱性磷酸酶(ALP)活性的变化.结果表明:在pH胁迫后第1天,pH4.5组...     

水温对氟苯尼考在中华绒螯蟹体内的药物代谢动力学影响

研究不同水温对氟苯尼考在中华绒螯蟹血淋巴、肌肉和肝脏组织中的代谢规律和组织分布的影响.在17℃和28℃水温下,以20 mg/kg剂量单次肌肉注射给药,于给药后不同时间点取各组织样品,氟苯尼考药物含量由高效液相色谱法测定.研究结果表明在两个温度下,血淋巴和肌肉组织中氟苯尼考含量瞬时达到峰值,而肝脏中氟苯尼考含量则随给药时间,呈先上升后下降的趋势.氟苯尼考在中华绒螯蟹体内代谢规律符合二室开放模型,吸收半衰期、消除半衰期、药时曲线下总面积均与温度呈负相关,而总体清除率与温度呈正相关.     

采用LC/MS~n法分析恩诺沙星在锯缘青蟹血浆中的代谢产物

采用电喷雾离子阱质谱法在正离子检测方式下,对锯缘青蟹口灌恩诺沙星给药后血淋巴中恩诺沙星和代谢产物进行定性分析。通过对血浆中恩诺沙星及其代谢产物的质谱裂解行为进行碰撞诱导解离分析,获得了各化合物的多级质谱信息,通过比较各化合物结构和质谱裂解途径的异同点,发现有三个代谢产物,其中峰面积最大的为环丙沙星,其次为羟基化恩诺沙星,另一代谢产物可能是加氧恩诺沙星;推测恩诺沙星在锯缘青蟹体内的代谢反应主要是脱乙基反应、羟基化反应和氧化反应。通过定量分析,血浆中恩诺沙星和代谢产物环丙沙星的浓度分别为19.1μg/ml和0.526μg/ml。     

Pharmacokinetic disposition of enrofloxacin in brown trout (Salmo trutta fario) after oral and intravenous administrations

In this study, the pharmacokinetic profile of enrofloxacin (EF) and its major metabolite, ciprofloxacin (CF), were investigated in brown trout (Salmo trutta fario) (n = 150) after intravenous (i.v.) and oral (p.o.) administrations of a single dose of 10 mg kg^− 1 body weight (b.w.) at 10 °C. The plasma concentrations of the drugs were determined by high-performance liquid chromatography (HPLC-UV) from 0.08 to 120 h. Pharmacokinetic parameters were described by the two-compartment open model for intravenous and oral administrations, respectively. After intravenous administration, the elimination half-life (t_1/2β), apparent volume of distribution at steady-state (V_ss) and total body clearance (Cl_tot) of enrofloxacin were 19.14 ± 1.51 h, 3.40 ± 0.18 L kg^− 1 and 0.14 ± 0.01 L kg h^− 1, respectively. After oral administration, the maximum plasma concentration (C_max), time of maximum concentration (t_max) and bioavailability (F%) were 2.30 ± 0.08 µg mL^− 1, 8 h and 78 ± 4%, respectively. Ciprofloxacin was not detected in the present study. The elimination half-life for enrofloxacin following oral administration was longer than values calculated for other animals. After oral administration, the mean plasma concentration was well above the minimum inhibitory concentrations (MICs)—that is, > 0.5 µg mL^− 1 at 36 h—for most gram-negative fish pathogens. It is possible and practical to obtain therapeutic blood concentrations of enrofloxacin in brown trout (S. trutta fario) using oral administration of 10 mg kg^− 1 body weight; therefore, it may be effective in the therapy for brown trout diseases.     

Biochemical composition of pond-reared and lake-stocked Chinese mitten crab Eriocheir sinensis (H. Milne-Edwards) broodstock

The morphological characteristics, tissue indices, proximate and fatty acid compositions of hepatopancreas, gonad and muscle of pond‐reared (PR) and lake‐stocked (LS) Eriocheir sinensis broodstock were compared. Lake‐stocked crabs are characterized by a blue carapace, white abdomen, golden legs and yellow setae while PR crabs have a brown carapace, grey abdomen, black legs and similar yellow setae. The carapace length and width of PR crabs were significantly less than those of LS crabs of the same wet weight (WW). The results showed that the hepatosomatic index (HSI) of PR males was significantly higher, but the gonadosomatic index (GSI) was significantly lower for PR females when compared with LS individuals of the same sex. Regardless of their origin, the muscular index (MI) of males was higher than that of females. The hepatopancreas of LS crabs had higher protein levels and lower lipid levels compared with PR crabs. The results of proximate analysis of male gonads from two different sources were similar, while the protein level in the ovary of LS females was higher than that of PR females. The tissues of males had higher water content but lower lipid and protein levels compared with females regardless of their source. The LS crabs had higher highly unsaturated fatty acid (HUFA) levels than PR crabs in their gonad and hepatopancreas. In conclusion, our results suggest that the nutritional quality of LS crabs is better than that of PR crabs, the nutritional quality of males is poorer than that of females. The reproductive performance and larval quality of crabs from these two rearing systems should be further verified.     

The effect of stocking density of Chinese mitten crab Eriocheir sinensis on rice and crab seed yields in rice-crab culture systems

Normally Chinese mitten crab Eriocheir sinensis were cultured for 2 years, 1 year is cultured from crab larvae to seeds or button sized crab in ponds, tanks or rice fields, the another year is cultured from seeds to market size in ponds, reservoirs or other waters. The present study was conducted to determine the effect of stocking density (0, 3.75, 15, 30 and 60 inds. per m², respectively) of Chinese mitten crab larvae on rice and crab seed yields in rice-crab culture systems using land-based enclosures (4 m × 7 m, with three replicates for each stocking density) at the Panjin Guanghe Fisheries Co. Ltd, in the Liaohe Delta, China. Zoobenthos biomass, production of crab and rice paddy, and economical profit among the treatments were determined. The experiment showed that zoobenthos biomass in rice-crab culture systems was relatively variant and not significantly correlated to these stocking density. The biomass of aquatic plant declined significantly with increasing stocking density. Specific growth rates (SGR) and survival rates of the crab were significantly higher at the lower stocking density (P < 0.05); in addition, the ratio of precocious crab and total crab significantly increased with the stocking density increasing (P < 0.05); There were not significant effect of presence of crab on yields of rice straw and rice paddy (P > 0.05). Net crab yield and net profit were the highest at the 15 treatment (P < 0.05), however, the net profits among 3.75, 15 and 30 treatments did not show significant difference (P > 0.05) economically. Based on the observations above, a stocking density of 3.75 to 30 inds. per m² was considered reasonable in rice-crab culture system.     

Therapeutic effects of enrofloxacin in an experimental infection with a luminescent Vibrio harveyi in Artemia franciscana Kellog 1906

Although a wide range and number of chemotherapeutants have been developed and applied in aquaculture, there have been no studies on the absorption of antibiotics by crustacean larvae and, consequently, none has been reported to treat an established infection.

The aim of this study was to evaluate the therapeutic effects of enrofloxacin during an experimental infection with the strain PN9801 identified as Vibrio harveyi in Artemia franciscana.

Four treatments were used: (1) Nauplii experimentally infected with V. harveyi; (2) Nauplii enriched with enrofloxacin; (3) Nauplii enriched with enrofloxacin and, 4 h later, experimentally infected with V. harveyi; and (4) Nauplii experimentally infected with V. harveyi and 24 h later enriched with enrofloxacin. Survival rates were estimated after 48 h and they were as it follows: 29%, 99%, 88% and 85% for treatments 1, 2, 3 and 4, respectively.

This study is the first one to show some evidence that antibiotics can, in fact, withhold the course of a bacterial infection already established in Artemia and possibly in crustaceans.     

长江中游三个湖泊中华绒螯蟹生长变化研究

选择长江中游的牛山湖、武湖和东汤逊湖三个典型的河蟹养殖湖区作为研究地点,从2009年2月起逐月调查湖泊放流河蟹体重和壳宽的生长变化,并同时记录投喂管理情况和收集试验湖区的水化特征及底栖生物资源状况。调查发现,河蟹在4、5、6月特定生长率最大,此时为河蟹生长的高峰期。在三个湖区间,生长有显著性差异,在收获季节,武湖河蟹的平均壳宽和体重均显著性大于另外两个湖区(P<0.05),牛山湖和东汤逊湖之间则无显著性的差异(P>0.05);这种情形可能主要与为湖泊的天然饵料资源的种类和丰度有关。     

超低温冷冻对中华绒螯蟹胚胎线粒体DNA的影响

为了解超低温冷冻对中华绒螯蟹(Eriocheir sinensis)胚胎线粒体DNA影响,选取细胞分裂期胚胎为研究对象,研究了4种玻璃化液处理和超低温冷冻对胚胎线粒体DNA的影响。结果表明:经过玻璃化液处理和冷冻后,胚胎线粒体上的Cytb基因能被稳定扩增,PCR电泳产物大小约1 600 bp,产物经回收后进行双向测序、比对和校正,获得10条Cytb基因全序列,长度为1 135 bp,其中有5个核苷酸变异位点,1 135 bp的Cytb基因序列一共编码378个氨基酸残基,从ATG第一位起始密码子开始到1 140位终止。经过玻璃化液处理和冷冻后,胚胎线粒体上的COI基因也能被稳定扩增,PCR电泳产物大小约1 600 bp,产物经回收后进行双向测序、比对和校正,获得10条COI基因全序列,长度为1 534 bp,其中有6个核苷酸变异位点,1 534 bp的COI基因序列一共编码511个氨基酸残基,从ATG第一位密码子开始到1 533位终止。经过玻璃化液处理和冷冻后,中华绒螯蟹胚胎线粒体上Cytb和COI基因共有11个位点发生了变异,但不同的玻璃化液处理和冷冻对碱基变异没有影响,发生的碱基变异中存在4种类型,即G→A、A→G、C→T、T→C,变异间只有转换发生,没有颠换发生,这些变异均未引起相应氨基酸残基的变异。     

中华绒螯蟹感染“颤抖病”病毒后血清免疫因子的变化

对中华绒螯蟹进行颤抖病病毒悬液和PBS(对照)注射感染,研究其血清免疫因子抗菌活性(Ua)、溶菌活性(UL)、凝集活性(HA)、酚氧化酶(PO)、过氧化酶(POD)、超氧化物歧化酶(S0D)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)活性的变化,并对实验的结果进行统计学分析。结果表明,在中华绒螯蟹人工感染颤抖病病毒后,蟹体内的免疫因子发生了明显的变化,血清中Ua、UL活性先是降低,然后上升至高于正常值水平。P0活性先上升然后下降至低于正常值水平,POD、HA、SOD活性逐渐升高至显著水平;ACP及AKP活性则显著下降。     

二氟沙星在大菱鲆(Scophthalmus maximus)体内的药代动力学特征

为给大菱鲆临床安全用药提供依据,对二氟沙星在健康大菱鲆体内的药代动力学特征进行了研究。在水温为14–17℃条件下,以20 mg/kg体重剂量静脉注射和口服二氟沙星,分别于给药后的15、30 min和1、2、4、6、8、12、16、24、36、48、72、96 h采血及各组织,利用高效液相色谱测定各组织和血浆中的二氟沙星含量,采用DAS 2.0自动药动学软件分析药代动力学参数。结果显示,静注给药后,血浆中二氟沙星的药-时曲线符合零级吸收二室开放模型,消除半衰期(t1/2β)和药时曲线下面积(AUC0-t)分别为30.02 h和440.37 h·μg/ml;口服给药后,血浆中二氟沙星的药-时曲线符合一级吸收二室开放模型,4 h时血药浓度达到最大值(Cmax)6.32μg/ml,t1/2β和AUC0-t分别为94.72 h和246.66 h·μg/ml,生物利用度(F)为56.01%,肾脏和肌肉内的药物浓度较高,而肝脏内相对较低。拟合的最佳口服给药方案为:按鱼体重每次给药11.94 mg/kg,每日1次。     

Kinetic analysis of oxytetracycline residues in Chinese mitten crab,Eriocheir sinensis,muscle following intramuscular administration

Crab culture is a very important economic industry in China. An epidemic of tremor disease of Chinese mitten crabs, Eriocheir sinensis, has become a serious problem in recent years. A spiroplasm has been proved to be the causative agent of this disease. Oxytetracycline (OTC) is used widely in aquaculture and was confirmed to be very effective against this pathogen. In this study, the distribution and depletion patterns of OTC in crab muscle were evaluated following single intramuscular doses of 2, 8 and 40 mg kg^?1 body weight. OTC was detected with a validated HPLC method. Concentration–time profiles were well described by a three‐compartment model with first‐order absorption after a single dose of 8 and 40 mg kg^?1. For comparison, a non‐compartment model was employed. A withdrawal time of 48.29 and 55.92 days was suggested prior to consumption after receiving 8 and 40 mg kg^?1. A recommended therapeutic dose of OTC in theory was calculated to be 36.37 mg kg^?1. OTC was distributed well throughout the body. The elimination of OTC in muscle was slower compared with fish and other crustaceans. A dose of 40 mg kg^?1 is suggested for practical use.     

Ingestion of Artemia nauplii by Chinese mitten crab Eriocheir sinensis zoea larvae

A series of ingestion trials were conducted to determine the ingestion rate of Artemia nauplii by Eriocheir sinensis zoea larvae with increasing densities of Artemia and with or without rotifers as a co-feed. At each zoeal stage, 10 groups of 10 larvae were reared individually in glass beakers and fed with increasing densities of newly hatched Artemia nauplii (0.5, 2.5, 5, 10 and 20 individual (ind.) mL⁻¹) with or without rotifers (15–25 ind. mL⁻¹) as a co-feed. The average number of ingested Artemia was measured over 24 h. In addition, the average larval development rate (Larval Stage Index, LSI) over a longer period (time needed for the best treatment to reach 100% moult or metamorphosis to the next larval stage) was compared. The results showed that Artemia ingestion rate of E. sinensis larvae increased with increasing prey densities and larval development, and had a significantly negative correlation with rotifer consumption for all zoeal stages. Rotifers as an alternative prey significantly affected the intake of Artemia at early larval stages (Z1 and Z2) and promoted LSI at a lower Artemia density. Further experiments are needed to clarify the effect of prey density on survival and larval development when larvae are reared communally.