复方中药制剂体外杀纤毛虫及毒性试验研究

研究3个复方中草药水煎浓缩液(1 g/mL)(复方Ⅰ:十大功劳、黄柏、半枝莲、苦参、百部、槟榔;复方Ⅱ:大黄、黄芩、野菊花、蛇床子、石榴皮、乌梅;复方Ⅲ:地丁、蒲公英、野菊花、地锦草、败酱草、龙胆草、鱼腥草、青蒿)体外杀水滴伪康纤虫和海洋尾丝虫的效果及对体质量18~20 g大菱鲆的毒性,旨在筛选出高效、低毒、少残留的治疗盾纤毛虫病的中药组合。体外杀虫试验在96孔细胞培养板的每孔中加入90μL药液和10μL离心后的虫液,药物剂量分别为0、5、10、15、20、25、30 mg/kg,每组3个平行,显微镜下观察盾纤毛虫的状态。体外杀虫试验结果显示,复方Ⅰ的剂量在0~30 mg/kg时对海洋尾丝虫无杀害作用;复方Ⅲ仅在药物剂量为30 mg/kg时对海洋尾丝虫有杀害作用,其他剂量时无效;复方Ⅱ20、25、30 mg/kg组1 h水滴伪康纤虫全部死亡,30 mg/kg组3 h海洋尾丝虫全部死亡。随即在循环水箱中进行为期7 d的大菱鲆毒性试验,随机分为4组,即空白对照组(0 mg/kg)、推荐剂量组(20 mg/kg)、3倍剂量组(60 mg/kg)、5倍剂量组(100 mg/kg),每组3个平行。试验结束时,鱼尾静脉采集全血和血清,使用血液细胞分析仪和全自动生化分析仪检测血常规和血液生化指标。大菱鲆毒性试验结果显示,复方Ⅱ药浴后,大菱鲆血液中白细胞、嗜酸性细胞等血常规指标以及葡萄糖、球蛋白含量等血液生化指标均无显著差异;各试验组大菱鲆的鳃、肠道、胃、肝脏和脾脏均无明显的病理变化。     

Effect of feed restriction on the growth performance of turbot (Scophthalmus maximus L.) juveniles under commercial rearing conditions

Two trials were performed to study the effect of periodic feed deprivation (trial 1) or feed restriction, followed by satiation feeding (trial 2) on the growth performance of turbot juveniles under commercial rearing conditions. In trial 1, duplicate groups of 350 fish with an initial weight of 62 g were fed a commercial diet to apparent visual satiation for 7, 6, 5 or 4 days a week for 83 days. At the end of the trial, fish weight was directly related to the number of feeding days but feed efficiency and protein efficiency ratio were not affected by treatments. At the end of the trial, there were no differences in whole‐body composition among groups. In trial 2, duplicate groups of 500 fish with an initial weight of 33 g were fed a commercial diet to satiation (100%) or feed restricted to 90%, 80% and 70% of satiation for 90 days. Thereafter, all groups were fed to satiation for 34 days. During the feed restriction period, growth was directly related to feed intake, while during the satiation feeding period, it was inversely related to the previous feeding level. At the end of the trial, the final weight was not different among groups. At the end of the feed restriction period, whole‐body lipid content showed a trend to decrease with an increase in the feed restriction level. The results of this study indicate that under practical conditions, turbot juveniles should be fed daily as even cycles of short periods of feed deprivation negatively affect growth, while not improving feed efficiency. On the contrary, even after a relatively long feed restriction period, fish shows compensatory growth, and this may be used as a feed management strategy for controlling fish production in commercial farms.     

Comparative study on the effects of chelated or inorganic manganese in diets containing tricalcium phosphate and phytate on the growth performance and physiological responses of turbot Scophthalmus maximus

A growth trial was conducted to evaluate the effects of chelated (Mintrex^? Mn, Mn‐M) or inorganic (MnSO₄·H₂O, Mn‐S) manganese (Mn) on growth, feed utilization, tissue Mn deposition and liver superoxide dismutase (SOD) activity in turbot Scophthalmus maximus. A semi‐purified basal diet was formulated to be deficient in Mn (3.7 mg kg^?1) and contained tricalcium phosphate and sodium phytate at levels of 20 and 5 g kg^?1, respectively. Ten other diets were made by adding five levels (5, 10, 20, 35 and 55 mg Mn kg^?1 diet) of either the Mn‐M or Mn‐S to the basal diet, respectively. The 11 experimental diets were fed to groups of turbot (mean initial weight: 4.6 g) for 8 weeks. Results showed that the specific growth rate (SGR), feed intake, whole body Mn/vertebra Mn concentration and Mn‐SOD activity in liver were significantly improved by Mn supplementation (P < 0.05). On the basis of SGR, vertebra Mn concentration or liver Mn‐SOD activity data, dietary Mn requirement was estimated to be 10.5, 46.3 or 12.9 mg kg^?1 for turbot fed Mn‐S, and the same was estimated to be 7.6, 43.0 or 22.5 mg kg^?1 for turbot fed Mn‐M, respectively. There was no significant difference in growth, feed intake, whole body Mn concentration or vertebra Mn concentration between the two dietary Mn sources (P > 0.05).     

Study of the distribution of active caspase‐3‐positive cells in turbot,Scophthalmus maximus (L.), enteromyxosis

Enteromyxosis caused by Enteromyxum scophthalmi is one of the parasitizations with a higher economic impact on turbot, Scophthalmus maximus (L.), aquaculture. This myxosporean produces severe catarrhal enteritis with abundant inflammatory infiltrates in the lamina propria‐submucosa (LP), epithelial detachment and leucocyte depletion of the lymphohaematopoietic organs. Some advances made on the pathogenesis pointed to a role of apoptosis in the enteromyxosis. Therefore, the main aim of this work was to employ the TUNEL assay and the anti‐(active caspase‐3) immunohistochemical assay to detect apoptotic cells in both healthy and E. scophthalmi‐infected turbot in order to establish the presence and distribution of apoptotic cells during development of the disease. More apoptotic cells located within the gastrointestinal epithelium were observed in the initial stages of the infection in E. scophthalmi‐infected turbot compared with non‐infected turbot. As the infection progressed, a higher degree of apoptosis occurred in the epithelium of folds heavily parasitized. In the severely infected turbot, apoptosis was also found among the leucocytes of the intestinal inflammatory infiltrates. Moreover, the number of active caspase‐3‐positive cells in the lymphohaematopoietic organs tended to increase with disease severity. In view of the results, increased apoptosis in the epithelium may favour the scaling that occurs during enteromyxosis and cell death of leucocytes in the intestinal LP, contributing to leucocyte depletion in severe cases.     

Immunohistochemical characterization of polyclonal antibodies against Enteromyxum leei and Enteromyxum scophthalmi (Myxozoa: Myxosporea), intestinal parasites of fish

The enteric myxozoan parasites Enteromyxum leei (Diamant, Lom et Dyková) and Enteromyxum scophthalmi Palenzuela, Redondo et Álvarez‐Pellitero are responsible for high weight loss in infected fish, which leads to subchronic disease and low mortality rates in gilthead sea bream (GSB), Sparus aurata L., and to high mortality rates in turbot, Psetta maxima (L.). The detection of initial parasite stages in histological sections is particularly difficult, but can be simplified by means of specific antibodies. Rabbit polyclonal antibodies (pAbs) were raised against E. scophthalmi and E. leei, and direct enzyme‐linked immunosorbent assay (ELISA) and immunohistochemistry were used to characterize their sensitivity and specificity. Both pAbs were adsorbed (apAb) with non‐infected intestines to avoid non‐specific labelling of fish tissues and to improve their specificity. The highest titre obtained in ELISA was 1: 32 000 for apAb‐Eleei and 1:16 000 for apAb‐Escoph. Working dilutions in immunohistochemistry were 1:1000 for apAb‐Eleei and 1:8000 for apAb‐Escoph. Both apAbs labelled proliferative and sporogonic stages with high specificity. apAb‐Escoph was very specific, whereas apAb‐Eleei cross‐reacted with Sphaerospora dicentrarchi Sitjà‐Bobadilla et Álvarez‐Pellitero and Sphaerospora testicularis Sitjà‐Bobadilla et Álvarez‐Pellitero, suggesting the presence of shared antigens. These pAbs stand as new tools for antigenic characterization and the diagnosis of both Enteromyxum species.     

Plankton composition and biomass development: a seasonal study of a semi‐intensive outdoor system for rearing of turbot

Plankton food web dynamics were studied during a complete production season in a semi‐intensive land‐based facility for rearing of turbot (Scophthalmus maximus) larvae. The production season was divided into three production cycles of 3–5 weeks. Phytoplankton biomass (using chlorophyll a as biomass proxy) peaked in each production cycle. However, the maximum biomass decreased from spring (18 μg chlorophyll a L^?1) to fall (ca. 7 μg chlorophyll a L^?1), simultaneous with a decline in the concentration of dissolved nitrogen in the inoculating water. During the three production cycles, we observed decreasing copepod nauplii concentration from spring to fall in the rearing tanks. The decreasing nitrogen gave increasing carbon to chlorophyll a ratio in the seston (from 23 in spring to 73 in fall). The pool of free amino acids in seston was constant. We suggest that the decreasing nitrogen input in the inoculating water reduces the quality of the phytoplankton and thus the growth potential of the prey for fish larvae, copepods.     

The effect of ultrafiltered fish protein hydrolysate level on growth performance,protein digestibility and mRNA expression of PepT1 in juvenile turbot (Scophthalmus maximus L.)

The intention of the study was to investigate the effect of ultrafiltered fish protein hydrolysate (UF) level on growth, feed utilization, apparent digestibility coefficients and proximal intestine peptide transporter 1 (PepT1) mRNA level for juvenile turbot (Scophthalmus maximus L.). Experimental diets (UF‐0, UF‐5, UF‐10, UF‐15 and UF‐20) were prepared containing about 68% plant protein, and fish meal protein was, respectively, replaced by 0%, 5%, 10%, 15% and 20% UF of dietary protein. Diet PP contained about 78% plant protein, and diet CAA contained about 10% crystalline amino acid mixture. All diets were fed to seven triplicate groups of turbot (initial weight 16.05 ± 0.03 g) for 68 days. Fish fed diet UF‐10 had an increasing tendency in growth compared with diets contained UF, while dietary UF level was not significantly correlated with specific growth rate and feed intake. Feed efficiency, protein efficiency ratio and protein productive value significantly correlated with dietary UF level, and fish fed diets contained low‐level UF had higher digestibility than that diets UF‐0, PP and CAA. There was a decreasing tendency in PepT1 expression level with dietary UF level. The results indicated that low‐level UF showed a positive effect on growth and feed utilization in juvenile turbot.     

Alleviation effect of dietary cerium and its complex with chitosan oligosaccharide on cadmium accumulation in juvenile turbot,Scophthalmus maximus L., under cadmium stress

An 8 weeks feeding trial was conducted to investigate the effect of dietary cerium (Ce) and its complex with chitosan oligosaccharide (COS‐Ce) on growth performance and cadmium (Cd) accumulation of turbot, Scophthalmus maximus L. under Cd stress. The basal diet (Diet 0) was formulated without Cd and cerium as the control. Seven other experimental diets (Diets 1–7) were formulated with supplementation of 50 mg Cd²⁺/kg feed, 50 mg Cd²⁺/kg and 50 mg Ce³⁺/kg feed, 50 mg Cd²⁺/kg and 100 mg Ce³⁺/kg feed, 50 mg Cd²⁺/kg and 200 mg Ce³⁺/kg feed, 50 mg Cd²⁺/kg and 50 mg COS‐Ce/kg feed, 50 mg Cd²⁺/kg and 100 mg COS‐Ce/kg feed, and 50 mg Cd²⁺/kg +200 mg COS‐Ce/kg feed. Results of the present study showed that, compared with the control group, the condition factor in fish fed the diet with 50 mg Cd²⁺/kg feed (Diet 1) was significantly lower, whereas the Cd concentration in liver and kidney of fish fed the diet with 50 mg/kg Cd²⁺ (Diet 1) was significantly higher (P < 0.05). The high Cd accumulation of fish fed diets with sole 50 mg/kg Cd (Diet 1) could be significantly decreased by 51.72% after supplementation of 200 mg COS‐Ce/kg in the diet (Diet 7). These results suggested that elevated COS‐Ce could effectively protect against dietary Cd accumulation in turbot.     

ICP-MS法测定大菱鲆饲料中微量元素的含量

采集辽宁葫芦岛地区10个大菱鲆养殖场的13份配合饲料样品和4份冰鲜野杂鱼样品,利用微波消解-电感耦合等离子体-质谱法(ICP-MS)检测样品中11种微量元素Cu、Zn、Fe、Mn、Co、Se、Cr、Pb、Cd、Hg、As的含量。结果表明:11种微量元素在大菱鲆配合饲料中的平均浓度顺序为Fe(719 mg/kg),Zn(122 mg/kg),Mn(32.4 mg/kg),Cr(8.35 mg/kg),Cu(7.31 mg/kg),Se(1.48 mg/kg),Pb(0.503 mg/kg),Cd(0.462 mg/kg),Co(0.422 mg/kg)和Hg(0.036 mg/kg)。除Cr外,配合饲料中微量元素水平均高于冰鲜鱼,差异极显著(P0.01);饲料质量评价结果显示,大菱鲆配合饲料中微量元素含量的变异程度比较稳定,潜在有毒元素Pb、Cd、Hg、As的含量均低于水产配合饲料安全限量,且大多数必需元素的最低含量均高于推荐剂量;相对于冰鲜野杂鱼,配合饲料能够为大菱鲆生长提供更为充足的必需元素,但Fe、Cr、Se存在超标现象。