Association of zinc with connective tissue in the digestive tract of common carp

ABSTRACT:   Zinc (Zn) concentration in the digestive tract of common carp is always >10 times higher than most animal tissues. In a previous paper, it was reported that this high Zn came from a 43 kDa Zn-binding membrane protein. In this present study it was further found that in the digestive tract of common carp, Zn content was closely associated with the amounts of extracellular macromolecules. The higher the Zn content, the more there is of collagen and glycosaminoglycans. An indirect immunoperoxidase staining method using antibody against the 43 kDa Zn-binding protein, or the fibroblast marker (Thy 1.1 protein) was applied to the sections of digestive tract of fish. It was found that the Zn-binding protein in the digestive tract of common carp is mainly located in the connective tissue of its lamina propria and submucosal layer. Connective tissue cells, probably fibroblasts, hold the 43 kDa Zn-binding protein. In the common carp Zn might be bound to the external side of the fibroblast. The present finding may have a significant meaning on extending the studies of Zn in biology to the field of Zn with extracellular matrix.     

Localization of Zn-binding protein in the digestive tract tissue of common carp

ABSTRACT: The concentrations of Zn and sulfhydryl (SH) groups in the digestive tract tissue of common carp and some aquatic animals were studied. It was found that Zn and bound SH groups could be used as indicators for detecting the Zn-binding protein in the digestive tract tissue of common carp. The digestive tract tissue of the fish underwent subcellular fractionation, and it was found that the nuclei/cell debris fraction contained most of the DNA (85%), Na⁺/K⁺-ATPase (82%), organic phosphate (90%) and the Zn-binding protein (79%), but only part of the 5'-nucletidase and alkaline phosphatase (<23%). The nuclei/cell debris fraction of the digestive tract tissue of common carp was treated with either collagenase type I or type IV, and subfractionated by sucrose density centrifugation. It was found that treatment with collagenase type IV could release more than 50% of the Zn-binding protein, Na⁺/K⁺-ATPase and organic phosphate from collagen. Sections of digestive tract tissue of common carp were stained for Zn. It was observed that Zn can be found mainly on the edge of the epithelial layer, and everywhere in the 'membrane-like' portion of the submucosal and muscular layers. It is proposed that most of the Zn-binding protein in the digestive tract tissue of common carp is located on the basolateral plasma membranes of the epithelial cells and on the surrounding muscle cells that are attached to the collagen type IV of basal laminae.     

鲫、草鱼外周血中淋巴细胞和单核细胞对鲤血管内皮细胞的粘附

借鉴哺乳动物内皮细胞体外培养技术，结合鱼类细胞自身特点，纯化培养鲤血管内皮细胞并传至3代，以此作为粘附材料。应用淋巴细胞分离液离心技术并结合玻璃粘附法分离纯化鲫、草鱼外周血中淋巴细胞、单核细胞，并与上述3代内皮细胞进行免疫粘附试验，同时进行免疫粘附动力学观察。结果显示，鲫、草鱼两类细胞对内皮细胞的粘附率分别为0.09±0.013，0.20±0.018；0.11±0.015，0.21±0.023。表明鲤科鱼类不同种的同类细胞粘附率差异不大，而同种不同类细胞则差异显著。动力学观察分析表明，淋巴细胞粘附较快，60min进入平台期；单核细胞粘附慢，120min进入平台期。初步证明鱼类内皮细胞具有免疫介导作用。     

Cloning and characterization of cDNA for carp matrix metalloproteinase 9

ABSTRACT: We have cloned a cDNA encoding the MMP-9 from a carp epidermal cell (EPC) cDNA library. The clone contains a 2025-base pair (bp) open reading frame encoding a protein of 674 amino acids. The deduced amino acid sequence shares 68% and 69% identity with medaka and Japanese flounder MMP-9. The hinge domain of the carp MMP-9, like those of the other non-mammalian species, lacks a type V collagen-like region that is typical of mammalian MMP-9. Gelatin zymography and immunoblot analysis of conditioned media of EPC cells and cDNA-transfected COS-7 cells detected a 76-kDa gelatinase. The apparent molecular mass of the carp zymogen is much smaller than those of its mammalian counterparts while almost identical with that of chicken 75-kDa gelatinase B-like enzyme. Although hypo-osmotic stress induced the elevation of MMP-9 mRNA level in EPC cells, no significant change in the protein in conditioned medium was detected during hypo-osmotic stress. Northern blot analysis detected a large amount of MMP-9 mRNA in carp kidney and spleen, suggesting the high expression of MMP-9 in blood cells, neutrophils, and macrophages. The smaller amount of MMP-9 mRNA was detected in gill, heart, fin, and eye, whereas none of the mRNA was detected in the hepatopancreas, intestine, brain, muscle, and skin.     

Binding characteristics of the Zn-binding membrane protein from common carp

ABSTRACT:    This study incorporated the 43 kDa Zn-binding membrane protein isolated from common carp into liposome. The specificity and strength of the binding of ⁶⁵Zn to the 43 kDa protein-liposomes, and the binding of the ⁶⁵Zn-labeled 43 kDa protein-liposomes to laminin were studied. It was found that ⁶⁵Zn was bound to the external side of the 43 kDa protein-liposomes. Specific binding of ⁶⁵Zn to the protein-liposomes was detected. The binding parameter of Zn to the protein was found to be: maximum binding site (N_max), 76.7 pmole/µg protein (approx. 3 mole of Zn²⁺/mole); and equilibrium dissociation constant (Kd), 0.19 µM. Of the cations introduced (Ca²⁺, Cd²⁺,Co²⁺, Cr²⁺, Cu²⁺, Fe²⁺, Hg²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺), only Co²⁺ competed significantly with Zn. The protein-liposomes were also found to bind specifically to laminin with a N_max of 1.1 pmole/µg laminin, and Kd of 4.79 µM. No significant protein-liposome binding occurred to other extracellular matrix proteins (fibronectin, fibrinogen or vitronectin). Furthermore, the binding was specifically inhibited by the Arg-Gly-Asp (RGD) peptide or GRGDSPG, while two other analogs (GRGESPG and GRADSPG) were without effect.     

High zinc in the erythrocyte plasma membranes of common carp Cyprinus carpio

ABSTRACT:   The distribution of zinc (Zn) concentration in the blood and erythrocytes of common carp, grass carp, silver carp and tilapia was studied. It was found that in whole blood, the average Zn concentrations in these four species of fish (approx. 6–14 μg/[ml whole blood]) were relatively similar to those in other species of fish and mammals. However, the mean Zn concentration in the erythrocytes of common carp is approximately two times higher than the other three species of fish (approx. 5 vs approx. 2 μg/[ml whole blood]). It was found that approximately 70% of the Zn in the common carp whole blood came from its erythrocytes. In addition, approximately 43% of the Zn in the erythrocytes of common carp was found to be located on its outer plasma membranes. When an antibody against a 43 kDa Zn-binding protein, isolated from the digestive tract tissue of common carp, was used, significant quantities of the protein were shown to be present on the erythrocyte plasma membranes of common carp by an indirect immunofluorescent staining. High Zn on the outer plasma membrane of the common carp erythrocyte most probably comes from the 43 kDa Zn-binding protein.     

Anoxia survival in common carp and crucian carp is related to high zinc concentration in tissues

ABSTRACT:   Ten lots of common carp and six lots of crucian carp (each lot of 100 fish) were treated under anoxia for five days at water temperatures of 25–31°C. The average per group mortality of common carp was 17%, but none of the 600 crucian carp died. The digestive tract tissues of the common carp that died had significantly lower zinc concentrations than those that survived (59 ± 41 vs 142 ± 60 μg/[g fresh tissue], P  < 0.001). The digestive tract tissues of the crucian carp had mean zinc concentrations of 652 ± 458 μg/(g fresh tissue). One lot of common carp that had low tolerance for anoxia was fed a high zinc diet (2000 mg zinc/kg diet) for 1, 2 or 6 months and then subjected to 5 days anoxia. The survival rates of those fed the high zinc diet 1 and 2 months increased from 0 to 50%, respectively; all of fish that had fed a high zinc diet for 6 months survived. Thus, anoxia survival in common carp and crucian carp is closely related to the high concentrations of zinc in their tissues.     

Accumulation of zinc from diet and its release in common carp

High zinc diet of 2000 mg kg^–1 was fed to common carp (Cyprinus carpio), grass carp (Ctenopharyngodon idellus) and tilapia (Oreochromis aureus) for 8 weeks to compare the accumulation of Zn in fish. It was found that accumulation of zinc from diet by grass carp and tilapia was low when compared to common carp. The largest difference occurred in digestive tract tissue. The digestive tract tissue of common carp had a large pool of stored zinc, the mean storable capacity is about 1500 g Zn g^–1 fresh tissue, and the time needed to saturate the tissue when feeding 2000 mg Zn kg^–1 diet is about 8 weeks. The accumulated Zn in the digestive tract tissue of common carp was released when the dietary Zn was reduced to normal level (50 mg kg^–1) or deficient level (4 mg kg^–1) for 4 weeks.Subcellular fractionation results indicate that Zn accumulated in digestive tract tissue of common carp was accumulated mainly in the nuclei/cell debris fraction. Exposure to a high Zn diet induced some metallothionein-like substance in the digestive tract tissue and the hepatopancreas of common carp, but the amount was very low when compared with the amount of Zn accumulated in the nuclei/cell debris fraction.     

Energy budget and metabolism in common carp, Cyprinus carpio L., fed on different dietary protein levels and at different ration levels

The total energy budget of 65 ± 8.0 g common carp, Cyprinus carpio L., was studied in the laboratory. The pattern of energy allocation varied with dietary protein content and ration levels. The energy lost as heat of metabolism, R, was found to increase with dietary protein level and total ration. Energy lost as faeces, F, varied from 19% to 24% of consumption, C, but did not appear to be related to either protein content or ration levels. The energy equivalent of nitrogenous excretion, U, as a proportion of C increased with an increase in dietary protein but decreased with an increase of ration level. Regression equations were developed from the data to allow prediction of respiratory energy losses, faecal losses and excretory losses. The balance of energy budgets compiled from experiments conducted over an 18-day period was between 66% and 82%. Observed growth was always less than predicted growth, so the predictive models overestimated growth. The data presented form the basis for the first reported study of total energy budgets in Cyprinus carpio.     

Marine microalgae from biorefinery as a potential feed protein source for Atlantic salmon,common carp and whiteleg shrimp

Two marine algal products MAP3 and MAP8 were examined for their suitability as fishmeal protein substitutes in feeds of three prominent farmed species, through short‐term feeding studies. Algal meals were tested at 5 and 10% protein replacement levels for Atlantic salmon and at 25 and 40% for common carp and whiteleg shrimp. At the end of the 12‐week period, the growth and feed performance of the two fish species did not reveal any significant difference between those fish offered the algae‐based feed and those offered the control feed. The whole body proximate compositions of Atlantic salmon fed the control and algae‐based feeds were not significantly different. In common carp, the lipid content in the fish fed higher level of MAP3 was significantly lower than that of the fish fed the control feed. In whiteleg shrimp, at the end of the 9‐week feeding period, growth performance and feed utilization did not differ between the treatment groups. Protein content in the shrimp fed the higher level of MAP8 was significantly lower than that of shrimp on the control feed. The three species could accept the algal meals in their feeds at the tested levels, though there were some noticeable effects on body composition at higher inclusion levels.     

用RAPD方法研究草鱼、柏氏鲤和3个地理种群鲤的亲缘关系

用２４种随机引物对雅罗鱼亚科（Ｌｅｕｃｉｓｃｉｎａｅ）的草鱼,鲤亚科（Ｃｙｐｒｉｎｉｎａｅ）的柏氏鲤和３个地理种群鲤（荷包红鲤、黑龙江野鲤和德国镜鲤）进行ＲＡＰＤ分析,构建了这５种鱼类的基因组指纹图谱。通过对获得的基因组指纹图谱的量化分析,利用ＵＰＧＭＡ重建了它们之间的亲缘关系。结果鲤种内３个地理种群之间的相似性高于鲤和柏氏鲤种间的相似性,而草鱼与另４种鲤亚科鱼类亚科之间的差异明显高于柏氏鲤、黑龙     

甲基汞对鲤鱼鳃组织及氯细胞的影响

水体中甲基汞(CH3ClHg)浓度(μg/L)分别为1、5、10、25、50、100、200和1、2．5、5、10、20、40、50染毒鲤鱼种24h和一周。鱼鳃石蜡切片观察结果：50μg/L染毒24h和20μg/L染毒1周鱼，鳃丝组织末见异常变化；100μg/L染毒24h和40μg/L染毒1周鱼，鳃丝出现轻微混乱且向上卷曲；200μg/L染毒24h和80μg/L染毒1周鱼，外表皮层从基膜脱落，鳃丝严重卷曲。鳃氯细胞的结构变化：100μg/L汞染毒24h40μg/L染毒1周鱼，氯细胞的细胞膜质分散，偶尔可见肿胀线粒体和空泡膜管系统；暴露于甲基汞200μg／L中24h和80μg/L1周鱼的氯细胞损伤明显：细胞质内空泡大量出现且无规律，膜管系统空泡增加，线粒体多数变成肿胀或已崩溃解体。     

鲤鲫杂交(♀)×鲫(♂)回交子代的形态特征研究

利用雌性鲤鲫杂交与雄性鲫鱼回交,制备了鲤鲫杂交回交子代鱼,并对其1龄鱼的形态特征和内部器官结构进行了测定。其主要性状为：回交子代的背鳍条Ⅲ,15～18;臀鳍条Ⅲ,4～6;侧线鳞27～36;侧线上鳞和侧线下鳞同为5～6;鳃耙28～33;体长为体高的(2.35±0.11)倍,为头长的(3.33±0.13)倍,为尾柄长的(6.36±0.54)倍;头长为眼径的(4.54±0.20)倍;为眼间距的(1.74±0.07)倍;为尾柄高的(1.96±0.10)倍;尾柄长为尾柄高的(0.90±0.28)倍;体高为头高的(1.59±0.09)倍。结果表明,该回交子代鱼在可数和可量性状上都有个别性状产生变易,但大多数性状相对比较稳定。     

The anaesthetic‐like effect of tea tree oil in common carp Cyprinus carpio L.

This study investigated the efficacy of tea tree oil (TTO) as an anaesthetic for common carp, Cyprinus carpio L., by estimation of behaviour changes and responsiveness to being taken out of the water. All applied concentrations (from 0.2 to 0.6 mL L⁻¹) resulted in sedation and immobilization. The lowest effective concentration (induction time ≤3 min, recovery time ≤10 min after 15 min of exposure) was 0.5 mL L⁻¹. Exposure in excess of 30 min at a concentration of 0.5 mL L⁻¹ caused mortality. TTO displayed distinct general anaesthetic‐like properties, fulfilling the basic requirements for a potent fish anaesthetic.     

Evaluation of carbohydrate rich diets through common carp culture in manured tanks

Four diets (T₀–T₃) were formulated reducing the fishmeal (Indian) component by 100 g kg^–1 from 300 to 0 g kg^–1 and including proportionately increasing quantities of maize. Diets were fed for 120 days at 50 g kg^–1 body weight to triplicate groups of common carp (av. wt. 2.11–2.18 g) stocked at 1 m^–2 in mud bottomed cement tanks (18 m²), fertilized with poultry manure. Fish growth, SGR and FCR in the different treatments were statistically not significantly different ( P  > 0.05). PER was lowest for the 300 g fishmeal kg^–1 diet treatment (diet T₀), increasing with decrease in dietary fishmeal content (diets T₁–T₃). Fish survival ranged from 96.29 to 100%. Diets influenced carcass composition and digestive enzyme activity. A significant increase in lipid deposition was recorded with increasing dietary carbohydrate content. Amylase, protease and lipase activities were higher in fish fed with diets T₂ and T₃. The protein sparing effect of dietary carbohydrate and the economic implication of eliminating fishmeal from the diet are discussed.     

转基因鲤放流试验湖鲫的年龄与生长

以鳞片作为年龄鉴定材料,研究转基因鲤(Cyprinus carpio)放流试验的人工湖中鲫(Carassius auratus)的年龄组成、大小与生长特性。结果显示:鲫种群由4个年龄组(1 a~4 a)构成。体重(W,g)与体长(L,mm)的关系式为:W=2.597×10-5L3.0133;体长与鳞径(R,mm)的关系式为:L=7.4+29.8R。生长模式可用von Bertalan-ffy生长方程描述:Lt=28.8[1-e-0.254(t+0.307)],Wt=668.9[1-e-0.254(t+0.307)]3.0133,体重生长拐点年龄ti=4.04 a。还对转基因鲤放流人工湖泊中鲫的生长数据同其它湖泊中的作了比较。这些数据为今后探讨转基因鲤种群的建立对鲫生长的影响积累了背景资料。     

Effects of feeding rates on growth and production of Nile tilapia, common carp and silver carp polycultured in fertilized ponds

Abstract In an attempt to identify appropriate feeding rates for multispecies of fish raised in fertilized earthen ponds, the present work was conducted over a 19‐week experimental period to establish the growth performance, production and body composition of Nile tilapia, common carp and silver carp fed 0.0%, 0.5%, 1.0%, 3.0%, 5.0% biomass and to apparent satiation (treatments). Twelve ponds were stocked with a similar number and weight of each fish species. Two ponds were assigned to each of the treatments, and a 25% protein pelleted fish feed was used to feed fish at the specified rate of feeding. At the end of the experiment, growth, weight gain, survival, yield and body composition of fish groups were affected by the treatments. The economic effectiveness also varied among treatments. The most conspicuous attribute of the feeding rates was its lack of influence on growth (g day^?1), weight gain (g per fish), yield (kg ha^?1) or body composition of silver carp. The results of whole‐body proximate analysis indicated that various feeding rates had either an irregular pattern or no effects on the protein and ash gain per 100 g of fish body weight (bw) gain. The most notable exceptions were significant (P < 0.05) increases in body fat and gross energy gains in Nile tilapia, common carp and silver carp accompanied by decreases in percentages of moisture (but not in silver carp) as feeding rate increased. Among the six different feeding levels, feeding to apparent satiation (feed amount was equivalent to 2.67% of fish bw day^?1) appeared to be optimal, as it significantly (P < 0.05) supported the highest fish production, income and net profit compared with all other treatments except for the 3% feeding level, for which the differences in those measurements were comparable.     

Study on the adaptability of common carp (Cyprinus carpio) to diets from the perspective of digestion and absorption

As an ecologically sustainable aquaculture mode, the rice‐fish approach has been paid more attention in recent years. In rice paddies, there are plant and animal diet items available to fish, but it is not clear how common carp adapt to different diets. In view of this, common carp (initial weight 492.6 ± 30 g, n = 270) were randomly divided into three groups and fed with earthworms (group A), earthworms + duckweed (group M) and duckweed (group P) respectively. After 8 weeks under these feeding regimes, the intestinal digestive enzymes activities were assayed, and intestinal tissue sections stained with HE and AB‐PAS were used to observe the morphology. The results showed that the activities of trypsin and lipase were highest in group A, followed by group M and group P. Group A had greater intestinal fold height, fold width and fold absorption area than the other groups (p < .05), especially in the foregut. Meanwhile, the total number of mucous cells was largest in group P, followed by the groups M and A. Totally, activities and distributions of digestive enzymes, fold height, fold width, fold absorption area and number and distribution of mucous cells of common carp were affected by the type of diet. Based on these results, it was found that common carp has strong adaptability to diets, and it was speculated that the majority of digestion and absorption of protein was concentrated in the foregut, while for starch and cellulose, it was spread out along the whole intestine.     

Improved healing of the deeply incisional wounds in partially scaled common carp by zinc sulphate bath

This study aimed to investigate the role of zinc immersion bath in promoting wound‐healing process in partially scaled common carp using different concentrations of zinc sulphate (ZnSO₄). After wound induction, three different doses of ZnSO₄ were used (1/20, 1/10 and 1/5 of the calculated LC50) for 10 days. After the end of the experimental period, tissue samples were collected from the area surrounding the wound for analysing the expression of the following genes: collagen type I alpha 1 (COL1α1), transforming growth factor (TGF)‐β1, vascular endothelial growth factor (VEGF)‐A, metallothionein (Met) and fibroblast growth factor (FGF)‐7. Moreover, the tissue antioxidant markers including superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and malondialdehyde (MDA) were also assessed. The extent of the healing process was also assessed by histopathological examination. The results revealed that the use of ZnSO₄ at 1/10 of the LC50 caused a significant improvement in tissue reepithelization, angiogenesis and tissue remodelling through overexpression of Col1α1, TGF‐β1, VEGF‐A and FGF‐7. Moreover, a significant improvement in antioxidant markers was also observed with ZnSO₄ immersion. In conclusion, this study showed that ZnSO₄ immersion at 1/10 of the calculated LC50 improved the healing of the incisional wound in partially scaled common carp via upregulation of the examined growth factor genes and improvement of the antioxidant status of fish.