黄鳝蛋白酶和淀粉酶活力的研究

黄鳝是我国重要的淡水经济鱼类之一,营养丰富、肉质鲜美,深受人们的喜爱。黄鳝的人工养殖受到人们越来越多的重视。成功的养殖,除了要解决好繁殖和苗种培育的问题外,还需要配制适宜的人工配合饲料。而黄鳝的配合饲料问题却一直没有得到很好的解决,限制了黄鳝的大规模养殖。对于黄鳝的研究,也主要集中在内分泌和繁殖方面,消化生理还无人做过。本实验对黄鳝的消化酶活力进行了测定,探讨了黄鳝的消化能力和消化特点,为更好地进行黄鳝人工饲料的配制,以及进一步了解黄鳝的生理生化特点提供一些参考数据。     

pH对黄鳝肠道和肝胰脏主要消化酶活力的影响

黄鳝（Monopterusalbus）是我国重要的淡水经济鱼类之一犤１犦，其营养丰富，肉质鲜美，以及独特的药用价值，深受国内外消费者的青睐，市场前景非常广阔。近年来由于天然资源的极度下降，供需矛盾日益突出，开展黄鳝规模化养殖已势在必行。目前对于黄鳝的生物学犤２犦和繁殖犤３，４犦等方面已有一定的研究，但对消化生理方面的研究只见零星报道。笔者通过黄鳝肠道和肝胰脏消化酶活力的测定，对黄鳝的消化能力和消化特点进行了探讨，以期为人工养殖提供一定的理论参考。１材料与方法１．１试验材料试验用黄鳝每尾平均重１１．２～３４．３…     

不同饲料对黄鳝消化酶活性的影响

进行了黄鳝喜食的天然动物性饵料蚯蚓、鲢肌肉和配合饲料对黄鳝消化酶活性影响的比较研究。结果表明：(1)黄鳝各消化组织中蛋白酶活性均以摄食蚯蚓组增加幅度最大，其次为摄食鲢肌肉组，而以摄食配合饲料的蛋白酶活性增加最小；(2)胰蛋白酶的活性变化则以摄食鲢肌肉组的增加幅度最大，以摄食蚯蚓组次之，摄取配合饲料组的增加幅度最小；(3)淀粉酶的活性以摄取配合饲料的黄鳝的增加幅度最大，其次为摄食鲢肌肉组，增加幅度最小的为摄取蚯蚓组个体；(4)脂肪酶的活性变化以摄取鲢肌肉的增加幅度最大，其次为摄食蚯蚓的，摄食配合饲料组增加幅度最小。这表明不同饲料对黄鳝消化器官消化酶的活性影响不同，这可能与不同饲料的营养组成、性质有关，食物中的不同营养物质会刺激黄鳝消化器官分泌相应的消化酶，以便更好地消化所摄取的食物。     

复合微生态制剂对黄鳝生长、免疫、抗氧化及消化酶的影响

正目前,在生产上黄鳝饲料主要以鱼浆和人工配合饲料搭配使用。本试验在实用饲料配方的基础上,通过饲料中添加不同含量的复合微生态制剂,探讨其对黄鳝的生长性能、消化酶活性及部分血清生理生化指标的影响,降低鱼浆使用量,为提高黄鳝的健康养殖提供一定的理论参考。     

黄鳝的消化酶活性及营养需求研究进展

黄鳝是我国重要经济淡水鱼类之一，因其营养丰富，肉质鲜美，可食部分达65％以上，深受消费者的青睐，近年来黄鳝养殖业也得到了重视，我国的学者通过对黄鳝的生长发育起着重要作用的消化酶酶活性和营养需求进行的研究表明，消化酶活性不仅与饲料的种类有关，而且还与饲料的营养组成及水平有关。     

越冬对黄鳝形体、肠道消化酶及免疫指标的影响

以初始体重(118.42±4.38)g的黄鳝(Monopterus albus)为对象,利用网箱研究越冬(时间为2012年11月20日至2013年3月25日)对黄鳝形体、肠道消化酶及部分生理生化指标的影响。试验结果表明,越冬后黄鳝的平均体重、脏体比显著下降,降幅分别为6.50%、19.84%;肝体比极显著下降,降幅32.93%;越冬对黄鳝的肥满度无显著影响。越冬显著降低黄鳝肠道胰蛋白酶活力,降幅46.37%;淀粉酶和脂肪酶活力虽下降,但不显著。越冬极显著降低了黄鳝血清中过氧化氢酶活力,降幅66.19%;显著降低了溶菌酶活力,降幅29.59%;血清中丙二醛含量虽有下降,但不显著;血清中补体C3活力极显著上升,升幅28.57%;补体C4无显著影响。越冬显著影响黄鳝形体指标,越冬期间黄鳝主要消耗自身储备来维持血糖水平提供能量,并通过降低过氧化氢酶、溶菌酶和肠道胰蛋白酶活力和提高补体C3活力达到免疫平衡。     

嗜水气单胞菌源黄鳝出血病的组织病理学研究

应用组织学方法研究了分离自患出血病黄鳝(Monopterus albus)体内的嗜水气单胞菌(Aermonas hydrophila)人工感染健康黄鳝后引起的组织病理学变化。结果显示:健康黄鳝感染嗜水气单胞菌后发病呈急性过程,感染后15~33 h为发病死亡高峰期。黄鳝人工感染后出现的临床症状与自然发病病例相似,主要表现:体表出现出血斑、肛门红肿、腹腔充血以及各实质性器官出血、充血。人工感染黄鳝的肝脏、脾脏和肾脏的病理变化明显:肝脏出现广泛的充血、出血,肝细胞出现空泡变性、坏死;肾脏肾小管上皮的肿胀、结构的消失,肾小球出血、变性坏死;脾脏充血、出血;肠粘膜上皮脱落,基底膜排列紊乱,大量出血。结果表明,嗜水气单胞菌感染黄鳝后的病理变化主要表现为肝脏、肾脏等实质性器官充血、出血,及广泛的组织细胞变性和坏死。     

饲料中添加胆碱对黄鳝生长、组织脂肪含量及消化酶活性的影响

研究了饲料中添用不同含量胆碱对黄鳝生长、饲料利用效率、肌肉和肝脏脂肪含量、肝体指数及消化器官4种消化酶活性的影响。结果表明，饲料中添加的胆碱含量不同，对黄鳝的影响不同，饲料中胆碱添加量在0～2.0％内，随着胆碱添加量的提高，黄鳝的生长速度会加快，饲料系数会逐步降低，肌肉、肝脏的脂肪含量及肝体指数降低，前肠、后肠和肝脏的蛋白酶、胰蛋白酶、淀粉酶和脂肪酶的活性均会相应的提高，而且饲料中的添加量达0.8%～1.0％时，这些变化显著，表明胆碱对黄鳝是不可缺少的，黄鳝饲料中胆碱的适宜添加量为0.8%～1.0％。     

不同规格黄鳝消化酶活性的比较

以相同环境条件下饲养2个月的黄鳝(Monopterus albus)为试验对象,以酶学方法研究3种规格[(143.81±36.27)g、(76.63 ±14.43)g、(31.35 ±5.82)g]黄鳝的肝脏、前肠、后肠蛋白酶、脂肪酶和淀粉酶的活性以及消化酶(蛋白酶、脂肪酶和淀粉酶)在肝脏、前肠、后肠中的活性分布.结果表明,黄鳝蛋白酶活性从高到低依次为前肠＞后肠＞肝脏;淀粉酶活性从高到低依次为后肠＞前肠＞肝脏;脂肪酶活性从高到低依次为后肠＞前肠＞肝脏;随着黄鳝体重的增加,肠道蛋白酶活性逐渐升高,而肝脏蛋白酶活性变化不明显;后肠、肝脏淀粉酶活性随着黄鳝体重的增加而升高,而前肠淀粉酶活性降低;黄鳝脂肪酶活性有随着规格变大先升高、后降低的趋势,以中规格黄鳝消化组织的脂肪酶活性最高.     

投喂水平对黄鳝(Monopterus albus)生长、肠道消化酶活性及部分血清生理生化指标的影响

为探究不同投喂水平对黄鳝(Monopterus albus)生长、肠道消化酶活性及血清生理生化指标的影响,分别以初始体重为(68.85±0.44)g和(26.67±0.17)g的2种规格黄鳝为研究对象,大规格按照体重的2.8%、3.6%、4.4%和5.2%投喂;小规格按照体重的3.0%、4.0%、5.0%、6.0%和7.0%投喂;每个处理组设置3个重复,大规格组50尾/箱,小规格组100尾/箱,实验持续56 d。实验结果显示,随着投喂水平的增加,2种规格黄鳝增重率、饲料系数、肝体比以及全鱼脂肪含量都显著升高(P0.05),但全鱼蛋白含量呈下降趋势。大规格黄鳝在投喂水平为4.4%和5.2%时增重率达到最高水平,且组间差异不显著(P0.05);大规格黄鳝肠道脂肪酶和胰蛋白酶活性随投喂水平的增加而显著升高(P0.05);大规格黄鳝血清超氧化物歧化酶(SOD)活性、总胆固醇(TC)含量先升后降,血清甘油三脂(TG)含量呈上升趋势;大规格黄鳝在投喂水平为4.4%时,SOD和溶菌酶(LZM)活性达到组间较高水平。随着投喂水平的增加,小规格黄鳝肠道胰蛋白酶活性显著升高(P0.05);血清TG、TC含量升高,SOD活性降低,血糖含量呈先升后降趋势。在投喂水平为6%时,小规格黄鳝的SOD和LZM活性均处在较高水平。本研究表明,当大规格黄鳝投喂水平为4.4%、小规格黄鳝投喂水平为5%–6%时,黄鳝的生长性能达到较佳状态。     

投喂水平对黄鳝(Monopterus albus)生长、肠道消化酶活性及部分血清生理生化指标的影响

为探究不同投喂水平对黄鳝(Monopterus albus)生长、肠道消化酶活性及血清生理生化指标的影响,分别以初始体重为(68.85±0.44)g和(26.67±0.17)g的2种规格黄鳝为研究对象,大规格按照体重的2.8％、3.6％、4.4％和5.2％投喂;小规格按照体重的3.0％、4.0％、5.0％、6.0％和7.0％投喂;每个处理组设置3个重复,大规格组50尾/箱,小规格组100尾/箱,实验持续56d.实验结果显示,随着投喂水平的增加,2种规格黄鳝增重率、饲料系数、肝体比以及全鱼脂肪含量都显著升高(P＜0.05),但金鱼蛋白含量呈下降趋势.大规格黄鳝在投喂水平为4.4％和5.2％时增重率达到最高水平,且组间差异不显著(P＞0.05);大规格黄鳝肠道脂肪酶和胰蛋白酶活性随投喂水平的增加而显著升高(P＜0.05);大规格黄鳝血清超氧化物歧化酶(SOD)活性、总胆固醇(TC)含量先升后降,血清甘油三脂(TG)含量呈上升趋势;大规格黄鳝在投喂水平为4.4％时,SOD和溶菌酶(LZM)活性达到组间较高水平.随着投喂水平的增加,小规格黄鳝肠道胰蛋白酶活性显著升高(P＜0.05);血清TG、TC含量升高,SOD活性降低,血糖含量呈先升后降趋势.在投喂水平为6％时,小规格黄鳝的SOD和LZM活性均处在较高水平.本研究表明,当大规格黄鳝投喂水平为4.4％、小规格黄鳝投喂水平为5％-6％时,黄鳝的生长性能达到较佳状态.     

黄鳝体内睾酮、雌二醇和炔诺酮含量的检测分析

检测并分析了黄鳝体内睾酮、雌二醇和炔诺酮的含量。结果显示，野生和人工饲养的黄鳝，其血清中睾酮、雌二醇的平均值为0．055ng／ml和22．43pg/ml，野生黄鳝中睾酮、雌二醇的含量普遍高于人工饲养的黄鳝，Student′↑s t检验差异显著。在野生及人工饲养的黄鳝体内均未检出炔诺酮。饲养试验的结果表明，如果黄鳝摄食了添加一定浓度炔诺酮的饲料，能检出其体内炔诺酮的残留。     

Isolation of heat-tolerant myoglobin from Asian swamp eel Monopterus albus

Myoglobin from Asian swamp eel Monopterus albus was purified from fish muscle using salt fractionation followed by column chromatography and molecular filtration. The purified Mb of 0.68?mg/g wet weight of muscle was determined for its molecular mass by MALDI-TOF-MS to be 15,525.18?Da. Using isoelectric focusing technique, the purified Mb showed two derivatives with pI of 6.40 and 7.12. Six peptide fragments of this protein identified by LC-MS/MS were homologous to Mbs of sea raven Hemitripterus americanus, yellowfin tuna Thunnus albacores, blue marlin Makaira nigicans, common carp Cyprinus carpio, and goldfish Carassius auratus. According to the Mb denaturation, the swamp eel Mb had thermal stability higher than walking catfish Clarias batrachus Mb and striped catfish Pangasius hypophthalmus Mb, between 30 and 60?(°)C. For the thermal stability of Mb, the swamp eel Mb showed a biphasic behavior due to the O(2) dissociation and the heme orientation disorder, with the lowest increase in both Kd(f) and Kd(s). The thermal sensitivity of swamp eel Mb was lower than those of the other Mbs for both of fast and slow reaction stages. These results suggest that the swamp eel Mb globin structure is thermally stable, which is consistent with heat-tolerant behavior of the swamp eel particularly in drought habitat.     

黄鳝血清转铁蛋白多克隆抗体的制备及检测

为实现黄鳝血清转铁蛋白基因的原核表达并制备其多克隆抗体,利用基因特异性引物从黄鳝肝脏cDNA中扩增黄鳝转铁蛋白的C端序列,亚克隆至原核表达载体pET-28a(+)中,构建pET/Tf-C重组表达载体;转化大肠杆菌BL21(DE3)后进行IPTG诱导。利用Ni离子亲和层析技术纯化Tf-C蛋白,并免疫新西兰兔制备多克隆抗体;通过间接ELISA技术和组织蛋白印迹对制备的多克隆抗体进行检测。试验结果表明,成功构建pET/Tf-C原核表达载体,并实现了蛋白的表达和纯化;制备的多克隆抗体效价大于1∶25 600,并能特异性地识别来源于黄鳝不同组织的血清转铁蛋白。研究结果对黄鳝血清转铁蛋白功能的研究奠定了基础。     

Identification of a gonad-expression differential gene insulin-like growth factor-1 receptor (Igf1r) in the swamp eel (Monopterus albus)

In vertebrate species, the biopotential embryonic gonad differentiation is affected by many key genes and key steroidogenic enzymes. Insulin-like growth factor-1 receptor (Igf1r) has been considered as an important sex-differentiation gene in mammals and could mediate the biological action of Igf1, an important regulator of key steroidogenic enzymes. However, Igf1r gene is still unknown in the swamp eel, an economically important fish. In our study, we identified Igf1r gene in the swamp eel, which was a 2,148-bp open-reading frame encoding a protein of 716 amino acids. The alignment and the phylogenetic tree showed that Igf1r of the swamp eel had a conservative sequence with other vertebrates, especial fishes. Western blotting of Igf1r showed that Igf1r expressed much more in ovotestis and testis than in ovary, indicating an important role of Igf1r during gonad differentiation. We analyzed ubiquitination of Igf1r by co-immunoprecipitation and found the amount of ubiquitinated Igf1r was increased from ovary, ovotestis to testis, which was reversely to the trend of Hsp10 expression during gonadal transformation. It was possible that Hsp10 could suppress Igf1r ubiquitination during gonadal development of the swamp eel.     

Aeromonas veronii caused disease and pathological changes in Asian swamp eel Monopterus albus

More and more diseases were occurred in the aquaculture of Asian swamp eel. Bacterial isolates were collected from the pathogenic organs of diseased Asian swamp eels. A dominant strain, Aeromonas veronii JL‐01, was isolated and identified from the liver and intestine tissue of diseased Asian swamp eels. The symptoms of fish infected artificially with A. veronii were the same as those observed under natural infection. The mortality rate was 40%–80% and infected Asian swamp eels died 2–3 days after infection. Drug sensitivity tests showed that the isolated bacterium was highly sensitive to seven kinds of antibiotics, including cephalosporins, aminoglycoside, quinolones, tetracyclines (except Tetracycline), pheniclos, nitrofurans and sulfonamides. The bacterium was resistant to penicillins (except Piperacillin), macrolides, polypeptides and lincomycin. The histological changes in Asian Swamp Eel induced by A. veronii infection mainly occurred in the liver, intestines and kidney. A. veronii showed strong pathogenicity towards the Asian swamp eel, causing tissue damage and death. Gentamycin, doxycycline and sulfamethoxazole could be used for preventing and treat this disease.     

黄鳝胃肠的生长抑素分泌细胞及超微结构

在肠卷石蜡法切片上，用ABC免疫组织化学方法，显示消化管全长的生长抑素分泌细胞（又称D细胞），结果只在胃体部显现，主要散在分布于胃腺中，个别出现在粘膜上皮。消化道内分泌细胞根据基部有否胞质突起，可分为4种类型，在黄鳝胃中均有，其中Ⅲ型数量最多，I、Ⅳ型次之，而Ⅱ型数量最少。由于产生生长抑素的D细胞在消化道内分泌细胞中作用多并且类型复杂，故黄鳝胃还是一个十分重要且复杂的内分泌器官。应用透射电镜，对黄鳝的胃肠作超薄切片观察显示在胃腺上皮细胞内含大量的微管泡系和一定量的酶原颗粒，表明其兼有泌酸及产生酶原的功能，属泌酸胃酶细胞。在肠上皮细胞游离面，含有大量微绒毛，但幼鳝比成鳝的长、密且整齐，提示幼鳝的肠上皮比成鳝有更强的吸收能力。     

Improving water quality following the death of Asian swamp eel (Monopterus albus) in cage culture through monitoring of ammonia nitrogen levels

Water quality management is important in cage culture of the Asian swamp eel (Monopterus albus). However, it is difficult to manage water quality after a large number of eels have died within the cages. The present study seeks to solve this problem by establishing a method to judge and maintain water quality. To this end, the effect of temperature and eel body weight on the water quality and microorganisms after the death of Asian swamp eels have been examined in the laboratory. The results showed that the changes in water quality parameters were similar at different temperatures and different eel weights. For example, at 26°C, the water quality started deteriorating at 5–7 days after death, which corresponded to the oxygen consumption stage. This was followed by the anaerobic decomposition stage at 11 days after death, at which point the self‐purification process of the water body was initiated. Under a lower temperature of 18°C, the oxygen consumption stage and, therefore, the overall decomposition process were extended. Proteobacteria, Bacteroidetes and Verrucomicrobia were the dominant phyla detected during the decomposition process, but the relative proportions of the dominant families differed across the stages. Based on these findings, we have provided innovative suggestions for water quality detection and management in Asian swamp eel production: (i) at a water temperature of 16–23°C, the following measures should be taken to deal with dead eels 15 days after the introduction of seedlings: (a) at an ammonia nitrogen level higher than 2 mg/L, the bodies present at the bottom of the cage need to be fished out with a net. However, when the ammonia nitrogen level is not higher than 2 mg/L, the whole cage should be sprayed with detoxifying substrate improver or Bacillus powder, according to the instructions, to improve water quality. (ii) At a water temperature of 23–30°C, the above measures should be taken on the 6th day after the seedlings are released, provided the critical value of ammonia nitrogen is 1.57 mg/L. An important limitation of this study is that water exchange was not possible under the laboratory experimental conditions, and the deterioration of water quality caused by eel death may have been alleviated due to water exchange between the inside and outside of the cage.     

Expression of pancreatic enzyme genes during the early larval stage of Japanese eel Anguilla japonica

ABSTRACT: To reveal the ontogeny of pancreatic exocrine function in the early larval stage of eel, cDNAs encoding major pancreatic enzymes, trypsinogen, amylase and lipase were identified from the Japanese eel Anguilla japonica and their expression pattern in larvae was analyzed. The cloned eel trypsinogen precursor consisted of 224 amino acids and showed 82.2% identity to trypsinogen-2 of winter flounder Pleuronectes americanus . The eel amylase precursor consisted of 512 amino acids and showed 77% identity to winter flounder amylase. Eel pancreatic lipase was composed of 470 amino acids and had 58.3% of identity to human pancreatic lipase. In the eel larvae, mRNA expression of trypsinogen and amylase was first detected at 6 days post-hatching (d.p.h.), and the expression level increased between 7 and 8 d.p.h. In contrast, mRNA expression of lipase was first detected at 8 d.p.h. Eel larvae start to feed actively at 8 d.p.h. Thus, it was indicated that eel pancreas starts to synthesize digestive enzymes at 6 d.p.h. and acquires full function by the onset of exogenous feeding at 8 d.p.h.     

Effects of salinity on osmoregulation,growth and survival in Asian swamp eel (Monopterus albus) (Zuiew 1793)

The Asian swamp eel Monopterus albus is normally considered a freshwater species, but can also occasionally be found in brackish water. It is an obligate air‐breather with highly reduced gills, making its osmoregulation physiology interesting because the gills normally represent the primary site of osmoregulation in teleosts. Being a popular fish for human consumption, the swamp eel is cultured extensively in freshwater ponds in the Mekong Delta of Vietnam. However, very little is known about its salinity tolerance, which is becoming topical due to the increasing salt‐water intrusions into tropical deltas. We therefore studied how increased salinity affects survival, growth, blood pressure, heart rate, blood osmolality and plasma ions. All eels survived prolonged exposure to 5 and 10 g L^?1, although plasma osmolality increased significantly at 10 g L^?1. Further elevation to 15 and 20 g L^?1 was associated with significantly elevated mortality, with a corresponding increase in plasma osmolality and ion con‐centrations. Our results show that swamp eels thrive in 0–10 g L^?1 with an optimum growth between 0 and 9 g L^?1, indicating that utilization of low saline brackish waters for aquaculture is possible.