【作者】 刘彩霞；

【导师】 罗智； 谭肖英；

【作者基本信息】 华中农业大学 ， 水产养殖， 2013， 硕士

【摘要】 胭脂鱼(Myxocyprinus asiaticus)是亚口鱼科在亚洲的唯一代表,被列为国家二级保护动物,在地理分类上都具有重要的研究价值。目前,国内外关于胭脂鱼的研究主要集中在其幼鱼的营养需求和繁殖技术等方面,尚未关于胭脂鱼消化系统发育和肉碱棕榈酰转移酶Ⅰ的酶动力学的研究。因此,本文拟对胭脂鱼消化系统的发育进行研究,并分析胭脂鱼发育阶段消化酶的活性和肉碱棕榈酰转移酶Ⅰ的酶动力学特征的变化，以期为胭脂鱼的形态学、营养学和发育生物学提供基础资料。其研究结果如下：1.胭脂鱼仔、稚鱼消化系统的发生本实验利用组织学和超微结构的方法,观察和研究了出膜后1-56日龄的胭脂鱼仔、稚鱼消化道及其附属结构(肝脏,胰脏和胆囊)的个体发生。以投喂模式为基础,并且分析了消化系统主要的组织学特点,出膜后胭脂鱼仔、稚鱼的发育主要分为三个阶段：阶段1(内源性营养期)：1-6日龄；阶段2(混合性营养期)：7-14日龄；阶段3(专一性外源性营养期)：15日龄以后。刚出膜时,仔鱼消化道是一条未分化的直管。4日龄时,仔鱼口打开,此时消化道分化为口咽腔,食道和肠道。7日龄仔鱼开始摄食。15日龄时卵黄囊吸收完全。56日龄稚鱼显示整齐排列的长微绒毛,丰富的空泡和蛋白质包涵体。从6日龄起,胭脂鱼仔鱼胰脏,肝脏和胆囊开始执行其功能,这使得仔鱼能够摄取、消化和吸收外源性食物。我们的研究表明,与其他有功能性胃的硬骨鱼相比,无胃胭脂鱼消化道发育完全相对较慢。2.胭脂鱼仔、稚鱼消化酶活性的变化本实验研究了胭脂鱼仔、稚鱼胰腺酶(胰蛋白酶和淀粉酶)和肠酶(碱性磷酸酶和氨肽酶N)从出膜到40日龄期间的发生。本实验所测定的几种酶在刚孵化出来的仔鱼当中都能检测到。淀粉酶活性在3至14日龄上升,这表明早期发育过程中淀粉酶不断生成且在这个过程中碳水化合物能被很好地分解。淀粉酶比活性在14至17日龄期间下降,在20日龄时又开始上升,这可能与其摄入的饵料种类相关。胰蛋白酶比活性在14至25日龄期间发生了显著性的变化。碱性磷酸酶活性在9至14日龄期间上升,表明肠细胞刷状缘的形成。碱性磷酸酶活性在14日龄和20日龄两个时期达到较高值,而在20-40日龄之间保持不变。基于以上观察所得,胭脂鱼消化系统似乎直到20日龄才完全发育成熟。此后胭脂鱼具有成鱼的消化模式,所以胭脂鱼从20日龄开始进行食性转变是可行的。3.胭脂鱼发育过程中肉碱棕榈酰转移酶Ⅰ动力学特征的变化从出膜到1龄,胭脂鱼肝脏中总肉碱浓度随着年龄的上升而逐渐上升。胭脂鱼肝脏中总肉碱浓度的增加表明肝脏组织合成肉碱的能力增强或吸收肉碱的能力提高。胭脂鱼从68日龄至1龄期间,肌肉组织中的游离肉碱浓度和总肉碱浓度都逐渐增加。肌肉组织中的游离肉碱浓度和总肉碱浓度的增加是为了促进脂肪酸的p-氧化,从而产生大量能量,满足胭脂鱼发育期间肌肉的快速增长对能量的需求。肠道中乙酰肉碱浓度和总肉碱浓度在发育期间波动较大,这是因为在胭脂鱼发育期间,肠道不断发育成熟,经历了一系列的变化。胭脂鱼肝脏组织中的AC／FC之比刚出膜时为1.03,4月龄时上升至1.61,但1龄时显著下降,但2龄时达到1.73,显著高于1龄时。胭脂鱼肌肉组织中的AC/FC之比最高时是在68日龄时,最低时是在4月龄时。刚出膜的胭脂鱼仔鱼的CPTI的表观Km值(以肉碱做底物)为4.51±0.16mM,肉碱棕榈酰转移酶Ⅰ的动力学参数Vmax为4.53±0.09nmol/min/mg protein.肠道组织中的肉碱棕榈酰转移酶Ⅰ的动力学参数Vmax随着年龄增长呈上升趋势。胭脂鱼不同时期不同组织(肝脏、肌肉和肠道)中的游离肉碱浓度都低于与之对应的表观Km。这些数据表明胭脂鱼需要在其饵料中添加肉碱以保证肉碱棕榈酰转移酶Ⅰ的活性。更多还原

【Abstract】 Chinese sucker(Myxocyprinus asiaticus) was the only species of the family Catostomidae in Asia, which had been an endangered species listed in the Category II of the endangered aquatic and land animal species in China. It had an important scientific value in geographic classification. At present, although several studies were involved in the nutrition of juvenile Chinese sucker and the production of the fish species, little information was available about the ontogeny of digesitive system and kinetics of carnitine palmitoyltransferase I in Chinese sucker (M. asiaticus). The aim of this study was to describe the ontogeny of digestive system, and analyse the development of digestive enzymes and the kinetics of carnitine palmitoyltransferase I (CPT I) in Chinese sucker, which would contribute to the information about morphology, nutriology and biology of development of this fish species. The main results are shown as follows.1. The development of digestive system in Chinese sucker (M. asiaticus)The present study was conducted to determine the ontogenetic development of the digestive tract and its accessory structures (liver, pancreas and gall bladder) in agastric larval Chinese sucker M. asiaticus with the histological and ultrastructural approaches from hatching to56days after hatching (DAH). Based on its feeding mode, and analysing the main histological features of the digestive system, larval development in Chinese sucker was divided into three stages from hatching:stage1(endotrophic period):1-6DAH; stage2(endoexotrophic period):7-14DAH; stage3(exclusively exotrophic period):from15DAH onwards. At hatching, the digestive tract of the larvae consisted of an undifferentiated straight tube. At4DAH, the mouth opened, and the digestive tract was differentiated into buccopharyngeal cavity, esophagus and intestine. At7DAH, fish started to feed exogenously. Yolk sac was completely exhausted at15DAH. Until56DAH, the digestive tract of the larvae displayed regularly arranged microvilli, abundant vacuoles and protein inclusion bodies. The pancreas, liver and gall bladder were functional from6DAH, which enabled larvae to ingest, digest, and assimilate the first exogenous food. In comparison with teleosts that have a stomach, the development of the digestive tract of the agastric Chinese sucker seemed relatively slow. 2. Developmental changes of selected digestive enzymes in Chinese sucker (M. asiaticus) during larval ontogenyThe ontogenesis and specific activities of pancreatic (trypsin and amylase) and intestinal enzymes (alkaline phosphatase and aminopeptidase N) were investigated in Chinese sucker M. asiaticus larvae from hatching to40day after hatching (DAH). All of assayed digestive enzymes were detected in newly hatched Chinese sucker larvae. However, the maximum activities varied among different digestive enzymes. From3to14DAH specific activity of amylase in Chinese sucker larvae increased, indicating the synthesis of amylase in early ontogenesis and the carbohydrates were actively catalyzed during the stage. The decrease of amylase from14to17DAH followed by an increase at20DAH in Chinese sucker larvae may be possibly due to progressive changes in the digestive tract and the subsequent response to composition and amount of the available food. The secretion level of amylase remained at the relatively low level between30and40DAH, suggesting the carnivorous feeding habitat for juvenile Chinese sucker. Significant changes of trypsin specific activities were observed from14to25DAH. The specific activity of alkaline phosphatase sharply increased between7and14DAH, indicating the formation of brush border enterocytes. Its activity decreased after peaking at14DAH, and remained constant between20and40DAH. The aminopeptidase N specific activity remained at a constant low level before12DAH. The activity of the enzyme reached the first peak at14DAH and a second peak on20DAH. Based on these observations above, the maturation of the digestive tract seemed fully achieved at20DAH in Chinese sucker. From this date, this species has an adult mode of digestion, so weaning at20DAH could be considered reasonable for Chinese sucker larvae.3. Changes of kinetics of CPT I in Chinese sucker (M. asiaticus)The total carnitine in liver tissues of Chinese sucker gradually increased from hatching to1year. The increase of hepatic total carnitine may be indicative of the enhanced biosynthesis capacity for carnitine in liver tissues. From68-day-old to1-year-old Chinese sucker, both free and total carnitine concentrations in muscle gradually increased. This increase would be a need to generate energy through β-oxidation of fatty acids for the rapid growth of the muscle during this developmental period. Both acyl and total concentrations in intestine at different stages changed violently, which may be explained by the fact that digestive tract went through a series of dramatic changes during the course of maturation. The ratio of AC/FC in liver progressively increased from hatching (1.03) to4months (1.61), but at the age of1year it significantly decreased to0.71, and then reached1.73by2years. In muscle tissues the ratio of AC/FC was highest at the age of68-days-old and lowest at4months. The whole Chinese sucker larvae at hatching had a high value of apparent Km (4.51±0.16mM) for carnitine; the kinetic parameter Vmax measured in the whole Chinese sucker larvae at hatching was4.53±0.09nmol/min/mg protein. I the kinetic parameter Vmax measured in intestine tissue showed an increasing trend with age. In our study, the free carnitine concentrations in the examined tissues (liver, muscle and intestine) at all stages of development were all less than the respective Km. The data imply that Chinese sucker may require supplemental carnitine to ensure the activity of CPT I.更多还原