【作者】 熊冬梅；

【导师】 谢从新；

【作者基本信息】 华中农业大学 ， 湿地资源与环境， 2010， 博士

【摘要】 本文以具有很高经济价值和养殖潜力的黑斑原鮡Glyptosternum maculatum (Regan,1905)为研究对象,系统地研究其消化系统的形态学、组织学和超微结构以及消化酶的种类、分布、理化性质和不同年龄组消化酶的差异,并测定胃肠道粗酶液对7种饲料原料的离体消化率,从而为黑斑原鮡人工饵料的配方提供一些理论依据,同时也丰富了鮡科鱼类消化生理的基础知识,主要研究结果如下：1.黑斑原鮡消化系统具有以下特点：(1)口下位,吻钝圆,唇具小乳突。口腔和口裂都较大；上下颌有细小的尖齿,齿尖朝里,密集排列形成齿带。舌退化,第一鳃弓外鳃耙数目为5-9。口咽腔顶壁和底壁为复层鳞状上皮,内含味蕾和杯状细胞。(2)食道粗短,肌层很发达,内壁上有较深的纵向褶,粘膜层有大量的杯状细胞。黑斑原鮡食道前段的粘膜层仍发现少量的味蕾。(3)胃呈囊状,U型,分为贲门、盲囊和幽门三部分,胃壁内有很深的纵向皱褶,贲门和胃底部有胃腺。向幽门处移动胃腺数量逐渐减少,胃肠交界处的幽门胃无胃腺。胃底部存在三种类型的细胞：粘液细胞、腺细胞和内分泌细胞。(4)肠管较短,肠道系数约为0.9。肠壁较厚,管腔从前肠到后肠逐渐变小。整个肠道没有任何膨大部分,盘旋1-2次。整个肠道内壁充满密集的纵向粘膜褶,粘膜皱褶高度从前肠向后肠逐渐降低。肠上皮主要有吸收细胞和杯状细胞。(5)肝胰脏是分离的,胰腺主要分布在胃、肠道前段和胆囊壁外周以及肠道系膜的脂肪中。肝脏覆盖在食道和胃前端的上面,分左右两叶,肝脏呈深红棕色,分别通过连接带与胸鳍基部皮下的腹腔外附肝相连。胆囊借结缔组织与肝脏左叶相连,呈深绿色。2.2.黑斑原鮡消化道蛋白酶活性以胃最高,前肠、主肝和附肝次之,胃和前肠以及胃和主肝、附肝之间差异显著(p<0.05),主肝和附肝之间差异不显著(p>0.05),中肠和后肠最低且两者差异不显著(p>0.05)；淀粉酶活性以前肠最高,主肝和附肝次之,前肠与后两者差异显著(p<0.05),但主肝和附肝之间差异不显著(p>0.05),再次为胃,胃与前肠淀粉酶活性差异显著(p<0.05),与两部分肝脏差异也不显著(p>0.05)；中肠和后肠的活性最低,与前肠的淀粉酶差异显著(p>0.05)；脂肪酶活性以前肠最高,主肝和后肠次之,三者之间差异显著(p<0.05),中肠、胃和附肝的脂肪酶活性最低,中肠与后肠的脂肪酶活性差异不显著(p>0.05),但二者都低于前肠(p<0.05),中肠与胃之间差异显著(p<0.05),主肝和附肝之间脂肪酶活性差异显著(p<0.05)；胰蛋白酶前肠最高,胰凝乳蛋白酶活性主肝和附肝最高,两种酶活性均以胃部最低；碱性磷酸酶活性前肠最高,中肠和后肠次之且两者无显著性差异(p>0.05),但与前肠差异显著(p<0.05),主肝、附肝和胃活性最低,三者无显著性差异(p>0.05)；亮氨酰氨基肽酶活性分布规律和碱性磷酸酶一致,前肠最高,中肠和后肠次之且两者无显著性差异(p>0.05),但与前肠差异显著(p<0.05),主肝、附肝和胃活性最低,三者无显著性差异(p>0.05)。3.黑斑原鮡胃蛋白酶最适温度为30℃,前肠、中肠和后肠为50℃；胃、前肠、中肠和后肠四个部位的淀粉酶最适温度均为30℃；胃脂肪酶最适温度为30℃,前肠最适反应温度为50℃,中肠和后肠的最适温度为40℃。胃蛋白最适pH为2.0,肠道最适pH为9.0-10.0；胃淀粉酶最适pH为6.0,肠道最适pH为7.0；胃脂肪酶最适pH为6.0,肠道最适pH为8.0。胃和肠道蛋白酶在20-50℃范围内均比较稳定。胃蛋白酶在pH 1.0-4.5之间都比较稳定,肠蛋白酶的pH稳定性范围为pH7.5-11.0。胃和肠道蛋白酶最适底物浓度分别为2.5%和2.0%干酪素,胃和肠淀粉酶最适底物浓度分别为2.5%和3.0%可溶性淀粉。Na+和Hg2+对胃蛋白酶有明显抑制作用,Cu2+和Co2+有明显激活效果；Cu2+、Zn2+和Fe3+对肠道蛋白酶有明显抑制作用,而Ca2+、Fe2+以及Mg2+对其有激活作用,EDTA对胃和肠道的蛋白酶均有强烈抑制作用。胃肠道蛋白酶明显受到PMSF、TPCK、TLCK、PepstatinA和β-巯基乙醇等的抑制。4.黑斑原鮡不同年龄组的消化酶活性有差异。蛋白酶和淀粉酶活性在9龄组最高,7龄组和15龄组最低,趋势是随年龄增加先增加后减小。碱性磷酸酶也随年龄增加先增加后降低。脂肪酶活性3龄组和5龄组最高,基本趋势随年龄增加而减小。胰蛋白酶和胰凝乳蛋白酶以及亮氨酰-氨基肽酶随年龄增加而降低。5.黑斑原鮡粗酶液对7种蛋白原料的干物质和粗蛋白的体外消化率大小顺序为：胃部>前肠>中肠>后肠,其中对动物性蛋白(如鱼粉)的消化率高于植物性蛋白(豆粕、菜粕和棉粕)。消化道粗酶液分解蛋白质所产生氨基酸的速率顺序为：前肠>中肠>胃>后肠。更多还原

【Abstract】 The aim of the study is to investigate the morphology, histology and ultrastructure of digestive tract of Glyptosternum maculatum, and the distribution and characteristics of main digestive enzymes, and the changes of digestive enzymes vary from age, also the in-vitro digestibility on seven feed ingredients of crude enzymes. Such information of digestive physiology of G. maculatum of notable economic importance and with high potential for controlled rearing is considered valuable, not only for adding to current knowledge on the biology of Sisoridae fishes, but also for understanding formulation of any artificial diets. The main results are below:1. The features of digestive tract of G. maculatum are summarized below:(1) Inferior mouth with thin papillate lips, blunt snout and broad mouth width. Premaxillary teeth are short and conical, arranged in irregular rows on as a patch. Tongue degenerate. The number of outer gill raker in the fist gill arch is 5-9. The mucosa of both upper and lower wall of buccopharynx is stratified squamous epithelium with goblet cells and taste bud.(2) The oesophagus is stubby with thick muscularis. The mucosa fold is longitudinal, lined with many goblet cells, and few taste buds are found in the forepart of oesophagus.(3) The U shape stomach was divided into three portions obviously:cardiac, fundic and pyloric parts. In the cardiac and fundic stomach, there were a great amount of gastric glands, whereas there is a gradual loss of glands towards the pyloric stomach, and no gastric glands are found in the posterior part of pyloric stomach. The mucosa fold of the stomach is longitudinal with zigzagging bend, and second fold was transverse or wavy. At the ultrastuctural level, three type cells (mucous, glandular and endocrine cell) were found in the stomach, and glandular cell with a great amount of pepsinogen granules.(4) The intestine is short with 1-2 times convolution, and the relative intestine length was 0.9. The wall of intestine tube is thick, and the height of mucosa fold and thickness of muscularis decrease from the anterior intestine to posterior intestine. The intestinal epithelium is composed of two main cell types:enterocytes and goblet cells. (5) Liver and pancreas is separate, and the pancreas mainly distribute close to stomach, anterior intestine and gall bladder, as well as the lipid tissue of mesenterium. Liver has right and left lobe, above the oesophagus and stomach, and gall bladder is attached to the left lobe of liver. The color of liver and gall bladder is puce and bottle green, respectively. The subdominant liver is under the skin of pectoral fin, with joint belt connected to dominant liver in the abdominal cavity.2. The descending order of protease activity in different digestive sections is: stomach, anterior intestine, dominant liver, subdominant liver, posterior intestine and middle intestine, and the activity in the stomach is significantly higher than that in the anterior intestine and two parts of liver (p<0.05), while there is no significant difference between dominant and subdominant liver, as well as between posterior intestine and middle intestine (p>0.05). The amylase activity is highest in anterior intestine, following in the dominant and subdominant liver, and the amylase in the in anterior intestine is significantly higher than that in two parts of liver (p<0.05). The amylase is lowest in the stomach, which had no significant differences compared to that in posterior intestine and middle intestine (p>0.05). Lipase activity is highest in anterior intestine, following in the dominant liver and posterior intestine, the lipase activity of three above digestive sections with significant differences (p<0.05). Lipase activity is lowest in the subdominant liver, and lipase activity has significant difference among the middle intestine, stomach and subdominant liver (p<0.05), as well as lipase activity between dominant and subdominant liver with significant difference (p<0.05). The highest trypsin activity is found in the anterior intestine, and highest chymotrypsin activity is in the two parts of liver, both digestive enzymes with lowest value in the stomach. Alkaline phosphatase activity is highest in the anterior intestine, following in the middle intestine and posterior intestine, and the activity in anterior intestine is significantly higher than that in the middle and posterior intestine (p<0.05), however with nosignificant difference between middle and posterior intestine (p>0.05). Alkaline phosphatase activity is lower in the stomach, dominant and subdominant liver and above three digestive sections with no significant difference. Leucine aminopeptidase activity is similar to alkaline phosphatase activity, highest activity in the anterior intestine, following in the middle intestine and posterior intestine, lower in the stomach, dominant and subdominant liver, and the activity in anterior intestine is significantly higher than that in the middle and posterior intestine (p<0.05). Leucine aminopeptidase activity of the stomach, dominant and subdominant liver has no significant difference (p>0.05).3. The optimal reaction temperature for pepsin is 30℃, and for protease in the anterior, middle and posterior intestine is 50℃. The optimal reaction temperature in four digestive sections is 30℃. The optimal temperature of lipase is 30℃in stomach,50℃in anterior intestine,40℃in middle and posterior intestine. The optimal pH for pepsin is 2.0, and pH 9.0-10.0 is the optimal pH for intestine protease. The optimal pH for amylase is 6.0 in the stomach, and 7.0 for three parts of intestine. The optimal pH for lipase is 6.0, and 8.0 for three parts of intestine. The thermal stability for gastrointestinal protease is ranged from 20 to 50℃. Pepsin is stable in the pH range 1.0-4.5, and intestine protease is stable in the pH range 7.5-11.0. The best concentration of substrate for pepsin and intestine protease is 2.5% and 2.0% casein, respectively. And the best concentration of substrate for stomach and intestine amylase is 2.5% and 3.0% soluble starch, respectively. Na+ and Hg2+promote pepsin activity, while Cu2+ and Fe3+ inhibit the pepsin activity. Ca2+, Fe2+ and Mg2+promote protease activity of intestine, while Cu2+, Zn2+and Fe3+ inhibit intestine protease activity. The protease activity in the stomach and intestine is inhibited by PMSF, TPCK, TLCK, Pepstatin A andβ-mercaptoethanol.4. Digestive enzymes vary from age. The highest protease and amylase is found the 9 age group, lowest value in 7 and 15 age groups, and the enzymes activity increase first then decrease with age increasing from 3 to 15. Alkaline phosphatase activity vary according to age change is similar to the trend of protease and amylase. The lipase activities are higher in the 3 and 5 age-groups than other age-groups, and the activity decrease with age increasing from 3 to 15. The trend of change of trypsin activity, chymotrypsin activity and leucine aminopeptidase activity also decrease with age increasing from 3 to 15. 5. The descending order of in-vitro protein and dry matter digestibility of crude enzymes of different digestive sections on seven feed ingredients was:stomach, anterior intestine, middle intestine and posterior intestine, and the in-vitro digestibility of crude enzymes on animal feed ingredients was higher than that of plant feed ingredients. The descending order of producing amino acid rate to the enzymolysis reaction by crude enzymes of different digestive sections was anterior intestine middle intestine, stomach and posterior intestine.更多还原