【作者】 郭占林；

【导师】 赵云龙；

【作者基本信息】 华东师范大学 ， 动物学， 2010， 硕士

【摘要】 红螯光壳螯虾(Cherax quadricarinatus),俗称澳洲淡水龙虾,我国于1992年在广州首次引进试养和养殖。近年来,随着该虾人工育苗技术的突破,成虾养殖规模的扩大,迫切需要了解红螯光壳螯虾的营养需求,但相关的研究资料较少,明显滞后于生产实践。国内外已有的研究多集中在红螯光壳螯虾幼虾对蛋白质、碳水化合物和维生素,或成虾对磷脂、胆固醇及长链多不饱和脂肪酸的需求上,对于红螯光壳螯虾幼体饲料脂肪源需求的研究报道较少。选择合适的脂肪源不仅可以有效提高虾体对饲料营养的利用率,促进个体生长,而且直接关系到幼体的成活率和饲料的生产成本。因此,本文在已有研究工作的基础上,采用电镜技术、生化、酶学等手段,综合组织学、发育生物学和营养学,较为系统地研究了不同脂肪源对红螯光壳螯虾仔虾及幼虾生长、消化酶活性、肝胰腺和腹部肌肉中脂肪酸组成及仔虾肝胰腺亚显微结构的影响比较,以期了解红螯光壳螯虾仔虾及幼虾生长对主要脂质的需求,并为其人工配合饲料的研制、养殖生产中的合理投饵,提供基础资料。主要研究结果如下：不同脂肪源对红螯光壳螯虾幼虾的体长增长率和特殊增长率影响不显著；但增重率各组间存在显著性差异,以豆油组最高,达到2332.93%,花生油组最低,为1839.50%；肝胰腺指数以豆油组显著高于其它各组,为0.75%。仔虾存活率以豆油组最高,花生油组次之,猪油组最低。幼虾存活率以豆油组较高,达到83.3%,鱼油组较低,仅为56.7%。不同的脂肪源对虾体生长指标的影响不尽相同,富含DHA和EPA的鱼油并没有占绝对优势,总体上豆油组表现出更高的饲料利用率,生长情况更好。采用生物化学的方法分别测定了红螯光壳螯虾仔虾和幼虾投喂不同脂肪源饲料后,肝胰腺消化酶：胃蛋白酶、脂肪酶、淀粉酶及纤维素酶的比活力。四种消化酶活性在不同组间表现出不同差异,体现了红螯光壳螯虾仔虾和幼虾对不同脂肪源饲料产生了不同的适应。不同脂肪源对仔虾的胃蛋白酶活力没有显著影响,但以豆油组的酶活最高；鱼油和豆油组的脂肪酶活力达到花生油和猪油组的2-3倍,且以鱼油组最高；不同脂肪源对淀粉酶活力影响更为显著,以猪油组>花生油>豆油组>鱼油组；纤维素酶在仔虾中表现出极低的活力,豆油和猪油组活力较高,为0.11μg/min,花生油组较低。结果表明,对于红螯光壳螯虾仔虾而言,对蛋白质和脂质的需求和利用高于碳水化合物,在养殖初期应该注重添加更多的蛋白。不同脂肪源对幼虾肝胰腺的消化酶活力在胃蛋白酶水平上无显著影响；脂肪酶活力花生油组显著高于其它实验组,为1177.23U/gprot;淀粉酶活力各组间差异显著,以鱼油组>对照组>豆油组>猪油组>花生油组；纤维素酶活力以花生油组较高,为61.14μg/min。结果暗示了使用植物油做脂肪源对红螯光壳螯虾幼虾的脂肪代谢具有促进作用,且豆油优于花生油,可以考虑将更多富含淀粉的成分添加到红螯光壳螯虾幼虾的饵料中来提高其利用率并降低饲料成本,但对糖类物质的有效利用率仍有待进一步研究。同一种脂肪源饲料对仔虾和幼虾的消化酶活力产生了不同影响。仔虾肝胰腺的胃蛋白酶活力高于幼虾；淀粉酶和纤维素酶活力明显低于幼虾。通过仔虾和幼虾消化酶活力的变化可以看出,红螯光壳螯虾仔虾在刚脱离母体时食性偏动物食性,随着个体的生长,逐渐向偏植食性转变。可见,在幼体的不同阶段也应可以根据其需求投喂合适成分的饲料,从而提高饲料效率。采用生物化学方法研究了不同脂肪源饲料对红螯光壳螯虾仔虾和幼虾肝胰腺和腹部肌肉中脂肪酸的组成,并通过透射电镜技术对不同饲料组仔虾肝胰腺的超微结构进行了比较。投喂不同脂肪源饲料,在红螯光壳螯虾仔虾的肝胰腺和腹部肌肉中共检测出16-22种脂肪酸。其中肝胰腺多不饱和脂肪酸以亚油酸(C18：2n6)为主,腹部肌肉中以亚油酸(C18：2n6)和EPA(C20：5n3)为主,此结果反映了仔虾生长期肝胰腺和肌肉的脂肪酸组成和利用特点。肌肉中多不饱和脂肪酸含量多于肝胰腺,说明红螯螯光壳螯虾仔虾期的脂肪酸尤其是亚油酸、亚麻酸和EPA等重要的多不饱和脂肪酸被优先转运到肌肉等组织中,供应其生长所需要的能量代谢。富含亚油酸的豆油既能促进脂肪酸代谢又能促进各种消化酶的活性,对红螯光壳螯虾仔虾的发育具有优良的脂肪酸价值。不同饲料组红螯光壳螯虾仔虾肝胰腺的细胞类型和结构基本相同，分四种类型：E、B、R和F细胞,但R细胞(Resorptive cell储存细胞)的超微结构发生显著变化。猪油组细胞膜的缺陷表明以猪油作为脂肪源不能满足红螯光壳螯虾仔虾生长的营养需要,导致代谢障碍,进而影响其存活率；豆油组仔虾肝胰腺的R细胞中含有许多脂肪滴的脂质储存,从而进一步为该组较高的存活率提供了依据。幼虾肝胰腺中饱和脂肪酸含量以猪油组显著高于其它组,与腹部肌肉中情况相似；单不饱和脂肪酸以花生油组含量最高,豆油组最低,而腹部肌肉中以动物油组高于植物油组；多不饱和脂肪酸则以豆油组含量最高,猪油组最低,这也与腹部肌肉相似。肝胰腺中EPA含量以鱼油组最高,为5.89%,花生油组次之,豆油和猪油组含量较低,仅为2.52%和2.77%；DHA以花生油组含量最高达5.62%,商业对照组次之,显著高于其它各组含量。腹部肌肉中亚油酸(C18：2n6)和亚麻酸(C18：3n3)含量均以豆油组显著高于其它四组,亚油酸在总脂肪酸中的比例最高,这与与肝胰腺一致。肝胰腺中实验组与对照组∑n-3脂肪酸含量均低于∑n-6脂肪酸含量,与肌肉中二者含量相反。亚油酸大量积累,在体成分组成上反映出红螯光壳螯虾幼虾生长的脂肪酸需求和利用的特点。研究结果表明,饲料中添加一定量的豆油作为脂肪源能够促进红螯螯虾幼虾体内∑n-3和必需脂肪酸的积累,较好地促进幼虾的正常生长,提高其存活率。更多还原

【Abstract】 Red claw crayfish (Cherax quadricarinatus) which was also called Australian freshwater crayfish was fisrt introduced to Guangzhou, China in 1992. It subordinated to Crustacea, Decapoda, Pleocyemata, Astacidea, Parastacoidea, Parastacidae, Cherax. Recent years, as the cultivation scale increased, it is urgent for us to know the nutrients need of crayfish, but little studies were focused on it. More studies were focused on the protein, carbon componds and vitamins needed for juvenile crayfish, or lecithoid, cholesterol and long-chain PUFA needed for adult crayfish. Choosing the right lipid sources for culturing can not only sufficiently enhance the growth and food utilization rate, but also reduce the cost and improve the surival rate of juvenile individuals. Based on the formal studies, in order to know the lipid need of juvenile crayfish, this study was conducted to evaluate the effects of dietary lipid sources (peanut oil, pork lard, fish oil and soybean oil) on growth, digestive enzyme activity, fatty acid composition and ultrastructure of hepatopancreas in post-larval and juvenile red claw crayfish using the methods of electron microscope, histology and enzymology. The data obtained would provide valuable information to the development of artificial diets for Cherax quadricarinatus.Using the biological measuring methods, we compared the length growth rate, special growth rate, weight gain, hepatopancreatic index and survival rate of red claw crayfish fed by different lipid sources. There was no significant effects (P> 0.05) on length growth rate and special growth rate among all treatments. However, the treatment with soybean oil had significantly (P< 0.05) higher weight gain (2332.93%) and hepatopancreatic index (0.75%) than other groups. But the lowest weight gain (1839.50%) was found in peanut oil group. Different lipid sources had different effects on growth indexs of red claw crayfish. Totally, the crayfish can better utilized the food with soybean oil, although fish oil was abundant in DHA and EPA. We used the biochemical methods to measure the effects of different lipid sources on digestive enzyme activity (pepsin, lipase, amylase and cellulase) in hepatopancreas of post-larval and juvenile red claw crayfish. They showed different digestive enzyme activity to different lipid sources. The result reflected that post-larval and juvenile red claw crayfish had a different adaptation to different diets.Analysis of digestive enzyme profiles from the hepatopancreas revealed a positive correlation between lipase, amylase, cellulase activities and the diet lipid sources, although there was no significantly different (P> 0.05) in pepsin activity. The treatment with fish oil had the highest lipase activity, and together with soybean oil, the lipase activity is 2-3 times higher than that of peanut oil and pork lard groups, but still lower than commercial group. Amylase activity was highest in pork lard group, but lowest in fish oil treatment. Crayfish larva showed low cellulase activity in any group, soybean oil and pork lard groups had higher activity (0.11μg/min) than other lipid sources. The results suggested that the post-larval red claw crayfish needed more protein and lipid than carbohydrate. So, we should add more protein to their food in the early days of culturing.Different lipid sources had no significant effects on pepsin activity of juvenile crayfish. Peanut oil group had the highest lipase activity (1177.23U/gprot). Amylase activity was significantly (P< 0.05) different in different diets, and fish oil group has the highest activity. Cellulase activity is higher in peanut oil group (61.14μg/min) than other groups. The result implied that vegetable oil had a role in promoting fat metabolism to juvenile red claw crayfish, and the soybean oil is better than peanut oil. So, maybe we could add more starch ingredients to the diet of juvenile crayfish, which can improve diet’s utilization and reduce the cost. However, the effective utilization of the carbohydrate needs further study.The same lipid source had a different effect on post-larval and juvenile red claw crayfish. Post-larval crayfish had a higher pepsin activity than juvenile crayfish, but amylase and cellulase activity were significantly lower in post-larval crayfish. Through the change of activity of digestive enzyme, we can see that feeding habits is partial animal feeding in post-larval crayfish but partial plant feeding with the growth of individual. So, larva should be fed diets containing suitable ingredients at different stages of individual to improve utilization of diets.We researched the effect of different lipid sources on fatty acid composition of hepatopancreas and tail muscle in post-larval and juvenile red claw crayfish using biochemistry methods. Fatty acids composition including Saturated fatty acids (SFA), Mono-unsaturated fatty acids (MUFA) and Poly-unsaturated fatty acids (PUFA) of in tail muscle and hepatopancreas were affected by different lipid sources. Meanwhile, we compared the ultra structure of hepatopancreas in post-larval crayfish fed different diets.Fed different lipid sources,16-22 fatty acids were detected in tail muscle and hepatopancreas. Polyunsaturated fatty acids is mainly based on linoleic acid (C18: 2n6) in hepatopancreas, but linoleic acid (C18:2n6) and EPA (C20:5n3) in tail muscle. The result reflected composition and using feature in post-larval crayfish growth. Content of polyunsaturated fatty acids in tail muscle were higher than hepatopancreas, which suggested that fatty acids, especially linoleic acid (C18:2n6), linolenic acid (C18:3n3) and EPA (C20:5n3), were transported to muscles first to supply energy metabolism require for growth. Soybean oil can promote not only fatty acids metabolism but also digestive enzyme activities, because of rich in linoleic acid (C18: 2n6) and good value of fatty acids.The ultra structures of hepatopancreas in post-larval crayfish were studied with electron microscopes. Cell type in hepatopancreas epithelium was same fed different diets, classified into E-cell, B-cell, R-cell and F-cell based on structure and function. But ultra structure of R-cell (Resorptive cell) was affected significantly in different treatment. Defect of membrane in R-cell of pork lard group suggested that as lipid source, pork lard couldn’t meet growing nutritional needs of post-larval red claw crayfish. Pork lard might lead to metabolic disturbances, and thereby affecting the survival rate. Rich store of lipid droplets in R-cell of soybean oil group was the reason for high survival rate.The content of fatty acids in tail muscle and hepatopancreas was obviously affected by the variety and content of fatty acids in dietary. Saturated fatty acid content in hepatopancreas to lard group was significantly higher than other groups, which is similar in the tail muscle. Monounsaturated fatty acid had the highest content to peanut oil group, and lowest to soybean oil group, but its content was higher in two animal oil groups than two plant oil groups. The content of linoleic acid (C18:2n6) and linolenic acid (C18:3n3) were highest in soybean oil group than all other four groups in tail muscle, which was consistent with hepatopancreas. Accumulation of linoleic acid showed needs and utilization feature of juvenile crayfish. The result suggested adding moderate soybean oil as lipid source could promote the accumulation of essential fatty acids, and improve growth and survival rate.更多还原