【作者】 王开来；

【导师】 徐永平；

【作者基本信息】 大连理工大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 刺参(Apostichopus japonicus)是我国北方最具经济价值的水产养殖品种之一。近年来,刺参的人工养殖蓬勃发展,但病害问题也日趋严重,成为困扰该产业的发展的瓶颈。抗生素的大量使用虽然在很大程度上达到了防病治病的目的,但是由此带来的负面效应也是显而易见的。因此,如何减少或者不使用抗生素而又使养殖对象保持较高抗病力的健康养殖方式成为现代养殖业的一种新理念。维生素C是一种重要的营养素,为动物生长和维持正常生理机能所必需,但多数水产动物不能自身合成,需要从饲料中获取,并且维生素C对提高免疫系统活力,增强抗病和抗应激能力起到积极作用,因而作为一种免疫增强剂在水产养殖中广泛使用。本课题旨在考察刺参是否具有自身合成维生素C的能力,以及不同剂量的维生素C对刺参生长、免疫和抗病能力的影响,以期探讨维生素C作为刺参免疫增强剂的应用效果,为实现刺参的健康养殖奠定基础。论文主要工作如下:(1)通过测定刺参体内维生素C生物合成途径的关键酶——L-古洛糖酸-γ-内酯氧化酶(GLO)的活力,考察刺参合成维生素C的能力。实验结果表明,刺参体内GLO酶活力极低,约为0.011～0.013μgAA/h·g tissue,仅为大鼠肝脏GLO活性的十万分之一,因而推测刺参自身不能够合成维生素C,其对维生素C的需求只能依靠从外界的食物中摄取。此外,通过分析野生刺参肠道内容物,考察刺参天然饵料中的维生素C含量。结果显示,刺参肠道内容物中的维生素C含量为108±16.0 mg/kg,说明刺参的天然饵料中含有丰富的维生素C,刺参可通过采食天然饵料来获取机体所需的维生素C。(2)通过60 d的养殖实验,考察了以Vc多聚磷酸酯剂型(LAPP)添加的5个维生素C水平:0、500、1500、5000和15000 mg/kg,对刺参稚参(120日龄,1.19±0.46 g)生长、胶原蛋白合成,以及组织维生素C积累量的影响。结果显示,500 mg/kg以上的维生素C可显著提高刺参增重速度和特定生长率(P＜0.05);5000 mg/kg维生素C可显著降低脏壁比,同时促进胶原蛋白合成(P＜0.05);在0～15000 mg/kg剂量范围内,刺参组织中维生素C积累量随饲料维生素C水平增加而递增。因此,以生长、胶原合成和组织维生素C积累量为评价指标,建议维生素C合适添加剂量为5000 mg/kg。(3)考查5个维生素C水平对刺参非特异性免疫和抗病力的影响。结果表明,5000mg/kg维生素C对提高碱性磷酸酶(AKP),酸性磷酸酶(ACP)、吞噬率、呼吸爆发活力作用最明显(P＜0.05);而15000 mg/kg维生素C对提高凝集素效价、酚氧化酶活力以及溶菌酶活力效果显著(P＜0.05);但维生素C对刺参SOD活力的影响不明显。维生素C对多数免疫指标的促进作用在20 d即可达到最高峰,继续投喂会出现免疫耐受现象。对刺参抗病力指标考察发现,1500～5000 mg/kg维生素C可显著提高刺参体表粘液和体腔液的抗菌活力(P＜0.05),5000 mg/kg维生素C能够显著降低灿烂弧菌攻毒后刺参的发病率和死亡率(P＜0.05)。综合以上结果,5000mg/kg维生素C对刺参免疫保护效果最佳,可作为刺参免疫增强剂的最佳添加剂量,其最佳投喂时间为20 d。更多还原

【Abstract】 The sea cucumber Apostichopus japonicus is one of the most valuable mariculture species in north China.In recent years,the rapid expansion and intensification of sea cucumber farming has led to the occurrence of various diseases,causing serious economic losses and becoming one of the limiting factors in the sustainable development of this industry.Although the use of antibiotics has gained certain effects in treating diseases and pathogens,the concomitant negative effects have been discovered evidently.At present,the applications of immunity stimulants to maintaining high resistance to pathogen in aquculture species is a preferred method in modern aquaculture industry.While the vitamin C is one of the essential nutrients which could promote immune response and used extensively as immunity stimulant in many aquculture species.And this study is aimed to observe the effect and requirement of vitamin C on growth,non-specific immunity and resistance in A. japonicus juveniles. An analysis of activity of the emzyme L-gulonol-γ-lactone oxidase(GLO) in bodywall and viscera has shown that the GLO activity in A.japonicus is extremely low,which is 0.011～0.013μgAA/h·g tissue,only one millionth of that in a rat liver.This proves that A.japonicus is unable to de-nove synthesis of ascorbic acid because of lacking the GLO activity;hence its demand for vitamin C can only rely on dietary intake.An analysis of intestinal contents of wild A.japonicus has shown that the vitamin C level in the intestinal contents is quite high,is almost 108mg/kg,which reveals that the natural baits of A.japonicus are rich in vitamin C,and the vitamin C demand of A.japonicus could be satisfied by its feeding behavior.A 60 day feeding trial was carried out to study effect of 5 different dieatary vitamin C level on the growth and non-specific immnity of A.japonicus.Five piratical feeds was desighed to contain 0,500,1500,5000 and 15000 mg/kg vitamin C,with the form of L-ascobyle-polyphosphate(LAPP).The results showed that over 500 mg / kg vitamin C has significantly effect on weight gain and specific growth rate(P＜0.05).5000 mg / kg vitamin C can significantly reduce the vescera-bodywall ratio,accelerate collagen synthesis and improve the alkaline phosphatase(AKP) activity,acid phosphatase(ACP) activity,phagocytic rate and respiratory burst activity(P＜0.05);at the same time,A.japonicus fed with this dietary level also had better resisitance to Vibrio Splendidus challenge and higher bactericidal activity in both coelomic fluid and skin mucus(P＜0.05).While 15000 mg / kg vitamin C has significant positive effect on promoting coelomocyte count,agglutination titre,phenol oxidase activity and the lysozyme activity(P＜0.05).And the tissue ascorbic acid accumulation in A.japonicus has been seen increase with the increment of dietary vitamin C level.But during the whole feed trial,the effect of vitamin C on SOD activity was not obvious.However,the immunity promoting effect of vitamin C concerning most immune parameters could reach the peak in 20 d,and some immune tolerance phenomenon could be observered with continue feeding of vitamin C.Therefore,taking the above parameters of growth,immunity and resistance as evaluation indicators,the suitable vitamin C level in maintain growth is 500 mg/kg and the dietary vitamin C level as immunity stimulant should be over 5000 mg/kg in the form of LAPP.And the immune enhancing effect of dietary vitamin C in A.japonicus could reach its peak within 20 days.更多还原