【作者】 卢素芳；

【导师】 何瑞国； 熊远著；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 磷脂是一种生物活性物质,具有独特的理化性质和营养价值,其应用前景十分广阔。虽然已有研究报道磷脂在水产动物幼体饲料中具有潜在的重要作用,但目前关于水产动物幼体需要磷脂的原因缺乏了解。本论文通过系统研究在黄颡鱼仔稚鱼人工饲料中添加磷脂对其生长、发育、存活、耐受力、脂类代谢、抗氧化功能及肠道和肝脏组织学结构的影响,从宏观和微观初步探讨仔稚鱼对磷脂需求的机理,为磷脂在仔稚鱼人工饲料中的应用开发提供参考。1黄颡鱼早期发育阶段受精卯和鱼体脂类及脂肪酸组成变化目的:研究黄颡鱼受精卵孵化期间和仔鱼发育阶段磷脂、脂肪含量和脂肪酸的组成变化规律。方法:采用常规化学分析方法和气相色谱法对黄颡鱼从鱼卵受精开始至仔鱼孵化后未投饵的7d内的脂类含量和脂肪酸组成进行测定。结果:受精卵在整个孵化期间脂肪含量有下降趋势,而磷脂含量在受精16h后略有增加。受精卵中不饱和脂肪酸含量大于饱和脂肪酸含量。受精卵在整个孵化期间各种脂肪酸含量无明显变化。仔鱼孵化后,鱼体总脂肪和磷脂含量急剧下降,总脂含量从0日龄的4.57%降低到7日龄的0.75%,磷脂含量则从1.32%下降到0.14%。进一步分析发现,0～5日龄,鱼体总脂肪含量的下降幅度明显大于磷脂。仔鱼在饥饿期间鱼体脂肪酸组成发生明显变化,单不饱和脂肪酸含量下降最为明显,尤其是C18:1。仔鱼在饥饿期间,脂肪酸按n-9＞n-6＞n-3顺序被先后利用,黄颡鱼仔鱼发育阶段主要以单不饱和脂肪酸作为能量代谢基质,而C20:4n6（AA）和C22:6n3（DHA）优先于C20:5n3（EPA）被保存下来。结论:磷脂是黄颡鱼仔稚鱼阶段代谢的重要能源,是仔稚鱼正常生长和发育所必需的重要营养素,仔稚鱼对磷脂的需求量很大。2人工饲料中添加磷脂对黄颡鱼仔稚鱼生长、存活及耐受力的影响目的:研究人工微粒饲料中添加不同水平磷脂对黄颡鱼仔稚鱼生长、存活及耐受力的影响。方法:通过生长试验,选取同批孵化的黄颡鱼仔鱼,随机分成5组,每组4个重复,每个重复10000尾鱼,其中四组饲喂人工饲料,另外一组饲喂轮虫,作为对照组,进行饲养试验,试验期21d。试验人工饲料均含94%的同一种基础饲料和6%脂肪源,脂肪源由不同添加水平的大豆磷脂（0%、2%、4%、6%）和鱼油、豆油（2:1）（6%、4%、2%、0%）构成。试验结束后,测定各试验组仔稚鱼生长性能、存活率及耐受力。结果:日粮磷脂含量显著影响黄颡鱼仔稚鱼生长（p＜0.05）。各组黄颡鱼的体重和全长随饲料中磷脂添加量的提高呈上升趋势,其中以添加6%磷脂的人工饲料组的仔稚鱼生长最佳,和轮虫组饲喂效果相当。14日龄和21日龄时,磷脂对黄颡鱼肥满度变化的影响均未达到显著水平（p＞0.05）。在本试验条件下饲喂人工饲料的黄颡鱼存活率都比较高,达到90%以上,除PL0组外,各组差异不显著。未添加磷脂组黄颡鱼的存活率低于其它各组且随着饲料中磷脂水平的增加有提高的趋势。7～14日龄PL6组和轮虫对照组黄颡鱼仔稚鱼的日增重、体长相对增长率、体重相对增长率、特定生长率显著高于其它试验组（p＜0.05）,而PL0组最低（p＜0.05）。14～21日龄各组日增重较7～14日龄高,仍以PL6组日增重最明显,而轮虫对照组此阶段日增重与7～14日龄期间相比却增长不多。14～21日龄阶段各组黄颡鱼体长相对增长率、体重相对增长率、特定生长率均比7～14日龄有所下降,尤其以轮虫对照组下降最为明显。但PL0组体重增长率和特定生长率却显著高于其它各组（p＜0.05）。从整个试验期来看,黄颡鱼的日增重、体长相对增长率、体重相对增长率、特定生长率均随饲料中磷脂水平的上升而提高。黄颡鱼仔稚鱼体重与体长的回归方程符合鱼类生长的一般模式。各组黄颡鱼仔稚鱼体重与体长关系式指数b在2.62～2.77之间。黄颡鱼仔稚鱼饲料系数随磷脂添加水平的增加呈下降趋势。磷脂对提高黄颡鱼仔稚鱼耐受力的影响效果不是很明显。结论:磷脂能有效促进黄颡鱼仔稚鱼生长,提高饲料效率,增加存活率。3人工饲料中添加磷脂对黄颡鱼仔稚鱼鱼体脂类和脂肪酸组成的影响目的:研究饲料中不同脂肪源及磷脂添加水平对黄颡鱼脂质代谢特别是脂肪酸相对含量的影响,进一步探讨磷脂促生长作用机理。方法:试验设计与第2部分相同,饲养结束后,测定各试验组黄颡鱼鱼体脂类成分及脂肪酸组成。结果:各组饲料在稳定性试验前后其脂肪酸组成并无明显增减变化。磷脂对人工饲料在水中的脂肪酸稳定性无显著影响。随饲料中磷脂添加量上升和日龄增长,黄颡鱼鱼体总脂肪含量有升高的趋势。PL4、PL6组鱼体脂肪含量显著高于PL0、PL2组（p＜0.05）。21日龄鱼体脂肪含量以PL6组最高,显著高于其它各组（p＜0.05）,其他两组间差异不显著（p＞0.05）。饲料中添加不同水平的磷脂对14日龄黄颡鱼鱼体磷脂含量有影响,14日龄时,PL4、PL6与轮虫对照组间鱼体磷脂含量差异不显著（p＞0.05）,均显著高于PL0组（p＜0.05）。21日龄各组间鱼体磷脂含量无显著性差异（p＞0.05）。21日龄与14日龄相比,PL0组鱼体磷脂含量增加幅度显著高于其他组（p＜0.05）。14日龄时,鱼体C18:2n6c、C18:3n6、C18:3n3、PUFA和n-6PUFA含量各组间有显著差异（p＜0.05）,且随磷脂添加水平增加而增加。PL6组EPA和DHA含量显著高于PL0组（p＜0.05）。鱼体的C18:0、SFA含量随磷脂添加而显著降低。21日龄时,鱼体的C18:2n6c、PUFA和n-6PUFA含量在添加磷脂组仍是显著升高（p＜0.05）,与此相反,C16:0、SFA含量降低。不同日龄鱼体脂肪酸组成比较,C18:2n6c、C18:3n3、EPA、DHA、PUFA和n-3PUFA含量有随日龄增长而呈上升的趋势,而C16:0、C20:4n6和SFA含量呈进一步降低的趋势。轮虫对照组鱼体脂肪酸组成与人工饲料各组比较,其C16:0、C18:0、EPA和SFA含量较高,C18:2n6c、DHA和PUFA含量相对较低,与轮虫中相应脂肪酸的含量存在着较明显的平行变化趋势。结论:饲料中添加磷脂,能够促进黄颡鱼仔稚鱼体内脂类沉积,改善饲料中的EFA的有效吸收与利用。4人工饲料中添加磷脂对黄颡鱼仔稚鱼抗氧化能力及ATP酶活性的影响目的:研究饲料中添加磷脂对黄颡鱼仔稚鱼抗氧化损伤能力及某些重要代谢酶的影响,以进一步探讨磷脂对仔稚鱼生长影响的机制。方法:试验设计与第2部分相同,饲养结束后,测定各试验组黄颡鱼鱼体中抗氧化酶活性及ATP酶活性。结果:随着饲料中磷脂水平的提高,黄颡鱼鱼体中的MDA含量逐渐下降,SOD、CAT和GST活性相应得到提高,GSH-PX活性也是以磷脂组显著高于未添加磷脂组（p＜0.05）,其中磷脂添加水平为4%时GSH-PX活性达到峰值,当磷脂添加水平达到6%时,GSH-PX活性不再提高。21日龄各组黄颡鱼鱼体中的SOD、CAT、GST、GSH-PX活性比14日龄时有所上升,而MDA含量进一步下降。添加适量磷脂可显著提高黄颡鱼Na⁺、K⁺-ATPase和Ca²⁺-ATPase活性。14日龄和21目龄时,添加磷脂组黄颡鱼Na⁺K⁺-ATPase和Ca²⁺-ATPase活性均显著高于未添加磷脂PL0组（p＜0.05）,其中以PL4组ATPase活性最高,而PL0组ATPase活性最低。不同日龄黄颡鱼ATPase活性比较,21日龄人工饲料各组鱼Na⁺K⁺-ATPase和Ca²⁺-ATPase活性比14日龄略有下降。结论:磷脂能够维持黄颡鱼仔稚鱼细胞膜的完整性,降低自由基对生物膜的过氧化损伤,提高机体抗氧化系统的活力和ATP酶活性。5饲料中不同磷脂添加水平对黄颡鱼仔稚鱼前肠和肝脏组织学的影响目的:研究在实际配方的人工微粒饲料中添加商品大豆磷脂对黄颡鱼仔稚鱼消化肠道肝脏器官发育及脂肪消化吸收的影响,进一步从组织学消化生理角度揭示磷脂对仔稚鱼促生长作用机理。方法:试验设计与第2部分相同,分别于14日龄和21日龄取样,制作组织切片,用病理成像系统和光学显微镜进行观察分析。结果:14同龄时PL0组黄颡鱼稚鱼前肠出现明显脂质沉积,细胞浆内有大量脂肪泡,肝脏固有结构不清,肝细胞索结构紊乱,肝窦消失,肝细胞肿胀,细胞浆呈空泡状。随着饲料中油脂添加量减少、磷脂添加量的增加,黄颡鱼稚鱼前肠粘膜上皮细胞中脂滴积聚现象逐渐减轻,杯状细胞数目增多,肝细胞空泡化程度降低。当饲料中添加6%磷脂时,黄颡鱼前肠上皮细胞和肝脏形态较正常,和轮虫组类似。21日龄黄颡鱼肠道和肝脏脂肪沉积程度较14日龄降低,肠粘膜上皮空泡化现象减少或消失,杯状细胞数目明显增多,肝脏脂肪沉积减少。结论:磷脂能减少黄颡鱼仔稚鱼前肠和肝脏脂肪积累,增强脂肪转运,促进仔稚鱼消化系统在发育过程中向成熟模式转化。随黄颡鱼仔稚鱼日龄增长,鱼体自身合成磷脂能力增强,磷脂作用随年龄增加而减弱,黄颡鱼对磷脂的需要量也随之降低。更多还原

【Abstract】 Phospholipid（PL） is a bioactive compound,has a broad application prospect because of its unique physico-chemical property and nutritional value.Dietary PL supplementation has potential importance for the formulation of practical larval diets,but the origin of the requirement is still unclear.The study was conducted to investigate the effects of PL supplementation in formulated microdiets on growth,survival,stress resistance,lipid metabolism,antioxygen ability and foregut and liver histological changes of Pelteobagrus fulvidraco larvae.This investigation was aimed to reveal the mechanisms of the PL requirement from macroscopic and microcosmic aspects,which can provide information on the practical use of PL in larval microdiets.1 Changes in lipids and fatty acid composition during development in Pelteobagrus fulvidraco fertilized eggs and larvaePurpose:To establish the changes which during embryogenesis and early larval development,fertilized eggs and larvae of Pe#eobagrus fulvidraco were examined for lipids and fatty acid compositions.Method:The lipids content and fatty acid composition in the fertilized eggs and larvae which did not feed for 7 days after hatching were analyzed by means of common chemical assay and gas chromatography（GC）.Result: The lipid content of fertilized eggs tended to increase during hatching period,while the PL content increased a little from 16h after fertilization.Fertilized eggs contained more polyunsaturated fatty acids（PUFA） and monounsaturated fatty acids（MUFA） than saturated fatty acids（SFA）.There was no significant change in the proximate fatty acid composition of eggs during hatching.The lipid and PL content of larvae decreased sharply after hatching.Total lipid decreased from 4.57%on day 0 to 0.75%on day 7 and PL content decreased from 1.32%to 0.14%.A further analysis showed that the reduction in lipid content of larvae was significantly larger than in the PL content from day 0 to day 5.The fatty acid composition in starved larvae changed significantly as starvation progressed.In starved larvae the MUFA content decreased significantly and the greatest decrease occurred in the C18:1 content.In this regard the degree of decreases was,n-9＞n- 6＞n-3,suggesting that MUFA were a major energy source for starving Pelteobagrus fulvidraco larvae,DHA and AA were conserved in preference to EPA.Conclusion:PL is a necessary nutrient for normal growth and development of larvae which served as an important energy source for metabolism during larval period.The larval fish have a huge PL requirement.2 Effect of phospholipid supplementation in formulated microdiets on growth,survival and stress resistance of Pelteobagrus fulvidraco larvaePurpose:The effect of supplementation of soybean PL to Pelteobagrus fulvidraco larvae diets on growth survival and stress resistance were investigated.Method:All Pelteobagrus fulvidraco larvae obtained following artificial fertilization were randomly divided into 5 groups with 4 replicates of 10000 larvae each.The larvae were fed from day 3 to 21 posthatch with four diets containing the same basal diet,coated with different lipid fractions（6%diet）.The lipid fractions consisted of increasing levels of soybean PL （0,2,4 or 6%of diet） and decreasing levels of mixed oil（fish oil:soybean oil,2:1）.A group of larvae was fed rotifers as a control.The growth performance,survival rate and stress resistance ability were determined after the experiment.Results:the PL content of microdiets significantly influenced the performance of larvae（p＜0.05）.The body weight and total length of larvae increased as a result of PL supplementation.Larvae fed diet supplemented with 6%PL exhibited the best growth performance and similar to those fed rotifers.The condition factor of larvae was not significantly affected by dietary PL level on day 14 and day 21（p＞0.05）.Survival was high and above 90%at the end of the feeding trial in all groups and did not differ significantly among the groups,except for group PL0.Survival of fish fed the diet with no supplementation of PL was lower than the other groups and tended to increase with increasing dietary PL levels.Larvae fed PL6 diet and rotifers had significantly higher average daily gain（ADG）,length gain rate（LGR）, weight gain rate（WGR） and specific growth rate（SGR） than those fed the other diets （p＜0.05） and larvae fed PL0 diet was the lowest（p＜0.05） during days 0-7.The ADG of larvae in each group during days 14-21 was higher than that during 0-7days.The larvae in group PL6 still had the highest ADG,while the ADG of larvae fed rotifers increased little during days 14-21.The LGR,WGR and SGR of larvae in each group were lower during days 14-21 than those during days 7-14 and those of the larvae in control group fed rotifers decreased significantly.The WGR and SGR of larvae in group PL0 were significantly higher than those in the other groups（p＜0.05）.During the whole test period, the ADG,LGR,WGR and SGR of larvae all increased with increasing dietary PL levels. The length-weight relation of Pelteobagrus fulvidraco larvae was consistent with the general growth model of fish.The index b of length-weight relation equation varied from 2.62 to 2.77.The feed coefficient（FC） of larvae tended to decreased with the increasing dietary PL levels.The stress resistance ability of larvae appears to be less affected by the dietary PL supply.Conclusion:PL had a growth-promoting effect on Pelteobagrus fulvidraco larvae,improved feed efficiency and enhanced survive rate.3 Effect of phospholipid supplementation in formulated microdiets on lipids and fatty acid composition of Pelteobagrus fulvidraco larvaePurpose:This study was undertaken to assess the effect of different lipid sources and dietary PL supplementation on lipid metabolism especially fatty acid composition of Pelteobagrus fulvidraco larvae and further investigated the mechanism of the growth-promoting effect of PL.Method:According to the design of part 2 above,the lipids content and fatty acid compositions in whole body of larval fish in each treatment were analyzed after feeding.Results:Dietary fatty acid loss or gain did not change significantly after the stability test.The fatty acid compositions of microdiets leached into the water during the stability test was not affected by dietary PL supplementation.The total lipid content of larvae tended to increased as a result of PL supplementation and the fish aged.The total lipid contents of group PL4 and PL6 were significantly higher than that of group PL0 and PL2（p＜0.05） and group PL6 was highest on day 21,though the difference was not significant from the other groups（p＞0.05）.The PL content of larvae was affected by dietary PL on day 14.The difference of PL content among group PL4, PL6 and control group fed rotifers was not significant（p＞0.05） and significantly higher than that in group PL0（p＜0.05）.The PL content of larvae did not differ significantly among the groups on day 21（p＞0.05）.Compared with day 14,the increase of PL content in group PL0 was significantly higher than that in the other groups on day 21（p＜0.05）. On day 14,the contents of C18:2n6c,C18:3n6,C18:3n3,PUFA and n-6PUFA showed significant difference（p＜0.05） and increased with increasing the dietary PL levels.The contents of DHA and EPA in group PL6 were significantly higher than those in group PL0 （p＜0.05）.The contents of C18:0 and SFA decreased significantly as a result of PL supplementation.On day 21,C18:2n6c,PUFA and n-6PUFA contents of larvae fed the diets with supplementation PL were increased significantly,while the 16:0 and SFA contents decreased.Comparison of fatty compositions of larvae at different ages of each group,the contents of C18:2n6c,C18:3n3,EPA,DHA,PUFA and n-3PUFA increased, while C 16:0,C20:4n6 and SFA decreased as the fish aged.The contents of C 16:0,C 18:0, EPA and SFA in control group fed rotifers were higher than those in groups fed microdiets,and C18:2n6c,DHA and PUFA were lower.The fatty acid compositions of larvae fed rotifers were characterized by the specific fatty acids related to the rotifer. Conclusion:The supplementation of PL to the diet could increased lipid retention in larval fish and enhanced the absorption and utilization of dietary EFA.4 Effect of phospholipid supplementation in formulated mierodiets on antioxygen ability and ATPase activity of Pelteobagrus fulvidraco larvaePurpose:This study was undertaken to assess the effect of dietary PL supplementation on antioxygen ability and ATPase activity of Pelteobagrus fulvidraco larvae and further investigated the mechanism of the growth-promoting effect of PL. Method:According to the design of part 2 above,antioxygen ability and ATPase activity of larval fish in each treatment were analyzed after feeding.Results:The content of MDA in larvae decreased and SOD,CAT and GST activities increased with increasing the dietary PL levels.The GSH-PX activity of larvae fed the diets with supplementation PL were higher than the PL0 group（p＜0.05）.The GSH-PX activity was the highest in the group fed 4%PL and did not improved by the addition of 6%PL.The SOD,CAT,GST and GSH-PX activities of larvae increased while the content of MDA decreased on day 21 compared with day 14.The addition of PL improved Na⁺,K⁺-ATPase and Ca²⁺-ATPase activities of larvae.The Na⁺,K⁺-ATPase and Ca²⁺-ATPase activities of larvae fed the diets with supplementation PL were significantly higher than the PL0 group fed the diet with no supplementation PL on day 14 and day 21（p＜0.05）.The ATPase activity of group PL4 was the highest and group PL0 was the lowest.Comparison of ATPase activities of larvae at different ages of each group,The Na⁺,K⁺-ATPase and Ca²⁺-ATPase activities of larvae fed microdiets decreased a little on day 21 compared with day 14.Conclusion:PL could maintain cellular membrane integrity,induce the oxidative damage to biomembrane by free radicle and improve antioxygen ability and ATPase activity of larval fish.5 Effect of phospholipid supplementation in formulated microdiets on foregut and liver histological changes of Pelteobagrus fulvidraco larvaePurpose:This study was undertaken to determine the effect of commercial PL supplementation in formulated microdiets on foregut and liver histological changes of Pelteobagrus fulvidraco larvae,which can further investigated the mechanism of the growth-promoting effect of PL with the study of histology and digestive physicalogy. Method:According to the design of part 2 above,Larvae were sampled from each group on days 14 and 21 for make tissue sections.The tissue sections were observed and analyzed by histological image system and light microscopy.Results:On day 14,larvae fed PL0 diet showed steatosis in the intestinal mucosa with a high incidence of lipid vacuoles and the hepatic tissue was in disorder with blurred hepatic trabecula,swollen hepatocytes contained numerous lipid cacuoles.The degree of lipid accumulation decreased and the number of goblet cells increased in the enterocytes of the anterior intestine with decreasing dietary oil supplementation level and with increasing PL supplementation.The enterocytes and hepatic tissue of larvae fed PL6 diet showed normal morphology,which similar to those fed rotifers.The degree of lipid accumulation in the anterior intestine and in the liver on day 21 was lower than that on day 14.The number of vacuoles in the anterior intestine decreased while goblet cells appeared more numerous.A considerable reduction of the vacuolization was also observed in the liver of fish fed the different experimental diets.Conclusion:The addition of PL to the diet caused a reduction in the degree of lipid accumulation,enhancing lipid transport and promoting the maturation of the digestive organs of larvae.As the fish aged,the ability of PL synthesis was enhanced and the effect of PL decreased,thus the PL requirement of fish decreased with age or development stage.更多还原