【作者】 张国斌；

【导师】 龚世园；

【作者基本信息】 华中农业大学 ， 水产养殖， 2009， 博士

【摘要】 本文以胭脂鱼（Myxocyprinus asiaticus）幼鱼为试验对象,研究涉及营养学和免疫学两部分,第一部分探讨了胭脂鱼幼鱼饲料中适宜的蛋白质需求量,第二部分探讨了3种中草药提取物的组方（包括蜂胶配淫羊藿、黄芪配甘草、陈皮）作为免疫增强剂对胭脂鱼幼鱼非特异性免疫力和抗病力的影响。主要研究结果如下:（1）饲料蛋白水平对胭脂鱼幼鱼生长和体组成的影响:以体重（13.5±1.1）g的胭脂鱼为试验对象,以白鱼粉作为主要蛋白源,按6个蛋白质水平梯度（30%、34%、38%、42%、46%、50%）设计等能的饲料,进行8周的饲养试验,探讨胭脂鱼幼鱼适宜的蛋白质需求量。结果显示,饲料蛋白质水平对增重率（WG）和特定生长率（SGR）的影响基本均呈先升后降的变化趋势,WG和SGR最大值均出现在蛋白质含量46%的饲料组,分别为（187.70±8.01）%和（1.89±0.11）%/天,与蛋白质含量42%及以下的饲料组比较有显著性差异（P＜0.05）,与蛋白质含量50%的饲料组间没有显著差异;根据折线模型计算出折点对应SGR的值为1.87%/天,对应的饲料蛋白质含量为46.5%;各组成活率（Survival）变化范围在71%～90%,不同饲料蛋白质含量对成活率无明显影响;随饲料蛋白质含量的增加,饲料系数（FCR）大体呈下降的趋势,最小值出现在46%饲料组;蛋白质效率（PER）和蛋白质利用率（PPV）均呈现先升高后降低的趋势,最大值均出现在46%饲料组;鱼体和肌肉粗蛋白的含量均随饲料蛋白质水平的升高而呈显著增加（P＜0.05）;随饲料蛋白质含量的增加,肥满度（CF）、肝体指数（HSI）、脏体指数（VSI）均呈大体升高的趋势。结果表明,本试验条件下,胭脂鱼幼鱼饲料适宜蛋白质含量为46.5%。（2）蜂胶配淫羊藿对胭脂鱼幼鱼非特异性免疫机能和抗病力的影响:醇提法制备中草药蜂胶提取物（总黄酮含量约37.1%）和淫羊藿提取物（总黄酮含量约42.6%）,二者按生药重量比3:1合用,按质量分数0%（对照组）、0.1%、0.5%和1.0%添加于基础饲料成鳗粉中,饲喂胭脂鱼幼鱼,分别于第10 d、20 d、30 d、40 d、50 d采样,不连续密度梯度分离法制备头。肾巨噬细胞,检测细胞免疫指标:采用中性红吞噬试验检测吞噬活性,流式细胞术检测呼吸爆发活力;取血清,检测体液免疫指标:利用溶壁微球菌（Micrococcus lysoleikticus）检测溶菌酶活性,利用嗜水气单胞菌（Aeromonas hydrophila）检测杀菌活性,利用兔血红细胞检测替代补体溶血活性(ACH₅₀)。结果显示,蜂胶配淫羊藿0.5%剂量组在第40 d和第50 d、1.0%剂量组在第30 d的头肾巨噬细胞吞噬活性均显著高于对照组（P＜0.05）;0.5%剂量组在第30 d和40 d的头肾巨噬细胞呼吸爆发活力与对照组比较均有显著性升高（P＜0.05）;0.1%剂量组在第40 d和第50 d、0.5%剂量组在第20 d和第40 d、1.0%剂量组在第10 d的血清溶菌酶活性均显著高于对照组（P＜0.05）;第40 d,0.5%剂量组的杀菌活性与对照组比较有显著升高（P＜0.05）;蜂胶配淫羊藿对ACHso则无明显影响。饲喂50 d后,各组随机取20尾鱼,用A.hydrophila腹腔注射进行攻毒试验,探讨蜂胶配淫羊藿对胭脂鱼幼鱼抗病力的影响,结果显示1周内对照组的累积死亡率为75%,1.0%和0.1%剂量组的累积死亡率分别为45%和55%,0.5%剂量组的累积死亡率40%则显著低于对照组（P＜0.05）。表明蜂胶配淫羊藿可开发为一种新型的免疫增强剂,饲料中适宜添加剂量为0.5%。（3）黄芪配甘草对胭脂鱼幼鱼非特异性免疫机能和抗病力的影响:醇提法制备黄芪提取物（黄芪多糖含量约为38.4%）和甘草提取物（甘草酸含量约为31.2%,总黄酮约为20.5%）,二者按生药重量比10:6合用,按质量分数0%（对照组）、0.1%、0.5%和1.0%添加于成鳗粉中,饲喂胭脂鱼幼鱼,分别于第10 d、20 d、30 d、40 d、50 d各取样一次,分离头肾巨噬细胞,检测吞噬活性和呼吸爆发活力等细胞免疫指标;分离血清,检测溶菌酶活性、杀菌活性和替代补体溶血活性等体液免疫指标。结果显示,1.0%剂量组在第30 d和第40 d、0.5%剂量组在第40 d以及0.1%剂量组在第50 d的头肾巨噬细胞吞噬活性均显著高于对照组（P＜0.05）;0.5%剂量组在第20 d和第40 d、1.0%剂量组在第40 d和第50 d的头肾巨噬细胞呼吸爆发活力与对照组比较均有显著性升高（P＜0.05）;1.0%剂量组在第20 d和第50 d、0.5%剂量组在第30 d以及0.1%剂量组在第40 d的血清溶菌酶活性均有明显增加,与对照组比较差异显著（P＜0.05）;1.0%剂量组在第30 d和第40 d、0.5%剂量组在第40 d和第50 d的杀菌百分率均有明显升高,与对照组比较有显著性差异（P＜0.05）;1.0%剂量组在第40和50 d、0.5%剂量组在第30 d以及0.1%剂量组在第50 d的ACH₅₀与对照组比较均有明显升高（P＜0.05）。饲喂50 d后,各组随机取20尾鱼,用A.hydrophila腹腔注射进行攻毒试验,探讨黄芪配甘草对胭脂鱼幼鱼抗病力的影响,结果显示1周内0.5%和0.1%剂量组的累积死亡率分别为45%和60%,1.0%剂量组的累积死亡率35%则显著低于对照组（P＜0.05）。结果表明,黄芪配甘草可开发为一种新型的免疫增强剂,对胭脂鱼幼鱼免疫功能的提高表现出剂量一效应关系,适宜添加剂量为1.0%。（4）陈皮对胭脂鱼幼鱼非特异性免疫机能和抗病力的影响:醇提法制备陈皮提取物（总黄酮含量约为3.3%,橙皮苷含量约为1.6%）,按质量分数0%（对照组）、0.1%、0.5%和1.0%添加于成鳗粉中,饲喂胭脂鱼幼鱼,分别于第10 d、20 d、30 d、40 d、50 d各取样一次,分离头肾巨噬细胞,检测吞噬活性和呼吸爆发活力;分离血清,检测溶菌酶活性、杀菌活性和替代补体溶血活性。结果显示,第50 d时,陈皮提取物1.0%剂量组的头肾巨噬细胞吞噬活性显著高于对照组（P＜0.05）;0.5%剂量组在第30 d、1.0%剂量组在第40 d的头肾巨噬细胞呼吸爆发活力与对照组比较均有显著性升高（P＜0.05）;第20 d,1.0%剂量组的血清溶菌酶活性比对照组有明显增加（P＜0.05）;陈皮提取物对血清杀菌活性和ACHso则无明显影响。饲喂50 d后,各组随机取20尾鱼,用A.hydrophila腹腔注射进行攻毒试验,探讨陈皮对胭脂鱼幼鱼抗病力的影响,结果显示1周内1.0%、0.5%、0.1%剂量组的累积死亡率分别为50%、55%、65%,与对照组比较差异并不显著（P＞0.05）。结果表明陈皮提取物能够在一定程度上增强胭脂鱼幼鱼的非特异性免疫力,关于陈皮中单一成分橙皮苷对胭脂鱼幼鱼非特异性免疫力和抗病力的影响尚需进一步研究。（5）中药提取物的体外免疫试验:以胭脂鱼幼鱼的头肾巨噬细胞为研究对象,探讨两种中药提取物组方（蜂胶配淫羊藿、黄芪配甘草）对离体培养细胞免疫活性的影响。不连续密度梯度分离法制备头肾巨噬细胞,纯化,接种到96孔培养板,加入终浓度依次为0、25、50、100、200、400μg/mL的中药提取物溶液,共培养24 h,NBT法检测头肾巨噬细胞的呼吸爆发活力。结果显示蜂胶配淫羊藿在浓度25、50、100μg/mL时,黄芪配甘草浓度为100、200μg/mL时,呼吸爆发活力值与空白对照组比较均有显著差异（P＜0.05）。这一结果从体外验证了2种中药提取物组方在胭脂鱼幼鱼养殖试验中的免疫增强效果。更多还原

【Abstract】 This study was conducted on juvenile Chinese sucker, Myxocyprinus asiaticus. In the first part, the dietary protein requirement of juvenile Chinese sucker was determined. In the second part, the effects of three traditional Chinese medicines （TCM） formulated from propolis and Herba Epimedii extracts, Radix Astragali and Glycyrrhiza uralensis extracts, Tangerine Peel extract respectively, on non-specific immune response of Chinese sucker were investigated. The results were as follows.（1） Growth performance and proximate composition of juvenile Chinese sucker fed the experimental diets containing different protein levels: Six fishmeal-based experimental diets containing various crude protein levels including 30%, 34%, 38%, 42%, 46% and 50% were fed to triplicate groups of 20 fish each （initial weight 13.5±1.1 g） for 56 days. Weight gain （WG） and specific growth rate （SGR） increased with increasing dietary protein level up to 46% （P < 0.05）. Based on broken-line regression analysis of SGR against dietary protein levels the optimal dietary protein requirement for juvenile Chinese sucker was estimated to be close to 46.5%. Survival was not affected by dietary protein level （overall survival 71 - 90%）. Feed conversion rate （FCR） generally showed a decline with higher protein levels （from 1.62 in 30% protein group to 1.13 in 50% protein group）. Protein efficiency ratio （PER） showed gradual improvements with increasing dietary protein up to 46%. A similar trend was found for protein productive value （PPV）. Among the proximate compositions of the whole body and muscle, crude protein contents all increased significantly with increasing dietary protein levels （P < 0.05）. Conditon factor （CF）, hepatosomatic index （HSI） and viscerosomatic index （VSI） generally showed an increase with higher protein levels. It was estimated that the optimal dietary protein requirement for juvenile Chinese sucker was close to 46.5%.（2） Effects of combination of propolis and Herba Epimedii extracts on the non-specific immune response and disease resistance of Chinese sucker: Propolis extract （Total flavone content: 37.1%） and Herba Epimedii extract （Total flavone content: 42.6%） was extracted using alcohol and mixed at the ratio of 3:1 （w/w）. Fish were fed diets containing 0 （control）, 0.1%, 0.5% or 1.0% TCM extracts for 50 days. Head-kidney and blood samples were collected at 10 d, 20 d, 30 d, 40 d and 50 d, respectively. Macrophages in head-kidney were separated by discontinuous density gradient centrifugation. The phagocytic activity of macrophage was measured using neutral red. The respiratory burst activity of macrophage was measured by flow cytometry （FCM）. The lysozyme, bactericidal and natural haemolytic complement (ACH₅₀) activities in blood serum were measured using Micrococcus lysoleikticus, Aeromonas hydrophila and RaRBC, respectively. Results of this study showed that feeding the fish with 0.5% TCM extracts significantly increased the phagocytic activity after 40 days （P < 0.05）. The enhanced phagocytic activity remained as compared to the control until the end of the experiment. Fish fed with 1.0% TCM extracts showed enhanced phagocytic activity on the 30 days compared to the control. The respiratory burst activity of macrophage showed a statistically significant increase on the 30 and 40 days in 0.5% dose group compared to the control （P < 0.05）. A significant increasing of lysozyme activity was found after 10 days in group fed with 1.0% TCM extracts compared to the control （P < 0.05）. Significant differences were also measured in 0.5% dose group on the 20 and 40 days and 0.1% dose group on the 40 days and the end of the experiment. Feeding the fish with 0.5% TCM extracts significantly increased the bactericidal activity after 40 days （P < 0.05）. There were no significant changes in ACH₅₀ during feeding. After 50 days of feeding fish were challenged with A. hydrophila and cumulative mortality was registered during one week. A cumulative mortality over the experimental period in the control group was 75%. All dosage of treated groups showed reduced mortality compared to the control. Cumulative mortalities in fish fed with 0.1% and 1.0% TCM extracts reached 45% and 55%, respectively. Feed containing 0.5% TCM extracts was the most effective with the mortality of the fish significantly reduced by 35% compared to the control. The results indicate that propolis and Herba Epimedii extracts in combination enhances the non-specific immune response and disease resistance of Chinese sucker against A. hydrophila. The optimal dosage added to feed was 0.5% （w/w）. （3） Effects of combination of Radix Astragali and Glycyrrhiza uralensis extracts on the non-specific immune response and disease resistance of Chinese sucker: Radix Astragali extract （Astragalus polysaccharides content: 38.4%） and Glycyrrhiza uralensis extract （Glycyrrhizic acid content: 31.2%; total flavone content: 20.5%） was extracted using alcohol and mixed at the ratio of 10:6 （w/w）. Fish were fed diets containing 0 （control）, 0.1%, 0.5% or 1.0% TCM extracts for 50 days. Head-kidney and blood samples were collected at 10 d, 20 d, 30 d, 40 d and 50 d, respectively. Macrophages in head-kidney were separated by discontinuous density gradient centrifugation. The phagocytic activity of macrophage was measured using neutral red. The respiratory burst activity of macrophage was measured by FCM. The lysozyme, bactericidal and ACH₅₀ activities in blood serum were measured using M. lysoleikticus, A. hydrophila and RaRBC, respectively. Results of this study showed that the phagocytic activity of macrophage showed a statistically significant increase on the 30 and 40 days in 1.0% dose group, 40 days in 0.5% dose group and 50 days in 0.1% dose group compared to the control （P < 0.05）. Feeding the fish with 1.0% TCM extracts significantly increased the respiratory burst activity of macrophage after 40 days. The enhanced activity remained as compared to the control until the end of the experiment （P < 0.05）. A significant increasing was also found on the 20 and 40 days in 0.5% dose group compared to the control. Significant differences of lysozyme activity was measured in 1.0% dose group on the 20 and 50 days, 0.5% dose group on the 30 days and 0.1% dose group on the 40 days compared to the control （P < 0.05）. Feeding the fish with 0.5% TCM extracts significantly increased the bactericidal activity after 40 days until the end of the experiment （P < 0.05）. A significant increasing was also found on the 30 and 40 days in group fed with 1.0% TCM extracts compared to the control. The ACH₅₀ activity showed a statistically significant increase on the 40 and 50 days in 1.0% dose group, 30 days in 0.5% dose group and 50 days in 0.1% dose group compared to the control （P < 0.05）. After 50 days of feeding fish were challenged with A. hydrophila and cumulative mortality was registered during one week. A cumulative mortality over the experimental period in the control group was 75%. All dosage of treated groups showed reduced mortality compared to the control. Cumulative mortalities in fish fed with 0.1% and 0.5% TCM extracts reached 60% and 45%, respectively. Feed containing 1.0% TCM extracts was the most effective with the mortality of the fish significantly reduced by 40% compared to the control （P < 0.05）. The results indicate that Radix Astragali and Glycyrrhiza uralensis extracts in combination enhances the non-specific immune response and disease resistance of Chinese sucker against A. hydrophila. The optimal dosage added to feed was 1.0% （w/w）.（4） Effects of Tangerine Peel extract on the non-specific immune response and disease resistance of Chinese sucker: Tangerine Peel extract （Total flavone content: 3.3%; Hesperidin content: 1.6%） was extracted using alcohol. Fish were fed diets containing 0 （control）, 0.1%, 0.5% or 1.0% Tangerine Peel extract for 50 days. Head-kidney and blood samples were collected at 10 d, 20 d, 30 d, 40 d and 50 d, respectively. Macrophages in head-kidney were separated by discontinuous density gradient centrifugation. The phagocytic activity of macrophage was measured using neutral red. The respiratory burst activity of macrophage was measured by FCM. The lysozyme, bactericidal and ACH₅₀ activities in blood serum were measured using M. lysoleikticus, A. hydrophila and RaRBC, respectively. Results of this study showed that the phagocytic activity of macrophage had a statistically significant increase on the 50 days in 1.0% dose group compared to the control （P < 0.05）. Significant differences of respiratory burst activity was measured in 1.0% dose group on the 40 days and 0.5% dose group on the 30 days compared to the control （P < 0.05）. Feeding the fish with 1.0% Tangerine Peel extract significantly increased the lysozyme activity in blood serum after 20 days （P < 0.05）. There were no significant changes in bactericidal and ACH₅₀ activities comparing to the control during the whole experiment. After 50 days of feeding fish were challenged with A. hydrophila and cumulative mortality was registered during one week. A cumulative mortality over the experimental period in the control group was 75%. Cumulative mortalities in fish fed with 0.1%, 0.5% and 1.0% Tangerine Peel extract reached 65%, 55% and 50%, respectively. The results indicate that Tangerine Peel extract enhances the non-specific immune response and disease resistance of Chinese sucker against A. hydrophila.（5） Effects of TCM extracts on respiratory burst activity of head-kidney macrophage of Chinese sucker in vitro: Macrophages in head-kidney of Chinese sucker were separated and purified by discontinuous density gradient centrifugation. Then head-kidney macrophages were incubated in a 96-well plate with different doses of TCM extracts （combination of propolis and Herba Epimedii extracts, combination of Radix Astragali and Glycyrrhiza uralensis extracts）, 0, 25, 50, 100, 200 and 400μg/mL, respectively, for 24 h. The respiratory burst activity of macrophage was measured by nitroblue tetrazolium （NBT）. Significant differences of respiratory burst activity was found in combination of propolis and Herba Epimedii extracts with doses of 25, 50 and 100μg/mL and combination of Radix Astragali and Glycyrrhiza uralensis extracts with doses of 100 and 200μg/mL compared to the control （P < 0.05）. The results indicate that combination of propolis and Herba Epimedii extracts and combination of Radix Astragali and Glycyrrhiza uralensis extracts could modulate the immune response of Chinese sucker in vitro.更多还原