【作者】 于明超；

【导师】 马甡； 李卓佳；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2007， 博士

【摘要】 本论文主要运用微生物学、生理学、免疫学、营养学和生态学等研究方法,研究了芽孢杆菌和中草药在凡纳滨对虾饲料中的应用,主要结果如下:1.本实验研究了不同浓度中草药制剂对地衣芽孢杆菌生长繁殖的影响。结果表明适当的中草药浓度能够促进地衣芽孢杆菌的生长繁殖,两者在饲料中共同添加使用是可能的。2.本实验分成生长实验和消化酶活性实验两部分,研究了饲料中单独或共同添加芽孢杆菌和中草药制剂对凡纳滨对虾存活、生长、消化酶活性及体成分的影响。实验分成5个处理,分别投喂5种不同的饲料:基础饲料,中草药饲料(在基础饲料中添加0.20%中草药制剂,M),芽孢杆菌饲料(在基础饲料中添加0.30%芽孢杆菌制剂,B),中草药和芽孢杆菌饲料1(在基础饲料中添加0.20%中草药制剂和0.30%芽孢杆菌制剂,BM1),中草药和芽孢杆菌饲料2(在基础饲料中添加0.10%中草药制剂和0.15%芽孢杆菌制剂,BM2)。实验结果,芽孢杆菌和中草药能够提高对虾的增重率, M、BM1、BM2组与对照组差异显著(P<0.05), B、BM1、M组之间差异不显著。BM2组特定生长率最高,BM1组次之,均与对照组差异显著(P<0.05)。对虾常规体成分除粗脂肪外,无显著性差异。BM1组脂肪含量最低,而BM2组最高。在实验过程中,每隔2周取样一次分析凡纳滨对虾肝胰脏的蛋白酶和淀粉酶活性,结果发现单独或共同添加芽孢杆菌和中草药制剂可以在一定程度上提高消化酶的活性。在第2周和第6周的取样中,BM2组的蛋白酶和淀粉酶均显著高于对照组(P<0.05);在第8周的取样中,B组的淀粉酶活性显著高于对照组(P<0.05)。根据本实验的结果,在基础饲料中添加0.10%中草药制剂和0.15%芽孢杆菌制剂使用效果最好。3.本实验研究了饲料中单独或共同添加中草药和芽孢杆菌对凡纳滨对虾(Litopenaeus vannamei)生长和肠道细菌的影响。以基础饲料为对照组(C),通过添加中草药和芽孢杆菌配制成4种实验饲料:0.2%中草药(M),0.20%中草药制剂+0.30%芽孢杆菌(BM1),0.10%中草药制剂+0.15%芽孢杆菌(BM2),0.30%芽孢杆菌(B)。实验共进行了56d,各组对虾的成活率都很高(95.83~98.33%),各处理间无显著差异(P>0.05)。投喂实验饲料的各组对虾终末体重均高于对照组(C),其中BM2与C差异性显著(P<0.05)。M、BM1和BM2的增重率和特定增长率显著高于C和B。M组的对虾肠道异养菌总数和弧菌数均高于其它各组,而共同添加组BM1和BM2的异养菌和弧菌数则最少。从各组随机挑取30株细菌进行分类鉴定,结果C、M、BM1、BM2和B组细菌属数分别为11、8、6、5和7个。饲料中添加中草药和芽孢杆菌促进了对虾的生长,且共同添加的效果好于单独添加,它们的使用改变了肠道细菌的数量和组成。4.本实验研究了饲料中添加不同水平的芽孢杆菌和中草药制剂对凡纳滨对虾生长、存活、体成分、消化酶活性以及血清生化指标的影响。以基础饲料为对照(Diet0),通过添加中草药和芽孢杆菌配制成6种实验饲料:0.10%中草药+ 0.10%芽孢杆菌(Diet11),0.10%中草药+0.20%芽孢杆菌(Diet12),0.10%中草药+0.30%芽孢杆菌(Diet13),0.20%中草药+ 0.10%芽孢杆菌(Diet21),0.20%中草药+0.20%芽孢杆菌(Diet22),0.20%中草药+0.30%芽孢杆菌(Diet23)。实验共进行56d,各组对虾的成活率76.19~84.76%,各处理间无显著差异(P>0.05)。对虾的终末体重、特定生长率(SGR)、蛋白质效率(PER)和蛋白质累积率(PPV)随中草药和芽孢杆菌浓度的增加而增加,饵料系数(FCR)随添加浓度增加有所降低。芽孢杆菌和中草药交互作用对终末体重和SGR影响显著,Diet23组的终末体重和SGR均与对照组差异显著(P<0.05)。添加中草药和芽孢杆菌对虾体的水分、粗蛋白、灰分等指标影响不大(P>0.05),对粗脂肪含量产生显著影响(P<0.05),Diet23组对虾的粗脂肪含量最高,显著高于Diet0、Diet13和Diet22组。添加中草药和芽孢杆菌提高了对虾肠的淀粉酶和蛋白酶以及肝胰脏的蛋白酶(P<0.05),对肝胰脏的淀粉酶活性作用不明显(P>0.05)。此外,中草药和芽孢杆菌交互作用显著影响了对虾肝胰脏的碱性磷酸酶和酸性磷酸酶的活性(P<0.05)。血清中的蛋白和葡萄糖含量随中草药和芽孢杆菌添加量的增加而增加,低浓度的中草药(Diet11、Diet12和Diet13组)可提高甘油三酯含量,高浓度的芽孢杆菌提高了虾血清中胆固醇含量高(P<0.05)。5.本实验研究了饲料中添加芽孢杆菌和中草药制剂对凡纳滨对虾免疫功能的影响。以基础饲料为对照(Diet0),通过添加中草药和芽孢杆菌配制成6种实验饲料:0.10%中草药+ 0.10%芽孢杆菌(Diet11),0.10%中草药+0.20%芽孢杆菌(Diet12),0.10%中草药+0.30%芽孢杆菌(Diet13),0.20%中草药+ 0.10%芽孢杆菌(Diet21),0.20%中草药+0.20%芽孢杆菌(Diet22),0.20%中草药+0.30%芽孢杆菌(Diet23)。实验共进行了56d,对血细胞数目、酚氧化酶活力(PO)、溶菌酶活力、超氧化物歧化酶(T-SOD)活力以及总抗氧化能力进行了测定。芽孢杆菌对血细胞数目产生显著影响,Diet12,Diet13和Diet23组对虾的血细胞数显著高于其余各组(P<0.05)。中草药以及交互作用对血清PO产生显著影响(P<0.05),高浓度的中草药可以提高血清PO。大多数实验组对虾溶菌酶活力高于对照组。中草药、芽孢杆菌以及它们的交互作用对T-SOD未产生显著影响(P>0.05)。Diet23组对虾血清的总抗氧化能力最高,除Diet21组外,与其余各组差异显著(P<0.05)。6.本实验研究了中草药和芽孢杆菌共同添加到不同营养水平的饲料中,对凡纳滨对虾生长、消化酶活性、体成分组成以及血清生化指标的影响。基础饲料1为鱼粉含量29%的高档饲料,基础饲料2是以豆粕和花生粕替代部分鱼粉的低档料。通过在两种饲料中添加芽孢杆菌和中草药制剂作为实验用料:基础饲料1 (Diet10),基础饲料1+0.10%中草药+0.15%芽孢杆菌(Diet11),基础饲料1+0.20%中草药+0.30%芽孢杆菌(Diet12),基础饲料2 (Diet20),基础饲料2+0.10%中草药+0.15%芽孢杆菌(Diet21),基础饲料2+0.20%中草药+0.30%芽孢杆菌(Diet22)。实验共进行56d,基础饲料2各组对虾成活率高于基础饲料1。数据分析显示,不同水平的饲料及添加剂均对终末体重、特定生长率(SGR)、饵料系数(FCR)和蛋白质效率(PER)产生了显著影响。摄食基础饲料1的各组对虾终末体重和特定增长率均显著高于摄食基础饲料2的各组对虾。对于同一种基础饲料,添加芽孢杆菌和中草药制剂可以提高蛋白质效率(PER)和蛋白质累积率(PPV),降低饵料系数(FCR)。饲料和添加剂对虾体的常规成分有一定的影响。中草药和芽孢杆菌能够提高对虾肠道和肝胰脏消化酶活性以及肝胰脏碱性磷酸酶活性。基础饲料1各组的碱性磷酸酶和酸性磷酸酶显著低于基础饲料2各组。基础饲料1组的对虾血清葡萄糖高于基础饲料2组对虾,但是胆固醇含量低于基础饲料2组对虾。添加剂能够提高对虾的血清葡萄糖和蛋白质含量。7.本实验研究了在不同饲料中添加芽孢杆菌和中草药制剂对凡纳滨对虾免疫功能的影响。实验以两种商用饲料原料为基础饲料,基础饲料1为鱼粉含量29%的高档饲料,基础饲料2是以豆粕和花生粕替代部分鱼粉的低档料。通过在基础饲料中添加不同量的芽孢杆菌和中草药制剂作为实验用料:基础饲料1 (Diet10),基础饲料1+0.10%中草药+0.15%芽孢杆菌(Diet11),基础饲料1+0.20%中草药+0.30%芽孢杆菌(Diet12),基础饲料2 (Diet20),基础饲料2+0.10%中草药+0.15%芽孢杆菌(Diet21),基础饲料2+0.20%中草药+0.30%芽孢杆菌(Diet22)。实验共进行了56d,对血细胞数目、酚氧化酶活力(PO)、溶菌酶活力、超氧化物歧化酶(T-SOD)活力以及总抗氧化能力进行了测定。饲料和添加剂对血细胞数目、溶菌酶活力产生显著影响,添加剂以及饲料和添加剂的交互作用对血清总抗氧化能力产生显著影响(P<0.05)。总体来讲,在不同饲料中添加中草药和芽孢杆菌对血细胞数目、溶菌酶活力、酚氧化酶活力(PO)、总抗氧化能力和超氧化物歧化酶(T-SOD)活力均有不同程度的提高。通过添加芽孢杆菌和中草药制剂可以显著提高基础饲料1的血细胞数目和总抗氧化能力以及基础饲料2的血细胞数目和溶菌酶活力。不同营养水平的饲料对免疫指标也有一定的影响,Diet20组的总抗氧化能力显著高于Diet10组。8.本实验从碳、氮、磷等营养物质的角度研究了中草药和芽孢杆菌共同添加到不同营养水平的饲料中,对凡纳滨对虾生长、摄食、粪便、非粪便流失、体成分,消化率等方面的影响。实验以两种商用饲料原料为基础饲料,基础饲料1为鱼粉含量29%的高档饲料,基础饲料2是以豆粕和花生粕替代部分鱼粉的低档料。通过添加不同量的芽孢杆菌和中草药制剂共制成6种实验用料:基础饲料1 (Diet10),基础饲料1+0.10%中草药+0.15%芽孢杆菌(Diet11),基础饲料1+0.20%中草药+0.30%芽孢杆菌(Diet12),基础饲料2 (Diet20),基础饲料2+0.10%中草药+0.15%芽孢杆菌(Diet21),基础饲料2+0.20%中草药+0.30%芽孢杆菌(Diet22)。实验共进行了56d,从生长结果可以看出,在饲料中添加芽孢杆菌和中草药添加剂可以促进对虾的生长,增加食物的摄入量,减少排便量,从而提高饲料转化率和降低单位体重增加流失量。从碳、氮、磷等营养物质来看,基本上也是增加了其摄入量和生长量,减少了粪便量。从各营养物质的生长、粪便以及非粪流失量与摄食量的比值来看,对虾摄食的各营养物质用于生长的部分增加了,而用于粪便和非粪便流失量的部分相应的减少了。摄食基础饲料1的各组对虾其摄入量、终末总重、增重量以及饲料转化率均相应的高于基础饲料2各组对虾,单位体重增加流失量也有所减少。虾体的碳、氮含量受饲料和添加剂的影响不大,磷受影响较大,而粪便中的碳、氮、磷含量则变化较大。添加剂提高了干物质及各营养物质的表观消化率,不同饲料对其也有所影响。更多还原

【Abstract】 A series of experiments were conducted to investigate the application of diatary Bacillus spp. and traditional Chinese medicine on shrimp Litopenaeus vannamei, based on microbiological, physiological, nutritional, immunological and ecological methods. The results are summarized as followed:1. A trial was conducted to investigate the effect of traditional Chinese medicines (TCM) on growth of Bacillus licheniformis. The growth of B. licheniformis could be promoted in some concentrations of TCM. Therefore, they were potential to be used together in the feed.2. The basal diet (C); with 0.20% TCM (M), with 0.30% Bacillus spp. and 0.20% TCM (BM1), with 0.15% Bacillus spp. and 0.10% TCM (BM2), with 0.30% Bacillus spp. (B) was used to fed white shrimp Litopenaeus vannamei (1.91±0.03 g) in order to assess the survival, growth, body composition and digestive enzyme activity. Growth measured as weight gain was significantly (P<0.05) higher in shrimp fed with BM2, BM1 and M compared to that of C. However no significant differences were found among B, BM1 and M. In the case of specific growth rate, the shrimp fed with BM1 and BM2 exhibited significantly (P<0.05) higher values than that of the C. The contents of body moisture, crude protein and ash seemed to be unaffected by the feed supplements, though lipid content was found to be significantly (P<0.05) different among the treatments. The shrimp fed with BM1 and BM2 had the lowest and highest lipid contents respectively. The digestive enzyme activity assessed using shrimp hepatopancreas revealed that the activities of protease and amylase in shrimp fed with BM2 were significantly (P<0.05) higher than those of the C at the end of 2nd and 6th week. However, better performance of the specific amylase activity was shown by the shrimp fed with B at the end of 8th week.3. A feeding experiment was conducted to investigate the effects of Bacillus and traditional Chinese medicines (TCM) on growth and intestinal bacterial flora of shrimp L. vannamei. Four experimental diets were formulated by adding the Bacillus and TCM to basal diet (C): 0.2% TCM (M), 0.20% TCM+0.30% Bacillus (BM1), 0.10% TCM+0.15% Bacillus (BM2), 0.30% Bacillus (B). The concentration of Bacillus was 109 CFU/g. The TCM consisted of a mixture of herbs and plant materials Isatis tinctoria L., Isatis indigodica Fort, Forsythia suspersa Vahl, Corydalis bungeana Turez, Pogostemon cablin (blanco) Benth and Astragalus menbranaceus (Fisch.) Bge. Acclimatized shrimp with an average initial weight 1.91±0.03 g were selected and stocked into 15 cement tanks at a stocking density of 80 shrimp. Each aquarium contained 1500-l of sand filtered seawater with a flow rate of 0.9-l min-1. The water was continuously aerated with two air stones. Each diet was given to shrimp four times (07:00, 12:00, 17:00, 23:00) a day to saturation for a period of 56 days. During the experimental period, the water temperature fluctuated between 19.6 and 25.2℃, salinity between 30 and 31. At the end of feeding trial, survival ranged from 95.83% to 98.33% with no significant difference (P>0.05) among all groups. Growth measured as final weight was slightly higher (P>0.05) in shrimp fed with BM1, M and B than that of C, and there was a significant difference (P<0.05) between BM2 and C. In case of weight gain (WG) and specific growth rate (SGR), the shrimp fed with M, BM1 and BM2 exhibited significantly (P<0.05) higher values than that of the C and B. Of the five diets, BM2 exhibited the best performance followed by BM1. The combination of Bacillus spp. and TCM, therefore, proved to be more effective and economically viable. The total bacteria counts and Vibrio counts in shrimp fed with M was the most, which fed with BM1 and BM2 was the least. In each group 30 bacterial strains were purified and identified. These bacterial strains belonged to 12, 8, 6, 5, 7 genera in C, M, BM1, BM2 and B respectively. Most of the bacteria were gram negative bacteria, they counted a percentage of 86% to 93% in each group. Vibrio spp., Photobacterium spp. and Pseudomonas spp were the predominant bacterial flora, however, their values varied in different groups. The results showed that the diets supplemented with TCM and Bacillus also changed the number and composition of intestinal bacterial flora.4. A feeding experiment was conducted to investigate the effects of Bacillus and traditional Chinese medicines (TCM) on the survival, growth, body composition, digestive enzyme activity and serum biochemical values of shrimp L. vannamei. Six experimental diets were formulated by adding the Bacillus and TCM to basal diet (Diet0, Control): 0.10% TCM+0.10% Bacillus (Diet11), 0.10% TCM+0.20% Bacillus (Diet12), 0.10% TCM +0.30% Bacillus (Diet13), 0.20% TCM+0.10% Bacillus (Diet21), 0.20% TCM+0.20% Bacillus (Diet22), 0.20% TCM +0.30% Bacillus (Diet23). After 56 days of feeding, survival ranged from 76.19% to 84.76% with no significant difference (P>0.05) among all groups. Growth measured as final weight, specific growth rate (SGR), protein efficiency ratio (PER), protein productive value (PPV) and feed efficiency ratio (FER) were improved as the addition of TCM and Bacillus increasing. In the case of final weight and SGR, the shrimp fed with Diet23 exhibited significantly (P<0.05) higher values than that of Diet0. The contents of body moisture, crude protein and ash seemed to be unaffected by the feed supplements, though lipid content was found to be significantly (P<0.05) different among the treatments. The shrimp fed with Diet23 had a higher lipid contents than which fed with Diet0, Diet13 and Diet22 (P<0.05). The digestive enzyme activity assessed using shrimp hepatopancreas and intestines revealed that the activities of protease and amylase in shrimp were significantly (P<0.05) enhanced by the feed supplements, but amylase of hepatopancreas was except. The alkaline phosphatease and acid phosphatease values were significantly affected by the interaction of Bacillus and TCM (P<0.05). Serum protein and glucose content were enhanced as the addition of TCM and Bacillus increasing. However, Serum triglyceride in shrimp of low TCM groups (Diet11, Diet12 and Diet13) and cholesterol in shrimp of high Bacillus groups (Diet12, Diet13 and Diet23) were significantly higher than that of Control (Diet0).5. A feeding experiment was conducted to investigate the effects of Bacillus and traditional Chinese medicines (TCM) on immunity of shrimp L. vannamei. Six experimental diets were formulated by adding the Bacillus and TCM to basal diet (Diet0, Control): 0.10% TCM+0.10% Bacillus (Diet11), 0.10% TCM+0.20% Bacillus (Diet12), 0.10% TCM +0.30% Bacillus (Diet13), 0.20% TCM+0.10% Bacillus (Diet21), 0.20% TCM+0.20% Bacillus (Diet22), 0.20% TCM +0.30% Bacillus (Diet23). After 56 days of feeding, the total haemocyte count (THC), phenoloxidase (PO), lysozyme activity, superoxide dismutase (SOD) and total antioxidant was measured. The data indicated that THC was significantly affected by Bacillus so that it was higher in shrimp fed with Diet 12, Diet13 and Diet23 than that of others (P<0.05). The value of PO was improved by high content of TCM. The lysozyme activity of shrimp in most experimental diet groups was higher than that of control. However, SOD seemed to be unaffected by the feed supplements. In the case of total antioxidant, the shrimp fed with Diet23 exhibited significantly (P<0.05) higher values than that of other groups except for Diet21.6. A feeding experiment was conducted to investigate the effects of Bacillus and traditional Chinese medicines (TCM) on the survival, growth, body composition, digestive enzyme activity and serum biochemical values of shrimp L. vannamei. Two practical diets were used as basal diets in the experiment, one of which is high quality diet contained 29% fish meal (basal diet 1), the other one of which is low quality diet contained 17% fish meal (basal diet 2). Six experimental diets were formulated by adding the Bacillus and TCM to the basal diets: Basal diet 1 (Diet10), Basal diet 1+0.10% TCM+0.15% Bacillus (Diet11), Basal diet 1+0.20% TCM+0.30% Bacillus (Diet12), Basal diet 2 (Diet20), Basal diet 2+0.10% TCM + 0.15% Bacillus (Diet21), Basal diet 2+0.20% TCM+0.30% Bacillus (Diet22). After 56 days of feeding, shrimp in the groups fed with basal diet 2 had a higher survival than that of basal diet 1. Growth measured as final weight, specific growth rate (SGR), protein efficiency ratio (PER), protein productive value (PPV) and feed efficiency ratio (FER) were significantly affected by the different basal diets and supplements. In the case of final weight and SGR, the shrimp in the groups fed with basal diet 1 exhibited significantly (P<0.05) higher values than that of basal diet 2. While for the same basal diet group, PER, PPV and FER of shrimp were improved by the supplement. The contents of body compositions were also affected by the feed supplements and different basal diets. The digestive enzyme activities assessed using shrimp hepatopancreas and intestines revealed that the activities of protease and amylase in shrimp were significantly (P<0.05) enhanced by the feed supplements. The alkaline phosphatease in shrimp hepatopancreas were significantly (P<0.05) enhanced by the feed supplements too. The alkaline phosphatease and acid phosphatease values were significantly higher in shrimp fed with basal diet 2 than basal diet 1. Serum cholesterol and glucose contents had a significant difference among different basal diet groups. However, Serum protein and glucose contents were enhanced by the supplements.7. A feeding experiment was conducted to investigate the effects of Bacillus and traditional Chinese medicines (TCM) on immunity of shrimp L. vannamei. Two practical diets were used as basal diets in the experiment, one of which is high quality diet contained 29% fish meal (basal diet 1), the other one of which is low quality diet contained 17% fish meal (basal diet 2). Six experimental diets were formulated by adding the Bacillus and TCM to basal diets: Basal diet 1 (Diet10), Basal diet 1+0.10% TCM+0.15% Bacillus (Diet11), Basal diet 1+0.20% TCM+0.30% Bacillus (Diet12), Basal diet 2 (Diet20), Basal diet 2+0.10% TCM + 0.15% Bacillus (Diet21), Basal diet 2+0.20% TCM + 0.30% Bacillus (Diet22). After 56 days of feeding, the total haemocyte count (THC), phenoloxidase (PO), lysozyme activity, superoxide dismutase (SOD) and total antioxidant were measured. The data indicated that THC and lysozyme activity were significantly affected by the feed and supplement, while total antioxidant was significantly (P<0.05) affected by the supplement and their interaction. As a whole, THC, PO, lysozyme activity, SOD and total antioxidant were enhanced more or less by adding the supplement in different diets. The values of THC and lysozyme activity in basal diet1 groups were significantly enhanced by the supplement. The values of THC and total antioxidant in basal diet2 groups were significantly enhanced by the supplement too. Total antioxidant were significantly (P<0.05) higher in Diet20 than in Diet10, which indicated that the immunity were also affected by the feed.8. A feeding experiment was conducted to investigate the effects of Bacillus and traditional Chinese medicines (TCM) on the growth, intake, faecal loss, non-faecal loss, body composition and apparent digestibility coefficients (ADCs) of shrimp L. vannamei. Two practical diets were used as basal diets in the experiment, one of which is high quality diet contained 29% fish meal (basal diet 1), the other one of which is low quality diet contained 17% fish meal (basal diet 2). Six diets were formulated by adding the Bacillus and TCM to the basal diets: Basal diet 1 (Diet10), Basal diet 1+0.10% TCM+0.15% Bacillus (Diet11), Basal diet 1+0.20% TCM+0.30% Bacillus (Diet12), Basal diet 2 (Diet20), Basal diet 2+0.10% TCM + 0.15% Bacillus (Diet21), Basal diet 2+0.20% TCM+0.30% Bacillus (Diet22). After 56 days of feeding, the results showed that growth, intake, feed conversion were increased while faecal loss and loss in unit weight gain were decreased by adding the supplement. The element of carbon, nitrogen and phosphorus in growth and intake were improved while which in faecal loss was reduced by adding the supplement. The value of intake, final weight, weight gain and feed conversion in shrimp of basal diet1 groups were higher than that of basal diet2 groups, while the value of loss in unit weight gain were reversed. Body carbon and nitrogen seemed not to be affected by the feed and supplement, while body phosphorus, faecal carbon, faecal nitrogen and faecal phosphorus were affected by them. ADCs of dry matter, carbon, nitrogen and phosphorus were improved by the supplement and also affected by the feed.更多还原