【作者】 赵彦翠；

【导师】 麦康森；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2011， 博士

【摘要】 本文以我国北方重要的水产养殖经济动物刺参(Apostichopus japonicus Selenka)为研究对象,研究主要的多糖类免疫增强剂(β-葡聚糖、肽聚糖和壳聚糖)对刺参生长、免疫及抗病力的影响。并运用微生物学方法,通过体外实验从刺参肠道、养殖水环境及养殖池底泥中筛选出对刺参有益的潜在益生菌,通过在体养殖实验,研究了筛选出的5株潜在益生菌(TMRC14、TC116、T13、TC22和EN25)对刺参稚参苗(初始平均体重0.2-0.4 g)生长、免疫及抗病力的影响,结果表明菌株T13、TC22和EN25对刺参稚参苗是有益的。因此选取T13、TC22和EN25三株细菌,通过养殖实验,研究在饲料中添加这3株益生菌对刺参幼参(初始平均体重3-5 g)生长、免疫及抗病力的影响,并探讨它们分别与低聚果糖复配的效果。同时对这3株细菌进行了鉴定,并对其最适的培养条件进行了初步摸索。主要研究内容和结果如下:1.以初始体重为(4.926±0.009) g的刺参为研究对象,探讨了在饲料中添加0 g/kg、1.25 g/kg、2.50 g/kg的β-葡聚糖对刺参生长、免疫及抗病力的影响。实验在循环水系统中进行,每个处理设3个重复。经过8周的养殖实验后,对刺参进行灿烂弧菌(Vibrio splendidus)攻毒。结果表明当饲料中添加1.25 g/kg的β-葡聚糖时,刺参的特定生长率显著高于对照组和2.50 g/kg添加组(P<0.05);后两者之间无显著差异(P>0.05)。饲料中添加β-葡聚糖能显著提高刺参体腔细胞的吞噬活性(P<0.05),而对其呼吸爆发活力的影响并不显著(P>0.05)。随着饲料中β-葡聚糖含量的升高,刺参体腔细胞内酚氧化酶活性显著升高(P<0.05)。刺参体腔细胞内酸性磷酸酶的活性随饲料β-葡聚糖含量升高而有升高的趋势,但各处理组间差异不显著(P>0.05)。攻毒实验表明,1.25 g/kg的β-葡聚糖可以显著降低灿烂弧菌对刺参的致病死亡率(P<0.05)。由此可见,饲料中添加适量的β-葡聚糖(1.25 g/kg)可以提高刺参的特定生长率、非特异性免疫力及抗病能力。2.以初始体重为(4.931±0.005) g的刺参为研究对象,在循环水系统中进行8周的养殖实验,探讨在基础饲料中分别添加0 mg/kg、200 mg/kg和500 mg/kg的肽聚糖对刺参生长、免疫及抗病力的影响。实验结果表明,随着饲料中肽聚糖含量的升高,刺参的特定生长率呈上升趋势,但各处理之间无显著差异(P>0.05)。饲料中添加肽聚糖显著提高了刺参体腔细胞的吞噬活性(P<0.05),而对其呼吸爆发活力的影响不显著(P>0.05)。饲料中添加200 mg/kg的肽聚糖可以显著提高刺参体腔细胞的酚氧化酶活性(P<0.05),而500 mg/kg添加组与对照组无显著差异(P>0.05)。随着饲料中肽聚糖添加量的升高,刺参体腔细胞内酸性磷酸酶活性呈下降趋势,其中,500 mg/kg肽聚糖添加组显著低于对照组和200 mg/kg的添加组(P<0.05)。攻毒实验表明,饲料中添加肽聚糖可以提高刺参对灿烂弧菌的抵抗能力,其中添加200 mg/kg的肽聚糖显著降低了刺参的致病死亡率(P<0.05)。由此可见,虽然饲料中添加肽聚糖对刺参的生长没有显著影响,但适量的肽聚糖(200 mg/kg)可以显著提高刺参的免疫力和抗病力。3.以初始体重为(3.734±0.016) g的刺参为研究对象,在循环水系统中进行8周的养殖实验,在基础饲料中分别添加0 g/kg、2.5 g/kg、5 g/kg和10 g/kg的壳聚糖,探讨其对刺参生长、免疫及抗病力的影响。实验结果表明,饲料中添加不同梯度的壳聚糖对刺参的生长无显著影响(P>0.05)。当壳聚糖的添加量为10 g/kg时,刺参体腔细胞密度和吞噬活性得到显著提高(P<0.05),但其它添加量组与对照组之间没有显著差异(P>0.05)。饲料中添加壳聚糖对刺参体腔细胞呼吸爆发活力有提高的作用,但影响不显著(P>0.05)。饲料中添加壳聚糖对刺参体腔细胞酚氧化酶活性没有显著影响(P>0.05),但显著降低了刺参体腔细胞内酸性磷酸酶的活力(P<0.05)。攻毒实验表明,饲料中添加壳聚糖并不能显著提高刺参对灿烂弧菌的抵抗能力(P>0.05)。因此,壳聚糖作为免疫增强剂在刺参上的应用需要进一步探讨。4.用2216E、TSB和MRS培养基,从健康刺参肠道、患病刺参肠道和体壁、刺参养殖池底泥和池水中分离得到细菌182株,以刺参“腐皮综合征”致病菌—灿烂弧菌和假交替单胞菌为指示菌,筛选出对灿烂弧菌或者假交替单胞菌有抑制作用的菌株19株,并对其菌落形态和革兰氏染色情况进行了观察。通过菌株16S rDNA的序列比对,确定19株细菌全部属于芽孢杆菌属。为了进一步筛选刺参潜在的益生菌,对19株细菌的产蛋白酶和淀粉酶性能进行了研究。根据细菌的分离来源及菌落形态和对病原菌的抑制作用情况,选取5株潜在的益生菌用于刺参的安全性实验。结果表明5株潜在的益生菌TMRC14、TC116、T13、TC22和EN25对刺参是安全的,可以成为刺参益生菌的候选菌株,用于后续的实验。5.以初始体重为(0.204±0.009) g的刺参为研究对象,探讨在基础饲料(鼠尾藻粉)中分别添加10~5、10~7、10~9 CFU/g的TMRC14或者TC116对刺参生长、免疫力及抗病力的影响。养殖实验一共进行30d,静水养殖,每天换水50%。养殖实验结束后,对刺参进行7d灿烂弧菌浸浴攻毒。结果表明,饲料中添加TMRC14对刺参的特定生长率(SGR)没有显著影响(P>0.05);添加10~5-10~7 CFU/g的TC116时,刺参的SGR和对照没有显著区别(P>0.05),但当添加量升高到10~9 CFU/g时,刺参的SGR显著下降(P<0.05)。饲料中添加TMRC14或者TC116对刺参体腔细胞密度没有显著影响(P>0.05)。当TMRC14的添加量为10~9CFU/g时,刺参体腔细胞的吞噬活性显著升高(P<0.05),刺参体腔细胞的呼吸爆发活性也在10~9CFU/g时达到最高,但与对照和其它添加组没有显著差异(P>0.05)。饲料中添加10~7CFU/g TC116时,体腔细胞吞噬活性显著高于对照和其它添加量组(P<0.05)。刺参体腔细胞呼吸爆发活性在TC116的添加量为10~7CFU/g时显著高于对照和其它添加量组(P<0.05),而10~5CFU/g和10~9CFU/g添加组与对照没有显著差异(P>0.05)。攻毒实验表明,饲料中添加10~7CFU/g和10~9CFU/g的TMRC14时,降低了刺参浸浴灿烂弧菌的死亡率,但与对照无显著差异(P>0.05)。饲料中添加10~5CFU/g和10~7CFU/g的TC116时,降低了刺参浸浴灿烂弧菌的死亡率,但与对照并无显著差异(P>0.05)。因此,TMRC14和TC116在刺参生长、免疫和抗病力促进方面的作用并不显著,在刺参稚参苗的生产养殖过程中应用价值不大。6.以初始体重为(0.204±0.009) g的刺参为研究对象,探讨在基础饲料(鼠尾藻粉)中分别添加10~5 CFU/g、10~7 CFU/g、10~9 CFU/g潜在的益生菌T13或TC22对刺参生长、免疫及抗病力的影响。以初始体重为(0.375±0.024) g的刺参为研究对象,探讨在基础饲料中添加10~5 CFU/g、10~7 CFU/g、10~9 CFU/g潜在的益生菌EN25对刺参生长、免疫及抗病力的影响。养殖实验共进行30d,静水养殖,每天换水50%。养殖实验结束后,对刺参进行7d的灿烂弧菌浸浴攻毒实验。结果表明,当饲料中分别添加10~9 CFU/g的T13或者TC22时,刺参的特定生长率显著升高(P<0.05),而添加EN25对刺参的生长没有显著影响(P>0.05)。饲料中添加T13、TC22、EN25对刺参体腔细胞数量没有显著影响(P>0.05)。饲料中添加10~9 CFU/g的T13或者TC22可以显著提高刺参体腔细胞吞噬活性、呼吸爆发活力和总一氧化氮合酶活力(T-NOS)(P<0.05),但对刺参体腔细胞内超氧化物歧化酶(SOD)、酸性磷酸酶(ACP)活力无显著影响(P>0.05)。当饲料中EN25的添加量为10~7 CFU/g时,刺参的体腔细胞吞噬活性、呼吸爆发活力和T-NOS显著高于对照组(P<0.05),而10~5-10~7 CFU/g的EN25对刺参体腔细胞内SOD、ACP无显著影响(P>0.05);添加量为10~9 CFU/g时,刺参体腔细胞内SOD显著降低(P<0.05),但对ACP无显著影响(P>0.05)。攻毒实验表明,饲料中分别添加10~9 CFU/g的T13或者TC22可以显著降低灿烂弧菌攻毒后刺参的死亡率(P<0.05)。饲料中添加10~7 CFU/g的EN25时,刺参抵御灿烂弧菌感染的能力显著高于对照组(P<0.05)。因此T13、TC22、EN25可以作为益生菌应用到刺参的育苗过程中。7.以初始体重(4.893±0.028) g的刺参为研究对象,在循环水系统中进行8周的养殖实验,在饲料中分别添加0、10~7、10~9 CFU/g的T13,并在每个T13添加水平,分别添加0、0.5%的果糖,以3×2的实验设计配制6种饲料,探讨饲料中添加T13和果糖及两者复配对刺参生长、免疫、肠道菌群及抗病力的影响。养殖实验结束后,每个重复取6头刺参用于免疫指标及肠道菌群分析,剩余刺参用于灿烂弧菌攻毒实验。结果表明,随着饲料中T13含量的升高,刺参SGR有升高的趋势,饲料中单独添加果糖或者与T13复配也提高了刺参的SGR,但所有处理组与对照差异不显著(P>0.05)。饲料中添加T13使刺参体腔细胞吞噬活性、呼吸爆发活性、酚氧化酶活性及一氧化氮合酶活性都得到提高,其中酚氧化酶活性显著高于对照组(P<0.05)。饲料中添加T13显著提高了刺参肠道可培养细菌总数(P<0.05),当T13添加量为10~9 CFU/g时,刺参肠道中弧菌总数显著高于对照(P<0.05),但当添加量为10~7 CFU/g时,刺参肠道弧菌总数与对照没有差异(P>0.05)。饲料中添加10~9 CFU/g T13显著降低了灿烂弧菌攻毒后刺参的死亡率(P<0.05)。饲料中添加0.5%的果糖可以显著提高刺参体腔细胞吞噬活性、呼吸爆发和酚氧化酶活性,并显著降低刺参攻毒后的死亡率(P<0.05)。饲料中果糖与T13复配与否,对刺参肠道可培养细菌总数没有显著影响(P>0.05)。单独添加果糖显著降低了刺参肠道弧菌总数(P<0.05),但果糖与T13复配对刺参肠道弧菌总数影响不显著(P>0.05)。饲料中0.5%果糖与T13复配时,刺参体腔细胞吞噬活性、呼吸爆发、酚氧化酶活性有所升高但和对照没有显著差异(P>0.05),而刺参一氧化氮合酶在果糖与10~9 CFU/g T13复配组显著高于对照组(P<0.05)。果糖与T13复配使刺参抗病力得到显著提高(P<0.05),但与单独添加果糖或者10~9 CFU/g T13相比并没有显著差异(P>0.05)。8.以初始体重(4.918±0.022)g的刺参为研究对象,在循环水系统中进行8周的养殖实验,在饲料中分别添加0、10~7、10~9 CFU/g的TC22,并在每个TC22的添加梯度,分别添加0、0.5%果糖,以3×2的实验设计配制成6种饲料,探讨饲料中添加TC22和果糖及两者复配对刺参生长、免疫、肠道菌群及抗病力的影响。养殖实验结束后,每个重复取6头刺参用于免疫指标及肠道菌群分析,剩余刺参用于灿烂弧菌攻毒实验。结果表明,在不同的TC22添加梯度下,无论添加果糖与否,刺参的特定生长率都与对照无显著差异(P>0.05)。随着饲料中TC22含量的升高,刺参体腔细胞吞噬活性、呼吸爆发活性及酚氧化酶活性都得到显著提高(P<0.05),而刺参体腔细胞一氧化氮合酶活性呈现先升高后降低的趋势,但与对照组没有显著差异(P>0.05)。饲料中添加10~9 CFU/g TC22显著提高了刺参肠道可培养细菌总数(P<0.05),但刺参肠道中弧菌总数并没有受到饲料中添加TC22和果糖的影响(P>0.05)。饲料中添加10~9 CFU/g TC22显著降低了灿烂弧菌攻毒后刺参的死亡率(P<0.05),添加0.5%的果糖可以显著提高刺参的免疫反应,降低刺参的死亡率(P<0.05)。饲料中0.5%果糖与10~9 CFU/g TC22复配时,刺参的免疫力和抗病力得到显著提高(P<0.05)。因此,益生菌TC22对刺参的健康有促进作用,并且和果糖复配具有正向的交互作用。9.以初始体重(3.127±0.012) g的刺参为研究对象,在循环水系统中进行8周的养殖实验,在饲料中分别添加0、10~7、10~9 CFU/g的EN25,并在每个EN25添加水平,分别添加0、0.5%的果糖,以3×2的实验设计配制成6种饲料,探讨饲料中添加EN25和果糖及两者复配对刺参生长、免疫、肠道菌群及抗病力的影响。养殖实验结束后,每个重复取6头刺参用于免疫指标及肠道菌群分析,剩余刺参用于灿烂弧菌攻毒实验。结果表明,当饲料中添加10~7 CFU/g EN25时,刺参SGR最高,但与对照和其它组差异不显著(P>0.05);饲料中添加果糖或者果糖与EN25复配对刺参SGR无显著影响(P>0.05)。饲料中单独添加EN25时,刺参体腔细胞吞噬活性、呼吸爆发活性显著高于对照组(P<0.05),但其酚氧化酶和一氧化氮合酶活性并没受到显著影响(P>0.05)。与对照相比,饲料中单独添加EN25对刺参肠道可培养细菌总数无显著影响(P>0.05),却显著降低了肠道弧菌总数(P<0.05)。饲料中单独添加EN25显著降低了灿烂弧菌攻毒后刺参的死亡率(P<0.05)。饲料中单独添加0.5%的果糖可以提高刺参体腔细胞吞噬活性、呼吸爆发、酚氧化酶和一氧化氮合酶活性,并显著降低刺参的死亡率(P<0.05)。饲料中0.5%果糖与EN25复配时,刺参体腔细胞吞噬活性、呼吸爆发、酚氧化酶和一氧化氮合酶活性有所升高,其中吞噬活性得到显著提高(P<0.05),呼吸爆发在果糖与10~7 CFU/g EN25复配组显著高于对照组(P<0.05)。饲料中果糖与EN25复配与否,对刺参肠道可培养细菌总数没有显著影响(P>0.05);而当果糖与10~7 CFU/g EN25复配时,刺参肠道弧菌总数显著降低(P<0.05)。果糖与EN25复配提高了刺参抵抗灿烂弧菌的能力,并在EN25的复配浓度为10~9 CFU/g时达到显著水平(P<0.05)。10~.通过Biolog微生物自动鉴定系统和16S rDNA序列的聚类分析相结合的方法,对筛选出的对刺参育苗和幼体养成都有有益作用的3株益生菌(T13、TC22和EN25)进行了鉴定并对其最适的摇瓶培养条件进行了简单研究,结果表明T13和枯草芽孢杆菌聚为一类,最适培养条件为:接种量2%、装液量50ml/250ml、初始最佳pH 7.0、温度32℃、转速180 r/min。TC22和地衣芽孢杆菌聚为一类,最适培养条件为:接种量4%、装液量75ml/250ml、初始最佳pH 7.2、温度30℃、转速180 r/min。EN25和蜡样芽孢杆菌聚为一类,最适培养条件为:接种量1%、装液量25ml/250ml、初始最佳pH 8.0、温度30℃、转速为180 r/min。更多还原

【Abstract】 Three feeding experiments were conducted to investigate the effects of three main polysaccharides immunostimulants includingβ-glucan, peptitoglycan and chitosan on growth, immunity and disease resistance in sea cucumber (Apostichopus japonicus Selenka). Nineteen potential probiotics were isolated from intestine of sea cucumber, culturing water and the mud of the pond. Five potential probiotics were selected for further study according to inhibitory ability against phathogen, colony morphologic, species and the source of isolation. Then, the effects of those five potential probiotics on growth, immunity and disease resisitance in juvenile sea cucumber (Initial weight 0.2-0.4 g) were studied. Three strains (T13, TC22 and EN25) of five potential probiotics were found to be beneficial to juvenile sea cucumber. Then three feeding experiments were conducted to investigate the effects of T13, TC22 and EN25 on growth, immunity, microflora and disease resisitance in young sea cucumber (Initial weight 3-5 g). The interaction between three probiotics and fructooligosaccharide respectively was also studied. At the end of the experiment, those three probiotics were identified and the better condition for growth of three probiotics was preliminarily studied. The results are summarized as follows:1. An 8 week feeding trial was conducted to determine the effects of dietary supplementation ofβ-glucan on the growth, immunity and resistance of sea cucumber against Vibrio splendidus infection. A basal diet was formulated to contain 20.6% crude protein and 4.8% crude lipid. Two levels (1.25 and 2.50 g/kg) ofβ-glucan were added to the basal diet to replace wheat. After the feeding trial, a V. splendidus injection challenge was executed to test the effects ofβ-glucan on disease resistance. Enhanced growth was observed in sea cucumber fed the diet supplemented with 1.25 g glucan/kg, but not in sea cucumber fed the diet supplemented with 2.50 g glucan/kg. The coelomocyte phagocytosis activity of sea cucumber fedβ-glucan supplemented diets was significantly (P<0.05) higher than those of sea cucumbers fed the basal diet. In addition, phenoloxidase activity of coelomocytes was significantly (P<0.05) enhanced by dietary supplementation ofβ-glucan. Sea cucumbers fed 1.25 g/kg glucan had a significant (P<0.05) increase in respiratory burst of coelomocytes, compared to sea cucumber fed diet containing 2.50 gβ-glucan/kg; however, coelomocyte respiratory burst of sea cucumber fed 2.50 gβ-glucan/kg diet was not significantly different from those of sea cucumber fed the basal diet. The challenge test showed that dietary supplementation ofβ-glucan at inclusion level of 1.25 g/kg conferred significant protection to sea cucumber against V. splendidus infection. However, protective effect ofβ-glucan supplementation at 2.50 g/kg was marginal. It is concluded that dietaryβ-glucan has potential for use in diet formulations of sea cucumber to limit the adverse effects of V. splendidus; however, dosage should be an important consideration in administration.2. An 8 week feeding experiment was conducted to determine the effects of dietary peptidoglycan on growth, immunity and resistance of sea cucumber against Vibrio splendidus infection. The basal diet was supplemented with 0, 200 and 500 mg/kg peptidoglycan to formulate three experimental diets. Results showed that growth of sea cucumber was improved with increasing dietary peptidoglycan. But no significant difference was observed between the control and peptidoglycan supplementation groups. The coelomocyte phagocytosis activity of sea cucumbers fed peptidoglycan supplemented diets was significantly (P<0.05) higher than those of sea cucumbers fed the basal diet. However, respiratory burst of sea cucumber coelomocytes was not significantly influenced by dietary peptidoglycan (P>0.05). Phenoloxidase activity of coelomocytes was significantly increased in animals fed the diet with 200 mg/kg peptidoglycan, but it was not affected by dietary peptidoglycan at 500 mg/kg compared to control (P>0.05). The acid phosphatase activity was decreased with increasing dietary peptidoglycan, and it was significantly lower in sea cucumbers fed the diet with 500 mg/kg peptidoglycan compared to animals fed other two experiment diets (P<0.05). The challenge test showed that dietary peptidoglycan at inclusion level of 200 mg/kg led to significant protection to sea cucumber against V. splendidus infection (P<0.05). It is concluded that dietary peptidoglycan has potential for use in diet formulations of sea cucumber to limit the adverse effects of V. splendidus.3. 8 week feeding experiment was conducted to determine the effects of dietary chitosan on growth, immunity and resistance of sea cucumber against Vibrio splendidus infection. The basal diet was supplemented with 0, 2.5, 5 and 10~ g/kg chitosan to formulate four experimental diets. Results showed that growth of sea cucumber was not significantly affected by dietary chitosan (P>0.05). The coelomocyte density and coelomocyte phagocytosis activity of sea cucumbers fed chitosan at 10 g/kg was significantly (P<0.05) higher than those of sea cucumbers fed the basal diet. However, respiratory burst of sea cucumber coelomocytes was not significantly influenced by dietary chitosan (P>0.05). There was no significant difference in phenoloxidase activity of coelomocytes (P>0.05). The acid phosphatase activity was significantly decreased in sea cucumbers fed diets with chitosan compared to animals fed the control diet (P<0.05). The challenge test showed that dietary chitosan conferred better protection to sea cucumber against V. splendidus infection, but no significant difference was observed compared to control group (P>0.05). Therefore, further studies on application of chitosan in sea cucumber are necessary.4. A total of 182 strains were isolated from the intestine of healthy sea cucumbers, intestine and skin of sea cucumbers affected by skin ulcer, mud and sea water of the pond cultured sea cucumbers by 2216E, TSB and MRS bacterial media. All selected isolates were tested for their inhibitory activity to two pathogens of sea cucumber, Vibrio splendidus and Pseudoalteromonas nigrifaciens by spot-on-lawn method. The 19 isolates of those strains were found to be inhibitory to both pathogens or one of them. Morphological analysis was conducted including colony morphologic and Gram coloration. The potential probiotics were identified by 16S rDNA preliminarily. The results showed that all 19 isolates were Gram-positive and Bacillus strains. All strains had amylase activity and 18 of them had proteinase activity. In the end, 5 isolates of 19 strains were selected for the safety test according to source of isolate, colony morphologic and inhibition against pathogens. The safety test of 5 selected potential probiotics showed that these 5 isolates could not induce disease and mortality of sea cucumber. Therefore, these 5 isolates (TMRC14, TC116, T13, TC22 and EN25) could be used for further study to investigate whether these 5 isolates can be applied for sea cucumber to increase the health of animals or not.5. The effects of potential probiotic TMRC14 and TC116 on growth, immune capacity, and disease resistance in juvenile sea cucumber Apostichopus japonicus were studied. Animals were fed with diet containing TMRC14 or TC116 at 0, 10~5, 10~7 and 10~9 CFU/g for 30 days. At the end of the feeding trial, fifteen sea cucumbers from each aquarium were sampled for immune indices measurement. Then twenty sea cucumbers of every replicate were challenged by immersion with Vibrio splendidus. The results revealed that administration of TMRC14 at all levels or TC116 at 10~5-10~7 CFU/g diet had no significant effect on the growth of sea cucumbers (P>0.05), but the growth of sea cucumber fed with diet containing TC116 at 10~9 CFU/g was significantly decreased (P<0.05). No statistical difference was found in the total coelomocytes counts in sea cucumbers fed the diet containing TMRC14 or TC116 (P>0.05). Phagocytosis of sea cucumber coelomocytes was significantly improved in animals fed with TMRC14 at 10~9 CFU/g diet or TC116 at 10~7 CFU/g diet (P<0.05). Dietary TMRC14 at 10~9 CFU/g increased the respiratory burst activity of sea cucumber coelomocytes, although there was no significant difference (P>0.05). Respiratory burst activity of sea cucumber fed the diet containing TC116 at 10~7 CFU/g was significantly higher than the control and other two groups. The cumulative mortality after V. splendidus challenge was decreased in the sea cucumbers fed with TMRC14 at 10~7-10~9 CFU/g or TC116 at 10~5-10~7 CFU/g, however no significant differences were observed between experimental and the control groups (P>0.05). Therefore, the effect of potential probiotic TMRC14 or TC116 on growth, immunity and protective effect against V. splendidus in sea cucumber were marginal.6. The effects of potential probiotic T13, TC22 and EN25 on growth, immune capacity, and disease resistance in juvenile sea cucumber Apostichopus japonicus were studied. Animals were fed the diet containing T13, TC22 and EN25 at 0, 10~5, 10~7 and 10~9 CFU/g for 30 days. At the end of the feeding trial, fifteen sea cucumbers from each aquarium were sampled for immune indices measurement. Then twenty sea cucumbers of every replicate were challenged by immersion with Vibrio splendidus. The results revealed that administration of T13 or TC22 at 10~9 CFU/g diet had significant effect on the growth of sea cucumbers (P<0.05), but dietary EN25 had no effect on the growth of animals (P>0.05). Phagocytosis, respiratory burst activity and total nitric oxide synthase (T-NOS) activity of sea cucumber coelomocytes were significantly improved in animals fed the diet with T13 or TC22 at 10~9 CFU/g diet or EN25 at 10~7 CFU/g diet (P<0.05). No statistical difference was found in the total coelomocytes counts and superoxide dismutase (SOD) activity in sea cucumbers fed with diet containing T13 or TC22 at 10~5-10~9 CFU/g or EN25 at 10~5-10~7 CFU/g (P>0.05), however, the SOD activity was significantly decreased in the group fed with diet containing EN25 at 10~9 CFU/g(P<0.05). Dietary supplementation of T13, TC22 or EN25 did not significantly influence acid phosphatase (ACP) activity of sea cucumber coelomocytes. The cumulative mortality after V. splendidus challenge was decreased significantly in the group fed the diet with T13 or TC22 at dose of 10~9 CFU/g feed or EN25 at 10~7 CFU/g feed (P<0.05). The results of present study confirmed the potential beneficial effects of T13, TC22 and EN25 as dietary probiotic in sea cucumber.7. The effects of probiotic T13 and the synergistic effects of T13 and fructooligosaccharide (FOS) on growth, immune response, microflora and disease resistance in sea cucumber Apostichopus japonicus (initial weight 4.893±0.028 g) were studied. Animals were fed on diet with T13 at doses of 0, 10~7, and 10~9 CFU/g feed with or without 0.5% FOS for 56d. At the end of the feeding trial, six sea cucumbers per tank were sampled for bacterial quantification and immune indices measurement. Then all the sea cucumbers left were challenged by injecting Vibrio splendidus. The results revealed that the SGR of sea cucumber fed the diet containing different doses of T13 with or without FOS was higher than control group, though no significant difference was observed (P>0.05). The phagocytosis activity, respiratory burst activity, phenoloxidase activity and total nitric oxide synthase of sea cucumber fed with diet containing T13 were improved, among these four indices, phenoloxidase activity was significantly higher than control (P<0.05). The total viable bacteria and Vibrio bacteria counts were enhanced significantly in sea cucumber fed with T13 at dose of 10~9 CFU/g feed (P<0.05). The cumulative mortality after V. splendidus challenge was decreased significantly in the group fed with T13 at dose of 10~9 CFU/g feed (P<0.05). The phagocytosis activity, respiratory burst activity and phenoloxidase activity of sea cucumbers fed with 0.5% FOS were increased significantly. And the sea cucumber fed with 0.5% FOS had a significant lower cumulative mortality than the control group (P<0.05). The total viable bacteria counts of sea cucumber were not influenced by dietary FOS with or without T13 (P>0.05). The total Vibrio bacteria counts was significantly decreased in sea cucumber fed with 0.5% FOS (P<0.05), however, it was not affected in sea cucumber fed diet containing FOS and T13 (P>0.05). The combination of 0.5% FOS with T13 increased the phagocytosis activity, respiratory burst activity and phenoloxidase activity of sea cucumbers without significant difference compared to control (P>0.05). The total nitric oxide synthase of sea cucumber fed with diet containing FOS and T13 at 10~9 CFU/g was significantly increased compared to control (P<0.05). The combination of 0.5% FOS with T13 improved disease resistance of sea cucumber compared with control (P<0.05).8. The effects of probiotic TC22 and the synergistic effects of TC22 and fructooligosaccharide (FOS) on growth, immune capacity, microflora and disease resistance in sea cucumber Apostichopus japonicus (initial weight 4.918±0.022 g) were studied. Animals were fed the diet with TC22 at doses of 0, 10~7, and 10~9 CFU/g feed with or without 0.5% FOS for 56d. At the end of the feeding trial, six sea cucumbers per tank were sampled for bacterial quantification and immune indices measurement. Then all the sea cucumbers left were challenged by injecting Vibrio splendidus. The results revealed that different doses of TC22 with or without FOS had no significant influence on sea cucumber growth (P>0.05). However, along with the increasing of TC22 dosage, the phagocytosis activity, respiratory burst activity and phenoloxidase activity of sea cucumber were improved significantly (P<0.05), while the total nitric oxide synthase acitivity of animals was not significantly influenced (P>0.05). The total viable bacteria counts was significantly enhanced in the sea cucmber fed with TC22 at dose of 10~9 CFU/g feed (P<0.05). But no significant difference was found on total Vibrio counts between trial treatment and the control (P>0.05). The cumulative mortality after V. splendidus challenge decreased significantly in the groups fed with TC22 at dose of 10~9 CFU/g feed (P<0.05). Animals fed with 0.5% FOS showed higher immune response and lower cumulative mortality than the control group (P<0.05). The combination of 0.5% FOS with TC22 at dose of 10~9 CFU/g feed produced significantly positive synergistic effects on sea cucumber immune responses and disease resistance compared with control group(P<0.05). Results of this experiment confirmed the potential of TC22 as dietary probiotic and the synergistic effects of TC22 and FOS in sea cucumber.9. The effects of probiotic EN25 and the synergistic effects of EN25 and fructooligosaccharide (FOS) on growth, immune response, microflora and disease resistance in sea cucumber Apostichopus japonicus (initial weight 3.127±0.012 g) were studied. Animals were fed the diet with EN25 at doses of 0, 10~7, and 10~9 CFU/g feed with or without 0.5% FOS for 56d. At the end of the feeding trial, six sea cucumbers per tank were sampled for bacterial quantification and immune indices measurement. Then all the sea cucumbers left were challenged by injecting Vibrio splendidus. The results showed that the SGR of sea cucumber was not significantly influenced by dietary EN25 with or without FOS (P>0.05). The phagocytosis activity and respiratory burst was significantly increased by dietary EN25 without FOS (P<0.05). Howerver, the phenoloxidase activity and total nitric oxide synthase of sea cucumber were not significantly influenced by dietary EN25 without FOS (P>0.05). Dietary EN25 without FOS did not produce significant influence on the counts of total viable bacteria of sea cucumber (P>0.05), but it significantly decreased the counts of Vibrio bacteria of animals (P<0.05). The cumulative mortality after V. splendidus challenge was decreased significantly in sea cucumber fed the diet containing EN25 without FOS (P<0.05). Sea cucumbers fed with 0.5% FOS had higher phagocytosis activity, respiratory burst activity, phenoloxidase activity and total nitric oxide synthase than control group, and had a significant lower cumulative mortality after challenged by V. splendidus (P<0.05). The combination of 0.5% FOS and EN25 increased the phagocytosis activity, respiratory burst activity, phenoloxidase activity and total nitric oxide synthase of sea cucumbers. Among these immune indices, the phagocytosis activity was significantly increased in sea cucumber fed FOS and EN25 (P<0.05) and respiratory burst activity was also significantly improved in sea cucumber fed on FOS combined with EN25 at 10~7 CFU/g (P<0.05). The total viable bacteria counts of sea cucumber intestine was not affected by dietary FOS with or without EN25 (P>0.05). The total Vibrio bacteria counts was significantly decreased in sea cucumber fed the diet with 0.5% FOS and EN25 at dose of 10~7 CFU/g (P<0.05). The disease resistance was significantly increased in sea cucumber fed on 0.5% FOS with EN25 at dose of 10~9 CFU/g.10~. The selected probiotics (T13, TC22 and EN25) were identified by Biolog microbes identification system and the cluster analysis of 16S rDNA. The optimum condition of culture for these three probiotics was preliminarily studied. The results showed that T13 was very similar to Bacillus subtilis and the optimum condition for growth was as follows: inoculation level 2%, liquid volumn 50ml/250ml, initial pH 7.0, temperature 32℃, rotating speed 180 r/min. TC22 was very similar to Bacillus licheniformis and the optimum condition for growth was as follows: inoculation level 4%, liquid volumn 75ml/250ml, initial pH 7.2, temperature 30℃, rotating speed 180 r/min. EN25 was very similar to Bacillus cereus and the optimum condition for growth was as follows: inoculation level 1%, liquid volumn 25ml/250ml, initial pH 8.0, temperature 30℃, rotating speed 180 r/min.更多还原