刺参（Apostichopus japonicus Selenka）高效免疫增强剂的筛选与应用
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摘要
本文选择我国北方海参养殖的主要种类刺参(Apostichopus japonicus Selenka)为研究对象,通过一系列在室内循环水养殖系统中开展的养殖实验,探讨了在刺参基础饲料中分别添加具有免疫促进作用的营养素(维生素C和维生素E)、多糖(β-葡聚糖)、低聚糖(低聚果糖、甘露寡糖)以及微生态制剂(枯草芽孢杆菌、硒酵母、酵母培养物)对刺参生长、免疫及抗病力的影响。主要研究内容如下：
1.以初始体重为(5.08±0.08)g的健康刺参(Apostichopus japonicus Selenka)为实验对象,采用3×3双因子设计,在基础饲料中分别添加0、500和2000 mg kg-1包膜维生素C,每种维生素C水平分别添加0、625和1250 mg kg-1β－葡聚糖,共制成9种实验饲料,在室内水循环系统中进行了为期9周的饲喂实验,探讨了饲料中不同含量的维生素C和β-葡聚糖对刺参生长性能、免疫力和抗病力的影响。研究结果表明：维生素C和β-葡聚糖对刺参的特定生长率都有显著影响,且同时添加高剂量维生素C和β-葡聚糖的实验组刺参的特定生长率显著高于对照组(P<0.05)；维生素C和β-葡聚糖对刺参体腔细胞的吞噬活性、呼吸爆发活性、酚氧化酶活性和酸性磷酸酶活性均有显著影响(P<0.05),当饲料中不添加维生素C时,β-葡聚糖的添加显著增强了刺参的呼吸爆发活性、酚氧化酶活性和酸性磷酸酶活性(P<0.05),当饲料中只添加500 mgkg-1维生素C时,添加625mg kg-1的β-葡聚糖可以增强显著刺参的吞噬活性和酚氧化酶活性(P<0.05),而添加1250 mg kg-1的β-葡聚糖可以显著增强刺参的呼吸爆发活性和酸性磷酸酶活性(P<0.05)。当饲料中添加2000 mg kg-1维生素C时,添加625 mg kg-1的β-葡聚糖可以显著增强刺参的酚氧化酶活性和酸性磷酸酶活性(P<0.05),而添加1250 mg kg-1的β-葡聚糖可以显著增强刺参的吞噬活性、呼吸爆发活性、酚氧化酶活性和酸性磷酸酶活性(P<0.05)。饲料中不同浓度的β-葡聚糖对刺参感染灿烂弧菌后的累积死亡率有显著影响(P<0.05),当饲料中添加500 mg kg-1和2000 mg kg-1的维生素C时,随着饲料中β-葡聚糖含量的升高,刺参的累积死亡率呈下降的趋势,尤其饲料中添加500 mg kg-1维生素C和高水平的β-葡聚糖(1250 mg kg-1)的实验组显著降低了刺参感染灿烂弧菌14d内的累积死亡率(P<0.05)。在本实验条件下,维生素C和β-葡聚糖对刺参体腔细胞的吞噬活性和呼吸爆发活性存在显著的交互作用(P<0.05)。
2.以初始体重为(5.09±0.03)g的刺参(Apostichopus japonicus Selenka)为实验对象,在室内水循环系统中进行了为期9周的饲喂实验,探讨了饲料中不同含量的维生素C(包膜维生素C)和维生素E(α-生育酚醋酸酯)对刺参生长性能、免疫力以及抗病力的影响。本实验采用3×3双因子实验设计,在基础饲料中分别添加0、500和2000 mg kg-1的维生素C,每种维生素C水平分别添加0、80和250 mg kg-1的维生素E,共制成9种实验饲料,每个处理3个重复。研究结果表明,维生素C和维生素E对刺参特定生长率均有显著影响(P<0.05),且当饲料中不添加维生素C或添加2000 mg kg-1维生素C时,添加250 mg kg-1的维生素E实验组刺参的特定生长率显著高于对照组(P<0.05)；维生素C和维生素E对刺参体腔细胞的吞噬活性、酚氧化酶活性和酸性磷酸酶活性都有显著的影响(P<0.05),当饲料中单独添加维生素C时,刺参的吞噬活性随着维生素C添加量的增加有上升的趋势,且2000 mg kg-1维生素C添加组刺参的吞噬活性显著高于对照组(P<0.05)。当饲料中不添加维生素C时,添加250 mg kg-1的维生素E实验组刺参的吞噬活性显著高于对照组(P<0.05)。当饲料中添加添加500 mg kg-1维生素C时,刺参的吞噬活性、呼吸爆发活性和酚氧化酶活性随着饲料中维生素E的添加有上升的趋势,且添加250 mg kg-1的维生素E实验组刺参的酚氧化酶活性显著高于对照组(P<0.05)。当饲料中添加2000 mg kg-1维生素C时,刺参的呼吸爆发活性、酚氧化酶活性和酸性磷酸酶活性随着饲料中维生素E的添加有上升的趋势,且添加250 mg kg-1的维生素E实验组刺参的吞噬活性、呼吸爆发活性、酚氧化酶活性和酸性磷酸酶活性都显著高于对照组(P<0.05)。饲料中不同浓度的维生素E对刺参感染灿烂弧菌后的累积死亡率有显著影响(P<0.05),在任何维生素C添加水平下,随着饲料中维生素E含量的升高,刺参的累积死亡率呈下降趋势,尤其饲料中同时添加高剂量的维生素C(2000 mg kg-1)和维生素E(250mg kg-1)的实验组显著降低了刺参感染灿烂弧菌14d内的累积死亡率(P<0.05)。在本实验条件下,维生素C和维生素E对刺参体腔细胞的吞噬活性表现出显著的交互作用(P<0.05)。
3.以初始体重为(5.06±0.10)g的刺参(Apostichopus japonicus Selenka)为实验对象,在室内水循环系统中进行了为期8周的饲喂实验,探讨了饲料中不同含量的低聚果糖和枯草芽孢杆菌对刺参生长性能、免疫力、肠道菌群以及抗病力的影响。本实验采用3×3双因子实验设计,在基础饲料中分别添加0、0.2%和0.5%的枯草芽孢杆菌,每种枯草芽孢杆菌水平分别添加0%、0.25%和0.5%的低聚果糖(有效含量为95%),共制成9种实验饲料,每个处理三个重复。研究结果表明,当不添加低聚果糖时,0.2%枯草芽孢杆菌显著提高了刺参的特定生长率(SGR)和刺参体腔细胞吞噬活性(P<0.05),显著降低了刺参感染灿烂弧菌后的累积死亡率(P<0.05)。当不添加枯草芽孢杆菌时,刺参SGR、PO和吞噬活性随着低聚果糖的添加而升高,且饲料中添加0.5%低聚果糖时,刺参体腔细胞破碎液中吞噬活性、酚氧化酶活性和酸性磷酸酶活性都显著高于对照组(P<0.05),当饲料中枯草芽孢杆菌含量为0.2%时,0.5%低聚果糖可以显著提高刺参的特定生长率、吞噬活性、PO活性(P<0.05),显著降低刺参肠道弧菌数及刺参感染灿烂弧菌后的累积死亡率(P<0.05)；当饲料中枯草芽孢杆菌含量为0.5%时,0.25%低聚果糖添加组显著提高了刺参的酚氧化酶活性和酸性磷酸酶活性(P<0.05),降低了刺参感染细菌后14d内的累积死亡率(P<0.05)。枯草芽孢杆菌和低聚果糖对刺参的吞噬活性、酚氧化酶活性、弧菌数目以及抗病力产生了明显的交互作用(P<0.05)。
4.以初始体重(3.04±0.01)g的刺参(Apostichopus japonicus Selenka)为研究对象,探讨饲料中添加低聚果糖或甘露寡糖对其生长、肠道菌群和非特异性免疫力的影响。分别向基础饲料中添加0%(对照组)、0.25%和0.5%低聚果糖或甘露寡糖,配制出5种实验饲料,在室内循环水系统中进行为期8周的养殖实验。每种饲料为一处理,每个处理设3个重复,每个缸(50 1)为一重复放养22头刺参。饲喂实验结束后测定实验刺参的生长及相关的免疫学指标,同时每个重复取13头刺参进行攻毒实验。结果表明：(1)0.5%低聚果糖组刺参的特定生长率(SGR)显著高于对照组(P<0.05),但与0.25%低聚果糖组差异不显著(P>0.05)；刺参肠道总菌数不受低聚果糖添加量的影响,但是两低聚果糖组刺参肠道中的弧菌数显著低于对照组(P<0.05)；随着饲料中低聚果糖含量的升高,刺参体腔细胞的吞噬活性、呼吸爆发活性、体腔细胞上清中的PO活性和ACP活性都有升高的趋势；攻毒实验结果显示,0.5%低聚果糖组刺参感染灿烂弧菌(V.splendidus)14d的死亡率显著低于对照组(P<0.05)；(2)饲料中甘露寡糖含量对刺参的SGR影响不显著(P>0.05)；饲料中添加甘露寡糖可以提高刺参肠道中的总菌数,降低肠道中的弧菌数,0.25%和0.5%甘露寡糖组刺参肠道中的总菌数均显著高于对照组(P<0.05),但只有0.5%甘露寡糖添加组刺参肠道中的弧菌数显著低于对照组(P<0.05)。0.25%甘露寡糖组刺参体腔细胞吞噬活性和呼吸爆发活性显著高于其他两组(P<0.05)；随着饲料中甘露寡糖添加量的升高,刺参体腔细胞上清中的酚氧化酶(PO)活性和酸性磷酸酶(ACP)活性都有升高的趋势,但是各处理组之间无显著差异(P>0.05)。0.25%甘露寡糖组刺参感染灿烂弧菌14d的累积死亡率显著降低(P<0.05)；(3)综上所述,饲料中分别添加0.5%的低聚果糖或者0.25%的甘露寡糖对刺参生长、免疫力和抗病力的效果较好。二者均可以作为安全高效的口服免疫增强剂应用于刺参的养殖生产。
5.以初始体重为(3.52±0.08)g的刺参(Apostichopus japonicus Selenka)为实验对象,在室内水循环系统中进行了为期8周的饲喂实验,探讨了饲料中不同含量的硒酵母和维生素E(VE)对刺参生长性能、免疫力以及抗病力的影响。本实验采用2×4双因子实验设计,在基础饲料中分别添加0和250 mg kg-1的VE,每种VE水平分别添加0、100、300和600 mg kg-1的硒酵母,共制成8种实验饲料,每个处理3个重复。研究结果表明,硒酵母和维生素E对刺参的特定生长率均有显著影响,饲料中添加硒酵母和维生素E的不同配伍均可显著提高刺参特定生长率(P<0.05),高剂量硒酵母(600 mg kg-1)和维生素E(250mg kg-1)添加组刺参的特定生长率最高；当饲料中不添加维生素E时,随着硒酵母添加量的增加,刺参的吞噬活性、呼吸爆发活性、酚氧化酶活性和酸性磷酸酶活性呈剂量依赖性升高,且600 mg kg-1硒酵母添加组刺参的吞噬活性、呼吸爆发活性、酚氧化酶活性和酸性磷酸酶活性均显著高于对照组(P<0.05)。当饲料中添加250 mg kg-1的维生素E时,添加600 mg kg-1硒酵母实验组刺参的吞噬活性、呼吸爆发活性和酚氧化酶活性高于其他各处理组,并显著高于对照组(P<0.05)；维生素E对过氧化氢酶活性(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活性、超氧化物歧化酶(SOD)活性都有显著的影响(P<0.05),而硒酵母仅对SOD和GSH-Px活性影响显著(P<0.05),且二者合用对刺参的SOD和GSH-Px活性有显著的交互作用(P>0.05)；当饲料中不添加维生素E时,饲料中单独添加硒酵母可以降低刺参感染灿烂弧菌后的14d内的累积死亡率,且随着添加量的增加刺参的累积死亡率有下降的趋势(P>0.05)。当饲料中添加添加250 mg kg-1的维生素E时,各水平硒酵母添加组的刺参感染灿烂弧菌后14d的累积死亡率显著低于对照组(P<0.05)。其中同时添加600 mg kg-1硒酵母和250 mg kg-1的维生素E的实验组显著提高了刺参的特定生长率、非特异性免疫力和抗氧化酶活力,显著降低了刺参感染灿烂弧菌后的累积死亡率,表现出较好的复合益生效果,是本研究最优的功能组合。
6.以初始体重为(3.49±0.06)g的刺参(Apostichopus japonicus Selenka)为实验对象,在室内水循环系统中进行了为期8周的饲喂实验,探讨了饲料中不同含量的酵母培养物(达农威益康XP)和枯草芽孢杆菌对刺参生长性能、免疫力、肠道菌群以及抗病力的影响。本实验采用2×4双因子实验设计,在基础饲料中分别添加0和0.2%的枯草芽孢杆菌,每种枯草芽孢杆水平分别添加0、0.1%、0.2%和0.4%的酵母培养物,共制成8种实验饲料,每个处理三个重复。研究结果表明,当不添加枯草芽孢杆菌时,酵母培养物显著提高了刺参的吞噬活性、呼吸爆发活性、ACP和SOD活性(P<0.05),显著降低了刺参感染灿烂弧菌14d内的累积死亡率(P<0.05)；当添加0.2%的枯草芽孢杆菌时,酵母培养物添加组的刺参特定生长率、肠道总菌数、呼吸爆发活性、ACP等的活性都显著高于对照组(P<0.05),且当饲料中酵母培养物的含量达到0.4%时,刺参体腔细胞的吞噬活性和呼吸爆发活性达到了最高水平。相应地,刺参感染细菌后14d内的累积死亡率也显著低于对照组(P<0.05)。双因素统计分析结果表明,酵母培养物(达农威益康XP)和枯草芽孢杆菌对刺参肠道总菌数和非特异性免疫力都产生了明显的交互作用(P<0.05)。
Six feeding trials were conducted in indoor circulating system to investigate the effects of dietary vitamin C,vitamin E,β-glucan, Bacillus subtilis, fructooligosaccharides (FOS), mannooligosaccharides (MOS), selenium yeast (Se-yeast) and yeast culture (Diamond V XP Yeast Culture, XP) on growth, immunity and disease resistance in sea cucumber (Apostichopus japonicus Selenka). The results are summarized as follows:
1.A 9-week feeding experiment was conducted to investigate the effect of vitamin C andβ-glucan on the growth performance, immunity and disease resistance of sea cucumber (A. japonicus Selenka).Five hundred and forty individuals (initial body weight:5.08±0.08) g, mean±S.E) were fed with nine practical diets according to a 3×3 factorial design:the basal diet as the control diet supplemented with three levels of vitamin C (0,500,2000 mg kg-1 diet), and each crossed with 0,625 or 1250 mg kg-1β-glucan. After 9 weeks, five sea cucumbers per tank were sampled for immune indices determination. Then all the sea cucumbers left were challenged by Vibrio splendidus. The results showed that dietary vitamin C andβ-glucan had significant effects on specific growth rate (SGR) of A. japonicus (P<0.05).SGR of the seacucumber with the combination of 2000 mg kg-1 vitamin C and 1250 mg kg-1β-glucan was significantly higher than the control group (P<0.05).Activities of phagocytosis, respiratory burst (RB), phenoloxidase (PO) and acid phosphatase (ACP) were significantly influenced by dietary vitamin C andβ-glucan (P<0.05).In the absence of dietary vitamin C, activities of RB,PO and ACP significantly increased with increasingβ-glucan (P<0.05). In the groups with 500 mg kg-1 vitamin C, sea cucumbers fed with 625 mg kg-1β-glucan had significant higher phagocytosis and PO activity than the control group (P<0.05),and those fed with 1250 mg kg-1β-glucan had significant higher RB activity and ACP activity (P<0.05).In the groups with 2000 mg vitamin C kg-1 diet,625 mg kg-1β-glucan supplementation remarkably increased PO activity and ACP activity (P<0.05), while 1250 mg kg-1β-glucan supplementation significantly enhanced phagocytosis, RB activity, PO activity and ACP activity (P<0.05). Moreover, dietaryβ-glucan had significant effect on cumulative mortality of sea cucumbers after 14 days following V. splendidus exposure (P<0.05).Cumulative mortality was significantly affected by dietaryβ-glucan (P<0.05), and cumulative mortality decreased with the increasing doses ofβ-glucan at 500 and 2000 mg kg-1 vitamin C level.The group fed diets supplemented with 500 mg kg-1 vitamin C and 1250 mg kg-1β-glucan had notably lower cumulative mortality compared to the control (P<0.05).Under the experimental conditions, dietary vitamin C andβ-glucan had a synergistic effect on enhancing phagocytosis and RB activity of sea cucumber(P<0.05).
2.A 9-week feeding experiment was conducted to investigate the effects of vitamin C and vitamin E on the growth performance, immunity and disease resistance of sea cucumber (A. japonicus Selenka).Five hundred and forty individuals (initial body weight:5.06±0.10 g, mean±S.E) were fed with nine practical diets according to a 3×3 factorial design:the basal diet as the control diet supplemented with three levels of vitamin C (0, 500,2000 mg kg-1 diet), and each crossed with 0,80 or 250 mg kg-1 vitamin E. After 9 weeks,five sea cucumbers per tank were sampled for immune indices determination. Then all the sea cucumbers left were challenged by Vibrio splendidus.The results showed that specific growth rate (SGR) of A. japonicus was significantly affected by both vitamin C and vitamin E (P<0.05), and SGR of the groups with 250 mg kg-1 vitamin E alone and 250 mg kg-1 vitamin E at 2000 mg kg-1 vitamin C level were significant higher than the control group (P<0.05). Dietary vitamin C and vitamin E had significant effects on phagocytosis, phenoloxidase (PO) activity and acid phosphatase (ACP) activity (P<0.05). In the groups with vitamin C alone, phagocytosis increased with the increasing doses of vitamin C and was significantly enhanced by 2000 mg kg-1 vitamin C(P<0.05).In the groups without vitamin C supplementation, phagocytosis of sea cucumbers fed with 250 mg kg-1 vitamin E was significantly higher than the control group(P<0.05).In the groups with 500 mg kg-1 vitamin C, phagocytosis, respiratory burst (RB) activity and PO activity showed increasing tendency with the increasing administration doses of vitamin E (P<0.05), and PO activity of the group fed with 250 mg kg-1 vitamin E significantly increased compared with the control (P<0.05).In the groups with 2000 mg kg-1 supplementation of vitamin C, RB activity and PO activity and ACP activity increased with the doses of vitamin E, and all the immune indices in the group fed with 250 mg kg-1 vitamin E was significant higher than the control group (P<0.05).Moreover, dietary vitamin E had significant effect on cumulative mortality of sea cucumbers after 14 days following V. splendidus exposure (P<0.05). Cumulative mortality decreased with the increasing doses of vitamin E at each vitamin C level.The group fed with diets supplemented with 2000 mg kg-1 vitamin C and 250 mg kg-1 vitamin E had notably lower cumulative mortality compared to the control (P<0.05).Under the experimental conditions, dietary vitamin C and vitamin E had a synergistic effect on enhancing phagocytosis of sea cucumber (P<0.05).
3.An 8-week feeding experiment was conducted to investigate the interaction of probiotic Bacillus subtilis and prebiotic fructooligosaccharide (FOS) on the growth performance, immunity, intestinal microflora and disease resistance of sea cucumber (A.japonicus Selenka).Five hundred and forty individuals (initial body weight:5.09±0.03 g, mean±S.E) were fed nine practical diets according to a 3×3 factorial design:the basal diet as the control diet supplemented with three levels of B. subtilis (0,0.2 or 0.5%),crossed with 0,0.25% or 0.50% FOS.After 8 weeks, five sea cucumbers per tank were sampled for bacterial quantification and immune indices determination. Then all remaining sea cucumbers were challenged by Vibrio splendidus.The results showed that dietary B. subtilis significantly increased the specific growth rate (SGR), phagocytosis and acid phosphatase (ACP) activity of sea cucumbers, the counts of total viable bacteria (TBC) and disease resistance to V.splendidus (P<0.05),whereas the counts of Vibrio (VBC) decreased (P<0.05).However, dietary B. subtilis had no significant effect on phenoloxidase (PO) activity (P>0.05).The SGR, PO activity, TBC and VBC were significantly affected by dietary FOS.In the groups with B. subtilis alone, SGR and phagocytosis of 0.2% B. subtilis administration significantly increased compared to the control group (P<0.05), while cumulative mortality of sea cucumbers significantly decreased (P<0.05).In the groups without B. subtilis, SGR, phagocytosis and PO activity increased with the doses of FOS,and phagocytosis, PO activity and ACP activity of the group fed with 0.5% FOS significantly enhanced compared the control (P<0.05). In the groups with 0.2% B. subtilis,0.5% FOS supplementation significantly increased the SGR, phagocytosis and PO activity of sea cucumbers, while notably decreased VBC and cumulative mortality (P<0.05).In the groups fed with 0.5% B. subtilis,0.25% FOS supplementation significantly increased the PO activity and ACP activity of sea cucumbers, while significantly decreased cumulative mortality. Under the experimental conditions, dietary B. subtilis and FOS had a synergistic effect on enhancing phagocytosis, PO activity and disease resistance of sea cucumber (P<0.05).
4.An 8-week feeding experiment was conducted to evaluate the effects of FOS and MOS on growth, non-specific immunity of sea cucumber (A. japonicus Selenka) as well as its resistance against V.splendidus.FOS and MOS were administered to sea cucumbers through the diets at three levels (0,0.25% and 0.5%, respectively). The basal diet without FOS or MOS was used as the control.Each diet was randomly allocated to triplicate groups of sea cucumbers in indoor 50 1 tanks with circulating seawater and constant aeration. And each tank was stocked with 22 sea cucumbers (initial average weight 3.04±0.06 g).The results showed that:Ⅰ. FOS supplemented at 0.5% significantly enhanced the specific growth rate (SGR) of sea cucumbers (P<0.05).However, no significant difference in SGR was observed among the group containing 0.25% FOS and the control groups (P>0.05).Vibrio bacteria counts (VBC) instead of total bacteria counts (TBC) were significantly affected by FOS administration (P<0.05). Phagocytosis, RB activity, PO activity and ACP activity significantly increased with the increasing doses of FOS, and all of these four immune indices were remarkably enhanced by 0.5% FOS supplementation (P<0.05).Ⅱ.Sea cucumbers fed with 0.25% and 0.5% MOS supplementation had no significant difference in SGR (P>0.05).The groups with two levels of MOS had higher TBC than the control group (P<0.05), and 0.5% MOS had lower VBC compared with the control (P<0.05). Phagocytosis and RB activity in sea cucumbers fed with 0.25% MOS was significant high when compared with the control and 0.5% MOS supplementation group (P<0.05).PO activity and ACP activity increased with the increasing doses of MOS, but there were no significant differences among the three treatments (P>0.05). The challenge experiment showed that sea cucumbers fed the diet with 0.25% MOS had significantly lower cumulative morbidity compared with the control group (P<0.05).Ⅲ.These results suggested that feeding FOS at a dose of 0.5% or MOS at a dose of 0.25% could enhance growth, non-specific immunity as well as resistance against V. splendidus of sea cucumber.
5.An 8-week feeding experiment was conducted to investigate the effect of selenium yeast (Se-yeast) and vitamin E on the growth performance, immunity and disease resistance of sea cucumber (A. japonicus Selenka).Six hundred individuals (initial body weight:3.52±0.08 g, mean±S.E) were fed eight practical diets according to a 2×4 factorial design: the basal diet as the control diet supplemented with two levels of vitamin E (0,250 mg kg-1 diet), crossed with 0,100,300 or 600 mg kg-1 selenium yeast (Se-yeast). The results showed that dietary Se-yeast and vitamin E had significant influence on specific growth rate (SGR) (P<0.05).And SGR of the sea cucumbers fed with the combination of 600 mg kg-1 Se-yeast and 250 mg kg-1 vitamin E was significantly increased when compared with the control group (P<0.05). Significant higher phagocytosis,respiratory burst (RB) activity, phenoloxidase (PO) activity and acid phosphatase (ACP) activity were observed in the group with 600 mg kg-1 Se-yeast alone compared with the control group (P<0.05). In the groups with 250 mg kg-1 vitamin E, sea cucumbers fed with 600 mg kg-1 Se-yeast had significant higher phagocytosis, RB activity and PO activity when compared with the control group (P<0.05).Dietary vitamin E had significant effect on catalase (CAT) activity,glutathione peroxidase (GSH-Px) activity and total superoxide dismutase (SOD) activity (P<0.05), but Se-yeast significantly influenced the activities of only GSH-Px and SOD (P<0.05), and there were significantly interactions between Se-yeast and vitamin E on GSH-Px activity and SOD activity (P<0.05). The challenge trial showed that dietary Se-yeast and vitamin E had significant effects on cumulative mortality after 14 days following V. splendidus exposure (P<0.05).In the absent of vitamin E, cumulative mortality showed increasing tendency with increasing Se-yeast, but there were no significant difference compared to the control (P>0.05).In the groups with 250 mg kg-1 vitamin E, sea cucumbers fed each Se-yeast levels had significant lower cumulative mortality compared with other groups and the control group (P<0.05). It could be concluded that the group with 600 mg kg-1 Se-yeast and 250 mg kg-1 vitamin E could significantly enhance the SGR and immunity, keep the antioxidant enzymes in a balanced level and decrease cumulative mortality of sea cucumbers. Therefore, combination of 600 mg kg-1 Se-yeast and 250 mg kg-1 vitamin E was the optimal group in the present study.
6.An 8-week feeding experiment was conducted to investigate the effect of yeast culture (Diamond V XP Yeast Culture, XP) and Bacillus subtilis on the growth performance, intestinal microflora, immunity and disease resistance of sea cucumber (A.japonicus Selenka).Six hundred individuals (initial body weight:3.49±0.06 g, mean±S.E) were fed eight practical diets according to a 2×4 factorial design:the basal diet as the control diet supplemented with two levels of Bacillus subtilis (0,0.2%), crossed with 0,0.1%, 0.2% or 0.4% yeast culture (XP).After 8 weeks, five sea cucumbers per tank were sampled for immune indices determination. Then all remaining sea cucumbers were challenged by Vibrio splendidus.The results showed that dietary XP had significant influence on specific growth rate (SGR), total bacteria counts (TBC) and Vibrio bacteria counts (VBC), activities of phagocytosis, respiratory burst (RB) activity, phenoloxidase (PO) activity and acid phosphatase (ACP) activity and cumulative mortality, but Bacillus subtilis significant influenced only SGR, counts of TBC and VBC, cumulative mortality (P<0.05).Evident increases of all the immune enzymes tested in the present study were noticed in sea cucumbers fed with o.4% XP in the groups without Bacillus subtilis (P<0.05). In the groups with 0.2% Bacillus subtilis, SGR, TBC, RB, ACP of each level of XP administration significantly increased compared to the group (P<0.05), while cumulative mortality of sea cucumbers significantly decreased (P<0.05).Under the experimental conditions, dietary vitamin XP and Bacillus subtilis had a synergistic effect on enhancing TBA, phagocytosis, RB activity, PO activity and ACP activity of sea cucumber (P<0.05).
1.以初始体重为(5.08±0.08)g的健康刺参(Apostichopus japonicus Selenka)为实验对象,采用3×3双因子设计,在基础饲料中分别添加0、500和2000 mg kg-1包膜维生素C,每种维生素C水平分别添加0、625和1250 mg kg-1β－葡聚糖,共制成9种实验饲料,在室内水循环系统中进行了为期9周的饲喂实验,探讨了饲料中不同含量的维生素C和β-葡聚糖对刺参生长性能、免疫力和抗病力的影响。研究结果表明：维生素C和β-葡聚糖对刺参的特定生长率都有显著影响,且同时添加高剂量维生素C和β-葡聚糖的实验组刺参的特定生长率显著高于对照组(P<0.05)；维生素C和β-葡聚糖对刺参体腔细胞的吞噬活性、呼吸爆发活性、酚氧化酶活性和酸性磷酸酶活性均有显著影响(P<0.05),当饲料中不添加维生素C时,β-葡聚糖的添加显著增强了刺参的呼吸爆发活性、酚氧化酶活性和酸性磷酸酶活性(P<0.05),当饲料中只添加500 mgkg-1维生素C时,添加625mg kg-1的β-葡聚糖可以增强显著刺参的吞噬活性和酚氧化酶活性(P<0.05),而添加1250 mg kg-1的β-葡聚糖可以显著增强刺参的呼吸爆发活性和酸性磷酸酶活性(P<0.05)。当饲料中添加2000 mg kg-1维生素C时,添加625 mg kg-1的β-葡聚糖可以显著增强刺参的酚氧化酶活性和酸性磷酸酶活性(P<0.05),而添加1250 mg kg-1的β-葡聚糖可以显著增强刺参的吞噬活性、呼吸爆发活性、酚氧化酶活性和酸性磷酸酶活性(P<0.05)。饲料中不同浓度的β-葡聚糖对刺参感染灿烂弧菌后的累积死亡率有显著影响(P<0.05),当饲料中添加500 mg kg-1和2000 mg kg-1的维生素C时,随着饲料中β-葡聚糖含量的升高,刺参的累积死亡率呈下降的趋势,尤其饲料中添加500 mg kg-1维生素C和高水平的β-葡聚糖(1250 mg kg-1)的实验组显著降低了刺参感染灿烂弧菌14d内的累积死亡率(P<0.05)。在本实验条件下,维生素C和β-葡聚糖对刺参体腔细胞的吞噬活性和呼吸爆发活性存在显著的交互作用(P<0.05)。
2.以初始体重为(5.09±0.03)g的刺参(Apostichopus japonicus Selenka)为实验对象,在室内水循环系统中进行了为期9周的饲喂实验,探讨了饲料中不同含量的维生素C(包膜维生素C)和维生素E(α-生育酚醋酸酯)对刺参生长性能、免疫力以及抗病力的影响。本实验采用3×3双因子实验设计,在基础饲料中分别添加0、500和2000 mg kg-1的维生素C,每种维生素C水平分别添加0、80和250 mg kg-1的维生素E,共制成9种实验饲料,每个处理3个重复。研究结果表明,维生素C和维生素E对刺参特定生长率均有显著影响(P<0.05),且当饲料中不添加维生素C或添加2000 mg kg-1维生素C时,添加250 mg kg-1的维生素E实验组刺参的特定生长率显著高于对照组(P<0.05)；维生素C和维生素E对刺参体腔细胞的吞噬活性、酚氧化酶活性和酸性磷酸酶活性都有显著的影响(P<0.05),当饲料中单独添加维生素C时,刺参的吞噬活性随着维生素C添加量的增加有上升的趋势,且2000 mg kg-1维生素C添加组刺参的吞噬活性显著高于对照组(P<0.05)。当饲料中不添加维生素C时,添加250 mg kg-1的维生素E实验组刺参的吞噬活性显著高于对照组(P<0.05)。当饲料中添加添加500 mg kg-1维生素C时,刺参的吞噬活性、呼吸爆发活性和酚氧化酶活性随着饲料中维生素E的添加有上升的趋势,且添加250 mg kg-1的维生素E实验组刺参的酚氧化酶活性显著高于对照组(P<0.05)。当饲料中添加2000 mg kg-1维生素C时,刺参的呼吸爆发活性、酚氧化酶活性和酸性磷酸酶活性随着饲料中维生素E的添加有上升的趋势,且添加250 mg kg-1的维生素E实验组刺参的吞噬活性、呼吸爆发活性、酚氧化酶活性和酸性磷酸酶活性都显著高于对照组(P<0.05)。饲料中不同浓度的维生素E对刺参感染灿烂弧菌后的累积死亡率有显著影响(P<0.05),在任何维生素C添加水平下,随着饲料中维生素E含量的升高,刺参的累积死亡率呈下降趋势,尤其饲料中同时添加高剂量的维生素C(2000 mg kg-1)和维生素E(250mg kg-1)的实验组显著降低了刺参感染灿烂弧菌14d内的累积死亡率(P<0.05)。在本实验条件下,维生素C和维生素E对刺参体腔细胞的吞噬活性表现出显著的交互作用(P<0.05)。
3.以初始体重为(5.06±0.10)g的刺参(Apostichopus japonicus Selenka)为实验对象,在室内水循环系统中进行了为期8周的饲喂实验,探讨了饲料中不同含量的低聚果糖和枯草芽孢杆菌对刺参生长性能、免疫力、肠道菌群以及抗病力的影响。本实验采用3×3双因子实验设计,在基础饲料中分别添加0、0.2%和0.5%的枯草芽孢杆菌,每种枯草芽孢杆菌水平分别添加0%、0.25%和0.5%的低聚果糖(有效含量为95%),共制成9种实验饲料,每个处理三个重复。研究结果表明,当不添加低聚果糖时,0.2%枯草芽孢杆菌显著提高了刺参的特定生长率(SGR)和刺参体腔细胞吞噬活性(P<0.05),显著降低了刺参感染灿烂弧菌后的累积死亡率(P<0.05)。当不添加枯草芽孢杆菌时,刺参SGR、PO和吞噬活性随着低聚果糖的添加而升高,且饲料中添加0.5%低聚果糖时,刺参体腔细胞破碎液中吞噬活性、酚氧化酶活性和酸性磷酸酶活性都显著高于对照组(P<0.05),当饲料中枯草芽孢杆菌含量为0.2%时,0.5%低聚果糖可以显著提高刺参的特定生长率、吞噬活性、PO活性(P<0.05),显著降低刺参肠道弧菌数及刺参感染灿烂弧菌后的累积死亡率(P<0.05)；当饲料中枯草芽孢杆菌含量为0.5%时,0.25%低聚果糖添加组显著提高了刺参的酚氧化酶活性和酸性磷酸酶活性(P<0.05),降低了刺参感染细菌后14d内的累积死亡率(P<0.05)。枯草芽孢杆菌和低聚果糖对刺参的吞噬活性、酚氧化酶活性、弧菌数目以及抗病力产生了明显的交互作用(P<0.05)。
4.以初始体重(3.04±0.01)g的刺参(Apostichopus japonicus Selenka)为研究对象,探讨饲料中添加低聚果糖或甘露寡糖对其生长、肠道菌群和非特异性免疫力的影响。分别向基础饲料中添加0%(对照组)、0.25%和0.5%低聚果糖或甘露寡糖,配制出5种实验饲料,在室内循环水系统中进行为期8周的养殖实验。每种饲料为一处理,每个处理设3个重复,每个缸(50 1)为一重复放养22头刺参。饲喂实验结束后测定实验刺参的生长及相关的免疫学指标,同时每个重复取13头刺参进行攻毒实验。结果表明：(1)0.5%低聚果糖组刺参的特定生长率(SGR)显著高于对照组(P<0.05),但与0.25%低聚果糖组差异不显著(P>0.05)；刺参肠道总菌数不受低聚果糖添加量的影响,但是两低聚果糖组刺参肠道中的弧菌数显著低于对照组(P<0.05)；随着饲料中低聚果糖含量的升高,刺参体腔细胞的吞噬活性、呼吸爆发活性、体腔细胞上清中的PO活性和ACP活性都有升高的趋势；攻毒实验结果显示,0.5%低聚果糖组刺参感染灿烂弧菌(V.splendidus)14d的死亡率显著低于对照组(P<0.05)；(2)饲料中甘露寡糖含量对刺参的SGR影响不显著(P>0.05)；饲料中添加甘露寡糖可以提高刺参肠道中的总菌数,降低肠道中的弧菌数,0.25%和0.5%甘露寡糖组刺参肠道中的总菌数均显著高于对照组(P<0.05),但只有0.5%甘露寡糖添加组刺参肠道中的弧菌数显著低于对照组(P<0.05)。0.25%甘露寡糖组刺参体腔细胞吞噬活性和呼吸爆发活性显著高于其他两组(P<0.05)；随着饲料中甘露寡糖添加量的升高,刺参体腔细胞上清中的酚氧化酶(PO)活性和酸性磷酸酶(ACP)活性都有升高的趋势,但是各处理组之间无显著差异(P>0.05)。0.25%甘露寡糖组刺参感染灿烂弧菌14d的累积死亡率显著降低(P<0.05)；(3)综上所述,饲料中分别添加0.5%的低聚果糖或者0.25%的甘露寡糖对刺参生长、免疫力和抗病力的效果较好。二者均可以作为安全高效的口服免疫增强剂应用于刺参的养殖生产。
5.以初始体重为(3.52±0.08)g的刺参(Apostichopus japonicus Selenka)为实验对象,在室内水循环系统中进行了为期8周的饲喂实验,探讨了饲料中不同含量的硒酵母和维生素E(VE)对刺参生长性能、免疫力以及抗病力的影响。本实验采用2×4双因子实验设计,在基础饲料中分别添加0和250 mg kg-1的VE,每种VE水平分别添加0、100、300和600 mg kg-1的硒酵母,共制成8种实验饲料,每个处理3个重复。研究结果表明,硒酵母和维生素E对刺参的特定生长率均有显著影响,饲料中添加硒酵母和维生素E的不同配伍均可显著提高刺参特定生长率(P<0.05),高剂量硒酵母(600 mg kg-1)和维生素E(250mg kg-1)添加组刺参的特定生长率最高；当饲料中不添加维生素E时,随着硒酵母添加量的增加,刺参的吞噬活性、呼吸爆发活性、酚氧化酶活性和酸性磷酸酶活性呈剂量依赖性升高,且600 mg kg-1硒酵母添加组刺参的吞噬活性、呼吸爆发活性、酚氧化酶活性和酸性磷酸酶活性均显著高于对照组(P<0.05)。当饲料中添加250 mg kg-1的维生素E时,添加600 mg kg-1硒酵母实验组刺参的吞噬活性、呼吸爆发活性和酚氧化酶活性高于其他各处理组,并显著高于对照组(P<0.05)；维生素E对过氧化氢酶活性(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活性、超氧化物歧化酶(SOD)活性都有显著的影响(P<0.05),而硒酵母仅对SOD和GSH-Px活性影响显著(P<0.05),且二者合用对刺参的SOD和GSH-Px活性有显著的交互作用(P>0.05)；当饲料中不添加维生素E时,饲料中单独添加硒酵母可以降低刺参感染灿烂弧菌后的14d内的累积死亡率,且随着添加量的增加刺参的累积死亡率有下降的趋势(P>0.05)。当饲料中添加添加250 mg kg-1的维生素E时,各水平硒酵母添加组的刺参感染灿烂弧菌后14d的累积死亡率显著低于对照组(P<0.05)。其中同时添加600 mg kg-1硒酵母和250 mg kg-1的维生素E的实验组显著提高了刺参的特定生长率、非特异性免疫力和抗氧化酶活力,显著降低了刺参感染灿烂弧菌后的累积死亡率,表现出较好的复合益生效果,是本研究最优的功能组合。
6.以初始体重为(3.49±0.06)g的刺参(Apostichopus japonicus Selenka)为实验对象,在室内水循环系统中进行了为期8周的饲喂实验,探讨了饲料中不同含量的酵母培养物(达农威益康XP)和枯草芽孢杆菌对刺参生长性能、免疫力、肠道菌群以及抗病力的影响。本实验采用2×4双因子实验设计,在基础饲料中分别添加0和0.2%的枯草芽孢杆菌,每种枯草芽孢杆水平分别添加0、0.1%、0.2%和0.4%的酵母培养物,共制成8种实验饲料,每个处理三个重复。研究结果表明,当不添加枯草芽孢杆菌时,酵母培养物显著提高了刺参的吞噬活性、呼吸爆发活性、ACP和SOD活性(P<0.05),显著降低了刺参感染灿烂弧菌14d内的累积死亡率(P<0.05)；当添加0.2%的枯草芽孢杆菌时,酵母培养物添加组的刺参特定生长率、肠道总菌数、呼吸爆发活性、ACP等的活性都显著高于对照组(P<0.05),且当饲料中酵母培养物的含量达到0.4%时,刺参体腔细胞的吞噬活性和呼吸爆发活性达到了最高水平。相应地,刺参感染细菌后14d内的累积死亡率也显著低于对照组(P<0.05)。双因素统计分析结果表明,酵母培养物(达农威益康XP)和枯草芽孢杆菌对刺参肠道总菌数和非特异性免疫力都产生了明显的交互作用(P<0.05)。
Six feeding trials were conducted in indoor circulating system to investigate the effects of dietary vitamin C,vitamin E,β-glucan, Bacillus subtilis, fructooligosaccharides (FOS), mannooligosaccharides (MOS), selenium yeast (Se-yeast) and yeast culture (Diamond V XP Yeast Culture, XP) on growth, immunity and disease resistance in sea cucumber (Apostichopus japonicus Selenka). The results are summarized as follows:
1.A 9-week feeding experiment was conducted to investigate the effect of vitamin C andβ-glucan on the growth performance, immunity and disease resistance of sea cucumber (A. japonicus Selenka).Five hundred and forty individuals (initial body weight:5.08±0.08) g, mean±S.E) were fed with nine practical diets according to a 3×3 factorial design:the basal diet as the control diet supplemented with three levels of vitamin C (0,500,2000 mg kg-1 diet), and each crossed with 0,625 or 1250 mg kg-1β-glucan. After 9 weeks, five sea cucumbers per tank were sampled for immune indices determination. Then all the sea cucumbers left were challenged by Vibrio splendidus. The results showed that dietary vitamin C andβ-glucan had significant effects on specific growth rate (SGR) of A. japonicus (P<0.05).SGR of the seacucumber with the combination of 2000 mg kg-1 vitamin C and 1250 mg kg-1β-glucan was significantly higher than the control group (P<0.05).Activities of phagocytosis, respiratory burst (RB), phenoloxidase (PO) and acid phosphatase (ACP) were significantly influenced by dietary vitamin C andβ-glucan (P<0.05).In the absence of dietary vitamin C, activities of RB,PO and ACP significantly increased with increasingβ-glucan (P<0.05). In the groups with 500 mg kg-1 vitamin C, sea cucumbers fed with 625 mg kg-1β-glucan had significant higher phagocytosis and PO activity than the control group (P<0.05),and those fed with 1250 mg kg-1β-glucan had significant higher RB activity and ACP activity (P<0.05).In the groups with 2000 mg vitamin C kg-1 diet,625 mg kg-1β-glucan supplementation remarkably increased PO activity and ACP activity (P<0.05), while 1250 mg kg-1β-glucan supplementation significantly enhanced phagocytosis, RB activity, PO activity and ACP activity (P<0.05). Moreover, dietaryβ-glucan had significant effect on cumulative mortality of sea cucumbers after 14 days following V. splendidus exposure (P<0.05).Cumulative mortality was significantly affected by dietaryβ-glucan (P<0.05), and cumulative mortality decreased with the increasing doses ofβ-glucan at 500 and 2000 mg kg-1 vitamin C level.The group fed diets supplemented with 500 mg kg-1 vitamin C and 1250 mg kg-1β-glucan had notably lower cumulative mortality compared to the control (P<0.05).Under the experimental conditions, dietary vitamin C andβ-glucan had a synergistic effect on enhancing phagocytosis and RB activity of sea cucumber(P<0.05).
2.A 9-week feeding experiment was conducted to investigate the effects of vitamin C and vitamin E on the growth performance, immunity and disease resistance of sea cucumber (A. japonicus Selenka).Five hundred and forty individuals (initial body weight:5.06±0.10 g, mean±S.E) were fed with nine practical diets according to a 3×3 factorial design:the basal diet as the control diet supplemented with three levels of vitamin C (0, 500,2000 mg kg-1 diet), and each crossed with 0,80 or 250 mg kg-1 vitamin E. After 9 weeks,five sea cucumbers per tank were sampled for immune indices determination. Then all the sea cucumbers left were challenged by Vibrio splendidus.The results showed that specific growth rate (SGR) of A. japonicus was significantly affected by both vitamin C and vitamin E (P<0.05), and SGR of the groups with 250 mg kg-1 vitamin E alone and 250 mg kg-1 vitamin E at 2000 mg kg-1 vitamin C level were significant higher than the control group (P<0.05). Dietary vitamin C and vitamin E had significant effects on phagocytosis, phenoloxidase (PO) activity and acid phosphatase (ACP) activity (P<0.05). In the groups with vitamin C alone, phagocytosis increased with the increasing doses of vitamin C and was significantly enhanced by 2000 mg kg-1 vitamin C(P<0.05).In the groups without vitamin C supplementation, phagocytosis of sea cucumbers fed with 250 mg kg-1 vitamin E was significantly higher than the control group(P<0.05).In the groups with 500 mg kg-1 vitamin C, phagocytosis, respiratory burst (RB) activity and PO activity showed increasing tendency with the increasing administration doses of vitamin E (P<0.05), and PO activity of the group fed with 250 mg kg-1 vitamin E significantly increased compared with the control (P<0.05).In the groups with 2000 mg kg-1 supplementation of vitamin C, RB activity and PO activity and ACP activity increased with the doses of vitamin E, and all the immune indices in the group fed with 250 mg kg-1 vitamin E was significant higher than the control group (P<0.05).Moreover, dietary vitamin E had significant effect on cumulative mortality of sea cucumbers after 14 days following V. splendidus exposure (P<0.05). Cumulative mortality decreased with the increasing doses of vitamin E at each vitamin C level.The group fed with diets supplemented with 2000 mg kg-1 vitamin C and 250 mg kg-1 vitamin E had notably lower cumulative mortality compared to the control (P<0.05).Under the experimental conditions, dietary vitamin C and vitamin E had a synergistic effect on enhancing phagocytosis of sea cucumber (P<0.05).
3.An 8-week feeding experiment was conducted to investigate the interaction of probiotic Bacillus subtilis and prebiotic fructooligosaccharide (FOS) on the growth performance, immunity, intestinal microflora and disease resistance of sea cucumber (A.japonicus Selenka).Five hundred and forty individuals (initial body weight:5.09±0.03 g, mean±S.E) were fed nine practical diets according to a 3×3 factorial design:the basal diet as the control diet supplemented with three levels of B. subtilis (0,0.2 or 0.5%),crossed with 0,0.25% or 0.50% FOS.After 8 weeks, five sea cucumbers per tank were sampled for bacterial quantification and immune indices determination. Then all remaining sea cucumbers were challenged by Vibrio splendidus.The results showed that dietary B. subtilis significantly increased the specific growth rate (SGR), phagocytosis and acid phosphatase (ACP) activity of sea cucumbers, the counts of total viable bacteria (TBC) and disease resistance to V.splendidus (P<0.05),whereas the counts of Vibrio (VBC) decreased (P<0.05).However, dietary B. subtilis had no significant effect on phenoloxidase (PO) activity (P>0.05).The SGR, PO activity, TBC and VBC were significantly affected by dietary FOS.In the groups with B. subtilis alone, SGR and phagocytosis of 0.2% B. subtilis administration significantly increased compared to the control group (P<0.05), while cumulative mortality of sea cucumbers significantly decreased (P<0.05).In the groups without B. subtilis, SGR, phagocytosis and PO activity increased with the doses of FOS,and phagocytosis, PO activity and ACP activity of the group fed with 0.5% FOS significantly enhanced compared the control (P<0.05). In the groups with 0.2% B. subtilis,0.5% FOS supplementation significantly increased the SGR, phagocytosis and PO activity of sea cucumbers, while notably decreased VBC and cumulative mortality (P<0.05).In the groups fed with 0.5% B. subtilis,0.25% FOS supplementation significantly increased the PO activity and ACP activity of sea cucumbers, while significantly decreased cumulative mortality. Under the experimental conditions, dietary B. subtilis and FOS had a synergistic effect on enhancing phagocytosis, PO activity and disease resistance of sea cucumber (P<0.05).
4.An 8-week feeding experiment was conducted to evaluate the effects of FOS and MOS on growth, non-specific immunity of sea cucumber (A. japonicus Selenka) as well as its resistance against V.splendidus.FOS and MOS were administered to sea cucumbers through the diets at three levels (0,0.25% and 0.5%, respectively). The basal diet without FOS or MOS was used as the control.Each diet was randomly allocated to triplicate groups of sea cucumbers in indoor 50 1 tanks with circulating seawater and constant aeration. And each tank was stocked with 22 sea cucumbers (initial average weight 3.04±0.06 g).The results showed that:Ⅰ. FOS supplemented at 0.5% significantly enhanced the specific growth rate (SGR) of sea cucumbers (P<0.05).However, no significant difference in SGR was observed among the group containing 0.25% FOS and the control groups (P>0.05).Vibrio bacteria counts (VBC) instead of total bacteria counts (TBC) were significantly affected by FOS administration (P<0.05). Phagocytosis, RB activity, PO activity and ACP activity significantly increased with the increasing doses of FOS, and all of these four immune indices were remarkably enhanced by 0.5% FOS supplementation (P<0.05).Ⅱ.Sea cucumbers fed with 0.25% and 0.5% MOS supplementation had no significant difference in SGR (P>0.05).The groups with two levels of MOS had higher TBC than the control group (P<0.05), and 0.5% MOS had lower VBC compared with the control (P<0.05). Phagocytosis and RB activity in sea cucumbers fed with 0.25% MOS was significant high when compared with the control and 0.5% MOS supplementation group (P<0.05).PO activity and ACP activity increased with the increasing doses of MOS, but there were no significant differences among the three treatments (P>0.05). The challenge experiment showed that sea cucumbers fed the diet with 0.25% MOS had significantly lower cumulative morbidity compared with the control group (P<0.05).Ⅲ.These results suggested that feeding FOS at a dose of 0.5% or MOS at a dose of 0.25% could enhance growth, non-specific immunity as well as resistance against V. splendidus of sea cucumber.
5.An 8-week feeding experiment was conducted to investigate the effect of selenium yeast (Se-yeast) and vitamin E on the growth performance, immunity and disease resistance of sea cucumber (A. japonicus Selenka).Six hundred individuals (initial body weight:3.52±0.08 g, mean±S.E) were fed eight practical diets according to a 2×4 factorial design: the basal diet as the control diet supplemented with two levels of vitamin E (0,250 mg kg-1 diet), crossed with 0,100,300 or 600 mg kg-1 selenium yeast (Se-yeast). The results showed that dietary Se-yeast and vitamin E had significant influence on specific growth rate (SGR) (P<0.05).And SGR of the sea cucumbers fed with the combination of 600 mg kg-1 Se-yeast and 250 mg kg-1 vitamin E was significantly increased when compared with the control group (P<0.05). Significant higher phagocytosis,respiratory burst (RB) activity, phenoloxidase (PO) activity and acid phosphatase (ACP) activity were observed in the group with 600 mg kg-1 Se-yeast alone compared with the control group (P<0.05). In the groups with 250 mg kg-1 vitamin E, sea cucumbers fed with 600 mg kg-1 Se-yeast had significant higher phagocytosis, RB activity and PO activity when compared with the control group (P<0.05).Dietary vitamin E had significant effect on catalase (CAT) activity,glutathione peroxidase (GSH-Px) activity and total superoxide dismutase (SOD) activity (P<0.05), but Se-yeast significantly influenced the activities of only GSH-Px and SOD (P<0.05), and there were significantly interactions between Se-yeast and vitamin E on GSH-Px activity and SOD activity (P<0.05). The challenge trial showed that dietary Se-yeast and vitamin E had significant effects on cumulative mortality after 14 days following V. splendidus exposure (P<0.05).In the absent of vitamin E, cumulative mortality showed increasing tendency with increasing Se-yeast, but there were no significant difference compared to the control (P>0.05).In the groups with 250 mg kg-1 vitamin E, sea cucumbers fed each Se-yeast levels had significant lower cumulative mortality compared with other groups and the control group (P<0.05). It could be concluded that the group with 600 mg kg-1 Se-yeast and 250 mg kg-1 vitamin E could significantly enhance the SGR and immunity, keep the antioxidant enzymes in a balanced level and decrease cumulative mortality of sea cucumbers. Therefore, combination of 600 mg kg-1 Se-yeast and 250 mg kg-1 vitamin E was the optimal group in the present study.
6.An 8-week feeding experiment was conducted to investigate the effect of yeast culture (Diamond V XP Yeast Culture, XP) and Bacillus subtilis on the growth performance, intestinal microflora, immunity and disease resistance of sea cucumber (A.japonicus Selenka).Six hundred individuals (initial body weight:3.49±0.06 g, mean±S.E) were fed eight practical diets according to a 2×4 factorial design:the basal diet as the control diet supplemented with two levels of Bacillus subtilis (0,0.2%), crossed with 0,0.1%, 0.2% or 0.4% yeast culture (XP).After 8 weeks, five sea cucumbers per tank were sampled for immune indices determination. Then all remaining sea cucumbers were challenged by Vibrio splendidus.The results showed that dietary XP had significant influence on specific growth rate (SGR), total bacteria counts (TBC) and Vibrio bacteria counts (VBC), activities of phagocytosis, respiratory burst (RB) activity, phenoloxidase (PO) activity and acid phosphatase (ACP) activity and cumulative mortality, but Bacillus subtilis significant influenced only SGR, counts of TBC and VBC, cumulative mortality (P<0.05).Evident increases of all the immune enzymes tested in the present study were noticed in sea cucumbers fed with o.4% XP in the groups without Bacillus subtilis (P<0.05). In the groups with 0.2% Bacillus subtilis, SGR, TBC, RB, ACP of each level of XP administration significantly increased compared to the group (P<0.05), while cumulative mortality of sea cucumbers significantly decreased (P<0.05).Under the experimental conditions, dietary vitamin XP and Bacillus subtilis had a synergistic effect on enhancing TBA, phagocytosis, RB activity, PO activity and ACP activity of sea cucumber (P<0.05).
引文
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