对虾与海参高效免疫激活物质的筛选与评价
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摘要
本文选择我国重要的海水养殖动物凡纳滨对虾(Litopenaeus vannamei)和刺参(Apostichopus japonicus Selenka)为研究对象,在室内循环水养殖系统中(200 l)进行为期8周的摄食生长实验。探讨(1)甘草酸和大豆异黄酮对凡纳滨对虾生产性能和免疫力的影响;(2)甘草酸、左旋咪唑、核苷酸以及乳铁蛋白对刺参生长、免疫及抗病力的影响。主要研究内容和结果如下:
1.选用初始体重为(0.233±0.000g)的凡纳滨对虾(L. vannamei)为研究对象,在基础饲料中分别添加50 mg kg-1、100 mg kg-1、150 mg kg-1、200 mg kg-1的甘草酸,制作出4种实验饲料,饲喂凡纳滨对虾8周。结果表明:各处理组对虾的成活率都在82.2-90.0%之间,相互之间没有显著差异。饲料中添加甘草酸可显著提高对虾的特定生长率(SGR) (P<0.05),150和200 mg kg-1添加组对虾特定生长率显著高于50和100 mg kg-1添加组(P<0.05)。饲料中添加甘草酸对凡纳滨对虾体常规成分含量没有产生显著影响(P>0.05)。饲料中添加200 mg kg-1的甘草酸可显著提高凡纳滨对虾血细胞计数(THC)和呼吸爆发活性(P<0.05);当饲料中甘草酸添加量为200 mg kg-1时,凡纳滨对虾血清酚氧化酶(PO)活性和超氧化物歧化酶(SOD)活性显著高于对照组和50 mg kg-1甘草酸添加组,但与150 mg kg-1甘草酸添加组差异不显著(P>0.05)。当饲料中甘草酸的添加量为150 mg kg-1时,凡纳滨对虾血清一氧化氮合酶(NOS)活性显著高于对照组及50和100mg kg-1甘草酸添加组,但与200 mg kg-1甘草酸添加组差异不显著。养殖实验结束后,通过注射对虾致病菌鳗弧菌(Vibrio alginolyticus)进行对虾攻毒实验,结果表明,饲料中添加甘草酸可以显著降低凡纳滨对虾攻毒后10日内的累计死亡率,其中150和200 mg kg-1甘草酸添加组的累计死亡率(10.0%和8.3%)显著低于50 mg kg-1甘草酸添加组(16.7%),但与100 mg kg-1甘草酸添加组(11.7%)差异不显著。本实验可得到以下结论:饲料中添加甘草酸可以提高凡纳滨对虾的特定生长率、免疫力和抗病力;甘草酸在凡纳滨对虾饲料中的建议添加量为200 mg kg-1饲料。
2.为研究饲料中添加大豆异黄酮对凡纳滨对虾(L. vannamei)生长、免疫及抗病力的影响,实验设五个处理,大豆异黄酮添加水平分别为0(对照组)、5、10、20和40 mg kg-1,养殖周期为56d。结果表明:各处理组对虾成活率在82.2%-91.1%之间,各处理之间无显著差异(P>0.05)。大豆异黄酮添加量在10 mg kg-1以上的3个处理组,凡纳滨对虾的特定生长率(SGR)显著高于对照组及5 mg kg-1处理组(P<0.05),且三个处理间差异不显著。饲料中添加大豆异黄酮对凡纳滨对虾体成分组成没有产生显著影响(P>0.05)。20和40 mg kg-1处理组,凡纳滨对虾的血细胞计数(THC)、呼吸爆发活性(NBT)、酚氧化酶(PO)活性、超氧化物歧化酶(SOD)活性及一氧化氮合酶(NOS)活性均显著高于对照组(P<0.05)。随着饲料中大豆异黄酮添加量的增加,凡纳滨对虾的THC、呼吸爆发活性、PO活性、SOD活性及NOS活性呈剂量依赖性升高,但当添加量为40 mg kg-1时,除NOS外其他各免疫比较均呈现出下降的趋势。攻毒实验结果表明,攻毒后的10天内对照组的累计死亡率显著高于4个添加组,4个添加组的免疫保护力为63%-72%,且相互之间差异不显著。本实验表明:1)饲料中添加大豆异黄酮可以提高凡纳滨对虾的特定生长率、免疫力和抗病力;2)大豆异黄酮在凡纳滨对虾饲料中的建议添加量为20 mg kg-1。
3.以初始体重为(6.80±0.00g)左右的刺参(A. japonicus Selenka)为研究对象,在基础饲料中分别添加0(对照组)、50、100和200 mg kg-1的甘草酸,配制4种实验饲料。结果表明:饲料中添加甘草酸对刺参的成活率没有显著影响,各处理组均为100%。饲料中添加200 mg kg-1甘草酸可显著提高刺参的特定生长率(SGR) (P<0.05)。饲料中添加甘草酸对刺参体常规成分含量没有产生显著影响。当饲料中甘草酸添加量为200 mg kg-1时,刺参体腔细胞一氧化氮合酶(NOS)活性和溶菌酶(LZM)活性显著高于其他添加组(P<0.05)。当饲料中甘草酸添加量≥100 mg kg-1时,刺参体腔细胞超氧阴离子产量(COP)、超氧化物歧化酶(SOD)活性、酸性磷酸酶(ACP)活性、NOS活性及LZM活性均显著高于对照组及50 mg kg-1添加组(P<0.05)。50 mg kg-1添加组以上各免疫指标与对照组差异不显著(P>0.05)。养殖实验结束后,通过注射刺参腐皮综合症致病菌灿烂弧菌(V. splendidus)进行刺参攻毒实验。攻毒后14d内对照组与50 mg kg-1添加组的累计发病率(分别为38.3%和36.7%)显著高于100和200 mg kg-1添加组(分别为30.0%和26.7%) (P<0.05)。本实验可得到以下结论:饲料中添加200 mg kg-1甘草酸可以提高刺参养殖的产量,同时可以提高刺参的非特异性免疫力和抗病力。
4.以初始体重为(6.80±0.01g)的刺参(A. japonicus Selenka)为研究对象,以基础饲料为对照组,在基础饲料中分别添加0.01%、0.02%和0.04%的左旋咪唑,配制4种实验饲料。实验结果显示,饲料中添加高含量的左旋咪唑(0.04%)显著提高了刺参的特定生长率(P<0.05),而低含量添加组(0.01%和0.02%)与对照组之间差异不显著(P>0.05)。饲料中添加左旋咪唑对刺参的成活率没有产生影响(P>0.05),各处理组成活率均为100%。饲料中添加左旋咪唑对刺参体常规成分含量没有产生显著影响(P>0.05)。饲料中添加高含量的左旋咪唑(0.04%)显著提高了刺参体腔细胞超氧阴离子产量(COP)、溶菌酶(LZM)活性和一氧化氮合酶(NOS)活性(P<0.05),低含量添加组(0.01%和0.02%)与对照组之间差异不显著(P>0.05)。当饲料中左旋咪唑添加量≥0.02%时,刺参体腔细胞的超氧化物歧化酶(SOD)活性和酸性磷酸酶(ACP)活性显著高于对照组,且0.04%添加组显著高于其余各处理组(P<0.05)。0.01%添加组上述免疫指标与对照组均差异不显著(P>0.05)。8周的饲养实验结束后,采用刺参腐皮综合症致病菌灿烂弧菌(V. splendidus)进行体壁注射攻毒实验。结果显示,饲料中添加0.04%的左旋咪唑显著降低了刺参攻毒后14 d的累计发病率(P<0.05),其余各添加组与对照组差异不显著(P>0.05)。上述结果表明,饲料中添加0.04%的左旋咪唑对刺参的生长、免疫力及抗病力具有较好的促进作用。
5.以初始体重为(6.80±0.00g)的刺参(A. japonicus Selenka)为研究对象,在配合饲料中分别加入0.00%(对照组)、0.03%、0.06%和0.12%的核苷酸制成4种实验饲料,在室内循环水系统中(200 l)进行为期8周的摄食生长实验。实验结果表明,饲料中添加核苷酸对刺参存活率没有产生影响,各处理组均为100%,但却显著影响其生长。当饲料中添加0.06%和0.12%的核苷酸时,刺参的特定生长率(SGR)显著高于对照组(0.00%)和低添加量组(0.03%)。0.03%添加组与对照组以及高核苷酸添加组间(0.06%添加组与0.12%添加组)的SGR均没有显著差异(P>0.05)。饲料中添加核苷酸对刺参体常规成分含量没有产生显著影响(P>0.05)。当饲料中核苷酸添加量≥0.06%时,刺参体腔细胞溶菌酶(LZM)活性和超氧化物歧化酶(SOD)活性均显著高于对照组和0.03%添加组(P<0.05),且0.06%添加组和0.12%添加组间差异不显著(P>0.05)。饲料中添加0.06%的核苷酸显著提高了刺参体腔细胞一氧化氮合酶(NOS)活性和酸性磷酸酶(ACP)活性(P<0.05),其余添加组与对照组差异不显著(P>0.05)。当饲料中核苷酸添加量为0.06%时,刺参体腔细胞超氧阴离子产量(COP)显著高于对照组与0.03%添加组(P<0.05),但与0.12%添加组差异不显著,0.03%添加组、0.12%添加组以及对照组间没有显著差异(P>0.05)。8周的饲养实验结束后,通过体壁注射刺参腐皮综合症致病菌灿烂弧菌(V. splendidus)进行刺参攻毒实验。在攻毒后的14天内,对照组和0.03%添加组刺参的累计发病率显著高于0.06%添加组和0.12%添加组(P<0.05)。0.03%添加组和对照组间以及0.06%添加组和0.12%添加组间刺参的累计发病率没有显著差异(P>0.05)。本研究结果表明,饲料中添加0.06%的核苷酸可以显著提高刺参的生长和非特异性免疫力,核苷酸可以作为一种安全高效的口服免疫增强剂应用于刺参的养殖生产。
6.以初始体重(6.80±0.02g)的刺参(A. japonicus Selenka)为研究对象,探讨饲料中添加乳铁蛋白对其生长和非特异性免疫力的影响。分别向每千克基础饲料中添加0(对照组)、50、100和200 mg乳铁蛋白,配制出4种实验饲料,在室内循环水系统中(200 l)进行为期8周的摄食生长实验。实验结果表明,饲料中添加乳铁蛋白对刺参的成活率没有产生影响,各处理组成活率均为100%。当饲料中添加100 mg kg-1乳铁蛋白时刺参特定生长率显著高于对照组(P<0.05)。饲料中添加乳铁蛋白对刺参体常规成分含量没有产生显著影响(P>0.05)。饲料中添加100 mg kg-1乳铁蛋白组刺参体腔细胞超氧阴离子产量(COP)著高于对照组,其余各添加组与对照组差异不显著。当饲料中乳铁蛋白添加量为100和200 mg kg-1时,刺参体腔细胞一氧化氮合酶(NOS)活性、超氧化物歧化酶(SOD)活性和酸性磷酸酶(ACP)活性显著高于对照组,且两个添加组间差异不显著。饲料中添加100 mg kg-1乳铁蛋白组刺参体腔细胞溶菌酶(LZM)活性显著高于对照组及其他添加组。50 mg kg-1乳铁蛋白组刺参体腔细胞上述免疫指标与对照组均没有显著差异。灿烂弧菌(V. splendidus)攻毒实验结果表明,饲料中添加100和200 mg kg-1乳铁蛋白组刺参的累计发病率显著低于对照组。因此,饲料中添加适宜含量的乳铁蛋白可以显著提高刺参生长率和非特异性免疫力。乳铁蛋白可以作为一种安全高效的口服免疫增强剂应用于刺参的养殖生产。
Six feeding trials were conducted in indoor circulating system to investigate the effects of dietary glycyrrhizin, soybean isoflavones, levamisole, nucleotide and bovine lactoferrin on growth, non-specific immunity and disease resistance for shrimp (L. vannamei) and sea cucumber (A. japonicus Selenka). The results are summarized as follows:
1. The growth response, total haemocyte count (THC), respiratory burst (release of superoxide anion), phenoloxidase (PO), superoxide dismutase (SOD), nitric oxide synthase (NOS) as well as resistance to the pathogen V. alginolyticus were measured in shrimps which had been fed diets supplemented with glycyrrhizin at 0, 50, 100, 150 and 200 mg kg-1 diet for 8 weeks. Dietary glycyrrhizin had no significant influence on survival rate of the shrimps (P>0.05). Significantly higher SGR was observed in the treatments with dietary glycyrrhizin supplementation than that in control (P<0.05). However, no significant differences in body composition were noticed among dietary treatments (P>0.05). Shrimps fed the diet with 200 mg kg-1 glycyrrhizin had significant higher THC, PO activity, respiratory burst activity and SOD activity than that in control (P<0.05). Increased THC, respiratory burst activity and NOS activity were also noticed in shrimps fed diets with 100 or 150 mg kg-1 glycyrrhizin (P<0.05). Significantly lower cumulative mortality after challenge was observed in shrimps fed the diets supplemented with glycyrrhizin (8.33 -16.67%) than that in control group (36.67%) (P<0.05). These results indicated that glycyrrhizin might elevate the function of nonspecific immunity and enhance disease resistance of L. vannamei as well as improve production in shrimp farming.
2. The growth response, total haemocyte count (THC), respiratory burst (release of superoxide anion), phenoloxidase (PO) activity, superoxide dismutase (SOD) activity, nitric oxide synthase (NOS) activity and resistance to the pathogen Vibrio alginolyticus were measured in the white shrimp (L. vannamei) which had been fed diets supplemented with soybean isoflavones (SI) at 0, 5, 10, 20, 40 mg kg-1 for 8 weeks. Dietary SI had no significant influence on survival rate of the shrimps (P>0.05). Significantly higher SGR was observed in treatments with 10, 20 and 40 mg kg-1 SI supplement than that in control (P<0.05). No significant differences in body composition were noticed among dietary treatments. Shrimps fed a diet with 20 mg kg-1 SI supplementation showed significant increase in THC, PO activity, respiratory burst activity, SOD activity as well as NOS activity (P<0.05). Increased THC, respiratory burst activity, SOD activity and NOS activity were also noticed in shrimps fed the diets with SI supplementation at 10 or 40 mg kg-1 (P<0.05). Significantly lower cumulative mortality after challenge with V. alginolyticus was observed in shrimps fed the diets with SI supplementation (10.0-13.3%) than that in control group (36.7%) (P<0.05). It is therefore concluded that oral administration of SI at 20 mg kg-1 for 8 weeks could enhance the immune ability of shrimps and improve production in shrimp farming.
3. An 8-week feeding experiment was conducted to evaluate the effects of glycyrrhizin on growth, non-specific immunity of sea cucumber (A. japonicus Selenka) as well as its resistance against V. splendidus. Glycyrrhizin was administered to sea cucumbers through the diets at four levels (0, 50, 100 and 200 mg kg-1 diet, respectively). The basal diet (0 mg kg-1 diet) was used as the control. Each diet was randomly allocated to triplicate groups of sea cucumbers in indoor fiberglass (200 l) tanks with circulating seawater and constant aeration. And each tank was stocked with 30 sea cucumbers (initial average weight 6.80±0.10g). The results showed that dietary glycyrrhizin had no significant influence on survival rate of sea cucumber. Glycyrrhizin supplemented at 200 mg kg-1 significantly enhanced the specific growth rate of sea cucumbers (P<0.05), whereas other supplementation did not (P>0.05). No significant difference in body composition was observed among dietary treatments. Sea cucumbers fed diet with 200 mg kg-1 glycyrrhizin had significantly higher nitric oxide synthase (NOS) activity and lysozyme (LZM) activity than that fed other diets(P<0.05). The intracellular O2- production (COP), LZM, NOS, superoxide dismutase (SOD) and acid phosphatase (ACP) activity in sea cucumbers fed diets with 100 and 200 mg kg-1 glycyrrhizin were significantly higher when compared with the control and 50 mg kg-1 supplementation groups (P<0.05). However, no significant difference of the above immunological parameters was observed between the control and 50 mg kg-1 glycyrrhizin groups (P>0.05). The challenge experiment showed that sea cucumbers fed the diet with 100 and 200 mg kg-1 glycyrrhizin had significantly lower accumulative morbidity compared with the control and 50 mg kg-1 supplementation groups (P<0.05). And no significant difference was observed between 100 and 200 mg kg-1 supplementation groups. These results suggested that feeding glycyrrhizin at a dose of 200 mg kg-1 could enhance growth, non-specific immunity as well as resistance against V. splendidus of sea cucumber (Apostichopus japonicus Selenka).
4. An 8-week feeding experiment was conducted to evaluate the effects of dietary levamisole on growth, non-specific immunity as well as resistance against V. splendidus of sea cucumber (A. japonicus Selenka) (initial body weight 6.80±0.01 g). Results showed that dietary levamisole has no influence on survival rate of A. japonicus (Selenka) (P>0.05). The specific growth rate (SGR) in sea cucumbers fed diet with 0.04% levamisole was significantly increased when compared with the control group (P<0.05). However, no significant difference in SGR was oberved among 0.01%, 0.02% and the control groups (P>0.05). Dietary levamisole had no significant influence on body composition of A. japonicus (Selenka) (P>0.05). Significantly higher intracellular O2- production (COP), lysozyme (LZM) activity, superoxide dismutase (SOD), nitric oxide synthase (NOS) activity and acid phosphatase (ACP) activity in coelomocyte were observed in sea cucumbers fed the diet with 0.04% levamisole when compared with control group (P<0.05). Increased SOD activity and ACP activity were also noticed in sea cucumbers fed diet containing 0.02% levamisole (P<0.05). The SOD activity and ACP activity in sea cucumbers fed a diet containing 0.04% levamisole were showed significantly higher when compared with the other treatments (P<0.05). After 8 weeks of feeding trial, a challenging test was conducted by injecting the sea cucumbers with Vibrio splendidus. Significantly lower (P<0.05) cumulative morbidity was observed during the following 14 days after challenge in sea cucumbers fed the diet containing 0.04% levamisole than that in the control group. The presents results suggested that 0.04% levamisole supplementation in diet could improve growth, non-specific immunity and disease resistance of sea cucumber.
5. An 8-week feeding trial was conducted to evaluate the effects of dietary nucleotide on growth, non-specific immunity and disease resistance against V. splendidus of sea cucumber, A. japonicus (Selenka). Nucleotide (GMP) was administered to sea cucumbers through the diets at four levels (0%, 0.03%, 0.06% and 0.12%, respectively). The basal diet (without nucleotide supplementation) was used as control. Each diet was allocated to triplicate groups of sea cucumbers stocked in indoor 200 l circular fiberglass tanks with circulating seawater and constant aeration. And each tank was stocked with 30 sea cucumbers (initial weight 6.80±0.00 g). The results of the feeding trial showed that dietary nucleotide had no significant influence on survival rate of A. japonicus (Selenka). Sea cucumbers fed diets containing 0.06% or 0.12% nucleotide proved to have a significantly better growth than those fed the diet supplemented with 0.03% nucleotide (P<0.05). No significant difference in body composition was observed among dietary treatments. Sea cucumbers fed the diet containing 0.06% dietary nucleotide showed a significant (P<0.05) increase in intracellular O2- production (COP), lysozyme (LZM) activity, superoxide dismutase (SOD), nitric oxide synthase (NOS) activity as well as acid phosphatase (ACP) activity in coelomocyte. Increased LZM activity and SOD activity were also noticed in sea cucumbers fed the diet supplemented with 0.12% nucleotide (P<0.05). No significant difference in the above immune parameters was observed between sea cucumbers fed control diet and fed diet with 0.03% nucleotide supplementation (P>0.05). The challenge trial showed that sea cucumbers fed the diets with 0.06% and 0.12% nucleotide had significantly lower cumulative morbidity compared with the control and 0.03% nucleotide groups (P<0.05), and no significant difference was observed between 0.06% and 0.12% nucleotide groups. These results suggested that feeding nucleotide at a dose of 0.06% could enhance growth, non-specific immunity as well as resistance against V. splendidus of sea cucumber Apostichopus japonicus Selenka.
6. An 8-week feeding experiment was conducted to evaluate the effects of bovine lactoferrin (LF) on growth as well as non-specific immunity of sea cucumber (Apostichopus japonicus Selenka). Lactoferrin was administered to sea cucumber through the diets at three levels (50, 100 and 200 mg kg-1 diet, respectively). The basal diet (without lactoferrin supplementation) was used as the control diet. The intracellular O2- production (COP), lysozyme (LZM) activity, superoxide dismutase (SOD), nitric oxide synthase (NOS) activity and acid phosphatase (ACP) activity in coelomocyte were measured at day 56. And the specific growth rate (SGR), survival and cumulative mortality after body injection with V. splendidus (100μl V. splendidus suspension at 1×109CFU ml-1 per sea cucumber) were assayed after 56 days feeding period. Dietary lactoferrin had no significant influence on survival rate of sea cucumber. Significantly higher SGR was observed in sea cucumber fed diet containing 100 mg kg-1 lactoferrin than that in control diet. No significant difference in body composition was observed among dietary treatments. Sea cucumbers fed the diet containing 100 mg kg-1 lactoferrin showed significant increase in COP, NOS activity, SOD activity, LZM activity as well as ACP activity (P<0.05). Increased NOS activity, SOD activity and ACP activity were also noticed in sea cucumbers fed the diet containing 200 mg kg-1 lactoferrin (P<0.05). Sea cucumbers fed the diet containing 100 mg kg-1 lactoferrin showed significantly higher LZM activity when compared with other treatments (P<0.05). The highest cumulative morbidity was observed in the sea cucumbers fed the control diet when challenged with injection of V. splendidus. Significantly lower cumulative morbidity at 14 days after challenge was observed in sea cucumber fed diets containing 100 or 200 mg kg-1 lactoferrin compared with the control (P<0.05). These results indicated that lactoferrin might elevate the function of nonspecific immunity and enhance disease resistance of A. japonicus (Selenka) as well as improve production in sea cucumber farming.
1.选用初始体重为(0.233±0.000g)的凡纳滨对虾(L. vannamei)为研究对象,在基础饲料中分别添加50 mg kg-1、100 mg kg-1、150 mg kg-1、200 mg kg-1的甘草酸,制作出4种实验饲料,饲喂凡纳滨对虾8周。结果表明:各处理组对虾的成活率都在82.2-90.0%之间,相互之间没有显著差异。饲料中添加甘草酸可显著提高对虾的特定生长率(SGR) (P<0.05),150和200 mg kg-1添加组对虾特定生长率显著高于50和100 mg kg-1添加组(P<0.05)。饲料中添加甘草酸对凡纳滨对虾体常规成分含量没有产生显著影响(P>0.05)。饲料中添加200 mg kg-1的甘草酸可显著提高凡纳滨对虾血细胞计数(THC)和呼吸爆发活性(P<0.05);当饲料中甘草酸添加量为200 mg kg-1时,凡纳滨对虾血清酚氧化酶(PO)活性和超氧化物歧化酶(SOD)活性显著高于对照组和50 mg kg-1甘草酸添加组,但与150 mg kg-1甘草酸添加组差异不显著(P>0.05)。当饲料中甘草酸的添加量为150 mg kg-1时,凡纳滨对虾血清一氧化氮合酶(NOS)活性显著高于对照组及50和100mg kg-1甘草酸添加组,但与200 mg kg-1甘草酸添加组差异不显著。养殖实验结束后,通过注射对虾致病菌鳗弧菌(Vibrio alginolyticus)进行对虾攻毒实验,结果表明,饲料中添加甘草酸可以显著降低凡纳滨对虾攻毒后10日内的累计死亡率,其中150和200 mg kg-1甘草酸添加组的累计死亡率(10.0%和8.3%)显著低于50 mg kg-1甘草酸添加组(16.7%),但与100 mg kg-1甘草酸添加组(11.7%)差异不显著。本实验可得到以下结论:饲料中添加甘草酸可以提高凡纳滨对虾的特定生长率、免疫力和抗病力;甘草酸在凡纳滨对虾饲料中的建议添加量为200 mg kg-1饲料。
2.为研究饲料中添加大豆异黄酮对凡纳滨对虾(L. vannamei)生长、免疫及抗病力的影响,实验设五个处理,大豆异黄酮添加水平分别为0(对照组)、5、10、20和40 mg kg-1,养殖周期为56d。结果表明:各处理组对虾成活率在82.2%-91.1%之间,各处理之间无显著差异(P>0.05)。大豆异黄酮添加量在10 mg kg-1以上的3个处理组,凡纳滨对虾的特定生长率(SGR)显著高于对照组及5 mg kg-1处理组(P<0.05),且三个处理间差异不显著。饲料中添加大豆异黄酮对凡纳滨对虾体成分组成没有产生显著影响(P>0.05)。20和40 mg kg-1处理组,凡纳滨对虾的血细胞计数(THC)、呼吸爆发活性(NBT)、酚氧化酶(PO)活性、超氧化物歧化酶(SOD)活性及一氧化氮合酶(NOS)活性均显著高于对照组(P<0.05)。随着饲料中大豆异黄酮添加量的增加,凡纳滨对虾的THC、呼吸爆发活性、PO活性、SOD活性及NOS活性呈剂量依赖性升高,但当添加量为40 mg kg-1时,除NOS外其他各免疫比较均呈现出下降的趋势。攻毒实验结果表明,攻毒后的10天内对照组的累计死亡率显著高于4个添加组,4个添加组的免疫保护力为63%-72%,且相互之间差异不显著。本实验表明:1)饲料中添加大豆异黄酮可以提高凡纳滨对虾的特定生长率、免疫力和抗病力;2)大豆异黄酮在凡纳滨对虾饲料中的建议添加量为20 mg kg-1。
3.以初始体重为(6.80±0.00g)左右的刺参(A. japonicus Selenka)为研究对象,在基础饲料中分别添加0(对照组)、50、100和200 mg kg-1的甘草酸,配制4种实验饲料。结果表明:饲料中添加甘草酸对刺参的成活率没有显著影响,各处理组均为100%。饲料中添加200 mg kg-1甘草酸可显著提高刺参的特定生长率(SGR) (P<0.05)。饲料中添加甘草酸对刺参体常规成分含量没有产生显著影响。当饲料中甘草酸添加量为200 mg kg-1时,刺参体腔细胞一氧化氮合酶(NOS)活性和溶菌酶(LZM)活性显著高于其他添加组(P<0.05)。当饲料中甘草酸添加量≥100 mg kg-1时,刺参体腔细胞超氧阴离子产量(COP)、超氧化物歧化酶(SOD)活性、酸性磷酸酶(ACP)活性、NOS活性及LZM活性均显著高于对照组及50 mg kg-1添加组(P<0.05)。50 mg kg-1添加组以上各免疫指标与对照组差异不显著(P>0.05)。养殖实验结束后,通过注射刺参腐皮综合症致病菌灿烂弧菌(V. splendidus)进行刺参攻毒实验。攻毒后14d内对照组与50 mg kg-1添加组的累计发病率(分别为38.3%和36.7%)显著高于100和200 mg kg-1添加组(分别为30.0%和26.7%) (P<0.05)。本实验可得到以下结论:饲料中添加200 mg kg-1甘草酸可以提高刺参养殖的产量,同时可以提高刺参的非特异性免疫力和抗病力。
4.以初始体重为(6.80±0.01g)的刺参(A. japonicus Selenka)为研究对象,以基础饲料为对照组,在基础饲料中分别添加0.01%、0.02%和0.04%的左旋咪唑,配制4种实验饲料。实验结果显示,饲料中添加高含量的左旋咪唑(0.04%)显著提高了刺参的特定生长率(P<0.05),而低含量添加组(0.01%和0.02%)与对照组之间差异不显著(P>0.05)。饲料中添加左旋咪唑对刺参的成活率没有产生影响(P>0.05),各处理组成活率均为100%。饲料中添加左旋咪唑对刺参体常规成分含量没有产生显著影响(P>0.05)。饲料中添加高含量的左旋咪唑(0.04%)显著提高了刺参体腔细胞超氧阴离子产量(COP)、溶菌酶(LZM)活性和一氧化氮合酶(NOS)活性(P<0.05),低含量添加组(0.01%和0.02%)与对照组之间差异不显著(P>0.05)。当饲料中左旋咪唑添加量≥0.02%时,刺参体腔细胞的超氧化物歧化酶(SOD)活性和酸性磷酸酶(ACP)活性显著高于对照组,且0.04%添加组显著高于其余各处理组(P<0.05)。0.01%添加组上述免疫指标与对照组均差异不显著(P>0.05)。8周的饲养实验结束后,采用刺参腐皮综合症致病菌灿烂弧菌(V. splendidus)进行体壁注射攻毒实验。结果显示,饲料中添加0.04%的左旋咪唑显著降低了刺参攻毒后14 d的累计发病率(P<0.05),其余各添加组与对照组差异不显著(P>0.05)。上述结果表明,饲料中添加0.04%的左旋咪唑对刺参的生长、免疫力及抗病力具有较好的促进作用。
5.以初始体重为(6.80±0.00g)的刺参(A. japonicus Selenka)为研究对象,在配合饲料中分别加入0.00%(对照组)、0.03%、0.06%和0.12%的核苷酸制成4种实验饲料,在室内循环水系统中(200 l)进行为期8周的摄食生长实验。实验结果表明,饲料中添加核苷酸对刺参存活率没有产生影响,各处理组均为100%,但却显著影响其生长。当饲料中添加0.06%和0.12%的核苷酸时,刺参的特定生长率(SGR)显著高于对照组(0.00%)和低添加量组(0.03%)。0.03%添加组与对照组以及高核苷酸添加组间(0.06%添加组与0.12%添加组)的SGR均没有显著差异(P>0.05)。饲料中添加核苷酸对刺参体常规成分含量没有产生显著影响(P>0.05)。当饲料中核苷酸添加量≥0.06%时,刺参体腔细胞溶菌酶(LZM)活性和超氧化物歧化酶(SOD)活性均显著高于对照组和0.03%添加组(P<0.05),且0.06%添加组和0.12%添加组间差异不显著(P>0.05)。饲料中添加0.06%的核苷酸显著提高了刺参体腔细胞一氧化氮合酶(NOS)活性和酸性磷酸酶(ACP)活性(P<0.05),其余添加组与对照组差异不显著(P>0.05)。当饲料中核苷酸添加量为0.06%时,刺参体腔细胞超氧阴离子产量(COP)显著高于对照组与0.03%添加组(P<0.05),但与0.12%添加组差异不显著,0.03%添加组、0.12%添加组以及对照组间没有显著差异(P>0.05)。8周的饲养实验结束后,通过体壁注射刺参腐皮综合症致病菌灿烂弧菌(V. splendidus)进行刺参攻毒实验。在攻毒后的14天内,对照组和0.03%添加组刺参的累计发病率显著高于0.06%添加组和0.12%添加组(P<0.05)。0.03%添加组和对照组间以及0.06%添加组和0.12%添加组间刺参的累计发病率没有显著差异(P>0.05)。本研究结果表明,饲料中添加0.06%的核苷酸可以显著提高刺参的生长和非特异性免疫力,核苷酸可以作为一种安全高效的口服免疫增强剂应用于刺参的养殖生产。
6.以初始体重(6.80±0.02g)的刺参(A. japonicus Selenka)为研究对象,探讨饲料中添加乳铁蛋白对其生长和非特异性免疫力的影响。分别向每千克基础饲料中添加0(对照组)、50、100和200 mg乳铁蛋白,配制出4种实验饲料,在室内循环水系统中(200 l)进行为期8周的摄食生长实验。实验结果表明,饲料中添加乳铁蛋白对刺参的成活率没有产生影响,各处理组成活率均为100%。当饲料中添加100 mg kg-1乳铁蛋白时刺参特定生长率显著高于对照组(P<0.05)。饲料中添加乳铁蛋白对刺参体常规成分含量没有产生显著影响(P>0.05)。饲料中添加100 mg kg-1乳铁蛋白组刺参体腔细胞超氧阴离子产量(COP)著高于对照组,其余各添加组与对照组差异不显著。当饲料中乳铁蛋白添加量为100和200 mg kg-1时,刺参体腔细胞一氧化氮合酶(NOS)活性、超氧化物歧化酶(SOD)活性和酸性磷酸酶(ACP)活性显著高于对照组,且两个添加组间差异不显著。饲料中添加100 mg kg-1乳铁蛋白组刺参体腔细胞溶菌酶(LZM)活性显著高于对照组及其他添加组。50 mg kg-1乳铁蛋白组刺参体腔细胞上述免疫指标与对照组均没有显著差异。灿烂弧菌(V. splendidus)攻毒实验结果表明,饲料中添加100和200 mg kg-1乳铁蛋白组刺参的累计发病率显著低于对照组。因此,饲料中添加适宜含量的乳铁蛋白可以显著提高刺参生长率和非特异性免疫力。乳铁蛋白可以作为一种安全高效的口服免疫增强剂应用于刺参的养殖生产。
Six feeding trials were conducted in indoor circulating system to investigate the effects of dietary glycyrrhizin, soybean isoflavones, levamisole, nucleotide and bovine lactoferrin on growth, non-specific immunity and disease resistance for shrimp (L. vannamei) and sea cucumber (A. japonicus Selenka). The results are summarized as follows:
1. The growth response, total haemocyte count (THC), respiratory burst (release of superoxide anion), phenoloxidase (PO), superoxide dismutase (SOD), nitric oxide synthase (NOS) as well as resistance to the pathogen V. alginolyticus were measured in shrimps which had been fed diets supplemented with glycyrrhizin at 0, 50, 100, 150 and 200 mg kg-1 diet for 8 weeks. Dietary glycyrrhizin had no significant influence on survival rate of the shrimps (P>0.05). Significantly higher SGR was observed in the treatments with dietary glycyrrhizin supplementation than that in control (P<0.05). However, no significant differences in body composition were noticed among dietary treatments (P>0.05). Shrimps fed the diet with 200 mg kg-1 glycyrrhizin had significant higher THC, PO activity, respiratory burst activity and SOD activity than that in control (P<0.05). Increased THC, respiratory burst activity and NOS activity were also noticed in shrimps fed diets with 100 or 150 mg kg-1 glycyrrhizin (P<0.05). Significantly lower cumulative mortality after challenge was observed in shrimps fed the diets supplemented with glycyrrhizin (8.33 -16.67%) than that in control group (36.67%) (P<0.05). These results indicated that glycyrrhizin might elevate the function of nonspecific immunity and enhance disease resistance of L. vannamei as well as improve production in shrimp farming.
2. The growth response, total haemocyte count (THC), respiratory burst (release of superoxide anion), phenoloxidase (PO) activity, superoxide dismutase (SOD) activity, nitric oxide synthase (NOS) activity and resistance to the pathogen Vibrio alginolyticus were measured in the white shrimp (L. vannamei) which had been fed diets supplemented with soybean isoflavones (SI) at 0, 5, 10, 20, 40 mg kg-1 for 8 weeks. Dietary SI had no significant influence on survival rate of the shrimps (P>0.05). Significantly higher SGR was observed in treatments with 10, 20 and 40 mg kg-1 SI supplement than that in control (P<0.05). No significant differences in body composition were noticed among dietary treatments. Shrimps fed a diet with 20 mg kg-1 SI supplementation showed significant increase in THC, PO activity, respiratory burst activity, SOD activity as well as NOS activity (P<0.05). Increased THC, respiratory burst activity, SOD activity and NOS activity were also noticed in shrimps fed the diets with SI supplementation at 10 or 40 mg kg-1 (P<0.05). Significantly lower cumulative mortality after challenge with V. alginolyticus was observed in shrimps fed the diets with SI supplementation (10.0-13.3%) than that in control group (36.7%) (P<0.05). It is therefore concluded that oral administration of SI at 20 mg kg-1 for 8 weeks could enhance the immune ability of shrimps and improve production in shrimp farming.
3. An 8-week feeding experiment was conducted to evaluate the effects of glycyrrhizin on growth, non-specific immunity of sea cucumber (A. japonicus Selenka) as well as its resistance against V. splendidus. Glycyrrhizin was administered to sea cucumbers through the diets at four levels (0, 50, 100 and 200 mg kg-1 diet, respectively). The basal diet (0 mg kg-1 diet) was used as the control. Each diet was randomly allocated to triplicate groups of sea cucumbers in indoor fiberglass (200 l) tanks with circulating seawater and constant aeration. And each tank was stocked with 30 sea cucumbers (initial average weight 6.80±0.10g). The results showed that dietary glycyrrhizin had no significant influence on survival rate of sea cucumber. Glycyrrhizin supplemented at 200 mg kg-1 significantly enhanced the specific growth rate of sea cucumbers (P<0.05), whereas other supplementation did not (P>0.05). No significant difference in body composition was observed among dietary treatments. Sea cucumbers fed diet with 200 mg kg-1 glycyrrhizin had significantly higher nitric oxide synthase (NOS) activity and lysozyme (LZM) activity than that fed other diets(P<0.05). The intracellular O2- production (COP), LZM, NOS, superoxide dismutase (SOD) and acid phosphatase (ACP) activity in sea cucumbers fed diets with 100 and 200 mg kg-1 glycyrrhizin were significantly higher when compared with the control and 50 mg kg-1 supplementation groups (P<0.05). However, no significant difference of the above immunological parameters was observed between the control and 50 mg kg-1 glycyrrhizin groups (P>0.05). The challenge experiment showed that sea cucumbers fed the diet with 100 and 200 mg kg-1 glycyrrhizin had significantly lower accumulative morbidity compared with the control and 50 mg kg-1 supplementation groups (P<0.05). And no significant difference was observed between 100 and 200 mg kg-1 supplementation groups. These results suggested that feeding glycyrrhizin at a dose of 200 mg kg-1 could enhance growth, non-specific immunity as well as resistance against V. splendidus of sea cucumber (Apostichopus japonicus Selenka).
4. An 8-week feeding experiment was conducted to evaluate the effects of dietary levamisole on growth, non-specific immunity as well as resistance against V. splendidus of sea cucumber (A. japonicus Selenka) (initial body weight 6.80±0.01 g). Results showed that dietary levamisole has no influence on survival rate of A. japonicus (Selenka) (P>0.05). The specific growth rate (SGR) in sea cucumbers fed diet with 0.04% levamisole was significantly increased when compared with the control group (P<0.05). However, no significant difference in SGR was oberved among 0.01%, 0.02% and the control groups (P>0.05). Dietary levamisole had no significant influence on body composition of A. japonicus (Selenka) (P>0.05). Significantly higher intracellular O2- production (COP), lysozyme (LZM) activity, superoxide dismutase (SOD), nitric oxide synthase (NOS) activity and acid phosphatase (ACP) activity in coelomocyte were observed in sea cucumbers fed the diet with 0.04% levamisole when compared with control group (P<0.05). Increased SOD activity and ACP activity were also noticed in sea cucumbers fed diet containing 0.02% levamisole (P<0.05). The SOD activity and ACP activity in sea cucumbers fed a diet containing 0.04% levamisole were showed significantly higher when compared with the other treatments (P<0.05). After 8 weeks of feeding trial, a challenging test was conducted by injecting the sea cucumbers with Vibrio splendidus. Significantly lower (P<0.05) cumulative morbidity was observed during the following 14 days after challenge in sea cucumbers fed the diet containing 0.04% levamisole than that in the control group. The presents results suggested that 0.04% levamisole supplementation in diet could improve growth, non-specific immunity and disease resistance of sea cucumber.
5. An 8-week feeding trial was conducted to evaluate the effects of dietary nucleotide on growth, non-specific immunity and disease resistance against V. splendidus of sea cucumber, A. japonicus (Selenka). Nucleotide (GMP) was administered to sea cucumbers through the diets at four levels (0%, 0.03%, 0.06% and 0.12%, respectively). The basal diet (without nucleotide supplementation) was used as control. Each diet was allocated to triplicate groups of sea cucumbers stocked in indoor 200 l circular fiberglass tanks with circulating seawater and constant aeration. And each tank was stocked with 30 sea cucumbers (initial weight 6.80±0.00 g). The results of the feeding trial showed that dietary nucleotide had no significant influence on survival rate of A. japonicus (Selenka). Sea cucumbers fed diets containing 0.06% or 0.12% nucleotide proved to have a significantly better growth than those fed the diet supplemented with 0.03% nucleotide (P<0.05). No significant difference in body composition was observed among dietary treatments. Sea cucumbers fed the diet containing 0.06% dietary nucleotide showed a significant (P<0.05) increase in intracellular O2- production (COP), lysozyme (LZM) activity, superoxide dismutase (SOD), nitric oxide synthase (NOS) activity as well as acid phosphatase (ACP) activity in coelomocyte. Increased LZM activity and SOD activity were also noticed in sea cucumbers fed the diet supplemented with 0.12% nucleotide (P<0.05). No significant difference in the above immune parameters was observed between sea cucumbers fed control diet and fed diet with 0.03% nucleotide supplementation (P>0.05). The challenge trial showed that sea cucumbers fed the diets with 0.06% and 0.12% nucleotide had significantly lower cumulative morbidity compared with the control and 0.03% nucleotide groups (P<0.05), and no significant difference was observed between 0.06% and 0.12% nucleotide groups. These results suggested that feeding nucleotide at a dose of 0.06% could enhance growth, non-specific immunity as well as resistance against V. splendidus of sea cucumber Apostichopus japonicus Selenka.
6. An 8-week feeding experiment was conducted to evaluate the effects of bovine lactoferrin (LF) on growth as well as non-specific immunity of sea cucumber (Apostichopus japonicus Selenka). Lactoferrin was administered to sea cucumber through the diets at three levels (50, 100 and 200 mg kg-1 diet, respectively). The basal diet (without lactoferrin supplementation) was used as the control diet. The intracellular O2- production (COP), lysozyme (LZM) activity, superoxide dismutase (SOD), nitric oxide synthase (NOS) activity and acid phosphatase (ACP) activity in coelomocyte were measured at day 56. And the specific growth rate (SGR), survival and cumulative mortality after body injection with V. splendidus (100μl V. splendidus suspension at 1×109CFU ml-1 per sea cucumber) were assayed after 56 days feeding period. Dietary lactoferrin had no significant influence on survival rate of sea cucumber. Significantly higher SGR was observed in sea cucumber fed diet containing 100 mg kg-1 lactoferrin than that in control diet. No significant difference in body composition was observed among dietary treatments. Sea cucumbers fed the diet containing 100 mg kg-1 lactoferrin showed significant increase in COP, NOS activity, SOD activity, LZM activity as well as ACP activity (P<0.05). Increased NOS activity, SOD activity and ACP activity were also noticed in sea cucumbers fed the diet containing 200 mg kg-1 lactoferrin (P<0.05). Sea cucumbers fed the diet containing 100 mg kg-1 lactoferrin showed significantly higher LZM activity when compared with other treatments (P<0.05). The highest cumulative morbidity was observed in the sea cucumbers fed the control diet when challenged with injection of V. splendidus. Significantly lower cumulative morbidity at 14 days after challenge was observed in sea cucumber fed diets containing 100 or 200 mg kg-1 lactoferrin compared with the control (P<0.05). These results indicated that lactoferrin might elevate the function of nonspecific immunity and enhance disease resistance of A. japonicus (Selenka) as well as improve production in sea cucumber farming.
引文
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