仿刺参幼参对不同刺激的应激机制及维生素C、E抗应激作用研究
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摘要
设置了不同剂量的维生素C（Vitamin C）和维生素E（Vitamin E）分别添加到仿刺参幼参饲料中，尝试降低其对不同刺激的应激反应。商品饲料作为对照日粮，在对照日粮基础上，另外分别添加3个浓度梯度的vitamin C1000,1500以及2000mg kg-1以及3个浓度梯度的vitamin E150、250、350mg kg-1分别获得vitamin C添加组日粮和vitamin E添加组日粮。试验动物选用初重约为2.5g的仿刺参幼参，分为对照组、vitamin C添加组（内有3个浓度梯度）、vitamin E添加组（内有3个浓度梯度），共计1个对照组和6个处理组，每组3个平行，每个平行放入500g幼参。试验养殖周期为45d，每15d设置一个应激试验时间点，在试验进行第0、15、30和45d取样。共设置两种刺激方式：亚硝酸盐和盐度变化。饲喂vitamin C饲料添加处理组的仿刺参幼参进行亚硝酸盐和盐度应激，vitamin E饲料添加处理组只进行亚硝酸盐应激试验。应激试验开始时，将对应饲料处理组的仿刺参幼参迅速放置到配好的浓度为0.5、1.0和1.5mg L-1亚硝酸盐溶液中以及盐度分别为20ppt和40ppt的海水中，应激进行12h，在0、4和8h进行取样，液氮速冻后置于-80℃冷藏备用。整个试验期间动物生长性能作为评价标准之一。测定氧自由基相关指标，如羟自由基（-OH）含量、丙二醛（MDA）和总抗氧化能力（T-AOC），以及抗氧化酶活性如超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性。响应面（Response surface method）分析法用作分析各变量相互作用以及vitamin C、E最优化添加量和养殖天数。结果获得了在不同亚硝酸浓度和盐度应激条件下，刺参幼参氧自由基和抗氧化酶活性变化情况：在亚硝酸盐应激条件下，-OH和MDA含量随应激时间延长和亚硝酸浓度升高而迅速升高，显著高于对照组(P＜0.05)，而T-AOC呈现出与之相反的趋势；抗氧化酶活性呈现先升高后降低的趋势。从生长性能看，添加Vitamin C或Vitamin E组均显著高于对照组。Vitamin C或Vitamin E添加后，-OH和MDA含量随养殖时间延长和添加量增加呈减少趋势，而T-AOC呈增加趋势。根据响应面二次回归方程，有效降低亚硝酸盐应激的Vitamin C添加量是1400–2000mgkg-1日粮，养殖时间是29–35d；而Vitamin E最优添加量则是180-260mg kg-1日粮，养殖时间是29-35d。
在不同盐度应激条件下，发现无论是在20ppt或在40ppt处理组时，-OH和MDA值均显著高于在30ppt (P＜0.05)，T-AOC则显著下降，此外，研究发现20ppt处理组-OH要显著高于40ppt处理组，而T-AOC则低于40ppt组，表明仿刺参幼参在低盐（20ppt）应激时氧化损伤可能要高于高盐（40ppt）应激，可能仿刺参幼参对高盐的抗性高于其对低盐的抗性。抗氧化酶SOD和CAT酶活性在应激时间12h内保持较高的酶活水平，均高于30ppt组。Vitamin C添加后，-OH和MDA值均出现降低趋势，T-AOC出现增加趋势，显示出Vitamin C添加对降低盐度应激氧化损伤起到了显著作用。根据响应面法二次回归曲线方程，获得能够有效降低盐度应激的Vitamin C最优补充添加量为1697-2000mg kg-1，养殖天数为34-37d。
总之，上述研究结果对研究刺参不同应激机制和有效降低应激反应，提供了理论依据，并对生产实践如何高效使用Vitamin C、Vitamin E降低应激反应提供了指导。
Different amounts of vitamin C，E were added to diets fed to juveniles(2.5±0.15g) of sea cucumber Apostichopus japonicus (Selenka) in an attempt toreduce the stress response of specimens exposed to stress. A commercial feed wasused as the control diet and three experimental diets were made by supplementing1000,1500, or2000mg vitamin C kg-1diet or150,250，350mg vitamin E kg-1diet tocontrol diet separately in a45-day experiment. Sea cucumbers were exposed to threedifferent levels (0.5,1.0, and1.5mg L-1) of nitrite stress or two levels salinity （20ppt and40ppt）for4,8, and12h at four time intervals (0,15,30, and45d). Growthof the animals was recorded during the experiment. Indices of reactive oxygen species(ROS)(i.e. hydroxyl free radical (–OH), malondialdehyde (MDA) and totalantioxidant capacity (T-AOC)) and antioxidant enzyme activities (i.e., superoxidedismutase (SOD) and catalase (CAT)) were measured. Response surface methodology(RSM) was used to analyze the effect of multiple factors on ROS indices and enzymeactivities. After45days feeding trial, weight gain (WG) and special growth rate (SGR)of both vitamin C and E supplementation groups were significantly higher than thoseof control group (P＜0.05). The results showed that vitamin C and Esupplementation could promote growth performance of sea cucumbers. The levels of–OH and MDA increased under exposure time extending and nitrite concentrationincreasing, whereas T-AOC level decreased. SOD and CAT activities increased at4and8h and decreased at12h. After a period during which the animal consumedexperimental diets, the levels of–OH and MDA decreased and that of T-AOC increased. This result suggests that diets containing vitamin C and E can reduce thenitrite stress response in the animals and increase their antioxidant capacity. Themultifactor regression equation of growth performance, ROS indices, and duration offeeding results suggest that vitamin C supplementation of1400–2000mg kg-1diet for29–35days and vitamin E supplementation of180-260mg kg-1diet for29-35d couldreduce the effects of nitrite exposure effectively.
During the salinity stress，the content of–OH and MDA increased significantlyin both20ppt and40ppt in contrast to30ppt, and the T-AOC level decreased.Additionally, the content of–OH of20ppt was higher than that of40ppt, and theT-AOC level lower than that of40ppt, may suggest that the oxidation impairment in20ppt was higher than that of40ppt. After vitamin C supplementation, the ROSindices were improved and the activities of antioxidant enzyme were maintained highlevel, suggesting vitamin C could effectively reduce the salinity stress. According tothe RSM equations, the optimal vitamin C supplementation was1697-2000mg kg-1diet and the feeding days were34-37d.
In conclusion， these results provide useful information for studying themechanism of stress and reducing the hazard of nitrite stress and acute salinitychanges in sea cucumber aquaculture.
在不同盐度应激条件下，发现无论是在20ppt或在40ppt处理组时，-OH和MDA值均显著高于在30ppt (P＜0.05)，T-AOC则显著下降，此外，研究发现20ppt处理组-OH要显著高于40ppt处理组，而T-AOC则低于40ppt组，表明仿刺参幼参在低盐（20ppt）应激时氧化损伤可能要高于高盐（40ppt）应激，可能仿刺参幼参对高盐的抗性高于其对低盐的抗性。抗氧化酶SOD和CAT酶活性在应激时间12h内保持较高的酶活水平，均高于30ppt组。Vitamin C添加后，-OH和MDA值均出现降低趋势，T-AOC出现增加趋势，显示出Vitamin C添加对降低盐度应激氧化损伤起到了显著作用。根据响应面法二次回归曲线方程，获得能够有效降低盐度应激的Vitamin C最优补充添加量为1697-2000mg kg-1，养殖天数为34-37d。
总之，上述研究结果对研究刺参不同应激机制和有效降低应激反应，提供了理论依据，并对生产实践如何高效使用Vitamin C、Vitamin E降低应激反应提供了指导。
Different amounts of vitamin C，E were added to diets fed to juveniles(2.5±0.15g) of sea cucumber Apostichopus japonicus (Selenka) in an attempt toreduce the stress response of specimens exposed to stress. A commercial feed wasused as the control diet and three experimental diets were made by supplementing1000,1500, or2000mg vitamin C kg-1diet or150,250，350mg vitamin E kg-1diet tocontrol diet separately in a45-day experiment. Sea cucumbers were exposed to threedifferent levels (0.5,1.0, and1.5mg L-1) of nitrite stress or two levels salinity （20ppt and40ppt）for4,8, and12h at four time intervals (0,15,30, and45d). Growthof the animals was recorded during the experiment. Indices of reactive oxygen species(ROS)(i.e. hydroxyl free radical (–OH), malondialdehyde (MDA) and totalantioxidant capacity (T-AOC)) and antioxidant enzyme activities (i.e., superoxidedismutase (SOD) and catalase (CAT)) were measured. Response surface methodology(RSM) was used to analyze the effect of multiple factors on ROS indices and enzymeactivities. After45days feeding trial, weight gain (WG) and special growth rate (SGR)of both vitamin C and E supplementation groups were significantly higher than thoseof control group (P＜0.05). The results showed that vitamin C and Esupplementation could promote growth performance of sea cucumbers. The levels of–OH and MDA increased under exposure time extending and nitrite concentrationincreasing, whereas T-AOC level decreased. SOD and CAT activities increased at4and8h and decreased at12h. After a period during which the animal consumedexperimental diets, the levels of–OH and MDA decreased and that of T-AOC increased. This result suggests that diets containing vitamin C and E can reduce thenitrite stress response in the animals and increase their antioxidant capacity. Themultifactor regression equation of growth performance, ROS indices, and duration offeeding results suggest that vitamin C supplementation of1400–2000mg kg-1diet for29–35days and vitamin E supplementation of180-260mg kg-1diet for29-35d couldreduce the effects of nitrite exposure effectively.
During the salinity stress，the content of–OH and MDA increased significantlyin both20ppt and40ppt in contrast to30ppt, and the T-AOC level decreased.Additionally, the content of–OH of20ppt was higher than that of40ppt, and theT-AOC level lower than that of40ppt, may suggest that the oxidation impairment in20ppt was higher than that of40ppt. After vitamin C supplementation, the ROSindices were improved and the activities of antioxidant enzyme were maintained highlevel, suggesting vitamin C could effectively reduce the salinity stress. According tothe RSM equations, the optimal vitamin C supplementation was1697-2000mg kg-1diet and the feeding days were34-37d.
In conclusion， these results provide useful information for studying themechanism of stress and reducing the hazard of nitrite stress and acute salinitychanges in sea cucumber aquaculture.
引文
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