循环水系统中基于鱼类福利学特征性指示因子的基础研究
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摘要
随着近海环境污染的加剧，地下海水资源的锐减，传统池塘和流水式养殖模式已经不能顺应产业可持续发展的需求。工业化循环水养殖模式以其可控性、产品质量可保证性、环境友好、资源节约等优点，正在迅速发展，正成为未来海洋农业的重要发展方向之一。为追求高产量、高品质的水产品，封闭循环水养殖模殖的鱼类福利学研究就变得尤为重要。本研究从养殖密度入手，从水质和养殖对象两方面筛选对鱼类福利、健康水平具有预警功能的敏感指示因子，探讨其跟养殖鱼类福利水平的耦合关系，以进一步推动封闭循环水式养殖模式的良性、健康和可持续发展。主要研究结果如下：
1.在12套独立的封闭循环水养殖系统中，当大菱鲆幼鱼（体重：13.84±2.74g）起始养殖密度为：低：0.66,中等：1.26,较高：2.56,高：4.00kg/m2时，经过10周养殖试验发现：
①密度与幼鱼生长存在极显著的负相关关系，但是同时其作为一种底栖鱼类，当水质优良、饵料充足情况下，在一定临界养殖密度范围（中等-较高密度），生长不再受到密度影响。
②随着养殖密度的增加，幼鱼摄食量会随着种内社会竞争的增加而减少（中等-较高密度没有差异），故摄食量可作为密度影响监测的敏感指示因子。同时建议，开发研究多层投饵装置，以减少饵料竞争压力。
③高密度下幼鱼摄食量最少，但是肠道的蛋白酶活力却最高；低密度下幼鱼饵料摄入量最多，但是肠道蛋白活力呈现最低的趋势。可能养殖鱼类在高密度下随着摄食量的减少，调动了补偿机制，提高营养物吸收效率。
④在高密度下，幼鱼血浆的过氧化氢酶活力出现了升高但是并不显著，可能高密度已开始给养殖对象带来氧化胁迫，故建议养殖密度低于试验中最高密度。
⑤不同养殖密度条件下，鱼体色变化非常明显：高密度下鱼体颜色发白而低密度下颜色更接近养殖背景色，且幼鱼一旦受到胁迫体色便会变深；同时，在不同养殖密度下，尽管常规水质参数基本一致，但是氧化还原电位（ORP）存在差异。故鱼体色和水体ORP水体可作为潜在敏感指示因子进行研究。
2.鱼体色变化与其福利水平的耦合关系
①通过不同颜色背景的9格试验系统发现，大菱鲆幼鱼出现在粉色、淡紫色背景下的频率明显高于黑色、红色，幼鱼对深色背景表现出明显厌恶。
②通过计算机视觉研究发现，幼鱼在黑色、红色背景下，体色明度变化速率明显要比在粉色、淡紫色背景下强烈，这也侧面证明了在黑色、红色背景下，幼鱼需要调动更多的激素来调整体色以适应背景色。
③不同浓度氨氮急性胁迫后，幼鱼在黑色背景色下体色明度变化速率随着胁迫氨氮浓度的增高而变小，故可通过监测大菱鲆幼鱼体色明度变化速率来判定其健康、福利水平。
3.养殖水体氧化还原电位与养殖水体水质、养殖对象福利水平的耦合关系。
①通过探讨不同ORP短期作用下，封闭循环水系统水质以及养殖对象福利水平的变化，确定海水封闭循环水系统水体中ORP最大应不高于320mV,否则较为危险容易引起鱼类死亡。
②随着ORP水平、养殖水体氧浓度的增高，即使经过长达2周的与对照组ORP、溶氧水平一致的恢复期，养殖对象全血的溶氧水平会显著增加、Hct（血红素浓度）和Hb（血细胞压积）含量会显著降低。
③当养殖水体ORP在300-320mV，养殖对象会出现饵料摄入量减少、生长缓慢的情况，故建议养殖海水ORP水平低于300-320mV。建议养殖水体ORP水平低于300mV。
④首次发现随着养殖水体ORP水平增高至300-320mV时，养殖对象更容易抵挡外来病原微生物的感染，抗病力更强。
Since the pollution on the coastal area and the reduction of the undergroundseawater resource, the traditional flow-through or pond culture style did not go withthe tide of the aquaculture development. Recirculating aquaculture system is growingto the most important culture way in the future sea-farming in view of its advantageon the stability of the system, safety of the product, and environmental pollution-free.In pursuit of high quality and quantity of seafood, the research on fish welfare is moreand more crucial. In this study, two potential fish welfare indicators were selectedfrom the primary density stress study. Then the relation between fish health andindicators was investigated. It aims to promote RAS style in a healthy, sustainable,smooth way. The main results are as follows,
1. Turbot (Scophthalmus maximus)(13.84±2.74g; average weight±SD) werereared at four different initial densities (low0.66, medium1.26, sub-high2.56,high4.00kg/m2) for10weeks in12independent recirculating systems (RAS) at23±1°C. Final densities were4.67,7.25,14.16, and17.47kg/m2, respectively,which translate to82,108,214, and282percent cover of the bottom of the tank.
①Density had both negative and independent impacts on growth. The final meanweight, specific growth rate (SGR), and voluntary feed intake significantlydecreased and the coefficient of variation (CV) of final body weight increasedwith increased stocking density. While as a benthic fish, with enough supplyof feed and good water quality, stocking density had no influence on fishgrowth within a certain density range.
②When density increased, feed intake decreased since the increased socialcompetition. Thus feed intake is an optimal indicator on density stressmonitoring. For culture at higher density, multi-level feeding devices aresuggested to ease feeding competition.
③The protease activity of the digestive tract at pH7,8.5,9, and10wassignificantly higher for the highest density group, whereas it had a tendency tobe lower (but not significantly) at pH4and8.5for the lowest density group.The intensity of protease activity was inversely related to feed intake at thedifferent densities possibly due to that fish at high density took compensatorystrategy to promote nutrition assimilations.
④Catalase activity was higher (but not significantly) at the highest density,perhaps because high density induced an oxidative effect in turbot. It issuggested the density is lower than the high density.
⑤Under different densities, fish body was obviously different visually. At thesame time, the regular water indexes were within an optimal range while ORP(oxidation-reduction potential) was different under different densities. Thus,body color and ORP would be two potential indicators for fish welfare.
2. The relation between fish body color and welfare,
①The experiment of9color backgrounds selection was conducted. Theappearance frequency of juvenile turbot in purple and pink background wassignificantly higher than that in black and red one.
②Body hue change rate under black and red backgrounds was more drastic andintensive than under pink and purple backgrounds based on computer visiontechnology. Results indicated that fish need more physiological adjustment toadapt backgrounds of different colors and it is better to use lighter color as thebackground rather than black or red color.
③Body brightness change rate under the black background decreasedsignificantly after acute ammonia stress. Thus turbot body brightness changerate was an optical indicator for welfare monitoring.
3. The relation between ORP and fish welfare, water quality in marine RAS,
①After several short-term ORP trials, ORP level in fish tank should be lowerthan320mV in marine RAS considering fish welfare and water quality inmarine RAS.
②With the increase of ORP and dissolved oxygen in fish tanks, the blood Hctand Hb would decrease accompanied with the increased bloody oxygen evenafter two weeks recovery (normal ORP and DO).
③When long-term ORP was kept between300and320mV, feed intake would decrease and growth was slowly. Thus for long-tem operation, ORP wassuggested around300mV.
④It is the first time to found that with the long-term promoted ORP at300-320mV, fish were more against vibrio infection.
1.在12套独立的封闭循环水养殖系统中，当大菱鲆幼鱼（体重：13.84±2.74g）起始养殖密度为：低：0.66,中等：1.26,较高：2.56,高：4.00kg/m2时，经过10周养殖试验发现：
①密度与幼鱼生长存在极显著的负相关关系，但是同时其作为一种底栖鱼类，当水质优良、饵料充足情况下，在一定临界养殖密度范围（中等-较高密度），生长不再受到密度影响。
②随着养殖密度的增加，幼鱼摄食量会随着种内社会竞争的增加而减少（中等-较高密度没有差异），故摄食量可作为密度影响监测的敏感指示因子。同时建议，开发研究多层投饵装置，以减少饵料竞争压力。
③高密度下幼鱼摄食量最少，但是肠道的蛋白酶活力却最高；低密度下幼鱼饵料摄入量最多，但是肠道蛋白活力呈现最低的趋势。可能养殖鱼类在高密度下随着摄食量的减少，调动了补偿机制，提高营养物吸收效率。
④在高密度下，幼鱼血浆的过氧化氢酶活力出现了升高但是并不显著，可能高密度已开始给养殖对象带来氧化胁迫，故建议养殖密度低于试验中最高密度。
⑤不同养殖密度条件下，鱼体色变化非常明显：高密度下鱼体颜色发白而低密度下颜色更接近养殖背景色，且幼鱼一旦受到胁迫体色便会变深；同时，在不同养殖密度下，尽管常规水质参数基本一致，但是氧化还原电位（ORP）存在差异。故鱼体色和水体ORP水体可作为潜在敏感指示因子进行研究。
2.鱼体色变化与其福利水平的耦合关系
①通过不同颜色背景的9格试验系统发现，大菱鲆幼鱼出现在粉色、淡紫色背景下的频率明显高于黑色、红色，幼鱼对深色背景表现出明显厌恶。
②通过计算机视觉研究发现，幼鱼在黑色、红色背景下，体色明度变化速率明显要比在粉色、淡紫色背景下强烈，这也侧面证明了在黑色、红色背景下，幼鱼需要调动更多的激素来调整体色以适应背景色。
③不同浓度氨氮急性胁迫后，幼鱼在黑色背景色下体色明度变化速率随着胁迫氨氮浓度的增高而变小，故可通过监测大菱鲆幼鱼体色明度变化速率来判定其健康、福利水平。
3.养殖水体氧化还原电位与养殖水体水质、养殖对象福利水平的耦合关系。
①通过探讨不同ORP短期作用下，封闭循环水系统水质以及养殖对象福利水平的变化，确定海水封闭循环水系统水体中ORP最大应不高于320mV,否则较为危险容易引起鱼类死亡。
②随着ORP水平、养殖水体氧浓度的增高，即使经过长达2周的与对照组ORP、溶氧水平一致的恢复期，养殖对象全血的溶氧水平会显著增加、Hct（血红素浓度）和Hb（血细胞压积）含量会显著降低。
③当养殖水体ORP在300-320mV，养殖对象会出现饵料摄入量减少、生长缓慢的情况，故建议养殖海水ORP水平低于300-320mV。建议养殖水体ORP水平低于300mV。
④首次发现随着养殖水体ORP水平增高至300-320mV时，养殖对象更容易抵挡外来病原微生物的感染，抗病力更强。
Since the pollution on the coastal area and the reduction of the undergroundseawater resource, the traditional flow-through or pond culture style did not go withthe tide of the aquaculture development. Recirculating aquaculture system is growingto the most important culture way in the future sea-farming in view of its advantageon the stability of the system, safety of the product, and environmental pollution-free.In pursuit of high quality and quantity of seafood, the research on fish welfare is moreand more crucial. In this study, two potential fish welfare indicators were selectedfrom the primary density stress study. Then the relation between fish health andindicators was investigated. It aims to promote RAS style in a healthy, sustainable,smooth way. The main results are as follows,
1. Turbot (Scophthalmus maximus)(13.84±2.74g; average weight±SD) werereared at four different initial densities (low0.66, medium1.26, sub-high2.56,high4.00kg/m2) for10weeks in12independent recirculating systems (RAS) at23±1°C. Final densities were4.67,7.25,14.16, and17.47kg/m2, respectively,which translate to82,108,214, and282percent cover of the bottom of the tank.
①Density had both negative and independent impacts on growth. The final meanweight, specific growth rate (SGR), and voluntary feed intake significantlydecreased and the coefficient of variation (CV) of final body weight increasedwith increased stocking density. While as a benthic fish, with enough supplyof feed and good water quality, stocking density had no influence on fishgrowth within a certain density range.
②When density increased, feed intake decreased since the increased socialcompetition. Thus feed intake is an optimal indicator on density stressmonitoring. For culture at higher density, multi-level feeding devices aresuggested to ease feeding competition.
③The protease activity of the digestive tract at pH7,8.5,9, and10wassignificantly higher for the highest density group, whereas it had a tendency tobe lower (but not significantly) at pH4and8.5for the lowest density group.The intensity of protease activity was inversely related to feed intake at thedifferent densities possibly due to that fish at high density took compensatorystrategy to promote nutrition assimilations.
④Catalase activity was higher (but not significantly) at the highest density,perhaps because high density induced an oxidative effect in turbot. It issuggested the density is lower than the high density.
⑤Under different densities, fish body was obviously different visually. At thesame time, the regular water indexes were within an optimal range while ORP(oxidation-reduction potential) was different under different densities. Thus,body color and ORP would be two potential indicators for fish welfare.
2. The relation between fish body color and welfare,
①The experiment of9color backgrounds selection was conducted. Theappearance frequency of juvenile turbot in purple and pink background wassignificantly higher than that in black and red one.
②Body hue change rate under black and red backgrounds was more drastic andintensive than under pink and purple backgrounds based on computer visiontechnology. Results indicated that fish need more physiological adjustment toadapt backgrounds of different colors and it is better to use lighter color as thebackground rather than black or red color.
③Body brightness change rate under the black background decreasedsignificantly after acute ammonia stress. Thus turbot body brightness changerate was an optical indicator for welfare monitoring.
3. The relation between ORP and fish welfare, water quality in marine RAS,
①After several short-term ORP trials, ORP level in fish tank should be lowerthan320mV in marine RAS considering fish welfare and water quality inmarine RAS.
②With the increase of ORP and dissolved oxygen in fish tanks, the blood Hctand Hb would decrease accompanied with the increased bloody oxygen evenafter two weeks recovery (normal ORP and DO).
③When long-term ORP was kept between300and320mV, feed intake would decrease and growth was slowly. Thus for long-tem operation, ORP wassuggested around300mV.
④It is the first time to found that with the long-term promoted ORP at300-320mV, fish were more against vibrio infection.
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