PROTECTIVE EFFECTS OF DIFFERENT C/N RATIOS FORMED BIOFLOCS ON ACUTE COPPER EXPOSURE OF RHYNCHOCYPRIS LAGOWSKII DYBOWSKI
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摘要: 实验旨在探索不同C/N生物絮团对急性铜暴露洛氏鱥免疫抑制、炎症反应与氧化应激的保护作用。挑选480尾洛氏鱥(Rhynchocypris lagowskii Dybowski)幼鱼(10±0.15) g, 随机分成4组, 每组3个重复, 每个重复40尾鱼。对照组饲喂商品料(C/N 10.8﹕1), 实验组选择葡萄糖为外添碳源调节C/N 15﹕1(Ⅱ组)、C/N 20﹕1(Ⅲ组)和C/N 25﹕1(Ⅳ组), 生长实验为56d, 之后进行为期96h的急性铜暴露胁迫实验。结果表明, 各实验组中免疫、抗氧化酶活性与炎症因子含量随着C/N的增加先升高后下降。其中, 与对照组相比, Ⅲ和Ⅳ组血清碱性磷酸酶(AKP)、酸性磷酸酶(ACP)、溶菌酶(LSZ)、一氧化氮合酶(NOS)、补体C3、C4和免疫球蛋白M(IgM)水平显著升高(P<0.05), 而Ⅲ和Ⅳ组之间补体C3和C4水平差异不显著(P>0.05); Ⅲ和Ⅳ组血清过氧化氢酶(CAT)、抗坏血酸(ASA)、超氧化物歧化酶(SOD)、总抗氧化能力(T-AOC)、谷胱甘肽过氧化物酶(GSH-PX)与谷胱甘肽还原酶(GR)酶活性显著高于对照组(P<0.05), 而丙二醛(MDA)含量显著低于对照组(P<0.05)。同时Ⅱ组的SOD和CAT酶活性也有所升高, 且与对照组差异显著(P<0.05); Ⅲ和Ⅳ组血清肿瘤坏死因子α(TNF-α)、白介素1β(IL-1β)与白介素-6(IL-6)含量显著低于对照组(P<0.05), 且它们两组之间无显著差异(P>0.05)。然而, Ⅱ、Ⅲ和Ⅳ组白介素-2(IL-2)含量均显著高于对照组, 且第Ⅲ组含量最高。在实验条件下, 当C/N≥15: 1时, 生物絮团能有效地增强急性铜暴露下洛氏鱥的免疫与抗氧化酶活力, 且在C/N为20﹕1时效果最为显著。Abstract: This study combined BFT with waterborne copper to explore the protective effects of different C/N ratios formed bioflocs of R. lagowskii on immunosuppression, inflammation and oxidative stress in acute copper exposure. The results showed that with the increase of C/N ratio, the activities of antioxidation, immune enzymes and anti-inflammation factor of each group increased at first and then decreased. Compared with the control group, the levels of serum alkaline phosphatase (AKP), acid phosphatase (ACP), lysozyme (LSZ), nitric oxide synthase (NOS), complement C3, C4 and immunoglobulin M (IgM) increased significantly in Ⅲ and Ⅳ groups (P<0.05). However, there was no significant difference in complement C3 and C4 content between Ⅲ and Ⅳ groups (P>0.05). The activities of serum catalase (CAT), ascorbic acid (ASA), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-PX) and glutathione reductase (GR) enzyme in Ⅲ and Ⅳ groups were significantly higher than those in the control (P<0.05), while the content of malondialdehyde (MDA) was significantly lower than that in the control group (P<0.05). At the same time, the activities of SOD and CAT in treatment Ⅱ were significantly higher than those in the control group (P<0.05). The serum levels of tumor necrosis factor-α (TNF-α), interleukin-1 β (IL-1β) and interleukin-6 (IL-6) in Ⅲ and Ⅳ groups were significantly lower than those in the control group (P<0.05) without significant difference between Ⅲ group and Ⅳ group (P>0.05). However, the content of interleukin-2 (IL-2) in Ⅱ, Ⅲ and Ⅳ groups were significantly higher than that of the control group (P<0.05) with the highest content in group Ⅲ. Overall, these findings suggest that bioflocs can effectively enhance the activities of immune and antioxidant enzymes under acute copper exposure when C/N≥15 with the most significant effect at a 20﹕1 C/N ratio. Therefore, this study provides a new idea for in-depth exploration of bioflocs, a new mitigation scheme for water environmental pollution and biological toxicity caused by heavy metals, and a theoretical basis for promoting the healthy and stable development of aquaculture.
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Keywords:
- Bioflocs /
- Copper exposure /
- Immunosuppression /
- Inflammation /
- Oxidative stress /
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Rhynchocypris lagowskii Dybowski
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随着工业的快速发展, 各种重金属及其类似物废水直接或间接地排入江河湖海之中, 对水环境造成了不可恢复的污染[1－3]。铜是水体中主要的重金属污染物之一, 也是维持生命正常发育和新陈代谢所必需的重要微量元素。以鱼类为主的水生生物对铜有一定的富集和积累能力, 然而过量的积累将扰乱水生动物的正常生命活动, 造成其急、慢性中毒甚至死亡[4, 5]。生物絮团技术(BFT)是近年来发展起来的一种环境友好型养殖模式, 它的基本原理主要是循环利用剩余的营养物质, 以产生微生物生物量, 这些微生物生物量既可以用来就地喂养养殖的鱼类, 也可以作为动物饲料的一种成分进行收获和加工[6－8]。同时, BFT不仅可以作为微生物的载体, 还能通过调节养殖池中微生物的组成, 利用微生物之间的相互作用, 转化对系统中生物有很高毒性的氨氮和亚硝酸盐, 来实现控制水质的目的[9, 10]。目前, 关于重金属铜对水体污染的研究较少, 主要体现在介绍铜对水生生物的危害上, 如: 组织积累、氧化应激、炎症反应及免疫抑制等[11－13]; 关于生物絮团的研究, 主要集中在: BFT对水生生物(鱼、虾等)营养与免疫的研究[7, 9]、对养殖水体水质的研究[8]、对肠道菌群的研究[14]及关于生物絮团C/N的研究[15－17]。而关于不同C/N生物絮团对急性铜暴露洛氏鱥的免疫、抗氧化酶活性及炎症反应的影响则鲜有研究。
本实验甄选病害少, 抗逆性强, 味道鲜美, 无肌间刺, 营养价值高, 深受广大养殖人员与消费者喜爱的我国土著杂食性小型名贵淡水经济鱼类——洛氏鱥(Rhynchocypris lagowskii Dybowski) [2, 18, 19], 探讨不同C/N行成的生物絮团对急性铜暴露洛氏鱥免疫抑制、炎症反应与氧化应激的保护作用。
1. 材料与方法
1.1 实验设计
洛氏鱥购于九台区泉源鱼苗养殖场, 饲养于吉林农业大学水产研究室玻璃水族箱中, 暂养2周使其适应环境, 期间投喂商品日粮(通威饲料有限公司, 粗蛋白36%、粗脂肪8.0%、粗纤维4.0%和粗灰分6.4%)使其适应基础料。根据我们课题组之前发表[2, 18－20]培养生物絮团与调节C/N的方法, 选择无水葡萄糖粉(99.97%)为外加碳源, 按照C/N不同设置4个实验组: 对照组(C/N 10.8﹕1)、Ⅱ组(C/N 15﹕1)、Ⅲ组(C/N 20﹕1)和Ⅳ组(C/N 25﹕1)。
1.2 饲养管理
在实验开始前, 停食24h, 选取480尾规格一致, 体质健壮的洛氏鱥幼鱼(10±0.15) g, 随机分入12个玻璃缸中(100 L水), 每缸40尾, 期间水温控制(18±2)℃, 按2%—3%投饵率日投喂2次(9:00和18:00), 每日19:00向鱼缸中添加葡萄糖粉。24h不间断曝气充氧, 每周测量溶解氧、pH、氨氮、亚硝酸盐和总磷等, 确保各项水质指标均符合洛氏鱥的生长条件。在8周饲养实验结束后, 进行96 h的急性攻毒胁迫实验, 根据前期研究, 铜暴露洛氏鱥的半致死浓度为1.03 mg/L[2], 向各实验组添加调配好浓度的无水硫酸铜(CuSO4)。攻毒期间的实验条件与饲养实验条件一致, 但是并未投喂。
1.3 样品收集及测定方法
水质指标与水体中铜浓度 实验期间使用多参数水质测试仪YSI556测定水质指标; 絮团体积采用 Imhoff 锥形管法测定; 总体悬浮物使用TOYO定量滤纸抽滤发测定; 在铜暴露实验结束前后, 分别从每个鱼缸中取5 mL养殖水体与10 mL浓硝酸(65%HNO3)混合, 经消化作用72h后, 4℃离心10min(12000×g), 最后, 取上清液用AA-6300原子吸收光谱仪测定铜的浓度。
生长性能 在饲养实验结束后, 停食24h, 每桶随机选取洛氏鱥10尾, 测其体长和体质量, 用于各项指标的测。特定生长率、增重率、存活率和饲料转化率的测试方法参考Long等[8]和Yu等[18－20]。
免疫、抗氧化与炎症反应指标 在胁迫实验结束后, 从每缸随机取洛氏鱥10尾, 经MS-222(200 mg/L)麻醉后用2.0 mL肝素钠注射器尾静脉采血, 血样 4℃离心10min(5000 r/min)收集血清, –20℃保存待测。
AKP、ACP(对硝基苯磷酸盐法)、LSZ(比浊法)、NOS(硝酸还原酶法)、C3、C4、IgM(免疫比浊法); CAT(可见光测定法)、ASA(比色法)、SOD(黄嘌呤氧化酶法)、T-AOC(ABTS速测法)、GSH-PX(二硫代二硝基苯甲酸法)、GR(紫外分光光度法)和MDA(TBA法); TNF-α、IL-1β、IL-2和IL-6采用双抗体夹心ELISA 法, 具体参照Yu等[2, 19, 20]。以上试剂盒均购买于南京建成生物工程研究所。
1.4 统计分析
结果以平均数±标准差表示。采用SPSS(20.0)软件对数据进行单因素方差分析(ANOVA), 以确定显著性差异, 再用Tukey’s多极差检验比较均值(P<0.05), 确定差异有统计学意义。
2. 结果
2.1 不同C/N生物絮团对养殖水体水质的影响
如表 1所示, 各组水温、胁迫后CuSO4浓度无显著差异(P>0.05); Ⅱ、Ⅲ和Ⅳ组pH、溶解氧、氨氮和亚硝酸盐显著低于对照组(P<0.05), 且各组之间差异不显著(P>0.05); Ⅲ和Ⅳ组总磷与透明度显著低于对照组(P<0.05), 其互相之间差异不显著(P>0.05)。相反的是: Ⅲ和Ⅳ组絮团体积与总体悬浮物显著高于对照组(P<0.05), 而Ⅲ和Ⅳ组之间无显著差异(P>0.05)。
表 1 不同C/N生物絮团对养殖水体水质的影响Table 1. Effects of different C/N ratios formed bioflocs on aquaculture water quality指标Index 对照组 Ⅱ组 Ⅲ组 Ⅳ组 水温Water temperature (℃) 18.54 ± 2.03 18.67±1.54 19.06±1.32 18.97±2.12 pH 7.52±0.36b 7.25±0.31a 7.21±0.29a 7.20±0.27a 溶解氧Dissolved oxygen (mg/L) 6.02±0.14b 5.49±0.11a 5.48±0.07a 5.41±0.09a 氨氮Ammonia nitrogen (mg/L) 0.43±0.05b 0.15±0.04a 0.12±0.06a 0.14±0.07a 亚硝酸盐Nitrite (mg/L) 0.13±0.03b 0.03±0.01a 0.01±0.01a 0.01±0.01a 总磷Total phosphorus (mg/L) 0.65±0.17b 0.61±0.18b 0.55±0.17a 0.58±0.2a 透明度Transparency (cm) 19.54±3.08c 14.77±2.56b 12.96±2.82a 10.47±2.19a 絮团体积Bioflocs volume (ml/L) 28.88±3.11a 33.18±2.96a 55.69±5.61b 68.73±5.19b 总体悬浮物Total suspended matter (mg/L) 324.98±17.93a 378±23.46a 580.6±40.26b 642.72±37.25b 添加CuSO4浓度Add concentration (mg/L) 1.030 1.030 1.030 1.030 实测CuSO4浓度Real concentration (mg/L) 1.010±0.020 1.007±0.031 1.020±0.020 1.013±0.021 胁迫后CuSO4浓度After stress concentration (mg/L) 1.009±0.014 0.997±0.152 0.980±0.026 0.987±0.152 注: 实验数据表示为“平均值±标准差”(n=3), 同行数据肩标有不同小写字母者表示差异显著(P<0.05), 相同小写字母或无字母表示差异不显著(P>0.05); 下表同Note: Values were expressed as “mean ± standard deviation” (n=3), and different lowercase letters were significant different (P<0.05). The same lowercase letter or no letter indicates not significant (P>0.05); the same applies below 2.2 不同C/N生物絮团对洛氏鱥生长性能的影响
如表 2所示, Ⅲ和Ⅳ组末体质量、特定生长率和增重率显著高于对照组, 而饲料转化率显著高低于对照组(P<0.05), 且Ⅲ和Ⅳ组之间差异不显著(P>0.05); Ⅱ和Ⅲ组的存活率显著高于对照组与Ⅳ组(P<0.05)。
表 2 不同C/N生物絮团对洛氏鱥生长性能的影响Table 2. Effects of different C/N ratios formed bioflocs on growth performance of R. lagowskii指标Index 对照组 Ⅱ组 Ⅲ组 Ⅳ组 初始体质量Initial body weight (g) 10.05±
0.0910.11±
0.1010.04±
0.1210.10±
0.15末体质量Final body weight (g) 29.71±
2.07a33.12±
2.85ab39.58±
3.72b40.17±
2.44b饲料转化率Feed conversion ratio 1.88±
0.11c1.74±
0.17c1.25±
0.12a1.36±
0.09b特定生长率Specific growth rate (%) 1.94±
0.08a2.12±
0.35ab2.46±
0.22bc2.47±
0.29c增重率Weight gain rate (%) 195.62±
20.37a227.59±
24.16ab294.22±
30.11b297.72±
32.66b存活率Survival rate (%) 90.00±
2.50a97.50±
2.50b99.17±
1.44b90.00±
2.50a2.3 不同C/N生物絮团对铜暴露洛氏鱥免疫酶活力的影响
由图 1可知, 生物絮团对铜暴露洛氏鱥免疫抑制具有一定的缓解作用, 其中: Ⅲ组各项免疫酶活力均显著高于对照组(P<0.05), Ⅳ组的ACP和补体C3, Ⅱ组的ACP、补体C4与IgM含量也显著高于对照组(P<0.05)。LSZ与AKP活力在Ⅱ、Ⅲ和Ⅳ组之间差异不显著(P>0.05); NOS活力在Ⅱ和Ⅳ组和对照组之间差异不显著(P>0.05)。
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图 1 不同C/N生物絮团对铜暴露洛氏鱥血清免疫酶的影响字母不同表示同一时间各实验组之间存在显著性差异(P<0.05); 下同Figure 1. Effects of different C/N ratios formed bioflocs on serum immune enzymes of R. lagowskii in waterborne copper exposureValues with the same letters are not significantly different at the same time (P<0.05); the same applies below2.4 不同C/N生物絮团对铜暴露洛氏鱥炎症因子的影响
由图 2可见, 不同C/N生物絮团对铜暴露洛氏鱥的炎症反应具有不同的缓解效果, 其中Ⅲ组和Ⅳ组TNF-α与IL-1β含量显著低于对照组, 且这两组之间差异不显著(P>0.05); IL-6含量在各实验组(Ⅱ、Ⅲ和Ⅳ组)中均显著低于对照组(P<0.05), 且Ⅱ、Ⅲ和Ⅳ组之间无显著差异(P>0.05); 关于IL-2的含量, Ⅱ、Ⅲ和Ⅳ组均显著高于对照组(P<0.05), 其中Ⅲ组含量最高, Ⅱ和Ⅳ组之间无显著差异(P>0.05)。
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图 2 不同C/N生物絮团对铜暴露洛氏鱥血清炎症因子的影响Figure 2. Effects of different C/N ratios formed bioflocs on serum inflammation factors of R. lagowskii in waterborne copper exposure2.5 不同C/N生物絮团对铜暴露洛氏鱥抗氧化酶的影响
由图 3可见, 生物絮团对铜暴露洛氏鱥氧化应激具有一定的缓解作用, 其中: Ⅲ和Ⅳ组SOD、GR和GSH-PX活性均显著高于对照组(P<0.05), 然而它们之间无显著差异(P>0.05)。与对照组相比, Ⅱ组的T-AOC和ASA含量差异不显著(P>0.05), 但是Ⅲ和Ⅳ组显著升高, 且Ⅲ组显著高于Ⅰ、Ⅱ和Ⅳ组(P<0.05); 各实验组(Ⅱ、Ⅲ和Ⅳ组)中的CAT活性均显著高于对照组(P<0.05), 其中Ⅲ组呈最大值, Ⅱ组与Ⅳ组无显著差异(P>0.05); MDA含量随着碳氮比的升高, 呈下降趋势, 当C/N 15(Ⅲ组)时达到最低值, 之后略有回升。与对照组相比, Ⅱ、Ⅲ和Ⅳ组均显著下降(P<0.05), 但是Ⅱ和Ⅳ组差异不显著, Ⅲ和Ⅳ组也无显著差异(P>0.05)。
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图 3 不同C/N生物絮团对铜暴露洛氏鱥血清抗氧化酶的影响Figure 3. Effects of different C/N ratios formed bioflocs on serum antioxidant enzymes of R. lagowskii in waterborne copper exposure3. 讨论
3.1 不同C/N生物絮团对洛氏鱥生长及养殖水体水质的影响
大量研究表明, 生物絮团可促进养殖动物的生长, 净化养殖水体, 当C/N适当时, 鱼类表现出最佳的生长趋势[6, 8, 16, 17]。本实验结果显示, 随着C/N的升高, 洛氏鱥的增重率和特定生长率呈上升趋势, 说明在本实验条件下, 高碳氮比形成的生物絮团有效地促进了洛氏鱥的生长; Ⅲ组饲料转化率显著低于对照组, 表明C/N20:1时形成的絮凝物可作为洛氏鱥的天然饵料, 并且可以连续在原位获得额外的蛋白质、脂肪、矿物质和维生素, 以加快生长进度, 与Wang等[17]所得结果基本吻合。Ⅳ组虽然在末体重、增重率与特定生长率上都出现最高值, 但在存活率上却与对照组不相上下, 究其原因可能是C/N过高, 导致异养菌大量增殖, 总体悬浮物含量过饱和, 堵塞鱼鳃, 不利于鱼类呼吸。
Avnimelech[21]研究表明, 当水体C/N为15.75以上时, BFT能显著净化水质污染问题。从本文结果来看: 随着C/N的升高, 溶解氧、pH、氨氮、亚硝酸盐、总磷、透明度及胁迫后CuSO4浓度均呈下降趋势。溶解氧下降是由于微生物繁殖生长消耗大量氧气[16]; pH下降可能是洛氏鱥和微生物生长, 呼吸放出的CO2增多, 导致废物累积和酸败[22]; Hari等[23]指出额外添加碳源可有效降低水体里的氨氮、亚硝酸盐与总磷的浓度, 原因可能是生物絮团对含氮污染物的快速异养转化[16], 与本文结果相似; 透明度持续下降是因为饲料的不断投入与异样菌大量繁殖[19]; 攻毒后CuSO4浓度下降, 可能是絮团对水体中铜的吸附、沉降等作用引起的[2]。
3.2 不同C/N生物絮团对铜暴露洛氏鱥免疫抑制的缓解作用
免疫指标是监测环境危害对鱼类健康潜在影响的重要参数, 作为抵御病原微生物的第一道防线, 非特异性免疫与先天免疫系统在保护鱼类健康方面起着至关重要的作用[2, 24－26]。LSZ作为非特异性免疫系统的重要组成部分, 能水解病原菌中的黏多糖, 分解细菌细胞壁上肽聚糖中N-乙酰胞壁酸和N-乙酰氨基葡萄糖之间的β-(1, 4)糖苷键, 在清除侵袭性病原体和细菌方面发挥着至关重要的作用[27], AKP和ACP是参与动物免疫的一种重要水解酶, 在免疫应答过程中能增强血细胞识别异物, 改变病原体表面结构[19, 20]。NOS能帮助巨噬细胞对抗免疫系统中的病原体[2]。在本实验中, 第Ⅲ组中的LSZ、AKP、ACP和NOS活力显著高于对照组, 表明当养殖水体中C/N为20﹕1时, 能显著缓解铜暴露洛氏鱥的免疫抑制, 增强其对环境胁迫的抗性。而当C/N为15﹕1与25﹕1时, LSZ、AKP和NOS活力与对照组无显著差异, 这可能是由于低C/N絮团或过高C/N絮团对铜暴露洛氏鱥的免疫酶影响不大。Liu等[28]研究生物絮团增强罗非鱼拥挤胁迫耐受能力, 结果显示C/N20﹕1组显著提高了LSZ和AKP活力, 与本实验结果大致吻合。补体C3和C4是参与体液免疫的大分子, 可以通过溶解免疫球蛋白复合物杀死病毒和细菌, 促进炎症反应[29]。IgM是衡量鱼类体液免疫功能的重要指标[30]。在本实验中, 在不同C/N生物絮团对洛氏鱥补体C3、C4和IgM的影响略有不同, 但总体趋势皆为先升高后趋于平缓。与对照组相比, 第Ⅲ组中的补体C3、C4和IgM水平显著升高。以上结果可能是由于, 鱼类摄食或生存于不同C/N形成的生物絮团环境中, 而一定范围之内, 高C/N生物絮团内含有多种微生物、益生菌和生物活性物质, 可以提高鱼类免疫酶活性, 并通过次生代谢产物使宿主受益[31, 32]。同时, 它们的细胞成分或代谢物(类胡萝卜素等)可以作为免疫刺激剂, 改善先天免疫系统并提供对宿主的保护[2, 9]。此外, 高C/N生物絮团还能通过促进鱼类生长, 诱导其消化道微生物区系的变化, 获得提高免疫力和抵御环境应激的能力[20]。
3.3 不同C/N生物絮团对铜暴露洛氏鱥炎症反应的缓解作用
鱼类的免疫功能与炎症密切相关, 炎症是由细胞因子介导和启动的, 已有实验表明重金属铜可诱导头肾免疫相关基因转录和细胞应激反应的改变[33]。细胞因子是一组多肽类细胞调节物质, 包括白细胞介素、肿瘤坏死因子和细胞刺激因子等, 主要由外周免疫细胞合成。众所周知, IL-1β和TNF-α是2种强大的促炎因子, 可通过调节其他细胞因子的表达来诱导炎症反应[34]。IL-2可促进T淋巴细胞增殖和分化, 诱导和增强致死性T细胞、单核细胞和巨噬细胞的活性; IL-6和IL-1共同促进T细胞增殖并参与炎症和发热[35]。在本研究中, 当C/N≥20﹕1时, BFT显著降低了铜暴露洛氏鱥血清的IL-1β、IL-6和TNF-α水平, 升高了IL-2水平。这表明高C/N生物絮团能有效缓解洛氏鱥铜暴露所引起的炎症反应, 与Yu等[18]利用BFT缓解氨胁迫引起的洛氏鱥炎症反应结果相似。究其结果可能是由于高碳氮比BFT系统中能形成大量有益菌群, 有益菌群通过产生有机酸、细菌素或争夺营养物质抑制有害菌的繁殖, 还可抑制有害菌产生内毒素和致癌物质, 降低炎症因子水平, 从而实现免疫调节作用[2, 9]。同时, BFT中的益生菌(芽孢杆菌、乳酸菌和丁酸梭菌等)能进入鱼类肠道, 通过代谢物或表面抗原刺激鱼类免疫体系, 与有害菌竞争营养和附着位点, 保护鱼类免受病原菌侵染, 进而增强鱼类非特异性免疫力[36, 37]。此外, BFT系统中水生微生物的微生态平衡可以有效控制氨氮和亚硝酸盐, 降低条件性病原微生物致病的几率 [38]。
3.4 不同C/N生物絮团对铜暴露洛氏鱥抗氧化应激的缓解作用
生物体具有复杂而又精密的抗氧化防御系统, 在一定范围内, 可以及时有效地清除重金属积累产生的活性氧(ROS), 减少脂质变化引起的细胞损伤, 保护细胞免受氧化应激的伤害[1－3, 26]。众所周知, 内源性抗氧化酶, 包括SOD、CAT、T-AOC、GSH-PX、GR和ASA是抵御氧化应激的第一道防线[2, 19, 20, 39]。Xu等[40]研究表明, 絮团组在血浆与肝胰脏中出现相对较高的SOD与T-AOC活性, 而GSH-PX活性与对照组差异不显著。Chen等[15]研究结果显示: 高C/N下生物絮团能显著提高仿刺参体液SOD、CAT和GSH-PX活力。Yu等[20]研究表明, 当C/N=20﹕1时, 生物絮团能有效提高黄金鲫肝胰脏、肠道与肾脏中SOD、CAT、GSH-PX与T-AOC活性, 降低MDA水平。且在我们实验组的前期发现中, C/N 20﹕1形成的生物絮团对洛氏鱥各个组织(鳃、脑、肾、脾、肝和肠)的抗氧化酶活性均有不同程度的提高[19]。在本实验中, 与对照组相比, 除MDA外, 铜暴露洛氏鱥血清抗氧化酶(Ⅲ和Ⅳ组)均显著提高, 相反的是, MDA含量显著降低。这表明在C/N≥20﹕1情况下, BFT可以有效提高抗氧化酶活性, 降低脂质变化引起的超氧化物损伤, 缓解洛氏鱥经铜暴露而引起的一系列氧化应激反应。这个发现可能是由于高C/N形成的生物絮团降低了洛氏鱥的脂质过氧化水平, 诱导洛氏鱥具有更强的抗氧自由基能力, 提高了其对抗环境胁迫的能力及成活率[2, 16, 25]。此外, BFT中含有大量的天然微生物和生物活性生长因子, 包括胡萝卜素、叶绿素、植物甾醇、多酚、多糖、牛磺酸和维生素, 也能增强鱼类的应激反应和抗氧化功能[19, 20, 22]。
4. 结论
综上, 在不同C/N水平下形成的生物絮团对铜暴露洛氏鱥的免疫抑制、炎症反应与氧化应激有不同的影响。当C/N=20﹕1时, 生物絮团能显著提高铜暴露洛氏鱥免疫酶(AKP、ACP、LSZ、NOS、C3、C4和IgM)活力和抗氧化酶(CAT、ASA、T-AOC、SOD、GSH-PX和GR)活力, 降低MDA含量, 稳定炎症因子(IL-1β、TNF-α、IL-2和IL-6), 从而有利于洛氏鱥缓解铜暴露引起的应激反应。
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图 1 不同C/N生物絮团对铜暴露洛氏鱥血清免疫酶的影响
字母不同表示同一时间各实验组之间存在显著性差异(P<0.05); 下同
Figure 1. Effects of different C/N ratios formed bioflocs on serum immune enzymes of R. lagowskii in waterborne copper exposure
Values with the same letters are not significantly different at the same time (P<0.05); the same applies below
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图 2 不同C/N生物絮团对铜暴露洛氏鱥血清炎症因子的影响
Figure 2. Effects of different C/N ratios formed bioflocs on serum inflammation factors of R. lagowskii in waterborne copper exposure
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图 3 不同C/N生物絮团对铜暴露洛氏鱥血清抗氧化酶的影响
Figure 3. Effects of different C/N ratios formed bioflocs on serum antioxidant enzymes of R. lagowskii in waterborne copper exposure
表 1 不同C/N生物絮团对养殖水体水质的影响
Table 1 Effects of different C/N ratios formed bioflocs on aquaculture water quality
指标Index 对照组 Ⅱ组 Ⅲ组 Ⅳ组 水温Water temperature (℃) 18.54 ± 2.03 18.67±1.54 19.06±1.32 18.97±2.12 pH 7.52±0.36b 7.25±0.31a 7.21±0.29a 7.20±0.27a 溶解氧Dissolved oxygen (mg/L) 6.02±0.14b 5.49±0.11a 5.48±0.07a 5.41±0.09a 氨氮Ammonia nitrogen (mg/L) 0.43±0.05b 0.15±0.04a 0.12±0.06a 0.14±0.07a 亚硝酸盐Nitrite (mg/L) 0.13±0.03b 0.03±0.01a 0.01±0.01a 0.01±0.01a 总磷Total phosphorus (mg/L) 0.65±0.17b 0.61±0.18b 0.55±0.17a 0.58±0.2a 透明度Transparency (cm) 19.54±3.08c 14.77±2.56b 12.96±2.82a 10.47±2.19a 絮团体积Bioflocs volume (ml/L) 28.88±3.11a 33.18±2.96a 55.69±5.61b 68.73±5.19b 总体悬浮物Total suspended matter (mg/L) 324.98±17.93a 378±23.46a 580.6±40.26b 642.72±37.25b 添加CuSO4浓度Add concentration (mg/L) 1.030 1.030 1.030 1.030 实测CuSO4浓度Real concentration (mg/L) 1.010±0.020 1.007±0.031 1.020±0.020 1.013±0.021 胁迫后CuSO4浓度After stress concentration (mg/L) 1.009±0.014 0.997±0.152 0.980±0.026 0.987±0.152 注: 实验数据表示为“平均值±标准差”(n=3), 同行数据肩标有不同小写字母者表示差异显著(P<0.05), 相同小写字母或无字母表示差异不显著(P>0.05); 下表同Note: Values were expressed as “mean ± standard deviation” (n=3), and different lowercase letters were significant different (P<0.05). The same lowercase letter or no letter indicates not significant (P>0.05); the same applies below 
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表 2 不同C/N生物絮团对洛氏鱥生长性能的影响
Table 2 Effects of different C/N ratios formed bioflocs on growth performance of R. lagowskii
指标Index 对照组 Ⅱ组 Ⅲ组 Ⅳ组 初始体质量Initial body weight (g) 10.05±
0.0910.11±
0.1010.04±
0.1210.10±
0.15末体质量Final body weight (g) 29.71±
2.07a33.12±
2.85ab39.58±
3.72b40.17±
2.44b饲料转化率Feed conversion ratio 1.88±
0.11c1.74±
0.17c1.25±
0.12a1.36±
0.09b特定生长率Specific growth rate (%) 1.94±
0.08a2.12±
0.35ab2.46±
0.22bc2.47±
0.29c增重率Weight gain rate (%) 195.62±
20.37a227.59±
24.16ab294.22±
30.11b297.72±
32.66b存活率Survival rate (%) 90.00±
2.50a97.50±
2.50b99.17±
1.44b90.00±
2.50a
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