渔业科学进展  2023, Vol. 44 Issue (1): 70-79  DOI: 10.19663/j.issn2095-9869.20210831005
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引用本文 

袁新程, 施永海, 徐嘉波, 税春, 杨明, 谢永德, 张忠华. 半咸水、淡水养殖刀鲚消化、抗氧化、非特异性免疫及代谢酶活力的比较分析[J]. 渔业科学进展, 2023, 44(1): 70-79. DOI: 10.19663/j.issn2095-9869.20210831005.
YUAN Xincheng, SHI Yonghai, XU Jiabo, SHUI Chun, YANG Ming, XIE Yongde, ZHANG Zhonghua. Analysis and Comparison of Digestive, Antioxidant, Nonspecific Immunity and Metabolic Enzyme Activities of Coilia nasus Cultured in Brackish Water and Freshwater[J]. Progress in Fishery Sciences, 2023, 44(1): 70-79. DOI: 10.19663/j.issn2095-9869.20210831005.

基金项目

上海市科技兴农项目(沪农科推字(2019)第2-5号)和上海长江口主要经济水生动物人工繁育工程技术研究中心项目(13DZ2251800)共同资助

作者简介

袁新程,E-mail: xcyuan2016@163.com

通讯作者

施永海,研究员,E-mail: yonghais@163.com

文章历史

收稿日期:2021-08-31
收修改稿日期:2021-11-02
半咸水、淡水养殖刀鲚消化、抗氧化、非特异性免疫及代谢酶活力的比较分析
袁新程 , 施永海 , 徐嘉波 , 税春 , 杨明 , 谢永德 , 张忠华     
上海市水产研究所 上海市水产技术推广站 上海 200433
摘要:为探究刀鲚(Coilia nasus)在半咸水(盐度为8.7~12.5)和淡水养殖条件下消化、抗氧化、非特异性免疫及代谢能力的差异,本研究采用池塘套养方式,以同批次人工繁育的刀鲚1+龄鱼为研究对象,进行为期210 d的养殖实验。结果显示,半咸水养殖刀鲚(以下简称半咸水刀鲚)的生长速度显著大于淡水养殖刀鲚(以下简称淡水刀鲚) (P < 0.05),其对蛋白质和淀粉的消化能力均高于淡水刀鲚,而对脂肪的消化有所减弱。其中,半咸水刀鲚的4种内脏器官的淀粉酶(AMS)活力均大于淡水刀鲚,但差异不显著(P > 0.05);胃和肝中蛋白酶活力均显著大于淡水刀鲚(P < 0.05),肝和盲囊中脂肪酶(LPS)活力均显著低于淡水刀鲚(P < 0.05)。半咸水刀鲚的AMS、蛋白酶活力大小顺序均为盲囊 > 肠 > 胃 > 肝,而LPS活力大小顺序为肠 > 盲囊 > 胃 > 肝;淡水刀鲚的AMS、蛋白酶和LPS活力大小顺序均为盲囊 > 肠 > 胃 > 肝。半咸水刀鲚的超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)活力及丙二醛(MDA)含量均显著升高(P < 0.05),而过氧化氢酶(CAT)活力显著降低(P < 0.05);其肝脏中碱性磷酸酶(AKP)和酸性磷酸酶(ACP)活力均显著低于淡水刀鲚(P < 0.05),半咸水对刀鲚谷草转氨酶(AST)活力无显著影响,但会显著降低谷丙转氨酶(ALT)活力。研究表明,半咸水养殖条件不仅促进了刀鲚的生长,提升了刀鲚对蛋白质和淀粉的消化能力,还通过提高SOD和GSH-PX活力来提升刀鲚免疫力,减少外界环境威胁。综上所述,刀鲚更适合在半咸水中进行养殖,建议今后可用半咸水(盐度为8.7~12.5)养殖1+龄刀鲚。
关键词刀鲚    半咸水    消化酶    抗氧化酶    非特异性免疫酶    代谢酶    
Analysis and Comparison of Digestive, Antioxidant, Nonspecific Immunity and Metabolic Enzyme Activities of Coilia nasus Cultured in Brackish Water and Freshwater
YUAN Xincheng , SHI Yonghai , XU Jiabo , SHUI Chun , YANG Ming , XIE Yongde , ZHANG Zhonghua     
Shanghai Fisheries Research Institute, Shanghai Fisheries Technical Extension Station, Shanghai 200433, China
Abstract: Coilia nasus is a precious migratory fish typical to the Yangtze River Basin of China. It is locally referred to as the "Three delicious fish of the Yangtze River, " with its fresh meat being particularly delicious and popular. In recent years, wild C. nasus numbers in the Yangtze River Basin have been decreasing due to environmental changes and illegal overfishing, among other reasons. To attenuate the availability of C. nasus, the Shanghai Fisheries Research Institute has successfully established an indoor artificial breeding and pond culture program in 2011. The research and development of C. nasus artificial breeding technologies have steadily progressed, though the artificial breeding yield of C. nasus remains relatively low. In addition, related research on the artificial cultivation of C. nasus is still in its infancy. Research on C. nasus in China and abroad mainly focuses on gonad development, breeding technology, muscle nutrient composition, and growth performance. No reports exist on the effects of brackish water culture conditions on the physiology of C. nasus. Given its economic importance, it would be of great value to evaluate the changes in the digestive, antioxidant, nonspecific immune capacity, and metabolic rates of C. nasus from brackish water (natural seawater in Hangzhou Bay, salinity 8.7~12.5) to freshwater aquaculture, to determine the breeding conditions of C. nasus, and to improve the artificial breeding yield and related technology. To investigate the differences in digestive capacity, antioxidant capacity, nonspecific immune capacity, and metabolic rates of C. nasus in brackish water and freshwater aquaculture conditions, the experiment adopted the pond interbreeding method. Two groups were established: A brackish aquaculture group and a freshwater aquaculture group (control group). Each group was housed in a single aquaculture pond with an area of 0.17 hm2. 500 C. nasus were stocked in each pond and moved to a Takifugu obscurus pond for a seven-month aquaculture experiment. At the end of the experiment, 12 C. nasus were randomly selected from each group and randomly divided into three replicates. Four C. nasus samples were selected from each replicate. The liver, intestine, stomach, and cecum of C. nasus were dissected on an ice plate and carefully removed to prepare a 10% homogenate. The activities of digestive enzymes, antioxidant enzymes, nonspecific immune enzymes, and metabolic enzymes and the total protein content were measured using a kit produced by the Nanjing Jiancheng Bioengineering Institute. The activities of digestive enzymes, antioxidant enzymes, nonspecific immunity enzymes, and metabolic enzymes in brackish water and freshwater aquaculture environments were compared and analyzed in a seven-month aquaculture experiment using the same batch of artificially bred C. nasus. The growth and survival rates of C. nasus cultured in brackish water (hereafter referred to as brackish water C. nasus) were significantly higher than that in the freshwater (hereafter referred to as freshwater C. nasus) (P < 0.05). The digestive ability of protein and starch in brackish water C. nasus was higher than that of freshwater C. nasus, while the digestive ability was weakened. Among them, the activity of amylase (AMS) in the four organs of brackish water C. nasus was higher than those in freshwater, but not significantly so (P > 0.05). The activities of protease in the liver and stomach were significantly higher than those in freshwater C. nasus (P < 0.05), and the activities of lipase (LPS) in the liver and cecum were significantly lower than those in freshwater C. nasus (P < 0.05). The order of AMS and protease activity of brackish water C. nasus was: cecum > intestine > stomach > liver, while the order of LPS activity was intestine > cecum > stomach > liver. The order of AMS, protease, and LPS activity of freshwater C. nasus was: cecum > intestine > stomach > liver. Brackish water had significant effects on antioxidant, nonspecific immunity, and metabolic abilities, and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and malondialdehyde (MDA) in the liver of C. nasus were significantly increased, while the activity of catalase (CAT) was significantly decreased (P < 0.05). The activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) in the liver of C. nasus in brackish water were significantly lower than those in freshwater (P < 0.05). Brackish water had no significant effect on the activity of aspartate aminotransferase (AST), but it significantly reduced the activity of alanine aminotransferase (ALT). In summary, brackish water aquaculture conditions promoted the growth of C. nasus; increased the survival rate; improved the ability of C. nasus to digest protein and starch; improved the immunity of fish; and reduced the influence of external stresses by decreasing the activities of AKP, ACP, and ALT enzymes and increasing the activities of SOD and GSH-PX enzymes. This study also found that C. nasus living in brackish water, as a result of maintaining the internal osmotic balance, significantly reduced ALT enzyme activity and reduced urea production and emission, thereby reducing the pollution of the direct environment. Therefore, brackish water is more suitable for the aquaculture of C. nasus, and it is recommended that brackish water (salinity of 8.7~12.5) be used for aquaculture of 1+ age C. nasus individuals in the future. The results of this study provide a scientific basis for the improvement of artificial breeding technology, improving the yield of C. nasus, and the development of special compound feed for C. nasus.
Key words: Coilia nasus    Brackish water    Digestive enzyme    Antioxidant enzyme    Nonspecific immunity enzyme    Metabolic enzyme    

刀鲚(Coilia nasus)俗称刀鱼,是我国长江流域一种典型的江海洄游名贵鱼类,自古至今一直享有“长江三鲜”的美誉(袁传宓等, 1976; 施永海等, 2014)。近年来,由于长江水域环境变化和非法过度捕捞等原因而导致刀鲚数量迅速减少,至2012年,长江刀鲚产量为5750 kg,降至历史最低点(徐钢春等, 2016)。鉴于长江流域野生刀鲚资源的不断减少和已不能形成优势种群,自2019年2月1日起,我国农业农村部已禁止天然刀鲚的生产性捕捞。同时,为填补长江刀鲚资源衰减的漏洞,上海市水产研究所一直致力于刀鲚人工繁育和养殖技术的研究,并于2011年成功突破了刀鲚室内全人工养殖技术,但刀鲚的养殖产量极低(施永海等, 2014; 徐钢春等, 2016)。为提高刀鲚的人工养殖产量,在实际养殖过程中,需要选择合适的养殖环境,以保障在安全养殖条件下获取最大渔产量。因此,探究刀鲚在半咸水和淡水养殖条件下的生长性能及消化、抗氧化、非特异性和代谢能力的变化,对确定刀鲚的养殖条件,提高刀鲚的人工养殖技术及产量具有重要意义。

目前,刀鲚人工养殖的相关研究还处于发展初级阶段(魏广莲等, 2012; 闻海波等, 2009),国内外有关刀鲚的研究主要集中于其性腺发育(Xu et al, 2011; 徐钢春等, 2011a)、苗种繁殖技术(徐钢春等, 2016; 施永海等, 2015)、肌肉营养成分(唐雪等, 2011; 李丽等, 2019)及生长性能(袁新程等, 2021; 董文霞等, 2014; 徐钢春等, 2011b)等方面,至今尚未报道半咸水养殖条件对刀鲚的消化、抗氧化、非特异性免疫和代谢能力影响的相关研究。因此,开展半咸水养殖对刀鲚生理、生化指标影响的研究具有十分重要的现实意义。本实验开展了半咸水和淡水养殖对刀鲚消化酶、抗氧化酶、非特异性免疫酶及代谢酶影响的研究,以期为刀鲚的人工养殖技术提供理论依据。

1 材料与方法 1.1 实验材料

实验所用刀鲚是上海市水产研究所奉贤科研基地(以下简称奉贤科研基地)于2019年全人工集约化繁育而成,并经过室外池塘养殖培育1年后的同一批鱼种。实验用水为经过池塘、蓄水池沉淀、60目筛网过滤的当地内河水和自然海水(杭州湾自然海水,盐度为8.7~12.5)。实验池塘呈长方形,为南北方向泥底池塘,面积为0.17 hm2,平均水深为1.8 m,每口池塘装配1台叶轮式增氧机(功率1.5 kW),实验开始前,使用漂白精进行清塘和消毒,清除杂鱼、杂虾。

1.2 实验设计与养殖管理

养殖实验于2020年3月17日—10月20日在奉贤科研基地的露天方形泥底池塘中进行,设置2个组,即半咸水养殖组(杭州湾自然海水,盐度为8.7~12.5)和淡水养殖组(对照组),每组1口养殖池塘(面积为0.17 hm2),共计2口池塘。每口池塘放养500尾刀鲚,套养于暗纹东方鲀(Takifugu obscurus)池塘中。在刀鲚放养之前,实验池塘先进行生物饵料的培养,在放养刀鲚的前7 d,向池塘进水至平均水深为180 cm,之后每天中午开启增氧机2 h,连续培养7 d,待每口池塘水体中的生物饵料(枝角类、桡足类、小糠虾等)量达到50 ind./L时开始放养刀鲚进行养殖实验。

实验期间,不投喂其他配合饲料,均以本池塘中活生物饵料为食,并每5 d对实验池塘水体中的活生物饵料进行1次检测,保证每口池塘水体中活饵料生物量>5 ind./L,当发现不足时,及时从其他培育池塘中捞取活生物饵料进行补给。养殖期间,每14 d换水1次,每次换水1/3。实验期间,每天18:30开启增氧设备至次日06:00将其关闭(下雨等恶劣天气,适当延长开增氧机的时间),自然水温为15.6~32.4℃,pH为8.22~8.63,溶解氧(DO)≥6.95 mg/L,总氨氮(TAN)≤0.25 mg/L,亚硝酸盐氮(NO2-N)≤0.13 mg/L。

1.3 取样与酶活力测定方法

养殖实验结束后,每组随机采取12尾刀鲚并随机分成3个重复,每个重复取4尾刀鲚为一个样本。先使用0.5 mg/L MS-222对取样刀鲚进行麻醉,再用清水清洗干净,擦干体表水分后放于冰盘之上,进行解剖并小心取出肝、肠、胃和盲囊4种器官,用事先冷却过的0.86%生理盐水(4℃)清洗后,使用干净滤纸擦干表面水分,整个操作过程均在冰盘上进行,将取出的样品分装后暂存在–80℃冰箱中待测。在酶活力测定前,先将冷冻样品置于4℃冰柜中进行解冻,然后移入事先预冷的5 mL离心管中,加入9倍体积的事先冷却过的0.86%生理盐水,在冰浴环境下进行研磨,制备10%匀浆液,并置于2500 r/min的低温离心机(4℃)中离心10 min,吸取上层液体,即刻测定其各酶活力指标和总蛋白含量。

刀鲚内脏样品中消化、抗氧化、非特异性免疫及代谢酶活力和其总蛋白含量的测定,均使用购自南京建成生物工程研究所生产的试剂盒。淀粉酶(Amylase, AMS)试剂盒(碘—淀粉比色法)、脂肪酶(Lipases, LPS)试剂盒(比色法)、胃蛋白酶(Pepsin, PPS)试剂盒、胰蛋白酶(Trypsin, TPS)测试盒、过氧化氢酶(Catalase, CAT)测试盒(可见光法)、超氧化物歧化酶(Superoxide dismutase, SOD)测试盒(羟胺法)、谷胱甘肽过氧化物酶(Glutathione peroxidase, GSH-PX)测试盒、A059-1碱性磷酸酶(Alkaline phosphatase, AKP)试剂盒(可见光比色法)、酸性磷酸酶(Alanine aminotransferase, ACP)测试盒(可见光比色法)、谷丙转氨酶(Alanine aminotransferase, ALT)测试盒(赖氏法)、丙二醛(Malondialdehyde, MDA)试剂盒(TBA法)以及谷草转氨酶(Aspartate aminotransferase, AST)试剂盒(比色法),使用考马斯亮蓝法检测刀鲚内脏样品中总蛋白质含量。本研究中,所有酶活力及总蛋白质含量测定步骤和计算方法均严格按照测试盒的说明书进行。

1.4 生长性能指标测定计算

生长性能参数计算参考敬庭森等(2021),具体方法如下:

$ 体长相对增长率(\text{relative length growth rate, LGR}, \%) = (L_{T} – L_{0})/L_{0} ×100\% $ (1)
$ 体重相对增长率(\text{relative weight gain rate, WGR}, \%) = (W_{t }– W_{0})/W_{0} ×100\% $ (2)
$ 肥满度(\text{condition factor, CF}) = W_{t }/(L_{t})^{3} × 100 $ (3)
$ 存活率(\text{survival rate, SR}, \%) = 100\%×(N_{T}/N_{0}) $ (4)
$ 特定生长率(\text{specific growth ratio, SGR}, \%/d)= [\ln(W_{t}) – \ln(W_{0})]/t×100\% $ (5)

式中,L0为实验开始时刀鲚的体长;Lt为实验结束时刀鲚的体长;W0为实验开始时刀鲚的体重;Wt为实验结束时刀鲚的体重;t为养殖时间;N0为实验初始时刀鲚的数量;Nt为实验结束时刀鲚存活的数量。

1.5 数据处理和统计

使用Excel 2007软件整理实验数据,运用SPSS 17.0软件对同一养殖条件下,刀鲚不同内脏器官间酶活力大小进行单因素方差(one-way ANOVA)分析及Duncan氏法多重比较,对2种养殖条件下刀鲚生长指标及酶活力进行独立样本t检验和比较其大小差异显著性。所得结果均以平均值±标准差(mean±SD)来表示,P < 0.05为差异显著。

2 结果与分析 2.1 半咸水养殖对刀鲚生长性能的影响

半咸水和淡水刀鲚池塘的养殖情况见表 1。从表 1可以看出,半咸水刀鲚和淡水刀鲚的初始体长和初始体重相比均无显著差异(P > 0.05),而经过213 d的养殖,半咸水刀鲚的末体长和末体重均显著大于淡水刀鲚(P < 0.05)。从表 2可以看出,半咸水刀鲚的LGR、WGR、CF和SGR均显著大于淡水刀鲚(P < 0.05),各实验组SR相比无显著差异。结果表明,在半咸水养殖条件下,刀鲚的生长速度较快。

表 1 半咸水和淡水池塘养殖刀鲚的基本情况 Tab.1 The basic condition of C. nasus cultured in brackish and fresh water ponds
表 2 半咸水和淡水养殖对刀鲚生长性能的影响 Tab.2 Effect of brackish water and fresh water culture on growth performance of C. nasus
2.2 半咸水养殖对刀鲚消化酶活力的影响

半咸水养殖对刀鲚内脏中AMS活力的影响见表 3。从表 3可以看出,半咸水刀鲚的盲囊和肠中AMS活力均明显大于肝和胃中AMS活力(P < 0.05),但盲囊和肠之间、肝和胃之间AMS活力相比均无显著差异(P > 0.05)。而半咸水养殖条件下的刀鲚各内脏器官中,AMS活力虽略有升高但相比均无显著差异(P > 0.05)。与半咸水刀鲚不同的是淡水刀鲚的4种内脏器官间相比均存在显著差异,盲囊中AMS活力显著大于肠(P < 0.05),肠中AMS活力显著大于胃(P < 0.05),胃中AMS活力显著大于肝(P < 0.05),半咸水和淡水刀鲚4种内脏器官AMS活力大小顺序均为盲囊 > 肠 > 胃 > 肝,表明AMS主要储存在盲囊和肠中,并对淀粉的消解起到重要作用。

表 3 半咸水和淡水养殖对刀鲚内脏器官中淀粉酶活力影响 Tab.3 Effect of brackish water and fresh water on AMS activities in visceral organs of C. nasus

半咸水和淡水刀鲚内脏中蛋白酶活力的影响见表 4。从表 4可以看出,盲囊中蛋白酶活力最高,均明显大于肝、肠、胃中蛋白酶活力(P < 0.05);其次,肠中蛋白酶活力也明显大于肝、胃中蛋白酶活力(P < 0.05),但肝和胃中蛋白酶活力相比无显著差异(P > 0.05)。2种刀鲚内脏中,蛋白酶活力大小顺序均为盲囊 > 肠 > 胃 > 肝脏,表明蛋白酶主要存在刀鲚的盲囊和肠中并发挥重要作用。而在2种不同养殖条件下,半咸水养殖条件均导致了刀鲚的蛋白酶活力升高,其中,半咸水刀鲚肝和胃中蛋白酶活力均显著大于淡水刀鲚,而盲囊和肠的蛋白酶活力相比无显著差异(P > 0.05)。表明半咸水养殖可提高刀鲚蛋白酶的活力,可能会引起刀鲚摄食习性和摄食量的改变。

表 4 半咸水和淡水养殖对刀鲚内脏器官中蛋白酶活力影响 Tab.4 Effect of brackish water and fresh water on protease activities in visceral organs of C. nasus

半咸水和淡水刀鲚内脏中LPS活力影响见表 5。从表 5可以看出,淡水刀鲚盲囊中LPS活力最高,并显著大于肝和胃中的LPS活力(P < 0.05),但与肠中LPS活力相比无显著差异(P > 0.05)。半咸水刀鲚中LPS活力最高的是肠,其次为盲囊,二者均显著大于肝脏的LPS活力,而与胃中LPS酶活力相比无显著差异(P > 0.05),表明LPS活力主要存在刀鲚的盲囊、肠中,并在其中发挥重要作用。而在4种内脏器官中,半咸水养殖对刀鲚的肝和盲囊具有显著影响,而对胃和肠均未产生显著影响。其中,半咸水刀鲚的肝和盲囊中,LPS活力均显著低于淡水刀鲚(P < 0.05),表明半咸水养殖条件可显著降低刀鲚肝和盲囊的LPS活力。

表 5 半咸水和淡水养殖对刀鲚内脏器官中脂肪酶活力影响 Tab.5 Effect of brackish water and fresh water on LPS activities in visceral organs of C. nasus
2.3 半咸水养殖对刀鲚肝脏抗氧化酶活力的影响

半咸水养殖对刀鲚肝脏中抗氧化酶活力的影响见表 6。从表 6可以看出,淡水刀鲚的CAT活力均值为111.24 U/mg prot,显著大于半咸水刀鲚(24.47 U/mg prot) (P < 0.05)。而半咸水刀鲚的SOD、GSH-PX活力和MDA含量均显著大于淡水刀鲚(P < 0.05),分别为148.35、26.67 U/mg prot和7.42 nmol/mg prot。结果表明,半咸水养殖条件可促进刀鲚机体中SOD和GSH-PX活力提高,可提升刀鲚鱼体的抗氧化力。

表 6 半咸水和淡水养殖对刀鲚肝脏中抗氧化酶活力影响 Tab.6 Effect of brackish water and fresh water on autioxidant enzyme activities in liver of C. nasus
2.4 半咸水养殖对刀鲚肝脏非特异性免疫酶和代谢酶活力的影响

半咸水养殖对刀鲚肝脏中AKP、ACP和ALT、AST活力的影响见表 7。从表 7可以看出,半咸水刀鲚肝脏中AKP和ACP活力均显著低于淡水刀鲚(P < 0.05),表明刀鲚在半咸水养殖条件下受到的威胁较少,所以,其肝脏中AKP和ACP活力较低。在半咸水和淡水2种养殖环境下,刀鲚肝脏中AST和ALT活力也发生了相应变化。从表 7还可以看出,半咸水刀鲚的AST活力大于淡水刀鲚,但差异不显著(P > 0.05);而ALT活力显著低于淡水刀鲚(P < 0.05)。表明在半咸水养殖条件下,对刀鲚的AST活力无显著影响,但会显著降低ALT活力。

表 7 半咸水和淡水养殖对刀鲚肝脏中非特异性免疫酶和代谢酶活力影响 Tab.7 Effect of brackish water and fresh water on nonspecific immunity enzyme and metabolic enzyme activities in liver of C. nasus
3 讨论 3.1 半咸水养殖对刀鲚生长性能和消化能力的影响

盐度作为影响鱼类生长和存活的重要外部因子,当鱼类处在适宜盐度条件下,其生长性能会达到最好状态(袁新程等, 2020)。本研究显示,在半咸水(盐度为8.7~12.5)养殖条件下,刀鲚的生长速度显著大于淡水刀鲚,SR相比无明显差别,这表明刀鲚更适于在半咸水中生长,这与罗海忠等(2015)研究的四指马鲅鱼(Eleutheronema tetradactylum)在盐度为2~10的水体中生长较适宜,而在淡水中生长缓慢的结果相似。

由于养殖水环境的不同会导致鱼体内消化酶活力改变(杨静雯等, 2019),而消化酶作为鱼类生理系统的重要参与者,其活力大小决定了鱼体对外界营养物质消化、吸收能力的大小,进而影响鱼类的正常生长(Lundstedt et al, 2004; 唐黎等, 2007)。因此,可通过检测鱼体内消化酶活力的大小,以确定在不同养殖条件下鱼体的消化生理状况。本研究发现,在半咸水刀鲚的内脏器官中,AMS活力均比淡水刀鲚的高,胃和肝脏中蛋白酶活力均显著增大,表明刀鲚在半咸水养殖条件下的消化能力强于淡水养殖,这与半咸水刀鲚生长速度快于淡水刀鲚的结果相一致。推测导致此结果的原因,一方面是在半咸水养殖条件下,刀鲚为了平衡机体内外盐度差异,需要机体提供更多能量进行渗透压调节,因此,半咸水养殖刀鲚的机体需要更高的消化酶来消化食物,所以,半咸水刀鲚内脏器官中的AMS和蛋白酶活力会升高;另一方面是半咸水水体中的活饵料[蒙古裸腹溞(Monia mongolica Daday)、糠虾、小野杂鱼等]和淡水水体中的活饵料[多刺裸腹溞(Moina macrocopa)、大型溞、小河虾等]不同,这可能引起了刀鲚摄食习性和摄食量的改变,从而导致消化酶活力的改变。这与杨静雯等(2019)研究的虹鳟(Oncorhynchus mykiss)和硬头鳟(Salmon gairdneri)均在盐度为5和10时的消化酶活力最大,袁新程等(2020)研究发现的褐菖鲉(Sebastiscus marmoratus)在盐度为7.5~10.0时,AMS、TPS和PPS活性较高的研究结果相似。同时,本研究也发现,半咸水和淡水刀鲚的盲囊和肠中AMS、蛋白酶、LPS活力均显著大于胃和肝脏,表明刀鲚体内消化酶主要储存和发挥作用的场所是盲囊和肠,并且不会因低盐度环境而改变,这与邓平平等(2016)研究的刀鲚不同消化组织中,消化酶活性总体上为肠 > 盲囊 > 胃的研究结果相似。因此,半咸水养殖条件较适宜刀鲚养殖。

3.2 半咸水养殖对刀鲚抗氧化能力的影响

肝脏作为鱼类重要的新陈代谢和免疫器官,不仅参与糖原的合成,而且在鱼体的抗氧化生理过程和自我防护系统中起到重要作用(张晨捷等, 2015; 郭本月等, 2017)。通常情况下,肝脏中含有3种主要的抗氧化酶即SOD、CAT和GSH-PX,其中,SOD的作用是消除鱼体内有害活性氧自由基(O2−),并通过歧化反应将其分解为H2O2和O2,而CAT或GSH-PX又均可通过催化反应将H2O2分解成无害的水,进而保护鱼体细胞免受自由基损害(丁金强等, 2013; Parihar et al, 1997)。本研究结果显示,半咸水刀鲚肝脏中SOD和GSH-PX活力均显著大于淡水刀鲚,表明半咸水养殖条件明显提升了刀鲚肝脏中SOD和GSH-PX的活力,但CAT活力出现了显著降低,造成此结果的原因,一方面是在不同盐度养殖条件下,CAT和GSH-PX活力优势存在显著不同(邓平平等, 2016),导致CAT和GSH-PX在参与H2O2降解反应中可能存在拮抗作用;另一方面可能是半咸水养殖条件下水体中的饵料生物发生改变,从而导致刀鲚体内营养结构的改变,而CAT活力对刀鲚体内营养结构改变的敏感性较强(刘永士等, 2014),所以导致CAT活力显著降低;也可能与半咸水养殖条件对刀鲚肝脏中CAT活力有抑制作用有关,这需要进一步研究验证。相似的研究结果也出现在余燕等(2009)的研究中。因此,CAT和GSH-PX活力在功能上虽存在相关性,但又相互独立的作用于不同的应激反应,对半咸水和淡水养殖条件下表现出不同的活力变化。

MDA含量作为鱼体内不饱和脂肪酸被氧化后的最后产物,其浓度高低不仅代表鱼体组织细胞受O2−损害的程度,同时,也反应了鱼体自身系统抗氧化水平的高低(Viarengo et al, 1995)。本研究中,半咸水刀鲚肝脏中MDA含量显著大于淡水刀鲚,这与SOD和GSH-PX活力均显著高于淡水刀鲚活性的结果相对应,表明半咸水养殖的刀鲚具有更高的抗氧化能力,抗逆性更强;此结果与鲜博等(2020)研究的在一定盐度养殖水体中,养殖50 d的刀鲚肝脏中MDA含量均显著大于淡水组的研究结果相一致。因此,对比半咸水刀鲚和淡水刀鲚肝脏中3种抗氧化酶(SOD、CAT和GSH-PX)活力和MDA含量可以看出,半咸水养殖条件下,刀鲚的抗氧化反应更加积极,抗氧化水平更高。因此,半咸水刀鲚比淡水刀鲚更能积极调动其自身的抗氧化系统来保护机体免受损害。

3.3 半咸水养殖对刀鲚非特异性免疫和代谢能力的影响

ACP和AKP作为鱼类体内非特异免疫系统中的2种特殊的水解酶(丁金强等, 2013),它们不仅能消解体外入侵的病原体,还能加快体内吞噬细胞的吞噬作用和有害物的降解作用,因此,ACP和AKP被称为2种特殊的非特异性免疫酶(Muta et al, 1996)。在本研究中,半咸水刀鲚肝脏中ACP和AKP活力均显著低于淡水刀鲚,表明刀鲚在半咸水养殖环境中受到外界有害病原体的威胁较少,未引起ACP与AKP活力升高,所以,相较于淡水养殖而言,刀鲚更适合在半咸水中生存。这与胡利华等(2011)研究的日本鳗鲡(Anguilla japonica)养殖在一定盐度的水体中,其AKP活力会降低的研究结果相似。

AST和ALT活力主要存于鱼类肝脏组织细胞内,是鱼体内蛋白质、氨基酸代谢过程中的2种关键酶,其活力大小反应了鱼体对氨基酸的生成和降解情况(文远红等, 2013; 向枭等, 2012)。AST活力越大,表明动物体内蛋白质、氨基酸生成速度增加,分解速度减慢,有助于氮营养素在动物体内的积累;而ALT活力越大,表明动物体内尿素产生速度增加,通过尿液排出体外,从而可降低蛋白质和氨基酸代谢产生的废物对水产动物机体的损害(向枭等, 2012)。本研究中,半咸水刀鲚肝脏中AST活力大于淡水刀鲚,但差异不显著,表明半咸水养殖的刀鲚机体内蛋白质合成代谢较快,有利于其生长,但未产生明显差异。而半咸水刀鲚中的ALT活力显著低于淡水刀鲚,造成此结果的原因是刀鲚在半咸水养殖条件下,体内外环境间存在渗透压差,所以,刀鲚体内渗透压调节系统被激发,对内外渗透压进行调节,减慢尿素产生速度,以维持内外渗透压平衡(严银龙等, 2019),从而导致刀鲚机体内ALT酶活力显著降低。

4 结论

半咸水养殖刀鲚生长较快,对蛋白质和淀粉的消化能力显著增强,具有更高的抗氧化能力和抗逆性,并通过调节代谢酶活力大小,减少尿素的产生和排放,维持内外渗透压平衡的同时,还减少了对养殖水质的污染。因此,相较于淡水养殖,刀鲚更适合在半咸水中养殖,建议今后可用盐度为8.7~12.5的半咸水进行刀鲚养殖。

参考文献
DENG P P, SHI Y H, WANG Y, et al. Effects of salinity on activities of non-specific immune and digestive enzymes in juvenile estuarine tapertail anchovy Coilia nasus. Journal of Dalian Ocean University, 2016, 31(5): 533-537 [邓平平, 施永海, 汪洋, 等. 盐度对长江刀鲚幼鱼非特异性免疫酶和消化酶活力的影响. 大连海洋大学学报, 2016, 31(5): 533-537]
DING J Q, LIU P, LI J, et al. Comparison of nonspecific immunity and the activities of antioxidant enzymes in different populations of Charybdis japonica. Journal of Fisheries of China, 2013, 37(2): 275-280 [丁金强, 刘萍, 李健, 等. 不同地理群体日本蟳非特异性免疫及抗氧化酶活力的比较. 水产学报, 2013, 37(2): 275-280]
DONG W X, TANG W Q, WANG L. Growth characteristics of reproductive population of Coilia nasus in the Yangtze River. Journal of Shanghai Ocean University, 2014, 23(5): 669-674 [董文霞, 唐文乔, 王磊. 长江刀鲚繁殖群体的生长特性. 上海海洋大学学报, 2014, 23(5): 669-674]
GUO B Y, HAN D D, ZHANG W B, et al. The effects of glucose stress on the activities of metabolism and digestive enzymes in liver and intestine of Japanese flounder Paralichthys olivaceus. Transactions of Oceanology and Limnology, 2017(3): 85-90 [郭本月, 韩冬冬, 张文兵, 等. 糖胁迫对牙鲆肝脏代谢和肠道消化相关酶活性的影响. 海洋湖沼通报, 2017(3): 85-90]
HU L H, YAN M C, ZHENG J H, et al. Effects of salinity on grow th and nonspecific immune enzyme activities of Anguilla japonica. Journal of Oceanography in Taiwan Strait, 2011, 30(4): 528-532 [胡利华, 闫茂仓, 郑金和, 等. 盐度对日本鳗鲡生长及非特异性免疫酶活性的影响. 台湾海峡, 2011, 30(4): 528-532]
JING T S, ZHOU M R, LI Z, et al. Effects of fish meal replacement with soy peptide protein on the growth performance, digestive enzymes, and antioxidant capacity of juvenile yellow catfish (Pelteobagrus fulvidraco). Progress in Fishery Sciences, 2021, 42(5): 149-157 [敬庭森, 周明瑞, 李哲, 等. 大豆小肽蛋白替代鱼粉对黄颡鱼幼鱼生长性能、消化酶活性和抗氧化功能的影响. 渔业科学进展, 2021, 42(5): 149-157]
LI L, TANG W Q, ZHANG Y. Changes of fatty acid content and its components in different tissues during spawning migration processes of female Coilia nasus in the lower reaches of the Yangtze River. Journal of Fisheries of China, 2019, 43(4): 790-800 [李丽, 唐文乔, 张亚. 长江下游雌性刀鲚生殖洄游过程中脂肪酸含量及其组分的变化. 水产学报, 2019, 43(4): 790-800]
LIU Y S, SHI Y H, ZHANG G Y, et al. Growth, digestive enzyme and antioxidant enzyme activities of tawny puffer (Takifugu flavidus) larvae. Journal of Zhejiang University (Agriculture and Life Sciences), 2014, 40(6): 688-696 [刘永士, 施永海, 张根玉, 等. 菊黄东方鲀仔稚鱼生长及其消化酶与抗氧化酶活性. 浙江大学学报(农业与生命科学版), 2014, 40(6): 688-696]
LUNDSTEDT L M, MELO J F B, MORAESG M. Digestive enzymes and metabolic profile of Pseudoplatystoma corruscans (Teleostei: Siluriformes) in response to diet composition. Comparative Biochemistry Physio1ogy Psty B: Biochemistry and Molecular Biology, 2004, 137(3): 331-339 DOI:10.1016/j.cbpc.2003.12.003
LUO H Z, LI W Y, FU R B, et al. The effects of salinity on the growth of juvenile Eleutheronema tetradactylum and Na+/K+-ATP enzyme. Progress in Fishery Sciences, 2015, 36(2): 94-99 [罗海忠, 李伟业, 傅荣兵, 等. 盐度对四指马鲅(Eleutheronema tetradactylum)幼鱼生长及其鳃丝Na+/K+-ATP酶的影响. 渔业科学进展, 2015, 36(2): 94-99]
MUTA T, IWANAGA S. The role of hemolymph coagulation in innate immunity. Current Opinion in Immunology, 1996, 8(1): 41-47 DOI:10.1016/S0952-7915(96)80103-8
PARIHAR M S, JAVERI T, HEMNANI T, et al. Responses of superoxide dismutase, glutathione peroxidase and reduxed glutathione antioxidant defenses in gills of the freshwater catfish (Heteropneu stes fossilis) to short-term elevated temperature. Journal of Thermal Biology, 1997, 22(2): 151-156 DOI:10.1016/S0306-4565(97)00006-5
SHI Y H, ZHANG G Y, ZHANG H M, et al. Complete artificial propagation and embryonic development of Coilia nasus. Journal of Shanghai Ocean University, 2015, 24(1): 36-43 [施永海, 张根玉, 张海明, 等. 刀鲚的全人工繁殖及胚胎发育. 上海海洋大学学报, 2015, 24(1): 36-43]
SHI Y H, ZHANG G Y, ZHANG H M, et al. Effects of compound feed and live feed on the growth, survival, and the activities of digestive enzyme, nonspecific immunity enzyme, metabolic enzyme, and antioxidant enzyme of young fish Coilia nasus. Journal of Fisheries of China, 2014, 38(12): 2029-2038 [施永海, 张根玉, 张海明, 等. 配合饲料和活饵料对刀鲚幼鱼生长、存活和消化酶、非特异性免疫酶、代谢酶及抗氧化酶活性的影响. 水产学报, 2014, 38(12): 2029-2038]
TANG L, WANG J Q, CHENG J C, et al. Study on digestive enzymes of aquatic animals. Feed Industry, 2007, 28(2): 28-31 [唐黎, 王吉桥, 程骏驰, 等. 水产动物消化酶的研究. 饲料工业, 2007, 28(2): 28-31]
TANG X, XU G C, XU P, et al. A comparison of muscle nutrient composition between wild and cultured Coilia nasus. Chinese Journal of Animal Nutrition, 2011, 23(3): 514-520 [唐雪, 徐钢春, 徐跑, 等. 野生与养殖刀鲚肌肉营养成分的比较分析. 动物营养学报, 2011, 23(3): 514-520]
VIARENGO A, CANESI L, GARCIA MARTINEZ P, et al. Pro-oxidant processes and antioxidant defence systems in the tissues of the Antarctic scallo (Adamussium colbecki) compared with the Mediterranean scallop (Pecten jacobeus). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1995, 111(1): 119-126
WEI G L, XU G C, GU R B, et al. Research progress of biology and artificial culture of Coilia nasus. Journal of Yangtze University (Natural Science), 2012, 9(7): 31-35 [魏广莲, 徐钢春, 顾若波, 等. 刀鲚的生物学及人工养殖研究进展. 长江大学学报(自然科学版), 2012, 9(7): 31-35]
WEN H B, ZHANG C X, XU G C, et al. Development of gonads in Coilia nasus from the Yangtze River and Artificial Pond. Chinese Journal of Zoology, 2009, 44(4): 111-117 [闻海波, 张呈祥, 徐钢春, 等. 长江刀鲚与池塘人工养殖刀鲚性腺发育的初步观察. 动物学杂志, 2009, 44(4): 111-117]
WEN Y H, CAO J M, HUANG Y H, et al. Effects of fish meal replacement by maggot meal on growth performance, body composition and plasma biochemical indexes of juvenile yellow catfish (Peltobagrus fulvidraco). Chinese Journal of Animal Nutrition, 2013, 25(1): 171-181 [文远红, 曹俊明, 黄燕华, 等. 蝇蛆粉替代鱼粉对黄颡鱼幼鱼生长性能、体组成和血浆生化指标的影响. 动物营养学报, 2013, 25(1): 171-181]
XIAN B, GAO J C, XU G C, et al. Influence of salinity on the growth, digestive ability, antioxidant ability and osmoregulation of Coilia nasus. Transactions of Oceanology and Limnology, 2020(2): 152-159 [鲜博, 高建操, 徐钢春, 等. 盐度对刀鲚生长、抗氧化应激和渗透压调节能力的影响. 海洋湖沼通报, 2020(2): 152-159]
XIANG X, ZHOU X H, CHEN J, et al. Effect of dietary replacement of fish meal protein w ith soybean meal protein on the grow th, body composition and hematology indices of Schizothorax prenanti. Journal of Fisheries of China, 2012, 36(5): 723-731 [向枭, 周兴华, 陈建, 等. 饲料中豆粕蛋白替代鱼粉蛋白对齐口裂腹鱼幼鱼生长性能、体成分及血液生化指标的影响. 水产学报, 2012, 36(5): 723-731]
XU G C, NIE Z J, DU F K, et al. Intensive culture and natural spawning of broodstock Coilia nasus under artificial feeding conditions. Acta Hydrobiologica Sinica, 2016, 40(6): 1194-1200 [徐钢春, 聂志娟, 杜富宽, 等. 长江刀鲚亲鱼强化培育及自然产卵规律研究. 水生生物学报, 2016, 40(6): 1194-1200]
XU G C, TANG X, ZHANG C X, et al. First studies of embryonic and larval development of Coilia nasus (Engraulidae) under controlled conditions. Aquaculture Research, 2011, 42(4): 593-601
XU G C, WAN J J, GU R B, et al. Morphological and histological studies on ovary development of Coilia nasus under artificial farming conditions. Journal of Fishery Sciences of China, 2011a, 18(3): 537-546 [徐钢春, 万金娟, 顾若波, 等. 池塘养殖刀鲚卵巢发育的形态及组织学研究. 中国水产科学, 2011a, 18(3): 537-546]
XU G C, XU P, GU R B, et al. Feeding habits and growth characteristics of pond-cultured Coilia nasus fingerlings. Chinese Journal of Ecology, 2011b, 30(9): 2014-2018 [徐钢春, 徐跑, 顾若波, 等. 池养刀鲚(Coilia nasus)鱼种的摄食与生长. 生态学杂志, 2011b, 30(9): 2014-2018]
YAN Y L, YUAN X C, SHI Y H, et al. Effects of salinity on growth, oxygen consumption rate and ammonia excretion rate of juvenile Sebastisucus marmoratus. Journal of Dalian Ocean University, 2019, 34(4): 545-551 [严银龙, 袁新程, 施永海, 等. 盐度对褐菖鲉幼鱼生长、耗氧率和排氨率的影响. 大连海洋大学学报, 2019, 34(4): 545-551]
YANG J W, ZHOU Y G, HUANG M, et al. Comparative studies on digestive and antioxidant enzyme activities between juvenile rainbow (Oncorhynchus mykiss) and steelhead trout (O. mykiss). Periodical of Ocean University of China (Natural Sciences), 2019, 49(3): 119-128 [杨静雯, 周演根, 黄铭, 等. 盐度对虹鳟和硬头鳟幼鱼消化酶和抗氧化酶活性的比较研究. 中国海洋大学学报(自然科学版), 2019, 49(3): 119-128]
YU Y, XU W N, LIU Z P, et al. Effect of low salinity stress on activities of digestive and anti-stress enzymes, and survival rate of juvenile Epinephelus malabaricus. Progress in Fishery Sciences, 2009, 30(4): 21-26 [余燕, 徐维娜, 刘兆普, 等. 低盐度胁迫对点带石斑鱼幼鱼消化酶、抗应激酶和存活率的影响. 渔业科学进展, 2009, 30(4): 21-26]
YUAN C M, LIN J B, QIN A L, et al. On the classification of the anchovies, Coilia, from the lower Yangtze Rivernd the southeast coast of China. Journal of Nanjing University (Natural Sciences), 1976, 12(2): 1-12 [袁传宓, 林金榜, 秦安舲, 等. 关于我国鲚属鱼类分类的历史和现状—兼谈改造旧鱼类分类学的几点体会. 南京大学学报(自然科学版), 1976, 12(2): 1-12]
YUAN X C, XIE Y D, LIU Y S, et al. Effects of two stocking densities on growth performance, digestion and non-specific immunity of the current year Coilia nasus. Journal of Shanghai Ocean University, 2021, 30(2): 222-230 [袁新程, 谢永德, 刘永士, 等. 两种养殖密度对刀鲚当年鱼种生长性能、消化及非特异性免疫能力的影响. 上海海洋大学学报, 2021, 30(2): 222-230]
YUAN X C, YAN Y L, SHI Y H. Effects of salinity on growth, body composition, digestive enzyme and antioxidant enzyme activities of juvenile Sebastisucus marmoratus. Acta Agriculturae Shanghai, 2020, 36(2): 114-119 [袁新程, 严银龙, 施永海. 盐度对褐菖鲉幼鱼生长、体成分、消化和抗氧化酶活力的影响. 上海农业学报, 2020, 36(2): 114-119]
ZHANG C J, ZHANG Y L, GAO Q X, et al. Effect of low salinity stress on antioxidant function in liver of juvenile Nibea albiflora. South China Fisheries Science, 2015, 11(4): 59-64 [张晨捷, 张艳亮, 高权新, 等. 低盐胁迫对黄姑鱼幼鱼肝脏抗氧化功能的影响. 南方水产科学, 2015, 11(4): 59-64]