黃條仔稚幼魚消化酶活性變化研究*

張正榮 柳學(xué)周 于 毅 史 寶 張言祥 徐永江 王 濱 姜 燕 孫冉冉

張正榮1,2柳學(xué)周1,2①于 毅3史 寶2張言祥3徐永江2王 濱2姜 燕2孫冉冉2

(1. 上海海洋大學(xué)水產(chǎn)與生命學(xué)院 上海 201306；2. 中國水產(chǎn)科學(xué)研究院黃海水產(chǎn)研究所 農(nóng)業(yè)農(nóng)村部海洋漁業(yè)可持續(xù)發(fā)展重點(diǎn)實(shí)驗(yàn)室 青島海洋科學(xué)與技術(shù)試點(diǎn)國家實(shí)驗(yàn)室海洋漁業(yè)科學(xué)與食物產(chǎn)出過程功能實(shí)驗(yàn)室 青島 266071；3. 大連富谷水產(chǎn)有限公司 大連 116400)

在魚類規(guī)模化育苗生產(chǎn)中，魚苗死亡率較高。Lauff等(1984)研究發(fā)現(xiàn)，消化酶是影響仔稚幼魚死亡率的重要因素。消化酶活力高低直接影響魚類的消化吸收，間接影響魚類的生長發(fā)育。因此，對(duì)仔稚幼魚消化酶活性的研究利于了解其消化生理特征，也有利于探索早期發(fā)育中魚苗大量死亡的原因。目前，研究的魚類消化酶主要包括脂肪酶、淀粉酶、胰蛋白酶和堿性磷酸酶等，脂肪酶主要由魚類的肝胰臟分泌，起分解脂肪的作用；胰蛋白酶主要由胰臟分泌，魚類早期發(fā)育蛋白消化主要靠胰蛋白酶完成；魚類各消化器官都有淀粉酶存在，分解碳水化合物；堿性磷酸酶主要存在于魚類腸道上皮細(xì)胞，與葡萄糖、Ca和P等的吸收有關(guān)，這些消化酶在魚類早期發(fā)育過程的消化生理中起重要作用。目前，對(duì)仔稚幼魚消化酶的研究日益增多，國內(nèi)外學(xué)者對(duì)大菱鲆()、半滑舌鰨()、金頭鯛()、古巴雀鱔()和日本黃姑魚()等仔稚幼魚消化酶的分泌規(guī)律已有研究(陳慕雁等, 2005; 常青等, 2005; Moyano, 1996; Comabella, 2006; 孫敏等, 2012)。

1 材料與方法

1.1 實(shí)驗(yàn)材料

1.2 實(shí)驗(yàn)方法

1.2.2 消化酶活性測(cè)定 使用碧云天的BCA蛋白濃度測(cè)定試劑盒，測(cè)量酶液中的蛋白濃度。脂肪酶用南京建成的脂肪酶試劑盒測(cè)定，活力單位定義：在37℃條件下，每毫升酶液在反應(yīng)體系中與底物反應(yīng) 1 min，消耗1 mmol底物為1個(gè)酶活力單位。淀粉酶用南京建成淀粉酶試劑盒測(cè)定，其活力單位定義：組織中每毫克蛋白37℃、pH 7.0條件下與底物作用 30 min，水解10 mg淀粉定義為1個(gè)淀粉酶活力單位。胰蛋白酶用南京建成胰蛋白酶試劑盒測(cè)定，其活力單位定義：在37℃、pH 8.0條件下，每毫克蛋白中含有的胰蛋白酶，每分鐘使吸光度變化0.003，即為 1個(gè)酶活力單位。堿性磷酸酶用南京建成堿性磷酸酶試劑盒測(cè)定，其活力單位定義：在37℃條件下，每克組織蛋白與基質(zhì)作用15 min產(chǎn)生1 mg酚為1個(gè)酶活力單位。

1.3 數(shù)據(jù)處理

采用SPSS 22.0軟件對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行分析，運(yùn)用單因子方差分析(One-way ANOVA)和Duncan氏檢驗(yàn)法對(duì)各組數(shù)據(jù)進(jìn)行顯著性差異分析和多重比較，顯著性水平為0.05，所有數(shù)值均采用平均值±標(biāo)準(zhǔn)差(Mean±SD)表示。

全長的平均日增長計(jì)算公式：

G=(2–1)/(2–1)

用指數(shù)函數(shù)分析全長和日齡的關(guān)系，公式：

=aebt

式中，為全長(mm)，為日齡(d)，、為常數(shù)。

2 結(jié)果

2.1 黃條仔稚幼魚的全長

=3.95e0.0358t，R=0.9788

孵化后60 d內(nèi)的仔稚幼魚全長隨著日齡的增加而加快增長，25 d后全長大幅增加。

圖1 黃條仔稚幼魚生長曲線和餌料投喂

2.2 黃條胚胎、仔魚消化酶比活力變化

圖2 黃條胚胎、仔魚脂肪酶的比活力變化

胚胎階段記為孵化后0 d。不同小寫字母表示消化酶比活力存在顯著差異(<0.05,=3)，下同

The embryonic stage is recorded as 0 d after hatching. Different lowercase letters mean significant differences in specific activities of digestive enzymes (<0.05,=3), the same as below

圖3 黃條胚胎、仔魚淀粉酶的比活力變化

圖4 黃條胚胎、仔魚胰蛋白酶的比活力變化

Fig.5 Specific activities of alkaline phosphatase during embryo and larval development of

2.3 黃條稚幼魚消化酶比活力變化

圖6 黃條稚幼魚脂肪酶的比活力變化

圖7 黃條稚幼魚淀粉酶的比活力變化

圖8 黃條稚幼魚胰蛋白酶的比活力變化

圖9 黃條稚幼魚堿性磷酸酶的比活力變化

2.4 不同發(fā)育階段4種消化酶比活力值的特征

圖10 不同發(fā)育階段黃條仔稚幼魚消化酶的比活力變化

不同小寫字母表示同一日齡不同消化酶存在差異顯著

Different lowercase letters mean significant differences at same age in different digestive enzymes

3 討論

3.1 早期階段消化酶的出現(xiàn)

3.2 黃條仔稚幼魚4種消化酶活力的變化

3.3 不同發(fā)育階段4種消化酶比活力值特征分析

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The Variations of Digestive Enzymes in Larval and Juvenile

ZHANG Zhengrong1,2, LIU Xuezhou1,2①, YU Yi3, SHI Bao2, ZHANG Yanxiang3, XU Yongjiang2, WANG Bin2, JIANG Yan2, SUN Ranran2

(1. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306; 2. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266071; 3. Dalian Fugu Fishery Co., Ltd., Dalian 116400)

To clarify the digestive and physiological characteristics in the early developmental stage of, the activity of different digestive enzymes (lipase, amylase, trypsin, and alkaline phosphatase) were studiedFertilized eggs and 1- to 60-day-old larvae and juveniles ofwere selected as the research objects. The relationship between total length and days after hatching was also studied. The results indicated that the total length of larvae and juveniles increased with age within 60 days after hatching and increased significantly after 25 days. Lipase, amylase, and alkaline phosphatase were detectable during the embryonic stage while trypsin was detected in newly hatched larvae, which indicated thatcan digest lipid, carbohydrate, and proteins before feeding. The specific activities of lipase and alkaline phosphatase increased rapidly after hatching (<0.05), reached a peak at (5.88±0.45) and (5.56±0.41) U/mg, respectively at first feeding. The amylase activity reached its peak at (1.59±0.02) U/mg on day 7. The specific activity of trypsin increased slowly after hatching, and reached the highest value ((1098.67±24.03) U/mg on day 15 (<0.05). Lipase, alkaline phosphatase, and trypsin were basically stable in the visceral mass of juvenile fish. However, theactivity of lipase, alkaline phosphatase, and trypsin increased with the development ofThe amylase activity in the visceral mass of juvenile fish showed a downward trend and was basically stable at a lower specific activity level. The results of this study showed significant changes in various digestive enzyme activities during the development of larvae and juveniles, which were closely related to the development stage and feeding habits. The digestive enzymes were detected in early larvae that had not yet eaten food which was considered that the digestive enzymes were maternal in origin and not from exogenous food. The lipase, alkaline phosphatase, and trypsin activities in juveniles significantly increased, which reflect the improvement of intestinal structure and digestive function and the increasing demand for fat and protein.

; Embryo; larvae and juvenile; digestive enzymes; Specific activities

S965.399

A

2095-9869(2020)02-0061-08

柳學(xué)周，研究員，E-mail: liuxz@ysfri.ac.cn

2018-12-11,

2019-02-14

*中國水產(chǎn)科學(xué)研究院中央級(jí)公益性科研院所基本科研業(yè)務(wù)費(fèi)專項(xiàng)資金(2018GH17; 2019GH15; 2019CY0204)、青島市民生科技計(jì)劃項(xiàng)目(17-3-3-61-nsh)、國家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2018YFD0901204; 2019YFD0900901)、青島海洋科學(xué)與技術(shù)國家實(shí)驗(yàn)室海洋漁業(yè)科學(xué)與食物產(chǎn)出過程功能實(shí)驗(yàn)室開放課題(2017-3A01)和現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系專項(xiàng)經(jīng)費(fèi)(CARS-47)共同資助[This work was supported by Special Scientific Research Funds for Central Non-Profit Institutes, Chinese Academy of Fishery Sciences (2018GH17; 2019GH15; 2019CY0204), Qingdao People's Livelihood Science and Technology Project(17-3-3-61-nsh), National Key Research and Development Program(2018YFD0901204; 2019YFD0900901), Opening Topic of Qingdao National Laboratory of Marine Science and Technology Functional Laboratory of Marine Fisheries Science and Food Output Process(2017-3A01), and China Agriculture Research System (CRAS-47)]. 張正榮, E-mail: 997197691@qq.com

10.19663/j.issn2095-9869.20181211002

http://www.yykxjz.cn/

Zhang ZR, Liu XZ, Yu Y, Shi B, Zhang YX, Xu YJ, Wang B, Jiang Y, Sun RR. The variations of digestive enzymes in larval and juvenile. Progress in Fishery Sciences, 2020, 41(2): 61–68

LIU Xuezhou, E-mail: liuxz@ysfri.ac.cn

(編輯 陳 輝)