張正榮 柳學周 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. 上海海洋大學水產與生命學院 上海 201306；2. 中國水產科學研究院黃海水產研究所 農業(yè)農村部海洋漁業(yè)可持續(xù)發(fā)展重點實驗室 青島海洋科學與技術試點國家實驗室海洋漁業(yè)科學與食物產出過程功能實驗室 青島 266071；3. 大連富谷水產有限公司 大連 116400)

在魚類規(guī)模化育苗生產中，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胞，與葡萄糖、Ca和P等的吸收有關，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，對仔稚幼魚消化酶的研究日益增多，國內外學者對大菱鲆()、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 實驗材料

1.2 實驗方法

1.2.2 消化酶活性測定 使用碧云天的BCA蛋白濃度測定試劑盒，測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定，活力單位定義：在37℃條件下，每毫升酶液在反應體系中與底物反應 1 min，消耗1 mmol底物為1個酶活力單位。淀粉酶用南京建成淀粉酶試劑盒測定，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個淀粉酶活力單位。胰蛋白酶用南京建成胰蛋白酶試劑盒測定，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個酶活力單位。堿性磷酸酶用南京建成堿性磷酸酶試劑盒測定，其活力單位定義：在37℃條件下，每克組織蛋白與基質作用15 min產生1 mg酚為1個酶活力單位。

1.3 數(shù)據(jù)處理

采用SPSS 22.0軟件對實驗數(shù)據(jù)進行分析，運用單因子方差分析(One-way ANOVA)和Duncan氏檢驗法對各組數(shù)據(jù)進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±標準差(Mean±SD)表示。

全長的平均日增長計算公式：

G=(2–1)/(2–1)

用指數(shù)函數(shù)分析全長和日齡的關系，公式：

=aebt

式中，為全長(mm)，為日齡(d)，、為常數(shù)。

2 結果

2.1 黃條仔稚幼魚的全長

=3.95e0.0358t，R=0.9788

孵化后60 d內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

Alvarez-González CA, Moyano-López FJ, Civera-Cerecedo R,. Development of digestive enzyme activity in larvae of spotted sand bass1. Biochemical analysis. Fish Physiology and Biochemistry, 2008, 34(4): 373–384

Babaei SS, Kenari AA, Nazari R,. Developmental changes of digestive enzymes in Persian sturgeon () during larval ontogeny. Aquaculture, 2011, 318 (1–2): 138–144

Cara JB, Moyano FJ, Cárdenas S,. Assessment of digestive enzyme activities during larval development of white bream. Journal of Fish Biology, 2003, 63(1): 48–58

Chang Q, Zhang XM, Chen SQ,. Variations in digestive enzymes activities in Tongue fishlarvae and juveniles. AdvancesinMarine Science, 2005, 23(4): 472–476 [常青, 張秀梅, 陳四清, 等. 半滑舌鰨仔稚魚消化酶活性的變化. 海洋科學進展, 2005, 23(4): 472–476]

Chen MY, Zhang XM, Lian JH. Development of some digestive enzymes and alkaline phosphatase activities in turbotlarvae and juveniles. Periodical of Ocean University of China (Natural Sciences), 2005, 35(3): 483–486 [陳慕雁, 張秀梅, 連建華. 大菱鲆仔稚魚期消化酶及堿性磷酸酶活性的變化. 中國海洋大學學報(自然科學版), 2005, 35(3): 483–486]

Comabella Y, Mendoza R, Aguilera C,. Digestive enzyme activity during early larval development of the Cuban gar. Fish Physiology and Biochemistry, 2006, 32(2): 147–157

Gisbert E, Giménez G, Fernández I,. Development of digestive enzymes in common dentexduring early ontogeny. Aquaculture, 2009, 287(3–4): 381–387

Ji H, Sun HT, Tian JJ,. Digestive enzymes activities during early larval development of the paddlefishActa Hydrobiologica Sinica, 2012, 36(3): 457–465 [吉紅, 孫海濤, 田晶晶, 等. 匙吻鱘仔稚魚消化酶發(fā)育的研究. 水生生物學報, 2012, 36(3): 457–465]

Kim BG, Divakaran S, Brown CL,. Comparative digestive enzyme ontogeny in two marine larval fishes: Pacific threadfin () and bluefin trevally (). Fish Physiology and Biochemistry, 2001, 24(3): 225–241

Lauff M, Hofer R. Proteolytic enzymes in fish development and the importance of dietary enzymes. Aquaculture, 1984, 37(4): 335–346

Li Q, Tang HY. Development of some digestive enzymes and alkaline phosphatase activities inlarvae and juveniles. Journal of Hydroecology, 2010, 3(5): 82–85 [李芹, 唐洪玉. 中華倒刺鲃仔稚魚期消化酶及堿性磷酸酶活性變化的研究. 水生態(tài)學雜志, 2010, 3(5): 82–85]

Liu YS, Shi YH, Zhang GY,. Growth, digestive enzyme and antioxidant enzyme activities of tawny puffer () larve. Journal of Zhejiang University(Agricultural and Life Science), 2014, 40(6): 688–696 [劉永士, 施永海, 張根玉, 等. 菊黃東方鲀仔稚魚生長及其消化酶與抗氧化酶活性. 浙江大學學報(農業(yè)與生命科學版), 2014, 40(6): 688–696]

Martínez I, Moyano FJ, Fernández-Díaz C,. Digestive enzyme activity during larval development of the Senegal sole (). Fish Physiology and Biochemistry, 1999, 21(4): 317–323

Moran D, Gara B, Wells RMG. Energetics and metabolism of yellowtail kingfish (Valenciennes 1833) during embryogenesis. Aquaculture, 2007, 265(1–4): 359–369

Moyano FJ, Díaz M, Alarcón FJ,. Characterization of digestive enzyme activity during larval development of gilthead seabream (). Fish physiology and biochemistry, 1996, 15(2): 121–130

Oozeki Y, Bailey KM. Ontogenetic development of digestive enzyme activities in larval walleye pollock,. Marine Biology, 1995, 122(2): 177–186

Pan L, Fang H, Zhang SC,. The variation of digestive enzymes in larval and juvenile. Progress in Fishery Sciences, 2013, 34(3): 54–60 [潘雷, 房慧, 張少春, 等. 大瀧六線魚仔、稚、幼魚期消化酶活力的變化. 漁業(yè)科學進展, 2013, 34(3): 54–60]

Poortenaar CW, Hooker SH, Sharp N. Assessment of yellowtail kingfish () reproductive physiology, as a basis for aquaculture development. Aquaculture, 2001, 201 (3–4): 271–286

Stuart KR, Drawbridge MA. Captive spawning and larval rearing of California yellowtail (). Aquaculture Research, 2013, 44(5): 728–737

Sun M, Chai XJ, Xu YJ,. Assessment of digestive enzymes activities during the early development of. Journal of Shanghai Ocean University, 2012, 21(6): 965– 970 [孫敏, 柴學軍, 許源劍, 等. 日本黃姑魚早期發(fā)育過程中消化酶活性變化研究. 上海海洋大學學報, 2012, 21(6): 965–970]

Tengjaroenkul B, Smith BJ, Caceci T,. Distribution of intestinal enzyme activities along the intestinal tract of cultured Nile tilapia,L. Aquaculture, 2000, 182(3): 317–327

Tong SY, Ling CH, Wang XT. Appraisement and analysis of nutrient compositions forDaday. Journal of Dalian Fisheries College, 1988(3、4): 29–33 [童圣英, 林成輝, 王雪濤. 蒙古裸腹溞營養(yǎng)成分分析與評價. 大連水產學院學報, 1988(3、4): 29–33]

Yang SG, Hur SW, Ji SC,. Morphological development of embryo, larvae and juvenile in yellowtail kingfish,. Development and Reproduction, 2016a, 20(2): 131–140

Yang SG, Ji SC, Lim SG,. Management of sexual maturation and natural spawning of captive-readed yellowtail kingfish,, in an indoor rearing tank. Development & Reproduction, 2016b, 20(2): 141–147

Zhang YL, Zhang HL, Wang LY,. Allometric growth and ontogenetic changes in nucleic acids and digestive enzymes during the early life stage in fish species: A review. Journal of Fishery Sciences of China, 2017, 24(3): 648–656 [張云龍, 張海龍, 王凌宇, 等. 魚類早期發(fā)育階段異速生長及核酸、消化酶變化的研究進展. 中國水產科學, 2017, 24(3): 648–656]

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

柳學周，研究員，E-mail: liuxz@ysfri.ac.cn

2018-12-11,

2019-02-14

*中國水產科學研究院中央級公益性科研院所基本科研業(yè)務費專項資金(2018GH17; 2019GH15; 2019CY0204)、青島市民生科技計劃項目(17-3-3-61-nsh)、國家重點研發(fā)計劃項目(2018YFD0901204; 2019YFD0900901)、青島海洋科學與技術國家實驗室海洋漁業(yè)科學與食物產出過程功能實驗室開放課題(2017-3A01)和現(xiàn)代農業(yè)產業(yè)技術體系專項經費(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)