王維鑫 崔愛君 徐永江 李海寧 柳學(xué)周王 濱 姜 燕 劉新富

王維鑫1,2崔愛君2徐永江2①李海寧3柳學(xué)周2王 濱2姜 燕2劉新富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)研究所青島海洋科學(xué)與技術(shù)試點國家實驗室深藍(lán)漁業(yè)工程聯(lián)合實驗室 山東 青島 266071；3. 東北林業(yè)大學(xué)生命科學(xué)學(xué)院 黑龍江 哈爾濱 150040)

1 材料與方法

1.1 實驗材料

實驗所需MS-222標(biāo)準(zhǔn)品(純度98%)和丁香油均購于國藥集團化學(xué)試劑有限公司。為提高丁香油的溶解率，使用時按照丁香油∶95%酒精=1∶10的比例溶解于酒精，再溶于水。

1.2 麻醉和復(fù)蘇過程分期及有效質(zhì)量濃度確定

根據(jù)Marking等(1985)的研究結(jié)果，本研究中麻醉劑有效質(zhì)量濃度標(biāo)準(zhǔn)定義為實驗魚在3 min內(nèi)進(jìn)入麻醉狀態(tài)(達(dá)到A6期)、5 min內(nèi)完全復(fù)蘇(恢復(fù)至B6期)。實驗魚進(jìn)入A7期麻醉狀態(tài)后再持續(xù)3 min，然后轉(zhuǎn)入清潔海水復(fù)蘇，成活率為100%。

1.3 不同水溫下MS-222和丁香油對黃條的麻醉實驗

在實驗開始前，實驗魚饑餓24 h。實驗容器為容積300 L的白色圓形塑料桶。設(shè)置溫度為20℃和24℃，使用加熱器控制水溫(精確度為±1℃)，MS-222濃度梯度為40、60、80、100和120 mg/L，丁香油濃度梯度為20、40、60、80、100和120 mg/L。麻醉實驗開始前，先將暫養(yǎng)的魚放到預(yù)先控制好溫度(20℃和24℃)的水桶中適應(yīng)2 h。每組隨機選取5尾魚，用于麻醉實驗，觀察每尾魚的麻醉情況，分別記錄每尾魚達(dá)到不同麻醉狀態(tài)(A1～A7期)所需要的時間。麻醉時間從實驗魚放入不同麻醉劑實驗組(A1期)開始計時，至A6期結(jié)束，精確到秒。麻醉后的實驗魚，轉(zhuǎn)移至清潔海水中復(fù)蘇，分別記錄每尾魚達(dá)到不同復(fù)蘇狀態(tài)(B1～B6期)所需要的時間，復(fù)蘇時間從實驗魚放入清潔海水中開始計時，至實驗魚行為達(dá)B6期狀態(tài)時結(jié)束，精確到秒。

1.4 血液樣品采集與血清激素指標(biāo)測定

表1 黃條麻醉和復(fù)蘇階段分期

表2 不同丁香油濃度和水溫對黃條麻醉時間和復(fù)蘇時間的影響

表3 不同MS-222濃度和水溫對黃條麻醉時間和復(fù)蘇時間的影響

1.5 數(shù)據(jù)處理

實驗數(shù)據(jù)均使用Excel 2016和SPSS 24.0軟件進(jìn)行處理。利用單因素方差分析法(one-way ANOVA)和Duncan多重分析法對麻醉時間、復(fù)蘇時間和血清激素和葡萄糖水平進(jìn)行顯著性分析，同一時間點2種不同溫度(20℃和24℃)條件下血清激素和葡萄糖水平采用成對樣本檢驗，所有結(jié)果以平均值±標(biāo)準(zhǔn)差(Mean±SD)表示，顯著性水平設(shè)定為0.05，當(dāng)<0.05時認(rèn)為差異顯著。

2 結(jié)果

2.1 不同水溫下麻醉劑有效濃度的確定

2.2 不同水溫下麻醉劑的效果

在同一麻醉濃度條件下，水溫的升高使實驗魚進(jìn)入A6期麻醉狀態(tài)的時間縮短，而對復(fù)蘇時間沒有明顯影響(表2和表3)。在2種不同水溫下，丁香油濃度為20 mg/L時，實驗魚進(jìn)入A6期的時間與其他各濃度均存在顯著性差異(<0.05)，其他各濃度組之間無顯著性差異(>0.05)，而MS-222濃度為100 mg/L和120 mg/L時，實驗魚進(jìn)入A6期的時間無顯著性差異(>0.05)，而與其他各濃度組之間均有顯著性差異(<0.05)。MS-222濃度為40 mg/L時，實驗魚在2種水溫下都不能進(jìn)入A6期麻醉狀態(tài)；MS-222濃度為60 mg/L時，20℃水溫下實驗魚僅能達(dá)到A5期狀態(tài)，而24℃水溫條件下實驗魚可達(dá)A6期麻醉狀態(tài)。

不同的水溫下，麻醉時間與復(fù)蘇時間之比隨丁香油濃度的升高整體呈下降趨勢，至120 mg/L降到最低。水溫為20℃時，丁香油濃度為20 mg/L和40 mg/L時與其他各濃度組均有顯著性差異；24℃時，僅有20 mg/L與其他各濃度組存在顯著性差異(<0.05)，其他濃度組之間無顯著性差異(>0.05)(圖1)。不同的水溫下，隨著MS-222濃度的增加，麻醉時間與復(fù)蘇時間之比顯著降低(<0.05)，MS-222濃度為100 mg/L和120 mg/L時，二者之間無顯著性差異(>0.05)，而與其他各濃度組均有顯著性差異(<0.05)(圖2)。

圖1 不同水溫與丁香油濃度下黃條的麻醉時間/復(fù)蘇時間比

圖2 不同水溫與MS-222濃度下黃條的麻醉時間/復(fù)蘇時間比

2.3 不同水溫下血清激素水平對麻醉劑的響應(yīng)

實驗開始前，20℃下對照組實驗魚血清腎上腺素水平為(59.17±0.5006) pg/mL，24℃下對照組血清腎上腺素水平為(60.36±0.2212) pg/mL。在2種溫度條件下，2種麻醉劑處理的實驗魚復(fù)蘇1 h后，血清腎上腺素水平均顯著升高(<0.05)(圖3A、B；圖4A、B)。在2種溫度條件下，丁香油麻醉處理的實驗魚復(fù)蘇后，實驗魚血清腎上腺素水平均在24 h達(dá)峰值，與其他各組均差異顯著(<0.05)(圖3A、B)。MS-222麻醉處理的實驗魚復(fù)蘇后，20℃下血清腎上腺素水平在24 h達(dá)峰值，與其他各組差異顯著(<0.05)(圖4A)，24℃下血清腎上腺素水平在12 h達(dá)峰值，且與其他各組差異顯著(<0.05)(圖4B)。在2種溫度條件下，丁香油和MS-222麻醉和復(fù)蘇處理的實驗魚，血清腎上腺素水平呈先上升后下降的趨勢，且高溫度組比低溫度組的腎上腺素水平上升快且恢復(fù)也快，復(fù)蘇72 h后均恢復(fù)到對照組水平以下(圖3A、B；圖4A、B)。

圖3 不同溫度下丁香油麻醉后黃條血清內(nèi)激素含量變化

圖4 不同溫度下MS-222麻醉后黃條血清內(nèi)激素含量變化

3 討論

3.1 麻醉劑的有效濃度和麻醉效果

麻醉劑的主要作用是抑制大腦皮質(zhì)的中樞神經(jīng)系統(tǒng)，從而對魚體產(chǎn)生一定的麻痹作用，在面對外界環(huán)境脅迫的刺激時，降低魚體應(yīng)激反應(yīng)，達(dá)到生理保護(hù)作用(Devi, 2019)。因此，麻醉劑的濃度直接影響魚類的麻醉效果，濃度過低起不到抗應(yīng)激作用，濃度過高則容易導(dǎo)致魚體受到生理脅迫傷害，甚至休克死亡。因此，在對魚類進(jìn)行不同的生產(chǎn)操作時需要明確不同的給藥劑量，合適的麻醉濃度有利于生產(chǎn)操作，還能起到?；钸\輸作用(Chanseau, 2002)。

3.2 水溫對黃條麻醉效果的影響

3.3 MS-222和丁香油對黃條血清生理指標(biāo)的影響

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Anesthetic Effect of MS-222 and Clove Oil on Yellowtail Kingfish

WANG Weixin1,2, CUI Aijun2, XU Yongjiang2①, LI Haining3, LIU Xuezhou2, WANG Bin2, JIANG Yan2, LIU Xinfu2

(1. School of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China; 2. Joint Laboratory for Deep Blue Fishery Engineering of Pilot National Laboratory for Marine Science and Technology (Qingdao), Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong 266071, China; 3. College of Life Science, Northeast Forestry University, Harbin, Heilongjiang 150040, China)

Yellowtail kingfishis a globally distributed, circumtropical pelagic fish of importance in both commercial fisheries and aquaculture. It swims fast and has fierce behavior under artificial culture conditions. To develop a suitable manipulation technique with low stress for yellow tail kingfish, the anesthetizing effects of two anesthetics, MS-222 and clove oil, on one-year-old yellowtail kingfish under different temperature conditions (20℃and 24℃) were studied. The optimal anesthesia times, recovery times, and anesthesia dosages of the two anesthetics were determined based on the discrimination of swimming behavior and investigation of serum hormones levels, including cortisol, adrenaline, and glucose. According to the behavioral characteristic changes of the yellowtail kingfish during anesthesia and recovery, the anesthesia process was divided into seven stages, whereas the recovery process was divided into six stages. The results showed that the optimal concentrations for MS-222 for anesthetizing yellowtail kingfish were 100～120 mg/L and 100 mg/L at water temperatures of 20℃ and 24℃, respectively. For clove oil, the optimal concentration for anesthetizing yellowtail kingfish was 40 mg/L at both temperatures. All the experimental yellowtail kingfish were anaesthetized within three minutes and recovered within five minutes. By increasing the anesthetic concentration, the time for the yellowtail kingfish to enter anesthesia was reduced and the recovery time prolonged. The increase in water temperature reduced the time to enter anesthesia, but had no obvious effect on the recovery time. When anesthetized with 40 mg/L clove oil at 20℃and 24℃, serum cortisol significantly peaked at 6 h and 12 h, respectively (<0.05). At 24℃, the serum adrenaline level significantly peaked at 12 h (<0.05) in yellowtail kingfish anesthetized with 100 mg/L of MS-222. The levels of serum adrenaline, glucose, and cortisol in the other experimental groups all significantly peaked after 24 h (<0.05) in fish anesthetized using both anesthetics. Under the two temperature conditions, the two anesthetics caused the three serum hormone levels to all decrease significantly (<0.05) to low levels that were lower than the initial levels after 72 h of recovery, indicating that yellowtail kingfish could be physiologically adaptive to the optimal treatment methods of clove oil and MS-222 under different temperatures. The results from the present study provide theoretical and technical support for the development of standard experimental and farming management technology for yellowtail kingfish.

Yellowtail kingfish; MS-222; Clove oil; Anesthetic effect; Physiological response

XU Yongjiang, E-mail: xuyj@ysfri.ac.cn

S948

A

2095-9869(2021)06-0205-10

10.19663/j.issn2095-9869.20210224001

http://www.yykxjz.cn/

WANG W X, CUI A J, XU Y J, LI H N, LIU X Z, WANG B, JIANG Y, LIU X F. Anesthetic effect of MS-222 and clove oil on yellowtail kingfish. Progress in Fishery Sciences, 2021, 42(6): 205–214

徐永江，研究員，E-mail: xuyj@ysfri.ac.cn

2021-02-24,

2021-03-31

*山東省支持青島海洋科學(xué)與技術(shù)試點國家實驗室重大科技專項(2018SDKJ0303-1)、國家重點研發(fā)計劃項目(2019YFD0900901; 2018YFD0901204)、中國水產(chǎn)科學(xué)研究院創(chuàng)新團隊(2020TD47)和財政部和農(nóng)業(yè)農(nóng)村部：國家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系(CARS-47)共同資助[This work was supported by Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology (Qingdao) (2018SDKJ0303-1), National Key R&D Program of China (2019YFD0900901; 2018YFD0901204), Science and Technology Innovation Team of Chinese Academy of Fishery Sciences (2020TD47), and China Agriculture Research System of MOF and MARA（CARS-47）].王維鑫，E-mail: wwxshou@163.com

(編輯 馮小花)