Anesthetic effect and tissue oxidative injury for Litopenaeus vannamei by two anesthetics
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摘要: 为明确丁香酚和MS-222对凡纳滨对虾 (Litopenaeus vannamei) 的麻醉效果及相同效果下造成的二次应激，为两种麻醉剂在凡纳滨对虾中的应用提供参考数据与实验依据。研究了不同质量浓度丁香酚 (20、30、40、60、80、100、120、140和160 mg·L−1) 和MS-222 (800、900、1 000、1 100、1 200、1 300、1 400、1 500和1 600 mg·L−1) 对体质量为 (14±3) g凡纳滨对虾的麻醉效果，以及相同效果下对其机体生理和组织形态的影响。结果表明：1) 丁香酚和MS-222的质量浓度分别为80和1 400 mg·L−1时，对虾在3 min内进入深度麻醉阶段，5 min内恢复正常；2) 使用这两种麻醉剂均会造成对虾的鳃和肝胰腺组织损伤，其中丁香酚组的鳃组织各项抗氧化指标早于MS-222组达到峰值；丁香酚组Caspase-3活性在麻醉复苏后6 h显著降低，而MS-222组则显著升高 (P<0.05)；丁香酚组和MS-222组的Na+/K+-ATP酶活性显著降低 (P<0.05)；组织形态分析显示两种麻醉剂均会引起对虾鳃和肝胰腺产生不同程度的变形或裂解损伤，而在麻醉复苏后6 h丁香酚组恢复正常。综上，丁香酚和MS-222对凡纳滨对虾均有麻醉效果，但MS-222所需浓度更高，且对机体造成的二次应激损伤更为严重。Abstract: To provide references for the application of two anesthetics in Litopenaeus vannamei, we studied the anesthetic effects of eugenol (20, 30, 40, 60, 80, 100, 120, 140, 160 mg·L−1) and MS-222 (800, 900, 1 000, 1 100, 1 200, 1 300, 1 400, 1 500, 1 600 mg·L−1) on L. vannamei with body mass of (14±3) g. Besides, we tested the secondary stress injury on the physiological functions and pathomorphologic changes by the two anesthetics. The results show that: 1) Eugenol of 80 mg·L−1and MS-222 of 1 400 mg·L−1 induced L. vannamei to enter the stage of deep anesthetic within 3 min, and return to the normal stage within 5 min. 2) The gills and hepatopancreas of L. vannamei were injured by the two anesthetics, and the antioxidant indexes of gills in the eugenol group reached the maximum value earlier than the MS-222 group. The Caspase-3 activity in the eugenol group decreased significantly after anesthesia recovery of 6 h, while that in the MS-222 group increased significantly (P<0.05). The Na+/K+-ATPase activity in both the groups decreased significantly (P<0.05). Pathomorphologic analysis shows that the two anesthetics could cause deformation or lysis of gills and hepatopancreas, and the eugenol group could recover after anesthesia recovery of 6 h. In conclusion, eugenol and MS-222 have anesthetic effects on L. vannamei, but the concentration of MS-222 is higher and the secondary stress damage to the body is more serious.
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Key words:
- Litopenaeus vannamei /
- Eugenol /
- MS-222 /
- Anesthetic effect /
- Stress injury
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图 1 丁香酚和MS-222麻醉对凡纳滨对虾鳃组织抗氧化酶活性影响
注：大写字母不同表示相同时间点上不同组间差异显著 (P<0.05)；小写字母不同表示同组间不同时间点间差异显著 (P<0.05)； 图2—图4同此。
Figure 1. Antioxidant capacity in gill tissue of L. vannamei exposed to combined stress of eugenol and MS-222
Note: Different uppercase letters indicate significant difference among different groups at the same time (P<0.05); different lowercase letters indicate significant difference among different time within the same group (P<0.05). The same case in Fig. 2–Fig. 4.
图 2 丁香酚和MS-222麻醉对凡纳滨对虾肝胰腺抗氧化酶活性影响
Figure 2. Antioxidant capacity in hepatopancreas tissue of L. vannamei exposed to combined stress of eugenol and MS-222
图 3 丁香酚和MS-222麻醉对凡纳滨对虾肝胰腺细胞凋亡相关指标影响
Figure 3. Apoptosis in hepatopancreas tissue of L. vannamei exposed to combined stress of eugenol and MS-222
图 4 丁香酚和MS-222麻醉对凡纳滨对虾渗透调节指标影响
Figure 4. Osmoregulation in tissue of L. vannamei exposed to combined stress of eugenol and MS-222
图 5 丁香酚和MS-222麻醉对凡纳滨对虾鳃部组织形态影响 (10×20倍)
注：a. 3 h对照组；b. 6 h对照组；c. 3 h丁香酚组；d. 6 h丁香酚组；e. 3 h MS-222组；f. 6 h MS-222组；图6同此。
Figure 5. Gill tissue of L. vannamei exposed to combined stress of eugenol and MS-222 (10×20 times)
Note: a. Control group at 3rd hour; b. Control group at 6th hour; c. Eugenol group at 3rd hour; d. Eugenol group at 6th hour; e. MS-222 group at 3rd hour; f. MS-222 group at 6th hour. The same case in Fig. 6.
图 6 丁香酚和MS-222麻醉对凡纳滨对虾肝胰腺组织形态影响 (10×20倍)
Figure 6. Hepatopancreas tissue of L. vannamei exposed to combined stress of eugenol and MS-222 (10×20 times)
表 1 麻醉与复苏阶段虾类行为特征
Table 1. Behavioral characteristics of shrimps at anaesthesia and recovery stages
状态 State 行为特征Behavioral characteristics 麻醉阶段Anaesthesia stage A0 正常 正常游动，对外界刺激反应迅速 A1 镇静 游动缓慢，对外界刺激反应迟钝 A2 轻度麻醉 游泳足缓慢摆动，对外界刺激反应微弱 A3 中度麻醉 游泳足轻微摆动，失去平衡，水底侧游 A4 深度麻醉 游泳足停止摆动，水底侧躺，无反应 A5 死亡 颚舟片摆动停止 复苏阶段 Recovery stage R1 轻度恢复 游泳足开始无规则摆动，对外界刺激无反应 R2 中度恢复 游泳足有规律摆动，外界刺激后有逃跑意识 R3 平衡恢复 恢复平衡，游动无规律 R4 正常 游动正常，对外界刺激反应灵敏 
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表 2 不同浓度丁香酚和MS-222对凡纳滨对虾的麻醉效果
Table 2. Anaesthesia and recovery effects of eugenol and MS-222 of different concentrations on L. vannamei
麻醉剂质量浓度Anesthetic mass concentration/ (mg·L−1) 进入不同麻醉阶段程度的时间Average time for reaching different anaesthesia stages/s 进入不同恢复阶段程度的时间Average time for reaching different recovery stages/s 复苏率Recovery rate/% 48 h后成活率Survival rate after 48 h/% A1 A2 A3 A4 A5 R1 R2 R3 R4 丁香酚Eugenol 20 87.6±24.3 182.1±20.7 — — — — — — — 100 100 30 86.8±15.6 156.2±33.9 — — — — — — — 100 100 40 55.7±4.6 126.8±33.8 132.7±4.1 — — 58.6±5.6 98.3±32.2 186.3±38.7 272.2±6.3 100 100 60 45.9±3.8 93.7±8.0 124.6±16.3 338.8±35.2 — 122.7±34 187.8±6.9 265.1±6.9 294.7±17.1 100 100 80 40.7±6.1 74.5±2.6 116.1±17.0 195.9±12.4 — 123.9±22.7 186.8±31.3 266.8±22.7 324.6±32.4 100 100 100 31.8±5.2 53.1±0.8 107.7±6.1 164.9±8.4 — 170.7±51.5 228.6±26.2 293.6±42.8 392.6±50.5 100 100 120 19.6±3.1 89.9±17.8 107.4±10.9 160.8±26.5 — 238.6±84.6 346.4±16.2 404.2±24.6 438.2±26.3 100 100 140 34.4±0.8 81.7±21.3 103.0±9.0 145.5±7.4 — 218.9±3.68 280.9±5.6 343.9±13.6 440.5±32.9 83.3 66.7 160 27.6±2.1 50.2±2.6 87.0±26.2 121.0±32.4 — 367.1±26.4 438.6±22.4 528.8±38.9 652.9±82.0 50 33.3 MS-222 800 74.9±3.5 237.5±24.9 — — — — — — — 100 100 900 78.3±21.1 200.5±22.8 632.4±68.4 — — — — 26.7±11.9 99.6±20.1 100 100 1 000 84.5±12.9 187.4±17.2 289.3±29.7 454.0±38.6 — 27.7±6.3 55.6±7.1 156.9±13.7 193.0±10.4 100 100 1 100 55.0±5.8 165.5±18.7 269.7±25.8 391.1±17.7 — 41.7±6.9 104.0±13.7 195.7±13.6 269.0±42.2 100 100 1 200 69.2±6.8 140.4±27.8 213.1±24.8 307.7±23.2 — 39.1±7.3 99.3±17.5 195.6±25.8 256.5±21.1 100 100 1 300 60.0±7.0 117.9±30.2 189.4±26.5 292.3±13.6 — 52.1±9.8 94.6±10.2 202.5±20.6 311.5±24.1 100 100 1 400 35.0±4.3 77.6±11.7 122.8±29.5 171.3±16.5 — 52.6±9.5 91.5±14.2 175.5±18.2 312.7±16.0 100 100 1 500 33.8±10.1 66.4±9.8 106.3±22.5 155.6±22.1 — 46.5±3.8 108.7±20.6 187.4±27.6 331.1±39.0 100 100 1 600 — 61.5±6.1 118.8±8.7 143.3±9.7 — 89.9±13.2 172.0±20.3 333.6±16.2 499.9±16.5 83.3 66.7 注：—. 未观察到相应的状态。 Note: —. No corresponding state was observed.
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