Essential oil of Magnolia denudata is an effective anesthetic for spotted seabass (Lateolabrax maculatus): a test for its effect on blood biochemistry, physiology and gill morphology

Essential oil of Magnolia denudata is an effective anesthetic for spotted seabass (Lateolabrax maculatus): a test for its effect on blood biochemistry, physiology and gill morphology | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Essential oil of Magnolia denudata is an effective anesthetic for spotted seabass ( Lateolabrax maculatus ): a test for its effect on blood biochemistry, physiology and gill morphology Xiangbing Zeng, Hongbiao Dong, Jingru Wu, Wenhao Wang, Yafei Duan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1475724/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Magnolia denudata is a well-known ornamental tree in China due to its beautiful blossoms, and it has been used as an analgesic to treat human headaches. This study investigates the anesthetic potential and physiological response of the essential oils of M. denudata flower on spotted seabass Lateolabrax maculatus . Fish (mean ± SD, 164.16 ± 15.40 g) were individually exposed to different concentrations of M. denudata essential oil (MDO, 10, 20, 40, 60, 80, 100 and 120 mg/L) and eugenol (10, 20, 30, 40, 50, 60 and 70 mg/L) to investigate anesthestic efficacy. Based on the time criteria of anesthesia induction (< 3 min) and post-recovery (< 10 min), the lowest effective concentration for spotted seabass was 100 mg/L for MDO and 60 mg/L for eugenol. The physiological and histopathological damages in the gill of L. maculatus after using essential oil and eugenol were also evaluated for the minimum dose inducing deep anesthetia, and 0, 6 and 24 h after recovery. These results showed that MDO and eugenol anesthesia alleviated the levels of cortisol, glucose and lactic dehydrogenase (LDH) activities by handling. Compared with the eugenol, MDO also caused secondary stress to the body, but MDO caused minor physiological responses and histological changes in the gills. This study suggests that MDO is an effective anesthetic for spotted seabass. Essential oil Anesthesia Fish Physiological Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction In the past decades, an increasing number of fish anesthetics have been used in animal welfare to reduce the trauma caused by handling (Wang et al., 2019 ; Jerez-Cepa I et al., 2018; Taheri Mirghaed et al., 2020; Aydın and Barbas 2020 ). Currently, fish anesthetics are classified as synthetic anesthetics or plant-derived anesthetics (Purbosari et al., 2019 ). However, chemically synthesized anesthetics have several adverse effects on fish, including reproductive suppression, liver function, and excessive mucus secretion (Purbosari et al., 2019 ; Dong et al., 2020a ; Wang et al., 2020 ; Teixeira et al., 2017 ; dos Santos Teixeira et al., 2021 ), and the FDA approved only tricaine methanesulfonate (MS-222) for food fish (Carter, Woodley and Brown, 2011 ). Moreover, there is a growing concern about their adverse impact in compromising human and fish health or the environment due to tissue residue. For the reasons stated above, less expensive, safer, and more natural anesthetics are required. Plant-derived anesthetics are antibacterial, antioxidant, anti-stress, and low risk to human health (Aydın and Barbas, 2020 ; Purbosari et al., 2019 ; Purbosari et al., 2021 ). The essential oil anesthetics have been considered most effective in plant-derived anesthetics (Aydın and Barbas, 2020 ). Plant essential oil is a type of volatile liquid with aromatic properties derived from aromatic plants such as leaves, flowers, fruits, seeds, buds, and stems (Baptista et al., 2020). Magnolia denudata is a well-known ornamental tree species due to its attractive flowers in China with a cultivation history of over 2500 years (Park et al., 2018 ). Its dried flower buds, known as Xin Yi in traditional Chinese herbal medicine, have been used as an analgesic agent for treating headaches (Wang et al., 2018). Furthermore, M. denudata flower can be eaten as a food (Lu, Li and Yin, 2016 ; Zhang et al., 2021 ). However, the M. denudata essential oils have not been assessed for their anesthetic properties in fish. Although anaesthetics are used to reduce stress, the anesthetic itself may also be a source of stress (Readman et al., 2013 ; Barata et al., 2016 ). When stress factors stimulate the body, it releases a large amount of catecholamine hormone and then activates the hypothalamus-pituitary-interregnal gland axis (HPI) to produce a neuroendocrine cascade reaction (Schreck, 2016 ). As a result, the corticosteroid-releasing hormone (CRH) and the pituitary gland-adrenocorticotropic hormone (ACTH) are released successively and pass through the blood circulation to reach the renal inter-renal tissues to stimulate the production of cortisol (Martos et al., 2014; Mommsen, Vijayan and Moon, 1999 ). Therefore, blood hematological and biochemical parameters are good indicators to evaluate fish stress and health (Roche and Bogé, 1996 ; Fazio, 2019 ). Multifunctional fish gills are essential for gas exchange, osmoregulation, acid-base regulation, and nitrogenous waste excretion (Evans et al., 2005 ; Jiao et al., 2019 ). Furthermore, the gill is the main organ to directly contact and absorb anesthetics (Hunn and Allen 1974; Ferreira et al., 1984). Many anesthetics have adversely impacted gills, such as lamellar epithelium hyperplasia, apoptosis of ionocytes, and necrosis (Wang et al., 2020 ; Afifi et al., 2000 ; Brandão et al., 2021 ; de Lima et al., 2021 ). Therefore, it is necessary to evaluate biochemical parameters in blood and the physiological changes on gills after anesthetic exposure. Dosage recommendations may differ between and within species, increasing the necessity for specific fish species research to prevent adverse effects from a high dose (Zahl, Samuelsen and Kiessling, 2012 ). The spotted seabass ( Lateolabrax maculatus ) is widely distributed in the water from China, Korea, and Japan. It has the advantages of fast growth, wide salinity, high breeding survival rate, delicious meat, and strong adaptability, and it is popular among farmers and consumers(Yokogawa et al., 1995; Dong et al., 2020b ). However, the poor stress tolerance of the spotted seabass, especially the stress caused by handling during culturing and transportation, has seriously affected its growth and survival (Sun et al., 2020 ; He et al., 2020). Tricaine methanesulfonate (MS-222) and eugenol are chemically synthesized anesthetics in aquaculture to reduce fish activities (Bahrekazemi and Yousefi, 2017 ; Skår et al., 2017 ; Aydın et al., 2015 ; Purbosari et al., 2019 ). Moreover, they have been tested on L. maculatus and have been shown to cause gill and liver damage in recent studies (Wang et al., 2018; Wang et al., 2020 ). In addition, no report conducts on the anesthetic effect of essential oil on L. maculatus . However, some previous studies have shown that eugenol is more effective and safer than MS-222 (Wang et al., 2018; Wang et al., 2020 ; Dong et al., 2020a ; Cao et al., 2019 ). Therefore, our study aimed to compare the anesthetic effects, blood parameters and gill physiological changes after exposure to the essential oil of M. denudate and eugenol. Material And Methods Fish maintenance and anesthetic agents Juvenile L. maculatus (164.16 ± 15.40 g) were purchased from a fish hatchery in Zhuhai, Guangdong, China. The fish were acclimated in plastic tanks (4 m×4 m ) for 2 weeks. The daily water exchange rate was 100% of the tank volume. The water quality parameters were maintained at dissolved oxygen 6.9 ± 0.2 mg/L, temperature 28 ± 0.5 o C and pH 7.7 ± 0.1. The natural photoperiod was used, and the water quality indicators were checked daily to maintain within the safety range. The eugenol (purity 99%; Wuhan Kangchun Perfume Co., Ltd. Wuhan, China) and essential oils of M. denudate (MDO, purity 98%; Wuhan Kangchun Perfume Co., Ltd. Wuhan, China) were first diluted 1:9 in 95% ethanol to better mix with the water. The MDO main components were linalool (47.8%), terpineol (9.9%), neral (7.0%), geranial (6.9%) and cinnamaldehyde (3.7%) determined by GC-MS. Experiment I: Anesthesia efficacy evaluation In this experiment, the MDO anesthetic efficacy was compared to eugenol. Before the experiment started, the fish fasted for 24 hours. Then, individual fish (n = 10) was individually observed and used only once during the test. Each fish was netted quietly and placed in an induction barrel (10 L) containing one of the seven MDO concentrations (10, 20, 40, 60, 80, 100 and 120 mg/L) or one of the seven eugenol concentrations (10, 20, 30, 40, 50, 60 and 70 mg/L). The L. maculatus staging criteria of the induction and recovery process was divided into five stages, according to Wang et al. (2018) and He et al. (2020) (Table 1). The induction and recovery time were recorded by using a digital stopwatch. After 0.5 hour, when the fish stopped breathing or was unable to enter more profound anesthesia, the fish was immediately transferred into the anesthetic-free and aerated in the recovery tank, and the time to restore regular swimming activity was recorded. The survival rate of induction, recovery, and 72 h after recovery were recorded. Experiment II: Anesthetic antistress and secondary stress evaluation The ideal anesthetic effect allows a fish to enter anesthesia in less than 3 minutes and recover in less than 10 minutes (Hanggono, 2006 ; Aydın and Barbas, 2020 ; Ross and Ross, 2008; Purbosari et al., 2019 ) because these concentrations have a short induction time and may not cause damage to the fish. In experiment I, the appropriate concentrations of MBO and eugenol for fast anesthesia (induction time < 180 s) were 100 and 60 mg/L. After fasting for 24 h, the fish were individually transferred to three aquaria with 100 mg/L MBO, 60 mg/L eugenol and without anesthetic (control). After anesthetic exposure to each fish lost body balance with slow opercular movement (deep anesthesia stage), five fish per aquarium were sampled at this time point. The remaining fish in each aquarium were then transferred to a new aquarium with anesthetic free to recover and were sampled at 0, 6, and 24 h after recovery. The fish of after recovery stages and those in the control group were euthanized via cranial concussion according to the protocol approved by the Animals Ethical Committee of the South China Sea Fisheries Institute. The fish head was hit by a mechanical hammer for cranial concussion, resulting in syncope but not death. This cranial concussion lasted about 30 s in air exposure. Subsequently, the fish was held with a damp cloth and blood was collected by caudal venipuncture. For later analyses, the gill samples were removed and immediately frozen at − 80°C. Serum biochemistry After centrifugation at 1000 g, at 4°C for 10 min, the serum was frozen and stored at − 20°C until further analysis. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), glucose, cortisol, and lactic dehydrogenase (LDH) were measured by using a BS-200 automatic biochemical analyses and a kit (Shenzhen Mindray Biomedical Electronics Co., Ltd. Shenzhen, Guangdong, China). Oxidative stress and physiological indicator assessments The gills were homogenized in 9 volumes (w/v) of 4°C sterile physiological saline and centrifuged at 6000×g at 4°C for 20 min. The antioxidant and physiological parameters were determined by a microplate reader (Bio-Rad, USA). In brief, the superoxide dismutase (SOD) activity was determined by the xanthine oxidase method, and the optical absorbance was determined at 450 nm. The peroxidase (POD) activity unit was measured and defined as the amount of enzyme that catalyzes 1 mL of sample to decompose 1µg of the substrate in 1 min at 37°C. Catalase (CAT) activity unit was defined as the amount of enzyme that catalyzes 1mg of protein to decompose 1µmol of H 2 O 2 in 1 s. The glutathione (GSH) concentration was measured by colorimetry via 5,5- dithiobis reacting with mercapto compounds to form yellow compounds. The malondialdehyde (MDA) concentration was measured by the thiobarbituric acid method. The MDA was condensed with thiobarbituric acid during lipid peroxide degradation to produce a red product for quantitative determination at the 532 nm optical absorbance. HSP70 and cytochrome C (Cyt-c) were determined with commercial test kits, respectively, based on the manufacturer's protocol (MLBio, Shanghai, China). The enzymatic activities of cysteine-aspartic proteases-9 (Caspase-9) and cysteine-aspartic proteases-3 (Caspase-3) were measured with Ac-LEHD-pNA and Ac-DEVD-pNA as the substrates, respectively (Beyotime, Haimen, China). Histopathology To further compared investigate the adverse effects of MDO and eugenol on L. maculatus , the gills were analyzed, and the histological changes of the gills were examined at 6 h. Gills were fixed with 10% formaldehyde and washed with a 4°C physiological saline solution. Subsequently, the samples were gradually dehydrated with ethanol (70% − 100%), made transparent with xylene, embedded in paraffin, and cut into 5–6 µm thick slices for H&E staining and neutral resin sealing. An optical microscope was used for staining observation and imaging. Image Pro Plus 6.0 software was used to process the images. The gill alterations were defined in three stages, stage I (alterations that do not affect organ functions), stage II (moderate alterations that affect the organ function) and stage III (severe and irreversible alterations), according to de Lima et al., 2021 . Statistical analyses All data were expressed as the mean ± standard deviation. Statistical analysis of the data was performed using SPSS 22.0 software. Based on the single-factor analysis of variance, the Duncan multiple comparison method was used for the analysis. In all cases, the minimum level of significance was set to p < 0.05. Results Anesthetic efficacy of MBO and eugenol No mortality was observed during the entire experimental period. When the anaesthetic concentration increased, the time of induction stages decreased, and recovery time was prolonged. All concentrations have induced the fish to enter the deep sedation stage. When the concentration of MBO increased to 100–120 mg/L, and the concentration of eugenol increased to 60–70 mg/L, the induction time could reach the criteria of ideal induction, which is less than 3 min and recovery less than 10 min. Not observed fish enter A4 stage at all the concentration of MBO, While the concentration of eugenol increased to 40 mg/L, fish opercular movements ceased in 0.5 hour. In addition, the recovery times of the experimental fish that were anesthetized by MDO (72–110 s) were shorter than those of the eugenol (170–269 s) anesthetized fish (Table 2 ). Serum biochemical parameters The serum biochemical parameters of MDO and eugenol are shown in Fig. 1 . The cortisol concentration significantly decreased at the deep anesthesia stage but increased at 0 h after recovery ( p < 0.05). The glucose of anesthetized fish by MDO and eugenol was significantly increased at 0 h, and the MDO group was higher than the control group at 6 h after recovery ( p < 0.05). LDH activity significantly decreased at the deep anesthesia stage and significantly increased at 0, 6, and 24 h in the eugenol group and 0 h in the MDO group ( p < 0.05). ALT activity increased at 6 h in eugenol ( p < 0.05). AST activity significantly increased at 6 h and 24 h after recovery in anesthetic groups. In addition, the eugenol group was higher than the MBO group at 6 h and 24 h ( p 0.05). Antioxidative stress and physiological response of gills The antioxidative stress parameters of L maculatus in gills after anesthesia with MDO and eugenol are shown in Fig. 2 . SOD activity significantly increased at 6 h after recovery in both eugenol and MDO groups, and eugenol group higher than MDO group ( p < 0.05). CAT activity significantly increased at 0 and 6 h after recovery in the eugenol group and MDO group at 6 h, and the eugenol group was higher than the MDO group at 6 h ( p 0.05). GSH concentration significantly increased at 6 h after recovery in both anesthetic groups, and the eugenol group was higher than the MDO group at 6 h., but the GSH concentration of both anesthetic groups significantly decreased at 24 h after recovery ( p < 0.05). The MDA concentration significantly increased at 6 h in both the eugenol and MDO groups and significantly increased at 24 h in the eugenol group ( p < 0.05). The physiological stress parameters of L maculatus in gills after anesthesia with essential oil of Magnolia denudate are shown in Fig. 3 . Caspase-3 activity significantly increased at 6 h after recovery in both anesthetic groups ( p < 0.05). Caspase-9 activity significantly increased at 6 h in both anesthetic groups and at 24 h after recovery in the eugenol group ( p < 0.05). Cyt-c concentration significantly increased at 0 h, 6 h and 24 h after recovery in the eugenol group and at 6 h after recovery in MDO group, and eugenol group higher than MDO group at 6 h ( p < 0.05). HSP70 concentration significantly increased at 6 h and 24 h in the eugenol and MDO groups at 6 h after recovery ( p < 0.05). Histopathology of gills The gill lamellas were intact in the control group, extending to both sides, and the pavement cells were regularly arranged on the gill lamella. The erythrocytes were distributed smoothly and evenly at the gill lamella. The gill structure showed a normal physiological condition, and no hyperplasia or edema was observed. A qualitative description of gill lesions found in fish anesthetized with the same effective concentrations of MBO and eugenol is shown in Table 3. The gills anesthetized with MBO had morphological alterations of the moderate response, such as the proliferation of chloride cells and hyperplasia and hypertrophy of epithelial cells, and the gill lamella was relatively neat. Hyperplasia and epithelial phenomena also appeared in the eugenol group. However, more severe damage such as epithelial rupture and necrosis was only observed in the eugenol group. In addition, the gill lamellas were not neatly arranged, and the blood vessels were swollen also were observed (Fig. 4 ). Discussion Anesthetic is useful for reducing stress and improving fish welfare during handling. However, only MS-222 and eugenol have been studied, and they could cause damage to the liver and gills of L. maculatus (Wang et al., 2018; Wang et al., 2020 ; Dong et al., 2020a ). Therefore, more anesthetics need to be tested on this species. Plant-derived anesthetics have become a hot spot contributed to their safety, gentleness, and low risk to humans. In these contexts, we have investigated the anesthesia and antistress efficacy of essential oil of M. denudate and the potential secondary stress to gills compared with eugenol on L. maculate. So far, a large number of essential oils have been reported to have anesthetic effects on fish, such as Lippia alba , Cymbopogum flexuosus , Myrcia sylvatica , Ocimum gratissimum , Aloysia triphylla , Lippia sidoides and Mentha piperita. The active ingredients in these essential oils are linalool, geranial, β-pinene, thymol, and menthol (Junior et al., 2017; dos Santos Batista et al., 2018 ; Brandão et al., 2021 ; Saccol et al., 2017 ; Boaventura et al., 2020 ; Souza et al., 2018). Souza et al., (2018) found that L. alba essential oil of linalool chemotype could induce fish to anesthesia stages. In addition, another study showed the use of C. flexuosus essential oil for the anesthesia in Oreochromis niloticus . Notely, the main active ingredients in the C. flexuosus essential oil are geranial and neral (Netto et al., 2017 ). Cinnamaldehyde, a Trpa1 agonist, acts as a local anesthetic by inhibiting voltage-gated sodium channels in sensory neurons (Boonen et al., 2014 ). Thus, we speculate that the MDO active substances on L. maculate anesthesia are linalool, neral, geranial, and cinnamaldehyde. It is critical to determine the effective concentration of anesthetics in each fish species to provide high welfare to both fish and the environment (Aydın and Barbas, 2020 ). The ideal anesthesia effect allows the fish to enter anesthesia in less than 3 minutes and recover in less than 10 minutes (Hanggono, 2006 ; Aydın and Barbas, 2020 ; Ross and Ross, 2008; Purbosari et al., 2019 ). Based on the time criteria of ideal, the lowest effective concentrations were 100 mg/L for MDO, 60 mg/L for eugenol on L. maculate juveniles. As the concentrations of the essential oils were increased, the time to induce anesthesia diminished and recovery time prolonged. In addition, MDO has a shorter recovery time compared with eugenol. This may indicate that MDO is easier to eliminate in fish. Besides, no mortality was observed during the entire experimental period. These results showed that MDO is a safe and efficient natural anesthetic for L. maculate handing. The blood biochemical parameters of fish are valuable markers and very sensitive to anesthesia protocols (Yousefi et al., 2018 ; Yousefi et al., 2019 ; Fazio 2019 ; Saccol et al., 2018 ). To further evaluate the anti-stress effect and potential secondary stress of MDO on fish, cortisol, glucose, LDH, AST, ALT and ALP levels in the blood have been investigated in the present study. Cortisol and glucose are the most common stress indicators in fish (Ainsworth, Bowser, and Beleau, 1985; Semenkova et al., 1999 ; Martínez-Porchas et al., 2009 ). Silva et al. ( 2015 ) found that the essential oil of Ocimum americanum effectively prevented cortisol from increasing in Rhamdia quelen after air exposure. Nile tilapia anesthetized with A. triphylla essential oil and then exposed to air for 1 minute lowered plasma cortisol levels (Teixeira et al., 2017 ). In the present study, fish in the deep anesthesia stage had lower cortisol levels than cranial concussion, confirming the stress-reducing properties of MDO and eugenol. We also found that anesthesia elevated cortisol and glucose levels after recovery, but these parameters declined back to control levels after 24h in the MDO group. Bodur et al. ( 2018 ) found that higher plasm cortisol was observed after exposed Origanum sp. and Eucalyptus sp. essential oils anesthetics at recovery stages. Toni et al. ( 2015 ) also found that L. alba essential oil induced higher cortisol at 4 h after recovery. These results suggest secondary stress of eugenol and MDO anesthesia on fish. ALT, AST and LDH activities can be used as sensitive biomarkers in ecotoxicology because they provide early warnings for aquatic organisms (Hoseini et al., 2016 ; Haschek et al., 2009 ). In the present study, the increased levels of ALT indicated hepatotoxicity. AST and LDH activities are indicators of not tissue-specific damage (Haschek et al., 2009 ). Our results showed that ALT, AST and LDH activities increased after MDO and eugenol exposure. Similar to our study, Toni et al. ( 2014 ) found that glucose level, AST and ALT activities were significantly increased during the recovery period after being exposed to different concentrations of essential oils of Hesperozygis ringens and L. alba . In addition, MDO has less adverse impact on L. maculatus than eugenol. In recent studies, eugenol also showed more serve impact than other anesthetics (Yousefi et al., 2018 ; Yousefi et al., 2019 ; de Lima et al., 2021 ). Antioxidant enzymes play a critical role in responding to oxidative stress during intracellular redox balance in vertebrates (Zhang et al., 2020 ; Wang et al., 2020 ; Jiao et al., 2019 ). SOD, CAT, and POD are important enzymes involved in antioxidant defense systems and represent the cell’s first line of defense against oxidative stress (Wang et al., 2020 ). In the present study, increasing antioxidant enzyme activity (POD, SOD, and CAT) implies that anesthesia stress stimulates the antioxidant system during the recovery period. Wang et al. ( 2020 ) found that SOD, CAT, and POD activities in the gills of L. maculatus were significantly increased at 6 h after eugenol and MS-222 treatment. The GSH is an important non-enzymatic antioxidant in cells, which can neutralize free radicals or oxidants (Galano et al., 2011). The MDA is the most important marker of lipid peroxidation (Liu et al., 2011 ). They have been commonly used as biomarkers for the evaluation of oxidative stress. In the present study, the high levels of GSH and MDA indicate that MDO and eugenol can give rise to oxidative stress, but MDO caused less oxidative stress in the gills of L. maculatus . Apoptosis, a type of cell death, is regulated by a series of molecules induced by environmental, physical, or chemical stress. These molecules can mediate phagocytosis and clearance of dead or infected cells and prevent autoimmunity (Cheng et al., 2018; Cifuentes-Rius et al., 2021). Cyt-c and Caspases play an essential role in cell apoptosis (Sun et al. 2020 ; Liang et al., 2019). In addition, HSP70 is a stress-induced molecular chaperone that aids in protein structural folding and transport in response to environmental stress (Tukaj, 2020; Maniya and Srivastava, 2020; Kurashova et al., 2020). Our study showed that eugenol and MBO induced apoptosis and HSP70 increased in the gills of L. maculatus. These results are consistent with another study on eugenol and MS-222 on the gills of spotted sea bass (Wang et al., 2020 ). In some studies, extensive histopathological abnormalities were observed in the gill, including hyperplasia, epithelial lifting, and curling of lamellae after exposure to anesthetics (essential oils of A. triphylla , L. sidoides , and M. piperita , MS-222, eugenol), indicating that the gill is a sensitive organ for responding to anesthetics (Brandão et al., 2021 ; Wang et al., 2020 ; Lima et al., 2021 ). In the present study, changes in gill morphology were identified using H&E staining and were classified in three degrees, according to de Lima et al. ( 2021 ). We observed lamellar epithelium hyperplasia in the gill after exposure to MDO and eugenol. The hypertrophy of the gill epithelium and hyperplasia of epithelium cells dilate the space between the exterior medium and the blood flow, reducing the direct contact of unwanted substances (Ghayyur et al., 2021 ). Furthermore, more severe pathology such as epithelial rupture and necrosis were found in gills exposed to eugenol, but MDO was not observed. These results are similar to a recent report of de Lima et al. ( 2021 ), which found that eugenol caused more irreversible damage than L. alba essential oils in gills on Potamotrygon wallacei . In another study, no irreversible damage was found in gills after exposure to three essential oil anesthetics (Brandão et al., 2021 ). Combined with the apoptotic and antioxidative results, we speculated that the use of eugenol caused more damage to the gills than MBO. In conclusion, the essential oils of M. denudata are efficient as sedatives and anesthetics in L. maculatus . The lowest effective concentration for L. maculatus was 100 mg/L for MDO. The MDO of 100 mg/L effectively reduced stress in handling and caused less secondary stress in gills than eugenol. These results showed that MDO is a safe and efficient natural anesthetic for L. maculate handing. Declarations Funding This research was supported by China - ASEAN Maritime Cooperation Fund Project "China - ASEAN Modern Marine Fishery Technical Cooperation and Industrial Development Demonstration", the fund of Central Public-interest Scientific Institution Basal Research Fund, South China Sea Fisheries Research Institute, CAFS (2021SD19), the fund of Guangdong province modern agricultural industrial technology system innovation team building project (2022KJ150), the fund of Guangzhou science and technology planning project (201904010169), the fund of National key research and development plan projects (2019YFD0900500), and the fund of Guangdong Province supporting town and villages technology commissioner project (KTP20210259). Conflicts of Interest The authors have declared no conflict of interest. Ethics approval The collection and handling of the animals in this study was approved by the Animal Care and Use Committee at the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (SCSFRI-CAFS), and all experimental animal protocols were carried out in accordance with national and institutional guidelines for the care and use of laboratory animals at the SCSFRI-CAFS. Consent for publication Not applicable Consent to participate Not applicable Data availability statement The authors confirm that all the data involved in the manuscript are available for publication. Code availability Not applicable Authors’ Contributions X.Z., H.D. and J.Z. designed the experiment. X.Z. and H.D. performed the experiments, data analyses, and drafted the manuscript.W.J., W.W., Y.D. and J.C. contributed with essential materials. 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Fish Shellfish Immunol. 99, 514–525. https://doi.org/10.1016/j.fsi.2019.11.056 Tables Table 1 Behavioral characteristics of L. maculatus during anesthesia and recovery stages. stage Behavioral characteristics A1 Deep sedation: equilibrium normal; total loss of reactivity to external stimuli. A2 Swimming ability disrupted and loss of equilibrium but fish respond to pressure on the caudal peduncle. A3 Deep anesthesia: completely loss of reflex activity or failure to respond to strong external stimuli. A4 Medullary collapse: Asphyxia; opercular movements cease Recovery Complete recovery of equilibrium; Ability to remain upright and normal swimming behavior Table 2 Induction and recovery times of fish anesthetized by MBO and eugenol. Concentration of anesthetic (mg/L) average time for reaching different anesthesia stages recovery time (R) survival rate after 72 hour A1 A2 A3 A4 MDO 10 515±144 _ _ _ _ 100% 20 210±35 _ _ _ _ 100% 40 57±12 275±33 * _ 72±13 100% 60 47±17 134±33 907±172 _ 76±13 100% 80 24±7 95±28 412±89 _ 78±16 100% 100 12±3 72±25 183±54 _ 90±19 100% 120 16±7 43±8 116±33 _ 110±33 100% Eugenol 10 129±29 * _ _ - 100% 20 50±23 83±18 677±108 _ 170±20 100% 30 27±5 51±11 295±36 _ 209±29 100% 40 26±6 40±12 225±42 _ 222±45 100% 50 22±6 34±12 220±36 1298±115 265±29 100% 60 19±4 26±5 167±24 777±86 269±33 100% 70 14±3 27±8 128±26 451±51 208±34 100% Note: Water temperature was 28±0.5℃. A1-A4 and recovery correspond to anesthesia stages in Table 1. “_” indicates that it wasn’t observed corresponding anesthesia state in the experiment within 0.5 h. “*” indicates that only a few go into anesthesia within 5h. Table 3 Qualitative analysis of the histological alterations observed in the gills of juveniles of L. maculatus exposed to eugenol and MDO. Histopathological alterations Degree anesthetics MDO eugenol Lamellar epithelium hypertrophy I + + Lamellar epithelium hyperplasy I + + Lamellar fusion I - - Epithelial detachment I - + Proliferation of chloride cells I + + Mucous cell proliferation I + + Edema I + + Epithelial rupture II - + Lamellar aneurysm II - - Necrosis III - + Note: +, - means to the occurrence of the phenomenon was observed or not. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1475724","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":95723617,"identity":"38739699-6588-41c7-863b-b4eaa06dea83","order_by":0,"name":"Xiangbing Zeng","email":"","orcid":"","institution":"Shanghai Ocean University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiangbing","middleName":"","lastName":"Zeng","suffix":""},{"id":95723618,"identity":"79e12dfe-86c6-4fb9-bd0c-146665662fda","order_by":1,"name":"Hongbiao Dong","email":"","orcid":"","institution":"South China Sea Fisheries Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongbiao","middleName":"","lastName":"Dong","suffix":""},{"id":95723619,"identity":"8c2925d5-38b9-4cc5-ab71-450f3fd05d78","order_by":2,"name":"Jingru Wu","email":"","orcid":"","institution":"Xuzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jingru","middleName":"","lastName":"Wu","suffix":""},{"id":95723620,"identity":"bd6437b6-0d58-48f0-823a-e26ad9e694d3","order_by":3,"name":"Wenhao Wang","email":"","orcid":"","institution":"South China Sea Fisheries Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wenhao","middleName":"","lastName":"Wang","suffix":""},{"id":95723621,"identity":"bcee955a-08bb-424d-9c56-bb00588eee0e","order_by":4,"name":"Yafei Duan","email":"","orcid":"","institution":"South China Sea Fisheries Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yafei","middleName":"","lastName":"Duan","suffix":""},{"id":95723622,"identity":"ec79193c-72e3-420e-8b9c-2b6f1b0fc589","order_by":5,"name":"Jian Chen","email":"","orcid":"","institution":"Yangjiang fisheries technical extension station","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Chen","suffix":""},{"id":95723623,"identity":"d402dde8-6415-4cbe-9c29-1a77954536b7","order_by":6,"name":"Jiasong Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIiWNgGAWjYBACxmYowwBEfGwAizUeIFoL48wGBgkg1YBXCxyAtDDzgrUwMODVwtzO/PjDxx219ubsZw+/tt1hU6fbfhhoS41NNG6HsZlJzjxznNmyJy/NOvdMmoTZmUSglmNpuQ24/WLGzNt2jM3gQI6ZcW7bYQmzA0AtjA2H8Whh//wZqIXH4PwbM2NLkJbzDwlp4TGQ5m2rkTC4kWP8mBGk5QZBW3jKJGe2HTAwuPHGjLG3LU1y2w2gLQl4/GLYf3zzh49tdfYG53OMP/xss+E3O5/+8MGHGhvcWiASh0EEmwRcOAGHchCQh1B1IIL5Ax6Fo2AUjIJRMIIBAHhLYtqWVdY1AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-9490-119X","institution":"South China Sea Fisheries Research Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jiasong","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2022-03-22 02:43:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1475724/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1475724/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":19989817,"identity":"6440310a-6cdd-4ffb-9a11-5213bb72413d","added_by":"auto","created_at":"2022-04-05 18:49:40","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":809276,"visible":true,"origin":"","legend":"\u003cp\u003eSerum parameters of \u003cem\u003eL. maculatus\u003c/em\u003e after anesthesia with essential oil of \u003cem\u003eM. denudata\u003c/em\u003e (MDO; 100 mg/L) and eugenol (60 mg/L). Different letters above the bars mean significant difference (n = 5; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). A: cortisol; B: glucose; C: LDH; D: ALT; E: AST; E: ALP; DA: deep anesthesia stage; R0: 0 h after recovery; R6: 6 h after recovery; R24: 24 h after recovery.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-1475724/v1/d6fe0f3e446e60dbe71544d9.jpeg"},{"id":19989530,"identity":"91b07f5a-7a02-4482-a76c-df5862030094","added_by":"auto","created_at":"2022-04-05 18:44:40","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":671292,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant stress parameters of \u003cem\u003eL. maculatus\u003c/em\u003e in gills after anesthesia with essential oil of \u003cem\u003eM. denudate\u003c/em\u003e (MDO; 100 mg/L) and eugenol (60 mg/L). Different letters above the bars mean significant difference (n = 5; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). A: SOD; B: CAT; C: POD; D: GSH; E: MDA; DA: deep anesthesia stage; R0: 0 h after recovery; R6: 6 h after recovery; R24: 24 h after recovery.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-1475724/v1/fc647ea8ba3284af23af2447.jpeg"},{"id":19989529,"identity":"d03b0962-766b-4537-ac4c-be49f95e8b6e","added_by":"auto","created_at":"2022-04-05 18:44:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":130976,"visible":true,"origin":"","legend":"\u003cp\u003ePhycological stress parameters of \u003cem\u003eL. maculatus\u003c/em\u003e in gills after anesthesia with essential oil of \u003cem\u003eM. denudate\u003c/em\u003e (MDO; 100 mg/L) and eugenol (60 mg/L). Different letters above the bars mean significant difference (n = 5; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). A: Caspase-3; B: Caspase-9; C: Cyt-c; D: HSP70; DA: deep anesthesia stage; R0: 0 h after recovery; R6: 6 h after recovery; R24: 24 h after recovery.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-1475724/v1/e8c9bdffa05b05dd38576c36.png"},{"id":19989531,"identity":"589db7fe-9370-477c-b01b-907ade453da4","added_by":"auto","created_at":"2022-04-05 18:44:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1036011,"visible":true,"origin":"","legend":"\u003cp\u003eObservation on gills in different groups\u003c/p\u003e\u003cp\u003ePVC: pavement cells; BV: blood vessel; ER: erythrocytes; Scale bar: 50 μm; (10×40 times); solid arrow (Epithelial rupture); dotted arrow (Edema); double solid arrow (Hyperplasia); triangle (Proliferation of chloride cells).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-1475724/v1/ae4e74d31c4e4ee35394900b.png"},{"id":19989818,"identity":"912a57e7-0d62-466b-aceb-413f32647275","added_by":"auto","created_at":"2022-04-05 18:49:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":667508,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1475724/v1/43b42833-986f-471c-99d6-0f6748cfd5f1.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eEssential oil of \u003cem\u003eMagnolia denudata\u003c/em\u003e is an effective anesthetic for spotted seabass (\u003cem\u003eLateolabrax maculatus\u003c/em\u003e): a test for its effect on blood biochemistry, physiology and gill morphology\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the past decades, an increasing number of fish anesthetics have been used in animal welfare to reduce the trauma caused by handling (Wang et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Jerez-Cepa I et al., 2018; Taheri Mirghaed et al., 2020; Aydın and Barbas \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Currently, fish anesthetics are classified as synthetic anesthetics or plant-derived anesthetics (Purbosari et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, chemically synthesized anesthetics have several adverse effects on fish, including reproductive suppression, liver function, and excessive mucus secretion (Purbosari et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dong et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Teixeira et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; dos Santos Teixeira et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and the FDA approved only tricaine methanesulfonate (MS-222) for food fish (Carter, Woodley and Brown, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Moreover, there is a growing concern about their adverse impact in compromising human and fish health or the environment due to tissue residue.\u003c/p\u003e \u003cp\u003eFor the reasons stated above, less expensive, safer, and more natural anesthetics are required. Plant-derived anesthetics are antibacterial, antioxidant, anti-stress, and low risk to human health (Aydın and Barbas, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Purbosari et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Purbosari et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The essential oil anesthetics have been considered most effective in plant-derived anesthetics (Aydın and Barbas, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Plant essential oil is a type of volatile liquid with aromatic properties derived from aromatic plants such as leaves, flowers, fruits, seeds, buds, and stems (Baptista et al., 2020). \u003cem\u003eMagnolia denudata\u003c/em\u003e is a well-known ornamental tree species due to its attractive flowers in China with a cultivation history of over 2500 years (Park et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Its dried flower buds, known as Xin Yi in traditional Chinese herbal medicine, have been used as an analgesic agent for treating headaches (Wang et al., 2018). Furthermore, \u003cem\u003eM. denudata\u003c/em\u003e flower can be eaten as a food (Lu, Li and Yin, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the \u003cem\u003eM. denudata\u003c/em\u003e essential oils have not been assessed for their anesthetic properties in fish.\u003c/p\u003e \u003cp\u003eAlthough anaesthetics are used to reduce stress, the anesthetic itself may also be a source of stress (Readman et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Barata et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). When stress factors stimulate the body, it releases a large amount of catecholamine hormone and then activates the hypothalamus-pituitary-interregnal gland axis (HPI) to produce a neuroendocrine cascade reaction (Schreck, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). As a result, the corticosteroid-releasing hormone (CRH) and the pituitary gland-adrenocorticotropic hormone (ACTH) are released successively and pass through the blood circulation to reach the renal inter-renal tissues to stimulate the production of cortisol (Martos et al., 2014; Mommsen, Vijayan and Moon, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Therefore, blood hematological and biochemical parameters are good indicators to evaluate fish stress and health (Roche and Bog\u0026eacute;, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Fazio, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Multifunctional fish gills are essential for gas exchange, osmoregulation, acid-base regulation, and nitrogenous waste excretion (Evans et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Jiao et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Furthermore, the gill is the main organ to directly contact and absorb anesthetics (Hunn and Allen 1974; Ferreira et al., 1984). Many anesthetics have adversely impacted gills, such as lamellar epithelium hyperplasia, apoptosis of ionocytes, and necrosis (Wang et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Afifi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Brand\u0026atilde;o et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; de Lima et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, it is necessary to evaluate biochemical parameters in blood and the physiological changes on gills after anesthetic exposure.\u003c/p\u003e \u003cp\u003eDosage recommendations may differ between and within species, increasing the necessity for specific fish species research to prevent adverse effects from a high dose (Zahl, Samuelsen and Kiessling, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The spotted seabass (\u003cem\u003eLateolabrax maculatus\u003c/em\u003e) is widely distributed in the water from China, Korea, and Japan. It has the advantages of fast growth, wide salinity, high breeding survival rate, delicious meat, and strong adaptability, and it is popular among farmers and consumers(Yokogawa et al., 1995; Dong et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). However, the poor stress tolerance of the spotted seabass, especially the stress caused by handling during culturing and transportation, has seriously affected its growth and survival (Sun et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; He et al., 2020). Tricaine methanesulfonate (MS-222) and eugenol are chemically synthesized anesthetics in aquaculture to reduce fish activities (Bahrekazemi and Yousefi, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sk\u0026aring;r et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Aydın et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Purbosari et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, they have been tested on \u003cem\u003eL. maculatus\u003c/em\u003e and have been shown to cause gill and liver damage in recent studies (Wang et al., 2018; Wang et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, no report conducts on the anesthetic effect of essential oil on \u003cem\u003eL. maculatus\u003c/em\u003e. However, some previous studies have shown that eugenol is more effective and safer than MS-222 (Wang et al., 2018; Wang et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Dong et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Cao et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, our study aimed to compare the anesthetic effects, blood parameters and gill physiological changes after exposure to the essential oil of \u003cem\u003eM. denudate\u003c/em\u003e and eugenol.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cp\u003eFish maintenance and anesthetic agents\u003c/p\u003e \u003cp\u003eJuvenile \u003cem\u003eL. maculatus\u003c/em\u003e (164.16\u0026thinsp;\u0026plusmn;\u0026thinsp;15.40 g) were purchased from a fish hatchery in Zhuhai, Guangdong, China. The fish were acclimated in plastic tanks (4 m\u0026times;4 m ) for 2 weeks. The daily water exchange rate was 100% of the tank volume. The water quality parameters were maintained at dissolved oxygen 6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mg/L, temperature 28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u003csup\u003eo\u003c/sup\u003eC and pH 7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1. The natural photoperiod was used, and the water quality indicators were checked daily to maintain within the safety range. The eugenol (purity 99%; Wuhan Kangchun Perfume Co., Ltd. Wuhan, China) and essential oils of \u003cem\u003eM. denudate\u003c/em\u003e (MDO, purity 98%; Wuhan Kangchun Perfume Co., Ltd. Wuhan, China) were first diluted 1:9 in 95% ethanol to better mix with the water. The MDO main components were linalool (47.8%), terpineol (9.9%), neral (7.0%), geranial (6.9%) and cinnamaldehyde (3.7%) determined by GC-MS.\u003c/p\u003e \u003cp\u003eExperiment I: Anesthesia efficacy evaluation\u003c/p\u003e \u003cp\u003eIn this experiment, the MDO anesthetic efficacy was compared to eugenol. Before the experiment started, the fish fasted for 24 hours. Then, individual fish (n\u0026thinsp;=\u0026thinsp;10) was individually observed and used only once during the test. Each fish was netted quietly and placed in an induction barrel (10 L) containing one of the seven MDO concentrations (10, 20, 40, 60, 80, 100 and 120 mg/L) or one of the seven eugenol concentrations (10, 20, 30, 40, 50, 60 and 70 mg/L). The \u003cem\u003eL. maculatus\u003c/em\u003e staging criteria of the induction and recovery process was divided into five stages, according to Wang et al. (2018) and He et al. (2020) (Table\u0026nbsp;1). The induction and recovery time were recorded by using a digital stopwatch. After 0.5 hour, when the fish stopped breathing or was unable to enter more profound anesthesia, the fish was immediately transferred into the anesthetic-free and aerated in the recovery tank, and the time to restore regular swimming activity was recorded. The survival rate of induction, recovery, and 72 h after recovery were recorded.\u003c/p\u003e \u003cp\u003eExperiment II: Anesthetic antistress and secondary stress evaluation\u003c/p\u003e \u003cp\u003eThe ideal anesthetic effect allows a fish to enter anesthesia in less than 3 minutes and recover in less than 10 minutes (Hanggono, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Aydın and Barbas, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ross and Ross, 2008; Purbosari et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) because these concentrations have a short induction time and may not cause damage to the fish. In experiment I, the appropriate concentrations of MBO and eugenol for fast anesthesia (induction time\u0026thinsp;\u0026lt;\u0026thinsp;180 s) were 100 and 60 mg/L. After fasting for 24 h, the fish were individually transferred to three aquaria with 100 mg/L MBO, 60 mg/L eugenol and without anesthetic (control). After anesthetic exposure to each fish lost body balance with slow opercular movement (deep anesthesia stage), five fish per aquarium were sampled at this time point. The remaining fish in each aquarium were then transferred to a new aquarium with anesthetic free to recover and were sampled at 0, 6, and 24 h after recovery. The fish of after recovery stages and those in the control group were euthanized via cranial concussion according to the protocol approved by the Animals Ethical Committee of the South China Sea Fisheries Institute. The fish head was hit by a mechanical hammer for cranial concussion, resulting in syncope but not death. This cranial concussion lasted about 30 s in air exposure. Subsequently, the fish was held with a damp cloth and blood was collected by caudal venipuncture. For later analyses, the gill samples were removed and immediately frozen at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003cp\u003eSerum biochemistry\u003c/p\u003e \u003cp\u003eAfter centrifugation at 1000 g, at 4\u0026deg;C for 10 min, the serum was frozen and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until further analysis. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), glucose, cortisol, and lactic dehydrogenase (LDH) were measured by using a BS-200 automatic biochemical analyses and a kit (Shenzhen Mindray Biomedical Electronics Co., Ltd. Shenzhen, Guangdong, China).\u003c/p\u003e \u003cp\u003eOxidative stress and physiological indicator assessments\u003c/p\u003e \u003cp\u003eThe gills were homogenized in 9 volumes (w/v) of 4\u0026deg;C sterile physiological saline and centrifuged at 6000\u0026times;g at 4\u0026deg;C for 20 min. The antioxidant and physiological parameters were determined by a microplate reader (Bio-Rad, USA). In brief, the superoxide dismutase (SOD) activity was determined by the xanthine oxidase method, and the optical absorbance was determined at 450 nm. The peroxidase (POD) activity unit was measured and defined as the amount of enzyme that catalyzes 1 mL of sample to decompose 1\u0026micro;g of the substrate in 1 min at 37\u0026deg;C. Catalase (CAT) activity unit was defined as the amount of enzyme that catalyzes 1mg of protein to decompose 1\u0026micro;mol of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in 1 s. The glutathione (GSH) concentration was measured by colorimetry via 5,5- dithiobis reacting with mercapto compounds to form yellow compounds. The malondialdehyde (MDA) concentration was measured by the thiobarbituric acid method. The MDA was condensed with thiobarbituric acid during lipid peroxide degradation to produce a red product for quantitative determination at the 532 nm optical absorbance. HSP70 and cytochrome C (Cyt-c) were determined with commercial test kits, respectively, based on the manufacturer's protocol (MLBio, Shanghai, China). The enzymatic activities of cysteine-aspartic proteases-9 (Caspase-9) and cysteine-aspartic proteases-3 (Caspase-3) were measured with Ac-LEHD-pNA and Ac-DEVD-pNA as the substrates, respectively (Beyotime, Haimen, China).\u003c/p\u003e \u003cp\u003eHistopathology\u003c/p\u003e \u003cp\u003eTo further compared investigate the adverse effects of MDO and eugenol on \u003cem\u003eL. maculatus\u003c/em\u003e, the gills were analyzed, and the histological changes of the gills were examined at 6 h. Gills were fixed with 10% formaldehyde and washed with a 4\u0026deg;C physiological saline solution. Subsequently, the samples were gradually dehydrated with ethanol (70% \u0026minus;\u0026thinsp;100%), made transparent with xylene, embedded in paraffin, and cut into 5\u0026ndash;6 \u0026micro;m thick slices for H\u0026amp;E staining and neutral resin sealing. An optical microscope was used for staining observation and imaging. Image Pro Plus 6.0 software was used to process the images. The gill alterations were defined in three stages, stage I (alterations that do not affect organ functions), stage II (moderate alterations that affect the organ function) and stage III (severe and irreversible alterations), according to de Lima et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eStatistical analyses\u003c/p\u003e \u003cp\u003eAll data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analysis of the data was performed using SPSS 22.0 software. Based on the single-factor analysis of variance, the Duncan multiple comparison method was used for the analysis. In all cases, the minimum level of significance was set to \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAnesthetic efficacy of MBO and eugenol\u003c/p\u003e \u003cp\u003eNo mortality was observed during the entire experimental period. When the anaesthetic concentration increased, the time of induction stages decreased, and recovery time was prolonged. All concentrations have induced the fish to enter the deep sedation stage. When the concentration of MBO increased to 100\u0026ndash;120 mg/L, and the concentration of eugenol increased to 60\u0026ndash;70 mg/L, the induction time could reach the criteria of ideal induction, which is less than 3 min and recovery less than 10 min. Not observed fish enter A4 stage at all the concentration of MBO, While the concentration of eugenol increased to 40 mg/L, fish opercular movements ceased in 0.5 hour. In addition, the recovery times of the experimental fish that were anesthetized by MDO (72\u0026ndash;110 s) were shorter than those of the eugenol (170\u0026ndash;269 s) anesthetized fish (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSerum biochemical parameters\u003c/p\u003e \u003cp\u003eThe serum biochemical parameters of MDO and eugenol are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The cortisol concentration significantly decreased at the deep anesthesia stage but increased at 0 h after recovery (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The glucose of anesthetized fish by MDO and eugenol was significantly increased at 0 h, and the MDO group was higher than the control group at 6 h after recovery (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). LDH activity significantly decreased at the deep anesthesia stage and significantly increased at 0, 6, and 24 h in the eugenol group and 0 h in the MDO group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). ALT activity increased at 6 h in eugenol (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). AST activity significantly increased at 6 h and 24 h after recovery in anesthetic groups. In addition, the eugenol group was higher than the MBO group at 6 h and 24 h (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). ALP activity was not affected by the treatments (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAntioxidative stress and physiological response of gills\u003c/p\u003e \u003cp\u003eThe antioxidative stress parameters of \u003cem\u003eL maculatus\u003c/em\u003e in gills after anesthesia with MDO and eugenol are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. SOD activity significantly increased at 6 h after recovery in both eugenol and MDO groups, and eugenol group higher than MDO group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). CAT activity significantly increased at 0 and 6 h after recovery in the eugenol group and MDO group at 6 h, and the eugenol group was higher than the MDO group at 6 h (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). POD activity was no change in the entire experimental period (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). GSH concentration significantly increased at 6 h after recovery in both anesthetic groups, and the eugenol group was higher than the MDO group at 6 h., but the GSH concentration of both anesthetic groups significantly decreased at 24 h after recovery (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The MDA concentration significantly increased at 6 h in both the eugenol and MDO groups and significantly increased at 24 h in the eugenol group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe physiological stress parameters of \u003cem\u003eL maculatus\u003c/em\u003e in gills after anesthesia with essential oil of \u003cem\u003eMagnolia denudate\u003c/em\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Caspase-3 activity significantly increased at 6 h after recovery in both anesthetic groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Caspase-9 activity significantly increased at 6 h in both anesthetic groups and at 24 h after recovery in the eugenol group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Cyt-c concentration significantly increased at 0 h, 6 h and 24 h after recovery in the eugenol group and at 6 h after recovery in MDO group, and eugenol group higher than MDO group at 6 h (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). HSP70 concentration significantly increased at 6 h and 24 h in the eugenol and MDO groups at 6 h after recovery (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistopathology of gills\u003c/p\u003e \u003cp\u003eThe gill lamellas were intact in the control group, extending to both sides, and the pavement cells were regularly arranged on the gill lamella. The erythrocytes were distributed smoothly and evenly at the gill lamella. The gill structure showed a normal physiological condition, and no hyperplasia or edema was observed. A qualitative description of gill lesions found in fish anesthetized with the same effective concentrations of MBO and eugenol is shown in Table\u0026nbsp;3. The gills anesthetized with MBO had morphological alterations of the moderate response, such as the proliferation of chloride cells and hyperplasia and hypertrophy of epithelial cells, and the gill lamella was relatively neat. Hyperplasia and epithelial phenomena also appeared in the eugenol group. However, more severe damage such as epithelial rupture and necrosis was only observed in the eugenol group. In addition, the gill lamellas were not neatly arranged, and the blood vessels were swollen also were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAnesthetic is useful for reducing stress and improving fish welfare during handling. However, only MS-222 and eugenol have been studied, and they could cause damage to the liver and gills of \u003cem\u003eL. maculatus\u003c/em\u003e (Wang et al., 2018; Wang et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Dong et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). Therefore, more anesthetics need to be tested on this species. Plant-derived anesthetics have become a hot spot contributed to their safety, gentleness, and low risk to humans. In these contexts, we have investigated the anesthesia and antistress efficacy of essential oil of \u003cem\u003eM. denudate\u003c/em\u003e and the potential secondary stress to gills compared with eugenol on \u003cem\u003eL. maculate.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eSo far, a large number of essential oils have been reported to have anesthetic effects on fish, such as \u003cem\u003eLippia alba\u003c/em\u003e, \u003cem\u003eCymbopogum flexuosus\u003c/em\u003e, \u003cem\u003eMyrcia sylvatica\u003c/em\u003e, \u003cem\u003eOcimum gratissimum\u003c/em\u003e, \u003cem\u003eAloysia triphylla\u003c/em\u003e, \u003cem\u003eLippia sidoides\u003c/em\u003e and \u003cem\u003eMentha piperita.\u003c/em\u003e The active ingredients in these essential oils are linalool, geranial, β-pinene, thymol, and menthol (Junior et al., 2017; dos Santos Batista et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Brand\u0026atilde;o et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Saccol et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Boaventura et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Souza et al., 2018). Souza et al., (2018) found that \u003cem\u003eL. alba\u003c/em\u003e essential oil of linalool chemotype could induce fish to anesthesia stages. In addition, another study showed the use of \u003cem\u003eC. flexuosus\u003c/em\u003e essential oil for the anesthesia in \u003cem\u003eOreochromis niloticus\u003c/em\u003e. Notely, the main active ingredients in the \u003cem\u003eC. flexuosus\u003c/em\u003e essential oil are geranial and neral (Netto et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Cinnamaldehyde, a Trpa1 agonist, acts as a local anesthetic by inhibiting voltage-gated sodium channels in sensory neurons (Boonen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Thus, we speculate that the MDO active substances on \u003cem\u003eL. maculate\u003c/em\u003e anesthesia are linalool, neral, geranial, and cinnamaldehyde.\u003c/p\u003e \u003cp\u003eIt is critical to determine the effective concentration of anesthetics in each fish species to provide high welfare to both fish and the environment (Aydın and Barbas, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The ideal anesthesia effect allows the fish to enter anesthesia in less than 3 minutes and recover in less than 10 minutes (Hanggono, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Aydın and Barbas, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ross and Ross, 2008; Purbosari et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Based on the time criteria of ideal, the lowest effective concentrations were 100 mg/L for MDO, 60 mg/L for eugenol on \u003cem\u003eL. maculate\u003c/em\u003e juveniles. As the concentrations of the essential oils were increased, the time to induce anesthesia diminished and recovery time prolonged. In addition, MDO has a shorter recovery time compared with eugenol. This may indicate that MDO is easier to eliminate in fish. Besides, no mortality was observed during the entire experimental period. These results showed that MDO is a safe and efficient natural anesthetic for \u003cem\u003eL. maculate\u003c/em\u003e handing.\u003c/p\u003e \u003cp\u003eThe blood biochemical parameters of fish are valuable markers and very sensitive to anesthesia protocols (Yousefi et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yousefi et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Fazio \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Saccol et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To further evaluate the anti-stress effect and potential secondary stress of MDO on fish, cortisol, glucose, LDH, AST, ALT and ALP levels in the blood have been investigated in the present study. Cortisol and glucose are the most common stress indicators in fish (Ainsworth, Bowser, and Beleau, 1985; Semenkova et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Mart\u0026iacute;nez-Porchas et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Silva et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) found that the essential oil of \u003cem\u003eOcimum americanum\u003c/em\u003e effectively prevented cortisol from increasing in \u003cem\u003eRhamdia quelen\u003c/em\u003e after air exposure. Nile tilapia anesthetized with \u003cem\u003eA. triphylla\u003c/em\u003e essential oil and then exposed to air for 1 minute lowered plasma cortisol levels (Teixeira et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In the present study, fish in the deep anesthesia stage had lower cortisol levels than cranial concussion, confirming the stress-reducing properties of MDO and eugenol. We also found that anesthesia elevated cortisol and glucose levels after recovery, but these parameters declined back to control levels after 24h in the MDO group. Bodur et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) found that higher plasm cortisol was observed after exposed \u003cem\u003eOriganum\u003c/em\u003e sp. and \u003cem\u003eEucalyptus\u003c/em\u003e sp. essential oils anesthetics at recovery stages. Toni et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) also found that \u003cem\u003eL. alba\u003c/em\u003e essential oil induced higher cortisol at 4 h after recovery. These results suggest secondary stress of eugenol and MDO anesthesia on fish. ALT, AST and LDH activities can be used as sensitive biomarkers in ecotoxicology because they provide early warnings for aquatic organisms (Hoseini et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Haschek et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In the present study, the increased levels of ALT indicated hepatotoxicity. AST and LDH activities are indicators of not tissue-specific damage (Haschek et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Our results showed that ALT, AST and LDH activities increased after MDO and eugenol exposure. Similar to our study, Toni et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) found that glucose level, AST and ALT activities were significantly increased during the recovery period after being exposed to different concentrations of essential oils of \u003cem\u003eHesperozygis ringens\u003c/em\u003e and \u003cem\u003eL. alba\u003c/em\u003e. In addition, MDO has less adverse impact on \u003cem\u003eL. maculatus\u003c/em\u003e than eugenol. In recent studies, eugenol also showed more serve impact than other anesthetics (Yousefi et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yousefi et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; de Lima et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAntioxidant enzymes play a critical role in responding to oxidative stress during intracellular redox balance in vertebrates (Zhang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jiao et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). SOD, CAT, and POD are important enzymes involved in antioxidant defense systems and represent the cell\u0026rsquo;s first line of defense against oxidative stress (Wang et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the present study, increasing antioxidant enzyme activity (POD, SOD, and CAT) implies that anesthesia stress stimulates the antioxidant system during the recovery period. Wang et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) found that SOD, CAT, and POD activities in the gills of \u003cem\u003eL. maculatus\u003c/em\u003e were significantly increased at 6 h after eugenol and MS-222 treatment. The GSH is an important non-enzymatic antioxidant in cells, which can neutralize free radicals or oxidants (Galano et al., 2011). The MDA is the most important marker of lipid peroxidation (Liu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). They have been commonly used as biomarkers for the evaluation of oxidative stress. In the present study, the high levels of GSH and MDA indicate that MDO and eugenol can give rise to oxidative stress, but MDO caused less oxidative stress in the gills of \u003cem\u003eL. maculatus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eApoptosis, a type of cell death, is regulated by a series of molecules induced by environmental, physical, or chemical stress. These molecules can mediate phagocytosis and clearance of dead or infected cells and prevent autoimmunity (Cheng et al., 2018; Cifuentes-Rius et al., 2021). Cyt-c and Caspases play an essential role in cell apoptosis (Sun et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liang et al., 2019). In addition, HSP70 is a stress-induced molecular chaperone that aids in protein structural folding and transport in response to environmental stress (Tukaj, 2020; Maniya and Srivastava, 2020; Kurashova et al., 2020). Our study showed that eugenol and MBO induced apoptosis and HSP70 increased in the gills of \u003cem\u003eL. maculatus.\u003c/em\u003e These results are consistent with another study on eugenol and MS-222 on the gills of spotted sea bass (Wang et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn some studies, extensive histopathological abnormalities were observed in the gill, including hyperplasia, epithelial lifting, and curling of lamellae after exposure to anesthetics (essential oils of \u003cem\u003eA. triphylla\u003c/em\u003e, \u003cem\u003eL. sidoides\u003c/em\u003e, and \u003cem\u003eM. piperita\u003c/em\u003e, MS-222, eugenol), indicating that the gill is a sensitive organ for responding to anesthetics (Brand\u0026atilde;o et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lima et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the present study, changes in gill morphology were identified using H\u0026amp;E staining and were classified in three degrees, according to de Lima et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We observed lamellar epithelium hyperplasia in the gill after exposure to MDO and eugenol. The hypertrophy of the gill epithelium and hyperplasia of epithelium cells dilate the space between the exterior medium and the blood flow, reducing the direct contact of unwanted substances (Ghayyur et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, more severe pathology such as epithelial rupture and necrosis were found in gills exposed to eugenol, but MDO was not observed. These results are similar to a recent report of de Lima et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which found that eugenol caused more irreversible damage than \u003cem\u003eL. alba\u003c/em\u003e essential oils in gills on \u003cem\u003ePotamotrygon wallacei\u003c/em\u003e. In another study, no irreversible damage was found in gills after exposure to three essential oil anesthetics (Brand\u0026atilde;o et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Combined with the apoptotic and antioxidative results, we speculated that the use of eugenol caused more damage to the gills than MBO.\u003c/p\u003e \u003cp\u003eIn conclusion, the essential oils of \u003cem\u003eM. denudata\u003c/em\u003e are efficient as sedatives and anesthetics in \u003cem\u003eL. maculatus\u003c/em\u003e. The lowest effective concentration for \u003cem\u003eL. maculatus\u003c/em\u003e was 100 mg/L for MDO. The MDO of 100 mg/L effectively reduced stress in handling and caused less secondary stress in gills than eugenol. These results showed that MDO is a safe and efficient natural anesthetic for \u003cem\u003eL. maculate\u003c/em\u003e handing.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp; \u0026nbsp;\u003c/strong\u003eThis research was supported by China - ASEAN Maritime Cooperation Fund Project \u0026quot;China - ASEAN Modern Marine Fishery Technical Cooperation and Industrial Development Demonstration\u0026quot;, the fund of Central Public-interest Scientific Institution Basal Research Fund, South China Sea Fisheries Research Institute, CAFS (2021SD19), the fund of Guangdong province modern agricultural industrial technology system innovation team building project (2022KJ150), the fund of Guangzhou science and technology planning project (201904010169), the fund of National key research and development plan projects (2019YFD0900500), and the fund of Guangdong Province supporting town and villages technology commissioner project (KTP20210259).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u0026nbsp; \u0026nbsp; The authors have declared no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eThe collection and handling of the animals in this study was approved by the Animal Care and Use Committee at the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (SCSFRI-CAFS), and all experimental animal protocols were carried out in accordance with national and institutional guidelines for the care and use of laboratory animals at the SCSFRI-CAFS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u0026nbsp; \u0026nbsp;\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u0026nbsp; \u0026nbsp; The authors confirm that all the data involved in the manuscript are available for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions \u0026nbsp;\u0026nbsp;\u003c/strong\u003e X.Z., H.D. and J.Z. designed the experiment. X.Z. and H.D. performed the experiments, data analyses, and drafted the manuscript.W.J., W.W., Y.D. and J.C. contributed with essential materials. All authors have given approval to the final version of the manuscript, decided to submit the work for publication.The authors like to thank all the laboratory members for experimental material preparation and technical assistance, and thank to Prof. Jian Qin, Flinders University, for providing revisions and suggestions in the manuscript writing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAfifi SH, Al-Thobaiti S, Rasem BM (2000) Multiple exposure of Asian sea bass, (\u003cem\u003eLates calcarifer\u003c/em\u003e, Centropomidae) to clove oil: a histopathological study. Ass Vet Med J 42(84):166\u0026ndash;174\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAydın B, Barbas L (2020) Sedative and anesthetic properties of essential oils and their active compounds in fish: A review. 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Fish Shellfish Immunol. 99, 514\u0026ndash;525. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fsi.2019.11.056\u003c/span\u003e\u003cspan address=\"10.1016/j.fsi.2019.11.056\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1\u003c/p\u003e\n\u003cp\u003eBehavioral characteristics of \u003cem\u003eL. maculatus\u003c/em\u003e during anesthesia and recovery stages.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.641744548286605%\"\u003e\n \u003cp\u003estage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"85.35825545171339%\"\u003e\n \u003cp\u003eBehavioral\u0026nbsp;characteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.641744548286605%\"\u003e\n \u003cp\u003eA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"85.35825545171339%\"\u003e\n \u003cp\u003eDeep sedation: equilibrium normal; total loss of reactivity to external stimuli.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.641744548286605%\"\u003e\n \u003cp\u003eA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"85.35825545171339%\"\u003e\n \u003cp\u003eSwimming ability disrupted and loss of equilibrium but fish respond to pressure on the caudal peduncle.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.641744548286605%\"\u003e\n \u003cp\u003eA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"85.35825545171339%\"\u003e\n \u003cp\u003eDeep anesthesia: completely loss of reflex activity or failure to respond to strong external stimuli.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.641744548286605%\"\u003e\n \u003cp\u003eA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"85.35825545171339%\"\u003e\n \u003cp\u003eMedullary collapse: Asphyxia; opercular movements cease\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.641744548286605%\"\u003e\n \u003cp\u003eRecovery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"85.35825545171339%\"\u003e\n \u003cp\u003eComplete recovery of equilibrium; Ability to remain upright and normal swimming behavior\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 2\u003c/p\u003e\n\u003cp\u003eInduction and recovery times of fish anesthetized by MBO and eugenol.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\" width=\"16.717791411042946%\"\u003e\n \u003cp\u003eConcentration of anesthetic (mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" width=\"61.65644171779141%\"\u003e\n \u003cp\u003eaverage time for reaching different anesthesia stages\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"9.815950920245399%\"\u003e\n \u003cp\u003erecovery time (R)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"11.809815950920246%\"\u003e\n \u003cp\u003esurvival rate after 72 hour\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"24.688279301745634%\"\u003e\n \u003cp\u003eA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"28.179551122194514%\"\u003e\n \u003cp\u003eA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.199501246882793%\"\u003e\n \u003cp\u003eA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.93266832917706%\"\u003e\n \u003cp\u003eA4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"7\" valign=\"top\" width=\"10.906298003072196%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eMDO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"5.837173579109063%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.2073732718894%\"\u003e\n \u003cp\u003e515\u0026plusmn;144\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.357910906298002%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.442396313364055%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.589861751152075%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.831029185867896%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.827956989247312%\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"6.551724137931035%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.06896551724138%\"\u003e\n \u003cp\u003e210\u0026plusmn;35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.482758620689655%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.96551724137931%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.620689655172413%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.03448275862069%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.275862068965518%\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"6.551724137931035%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.06896551724138%\"\u003e\n \u003cp\u003e57\u0026plusmn;12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.482758620689655%\"\u003e\n \u003cp\u003e275\u0026plusmn;33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.96551724137931%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.620689655172413%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.03448275862069%\"\u003e\n \u003cp\u003e72\u0026plusmn;13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.275862068965518%\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"6.551724137931035%\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.06896551724138%\"\u003e\n \u003cp\u003e47\u0026plusmn;17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.482758620689655%\"\u003e\n \u003cp\u003e134\u0026plusmn;33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.96551724137931%\"\u003e\n \u003cp\u003e907\u0026plusmn;172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.620689655172413%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.03448275862069%\"\u003e\n \u003cp\u003e76\u0026plusmn;13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.275862068965518%\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"6.551724137931035%\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.06896551724138%\"\u003e\n \u003cp\u003e24\u0026plusmn;7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.482758620689655%\"\u003e\n \u003cp\u003e95\u0026plusmn;28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.96551724137931%\"\u003e\n \u003cp\u003e412\u0026plusmn;89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.620689655172413%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.03448275862069%\"\u003e\n \u003cp\u003e78\u0026plusmn;16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.275862068965518%\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"6.551724137931035%\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.06896551724138%\"\u003e\n \u003cp\u003e12\u0026plusmn;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.482758620689655%\"\u003e\n \u003cp\u003e72\u0026plusmn;25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.96551724137931%\"\u003e\n \u003cp\u003e183\u0026plusmn;54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.620689655172413%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.03448275862069%\"\u003e\n \u003cp\u003e90\u0026plusmn;19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.275862068965518%\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"6.551724137931035%\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.06896551724138%\"\u003e\n \u003cp\u003e16\u0026plusmn;7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.482758620689655%\"\u003e\n \u003cp\u003e43\u0026plusmn;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.96551724137931%\"\u003e\n \u003cp\u003e116\u0026plusmn;33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.620689655172413%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.03448275862069%\"\u003e\n \u003cp\u003e110\u0026plusmn;33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.275862068965518%\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"7\" valign=\"top\" width=\"10.906298003072196%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eEugenol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"5.837173579109063%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.2073732718894%\"\u003e\n \u003cp\u003e129\u0026plusmn;29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.357910906298002%\"\u003e\n \u003cp\u003e*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.442396313364055%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.589861751152075%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.831029185867896%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.827956989247312%\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"6.551724137931035%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.06896551724138%\"\u003e\n \u003cp\u003e50\u0026plusmn;23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.482758620689655%\"\u003e\n \u003cp\u003e83\u0026plusmn;18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.96551724137931%\"\u003e\n \u003cp\u003e677\u0026plusmn;108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.620689655172413%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.03448275862069%\"\u003e\n \u003cp\u003e170\u0026plusmn;20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.275862068965518%\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"6.551724137931035%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.06896551724138%\"\u003e\n \u003cp\u003e27\u0026plusmn;5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.482758620689655%\"\u003e\n \u003cp\u003e51\u0026plusmn;11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.96551724137931%\"\u003e\n \u003cp\u003e295\u0026plusmn;36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.620689655172413%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.03448275862069%\"\u003e\n \u003cp\u003e209\u0026plusmn;29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.275862068965518%\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"6.551724137931035%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.06896551724138%\"\u003e\n \u003cp\u003e26\u0026plusmn;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.482758620689655%\"\u003e\n \u003cp\u003e40\u0026plusmn;12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.96551724137931%\"\u003e\n \u003cp\u003e225\u0026plusmn;42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.620689655172413%\"\u003e\n \u003cp\u003e_\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.03448275862069%\"\u003e\n \u003cp\u003e222\u0026plusmn;45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.275862068965518%\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"6.551724137931035%\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.06896551724138%\"\u003e\n \u003cp\u003e22\u0026plusmn;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.482758620689655%\"\u003e\n \u003cp\u003e34\u0026plusmn;12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.96551724137931%\"\u003e\n \u003cp\u003e220\u0026plusmn;36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.620689655172413%\"\u003e\n \u003cp\u003e1298\u0026plusmn;115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.03448275862069%\"\u003e\n \u003cp\u003e265\u0026plusmn;29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.275862068965518%\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"6.551724137931035%\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.06896551724138%\"\u003e\n \u003cp\u003e19\u0026plusmn;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.482758620689655%\"\u003e\n \u003cp\u003e26\u0026plusmn;5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.96551724137931%\"\u003e\n \u003cp\u003e167\u0026plusmn;24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.620689655172413%\"\u003e\n \u003cp\u003e777\u0026plusmn;86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.03448275862069%\"\u003e\n \u003cp\u003e269\u0026plusmn;33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.275862068965518%\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"6.551724137931035%\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.06896551724138%\"\u003e\n \u003cp\u003e14\u0026plusmn;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.482758620689655%\"\u003e\n \u003cp\u003e27\u0026plusmn;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.96551724137931%\"\u003e\n \u003cp\u003e128\u0026plusmn;26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.620689655172413%\"\u003e\n \u003cp\u003e451\u0026plusmn;51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.03448275862069%\"\u003e\n \u003cp\u003e208\u0026plusmn;34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.275862068965518%\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: Water temperature was 28\u0026plusmn;0.5℃. A1-A4 and recovery correspond to anesthesia stages in Table 1. \u0026ldquo;_\u0026rdquo; indicates that it wasn\u0026rsquo;t observed corresponding anesthesia state in the experiment within 0.5 h. \u0026ldquo;*\u0026rdquo; indicates that only a few go into anesthesia within 5h.\u003c/p\u003e\n\u003cp\u003eTable 3\u003c/p\u003e\n\u003cp\u003eQualitative analysis of the histological alterations observed in the gills of juveniles of \u003cem\u003eL. maculatus\u003c/em\u003e exposed to eugenol and MDO.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"28.037383177570092%\"\u003e\n \u003cp\u003eHistopathological alterations\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"17.133956386292834%\"\u003e\n \u003cp\u003eDegree\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003eanesthetics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eMDO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eeugenol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.037383177570092%\"\u003e\n \u003cp\u003eLamellar epithelium hypertrophy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.133956386292834%\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.037383177570092%\"\u003e\n \u003cp\u003eLamellar epithelium hyperplasy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.133956386292834%\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.037383177570092%\"\u003e\n \u003cp\u003eLamellar fusion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.133956386292834%\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.037383177570092%\"\u003e\n \u003cp\u003eEpithelial detachment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.133956386292834%\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.037383177570092%\"\u003e\n \u003cp\u003eProliferation of chloride cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.133956386292834%\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.037383177570092%\"\u003e\n \u003cp\u003eMucous cell proliferation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.133956386292834%\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.037383177570092%\"\u003e\n \u003cp\u003eEdema\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.133956386292834%\"\u003e\n \u003cp\u003eI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.037383177570092%\"\u003e\n \u003cp\u003eEpithelial rupture\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.133956386292834%\"\u003e\n \u003cp\u003eII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.037383177570092%\"\u003e\n \u003cp\u003eLamellar aneurysm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.133956386292834%\"\u003e\n \u003cp\u003eII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.037383177570092%\"\u003e\n \u003cp\u003eNecrosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.133956386292834%\"\u003e\n \u003cp\u003eIII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.414330218068535%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: +, - means to the occurrence of the phenomenon was observed or not.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"fish-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fish","sideBox":"Learn more about [Fish Physiology and Biochemistry](https://www.springer.com/journal/10695)","snPcode":"10695","submissionUrl":"https://submission.nature.com/new-submission/10695/3","title":"Fish Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Essential oil, Anesthesia, Fish, Physiological","lastPublishedDoi":"10.21203/rs.3.rs-1475724/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1475724/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eMagnolia denudata\u003c/em\u003e is a well-known ornamental tree in China due to its beautiful blossoms, and it has been used as an analgesic to treat human headaches. This study investigates the anesthetic potential and physiological response of the essential oils of \u003cem\u003eM. denudata\u003c/em\u003e flower on spotted seabass \u003cem\u003eLateolabrax maculatus\u003c/em\u003e. Fish (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, 164.16\u0026thinsp;\u0026plusmn;\u0026thinsp;15.40 g) were individually exposed to different concentrations of \u003cem\u003eM. denudata\u003c/em\u003e essential oil (MDO, 10, 20, 40, 60, 80, 100 and 120 mg/L) and eugenol (10, 20, 30, 40, 50, 60 and 70 mg/L) to investigate anesthestic efficacy. Based on the time criteria of anesthesia induction (\u0026lt;\u0026thinsp;3 min) and post-recovery (\u0026lt;\u0026thinsp;10 min), the lowest effective concentration for spotted seabass was 100 mg/L for MDO and 60 mg/L for eugenol. The physiological and histopathological damages in the gill of \u003cem\u003eL. maculatus\u003c/em\u003e after using essential oil and eugenol were also evaluated for the minimum dose inducing deep anesthetia, and 0, 6 and 24 h after recovery. These results showed that MDO and eugenol anesthesia alleviated the levels of cortisol, glucose and lactic dehydrogenase (LDH) activities by handling. Compared with the eugenol, MDO also caused secondary stress to the body, but MDO caused minor physiological responses and histological changes in the gills. This study suggests that MDO is an effective anesthetic for spotted seabass.\u003c/p\u003e","manuscriptTitle":"Essential oil of Magnolia denudata is an effective anesthetic for spotted seabass (Lateolabrax maculatus): a test for its effect on blood biochemistry, physiology and gill morphology","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-04-05 18:44:38","doi":"10.21203/rs.3.rs-1475724/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2022-08-20T15:59:51+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2022-06-28T13:21:24+00:00","index":0,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-04-04T06:16:33+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-04-03T15:18:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-03-24T06:14:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Fish Physiology and Biochemistry","date":"2022-03-21T22:42:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"fish-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fish","sideBox":"Learn more about [Fish Physiology and Biochemistry](https://www.springer.com/journal/10695)","snPcode":"10695","submissionUrl":"https://submission.nature.com/new-submission/10695/3","title":"Fish Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3622c18b-c566-45df-b028-6189e086334f","owner":[],"postedDate":"April 5th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-09-09T09:19:23+00:00","versionOfRecord":[],"versionCreatedAt":"2022-04-05 18:44:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1475724","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1475724","identity":"rs-1475724","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}