Method of using plant essential oil in anesthetic treatment of spotted seabass

Abstract

To provide a method for using Antrodia cinnamomea essential oil for anesthetic treatment of spotted seabass, in which a problem to be solved is the feasibility of using Antrodia cinnamomea essential oil, which is a natural plant extract, as a new anesthetic to anesthetize live fish, and reduction in stress reaction and tissue damage in the process of anesthetizing live fish.SOLUTION: After spotted seabass is anesthetized by adding Antrodia cinnamomea essential oil in water, the morphological structure of the gill tissue of the spotted seabass and the influence on physiological, biochemical, and oxidative stress are studied, and the blood biochemical stress level, antioxidant enzyme activities, and immune indexes of the spotted seabass are measured to provide a theoretical basis for the anesthetic treatment of the sea fish. As compared with the expensive MS-222 and linalool, the price of Antrodia cinnamomea essential oil is 1 / 10 thereof, which can effectively reduce the cost of anesthesia, is particularly suitable for vaccination, blood collection, weighing, and transportation of live fish, etc., and has a higher economic benefit.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to the field of fish anesthesia, and more particularly to a method for anesthetizing saltwater fish, particularly sea bass. [Background technology]

[0002] Sea bass (Lateolabrax maculatus) belongs to the Osteichthyes, Perciformes, Perciformes, Culicidae, and Ophiopogon genus, and is the main species of marine aquaculture fish in China. Sea bass is rich in protein, has fine flesh, and is highly edible. However, sea bass are highly susceptible to stress, and have coarse scales and sharp dorsal fins. These make them prone to violent thrashing and mechanical injury during blood collection, weighing, and transportation, which can promote stress responses in live fish and lead to fish deaths, limiting the market size for sea bass sales.

[0003] Several programs in commercial aquaculture and fisheries require anesthesia to improve animal welfare, including reducing fish pressure and injury during processes such as culling, vaccination, bleeding, weighing, and transportation. Fish anesthetics are divided into two categories: synthetic and natural (plant-based) products. The most commonly used synthetic anesthetic is MS-222 (the chemical name for MS-222 is m-aminobenzoic acid ethyl methanesulfonate, and the molecular formula is C). 10 H 15 These include NO5S (used as an anesthetic for transporting live fish), benzozocaine, quinaldine, and 2-phenoxyethanol. Some synthetic anesthetics are expensive, stressful, or have adverse side effects, such as excessive mucus secretion, gill irritation, immune system inhibition, and increased muscle tone. In recent years, essential oils have been investigated as anesthetics for fish and have become suitable alternatives to synthetic anesthetics.

[0004] The physiological, biochemical, and histopathological effects of botanical anesthetics on fish are increasingly being evaluated. The advantages and disadvantages of different botanical anesthetics for each species should be clarified, as they may affect the health and welfare of fish. Anesthetics strongly affect fish blood biochemistry, such as blood cell counts, glucose and lactate levels, liver enzymes, and immune status. After exposure of niloferalis (Oreochromis niloticus) to ginger essential oil, blood glucose and cortisol levels remained essentially normal, whereas those exposed to MS-222 were relatively high. Furthermore, plant essential oils used as anesthetics are particularly important for the histopathological effects of fish. In anesthesia studies, the gills are the organ most severely affected. Exposure of red sea bream to eugain oil resulted in changes in gill histology. However, upon exposure to eugain oil, the extract accumulates in the gill tissue of Odopus odopus.

[0005] The Tilia chinensis tree, a member of the Laurylaceae family, has significant biological value and is used worldwide for medicinal, industrial, and fragrance purposes. Camphor tree essential oil is isolated from the branches and leaves of the Tilia chinensis tree. Its main component is linalool, and research has shown that linalool has anesthetic and sedative effects on fish. However, commercially available pure linalool is expensive (205.9 yuan per 1 mL, Arabia Reagent Co., Ltd.). Linalool is heat-sensitive, making the process of separating and purifying linalool from plant essential oils difficult, and the purified product is prone to contain other impurities. Therefore, there is an urgent need to find an economical and safe plant essential oil for fish anesthesia.

[0006] At present, no domestic or foreign scholars have reported on the use of Camphor oil in marine fish anesthesia and its effects on organ function and stress response. Summary of the Invention

[0007] This invention provides a method for using camphor tree essential oil to anesthetize sea bass. The problem to be solved is the feasibility of using camphor tree essential oil, a natural plant extract, as a new anesthetic agent to anesthetize live fish, and to reduce stress responses and tissue damage during the anesthesia process of live fish. After anesthetizing sea bass with camphor tree essential oil in water, the effects on the morphology, structure, physiology, biochemistry, and oxidative stress of the gill tissue of the sea bass were studied, and the biochemical stress levels, antioxidant enzyme activity, and immune indexes of the sea bass's blood were measured, providing a theoretical basis for anesthetizing sea bass.

[0008] The problem to be solved by the present invention is achieved by the following technical means.

[0009] A method for anesthetizing sea bass with camphor essential oil, comprising the following steps (1) to (4): (1) Sea bass temporary rearing: Sea bass are reared in an aquarium for 24 hours before anesthesia, and the water temperature in the aquarium during the rearing period is 22±1°C, the salinity of the water in the aquarium is 16‰, the dissolved oxygen of the water in the aquarium is 6mg / L, the pH is 7.5-8.0, and the fish-water ratio is 1:50. During the rearing period of the sea bass in the aquarium, metabolic waste products are excreted from the body; (2) Camphor essential oil emulsification: Camphor essential oil, ethanol, and polysorbate-18 are emulsified and dissolved in the appropriate ratio. (3) Anesthesia: the emulsified camphor oils are placed in aquariums, and the water temperature of the aquarium is controlled at 22±1°C. The concentration of the camphor oil in the water body of the aquarium is in the range of 10-200mg / L. The sea bass are transferred to the aquariums containing the essential oils of different concentrations, and the concentration of the camphor oil in the aquarium and the anesthetization time of the sea bass are recorded. (4) Recovery: After the sea bass enters the deep anesthesia stage, the sea bass is placed in an aquarium containing clean water to recover. If the sea bass does not roll over, swims normally and is active, it is determined to have recovered normally, and the recovery time is recorded.

[0010] Furthermore, the concentration of the camphor essential oil is 150 mg / L.

[0011] Furthermore, the emulsification step of the camphor essential oil includes the following steps 1 and 2: In step 1, 10 ml of camphor essential oil, 200 ml of 50% ethanol, and 1 ml of polysorbate-80 are mixed in a volume ratio of 10:200:1, and then slowly added dropwise to 500 ml of water to form a first mixed solution; In step 2, after stirring with a magnetic stirrer, the first mixture is evaporated and concentrated to 100 ml.

[0012] Furthermore, the temperature of the evaporation is 20-40°C.

[0013] Furthermore, the emulsification temperature of the camphor essential oil is preferably 25°C.

[0014] Furthermore, the water temperature in the aquarium was 22±1°C, the salinity was 16‰, the dissolved oxygen was 6mg / L, and the pH was 7.5.

[0015] In a preferred embodiment, the sea bass is a fish with a weight of 500±25g, a length of 30±1cm, and a respiratory rate of 60-65 times / min (the number of times the sea bass's gill operculum opens and closes within 1 minute).

[0016] In the most preferred embodiment, the camphor essential oil is purchased from Jiangxi Xuesong Natural Medicinal Oil Co., Ltd. (China), and the polysorbate-80 is purchased from Shanghai Anpu Experimental Technology Co., Ltd.

[0017] Beneficial Effects of the Invention The essential oil anesthesia method provided in the present invention can effectively reduce tissue damage and stress response of sea bass during the anesthesia process. Compared with expensive MS-222 and linalool, the price of Camphor tree essential oil is 1 / 10 of that of them, which can effectively reduce the anesthesia cost, and is especially applicable to vaccination, blood sampling, weighing and transportation of live fish, etc., with higher economic benefits. During the anesthesia period of the sea bass, camphor oil can reduce the oxidative stress and metabolic disorder of the sea bass during the anesthesia process, alleviate the damage to the gills caused by anesthesia, reduce the increase in blood glucose and lactic acid of the sea bass, and not inhibit the activities of alkaline phosphatase, immunoglobulin M and lysozyme of the sea bass, so as to maintain the immune strength of the sea bass during anesthesia. [Brief explanation of the drawings]

[0018] [Figure 1] Pathological photographs of sea bass gill tissue caused by MS-222 of the present invention. 1. Chloride cell hyperplasia; 2. Gill stratified epithelial swelling; 3. Gill stratified epithelial hyperplasia. A. Sea bass gill tissue section before anesthesia; B. Sea bass gill tissue section after anesthesia with MS-222; C. Sea bass gill tissue section after 24 hours of recovery from anesthesia with MS-222. [Figure 2] Pathological photographs of sea bass gill tissues treated with camphor essential oil of the present invention. A: Sea bass gill tissue section before anesthesia; B: Sea bass gill tissue section after 24 hours of recovery from anesthesia with camphor essential oil; C: Sea bass gill tissue section after 24 hours of recovery from anesthesia with linalool. [Figure 3] Pathological photographs of sea bass gill tissues treated with linalool according to the present invention. 4. Gill stratified epithelial desquamation; 5. Gill stratified epithelial swelling; 6. Gill stratified epithelial desquamation; A. Sea bass gill tissue section before anesthesia; B. Sea bass gill tissue section after anesthesia with camphor essential oil; C. Sea bass gill tissue section after anesthesia with linalool. [Figure 4] The effects of anesthesia with MS-222 of the present invention, camphor oil, and linalool on red blood cell count (A), hematocrit (B), and hemoglobin (C) in sea bass blood. [Figure 5] The effects of anesthesia using MS-222, camphor oil, and linalool on glucose content (A) and lactic acid content (B) in sea bass. [Figure 6] The effects of MS-222 of the present invention, camphor oil, and linalool on catalase (A), glutathione peroxidase (B), and superoxide dismutase (C) in sea bass. [Figure 7]The effects of MS-222 of the present invention, camphor oil, and linalool on alkaline phosphatase (A), immunoglobulin M (B), and lysozyme (C) in sea bass. DETAILED DESCRIPTION OF THE INVENTION

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. The present invention will be further described below through specific examples. Based on the embodiments of the present invention, any other embodiments that can be obtained by those skilled in the art without creative efforts are all included in the scope of protection of the present invention.

[0020] The camphor essential oil described in this invention is purchased from Jiangxi Xuesong Natural Medicinal Oil Co., Ltd. (China). Linalool is purchased from Aladdin Reagent (Shanghai) Co., Ltd. MS-222 is purchased from Merck Biotech GmbH (Germany). MS-222 is a chemically synthesized anesthetic (chemical name: 3-aminobenzoic acid ethyl methanesulfonate; molecular formula: C10H15NO5S) and is used as an anesthetic for transporting live fish. Polysorbate-80 is purchased from Shanghai Anpu Experimental Technology Co., Ltd.

[0021] Detection of essential oil components GC-MS analysis of chemical properties of Fangzhou essential oil Detection was performed using an FID detector, Agilent (registered trademark) GC-7890. The GC / MS analysis conditions were as follows. Column: HP-5 Inlet temperature: 140℃ Diversion ratio: 60:1 Heater: 280℃ Chromatography conditions: 40°C, hold for 5 minutes, increase temperature at 10°C / min to 250°C, increase temperature from 5°C to 280°C, hold for 5 minutes Extraction conditions: 30 mL of sample was taken, 30 mL of ethyl acetate was added, and the supernatant was taken after extraction. The above process was repeated three times. The supernatant was concentrated to 2 mL, and the concentrate was filtered through a 0.22 μm membrane. 1 μL was then injected into the GC-MS system. The components of the obtained camphor tree essential oil are shown in Table 1.

[0022] JPEG2025149773000002.jpg39131

[0023] In the following example, sea bass of uniform size, healthy constitution, and consistent respiratory rate were selected and then temporarily reared under 24-hour fasting conditions. During the rearing period, the water temperature was 22±1°C, the salinity was 16‰, the dissolved oxygen was 6mg / L, the pH was 7.5, and the fish / water ratio was 1:50. During the fasting period, metabolic waste products were excreted from the sea bass's body.

[0024] Example 1 Preparation of essential oil emulsion: 200 mL of 50% ethanol, 1 mL of polysorbate-80, and 10 mL of camphor essential oil were mixed in a ratio of V (essential oil):V (50% ethanol):V (polysorbate-80) = 10:200:1, and the mixture was slowly added dropwise to 500 mL of water. The mixture was stirred magnetically at 20°C for 100 minutes and concentrated to 100 mL using a rotary evaporator.

[0025] Example 2 Preparation of essential oil emulsion: 200 mL of 50% ethanol, 1 mL of polysorbate-80, and 10 mL of camphor essential oil were mixed in a ratio of V (essential oil):V (50% ethanol):V (polysorbate-80) = 10:200:1, and the mixture was slowly added dropwise to 500 mL of water. The mixture was stirred magnetically at 25°C for 100 minutes and concentrated to 100 mL using a rotary evaporator.

[0026] Example 3 Preparation of essential oil emulsion: 200 mL of 50% ethanol, 1 mL of polysorbate-80, and 10 mL of camphor essential oil were mixed in a ratio of V (essential oil):V (50% ethanol):V (polysorbate-80) = 10:200:1, and the mixture was slowly added dropwise to 500 mL of water. The mixture was stirred magnetically at 30°C for 100 minutes and concentrated to 100 mL using a rotary evaporator.

[0027] Example 4 Preparation of essential oil emulsion: 200 mL of 50% ethanol, 1 mL of polysorbate-80, and 10 mL of camphor essential oil were mixed in a ratio of V (essential oil):V (50% ethanol):V (polysorbate-80) = 10:200:1, and the mixture was slowly added dropwise to 500 mL of water. The mixture was stirred magnetically at 40°C for 100 minutes and concentrated to 100 mL using a rotary evaporator.

[0028] Example 5 Preparation of essential oil emulsion: 200 mL of 50% ethanol, 1 mL of polysorbate-80, and 10 mL of camphor essential oil were mixed in a ratio of V (essential oil):V (50% ethanol):V (polysorbate-80) = 10:200:1, slowly added dropwise to 500 mL of water, magnetically stirred at 19°C for 100 minutes, and concentrated to 100 mL using a rotary evaporator.

[0029] Example 6 Preparation of essential oil emulsion: 200 mL of 50% ethanol, 1 mL of polysorbate-80, and 10 mL of camphor essential oil were mixed in a ratio of V (essential oil):V (50% ethanol):V (polysorbate-80) = 10:200:1, slowly added dropwise to 500 mL of water, magnetically stirred at 42°C for 100 minutes, and concentrated to 100 mL using a rotary evaporator.

[0030] The emulsification temperature of camphor oil is controlled between 20-40°C. If the temperature is below 20°C, the solubility of the emulsifier (polysorbate-80) is low and emulsification is insufficient. If the temperature is above 40°C, camphor oil is prone to volatilization. JPEG2025149773000003.jpg43145

[0031] 1. The emulsified camphor oils were placed in aquariums, the water temperature was controlled at 22±1℃, and the concentrations of camphor oil were set at 10, 20, 30, 50, 100, 150, and 200 mg / L, respectively, to prepare MS-222 anesthetic solution and linalool solution of the same concentration. 2. The sea bass were placed in aquariums (5 bottles per box) containing different concentrations of camphor oil, MS-222 and linalool anesthetics, and the anesthetic concentration, sedative concentration and duration of anesthesia were recorded. The fish was considered to be in an anesthetized state when it rolled over. 3. After entering the deep anesthesia period, the fish were placed in an aquarium containing clean water for recovery. If the sea bass did not roll over, swam normally, and showed high activity, it was considered to have recovered normally, and the recovery time was recorded. 4. The optimal concentration range was obtained, and indicators such as oxidative stress and tissue damage were measured.

[0032] JPEG2025149773000004.jpg45130

[0033] (7) Optical microscope observation Gill filaments were fixed in 4% formaldehyde solution for 24 h, then rinsed three times in phosphate-buffered saline (PBS, pH 7.4). They were then dehydrated through a gradient of ethanol solutions (30%, 50%, 70%, 80%, 90%, 95%, and 100%). They were then filtered through xylene in a fume hood, embedded in paraffin, and cut into 4 μm sections using a slicer. Gill pieces were stained with hematoxylin and eosin (Sigma-Aldrich), rehydrated through gradient alcohols, dewaxed in xylene, and mounted with rhamsan gum according to the manufacturer's instructions. Images were recorded using an Olympus BX-43 light microscope (Japan).

[0034] Results and analysis of the examples 1. Sedative concentration, anesthesia induction concentration and recovery time of different essential oils As can be seen from Table 2, the time it takes for sea bass to reach the same anesthesia stage decreases with increasing mass concentrations of camphor oil, MS-222, and linalool, and the recovery time of sea bass increases with increasing anesthetic concentration. Sea bass ultimately reach a deep sedation stage in anesthetic solutions containing 20 and 30 mg / L camphor oil. Deeply sedated fish have a low stress response to external stimuli, a low metabolic rate, and can still maintain their equilibrium. This state is optimal for live transport. When the mass concentration of camphor oil is 150 mg / L, sea bass reach the fourth stage of anesthesia, becoming anesthetized within 3 minutes and recovering within 4 minutes. This essential oil concentration is the optimal anesthetic concentration for sea bass. The optimal anesthetic concentration for MS-222 for sea bass is 100 mg / L. Compared to the optimal anesthetic concentration of camphor oil, the difference in the time required to reach anesthesia is relatively small, and the fish require a longer recovery time. The optimal anesthetic concentration for sea bass is 100 mg / L, and at this concentration, sea bass are anesthetized within 3 minutes, but the recovery time is much longer than that of sea bass anesthetized with camphor oil or MS-222, indicating that linalool is not an ideal anesthetic for fish.

[0035] JPEG2025149773000005.jpg117164

[0036] 2. Changes in tissue damage Histopathological studies are essential for verifying the sensitivity of organs, primarily the gill epithelium, which is the primary site of gas exchange and osmoregulation, to toxic substances. Because the safe use of anesthetics is crucial, we analyzed the safety of anesthetics using stratified gill structure as the best indicator of health. Figure 1, A, shows a tissue section of a sea bass gill before anesthesia. After anesthesia with MS-222, as seen in Figure 1, B, there was significant chloride cell hyperplasia (1), swelling of the gill tissue stratified epithelium (2), and hyperplasia of the stratified epithelium (3). In Figure 2, no significant changes were observed after the use of camphor essential oil (e.g., Figure 2, B and C). In Figure 3, after anesthetizing sea bass with linalool, the gill tissue of the fish exhibited swelling of the stratified epithelium (5), as shown in Figure 3B, and epithelial hyperplasia (4) and (6), as shown in Figure 3B. As can be seen in Figure 1A, sea bass anesthetized with MS-222 showed relatively severe swelling of the gill stratified epithelium (2), along with chloride cell hyperplasia (1) and stratified epithelial hyperplasia (3). In contrast, sea bass anesthetized with linalool exhibited desquamation of the gill stratified epithelium (4) and (6), as shown in Figure 3. This is because the fish undergo stress during the anesthesia process, and the gill tissue changes to adapt to the new conditions. These morphological changes indicate that the fish have developed adaptive strategies to protect important physiological functions, such as gas exchange, acid-base balance, osmoregulation, and excretion of nitrogen compounds. Because the gill is directly exposed to the aquatic environment, it is susceptible to various types of damage (traumatic, toxic, and infectious), and fish tissues can only respond in a limited manner to harmful stimuli. This also explains the swelling and hyperplasia of the gill stratified epithelium observed in gill tissue sections under anesthesia. After 24 hours of recovery, the histopathological changes in each experimental group of sea bass recovered to varying degrees. This indicates that the gill tissue damage caused by these three anesthetics in sea bass is reversible. This is a defense mechanism used by fish to cope with external stimuli and is a repairable nonspecific response. Compared to MS-222 and linalool, sea bass anesthetized with camphor oil suffered less stress damage during the anesthesia and recovery process and showed a faster recovery rate after anesthesia, as shown in Figure 2C.

[0037] 3, blood analysis Changes in hematological levels are an indicator of the degree of pressure stress in fish. Different anesthetics cause different changes in the blood levels of sea bass. As shown in Figure 4, after sea bass were anesthetized with MS-222, camphor oil, and linalool, the red blood cell count, hemoglobin, and hematocrit content all showed an increase. This is due to the slowed gill movement and oxygen deficiency of fish exposed to anesthetics. The specific cell volume percentage reflects the ratio of red blood cells to white blood cells and plasma in the blood. The increase in hematocrit after anesthesia induction may lead to a decrease in ventilation frequency and oxygen deficiency due to anesthesia. After anesthesia, the red blood cell counts of sea bass in the MS-222 and linalool-treated groups were significantly higher than those in the camphor oil-treated group. The increase in red blood cell counts may be due to the increased energy demand caused by the pressure of these anesthetics, which requires more oxygen transport. Higher hemoglobin concentrations indicate higher oxygen transport capacity of red blood cells. Research has shown that these changes occur to respond to increased physiological needs due to malfunction. Under stressful conditions, more oxygen is required to meet the organism's high energy demands, and hemoglobin levels increase to transport more oxygen to the blood. Generally, such short-term stress is beneficial to the organism because it regulates and redistributes energy reserves to respond to stress. During the post-anesthesia recovery process, the contents of these three components decrease and gradually return to their initial levels. This indicates that anesthetic pressure can be regulated by physiological changes. However, after 24 hours of recovery, sea bass anesthetized with MS-222 had higher red blood cell counts and lower hematocrits than their initial values. However, this is not entirely harmful to the fish and may be a typical physiological change that allows fish to self-regulate external pressures. At the same time, the 24-hour recovery period indicates that fish anesthetized with MS-222 are insufficient to completely offset the stress pressure caused by anesthesia and recover to an optimal state.

[0038] 4. Energy metabolism Glucose (GLU) is an important energy source for various vital activities in fish. Blood glucose levels affect the stress level of fish by regulating cortisol release and glucose homeostasis through glycogen metabolism and glycolysis. The glucose content of anesthetized sea bass showed an increasing trend (see Figure 5). The increase in blood glucose content after anesthesia indicates that anesthesia caused stress to the sea bass. The increase in blood glucose content is due to increased irritability and hypoxia during anesthesia. Compared to MS-222 and linalool, the change in glucose content of sea bass anesthetized with camphor essential oil was minimal, at 2.80%. After 24 hours of recovery for each anesthetized sea bass, the glucose content gradually returned to its initial level. Lactic acid can reflect the oxygenation and metabolic status of tissues. Under pressure, fish activate the hypothalamic-pituitary-renal axis, which causes changes in blood hormone levels and metabolic changes such as increased glucose and lactate levels and impaired osmoregulation. Sea bass anesthetized with MS-222, camphor oil, and linalool showed elevated lactic acid levels, with the linalool-anesthetized group showing the highest lactic acid levels. Research has shown that anesthesia inhibits the sensory nerve function of fish, thereby increasing plasma lactate levels. Furthermore, even under hypoxic conditions during anesthesia, fish generate energy through glycolysis, which is accompanied by lactate production. An increase in the level of lactate in fish indicates that the fish is unable to maintain its internal balance and is actively adapting to environmental changes. Among MS-222, camphor oil and linalool, the lactic acid content of sea bass anesthetized with MS-222 and camphor oil gradually returned to the initial level after 24 hours of recovery, indicating that the metabolic changes caused by anesthesia with camphor oil are reversible and will not cause metabolic disorders in fish.

[0039] 5. Oxidative stress The presence of oxidants in cells stimulates the body's antioxidant defense system and enhances antioxidant enzyme activity. Oxidative stress responses are triggered by an imbalance between prooxidant and antioxidant concentrations in organisms experiencing biotic or abiotic stress. Superoxide dismutase (SOD) is one of the antioxidant enzymes responsible for removing reactive oxygen species (ROS) generated in cells. SOD converts ROS into hydrogen peroxide (H2O2) and water (H2O), which is then converted into H2O and O2 by the action of catalase (CAT) and glutathione peroxidase (GSH-Px). SOD, CAT, and GSH-Px activities in sea bass anesthetized with MS-222, camphor oil, and linalool all tended to increase after anesthesia. Here, the camphor oil experimental group showed the smallest post-anesthesia changes in sea bass, with changes in SOD, CAT, and GSH-Px of 6.90%, 9.77%, and 4.53%, respectively. In defining an ideal anesthetic, the positive effect of anesthetics on the antioxidant system has been emphasized. Anesthetics can alter the oxygen activity in tissues and have antioxidant effects at appropriate concentrations. For example, anesthetizing fish with chamomile oil or eucalyptus oil can improve their antioxidant capacity. In contrast, camphor oil has a certain antioxidant capacity, reducing the anesthetic pressure of sea bass and minimizing oxidative stress. The sea bass in the MS-222 and linalool experimental groups showed significant changes in SOD, CAT, and GSH-Px activities. After 24 h of recovery, the SOD and CAT activities of the sea bass in the MS-222 and camphor oil anesthetized group returned to their initial values, while the GSH-Px activity of the sea bass in the camphor oil anesthetized group returned to its initial level. The SOD and GSH-Px activities of the sea bass in the linalool-treated group were still higher than their initial levels, indicating the continued existence of oxidative stress (Fig. 6).

[0040] 6. Immune response Nonspecific immunity plays an important role in fish immunity. When external pressure breaches the antioxidant system, immune enzymes and immune-activating substances begin to be activated. Acid phosphatase (ACP) is an important hydrolase in the fish liver detoxification system and can reflect the immune strength of the organism. As shown in Figure 7, during the anesthesia and recovery periods, the ACP activity of sea bass anesthetized with linalool was significantly lower than that of sea bass anesthetized with MS-222 and camphor oil. The ACP activity of sea bass in the camphor oil treatment group was not significantly different from that of the MS-222 group, indicating that anesthetization with MS-222 and camphor oil did not cause any changes in ACP activity in sea bass and maintained the stability of their immune system. Immunoglobulin M (IgM) is an important immunoglobulin in fish, protecting them from infection and maintaining mucosal homeostasis. There was no significant difference in IgM activity between sea bass anesthetized with MS-222, camphor oil, and linalool. Lysozyme (LZM) is a nonspecific immunolabeling enzyme that can lyse bacterial cells, eliminate harmful bacteria from the body, and maintain tissue and organ homeostasis. Changes in LZM activity can reflect the state of fish immune function. Sea bass anesthetized with camphor oil showed increased LZM activity, whereas sea bass anesthetized with MS-222 showed no significant change. However, sea bass anesthetized with linalool showed decreased LZM activity, which was significantly lower than that of sea bass treated with camphor oil and MS-222. Studies have shown that anesthetics can cause immunosuppression in fish, and the inhibitory response is related to the type and dose of fish anesthetic. When anesthetized with camphor oil, it had no inhibitory effect on LZM activity in sea bass, indicating that camphor oil is a safer anesthetic.

[0041] The plant essential oil provided by the present invention can be used in the anesthesia treatment method for sea bass, which can effectively reduce the oxidative stress and immunosuppression during the anesthesia process of sea bass, effectively alleviate the damage to gills caused by anesthesia, and maintain the homeostasis of hematological level.In addition to obtaining good and safe anesthetic effect, compared with the expensive MS-222, the price of Camphor tree essential oil is only 1 / 10, which can effectively reduce the anesthesia cost, and can be applied to the processes of live fish vaccination, blood sampling, weighing and transportation, etc., and achieve higher economic benefits.

[0042] The above-described embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Those skilled in the art may modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical ideas disclosed in the present invention should be covered by the claims of the present invention.

Claims

1. A method for anesthetizing sea bass with camphor essential oil, comprising the following steps (1) to (4): Step (1) in the temporary rearing of sea bass, the sea bass are fasted for 24 hours in the aquarium before anesthesia, and reared temporarily, during the temporary rearing period, the water temperature of the aquarium is 22±1°C, the salinity of the water body in the aquarium is 16‰, the dissolved oxygen of the water body in the aquarium is 6mg / L, the pH is 7.5-8.0, and the fish-water ratio is 1:50, and during the temporary rearing period of the sea bass in the aquarium, metabolic waste products are excreted from the body; Step (2) in emulsifying camphor essential oil, emulsifying and dissolving camphor essential oil, ethanol, and polysorbate-18 in the appropriate ratio; Step (3) for anesthesia, the emulsified camphor oils are respectively placed into aquariums, the water temperature of the aquarium is controlled to 22±1°C, the concentration of the camphor oil in the water body of the aquarium is in the range of 10-200mg / L, the sea bass are respectively transferred to the aquariums containing essential oils of different concentrations, and the concentration of the camphor oil in the aquariums and the anesthetized time of the sea bass are recorded; In step (4) of recovery, after the sea bass enters the deep anesthesia stage, the sea bass is placed in an aquarium containing clean water to recover. If the sea bass does not roll over, swims normally and is active, it is determined to have recovered normally, and the recovery time is recorded.

2. 2. The method according to claim 1, wherein the concentration of the camphor essential oil is 150 mg / L.

3. The emulsification step of the camphor essential oil includes the following steps 1 and 2: In step 1, 10 ml of camphor oil, 200 ml of 50% ethanol, and 1 ml of polysorbate-80 are mixed in a volume ratio of 10:200:1, and then slowly added dropwise to 500 mL of water to form a first mixed solution; 2. The method of claim 1, wherein in step 2, the first mixture is evaporated and concentrated to 100 ml after stirring with a magnetic stirrer.

4. 4. The method according to claim 3, wherein the temperature of the evaporation is 20-40°C.

5. The method according to claim 4, wherein the emulsification temperature of the camphor essential oil is 25°C.

6. 3. The method according to claim 2, wherein the water temperature in the aquarium is 22±1°C, the salinity is 16‰, the dissolved oxygen is 6 mg / L, and the pH is 7.5.

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