Method for anesthetizing grass carp with CO2 assisted by ethanol and application

Abstract

The invention relates to the technical field of fish anesthesia treatment, in particular to a method for anesthetizing grass carps through CO2 assisted by ethyl alcohol and application. The method comprises the steps that grass carps are placed in anesthetic liquid to be anesthetized, the anesthetized grass carps are placed in clear water to be resuscitated, dissolved oxygen of the clear water used in the resuscitation process is larger than 8.5 mg / L, the temperature of the clear water is 20-25 DEG C, oxygen is continuously conveyed into the clear water in the resuscitation process, the anesthetic liquid is 2%-4% ethanol aqueous solution with saturated CO2, and the concentration of the ethanol aqueous solution is 1%-2%. The temperature of the anesthesia liquid is 8-24 DEG C, and the anesthesia time does not exceed 4 min; the variation amplitude of biochemical and antioxidant indexes of the grass carp anesthetized by the method disclosed by the invention is effectively weakened, and the shortening of the time of the grass carp in the anesthetic solution has a positive effect on reducing the stress of the grass carp.

Description

Technical Field

[0001] The invention relates to the technical field of fish anesthesia treatment, and in particular to a method and application of an ethanol-assisted CO2 anesthesia method for grass carp. Background Art

[0002] Grass carp, a stress-sensitive, common freshwater fish, exhibit various behaviors when stressed, such as jumping out of the water and swimming in circles. During grass carp transport and harvesting, hypothermia, anesthetics, and CO2 are often used to induce a deep dormancy state, thereby reducing the fish's stress response. Commonly used anesthetics in the aquatic market include MS-222, eugenol, plant extracts, 2-phenoxyethanol, and benzocaine. Studies have shown that the addition of synthetic chemicals can leave residues in the body, leading many countries to stipulate that aquatic products must undergo a withdrawal period before entering the market. For example, the US Food and Drug Administration (FDA) requires a 21-day withdrawal period for MS-222, the only fish anesthetic, and it is considered carcinogenic. Summary of the Invention

[0003] In view of the above-mentioned deficiencies in the prior art, the present invention aims to provide a method and application of ethanol-assisted CO2 anesthesia of grass carp, so as to reduce the time and side effects of grass carp anesthesia.

[0004] In order to solve the above problems, the present invention adopts the following technical solutions:

[0005] In a first aspect, the present invention provides a method for ethanol-assisted CO2 anesthesia of grass carp, comprising: placing the grass carp in an anesthetic solution for anesthesia; wherein the anesthetic solution is a 2%-4% ethanol aqueous solution with saturated CO2, the temperature of the anesthetic solution is 8-24°C, and the anesthesia time does not exceed 4 minutes.

[0006] Furthermore, the method includes placing the anesthetized grass carp in clean water for resuscitation, wherein the dissolved oxygen in the clean water used during the resuscitation process is greater than 8.5 mg / L, the temperature of the clean water is 20-25°C, and oxygen is continuously supplied to the clean water during the resuscitation process.

[0007] Furthermore, the temperature of the anesthetic solution is 12° C., and the concentration of the ethanol aqueous solution is 2%.

[0008] Furthermore, the volume ratio of the mass of the grass carp to the anesthetic solution is 1:3.

[0009] Furthermore, before the grass carp is placed in the anesthetic solution for anesthesia, the water temperature of the water environment in which the grass carp is located is lowered to the temperature of the anesthetic solution at a rate of 3°C / h.

[0010] In a second aspect, the present invention provides the use of ethanol and CO2 in preparing grass carp anesthetic.

[0011] Furthermore, ethanol-assisted CO2 anesthesia of grass carp (compared to saturated CO2 anesthesia) promoted the increase of AKP content in the fish during the recovery phase.

[0012] Furthermore, ethanol-assisted CO2 anesthesia of grass carp (compared to saturated CO2 anesthesia) shortens the anesthesia time of grass carp by reducing the MDA content in the fish during the anesthesia stage, and reduces the oxidative stress response of the fish by promoting the decrease rate of SOD and CAT enzyme activities during the recovery stage.

[0013] Furthermore, ethanol-assisted CO2 anesthesia and resuscitation of grass carp (compared to saturated CO2 anesthesia) reduced the amount of nuclear lysis, the number of red blood cell aggregation areas, and the number of vacuolar areas in the fish liver tissue;

[0014] Furthermore, after grass carp was anesthetized with ethanol-assisted CO2 and resuscitated (compared with saturated CO2 anesthesia), the decrease in the content of umami and sweet amino acids was reduced, and the decrease in the content of bitter amino acids was promoted.

[0015] The beneficial effects of the present invention are as follows: the present invention has a better effect on anesthesia of grass carp using auxiliary CO2 with a concentration of 2% ethanol at 12°C; the anesthesia time of the low-concentration ethanol-assisted group is significantly shorter than that of the saturated CO2 anesthesia; the change amplitudes of its biochemical and antioxidant indicators are weaker than those of the saturated CO2 anesthesia group; shortening the time the grass carp spends in the anesthetic solution has a positive effect on reducing the stress of the grass carp; and a longer immersion in the saturated CO2 anesthetic solution has a significantly stronger effect on the stress level of the grass carp than appropriately adding ethanol to shorten the stress level of the immersion in the anesthetic solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing changes in AST of grass carp under different anesthesia and resuscitation times in an embodiment of the present invention.

[0017] Figure 2 This is a graph showing changes in ALT of grass carp under different anesthesia and recovery times in an embodiment of the present invention.

[0018] Figure 3 This is a graph showing changes in AKP of grass carp under different anesthesia and resuscitation times in an embodiment of the present invention.

[0019] Figure 4 This is a graph showing changes in COR of grass carp under different anesthesia and resuscitation times in an embodiment of the present invention.

[0020] Figure 5 This is a graph showing changes in GLU of grass carp under different anesthesia and resuscitation times in an embodiment of the present invention.

[0021] Figure 6 This is a graph showing changes in SOD of grass carp under different anesthesia and recovery times in an embodiment of the present invention.

[0022] Figure 7This is a graph showing changes in CAT of grass carp under different anesthesia and resuscitation times in an embodiment of the present invention.

[0023] Figure 8 This is a graph showing changes in GSH-Px in grass carp under different anesthesia and resuscitation times in an embodiment of the present invention.

[0024] Figure 9 This is a graph showing changes in MDA of grass carp under different anesthesia and resuscitation times in an embodiment of the present invention.

[0025] Figure 10 This is a graph showing changes in liver glycogen in grass carp under different anesthesia and recovery times in an embodiment of the present invention.

[0026] Figure 11 This is a graph showing changes in grass carp muscle glycogen under different anesthesia and recovery times in an embodiment of the present invention.

[0027] Figure 12 This is a graph showing changes in liver lactic acid in grass carp under different anesthesia and recovery times in an embodiment of the present invention.

[0028] Figure 13 This is a graph showing changes in grass carp muscle lactate under different anesthesia and recovery times in an embodiment of the present invention.

[0029] Figure 14 This is a diagram showing the texture changes of grass carp under anesthesia and different recovery times in an embodiment of the present invention.

[0030] Figure 15 Graph showing the effect of hypoxia stress on the liver tissue structure of grass carp in an embodiment of the present invention. DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below with reference to specific embodiments.

[0032] It should be noted that these embodiments are only used to illustrate the present invention, rather than to limit the present invention. Simple improvements to the method based on the concept of the present invention fall within the scope of protection claimed by the present invention.

[0033] Anesthesia and resuscitation methods

[0034] Grass carp were purchased from an aquaculture farm, weighing 1200 (±100) g and 43 (±2) cm in length. They had no surface injuries and were transported to the laboratory in aerated water. The fish were placed in pre-aerated water (25°C) for 2 h to relieve stress, with continuous oxygen supply.

[0035] Anesthetic solution preparation

[0036] The microporous aeration tube was coiled and bent at the bottom of a white anesthesia barrel with a diameter of 38 cm and a height of 54 cm, fixed with a suction cup, and the pressure reducing valve was adjusted to 0.4 MPa to fill CO2 gas into the water to form an anesthetic solution. The CO2 gas was dispersed in the water through the pressure-resistant airway tube and the microporous aeration tube to form fine bubbles. The dissolved oxygen meter and pH sensor of the Multi3630 IDS portable multi-parameter water quality analyzer were used for detection. The anesthetic solution was considered saturated if the dissolved oxygen meter showed no dissolved oxygen, and the pH was 4.7 (±0.2). The pressure of the pressure reducing valve was adjusted to 0.2 MPa to maintain the saturation of the CO2 anesthetic solution.

[0037] Preparation of low-concentration ethanol-assisted CO2 anesthetic solution: After forming a saturated CO2 anesthetic solution, calculate the required volume of edible alcohol according to different concentrations to form a mixed anesthetic solution.

[0038] Effects of different temperatures on CO2 anesthesia

[0039] Prepare five water tanks, using a chiller to control and maintain the saturated anesthetic solution at 24, 20, 16, 12, and 8°C, respectively. The chiller lowers the water temperature to the same level as the anesthetic solution at a rate of 3°C / h. Grass carp are then reeled in with a dip net and placed in the saturated anesthetic solution. The fish-to-water ratio (kg:L) is 1:3. Five fish are reeled in at each temperature, repeated three times. The anesthetic state (entry and recovery time) of the grass carp is observed and recorded, and the anesthetic efficacy is evaluated. After anesthesia, the grass carp are reeled in clean water (room temperature) for resuscitation. The dissolved oxygen in the resuscitation tank is greater than 8.5 mg / L, and the water temperature is 20-25°C. Oxygen is continuously supplied during the resuscitation process. The recovery of the grass carp and the survival rate after different resuscitation times are observed to evaluate the resuscitation efficacy.

[0040] Effects of different ethanol concentrations on CO2 anesthesia

[0041] By observing and recording the anesthetic effect, anesthesia and recovery time of grass carp at different temperatures, edible alcohol was added to 15L of saturated anesthetic solution at 12°C to form a low-concentration saturated CO2 anesthetic solution with a gradient of 1%, 2%, 3%, 4%, and 5% (volume ratio).

[0042] Anesthesia stage determination

[0043] Combined with the anesthesia status of grass carp in the experiment, the different behavioral characteristics of the fish during the anesthetic recovery process are analyzed as shown in Table 1.

[0044] Table 1 Behavioral characteristics of grass carp anesthesia and resuscitation stages

[0045]

[0046]

[0047] Experimental sampling

[0048] The grass carp whose water temperature was lowered to 12℃ at 3℃ / h was used as the control group, the anesthesia group was anesthetized with saturated CO2 and the anesthesia group was assisted by CO2 + 2% ethanol, and the resuscitation group was anesthetized with two different methods for 0h, 6h, and 24h. Blood, muscle and liver were collected.

[0049] Muscle texture index determination

[0050] Fish dorsal muscle was collected and trimmed into a regular 4 × 3 × 1 cm shape. A texture analyzer with a flat-bottomed cylindrical probe (P / 44) was used for the measurement. The test procedure was configured as follows: two cycles of compression in the texture multi-faceted profiling mode, a test speed of 1 mm / s, a trigger load of 5.0 g, and a compression depth of 5 mm. Under these conditions, the firmness, adhesiveness, cohesiveness, elasticity, chewiness, and stickiness of fish meat were measured under different anesthesia and resuscitation conditions. Six fish fillets were collected for each group, and four points were tested on each fillet.

[0051] The effects of temperature and ethanol concentration on the anesthetic effect of grass carp are shown in Table 2.

[0052] Table 2 CO2 anesthesia effect of grass carp at different temperatures

[0053]

[0054] Note: Items marked with different letters in the same column indicate significant differences between the groups (P<0.05), and items marked with the same letter indicate no significant differences between the groups (P>0.05).

[0055] The effect of temperature on CO2 anesthesia was investigated by examining the induction time of grass carp in saturated CO2 anesthetic solution at different temperatures and the recovery time in clear water. Table 2 shows that as the anesthetic solution temperature increased from 8°C to 24°C, the induction time of the fish gradually increased, with the longest anesthesia time exceeding 4 minutes at 24°C. There were no significant differences in induction and recovery time between 12°C, 16°C, and 20°C. According to the standard for fishery anesthetics: induction within 3 minutes and recovery within 5 minutes, grass carp induction and recovery time were the shortest at 12°C. The survival rate of grass carp after 48 hours of recovery was 100%. CO2 is recognized as a green, residue-free anesthetic for fishery use. However, prolonged exposure to saturated anesthetic solution causes hypoxia, decreased respiratory rate, and respiratory failure, leading to death. Therefore, temperature and duration of anesthesia are critical control points in CO2 anesthesia. In this experiment, the average induction and recovery time at 12°C was shorter than in other groups. This suggests that hypothermia and CO2 anesthesia have a synergistic effect during this anesthesia process, accelerating the induction and induction of grass carp.

[0056] Table 3 Anesthetic effect of low concentration ethanol assisted CO2 anesthesia on grass carp

[0057] Ethanol concentration / % Time to enter numbness / s Recovery time / s 24h survival rate / % 48h survival rate / % 0 <![CDATA[132±17 a ]]> <![CDATA[138±16 b ]]> 100 100 1 <![CDATA[111±11 a ]]> <![CDATA[145±16 ab ]]> 100 100 2 <![CDATA[96±6 b ]]> <![CDATA[107±7 c ]]> 100 100 3 <![CDATA[93±11 b ]]> <![CDATA[178±25 ab ]]> 100 100 4 <![CDATA[92±5 b ]]> <![CDATA[192±31 a ]]> 100 100 5 <![CDATA[60±9 c ]]> <![CDATA[190±17 a ]]> 100 100

[0058] As shown in Table 3, based on the induction and recovery times of grass carp at 12°C, different volumes of edible alcohol were added to the anesthetic solution to create ethanol-CO2 composite anesthetic solutions with varying concentrations, ranging from 1% to 5%. The experiment showed that the induction time of grass carp was inversely proportional to the ethanol concentration of the anesthetic solution and directly proportional to the recovery time. Comparing the induction and recovery times at different ethanol concentrations, no significant differences were observed in the induction time within the 2% to 4% ethanol concentration range. The recovery time of grass carp at 2% concentration was significantly lower than that at 3% and 4%. Therefore, low-concentration ethanol-assisted CO2 anesthesia can shorten induction and recovery times.

[0059] Changes of serum biochemical parameters in grass carp under anesthesia and different recovery times

[0060] Depend on Figure 1-5 It is known that the indicators showed a trend of increasing first and then decreasing compared with the control group. Comparing the two different anesthesia methods, it was found that the biochemical indexes in the 12℃ saturated CO2 anesthesia group were significantly higher than those in the mixed anesthesia solution using 2% ethanol concentration to assist CO2. AST (aspartate aminotransferase) ( Figure 1 )、ALT(alanine aminotransferase)( Figure 2 ), AKP (alkaline phosphatase) ( Figure 3 )、COR(cortisol)( Figure 4 ) and GLU (blood sugar) Figure 5 ) levels were 208.6 (±3.2) U / L, 28.7 (±0.8) U / L, 44.8 (±1.3) U / L, 2245.1 (±44.2) ng / L, and 13.6 (±0.6) mmol / L, respectively. These were 2.3, 4, 4, 2.9, and 6.2 times those of the control group, and significantly higher than those in the mixed anesthetic solution group. CO2 anesthetic solution was the primary anesthetic agent in this experiment. The levels of these indicators gradually decreased with prolonged resuscitation. The differences in serum biochemical parameters between fish resuscitated 24 hours after saturated CO2 anesthesia and the control group were smaller than those in the mixed anesthetic solution group. COR levels significantly increased and then decreased after saturated CO2 anesthesia and at 0 and 6 hours of resuscitation, whereas no significant decrease was observed in the mixed anesthetic solution group. GLU and AKP levels were significantly higher in the 2% ethanol-assisted anesthesia group at 6 and 24 hours after resuscitation than in the saturated CO2-assisted anesthesia group, indicating that low-concentration ethanol-assisted anesthesia slows the recovery of various tissues and organs in grass carp. During anesthesia, CO2 anesthetic fluid acts as a stressor, causing short-term hypoxia due to insufficient ventilation. CO2 anesthesia activates the HPI axis to release corticosteroids, leading to elevated serum cortisol levels. This, in turn, stimulates the fish's metabolic pathways, resulting in elevated serum GLU levels. As the fish gradually recovers, COR levels gradually decrease, and GLU levels also decrease. AKP is a marker of fish nonspecific immunity, and its high level indicates that low-concentration ethanol-assisted anesthesia significantly affects the nonspecific immune capacity of grass carp.

[0061] Changes in antioxidant indices of grass carp under anesthesia and different recovery times

[0062] Depend on Figure 6-9 SOD (superoxide dismutase) Figure 6 ), CAT (catalase) ( Figure 7 )、GSH-Px(glutathione peroxidase) Figure 8 ) enzyme activity and MDA (propylene glycol) ( Figure 9 ) content first increased and then decreased during the anesthesia and resuscitation process. Carbon dioxide anesthesia produces strong hypoxic stress, and enters A3 within 2 minutes. CAT and GSH-Px enzyme activities were significantly higher during the resuscitation process than those under saturated CO2 anesthesia, indicating that the addition of ethanol to the anesthetic solution caused greater short-term oxidative stress in the fish body. The decrease in the activities of various antioxidant enzymes and MDA content with prolonged resuscitation time indicated that the stress in grass carp gradually weakened. The MDA content of grass carp resuscitated for 24 hours after saturated CO2 anesthesia was significantly higher than that of the mixed anesthetic solution group, indicating that anesthetizing grass carp in mixed anesthetic solution significantly shortened the time to anesthesia. Although a strong stress response was produced, compared with the oxidative stress damage caused by long-term hypoxia, the oxidative damage to the body caused by this stress response after mixed anesthesia recovered faster with prolonged resuscitation time. In addition, the differences in SOD and CAT enzyme activities during the resuscitation of grass carp under two different anesthesia methods show that the enzyme activity and lipid peroxidation end product content decreased faster after resuscitation with mixed anesthetic solution. Therefore, it is believed that the mixed anesthetic solution containing 2% ethanol significantly shortens the anesthesia time and helps to reduce the oxidative stress of the fish. After 24 hours of resuscitation, the CAT enzyme activity of grass carp dropped to a level that was not significantly different from that of the control group, and the SOD and GSH-Px enzyme activities also decreased, but were significantly higher than those of the control group, indicating that there was still a stress response in the body at this time.

[0063] Changes in metabolic indicators of grass carp under anesthesia and different recovery times

[0064] Figure 10-13 The changes of glycogen and lactic acid content in the liver and muscle tissue of grass carp after anesthesia and recovery for different periods of time are shown in Figure 2. Figure 10-11It is known that the GLY (glycine) content in grass carp liver and muscle tissue shows a significant downward trend. When the fish undergoes a stress response, liver glycogen breaks down into glucose to provide energy to maintain balance. In addition, COR release leads to gluconeogenesis, which significantly increases GLU content, generating heat to resist hypothermia and hypoxia. Over time, glycogen content decreases. GLY is present in both the liver and muscle. In the liver, glycogen maintains blood sugar stability; in the muscle, glycogen provides the energy required for muscle movement. At 12°C, the GLY content in the liver and muscle of the control group was 6.7 mg / g and 1.9 mg / g, respectively. After CO2 anesthesia, the GLY content in the liver and muscle decreased to 5.7 mg / g. In grass carp anesthetized with CO2, the GLY content in liver and muscle decreased to 1.6 mg / g and 0.7 mg / g, respectively, 24 hours after resuscitation, representing decreases of 76.1% and 63.2%, respectively, compared to the control group. After resuscitation with a 2% ethanol-containing CO2 mixed anesthetic solution, the GLY content in liver and muscle decreased to 2.6 mg / g and 0.8 mg / g, respectively, representing decreases of 61.2% and 57.9%, respectively, compared to the control group. Therefore, a comparison of muscle and liver GLY content 24 hours after resuscitation with saturated CO2 anesthesia revealed a greater decrease in liver GLY content in grass carp anesthetized with the two different anesthesia methods. Furthermore, a comparison of the rate of decrease in GLY content in tissues revealed a greater depletion of liver glycogen after 24 hours of resuscitation under both anesthesia methods, indicating that the decomposition of liver GLY during resuscitation of anesthetized grass carp provides the body with energy to cope with stress.

[0065] Figure 12-13 This study shows the metabolism of lactic acid (LD) in the liver and muscle of grass carp after different anesthesia methods and resuscitation times. LD is an important indicator of anaerobic metabolic intensity. In this metabolic pathway, LD and ATP are produced for energy. LD produced in the liver is primarily produced through the breakdown of glycogen. Glycogen, a polysaccharide, is broken down into glucose by the liver, which is then metabolized to LD. In the liver, LD is primarily involved in blood glucose regulation and energy metabolism. In muscle, LD is produced by muscle cells through anaerobic glycolysis. When muscle lactate reaches a threshold, it leads to adverse physiological reactions such as muscle soreness. During anesthesia, LD content in grass carp gradually decreases with prolonged resuscitation. The highest LD content was observed in grass carp muscle in the anesthesia group, reaching 12.8 mol / g and 10.6 mol / g, respectively, under the two different anesthesia methods. LD content in the liver and muscle of grass carp anesthetized with saturated CO2 was significantly higher than that in the mixed anesthetic group. Liver LD content reached its maximum at 0 hours after resuscitation, reaching 26.3 mol / g and 24.7 mol / g, respectively, under the two different anesthesia methods. This content then gradually decreased.

[0066] Texture changes of grass carp under anesthesia and different recovery times

[0067] Texture is often used to evaluate the nutritional value of food. Using a texture analyzer to simulate the human oral process of chewing food, various evaluation indicators are quantified into experimental data to reflect the various states of food. The quality of fish meat is directly related to its texture properties. Changes in muscle structure lead to tissue changes, which can deteriorate the quality of fish meat.

[0068] The hardness index of muscle reflects its ability to maintain its original shape when subjected to deformation force; the ability of fish meat to quickly recover deformation under pressure can be reflected by its resilience; the elasticity of muscle can reflect the strength of the binding force between muscle protein and water molecules; cohesion and viscosity comprehensively reflect the strength of the binding force between muscle cells; chewiness refers to the fish meat's continued resistance to chewing, which can simulate the chewing situation in the human mouth. These indicators can be used to evaluate and describe the texture and taste characteristics of muscle. Figure 14(Group A: CO2 anesthesia; Group B: 2% ethanol + CO2 anesthesia; Group C: control; Group D: CO2 anesthesia resuscitation for 6 hours; Group E: CO2 anesthesia resuscitation for 24 hours; Group F: 2% ethanol + CO2 anesthesia resuscitation for 6 hours; Group G: 2% ethanol + CO2 anesthesia resuscitation for 24 hours; Group H: CO2 anesthesia resuscitation for 0 hours; Group I: 2% ethanol + CO2 anesthesia resuscitation for 0 hours) Changes in various textural indicators of grass carp after different anesthesia methods and resuscitation times were observed. The results showed that compared to the low-temperature control group, the hardness and viscosity of grass carp decreased after anesthesia. Subsequently, the hardness increased with the extension of the resuscitation time. After 24 hours of resuscitation, the hardness of grass carp was slightly higher than that of the control group, while the viscosity was still lower than that of the control group after 24 hours of resuscitation. The cohesiveness of grass carp in the anesthesia group was higher than that in the control group. With increasing resuscitation time, cohesion decreased slightly after 24 hours. Elasticity remained unchanged in the anesthesia group and at 0, 6, and 24 hours after resuscitation, but increased slightly after 24 hours. Neither adhesiveness nor chewiness changed significantly in the anesthesia group, but increased slightly after 24 hours. Improving firmness, elasticity, chewiness, and cohesiveness can improve taste and muscle quality. In this study, anesthesia with low-temperature saturated CO₂ and 2% ethanol-CO₂ saturated anesthetic solution improved overall muscle quality. However, muscle firmness and stickiness decreased in the anesthesia group compared to the control group. This is believed to be due to the stress caused by the sudden onset of hypothermia and hypoxia, which rapidly degrades glycogen to combat stress, leading to a decrease in muscle water content and, consequently, firmness. Following anesthesia and varying lengths of resuscitation, stress perception diminishes or hypoxia resolves, leading to a significant accumulation of lactic acid in the muscle, resulting in increased firmness. The changing trends of the textural properties of grass carp after anesthesia and resuscitation for different lengths with two different anesthesia methods were basically the same. Among them, the viscosity of the 2% ethanol concentration CO2 mixed anesthetic solution after 24 hours of resuscitation was lower than that after 24 hours of resuscitation with CO2 anesthesia. It is believed that low-concentration ethanol penetrates into the fish body and dissolves certain components in the fish meat, such as protein and muscle fibers, due to its dissolving effect and moisture regulation response. This may lead to changes in the viscosity of the fish meat, and adjust the moisture content in the fish meat by adsorbing or releasing water, and changes in moisture content affect the viscosity of the fish meat.

[0069] Tissue section structural analysis

[0070] Preparation of tissue sections: An appropriate volume of liver tissue was fixed with 4% paraformaldehyde (volume ratio 1:15) for one day, then dehydrated with different concentrations of ethanol (volume fractions of 50%, 70%, 80%, 95% and 100%), transparentized with xylene and embedded in wax, cut into thin sections (4 μm) with a microtome, and stained with hematoxylin-eosin solution. The above steps were repeated again. After the sections were air-dried, they were mounted with neutral resin and examined under a microscope for image acquisition and analysis.

[0071] Effects of different anesthesia methods on muscle free amino acids

[0072] Grind the fish muscle, weigh 1.5 g of muscle into a 10 mL centrifuge tube, add 5.0 mL of 0.01 M hydrochloric acid (or pure water), mix well, boil in a boiling water bath for 30 minutes, and centrifuge at 10,000 rpm for 10 minutes; take the supernatant, add 4 mL of 0.01 M hydrochloric acid to precipitate, resuspend and sonicate for 5 minutes, centrifuge, combine the supernatants, make up to 10 mL, and pass through a membrane for determination.

[0073] Effects of different anesthesia methods on the liver tissue structure of grass carp

[0074] The liver is the largest digestive gland in fish and has multiple functions. The bile secreted by the liver can promote the decomposition and absorption of fat, and participates in the synthesis, storage, metabolism, conversion and decomposition of various substances. Liver cells are rich in substances such as glycogen, lipid droplets and pigments, and their content varies with the physiological state of the body. The hepatic epithelium of unstressed fish is composed of a single layer of Solomon-shaped squamous epithelial cells and connective tissue. The cytoplasm of the hepatocytes is uniform and regular, with rich cytoplasmic contents. The hepatocytes are oval, with a clear nucleus in the center, and are arranged radially with the central vein as the center. The intercellular spaces are distributed with hepatic sinusoids and branches of the central vein, and the boundaries between the liver plates are clear. By Figure 15 (A and B are 12°C hypothermic hibernation groups, 200× and 400×; C and D are saturated CO2 hypoxia stress groups, 200× and 400×; E and F are 2% ethanol-assisted CO2 hypoxia stress groups, 200× and 400×; (CV: central vein; LC: hepatocyte; N: nucleus; VS: vacuoles; BC: erythrocyte; HS: hepatic sinusoids; HP: hepatic plates)) HE-stained liver sections of grass carp from different groups show that red blood cells aggregate in the 12°C hypothermic control group, indicating stress. Vacuoles appear in some areas, and the nuclei of a few hepatocytes are close to the cell edges, with clear hepatic plates. Hypoxia stress induced by the two different anesthesia methods can be observed in liver tissue sections, with large numbers of red blood cells aggregated, nuclei dissolved, cells fused, and a clear trend of hepatocyte atrophy and shrinkage. A comparative analysis of liver tissue sections after saturated CO2 stress and low-concentration ethanol-assisted CO2 stress showed that under hypoxic stress caused by saturated CO2, cell nuclei dissolved more, there were more areas of red blood cell aggregation, and more vacuolar areas appeared than under hypoxic stress caused by low-concentration ethanol-assisted CO2. In addition, it was observed that after hypoxic stress, the nuclei of hepatocytes were mostly at the edges of the cells rather than in the center, their liver plates dissolved, the boundaries between cells were unclear, the hepatic sinusoids expanded, and the volume of hepatocytes decreased.

[0075] Effects of different anesthesia methods on the free amino acid content in grass carp muscle

[0076] Improving fish muscle quality helps ensure food quality, as muscle is the primary edible part of fish. Aquatic organisms typically adapt to environmental changes by regulating their osmotic pressure. This can be achieved by adjusting the content of organic matter, such as free amino acids, to balance the osmotic pressure between tissues and hemolymph. Amino acids are categorized as essential and non-essential. The main umami and sweet amino acids include aspartic acid and glutamic acid, while some bitter amino acids include histidine and tyrosine. The content and ratio of these amino acids in food can influence the taste characteristics of the food. As shown in Table 4, free amino acid analysis in grass carp meat revealed high levels of His, Gly, and Val. After 24 hours of resuscitation using both anesthesia methods, the content of umami and sweet amino acids decreased significantly. The decrease in umami and sweet amino acids using saturated CO2 anesthesia was approximately 21.1% compared to the hypothermia control group, a much greater decrease than using 2% ethanol-assisted CO2 anesthesia. Overall, the content of bitter amino acids in both anesthesia groups was significantly reduced compared to the hypothermia group. The content of bitter amino acids in grass carp decreased by 22.9% and 36.6%, respectively, after 24 hours of resuscitation using the two different anesthesia methods. There was no significant change in the essential amino acid content of grass carp after anesthesia with CO2 and compound anesthetic solution.

[0077] Table 4 Changes in free amino acid content in grass carp meat (wet basis) under different anesthesia methods

[0078]

[0079]

[0080] The present invention shows that there is no significant difference in the anesthesia and recovery time of grass carp when anesthetized with saturated CO2 at 12-16°C. The average anesthesia and recovery time required for anesthesia at 12°C is shorter than that at other temperatures. At 12°C, 2% low-concentration ethanol assisted CO2 anesthesia will shorten the anesthesia and recovery time. The anesthesia time of grass carp gradually shortens with increasing ethanol concentration. Grass carp anesthesia with 2% ethanol at 12°C has a better effect. Comparison of various indicators after different anesthesia methods and different resuscitation times revealed that grass carp gradually recovered with prolonged resuscitation time, which was significantly different from the low-temperature control group, indicating that hypoxia caused stress damage to grass carp. However, after short-term hypoxia stress, various biochemical indicators could slowly recover. The anesthesia time of the low-concentration ethanol-assisted group was significantly shorter than that of saturated CO2 anesthesia, and the changes in its biochemical and antioxidant indicators were weaker than those of the saturated CO2 anesthesia group. Shortening the time grass carp was in the anesthetic solution had a positive effect on reducing the stress of grass carp. Longer immersion in saturated CO2 anesthetic solution caused a significantly greater stress level for grass carp than appropriately adding ethanol to shorten the immersion in the anesthetic solution. HE staining of grass carp liver tissue sections comparing the effects of hypoxia stress on the control group, low-temperature group, and anesthesia groups with different anesthesia methods revealed that both hypoxia and hypoxia stress caused a significant stress response in grass carp. Red blood cell aggregation, blurred liver plates, and hepatocyte nuclear displacement were all manifestations of significant stress in the fish. Analysis of grass carp muscle texture at different stages of post-anesthesia resuscitation revealed no significant differences between the two anesthesia methods. However, with prolonged resuscitation, the hardness, elasticity, chewiness, and adhesiveness of the grass carp increased, indicating improved overall muscle quality after anesthesia. Analysis of free amino acids in grass carp muscle (wet basis) 24 hours after resuscitation using different anesthesia methods revealed no significant changes in essential amino acid content after anesthesia.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described with reference to the preferred embodiments of the present invention, it should be understood by those skilled in the art that various changes can be made in form and details without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A method for ethanol-assisted CO2 anesthesia of grass carp, characterized in that: include: The grass carp is placed in an anesthetic solution for anesthesia; wherein the anesthetic solution is a 2%-4% ethanol aqueous solution with saturated CO2, the temperature of the anesthetic solution is 8-24°C, and the anesthesia time does not exceed 4 minutes.

2. The method for ethanol-assisted CO2 anesthesia of grass carp according to claim 1, wherein: The method includes placing the anesthetized grass carp in clean water for resuscitation. The dissolved oxygen in the clean water used during the resuscitation process is greater than 8.5 mg / L, the temperature of the clean water is 20-25°C, and oxygen is continuously supplied to the clean water during the resuscitation process.

3. The method for ethanol-assisted CO2 anesthesia of grass carp according to claim 1, characterized in that: The temperature of the anesthetic solution is 12° C., and the concentration of the ethanol aqueous solution is 2%.

4. The method for ethanol-assisted CO2 anesthesia of grass carp according to claim 1, wherein: The volume ratio of the grass carp mass to the anesthetic solution is 1:

3.

5. The method for ethanol-assisted CO2 anesthesia of grass carp according to claim 1, characterized in that: Before placing the grass carp in the anesthetic solution for anesthesia, the water temperature of the water environment where the grass carp is located is lowered to the temperature of the anesthetic solution at a rate of 3°C / h.

6. Application of ethanol and CO2 in the preparation of grass carp anesthetics.

7. The use according to claim 6, characterized in that After the grass carp was anesthetized with ethanol-assisted CO2, the content of AKP in the fish body was promoted during the recovery stage.

8. The use according to claim 6, characterized in that Ethanol-assisted CO2 anesthesia of grass carp was used to shorten the anesthesia time of grass carp by reducing the MDA content in the fish during the anesthesia stage, and to reduce the oxidative stress response of the fish by promoting the decreasing rate of SOD and CAT enzyme activities during the recovery stage.

9. The use according to claim 6, characterized in that After ethanol-assisted CO2 anesthesia and resuscitation of grass carp, the amount of cell nuclear lysis, the number of red blood cell aggregation areas and the number of vacuolar areas in the fish liver tissue were reduced.

10. The use according to claim 6, characterized in that After ethanol-assisted CO2 anesthesia and resuscitation of grass carp, the decrease in the content of umami and sweet amino acids was reduced, and the decrease in the content of bitter amino acids was promoted.

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