Effect of Vanillin on the Anaesthesia of Crucian Carp: Effects on Physiological and Biochemical Indices, Pathology, and Volatile Aroma Components

The anaesthetic effect of vanillin on crucian carp was investigated using different concentrations of vanillin, with a nonvanillin control. The effective concentration range of vanillin anaesthesia was determined from the behavioural characteristics of crucian carp during the anaesthesia onset and recovery phases. Physiological and biochemical indices, and the electronic nose response to the fish muscle, were measured over the range of effectiveanaestheticc concentrations. An increased concentration of vanillin shortened the time taken to achieve deep anaesthesia but increased the recovery time. The levels of white blood cells, red blood cells, haemoglobinn, platelets, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, phosphorus, potassium, magnesium, total protein, and serum albumin were lower than the control in the vanillin treatment group. Triglycerides and total cholesterol were not significantly affected. Histology showed no effect of vanillin on the liver, except at 1.00 g/L vanillin. Vanillin resulted in a nondose-responsive effect on the gill tissue, increasing the width and spacing of the gill lamellae. E-Nose analysis of the carp-muscle flavour volatiles was able to distinguish between different vanillin treatment concentrations. GC-IMS identified 40 flavour compounds, including 8 aldehydes, 11 alcohols, 10 ketones, 2 esters, and 1 furan. Vanillin had aanaestheticic effect on crucian carp and these findings provide a theoretical basis for improving the transport and experimental manipulation of crucian carp.


Introduction
Freshwater fish are popular with consumers for their high-protein and low-fat contents, and there is increasing consumer demand for high-quality fish. During the production and transportation of fish, trauma and stress often occur, affecting their quality and survival rate. Fish anaesthetics are widely used, as they have a sedative effect during transport, and effectively reduce fish mortality. Commonly used fish anaesthetics in China are ethyl mamino benzoate (MS-222), eugenol, and 2-phenoxyethanol, however, all three anaesthetics have undesirable side effects. MS-222 solution is weakly acidic, resulting in the plasmacortisol content increasing after the fish recover from deep anaesthesia; exposure to direct sunlight may produce toxicity to fish when using MS-222 [1]. Eugenol is volatile and its effect diminishes during anaesthesia [2,3]; 2-Phenoxyethanol is hazardous to fishery When the anaesthetic concentration is low, the fish cannot enter anaesthesia quickly; when the anaesthetic concentration is high, the fish enter anaesthesia too quickly and the behavioural characteristics of the anaesthesia are difficult to distinguish. To observe stable and distinct anaesthetic staging characteristics, an appropriate anaesthetic concentration is required. Based on the results from Section 2.2.1, the behavioural characteristics of the carp during anaesthesia were observed in a 1.00 g/L vanillin solution. The anaesthesia (A 0 -A 4 ) and recovery (R 1 -R 4 ) periods were classified according to the criteria of Mirghaed et al. [21] and correlated with the behavioural characteristics of the different periods.

Effect of Vanillin Concentration on Blood and Serum Parameters of Crucian Carp
Three crucian carp were randomly placed in treatment (different vanillin concentrations) and control tanks, then each fish was tested for anaesthesia (each fish was tested only once). The fish were kept in a state of complete anaesthesia for 10 min, and then blood samples (5 mL) were drawn with a syringe from the tail vein. Part of the sample was mixed with an anticoagulant and used for the determination of the physiological parameters, red blood cells (RBC), white blood cells (WBC), haemoglobin (HGB), and platelets (PLT); the other part was centrifuged at 4 • C for 20 min and 4000 rpm. Then, the supernatant was stored at −80 • C until needed for the determination of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), inorganic phosphate (P i ), magnesium (Mg 2+ ), potassium (K + ), sodium (Na + ), glucose (GLU), total protein (TP), albumin (ALB), cholesterol (CHO), and triglycerides (TG).

H&E Staining and Histological Examination of Liver and Gill Tissues
The carp were put under complete anaesthesia with different concentrations of vanillin, maintained for 10 min and then gently removed and placed on a dissecting table to remove the liver and gill parts for H&E staining. All samples were cut into 5-6 µm thick slices. Then H&E was stained for microscopic observation. Dehydration and transparency were performed in ethanol and xylene, and then cover slipped. The stained sections were observed and photographed using a light microscope (Nikon, DS-Fi2) and spliced into a complete image using ImageJ software.

E-nose Analysis
The carp were put under complete anaesthesia with different concentrations of vanillin, maintained for 10 min, and then gently removed and placed on a dissecting table to remove the muscle for the electronic nose test. The E-Nose (FOX4000, Alpha MOS, Toulouse, France) sampling was performed as described previously [22], with some modifications. Muscle tissue (5.0 g) was homogenized, then sealed into 10 mL sample vials and analyzed each sample 4 times. The samples were analyzed as follows: carrier gas velocity, 150 mL/min; headspace generation temperature, 40 • C; injection volume, 2 mL; injection speed, 2 mL/s; headspace generation time, 600 s; data acquisition time, 120 s; delay time, 400 s. Radar charts and linear discriminant analysis (LDA) were used to analyse the data and remove outliers. The response characteristics of each E-Nose sensor are shown in Table 1.

Gas Chromatography-Ion Mobility Spectrometry (GC-IMS) Analysis of Flavour Volatile Compounds
The carp were put under complete anaesthesia with different concentrations of vanillin, maintained for 10 min and then gently removed and placed on a dissecting table to extract muscle parts for GC-IMS studies. The analysis was performed with a FlavourSpec flavour analyser (G.A.S., Dortmund, Germany). The control software has built-in NIST and IMS databases, which were used for analyte identification. A 4.0 g sample was placed in a 20.0 mL headspace vial and incubated for 15 min at 60 • C. Headspace sampling conditions were as follows: headspace temperature, 60 • C; incubation time, 15 min; heating mode, oscillation; headspace injection needle temperature, 85 • C; injection volume, 500 µL; nonsplit mode; carrier gas, N 2 (purity ≥ 99.999%); and wash time 0.50 min. Chromatography conditions were as follows: column temperature, 60 • C; run time, 15 min; carrier gas, N 2 (≥99.999%); flow rate: initially 5.0 mL/min, held for 10 min, and then linearly increased to 150 mL/min over 5 min. Detection conditions were as follows: drift tube length, 5 cm linear voltage in the tube, 400 V/cm; drift tube temperature, 40 • C; drift gas, N 2 , (≥99.999%); flow rate, 150 mL/min; and IMS detector temperature, 45 • C.  26.0 (SPSS Inc., Chicago, IL, USA) was used for data analysis. Data are expressed as the mean ± standard deviation, according to an analysis of variance (ANOVA) and Duncan's multiple-range test (p < 0.05). The figures were plotted using OriginPro 2021 (OriginLab, Northampton, MA, USA). GC-IMS data were collected and analyzed from different perspectives using the Laboratory Analytical Viewer (LAV) software and three plug-ins (Reporter, Gallery Plot, Dynamic PCA) as well as the GC-IMS Library Search qualitative software.

Determination of the Effective Vanillin Anaesthesia Concentration Range for Crucian Carp
As the concentration of vanillin increased, the time taken to achieve deep anaesthesia (stage A5; anaesthesia time) decreased and the time for complete recovery increased (Table 2, Figure 1); the anaesthesia time with 0.25 g/L vanillin was markedly longer than at other concentrations. The average anaesthesia time at 1.50 g/L was 50% of that at 0.50 g/L, whereas the average recovery time was 281% of that at 0.50 g/L. The recovery rate of carp was 100% at all six vanillin concentrations, indicating that, within the concentration range of 0.50-1.50 g/L, the vanillin bath was safe for the fish. As the vanillin concentration increased, the anaesthesia time decreased, in a dose-responsive, though nonlinear manner ( Table 2). Taking market application as the starting point, the short average anaesthesia time is the focus of market demand, and it can be seen that the anaesthesia efficiency is higher when the test concentration is 0.50 g/L~1.50 g/L. range of 0.50-1.50 g/L, the vanillin bath was safe for the fish. As the vanillin concentration increased, the anaesthesia time decreased, in a dose-responsive, though nonlinear manner (Table 2). Taking market application as the starting point, the short average anaesthesia time is the focus of market demand, and it can be seen that the anaesthesia efficiency is higher when the test concentration is 0.50 g/L~1.50 g/L.   The timing and behavioural characteristics at each stage of anaesthesia (A 0 -A 4 ) and recovery (R 1 -R 4 ) of crucian carp were determined using 1.00 g/L vanillin (Table 3). When a fish stopped swimming, completely lost the ability to respond, and the operculum opened weakly and slowly, it was defined as being at the A 3 stage of the anaesthesia period, and when the fish body was balanced and responsive, and the operculum opened normally, it was defined as being at the R 3 stage of recovery. Table 3. Behavioural characteristics of crucian carp during the successive stages of anaesthesia and recovery.

Stages Behavioural Characteristics Minute
A 0 stress period A stress response occurs, swimming is accelerated, and operculum opening and closing are accelerated 0.40 ± 0.06 The response to external stimuli is weakened, the ability to swim is weakened, the body is slightly out of balance, and the breathing rate is further increased 0.75 ± 0.12 The body rolls on its side, the ability to respond to external stimuli continues to weaken, swimming slowly, and the rate of operculum opening and closing decreases The control WBC count was 809.76 ± 21.51 × 10 9 /L; the vanillin treatment WBC count initially markedly decreased, then slightly increased, with the increased vanillin concentration (Figure 2A). The control RBC count was 0.17 ± 0.10 × 10 12 /L and markedly decreased at all vanillin treatment concentrations, with a minimum of 0.75 g/L ( Figure 2B). The control HGB content was 95.11 ± 4.59 g/L and moderately decreased, then slightly increased, with increased vanillin treatment concentration ( Figure 2C). The control blood PLT content was 27.22 ± 4.63 × 10 9 /L and markedly decreased at all vanillin treatment concentrations; the PLT content was not significantly different between the five vanillin concentrations ( Figure 2D).

Effect of Vanillin Concentration on Blood Serum Enzymes in Crucian Carp
The control serum ALT concentration was 97.73 ± 58.18 U/L; the vanillin treatment ALT concentration initially markedly decreased, then slightly, though not significantly, increased, with increased vanillin concentration, except for a marked increase at 1.50 g/L vanillin, to 111.77 ± 21.97 U/L ( Figure 3A). The control serum AST concentration was 440.4 ± 177.39 U/L; the vanillin treatment AST concentration markedly increased, though remained relatively constant, at all vanillin treatment concentrations ( Figure 3B). The control serum ALP concentration was 26.67 ± 12.66 U/L; the vanillin treatment ALT concentration initially markedly decreased, then gradually increased, with the increased vanillin concentration ( Figure 3C). The control serum LDH concentration was 2560.02 ± 7.23 U/L and moderately decreased at all vanillin treatment concentrations, with a minimum of 1.00 g/L ( Figure 3D).

Effect of Vanillin Concentration on Blood Serum Enzymes in Crucian Carp
The control serum ALT concentration was 97.73 ± 58.18 U/L; the vanillin treatment ALT concentration initially markedly decreased, then slightly, though not significantly, increased, with increased vanillin concentration, except for a marked increase at 1.50 g/L vanillin, to 111.77 ± 21.97 U/L ( Figure 3A). The control serum AST concentration was 440.4 ± 177.39 U/L; the vanillin treatment AST concentration markedly increased, though remained relatively constant, at all vanillin treatment concentrations ( Figure 3B). The control serum ALP concentration was 26.67 ± 12.66 U/L; the vanillin treatment ALT concentration initially markedly decreased, then gradually increased, with the increased vanillin concentration ( Figure 3C). The control serum LDH concentration was 2560.02 ± 7.23 U/L and moderately decreased at all vanillin treatment concentrations, with a minimum of 1.00 g/L ( Figure 3D).

Effect of Vanillin Concentration on the Blood Serum Ion Concentrations of Crucian Carp
The PO 4 3− concentration gradually decreased from the control value with the increased vanillin concentration, with a minimum of 1.25 g/L, and a significant increase at 1.50 g/L ( Figure 4A). The K + concentration markedly decreased from the control value with increased vanillin concentration, with a minimum of 1.00 g/L, and a significant increase at 1.50 g/L ( Figure 4B). The Mg 2+ concentration gradually decreased from the control value with increased vanillin concentration, with a minimum of 1.25 g/L, and a slight increase of 1.50 g/L ( Figure 4C). The Na + concentration significantly increased from the control value at all vanillin treatment concentrations, with a gradual increase with increased vanillin concentration to a maximum of 1.25 g/L, and a small decrease at 1.50 g/L ( Figure 4D).

Effect of Vanillin Concentration on the Blood Serum Concentrations of Organic Components in Crucian Carp
The control TP concentration of 76.33 ± 6.79 g/L decreased gradually with increasing vanillin treatment concentration to a minimum of 1.00 g/L vanillin, then gradually increased; the variation in ALB content was very similar to that of the TP content ( Figure  5A). The CHO concentration of the vanillin treatments was slightly higher than the control ( Figure 5B). The GLU concentration varied widely with increased vanillin concentration, increasing markedly at 0.50 g/L with a further increase at 0.75 g/L, followed by a marked decrease to a minimum at 1.25 g/L, and then a small increase at 1.50 g/L ( Figure  5C). The control TG concentration slightly increased at 0.50 g/L vanillin, then slightly decreased with increasing vanillin concentration ( Figure 5D).

Effect of Vanillin Concentration on the Blood Serum Concentrations of Organic Components in Crucian Carp
The control TP concentration of 76.33 ± 6.79 g/L decreased gradually with increasing vanillin treatment concentration to a minimum of 1.00 g/L vanillin, then gradually increased; the variation in ALB content was very similar to that of the TP content ( Figure 5A). The CHO concentration of the vanillin treatments was slightly higher than the control ( Figure 5B). The GLU concentration varied widely with increased vanillin concentration, increasing markedly at 0.50 g/L with a further increase at 0.75 g/L, followed by a marked decrease to a minimum at 1.25 g/L, and then a small increase at 1.50 g/L ( Figure 5C). The control TG concentration slightly increased at 0.50 g/L vanillin, then slightly decreased with increasing vanillin concentration ( Figure 5D).

Histopathological Examination
Liver tissue sections ( Figure 6) showed that the hepatocytes in the control and treatment groups were evenly arranged, intact and clear, except at 1 g/L vanillin, and vacuolation in the treatment groups was more marked than in the control. At 1.50 g/L vanillin, the accumulation of hepatocyte nuclei was greater than in the control, though their integrity was better.

Histopathological Examination
Liver tissue sections ( Figure 6) showed that the hepatocytes in the control and treatment groups were evenly arranged, intact and clear, except at 1 g/L vanillin, and vacuolation in the treatment groups was more marked than in the control. At 1.50 g/L vanillin, the accumulation of hepatocyte nuclei was greater than in the control, though their integrity was better.

Histopathological Examination
Liver tissue sections ( Figure 6) showed that the hepatocytes in the control and treatment groups were evenly arranged, intact and clear, except at 1 g/L vanillin, and vacuolation in the treatment groups was more marked than in the control. At 1.50 g/L vanillin, the accumulation of hepatocyte nuclei was greater than in the control, though their integrity was better.  Gill tissue sections (Figure 7) showed that the control gill lamellae (GL) were flattened, vesicular, elongated, and arranged in a comb-like pattern, they consisted of a single layer of epithelial cells, capillaries, and columnar support cells. The epithelial cells were flattened with spindle-shaped nuclei, and the columnar support cells had large, round nuclei and were attached to the basement membrane. The filaments and lamellae contained small numbers of chlorine-secreting cells. Compared to the control, the filaments and lamellae were swollen at 1.50 g/L vanillin; there was a proliferation of flattened and columnar cells in the vascular walls of the filaments, an increase in chlorine-secreting cells, curling and fusion of the lamellae, and detachment of the supragillar cells. Microstructural measurements showed a slight swelling of the chlorine-secreting cells at all treatment concentrations, which was not significantly different from the control, except at 1.00 g/L, at which the width and spacing of the gill lamellae increased significantly. Gill tissue sections (Figure 7) showed that the control gill lamellae (GL) were flattened, vesicular, elongated, and arranged in a comb-like pattern, they consisted of a single layer of epithelial cells, capillaries, and columnar support cells. The epithelial cells were flattened with spindle-shaped nuclei, and the columnar support cells had large, round nuclei and were attached to the basement membrane. The filaments and lamellae contained small numbers of chlorine-secreting cells. Compared to the control, the filaments and lamellae were swollen at 1.50 g/L vanillin; there was a proliferation of flattened and columnar cells in the vascular walls of the filaments, an increase in chlorinesecreting cells, curling and fusion of the lamellae, and detachment of the supragillar cells. Microstructural measurements showed a slight swelling of the chlorine-secreting cells at all treatment concentrations, which was not significantly different from the control, except at 1.00 g/L, at which the width and spacing of the gill lamellae increased significantly.

Radar Fingerprinting of the E-Nose Sensor Responses to Carp-Muscle Flavour Volatiles
The muscle aroma of the anaesthetized crucian carp was analyzed at different vanillin treatment concentrations. The 18 sensors of the FOX4000 E-Nose are sensitive to different chemical classes of volatile compounds and the ratio of relative conductivity G/G0 is proportional to the concentration of the corresponding compound class. When G/G0 > 1, the volatile concentration is detectable and when G/G0 ≤ 1 the volatile concentration is below the detection limit [23]. There was a wide variation in sensor response (Figure 8), with six sensors giving no response and those of T30/1, P10/1, PA/2, and P30/1 being relatively strong. However, the differences between the control and the six vanillin treatment concentrations were very small. It appears that vanillin treatment has a negligible effect on fish flavour, which is highly desirable from a commercial viewpoint.

Radar Fingerprinting of the E-Nose Sensor Responses to Carp-Muscle Flavour Volatiles
The muscle aroma of the anaesthetized crucian carp was analyzed at different vanillin treatment concentrations. The 18 sensors of the FOX4000 E-Nose are sensitive to different chemical classes of volatile compounds and the ratio of relative conductivity G/G 0 is proportional to the concentration of the corresponding compound class. When G/G 0 > 1, the volatile concentration is detectable and when G/G 0 ≤ 1 the volatile concentration is below the detection limit [23]. There was a wide variation in sensor response (Figure 8), with six sensors giving no response and those of T30/1, P10/1, PA/2, and P30/1 being relatively strong. However, the differences between the control and the six vanillin treatment concentrations were very small. It appears that vanillin treatment has a negligible effect on fish flavour, which is highly desirable from a commercial viewpoint.

Linear Discriminant Analysis (LDA) of E-Nose Data
LDA is a statistical method that uses samples of known categories to establish a discriminant model and discriminate samples of unknown categories [24]. The E-Nose data were subjected to an LDA dimension reduction analysis (Figure 9). The contributions of LDA1 and LDA2 were 77.29% and 19.69%, respectively, and the combined contribution was 96.98%. LDA can distinguish the treatment and control samples, though there was no dose response to the vanillin treatment.

Linear Discriminant Analysis (LDA) of E-Nose Data
LDA is a statistical method that uses samples of known categories to establish a discriminant model and discriminate samples of unknown categories [24]. The E-Nose data were subjected to an LDA dimension reduction analysis (Figure 9). The contributions of LDA1 and LDA2 were 77.29% and 19.69%, respectively, and the combined contribution was 96.98%. LDA can distinguish the treatment and control samples, though there was no dose response to the vanillin treatment.

Linear Discriminant Analysis (LDA) of E-Nose Data
LDA is a statistical method that uses samples of known categories to establish a discriminant model and discriminate samples of unknown categories [24]. The E-Nose data were subjected to an LDA dimension reduction analysis (Figure 9). The contributions of LDA1 and LDA2 were 77.29% and 19.69%, respectively, and the combined contribution was 96.98%. LDA can distinguish the treatment and control samples, though there was no dose response to the vanillin treatment.

GC-IMS Analysis of Aroma Volatiles from Crucian Carp Muscle at Different Vanillin Treatment Concentrations
The GC-IMS 3D spectra of carp-muscle flavour volatiles resulting from different vanillin treatment concentrations ( Figure 10) are not easily distinguished by the eye.

GC-IMS Analysis of Aroma Volatiles from Crucian Carp Muscle at Different Vanillin Treatment Concentrations
The GC-IMS 3D spectra of carp-muscle flavour volatiles resulting from different vanillin treatment concentrations ( Figure 10) are not easily distinguished by the eye.  Table 4), including eight aldehydes, 11 alcohols, 10 ketones, two esters, and one furan.

GC-IMS Analysis of Aroma Volatiles from Crucian Carp Muscle at Different Vanillin Treatment Concentrations
The GC-IMS 3D spectra of carp-muscle flavour volatiles resulting from different vanillin treatment concentrations ( Figure 10) are not easily distinguished by the eye.  Table 4), including eight aldehydes, 11 alcohols, 10 ketones, two esters, and one furan.   Table 4. Table 4. Volatile organic compounds were identified from crucian carp muscle, after treatment with vanillin.  Table 4. To analyze differences in aroma profiles resulting from different vanillin treatment concentrations, a flavour profile fingerprint was generated from the triplicate analyses of the control and vanillin treatment samples ( Figure 13). Some compounds, such as 3-hydroxy-2-butanone, 2,3-butanedione, 2,3-pentanedione, 2,3-butanediol, ethanol, and 2-methylbutanol were present in similar amounts in all samples. Some other compounds varied in content greatly between different samples, such as hexanal, E-2-hexenal, methyl acetate, 2-heptanone, 3-octanone, 4-methyl-3-penten-2-one, 2-pentylfuran, and 5-methylfuran, which were higher in A or D and lower in C, E, and F. The difference in muscle volatile flavour substances between group A and group D was not significant, indicating that vanillin with a mass concentration of 1 g/L had little effect on the muscle flavour of the crucian carp. methylbutanol were present in similar amounts in all samples. Some other compounds varied in content greatly between different samples, such as hexanal, E-2-hexenal, methyl acetate, 2-heptanone, 3-octanone, 4-methyl-3-penten-2-one, 2-pentylfuran, and 5methylfuran, which were higher in A or D and lower in C, E, and F. The difference in muscle volatile flavour substances between group A and group D was not significant, indicating that vanillin with a mass concentration of 1 g/L had little effect on the muscle flavour of the crucian carp.

Discussion and Conclusions
Vanillin had an anaesthetic effect on crucian carp when added to the water in which the fish were present. With an increasing vanillin concentration, the time taken to achieve deep anaesthesia decreased in an exponential, dose-responsive manner and the recovery time of the fish, when removed to vanillin-free water, increased. All the test fish recovered completely and there was no evidence of adverse effects. This is consistent

Discussion and Conclusions
Vanillin had an anaesthetic effect on crucian carp when added to the water in which the fish were present. With an increasing vanillin concentration, the time taken to achieve deep anaesthesia decreased in an exponential, dose-responsive manner and the recovery time of the fish, when removed to vanillin-free water, increased. All the test fish recovered completely and there was no evidence of adverse effects. This is consistent with reports on the anaesthetic effect of eugenol on juvenile yellow-spotted basketfish (Siganus oramin) [25], magnolia essential oil on spotted seabass (Lateolabrax maculatus) [26], and carbon dioxide on juvenile oval pompano trevally (Trachinotus ovatus) [27].
Blood physiological indices in fish are good indicators of their metabolic state, nutritional status, and health. White blood cells (WBC) are an essential part of the immune system [28]; red blood cells (RBC) deliver oxygen to body tissues [29]; platelets (PLT) are mainly involved in blood clotting, though they also have immunomodulatory effects; and haemoglobin HGB is the oxygen-binding protein in RBCs [30]. Liang et al. [31] analysed the anaesthetic effect of clove oil on the blood indexes of tilapia; the content of WBC, RBC, HGB, and PLT increased after anaesthesia. In this study, vanillin-induced anaesthesia moderately reduced the contents of WBC and HGB, though more than halved the contents of RBC and PLT.
Blood serum indices in fish are also good indicators of their metabolic state, nutritional status, health, and of cell membrane integrity [32]. Alanine and aspartate aminotransferases (ALT and AST) are important mitochondrial enzymes and are abundant in hepatocytes [33]. Under normal conditions, only small amounts of aminotransferases are released into the blood from hepatocytes and, therefore, serum aminotransferase activity is low. Elevated serum levels of ALT and AST are indicators of liver damage. Alkaline phosphatase (ALP) is involved in metabolism and the immune system [34]. Lactate dehydrogenase (LDH) is abundant in cardiac muscle cells and increased activity is an indicator of damage to the cardiomyocytes [35]. In this study, the ALT activity of carp anaesthetized with vanillin was reduced, whereas the AST activity markedly increased, indicating that there may have been some liver damage in the fish, which is consistent with the effect of anaesthesia with Buddleja lindleyana on serum AST in crucian carp [36]. The LDH activity was moderately reduced, suggesting that anaesthesia reduced myocardium damage, consistent with the response of MS-222 anaesthesia on bream (Parabramis pekinensis) [37]. The ALP activity of crucian carp anaesthetized by the various concentrations of vanillin solution was lower than that of the control group, indicating that the vanillin aqueous solution contained components that inhibited ALP activity, indicating that vanillin had less effect on ALP in crucian carp.
Inorganic ions such as PO 4 3− , Mg 2+ , K + , and Na + in serum are important for maintaining osmolality, acid-base balance, and overall homeostasis in fish, and their content changes after environmental stress [38]. Elevated serum PO 4 3− and Mg 2+ levels in fish can indicate kidney damage [39]. In this study, the levels of PO 4 3− and Mg 2+ in anaesthetized crucian carp were reduced, indicating that vanillin did not cause kidney damage. The increase in serum Na + and the decrease in serum K + suggest increased permeability of the gill epithelium, which allows leakage of Na + and ingress of K + . In this study, with the increase in the vanillin mass concentration, the Na + content of each concentration of the anaesthesia group increased compared with the control group, and the K + content decreased compared with the control group, and it was speculated that the higher concentrations of vanillin caused increased gill tissue cell deformation. This is consistent with the anaesthesia of tilapia with MS-222, which decreased serum K + in a dose-responsive manner, whereas the Na + concentration remained relatively stable [40].
Glucose in the bloodstream supplies energy for various vital activities in fish [41] and its concentration is regulated by insulin and epinephrine and fluctuates in response to environmental factors [42]. In this study, the treatment group's blood glucose levels were higher than the control in different proportions, possibly due to less dissolved oxygen in the water, which lowered the metabolic rate and/or because of the reduced physical activity of the anaesthetized fish. The increases in GLU concentration after anaesthesia may result from a need for increased energy associated with the stress response, which is met by increased glycogenolysis. At higher vanillin concentrations, there was an irregular decrease in blood glucose, consistent with more rapid anaesthesia, which is consistent with a previous report on electroanesthetized pearl gentian grouper [43]. In contrast, blood glucose concentrations decreased after CO 2 anaesthesia of the grouper [44]. Serum albumin (ALB) is the most abundant serum protein and acts as a transporter for insoluble fatty acids [45]. The ALB concentration was slightly decreased by vanillin treatment. Cholesterol (CHO) is an important component of the cell membrane and is transported in the bloodstream by lipoproteins in fish [46]. Triglycerides, cholesterol, and total protein levels are affected by protein catabolism and hepatic glycogenolysis. In this study, except for triglycerides, the TP and ALB contents of the crucian carp after anaesthesia were lower than those in the control group, indicating that the protein breakdown and liver glycogen decomposition of crucian carp decreased after anaesthesia.
Fishery anaesthetics are a class of substances that inhibit the sensory centres of the fish brain to varying degrees, causing the fish to lose the ability for reflex action. The mechanism of action is to first inhibit the brain cortex (tactile loss phase), then act on the basal ganglia and cerebellum (excitation phase), and finally on the spinal cord (anaesthesia phase) [47]. An appropriate dose of anaesthetic reduces oxygen consumption and ammonia excretion, inhibits excessive stress in the fish, and effectively reduces injury, or mortality during handling. The liver is an important glandular and digestive-metabolic organ in fish and is involved in bile secretion, metabolism, detoxification, and defence [48]. The gills are the main respiratory organ of fish and excrete metabolic wastes, such as ammonia and nitrogen [49]. Anaesthesia, with 40 mg/L MS-222 for 24 h, may cause some damage to the liver tissue of the Larimichthys crocea [50]; eugenol anaesthesia of the carp (Cyprinus carpio) did not result in detectable liver or kidney damage [51]; deep anaesthesia of rainbow trout with Coriandrum sativum essential oil did not result in gill or liver damage [52]. In this study, vanillin caused no detectable pathological effects on the liver. All concentrations of vanillin, except for 1.00 g/L, had some effect on the gill tissue, increasing the width and spacing of the gill lamellae. Further research will be needed to determine whether this is harmful to the fish and the mechanism of any harm.
The electronic nose (E-Nose) is an array of gas sensors that mimics the human olfactory system and characterizes the aroma of a sample, which has low cost, ease of operation, and high accuracy [53]. E-Nose analysis of carp muscle after vanillin treatment indicated that the most abundant flavour volatiles are alcohols, amines, and hydrocarbons. Linear discriminant analysis separated the control and the different vanillin concentration treatments, though there was no clear dose response in the separation, indicating that, although the E-Nose could discriminate the control and treatment samples, there was little difference in the flavour profiles between the different vanillin treatment concentrations. The response of different concentrations of vanillin to the muscle of crucian carp. Gas-phase ion mobility spectrometry (GC-IMS) combines gas chromatography and ion mobility spectrometry [54] and has the advantages of the high resolution of ion mobility spectrometry and the high sensitivity of gas chromatography, resulting in richer chemical information than GC-mass spectroscopy [55]. In this study, the GC-IMS was used to analyze differences in carp-muscle flavour profile after anaesthesia with different vanillin concentrations. A total of 40 flavour compounds were identified, including eight aldehydes, 11 alcohols, 10 ketones, two esters, and one furan. Of these, 3-hydroxy-2-butanone, 2,3-butanedione, 2,3-pentanedione, 2,3-butanediol, ethanol, and 2-methylbutanol were present in all samples with similar signal intensities, suggesting that these compounds are the main contributors to the overall flavour of crucian carp muscle tissue after anaesthesia. Moreover, the difference between the control group and the vanillin concentration of 1 g/L on muscle volatile flavour substances was not significant, indicating that vanillin with a mass concentration of 1 g/L had little effect on the muscle flavour of crucian carp.
In conclusion, the effective concentration range of vanillin to anaesthetize crucian carp is 0.50-1.50 g/L, which resulted in relatively small changes to blood biochemistry, and no detectable liver damage, though minor, nonlethal damage to the gills. Future research on the anaesthetic effect of vanillin should include an examination of molecular toxicology and protein metabolism. GC-IMS proved very useful for the identification of carp-muscle flavour volatiles, however, the GC-IMS database is not yet complete for fish muscle flavour compounds, so more information on these compounds will be needed.